The invention provides methods for sorting polynucleotides from a population based on predetermined sequence characteristics. In one aspect, the method of the invention is carried out by extending a primer annealed polynucleotides having predetermined sequence characteristics to incorporate a predetermined terminator having a capture moiety, capturing polynucleotides having extended primers by a capture agent that specifically binds to the capture moiety, and melting the captured polynucleotides from the extended primers to form a subpopulation of polynucleotides having the predetermined sequence characteristics. In another aspect, the method of the invention is carried out on a population of tagged polynucleotides so that after a subpopulation is selected, the members of the subpopulation may be simultaneously analyzed using the unique tags on the polynucleotides to convey analytical information to a hybridization array for a readout.
This application claims priority from U.S. provisional application Ser. No. 60/543,887 filed 12 Feb. 2004, which is incorporated herein by reference in its entirety.
The invention relates generally to methods and compositions for analyzing complex populations of polynucleotides, and more particularly, to methods and compositions for partitioning a population of polynucleotides into one or more subpopulations of lesser complexity.
A major goal in genetics research is to understand how sequence variations in the genome relate to complex traits, particularly susceptibilities for common diseases such as diabetes, cancer, hypertension, and the like, e.g. Collins et al, Nature, 422: 835-847 (2003). The draft sequence of the human genome has provided a highly useful reference for assessing variation, but it is only a first step towards understanding how the estimated 10 million or more common single nucleotide polymorphisms (SNPs), and other polymorphisms, such as inversions, deletions, insertions, and the like, determine or affect states of health and disease. Many powerful analytical approaches have been developed to address this problem, but none appear to have adequate throughput or flexibility for the types of studies required to associate traits practically and reliably with genomic variation, e.g. Syvanen, Nature Reviews Genetics, 2: 930-942 (2001). For example, it would be desirable to carry out trait-association studies in which a large set of genetic markers from populations of affected and unaffected individuals are compared. Such studies depend on the non-random segregation, or linkage disequilibrium, between the genetic markers and genes involved in the trait or disease being studied. Unfortunately, the extent and distribution of linkage disequilibrium between regions of the human genome is not well understood, but it is currently believed that successful trait-association studies in humans would require the measurement of 30-50,000 markers per individual in populations of at least 300-400 affected individuals and an equal number of controls, Kruglyak and Nickerson, Nature Genetics, 27: 234-236 (2001); Lai, Genome Research, 11: 927-929 (2001); Risch and Merikangas, Science, 273: 1516-1517 (1996); Cardon and Bell, Nature Reviews Genetics, 2: 91-99 (2001).
One approach to dealing with such whole-genome studies is to create subsets of genomic DNA having reduced complexity with respect to the genomes being analyzed in order to simplify the analysis, e.g. Lisitsyn et al, Science, 259: 946-951 (1993); Vos et al, Nucleic Acids Research, 23: 4407-4414 (1995); Dong et al, Genome Research, 11: 1418-1424 (2001); Jordan et al, Proc. Natl. Acad. Sci., 99: 2942-2947 (2002); Weissman et al, U.S. Pat. No. 6,506,562; Sibson, U.S. Pat. No. 5,728,524; Degau et al, U.S. Pat. No. 5,858,656. Unfortunately, most of these techniques rely on some form of subtraction, sequence destruction, or direct or indirect size selection to create subsets, which are difficult to implement and reduced sensitivity.
In view of the above, the field of genetic analysis would be advanced by the availability of a method for converting a highly complex population of DNA, such as a mixture of genomes, into subsets having reduced complexity without requiring subtraction, or other sequence destroying, steps.
The invention provides methods and compositions for sorting polynucleotides from a population based on predetermined sequence characteristics. In one aspect, the method of the invention is carried out by the following steps: (i) extending a primer annealed polynucleotides having predetermined sequence characteristics to incorporate a predetermined terminator having a capture moiety, (ii) capturing polynucleotides having extended primers by a capture agent that specifically binds to the capture moiety, and (iii) melting the captured polynucleotides from the extended primers to form a subpopulation of polynucleotides having the predetermined sequence characteristics.
In another aspect, the population of polynucleotides comprises fragments from a population of genomes, wherein the fragments from each genome has the same unique oligonucleotide tag attached. In this aspect, the invention includes a method of determining a frequency of a nucleotide at a predetermined locus in a population of genomes, such method comprising the following steps: (i) separately generating fragments of each genome of the population; (ii) attaching a unique oligonucleotide tag to each genome; (iii) selecting fragments from each genome that contains the predetermined locus; (iv) generating a labeled oligonucleotide tag from each unique oligonucleotide tag, the labeled oligonucleotide tag generating a signal indicative of the nucleotide at the predetermined locus; and (v) determining the frequency of the nucleotide at the predetermined locus by detecting the signals generated by the labeled oligonucleotide tags specifically hybridized with their respective tag complements, the respective tag complements being attached in spatially discrete regions on the one or more solid phase supports.
FIGS. 1A-1F illustrate the selection of particular fragments by common sequence elements.
FIGS. 2A-2D illustrate the application of the invention for selecting particular haplotypes.
FIGS. 3A-3D illustrate hybridization tags with "commas" and a hybridization tag with the "comma-less" property.
FIG. 4 lists melting temperatures of selected tags consisting of four words each having the comma-less property.
"Addressable" in reference to tag complements means that the nucleotide sequence, or perhaps other physical or chemical characteristics, of a tag complement can be determined from its address, i.e. a one-to-one correspondence between the sequence or other property of the tag complement and a spatial location on, or characteristic of, the solid phase support to which it is attached. Preferably, an address of a tag complement is a spatial location, e.g. the planar coordinates of a particular region containing copies of the tag complement. However, tag complements may be addressed in other ways too, e.g. by microparticle size, shape, color, frequency of micro-transponder, or the like, e.g. Chandler et al, PCT publication WO 97/14028.
"Allele frequency" in reference to a genetic locus, a sequence marker, or the site of a nucleotide means the frequency of occurrence of a sequence or nucleotide at such genetic loci or the frequency of occurrence of such sequence marker, with respect to a population of individuals. In some contexts, an allele frequency may also refer to the frequency of sequences not identical to, or exactly complementary to, a reference sequence.
"Amplicon" means the product of an amplification reaction. That is, it is a population of polynucleotides, usually double stranded, that are replicated from one or more starting sequences. The one or more starting sequences may be one or more copies of the same sequence, or it may be a mixture of different sequences. Amplicons may be produced in a polymerase chain reaction (PCR), by replication in a cloning vector, or by linear amplification by an RNA polymerase, such as T7 or SP6, or by like techniques.
"Analyte" means any molecule, including organic, inorganic, or biomolecule, whose presence or absence or quantity or concentration in a sample is to be determined in an assay. In particular, biomolecule analytes include oligonucleotides, polynucleotides, genomic fragments, messenger RNAs (mRNAs), proteins, antibodies, enzymes, complementary DNAs (cDNAs), and like compounds.
"Complement" or "tag complement" as used herein in reference to oligonucleotide tags refers to an oligonucleotide to which an oligonucleotide tag specifically hybridizes to form a perfectly matched duplex or triplex. In embodiments where specific hybridization results in a triplex, the oligonucleotide tag may be selected to be either double stranded or single stranded. Thus, where triplexes are formed, the term "complement" is meant to encompass either a double stranded complement of a single stranded oligonucleotide tag or a single stranded complement of a double stranded oligonucleotide tag.
Further sets of words for constructing comma-less tags are listed in FIG. 4.
Preferably, tag complements are synthesized on the surface of a solid phase support, such as a microscopic bead or a specific location on an array of synthesis locations on a single support, such that populations of identical, or substantially identical, sequences are produced in specific regions. That is, the surface of each support, in the case of a bead, or of each region, in the case of an array, is derivatized by copies of only one type of tag complement having a particular sequence. The population of such beads or regions contains a repertoire of tag complements each with distinct sequences. As used herein in reference to hybridization tags, tag complements, and synthesis tags, the term "repertoire" means the total number of different tags or tag complements in a given set or population.
Solid phase supports containing tag complements may take a variety of forms, e.g. particulate, single-piece and planar, such as a glass slide, and may be composed of a variety of materials, e.g. glass, plastic, silicon, polystyrene, or the like. Particulate solid phase supports include microspheres, particularly fluorescently labeled microspheres, e.g. Han et al, Nature Biotechnology, 19: 631-635 (2001); Kettman et al, Cytometry, 33: 234-243 (1998); and the like. Preferably, hybridization tags are detected by hybridizing them to their complementary sequences on a conventional microarray. Such microarrays may be manufactured by several alternative techniques, such as photo-lithographic optical methods, e.g. Pirrung et al, U.S. Pat. No. 5,143,854, Fodor et al, U.S. Pat. Nos. 5,800,992; 5,445,934; and 5,744,305; fluid channel-delivery methods, e.g. Southern et al, Nucleic Acids Research, 20: 1675-1678 and 1679-1684 (1992); Matson et al, U.S. Pat. No. 5,429,807, and Coassin et al, U.S. Pat. Nos. 5,583,211 and 5,554,501; spotting methods using functionalized oligonucleotides, e.g. Ghosh et al, U.S. Pat. No. 5,663,242; and Bahl et al, U.S. Pat. No. 5,215,882; droplet delivery methods, e.g. Caren et al, U.S. Pat. No. 6,323,043; Hughes et al, Nature Biotechnology, 19: 342-347 (2001); and the like. The above patents disclosing the synthesis of spatially addressable microarrays of oligonucleotides are hereby incorporated by reference. Microarrays used with the invention contain from 50 to 500,000 hybridization sites; or from 100 to 250,000 hybridization sites; or from 100 to 40,000 hybridization sites; and preferably, they contain from 100 to 32,000 hybridization sites; or from 100 to 20,000 hybridization sites; or from 100 to 10,000 hybridization sites.
Guidance for selecting conditions and materials for applying labeled oligonucleotide probes to microarrays may be found in the literature, e.g. Wetmur, Crit. Rev. Biochem. Mol. Biol., 26: 227-259 (1991); DeRisi et al, Science, 278: 680-686 (1997); Wang et al, Science, 280: 1077-1082 (1998); Duggan et al, Nature Genetics, 21: 10-14 (1999); Schena, Editor, Microarrays: A Practical Approach (IRL Press, Washington, 2000); Hughes et al (cited above); Fan et al, Genomics Research, 10: 853-860 (2000); and like references. These references are hereby incorporated by reference. Typically, application of hybridization tags to a solid phase support includes three steps: treatment with a pre-hybridization buffer, treatment with a hybridization buffer that includes the probes, and washing under stringent conditions. A pre-hybridization step is employed to suppress potential sites for non-specific binding of probe. Preferably, pre-hybridization and hybridization buffers have a salt concentration of between about 0.8-1.2 M and a pH between about 7.0 and 8.3. Preferably, a pre-hybridization buffer comprises one or more blocking agents such as Denhardt's solution, heparin, fragmented denature salmon sperm DNA, bovine serum albumin (BSA), SDS or other detergent, and the like. An exemplary pre-hybridization buffer comprises 6×SSC (or 6×SSPE), 5× Denhardt's solution, 0.5% SDS, and 100 μg/ml denatured, fragmented salmon sperm DNA, or an equivalent defined-sequence nucleic acid. Another exemplary pre-hybridization buffer comprises 6×-SSPE-T (0.9 M NaCl, 60 mM NaH2PO4, 6 mM EDTA (pH 7.4), 0.005% Triton X-100) and 0.5 mg/ml BSA. Pre-hybridization and hybridization buffers may also contain organic solvents, such as formamide to control stringency, tetramethylammonium chloride to negate base-specific effects, and the like. An exemplary hybridization buffer is SSPE-T and the desired concentration of isostringency probe. After hybridization, unbound and non-specifically bound isostringency probe is removed by washing the detection support under stringent conditions. Preferably, stringency of the wash solution is controlled by temperature, organic solvent concentration, or salt concentration. More preferably, the stringency of the wash conditions are determined to be about 2-5° C. below the melting temperature of the isostringency probes at the salt concentration and pH of the wash solution. Preferably, the salt concentration of the wash solution is between about 0.01 to 0.1 M.
Instruments for measuring optical signals, especially fluorescent signals, from labeled tags hybridized to targets on a microarray are described in the following references which are incorporated by reference: Stern et al, PCT publication WO 95/22058; Resnick et al, U.S. Pat. No. 4,125,828; Kamaukhov et al, U.S. Pat. No. ,354,114; Trulson et al, U.S. Pat. No. 5,578,832; Pallas et al, PCT publication WO 98/53300; Brenner et al, Nature Biotechnology, 18: 630-634 (2000); and the like.
When tag complements are attached to or synthesized on microbeads, a wide variety of solid phase materials may be used with the invention, including microbeads made of controlled pore glass (CPG), highly cross-linked polystyrene, acrylic copolymers, cellulose, nylon, dextran, latex, polyacrolein, and the like, disclosed in the following exemplary references: Meth. Enzymol., Section A, pages 11-147, vol. 44 (Academic Press, New York, 1976); U.S. Pat. Nos. 4,678,814; 4,413,070; and 4,046;720; and Pon, Chapter 19, in Agrawal, editor, Methods in Molecular Biology, Vol. 20, (Humana Press, Totowa, N.J., 1993). Microbead supports further include commercially available nucleoside-derivatized CPG and polystyrene beads (e.g. available from Applied Biosystems, Foster City, Calif.); derivatized magnetic beads; polystyrene grafted with polyethylene glycol (e.g., TentaGel™, Rapp Polymere, Tubingen Germany); and the like. Generally, the size and shape of a microbead is not critical; however, microbeads in the size range of a few, e.g. 1-2, to several hundred, e.g. 200-1000 μm diameter are preferable, as they facilitate the construction and manipulation of large repertoires of oligonucleotide tags with minimal reagent and sample usage. Preferably, glycidal methacrylate (GMA) beads available from Bangs Laboratories (Carmel, Ind.) are used as microbeads in the invention. Such microbeads are useful in a variety of sizes and are available with a variety of linkage groups for synthesizing tags and/or tag complements.
where H1 and H2 are words of a hybridization tag as described above, for example 4-mer words. Such words may vary in length depending on the embodiment, but generally are in the range of from 2 to 10 nucleotides in length; or they may be in the range of from 3 to 6 nucleotides in length. One factor in selecting word length is whether they are processed by restriction enzymes, such as type IIs restriction enzymes, whose recognition and cleavage characteristics may dictate word length. Using an eight-word set described above, 64 such di-words are constructed, cloned in conventional vectors, and the DNA can be obtained thereafter by PCR. These reagents containing pairs of hybridization "words" are used to form word-pair conversion adaptors, described more fully below.
where the "Z2's" are the nucleotides of the next word, and the "Zx's" are the nucleotides of all the possible cleavage products. The cleavage product includes ends complementary to all of the possible ends of the cleavage product of formula (II). Thus, ligation of the two products permits combinatorial synthesis of the tags.
In one aspect of the invention, all fragments of each genome of a population of genomes are labeled with one combination of words selected from a repertoire of 32,768 (=85) five-word oligonucleotide tags. Once each genome has a unique tag, then common-sequence fragments, e.g. a restriction fragment from a particular locus, can be selected using the method of the invention. The tags may then be used to convey information about the fragments, e.g. the identity of a nucleotide at a particular locus, to a hybridization array for a readout. One of ordinary skill in the art understands that the selection of 5-word oligonucleotide tags of five nucleotides each and the use of commaless tags are design choices that may be varied depending on the goals and constraints of any particular application. In one embodiment the following eight-word minimally cross-hydridizing set may be used to construct the above repertoire. As described below, preferably, each word is cloned in a plasmid with additional elements for aiding in the construction of oligonucleotide tags.
A. Single-Word Library and Counting Array Element.
In one embodiment, the single word library contains a ten-base sequence [G/T; G/T; A/T]3G/T, where "x/T" is an equal mixture of the two bases "x" and "T" at a particular locus. This element encodes a repertoire of 1024 (=210) different sequences that permits sequences to be counted by hybridization of copies of the sequence to an array of complementary sequences, i.e. a "counting" array. This element is referred to herein as the "Counting Array" or "CAR" element. In this embodiment, about 30 copies of each genome are tagged and each is labeled with one unique sequence. Thus, if any sorted molecule is found to have a unique sequence for this array, it is not a genome difference that should have multiple sequences, and is likely to represent an error in the process which has resulted in an altered molecule. Note that however much any fragment is amplified that it will always possess the original sequences in the counting array, preserving cardinality as distinct from the concentration of DNA.
Using the primer "5′-NNNAGGCCT[N15]GGGAC" (SEQ ID NO: 13) the above is copied, cleaved with KpnI and BspE1, and cloned into each of the single-word plasmids. 104 clones of each are isolated to make sure that all the sequences of the counting array are in the library.
5′- . . . NN GATCGAAGAGC . . .
. . . NNTAGp GCTTCTCG . . .
permits efficient and directional cloning of fragments.
were "N" are arbitrarily selected nucleotides and "CAR" is a counting array element, as described above.
Here a library of 64 vectors is disclosed each containing one of the 64 possible two-word, or "di-word," concatenations of words from the 8-word library flanked by primer binding sites. This double-word library is then used essentially as described in Brenner and Williams (cited above) to construct oligonucleotide tags. In this embodiment, the first flanking primer binding site is that shown above as "Primer X," and the other contains a recognition site for FokI, 5′-GGATG(9/13), which contains "GG" and therefore cannot cut any of the words described above.
where it is understood that the "word 1" and "word 2" refer to both word sequences and their respective complements. After annealing the above fragments to form a doublestranded element, it is cloned into a plasmid digested with ApaI and BamHI. To assure the accuracy of the incorporation, several clones of each "double word" vector are selected and sequenced. Copies of di-words may be conveniently obtained by PRC using a biotinylated X primer and another primer.
About 1 ng of human DNA (about 30 copies of the haploid genome) is digested with Bst Y1 to give fragments of an average size of 1 Kb, after which ends are filled in with dGTP to give 3-base ends as described above.
The single-word fragments are ligated to the genome fragments to give genome fragments having single-word fragments on both ends. These fragments are processed as follows so that a single-word fragment is on only one end. First, the reaction constituents from every vessel in each row are pooled so that eight mixed samples are obtained.
AGGCCTN15GGGAC [frag- [CAR]GTC(Me)C(Me)C(Me) . . .
TCCGGAN15CCCTG [frag- [CAR]CAG G G . . .
where all eight words are represented in the overhang and "W" represents a nucleotide of a word or its complement. Next the di-word libraries are pooled, cleaved with FokI, then ligated to the above fragment to add the next word. The process is continued until the desired number of words is added to the genomic fragments to complete the tags.
melting the captured polynucleotides from the extended primers.
melting the primer-polynucleotide duplexes to form a subpopulation of polynucleotides having a complexity less than that of the parent population.
(e) repeating steps (a) through (d) until a selected population of the desired complexity is obtained.
4. The method of claim 3 further comprising a step of replicating said selected population after said step of melting.
5. The method of claim 4 wherein during each said step of repeating steps (a) through (d), said primer anneals to a different primer binding site on said polynucleotides of said parent population or said selected population.
6. The method of claim 5 wherein in each successive step of repeating steps (a) through (d), said different primer binding site is shifted along said polynucleotides at least one nucleotide in a primer extension direction.
7. The method of claim 5 wherein in each successive step of repeating steps (a) through (d), said different primer binding site is at a different and non-overlapping locus of said polynucleotides.
8. The method of claim 7 wherein said different and non-overlapping locus is adjacent to and upstream of a single nucleotide polymorphism site.


