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South African Journal of Animal Science
versão On-line ISSN 2221-4062
S. Afr. j. anim. sci. vol.47 no.2 Pretoria 2017
http://dx.doi.org/10.4314/sajas.v47i2.6
Modelling functional and structural impact of non-synonymous single nucleotide polymorphisms of the DQA1 gene of three Nigerian goat breeds
A. YakubuI, II, *; M. De DonatoII, III; I. G. ImumorinII, *
IDepartment of Animal Science, Nasarawa State University, Keffi, Shabu-Lafia Campus, P.M.B., 135, Lafia, 950101, Nigeria
IIAnimal Genetics and Genomics Laboratory, International Programs, College of Agriculture and Life Sciences, Cornell University, Ithaca, NY 14853, USA
IIILaboratorio Genetica Molecular, IBB, Universidad de Oriente, Cumana, Venezuela
The DQA1 gene is a member of the highly polymorphic MHC class II locus that is responsible for the differences among individuals in immune response to infectious agents. In this study, the authors performed a comprehensive computational analysis of the functional and structural impact of non-synonymous or amino acid-changing single nucleotide polymorphisms (SNPs) (nsSNPs) that are deleterious to the DQA1 protein in Nigerian goats. A 310-bp fragment of exon 2 of the DQA1 gene was amplified and sequenced in 27 unrelated animals that are representative of three major Nigerian goat breeds (nine each of West African Dwarf, Red Sokoto, and Sahel of both sexes) using genomic DNA. Forty-two nsSNPs were identified from the alignment of the deduced amino acid sequences. Based on the PANTHER, PROVEAN and PolyPhen-2 algorithms, there was consensus in identifying the mutants I26D, E114V and V115F as being deleterious. Further, differences between the native and the mutant proteins in the subsequent molecular trajectory analysis (stabilizing and flexible residue composition, total grid energy, solvation energy, coulombic energy, solvent accessibility, and protein-protein interaction properties) revealed E114V and V115F to be highly deleterious. Combined mutational analysis comparing the amutant (I26D, E114V and V115F mutations collectively) with the native protein also showed changes that could affect protein function and structure. Further wet-lab confirmatory analysis in a pathological association study involving a larger population of goats is required at the DQA1 locus. This would lay a sound foundation for breeding disease-resistant individuals in the future.
Keywords: Goats, in silico, mutants, protein, tropics
Single nucleotide polymorphisms (SNPs) are a common form of genetic variation among individuals (Li et al., 2009). They are amenable to high-throughput automated genotyping, and therefore are a preferred choice as genetic markers in research on diseases and their corresponding drugs (Doss et al., 2008). Non-synonymous SNPs (nsSNPs) alter the transcribed amino acid residues and result in functional diversity of the encoded proteins (Yates & Sternberg, 2013). One of the major purposes of genetics studies is to distinguish functionally neutral mutations from those that contribute to differences in phenotypes (Alshatwi et al., 2012). An abundance of diverse biological data from various sources constitutes a rich fund of knowledge, which has the power to advance our understanding of organisms. Computational methods allow the integration and effective use of these data to elucidate local and genome-wide functional connections between protein pairs, thus enabling functional inferences for uncharacterized proteins (Gray et al., 2012). Next generation sequencing techniques generate large volumes of data related to SNPs, but evaluation of biologically functional SNPs using in vitro studies is tedious, time consuming and expensive. However, the consequences of mutations and their effects on biological pathways can be predicted in silico (Patel et al., 2015; Chandramohan et al., 2015; AbdulAzeez & Borgio, 2016).
The major histocompatibility complex (MHC) is a large genomic region or gene family, which is found in most vertebrates and is highly polymorphic (Amills et al., 2005; IIhan et al., 2016). Molecules encoded by the MHC play an important role in the immune system and autoimmunity (Amills et al., 1998).
The DQA1 gene is a member of the MHC (Zhou & Hickford, 2004). Sequencing of the DQA1 complementary DNA (cDNA) revealed a single 768 bp open reading frame consisting of four exons and encoding a 255 amino acid protein (Amills et al., 2005; Plasil et al., 2016). Among MHC genes, DRB and DQA are most polymorphic (Subramani et al., 2016). This extensive structural polymorphism is responsible for the differences among individuals in immune response to infectious agents (Vandre et al., 2014). Most of the functionally important polymorphisms in DQA1 are concentrated in exon 2, and this diversity is correlated with pathogen richness (Wegner et al., 2003).
Although previous studies have examined the sequence variability of the MHC DQB1 and DRB genes (Yakubu et al., 2013; Yakubu et al., 2017), there is a dearth of information on nsSNPs of the MHC DQA1 gene of Nigerian goats. Such information could help to unravel the possible genotypes that affect livestock diseases in future association studies. Therefore, the present investigation aimed at identifying and screening deleterious nsSNPs of the DQA1 gene of West African Dwarf, Red Sokoto and Sahel goats of Nigeria, which are likely to affect the structure and function of the transcribed protein.
