We provide insights into the function of the hrpB gene in Xanthomonas citri subsp. citri by investigating the roles of hrpB in biofilm formation on abiotic surfaces and host leaves, cell motility, host virulence of the citrus canker bacterium and growth in planta.RNA helicases are enzymes that catalyze the separation of double-stranded RNA (dsRNA) using the free energy of ATP binding and hydrolysis. DEAD/DEAH families participate in many different aspects of RNA metabolism, including RNA synthesis, RNA folding, RNA-RNA interactions, RNA localization and RNA degradation. Several important bacterial DEAD/DEAH-box RNA helicases have been extensively studied. In this study, we characterize the ATP-dependent RNA helicase encoded by the hrpB gene is highly conserved in the sequenced strains of Xanthomonas. Mutation of the hrpB gene (∆hrpB) resulted in a significant reduction in biofilms on abiotic surfaces and host leaves. ∆hrpB also exhibited increased cell dispersion on solid medium plates. ∆hrpB showed reduced adhesion on biotic and abiotic surfaces and delayed development in disease symptoms when sprayed on susceptible citrus leaves. Quantitative reverse transcription-PCR assays indicated that deletion of hrpB reduced the expression of four type IV pili genes. The transcriptional start site of fimA (XAC3241) was determined using rapid amplification of 5′-cDNA Ends (5′RACE). Based on the results of fimA mRNA structure predictions, the fimA 5′ UTR may contain three different loops. HrpB may be involved in alterations to the structure of fimA mRNA that promote the stability of fimA RNA.The hrpB is involved in adherence of Xanthomonas citri subsp. citri to different surfaces. In addition, to the best of our knowledge, this is the first time that a DEAH RNA helicase has been implicated in the regulation of type IV pili in Xanthomonas.Our data show that The online version of this article (doi:10.1186/s12866-016-0655-1) contains supplementary material, which is available to authorized users. RNA helicases are enzymes that catalyze the ATP-dependent separation of double-stranded RNA (dsRNA). RNA helicases are found in all kingdoms of life , 2. DEADBacillus subtilis, two cold-induced putative RNA-helicases, CshA and CshB, are thought to be essential for cold adaption, during which they work in conjunction with cold-shock proteins to rescue misfolded mRNA molecules and to maintain proper initiation of translation at low temperatures [cshA DEAD-box gene in Staphylococcus aureus resulted in the dysregulation of biofilm formation and hemolysis via modulation of agr mRNA stability [Lactobacillus reuteri. In Listeria monocytogenes, four putative DEAD-box RNA helicases  are required for growth and motility [DExD/H proteins participate in many different parts of RNA metabolism, including RNA synthesis, RNA folding, RNA-RNA interactions, RNA localization and RNA degradation –9. Severeratures . Mutatiotability . Furthertability  in LactoEscherichia coli, is involved in processing daa mRNA from a fimbrial operon. This processing event results in a stable mRNA and the up-regulation of daa expression relative to the levels of other proteins that are encoded by the polycistronic transcript [E. coli either directly or indirectly through RNA interactions [HrpA, a DEAH-box RNA helicase in anscript . The Hrpractions , 15, conractions .Xanthomonas citri subsp. citri (X. citri) for mutants that were associated with effects on biofilm and identified hrpB (XAC0293), which encodes a probable DEAH-box RNA helicase. The function of RNA helicases in the Gram negative bacteria X. citri has not been explored. X. citri causes citrus canker, one of the most economically damaging diseases that affects citrus [X. citri forms biofilms on leaf surface [X. citri [In our previous study, we screened s citrus , 17. It s citrus . Previou surface –21, whicX. citri , 22.X. citri strain 306 contains a functional T4P [Biofilms are communities of bacterial cells that are embedded in a matrix of extracellular polymeric compounds that are attached to a surface . Biofilmonal T4P . The Fimonal T4P , are proonal T4P . Pilus ronal T4P .hrpB (XAC0293) gene plays an important role in adherence and biofilm development in X. citri and that its deletion reduced the expression of type IV pili genes. Our study sheds light on the involvement of DEAH-box proteins in adhesion, biofilm formation and pathogenicity in plant-pathogen bacteria.In this study, we