Ferroportin 3 is a dual-targeted mitochondrial/chloroplast iron exporter necessary for iron homeostasis in Arabidopsis.

Mitochondria and chloroplasts are organelles with high iron demand that are particularly susceptible to iron-induced oxidative stress. Despite the necessity of strict iron regulation in these organelles, much remains unknown about mitochondrial and chloroplast iron transport in plants. Here, we propose that Arabidopsis ferroportin 3 (FPN3) is an iron exporter that is dual-targeted to mitochondria and chloroplasts. FPN3 is expressed in shoots, regardless of iron conditions, but its transcripts accumulate under iron deficiency in roots. fpn3 mutants cannot grow as well as the wild type under iron-deficient conditions and their shoot iron levels are lower compared with the wild type. Analyses of iron homeostasis gene expression in fpn3 mutants and inductively coupled plasma mass spectrometry (ICP-MS) measurements show that iron levels in the mitochondria and chloroplasts are increased relative to the wild type, consistent with the proposed role of FPN3 as a mitochondrial/plastid iron exporter. In iron-deficient fpn3 mutants, abnormal mitochondrial ultrastructure was observed, whereas chloroplast ultrastructure was not affected, implying that FPN3 plays a critical role in the mitochondria. Overall, our study suggests that FPN3 is essential for optimal iron homeostasis.

Lossless immunocytochemistry using photo-polymerized hydrogel thin-films.

Immunocytochemistry (ICC), or immunofluorescence microscopy, is an essential biological technique for phenotyping cells in both research and diagnostic applications. Standard ICC methods often do not work well when the cell sample contains a small number of cells (<10 000) because of the significant cell loss that occurs during washing, staining, and centrifugation steps. Cell loss is particularly relevant when working with rare cells, such as circulating tumor cells, where such losses could significantly bias experimental outcomes. In order to eliminate cell loss in ICC protocols, we present a method to encapsulate the cell sample in a photo-polymerized hydrogel thin-film. The hydrogel thin-film is permeable to antibodies and other ICC reagents, thereby allowing the use of standard ICC protocols without modification. The cell sample is physically constrained by the hydrogel at the bottom surface of a standard (unmodified) imaging microtiter plate, thereby enabling the acquisition of high-quality micrographs regardless of the properties of the cell sample or staining reagents. Furthermore, while standard ICC requires several centrifugation steps during staining and washing, our hydrogel encapsulation method requires only a single centrifugation step. This property greatly reduces the time required to perform ICC protocols and is more compatible with robotic platforms. In this study, we show that standard ICC and Cytospin protocols are extremely lossy (>70% loss) when the sample contains less than 10 000 cells, while encapsulating the cells using a permeable hydrogel thin-film results in a lossless ICC process.

Simultaneous Detection of Protein and mRNA in Jurkat and KG-1a Cells by Mass Cytometry.

Mass cytometry uniquely enables high-dimensional single-cell analysis of complex populations. This recently developed technology is based on inductively coupled time-of-flight mass spectrometry for multiplex proteomic analysis of more than 40 markers per cell. The ability to characterize the transcriptome is critical for the understanding of disease pathophysiology, medical diagnostics, and drug discovery. Current techniques allowing the in situ detection of transcripts in single cells are limited to a small number of simultaneous targets and are generally tedious and labor-intensive. In this report, we present the development of a multiplex method for targeted RNA detection by combining the mass cytometry and RNAscope® platforms. This novel assay, called Metal In Situ Hybridization (MISH), includes the hybridization of RNA-specific target probes followed by signal amplification achieved through a cascade of hybridization events, ending with the binding of amplifier-specific detector probes. The detector probes are tagged with isotopically pure metal atoms used for detection by mass cytometry. Proof-of-principle experiments show the simultaneous detection of three mRNA targets in Jurkat cells in suspension cell assay mode. The localization of transcripts was also investigated using the imaging mass cytometry platform in Jurkat and KG-1a cells. In addition, we optimized the antibody staining procedure to allow the co-detection of mRNA and cell surface markers. Our data demonstrate that MISH can be used to complement protein detection by mass cytometry as well as to investigate gene transcription and translation in single cells. © 2017 International Society for Advancement of Cytometry.

Fully Automated RNAscope In Situ Hybridization Assays for Formalin-Fixed Paraffin-Embedded Cells and Tissues.

