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An apparatus and method are provided for shipping, storing, and high-throughput processing of microarrays. An exemplary microarray cartridge according to the present invention includes a body having a cavity defined by an outer surface and two dimple features in fluid communication with the cavity. The cavity includes at least one ledge for supporting a microarray of biological probes, and a reaction chamber defined at least in part by the ledge. A plate covers the cavity and sealingly attaches to the outer surface of the body. Ports in the plate or the dimple features allow introduction of sample, reaction and wash solutions into the reaction chamber such that the solutions contacts the probes on the microarray. The disposable microarray cartridges of the present invention are used to package and store microarrays prior to use and to process microarrays in a high-throughput manner. The footprint of the cartridge is designed to be compatible with standard robotic formats or standard re-formatting approaches.
1. A microarray cartridge, comprising:
a body having a wall forming a cavity surrounded by a mating surface, said body comprising a reaction chamber and at least one microarray support contained within the cavity, said at least one microarray support supporting a microarray slide within the cavity such that a surface of the slide covers the reaction chamber; and
a cover that (a) is covering the cavity such that said slide is disposed between said cover and the reaction chamber, and (b) is sealingly adhered with the mating surface of said body by non-removable adhering means on all but one edge of the mating surface, said all but one edge being unsealed to said cover.
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This application claims benefit of U.S. provisional patent application Ser. No. 60/296,495, filed Jun. 6, 2001, which is incorporated by reference herein in its entirety.
The present invention relates generally to the processing of microarrays, and particularly to a method and apparatus for storing, shipping and high-throughput processing of microarrays.
Microarrays for high-throughput analysis of biological compounds are known in the biotechnology field. Microarrays generally consist of a substrate, such as a slide or chip made of glass, plastic or silicon, upon which is attached an array of biological probes representing discrete binding or reaction sites for target biological compounds. Various types of probes are known, including nucleic acids, proteins, ligands, antibodies or other cellular proteins. For example, a typical DNA microarray includes an array, or matrix, of DNA probes representing discrete binding sites for at least some of the genes or gene products (e.g., cDNAs, mRNAs, cRNAs, polypeptides, and fragments thereof) in an organism's genome.
Microarrays have various applications, depending upon the probes used and the target compounds to be analyzed. For example, DNA microarrays have been used to study gene expression profiles, such as analyzing the transcriptional state of a cell exposed to graded levels of a drug of interest. Protein microarrays, including proteome microarrays representing most or at least some encoded proteins for a particular organism, may be used to study a wide variety of biochemical activities, such as protein-protein interactions, protein-lipid interactions, identifying the substrates of protein kinases, or identifying the protein targets of small molecules (MacBeath and Schreiber, 2000, Science 289:1760-1763; Zhu et al., 2001, Science 293:2101-2105). Similarly, a microarray having an ordered array or matrix of antibodies may be used for high-throughput screening of antibody-antigen interactions (de Wildt et al., 2000, Nature Biotechnology 18:989-994).
While microarrays are generally known and used in a wide variety of applications, available apparatus and systems for storing and processing microarrays have several shortcomings. For example, microarrays are typically placed in conventional microscope slide boxes or similar containers, which may not provide adequate protection during shipping and storage. Moreover, there is a potential to damage the microarray substrate or disrupt the array of biological probes when placing microarrays into, or removing them from, such a container.
During high-throughput processing of biological samples using microarrays, a common bottleneck is the selective binding or other reaction step between biological probes on a microarray and target compounds in the biological sample of interest. In the case of nucleic acid microarrays, for example, the selective binding step is often called the "hybridization" step, and is the process by which cDNA, cRNA or other nucleic acids in a sample bind selectively (i.e., hybridize) to complementary nucleic acid probe sequences (e.g., DNA or RNA) on the substrate. Preferable experimental protocols are best implemented if microarrays can be processed in pairs (sample vs. control or reference) or groups. Prior to the present invention, however, no suitable microarray processing apparatus or methods were available to handle microarray hybridization or other reactions in pairs or groups (analogous to 96-well plates, microcentrifuge tubes, or multi-well strips) for high-throughput sample processing.
In addition to their inability to process groups of samples, previously disclosed microarray processing systems have several other disadvantages. For example, many of the available hybridization devices utilize mechanical sealing mechanisms and fasteners, such as removable screws, clips, latches, screw caps and gaskets. Such devices are not desirable or practical for high-throughput processing of microarrays as they are generally closed systems, relatively complicated to use, expensive to manufacture, and/or they are not configurable with standard format footprints. Alternatively, systems employing plastic bags are relatively simple and inexpensive, however they do not provide sufficient rigidity for use with liquid dispensing robots. Other relatively simple systems utilize coverslip-type hybridization chambers placed on a microarray slide, however such systems have insufficient hybridization solution volumes and do not provide protection for microarray slides during processing or storage. Thus, there exists a need for efficient, cost-effective apparatus and methods for shipping, storing, and high-throughput processing of all types of microarrays.
Discussion or citation of a reference herein shall not be construed as an admission that such reference is prior art to the present invention.
An apparatus and method are provided for shipping, storing, and high-throughput processing of microarrays. In particular, the present invention is directed to a dual purpose packaging/reaction cartridge for performing high-throughput hybridization of microarrays and associated processes. An exemplary microarray cartridge according to the present invention includes a body having a reaction chamber within a central cavity. The central cavity is adapted to hold a microarray slide and includes at least one ledge or other feature that supports the microarray and defines, at least in part, the volume of a reaction chamber. The geometry of the ledge or other support also aids in minimizing sample capture behind the microarray substrate. The body optionally includes one or more dimple features in fluid communication with the cavity for adding and/or removing fluids. The dimples preferably form a passage for fluid to pass around one or more edges of the microarray, and also facilitate mixing of fluids within the chamber. A plate covers the cavity and sealingly adheres to the outer surface of the body by a heat seal or other non-removable sealing means, without the aid of screws clips, clams, latches, gaskets, or other mechanical fasteners. Optional access sites, such as ports or pierceable walls, in the plate or the dimple features facilitate introduction of sample and hybridization solutions into the reaction chamber such that the solutions contact the probes on the microarray.
The disposable microarray cartridges of the present invention are used to package and store microarrays prior to use and to process microarrays in an efficient high-throughput manner. The cartridge is configured and dimensioned to be compatible with standard robotic formats or standard re-formatting approaches. The size, shape, and construction of the chamber portion of the microarray cartridge is designed to be inexpensive to manufacture and easy to use, and to provide optimal conditions for reactions between biological probes on a microarray and molecules in a biological sample. For example, the cartridges of the present invention provide for optimal sample volumes, mixing during sample exposure, and post-reaction washing in a high-throughput mode.
A preferred embodiment of the invention includes a multi-microarray cartridge having reaction chambers for a plurality of microarrays. The multi-microarray cartridge is designed to be compatible with a high-throughput robotic system with multi-channel liquid dispensing capability for injecting sample solutions, including, for example, a biological sample in a solution with one or more reagents, into the individual microarray cartridges. Exemplary robotic systems include at least four, preferably eight or more, channels. Dimples, ports, or other features can be incorporated into the cartridge design to provide suitable tolerance clearances and zones of rigidity for pipette tips or injection needles. With appropriate fixturing, samples can be transferred from 8�12 (96 well) plates or 8-strip wells directly into the multi-microarray cartridge.
An advantage of the microarray cartridges of the present invention is that microarrays can be placed directly into the cartridge by a manufacturer for shipping or they can be used to group and store microarrays in inventory for specific projects. Cartridge and microarray are ready for use with no additional handling of microarray required, thus limiting possible damage to the microarray. By using a semi-rigid design, the cartridges of the present invention are robust enough to interface with standard robotic pipetting and, as open platforms, allow for reconfigurable reaction, washing and scanning protocols. At the same time, the semi-rigid cartridges of the present invention are designed to have relatively thin walls and a minimal number of parts, thereby optimizing cost, ease-of-use, and performance characteristics (such as fluid mixing) within the reaction chamber. The relatively thin-walled construction of the disposable cartridges also allows access to the reaction chamber by piercing or cutting through the cartridge.
For a better understanding of the nature and details of the invention, reference should be made to the following detailed description taken in conjunction with the accompanying drawings, in which:
Like reference numerals refer to corresponding parts throughout the several views of the drawings.
This section presents a detailed description of the apparatus of the present invention and methods of their use. In particular, Section 5.1 describes various exemplary embodiments of the microarray cartridges of the present invention, including a single microarray cartridge, a multi-microarray cartridge, and a cartridge designed to enhance fluid motion over a microarray. Section 5.2 describes microarrays generally and their methods of preparation. Section 5.3 generally describes high-throughput methods of processing microarrays using the apparatus of the present invention.
Referring to
Cavity 24 includes a reaction chamber 30, that is bordered, at least in part, by ledges 26, 28 and dimples 32, 34. Microarray 40 is removably held within cavity 24 by ledges 26, 28 with the probe side of the microarray facing reaction chamber 30. The volume of reaction chamber 30 is defined, at least in part, by the geometry of ledges 26, 28 and the dimensions of cavity 24.
Body 20 optionally includes one or more dimple features 32, 34. Each dimple feature 32, 34 forms a passage for communication of fluid around slide 42, through ledges 26, 28 and into reaction chamber 30. Such dimple features may be used, for example, to introduce a sample fluid onto the probe side of microarray 40 or to wash the probes by flowing a buffer or other wash solution over microarray 40.
Cover 50 comprises a flat plate that mates with an outer portion, or mating surface, 22 of body 20. Cover 50 optionally includes access sites, such as ports 52 and 54, that correspond and co-locate with dimples, 32 and 34, respectively. Ports 52, 54 facilitate proper alignment of body 20 and cover 50 before the two are joined by a water-tight seal. Ports 52 and 54 allow introduction of sample solution, buffer, and/or other fluids into reaction chamber 30 via dimple features 32, 34. Ports 52, 54 are shown as open ports that allow introduction of fluids from robotically controlled pipettes. Alternatively, ports 52, 54 may be covered with a septum compatible with a needle for injecting solutions into ports 52, 54, where the septum re-seals after a needle is withdrawn. In a preferred embodiment of the invention, cover 50 does not include ports 52, 54; rather, samples are introduced through dimple features 32, 34, through an un-sealed edge between body 20 and cover 50, or by piercing body 20. Any such site for introducing samples is herein referred to as an "access site". After introduction of samples, the access site (e.g., an access port, an unsealed edge, or a pierced body or cover) may be sealed using adhesive, heat sealing, or the like. Cover 50 is preferably transparent, allowing bar code 44 to be scanned while microarray slide 40 is sealed in cartridge 10.
