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In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

The Total Solar Irradiance (TSI) primarily varies on an 11-year time scale and is governed by features such as sunspots and associated decay products such as plage and faculae. These short-lived physical features can also modulate the solar irradiance at intermediate and short temporal scales. Here we investigate the periodic variations, at solar-surface-rotation time scales, of photometric indices derived from images obtained at the San Fernando Observatory (SFO), and we compare them to the properties of the contemporaneous TSI as measured by the Total Irradiance Monitor (TIM) onboard the SOlar Radiation and Climate Experiment (SORCE) spacecraft. Both the daily ground- and space-based data, which span from early 2003 to late 2018, present missing pixels. We use an autoregressive gap-filling method to construct continuous time series to be analyzed via Fourier and wavelet spectral techniques. Lomb–Scargle periodograms, which can handle time series with missing data, are used for comparison. Both the Fourier spectral power and the periodograms yield compatible results with statistically significant periodicities in the range 25 – 35 days. All of the time series have maximum power at 27 days. Significant secondary periods are found at 29 – 30 days and 34 – 35 days. Wavelet analyses of the full time series show that the photometric index resulting from the red-continuum photometric sum $[\Sigma _{\mathrm{{r}}}]$ and the TSI exhibit common high power at surface-solar-rotation scales during the active part of the solar cycle. The phase relation at the surface-solar-rotation scales is not definite. During the solar minimum interval between Solar Cycles (SCs) 23 and 24, variations in the TSI are found to be related to variations both in the photometric index $\Sigma _{\mathrm{{K}}}$, calculated from Ca ii K-line photometric sums and in the magnetic flux in the solar activity latitudinal band (as found in previous work). This suggests that the TSI changes during the minimum are caused by the reduced line-blanketing effect of diffused magnetic field.

Systemic amyloidosis is a fatal disorder caused by pathological extracellular deposits of amyloid fibrils that are always coated with the normal plasma protein, serum amyloid P component (SAP). The small-molecule drug, miridesap, [(R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC)] depletes circulating SAP but leaves some SAP in amyloid deposits. This residual SAP is a specific target for dezamizumab, a fully humanized monoclonal IgG1 anti-SAP antibody that triggers immunotherapeutic clearance of amyloid. We report the safety, pharmacokinetics, and dose-response effects of up to three cycles of miridesap followed by dezamizumab in 23 adult subjects with systemic amyloidosis (ClinicalTrials.gov identifier: NCT01777243). Amyloid load was measured scintigraphically by amyloid-specific radioligand binding of 123I-labeled SAP or of 99mTc-3,3-diphosphono-1,2-propanodicarboxylic acid. Organ extracellular volume was measured by equilibrium magnetic resonance imaging and liver stiffness by transient elastography. The treatment was well tolerated with the main adverse event being self-limiting early onset rashes after higher antibody doses related to whole body amyloid load. Progressive dose-related clearance of hepatic amyloid was associated with improved liver function tests. 123I-SAP scintigraphy confirmed amyloid removal from the spleen and kidneys. No adverse cardiac events attributable to the intervention occurred in the six subjects with cardiac amyloidosis. Amyloid load reduction by miridesap treatment followed by dezamizumab has the potential to improve management and outcome in systemic amyloidosis.

A low quadriceps slow-twitch (ST), oxidative (relative to fast-twitch) fiber proportion is prevalent in chronic diseases such Chronic Obstructive Pulmonary Disease (COPD) and is associated with exercise limitation and poor outcomes. Benefits of an increased ST fiber proportion are demonstrated in genetically modified animals. Pathway analysis of published data of differentially expressed genes in mouse ST and FT fibers, mining of our microarray data and a qPCR analysis of quadriceps specimens from COPD patients and controls were performed. ST markers were quantified in C2C12 myotubes with EGF-neutralizing antibody, EGFR inhibitor or an EGFR-silencing RNA added. A zebrafish egfra mutant was generated by genome editing and ST fibers counted. EGF signaling was (negatively) associated with the ST muscle phenotype in mice and humans, and muscle EGF transcript levels were raised in COPD. In C2C12 myotubes, EGFR inhibition/silencing increased ST, including mitochondrial, markers. In zebrafish, egfra depletion increased ST fibers and mitochondrial content. EGF is negatively associated with ST muscle phenotype in mice, healthy humans and COPD patients. EGFR blockade promotes the ST phenotype in myotubes and zebrafish embryos. EGF signaling suppresses the ST phenotype, therefore EGFR inhibitors may be potential treatments for COPD-related muscle ST fiber loss.

Skeletal muscle dysfunction is a frequent extra-pulmonary manifestation of Chronic Obstructive Pulmonary Disease (COPD) with implications for both quality of life and survival. The underlying biology nevertheless remains poorly understood. We measured global gene transcription in the quadriceps using Affymetrix HuGene1.1ST arrays in an unselected cohort of 79 stable COPD patients in secondary care and 16 healthy age- and gender-matched controls. We detected 1,826 transcripts showing COPD-related variation. Eighteen exhibited ≥2fold changes (SLC22A3, FAM184B, CDKN1A, FST, LINC01405, MUSK, PANX1, ANKRD1, C12orf75, MYH1, POSTN, FRZB, TNC, ACTC1, LINC00310, MYH3, MYBPH and AREG). Thirty-one transcripts possessed previous reported evidence of involvement in COPD through genome-wide association, including FAM13A. Network analysis revealed a substructure comprising 6 modules of co-expressed genes. We identified modules with mitochondrial and extracellular matrix features, of which IDH2, a central component of the mitochondrial antioxidant pathway, and ABI3BP, a proposed switch between proliferation and differentiation, represent hubs respectively. COPD is accompanied by coordinated patterns of transcription in the quadriceps involving the mitochondria and extracellular matrix and including genes previously implicated in primary disease processes.

