The Potential Role of Small-Molecule PERK Inhibitor LDN-0060609 in Primary Open-Angle Glaucoma Treatment.

Primary open-angle glaucoma (POAG) constitutes the most common type of glaucoma. Emerging evidence suggests that Endoplasmic Reticulum (ER) stress and the protein kinase RNA-like endoplasmic reticulum kinase (PERK)-mediated Unfolded Protein Response (UPR) signaling pathway play a key role in POAG pathogenesis. Thus, the main aim of the study was to evaluate the effectiveness of the PERK inhibitor LDN-0060609 in cellular model of glaucoma using primary human trabecular meshwork (HTM) cells. To evaluate the level of the ER stress marker proteins, Western blotting and TaqMan gene expression assay were used. The cytotoxicity was measured by XTT, LDH assays and Giemsa staining, whereas genotoxicity via comet assay. Changes in cell morphology were assessed by phase-contrast microscopy. Analysis of apoptosis was performed by caspase-3 assay and flow cytometry (FC), whereas cell cycle progression by FC. The results obtained have demonstrated that LDN-0060609 triggered a significant decrease of ER stress marker proteins within HTM cells with induced ER stress conditions. Moreover, LDN-0060609 effectively increased viability, reduced DNA damage, increased proliferation, restored normal morphology, reduced apoptosis and restored normal cell cycle distribution of HTM cells with induced ER stress conditions. Thereby, PERK inhibitors, such as LDN-0060609, may provide an innovative, ground-breaking treatment strategy against POAG.

Inhibition of the PERK-Dependent Unfolded Protein Response Signaling Pathway Involved in the Pathogenesis of Alzheimer's Disease.

OBJECTIVES: There is a body of evidence that neurodegenerative disease entities are directly correlated with the perturbations on the molecular level. Hence, the ER stress-mediated Unfolded Protein Response (UPR) is activated resulting in PERK-dependent phosphorylation of the Eukaryotic initiation factor 2 (eIF2α). Thus, the levels of ATF4 and CHOP proteins are significantly increased, which subsequently switches the pro-adaptive branch of the UPR into the pro-apoptotic directly leading to neuronal loss and initiation of the neurodegenerative process. The aim of the presented study was the evaluation of the biological activity of highly specific, small-molecule inhibitors of the PERKdependent UPR signaling pathway. METHODS: The study was conducted on rat astrocytic DI TNC1 cell line. The level of p-eIF2α was measured by Western blot technique, the cytotoxicity of the investigated compound was assessed by the MTT assay and using the FITC-conjugated Annexin V (Annexin V-FITC) to indicate apoptosis and propidium iodide (PI) to indicate necrosis. The effect of tested compound on cell cycle progression was measured by flow cytometry, where the PI-labelled nuclei were analysed for DNA content. RESULTS: As a result one of the investigated compound LDN-0060609 triggers a significant inhibition of the eIF2α phosphorylation in DI TNC1 cell line. Moreover, we showed that compound LDN-0060609 is non-cytotoxic and has no effect on cell cycle progression. CONCLUSION: In conclusion, LDN-0060609 may constitute a novel, targeted treatment approach against neurodegenerative diseases, including Alzheimer's disease (AD), where pathogenesis and progression are closely associated with the overactivation of the PERK-dependent UPR signaling pathway.

Cerebral Oximetry for Detecting High-mortality Risk Patients with Cryptococcal Meningitis.

