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A group of 20 cellulosic biofuels trade groups sent a letter to the Environmental Protection Agency urging it to consider corn kernel fiber-derived cellulosic ethanol as it develops the proposed Renewable Fuel Standard renewable volume obligations for 2019. "Investments in cellulosic biofuel production are paying off, particularly through innovations in use of corn kernel fibers," said Brent Erickson, Biotechnology Innovation Organization executive vice president and head of the Industrial Biotechnology section. Data from the Energy Information Administration showed that ethanol production in the US declined by 41,000 barrels per day from the previous week to reach an average of 1.016 million barrels per day, a five-week low. The four-week average also dropped to 1.044 million barrels per day for an annualized rate of 16 billion gallons. A study published in Science showed how Broad Institute researchers David Liu and Weixin Tang used the CRISPR-Cas9 gene-editing technology to create a cellular recorder from DNA molecules called plasmids and inspired by flight-data recorders found in airplanes. Dubbed as CAMERA1, the device could read information from as few as ten bacterial cells, according to the study. Tire manufacturer Bridgestone Americas has entered into several agreements related to the development of natural rubber and byproducts made from guayule, a desert shrub. Bridgestone and Italian firm Versalis will develop and distribute a technology package to market guayule as a sustainable source of natural rubber and as an important addition to the renewable chemical sector, while its collaboration with genomics firm NRGene aims to improve the shrub's breeding. Sustainability accounting systems help companies document the effects of their environmental, social and governance efforts while providing investors with a better understanding of a firm's overall performance, according to Sustainability Accounting Standards Board Deputy Director of Research David Parham. "There is increasing recognition of -- and demand for -- standardized reporting on material factors related to company performance on ESG impacts," Parham said. Renewable energy will account for more than 50% of global power generation growth by 2040, according to BP's 2018 Energy Outlook. In 2040, renewables should comprise about 14% of the world's energy mix and 25% of its power generation mix. EDF, Orsted and BP are among the energy companies that have recently decided to expand their clean energy investments. "Renewables (and storage) are where the future growth opportunities lie," writes Peter Kelly-Detwiler. Wind is expected to reach grid parity with fossil fuels in the US and Denmark in 2025, according to Lloyd's Register 2018 Technology Radar, an opinion-led survey of 800 industry experts. Seventy-one percent of participants said they expect technology to shape the renewables industry more than policy or regulations over the next five years. IKEA has partnered with Big Clean Switch to offer UK customers the lowest possible prices for renewable energy. Interested customers can sign up for the program, with the eventual energy costs to be based on the number of enrollments. The EU can double the proportion of renewables in its energy portfolio while realizing annual cost savings of up to $139 billion per year by 2030, according to an International Renewable Energy Agency report. By increasing renewables to account for 34% of its energy mix, the EU could also reduce emissions by an additional 15%, the report states.
× Thanks for reading! Log in to continue. Enjoy more articles by logging in or creating a free account. No credit card required. Log in Sign up {{featured_button_text}} DES MOINES -- Gov. Kim Reynolds signed a bill that will expand the role and abilities of physician assistants in Iowa, her office announced Wednesday. The bill allows physician assistants to prescribe or dispense prescription drugs and medical devices, allows for legal protections similar to others in the field and allows them to be reimbursed by Medicaid, among other changes. "Enabling physician assistants to better serve the health care needs of Iowans will result in better care in a more timely fashion," Reynolds said in the release. "As the state continues to combat the spread of COVID-19 and the strains it will place on our health care system, this will be yet another tool in our arsenal." The full text of the bill can be found here. Concerned about COVID-19? Sign up now to get the most recent coronavirus headlines and other important local and national news sent to your email inbox daily. Sign up! * I understand and agree that registration on or use of this site constitutes agreement to its user agreement and privacy policy.
BEST breakfast ever!. When in Portland last month, our visit to this cheerful diner was a highlight. Arrived 9:30am on a Saturday and were greeted with a smile and brought directly to the last booth. Our waters and my coffee arrived immediately while we perused the menu. It was the best coffee, and I happily accepted a couple of refills! I loved the fun décor and our highly capable waitress...
Advances in understanding and assessing malnutrition. To describe the varying views on the pathophysiology of malnutrition in different populations and the definitions that result from these views. To propose an umbrella definition for different malnutrition syndromes and principles of assessment of nutritional state. At present, tacitly or openly inflammatory activity is considered to contribute to the malnourished state. The malnourished state, therefore, arises from a combination of inflammation and a disturbed nutrient balance (undernutrition or overnutrition). The undernourished category of malnutrition leads to loss of body cell mass, which, together with inflammation diminish host response and quality of life. On the basis of these findings, malnutrition may be assessed by estimating nutrient balance but, subsequently, to measure body composition (muscle mass), inflammatory activity (plasma albumin and C-reactive protein) and muscle endurance and force. Changes in muscle function in patients with chronic obstructive pulmonary disease are associated with changes in fiber composition. Few data exist in other malnourished states. There is an increasing acknowledgement of the fact that malnutrition is caused by disturbances in nutrient balance and inflammatory activity. This leads to changes in body composition and diminished function. An umbrella definition has been proposed including the pathogenetic factors, underlying the different malnutrition syndromes and dictating the methods to assess malnutrition.
Persistent expression of an unproductive immunoglobulin heavy chain allele with DH-JH-gamma configuration in peripheral tissues. Genomic recombination events, including VDJ recombination (VDJR) and class-switch recombination (CSR), are indispensable for the adaptation and progression of the acquired immune system. These processes are completed by orderly, temporal onsets of the gene rearrangements along with B-cell differentiation. The presence of various premature transcripts of immunoglobulin heavy chain (IgH) alleles has been demonstrated during B-cell ontogeny. These include D(H)-J(H) (DJ)-mu, J(H)-mu, and sterile transcripts of C(H). Since these transcripts can be detected during the onset of VDJR and CSR, their presence is believed to reflect a structural change in the genome, favoring VDJR and CSR. This report presents evidence of persistent DJ transcription and onset of CSR on an unproductive IgH allele in peripheral tissues. Nucleotide sequence analysis revealed that these transcripts showed DJ-gamma (Dgamma) configuration and that characteristics of the variable region were essentially the same as those of the DJ-mu transcript previously described. It was noted that the small intestine abundantly expresses Dgamma transcripts with gamma2b and gamma1 isotypes of the IgH constant region. The present findings indicate the onset of CSR preceding V(H) to DJ joining in an unproductive IgH allele of the peripheral B cell and the specificity for the gut-associated condition for B-cell differentiation in the small intestine.
Avian infectious bronchitis virus (IBV), a member of family Coronaviridae, order Nidovirales, causes a highly contagious respiratory and sometimes urogenital disease of chickens that is characterized by respiratory signs, nephritis, or reduced egg production and quality in layer chickens. IBV is a major poultry pathogen that is endemic worldwide and leads to serious economic losses. The main method of protecting poultry from infectious bronchitis (IB) is the administration of live or killed vaccines. However, IB continues to cause economic losses in the poultry industry despite intensive vaccination programs in many countries ([@R1]--[@R5]). Outbreaks of IB often are due to infections with strains serologically different from those used for vaccination ([@R2],[@R6]). Since IBV was first described in 1931, a large number of serotypes or variants have emerged, and some have become endemic worldwide ([@R4],[@R7]--[@R12]). That little or no cross-protection occurs between different serotypes of IBV is well known ([@R13]). Therefore, continuous determination of the epidemic serotype and production of new generations of vaccines are crucial for controlling IB in each geographic region or country. IBV was first isolated in the People's Republic of China in the early 1980s; since then, the Massachusetts-type (e.g., H120, H52, Ma5, W93, and 28/86) or Connecticut-type live attenuated IB vaccines and the inactivated killed oil-emulsion vaccine have been used to prevent and control the disease. However, the serotypes of the vaccines used have been epidemiologically determined to differ from those of the predominant IBV isolates in China that form 2 large groups of unique strains, named A2-like and QXIBV-like strains ([@R5],[@R6],[@R14]--[@R18]). Therefore, because of the lack of a vaccine against endemic strains of IBV in China, IBV infection has remained a problem in the Chinese poultry industry. We isolated a virulent IBV strain from 30-day-old broiler chickens in the Yunnan Province and characterized it using sequence alignment, phylogenetic analysis, pathogenicity studies, histopathologic observation, and immunohistochemical (IHC) examination. The results indicate that the isolate is genetically similar to most of the prevalent strains of IBV found in China. We also showed that IBV YN isolate displays more severe pathogenicity than previously characterized strains in China. Methods ======= Virus and Animals ----------------- The YN strain was isolated in 2005 from 30-day-old broilers that had been inoculated oculonasally with H120 vaccines of IBV on days 1, 7, and 21, respectively. The sick birds had respiratory signs, severe renal disease, and a death rate of 30%. The virus was propagated in 10-day-old specific-pathogen--free (SPF) embryonated chicken eggs at 37°C for 40 h. Allantoic fluid was recovered from infected eggs and stored at −80°C. All animal research was approved by the Beijing Administration Committee of Laboratory Animals under the leadership of the Beijing Association for Science and Technology (approval ID is SYXK \[Beijing\] 2007--0023). Viral RNA Extraction, Reverse Transcription PCR, and DNA Sequencing ------------------------------------------------------------------- On the basis of IBV nucleotide sequences (M41, A2, BJ, ZJ971, and SC021202; GenBank accession nos. DQ834384, AY043312, AY319651, AF352313, and AY237817, respectively), 22 pairs of specific primers ([Table 1](#T1){ref-type="table"}) were designed to amplify the complete genome (excluding the 5′- and 3′-terminal segments) of the YN strain. Viral RNA was extracted from virus-infected allantoic fluid by using Trizol Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Reverse transcription (RT) was performed at 37°C for 1 h by using 3 μg total RNA, 1 μL random primers (500 μg/mL, random hexadeoxynucleotides; Promega, Madison, WI, USA) and 0.5 μL M-MLV RT (200 U/μL) (Promega). For PCR, 1 U Taq DNA Polymerase (Promega) and 10 pmol of each primer were added to 100 ng cDNA as template in a total of 20 μL reaction volume. PCR was performed at 95°C for 5 min, followed by 35 cycles of denaturation (95°C, 45 s), annealing (53°C or 55°C, 45 s), and polymerization (72°C, 2 min), and the postpolymerization step was performed at 72°C for 10 min. Amplified sequences were analyzed by 1.2% agarose gel electrophoresis. ###### Primers used to amplify the complete genomic sequence of avian infectious bronchitis virus YN strain, People's Republic of China Primer\* Location, bp Upstream primer Downstream primer Length, bp ---------- ---------------- ----------------------- ----------------------- ------------ 1 22--1,475 ATATCATACATACTAGCCTTG AGTTAAGTCGTTTCGCATG 1,454 2 1,302--2,411 CCAACTGGTTTTATGGGTGC TGGAAGTGTCACTGCCTCG 1,110 3 2,240--3,784 ACTTGGTAGAGTTTCTGGGG TTGACATACGAAGGTGTGACA 1,545 4 3,635--5,161 TGTAAACGCCGCAAATGAG GGCAACTTGGAATCCTCTT 1,527 5 5,040--6,636 GATCTTACTGACTTTGAAC CACTGAAACACTTAACTG 1,597 6 6,424--7,936 GGAATTGTGACGAGTATGG TGAAAGTATACCCTATGAGGA 1,513 7 7,746--9,246 GAAATTGTCGGTTACACTCAG TAGAACGCATAGTAACAGGG 1,501 8 9,130--9,985 CTAACGCTGAAACTCCA GTTGGCAATAGGAAAGTA 856 9 9,082--10,645 TCAGTAGGCGGATAAAAGG ATAGGCAACACACGGTCG 1,564 10 10,516--12,092 ATTTCAAAGTTTCGGTTGACC CACAGAAGCTCCTCCATAG 1,577 11 11,861--13,471 GTTTTACAATCTAAAGGTCAT AAGAGCGGGATCTCCTAC 1,611 12 13,270--14,908 CAACATTCTTTTCTCTACAC GATATAACGCTCCATAACT 1,639 13 14,712--16,138 CTGATTCTAAGTGTTGGGTTG TCCTTTGAGGTACTATGCGA 1,427 14 16,027--17,539 TGCTCGTGTTGTTTTTACTGC CACTTGCTCCTTGCCTATTTT 1,513 15 17,362--18,993 CCACTTGAGGGCTTTGT TAAACATACAGGTTCGCT 1,632 16 18,945--20,442 AAGCGGTATYCNTATGTAGAA ATAGTRCAVACAAAAKRGTCA 1,498 17 20,336--21,920 ACTGAACAAAAGACMGACTT CCACCAGAAACTACAACT 1,585 18 21,873--22,919 AAGGTTAATCCCTGTGAAG AGTYTCVGTAAGAATAGCA 1,047 19 22,723--23,837 CTTTTGCHACTCAGATDCA AGATTTCTTACCACACTTACT 1,115 20 23,435--24,898 ATTTGTAGAAGATGACGAT CATTGTTGACCATTAGTTA 1,464 21 24,747--26,141 GCAGCGATAATACTTACCGTG GCTTGGCGTCTCCAGTATC 1,395 22 25,965--27,650 TATCAAACTAGGAGGACCA GCTCTAACTCTAAACTAGCCT 1,686 \Primer locations are listed according to strain SC021202 (GenBank accession no. EU714029). The amplified DNA products were purified by using an AxyPrep DNA Gel Extraction Kit (AxyGEN, Union City, CA, USA), and the purified products were ligated to the pMD18-T Easy Vector system (Promega). Recombinant plasmids were extracted from positive clones by using the E.Z.N.A.R. Plasmid Miniprep Kit (Omega, Norcross, GA, USA) and identified by EcoRI restriction digestion (Promega). Nucleotide sequencing reactions were performed by Sunbio Biotech (Bejjing, China). Sequence and Phylogenetic Analysis ---------------------------------- Complete genome or S1 gene sequences of IBV were obtained from GenBank, and these IBV sequences and the complete coding sequence of the IBV YN isolate were aligned and analyzed by using the ClustalW multiple alignment algorithm in the MegAlign program of the DNASTAR software suite (version 3.1; DNAstar, Madison, WI, USA). The phylogenetic tree of S1 gene or complete genomic sequences was constructed by using MEGA4.0 software ([www.megasoftware.net](www.megasoftware.net)) by the neighbor-joining method (1,000 bootstrap replicates). Evolutionary distances were computed by the pairwise distance method using the maximum composite likelihood model ([@R19]). Clinicopathologic Assessment in Chickens ---------------------------------------- Forty 30-day-old SPF white leghorn chickens were randomly divided into 2 groups of 20 birds each. All birds in 1 group were challenged intranasally with at least 10^5^ 50% egg infectious dose per 0.2 mL of IBV YN strain. Birds in another group were inoculated with 0.2 mL phosphate-buffered saline (PBS) as noninfected controls. Birds were housed in isolators and provided feed and water ad libitum. All birds were observed daily for signs of disease (e.g., disheveled feathers, depression, respiratory signs, or diarrhea) and death for 21 days. Gross pathologic changes were observed, and tissues (trachea, lung, kidney, bursa, liver, and brain) were collected for RT-PCR from 10 randomly selected chickens that died within 4--10 days after inoculation, virus isolation, and histopathologic and IHC analyses. Serum samples collected from birds that survived were tested for IBV antibodies by a commercial ELISA kit (IDEXX Laboratories, Westbrook, ME, USA). The endpoint titers were calculated according to the manufacturer's instructions, and titers \>396 were considered positive for IBV antibody. RT-PCR of Tissue Samples ------------------------ Tissue samples from deceased birds were collected and used for RT-PCR to determine tissue distribution of the virus. Total RNA was obtained by using Trizol Reagent (Invitrogen) as recommended for tissue samples. RT-PCR was performed as described above by using a pair of primers (forward: 5′-TTTTGGTGATGACAAGATGAA-3′; reverse: 5′- CGCATTGTTCCTCTCCTC-3′), which amplify and detect a 403-bp fragment of the S1 gene of IBV. PCR products were analyzed on 1.5% agarose gels. Virus Re-isolation from Infected Tissue Samples ----------------------------------------------- Tissue samples collected after challenge were used for virus isolation. Briefly, at least 6 SPF embryos were inoculated through the allantoic cavity, with each sample, containing 10,000 U/mL penicillin and 10,000 µg/mL streptomycin (0.2 mL/egg). The eggs were candled daily, and allantoic fluids from 3 inoculated embryos were collected 48 h after inoculation for RT-PCR amplification. The remaining embryos were examined 6 days after inoculation for characteristic IBV lesions, such as dwarfing, stunting, or curling of embryos. Histopathology -------------- Tissues (trachea, lung, kidney, bursa, liver, and brain) were collected and fixed by immersion in 10% neutral formalin at room temperature for 48 h. Tissue was then routinely processed, embedded in paraffin wax, and cut into 5-μm sections. The sections were stained with hematoxylin and eosin and examined by light microscopy for lesions resulting from IBV infection. Immunohistochemistry -------------------- All sampled tissues were examined by IHC analysis to detect viral antigen. Briefly, 5-μm tissue sections were subjected to antigen retrieval ([@R20]) and were then incubated in 10% normal goat serum in PBS for 30 min to block nonspecific binding sites. Slides were further incubated with chicken anti-IBV hyperimmune serum at 1:500 dilution in PBS for 2 h, followed by incubation with a horseradish peroxidase--conjugated rabbit chicken IgG for 1 h. The reaction was visualized by the addition of 3,3-diaminobenzidine (DAB; Sigma, St. Louis, MO, USA) for 15 min. After IHC staining, sections were counterstained with hematoxylin, air dried, and examined by light microscopy. Results ======= Genome Sequencing ----------------- The complete genome sequence (excluding the 5′- and 3′-terminal segments) of YN strain was obtained by assembling 22 overlapping sequences that ranged from 856 bp to 1,686 bp by the DNAstar software. The nucleotide sequences were submitted to GenBank under accession no. JF893452. The complete genomic sequences (excluding the 5′- and 3′-terminal segments) of YN isolate comprised 27,635 nt, which putatively contains 6 different genes, each containing single or multiple open reading frames. Phylogenetic Analysis --------------------- Phylogenetic trees for the genome and each gene of IBV reinforced the viral nucleotide sequencing results, suggesting that the YN isolate shares an immediate ancestor with the SC021202 strain ([Figure 1](#F1){ref-type="fig"}). To elucidate the phylogenetic relationships of China IBV strains, we further analyzed S1 genes of 70 IBVs, including 51 China isolates and 19 standard strains or vaccine strains ([Figure 1](#F1){ref-type="fig"}, panel B). The data indicated that China isolates could be differentiated into 3 distinct genetic groups or genotypes. Group I included 41 of the 51 field isolates, which were tentatively named A2-like viruses. Four China field isolates were included in group II, which were tentatively named LSD-like viruses. China group III comprised 6 isolates, which were grouped with the Massachusetts serotype. These results showed that at least 3 distinct groups existed in chicken flocks in China, and A2-like and M41-like viruses were mainly responsible for the IB panzootic, consistent with findngs of our previous studies ([@R5],[@R16]). ![Phylogenetic tree of avian infectious bronchitis virus based on the nucleotide sequences of the complete genome (A), S1 gene (B), S2 gene (C), E gene (D), M gene (E), and N gene (F). The phylogenetic tree was constructed by the neighbor-joining method with 1,000 bootstrap replicates (bootstrap values are shown on the tree). The isolate sequenced in this study is indicated with a black dot.](12-0552-F1){#F1} Sequence Comparisons -------------------- Ten virus sequences were selected for pairwise comparisons with the YN strain according to the phylogenetic analysis for S1 ([Figure 1](#F1){ref-type="fig"}, panel B), which included 2 common vaccine strains in China (M41 and H120) and 8 China isolates from 3 different gene clusters (DY07, LX4, A2, and SC021202 from cluster I; CK/CH/LSD/05I and CK/CH/LHB/100801 from cluster II; CK/CH/LHLJ/07VII and CK/CH/LDL/101212 from cluster III) ([Table 2](#T2){ref-type="table"}). Overall, the complete genomic sequences (excluding the 5′- and 3′-terminal segments) were 87.1%--98.0% identical among the isolates, and the YN strain had the highest nucleotide identity (98.0%) to the SC021202 strain isolated in southern China (GenBank accession no. EU714029). For each gene, the identity hanged from 93.7% to 100% between YN and SC021202 strains ([Table 2](#T2){ref-type="table"}). ###### Percent sequence identities between the YN isolate and 10 selected IBV strains, People's Republic of China Strain\ Gene 1 Gene 2 ------------------ ---------- ---------- ---------- --------- --------- --------- ---------- ---------- ---------- --------- --------- --------- -- ----------------- ----------------- DY07 90.3 85.9 88.2 94.8 88.1 92.5 91.3 92.1 89.9 89.2 88.4 91.2 79.2 (77.9) 93.4 (94.3) LX4 82.2 91.9 97.3 97.6 94.8 100 97.3 97 96.0 96.3 92.3 96.4 82.1 (81.2) 89.7 (92.6) A2 81.8 89.9 90.2 97.6 92.4 99.1 94.5 98 96.0 89.5 90.0 94.7 91.5 (90.0) 92.7 (94.2) SC021202 97.7 99.0 99.0 100 100 100 99.0 97.9 99.0 100 100 100 99.3 (98.6) 99.5 (99.5) CK/CH/LSD/05I 84.9 92.9 92.5 96.4 94.3 99.1 95.9 96.2 96.5 96.2 94.1 95.4 82.9 (82.6) 89.3 (92.5) CK/CH/LHB/100801 86.1 94.2 92.2 100 94.3 99.1 95.9 96.6 96.0 96.2 94.4 96.4 82.9 (82.6) 89.3 (92.5) CK/CH/LHLJ/07VII 86.7 86.1 88.7 93.6 87.3 91.3 90.8 88.4 90.7 89.7 88.3 88.0 69.1 (75.4) 84.7 (87.4) CK/CH/LDL/101212 84.9 88.2 89.0 95.2 89.5 91.9 91.0 87.6 90.9 89.8 88.0 88.3 80.7 (77.8) 84.6 (87.7) M41 87.1 93.8 91.5 96.4 95.3 98.2 99.0 96.6 96.3 97.1 92.3 92.1 81.8 (78.2) 86.1 (89.1) H120 86.6 93.5 90.8 96.4 95.3 98.2 99.0 97.0 96.2 96.7 92.6 91.1 81.6 (78.2) 85.8 (88.8) Gene 3 Gene 4 Gene 5 Gene 6 3′ UTR Genome ------------------ ----------------- ----------------- ----------------- -- --------------------- -- ----------------- ----------------- -------- ---------------- -- ---------- ---------- DY07 92.5 (91.2) 76.6 (65.1) 87.8 (88.9) 92.8 (96.9) 92.4 (92.3) 91.2 (85.4) 86.4 (92.2) 96.7 89.5 LX4 93.1 (91.5) 74.1 (66.7) 87.5 (88.2) 91.3 (93.0) 89.9 (87.9) 90 (89.3) 85.9 (90.3) 76.9 87.5 A2 87.4 (80.7) 78.4 (67.2) 87.5 (88.2) 91.9 (93.8) 83.9 (78.8) 91.2 (86.9) 85.9 (90.0) 95.2 87.9 SC021202 96.6 (93.2) 93.7 (93.6) 98.2 (98.2) 95.6 (98.7) 99.5 (98.5) 99.6 (98.8) 99.7 (100) 98.7 98.0 CK/CH/LSD/05I 84.4 (68.4) 83.7 (70.3) 87.2 (88.2) 89.8 (92.1) 86.5 (81.8) 94.0 (91.7) 88.3 (93,7) 89.4 88.2 CK/CH/LHB/100801 85.2 (84.7) 83.7 (71.9) 87.2 (88.2) 89.7 (93.4) 86.5 (81.8) 94.0 (91.7) 88.1 (91.0) 93.2 87.7 CK/CH/LHLJ/07VII 82.18 (63.2) 82.1 (79.7) 89.0 (88.1) 89.1 (92.9) 81.8 (75.4) 93.6 (89.0) 87.0 (91.2) 98.7 92.5 CK/CH/LDL/101212 81.0 (73.7) 82.1 (78.1) 88.1 (83.5) 89.7 (93.3) 84.9 (80.0) 93.6 (89.0) 87.0 (91.9) 94.6 87.1 M41 82.3 (72.9) 82.6 (79.7) 89.3 (89.1) 89.4 (93.8) 81.4 (74.2) 94.0 (89.3) 88.0 (91.5) 94.2 87.1 H120 81.2 (73.7) 82.6 (78.1) 88.1 (84.5) 89.7 (93.4) 85.5 (80.3) 93.6 (89.3) 87.2 (92.2) 78.9 87.2 \Except for the M41 and H120 strains which are in the IBV vaccine, all other strains are China isolates from different groups. Highest identities are indicated in boldface. IBV, infectious bronchitis virus; UTR, untranslated region. †Percentage of amino acid identity is shown in the parenthesis. The S glycoprotein is the major functional protein for IBV; thus, the putative differences between the YN strain and other IBVs were investigated. Analyses showed that a single amino acid insertion was present at position 22 ([Figure 2](#F2){ref-type="fig"}, panel A) in addition to a 7-aa insertion at positions 74--81 ([Figure 2](#F2){ref-type="fig"}, panel B) in the S1 gene of the YN isolate, in which a putative N-glycosylation site was found. Among available sequences in GenBank, strains A2, BJ, CQ04--1, SC021202, and SAIBK had the same insertion in the S1 gene. Compared with other available strains, 9-aa deletions were located at the N terminal of the S2 gene ([Figure 2](#F2){ref-type="fig"}, panel C) from a base transition (G→T) at position 1,849 nt, resulting in premature stop codon in the open reading frame. ![Amino acid sequence alignment for the S1 gene (A, B) and S2 gene (C) of 14 strains of avian infectious bronchitis virus. Strains YN, SC021202, A2, and SAIBK have a 7-aa insertion in the S1 gene and a 9-aa deletion in the S2 gene, compared with most strains.](12-0552-F2){#F2} Clinicopathologic Assessment in Chickens ---------------------------------------- Clinically, some birds appeared depressed with ruffled feathers; these birds had increased water intake and huddled together in YN-inoculation group at 2 days postinfection (dpi). At 4 dpi, IB-like signs became obvious in all infected chickens, and some birds were euthanized because of severe disease. Deaths occurred until 21 dpi; the death rate reached 65% ([Figure 3](#F3){ref-type="fig"}, panel A). No deaths or clinical signs were observed in the control group. ![Seroconversion and percentage survival of chickens experimentally infected with infectious bronchitis virus (IBV), People's Republic of China. A) Survival of chickens after inoculation with IBV YN strain. B) Detection of IBV antibodies by ELISA at 21 days postinoculation. Cutoff titer = 396.](12-0552-F3){#F3} Slight hemorrhage with serous catarrhal exudate could be seen in the trachea of all euthanized birds. Air sac lesions also were present, characterized by marked thickening of the air sac wall and a yellow caseous exudate. All euthanized chickens showed typical kidney lesions. The affected kidneys were substantially enlarged, and deposits of pale urate were frequently observed in the tubules and ureters ([Figure 4](#F4){ref-type="fig"}). No gross lesions were observed in any birds in the control group. ![Gross lesions from kidney tissues from chickens experimentally infected with infectious bronchitis virus (IBV). A) Kidney tissue of an uninfected control chicken. B) Obvious enlargement and urate deposition in the kidney of a chick infected with the IBV YN strain at 7 days postinfection.](12-0552-F4){#F4} Antibody responses in birds that survived infection were measured by a commercial ELISA kit (IDEXX Laboratories). All but 1 chicken showed a positive reaction ([Figure 3](#F3){ref-type="fig"}, panel B) by ELISA, and the mean titer induced by the YN strain was 1,250.35 at 21 dpi. RT-PCR and Virus Isolation on Tissues ------------------------------------- To further examine the pathogenicity of IBV YN, the tissue tropism of the strain was investigated by RT-PCR and virus isolation by using tissue suspensions from dead birds. YN strain replicated well in the various organs, including the kidneys, trachea, lungs, and bursa ([Table 3](#T3){ref-type="table"}; [Technical Appendix](#SD1){ref-type="local-data"}). ###### Tissue tropism of YN strain of avian IBV, People's Republic of China\ Method Tissue examined† ------------------ ------------------ ------- ------- ------ ------ ------ RT-PCR‡ 10/10 10/10 8/10 7/10 1/10 1/10 Virus isolation§ 7/10 8/10 10/10 9/10 ND ND \Thirty-day-old SPF chickens were inoculated intranasally with YN IBV (10^5^ 50% egg infectious dose per bird). IBV, infectious bronchitis virus; SPF, specific-pathogen free; RT-PCR, reverse transcription PCR; ND, not detected. †Ten tissue samples from euthanized birds were randomly collected and examined. ‡RT-PCR was directly performed on collected tissues. §Tissue samples were collected for re-isolation of the challenge virus using SPF embryos. Data are the number of tissues from which the virus was isolated/number of tissues examined. Histopathologic and IIHC Analyses --------------------------------- Microscopic examination of the tracheal tissues revealed extensive degeneration and necrosis of the ciliated epithelial cells, sometimes with pseudoacinar structures resulting from dropout of dead cells ([Figure 5](#F5){ref-type="fig"}, panel B). Viral antigen was detected at high levels in the epithelial cells of the tracheal mucosa ([Figure 6](#F6){ref-type="fig"}, panel B). Lung lesions were characterized by hemorrhage, congestion, and lymphocytic infiltration in the alveolar lumen ([Figure 5](#F5){ref-type="fig"}, panel D), and viral antigen was detected in alveolar cells ([Figure 6](#F6){ref-type="fig"}, panel D). Severe renal lesions, including degeneration and necrosis of renal tubular epithelial cells, lymphocytic infiltration in the interstitium, exfoliated renal tubular epithelial cells, and erythrocytes were frequently observed ([Figure 5](#F5){ref-type="fig"}, panel F). Viral antigens were detected extensively in the renal tubular epithelial cells ([Figure 6](#F6){ref-type="fig"}, panel F). Serious atrophy of lymphoid follicles and widening of the interstitium were observed in the bursa of Fabricius ([Figure 5](#F5){ref-type="fig"}, panel H), and viral antigens were detected widely in the mucosal epithelium of the bursa of Fabricius ([Figure 6](#F6){ref-type="fig"}, panel H). Sporadic congestion also was observed in the liver and cerebrum. ![Histopathologic analysis (hematoxylin and eosin stain) of tissues from 30-day-old chickens infected with infectious bronchitis virus YN strain. Panels A, C, E, and G, correspond to control tissues. B) Trachea, extensive dropout, degeneration, and necrosis of the ciliated epithelial cells (black arrow). Scale bar = 100 μm. D) Lung tissue with hemorrhage (black arrow), congestion, and lymphocytic infiltration in alveolar lumen (white arrow). Scale bar = 50 μm. F) Kidney tissue with severe renal lesions, including degeneration (white arrow), and necrosis of renal tubular epithelial cells, lymphocytic infiltration in the interstitium (black arrow), exfoliated renal tubular epithelial cells and erythrocytes were observed extensively. Scale bar = 50 μm. H) Bursa tissue with serous atrophy of lymphoid follicles and widening of the interstitium were observed in bursa of Fabricius (black arrow). Scale bar = 200 μm.](12-0552-F5){#F5} ![Immunohistochemical detection of avian infectious bronchitis virus (IBV) antigens in tissues after experimental infection with IBV YN strain. Panels A, C, E, and G correspond to control tissues. B) Tracheal tissue with viral antigen detected extensively in the epithelial cells of the tracheal mucosa (black arrow). Scale bar = 50 μm. D) Lung tissue with viral antigen detected in alveolar cells (black arrow). Scale bar = 50 μm. F) Kidney tissue with viral antigens detected widely in the renal tubular epithelial cells (black arrow). Scale bar = 50 μm. H) Bursa tissue with viral antigens detected at high levels in mucosal epithelium in the bursa of Fabricius (black arrow). Scale bar = 100 μm.](12-0552-F6){#F6} Discussion ========== IBV infection leads to severe economic losses in the poultry industry because of poor weight gain and feeding efficiency in broilers or reduced egg production and egg quality in laying birds. Death rates are often low (\<30%) unless secondary bacterial infections cause increased deaths of birds ([@R21],[@R22]). Evaluation of the pathogenicity of IBV was based on several criteria, including: clinical signs, gross pathologic lesions, histopathologic changes, and tissue tropism. In this study, a China IBV strain isolated from a 30-day-old vaccinated broiler flock that had a history of respiratory signs, severe renal disease, and higher than expected mortality rate was analyzed by clinical observation, histopathologic examination, RT-PCR, virus isolation, and immunohistochemistry. Analyses revealed that the YN strain induced severe pathogenicity in 30-day-old SPF chickens with a 65% death rate and substantial kidney lesions. Sequence analysis of the S1 gene of YN isolate showed high nucleotide similarities to CQ04--1 (99.5% nt and 98.8% aa identities) and SC021202 (99.3% nt and 98.6% aa identities). Phylogenetic analysis further indicated that at least 3 distinct genetic clusters of IBV were present in chicken flocks in China, named A2-like, LSD-like, and M41-like strains. Most China IBV field isolates belonged to the same genetic cluster (A2-like), but the serotypes of these prevalent IBV strains differed from those of the currently used vaccine strains in China (M41-like, e.g., H120, Ma5, 28/86, and W93), which are considered the main cause of disease outbreaks ([@R5],[@R15]--[@R17]). An attenuated or inactivated vaccine that matches with pandemic strains is urgently needed in China to control IBV infection. IBV has been shown to replicate in many respiratory tissues (including trachea, lungs, and air sac), causing respiratory disease; in some urogenital tissues (including kidney), causing minor or major nephritis; and in many parts of the alimentary tract (including esophagus, proventriculus, and intestine) ([@R23]--[@R25]). As expected, YN was most often detected in the trachea, lung, kidney, and bursa by RT-PCR, virus isolation, and IHC analysis. In addition, we found YN antigens partly from liver (1/10) and brain (1/10). Among 4 major structural proteins, the S glycoprotein is known to contain regions that induce neutralizing, serotype-specific, membrane fusion, attachment, and hemagglutination-inhibiting antibodies ([@R5],[@R26]--[@R28]). In addition, the S protein is a determinant of cell tropism ([@R29],[@R30]). Our data showed a 7-aa insertion between positions 72 and 78 ([Figure 2](#F2){ref-type="fig"}, panel A) in the S1 gene of the YN strain and a 9-aa deletion in the N terminal of the S2, generating a stop codon. These changes are possibly related to the increased virulence observed for YN IBV, and reverse genetic analyses are needed to confirm these findings. In conclusion, we showed that YN-like viruses are a predominant strain in China and that YN IBV exhibits broad tissue tropism and severe pathogenicity in chickens. To better control IBV in China, more detailed analysis of the biologic and antigenic characteristics of the predominant IBV isolates is warranted, and assessments of the efficacy of current vaccines against these isolates are needed. ###### Technical Appendix Results of reverse transcription PCR analysis of tissues from chickens experimentally infected with infectious bronchitis virus. Suggested citation for this article: Feng J, Hu Y, Ma X, Yu Q, Zhao J, Liu X, et al. Virulent avian infectious bronchitis virus, People's Republic of China. Emerg Infect Dis \[Internet\]. 2012 Dec \[date cited*\]. <http://dx.doi.org/10.3201/eid1812.120552> This work was supported by Beijing Research Group for Poultry Production Technology System. Ms Feng is a postgraduate student at the College of Veterinary Medicine, People's Republic of China Agricultural University, in Beijing. Her scientific interests include the epidemiology and pathogenicity of avian infectious bronchitis viruses.
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1. Field of the Invention The invention pertains to a carrier tape reel assembly which includes two reel halves with detent locks. The two reel halves can be disassembled and stacked in a compact configuration. 2. Description of the Prior Art In the prior art, it is known to use a two-piece reel for the storing and shipping of flexible sheet media. The flexible sheet material can be film, tape, or elongated ribbons. The flexible sheet material can likewise be laminated sheet material for packaging a plurality of parts, electrical components, and the like, as described in U.S. Pat. No. 4,726,534 entitled xe2x80x9cConvertible Reel Assemblyxe2x80x9d issued on Feb. 23, 1988 to Chenoweth. This reference uses inner and outer circles of interengaging ears along with a circle of locks which have interengaging lips which prevent reverse or backward rotation of the reel sections thereby maintaining the interlocking ears of the reel section in holding relationship with each other. However, the rotational movement required for assembly is inconvenient and may result in a strength of connection which is less than satisfactory. Additionally, this reference does not appear to provide any mechanism to assure alignment of the open tape slots between the two reel portions. Reel Services manufactures a reel which includes reel halves with detent elements which are assembled with a longitudinal motion. However, the detent elements are not positioned rotationally symmetrically. That is, the two male detent elements on a reel half are separated from each other by ninety degrees. Likewise, the two female detent elements are separated from each other by ninety degrees. Other prior art references have similar deficiencies such as complicated assembly procedures with rotational movements, rotational engagements which may not be adequately secure against inadvertent disengagement or may be difficult to separate when desired. Similarly, some prior art references may not provide for simple stacking and shipping of the reel halves prior to assembly. Moreover, some prior art references may have complicated designs or protrusions in the locking mechanisms which are easily damaged. Some prior references include U.S. Pat. No. 5,941,477 entitled xe2x80x9cVirtually Fully Engagable Stacking Reel Componentxe2x80x9d issued on Aug. 24, 1999 to Basili et al.; U.S. Pat. No. 5,779,186 entitled xe2x80x9cMulti-Part Reel for Electrical Terminals and the Likexe2x80x9d issued on Jul. 14, 1998 to Bakker et al.; U.S. Pat. No. 5,531,399 entitled xe2x80x9cTape Reelxe2x80x9d issued on Jul. 2,1996 to Weisburn et al.; U.S. Pat. No. 5,524,850 entitled xe2x80x9cCarrier Tape Reel Assemblyxe2x80x9d issued on Jun. 11, 1996 to Liao; U.S. Pat. No. 4,226,381 entitled xe2x80x9cTape Reelxe2x80x9d issued on Oct. 7, 1980 to Katata; U.S. Pat. No. 4,002,309 entitled xe2x80x9cBobbin for Cinematographic Filmsxe2x80x9d issued on Jan. 11, 1977 to Ruiz-Barbotteau; U.S. Pat. No. 2,494,612 entitled xe2x80x9cSeparable Moving Picture Film Reelxe2x80x9d issued on Oct. 31, 1947 to Goldberg et al.; and U.S. Pat. No. 1,819,337 entitled xe2x80x9cReelxe2x80x9d issued on Aug. 18, 1931 to Pevear. It is therefore an object of the present invention to provide a tape reel assembly with reel halves which can be assembled quickly and easily. It is therefore a further object of the present invention to provide a tape reel assembly with reel halves which are secure against relative rotation after assembly, but which can be easily disassembled when desired. It is therefore a still further object of the present assembly to provide a tape reel assembly with reel halves which include structure to assure alignment of the open tape slots of the reel halves. It is therefore a still further object of the present invention to provide a tape reel assembly with two reel halves with engagement structure which is not susceptible to damage. It is therefore a still further object of the present invention to provide a tape reel assembly with reel halves which can be stacked easily for storage and shipment prior to assembly. It is therefore a still further object of the present invention to provide a tape reel assembly with reel halves which can accommodate a number of different widths of media by varying the total thickness of the hub formed by the two reel halves. It is therefore a still further object of the present invention to provide a tape reel assembly with reel halves wherein the detent elements of like gender are placed rotationally symmetrically with respect to each other. It is therefore a still further object of the present invention to provide a tape reel assembly with a simple design, which is economical to manufacture. These and other objects are attained by providing a tape reel assembly comprised of two halves. Each half is substantially identical to the other, although halves with different hub thicknesses can be joined to vary the resulting hub thickness for different widths of media. The halves each include two longitudinally oriented male detent elements which are opposed 180 degrees apart from each other. Similarly, two longitudinally oriented female detent elements are opposed 180 degrees apart from each other and 90 degrees apart from each male detent element. While the detent elements are positioned rotationally symmetrically about the reel halves, the male detent elements themselves include rotationally asymmetric ridges which align with rotationally asymmetric grooves in the female detent elements to assure that the open tape slots of the two reel halves are properly aligned. The longitudinal orientation of the detent elements provides for the reel halves to be secured to each other by a single straight-line motion of the reel halves without need for rotation during attachment. Moreover, the longitudinal orientation of these elements eliminates any reasonable possibility of relative rotation of the reel halves after proper engagement.
Oreca 03 The Oreca 03 is a Le Mans Prototype built by Oreca in 2011. It is built within the revised 2011 ACO regulations for the 24 Hours of Le Mans. The car made its début at the 12 Hours of Sebring run by Signatech Nissan and is set to race at the 2011 24 Hours of Le Mans. It is the third car that Oreca has produced after the 01 and FLM09. 24 Oreca 03 were built. Development To meet the new regulations brought in by the ACO for endurance racing, the 03 has been cost-capped along with all 2011 LMP2 cars with a price of €345,000. The engines used in LMP2 cars for 2011 are production-based and there is a wide variety of engines to choose from that can be put into an 03. The five teams racing it in 2011 have chosen two of these engine. Most of the teams, including Oreca themselves, have chosen the Nissan-powered 4.5 L V8 powerplant which is similar to the engine used by Nismo in Super GT. The Swiss team of Race Performance have opted for a Judd BMW-powered 3.6 L V8 engine based on the BMW M3. Both engines produce a limited . The 03 is built around a light carbon fibre monocoque chassis with an open top bodywork. Suspension is by double wishbones and push-rods on all four corners. Stopping power is provided by carbon-ceramic disc brakes. The Oreca 03 uses the same body and aero package developed by Oreca's chief-engineer David Floury for the LMP1 car campaigned during the 2009 and 2010 season by the works team. The blade-shaped roll-over structure is still a direct descendant of the original Courage LC70 design. Racing History 2011 Five 03's have been built, four of which have competed in races. Team Oreca have not entered a race yet but plan to race at the 1000 km of Spa and Le Mans. The first race the Oreca 03 competed in was the 2011 12 Hours of Sebring, the first round of the American Le Mans Series and Intercontinental Le Mans Cup, where the French team of Signatech Nissan competed for ILMC glory in the LMP2 class with drivers Franck Mailleux, long term Oreca driver Soheil Ayari and 2009 GT Academy winner Lucas Ordoñez. The trio had been quick throughout practice and took pole at the Sebring International Raceway, almost four seconds quicker than the next nearest LMP2 class car. Unfortunately, because the car was brand new, problems occurred including continuous gearbox problems. In the end, the No. 26 car finished 30th overall and second in class as there were very little LMP2 entrants. The next race on the calendar was the 6 Hours of Castellet, round one of the 2011 Le Mans Series season. There were three 03's present at this race with TDS Racing, Boutsen Energy Racing and Race Performance competing in their respective chassis'. It was the TDS Racing team of Mathias Beche, Pierre Thiriet and Jody Firth that took pole position in the LMP2 class with the Boutsen car qualifying second in class and Race Performance qualifying fourth. Once again the 03's encountered mechanical issues with the TDS car retiring after 91 laps. The other two cars finished the race with the Boutsen Energy drivers of Dominik Kraihamer and Nicolas de Crem finishing fourth place in class and the Race Performance drivers of Michel Frey, Ralph Meichtry and Thor-Christian Ebbesvik finishing sixth. References External links Oreca 03 Technical Specs Category:24 Hours of Le Mans race cars Category:Le Mans Prototypes Category:Sports prototypes
So, about that fiscal crisis — the one that would, any day now, turn us into Greece. Greece, I tell you: Never mind. Over the past few weeks, there has been a remarkable change of position among the deficit scolds who have dominated economic policy debate for more than three years. It’s as if someone sent out a memo saying that the Chicken Little act, with its repeated warnings of a U.S. debt crisis that keeps not happening, has outlived its usefulness. Suddenly, the argument has changed: It’s not about the crisis next month; it’s about the long run, about not cheating our children. The deficit, we’re told, is really a moral issue. There’s just one problem: The new argument is as bad as the old one. Yes, we are cheating our children, but the deficit has nothing to do with it. Before I get there, a few words about the sudden switch in arguments. There has, of course, been no explicit announcement of a change in position. But the signs are everywhere. Pundits who spent years trying to foster a sense of panic over the deficit have begun writing pieces lamenting the likelihood that there won’t be a crisis, after all. Maybe it wasn’t that significant when President Obama declared that we don’t face any “immediate” debt crisis, but it did represent a change in tone from his previous deficit-hawk rhetoric. And it was startling, indeed, when John Boehner, the speaker of the House, said exactly the same thing a few days later. What happened? Basically, the numbers refuse to cooperate: Interest rates remain stubbornly low, deficits are declining and even 10-year budget projections basically show a stable fiscal outlook rather than exploding debt. So talk of a fiscal crisis has subsided. Yet the deficit scolds haven’t given up on their determination to bully the nation into slashing Social Security and Medicare. So they have a new line: We must bring down the deficit right away because it’s “generational warfare,” imposing a crippling burden on the next generation. What’s wrong with this argument? For one thing, it involves a fundamental misunderstanding of what debt does to the economy. Contrary to almost everything you read in the papers or see on TV, debt doesn’t directly make our nation poorer; it’s essentially money we owe to ourselves. Deficits would indirectly be making us poorer if they were either leading to big trade deficits, increasing our overseas borrowing, or crowding out investment, reducing future productive capacity. But they aren’t: Trade deficits are down, not up, while business investment has actually recovered fairly strongly from the slump. And the main reason businesses aren’t investing more is inadequate demand. They’re sitting on lots of cash, despite soaring profits, because there’s no reason to expand capacity when you aren’t selling enough to use the capacity you have. In fact, you can think of deficits mainly as a way to put some of that idle cash to use. Yet there is, as I said, a lot of truth to the charge that we’re cheating our children. How? By neglecting public investment and failing to provide jobs. You don’t have to be a civil engineer to realize that America needs more and better infrastructure, but the latest “report card” from the American Society of Civil Engineers — with its tally of deficient dams, bridges, and more, and its overall grade of D+ — still makes startling and depressing reading. And right now — with vast numbers of unemployed construction workers and vast amounts of cash sitting idle — would be a great time to rebuild our infrastructure. Yet public investment has actually plunged since the slump began. And why are we shortchanging the future so dramatically and inexcusably? Blame the deficit scolds, who weep crocodile tears over the supposed burden of debt on the next generation, but whose constant inveighing against the risks of government borrowing, by undercutting political support for public investment and job creation, has done far more to cheat our children than deficits ever did. Fiscal policy is, indeed, a moral issue, and we should be ashamed of what we’re doing to the next generation’s economic prospects. But our sin involves investing too little, not borrowing too much — and the deficit scolds, for all their claims to have our children’s interests at heart, are actually the bad guys in this story
+ 5l - 4l + 15, 4c - 26 = -2l for c. 4 Solve i + 5b + 17 + 14 = 0, -30 = 5b for i. -1 Solve z = 4c + 4, 0 = 3z - 4z - 5c - 5 for z. 0 Solve -3a - 9 = -0a, 1 = p - 2a for p. -5 Solve -5y + 3 = -f - 8, -6 = -y + 4f for y. 2 Solve -89q + 94q + 3 = -p, 2q + p + 3 = 0 for q. 0 Solve -4u + 5 = -5o - 1, -3o = 6 for u. -1 Solve -2m = -5w + 18, -3w - 329m = -330m - 11 for w. 4 Solve -5d + 20 = -f + 4f, -d = -5f - 4 for f. 0 Solve 3v + r + 16 = 0, 69v - 2r + 34 = 64v for v. -6 Solve -3v = 5s - 4v - 18, 5v - 10 = 0 for s. 4 Solve 2d - 6 = -d - 5y, -5d + 3y + 10 = 0 for d. 2 Solve 4n + 4 = 282l - 278l, 0 = 5n + 2l - 2 for n. 0 Solve -n - 11b + 7 = -10b, 2n - 5b + 21 = 0 for n. 2 Solve 4i + 3b + 5 = 0, 4b + 7 = -5i - 0 for i. 1 Solve 3p + 27 = 5a, 9 = 2a - 3p - 9 for a. 3 Solve 0 = 5z - 4m + m - 18, -12 = -5z - 3m for z. 3 Solve -2i - 12 + 7 = -5a, -4a - 2i + 22 = 0 for a. 3 Solve 5b + 21 = 2x, b + 0b = 3x + 1 for x. -2 Solve 12g - 15g = -4h - 35, 0 = -g - 3h - 10 for g. 5 Solve -2n = -3u, n = -0n - 5u for n. 0 Solve -4g = -5n + 7 - 17, 5g - 3n - 19 = 0 for g. 5 Solve 4h + 3r - 9 = 0, 2h + 2r - 6 = 3h for h. 0 Solve -r = 4x, -21r + 22r = -3x + 1 for x. -1 Solve -15 = 5h - 5s, 0h - s = -5h - 11 for h. -2 Solve -4q = -6n + n + 2, -3q = 4n + 17 for q. -3 Solve -5a - 5 = 0, 11a - 2 = -5r + 13a for r. 0 Solve -g - 15 = 2l, 0 = -3l - 2g - 12 - 14 for l. -4 Solve -3w + 2s = s - 2, -w - 5s - 26 = 0 for w. -1 Solve 4v - 6 = -5b, -4b + 0b = -v + 12 for b. -2 Solve 0s = 2s - 2o + 4, 3o - 4 = 2s for s. -2 Solve o - 2w = 5o - 14, 4o = 3w + 19 for o. 4 Solve -3w + 9 = 0, c - w + 4 + 1 = 0 for c. -2 Solve -z = 5h + 480 - 473, 3z - 3h = 15 for z. 3 Solve -s - 8 = 6r - r, -2s = 5r + 11 for s. -3 Solve 0 = -x - q - 2, -13q + 6 = 5x - 16q for x. 0 Solve 0 = 3y + 4j - 11, 5j - 34 = 5y - 2y for y. -3 Solve 0 = -a - 5y - 21 + 17, 4a - 2y = -16 for a. -4 Solve -4x + 22 = -2v, -2x - 2v = 141 - 137 for x. 3 Solve 4a + 5 = -3c, c - 4a - 28 = -51 for c. -7 Solve -6 = 7g - 4g - 4k, 3k - 15 = -3g for g. 2 Solve -u + 2w = -13, u + 10w - 7w + 12 = 0 for u. 3 Solve -3i + 4x = -6i + 11, 5i = -3x + 22 for i. 5 Solve 0 = -3j - j + 16, 2s + 5j = 24 for s. 2 Solve -4x = v + 15, -6v + 3x + 9 = -9v for v. 1 Solve -5a + 4b = -b + 10, -12 = -3b for a. 2 Solve 0 = 2w + i - 6, -56w + 51w + 2i = -24 for w. 4 Solve 0 = -4r + 4, 15b - r + 5 = 19b for b. 1 Solve 7a + 9 = p + 3a, a = p - 3 for p. 1 Solve 3s = 3v - 12, 0 = -2v + 4s + 5 + 11 for v. 0 Solve -2g - 16 = -6q, 4g + 15q - 10q = -15 for g. -5 Solve -3 = -r, -t - 7r + 9r - 9 = 0 for t. -3 Solve 0 = -2i + 4w + 26, -3i = -7w + 10w - 3 for i. 5 Solve -30i + 20 = 4b - 34i, 0 = -3b + 2i + 16 for b. 6 Solve 0 = -f - 2p + 4, 2f = -23p + 26p - 6 for f. 0 Solve 2r + 12 = 264u - 261u, 0 = 5u - 2r - 20 for u. 4 Solve -2d + 8g = 5g - 11, g = -1 for d. 4 Solve 0 = -3n - 6, -29d + 26d - 2n = 13 for d. -3 Solve -8j + 7j = -2s - 3, 0 = j - 5s - 3 for j. 3 Solve 0 = -4q - 21y + 18y - 3, q + 6 = y for q. -3 Solve 0 = -2w + 3w + 5, 3n = 3w + 6 for n. -3 Solve -64a + 66a - 6 = -5j, 2a - 2j + 8 = 0 for a. -2 Solve 3a = 2d + 4, -5 = 8d - 7d - 3a for d. 1 Solve -5u + 3 - 2 = -2w, -u = -2w - 5 for w. -3 Solve -4s - 5v + 79 - 88 = 0, 3s + 4 = -v for s. -1 Solve 4q - 2b = 5 + 1, 2b - 10 = 0 for q. 4 Solve 28o - 25o - 32 = -4p, -5o = -20 for p. 5 Solve 3z + 0z = -4c - 11, z = 3c - 8 for c. 1 Solve -n + 8 = -6n + 2p, -4p = 4n + 40 for n. -4 Solve -3a - 7 - 8 = k, -3a = -4k + 15 for a. -5 Solve -4o + 5r + 9 = 0, 23 = 5o - 7o - 3r for o. -4 Solve -2k - 4b = -6, -4k + 6 = -2k - b for k. 3 Solve -10j + 6j + 18 = -5c, 8 = 3j - c for j. 2 Solve -3k - 2h + h = 9, 0 = 3k - 2h for k. -2 Solve -5d + 35 = -5r, 0 = 4r - r + d + 13 for r. -5 Solve -5u + 6 = -4m + m, -m - 5u + 18 = 0 for m. 3 Solve -71g + 76g + 5a + 20 = 0, -19 = g + 4a for g. 1 Solve 0 = 5t + 3q - 32, -5q = -3t - 8 for t. 4 Solve -3c = 3d - 5d - 23, -3d - 2c = 2 for d. -4 Solve 0 = 3l + 15, 41 = -2n - 3l + 16 for n. -5 Solve 2g + 3q = -3, -18 = 7g - 11g + 2q for g. 3 Solve -2t - 3h = 3t + 26, 3h + 10 = -t for t. -4 Solve -k - 13 = 2y, -591k + 589k - 18 = 2y for k. -5 Solve 34 = -15s + 13s - 4u, -3s + u - 2 = 0 for s. -3 Solve -5q - 30 = 3l, 4l - 8l = 2q + 12 for q. -6 Solve -3g - 11 = -4g - 5x, 2x + 10 = -4g for g. -4 Solve 16 = 5o + m, -30o - 17 = -34o - 5m for o. 3 Solve -4p = 12, 5p + 3 = -2f - 2f for f. 3 Solve 0 = -2h + 2w - 6, -63w - 12 = 5h - 65w for h. -2 Solve -46v + 5o + 10 = -51v, -v = 4o + 11 for v. 1 Solve 9v = 2c + 8v - 4, -3v - 12 = -5c for c. 0 Solve 0 = 2t + 2t - 2u + 2, 0 = -5t + 2u - 3 for t. -1 Solve 3l - 6 = -8k + 3k, 4k + 2l = 6 for k. 3 Solve 0 = o + 4q + 13, -4o + q - 5q - 4 = 0 for o. 3 Solve -7k + 20 = -4k - 2i, 4i + 36 = 5k for k. 4 Solve 0 = -5w - 5t - 5, 0 = -4w - 5t + 1 - 5 for w. -1 Solve n - 4n - 4a + 21 = 0, -4a = -4n for n. 3 Solve -6u - 4p = -10u - 20, -2p + 4 = u for u. -2 Solve -4v = 5a - 0v - 10, 0 = -5a + 5v + 10 for a. 2 Solve -5b + 2l + 91 = 114, 5b = 3l - 22 for b. -5 Solve r - 5 = 3m, -2 = 3r - m - 9 for r. 2 Solve -5p = 5t + 40, 3p - 5t - 16 = -0p for p. -3 Solve -2p - 6 = p - n, -p + 5n - 2 = 0 for p. -2 Solve -3l + 29 = -4z, 2z - 20 = 6z for l. 3 Solve 9 - 3 = 3k + 2x, 15 = 3k + 5x for k. 0 Solve 0 = -4g + 7a - 5a - 10, -a = -4g - 5 for g. 0 Solve -v - y = 3, -5v - 10y = -13y - 9 for v. 0 Solve 31j - 1 = -5k + 29j, -j = 2k for k. 1 Solve -v - 19 = -20k + 17k, 4k - 57 = -5v for v. 5 Solve 32x + 6 = 31x - 5v, 5x + 3 = 2v for x. -1 Solve 3s + 3j = j + 16, 3s - 2j - 8 = 0 for s. 4 Solve 36b - 19 = 41b - 2q, 0 = -3b - 5q - 30 for b. -5 Solve -5o + 3n - 20 = -15, -4o - 4 = 3n for o. -1 Solve -5p + p - 20 = -2h, 4h = -3p - 15 for p. -5 Solve 5p = 0, -17a - 3 = -14a + 4p for a. -1 Solve -5n - 25 = -5i, 0 = -2i + 3n - 7n - 2 for i. 3 Solve -n - 2n - 5 = -h, 0 = 2n - 2h + 6 for n. -1 Solve 0 = -4r + 2t + 20, 0r - r + t = -5 for r. 5 Solve -420k = -418k - 2j - 4, -2k = 2j for k. 1 Solve -3 = 3t + 5y + 9, 12 = -3t + y for t. -4 Solve -j + 8 = -3t + 23, 5 = 3j + t for j. 0 Solve 3h - 3k - 1 = 5, 4h = -3k + 1 for h. 1 Solve -5n + p = -2n - 13, -5n + 15 = 5p for n. 4 Solve -17n - 24 = 3z - 14n, -2z = -3n - 4 for z. -4 Solve -5q + 0q - 5m = -20, -16 = q - 4m for q. 0 Solve o + 4h = 4o - 7, -5o + h + 6 = 0 for o. 1 Solve -32 = -2q - 2q - 4j, 0 = 3q + 4j - 29 for q. 3 Solve -10 = -3c - 5n, 3n = 3c + 171 - 189 for c. 5 Solve 3p + d - 3 = 0, 4d = -p + 9 + 3 for p. 0 Solve 4x - 20 = 0, 3l + 5x - 25 = -2l for l. 0 Solve -4f = -4d + 4, 69d = 72d + 5f + 13 for d. -1 Solve r = -2v - 12, -6v = -2r - v + 21 for r. -2 Solve -4n = 3i + 6, -3n + 18 = -3i + 12 for i. -2 Solve 11 = 3o + 4x, -9 = -o - 2x - 2 for o. -3 Solve -3n + 15 = 0, -17n + 16n = -4r + 11 for r. 4 Solve 7 = 2t - m, 8 = t + 2m + 2 for t. 4 Solve -4z + 20 = -8z, -4k - 4z - 24 = 0 for k. -1 Solve -4o - 3t = -16, -2o + 9 = t + 3 for o. 1 Solve 0 = -4q - 2j - 8, 0 = 4q - 0j - 2j + 24 for q. -4 Solve -3n - 2n + 7 = 3r, n + 2r = 0 for n. 2 Solve 4k + 5 = 3p, -2p + 5k = -3 + 2 for p. 3 Solve 11m - 4 = -4g + 7m, 0 = -4g + 4m - 28 for g. -3 Solve w = -4n + 16, -20n + 5w = -21n - 15 for n. 5 Solve 5g + 3h = 20, h = -4h for g. 4 Solve -5g - 28 = 3a, -5g + 8g + 20 = -5a for g. -5 Solve 3h + 4d + 8 = 0, 2h + 3h - 2d + 22 = 0 for h. -4 Solve i + i - t + 8 = 0, 5i - 5t = -25 for i. -3 Solve n - 4v = 0, n + n + 9 = -v for n. -4 Solve -6o = -a - 3o + 2, 5a - o = -4 for a. -1 Solve 5r - 4i = -3, -4r + 4 - 2 = -i for r. 1 Solve -t = -4k + 11, -3k + 11 - 2 = -t for k. 2 Solve -2w = -4b + 22, 4w + 3 = w for b. 5 Solve 3o = -5m - 0 - 7, 3 = -o - m for o. -4 Solve 3y + 6n - 4n + 4 = 0, -4y - 5n = 10 for y. 0 Solve -5p - 17 = 2h, 2h - 12 = 4p + 16 for p. -5 Solve -4d - 13 = 5
Chris Christie TIME.JPG The new issue of Time magazine, out Friday, featuring Gov. Chris Christie. (Courtesy of Time magazine) TRENTON — With re-election in the bag, Gov. Chris Christie on Wednesday insisted he wanted to focus on his second-term legislative agenda in Trenton, saying he is "thrilled to have the campaign behind me and to get back to governing." The political world has different ideas. In an issue that hits newsstands Friday, Time magazine put him on the cover with the words, "The Elephant in the Room" and with a package of stories asking if he is the savior of the Republican Party. Network television reporters flocked to New Jersey to ask whether his 22-point win will translate to a national audience. And, in a sure-fire sign that he's dominating a Republican Party itching to retake the White House, Christie's potential rivals for the 2016 presidential nod coolly downplayed his landslide victory. "I think we need to understand that some of these races don’t apply to future races," U.S. Sen. Marco Rubio of Florida told CNN. "Every race is different. It has a different set of factors. But I congratulate (Christie) on his win." "All year long, Christie has presented this character he has created as the savior for the Grand Old Party. His point now is that ideas alone don’t win elections. ... From Parsippany to Cape May, Christie’s message, often devoid of policy or ideology, was designed and delivered for national consumption." With the attention of national media, Christie on Wednsday brushed off 2016 talk and explained his political philosophy, honed over a lifetime in the thick of Jersey politics. "We won the Latino vote last night," Christie said. "Find another Republican who’s won the Latino vote recently. Why? It’s about the relationships." Echoing the theme of his acceptance speech, Christie said politicians can’t expect to win votes by showing up in minority communities six months before an election. They have to lay the groundwork with relationships based on trust. Exit polls show the strategy worked: He won 51 percent of Hispanic voters and 21 percent of African Americans. When Christie takes over as chairman of the Republican Governors Association in two weeks, he’ll apply this vision to the 2014 races. "I can’t speak to how it will translate nationally. I don’t know," he said. "Sometimes I think people make politics too complicated. It’s about personal relationships. I mean personal relationships first and foremost with the voters. Second, with the people you work with each and every day. … Third, with the people in the legislative branch." Christie strode into the school auditorium with Union City Mayor Brian Stack, a Democrat, by his side. After basking in a standing ovation, Christie thanked Stack, who also won re-election as a state senator. "When you’re a Republican, you know you’re on your way to a good night when you win Union City by 2,000 votes," Christie said. Star-Ledger staff writer Brent Johnson contributed to this report. Gov. Christie visits Union City day after winning re-election, Nov. 6, 2013 22 Gallery: Gov. Christie visits Union City day after winning re-election, Nov. 6, 2013 RELATED COVERAGE • With big win, Christie set to wield power on national stage • Complete coverage of the 2013 New Jersey governor's race FOLLOW STAR-LEDGER POLITICS: TWITTER • FACEBOOK • GOOGLE+
Q: how to give commands to the running instance of maya with mayapy.exe? I want to create a simple python script which will directly transfer objects from blender to Maya. I created a python script which exports the object from blender to a temp folder. now I want to import that object into Maya without actually going to Maya>file>import. I searched for the solution for a while and found out that I can create a standalone instance of Maya with mayapy.exe and work in non-GUI instance of Maya. but what I want to do is import the object into an already running instance of Maya(GUI version) as soon as the exporting script is done running. A: as suggested by Andrea, I opened the commandPort of maya and connected to it using socket in python script. now i can send commands to maya using that python script as long as the maya commandPort is open.