A 310-bp fragment of exon 2 of the MHC Class II DQA1 gene was amplified in 27 unrelated animals from three major Nigerian goat breeds (nine each of West African Dwarf (WAD), Red Sokoto (RS) and Sahel (SH)] using genomic DNA. Primers were designed using data from Amills et al. (2005). Primer sequences were DQA/FW, 5'GAAGCCCACAATGTTTGATAGTCA-3' and DQA/REV, 5'-GGGGAAGAACAACAAAGAGAGGCAG-3'. Primer-BLAST of NCBI (National Center for Biotechnology Information) was employed. The organism used was Capra hircus (taxid: 9925). The length of the forward primer was 24 bp with Tm (melting temperature) and GC (guanine-cytosine) values of 59.54 oC and 41.67%, respectively, while the corresponding length, Tm, and GC contents for the reverse primer were 25 bp, 63.73 oC and 52.00%. Primer dilution was done by adding 302.6 μL of 1 x TE buffer to DQA1 FW and 109 μL of 1 x TE buffer to DQA1 REV. Polymerase chain reaction (PCR) amplifications were carried out in a C1000TM thermal cycler (Bio-Rad, USA) in a total reaction volume of 20 μL containing about 20 ng DNA, 10 pmol of each primer in 10 μL Syd Lab PCR Premix (Syd Labs, Inc., Malden, USA) containing Taq DNA polymerase, dNTPs, MgCl2, reaction buffer, PCR stabilizer and enhancer at optimal concentrations. The thermal profile for amplifying the DQA1 exon 2 involved 35 cycles of initial denaturation at 94 °C for 4 min, denaturation at 94 °C for 30 s, annealing at 62 °C for 30 s, extension at 72 °C for 30 s, and elongation at 72 °C for 10 min. Every other protocol, including sequencing of the gene, is as described in Yakubu (2015). The ethical guidelines of the International Council for Laboratory Animal Science and Cornell University, Ithaca, NY, USA, were followed strictly. For nsSNP (missense variant) identification, sequence alignment, translations, and comparisons were carried out with the ClustalW option of MEGA 5 (Tamura et al., 2011), using the Capra_hircus_DQA1_AY464652.1 complete coding sequence (CDS) from the GenBank as the reference.
Functional effects of nsSNPs were predicted in silico using the PANTHER, PROVEAN, and PolyPhen-2 algorithms. PANTHER (Thomas et al., 2003) estimates the likelihood of a particular non-synonymous amino acid change having a functional impact on the protein. The web-based tool PROVEAN predicts the effects of an amino acid substitution or indel on the biological function of a protein, based on sequence clustering and alignment-based scoring. Variants with scores less than -2.5 were considered deleterious (Choi & Chan, 2015). PolyPhen-2 predicts the potential impact of an amino acid substitution on the function and structure of a protein (Adzhubei et al., 2010; Adzhubei et al., 2013). Where there was consensus by the three algorithms on the deleterious effect of a nsSNP, further confirmatory analyses were carried out. Additionally, a combined mutational analysis was conducted, incorporating all of the deleterious nsSNPs simultaneously (the term 'amutant' was coined to refer collectively to the nsSNPs) to reduce the possibility of false positives and exploit the effect of correlated mutations because these may enhance or diminish the functional properties of a protein.
Effects of nsSNP on protein stability were assessed with I-Mutant2.0 (Capriotti et al., 2005) and MuStab (Teng et al., 2010). I-Mutant2.0 predicts whether a single site mutation stabilizes or destabilizes the protein in 80% of the cases in which the three-dimensional structure is known and 77% of the cases in which only the protein sequence is available. MuStab, on the other hand, encodes the input sequence with the optimal feature subset, and then calls the svm_classify program of SVM-Light software package to classify the protein stability changes after the amino acid substitution (Teng et al., 2010).
Structural models for the native protein and mutant proteins I26D, E114V, V115F, and amutant were generated with Phyre2 (Kelley et al., 2015). This suite of tools aligns an input target with pre-existing templates to generate a series of predicted models. Structural similarity of alternative protein models was quantified by template modelling (Tm) scores and root mean square deviation (RMSD) in Angstroms (A) using Tm-Align. A Tm-score <0.2 is equivalent to a random structure from the PDB (Protein Data Bank) and a Tm-score of 0.5 or greater indicates that the proteins have a high probability of being in the same SCOP/CATH (structural classification of proteins/class architecture topology homology) fold (Zhang & Skolnick, 2005). An RMSD >2.0 has a negative effect on the stability and function of the protein (Han et al., 2006). Total energy after energy minimization was calculated for the native and each altered model using the Groningen Molecular Simulation (GROMOS) computer program package implementation of DeepView v 4.1 (Guex & Peitsch, 1997).
The proposed protein structures were validated with ERRAT and ProSA (Sippl, 1995; Wiederstein & Sippl, 2007). ERRAT is a program for verifying protein structures determined by crystallography (Colovos & Yeates, 1993). Error values are plotted as a function of the position of a sliding 9-residue window. The error function is based on the statistics of non-bonded atom-atom interactions in the reported structure compared with a database of reliable high-resolution structures. ProSA returns a z-score that indicates overall model quality based on the Co positions in 3-D space.
Stabilizing residues of the native and mutant DQA1 proteins of Nigerian goats were identified with SRide (Gromiha et al., 2004). The option selected was the PSSM-based encoding from the PDB file. The analysis was based on parameters such as surrounding hydrophobicity, long range order (LRO), stabilization centre and conservation score.
Residue positions that could change the conformation of the DQA1 protein were predicted with FlexPred. The software uses solvent accessibility of a protein sequence to identify the positions and thus change kinetic energy and potentially cause pathogenic disorders (Kuznetsov, 2008; Kuznetsov & McDuffie, 2008).