BACKGROUND: Cryptococcus is the commonest cause of adult meningitis in Africa, with 50%-70% experiencing increased intracranial pressure. Cerebral oximetry is a noninvasive near-infrared spectroscopy technology to monitor percent regional cerebral tissue oxygenation (rSO2). We assessed if cerebral oximetry predicts meningitis mortality. METHODS: We performed cerebral oximetry within 14 days of cryptococcal meningitis diagnosis on 121 Ugandans from April 2016 to September 2017. We evaluated baseline rSO2 association with mortality by multivariable logistic regression and correlation with other clinical factors. We compared groups formed by initial rSO2 <30% vs ≥30% for longitudinal change with mixed effects models. We measured change in %rSO2 before and after lumbar puncture (LP). RESULTS: The median initial rSO2 (interquartile range) was 36% (29%-42%), and it was <30% in 29% (35/121). For 30-day mortality, the unadjusted odds ratio (per 5% increase in rSO2) was 0.73 (95% confidence interval [CI], 0.58 to 0.91; P = .005). Those with initial rSO2 <30% had 3.4 (95% CI, 1.5 to 8.0) higher odds of 30-day mortality than those with initial rSO2 ≥30%. Hemoglobin correlated with initial rSO2 (rho = .54; P < .001), but rSO2 did not correlate with pulse oximetry, intracranial pressure, cerebral perfusion pressure, or quantitative cerebrospinal fluid culture, and rSO2 was unchanged pre/post-lumbar punctures. The longitudinal rSO2 measurements change was 15% (95% CI, 12% to 18%) lower in the group with initial rSO2 <30%. CONCLUSIONS: Individuals with cryptococcal meningitis and low cerebral oximetry (rSO2 < 30%) have high mortality. Cerebral oximetry may be useful as a prognostic marker of mortality. Targeted interventions to improve rSO2 should be tested in trials to try to decrease mortality in meningitis.

Microwave photonic delay line signal processing.

This paper provides a path for the design of state-of-the-art fiber-optic delay lines for signal processing. The theoretical forms for various radio-frequency system performance metrics are derived for four modulation types: X- and Z-cut Mach-Zehnder modulators, a phase modulator with asymmetric Mach-Zehnder interferometer, and a polarization modulator with control waveplate and polarizing beam splitter. Each modulation type is considered to cover the current and future needs for ideal system designs. System gain, compression point, and third-order output intercept point are derived from the transfer matrices for each modulation type. A discussion of optical amplifier placement and fiber-effect mitigation is offered. The paper concludes by detailing two high-performance delay lines, built for unique applications, that exhibit performance levels an order of magnitude better than commercial delay lines. This paper should serve as a guide to maximizing the performance of future systems and offer a look into current and future research being done to further improve photonics technologies.

Consequence of the tumor-associated conversion to cyclin D1b.

Clinical evidence suggests that cyclin D1b, a variant of cyclin D1, is associated with tumor progression and poor outcome. However, the underlying molecular basis was unknown. Here, novel models were created to generate a genetic switch from cyclin D1 to cyclin D1b. Extensive analyses uncovered overlapping but non-redundant functions of cyclin D1b compared to cyclin D1 on developmental phenotypes, and illustrated the importance of the transcriptional regulatory functions of cyclin D1b in vivo. Data obtained identify cyclin D1b as an oncogene, wherein cyclin D1b expression under the endogenous promoter induced cellular transformation and further cooperated with known oncogenes to promote tumor growth in vivo. Further molecular interrogation uncovered unexpected links between cyclin D1b and the DNA damage/PARP1 regulatory networks, which could be exploited to suppress cyclin D1b-driven tumors. Collectively, these data are the first to define the consequence of cyclin D1b expression on normal cellular function, present evidence for cyclin D1b as an oncogene, and provide pre-clinical evidence of effective methods to thwart growth of cells dependent upon this oncogenic variant.

Even-order harmonic cancellation for off-quadrature biased Mach-Zehnder modulator with improved RF metrics using dual wavelength inputs and dual outputs.

We present a technique using a dual-output Mach-Zehnder modulator (MZM) with two wavelength inputs, one operating at low-bias and the other operating at high-bias, in order to cancel unwanted even-order harmonics in analog optical links. By using a dual-output MZM, this technique allows for two suppressed optical carriers to be transmitted to the receiver. Combined with optical amplification and balanced differential detection, the RF power of the fundamental is increased by 2 dB while the even-order harmonic is reduced by 47 dB, simultaneously. The RF noise figure and third-order spurious-free dynamic range (SFDR(3)) are improved by 5.4 dB and 3.6 dB, respectively. Using a wavelength sensitive, low V(pi) MZM allows the two wavelengths to be within 5.5 nm of each other for a frequency band from 10 MHz to 100 MHz and 10 nm for 1 GHz.

Is it cost-effective to require recreational ice hockey players to wear face protection?