The amyloid deposits that cause disease in systemic amyloidosis always contain the normal plasma protein, serum amyloid P (SAP) component. SAP is the target of a novel immunotherapy approach now being developed to eliminate amyloid deposits. The treatment is enabled by, and critically depends on, the use of the drug (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC, GSK2315698, Ro 63-8695), which depletes circulating SAP almost completely but leaves some SAP in amyloid deposits for specific recognition by subsequently administered therapeutic anti-SAP antibodies. Herein, we report a mechanistic model that predicts, with clinically acceptable precision, the exposure-response relationship for CPHPC, both in healthy individuals and in patients with systemic amyloidosis. The model covariates are gender, renal function, total amyloid load, and presence of hepatic amyloid, all of which are known at baseline. The model is being used to predict individualized dosing regimens in an ongoing, first-in-human study with anti-SAP antibodies.

There has been much speculation about the extended minimum between Solar Cycles 23 and 24. Cycle 24 itself has been unusually weak compared with recent cycles. We present quantitative evidence for the weakness of both Cycles 23 and, particularly, 24. The data are objective indices derived from precision photometric images obtained on a daily basis at the San Fernando Observatory. These data form the longest running, homogeneous photometric record known to us. We show sunspot areas from red images and facular/network areas from Ca ii K-line images. Spot and facular area are a simple and direct measurement of the strength of solar activity. The data clearly show the decline in the amplitude of sunspot maxima for Cycles 23 and 24 compared with Cycle 22. The relative amplitudes of mean spot area for Cycles 22 through 24 are 1.0, 0.74, and 0.37, respectively. There is also an indication that the facular-to-spot area ratio has increased in Cycle 24.

We model total solar irradiance (TSI) using photometric irradiance indices from the San Fernando Observatory (SFO), and compare our model with measurements compiled from different space-based radiometers. Space-based measurements of TSI have been obtained recently from ACRIM-3 on board the ACRIMSAT. These data have been combined with other data sets to create an ACRIM-based composite. From VIRGO on board the Solar and Heliospheric Observatory (SOHO) spacecraft two different TSI composites have been developed. The VIRGO irradiance data have been combined by the Davos group to create a composite often referred to as PMOD (Physikalisch-Meteorologisches Observatorium Davos). Also using data from VIRGO, the Royal Meteorological Institute of Belgium (RMIB) has created a separate composite TSI referred to here as the RMIB composite. We also report on comparisons with TSI data from the Total Irradiance Monitor (TIM) experiment on board the Solar Radiation and Climate Experiment (SORCE) spacecraft. The SFO model correlates well with all four experiments during the seven-year SORCE interval. For this interval, the squared correlation coefficient R 2 was 0.949 for SORCE, 0.887 for ACRIM, 0.922 for PMOD, and 0.924 for RMIB. Long-term differences between the PMOD, ACRIM, and RMIB composites become apparent when we examine a 21.5-year interval. We demonstrate that ground-based photometry, by accurately removing TSI variations caused by solar activity, is useful for understanding the differences that exist between TSI measurements from different spacecraft experiments.

In order to test for human histocompatibility leucocyte antigens (HLA) class II restriction of IgE responses, 431 subjects from 83 families were genotyped at the HLA-DR and HLA-DP loci and serotyped for IgE responses to six major allergens from common aero-allergen sources. A possible excess of HLA-DR 1 was found in subjects who were responsive to FeldI compared with those who were not (Odds Ratio (OR) = 2, P = 0.002), and a possible excess of HLA-DR4 was found in subjects responsive to Alt a I (OR = 1–9, P = 0.006). Increased sharing of HLA-DR/DP haplotypes was seen in sibling pairs responding to both allergens. Der p I, Der p II, Phi p V and Can f I were not associated with any definite excess of HLA-DR alleles. No significant correlations were seen with HLA-DP genotype and reactivity to any of the allergens. The results suggest class II HLA restriction is insufficient to account for individual differences in reactivity to common allergens.

Total solar irradiance (TSI) measurements have been available from the TIM instrument on the SORCE spacecraft since 2003. We compare TSI data, both 24-h and 6-h averages, with photometric indices from red and K-line images obtained on a daily basis at the San Fernando Observatory (SFO). For 1253 days of data from 2 March 2003 to 5 May 2010 we compare the data in linear multiple regression analyses. The best results come from using two photometric indices, the red and K-line photometric sums, and SORCE TSI 6-h averages interpolated to the SFO time of observation. For this case, we obtain a coefficient of multiple determination, R 2, of 0.9495 and a quiet-Sun irradiance S 0 = 1360.810 ± 0.004 W m−2. These results provide further support for the hypothesis that the quiet Sun is constant over time.

The amyloid fibril deposits that cause systemic amyloidosis always contain the nonfibrillar normal plasma protein, serum amyloid P component (SAP). The drug (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC) efficiently depletes SAP from the plasma but leaves some SAP in amyloid deposits that can be specifically targeted by therapeutic IgG anti-SAP antibodies. In murine amyloid A type amyloidosis, the binding of these antibodies to the residual SAP in amyloid deposits activates complement and triggers the rapid clearance of amyloid by macrophage-derived multinucleated giant cells.We conducted an open-label, single-dose-escalation, phase 1 trial involving 15 patients with systemic amyloidosis. After first using CPHPC to deplete circulating SAP, we infused a fully humanized monoclonal IgG1 anti-SAP antibody. Patients with clinical evidence of cardiac involvement were not included for safety reasons. Organ function, inflammatory markers, and amyloid load were monitored.There were no serious adverse events. Infusion reactions occurred in some of the initial recipients of larger doses of antibody; reactions were reduced by slowing the infusion rate for later patients. At 6 weeks, patients who had received a sufficient dose of antibody in relation to their amyloid load had decreased liver stiffness, as measured with the use of transient elastography. These patients also had improvements in liver function in association with a substantial reduction in hepatic amyloid load, as shown by means of SAP scintigraphy and measurement of extracellular volume by magnetic resonance imaging. A reduction in kidney amyloid load and shrinkage of an amyloid-laden lymph node were also observed.Treatment with CPHPC followed by an anti-SAP antibody safely triggered clearance of amyloid deposits from the liver and some other tissues. (Funded by GlaxoSmithKline; ClinicalTrials.gov number, NCT01777243.).