The molecular dynamic simulation was performed to calculate the total energy difference in solvated condition using the Poisson-Boltzmann equation (PBE) solver online tool (Smith et al., 2012; Sarkar et al., 2013). The simulation was performed by placing the protein under these conditions: i) interior dielectric constant: 4.00, ii) exterior dielectric constant: 80.00, iii) percentage fill: 80, grid scale: 2.00, iv) salt concentration: 0.10, v) probe radius: 1.40, and vi) boundary conditions: 2.00.
Solvent accessibility of residues of alternative amino acid sequences of the DQA1 protein were predicted with Weighted Ensemble Solvent Accessibility (WESA) (Shan et al., 2001; Chen & Zhou, 2005). The software predicts a residue as being buried or exposed (defined as having a surface area greater than 20% of the maximum area for that type of amino acid) with an expected accuracy of 80%.
Potential protein-protein interaction effects were predicted with InterProSurf (Zhou & Shan, 2001; Negi et al., 2006; Negi & Braun, 2007; Negi et al., 2007) and consensus Protein-Protein Interaction Site Predictor (cons-PPISP) (Chen & Zhou, 2005). InterProSurf predicts interacting amino acid residues in proteins that are most likely to interact with other proteins, given the 3D structures of subunits of a protein complex. The prediction method is based on the solvent accessible surface area of residues in the isolated subunits, a propensity scale for interface residues and a clustering algorithm to identify surface regions with residues of high interface propensities. cons-PPISP is a consensus neural network method that is trained to predict whether a surface residue is in the interaction site. Given the structure of a protein, cons-PPISP will predict the residues that would probably form the binding site for another protein.
Forty-two nsSNPs of the caprine DQA1 alleles were obtained from the alignment of the deduced amino acid sequences of Nigerian goats (Table 1). Of these, five, seven and thirteen were predicted to be deleterious by PANTHER, PROVEAN and PolyPhen2, respectively (Table 1). All three algorithms identified I26D, E114V and V115F as being harmful, indicating that these amino acid substitutions negatively affect the function of the DQA1 protein. Two of the three nsSNPs that were predicted to be collectively deleterious were found to decrease protein stability, with the amino acid substitution E114V being the exception. Although the stability of E114V increased, its reliability index of 1 and 24.64% prediction confidence were very low. In contrast the reliability indexes were 8 and 9, and the prediction confidence was 94% and 88% for I26D and V115F, respectively.
There were 181 residues (71% of DQA1 sequence) modelled with 100.0% confidence by the single highest scoring template for native, I26D, E114V, V115F amino acid substitutions and the amutant protein, respectively (Figure 1). That is, the percentage identity between the DQA1 sequence of the native protein and the mutations (above) in Nigerian goats and the template used to generate the structures is high, conferring accuracy to the 3D structures. This means all the five DQA1 structures were correctly predicted.
For all of the amino acid substitutions that were deleterious, including amutant, RMSD = 0.0 and TM = 1.00. However, energy of minimization varied from one amino acid substitution to another. The values recorded for V115F and amutant (-7046.844 and -7048.283kJ/mol, respectively) were greater than for the native protein (-7208.369 kJ/mol) and I26D (-7208.369 kJ/mol) and E114V (-7209.886 kJ/mol).
The native, I26D and E114V protein structures were of higher quality (61.6%) compared with V115F and amutant (55.8%), based on the ERRAT analyses. The overall structure quality also differed between the native and the mutant proteins using ProSA [Z score = -5.57 (native), -5.57 (I26D), -5.51 (E114V), -5.54 (V115F) and -5.49 (amutant).
Seven stabilizing residues were predicted for the native and mutant I26D, six residues for E114V, five for V115F, and eight for amutant (Table 2). Variation in flexibility was also observed in the native and the variants. Residues 110 and 155 were unique to the native protein. The amino acid substitution E114V has a unique flexible residue at position 244, while flexible residues at positions 121 and 125 were found only in V115F (Table 3). It was not possible to calculate flexible residues for the amutant.
The DelPhi results showed that the native and the mutants (with the exception of I26D) differed in their average total grid energy, solvation energy and coulombic energy (Table 4).
In terms of solvent accessibility, the mutant I26D was exposed, while variants E114 and V115F were found in a buried region of the protein (Table 5).
The protein-protein interaction patterns varied among the native and E114V, V115F, and amutant, respectively (Table 6). There were more buried residues in V115F and amutant (543) than in the native protein (539).
The cons-PPISP prediction of the residue contact was negative for E114V (neural network score: 0.076) and buried for V115F (Neural network score: 0.00). There was no result for the mutant I26D.
The use of polymorphisms within genes is fast gaining attention as a complement to the current methods of selection because of their association with traits of interest in animals, especially those that affect livestock diseases (Miyasaka et al., 2011; Yakubu, 2015). However, the effects of nsSNPs in the DQA1 gene in goats have not been predicted to date in silico. Amino acid substitutions in DQA1 may induce structural and functional damage. Normal protein function can be altered by deleterious nsSNPs, through geometric constraint changes (Gromiha & Ponnuswamy, 1995), hydrophobic changes (Rose & Wolfenden, 1993), and disruption of salt bridges or hydrogen bonds (Shirley et al., 1992; Michels et al., 2011). These may have pathological phenotypic consequences (Kumar et al., 2012; Yates & Sternberg, 2014). The differences in prediction capabilities of the three algorithms used in the present study may be connected with their differing alignment procedures. The difference in the results of computational tools may be due to differences in features utilized by the methods and therefore dissimilar predictions might be expected (de Alencar & Lopes, 2010).