Biomarkers such as DNA, RNA, and protein are powerful tools in clinical diagnostics and therapeutic development for many diseases. Identifying RNA expression at the single cell level within the morphological context by RNA in situ hybridization provides a great deal of information on gene expression changes over conventional techniques that analyze bulk tissue, yet widespread use of this technique in the clinical setting has been hampered by the dearth of automated RNA ISH assays. Here we present an automated version of the RNA ISH technology RNAscope that is adaptable to multiple automation platforms. The automated RNAscope assay yields a high signal-to-noise ratio with little to no background staining and results comparable to the manual assay. In addition, the automated duplex RNAscope assay was able to detect two biomarkers simultaneously. Lastly, assay consistency and reproducibility were confirmed by quantification of TATA-box binding protein (TBP) mRNA signals across multiple lots and multiple experiments. Taken together, the data presented in this study demonstrate that the automated RNAscope technology is a high performance RNA ISH assay with broad applicability in biomarker research and diagnostic assay development. J. Cell. Biochem. 117: 2201-2208, 2016. © 2016 Wiley Periodicals, Inc.

Continuous Flow Deformability-Based Separation of Circulating Tumor Cells Using Microfluidic Ratchets.

Circulating tumor cells (CTCs) offer tremendous potential for the detection and characterization of cancer. A key challenge for their isolation and subsequent analysis is the extreme rarity of these cells in circulation. Here, a novel label-free method is described to enrich viable CTCs directly from whole blood based on their distinct deformability relative to hematological cells. This mechanism leverages the deformation of single cells through tapered micrometer scale constrictions using oscillatory flow in order to generate a ratcheting effect that produces distinct flow paths for CTCs, leukocytes, and erythrocytes. A label-free separation of circulating tumor cells from whole blood is demonstrated, where target cells can be separated from background cells based on deformability despite their nearly identical size. In doping experiments, this microfluidic device is able to capture >90% of cancer cells from unprocessed whole blood to achieve 10(4) -fold enrichment of target cells relative to leukocytes. In patients with metastatic castration-resistant prostate cancer, where CTCs are not significantly larger than leukocytes, CTCs can be captured based on deformability at 25× greater yield than with the conventional CellSearch system. Finally, the CTCs separated using this approach are collected in suspension and are available for downstream molecular characterization.

Simultaneous flow cytometric analysis of IFN-γ and CD4 mRNA and protein expression kinetics in human peripheral blood mononuclear cells during activation.

The application of fluorescently-labeled antibodies for flow cytometric identification and characterization of specific cell types within heterogeneous populations by their protein expression profile is well established. While detection of proteins is informative, concomitant transcript analysis in the same cells would provide a more complete and comprehensive view of intracellular signaling events. We recently reported on the efficient detection of RNA in suspension cells for flow cytometric analysis. The improved RNA flow cytometry procedure described here allows for the specific labeling of multiple RNA species, and is compatible with antibody-based targeting of extracellular and intracellular antigens for multiplexing purposes. To show proof of concept, human peripheral blood mononuclear cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin for a maximum of 5 h, during which their CD4 and interferon-gamma (IFN-γ) transcript and protein levels were monitored. Substantial and increasing numbers of IFN-γ mRNA+ cells were detected within 30 min after initiation of induction, while IFN-γ protein+ cells could only be discerned at 1 h and beyond. Surprisingly, resting lymphocytes contained less CD4 mRNA but more of the protein per cell compared with monocytes, revealing a difference in the relationship of transcript and protein levels in these two cell types. We additionally applied monensin, which is commonly used to block cytokine secretion, and found that IFN-γ mRNA can still be analyzed consistently using the improved RNA flow cytometry staining method. Notably, a subset of IFN-γ mRNA(-)/protein+ cells that were not observed in the absence of monensin became apparent at the 5-h mark. This subset probably represents cells that have accumulated IFN-γ protein, but no longer transcribe mRNA. Collectively, the results described here exemplify how the improved RNA flow cytometry labeling procedure can be applied to simultaneously assess mRNA and protein dynamics to gain insight into the regulation of gene transcription and translation in individual cells.

The SATB1-MIR22-GBA axis mediates glucocerebroside accumulation inducing a cellular senescence-like phenotype in dopaminergic neurons.