Genome-wide association studies (GWAS) have been a standard practice in identifying single nucleotide polymorphisms (SNPs) for disease susceptibility. We propose a new approach, termed integrative GWAS (iGWAS) that exploits the information of gene expressions to investigate the mechanisms of the association of SNPs with a disease phenotype, and to incorporate the family-based design for genetic association studies. Specifically, the relations among SNPs, gene expression, and disease are modeled within the mediation analysis framework, which allows us to disentangle the genetic effect on a disease phenotype into two parts: an effect mediated through a gene expression (mediation effect, ME) and an effect through other biological mechanisms or environment-mediated mechanisms (alternative effect, AE). We develop omnibus tests for the ME and AE that are robust to underlying true disease models. Numerical studies show that the iGWAS approach is able to facilitate discovering genetic association mechanisms, and outperforms the SNP-only method for testing genetic associations. We conduct a family-based iGWAS of childhood asthma that integrates genetic and genomic data. The iGWAS approach identifies six novel susceptibility genes (MANEA, MRPL53, LYCAT, ST8SIA4, NDFIP1, and PTCH1) using the omnibus test with false discovery rate less than 1%, whereas no gene using SNP-only analyses survives with the same cut-off. The iGWAS analyses further characterize that genetic effects of these genes are mostly mediated through their gene expressions. In summary, the iGWAS approach provides a new analytic framework to investigate the mechanism of genetic etiology, and identifies novel susceptibility genes of childhood asthma that were biologically meaningful.

Higher levels of focal adhesion kinase (FAK) are expressed in colon metastatic carcinomas. However, the signaling pathways and their mechanisms that control cell adhesion and motility, important components of cancer metastasis, are not well understood. We sought to identify the integrin-mediated mechanism of FAK cleavage and downstream signaling as well as its role in motility in human colon cancer GEO cells. Our results demonstrate that phosphorylated FAK (tyrosine 397) is cleaved at distinct sites by integrin signaling when cells attach to collagen IV. Specific blocking antibodies (clone P1E6) to integrin alpha2 inhibited FAK activation and cell motility (micromotion). Ectopic expression of the FAK C-terminal domain FRNK attenuated FAK and ERK phosphorylation and micromotion. Calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal blocked FAK cleavage, cell adhesion, and micromotion. Antisense approaches established an important role for mu-calpain in cell motility. Expression of wild type mu-calpain increased cell micromotion, whereas its point mutant reversed the effect. Further, cytochalasin D inhibited FAK phosphorylation and cleavage, cell adhesion, locomotion, and ERK phosphorylation, thus showing FAK activation downstream of actin assembly. We also found a pivotal role for FAK Tyr(861) phosphorylation in cell motility and ERK activation. Our results reveal a novel functional connection between integrin alpha2 engagement, FAK, ERK, and mu-calpain activation in cell motility and a direct link between FAK cleavage and enhanced cell motility. The data suggest that blocking the integrin alpha2/FAK/ERK/mu-calpain pathway may be an important strategy to reduce cancer progression.

Recently, we showed that autocrine transforming growth factor alpha (TGFalpha) controls the epidermal growth factor receptor (EGFR)-mediated basal expression of integrin alpha2, cell adhesion and motility in highly progressed HCT116 colon cancer cells. We also reported that the expression of basal integrin alpha2 and its biological effects are critically controlled by the constitutive activation of the ERK/MAPK pathway (Sawhney, R. S., Sharma, B., Humphrey, L. E., and Brattain, M. G. (2003) J. Biol. Chem. 278, 19861-19869). In the present report, we further examine the downstream signaling mechanisms underlying EGFR/ERK signaling and integrin alpha2 function in HCT116 cells. Selective MEK inhibitors attenuated TGFalpha-mediated basal activation of p70S6K (S6K) specifically at Thr-389, indicating that this S6K site is downstream of ERK/MAPK signaling. Cells were treated with the selective protein kinase C (PKC) inhibitor bisindolylmaleimide to determine the role of PKC in S6K activation. The Thr-421 and Ser-424 phosphorylation sites of S6K were specifically inhibited by bisindolylmaleimide, which also blocked integrin alpha2 expression, cell adhesion, and motility. These data establish a novel cell motility function of S6K via PKC activation in a cancer cell. In addition, we examined whether mammalian target of rapamycin signaling controls S6K activation. Rapamycin inhibited constitutive S6K phosphorylation specifically at Thr-389, Thr-421, and Ser-424 sites. The assignment of these phosphorylation sites on S6K to biological functions was unequivocally confirmed by transfection of cells with specific single phosphorylation site dominant negative mutants. These experiments show for the first time that autocrine TGFalpha regulates cell adhesion function by multiple signaling pathways via specific phosphorylation sites of S6K in cancer cells.