A structural model of a protein is important in understanding biological processes at molecular level (Wiederstein & Sippl, 2007). The statistic RMSD is commonly used to evaluate the similarity of protein structures to their templates and to determine the accuracy of the alignment of the residues of two structures (Kufareva & Abagyan, 2012). In the present study, the structures of the native and mutant proteins, which were transcribed from the DQA1 gene, appeared similar, based on RMSD values. However, energy of minimization differed between the native protein and the mutants. It implies that the higher energy of minimization of V115F and amutant might impact the protein negatively because high energy configurations may lead to physical perturbation and instability. This difference was also observed in the overall structure quality of V115F and amutant as revealed by ERRAT, where the model quality of the native protein was higher. The different energy profiles of the native and the mutants, as found in ProSA, indicate varying structural quality of the DQA1 protein of the present study. This is because the z-score of ProSA indicates overall model quality and measures the deviation of the total energy of a structure with respect to an energy distribution derived from random conformations (Sippl, 1995; Zhang & Skolnick, 1998). The more negative the z-score, the better the model quality (Saha et al., 2013). The energy separation between the native fold and the average of an ensemble of miss-folds also indicated that the conformational energy landscape of the E114V, V115F and amutant proteins differed from that of the native protein. The observed variation in the total grid energy, solvation energy, and coulombic energy of the native and the mutants could influence the structure and biological functions of the proteins. This is consistent with the submission of Smaoui et al. (2016) that mutations that increase the stability of the structure have the lowest energies, while mutations that destabilize the structure increase its energy. The varying composition of the stabilizing residues in the native and especially variants E114V, V115F and combined I26D, E114V, and V115F amino acid substitutions could influence the structure of the protein, thereby affecting its stability and function. Some of the flexible residues were found around the point of mutation in the present study. These variations may contribute to differences in folding of the protein at these positions. An inherent property of macromolecular structure is conformation flexibility (Ramesh et al., 2013). Changes in protein folding may be involved in various biological functions, such as signal transduction, catalysis, macromolecular recognition, locomotion, and many pathogenic disorders (Kuznetsov & McDuffie, 2008).
Hydrophobic core residues are probable sites of deleterious mutations (Zhangab & Wanga, 2014; David & Sternberg, 2015; Sahoo et al., 2015). Replacing a hydrophobic residue with another hydrophobic residue, as observed in mutant V115F, may induce a volume change (Eriksson et al., 1992) and thereby decrease the stability of the protein. This was supported by the submission of Ohkuri & Yamagishi (2003) that residues with larger Van der Waals volume may introduce stress in the conformation of side chains at the subunit interface. The mutant E114V is a change from a polar/hydrophilic, H-bonding, acidic residue to a non-polar/hydrophobic residue. It is possible that this change could result in loss of hydrogen bonds and, consequently, disturb correct folding (Doss & NagaSundaram, 2012). Doss et al. (2008) reported that a change from exposed to buried state could be considered functionally significant in a mutant protein. Accordingly, change in amino acid may affect polar-polar interactions within the protein molecule itself, alter the energy of stabilization, and further destabilize the protein (Peng et al., 2005). The difference in charge between hydrophobic and acidic residues, as observed in the present study, would probably disturb the ionic interaction in the native protein.
The prediction of residue solvent accessibility could assist in a better understanding of the relationship between structure and sequence (Alanazi et al., 2011). The cost of burying a hydroxyl group depends on solvent accessibility (Blaber et al., 1993). If the residue is exposed, the mutant is destabilized by <0.5 kcal/mol. If the residue is fully buried, the mutant is destabilized by ~ 1- 3 kcal/mol (Blaber et al., 1993). Mutants E114V and V115F were predicted to be buried in this study, and therefore may disturb interactions with other molecules or other parts of the protein.
Identified structural and functional changes in the DQA1 protein supported predictions of the nsSNPs that resulted in I26D, E114V and V115F being deleterious. The combined mutational analysis carried out with them exposed the way in which amutant is different from other individual mutations compared with the native. The difference between results of the trajectory analysis and the RMSD values may be connected with the drawback of RMSD as a positional distance-based measure compared with measurements that are contact-based. Protein-protein interaction (PPI) properties of variants E114V and V115F and amutant differed from those of the native protein. Such PPI can be strengthened or weakened by nsSNPs, which may cause loss of salt bridges, steric clashes and later post-translational modifications, among other effects. Changes in the interactions among proteins can lead to rewiring (i.e. acquisition or loss of co-complex memberships) of the PPI network and thus alter phenotype. These results could contribute to explaining susceptibility to disease in Nigerian goats, pending their confirmation in future wet lab and pathogenic population-based association studies.
This project was funded by the College of Agriculture & Life Sciences through National Research Initiative Competitive Grant Programme (Grant No. 2006-35205-16864) from the USDA National Institute of Food and Agriculture and is gratefully acknowledged. The research visit of AY to Cornell was made possible through the fellowship from the Nigerian Government Education Trust Fund (ETF), now known as Tertiary Education Trust Fund (TETFUND) (ETF/DOPS/AST&D/UNIV/KEFFI/VOL.2).
The work was designed by AY and IGI. AY, MDD and IGI did the laboratory work and statistical analysis. AY wrote the manuscript. All authors read and approved the final manuscript.
Conflict of interest statement
The authors declare that they have no conflict of interest.