Body 20 and cover 50 are preferably sealingly adhered by non-removable adhering means, preferably a heat seal or adhesive, about the mating surface 22 of the body. As used herein, the term "non-removable adhering means" includes, but is not limited to, heat seals, adhesives, ultrasonic bonding, pressure stamping, and combinations thereof. Body 20 and cover 50 are joined together by any of such non-removable adhering means to create an adhering seal about mating surface 22 without the aid of screws, clips, clamps, latches, removable gaskets, or other mechanical fasteners or complex sealing mechanisms. Such mechanical fasteners and/or sealing mechanisms are not desirable for use with inexpensive, disposable, high-throughput systems such as the microarray cartridges of the present invention.
Components of cartridges of the present invention can be constructed from a variety of materials that are compatible with biological sample solutions. Suitable materials include, but are not limited to, polypropylenes, polycarbonates, polyethylenes, polystyrenes, polyvinylchlorides, polyacrylics, polyesters, ABS, acetal, polyether ether keytones (PEEK), polyamides, fluoro polymers, polyethylene terephthalate (PET), polyethylene terephthalate glycol (PETG), styrene acrylonitriles, and co-polymers of the above. Methods for manufacturing the cartridges include thermoforming (e.g., pressure forming or vacuum forming), injection molding, stamping, and machining, among others.
In a preferred embodiment of the present invention, cartridge 10 is a relatively thin, semi-rigid, thermoformed structure. The cartridge is preferably thermoformed from a single sheet of polymer material, such as polypropylene, a polyethylene or a polyvinylchloride. Using known thermoforming process, cartridge 10 can be made to have wall thicknesses between 0.003 and 0.065 inches, preferably between 0.005 and 0.025 inches, and most preferably between 0.010 and 0.015 inches. The resulting cartridge is rigid enough to withstand microarray packaging, handling, and sample processing by robotic instruments without damaging microarray 40. At the same time, the relatively thin-walled construction has several advantages. For example, the materials are low cost, easy to form, and provide crisp and reliable definition of features and dimensions such as reaction chamber 30 volume, microarray supports 26, 28, dimple features 32, 34 and flange features 56, 58.
The relatively thin nature of the walls of cartridge 10 may also help optimize mixing and flow characteristics within the reaction chamber 30 during hybridization or other reactions. Still another benefit of the semi-rigid and relatively thin walls of the cartridge of the present invention is that they may be easily punctured by a needle or other sharp object to allow introduction of the sample solution, or for washing or rinsing of the microarray inside the chamber. In such applications, dimple features 32, 34 provide additional zones of rigidity whereby a needle or similar device can pierce the wall of body 20 or cover 50 without collapsing any part of cartridge 10 against the microarray 40. The thin-walled, semi-rigid construction of the cartridges according to the present invention also allows easy formation of an adhering seal, such as a heat seal or compression seal, between mating surface 22 of body 20 and cover 50 without the aid of gaskets, screws, clips, clamps, latches, or other mechanical fasteners. Still another advantage of the relatively thin, semi-flexible walls of the cartridges of the present invention is that they deflect to allow entry of a pipette or other delivery device between an unsealed edge of cartridge, for example in embodiments having an opening between body 20 and cover 50 at flanges 56, 58.
In another embodiment, cartridge 10 of the present invention is formed by injection molding to produce wall thicknesses of 0.016 to 0.100 inches. Injection molding is generally known in the art. A preferred wall thickness range for an injection molded cartridge is between 0.032 and 0.075 inches, more preferably between 0.040 and 0.060 inches. In such embodiments, potential access sites for adding or removing fluids to chamber 30 include open ports (e.g., ports 52, 54) on cover 50 or body 20, an unsealed end of cartridge 10 (e.g. between flange features 56, 58) or through a relatively thin puncture site, for example a puncture site having a thickness of less than about 0.010 inch, preferably about 0.003 inch (not shown). Similarly, in injection molded (or machined) embodiments employing heat seals to adhere body 20 and cover 50, cartridge material thicknesses in the heat sealed area are preferably between 0.005 and 0.040 inches.
Referring to
Snap features 62, 64 are preferably located along an edge or corner opposite hinge feature 60; however, the type, number and location of snap features 62, 64 may vary. The mating ends of body 20 and cover 50 each optionally include a flange 56, 58, respectively, that form a fluted opening when cover 50 is closed (e.g. see
Referring to
One skilled in the art will appreciate that the volume capacity of chamber 30 of cartridge 10 is distinct from commercially available hybridization or reaction chambers, which are designed for small volumes (generally about 40 μL to 400 μL). Indeed, most of the commercial chambers are more accurately described as humidity control devices as they are really designed to prevent excessive evaporation of the small volume of biological sample solution that is pipetted under a microscope coverslip which covers the microarray probe area. In such systems, the sample solution is held in contact with the microarray throughout the hybridization or other reaction step by capillary action.
Comparing the schematic cross-sectional view of cartridge 10 in
Referring again to
As shown in
Aside from the advantage of using robots for high throughput injection of solutions, the multi-microarray cartridge 400 of
While multi-microarray cartridge 400 of
Referring to
While one skilled in the art will appreciate that various types of microarrays may be used in the cartridges of the present invention, including but not limited to nucleic acid microarrays, proteome microarrays (Zhu et al., 2001, Science 293:2101-2105), other protein microarrays (MacBeath and Schreiber, 2000, Science 289:1760-1763) and antibody microar-rays (de Wildt et al., 2000, Nature Biotechnology 18:989-994), the following description of the general characteristics and preparation of microarrays utilizes nucleic acid microarrays as a primary example. For a more detailed description of nucleic acid microarrays, see U.S. Pat. No. 6,203,987 and International Patent Publications WO 99/66024, published Dec. 23, 1999 and WO 01/05935, published Jan. 25, 2001.
A nucleic acid microarray typically comprises a surface with an ordered array of reaction (e.g., hybridization) sites for products of many of the genes in the genome of a cell or organism, preferably most or almost all of the genes. The surface is typically a solid support such as slide 42 of microarray 40, but may be any non-porous or porous substrate including, but not limited to, a glass or plastic surface or a nitrocellulose or nylon membrane or filter.
Microarrays can be made in a number of ways, of which several are described below. However produced, microarrays share certain characteristics: The arrays are reproducible, allowing multiple copies of a given array to be produced and easily compared with each other. Preferably, a given reaction site or unique set of reaction sites (i.e., probes) in the microarray will specifically bind or otherwise react to the presence or condition of a specific target compound in a biological sample. In the case of nucleic acid microarrays, for example, each probe in the array will preferably hybridize with the product of a single gene in a cell, for example a specific mRNA or a specific cDNA derived therefrom. However, in general other, related or similar sequences will cross hybridize to a given binding site. Although there may be more than one physical binding site per specific target compound or gene product, for the sake of clarity the discussion below will assume that there is a single, completely complementary binding site.
Nucleic acid microarrays typically include one or more test probes, each of which has a polynucleotide sequence that is complementary to a subsequence of RNA or DNA to be detected. Each probe preferably has a different nucleic acid sequence. The position of each probe on the solid surface is preferably known. In one embodiment, the microarray is a high density array, preferably having a density greater than about 60 different probes per 1 cm2. In one embodiment, the microarray is an array (i.e., a matrix) in which each position represents a discrete binding site for a product encoded by a gene (i.e., an MRNA or a CDNA derived therefrom), and in which binding sites are present for products of most or almost all of the genes in the organism's genome. For example, the binding site can be a DNA or DNA analogue to which a particular RNA can specifically hybridize. The DNA or DNA analogue can be, e.g., a synthetic oligomer, a full-length cDNA, a less-than full length cDNA, or a gene fragment.
Although in a preferred embodiment the nucleic acid microarray contains binding sites for products of all or almost all genes in the target organism's genome, such comprehensiveness is not necessarily required. Usually the microarray will have binding sites corresponding to at least about 50% of the genes in the genome, often to about 75%, more often to at least about 85%, even more often to about 90%, and still more often to at least about 99%. Preferably, the microarray has binding sites for genes relevant to the action of a drug of interest or in a biological pathway of interest. A "gene" is identified as an open reading frame ("ORF") which encodes a sequence of amino acids (e.g. 50, 75, 99 amino acids or more) from which a messenger RNA is transcribed in the organism or in some cell in a multicellular organism. The number of genes in a genome can be estimated from the number of different mRNAs expressed by the organism, or by extrapolation from a well characterized portion of the genome. When the genome of the organism of interest has been sequenced, the number of ORF's can be determined and MRNA coding regions identified by analysis of the DNA sequence. For example, the genome of Saccharomyces cerevisiae has been completely sequenced, and is reported to have approximately 6275 ORFs longer than 99 amino acids. Analysis of the ORFs indicates that there are 5885 ORFs that are likely to encode protein products (Goffeau et al., 1996, Science 274:546-567). In contrast, the human genome is estimated to contain approximately 30,000 to 40,000 genes (see Nature 409:814-816, 2001).
As noted above, the "probe" in a nucleic acid microarray to which a particular polynucleotide molecule specifically hybridizes according to the invention is usually a complementary polynucleotide sequence. In one embodiment, the probes of the microarray are DNA or DNA "mimics" (e.g., derivatives and analogues) corresponding to at least a portion of each gene in an organism's genome. In another embodiment, the probes of the microarray are complementary RNA or RNA mimics.
DNA mimics are polymers composed of subunits capable of specific, Watson-Crick-like hybridization with DNA, or of specific hybridization with RNA. The nucleic acids can be modified at the base moiety, at the sugar moiety, or at the phosphate backbone. Exemplary DNA mimics include, e.g., phosphorothioates.