Idiopathic Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, which is associated with neuroinflammation and reactive gliosis. The underlying cause of PD and the concurrent neuroinflammation are not well understood. In this study, we utilize human and murine neuronal lines, stem cell-derived dopaminergic neurons, and mice to demonstrate that three previously identified genetic risk factors for PD, namely SATB1, MIR22HG, and GBA, are components of a single gene regulatory pathway. Our findings indicate that dysregulation of this pathway leads to the upregulation of glucocerebrosides (GluCer), which triggers a cellular senescence-like phenotype in dopaminergic neurons. Specifically, we discovered that downregulation of the transcriptional repressor SATB1 results in the derepression of the microRNA miR-22-3p, leading to decreased GBA expression and subsequent accumulation of GluCer. Furthermore, our results demonstrate that an increase in GluCer alone is sufficient to impair lysosomal and mitochondrial function, thereby inducing cellular senescence. Dysregulation of the SATB1-MIR22-GBA pathway, observed in both PD patients and normal aging, leads to lysosomal and mitochondrial dysfunction due to the GluCer accumulation, ultimately resulting in a cellular senescence-like phenotype in dopaminergic neurons. Therefore, our study highlights a novel pathway involving three genetic risk factors for PD and provides a potential mechanism for the senescence-induced neuroinflammation and reactive gliosis observed in both PD and normal aging.

Chemical reprogramming takes the fast lane.

Small molecule-induced cell fate transitions are characterized by low efficiency and slow kinetics. An optimized chemical reprogramming approach now facilitates the robust and rapid conversion of somatic cells to pluripotent stem cells, unlocking exciting avenues to study and manipulate human cell identity.

The SATB1-MIR22-GBA axis mediates glucocerebroside accumulation inducing a cellular senescence-like phenotype in dopaminergic neurons.

Idiopathic Parkinson's Disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta, which is associated with neuroinflammation and reactive gliosis. The underlying cause of PD and the concurrent neuroinflammation are not well understood. In this study, we utilized human and murine neuronal lines, stem cell-derived dopaminergic neurons, and mice to demonstrate that three previously identified genetic risk factors for PD, namely SATB1, MIR22HG, and GBA, are components of a single gene regulatory pathway. Our findings indicate that dysregulation of this pathway leads to the upregulation of glucocerebrosides (GluCer), which triggers a cellular senescence-like phenotype in dopaminergic neurons. Specifically, we discovered that downregulation of the transcriptional repressor SATB1 results in the derepression of the microRNA miR-22-3p, leading to decreased GBA expression and subsequent accumulation of GluCer. Furthermore, our results demonstrate that an increase in GluCer alone is sufficient to impair lysosomal and mitochondrial function, thereby inducing cellular senescence dependent on S100A9 and stress factors. Dysregulation of the SATB1-MIR22-GBA pathway, observed in both PD patients and normal aging, leads to lysosomal and mitochondrial dysfunction due to the GluCer accumulation, ultimately resulting in a cellular senescence-like phenotype in dopaminergic neurons. Therefore, our study highlights a novel pathway involving three genetic risk factors for PD and provides a potential mechanism for the senescence-induced neuroinflammation and reactive gliosis observed in both PD and normal aging.

See-N-Seq: RNA sequencing of target single cells identified by microscopy via micropatterning of hydrogel porosity.

Single cell RNA sequencing has the potential to elucidate transcriptional programs underlying key cellular phenotypes and behaviors. However, many cell phenotypes are incompatible with indiscriminate single cell sequencing because they are rare, transient, or can only be identified by imaging. Existing methods for isolating cells based on imaging for single cell sequencing are technically challenging, time-consuming, and prone to loss because of the need to physically transport single cells. Here, we developed See-N-Seq, a method to rapidly screen cells in microwell plates in order to isolate RNA from specific single cells without needing to physically extract each cell. Our approach involves encapsulating the cell sample in a micropatterned hydrogel with spatially varying porosity to selectively expose specific cells for targeted RNA extraction. Extracted RNA can then be captured, barcoded, reverse transcribed, amplified, and sequenced at high-depth. We used See-N-Seq to isolate and sequence RNA from cell-cell conjugates forming an immunological synapse between T-cells and antigen presenting cells. In the hours after synapsing, we found time-dependent bifurcation of single cell transcriptomic profiles towards Type 1 and Type 2 helper T-cells lineages. Our results demonstrate how See-N-Seq can be used to associate transcriptomic data with specific functions and behaviors in single cells.