showed that the http://web.expasy.org/compute_pi/), respectively. The protein XAC0293 shares 42 % identity with the Escherichia coli RNA helicase HrpB, the function of which has not been described. A domain structure analysis performed using the Pfam database showed that XAC0293 contains four domains that are associated with DEAD/H-box RNA helicase proteins, including a DEAD-like helicase superfamily domain (DEXDc) at the N-terminal, a helicase superfamily c-terminal domain (HELICc), a helicase-associated domain (HA2), and an ATP-dependent helicase C-terminal domain (HrpB_c) at its C-terminal. The HrpB protein also contains two predicted ATP-binding sites, one Mg++-binding site and one nucleotide-binding region. Protein BLAST showed that XAC0293 is highly conserved in other Xanthomonas species , X. oryzae pv. oryzae KACC 10331 (95 % identity), X. campestris pv. campestris str. ATCC 33913 (91 % identity), and Xylella fastidiosa (72 % identity)  is 2501 bp in length and is located within the genome at position 348799–351300  of X. citri strain 306, which was confirmed using PCR. The growth curve of the mutant was not different from that in the wild-type (data not shown). Biofilm development was examined in polystyrene plates and glass tubes and on sweet orange leaves. A significant reduction in biofilm formation was observed in ∆hrpB after 48 h of growth in NB medium containing 1 % glucose compared to the wild-type and complemented strains  than the wild-type strain (OD590 = 2.19 ± 0.45), and the complemented strain, ∆hrpB-p53hrpB, formed levels similar to the wild-type strain . These findings suggest that the hrpB gene is involved in cell adhesion and, consequently, biofilm formation in X. citri.To study the function of ins Fig. . Crystalain Fig. . No diffhrpB and wild-type strains were both tested to determine their motility on 0.5 % agar SB medium, which is used in sliding analyses in X. citri. In X. citri, sliding motility was promoted by EPS and inhibited by type IV pili [hrpB increased cell dispersion in X. citri on SB medium plates   Fig. . On the  °C Fig. . Swimmin °C Fig. . Growth X. citri on its sweet orange host. Populations of different strains were quantified at different post-spraying time points on citrus leaf surfaces. At seven days after the initial inoculation, the ∆hrpB population was smaller than the population of the wild-type strain 306 , the number of canker lesions on leaves infected with ∆hrpB was significantly less than the number on leaves inoculated with the wild-type strain  based on our results and previous studies that showed that mutations in type IV pilus gene are associated with increased motility and decreased biofilms [fimAXAC3241 and fimA2XAC3240, which encode for the major pilin subunit [pilBXAC3239, an ATPase gene that is required for T4P polymerization [hrpB strain than in the wild-type strain on NB medium . The RACE PCR product was sequenced to identify the specific transcriptional start site of fimA, which is located 113 nucleotides upstream of the first codon  transcript in the wild-type and ∆hrpB strains. To measure the stability of the fimA mRNA in the wild-type and ∆hrpB strains, the abundance of the fimA transcript was analyzed using the gyrA transcript as a control in RT-PCR. Prior to the reactions, transcription was blocked by adding 10 μg/mL ciprofloxacin  to the X. citri cell cultures. The relative abundance of the fimA transcripts was estimated from the specific PCR products that were observed on the agarose gels in comparison to the abundance of the control. The data showed that there was a significant reduction in the abundance of the fimA mRNA after 10 and 15 min in ∆hrpB compared to the wild-type strain , by the HrpB protein in wild-type X. citri. Our results show that the predicted HrpB protein in X. citri exhibits several characteristics that are considered typical for RNA helicases [hrpB mutants may be caused by the presence of multiple DEAD-box RNA helicases in X. citri, such as Xac0293 and Xac0442. Interestingly, four DEAD-box helicases, SrmB, RhlE, CsdA, and RhlB, are present in E.coli, and RhlB, RhlE, and CsdA are interchangeable for certain functions [Stable adhesion to an appropriate surface is the first step in biofilm formation . The abinfection , 21. Theelicases . The obsunctions .X. citri [X. citri [hrpB (XAC0293) has not been previously reported to play a role in biofilm formation in Xanthomonas. In X. citri, deletion of