DNA can be obtained, e.g., by polymerase chain reaction ("PCR") amplicafication of gene segments from genomic DNA, cDNA (e.g., by RT-PCR), or clones sequences. PCR primers are preferably chosen based on known sequences of the genes or cDNA that result in amplification of unique fragments (i.e., fragments that do not share more than 10 bases of contiguous identical sequence with any other fragment on the microarray). Computer programs that are well known in the art are useful in the design of primer with the required specificity and optimal amplification properties, such as Oligo version 5.0 (National Biosciences). Typically, each probe of the microarray will be between about 20 bases and about 12,000 bases, and usually between about 300 bases and about 2,000 bases in length, and still more usually between about 300 bases and about 800 bases in length. PCR methods are well known in the art, and are described, for example, in Inis et al., eds., 1990, PCR Protocols: A Guide to Methods and Applications, Academic Press Inc., San Diego, Calif. It will be apparent to one skilled in the art that controlled robotic systems are useful for isolating, purifying and amplifying nucleic acids.
An alternative means for generating the polynucleotide probes of the microarray is by synthesis of synthetic polynucleotides or oligonucleotides, e.g., using N-phosphonate or phosphoramidite chemistries (Froehler et al., 1986, Nucleic Acid Res. 14:5399-5407; McBride et al., 1983, Tetrahedron Lett. 24:246-248). Synthetic sequences are typically between about 15 and about 500 bases in length, more typically between about 20 and about 60 bases. In some embodiments, synthetic nucleic acids include non-natural bases, such as, but by no means limited to, inosine. As noted above, nucleic acid analogues may be used as binding sites for hybridization. An example of a suitable nucleic acid analogue is peptide nucleic acid (see, e.g., Eghohm et al., 1993, Nature 363:566-568; U.S. Pat. No. 5,539,083).
In alternative embodiments, the reaction sites (i.e., the probes) are made from plasmid or phage clones of genes, cDNAs (e.g., expressed sequence tags), or inserts therefrom (Nguyen et al., 1995, Genomics 29:207-209).
In other alternative embodiments, the probes are proteins attached covalently onto chemically derivatized glass or plastic slides, wherein the proteins retain their folded conformations and thus their ability to react with other proteins, substrates of protein kinases and/or other small molecules, as described in MacBeath and Schreiber, 2000, Science 289:1760-1763. Similarly, proteome microarrays (also called "proteome chips") having a large number of encoded proteins for a particular organism arrayed on the surface of a slide or chip may be used, for example, to screen for protein-drug interactions or to detect posttranslational modifications, as described in Zhu et al, 2001, Science 293:2101-2105. In still other alternative embodiments, the probes are antibodies attached in an array to a slide or chip, where each antibody probe selectively reacts with a particular antibody as described in de Wildt et al., 2000, Nature Biotechnology 18:989-994.
The probes are attached to a solid support or surface, which may be made, e.g., from glass, plastic (e.g., polypropylene, nylon), polyacrylamide, nitrocellulose, or other materials. A preferred method for attaching the probes to a surface is by printing on glass plates, as is described generally by Schena et al, 1995, Science 270:467-470. This method is especially useful for preparing microarrays of cDNA (See also, DeRisi et al., 1996, Nature Genetics 14:457-460; Shalon et aL, 1996, Genome Res. 6:689-645; and Schena et al., 1995, Proc. Natl. Acad. Sci. U.S.A. 93:10539-11286). Blanchard discloses the use of an ink jet printer for oligonucleotide synthesis using propylene carbonate as solvent (U.S. Pat. No. 6,028,189 and WO 98/41531, published Sep. 24, 1998).
A second preferred method for making microarrays is by making high-density oligonucleotide arrays. Techniques are known for producing arrays containing thousands of oligonucleotides complementary to defined sequences, at defined locations on a surface using photolithographic techniques for synthesis in situ (see, Fodor et al., 1991, Science 251:767-773; Pease et al., 1994, Proc. Natl. Acad. Sci. U.S.A. 91:5022-5026; Lockhart et al., 1996, Nature Biotechnology 14:1675; U.S. Pat. Nos. 5,578,832; 5,556,752; and 5,510,270) or other methods for rapid synthesis and deposition of defined oligonucleotides (Blanchard et al., Biosensors & Bioelectronics 11:687-690). When these methods are used, oligonucleotides (e.g., 20-mers) of known sequence are synthesized directly on a surface such as a derivatized glass slides. Usually, the array produced is redundant, with several oligonucleotide molecules per RNA. Oligonucleotide probes can be chosen to detect alternatively spliced mRNAs. In a specific embodiment, the oligonucleotides probes include 40-70 nucleotides, preferably 50-60 nucleotides (Hughes et al., Nat. Biotech. 19:342-347, 2001).
In the case of protein microarrays, for example, protein probes may be printed on glass or plastic slides using a high-precision contact-printing robot (e.g., GMS 417 Arrayer, Affymetrix, Santa Clara, Calif.) to deliver nanoliter volumes of hydrated protein samples to the slides. Depending upon the proteins used, it may be preferable to utilize treated slides such as aldehyde slides or BSA-N-hydroxysuccinimide slides (MacBeath and Schreiber, 2000, Science 289:1760-1763).
Other methods for making microarrays, e.g., by masking (Maskos and Southern, 1992, Nuc. Acids. Res. 20:1679-1684), may also be used. In principle, any type of array, for example, dot blots on a nylon hybridization membrane (see Sambrook et al., supra) could be used. However, as will be recognized by those skilled in the art, very small arrays will frequently be preferred because hybridization volumes will be smaller.
The polynucleotide molecules which may be analyzed by nucleic acid microarrays within the cartridges of the present invention (the "target polynucleotide molecules") may be from any source, but are expressed RNA or a nucleic acid derived therefrom (e.g., cDNA or amplified RNA derived from cDNA that incorporates an RNA polymerase promoter), including naturally occurring nucleic acid molecules, as well as synthetic nucleic acid molecules. In one embodiment, the target polynucleotide molecules comprise RNA, including, but by no means limited to, total cellular RNA, poly(A)+messenger RNA (MRNA) or fractions thereof, cytoplasmic mRNA, or RNA transcribed from cDNA (i.e., cRNA; see, e.g., Linsley & Schelter, U.S. patent application Ser. No. 09/411,074, filed Oct. 4, 1999, or U.S. Pat. Nos. 5,545,522, 5,891,636, or 5,716,785). Methods for preparing total and poly(A)+RNA are well known in the art, and are described generally, e.g., in Sambrook et al., supra. In one embodiment, RNA is extracted from cells of the various types of interest in this invention using guanidinium thiocyanate lysis followed by CsCl centrifugation (Chirgwin et al, 1979, Biochemistry 18:5294-5299). In another embodiment, total RNA is extracted using a silica gel-based column, commercially available examples of which include RNeasy (Qiagen, Valencia, Calif.) and StrataPrep (Stratagene, La Jolla, Calif.). In an alternative embodiment, which is preferred for S. cerevisiae, RNA is extracted from cells using phenol and chloroform, as described in Ausubel et al. (Ausubel et aL, eds., 1989, Current Protocols in Molecular Biology, Vol III, Green Publishing Associates, Inc., John Wiley & Sons, Inc., New York, at pp. 13.12.1-13.12.5). Poly(A)+ RNA can be selected, e.g., by selection with oligo-dT cellulose or, alternatively, by oligo-dT primed reverse transcription of total cellular RNA. In one embodiment, RNA can be fragmented by methods known in the art, e.g. by incubation with ZnCl2, to generate fragments of RNA. In another embodiment, the polynucleotide molecules analyzed by the invention comprise cDNA, or PCR products of amplified RNA or cDNA.
In a preferred embodiment, total RNA, MRNA, or nucleic acids derived therefrom, from a wide number of diverse types of cells of the genome of interest are contacted with the genomic microarrays of the invention, so that all, or substantially all exons in a given cell's genome are detected, since different cell types express different levels of RNAs. Cells of interest include, but are by no means limited to, wild-type cells, mutant cells, drug-exposed wild-type cells, drug-exposed mutant cells, primary cells, cell lines, cells of different tissues or developmental stages, modified cells, diseased cells and cancer cells. Target polynucleotide molecules that are poorly expressed in particular cells may be enriched using normalization techniques (Bonaldoi et al., 1996, Genome Res. 6:791-806).
As described above, the target polynucleotides are detectably labeled at one or more nucleotides. Any method known in the art may be used to detectably label the target polynucleotides. Preferably, this labeling incorporates the label approximately uniformly along the length of the RNA, and more preferably, the labeling is carried out at a controlled, reproducible efficiency. One embodiment for this labeling uses oligo-dT primed reverse transcription to incorporate the label; however, conventional methods of this method are biased toward generating 3′ end fragments. Thus, in a preferred embodiment, random primers (e.g., 9-mers) are used in reverse transcription to uniformly incorporate labeled nucleotides over the full length of the target polynucleotides. Alternatively, random primers may be used in conjunction with PCR methods or T7 promoter-based in vitro transcription methods in order to amplify the target polynucleotides.
In a preferred embodiment, the detectable label is a luminescent label. For example, fluorescent labels, bio-luminescent labels, chemi-luminescent labels, and colorimetric labels may be used in the present invention. In a highly preferred embodiment, the label is a fluorescent label, such as a fluorescein, a phosphor, a rhodamine, or a polymethine dye derivative. Examples of commercially available fluorescent labels include, for example, fluorescent phosphoramidites such as FluorePrime (Amersham Pharmacia, Piscataway, N.J.), Fluoredite (Millipore, Bedford, Mass.), FAM (ABI, Foster City, Calif.), and Cy3 or Cy5 (Amersham Pharmacia, Piscataway, N.J.). In another embodiment, the detectable label is a radiolabeled nucleotide.
In a further preferred embodiment, target polynucleotide molecules from two or more different sources are distinguishably labeled, such as with different color labels. In a highly preferred embodiment, these different sources are chosen so as to maximize the differential expression of genes of interest. The skilled artisan will appreciate that differentially expressed exons are easier to detect in this manner. For example, target polynucleotide molecules can be isolated from diverse tissues or growth conditions and distinguishably labeled with different color labels for each source of molecules. In some embodiments of the invention, at least 2, 5, 10, 20, or 100 dyes of different colors can be used for labeling.
