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  1. Viral Production at Addgene

    Type
    Blog Post
    ...Next-generation sequencing is performed on viral genomes isolated from the final AAV preparation. Sequencing results...of AAV preparations is assayed by comparing the relative stoichiometric ratios of the viral capsid proteins...SYPRO Ruby staining and the molecular weight and relative intensity of the viral capsid proteins are analyzed...Viral capsid proteins VP1, VP2, and VP3 are shown relative to the total protein present in the sample. ...are added to cells in culture. Three to five days later, cell cultures are inspected for sterility. Transducibility...-AAVrg) alone at 1.1E6 vg/mL, or both. Two days later, Cre-dependent GFP expression was detected with ...vectors consist of full particles (white arrowhead) relative to empty particles (black arrowhead). Scale bar...

  2. Antibiotic Resistance: An Old Solution but a New Problem

    Type
    Blog Post
    ...been battling for centuries. As such, resistant populations can arise very quickly following the tried and...showing bacteria evolve antibiotic resistance on a plate! Antibiotic resistance can arise, and has arisen...antibiotics These scenarios expose bacterial populations to antibiotics at unnecessary times, putting ...pressure on resistant bacteria to over-take the population and making them more difficult to treat in the...susceptible bacteria that will be more difficult to treat later. A similar situation can occur if you treat a bacterial...because scientists in the laboratory will need to isolate and grow whatever bacteria are causing the infection...before they can test for resistance. Growing and isolating the infecting bacteria can take 24 hours or more...

  3. CRISPR Activation: A Practical Guide

    Type
    Blog Post
    ...transcriptional activators. These constructs allow for the upregulation of essentially any gene by fusing mutants of...cause differentiation and to reverse HIV viral latency (Bialek et al., 2016). Additionally, CRISPR-based...factors that may prevent your gene from being up-regulated. For instance, generally, the more highly expressed...Additionaly, cDNAs cannot be used to study in cis regulation and also suffer from an inability to easily deliver...CRISPR-based tools for altering DNA sequence or regulating gene expression.     References Behan FM, Iorio...Stucka R, Hauber J, Lange UC (2016) Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional...for inducible genome editing and transcription modulation. Nat Biotechnol 33:139–142 . https://doi.org/...

  4. Fueled by Coffee at #SfN14

    Type
    Blog Post
    ...sessions, exhibit booths, happy hour socials, and late, late night socials. As a newbie to the SfN conference... global coffee supply… My back-of-the-napkin calculations put the daily consumption here at ~ 60,000 cups...students and postdocs, which accounts for many, many lattes and espressos. The energy boost is well needed ... Investigator Awards and we couldn’t wait to congratulate him on his successes. We've been lucky enough...SfN14 on Twitter for live tweeting updates and the latest news from attendees. And for those of you presenting...

  5. A Scientist's Guide to Ebola

    Type
    Blog Post
    ...have similarly curated their Ebola-related content. -And the latest updates on the ongoing outbreak from... underestimate. Given all the excitement and speculation, we thought we’d do a little digging and assemble...assemble a rough guide to the science of Ebola and related resources. Ebola Basics A few basics: Ebolavirus... data on five strains of Ebola representing the latest outbreak and earlier ones, along with links to ...the current Ebola crisis, Science and Science Translational Medicine have made their complete collection...

  6. New Optogenetic Tools for Cytoskeleton and Membrane Control

    Type
    Blog Post
    ... target to specific subcellular regions, laser ablation can damage other cellular structures, and genetic...genetic mutations are not easily reversible. Late last year, the Akhmanova Lab published a new optogenetic...for a wide variety of experiments. Opto-RhoGEFs manipulate cell morphology and adhesion But what if you’... Taking control of a RhoGEF is a great way to stimulate and study these physical processes. Joachim Goedhart...of membrane-recruitment optogenetic tools for manipulating Rho GTPases based on BcLOV4. These single-component...membranes) (Figure 4). The authors used SuperPLD to manipulate phosphatidic acid signaling (such as Hippo, AMPK...minutes of blue light illumination, substantial PA accumulates in the plasma membranes, indicated by the PA ...