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February 23, 2015: Weekly News Roundup
by | Feb 22, 2015 | Uncategorized |
Protests Continue Following Police Shooting in Washington State
The fatal police shooting of Antonio Zambrano-Montes on February 10 has spurred daily protests in Pasco, Washington. The police claim Zambrano-Montes, a Mexican immigrant, was throwing rocks at the officers, but the situation is still under investigation. Protestors have been gathering at the Pasco City Hall every day this week, and people around the nation are drawing parallels between this shooting and the police shootings of Michael Brown and Eric Garner.
Confusion in Alabama Continues Regarding Same-Sex Marriage Licenses
Two conservative groups filed an emergency petition to the Alabama Supreme Court, requesting state and federal courts in Alabama to stop issuing same-sex marriage licenses. This comes in the wake of the U.S. District Court for the Southern District of Alabama deciding that the state's ban on gay marriage was unconstitutional. While the conservative organizations hoped this petition would stop gay marriages from taking place in Alabama, many probate judges continue to issue marriage licenses to same-sex couples. Republicans fill every seat on the Alabama Supreme Court, and activists on both sides of this issue are eagerly awaiting a decision.
Wal-Mart Raising Wages for Employees to $9 an Hour
On Tuesday, Wal-Mart announced that it is raising its wages to at least $9 an hour for a half-million employees. A White House spokesman said, "Today's announcement is another example of businesses, along with cities and states, taking action on their own to raise wages for their workers, recognizing that doing so can raise productivity, reduce turnover and improve morale." This move could also encourage other retailers to increase wages for their employees.
Incorrect Tax Information Regarding Obamacare Impacting 800,000 Americans
On Friday, the Obama administration announced that the government sent incorrect tax information to 800,000 Americans who had signed up for insurance policies via HealthCare.gov. While the Affordable Care Act has been widely declared a success due to its strong enrollment numbers, this mistake could hurt many low-income Americans who must now wait to file their taxes and receive their refunds. The government decided to postpone the deadline to sign up for health insurance, in order to allow people to avoid any tax penalties. Critics of the Affordable Care Act have jumped upon this mistake, claiming that "Obamacare continues to frustrate and confuse Americans." However, the White House claims this will only impact "a very small fraction" of taxpayers and that they still have plenty of time to file before the tax deadline.