OBJECTIVE: The purpose of this study was to determine the cost-effectiveness of requiring all recreational hockey players to wear facial protection. METHODS: The authors randomly surveyed recreational hockey players at two indoor hockey rinks in Evendale, Ohio. Data were collected on face protection, injuries, demographic variables and attitudes about protective gear from 11/2005 to 03/2006. RESULTS: We surveyed 190 players. The mean age was 34 +/- 8.7 years and 99% were male. The average hockey experience was 17 years. Forty-six percent of respondents reported at least one serious hockey injury in the last five years. Twenty-four percent of the surveyed population chose to not wear face protection (46/190). The average cost of face protection was $48. The cost to purchase two shields for the 46 players would be $4416. Individuals with face protection reported significantly more sprains and strains that resulted in significantly more physician office visits and specialty physician visits. The extra physician visits would add approximately $4590 for the 46 people needing face protection. Those with face protection reported significantly fewer facial lacerations and facial bone fractures. Requiring face protection should prevent seven facial lacerations and three facial bone fractures over five years. The savings would be approximately $15,000. The net savings by requiring face protection would be $6,000/5 years. In our population, with 24% choosing to not wear face protection, requiring face protection would save $250/5 years/person needing protection. CONCLUSION: It is cost-effective to require facial protection in all recreational hockey players.

In vivo regulation of experimental autoimmune encephalomyelitis by NK cells: alteration of primary adaptive responses.

Innate immune responses provide the host with its first line of defense against infections. Signals generated by subsets of lymphocytes, including NK cells, NKT cells, and APC during this early host response determine the nature of downstream adaptive immune responses. In the present study, we have examined the role of innate NK cells in an autoimmune model through the use of primary immunization with the myelin oligodendrocyte glycoprotein peptide to induce experimental autoimmune encephalomyelitis (EAE). Our studies have shown that in vivo depletion of NK cells can affect the adaptive immune responses, because NK cells were found to regulate the degree of clinical paralysis and to alter immune adaptive responses to the myelin oligodendrocyte glycoprotein peptide. The requirement for NK cells was reflected by changes in the T cell responses and diminished clinical disease seen in mice treated with anti-NK1.1, anti-asialo GM1, and selected Ly49 subtype-depleted mice. In addition to alteration in T cell responses, the maturational status of dendritic cells in lymph nodes was altered both quantitatively and qualitatively. Finally, examination of TCR Vbeta usage of the brain lymphocytes from EAE mice indicated a spectra-type change in receptor expression in NK- depleted mice as compared with non-NK-depleted EAE mice. These findings further establish a recently postulated link between NK cells and the generation of autoreactive T cells.

Determination of total biodiesel fatty acid methyl, ethyl esters, and hydrocarbon types in diesel fuels by supercritical fluid chromatography-flame ionization detection.

A method for determining total biodiesel methyl and ethyl ester content in diesel fuels by supercritical fluid chromatography-flame ionization detection (SFC-FID) is developed. A silica column typically used for determining aromatics in conventional diesel fuels by ASTM D5186 is back-flushed after separation of the hydrocarbons to allow elution of the various esters as a single "total biodiesel" distinct peak. The modification concurrently allows the determination of total aromatic hydrocarbons and their distribution as mono- and polynuclear compounds, as described in the current version of D5186. The instrument response is linear from 1.0% to 50% biodiesel esters with a signal-to-noise ratio of 25 at the 0.1% level. The short-term relative standard is 0.8%. Normalized percent quantitation using a hydrocarbon response factor of 1.00 and an ester response factor of 1.19 provide an average percentage error of 1.8% when measuring actual biodiesel/hydrocarbon fuel blends. The ester response factor is the average of the response factors of 10 pure ester compounds. These responses are calculated from respective solutions of each ester and the four compounds, hexadecane, toluene, tetralin, and naphthalene, as used for the D5186 response factor mixture.

Face protection in recreational hockey players.

