Chimeric Antigen Receptors (CARs) are engineered immune receptors that underpin a promising new form of cellular immunotherapy for the treatment of cancer. Recently there have been two landmark FDA approvals for the use of Chimeric Antigen Receptor (CAR) T cell therapy for B cell malignancies, and with more than 300 CAR T cell clinical trials globally, it has become more important than ever to investigate the cell biology of these new “living” immunotherapies. The rapid pace at which these therapies have been translated, means that we still have a way to go in understanding how triggering T cells via a synthetic engineered receptor might alter the cell biology, function and persistence of CAR T cells. We have previously shown that CAR T cells have the ability to be serial killers, a property likely to be a key requirement for effective anti-tumour therapy and ultimately influencing the numbers of CAR T cells required for effective therapy .Activation of cytotoxic T cells is initiated after formation of an immune synapse, a highly organized structure formed at the interface of the effector and target cell. The synapse is a dynamic structure where T cell signaling occurs, serine kinases are recruited and key effector proteins perforin and granzymes are secreted into, inducing target cell apoptosis. The synapse is comprised of a series of concentric rings or SupraMolecular Activating Clusters (SMACs) that have been likened to a “Bull's eye” structure. T cell receptor (TCR) signaling and termination occurs in the central SMAC, whilst the peripheral SMAC provides adhesion, and actin clears away to the distal SMAC .We recently examined how triggering CAR T cells via CAR would alter the immune synapse. We utilized a dual-receptor transgenic mouse model, where the OTI TCR specific for SIINFEKL peptide and H-2Kb and a second generation anti-Her2 CAR CD28-CD3ζ) were expressed by the same T cell ζ were ex. The strIn our current study, we demonstrate that faster proximal signaling occurs via CAR compared to TCR, which raises the possibility that high affinity CAR design could be further fine-tuned . CAR T cTaken together, our recent study highlights the need to further understand the mechanisms by which CAR T cells kill their target cells which will also provide insight to increase efficiency. Whilst CAR T cells have become a highly effective form of therapy in some haematological cancer, therapeutic application to solid tumours presents additional challenges. As CAR T therapy matures, further clinical investigations are underway combining CAR T therapy with checkpoint inhibition via anti-PD-1/CTLA- 4 and other chemotherapeutic drugs. Current CAR T cell clinical trials utilise a variety of CAR designs, therefore varying affinity and CAR design may result in different signalling thresholds and functional outcomes. As such CAR T cell functional ability will be influenced by their fundamental molecular design.


Movile Cave  is a unique ecosystem where the food web is sustained by microbial primary production, analogous to deep-sea hydrothermal vents. Specifically, chemoautotrophic microbes deriving energy from the oxidation of hydrogen sulphide and methane form the basis of the food web.Methylomonas sp. LWB, a new species and representative of Movile Cave microbial mat samples. While previous research has suggested a prevalence of anoxic conditions in deeper lake water and sediment, using small-scale shotgun metagenome sequencing, we show that metabolic genes encoding enzymes for aerobic methylotrophy are prevalent in sediment metagenomes possibly indicating the presence of microoxic conditions. Moreover, this study also indicates that members within the family Gallionellaceae  were the dominant taxa within the sediment microbial community, thus suggesting a major role for microaerophilic iron-oxidising bacteria in nutrient cycling within the Movile Cave sediments.Here, we report the isolation of the first methane-oxidizing bacterium from the Movile Cave ecosystem, Candidatus In this study, based on phylogenetic and metabolic gene surveys of metagenome sequences, the possibility of aerobic microbial processes  within the sediment is indicated. We also highlight significant gaps in our knowledge on biogeochemical cycles within the Movile Cave ecosystem, and the need to further investigate potential feedback mechanisms between microbial communities in both lake sediment and lake water.The online version of this article (10.1186/s40168-017-0383-2) contains supplementary material, which is available to authorized users. Harpacticoid copepods , gammarids, isopods, acarids, rotifers, and gastropods occurred in very low densities [Lake Room, due to the almost normal atmospheric level of O2, when present, the biofilm is barely visible, being very thin and fragile . In contrast, in Airbell II, the atmosphere is poor in O2 but unusually rich in CO2 and CH4, conditions that enable substantial microbial biofilm formation [2. The microbial mats are white, grey or yellowish and floats on the surface due to the CH4 bubbles. Our understanding of microbial life within Movile Cave ecosystem is based only on the analysis of microbial mats (reviewed in 3). A previous study reported dissolved O2 concentration ranges between 9 and 16 μM at the water surface, reducing to less than 1 μM, a few centimeters below, with anoxic conditions likely in deeper lake water and sediment [Despite the lack of photosynthetically-fixed carbon, the Movile Cave ecosystem  hosts a diverse range of invertebrates  that are adapted to life in the dark. Life within this ecosystem is supported by chemoautotrophic primary producers that derive energy from the oxidation of inorganic compounds such as hydrogen sulphide (H4 and methylated amines (MA), and autotrophy are the major microbial processes that fix carbon and form the basis of the food web in this ecosystem [13CH4, and DNA—stable isotope probing (SIP)—Hutchens and colleagues [Methylomonas, Methylococcus, and Methylocystis. Previous cultivation-dependent studies in Movile Cave ecosystem have resulted in isolation of both sulphur oxidizers and reducers [Aerobic methylotrophy, i.e., utilisation of one-carbon C1) compounds, such as CH compoundcosystem , 6. Usinreducers , 8, methreducers , and metreducers , 11. DesMethylomonas sp. LWB from Movile Cave microbial mats and preliminary insights on its metabolic potential from the draft genome sequence. We also compare relative abundance of aerobic methylotrophs within mat and sediment communities and provide preliminary evidence (based on detection of specific gene signatures) that suggests the presence of microoxic conditions in sediments and also bacteria that can use inorganic energy sources such as iron.In this study, we describe the isolation of the first aerobic MOB representative, Candidatus 2-free nitrogen and amended with 7% O2 and 2.5% CO2. Methane (10%) was introduced to the serum vial headspace as carbon source for enrichment of aerobic MOB. Following 2 weeks of enrichment, 50 μl aliquots, including a dilution series of 1:10 and 1:100 of the culture were spread onto Dilute Basal Salts (DBS) agar plates [4 in the atmosphere and monitored for colony formation. Selected colonies were streaked onto fresh DBS agar plates and sub-culturing was performed until the cultures were deemed to be pure. Purity was determined by phase contrast microscopy (×1000) and lack of growth on R2A agar (Oxoid) plates were used to confirm the purity of the culture. Initial identification of the MOB isolate was determined by sequencing the 16S rRNA gene as described in [Enrichment cultures with both microbial mats and lake water (20 ml) collected from Movile Cave (April 2010) were set up in 120 ml serum vials. In order to reproduce the atmosphere found in the air bells, the headspace of the serum vials was flushed with Oribed in .Methylomonas sp. LWB was performed on DNA extracted, based on the protocol described previously [Microbial mat and sediment samples  for shotgun metagenome sequencing were collected in October 2013. The samples were transported to Norwich (UK) in RNAlater reagent (Qiagen) and stored at -20 °C until further downstream analysis. Community DNA from the sediment sample was extracted using the FastDNA SPIN Kit . DNA from microbial mats was extracted according to the protocol described by Neufeld et al. [pmoA, mmoX, mxaF/xoxF, mauA, gmaS) involved in aerobic methylotrophy, alongside other genes involved in the major biogeochemical processes in the Movile Cave i.e. nitrogen cycling  and sulphur cycling (soxB and dsrA/B). Hidden Markov Model (HMM) profiles for metabolic genes were obtained from the fungene repository [gmaS, mauA, and mxaF/xoxF), the profiles were created based on curated reference sequences using the HMMER tool. The number of hits were normalized to the relative abundance were RecA gene abundance and are presented as percentage of total RecA sequences. Retrieved metabolic sequences were aligned to an existing alignment of curated sequences using HMMER  and the aligned sequences were used to construct an approximate maximum-likelihood tree (JTT model with 1000 bootstraps) using FastTree 2 [gmaS sequences, homologues related to glnA (glutamine synthetase) sequences were identified and subsequently removed from the data used to compare the relative abundance of metabolic genes between mat and sediment metagenomes. Genome assembly of the isolate LWB was performed as described in [www.img.jgi.doe.gov) for downstream analysis. The neighbour-joining phylogenetic trees (1000 bootstrap replicates and Poisson correction method for computing evolutionary distances) for both 16S rRNA and metabolic genes were constructed using the MEGA 7 software as described in [Metagenome sequence statistics for raw (duplicate) microbial mat (ERR 1198911 and ERR 1198912) and sediment (ERR 1198913 and ERR 1198914) metagenome sequences are available in Additional file pository ; when avstTree 2 . Retrievribed in . Brieflyribed in , visualiribed in .pmoA gene phylogeny the MOB isolate can be classified to the genus Methylomonas  indicating the possibility that this may be a new species  enzyme) and mmoX  enzyme; Fig. Microbial mat with lake water samples collected from Movile Cave were used for isolating MOB in enrichments supplemented with methane as a sole carbon source. We identified an isolate that was a genuine MOB and DNA from this isolate was subsequently used to obtain its draft genome sequence using the Illumina Miseq sequencing platform. The draft genome sequence of the isolate is 5.36 Mb in size  and includes 5225 protein coding genes, 18 rRNA and 53 tRNAs . Based oies Fig. . The draProteobacteria in both of the metagenomes  while sequences affiliated to the family Comamonadaceae  were more prevalent in the mat metagenome.Phylogenetic assignment of metagenome sequences  and other dominant microbial processes  within the Movile Cave , Methylococcus , and Methylocella (which only contain mmoX)  Fig. .Fig. 2ComxaF and mxaI and the recently identified homologue of the mxaF gene, xoxF that requires rare earth elements such as lanthanum or cerium for activity [mxaF/xoxF sequences between the mat and sediment metagenomes  compared to the sediment metagenome .The enzyme methanol dehydrogenase (MDH) catalyses the conversion of methanol to formaldehyde in MOB and non-methane oxidising methylotrophs. Two homologues of MDH have been reported; the calcium-dependent classical MDH encoded by activity . In the Methylomonas sp. LWB, while one of the sets of pMMO genes conformed to the expected gene arrangement of pmoCAB the other had the gene arrangement of pmoABC (designated as PxmA). Since the pmoA gene from the pmoCAB operon in the genome was shorter  than the expected size , we used the pmoA sequence retrieved from the isolate DNA (amplified using the pmoA primer set A189/Mb661) to infer phylogeny [pmoA and pxmA sequence  have so far only been identified within Gammaproteobacterial MOB. It has been suggested that pXMO could possibly be involved in oxidation of other compounds such as ethane or ammonia, however the exact role of this enzyme in the environment is yet to be confirmed [mmoR and mmoG and interestingly the mmoX sequence