Over the past 20 years, protein engineering has been extensively used to improve and modify the fundamental properties of fluorescent proteins (FPs) with the goal of adapting them for a fantastic ...range of applications. FPs have been modified by a combination of rational design, structure-based mutagenesis, and countless cycles of directed evolution (gene diversification followed by selection of clones with desired properties) that have collectively pushed the properties to photophysical and biochemical extremes. In this review, we provide both a summary of the progress that has been made during the past two decades, and a broad overview of the current state of FP development and applications in mammalian systems.
Monomeric red and far-red FPs and indicators now perform nearly as well as the best green FPs (and indicators).
Reversible and irreversible photochromism in FPs can be exploited to increase optical resolution and improve contrast compared with traditional fluorescence microscopy.
Infrared FPs (IFPs) are becoming ever more useful as labels for various proteins that allow correct localization and whole-animal imaging. IFPs can serve as an additional fluorescent ‘color’ for simultaneous imaging with visible FP-labeled proteins.
Bacterial phytochrome (BphP)-based IFPs provide a new scaffold for engineering fluorogenic indicators, which are ideal to visualize spatiotemporal dynamics of cell signaling in vivo.
Small ultra-red FP (smURFP) is the brightest far-red nonprototypical FP (comparable with EGFP) and is extremely photostable. smURFP may prove particularly useful as a photostable FP for super-resolution imaging and as a FRET acceptor for biosensing applications.
The engineering of new fluorescent indicators that combine features of prototypical FP-based indicators with photochromic proteins can reveal the cellular maps of biochemical activities in super-resolution.
FPs can be used as optogenetic actuators to manipulate cellular and protein functions through chromophore-assisted light inactivation or light-controlled protein oligomerization.
In October 2017, the FDA granted regular approval to axicabtagene ciloleucel, a CD19-directed chimeric antigen receptor (CAR) T-cell therapy, for treatment of adult patients with relapsed or ...refractory large B-cell lymphoma after two or more lines of systemic therapy. Efficacy was based on complete remission (CR) rate and duration of response (DOR) in 101 adult patients with relapsed or refractory large B-cell lymphoma (median 3 prior systemic regimens) treated on a single-arm trial. Patients received a single infusion of axicabtagene ciloleucel, preceded by lymphodepleting chemotherapy with cyclophosphamide and fludarabine. The objective response rate per independent review committee was 72% 95% confidence interval (CI), 62-81, with a CR rate of 51% (95% CI, 41-62). With a median follow-up of 7.9 months, the median DOR was not reached in patients achieving CR (95% CI, 8.1 months; not estimable, NE), whereas patients with partial remission had an estimated median DOR of 2.1 months (95% CI, 1.3-5.3). Among 108 patients evaluated for safety, serious adverse reactions occurred in 52%. Cytokine release syndrome and neurologic toxicities occurred in 94% and 87% of patients, respectively, leading to implementation of a risk evaluation and mitigation strategy.
Gout is a common inflammatory arthritis caused by precipitation of monosodium urate (MSU) crystals in individuals with hyperuricemia. Acute flares are accompanied by secretion of proinflammatory ...cytokines, including interleukin-1β (IL-1β). Clonal hematopoiesis of indeterminate potential (CHIP) is an age-related condition predisposing to hematologic cancers and cardiovascular disease. CHIP is associated with elevated IL-1β, thus we investigated CHIP as a risk factor for gout. To test the clinical association between CHIP and gout, we analyzed whole exome sequencing data from 177 824 individuals in the MGB Biobank (MGBB) and UK Biobank (UKB). In both cohorts, the frequency of gout was higher among individuals with CHIP than without CHIP (MGBB, CHIP with variant allele fraction VAF ≥2%: odds ratio OR, 1.69; 95% CI, 1.09-2.61; P = .0189; UKB, CHIP with VAF ≥10%: OR, 1.25; 95% CI, 1.05-1.50; P = .0133). Moreover, individuals with CHIP and a VAF ≥10% had an increased risk of incident gout (UKB: hazard ratio HR, 1.28; 95% CI, 1.06-1.55; P = .0107). In murine models of gout pathogenesis, animals with Tet2 knockout hematopoietic cells had exaggerated IL-1β secretion and paw edema upon administration of MSU crystals. Tet2 knockout macrophages elaborated higher levels of IL-1β in response to MSU crystals in vitro, which was ameliorated through genetic and pharmacologic Nlrp3 inflammasome inhibition. These studies show that TET2-mutant CHIP is associated with an increased risk of gout in humans and that MSU crystals lead to elevated IL-1β levels in Tet2 knockout murine models. We identify CHIP as an amplifier of NLRP3-dependent inflammatory responses to MSU crystals in patients with gout.
•TET2-mutant clonal hematopoiesis is associated with increased risk of incident gout.•Monosodium urate crystal treatment exacerbates Nlrp3-dependent IL-1β secretion and functional impairment in transplanted Tet2-knockout mice.