hrpB resulted in a decrease in cell adhesion and consequently less biofilm compared to a wild-type strain on both abiotic and biotic surfaces  and pilB (XAC3239) mutants showed increased sliding motility and reduced biofilm formation [hrpB mutation resulted in the repression of TP4 genes, a reduction in adhesion and an increase in movement  assists in protecting mRNAs from attack by ribonucleases [E. coli and B. subtilis have shown that a variety of mRNAs can be significantly destabilized by mutations in the Shine-Dalgarno element that interfere with ribosome binding by markedly reducing complementarity to 16S rRNA [hrpB would be that HrpB is associated with the recruitment of other regulatory factors. In studies of other DExD/H RNA helicases, it has been proposed that when one of these factors binds to a 5′UTR, it may modulate its activity physically to recruit a complex of proteins, which could potentially defend the 5′mRNA against decay [fimA mRNA stability, the factors that interact with this putative ATP-dependent RNA helicase need be identified and characterized. The third hypothesis involves the affinity of some ribonucleases to double-stranded RNA. Double-stranded RNAs are targeted by specific ribonucleases, such as RNAse III [fimA mRNA, the putative ribonuclease would not be able to bind to its target, and HrpB could thereby prevent the degradation of the mRNA.Translational repression often leads to the rapid decay of mRNA . When thype Fig. , which wucleases , 46. Thiucleases . For exa16S rRNA . Convers16S rRNA . Furtherst decay . HoweverNAse III . If HrpBfimA mRNA in addition to other genes in the same operon that are involved in adherence, biofilm formation, motility and the development of citrus canker disease. Further biochemical studies are required to determine the mechanisms involving the HrpB protein in this process. To the best of our knowledge, this is the first time that a DEAH-box RNA helicase has been implicated in the regulation of type IV pili genes in Xanthomonas and the first time that a RNA helicase has been shown to be important for motility, biofilm formation and epiphytical survival.The ATP-dependent RNA helicase HrpB appears to positively regulate the hrpB gene, which encodes an ATP-dependent RNA helicase, in X. citri. We demonstrate that the HrpB protein in X. citri is involved in biofilm formation, motility and survival on leaf tissues. Quantitative reverse transcription-PCR assays indicated that deletion of hrpB reduced the expression of three IV pili genes, including fimA. The fimA mRNA predicted structure indicated that the fimA 5′ UTR may contain three different loops. The ATP-dependent RNA helicase HrpB appears to positively regulate the abundance of the mRNA of fimA by promoting the stability of the fimA RNA.In this work, we characterized the E. coli DH5α cells were grown at 37 °C in Luria-Bertani (LB) medium (1 (w/v) tryptone, 0.5 (w/v) yeast extract, and 1 % (w/v) sodium chloride, pH 7.5) shaking at 200 rpm or on plates. X. citri wild-type (ampicillin-resistant) [The bacterial strains and plasmids used in this study are listed in Table sistant)  and mutaBacterial genomic DNA and plasmid DNA were extracted using a Wizard genomic DNA purification kit and a Wizard miniprep DNA purification system according to the manufacturer’s instructions . The concentration and purity of the DNA were determined using a Nanodrop ND-1000 spectrophotometer . PCR was performed using standard procedures  with PfuhrpB deletion mutant, approximately 1 kb of the upstream and downstream regions of the hrpB gene (XAC0293) was amplified using PCR from genomic DNA obtained from X. citri strain 306 using the following primer pairs: 0293mF1Hind (5′ CGTGTTCACCGAGGACAGTGGCCTG 3′) and 0293mR1Bam (5′ ATAAGGATCCAAAAGCGGGGTCGGTCATGC 3′); and 0293F2Bam (5′ CTATGGATCCGGCACCTGAAACACATGGACC 3′) and 0293mR2Hind (5′ GAATAAGCTTTGCTGCCGGTGGTGGATTGTG 3′), respectively. The PCR products were digested with BamH1, and both fragments were ligated to produce a deletion construct. The resulting fragment was cloned into the pNPTS138 suicide vector  to generate pNPTS_hrpB  and 0293_p53_R (5′AGGAAGGATCCTGCGGTACCGGTGCCACGTGGTTTTGCTCTGT 3′). The amplified DNA fragment was cloned into pUFR053 [KpnI restriction site to obtain the recombinant plasmid p53_hrpB   was incubated on the abaxial surface of Valencia sweet orange leaves, which were maintained at 28 °C in a humidified chamber. At 24, 48 and 72 h after