Introduction {#s1} ============ Assessment of the number of individuals with early HIV infection is needed for evaluation of the current HIV epidemic and preventive efforts targeted at the different transmission risk populations. Consequently, serologic testing algorithms for recent HIV seroconversion (STARHS) have been developed. These tests utilize the fact that both the concentration and affinity of HIV antibodies in early infection are lower than at later stages \[[@pmed-0040343-b001],[@pmed-0040343-b002]\]. The fact that STARHS requires a special assay, which has a deliberately reduced sensitivity compared to HIV screening tests, restricts STARHS to epidemiologic studies. However, for systematic epidemiologic monitoring it would be advantageous if "recency" information (i.e. how recently the infection was acquired) could be simultaneously gained from the same tests being used to diagnose HIV infection. Here we have investigated whether a second-generation Western blot assay (line immunoassay) provides recency information similar to that of a commercially available STARHS test, which is used for estimating the incidence of recent infections in a population and was originally developed by researchers at the US Centers for Disease Control and Prevention (CDC) \[[@pmed-0040343-b003]\]. The line immunoassay features standardized antigens of both HIV-1 and HIV-2 and three internal controls. It is currently being used increasingly as a replacement for the traditional first-generation HIV Western blot developed more than 20 years ago \[[@pmed-0040343-b004],[@pmed-0040343-b005]\]. The test has recently become mandatory for confirmation of HIV infection and differentiation of HIV-1 and HIV-2 in Switzerland \[[@pmed-0040343-b006]\]. Methods {#s2} ======= Patients and Specimens {#s2a} ---------------------- It is estimated that several hundred thousand HIV screening tests are performed annually in Switzerland. Serum or plasma specimens from all individuals in whom HIV infection was newly diagnosed in Switzerland between 1 July 2005 and 30 June 2006 were selected for the study. By federal ordinance, an epidemiologic questionnaire on each newly diagnosed HIV infection has to be forwarded under code to the Swiss Federal Office of Public Health (SFOPH) by the patient\'s physician. Of relevance to the present study, the questionnaire contains items about clinical or laboratory signs of recent infection (e.g., symptoms of primary HIV infection, CDC staging \[[@pmed-0040343-b007]\], and seroconversion). Definition of Recent or Older Infection {#s2b} --------------------------------------- For the purpose of this study an HIV infection was defined as recent on the basis of the physicians\' information reported to the SFOPH and by the laboratory results obtained at the time of diagnosis of the HIV infection. Specifically, a recent infection was required to meet one or more of the following conditions. Definition 1: signs of primary HIV infection (PHI) at the time of diagnosis \[[@pmed-0040343-b008]\]; definition 2: a self-reported or documented negative result of a HIV screening test within 12 mo before HIV diagnosis; or definition 3, laboratory evidence of seroconversion at the time of diagnosis, i.e., a reactive fourth-generation HIV-1/2/O antibody/antigen combination screening test and a positive virus component test (HIV-1 p24 antigen or HIV-1 RNA) combined with a negative or indeterminate antibody assay (third-generation HIV-1/2/O antibody enzyme immunoassay, Western blot, or INNO-LIA). The time window during which an infection was defined as being recent thus was 12 mo. An infection was defined as older if symptoms of CDC stages B or C were present at the time of diagnosis and reported to the SFOPH \[[@pmed-0040343-b007]\]. Report of no symptoms (CDC stage A) or lack of staging information remained undefined and were either excluded from or included in the analysis depending on the respective evaluation as described under results. Tests for Recent HIV-1 Infection {#s2c} -------------------------------- All study samples were tested prospectively by the INNO-LIA HIV I/II Score assay (Innogenetics, Ghent, Belgium) in one of the 11 accredited Swiss HIV Confirmatory Labs or the Swiss National Center for Retroviruses (SNCR), the later being commissioned by the SFOPH to serve as the national HIV reference laboratory. INNO-LIA tests were conducted as a mandatory component of the SFOPH HIV testing system \[[@pmed-0040343-b006]\]. The INNO-LIA HIV I/II Score assay (INNO-LIA) is a Western blot--like line immunoassay that measures antibodies against recombinant proteins or synthetic peptides of HIV-1, HIV-1 group O, or HIV-2, which are coated as discrete lines on a nylon strip with plastic backing. As each test strip also contains three quantitative internal standards, a semiquantitative ranking of the different antibody reactions is possible \[[@pmed-0040343-b009],[@pmed-0040343-b010]\]. All INNO-LIA assays were performed by trained laboratory personnel using the manufacturer\'s 16-h sample incubation protocol. Antibody reaction to each of the seven HIV antigen bands present on the INNO-LIA test strip (sgp120 \[including group O peptides\], gp41, p31, p24 and p17 of HIV-1, and sgp105 and gp36 of HIV-2) was assessed visually in nine laboratories and by the automated scanner--based LiRAS system (Innogenetics) in three laboratories. Based on the three internal standards, which define reaction levels of 0.5 (+/−), 1, and 3, the antibody reaction to each HIV antigen was classified into one of six possible intensity scores (0, 0.5 \[for +/−\], 1, 2, 3, or 4) according to the manufacturer\'s instructions. Frozen aliquots (−20 °C) of all samples were furthermore sent under code to the SNCR and tested prospectively by the HIV-1 BED Incidence enzyme immunoassay (BED-EIA, Calypte Biomedical Corporation, Lake Oswego, Oregon, United States). BED-EIA was performed as a part of an ethically approved nationwide epidemiologic study (CH.A.T. survey) commissioned by the SFOPH. It aimed at elucidating the circumstances under which new HIV infections in Switzerland occurred (see <http://www.bag.admin.ch/hiv_aids/00829/03471/index.html?lang=de> for further information). All BED-EIA testing was performed in blinded fashion by a single, highly experienced person and using a Microsoft Excel-based program for automated result interpretation prepared for the study according to Calypte\'s instructions, which specify a cutoff of 0.8 normalized optical density units (OD~n~). Determination of INNO-LIA Window Periods {#s2d} ---------------------------------------- Window periods for INNO-LIA recency algorithms were determined based on results with 15 seroconversion panels with a total of 105 specimens (Boston Biomedica, West Bridgewater, Massachusetts, United States; panels PRB 903--904, 909--912, 916--919, 922--925, and 927). The INNO-LIA results on these panels were provided by Innogenetics. The raw window period was calculated as the difference in days between the first sample reactive in the most sensitive third-generation HIV-1/2 screening test (based on data from Boston Biomedica) and the first sample ruled "older" by the respective INNO-LIA recency algorithm. To these raw window periods was added a constant of 22 d, which corresponds to the estimated median seroconversion window of sensitive third-generation HIV-1/2 screening assays \[[@pmed-0040343-b011]\]. Statistical Evaluation {#s2e} ---------------------- Differences in INNO-LIA reaction intensity in different study subgroups, as assessed in [Figure 1](#pmed-0040343-g001){ref-type="fig"}, were compared by unpaired t-test. Specificity, sensitivity, diagnostic odds ratio, and logistic likelihood ratio of the BED-EIA and various tentative INNO-LIA HIV I/II Score algorithms for recency, as presented in [Tables 1](#pmed-0040343-t001){ref-type="table"}--[3](#pmed-0040343-t003){ref-type="table"}, were calculated by means of contingency tables and univariate and multivariate logistic regression analysis, as implemented by the StatView 5.0 program for Macintosh (SAS Institute, Cary, North Carolina, United States). Physicians\' information was used as the reference standard for recency in all such evaluations. Estimates for the recent infection rates in [Table 4](#pmed-0040343-t004){ref-type="table"} were obtained by the equation Recency Rate = (n ~tested\ recent~/n ~tested~ + %Specificity/100 − 1) / (%Sensitivity/100 + %Specificity/100 − 1). This equation is based on the relationship n ~tested\ recent~ = n ~true-recent~ + n ~false-recent~, wherein n ~true-recent~ = n ~tested~ × Recency Rate × %Sensitivity/100 and n ~false-recent~ = n ~tested~ × (1 − Recency Rate) × (1 − %Specificity/100). Window-based estimates of the recent infection rate were performed by the equation (n ~recent~/748) × (365/Window), shown with [Table 5](#pmed-0040343-t005){ref-type="table"}. ![Antibody Reaction as Assessed by the INNO-LIA in Different Groups of Newly Diagnosed HIV-1 Patients\ (A) Sum of antibody scores to env antigens sgp120 and gp41 (top graph), gag antigens p24 and p17 (middle graph) and pol antigen p31 (bottom graph); bars represent means and error bars their 95% confidence intervals. Reaction is stratified according to recency definitions 1, 2, or 3 and other reported staging information (see [Methods](#s2){ref-type="sec"}).\ (B) Correct order of recency definitions based on increasing antibody development. Box plots show sum of antibody scores to all five HIV-1 antigen bands. Boxes indicate the median and the 25th and 75th percentile. Horizontal lines located outside of, but closest to the boxes represent the 10th and 90th percentile, respectively. Outliers are plotted individually. The medians of the groups consisting of infections not classified coincide with the 75th percentile (upper limit of box). p-Values on top denote differences between subgroups of recent infections.](pmed.0040343.g001){#pmed-0040343-g001} ###### Performance of Various Algorithms for Detecting Recent Infection among Cases of Defined Recency Status ![](pmed.0040343.t001) ###### Diagnosis of Recent Infection and Test Concordance in Dependence of Stage ![](pmed.0040343.t002) ###### Percent Sensitivity of Various Recency Algorithms at Different Phases of Recent Infection ![](pmed.0040343.t003) ###### Estimation of the Overall Recent Infection Rate in the Study ![](pmed.0040343.t004) ###### INNO-LIA Window Periods Derived from Seroconversion Panels and Estimates for the Recent Infection Rate ![](pmed.0040343.t005) Results {#s3} ======= A total of 765 newly diagnosed HIV infections were reported to the SFOPH during the 12 mo of the study (July 05--June 06). Seventeen cases were excluded due to missing INNO-LIA results, thus leaving a total of 748 patients for evaluation (97.8%). All infections were by HIV type 1. A total of 195 patients met the definition of a recent infection as described in [Methods](#s2){ref-type="sec"}, and 161 patients met the definition of an older infection. Of these, 84 were reported to be in CDC stage B and 77 in stage C. Remaining unclassified were 392 patients, 252 without symptoms (stage A) and 140 with no information provided. The main characteristics of the study population are summarized in [Table 6](#pmed-0040343-t006){ref-type="table"}. ###### Characteristics of the Study Population ![](pmed.0040343.t006) INNO-LIA Reactivity in Dependence of Stage {#s3a} ------------------------------------------ INNO-LIA reactivity to env bands sgp120 + gp41, gag bands p17 + p24, and pol p31 in the different groups is summarized in [Figure 1](#pmed-0040343-g001){ref-type="fig"}A. Infections defined as recent had lower mean antibody intensities to all antigens compared to all other groups (p \< 0.001 in all comparisons). Those with both symptoms of PHI and a reported negative HIV test within the past 12 mo (thus meeting both recency definitions 1 and 2) had lower antibodies on average than those with reported PHI but no negative HIV test (recency definition 1), and the latter in turn had lower antibody levels than those with a history of a negative HIV test but no PHI symptoms (recency definition 2). Thus, those with both symptoms of PHI and a reported negative test represented an early phase, those with PHI symptoms alone an intermediate phase, and those with a documented negative test alone a late phase of recent infection ([Figure 1](#pmed-0040343-g001){ref-type="fig"}B). Moreover, infections defined as recent based on their negative or indeterminate results in HIV antibody tests at the time of diagnosis (recency definition 3) showed the lowest antibody reactivity of all, thus classifying themselves as the most recent infections. [Figure 1](#pmed-0040343-g001){ref-type="fig"}B also shows the p-values for the differences between the various subgroups of recent infection. Antibody reactions in CDC stage A were similar to those in stages B or C except that antibodies to gag antigens were slightly higher than in stage C (p = 0.01), in agreement with established knowledge \[[@pmed-0040343-b012],[@pmed-0040343-b013]\]. The 140 cases with no information were indistinguishable as a group from CDC stage A ([Figure 1](#pmed-0040343-g001){ref-type="fig"}B) and like A exhibited a trend for higher antibodies to gag antigens compared to CDC stage C (for p17, p = 0.053). Prediction of Recent Infection in Patients with Defined Recency Status {#s3b} ---------------------------------------------------------------------- A first evaluation of recency prediction by BED-EIA or INNO-LIA was based on the 356 cases classified either as recent or older as defined under [methods](#s2){ref-type="sec"}. The BED-EIA classified 127 of the recent 195 infections as recent, exhibiting 65% sensitivity. The low sensitivity of the BED-EIA is due to the longer duration of the interval defined recent (12 mo in this study compared to 153 d in other studies using this test) \[[@pmed-0040343-b002],[@pmed-0040343-b003],[@pmed-0040343-b014]\]. The BED-EIA also classified 129 of the 161 older infections as older, exhibiting 80% specificity. Overall correct results amounted to 72%, the diagnostic odds ratio was 7.4, and the logistic likelihood ratio (LLR) Chi-square was 78.7 ([Table 1](#pmed-0040343-t001){ref-type="table"}). Univariate logistic regression analysis showed that a low antibody score to any of the five HIV-1 antigen bands of the INNO-LIA strip was associated significantly with recent infection (p \< 0.0001 for all, data not shown). Algorithms (Alg) 2--6 in [Table 1](#pmed-0040343-t001){ref-type="table"} demonstrate the best power to discriminate between recent and older infection, as indicated by the highest LLR Chi-square achieved when varying the band intensity cut-off. Some of these algorithms exhibited similar or superior strength compared to BED-EIA, as shown by higher diagnostic odds ratios and LLRs. Alg2--6 were considerably more specific than was BED-EIA, but their sensitivities were lower, thus resulting in an overall lower percentage of correct results for INNO-LIA than for BED-EIA. When using bands in combination and again optimizing cutoffs by means of logistic regression analysis, algorithms with around 98% specificity and 40% sensitivity were developed (Alg7--9). Based on the observation that seroconversion to p31 in Western blot occurs consistently later than to p24 \[[@pmed-0040343-b015],[@pmed-0040343-b016]\], we also assessed this criterion alone (Alg10) or in combination (Alg11--13). Despite being a rather weak criterion on its own, Alg10 strongly increased sensitivity, albeit at some cost to specificity, when added to other criteria. Overall correct results by Alg11--13 amounted to around 70%, which was comparable to BED-EIA (72%). Multivariate logistic regression analysis adjusting for sex, transmission risk, and INNO-LIA reading mode (visual or automated) demonstrated no impact of these variables on the strength of the algorithms (unpublished data). Moreover, the reading mode had no significant impact on the INNO-LIA result. Although automated reading was associated with overall slightly lower intensity scores (p = 0.005, Mann-Whitney U test), probably due to the fact that the Liras software counts a score of 4 as 3, this reduction in mean scores had no impact on an algorithm\'s outcome, as an algorithm\'s cutoff is always placed at lower scores. For Alg12, for example, the odds ratio for automated versus visual reading for ruling an infection as recent was 1.11 (95% confidence interval \[CI\] 0.76--1.62; p = 0.60). There was also no difference in the case of single-band algorithms. Recency Rates in Different Patient Groups and Test Concordance {#s3c} -------------------------------------------------------------- The proportion of patients ruled to have been infected recently by BED-EIA or INNO-LIA and the concordance of the two procedures in the different patient groups is shown in [Table 2](#pmed-0040343-t002){ref-type="table"}. Alg12 was selected for this comparison due to its overall highest rate of correct results (compare to [Table 1](#pmed-0040343-t001){ref-type="table"}). In all groups, BED-EIA found considerably more samples recent than did Alg12. Concordance between the two procedures was lowest among recent infections (74%) and highest in CDC stage C (86%). Note again that the relatively low sensitivity of the BED-EIA compared to other studies is due to the longer duration of the interval defined recent in this study. We next investigated the sensitivity of selected algorithms of [Table 1](#pmed-0040343-t001){ref-type="table"} for detection of the early, intermediate, or late phase of recent infection, as established in [Figure 1](#pmed-0040343-g001){ref-type="fig"}B. The sensitivity of BED-EIA and all INNO-LIA algorithms decreased from early to intermediate to late phase ([Table 3](#pmed-0040343-t003){ref-type="table"}). In the early phase, INNO-LIA algorithms were similar in sensitivity to BED-EIA. In the intermediate and particularly the late phases of recent infection, INNO-LIA was considerably less sensitive than BED-EIA. Thus, the overall higher sensitivity of BED-EIA in [Table 1](#pmed-0040343-t001){ref-type="table"} was due to its better recognition of the intermediate and late phases of recent HIV infection. Estimation of the Recent Infection Rate in the Entire Study Population {#s3d} ---------------------------------------------------------------------- Given the more than 50% cases without reference recency information, the true proportion of recent infections (infections that occurred within the past 12 mo) in the study population was unknown. We estimated this value on the basis of the results of the different recency tests in the entire study population. The equation used for these estimates (see [Methods](#s2){ref-type="sec"}) adjusts for a test\'s sensitivity and specificity, as determined in [Tables 1](#pmed-0040343-t001){ref-type="table"} and [3](#pmed-0040343-t003){ref-type="table"}. The BED-EIA ruled 262 (35.3%) of the 748 infections as recent. When entering the sensitivity and specificity determined in [Table 1](#pmed-0040343-t001){ref-type="table"} into the equation, a proportion of 33% recent infections corresponding to 250 cases was calculated ([Table 4](#pmed-0040343-t004){ref-type="table"}, model 1). Recency estimates by Alg12 and Alg9 were markedly lower, but yielded identical results to each other (27%, 204 cases), despite their considerable difference in the actual number of infections diagnosed as recent (130 versus 88). For Alg3 which according to [Table 3](#pmed-0040343-t003){ref-type="table"} identifies only early recent infections and did not detect any further recent cases outside the group defined as recent, the estimate was remarkably close (26%). Application of the sensitivity and specificity rates of the reference group of [Table 1](#pmed-0040343-t001){ref-type="table"} to the unknown group is, however, feasible only if both groups are composed similarly; otherwise bias is introduced. [Figure 1](#pmed-0040343-g001){ref-type="fig"}B shows that there is indeed considerable heterogeneity. Regarding their overall antibody reaction the unknown group differs significantly from the reference group of [Table 1](#pmed-0040343-t001){ref-type="table"} (Mann-Whitney U test, p \< 0.001). The unknown group has very little overlap with the early and intermediate phases of recent infection ([Figure 1](#pmed-0040343-g001){ref-type="fig"}B). Thus, if the nonclassified infections include any recent infections at all, these are likely to be in the late phase of recent infection. It is therefore appropriate to use the reduced sensitivity rates shown in the last column of [Table 3](#pmed-0040343-t003){ref-type="table"} for this group. Specificity, in contrast, was very similar among the different groups, in particular as regards INNO-LIA (cf. [Table 2](#pmed-0040343-t002){ref-type="table"}). Hence, by doing a mixed calculation, i.e., applying the sensitivity of [Table 1](#pmed-0040343-t001){ref-type="table"} to the reference group and the reduced sensitivity of late recent infection of [Table 3](#pmed-0040343-t003){ref-type="table"} to the unknown group while applying the specificity of [Table 1](#pmed-0040343-t001){ref-type="table"} to both groups, we obtained more appropriate estimates for the recent infection rate of the entire study ([Table 4](#pmed-0040343-t004){ref-type="table"}, model 2). Compared with model 1 the adjusted model 2 showed an only 12% higher rate of recent infections for the BED-EIA, but as much as 20%--30% higher rates for Alg9 and Alg12, in agreement with the fact that the sensitivity of BED-EIA is reduced less in the late phase of recent infection than is the sensitivity of INNO-LIA (compare to [Table 3](#pmed-0040343-t003){ref-type="table"}). With recency rates of 0.35 and 0.33, respectively, Alg12 and Alg9 now showed good agreement with BED-EIA (0.37), while the estimate by Alg3, which does not detect cases in the late phase of recent infection, remained unchanged at 0.26. Alg3 clearly underestimated recent infection. Window Periods and Window-Based Estimation of the Recency Rate {#s3e} -------------------------------------------------------------- In order to determine the window periods of the different INNO-LIA algorithms we analyzed INNO-LIA results of 15 seroconversion panels ([Table 5](#pmed-0040343-t005){ref-type="table"}; see also [Methods](#s2){ref-type="sec"}). Alg3 (gp41) was associated with the shortest window period among the single band algorithms, followed by Alg5 (p24), Alg6 (p17), Alg2 (sgp120), and Alg4 (p31). The time span of many panels was shorter than the window period of many algorithms. The resulting window information is thus based on too few measurements and not reliable. Nevertheless, we tentatively used this information for an independent estimation of the recent infection rate in our study population. With this approach, BED-EIA yielded a rate of 0.41 compared to 0.37 obtained by model 2 of [Table 5](#pmed-0040343-t005){ref-type="table"}. Alg3 (gp41) and Alg5 (p24), whose window periods are the most reliable, both yielded a rate of 0.53. Other INNO-LIA algorithms supported less well by seroconversion panel results yielded rates of up to 0.98, thus confirming that the respective windows are indeed too short. Discussion {#s4} ========== We demonstrate that a patient\'s antibody reaction in the INNO-LIA HIV I/II Score assay contains information which, similar to the Calypte BED-EIA, allows a distinction to be made between recent and older HIV-1 infection, thus providing a tool for estimating HIV-1 incidence in a population. Because antibody reactions to the five HIV-1 antigens measured by the INNO-LIA evolve over time after the infection ([Figure 1](#pmed-0040343-g001){ref-type="fig"}), we evaluated various algorithms for their ability to distinguish between recent and older infection in a cohort of 748 unselected HIV-1 infected patients newly diagnosed during a period of 12 mo in Switzerland. The best INNO-LIA algorithms ruled 40%--50% of the 195 infections defined as being recent (up to 12 mo duration of infection) as recent, while ruling 95%--99% of the 161 infections defined as being older as older. In comparison, a dedicated commercial recency test, the BED-EIA, ruled 65% of the recent samples as recent and 80% of the older samples as older ([Table 1](#pmed-0040343-t001){ref-type="table"}). Concordance of the two tests was 74%--86% based on stage ([Table 2](#pmed-0040343-t002){ref-type="table"}). Both tests exhibited about 80% sensitivity for the early phase of recent infection, while showing reduced sensitivity for the intermediate and, particularly, late phases of recent infection ([Table 3](#pmed-0040343-t003){ref-type="table"}). Based on the sensitivity and specificity rates thus obtained from patients of known recency status we estimated the proportion of recent infections in the entire cohort of 748 patients. Utilizing an adjusted model we obtained recency rates of 0.33 and 0.35 for two INNO-LIA algorithms and of 0.37 for the BED-EIA ([Table 4](#pmed-0040343-t004){ref-type="table"}), which was similar to the window-based estimate of 0.41 for the BED-EIA ([Table 5](#pmed-0040343-t005){ref-type="table"}). There was higher discrepancy for the INNO-LIA algorithms, due to still-unreliable window period estimates. All STARHS reported to date utilize window periods, i.e., the mean time from infection to the first positive result of a test. Based on the number of cases ruled recent in a tested population and the length of the window period the annual incidence can be calculated. The approach in our study is fundamentally different. Based on reference information obtained from the treating physicians we first defined two subsets of patients. One subset consisted of patients infected within 12 mo prior to HIV diagnosis (recent infection), and the other of patients who had older infections based on symptoms of CDC stage B or C. We used these two reference subsets to determine the sensitivity and specificity of various recency algorithms ([Tables 1](#pmed-0040343-t001){ref-type="table"}--[3](#pmed-0040343-t003){ref-type="table"}). Based on the now-known sensitivity and specificity and the number of cases ruled recent by a given algorithm the proportion of recent infections in the entire study population was calculated ([Table 4](#pmed-0040343-t004){ref-type="table"}). Thus, while the classical STARHS approach requires reliable window information for the test but no clinical information on the patients tested, our approach requires reliable clinical reference information for a subgroup of patients but no window information for the test. During seroconversion, an HIV-infected person progresses from seronegativity to a pattern of full reactivity to all major HIV proteins \[[@pmed-0040343-b015]\]. Defining HIV seroconversion or recent infection as anything less than a full antibody reaction pattern may thus seem trivial. However, as disease progression is associated with decreasing concentrations of many antibodies, there is a second, late phase in HIV infection with incomplete patterns or submaximal concentrations of antibodies \[[@pmed-0040343-b012],[@pmed-0040343-b013],[@pmed-0040343-b017]--[@pmed-0040343-b020]\]. The detection of patients in recent infection by means of differential antibody reactivity thus requires a careful optimization of candidate algorithms with the aim to maximize correct assessment. The best INNO-LIA algorithms compared well with the BED-EIA regarding power to discriminate between recent and older infection and were considerably more specific than the BED-EIA ([Table 1](#pmed-0040343-t001){ref-type="table"}). Unlike the BED-EIA, which due to its deliberately reduced sensitivity compared to HIV screening tests has no role in the diagnosis of HIV infection, the INNO-LIA is an excellent test for confirmation of HIV infection and is superior to (and less expensive than) Western blot for differentiating between HIV-1 and HIV-2 infection \[[@pmed-0040343-b009],[@pmed-0040343-b010]\]. Early identification of HIV-2 infection is becoming increasingly important, as HIV-2 cannot be quantitated by any of the FDA-approved tests for HIV-1 RNA measurement \[[@pmed-0040343-b021]\] and is naturally resistant to several commonly used antiretroviral drugs \[[@pmed-0040343-b022]--[@pmed-0040343-b025]\]. The INNO-LIA HIV I/II Score assay is CE marked, i.e., approved for diagnostic use in the European Union, but currently cannot be used in the US, as the test is not FDA approved. INNO-LIA based STARHS is not a reasonable option for low-resource countries that use less expensive combinations of HIV screening tests for confirmation of HIV infection. The recency information provided by the INNO-LIA comes at no additional costs and can be integrated easily into existing national HIV reporting systems. The number of cases for which recency information is available will thus be higher than in systems in which recency information must be gained from costly epidemiologic studies. This fact should result in HIV-1 incidence estimates of overall higher quality. The multicenter nature of the present study and the employment of both visual and automated strip evaluation in the 12 collaborating labs, with no apparent impact on the result, suggests that INNO-LIA-based recency assessment can be transferred to other labs without loss of quality. Studies to clarify this point and to determine whether automated reading is advantageous are currently underway. In order to obtain an independent estimate of the recent infection rate in our study we also used a window-based approach ([Table 5](#pmed-0040343-t005){ref-type="table"}). The resulting infection rate of 0.41 of the BED-EIA was remarkably close to the 0.37 obtained by the adjusted model in [Table 4](#pmed-0040343-t004){ref-type="table"}. This agreement of results suggests that, by and large, the information from the physicians was reliable, although we cannot exclude the possibility of misclassification. For example, some PHI cases presenting with severe clinical manifestations may have been misclassified as older infections \[[@pmed-0040343-b026]--[@pmed-0040343-b029]\]. Altogether, the window based recency assessment by the BED-EIA however validates the sensitivity/specificity-based approach. As the time span covered by many of the seroconversion panels was too short, sufficiently accurate window information is still lacking for most INNO-LIA algorithms. For Alg3 (gp41) and Alg5 (p24), window periods of 36 d and respectively 38 d were obtained, which is considerably shorter than the 153 d of the BED-EIA and consistent with the low sensitivity of INNO-LIA for the intermediate and late phases of recent infections ([Table 3](#pmed-0040343-t003){ref-type="table"}). Alg3 and Alg5 yielded an identical recent infection rate of 0.53, which is somewhat higher than that of BED-EIA (0.41). As the window periods are very short, a few days\' difference can have a big effect when calculating an annual rate. For example, increasing the window periods of Alg3 or Alg5 by 6 d, as would result from using an average seroconversion interval of 28 d instead of 22 d (see [Methods](#s2){ref-type="sec"}), would reduce the rate from 0.53 to 0.45. Testing of further panels covering extended time periods thus is urgently needed to define longer INNO-LIA windows with sufficient accuracy. Once established, window-based recency estimates will also be feasible for INNO-LIA algorithms and probably represent the preferred method. Different algorithms will also be usable in combination, which should result in more reliable estimates. Limitations {#s4a} ----------- A possible current limitation of INNO-LIA-based recency testing may derive from the influence of infections by non-B subtypes of HIV-1. Like other European countries, Switzerland has a high rate of non-B infections \[[@pmed-0040343-b030]\] and the proportion of such viruses among newly diagnosed HIV-1 infections has now reached proportions of two-thirds in women and one-third in men (unpublished data from the Swiss genotypic resistance test database). Patients infected with such viruses may produce antibodies of reduced avidity to the subtype B antigens frequently employed in diagnostic tests, thus leading to low-reactivity patterns when tested and subsequent false diagnosis of recent infections. Such an effect has been observed with several early STARHS tests \[[@pmed-0040343-b031],[@pmed-0040343-b032]\]. The BED-EIA, developed in order to overcome this problem, therefore features a branched peptide consisting of the immunodominant gp41 B-cell epitope of subtypes B, D, and E \[[@pmed-0040343-b003]\]. The fact that all INNO-LIA based algorithms were considerably more specific than the BED-EIA suggests that subtype interference should be less a problem for the INNO-LIA than it is for the BED-EIA. Systematic studies are planned to clarify this point. Another known factor which may impair the specificity of STARHS consists of the reduced antibody concentrations associated with advanced disease stage ([Figure 1](#pmed-0040343-g001){ref-type="fig"}). A further problem consists of cases that never reach a sufficiently high optical density to qualify as older infection \[[@pmed-0040343-b033]\]. The Joint United Nations Programme on HIV/AIDS Epidemiology Reference Group has recently issued a warning that the BED-EIA may overestimate HIV-1 incidence and should not be used for routine surveillance applications, and other investigations have also reported overestimation \[[@pmed-0040343-b014],[@pmed-0040343-b034]\]. As the specificity of INNO-LIA based algorithms in patients with reported symptoms of AIDS is already high ([Table 2](#pmed-0040343-t002){ref-type="table"}), this concern should not be a problem for INNO-LIA. Nevertheless, studies addressing this question by means of well characterized patients of the Swiss HIV Cohort Study (SHCS) are planned. As shown by [Figure 1](#pmed-0040343-g001){ref-type="fig"}B, the nonclassified infections differ as group from the classified infections. Application of the sensitivity and specificity rates of the reference classified group to the unknown group will thus introduce bias. As a consequence, model 1 of [Table 4](#pmed-0040343-t004){ref-type="table"}, which does not correct for such bias, clearly underestimates the overall rate of recent infections. Although model 2 of [Table 4](#pmed-0040343-t004){ref-type="table"} adjusts for the discrepancy, it is possible that the model still underestimates the true proportion of recent infections in the unknown group and, thus, the entire study population. Given the small difference between window-based and sensitivity/specificity-based recency rate estimates (0.41 compared to 0.37 for the BED-EIA), the influence of such possible further bias in the present study should be small, however. Nevertheless, as window-based recency assessment does not require knowledge of group composition, it should be the preferred method of INNO-LIA based recency assessment once INNO-LIA windows have been established with sufficient precision. Window-based recency assessment should also facilitate transfer of the method to other populations. In conclusion, we provide proof of principle that a well-standardized line immunoassay for HIV confirmation and type differentiation also contains information on whether an HIV-1 infection is recent. The recency information provided by the INNO-LIA HIV I/II Score is available at no additional expenditure to countries in which this test is routinely used for confirmation of an HIV infection and HIV type differentiation. Utilization of this information could improve HIV surveillance in such countries. Because reliable window periods for INNO-LIA algorithms have not yet been established, INNO-LIA based recency assessment is currently restricted to the sensitivity/specificity approach described here. As the sensitivity and specificity of INNO-LIA algorithms may vary between different populations, it will be necessary to assess them anew when transferring the method to a different study population. Additional studies are needed to establish reliable window periods for the different INNO-LIA algorithms, to clarify the influence of HIV-1 subtype, and to further assess the specificity of the method in advanced disease. Supporting Information {#s5} ====================== ###### Flow Chart (26 KB PPT) ###### Click here for additional data file. ###### STARD Checklist (258 KB PPT) ###### Click here for additional data file. The authors acknowledge the assistance of Katrin Steinbeck in this work and are grateful to Dr. Leslie R. Bisset for critical reading of the manuscript. The corresponding author, Jörg Schüpbach, supplied the information regarding Bruno Güntert\'s contribution to the manuscript and his competing interests and it is correct to the best of his knowledge. Author contributions. JS, RS, and PV designed the study. JS, MDG, DS, and PV analyzed the data. JS, ZT, CN, SY, PB, LM, MG, RD, DS, IS, CA, BM, and BG enrolled patients. JS, SY, PB, LM, MG, DS, and PV contributed to writing the paper. MDG, ZT, CN, SY, PB, LM, MG, RD, DS, IS, CA, GM, BG, SD, and PV collected data or performed experiments for this study. MDG contributed the clinical data (from surveillance) needed for the analysis. Patients included in the present study are based on patients enrolled in the CHAT study, which included all individuals with a newly diagnosed HIV infection for the duration of 1 year. SD and PV were responsible for the design, data collection, and summary of the CHAT study, of which some results are presented in the present study. Funding: This study was funded by the Swiss Federal Office of Public Health (SFOPH) contracts No. 04.002598 / 2.24.01.-849 (CHAT Survey), Nos. 05.000323/2.25.02.-23 and 06.000487/2.25.01.-989 (to Swiss HIV Confirmatory Labs), and No. 03.000571/2.31.02.-3 (to Swiss National Center for Retroviruses). The SFOPH had no direct role in the design of this study, but provided anonymized clinical reporting data on the patients investigated in the study. The funders did not have any role in data analysis or the decision to publish the manuscript. Competing Interests: The authors have declared that no competing interests exist. CI : confidence interval EIA : enzyme immunoassay LLR : logistic likelihood ratio PHI : primary HIV infection SFOPH : Swiss Federal Office of Public Health STARHS : serologic testing algorithms for recent HIV seroconversion.
Introduction ============ Epigenetic modification of DNA has been increasingly recognized as performing an important role in carcinogenesis. However, there are surprisingly few studies of genome-wide methylation and its effects in colorectal cancer (CRC). Initial studies of gene-specific promoter methylation [@b1] described the phenomenon of the CpG island phenotype (CIMP), in which multiple tumour suppressor genes, such as CDKN2A and MLH1, were aberrantly methylated at their promoter regions. CIMP tumours are known to have distinct clinical and pathological characteristics. These include [@b2] high rates of microsatellite instability (MSI) and BRAF mutations, as well as being predominantly in the right side of the colon. Extensive work has been carried out to investigate the characteristics of CIMP tumours. Ogino et al [@b2] examined DNA methylation in five gene promoter regions (CACNA1G, CDKN2A, CRABP1, MLH1 and NEUROG1) in a large set of 840 population-based colorectal cancer samples. They defined several types of CIMP, including CIMP-low (1--3/5 promoter regions methylated). CIMP-low tumours had significantly more KRAS mutations as compared to other groups, CIMP-high (≥ 4/5 promoter regions methylated) or CIMP-negative (0/5 promoter regions methylated). Nosho et al [@b3] verified these categories, also validating the finding that CIMP-high tumours were predominantly poorly differentiated, proximally located, microsatellite unstable and frequently had BRAF mutations. In examining CIMP-negative tumours, Ogino et al [@b4] and Goel et al [@b5] found that tumours belonging to this group frequently demonstrated loss of heterozygosity at 18q, indicating chromosomal instability (CIN). This phenomenon and the associations observed with CIMP-low and CIMP-high tumours have been reproduced independently by other groups [@b6]--[@b9]. Yagi et al [@b10] carried out methylated DNA immunoprecipitation-on-chip (MeDIP-Chip) analysis of HCT116 and SW480 CRC cell lines and coupled this with whole-genome expression array analysis in a set of primary CRCs. Although they demonstrated some associations between low levels of CIMP and KRAS mutation, their study had a very high level of bias, due to the use of only two cell lines, with potential bias due to methylation differences in cell lines. Ang et al [@b11] utilized an Illumina Goldengate methylation array to examine the methylation status of 1505 CpG islands. Examining fresh-frozen tissue from 91 CRC samples against 28 randomly selected, normal colorectal tissue samples, they found that 202 CpG islands were differentially methylated in tumours. Their top four differentially methylated genes -- comparing methylation between tumour and normal mucosa -- were EYA4, HS3ST2, TFPI2 and SLIT2. Using unsupervized hierarchical clustering analysis, they found that differential methylation segregated into CIMP-H, -M and --L; however, their findings have not been independently reproduced and their study may have experienced bias due to small sample size. Hinoue et al [@b12] analysed 120 tumour samples, a minority of which were paired with corresponding normal tissues, utilizing the Illumina HumanMethylation27 platform. They described three biologically distinct groups. The first, CIMP-H, showed a strong association with hypermethylation of MLH1 and the BRAF V600E mutation. The second, CIMP-L, was enriched for KRAS mutations. In the third group, two distinct subgroups were found, the first with a high frequency of TP53 mutations and association with the distal colon, and the second with low-level DNA hypermethylation and an association with the rectum. This study was primarily geared towards analysis of patterns of gene expression in relation to CpG island hypermethylation. A paired analysis of methylation was not carried out, and no premalignant lesions were analysed. Yamauchi et al [@b13] recently carried out a study which examined the rates of CIMP, KRAS and BRAF mutations and microsatellite instabilities (MSIs) compared to the location of tumours in the colon or rectum. They found that rates of CIMP, MSI and BRAF mutation increased from the distal rectum towards the proximal colon. They suggested that these changes demonstrated that methylation occurs as a continuum in the colon, rather than a simple proximal/distal tumour divide, as previously described. They also found that cecal tumours represented a unique subtype of colorectal cancer, possessing high KRAS mutation frequency but lower CIMP-high frequency than in ascending colon tumours. A further study by the Cancer Genome Atlas Network [@b14] examined methylation patterns in 276 colorectal tumours as part of a larger study. Using unsupervised hierarchical clustering, it found that tumours segregated into four groups. The first two, CIMP-high and CIMP-low, were identical to those previously described. It additionally described two other groups, which consisted of tumours that were not hypermethylated but had a higher frequency of APC and TP53 mutations than the CIMP-high and -low groups. We aimed to carry out a whole-genome methylation analysis of paired CRC and normal tissue, as well as colorectal adenomas to identify changes in patterns of methylation in each of these lesions. Materials and methods ===================== Patient recruitment ------------------- Adenomas were sampled at Hammersmith Hospital from patients undergoing colonoscopy. CRCs and paired normal tissues were from patients at St George\'s Hospital undergoing a resection of their tumours. The clinical and pathological characteristics of the cancers and adenomas are shown in [Tables 1](#tbl1){ref-type="table"} and [2](#tbl2){ref-type="table"}. At the time of surgery, resection specimens were immediately placed on ice and conveyed to a specialist histopathologist, where the specimen was opened and a representative sample of adenoma/cancer taken. For the cancers, paired normal colonic mucosa was taken as far as possible from the site of the tumour. Prior to freezing, confirmatory H&E-stained frozen sections were taken to confirm that tumour or normal tissue was present. The specimens were immediately snap-frozen in liquid nitrogen and then stored at −80 °C until use. Ethical approval was obtained from Oxfordshire Research Ethics Committee (MREC 05/Q1605/66). ###### Details of carcinomas Tumour ID Age TNM stage Duke stage Site --------------- --------- --------------- ---------------- ------------------ 20 T 85 T4N0M0 B Caecum 21 T 77 T2N2M1 D Rectum 22 T 85 T3N0M0 B2 Caecum 23 T 94 T2N0M0 B1 Rectum 24 T 84 T3N2MX C2 Sigmoid 26 T 51 T4N2MX C2 Rectum 27 T 66 T3N1M0 C2 Sigmoid 28 T 80 T4N0M0 B Descending colon 29 T 58 T3N0M0 B2 Sigmoid 30 T 48 T3N0M0 B2 Caecum ###### Details of adenomas ID Age Dysplasia Location Morphology -------- --------- --------------- -------------- ---------------- Ad1 74 Severe Rectum Tubulovillous Ad2 64 Mild Colon Tubulovillous Ad3 84 Moderate Colon Tubulovillous Ad4 84 Moderate Colon Tubulovillous Ad5 54 Moderate Colon Tubulovillous DNA extraction and processing ----------------------------- DNA was extracted using proteinase K digestion at 55 °C over 2 days in 180 µl buffer ATL and was purified using a Qiagen DNEasy kit. DNA samples were quantified using spectrophotometry. DNA samples were diluted to 50 ng/µl, using nuclease-free water, and stored at −20 °C until use. Bisulphite conversion of DNA was carried out a EZ Methylation Kit (Zymo Labs) according to the manufacturer\'s protocol. Quantitation of converted bisulphite DNA and assessment of completeness of conversion were carried out using qRT--PCR, according to the method of Campan et al [@b15], using primers specific for ALU repeats and for 0% and 100% bisulphite conversion, against a 100% methylated DNA and 100% unmethylated DNA reference. Illumina methylation array analysis ----------------------------------- Whole-genome methylation analysis was carried out using the Illumina HumanMethylation27 array system. 1.1 µg tumour/adenoma/normal DNA was bisulphite-converted according to the recommended protocol, quantified and quality-checked as above. The converted DNA was whole-genome amplified and hybridized to the array, which was processed according to the manufacturer\'s protocol. The arrays were then scanned using the Illumina BeadArray system. Standard array quality checks were performed post-scanning, using Illumina GenomeStudio software, and only samples that passed internal QC were taken forward for analysis. Raw probe data were exported from GenomeStudio into R 2.9.0. The percentage methylation β (β, %methylation) at each CpG island was calculated using the internal proprietary algorithm and normalization carried out initially using the IlluminaCustom model, and with samples being divided into two groups, tumours versus normals and adenomas versus normals. Probes that failed quality control (detection p value \> 0.05) were removed, as well as all X chromosome probes, due to hemi-methylation of these probes in female patients. Data were exported from GenomeStudio into R and the data normalized successfully, using the normalizeMethyLumiSet function of methylumiR. A least-squares linear fit model was performed on the methylation dataset for each group. The model estimates were then transformed into moderated t-statistics and log-odds of differential expression by empirical Bayes shrinkage of the standard errors towards a common value. For cancers and normals this was carried out as a paired analysis, but for adenomas this analysis was carried out in an unpaired fashion, as no paired samples existed for adenomas and normals, reducing the power of the analysis. The top 100 probes based on the highest BF were identified and QQ and volcano plots produced for the entire probe set. Hierarchical clustering (HC) of the datasets was performed using the clustering function of MultiExperiment Viewer (TMEV), part of the TM4 Microarray Software Suite [@b16]. GSEA was carried out using GSEA v 2.1 [@b17] (Broad Institute, MIT, USA). KRAS, BRAF and MSI status ----------------------------- Mutation analysis of codons 12,13 and 146 of KRAS and codon 600 of BRAF was carried out by direct sequencing (details available on request). For microsatellite instability (MSI) analysis, only the BAT25 and BAT26 alleles were studied. Samples were said to be MSI if they possessed one or more additional alleles at either locus compared with a control microsatellite-stable DNA. Results for KRAS, BRAF and MSI status are shown in [Table 3](#tbl3){ref-type="table"}. ###### Mutation status of tumours analysed Sample MSI *KRAS* codon 12 *KRAS* codon 13 *KRAS* codon 146 *BRAF* V600E ------------ --------- ------------------------- ------------------------- -------------------------- ------------------------- 20 T MSS c.35 G \> A (p.G12D) WT WT WT 21 T MSS WT WT WT WT 22 T MSS c.35 G \> A (p.G12D) WT WT WT 23 T MSS WT WT WT WT 24 T MSS WT WT WT WT 26 T MSS WT WT WT WT 27 T MSS WT WT WT WT 28 T MSS WT WT WT WT 29 T MSI WT WT WT c.1799 T \> A (p.V600E) 30 T MSS WT c.38 G \> A (p. G13D) WT WT Ad1 MSS WT WT WT WT Ad2 MSS WT WT WT WT Ad3 MSS WT WT WT WT Ad4 MSI c.35 G \> A (p.G12D) WT WT V600E Ad5 MSS WT WT WT WT Correlation between promoter methylation and expression of genes ---------------------------------------------------------------- In order to ascertain whether observed differential methylation changes correlated with changes in expression of the gene, we used publically available datasets for methylation and expression from Hinoue et al [@b12]. This dataset used the Illumina HumanMethylation27 array to measure whole-genome methylation (GEO Accession No. GSE25062) and the Illumina Ref-8 whole-genome expression array to measure expression (GEO Accession No. GSE25070). Expression and methylation values were correlated using linear regression. Results ======= When comparing carcinomas and paired normal tissue, we found that \> 2000 genes were differentially methylated at p~corrected~ \< 0.05 ([Figure 1](#fig01){ref-type="fig"}a). Overall results are shown in [Tables S1](#SD4){ref-type="supplementary-material"}--[S3](#SD7){ref-type="supplementary-material"} (see Supplementary material). On examining the volcano plots of each cohort, a number of interesting features were found. Comparing adenomas with normal tissue, the majority of genes did not change significantly or became hypomethylated in the transition from normal tissue to adenoma. In contrast, however, comparing normal tissue with carcinoma, there was a shift towards increased methylation in promoter regions in the latter, an observation confirmed by the larger proportion of genes that were methylated in carcinomas versus adenomas. These results suggest that the bulk of promoter methylation occurs around the time of transition from colorectal adenoma to carcinoma. ![QQ and volcano plots of Bayesian analyses of differential methylation for cancers versus normal samples (a), adenomas versus normal samples (b) and cancers versus adenomas (c). The QQ plots (left) demonstrate probes that are hypermethylated (above the black normal distribution line on the left of the plot) and hypomethylated (below the normal line on the left of the plot). The volcano plot (right) demonstrates that both significantly hypomethylated and hypermethylated probes exist when comparing cancers with normal tissues, The top five probe identifiers are shown in blue text on the volcano plots, and that the five most significantly differentially methylated probes all become hypermethylated.](path0229-0697-f1){#fig01} The highest-rated individual gene for differential methylation in carcinomas versus normals was GRASP (p~adjusted~ = 1.59 × 10^--5^, BF = 12.62), which encodes the general receptor for phosphoinositides-1-associated scaffold protein. GRASP was also the highest-rated gene when comparing adenomas and normal tissue (p~adjusted~ = 1.68 × 10^--6^, BF = 14.53). The highest-rated gene when comparing carcinomas versus adenomas was ATM (p~adjusted~ = 2.0 × 10^--4^, BF = 10.17). Volcano and QQ plots for each three groups of methylation comparisons are shown in [Figure 1](#fig01){ref-type="fig"}a--c. Comparison of methylation and expression for GRASP was carried out using the dataset of Hinoue et al [@b12]. This demonstrated a strong negative correlation (coef~logexpr~^GRASP^ = −2.95, t = −2.66, p = 0.01), ie promoter methylation of GRASP led to reduced expression. We found no correlation between ATM promoter methylation and expression, in common with many other studies showing that ATM expression may not be controlled by methylation in the ATM promoter [@b18]--[@b20]. Hierarchical clustering (HC) into CIMP groups --------------------------------------------- We studied the effectiveness of commonly-used CIMP gene sets in ascertaining whether CIMP was a true reflection of whole-genome methylation and whether methylation patterns in our study segregated into CIMP, as previously observed. CIMP genes were defined, as those given in the studies of Toyota [@b1], Yagi [@b10] and Weisenberger [@b21], as CACNA1G, CDKN2A, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1. Because of their position outside CpG islands and lack of coverage on the HumanMethylation27 array, MINT-1, MINT-2 and MINT-31 were not included in the analysis. A median methylation value (β~median~) was calculated for all the normal mucosa samples in the dataset, and each β~tumour~ value was subtracted from this. Hierarchical clustering was then performed on the samples, using a Pearson correlation metric and average linkage clustering to generate a heat map and clusters for the samples. The heat map for clustering by CIMP probes is shown in [Figure 2](#fig02){ref-type="fig"} for carcinomas and adenomas. In the cancer group, there appeared to be three distinct clusters, with a low methylation cluster (cancers 28 T and 29 T), an intermediate methylation cluster (24 T) and a high methylation cluster (20 T, 22 T, 23 T, 26 T, 27 T and 30 T). ![Hierarchical clustering diagram of CIMP probes for cancers and adenomas. In cancers (left) there is clustering into two groups, which correspond to the CIMP-intermediate and CIMP-low groups previously described by Yagi et al [@b10]. In the adenomas (right) there is clustering into two groups, with a single sample (Ad1) having extensive methylation compatible with CIMP and another group demonstrating low levels of methylation: red, hypermethylation; green, hypomethylation; black, no change.](path0229-0697-f2){#fig02} For the cancer samples, we initially compared our clusters with the conventional definition of CIMP of Weisenberger et al [@b21] and Toyota et al [@b1], viz. that ≥ 3/5 CIMP-related genes should be methylated to call a sample CIMP-H, a trait that is usually associated with BRAF mutation. In our group of tumours, no sample fitted this definition of CIMP-H using ab initio hierarchical clustering. We observed that the CIMP-intermediate phenotype observed by Yagi et al [@b10] would correspond with the group of tumours that clustered into a high methylation group seen in our series. Within these cancers, 20 T and 22 T possessed KRAS mutations, an association also observed by Yagi in the CIMP-intermediate group. We also found that in tumour 29 T, although it was BRAF mutant and microsatellite unstable, there were low levels of methylation, in contrast to other studies. In the adenoma dataset, the pattern of hierarchical clustering was clearer, with segregation into two main clusters, one with high levels of methylation, which fitted with the definition of CIMP-H by Weisenberger and Toyota. There was one other cluster with lower levels of methylation, conforming to the CIMP-L epigenotype. In this dataset, the MSI^+^, BRAF-mutant adenoma (Ad4) was not associated with high levels of methylation (Ad1). Gene set enrichment analysis (GSEA) ----------------------------------- GSEA to determine sets of genes that tended to be hypo/hypermethylated was performed for carcinomas versus normal tissue and adenomas versus normal tissue. The C5 GO gene set was chosen for the analysis, which consists of 1454 curated gene sets divided by molecular function. As more than one probe was present for each CpG island, and because there may have been multiple probes for each gene, probes for the same gene were collapsed in the analysis, and 1000 GSEA iterations were carried out. GSEA comparing carcinomas versus normal tissue ---------------------------------------------- In total, 569/938 gene sets were hypermethylated and 369 gene sets were hypomethylated. The top 10 hypermethylated sets (by normalized enrichment score, NES) are shown in [Table S4](#SD7){ref-type="supplementary-material"} (see Supplementary material). The overall false-discovery rate (FDR) statistic was 0.31, missing the threshold for significance set by Subramanian et al [@b17] of FDR \< 0.25. One gene set was significantly enriched at p \< 0.01 (FDR = 0.24) and 11 gene sets were significantly enriched at p \< 0.05 (FDR = 0.31). The top-ranked gene set was the extracellular matrix structural constituent gene set and the top-ranked gene within this was FBN2 (Fibrillin-2), which has been proposed previously as a marker for tumour methylation by Yagi et al [@b10]. Within the third-ranked gene set, the GRM series of metabotropic glutamate receptors was highly ranked, a set of genes which has been identified as being associated with altered responses to 5-FU chemotherapy [@b22]. A leading-edge analysis was then carried out in GSEA (leading-edge plots in [Figures S1](#SD1){ref-type="supplementary-material"} and [S2](#SD2){ref-type="supplementary-material"}; see Supplementary material) and the top 20 datasets ranked by NES were chosen. The top-ranked gene in the leading-edge analysis was SLIT2 (slit homologue 2), appearing in six gene sets. SLIT2 interacts with netrin-1 (also known as Deleted in Colorectal Cancer), a possible tumour suppressor [@b23] that has previously been identified in other methylation studies of CRC [@b11]. Other genes of interest in CRC seen in the leading-edge analysis included SLIT1, TGFBR2, PAX2, UNC5C, OTX2, NGNT1 and GDNF1. GSEA comparing adenomas and normal tissue ----------------------------------------- GSEA was then carried out comparing adenomas with normal mucosa. 118/938 gene sets were hypermethylated and 118/938 became hypermethylated, with no gene sets meeting the FDR \< 25% threshold. However, 21 hypomethylated gene sets were significantly enriched at p \< 0.01 and 98 were enriched at p \< 0.05. The top 20 sets are shown in [Table S5](#SD8){ref-type="supplementary-material"} (see Supplementary material). In these sets, the top-ranked set was Viral Genome Replication, a set that includes UBP1 and EIF5A (eukaryotic translation initiation factor 5A). The 98 significantly enriched gene sets were then taken forward into a leading-edge analysis to identify common genes that were hypomethylated. The genes of interest within this subset included the top-rated NDUFA13 (NADH dehydrogenase ubiquinone 1α subcomplex subunit 13), represented in 20 gene sets. This gene has been shown to interact with STAT5 [@b24] and therefore the JAK--STAT--EGFR pathway, which has been associated with a worse prognosis in CRC [@b25]. Discussion ========== We have carried out whole-genome methylation analysis of paired colorectal tumour and normal tissues, and have undertaken a comparison with unpaired colorectal adenomas. A strength of our study was the use of paired tumour--normal mucosa as well as premalignant adenomas. Our study suffers from small sample numbers, due to the difficulties of obtaining this type of material, and therefore introduces potential bias. Our study also focused solely on human tumour material, and did not take account of the tumour microenvironment (ie tumour-associated fibroblasts), as described by Allen et al [@b26]. This microenvironment could have modulatory effects on tumour behaviour in addition to promoter methylation and could also contaminate any methylation analysis. Using Bayesian analysis, we found a strong evidence of GRASP promoter methylation in carcinomas and adenomas. We have also shown that GRASP promoter methylation is significantly correlated with GRASP expression. As we found that GRASP becomes methylated in both adenomas and carcinomas, it is reasonable to assume that its promoter methylation occurs at an early stage in the adenoma--carcinoma sequence. GRASP is located at chromosome 12q13.13 and encodes a 395 amono acid protein involved in p14ARF signalling [@b27]. The normal function of GRASP is to promote ARF--Rac signalling. p14ARF, which is encoded by the CDKN2A (INK4a) tumour suppressor gene, acts as a checkpoint within the ARF--MDM2--p53 pathway [@b28], in that normal expression of p14ARF is required to activate and stabilize p53, which can interrupt the cell cycle if needed when a mutagenic event occurs [@b29], as shown in [Figure S3](#SD3){ref-type="supplementary-material"} (see Supplementary material). We also found that the promoter region of ATM is significantly methylated in the transition from adenoma to carcinoma; however, this does not correlate with expression of ATM. Not all gene promoter regions control expression of that gene and it is possible that control of expression of ATM is via another mechanism, given the heterogeneity of data [@b18]--[@b20] comparing ATM promoter methylation and expression. We found that the bulk of promoter methylation occurs in the transition from adenoma to carcinoma, rather than in that from normal tissue to adenoma. Moreover, hypomethylation occurs in the transition from normal tissue to adenoma, whereas hypermethylation is mostly associated with the transition from adenoma to carcinoma. A few other studies have examined the temporal relationship of promoter methylation to colorectal carcinogenesis, but these have mainly examined CIMP genes. For example, Ahlquist et al [@b30] examined the methylation of a set of 11 genes made up of a mixture of CIMP genes and others identified as significant in methylation studies. They found that the average number of methylated genes was 0.4 in normal mucosa, but 2.2 in adenomas and 3.9 in carcinomas. They suggested that the bulk of methylation occurs during adenoma formation. However, our cluster data suggest that CIMP is not fully representative of whole-genome methylation. None of the cancers in this study reached the conventional threshold for CIMP positivity [@b21], although we did observe the reported association between KRAS mutation and a \'CIMP-low\' phenotype [@b10]. We also agreed with the findings of Yamauchi et al [@b13], as all our cecal tumours possessed KRAS mutations, with variable levels of methylation. One of the main roles of GSEA is that it can highlight groups of biological pathways for further investigation. For example, in the GSEA for the normal--carcinoma group, cellular pathways concerned with signalling (cAMP, tyrosine kinase and G-protein-coupled receptors) were significantly enriched in the tumours. The metabotropic glutamate receptors (GRM) were significantly over-represented in the GSEA analysis, and these have been implicated in chemotherapy resistance in a number of tumours, including CRC [@b22], melanoma [@b31] and glioblastoma [@b32]. The leading-edge analysis of the CRC methylation dataset revealed that extensive methylation occurs in the DCC--netrin pathway. SLIT2 (SLIT2 homologue 2 protein), as well as UNC5C, are methylated in the CRC samples in this study. SLIT2 has been shown to be a negative regulatory component of the DCC--netrin pathway [@b33], and normal expression of SLIT2 has been shown to repress growth and metastasis of squamous cell carcinomas and fibrosarcomas [@b23]. In conclusion, the use of whole-genome methylation analysis has highlighted a number of genes and pathways, some of which have not been suggested to have a role in CRC. GRASP is one such interesting candidate. We have also demonstrated that promoter methylation occurs mainly in the transition from adenoma to carcinoma, suggesting that epigenetic events may be more important in promoting than initiating colorectal tumorigenesis. We thank Professor D Kumar, Mr S Ganapathy and Mr S Nizar of St George\'s Hospital for assistance in obtaining tumour material. ADB was funded by a Mason Medical Foundation Research Fellowship and grants from the Peel Medical Research Trust and St George\'s Hospital Charity; IPMT acknowledges funding from Cancer Research UK and a core centre grant from the Wellcome Trust (No. 090532/Z/09/Z). Author contributions ==================== Planned study, ADB, SVH and IPMT; molecular analysis, ADB and AJ; patient recruitment, ADB, ME-B, MA and SVH; manuscript, ADB, SVH and IPMT. SUPPORTING INFORMATION ON THE INTERNET ====================================== The following supporting information may be found in the online version of this article. Figure S1. Leading edge diagram for carcinoma versus normal tissue, demonstrating the number of times a gene has a significant change in methylation in each set. SLIT2, the highest-ranked gene, is noted to be significantly hypermethylated in six different gene sets. Figure S2. Leading edge diagram of adenoma versus normal tissue, demonstrating the number of times a gene is significantly hypomethylated within a gene set. The top-ranked gene, NDUFA13, occurs in 20 different gene sets. Figure S3. Representation of GRASP/CDKN2A (p14ARF)/TP53 pathway. Table S1. Top 25 differentially methylated genes from Bayesian model of carcinomas versus normal tissue. Table S2. Top 25 differentially methylated genes from Bayesian model of adenomas versus normal tissue. Table S3. Top 25 differentially methylated genes from Bayesian model of carcinomas versus adenomas. Table S4. Table of gene set ranked by NES score for cancers versus normals in methylated probes. Table S5. Table of gene sets in adenomas ranked by NES score for adenomas versus normals in demethylated probes. [^1]: No conflicts of interest were declared.
Insomnia at 7 weeks? 7 weeks pregnant and it seems anytime I wake up in the night to pee I can not get back to sleep for hours after. My mind is just soooo awake. I normally eat a little cereal then lay awake for a couple hours then sleep for an hour or so more before work. Anyone else?
Q: Specific CSS on Wordpress index page I have a Wordpress site and I want to change certain CSS values in the header.php on the index page only. I have a div which is 100% width and border bottom to add an underline to the header.php <div class="nav"> ...content to create nav-bar ... <div class="hr"></div> <!-- remove on index.php --> </div> I don't want it to show, or affect anything on the index.php. (I don't want to target another header.php file for this one small thing) A: Wordpress has a template tag called body_class which is used to create contextual CSS hooks. <body <?php body_class(); ?>> You can then use it in your CSS as follows: .blog {} .blog.home {} / if blog index is a page / .page.blog {} see the documentation for the rather extensive list of possible output.
Strategic Plan 2006-2010 The Hood Museum of Art’s strategic plan maps the purpose, objectives, key strategies, and direction of the Hood Museum of Art for the years 2006 through 2010 within the context of planning for reaccreditation by the American Association of Museums (AAM) in 2007 and the twenty-fifth anniversary of the Hood Museum of Art in 2010, both opportunities to measure the museum’s success. The plan is framed to be consistent with the purpose, intellectual character, and core values of Dartmouth College, including especially its commitments to academic and student life and to diversity. Purpose The purpose of the Hood Museum of Art at Dartmouth College is to inspire, educate, and collaborate with our college and broader communities about creativity and imagination, through direct engagement with works of art of historic and cultural significance, by making effective use of our collections and staff. The Hood Museum of Art seeks to provide access to works of art and to information about them. Objectives The strategic plan builds on the museum’s achievements and gives direction for the management and development of our collections, audiences, and resources. Objective 1: Manage and Develop Our Collections To acquire works of art by purchase and gift to enhance the aesthetic and cultural impact of the Hood Museum of Art’s collections and build on their strengths, and to manage and protect all of the works in the museum’s care. Key Strategies: MANAGE —Maintain the collections catalogue through research, imaging, and documentation, using appropriate technologies —Research, document, and review the collections, identifying strengths and gaps and evaluating future acquisitions needs —Build on our role as a museum in an academic environment by promoting our dual provision of public galleries and study storage areas DEVELOP —Conserve, protect, and secure the collections by reorganizing, improving, and expanding storage facilities —Identify and acquire works of art by purchase, gift, and loan to build on strengths and fill gaps in the collections —Identify potential donors and acquire works of art and acquisitions funds from them in collaboration with the College’s development office Objective 2: Manage and Develop Our Audiences To provide access to works of art through displays, exhibitions, programs and activities, publications, the Internet, and promotion. Key Strategies: MANAGE —Engage our audiences by promoting our collections and works of art on loan through lively and diverse exhibitions, displays, and publications —Identify and evaluate our College and community audiences’ knowledge of and access to our collections, exhibitions, and programs —Make the most effective use of museum facilities, including the public galleries, study storage, classroom spaces, and information technology for our diverse audiences DEVELOP —Inspire, educate, and collaborate with College and broader audiences through direct engagement with exciting and provocative works of art —Attract new audiences by developing and promoting the relationships between academic programming and other educational programming at the College and in the community through collaboration and partnerships —Increase our capacity to engage College and broader audiences through enhanced facilities, promotion, and information technology services Objective 3: Manage and Develop Our Resources MANAGE —Organize and manage our staff, facility, and information technology resources to make effective use of our collections in support of our objectives —Manage and evaluate our resources to provide the most effective access for our diverse audiences —Realize the potential of our many sources of support to fund our objectives DEVELOP —Secure funds for the development of our collections, staffing, facilities, and information technologies resources —Develop our resources to attract, inspire, and engage our audiences with curricular and non-curricular programming —Expand and develop new sources of support to fund our objectives
Trace elements and alcohol. In our study alcoholic patients with and without cirrhosis have a decreased serum zinc. They also have increased serum copper and iron with an increase in the serum ferritin. There is no evidence of selenium deficiency in either alcoholic group. Alcohol when given with zinc in a single dose to normal volunteers increases the serum zinc and therefore appears to increase the absorption of zinc.
Berry Alvis's Page √ New Stuff "Our church (Experience Life in Lubbock, Tx) begins each year with a weekend corporate prayer service instead of regular services and then we invite all to join us throughout the year at our corporate prayer services for that campus each week.…" "long winded praying long winded speakers lack of direction not praying for most impt things: salvations, missions, revival, vision lack of creativity not connected with churches vision putting people on the spot rather than instructing them step by…" "At our weekly Monday night prayer gathering we try and make sure our hearts are set on how big God is verses how big the problems are we are praying for. With that in mind we begin the evening for the most part with Adoration (worship) or…" "We have a connection card that is perforated on our bulletin. It has a place for prayer requests. We put those requests in a spreadsheet and intergrate it with business card software. We print the request off on business cards…" For those who coordinate, facilitate, motivate individual prayer and corporate praying.✅ PrayerLeader.com ... Our heart beats for prayer. Let Us Help You . . .— Go deeper in your prayer connection with Jesus— Be a prayer encourager to others— Be a catalyst and leader for prayer in your churchSee More
Iranian nuclear scientist arrives in Pakistani Embassy in Washington Published July 13th, 2010 - 15:23 GMT Iranian nuclear scientist Shahram Amiri, who was abducted by the US last year, has been escorted by American forces to Iran's interest section in Washington. Iranian nuclear scientist Shahram Amiri, who was abducted by the US last year, has been escorted by American forces to Iran's interest section in Washington. According to an Iranian TV report on Tuesday, Amiri took refuge in Iran's interest section in Washington, urging an "immediate return" to Iran. The Pakistani Embassy in Washington preserves Iran's interests in the United States, since the two countries have no diplomatic ties. Iran says that in collaboration with Saudi Arabia, US agents kidnapped Amiri while he was on a pilgrimage in June 2009 and took him to America. Since then, two videos and one audio message featuring him have surfaced. In the first video, Amiri said that he was abducted "in a joint operation by terror and kidnap teams from the US intelligence service, CIA and Saudi Arabia's Istikhbarat" from Medina. In the second video, he contradicted his earlier statements, saying that he was in the US of his own free will to further his education, dismissing all rumors about his defection.
# walt-link Linker for `walt` WebAssembly Programs. ## About Wraps `.walt` file with a JavaScript module, with all the dependencies linked. Intented to be used in a _node environment_. Not yet implemented in a loader. Still under development, but the API wont change. Notes * Any import with a leading dot(`.`) will be linked into the final binary * Anything without a leading dot(`.`) will be treated as an environment import and is left up to the user to implement. * The top-level function returns a factory method which returns a function taking an import object. Every call to the method returns a brand new module * _importObj_ used in the factory method is _shared accross all modules_. * Walt Dependencies are shared within a single WebAssembly module instance. Each import is a stand alone module linked by the linker. This is useful if you want shared module state across imports, similar to node modules. TODOs - ## Usage ```js const { link } = require("walt-link"); const path = require("path"); const factory = link(path.resolve(__dirname, "./index.walt")); factory({ env: { memory, }, }).then(wasmModule => { /* run your code here */ }); ```
All teams finish their Conference schedule with two games of one at home and one away. Nearly all teams have two home games and two away games in the first half of the Conference schedule as well as the second half of the Conference schedule; and At least one conference game in all but the opening weekend of the season. 2018 CONFERENCE USA COMPOSITE FOOTBALL SCHEDULE (as of January 23, 2018) SATURDAY, AUGUST 25 FRIDAY, AUGUST 31 SATURDAY, SEPTEMBER 1 SATURDAY, SEPTEMBER 8 SATURDAY, SEPTEMBER 15 SATURDAY, SEPTEMBER 22 SATURDAY, SEPTEMBER 29 SATURDAY, OCTOBER 6 SATURDAY, OCTOBER 13 SATURDAY, OCTOBER 20 SATURDAY, OCTOBER 27 SATURDAY, NOVEMBER 3 SATURDAY, NOVEMBER 10 SATURDAY, NOVEMBER 17 SATURDAY, NOVEMBER 24 SATURDAY, DECEMBER 1 All games and dates subject to change Conference USA has announced its football schedule for the 2018 season. The 14-team alignment for the 2018 season will again feature each team playing eight conference games, along with four non-league contests. A total of 169 games are featured over 14 regular season playing weeks.The 2018 alignment will feature seven teams in the East Division and seven teams in the West Division. Charlotte, FIU, Florida Atlantic, Marshall, Middle Tennessee, Old Dominion and WKU comprise the East Division, while Louisiana Tech, North Texas, Rice, Southern Miss, UAB, UTEP and UTSA make up the West Division.Each school will play every team in its division once for six games, while playing two cross-over opponents from the opposite division. Each team will reverse the location of its 2017 conference opponents, both inter-division opponents and intra-division foes. The conference is in the second year of a rotation between cross-division opponents that will rotate every two years until 2024, when each school has played every opponent in the opposite division once home and away.The 2018 schedule complies with C-USA scheduling policies as approved by the Board of Directors. In addition, the schedule provides the following:It is important to note that all games are subject to date changes and some dates are expected to move in the coming weeks in order accommodate national television.A schedule of televised games will be released at a later date. Conference USA’s 14th annual Championship Game, pitting the champions of the East and West Divisions, is scheduled for Saturday, December 1.Prairie View A&M at RiceWKU at WisconsinFordham at CharlotteIndiana at FIUFlorida Atlantic at OklahomaLouisiana Tech at South AlabamaMarshall at Miami (Ohio)Middle Tennessee at VanderbiltSMU at North TexasOld Dominion at LibertyHouston at RiceJackson State at Southern MissSavannah State at UABNorthern Arizona at UTEPUTSA at Arizona StateAppalachian State at CharlotteAir Force at Florida AtlanticSouthern at Louisiana TechEastern Kentucky at MarshallTennessee-Martin at Middle TennesseeIncarnate Word at North TexasRice at Hawai’iLouisiana-Monroe at Southern MissUAB at Coastal CarolinaUTEP at UNLVBaylor at UTSAMaine at WKUMassachusetts at FIUBethune-Cookman at Florida AtlanticMarshall at South CarolinaMiddle Tennessee at GeorgiaNorth Texas at ArkansasSouthern Miss at Appalachian StateTulane at UABUTEP at TennesseeUTSA at Kansas StateWKU at LouisvilleCharlotte at MassachusettsFIU at Miami (Fla.)Florida Atlantic at UCFLouisiana Tech at LSUNC State at MarshallNorth Texas at LibertyVirginia Tech at Old DominionNew Mexico State at UTEPTexas State at UTSAWKU at Ball StateArkansas-Pine Bluff at FIUOld Dominion at East CarolinaRice at Wake ForestSouthern Miss at AuburnCharlotte at TennesseeLouisiana Tech at Mississippi StateMiddle Tennessee at KentuckyVMI at Old DominionRice at LSUUAB at Texas A&MC-USA Championship Game
James Estrin/The New York Times The Great Recession was the worst downturn since the Great Depression. And yet, throughout the recent decline and today’s sluggish recovery, conditions have never seemed as bad as they were in the 1930s. Breadlines, for example, have not been commonplace. That may be about to change. In an article published on Monday, The Times’s Patrick McGeehan described a line snaking down Fulton Street in Brooklyn last week, with people waiting to enter a food pantry run by the Bed-Stuy Campaign Against Hunger. The line was not an anomaly. Demand at all of New York City’s food pantries and soup kitchens has spiked since federal food stamps were cut on Nov. 1. The cut — which affects nearly all of the nation’s 48 million food stamp recipients — amounts to a loss of $29 a month for a New York City family of three. On the shoestring meal budgets of food stamp recipients, that’s enough for some 20 individual meals, according to the New York City Coalition Against Hunger. The food stamp cuts are occurring even though need is still high and opportunity low. In a report released today, the Coalition estimates that one-sixth of the city’s residents and one-fifth of its children live in homes without enough to eat. Those numbers have not improved over the past three years. The lack of economic recovery for low income New Yorkers is at odds with gains at the top of the income ladder, reflected in soaring real estate prices, rising stock prices and big Wall Street bonuses. And there are more food-stamp cuts to come. House Republicans have proposed to cut the program by $40 billion over 10 years in the pending farm bill; the Senate has proposed a $4 billion reduction. With Congress framing its task not as whether to cut the program, but how much, is there any doubt that food lines will soon be getting longer — and children hungrier? If the current downturn has not mirrored the Great Depression, that’s thanks to safety net programs that grew out of the Depression and other hard times. Breadlines have, by and large, been replaced by food stamps, while income from Social Security, unemployment benefits and the earned income tax credit has kept many people from falling irretrievably into the economic abyss. Without those supports, conditions — which are bad enough — would be much worse, as is clear from the Census Bureau’s latest poverty measures. According to the bureau: Poverty among children, at 18 percent in 2012, would have been nearly 21 percent, but for food stamps; it would have been nearly 25 percent but for household income from tax credits like the one for earned income. Poverty among the elderly, at 14.8 percent, would be nearly 55 percent, but for Social Security. Poverty among non-elderly adults, at 15.5 percent, would be 16.4 percent, but for unemployment benefits. Now is not the time to cut back. Now is the time to provide.
Q: link up unanswered questions from a launchpad mailing list to askubuntu? Is there a way to hook up unanswered questions from a launchpad mailing list to askubuntu? I thought the poor man's solution would be to just copy+paste the mailing list question to askubuntu and reply to the mailing list with the askubuntu URL, but maybe there is something better out there. A: LP Answers currently doesn't have a way to connect to an external source (such as AU) to a project. We've discussed this at a UDS and currently it's on a "wow this would be great to have" column, but it wouldn't be useful if we were trying to do this by hand as it would be confusing to people unless we did it right. What was discussed, and what was the outcome of the Ask Ubuntu UDS-N session?
North Korea will soon allow foreign visitors access to the internet via the nation's Koryolink 3G service, according to reports from the Associated Press Pyongyang bureau. The Egyptian firm that built North Korea's cell tower infrastructure, Orascom Telecom, reportedly told foreign residents in Pyongyang that internet service would launch no later than March 1. The news comes just weeks after NK NEWS exclusively revealed that the KoryoLink network had passed new measures that would allow foreigners to use their cellphones for the first time during visits to North Korea. The move is a logical next step for KoryoLink, a joint venture company that has been developing its 3G internet-compatible cell infrastructure in North Korea for over four years now.
The Young and the Restless Spoilers (YR): Will Mariah and Devon CRASH and BURN? The Young and the Restless spoilers (YR) reveal one of Genoa City’s hottest couples is either going to heat up–or fizzle out as quickly as they began. Falling Flat? From day one, Mariah (Camryn Grimes) and Devon (Bryton James) were an unlikely duo. Their progression from friends to lovers threw some fans through a loop, but their sweet nature quickly won them over. They work well together on the surface, but their chemistry isn’t exactly over-the-top. Revenge Relationships Never Last Hooking up with Devon always did seem like quite the slap in Hilary’s (Mishael Morgan) face. The divorce wasn’t easy for the superstar and having to see her co-host with her ex is still a sore spot. Of course, Hilary’s feelings didn’t Mariah and Devon from doing what they wanted. In fact, the more it bothered Hilary, the closer and more public they became. Running From Reality Adding more complications, to say Mariah’s been out of sorts since her kiss with Tessa (Cait Fairbanks) is an understatement. The moment has her running scared and saying things she doesn’t mean just to keep hanging onto Devon. But, I (Fake) Love You… Not only is Mariah clinging a little too hard, but her shocking admission of love rightfully fell flat. It was an act of sheer desperation and Devon saw right through it. But, that’s still not enough to end this relationship. One More Chance Despite all the obstacles, Devon insists Mariah’s the right woman for him. He wants to give this relationship his all and move forward with a fresh outlook. While Mariah agrees to try again, the reluctance in her eyes proves this is one love affair that may not last too much loner.
#ifndef __ASM_POWERPC_PCI_H #define __ASM_POWERPC_PCI_H #ifdef __KERNEL__ /* * This program is free software; you can redistribute it and/or * modify it under the terms of the GNU General Public License * as published by the Free Software Foundation; either version * 2 of the License, or (at your option) any later version. / #include <linux/types.h> #include <linux/slab.h> #include <linux/string.h> #include <linux/dma-mapping.h> #include <asm/machdep.h> #include <asm/scatterlist.h> #include <asm/io.h> #include <asm/prom.h> #include <asm/pci-bridge.h> #include <asm-generic/pci-dma-compat.h> / Return values for ppc_md.pci_probe_mode function / #define PCI_PROBE_NONE -1 / Don't look at this bus at all / #define PCI_PROBE_NORMAL 0 / Do normal PCI probing / #define PCI_PROBE_DEVTREE 1 / Instantiate from device tree / #define PCIBIOS_MIN_IO 0x1000 #define PCIBIOS_MIN_MEM 0x10000000 struct pci_dev; / Values for the `which' argument to sys_pciconfig_iobase syscall. / #define IOBASE_BRIDGE_NUMBER 0 #define IOBASE_MEMORY 1 #define IOBASE_IO 2 #define IOBASE_ISA_IO 3 #define IOBASE_ISA_MEM 4 / * Set this to 1 if you want the kernel to re-assign all PCI * bus numbers (don't do that on ppc64 yet !) / #define pcibios_assign_all_busses() \ (ppc_pci_has_flag(PPC_PCI_REASSIGN_ALL_BUS)) static inline void pcibios_set_master(struct pci_dev dev) { /* No special bus mastering setup handling / } static inline void pcibios_penalize_isa_irq(int irq, int active) { / We don't do dynamic PCI IRQ allocation / } #define HAVE_ARCH_PCI_GET_LEGACY_IDE_IRQ static inline int pci_get_legacy_ide_irq(struct pci_dev dev, int channel) { if (ppc_md.pci_get_legacy_ide_irq) return ppc_md.pci_get_legacy_ide_irq(dev, channel); return channel ? 15 : 14; } #ifdef CONFIG_PCI extern void set_pci_dma_ops(struct dma_map_ops dma_ops); extern struct dma_map_ops get_pci_dma_ops(void); #else /* CONFIG_PCI / #define set_pci_dma_ops(d) #define get_pci_dma_ops() NULL #endif #ifdef CONFIG_PPC64 / * We want to avoid touching the cacheline size or MWI bit. * pSeries firmware sets the cacheline size (which is not the cpu cacheline * size in all cases) and hardware treats MWI the same as memory write. / #define PCI_DISABLE_MWI #ifdef CONFIG_PCI static inline void pci_dma_burst_advice(struct pci_dev pdev, enum pci_dma_burst_strategy strat, unsigned long strategy_parameter) { unsigned long cacheline_size; u8 byte; pci_read_config_byte(pdev, PCI_CACHE_LINE_SIZE, &byte); if (byte == 0) cacheline_size = 1024; else cacheline_size = (int) byte * 4; strat = PCI_DMA_BURST_MULTIPLE; strategy_parameter = cacheline_size; } #endif #else /* 32-bit / #ifdef CONFIG_PCI static inline void pci_dma_burst_advice(struct pci_dev pdev, enum pci_dma_burst_strategy strat, unsigned long strategy_parameter) { strat = PCI_DMA_BURST_INFINITY; strategy_parameter = ~0UL; } #endif #endif /* CONFIG_PPC64 / extern int pci_domain_nr(struct pci_bus bus); /* Decide whether to display the domain number in /proc / extern int pci_proc_domain(struct pci_bus bus); /* MSI arch hooks / #define arch_setup_msi_irqs arch_setup_msi_irqs #define arch_teardown_msi_irqs arch_teardown_msi_irqs #define arch_msi_check_device arch_msi_check_device struct vm_area_struct; / Map a range of PCI memory or I/O space for a device into user space / int pci_mmap_page_range(struct pci_dev pdev, struct vm_area_struct vma, enum pci_mmap_state mmap_state, int write_combine); / Tell drivers/pci/proc.c that we have pci_mmap_page_range() / #define HAVE_PCI_MMAP 1 extern int pci_legacy_read(struct pci_bus bus, loff_t port, u32 val, size_t count); extern int pci_legacy_write(struct pci_bus bus, loff_t port, u32 val, size_t count); extern int pci_mmap_legacy_page_range(struct pci_bus bus, struct vm_area_struct vma, enum pci_mmap_state mmap_state); #define HAVE_PCI_LEGACY 1 #if defined(CONFIG_PPC64) \|\| defined(CONFIG_NOT_COHERENT_CACHE) /* * For 64-bit kernels, pci_unmap_{single,page} is not a nop. * For 32-bit non-coherent kernels, pci_dma_sync_single_for_cpu() and * so on are not nops. * and thus... / #define DECLARE_PCI_UNMAP_ADDR(ADDR_NAME) \ dma_addr_t ADDR_NAME; #define DECLARE_PCI_UNMAP_LEN(LEN_NAME) \ __u32 LEN_NAME; #define pci_unmap_addr(PTR, ADDR_NAME) \ ((PTR)->ADDR_NAME) #define pci_unmap_addr_set(PTR, ADDR_NAME, VAL) \ (((PTR)->ADDR_NAME) = (VAL)) #define pci_unmap_len(PTR, LEN_NAME) \ ((PTR)->LEN_NAME) #define pci_unmap_len_set(PTR, LEN_NAME, VAL) \ (((PTR)->LEN_NAME) = (VAL)) #else / 32-bit && coherent / / pci_unmap_{page,single} is a nop so... / #define DECLARE_PCI_UNMAP_ADDR(ADDR_NAME) #define DECLARE_PCI_UNMAP_LEN(LEN_NAME) #define pci_unmap_addr(PTR, ADDR_NAME) (0) #define pci_unmap_addr_set(PTR, ADDR_NAME, VAL) do { } while (0) #define pci_unmap_len(PTR, LEN_NAME) (0) #define pci_unmap_len_set(PTR, LEN_NAME, VAL) do { } while (0) #endif / CONFIG_PPC64 \|\| CONFIG_NOT_COHERENT_CACHE / #ifdef CONFIG_PPC64 / The PCI address space does not equal the physical memory address * space (we have an IOMMU). The IDE and SCSI device layers use * this boolean for bounce buffer decisions. / #define PCI_DMA_BUS_IS_PHYS (0) #else / 32-bit / / The PCI address space does equal the physical memory * address space (no IOMMU). The IDE and SCSI device layers use * this boolean for bounce buffer decisions. / #define PCI_DMA_BUS_IS_PHYS (1) #endif / CONFIG_PPC64 / extern void pcibios_resource_to_bus(struct pci_dev dev, struct pci_bus_region region, struct resource res); extern void pcibios_bus_to_resource(struct pci_dev dev, struct resource res, struct pci_bus_region region); extern void pcibios_claim_one_bus(struct pci_bus b); extern void pcibios_finish_adding_to_bus(struct pci_bus bus); extern void pcibios_resource_survey(void); extern struct pci_controller init_phb_dynamic(struct device_node dn); extern int remove_phb_dynamic(struct pci_controller phb); extern struct pci_dev of_create_pci_dev(struct device_node node, struct pci_bus bus, int devfn); extern void of_scan_pci_bridge(struct device_node node, struct pci_dev dev); extern void of_scan_bus(struct device_node node, struct pci_bus bus); extern void of_rescan_bus(struct device_node node, struct pci_bus bus); extern int pci_read_irq_line(struct pci_dev dev); struct file; extern pgprot_t pci_phys_mem_access_prot(struct file file, unsigned long pfn, unsigned long size, pgprot_t prot); #define HAVE_ARCH_PCI_RESOURCE_TO_USER extern void pci_resource_to_user(const struct pci_dev dev, int bar, const struct resource rsrc, resource_size_t start, resource_size_t end); extern void pcibios_setup_bus_devices(struct pci_bus bus); extern void pcibios_setup_bus_self(struct pci_bus bus); extern void pcibios_setup_phb_io_space(struct pci_controller hose); extern void pcibios_scan_phb(struct pci_controller hose, void sysdata); #endif /* __KERNEL__ / #endif / __ASM_POWERPC_PCI_H */
Answer: 22222 Difficulty: 1 # Hint The macro is counting how many "tokens" are in its input. # Explanation All five invocations of `m!` pass two tokens as input: a minus sign followed by an integer or floating point literal token. The floating point literals `1.`, `1.0`, `1.0e1`, `1.0e-1` are each a single atomic token. The parser built into the Rust compiler always parses a negative sign as a separate token from the numeric literal that is being negating. However, it is possible for a user-defined parser within a [procedural macro] to construct a negative number as a single token by passing a negative integer or negative floating point value to one of the constructors of [`proc_macro::Literal`]. If such a negative literal ends up in the input of a subsequent procedural macro invocation, it is up to the compiler whether to rewrite into a pair of tokens or keep them as one. [procedural macro]: https://github.com/dtolnay/syn [`proc_macro::Literal`]: https://doc.rust-lang.org/proc_macro/struct.Literal.html The behavior of the compiler's parser is observable in the surface language as well, not only in macros. For example the following code prints `-81` because the expression is parsed as `-(3i32.pow(4))` rather than `(-3i32).pow(4)`. ```rust fn main() { let n = -3i32.pow(4); println!("{}", n); } ```
Q: openvpn radius-plugin does not assign framed-ip-address from freeradius to clients I am new to openvpn. I have an openvpn setup on ubuntu 14.04 which has a radius AAA backend for authentication, authorization and accounting. In addition to this, we have configured freeradius to assign ips from a pool as framed-ip-address. From radius logs, it appears that freeradius returns framed-ip-address in response to access-request message when authentication and authorization is successful but openvpn seems to ignore it and uses its own ip pool specified in the server directive. What i want is that openvpn respects framed-ip-address returned by freeradius but it does not. Since i don't have prior experience with openvpn, i would appreciate help from experts. Here is my openvpn side of configuration which i think has a problem. OpenVPN configuration: local 192.168.7.100 mode server port 443 proto tcp dev tun tcp-queue-limit 256 tun-mtu 1500 mssfix 1460 sndbuf 0 rcvbuf 0 cipher AES-256-CBC ca /etc/openvpn/easy-rsa/keys/ca.crt cert /etc/openvpn/easy-rsa/keys/server.crt key /etc/openvpn/easy-rsa/keys/server.key dh /etc/openvpn/easy-rsa/keys/dh2048.pem tls-auth /etc/openvpn/easy-rsa/keys/ta.key 0 plugin /etc/openvpn/rad/radiusplugin.so /etc/openvpn/rad/443.cnf client-cert-not-required username-as-common-name server 10.10.0.0 255.255.0.0 ;ifconfig-pool 10.0.0.0 10.1.255.254 ifconfig-pool-persist ipp.txt push "redirect-gateway def1" push "dhcp-option DNS 8.8.8.8" push "dhcp-option DNS 8.8.4.4" reneg-sec 0 keepalive 60 120 tcp-nodelay comp-lzo persist-key persist-tun status /etc/openvpn/443.log 1 status-version 1 verb 4 management 192.168.7.100 7505 Radius Plugin: NAS-Identifier=openvpn_tcp_443 Service-Type=5 Framed-Protocol=1 NAS-Port-Type=5 NAS-IP-Address=192.168.7.100 OpenVPNConfig=/etc/openvpn/tcp_443.conf subnet=255.255.255.0 overwriteccfiles=true nonfatalaccounting=false server { acctport=1813 authport=1812 name=192.168.7.102 retry=1 wait=1 sharedsecret=--redacted-- } server { acctport=1813 authport=1812 name=192.168.7.103 retry=1 wait=1 sharedsecret=--redacted-- } Thankyou. ====================== Update I have made the following changes to config. topology subnet push "topology subnet" client-config-dir clients After adding client-config-dir, the framed-ip-address is being assigned. As mentioned earlier, now from my clients, I cannot browse anything. Seems the traffic cannot be routed properly. There seems to be a routing issue or the topology or something else that i expect openvpn to handle but it does not. Here is the relevant information that can help it understand. The ip assigned to openvpn client is a public ip whose routing is set on the gateway of the machine. The same client ip works properly when used with strongswan. Which means it is properly routable. But with openvpn, it does not. relevant output from ifconfig on server machine. tun0 Link encap:UNSPEC HWaddr 00-00-00-00-00-00-00-00-00-00-00-00-00-00-00-00 inet addr:10.10.0.1 P-t-P:10.10.0.1 Mask:255.255.255.0 UP POINTOPOINT RUNNING NOARP MULTICAST MTU:1500 Metric:1 RX packets:0 errors:0 dropped:0 overruns:0 frame:0 TX packets:0 errors:0 dropped:0 overruns:0 carrier:0 collisions:0 txqueuelen:100 RX bytes:0 (0.0 B) TX bytes:0 (0.0 B) relevant output of route -n 0.0.0.0 --redacted-- 0.0.0.0 UG 0 0 0 em2 10.10.0.0 0.0.0.0 255.255.255.0 U 0 0 0 tun0 --redacted-- 0.0.0.0 255.255.255.248 U 0 0 0 em2 the rp_filter information: root@us1-ps1:~# sysctl -a \| grep rp_filter net.ipv4.conf.all.arp_filter = 0 net.ipv4.conf.all.rp_filter = 1 net.ipv4.conf.default.arp_filter = 0 net.ipv4.conf.default.rp_filter = 0 net.ipv4.conf.em1.arp_filter = 0 net.ipv4.conf.em1.rp_filter = 0 net.ipv4.conf.em2.arp_filter = 0 net.ipv4.conf.em2.rp_filter = 0 net.ipv4.conf.lo.arp_filter = 0 net.ipv4.conf.lo.rp_filter = 0 net.ipv4.conf.tun0.arp_filter = 0 net.ipv4.conf.tun0.rp_filter = 0 root@us1-ps1:~# ping 10.10.0.1 PING 10.10.0.1 (10.10.0.1) 56(84) bytes of data. 64 bytes from 10.10.0.1: icmp_seq=1 ttl=64 time=0.058 ms 64 bytes from 10.10.0.1: icmp_seq=2 ttl=64 time=0.037 ms Internet is working properly. root@us1-ps1:~# ping google.com PING google.com (172.217.0.46) 56(84) bytes of data. 64 bytes from lga15s43-in-f14.1e100.net (172.217.0.46): icmp_seq=1 ttl=51 time=20.3 ms 64 bytes from lga15s43-in-f14.1e100.net (172.217.0.46): icmp_seq=2 ttl=51 time=20.2 ms 64 bytes from lga15s43-in-f14.1e100.net (172.217.0.46): icmp_seq=3 ttl=51 time=20.2 ms ^C --- google.com ping statistics --- 3 packets transmitted, 3 received, 0% packet loss, time 2002ms rtt min/avg/max/mdev = 20.216/20.279/20.338/0.171 ms default gateway can also be pinged. let me know if other information you want to print. Thankyou A: Specify the client-config-dir directive. Is where the radius plugin will put the files who will be used by openvpn to assign the ip. Eg. client-config-dir clients
A BATTLE has been launched to restore the founding principles behind the NHS and halt creeping privatisation. The National Health Service Reinstatement Bill is due to have its second reading in Parliament early next year. It was tabled by Brighton Pavilion MP Caroline Lucas, who has called on MPs from all parties to back it. There has been growing anger at sweeping changes made to the NHS in Sussex and nationally which have led to more private companies taking over services. Opponents have said this goes against the whole ethos of the NHS and more focus should be spent on supporting existing services instead. Health bosses have also been struggling to meet savings targets while dealing with rising prices and a growing demand for services. Council funding cuts have increased pressure on community services and beds, putting hospitals under more pressure. Ms Lucas MP said: “The NHS we love is facing an existential threat, with a quarter of a century of creeping marketisation bringing increased fragmentation and inefficiencies into our health service. “I tabled the NHS Reinstatement Bill because I believe that it’s time to take a stand for our most valued public asset. “I’m honoured to be presenting the bill with such strong cross-party support but to take the campaign for a truly public NHS to the next level, more MPs from all parties need to back it.” The bill aims to roll back NHS privatisation and reinstate its principles of being truly public, fully protected and free at point of delivery. It would reinstate the secretary of state’s responsibility for the health of UK citizens, something which was removed in the controversial Health and Social Care Act of 2012. The bill would also abolish bodies such as NHS trusts, NHS foundation trusts and clinical commissioning groups, allowing commercial companies to provide services only if they were essential to patient welfare and the NHS could not do so itself. Peter Roderick, barrister and one of the bill’s authors, said: “Unless the Westminster Parliament passes a law to remove the market from the NHS in England, the top-down disorganisation introduced in 2012 will continue, services will be reduced and piecemeal devolution will fragment the system further. “Unfortunately, most MPs don’t support the bill so the chances of it becoming law before the next general election are very slim.” Brighton's Royal Sussex County Hospital accident and emergency consultant Rob Galloway said: "The NHS has been damaged by the 2012 bill and interest of big business has become more important than the interest of the patients." The Government has said the NHS needs change in order to cope with the growing number of people living longer . Ministers said keeping the NHS running as it was, was unsustainable and unaffordable and efficiency needed to be improved. The bill has been supported by MPs from Labour and the Liberal Democrats as well as from members of the British Medical Association.