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Dysregulated vascular inflammation is the underlying cause of acute lung inflammation/injury (ALI). Bacterial infections and trauma cause ALI that may rapidly lead to acute respiratory distress ...syndrome (ARDS). There are no pharmacological therapies available to patients with ALI/ARDS, partially as drugs cannot specifically target the lungs. Herein, we developed a stimuli-responsive nanoparticle (NP) to target inflammatory lungs for ALI therapies. The NP is composed of a sharp acid-sensitive segment poly(β-amino esters) as a core for drug loading and controlled release and a polyethylene glycol-biotin on the particle surface available for bioconjugation, enabling lung targeting and extended circulation. The studies on dissipative particle dynamics simulation and characteristics of NPs suggest that anti-ICAM-1 antibodies can be coated to the particle surface and this coating is required to enhance lung targeting of NPs. A model drug of anti-inflammatory agent TPCA-1 is encapsulated in NPs with a high drug-loading content at 24% (w/w). In the mouse ALI model, our TPCA-1-loaded NPs coated with anti-ICAM-1 can target inflamed lungs after intravenous injection, followed by drug release triggered by the acid environment, thus mitigating lung inflammation and injury. Our studies reveal the rational design of nanotherapeutics for improved therapy of ALI, which may be applied to treating a wide range of vascular inflammation.
A novel, common, and potent cardiovascular risk factor has recently emerged: clonal hematopoiesis of indeterminate potential (CHIP). CHIP arises from somatic mutations in hematopoietic stem cells ...that yield clonal progeny of mutant leukocytes in blood. Individuals with CHIP have a doubled risk of coronary heart disease and ischemic stroke, and worsened heart failure outcomes independent of traditional cardiovascular risk factors. The recognition of CHIP as a nontraditional risk factor challenges specialists in hematology/oncology and cardiovascular medicine alike. Should we screen for CHIP? If so, in whom? How should we assess cardiovascular risk in people with CHIP? How should we manage the excess cardiovascular risk in the absence of an evidence base? This review explains CHIP, explores the clinical quandaries, strives to provide reasonable recommendations for the multidisciplinary management of cardiovascular risk in individuals with CHIP, and highlights current knowledge gaps.
Clonal hematopoiesis (CH) can be transmitted from a donor to a recipient during allogeneic hematopoietic cell transplantation. Exclusion of candidate donors with CH is controversial since its impact ...on recipient outcomes and graft alloimmune function is uncertain.
We performed targeted error-corrected sequencing on samples from 1,727 donors age 40 years or older and assessed the effect of donor CH on recipient clinical outcomes. We measured long-term engraftment of 102 donor clones and cytokine levels in 256 recipients at 3 and 12 months after transplant.
CH was present in 22.5% of donors, with
(14.6%) and
(5.2%) mutations being most common; 85% of donor clones showed long-term engraftment in recipients after transplantation, including clones with a variant allele fraction < 0.01.
CH with a variant allele fraction ≥ 0.01, but not smaller clones, was associated with improved recipient overall (hazard ratio HR, 0.79;
= .042) and progression-free survival (HR, 0.72;
= .003) after adjustment for significant clinical variables. In patients who received calcineurin-based graft-versus-host disease prophylaxis, donor
CH was associated with reduced relapse (subdistribution HR, 0.59;
= .014), increased chronic graft-versus-host disease (subdistribution HR, 1.36;
= .042), and higher interleukin-12p70 levels in recipients. No recipient of sole
or
-CH developed donor cell leukemia (DCL). In seven of eight cases, DCL evolved from donor CH with rare
or splicing factor mutations or from donors carrying germline
mutations.
Donor CH is closely associated with clinical outcomes in transplant recipients, with differential impact on graft alloimmune function and potential for leukemic transformation related to mutated gene and somatic clonal abundance. Donor
-CH is associated with improved recipient survival because of reduced relapse risk and with an augmented network of inflammatory cytokines in recipients. Risk of DCL in allogeneic hematopoietic cell transplantation is driven by somatic myelodysplastic syndrome-associated mutations or germline predisposition in donors.
Genetically encoded sensors based on fluorescence resonance energy transfer (FRET) are powerful tools for reporting on ions, molecules and biochemical reactions in living cells. Here we describe the ...development of new sensors for Zn²⁺based on alternate FRET-pairs that do not involve the traditional CFP and YFP. Zn²⁺ is an essential micronutrient and plays fundamental roles in cell biology. Consequently there is a pressing need for robust sensors to monitor Zn²⁺ levels and dynamics in cells with high spatial and temporal resolution. Here we develop a suite of sensors using alternate FRET pairs, including tSapphire/TagRFP, tSapphire/mKO, Clover/mRuby2, mOrange2/mCherry, and mOrange2/mKATE. These sensors were targeted to both the nucleus and cytosol and characterized and validated in living cells. Sensors based on the new FRET pair Clover/mRuby2 displayed a higher dynamic range and better signal-to-noise ratio than the remaining sensors tested and were optimal for monitoring changes in cytosolic and nuclear Zn²⁺. Using a green-red sensor targeted to the nucleus and cyan-yellow sensor targeted to either the ER, Golgi, or mitochondria, we were able to monitor Zn²⁺ uptake simultaneously in two compartments, revealing that nuclear Zn²⁺ rises quickly, whereas the ER, Golgi, and mitochondria all sequester Zn²⁺ more slowly and with a delay of 600-700 sec. Lastly, these studies provide the first glimpse of nuclear Zn²⁺ and reveal that nuclear Zn²⁺ is buffered at a higher level than cytosolic Zn²⁺.