of LWB aligned closely with that of a Methylomicrobium species, possibly indicating a divergent mmoX sequence from the other members of the genus Methylomonas  involved in the ribulose monophosphate pathway (RuMP) indicating that the isolate LWB probably assimilates formaldehyde through the ribulose monophosphate pathway (RuMP), similar to other members of the genus Methylomonas including M. methanica MC09 [Methylomonas koyamae alongside detailed experiment to characterize its physiology. The availability of an extant MOB from this ecosystem will certainly improve our understanding of the physiological and molecular adaptations of MOB to extreme conditions within this unusual cave ecosystem.In the genome of isolate Candidatus  750 bp; 0 aa, we onfirmed . We alsoica MC09 . It shoupmoA, mmoX and gmaS; Fig. proteobacterial methylotrophs, within the sediment that is perceived to be anoxic. One hypothesis for the detection of gene sequences involved in aerobic microbial processes could be due to the retrieval of DNA from remnant cells that sunk with microbial mat into sediments and thus could represent an inactive population. However, the detection of aerobic neutrophilic iron-oxidizing bacteria (FeOB), belonging to genera Sideroxydans and Gallionella  oxidation to oxygen reduction at microoxic niches [One of the key observations, particularly in the sediment metagenome was the detection of gene signatures , we recently identified active methylotrophs in Movile Cave that included members of the genera Methylotenera and also isolated two novel facultative methylotrophs, Gemmobacter sp. LW1 and Mesorhizobium sp. 1 M-11 and obtained draft sequences of their genomes [mauA and gmaS have been used as metabolic markers genes for these pathways, respectively [gmaS gene sequences were relatively more abundant than mauA sequences in both the mat and sediment metagenomes  and facultative methylotrophs  that were previously identified as active organisms that can use MMA as a carbon source [Methylobacterium extorquens strains that possess genes for both MaDH and NMG pathway had greater fitness compared to strains with only the NMG pathway [Gemmobacter, and Mesorhizobium isolated from Movile Cave possess both the MMA utilisation pathway [Methylated amines (MA) can serve as both C and N substrate for microbes. Degradation of microbial mats could be a potential source of MA and indeed using  genomes . The in  genomes , which mectively , 32. In es Figs.  indicatilotrophs . The retn source . By usin pathway . While,  pathway , the eco0/00) compared to the ammonium-N in the cave water (δ15N = +19.90/00) and it might be possible that nitrifiers could contribute to the primary productivity [Methylophilaceae  are known to link methanol oxidation to denitrification [mxaF/xoxF sequences affiliated to Candidatus Methylomirabilis oxyfera (NC10 phylum), a denitrifying MOB that can couple denitrification with methane oxidation in anoxic conditions [2 is widespread among bacteria present in Movile Cave as revealed by the recovery of wide diversity of nifH gene sequences belonging to methylotrophic taxa . Fixing N2 is an energy expensive process for bacteria and given the fact that high ammonium concentrations are observed in cave water, it is essential to identify whether there are nitrogen-depleted niches where N fixation occurs. Nitrogen cycling within the Movile Cave ecosystem remains under-explored, in particular the influence of methylotrophs on nitrogen turnover needs to be examined in detail.Microbial nitrogen cycling within Movile Cave has been largely unexplored. Sarbu and colleagues , observeuctivity . While iuctivity . It mighfication . Wischerδ15N = +1.90/00 annditions . The abiIn summary, our reconnaissance study based on small-scale metagenome libraries elucidated the distribution of aerobic methylotrophic organisms within Movile Cave microbial mats and sediments. This study clearly indicates the possibility of ecological niches  within sediments that was perceived to be anoxic. It should be noted that these observation (i.e. gene signatures) should be followed up with process measurements to ascertain function. This study also highlights the fact that there are still significant gaps in our knowledge on microbial nutrient cycling within the Movile Cave ecosystem, such as Fe-cycling in sediments or feedback mechanisms, if any, between microbial communities in sediments and lake water. Future research should build on these preliminary observations  with process-based measurements to assess whether the presence of these metabolic genes (in either the metagenome or isolate genome) correlate with biological activity.Additional file 1: Figure S1.Microbial mat floating in the lake room (A) and air bell 2 . (TIFF 4369 kb)Additional file 2: Table S1.Sequence statistics of raw metagenome sequences from DNA isolated from Movile Cave microbial mat and sediment. (DOCX 55 kb)Additional file 3: Figure S2.Krona chart representing the phylogenetic distribution of proteobacterial sequences in the microbial mat metagenome (A) and sediment metagenome (B). (TIFF 1884 kb)Additional file 4: Figure S3.An approximately maximum-likelihood tree constructed using partial MxaF/XoxF peptide sequences retrieved from both microbial mat and sediment metagenomes, ratified XoxF peptide sequences (represented in blue font) and MxaF sequences (represented in red font). Nodes with only MxaF/XoxF peptide sequences from the microbial mat metagenomes are collapsed (grey triangles). Scale bar = 1 change per base position. (TIFF 5972 kb)


Human embryonic stem cells (hESCs) and induced pluripotent cells (iPSCs) have the potential to differentiate into any somatic cell, making them ideal candidates for cell replacement therapies to treat a number of human diseases and regenerate damaged or non-functional tissues and organs. Key to the promise of regenerative medicine is developing standardized protocols that can safely be applied in patients. Progress towards this goal has occurred in a number of fields, including type 1 diabetes mellitus (T1D). During the past 10 years, significant technological advances in hESC/iPSC biochemistry have provided a roadmap to generate sufficient quantities of glucose-responsive, insulin-producing cells capable of eliminating diabetes in rodents. Although many of the molecular mechanisms underlying the genesis of these cells remain to be elucidated, the field of cell-based therapeutics to treat T1D has advanced to the point where the first Phase I/II trials in humans have begun. Here, we provide a concise review of the history of cell replacement therapies to treat T1D from islet transplantations and xenotranplantation, to current work in hESC/iPSC. We also highlight the latest advances in efforts to employ insulin-producing, glucose-responsive β-like cells derived from hESC as therapeutics. There remains an urgent and critical need for new treatments for type 1 diabetes (T1D). A current prevailing hypothesis is that cell replacement for pancreatic β-cells destroyed by autoimmune attack will optimally restore euglycemia. While efforts to augment endogenous populations of a patient’s residual β-cells or pancreatic specific stem cells remains an active avenue of research, efforts to enhance proliferation of these populations of cells without further autoimmune damage has had limited success. Conversely, the development of in vitro generated populations of insulin-producing, glucose-responsive cells has overcome many significant challenges, including generation of chemically defined conditions for reproducibly differentiating hESCs into endocrine precursors (EPs) and, the development of strategies to purify these precursor cells to avoid the development of benign tumors such as teratomas. With the basic protocols in place other pressing issues will move to the forefront, including prevention of cell destruction following transplantation, encapsulation, and the application of newer anti–rejection therapies.The modern age of islet transplantation was ushered in by pioneering studies of Lacy and Kostianovsky who developed a method to isolate and purify islets from rat pancreas . BuildinOver 20 years ago, our laboratory identified the combination of hepatocyte growth factor/scatter factor (HGF/SF) and the HTB-9 extracelluar matrix from human bladder carcinoma cells, as potent stimuli for β cell replication . Cells cInvestigations using human fetal pancreatic cells from the mid 1980s to the 1990s first provided evidence that human fetal tissues could be manipulated in vitro to advance differentiation to β-cells from endocrine precursors previously identified in experiments performed during pancreas development in rodents. Full appropriate response to glucose in terms of insulin release was not obtained until the cells were transplanted into immune-deficient mice for periods of 10 to 12 weeks –21. ThesAlthough much has been published about the potential for β-cell regeneration through replication of existing β-cells and/or differentiation from putative precursors, clinical protocols for their use are not available since the positive results reported in small animals models have not been possible to translate to humans.Abnormalities of the exocrine pancreas in T1D have been described since 1940 but still scant attention has focused on research in this area. A recent review describes subclinical exocrine dysfunction associated with the characteristic findings in the islets of T1D subjects, but it remains unclear whether the two are related to the autoimmune response after the onset of T1D . BecauseXenotransplantation provides another alternative for the limited supply issue faced from the too few human islets available from donated human pancreases. In 2006, two groups reported long-term survival of porcine islets in non-human primates , 30. RecAn effective cell-based therapeutic for T1D requires cells that sense glucose fluctuations and respond with appropriate insulin secretion. While islet transplantation has shown promise as a treatment for type T1D, a major obstacle to this approach is the shortage of the islets containing insulin-producing cells. Over the past decade, hESCs/iPSCs have emerged as promising sources for the pancreatic β-cells lost in T1D. hESCs are characterized by their capacity for self-renewal and differentiation to almost any specific cell type in the human body. The first study demonstrating functional and meaningful secretion of insulin after transplantation into mice of β-cells generated from hESC was published in 2008 , less thAlthough critical gaps in the understanding of the molecular mechanisms that drive the genesis of insulin producing cells from hESCs/iPSCs exist, many of the essential growth factors and inhibitors have been identified. Below, we briefly highlight the various steps in hESC differentiation toward glucose-responsive, insulin secreting cells.Pluripotency of hESCs initially required co-culture with a fibroblast feeder layer that secreted unidentified soluble factors that helped maintain pluripotency. Unlike mouse ES cells, hESC were unable to maintain pluripotency by incubation with leukemia inhibitor factor (LIF) . In 2005Xenopus and rodents [Over the past decade many different groups have contributed to generate differentiation protocols that mimic pancreas development . From t rodents , 43, 44. rodents . ViaCyte rodents . In more rodents . Recentl rodents , Rezania rodents , and Rus rodents . In all  rodents . Additio rodents . Sixteen rodents . Similar rodents . hESCs d rodents . β-like  rodents .Careful examination of the similarities and differences between the four protocols reveals limitations between the different methods to generate glucose-responsive, insulin-producing cells from hESCs. Rezania and Pagliuca relied upon previously established protocols for generation of primitive gut tube and posterior foregut and primarily focused on optimization of the later stages of hESC differentiation. Russ found that elimination of Noggin during posterior foregut formation (high retinoic acid levels) followed by combined treatment with EGF/KGF enhanced PDX1/NKX6.1 expression and reduced the number of polyhormonal cells. This work emphasizes the importance