  7. CRISPR 101: Engineering the Plant Genome Using CRISPR/Cas9

    Type
    Blog Post
    ...often requiring several rounds of selection to isolate plants with the phenotype of interest. Genome engineering... modification of known or suspected genes that regulate a desired phenotype. In fact, CRISPR has already...variants have been created in an effort to increase translation in a particular plant species or cell type (Belhaj...driven by plant-derived RNA pol II promoters which regulate expression of longer RNAs (such as mRNAs for gene...plant cytorhabdovirus as versatile expression platforms for planthopper and cereal genomic studies. New...Multiplexed Plant Genome Editing and Transcriptional Regulation. Plant Physiol 169:971–985 . https://doi.org/...(2016) Gene-edited CRISPR mushroom escapes US regulation. Nature 532:293–293 . https://doi.org/10.1038...

  8. Evolution of Brainbow: Using Cre-lox for Multicolor Labeling of Neurons

    Type
    Blog Post
    ... be the hot new way to manipulate gene expression, but other gene manipulation systems remain valuable...To achieve even cell labeling, they generated farnesylated derivatives of the fluorescent proteins, which...trafficked to cell membranes. Membrane trafficking of farnesylated derivatives enables labeling of delicate axonal... default fluorescence due to the addition of translation-blocking STOP cassette immediately following ...woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), commonly used to increase transgene...and Flpbow can be used to label distinct cell populations. To decrease the animal breeding necessary to...

  9. Optogenetics + CRISPR, Using Light to Control Genome Editing

    Type
    Blog Post
    ...formed 20-40 min later. The CASANOVA system is versatile in that it can be adapted to regulate gene editing...modifications by NHEJ and HDR using split systems Later in 2015, the Sato lab unveiled a photoactivatable...opens intriguing possibilities for optogenetic manipulation of CRISPR-based systems in animal models. Use... prevent DNA binding in the dark. Upon light stimulation, the pdDronpa moieties move apart, allowing DNA...LOV2 has a C-terminal helix that unfolds when stimulated by blue light. When fused to the catalytic site...genome and chromatin architecture, it is useful to regulate the time in which dCas9 binding occurs. Bubeck...Niopek D (2020) Optogenetics and CRISPR: A New Relationship Built to Last. In: Methods in Molecular Biology...

  10. Scientist Networking: What is an Informational Interview?

    Type
    Blog Post
    ... more open to developing a relationship. The time to develop relationships is before you need them (well...doing interesting things. I call this building relationships because networking has gotten a bad reputation...Scientists enjoy learning new things. Building new relationships is all about learning new things from other ...networking for a job connection with ongoing relationship building through Informational Interviews. Making...is always best to meet in person to develop relationships, but if it is not possible consider a phone ...

  11. Easing the Protein Purification Process with pCri

    Type
    Blog Post
    ...has the capacity to glycosylate proteins and may recapitulate the post-translational modifications added...requires a substantial amount of properly folded, relatively pure protein, but getting to this stage is often...one’s protein of interest can save a lot of time later. Protein purification specialists recommend doing...the MISTIC (membrane-integrating sequence for translation of integral-membrane protein constructs) protein...

  12. CRISPR Protocol for Genomic Deletions in Mammalian Cell Lines [Video]

    Type
    Blog Post
    ...non-coding regulatory elements. Regulatory DNA function is typically inferred based on correlative biochemical...of the method. One observation was an inverse relationship between deletion size and frequency [3]. There...interested in exploring the function of non-coding regulatory elements in their native chromatin context. An...   Like the video? The Orkin and Bauer lab's related paper describes their methods for CRISPR design...

  13. Scientific Sharing in the Time of COVID-19: Databases and Resources

    Type
    Blog Post
    ...asked the individuals behind some of these new platforms what their motivations were for developing these...knowledge that could help fight COVID-19. They’ve also collated a list of useful research resources.  "We realized...Canadian volunteers, and features over 200 COVID-19 related research projects, funding opportunities, and community...community response initiatives. Accelerate the translational impact of research Universities have also been...creating research synergies and accelerating the translational impact of research activities. By streamlining...research in their field including datasets and platforms for discussing new developments, and more. As ...