<?php namespace Everyman\Neo4j\Cypher; class QueryTest extends \PHPUnit_Framework_TestCase { protected $client = null; protected $query = null; protected $template = null; protected $vars = null; public function setUp() { $this->client = $this->getMock('Everyman\Neo4j\Client', array(), array(), '', false); $this->template = 'START a=({start}) RETURN a'; $this->vars = array('start' => 0); $this->query = new Query($this->client, $this->template, $this->vars); } public function testGetQuery_ReturnsString() { $result = $this->query->getQuery(); $this->assertEquals($result, $this->template); } public function testGetParameters_ReturnsArray() { $result = $this->query->getParameters(); $this->assertEquals($result, $this->vars); } public function testGetResultSet_OnlyExecutesOnce_ReturnsResultSet() { $return = $this->getMock('Everyman\Neo4j\Query\ResultSet', array(), array(), '', false); $this->client->expects($this->once()) ->method('executeCypherQuery') ->will($this->returnValue($return)); $this->assertSame($return, $this->query->getResultSet()); $this->assertSame($return, $this->query->getResultSet()); } public function testGetResultSet_ClientReturnsFalse_ReturnsFalse() { $return = false; $this->client->expects($this->once()) ->method('executeCypherQuery') ->will($this->returnValue($return)); $this->assertFalse($this->query->getResultSet()); $this->assertFalse($this->query->getResultSet()); } }
But we are going to create thousands of green jobs. That makes sense, right? 3:09 pm August 12, 2009 Sean wrote : Lets just look at MI where the recession has caused the unemployment rate to jump to 15.4% and the senator from that state looks at climate change as her #1 priority. In the US, the recession has led to a very large decrease in our CO2 emmissions (even though it increased in the rest of the world where they still make things). It appears that the senator from MI has delivered on her #1 priority just has Waxman Markey will for the rest of the nation. 3:51 pm August 12, 2009 "If I sound fishy please don't turn me in" wrote : Don't worry green jobs already make up .05 of 1% of American jobs. That should offset those 2 million unemployed. 4:25 pm August 12, 2009 Terry wrote : Sean, as someone who has worked in wind power for a while now, I can't imagine a worse place than Michigan to develop renewable energy. Between the high cost labor, absurdly crafted RPS and overwhelming power of the utilities, I think I'll stick to Texas. 4:59 pm August 12, 2009 Andrea B wrote : I think it's important to recognize that NAM isn’t speaking for all manufacturers. An increasing number of U.S. manufacturers and other businessess believe that clean energy and climate policies will create jobs, not the reverse. They believe this because it’s already happening in places like Indiana, Michigan and Ohio, where dozens of manufacturing firms are producing components for solar panels, wind turbines, hybrid cars, and the like. Just last week, more than 150 businesses came out in favor of clean energy legislation that U.S. Sen. Sherrod Brown is sponsoring, which would boost domestic clean energy manufacturing and ensure that new clean energy manufacturing jobs stay in America. Brown's bill is called the IMPACT Act -- Investments for Manufacturing Progress and Clean Technology. Identical clean energy manufacturing language was already included in ACES -- the clean energy and climate bill passed by the U.S. House of Representatives. 5:42 pm August 12, 2009 wake up wrote : Andrea: I am sorry, but you are clueless. The cap and trade tax will be the final nail in the coffin for manufacturing in the United States. Labor costs and regulation in places like China, India, and even Mexico are a fraction of what they cost here in the US. Once the cap and tax passes, energy costs will go up significantly and the remaining manufacturers will move oversees where they can have cheap energy powered by dirty coal. As for "green jobs", the vast majority of "green" manufacturing jobs are already in China. The only real green jobs in the US is the people who invent the techonolgy and the people who install the "green" machines. This number of people is not significant and any growth in these jobs will not be enough to offset the lost manufacturing jobs shipped oversees, not to mention all of the people employed by the coal and oil industry. It's naive and ignorant to suggest the cap and tax will create a net postive in jobs. That is patently false. 2:13 am August 13, 2009 tom a taxpayer wrote : The mindless destruction of productive assets mandated by the "Cash for Clunkers" is a tiny taste of the destruction the Cap and Trade whirlwind will reap. Scrapping cars years before the end of their useful life is the height of profligate and wasteful consumption. The thousands of pounds of energy-intensive, highly-processed materials and advanced engineering equipment in cars is an enormous investment based on massive inputs of energy, mining, water, labor and materials. It makes no energy, environmental or economic sense to destroy the cars engines and render the investment useless years before the end of their useful life. By disabling the engines and scrapping perfectly good used cars, the "Cash for Clunkers" program is hurting the used cars businesses and the poor and moderate income folks who can only afford used cars. The "Cash for Clunkers" is largely benefitting the haves and hurting the have-nots. The same arrogant, ignorant environ-mentalmidgets in Congress and the Obama administration who created "Cash for Clunkers" have created a far grander Clunker called "Cap and Trade". The Malarkey-Waxman cap-and-trade bill that passed the House of Reprehensibles is a stealth Value Added Tax (VAT). It will levy major costs on every fossil fuel component in the supply chain. It will be a balloon tax on all the essentials that the poor and middle class pay a higher percentage of their income on (food, electricity, heating, transportation, shelter, etc.). The dream of a significant increase in wind and solar renewable energy in the U.S. will not be realized. Why? Who is the biggest obstacle to actually building significant wind or solar projects? Answer: the environmental hypocrites who protest, appeal, delay, fight and file lawsuits to prevent significant wind or solar projects. The environmental hypocrites always find some environmental effect (often a litany of effects) to prevent significant wind or solar projects. They also try to create a negative reaction in communities considering significant wind or solar projects by branding the projects as, for example, "industrial wind". The environmental hypocrites can not accept the reality that if you want renewable energy, such as wind and solar, to make a significant reduction in the Nation's fossil fuel use, there will be significant environmental effects from installing a significant renewable energy infrastructure. Perhaps the Senate climate bill will include provisions to require environmental organizations to buy the carbon credits to offset each carbon-reducing renewable energy project stopped by the environmental organizations. 10:30 am August 13, 2009 Sam wrote : Keith- Pretty shameful to run a "report" before you understand the underlying assumptions used to determine their numbers. This is especially shameful after the blatant lying in the Bonner fraud scandal (not to mentioned the misinformation being pushed around health care). Also, probably important to remember that NAM members are responsible to offshoring most of the manufacturing jobs we've lost in the last few decades. They're a lobby created to boost corporate profits, not protect jobs. 11:36 am August 13, 2009 Matt wrote : Somehow, I think the EIA, which developed NEMS and uses it all the time, would know how to model the policy better than a self-interested industry group. Looking at the sources, I think it's pretty obvious who is more likely to have the better result. 12:04 pm August 13, 2009 AkaDad wrote : Well now, if the National Association of Manufacturers says it, it must be true. 1:18 pm August 13, 2009 CTF wrote : We still need to raise the cost of carbon based fuels even if Waxman-Markey has turned into a boondoggle. Congress needs to start looking at what this country's leading economists and scientists have been advocating since the beginning: a revenue-neutral carbon tax. Add a Comment Error message Name We welcome thoughtful comments from readers. Please comply with our guidelines. Our blogs do not require the use of your real name. Comment About Environmental Capital Environmental Capital provides daily news and analysis of the shifting energy and environmental landscape. The Wall Street Journal’s Keith Johnson is the lead writer. Environmental Capital is led by Journal energy reporter Russell Gold, and includes contributions from other writers at the Journal, WSJ.com, and Dow Jones Newswires. Write us at environmentalcapital@wsj.com.
1. Field of the Invention This invention relates to an intake manifold structure for a V-type engine. 2. Description of the Prior Art Recently there have been made various attempts to effectively utilize the inertia effect of engine intake air in order to improve the volumetric efficiency, thereby improving the output power of an engine. The inertia effect of intake air is enhanced with the reduction in the flow resistance in the intake passage and the increase in the length of the intake passage. Accordingly, in order to sufficiently improve the volumetric efficiency by the inertia effect of intake air, it is required that the intake manifold is as straight and long as possible. In the case of an in-line engine, a relatively large space remains in the engine compartment at the opposite sides of the engine, and accordingly, an intake manifold which can satisfy such a requirement can be easily mounted. On the other hand, in the case of a V-type engine, a pair of cylinder banks are disposed in the engine compartment spaced transversely from each other, and accordingly, the space on the opposite sides of the engine is relatively small and is not sufficient to accommodate an intake manifold which can satisfy the requirement described above. Therefore, in the V-type engine, the intake manifolds for the right and left cylinder banks are arranged to cross each other and are disposed above the space between the cylinder banks in order to satisfy the requirement described above as disclosed, for instance, in Japanese Unexamined Patent Publication No. 59(1984)-115460. It is preferred that the intake manifolds for the right and left cylinder banks be integrally formed in order to facilitate incorporation of the intake manifolds in the engine. Therefore, conventionally, the intake manifolds are generally formed in one piece by cast molding. However, in the case that the integral intake manifolds are molded by molds having no core, many undercut portions are required in the molds since the parting line of the molds is complex, and accordingly the number of post-processing steps is increased, canceling out the advantage of cast molding. On the other hand, when the integral intake manifolds are molded by molds having cores for forming the undercut portions, the molding operation itself is complicated, making mass production thereof difficult, thereby adding to the manufacturing cost.
There is another aspect as to why the Cubs don't bring up players like Armando Rivero and Kris Bryant -- and that aspect is roster management. There is a finite amount of space on a roster and you have to manage that space as best you can. Sometimes that means delaying one call-up so that you can protect a player later so that you don't lose him in the Rule 5 draft. With the Cubs increasing their talent level the past 3 years, roster management becomes more important than ever. In the past they have lost utility infielders Marwin Gonzalez and Ryan Flaherty, but that could change. They don't want to be the team that loses a Hector Rondon-type player. Perhaps less direct is the missed opportunity to develop a player. Players gets drafted before they become a finished product. It's possible that player could have developed differently had he stayed in the minors -- and even if he didn't have a future with the team, there is the possibility that he could have developed some trade value in the minors. Let's use a player to illustrate what I mean. What if, for example, Marco Hernandez takes a step forward in AA with his offense? A slick fielding SS with even an average bat has value on the market. But if you lose that player then you lose that value as well. A good organization is not in the business of giving away value for nothing in return. This isn't a case of choosing between two players and deciding who is more valuable. Kris Bryant is obviously more valuable than Marco Hernandez. You aren't sacrificing Bryant for the sake of Hernandez, you are sacrificing one month's worth of Bryant in a non-contending season for Hernandez's career in the organization and/or potential future trade value. Considering that month also has significant financial ramifications for the Cubs and their ability to control payroll costs, it becomes a rather easy decision to not call-up Bryant. Rivero is a better example, however, because the delay in his call-up has nothing to do with finances. It is strictly about conserving roster space. The Cubs don't need to have a month of Rivero this season at the expense of losing a player like Hernandez in December. Now, I am only using Hernandez as an example but we can use any number of Rule 5 eligible players to illustrate. Let's take a look at some of the more talented players who are vulnerable to the Rule 5 draft this year. You can see the full list at The Cub Reporter. Keep in mind that the Cubs have already decided to protect would be eligibles Kyle Hendricks, Eric Jokisch, and Rafael Lopez by virtue of their early call-ups. We have mentioned often that there are two basic types of players to protect. Near MLB ready players who could contribute and have some upside, probably as role players. Higher upside players who are not MLB ready but have some useful skills (i,e. speed, versatility, defense) that makes them useful as a 25th man until the team can send them back down the next season. Here are the players that best fit those descriptions... Gioskar Amaya, 2B, Daytona (A+) Jeffrey Baez, OF, Kane County (A) Zach Cates, RP, Tennessee (AA) Hunter Cervenka, RP (L), Tennessee Gerardo Concepcion, RP (L), Daytona Willson Contreras, C, Daytona CJ Edwards, SP, Tennessee Marco Hernandez, SS, Daytona Andrew McKirahan, RP (L), Tennessee Carlos Penalver, SS, Kane County Ivan Pineyro, SP, Tennessee Won't be protected: Jeffery Baez is too raw and does not give enough value on defense as a corner OF'er to have any chance of sticking on a 25 man roster. He has some upside but not enough for a team to give up full year's worth of a roster space while taking a year away from much needed development. Willson Contreras' appeal comes from his athleticism, raw talent, and the scarcity of catching prospects. An off year makes it pretty certain the Cubs won't protect him, especially since the Cubs have since acquired Kyle Schwarber, Mark Zagunis, and Victor Caratini to fill the gap, not to mention the emergence of Will Remillard and Cael Brockmeyer as potential prospects. It's unlikely a team takes Contreras given his poor hitting performance, but had he hit well, his athleticism, surprising speed and ability to play the OF might have made him interesting as a 3rd catcher/25th man. Carlos Penalver is a slick fielder but he did not hit at the low A ball level and is still too young and erratic to be of much value as a defensive replacement. On the bubble: Gioskar Amaya is a mature beyond his years player who can be an asset defensively at 2B. He has a good approach at the plate and enough bat speed to hit MLB quality fastballs with regularity. He has not shown much extra base power this year but does have the potential for gap power, especially to his pull side. Amaya is respected in scouting circles and some team may take a chance. What works against him is that he is essentially a 2B only and doesn't have great use as a utility if he has to come off the bench. There is some risk of losing him and he has value in this organization, so the Cubs may choose to protect him even if he isn't close to being MLB ready and appears to have his path to the big leagues blocked by a number of more highly regarded prospects. Zach Cates showed tremendous progress with his fastball command at Daytona and was one of the team's most reliable relievers in the first half. He struggled some at Tennessee but showed flashes there as well. Cates can hit 96 mph to go with above average athleticism. That alone will draw some attention from scouts. The Cubs will get a longer look in the AFL to determine whether they should protect him or take their chances. Hunter Cervenka has a power arm (low 90s with sink/tail, high 80s cutter) and he's lefty, so that alone makes him a risk to be taken. He has been exceptionally tough on LH hitters this year, making him attractive as a potential LOOGY for now. The key for him is command/control. Cervenka showed some progress in that area but was streaky in that regard. Gerardo Concepcion has come a long way and has been in the low 90s at times this year, flashing an occasionally good breaking ball that drew swings and misses when I saw him at Kane County. He was an effective starter in Cuba's highest league, the Serie Nacional, so he is not your typical A ball prospect. He has a good chance of holding his own as a lefty reliever in the short term. The Cubs are sending him to the AFL to get an extended look and to make up for some lost innings. If he can hold up physically, he has the potential to be a back-end starter, so that may increase his value even more. Marco Hernandez isn't ready for the bigs but he is a talented defender and good athlete with some speed. That alone gives him pretty good utility value for a year, after which a team can send him down again and hope his bat develops enough to make him a starter. Hernandez made some great strides in terms of his maturity and his hit tool this season. Is it enough to make him a potential Rule 5 casualty? That's a decision the Cubs will have to make. Andrew McKirahan has a live fastball (I saw him sit 92-93 at Kane) and a good breaking ball. He misses bats with regularity (just under 8 Ks/9 IP) and this year he has kept the walks down to under 2 per 9 IP (around the 5% range). The difference is that he stayed healthy this year. His dominance at Daytona (0.99 ERA/2.76 FIP) and continued strong performance at Tennessee (3.45 ERA/3.50 FIP) certainly drew some attention and his left-handedness will probably get him a look. Ivan Pineyro was injured much of the season (shoulder) and didn't pitch well when he was on the mound (5.55 ERA/4.85 FIP). He is a finesse starter with a high 80s fastball and a good change, so he depends on good command. That did not happen this year and Pineyro got hit hard. The Cubs liked Pineyro when they acquired him and sending him to the AFL is in part to give him another look and also to make up for lost innings. My guess is he won't be protected as many teams have similar pitchers and Pineyro hasn't done much to convince anyone he is close to being ready. Most would agree he has the poise and maturity to handle the jump, but physically he needs development, particularly with his command and breaking stuff. The lock: CJ Edwards: Do I need to explain why? Edwards is a top 100 prospect with electric stuff when healthy and the central piece in the deal that sent Matt Garza to Texas. The Cubs probably have a good idea of who they'd like to protect but it is impossible to say with any certainty how much space they will need this offseason. Will they want to leave a space open to facilitate an early free agent signing? Can they get what they want without losing a roster spot via a trade? There are still questions to be answered. Those questions include the players the Cubs may remove from the roster to make room. Players who will come off the 40 man roster Carlos Villanueva is a free agent and he will open up a spot, so we need not worry about protecting CJ Edwards, who is next in line for a roster spot. The rest of the roster composition will depend on the following decisions.... Here are the other players who could be on the outside looking in when decision time rolls around... Josh Vitters: The Cubs have cut the cord with their other former Pre-Theo top prospect, Brett Jackson. Could Vitters be far behind. A poor season at AAA which showed no improvement in his plate discipline or defense makes it likely that the Cubs consider him a poor fit for their 25 man roster. I have questioned his commitment to development in the past though I don't know if the Cubs feel the same way. Add that he is out of options in 2015 and this is close to a no-brainer for a non-tender. Chris Rusin: His non-call speaks volumes, though publicly the Cubs would probably say he reached his innings limit. That may be true, but if Rusin was a part of the plan the Cubs wouldn't have let it get to that point. He also has less innings than other pitchers who did get the call, most notably Eric Jokisch. The addition of Jokisch and Felix Doubront as LHPs to the 40 man roster give even more indication that the writing may be on the wall here. Chris Valaika: Valaika got a much deserved reward with a call-up but the job security of replacement level utility infielders with no options on 40 man rosters is tenuous. The Cubs will not hold on to Valaika if roster space is needed. They can either bring him back on a minor league deal or find a similar player who will. Kyuji Fujikawa: He had a vesting option which did not vest. Now it becomes a club option for $5.5M. The Cubs have little to show for their investment because of Fujikawa's unfortunate early injury, but the option presents little value for them right now. The guess here is they decline that option and allow him to become a free agent. They may still bring him back, but the free agency at least opens up some short term space. Zac Rosscup may be getting an audition for his roster spot this September. You have to like the 90-94 mph FB and the slider, both of which are swing and miss pitches, but his inability to throw strikes makes him a question mark. The Cubs have another lefty in McKirahan who doesn't miss quite as many bats, but does throw hard and throws more strikes. Still, it's hard to give up on a lefty with Rosscup's good stuff. He would undoubtedly get picked up quickly by a team that thinks they can coach him up. John Baker: The popular backup is great in the clubhouse, but below replacement level on the field. That makes him a non-tender candidate that the Cubs could try to bring back on another minor league deal. The Cubs could easily open up as many as 5 spaces without affecting the 2015 roster. It's possible they will leave one open, either for the Rule 5 Draft or to give them the flexibility to sign a free agent quickly. But they could always wait to make those changes until it's needed. It's difficult to know how many players the Cubs will protect but CJ Edwards is certain to be one of them. The Cubs will then have 3-5 spots to consider with Gerardo Concepcion, Andrew McKirahan, Gioskar Amaya, Marco Hernandez, and Zach Cates as my early favorites, but things are certain to change and we'll learn more as the season/offseason unfolds.
House and Senate Democrats are teaming up in an attempt to increase pressure on the Trump administration in the coming days over its controversial decision to not immediately implement sanctions against Russia last month, as mandated under legislation that Congress passed overwhelmingly. This week, House Democrats are filing a resolution aimed at compelling the administration to implement those sanctions under the Countering America’s Adversaries Through Sanctions Act (CAATSA). A copy of the resolution was obtained by The Daily Beast. “Congress passed a pretty comprehensive sanctions bill with respect to Iran, North Korea, and Russia last year—and the president has not implemented any of those sanctions. So we need to put our foot to the gas pedal,” Rep. Gerald Connolly (D-VA), who is introducing the House resolution, said in an interview. “If the president’s not going to do it, then Congress needs to.” The resolution names Russia’s “continued aggression in Ukraine and forcible and illegal annexation of Crimea and assault on democratic institutions around the world, including through cyberattacks.” It mirrors a Senate resolution introduced this month by Sens. Ben Cardin (D-MD), Bob Menendez (D-NJ), and Sherrod Brown (D-OH). The House and Senate efforts are largely symbolic, lawmakers acknowledge. And they’re unlikely to force real action absent Republican support. But Democrats are hoping that the effort will keep the spotlight on the sanctions as Moscow continues to destabilize Eastern Europe and the Middle East, and as Trump and his associates remain under investigation by the special counsel. When CAATSA became law last August, Putin’s foes in Eastern Europe commended Trump and members of Congress. While American lawmakers were crafting the legislation, they consulted and met with top Ukrainian officials to hear their concerns—both in Washington and in Kiev, U.S. and Ukrainian officials told The Daily Beast. The significance of the CAATSA legislation extends beyond the halls of the Capitol. “When you look at the comprehensiveness of this legislation, I must say that it’s the first international legislation which is seriously hitting the Russian Federation,” Dmytro Shymkiv, a top aide to Ukrainian President Petro Poroshenko, told The Daily Beast in a recent interview. “It’s a very important step for countering Russian aggression in Ukraine, but also in the U.S. and other European states.” That’s why lawmakers, mostly Democrats, were outraged when the administration informed Congress last month that it would not impose new sanctions against Russia by the Jan. 31 deadline. A State Department spokeswoman told The Daily Beast at the time that it felt that the mere threat of additional sanctions was already acting as a “deterrent” against foreign investment in Russia’s defense and intelligence sectors. “The threat of sanctions is not a substitute for real sanctions that can hopefully deter behavior and change it,” Connolly said, referring to U.S. intelligence officials’ warnings that Russia is poised to interfere in the midterm elections later this year. State Department spokeswoman Heather Nauert said at the time that no further sanctions were required because foreign governments “have abandoned planned or announced purchases of several billion dollars in Russian defense acquisitions.” The sanctions, if the administration had implemented them, would have hit foreign government and other entities that were doing business with those Russian companies that the State Department named in October. But many pointed out that the impetus for the CAATSA legislation was to punish Russia for its incursions into Eastern Europe and its interference in the U.S. presidential election in 2016, arguing that the Trump administration was abandoning its responsibilities under a law that passed in the House 419-3 and in the Senate 98-2. As written, the legislation—which President Donald Trump reluctantly signed after his administration tried to weaken a core tenet of the bill—aims to punish Russia’s defense and intelligence sectors in particular. “We want concrete actions that put Russia back into the norms of international behavior—stop aggression against Ukraine, stop meddling with the political system in Europe and in Eurasia and other places, and become a decent political entity,” Shymkiv said. The formal introduction of the House resolution comes as Secretary of State Rex Tillerson is due to testify in front of the House Foreign Affairs Committee on Tuesday. Democrats are planning to use much of their time questioning Tillerson about the sanctions implementation and, in particular, what the federal government is doing to prevent Russian President Vladimir Putin from seeking to interfere in American elections later this year and beyond. Tillerson’s relationships with lawmakers on both sides of the aisle became strained in October after the administration missed the first deadline for issuing guidance about the implementation of the sanctions. Furthermore, frustrated Democratic and Republican senators told The Daily Beast at the time that they were not getting straight answers from Tillerson about the delay. The episode led to a breakdown of State Department-Capitol Hill relations. Connolly, along with House Foreign Affairs Committee ranking member Eliot Engel (D-NY), introduced legislation last year aimed at sanctioning individuals and entities that meddle in a federal election. The legislation, which remains stalled, would cover the 13 Russian individuals and other groups that were named in special counsel Robert Mueller’s indictment this month who are charged with conspiracy to defraud the United States. While most Republicans have largely taken no issue with the administration’s sanctions regime, Sen. John Kennedy (R-LA) took Trump to task over the issue last month after the president delivered his State of the Union address. “I wish he’d talked about sanctions on the Russians and explained to us why he is not immediately imposing the sanctions, because I think President Putin has acted for the past five years like a thug,” Kennedy said during a CNN interview. Kennedy sits on the Senate Banking Committee, which has formal jurisdiction over the Treasury Department’s implementation of sanctions. Treasury Secretary Steven Mnuchin faced tough questioning from that panel last month, and senators were particularly concerned about a Treasury report on Russian oligarchs that was made public the same day, as mandated under CAATSA. The public version of that report appeared to be cribbed from a Forbes list of Russian billionaires and those with deep connections to the Kremlin. The full report was delivered to the House Foreign Affairs Committee and the Senate Foreign Relations Committee by the Jan. 31 deadline, but the document is classified and is not expected to be made public, three sources with direct knowledge of the matter told The Daily Beast.
Performance of physical activities by adolescents with cerebral palsy. Mobility and self-care are important considerations for successful transition of adolescents with cerebral palsy to adulthood. The purpose of this study was to characterize performance of physical activities from the perspective of adolescents themselves. The subjects were 156 adolescents with cerebral palsy, 11.6 to 17.7 years of age. A therapist completed the Gross Motor Function Classification System (GMFCS). Adolescents completed the Activities Scale for Kids-Performance Version (ASKp) twice over a 1-year period. A repeated-measures analysis of variance indicated a main effect for GMFCS level. The main effect for time and the GMFCS level x time interaction were not significant. Post hoc comparisons indicated that ASKp scores differed among all GMFCS levels. Performance of physical activities by adolescents with cerebral palsy differed based on GMFCS level and did not change over 1 year. The ASKp scores of adolescents in levels II through V suggest the need for physical assistance at times throughout the day. The results have implications for the role of the physical therapist in transition planning.
Sabit Hadžić Sabit Hadžić (7 August 1957 – 3 March 2018) was a Bosnian basketball player who competed for Yugoslavia in the 1984 Summer Olympics. He also worked as a basketball coach. He died on 3 March 2018 after a reported stroke. He was 60. References Category:1957 births Category:2018 deaths Category:Bosnia and Herzegovina men's basketball players Category:Yugoslav men's basketball players Category:Olympic basketball players of Yugoslavia Category:Basketball players at the 1984 Summer Olympics Category:Olympic bronze medalists for Yugoslavia Category:Olympic medalists in basketball Category:Bosnia and Herzegovina basketball coaches Category:Medalists at the 1984 Summer Olympics Category:Mediterranean Games silver medalists for Yugoslavia Category:Mediterranean Games medalists in basketball Category:Competitors at the 1979 Mediterranean Games
Cardif has released the results of its fourth annual Barometer of Independent Financial Advisors (IFAs). The survey was done in France between May 26 and June 22, 2010 in conjunction with TNS Sofres, the French leader in market research and opinion surveys. The Barometer covers a representative sample group of 501 IFAs. Commenting on the results of the latest survey, Roger Mainguy, Director of the Cardif Networks and Partnerships in France, says: “This year we asked IFAs not only about their profession, but also their clientele. The crisis has impacted the attitudes of IFAs' clients, who expect more advice and proactive initiative, even though they are not prepared to pay more for this service. This should push IFAs to change the way they work and charge fees that reflect the value of their expertise.” “Just as notaries, certified accountants and legal advisors bill their services, the time has come for IFAs to be compensated for the value of the wealth management services and advice they provide. Maintaining the status quo could in the medium term weaken the stability of their activity unless they make an effort to adjust their business model,” Mr. Mainguy adds. More demanding client expectations for consulting and support create a challenging paradox The clients of IFA are primarily non-salaried and self-employed professionals. Over 40 percent of them are under 50 years old. This clientele is fairly young compared with wealth management clients in general. They also have an entrepreneurial mindset, are very demanding, and are very careful about choosing their financial consultants.They have amassed savings mainly from regular revenues, both from professional activities and rental income (cited by 88 percent of the IFAs), as well as from the sale of assets (84 percent) or an inheritance (79 percent). They have long-term investment objectives, mainly planning for their retirement, which was the primary client motivation cited by 60 percent of the IFAs surveyed. IFAs have seen major changes in client expectations and needs due to the crisis (cited by 71 percent of IFAs). Clients have turned to their IFAs for solutions to diversify their assets and management strategies, with strong expectations in three key areas:- Advice: 85 percent of the IFAs have seen greater expectations in terms of the advisory service they provide. The top three competitive advantages for IFAs remain unchanged, namely the quality of their advice, their availability and the stability of the clients' contact. - Security: Client objectives for their wealth management are linked to long-term horizons and protection of assets. Yield objectives are not cited as frequently. For life insurance vehicles, IFAs report that their clients place a priority on security. The solidity of the company is the number one criteria (cited by 56 percent of the IFAs), well ahead of the quality of the range of investment vehicles (37 percent). Results show that the effective yield ranks a distant third, cited by only 25 percent. In this context, clients tend to prefer the security of euro fund instruments: 71 percent of the IFAs say they have not seen any renewed interest among their clients for unit-linked funds. - Simplicity:76 percent of the IFAs say their clients are turning to the more basic products. Asked about the commercial relationship, 82 percent of them cite a need for better understanding of the products offered. Introducing fees poses a real challenge The IFAs say that getting clients to accept fees for their services ranks fourth on the list of challenges they need to address. The number of respondents citing fees as a key challenge rose 9 points compared with the last four years. “In terms of fees, the results of the survey underline two paradoxes,” says Roger Mainguy, Director of the Cardif Networks and Partnerships in France. “The first paradox is that of all the European savings clients, the French have the greatest demand for advice and security, but are least willing to pay for it. Fifty-three percent of the IFAs say that their clients are taking a closer look at the cost of their services.”“The second paradox,” says Mr. Mainguy, “concerns the IFAs themselves. These are professionals whose expertise is recognized by their clients. Their ability to provide pertinent advice is a pillar of their marketing strategy, which makes it surprising that they bill nothing or very little for their services. And yet charging fees is clearly the best way to leverage the value of their advisory role.” Although 66 percent of the IFAs do bill fees to their clients—especially for a comprehensive review of their assets, or for tax advice—fees account for only 17 percent of their compensation (behind fees on assets under management and initial sales charges). Compensation from the volume of assets under management accounts for the vast majority of their revenues, which explains the difficulty the IFAs have in transforming their advantages (personal relations, advice, information, proactive responsiveness, availability, etc.) into financial compensation. There are however indicators that IFAs can use to take a more offensive strategy in explaining to clients why charging fees for their services is legitimate: - Current financial trends show favorable momentum.- IFA clients invest with a long-term horizon, which is propitious to building a relationship anchored in confidence. - The regulatory environment, identified by 92 percent of the IFAs as a major challenge, continues to change and could alter the context. In the years ahead France could require the same transparency regarding fees as its European neighbors (the Netherlands, the UK and other countries). IFAs remain fairly optimistic regarding their profession After continued turbulence in 2009, a year that saw a significant drop in net assets gathered (the average was 2.7 million euros, compared with 3.2 million euros the previous year), IFAs remain optimistic regarding the future of their business. Eight out of 10 IFAs say their business is doing well, even though this overall optimism is mitigated since this is the lowest percentage of positive assessments since 2007. - The IFAs are generally upbeat about the first half of 2010: 77 percent of them say their financial situation is identical or better compared with 2009. At the same time, the number of IFAs who believe their situation is “clearly not as good” declined from 55 percent in 2009 to just 21 percent in 2010.- In the wake of the crisis, the majority of the IFAs added new clients (58 percent). These new clients, some of them disappointed by their relation with major branch banking networks are interested in diversifying their assets. Over 80 percent of these new clients came from bank networks and 7 percent form insurance networks. - Projections for 2010 confirm this favorable outlook since over half of the IFAs expect an increase in assets gathered. This confidence is mitigated by the fact that 18 percent of the respondents do not see a positive development outlook. The expectations of IFAs are reflected at several levels: - Recruitment: 21 of the firms surveyed hired staff, compared with just 16 percent the previous year, led by larger firms (over 5 employees). - Acquisitions: only 39 percent of the IFAs see acquisition opportunities in the coming five years. Opinions are divided concerning the advantages of joining larger groups. The IFAs reaffirmed their confidence in the future of independent structures and thus in their business development model. Methodology: Telephone survey by TNS Sofres Finance Department between May 26 and June 22, 2010 covering a representative nationwide sample group of 501 IFAs. Interviewees were selected from a list of 3,900 Independent Financial Advisory firms representative of the IFA segment, or 13 percent of the total population (the representative nature of sample group was validated using quotas for the sizes of the firms and the region). About CardifCardif (www.cardif.com) is the insurance unit of BNP Paribas Assurance. It is rated, AA by Standard & Poor's. Cardif develops and markets savings and protection solutions which are distributed via diverse channels. With a diversified geographic footprint, Cardif enjoys strong positions in Europe, Asia and Latin America. Cardif's roster of partners now includes more than 35 of the world's top 100 banks.In France, Cardif has offices in 20 major cities and has worked closely for 27 years with over 2,500 independent financial advisors, providing them with personalized support through a network of 150 local staff dedicated to IFAs. BNP Paribas Assurance (www.assurance.bnpparibas.com) is the Life and Property & Casualty insurance unit of BNP Paribas. It counts over 8,000 employees, 74 percent of them outside France. BNP Paribas Assurance had gross written premiums of 20.7 billion euros in 2009, of which 41 percent was generated outside France. BNP Paribas Assurance is the world leader in creditor insurance and the fourth-largest life insurance company in France. BNP Paribas Assurance is actively committed to exemplary Corporate Social Responsibility. It has adopted a Socially Responsible Investment program, encourages diversity throughout the enterprise (earning the "Gender Equality at Work" label in France) and supports local economic development in the markets where it operates. BNP Paribas is equally committed to environmental responsibility and has deployed an action plan to achieve a 10-percent reduction in the company's carbon emissions.
CALGARY—When Steve Durrell, the 29-year-old NDP candidate in Airdrie-Cochrane, found himself the focus of a video circulating on social media where Jason Kenney called him a 19-year-old, he was surprised to say the least. But Durrell’s surprise turned to disappointment, not at being targeted or misrepresented, but instead for Kenney’s insinuation that being young should be considered a negative factor in politics. “I like to think that having young people involved in politics is a good thing,” Durrell said. “I don’t really think that it was something worthy of a punchline for a cheap laugh.” The video, which has caused a stir online and received some backlash, shows the United Conservative Party leader speaking at an event and referring to Durrell as a 19-year-old, drawing laughter from the crowd. On Saturday, UCP spokesperson Matt Solberg said Kenney had been misinformed about Durrell’s age and hadn’t intended any offence. Instead, Solberg said Kenney merely meant to draw attention to the “gulf of experience” between Durrell and the UCP’s own candidate in this riding, Peter Guthrie. But Durrell also challenged that notion. He cited his work managing and volunteering on other campaigns, his familiarity with his hometown of Cochrane, and the fact that Jason Kenney was the same age when he was first voted into parliament, all arguments he had in favour of his level of experience. “When (youth) are mocked for getting involved in politics and entering the political field, it is definitely a barrier,” Durrell said. “When people throw up a wall in front of them, it discourages them from getting involved.” But Kenney’s comments don’t reflect the entire group of candidates that will run for the party this spring. Hannah Presakarchuk, the 24-year-old UCP candidate in Edmonton-Rutherford, said she couldn’t speak to Kenney’s comments, but that she herself felt supported by the party and had noticed many other young people getting involved with the UCP. “As far as talking with them, they haven’t found barriers. They’ve been supported, they’ve been encouraged. I think there’s more of a grassroots movement going on and young people are hopping onto that,” Presakarchuk said. But the connection between age and experience that Kenney’s comments implied did bother Sam Goertz, a 21-year-old City of Edmonton Youth Council member. He said connecting the two is often unfair. “I think we’re more experienced than ever as young people, but that hasn’t caught up with older folks. And I think that’s just ageism for young people. If you’re looking at the number and not the candidate’s experience, I think that’s such a huge problem,” Goertz said. Goertz also sits on the University of Alberta senate and works for an Edmonton city councillor. He said in his experience, he often tries to downplay his age, having experienced how some won’t listen to what he has to say if they know how young he is. But Goertz said it’s important for young people to be represented in politics to truly voice his generation’s concerns. “This is a demographic that’s often underserved by the government and you’re not going to hear about their concerns necessarily in such a fulsome way through polls,” Goertz said. “That’s something that you get by having young people in the legislature, in councils, in parliament, and that’s not really something you can get through an opinion poll or Facebook Live. I think the politics of presence matters.” Thomas Dang knows firsthand about the doubts people can have for young political candidates. The Edmonton—South West NDP MLA had just turned 20 years old when he was elected to office and remembers meeting people when he was door-knocking in 2015 who told him they wanted to vote NDP, but wished the party had found an older candidate instead of him. But since 2015, Dang said he believes his age has provided him with a valuable perspective to bring to the legislature. In his work, he strives to talk about issues young people care about that have far-reaching impacts on Alberta’s future and to engage more young candidates to run for office. Loading... Loading... Loading... Loading... Loading... Loading... “Too often, politics is relegated to the old boys club, and it’s something that happens far away,” Dang said. “But really being able to engage youth and say these issues affect your life and not just right now but for many years in the future, getting people to understand and feel encouraged they can make that difference is something we can all do a better job of talking to people about.” Read more about:
Fatigue Other Topics Fatigue comes in many forms, and has many causes. Severe debilitating fatigue should be reviewed by a medical doctor to rule out the more serious causes. Other causes of fatigue include obesity, sleep disorders, adrenal malfunction, blood sugar imbalances, hormonal imbalances, digestive system disorders, improper diet, food allergies, latent viruses (chronic fatigue, Esptein Barr) and the list goes on and on. The most important factor in treating fatigue is determining the imbalance in the body that is the primary cause, and using acupuncture, nutrition, hormone profiling, or supplementation to correct it. General lifestyle tips for beating simple fatigue include obtaining adequate rest (this is an obvious one, but most people do not get the necessary 7 hours of sleep per night), balance stress, exercise moderately, eat a healthy balanced diet, and maintain a healthy weight. If making these changes does not improve your lack of energy, additional help from acupuncture and natural medicine may be necessary. Try to map out your fatigue, notice if it comes on at a certain time every day. If energy improves after 3 p.m. with difficulty falling asleep, the adrenal glands may be imbalanced. If fatigue occurs after meals, a digestive disorder or food allergy could be implicated. If fatigue is constant and unrelenting, a chronic virus or hormonal imbalance may be at fault.
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Thursday, May 29, 2008 Ellery Queen Recently, I’ve dug back through the video archives here at the Library and watched most of the episodes of ELLERY QUEEN, the 1975 TV series starring Jim Hutton and David Wayne. A marvel of casting, Levinson and Link's ELLERY QUEEN presented the sometimes annoying Hutton the opportunity for a splendidly absent-minded genius characterization and he ran with it! At times reminiscent of Jimmy Stewart’s Elwood P. Dowd in HARVEY, Ellery is endearingly quirky but inevitably sharp. As his father, the police inspector, David Wayne (whom my mother misinformed me was John Wayne’s son when he had appeared on BATMAN as the Mad Hatter!) is equally well-served and plays it as gruff but loving. The two of them come across so well as father and son that one is able to suspend disbelief easily. Ken Swofford as a FRONT PAGE-style reporter and a pre-MAGNUM PI John Hillerman as a supercilious radio crimesolver appear from time to time as foils for "the Maestro." As with any good "fair play" mystery, the audience is given all of the same clues as the detective. The gimmick here was that Ellery, toward the end of every episode, would break the fourth wall and address the audience as to whether they (we) had yet solved the mystery. This was a variation on the forties radio series in which a guest panel would be onstage and asked to give their theories on the episode’s crime before Ellery solved it.Preceded by a TV movie and an even earlier (and horribly cast) pilot with Peter Lawford (!), the series, in spite of its quality and good reviews (as seen here Cleveland Amory’s from TV GUIDE) didn’t last and has yet to be issued on DVD. It does turn up in reruns from time to time, though, and is highly recommended. I tried to read some of the ELLERY QUEEN books while imagining Hutton in the lead but it didn't work. The literary Ellery comes across as pompous and lacks the quirks Hutton brought to the role. Hutton would appear a year later in a memorable 4 part episode of ONE DAY AT A TIME but then took ill and passed away in 1979. 5 comments: Have you seen the comics-themed episode Cleveland Amory reviews in this column? I vividly remember it from when it aired -- Amory pretty nearly gives away the ending, such as it is -- and liked it at the time, but I wonder how well the episode would stand up today. Steve, Give the Ellery books another chance. In the earlier ones he was portrayed as pompous and arrogant - more like Philo Vance than the Ellery we love. He even wore pince-nez. Starting in the late 30's a more accesible Ellery appeared. Check out "Four of Hearts" and "Devil To Pay". Also "Cat of Many Tails" is a great thriller about Ellery's hunt for a serial killer in 1940's Manhattan. Land of the Lost Actress Kathy Coleman's Story! Written With Booksteve! About Me First published in 1968 (I was 9!), I have been writing professionally part-time for more than two decades. I have been freelancing for various authors, editors and publishers for the past three years on the behind-the-scenes tasks of writing.
Treasury to sell 30 million shares of GM stock ASSOCIATED PRESS June 5, 2013 Photo: David Goldman, STF FILE - In this Thursday, Jan. 10, 2013 file photo, the logo for General Motors decorates the entrance to a former UPS facility as GM announced plans to open an information technology center in the building that would create about 1,000 jobs, in Roswell, Ga. The U.S. government is taking advantage of the recent run-up in General Motors stock to sell off another 30 million shares in the auto giant that it acquired in a bailout, according to the Treasury Department, Wednesday, June 5, 2013. (AP Photo/David Goldman, File) FILE - In this Thursday, Jan. 10, 2013 file photo, the logo for... DETROIT - The U.S. government plans to sell another 30 million shares of General Motors stock in a public offering on Thursday as it speeds up efforts to divest itself from a stake that it got in a bailout four years ago. The Treasury Department will set the price for the shares after the markets close Thursday, with the sale taking place shortly thereafter. A United Auto Workers retiree health care trust fund will join the sale and sell 20 million shares. General Motors Co. stock fell 2.6 percent to $34.02 Wednesday, but it has seen huge growth recently. The stock hit $35.49 on Tuesday, the highest point since December 2010, according to FactSet. Shares are up about 19 percent since the beginning of the year. The public offering will help accelerate the government's exit from owning GM stock, which it got in exchange for a $49.5 billion bailout in 2008 and 2009. Translator To read this article in one of Houston's most-spoken languages, click on the button below. GM has longed for the government to exit partial ownership of the company, hoping to shed the derisive nickname "Government Motors," as well as attract customers who have shunned GM cars because it took government money. GM nearly ran out of cash late in 2008 and needed the government cash to make it through bankruptcy protection. While under government ownership, the company also falls under executive pay caps that it says hinder recruiting of top talent. After Thursday's sale, the government would still own around 211 million shares, although it likely sold additional shares on the open market in May that haven't been disclosed yet. The Treasury Department said it still plans to sell its entire stake in GM by early next year. The company's stock has been gradually rising since early May, when it reported solid first-quarter earnings.
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Incidental pheochromocytoma: fifteen-year experience at a community hospital. Pheochromocytoma is a well-recognized neuroendocrine tumor. Classical symptoms are well described, but recent studies have suggested that many patients are diagnosed incidentally. No studies have evaluated incidental pheochromocytoma with respect to year of diagnosis. A retrospective study was performed from January 1992-November 2006 to evaluate the frequency of incidental pheochromocytoma. Patients were included if pathological specimens were available. 21 patients were included. 15/21 (71.4%) cases were incidentally discovered. 11/15 (73.3%) were diagnosed after 2000, while 4/15 (26.7%) were diagnosed before 2000. 15/21 (71.4%) patients had hypertension, and all 6 patients without hypertension had pheochromocytoma diagnosed incidentally. Only 3/21 (14.3%) patients were diagnosed by biochemical testing. This observational study suggests an increasing number of incidental pheochromocytomas. Several possibilities for this observation include increased usage of imaging studies, decreased use of biochemical testing, and an increase in referring patients to surgeons for resection without an appropriate endocrine work-up. Referring physicians and surgeons alike should take note of this.
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There are many lift mechanisms in use today. Elevators are one form, lifting and lowering people and other loads, usually within buildings. Another form of lift is a hydraulic lifts that is used to raise vehicles in service stations, allowing mechanics to work from beneath the vehicles. Jacks are also lifts that raise vehicles allowing for changing of tires. The list continues, but in general, the lift mechanisms in use today make space/speed tradeoffs that limit usability in certain applications such as watercraft. For example the hydraulic lifts used to hoist vehicles in your neighborhood garage performs well for its intended purpose, but will not perform well as a lift on a watercraft for several reasons. The first reason is speed. Such lifts are very slow. In many at-sea situations, there are often reasons for quick operation. It is often important to deploy a life raft or return a dingy to the deck and due to emergencies or high surf, the operation must be performed relatively quickly without precluding the use of a service station type of lift that often requires several minutes to lift an object eight feet. The next reason why a garage-type lift will not function in a watercraft is vertical displacement. For example, to lift a vehicle eight feet, the hydraulic cylinder must be set at least eight feet into the floor, and likely at least ten feet. This is easily accomplished beneath the floor of a service station, but in many of watercraft, there is insufficient clearance between the deck of the watercraft and the hull of the watercraft. Many a watercraft do not have sufficient vertical displacement for a garage-type lift, especially in areas of the watercraft towards the bow where the hull slopes upward, closer to the deck, for cutting through waves. The next reason why a garage-type lift will not function in a watercraft is weight. The overall weight of such a hydraulic cylinder and the hydraulic fluid needed to lift the requisite distance will be a burden to many a watercraft and even if the watercraft is large enough to support the weight, the excess weight will impact fuel economy and the ability to bring the watercraft up on plane. Another reason why a garage-type lift will not function in a watercraft is stability. Such a lift operates well on stable ground, but in a watercraft, wave motion and winds create instability. When operating certain payloads on a garage-type lift within a watercraft, stability is often required. For example, when extending a hoist to lift a dingy out of the sea, sudden movement of the hoist due to movement of the lift mechanism is often disastrous. Certain movement results in damage to the dingy and/or sinking of the dingy. Other lift mechanisms are not suited for watercraft for similar or different reasons. For example, elevators are not practical because such require overhead pulley systems which are not feasible on most watercraft. What is needed is a lift system that will quickly deploy and retract a payload while occupying minimal vertical space and adding minimal weight to a vehicle such as a watercraft.
11/14/15 Update: Yesterday, the LDS Church released a clarification that directly affects the family described in this story. Read here for information on the November 13 First Presidency letter, more details on the Church's policy toward same-sex families, and Alyssa Paquette's reaction. This time last week, Alyssa Paquette’s twelve-year-old stepson was preparing to be ordained to the priesthood in the LDS Church. Now that has all changed. On November 5, the Church confirmed a new policy that forbids baby blessings, baptisms, and priesthood ordinations for minor children who reside at least part of the time in a home where a parent is in a same-sex marriage. The sadness has been palpable. After a crushing weekend spent trying to understand what the Church’s new policy means for him, the boy* is crestfallen. “Usually he is so positive and easygoing, but ever since these policies hit and we learned he would not be ordained, he has been depressed and anxious,” says Paquette, 35, a Mormon mother living in Oregon. He’s not the only one. Their whole blended family has been suffering since the news hit last Thursday. Their family configuration is complicated but loving: The twelve-year-old is Paquette’s husband’s son from a prior relationship. The boy’s mother subsequently began cohabiting with a woman. The father joined the LDS Church, married Paquette, and had three more children with her, now ages 8, 6, and 2. The boy's two families share equal custody and his biological mother has been accepting of the decision to raise him as a Mormon, despite her reservations about its teachings on LGBT issues. “All of his parents were there” at his baptism four years ago, says Paquette. “It was a great experience because we all came together to support our son during an important time.” Now Paquette expresses shock and grief that on the eve of their son’s ordination, he’s being rejected. “It feels like a mourning process, like someone has actually died. The church is such a huge part of our lives, and to have that suddenly taken away from him is really challenging,” she says. Paquette notes that her family has been deluged with “an outpouring of love and support” from their local ward, and that the bishop reached out to them immediately. “He was very sympathetic and full of love, and struggling to find the right words for us.” But, she says, “He was also at a loss for how the new policies would apply in our situation. It sounds like he hasn’t been given much guidance other than what’s in the handbook.” At first, the family hoped that an exception might be made because their son is already a baptized member of the church, and so close in age to his planned ordination. However, that was impossible since he is legally required to live part-time in his biological mother’s home according to the terms of their joint custody agreement. Under the new policy, this makes him ineligible for most of the church’s rites until he becomes an adult—and even then only if he disavows his mother’s same-sex marriage. “That is just not an option for us,” Paquette says. “My husband and I feel that it would be wrong to have him disavow half of his family.” The shock wave of this policy change doesn’t just affect her son, though. Her eight-year-old daughter was scheduled to be baptized next week, and now that will not be happening. “Even though our three other children aren’t precluded from being baptized, we feel like we can’t continue to participate in church with the policy as it stands,” she says. “We have a strong conviction that it’s wrong.” The Paquettes have decided that they will either attend church together as a family, with all of their children treated equally, or they will find somewhere that is a “safe place” for the six of them. Paquette breaks down in tears at the thought of not being Mormon, which is “a huge part” of her identity. She does not want to have the family’s names removed from the rolls—“that would be really drastic, and would close a door”—but she won’t choose the church over keeping her family whole. “When you’re raised in the church, you’re raised to sustain your leaders. You’re taught that anything that comes from the church is from God, and to not sustain them is to not sustain God,” she says. “But I don’t know how we’re supposed to sustain something that tears our family apart.” RELATED STORIES OF FAMILIES AFFECTED BY THE POLICY CHANGE: "If and Then," by Exponent II"s Meghan Raynes, who would not have been permitted to be baptized as a child if this policy change had come years ago instead of now From Patheos: "Familes Affected by New Policy" and "More Families Affected by New Policies," compiled by mental health professional Natasha Helfer Parker Openly gay Mormon Mitch Mayne writes in the Huffington Post about how the outcry over this policy may affect moderate Mormons: "We have, in essence, pushed the fringe to the outside, and the middle to the fringe" * Children’s names have been omitted from this story at the request of the family.
Introduction ============ Anxiety Disorders and Adolescence --------------------------------- The term anxiety disorder (AD) represents a category of psychological disorders characterized by feelings of anxiety about future events, and fear reactions to current events \[[@ref1]\], in addition to increased attentional biases toward threat detection \[[@ref2]\]. ADs are the most prevalent of the psychiatric disorders \[[@ref3]\], affecting approximately 117 million young people worldwide; it is the sixth leading cause of disability, with the largest longevity among young people aged 15-34 years \[[@ref4]\], with a range of factors such as misdiagnosis, health care avoidance behaviors, and hardiness, meaning these statistics change relentlessly. The extant evidence suggests that the proportion of adolescents suffering from ADs has increased by up to 70% since the mid-1980s and that nearly 300,000 young people in the United Kingdom have a diagnosable AD \[[@ref5]\]. The onset of AD increases significantly during the adolescent years \[[@ref6]\], in part as a result of conflicts regarding existential identity \[[@ref7]\], educational pressures and high self-expectations \[[@ref8]\], negative peer comparisons or perceived relational victimization \[[@ref9]\], and over-demanding intrusive parenting \[[@ref10],[@ref11]\]. Experience of AD in early life is associated with negative short- and long-term implications for social, academic, financial, and health performance \[[@ref12]\] and predicts adult anxiety and substance abuse disorders \[[@ref13]\]. For the individual, adolescence is both a source of increased opportunity and increased pressure and risk \[[@ref14]\]. Increased social expectations of developing autonomy in self-regulation and self-determination of behavior, coinciding with diminishing assistance from adults, require the adolescent individual to develop and coordinate effective emotional and cognitive capabilities in relatively short time frames. These time frames, however, do not always correlate well with progress made in brain maturation \[[@ref15],[@ref16]\]. Functional magnetic resonance imaging data also point to a tendency toward an increased response to emotionally loaded stimuli at this age \[[@ref17],[@ref18]\]. Neuroimaging data point to a biomaturational explanation for the increased prevalence of AD in the adolescent years \[[@ref19]\]. Furthermore, naturally occurring consolidation of neural pathways during adolescence may explain the tendency of experience of AD at this age to lead to negative outcomes in later life, leading some to describe AD as a potential gateway disorder \[[@ref20]\]. Therefore, effective treatment of adolescent AD is critical in the mitigation of both its impact at the point of experience and the potential long-term ramifications \[[@ref21]\]. Therapeutic Interventions for Anxiety Disorders ----------------------------------------------- Cognitive behavioral therapy (CBT) has been shown to be highly effective in the treatment of ADs \[[@ref22]\], reducing or eliminating symptoms through the development of effective behavioral adjustment and coping strategy enhancement. Attention bias modification (ABM), an emerging technique derived from neurocognitive models of anxiety, has also been noted for having significant potential to enhance both pharmacological and psychological interventions for anxiety, as well as being an effective standalone intervention \[[@ref23]\]. However, although evidence-based early intervention strategies reduce the probability of negative life outcomes \[[@ref24]\], practical barriers to treatment (eg, cost) and social barriers to treatment (eg, stigma) mean as many as 50% of people in the United Kingdom experiencing anxiety do not seek treatment \[[@ref25]\]. For those who seek treatment, waiting lists via Improving Access to Psychological Therapies (IAPT) referrals can be lengthy, leading to high dropout \[[@ref26]\]. In addition, educational institutions and universities often fail to provide adequate support \[[@ref27]\], with the existing services unable to meet the rising demand \[[@ref28]\]. As a result, in adolescents, it is estimated that less than 20% of individuals affected by ADs receive treatment \[[@ref29]\], with fewer than 20% of those seeking and receiving treatment being provided with interventions supported by scientific evidence \[[@ref30]\]. The growing discrepancy between demand for, and available provision of, mental health services has led to the development of a range of alternative methods for delivering clinical interventions for anxiety \[[@ref31]\]. eHealth (health care practice supported by electronic processes and communication) and mHealth (versions of eHealth using mobile devices) models (eg, computerized cognitive behavioral therapy) aim to mitigate the impact of both practical and social barriers to treatment by utilizing ubiquitous mediums to broaden the reach of clinical models \[[@ref32]-[@ref34]\]. One such medium is therapeutic video games, a derivative of serious games. Due to the improved realism in simulated artificial environments and capabilities of contemporary hardwares, Web-based therapies are more comparable than ever to in vivo forms of treatment \[[@ref35]\]. The gamification of clinical models may be particularly suitable for younger people, as they often reflect the typically more visual, rapid, and multi-tasking learning styles of a generation with a lifelong exposure to and familiarity with technology \[[@ref36],[@ref37]\]. Therapeutic games afford a flexible and personalized learning environment that allows for exploratory learning and behavior practice \[[@ref38]\], allowing Web-based environments to be adapted in terms of content and challenge to the requirements of the user, which is likely to be conducive to an enhanced learning experience \[[@ref39],[@ref40]\]. As games utilize both intrinsic and extrinsic motivational elements in active and realistic learning opportunities with immediate opportunities for feedback, they have already been shown to be capable of eliciting improvements in self-awareness and self-management behaviors in people with chronic physical health conditions \[[@ref41]\]. In terms of the benefits of therapeutic games for mental health and well-being, the extant evidence suggests they may be effective across a variety of disorders, including reducing psychopathological symptoms associated with gambling disorders \[[@ref42]\], and as an effective preliminary treatment to CBT for bulimia nervosa \[[@ref43]\]. Although the current literature presents conflicting evidence regarding the health benefits versus health hazards of video game platforms \[[@ref35],[@ref44]\], therapeutic games utilize a popular platform to achieve clinically measurable health improvements and behavioral changes \[[@ref45]\]. Research Questions ------------------ Therapeutic games provide young people with a dynamic, adaptable, and personalized learning environment in which they are afforded an opportunity to seek relevant information and guidance in an exploratory manner, receive immediate feedback, and utilize unlimited opportunities for repeat engagement \[[@ref38]\]. As a result, therapeutic games should offer a more accessible platform for rapid learning and adoption of scientifically validated therapeutic techniques. Although some evidence exists to suggest that therapeutic games can be linked with reductions in subjective anxiety and observed stress reactivity \[[@ref46]\], research to date often combines adolescent samples with either child or adult participants, limiting the capacity of the current data in terms of its applicability to the unique nature of anxiety experienced at this life stage. Furthermore, to the researchers' knowledge, there is currently no stated set of guidelines available for the development of therapeutic games, to which developers are required to adhere, nor is there a definitive protocol established for their scientific evaluation. Consequently, it is unclear whether the potential benefits of therapeutic games establish themselves in anxiety in adolescents, and if so, whether any benefits are modulated by the therapeutic framework employed. A systematic review was conducted to assess the effectiveness of therapeutic games in enhancing engagement with clinical interventions, and their efficacies in making clinically measurable reductions in AD symptoms in adolescent samples. Methods ======= Databases Searched ------------------ Relevant papers were identified by performing a comprehensive literature search of the following databases: Journal of Medical Internet Research, Journal Storage, Psychology Articles, Psychology Info, ScienceDIRECT, and Scopus. Search Terms and Selection of Papers for Inclusion -------------------------------------------------- The following search terms were used to address the variety of games that might be played, and the variation in terms used to describe them: all ("serious game" OR "video game" OR "therapeutic game" OR "online game") AND all("adolescen\" OR "teenage" OR "youth" OR "young adult\") AND "anxi\." For further detail regarding search terms, definitions, and variation of input, see [Multimedia Appendix 1](#app1){ref-type="supplementary-material"}. Paper abstracts were initially scanned to determine eligibility. If eligibility could not be determined from the abstract alone, or if the paper was deemed as potentially relevant from the abstract, the full-text paper was studied for its relevance to the review. Inclusion Criteria ------------------ In line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, clear inclusion criteria were established to determine the eligibility of papers for inclusion in the review. Only studies meeting the following criteria were considered eligible for inclusion: papers linked to therapeutic games for ADs; studies conducted on adolescent samples; empirical research using a randomized controlled trial (RCT) design, quasi-experimental design, or correlational design; and studies using a control group or pre- versus posttest design to allow for comparison. All searches were limited to anxiety. Adolescent sample was defined using American Psychiatric Association criteria (10-19 years). As a result, papers that used a sample of participants aged between 10 and 19 years were considered eligible. Papers that used samples including participants aged 8 or 9 years were considered eligible if the mean age for the sample was over or very close to 10 years. Papers published in English or German were selected and subjected to the inclusion criteria as outlined above. In line with PRISMA guidelines, a specific date range was established. Studies published between January 1990 and July 2017 were selected. This date frame was chosen as papers first studying the effect of video games in the context of health education were published in the 1990s \[[@ref41]\]. Exclusion Criteria ------------------ Papers regarding opinion pieces, existing literature reviews, conference posters, and design documents for therapeutic games were excluded from the review. Study protocols were also eliminated from the review as they would be unable to provide outcome measures. Pilot studies were included in the review provided the 4 criteria discussed above were met. Quality Assessment ------------------ For the purposes of consistency, one researcher oversaw the initial coding of the papers. Quality assessment of papers meeting the inclusion criteria was assessed using the mixed methods appraisal tool (2011) \[[@ref47]\]. The mixed methods appraisal tool is designed for systematic reviews, including a combination of quantitative, qualitative, and mixed methods studies, and has been noted for its reliability and efficiency as a quality assessment protocol, and capability to concomitantly appraise methodological quality across a variety of empirical research \[[@ref48]\]. In line with PRISMA guidelines, an interrater process was adopted and the degree of agreement was assessed, to reduce risk of bias. Primary Outcome Measure ----------------------- To assess the extent to which therapeutic games elicit reductions in AD symptoms, papers were studied for comparisons between measures of anxiety symptoms at pre- versus postintervention. Results ======= Papers Meeting Inclusion Criteria --------------------------------- A total of 2259 records were identified through database searches. After papers published in languages other than English or German, and duplicate instances of papers were removed, remaining papers were assessed using the inclusion and exclusion criteria outlined above (N=2222). Initially, abstracts were searched to assess a paper's eligibility for inclusion. If abstract information alone was not sufficient to determine whether a paper met the criteria, the entire paper was studied. [Figure 1](#figure1){ref-type="fig"} shows the number of academic papers from each database identified using the search terms and the number of papers meeting the inclusion criteria. ![Outcome of literature search.](games_v6i1e3_fig1){#figure1} Quality Assessment Outcomes --------------------------- Papers meeting all of the inclusion criteria (N=5) were then quality assessed using the procedure described above. Subsequent to these papers meeting the quality criteria, they were included in the review. The mean rating for the papers was 75%, and the modal rating for the papers was 75%. Owing to the small number of papers meeting the inclusion criteria, all papers included in the review were subsequently coded again for interrater reliability. The interrater reliability for the total scores was 0.8, showing good agreement between the 2 coders regarding paper quality. Overview of Papers Included in the Review ----------------------------------------- Of the 5 studies selected as meeting the criteria for inclusion in the review, 2 relied solely on quantitative measures \[[@ref49],[@ref50]\] and 3 used a mixed-methods approach \[[@ref51]-[@ref53]\], with differing balances of reliance on quantitative versus qualitative data. Of the papers reporting quantitative data (N=5), 2 used an RCT design \[[@ref49],[@ref52]\], 1 used a quasi-experimental design \[[@ref50]\], and 2 were exploratory evaluations and usability studies \[[@ref50],[@ref53]\]. Of the papers reporting qualitative data (N=3) \[[@ref51]-[@ref53]\], all used interview techniques to obtain varying amounts of qualitative feedback. Of the 2 papers focusing on the therapeutic game "Dojo" \[[@ref51],[@ref52]\], one study focused on a pilot study to evaluate perceptions and feasibility of the game \[[@ref51]\], whereas the other paper concerned an RCT comparing the game to "Rayman 2: The Great Escape," in terms of their relative abilities to reduce adolescent anxiety \[[@ref52]\]. In terms of quantitative data, one paper focused on the neurofeedback game "MindLight" \[[@ref49]\] using an RCT to compare the game to "Max and the Magic Marker" in terms of their relative abilities to reduce adolescent anxiety. One paper used a mixed-methods approach to qualitatively evaluate impressions of the game "gNats Island" using a sample of 6 adolescents \[[@ref53]\]. Pre- and posttreatment, and 6-week follow-up, measures of anxiety were assessed quantitatively using a range of clinical measures to indicate symptom levels for anxiety and a range of other mental health conditions. The final paper included in this review utilized a quantitative approach to assess the efficacy of an augmented reality (AR) therapeutic game \[[@ref50]\]. A quasi-experimental design was adopted for this study, recruiting participants to experimental (AR exposure therapy) and control (no game exposure) conditions in 2 separate phases. Outcomes of Therapeutic Games on Adolescent Anxiety --------------------------------------------------- Schoneveld et al \[[@ref49]\] utilized an RCT design to examine the anxiety-reduction effects of the game "MindLight"---a game designed for children and adolescents using a combination of CBT and ABM \[[@ref49]\]. In this game, users are guided through relaxation techniques (CBT) and must use a glowing light on their headset (their "mind light"), which reacts to activity collected from an electroencephalogram (EEG) they wear during play, to help them navigate the in-game world and defeat monsters they encounter. Nonplayable characters (NPCs) become gradually more difficult to ignore, and players must remain calm (keep their "mind-light" bright) to "decloak" the threats (eg, turn a scary cat into a friendly kitten). The game also rewards players for attending to and quickly responding to positive stimuli and disattending or moving away from negative stimuli (ABM). A total of 136 children aged between 8 and 13 years (mean 9.93 \[SD 1.33\]) were selected after screening for elevated anxiety and randomized to either an experimental or control condition. Experimental participants took part in five 1-hour sessions, scheduled twice a week, in which they played the game "MindLight" in groups of 7-19 participants at a time. Control participants undertook the same program in terms of time allocated to game playing, and group size, but the therapeutic game was substituted for a control game "Max and the Magic Marker." Self- and parent-reported anxiety levels were assessed pre- and postintervention, followed by a 3-month follow-up, using the child and parent versions of the Spence Children's Anxiety Scale (SCAS-C and SCAS-P) \[[@ref54]\]. Latent growth curve modeling revealed a significant slope for all models, indicating levels of anxiety decreased significantly over time. Intention-to-treat linear regression analysis found no significant effect, however, of game condition on anxiety outcome. Qualitative feedback revealed that "MindLight" was more anxiety inducing than "Max and the Magic Marker," suggesting "MindLight" was successful in achieving its intended emotional exposure effects. Furthermore, no difference was found in perceived difficulty of the 2 games studied, or their perceived appeal to other children. "MindLight," however, was reported by the participants as less appealing to themselves and less likely to induce flow, a common issue with serious games when they are compared with their more entertainment-focused counterparts \[[@ref55]\]. The therapeutic game "Dojo" appeared in 2 of the papers selected for review. "Dojo" is a first-person emotion-management game that takes place in a secret temple below an urban subway. The player assumes the role of a young person experiencing a difficult time, and navigates 3 rooms, headed by a "dojo master" thematically designed to represent different emotions: anger, frustration, and fear. In each room, the "dojo master" trains the player in a relevant coping skill for the emotion, after which the player undertakes a task (eg, in the fear room, the "dojo master" trains the player in deep-breathing exercises), then challenges them to collect bones in a labyrinth while attempting to evade a powerful and frightening angry ghost. The player's heart rate can be monitored and used by the game to increase or decrease game difficulty. In one study, "Dojo" was assessed by means of a pilot study using a mixed-methods approach \[[@ref51]\]. This study was conducted in 2 residential treatment centers offering 24-hour care for youths with severe mental health problems. A total of 8 adolescents (mean~age~ 14.38 \[SD 1.60\]) took part in eight 30-min sessions playing "Dojo" on a laptop. These sessions took part twice a week for 4 consecutive weeks---though 3 participants experienced a 2-week break due to scheduling conflicts. The participants rated statements regarding game satisfaction on a 5-point scale, and they were also offered the opportunity to provide comments. The Dutch language version of the SCAS was used to measure anxiety. Reported satisfaction with "Dojo" was high, and both participants and mentors reported high compliance and positive changes in anxiety. The participants did, however, suggest that "Dojo" became repetitive and would have preferred more game rooms. Following this study, Scholten et al \[[@ref52]\] then utilized an RCT design to examine the anxiety-reduction potential of "Dojo" in comparison with "Rayman 2: The Great Escape," which was chosen as a control game. A total of 138 adolescents (11-15 years, mean~age~ 13.87 \[SD 0.91\]) were tested both pre- and postintervention, with a 3-month follow-up (N=126) using the SCAS-C. The participants also provided feedback about game experience and game expectations before the intervention. The intervention took place over 3 weeks, consisting of two 1-hour sessions a week. All the participants accessed their games after school hours in the same room regardless of condition, using separate computer terminals and headphones to hear game sound and diminish distractions. Results indicated that anxiety symptoms significantly decreased at follow-up in both conditions (total anxiety symptoms: beta=.70, SE=0.04, P\<.001; personalized anxiety symptoms: beta=.63, SE=0.05, P*\<.001). Latent growth curve models revealed a steeper decrease of personalized anxiety symptoms in "Dojo," but not total anxiety symptoms. Coyle et al \[[@ref53]\] studied the therapeutic game "gNats Island" using a series of trials. "gNats Island" is a gamified CBT intervention derived from a paper-based CBT manual for 19 adolescents (total sample aged 11-16 years, 12 males, 7 females). Players navigate a 3D animated tropical island in which they meet a series of NPCs, which introduce mental health concepts using a spoken conversation, embedded animations, videos, and questions regarding the player's own situation (to which players can respond by a multiple-choice question). Players carry an in-game notebook to answer further questions posed by NPCs and record new ideas. Negative automatic thoughts are represented in-game as "gNats," which sting players to cause negative thinking. Catching, trapping, and swatting gNats are used to represent identification and challenging of negative thinking. In one study, therapists independent of the design team used "gNats Island" with adolescents referred to the psychology team at the participating hospital experiencing clinical ADs. Adolescents played the game alongside a clinician who acted as a partner. Results from questionnaire feedback indicated that adolescents found the game more fun and engaging than "just talking," and assisted in avoiding perceptions of confrontation in direct face-to-face interaction. Quantitative data indicated a decrease in anxiety scores both during and postintervention. No further statistical data are provided in this study. In a second study, a member of the design team used the game with 15 adolescents experiencing issues including anxiety, but also depression, anger management, and issues relating to autism spectrum conditions. Although some participants used the game in a structured manner, in six 1-hour sessions over 6 weeks, others used the game flexibly as determined by clinician assessments of their individual needs. Qualitative feedback showed that after the intervention stage had finished, the participants preferred to explore the Web-based world, suggesting "winding down" time may be as important as engagement with therapeutic elements of game play. The participants also rated modules 4 and 5 of the game lower than previous elements. Notably, difficulty levels of the game at this point had increased, and a core component of CBT had been introduced, suggesting pacing at this stage of the game may not have been as effective as required. Low graphical fidelity of the game was noted by users, but not reported as a barrier. The final study eligible for inclusion in this review was conducted by Li et al \[[@ref50]\] and investigated the effectiveness of "PlayMotion" hardware (which creates Web-based environments via AR) using a quasi-experimental design. A total of 122 children aged between 8 and 16 years (mean~age~ 11.85 \[SD 2.20\]) admitted to an oncology ward in a large hospital in Hong Kong were assigned either to experimental (PlayMotion) or control (routine nursing care with no engagement in Web-based interactions) conditions. The participants were recruited in 2 phases, with all patients admitted in phase 1 assigned to control (N=70), followed by a 1-month washout, followed by phase 2, in which all patients admitted were assigned to the experimental condition (N=52). Engagement with the Web-based intervention consisted of 30-min sessions for 5 days a week, with 4 participants per group. Anxiety was measured using the SCAS-C, with results indicating no change in anxiety symptoms after 7 days. Discussion ========== Principal Findings ------------------ Although therapeutic games show early signs of promise in helping to alleviate symptoms of anxiety in adolescent samples, a number of issues and limitations of the extant evidence have emerged. First, although some evidence utilizes RCT protocols to establish a clear comparison between therapeutic games designed specifically for anxiety reduction and control games designed without this primary purpose in mind, other research is based on clinician's impressions of games, or feedback from adolescents while in the presence of a clinician, who may also be a member of the design team, creating the potential for bias. Of the RCTs that exist, these are limited in number (N=2) \[[@ref49],[@ref52]\]. Furthermore, both these studies reported reductions in anxiety in control groups, with no significant differences in anxiety reduction found between the 2 conditions. Authors note that control condition games may have inadvertently utilized game mechanics that trained resilience and coping skills despite this not being their intended or primary purpose \[[@ref49]\], or that participants may have vicariously acquired coping strategies from their peers in the experimental group, playing their games in the same room at the same time \[[@ref49],[@ref52]\]. In addition, as neither study utilized a waiting-list control group or comparison to established nongaming therapy for further comparison, and although the therapeutic games tested seem to have potential, it is difficult in either case, despite their healthy sample sizes, to establish the extent to which specifically designed therapeutic games may have additional capacities for anxiety reduction in adolescents. As other findings offer no follow-up results, offer pre- versus posttest as control \[[@ref53]\], or offer a "wait-list" control but use a game designed for multiple conditions \[[@ref50],[@ref53]\], there is a scope for further research to further examine the capabilities of specifically designed therapeutic games to reduce anxiety in both short term and long term. Current research has also focused on the evaluation and exploration of the benefits of therapeutic games in relatively controlled environments using hardwares such as EEG, AR hardware, and heart rate monitoring, which are impractical in everyday environments. Intervention sessions in this field are routinely scheduled in classrooms \[[@ref49],[@ref52]\] or clinical environments, sometimes with a practitioner present to guide the interaction \[[@ref50],[@ref51],[@ref53]\]. Although initial findings suggest that engagement with therapeutic gaming may assist in clinically measurable reductions in anxiety symptoms over time, it is not clear how effective such games may be in real-life environments. It is also unclear how such games may have potential to assist at the point of symptom experience, either as a distraction technique or coping mechanism, in an everyday manner. As highlighted previously, access to, and efficacy of, clinically proven interventions for anxiety is limited by practical and social barriers to treatment \[[@ref24]\], lengthy IAPT referrals \[[@ref26]\], or inadequate support from their educational institution \[[@ref27],[@ref28]\]. As a result, as many as 50% of individuals who may benefit do not receive any form of therapy \[[@ref25]\], with figures significantly higher for adolescents \[[@ref29]\]. Therapeutic games that successfully manage to breach these practical and social barriers to treatment, overcoming the need for hardwares impractical to day-to-day life, may be significant in helping to mitigate the short- and long-term implications of one of the most prevalent psychological disorders in a population that is difficult to treat. A further theme of the current research concerned the structure and format of delivery of the existing interventions. For instance, in Scholten et al's \[[@ref52]\] RCT to assess "Dojo," participants reported that the duration of the intervention was "too long," specifically with regard to maintaining concentration and motivation after approximately 4 sessions. Authors suggested that this was potentially a product of "Dojo" being a small game. Once adolescents finished the available rooms, as most did after 3-4 sessions, they were required to repeat the rooms until the end of the intervention period. Although repeating the rooms may be seen as a reinforcement of their learning, this repetitive play may also have caused boredom. Murphy et al \[[@ref56]\] suggest that although some degree of repetition may be beneficial in improving learning and future experience of flow in video games, repetition without learning anything new, or repetition without experience of even subtle changes in gameplay, can disrupt flow and inhibit perceptions of "mastery." This may explain wider research in the field, which suggests that programs with shorter durations tend to have better outcomes \[[@ref57]\]. Limitations ----------- Despite the initial number of studies found through search terms being extensive, the final number of papers successfully meeting the inclusion criteria was low (N=5). Accordingly, although this was somewhat expected due to the stringency of the criteria used in this review, there is further research using therapeutic games for young adults, which may be of interest in the development of games for anxiety aimed at older adolescents. In addition, while the mean age of participants in papers included in this review was between (or just below) 10-19 years, the studies used individual participants aged below 10. As a result, the data are partially affected by the presence of participants who do not qualify as adolescents, but rather as children, making current research to date problematic in establishing the extent to which currently available therapeutic games may be beneficial for older adolescents on the brink of early adulthood. Furthermore, this review considered papers with a publication date of up to and including July 2017. Due to the fast-paced nature of technology development, particularly with regard to software applications, of which games are an example, the relevance of this review in terms of its ability to answer the research questions posed may be time-limited. Future Research --------------- As noted by authors of papers included in this review, this is an emerging area of interest in the field, and subsequently, there are several avenues of exploration yet to be fully explored. Further research would benefit from full RCT studies ensuring appropriate control games are selected, or by using a waiting-list control as a second control condition, to allow for more rigorous comparison. Furthermore, although some studies to date have utilized a follow-up time point, others have not. As a result, the long-term benefits of the use of therapeutic games for adolescent anxiety is currently unclear. Future research would also benefit from further consideration of the applicability and efficacy of therapeutic games in more ecologically valid settings. Current research to date has explored the use of therapeutic gaming in systematically controlled environments. Consequently, the potential of using games in day-to-day life or at the point of symptom experience remains unknown. As noted previously, therapeutic games that successfully manage to breach practical and social barriers to treatment, with engaging games capable of repeating concepts of clinical value while maintaining flow, will be of value to the field in establishing the potential of this protocol. Finally, no current legislative body or code of conduct exists for the development and regulation of "therapeutic games," nor is there currently a standardized procedure or empirical protocol for their scientific evaluation. In such an unregulated environment, there is substantial potential, therefore, for misuse of the term "therapeutic game" on the part of a more commercially driven developer. The capricious nature of the results presented in the investigations included in this review could be argued to be a product of such methodological variability, rather than an indication of inconsistencies of therapeutic games as effective treatments for anxiety symptoms. Consequently, future research should aim to establish a valid and reliable model for the assessment and verification of therapeutic games, with the view to developing a trustworthy quality-approved protocol. Conclusions ----------- This review aimed to assess the effectiveness of therapeutic games in making clinically measurable reductions in AD symptoms in adolescent samples. Although research in this field appears to be extremely limited, as demonstrated by the small number of papers meeting the inclusion criteria for this review, early findings suggest that therapeutic games have potential in helping to reduce anxiety levels in adolescents. By utilizing this protocol in a medium that facilitates overcoming the existing barriers to treatment, therapeutic games may be a valuable facilitator in reducing the short- and long-term implications of one of the most prevalent psychological disorders in a population that is difficult to treat. The authors would like to thank the authors of all data used in the review. Conflicts of Interest: None declared. Search term definitions and variations of input. ABM : attention bias modification AD : anxiety disorder AR : augmented reality CBT : cognitive behavioral therapy eHealth : health care practice supported by electronic processes and communication IAPT : improving access to psychological therapies mHealth : versions of eHealth using mobile devices NPC : nonplayable character RCT : randomized controlled trial SCAS-C : Spence Children's Anxiety Scale-Child version SCAS-P : Spence Children's Anxiety Scale-Parent version
Q: DataTables - hide buttons on second page I try to hide buttons in my table when certain value is not "error". It works perfectly fine on the first page, but due to pagination the buttons are not hidden on the second page. JSFiddle $(function() { $('#table1').each(function() { var Cell = $(this).find('td:eq(2)'); debugger; if (Cell.text() !== 'error' ) { $(this).find('button').hide(); } }); }); A: Like you have done with the initComplete callback, you can pass a drawCallback function through the DataTable options. This will run everytime the table is drawn. Just add the following to the options. JSFiddle See: https://datatables.net/reference/option/drawCallback drawCallback: function (settings) { $('#table1').each(function () { var Cell = $(this).find('td:eq(2)'); debugger; if (Cell.text() !== 'error') { $(this).find('button').hide(); } }); }
Artificial Intelligence (AI) is the most anticipated concept ever introduced in the world of technology. We hear about AI quite frequently these days. Ever thought why humans have fallen for Artificial Intelligence? To find the answer, we need to go back to the golden days where it started from. Artificial Intelligence came into existence in the 1950s and back then it was only limited to academic disciplines. It was never assumed that AI will become an integral part of our day to day life. Tech giants like Google, Facebook, and Microsoft are also turning their way towards Artificial Intelligence. For example, Google made a breakthrough in machine-generated speech through its acquired AI company Deepmind technologies limited. Google is also using Deepmind's AI to slash down it's giant electricity bill. Microsoft has also made some advancement in deep learning with the release of its Cognitive toolkit. Artificial Intelligence has undoubtedly changed and accelerated growth of the companies. At the same time, it has also increased the unemployment. Recently, a news published in The Guardian, A Japanese company has replaced its 34 employees with IBM Watson. Let's have a look at the emerging trends in 2017:- 1. Chat Bots These are also known as Artificial Conversational Entity. Chatbots are computer programs which are designed in a way to stimulate human behavior as a conversational partner. If something is there which tech giants like Google, Apple, Amazon have in common, this is it. Most of you must have asked Siri or Cortana about today's weather. If you have done that then unknowingly you had a conversation with a chatbot dubbed as a virtual assistant. In 2016, we saw companies opening their Chatbots API to third party developers in order to develop smart and task-oriented bots. In coming years, be prepared to welcome new chatbots as we enter the age of interaction between humans and technology. 2. Artificial Intelligence in App Development In early days, applications developed for Android were highly fragmented. Now, the whole ecosystem has become much more integrated. Today integrating voice interfaces into the applications have become an essential part of the mobile ecosystem. This has happened because IoT has grown tremendously over the years. It was reported that nearly four billion connected things were in use in 2014. Developers have now started adding virtual assistant support to their applications. Some of the famous applications which are using AI - Prisma, Google Allo and more!! 3. AI hardware To reinvent IT many companies like Intel, Google, Microsoft has taken their way towards Artificial Intelligence. These companies are investing heavily on ML/AI with hardware designs to accelerate next-generation application development. Intel recently introduced Knight Mill, a new line of CPU aimed at Machine Learning applications. The company is looking to make some differences because PC market has seen some downfall in recent years. Google has also done some huge investments in ML/AI market with the introduction of frameworks like TensorFlow. With the introduction of the frameworks they have also come up with the hardware implementation - Tensor Processing Unit - to accelerate specific machine learning functions. IBM is also having its own machine learning toolkit called PowerAI. This new ML toolkit runs on IBM's processor and Nvidia GPUs connected together using a new proprietary hardware. 4. Internet of Things (IoT) Internet of Things have got a massive coverage in recent years and people are also getting used to it. But without Artificial Intelligence a device doesn't become a smart device. To put it in a simple way, AI + IoT = Smart Device. Smart devices will also have human interactions. Interacting with humans involves not only recognizing irregular behavior but also facial and verbal expressions. Thus Artificial Intelligence plays an important role in converting a device to smart device. The concept of smart homes is going to be huge in coming years. Who doesn't want his/her appliances connected in a cohesive network and assisting it verbally? 5. AI Startups Corporate giants like Google, Microsoft, Intel, and Apple are competing with each other in the race of achieving complete dominance in Artificial Intelligence industry. The technology world is already showing signs of the acceptance and success of AI/ML applications in almost every modern technology. Nearly 140 companies working behind artificial intelligence got acquired in 2011. To name a few acquisitions - Google acquired DeepMind, AOL made an acquisition of Convertro, and IBM took over Cogenea.