of temporal activation of signaling pathways during hESC differentiation.Reproducible protocols to generate cells that can ablate diabetes in mice have led to questions about how these cells can be used therapeutically. Similar to the situation with islets, transplanted stem cells also face the problem of rejection. Cell encapsulation has been actively pursued as a means of abrogating rejection by protecting the encapsulated cells from the immune system while allowing adequate oxygenation, nutrient delivery, and glucose and insulin transport across the barrier. Although no successful and reproducible human islet encapsulation lasting more than a few weeks has been reported in patients with T1D (See review ), ViaCythttps://clinicaltrials.gov/show/NCT02239354).ViaCyte has obtained FDA approval and initiated a phase I/II clinical trial for the implantation of encapsulated endocrine progenitor cells derived from hESCs from their VC-01cell line. . Preliminary data suggests that significant differences in expression of receptor tyrosine kinases and G-protein coupled receptors exist on the different cell lines that significantly alter signaling, proliferation, apoptosis, expression of transcription factors and epigenetic modifications. These biochemical differences in cell lines could have multiple repercussions in the interpretation of results with clinical trials. ViaCyte has demonstrated a reproducible in vitro hESC expansion and banking method for their VC-01 cell line that achieves 50–100 fold expansion per week . ScalabiLike hESCs, iPSCs have infinite self-renewal capacity and the ability to differentiate into any cell type, providing the hope of patient-specific cells for therapeutic use. iPSCs can be generated from virtually any adult somatic and peripheral blood cell through reprogramming by the addition of integrating retroviral vectors containing four pluripotency transcription factors, OCT4, SOX2, KLF4, and c-MYC . iPSCs bRedifferentiation of iPSCs from diabetic patients into pancreatic islets has the potential to allow for patient-specific cell replacement therapy. This field has been recently reviewed by Neofytou et al. , pointinThis review has focused upon current state of cell replacement therapeutics in T1D. These cells are designed to function when transplanted into humans without eliciting an immune response. While considerable headway is being made in this respect, other factors that may influence the progression of type 1 diabetes cannot be ignored. For example, loss of β-cell function has been observed in NOD mice before the onset of hyperglycemia , possibl


Transfection is one of the most frequently used techniques in molecular biology that is also applicable for gene therapy studies in humans. One of the biggest challenges to investigate the protein function and interaction in gene therapy studies is to have reliable monospecific detection reagents, particularly antibodies, for all human gene products. Thus, a reliable method that can optimize transfection efficiency based on not only expression of the target protein of interest but also the uptake of the nucleic acid plasmid, can be an important tool in molecular biology. Here, we present a simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency at the single cell level while overcoming limitations of prior established methods that quantify transfection efficiency. By using optimized ratios of transfection reagent and a nucleic acid (DNA or RNA) vector directly labeled with a fluorochrome, this method can be used as a tool to simultaneously quantify cellular toxicity of different transfection reagents, the amount of nucleic acid plasmid that cells have taken up during transfection as well as the amount of the encoded expressed protein. Finally, we demonstrate that this method is reproducible, can be standardized and can reliably and rapidly quantify transfection efficiency, reducing assay costs and increasing throughput while increasing data robustness. Transfection is one of the most common used techniques in molecular biology , 2. TranResearchers often use easily tractable reporter assays for determining transfection efficiency and their downstream applications , 2. Comm® Tracker TM Kits, any plasmid can be custom labeled in a simple one-step chemical reaction before introduction into mammalian cells [An alternative and more direct method to using fluorescent reporter genes is to directly label nucleic acids with fluorescent dyes to track their intracellular delivery . Non-radan cells . Thus, ban cells , 18. Thian cells , 16, 18.® TrackerTM. This method does not depend on co-transfection of two different plasmids and simultaneously quantifies cell death, uptake of the labeled plasmid during transient transfection, and expression of the target protein. We demonstrate that this method can be used as a tool to i) optimize transfection efficiency in a standard cell line ii) to quantify cellular toxicity of different transfection methods iii) to determine uptake of DNA into difficult to transfect cells via electroporation without the need to use co-transfection of GFP plasmid that can further reduce the efficiency of transfection. This flow cytometric method can be directly applied to optimize several transfection methods including gene therapy strategies (e.g. CRISPR/Cas system).Herein, we demonstrate the development of a flow-cytometric assay to determine transfection efficiency by labeling a reporter plasmid with Label IT2.293T cells were maintained with Dulbecco's modified Eagle medium (DMEM)  supplemented with 10% fetal bovine serum (FBS)  and penicillin and streptomycin (Invitrogen). Jurkat E6-1  were culpNL4-3 is a full-length, replication and infection competent chimeric HIV DNA plasmid (14825 bp) that was received from AIDS reagent program . pUltraH™  or Stellar  bacteria using the PureLink HiPure Midiprep Kit . The concentration was determined using spectrophotometry and the purity was between 1.8–2.0 (260/280 ratio). Plasmid DNA was labeled with DNA LabelIT Tracker  the day before transfection using 0.5 ul FITC/1 μg DNA according to the manufacture`s protocol. Unreacted LabelIT Tracker reagent was removed using ethanol precipitation. According to the manufacturer 1 ul of labeling dye per 1 μg plasmid DNA will yield labeling efficiencies of approximately one Label molecule every 40 base pairs (on average) of double-stranded DNA. The purity and concentration of labeled DNA was determined by spectrophotometry.Plasmid DNA for transfection was extracted from Stbl3The following commercially available transfection reagents were purchased: TransIT-X2 , Jet Prime , Lipofectamine 2000  and Fugene HD . All reagents were used at recommended reagent to DNA ratio to transfect a constant amount of 1 μg DNA for comparison.5 293T cells passaged less than 20 times were plated into 12 well plates the day before transfection. At this density, cells were attached to the well bottom after 24 h at a confluency of 70%–80%. Twenty-four hours after seeding, cells were transfected with FITC-labeled and unlabeled pNL4-3 or pUltraHot encoding mCherry. The transfection was performed using 1 μg plasmid DNA and 3 μl TransIT X2 in serum free media per well, according to the manufactures`protocol. Cells were cultured in complete media. Transfection complexes were removed after 6 hours by replacing all of the media in the well. For time course experiments, the cells were transfected at the same time and harvested at 0, 6, 12, 24, 36, 48 and 72hrs. For experiments comparing different transfection reagents, the cells were transfected with 1 μg DNA/well with varying amounts of transfection reagents to facilitate the recommended ratio of transfection reagent to DNA for each reagent: 3 μl TransIT X2, 4 μl Lipofectamine 2000, 2 μl Jet Prime and 3 μl Fugene HD. Cells were harvested at 24 hrs post-transfection and stained with a viability dye and for intracellular target protein. All experiments were performed in triplicates.For transfection experiments, 3x10Jurkat E6-1 cells were washed 2 times in OptiMem  (without antibiotics) before being resuspended in OptiMem at 1x10^6 cells/ 100 μl. Cells were transferred into 4mm cuvettes  and 4 μg of labeled or unlabeled pUltraHot plasmid were added to the suspension. Jurkat E6-1 cells were electroporated with a BioRad Gene Pulser Xcell electroporation system using the exponential protocol with 250 volts, 350uF capacitance, 1000 ohm Resistance. Cells were transferred immediately into pre-warmed media and incubated for 24 hours before staining for viability and flow cytometric analysis.Anti-HIV-1 p24 monoclonal antibody (71–31) was obtained through the NIH AIDS reagent program #530 and was conjugated with a CF647 dye . The matching human IgG1 control was purchased from Biolegend  and conjugated with a CF647 dye . Amine reactive (Ghost 780 Live/dead dye) and nucleic acid dye (7-AAD: 7-Aminoactinomycin D) were obtained from Tonbo Biosciences .For flow cytometry, 293T cells were harvested by detaching and washing once in PBS. Cells were fixed in 2% PFA for 15 min and permeabilized in 0.2% Tween/PBS for 15 min. Cells were then stained for HIV-1 p24 protein with 1 μg anti-p24 antibody conjugated to CF647 in 100 μl staining medium containing PBS/2% BSA for 30 min at 4 C, washed and fixed in 1% PFA. Cells transfected with pUltraHot were stained with 1 μl Ghost Violet 450 Live/dead dye in 1 mL PBS. The cells were then washed with 1% FBS in PBS and fixed in 2% PFA for 15 min.™ Software (BD Biosciences). The flow cytometer was equipped with 405, 488, 561 and 635 nm lasers, and emission filters for Pacific blue , Alexa fluor-488 , PE , mCherry (LP: 600 BP: 610/20), PerCP-Cy5.5 , APC . The cytometer was routinely calibrated with BD cytometer setup and tracking beads (BD Biosciences). Gating was conducted as shown in y-axis) is plotted on a log scale against the fluorescence intensity (x-axis) of fluorochrome that is used to quantify protein expression (e.g. mCherry)  with BDFACSDiVamCherry) . Backgrot-test, or non-parametric equivalent as appropriate. Correlations were assessed with the Spearman rank correlation coefficients. Statistical analyses were performed using GraphPad Prism. Significant differences were accepted when p≤0.05.Experiments were performed in three independent cell culture preparations  unless stated otherwise in the figures. Differences of MFI between different transfection variations were analyzed by two-way analysis of variance (ANOVA) or by Student's To develop a robust flow cytometric method that can be used to reliably quantify transfection efficiency at the single cell level and to avoid limitations of GFP-based flow cytometric methods that can compromise accuracy of determination of transfection efficiency , 9–15, wHowever, our novel flow cytometric method can also be used as a tool to optimize transfection efficiency in difficult to transfect human cells without the need to co-transfect a GFP reporter plasmid that can further compromise transfection efficiency . We valiToxicity of DNA and the transfection method per se should also be taken into consideration during transfection  but mostUsing the approach described in There is limited published evidence regarding the optimal timing after transfection for determination of transfection efficiency , 10, 22.To further optimize transfection efficiency, we directly compared four major commercially available nonviral reagents of chemical transfection using the same approach as in The MESF readouts (at 24 hours after transfection) for labeled plasmid and expressed protein, correlated better compared to the % positivity for labeled plasmid and expressed protein . In addiHere we provide a relatively simple, rapid and robust flow cytometric method that can be used as a tool to optimize transfection efficiency at the single-cell level. This method takes into consideration cell toxicity as a direct result of the transfection and the nucleic acid per se and uses two independent readouts of transfection efficiency: a) the amount of plasmid nucleic acid that cells have taken up during transfection b) the amount of the encoded expressed protein. It is important to emphasize that any nucleic acid plasmid can be labeled with