BACKGROUND AND OBJECTIVES: Our primary objective was to determine the percentage of recreational hockey players who do not wear face protection while playing hockey. METHODS: We randomly surveyed recreational hockey players at two indoor hockey rinks in Evandale, Ohio. Data were collected on face protection, injuries, demographic variables, and attitudes about protective gear. RESULTS: We surveyed 190 players. Their mean age was 34 +/- 8.7 years, and 99% were male. The average years of hockey experience were 17. Twenty percent of respondents reported using no facial protection either at the time of a past serious injury or currently. Those without face protection were significantly more likely to report having had a past facial laceration (odds ratio [OR]=3.40, 95% confidence interval [CI]=2.0--5.8) or facial bone fracture (OR=10.1, 95% CI=1.23--83.4). On the other hand, 69% of those wearing face protection reported that they felt they could "play more aggressive" with the protection, and they had a higher rate of past injuries other than facial injuries. CONCLUSIONS: Twenty percent of the recreational hockey players reported using no facial protection at the time of a prior serious injury, and players not using facial protection were more likely to report a facial laceration or facial bone fracture. Most players who wore face protection, on the other hand, reported that they played more aggressively and, over time, experienced more serious injuries.

Mucosal tolerance to E-selectin and response to systemic inflammation.

Mucosal tolerance to E-selectin has been shown to prevent stroke and reduce brain infarcts in experimental stroke models. However, the effective E-selectin dose range required to achieve mucosal tolerance and the precise mechanisms of neuroprotection remain unclear. We sought to examine the mechanisms of cytoprotection using gene expression profiling of tissues in the setting of mucosal tolerance and inflammatory challenge. Using spontaneously hypertensive rats (SHRs), we achieved immune tolerance with 0.1 to 5 microg E-selectin per nasal instillation and observed a dose-related anti-E-selectin immunoglobulin G antibody production. We also show the distinct patterns of gene expression changes in the brain and spleen with the different tolerizing doses and lipopolysaccharide (LPS) exposure. Prominent differences were seen with such genes as insulin-like growth factors in the brain and downregulation of those encoding the major histocompatibility complex class I molecules in the spleen. In all, mucosal tolerance to E-selectin and subsequent exposure to LPS resulted in significant tissue changes. These changes, while giving an insight to the underlying mechanisms, serve as possible targets for future studies to facilitate translation to human clinical trials.

An imprinted locus epistatically influences Nstr1 and Nstr2 to control resistance to nerve sheath tumors in a neurofibromatosis type 1 mouse model.

Cancer is a complex disease in which cells acquire many genetic and epigenetic alterations. We have examined how three types of alterations, mutations in tumor suppressor genes, changes in an imprinted locus, and polymorphic loci, interact to affect tumor susceptibility in a mouse model of neurofibromatosis type 1 (NF1). Mutations in tumor suppressor genes such as TP53 and in oncogenes such as KRAS have major effects on tumorigenesis due to the central roles of these genes in cell proliferation and cell survival. Imprinted genes expressed from only one parental chromosome affect tumorigenesis if their monoallelic expression is lost or duplicated. Because imprinted loci are within regions deleted or amplified in cancer, the parental origin of genomic rearrangements could affect tumorigenesis. Gene polymorphisms can vary tumor incidence by affecting rate-limiting steps in tumorigenesis within tumor cells or surrounding stroma. In our mouse model of NF1, the incidence of tumors mutant for the tumor suppressor genes Nf1 and Trp53 is strongly modified by a linked imprinted locus acting epistatically on two unlinked polymorphic loci, Nstr1 and Nstr2. This interaction of an imprinted locus and polymorphic susceptibility loci has profound implications for human mapping studies where the parental contribution of alleles is often unknown.

Determination of aromatic hydrocarbons in gasolines by flow modulated comprehensive two-dimensional gas chromatography.

Valve based/flow modulated comprehensive two-dimensional gas chromatography-flame ionization detection (GC x GC-FID) was used for quantification of C6 through C12 aromatic hydrocarbons by carbon number in gasolines. A 0.53 mm i.d. non-polar first dimension column was coupled to a 0.53 mm i.d. polar second dimension column through a double loop eight port valve modulator. Depending on the sample type, normalized percent and internal standard (I.S.) quantification was performed. For normalized percent quantification, a one-point calibration performed with one aromatic compound per carbon number/class provided an average % accuracy of 2.1% and a short-term n--1 relative standard deviation of 1.0%. For total aromatic compounds good agreement with the more complex conventional multidimensional GC technique was obtained. However, GC x GC has certain advantages over most other methods, mainly increased selectivity for total and carbon number aromatic content. The identification of the aromatic hydrocarbons was confirmed by GC x GC-MS.