<main class="main-container ng-scope" ng-view=""><div class="main receptacle post-view ng-scope"><article class="entry ng-scope" ng-controller="EntryCtrl" ui-lightbox=""><header><h1 class="entry-title ng-binding">应对CC攻击的自动防御系统——原理与实现</h1><div class="entry-meta"><a target="_blank" class="author name ng-binding">燕云</a> <span class="bull">·</span> <time title="2014/12/03 11:03" ui-time="" datetime="2014/12/03 11:03" class="published ng-binding ng-isolate-scope">2014/12/03 11:03</time></div></header><section class="entry-content ng-binding" ng-bind-html="postContentTrustedHtml"><p></p><h1>0x00 系统效果</h1><hr><p>此DDOS应用层防御系统已经部署在了<a href="http://www.yfdc.org" title="http://www.yfdc.org">http://www.yfdc.org</a>网站上(如果访问失败,请直接访问位于国内的服务器<a href="http://121.42.45.55" title="http://121.42.45.55">http://121.42.45.55</a>进行在线测试)。</p><p>此防御系统位于应用层，可以有效防止非法用户对服务器资源的滥用:</p><pre><code>只要是发送高频率地、应用层请求以实现大量消耗系统资源的攻击方式，皆可有效防御。 </code></pre><p>其实现的基本思想是:</p><pre><code>定期分析所有访问用户在过去各个时间段内的请求频率，将频率高于指定阈值的用户判定为资源滥用者，将其封杀一段时间，时效过后，防御系统自动将其解封。 </code></pre><p><strong>在线效果测试:</strong></p><p>进入<a href="http://www.yfdc.org" title="http://www.yfdc.org">http://www.yfdc.org</a> -> 点击右上侧<strong>在线查询</strong>，此时将会进入<code>/shell/yf</code>域内，<code>/shell/yf</code>是一个用<code>bash scripts</code>写的动态<code>web-cgi</code>程序，用户每一次提交信息，此程序将会执行一些服务器端的查询操作，然后将数据处理后返回到客户端。</p><p>为了防止非法用户高频率地访问这个程序，而影响到其他正常用户的访问，故需要进行一些保护措施。</p><p>最终效果:</p><p><img alt="enter image description here" img-src="0d559aea7a0b6b3f6f2f87c91556f1d544656856.jpg"></p><p><code>被封信息页面</code></p><pre><code>在/shell/yf域内，按住F5不放，一直刷新，几秒后松开，就能看到被封信息和解封时间。只要某个用户对/shell/yf的访问超过了正常的频率，服务将会对这个用户关闭一段时间，期满后自动解封。 </code></pre><h1>0x01 系统原理</h1><hr><p><code>操作系统: CentOS 6.5 x86_64</code> <code>开发语言: Bash Shell Scripts</code> <code>Web服务器: Apache Httpd</code></p><p><img alt="enter image description here" img-src="0b586875fb32a0d2c6bba4722ee67b79a6ffbd60.jpg"></p><p><code>(此图为系统结构的鸟瞰图可存至本地后放大查看)</code></p><h2>2.1 自定义日志:/etc/httpd/logs/yfddos_log</h2><hr><p><img alt="enter image description here" img-src="41f2a11838d62d74a49d10ac8e92ab79d3794ac9.jpg"></p><p><code>(自定义日志文件的格式)</code></p><p>在httpd.conf的日志参数中，加入如下两行：</p><pre><code>LogFormat "%a \"%U\" %{local}p %D %{%s}t " yfddos CustomLog logs/yfddos_log yfddos </code></pre><p>我们接下来重点分析日志文件/etc/httpd/logs/yfddos_log.</p><p><code>LogFormat "%a \"%U\" %{local}p %D %{%s}t " yfddos</code></p><p>解释:</p><pre><code>%a -> 用户的IP %U -> 请求的URL地址，但并不包含query string（The URL path requested, not including any query string.） %{local}p -> 用户请求的服务器端口（一般为80） %D -> 这个请求共消耗了服务器多少微秒（The time taken to serve the request, in microseconds.） %{%s}t -> 服务器收到这个请求时，时间戳的值（seconds since 1970-01-01 00:00:00 UTC） </code></pre><p>例子:</p><pre><code>192.168.31.1 "/shell/yf" 80 118231 1417164313 </code></pre><p>译为:<code>IP为192.168.31.1的主机,在时间戳为1417164313的时候，访问了/shell/yf，并由服务器的80端口向其提供服务，共耗时118231微秒</code></p><p>或为:<code>IP为192.168.31.1的主机,在2014-11-28 16:45:13的时候，访问了/shell/yf，并由服务器的80端口向其提供服务，共耗时0.118231秒</code></p><p>至于为什么不使用httpd.conf中官方定义的日志，原因如下:</p><pre><code>- 用户访问日志的一条记录可大约控制在60Bytes以内，数据量小，便于后期分析，官方定义的日志太过臃肿，影响分析速度 - 使用时间戳标志时间，便于后期分析，官方定义的日志时间参数为常规的表达方式，不便于直接进行处理 - httpd的日志系统本身就是从旧到新进行排序记录的，所以/etc/httpd/logs/yfddos_log日志条目的时间戳，亦为从小到大进行排序的，数据记录更加鲜明 </code></pre><h2>2.2 yfddosd黑名单文件格式</h2><hr><p><img alt="enter image description here" img-src="80b5741891c44a97eeae46606c8fd9c68f813e98.jpg"></p><p><code>黑名单文件格式</code></p><p>yfddosd黑名单文件/etc/yfddos/web-yf-search.b格式如下:</p><pre><code># ip add-stamp rmv-stamp 1.2.3.4 1416046335 1416046395 1.2.3.5 1416046336 1416046396 1.2.3.6 1416046339 1416046399 </code></pre><p>每一行为一个黑名单条目，上面第一个条目的意义为：</p><pre><code>IP地址 :1.2.3.4 开始时间:时间戳1416046335，即 2014-11-15 18:12:15 终止时间:时间戳1416046395，即 2014-11-15 18:13:15 </code></pre><p>直观意义为:</p><p><code>IP地址:1.2.3.4，从2014-11-15 18:12:15开始，被封杀1分钟，在2014-11-15 18:13:15时自动解封。</code></p><p>这个文件将由驻留在系统中的daemon守护进程yfddosd进行维护更新。</p><h2>2.3 守护进程yfddosd:防御系统的逻辑核心</h2><hr><p><img alt="enter image description here" img-src="8afab64c28e8ca8286ef4135fb5e5617c80e947c.jpg"></p><p><code>守护进程的原理图</code></p><p>守护进程yfddosd是整个CC防御系统的核心，而<code>function analyze_and_insert_black()</code>则是yfddosd的核心。</p><p>yfddosd的配置参数:</p><pre><code>yfddos_blackfilePath='/etc/yfddos/web-yf-search.b' yfddos_accesslogPath='/etc/httpd/logs/yfddos_log' function analyze_and_insert_black() { # analyze_and_insert_black() : # $1:max frequency(seems as abuse if above that) $2:blackip-ttl,time to live,unit is seconds (s) # $3:the access log ${3} seconds before will be analyzed to generate the abuse ip lists that we will block # example : analyze_and_insert_black "limit" "ttl" "time" # example : analyze_and_insert_black "4" "10" "5" # 分析在过去5s内的用户访问日志如果有人在这5s内访问量>=4 系统将视其为资源滥用者将其加入服务黑名单 # 一条黑名单的作用时间为10s 即在10s之后系统自动删除此黑名单条目服务则继续向其开放 # global vars: # stamp logtmpfile yfddos_blackfilePath # ...... } </code></pre><p>函数<code>analyze_and_insert_black</code>有三个输入参数：</p><p>例子: <code>analyze_and_insert_black "4" "10" "5"</code></p><pre><code>解释: 分析日志文件/etc/httpd/logs/yfddos_log中,在过去5s内的用户访问日志,如果有IP在这5s内访问量>=4,守护进程yfddosd将视其为资源滥用者，然后将这个IP加入到黑名单文件/etc/yfddos/web-yf-search.b中，此条黑名单的作用时间为10s，在10s之后，守护进程yfddosd将删除此黑名单条目。 </code></pre><p>例子: <code>analyze_and_insert_black "150" "2700" "905"</code></p><pre><code>解释: 分析日志文件/etc/httpd/logs/yfddos_log中,在过去905s内的用户访问日志,如果有IP在这905s内访问量>=150,守护进程yfddosd将视其为资源滥用者，然后将这个IP加入到黑名单文件/etc/yfddos/web-yf-search.b中，此条黑名单的作用时间为2700s，在2700s之后，守护进程yfddosd将删除此黑名单条目。 </code></pre><p>简记为: <code>analyze_and_insert_black "limit" "ttl" "time"</code></p><pre><code>解释: 分析日志文件/etc/httpd/logs/yfddos_log中,在过去(time)s内的用户访问日志,如果有IP在这(time)s内访问量>=limit,守护进程yfddosd将视其为资源滥用者，然后此IP将会被加入到黑名单文件/etc/yfddos/web-yf-search.b中，作用时间为(ttl)s，在(ttl)s之后，守护进程yfddosd将自动删除此条目。 </code></pre><p>从上述中可看出，守护进程yfddosd至少需要完成如下三个任务:</p><ul><li>分析日志文件/etc/httpd/logs/yfddos_log中指定时间内的用户访问记录</li><li>将资源滥用者的IP加入文件/etc/yfddos/web-yf-search.b，并设置封杀TTL参数值</li><li>将/etc/yfddos/web-yf-search.b中已经过期的条目全部及时删除</li></ul><p>守护进程<code>yfddosd</code>是如何实现上面三个逻辑的:</p><ul><li><p>分析日志文件/etc/httpd/logs/yfddos_log中指定时间内的用户访问记录:</p><pre><code>(1) 取出/etc/httpd/logs/yfddos_log中过去time秒的访问日志数据，使用二分法将这一操作的时间复杂度压缩到Klog2(N)以内，其中N为/etc/httpd/logs/yfddos_log中日志总行数，K为一次测试的耗时量，一般为1ms以内，即如有1048576条访问记录，这一操作将仅需要201ms (2) 使用正则RE对这些数据进行二次处理，过滤出所有访问指定URL的用户IP(这个URL为想要防御的http服务url，例如在http://www.yfdc.org系统中，所防御的就是/shell/yf,这个服务向访问者提供信息的search与get服务)，再次使用sort与uniq对这些IP进行处理，以统计出每个IP的访问次数并进行高低排序 </code></pre></li><li><p>将资源滥用者的IP加入文件/etc/yfddos/web-yf-search.b，并设置封杀TTL参数值</p><pre><code>将所有访问次数超过阈值limit的IP更新到黑名单文件/etc/yfddos/web-yf-search.b中,每个黑名单条目的封杀时间为ttl秒 </code></pre></li><li><p>将/etc/yfddos/web-yf-search.b中已经过期的条目全部及时删除</p><pre><code>遍历/etc/yfddos/web-yf-search.b中所有黑名单条目，结合当前时间戳，将所有已经过期的条目一一删去 </code></pre></li></ul><p>下面是守护进程yfddosd状态机的伪代码:(略去了一些处理细节)</p><pre><code>#init and FSM start work... counter=0 while true do sleep 5 counter=counter+1 delete obsolete items #将/etc/yfddos/web-yf-search.b中已经过期的条目全部删除 if # every 5 seconds : 5s then analyze_and_insert_black "6" "10" "5" # 分析在过去5s内访问的用户如果有人其访问量大于等于6 系统将视其为资源滥用者 # 遂将其加入服务黑名单其作用时间为10s 在10s之后 daemon进程自动删除这个ip黑名单条目 fi if #every 53 seconds : 15s then analyze_and_insert_black "14" "45" "15" fi if #every 534+5 seconds : 65s then analyze_and_insert_black "40" "840" "65" fi if #every 53435+5 seconds : 905s : 15min then analyze_and_insert_black "150" "2700" "905" fi if #every 534354+5 seconds : 3605s : 1h then analyze_and_insert_black "300" "7200" "3605" fi if #every 5343543+5 seconds : 10805s : 3h then analyze_and_insert_black "400" "21600" "10805" if #在每天的00:01-04:59时间区间一天仅执行一次 then #备份日志 fi fi done </code></pre><p>防御者应斟酌调整每个检测时间点的参数值(封杀时间ttl与判定阈值limit)，以调节系统应对CC攻击到来时的反应时间。</p><h1>0x02 源代码</h1><hr><pre><code>#!bash ##################################### vim /usr/local/bin/yfddosd.sh : ##################################### nohup bash /usr/local/bin/yfddosd.sh &>"/etc/yfddos/""yfddosd-log-`date +%Y-%m-%d`" & ##################################### yfddos daemon mkdir /etc/yfddos yfddos_blackfilePath='/etc/yfddos/web-yf-search.b' yfddos_accesslogPath='/etc/httpd/logs/yfddos_log' ### refresh tll logtmpfile=`mktemp` stamp=`date +%s` touch "$yfddos_blackfilePath" if grep -Po '[0-9]+\.[0-9]+\.[0-9]+\.[0-9]+' "$yfddos_blackfilePath" &>/dev/null then cat "$yfddos_blackfilePath" \| while read i do deadstamp=`echo "$i" \| grep -Po '[0-9]+$'` if [ "$stamp" -le "$deadstamp" ] then echo "$i" >>"$logtmpfile" fi done fi chmod o+r "$logtmpfile" mv -f "$logtmpfile" "$yfddos_blackfilePath" if ! grep -Po '[0-9]+\.[0-9]+\.[0-9]+\.[0-9]+' "$yfddos_blackfilePath" &>/dev/null then echo '255.255.255.255 0 0' >> "$yfddos_blackfilePath" fi function analyze_and_insert_black() { # analyze_and_insert_black() : # $1:max frequency(seems as abuse if above that) $2:blackip-ttl,time to live,unit is seconds (s) # $3:the access log ${3} seconds before will be analyzed to generate the abuse ip lists that we will block # example : analyze_and_insert_black "limit" "ttl" "time" # example : analyze_and_insert_black "4" "10" "5" # 分析在过去5s内的用户访问日志如果有人在这5s内访问量>=4 系统将视其为资源滥用者将其加入服务黑名单 # 一条黑名单的作用时间为10s 即在10s之后系统自动删除此黑名单条目服务则继续向其开放 # global vars: # stamp logtmpfile yfddos_blackfilePath local threshold="$1" local ttl="$2" local stamp_pre="$3" local i=0 local num="" local fre=0 local ip=0 local localbuf=0 local linenum=0 local deadstamp=0 stamp_pre="$((stamp-stamp_pre))" #二分查找初始化 local temp=0 local yf_x='1' local yf_y=`cat "$logtmpfile" \| wc -l` if [ "$yf_y" -le "1" ] then yf_y=1 fi local yf_I=$(((yf_x+yf_y)/2)) temp=`cat "$logtmpfile" \| wc -l` if [ "$temp" -gt "0" ] then temp=`sed -n '$p' "$logtmpfile" \| grep -Po '[0-9]+ $'` if [ "$temp" -lt "$stamp_pre" ] then num="" else while true #使用二分查找的方法快速地分析访问日志 do temp=`sed -n "${yf_x}p" "$logtmpfile" \| grep -Po '[0-9]+ $'` if [ "$temp" -ge "$stamp_pre" ] then break fi if [ "$((yf_y-yf_x))" -le "1" ] then yf_x="$yf_y" break fi temp=`sed -n "${yf_I}p" "$logtmpfile" \| grep -Po '[0-9]+ $'` if [ "$temp" -lt "$stamp_pre" ] then yf_x="$yf_I" yf_y="$yf_y" yf_I="$(((yf_x+yf_y)/2))" continue fi yf_x="$yf_x" yf_y="$yf_I" yf_I="$(((yf_x+yf_y)/2))" continue done temp=`sed -n "${yf_x}p" "$logtmpfile" \| grep -Po '[0-9]+ $'` if [ "$temp" -ge "$stamp_pre" ] then num="$yf_x" else num="" fi fi if [ -n "$num" ] then sed -n "${num},\$p" "$logtmpfile" \| grep -Po '^[0-9]+\.[0-9]+\.[0-9]+\.[0-9]+' \| sort -n \| uniq -c \| sort -rn \| while read i do fre=`echo "$i" \| grep -Po '[0-9]+' \| head -1` ip=`echo "$i" \| grep -Po '[0-9]+\.[0-9]+\.[0-9]+\.[0-9]+' ` if [ "$fre" -ge "$threshold" ] then #insert illegal ips : cat "$yfddos_blackfilePath" # ip add-stamp rmv-stamp #1.2.3.4 1416046335 1416046395 temp=`grep -Pn "${ip//./\\.} " "$yfddos_blackfilePath"` if [ -n "$temp" ] then linenum=`echo "$temp" \| grep -Po '^[0-9]+' \| head -1` deadstamp=`echo "$temp" \| grep -Po '[0-9]+$' \| sort -rn \| head -1 ` if [ "$((stamp+ttl))" -gt "$deadstamp" ] then sed -i "${linenum}s/./${ip} ${stamp} $((stamp+ttl))/g" "$yfddos_blackfilePath" fi else sed -i "\$a ${ip} ${stamp} $((stamp+ttl))" "$yfddos_blackfilePath" fi else break fi done fi fi } #init and yfddosd's FSM start work... counter=0 while true do sleep 5 counter=$((counter+1)) echo -n `date +%Y-%m-%d\ %H:%M:%S`" ""counter ${counter}:"`cat /proc/uptime \| grep -Po '[0-9\.]+' \| head -1`" " echo -n "refresh tll:"`cat /proc/uptime \| grep -Po '[0-9\.]+' \| head -1`" " ### refresh tll #refresh ttl: analyze file: "$yfddos_blackfilePath" if some items'ttl has been reach the date , we will remove it and open service to the ip had been banned before. #insert illegal ips : cat "$yfddos_blackfilePath" # ip add-stamp rmv-stamp #1.2.3.4 1416046335 1416046395 #sed -i "/^. $((stamp-5))$/d;/^.* $((stamp-4))$/d;/^.* $((stamp-3))$/d;/^.* $((stamp-2))$/d;/^.* $((stamp-1))$/d;/^.* $((stamp))$/d;/^$/d" "$yfddos_blackfilePath" logtmpfile=`mktemp` stamp=`date +%s` touch "$yfddos_blackfilePath" if grep -Po '[0-9]+\.[0-9]+\.[0-9]+\.[0-9]+' "$yfddos_blackfilePath" &>/dev/null then cat "$yfddos_blackfilePath" \| while read i do deadstamp=`echo "$i" \| grep -Po '[0-9]+$'` if [ "$stamp" -le "$deadstamp" ] then echo "$i" >>"$logtmpfile" fi done fi chmod o+r "$logtmpfile" mv -f "$logtmpfile" "$yfddos_blackfilePath" if ! grep -Po '[0-9]+\.[0-9]+\.[0-9]+\.[0-9]+' "$yfddos_blackfilePath" &>/dev/null then echo '255.255.255.255 0 0' >> "$yfddos_blackfilePath" fi logtmpfile=`mktemp` stamp=`date +%s` cat "$yfddos_accesslogPath" \| grep -P ' "/shell/yf" ' >"$logtmpfile" if true # every 5 seconds : 5s then echo -n "analyze_and_insert_black 6 10 5:"`cat /proc/uptime \| grep -Po '[0-9\.]+' \| head -1`" " #analyze yfddos log : analyze_and_insert_black() $1:max frequency(seems as abuse if above that) $2:blackip-ttl $3:the access log ${3} seconds before will be analyzed to generate the abuse ips that we will block analyze_and_insert_black "6" "10" "5" # 分析在过去5s内访问的用户如果有人其访问量大于等于6 系统将视其为资源滥用者遂将其加入服务黑名单其作用时间为10s 在10s之后 daemon进程自动删除这个ip黑名单条目 fi if [ "$((counter%(3)))" -eq "0" ] #every 53 seconds : 15s then echo -n "analyze_and_insert_black 14 45 15:"`cat /proc/uptime \| grep -Po '[0-9\.]+' \| head -1`" " # example : analyze_and_insert_black "limit" "ttl" "time" analyze_and_insert_black "10" "45" "15" fi if [ "$((counter%(34+1)))" -eq "0" ] #every 534+5 seconds : 65s then echo -n "analyze_and_insert_black 40 840 65:"`cat /proc/uptime \| grep -Po '[0-9\.]+' \| head -1`" " # example : analyze_and_insert_black "limit" "ttl" "time" analyze_and_insert_black "25" "840" "65" fi if [ "$((counter%(3435+1)))" -eq "0" ] #every 53435+5 seconds : 905s : 15min then echo -n "analyze_and_insert_black 150 2700 905:"`cat /proc/uptime \| grep -Po '[0-9\.]+' \| head -1`" " # example : analyze_and_insert_black "limit" "ttl" "time" analyze_and_insert_black "150" "2700" "905" fi if [ "$((counter%(34354+1)))" -eq "0" ] #every 534354+5 seconds : 3605s : 1h then echo -n "analyze_and_insert_black 300 7200 3605:"`cat /proc/uptime \| grep -Po '[0-9\.]+' \| head -1`" " # example : analyze_and_insert_black "limit" "ttl" "time" analyze_and_insert_black "300" "7200" "3605" fi if [ "$((counter%(343543+1)))" -eq "0" ] #every 534354*3+5 seconds : 10805s : 3h then echo -n "analyze_and_insert_black 400 21600 10805:"`cat /proc/uptime \| grep -Po '[0-9\.]+' \| head -1`" " # example : analyze_and_insert_black "limit" "ttl" "time" analyze_and_insert_black "400" "21600" "10805" #### "${yfddos_accesslogPath}" backup : 在每天的00:01-04:59时间区间内备份日志一次 if [ "`date +%H`" -le "5" ] && ! [ -f "${yfddos_accesslogPath}-`date +%Y-%m-%d`" ] then service httpd stop mv "${yfddos_accesslogPath}" "${yfddos_accesslogPath}-`date +%Y-%m-%d`" service httpd start fi fi rm -fr "$logtmpfile" echo "sleep:"`cat /proc/uptime \| grep -Po '[0-9\.]+' \| head -1`" " done </code></pre><p></p></section></article><div class="entry-controls clearfix"><div style="float:left;color:#9d9e9f;font-size:15px"><span>©乌云知识库版权所有未经许可禁止转载</span></div></div><div class="yarpp-related"><h3>为您推荐了适合您的技术文章:</h3><ol id="recommandsystem"><li><a href="http://drops.wooyun.org/tips/2014" rel="bookmark" id="re1">批量网站DNS区域传送漏洞检测——bash shell实现</a></li><li><a href="http://drops.wooyun.org/papers/653" rel="bookmark" id="re2">搭建基于Suricata+Barnyard2+Base的IDS前端Snorby</a></li><li><a href="http://drops.wooyun.org/papers/8413" rel="bookmark" id="re3">PfSense命令注入漏洞分析</a></li><li><a href="http://drops.wooyun.org/tips/411" rel="bookmark" id="re4">各种环境下的渗透测试</a></li></ol></div><div id="comments" class="comment-list clearfix"><div id="comment-list"><div class="note-comment"><img class="avatar" alt="30" src="http://wooyun.b0.upaiyun.com/wooyun_job/avatar/default.png"><div class="content"><div class="comment-header"><span class="author-link">燕云</span> <span class="reply-time">2015-04-27 11:08:18</span></div><p></p><p>@han 你好！<br>黑名单存放在/etc/yfddos/web-yf-search.b 文中有介绍此文件的格式 web应用程序只需要在每一次接入新的连接时判定一下即可</p><p></p></div></div><div class="note-comment"><img class="avatar" alt="30" src="http://wooyun.b0.upaiyun.com/wooyun_job/avatar/default.png"><div class="content"><div class="comment-header"><span class="author-link">han</span> <span class="reply-time">2015-02-05 15:05:51</span></div><p></p><p>没看到怎么封锁客户端ip的代码</p><p></p></div></div><div class="note-comment"><img class="avatar" alt="30" src="http://wooyun.b0.upaiyun.com/wooyun_job/avatar/default.png"><div class="content"><div class="comment-header"><span class="author-link">燕云</span> <span class="reply-time">2014-12-17 17:06:47</span></div><p></p><p>我之前一直用C开发工具，后来经历了Python->shell scripts<br>一个人和一个团队是不一样的，时间成本问题</p><p>我测过效率真的没有你想的那样糟 :)</p><p></p></div></div><div class="note-comment"><img class="avatar" alt="30" src="http://wooyun.b0.upaiyun.com/wooyun_job/avatar/default.png"><div class="content"><div class="comment-header"><span class="author-link">燕云</span> <span class="reply-time">2014-12-17 17:02:44</span></div><p></p><p>你好！</p><p>因为系统有几个监测时间点，如果你在第一个时间段已经访问了很多次，当到达第二个时间段时，依旧被判定为了 abuse ，所以出现了上面的现象 :)</p><p></p></div></div><div class="note-comment"><img class="avatar" alt="30" src="http://wooyun.b0.upaiyun.com/wooyun_job/avatar/default.png"><div class="content"><div class="comment-header"><span class="author-link">Sd_red</span> <span class="reply-time">2014-12-16 09:57:25</span></div><p></p><p>已加入收藏夹，还没仔细看。我有时间测试下效率问题。因为应对CC如果用shell的话，计算效率是很低的。。。。总之，很感谢作者分享的文章。让我受益很多。</p><p></p></div></div><div class="note-comment"><img class="avatar" alt="30" src="http://wooyun.b0.upaiyun.com/wooyun_job/avatar/default.png"><div class="content"><div class="comment-header"><span class="author-link">cc</span> <span class="reply-time">2014-12-11 10:05:54</span></div><p></p><p>解封后点一次页面就被封</p><p></p></div></div><div class="note-comment"><img class="avatar" alt="30" src="http://wooyun.b0.upaiyun.com/wooyun_job/avatar/default.png"><div class="content"><div class="comment-header"><span class="author-link">燕云</span> <span class="reply-time">2014-12-04 00:18:38</span></div><p></p><p>ps: 这里的env指的是cgi环境的env</p><p></p></div></div><div class="note-comment"><img class="avatar" alt="30" src="http://wooyun.b0.upaiyun.com/wooyun_job/avatar/default.png"><div class="content"><div class="comment-header"><span class="author-link">燕云</span> <span class="reply-time">2014-12-04 00:16:48</span></div><p></p><p>Apache Module mod_log_config:<br>%{FOOBAR}e : The contents of the environment variable FOOBAR<br>使用这个选项所有http连接参数都能写到日志中 cookie信息自然也可以了 :)</p><p></p></div></div><div class="note-comment"><img class="avatar" alt="30" src="http://wooyun.b0.upaiyun.com/wooyun_job/avatar/default.png"><div class="content"><div class="comment-header"><span class="author-link">DBA</span> <span class="reply-time">2014-12-03 22:01:01</span></div><p></p><p>未考虑NAT网络的情况。token+ip 这应该是程序里才能实现的,仅仅利用Apache的日志怕是不行了。</p><p></p></div></div><div class="note-comment"><img class="avatar" alt="30" src="http://wooyun.b0.upaiyun.com/wooyun_job/avatar/default.png"><div class="content"><div class="comment-header"><span class="author-link">燕云</span> <span class="reply-time">2014-12-03 20:59:39</span></div><p></p><p>笔者是14本科毕业生，现正找一份关于Linux系统维护的工作，<a class="__cf_email__" href="/cdn-cgi/l/email-protection" data-cfemail="43a5dfcaa5c7ccabc3c6abecf4abc2d7a4f0f83a362d372b222d22372c30037275706d202c2e">[email protected]</a></p><p></p></div></div><div class="note-comment"><img class="avatar" alt="30" src="http://wooyun.b0.upaiyun.com/wooyun_job/avatar/default.png"><div class="content"><div class="comment-header"><span class="author-link">燕云</span> <span class="reply-time">2014-12-03 20:57:58</span></div><p></p><p>使用IP进行封杀确实存在一定误杀几率，现在有如下若干解决方案：<br>1.验证码：这种方法在面对用户需要经常访问的服务面前，只能作为辅助手段，否则影响用户体验<br>2.Cookie:因为Cookie非常容易伪造，这种方法只能阻碍住一般水平的攻击<br>3.usr-token+ip：采用用户登录后为其发放令牌，令其与用户IP进行绑定的方法，使用（usr-token，ip）二元组，这种方法最为理想，但需要用户先进行登录系统<br>因为目前yfdc网站上并无用户系统，所有并未实行第三种方案。<br>要实行第三种方法，只需要在yfddos_log里增加usr-token数据项，在后续的处理中对（usr-token，ip）一同处理即可。</p><p>另外，mod_security确实很强大</p><p></p></div></div><div class="note-comment"><img class="avatar" alt="30" src="http://wooyun.b0.upaiyun.com/wooyun_job/avatar/default.png"><div class="content"><div class="comment-header"><span class="author-link">燕云</span> <span class="reply-time">2014-12-03 20:55:37</span></div><p></p><p>这个系统只是快速建立的“第一个系统”原型，第一个系统顺利运行成功后，以后的优化和改进方向便更加清晰明了(惭愧自己动手之前确实不知道nginx/tengine的limit_req_zone功能)<br>日志文件很小，对于每5s一次的分析任务，这点消耗服务器还是顶得住吧 :)</p><p></p></div></div><div class="note-comment"><img class="avatar" alt="30" src="http://wooyun.b0.upaiyun.com/wooyun_job/avatar/default.png"><div class="content"><div class="comment-header"><span class="author-link">燕云</span> <span class="reply-time">2014-12-03 20:51:17</span></div><p></p><p>代码好用，应经部署在了yfdc.org上，可自行测试</p><p></p></div></div><div class="note-comment"><img class="avatar" alt="30" src="http://wooyun.b0.upaiyun.com/wooyun_job/avatar/default.png"><div class="content"><div class="comment-header"><span class="author-link">Forever80s</span> <span class="reply-time">2014-12-03 16:44:43</span></div><p></p><p>100人用一个外网ip会不会false positive？<br>记得mod_security很强大</p><p></p></div></div><div class="note-comment"><img class="avatar" alt="30" src="http://wooyun.b0.upaiyun.com/wooyun_job/avatar/default.png"><div class="content"><div class="comment-header"><span class="author-link">missdiog</span> <span class="reply-time">2014-12-03 15:47:20</span></div><p></p><p>shell实现的<br>nginx ／tengine 的limit_req_zone 功能</p><p>通过日志获取频率，于磁盘IO，CPU都不好，性能太低了，高并发死得快，还是nginx好点，漏桶原理，动作很快，姿势很帅</p><p></p></div></div><div class="note-comment"><img class="avatar" alt="30" src="http://wooyun.b0.upaiyun.com/wooyun_job/avatar/default.png"><div class="content"><div class="comment-header"><span class="author-link">wangy3e</span> <span class="reply-time">2014-12-03 13:18:54</span></div><p></p><p>代码好用么???~`</p><p></p></div></div><div class="note-comment"><img class="avatar" alt="30" src="http://wooyun.b0.upaiyun.com/wooyun_job/avatar/default.png"><div class="content"><div class="comment-header"><span class="author-link"><a class="__cf_email__" href="/cdn-cgi/l/email-protection" data-cfemail="b4dcd5d7dfd1c6f4c7dddad59ad7da">[email protected]</a></span> <span class="reply-time">2014-12-03 12:37:40</span></div><p></p><p>Mark！</p><p></p></div></div></div></div></div></main>
[Posttranslational p53 phosphorylation in non small cell lung cancer cells after radio/chemotherapy]. p53 protein is a critical regulator of cell cycle and apoptosis. Many stimuli change its expression and modify its functions. The aim of our work was to determine stability and chosen posttranslational modification of p53 protein during the treatment of lung cancer. We investigated levels of poly-ADP-ribose- a marker of cellular DNA damage, DNA ploidy, Ki-67 expression, wild type and mutated p53 protein expression and intensity of phosphorylation of chosen p53 serine sites: C-terminal Ser392, and N-terminal Ser15, and Ser20 in fiberoptic bronchoscopy biopsy samples taken from patients suffering from recurrent squamous cell lung cancer before and after radio/chemotherapy. Analysis was based on results obtained from 23 patients after surgery in I-IIIA clinical stage of the disease. Therapy lowered the number of G2/M cells, but increased S fraction cell population in about 50%. Therapy increased p53 expression and p53 phosphorylation at Ser392 and Ser20, and these changes correlated with poly-ADP-ribose levels and Ki-67 expression. Our results indicate that apart from changes in p53 quantity, p53 posttranslational phosphorylation play a role in regulation of neoplastic cells proliferation in response to drugs.
When it comes to Election Day problems in St. Louis, the politicians' rhetoric doesn't match the reality Shortly after the polls closed on Election Night, the statewide Republican "victory" party hummed along at the Marriott West in Town & Country. It was the lull between the closing of the polls and the trickling in of any meaningful results, and the buzz was all about the fiasco with the elections in the city of St. Louis. Earlier in the day, Democrats in the city of St. Louis had marched into state circuit court demanding that the polls in the city of St. Louis be kept open until 10 p.m. because hundreds, if not thousands, of voters in the city of St. Louis were being denied their right to vote by an incompetent election commission that had screwed up voter lists. And a circuit judge in the city of St. Louis had consented. At a primarily white suburban Republican affair, it didn't need to be said that most of the protesters, as well as the politician who led the court fight and the judge who ruled, were black Democrats. Then U.S. Sen. Christopher "Kit" Bond stepped up to the podium. Sen. Christopher "Kit" Bond went ballistic after learning that a judge had ordered that the polls remain open. "Can you believe?" he thundered into the microphone. "Can you believe that anybody would say that a Democratic election board, appointed by a Democratic governor in a Democratic city, dominated by Democrats, was trying to keep Democrats from voting?" He then straightened himself up and roared, "That's an outrage!" Then he began banging the podium with his right fist. "That is -- thump! -- absolutely -- thump! thump! -- an outrage!" he bellowed. The crowd, predictably, went wild. This histrionic highlight was followed by a less effective but very much on-message rant by Missouri Republican Party chairman Ann Wagner: "We are not going to let the city of St. Louis play by a different set of rules than the rest of this great state!" Given the heightened passions of an election night, one might excuse Bond and Wagner's shrill emotional pitch. By Thursday, of course, things had changed. The Republicans had lost all the statewide offices except secretary of state. But although Bond's voice had softened, his message hadn't. He announced that he had asked the U.S. Attorney and the FBI to investigate what he called "a major criminal enterprise designed to defraud voters" -- in the city of St. Louis, of course. In his letters to the two federal agencies, Bond wrote that although the circuit judge's order to keep the polls open until 10 p.m. was overturned at the appellate level by 7:45 p.m., "there is abundant evidence that the appellate order was ignored and widespread voter fraud occurred throughout the city of St. Louis." He wrote of a "deliberate scheme" planned in advance so unregistered voters could vote illegally: "There is reason to believe that collusion existed to commit voter fraud and voter fraud occurred on a wide scale throughout the city of St. Louis." Unlike his election-night tirade, his letters did not mention the words "Democrat" and "Democratic." Nor did they make any reference to "blacks" or "African-Americans." Needless to say, in Mr. Bond's milieu, the words "city of St. Louis" convey all that quite well. Talking of words, let's try separating the rhetoric from the reality. First, let's put to rest Bond's misstatement about the Board of Election Commissioners in St. Louis. It isn't a "Democratic election board." By law, two of the four commissioners are Democrats, the other two Republicans. Of the 28 employees, 14 are from each party. Yes, Bond was right -- and redundant -- when he said, "in a Democratic city, dominated by Democrats." In last week's elections, Democratic candidates beat their Republican opponents by a ratio of roughly 4-to-1 in the city. But Bond's primary thesis -- that it makes no sense that Democrats would prevent Democrats from voting -- is easily refuted. Half the board is Republican, but nothing suggests party affiliation had anything to do with problems voters were having. All you had to do was turn to your TV on Election Night to see angry voters complaining of delays and denial of voting rights. Here at the Riverfront Times, at least two employees were registered and had not moved since voting in the last election but found at their usual polling places that their names were not on the list. This started the interminable process of election judges' trying to get through on the phone lines to the election-board headquarters downtown. Some voters, figuring they'd save time by going to the downtown office themselves, found long lines there as well. When the Democrats went to court, led by state Sen. (and congressional candidate) William "Lacy" Clay Jr., and requested that the polls be kept open late, the election board opposed the request. And when Circuit Judge Evelyn Baker -- appointed by Bond himself in 1983 -- consented to keeping the polls open until 10 p.m., the election board favored a reversal of her decision. "We went to the appellate court to close the polls down," says an exasperated Floyd Kimbrough, the election-board chairman, a Democrat and an ally of Clay's father, retiring Congressman Bill Clay. Kimbrough disputes both the Republican allegation of fraud and the Democratic allegation that thousands of registered voters were denied the right to vote. He calls each allegation "an outright lie." Nevertheless, Bond and his Republican cohorts maintain that there was a premeditated plan to keep the polls open late and get unregistered voters to vote. They base it primarily on three things: Clay said on the day before the election that voters might have problems and that if they did, he and the Democratic Party would go to court to keep polls open late. Shortly after Judge Baker's decision at about 6 p.m., a message from the Rev. Jesse Jackson was phoned to voters, telling them the polls would be open until 10 p.m. "Numerous witnesses" said unregistered voters were allowed to vote after the shutdown order from the appellate court. Clay readily concedes that he and Democratic Party officials anticipated problems in the city. "We knew there were going to be problems, because we sent 60 people down to vote (absentee) one week before the election -- they were listed as inactive," he says. Clay is quite conscious of the fact that there have been reports nationwide of inner-city blacks' being denied the right to vote through purges of voter lists. As for Jackson's taped phone message being "prepared in advance," one has only to listen to the actual message to make a judgment. Jackson tells recipients of his call that polls will remain open "until 10 p.m. in your neighborhood and until midnight downtown at the board of elections" -- precisely what Judge Baker ruled. It could only have been recorded after Baker's ruling. Unless, of course, one were inclined to believe in a vast left-wing conspiracy -- which, in this case, would have to include both the reverend and the judge. There remains the Republican allegation that unregistered voters ended up voting. They see it as a result of the "deliberate scheme," which led to intentional fraud. The Democratic allegation, meanwhile, is that thousands of registered voters found that their names were not on the list at polling places and either experienced long delays or were denied the right to vote. They see it as an inept and incompetent election-board operation. There is some truth to both assertions -- not a whole lot, but some. Kevin Coan, the Republican director of elections for the city, says the board had projected 70 percent turnout in the city; it got 68 percent, with about 129,000 of the city's 190,000 registered voters actually voting. The board deployed 1,500 voting machines at approximately 260 locations, says Coan, a lower voters-per-machine ratio than St. Louis County had. The board had conducted a canvass of voters in the spring, mailing cards to more than 200,000 voters. About 33,000 were returned by the U.S. Postal Service as "undeliverable," meaning that the recipient was not living at the address or had moved. Those 33,000 names were placed on an "inactive" list. Between spring and October, about 30,000 voters registered, many of them the same people placed on the "inactive" list. Coan downplays problems with people experiencing delays. What he objects to is what Clay and his allies did, starting about midday. Clay came to the election-board offices downtown and -- after failing to get the board to approve voters who, says Coan, were not registered -- called in the city's circuit judges to authorize unregistered voters to vote. It was then, in the early afternoon, that Presiding Judge Michael Calvin and six other judges approved 233 people to vote. On that point, Kimbrough agrees with Coan. Once the judges authorized voters to vote, the election board couldn't stop them from voting. Then came the hearing before Judge Baker. The hearing, says Coan, "was a complete farce." Clay testified before the judge, as did his sister, Michelle Clay, an attorney who heads the NAACP Voter Education Fund, along with Steve Englehardt, Clay's assistant. All three, along with a fourth city resident, maintained that registered voters were having to wait hours to get clearance to vote and that the polls needed to remain open late to allow people to vote. They also gave Judge Baker signed affidavits from several voters who said they were registered but were told their names were not on the lists at the polling places. The way Coan sees it, Clay engineered the whole thing. Baker says she based her decision on "the overwhelming weight of the testimony at the hearing from registered voters who had voted at the same polling places, who had not changed their addresses, who arrived at the polls in a timely fashion in the morning to vote and had been told that their names had been removed from the rolls." Baker adds that the election board was unprepared for the volume of voters and that the problem could have been alleviated, if not solved, had election judges been given the "inactive" voter lists instead of having to call the election-board offices downtown to verify the names on the "inactive" lists. As for Bond's calling on the feds to investigate a planned scheme to commit fraud, Baker says: "I think Sen. Bond needs to get a grip. I don't think stupidity rises to the level of a federal investigation." All rhetoric aside, a clearer view of the reality on Election Day in St. Louis comes from Michael Chance, a Republican election deputy who worked with a Democratic deputy overseeing the precincts in the 3rd Ward. Minutes after the polling places opened at 6 a.m., he says, judges were dialing the election board to verify names but kept getting busy signals. This continued the rest of the day at all the precincts in the ward, resulting in frustrated voters' having to wait to vote. "There was a constant complaint from judges -- they just couldn't get through," Chance says. He also says he stopped about five people from voting because they said they lived in the county but figured that because they had previously been registered in the city, they could vote. Others were turned away because they registered after the Oct. 11 deadline, he says. Better-prepared voters who know the law would go a long way toward a more orderly Election Day, he adds. Chance also says too many election judges are simply too old to do the job. "It's a long, grueling day with a lot of cranky and angry people you're dealing with," he says. "Quite frankly, a lot of these people are not capable, physically or mentally, to do the job." Election judges are paid $79 for the day, which translates to barely minimum wage for the 14 or 15 hours they work. Kimbrough agrees that too many elderly people work as judges, slowing the process when turnout is large. "If you would double the pay, we would get more people applying," he says. He also notes that the election-board offices, at 300 N. Tucker Blvd., are far too small and have only three windows to serve voters. The board's previous office, across from City Hall, had 28 windows. Kimbrough says he's absolutely certain that no fraud took place, that no more than 100 people were allowed to vote after the polls were ordered closed by the appellate court and that although voters experienced long delays, not that many were denied their right to vote. In any case, nobody is alleging that the outcome of any race would have changed had there been perfectly run city elections. All of this goes to show that Bond and his suburban Republican colleagues have blown a relatively minor problem out of proportion and that Clay and his Democratic colleagues overstated the extent of the disenfranchisement. As for the politicians who are promising to overhaul the election board, including Mayor Clarence Harmon and Aldermanic President Francis Slay, they might be better off using their power to leverage more money -- so the election board can get more and better workers by offering more than a minimum wage and so it can set up a better phone system, staffed by more workers, to cut down delays. That way, we won't have to suffer another court fight led by Democrats in the city of St. Louis. Or a ballistic Kit Bond spouting nonsense on the next Election Night.
1. Field of the Invention The present invention relates to a method of manufacturing a micro machine member and the like from a silicon wafer according to a semiconductor process, and, more particularly to a silicon processing method for manufacturing a silicon structure from a wafer, a silicon substrate with etching mask, and the like. The structure is used as, for example, an elastic member and configures a micro oscillator that performs oscillation. The oscillator can be used for optical apparatuses such as a light deflector and an image forming apparatus using the light deflector, sensors such as an acceleration sensor and an angular velocity sensor, and the like. 2. Description of the Related Art Conventionally, micro machine members manufactured from a wafer according to a semiconductor process can be processed in a micrometer order. Various micro functional elements are realized by using the micro machine members (see, Japanese Patent Application Laid-Open No. H06-232112). As one of such methods of finely processing silicon, there is a method of performing anisotropic etching with an alkali solution after forming an etching mask on a wafer. The anisotropic etching is a method of forming, making use of the fact that an etching rate of a (111) surface of silicon and a crystal surface equivalent thereto (these are collectively referred to as (111) equivalent surface as well) is lower than other crystal surfaces, a structure mainly including the (111) surface and the crystal surface equivalent thereto. This is a simple processing method of immersing wafers in the alkali solution. A large number of wafers can be collectively processed. Therefore, inexpensive micro-order processing is possible. More specifically, the anisotropic etching is etching performed by using etchant having a characteristic that the etching does not proceed in a specific crystal orientation. A microstructure with a specific crystal surface set as a reference, i.e., a structure specified by the crystal surface can be created at extremely high processing accuracy. Examples of anisotropic etchant include KOH (potassium hydroxide), TMAH (tetramethyl ammonium hydroxide solution), EDP (ethylene diamine pyrocatechol+water), NaOH (sodium hydroxide) and hydrazine. In a silicon micro functional element, a structure subjected to stress is often used. With the anisotropic etching, a member having a smooth processed surface can be obtained. In particular, when the structure subjected to stress is formed, since the smooth surface can avoid stress concentration, a structure having satisfactory durability can be manufactured. A light deflector that performs optical scanning by torsionally oscillating a reflection surface with a micro oscillator formed by such a technique (see U.S. Pat. No. 4,317,611) has, for example, the following characteristics compared with an optical scanning optical system that uses a rotary multisurface mirror such as a polygon mirror: the light deflector can be reduced in size and power consumption is small. In particular, power consumption can be further reduced by driving the light deflector near a resonant frequency of the torsional oscillation of the micro oscillator. However, as one of problems of the method of manufacturing a structure from a wafer according to the anisotropic etching, an error occurs in a processing dimension between a formed etching mask and a crystal axis direction of the wafer because of an alignment error. For example, when such a structure is used as a spring to manufacture a micro oscillator, a spring constant error occurs because of the processing dimension error. This is likely to lead to a manufacturing error of a resonant frequency of the micro oscillator.
LED ZEPPELIN SAYS NO EVEN TO BILL CLINTON NEW YORK (AP) – Bill Clinton can bring together all sorts of warring parties, but couldn’t do it for Led Zeppelin. David Saltzman of the Robin Hood Foundation tells the “60 Minutes Overtime” webcast that he and executive Harvey Weinstein flew to Washington to ask Clinton to ask the surviving members of Led Zeppelin to reunite for the Superstorm Sandy benefit concert. Clinton asked, and they said no. Led Zeppelin was in Washington just before the Sandy concert to accept their Kennedy Center Honors.
441 S.W.2d 485 (1969) Frank BROWN, Plaintiff-in-Error, v. STATE of Tennessee, Defendant-in-Error. Court of Criminal Appeals of Tennessee. April 16, 1969. Certiorari Denied June 2, 1969. 486 James G. Nave, Cleveland, for plaintiff in error. George F. McCanless, Atty. Gen. of Tenn., Lance D. Evans, Asst. Atty. Gen., Nashville, Tom J. Taylor, Dist. Atty. Gen., Cleveland, for defendant in error. Certiorari Denied by Supreme Court June 2, 1969. OPINION OLIVER, Judge. Frank Brown, the plaintiff in error and defendant below, was convicted in the Criminal Court of Bradley County of assault with intent to commit second degree murder and was sentenced to imprisonment for one year in the State Penitentiary. His motion for a new trial being overruled, the defendant excepted and prayed and was granted and has perfected an appeal in the nature of a writ of error to this Court. The first two Assignments of Error challenge the sufficiency of the evidence to warrant and sustain the verdict of the jury. In examining this contention, we are bound by the well-established rule, settled by numerous decisions of the Supreme Court of Tennessee, that a verdict of guilt, approved by the trial judge, accredits the testimony of the State's witnesses, resolves all conflicts in the testimony in favor of the State and establishes the State's theory of the case; that under such a verdict the presumption of innocence which the law throws around an accused and which stands as a witness for him in his trial, disappears, and upon appeal that presumption of innocence is replaced by a presumption of guilt; that this Court is not permitted to reverse a conviction upon the facts unless the evidence clearly preponderates against the verdict of the jury and in favor of the innocence of the accused; that we may review the evidence only to determine whether it preponderates against the verdict; and that the defendant has the burden of showing on appeal that the evidence preponderates against the verdict and in favor of his innocence. Turner v. State, 216 Tenn. 714, 394 S.W.2d 635; Chico v. State, 217 Tenn. 19, 394 S.W.2d 648; Johnson v. State, 217 Tenn. 234, 397 S.W.2d 170; Brenner v. State, 217 Tenn. 427, 398 S.W.2d 252; Owens v. State, 217 Tenn. 544, 399 S.W.2d 507; Harris v. State, 217 Tenn. 582, 399 S.W.2d 749; Pryor v. State, 217 Tenn. 695, 400 S.W.2d 700; Monts v. State, 218 Tenn. 31, 400 S. W.2d 722; Patterson v. State, 218 Tenn. 80, 400 S.W.2d 743; Carroll v. State, 212 Tenn. 464, 370 S.W.2d 523; McBee v. State, 213 Tenn. 15, 372 S.W.2d 173; Gulley v. State, 219 Tenn. 114, 407 S.W.2d 186; Jamison v. State, 220 Tenn. 280, 416 S.W.2d 768. 487 This rule governing appellate review of criminal convictions makes unnecessary and, indeed, inappropriate, any detailed discussion of the evidence pro and con. Hargrove v. State, 199 Tenn. 25, 28, 281 S.W. 2d 692, 694; Morrison v. State, 217 Tenn. 374, 397 S.W.2d 826, 400 S.W.2d 237. The evidence accredited by the jury may be summarized briefly. Mike Westfield, who was fifteen, was driving his father's car in the late afternoon of September 28, 1966 in Cleveland, Tennessee. With him were his cousin Isbella Westfield and Alvin Porter. The motor stopped on Twentieth Street near the defendant's property. Unable to get it started again immediately, it was maneuvered to the side of the street by letting it roll backwards. Mike was acquainted with this misbehavior of the vehicle and knew that it would start again after letting it cool off. So the three youngsters sat inside the car and talked for about twenty minutes. The defendant operated a junkyard on the adjacent property, and a roadway which was formerly a street ran through his property. While Mike was outside the car checking it, the defendant and an employee, J.B. Martin, were seen approaching on this roadway. The defendant yelled twice at him to stop. Seeing that the defendant had a shotgun, Mike became frightened and tried to get back into the car. As he was doing so the defendant shot him, shots striking him under his right arm and in the lower portion of his back. After Mike got into the rear seat of the car the defendant fired again, breaking out the rear window and hitting him in the head. The defendant then turned and walked back toward his house. Isbella Westfield then succeeded in getting the car started and took Mike to the hospital. In his testimony, the defendant maintained that thieves had stolen much of his property from the junkyard; that he had repeatedly complained to the Sheriff's office to no avail; that Mike Westfield and Isbella Westfield were stealing five-gallon buckets of his plumbing fixtures and fittings and were putting them in the car, and that he fired to stop the theft when Mike ignored his demand to stop. His attitude in the whole matter is expressed in his testimony: "I think it is an awful good remedy. It must have got the job done." Mike and Isbella denied that they were stealing or loading any of the defendant's property into the car. The defendant did not offer J. B. Martin as a witness, whom he had hired to help him protect his property, and was most reluctant to give his namegiving as his reason that Martin did not want to become involved. His failure to produce Martin as a witness, without any showing of an effort to do so, gives rise to a presumption that if he had been presented as a witness his testimony would have been unfavorable to the defendant. Ford v. State, 184 Tenn. 443, 449, 201 S.W.2d 539; Harless v. State, 189 Tenn. 419, 422, 225 S.W.2d 258. Clearly, the defendant has failed to carry the burden of demonstrating here that the evidence preponderates against the verdict of the jury and in favor of his innocence. In his third and fourth Assignments of Error, the defendant insists that the trial court erred in not allowing him to testify about his property having been continually stolen over a period of time and that he had been unsuccessful in obtaining the protection of the law from the Sheriff. Notwithstanding that the trial court repeatedly sustained objections by the State to such testimony, defense counsel persisted in pursuing the matter through interrogation of the defendant and thereby effectively presented those contentions before the jury. Moreover, those matters were wholly irrelevant and immaterial and such testimony was incompetent and inadmissible for any purpose, as the trial court correctly held. 488 The defendant's fifth and sixth Assignments of Error may be considered together. His fifth Assignment is that the trial court erroneously charged the jury: "Gentlemen, no one has the right to shoot another man for the commission of larceny or the commission of any other crime. The law never allows any man to judge or inflict any punishment upon his fellow man, except by due process of law. * * * * * "A person does not have the right to shoot another or inflict punishment upon another under the guise of making an arrest or under the guise of self defense. "A person does not have a right to shoot another person merely in the protection of his property, but if a person came to rob the owner and the larceny was directed more toward the person than to the property, a person may shoot in self defense. That is, he may be justified in shooting if in protecting his property he also was protecting his self." The sixth Assignment relates to the refusal of the trial court to charge the following special request: "If you find that Mike Westfield was in the actual perpetration of a felony, that is that of larceny, when he was shot, then you should acquit the defendant." There is no merit in these last two Assignments of Error. They represent some misunderstanding of the applicable law. In 40 C.J.S. Homicide § 93, p. 954 it is said: "An assault with intent to kill cannot be justified on the ground that it was necessary to prevent a mere trespass on property or to expel an actual trespasser; but a person may lawfully repel force by force in defense of his property against one who manifestly intends, by violence or surprise, to commit a felony against it, and, if in making such defense, he takes or endangers the life of the felonious aggressor, the act is justifiable. Under a statute regarding resistance of an attempt to take property by `force,' an attempted taking need not be by violence to justify forcible resistence. "The owner may lawfully use such reasonable force as is necessary, under the circumstances, to protect or regain possession of his property, and what is such reasonable force is a question of fact for the jury. The criterion of necessity which justifies the use of force by the property owner in defense of his property is the apparent, and not the actual, necessity, and he will be judged by what a reasonably prudent man in good faith would do under such circumstances." The subject is treated as follows in 40 Am.Jur.2d, Homicide, § 180, pp. 464-465: "While the law justifies or excuses the taking of life when necessary to prevent commission of felonies, or in the defense or protection of one's dwelling house or habitation, one cannot defend his property, other than his habitation, to the extent of killing or inflicting serious bodily injury upon the aggressor for the mere purpose of preventing a trespass, where the invasion is made without force. While a person aggrieved by a trespass may repel the intruder by such force as may be reasonably necessary, short of taking human life or inflicting great bodily harm, only in extreme cases can one endanger human life or do great bodily harm in defense of property. Although there is a suggestion in some of the cases that a destruction of property may justify a taking of life, or that the right to take life in defense of property may be a `natural right,' yet its exercise is limited strictly to the prevention of forcible and atrocious crimes. "In general, it may be said that the law countenances the taking of human life in connection with the defense of property only where an element of danger to the person of the slayer is present or where the slaying is necessary to prevent the 489 commission of a felony. While an owner or lawful occupant of land may resist a forcible trespass upon the land, he cannot justify or excuse his act in killing another to prevent a mere trespass upon his property other than his habitation if the homicide was not necessary to prevent a forcible felony, or to protect his own life from the trespasser, or if the trespass was unaccompanied by any attack on or danger to the person of the slayer." The law upon this subject is also given in 1 Wharton's Criminal Law and Procedure, Homicide, § 223, pp. 492, 495: "Except to the extent that one may kill to prevent the commission of a felony by force or surprise, or to apprehend a felon, or in defense of one's habitation or place of business, there is no right, apart from statute, to kill in defense of property. While an owner may lawfully use reasonable force to protect his property, if he kills in order to protect it, he is guilty of felonious homicide. * * * * * "While a man may use as much force as is necessary in the defense of his property, it is generally held that in the absence of the use of force on the part of the intruder, he is not justified in the use of such force as to inflict great bodily harm or to endanger life." (Ibid. p. 495) This subject is further elaborated in the author's treatment of assault, battery and mayhem in 1 Wharton's Criminal Law and Procedure, § 354, p. 708: "A person may use as much force as is reasonably necessary in the defense of his property. It is generally held that in the absence of the use of force on the part of the intruder, the owner is not justified in inflicting great bodily harm or endangering life. "The preservation of human life and limb from grievous harm is of more importance to society than the protection of property. Hence, when a person uses more force in the protection of his property than the circumstances of the attack thereon warrant, he is chargeable criminally for the assault and battery." The rule is also stated in 1 Warren on Homicide, § 141, p. 614, in the author's treatment of the subject of assault with intent to kill: "One in possession of land may resist an entry thereon. But an assault with intent to kill can not be justified on the ground of its necessity in defense of property, unless there is a statute making it justifiable. Therefore it is no defense to a prosecution for assault with intent to murder that the one assaulted was a trespasser on the grounds of defendant." The defendant does not claim that he was being robbed, nor that Mike Westfield was threatening or attempting to assault or inflict upon him any bodily harm, nor that he acted in his own necessary self defense. Clearly, the jury rejected the defendant's contention that Mike Westfield and those with him were stealing and carrying off his property. However, even if they were doing so, the defendant acted unlawfully and became criminally liable when he deliberately shot Mike Westfield with a shotgun as he entered his vehicle and again after he was seated inside. And when the defendant did so, under the facts and circumstances shown in this record, there can be no doubt that he thereby intended to kill the boy then and there. All Assignments of Error are overruled. The judgment of the trial court is here modified by vacating that portion thereof which erroneously rendered the defendant infamous. T.C.A. § 40-2712. As thus modified, the judgment of the trial court is affirmed. GALBREATH, J., did not participate in this case.
Muhammad, who played the last three games for Minnesota, was coming off an injury that kept him out of the lineup for 16 consecutive games. Muhammad's season ends averaging 13.5 points and 4.1 rebounds in 38 games. Timberwolves' Shabazz Muhammad out for Sunday's game by Shawn Krest \| CBSSports.com (2/21/15) Timberwolves F Shabazz Muhammaddidn't make the trip to Houston for Sunday's game against the Rockets, according to StarTribune.com's Kent Youngblood. Muhammad played Friday with a sprained middle finger. He scored seven points in 13 minutes, but the finger will keep him out of Sunday's game. Muhammad is averaging 13.6 points and 4.1 rebounds in 37 games. Timberwolves' Shabazz Muhammad (finger) set to play Friday by R.J. White \| CBSSports.com (2/20/15) Timberwolves small forward Shabazz Muhammad is set to play Friday against the Suns, the team announced. Muhammad was listed as questionable for Friday's game due to a sprained middle finger. He averagd 13.6 points and 4.1 rebounds in 37 games during his second season. The Timberwolves are 1 1/2-point favorites at home Friday. Timberwolves' Shabazz Muhammad questionable Friday Muhammad missed about a month before the break due to an oblique injury but scored 24 points and grabbed 12 rebounds in the team's final two games heading into the All-Star festivities. He's averaged 13.6 points and 4.1 rebounds in 37 games. 03/03/2015 12:2110 players under 20 percent At the Fantasy Basketball Today blog, Chris Towers looks at the lower end of the Fantasy tiers for players who might help out down the stretch.