Human pain models invoking central sensitization, one of the key mechanisms of chronic pain, may be useful for characterizing new analgesics. A new model of electrical hyperalgesia can detect the efficacy of several analgesic mechanisms. Because IV adenosine can alleviate neuropathic pain, we investigated its effect on experimental sensitization. This was a double-blinded, randomized, two-period crossover study in 20 healthy volunteers. Current pulses (0.5 ms; 1 Hz) were applied intracutaneously to achieve pain rating of approximately 5 on a 0-10 numeric rating scale. Pain, areas of pinprick hyperalgesia, and tactile allodynia were assessed during the 2.5-h stimulation period. Adenosine (50 microg. kg(-1). min(-1)) and placebo were infused IV over 60 min. Additional testing was performed 24 h after each treatment. Adenosine reduced the area of pinprick hyperalgesia during the infusion compared with placebo; there was no significant effect on tactile allodynia or pain rating. The effect on hyperalgesia developed over 15 min and was significant (P < or = 0.05) for the rest of the infusion period. There was no difference between treatments at 24 h. Thus, in accordance with reports on neuropathic pain, adenosine reduced central sensitization in the human model of electrical hyperalgesia. However, adenosine did not have the long-term effects seen in patients. The model can investigate mechanisms of drugs for the treatment of chronic pain.

The latest SOHO VIRGO total solar irradiance (TSI) time series is analyzed using new solar variability measures obtained from full-disk solar images made at the San Fernando Observatory and the Mg II 280 nm index. We discuss the importance of solar cycle 23 as a magnetically simpler cycle and a variant from recent cycles. Our results show the continuing improvement in TSI measurements and surrogates containing information necessary to account for irradiance variability. Use of the best surrogate for irradiance variability due to photospheric features (sunspots and faculae) and chromospheric features (plages and bright network) allows fitting the TSI record to within an rms difference of 130 ppm for the period 1986 to the present. Observations show that the strength of the TSI cycle did not change significantly despite the decrease in sunspot activity in cycle 23 relative to cycle 22. This points to the difficulty of modeling TSI back to times when only sunspot observations were available.

Summary Background Polymorphisms within the β subunit of the high-affinity receptor for IgE (FceR1-β) on chromosome 11q13 have been related to atopy and asthma and the lymphotoxin α (LTα) gene on chromosome 6 is implicated in asthma. Objective To elucidate the association of polymorphisms in the FceR1-β and LTα genes to IgE responses and asthma in a family-orientated rural population. Methods A total of 461 adult farmers, who participated in an epidemiological follow-up study on respiratory symptoms among farmers on the Swedish island of Gotland, were examined. The traits assessed included serum total IgE, IgE antibody responses to 21 common inhalant allergens and asthma. Results The 237G mutation was only detected in seven persons. Atopy was found to be associated with the RsaI-ex7 AB-genotype (OR = 1.9; P = 0.04). The RsaI-ex7 B allele had a significant influence on IgE responses to pollens and dust mites (OR = 5.5; P = 0.03 and OR = 5.2; P = 0.049, respectively). The influence of this allele was stronger when the association towards single dust mite species (Lepidoglyphus destructor) was estimated (OR = 7.1, P = 0.03) and the association increased even more when the major allergen of L. destructor (rLep d 2) was analysed (OR = 11.2, P = 0.02). These associations were independent of sex, age and smoking, and the estimates of RsaI-in2 independent of RsaI-ex7. RsaI-in2, RsaI-ex7 and LTα genotypes were unassociated with total serum IgE. No significant difference in the distribution of RsaI-in2, RsaI-ex7 and LTα genotypes was found among subjects with atopy or asthma compared to healthy controls. Conclusion This study supports the notion that polymorphisms in the FceR1-β gene have significant effects on IgE responsiveness. Secondly, dust mites in rural populations influence the expression of genes on chromosome 11q13.

We report new results on the ratio of facular area to sunspot area from a program of continuing photometric observations using the Cartesian Full Disk Telescope No. 1 (CFDT1) at the San Fernando Observatory (SFO). The facular areas are determined from images obtained with a 1 nm bandpass Ca II K line filter, and sunspot areas are determined from red images at 672 nm with a 10 nm bandpass filter. On the K line images faculae were identified by pixels that had a contrast equal to or greater than 4.8% divided by μ. Previously, we found that the average facular-to-spot area ratio was 16.7 ± 0.5 during the latter part of solar cycle 22 and that there was a small but statistically significant rise in the ratio with time. If we take an average from the beginning of the K line data (mid-1988) until the middle of 1996, excluding days of zero sunspot area, the average ratio is 16.4 ± 0.4. The average ratio from mid-1996 to the end of 1999 November is 12.6 ± 0.5. Including days of zero sunspot area for these same intervals we find average ratios of 16.8 ± 0.5 and 13.2 ± 0.6, respectively. We have recently reprocessed our K line images, which have been photometrically "cleaned." We can now reliably identify facular pixels with a contrast criterion of 2.4%, resulting in an increase in the average facular-to-spot ratio of approximately 3. The average facular and sunspot areas for cycle 23 are significantly lower than for cycle 22.

Analysis of the current solar cycle 23 shows a greater increase in total solar irradiance (TSI) for the early phase of this cycle than expected from measurements of the total magnetic flux and traditional solar activity indices, which indicate that cycle 23 is weaker than cycle 22. In contrast, space observations of TSI from the Solar and Heliospheric Observatory/VIRGO and the Upper Atmospheric Research Satellite/ACRIMII show an increase in TSI of about 0.8-1.0 W m-2 from solar minimum in 1996 to the end of 1999. This is comparable to the TSI increase measured by Nimbus 7/ERB from 1986 to 1989 during the previous cycle. Thus, solar radiative output near the maximum of the 11 yr cycle has been relatively constant despite a factor of 2 smaller amplitude increase for cycle 23 in sunspot and facular areas determined from ground-based observations. As a result, empirical models of TSI based on sunspot deficit and facular/network excess in cycle 22 underestimate the TSI measurements in 1999. This suggests either a problem in the observations or a change in the sources of radiative variability on the Sun.