  14. CUT&RUN: An Improved Method for Studying Protein-DNA Interactions

    Type
    Blog Post
    ...organisms and cell lines. Although ChIP-seq is a relatively simple and robust protocol it does have limitations...study how differential access to transcriptional regulatory elements is controlled during development using...fused micrococcal nuclease (MNase) to specifically isolate DNA that is bound by a protein of interest. First...First, nuclei from tissue or cell culture are isolated using lectin-coated magnetic beads. Nuclei are then...MNase requires for its nuclease activity and is chelated from the reaction up until this point. The nuclease...simple lab protocols Browse CRISPR plasmids for isolating a genomic locus Browse FRET plasmids for studying...

  15. Arabidopsis in Education: How the Arabidopsis Biological Resource Center Brings Plant Science to Life

    Type
    Blog Post
    ...seeds, DNA and other resources for Arabidopsis and related species. Located at The Ohio State University in...member of the Brassicaceae family, Arabidopsis is related to a variety of food crops such as broccoli, cabbage...utility to science, and its well-known edible relatives, it is fair to say that most people do not recognize...teaching community through the TRAINED initiative (Translating Research on Arabidopsis Into a Network of Educational...through TAIR with a new ABRC database and ordering platform soon to be released. For individuals interested...Arabidopsis is small, has a short life cycle, and is relatively easy to grow, it is an ideal organism for teachers...

  16. Plasmids 101: Origin of Replication

    Type
    Blog Post
    ...balance between positive and negative regulation and can be manipulated with mutations in the replicon. For...produce just a few copies depending on how they are regulated. Generally, control of replication is referred...stringent" depending on whether the ori is positively regulated by RNA or proteins, respectively. A plasmid's ...plasmid copy number  Although the sequence and regulation of the ori dramatically affect the copy number...SB, Furman JL, Xiao H, Schultz PG. A versatile platform for single- and multiple-unnatural amino acid ...

  17. Viral Vectors 101: Parts of the AAV Transfer Plasmid

    Type
    Blog Post
    ...polyA  The polyadenylation or polyA signal aids in the nuclear export of RNA and RNA translation, and promotes...plasmid. Parts shown in grey are optional cis-regulatory elements.   Key elements of an AAV plasmid...plasmid harbors non-coding sequences. Other cis-regulatory elements Next let’s talk about some of the elements...Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) forms a tertiary structure when...Technologies and Methods for Targeted Neuronal Manipulation. Front Neuroanat 13: . https://doi.org/10.3389...

  18. Hot Plasmids and Viral Preps - September 2021

    Type
    Blog Post
    ...viral preps   Improved cell-free transcription/translation system to examine biosynthetic pathways  By Gabrielle...Gabrielle Clouse Recently, cell-free transcription/translation systems (also referred to as TX-TL systems) have...In summary, this cell free system allows for a relatively simple, controlled “one-pot” reaction capable...plasmids that helps identify base edited cell populations without end-point sequencing assays. These plasmids..., or XMAS-TREE, to identify base edited cell populations without end-point sequencing assays.  This tool...different vectors and flow cytometry is used to sort populations by mCherry and GFP expression to check for ABE...

  19. Plasmid Cloning by PCR

    Type
    Blog Post
    ...DNA, a cDNA library, or by RT-PCR. Isolate the PCR product Isolate your PCR product from the rest of the...function in the same buffer, as this will save time later In our example, we will use EcoRI and NotI to ...TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3’) into this calculator we get a final Reverse Primer sequence of 5’-...If you are amplifying from a plasmid or simple template, there is very little chance for mis-priming, ...now ready for restriction digestion. As such, the later steps in this process are the same as those discussed...

  20. Live and Let Dye: Self-Labeling Protein Tags

    Type
    Blog Post
    ...BG) (Keppler et al., 2003). This engineered hAGT later became known as SNAP-tag. There is also a fast-reacting...demonstrated in 2005 that proteins fused with E. coli dihydrofolate reductase (eDHFR) could be labeled with fluorescent...Although this tight interaction is not covalent, later work showed that introducing a Leu28Cys mutation...SNAP-OMP25 with Atto590-BG (green), imaged with stimulated emission depletion (STED) nanoscopy. Scale bar...different ligands to the same sample to label sub-populations of your protein of interest. For example, the...-molecule tracking of actin filament assembly-regulating proteins in vitro reveals complex events. a) ...Gaska, I., & Shekhar, S. (2023). Multicomponent regulation of actin barbed end assembly by twinfilin, formin...