the LabelIT Tracker, making this method very versatile and applicable to any transfection method. We observed a higher transfection efficiency based on DNA FITC signal and protein expression with a smaller plasmid, which is in line with the nature of large plasmid requiring more transfection reagent to complex the DNA and being more difficult to be transported across the plasma membrane than smaller constructs. We here also discuss important considerations required to successfully implement this method that can be used to optimize several variables that can affect transfection efficiency such as cellular toxicity related to the transfection method, the time of exposure of a standard cell line to specific nucleic acid plasmid and the type of transfection reagent. This method overcomes limitations of prior established methods such as dual reporter systems , 9–15 anTransfection is an important tool in molecular biology that is applicable for gene therapy studies in humans using methods such as LTR-Specific Tre-Recombinase , the SleThe measure of transfection efficiency, the percentage of transfected cells in a cell population, is a subjective measure prone to many variable factors, such as cell cycle progression, the circadian rhythm of gene expression activity, promoter activity, and general activity of a given cell type. All of these factors can inhibit a cell from actually expressing the transfected protein. Commonly used methods to quantify transfection efficiency such as the luciferase assay, require cell lysis, do not quantify overall cell toxicity, and thus the determination of the percentage of transfected cells in a cell population may not be accurate . Indeed,ex vivo genetic modification of human tissue/cell types for therapeutic purposes has been limited by the low efficiency of currently available plasmid transfection systems and their cell toxicity [We also used this method to demonstrate that it can be used as a tool to detect transfected DNA in difficult to transfect human cells without the need to co-transfect plasmids for reporters that can further compromise transfection efficiency. The use of plasmid vectors for the toxicity –32. SincOne of the limitations of nonviral gene delivery systems is also their toxicity that can significantly compromise transfection efficiency. Other common methods that quantify transfection efficiency such as luciferase assay do not quantify cell toxicity of the transfection method . Our assIncubation time for optimal protein detection after transfection varies (3–48 hours) between different plasmids and transfection methods depending on the protein of interest and its post-translational modifications, location and turnaround time . Due to ™ 2000 (Invitrogen) transfection reagent, is based on the lipofection method [™ , which is a nonliposomal, polymer-based transfection reagent. Both lipofection and polymer transfections, like JetPEI [TransIT-X2® Dynamic Delivery System an advanced non-liposomal system of proprietary polymers that aid in nucleic acid complexation, uptake and endosomal release thereby enabling superior transfection of plasmid DNA and smaller nucleic acids. Finally, the fourth transfection reagent was FuGENE® HD Transfection Reagent a nonliposomal formulation designed to transfect DNA into a wide variety of cell lines with high efficiency and low toxicity. While these four different transfection reagents only represent a small fraction of commercially available products, our flow cytometric assay is a valuable tool for future evaluation of any transfection reagent in specific cell types.There is limited published evidence regarding direct comparison of the major commercially available transfection reagents (for the same plasmid and cell type). With oun method . Lipofecn method . The sece JetPEI  and JetP®3, Cy®5, TM-Rhodamine) that are not as pH-sensitive dyes as FITC can be used to label plasmid nucleic acid (RNA or DNA)[®) that are optimized to allow escape of the nucleic acid plasmid from endosomes/lysosomes should be preferred when our flow cytometric method is used to quantify efficiency of chemical transfection. Finally, possible biochemical interactions with individual fluorochromes and the DNA LabelIT Tracker need to be individually tested for each transfection method.Interestingly, a drastic reduction in the detection of the FITC labeled DNA was seen when Lipofectamine 2000 and Fugene HD were used for chemical transfection. It is possible that quenching of FITC in endosomes/lysosomes may be an explanation for our findings given the nature of the transfection reagents and that FITC is a pH-sensitive fluorescent dye , 43. It A or DNA)–46. The ®3, Cy®5, TM-Rhodamine)[Any nucleic acid (DNA or RNA) plasmid can be labeled with the LabelIT Tracker, making this method very versatile and applicable to any transfection method. This method is applicable to transfection methods that have revolutionized the field of gene therapy such as the bacterial CRISPR/Cas9 system. CRISPR consists of two components: a “guide” RNA (gRNA) and a non-specific CRISPR-associated endonuclease (Cas9) –50. The hodamine)–46 can bTaken together, the described relatively simple, rapid and robust flow cytometric method can be used as a tool to optimize transfection efficiency at the single-cell level. This method overcomes limitations of prior established methods  to quantify transfection efficiency. Any DNA or RNA template can be labelled and is suitable for a wide range of applications. The one-step chemical method is easy and allows consistent control of the labeling reactions and high sensitivity of detection of plasmid DNA can be achieved with optimally labeled DNA and RNA. In addition, the fluorescent labels do not impact hybridization performance and the covalent mechanism allows permanent, non-destructive modification of nucleic acid residues that is ideal for many diverse applications. Given the importance of transfection in molecular biology that has set the basis for gene therapy studies in humans, this novel flow cytometric method can be an important tool in molecular biology and gene therapy studies using methods such as the Sleeping Beauty (SB) transposon system, the CRISPR technology and in cases where reliable monospecific detection methods for human gene products (protein) do not exist.


Escherichia coli cells with multiple genomic knockouts to increase cellular accumulation of various metals. Lastly to explore further protein candidates for biomagnetism, we characterized members of the DUF892 family using the iron sensor and magnetic columns, confirming their intracellular iron sequestration that results in increased cellular magnetization.Genetically encoding the synthesis of functional nanomaterials such as magnetic nanoparticles enables sensitive and non-invasive biological sensing and control. Via directed evolution of the natural iron-sequestering ferritin protein, we discovered key mutations that lead to significantly enhanced cellular magnetism, resulting in increased physical attraction of ferritin-expressing cells to magnets and increased contrast for cellular magnetic resonance imaging (MRI). The magnetic mutants further demonstrate increased iron biomineralization measured by a novel fluorescent genetic sensor for intracellular free iron. In addition, we engineered  Inorganic nanomaterials have been used in a wide range of biological applications including fluorescent or plasmonic labelling for imaging, magnetic labelling for extraction and high throughput sequencing and drug-delivery12Magnetospirillum naturally synthesize crystalline magnetite nanoparticles and align them as a passive navigation compass for the cell in its natural environment5689102121314151617192022232425262728293031323334E. coli. In addition, a novel genetic biosensor for cytoplasmic free iron was developed to easily measure biomineralization efficiency in vivo, combined with genetic manipulations of metal transporters to optimize intracellular metal levels36373839There are few examples of bio-synthesized inorganic nanomaterials in Nature. Certain species of bacteria and archaea can mineralize nanoparticles via proteins or metabolites that reduce toxic metal cations1E. coli ferritin (ftnA) mutants was cloned and transformed into E. coli. Magnetic cells from this population were enriched through ten iterative rounds of growth followed by passage through high-gradient magnetic columns. Eventually, the selected population of cells was plated and sequenced, and the most frequent mutations were identified and validated in freshly cloned and transformed cells  were knocked-out to prevent interference with the magnetic evolution approach. Changes in cellular magnetism were compared via measurement of the retention fraction of cells in high-gradient magnetic columns. The results show that the majority of mutants, compared to wild-type ferritin, significantly increase levels of magnetism of cells . Fur bound to Fe2+ represses downstream expression of GFP on a low-copy plasmid (p15A origin)  allows conversion of culture density-normalized fluorescence to free iron concentrations per cell  sulfate concentrations (0 to 5 mM) over a range of ferritin induction levels (0% to 0.2% rhamnose). The time courses show rapid sequestration of intracellular iron under high ferritin inductions and decreased time to reach peak iron concentrations (E. coli (~10 mM). Comparing the final readout at 15 h between cells expressing wild-type and the most magnetic mutant (m1) shows that the mutant sequesters more iron, especially at low induction levels below 0.01% rhamnose (We further developed a genetic fluorescent iron sensor that demonstrates an increase in cytosolic iron sequestration in strains expressing mutant ferritins with increased magnetism. The iron sensor employs the  origin) . Hence tper cell . This cotrations . Withoutrhamnose . We alsorhamnose . Considerhamnose , iron seCellular magnetism and mineralization were further confirmed by a range of characterization methods. The increased magnetic moment of the mutant cells was visualized directly by growing cells in LB rich medium in titer plate over ring-shaped permanent magnetsE. coli with knockouts in three divalent cation exporters  and also the iron master regulator fur, causing increased metal accumulation in the cells, particularly for iron, zinc and cobalt  significantly increased cellular iron levels as measured by inductively coupled plasma mass spectrometry (ICP-MS). We also found co-expressing importers could increase iron levels and cellular magnetism (data not shown). We then tested several elements known to absorb to or alloy with iron oxide: cobalt, nickel, cadmium and arsenic. Cells were exposed to varying concentrations of these elements. Only strains expressing magnetic mutant ferritins exhibited a decreased growth defect, suggestive of toxic metal sequestration by over-expressed ferritin mutants  proteins, which fold into a similar 4-helix bundle as ferritins and are thus distantly related in structure-based phylogenylization . These rE. coli that fluorescently detects variations in intracellular free iron levels to validate iron sequestration by ferritin mutants and other previously uncharacterized iron-sequestering proteins.In this study, we used directed evolution by magnetic selection to discover point mutations in ferritin that enhance iron sequestration and the magnetic phenotype of cells expressing ferritin mutants. Increased cellular magnetism was shown to lead to physical attraction to magnetic field gradients and improved MRI contrast. We further developed a new genetic iron sensor in E. coli increases sub-linearly with increasing iron supplement in culture media until around 2 mM, above which the sensor displays a sudden increase in intracellular free iron levels, coincident with significant growth defect and death  ions. Histidine has a larger side chain compared to leucine. On the other hand, threonine at position 64 could serve as a C-terminal cap of the second major helix via hydrogen bonding, hence its replacement could change pore structure to favor ion entry. This in turn may affect not only the concentration but also the balance of Fe(II) and oxidized