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Received 11 December 2016
Accepted 21 January 2017
First published online 3 February 2017
* Corresponding authors: abdulmoiyak@gmail.com, igi2@cornell.edu
Todo o conteúdo deste periódico, exceto onde está identificado, está licenciado sob uma Licença Creative Commons
South African Society for Animal Science (SASAS)
P O Box 13884, Hatfield, Pretoria, Gauteng, ZA, 0028,
sajas.eic@gmail.com


learn about this party theme!
Celebrate your next special day with us!
Several party options available including extra awesome theme parties such as Amazing Animals, Bubbles, and Polymers. Parties are held on weekends only and are 2 hours long.
Call (413) 572-4014 today to reserve your party – time slots fill up quickly!
A non-refundable $65.00 deposit is required within 3 days of booking to hold your date. APCM accepts cash, check or credit cards.
Price includes up to 10 children and 2 hours in party room.
Up to 15 children, 1 adult per child is free.
Price includes up to 20 children and 2 hours in party room.
Up to 25 children, 1 adult per child is free.
After leaving the party room guests can stay in the museum until we close at 4.
Add A Party Theme For Extra Fun!
Make your special day extra special!
Ooey, gooey, slimy, slippery and just plain fun! Learn all about the fascinating world of Polymers. Make your own Slime to take home.
Learn about the fascinating world of bubbles! Discover the science behind our secret bubble solution, why are bubbles round, how they float and why can we see colors on the bubble film? All these questions and more will be answered! Make your very own bubble wand to take home and our very own bubble recipe to continue the fun at home.
Animals are awesome! Have you ever wanted to hold a snake, feel how soft a guinea pig/hamster is, feed a lizard, or time a turtle's speed? With our animals theme here at APCM, you can do all of the above! Pick two of our amazing animals for your party, and you'll be able to not only learn about the animals you chose, but you'll also get the opportunity to interact with them.
For detailed information & reservations, please call APCM at 413-572-4014. Make sure to contact the museum at minimum one week in advance to book your party. We recommend booking a month prior to ensure getting your preferred spot.
Parties are held at 10am-12pm and 1pm-3pm; on Saturday & Sunday only.
You may bring cake, ice cream, pizza or other foods for your party. We are happy to refrigerate or freeze any refreshments you bring in the kitchen adjacent to the party room.
Please be aware that APCM is a NUT FREE facility.
You are advised to bring your own paper products and decorations. Please bring all necessary utensils and serving dishes. APCM does not have utensils, platters or bowls for the birthday parties.
You may arrive 30 minutes prior to your scheduled party time. You and your guests are allowed to stay within the museum until the museum closes. However all belongings must be removed from the party room 15 minutes after your scheduled party time. Free parking located behind our facility for you and your guests.
We do NOT allow the use of pinatas, confetti, helium tanks or other pressurized tanks such as the kind used to fire confetti canons.


text:
Q: Signal reflections inside CPU? Given the fact that the clock frequency is about one GHz and the corresponding wavelength is about the same order of magnitude (velocity of propagation is lower than 3 * 10^8 m/s inside the chip) with chip dimensions, is care taken care of to suppress signal reflections inside the chip?

A: Most traces inside of dies are pretty short relative to the wavelength. An individual x86 processor core at 14nm is only 3x4 mm, while even the longer traces connecting things like L3 cache slices between cores in the Intel die photos look to be only about 4-5 mm long.  Compared to the wavelength (>40mm), that is pretty short.  My guess is they're not terminated, and they've picked that spacing and die orientation specifically to avoid it, but I could be wrong.
Conversely things like off die links (memory, PCIe, etc) are impedance matched because the distances are much longer.  AMD for example shows how they use on-die termination resistors for their chip to chip links in this diagram:
https://en.wikichip.org/wiki/File:amd_if-ifop-serdes.png

A: hard to get reflections, when skin effect causes current crowding and the higher frequency edges are attenuated.
Skin depth in copper is about 35 microns, at 4MHz.
At 400 MHz, the depth is sqrt(100) smaller, at 3.5 microns.
At 100 * 400MHz, or 40GHz or 25 picosecond edge rates which is about what the faster MCUs have internally, the skin depth is sqrt(100) smaller still, at 0.35 microns.
===================
Now we should consider the aspect ratios of modern metalizations, where plasma etching is used ----- very steep sidewalls.

redpajama_set_name: RedPajamaStackExchange


RPSC Recruitment 2015: The Rajasthan Public Service Commission has declared the notification for the Tehsil Revenue Accountant (TRA) 279 Vacancies. The candidates apply for online application before the 15/05/2015 till 12:00 midnight. Now candidates go to visit the official site rpsc.rajasthan.gov.in to apply online. Then candidates apply the application and they will annouce to provide the admit card for the RPSC Recruitment 2015 exam. Here we have published the detailed notification like as age, qualification, dates others information given below.
The candidate's should be completed the degree in Arts, Science, Commerce or Agriculture from the recognized university established by Law in India & "O' or Higher Level Certificate Course conducted by DOEACC under control of the department of electronics.
Candidates age limit must be minimum age 21 years and maximum age 35 years. The age relaxation will be available for the candidates as per the rules.
The selection process based upon the candidates will have to appear in the written exam and later the shortlisted candidates called for interview.
Eligible candidates apply through the online official site rpsc.rajasthan.gov.in 15/05/2015 till 12:00 Midnight.