In one embodiment, such diverse conditions can be determined using a large collection of known genes and examining the correlation of their expression in pairwise combinations of experimental conditions. For example, the correlation data may be based on expression data from at least 2, 5, 10, 100, 500, 2,000, 10,000, or 50,000 different conditions. Conditions of interest include, but are by no means limited to, wild-type cells, mutant cells, drug-exposed wild-type cells, drug-exposed mutant cells, primary cells, cell lines, cells of different tissues or developmental stages, modified cells, diseased cells and cancer cells. The correlation data are used to assemble clusters of related conditions (Ross et al., Nature Genet. 24, 227-35, 2000; Friend et al., International Patent Publication WO 00/24936, published May 4, 2000) and the most informative members of each cluster are used in subsequent steps. Informative conditions may be picked via the intensity of the observed profile of the experimental condition compared to the profile of a reference condition in a two color assay with the expressed nucleic acids from the reference condition labeled with a first fluorescent dye, and the expressed nucleic acids from the experimental condition labeled with a second, distinguishable fluorescent dye. Alternatively, a single color assay can be performed using the appropriate normalization controls, and the ratios of all possible combinations can be generated using a computer.
The cartridges of the present invention may be used in a variety of ways, including, but not limited to, packaging, shipping, storing, and high-throughput processing of microarrays. Examples of methods of using the microarray cartridges of the present invention follow. In many cases, the exemplary methods of use are described in the context of processing nucleic acid microarrays; however one skilled in the art will appreciate that the cartridges of the present invention are suitable for packaging, shipping, storing, and high-throughput processing of any type of microarray, including, but not limited to, those having proteins, ligands, antibodies, adhesion molecules, cellular receptors or other cell fragments or molecules as probes attached to a slide or other suitable substrate.
As stated previously, conventional microarrays are typically placed in microscope slide boxes for shipping and storage, often resulting in damaged or lost microarrays. Thus, an advantage of the cartridges of the present invention is that they can also serve as a shipping container and storage container, with the further advantage that the package is ready for use with automated liquid dispensing robots straight from the manufacturer without costly and time consuning customization. In a preferred method of the present invention, microarrays are placed in cartridges and secured and/or sealed in cartridges during storage. For example, referring to
Examples of methods of using the cartridges of the present invention are detailed in the process flows 600, 800 and 900 of
Referring to
In step 618, the microarrays are prepared as described above in section 5.2 above. Nucleic acid microarrays containing double-stranded probe DNA situated thereon (e.g., 'spotter arrays') are preferably subjected to denaturing conditions to render the DNA single-stranded prior to contacting with the target polynucleotide molecules. Arrays containing single-stranded probe DNA (e.g., synthetic oligodeoxyribonucleic acids) may need to be denatured prior to contacting with the target polynucleotide molecules, e.g., to remove hairpins or dimers which form due to self complementary sequences. Also in step 618, the microarrays are placed in the microarray cartridge (e.g. cartridge 10 of
In step 622, the processed sample solution is added to the microarray in the reaction chamber 30, for example using a robotically controlled pipette or other delivery device inserted through an open edge of cartridge 10 of
After adding the processed sample, any unsealed edges, ports or holes of the microarray cartridge are sealed in step 626 to prevent leakage of the processed sample solution during incubation. For example, unsealed edges are sealed by heat sealing or adhesive. If samples were added through ports or by piercing the cartridge, any holes are sealed with adhesive, a plug, a heat sealant, etc., preferably by the robotic processing system. For example, the robotic system could be configured with a gasket and/or other sealing device that seals the entry port automatically after the delivery device (e.g., pipette or injection tip) is withdrawn from the cartridge. Alternatively, the ports may consist of resealable elastomeric septums through which the sample solution is injected.
After adding processed sample solution to the microarray and sealing the cartridge, one or more cartridges are placed in an oven in step 630. Preferably, a number of cartridges are placed in a mixing device, such as a rotating wheel or a rocker platform, and the entire device is placed inside an oven. For example,
Step 634 entails incubating the arrays within the cartridges inside the oven at a particular temperature while mixing and flowing the processed sample over the array. For example, wheel 700 is rotated about its central axis 730 at a set rate, e.g. 20 rpm, to provide continuous mixing and flow of the processed samples over the arrays. Frequency of rotation or rocking may be modified to optimize reaction conditions, and obstacles (e.g. obstacles 560 of
Reactions are typically performed in the range of 40� C. to 65� C., depending upon the nature of the microarray probes. An exemplary embodiment uses 60mer oligonucleotides probes selected using the oligo design principles articulated in Hughes et aL, 2001, Nat. Bio. 19:342-347. Using such 60 mer microarrays, for example, the hybridization reaction is performed at 40� C. for 24 hours.
In order to achieve high quality reaction data, it is advantageous to incubate microarray reactions in a fashion that causes the biological sample solution to be continuously mixed as it flows over the microarray probe surface as describe above. Reaction chamber volume in the cartridges of the present invention are dimensioned to provide optimal flow and mixing for a given sample solution volume using the principles taught herein. In addition, quality and uniformity of reactions over the entire microarray probe matrix may be improved by constructing the cartridge of material that flexes as the sample solution washes from side to side in the chamber as the cartridge is rotated or rocked in the oven. Minimizing the volume of solution that gets trapped behind the array also tends to lead to better reactions. Thus, the supports (e.g. ledges 26, 28 of
To achieve higher microarray throughput, the present invention contemplates stacking cartridge packs on a fixture, such as a rocker platform or a rotating wheel, and either rocking or rotating the whole fixture in an oven. In this manner, at least 64 to 128 samples may be analyzed in a single oven, as compared with a maximum of 96 per oven using a conventional hybridization bag method. Alternative approaches include custom heat strips or trays with temperature controls, dipping cartridges into temperature-controlled water baths, or setting the cartridges in a temperature-controlled air stream.
After incubating the arrays in step 634, the cartridges are removed from the oven in step 638. The sample is then drained from each cartridge in step 642 and each array is removed from its reaction chamber in step 646. Draining the sample may be performed by piercing the reaction chamber, e.g. at a dimple feature, or by simple cutting off an edge of the chamber to expose the array.
In step 650, microarrays are washed with buffers as a final processing step prior to scanning in step 654. Washing removes background signal and provides a final level of stringency for quality data. In process 600, washing takes place with microarrays removed from cartridges, e.g. by washing buffers over the microarrays (either by bath dipping or in flow streams). Because the microarrays of the present invention preferably have hydrophobic surfaces outside the array dots, water does not adhere well to the surface, a drying step is not typically needed. However, if desired, an air knife or flow could be developed to dry the microarrays in batch. For example, the arrays may be dried using nitrogen or a centrifuge to spin-dry. There is also the potential to scan the microarrays in the cartridge without washing, if a confocal microscope can reduce background sufficiently. In step 654 the processed microarrays are placed into a suitable microarray scanning instrument and scanned as mentioned above and as detailed in U.S. Pat. No. 6,225,645.
Referring to
Referring to
In general, nucleic acid hybridization and wash conditions are chosen so that the polynucleotide molecules to be analyzed by the invention "specifically bind" or "specifically hybridize" to the complementary polynucleotide sequences of the array, preferably to a specific array site, wherein its complementary DNA is located.
Optimal hybridization conditions will depend on the length (e.g., oligomer versus polynucleotide greater than 200 bases) and type (e.g., RNA or DNA) of probe and target nucleic acids. General parameters for specific (i.e., stringent) hybridization conditions are described in Sambrook et al. (supra), and in Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York. When the cDNA microarrays of Schena et al. are used, typical hybridization conditions are hybridization in 5�SSC plus 0.2% SDS at 65� C. for four hours, followed by washes at 25� C. in high stringency wash buffer (0.1�SSC plus 0.2% SDS) (Shena et al., 1996, Proc. Natl. Acad. Sci. U.S.A. 93:10614). Useful hybridization conditions are also provided, e.g., Tijessen, 1993, Hybridization With Nucleic Acid Probes, Elsevier Science Publishers B.V.; and Kricka, 1992, Nonisotopic DNA Probe Techniques, Academic Press, San Diego, Calif.
While the exemplary methods of the invention are described in the context of hybridizing nucleic acid microarray probes with nucleic acid samples, one skilled in the art will appreciate that the apparatus and methods described herein encompass the use of other probe-sample combinations, including but not limited to, protein-protein, protein-ligand, enzyme-substrate, antibody-antigen, and other combinations.
All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
While the invention is susceptible to various modifications and alternative forms, specific examples thereof have been shown in the drawings and are herein described in detail. It should be understood, however, that the invention is not to be limited to the particular forms or methods disclosed, but rather the invention is to cover all modifications, equivalents and alternatives falling within the spirit and scope of the appended claims.
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Re "Should every officer get mental illness training?" (The Conversation, June 29): Police step in when one condition has been met: the mental health system has failed. California's mental health system only serves those well enough to recognize their need for treatment. They turn the others over to police, shelters, prisons and morgues.
About 90 percent of those in California state psychiatric hospitals got there through criminal justice. California has more people incarcerated for mental illness than hospitalized. Proposition 63 made California's mental health system very well-funded.
However, until mental health directors and programs are trained, indeed required, to use those funds to provide real treatment for the really sick, the police will continue to run their shadow mental health system.
-- DJ Jaffe, New York City

subset: commoncrawl


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Growing up in Boston, the MBTA Green Line was a lifeline to many activities in the city. My favorite green line station is KENMORE or Kenmore Square - home of Fenway Park and the Boston Red Sox.
H/t Leah Harnack....Read more
Growing up in Boston, the MBTA Green Line was a lifeline to many activities in the city. My favorite green line station is KENMORE or Kenmore Square - home of Fenway Park and the Boston Red Sox.
H/t Leah Harnack....Read more
Seems a trolley breathed its last around 8:30 a.m. near Park Street and gummed everything up. Caitlin Drown reports:
It's taken almost 20 mins going from Hynes to just past Arlington.Read more
Shortly before noon at Arlington, now riders between Kenmore and Haymarket get to enjoy above-ground scenery in shuttle buses.Read more
Paul Levy discusses Green Line trolleys that get delayed at stations because they have a little rubber thing at the bottom of doors that catches on the nubbly yellow warning strips on platforms, forcing the drivers to get out and whack the doors - and how complaining about the problem doesn't seem to get anything done.