redpajama_set_name: RedPajamaCommonCrawl


text:
Common pesticides are indirectly killing bees
Posted by Laura Boness on April 2, 2012 in Insects, Nature, News, Science · 0 Comments
Bumblebee populations are also declining. Image: Shutterstock
Sublethal doses affect bees by impairing their ability to find their way home.
Two new studies from France and the UK have suggested that neonicotinoid insecticides may be wiping out bees by reducing the numbers of queens and affecting the workers' ability to gather food.
Neonicotinoid insecticides get rid of pests by affecting their central nervous systems and have become one of the most widely used crop pesticides in the world since their introduction in the 1990s. Both studies, published in Science, have shown that they can spread to nectar and pollen of flowering crops and affect bees.
It isn't just honeybee populations that are plummeting; bumblebees have also been affected, according to Dave Goulson of The University of Stirling in the UK. "In North America, several bumblebee species which used to be common have more or less disappeared from the entire continent," he said.
"In the U.K., three species have gone extinct."
One effect of these insecticides on bumblebee colonies is that they don't bring in as much food, according to researchers from the University of Stirling. By weighing the bees at the beginning and end of a six-week foraging period, they found that the treated colonies were eight to 12 per cent smaller than the control colonies.
The treated colonies also produced 85 per cent fewer queens, which would affect the establishment of new nests and the colony's survival. "The use of neonicotinoid pesticides on flowering crops clearly poses a threat to their health, and urgently needs to be re-evaluated," said Goulson.
Researchers from the French National Institute for Agricultural Research (INRA) focused on honeybees and found that exposure to the pesticide thiamethoxam impaired the bees' homing ability. By tracking the bees with RFID microchips, they found that the bees were two to three times more likely to die away from their nests.
Collecting honeybees at the entrance of an RFID beehive. Image © Science/AAAS
The researchers then incorporated this data into a mathematical model that simulated population dynamics. The model predicted that populations affected by the pesticide would drop to a point from which it would be difficult to recover.
The doses the bees were given in both cases were sublethal. So while manufacturers keep pesticide doses below lethal limits, smaller doses may kill the bees indirectly.
This raises important issues for pesticide authorisation procedures, Mikaël Henry of INRA said. "So far, they mostly require manufacturers to ensure that doses encountered on the field do not kill bees, but they basically ignore the consequences of doses that do not kill them but may cause behavioural difficulties."
Source: Eureka Alert
Old termites go on suicide missions
Climbing in the rain
Mischievous rodents the reason for tropical tree survival
Fruit flies change after sex
Tags: bees, insects, nature, News, science
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Posted on April 5, 2016 by revivewa	in News.
It is everyone's responsibility to provide an environment that is safe from sexual abuse for all our children.
The media coverage in February of The Royal Commission into Institutional Responses to Child Sexual Abuse hearings from Rome, has once again focussed the attention of many.
In Western Australia, the Synod's Royal Commission Task Group continues to meet regularly, refining our current practices and policies, as well as identifying areas and actions for future improvement. Coupled with this is the need to ensure all aspects of the church's life are compliant of the new requirements.
As an individual, what can you do?
Most importantly, be actively involved in ensuring the safety of everyone participating in our church community, particularly children. You may need to speak-up, if you see or hear anything which makes you uncomfortable or doesn't seem quite right.
You can also attend a Safe Church Awareness Workshop to learn the steps and processes to keep your church safe. Anecdotal evidence shows that the more people in a congregation that attend training, the more likely it is that the congregation will be a safe place for all. Workshops will be held on Saturday 28 May, Kalamunda Uniting Church; Saturday 23 July, Willetton Uniting Church; and Saturday 15 October, St Peter Emmaus Community Church.
Discussions are underway to provide training in rural and remote congregations.
Everyone involved, or likely to be involved, in contact with children is required by Western Australian law to have a Working with Children Check (WCC), although some exemptions apply. Please ask questions about the WCC and the practices about who has them within your church community. You may also like to show personal support for those who have been sexually abused as children by tying ribbons to fences and letterboxes etc as part of the 'Loud Fence' campaign.
Cindy Gorton, Safe Church/Royal Commission officer, Robert Watson, WA Synod Task Group chairperson, Rev David de Kock, general secretary and Rev Steve Francis, moderator are here to walk beside you on this journey. Please contact them at the Uniting Church Centre on 9260 9800 if you have any questions or concerns.
For further information, please visit http://unitingchurchwa.org.au/safe-church/safe-church-training; https://workingwithchildren.wa.gov.au; and http://www.facebook.com/loudfence.