Allergies in Children Published on May 21, 2019 Children with allergies Allergies are very common in children. It is estimated that up to 40% of children in Australia and New Zealand will be affected by allergies at some point in their lives. The most common allergic conditions are food allergies, eczema (skin allergy), and hayfever1. Why do children develop allergies? Children with allergies have an immune system that is overactive. It has at some stage mistakenly identified something harmless, such as dust mites or pollen, as a threat. After exposure to this ‘threat,’ the body tries to defend itself by producing antibodies and substances such as histamine. It’s the excessive production of histamine that causes the symptoms of allergies. How do I know if my child has allergies? Allergy symptoms are different for every child. Symptoms in children can be unpredictable and each allergic reaction can differ in severity. An initial mild reaction does not mean that all future reactions will also be mild. Allergy symptoms from airborne allergens Allergies caused by airborne allergens such as mould spores, animal dander, dust mites and pollen are very common. They are known as allergic rhinitis and these types of allergies usually develop during childhood and may start to diminish later in life. Typical symptoms of allergies caused by airborne allergens can include: Sneezing Itchy nose Itchy throat Blocked nose Runny nose Coughing Itchy, watery and/or red eyes – known as allergic conjunctivitis Puffy blue tinged skin around the eyes – sometimes known as “allergic shiners” Allergy symptoms from food, medicine, or insect venom Children with allergies to food, medicine or insect venom may experience the following symptoms: Remove soft toys from the bedroom or at least wash them in very hot water weekly. Consider replacing carpet with hard flooring like wood, tiles, or linoleum if possible. Antihistamines Give your child a suitable antihistamine to help relieve the symptoms of hayfever allergies. Telfast Oral Liquid is an oral liquid antihistamine that is recommended for children between the ages of 2 and 11 for hayfever, and provides fast‐acting non‐drowsy relief from sneezing, a runny and itchy nose, itchy and watery eyes and an itchy throat. It is gluten and lactose free and comes in a raspberry flavour that children will love. Contact your healthcare professional if you have any questions or concerns, your child becomes worse, or if your child’s hayfever symptoms are interfering with school or their day to day activities after two days of taking antihistamines. Purchase now from selected retailers The liability of the Company or of SANOFI shall not be incurred by a third party site that can be accessed via the Site. We do not have any way of controlling the content of such third party sites which remain entirely independent of the Company. Moreover, the existence of a link between the Site and a third party site does not under any circumstances mean that the Company approves the content of that site in any way whatsoever and in particular the use that may be made of it. Contact Us If you’d like to know which product might be suitable for your needs, or if you have questions relating to any of our products, please fill in the form below. We’d love to hear from you. To speak with a team member or report an adverse event, please call 1800 818 806 within Australia. Thank you for your submission. A representative will be in contact shortly. Yes! I want to receive future offers, communications and product information from Telfast.
497 S.W.2d 422 (1973) Virginia HARRIS and Jerry L. Reese, Administrator of the Estate of Ruth Reese, Deceased, Appellants, v. Ross W. THOMPSON, Jr. and William B. Sechrest, Appellees. Court of Appeals of Kentucky. June 29, 1973. 424 Charles V. Collins, Lexington, Richard M. Compton, Georgetown, for appellants. V. A. Bradley, Jr., Bradley & Bradley, Virgil F. Pryor, McKnight & Pryor, Georgetown, Ross Thompson, Jr., pro se, for appellees. PALMORE, Chief Justice. Just before the break of dawn on February 16, 1968, an automobile being driven by the appellee William B. Sechrest, Jr., southward on U. S. Highway 25 from his home in Georgetown toward his place of employment in Lexington passed over a patch of ice on the road, went out of control, turned over and struck two ladies standing by a fence near the east or northbound side of the highway. One of the ladies, Mrs. Reese, was killed and the other, Mrs. Harris, was severely injured. Mrs. Harris and the administrator of Mrs. Reese's estate brought suit against Sechrest and the appellee Ross W. Thompson, Jr., who was the owner of a house on the west side of the highway. The claims against Thompson proceeded on the theory that the ice on the road was formed of water that had escaped from a broken pipe in his house. A jury returned a unanimous verdict for Sechrest and a nine-man verdict for Thompson. The administrator and Mrs. Harris appeal from the ensuing judgment dismissing their complaints. They assert prejudicial error by the trial court in the following respects, which (except for point 7, which may not and need not 425 arise upon retrial of the case) we shall discuss in order: 1. In giving a contributory negligence instruction. 2. In giving an unavoidable accident instruction. 3. In allowing evidence of other accidents at the same place as the accident in question. 4. In denying appellants a directed verdict. 5. In denying a motion for new trial on the ground that the verdict was not supported by sufficient evidence and was contrary to law. 6. In failing to define agency in an instruction relating to the possible negligence of Thompson's brother in looking after the house in which the water pipe broke. 7. Misconduct of Sechrest's counsel. Mrs. Harris and Mrs. Reese had left Georgetown at or shortly after 6:30 A.M. in a car driven by Mrs. Ella Moore. They were on their way to work in Lexington. At a point on U.S. Highway 25 about three miles south of Georgetown the road crosses a bridge known as Three-Mile Bridge and then slopes upward to the crest of a hill. The Thompson house and the intersection of a road called Coleman's Lane are situated on the west side of the highway between the bridge and the crest of the hill. U.S. 25 is a two-lane highway. According to a deputy sheriff, the ice patch was in the southbound lane of travel only, was "a little bit this way toward Georgetown" from the Thompson house, and ran to the center of Coleman's Lane. There was other testimony, given by Thompson, to the effect that on the west side of the road about half way between the bridge and the crest of the hill there were an old rock quarry and a wet-weather spring from which water sometimes drained onto the highway. When Mrs. Moore approached this area it was dark and her headlights were on. She said she was travelling at 40 to 45 m. p. h. but could have been going 50 m. p. h. The road was dry. She did not see the ice until she was "right on" it. Her car went out of control on the ice, spun around and came to rest facing back toward Georgetown. According to Mrs. Moore, her car stopped on the west side of the highway with two wheels off the pavement in a shallow ditch bordering the highway, but according to Mrs. Harris it came to rest on the opposite or northbound side of the highway. All three of the ladies got out of the car to see what had happened to them. Mrs. Moore said the car had travelled some 200 feet to 300 feet beyond the point at which it went out of control, though Mrs. Harris testified it had slid only "20 to 25 feet." At any rate, Mrs. Moore got back in the car and drove it toward Georgetown in order to find a convenient turning place and then come back to pick up Mrs. Reese and Mrs. Harris. Meanwhile, Mrs. Reese and Mrs. Harris walked down the side of the road toward Lexington for a distance of about 100 feet, "wanting to get away from the ice," and got over next to the fence. While they were in this position Sechrest's automobile approached from the north, hit the stretch of ice, went out of control, crossed the highway and struck Mrs. Reese and Mrs. Harris, and careened through the fence. According to the evidence for the plaintiffs, the place at which the two ladies were struck was about 320 feet from the southerly end of the ice slick. The deputy sheriff estimated that it was 160 feet from the ice to the crest of the hill and another 160 feet to the place at which the Sechrest automobile went through the fence. An engineer appearing for Sechrest testified that a person standing beside the fence at a point 336 feet south of Coleman's Lane and looking to the north could see approaching vehicles the entire distance from a point north of Three-Mile Bridge. Mrs. Harris did not, however, see the Sechrest 426 car approaching and had only a "vague recollection" of seeing it just before she was hit. Sechrest testified that the accident happened about 6:45 A.M., that although dawn was beginning to break it was still dark and his lights were on, and that he was travelling at a speed of 45 to 50 miles per hour. He did not see the ice, did not know it was there, said he had never seen any ice or water there before, and did not slow down. He estimated that he was 5 or 6 feet north of Coleman's Lane when he realized he had hit something, after which he was unable to bring his car back under control. He did not see the two ladies before they were struck. The speed limit was 50 m. p. h., although road signs were present indicating for southbound travelers that there was a curve and a side road. All of the witnesses agreed that this was a very hazardous stretch of highway even under normal conditions. The appellee Thompson testified that he owned a house situated on a bank at the southwest corner of U.S. 25 and Coleman's Lane. It had been rented out for about three months to a man named Stone, who on February 1, 1968, had paid the rent for the month of February. Thompson had not been on the premises since some time in January. On the day of the accident he learned from his brother, Burnley Thompson, who lived a quarter of a mile up the road (U.S. 25), that Stone had moved out and that a water pipe in the house was broken. Neither Ross Thompson nor his brother Burnley had any previous knowledge or notice that the water pipe was broken. Burnley usually "watched over" his brother's property "just . . . to help him out," but had not been hired and was not under any contractual obligation to do so. Some "two or three days" before the accident he noticed that the window curtains had been taken down and heard from someone in town that the tenant had moved, but he made no investigation of the premises and did not at that time communicate this information to Ross. The tenant had never complained about the condition of the house, and there was no evidence that any of its water pipes had been frozen or broken before. There were several possible sources from which water could have flowed onto the highway near the mouth of Coleman's Lane, but the evidence was sufficient to support a finding that at least some of that which formed the ice patch on February 16, 1968, had come from the broken pipe in the Thompson house. Other pertinent evidence will be mentioned as it becomes appropriate in connection with our analysis of the questions presented on the appeal, which follows: 1. The contributory negligence instruction. According to the testimony of the engineer, the fence by which the two victims of this tragic accident were standing when struck by the Sechrest vehicle was less than 10 feet from the paved surface of the highway. Even though they may have been in a better position to anticipate the accident, having just experienced a similar though less serious misadventure, it is difficult to see any possible theory of negligence on their part except that during the brief interval in which they were awaiting Mrs. Moore's return they should have gone back toward Georgetown, placing themselves between the highway and oncoming traffic from the north, rather than toward Lexington. To demand such sagacity on the part of two ladies stranded in the dark on the side of the highway would, in our opinion, be too much. It is our conclusion that the instruction should not have been given. It will be recalled that the jury returned a unanimous verdict for Sechrest and only a nine-man verdict for Thompson, which indicates, say the appellees, that the verdicts were not based upon contributory 427 negligence and that the error was not prejudicial. It may be improbable that the verdicts were affected by the contributory negligence instruction, but assuredly it is not impossible, and we cannot speculate as to whether some of the jurors who voted with the majority did so on the basis of contributory negligence. Consequently, it is necessary that a new trial be granted. 2. The unavoidable accident instruction. In Sloan v. Iverson, Ky., 385 S.W.2d 178, 179 (1964), we expressed "serious doubt whether an `unavoidable accident' instruction should ever be given in an automobile collision case." Proof that an accident was unavoidable is neither more nor less than proof of non-negligence. It does not call for any modification or qualification of the duties applicable under the circumstances of the case. The instruction here under attack reads as follows: "If the jury believe from the evidence that the defendant, Sechrest, while operating his automobile in a careful and prudent manner within the meaning of these instructions, lost control of his automobile as a result of suddenly coming upon ice on the highway which he did not see and could not in the exercise of reasonable care have seen in sufficient time, and therefore lost control of his automobile so that he did not and could not with reasonable care have regained control of same in time to have avoided the accident then and in that event the law is for the defendant Sechrest and the jury should so find." The propriety of this instruction must be determined in relation to the preceding instruction, which enumerated the specific duties embraced within Sechrest's general duty of ordinary care in the operation of his automobile. One of these specific duties was "to have his automobile under reasonable control and not to operate it at a greater speed than was reasonable, considering the condition and use of the highway, insofar as the condition of the highway was known to him or should have been known to him in the exercise of reasonable care." This was appropriate to the circumstances of the case in conditioning Sechrest's duties upon what he should have known with respect to the existing condition of the highway. Cf. Tirey v. Tirey, Ky., 420 S.W.2d 564, 566 (1967). The next following duty, which was to operate his automobile on the right-hand side of the highway "and not to pass over the center medium [sic] at the point where the accident herein occurred," was not modified so as to allow for an unanticipated emergency which, without fault on his part, may have prevented his so doing. As given in this instruction the duty was absolute, hence in the absence of some qualifying language appearing elsewhere in the instructions it would have amounted to a directed verdict against Sechrest, because obviously he did cross over the center line of the highway on the way toward striking the two pedestrians. The separate instruction quoted above had the effect of providing the essential qualification of this specific duty imposed upon Sechrest. It told the jury in effect that if without negligence Sechrest was confronted with a sudden emergency which prevented his avoiding the accident by the exercise of reasonable care he was not liable. It is therefore our opinion that the instruction was not improper, though in the event of a new trial it is recommended that the sudden emergency principle be cast in the form of a qualification immediately following the enumeration of the specific duties of ordinary care and as part of the same instruction, along the following lines: ". . . all of the foregoing duties being subject however, to this qualification: that if immediately before the accident the defendant was suddenly and unexpectedly confronted with an emergency 428 by the presence of ice on the surface of the highway and such emergency was not brought about by any failure on his part to perform the duties above set forth, he was required thereafter to exercise only such care as an ordinarily prudent person would exercise under the same conditions and circumstances." The appellants evidently feel that our opinions in Jones v. Carr, Ky., 382 S.W.2d 853 (1964), and Sloan v. Iverson, Ky., 385 S.W.2d 178, 179 (1964), and possibly Hettrick v. Willis, Ky., 439 S.W.2d 942, 944 (1969), have eroded the effect of Hall v. Ratliff, Ky., 312 S.W.2d 473 (1958), in which the defendant's vehicle had struck a patch of ice and slid off the road and this court held he was entitled to a sudden emergency instruction. Perhaps a good deal of confusion on this subject has arisen from a failure to distinguish between the theories of sudden emergency and unavoidable accident, and also from a tendency to pay too much regard to the label instead of the basic theory. As we have indicated, that an accident was unavoidable merely negates the existence of negligence as a causative factor. As such, it does not require an instruction, and the giving of one has the possible prejudicial effect of placing undue emphasis on the defendant's evidence tending to explain why he was not negligent. But when a defendant is confronted with a condition he has had no reason to anticipate and has not brought on by his own fault, but which alters the duties he would otherwise have been bound to observe, then the effect of that circumstance upon these duties must be covered by the instructions. It is not a matter of defense which must be pleaded (though in fact it was pleaded in Hall v. Ratliff), but a matter of what his duties were under each state of facts inferable from the evidence, and it is of course the plaintiff's burden to persuade the jury as to which of those factual states existed. To summarize this phase of the discussion, whether the instruction on a motorist's duties should be qualified by a proviso such as the sudden emergency theory does not depend upon whether the particular circumstance might be characterized in common parlance as a "sudden emergency," but whether it changes or modifies the duties that would have been incumbent upon him in the absence of that circumstance. In this case the qualification was made necessary because by not remaining on the right side of the road Sechrest violated a specific duty unless the exceptional circumstance of the ice on the road had the effect of relieving him from it. Had the accident taken place in his own lane of travel, or on the right side of the highway, it would not have been necessary, because then the unexpected presence of the ice would have amounted to no more than a condition bearing upon the question of whether the accident resulted from a failure on his part to comply with the more generalized duties of ordinary care. The proper criterion is whether any of the specific duties set forth in the instruction would be subject to exception by reason of the claimed emergency. We do not accede to the appellants' argument that under the cases above cited the presence of ice on a highway cannot be the occasion for inclusion of the sudden emergency theory in the instructions. 3. The evidence of other accidents. At 4:45 A.M., some two hours before the accident in question, the appellee Sechrest's witness Roy Pollett, driving southward at a speed of 40 m. p. h. with his headlights on, had encountered the ice in the vicinity of Coleman's Lane and lost control of his automobile, which went off the highway, turned around and struck a telephone pole. He did not see the ice before he was upon it. A few minutes after the Sechrest accident the witness Gail Tackett, also driving southward at the same place and at a speed of 40 m. p. h., had a similar experience, 429 and had not observed the ice before he skidded. Another witness, W. J. Jones, whose automobile was struck from the rear by the Tackett vehicle, gave testimony substantially to the same effect. The appellants objected to all of this evidence of other accidents, claiming among other arguments that they were not shown to have occurred under similar circumstances (for example, speed) as the Sechrest accident. Evidence of the occurrence or nonoccurrence of other accidents or injuries under substantially similar circumstances is admissible when relevant to certain limited issues, such as the existence or causative role of a dangerous condition, or a party's notice of such a condition. McCormick on Evidence (2d ed.) § 200; Wigmore on Evidence (3d ed.) § 458; 29 Am.Jur.2d, Evidence, §§ 305 et seq.; and Annotations at 31 ALR2d 190 and 70 ALR2d 167. Most usually, as indicated by the authorities collected in these texts, the case in which it is admitted involves a claim against the person or persons responsible for the condition alleged to have been the cause of the accident. In the instant proceeding it was of course unnecessary for these particular purposes, because there was no real issue as to whether the patch of ice on an otherwise dry highway constituted a dangerous condition or whether that condition was a causative factor in the accident, and there was no contention that any of the accidents were relevant to the issue of notice. Hence the only purpose that could have been served by this evidence was to show whether Sechrest was negligent by comparison with the experience of other persons under similar circumstances. As we all recognize, in a negligence case the comparison to be made is between the party alleged to have been negligent and that imaginary ideal, the ordinarily prudent person acting under similar circumstances. Without any way to prove or to judge whether another person who did or did not have an accident at the same place and under the same circumstances was himself an ordinarily prudent person, or was above or below average in that respect, we are forced to the conclusion that such evidence cannot be competent on the narrow issue of negligence.[1] Cf. 29 Am.Jur.2d, Evidence, § 308. Our opinions on the subject disclose a certain degree of ambivalence. The general rule stated in Louisville & N. R. Co. v. Loesch, 215 Ky. 452, 284 S.W. 1097, 1100, 47 A.L.R. 347 (1926), is "that evidence of a previous accident at the same place is incompetent upon the investigation of the cause of a subsequent one." Yet in Louisville & N. R. Co. v. Jackson's Adm'r, 250 Ky. 92, 61 S.W.2d 1104, 1105 (1933), and Phelps v. Henkels & McCoy, Inc., Ky., 435 S.W.2d 83, 86 (1968), "the absence of contributory negligence on the part of plaintiff's decedent" was cited as an issue on which such evidence may be admitted. In neither of those cases, however, was it necessary so to decide, because in each of them the evidence was admissible as tending to show both the dangerous tendencies of the condition and notice to the defendant who was alleged to be responsible for it, and to the extent that they stand for the proposition that the occurrence or nonoccurrence of other accidents may be shown for the sheer purpose of comparison with the conduct of a party alleged to have been negligent in a similar accident they are overruled. Although we hold the evidence of other accidents inadmissible on the issue of Sechrest's negligence, vel non, its reception by the trial court was not only justified but made necessary by testimony introduced earlier, over objections of the appellees, by the appellants themselves. For example, the witness Libby Abrams testified that shortly before the accident 430 she had driven over the same patch of ice but was able to see it in time to reduce her speed from 45 m. p. h. and pass over it without accident. The witness Luther Mason came along after the accident, did not notice the ice at all, topped the crest of the hill at 40 to 50 m. p. h. without having skidded, and then stopped upon seeing a lady he knew standing by the side of the road. The witness Frank Sargent drove past the scene of the accident at 7:30 or 8:00 A.M., in the daylight, at a speed of 40 to 50 m. p. h., saw the ice at a distance of 50 to 60 feet, continued across it without slackening speed, and had no trouble. Four other occupants (one of them being the husband of Mrs. Harris) of a car that drove by in the same direction within minutes after the accident were permitted to testify that they saw the ice from a distance of three or four car lengths and that the driver slowed down from 40 to 45 m. p. h. to 5 m. p. h., or "as much as possible," and passed on with no difficulty. The admission, over Sechrest's objection, of this array of evidence to the effect that some people got by without accident entitled him to rebut it by evidence that other motorists were not so fortunate. Claycomb v. Howard, Ky., 493 S.W.2d 714, 717 (1973). In short, the appellants, having opened the book on the subject, were not in a position to complain when their adversaries sought to read other verses from the same chapter and page. We do not perceive any substantial dissimilarity in the circumstances. 4. Denial of a directed verdict. This contention is based on the argument that as a matter of law Sechrest was negligent and Mrs. Reese and Mrs. Harris were not. As heretofore indicated, we conclude that there was no submissible issue of contributory negligence, but we cannot agree that Sechrest was negligent as a matter of law. 5. Denial of the motion for a new trial. The argument on this point runs along the same lines as the contention that the appellants were entitled to a directed verdict. With respect to Thompson, it is said that under the rule of Rylands v. Fletcher, LR 1 Exch. 265, LR 3 HL 330, 4 Hurlst & C 263, 1 Eng.Rul.Cas. 236, the owner of premises from which water escapes and damages another is liable as a matter of law. The same argument was made and found wanting in United Fuel Gas Co. v. Sawyers, Ky., 259 S.W.2d 466, 38 A.L.R.2d 1261 (1953). See Louisville Refining Company v. Mudd, Ky., 339 S. W.2d 181 (1960). Certainly by no stretch of the imagination could the maintenance of water pipes and a system of running water be considered an unreasonable use of property within the nuisance theory. Accidental damage resulting from an unanticipated malfunctioning of the system could be actionable only on the theory of negligence, and in such a case the test of liability is whether by the exercise of ordinary care the defendant should have foreseen and provided against the misadventure or should have discovered and remedied it. Cf. Stein v. Louisville Water Co., Ky., 249 S.W.2d 750, 752 (1952). There was no basis in this case for holding Thompson liable as a matter of law. A better question, which we do not reach, is whether the converse is true, and a verdict directed for Thompson. Sechrest, the appellants submit, is liable as a matter of law under the doctrine of res ipsa loquitur, but there is no such magic in that doctrine, at least as it is conceived in this jurisdiction. See discussion in Bell & Koch v. Stanley, Ky., 375 S.W.2d 696 (1964). The effect in this case was the same as in Vernon v. Gentry, Ky., 334 S.W.2d 266, 268, 79 ALR 2d 1 (1960), except that the jury found the other way. That is, the facts were sufficient to entitle the plaintiffs to a directed verdict 431 in the absence of an explanation by the defendant tending to rebut the prima facie case of negligence arising from the evidence adduced by the plaintiffs, but Sechrest's explanatory evidence had the effect of reducing it to a permissible inference. The burden being on the plaintiffs to prove negligence, it was necessary for them to persuade the jury that the presence of ice on the highway did not exculpate him. Again, we reject the contention that as a matter of law an automobile cannot slide across a highway without negligence on the operator's part. 6. The failure to define agency. An instruction was given to the effect that if the accumulation of ice on the highway resulted from the flow of water from the Thompson house, that Thompson knew or by the exercise of ordinary care could have known of the danger thus created, and that his failure to prevent or remedy it was a proximate cause of the accident, the jury should find against him. The same instruction went on to say that he was liable also for a similar negligent failure on his brother Burnley's part if the jury should believe from the evidence that Burnley was his agent for the purpose of inspecting the premises. There was no objection to this instruction. However, after retiring to deliberate on the case the jury asked the trial court, in the presence of counsel, for a definition of agency as mentioned in the instruction. The court declined to give it. Again no objection was presented, nor was any question raised by counsel until the motion for new trial was made. Without implying that there was enough evidence to submit the case against Thompson to the jury in the first place, we think the obvious answer on this point is that if the appellants were not satisfied with any phase or portion of the instructions the time to speak was before they were given to the jury. C.R. 51. We perceive no good to be accomplished by departing from the letter of the Rule. In conclusion, it will be noted that it may very well have been unnecessary to reverse this judgment if it had been possible to tell whether the verdict was based to some extent upon contributory negligence. When instructions are given on both negligence and contributory negligence, this type of "blind spot" can be forestalled by using a form of instructions in which, following the enumeration of each party's duties, the jurors are required to answer the following interrogatory: Do you believe from the evidence that ______ failed to comply with any one or more of these duties? YES NO (Strike one) If your answer is "Yes," do you further believe from the evidence that such failure was a substantial factor in causing the accident? YES NO (Strike one) See C.R. 49.01 and note, The Case for Interrogatories Accompanying a General Verdict, 52 Ky.L.J. 852 (1964). The judgment is reversed and the cause remanded for a new trial. PALMORE, C.J., and MILLIKEN, OSBORNE, REED, STEINFELD and STEPHENSON, JJ., sitting. All concur. NOTES [1] Obviously the same argument can be made with respect to the issue of whether a particular condition was abnormally dangerous, but that question is not before us in this case.
Hey! So let's A some Qs! Tara Carson asks about roommate relationships. (On-screen comment: What advice do you have for roommate relationships? How to pick a roommate, or just how to get along?) Now I've only had a roommate in the context of a college dorm where we had to fill out a roommate agreement at the beginning of the year à la Sheldon Cooper. Although we kind of scoffed at it at the time, I think the basic principle of a roommate agreement is a really good idea. And if not drafting it all up formal-like, at least discussing some of that stuff at the beginning can be very helpful. (On-screen text: For example...) How often do we clean the bathroom/kitchen/shared common areas? What's our policy on overnight guests? Do we have quiet hours? Can we use each others stuff/eat each others food, with permission, without permission, never? Above all, keep the lines of communication open, if your roommate's 2am Bohemian Rhapsody Air Guitar seshes are driving you a little bit crazy. (sings tune of bohemian rhapsody). Let them know instead of letting it build up and fester and just passive-aggressively drinking their orange juice. kbwm1212 asks for advice on how to flirt. (on-screen comment: This is probably really embarrassing, but I have come to the realization that I am an adult and I don't know how to flirt. Granted, I rarely meet guys that I WANT to flirt with, but if I did, I wouldn't know what to do! I'm a female seeking a male, so any ideas would be helpful!) Okay, just for funsies, I went to cosmo.com to see what they had to say on this topic, and I found some tips, such as the following. "Check out a cutie on the subway, look at him for two seconds, look away, and then look back through lowered lashes." And that brought up a lot of questions. First of all: do I count these seconds? "One Mississippi, two Mississippi" Is he looking back at me when this is happening? How long do I look away for, and when I look back, I'm pretty sure that if my eyelashes are lowered, my eyes are closed. I have yet to figure out the logistics of that. When it comes to flirting, I am no expert, but I have noticed, in my flirting gambits, that some of the same stuff comes up. I usually try to do three things. 1. Paying the other person compliments, 2. Asking the other person questions and 3. Trying to make the other person laugh. If you haven't seen our episode "How to Ask Someone Out" I will link you to that below. In that episode, we presented the indirect method for asking someone out, and some people in the comments section said "Alright, STOP, collaborate and listen!" Some people thought that the indirect method might be convenient for the asker, but it might not be fair to the askee to not present the situation as a date from the very beginning. And I think that's a very valid point, and I thank you for bringing it up, so if you go and check out that video, make sure to scroll down to the comments section and check out some of the conversation that's occurring there. jassmlim asks are there certain questions I should ask my landlord before I sign a lease? (On-screen comment: Advice on the relationship with your landlord? I'm about to go flatting for the first time in my life and any advice would be wonderful. Are there certain questions I should ask him before I sign my lease? TEACH ME HOW TO ADULT PLEASE!) So this is gonna be an Emma and Mike tag-team duo. Like the roommate question, we're gonna answer this question with a series of questions. How much is a security deposit? Do they want first-month and last-month's rent when you sign the lease or do they just want one of those two? Mike: When you're moving in you should also be clear about the financial obligations you will face if you break your lease, some states you have to pay rent until someone else gets in the apartment, sometimes you just lose a deposit. E: What's the policy on parking, do you have to pay for a space, is there covered parking, if you live in a place where it snows, are they gonna plow the parking lot? What's included in the utilities, do you get water, electricity, sewerage, trash, is there a pet feed? Do you have to pay to have a pet? Can you have dogs over a certain size? M: Before you move in, if it's important to you, you can usually also check crime stats for the neighbourhood online, also in that same vein, I would recommend actually going to the apartment at two different times of day, like one during the day, and then drive past in the evening or at night. On the day of the move-in, you should also get written documentation of any damages there are in the apartment. You don't want to be on the hook to pay for the damages that were already there. bloodandkoolaid asks about building rapport and/or friendship with professors in college. (On-screen comment: Relationship question! Since I am now an undergraduate and you are a graduate student, maybe you can help me out. How do you build rapport and/or friendship with people of way higher status than you (like professors)? I feel like I vacillate between being overly formal and overly intimate and I'm not sure what's appropriate! Is it super weird to ask my supervisor or a friendly professor to coffee? Drinks? Please help!) I actually consulted a professor on this one, and the professor said that probably rather than a friendship, what you should pursue with a professor in college is a mentorship. Make an appointment to meet with your professor outside of class, in their office if they have one, and bring specific things to talk about at that meeting. For example, you could say: "I really liked your lecture on xyz, I would love to talk about it with you more... further." Say it better! Cause this is an academic environment! Professor Consultant also recommended exchanging emails with that professor so that you can keep in touch with them and let them know what you're up to. Professor Consultant finished up by saying, and I quote: "It never hurts to bring a food bribe." Just putting that out there. Starius2 asks "How would I go about asking Jennifer Lawrence out?" E: I've on- I can only think of two, and not even full pick-up lines. M: Okay E: But just something to do with loins catching fire M: (laughs whilst making disgusted face) E: And then something about "I'm an American, but this is no Hustle." M: You're famous, you're Jennifer Lawrence, I'm just X-man, but I'm first class. E: (whispers) Oh my God... (starts laughing) M: (laughs) E: And that's all we've got for you today, if you've got thoughts, comments, questions, more advice, different or better advice on any of the stuff discussed here today, please leave it for us in the comment section below. We love to hear from you! In the meantime, imagine the best catchphrase in the history of spoken word and imagine me saying it right now! E: But we didn't even touch on pick-up lines in our How to Ask Someone out video. M: Have you ever had, like, used one? or.. E: No. M: Had one? No, I've never either. E: No. I mean one time someone was like "we should hang out sometime" which is not like a pick-up line. M: Yeah E: I know it's Josh Sundquist's, like, great way to ask someone out, which I think is a good way to ask someone out. M: Funniest kid I've ever met is my 12 year-old cousin, I play Xbox with him all the time. The first day of first grade, he got detention. Do you wanna know why? E: Yes M: He went up to the lunchlady and said "So, you come here often?" E: (laughs) M: (laughs)
1. Introduction {#sec1-molecules-25-00120} =============== The use of fining agents in winemaking is widely known as a processing aid to clarify, enhance the wine stability, remove off-flavors, and, particularly in red wines, soften sensory properties such as bitterness and astringency by modulating phenolic composition \[[@B1-molecules-25-00120]\]. Traditional fining agents based on proteins of animal origin, including casein, egg albumin, gelatin, and isinglass, are commonly used to remove protein-reactive phenolic compounds \[[@B2-molecules-25-00120]\]. However, color losses and unpleasant aromas can sometimes occur in the wine after the fining treatment \[[@B3-molecules-25-00120]\]. From the health point of view, animal proteins have allergenic or intolerant potential and their residual presence in the wine may pose an important risk in sensitive individuals \[[@B1-molecules-25-00120],[@B4-molecules-25-00120]\]. In this sense, according to EU regulation No. 579/2012 \[[@B5-molecules-25-00120]\], all potentially allergenic fining agents present in the wine at a concentration higher than 0.25 mg/L have to be declared on the wine label. In the last recent years, cultural and ideological aspects are also increasingly influencing the wine market. Thereby, the global increase in vegetarian and vegan consumers has highlighted the necessity to make wines using fining products that represent a possible alternative to animal-derived fining agents. For this purpose, vegetal proteins have been already isolated from cereals, legumes, potatoes \[[@B6-molecules-25-00120],[@B7-molecules-25-00120]\], seaweeds, grape seed extracts \[[@B8-molecules-25-00120],[@B9-molecules-25-00120]\], and yeasts \[[@B10-molecules-25-00120],[@B11-molecules-25-00120]\]. Other vegetal non-proteinaceous products, such as polysaccharide-based agents isolated from cell wall material of fresh apples and grapes or their respective pomaces, have shown great potential for wine fining purposes \[[@B12-molecules-25-00120],[@B13-molecules-25-00120],[@B14-molecules-25-00120],[@B15-molecules-25-00120]\]. Focusing on protein-based fining agents, they can have a different affinity to different types of phenolic compounds. Proteins interact with wine phenolic compounds initially through hydrogen bonding and hydrophobic interactions, forming soluble complexes. In fact, gelatin is a highly efficient fining agent because of its potential hydrogen binding sites \[[@B3-molecules-25-00120]\]. Subsequently, phenolic compounds are removed by precipitation through self-association of the complexes formed \[[@B16-molecules-25-00120]\] or formation of insoluble protein aggregates (cross-linkages between proteins) incorporating the target species \[[@B17-molecules-25-00120]\]. This precipitation induces reduced astringency and depends on the protein to tannin ratio as well as on the amino acid composition, tertiary structure, hydrophobicity, and molecular mass of proteins \[[@B2-molecules-25-00120],[@B18-molecules-25-00120]\]. It is well-known that proline-rich proteins have a strong affinity to phenolic compounds and therefore they are of great interest for wine fining \[[@B19-molecules-25-00120]\]. Concretely, aromatic ring structures of phenolic compounds have hydrophobic properties and the presence of galloylated units enhances the hydrophobic interactions with proline-rich proteins \[[@B20-molecules-25-00120]\]. Among allergen-free vegetal proteins isolated from cereals, corn zeins are a heterogeneous protein mixture made of polypeptides differing in molecular mass, aggregation state, and isoelectric point. The effectiveness of zein as a fining agent could be due to their relative hydrophobicity, which is associated with the high content of non-polar amino acids \[[@B21-molecules-25-00120],[@B22-molecules-25-00120]\]. In Cabernet Sauvignon, Merlot, and Valpolicella red wines, these authors reported that corn protein doses ranging from 5 to 15 g/hL are able to decrease wine turbidity and remove phenolic compounds, such as anthocyanins (−2.90--11.64%) and proanthocyanidins (−5.40--19.26%) after settling for 48 h at 20 °C, in a similar way to animal gelatin while preserving red wine color. Higher variability is found in the scientific literature in wine fining effectiveness for rice-based proteins. They have relatively low proline content and therefore only reduce slightly total tannins and astringency, showing also a low influence on the Cabernet Sauvignon wine color after settling for 48 h at 20 °C \[[@B7-molecules-25-00120]\]. However, the use of molecular mass fractions ranging from 10 to 32 kDa promotes the removal of phenolic compounds that contribute to bitter and astringent perception and, at the same time, preserves the color of Samtrot and Lemberger red wines after storing in the dark at 8 °C for 24 h \[[@B23-molecules-25-00120]\]. Grape-endogenous proteins sourced from grape seed extracts (GSE) have also shown good performance in decreasing Chardonnay white wine turbidity and increasing oxidative stability. In the Raboso red wine, a reduction in some anthocyanins (−1.0--4.0%) and proanthocyanidins (−3.2--5.9%) was observed, similarly to other animal- and vegetal-derived fining agents tested, without markedly affecting wine chromatic characteristics (less than −5%). Nevertheless, the most important impact of GSE corresponds to the sensory attributes, reducing the vegetal notes in the Raboso rosé wine and both acidity and astringency in the Raboso red wine \[[@B8-molecules-25-00120]\]. The dosages used were 5, 10, and 20 g/hL of GSE, corresponding respectively to 2.2, 4.4, and 8.7 g/hL of protein. Despite the effort made by various research groups in relation to the use of vegetal proteins as wine fining agents, few have been commercialized until now. Some of these products are based on Patatin P. It is a major 39--45 kDa glycoprotein from potato, with low proline content, whose fining ability has been demonstrated in red wines. Different studies have evidenced that proteins from potato reduce total tannin content and astringency similarly to gelatin, and even better than other fining agents in Aglianico and Cabernet Sauvignon red wines \[[@B6-molecules-25-00120],[@B7-molecules-25-00120]\]. Nevertheless, these two studies reported contrasting results in relation to the wine color. The use of patatin for wine fining during one week at 14 °C preserves the chromatic characteristics of red wine, even though it removes 30% of monomeric anthocyanins at 30 g/hL dose in Aglianico red wine \[[@B6-molecules-25-00120]\], whereas a significant decrease in the hue parameter is found in Cabernet Sauvignon after settling for 48 h at 20 °C \[[@B7-molecules-25-00120]\]. Therefore, the wine phenolic composition and the fining conditions (dose, temperature, and time) may influence the impact of fining agents. In addition, other studies highlighted that potato protein with molecular mass fractions between 10 and 32 kDa reduces efficiently non-polymeric bitter and astringent marker compounds in red wines, such as caftaric acid, (+)-catechin, and (−)-epicatechin, with a minor impact on color intensity \[[@B23-molecules-25-00120]\]. Other commercialized products are legume-derived proteins extracted from pea, although lentil, soybean, and lupine have been also studied as possible sources of vegetal protein. All of them are efficient fining agents when compared to gelatin, but soybean and lupine are considered allergenic substances \[[@B1-molecules-25-00120]\]. In Aglianico red wine, either young or aged for twelve and twenty-four months, a recent study has reported that pea protein shows limited interaction with proanthocyanidins after settling for 168 h at 20 °C \[[@B18-molecules-25-00120]\]. However, selective enzymatic hydrolysis, reducing the protein size but keeping hydrophobic binding sites intact, could enhance the accessibility to phenolic compounds and therefore improve the fining efficiency of pea protein. Lentil and soybean proteins, followed by hydrolyzed pea protein, remove efficiently proanthocyanidins in red wines (about 30--40% for monomers and 70--80% for proanthocyanidin dimers and trimers). An important advantage of pea protein isolate is the preservation of anthocyanins, whose losses occur and are particularly noticeable in young red wine so color tonality is not influenced by wine fining with pea, lentil, soybean, or gelatin proteins \[[@B18-molecules-25-00120]\]. Other authors confirmed the lower efficiency of pea proteins to remove tannins when compared to gelatin and potato proteins \[[@B7-molecules-25-00120]\], but they evidenced that the three vegetal-derived proteins reduce similarly the astringency of Cabernet Sauvignon wine. Similarly, the pea protein had a minor impact on proanthocyanidins in Catalanesca white wine \[[@B24-molecules-25-00120]\]. In white wine, another study reported that pea protein could be an alternative to potassium caseinate by decreasing similarly total phenols (about −9%) and wine color (about −11% to --12%) at 0.4 g/L dose, being pea protein less efficient at reducing the browning potential \[[@B25-molecules-25-00120]\]. There are few commercialized products alternative to the use of animal allergenic proteins for wine fining and some studies have provided contradictory results. Considering the increasing demand for the production of wines suitable for allergen-sensitized, vegetarian, and vegan consumers, the study aimed to compare the effectiveness of commercialized vegetable proteins derived from pea and potato, and the traditionally used fining agent (i.e., animal gelatin) at two different doses. Concretely, their ability to both remove monomeric, oligomeric, and polymeric flavanols that are involved in the wine astringency perception and preserve anthocyanins and color characteristics was assessed. The study was carried out on four monovarietal red wines, characterized by different phenolic content and composition, to assess the possibility of adapting the fining agent and dosage to a specific type of wine and/or phenolic profile. To our knowledge, no scientific study has been published on vegetal proteins derived from pea and potato for the fining of wines with different phenolic composition. 2. Results and Discussion {#sec2-molecules-25-00120} ========================= The wines were selected on the basis of the different phenolic composition, particularly the content of oligomeric and polymeric flavanols (FRV and PRO, respectively), as well as the content of anthocyanins (TA) (control wines, [Table 1](#molecules-25-00120-t001){ref-type="table"} and [Table 2](#molecules-25-00120-t002){ref-type="table"}). The lower FRV/PRO ratio corresponds to Montepulciano wine (0.38), followed by Primitivo (0.41), and this ratio increases in Syrah and Nebbiolo wines (0.54 and 0.59, respectively). Regarding anthocyanins, Nebbiolo wine is characterized by a low content (112 mg/L) whereas Syrah is the richest wine in these red pigments (367 mg/L), followed by Montepulciano and Primitivo (275 and 255 mg/L, respectively). 2.1. Total Phenolic Compounds and Flavanols {#sec2dot1-molecules-25-00120} ------------------------------------------- Regarding Primitivo wine, total phenolic compounds evaluated through the A~280~ parameter only showed a significant difference for the high dose of GE (gelatin, conventional treatment) with respect to untreated wine (−8.6%, p \< 0.001; [Table 1](#molecules-25-00120-t001){ref-type="table"}). By contrast, using more comprehensive parameters such as the content of polymeric and oligomeric flavanols (PRO and FRV, respectively), a significant decrease was found for several fining treatments when compared to control wine. PRO content was significantly reduced when the wine was treated with gelatin independently on the dose (−10.3% and −13.9%, p \< 0.001 for low and high dose, respectively), as occurred with the PT1 treatment (potato-based) at low dose and PE1 (pea-based) at the two doses studied (−7.8% and −8.8%, p \< 0.001 for PT1L and PE1H, respectively). PE1L treatment reduced both PRO and FRV contents (−7.1%, p = 0.004 and −11.1%, p \< 0.001, respectively), and it was the treatment that most significantly affected FRV. This decrease could be exploited in wine production because low molecular fractions of flavanols contribute greatly to wine bitterness \[[@B26-molecules-25-00120]\]. In Montepulciano wine, even if no significant differences in total phenolic compounds (assessed by A~280~) occurred using different fining agents, variations in the flavanol content were found for both FRV and PRO values ([Table 1](#molecules-25-00120-t001){ref-type="table"}). GE treatments significantly reduced both parameters with respect to the control (−12.1% and −18.9%, p \< 0.001 for PRO, and −7.1%, p = 0.006 and −17.9%, p \< 0.001 for FRV, for low and high dose, respectively), highlighting their effectiveness. The only vegetal fining agent that allowed a decrease of both PRO and FRV contents was PT1, giving a significant removal of PRO (−11.6%, p \< 0.001 and −9.5%, p = 0.006 for low and high dose, respectively) and FRV (−11.2% and −12.8%, p \< 0.001 for low and high dose, respectively). Instead, PE1 treatments resulted to be effective mainly on the reduction of low molecular flavanol fraction (FRV, −7.0%, p = 0.006 and −10.8%, p \< 0.001 for low and high dose, respectively) while PRO contents showed a non-significant decrease when compared to control wine. In the case of Syrah wine, total phenolic compounds (A~280~) determination after wine fining unveiled a significant decrease only using the high dose of gelatin with respect to the control (GEH, −8.0%, p = 0.002; [Table 1](#molecules-25-00120-t001){ref-type="table"}). However, the more detailed flavanol estimation using PRO and FRV parameters evidenced, for GE and PT2 at high dose, a decrease of both PRO (−12.6%, p \< 0.001 and −6.7%, p = 0.004, respectively) and FRV (−9.9%, p \< 0.001 and −9.2%, p = 0.003, respectively). In addition, PE2 and PT1 at high dose, as well as PE1 at a low dose, had a significant impact only on PRO contents (−11.7%, −15.3%, and −7.4%, respectively, p \< 0.01), the second fining condition (PT1H) presenting the highest PRO decrease observed among treatments. This aspect is of particular relevance because wine astringency is primarily driven by polymeric flavanols \[[@B26-molecules-25-00120]\]. Moreover, the FRV parameter showed a significant decrease (−10.0%, p \< 0.001) also after PT1 treatment at a low dose when compared to the control wine. The main differences found in this study concerned Nebbiolo wine because it was strongly affected by the different fining treatments in terms of A~280~, PRO, and FRV (all p \< 0.001; [Table 1](#molecules-25-00120-t001){ref-type="table"}). The unusual phenolic profile of this wine, which is characterized by a limited anthocyanin content and a typically high flavanols content with respect to the other varieties evaluated, undoubtedly played a major role in this outcome. A significant reduction of A~280~ was observed after all treatments (from −3.3% up to −8.6%, p \< 0.01). In fact, this decisive reduction of A~280~ could be justified by the removal of oligomeric and polymeric flavanols (FRV and PRO, respectively). The higher decrease in A~280~ with respect to the control wine was found for GE treatment at both low and high doses and for PT1 at high dose (−7.5%, −8.6%, and −7.4%, p \< 0.001 for GEL, GEH, and PT1H, respectively). In accordance with the A~280~ parameter, PRO content was significantly reduced by all treatments with respect to the control (from −5.8% up to −15.4%, p \< 0.01) with the exception of PT2 at both low and high doses. In particular, GEH was the most effective fining agent in the removal of both PRO and FRV with respect to the control (−15.4% and −13.8%, respectively, p \< 0.001). As well, also PE1 reduced these two parameters (−9.6% and −9.7%, p \< 0.001 for PRO, −7.4% and −10.4%, p \< 0.001 for FRV, for low and high dose, respectively) as also PT1 at low dose did (−8.5% and −7.5%, p \< 0.001 for PRO and FRV, respectively). By contrast, PE2, GEL, and PT1H treatments led to a PRO decrease (p \< 0.01) without significantly affecting the low molecular flavanol component (FRV). In the present study, the removal of flavanols during wine fining was similar to or even higher than that previously published for the use of gelatin, potato-based proteins (about −9% for PRO using both patatin and gelatin at 10 and 30 g/hL dose, FRV ranging from −1.1% to −13.9% for patatin and from −7.1% to −18.8% for gelatin at 10 and 30 g/hL \[[@B6-molecules-25-00120]\]), and pea-derived proteins (−6.8% for PRO at 10 g/hL dose \[[@B8-molecules-25-00120]\]; −3--5% of catechin and epicatechin and −15--22% of oligomeric proanthocyanidins at 20 g/hL dose \[[@B18-molecules-25-00120]\]). The effectiveness of GEH fining agent to remove oligomeric and polymeric flavanols is undoubted as this treatment reduced PRO and FRV contents more than vegetal-derived proteins in Montepulciano and Nebbiolo wines, which are richer in polymeric proanthocyanidins, and was highly effective in Primitivo and Syrah wines. However, this study highlighted for the first time that both the effectiveness of vegetal-derived proteins and the target compounds are influenced by the wine phenolic composition. Specifically, the PE1L treatment reduced FRV content more efficiently than gelatin in Primitivo wine whereas PT1H removed a significantly higher PRO content than gelatin in Syrah wine. In Montepulciano and Syrah wines, which are characterized by the greatest richness in total phenolic compounds (expressed as A~280~, [Table 1](#molecules-25-00120-t001){ref-type="table"}), PT1H treatment showed a decrease in low molecular flavanols similar to GEH. Instead, the same PT1H treatment allowed to reduce the PRO content in Syrah and Nebbiolo wines having a high FRV/PRO ratio. A decrease in FRV content not significantly different from that obtained for GE1H was achieved using PT1L and PE1H in Syrah and Nebbiolo wine, respectively. Fining trials showed that polymeric proanthocyanidins are reduced significantly by a higher number of vegetal fining agents with respect to low molecular mass flavanols, probably due to they are firstly precipitated. The higher number of phenolic rings present in the more polymerized proanthocyanidins increases their hydrophobicity and therefore facilitates flavanol-protein interactions \[[@B17-molecules-25-00120]\]. In addition to polymerization degree, other characteristics of flavanols such as galloylation percentage and conformational flexibility may play an important role in removing these phenolic compounds. In fact, galloylated proanthocyanidins are removed in a preferential way \[[@B17-molecules-25-00120]\]. Moreover, hydrophobic interactions could be mainly responsible for precipitation by vegetal proteins derived from pea and potato, and some studies have confirmed the relatively high surface hydrophobicity of patatin \[[@B17-molecules-25-00120],[@B27-molecules-25-00120]\]. 2.2. Total Anthocyanins and Color Parameters {#sec2dot2-molecules-25-00120} -------------------------------------------- A side effect of wine fining is the removal of colored pigments, which may lead to a wine with reduced color quality. Different results were obtained depending on the fining agent used, the dose applied, and the variety in terms of total anthocyanins (TA), color intensity (CI), and tonality (hue) as reported in [Table 2](#molecules-25-00120-t002){ref-type="table"}. In Primitivo wine, significant differences between the wines treated with different fining agents and the untreated wines (control) were found for TA and CI parameters whereas the hue of the treated wines agreed with that of the control wine. TA content was significantly decreased with respect to the control sample when the wine was treated with gelatin at both low and high doses (−6.8%, p = 0.004 and −7.0%, p = 0.037 for GEL and GEH, respectively). As well, the high dose of PE1 (pea-based) reduced significantly anthocyanins, leading to a decrease of −7.7% for PE1H (p = 0.014). In the case of gelatin, also a CI decrease was reported with respect to the control (−6.8% and −12.8% for GEL and GEH, respectively, both p \< 0.001), in agreement with the anthocyanins loss. In contrast, even if there was no significant reduction of TA content, PT1 treatment at a high dose led to a decrease of CI value with respect to the control (−9.5%, p \< 0.001 for PT1H). Although the anthocyanin content mainly determines wine color, the CI parameter can be magnified by the presence of co-pigments, such as flavonols and flavanols, which may have been removed by the protein-based treatment. In general, a similar effect of fining treatment was observed on the three color components (yellow, red, and blue as A~420~, A~520~, and A~620~, respectively). However, GEL, GEH, and PT1H samples evidenced a higher decrease in the blue color component (A~620~, data not shown), which is related to co-pigmentation effects \[[@B28-molecules-25-00120]\]. As found for Primitivo, lowered TA content was evidenced in Montepulciano treated wines depending on the fining treatments, whereas CI value was significantly reduced in all cases with the exception of PE2 at a low dose (from −2.7% up to −11.7%, p \< 0.01) when compared to untreated wine. GEH and PT1H treated wines reported the lowest TA content, with a loss of −6.6% and −6.2%, respectively, compared to the control wine (both p \< 0.001), followed by GEL and PE2H treatments with a loss of −3.7% and −3.5% (both p \< 0.001) in TA content, respectively. This anthocyanin loss resulted in an increase in the hue parameter for PT1H treatment (+1.0%, p \< 0.001). Even if to a lesser extent, also PT1L and PE1H showed lower TA content with respect to the control wine (both −2.8%, p = 0.002). In Montepulciano, a significant loss of anthocyanins resulted in lower color intensity. Anyway, the CI value was also reduced when TA content was not affected. The removal of oligomeric flavanols can support this reduction in PE1L sample ([Table 1](#molecules-25-00120-t001){ref-type="table"}), however, the blue color component was more strongly affected than yellow and red components in wines treated with PE1L, PT2L, and PT2H, evidencing that the removal of other co-pigments, such as flavonols, may have occurred. In Syrah wine, there was no significant loss of anthocyanins, but CI and hue were changed significantly for several fining treatments (both p \< 0.001). In particular, CI value was reduced in all treated wines, with the exception of PT2L treatment, decreasing from −3.1% up to −9.0% (all p \< 0.01) with respect to the control samples. The highest color losses corresponded to GEH and PT1H treatments (−9.0%, p \< 0.001), followed by PT1L (−5.8%, p \< 0.001) and GEL (−4.7%, p \< 0.001). Interestingly, hue value was often lower in treated samples (decreasing from −0.6% to −1.0%, p \< 0.01; except for PE2L, PT1L, and PT2H), meaning a lower yellow color component (A~420~) with respect to the red component (A~520~) and, therefore, the wine shifted to a red hue instead of yellow one. Considering that no change affected monomeric anthocyanins (data not shown), it is possible to hypothesize a decrease in anthocyanin-derived pigments, such as pyranoanthocyanins, which contribute to the orange hue of wines \[[@B29-molecules-25-00120]\]. Granato et al. \[[@B18-molecules-25-00120]\] reported losses of individual anthocyanin-derived pigments in Aglianico red wine treated with plant-based proteins and evidenced that gelatin removed monomeric anthocyanins and anthocyanin-flavanol adducts whereas pea-based proteins were ineffective. Nebbiolo wine was strongly influenced by all fining treatments: a significant reduction from −8.0% to −21.5% (p \< 0.01) of TA was found with respect to the control sample. The higher anthocyanin loss was reported for PE2 treatment at high dose, followed by gelatin at both the low and high doses, and PT1 at high dose (−21.5% for PE2H, −18.1% for GEL, GEH, and PT1H, all p \< 0.001). This loss was reflected also in a significant decrease of CI value in all treated samples with respect to the control (from −3.9% to −15.6%, p \< 0.05), which was particularly evident for GE treatment at both low and high dose, and PT1 at high dose (−10.1%, −15.6%, and −10.7% for GEL, GEH, and PT1H, respectively, all p \< 0.001). Nevertheless, the highest loss of anthocyanins is not in correspondence with the highest decrease in color intensity. Despite the differences in CI and TA parameters, the hue was significantly increased only in PT1H wine with respect to the control (+1.3%, p = 0.011). In Nebbiolo, as well as in Montepulciano, the increased hue, in contrast with Syrah treatments' effect, could be justified by the TA decrease, leading to a loss of red color component (A~520~), which was less observed in Syrah. This behavior in Syrah could be linked to the greatest richness in anthocyanins and to an anthocyanin profile prevalent in malvidin-3-glucoside with a high ratio of acylated derivatives, giving high anthocyanin stability \[[@B30-molecules-25-00120]\]. In this aspect, Nebbiolo wines are particularly different because of their lower anthocyanin content and less abundance of acylated derivatives \[[@B31-molecules-25-00120]\]. Anthocyanin losses during wine fining have been also reported in many studies, but their magnitude is variable. In Aglianico red wine, Gambuti et al. \[[@B6-molecules-25-00120]\] reported a removal of total anthocyanins lower than −4% for patatin and gelatin at doses between 10 and 30 g/hL, whereas Granato et al. \[[@B18-molecules-25-00120]\] evidenced losses of about −31% for gelatin and of −4% for pea proteins at doses of 20 g/hL. According to these studies, gelatin (animal origin) and potato-based vegetal proteins reduced more significantly the content of petunidin-, peonidin-, and malvidin-3-glucoside \[[@B6-molecules-25-00120]\] whereas cyanidin-3-glucoside was negatively influenced by pea-derived proteins \[[@B18-molecules-25-00120]\]. In the present work, the results obtained are in agreement with these findings. In fact, the anthocyanin losses in Primitivo and Nebbiolo wines, which are richer in cyanidin-3-glucoside than Montepulciano and Syrah, were higher after fining treatments with pea-derived proteins (PE1H and PE2H, respectively) whereas the lowest contents of anthocyanins corresponded to both GEH and PT1H treatments in Montepulciano and Syrah wines. 2.3. Comparison between Flavanol Reduction and Color Modification {#sec2dot3-molecules-25-00120} ----------------------------------------------------------------- The first aim of red wine fining is to deplete an unbalanced astringency, which may be given from an excess of flavanols. However, a strong color loss affects negatively the wine quality. Therefore, an important key to the fining treatment is the selective reduction of unbalanced polymeric flavanol content combined with the lowest anthocyanin loss and, as a consequence, the highest final color intensity. In [Figure 1](#molecules-25-00120-f001){ref-type="fig"}, the polymeric flavanol (PRO) reduction was compared to the anthocyanin (TA) loss and to the color intensity (CI) decrease, expressed as a percentage. Gelatin treatments were in general efficient in removing polymeric flavanols, with a dose-dependent reduction not only for PRO but also for CI in all the varieties. As well, TA removal was related to the dose in Montepulciano, whereas a higher gelatin dose was not corresponded by a higher TA decrease in Nebbiolo and Primitivo wines. Interestingly, in Syrah treated wines, the TA decrease was not significantly different for any of the investigated protein fining agents, in agreement with TA contents ([Table 2](#molecules-25-00120-t002){ref-type="table"}), although CI variations were observed. Pea-based proteins had a different effect depending on the formulation, the dose, and the variety evaluated ([Figure 1](#molecules-25-00120-f001){ref-type="fig"}). PE1 formulation gave interesting results for PRO removal in Primitivo and Nebbiolo wines. When PE1 was applied at a high dose, the PRO removal was similar to gelatin (GEH), even though differences were observed on the anthocyanin loss. In Primitivo wines, PE1H and GEH treatments caused similar TA reduction (−7.7% and −7.0% for PE1H and GEH, respectively), whereas in Nebbiolo the removed quantity of anthocyanins by PE1H was significantly lower than that reduced by gelatin treatments (−8.9% and −18.1%, corresponding to 29.3 mg and 36.7 mg of TA removed/g of PRO removed, for PE1H and GEH, respectively). On the other hand, the PRO removal by PE1 treatment at low dose was not significantly different to that corresponding to the high dose in Primitivo (−7.1% and −8.8% of PRO for PE1L and PE1H, respectively) but preserving anthocyanins, whereas in Nebbiolo wines similar removal of PRO and TA was obtained by using both low and high dose (27.3 mg and 29.3 mg of TA removed/g of PRO removed for PE1L and PE1H, respectively). In any case, a reduced loss of CI was observed for PE1L and PE1H treatments with respect to GEH in Nebbiolo and Primitivo wines. For PE1 formulation, the dose-response effect was negative on PRO removal for Syrah, namely the higher dose gave poorer results than the lower dose (−7.4% and −4.7% for PRO, resulting in 26.9 mg and 77.3 mg of TA removed/g of PRO removed for PE1L and PE1H, respectively). Regarding CI reduction, no significant difference was evidenced for all pea-based treatments in Syrah wines. The PE2H treatment was interesting in Syrah because it removed PRO similarly to gelatin (GEH) but preserving anthocyanins (−11.7% and −12.6% of PRO for PE2H and GEH, respectively). However, in Nebbiolo, the same treatment showed both low PRO removal (−7.9% for PE2H) and the highest removed amount of TA (−21.4% for PE2H, corresponding to 84.6 mg of TA removed/g of PRO removed), although there was not the same remarkable effect on the color intensity when compared to the other pea-based treatments. Among potato-based treatments, high variability in the efficiency was found, particularly when applied to the different wines. Regarding the PRO removal, PT1 formulation at high dose gave similar results to gelatin (GEH) in Nebbiolo and Syrah wines, but anthocyanin losses and CI reduction were also similar (43.5 mg and 31.9 mg of TA removed/g of PRO removed for PT1H, 36.7 mg and 41.5 mg of TA removed/g of PRO removed for GEH, respectively, for Nebbiolo and Syrah). PT2H treatment also provided results in agreement with GEH for Syrah (42.6 mg of TA removed/g of PRO removed) with reduced CI decrease. In Nebbiolo wines, PT1L treatment reduced anthocyanin losses when compared to gelatin (−18.1%, −18.1%, and −8.0% for GEH, GEL, and PT1L, respectively) and therefore preserved better the wine color. In Primitivo and Montepulciano wines, also PT1L treatment gave the best results for potato-derived proteins, being worse the performance in relation to GEH and similar to GEL (--7.8% and --11.6% of PRO for PT1L; --10.3% and --12.0% of PRO for GEL, respectively, for Primitivo and Montepulciano wines). A non-significant dose-dependent reduction of PRO was observed for most of the potato-based formulations, but this relation was different depending on the variety evaluated. In fact, a smaller PRO removal was evidenced for PT1 at the high dose in Primitivo and Montepulciano, when compared to a low dose, whereas the PRO decrease was higher for high dose in Syrah and Nebbiolo wines. In most cases, PT2 formulation showed the lowest effectiveness in PRO removal for all the varieties investigated, being it remarkably lower with respect to gelatin. Taking into account that the dose recommended by the supplier depends on the protein content, probably this formulation requires a much higher dose to appreciate its efficiency. In fact, only the highest recommended dose permitted the significant removal of oligomeric and polymeric flavanols in Syrah wine. However, it was evident that, even at low doses, anthocyanin contents were significantly reduced in Nebbiolo (up to −16.1%, p \< 0.001, corresponding to a removal of 145 mg and 219 mg of TA/g of PRO for PT2L and PT2H, respectively) and a significant reduction of color intensity was also observed in Montepulciano, Syrah, and Nebbiolo with respect to control ([Table 2](#molecules-25-00120-t002){ref-type="table"}). Therefore, this negative impact on wine color could be the limiting factor to an increased dosage of PT2. Interestingly, the present study highlighted that the protein features, such as the proline content and the molecular mass \[[@B20-molecules-25-00120],[@B23-molecules-25-00120]\], although of great relevance, are not the only ones influencing the effectiveness of red wine fining. In fact, earlier studies have evidenced that proline-rich proteins, such as pea-derived proteins, could interact more efficiently with galloylated flavanic derivatives \[[@B20-molecules-25-00120]\]. In addition, molecular mass fractions ranging from 10 to 32 kDa act more specifically on astringent compounds, i.e., polymeric flavanols, whereas fractions between 30 and 42 kDa could be responsible for the pigment losses \[[@B23-molecules-25-00120]\]. Therefore, the phenolic profile and concentration can play a key role in the hydrophobic interactions governing the insolubilisation and therefore the removal of target phenolic compounds involved in the perception of astringency and color. This was previously suggested by other authors but it had never been confirmed before. To better investigate the efficiency of protein treatments and the involved variables, a comparison of the different treatments was performed by PCA ([Figure 2](#molecules-25-00120-f002){ref-type="fig"}), with the aim of understanding if a general trend occurred for the same treatment (GE, PE1, PE2, PT1, and PT2), or if there was a stronger effect of the variety of features in the studied wines (Montepulciano, Primitivo, Nebbiolo, and Syrah). To minimize the variety differences, the multivariate comparison was done on the normalized data of each parameter reduction with respect to the control. Dimension 1 (D1) accounted for 42.1% of the explained variance, whereas dimension 2 (D2) explained 22.0% (total explained variance by the first two components = 64.1%). Dimension 1 was positively correlated, in order, with the decrease of PRO, CI, TA, FRV, and A~280~ (0.858, 0.706, 0.660, 0.656, and 0.651, respectively, all p \< 0.001). Dimension 2 was strongly influenced by color parameters with a positive correlation with hue (0.893, p \< 0.001) and a negative correlation with TA (−0.557, p \< 0.001). As well, a less significant positive correlation was also found between D2 and FRV reduction (0.441, p \< 0.001). Regarding the variety effect ([Figure 2](#molecules-25-00120-f002){ref-type="fig"}A), variety features seem to have an important effect on the removal of phenolic compounds, being Syrah well separated from the other varieties, in particular from Nebbiolo, and D2 parameters (mainly hue loss) contributed strongly to the differentiation. Syrah wines, being rich in stable compounds from the chromatic point of view \[[@B30-molecules-25-00120]\], fining treatments caused the shift to a red hue instead of yellowish ([Table 2](#molecules-25-00120-t002){ref-type="table"}). Nebbiolo wines, having a higher ratio of di-substituted anthocyanin forms, are more prone to anthocyanin loss ([Table 2](#molecules-25-00120-t002){ref-type="table"}) and consequently, the decrease of red color component (A~520~) was evidenced. Regarding vegetal fining agents, PT1 treatments (potato-derived protein, [Figure 2](#molecules-25-00120-f002){ref-type="fig"}B) were the most effective in the reduction of flavanol compounds, followed by PE1 (pea-based protein). PT1 treatments removed flavanols in a relatively similar way to gelatin whereas PE1 formulation preserved better-colored pigments. An important aspect to consider is the strong influence of formulation for the potato-based fining agents (PT1 and PT2) because PT2 treatment caused only a minor removal of flavanols and few changes in color parameters. Therefore, great variability in potato-based fining agents on the market can be assumed because of different sources, extraction conditions, extract preparation, and possible protein hydrolysis \[[@B1-molecules-25-00120]\]. Undoubtedly, this leads to a different binding affinity and efficiency \[[@B23-molecules-25-00120]\]. 