Fe(III) species in the core. Natural ferritin mineralizes mostly Fe(III) ions into Fe2O3, which may exhibit antiferromagnetic coupling of iron atom spins resulting in a very low magnetic moment. However, magnetite (Fe3O4), the most magnetic form of iron oxide, contains mixed valence atoms of Fe(II) and Fe(III). Hence the influx of Fe(II) into the ferritin core without oxidation could change the stoichiometry of iron valencies inside the nanoreactor to favor mineralization of the more magnetic magnetite. The increased iron-sequestration and cellular magnetism of mutants resulting from the directed mutation of residues His34 and Thr64 at the B-channel to alanine supports this hypothesis. Interestingly, we found that certain mutants with N-terminal “SpyTag” also increased magnetization  to first localize to and oxidize at the ferroxidase active site close to the center of the 4-helix bundle. The role of the B-type channel has not been highlighted until recentlytization . SpyTag in vivo imaging, both T2 and T2* imaging modes can be used to detect contrast generated from endogenous magnetic particles. The ferritin mutants could allow noninvasive reporting of in vivo biological signals from engineered cells such as bacteria in the gut, or immune cells targeted to cancer tumors. There are further potential application of engineered intracellular magnetic particles as magnetic force or magneto-thermal actuators to manipulate proteins such as ion channels or subcellular compartments to control cellular behavior. Such applications include control of calcium-based signaling in mammalian cells, particularly neurons to induce or inhibit action potentials (e.g. “magneto-genetics”)33Improving biomineralization and biomagnetism can lead to diverse applications. Cells genetically modified to accumulate and store toxic or valuable metals can enable cell-based bioremediation or mining. On the other hand, the magnetic particles from ferritin mutants could serve as noninvasive, genetically-encoded reporters of biological activity in deep tissues transparent to magnetic fields, as demonstrated by their enhanced MRI relaxation rate in T2* MRI. T2 imaging using spin-echo sequence is more commonly reported for magnetic particles. However, spin-echo sequences are designed to cancel the dispersion in transverse relaxation due to magnetic field inhomogeneity produced by the ferritin iron-oxide particles in the static limit. Hence the T2 signal change is dependent not only on the magnetic moment but also on change in the particles’ local magnetic environment due to diffusion during measurement. Hence T2 measurement is likely dependent on measurement parameters like sequence duration, whereas T2* gradient echo is not. For E. coli BW25113 as the background to introduce plasmids expressing recombinant proteins or conducting sequential knock-out of genes via P1 transduction from knock-out strains in the KEIO collection of non-essential E. coli knock-out strains. Plasmid pCP20 was electroporated into the transduced cells and grown at 37 °C on LB agar plate to induce expression of recombinase to that flip out the Kanamycin resistance cassette used for transduction selection, as well as loss of the pCP20 plasmid. The genes knocked out include genes that serve as endogenous iron storage proteins , genes that serve as exporters of metal cations  and the iron master regulator fur. BW25113 with all ftnA, bfr and dps served as the background strain for expressing recombinant ferritins so as to minimize the small background of endogenous iron storage proteins. BW25113 with all fief, rcnA, zntA and fur knockous served as the metal over-accumulation strain to minimize metal export and down-regulation of import by fur in a metal-rich environment.We chose E. coli ferritin gene ftnA was cloned into a high copy-number plasmid (pUC Origin of replication) with rhamnose inducible promoterrhaPBAD, with native E. coli transcription factors RhaS and RhaR) and kanamycin resistance cassette via Gibson AssemblyE. coli BW25113 cells via electroporation. Expression of ftnA was induced in cells by adding rhamnose to cell culture (maximum 0.2%) during log-phase growth (OD600~0.4).E. coli ferritin (ftnA) sequence with on average one mutation per copy. The PCR products were cloned into the vector with rhamnose promoter and transformed via electroporation into E. coli strain containing the GFP-based genetic iron sensor and triple knockout of endogenous iron sequesterers . This library of cells each expressing a particular ftnA mutant was grown and induced in log-phase with 0.2% rhamnose to initiate high level expression of ferritins and simultaneously exposed to 100 uM iron (II) sulfate as supplement. The cells were cultured with good oxygenation in deep-well plates shaken at 900 rpm. In saturation phase , the cells were filtered through the magnetic column (Miltenyi LD column) placed between neodymium permanent magnets  to create a high magnetic field gradient that help to retain cells that are more magnetic in the column whereas the cells expressing less magnetic ferritin mutants are flushed out. Subsequently, the permanent magnets are removed and the previously retained magnetic cells were eluted separately, grown again to log-phase, and re-induced and exposed to iron in fresh media to iterate another day of growth and magnetic selection. In total, 10 days or iterations of magnetic selection were conducted before the final eluted magnetic cells were plated. Around 100 colonies were picked and sequenced by Sanger sequencing (Genewiz). A Python script was used to analyze the sequences to determine the most frequent or representative mutations. The top mutations were re-constituted in wildtype ftnA plasmid (NEB Q5 Site-Directed Mutagenesis Kit) and verified for increased iron sequestration (via GFP-based genetic iron sensor) and magnetic column retention relative to overexpressing the wildtype ftnA.Error-prone PCR (Agilent GeneMorph II Random Mutagenesis Kit) was used to generate random mutants of the A high-gradient magnetic column (Miltenyi LD columns) was sandwiched between two neodymium permanent magnets  to create high magnetic field gradients inside the column. The column is first wetted by passage of 2 ml of PBS 1X buffer. Then 500 μl of cells re-suspended in PBS 1X buffer were added and flowed through by gravity into the elution tube, followed by addition of 3 ml of PBS 1X buffer to wash through any unbound cells into the elution tube. Once dry, the column is removed from the permanent magnets, and 3 ml of PBS 1X buffer is pushed through the column to extract the magnetically retained cells into a separate retention tube. Measuring OD600 of the elution and retention tubes allow estimation of cell counts and the percentage of total cells retained by the magnetic column.E. coli fiu promoter was cloned along with a super-folder GFP (sfGFP) reporter via Gibson Assembly into a low copy (p15A origin), chloramphenicol-resistance plasmid compatible with the ferritin-expressing plasmid. Iron levels were measured for cells containing the ferritin-expression and iron sensor plasmids by taking the GFP fluorescence of the culture of cells  in 96-well plate format using the BioTek NEO plate-reader. For calibration, known concentrations of iron sequesterer bi-pyridine were added to cell cultures. The fluorescence measured were normalized to culture density by dividing by OD600 measured by the same plate-reader. The increase in normalized fluorescence of the cells was plotted against the increase in bipyridine (or consequent decrease in free iron) and modeled  to lyse and digest organic material. The solution is then fed to Perkin Elmer 6100 ICP-MS machine  to determine the concentration of a variety of elements  Prior to sample measurement, machine calibration was performed using solutions containing a linear range of known concentrations of the measured metals.MRI characterization was performed on a Brucker 9.4 T MRI system at the MRI preclinical core of the Beth Israel Deaconess Medical Center. Cells were first washed of metal in culture and resuspended in metal-free PBS 1X buffer and subsequently loaded into NMR tubes. The NMR tubes were further immersed in DI water in a container to prevent artefacts due to susceptibility mismatch at the glass/air interface. Once samples were loaded into the MRI system, T2* weighted relaxation times were obtained from fits of the signal decay after a gradient-echo pulse sequence and compared among samples.Cells were first washed by DI water three times to remove excess iron, then pelleted by high-speed centrifuge (4000 g). The cell pellets were flash-frozen by dipping into liquid nitrogen, followed by sublimation in a lyophilizer for one hour to remove water. The desiccated pellets were weighed and loaded into the Quantum Design MPMS SQUID and the sample magnetization (moment) was measured as magnetic field was varied in steps.E. coli cells induced to express ferritins were grown to saturation in LB media supplemented with 100 μM iron and back diluted 1:5 with fresh LB media with 20% OptiPrep Density Gradient Medium to help suspend the cells. The cultures were dispensed into wells of a 6-well titer plate, where ring magnets were placed directly under the bottom of the wells, and left undisturbed. Plates were imaged by a digital camera after overnight growth to capture visible aggregation of cells toward the magnets.E. coli cells in culture were resuspended in PBS 1X and fixed with glutaraldehyde overnight. The samples were subsequently dried, embedded in resin, and sectioned without additional heavy metal staining. The sections were imaged using the Tecnai G2 Spirit BioTWN microscope at variable acceleration voltage and magnification to maximize contrast of the electron dense nanoparticles in the cell cross-sections.E. coli cells were resuspended in SDS Buffer (NuPAGE LDS Buffer) with reducing agent, followed by two cycles of boiling at 95 °C for 5 minutes and vigorous vortexing to lyse cells and denature proteins. The lysate was centrifuged to pellet cell debris, and the protein suspension was diluted and added to NuPAGE 4–12% Bis-Tris gel with MES buffer. For calibration of protein concentration via densitometry analysis, known dilutions of BSA protein in the same buffer and similarly denatured were added to the same gel containing the samples. Empty lanes in the gel were filled with equal volume of SDS buffer. After running at 200 V for 35 minutes, the gel was removed and stained with Coomassie Orange dye for one hour and subsequently imaged for dye fluorescence on a Typhoon Imager. Densitometry analysis of the gel bands was conducted using ImageJ.How to cite this article: Liu, X. et al. Engineering Genetically-Encoded Mineralization and Magnetism via Directed Evolution. Sci. Rep.6, 38019; doi: 10.1038/srep38019 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Monitoring of pulmonary physiology is fundamental to the clinical management of patients with Cystic Fibrosis. The current standard clinical practise uses spirometry to assess lung function which delivers a clinically relevant functional readout of total lung function, however does not supply any visible or localised information. High Resolution Computed Tomography (HRCT) is a well-established current ‘gold standard’ method for monitoring lung anatomical changes in Cystic Fibrosis patients. HRCT provides excellent morphological information, however, the X-ray radiation dose can become significant if multiple scans are required to monitor chronic diseases such as cystic fibrosis. X-ray phase-contrast imaging is another emerging X-ray based methodology for Cystic Fibrosis lung assessment which provides dynamic morphological and functional information, albeit with even higher X-ray doses than HRCT. Magnetic Resonance Imaging (MRI) is a non-ionising radiation imaging method that is garnering growing interest among researchers and clinicians working with Cystic Fibrosis patients. Recent advances in MRI have opened up the possibilities to observe lung function in real time to potentially allow sensitive and accurate assessment of disease progression. The use of hyperpolarized gas or non-contrast enhanced MRI can be tailored to clinical