Photometric images are used to measure the temperature of sunspots at different wavelengths. Images at 672.3 nm and 472.3 nm are obtained at the San Fernando Observatory using the CFDT2 (2.5” x 2.5” pixels). Images at 607.1 nm and 409.4 nm are obtained by the PSPT at Mauna Loa Observatory. Monochromatic intensities are converted to temperatures as in Steinegger et al (1990). The pixel by pixel temperature for a sunspot is converted into a bolometric contrast for that sunspot according to Chapman et al (1994). Sunspot temperatures, i.e., their bolometric contrasts, are calculated from both red (672.3 nm) and blue wavelengths (472.3 nm) and compared.

BACKGROUND Tumour necrosis factor (TNF) is a potent pro-inflammatory cytokine which is prominent in asthmatic airways. TNF shows genetic variations in secretion which are linked to polymorphisms in the TNF gene complex and the surrounding major histocompatibility (MHC) locus. These polymorphisms do not seem to be themselves functionally important. In these circumstances, the identification of disease associated haplotypes (combination of alleles on individual chromosomes) may narrow the search for polymorphisms which alter gene function. METHODS TNF-308, LTα Nco I, and HLA-DRB1 polymorphisms were investigated for association with asthma, bronchial responsiveness, and medication use in 1004 subjects in 230 families from a general population sample. RESULTS The common LTα Nco I*1/TNF-308*2/HLA-DRB1*03 haplotype, which was present in 11% of unrelated individuals, was weakly associated with asthma (OR = 1.38, p = 0.016, corrected for familial correlation). The rarer LTα Nco I*1/TNF-308*2/HLA-DRB1*02 haplotype, which was found in 0.6% of unrelated subjects, was more strongly associated with asthma (OR = 6.68, p = 0.002). This haplotype also showed association with bronchial hyperresponsiveness (OR = 21.9, p = 0.0000) and the use of inhaled or oral steroids (OR 8.0, p = 0.04). CONCLUSIONS The results of this study show only two extended TNF/HLA-DR haplotypes to be associated with asthma. The search for functional alleles responsible for an increased risk of asthma should concentrate on the LTα Nco I*1/TNF-308*2/HLA-DRB1*02 haplotype.

Stainless steel (SS) samples were irradiated with protons at 400°C and strained in 288°C water to examine the role of oxide particles in the irradiation-assisted stress corrosion cracking ...

Daily, photometric, full-disk digital solar images have been taken at the San Fernando Observatory (SFO) at two resolutions and in several wavelengths for more than eleven years. We describe the standard data processing techniques used for these images, including: calibration, limb fitting, geometric correction, and production of a solar contrast map by limb-darkening removal. The resulting contrast maps have a photometric accuracy which is often a few tenths of a percent. We show that the geometric accuracy of our images, as measured by the reproducibility of disk and sunspot areas, is very high as well. The techniques described in this paper should be applicable to any instrument producing full-disk photometric images.

The total irradiance of the Sun has been found to vary mostly because of changes in the areas of dark sunspots and bright faculae. Improved observations, such as those discussed in this paper, are needed to understand better the interplay between these two competing features. In this paper, faculae are determined by observations using a filter centered at the Ca II K line (393.4 nm) with a bandpass of 0.9 nm. This filter allows the detection of faculae across the entire solar disk rather than just at the limb, as is the case for white-light faculae. Sunspots are detected with a filter at 672.3 nm with a bandpass of 9.7 nm. The mean ratio of facular to sunspot area was found to be 16.7 ± 0.51 for a 7½ year period during solar cycle 22 but showed a significant increase as the solar cycle progressed. This ratio suggests that the irradiance excess associated with faculae outweighs the irradiance deficit associated with sunspots by about 50%. The facular area also exhibited a quadratic dependence on sunspot area, as suggested by Foukal, but there is no clear evidence of a turnover in facular area at large sunspot areas. Lagged cross-correlations between facular and sunspot areas showed a clear rotational modulation extending to lags of five to six rotations when spots led faculae. Lags in the opposite direction, however, showed the rotational modulation falling abruptly after about two rotations.

Prostate cancer is the highest-incidence cancer and second-leading cancer killer of men in the U.S. Diagnosis now relies virtually exclusively on core-needle biopsy, guided by transrectal ultrasound (TRUS). Because of the limitations of TRUS in detecting suspicious regions, biopsy often fails to sample cancer that is present or to determine that extracapsular cancer exists, which results in false-negative biopsies or inappropriate prostatectomies. Therefore, the authors conducted this study to investigate the use of spectrum analysis of radio frequency (RF) echo signals as a possible means of reducing the number of false-negative biopsies and inappropriate prostatectomies. This method utilizes databases of parameters derived from normalized power spectra of RF echo signals and histologically proven tissue types to determine ranges of parameter values associated with tissue types of interest. Typing an unknown tissue is performed by comparing the parameter values of the unknown to the value ranges of specific tissue types in the database. The authors' results provide encouraging preliminary discriminant-function distributions that suggest an excellent potential for differentiating cancerous from noncancerous prostate tissue far superior in terms of sensitivity and specificity than means now used to determine whether biopsy is required. In addition, the authors developed images using color to indicate the most likely tissue type throughout the tissue cross section as determined by comparisons with database parameter values. These images showed excellent correlation with histology.