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Erdogan Blames Foreign Hand in Protests
Tulin Daloglu June 3, 2013
Turkish Prime Minister Recep Tayyip Erdogan accused foreign intelligence services of being behind the street demonstrations in Turkey.
REUTERS/Umit Bektas
A protester throws a gas canister back at the riot police during a demonstration in Ankara, June 3, 2013.
Although the ongoing street protests in Turkey cannot and should not be considered as being the same as the developments in some Arab countries rocked by popular uprisings, Turkish Prime Minister Recep Tayyip Erdogan provided a most-unexpected comparison on Monday.
Just like Syrian President Bashar al-Assad, who initially blamed foreign intelligence services as being behind the protests in Syria, Erdogan, too, accused foreign agents of supporting the ongoing protests asking for his resignation.
"Our intelligence work is ongoing [to determine the foreign actors behind the protests]," Erdogan said before departing for Morocco on Monday. "It's not possible to reveal their names. But we will have meetings with their bosses."
This suggests once again that Erdogan does not really get it. He continues to play the tough guy, and blames the opposition or, now, foreign agents for being responsible for the protests on the streets, as if he has had no role to play in them. Or, in other words, you are with me or against me — as if the protesters are the enemy.
I explained in "Erdogan weakened by protests" and "Turks Rally Against Erdogan Rule" what really triggered these massive demonstrations on Turkey's streets. Erdogan's accusation of foreign agents is disappointing, even by what we have come to expect these days in Turkey. While one can consider Erdogan's rhetoric addressing these demonstrations as a blatant provocation triggering the further escalation of the unrest, provocateurs could also alter the protests for the worse. The majority of protesters are against vandalizing public or private property. None applauded the news that a masked group put the AKP [Erdogan's Justice and Development Party] building in Izmir on fire. The protesters on the street just want to make a statement to the prime minister that he is acting as if they don't exist, and to remind him that they do.
These people on the streets are not politically organized. Many of those I chat with on the streets of Ankara also slam the opposition parties for their inability to govern. There was growing tension in the capital on Monday, the first work day since the demonstrations in Istanbul spread to Ankara and other cities on Friday. The roads to the prime minister's offices were all blocked and police did not allow anyone to go through. Public transportation as well as the rest of the traffic had to alter their routes. One pedestrian told me, "They're afraid of their own shadows." There were also others, though, supporters of Erdogan, who stayed distant, but made their body language seen. Both sides are somehow in shock in trying to comprehend what is really going on. And police continued their brutal crackdown.
A huge amount of pepper and tear gas was sprayed on people, in addition to vast streams of water sprayed by water cannons to disperse the crowds. Around midday Monday, high school students gathered in downtown Ankara in protest of the government. The police intervention was intense. It looks like it won't be the last, as it appears these protests will be continuing a little longer. The more they continue, however, the government risks their taking a much more violent turn. A doctor friend told me that there were a handful of protesters who were urgently taken into surgery due to brain trauma and remain in critical condition. There are thousands injured, and an equal number arrested. It's an absolute mess.
Turkish President Abdullah Gul met Monday with Kemal Kilicdaroglu, the main opposition party Republican People's Party (CHP) leader, at Kilicdaroglu's request. "The prime minister's rhetoric is not helping to de-escalate the situation," he said, drawing attention to the excessive use of force. "We have to first accept that there is excessive use of force. I told the president that he has powers." In other words, Kilicdaroglu appealed for help to Gul to intervene and help calm the streets.
No one needs to be an Einstein to understand that if this tension continues on the streets, this will turn more violent. No one also needs to ignore that if the demonstrations continue, people need to channel this social movement into a political activity. Things are not there yet, but Erdogan's harsh rhetoric may push people in such a direction. And it's all unprecedented what might follow next.
European diplomats are also on the streets, closely observing and witnessing the events. The reason is simple — to find out for themselves what is happening.
It's a shame that the broadcast media covered Erdogan's speeches from beginning to end, but not the protests. Thousands gathered in front of NTV, Turkey's first news channel, to protest its nonexistent coverage of the demonstrations. And the European diplomats are basically making sure by quietly attending these demonstrations that they can witness what the police and demonstrators are actually doing on the streets. "We call for restraint from the security forces," a European diplomat told Al-Monitor. "We will, however, wait for the coming days to see how these events evolve. Our statements may change based on these developments, such as if there is a casualty."
Erdogan might do well for the nation if he starts considering his own role in all of this. As the night sinks in while I write this article for the fourth night in a row, people again came out in Ankara's suburbs banging their pots and pans and honking their cars. There is an outpouring of reaction against the AKP government, and this must have a meaning beyond blaming the opposition or foreign agents.
Tulin Daloglu is a contributor to Al-Monitor's Turkey Pulse. She has also written extensively for various Turkish and American publications, including The New York Times, International Herald Tribune, The Middle East Times, Foreign Policy, The Daily Star (Lebanon) and the SAIS Turkey Analyst Report. On Twitter:@TurkeyPulse
Found in: turkish nationalism, turkey
Tulin Daloglu has written extensively for various Turkish and American publications, including The New York Times, International Herald Tribune, The Middle East Times, Foreign Policy, The Daily Star (Lebanon) and the SAIS Turkey Analyst Report.
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Home>Highlight>Beto O'Rourke Addresses Enthusiastic But Smaller-Than-Expected Launch Crowd
Highlight Media National
Beto O'Rourke Addresses Enthusiastic But Smaller-Than-Expected Launch Crowd
MAGA hats ubiquitous at Democrat hopeful's kickoff rally
By Ken Kurson, March 31, 2019 3:12 pm
31 Mar 2019 3:12 pm
Beto O'Rourke addresses the crowd at his kick-off rally in Austin, March 30, 2019.
AUSTIN—Former congressman and current heartthrob Beto O'Rourke took the stage in front of the Capitol building over an hour and a half late. But if any of the several thousand people who endured an unexpectedly chilly early spring evening were disappointed, they certainly didn't show it.
The Bobby Kennedy look-alike became a household name throughout the country by giving Senator Ted Cruz a run for his money in blood-red Texas. He capped a day of three appearances across the state with a rally in the state's most progressive city. Young people piled onto the annoying rideshare scooters that are plaguing Austin and pressed into Capitol Square to hear the candidate discuss, among other well-delivered talking points, a border situation that seems to be worsening by the day.
Hundreds of Trump supporters, many toting semi-automatic rifles, joined the Austin crowd for Beto O'Rourke's third rally of his campaign kickoff day. (Ken Kurson for California Globe)
As they waited, they were warmed up by local queen of soul Tameca Jones, whose twin 17-year-old daughters were in attendance. Her credible version of "Benny and the Jets" broke with the traditional American classics usually performed at campaign rallies, but that's in keeping with a candidate who takes the stage to "Clampdown" by Elton John's countrymen The Clash.
When the candidate finally took the stage at 10:35 pm — no tie, a blue shirt, and rolled-up sleeves — he told the crowd, "It is good to be back here in Austin, the source of what is good for Texas. And for the United States of America." He discarded his fleece, rolled up his sleeves even further and pounced around the stage with pogo-stick energy, addressing all sides.
With his voice showing all the strain of a third rally, O'Rourke thundered, "The only answer is a vigorous democracy – both a political democracy and an economic democracy."
Meanwhile, young volunteers and staff scurried to register voters and hand out buttons at tents advertising "Beto Para Presidente." But the planners of the rally were clearly expecting a much larger turnout.
'Finish the Wall' was a popular placard. (Ken Kurson for California Globe)
Rallies at the Capitol Square are judged by how many streets back they reach, with the largest extending all the way down Congress St. to the rockin' bars and Voodoo Donuts on 6th Street. In fact, O'Rourke's staffers put up speakers extending that far but the actual crowd petered out before 9th St. A disappointed volunteer told the Globe, "We thought we'd have way more people, but it's so cold and it's so late."
One contingent evidently not put off by the chill wind were the surprising number of Trump supporters. Dozens of MAGA hat wearing Texans —many with semi-automatic rifles strapped across their chests in celebration of the state's open-carry policy—showed up carrying signs in support of the GOP incumbent the majority of the rally-goers are hoping their guy will earn the right to face. Several critics were spotted with signs mocking O'Rourke's cultural appropriation. "His name is Robert O'Rourke NOT BETO" read many professionally produced placards. "He's 100% Irish-American / He's 0% Hispanic."
A well-armed, well-behaved Trump supporter. (Josh Jacobs for California Globe)
Other Trump supporters advocated to "Finish the Wall" and "Keep America Great."
With a field of candidates quickly approaching two dozen, O'Rourke joins all the candidates in facing a talented, crowded field. And unlike others in the field, who include Senators, Governors and a possible two-term Vice President, O'Rourke hasn't developed much of a record in his three terms as a Congressman. Meanwhile, some are questioning whether a party increasingly vocal about identity politics ought to be running a straight, white male as its champion.
O'Rourke himself looked to address that easy line of attack, telling the cheering crowd, "We're gonna make sure that your family, your gender, your race, your sexual orientation does nothing to prevent your advancement."
Whether it's enough to talk about these groups instead of be among them in a Democratic primary remains to be seen.
Still, the presence of so many Trump supporters indicates that people are paying attention.
Trump supporters made their presence felt on a chilly evening in the Texas capital. (Ken Kurson for California Globe)
Ken Kurson
Ken Kurson is the founder of the Globe suite of sites. He is also the founder of Green Magazine and greenmagazine.com and covered finance for Esquire magazine for almost 20 years. Ken is the author of several books, including the New York Times No. 1 bestseller Leadership.
Latest posts by Ken Kurson (see all)
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Tagged Beto O'Rourke, Donald Trump, Tameca Jones
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2 thoughts on "Beto O'Rourke Addresses Enthusiastic But Smaller-Than-Expected Launch Crowd"
Ken…..you need to stop. You're going to leave a hickey on Butto's butthole if you don't quit all that asskissing. Your little heart throb is a degenerate criminal from a criminal family as well as a communist.
Mark Young says:
Thank you for acknowledging the fake Hispanic has appropriated another culture. I would like to point out that our forefathers saw corectly that democracy is two wolfves and a sheep voting on dinner. That is why America is a constitutional republic. Also the Texas LAW which allows open carry of rifles dates back to post civil war. It is not a policy.
I would like to congraduate California in overturning their unconstitutional gun magazine law.