needs. While MRI offers significant promise it still suffers from poor spatial resolution and the development of an objective scoring system especially for ventilation assessment. Cystic Fibrosis (CF) is an autosomal recessive non-curable condition caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, located on human chromosome 7. The CFTR protein is a chloride ion channel and is expressed in many different organs. Deletion of CFTR activity within the lungs leads to dehydration of the airway surface liquid layer, reduction in mucociliary clearance and chronic bronchopulmonary infection , 2. ChroIn Europe Cystic Fibrosis affects 1 to 3500 new-borns . NeverthThe overall goal of this review is to understand how to improve respiratory diagnosis and treatment in patients with Cystic Fibrosis in two contexts: (i) clinical respiratory imaging research techniques, and (ii) novel respiratory imaging research applications.standards of caremagnetic resonance imaging (MRI),He-3 MRI,Xe-129 MRI,non-contrast enhanced MRI,phase contrast MRI.A systematic search of PubMed database was performed for years from 2007 to 2016. We looked for records which involved the phrase ‘cystic fibrosis’ and additional phrases:Papers were included if they mentioned respiratory function assessment in quantitative manner or described the standard clinical tests used to assess cystic fibrosis pulmonary disease. Papers were excluded if the information about material, methods and results was insufficient (did not include quantitative data) or was not written in English.1) is the main indicator of disease progression [There are many different methods to evaluate pulmonary function in patients with CF with the most common method being spirometry. According to the European Cystic Fibrosis Society spirometry should be performed at least once a year in CF patients. From all obtained values, forced expiratory volume in one second  and residual volume (RV) and it can assess volume of trapped air which is increased in CF lung disease . Body plA more sensitive method to detect small changes in the airways is the inert gas washout test. It measures heterogeneity of ventilation during tidal breathing, and is simple to perform especially in young children. The most studied index for the multiple breath washout (MBW) test in patients with CF is the lung clearance index (LCI). LCI is defined as the number of lung turnovers  required to reduce alveolar gas concentration of nitrogen to less than 2% . LCI hasChest X-Ray is a fast and widely used method of assessing anatomical changes in the lungs. For patients with Cystic Fibrosis it is recommended to perform a chest X-Ray once a year . HoweverHigh-Resolution Computed Tomography (HRCT) in patients with Cystic Fibrosis was performed for the first time almost 30 years ago . Since tTo reduce movement artefact, especially in infants, researchers use controlled ventilation high resolution computed tomography (CV-HRCT). The images are comparable with those from adults undertaking the breath-hold test , 21, andMagnetic Resonance Imaging of the lung is a significant challenge due to a low physical and proton density in the lungs and the air-tissue interfaces that result in weak and/or distorted MR signals. Whilst the field of pulmonary MRI is rapidly developing, intravenous contrast-enhanced lung MRI (IV-CEMRI) with gadolinium based agents still remains the most common pulmonary imaging modality. MRI data sets have spatial resolution which enables analysis of pulmonary perfusion down to the segmental level and provide very high temporal resolution with less than 1.5 s acquisition times. Pulmonary perfusion MRI is suitable to assess bronchoarterial perfusion changes and to localize perfusion defects in Cystic Fibrosis .1 even until 4 years [n = 26) and CF (n = 50) infants and preschool children. All subjects were examined with a morphological MRI protocol  and an IV-CEMRI perfusion protocol (3D fast low angle shot /FLASH/ sequence). Abnormal lung structure and perfusion defects were clearly visible in MR images in the CF group [For paediatric patients it is essential to monitor lungs as early as possible because morphological changes (i.e. mucus plugging and airways inflammation) occur during the first few months of life and are usually not reflected in noticeable functional measures, e.g. FEV 4 years . A signiCF group . A recenCF group .For adult CF patients, systematic monitoring of lung changes is crucial. HRCT and CXR deliver a radiation dose which is cumulative over time and becomes more important with an increasing life expectancy of CF patients. Similar to paediatric MRI, a comparison of CT and CXR with IV-CEMRI outcomes in adult CF patients demonstrated approximately 80% concordance for bronchiectasis and mucus plugging . IntraveTo effectively image ventilation, hyperpolarized gases  can be administered prior to subsequent magnetic resonance imaging. The hyperpolarized spin state is generated at a very low spin temperature and is in disequilibrium with the temperature of the sample. The most popular method to produce polarized noble gases is via spin exchange optical pumping (SEOP) utilizing vaporized alkali metal .3He MRI) has been examined in multiple settings and has shown high sensitivity for the detection of ventilation impairments [3He MRI results correlate strongly with spirometry values  and HRCT , and there is also a trend to correlate with chest X-ray  in adults with Cystic Fibrosis. [1) values correlate better with HP 3He MRI than with CXR measures [3He MRI correlate positively while the ventilation defect volume (VDV) and the ventilation defect percentage (VDP) negatively correlate with equivalent spirometry and HRCT results in CF patients [Imaging of lung ventilation in patients with Cystic Fibrosis using hyperpolarized helium-3 magnetic resonance imaging was first published in 1999 . Followiairments –44. Thisairments , 36. Theibrosis. . Moreovepatients .3He MRI is useful to assess different treatments and progression of CF disease by measuring ventilation volumes. The effectiveness of an eleven day intervention with intravenous tobramycin, beta-lactam antibiotic, inhalation of 2.5 mg of recombinant human DNase administrated daily, inhalation of 4 mL of 7% hypertonic saline and chest physiotherapy twice a day was monitored by HP 3He MRI and showed a 25% increase in VV in treated patients [1 [3He MRI may provide greater sensitivity in detecting changes. Unfortunately, there were no noticeable effects of a single bout of chest physical therapy (CPT), however one CPT session may not have been sufficient to visualise effects as effective physiotherapy is an ongoing process and not a single event [1/FVC) ratio. Moreover, they observed a strong correlation between HP 3He MRI and spirometry outcomes  [HP patients . Improveients [1 , 41 indile event . A study< 0.001) .3He MRI lung images and gives little information about the heterogeneity of ventilation. However, in 2014 Horn and co-workers  presented a new study protocol based on a combination of hyperpolarized helium-3 MRI and the multiple breath washout (MBW) test. This approach allows the imaging of regional fractional ventilation (RFV) and provides more information about ventilation quality itself, instead of the lung ventilated area only. Nevertheless, the method requires further testing and adjustments and only one CF patient was tested to check the tolerability of the protocol  allows the visualisation and quantitation of 129 Xe gas diffusion into the tissue. ADC measurements are also possible using 3He, however 129Xe MRI outcomes correlate better with spirometry values  and 129Xe has a six-fold smaller self-diffusion coefficient which means smaller field gradients can be used [129Xe allows MR imaging of the gas transition from the alveolar air spaces across the lung wall into red blood cells (RBC). The relative signal intensity from129Xe dramatically reduces as 129Xe transitions from air to dissolution in interstitial fluid and blood. This causes issues for imaging the transition as the signal is almost immediately overlapped by the diffusion signal and it is difficult to separate the barrier dissolution phase from the RBC dissolution phase due to the small chemical shift difference (19 ppm) and short relaxation time (2 ms) [Diffusion weighted imaging of hyperpolarized gas is possible within the pulmonary system, and is another modality which is very useful for assessing alveolar space . Free di be used . The unie (2 ms) .129Xe distribution were acquired and a homogenous pattern was found in all three images among healthy volunteers. In IPF patients only ventilation and barrier 129Xe images were homogenous and RBC image intensities were lower, mainly in peripheral and basal lung. The calculated gas-transfer defect percentage for IPF patients (22.4% ± 5.4%) was almost two times higher than in the healthy group (11.5% ± 1.3%) [129Xe is longer in emphysematous mouse lungs compared to healthy controls. While this method demonstrated that it is feasible to assess ventilation defects, it will be difficult to use in humans due to xenon’s anaesthetic properties and the requirement for constant infusion [129Xe MRI in Cystic Fibrosis patients (3 adults) demonstrated that 500 ml 129Xe + 500 ml 4He, 12–14 s breath-hold, was well tolerated by all participants and showed no significant changes in oxygen saturation and heart rate before, during and after 129Xe administration [A new imaging protocol based on an adapted Dixon technique (water/fat separation) called xenon alveolar capillary transfer imaging (XACT) can be used to differentiate barrier and RBCs dissolved phases . A modif ± 1.3%) . The timinfusion . A smallstration .Even very well tolerated contrast agents may cause severe allergic reactions in rare cases, therefore the development of new techniques allowing the measurement of pulmonary perfusion without contrast agents would be extremely useful in these situations . Despiter = 0.84, p < 0.0001) [p = 0.02, p < 0.05) and the values were even lower for patients older than 15 years  which demonstrates the ability of ASL based techniques to assess disease progression [Arterial Spin Labelling (ASL) is a useful, non-contrast-enhanced MRI technique. It allows assessment of lung perfusion by magnetically labelling water protons within arteries supplying the lungs. Perfusion is measured using a flow-sensitive alternating inversion recovery (FAIR) MRI sequence and then calculated pixel-by-pixel with modified Bloch equations allowing perfusion homogeneity and assessment during tidal, imaging-synchronized breathing. The ASL protocol is well-tolerated by young patients (mean age: 13 ± 5 years) and the outcomes correlate strongly with FEV1 values ( 0.0001) . Mean pegression .1 level and FEV1/FVC ratio from spirometry. However, this can be insufficient for patients without clear symptoms of airflow obstruction. The ASL method can be applied to assess pulmonary perfusion in patients after bilateral lung transplant. To evaluate heterogeneity of pulmonary blood flow using MRI, the FAIRER  technique can be used and the comparison of two images enables the calculation of the relative dispersion (RD). Patients with BOS 3 (FEV1 < 50%) have greater perfusion heterogeneity (RD = 1.41) than patients with BOS 0, 1 and 2 . However patients with the classification of probable BOS  have similar perfusion heterogeneity to BOS 3 patients (RD = 1.61). These results suggest this group may be following the RAS pathway where the abnormality is primarily in the pulmonary vasculature with the airways being relatively well preserved [The ASL technique has also been used to assess pulmonary perfusion after lung transplantation. Lung transplantation is an established treatment for patients with end-stage pulmonary disease. However even though early outcomes show significant benefit the patient is at significant risk of lung rejection known as chronic lung allograft dysfunction (CLAD) . The rejreserved . MonitorFourier Decomposition MRI is another technique that has been developed for perfusion evaluation as well as ventilation assessment. It is based on registering the amplitude of local signal strength alterations generated by blood