Pooled screening of CAR T cells identifies diverse immune signaling domains for next-generation immunotherapies.

Chimeric antigen receptors (CARs) repurpose natural signaling components to retarget T cells to refractory cancers but have shown limited efficacy in persistent, recurrent malignancies. Here, we introduce "CAR Pooling," a multiplexed approach to rapidly identify CAR designs with clinical potential. Forty CARs with signaling domains derived from a range of immune cell lineages were evaluated in pooled assays for their ability to stimulate critical T cell effector functions during repetitive stimulation that mimics long-term tumor antigen exposure. Several domains were identified from the tumor necrosis factor (TNF) receptor family that have been primarily associated with B cells. CD40 enhanced proliferation, whereas B cell-activating factor receptor (BAFF-R) and transmembrane activator and CAML interactor (TACI) promoted cytotoxicity. These functions were enhanced relative to clinical benchmarks after prolonged antigen stimulation, and CAR T cell signaling through these domains fell into distinct states of memory, cytotoxicity, and metabolism. BAFF-R CAR T cells were enriched for a highly cytotoxic transcriptional signature previously associated with positive clinical outcomes. We also observed that replacing the 4-1BB intracellular signaling domain with the BAFF-R signaling domain in a clinically validated B cell maturation antigen (BCMA)-specific CAR resulted in enhanced activity in a xenotransplant model of multiple myeloma. Together, these results show that CAR Pooling is a general approach for rapid exploration of CAR architecture and activity to improve the efficacy of CAR T cell therapies.

Performance evaluation of a non-invasive one-step multiplex RT-qPCR assay for detection of SARS-CoV-2 direct from saliva.

Polymerase chain reaction (PCR) has proven to be the gold-standard for SARS-CoV-2 detection in clinical settings. The most common approaches rely on nasopharyngeal specimens obtained from swabs, followed by RNA extraction, reverse transcription and quantitative PCR. Although swab-based PCR is sensitive, swabbing is invasive and unpleasant to administer, reducing patient compliance for regular testing and resulting in an increased risk of improper sampling. To overcome these obstacles, we developed a non-invasive one-step RT-qPCR assay performed directly on saliva specimens. The University of Nottingham Asymptomatic Testing Service protocol simplifies sample collection and bypasses the need for RNA extraction, or additives, thus helping to encourage more regular testing and reducing processing time and costs. We have evaluated the assay against the performance criteria specified by the UK regulatory bodies and attained accreditation (BS EN ISO/IEC 17,025:2017) for SARS-CoV-2 diagnostic testing by the United Kingdom Accreditation Service. We observed a sensitivity of 1 viral copy per microlitre of saliva, and demonstrated a concordance of > 99.4% between our results and those of other accredited testing facilities. We concluded that saliva is a stable medium that allows for a highly precise, repeatable, and robust testing method.

Direct RT-qPCR Assay for the Detection of SARS-CoV-2 in Saliva Samples.

Since mid-2020 there have been complexities and difficulties in the standardisation and administration of nasopharyngeal swabs. Coupled with the variable and/or poor accuracy of lateral flow devices, this has led to increased societal 'testing fatigue' and reduced confidence in test results. Consequently, asymptomatic individuals have developed reluctance towards repeat testing, which remains the best way to monitor COVID-19 cases in the wider population. On the other hand, saliva-based PCR, a non-invasive, highly sensitive, and accurate test suitable for everyone, is gaining momentum as a straightforward and reliable means of detecting SARS-CoV-2 in symptomatic and asymptomatic individuals. Here, we provide an itemised list of the equipment and reagents involved in the process of sample submission, inactivation and analysis, as well as a detailed description of how each of these steps is performed.

Control of the fluorescence of dye-antibody conjugates by (2-hydroxypropyl)-ß-cyclodextrin in fluorescence microscopy and flow cytometry.

