.... The overlap here is that it is unethical to use scientifically unproven technologies to edit a human...human clinical trials. This model also demonstrates that when we are at the point of scientifically rigorous...throughout the history of medical research, there have always been people who unethically experiment on humans...Cas9 genome editing is not far enough along, scientifically, to be doing in humans; We have not had a broad... recently published a report on the social and ethical issues of genome editing and human reproduction...research into both novel and previously established ethical issues raised around mitochondrial replacement ...respective mandates, to tackle a controversial and ethically fraught rapidly emerging technology. We could ...

...process allows you to physically magnify a sample so you don’t have to optically magnify quite so much...compatible with conventional antibodies, chemical dyes, and genetically encoded fluorescent proteins (Gambarotto... and the physical properties of the sample itself. For example, cultured cells are typically easier to...promises an easier, more accessible way to image biological features previously only visible via techniques...techniques also require specialized instruments and technical expertise, limiting their accessibility (Prakash... bigger? In ExM, and its various derivatives, biological samples are enmeshed in a gel of a highly absorbent...be expanded to ~4-10x linearly! This expansion physically separates molecules while maintaining their relative...

...the tumor, this results in frequent physiological and morphological changes. Tumors differ in their genotypic...their level of progression, anatomical location, characteristic (solid localized tumors or systemic tumors...favorably attaining the equilibrium stage. This can be called “stalemate equilibrium” where the cancer stops ...either due to age, infections, or other strenuous biological changes. In contrast to the acute immune responses...themselves from attack. It is a region of incessant chemical activity, and consists of other cell types, especially...attack the cancer cells are also suppressed by chemical signals such as IL-8, IFNγ, and colony stimulating...contributions from Madame Curie), chemotherapy, and surgical removal of the tumor. However, these conventional...

...coverslip creates mechanical tension, and the luminescence colocalizes with the focal adhesion marker paxillin... used the reporter to visualize biologically relevant forces at focal adhesions in living cells in several...Sensors for Bioluminescent Readout of Mechanical Forces in Biological Systems. ACS Sensors, 9(7), 3489–3495...disruption in M. abscessus  Bioluminescent reporter of mechanical forces   Epitope tag antibody collection grows... forms that limit its functionality. To make biophysical and structural studies more feasible, the lab...properties, and is amenable to a wide range of biophysical assays (Kroupova et al., 2024). They were able... Cereblon construct for crystallographic and biophysical studies of protein degraders. Nat Commun., 15...

...of human (and most eukaryotic) proteins localize to chemically distinct subcellular environments, including...sequence intended to localize them to the secretory pathway, a dark pool that is mislocalized to the ER can ...in the eukaryotic secretory pathway."Biochemical and biophysical research communications 430.3 (2013):...While Vader was referring to the evil side of a mystical "Force," this quote is equally applicable to many...microscopy experiments with fluorescent proteins (FPs) localized to compartments other than the cytoplasm. That...resident endoplasmic reticulum (ER) protein instead localizes to the cytoplasm? Or more troubling, what if a...properly, FPs enable researchers to investigate the localization and dynamics of fluorescent protein fusions ...

...transmitted transsynaptically). This is because the recombinant forms of these viruses typically used in ... became foundational later on when we studied biological processes, like how our stem cells are housed... thus, are a major part of the answer to many biological questions about the brain. To address this, molecular...enter cells and deliver a variety of cargoes, genetically engineered neurotropic viruses emerged as powerful... (Nassi et al. 2015). The recombinant viruses typically used in neuroscience research (e.g., AAV) are ...spread is useful for neuronal tracing, it has two practical limitations. First, these high levels of viral...starter cell types, as well as to only spread monosynaptically from these cells. These modifications take...

..., an optogenetically controlled phospholipase D (PLD). Now they present a set of variants called SuperPLDs...membranes, indicated by the PA probe colocalizing with SuperPLDx30. Scale bar 10 µm. Images adapted from Tei...components of the cytoskeleton. They provide cells with physical structure, separate chromosomes during mitosis...Akhmanova Lab published a new optogenetic tool for localized microtubule disassembly, Opto-katanin (Meiring...Figure 1). Figure 1: Opto-katanin induces fast, localized microtubule severing. A) Schematic of Opto-katanin...Katanin is of course named for the samurai sword called a katana. Opto-katanin consists of two separate...a stable cell line. This design allows highly localized, rapid and reversible microtubule severing and...