My friend and I both reported problems with...Read more
Just got this e-mail from Keolis
Service Recovery Update Effective March 23, 2015...Read more
Starting Monday, March 23rd, we will restore service on an additional 63 MBTA Commuter Rail trains, including 17 rush hour train trips, with full service scheduled to be restored on or before March 30. This marks the largest number of commuter trains restored to service since the recovery schedule was
Getting around the Seaport District by car can be a royal pain, especially during the evening commute. According to the Boston Herald, the Massachusetts Convention Center Authority (MCCA) thinks it has the solution. It wants to get into the shuttle bus business.
Many of the area's businesses already offer shuttle bus services for their employees. Fidelity (through Boston Coach) and Vertex, for example,...Read more
Last May, the MBTA released a video showing a Transit Police detective rescuing a man from jumping in front of a train at Park Street. WCVB reports the man now has a lawyer - who had to sue to get the complete video, which shows the two struggled on the platform and the detective punching the man to subdue him....Read more
Transit Police report arresting Abdulkadir Abbas, 33, for allegedly punching out a window on an Orange Line train at Ruggles last night.
Police say Abbas punched the window around 10:15 p.m., then got on the train - where passengers promptly pointed him out to arriving officers.
Abbas is scheduled for arraignment today in Roxbury District Court on a charge of malicious destruction of property...Read more
Although the T hasn't told us one way or another, fans of the 1960's rec-room look probably have to resign themselves to losing the faux wood on the Orange Line trains once the new cars get here in, well, whenever.
Patrick Baxter gives us a taste of an Orange Line car without the rich, dark wood, thanks to an ad campaign that involves wrapping...Read more
There are some things you just shouldn't be seeing first thing in the morning at South Station, and Serena Tang reports this group of black-clad symbolic cats is one of them. And she reports they could sense her get-away-from-me-ness:
They started meowing at me.Read more
The MBTA reports that with the addition of some Green Line cars this morning, all four subway lines are now back at their normal car numbers for the first time since Jan. 22. The T reports that Green Line maintenance crews "have been working around the clock to restore service to pre-blizzard levels."
Commuter rail? Well, that's another matter.
MassDOT reports it's started accepting delivery of the first of 86 Type 7 trolleys being rebuilt in a factory in upstate New York.
The cars, which went into service in 1986 and 1987, were nearing the end of their intended lifespans. Unlike with Orange and Red Line cars that are now well past theirs, the state was able to find a company willing to rehab...Read more
After seeing Alex Wheeler's photo of Green Line parking skill, the judges have closed this week's round.Read more
Update, Wednesday, 6:20 a.m. The MBTA reports the D Line is back in service.
Grace Holley reports tonight from Brookline Hills, where a loud explosion shortly after 8:30 p.m. was followed by a torrential waterfall from a newly burst water main right above the tracks.
Normally I love water rides but this is a nightmare.
A second Green Line train approached waterfall but went...Read more
A Braintree man admitted today he walked across the tracks from one platform to the other at Andrew Square station on Sunday - narrowly avoiding getting flattened by a train entering the station at the same time.
In exchange, a South Boston Municipal Court judge continued Patrik Finnerty's trespassing case for three months, during which he has to perform 25 hours of community service...Read more
UPDATE, 3:15 p.m., Tuesday: Finnerty found, shows up in court, has case continued without a finding pending three-month period.
UPDATE: Warrant issued for his arrest after he failed to show up at his arraignment, the Suffolk County District Attorney's office reports.
Transit Police report arresting a Braintree lad who, on finding himself on the inbound platform at Andrew last night, allegedly thought the...Read more
The Boston Globe reported today that in exchange for an $8M contribution to build the West Station commuter rail station adjacent to its campus, Boston University sought "enforceable language prohibiting the use of BU's West Campus as a route for buses or other vehicular traffic to carry commuters to or from West Station."
If a new commuter rail station is going to be built...Read more
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Cheap ARC to Anchorage flights
Popular Flights from Arctic Village, Alaska
Top Arctic Village Flights to US Destinations
Popular Flights from Anchorage, Alaska
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Arctic Village to Anchorage Flights, Stats and Airplanes
Route information for flights from Arctic Village AK to Ted Stevens Anchorage Intl..
496.69 miles
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Browse the most popular airlines servicing the Anchorage (ANC) airport. Visit their flights pages to learn more about the most popular routes these carriers fly.


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a new commercial space for rent, located near, snr, walter mart, munoz market.. ideal for office, clinic, laundry shop, internet shop, salon business etc. just contact me for more details, 09228212593, 925-15-24
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You can text, or tweet, or just put on this T-shirt - just thinking of God's amazing love for us should bring a smile to your face, and this design is the perfect way to share that! For God so loved the world that he gave his one and only Son, that whoever believes in him shall not perish but have eternal life.--John 3:16
If you want to:
6,000 + (and growing) factories in our existing network are eager to make it for you.
When it comes to selling in stock products, we are no difference from many other Internet retailers, but we take pride in going extra miles to do something special for you: Have some products custom made for you.
We cannot do everything, but we usually can accommodate a certain changes in product options such as color, size, material, or minor design twist. We primarily work with vendors or factories, not individual artist or craftsman. For custom made products, we usually only cater to bulk size order that meets the minimum quantity requirement of a single factory run. Don't be shy. Ask. What we can accomplish may surprise you!
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March 24th, 2014 11:37 AM #1Member
- Join Date
- Oct 2013
- Posts
- 96
What about the name Evan for a girl's MN?
I love my grandma so much. Her maiden name is Evans. We have plans for boy names already that honor my grandpa & my husband's dad.
Tell me what you think about these names...
Lila Evan
Lily Evan
Nora Evan
I think Evalyn is pretty, but I really love short names. I think Evan is different & I like the way it sounds with girly first names.Presley, Lily, Evan, Mae, Nora, Evalyn, Evie, Olive, Jewel, Eloise
Lennox, Lincoln, Evan, Finn, Jude, Quillan, Jonah, Noah, Owen
March 24th, 2014 12:27 PM #3
Strangely enough, I actually know a girl with Evan as a first name. I think there was a family connection behind her name, too. If she made it work as a first name, you can definitely make it work as a middle name. I mean, middles are a great place to have a meaningful name if you don't want to use it as a first name.
I love the name Nora, but it doesn't have the best flow with Evan. Nora Evalyn would be better if you really loved Nora. I love the way that Lily Evan and Lila Evan sound together!
March 24th, 2014 12:29 PM #5
March 24th, 2014 12:54 PM #7Olympia
Russian Lit student, bookworm, and arts geek.
• Frances Birdie Jane • Emmeline Twila Alice • Ramona Juniper Monet • Esme Helena Mazarine • Sibyl Jemima Iris
• Nathaniel Roscoe Wilde • Grover Elijah St John • Oscar Ignatius Cosmo • Robin Hector Barnaby • Axel Isidore Sage
Apollo Amy Atlas Celia Dashiell Constance Jarvis Eugenie Raphael Magnolia Magnus Marigold Seymour Rosario Wilbur
March 24th, 2014 01:18 PM #9Mickie
| Camille | Élise | Sophie-Anne |
| Benoit | Cédric | Luca | Sébastien |
- Celia - Éliane - Jasmine - Juliette - Laurianne - Mireille -
- Aldéric - Éliam - Emeric - Henrik - Nikolas - Theodore -


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The N.C. Department of Transportation has some money tucked away in its coffers, although we don't seem to see it in action much around here.
Two of the newest road improvements are the new superstreet designs at the busy intersections of U.S. 17 and Mount Pisgah and Ocean Isle Beach roads. If you can't remember where you've heard the term superstreet before—think the year-old intersections on U.S. 17 in Leland.
Apparently no one, not even DOT, could argue these busy intersections didn't need major improvements.
The smell was burning my nostrils as I waited for the pump to cut off. I watched as the meter passed $30, then $40, and finally $50. At $52 it stopped.
My receipt printed, and I could be on my way—for another four days at least. Then I would be back to the dreaded pump, watching my as my money rushed out of my bank account and into my gas tank.
Perhaps it would be wise to do research before naming the new middle school in the Cedar Grove community. Once a school is named, it is open to scrutiny as to why the name was chosen. Surrounding counties are research-savvy when it comes to Brunswick County, and some of their recent findings have not been positive.
Schools in Brunswick County have received stinging press reviews, which include poor test scores and several inappropriate teacher/student relationship allegations.
Last week's celebration honoring Shallotte firefighters and the open house that followed, giving the public a chance to tour the new fire station, were the culmination of many years of hard work.
More than once during the event, the Shallotte Fire Department was called the best in the county, a far cry from what it was just 10 years ago when most people would have said it was one of the worst.
First of all, I don't care what you say, at the end of the day, prefacing your sentences with meaningless phrases is annoying. (See?)
If you need time to work on how you're going to word your statement correctly, just work it out in your head before opening your mouth, then go directly to your statement.
Twelve or more years of hard work and hassle came to a dramatic, yet simple conclusion last week as hundreds of Brunswick County graduates picked up their tassels and moved them from one side of their mortarboards to the other.
In that gesture, often accentuated by the ceremonial tossing of the mortarboard into the air, students made the transition from being kids to almost, but not quite, grown up. After pomp and circumstance, they now find themselves lingering on the outskirts of the "real world."
The heat may be blazing and corn and gas prices rising higher than giraffes' eyes.
What better time than summer, then, which officially clocks in at 7:59 p.m. EDT this Friday, June 20, than to stop and be thankful it's finally here.
Never mind the heat or the humidity.
It's el solsticio de verano, which only comes but once a year. I just hope it doesn't bring any el ninos, la ninas or el hurrican-os along with it.
Congratulations class of 2008, you made it! Now you're in a new class—class of 2012.
As most of you begin to prepare for the first move-in day at your new college or university, I'm sure you're hearing the same things from your moms and dads.
"Don't party too much; do your homework; don't stay out too late," blah, blah, blah.
Having graduated from college only two years ago myself, I believe to still be in tune with the college scene and have a pretty good idea of what to do and what not to do.