The competition is on the rise, especially with the advent of the online freelance industry. The sprawling presence of this industry has brought together people from all over the world and enabled them to compete with one another in a more open and transparent platform.
In order to stand out in this industry, a determination will not suffice. You will be required to develop and hone skills that are most crucial to the freelance industry.
While there are all kinds of skills operational in this industry, there are a few whose demand is phenomenally high. Thus, if you work on these few skills, you might be able to get access to greater and better work opportunities.
In this article, we have shortlisted ten skills that are in huge demand in the freelance industry. What is notable about these skills is that there are many with these skills but only the best survive the harshness of the competition. So, if you think you can be one of them, you should start working hard.
Here are those ten skills you should know about in order to brighten your freelance career and take it to newer heights.
Charting at number one, Content Writer is a universally acclaimed and the most wanted freelance skill in the present freelance industry. Thus, content writing invites a lot of lucrative opportunities for freelancers.
You get paid for every word, and all you need to do is sit in front of a computer and write a batch of articles by following a prescribed manner. You will never fall short of the platforms for which you can write—newspapers, blogs, YouTube, Facebook pages, promotional pages, entertainment websites such as 9gag and Buzzfeed, etc.
In order to establish a sound and profitable career in content writing, start working on your basics, style of writing and a couple of other things such as sticking to the guidelines.
You might be surprised to know that an average Indian freelance content writer earns about 1.8 lacs annually, but the figure is higher since there are those with many years of experience and talent for profiting from this business smartly.
Blogging is part of the content writing, but it deserves a separate mention because blogging is huge enough to define itself as an industry.
Website owners need bloggers to advertise their portals on various platforms. Those with good blogging skills will be able to direct the audience's attention to the relevant content and increase the number of visitors to the website.
If you are wondering about the earnings, you should know that bloggers make as much as $100k every year!
Graphic designing is huge in demand in the freelance industry. Several digital marketing companies, magazines, newspapers, and blogs require talented and punctual graphic designers to cater to their graphics-related needs in minimum time.
In order to make big as a graphic designer, you must polish your computer skills, especially the use of Photoshop or other editing software. Plus, you should be flexible enough to accommodate brighter and out-of-the-box ideas to give more meaning to your designs.
It is recommended that you maintain a proper portfolio of all your designs in order to increase your employment chances.
The Internet is about websites; without websites, the gigantic Internet will be no fun. It, therefore, becomes important that the Internet keeps on growing with more productive and interactive websites.
Getting on board web developers is not always easy, but the freelance industry has done away with several hassles. Freelance web developers are greatly in demand. As business is growing, websites become backbones, and only a good website will be able to contribute to the business.
If you are a web developer, you should start building a portfolio and applying. You can earn a significant sum daily by investing only a couple of hours into the job.
A side tip: If you happen to work for bigger companies, you can end up earning more than you could ever have imagined. So, keep trying!
Voice-over artists have become trending artists lately. With the rise of documentaries, international broadcast of shows such as anime, Asian dramas, English movies and whatnot, voice-over artists are direly needed.
You can do a lot of things: work at a radio station or dub regional shows in different languages. All you are expected to have are great voice modulation, clarity, and enunciation. Moreover, your ability to emotionally connect to the character you are providing your voice to is also material.
There is no age requirement to prevent you from venturing into voice-overs. Your voice will make up an excellent skill!
Known more popularly by its acronym SEO, Search Engine Optimisation is a technique aimed at increasing the website traffic.
Why is SEO a great skill? Well, the answer is simple and valid. If you wish to survive and make money online, you will have to attract traffic. Most of the revenues earned online is through advertisements following the "pay-per-click" basis of payment.
Owners of search engines and websites often hire freelancers to optimise searches and increase traffic. However, it is to be understood that SEO skills need a lot of practice. For example, if you are an SEO article writer, you should know what keywords will most likely be inputted into the search engine and you on only learn them with experience.
The Internet has ushered in the world of cosmopolitanism. Now every person can have access to international content, and this begs the need to translate. The linguistic differences are done away with the help of translators and you can find most of them working as freelancers.
Translation is a coveted skill today. There is a non-stop transmission of international content such as English movies, Japanese animation and whatnot, and this requires the need to translate in regional languages for better access.
Freelance translation assignments are lucrative. From private companies to government institutions, all of them require translators to expand their audience.
If you go to Playstore, you will immediately realize the importance of Apps in the digital world today. In this world of Internet of things, Apps are utterly important.
App development is a skill companies are desperately looking for. There are many App developers in the world but only a selective portion of them hand over quality applications. Thus, the tragedy is that despite many App developers, creativity is still confined to not many.
Naturally, App developers will face competition, but they will always be open to lucrative opportunities. App developers are paid heavily and thus, to make best of your skill, polish it.
Companies want to work with best people, and you can help them find such people. Many companies hire freelance Executive Search Specialist to help them find suitable candidates for particular roles and pay the latter accordingly.
The contribution would be a relatively less-pressurized HR system contributing to the efficiency of the overall organization of the company.
The clients are usually medium-sized and large-sized companies and hence, you are sure to make handsome earnings. Just work on your professional connections and use them in these assignments for your own good.
If you are a social media addict, you can profit from your addiction. As noted earlier, to survive in the digital market, you will need to attract traffic—naturally, you will have to advertise.
Advertisement can be complex, and to ease the entire process a little bit, freelance social media marketers are roped in to do the job. All you need to do is to share the website or any other information on popular social media platforms such as LinkedIn, Pinterest, Reddit, Instagram, etc and you can earn a huge amount.
Companies want candidates who are well-connected because if they are reclusive, there will be no point in making them share information.