text:
https://www.heraldandnews.com/ap_news/oregon/congress-oks-bill-to-allow-killing-sea-lions-to-help/article_b4c17611-b9cc-5ca2-8e22-cbd843b05245.html
Congress OKs bill to allow killing sea lions to help salmon
Dec 12, 2018 , Herald and News
PORTLAND, Ore. (AP) — Congress has agreed to make it easier to kill sea lions threatening fragile runs of salmon in the Northwest.
Oregon Public Broadcasting reports that a bill approved by the House Tuesday changes the Marine Mammal Protection Act to lift some of the restrictions on killing sea lions to protect salmon and steelhead in the Columbia River and its tributaries. The measure had previously passed the Senate.
Wildlife managers say sea lion populations have grown so large that they no longer need all the protections that were put in place for them in 1972.
The measure would usher in a more streamlined process for Washington, Idaho, Oregon and several Pacific Northwest tribes to capture and euthanize sea lions. Sea lions deemed to be a problem are captured and euthanized.
Supporters, including the governors of Oregon, Washington and Idaho, fishing groups and tribes, have said the bill will give wildlife managers greater flexibility in controlling California sea lions that dramatically increased from about 30,000 in the 1960s to about 300,000 following enactment of the 1972 Marine Mammal Protection Act.
Jaime Pinkham, executive director of the Columbia River Inter-Tribal Fish Commission, said in a statement that he was "grateful Congress worked in a bipartisan manner to give us the local flexibility to protect the tribal treaty resources we share with others in the Columbia and Willamette rivers."
Critics called the move by Congress ill-conceived and say it will not solve the problem of declining salmon, which also face other problems such as habitat loss and dams.
While there are several thousand California sea lions in the Columbia River estuary, only about 200 to 300 swim more than 100 miles (160 kilometers) upriver from the Pacific Ocean and would be eligible for removal, state wildlife officials say.
An orca task force convened by Washington Gov. Jay Inslee, a Democrat, also backed the legislation to boost the fish for the struggling population of southern resident killer whales.
In accordance with Title 17 U.S.C. section 107, any copyrighted material herein is distributed without profit or payment to those who have expressed a prior interest in receiving this information for non-profit research and educational purposes only. For more information go to: http://www.law.cornell.edu/uscode/17/107.shtml
Page Updated: Sunday December 16, 2018 12:35 AM Pacific
Copyright © klamathbasincrisis.org, 2001 - 2018, All Rights Reserved

redpajama_set_name: RedPajamaCommonCrawl


It isn't easy to start school again after vacation and a week of adequate sleep.
I think of the nights when I felt like punching out the blinking computer screen after staying alive on tea and carbonated drinks. I can just feel my clammy hands when I recall those Mondays when I had three tests in a row. Those Sunday nights were even worse.
School is starting again and so is the race to the top where only the superhuman teens with perfect GPAs, SAT scores, and 20 extra-curriculars will survive. Well, that's what it seems like, anyway.
I'm a sophomore, and many juniors and seniors tell me, "It's never too early to start thinking about college." These words have often crept into my nightmares as I sleep through a torrent of SAT words and college applications.
I actually had a horrible dream that I couldn't get into any of the colleges I applied to. I woke up in a sweat, feeling like it was already too late to do anything about my future.
I go to Hunter High School, a specialized school. There are a lot of things that are really great about Hunter, including the fact that there are only about 200 people in my grade and it's easy to get to know almost everybody. Plus the quality of our education is good.
But about every two months, all the teachers simultaneously give "hell week" and we are loaded with assignments, projects, tests, term papers, and essays from six or seven different classes.
My friend, Judy Lin, 15, also a sophomore at Hunter, agrees that stress is, by all means, a way of life. She slaves away for three and a half hours a night on mindless homework so she can go to the college of her choice. She's afraid that if she doesn't, she'll be lost.
In addition, Judy gets a lot of stress from her family.
"My parents are the first generation in America," she said, "and we're not doing so well." She explained that because her parents couldn't really speak English and knew only Chinese customs, it was hard for them to get good jobs.
But while Judy and I have both been stressing over our huge workload, I know other teens face a different kind of academic stress.
Baudilio Lozado, 15, used to attend Smith Vocational High School in the Bronx but transferred out.
Baudilio aspires to be a writer, but he said he doesn't know of any advanced placement or accelerated classes in his school, and he wasn't getting the kind of education that he wanted.
He guessed that only 3 out of 10 of his friends would graduate from high school. No one seemed to care about school, least of all his friends. "They talked about everything except academics," he said.
Baudilio felt that if he failed, it wouldn't matter that much because he would become "just another story." But Baudilio also didn't want to fail.
Unfortunately, Baudilio didn't make it to Bronx Science, but he applied to Urban Academy, an alternative high school located in Manhattan. He knew that if he stayed at his old school, he wouldn't have opportunities to pursue a career in writing.
Even though Baudilio's experience at school has been so different from mine, talking to him made me see that we were both stressed because we both cared about our futures.
Just out of curiosity, I asked Baudilio to tell me one wish that would make all his stress go away. He replied, "I want to be fearless." Tests, colleges, and interviews all make him nervous, and he just wants to be able to face them.
Before I wrote this story, I knew that I wasn't the only student in this world who had academic stress, but I didn't know anything about how others experienced it or why students put up with it.
I was surprised when I found that most of the teens I talked to said they felt they had no choice but to deal with the stress. We're stressed because, in one way or another, we're motivated to do well and because we have goals that we want to achieve.