...holding a physical model of the protein in your hand – you will understand the power of a physical model.... researchers use physical models of protein structure? We have been creating physical models of proteins...work closely with local researchers to learn about their research while creating physical models of proteins... your hand? At 3D Molecular Designs, we create physical models of proteins using 3D printing technology...and I encourage you to hold one. Why do we need physical models of protein structure? Structural biologists...problem with the development of Byron’s Bender – a mechanical device that could be used to bend 1/8th inch ... example, when Tom Steitz was presented with a physical model of his large ribosomal subunit, he kept ...

...data The result was a fusion protein called CalFlux VNT. CalFlux VNT is composed of Venus fluorescent ...and NanoLuc® Luciferase. CalFlux VNT responds to changes in intracellular calcium and is useful for direct... requires long acquisition times that may be impractical or infeasible for certain experimental setups... phototoxicity, autofluorescence, and perturb biological phenomena. The technique of coupling FPs with...RLuc8 moieties in their Nano-Lanterns with NLuc and called their improved creations Enhanced Nano-Lanterns...luciferase Many intracellular sensors of events like calcium release have been made using fluorescent resonance...generally consist of a responsive protein (e.g. calcium binding protein) fused to two fluorescent proteins...

... techniques in which non-canonical amino acids (ncAAs, also sometimes called unnatural amino acids) are...placed in a protein site-specifically to alter its function, mimic a biologically relevant modification,...proteins, you’re probably quite interested in the canonical 20 amino acids. But in your quest to learn more...modification, or add in fluorescent or chemically reactive side chains, all of which can help determine structure...structure and function relationships or, in more practical applications, allow a protein to be more effectively...was developed into an E. coli system, with the noncanonical amino acid in the media and the tRNAs and synthetase...use. First, and most obviously, you need a non-canonical amino acid, which is usually added to the media...

...ll use calcium biosensors as an example. Many calcium biosensors include GFP fused to calmodulin (CaM) ...content of this article. Biosensors (‘biological sensors’) are biological tools that monitor a process or detect...and the chicken myosin kinase calmodulin binding region RS20. When calcium is absent, the construct does...a reporter signal. Reporter signals may be electrochemical or light-based, with luminescent and fluorescent...like arabinose or hydrogen peroxide or ions like calcium and zinc. Some biosensors contain a single fluorescent...does not fluoresce; when present, calcium induces CaM/RS20 interaction, thereby changing the FP’s structural... biosensors from Robert Campbell’s lab display calcium-dependent increases in fluorescence of up to 11,000...

...editor-based clinical trial for SCD in 2022 and Editas Medicine starting a Cas12-based clinical trial for... no longer an accurate descriptor; progress in clinical therapy is more fitting, so it is time to update... approved or have made significant progress in clinical trials.     Figure 1: Types of delivery...some solid tumors are currently in early-phase clinical trials and have shown promising preliminary results...introduction of the cancer-fighting component, CAR. Clinical trials are underway to explore CRISPR’s capacity...for these diseases was sponsored by Vertex Pharmaceuticals and CRISPR Therapeutics. Data shared from these...-Cas construct targeting the diseased allele specifically. Early-stage trials showed a high success rate...

...Institute for Biological Studies) Multiple uses: From multiplexing to multiscaling NIR-shifted spectrum...and iGECI calcium indicator (Shemetov et al., 2021). In addition to FRET, intensiometric calcium indicators...biosensors for multiscale imaging. Nat Commun 7, 12405 (2016). Qian, Y. et al. A genetically encoded near-infrared...Shemetov, A.A. et al. A near-infrared genetically encoded calcium indicator for in vivo imaging. Nat Biotechnol...photoreceptors incorporating biliverdin IXα (BV) autocatalytically. Fortunately for probe developers and users...NIR FPs and biosensors can be imaged at spatial scales from sub-cellular to whole-body in small animals...performance, and applications of near-infrared calcium indicators in this recent review (Shcherbakova ...