They are the largest, deadliest storms on Earth, and 14 of them have affected the Brunswick County coast since 1900.
Hurricanes are a constant threat for the residents of Brunswick County, and last Thursday evening Brunswick County Emergency Services sponsored a public forum to help residents understand how to prepare for these storms.
With an expert panel of speakers that included representatives from FEMA, the state department of crime and public safety and the National Weather Service, the message seemed to be the same—be prepared.
I'm sure global warming supporters are jumping for joy with the temperatures soaring faster than gas prices.
You know it's hot out there when the conversation turns from rising gas prices to rising temperatures.
On a recent 99-degree day in Wilmington, my brother was complaining to me that it was even hotter and more humid in his D.C. suburb, missing the coastal breeze of Southeastern North Carolina.
With normal early-June highs teetering around the mid-80s mark in our part of the world, the recent temperatures have people talking.
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The Brunswick Beacon is your source for local news, sports, events and information in Brunswick County, North Carolina and surrounding areas.


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Road Warrior
Joined: Nov 2011
Location: PanAmerican Highway
Oddometer: 97
Sorry it has been so long since we have updated, We have been caught up in a whirlwind of travel lately. (This is a good thing!) We have now settled down in a beautiful place called San Pedro De Laguna, Guatemala. We found a great spanish school that rents out nice little cabanas for $25 a week! We are right on the water and loving it here. I am lounging in the shady hammock outside, typing this up and listening to the birds chirp in the trees. Behind us about 100 yards is gigantic lagoon ringed by 3 massive dormant volcano mountains. Have we found paradise already!? Perhaps Needless to say we have decided to stay here for a month taking spanish lessons and slowing down the pace.
Now back to our regular scheduled programing!
After our hectic day in Oaxaca we decided to put some miles down. My friend Doug runs a community center for a small barrio in Cancun. We had told him we were going to stop by and help him out so we set our sights for the tip of the Yucatan peninsula. As we descended from the top of the Oaxcan mountain range towards the isthmus of Mexico the change was immediately apparent. The pine trees gave way to jungle and the the cool dry air was now thick with humidity. Toto Were not in Kansas anymore.
On our way up to Cancun we stopped into the city of Villahermosa. A primarily industrial city with not much in the way of scenery. However it did have a nice museum/zoo combo that sounded interesting. The Parque Mueso La Venta combined both native Yucatan animals and excavated artifacts from the nearby Olmec ruins of La Venta into one attraction. Plus it was only $3 which the budget surely appreciates.
Yucatan Crocodiles. Vicious little guys. Note the croc is already missing one foot.
Lauren goes to the bathroom and when she gets back she hurriedly tells me I think something escaped from the zoo! and drags me to come look. Figuring she has been standing in the sun too long I reluctantly follow, and sure enough something did escape!
or so we thought We went and grabbed some employees and drug them over to look. They just took a glance at this obviously escaped zoo convict and started laughing. Ummm hello? Aren't you going to put it back in the cage!? Well it turns out these odd looking foreign creatures are basically a Yucatan raccoon and are more of a pest than a zoo exhibit. As we walked around the rest of the zoo we ended up seeing tons of them digging and climbing all around in the jungle. Man we are such gringos.
The Olmec artifacts were very interesting, the La Venta ruins site is just up the road from Villahermosa. In the 1950s they were planning to bulldoze the ruin area for crop land. An archeologist took charge, relocated most of the ruins to Villahermosa, and started the Museo Venta to educate people on the ruins site and Olmec heritage.
Magnificent Olmec heads weighing over 9 tons.
Growing up in Florida I have seen my fair share of Gators, I've seen the Worlds Largest Gator at least 4 different times in 4 different tourist traps. But I think I may have finally found the actual Worlds Biggest Gator. Rumor has it that this thing eats Coatimundis by the bakers dozen, as the zoo keepers try to rid the park of the pests they toss them into the gator pit for dinner. He was a BEAST. I would say easily 17ft-20ft long.
Note the turtles in the pic are huge snapping turtles, not any baby sized Red Slider nonsense.
We packed up from Villahermosa and headed deeper into the Peninsula. We have visited a few ruins on the trip so far but we have heard that Palenque was one of the larger more magnificent ruins in Mexico. After learning about the Mayan Emperor Pakal, his tomb, and his jade mask in Mexico City, we were excited to see where it was all discovered.
The Palenque ruins were discovered in the 1800s, explored and excavated over the centuries by a few different archeological groups. It is a beautiful Mayan site set deep in the jungle. They have done a great job with the excavation and restoration. The site and grounds are wonderful to tour around.
Although the site has been worked on for 200 some years, It wasnt until the 1950s that Alberto Lluhlier discovered Pakals tomb buried deep inside the temple. When he removed the (7 ton!) sarcophagus lid he discovered Pakals body dyed a deep maroon red and covered in magnificent jade jewelry. It was one of the largest archeological discoveries ever made on the Yucatan peninsula.
Pakals temple
I heard heard rumor that there were Mayan bathrooms at the site. I think this is a ancient Mayan shittter. Either that or I just desecrated thousands of years of history to make a poop joke.
Read more on the blog... http://homeonthehighway.com/getting-...gley-out-here/
Joined: Jun 2007
Location: Madison, Wisconsin and/or Panama, Panama
Oddometer: 7,769
If you do too much off-road stuff, especially river crossings, then you'll never make it to ushuaia. Good luck, though.
ps: my $.02... a totally stock, un-lifted truck would be better, on account of the twisty mountains. Sure, a lifted truck is better off-road, but for all purpose use, a stock truck would be best. You don't even need big tires.
I am Charlie.
Road Warrior
Joined: Nov 2011
Location: PanAmerican Highway
Oddometer: 97
we do a good mix of highway and offroad, more fun that way
Joined: Apr 2009
Location: Nashville TN, no chit, police state.
Oddometer: 3,858
but for that "little bit" I recommend 4Crawlers Ball Joint Spacers. About as complicated as a shovel, one piece and over-all doesn't effect much.
Depending on what you like, BFG 33/10.50's, 4:56's or 4:88's from an Automatic, and some traction diffs maybe even a retro fitted TRD and you'll embarrass poser 4WD's. And have 10x the driveability.
Everyday IS 3/11!
Everybody's always talking out the side of their mouths about our "dependency on foreign oil", what about our dependency on cheap china crap? Who exactly again is killing our dollar?
Road Warrior
Joined: Nov 2011
Location: PanAmerican Highway
Oddometer: 97
I spent 20 minutes trying to come up with a clever title, not much rhymes with Yucatan. I thought maybe YuCAtaN DO IT! but then gave up on that. I considered making up a fictional character named Yucatan Dan who lives in the jungle and grants 3 wishes to lost gringos. I wish for 20 gallons of DEET bugspray Perhaps Doin our thang in the Yucatang I dont know! I have spanish class in 30 minutes and need to get this post done. Focus James!
We pushed further down the Caribbean coast headed towards a concentration of Mayan ruins called The Ruta Puuc. The Ruta Puuc is about 25 miles of backroad that connect 6 different Mayan ruins together. In Mayan times there was actually a road of limestone running through the jungle connecting many of the large sites. Unfortunately this road is long gone and were stuck to boring ol tarmac.
We drove on and on through the jungle on the paved roads eventually arriving near the Mayan site of Uxmal. The Sun was setting and we needed to find a camping site quick. We pulled our usual maneuver of scoping out the surrounding areas for cutty backroads, eventually finding one that looked good and turned off into the deep jungle.
We followed this trail for miles, passing 2 small bee farms and not much else. Intrigued as to what the hell this random road in the jungle leads to we pushed on further. Slowly the road deteriorates to little more than a single track ATV trail. After 10 miles of slow going through the jungle we stumble upon a very small, very creepy camp.
Hmmm Skulls, dirty old clothes, random stick structures, 15 miles deep in the jungle. Me thinks we should NOT camp here.
Next morning we woke up and headed to Uxmal. Uxmal is a magnificent Mayan site. Estimated to have supported over 15,000 inhabits at its height in 900 A.D. or so. The site is one of the finest examples of Mayan construction, relying on precisely cut stone blocks for the exterior of the buildings rather than plaster which wears away quickly.
The carvings here were still in great shape and easily recognizable.
The site is surrounded by dense jungle as far as the eye can see, we were driving around somewhere in that mess the day before.
They had a wonderfully intact ancient mayan ball court as well.
The goal. I believe the way the game is played is the players are allowed to use any part of there body aside from there hands and feet. The game is over when someone sends the ball through the hoop. Rumor has it that the captain of the losing team is sacrificed. Now thats some team motivation!
They also had this weird shrine to penises, or is it penii?
Fractals everywhere, endlessly repeating patterns, the Mayans were definitely spacing out on something
We spent about 1/2 of the day touring Uxmal then headed down the road towards another site named Kabah
Kabah is home to the Codz Poop
Surprisingly enough. to me, Codz Poop is in fact not petrified Mayan doo, but in fact a hugely impressive Palace of Masks. The entire face of the building is compiled of hundreds of repeating Chac Mool (The Rain God) carvings.
Read the whole story on the blog Home on the Highway
Road Warrior
Joined: Nov 2011
Location: PanAmerican Highway
Oddometer: 97
It seems obligatory for all overlanders to write a Reflections on Mexico post after their travels through the country are completed. Not only for collecting their own thoughts but also to share the truth about this dangerous country. While Lauren and I were preparing for this trip many people thought we were insane. Oddly, It wasnt for the fact that we were planning to drive 25,000 miles to the bottom of the world. We were primarily labeled insane for simply wanting to drive into Mexico.
Cant you just skip Mexico?
Have you thought about shipping around Mexico straight to South America?
I have heard/read/seen that you are going to be beheaded, raped, robbed, scammed, schemed, murdered, sold into slavery; the instant you step across the border to Mexico.
Honestly, I cannot really blame them. The media blasts us with reports of daily beheadings, bodies lining the streets, downright bloodbaths. Momentarily while preparing for the trip we would feel a shimmer of trepidation as well. What if our years of research, planning, and reports from people who are actually IN Mexico were wrong and they were all right? Would there be banditos waiting to have their way with our innocent American flesh?
We pushed aside these fears and stood strong, after all hard facts beat out hearsay everyday.