But we should also know what our priorities are—no matter what, our own well-being and health should come first. There are times when academics are just not worth the stress, or when stress may get to be too much for some of us. We need to remember that we're not supposed to be perfect, and that failing a test or even a class doesn't mean that we'll never achieve our goals in life.
Still, we choose to face stress every day because we have a place we want to go, a somebody we want to be.
I know now that the next time I feel like crawling under the blankets and never, ever coming out, well...I'll lie there for five minutes, max. Then I'll face that stupid computer and start typing again.


text:
A Sugar Rush That Could Fuel The Indian Economy
India should take its cue from Brazil and invest in ethanol as a viable commercial substitute for costly petrol, argues by R. Viswanathan. An excellent analysis from which not just India but Pakistan and other countries with strong agriculture can immensely benefit.
By R. Viswanathan | Via The Hindu
(Photo by US department of Agriculture)
The public and media were outraged recently after a suggestion that petrol (gas) stations could be closed from 8 p.m. to 8 a.m. to curb consumption. Oil import is the heaviest burden on India's foreign exchange, at $144 billion last year. The situation could get worse, given the potential for an increase in crude prices with further destabilization in West Asia. India needs to think beyond 8 p.m. to 8 a.m. and look for long-term, indigenous, sustainable solutions — solutions innovated in Brazil.
Flex-fuel engines
One way to reduce petrol (gas) consumption is to use ethanol as a fuel. Brazil has already done it successfully, reducing petrol (gas) consumption by 30 per cent. All new cars produced in Brazil have flex-fuel engines which run on 100 per cent ethanol or 100 per cent petrol (gas) or any combination of the two. In addition, there is a 20 per cent mandatory ethanol addition to petrol.
Brazil is the global leader with 16 million of the world's total 27 million flex-fuel vehicles in December 2011. Brazil uses ethanol in motorcycles, light commercial vehicles and even agricultural aircraft. It is now experimenting with ethanol in buses, trucks, tractors and harvesters. Indigenously-developed sugarcane diesel is now used in some Sao Paulo buses. The Brazilians are now moving into cellulosic ethanol technology, which uses waste material and bagasse from sugar mills. The use of fuel ethanol has created a synergy between the agriculture and energy sectors and a win-win for farmers, industry and government. It has also improved the quality of air in Brazil's cities as ethanol is less polluting than petrol (gas).
Oil crisis
How did Brazil do it? In 1974, hurt by the oil crisis, the government brought together all stakeholders — sugarcane farmers, sugar mills, car manufacturers and oil companies besides the ministries of agriculture, industry and energy — and formulated a policy that would ensure energy independence. The government then oversaw its implementation rigorously as a strategic priority. It provided three important initial drivers: guaranteed purchases of ethanol by the state-owned oil company Petrobras, low-interest loans for sugarcane farming and ethanol production, and fixed gasoline and ethanol prices. The policy has evolved over the years through learning from experience. Today, fuel ethanol is the centerpiece of Brazil's energy, agricultural and industrial strategy and Brazil is a role model for the world.
The success of the policy has encouraged the oil companies to become stakeholders in ethanol production in Brazil. Multinational oil companies such as Shell and traders like Louis Dreyfus and Bunge have invested in ethanol, besides Petrobras, the state oil company.
Conscious of the cyclical nature of sugarcane production and weather risks, the government has a flexible policy of exporting the product when there are surpluses and importing during times of shortage. Brazil is working quietly to create a global ethanol market with standardization of specifications and market mechanisms in collaboration with the U.S. It is also encouraging and collaborating with other Latin American and Caribbean countries to produce fuel ethanol; Brazilian ethanol companies have started investing in other countries.
India has the potential to replicate the success of Brazil; we are the second largest producer of sugarcane in the world. And the risk and investment involved in ethanol production is far lower than in oil exploration and production.
Hostage to policy
Ethanol has been considered before. In 2006, the government of India launched a program of five per cent mandatory ethanol blending. But it has not taken off largely because the oil companies have shown little interest in ethanol. They offer unattractive prices to local ethanol suppliers even as they pay top dollar for foreign oil supplies. The ethanol producers naturally prefer the better prices offered by chemical and alcohol companies as well as foreign importers.
It is a mistake for our government to have held the ethanol policy hostage to this conflict of interest between the oil and the sugar companies. On their part, neither see the national interest. The petroleum ministry is biased in favor of the oil companies.
This can change if the government as a whole — and not just the oil ministry — takes charge of the new idea, and like Brazil, brings all the stakeholders together and evolve a long-term policy, treating it as a national energy security imperative. The mandatory blending should be increased to 10 per cent immediately, with the target of 20 per cent in the next three years. New Delhi can compel the oil companies to invest in ethanol production, so they have a stake in its development. The car manufacturers too must be brought on board to modify the engines. Some foreign car makers in India such as Ford, GM, Volkswagen, Honda, Toyota, Renault and Fiat are already producing flex-fuel cars in Brazil, and can easily do so in and for India.
The use of ethanol as fuel has multiple benefits for India besides reducing petrol consumption — all politically positive. We will save on foreign currency — money will stay with Indian farmers and industry in rupees instead of the dollars that foreign oil suppliers receive. Ethanol will help farmers sustain their income during the cyclical bumper harvests and their lower sugar prices, as is the prevailing situation this year. India will reduce its trade deficit and foreign exchange outflow. And there will be less pollution. Most importantly, fuel ethanol will be a sustainable, long term, India-centric solution.