  21. Quick Guide to All Things Lentivirus

    Type
    Blog Post
    ...of interest as well as sequences necessary to encapsulate that insert into a viral particle (the packaging...lentiviral vector system is the tetracycline (Tet)-regulated system. You can choose either the Tet-off system...rtTA (FUW-M2rtTA). In this kind of approach, a population of cells might express only the tTA or your transgene...proto-oncogenes or oncogenes for instance should be manipulated with care. See the NIH’s biosafety guidelines...viral capsid antigen p24, the FACS dosing method correlates viral titre to the number of cells transduced...order to aid viral binding. This method, called spinoculation, has been found very effective for T cells transduction...Yasutsugu Suzuki and Youichi Suzuki (2011). Gene Regulatable Lentiviral Vector System, Viral Gene Therapy,...

  22. Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

    Type
    Blog Post
    ...Stable Cell Lines with Lentivirus Isolating a Monoclonal Cell Population by Limiting Dilution Lentivirus...high Cas9 MOI considering that A549 cells are relatively easy to transduce. I ended up with too many Cas9...be uniform editing across all the cells in the population, so some of the cells will still appear green...efficiently editing, don’t expect a 100% knockout population. BRAF gRNA delivery  Overview: In practice you...on genomic DNA. The surveyor assay itself was relatively easy. Figure 4: Detection of edited BRAF...get an idea of the percentage of cells in the population that have an edit. I didn’t do this, but to do...

  23. "Build Your Plasmid": The Game - Play to Teach

    Type
    Blog Post
    ...genetic manipulation more hands on While teaching courses about gene therapy and genetic manipulation, I and...on genetic manipulation of human stem cells when students are not even allowed to manipulate prokaryotic...transfer plasmid that could be used to genetically manipulate human stem cells. Then they had 10 minutes to...explain why they selected a certain target cell population. Who is the winner? Ultimately, society should...

  24. FPbase: A new community-editable fluorescent protein database

    Type
    Blog Post
    ...Kurt Thorn (UCSF at the time) began an effort to collate some of the current “best” FPs, and I collaborated...using simple web forms) while also maintaining a relatively strict database design (schema).  Registered ...allows for visual comparison of FPs and how they relate to one another. .  In addition, the FPbase Fluorescence...feature filter spectra and collection efficiency calculations.  With ~80 FP spectra (and rapidly growing, ... page with all the information in the database relating to that specific protein (for instance: EGFP)....retrieve all of the information in the database relating to that set of proteins.  For computer-savvy users...

  25. Hot Plasmids - September 2019 - CRISPR, Auxin Inducible Degradation, MerMAID Channelrhodopsins, and Genome Looping

    Type
    Blog Post
    ...technology (Yesbolatova et al., 2019). Find the mAID tagging toolkit at Addgene Yesbolatova et al., Methods...ubiquitin ligase, the Kanemaki lab was able to translate AID to yeast, as well as mammalian and human cell...tagging of proteins with mAID and other tags (Yesbolatova et al., 2019) using CRISPR. This toolkit makes...thought to play a key role in the transcriptional regulation of many genes. A number of groups have designed...synthetic looping factors developed thus far for these latter experiments, such as transcription factors tethered...

  26. Quantifying DNA? Here are Five DNA Quantification Methods to Consider

    Type
    Blog Post
    ...might not have to do the calculation yourself as many fluorometers will calculate your sample concentration...than 1.5. Then, using the A260 reading, you can calculate the DNA concentration. Generally, A260 of 1.0 ... start by pouring your gel containing a DNA intercalating dye (ex: ethidium bromide) and choosing a DNA...quantify based on band intensities of your DNA relative to the intensity in the ladder. It’s best to compare...quantified over time using a fluorescent dye that intercalates into the DNA such that smaller fragments are...

  27. Plasmids 101: How to Verify Your Plasmid Using a Restriction Digest Analysis

    Type
    Blog Post
    ...Watch out for methylation issues. Enzymes like XbaI and ClaI are sensitive to methylation and their activity... Congratulations, you have a plasmid expressing your gene of interest (YGOI) and are ready to dive into...the reaction on an agarose gel to determine the relative sizes of the resulting DNA fragments. Restriction...will need to purify your DNA from a dcm or dam methylation-deficient bacterial strain such as JM110 or INV110...enzymes are working. When uncut plasmid DNA is isolated and run on an agarose gel, you are likely to see...