The coupling of supercritical fluid chromatography and field ionization time-of-flight high-resolution mass spectrometry for rapid and quantitative analysis of petroleum middle distillates.

We report the first coupling of supercritical fluid chromatography (SFC) with field ionization time-of-flight high-resolution mass spectrometry (FI-ToF HRMS), in parallel with ultraviolet (UV) detection and flame ionization detection (FID), for rapid and quantitative analysis of petroleum middle distillates. SFC separates petroleum middle distillates into saturates and 1- to 3-ring aromatics. FI generates molecular ions for hydrocarbon species eluted from the SFC. The high resolution and exact mass measurements by ToF mass spectrometry provide elemental compositions of the molecules in the petroleum product. The amounts of saturates and aromatic ring types were quantified using the parallel SFC-FID assisted by SFC-UV. With a proper carbon-number calibration, the detailed composition of the petroleum middle distillate was rapidly determined.

Chromosomal location, structure, and temporal expression of the platelet-activating factor receptor (PAFr) gene in porcine endometrium and embryos relative to estrogen receptor alpha gene expression.

Although platelet-activating factor receptor (PAFr) gene was well characterized in the human, little was known about it in domestic animals. Porcine PAFr gene was mapped using fluorescence in situ hybridization (FISH). The structure of this gene was investigated using a 5' rapid amplification of cDNA ends (RACE) technique. Temporal expression of PAFr and estrogen receptor alpha genes (ER), and distribution of the PAFr transcripts in porcine endometrial and embryonic tissues on days 0, 10, 12, 14, 16, and 18 were analyzed using DNA competitors and reverse transcription and polymerase chain reaction (RT-PCR). The porcine PAFr gene was mapped to SSC6q26-27. Alternative splicing of primary transcripts of the PAFr gene produced two different transcripts. Transcript 1 was expressed in all tissues and cells, and transcript 2 was detected in all tissues but white blood cells. The temporal expression of the PAFr gene in endometrial (P > 0.05) and embryonic (P < 0.05) tissues of pregnant sows increased from day 10 to 16. The temporal expression of ER genes in endometrial tissues of pregnant sows decreased from day 10 to 18 (P < 0.05). In addition, ER expression was detectable in 20-60% of embryonic tissue samples, which generally decreased. In combination with previously obtained data on PAF and estradiol-17beta (E(2)) concentrations in pregnant uterine luminal fluids (pULF), endometrial and embryonic tissues, the present results indicated that the increasing PAFr transcripts were positively associated with increasing levels of PAF. Both ER transcripts and E(2) found in pULF decreased correspondingly from day 13 to 16. These results indicate that via PAFr, PAF could play a dominant role in peri-implantation development in pigs as compared to E(2).

Organization of porcine platelet-activating factor receptor gene.

Four exons of porcine platelet-activating factor receptor (PAFr) gene expressing transcript 1 and transcript 2 were determined previously. In this study, we cloned and sequenced a new exon, which also initiates transcript 2, and determined the order of 5 exons in the PAFr gene. In addition, two other variants of transcript 2 were found, but no additional variants of transcript 1 were found. Transcript 2 has three variants that were detected in porcine tissues other than in white blood cells.

The pig platelet-activating factor receptor gene is expressed at the mRNA level in different tissues and is mapped to chromosome 6.

After the pig platelet-activating factor receptor (PAFr) gene was cloned and sequenced, the chromosomal location of this gene was studied using a pig/rodent somatic cell hybrid panel containing 27 cell lines. The results indicated that the pig PAFr gene is located on SSC6q22-23. Platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is thought to be very important in the animal reproductive processes. Its function is mediated through a membrane-bound receptor. Pig PAFr mRNA distribution in different tissues was tested using reverse transcription and PCR (RT-PCR) reactions. All tissues examined expressed PAFr. Using a pig PAFr gene DNA competitor, PAFr expression was quantificated. The pig PAFr mRNA expression level was estimated to be from 1 x 10(2) to 1.2 x 10(4) copies of complementary DNA (cDNA) per 50 ng of total RNA. The highest level was found in lung, and the lowest in the skeletal muscle. These results demonstrated that PAFr was differentially expressed in pig tissues.