the start of incubation, biofilm formation was visualized on the leaf surfaces using crystal violet staining. Leaf discs from staining spots were excised, dissolved in 1 mL ethanol:acetone  and quantified by measuring the optical density at 590 nm. Data from both experiments were statistically analyzed using one-way analysis of variance (ANOVA) (p < 0.05), and values are expressed as the means ± standard deviations.Biofilms that formed on polystyrene and glass surfaces were examined as previously described , with moescribed . BrieflyX. citri strains. The experiments were repeated three times with three replicates each time. The diameter measurements were statistically analyzed using one-way analysis of variance (ANOVA) (p < 0.05), and the values are expressed as the means ± standard deviations of three independent experiments.To test cell motility, bacteria were grown overnight in NB medium. A 3 μL volume of bacterial cultures with OD 600 = 0.3 was then spotted onto the surface of a plate containing SB medium plus 0.5 % (wt/vol) agar   for the Citrus sinensis cv. Valencia) plants that were prepared in a quarantined greenhouse at the Citrus Research and Education Center . X. citri strains  that were grown in selective antibiotic NB medium overnight at 28 °C were centrifuged at 4800 rpm and then resuspended in 1 % phosphate buffer (pH 7.0). Bacterial suspensions from each strain were inoculated by pressure infiltration (105 CFU/ml) and spraying (108 CFU/mL). Phosphate buffer was used as the control in non-infected plants. All plant inoculations involved a minimum of three immature leaves from each plant, and three plants were inoculated for each bacterial strain. The plants were kept in a greenhouse at the Citrus Research and Educational Center  at a temperature of 28 ± 4 °C in high humidity for 21 days. Disease symptoms were photographed at 7, 14 and 21 days post-inoculation, and both tests were independently repeated two times.Pathogenicity assays were performed as previously described . BrieflyTo obtain measurements to analyze bacterial epiphytic survival, populations of the pathogen were isolated from the leaves that were inoculated by spraying as described above. At 0, 7, 14 and 21 days post-inoculation, three leaves with similar sizes were randomly collected from three different plants. The leaves were immersed in 10 mL of 1 % phosphate buffer in Falcon flasks (50 mL). Bacterial cells were collected and then vortexed for three minutes to homogenize the tissue. Epiphytic bacterial numbers were determined in serial dilutions of these suspension and then plated on NA medium with the appropriate antibiotics. Colonies were counted after 2 days of incubation at 28 °C. The assays were independently repeated two times with three replicates.X. citri wild-type and hrpB mutant (ΔhrpB) strains using two methods: NB medium and the leaves of sweet orange plants that were inoculated by spraying. For the experiments performed using NB medium, the strains were grown in 10 mL of NB at 28 °C while shaking, and both bacterial cultures were collected in the middle exponential stage (OD = 0.8). RNA was immediately stabilized by mixing it with 2 volumes of RNA-protecting bacterial reagent . For the epiphytical assays, 3 different plants were inoculated with each strain at 108 CFU/mL. At different timepoints after infection , three leaves were collected from each plant to perform RNA extraction using a RNase plant mini kit . Contaminated genomic DNA was removed from the RNA during both experiments by processing the preparation using a TURBO DNA-free kit , and RNA purity and quantity was determined using a ND-8000 Nanodrop spectrophotometer . For qRT-PCR assays, 4 genes in the T4P system  were chosen for gene expression analyses using the primers shown in Table –∆∆CT method. gyrA was used as the endogenous control. Both types of quantitative real-time PCR experiments were performed using three biological replicates.To quantitatively analyze gene expression, we used RNA obtained from fimA (XAC3241) was determined using a 3′/5′ RACE Kit (Roche) according to the manufacturer’s instructions. Briefly, total RNA was obtained from X. citri wild-type cell cultures that were grown in NB medium to an OD 600 of 1.0. After treating the RNA using a TURBO DNA-free kit , the RNA was reverse-transcribed using a gene-specific primer  were grown at 28 °C in NB medium to an OD 600 nm of 0.5 and then treated with ciprofloxacin at