In this study, we have attempted to model the variations in total solar irradiance from two spacecraft. Specifically, we have modeled the Earth Radiation Budget on the Nimbus 7 spacecraft and the active cavity radiometer irradiance monitor (ACRIM-I) on the Solar Maximum Mission (SMM) spacecraft using ground-based photometry of sunspots and faculae from the San Fernando Observatory (SFO). Additionally, for some cases, solar backscatter ultraviolet/2 data on the Mg II core-to-wing ratio from the NOAA 9 spacecraft was used. We have found that most of the solar cycle variation in the total solar irradiance can be accounted for by sunspots and faculae/network. The unexplained variation is not greater than approximately 0.0022% (22 ppm) per year for most of solar cycle 22. Using Nimbus 7 data from March 2, 1985, to December 13, 1993 (1281 data points), as the dependent variable, with the SFO photometric sunspot index (PSI) and the NOAA 9 Mg II core-to-wing ratio for the faculae/network as independent variables (the best model for this interval), we obtained a multiple correlation coefficient squared (R2) of 0.848. The rms noise in the residuals is approximately 0.221 W m−2 (162 ppm). This rms noise appears to be dominated by noise in the spacecraft data. For the same model, but for the time interval from March 2, 1985, to July 14, 1989, we obtained an R2 of 0.838 for 718 data points. The same type of model for this same interval, substituting SMM/ACRIM-I total irradiance for Nimbus 7, gave an R2 of 0.857 for 685 data points. Our best correlation, however, came from a three-parameter model, fitting Nimbus 7 data to the SFO digital PSI, the SFO facular index PFIFA, and the NOAA 9 data for the interval May 30, 1988, to December 13, 1993, giving an R2 of 0.887 (745 data points). These strong correlations suggest that most of the variation in solar irradiance is associated with known solar magnetic features. Whether or not these magnetic features can explain all of the solar irradiance variability will require more stable and accurate long-term measurements from space and the ground.

Daily photometry of the full solar disk began at the San Fernando Observatory in mid-1985. At present, observations with two photometric telescopes produce images in the red, blue and CaII K -line. The smaller telescope obtains images that are 512 × 512 pixels. The larger one obtains images that are 1024 × 1024 pixels. In addition, the larger telescope produces images with a narrower K -line and an IR filter. Images are processed to determine a number of photometric quantities including sunspot deficits and facular/network excesses. These photometric quantities are highly correlated with fluctuations in the total solar irradiance (TSI) from spacecraft experiments.

A facility has been established at the Michigan Ion Beam Laboratory for the study of radiation effects on materials. The capabilities include a broad range of materials (metals, ceramics and polymers), radiation damage rates (10−8 to 10−3 dpa/s) and irradiation temperatures (−196°C to 600°C). The key to the utility of this facility is the control of irradiation dose, dose uniformity, and sample temperature during irradiation. Temperature stability is maintained by simultaneous heating and cooling of the sample stage, and use of a liquid metal interface (for metal samples). The temperature of individual samples in the irradiated area is measured via an infrared pyrometer and thermocouples. Temperature uniformity is confirmed by the pyrometer, while dose uniformity is provided by a split aperture. A total of eight input channels transmit temperature and beam current signals to a 486DX computer to provide feedback to the operator and to record the irradiation history at a frequency of 1 point per second. Continuous irradiations lasting up to 120 hours have been successfully conducted.

In systemic amyloidosis, disease is caused by the extracellular accumulation of amyloid fibrils which do not elicit the normally efficient mechanisms of clearance of interstitial debris, and which progressively disrupt tissue architecture and function. Diagnosis is often late, with advanced organ dysfunction and major morbidity. The condition is usually fatal despite organ support and efforts to reduce production of the fibril precursor protein. In the most common type, AL, caused by monoclonal gammopathy, treatment with cytotoxic chemotherapy can slow or arrest amyloid deposition in some patients, and amyloid deposits may sometimes slowly regress but about 20% of patients still die within 6 months of diagnosis. No specific interventions exist for many of the rarer forms of systemic amyloidosis. Directly targeted measures are required to specifically remove amyloid deposits in order to preserve and possibly restore organ function. We have identified the normal plasma protein, serum amyloid P component (SAP), as a therapeutic target for this purpose. SAP binds to amyloid fibrils of all types and is thus always present in human amyloid deposits. Acute administration of (R)-1-[6-[(R)-2-carboxy-pyrrolidin-1-yl]-6-oxo-hexanoyl]pyrrolidine-2-carboxylic acid (CPHPC), swiftly depletes circulating SAP but leaves some SAP in amyloid deposits as an amyloid-specific antigen target. In patients with systemic AL, AA and AApoAI amyloidosis, we have lately reported that, following depletion of circulating SAP by CPHPC, a single dose of humanized monoclonal anti-SAP antibody substantially reduced the amyloid load, especially from the liver (D.B. Richards et al, New England Journal of Medicine, July 15, 2015; DOI: 10.1056/NEJMoa1504942). Here we show that repeated administration of the obligate therapeutic partnership of CPHPC with appropriate doses of anti-SAP antibody, progressively removed amyloid from the liver and other organs, including the kidney. Up to three antibody doses were given at intervals to patients, mostly with AL or AFib amyloidosis, in this second part of a phase I dose-ascending study. Treatments were generally well tolerated. Infusion reactions were largely mitigated by hydrocortisone and antihistamine premedication. Pharmacodynamic responses were associated with an early transient inflammatory cytokine response and increased CRP and SAA production, followed by substantial depletion of plasma C3 and a less marked fall in C4 and CH50. Pharmacodynamic responses were sometimes associated with self-limiting cutaneous rashes, especially in subjects without hepatic amyloidosis. Reduced amyloid load was demonstrated by radiolabelled SAP scintigraphy (liver, spleen, kidneys), extracellular volume measurement by equilibrium MRI (liver, spleen) and liver stiffness determined by transient elastography. Amyloid removal was not associated with detectable additional organ dysfunction. Abnormal liver function tests improved following clearance of hepatic amyloid. Renal parameters were stable but follow up is still too short to ascertain long term renal effects. In the preclinical mouse model, amyloid clearance mediated by anti-SAP antibody requires complement and macrophages and is effected by multinucleated giant cells. Consistent with this mechanism, the extent of amyloid clearance in patients depended on the dose of anti-SAP antibody in relation to the whole body amyloid load. In patients with hepatic and splenic amyloid, the anti-SAP antibody rapidly disappeared from the circulation, consistent with its easy access to these organs via their sinusoidal endothelium. Liver and spleen amyloid were cleared first but, after major reduction of liver and spleen load, renal amyloid was cleared by subsequent antibody doses. These preliminary observations demonstrate that progressive amyloid removal can probably be achieved in all types of systemic amyloidosis by repeated courses of CPHPC and anti-SAP antibody. A phase II study is now planned. This program is funded by GlaxoSmithKline. Figure 1. Figure 1. Disclosures Pepys: Pentraxin Therapeutics Ltd: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; UK NIHR: Research Funding; UK MRC: Research Funding. Cookson:GlaxoSmithKline: Employment, Equity Ownership. Barton:GlaxoSmithKline: Employment, Equity Ownership. Berges:GlaxoSmithKline: Employment, Equity Ownership. Moon:GlaxoSmithKline: Consultancy, Research Funding. Richards:GlaxoSmithKline: Employment, Equity Ownership.