...Volume: < 0.05 ml Skill required: advanced Intrathecal Intrathecal (IT) injections are probably the most advanced...location requires a lot of skill and anatomical knowledge. Intrathecal injections are used for a wide array...experiments. Some of these techniques are what we call “beginner”, or injections that are easy to pick ...easier of the two, but it actually requires more technical skill to place the needle in the correct vein ...institutional oversight is required. Restraint: mechanical restraint or anesthesia Location: tail vein or...you might after getting a vaccine. Restraint: mechanical restraint Location: thigh Gauge: 25–27 Volume...xenograft models. Restraint: back scruffing or mechanical restraint Location: back or hind legs Gauge: ...

...per gene approach that has been the historical standard. Specifically, in our recent publications, we used...these “variants of unknown significance” (sometimes called “VUS”) function is unknown. How can we make a decision... if the majority of information on each cancer clinical sequencing report includes rare variants that ...characterization For many years, we’ve hoped that large scale laboratory-based functional experiments that classify...from testing this hypothesis: There was no large scale, broadly available collection of mutant cDNA clones... variants across a large number of genes as systematically as possible. In developing these assays, we...tumorigenesis assays, drug epistasis assays, and morphological profiling can be used as generalizable strategies...

...understanding of complex biological processes. Many tools exist to perturb biological function in a direct...However, recent work has called this prevalent assumption into question. Specifically, CRISPR-generated mutants...limitations of any technique in the context of the biological question. For example, while gene knockouts have...makes traditional knockouts unattractive from a practical perspective. Therefore, techniques that function...Figure 1: Schematic of an RNAi-inducing transgene. Typically, the desired target of RNAi is expressed as inverted...cannot be used to investigate essential genes. Historically, gene silencing methods were thought to be afflicted...genes of interest to trigger RNAi activation. A typical RNAi experiment can be divided into three fundamental...

...their localization and expression. Qiu saw that inhibition of the PV neurons resulted in mechanical allodynia...Glutamate sensing reporters Abhi Aggarwal is a postbaccalaureate research technician in Kaspar Podgorski’s ... post-transfection, excited at 505 nm (right). Scalebar represents 100 µm. Image from Abhi Aggarwal. ...fourth year Ph.D. candidate at the University of California, Irvine where she studies opioid use disorder...will image these neurons using GCaMP7, measuring calcium signaling related fluorescence during morphine ... disorder,” says Hubbard. Figure 3: 20x confocal image of the periaqueductal gray (PAG), with red...Elizabeth Hubbard.   Haoyi Qiu: Pain response in mechanical allodynia Haoyi Qiu is a M.Sc. student at McGill...

...of the FRET pair. Since many biological processes occur within the typical FRET range, FRET efficiency ...nanometers of each other, or measure the tension in the helical structure of spider silk, or the activity of a ...Fluorescence Resonance Energy Transfer. Unlike the typical excitation and emission of an excited fluorophore...donor fluorophore to the acceptor fluorophore. The typical steps in FRET are: 1. Donor fluorophore excitation... between the fluorophores and serves as a small-scale ruler to measure distances that are too miniscule...Experiments using intermolecular FRET probes are typically used for studying protein-protein interactions...with an intermolecular probe is often either impractical (for biomolecules that are not proteins) or would...

...full support of local law enforcement, and, although there was at least one person vocally protesting us...Stephanie Hays, a researcher at the University of California Berkeley. It’s been half a year since the march...Alina Chan is a postdoctoral researcher at Harvard Medical School working on human artificial chromosomes....rains, it was weather befitting of the ominous political climate in which American scientists find themselves...don't line up with the scientific consensus and calling for billions of dollars in cuts to important research...science, I loved the idea and decided to go. At my local March for Science in Albany, NY, many tables were...scientist at the University of Colorado, Anschutz Medical Campus.  He studies viruses including HIV and is...

...the undesirable side products generated during chemical synthesis of these molecules. To make large amounts...will get you started (Obst et al., 2017)! It is specifically curated for protein expression in K. phaffii... on using K. phaffii, then the MoClo YTK Kit specifically designed for S. cerevisiae is at your service...plant-specific parts you‘ll need, including sub-cellular localization signals, plant markers, and reporters (Engler... arena! The Mammalian Toolkit was engineered specifically for the types of expression and integration ... AAV delivery. While Fragmid developers were specifically testing CRISPR tools, it should be easy enough... MK system was used successfully for protein localization and transport studies in HEK cells, recombinant...