  28. Choosing a CRISPR Nuclease: Site Accessibility, Specificity, and Sensitivity

    Type
    Blog Post
    ... Sensitivity Cas9 sensitivity is often closely related to specificity. Sensitivity is a measure of the...site. A specificity ratio can be measured by calculating the on-target activity divided by the off-target... SpCas9 described the PAM specificity of Cas9s isolated from different organisms and engineered Cas9 variants...the enzymes showed decreased off-target activity relative to Cas9. Since then, the Joung and Jennifer Doudna...showed decreased or similar off-target activity, relative to eSpCas9 and SpCas9-HF across a number of different...research due to its small size and specificity relative to Cas9. As scientists continue to characterize...

  29. Sharing is Caring: But How to Distribute Open Hardware?

    Type
    Blog Post
    ...devices for research. There are a number of new platforms (such as openUC2, which I am a co-founder of),...that make everyday microscopy much easier. On platforms such as Github, users can download the software...example, can also be printed quickly in Madagascar. Platforms that allow for on-going creativity with the construction...organizations and companies to provide problem-solving platforms.     Fig. 3: Different workflows involved...their work on the shoulders of giants. The still relatively young movement of sharing open hardware has a...Prakash, "Squid: Simplifying Quantitative Imaging Platform Development and Deployment." bioRxiv. 2020. Diederich...

  30. Plasmids 101: Yeast Vectors

    Type
    Blog Post
    ...limited to: rapid growth, ease of replica plating and mutant isolation, a well-defined genetic system, and ...and animals. Unlike bacteria, yeast can post-translationally modify proteins yet they still share many of...a previous post, we briefly discussed how the regulation of bacterial ORIs determines plasmid copy number...these vectors can also be controlled if specific regulatable elements are included (reviewed here). Plasmids...complementation is weak. yes   LYS2 L-lysine yes - Grow on plates containing alpha-aminoadipate in the absence of...

  31. Antibodies 101: Designing Your First Flow Panel

    Type
    Blog Post
    ...brightness for a few commonly used fluorophores in relation to the laser type. Table adapted from: https:/...are absorbed and then emitted, which creates a relatively stronger — or brighter — signal.   Figure...use brighter fluorophores for markers that are relatively lower in abundance. In contrast, when you expect...the spectra of enhanced GFP (eGFP) and FITC, the latter being a commonly used fluorophore for antibody-...again. In the beginning, we talked about the relationship between a photon's wavelength and energy, how... into different levels of brightness and their relation to the fluorophore's excitation efficiency, and.... His projects include investigating negative regulators of T cells and cytokine signaling in tumor immunity...

  32. Stabilized Bacterial Promoters: Constant Gene Expression at any Copy Number

    Type
    Blog Post
    ...Unfortunately, plasmid copy numbers vary within cell populations and over time resulting in variable gene expression... use of stabilized promoters. In contrast to unregulated promoters that are always on, stabilized promoters...expression. However, if you introduce negative regulation by using a promoter that is responsive to a repressor...that the stabilized promoters were able to better modulate gene expression during shifts in growth rate as... as compared to a constitutive promoter. To simulate the increase in plasmid copy number during the transition...generation of the desired product while avoiding the accumulation of toxic intermediates or the shuttling of the...

  33. Seven Tips for Using LinkedIn as a Scientist

    Type
    Blog Post
    ...is the question. When presenting on building relationships (also known as “networking”), one of the most...networking to start, build, and track professional relationships. Here are my seven best tips beyond the basics... “Joanne Kamens, PhD viewed your profile.”  The latter is much less creepy. If we’ve met before, I’d be...on LinkedIn!” resulting in just a little more relationship. Pro tip: Once you are connected with someone...connection requests LinkedIn is a place to build relationships. If you don’t add a personal note, you waste...volunteer organizational work. LinkedIn is not the platform for politics, family updates, or click bait of...