flow during free breathing using short echo-time sequences. Subsequently, collected images are transformed with Fourier analysis to separate the signal from lung parenchyma and blood flow and obtain perfusion and ventilation images. Fourier Decomposition MRI provides results comparable to contrast-enhanced MRI and single photon emission CT (SPECT) , 60. Whe1 and nT1 values  and good agreement was observed between nT1 and ASL to determine regions of oxygen inhomogeneity [Normalized T1 MRI (nT1-MRI) utilizes T1-weighted images, obtained through rapid Look-Locker acquisition to generate a quantitative T1 map of the lung and is able to detect early-stage CF-related lung disease. nT1-MRI is based on the differences in T1-relaxation times between soft tissue (relatively short) and body fluids (relatively long) which gives information about the homogeneity of the dissolution phase within the lungs . A recenogeneity , 62.r = 0.79, p < 0.0001) and FEV1  values with the highest spatial agreement between mucus plugging and areas with elevated diffusion called hotspots (60%) [Diffusion weighted imaging (DWI) is used extensively to measure water proton diffusion and image inflammation in the brain or abdomen , 64. Howts (60%) .The techniques described above offers promise for clinical lung imaging however all require further refinement to improve spatial resolution and disease correlation. The outcomes generally correlate well with standard pulmonary measures and further refinement will improve the spatial resolution, reduce artefacts and allow discrimination of localised lesions within Cystic Fibrosis lungs.High energy phase-contrast X-ray imaging (PCXI) offers the highest resolution possible for morphological and functional lung imaging. To obtain high-quality images with this technique it is necessary to use X-ray sources with high spatial and temporal coherence. These ultrahigh quality images typically require a synchrotron-based X-ray source . TraditiIn patients with CF the dysfunctional CFTR ion channel in the airway cells causes a reduction in the airway surface liquid (ASLiq) volume. The ASLiq layer decrease is associated with a reduction in mucociliary clearance and an exacerbation of disease . DespiteTo assess aerosol treatment efficacy, the nasal passages and trachea of anesthetised mice were imaged using a propagation-based phase-contrast X-ray technique before, during and after nebulization with hypertonic saline. Three of seven mice showed significant improvement in ASLiq layer depth (2-5 μm). In contrast to standard X-ray imaging, the PCXI technique can detect borders between different tissues allowing differentiation between the airway lumen and the ASLiq layer .p < 0.05) at the micrometre scale  were noted from 9, 12 and 15 min acquisitions compared to isotonic saline response [Propagation-based PCXI has a relatively high ability to distinguish ASLiq-to-air differences however it is difficult to distinguish ASLiq and tissue in vivo. Morgan et al. 2013) placed a single absorption grid between the X-ray source and the sample for the ex vivo imaging of murine trachea and were able to determine ASLiq layer volume changes after a hypertonic saline treatment. A statistically significant improvement of ASLiq volume was observed in the treated tracheas ( placed aresponse .Fig. 3PhPXCI offers extraordinary detail for the assessment of CF lung disease progression, and while the application of the above techniques in humans is certainly challenging, it offers insight into the mechanisms in pre-clinical animal models and can be used to provide excellent spatial resolution and track the effectiveness of applied therapies.A summary of the techniques can be found in Table There is a strong rationale to monitor the lungs of patients with Cystic Fibrosis at least annually . However3He and 129Xe are currently used by many researchers, preparing the gas mixture is still challenging since it has to give enough contrast during the examination while being safe for a patient with compromised lungs at the same time. More research investigating the acceptable doses for patients or in pre-clinical models with diseased lungs needs to be performed before effective clinical protocols can be developed. Improvements in the production of hyperpolarized gas would be extremely beneficial, small commercial systems are available however they are not amenable to clinical research and many remain in testing phases and have to be improved before they are ready for more straightforward usage.In comparison to X-ray based methodologies MRI has lower spatial resolution for lung imaging. To achieve acceptable signal to noise in an MRI image of the lung it is often essential to use a contrast agent. Lung imaging techniques using non-CE MRI are largely focused on documenting perfusion changes, with poor structural detail available due to the low density of protons and the air-surface interface. To accurately assess ventilation parameters, the use of hyperpolarised gas contrast agent is essential. Although hyperpolarised Despite the great value of developed methods, the available MRI sequences need significant refinement and testing to minimise the artefacts associated with cardiac and breathing movement. Lung MRI typically involves up to 18 s breath hold which is often a great challenge, especially for young patients. Sequences which allow images to be obtained during free breathing would be of great value for children and patients with advanced pulmonary disease.Although many MRI techniques have been developed and successfully used for patients with CF, there is still a strong need to perform longitudinal imaging studies to establish clinical protocol. Moreover, the analysis of the images is still problematic, especially for clinical purposes, the use of complicated and time-consuming methods is not practical. Therefore, standardisation of image analysis tools and development of clinically relevant disease markers together with comprehensive clinical tests should be key goals in the future research.Nevertheless, it is worth noting that MRI allows for dynamic imaging which is critical for CF patients and which provides a unique opportunity to gain insight into lung function in real time.129Xe MRI is a poorly developed technique that requires more thorough investigation. Many challenges remain before MRI of the lungs, with or without IV or hyperpolarised gas contrast becomes a routine clinical assessment tool, however MRI appears to have the most potential to become the “gold standard” for regional assessment of cystic fibrosis lung pathology in the future.The ultimate goal of clinical lung imaging methods in CF is to provide diagnostic indicators and treatment information to clinicians and associated health care workers. Unfortunately, none of the currently available methodologies is simple and detailed enough to fulfil all these objectives. Clearly there is no ‘ideal’ method which allows for the assessment of all CF-associated pulmonary pathology with a single or a few simple modalities. Whilst lung imaging in patients with Cystic Fibrosis is a well-established discipline, there are still many opportunities to improve the imaging methods used. Methods based on ionising radiation should be compared with and if possible replaced by ionising radiation-free methods as any reduction in radiation exposure in CF studies is an important goal to achieve. Magnetic Resonance Imaging requires substantial investment and investigation in CF patients of all ages to improve its clinical relevance. In particular, hyperpolarized


Pain is a hallmark feature of sickle cell disease (SCD). Recurrent and unpredictable acute pain due to vaso-oclussive crises (VOC) is unique to SCD; and can be superimposed on chronic pain. To examine the mechanisms underlying pain in SCD, we performed RNA sequencing of dorsal root ganglion (DRG) of transgenic sickle mice and their age-matched control mice expressing normal human hemoglobin A, at 2 and 5 months of age. Sickle and control mice of both ages were equally divided into hypoxia/reoxygenation (to simulate VOC) and normoxia treatment, resulting in eight groups of mice. Each group had at least six mice. RNA isolated from the DRG was sequenced and paired-end 50 bp sequencing data were generated using Illumina’s HiSeq 2000. This large dataset can serve as a resource for examining transcriptional changes in the DRG that are associated with age and hypoxia/reoxygenation associated signatures of nociceptive mechanisms underlying chronic and acute pain, respectively. Under low-oxygen conditions, hemoglobin with mutated β-globin polymerizes resulting in sickle shaped red blood cell (RBCs). Sickle RBCs stick and occlude capillaries leading to vaso-occlusive crises (VOCs), leading to impaired oxygen supply to the tissues, inflammation, ischemia/reperfusion injury, oxidative stress, end organ damage and severe pain. In addition to acute pain due to VOC, SCD can be accompanied by severe chronic pain which can start in infancy. Pain in SCD is considered to be worse than labor pain, often requiring hospitalization, reduced survival and poor quality of life2. Although both chronic and acute pain are observed in adult patients, infants and children are reported to mostly suffer from acute pain. Transition from acute to chronic pain is likely to occur during the adolescence and remains poorly understood4. Elucidating the mechanisms underlying the transition from acute to chronic pain could facilitate better pain management and targeted interventions with improved outcomes.Sickle cell disease (SCD) was the first molecular disease to be identified, which occurs due to a point mutation leading to a single amino acid substitution from Glutamic acid to Valine on position 6 of the β-globin chain of hemoglobin5. Thus, differences in gene expression are expected to reflect the mechanisms underlying acute and chronic pain in SCD. Therefore, we obtained the transcriptomic profile of the DRG of transgenic sickle mice under different age and pain conditions which can provide insights into the molecular changes. We chose to use HbSS-BERK sickle mice because of their similarity to human SCD with respect to pain characteristics. Here we report the transcriptomic analysis carried out among different cohorts and subsequent validation of some of the differentially expressed genes.Dorsal root ganglion (DRG) houses neuronal cell bodies of the afferents involved in transmission of action potentials from the periphery to the spinal cord. Since, pain can start early in age in SCD, it is likely to influence the genetic architecture of the primary sensory neurons. This may lead to the hyper-excitability of spinal neurons and chronic hyperalgesia observed by us in sickle micen=6 in each group) qualifying the study design to evaluate the effect of age and transition from acute to chronic pain. RNA was extracted from the isolated DRG of these animals and sequenced using Illumina’s RNA-seq platform. The scope and scale of the study design will allow identification of a large array of differentially expressed genes (DEGs) that contribute to SCD pain and transition from acute to chronic pain state. This will also allow for identification of protective mechanisms/genes unknown/ not yet discovered in this disease state. We provide an example in the usage notes section to depict how this large data set can enable us to identify important genetic targets with the potential to improve the quality of life of SCD patients. Considering the comprehensive study design of this large data-set, further downstream data processing and pathway analysis will allow identification of genetic variations and associated molecular events underlying SCD pain.As illustrated in the study design, two transgenic mouse models were used: [i] HbSS-BERK (sickle) mice that express human sickle hemoglobin and simulate human SCD phenotype, including pain, and [ii] HbAA-BERK (control) mice which express normal human hemoglobin A . Each moAll mice are bred in the housing at the University of Minnesota, approved by Association for the Accreditation and Assessment of Laboratory Animal Care, Int (AAALAC); and maintained under controlled environmental conditions . All