Newcastle Central MP calls for greater government scrutiny of Newcastle Utd's finances under owner Mike Ashley.
It's reported the Sports Direct owner has handed an offer to administrators for the high street music chain, which has more than 100 stores.
Diamé has warned Newcastle will have to be even more miserly if they are to deny Premier League leaders Liverpool victory on Boxing Day.
Asked if a deal being struck before 2019 was realistic, Ashley said the word "possible" would be a more accurate description.
It's been reported that a former Manchester United Chief Executive is putting together a bid to buy Newcastle United football club.
It's believed Peter Kenyon, who also spent time at Chelsea, is working with a New York based capital management firm on the bid.
If they're successful, it would end Mike Ashley's time as Newcastle's owner.
The club was unable to comment.
Fans arriving for Saturday's game against Leicester at St.James Park told ITV Tyne Tees they largely welcomed the news.
Hundreds of Newcastle fans have lined Northumberland Street to protest against the running of the club by owner Mike Ashley.
An agreement has been formalised between the Magpies and Muto's current club, FSV Mainz 05, and the player has undergone a medical.


Black denim Spray On fit jeans, can be back or full zip. Sold as seen.
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Strict rest may not be the best medicine for kids with concussions, a Canadian study found, challenging the idea that physical activity should be avoided until symptoms disappear.
A month after their concussions, ongoing or worse symptoms were more common in children and teens who were inactive during the week following injury, compared with those who engaged in physical activity during that first week.
Activity was mostly light exercise including walking and swimming.
The results were similar even among those who early on had three or more concussion symptoms, which can include nausea, headaches and confusion.
Physical activity still seemed to reduce chances for lingering symptoms a month after the concussion.
'It's still important to have caution in the immediate post-injury period,' said lead author Dr. Roger Zemek, an emergency medicine specialist at Children's Hospital of Eastern Ontario.
To avoid re-injury, kids should generally be sidelined from the sport or activity that led to the concussion until a doctor clears them to return, he said.
But the study results suggest they can resume sports or other physical activities sooner than previously thought, he said.
The researchers surveyed about 2,400 kids aged 5 to 18 treated for concussions in nine emergency departments in Canada.
Most were sports-related injuries and most kids had at least one concussion symptom in the first week.
Current guidelines recommend rest until symptoms disappear to avoid additional concussions, which can increase chances for permanent brain damage.


Így jelent meg, hogy küldtem egy üzit. Valójában 18:59-kor küldtem. 2 órát késik.
How many people can go on the trail?
Hi, we increased the limit to 300!
I dont find any e-mail on you, thats why I have to write this comment. I would like to log out from this trail for personal reasons. Thank you for understanding.


text:
Considering the failure of Congress to address the thorny issue of illegal immigration states have been left to establish their own laws aimed at curbing illegal immigration. This approach will surely create a new patchwork of state regulations that could blow up in the face of Congress if it attempts to exert its authority and pass immigration reforms that supersede existing state established reforms. Action from lawsuits claiming violation of Due process of law and/or violation of the 10th amendment of the U.S. Constitution addressing state's rights to violence could accompany reaction to any ruling or movement on the immigration issue. A ruling by the U.S. Supreme Court is highly unlikely, however; the high court's recent history of basing rulings on international law could put the illegal immigration issue on center stage.
An H1B1 visa is defined as a national of Chile or Singapore coming to the Unites States to work temporarily in a specialty occupation. The law defines an H1B1 specialty occupation as a position that requires theoretical and practical application of a body of specialized knowledge. The H1B1 visa program is used by some U.S. employers to employ foreign workers in specialty occupations that require theoretical or technical expertise in a specialized field and a bachelor's degree or its equivalent. Typical H1B1 occupations include architects, engineers, computer programmers, accountants, doctors and college professors.
The Obama administration has said that it strongly supports expansion of H1B1 visas as a method of economic stimulus and as a means of reforming U.S. immigration status giving educated foreign workers a path to citizenship. Proponents of such a policy have argued that as the U.S. economy improves it will create jobs that demand high tech skills. However, the question of whether such proponents are aware of the economic reality known as structural unemployment remains. The principal states that a certain number of individuals will not achieve the goal of obtaining a job in their field as it is a trait of a free-market economy that the economy is not capable of producing the kind of jobs society demands in the quantity demanded to match the skills one possesses. Will the new administration expand the H1B1 visa program? Will such expansion bring about the displacement of a greater number of American workers by outsourcing American jobs to foreign countries? An answer to the latter depends on the answer to the former thereby rendering the final answers ignotus. What is known is that if in the future an American worker wonders to which foreign country the job he once had is being outsourced he may not need to look farther than those nations most negatively affected by U.S. policy reforms aimed at combating illegal immigration.
htmlhttp://singapore.usembassy.gov/fta_visas.
htmlhttp://chile.usembassy.gov/h1b1-visa.
htmlhttp://www.haranlaw.com/blog/http://www.zazona.com/ShameH1B/Links.
htmhttp://www.supremecourt.gov/
http://www.house.gov/house/Educate.shtml
The United States Has to Do Something About Reforming Our Broken Immigration System to Insure Fairness Through Comprehensive Immigration Reform.
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Glass Vessel Sink in Amber Sunset with Faucet in Chrome is rated 4.8 out of 5 by 52.
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The "Thrive with Me" (TWM) intervention leverages enhanced peer-to-peer interaction, adherence to antiretroviral therapy (ART) reminders and self-monitoring, and ART and HIV informational content to improve ART adherence.
Researchers at Baylor College of Medicine and Texas Children's Hospital are conducting a voluntary research study about slow stomach emptying and how it affects children.
The purpose of this study is to find the largest safe dose of donor-derived tumor protein multi-TAA-specific T cells for patients with AML or MDS.
The purpose of this study is to find the largest safe dose of chimeric T cells, to learn what the side effects are, and to see whether this therapy might help patients with sarcoma.
The purpose of this study is to collect health and treatment information on patients previously diagnosed with atypical hemolytic uremic syndrome (aHUS).
The purpose of this study is to compare at-home sleep with in-hospital sleep using a portable sleep device to help monitor quality of sleep.
The purpose of this project is to examine which body tissues (such as hair, fingernails, urine or blood) are needed to accurately study how gene activity is affected by prenatal factors.


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Q: Can you use a context manager to write a pandas DataFrame to sqlite When writing a pandas DataFrame to sql, would it be possible to use the with statement in the following way?
import sqlite3
import pandas as pd

with sqlite3.connect('database.db') as conn:
    df = pd.read_sql("SELECT * FROM table", conn)

    # add change to db

    df.to_sql('table', conn, if_exists='replace', index=False)

A: From the documentation

Connection objects can be used as context managers that automatically commit or rollback transactions. In the event of an exception, the transaction is rolled back; otherwise, the transaction is committed

Example:
import sqlite3
import pandas as pd

con = sqlite3.connect("database.db")

# Return the result as a list
with con:
    result = con.execute("SELECT * FROM table").fetchall()

# Pass as engine to pandas
with con:
    df = pd.read_sql("SELECT * FROM table", con)

# Close connection
con.close()

Edit
As @Parfait has rightly pointed out the context manager is on transactions made by the connection, thus the con object should closed manually.

redpajama_set_name: RedPajamaStackExchange


text:
GUINESS WORLD RECORDS: SA Woman Births 10 Children At Once
Africa, News / By Oluwamayowa Akinyemi
It sounds untrue, appearing like a click bait, but 37-year-old Gosiame Thamara Sithole, a South African woman has given birth to 10 babies at once.
This sets a new world record topping Halima Cisse — a Malian woman who gave birth to nonuplets in Morocco in May.
A caesarian operation was carried out on Sithole to give birth to 3 girls and 7 boys successfully. As a matter of fact, what could have turned out to be octuplets became decuplets.
Gosiame Thamara Sithol and husband,
Initially, Sithole and her husband were expecting 8 babies after she was first scanned. But somehow, 2 amazing babies were playing hide-and-seek and missed out while in the wrong tube.
"I am shocked by my pregnancy. It was tough at the beginning. I was sick. It was hard for me. It's still tough but I am used to it now. I don't feel the pain anymore, but it's still a bit tough. I just pray for God to help me deliver all my children in a healthy condition, and for me and my children to come out alive. I would be pleased about it," Sithole said.
With a successful delivery, she is now the first Guinness Record-holder woman for the most children birthed at once.
Some people still find this unbelievable saying it is unnatural. But her husband cleared the air saying his wife wasn't even on any fertility treatment of sorts. She gave birth 29 weeks into her pregnancy naturally. It is rare for a human but certainly not unnatural.
This isn't the first time Sithole is giving birth to more than a baby at once. The couple have been raising 6-year-old twins before she made her way into the Guinness book of record.
Before Gosiame Thamara Sithole, Halima Cisse held the record for the most children birthed by one woman at once, and before her there was Octomom, Nadya Suleman, who gave birth to octuplets 12 years ago.
As the world continues to change, and life on Earth continues to evolve, change remains constant with us 🙂
By Elijah Christopher
Elijah Christopher is a journalist at A New Touch Of Africa, is also a creative writer, a poet, and an IT enthusiast. He contributed to the collaborative poem written in celebration of Edwin Morgan Centenary, the first Glasgow poet laureate and Scottish national poet from the University of Glasgow. He loves meeting people and learning about new places, cultures, events, and lifestyles.