...Spatially Confined Persistent Diradical Emitter. Journal of the American Chemical Society, 145(25), 13615–13622... Andrew York, Maria Ingaramo, and their team at Calico Life Sciences recently set out to do just that ... many organisms) are opaque, which makes most biomedical applications of these tools difficult or impossible...some previous attempts at magnetic control of biological systems (Wheeler et al., 2016; Duret et al., ... were very small or were possible only in non-biological conditions. So, they decided to search for a ...removed (Figure 1). At first, Andrew and Maria were skeptical. The team was careful to test the possibility ...record of developing new microscopy tools for biological applications across a wide range of topics. He...

...Foundation, an organization working to advance biological engineering openly so as to benefit all people...with the BioBricks Foundation to distribute the physical DNA samples, for when that's the preferred mode...parts must flow.' People must be able to use genetically encoded functions - the words in our language...operating system for engineering an entire cell. Typically when people use plasmids, they are trying to do...the entirety of a cell. That's a big long-term technical challenge. We have a strategy that organizes how... engineering of the central dogma at the genome scale, it's going to be tricky to tame the rest of a cell...gates collection of parts implements the basic logical functions, which we know and love. Boolean is in...

...the gut side of things, the authors optigenetically and chemically excited a class of enteroendocrine ...behavior, or the animals being monitored were physically hooked up to invasive, cumbersome systems during...and stimulate or manipulate responses through neurological means like electric stimulation, drug administration...and whistles. The fibers are made from a process called thermal drawing, essentially where blocks of polymers... added - the ‘bells and whistles’ – including microscale light emitting diodes, thermal sensors, and microfluidic... previous implementation of optogenetic tools, calcium sensors, and other common neuroscience tools into...programmable microcontroller, NeuroStack coordinates optical stimulation of its associated fibers across multiple...

...higher specificity and reduced technical variability compared to chemical tracers. rAAV2-retro also has...memory, a special type of memory which includes autobiographical events (see Jin & Maren, 2015). Directional...Further, the connections between regions are often reciprocal, forming a neuronal loop which is activated and...pathways in neurons. Anterograde transport is typically used for the trafficking of organelles, such as... with other neuroscience methods such as electrophysiological recordings. The origins of these methods...serotypes, have been found naturally or have been genetically engineered to have differences in their capsid...plasmids at Addgene). The Gradinaru Lab at the California Institute of Technology generated novel AAVs ...

...Miller-Rhodes from the University of Rochester Medical Center.  You’ve probably heard of Forster Resonance...used the method to interrogate the subcellular localization of interactions between basic leucine zipper...it requires can lead to aberrant subcellular localization of the studied PIPs. The straightforward nature...2018), the generation of such resources in less genetically tractable organisms lags behind. BiFC can also...to the N- and C-terminal end of each PIP and empirically test which combination provides the best fluorescent...counterparts using western blotting. The subcellular localization of BiFC constructs should also be compared against...example), as this can also be perturbed by supraphysiological expression levels. BiFC quantification This...

...D) teams at pharma companies carry out preclinical and clinical research to discover and test the best...molecular behaviors of chemicals and proteins to learn how a particular drug or medical device works. This... consulting? Healthcare consultants advise pharmaceutical companies, biotech start-ups, health insurance...efficient ways of delivering healthcare. Clients typically engage consultants as short-term experts to work...different kinds of challenges that consultants get called upon to tackle, depending on the part of the company...acquisitions of or collaborations with other companies. Medical Affairs groups work on building awareness among...among physicians and scientists about clinical research, drugs, the diseases they can treat, and diagnostic...

... patients, specifically children and patients with skin disorders for research and clinical trials, as...system thus provides a practical way to generate transgene-free, virus-free clinical grade hiPSCs from easily...22422317. 6. Diecke, S., et al., Recent technological updates and clinical applications of induced pluripotent...Their work opened up vast possibilities for the clinical and therapeutic applications of iPSCs. The generation...SOX2, KLF4, and C-MYC [1]. For the derivation of clinical grade hiPSCs, reprogramming methods that do not...therapy trials and remains a safety concern in the clinical field [4]. Three major reprogramming methods are... are considered when generating hiPSCs for clinical purposes: mRNA transfection- this allows transient...