2.4. Astringency and Visual Color Assessment {#sec2dot4-molecules-25-00120} -------------------------------------------- The effect of the fining treatment on wine astringency was attempted by means of chemical and sensory methods in order to investigate if the significant reduction in the content of phenolic compounds effectively influenced astringency. The results obtained from the BSA precipitation index, as described by Boulet et al. \[[@B32-molecules-25-00120]\], and from tasting sessions by a trained panel on astringency are shown in [Table 3](#molecules-25-00120-t003){ref-type="table"}. Astringency determinations gave different results depending on the analytical method: the BSA index highlighted significant differences whereas the sensory-perceived intensity by the trained panel had not shown relevant differences in terms of astringency among fining treatments. For Syrah wine, a significant reduction in the BSA index was observed for gelatin at both low and high dose (--26.7%, p = 0.006 and --27.4%, p = 0.005 for GEL and GEH, respectively), and for PT1, PE1, and PE2 at high dose (--35.0%, p \< 0.001, --25.2%, p = 0.010, and --25.8%, p = 0.009 for PT1H, PE1H, and PE2H, respectively) with respect to control wine. The highest decrease in the BSA index may be due to the greatest removal of polymeric flavanols (PRO, [Table 1](#molecules-25-00120-t001){ref-type="table"}). Regarding Primitivo wine, a significantly lower BSA index was found after PT1H fining treatment (--34.6%, p \< 0.001) when compared to control samples, although no reduction of A~280~, PRO, and FRV was reported ([Table 1](#molecules-25-00120-t001){ref-type="table"}). As well, in Montepulciano and Nebbiolo wines, GEH and PT1H treatments also gave the lowest value of the BSA index, even though it is not significantly different from the control samples. In this case, the reductions were in accordance with a high decrease of FRV and PRO contents in Montepulciano, and of A~280~ and PRO in Nebbiolo ([Table 1](#molecules-25-00120-t001){ref-type="table"}). Furthermore, in both Montepulciano and Nebbiolo wines, the trained panel perceived a lower intensity of astringency in the wines treated with PT1H fining agent with respect to the control, although the differences were not significant. The results obtained for PT1H formulation are in agreement with the effectiveness reported for potato protein in diminishing astringency of Aglianico wine \[[@B6-molecules-25-00120]\]. The removal of flavanols, oligomeric or polymeric, is not always directly related to a reduction of astringency, either sensory perceived or chemically assessed. This could be due to the complexity of astringency sensation, which can be mainly triggered by polymeric flavanols of different subunit composition and modified by wine features such as alcohol content, acidity, and pH \[[@B32-molecules-25-00120],[@B33-molecules-25-00120]\]. It is well known that the perceived astringency is increased at lower pH values. Furthermore, Quijada-Morín et al. \[[@B34-molecules-25-00120]\] highlighted that polysaccharides, mainly mannoproteins and rhamnogalacturonan-II (RG-II), reduce astringency perception whereas the presence of mannose and galactose residues in the oligosaccharide fraction increases the perceived astringency. Regarding visual color assessment, CIE L\a\b\* coordinates ([Table S1](#app1-molecules-25-00120){ref-type="app"}) were used to calculate the corresponding color on the RGB scale and the results are reported in [Figure 3](#molecules-25-00120-f003){ref-type="fig"} for control and treated wines. In agreement with the significant changes observed in color intensity ([Table 2](#molecules-25-00120-t002){ref-type="table"}), CIE L\a\b\* parameters were strongly affected by fining treatments (L\, b\, p \< 0.001 for all the varieties; a\, p* \< 0.01 for Nebbiolo and p \< 0.001 for the other varieties, [Table S1](#app1-molecules-25-00120){ref-type="app"}). This was particularly evident for GEH and PT1H treatments in [Figure 3](#molecules-25-00120-f003){ref-type="fig"} for all the varieties evaluated. Lightness (L\) increased in the treated wines with respect to control samples, even though in different extent depending on the variety, the fining agent used, and the dose applied. The a\ and b\* coordinates (red/green color and yellow/blue color, respectively) also increased for Primitivo, Montepulciano, and Syrah wines, indicating a displacement towards reddish and yellowish hue, respectively. In contrast, b\* values decreased in Nebbiolo wines treated with gelatin, PE1L, and PT1H showing a hue shifting to blue whereas a significant reduction in a\values were only found in PT1H treatment, thus reducing the red color component. In addition, CIE L\a\b\ parameters highlighted the variety-dependent effect of the fining treatments on color modification. The three CIE L\a\b\* coordinates increased significantly with GEL, GEH, and PT1H treatments in Primitivo wines, with all formulations at the high dose in Montepulciano, and with all fining treatments with the exception of PT2L in Syrah. In Nebbiolo wines, the three color coordinates were differently affected by the fining treatments. To quantify the differences found in the wine color at the end of each fining treatment, ΔE\* values were calculated with respect to control. This parameter was previously used to underline the ability of tasters to detect even small color differences in wines. While differences of one unit in this parameter could be detectable by tasters when the wine is directly observed \[[@B35-molecules-25-00120]\], the ΔE\* threshold suggested to correctly detect a wine color difference by the human eye was about 3 \[[@B36-molecules-25-00120]\] or 5 units \[[@B37-molecules-25-00120]\] probably as a consequence of color observation through a glass. As can be observed in [Figure 3](#molecules-25-00120-f003){ref-type="fig"}, ΔE\* data evidenced several important trends among fining agents for all the varieties evaluated. Gelatin treatments at high dose (GEH) gave the highest ΔE\* values of the study, the resulting wines consistently achieving ΔE\* values above 4.8 units. Only PT1 (potato-based) fining agent also at the high dose was found to cause a similar color reduction with ΔE\* ranging from 3.3 to 5.2; these values being higher than those for GEH only in the case of Syrah wines. Furthermore, Primitivo and Nebbiolo wines treated with gelatin at a low dose (GEL) showed a visually perceived color reduction, as well for PT1 at a low dose (PT1L) in Syrah wine (ΔE\* values between 3.2 and 3.5). No other pea and potato-based fining agent reached ΔE\* values above three units, regardless of the dose considered. The results obtained are consistent with previous studies on the impact on young wine color of fining treatments using gelatin and vegetal proteins from gluten \[[@B38-molecules-25-00120]\]. In the cited study, darker colors were found in control wines, although lightness (L\) was not always significantly affected by the gelatin or gluten protein treatments. 3. Materials and Methods {#sec3-molecules-25-00120} ======================== 3.1. Reagents and Standards {#sec3dot1-molecules-25-00120} --------------------------- Chemicals of analytical reagent grade, cyanidin chloride, (+)-catechin, and bovine serum albumin were supplied by Sigma-Aldrich (St. Louis, MO, USA). The standard of malvidin-3-O-glucoside chloride was acquired from Extrasynthese (Genay, France). The solutions were prepared in deionized water produced by a Milli-Q system (Merck Millipore, Darmstadt, Germany). 3.2. Wines {#sec3dot2-molecules-25-00120} ---------- Four young red wines (one wine for each variety, all belonging to vintage 2016) from Italian wineries were used in the present study for the fining trials: Primitivo, Montepulciano, Syrah, and Nebbiolo. They were selected on the basis of their different ratios between oligomeric and polymeric flavanols because of the impact on bitterness and astringency perception. The chemical characteristics of these four wines are shown in [Table S2](#app1-molecules-25-00120){ref-type="app"}. 3.3. Wine Fining Trials {#sec3dot3-molecules-25-00120} ----------------------- The fining agents used in this study were four commercially available and allergen-free vegetal-derived protein extracts; two from pea (PE1 and PE2) and other two from potato (PT1 and PT2). The study also included one animal gelatin (GE). For each fining agent, a stock solution of 10% w/v* was prepared in deionized water with the exception of PT2, for which a stock solution of 2.5% w/v was done. Wine fining trials were carried out in completely filled 1-L bottles where two different dosages (Low and High) were added for each fining agent to each wine. Low (L) corresponds to the minimum dose +20% (i.e., +1/5) of the recommended dosage range whereas high (H) is the maximum dose --20% (i.e., --1/5) of the recommended dosage range ([Table 4](#molecules-25-00120-t004){ref-type="table"}), representing doses commonly used for wine fining in industrial winemaking. For each wine, the control (CO) was prepared by adding deionized water instead of the fining agent. All fining experiments were carried out in triplicate, by setting for 7 d at 18 °C. At the end of the fining treatment, wine samples were filtered on 40 μm cellulose filters (VWR International SAS, Leuven, Belgium). For each replicate, a 50 mL-aliquot of filtered wine was further centrifuged in a PK 131 centrifuge (ALC International, Milan, Italy) for 15 min at 3000× g at 20 °C and then used for chemical analysis. The remaining wine from each of the three replicates was homogenized, bottled, stored at 18 °C, and then used for sensory analysis. 3.4. Chemical Analysis after Wine Fining {#sec3dot4-molecules-25-00120} ---------------------------------------- Wine phenolic composition was determined through spectrophotometric methods using a UV-1800 spectrophotometer (Shimazdu Corp., Kyoto, Japan). In particular, total phenolics were assessed by the measurement of absorbance at 280 nm in deionized water (A~280~). Proanthocyanidins (PRO) were determined after acid hydrolysis at 100 °C using a ferrous salt (FeSO~4~) as catalyst according to the Bate-Smith reaction and expressed as mg of cyanidin chloride/L of wine. Monomeric and oligomeric forms of flavanols were evaluated as flavanols reactive to vanillin (FRV) and expressed as mg of (+)-catechin/L of wine. Total anthocyanins were determined by measuring absorbance at 536--540 nm after dilution with a hydroalcoholic solution composed of ethanol: water: 37% hydrochloric acid (70:30:1, v/v) and expressed as mg of malvidin-3-glucoside chloride/L of wine \[[@B39-molecules-25-00120]\]. Flavanols contributing to astringency perception were evaluated following the Adams--Harbertson protein precipitation assay \[[@B40-molecules-25-00120]\], modified by Boulet et al. \[[@B32-molecules-25-00120]\]. Briefly, the wine was diluted with a buffer solution adjusted to pH 3.2, containing 12% v/v of ethanol and 5 g/L of tartaric acid. Then, 0.5 mL of diluted wine was added either with 1 mL of a buffer solution at pH 4.9 composed by 200 mM of acetic acid and 170 mM of NaCl or with 1 mL of the same buffer solution at pH 4.9 containing also 1 mg/mL of bovine serum albumin (BSA). After incubation at room temperature for 15 min with continuous agitation, the sample was centrifuged at 13,500× g for 5 min. The supernatants were diluted with 2% of hydrochloric acid and absorbance was measured at 280 nm. The BSA index was calculated as the difference between the two absorbance values and expressed as mg of (+)-catechin/L of wine. 3.5. Color Characteristics after Wine Fining {#sec3dot5-molecules-25-00120} -------------------------------------------- After the acquisition of visible spectra of undiluted samples using 1-mm optical path cuvettes, color intensity was calculated as the sum of absorbance measured at 420, 520, and 620 nm (A~420~ + A~520~ + A~620~ on an optical path of 10 mm) and hue was obtained as the ratio of absorbances measured at 420 and 520 nm (A~420~/A~520~) following the method OIV-MA-AS2-07B \[[@B41-molecules-25-00120]\]. The wine color was also evaluated by the CIE L\a\b\* parameters, namely lightness (L\), red/green color coordinate (a\), and yellow/blue color coordinate (b\), according to the method OIV-MA-AS2-11. The total color difference (ΔE\) between two samples (for example between a fined wine and the respective control wine) was calculated using the following expression: ΔE\* = \[(ΔL\)^2^ + (Δa\)^2^ + (Δb\)^2^\]^1/2^ \[[@B41-molecules-25-00120]\]. A UV-1800 spectrophotometer (Shimazdu Corporation) was used. 3.6. Sensory Analysis after Wine Fining {#sec3dot6-molecules-25-00120} --------------------------------------- Sensory analysis was conducted by nine tasters (6 males and 3 females) aged between 30 and 55 years from the University of Turin researchers and professors with experience in wine sensory analysis. Panelists were trained during twelve sessions (1 h per session, three times per week) to recognize, standardize, and rate the perceived intensity of astringency. During the training sessions, panelists were asked to mark when they began to feel some stimuli and to order the samples on the basis of the perceived intensity. Triangle, duo-trio, and intensity tests on structured (1--10) and unstructured (10 cm) scales were performed using enological tannin (0.1--1 g/L) dissolved in water and wine. Wine samples were then evaluated in five formal sessions to identify and rate the astringency perception. In the first session, the control wines were assessed to establish the astringency intensity of each non-treated wine. Then, all wines (control and fined) from the same variety were arranged in one session of 1 h, randomly presented to avoid a likely effect of context stimuli on ratings, and identified with three-digit random codes. A constant volume of 30 mL for each wine was evaluated in wine-taster glasses at 12 °C as described by the International Organization for Standardization (ISO) 3591 Norm (1977) \[[@B42-molecules-25-00120]\]. Panelists recorded their perceived intensity on a 10-cm unstructured linear scale. To minimize carryover effects, panelists eat crackers between samples and then rinsed the mouth with natural water. 3.7. Statistical Analysis {#sec3dot7-molecules-25-00120} ------------------------- Statistical analysis was conducted using R statistic software (R Core Team \[[@B43-molecules-25-00120]\]). For each studied variable, one-way analysis of variance (ANOVA) using the Tukey HSD post-hoc test was used to evaluate significant differences among treatments. Levene's and Shapiro--Wilk's tests were applied for assessing the homogeneity of variance and normality of ANOVA residuals, respectively. Moreover, multiple comparison Dunnett's test was performed to investigate significant differences between each fined wine and the respective control. Principal component analysis (PCA) using the R package "factoextra" \[[@B44-molecules-25-00120]\] was performed on the ANOVA significant chemical parameters determined (p* \< 0.05) and therefore A~280~, PRO, FRV, TA, color intensity, and hue were the variables. To compare the effect of fining agents in varieties with different phenolic composition while minimizing the contribution of different phenolic content and profile, each value of the chemical parameters in fined wines was subtracted to that of the respective control wine and the difference was then normalized as z-scores before multivariate analysis. 4. Conclusions {#sec4-molecules-25-00120} ============== Gelatin is the most commonly used fining agent in wine production, and results showed its ability and efficiency in the reduction of wine flavanol components, counter parted by a loss of anthocyanins to a different extent depending on the studied variety characteristics and the treatment dose. Plant-derived protein finings have been proposed to be a possible alternative. In particular, those derived from potato and pea could be useful for vegan-friendly, allergen-free wine production. The study tested the performance of several plant-derived agents on the red wine fining compared to gelatin. In this study, fining agents derived from plants gave different results on phenolic compounds reduction, depending on their origin, formulation, dose applied, and also on the studied wine characteristics in terms of phenolic content and profile. Indeed, using four different monovarietal red wines exhibiting different phenolic features allowed us to highlight the effect of the wine composition, particularly on the phenolic characteristics, on the effectiveness of the fining treatment. Furthermore, the results suggest the necessity of preliminary trials for wine fining, possibly accompanied by instrumental measurements in terms of flavanols, astringency, and color. Sample Availability: Samples of the compounds are not available from the authors. ###### Click here for additional data file. The following are available online, chemical composition of initial wines and CIE L\a\b\* coordinates of control and treated wine. L.R. and V.G. designed the experiments and revised the manuscript; S.R.S., M.V., and S.G. performed the experiments; S.R.S. and M.A.P. analyzed the data and wrote the manuscript. All authors read and approved the final manuscript. All authors have read and agreed to the published version of the manuscript. This research received partial funding from the University of Torino Local Research Initiative (Torino, Italy). The authors declare no conflict of interest. ![Reduction percentage of wine proanthocyanidins (PRO) after fining treatments with proteins of animal origin like gelatin (GE) and of vegetal origin from pea (PE1, PE2) and potato (PT1, PT2) with respect to the untreated wine, compared to reduction percentage of total anthocyanins (TA) and color intensity (CI). All data are expressed as the average value of individual treatments with respect to the control ± standard deviation (n = 3). Different lowercase Latin letters for PRO, different uppercase Latin letters for TA, and different lowercase Greek letters for CI indicate significant differences among treatments according to Tukey test (p \< 0.05), respectively. Sign: \\, \\\, and ns indicate significance at p* \< 0.01, 0.001, and not significant, respectively. GE = Gelatin, PE1 = Pea 1, PE2 = Pea 2, PT1 = Potato 1, PT2 = Potato 2, L = low dose, H = high dose, (nr) = no reduction occurred.](molecules-25-00120-g001){#molecules-25-00120-f001} ![Principal component analysis (PCA) of phenolic compounds and color data obtained from wines after fining treatments with proteins of animal origin like gelatin (GE) and of vegetal origin from pea (PE1, PE2) and potato (PT1, PT2). PCA was performed on each parameter reduction with respect to the control. Confidence ellipses (p \< 0.05) were drawn for variety (A) and fining agents formulation (B). Treatments corresponded to GE = Gelatin, PE1 = Pea 1, PE2 = Pea 2, PT1 = Potato 1, PT2 = Potato 2, L = low dose, H = high dose. Variety: Pr = Primitivo, Mo = Montepulciano, Sy = Syrah, Ne = Nebbiolo.](molecules-25-00120-g002){#molecules-25-00120-f002} ![Wine color detected at the end of the treatment. Each wine color was acquired by spectrophotometry, expressed in CIE L\a\ b\* coordinates, and then converted to 8-bit RGB values for journal compatibility purposes. For each variety, the CO (Control) sample was extended on the left and top sides of the color bar to facilitate comparisons with treated wines in terms of colors. Treatments corresponded to GE = Gelatin, PE1 = Pea 1, PE2 = Pea 2, PT1 = Potato 1, PT2 = Potato 2, L = low dose, H = high dose.](molecules-25-00120-g003){#molecules-25-00120-f003} molecules-25-00120-t001_Table 1 ###### Phenolic composition of red wines untreated (CO) and treated with fining agents of animal origin (GE) and vegetal origin from pea (PE1, PE2) and potato (PT1, PT2). Primitivo Montepulciano Syrah Nebbiolo ------------------------------------------------------- --------------- --------------- -------------- ------------ -------------- ------------- -------------- --------------- --------------- --------------- --------------- ------------- CO 68.8 ± 2.1ab 3254 ± 83a 1323 ± 42abc 84.1 ± 1.1 3440 ± 40a 1330 ± 39a 72.5 ± 1.4a 3094 ± 19a 1659 ± 9abc 56.7 ± 0.5a 3589 ± 16a 2129 ± 33ab GEL 65.1 ± 2.1bc 2919 ± 76cd 1318 ± 59abc 84.7 ± 0.6 3025 ± 33c 1236 ± 55bc 70.9 ± 1.4ab 2991 ± 43ab 1587 ± 35abcd 52.4 ± 0.4cd 3330 ± 81cd 2085 ± 26b GEH 62.9 ± 1.0c 2801 ± 66d 1229 ± 4cd 82.1 ± 0.9 2788 ± 44d 1092 ± 10d 66.7 ± 1.3b 2704 ± 30de 1495 ± 39d 51.8 ± 1.1d 3038 ± 0f 1836 ± 45e PE1L 68.2 ± 1.4abc 3022 ± 87bc 1176 ± 25d 83.2 ± 0.2 3209 ± 79abc 1237 ± 50bc 70.5 ± 1.3ab 2863 ± 75bcd 1649 ± 6a 53.9 ± 0.8bc 3243 ± 77de 1971 ± 49cd PE1H 68.5 ± 2.1ab 2969 ± 30bcd 1235 ± 13bcd 83.4 ± 2.1 3134 ± 71abc 1186 ± 31c 69.0 ± 2.4ab 2947 ± 33ab 1591 ± 30abcd 53.8 ± 0.4bc 3240 ± 100de 1907 ± 33de PE2L 67.3 ± 1.9abc 3172 ± 64ab 1347 ± 20ab 85.1 ± 1.4 3262 ± 80ab 1304 ± 3ab 70.0 ± 1.5ab 2910 ± 30abc 1621 ± 40abc 54.8 ± 0.5b 3380 ± 14bcd 2196 ± 36a PE2H 69.0 ± 0.4ab 3234 ± 43a 1255 ± 9bcd 84.9 ± 1.0 3178 ± 49abc 1306 ± 17ab 69.7 ± 2.0ab 2732 ± 103cde 1544 ± 40bcd 53.7 ± 0.3bc 3306 ± 75cde 2190 ± 22a PT1L 71.4 ± 2.0a 3000 ± 56bcd 1337 ± 82abc 85.2 ± 1.2 3041 ± 30c 1181 ± 14c 70.0 ± 1.9ab 2935 ± 95ab 1492 ± 39d 54.6 ± 0.5b 3284 ± 103cde 1970 ± 47cd PT1H 65.5 ± 1.0bc 3159 ± 81ab 1380 ± 39a 82.4 ± 2.2 3112 ± 57bc 1160 ± 5cd 68.0 ± 1.9ab 2620 ± 82e 1527 ± 14cd 52.5 ± 0.5cd 3125 ± 68ef 2058 ± 14bc PT2L 68.6 ± 2.5ab 3182 ± 20ab 1372 ± 32a 84.4 ± 0.3 3334 ± 14a 1367 ± 19a 69.9 ± 1.0ab 2988 ± 56ab 1632 ± 9ab 54.1 ± 0.3bc 3464 ± 68abc 2151 ± 8ab PT2H 69.8 ± 2.3ab 3125 ± 30abc 1261 ± 5bcd 85.7 ± 0.5 3212 ± 44abc 1312 ± 24ab 70.8 ± 1.4ab 2885 ± 62bcd 1507 ± 50d 53.3 ± 0.9bcd 3536 ± 47ab 2060 ± 24bc ANOVA \\\* \\\* \\\* ns \\\* \\\* \* \\\* \\\* \\\* \\\* \\\* Contrasts' significance p-values respect to control CO vs. GEL 0.076 0.000 1.000 0.999 0.000 0.006 0.789 0.309 0.627 0.000 0.001 0.530 CO vs. GEH 0.001 0.000 0.021 0.290 0.000 0.000 0.002 0.000 0.001 0.000 0.000 0.000 CO vs. PE1L 1.000 0.004 0.000 0.939 0.201 0.006 0.610 0.002 0.991 0.000 0.000 0.000 CO vs. PE1H 1.000 0.000 0.193 0.983 0.015 0.000 0.095 0.064 0.727 0.000 0.000 0.000 CO vs. PE2L 0.937 0.928 0.799 0.945 0.711 0.873 0.369 0.013 1.000 0.009 0.008 0.134 CO vs. PE2H 1.000 1.000 0.189 0.983 0.074 0.914 0.263 0.000 0.049 0.000 0.000 0.205 CO vs. PT1L 0.268 0.001 1.000 0.874 0.000 0.000 0.376 0.038 0.001 0.003 0.000 0.000 CO vs. PT1H 0.154 0.807 0.101 0.458 0.006 0.000 0.020 0.000 0.014 0.000 0.000 0.100 CO vs. PT2L 1.000 0.682 0.197 1.000 1.000 0.590 0.335 0.280 1.000 0.000 0.199 0.974 CO vs. PT2H 0.973 0.359 0.296 0.592 0.220 0.985 0.765 0.004 0.003 0.000 0.933 0.116 All data are expressed as average value ± standard deviation (n = 3). Different Latin letters within the same column indicate significant differences among treatments according to Tukey test (p \< 0.05). Sign: \, \\\, and "ns" indicate significance at p \< 0.05, 0.001, and not significant, respectively. Contrasts values in bold face are significantly different from the control according to Dunnett test (p \< 0.05). A~280~ = Absorbance measured at 280 nm, PRO = Proanthocyanidins expressed as mg of cyanidin chloride/L of wine, FRV = Flavanols reactive to vanillin expressed as mg of (+)-catechin/L of wine. CO = Control, GE = Gelatin, PE1 = Pea 1, PE2 = Pea 2, PT1 = Potato 1, PT2 = Potato 2, L = low dose, H = high dose. molecules-25-00120-t002_Table 2 ###### Total anthocyanins and color parameters of red wines untreated (CO) and treated with fining agents of animal origin (GE) and vegetal origin from pea (PE1, PE2) and potato (PT1, PT2). Primitivo Montepulciano Syrah Nebbiolo ------------------------------------------------------- ------------- ---------------- ----------------- ------------ ---------------- ----------------- ----------- ---------------- ------------------ ----------- --------------- ------------------ CO 255 ± 1abc 13.97 ± 0.20a 0.708 ± 0.002ab 275 ± 4a 15.12 ± 0.06a 0.664 ± 0.002b 367 ± 11a 13.73 ± 0.14a 0.677 ± 0.001a 112 ± 4a 4.73 ± 0.06a 1.028 ± 0.005bc GEL 238 ± 3d 13.02 ± 0.01bc 0.708 ± 0.001ab 265 ± 2c 14.22 ± 0.12de 0.669 ± 0.002ab 367 ± 15a 13.08 ± 0.01bc 0.673 ± 0.001bc 92 ± 3de 4.25 ± 0.03de 1.028 ± 0.003bc GEH 238 ± 2cd 12.19 ± 0.10d 0.709 ± 0.001a 257 ± 2d 13.35 ± 0.08f 0.668 ± 0.001ab 351 ± 7a 12.50 ± 0.12d 0.672 ± 0.001bc 92 ± 2de 3.99 ± 0.04f 1.022 ± 0.000c PE1L 261 ± 3a 13.68 ± 0.05a 0.707 ± 0.001ab 271 ± 2abc 14.69 ± 0.07bc 0.666 ± 0.001b 361 ± 11a 13.31 ± 0.23bc 0.672 ± 0.001bc 102 ± 3b 4.45 ± 0.09c 1.030 ± 0.005abc PE1H 236 ± 2cd 13.55 ± 0.05a 0.706 ± 0.001b 267 ± 0bc 14.45 ± 0.07cd 0.665 ± 0.002b 355 ± 9a 13.20 ± 0.13bc 0.672 ± 0.002bc 102 ± 4bc 4.42 ± 0.03cd 1.022 ± 0.003c PE2L 253 ± 2abcd 13.59 ± 0.12a 0.707 ± 0.001ab 270 ± 2abc 14.82 ± 0.08ab 0.665 ± 0.001b 360 ± 10a 13.20 ± 0.06bc 0.674 ± 0.001ab 96 ± 1bcd 4.64 ± 0.05ab 1.036 ± 0.003ab PE2H 243 ± 5bcd 13.39 ± 0.02ab 0.706 ± 0.001b 265 ± 3c 14.45 ± 0.16cd 0.664 ± 0.001b 371 ± 6a 13.17 ± 0.08bc 0.672 ± 0.001bc 88 ± 2e 4.46 ± 0.06bc 1.034 ± 0.008abc PT1L 248 ± 8abcd 13.18 ± 0.06ab 0.708 ± 0.001ab 267 ± 0bc 14.48 ± 0.11cd 0.669 ± 0.002ab 364 ± 15a 12.94 ± 0.10c 0.674 ± 0.002abc 103 ± 3b 4.46 ± 0.08bc 1.035 ± 0.004ab PT1H 245 ± 2bcd 12.65 ± 0.04cd 0.707 ± 0.001ab 258 ± 2d 13.90 ± 0.24e 0.672 ± 0.005a 352 ± 6a 12.50 ± 0.11d 0.670 ± 0.002c 92 ± 2de 4.22 ± 0.11e 1.041 ± 0.005a PT2L 255 ± 2ab 13.67 ± 0.04a 0.709 ± 0.000ab 275 ± 1a 14.71 ± 0.07bc 0.666 ± 0.002b 363 ± 6a 13.43 ± 0.11ab 0.673 ± 0.001bc 94 ± 1cde 4.54 ± 0.03bc 1.035 ± 0.005ab PT2H 251 ± 5abcd 13.48 ± 0.10ab 0.708 ± 0.000ab 272 ± 1ab 14.57 ± 0.08bc 0.666 ± 0.001b 358 ± 8a 13.20 ± 0.23bc 0.674 ± 0.002ab 100 ± 4bc 4.41 ± 0.01cd 1.031 ± 0.000abc ANOVA \\\* \\\* \* \\\* \\\* \\ ns \\\* \\\* \\\* \\\* \\\* Contrasts' significance p-values respect to control CO vs. GEL 0.004 0.001 1.000 0.000 0.000 0.081 1.000 0.000 0.006 0.000 0.000 1.000 CO vs. GEH 0.037 0.000 0.635 0.000 0.000 0.240 0.295 0.000 0.005 0.000 0.000 0.413 CO vs. PE1L 0.081 1.000 0.606 0.122 0.001 0.763 0.980 0.008 0.002 0.004 0.000 0.999 CO vs. PE1H 0.014 0.997 0.189 0.002 0.000 0.999 0.671 0.001 0.001 0.002 0.000 0.342 CO vs. PE2L 1.000 1.000 0.395 0.033 0.028 0.999 0.949 0.001 0.128 0.000 0.435 0.271 CO vs. PE2H 0.339 0.405 0.251 0.000 0.000 1.000 0.998 0.000 0.002 0.000 0.000 0.612 CO vs. PT1L 0.843 0.013 1.000 0.002 0.000 0.048 1.000 0.000 0.093 0.006 0.000 0.322 CO vs. PT1H 0.373 0.000 0.635 0.000 0.000 0.000 0.364 0.000 0.000 0.000 0.000 0.011 CO vs. PT2L 0.939 1.000 0.999 1.000 0.002 0.912 1.000 0.073 0.010 0.000 0.012 0.413 CO vs. PT2H 1.000 0.852 0.996 0.670 0.000 0.827 0.864 0.001 0.128 0.000 0.000 0.996 All data are expressed as average value ± standard deviation (n = 3). Different Latin letters within the same column indicate significant differences among treatments according to Tukey test (p \< 0.05). Sign: \, \\, \\\, and "ns" indicate significance at p \< 0.05, 0.01, 0.001, and not significant, respectively. Contrasts values in bold face are significantly different from the control according to Dunnett test (p \< 0.05). TA = Total anthocyanins expressed as mg of malvidin-3-glucoside chloride/L of wine. CO = Control, GE = Gelatin, PE1 = Pea 1, PE2 = Pea 2, PT1 = Potato 1, PT2 = Potato 2, L = low dose, H = high dose. molecules-25-00120-t003_Table 3 ###### Astringency determined by chemical and sensory analysis of red wines untreated (CO) and treated with fining agents of animal origin (GE) and vegetal origin from pea (PE1, PE2) and potato (PT1, PT2). Treatment Primitivo Montepulciano Syrah Nebbiolo ------------------------------------------------------- ------------- --------------- ------------- -------------- ------------- ------------ ------------- ------------ CO 6.54 ± 0.47 1008 ± 113abc 5.43 ± 0.33 601 ± 74abc 4.75 ± 0.67 889 ± 68a 7.54 ± 0.43 585 ± 67ab GEL 5.25 ± 0.65 957 ± 59bc 4.70 ± 0.84 431 ± 47c 3.69 ± 1.02 652 ± 85b 6.16 ± 0.39 723 ± 49a GEH 4.89 ± 0.47 912 ± 28c 4.69 ± 0.43 539 ± 62abc 3.51 ± 0.79 646 ± 85b 7.06 ± 0.44 527 ± 27b PE1L 4.79 ± 0.61 1189 ± 83ab 4.54 ± 0.67 744 ± 87a 4.50 ± 0.80 681 ± 63ab 6.47 ± 0.53 631 ± 68ab PE1H 4.73 ± 0.86 1057 ± 151abc 4.37 ± 0.60 467 ± 111bc 4.38 ± 1.02 665 ± 105b 6.63 ± 0.79 594 ± 15ab PE2L 3.70 ± 0.63 1035 ± 144abc 3.94 ± 0.65 477 ± 61bc 3.84 ± 0.95 712 ± 33ab 6.59 ± 0.45 585 ± 34ab PE2H 4.35 ± 0.70 1214 ± 27a 4.52 ± 0.80 494 ± 44abc 4.24 ± 0.80 660 ± 79b 5.60 ± 0.82 586 ± 86ab PT1L 6.04 ± 0.46 889 ± 96cd 4.20 ± 0.62 630 ± 74abc 4.33 ± 0.69 675 ± 75ab 6.57 ± 0.42 615 ± 20ab PT1H 5.39 ± 0.68 660 ± 92d 3.41 ± 0.64 417 ± 102c 4.19 ± 0.96 578 ± 76b 6.02 ± 0.38 570 ± 24b PT2L 5.04 ± 0.73 968 ± 46bc 4.26 ± 0.76 718 ± 77ab 3.38 ± 1.13 693 ± 50ab 6.68 ± 0.65 597 ± 25ab PT2H 4.95 ± 0.49 835 ± 98cd 3.60 ± 0.64 646 ± 182abc 3.66 ± 0.90 760 ± 95ab 6.30 ± 0.38 573 ± 69b ANOVA ns \\\* ns \\ ns \\ ns \* Contrasts' significance p-values respect to control CO vs. GEL 0.637 0.981 0.981 0.193 0.972 0.006 0.375 0.019 CO vs. GEH 0.352 0.640 0.978 0.972 0.932 0.005 0.996 0.663 CO vs. PE1L 0.289 0.075 0.937 0.344 1.000 0.019 0.666 0.843 CO vs. PE1H 0.254 0.984 0.843 0.418 1.000 0.010 0.820 1.000 CO vs. PE2L 0.015 1.000 0.515 0.500 0.990 0.056 0.781 1.000 CO vs. PE2H 0.105 0.035 0.928 0.663 1.000 0.009 0.086 1.000 CO vs. PT1L 0.998 0.413 0.721 1.000 1.000 0.015 0.761 0.984 CO vs. PT1H 0.751 0.000 0.189 0.135 1.000 0.000 0.277 1.000 CO vs. PT2L 0.461 1.000 0.764 0.577 0.884 0.029 0.855 1.000 CO vs. PT2H 0.396 0.097 0.280 0.996 0.968 0.256 0.503 1.000 Sensory data are expressed as average value ± error calculated as sd/(n)^1/2^, where sd is the standard deviation and n is the number of panelists (n = 9). BSA data are expressed as average value ± standard deviation (n = 3). Different Latin letters within the same column indicate significant differences among treatments according to Tukey test (p \< 0.05). Sign: \, \\, \\\, and "ns" indicate significance at p \< 0.05, 0.01, 0.001, and not significant, respectively. Contrasts values in bold face are significantly different from the control according to Dunnett test (p \< 0.1). CO = Control, GE = Gelatin, PE1 = Pea 1, PE2 = Pea 2, PT1 = Potato 1, PT2 = Potato 2, L = low dose, H = high dose. molecules-25-00120-t004_Table 4 ###### Fining agents used in this experiment. Code Origin Recommended Dose (g/hL) Low Dose (g/hL) High Dose (g/hL) ------ -------- ------------------------- ----------------- ------------------ CO \- \- \- \- GE Animal 5--30 10 25 PE1 Pea 10--20 12 18 PE2 Pea 5--20 8 17 PT1 Potato 5--30 10 25 PT2 Potato 2--5 2.6 4.4 [^1]: These authors contributed equally to the study.
Ten months after being sworn in as the first openly gay man to serve as a public official in the state of California, Harvey Milk, along with Mayor Moscone, was shot and killed in his office by Dan White, a former member of the San Francisco Board of Supervisors at the time. White avoided being convicted of murder with what is now known as the "Twinkie Defense". After being released from prison less than two years later, White killed himself, and was often referred to as "perhaps the most hated man in San Francisco's history."
If this is your first visit, be sure to check out the FAQ by clicking the link above. You may have to register before you can post: click the register link above to proceed. To start viewing messages, select the forum that you want to visit from the selection below. Christians outpace FEMA in aid to hurricane victims By Cheryl K. Chumley - The Washington Times - Tuesday, September 12, 2017 ANALYSIS/OPINION: Faith-based groups — Christian nonprofits, specifically — have been busy bees of late, providing more aid to hurricane victims than even FEMA, the federal agency that’s supposed to swoop to the scenes of natural disasters, assess the situation and speed the recovery and rebuilding process. Just goes to show: Where charity exists, government is not needed. Look at this, from the Daily Caller: “Faith-based relief groups are responsible for providing nearly 80 percent of the aid delivered thus far to communities with homes devastated by the recent hurricanes.” The piece cited USA Today, which ran a headline: “Faith groups provide the bulk of disaster recovery, in coordination with FEMA.” Imagine that. When disaster strikes, it’s Americans — specifically, Americans of faith — who lend the quickest hands, who provide the most assistance. As USA Today noted, the Seventh Day Adventists took charge of disbursing bottles of water, diapers, clothing and other material needs. The United Methodist Committee on Relief sent out the after-disaster work crews to help with cleanup — which includes everything from pulling mud from homes to assisting victims file their claims forms to government and insurance entities. The Convoy of Hope helped feed the masses, arriving on scene in the south with trucks upon trucks of food, even before Irma hit. Samaritan’s Purse, the Rev. Franklin Graham’s organization, brought everything from food to chainsaws. Post-storm, the same group will help victims rebuild their homes. And while FEMA is on scene, too, it’s the Christian groups and faith-based organizations who’ve taken the lead. “FEMA, they have been a big blessing to us, they’re an assistance to us,” said Luther Harrison, vice president of North American Ministries at Samaritan’s Purse, in USA Today. “For Hurricane Irma, the majority of our equipment across the border and FEMA was instrumental in helping us clear that with customs and getting all the paperwork done.” And perhaps most crucial to the cleanup is manpower — something the faith groups bring in droves. United Methodist, for example, has 20,000 or so trained volunteers around the nation, standing at the ready to serve as early responders. All they need is the call — all they need is to be activated — and they’re there. There’s a critical point to note here, and it goes like this: Big Government types would have it believed that government, and only government, can save the citizens from disasters. But before Big Government, there was the Citizenry. And people who put God first, people who are committed to serving Jesus, people who are driven by a moral compass that comes from above, are the real doers and shakers and movers — the ones who see a need and respond. The Big Government types? They see a need and dial their lawmaker — call for a committee hearing — petition for a study. It’s only after they navigate the hoops, and fill out the proper forms, they respond. America’s greatness was, is and always will be rooted in the fact that our rights come from God, not government. With that, comes a responsibility — that we conduct ourselves on an individual basis as if we believe in God. Bluntly put, it’s what the founders believed; it’s how they envisioned a moral and limited government not just shaping, but staying around a while. Happily, it’s what a large portion of America’s population today still believes. But let’s not miss the even bigger lesson that can be gleaned here. If you want to reel in government and curb the bureaucracy, you have to make the services government provides irrelevant. And the way to do that is provide them privately. Government more or less doesn't address the problem....it is the problem. Christian and other charity organizations don't have miles of red tape and regulations to jump through. They just do it and get it done. Every government agency has so many rules and statutes that they have to adhere to that it's amazing that they can accomplish almost anything at all. What is the lake of fire? What is it's purpose? Is the lake of fire eternal hell? Is there any hope of escape for those cast into this lake?http://bible-truths.com/lake1.html Come on now, you guys are lying. It isn't those right wing Bible thumping hypocrites doing all the work, it is the "compassionate" social groups. You know, like the "ANTIFA Relief Organization" and "Atheists Helping the People They Claim to Love" and "Black Lives Matter - Hurricane Relief to the Black Community" and, let's not forget, "Homosexuals United to Love Storm Survivors Because 'Love Wins.'" I'm sure there are others, but I think everyone gets the picture about all these left wing organizations who claim love and that they do so much for humanity. God uses everything to His Glory. I bet ol nick is just spitting at all the Christians helping people and showing that the demonic swamp critters are not only not necessary, but essentially a hindrance it getting things done. Hopefully many eyes are opened to the truth and find their way to Christ. President Trump is busting his ass to allow faith based groups to be treated like other groups and be reimbursed for their rescue aid. It may be the law, but the .gov needs to be firmly guided in non discrimination against religious groups. The .gov can handle bridges and big highways..etc. but they are, by necessity slow. A church, with a few brothers and sisters can get what is needed, where is needed and fast. Of course, the crooks and scamsters also are fast. Sometimes the .gov works best when it gets out of the way. SS . “Then the creatures of the high air answered to the battle, .., and the woods trembled and the wind sobbed telling them, the earth shook,; the witches of the valley, and the wolves of the forests, howled from every quarter and on every side of the armies, urging them against one another.” ― Lady Gregory, Gods and Fighting Men: The Story of the Tuatha De Danaan and the Fianna of Ireland Absolutely true about Christian groups being the vanguard of relief, and I saw this firsthand while doing relief efforts in late 2005 after Hurricane Katrina hit New Orleans and the Gulf shore of Mississippi. Our church went to the Biloxi, MS area to help out with cleanup and recovery efforts in an area that was hard hit but got very little press coverage (New Orleans got most of the attention on the media). We and other Christian groups were really the ONLY people actually on the ground doing real, practical relief work. It was really great to witness cooperation between Christians who did not know each other before and had just met on site, as well as setting aside denominational differences to work together. It's a big part of what being a part of the body of Christ is about! Christians have been doing this sort of thing in various ways since the beginning of the church in the first century AD. NOTICE: Timebomb2000 is an Internet forum for discussion of world events and personal disaster preparation. Membership is by request only. The opinions posted do not necessarily represent those of TB2K Incorporated (the owner of this website), the staff or site host. Responsibility for the content of all posts rests solely with the Member making them. Neither TB2K Inc, the Staff nor the site host shall be liable for any content. All original member content posted on this forum becomes the property of TB2K Inc. for archival and display purposes on the Timebomb2000 website venue. 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Francis O'Reilly Francis Joseph Charles O'Reilly (13 November 1922 – 11 August 2013) was an Irish businessman, noted for his work in the reviving the Irish distillery industry and modernising Ireland's banking. He served as Chancellor of the University of Dublin from 1985 to 1998. Biography O'Reilly was born into an affluent family in Dublin amidst the Irish Civil War, the son of a physician. Descended from John Power of the Powers Gold Label whiskey, he was educated at St Gerard's School, Bray, Ampleforth College in Yorkshire and Trinity College Dublin, earning an engineering degree in 1943. During the Second World War, O'Reilly served with the British Army's Royal Engineers from 1943 to 1946, part of which he spent with the 7th Indian Infantry Division during the liberation of Burma from Imperial Japanese occupation in 1944–45. O'Reilly joined Powers in 1946, during a time when there were only four Irish distilleries left: Powers, Cork Distilleries Company, Jameson Irish Whiskey, and Williams of Tullamore. Since the First World War, the Irish whiskey industry was struggling to stay afloat in the small domestic market, as they had been unable to do normal business in the U.S. because of Prohibition in the United States (1920–33) and the Anglo-Irish Trade War. By the 1960s, O'Reilly was joint managing director of Powers with his cousin John A. Ryan, but whiskey sales were still in decline. O'Reilly realised the industry needed to be radically reorganised. Powers, which had acquired Tullamore in the 1950s, merged with rivals Cork and Jameson to form Irish Distillers, Ltd. (IDL) in 1966, with O'Reilly serving as its first chairman. O'Reilly was credited with several key decisions that made a lasting contribution to Ireland's economy. In particular, O'Reilly was instrumental in selling IDL to the French company Pernod Ricard in 1988, avoiding a hostile takeover by the UK's Grand Metropolitan group. Other activity O'Reilly was president of the Royal Dublin Society (1986–89) and served as the 23rd Chancellor of the University of Dublin (1985–98). He was also a director of Ulster and National Westminster Banks. Equestrian sport O'Reilly was very involved in equestrian sport, and was a member of the National Hunt Steeplechasing Committee, president of the Equestrian Federation of Ireland, chairman of Punchestown Racecourse and of the Kildare Hunt, and steward and trustee of the Turf Club. O'Reilly was instrumental in Dublin being selected as the site of the 10th Show Jumping World Championships in 1982. These world championships were the first to include a compulsory horse inspection prior to the event. Honours O'Reilly was conferred an honorary degree of doctor of laws by Dublin University (1978) and the National University of Ireland (1986). He was also an honorary fellow of his alma mater, Trinity College Dublin. In 2002, O'Reilly was invested as a Knight Commander of the Equestrian Order of St Gregory the Great by Pope John Paul II in recognition of his outstanding contribution to the restoration of the Irish College in Paris. Personal life In 1950, O'Reilly married Teresa "Tess" Williams, of the Tullamore distilling family. They had 10 children: Mary, Charles, Jane, Olivia, Margaret, Rose, Louise, Peter, Paul and Julie. He died at Rathmore, County Kildare at age 91, survived by his wife and 10 children, and several grandchildren and great-grandchildren. References Category:1922 births Category:2013 deaths Category:Alumni of Trinity College Dublin Category:Chancellors of the University of Dublin Category:Disease-related deaths in Ireland Category:People educated at Ampleforth College Category:People from County Dublin Category:Knights Commander of the Order of St Gregory the Great Category:People educated at St Gerard's School, Bray
Vanishing Communities The world’s indigenous suicide crises. Indigenous people generally have a lower life expectancy than their non-native counterparts, In the US, native Americans have the highest rates of suicide for any ethnic group for the 18 to 24 age range. The suicide rate among the Guaraní-Kaiowa indigenous is 34 times higher than the national average. By Beenish Ahmed. ThinkProgress. Published on April 21, 2016 The world’s indigenous suicide crises. Guaranies. Photo: Sebastião Salgado One of the most effective ways to prevent suicide among indigenous communities is instilling a sense of their heritage. A state of emergency was declared after 11 members of a single, remote community of Aboriginal Canadians tried to take their lives earlier this month. But as many indigenous and political leaders noted, the issue isn’t isolated to Attawapiskat Canada — it isn’t even limited to Canada. According to a report by Survive International, interference by outside forces has effected indigenous communities around the world for the worst. “Forcing development on tribal peoples never brings a longer, happier life, but a shorter, bleaker existence only escaped in death,” the reports’ authors wrote last year. In fact, one of the most effective ways to prevent suicide among indigenous communities is instilling a sense of their heritage, or “cultural continuity,” as Christopher Lalonde, a University of Victoria psychologist who has studied the issue, put it. “The government spent 150 years trying to get rid of cultural resiliency, so it becomes a precious commodity,” Lalonde told the CBC. “The notion that ‘My culture is alive, and I am my culture’ is just deeply important.” Here’s a sense of how deeply suicide impacts indigenous communities around the world. Indigenous people generally have a lower life expectancy than their non-native counterparts, according to a new report that looked at half of the world’s indigenous population. The difference is most stark among the Baka in Cameroon who are expected to live 21.5 less years than others in Cameroon. Suicide rates among Canadian aboriginal youth were at least 10 times higher than for the general population of young people, according to the country’s health minister Jane Philpott. Suicide is the leading cause of death for indigenous people under the age of 45. Although suicide has been called an “epidemic” among Canada’s First Nations, it is isolated to certain communities. Just 10 percent of First Nations communities in British Columbia accounted for more than 90 percent of suicides, according to Lalonde’s findings. In the United States, suicide primarily claims the lives of middle-aged white people, but 40 percent of Native Americans who commit suicide are between the ages of 15 and 24. Native Americans have the highest rates of suicide for any ethnic group for the 18 to 24 age range. Native Alaskans had a suicide rate of 51 percent between 2003 and 2006, compared to a 16.9 percent rate among non-Native Alaskans. Greenland, a country where 88 percent of the population is comprised of indigenous Inuits, has the highest suicide rate in the world — six times that of the United States. The suicide rate among Aboriginal and Torres Strait Islanders was 2.6 times higher than the suicide rate for non-Indigenous Australians. The suicide rate among the Guaraní-Kaiowa (Paraguay, the Brazilian state of Mato Grosso do Sul and northeastern Argentina) indigenous is 34 times higher than the national average. ##
Open XML Paper Specification Open XML Paper Specification (also referred to as OpenXPS) is an open specification for a page description language and a fixed-document format. Microsoft developed it as the XML Paper Specification (XPS). In June 2009, Ecma International adopted it as international standard ECMA-388. It is an XML-based (more precisely XAML-based) specification, based on a new print path (print processing data representation and data flow) and a color-managed vector-based document format that supports device independence and resolution independence. In Windows 8 .xps was replaced with the ECMA standard .oxps format which is not natively supported in older Windows versions. Format The XPS document format consists of structured XML markup that defines the layout of a document and the visual appearance of each page, along with rendering rules for distributing, archiving, rendering, processing and printing the documents. Notably, the markup language for XPS is a subset of XAML, allowing it to incorporate vector-graphic elements in documents, using XAML to mark up the Windows Presentation Foundation (WPF) primitives. The elements used are described in terms of paths and other geometrical primitives. An XPS file is a ZIP archive using the Open Packaging Conventions, containing the files which make up the document. These include an XML markup file for each page, text, embedded fonts, raster images, 2D vector graphics, as well as the digital rights management information. The contents of an XPS file can be examined by opening it in an application which supports ZIP files. There are two incompatible XPS formats available. The original document writer printed to .xps in Windows 7 and earlier (i.e., Windows Vista and Windows XP). Beginning with Windows 8, the document writer defaults to the OpenXPS .oxps format. Trying to open .oxps files in Windows 7 or lesser without an .oxps-to.xps converter will result in an error. Microsoft provides two free converters. XpsConverter converts documents between .xps and .oxps format, while OxpsConverter converts documents from .oxps to .xps format. Features XPS specifies a set of document layout functionality for paged, printable documents. It also has support for features such as color gradients, transparencies, CMYK color spaces, printer calibration, multiple-ink systems and print schemas. XPS supports the Windows Color System color management technology for color conversion precision across devices and higher dynamic range. It includes a software raster image processor (RIP) (downloadable separately). The print subsystem supports named colors, simplifying color definition for images transmitted to printers supporting those colors. XPS supports HD Photo images natively for raster images. The XPS format used in the spool file represents advanced graphics effects such as 3D images, glow effects, and gradients as Windows Presentation Foundation primitives, which printer drivers could offload their rasterization to the printer in order to reduce computational load if the printer is capable of rasterizing those primitives. Similarities with PDF and PostScript Like Adobe Systems's PDF format, XPS is a fixed-layout document format designed to preserve document fidelity, providing device-independent document appearance. PDF is a database of objects that may be created from PostScript or generated directly from applications, whereas XPS is based on XML. Both formats are compressed, albeit using different methods. The filter pipeline architecture of XPS is also similar to the one used in printers supporting the PostScript page description language. PDF includes dynamic capabilities purposely not supported by the XPS format. Viewing and creating XPS documents XPS is supported on several versions of Windows. Because the printing architecture of Windows Vista uses XPS as the spooler format, it has native support for generating and reading XPS documents. XPS documents can be created by printing to the virtual XPS printer driver. The XPS Viewer is installed by default in Windows Vista, Windows 7, Windows 8, and Windows 10 (until version 1709). The viewer is hosted within Internet Explorer (IE) in Windows Vista, but is a native application in 7 and 8 which supports digital signatures. Windows 8 also has a Reader application that reads XPS, OXPS, and PDF files. The IE-hosted XPS viewer and the XPS Document Writer are also available to Windows XP users when they download the .NET Framework 3.0. The IE-hosted viewer supports digital rights management and digital signatures. Users who do not wish to view XPS documents in the browser can download the XPS Essentials Pack, which includes a standalone viewer and the XPS Document Writer. The XPS Essentials Pack also includes providers to enable the IPreview and IFilter capabilities used by Windows Desktop Search, as well as shell handlers to enable thumbnail views and file properties for XPS documents in Windows Explorer. The XPS Essentials Pack is available for Windows XP, Windows Server 2003, and Windows Vista. Installing this pack enables operating systems prior to Windows Vista to use the XPS print spooler instead of the GDI-based WinPrint, which can produce better quality prints for printers that support XPS in hardware (directly consume the format). The print spooler format on these operating systems when printing to older, non-XPS-aware printers, however, remains unchanged. Third-party support Software Hardware XPS had the support of printing companies such as Konica Minolta, Sharp, Canon, Epson, Hewlett-Packard, and Xerox and software and hardware companies such as CSR (formerly Zoran), and Global Graphics. Native XPS printers were introduced by Canon, Konica Minolta, Toshiba, and Xerox. Devices at the Certified for Windows level of Windows Logo conformance certification were required to have XPS drivers for printing since 1 June 2007. Licensing Microsoft released XPS under a royalty-free patent license called the Community Promise for XPS, allowing users to create implementations of the specification that read, write and render XPS files as long as they included a notice within the source that technologies implemented may be encumbered by patents held by Microsoft. Microsoft also required that organizations "engaged in the business of developing (i) scanners that output XPS Documents; (ii) printers that consume XPS Documents to produce hard-copy output; or (iii) print driver or raster image software products or components thereof that convert XPS Documents for the purpose of producing hard-copy output, [...] will not sue Microsoft or any of its licensees under the XML Paper Specification or customers for infringement of any XML Paper Specification Derived Patents (as defined below) on account of any manufacture, use, sale, offer for sale, importation or other disposition or promotion of any XML Paper Specification implementations." The specification itself was released under a royalty-free copyright license, allowing its free distribution. On September 13, 2011, Monotype Imaging announced it had licensed its XPS-to-PCL 6 and XPS-to-PostScript vector conversion filters to Microsoft for use in the next version of Windows. History and standardization In 2003, Global Graphics was chosen by Microsoft to provide consultancy and proof of concept development services on XPS and worked with the Windows development teams on the specification and reference architecture for the new format. Microsoft submitted the XPS specification to Ecma International. In June 2007 Ecma International Technical Committee 46 (TC46) was set up to develop a standard based on the Open XML Paper Specification (OpenXPS). At the 97th General Assembly held in Budapest, June 16, 2009, Ecma International approved Open XML Paper Specification (OpenXPS) as an Ecma standard (ECMA-388). TC46's members included: Autodesk Brother Industries Canon CSR (formerly Zoran) Fujifilm Fujitsu Global Graphics Hewlett Packard Konica Minolta Lexmark Microsoft Monotype Imaging Océ Technologies Panasonic/Matsushita Ricoh Software Imaging Toshiba Xerox See also Comparison of OpenXPS and PDF Windows Vista printing technologies Functional specification References External links XML Paper Specification Version 1.0 (via Internet Archive; from 2006-11-17) Microsoft XPS Development Team Blog Standard ECMA-388 Open XML Paper Specification View and Generate XPS Documents (via Internet Archive; from 2006-12-05) View and Generate XPS Documents (via Internet Archive; from 2009-12-14) Xps to Pdf Conversion online Category:Computer printers Category:Digital press Category:Document-centric XML-based standards Category:Ecma standards Category:Office document file formats Category:Page description markup languages Category:XML Category:XML-based standards Category:Open formats
While ostensibly commissioning a new naval warship, Donald Trump couldn't help but plead for political support from the audience, as his presidency sinks like a stone. Donald Trump may have gotten a lot of use out of the "I'm not a politician" line, but at the ceremony Saturday for a new naval warship, he sure acted like a politician. While speaking to United States Navy service members and guests, prior to the commissioning of the USS Gerald R. Ford, Trump praised the ship in typical Trump fashion. Calling it "the greatest ship anywhere in the world," Trump extolled "this wonderful, beautiful, but very, very powerful" ship which would cause "our enemies [to] shake with fear." "We will win, win, win. We will never lose. We will win," continued his profound remarks. Trump also did take time to praise the men and women in uniform before him, as well as former President Ford and his wife. But of course, he could not resist an opportunity to lash out at the previous administration, to put himself forth as some kind of savior, and to demand political support for his disastrous causes. He railed against the budget sequester from 2013 — for which he could lay blame on his own party, though of course he only referred to "our government" as being at fault — declaring, to members of our military, that "It's been a very, very bad period of time for our military." He then referenced his proposed budget, nagging at Congress to "do its job" and pass it — a budget that callously targets veterans, would hobble counterterrorism efforts, and would, in fact, "kill a lot of people." Slipping bizarrely into third person, he pointedly stated of his promised funding increases, "President Trump, I will tell you, you will get it." But then he got even stranger, exhorting the audience to call their elected officials and offer support for his budget — oh, and also, ask them to pass their health care repeal bill that is a daily increasing disaster. For years, our government has subjected the military to unpredictable funding and a devastating defense sequester — you remember that, sequester, not good. This has led to deferred maintenance, a lack of investment in new equipment and technology, and a shortfall in military readiness. In other words, it's been a very, very bad period of time for our military. That is why we reached a deal to secure an additional $20 billion for defense this year, and it is going up. And why I asked Congress for another $54 billion for next year. Now we need Congress to do its job and pass the budget that provides for higher, stable, and predictable funding levels for our military needs that our fighting men and women deserve. And you will get, believe me. President Trump, I will tell you, you will get it. Don't worry about it. But I don't mind getting a little hand, so call that congressman and call that senator and make sure you get it. [applause] And by the way, you can also call those senators to make sure you get health care. That Trump could, and would, take a pleasant and ceremonial occasion such as this and turn it nearly into a stump speech is odious. And it goes to show that the "not a politician" sheen wears off quickly once one occupies the White House. And pleading for support for the failures that are his budget and the GOP health care bill makes the downward spiral in which his presidency is foundering even more obvious. As of the recent six-month mark of his time in office, Trump had the lowest approval ratings of any president going back through the entire 70 years of modern polling. In the growing shadow of the Russia scandal, he has been avoiding the press, spending his time golfing — golfing a lot — and babbling on Twitter. And now, instead of giving a nice ceremonial speech and leaving politics at the gangway, Trump could not resist trying to make his predecessor look bad and begging for the audience to help make himself look better.
1. Introduction {#sec1-molecules-23-00506} =============== Persistent inflammation is a central component of numerous widespread and devastating chronic diseases, including osteoarthritis, atherosclerosis, cancer, type 2 diabetes, and Alzheimer's disease \[[@B1-molecules-23-00506]\], that dominate in older people \[[@B2-molecules-23-00506]\]. The pro-inflammatory eicosanoids prostaglandin (PG) E~2~ and leukotrienes (LT), produced by cyclooxygenase (COX) and 5-lipoxygenase (5-LO) pathways from arachidonic acid (AA), as well as secreted proteases from innate immune cells, significantly contribute to the inflammatory response \[[@B3-molecules-23-00506]\]. Non-steroidal anti-inflammatory drugs (NSAIDs) suppress inflammation, essentially by interference with eicosanoid biosynthesis, and they are the most frequently used therapeutics to treat chronic inflammatory diseases \[[@B4-molecules-23-00506]\]. Moreover, NSAIDs are considered as the most commonly self-prescribed class of anti-inflammatory and anti-nociceptive drugs in the elderly population \[[@B5-molecules-23-00506]\]. However, long-term use of NSAIDs is associated with frequent and severe side effects, such as gastric and renal toxicities \[[@B4-molecules-23-00506]\], and they can reduce the efficacy of diuretics and ACE inhibitors, drugs that are commonly used by elderly patients \[[@B5-molecules-23-00506]\]. The gum resin from various Boswellia species, termed frankincense, is a traditional Ayurvedic medicine that has experienced increasing popularity also in Western countries during the past decades \[[@B6-molecules-23-00506]\]. Frankincense is a natural and rich source for a variety of triterpene acids, including boswellic acids (BAs), tirucallic acids, roburic acids, and lupeolic acids \[[@B7-molecules-23-00506],[@B8-molecules-23-00506],[@B9-molecules-23-00506]\]. In folk medicine, lipophilic frankincense extracts are used as alternative to anti-inflammatory steroidal drugs (i.e., glucocorticoids) or NSAIDs for treatment of inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, asthma, atopic dermatitis, and inflammatory bowel diseases \[[@B6-molecules-23-00506]\]. The 3-O-acteyl-11-keto-β-BA (AKBA 1), 11-keto-β-BA (KBA 2), β-BA 3, and 3-O-acteyl-β-BA (ABA 4) are pentacyclic triterpene acids that represent major ingredients in lipophilic frankincense extracts, reaching 14 to 25% (m/m) \[[@B7-molecules-23-00506],[@B10-molecules-23-00506]\]. Previous studies have shown that BAs display potent anti-inflammatory properties by interfering with multiple targets that are involved in the initiation and maintenance of inflammation \[[@B6-molecules-23-00506],[@B11-molecules-23-00506]\]. For example, BAs were reported to inhibit the key enzymes 5-LO \[[@B12-molecules-23-00506],[@B13-molecules-23-00506]\], COX \[[@B14-molecules-23-00506]\], and microsomal prostaglandin E~2~ synthase (mPGES)-1 \[[@B15-molecules-23-00506]\] in pro-inflammatory LT and prostaglandin (PG)E~2~ biosynthesis, the neutrophil proteases elastase \[[@B16-molecules-23-00506]\] and cathepsin G \[[@B17-molecules-23-00506]\], the immunoregulating antimicrobial peptide LL-37 \[[@B18-molecules-23-00506]\], inhibitor of NFκB (IkB) kinases \[[@B19-molecules-23-00506]\] and lipopolysaccharide (LPS) activity \[[@B20-molecules-23-00506]\]. Inhibition of 5-LO, mPGES-1 and cathepsin G received much attention as major targets of BAs being responsible for suppressing inflammation, and animal studies as well as clinical trials confirmed that isolated BAs or frankincense extracts suppress PGE~2~ formation and cathepsin G activity in vivo \[[@B15-molecules-23-00506],[@B17-molecules-23-00506],[@B21-molecules-23-00506]\]. Notably, the potencies of the four different BAs varied depending on the nature of the target, and in many cases (e.g., for 5-LO, cathepsin G, and mPGES-1), AKBA was the most potent derivative \[[@B11-molecules-23-00506],[@B12-molecules-23-00506],[@B13-molecules-23-00506],[@B15-molecules-23-00506],[@B17-molecules-23-00506]\]. We previously showed that for mPGES-1, other triterpene acids from frankincense than BAs inhibit the enzyme with even superior potencies versus BAs \[[@B9-molecules-23-00506]\]. Thus, the tetracyclic tirucallic acids 14--17 and the pentacyclic lupeolic acid derivative 12 were found to be up to 8-fold more potent than AKBA in vitro \[[@B9-molecules-23-00506]\], implying that these triterpene acids may contribute to the pharmacological/anti-inflammatory properties of crude frankincense extracts as well. Since only BAs have been evaluated for inhibition of 5-LO and cathepsin G, we here performed a comprehensive analysis of frankincense-derived tetra- and pentacyclic triterpene acids that we investigated for interference with 5-LO and cathepsin G. Moreover, a panel of novel semi-synthetic BA-derivatives were synthesized and examined along these lines, to further explore the critical pharmacophores. 2. Results {#sec2-molecules-23-00506} ========== 2.1. Isolation and Semi-Synthesis of the Triterpene Acids {#sec2dot1-molecules-23-00506} --------------------------------------------------------- The four BAs 1--4, the three roburic acids 5--7, dihydro-nyctanthic acid (DHNA 8), the four lupeolic acids 9--12, and the five tirucallic acids 13--17 ([Table 1](#molecules-23-00506-t001){ref-type="table"}) were isolated from gum resins of different Boswellia species by preparative high-performance liquid chromatography (HPLC), as described previously \[[@B9-molecules-23-00506],[@B22-molecules-23-00506]\]. The R- and S-configured ally-alcohols 18 and 19 were synthesized from KBA and AKBA using a combination of LiBr and NaBH~4~ in diglyme at reflux temperature, as described \[[@B23-molecules-23-00506],[@B24-molecules-23-00506]\]. The ABA and AKBA derivatives with modified C3 position were prepared as follows: β-BA 3 or KBA 2 were treated with oxalyl chloride to form the half-ester oxaloyl-BA 20 and oxaloyl-KBA 21; β-BA 3 or KBA 2 were treated with succinic or glutaric anhydride to form the half-ester succinoyl-BA 22 and succinoyl-KBA 23 as well as glutaroyl-BA 24 and glutaroyl-KBA 25; the carboxymethyl-BA 26 and carboxymethyl-KBA 27 were synthesized from β-BA 3 or KBA 2 using 2-chloro acetic acid and NaH in THF over night as described earlier \[[@B23-molecules-23-00506],[@B24-molecules-23-00506]\]. All compounds were analyzed by ^1^H- and ^13^C-NMR, as well as by mass spectrometry. 2.2. Inhibition of 5-LO by Natural Occurring Triterpene Acids {#sec2dot2-molecules-23-00506} ------------------------------------------------------------- In analogy to our previous SAR study on 17 triterpene acids from frankincense that were tested for inhibition of mPGES-1 activity in a cell-free assay \[[@B9-molecules-23-00506]\], we analyzed the effects of these compounds for their inhibitory effects against human isolated 5-LO in a suitable cell-free test system, where 20 µM arachidonic acid was exogenously added as substrate \[[@B25-molecules-23-00506]\]. The well-recognized 5-LO inhibitor BWA4C \[[@B25-molecules-23-00506]\] was used as reference drug that inhibited 5-LO with IC~50~ = 0.04 µM. In agreement with previous data \[[@B13-molecules-23-00506]\], AKBA 1 and KBA 2 efficiently inhibited 5-LO activity with IC~50~ = 3.0 and 4.6 µM, respectively, while β-BA 3 and ABA 4 suppressed 5-LO activity at 10 µM by only 21--26% ([Table 1](#molecules-23-00506-t001){ref-type="table"}). Among the three roburic acids 5--7, only 7 caused some (27%) inhibition at 10 µM, while 5 and 6 failed in this respect. Similarly, DHNA 8 and the lupeolic acids 9 and 10 did not markedly inhibit 5-LO, but the hydroxylated lupeolic acids 11 and 12 were significantly active, with IC~50~ = 10 and 8.3 µM, respectively ([Table 1](#molecules-23-00506-t001){ref-type="table"}). Among the tirucallic acids (13--17), 14 and 15 caused weak 5-LO inhibition at 10 µM (34 and 36%, respectively), and other members were hardly active. Together, AKBA 1 and KBA 2 are clearly the most potent direct 5-LO inhibitors out of the 17 triterpene acids from frankincense, which underlines that different structural requirements of triterpene acids are required for efficient inhibition of 5-LO versus mPGES-1 \[[@B9-molecules-23-00506]\]. Several studies reported that frankincense extracts, as well as AKBA 1 and KBA 2, are superior inhibitors of 5-LO in intact cells as compared to cell-based assays, for multiple possible reasons that remain unclear \[[@B6-molecules-23-00506],[@B12-molecules-23-00506],[@B13-molecules-23-00506],[@B26-molecules-23-00506]\]. Thus, we studied the triterpene acids also against 5-LO activity in a well-established cell-based assay using human neutrophils stimulated with A23187 \[[@B25-molecules-23-00506]\]. The reference 5-LO inhibitor BWA4C was about 4-fold less active in neutrophils (IC~50~ = 0.16 µM) as compared to cell-free assay conditions. Both AKBA 1 and KBA 2 were somewhat more potent in intact cells (IC~50~ = 3.0 and 3.5 µM) versus isolated 5-LO, and also for β-BA 3, but not for ABA 4, where slightly more efficient 5-LO inhibition was observed (47% inhibition at 10 µM, [Table 1](#molecules-23-00506-t001){ref-type="table"}). Again, roburic acids 5 and 6 as well as DHNA 8 were hardly active, but 7 caused potent suppression of 5-LO with IC~50~ = 4.3 µM, being almost equally effective as AKBA 1 and KBA 2. Along these lines, also, the lupeolic acids 9, 11, and 12 (but not 10) suppressed 5-LO product formation, with IC~50~ = 4.0, 4.6, and 5.1 µM. Although tirucallic acids 13--17 failed to inhibit isolated 5-LO, 13--16 were quite effective in intact cells, with 14 being about 3-fold more potent (IC~50~ = 1.1 µM) than AKBA 1, and with 13 and 15 displaying similar potencies (IC~50~ = 2.9 and 3 µM). In conclusion, among the 17 triterpene acids, the tirucallic acid 14 is the most potent inhibitor of 5-LO product biosynthesis in neutrophils, thus outperforming AKBA, despite lack of pronounced effectiveness against isolated 5-LO. 2.3. Inhibition of Cathepsin G by Natural Occurring Triterpene Acids {#sec2dot3-molecules-23-00506} -------------------------------------------------------------------- Since cathepsin G is a pharmacological relevant target for BAs and frankincense with high affinities to BAs \[[@B17-molecules-23-00506]\], we analyzed also the effects of the 17 triterpene acids for inhibition of cathepsin G activity in a cell-free assay. Since cathepsin G is a secreted protease that acts outside the cell upon degranulation, cell-based assays are not feasible for cathepsin G inhibitor studies as compared to 5-LO. JNJ-1031795 was used as reference drug \[[@B27-molecules-23-00506]\] that blocked cathepsin G activity with IC~50~ = 0.06 µM. The rank order for cathepsin G inhibition by the BAs was β-BA 3 \> AKBA 1 \> ABA 4 \> KBA 2, with IC~50~ = 0.5, 0.6, 2.0, and 4.1 µM, respectively. Among all other triterpene acids tested at 10 µM, only the lupeolic acid 10 exhibited \>50% inhibition of cathepsin G with an IC~50~ = 7.5 µM. Nevertheless, marked suppression of cathepsin G activity (i.e., 27--44% inhibition at 10 µM) was found for all roburic acids (5--7), DHNA 8, lupeolic acids 9 and 11, and tirucallic acid 13, 14, 16, and 17. Only 12 and 15 failed to markedly inhibit cathepsin G. Together, cathepsin G is a clearly preferred target for BAs (particularly β-BA 3 and AKBA 1), with much less susceptibility for all other triterpene acids tested. 2.4. Effects of Semi-Synthetic BAs against 5-LO and Cathepsin G {#sec2dot4-molecules-23-00506} --------------------------------------------------------------- Next, we investigated a set of semi-synthetic BA derivatives for inhibition of 5-LO and cathepsin G to further explore the structure-activity relationships (SARs) of BAs. First, the 11-keto moiety of KBA was reduced to hydroxy, yielding the R- and S-configured ally-alcohols 18 and 19. Of interest, 18 was somewhat more efficient to inhibit 5-LO in neutrophils (IC~50~ = 2.8 µM) versus parental KBA 2 while 19 was less active, and both compounds exhibited only weak effects against 5-LO in cell-free assays (\<25% inhibition at 10 µM). Also, cathepsin G activity was less pronouncedly affected by 18 and 19. Then, we replaced the 3-O-acetyl moiety of AKBA 1 or ABA 4 by acidic moieties, such as oxaloyl, succinoyl, glutaroyl, and carboxymethyl groups. All these compounds were not or only weak 5-LO inhibitors, with \<25% inhibition at 10 µM in cell-free assays. Similarly, in intact cells, 5-LO product formation was not or barely affected (IC~50~ \> 10 µM); the most potent compound was the carboxymethyl-BA 26 that inhibited 5-LO activity by 42% at 10 µM. By contrast, pronounced cathepsin G inhibition was observed for BA derivatives lacking the 11-keto moiety, such as oxaloyl-BA 20, glutaroyl-BA 24, and carboxymethyl-BA 26, with IC~50~ = 4.7, 3.6, and 3.4 µM. Of interest, even though AKBA 1 (an 11-keto derivative carrying a 3-acetoyl moiety) was quite potent (IC~50~ = 0.6 µM), the 11-keto derivatives oxaloyl-KBA 21, succinoyl-KBA 23, and carboxymethyl-KBA 27 were not active, and glutaroyl-KBA 25 (IC~50~ = 8.6) was less efficient than the respective counterpart, 24, lacking the 11-keto moiety. Taken together, modification of the acetoyl group of AKBA 1 or ABA 4 towards acidic residues is strongly detrimental for inhibition of 5-LO, and to a varying extent, also for interference with cathepsin G, depending on the presence of the 11-keto moiety. 3. Discussion {#sec3-molecules-23-00506} ============= Here, we report the SAR of various natural occurring triterpene acids from frankincense, and of novel semi-synthetic BA derivatives for inhibition of the two molecular targets, 5-LO and cathepsin G. For 5-LO, the SARs depend on the assay type: for direct interaction with 5-LO in the cell-free assay, AKBA 1 and KBA 2 were most potent, and neither natural nor semi-synthetic triterpene acids were comparably active. Modification of the acetoyl group of AKBA 1 towards acidic residues is clearly detrimental for interference with 5-LO. For inhibition of 5-LO product formation in neutrophils, however, several of the natural triterpene acids were efficient and outperformed AKBA 1, such as the tirucallic acids 13 and 14. Since tirucallic acids were shown to interfere with various signaling pathways \[[@B28-molecules-23-00506],[@B29-molecules-23-00506]\], among others, Ca^2+^ mobilization and mitogen-activated protein kinase kinase (MEK)-1/2, that could also activate 5-LO in intact cells, one may speculate that tirucallic acids and possibly also roburic and lupeolic acids interfere with the activation of 5-LO, and thus, with 5-LO product biosynthesis. In fact, lupeolic acids (e.g., 12) were shown to suppress the biosynthesis of various eicosanoids by interference with cytosolic phospholipase A2 \[[@B8-molecules-23-00506]\] that provides substrate to 5-LO. In analogy to interference with 5-LO, inhibition of cathepsin G activity was also most efficient for BAs, particularly for β-BA 3 and AKBA 1, with submicromolar IC~50~ values. Among the natural triterpene acids, only the lupeolic acid 10 reached an IC~50~ below 10 µM, even though several other triterpene acids significantly inhibited cathepsin G at 10 µM, albeit less than 50%. Among the semisynthetic BAs lacking the 11-keto moiety, oxaloyl-BA 20, glutaroyl-BA 24, and carboxymethyl-BA 26 were efficient cathepsin G inhibitors, indicating that the 11-keto moiety is dispensable for cathepsin G interference. In summary, among the 17 investigated natural occurring triterpene acids from frankincense, the BAs provide the most potent inhibitors of 5-LO (i.e., AKBA 1) and cathepsin G (i.e., β-BA 3). Nevertheless, some lupeolic acids are efficient as well, and for inhibition of 5-LO product formation in intact cells, diverse representatives from all types of triterpene acids are active, even with superior potency (e.g., tirucallic acid 14) than BAs. Notably, our previous SAR study on the same 17 triterpene acids for inhibition of mPGES-1 revealed opposite findings: thus, all six tirucallic acids were more potent than the BAs, and also, the lupeolic acid 12 and the roburic acid 7 were clearly superior \[[@B9-molecules-23-00506]\]. Together, dual interference with cathepsin G and 5-LO product formation in human neutrophils, on top of mPGES-1 inhibition, represents a beneficial pharmacological profile, and may qualify triterpene acids as anti-inflammatory natural products and pharmacological leads for intervention with inflammatory diseases. 