Designing suitable material systems to construct artificial synapses and exploring novel synaptic functions is a crucial step toward the realization of efficient large‐scale bioinspired neuromorphic ...systems. In this work, flexible and insoluble bio‐memristor devices are fabricated by precisely engineering the molecular structures of wool keratin. This flexible Ag/keratin/indium tin oxide‐polyethylene naphthalate synaptic device possesses enhanced mechanical resistance, which is achieved by photo‐cross‐linking keratin molecules, and can withstand a bending radius of up to 1.2 mm. This device is promising for implantable applications because it is water‐resistant. When modulated by triangle‐wave DC voltages and pulsed voltages, this flexible electronic device emulates typical memristor characteristics and synaptic functions, including potentiation/depression, spike timing dependent plasticity, and long‐term/short‐term plasticity. Simulation results indicate that a memristor network made by this wool‐keratin based device has ≈95.8% memory learning accuracy and capability for pattern learning. Combined, these features prove that the cross‐linked wool‐keratin based device has potential in wearable and flexible neuron computing systems.
A cross‐linked wool keratin based flexible artificial synapse is fabricated by precisely engineering molecular structures, which achieve synaptic functions successfully, including potentiation/depression, STDP, LTP/STP, PPF. After large numbers of bending and long‐term water immersion, the device remains functional. The results reveal that the resistance and synaptic behaviors of keratin are enhanced by chemical cross‐linking treatment.
Intratumor heterogeneity remains a major obstacle to effective cancer therapy and personalized medicine. Current understanding points to differential therapeutic response among subpopulations of ...tumor cells as a key challenge to successful treatment. To advance our understanding of how this heterogeneity is reflected in cell-to-cell variations in chemosensitivity and expression of drug-resistance proteins, we optimize and apply a new targeted proteomics modality, single-cell western blotting (scWestern), to a human glioblastoma cell line. To acquire both phenotypic and proteomic data on the same, single glioblastoma cells, we integrate high-content imaging prior to the scWestern assays. The scWestern technique supports thousands of concurrent single-cell western blots, with each assay comprised of chemical lysis of single cells seated in microwells, protein electrophoresis from those microwells into a supporting polyacrylamide (PA) gel layer, and in-gel antibody probing. We systematically optimize chemical lysis and subsequent polyacrylamide gel electrophoresis (PAGE) of the single-cell lysate. The scWestern slides are stored for months then reprobed, thus allowing archiving and later analysis as relevant to sparingly limited, longitudinal cell specimens. Imaging and scWestern analysis of single glioblastoma cells dosed with the chemotherapeutic daunomycin showed both apoptotic (cleaved caspase 8- and annexin V-positive) and living cells. Intriguingly, living glioblastoma subpopulations show up-regulation of a multidrug resistant protein, P-glycoprotein (P-gp), suggesting an active drug efflux pump as a potential mechanism of drug resistance. Accordingly, linking of phenotype with targeted protein analysis with single-cell resolution may advance our understanding of drug response in inherently heterogeneous cell populations, such as those anticipated in tumors.
A dearth of protein isoform-based clinical diagnostics currently hinders advances in personalized medicine. A well-organized protein biomarker validation process that includes facile measurement of ...protein isoforms would accelerate development of effective protein-based diagnostics. Toward scalable protein isoform analysis, we introduce a microfluidic "single-channel, multistage" immunoblotting strategy. The multistep assay performs all immunoblotting steps: separation, immobilization of resolved proteins, antibody probing of immobilized proteins, and all interim wash steps. Programmable, low-dispersion electrophoretic transport obviates the need for pumps and valves. A three-dimensional bulk photoreactive hydrogel eliminates manual blotting. In addition to simplified operation and interfacing, directed electrophoretic transport through our 3D nanoporous reactive hydrogel yields superior performance over the state-of-the-art in enhanced capture efficiency (on par with membrane electroblotting) and sparing consumption of reagents (ca. 1 ng antibody), as supported by empirical and by scaling analyses. We apply our fully integrated microfluidic assay to protein measurements of endogenous prostate specific antigen isoforms in (i) minimally processed human prostate cancer cell lysate (1.1 pg limit of detection) and (ii) crude sera from metastatic prostate cancer patients. The single-instrument functionality establishes a scalable microfluidic framework for high-throughput targeted proteomics, as is relevant to personalized medicine through robust protein biomarker verification, systematic characterization of new antibody probes for functional proteomics, and, more broadly, to characterization of human biospecimen repositories.
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