a final concentration of 10 mg/ml to inhibit transcription. Samples were collected at 0, 5, 10 and 15 min after treatment with ciprofloxacin. The cells were harvested by centrifugation at 5,000 rpm and used immediately to extract RNA using a RNeasy Mini kit . Total RNA samples were treated with Turbo RNase-free DNase (Ambion) and quantified using a Nanodrop. A total of 2 μg of treated RNA was used for reverse transcription using an iScript cDNA Synthesis kit (Bio-Rad) according to the manufacturer’s protocol. Reactions were subjected to PCR amplification for 22 cycles using 0.3 mM of each of two primers, fimAF (5′ GAAGCAACAGGGTTTCACGC 3′) and fimAR (5′TATGTTGCCAAGTCGCAGG 3′), and Taq 2x Master Mix (Biolabs). Ten microliters of each reaction was resolved in a 1.5 % agarose gel. Analyses of gyrA were performed and used as the controls for normalizing the fimA amplified products.Bacterial cultures of


We examined the demographic and health characteristics of people aged 45 years or older in 21 states with self-reported increased confusion or memory loss (ICML)  by whether or not they also reported functional difficulties related to ICML. We used data from the 2011 Behavioral Risk Factor Surveillance System optional module on impact of cognitive impairment. After adjusting for demographic differences, we found that respondents with ICML and functional difficulties were significantly more likely than those with ICML and no functional difficulties to report frequent poor physical health, frequent poor mental health, limited activity due to poor physical or mental health, and a need for more help. Further understanding of the implications for long-term services and supports is needed. In response to national recognition of the importance of perceptions about cognitive functioning and its effect on functioning and well-being ,2, severBRFSS is a state-based random-digit–dialed telephone survey of noninstitutionalized US adults aged 18 or older that examines perceptions about health and health behaviors . In 2011The BRFSS core survey included questions on age, sex, race, ethnicity, education level, and employment status. Respondents were classified as living alone based on BRFSS screening procedures that determine household composition. Disability status was ascertained from reported activity limitations, use of special equipment, or both. Having 1 or more chronic condition was determined by asking about 6 conditions named in the BRFSS core survey: arthritis, asthma, cardiovascular disease, cancer, chronic obstructive pulmonary disease, and diabetes. Physically and mentally unhealthy days and activity-limited days were classified as being frequent if respondents reported 14 days or more in the previous month  of well-being for those with functional difficulties and those without such difficulties in 5 models, each adjusted for categorical age, race/ethnicity, and education  and Stata 12.1 (StataCorp LP) and accounted for the complex sampling design. Analyses were restricted to landline respondents because only 7 states included cellular telephone respondents in the module and evidence suggests that cellular telephone respondents differ from landline respondents . Weighteducation .The sample consisted of 10,583  respondents who reported ICML. Among these, 10,412 responded to the questions on functional difficulties; 42.9% were classified as ICML with functional difficulties and 57.1% as ICML with no functional difficulties. Respondents who reported ICML with functional difficulties tended to be younger and have less education than those who reported ICML and no functional difficulties . We founAdjusted analyses showed that respondents with ICML and functional difficulties were significantly more likely than those with ICML and no functional difficulties to report frequent poor physical health , frequent poor mental health , and limited activity due to poor physical or mental health . Respondents with ICML and functional difficulties also were significantly more likely than respondents with ICML and no functional difficulties to report needing help related to ICML  and to always or usually receive care from a family member or friend related to cognitive decline .Although ICML is commonly regarded as a concern only among older adults, we found that those with ICML and functional difficulties tended to be younger than those with ICML and no functional difficulties. Studies of adults aged 60 or older