4. Materials and Methods {#sec4-molecules-23-00506} ======================== 4.1. Chemistry {#sec4dot1-molecules-23-00506} -------------- General information. All reactions were run in dried solvents under N~2~ in flame dried glassware. THF and diethyl ether were dried with Na/benzophenone under N~2~. Flash chromatography was done with silica gel (Merck, Darmstadt, Germany) with particle size 40--63 µm and with distilled solvents. Reagents were bought from chemical suppliers and used without further purification. NMR spectra (^1^H, ^13^C, Distortionless Enhancement of Polarization Transfer (DEPT), H,H-Correlation Spectroscopy (H,H-COSY), Hetero Single Quantum Coherence (HSQC), Hetero Multiple Bond Correelation (HMBC), Nuclear Overhauser Effect Spectroscopy (NOESY) were recorded with a Bruker AV 500 (Bruker, Rheinstetten, Germany) at room temperature (rt). Mass spectra (MS, HRMS) were recorded on a Finnigan MAT 95S (Thermo Fisher Scientific, Waltham, MA, USA). Optical rotations were measured on a Perkin Elmer polarimeter type 241MC (Perkin Elmer, Rodgau, Germany) Melting points were measured with a Büchi melting point apparatus (Dr. Tottolli) (Büchi Labortechnik GmbH, Essen, Germany) and are uncorrected. The following original data on the chemistry of compounds 18--27 are reported also in the doctoral thesis by Nicole Kather, 2007 \[[@B24-molecules-23-00506]\]. 11α-Hydroxy-β-boswellic acid 18 and 11β-hydroxy-β-boswellic acid 19. NaBH~4~ (121 mg, 3.2 mmol) was dissolved in dry diglyme (3.5 mL). LiBr (278 mg, 3.2 mmol) was added with stirring at rt. After 30 min, β-KBA 2 (300 mg, 0.64 mmol) was added and the mixture was refluxed for 1 h. After cooling to rt, the mixture was diluted with cold water (approx. 5 mL) and acidified carefully to pH 4 with 1 N HCl. The mixture was extracted three times with diethyl ether (ca. 10 mL each). The combined ethereal extracts were washed with water and brine, dried with MgSO~4~, and filtered, and the solvent was evaporated in vacuo at rt. The crude product was purified by flash chromatography (pentane/diethyl ether 1:1 + 1% acetic acid) to yield 83 mg of 18 and 141 mg of 19 as white solids. 11-α-Hydroxy-β-boswellic acid 18: ^1^H-NMR ((CD~3~)~2~CO, 500.13 MHz): δ \[ppm\] = 5.22 (d, J = 2.1 Hz, 1H, H-12), 4.21 (d, J = 9.1 Hz, 1H, H-11), 3.98 (bs, 1H, H-3), 2.22--2.06 (m, 3H, H-1β, H-2β, H-16α), 1.94--1.86 (m, 1H, H-6β), 1.84--1.75 (m, 1H, H-15β), 1.75--1.68 (m, 2H, H-6α, H-9), 1.68--1.60 (m, 2H, H-1α, H-5), 1.60--1.50 (m, 1H, H-7α), 1.50--1.30 (m, 8H, H-2α, H-7β, H-18, H-19, H-21α, H-21β, H-22α, H-22β), 1.29 (s, 3H, H-23), 1.22 (bs, 4H, H-15α, H-27), 1.14 (s, 3H, H-26), 1.09 (s, 3H, H-25), 0.93 (bs, 5H, H-16β, H-20, H-30), 0.90 (d, J = 6.2 Hz, 3H, H-29), 0.83 (s, 3H, H-28). ^13^C-NMR ((CD~3~)~2~CO, 125.76 MHz): δ \[ppm\] = 180.3 (C-24, COOH), 142.2 (C-13, H\>C=C\<), 132.8 (C-12, H\>C=C\<), 71.7 (C-3, HO\>CH-), 69.6 (C-11, HO\>CH-), 60.3 (C-18, \>CH-), 55.4 (C-9, \>CH-), 50.7 (C-5, \>CH--), 49.3 (C-4, \>C\<), 45.0 (C-8, \>C\<), 44.1 (C-14, \>C\<), 43.2 (C-22, --CH2--), 41.4 (C-19, \>CH--), 41.4 (C-20, \>CH--), 40.5 (C-10, \>C\<), 37.8 (C-1, --CH~2~--), 36.1 (C-7, --CH~2~--), 35.5 (C-17, \>C\<), 32.9 (C-21, --CH~2~--), 30.2 (C-28, --CH~3~), 29.8 (C-16, --CH~2~--), 28.4 (C-15, --CH~2~--), 28.3 (C-2, --CH~2~--), 26.2 (C-23, --CH~3~), 24.3 (C-27, --CH~3~), 22.6 (C-30, --CH~3~), 21.6 (C-6, --CH~2~--), 19.7 (C-26, --CH~3~), 18.9 (C-29, --CH~3~), 15.8 (C-25, --CH~3~). MS (EI, 70 eV): m/z (%) = 454 (100) \[M-H~2~O\]^+^, 439 (8), 421 (12), 325 (4), 301 (12), 269 (8), 255 (45), 253 (6), 237 (4), 215 (5). HRMS (EI, 70 eV): calculated: 472.3587 for C~30~H~48~O~4~; found: 472.3570. \[α\]^D^: +51.7° (c = 1.61, acetone). m.p. 166--169 °C (dec). 11-β-Hydroxy-β-boswellic acid 19: ^1^H-NMR ((CD~3~)~2~CO, 500.13 MHz): δ \[ppm\] = 5.30 (d, J = 4.3 Hz, 1H, H-12), 4.45 (t, J = 4.7 Hz, 1H, H-11), 4.00 (t, J = 2.4 Hz, 1H, H-3), 2.33--2.25 (m, 1H, H-2β), 2.12--2.05 (m, 1H, H-16α), 2.00--1.88 (m, 2H, H-6β, H-15β), 1.84--1.78 (m, 1H, H-1β), 1.76--1.70 (m, 1H, H-6α), 1.70--1.60 (m, 2H, H-1α, H-7α), 1.58--1.50 (m, 3H, H-2α, H-5, H-9), 1.47--1.43 (m, 1H, H-22α), 1.43 (s, 3H, H-25), 1.43--1.36 (m, 3H, H-18, H-19, H-21β), 1.36--1.33 (m, 3H, H-7β, H-21α, H-22β), 1.33 (s, 3H, H-26), 1.28 (s, 3H, H-23), 1.15--1.10 (m, 1H, H-15α), 1.09 (s, 3H, H-27), 1.09--1.06 (m, 1H, H-16β), 0.93 (s, 3H, H-30), 0.93--0.87 (m, 1H, H-20), 0.87 (s, 3H, H-28), 0.84 (d, J = 5.9 Hz, 3H, H-29). ^13^C-NMR ((CD~3~)~2~CO, 125.76 MHz): δ \[ppm\] = 180.2 (C-24, COOH), 142.1 (C-13, H\>C=C\<), 131.7 (C-12, H\>C=C\<), 71.8 (C-3, HO\>CH--), 66.8 (C-11, HO\>CH--), 60.8 (C-18, \>CH--), 53.9 (C-9, \>CH--), 51.7 (C-5, \>CH--), 48.7 (C-4, \>C\<), 44.4 (C-14, \>C\<), 43.3 (C-22, --CH~2~--), 41.6 (C-8, \>C\<), 41.4 (C-19, \>CH--), 41.4 (C-20, \>CH--), 40.6 (C-10, \>C\<), 35.6 (C-1, --CH~2~--), 35.4 (C-17, \>C\<), 35.4 (C-7, --CH~2~--), 32.9 (C-21, --CH~2~--), 30.4 (C-28, --CH~3~), 29.8 (C-16, --CH~2~--), 29.0 (C-15, --CH~2~--), 28.1 (C-2, --CH~2~--), 25.8 (C-23, --CH~3~), 23.9 (C-27, --CH~3~), 22.3 (C-30, --CH~3~), 22.1 (C-6, --CH~2~--), 20.7 (C-26, --CH~3~), 18.7 (C-29, --CH~3~), 17.7 (C-25, --CH~3~). MS (EI, 70 eV): m/z (%) = 472 (4) \[M\]^+^, 454 (100), 421 (8), 273 (2), 255 (6), 234 (3), 203 (3). HRMS (EI, 70 eV): calculated: 472.3552 for C~30~H~48~O~4~; found: 472.3552. \[α\]^D^: +117.5° (c = 0.63, acetone). m.p. 125--128 °C (dec). 3-O-Oxaloyl-β-boswellic acid 20. β-BA 3 (150 mg, 0.33 mmol) was dissolved in dry THF (2.3 mL). This solution was added dropwise with stirring to a solution of oxaly dichloride (288 μL, 3.30 mmol) in THF (1 mL). After 30 min at rt, cold water (ca. 50 mL) was added, and the mixture was extracted three times with diethyl ether (ca. 10 mL each). The combined ethereal extracts were washed with H~2~O and brine, and dried with MgSO~4~. After filtering, the MgSO~4~ the solvent was evaporated in vacuo to yield 140 mg of 20. ^1^H-NMR ((CD~3~)~2~CO, 500.13 MHz): δ \[ppm\] = 5.40 (t, J = 2.6 Hz, 1H, H-3), 5.20 (t, J = 3.5 Hz, 1H, H-12), 2.31--2.22 (m, 1H, H-2β), 2.12--2.08 (m, 1H, H-16α), 2.01--1.85 (m, 4H, H-6β, H-11α, H-11β, H-15β), 1.82--1.76 (m, 1H, H-6α), 1.75--1.65 (m, 2H, H-2α, H-9), 1.65--1.53 (m, 3H, H-1β, H-5, H-7α), 1.48--1.28 (m, 7H, H-1α, H-7β, H-18, H-19, H-21β, H-22α, H-22β), 1.28 (bs, 4H, H-21α, H-23), 1.15 (s, 3H, H-27), 1.10 (s, 3H, H-26), 1.08--1.04 (m, 1H, H-15α), 0.99 (s, 3H, H-25), 0.93 (bs, 4H, H-20, H-30), 0.83 (s, 3H, H-28), 0.83 (t, J = 6.0 Hz, 3H, H-29). ^13^C-NMR ((CD~3~)~2~CO, 125.76 MHz): δ \[ppm\] = 178.5 (C-24, COOH), 160.3 (C-32, COOH (oxalyl)), 160.0 (C-31, \>C=O (oxalyl)), 141.4 (C-13, H\>C=C\<), 126.5 (C-12, H\>C=C\<), 78.5 (C-3, HO\>CH--), 61.0 (C-18, \>CH--), 52.1 (C-5, \>CH--), 48.6 (C-9, \>CH--),48.2 (C-4, \>C\<), 44.0 (C-8, \>C\<), 43.2 (C-22, --CH~2~--), 41.8 (C-14, \>C\<), 41.5 (C-19, \>CH--), 41.4 (C-20, \>CH--), 39.1 (C-10, \>C\<), 36.2 (C-1, --CH~2~--), 35.5 (C-17, \>C\<), 34.8 (C-7, --CH~2~--), 32.9 (C-21, --CH~2~--), 30.2 (C-28, --CH~3~), 29.8 (C-16, --CH~2~--), 28.2 (C-15, --CH~2~--), 25.1 (C-2, C-11, --CH~2~--), 25.0 (C-23, --CH~3~), 24.7 (C-27, --CH~3~), 22.6 (C-30, --CH~3~), 21.5 (C-6, --CH~2~--), 18.9 (C-29, --CH~3~), 18.4 (C-26, --CH~3~), 14.8 (C-25, --CH~3~). MS (CI, 150 eV): m/z (%) = 484 (5) \[M-CO~2~\]^+^, 447 (12), 394 (7), 307 (14), 231 (4), 218 (100), 203 (17). HRMS (CI, 150 eV): calculated: 484.3495 for C~31~H~48~O~4~ (C~32~H~48~O~6~-CO~2~); found: 484.3524. \[α\]^D^: +56.5° (c = 0.78, acetone). m.p. 212--215 °C (dec). 3-O-Oxaloyl-11-keto-β-boswellic acid 21 was prepared analogously to 20 (see above). ^1^H-NMR (CDCl~3~, 500.13 MHz): δ \[ppm\] = 5.57 (s, 1H, H-12), 5.43 (bs, 1H, H-3), 2.61--2.56 (m, 1H, H-1β), 2.46 (s, 1H, H-9), 2.35--2.25 (m, 1H, H-2β), 2.15--2.05 (m, 1H, H-16α), 1.96--1.84 (m, 2H, H-6β, H-15β), 1.80--1.65 (m, 3H, H-2α, H-6α, H-7α), 1.57--1.37 (m, 6H, H-5, H-7β, H-18, H-19, H-21β, H-22α), 1.34 (s, 3H, H-27), 1.30 (s, 3H, H-23), 1.30--1.24 (m, 3H, H-1α, H-21α, H-22β), 1.19 (bs, 4H, H-15α, H-26), 1.16 (s, 3H, H-25), 1.05--0.98 (m, 1H, H-16β), 0.94 (bs, 4H, H-20, H-30), 0.82 (s, 3H, H-28), 0.79 (d, J = 6.3 Hz, 3H, H-29). ^13^C-NMR (CDCl~3~, 125.76 MHz): δ \[ppm\] = 199.7 (C-11, \>C=O), 180.8 (C-24, COOH), 165.9 (C-13, H\>C=C\<), 158.8 (C-32, COOH (oxalyl)), 157.8 (C-31, \>C=O (oxalyl)), 130.2 (C-12, H\>C=C\<), 77.3 (C-3, HO\>CH--), 60.1 (C-9, \>CH--), 59.1 (C-18, \>CH--), 50.1 (C-5, \>CH--), 46.6 (C-4, \>C\<), 45.1 (C-8, \>C\<), 43.8 (C-14, \>C\<), 40.9 (C-22, --CH~2~--), 39.3 (C-19, \>CH--), 39.3 (C-20, \>CH--), 37.3 (C-10, \>C\<), 34.2 (C-1, --CH~2~--), 34.0 (C-17, \>C\<), 32.7 (C-7, --CH~2~--), 30.9 (C-21, --CH~2~--), 28.9 (C-28, --CH~3~), 27.5 (C-16, --CH~2~--), 27.2 (C-15, --CH~2~--), 23.9 (C-23, --CH~3~), 23.3 (C-2, --CH~2~--), 21.1 (C-30, --CH~3~), 20.5 (C-27, --CH~3~), 18.7 (C-6, --CH~2~--), 18.4 (C-26, --CH~3~), 17.4 (C-29, --CH~3~), 13.3 (C-25, --CH~3~). MS (CI, 150 eV): m/z (%) = 499 (100) \[M-CO~2~ + H\]^+^, 453 (42), 409 (44), 408 (16), 393 (6), 299 (5), 287 (3), 273 (90), 232 (91). HRMS (CI, 150 eV): calculated: 498,3395 for C~31~H~46~O~5~ (C~32~H~46~O~7~-CO~2~); found: 498.3370. \[α\]^D^: +67.0° (c = 0.98, acetone). m.p. 188--191 °C (dec). 3-O-Succinoyl-β-boswellic acid 22. β-BA 3 was dissolved in dry pyridine (2.2 mL). Succinic anhydride (220 mg, 2.2 mmol) was added, followed by 4-pyrrolidino pyridine (36.2 mg, 0.22 mmol). The reaction mixture was refluxed for 7 h. After cooling to rt, the dark brown mixture was diluted with diethyl ether (ca. 20 mL) and was washed three times with 1 N HCl (ca. 10 mL each). The organic phase was washed with H~2~O and brine, and dried with MgSO~4~. After filtration, the solvent was removed in vacuo to yield an orange-brown solid which was purified by flash chromatography (pentane/diethyl ether 2:1 + 1% HOAc) to give 87 mg of 22 as a white solid. ^1^H-NMR (CDCl~3~, 500.13 MHz): δ \[ppm\] = 5.36 (t, J = 2.3 Hz, 1H, H-3), 5.16 (t, J = 3.4 Hz, 1H, H-12), 2.78--2.62 (m, 4H, H-32, H-33 (Succinyl)), 2.17--2.10 (m, 1H, H-2β), 2.05--1.99 (m, 1H, H-16α), 1.95--1.90 (m, 2H, H-11α, H-11β), 1.88--1.77 (m, 2H, H-6β, H-15β), 1.72--1.67 (m, 1H, H-6α), 1.64--1.56 (m, 2H, H-2α, H-9), 1.56--1.47 (m, 2H, H-1β, H-7α), 1.47--1.36 (m, 4H, H-5, H-7β, H-21β, H-22α), 1.36--1.30 (m, 2H, H-18, H-19), 1.30--1.22 (m, 2H, H-21α, H-22β), 1.22 (bs, 4H, H-1α, H-23), 1.12 (s, 3H, H-27), 1.05 (s, 3H, H-26), 1.05--0.98 (m, 1H, H-15α), 0.94--0.86 (m, 8H, H-16β, H-20, H-25, H-30), 0.81 (s, 3H, H-28), 0.81 (bs, 3H, H-29). ^13^C-NMR (CDCl~3~, 125.76 MHz): δ \[ppm\] = 182.3 (C-24, COOH), 177.9 (C-34, COOH (succinyl)), 171.1 (C-31, \>C=O (succinyl)), 139.6 (C-13, H\>C=C\<), 124.5 (C-12, H\>C=C\<), 73.8 (C-3, HO\>CH--), 59.2 (C-18, \>CH--), 50.6 (C-5, \>CH--), 46.8 (C-9, \>CH--), 46.8 (C-4, \>C\<), 42.3 (C-14, \>C\<), 41.6 (C-22, --CH~2~--), 40.1 (C-8, \>C\<), 39.8 (C-19, \>CH--), 39.6 (C-20, \>CH--), 37.4 (C-10, \>C\<), 34.6 (C-1, --CH~2~--), 33.8 (C-17, \>C\<), 33.1 (C-7, --CH~2~--), 31.3 (C-21, --CH~2~--), 29.3 (C-32\/C-33\, --CH~2~-- (succinyl)), 29.0 (C-32\/C-33\, --CH~2~-- (succinyl)), 28.8 (C-28, --CH~3~), 28.2 (C-16, --CH~2~--), 26.6 (C-15, --CH~2~--), 23.7 (C-23, --CH~3~), 23.7 (C-2, --CH~2~--), 23.4 (C-11, --CH~2~--), 23.2 (C-27, --CH~3~), 21.4 (C-30, --CH~3~), 19.6 (C-6, --CH~2~--), 17.5 (C-29, --CH~3~), 16.9 (C-26, --CH~3~), 13.4 (C-25, --CH~3~); \ambiguous. MS (CI, 150 eV): m/z* (%) = 556 (8) \[M\]^+^, 439 (32), 394 (13), 379 (4), 231 (6), 218 (100), 203 (10). HRMS (CI, 150 eV): calculated: 556.3780 for C~34~H~52~O~6~; found: 556.3772. \[α\]^D^: +57.0° (c = 2.02, chloroform). m.p. 176--179 °C (dec). 3-O-Succinoyl-11-keto-β-boswellic acid 23 was prepared analogously to 22. ^1^H-NMR (CDCl~3~, 500.13 MHz): δ \[ppm\] = 5.57 (s, 1H, H-12), 5.35 (bs, 1H, H-3), 2.75--2.63 (m, 4H, H-32, H-33 (succinyl)), 2.57--2.52 (m, 1H, H-1β), 2.43 (s, 1H, H-9), 2.25--2.05 (m, 2H, H-2β, H-16α), 1.95--1.85 (m, 2H, H-6β, H-15β), 1.75--1.65 (m, 2H, H-6α, H-7α), 1.61--1.36 (m, 7H, H-2α, H-5, H-7β, H-18, H-19, H-21β, H-22α), 1.35 (s, 3H, H-27), 1.35--1.30 (m, 2H, H-21α, H-22β), 1.22 (bs, 4H, H-15α, H-23), 1.19 (bs, 4H, H-1α, H-26), 1.14 (s, 3H, H-25), 1.04--0.98 (m, 1H, H-16β), 0.95 (bs, 4H, H-20, H-30), 0.83 (s, 3H, H-28), 0.82 (d, J = 6.5 Hz, 3H, H-29). ^13^C-NMR (CDCl~3~, 125.76 MHz): δ \[ppm\] = 199.4 (C-11, \>C=O), 181.7 (C-24, COOH), 177.8 (C-34, COOH (succinyl)), 170.8 (C-31, \>C=O (succinyl)), 165.1 (C-13, H\>C=C\<), 130.5 (C-12, H\>C=C\<), 73.6 (C-3, HO\>CH--), 60.3 (C-9, \>CH--), 59.1 (C-18, \>CH--), 50.4 (C-5, \>CH--), 46.6 (C-4, \>C\<), 45.1 (C-8, \>C\<), 43.8 (C-14, \>C\<), 40.9 (C-22, --CH~2~--), 39.3 (C-19, \>CH--), 39.3 (C-20, \>CH--), 37.4 (C-10, \>C\<), 34.6 (C-1, --CH~2~--), 34.0 (C-17, \>C\<), 32.9 (C-7, --CH~2~--), 30.9 (C-21, --CH~2~--), 29.4 (C-32\/C-33\, --CH~2~-- (succinyl)), 29.1 (C-32\/C-33\, --CH~2~-- (succinyl)), 28.9 (C-28, --CH~3~), 27.6 (C-16, --CH~2~--), 27.3 (C-15, --CH~2~--), 23.8 (C-23, --CH~3~), 23.6 (C-2, --CH~2~--), 21.1 (C-30, --CH~3~), 20.5 (C-27, --CH~3~), 18.7 (C-6, --CH~2~--), 18.4 (C-26, --CH~3~), 17.4 (C-29, --CH~3~), 13.3 (C-25, --CH~3~). MS (CI, 150 eV): m/z (%) = 571 (13) \[M + H\]^+^, 471 (4), 453 (17), 409 (100), 408 (38), 393 (7), 379 (15), 273 (36), 232 (19), 218 (10). HRMS (CI, 150 eV): calculated: 570.3666 for C~34~H~50~O~7~; found: 570.3611. \[α\]^D^: +79.7° (c = 1.64, chloroform). m.p. 169--172 °C (dec). 3-O-Glutaroyl-β-boswellic acid 24 was prepared analogously to 22. ^1^H-NMR ((CD~3~)~2~CO, 500.13 MHz): δ \[ppm\] = 5.30 (t, J = 2.4 Hz, 1H, H-3), 5.20 (t, J = 3.4 Hz, 1H, H-12), 2.46 (t, J = 7.4 Hz, 2H, H-34 (glutaroyl)), 2.40 (t, J = 7.4 Hz, 2H, H-32 (glutaroyl)), 2.21--2.14 (m, 1H, H-2β), 2.11--2.07 (m, 1H, H-16α), 2.00--1.86 (m, 6H, H-6β, H-11α, H-11β, H-15β, H-33 (glutaroyl)), 1.78--1.75 (m, 1H, H-6α), 1.70--1.67 (m, 1H, H-9), 1.64--1.58 (m, 2H, H-2α, H-7α), 1.55--1.49 (m, 2H, H-1β, H-5), 1.47--1.26 (m, 8H, H-1α, H-7β, H-18, H-19, H-21α, H-21β, H-22α, H-22β), 1.23 (s, 3H, H-23), 1.15 (s, 3H, H-27), 1.09 (s, 3H, H-26), 1.07--1.02 (m, 1H, H-15α), 0.98 (s, 3H, H-25), 0.94-0.89 (m, 5H, H-16β, H-20, H-30), 0.84 (s, 3H, H-28), 0.84 (d, J = 5.8 Hz, 3H, H-29). ^13^C-NMR ((CD~3~)~2~CO, 125.76 MHz): δ \[ppm\] = 179.0 (C-24, COOH), 175.1 (C-35, COOH (glutaroyl)), 173.6 (C-31, \>C=O (glutaroyl)), 141.4 (C-13, H\>C=C\<), 126.6 (C-12, H\>C=C\<), 75.0 (C-3, HO\>CH--), 61.1 (C-18, \>CH--), 52.3 (C-5, \>CH--), 48.7 (C-9, \>CH--), 48.2 (C-4, \>C\<), 44.0 (C-8, \>C\<), 43.3 (C-22, --CH~2~--), 41.9 (C-14, \>C\<), 41.5 (C-19, \>CH--), 41.4 (C-20, \>CH--), 39.1 (C-10, \>C\<), 36.5 (C-1, --CH~2~--), 35.5 (C-17, \>C\<), 35.1 (C-34, --CH~2~-- (glutaroyl)), 34.9 (C-7, --CH~2~--), 34.3 (C-32, --CH~2~-- (glutaroyl)), 32.9 (C-21, --CH~2~--), 30.3 (C-28, --CH~3~), 29.8 (C-16, --CH~2~--), 28.3 (C-15, --CH~2~--), 25.2 (C-23, --CH~3~), 25.4 (C-2, --CH~2~--), 25.1 (C-11, --CH~2~--), 24.7 (C-27, --CH~3~), 22.7 (C-30, --CH~3~), 22.1 (C-33, --CH~2~-- (glutaroyl)), 21.6 (C-6, --CH~2~--), 18.9 (C-29, --CH~3~), 18.4 (C-26, --CH~3~), 14.9 (C-25, --CH~3~). MS (EI, 70 eV): m/z (%) = 570 (8) \[M\]^+^, 471 (8), 454 (12), 438 (28), 423 (19), 394 (25), 379 (15), 218 (100), 203 (33), 189 (22). HRMS (EI, 70 eV): calculated: 570.3982 for C35H54O6; found: 570.3951. \[α\]^D^: +56.7° (c = 0.72, acetone). m.p. 137--140 °C (dec). 3-O-Glutaroyl-11-keto-β-boswellic acid 25 was prepared analogously to 22. ^1^H-NMR ((CD~3~)~2~CO, 500.13 MHz): δ \[ppm\] = 5.49 (s, 1H, H-12), 5.29 (t, J = 2.6 Hz, 1H, H-3), 2.53--2.48 (m, 2H, H-1β, H-9), 2.44 (t, J = 7.4 Hz, 2H, H-34 (glutaroyl)), 2.39 (t, J = 7.4 Hz, 2H, H-32 (glutaroyl)), 2.27--2.16 (m, 2H, H-2β, H-16α), 1.97--1.88 (m, 4H, H-6β, H-15β, H-33 (glutaroyl)), 1.80--1.74 (m, 2H, H-6α, H-7α), 1.61--1.43 (m, 7H, H-2α, H-5, H-7β, H-18, H-19, H-21β, H-22α), 1.39 (s, 3H, H-27), 1.39--1.35 (m, 2H, H-21α, H-22β), 1.33--1.25 (m, 2H, H-1α, H-15α), 1.23 (s, 3H, H-23), 1.21 (s, 3H, H-26), 1.18 (s, 3H, H-25), 1.07--1.02 (m, 1H, H-16β), 0.96 (bs, 4H, H-20, H-30), 0.86 (s, 3H, H-28), 0.83 (d, J = 6.4 Hz, 3H, H-29). ^13^C-NMR ((CD~3~)~2~CO, 125.76 MHz): δ \[ppm\] = 199.8 (C-11, \>C=O), 178.8 (C-24, COOH), 175.1 (C-35, COOH (glutaroyl)), 173.7 (C-31, \>C=O (glutaroyl)), 165.8 (C-13, H\>C=C\<), 132.2 (C-12, H\>C=C\<), 74.9 (C-3, HO\>CH--), 62.0 (C-9, \>CH--), 60.8 (C-18, \>CH--), 52.0 (C-5, \>CH--), 48.1 (C-4, \>C\<), 46.7 (C-8, \>C\<), 45.5 (C-14, \>C\<), 42.7 (C-22, --CH~2~--), 41.1 (C-19, \>CH--), 40.9 (C-20, \>CH--), 39.2 (C-10, \>C\<), 36.4 (C-1, --CH~2~--), 35.7 (C-17, \>C\<), 35.1 (C-34, --CH~2~-- (glutaroyl)), 34.5 (C-7, --CH2--), 34.3 (C-32, --CH~2~-- (glutaroyl)), 32.6 (C-21, -CH~2~-), 30.2 (C-28, -CH~3~), 29.2 (C-16, -CH~2~-), 28.9 (C-15, --CH2--), 25.3 (C-23, --CH3), 25.2 (C-2, --CH2--), 22.4 (C-30, --CH3), 22.1 (C-33, --CH~2~-- (glutaroyl)), 21.9 (C-27, --CH~3~), 21.9 (C-6, --CH~2~--), 19.9 (C-26, --CH~3~), 18.7 (C-29, --CH~3~), 14.8 (C-25, --CH~3~). MS (EI, 70 eV): m/z (%) = 584 (4) \[M\]^+^, 452 (15), 408 (18), 273 (19), 232 (39), 228 (60), 182 (100). HRMS (EI, 70 eV): calculated: 584.3713 for C~35~H~52~O~7~; found: 584.3713. \[α\]^D^: +67.7° (c = 0.64, acetone). m.p. 139--142 °C (dec). 3-O-Carboxymethyl-β-boswellic acid 26. β-BA 3 (300 mg, 0.66 mmol) was dissolved in dry THF (2 mL). This solution was added dropwise to a suspension of NaH (154 mg, 6.4 mmol) in dry THF (3 mL). After 5 min a solution of 2-chloroacetic acid (242 mg, 2.56 mmol) in dry THF (1.6 mL) was added dropwise and the mixture was refluxed overnight. After cooling to rt, the reaction mixture was acidified with 1 N HCl to pH 3 and extracted three times with diethyl ether (ca. 10 mL each). The combined organic extracts were washed with H~2~O and brine and were dried with MgSO~4~. After filtering the MgSO~4~ the solvent is removed in vacuo. The white solid is further purified by flash chromatography with pentane/diethylether 1:1 + 1% HOAc and a second flash chromatography with dichloromethane/MeOH 15:1 yielding 157 mg of 26 as white solid. ^1^H-NMR (CDCl~3~, 500.13 MHz): δ \[ppm\] = 5.14 (t, J = 3.4 Hz, 1H, H-12), 4.21 (d, J = 16.9 Hz, 1H, H-31 (carboxymethyl)), 4.08 (d, J = 16.9 Hz, 1H, H-31 (Carboxymethyl)), 3.78 (bs, 1H, H-3), 2.06--1.96 (m, 2H, H-2β, 16α), 1.95--1.88 (m, 2H, H-11α, H-11β), 1.88--1.79 (m, 2H, H-6β, H-15β), 1.79--1.67 (m, 2H, H-2α, H-6α), 1.62--1.56 (m, 1H, H-9), 1.56--1.45 (m, 3H, H-1β, H-5, H-7α), 1.45--1.42 (m, 1H, H-22α), 1.42 (s, 3H, H-23), 1.42--1.36 (m, 2H, H-7β, H-21β), 1.35--1.30 (m, 2H, H-18, H-19), 1.30--1.20 (m, 3H, H-1α, H-21α, H-22β), 1.10 (s, 3H, H-27), 1.04 (s, 3H, H-26), 1.04--0.98 (m, 1H, H-15α), 0.92 (d, J = 6.5 Hz, 3H, H-30), 0.91 (s, 3H, H-25), 0.91--0.88 (m, 2H, H-16β, H-20), 0.80 (s, 3H, H-28), 0.79 (d, J = 5.7 Hz, 3H, H-30). ^13^C-NMR (CDCl~3~, 125.76 MHz): δ \[ppm\] = 183.3 (C-24, COOH), 174.6 (C-32, COOH (carboxymethyl)), 139.6 (C-13, H\>C=C\<), 124.4 (C-12, H\>C=C\<), 80.1 (C-3, HO\>CH--), 66.2 (C-31, --CH~2~-- (carboxymethyl)), 59.2 (C-18, \>CH--), 49.9 (C-5, \>CH--), 47.9 (C-4, \>C\<), 46.7 (C-9, \>CH--), 42.3 (C-14, \>C\<), 41.5 (C-22, --CH~2~--), 40.0 (C-8, \>C\<), 39.7 (C-19, \>CH--), 39.6 (C-20, \>CH--), 37.5 (C-10, \>C\<), 34.1 (C-1, --CH~2~--), 33.8 (C-17, \>C\<), 33.0 (C-7, --CH~2~--), 31.3 (C-21, --CH~2~--), 28.8 (C-28, -CH~3~), 28.1 (C-16, --CH~2~--), 26.5 (C-15, --CH~2~--), 24.2 (C-23, --CH~3~), 23.4 (C-11, --CH~2~--), 23.3 (C-27, --CH~3~), 21.4 (C-2, --CH~2~--), 21.4 (C-30, --CH~3~), 19.6 (C-6, --CH~2~--), 17.5 (C-29, --CH~3~), 16.9 (C-26, --CH~3~), 13.6 (C-25, --CH~3~). MS (CI, 150 eV): m/z (%) = 514 (76) \[M\]^+^, 499 (12), 470 (29), 439 (100), 393 (38), 379 (8), 218 (8). HRMS (CI, 150 eV): calculated: 514.3699 for C~32~H~50~O~5~; found: 514.3679. \[α\]^D^: +61.7° (c = 0.72, methanol). m.p. 265--269 °C (dec). 3-O-Carboxymethyl-11-keto-β-boswellic acid 27 was prepared analogously to 26. ^1^H-NMR (CDCl~3~, 500.13 MHz): δ \[ppm\] = 5.55 (s, 1H, H-12), 4.20 (d, J = 16.8 Hz, 1H, H-31 (carboxymethyl)), 4.07 (d, J = 16.9 Hz, 1H, H-31 (carboxymethyl)), 3.78 (bs, 1H, H-3), 2.54--2.46 (m, 1H, H-1β), 2.41 (s, 1H, H-9), 2.12--2.04 (m, 2H, H-2β, H-16α), 1.94--1.82 (m, 2H, H-6β, H-15β), 1.77--1.62 (m, 3H, H-2α, H-6α, H-7α), 1.55--1.42 (m, 4H, H-5, H-7β, H-18, H-22α), 1.42 (s, 3H, H-23), 1.42-1.32 (m, 2H, H-19, H-21β), 1.32 (s, 3H, H-27), 1.32--1.18 (m, 4H, H-1α, H-15α, H-21α, H-22β), 1.18 (s, 3H, H-26), 1.14 (s, 3H, H-25), 1.05--0.98 (m, 1H, H-16β), 0.94 (bs, 4H, H-20, H-30), 0.82 (s, 3H, H-28), 0.80 (d, J = 6.3 Hz, 3H, H-29). ^13^C-NMR (CDCl~3~, 125.76 MHz): δ \[ppm\] = 199.5 (C-11, \>C=O), 182.4 (C-24, COOH), 174.2 (C-32, COOH (carboxymethyl)), 165.3 (C-13, H\>C=C\<), 130.4 (C-12, H\>C=C\<), 79.9 (C-3, HO\>CH--), 66.1 (C-31, --CH~2~-- (carboxymethyl)), 60.3 (C-9, \>CH--), 59.0 (C-18, \>CH--), 49.6 (C-5, \>CH--), 47.7 (C-4, \>C\<), 45.1 (C-8, \>C\<), 43.8 (C-14, \>C\<), 40.9 (C-22, --CH~2~--), 39.3 (C-19, \>CH--), 39.3 (C-20, \>CH--), 37.4 (C-10, \>C\<), 34.0 (C-1, --CH~2~--), 34.0 (C-17, \>C\<), 32.8 (C-7, --CH~2~--), 30.9 (C-21, --CH~2~--), 28.9 (C-28, --CH~3~), 27.5 (C-16, --CH~2~--), 27.2 (C-15, --CH~2~--), 24.4 (C-23, --CH~3~), 21.1 (C-2, --CH~2~--), 21.1 (C-30, --CH~3~), 20.6 (C-27, --CH~3~), 18.8 (C-6, --CH~2~--), 18.4 (C-26, --CH~3~), 17.4 (C-29, --CH~3~), 13.5 (C-25, --CH~3~). MS (CI, 150 eV): m/z (%) = 529 (91) \[M + H\]^+^, 453 (13), 408 (7), 299 (5), 287 (7), 273 (100), 232 (97). HRMS (CI, 150 eV): calculated: 528.3467 for C~32~H~48~O~6~; found: 528.3459. \[α\]^D^: +94.4° (c = 0.91, chloroform). m.p. 215--217 °C (dec). 4.2. Cells and Cell Isolation {#sec4dot2-molecules-23-00506} ----------------------------- Neutrophils were isolated from peripheral blood (University Hospital Tübingen, Germany), collected from fasted healthy adult donors that were informed about the aim of the study and gave written consent. Leukocyte concentrates were obtained by centrifugation (4000× g, 20 min, 20 °C) of heparinized blood preparation, and neutrophils were immediately isolated as described before \[[@B30-molecules-23-00506]\]. Briefly, leukocyte concentrates were subjected to dextran sedimentation and centrifuged on lymphocyte separation medium (LSM 1077, PAA, Coelbe, Germany). For isolation of pelleted neutrophils, remaining erythrocytes were removed by hypotonic lysis, washed twice with ice-cold phosphate-buffered saline (PBS) and finally resuspended in PBS buffer containing 0.1% glucose and CaCl~2~ (1 mM) (PGC buffer). 4.3. Expression and Purification of Human Recombinant 5-LO {#sec4dot3-molecules-23-00506} ---------------------------------------------------------- Human recombinant 5-LO was expressed in Eschericha coli BL21 transformed with pT3-5-LO plasmid at 30 °C overnight, as described elsewhere \[[@B31-molecules-23-00506]\]. Cells were lysed in lysis buffer containing triethanolamine (50 mM, pH 8.0), EDTA (5 mM), phenylmethanesulfonyl fluoride (1 mM), soybean trypsin inhibitor (60 µg/mL), dithiothreitol (2 mM), and lysozyme (1 mg/mL), and homogenized by sonification (3 × 15 s). 5-LO was then purified from 40,000× g supernatant (20 min, 4 °C) using an ATP-agarose column (Sigma-Aldrich, Deisenhofen, Germany), diluted with PBS buffer containing 1 mM EDTA, and immediately used for 5-LO activity assays. 4.4. Determination of 5-LO Activity in a Cell-Free Assay {#sec4dot4-molecules-23-00506} -------------------------------------------------------- To determine 5-LO activity, aliquots of purified 5-LO (0.5 µg 5-LO in 1 mL PBS plus 1 mM EDTA) were pre-incubated with the test compounds or vehicle (0.1% dimethyl sulfoxide, DMSO) on ice for 15 min, pre-warmed for 30 s at 37 °C in a water bath, and then stimulated with 20 µM AA and CaCl~2~ 2 mM for 10 min at 37 °C. The reaction was stopped with one volume of ice-cold methanol and 5-LO products (including all-trans-isomers of LTB~4~ and 5-hydroperoxyeicosatetraenoic acid (5-HpETE), as well as its corresponding alcohol 5-hydroxyeicosatetraenoic acid (5-HETE)) were analyzed by reversed phase (RP)-HPLC (Elite LaChrom, VWR, Radnor, PA, USA) as previously described \[[@B32-molecules-23-00506]\]. In brief, acidified PBS and 200 ng of internal PGB~1~ standard were added and solid phase extraction using C18 RP-columns (100 mg, UCT, Bristol, PA, USA) was performed. After elution with methanol, samples were analyzed by RP-HPLC using a C-18 Radial-PAK column (Waters, Eschborn, Germany). 4.5. Determination of 5-LO Product Formation in Neutrophils {#sec4dot5-molecules-23-00506} ----------------------------------------------------------- 5-LO product formation in intact human neutrophils was performed using 5 × 10^6^ freshly isolated cells that were resuspended in 1 mL PGC buffer. Cells were pre-incubated with the test compounds or vehicle (0.1% DMSO) at 37 °C for 15 min prior to stimulation with 2.5 µM Ca^2+^-ionophore A23187 for 10 min (37 °C). 5-LO product formation was stopped by addition of one volume of ice-cold methanol, samples were subjected to solid phase extraction after addition of 200 ng PGB~1~ as internal standard, and 5-LO products (LTB~4~, its trans-isomers and 5-HETE) were analyzed by RP-HPLC as described above. 4.6. Determination of Cathepsin G Activity in a Cell-Free Assay {#sec4dot6-molecules-23-00506} --------------------------------------------------------------- Commercially available purified cathepsin G (0.2 µg) from human neutrophils (Calbiochem, La Jolla, CA, USA) was mixed with test compounds or DMSO (vehicle control) in 200 µL 0.1 M HEPES pH 7.4, 0.5 M NaCl, and 10% DMSO in a 96-well plate (Greiner Bio-One, Frickenhausen, Germany) and pre-incubated for 20 min at 25 °C. Then, N-Suc--Ala--Ala--Pro--Phe--pNA (Suc--AAPF--pNA) (1 mM) as substrate for cathespin G was added, and the absorbance was measured at 410 nm at 25 °C using a Victor^2^ plate reader (PerkinElmer, Waltham, MA, USA). The enzymatic activity was determined by the progress curve method, and cathepsin G activity is given as percentage of the vehicle (DMSO) control. 4.7. Statistics {#sec4dot7-molecules-23-00506} --------------- Results are presented as mean ± standard error of the mean (SEM) out of n independent experiments, where n represents the number of experiments performed on different days or with different donors. IC~50~ values were calculated from at least 5 different concentrations using a nonlinear regression interpolation of semi-logarithmic graphs in GraphPad Prism (Graphpad Software Inc., San Diego, CA, USA). Statistical evaluation was performed by two-tailed student t-test for single comparisons. P-values \< 0.05 were considered as significant. This work was partially funded by the Free State of Thuringia and the European Social Fund (2016 FGR 0045) and the Deutsche Forschungsgemeinschaft SFB1278 Polytarget. Sample Availability: Only limited samples of the compounds (AKBA 1 and KBA 2) at amounts less than 1 mg are available from the authors. A.K., J.J., D.F. and O.W. conceived and designed the experiments; A.H., M.V., L.T., S.S., M.P., N.K. and S.K. performed the experiments; A.K., A.H., M.V., L.T., S.S., M.P., N.K. and S.K. analyzed the data; A.K., J.J. and O.W. wrote the paper. The authors declare no conflict of interest. molecules-23-00506-t001_Table 1 ###### Effects of triterpene acids on 5-lipoxygenase (5-LO) and cathepsin G. ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Cmpd. Structure 5-LO Activity \[% Control\]\ Cat. G Activity \[% Control\]\ (IC~50~ in µM) (IC~50~ in µM) ---------------------------------- ---------------------------------- ---------------------------------- -------------------------------- -------------------- ----------------- --------------- ![](molecules-23-00506-i001.jpg) 1 R = --CO--CH~3~\ 23.1 ± 9.8 \\\ 40.7 ± 10.8 \\ 14.8 ± 0.9 \\\\ X = C=O (3.0 µM) (3.0 µM) (0.6 µM) 2 R = --H\ 25.7 ± 7.3 \\\ 31.1 ± 5.0 \\\\ 29.4 ± 3.5 \\\\ X = C=O (3.5 µM) (4.6 µM) (4.1 µM) 3 R = --H\ 53.0 ± 7.6 \* 78.7 ± 12.6 27.7 ± 1.8 \\\\ X = CH~2~ (0.5 µM) 4* R = --CO--CH~3~\ n.i. 73.5 ± 13.3 28.1 ± 4.9 \\\\ X = CH~2~ (2.0 µM) ![](molecules-23-00506-i002.jpg) 5* R = ![](molecules-23-00506-i003.jpg) 74.5 ± 3.7 \\ n.i. 69.8 ± 8.0 X = CH~2~ 6 R = ![](molecules-23-00506-i004.jpg) n.i. n.i. 70.4 ± 3.9 \\ X = CH~2~ 7 R = ![](molecules-23-00506-i005.jpg) 7.1 ± 3.3 \\\\ 73.0 ± 8.1 53.8 ± 8.1 \ (4.3 µM) X = C=O 8 ![](molecules-23-00506-i006.jpg) n.i. 89.1 ± 7.5 59.2 ± 4.0 \\ ![](molecules-23-00506-i007.jpg) 9 R~1~ = --H\ 36.2 ± 5.1 \\\\ 76.6 ± 12.2 58.1 ± 3.6 \\* R~2~ = --H (4.0 µM) 10 R~1~ = --CO--CH~3~\ 88.7 ± 6.7 n.i. 35.9 ± 2.5 \\\\ R~2~ = --H (7.5 µM) 11* R~1~ = --H\ 24.0 ± 7.2 \\\ 52.7 ± 7.1 \\ 68.1 ± 6.5 \* R~2~ = --OH (4.6 µM) 12 R~1~ = --CO--CH~3~\ 28.4 ± 11.1 \\ 41.8 ± 3.4 \\\\ 87.2 ± 2.5 \* R~2~ = --OH (5.1 µM) (8.3 µM) 13 ![](molecules-23-00506-i008.jpg) 8.2 ± 2.2 \\\\ 89.6 ± 8.5 72.3 ± 3.6 \\* (2.9 µM) ![](molecules-23-00506-i009.jpg) 14 X = ![](molecules-23-00506-i010.jpg) (S) 3.1 ± 0.6 \\\\ 66.0 ± 15.1 73.0 ± 7.3 (1.1 µM) 15* X = ![](molecules-23-00506-i011.jpg) (R) 5.2 ± 1.0 \\\\ 64.1 ± 12.4 80.1 ± 6.2 (3.0 µM) 16* X = C=O 37.8 ± 2.4 \\\\ 77.1 ± 8.7 53.1 ± 5.5 \\* (7.1 µM) 17 X = ![](molecules-23-00506-i012.jpg) 75.5 ± 2.1 \\ 75.6 ± 3.0\\ 66.7 ± 4.5 \\ ![](molecules-23-00506-i013.jpg) 18 X = ![](molecules-23-00506-i014.jpg) (R) 18.3± 2.7 \\\\ 72.8 ± 7.1 62.9 ± 6.6 \ (2.8 µM) R = --H 19 X = ![](molecules-23-00506-i015.jpg) $S$ 58.2 ± 0.9 \\\* 87.2 ± 1.8 \\ 82.7 ± 9.0 R = --H 20 X = CH~2~\ 77.3 ± 5.7 \* n.i. 38.1 ± 6.4 \\\\ R = --CO--COOH (4.7 µM) 21* X = C=O\ 70.3 ± 6.9 \* n.i. n.i. R = --CO--COOH 22 X = CH~2~\ 60.3 ± 8.0 \* 85.9 ± 11.1 59.4 ± 7.6 \* R = --CO-- (CH~2~)~2~--COOH 23 X = C=O\ 80.8 ± 6.8 75.0 ± 4.8\* n.i. R = --CO-- (CH~2~)~2~--COOH 24 X = CH~2~\ 65.3 ± 8.0 \* n.i. 24.5 ± 1.7 \\\\ R = --CO-- (CH~2~)~3~--COOH (3.6 µM) 25* X = C=O\ n.i. n.i. 49.1 ± 11.0 \\ R = --CO-- (CH~2~)~3~--COOH (8.6 µM) 26* X = CH~2~\ 57.6 ± 5.3 \\ 78.1 ± 7.4 28.8 ± 7.4 \\\ R = --CH~2~--COOH (3.4 µM) 27 X = C=O\ 68.4 ± 2.1 \\\* n.i. n.i. R = --CH~2~--COOH ------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------ Residual activity (% control) and IC~50~ values (µM) are given as mean ± SEM of single determinations obtained in 3--4 independent experiments. (\) P* \< 0.05, (\\) P \< 0.01, (\\\) P* \< 0.001; student t-test. n.i., no inhibition.
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Awarded to an organization working in the fields of sustainability, conservation, or natural resources protection UNDER A DARK NORTHWEST canopy, verdant sword ferns and wild oxalis sprout in the underbrush while fearless baby Douglas squirrels scurry down their towering namesake trees. This isn’t a stretch of Forest Park or Oaks Bottom Wildlife Refuge, though. It’s the lawn of West Hills homeowner Lorena O’Neill, and it has earned her the highest honor in the Backyard Habitat Certification Program: platinum. In a partnership, the Columbia Land Trust and Audubon Society of Portland first piloted the program in 2006 with a handful of homeowners pledging to eradicate the worst invasive plants from their yards (top of the hit list: ivy and blackberry) and to garden solely with Willamette Valley natives. Flash-forward to today: more than 1,750 properties are enrolled across Portland, with at least 50,000 native plants distributed at wholesale prices from local nurseries. Backyards are surveyed by expert technicians, reassessed by an army of trained volunteers, and given a certification level from silver to platinum, with a prominent sign in the front yard championing the achievement. But habitat certification is about more than one-upping your neighbors or putting a fence around a wild piece of land. “You experience wildlife and preservation at a very personal level in your own backyard,” says O’Neill as she gives a tour, proffering the tart, bright fruits from a thimbleberry shrub.“The program essentially relies on community-based social marketing,” says program manager Gaylen Beatty, “and it’s incredibly effective.” The backyard certification community has grown so rapidly, that the total acreage now amounts to around 330. In comparison, Hoyt Arboretum has 187 acres. Eventually, Beatty hopes, the program will form entirely native corridors throughout the city. Impact at a Glance Year Founded 1994Hours Volunteered In 2011 1,650Pounds of Garbage Removed In 2011 3,000 When Lewis and Clark first traveled down the Columbia in 1805, the explorers wrote that they were “kept up during the whole of the night by swans and geese.” But by 1955, part of that floodplain, the Columbia Slough, once rich with wildlife, sat clogged with tires, sewage, and garbage. “It was treated like a dump,” says Jane Van Dyke, executive director of the Columbia Slough Watershed Council. For 20 years, Van Dyke’s group—with the help of thousands of volunteers—has removed umpteen tons of garbage (3,000 pounds last year alone), planted native alder and cottonwood trees along denuded banks, and removed invasive weeds, such as reed canary grass, that can choke out native species. Perhaps even more important, the council has helped restore the slough’s reputation as a vital part of the Columbia River ecosystem. (That has been a challenge, considering that the waterway remains largely hidden behind industrial buildings and homes—feeding an out-of-sight, out-of-mind mentality.) Last year, the nonprofit’s Slough School brought 6,157 students to its waters for science-based field trips, and 2,100 people participated in the council’s annual boat trips, walking and paddling tours, and workshops. Those who ply the slough’s highly managed waters by canoe or kayak today may not hear the cacophony of birds that Lewis and Clark did, but they are rewarded by experiencing what many in Portland do not: a hidden oasis that is home to sensitive species like bald eagles, peregrine falcons, and western pond turtles, all harbingers of a waterway that is on the right track. 2011: The Wetlands Conservancy The next time you sit down to a plate of cedar-plank Pacific salmon for dinner, be sure to raise a glass to the Wetlands Conservancy. Without the group’s effort to educate the public about the importance of wetlands in everything from preventing flooding to their essential role in providing healthy habitats for salmon, your plate might well be empty. Over the course of its 30-year history, the Wetlands Conservancy has helped raise awareness about these sensitive marshy areas with posters, TV spots, and even on-the-ground guerrilla efforts like the neighborhood gathering it organized in June to educate Tigard residents with yards adjacent to the Roger Hart Wetland Preserve about how to plant and care for their land with the wetland in mind (not using pesticides that will run off into the fragile ecosystem, for example). One year, the group ran an Aqua Plate Special at local restaurants and grocery stores to raise awareness, identifying seafood that depends on healthy Oregon estuaries. The effort also netted $9,000 in donations. “We want people to understand how wetlands are more than just really cool spaces,” says the group’s executive director, Esther Lev. “They provide functions that intersect with people’s lives.” The Wetlands Conservancy isn’t just sis-boom-bahing wetland preservation, though; it’s an active player in acquiring land. Since the conservancy was founded three decades ago, it has procured more than 1,600 acres around the state, including a preserve in Yaquina Bay and 56 acres in Tualatin. “The Wetlands Conservancy plays a hugely important role in conservation in Oregon,” says Bob Sallinger, conservation director for the Audubon Society of Portland. “There are a lot of land trusts out there, but the Wetlands Conservancy stands out because they bring science and stewardship to the lands they protect.” 2011: Multnomah Education Service District Outdoor School Hands up: who’s licked a slug? If you attended the sixth grade in Multnomah County, we’re betting you may have … or that you brought back some other camp stories from Outdoor School, a sleepaway environmental education program that’s become a legendary institution since its inception in 1966. Free to most participants and part of the annual sixth-grade curriculum throughout the county, Outdoor School and its annual team of more than 1,300 high school volunteer counselors ensure every student experiences and learns about the natural world at camps near Corbett and Sandy. Students make leaf rubbings, observe migratory birds, and examine invertebrates under a microscope. (And on a dare, some might even sample “Oregon escargot.”) There are other lessons too: away from home and outside of embedded social patterns, Outdoor School creates a safe space for personal growth. “Even the students who were troublemakers in the classroom opened up and had the time of their lives,” says Katie Kramer, who spent five springs as an Outdoor School counselor and special-needs volunteer. “They got a taste of being part of something bigger than themselves: a community.” 2010: NORTHWESTEARTHINSTITUTE Twenty years ago, Dick Roy quit his job at a law firm to form the Northwest Earth Institute (NWEI) with his wife, Jeanne. Their goal? To inspire mainstream workplaces to initiate eco-friendly programs in the office. With the help of an NWEI volunteer, groups of about 10 people gather once or twice a week for an hour to discuss ways to take responsibility for both their own well-being and the planet’s. In the first year, 97 workplace groups signed up for the inaugural course, “Exploring Deep Ecology.” “Nature isn’t just a place to go on vacation—it’s the air we breathe, the water we drink, and the food we eat,” says NWEI’s executive director, Mike Mercer. Clearly, the message spoke to the masses: this year, 10,000 people participated in eight of NWEI’s programs, and courses are popping up all over the country in private homes, faith centers, nonprofits, and universities. NWEI also recently celebrated its second successful EcoChallenge, in which teams take active steps toward a healthy future by tackling two-week eco-goals like saving water, finding sustainable food solutions, and generating less waste. “Sustainable choices don’t just benefit polar bears and glaciers,” Mercer says. “What’s good for the planet is also good for your family.” And for your soul. File Under: RELATED CONTENT Please help us keep this community civil. We retain the right to remove or edit comments containing personal attacks or excessive profanity, and comments unrelated to the editorial content. Consult our Terms of Use for more details.
1. Field of the Invention The present invention relates to a digital measuring instrument. Specifically, the present invention relates to a digital measuring instrument whose display surface is arranged in an attitude substantially orthogonal to a moving direction of a spindle. 2. Description of Related Art Conventionally, there has been known a digital indicator as the digital measuring instrument which measures dimensions, such as thickness, of a workpiece from a moving amount of a spindle and digitally displays the measured result. The conventional digital indicator includes a body case, a spindle provided to the body case, the spindle being movable in the axial direction, a displacement detector that detects a moving amount of the spindle, and a digital display that digitally displays the moving amount detected by the displacement detector. Since a display surface of the digital display is typically provided on a front surface of the body case, namely on a surface parallel with a moving direction of the spindle movably provided to the body case, when a measurement is performed in a condition where it is difficult to visually check the display surface from the front side of the display surface, the measured value displayed on the display surface will be difficult to read. To solve this problem, there has been developed a so called “back-plunger” type digital dial gauge (refer to U.S. Pat. No. 5,768,798, FIGS. 4 to 6, etc.). In the back-plunger type digital dial gauge, a body case having a digital display is movably provided with a spindle in the direction orthogonal to the display surface of the digital display, and a movable member movable in the direction orthogonal with the moving direction of the spindle. Also, a conversion mechanism is provided between the spindle and the movable member for converting a moving amount of the spindle into the moving amount of the movable member. However, in the back-plunger type digital dial gauge described in the document, since the digital display is arranged on the body case in a position right above the spindle so as to be orthogonal to the moving direction of the spindle, the moving stroke of the spindle is restricted by the digital display, and therefore it is difficult to obtain a digital dial gauge having a spindle with a long moving stroke.
&QUOT;CASTING&QUOT; CALL "Casting", "Flipping", "Scooping", or "Early Release": All of these terms describe a golfer's tendency to be over-active with their hands through impact. If this describes you, (or even if it doesn't) here is a five-step, 30-minute practice plan that should be repeated several times a week for several weeks to help you ingrain good release fundamentals for the New Year (all tips are written with a right-handed perspective)! Hit 20 bump-and-run chip shots holding a rod alongside your grip that extends up the left side of your body. If you cast, you will whack your left side with the rod — ouch! Hit twenty 10-yard pitch shots (medium-high trajectory) with only your right hand. If you cast, you will probably hit a low bladed shot or possibly scoop right under the ball and get limited distance. Hit twenty half-swing punch shots on the range with a short iron stopping your finish at hip height with arms and wrists straight pointing to the target. If you have an impact bag, I would alternate between hitting the bag and doing these punch shots for added benefit. If not, just make sure in your finish position your trying to hold back your right wrist so you maintain a slight angle in it (forward press). Hit 20 full swing shots with your right-arm only using a short iron. Do 10 sets of the following: Line up four balls in a row and hit them in this order — half swing with right hand, half swing punch shot with both hands, full swing with right hand only, full swing with two hands, normal finish. This will be an intense workout if done properly, especially for your right hand/arm. If you have energy left, I would suggest going through different clubs in your bag and applying the new sensations these drills can provide, however integrate your pre-shot routine to a specific target just as you would on the course. Good luck and have fun! If you follow this regiment you'll be on your way to a cast-free swing!! Erika Larkin is the Director of Instruction at Larkin Golf Learning Community, at Stonewall Golf Club in Gainesville, Virginia. She was named the 2012 Middle Atlantic PGA "Teacher of the Year" and the 2011 "Top Golf Pro" by Washingtonian Magazine — and she's SkyGolf's newest columnist! She writes on a variety of topics including instruction, so if you have a question for her or an idea for a column, e-mail her at ErikaLarkin@pga.com. Enjoy! [ comments ] aaravydv says:The goal of duck life 4 free is to discover where all the mines (bombs) are within a particular field of squares. 3/15/18 senazuch says:I really love golf. I wrote a thesis with supreme dissertations about golf. Several years ago I practiced in golf every day. It was a 30-minute intensive task, every day. 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I hope that you will keep it up and we will have more informative and helping news from you. They are enjoyable and easy. Net games are designed to be not solely fun, but additionally very easy to grasp. Each child or everyone else fascinated with playing games online www.minifrivgames.co.uk/ will discover them very straightforward to master. 1/21/19 kairaadvani says:The kfc feedback survey is meant to provide the company with valuable information on issues where the customers are not satisfied with the products or service of the company. 1/22/19 jannetjoy1231 says:I think this is a very important point Hit twenty half-swing punch shots on the range with a short iron stopping your finish at hip height with arms and wrists straight pointing to the target. If you have an impact bag, I would alternate between hitting the bag and doing these punch shots for added benefit. If not, just make sure in your finish position you're trying to hold back your right wrist so you maintain a slight angle in it. best cloud VPS hosting help you for website hosting services. You can get a free Go Cup coupon subsequent to finishing the KFC input review. Browse inside 4 distinctive Go Cup alternatives. You can likewise enter sweepstakes to win gift vouchers and different prizes. overview is intended to give the organization important data on issues where the clients are not happy with the items or administration of the organization. You can get a free Go Cup coupon subsequent to finishing the KFC input review. Browse inside 4 distinctive Go Cup alternatives. You can likewise enter sweepstakes to win gift vouchers and different prizes 2/11/19 iamannastanley says:This article gives the light in which we can observe the reality. This is very nice one and gives indepth information. Thanks for this nice article Write My Essay For Me
--- abstract: 'Understanding phononic heat transport processes in molecular junctions is a central issue in the developing field of nanoscale heat conduction and manipulation. Here we present a Stochastic Nonequlibrium Molecular Dynamics simulation framework to investigate heat transport processes in molecular junctions in and beyond the linear response regime. We use extended molecular models which filter Markovian heat reservoirs through an intermediate substrate region, to provide a realistic and controllable effective bath spectral density. The results obtained for alkanedithol molecules connecting gold substrates agree with previous nonequilibrium Green’s function calculations in frequency domain, and match recent experimental measurements (e.g. thermal conductance around 20 pW/K for alkanedithiols in single molecular junctions) Classical MD simulations using the full molecular forcefield and quantum Landauer-type calculations based on the harmonic part of the same forcefield are compared, and the similarity of the results indicate that heat transport is dominated by modes in the lower frequency range. Heat conductance simulations on polyynes of different lengths illuminates the effects of molecular conjugation on thermal transport.' author: - Inon Sharony - Renai Chen - Abraham Nitzan bibliography: - 'bib/j.bib' - 'bib/references.bib' - 'bib/achemso-demo.bib' --- ![](img/TableOfContent.pdf){width="100.00000%"} Introduction ============ Heat conduction in molecular junctions[@Segal2016arpc] has become a subject of increasing interests as a sub-field of nanoscale energy dissipation and transport over the last decade[@Pop2010], driven by technological considerations of both stability and functionality of envisioned molecular electronic devices as well as the need to understand the fundamentals of heat transport in nanosize systems[@Li2012rmp; @Dubi2011]. The most common approach to such calculations is based on classical MD simulations with substrate temperature controlled by generalized Langevin baths, with the obvious deficiency of misrepresenting the dynamics of high frequency modes, relying on the assumption that molecular heat conduction is dominated by modes in the lower frequency regime. Alternatively, quantum calculations were done, mostly based on the non-equilibrium Green’s Function (NEGF) [@Yamamoto2006; @Wang2006prb] methodology usually using the harmonic part of the molecular force field, leading to Landauer-type expressions[@landauer1957] for the molecular heat conduction in the harmonic approximation analogous to its counterpart in the problem of molecular electronic transport using free electron models. Given the different ranges of applicability of classical dynamics on the one hand and harmonic quantum dynamics on the other, comparing their performance in evaluating and predicting heat molecular conduction is obviously of interest. Another powerful tool to investigate many-body interactions, without necessitating the harmonic approximation, is using atomistic Molecular Dynamics (MD) simulations. MD simulation of an ergodic system allows calculation of Statistical Mechanical properties of a system (e.g. thermal conductivity) by analysis of the atomic trajectories. MD simulations from previous reports, however, are mostly focused on specific systems such as liquids[@Zhang2005jpcb], thin-films[@Lukes2000jht], Graphene[@Berber2000prl], or one-dimensional metal/semi-metal chains or wires[@Ness2017jcp]. It is unclear how applicable these system-tailored simulations are to the thermal conduction in Single Molecule Junctions (SMJ). A fully-functional MD simulation tool to study the structural dependence of molecular heat conduction, in which a full force-field is applied without particular system restrictions, is still lacking. Focusing on classical simulations, equilibrium MD (EMD) is one of the easiest approaches to implement. One essentially applies the Green-Kubo formula to the time-autocorrelation of the current to get the thermal conductivity in the linear response regime [@Ladd1986prb; @Volz2000prb; @Che2000jcp; @Schelling2002prb; @McGaughey2004ijhmt1; @McGaughey2004ijhmt2; @Chen2012jap; @Sellan2010prb]. Besides its limitation to linear response approximation, the method also suffers from slow convergence and limited applicability to heterogeneous systems[@Schelling2002prb]. Alternatively, under the nonequilibrium MD (NEMD) approach [@Baranyai1996; @Poetzsch1994; @Oligschleger1999; @Berber2000prl; @Schelling2002prb; @Jiang2010jap] one creates a temperature gradient by separating the simulated system into “slabs”, and rescaling the atomic velocities at the “heat source” and “sink” slabs to set the temperature boundary conditions. In implementing this methodology care has to be taken for the finite-size effects associated with the so-imposed boundary conditions[@Schelling2002prb; @Sellan2010prb]. Plus, the thermal bath effects are relatively obscure for this method. Another popular tool is the so-called Reversed Nonequilibrium MD (RNEMD) [@Jund1999; @Muller-Plathe1997; @Muller-Plathe1999; @Zhang2005jpcb; @Bagri2011nl; @Dong2014scirep; @Tang2013apl; @Si2017ijhmt] in which the effect (fluxes) and the cause (temperatures) are reversed: one creates temperature differences by separating the simulated system into “slabs”, and enforces a given heat flux on the system by taking a certain amount of kinetic energy from the “heat source” slab and put it into the “heat sink” slab, until the system reaches steady state at which the temperature at the source and sink sides is determined. Since in this approach non-equilibrium is imposed by a constant heat flux, it is limited to steady-state calculations. The stochastic nonequilibrium MD (SNEMD) methodology used in the present work is a variant of the NEMD outlined above, in which velocity rescaling reflects the interaction with a generic (white) thermal bath in a way consistent with the fluctuation-dissipation theorem. To account substrate actual spectral properties a section adjacent to the molecule is modeled explicitly and filters the effects of the generic stochastic dynamics[@Goga2012jctc] applied to bulk layers further from the molecular bridge. We show the stability and applicability of our method in calculating the temperature distribution, heat current, and thermal conductance in various molecular junction settings. Under our approach, the concept of temperature and build-up of thermal bias come in naturally, without manually perturbing the system at each simulated time-step or reversing causality. This paper is the first in a series in which we plan to study the interplay between molecular composition and structure and its heat transport properties. For this purpose we have developed a numerical tool (described below) that can be readily adapted to different molecules and structures. Here we apply our tool to the study of heat transport in single alkane chains, a system that has been studied numerically[@Segal2003; @Kloeckner2016] and experimentally (mostly for alkane layers[@Wang2006apl; @Meier2014prl; @Majumdar2015nl], but very recently, for the first time, also for single alkane chains[@Cui2019nature]). Our results serve to test our calculation against previous calculation and most importantly against recent experimental results as well as Landauer based harmonic quantum calculations, and demonstrate the applicability robustness of these calculations. Furthermore, we present heat conduction results also for a series of conjugated carbon chains –- better candidates for molecular electronic transport applications[@Crljen2007prl; @Garner2018jpcc] but, as be find, similar to their saturated counterparts in their heat transport behavior. Section 2 provides details on our simulation technique and our code. Section 3 discuss the results of heat conduction properties of different types of hydrocarbon chains within molecualr junctions using such approach, and compare them to the existing theoreical and experimental data. In Section 4 we conclude and give future directions of research in this series. Model and Calculations ====================== While the energy spectrum of molecular vibrations encompass a relatively large ( 0 – 0.5 eV) frequency range, high-frequency vibrations tend to be spatially localized and energetically above the cutoff frequency of many solid-state substrates. For these reasons, and also because such modes are not populated at room temperature, they contribute little to molecular heat transport at that temperature[@Segal2003]. Molecular heat transport is therefore dominated by lower frequency vibrations, for which classical dynamics provide a reasonable approximation. Molecular Force-Fields (FF) allow efficient representation of a classical, anharmonic molecular Potential Energy Surface (PES) which is the input to the SNEMD studies described below. In the present work we chose to represent the attributes of the thermal environment using an explicit thermal bath. As shown in figure 2, we extend our molecular system with one or more atomic layers of the substrate, while the bulk atoms furthest from the molecular system are subjected to Markovian white noise which is thus filtered by the explicit substrate layers. Specifically, the interfaces between the molecule and the baths (Region III) on either side of it are denoted as Region II in the diagram (Figure \[fig:schematic\]). They are modeled using the same Molecular Mechanical Force Fields as the molecular system, which are optimized for small organic and organometallic molecules (more details later in this section). The interfaces are comprised of an explicit part of the bulk, which can be seen as the tips of the measuring apparatus, and are usually composed of layers of metallic materials (e.g gold, platinum). ![A schematic diagram of the explicit bath model. Region I is the molecular system (including thiol groups); Region II represents the interface and is comprised of explicit layers of metallic materials; Region III are implicit baths representing the infinitely large thermal reservoirs, exerting white noise.[]{data-label="fig:schematic"}](img/schematic.png){width="100.00000%"} The calculation of the heat current starts by representing the potential energy of the whole system as a sum of individual interaction terms $V_\tau$, where $\tau$ refers to different interaction types, for example, the two-body interaction between atom 1 and 2 or the three-body interaction between atoms 1, 2, and 3. We assume an interaction term $V_\tau$ can be further separated as a weighted sum over the atoms connected by it, weighted according to some partition scheme. A generalized analytic formalism has been explored by Torii et al.[@Torii2008jcp], explicitly $$\label{eqn:potential_partition} \begin{split} E_{tot}&=\sum_i^{N}\frac{1}{2}m_i\mathbf{v}^2_i+\sum_\tau V_\tau \\V_\tau&=\sum_j^{n(\tau)}U_{\tau,j}, \\ U_{\tau,j}(\{\mathbf{r}_1\ldots\mathbf{r}_{n(\tau)}\})&=C_{\tau,j}V_\tau(\{\mathbf{r}_1\ldots\mathbf{r}_{n(\tau)}\}),\\ \sum_j^{n(\tau)}C_{\tau,j}&=1, \end{split}$$ where $n(\tau)$ is the number of atoms connected by the interaction $V_\tau$. The fraction, $C_{\tau,j}$, of potential energy from $V_\tau$ assigned to atom $j$ is termed as $U_{\tau,j}$. Assigning specific energies to individual atoms is necessary in order to define atomic energies and energy flows between atoms, but is obviously somewhat arbitrary. In our modeling we chose to assign equal partitioning of each potential energy term between the individual participating atom. With such partitioning defined, the heat flux associated with a given atom i in the molecular system, is given by $$\label{eqn:heat_flux} J_i\equiv\frac{dE_i}{dt}=\frac{d}{dt}\left( \frac{1}{2}m_i\mathbf{v}^2_i+\sum_\tau U_{\tau,i}\right) \\ =\sum_\tau\sum_{j=1}^{n(\tau)} J_{\tau,i j},$$ where the heat flux going from atom $j$ to atom $i$ which are connected by $V_\tau$ is defined as, $$\label{eqn:heat_flux_tau} J_{\tau,i j} = C_{\tau,j}\mathbf{f}_{\tau,i}\cdot \mathbf{v}_i-C_{\tau,i}\mathbf{f}_{\tau,j}\cdot \mathbf{v}_j.$$ We have defined $\mathbf{f}_{\tau,j}$ as the force derived from interaction $U_{\tau,j}$. This is the core expression we use to calculate the inter-atomic heat currents. In addition to the inter-atomic force fields we add the effect of the thermal baths through damping forces and random fluctuations from Langevin Dynamics. We expect heat current values to plateau as the system tends towards steady-state. A customized Molecular Dynamics (MD) package built around GROningen MAchine for Chemical Simulations (GROMACS) 4.5[@Gromacs4.5] is developed and utilized to conduct the simulations. The leap-frog algorithm (provided by GROMACS) is used for propagation of the deterministic parts of the system, while Langevin dynamics are used to propagate the stochastic parts of the simulation[@Goga2012jctc]. Unless otherwise stated, the time step is always 1 fs for all runs and the coupling strength between the Markovian bath and outermost layer of explicit bulk (region is 1) is $ps^{-1}$. First, different utilities, are used to prepare the initial conditions for the simulation. These include open source software Open Babel[@OpenBabel], Avogadro[@Avogadro] and other homemade programs and scripts for creating input topologies and indices. The Universal Force Field (UFF) [@UFF] parameters are chosen throughout the simulations. UFF is one of a few force fields that includes most of the atomic types and bonds across the periodic table, and thus is suitable for organometallic junctions. As the high frequency carbon-hydrogen bonds often contribute little to the overall vibrational heat conduction, it is reasonable to compare side-by-side the effect of with and without hydrogen explicitly appear in the force field. This will in principle determine whether it is a good approximation to use unified-atom (ua) version of UFF over all-atom(aa) UFF. ![Temperature profile for 1,6-hexanedithiol, comparing UFF all-atom and UFF unified-atom force fields. The horizontal axis is labeled according to the index of the backbone atoms (Sulfur atoms included), and also including the layer of Gold atoms nearest the molecule (the leads), to the left most and right most. The temperatures of the left and right leads are set at 350K and 300K respectively. The error bars represent the standard error.(see also Caption in Figure \[fig:conductance\_layers\])[]{data-label="fig:T_UFF"}](img/Tprofile_aa_vs_ua.pdf){width="100.00000%"} Conductance SD SE. -------------- ------------- ------ ------ -- All-Atom 20.73 7.44 0.74 Unified-Atom 21.77 6.40 0.64 : Heat conductance difference between UFF all-atom and UFF unified-atom force fields, for 1,6-hexanedithiol with three layers of Gold electrodes on each side. SD stands for standard deviation, and SE for standard error. All values are given in units of pico-Watts per Kelvin.[]{data-label="table:UFF"} The local temperatures of the backbone carbons (together with sulfurs and first layer of gold) are reasonably unchanged when change the UFF-aa to UFF-ua (\[fig:T\_UFF\]). Table \[table:UFF\] compares the effect of force-field choice between all-atom and a unified-atom approximation of UFF, on the heat conduction of the hexanedithiold molecule. The relative symmetric difference[^1] between the calculation results for the two force-fields is 4.89%, and Welch’s t-test[^2] is 13%. Therefore, we conclude that the unified-atom approximation is acceptable for our purposes. The simulation begins by preparing the desired molecular state through building of the junction structure, and optimizing its geometry to be at the configuration of minimal energy. The structure is equilibrated to the average temperature of the baths, and then propagated under the boundary conditions of the required temperature bias (e.g. 300K and 350K) until it reaches steady state (usually about a few nanosecond). The steady state trajectories are sampled under this specific temperature bias. Pairwise forces between atoms, and between each bath and the atoms coupled to it, are also sampled. Finally, the heat currents are calculated from the trajectories and forces. The heat currents are then time-averaged. Ensemble-averages are performed to obtain statistically sound final currents and conductance. As for Landauer-type calculations, a detailed description of the formalism is provided in the Supporting Information (SI), together with other relevant data and figures. The heat current equations and computational apparatus described above were used to calculate individual heat currents between any two atomic pair within the molecular systems. This is not limited to nearest-neighbour bonded atoms (bond stretching interactions), but also applies to atoms that are three or four sites apart but still interconnected by other interactions parameterized in the force fields (e.g., angle bending, torsion, etc.) The heat current flowing from one heat bath to the other bath in the molecular junction, can be measured by setting up an imaginary plane which is perpendicular to the longitudinal axis of the molecule and sum over all the inter-atomic heat currents going from one side of the plane to the other side of the plane. In steady-state, the heat current through the molecule will be measured the same, regardless of where we chose to draw this imaginary plane. For computational simplicity, we chose to draw it between region I in figure \[fig:schematic\] (molecular) and region II (the substrate interface). The average thermal conductance is defined as the ratio between this quantity and the temperature bias between the hot and cold baths, $$\kappa=\frac{J_{tot}}{T_\text{hot}-T_\text{cold}}.$$ In addition to heat fluxes, the local temperature of each atom in the conducting molecule is calculated from the statistically averaged kinetic energy of the atoms. More details regarding our methodology are given at the end of this article and in the Supporting Information (SI). Results and discussion ====================== The system under investigation is a Single Molecule Junction comprising an alkanedithiol as a molecular bridge connecting several layers of explicit bulk atoms. We compared alkanedithiols of various lengths (measured by the number of Carbon atoms in the alkane backbone), and explicit bulk comprised of one to four layers of gold atoms which are further connected to bulk substrates. Some examples are illustrated in Figure \[fig:alkanesB\]. For each molecular species, we performed MD simulations of the non-equilibrium molecular junction, evaluating its steady-state heat transport behavior following the procedure described in Section 2. ![Illustration of some of the alkane molecules studied in the simulation, with three layers of explicit gold bulk. The white noise thermostats are only attached to the layer of gold atoms furthest from the alkane bridge. (a) 1,4-butanedithiol(); (b) 1,8-octanedithiol().[]{data-label="fig:alkanesB"}](img/alkanes_drawings_on_Black_background.pdf){width="100.00000%"} For each molecular species, we performed MD simulations of the molecular junction at Nonequilibrium Steady-State (NESS). More computational details are given in the SI. In order to ascertain the relevance of the explicit baths modelling, we compared results for molecular heat conductance using different numbers of explicit gold layers to represent the bulk. Specific simulations are performed on hexanedithiol (Figure \[fig:conductance\_layers\]). The calculated conductance appears to converge when three layers of explicit gold are used in the substrate representation. A similar saturation of layer effect was found also in the other hydrocarbon molecules (See SI for results of more alkanedithiols). In agreement with this observation, a study by Zhang et. al. on self-assembled monolayers showed that the effect of the baths on the molecular system is mainly due to the first few layers of gold substrate[@Zhang2010pccp]. As seen in Fig. \[fig:auto\], the observed convergence reflects the convergence in spectral density properties of the gold clusters used to represent the thermal baths. It should be noted that to enforce the junction geometry, the explicit bulk is position-restrained by a harmonic force acting on the layer of atoms furthest from the molecule. The spectral densities in Fig. \[fig:auto\] are calculated for the position-restrained clusters since the position restraint force is part of the spectral density affecting the molecule. The signature of the harmonic position-restraining force is evident at its frequency of about 40 wavenumbers. The rest of the spectrum shows frequencies in the range of tens to a few hundred wavenumbers, which agrees with experimental measurements of vibrational DOS for gold nanoparticles[@Carles2016scirep]. ![Heat conductance for the molecule given different numbers of gold layers as the explicit bulk. The temperature bias is set at 300K to 350K. The bars shown in the figure are the standard errors (SE) of the conductance measurements. (SE=Standard Deviation (SD) / square root of the sample size, is a statistical uncertainty indicator of the estimated mean value of the conducted measurements.[@SE]) []{data-label="fig:conductance_layers"}](img/conductance_vs_num_gold_layers.pdf){width="100.00000%"} ![Velocity-velocity autocorrelation functions of the only atom in the first layer of each of four different gold clusters. The outer-most layers are attached to Markovian thermal reservoirs at temperature of 300K and the clusters are allowed sufficient time (e.g. a few nanoseconds) to relax to the temperature of the bath. Column (a): Velocity time-autocorrelations, $C_{vv}(t)$, which are normalized to the value at $t=t_1-t_2=0$; Column (b): The Fourier transforms of the corresponding correlations, normalized across the whole spectra; Column (c): Artistic representation of the corresponding gold clusters[]{data-label="fig:auto"}](img/auto_fourier_molecule_ps.pdf){width="100.00000%"} To study the effect of the molecular structure on the junction conductance, we compared molecules of various lengths and of different degrees of saturation. Namely, we compared molecules with an alkane backbone (saturated) against molecules with a conjugated polyyne backbone (unsaturated)[^3]. Unless otherwise specified, the simulation results displayed below were obtained using three explicit atomic layers for the gold substrates. Figure \[fig:conductance\_conjugated\] shows that heat conductance of shorter molecules ($n < 6$) is not strongly affected by carbon bond saturation, however longer unsaturated chains exhibit lower conductance than their saturated counterparts. This observation stands in contrast to the higher electronic transport properties of conjugated chain molecules[@Crljen2007prl], and may be explained by the difference in current carriers of thermal and electronic transport in these systems: While the delocalized electrons in conjugated molecules may contribute much to the overall electronic conduction, heat conduction, which is dominated by phonon transport, is mostly determined by bond structure and vibrational modes in the molecular system.The steady state temperature profiles associated with the results of Figure \[fig:conductance\_conjugated\] are displayed in Figure \[fig:T\_alkane\]. The bias (300K - 350K) clearly exceeds the regime of validity of linear response, yet is more realistic with respect to existing experimental setups [@Meier2014prl; @Wang2006apl; @Majumdar2015nl; @Cui2019nature]. The temperature profiles show that most of the thermal resistance is interfacial. Even the longer molecules are homogeneous enough that the temperature profile does not slope significantly, which could point to a ballistic regime of heat conduction in the molecular bridge. This can be ascribed to the explicit modelling of the molecule-bulk interface at the atomic level. The features of interfacial temperature already show some filtering effects from the white baths, noting the first layer of gold bulk on the left is about 10 degrees lower than external hot reservoir and right first layer 10 degrees higher than the external cold reservoir. ![Length-dependent heat conductance of alkane chains (saturated) and triple-bond conjugated hydrocarbon chain (unsaturated) molecules for a temperature bias of 300K and 350K. The error bars represent the SE (see Caption in figure \[fig:conductance\_layers\]) of the conductance measurements.[]{data-label="fig:conductance_conjugated"}](img/conductance_alkanes_saturation_upto12.pdf){width="100.00000%"} ![Temperature profile for non-branching alkanedithols of various lengths. In the legend the ones with and without hydrogen atoms are alkanes and conjugated polyyenes respectively. The horizontal axis is labeled according to the index of the backbone atoms (Sulfur atoms included), and also including the layer of gold atoms nearest the molecule (the leads), to the left most and right most. The temperatures of the left and right leads are set at 350K and 300K respectively. That is, atom zero is always the gold atom to the left of the alkanedithiol, atom one is the left Sulfur atom, and the same on the right. The error bars represent the SE (see Caption in figure \[fig:conductance\_layers\]) the temperature measurements.[]{data-label="fig:T_alkane"}](img/Tprofile_alkane_polyyne248.pdf){width="100.00000%"} Next, consider the results obtained from the quantum-mechanical harmonic model calculation. The theoretical and numerical details of the Landauer approach for calculating heat conductance has been specified in the Support Information. Figure \[fig:conductance\_MD\_vs\_Landauer\] compares the results of this model to those obtained from the classical MD simulation. While a certain degree of divergence occurs of these two methods in the results of alkanedithiolds (which we will explain in the later parts in the section), the remarkable agreement in polyynes molecules indicates that heat conduction in these systems is determined by the harmonic part of the force field and dominated by modes from the lower frequency range of the molecular spectrum. ![Length-dependent heat conductance of alkane chains (saturated, in the upper panel) and triple-bond conjugated hydrocarbon chain (unsaturated, in the lower panel) molecules, obtained with GROMACS MD simulations and Landauer-type quantum calculations, respectively. In all cases, thiol head-groups are attached, and the currents are calculated across a temperature bias from 350K to 300K, with conductance defined as the ratio between heat current and temperature bias (50K).[]{data-label="fig:conductance_MD_vs_Landauer"}](img/conductance_MD_vs_Landauer_2panel.pdf){width="100.00000%"} Finally, consider the absolute values of the calculatioted heat conductions. In general, our results are in agreement with the most recent experimental measurements for heat conduction in SMJs [@Cui2019nature]. For the saturated alkanes, the Landauer conductance starts to fluctuate and then goes down as the chain grows even longer (See SI for more data), while the MD results decrease slowly and stay relatively stable around the value 20 pW/K. Though the non-monotonic behavior of the Landauer calculations align with an independent ab initio Landauer-type calculation[@Kloeckner2016], MD simulations align more closely with the experimental data and trend[@Cui2019nature], indicating anharmonicity plays a tunning role in molecular thermal transport. It is interesting and intuitive to see heat conduction from a normal mode harmonic perspective (i.e. the normal mode density, localization, and transmission probablity, see SI for detailed analysis), but at room temperature classical MD seems do better in taking into account both harmonic and anharmonic effects by utlizing full-force-field interactions of the molecular potential energy. Conclusion ========== In summary, we have presented results of classical MD simulations using stochastic Langevin thermal baths, as well as results of a quantum calculation based on the harmonic part of the molecular force field, for the steady-state heat conduction of molecular junctions comprising saturated and conjugated hydrocarbon chains connecting gold leads. The multiple layers of explict gold substrates act as filters of larger environmental white noise and bring characteristic bath effects to the heat conducting molecular systems under investigation. The high degree of agreement between our simulations and the most recent experimental measurements[@Cui2019nature] also validates the methods and numerical tools we use. For the alkanedithiols in particular, MD simulation agrees better with the experiment than Landauer-type quantum calculation in conditions of room temperature and with large bias. This may hint that at ambient conditions, the explicit treatment of quantum effects is less relevant than explicit treatment of anharmonicity, and linear response less relevant than the behavior of systems far-from-equilibrium. For conjugated hydrocarbons, while previous studies have shown unusually high electronic conduction of polyynes[@Crljen2007prl], our study shows that they have a lower thermal conductance than their saturated counterparts. which might indicate they are potentially good candidates for thermoelectric nanomaterials. Still the effects of complex molecular structures and topologies on nanoscale heat conduction are still not clear, and will be future directions of our research in the group by making full use of the atomistic-resolution afforded by our approach. The research of AN is supported by the Israel-U. S. Binational Science Foundation, the German Research Foundation (DFG TH 820/11-1), the U. S. National ScienceFoundation(GrantNo. CHE1665291),and the University of Pennsylvania. [^1]: The relative symmetric difference is $\delta(x,y) = \frac{\|x-y\|}{(x+y)/2}$ [^2]: Welch’s t-test is $\eta(x,y)=\frac{\|\mathbf{E}[x]-\mathbf{E}[y]\|}{\sqrt{\sigma_x^2+\sigma_y^2}}$, where $\sigma_x$ is the Standard Deviation in random variable $x$ [^3]: A polyyne is an organic compound with alternating single and triple bonds; that is, a series of consecutive alkynes, $\left(- C \equiv C - \right)_n$ with $n$ greater than 1.