(R. Viswanathan, former Indian Ambassador to Argentina, Venezuela, and India's first consul general in Sao Paulo, is a distinguished fellow, Latin America Studies, at Gateway House: Indian Council on Global Relations.)
This article first appeared in The Hindu, one of India's leading English dailies. Click here to goto the original.
Brazil Caribbean ethanol Flex-Fuel Latin American	2013-09-27
Previous Obamacare Will Cost Much Less Than Expected
Next Enforcing Peace in Pakistan's Largest City
Former US Ambassador to Pakistan Cameron Munter, Senior Research Professor at John Hopkins University Dr. Daniel Markey, Ikram Sehgal and other foreign policy experts hold a candid discussion on Pakistan-US relations.
Year after year, Moscow is moving east, enhancing its ties to China. Year after year, New Delhi is moving west, building stronger links to the US. If this trend continues into the mid-term future, the two friendly countries might ultimately find themselves in the opposite geopolitical and economic blocks, and the Eurasian space will split into two pieces. Over time, Moscow and New Delhi will find it more challenging to maintain their bilateral cooperation even at the current levels, not to mention it is further deepening and broadening.


text:
Trump Urges Roy Moore Not to Run for Alabama Senate Seat
FILE - U.S. Senate candidate Roy Moore speaks at a campaign rally in Midland City, Ala., Dec. 11, 2017. Moore is going to court to try to stop Alabama from certifying Democrat Doug Jones as the winner of the U.S. Senate race.
President Donald Trump warned Wednesday the "consequences will be devastating" if Alabama Republican Roy Moore, whose 2017 U.S. Senate campaign was battered by allegations of long-ago sexual harassment of teenagers, seeks the seat again in 2020.
Moore lost in the once-reliably red state in a 2017 special election amid the sexual misconduct allegations, which he denied. He told The Associated Press earlier this month he is considering another campaign next year.
Trump, who backed Moore in 2017 despite the allegations, tweeted "I have NOTHING against Roy Moore," but warned that "Roy Moore cannot win." He adds that if Democrat Doug Jones retains the seat in 2020 "many of the incredible gains that we have made during my Presidency may be lost."
Trump's comments come as national Republicans have tried to keep Moore out of the race.
Moore's nomination could have national repercussions, allowing Democrats to accuse the GOP of ignoring the #MeToo movement and coddling a man accused of sexual misconduct. Moore says he expects to announce a decision in mid-June.
Republicans control the U.S. Senate 53-47 and view defeating Jones as a top priority. Jones, 65, is considered the most endangered Democratic incumbent facing re-election in 2020, a year when several GOP senators are vulnerable and control of the chamber will be at stake.
Jones defeated Moore in 2017 by 22,000 votes out of 1.3 million cast in a special election to fill the seat previously held by Jeff Sessions, who became Trump's attorney general.
2020 USA Votes
Small Numbers of Protesters Gather at Fortified US Statehouses


Working independently, the technologist ensures a proper environment for testing in the laboratory, in the wards, Emergency Room (ER) or Intensive Care Units (ICUs). Obtains medical history of patient and summarizes relevant data. Puts patient at ease and explains procedure. Precisely measures and applies multiple electrodes carefully and securely to predetermined anatomic sites. Based on patient's age, diagnosis, current state and medical history, performs Digital Video Electroencephalography (EEG), Visual, Brainstem Auditory and Somatosensory Evoked Potentials, Nerve conduction Studies, Sleep studies, Pulse Oximetry, Autonomic Studies and Long Term Video/Digital Encephalographic monitoring (LTM), in both adult and paediatric patients. Safely performs auditory, visual or electrical stimulation of the nervous system. Adjusts settings of equipment to ensure proper and accurate test results. Recognizes and eliminates artefacts to produce meaningful diagnostic information.
Performs urgent diagnostic tests on critically ill patients in ICUs and the ER on mobile equipment or in the laboratory setting, coordinating with nursing and respiratory therapy personnel.
Monitors patients with surgically implanted intracranial electrodes.
Assists physicians with invasive procedures in Intraoperative Neurophysiological Monitoring, Electrocorticography and Electromyography.
Monitors patients intraoperatively. Performs complex pre-operative setups with electrodes on multiple anatomic sites. Watches and assesses the quality of the neurophysiology waveforms. Follows the patient's systemic and anaesthetic state while keeping track of surgical activities. Communicates with and gives feedback to Neurophysiologist as necessary.
Observes patient reactions clinically and electrographically during testing. Notes signs of distress, medical emergencies, or seizures, takes remedial action, notifies nurse and notifies physician if necessary.
Makes preliminary analysis and description of the waveforms or potentials, ensuring that it is meaningful and acceptable. Notes pertinent data for physician review.
Ensures safety of patient and safe operation of equipment. Informs supervisor in writing of equipment faults. Maintains work area. Ensures adequate levels of stocks and supplies in procedure areas.
Cleans and disinfects clinical equipment, following infection control policies and guidelines.
Works as team leader in procedural areas, on a rotational basis.
Participates in self and others education, training and development. Attends department education activities and participates in laboratory inservice program.
Participates in laboratory quality improvement programs.
Follows all hospital policies and procedures. Follows all laboratory procedures and guidelines.
Three years of experience as a Neurophysiology Technologist (post qualification) in an acute care hospital setting in North America, or Western Europe, or Australia/New Zealand.