experiments were performed following approved protocols from the University of Minnesota’s Institutional Animal Care and Use Committee and conform to the statutes of the Animal Welfare Act and the guidelines of the Public Health Service as issued in the Guide for the Care and Use of Laboratory Animals.6. HbSS-BERK mice are homozygous for knockout of both murine α and β globins and carry the linked transgenes for human α and βS globins. Thus, the HbSS-BERK mice express human sickle hemoglobin, and demonstrate severe disease including reticulocytosis, hemolysis, anemia, organ damage, hyperalgesia, and a shortened life span6–8. The HbSS-BERK mice exhibit chronic hyperalgesia with characteristic features of sickle pain, including, mechanical, deep-tissue and thermal hyperalgesia8. Additionally, VOC-like pain can be evoked in this model by hypoxia/reoxygenation treatment described below. Control HbAA-BERK are bred by backcrossing HbAA-BERK with HbSS-BERK and therefore have the same mixed genetic background as HbSS-BERK, but exclusively express normal human hemoglobin A  and no murine globins. The HbAA-BERK mice do not exhibit constitutive hyperalgesia.Transgenic sickle mice (HbSS-BERK) expressing >99% human sickle hemoglobin and controls (HbAA-BERK) expressing >99% normal human hemoglobin A were used7. Mouse genotypes for the transgenes for human hemoglobin A (Hba-5 Tg), human hemoglobin S (HuHbs-1 Tg), and murine α and β globins  were determined using real time PCR .All mice were phenotyped for sickle and normal human hemoglobin by iso-electric focusing2 and 92% N2 for 3 h, followed by reoxygenation for 1 h in the room air8. H/R treatment was repeated after one week, thus exposing the mice to two recurrent H/R treatments. DRG were collected 24 h after the second hypoxia reoxygenation treatment.The mice in small rodent cages are placed in a chamber with the atmosphere controlled by a Proox Model 110 gas controller . These mice are exposed to hypoxia with 8% OMice were euthanized and DRG were collected and placed in RNA later before proceeding to RNA isolation. H/R-treated mice were sacrificed after 24 h post-2nd H/R treatment. RNA isolation was performed using TRIzol Reagent (Applied Biosystems (ABI), Foster City, CA) followed by purification using the RNeasy mini kit . The purity of RNA was checked with Nanodrop (Thermo Fisher Scientific).RNA sequencing from the collected samples was performed at the University of Minnesota Genomics Center (UMGC) using Illumina HiSeq 2000 sequencer platform. The details of the library preparation and sequencing are provided below.For the RNA samples to pass the initial quality check before preparing the sequencing library, each RNA sample needs to be more than 1 μg and must have an RNA integrity number (RIN) of 8 or greater. The total amount of RNA in each sample is quantified using a fluorimetric RiboGreen assay (R11490 Quant-iT from Thermo-Fischer) and RIN is generated for each sample by performing capillary electrophoresis using Agilent BioAnalyzer 2100.www.illumina.com for a detailed list of kit contents and methods. In brief, 1 μg of total RNA from each sample is purified using oligo-dT coated magnetic beads (part of RNA prep kit). Next they are fragmented and then reverse transcribed into cDNA. The cDNA is blunt-ended, A tailed and ligated to indexed (barcoded) adaptors and amplified using 15 cycles of polymerase chain reaction (PCR) (Biorad DNA Engine Tetrad 2). Final library size distribution is validated using capillary electrophoresis and quantified using fluorimetry (PicoGreen) and via qPCR. Indexed libraries are then normalized, pooled and then size selected to 320 bp+/−5% using Caliper’s XT instrument (Perkin Elmer).Total RNA samples are converted to Illumina sequencing libraries using Illumina’s Truseq RNA Sample Preparation Kit (Cat.# RS-122-2001). Please see Truseq libraries are hybridized to a paired end flow cell and individual fragments are clonally amplified by bridge amplification on the Illumina cBot. Once clustering is complete, the flow cell is loaded on the HiSeq 2000 and sequenced using Illumina’s sequencing by synthesis (SBS) chemistry to generate 50 bp paired end reads.Base call (.bcl) files for each cycle of sequencing are generated by Illumina Real Time Analysis (RTA) software during the sequencing run on HiSeq 2000. Primary analysis and de-multiplexing are performed using Illumina’s CASAVA software 1.8.2. The end result of the CASAVA workflow is de-multiplexed FastQ files that are subjected to subsequent analysis described below. The CASAVA software assigns quality scores to the FastQ files.The Illumina read files were processed via a pipeline developed by the University of Minnesota Informatics Institute (UMII) in collaboration with the Minnesota Supercomputing Institute (MSI) and the UMGC. Briefly, FastQ files were trimmed via trimmomatic using the following parameters: -phred33 -threads 8, ILLUMINACLIP, LEADING:3 TRAILING:3 SLIDINGWINDOW:4:16 MINLEN:25. After trimming, mapping was performed via TopHAT (v2.0.13) using bowtie (v2.2.4.0). This mapping results in output of one binary alignment/map (BAM) file for each sample matched to a reference mouse genome. Finally, fragments per kilobase of transcript per million mapped reads (FPKM) values were calculated as expression data via cuffquant . ‘featureCounts’ was used to count the reads. The primary output files from the analysis pipeline are FastQC files, raw count expression tables as text files and expression data as FPKM values.P values for both false discovery rate (FDR) and Bonferroni corrections. We chose Bonferroni corrections (P<0.05) to be our selection criteria which provides more stringent statistical significance for the data. For our analyses, we selected the genes with equal to or greater than two-fold changes in the comparison with this Bonferroni correction criteria.Most DGE analysis platforms use raw read counts as inputs. We performed DGE analysis among different treatment groups using CLC genomics workbench  which utilizes a platform called empirical analysis of DGE in R (edgeR) from Bioconductor. EdgeR performs the statistical analysis of raw counts from high-throughput sequencing of multi-factorial comparative experimental designs. This platform exploits the advantage of using negative binomial distribution to model the transcript raw counts so that the biological variation is distinguished from technical variations. In other words, the variability within each of the two groups in comparison are taken into account assuming that the dispersion in each group is identical, which is usually not true in case of original data. As EdgeR separates technical variance from biological variance, DGE can be reliably estimated between groups with small number of samples. We imported the raw read count files from two groups of interest into the CLCGWB interface to analyze the DGE of each such comparison. The resultant file contains a list of genes that are differentially expressed in these two groups with their statistically significant 9. The PCR primers were as follows:To validate the expressions of the genes of interest obtained from our DGE analyses, we independently collected samples from sickle mice and matched controls. For validation experiments, RNA was isolated and the purity of the RNA was checked as mentioned above. Thereafter, up to 1 μg of RNA was converted into cDNA by using a high capacity RNA to cDNA kit  and the resultant cDNA was diluted 20-fold to be used in q-PCR reaction mix. Quantifications of RNA expression levels were performed in an Applied Biosystems 7000 RT-PCR system. GAPDH was used for normalization. The total volume of reaction mixture was 25 μl containing 12.5 μl Power Sybr Green PCR master mix , 2 μl of diluted cDNA template, 0.1 μl of sense primer, 0.1 μl of anti-sense primer and 10.3 μl of distilled water. All the primers were synthesized by Integrated DNA Technologies (IDT). Thereafter, the calculations of relative gene expression were done using the 2(-Delta Delta C(T)) methodSmall proline rich protein 1A (Sprr1a): Sense: 5′-GCT GTC TAT CCT GCT TAT GAG TC-3′; Anti-sense: 5′-CTT TGG GCA ATG TTA AGA GGC-3′SerpinA1e: Sense: 5′-CCC TAT CTT GCA CTT CAA CCG-3′; Anti-sense: 5′-CAG CCC GTG TTT AAT GGA AA-3′SerpinA3g: Sense: 5′-GTG TCG GAT GTT GTG CAG TT-3′; Anti-sense: 5′-CAC CAT TTG GGA AGT TCA T-3′Glyceraldehyde 3-phosphate dehydrogenase (GAPDH): Sense: 5′-ATG TGT CCG TCG TGG ATT TGA-3′; Anti-sense: 5′-ATG CCT GCT CTA CCA CCT TCT T-3′RNA-seq data files in FastQ format were deposited in the Gene Expression Omnibus  under the accession number GSE86418 (Data Citation 1). This accession contains a total of 98 FastQ files resulting from the forward and reverse paired-end runs for each of the 49 samples. The FastQ format data serves as the raw data from the sequencing, which are subjected to further downstream processing. Additionally, the ISA-tab file provided with this publication includes detailed metadata for the study and standardized representation of the study design. Examples of processed data in two .xlsx files obtained from UMII-MSI-UMGC analysis pipeline are also stored in this accession: a) Raw_Read_Count_all samples.xlsx and b) FPKM_Values_all samples.xlsx. As the names suggest they contain the raw read counts and FPKM expression values obtained from this data. The metadata for these files are also presented in ISA-tab file provided with this publication. Three excel files named ‘edgeR_Age_Comparison_Output files’, ‘edgeR_Genotype_Comparison_Output files’ and ‘edgeR_Treatment_Comparison_Output files’ have been provided in figshare with full outputs from edgeR DGE analysis (Data Citation 2).In this study, best practices of NGS analysis were used to assess the quality of the reads and align them against a reference genome as described in the methods section. FastQ files were fed into the UMII-MSI-UMGC analysis pipeline that generates information on the mapping and gene quantification of the reads.The representative quality plots from the filtered fastQ files of this data-set are shown in 10. The large number of biological replicates (6) for each group along with high read depth enhanced the statistical power of these dataset and resulted in a very low coefficient of variance11. Consequently, the number of false positives expected during the differential gene expression promises to provide specific variations even with small fluctuations at the transcript level11. Therefore, robustness of this dataset is reflected from the above parameters and the DGE analysis performed using this dataset will be prone to error at the least possible level and the results should be of high accuracy and power.Another quality control measure of the data originates from the fact that the sequencing read depths used in this study ranged from 20–30 millions . These h12, MapSplice13, Cufflinks14 and others15 can be used to align and map reads from this dataset. Downstream quantification of transcript abundance can be performed using multiple platforms16, of which, we used cufflinks in our analysis. From the counts of the raw reads of the expression values or FPKM values, DGE analysis can be executed using a platform of choice from an array of developed packages such as DESeq2  groups of comparisons. From these 56 groups, there will be a much lesser number of comparisons which are of meaningful interpretation. The DGE analysis was performed using edgeR as this platform applies a normalization of the raw read counts which ensures that the genes with low abundance but highly variable read counts will have same variance as the genes with high abundance. Therefore, while edgeR provides more strict bounds for the calculations of DGE, it also enables decoupling of the technical and biological variances, even with small set of replicates, enhancing the overall statistical power of the comparisons.We used edgeR from CLCGWB software package for performing DGE analysis from the raw read counts obtained after TopHat-Bowtie mapping and subsequent quantification of the gene expression. From the set of 8 groups of samples, if we compare two (2) groups at a time, we can have a total of 56 .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.