We have compared total solar irradiance from Nimbus-7 with ground-based photometry from the San Fernando Observatory (SFO) for 109 days between June 1 and December 31, 1988. We have also included in some analyses NOAA-9 SBUV2 data orF10.7 radio flux. The Nimbus-7 data are from orbital samples, averaged to the mean time of observation at SFO. Using the same parameters as in Chapmanet al. (1992), the multiple regression gives anR2 = 0.9131 and a ‘solar minimum’ irradiance,S0, = 1371.76 ± 0.18 W m−2 for the best fit.

Growth-receptor-bound protein (Grb)7 is an adapter protein aberrantly overexpressed, along with the erbB-2 receptor in breast cancer and in other cancers. Normally recruited to focal adhesions with a role in cell migration, it is associated with erbB-2 in cancer cells and is found to exacerbate cancer progression via stimulation of cell migration and proliferation. The G7-18NATE peptide (sequence: WFEGYDNTFPC cyclized via a thioether bond) is a nonphosphorylated peptide that was developed for the specific inhibition of Grb7 by blocking its SH2 domain. Cell-permeable versions of G7-18NATE are effective in the reduction of migration and proliferation in Grb7-overexpressing cells. It thus represents a promising starting point for the development of a therapeutic against Grb7. Here, we report the crystal structure of the G7-18NATE peptide in complex with the Grb7-SH2 domain, revealing the structural basis for its interaction. We also report further rounds of phage display that have identified G7-18NATE analogues with micromolar affinity for Grb7-SH2. These peptides retained amino acids F2, G4, and F9, as well as the YDN motif that the structural biology study showed to be the main residues in contact with the Grb7-SH2 domain. Isothermal titration calorimetry measurements reveal similar and better binding affinity of these peptides compared with G7-18NATE. Together, this study facilitates the optimization of second-generation inhibitors of Grb7.

NIDDM has a strong genetic component, as evidenced by the high level of concordance between identical twins. The nature of the genetic predisposition has remained largely unknown. Recently, the glucokinase gene locus on chromosome 7p has been shown to be linked to a subtype of NIDDM known as MODY in French and British pedigrees, and glucokinase mutations have been identified. To study the relationship between the glucokinase gene and NIDDM, we performed a linkage analysis in 12 Caucasian pedigrees ascertained through a proband with classical NIDDM. The LINKAGE program was used under four models, including autosomal dominant and recessive, with individuals with glucose intolerance counted as either affected or of unknown status. Linkage was significantly rejected with the dominant models (LOD scores −4.65, −4.25), and was unlikely with the recessive model when glucose intolerance was considered as affected (LOD score −1.38). These findings suggest that mutations in or near the glucokinase gene are unlikely to be the major cause of the inherited predisposition to NIDDM in Caucasian pedigrees, but do not exclude a role for this locus wit a polygenic model, or a major role in some pedigrees.

This paper presents the results of a survey of alcohol use by 604young male convicted offenders. From the internal consistency of the data, and from verification from another sample, it was concluded that trainees in Young Offender Institutions were reporting on their drinking behaviour with a fair degree of accuracy. A significant association was found between drinking at the time of the current offence and offences of a violent nature. A significant association was also found between violent current offences and the consumption of excessive amounts of spirits. Drunkenness was found to be related to large and excessive consumption of beer, cider, or lager. Habitual drunkenness was associated with self-reports of all types of delinquency. Previous work by the Young Offender Psychology Unit (YOPU) of the Home Office Prison Department (Thornton et al. 1990) has shown that Young Offender Institution inmates with violent current offences are more likely to claim to have been under the influence of alcohol at the time of the offence than are inmates with acquisitive current offences. The research reported here is a continuation of the earlier work in that it attempts to explore this link further. Sample The sample consisted of604 young (17-21-year-old) convicted male offenders. A team of psychologists visited all Young Offender Institutions in England (excluding juvenile and short-term establishments) over a period of a year beginning in 1988, and interviewed twenty-four randomly selected trainees in each. These trainees, having been sentenced to detention in a custodial institution for a period of at least four months, are representative not of young offenders in general, but of those who have committed the more serious offences or who have previous convictions. In one section of the interview, trainees were asked a number of questions about consumption of alcohol at the time of the current offence, and also about their normal drinking habits. In another section they were asked how many times they had committed various types of crime. Data were also collected on each trainee's current offence and any previous convictions.