... tools for all-optical electrophysiology A major challenge in the field of all-optical electrophysiology...response to blue light, which will ultimately co-localize the VP64 transactivator with a dCas9 that has ...expression Brilliant technologies adopt basic biological concepts and employ them in creative and innovative...protein multimerization is seen in many important biological contexts, such as the enhancement of transcriptional... of a transcription factor to a promoter and localization of a protein via the presence of multiple copies...system occurs via antibody-peptide labeling; specifically, cognate peptide epitopes fused to the protein...fluorescent and luminescent siganls and a ratio is calculated (the BRET ratio), which allows for compensation...

...next logical step after technical mastery. However, being a great scientist doesn’t automatically translate...transition While a PhD equips you with exceptional technical skills, transitioning into management often means...stepping into unfamiliar territory. Unlike solving technical problems in the lab, management involves a different...time, I discovered that I not only enjoyed the technical aspects of my work but also found fulfilment in...your team's—can lead to frustration. Clinging to technical tasksDelegation can feel unnatural for those accustomed...myself to focus on my core responsibilities, which typically revolved around the broader vision rather than...insights, lessons from their experiences, and practical advice for overcoming challenges. Experimenting...

...revolves around synthetic humans called replicants which are genetically engineered by a powerful corporation...fused a catalytically impaired Cas9 protein to a DNA-editing enzyme to generate the prototypical base editor...STUDIES ON THE CHEMICAL NATURE OF THE SUBSTANCE INDUCING TRANSFORMATION OF PNEUMOCOCCAL TYPES. The Journal...Rallapalli, a graduate student at the University of California, San Diego. The revolution in genetic engineering...technology led to the development of many important medical and agricultural products. But it also made SciFi...break at the target genomic location. This is typically followed by non-homologous end-joining that results...sees the USS Enterprise crew fight against a genetically engineered superhuman Khan. The novel, Inferno...

...intramuscular (IM), intrathecal (IT), and intravenous delivery (IV). In mice, IV injections are typically administered...rather than being geographically constrained to the brain. In cases where it’s critical to minimize or eliminate...crossing into the brain. While helpful from a biological standpoint, it makes delivering AAVs to the brain...throughout the organism to target large or diffuse biological structures. Of primary interest here are systemic...peripheral nervous system (PNS). (Capsids, if you recall, are the outer shell of an AAV vector, which determine...Where do they come from? Systemic capsids are typically derived from AAV9, which is one of a handful of...capsid applications. For example, AAV-PHP.eB is typically administered at a total dose between 1 x 1011 ...

...the nucleus, also called the 4D nucleome. Visualizing specific loci has historically been difficult, as...successfully resolved loci with physical map distances of 2 and 75 Mbp, with the calculated fluorescent distances...to label certain loci, but this approach is not scalable for every locus...unlike CRISPR. Early CRISPR ...Figure 1: CRISPRainbow was used to track the localization of 6 chromosome-specific loci in the U20S cancer... every genomic locus, and to do so in vivo. Catalytically dead Cas9 (dCas9) cannot induce gene editing... to a given genomic locus, you can track the localization and movement of this locus in live cells! This...distances correlating with the previously established physical map. In comparing pairs of targets ~2 Mbp apart...

...plates and gels, which either don’t scale or have limited scaling. The downside of high-throughput work...Designing precise calculations for any of the numbers in this post is a mathematical story well beyond... Addgene provides large-scale savings. How large? Using Doench’s calculations and the 2018 numbers for...Debate of ’18, Blue Flame depositor John Doench calculated the time to make a plasmid. Doench’s lab in the...time it takes to make a plasmid could increase dramatically if you are piecing together several PCR products...long does it take in total? While much harder to calculate, in both our 2018 and 2023 poll, most people said...much does it cost to make a plasmid? Doench also calculated the cost of consumable reagents used to make ...

... a slightly slower scale.  All living cells with a membrane possess an electrical state. This state, ...potential (Vmem), can be measured as the imbalance of electrical charge between the inside and the outside of ...bioelectricity is revealing the important role of bioelectrical signaling mediated by ion-channels and pumps...affect patterning and in some cases even override genetically encoded patterning. Based upon trends in studies...formation of whole eyes from cell types that don’t typically give rise to eyes (Pai et al., 2011).  Additionally...head and a tail, and when gap junction-mediated electrical communication is briefly inhibited in the entire... two-headed worm, despite the fact that it’s genetically wild type  (Oveido et al., 2010). One of the ...