At Home in the World The Most Rejuvenating Natural Hot Springs Around the World Sometimes, in an effort to climb that mountain, see that sight, and eat at that restaurant, we end up needing a vacation from our vacation. Not so at nature’s own super spa: the hot spring. These mineral-rich waters from Palm Springs to Chilean Patagonia have nourished for centuries; there's no better way to soak your body and free your mind. The Most Rejuvenating Natural Hot Springs Around the World Sometimes, in an effort to climb that mountain, see that sight, and eat at that restaurant, we end up needing a vacation from our vacation. Not so at nature’s own super spa: the hot spring. These mineral-rich waters from Palm Springs to Chilean Patagonia have nourished for centuries; there's no better way to soak your body and free your mind. The Spring, Palm Springs, California Taking a dip in a warm pool of water in the middle of the desert may not seem that refreshing, but remember: The hotter the water, the greater the mineral content. The water at The Spring is said to be some of the most restorative in America, rich with minerals such as sodium chloride (helps with joint pain) and sulfur (reduces inflammation). Starting off as snow melt from the mountains, the water runs underground until it bubbles back up at 170 degrees. It is then cooled to three different temperatures in three separate pools surrounded by both palm trees and snow-capped mountains. Baden-Baden, Germany This small German town on the edge of the Black Forest has been drawing people to its curative hot springs for centuries. Up from some 6,500 feet below, the water from these 12 springs can relax and regenerate the body: Lithium, cesium, silica, boric acid, and magnesium will defuse any cardiovascular, metabolic, and respiratory problems, while the heat helps with circulation and relieves pain in muscles and joints. The Caracalla Spa, which has springs that are between 12,000 and 17,000 years old, is built around Roman bath ruins and has a saltwater inhalation room (to help clear mucous membranes). The Friedrichsbad Baths sit atop Roman bath ruins (it has Roman walls to prove it) and follows a 17-step Roman ritual, which includes a series of hot baths, steam rooms, and brush massages. Fontana, Iceland Bathing in hot springs is an Icelandic tradition that’s been around since the Vikings. Skip the overcrowded Blue Lagoon, which has become the Times Square of Icelandic pools, and make your way to the lesser known spa, Laugarvatn Fontana. It has views of Lake Laugarvatn and, if you’re lucky, the Northern Lights. The village of Laugarvatn was established following the discovery of the hot springs; the water could be used for cooking and bathing, and the local municipality uses it for geothermal house heating. In addition to the three outdoor pools, there are steam baths, which locals have been enjoying since 1929. Built over natural hot springs, the steam simmers up through the grids of the cabin floors, creating a steam bath—ideal after a day of hiking through the wild Icelandic landscape. Dunton Hot Springs, Dolores, Colorado A ghost town turned luxe resort in the San Juan National Forest, Dunton Hot Springs is romantic Americana at its best: cozy log cabins, steaming hot spring pools, and Rocky Mountain views. This former mining town now draws the well-heeled with its pools of iron-, manganese-, and lithium-rich water. In addition to the outdoor pools (one of which is on the river), there is a 19th-century indoor bathhouse, plus two private pools for guests staying in the Dunton Store and Well House cabins. Come sun, rain, or snow: The pools are open year-round. The Green Leaf Onsen, Niseko Village, Japan In a volcano-covered country like Japan, bathing in an onsen (Japanese hot spring) is very much a part of the culture. Onsens are believed to have healing powers (it's that high mineral content again), while bathing in a naked communion helps break down barriers between people. After a hard day of skiing in the small ski village of Niseko, there is nothing more therapeutic than luxuriating in a natural rock pool with 104 degree water. In true Japanese tradition, the pools don’t allow bathing suits, but the water feeds into indoor and outdoor onsens separated for gender. With pine trees and snow-covered boulders as the backdrop, these pools are as good for your body as they are for your mind. Therme Vals, Switzerland Trust Switzerland to boast the most sophisticated springs. Therme Vals is a chic, minimalist hotel and bathhouse in the small Swiss alpine village of Vals, designed by Pritzker Prize-winning architect Peter Zumthor. Originally opened in 1893 as a bathhouse, the space was reborn in 1996 as a hotel-and-hot springs getaway. Sitting over the only thermal springs in the Graubünden district, Therme Vals was built with stones from the surrounding area and mimic a quarry-like structure, fitting seamlessly into the mountainous landscape. The Banjaran, Malaysia Hidden in a valley surrounded by tropical rainforest and dramatic limestone hills, the Banjaran Hot Spring Retreat is ideal for those who want to completely disconnect from the outside world. The grounds are home to hot springs and naturally formed limestone caves that fill with steam. Submerge in a private plunge pool or hot spring tub filled with geothermal water, or enjoy one of the two indoor geothermal pools on the property, one of which is concealed in a cave. The water temperature is around 104 degrees and contains calcium, lithium, and sulfur, which are known to reduce pain caused by arthritis. Puyuhuapi, Chile On the edge of Dorita Bay in a remote region of Chilean Patagonia, Puyuhuapi Hot Springs take mineral-rich water from three different sources: the sea, waterfalls, and thermal springs. There are three large outdoor thermal pools as well as two smaller outdoor pools, one filled with sea water and one filled with mountain water. The thermal water contains minerals used in the spa's signature massage treatment to help with physical fatigue and to calm the mind. But perhaps the most remarkable feature of this retreat its isolation: soothing with its rainforest-covered mountains and views that go on forever.
We'll keep track of the day's updates on Masahiro Tanaka right here: ESPN's Jayson Stark tweets that it was amazing how many owners at the quarterly owners meetings in Arizona were convinced that the Cubs were preparing to blow away the rest of the field with their offer to sign Tanaka. Earlier Updates
Michelle LaVigne Associate Professor Michelle LaVigne's writing/research focuses on the intersection of dance, rhetoric, and performance. In particular, she writes about the persuasive qualities of dance movements and aesthetics, and how practices of rhetoric might be rethought from the movements of dance. In addition to speaking at national and international conferences, she has published reviews in the Quarterly Journal of Speech and Journal of Aesthetics and Art Criticism.
1989–90 Albanian Superliga The 1989–90 Albanian Superliga was contested by 12 teams, and Dinamo Tirana won the championship. League table Note: '17 Nëntori' is Tirana, 'Lokomotiva Durrës' is Teuta, 'Labinoti' is Elbasani References Albania - List of final tables (RSSSF) Category:Albanian Superliga seasons 1 Albanian Superliga
#N canvas 432 53 448 643 10; #X obj 1 1 cnv 15 445 20 empty \$0-pddp.cnv.header midi_flags 20 10 1 18 -261106 -33289 0; #X obj 407 2 pddp/pddplink http://puredata.info/dev/pddp -text pddp ; #X text 40 79 pd -midiindev 1 -midioutdev 2; #X text 39 201 pd -mididev 1; #X text 19 223 This will use the first port for both MIDI input and output.; #X text 19 291 There is also a little bit of confusion about OSS midi (which uses the raw midi - /dev/midi* - devices by default) and ALSA (which is becoming very popular). ALSA uses /dev/snd/midiCD as their raw midi devices. The C* (* = some number) is the device number while D* (* = some number) is the port number. Normally the ALSA driver links its raw devices to the corresponding /dev/midi* automatically (i.e.: /dev/snd/midiC0d0 -> /dev/midi00 \, /dev/snd/midiC0D1 -> /dev/midi01 etc).; #X text 19 411 MIDI objects in Pd (the ones that read and write to MIDI ports) take a MIDI channel as an argument. Channels 1-16 use the first MIDI device \, 17-32 use the second MIDI device and so on. For this to work one must specify which devices Pd is supposed to use: ; #X text 38 478 -midiindev 1 \, 2 -- use devices 1 and 2 for input; #X text 38 493 -midioutdev 1 \, 2 -- use devices 1 and 2 for output ; #X text 38 508 -mididev 1 \, 2 -- use devices 1 and 2 for input and output; #X text 19 526 NOTE (Windows): -listdev command line option lists all available MIDI and audio devices.; #X text 19 558 A couple more useful command line options (related to MIDI):; #X text 39 573 -nomidiin -- do not listen to any midi input; #X text 39 588 -nomidiout -- do not send any midi output; #X text 39 603 -nomidi -- do not use any MIDI; #X text 20 36 MIDI in Pd is handled through the 'raw' midi devices (such as /dev/midi*). Specify which MIDI port Pd is supposed to use through a command line switch when starting Pd \, i.e.:; #X text 19 101 The above will use the first MIDI device for MIDI input (it could be a MIDI controller such as a keyboard \, midi-guitar \, midi knob box \, etc.) and device #2 will be used for outputting the MIDI (re)generated or sent by Pd.; #X text 19 158 To use the same device for MIDI input AND output (e.g. \, a MIDI keyboard which is also a synthesizer) use the following command line switch:; #X text 19 238 NOTE (Linux): Pd counts the MIDI devices starting from one. Usually \, the operating system counts them starting with 0 (zero) so if you want to use /dev/midi00 \, start Pd with "-mididev 1". Always add 1 to the device number.; #X obj 1 621 cnv 15 445 20 empty \$0-pddp.cnv.footer empty 20 12 0 14 -233017 -33289 0; #N canvas 381 532 494 143 META 0; #X text 12 85 HELP_PATCH_AUTHORS Dave Sabine \, May 5 \, 2003 . Jonathan Wilkes revised the patch to conform to the PDDP template for Pd version 0.42.; #X text 12 65 LIBRARY PDDP; #X text 12 45 DESCRIPTION list of command line flags relating to MIDI ; #X text 12 25 KEYWORDS MIDI all_about_pd; #X restore 392 623 pd META; #N canvas 220 305 428 342 Related_objects 0; #X obj 22 41 dbtopow~; #X obj 76 41 dbtorms~; #X obj 131 41 rmstodb~; #X obj 186 41 powtodb~; #X obj 241 41 mtof~; #X obj 278 41 ftom~; #X obj 22 68 expr; #X obj 57 68 expr~; #X obj 97 68 sig~; #X obj 131 68 snapshot~; #X text 19 98 [rmstopow~]; #X text 99 98 [powtorms~]; #X obj 22 169 db2v; #X obj 60 169 f2note; #X obj 108 169 t3_sig~; #X obj 164 169 m2f~; #X text 19 226 These objects are offered in Pd only if you have downloaded and properly installed the appropriate library. These objects may or may not exist in a single library.; #X text 19 266 The best places to find information about Pd's libraries is:; #X text 20 286 www.puredata.org and click on "Downloads" then "Software" ; #X text 20 301 or; #X text 19 316 iem.kug.ac.at/pdb/; #X text 18 198 [b2db]; #X text 61 198 [tmtof]; #X text 20 142 Externals; #X obj 1 1 cnv 15 425 20 empty \$0-pddp.cnv.subheading empty 3 12 0 14 -261106 -33289 0; #X text 7 2 MIDI flags- Related Objects; #X restore 103 623 pd Related_objects; #X obj 6 623 pddp/pddplink all_about.pd -text All About Pd;
Commentary: Reid ignoring the inevitable Philadelphia Eagles coach Andy Reid speaks with members of the media during a news conference at the team's NFL football training facility, Wednesday, Dec. 19, 2012, in Philadelphia. (AP Photo/Matt Rourke) PHILADELPHIA — The Eagles had finished their morning practice, and Andy Reid was surrounded by cameras and recorders. So he began with the injuries and ended by pretending he wasn’t sure what Eagles fans were thinking. Fourteen-plus years later, that wouldn’t change. It’s why, high among other reasons, there won’t be a 15th. Sunday, the Birds will play their final home game. That will make it the last time Reid will be on the Linc’s home sideline. That will also make it the last chance for the fans to boo him, or cheer him, or ignore him, or heckle, serenade or stand for him and for what he has achieved. Yet there was Reid Wednesday, with a chance to begin the farewells. As if. Advertisement “I haven’t even gone there,” he said. “I am just concentrating on the Washington Redskins and keeping it at that.” If he was concentrating only on the Redskins and how to improve from 4-10 to 5-10, he would not have crammed an available four-time Pro Bowl quarterback into the No. 3 hole, two spots behind a 1-and-4 rookie. But to the end, he would do one thing and expect fans to see another. He is going to insist that he has not considered that he will be coaching his last game in front of fans he had always said out loud that he appreciated and for an owner who has stuck with him so long that it has done his own professional reputation grave damage. It’s always been that way with Reid. If the fans chant negative things about him, he says he is too immersed in his job to listen. But if the Eagles win, he is quick to credit their support. So there he was, deep into run-out-the-schedule time, acting surprised that someone would wonder about his view of his last Linc game. “I really haven’t thought about it,” he said. “I’d like to tell you a different answer but I haven’t really gone there.” So it will be, to the end. It’s how he’s been, obsessed only with the next game. More often than not, that has been to the Eagles’ ultimate benefit. But now? Really? Maybe there is a financial incentive for Reid to be so coy. He is signed for next season and would deserve that cash if fired. Maybe that’s why he can’t say that he is thinking about his last home game. Maybe. Inside the locker room, where there will be plenty of changes, too, many of the players were on edge. Unless they are reading the standings from the bottom up, they know the deal: They will work just two more Sundays for Reid. Yet Reid still has such a command of the room that the subject of his employment situation — and likely Sunday sendoff — remains taboo. “Hey, I don’t know about the sendoff or not,” Trent Cole said. “I don’t know if they say he is going or not. I don’t know what you’ve heard, but I hope he is here. But anyway at that, we are out to win. That’s what it is. We have the faith in Coach Reid that he is going to put us in a position to go out there and win. And that’s pretty much it.” Once, there was reason for that faith. But that was too long ago to matter. The Eagles have become a turnover-prone, disorganized slapstick act, and for that, Reid soon will be working elsewhere, no matter how thick the in-house denial. “We’re not thinking that way and Andy’s not thinking that way,” Evan Mathis said. “He’s taught us to stay focused on the task at hand. Those external circumstances are not anything that we should be worried about. It would be just a distraction to our preparation.” So, the Eagles prepare — for the games, if not the reactions. “Since everybody is saying he won’t be back next year,” Brandon Graham said, “I want to send him out with a W here and a W away in New York.”
Springspiration│Decoration and garden edition Hello everyone! Back today with a springspiration post! I have a decoration and garden edition planned for today. Something a bit different, but I love to change it up once in a while. Enjoy the pretty pictures guys! Decoration These pillows are giving me life. I love the patterns on it! They give me a happy feeling. Credit: Pinterest There is very much light in this bedroom. It looks classy and sophisticated. I love how the makers of this interior created so much light. The colors blend in very well. Credit: Pinterest This is a very creative idea. I wouldn’t come up with something so smart as this! I like the color of the paint. I would love to hang this decoration in my house. Credit: Pinterest Candy colors are all over the place in spring. Pastel and candy are having the time of their lives lately. These butterflies are enjoying their time in the living room! Credit: Pinterest Garden I am so happy to see the flowers back. They make me so happy and delighted. They literally put a smile on your face and wipe away your winterblues. These butterflies are flying around in their favorite flowers. Credit: Pinterest An old trend that is making its comeback this year. The flowerbucket trend. I saw my mom doing this trend when I was still a little child. She’ll be happy to know the trend is back! Credit: Pinterest Can we just talk about the candy pink color of these boots? So freaking pretty. The tulips in it are such a great idea. It breaths spring! Credit: Pinterest That was it for today everyone! I hope you enjoyed and keep an eye out for the next one in these series, because there are still two coming up! See you next time! Your Belgian blogger, xoxo, Tessa Disclaimer: Not sponsored. These are all my honest opinions. Pictures aren’t mine, a credit is always mentioned. Please note that I am not a professional.
WE ARE TWIN SISTERS who are IDENTICAL in some areas and SO DIFFERENT in others. we enjoy blogging about being mommies and about the things we love: music, books, art, design, crafts... Saturday, June 14, 2008 gone daddy gone my husband is a great dad. it's very nice for me that it worked out that way. at our family reunion this past week, we had a little discussion about fathers and we all listed qualities that we thought were important in being a good dad. one of the things i listed was an attribute that mr. mama has in spades: a willingness to be silly. maybe that doesn't sound very important, but can you imagine a father who would never get down on the floor and play with his kids? never make them laugh by pulling funny faces at them or inventing silly songs with their names? never make up child-friendly versions of the board games his 4 year old wants to play but can't understand yet? i'm spoiled because i have a husband who does all this and so much more. his affection for his children is so genuine, so matter-of-fact, that i'm kind of at a loss as to what to say about it. i'm just grateful for it.daddy and the little self-dresser, m. in her heart outfit: heart shirt, heart dress, heart leggings, and heart socks. at the family reunion: little c had just got up from a nap, daddy was trying to copy his pointing. I think this is one of the most important things. When I spend the night at my parents, my dad still wakes me up by coming into my room, grabbing my feet and tickling them until I can't breathe. It's crazy! My dad was always playing with us on the floor, always telling us stories, always taking us camping. A good dad is a great thing! You've got a great husband Jo! I knew it when you guys were dating, and I can see it more even now. sugar, i'm so sorry that you didn't have that kind of positive impact in your life. i don't know what to say except that.d'arcy, your dad sounds great! and thanks for saying what you did about my hub. sometimes it's easy for me to focus on what i think he should be doing better, so it's nice to hear from someone who was there in the very beginning, what a great guy you think he is.
1. Introduction {#sec1} =============== Dengue viruses are transmitted by mosquitoes and infect \~50 million people annually with an additional 2.5 billion people at risk living in tropical areas \[[@B1]--[@B3]\]. Expanding mosquito habitats are increasing the range of dengue virus outbreaks and the occurrence of severe diseases with 5--30% mortality rates: dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) \[[@B1]--[@B3]\]. The majority of patients are asymptomatic or display mild symptoms of dengue fever (DF) which include rapid onset of fever, viremia, headache, pain, and rash \[[@B4]\]. Patients with DHF and DSS display symptoms of DF in addition to increased edema, hemorrhage, thrombocytopenia, and shock \[[@B1]--[@B3]\]. Although patient progression to DHF and DSS is not fully understood \[[@B3], [@B5]\], antibody-dependent enhancement (ADE) of dengue infection increases the potential for DSS and DHF \[[@B3], [@B6], [@B7]\]. There are four dengue virus serotypes (types 1--4) and infection by one serotype predisposes individuals to more severe disease following a subsequent infection by a different dengue serotype. The circulation of serotype-specific cross-reactive antibodies or preexisting maternal antibodies may contribute to progression to DHF/DSS by facilitating viral infection of immune cells and eliciting cytokine and chemotactic immune responses. In a murine antibody dependent enhancement model of dengue disease it was observed that a dramatic increase in infected hepatic endothelial cells (ECs) coincides with the onset of severe disease \[[@B8]\] and suggests a role for the endothelium in an immune-enhanced disease process during dengue infection. The major target tissues for dengue virus infection have been difficult to determine but virus has been isolated from human blood, lymph node, bone marrow, liver, heart, and spleen \[[@B9]--[@B14]\]. Blood samples are more easily obtained from dengue patients than tissues and yield a wide array of information about cytokine responses elicited by dengue virus infection \[[@B1]--[@B3], [@B14]--[@B18]\]. While many of these cytokines are present in DF patients, the majority of them are increased during DHF. Overall, DHF responses include greater cytokine production, T- and B-cell activation, complement activation, and T-cell apoptosis \[[@B3]\]. Complement pathway activation and elevated levels of complement proteins C3, C3a, and C5a are significant in that they can direct opsonization, chemotaxis of mast and other immune cells, and direct the localized release of the vascular permeability factor histamine from mast cells \[[@B17], [@B19]--[@B23]\]. Importantly, cytokines and complement factor responses all act on the endothelium and alter normal fluid barrier functions of ECs. The ability of dengue virus to infect immune, dendritic, and endothelial cells fosters a role for immune responses to act on the endothelium and increase capillary permeability \[[@B5], [@B24]--[@B29]\]. However, the redundant nature of capillary barrier functions suggests that permeability is likely to be multifactorial in nature with many factors working in concert to modulate EC responses and permeabilize the endothelium. Dengue infected ECs are observed in DHF/DSS patient autopsy samples and in murine dengue virus disease models \[[@B8], [@B9], [@B14], [@B30]\]. This suggests that dengue infected ECs may also contribute directly to pathogenesis by increasing viremia, secreting cytokines, modulating complement pathways, or transforming the endothelium into an immunologic target of cellular and humoral immune responses. Plasma constituents contain factors secreted by an estimated \~10^13^ ECs present in the body, and autopsy samples and murine dengue disease models clearly demonstrate that vascular ECs are infected \[[@B8], [@B9], [@B30], [@B31]\]. The endothelium is the primary fluid barrier of the vasculature and dengue virus-induced responses resulting in edema or hemorrhagic disease ultimately cause changes in EC permeability. Unique EC receptors, adherens junctions, and signaling pathways respond to cytokines, permeability factors, immune complexes, clotting factors, and platelets, normally acting in concert to control vascular leakage \[[@B5], [@B32]--[@B36]\]. Virally induced changes in endothelial or immune cell responses have the potential to alter this orchestrated balance with pathologic consequences \[[@B5], [@B32]--[@B35]\]. However, very little is known about the role of dengue virus-infected ECs in disease or the kinetics, timing, and replication of dengue viruses within patient ECs. The inability to kinetically study the endothelium in dengue patients and the relative ease of assessing blood components has resulted in a focus on immune cells instead of ECs. Yet, the endothelium is the ultimate target of permeabilizing responses. Here, we discuss studies of dengue infected ECs and the potential for the dengue infected endothelium to contribute to dengue pathogenesis. 2. Human Responses to Dengue Virus Infection {#sec2} ============================================ DHF and DSS are severe manifestations of dengue virus infection that result in increased vascular permeability, hemorrhage, and shock \[[@B3]\]. The presence of preexisting antibodies to dengue virus predisposes patients to severe disease following infection by a second dengue serotype \[[@B3]\]. A myriad of responses are associated with dengue infection that may contribute to disease, but the pathogenic mechanisms that result in DHF and DSS remain ambiguous \[[@B1]--[@B3], [@B26]\]. One common element of the dengue disease process is that enhanced immune responses increase vascular permeability by acting on the endothelium. Although it is clear that immune cells and their responses contribute to pathogenesis, the endothelium, which regulates vascular leakage, has not been considered a significant component of DHF and DSS \[[@B2], [@B5], [@B34], [@B35], [@B37]\]. 2.1. Patient Studies {#sec2.1} -------------------- The ability of dengue virus to infect human ECs has been documented in autopsy samples of the heart, liver, and lung \[[@B9], [@B14]\]. In a postmortem study, Jessie et al. reported dengue virus antigen in sinusoidal ECs in the liver as well as macrophages, lymphoid cells in the spleen, the vascular endothelium of the lung, monocytes within the blood, and kidney tubules \[[@B9]\]. Salgado et al. also demonstrated the presence of viral antigen in endothelial cells within the heart and small myocardial vessels of a patient postmortem \[[@B14]\]. Depressed myocardial function has been associated with hemorrhagic forms of dengue virus infection \[[@B38]\]. Although no dengue virus RNA was detected in these cells by in situ hybridization, viral antigen uptake was also not confirmed. Additionally, no morphological damage to the endothelium was observed that might explain vascular leakage through disruption of the endothelium. However, the presence of circulating ECs, EC markers (VCAM and ICAM), and increased von Willebrand factor antigen and procoagulants, specifically in DHF patients, has been reported in other cases \[[@B39], [@B40]\]. Nevertheless, autopsy samples do not take into account contributions of dengue virus infection of ECs at earlier time points that may contribute to viremia and immune enhancing responses. Kinetic analysis of the endothelium in patients is invasive and has not been addressed. In general, little clinical data has been obtained between viral inoculation and onset of fever and viremia \[[@B3]\]. Thus, findings that ECs are infected with dengue have been marginalized since immune enhancing responses are presumed to be derived solely from immune cells. However, a variety of mechanisms exist for ECs to elicit immune enhancing cytokine and complement responses that recruit immune cells to the endothelium or directly alter barrier functions of EC adherens junctions \[[@B3]--[@B5], [@B41]\]. Finding that ECs are infected in patients suggests a direct means by which the infected endothelium may be altered by dengue virus. Additionally, preexisting antibodies may target DV antigen within infected ECs, further contributing to immune-enhanced permeability deficits observed in DHF and DSS. 2.2. Patient Responses to Infection and Markers of DHF/DSS {#sec2.2} ---------------------------------------------------------- A hallmark of severe dengue disease is the presence of elevated levels of cytokines and chemokines including IP-10, ITAC, IL-1β, IL-2, IL-6, IL-8, IL-10, IL-12, IL-13, TNFα, IFNα, IFNγ, MIF, RANTES, histamine, and complement proteins C3, C3a, and C5a within blood and tissues \[[@B1]--[@B3], [@B14]--[@B18]\]. In patients, complement activation and an increase in complement protein products correlate with the severity of disease \[[@B42]--[@B45]\]. In a study by Avirutnan et al., C5b-9 complexes, complement-activated membrane-attack complexes, and C3a were formed on dengue-infected ECs in the presence of antibody---containing immune serum, though they did not direct complement-mediated cell lysis \[[@B26]\]. C3a is an anaphylatoxin that recruits mast cells and directs histamine release that locally increases vascular permeability \[[@B17], [@B19]--[@B22]\]. Elevated C3a, C5a, and histamine have been associated with severe permeability deficits in dengue virus patients and in the development of DHF and DSS \[[@B2], [@B3], [@B17], [@B26], [@B42]\]. Their presence in the blood of patients with severe dengue disease is significant since these anaphylatoxins direct lysis of infected cells and mast cell degranulation, leading to histamine release. Importantly, cytokines, chemokines, and complement-activating factors can all be secreted by and act on the endothelium, influencing EC regulation of fluid barrier function and vascular leakage \[[@B5], [@B32]--[@B35]\]. The ability of dengue virus to infect the endothelium intimates that additional mechanisms could contribute to vascular permeability deficits through both direct- and immune-enhanced disease processes. Dengue infected ECs may elicit chemokine and cytokine responses that further activate or recruit immune cells to the endothelium and preexisting dengue antibodies may target viral proteins displayed on infected endothelium. Recent analysis of primary EC transcriptional responses indicates that dengue virus strongly induces secretion of immune cell activating cytokines, chemokines, and complement factors that are likely to contribute to an immune enhanced disease process \[[@B46]\]. Since permeability is ultimately the result of responses that act on the endothelium, dengue infected ECs are key elements in DSS and DHF that must be considered more fully within animal and in vitro models. 2.3. Roles of NS1 and Cross-Reactive NS1 Antibody {#sec2.3} ------------------------------------------------- Dengue proteins may also play critical roles in enhancing DV pathogenesis within ECs through a variety of mechanisms. In particular, the NS1 protein is uniquely expressed in three forms: cytosolic, cell-surfaced expressed, and secreted \[[@B47]--[@B49]\]. Soluble secreted NS1 is both highly abundant and highly antigenic \[[@B50]\]. Likewise, NS1 antibodies are also present in high quantities and have been shown to bind the surface of platelets and ECs \[[@B51]\]. Because ECs are in steady contact with blood, they are susceptible to enhanced immune cell targeting promoted by adherent cross-reactive NS1 antibodies \[[@B51]--[@B53]\]. This targeting, as well as intracellular signaling triggered by direct NS1 antibody binding, may contribute to tissue-specific endothelial dysfunction and vascular leakage through EC activation \[[@B5], [@B51]--[@B53]\]. However, despite the abundance of NS1 antibodies that could promote leakage, vascular permeability is transient and additional contributions of NS1 have been poorly explored within humans and mouse models. Secreted NS1 can also bind cellular heparan sulfate E present on primary liver and lung ECs \[[@B54]\] and, along with the cell surface form of NS1, may further recruit circulating antibodies and immune factors to dengue infected ECs \[[@B55], [@B56]\]. As a secreted protein, the dengue NS1 protein modulates classical complement activation by binding to the C4b binding protein, thereby inactivating C4b \[[@B57]\]. Thus, cell-surface expressed NS1 on ECs could serve as a platform for C4b inactivation and antagonize classical complement activation pathways. Secreted NS1 may similarly attenuate complement activation by binding C1s/proC1s and C4 in a complex that reduces C4 levels required for complement pathway activation \[[@B58]\]. Together, NS1 and NS1 antibodies form a potent combination within DHF and DSS patients capable of eliciting or regulating immune and complement responses that act on the endothelium and contribute to dengue pathogenesis \[[@B59]\]. Curiously, the alternative complement pathway activator complement factor B, transcriptionally induced in dengue-infected endothelial cells \[[@B46]\], may induce C3a- and C5a-directed chemotaxis and histamine release by bypassing NS1 complement regulation. Antibody targeting of factor D, which activates factor B through cleavage, inhibits complement and leukocyte activation in nonhuman primates and several therapeutics have been developed that antagonize C3a and C5a receptor binding \[[@B60]--[@B63]\]. These advances suggest that the alternative pathway may be a new potential target for therapeutically reducing the severity of DHF and DSS diseases. Additional barrier stabilizing effectors that target the endothelium may also be considered as a means of therapeutically reducing vascular leakage and inflammation that contribute to dengue pathogenesis \[[@B64], [@B65]\]. 3. In Vivo Animal Models of Dengue Virus Infection {#sec3} ==================================================== 3.1. Mouse Models of Dengue Virus Infection {#sec3.1} ------------------------------------------- Progress in understanding dengue pathogenesis has been hampered by the lack of suitable mouse models that replicate human cellular tropism and disease symptoms. In normal mice, dengue infection results in limb paralysis and little mortality \[[@B31]\]. Recently mouse-adapted dengue strains that mimic aspects of severe human disease in interferon (IFN) receptor knockout AG129 mice have been used as a dengue virus animal model \[[@B8], [@B31], [@B66]--[@B68]\]. Organ damage, hemorrhage, vascular leakage, viremia, and elevated cytokine levels analogous to that in humans are observed following dengue infection of AG129 mice \[[@B66], [@B67]\]. In one study, high titer inoculation of mice initiated TNFα-induced apoptosis of ECs, leading to vascular leakage \[[@B69]\]. However, IFN defective murine models further complicate our understanding of the dengue disease process since they do not fully mimic human responses to infection and lack IFN responses that limit dengue spread and induce EC proliferation and repair. Despite these limitations, current models have provided new insight into dengue virus pathogenesis and allow for kinetic studies of dengue virus infection of ECs. Vascular leakage occurs in AG129 mice infected with mouse-adapted dengue strains and several studies have isolated murine-infected ECs within the spleen and liver \[[@B8], [@B30]\]. In support of a role for infected ECs in mediating severe dengue disease, Zellweger et al. recently reported that in the presence of subneutralizing levels of dengue-specific antibodies (ADE-mediated infection), a large percentage of infected liver sinusoidal ECs (LSECs) were detected and correlated directly with disease severity \[[@B8]\]. No evidence for ADE-mediated infection of ECs exists in vitro \[[@B70]\], although liver sinusoidal cells reportedly express Fcγ receptors that may contribute to immune enhanced infection of liver ECs \[[@B71]\]. In the same study, infection of mucosal macrophages was not enhanced by the presence of dengue antibodies and occurred after detection of infected LSECs, suggesting that increased viral loads from LSEC infection, but not ADE, enhanced immune cell infection \[[@B8]\]. Although a mechanism for this occurrence has yet to be determined, these findings give importance to the role of ECs in mediating dengue pathogenesis in the mouse animal model. Therefore, in addition to increasing viremia, the ability of dengue virus to infect ECs in vivo may provide a means for infection to alter capillary permeability and induce cytokine responses from ECs that recruit immune cells and contribute to dengue pathogenesis. 3.2. Mouse Model Responses Influenced by IFN {#sec3.2} -------------------------------------------- Since IFN plays a significant role in the regulation of viral spread and the growth and repair of the endothelium \[[@B3], [@B72]--[@B76]\], it is important to consider the consequences of dengue infections occurring in IFN unresponsive mouse models. Since IFN reportedly stimulates EC proliferation \[[@B76]\], IFN secretion by dengue infected cells is also likely to contribute to vascular repair following dengue infection, and the absence of the IFN-signaling response may explain the enhanced pathogenesis of dengue infections in IFN receptor knockout AG129 mice \[[@B31], [@B67], [@B77]\]. This absence abrogates antiviral responses that may naturally curb infection \[[@B72], [@B74]\]. Likewise, the dengue NS5 protein, which interferes with downstream IFN signaling to permit virus replication through STAT2 degradation, is unable to bind mouse STAT2 \[[@B78], [@B79]\]. These differences cloud interpretation of results from dengue-infected mice and may in fact contribute significantly to hemorrhagic responses that may or may not reflect normal pathogenic mechanisms. Current work is ongoing to address the lack of IFN responses within mice and create knock-in mice, which harbor functional human STAT2 \[[@B79]\]. 3.3. Nonhuman Primate Models {#sec3.3} ---------------------------- Limited studies have also been conducted on nonhuman primates (NHPs) as an animal model. However, NHPs display almost no human symptoms of DF/DSS/DHF despite detectable virus replication \[[@B80]\]. Gene profiling following infection in NHPs revealed a potent antiviral response yet, in contrast to humans, almost no production of type I or II interferons or inflammatory cytokines \[[@B81]\]. Thus, murine models still appear to be the most suitable animal model for studying dengue infection, specifically in relation to cellular tropism and EC responses. Further work continues to explore new mouse adaptations that may one day produce animal models that fully mimic human responses to dengue infection. 4. In Vitro Infection of Endothelial Cells {#sec4} ============================================ 4.1. Use of Endothelial Cell Lines for Studying Dengue Virus Infection {#sec4.1} ---------------------------------------------------------------------- The difficulty of analyzing infections of the endothelium in vivo has driven the in vitro exploration of dengue infection of ECs. In vitro, ECs from various sources are permissive for dengue virus infection and have been used to study pathophysiological changes occurring within the endothelium following dengue virus infection. However, not all EC lines are equivalent and this has led to confusing and often contradictory results \[[@B82], [@B83]\]. Cell lines derived from endothelial and epithelial cell fusions are not representative of primary ECs and the ECV304 endothelial cell line has been shown to be bladder carcinoma and not endothelial in nature \[[@B84]\]. However, early passage primary human ECs permit investigation of dengue virus infection that approximates the human endothelium for analysis of dengue-altered EC responses. 4.2. Replication and Receptors for Dengue Virus on ECs {#sec4.2} ------------------------------------------------------ Dengue virus replicates within HUVECs (human umbilical vein ECs), LSECs, HPMEC-ST1.6R cells (human pulmonary EC line), ECV304 (endothelial cell line), and HMEC-1 cells (human microvascular EC line) \[[@B26], [@B27], [@B70], [@B85]--[@B89]\]. Recent in vitro studies demonstrated that efficient infection of primary ECs by dengue virus occurs as a result of attachment to heparan sulfate-containing cell surface proteins (HSPGs) \[[@B88]\]. HSPGs, specifically heparan sulfate glycoproteins of syndecan 2, also mediate attachment of dengue virus to K562 monocytes \[[@B90]\]. Although more specific HSPGs on ECs still need to be defined, an EC receptor blockade has the potential to reduce viremia, immune targeting of dengue virus infected ECs, and dengue virus-induced changes in ECs that contribute to pathogenesis. 4.3. Responses Elicited by Dengue-Infected ECs In Vitro {#sec4.3} --------------------------------------------------------- Several studies have focused on changes in the levels of cellular molecules or markers of EC activation, including VCAM and ICAM \[[@B91], [@B92]\]. Dengue virus infection upregulates cell surface markers of EC activation which can trigger the expression and release of various cytokines, chemokines, and complement factors that act on neighboring tissues, ECs, and circulating immune cells. Analysis of dengue-induced permeability responses suggested that EC permeability was increased in vitro \[[@B93]\]. Although a productive infection was not verified in this study, permeability occurred in conjunction with a decrease in VE-cadherin, which regulates the fluid barrier function of adherens junctions \[[@B93], [@B94]\]. Additional studies examined the induction or secretion of cytokines following dengue virus infection of primary HUVECs and ECV304, LSEC-1, HMEC-1, or HPMEC-ST1.6R cell lines \[[@B26], [@B27], [@B37], [@B85], [@B87], [@B89], [@B95]\]. Dengue infection of the HPMEC-ST1.6R cell line increased IL-6 and IL-8 (6--8 days p.i.) as well as vascular endothelial cell growth factor (VEGF) \[[@B27], [@B95]\]. Other studies have also singled out RANTES, IL-6 and/or IL-8 as cytokines elicited by dengue-infected ECs \[[@B26], [@B37], [@B46], [@B87]\], thus promoting the endothelium as a source of potent chemotactic cytokines in DSS/DHF patients. Both IL-8 and RANTES are chemotactic agents that can increase vascular permeability by localized attraction of neutrophils \[[@B96], [@B97]\]. Analyses of transcriptional changes elicited in response to dengue infection also show small increases in additional cytokines and antiviral IFN-stimulated genes \[[@B27], [@B37], [@B46]\]. IFN is highly induced in dengue patients and these findings suggest that ECs are likely to contribute to circulating IFN responses. 4.4. Kinetics of Dengue Virus Infection In Vitro {#sec4.4} -------------------------------------------------- Dengue infected endothelial cell lines reportedly induce a low level of infection (\<10%) and coordinately low-level transcriptional responses \[[@B26], [@B85]\]. However, analysis of \>90% uninfected cells at late time points after infection (3--8 days) makes it difficult to accurately assess the contribution from dengue infected cells. This is compounded by innate antiviral responses elicited by infecting a small number of ECs and the stimulation of IFN responses by \>90% uninfected ECs. A recent kinetic analysis of primary EC responses to dengue virus infection paints an important picture of the endothelium\'s role in dengue disease. Primary EC monolayers are \~80% infected by dengue virus and rapidly produce virus by 24 hours after infection \[[@B88]\]. Both the production of progeny virus and the number of infected ECs within monolayers decrease 2-3 days after infection \[[@B88]\]. Dengue virus titers following EC infection were nearly identical to viral titers observed in IFN-deficient VeroE6 cells \[[@B88]\], suggesting that virus regulates early cellular interferon responses of ECs. In fact, the lack of viral spread reported in some studies, where fewer than 10% of ECs were infected \[[@B37], [@B70]\], is consistent with the paracrine effect of interferon produced by a small number of initially infected cells. Thus, in vitro studies point to the infected endothelium as a likely source of viremia following dengue virus infection. These findings also highlight the importance of assessing the impact of EC infections at early times following in vivo infection, something more easily achieved within mouse models than patients. Recovering DHF patients regain normal endothelial function quickly, implying a transient effect and early recovery of EC functions following infection \[[@B3]\]. A rapid productive infection of the endothelium is difficult to assess in humans, but in vitro studies show that infected ECs rapidly release virus. This suggests that dengue infected ECs may contribute to early viremia in dengue patients and present dengue virus antigens on EC surfaces that may be targeted by immune cells \[[@B88]\]. Infected ECs may also elicit cytokine and chemokine responses that can act directly on the endothelium \[[@B26], [@B27], [@B37], [@B85], [@B87], [@B89], [@B95]\]. Endpoint sampling of ECs at autopsy or analysis of endothelial function within recovering patients does not take into consideration an early or transient infection of ECs as is observed in vitro. The lack of apparent spread within EC monolayers and the apparent decrease in infected cells 2-3 days after infection \[[@B46]\] also suggest that EC-elicited IFN responses may limit dengue virus spread in vitro and contribute to high levels of circulating IFN in dengue patients. Interestingly, IFN treatment directs EC proliferation and may be a response that both limits viral spread and activates the EC repair process \[[@B76]\]. In fact, EC proliferation in response to IFN may explain the absence of endothelial damage within DHF patients and the presence of vascular leakage in IFN receptor deficient mouse models. 5. Conclusions {#sec5} ============== The endothelium is not a static channel that simply separates the vasculature from surrounding tissue \[[@B33]--[@B35]\]. The endothelium dynamically elicits responses that may contribute to immune enhancement and vascular permeability during dengue virus infection. Several hypotheses have been offered to explain the development of severe dengue disease and immune enhanced responses clearly impact barrier functions of the endothelium, but pathogenic mechanisms that result in DHF and DSS remain vague \[[@B1]--[@B3], [@B26]\]. Leakage of the vascular endothelium is a central component of dengue virus disease and studies discussed here suggest that the dengue infected endothelium may contribute to pathogenic immune responses and immune targeting of the endothelium. However, the role of dengue virus infected ECs in pathogenesis requires definitive in vivo kinetic studies which are difficult to perform in patients. The ability of the endothelium to respond to immune cells resulting in capillary permeability further highlights the importance of dengue infected ECs in pathogenesis. The central importance of the endothelium in dengue disease suggests that stabilizing fluid barrier functions of the endothelium may be a therapeutic approach for reducing vascular leakage in DHF and DSS patients \[[@B64], [@B65]\]. The authors thank Irina Gavrilovskaya, Valery Matthys, and Elena Gorbunova for critical paper review and helpful discussions. This work is supported by NIH, NIAID Grants R01AI47873, PO1AI055621, R21AI1080984, and U54AI57158 (NBC-Lipkin). [^1]: Academic Editor: Sujan Shresta
Why doesn’t this site have ads? In order to maintain our integrity, Truthout doesn’t accept any advertising money. Help us keep it this way — make a donation to support our independent journalism. Last weekend, while Vice-President and former Indiana governor Mike Pence was giving the commencement address at Notre Dame University, over 100 students walked out in protest over his anti-LGBTQ and anti-refugee policy positions. Pence used this opportunity to give a 15-minute lecture about free speech on campuses, condemning what he calls “speech codes, safe spaces, tone policing, administration-sanctioned political correctness — all of which amounts to nothing less than suppression of the freedom of speech.” In contrast, he extolled the virtues of civility, open debate, the pursuit of knowledge, and the free exchange of ideas. Pence’s arguments, which sound lofty and noble, conceal as much as they reveal about the role of free speech in educational contexts today. Also see: The Home of Free Speech™: A Critical Perspective on UC Berkeley’s Coalition With the Far-Right Much has been written in the past several months about dramatic conflicts at universities, especially those between protesters and high-profile far right figures like Ann Coulter, Milo Yiannopoulos, and Richard Spencer, bringing the issue of student activism and free speech to the forefront. While the recent focus has been on these so-called “alt right” celebrities and the growing role of groups like the Young America’s Foundation (YAF), there is a much longer history of conservative speakers being invited to campuses under the banner of free speech. Here I examine the groundwork laid by the Federalist Society, a long-standing legal organization which has been sending reactionary speakers to universities for nearly 40 years. Drawing connections between arguments used by liberal proponents of free speech and the rhetoric of the alt right, I examine how the free speech and open debate arguments being used today to defend the hateful messages of far right speakers have been established over a long period and need to be explored in the context of rising fascism, white supremacy, and extreme social inequality. From this perspective, the comments of Pence (himself an affiliate of the Federalists) take on a deeper and more ominous meaning. The Federalist Society Outside the legal profession, most people know very little about the Federalist Society, a group that has been called “quite simply the best-organized, best-funded, and most effective legal network operating in the country.”[1] As the political right gains traction under the Trump administration, it is worth exploring the mission and history of this group, which has played a critical role in the conservative shift of law and politics over the past 35 years. One of the ways the Society has spread its ideas and found new members is through its long-standing debate program, in which far right attorneys are sent to speak at law schools. According to their latest annual report, the Federalists spent $2.5M on student debates and hosted 1,100 events at law schools across the country in 2016 alone. The Federalist Society was founded in 1980 by law students and faculty who felt alienated by the allegedly liberal atmosphere of law schools. Since then, the organization has been enormously successful in translating its ideas into law and policy, and has done so while remaining mostly outside the attention of media and the general public. In their recent book, The Federalist Society: How Conservatives Took the Law Back From Liberals, Michael Avery and Danielle McLaughlin show how unrestricted funding provided by billionaires like the Koch brothers and John Olin has allowed the Federalists to promote extremely conservative legal positions which privilege private property rights, criticize government interventions in social and economic problems, and target the rights of women, immigrants, people of color, and gay and trans individuals and communities. Since its founding, the Society has grown exponentially. From four law school chapters in 1982, it has expanded to over 60,000 members in its 300+ student, lawyer, faculty, and alumni divisions.[2] However, not all “members” pay dues and the organization’s claims that they have active chapters at every law school are exaggerated. Regardless of actual numbers, the ideas of the Federalists have spread rapidly through members’ prolific publications, presentations, and influential public positions. With an annual budget ranging from $10-15M, the Federalists have developed a powerful network of think tanks, law firms, faculty, judges, and politicians.[3] The Federalist Society has been extremely successful in getting its members into powerful positions while keeping its influence out of public view. Those unfamiliar with the Society may be surprised to learn that its members are represented at every level of government and the judiciary, including four current Supreme Court justices (Clarence Thomas, John Roberts, Samuel Alito, and most recently, Neil Gorsuch). Every Federal Judge appointed by Presidents Bush (Jr. and Sr.) was a member of the Society or of an approved affiliate of the organization, and every Republican administration since Reagan has included prominent Society members.[4] This trend continues and has become even more pronounced with the Trump administration. During his election campaign, Trump promised that all of his judicial nominees would be “picked by the Federalist Society” and since becoming President he has consulted with both the Federalists and conservative think tank The Heritage Foundation in making lists to fill the 120+ currently vacant federal court positions. Federalist Society members argue that they do not have a specific “agenda” and that there is nothing clandestine or nefarious about their organization. Indeed, the Society is very public about its mission, its focus on ideas, and its commitment to speaking openly about conservative legal perspectives. Furthermore, given its alliance of libertarians, economic conservatives, social conservatives, and Christians, it is true that the Federalists cannot be said to be an ideological monolith. In fact, the organization itself does not lobby or take public policy positions, but rather relies on its individual members and allied organizations to pursue goals such as rolling back affirmative action and identity-based discrimination laws, contesting government regulation of the economy and environment, removing access to legal remedies for workers and consumers, expanding state support for religious institutions, opposing abortion, protecting private property, challenging protections for immigrants, and limiting the size of the federal government. The overall impact of these various (sometimes disparate) positions is to provide advantages to the already wealthy, while leaving the rest of society poorer and increasingly disenfranchised. Although the Society presents itself as simply an intellectual forum, in reality it holds an immense amount of power and influence. Free Speech and Its Discontents For decades, the Federalist Society has sponsored debates at law school campuses in which their members argue the various positions described above. Organizing debates is a key strategy of the Society, which allows it to present itself as offering a dialogue of perspectives in order to provide a platform for what is often dehumanizing and far right rhetoric. In recent years, the Federalists have organized events featuring right luminaries such as John Yoo (author of the “torture memos“), Ryan Anderson (Fellow at the Heritage Foundation who calls gay rights “make believe” and defends conversion therapy), Roger Clegg (President of the Center for Equal Opportunity who argues that affirmative action discriminates against whites), Ilya Shapiro (Fellow at the CATO Institute who claims that corporate donations to political campaigns are not a problem), and Edward Whelan (President of the Ethics and Public Policy Center and proponent of the controversial “Bathroom Bill” in North Carolina, who argues that transgender activism has produced legal absurdities). During this time of controversy on campuses over the place of free speech within current political struggles, the role of the Federalist Society provides an example of how the conservative movement successfully legitimizes itself and spreads its message. Despite the conservative atmosphere of almost all law schools, and the current far-right influence in politics more generally, law student members of the Federalist Society still claim to feel silenced within the “liberal” context of their schools. Student groups and administrators invite far-right speakers under the banner of free speech, viewpoint diversity, and healthy debate, and portray challenges to or dissent against these speakers as attacks on the First Amendment (rather than seeing the protests themselves as protected forms of speech). While the Federalist Society does at least offer other perspectives by framing their events as debates, events sponsored by groups like YAF and College Republicans have increasingly been inviting provocative far right speakers the New York Times has described as “edgier, more in-your-face and sometimes even mean-spirited.” Competing perspectives on free speech across the spectrum of the left are worth examining at this fraught political moment. One popular approach, exemplified by our allies at the ACLU, argues that even hateful speech is constitutionally protected. From this perspective, speech that attacks individuals and groups based on race, gender, ethnicity, religion, or sexual orientation is both legal and defendable. The ACLU and many liberal-minded people assume that allowing all speech under any circumstances will ensure that the best ideas win out and that it is ideal to have even potentially dangerous ideas out in the open where they can be challenged. They question attempts by universities to adopt codes and policies prohibiting hate speech, arguing that this well-intentioned response is incorrect and akin to censorship. Rather than restrict the right to use racist, sexist, transphobic, ableist, or other such speech on campuses, the ACLU recommends an educational approach that offers less intolerant viewpoints from which individuals can choose. A final important argument from this perspective points out that the limiting of speech on one end of the political spectrum can produce limitations on any speech found to be controversial, and will inevitably lead to greater restrictions on the other end. This approach may seem logical and commonsense to many, and this line has certainly been taken up by the far right, who complain that the failure to include conservative views alongside liberal perspectives is a violation of free speech. On university campuses, reactionary student groups and their supporters draw on First Amendment arguments to promote agendas that are openly racist, sexist, homophobic, transphobic, xenophobic, and ableist. They claim that any resistance from the administration or the student body to these hateful ideologies is in violation of legally protected speech, and even ostensibly progressive universities have given in to this pressure by monitoring and censoring opposition. Extreme right fascist and white nationalist groups outside of universities also rely on the discourse of free speech to claim their views are valid and protected. While complaining about the “politically correct snowflakes” on the left, these far right speakers and their supporters actively cultivate their status as victims by attacking the vulnerable through their hateful speech and then claiming persecution when challenged. From the commonsense liberal approach described above, the best way to address these kinds of speakers would be to let them express their views so others can decide if they agree or not. If all sides are debated openly, advocates of this perspective contend, the best one will obviously succeed. However, far right conservative and fascist ideology is not simply based on logical and reasonable arguments; rather, these movements depend on the irrational mobilization of hate, fear, and anger against some of the most marginalized and vulnerable populations. Offering them an open forum and vigorously defending their right to promote harmful speech confers legitimacy on their positions as being equally as acceptable as any other. Another problem with the liberal free speech model is that is does not take into account the asymmetry of different positions and the reality of unequal power relations. Arguments about free speech rarely address the significant imbalances in power that exist between, for example, a wealthy white speaker with the backing of a multi-million dollar organization and members of the populations affected by their words (i.e. immigrants, people of color, queer and trans people, low-wage workers, etc.). What are lost in the abstract notion of free speech are the rights of those who do not have the connections or wealth to equally participate in public discourse. The “marketplace of ideas” is like any other marketplace; those with the most resources dominate. Finally, the trend of students and local community members protesting reactionary speakers at universities has led to outcry about the “intolerant left” violating the free speech of the far right. But those who are so determined to protect the free speech of fascists, white supremacists, and other hate groups should be equally as concerned with protecting the right of dissidents to protest these viewpoints. While giving a speech attacking individuals and groups based on their race, sexuality, or immigration status is considered legal and acceptable by universities, the protests of those who find these viewpoints reprehensible are often censured or punished by the same institutions. It should give us pause that recent model legislation to protect “free speech” on campuses and to discipline those who protest controversial speakers comes from conservative think tanks The Heritage Foundation, The Goldwater Institute, and The Ethics and Public Policy Center. Strategic Interventions Since the 1980s, when the Federalist Society began sending extremely conservative speakers to law schools, concerned law students and faculty have responded in various ways. In 2001, the American Constitution Society was formed to help counter-balance the effect of the Federalists in law schools. The ACS position aligns with the general liberal perspective described above and held by the ACLU. By taking part in the Society’s debates, and regularly co-sponsoring them, they hope to provide other, less harmful perspectives. NLG faculty members have also taken part in these exchanges, although Guild members are generally more cautious about participating in debates that are framed in biased or oppressive ways. While there are advantages to debating conservative speakers head on, this approach also comes with the danger of legitimizing or validating the terms of the debate. However, taking part and challenging the framing of the debate itself can be a politically useful strategy under some circumstances. Finally, it is important to acknowledge the reality that Federalist Society speakers have access to resources that make it far easier for them to have a platform than many ACS or NLG speakers. While the Federalists can afford to pay for travel, expenses, and honorariums for their spokespeople, many progressive speakers have to turn down speaking engagements for lack of funds. Federalist Society speakers have often been met with protests from law student groups like the NLG, OutLaws, and If/When/How. Challenges to reactionary speakers have included putting up flyers with information about the speakers and their background, circulating petitions to have the event cancelled, organizing counter-events and speakers, writing op-ed pieces for campus and local publications, sending students to the event with a list of critical questions, and protesting outside or within the event by walking out or holding signs. University administration responses to these kinds of interventions have often been to stifle the protest, although these activities also fall under the banner of protected speech. Law students report having their fliers removed from the campus, being threatened with disciplinary sanctions, or even being told that protesting will lead to negative evaluations on the Character and Fitness Exam required for the bar. While the rights of dissenting students are suppressed, the ability of far right speakers to disseminate hateful rhetoric is protected through claims of the right to free speech. These are only some strategies for confronting harmful speech in educational settings. While liberal advocates are quick to invoke First Amendment arguments to allow all speech, there are other considerations to take into account as well, such as: Who is able and allowed to speak, under what conditions and with what consequences? What voices are silenced and what forms of dissent are possible (or not)? Universities can use free speech principles to justify invitations to xenophobic and hate-mongering speakers, but not inviting or funding these people is not necessarily a violation of their free speech, especially when they have many other platforms for getting their message out. Private schools, for example, are not bound by the First Amendment in the same ways as public schools, and can therefore make policies about hate speech that limit invitations and/or funding to reactionary speakers and groups. When the views of speakers are actually dangerous to other people, universities should consider the implications and balance the need for a diversity of viewpoints with the consequences of invalidating the humanity or rights of entire groups of already disadvantaged people. [1] Jerry M. Landay, “The Conservative Cabal That’s Transforming American Law,” Washington Monthly, March 2000. [2] For more information, see the Federalist Society website. Background information can be found at fed-soc.org/aboutus/page/our-background. [3] Michael Avery and Danielle McLaughlin, The Federalist Society: How Conservatives Took the Law Back From Liberals (Vanderbilt University Press, 2016). [4] Ralph G. Neas, “The Federalist Society from Obscurity to Power: The Right Wing Lawyers Who Are Shaping the Bush Administration’s Decisions on Legal Policies and Judicial Nominations,” Report of the People for the American Way Foundation, 2001. Available at: https://files.pfaw.org/uploads/2017/01/federalist-society-report.pdf.
Losing my mind one day at a time… Main menu Post navigation The year the plan expired Life is full of moments. This is one now and that was one then. If you walked into that Dublin hotel you could, as a non-privy spectator, have mistaken it for a wedding reception, such was the crowd. The same crowd that could confuse would also, on closer inspection, give the game away. The colours were all wrong. Dark shades and black ties had won out. Hands were busy, holding either hot drinks or each other, offering a consoling hand on the shoulder or a hug to those who needed something more. Caffeine trumped alcohol as we talked with family and friends, reminiscing about my father, whose funeral had been that morning. 2007 had an awful lot to answer for. With a breaking heart and a muddled brain my phone rang, and I answered it. I excused myself and confirmed it. As planned, the phone line would be connected tomorrow. No ifs or buts. And no father. He had visited the busy construction site before and we pointed out the place we believed to be ours. I hope he saw the one we meant. I know he felt our excitement. The year could not, would not, end this way. We finally had our keys and, after two years, couldn’t wait a minute longer to move in. Our relationship was going from strength to strength. Our subsequent desire to make a ‘grown-up’ decision was to put us in the company of thousands of others – not of developers, or investors, but of couples in love who wanted nothing more than the opportunity to turn a house into a home, our home. In the five years that passed, it never became that to me. I couldn’t bring myself to let it. The property crash, and the gradual realisation that we were to become a statistic in the mess and little more, led to a deep inward resentment. It goes without saying that I, like many many others, did not see the crash coming. There are plenty who will say that they did, but had they been of a certain age, means and motive, would have done similar. They now have hindsight, without the bittersweet aftertaste, and can rewrite their own personal history as they see fit. The truth be told, we did our best to be pragmatic, making sure to stick below the €317,500 in order to avoid paying stamp duty. That additional outlay is now but a small percentage of what we owe, which will never be recouped by what we own. Watching things pan out in a way you never expected, with each twist and turn making it painfully clear how little control we have over this situation has proven to be gut wrenching and too often, gut wrenching on a daily basis. The five-year plans of many homeowners have by this stage fallen by the wayside. 2012 was our expiration date. Wearing the now tattered and ill-fitting clothing that is negative equity does little to compliment the outfit that is our life in 2013, and what it has become. The term ‘long-term’ has for too long held only negative connotations for me. My imagination, vivid as it is, would struggle to visualise a different property in our future, the property that would become our real house, the one that would allow for pet ownership, better amenities and a shorter commute. Thankfully, the starting of a family, whenever that will happen and however it’ll be funded, is considered a compromise too far by both of us. There will come a point, however, when space, or the lack of it where we are, may come to play a part in that as well. By virtue of the things this real house would allow for, it couldn’t but become a home. Couldn’t it? As a journalist I spend a great deal of each day being objective. My own inner newsreel does not benefit from any such objectivity, however, when thoughts of signing on the dotted line 2005 are replayed. No balance. Just bias. And let’s not forget the misplaced self-criticism at not being able to foresee what the future would hold. In the years that have passed, life has gone on in this house. Ten birthdays and six Christmases have been celebrated here. Having never missed a mortgage payment and with two incomes (for now), we remain the lucky ones. There are those who can no longer afford their mortgage repayments, those whose homes have been effected by pyrite, and those from Priory Hall who remain in limbo. I can only write about my reality, however, and the reality is that holding onto that five-year plan and watching it count down to zero was a waste of time and energy. When everything had changed but the self-imposed deadline which justified the purchase, simple math rendered our best intentions redundant. It’s time for a new plan. What is common to us all, regardless of our financial burdens – past, present, or future – is that each of us remain in possession of something that has never dropped in value, and never will. If you’re still struggling with your New Year’s resolution for 2013 and, more importantly, if you find yourself struggling in general, adopt the one that I should have adopted in 2009 – the one that I am adopting now. Be a better friend to yourself. Feel the fear, do it anyway, but accept yourself – always and without condition. Be your own best friend and safeguard the only thing you will ever own that is truly priceless.
This month (May 2019), Microsoft successfully completed the service updates on Auto Attendant and Call Queue for Microsoft Teams & Skype for Business Online. Curious about what’s changed? We’ll talk you through it with four questions and answers: There’s no one-size-fits-all formula that tells you how to communicate optimally during your change process, but our change consultants do follow a set of basic principles and tools when supporting your project. The following three tips will help you set up your communication plan. In the previous blog post in our ‘6 steps to the cloud’ series, we discussed ARM templates and Kubernetes manifests. In this post, we’ll use templates to roll out the Kubernetes infrastructure and our applications in Azure. Which templates do you use to roll out apps and infrastructure using code? We recommend templates which contain a desired state and which you can roll out idempotently (this means you can execute the template multiple times without impacting the current state). Think of ARM templates and Kubernetes manifests, for example. Do you want to offer your end users reliable containers? A container orchestrator is just what you need. The most common one is Google’s open-source container orchestrator, Kubernetes. Since almost all public clouds work with it, it’s often considered the standard solution. Microsoft offers Kubernetes as a service through Azure Kubernetes Service (AKS). On Wednesday April 17, Microsoft and Steelcase launched the new Microsoft Surface Hub 2S solutions at the ‘New Work, New Rules’ event in New York City. Let's take a look at the technological details, the price and the product roadmap.
Seat Stretch For people who spend most of their work day sitting down, being sedentary for most of your day can only hurt your posture and back. Without the chance to move around, our bodies become stiff and inflexible, which is bad news for our muscles. The best thing you can do for your body is to take stretch breaks throughout the day. Not only will it keep you more alert and help you focus on your work, but you can perform these stretches right in your cubicle without bothering any of your coworkers. What to expect from seated stretch There are plenty of stretches you can perform from your office chair. You can stretch out your neck, which does not often get the exercise attention it needs. Neck exercises are devastatingly simple. You just stretch backwards, forwards, to the sides, and by looking over each shoulder. The chest and biceps stretch can be performed standing or sitting, since you’re only holding your hands behind your back and stretching them upwards. A simple shoulder stretch merely entails holding one arm across your chest as you keep your shoulders relaxed, and you can stretch your torso by holding your hands together and reaching them straight up to the ceiling. Make sure you bring your hands down slowly after each rep. Each of these exercises demands straight posture, both so you can breathe easily during them and so your muscle groups are not strained from slouching and thus reap no benefits, or get injured. How to prepare for the seated stretch Stretching in your seat is great not just for people who work in an office, but for those who do not have great balance and so prefer to be seated. Just make sure that your chair is sturdy enough to stay upright if you wiggle around a bit. Before you begin you should probably scooch your chair back from the desk a bit, so you don’t accidentally knock anything over or hit your arm on the wall. If you want to bring in some equipment, you can trade your chair of a stability ball, or use a hand gripper to exercise your hands as you read from your computer monitor. Muscles you'll engage with the seated stretch Seat stretching mainly works out the muscles above your waist since those are the easy muscles to move around as you sit. The stretches listed above work out your neck, shoulders muscles, back muscles, chest, biceps, and torso. Stretching out your legs is probably best left for at home, where you can spread out on the floor and stretch out your hamstrings and such. Your muscles above the waist need to be loosened up the most because those are the areas that can be compromised while you are working at a desk. You’re probably slouching, and your arms aren’t getting much movement as your fingers type, and since most people do not have their computer screens and chairs at the correct height you are probably straining your neck more than you have to. Compared to that, your leg muscles are fine and can wait until you get home. But your upper-body muscles are key to your comfort, and as bad posture persists it begins to affect each muscle group in a domino-like effect. You won’t just be easing your pain, but pumping energetic life back into those muscles! Tips for success with the seated stretch Remember that you should only hold each stretch for about 30 seconds, and stop immediately if you begin to feel pain. To keep the blood flowing throughout your body, you should try to stand up and take a few steps every half-hour, even if you are just walking to the kitchen and back. Whenever you feel yourself starting to lose focus or wait for your slow internet to load, you can push out a few stretches. You’ll feel better if you keep a water bottle nearby to sip through regularly, and make sure you are taking good deep breaths as you exercise.
Complications After Body Contouring Surgery in Postbariatric Patients. Over recent years, body contouring procedures in postbariatric patients have been in exponentially growing demand resulting in high complication rates rendering a variety of ciphers in the literature. The purpose of this study is to determine the complication rate in patients who have undergone body contouring surgery after bariatric surgery between June 2012 and March 2015 at Hospital de San José. A cohort study including 153 individuals who underwent a total of 198 body contouring procedures after massive weight loss following bariatric surgery was conducted. Data on variables, such as complication rate according to the type of body contouring surgery, major and minor complications, weight of resected tissue, or intraoperation time, among other variables, were analyzed. A total of 198 procedures were performed in 153 patients. The mean (SD) age of the patients was 43.93 years (9.4 years). Of 198 procedures, 110 (55.5%) had complications. The rate for major complications was 13%, and for minor complications, 87%. Complication rates according to the type of operation were as follows: circumferential abdominal lipectomy, 55.7%; extended abdominal lipectomy, 53.7%; cruroplasty, 69%; breast surgery, 57%; and brachioplasty, 40%. Patients who presented with bleeding enough to require transfusion (P = 0.000) and with weight of the resected tissue greater than 2700 g in abdominoplasty (odds ratio, 3.26; 95% confidence interval, 1.48-7.1) had a higher complication rate. There were no thromboembolic events among this population. The overall complication rate was 55.5%. The great majority were minor complications. The thromboprophylaxis regimen used was 100% effective.
PaRappa chief artist Rodney Greenblat kindly offered up some of his free time to answer our pressing questions. Enjoy! PaRappa the Rapper is one of the most iconic characters of the early PlayStation era. Can you explain to our readers how the opportunity to design this great character first came about? My agent helped me get a contract with a licensing division of Sony in Japan called Sony Creative Products. I was working for them designing cute characters for printed items and toys. The wonderful team at Sony Creative introduced me to Matsuura and his game project. Matsuura and his team were already fans of my children’s books and CD-ROM productions. They asked me to design the characters and world for the new mysterious and fantastic Playstation 1. PaRappa was a huge success and helped create a whole new genre of rhythm games. How does it feel that you helped create such an important character and were you surprised at how well the game sold? Sony and the whole Parappa 1 team were completely surprised by the runaway sales in Japan. I think what was great about making Parappa 1 was that we had few expectations, and were just trying to make something fun. When it turned out to be a hit, I was overcome with joy! Can you explain the inspiration behind your great artwork for PaRappa and his many friends from the series? When I was a kid I loved TV cartoons like Bullwinkle and Speed Racer. I also wanted to imitate Picasso and Matisse. So mix that all together. Sony Creative really understood my style, and their confidence along with the game team’s incredible creativity made it easy for me to make the best characters. Out of all the characters in the game, do you have a personal favourite and can you explain why (I love Chop Chop Master Onion)? I’ve always been partial to PJ Berri. He sleeps late every day, works in an old style record store and is a DJ at Club Fun at night. He loves to eat. What better life is there? Was PaRappa the first video game you helped create artwork for? Yes. My previous interactive projects were CD-ROM works, Rodney’s Funscreen, Wonder Window and Dazzeloids. Um Jammer Lammy again proved a huge hit, especially in Japan. Why do you think Japanese audiences really took to the games and their great style? Lammy has everything that Parappa 1 has, but is more complex and challenging to play. Gamers at the time wanted a more difficult game play. The style of both really fit the fashion of Tokyo at the time. Who would you rather rap/jam with? PaRappa the Rapper or Um Jammer Lammy? I have no sense of rhythm myself, so better not to rap. I might be able to sing along in the background with Milk Can. PaRappa is making a bit of a comeback with a new remastered version of the game now available on the PS4. Do you feel there is a chance a brand new game could be on the cards? I hope so! Were you involved in the TV series and are you a fan of it? No, I was not involved in the Parappa Anime, except I designed a few new characters for the new setting. The show is fun, but I always thought it could have been better, sticking to the concepts and style of the games a little more. Your great artwork has also been heavily used in many other types of media, from books to music. Do you have a particular favourite piece of artwork you have ever created? Right now I’m most proud of my book “Dharma Delight.” It available on Amazon.com and at many book stores. It’s a compilation of many of my recent artworks and an introduction to Buddhism and Zen. What projects/games are you currently working on? Right now I’m preparing to open my first retail shop. It called THE RODNEY SHOP and it opens May 20th, 2017 in Catskill NY (sorry for the lateness of the article all! – Ed). I’ll be selling artwork, prints and posters, plus my back stock of vintage 1990’s Japanese products. My hours will be Friday and Saturday 10 to 5, and Sunday 10 to 4. We’ll see you there! One more question before you shoot off, if you could share a few drinks with a video game character who would you choose and why? I think I’d have a beer with Mr. Prince Feaswallow and chat about the retail business. Nice! Thanks so much for stopping by Rodney, some great stories there! Readers, please visit Rodney’s website whimsyload.com Adrian
1. Field of the Invention The present invention relates to the field of flat panel display technology, and in particular to a method for manufacturing a thin-film transistor and a thin-film transistor manufactured with the method. 2. The Related Arts Liquid crystal displays (LCDs) have numerous advantages, such as thin device body, less power consumption, and being free of radiation, and is thus widely used. Most of the liquid crystal displays available in the market are backlighting liquid crystal displays, which comprise a liquid crystal display panel and a backlight module. The operation principle of the liquid crystal display panel is that liquid crystal molecules are arranged between two parallel glass substrates and electricity is selectively applied to the glass substrates to control change of the orientation of the liquid crystal molecules in order to refract out the light from a backlight module for formation of an image. A liquid crystal display panel is generally composed of a color filter (CF) substrate, a thin-film transistor (TFT) substrate, liquid crystal (LC) interposed between the CF substrate and the TFT substrate, and a sealant. A general manufacturing process comprises a front stage of array process (including thin film, yellow light, etching, and film stripping), an intermediate stage of cell process (including bonding TFT substrate and the CF substrate), and a rear stage of assembling process (including mounting drive ICs and printed circuit board). The front stage of array process generally makes the TFT substrate for controlling the movement of liquid crystal molecules. The intermediate stage of cell process generally introduces liquid crystal between the TFT substrate and the CF substrate. The rear stage of assembling process generally mounts the drive ICs and combining the printed circuit board to effect driving the liquid crystal molecules to rotate for displaying images. The TFT substrate generally comprises a glass substrate and TFTs formed on the glass substrate. The thin-film transistors are generally of three structures, which are a coplanar structure, an island-stop structure, and a BCE (back channel etch) structure. The currently available oxide thin-film transistors primarily belong to the coplanar structure and the island-stop structure. This is because the BCE structure may cause damage to the oxide semiconductor layers in the manufacturing process and thus affect the electrical property thereof. Referring to FIG. 1, a schematic view is given to illustrate the structure of a BCE structure thin-film transistor that is formed on a glass substrate 100 through a five-masking-operation manufacturing process and comprises a gate terminal 101 formed on the glass substrate 100, a gate insulation layer 103, a oxide semiconductor layer 105 formed on the gate insulation layer 103, and a source/drain terminal 107 formed on the oxide semiconductor layer 105. The oxide semiconductor layer 105 comprises at least one of zinc oxide (ZnOx), tin oxide (SnOx), indium oxide (InOx), and gallium oxide (GaOx) and indium gallium zinc oxide (IGXO) is commonly used. The source/drain terminal 107 is generally composed of an aluminum (Al) layer and a molybdenum (Mo) layer sequentially formed on the oxide semiconductor layer 105 through sputtering, followed by coating of photoresist material, exposure, development, etching, and removal of the photoresist material. An etching operation is applied in processing the aluminum layer and the etchant solution used is generally a mixed acid of phosphoric acid (H3PO4) and nitric acid (HNO3). However, the mixed acid of phosphoric acid and nitric acid may also react with the oxide semiconductor layer 105, making the oxide semiconductor layer 105 etched away and thus affecting the electrical property of the thin-film transistor and deteriorating the quality of the thin-film transistor.
<component name="libraryTable"> <library name="Maven: org.apache.hbase:hbase-client:1.2.0-cdh5.11.0"> <CLASSES> <root url="jar://$MAVEN_REPOSITORY$/org/apache/hbase/hbase-client/1.2.0-cdh5.11.0/hbase-client-1.2.0-cdh5.11.0.jar!/" /> </CLASSES> <JAVADOC> <root url="jar://$MAVEN_REPOSITORY$/org/apache/hbase/hbase-client/1.2.0-cdh5.11.0/hbase-client-1.2.0-cdh5.11.0-javadoc.jar!/" /> </JAVADOC> <SOURCES> <root url="jar://$MAVEN_REPOSITORY$/org/apache/hbase/hbase-client/1.2.0-cdh5.11.0/hbase-client-1.2.0-cdh5.11.0-sources.jar!/" /> </SOURCES> </library> </component>
Q: How can i transfer Array to table in TodayExtansion (Swift) How can i transfer array to table in Today Extansion (Swift) I'm new to this and nothing happens In main app i have a table with data In Today Extansion i have a table (in which the data should be displayed) its work with TEXT but not work with ARRAY var myData: [String] = [] MainVC.swift if let userDefaults = UserDefaults(suiteName: "group.Name1") { userDefaults.set("Test12345" as AnyObject, forKey: "key3") userDefaults.synchronize() } TodayExtVC.swift In ViewDidLoad: if let userDefaults = UserDefaults(suiteName: "group.Name1") { let value2 = userDefaults.string(forKey: "key3") print("\(value2)") } A: Set in MainVC.swift let array = ["horse", "cow", "camel", "sheep", "goat"] userDefaults.set(array, forKey: "key3") get in TodayExtVC.swift let myarray = userDefaults.stringArray(forKey: "key3") ?? [String]()
Radiographic localization of unerupted teeth: further findings about the vertical tube shift method and other localization techniques. The parallax method (image/tube shift method, Clark's rule, Richards' buccal object rule) is recommended to localize unerupted teeth. Richards' contribution to the development of the parallax method is discussed. The favored method for localization uses a rotational panoramic radiograph in combination with an occlusal radiograph involving a vertical shift of the x-ray tube. The use of this combination when localizing teeth and supernumeraries in the premolar region is illustrated. When taking an occlusal radiograph to localize an unerupted maxillary canine, clinical situations are presented where modification of the vertical angulation of the tube of 70 degrees to 75 degrees or of the horizontal position of the tube is warranted. The limitations of axial (true, cross-sectional, vertex) occlusal radiographs are also explored.
Byron Wesley Byron Wesley Julie Jacobson / Associated Press USC's Byron Wesley, right, gets his hand on a shot by Colorado's John Hopkins during the Trojans' season-ending loss in the Pac-12 tournament. Wesley plans to graduate from USC this summer and use his final season of eligibility at another school. USC's Byron Wesley, right, gets his hand on a shot by Colorado's John Hopkins during the Trojans' season-ending loss in the Pac-12 tournament. Wesley plans to graduate from USC this summer and use his final season of eligibility at another school. (Julie Jacobson / Associated Press) USC's Byron Wesley, right, gets his hand on a shot by Colorado's John Hopkins during the Trojans' season-ending loss in the Pac-12 tournament. Wesley plans to graduate from USC this summer and use his final season of eligibility at another school.
Q: Intel TBB license I'm a bit confused over the Intel Threading Building Blocks commercial vs open source license. The open source version is licensed under GPLv2 with the runtime exception, but what does that imply in plain english? Can it be used in a commercial, closed source application as long as it just links with the unmodified .dlls? A: I assume that you are referring to the license for http://threadingbuildingblocks.org/ : it uses the same wording as the libstdc++ exception: http://gcc.gnu.org/onlinedocs/libstdc++/manual/bk01pt01ch01s02.html In general, this exception allows use of the library in proprietary applications. Per http://www.gnu.org/licenses/old-licenses/gpl-2.0-faq.html#LibGCCException : Does the libstdc++ exception permit dynamic linking? Yes. The intent of the exception is to allow people to compile proprietary software using gcc. Per http://gcc.gnu.org/onlinedocs/libstdc++/faq.html#faq.license : 2.2.So any program which uses libstdc++ falls under the GPL? No. The special exception permits use of the library in proprietary applications. Libstdc++ is widely used in proprietary applications. Read also: http://threadingbuildingblocks.org/wiki/index.php?title=Licensing which is less explicit imho. There is some commercial incentive to a quid pro quo, tilting the balance towards inciting to buy a commercial license in case of doubt. When/if the library is modified, the situation may be different. Ask a lawyer in case of doubt. /HTH, IANAL, TINLA

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