Background: Initial studies by positional cloning have identified the genes encoding the plant homeodomain zinc finger protein 11 (PHF11) and dipeptidyl-peptidase 10 (DPP10) as asthma susceptibility genes. The variants in the two genes have been associated with asthma in several populations of European or Latin American origin. Objective: The aim of this study was to assess the common PHF11 and DPP10 polymorphisms for associations to asthma and asthma-related traits in a Han Chinese population. Methods: We genotyped six polymorphic markers in PHF11 and five polymorphic markers in DPP10 in a Han Chinese case-control cohort consisting of 408 asthma patients and 288 unrelated disease-free controls recruited from the Northern region of China. We analyzed the association between these markers and asthma as well as a number of intermediate, asthma-related traits. Linkage disequilibrium and haplotype patterns were also evaluated. Results: Significant associations were identified between two makers in PHF11 (rs1046295 and rs16659) and asthma susceptibility (odds ratio, OR = 1.32, 95% con fidence interval, CI = 1.06–1.65, p = 0.0096, for rs1046295, and OR = 1.41, 95% CI = 1.12–1.75, p = 0.0026, for rs16659). A strong association was observed between an SNP in DPP10 (rs10208402) and loge-transformed total IgE (p = 0.0003) and the percentage of peripheral blood eosinophils (p = 0.0023). A weak association between rs1430090 in DPP10 and forced expiratory volume in 1 s was also observed (p = 0.048). Haplotype analysis revealed two protective haplotypes in PHF11 against asthma. Conclusion: The results provide supporting evidence for genetic variants in PHF11 and DPP10 genes underlying asthma susceptibility and asthma-related quantitative traits in a Han Chinese population.

A technique is developed which addresses the problem of irradiation assisted stress corrosion cracking of stainless steels in light water reactors using high energy protons to induce grain boundary segregation. These results represent the first grain boundary segregation measurements in bulk produced by proton irradiation of stainless steel. The technique allows the study of grain boundary composition with negligible sample activation, short irradiation time, rapid sample turnaround and at minimal cost. Scanning Auger electron microscopy is used to obtain grain boundary composition measurements of irradiated and unirradiated samples of ultra high purity (UHP) type 304L stainless steel and UHP type 304L steels with the additions of phosphorus (UHP + P) and sulphur (UHP + S). Results show that irradiation of all three alloys causes significant Ni segregation to the grain boundary and Cr and Fe away from it. Irradiation of the UHP + P alloy also results in segregation of P at the grain boundary from...

Grb7 has potential importance in the progression of cancer. We have previously identified a novel peptide that binds to the SH2 domain of Grb7 and inhibits its association with several different receptor tyrosine kinases. We have synthesised the Grb7 peptide, G7-18NATE, with two different cell penetrating peptides, Penetratin and Tat. In this study, we have shown that both Penetratin- and Tat-conjugated G7-18NATE peptides are able to inhibit the proliferation of SK-BR-3, ZR-75-30, MDA-MB-361 and MDA-MB-231 breast cancer cells. There was no significant effects on breast cancer MCF-7cells, non-malignant MCF 10A or 3T3 cells. In addition, there was no significant inhibition of proliferation by Penetratin or Tat alone or by their conjugates with arbitrary peptide sequence in any of the cell lines tested. We determined the EC50 of G7-18NATE-P peptide for SK-BR-3 cell proliferation to be 7.663 x 10(-6) M. Co-treatment of G7-18NATE-P peptide plus Doxorubicin in SK-BR-3 breast cancer cells resulted in an additional inhibition of proliferation, resulting in 56 and 84% decreases in the Doxorubicin EC50 value in the presence of 5 x 10(-6) and 1.0 x 10(-5) M G7-18NATE-P peptide, respectively. Importantly, the co-treatment with Doxorubicin and the delivery peptide did not change the Doxorubicin EC50. Since Grb7 associates with ErbB2, we assessed whether the peptide inhibitor would have a combined effect with a molecule that targets ErbB2, Herceptin. Co-treatment of Herceptin plus 1.0 x 10(-5) M G7-18NATE-P peptide in SK-BR-3 cells resulted in a 46% decrease in the Herceptin EC50 value and no decrease following the co-treatment with Herceptin and penetratin alone. This Grb7 peptide has potential to be developed as a therapeutic agent alone, in combination with traditional chemotherapy, or in combination with other targeting molecules.

Higher levels of focal adhesion kinase (FAK) are expressed in colon metastatic carcinomas. However, the signaling pathways and their mechanisms that control cell adhesion and motility, important components of cancer metastasis, are not well understood. We sought to identify the integrin-mediated mechanism of FAK cleavage and downstream signaling as well as its role in motility in human colon cancer GEO cells. Our results demonstrate that phosphorylated FAK (tyrosine 397) is cleaved at distinct sites by integrin signaling when cells attach to collagen IV. Specific blocking antibodies (clone P1E6) to integrin alpha2 inhibited FAK activation and cell motility (micromotion). Ectopic expression of the FAK C-terminal domain FRNK attenuated FAK and ERK phosphorylation and micromotion. Calpain inhibitor N-acetyl-leucyl-leucyl-norleucinal blocked FAK cleavage, cell adhesion, and micromotion. Antisense approaches established an important role for mu-calpain in cell motility. Expression of wild type mu-calpain increased cell micromotion, whereas its point mutant reversed the effect. Further, cytochalasin D inhibited FAK phosphorylation and cleavage, cell adhesion, locomotion, and ERK phosphorylation, thus showing FAK activation downstream of actin assembly. We also found a pivotal role for FAK Tyr(861) phosphorylation in cell motility and ERK activation. Our results reveal a novel functional connection between integrin alpha2 engagement, FAK, ERK, and mu-calpain activation in cell motility and a direct link between FAK cleavage and enhanced cell motility. The data suggest that blocking the integrin alpha2/FAK/ERK/mu-calpain pathway may be an important strategy to reduce cancer progression.