There is no doubt that there is truly a war going on in Mexico. A bloody drug war, a struggle for power between the all-powerful gangs and the quickly dwindling police and military forces of Mexico. However this war is concentrated primarily to the border areas and rarely involves anyone that is not poking their nose where it shouldnt belong. I will leave my opinions on Americas drug/immigration policies being a primarily catalyst for this war for another time
The MAJORITY of Mexico is a beautiful, peaceful, tranquil place. We found plenty of farmers/fisherman/working class people quietly going about their business. We found zero insane banditos hellbent on attacking innocent gringos. We primarily encountered children laughing and playing in the streets, women washing clothes and making fresh tortillas by the roadside, and hombres walking their cows from field to field.
We also found lots of police and military checkpoints searching for drugs and guns along our way. We saw 50-cal machine guns mounted to police truck cabs. It was not rare to be shopping in a supermarket picking up some milk next to a sawed-off shotgun wielding security guard. However after the initial gun shock wears off these things start to just be part of the routine.
Never in our entire month journey all throughout the far corners of Mexico did we ever feel remotely in danger. Lost, confused, frustrated, yes. In danger? Never.
Mexico is getting a raw deal. There are tons of RV parks, campgrounds, and other tourist attractions that are drying up and going to waste down here. The media has put a downright HALT to peoples plans to visit this beautiful country. Every single traveler we ran into had the same story to tell, zero problems whatsoever, great people, great food. We are here to tell you, do not be afraid. It is a wonderful place, full of wonderful people, with a rich and vibrant culture.
Fear is a strangle-hold on life, Do not let the fear of the unknown keep you from exploring outside your comfort zone. We have found that good research, a sharp mind, and a easy smile will keep you out of trouble 99% of the time.
Next time you hear someone talking about how dangerous Mexico is, Ask them the last time they were there? And then point them to our blog.
Precious Snowflake
Joined: Mar 2007
Location: Missouri
Oddometer: 390
I really enjoy your trip report. Thank you for taking the time to share it with us.
"Bad planning is the mother of adventure." - Richard Grant
Road Warrior
Joined: Nov 2011
Location: PanAmerican Highway
Oddometer: 97
DC GSer
Joined: Dec 2002
Location: Washington DC
Oddometer: 10,895
Support DC voting rights.
Gnarly Adventurer
Joined: Jun 2011
Location: Apopka, FL
Oddometer: 404
Excellent update about Mexico. I believe that you can get killed in Mexico or in your own home town depending where you are located. I live in Orlando and we have some part of towns that leave a lot to be desired and people that usually get killed or mugged there is because they are doing things they shouldn't do in the first place. Heck Orlando was named one of the top ten cities in the US with the highest rate of crime and other things not long ago and my wife and I have yet to feel unsafe or threathen in any way. Thank you for clarifying about Mexico so other people like you guys take the plunge and venture in to Mexico without that fear.
Road Warrior
Joined: Nov 2011
Location: PanAmerican Highway
Oddometer: 97
Road Warrior
Joined: Nov 2011
Location: PanAmerican Highway
Oddometer: 97
After a great night in Bakalar, Mexico we headed south to the Belize/Mexico border. Unsure of what to expect we checked out our friends Life Remotely blog who recently crossed the border and posted a great detailed report explaining the crossing in detail.
It turned out to be a simple affair. We found the Mexican customs office, relinquished our Mexican visas and stopped at the Banjercito to check-out the truck. We received an exit stamp in our passport and they removed our Mexican vehicle import sticker from the trucks windshield. We were officially in No Mans Land. The area that exists while you are checked out from one country but not checked into the next. You may know it by the term duty-free zone. They had a large mall here where you could pick up cheap booze, cigarettes, and crappy knock-off brand name clothes.
After stocking up on junk we hit the Belize border. We had just learned the day before that Belizes official language is actually English. Quite a surprise to us. It took a while to get used to saying, Thank you, instead of Gracias and Yes, instead of Si. But man were we happy to finally be able to have a full-on conversation with people instead of standing there like idiots trying to communicate.
The check-in process to Belize was simple, a few stamps in the passport, a cursory check of the vehicle and we were in. Welcome to Belize! We picked up vehicle insurance just past the border, $23 for 2 weeks.
While we were in Bakalar we met up with "Team Equipt" who gave us the line on a great campsite just past the Belize border, We headed off towards the GPS coords. The road was not on our map but Ben assured me, we could make it. ;)
Cruising down the road we hit a river with a ferry crossing. This was no ordinary ferry, an ancient hand-cranked job which looked as if it would sink at any moment. (I later learned that it actually did sink about 3 weeks before ) It could hold about 3 cars at a time, apparently it runs 24/7. The conductor sleeps on a wooden bench in the ferry.
We met some cool Mennonites on the ferry who were partying it up, we shared a few beers while we took turns cranking the ferry across the river. Hard working farming folk, there is a large Mennonite community in Belize. Apparently they got fed up with U.S religious policy and a large population relocated to Belize in the 1950s. Most are still very religious leading an almost Amish lifestyle, preferring horsedrawn buggies to automobiles. We met some of the more "progressiveboys. Ha!
We crossed the river, continued down the road, eventually hitting another hand-cranked ferry.
Pressing on towards the GPS coords we eventually found the spot. And it was worth every mile! Thanks again
From our cove we headed towards a small town in Northern Belize by the name of Sartenja. Sartenja, Belize is home to the Backpackers Paradise A great little hostel/restaurant run by an amazing French and Swiss couple. They have carved out their own little piece of paradise here. They rent out cabins, tents, and hammocks to travelers for great rates. Natalie also can cook like nobodies business, we had amazing French/Belizean fusion meals for dinner every night.
The common area. No shortage of hammocks to go around. Lauren and I spent most of our nights here lounging in the hammocks listening to the rain and crickets chirping outside.
Read more on the blog... http://homeonthehighway.com
Road Warrior
Joined: Nov 2011
Location: PanAmerican Highway
Oddometer: 97
We left Caye Caulker behind and headed back towards Sarteneja where we had left our truck. We took a quick pitstop in Ambergris Caye as we waited for the next ferry.
We made friends with a Coatimundi (You may remember these guys as Crock Snacks in Mexico. Now I feel kinda bad, they are awfully cute.
We picked the truck up and headed deeper into the interior of Belize. We had heard good things about The Belize Zoo and went to check it out.
The entry fee for the zoo was a bit steep ($15US per person??) but all the animals were rescues so we figured it was for a good cause. It turned out to be a great little zoo, with lots of native Belizean animals we have never seen before.
The Jabiru Stork, largest bird in Central/South America, 2nd largest wingspan in the world. Over 9ft wide!
THE HARPY EAGLE! The largest and most powerful eagle in the Americas. This thing eats Coatimundis for lunch. (Coatis got it rough ) The harpy eagles are practically extinct in Central America due to deforestation.
Harpy eagle attacking some poor zookeeper!
Junior the jaguar, It was great how little concerns for safety the zoo had, You could stick you arm in the cages and pet the jaguar
Hahahaha, Lauren was shooting shots of this Tapir when all of the sudden it turned around and shot a 10FT firehose stream of urine (At least we hope it was urine ) all over her pants and shoes.
After Lauren burned her clothes we jumped back in the truck and headed towards Barton Creek Outfitters. A small hostel deep in the jungle of Belize.
Adios pavement
A fun river crossing
More pictures and the rest of the story on the blog... http://homeonthehighway.com
Joined: Feb 2006
Location: Des Moines, IA
Oddometer: 367
Thanks for the update. Safe travels!
Gnarly Adventurer
Joined: Aug 2011
Location: the other Redmond
Oddometer: 204
Ha that Tapir hosed down my my girlfriend too. I think for a fee you can get in the Jaguar cage. Looks like a great trip.
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Links Provider » Business & Economy » Services » Catering and Food
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UK based promotional apron and tabard manufacturers with secure online ordering and speedy delivery. - Details
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Michele shares her favorite quick, easy and nutritious recipes thru her cooking videos. - Details


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Paul A. Smith offers news, notes and perspective on the great outdoors.
Hunter education classes available
Hunters who need to obtain a hunter education certificate prior to the fall seasons should register for a class in the near future.
"Nearly all of the volunteer hunter education instructors are hunters themselves and enjoy hunting in the fall," said conservation warden Tim Lawhern, who also serves as the state's hunting education administrator. "The hunter education program offers about 1,200 courses every year, but very few of them are offered from October through December."
Lawhern said each year he fields calls two weeks before the gun-deer season with hunter-hopefuls looking to fulfill the mandatory hunter education course. But more than 99 percent of the courses have been offered by that time, said Lawhern.
Hunters born on or after Jan. 1, 1973, must have completed a hunter education course and show the certificate to purchase any hunting license in Wisconsin. Also, recreational safety students are required to obtain a Wisconsin DNR Customer ID Number before the completion of any recreational safety class and must provide that Customer ID Number to the instructor.
"In Wisconsin, we've reduced hunting accidents by 90 percent since the hunter education program began," Lawhern said. "Hunting is safe -- and getting safer -- because of the volunteer instructors who teach hunter education and the number of our hunters who have now graduated from our courses."
The information listed below shows contact information for classes in southeastern Wisconsin and the date of the first session in each class. Call the listed contact for additional details. A list of statewide classes is also available on the DNR's website.
Milwaukee County:
Milwaukee, 5476 S. 13th Street, Moose Lodge #49, Michael Kiedrowski, (414) 460-4553. July 29, 9:30 a.m. Class Notes: All students must register on July 29, 2010 between 9:30am & Noon at the Moose Lodge.
Waukesha County:
Mukwonago, S93 W30850 County Road NN, Our Saviors Wesleyan Church, Patrick Kaboskey, (262) 363-8025. July 22, 9 a.m. Class Notes: Need to pre register. Call Jeanette at the church.
Oconomowoc, 2911N Dousman Road, Summit Town Hall, Daniel Supple, (262) 567-1134. July 19, 6 p.m. Class Notes: Phone Registration Only at 262-567-1134. Registration begins on June 28 and ends on July 19.