With the rapid replacement of the phone, Samsung has released its flagship phone – Galaxy Note 5. Samsung Galaxy Note 5 continues the appearance of the Galaxy S6 design style, the back of the glass material, camera raised, the edge of the fuselage using a rounded handle, better grip. The phone is equipped with a 16000000 pixel rear camera, and supports 4K, 1080p video shooting. This article will give you an introduction to how to spy the Note Galaxy 5 with the Android tracking Software.
When it comes to monitoring on Galaxy Note 5, you may have a lot of reasons. Perhaps you would like to know about your child's activities, browse unhealthy websites, fear that they will play with the bad boys, and the test results will fall. You need to spy on your child's Galaxy Note 5. Perhaps your spouse always sneaking, your husband always call a woman in the room next door? In order to understand the details of the call, you have to spy on his Galaxy Note Note 5. Otherwise you may have the risk of losing your family. If Galaxy Note 5 is the main communication tool for your employees, it is more necessary to monitor their every move. Of course, you can also hire a private detective to perform your surveillance tasks, but it would be a big expense. Why don't let the Samsung Galaxy Note 5 monitoring software work for you?
Trouble for can not find a suitable monitoring software to track on Note 5? Samsung Galaxy Note 5 Spy Software is the best solution for you. As long as you install this monitoring software to Galaxy Note 5, the phone seems to be in your hands. It will give you feedback on all activities of the Samsung Galaxy Note 5. Including caller number, all the received and received text message, web pages have been visited, videos and pictures taken by mobile phone, GPS, Skype, Facebook, iMessage, Whats App, installed Applications, etc. The software has all the features you want. Even you can record the phone calls. Of course, no one can know the existence of it.
Free try Galaxy Note 5 Spy Software here.
Supported Samsung Devices: You can monitor any Samsung Galaxy devices including Android smartphones and tablets. Such as Galaxy Note 5, Galaxy S6, Galaxy S7, Galaxy TabPro S, Galaxy Note 8, Galaxy S9, Galaxy Note 9, etc.
How the Galaxy Note 5 Spy Software Works?
You can purchase our Galaxy Note 5 spy app directly from this website. Choose the subscription and fill in the order form. After your payment has been confirmed, installation guidelines will be sent to you.
After the purchase is completed you need to download our Galaxy Note 5 tracking application and install it onto the target Android device. Galaxy Note 5 Spy Software installation is so simple and intuitive that you can complete it in several minutes.
Simply log in to your Control Panel and our Galaxy Note 5 tracker will start tracking text messages, call history, GPS location details, and any other activity taking place on the target device. Our Galaxy Note 5 monitoring app will capture the information on every activity of your target Note 5 user.
Tip: It's best not to monitor anyone, they will hate you, and you will have a crisis. Before you spy on their Galaxy Note 5, you need to tell them and make sure that you get their permission. For your children, familiy and company, you have to learn to self-correcting, with a positive attitude to deal with the problem!
Other than WHATSAPP, what other instant messaging apps can this software track?
Including WhatsApp, iMessage, Social Network, Viber, Snapchat, Line, etc.
If the phone get factory restarted what's happen ? Loss the app do I need to rooter the phone.
You need not to rooter the phone. But if you wish to monitor Whatsapp, Viber, Facebook, Skype, Line, Snapchat and Gmail, your target Android phone should be rooted. You will set the user name and password in the installation process. Once Galaxy Note 5 Spy App is set up on the monitored device and is connected to our server, the information will be automatically displayed in the Control Panel of your personal account that can be accessed from any browser.
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Temperatures ranging from 14 to 18 degrees over the day with best temperatures in the early afternoon.
Tomorrow looks much the same, very cloudy and grey, but warm.


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text:
Сан-П'єр-Нічето (, ) — муніципалітет в Італії, у регіоні Сицилія,  метрополійне місто Мессіна.

Сан-П'єр-Нічето розташований на відстані близько 490 км на південний схід від Рима, 175 км на схід від Палермо, 18 км на захід від Мессіни.

Населення —   (2014).
Щорічний фестиваль відбувається 29 червня. Покровитель — святий Петро.

Демографія

Сусідні муніципалітети

 Кондро
 Фьюмедінізі
 Гуальтієрі-Сікаміно
 Монфорте-Сан-Джорджо
 Паче-дель-Мела
 Санта-Лучія-дель-Мела

Див. також
 Список муніципалітетів метрополійного міста Мессіна

Примітки

redpajama_set_name: RedPajamaWikipedia


By Anuradha Kher, Online News EditorAlisa Viejo, Calif.–The real estate industry just got its own Wikipedia. Realestatewiki.com, a privately funded, non-profit, non-bias venture similar to Wikipedia, and founded by a group of 12 real estate professionals, provides a free real estate encyclopedia and resource for everyone to use.Stefan Swanepoel, co-founder of realestatewiki.com, tells MHN, "Many of us were frustrated with the fact that there is a lack of information on real estate available online. We all know it's there, so we decided to consolidate it."In addition to the 7000 entries added by the founding members, the Web site has produced—through voluntary contributions—3,000 definitions, 1,000 answers to frequently asked questions about home buying, selling and financing, more than 500 acronyms, over 400 bios of real estate leaders and authors, a list of more than 600 real estate brokerage companies and over 800 real estate associations, institutes, councils, designations, courses, blogs, social networks and publications."Our objective is to provide neutral, objective information on the subject. We edit all the material submitted to us and make it free of marketing type or negative language," Swanepoel tells MHN.An estimated 10 million people work in the real estate industry (construction, brokerage, financial, insurance, appraisal, etc.) and real estate is the number one search topic on Google, according to realestatewiki.comSince its beta launch in January this year, realestatewiki.com has added 3,000 new entries in categories such as "Who's Who in Real Estate," "Real Estate Newspapers and Research," "Real Estate Training Schools" and "Real Estate Brokerage Companies."Realestatewiki regularly gets requests from people in the real estate industry, who want to contribute to the Web site.