  34. To Each HIS Own

    Type
    Blog Post
    ...purification. In this strategy a single protein can be isolated from a complex mix using affinity chromatography...variety of methods can be used to successfully isolate proteins, this blog will focus on one of the most...the protein of interest and it is successfully isolated.   Prior to the polyhistidine tag, alternative...binding of untagged proteins. Despite being a relatively low frequency amino acid, proteins containing...questions about polyhistidine tags. For more antibody-related information, sign up to receive alerts for antibody...Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent. Bio/Technology. 1988; 6: 1321–1325. ...

  35. The Future of Research Symposium Boston 2015

    Type
    Blog Post
    ...future prospects. Among the topics discussed, many related to the structure of the biomedical workforce. Quotes...results, not scientists”  These comments are well correlated with published data. For example, in 2010, less...compulsory internship to be taken in an industry, regulatory, or clinical environment. It remains contested...6). Academic data and the labor market for PhDs Later this week, postdocs and students will again gather...grants through the Sloan foundation for postdoc-related data collection and analysis. He is also a strong...training to reflect the workforce." Science Translational Medicine (2015): 285ED4. Pubmed PMID: 25925677...

  36. Plasmids 101: Cre-lox

    Type
    Blog Post
    ...have adapted the components for use in genome manipulation. Cre recombinase, originally named because ...because it “causes recombination” (although later referred to as the “cyclization recombinase”), is a 38 kDa... in a DNA sequence allows for very specific manipulations as discussed below. How does it work? As alluded...expression – placing a series of transcriptional and translational termination features with loxP sites on either... can be used to easily perform this eviction. Regulated Cre expression – placing Cre downstream of promoters...express Cre under a variety of ubiquitous and regulated promoters, and many loxP-containing alleles have...

  37. A Look at Addgene's QC Process

    Type
    Blog Post
    ...Nobody’s perfect! This is a platitude I’m sure we have all heard. It’s a truth that extends to both people... of tools that rely on DNA amplification and manipulation to produce and edit plasmids. These are prone...polymerases can make mistakes and lead to the accumulation of errors in DNA sequences. In research, these...and identifying mixed plasmid and nucleotide populations. All of these topics require additional considerations...sequence and assemble using the Illumina MiSeq platform, such as GC-rich regions and highly repetitive...Plasmidsaurus for long-read nanopore sequencing. This platform sequences the entire plasmid in one read and thus...

  38. Viral Vectors 101: Chemogenetics

    Type
    Blog Post
    ...field as it provides a noninvasive method for manipulating cell activity, especially neuronal firing. ...approved drugs so that your results are viable for translation to human patients. Picking receptors The receptor...dependent on what cellular response you aim to manipulate. It is important to consider that the same receptor... in. The most common target for chemogenetic manipulation are neurons where chemogenetic receptors can...control ion flux. The system allows you to directly manipulate ion channels. When working with PSAM receptors...BL, Jin J (2015) The First Structure–Activity Relationship Studies for Designer Receptors Exclusively Activated...

  39. A Look Back at One Year of Plasmid Sharing for COVID-19 Research

    Type
    Blog Post
    ...received over 13,000 plasmids requests for COVID-19 related plasmids. These plasmids have reached labs in over...expression and pseudotyping efficiency. This plasmid was later used by another team of researchers for neutralization...opportunities to evolve. Several variants are now circulating and plasmids containing these mutations have ...are inhibitors of SARS-CoV-2 entry and become upregulated during viral infection.  Expression of SARS-CoV...their work, head over to their blog post. Months later, we partnered with Ginkgo Bioworks to provide synthesized...screens identify mucins as critical host factors modulating SARS-CoV-2 infection. bioRxiv. https://doi.org...

  40. Tips for Technical Support Calls

    Type
    Blog Post
    ...calls, I feel compelled to share my years of accumulated wisdom so that you, the customer, can get the...these tips and tricks will apply to any biology-related customer support interaction. Before contacting...paperwork associated with a product only to realize later that everything I could have wanted to know was ... science, but we don’t have the resources to recapitulate the experiments performed by our thousands of...use a particular reagent.  We also have links to later papers that successfully used said plasmids. We ...support scientists wear many hats at this still relatively small nonprofit. If you do go longer than a few...
Showing: 361 - 400 of 974 results