New Berlin, 18200 West Cleveland Avenue, Milwaukee Casting Club, Robert Belling, (414) 321-6797. August 17, 6:15 p.m. Class Notes: A field day will be held Sept. 11. No Calls After 9 p.m. please.
Dousman, South Main Street, Dousman Gun Club, Bradley Graf, (262) 965-3792. August 9; 6 p.m.
Washington County:
West Bend, Highway 45 and Trap Shoot Lane, West Bend Barton Sportmens Club, Stephen Bieszki, (920) 398-2801. August 2, 7 p.m. Class Notes: Class size is limited.
West Bend, 500 Schmidt Road, Washington County Sheriffs Dept., Scott Sager, (262) 707-1731. August 17, 7 p.m. Class Notes: Required completion of the IHEA internet tutorial prior to class. Pre-registration required; contact instructor for information.
Hartford, 110 N. Johnson Street, Hartford City Hall Community Room, Timothy Rohrer, (262) 673-8226. August 1; 6:15 p.m. Class Notes: Students must pre register at the Hartford Recreation Department. There is a field day on Sept 11.
Hubertus, 4694 Highway 167, Daniel Boone Conservation League, Jerry Schmidt. Sept 8, 6:30 p.m. Class Notes: Mandatory Field Day October 16, 8 a.m. to 2 p.m. Students to use email address to contact or enroll in the class: firstname.lastname@example.com
Newburg, Highway MY, Newburg Sportsmen, Ralph Bancroft, (262) 334-0512. August 2, 7 p.m.
Racine County:
Wind Lake, 6419 Heg Park Road, Norway Town Hall, Joshua Heinze, (262) 895-2834. August 9, 6 p.m.
Comments »
Read and share your thoughts on this story.


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National Credit Union Foundation Executive Director Gigi Hyland publishes an impressive "foodie" blog.
See a round up of the latest credit unions and industry organizations to join the national ALS Ice Bucket Challenge.
CUNA paid its CEO more than double the salary that NAFCU did in 2013, according to forms filed with the IRS.
The former CEO earned a total package of $1,310,479 in 2013. Learn what other CUNA executives earned last year.
The $183 million Aspire Federal Credit Union in Clark, N.J., hired Joseph Riccardo as its first chief information officer. Riccardo's hiring follows the credit union's decision to convert to the Corelation KeyStone core processing platform next year, Aspire said. Riccardo has more than 30 years of financial and technology experience,...
John McCloy promoted from within in Colorado, and other industry career news.
Today's new and soon-to-be retirees are deeper in debt than previous generations.
The NCUA's new rule on CDAs benefits industry charities as credit unions invest in the vehicles.
The $1.1 billion Jeanne D'Arc Credit Union in Lowell, Mass, hired Matthew Bernard as assistant vice president for network operations. He has more than 15 years of experience in the technology industry and was awarded two Purple Stars for wounds he received in combat while serving as an infantryman in...
All 158 Children's Miracle Network hospitals receive Biz Kid$ DVDs following donation drive.


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The Limits of Agile
The problems faced by teams that are attempting Agile in non-traditional settings aren't that Agile principles are inapplicable, nor that the feedback cycle is doomed to failure; but rather, outside of a certain Agile sweet-spot there are additional barriers and costs to applying Agile techniques. None of these obstacles prevents Agile in itself but each increases the cost of getting to Agile.
A Tester's Learning Journey
The software industry is changing fast. More and more teams put testing up front and center; they use tests to drive development. In this article, Lisa Crispin talks about how her attitude and curiosity have shaped her career and kept her passion for testing software fresh.
Making TDD Stick: Problems and Solutions for Adopters
Mark Levison observed that, after solid classroom training, teams in larger companies still struggle to adopt TDD. To better understand the problem he surveyed team members. In this article he shares the problems he uncovered and his own comprehensive strategy, designed to help anyone introducing TDD into an organization.
Iterative, Automated and Continuous Performance
Iterative and continuous are terms that are often used in reference to testing of software. This new InfoQ article takes a look at whether the same concepts can be applied to performance tuning. 16
Test Driven Development with Visual Studio for Database Professionals
Cory Foy walks through the process to implement Test Driven Development for business logic implemented in the database. 4
Book Excerpt: Continuous Integration means Continuous Testing
Continuous Integration has become a standard development best practice - but it's not always done well. InfoQ presents advice and examples in Chapter 6: Continuous Testing from a new CI book. 4
Agile, Architecture and the 5am Production Problem
Can refactoring and TDD create "working software" that survives real life? Michael Nygard suggests that "leaky abstractions" ambush us at 5AM when we've paid too little attention to architecture. 25
Unit-Testing XML
Today, almost every modern application consumes or produces XML. Stefan Bodewig explains the challenges in testing XML-based applications, and shows how to use the XMLUnit Java Framework to do so. 8
TDD with Selenium and Castle
TDD principals provide architects a way to quickly jump into active development early in the application development lifecycle. Dan Bunea demonstrates how TDD can be applied in .NET. 5
Case Study: Targeted Practice Adoption using Patterns
Teams new to Agile wonder which practices to start with, and which will most benefit their own context. This article proposes a customized adoption approach to help teams decide where to focus first.
Adopting Agile Development Practices: Using Patterns to Share our Experiences
Amr Elssamadisy tells how participants at XPday Montreal focused on the dynamics of adoption, rather than the structure resulting from adoption, to compile Agile adoption patterns helpful to novices. 1
Experience Report: Agile Development Apprenticeship at NMHU
The story of a unique work-study degree program at New Mexico Highlands University, begun in 2004-2005: an apprentice shop where students and customers used Agile practices.


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Today will be sunny with a high near 66. Winds will be from the west-southwest at 10 to 20 mph, with gusts as high as 25 mph.
Tonight, expect to see clear skies with a low around 40, and south-southwestern winds at 10 to 15 mph.
Learn more at weather.gov.
[1] mailto:firstname.lastname@example.com
[2] http://www.sweetwaterreporter.com/sites/default/files/weather forecast_6.jpg


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They feel bad, so they found you these other deals to make up for it.
Select Local Merchants
Flamig FarmRead MoreSince 1907, Flamig Farm has developed into a reputable educational-resource facility complete with an extensive petting zoo. Visitors can frolic with emus, ducks, and sheep, then cuddle with bunnies and piglets. Though not included in this Groupon, the farm offers several other activities, including pony rides, hikes, and hayrides. The farm closes when the weather gets cold, so be sure to visit before animals migrate to Hollywood and resume their winter jobs as fast-food commercial spokesmen.7 Shingle Mill RdWest Simsbury, CT US
Ioka Valley FarmRead MoreIn 1936, Robert and Dorothy Leab drove their 13 head of cattle over Brodie Mountain and into Ioka Valley, where they broke ground on their new home. Despite the poor quality of the farm's soil, their hard work gradually resulted in bountiful harvests. Decades later, the third generation of the Leab family still tills the land, planting assorted crops and opening the farm to visitors for year-round activities. Each season brings new life to the farm, from the pastel buds and new shoots of spring to summer's vibrant strawberries, which are grown on raised beds so visitors can pick their own pints. Kids frolic in Uncle Don's Barnyard all summer, petting tame rabbits and llamas and whooshing down a 40-foot pipeline slide. Fall festival activities include hayrides and pumpkin picking, and during the winter, snow-covered Christmas trees can be carted home to add holiday cheer or provide a new project for the family's pet beaver. Maple season stretches from February to April in the sugar house, occupied by 5,000 taps and two boilers. The farm churns out deep maple syrup that is served over pancakes and waffles in the Calf-A, a calf barn converted into a café. The farm's cattle herds are pasture-raised during warm months, with their diet supplemented by the farm's own corn, before becoming hormone-free, all natural beef.3475 Route 43Hancock, MA US
Animal AdventuresRead MoreA haven for exotic animals rescued from neglect or abandonment, Animal Adventures lets visitors interact with its furry and scaly refugees, teaching them firsthand to appreciate and respect the earth's diverse wildlife. Working with a rotating cast of mammals, reptiles, amphibians, and marsupials, animal experts regale audiences with facts and the backstories of each animal, such as how they were rescued and which cartoon representative of their species best captures their regional accent. Though its denizens regularly find new homes, Animal Adventures's altruistic menagerie has included a massive alligator snapping turtle, a canadian lynx, and an asian water monitor. The sanctuary also offers day camps for youngsters looking to get closer with the animals, and an animal-adoption program for adults looking to support the cause by taking a critter home and putting it through college.336 Sugar Rd.Bolton, MA US
The Zoo in Forest Park and Education CenterRead MoreVisiting The Zoo in Forest Park and Education Center is a lot like stepping into a nature documentary. Guests can take a self-guided journey to meet more than 200 creatures from across the world. They may stop by the habitats of the black and white ruffed lemur, the western bobcat, and the spotted leopard. Along the way, guests might learn a lot: for instance, that the Bennett's wallaby carries its young in a pouch, and that the critically-endangered cotton-top tamarin has lost more than 75% of its native habitat. But in at least one way, the zoo accomplishes something that David Attenborough never could. Visitors can actually reach out and touch a creature during discovery programs. They can even adopt certain animals, perhaps helping provide tasty grasses and career guidance to a red kangaroo. These programs exemplify the nonprofit zoo's dedication to wildlife education and awareness, something they hope to instill in their visitors from an early age. In the summer, educators spin "Animal Tales" for rapt young audiences and hold a Zoo Camp, where kids start to learn about diet and animal care. As kids' love of animals grows, the zoo invites them to volunteer as Crew in Training members. Once they hit college, students can become interns working on projects such as field studies of the patas monkey.302 Sumner Ave.Springfield, MA US
Mass Audubon SocietyRead MoreOne of the largest conservation organizations in New England, Mass Audubon cares for 34,000 acres of natural land in a network of more than 50 wildlife sanctuaries across the state. Its members receive free admission to these pacific preserves, where, alongside more than 150 endangered or threatened native species, they can breathe in Mother Nature?s perfume or have a good cry on her mossy bosom. During bird-migration season, alert gazes can capture some 300 species of sky surfer at Allens Pond on the South Coast, and visitors to Lincoln?s Drumlin Farm can re-enact Charlotte's Web with a motley band of sheep, cows, goats, and pigs.127 Combs RdEasthampton, MA US