... brainstorming new technological innovations. Let’s take a look at three historical techniques: southern...computers, cell phones, and cars become more technologically advanced leaving earlier versions obsolete,...that save both time and money. Yet, knowledge of historical techniques comes in handy whether you are perusing...cloning or knockout experiments to forensics and clinical diagnostics. One example of southern blotting ...fragments of a DNA sequence of interest to create a physical map of the DNA. Starting in the 1970s, restriction...keep restriction enzymes on hand for cloning and analytical digests. A researcher can test the orientation...). Sanger sequencing uses modified nucleotides, called dideoxynucleotides (ddNTPs), lacking the 3’ hydroxyl...

...Sensitivity in Western Blotting. Journal of Pharmacological and Toxicological Methods, 64, 233–237. https://doi....to get right. If you’ve thoughtfully done the technical design of your blot, but are still finding yourself... be done at 4 °C or on ice, whichever is more practical. If your lysing method is not resulting in your...Most people will simply load an extra ladder or technical replicate (ie, leftover sample) to fill the relevant...you may want to consult Tables 1 and 2 in our Technical Design of a Western Blot post to make sure you...transfer. Troubleshooting The two most common (non-technical) issues with transferring are smaller proteins...than just high background, your issue is likely technical. White circles, for example, could indicate that...

...UCIBIO) focused on understanding the physiological and pathological roles of human cell glycans. Accordingly... biosynthetic pathway and have a wide range of clinical manifestations. PMM2-CDG is the most frequent ...reported that PMM2-CDG patients suffer from immunological defects. These are characterized by a predisposition...these lines to study how immune processes and biological pathways are altered in PMM2-CDG. These studies...reducing their risks for infection and other pathological conditions. References: Monticelli, M., Ferro... J., dos Reis Ferreira, V. & Videira, P. A. Immunological aspects of congenital disorders of glycosylation...Biology at the University of Colorado Anschutz Medical Campus in Aurora, Colorado, USA. The primary focus...

...notable in recent years. A variant of V-U, paradoxically called V-K, is a CRISPR-associated transposase ... CRISPR-Cas systems. Philosophical Transactions of the Royal Society B Biological Sciences, 374(1772),...What’s in a type? That which we call CRISPR, by any other name would…probably still edit genomes. Understanding...function and composition; subtypes and variants are typically based on species of origin. There are currently...systems. Type III systems are unique, as they can technically target both RNA and DNA, though it is largely...systems function, a III-E effector was developed, called Cas7-11. Despite being in Class 1, Cas7-11 is a... that other CRISPR types do, due to a lack of canonical CRISPR features (Pinilla-Redondo et al., 2019;...

...signal is in hot and the anatomical B-mode images are in greyscale. (scalebar = 2mm)   We hope you...the tumors was overlaid with the anatomical B-mode images in greyscale.   ARG expression in mammalian...production of air-filled protein nanostructures called gas vesicles, which scatter sound waves and thereby...contrast in E. coli. We deleted an unannotated hypothetical protein, improving expression, and named this...of the mARG plamsids into HEK293T cells using a chemical transfection reagent (e.g. PEI-MAX). Simply cotransfect...images of GV expressing MDA-MB-231 cells (top, scalebar = 10µm) and TEM of GVs purified from MDA-MB-231...cell lysate after 4-days of expression (bottom, scalebar = 0.5µm). (d) Ultrasound imaging of MDA-MB-231...

...said to be representative or the statistical variation is calculated and placed in the legend. Widely ...publication called Scientific Data, which aims to act as a peer-reviewed repository for “scientifically valuable... errors in assumptions, use of inappropriate statistical methods, omission of some data, etc.  The firestorm...months or years before others. In some cases, typically consortia involving genomic screens, the data ...  This means that supporting datasets must be logically annotated and arranged, as in an actual publication...published, this data is usually condensed into a statistically evaluated set of values.  However, to evaluate...likely to improve research. What is published is typically a tiny fraction of what was actually carried out...