When proteins are conjugated to fluorescent organic dyes, fluorescence emission of the dye molecules is usually decreased, sometimes up to 50-70%. This quenching phenomenon has been acknowledged for decades, but as yet, there are no simple, practical methods to control the fluorescence of dyes conjugated to proteins, especially for dyes conjugated to immunoglobulins. Here, we report that the addition of (2-hydroxypropyl)-ß-cyclodextrin (HPßCD) to dye-antibody conjugates can increase fluorescence up to 2.5-fold in cell imaging and flow analysis. This method may be an effective way to increase the sensitivity of detection of fluorescent organic labels used in immunology, histochemistry, and cell biology.

Methods for Quantitative Assessment of Protein Degradation.

Assessment of small molecules that promote selective protein degradation (degraders) requires detailed characterization and measurement of protein levels in cells. Here we describe ratio-metric methods based on a dual fluorescent GFP/mCherry reporter system to quantify cellular protein levels. We further develop a kinetic framework for the analysis of such data. We describe two methods of generating the stable GFP-protein of interest (POI)/mCherry reporter cell lines, alternative readout methods by FACS and Laser Scanning Cytometry as well as the corresponding tools used for processing and analysis of such data. Finally, we show that the commonly used half-maximal degradation constant (DC50) or maximum degradation efficacy (Dmax) metrics are time-dependent and propose a time-invariant Michaelis-Menten-like analysis of degradation kinetics with analogous key parameters Km app and Vmax app.

A systematic review of neuropsychiatric and cognitive assessments used in clinical trials for amyotrophic lateral sclerosis.

BACKGROUND: Up to 50% of people with amyotrophic lateral sclerosis (ALS) experience cognitive dysfunction, whilst depression and anxiety are reported in up to 44% and 33%, respectively. These symptoms impact on quality of life, and are associated with a poorer prognosis. Historically, outcomes in clinical trials have focused on the effect of candidate drugs on physical functioning. METHODS: We reviewed the past 25 years of clinical trials of investigative medicinal products in people with ALS, since the licensing of riluzole, and extracted data on frequency and type of assessment for neuropsychiatric symptoms and cognitive impairment. Trial registry databases, including WHO International Trials Registry, European Clinical Trials Register, clinicaltrials.gov, and PubMed, were systematically searched for Phase II, III or IV trials registered, completed or published between 01/01/1994 and 31/10/2019. No language restrictions were applied. Outcome measures, exclusion criteria and assessment tool used were extracted. RESULTS: 216 trials, investigating 26,326 people with ALS, were reviewed. 35% assessed neuropsychiatric symptoms, and 22% assessed cognition, as Exclusion Criteria or Outcome Measures. 3% (n = 6) of trials assessed neuropsychiatric symptoms as a Secondary Outcome Measure, and 4% (n = 8) assessed cognition as Outcome Measures; only one trial included assessments for both cognition and neuropsychiatric symptoms as Outcome Measures. Three ALS-specific assessments were used in six trials. CONCLUSIONS: Trials for people with ALS have neglected the importance of neuropsychiatric symptoms and cognitive impairment. Evaluation of these extra-motor features is essential to understanding the impact of candidate drugs on all symptoms of ALS. PROPSERO REGISTRATION: CRD42020175612.

PRC2-mediated H3K27me3 modulates shoot iron homeostasis in Arabidopsis thaliana.

Plants use intricate mechanisms to adapt to changing iron conditions because iron is essential and also one of the most limiting nutrients for plant growth. Furthermore, iron is potentially toxic in excess and must be tightly regulated. Previously, we showed that chromatin remodeling via histone 3 lysine 27 trimethylation (H3K27me3) modulates the expression of FIT-dependent genes under iron deficiency in roots. This study builds on our previous findings, showing that H3K27me3 also modulates iron regulation in shoots. In the clf mutant, which lacks the predominant H3K27 tri-methyltransferase, we detected increased iron translocation to shoots under iron deficiency as compared to wild type. Transcriptomic analysis of shoots also revealed differential expression of genes consistent with higher iron levels in clf shoots than wild type shoots under iron-deficient conditions. In addition, we verify that YSL1 and IMA1, two genes involved in signaling iron status from shoots to roots, are direct targets of H3K27me3 and reveal iron-dependent deposition of H3K27me3 on these loci. This study contributes to a better understanding of the molecular mechanisms behind iron regulation in plants, as the effect of PRC2-mediated H3K27me3 on iron homeostasis genes expressed in the shoots has not been previously reported to our knowledge.