text:
Plan-d'Aups-Sainte-Baume
French Version : http://www.cartesfrance.fr/carte-france-ville/83093_Plan-d'Aups-Sainte-Baume.html
> Var
> Plan-d'Aups-Sainte-Baume
Town of Plan-d'Aups-Sainte-Baume
The village of Plan-d'Aups-Sainte-Baume is a small french village located south east of France. The town of Plan-d'Aups-Sainte-Baume is located in the department of Var of the french region Provence-Alpes-Côte d'Azur.	The town of Plan-d'Aups-Sainte-Baume is located in the township of Saint-Maximin-la-Sainte-Baume part of the district of Brignoles. The area code for Plan-d'Aups-Sainte-Baume is 83093 (also known as code INSEE), and the Plan-d'Aups-Sainte-Baume zip code is 83640.
Geography and map of Plan-d'Aups-Sainte-Baume :
The altitude of the city hall of Plan-d'Aups-Sainte-Baume is approximately 686 meters. The Plan-d'Aups-Sainte-Baume surface is 24.91 km ². The latitude and longitude of Plan-d'Aups-Sainte-Baume are 43.33 degrees North and 5.717 degrees East. Nearby cities and towns of Plan-d'Aups-Sainte-Baume are : Riboux (13780) at 4.24 km, Cuges-les-Pins (13780) at 6.06 km, Saint-Zacharie (83640) at 6.31 km, Nans-les-Pins (83860) at 7.09 km, Gémenos (13420) at 7.94 km, Auriol (13390) at 8.15 km, Roquevaire (13360) at 9.32 km, La Destrousse (13112) at 10.43 km.
(The distances to these nearby towns of Plan-d'Aups-Sainte-Baume are calculated as the crow flies)
Population and housing of Plan-d'Aups-Sainte-Baume :
The population of Plan-d'Aups-Sainte-Baume was 764 in 1999, 1 125 in 2006 and 1 176 in 2007. The population density of Plan-d'Aups-Sainte-Baume is 47.21 inhabitants per km². The number of housing of Plan-d'Aups-Sainte-Baume was 615 in 2007. These homes of Plan-d'Aups-Sainte-Baume consist of 422 main residences, 164 second or occasional homes and 29 vacant homes.
I like Plan-d'Aups-Sainte-Baume !
Administrative Information of Plan-d'Aups-Sainte-Baume
Region : Provence-Alpes-Côte d'Azur (93)
Departement : Var (83)
District : Brignoles
Township : Saint-Maximin-la-Sainte-Baume
Area code : 83093
Nearby towns of Plan-d'Aups-Sainte-Baume
Here are maps and information of the close municipalities near Plan-d'Aups-Sainte-Baume.
See all the towns of Var (Distances are calculated as the crow flies)
Riboux (83) at 4.24 km Cuges-les-Pins (13) at 6.06 km
Saint-Zacharie (83) at 6.31 km Nans-les-Pins (83) at 7.09 km
Gémenos (13) at 7.94 km Auriol (13) at 8.15 km
Roquevaire (13) at 9.32 km La Destrousse (13) at 10.43 km
Roquefort-la-Bédoule (13) at 11.40 km La Bouilladisse (13) at 11.97 km
Signes (83) at 12.54 km Aubagne (13) at 12.73 km
Rougiers (83) at 12.85 km Peypin (13) at 12.88 km
Trets (13) at 13.50 km Carnoux-en-Provence (13) at 14.43 km
Ceyreste (13) at 14.57 km Peynier (13) at 14.62 km
Belcodène (13) at 15.00 km Le Castellet (83) at 15.01 km
Geography of Plan-d'Aups-Sainte-Baume
Area : 24.91 km² (2 491 hectares)
Minimum altitude : 510 m
Maximum altitude : 1 148 m
Average altitude : 829 m
Altitude of the city hall : 686 m
Sexagesimal geographical coordinates (WGS84): Latitude: 43° 19' 48'' North
Longitude: 05° 43' 03'' East
Decimal geographical coordinates : Latitude: 43.33 degrees (43.33° North)
Longitude: 5.717 degrees (5.717° East)
Lambert 93 geographical coordinates : X: 9 205 hectometers
Y: 62 517 hectometers
Population and housings of Plan-d'Aups-Sainte-Baume
Inhabitants in 1999 : 764 inhabitants
Inhabitants in 2006 : 1 125 inhabitants
Population data : Population Plan-d'Aups-Sainte-Baume
Population density : 47.21 hab/km²
Variation of population density : +5.54 %/year
Housings in 1999 : 503 logements
Housings data : Housing Plan-d'Aups-Sainte-Baume
Hotel Plan-d'Aups-Sainte-Baume
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Find all hotels of Plan-d'Aups-Sainte-Baume :
Guest accommodation : Zenitude En Provence
Set in Plan d'Aups, Zenitude En Provence offers a private pool. This self-catered holiday home features a garden and barbecue facilities. The air-conditioned holiday home consi...
Address : 16 impasse des violettes Chemin de la Morguette 83640 Plan d'Aups
Distance Guest accommodation - Plan-d'Aups-Sainte-Baume : 0.59 km
Guest accommodation : JAS en Provence
JAS en Provence is situated in Plan d'Aups. The holiday home has mountain views and is 44 km from Marseille. The holiday home includes 1 bedroom and a kitchenette with a fridge...
Contact : 754 Allée du Jas 83640 Plan d'Aups
Guest accommodation : Maison Rouge
Maison Rouge is a wooden Guest House surrounded by mountainous countryside in La Sainte Baume. The property features a ground level terrace with sun loungers, a garden and a com...
Contact : 1020 route de Nans 83640 Plan d'Aups
Guest accommodation : Appart'Hôtel Les Rochers
Appart'Hôtel Les Rochers offers self-catering apartments and studios, just 2 km from Auriol. It provides air conditioning, free Wi-Fi access and free private parking. Cassis and...
Contact : 1156 Route de la Sainte-Beaume 13390 Auriol
Guest accommodation : Zacharie
Address : 115 Chemin du Moulin de Redon 83640 Saint-Zacharie
Apartment : Apartment Carraire Delvieux Sud
Featuring an outdoor pool and pool views, Apartment Carraire Delvieux Sud is located in Nans-les-Pins. Guests benefit from a balcony and a barbecue. The apartment includes a ki...
Contact : 132 Carraire Delvieux Sud 83860 Nans-les-Pins
Distance Apartment - Plan-d'Aups-Sainte-Baume : 6.56 km
Guest accommodation : Mas Des Sentes
Located in the Provençal town Nans-les-Pins, this villa features a private swimming pool, a quiet garden area and a terrace. Aix-en-Provence is 45 km away and the 13th century B...
Contact : 13 Chemin des Sentes - Quartier Vandegale 83860 Nans-les-Pins
Guest accommodation : Les Villagines
Located in Auriol in the Provence-Alpes-Côte d'Azur region, Les Villagines provides accommodation with free WiFi. All units come with a terrace, a kitchen with an oven and a mi...
Contact : Avenue du 19 Mars 1962 13390 Auriol
Bed and Breakfast : La Grande Maison De Nans
La Grande Maison De Nans Guest House, located 44 km from Marseille in Nans-les-Pins, offers an outdoor pool, a garden and a terrace. The guest rooms and suites feature free WiF...
Address : 1 rue du Vieux Nans 83860 Nans-les-Pins
Distance Bed and Breakfast - Plan-d'Aups-Sainte-Baume : 6.80 km
Guest accommodation : Holiday Home Villa Le Jardin 28
Situated in Nans-les-Pins, Holiday Home Villa Le Jardin 28 features accommodation with a terrace and free WiFi. Guests staying at this holiday home have access to a fully equipp...
Contact : 83860 Nans-les-Pins
Find another hotel near Plan-d'Aups-Sainte-Baume :
Plan-d'Aups-Sainte-Baume hotels list
Map of Plan-d'Aups-Sainte-Baume
Map of Plan-d'Aups-Sainte-Baume :
At right you can find the localization of Plan-d'Aups-Sainte-Baume on the map of France. Below, this is the satellite map of Plan-d'Aups-Sainte-Baume. A road map, and maps templates of Plan-d'Aups-Sainte-Baume are available here : "road map of Plan-d'Aups-Sainte-Baume". This map show Plan-d'Aups-Sainte-Baume seen by the satellite of Google Map. To see the streets of Plan-d'Aups-Sainte-Baume or move on another zone, use the buttons "zoom" and "map" on top of this dynamic map.
Search on the map of Plan-d'Aups-Sainte-Baume :
To search hotels, housings, tourist information office, administrations or other services, use the Google search integrated to the map on the following page : "map Plan-d'Aups-Sainte-Baume".
Hotels of Plan-d'Aups-Sainte-Baume are listed on the map with the following icons:
The map of Plan-d'Aups-Sainte-Baume is centred in the following coordinates :
Latitude : Longitude :
Print the map of Plan-d'Aups-Sainte-Baume
Print the map of Plan-d'Aups-Sainte-Baume : map of Plan-d'Aups-Sainte-Baume
Photos Plan-d'Aups-Sainte-Baume
Most popular and closest pictures of Plan-d'Aups-Sainte-Baume town hall :
Le Castellet-M.J. 200 1977 (1)
Sep '20: OK Corral
Photos are copyrighted by their owners.
See all photos of Plan-d'Aups-Sainte-Baume :
Other photos of Plan-d'Aups-Sainte-Baume and nearby towns can be found here: photos Plan-d'Aups-Sainte-Baume
Plan-d'Aups-Sainte-Baume weather forecast
Find next hours and 7 days weather forecast for Plan-d'Aups-Sainte-Baume here : weather Plan-d'Aups-Sainte-Baume (with english metrics)
This is the last weather forecast for Plan-d'Aups-Sainte-Baume collected by the nearest observation station of Brignoles.
Week Forecast: No precipitation throughout the week.
The latest weather data for Plan-d'Aups-Sainte-Baume were collected Monday, 17 January 2022 at 17:27 from the nearest observation station of Brignoles.
15°C -2°C
0% 5%
Clear throughout the day.
Temperature Max: 15°C Temperature Min: -2°C
Pressure: 1027.4 mbars
Percentage of sky occluded by clouds: 5%
Wind speed: 2 km/h South-East
Wind Gust Speed: 3 km/h
Visibility: 16.093 km Humidity: 55%
Dew Point: -6° UV Index: 2
Sunrise: 08:05 Sunset: 17:28
Precipitation Probability: 5% rain
Percentage of sky occluded by clouds: 23%
Wind speed: 2 km/h Sout-East
Dew Point: 1° UV Index: 2
Partly cloudy throughout the day.
Temperature Max: 13°C Temperature Min: 3°C
Wind Gust Speed: 13 km/h
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