found similar trends . EnsurinA growing body of evidence suggests a greater burden of multiple chronic health conditions among adults aged 60 years or older who report memory limitations than among those who report no such limitations ,10. We fThis study has several limitations. Data are from 21 states, and therefore the sample does not represent the United States as a whole. Although questions underwent multiple rounds of cognitive testing, ICML is self-reported, might be subject to recall bias, and has not been validated by clinical assessment. The study design is cross-sectional, and causality cannot be inferred. We examined functional limitations related to ICML but did not account for all causes of functional disabilities. BRFSS respondents were drawn from households with a landline telephone, thereby limiting the sample to those who can afford one, and the survey also excludes adults in noninstitutionalized settings.Our findings highlight the association between physical and cognitive health among a large group of community-dwelling respondents. That people with ICML and functional difficulties were younger than those with ICML and no functional difficulties calls for examination of cognitive health issues at younger ages. Public health surveillance monitors trends, identifies disparities, and informs public health service needs and research gaps. Another key aspect of work in this area concerns the prevalence of ICML in households; Deokar and colleagues examine ICML in households using 2011 Behavioral Risk Factor Surveillance System data from 13 states (


Deregulation of RNA editing by adenosine deaminases acting on dsRNA (ADARs) has been implicated in the progression of diverse human cancers including hematopoietic malignancies such as chronic myeloid leukemia (CML). Inflammation-associated activation of ADAR1 occurs in leukemia stem cells specifically in the advanced, often drug-resistant stage of CML known as blast crisis. However, detection of cancer stem cell-associated RNA editing by RNA sequencing in these rare cell populations can be technically challenging, costly and requires PCR validation. The objectives of this study were to validate RNA editing of a subset of cancer stem cell-associated transcripts, and to develop a quantitative RNA editing fingerprint assay for rapid detection of aberrant RNA editing in human malignancies.in vitro RNA editing model and developed a sensitive RNA editing fingerprint assay that employs a site-specific quantitative PCR (RESSq-PCR) strategy. This assay was validated in a stably-transduced human leukemia cell line, lentiviral-ADAR1 transduced primary hematopoietic stem and progenitor cells, and in primary human chronic myeloid leukemia stem cells.To facilitate quantification of cancer stem cell-associated RNA editing in exons and intronic or 3'UTR primate-specific Alu sequences using a sensitive, cost-effective method, we established an In lentiviral ADAR1-expressing cells, increased RNA editing of MDM2, APOBEC3D, GLI1 and AZIN1 transcripts was detected by RESSq-PCR with improved sensitivity over sequencing chromatogram analysis. This method accurately detected cancer stem cell-associated RNA editing in primary chronic myeloid leukemia samples, establishing a cancer stem cell-specific RNA editing fingerprint of leukemic transformation that will support clinical development of novel diagnostic tools to predict and prevent cancer progression.RNA editing quantification enables rapid detection of malignant progenitors signifying cancer progression and therapeutic resistance, and will aid future RNA editing inhibitor development efforts.The online version of this article (doi:10.1186/s12967-014-0370-3) contains supplementary material, which is available to authorized users. Dormant cancer stem cells (CSCs) are primary arbiters of cancer progression and a major focus of targeted therapy development efforts. Deregulation of RNA editing by adenosine deaminases acting on double-stranded (ds) RNA (ADARs) has been implicated in the progression of diverse human malignancies, including chronic and acute forms of leukemia -3, lobulADAR), ADAR2 (ADARB1), and ADAR3 (ADARB2). In mouse hematopoietic development, ADAR1 plays a key role in hematopoietic stem cell (HSC) survival [Of particular relevance to the pathogenesis of human disease, over 90% of RNA editing occurs in primate-specific Alu sequences that form dsRNA secondary structures , often wsurvival  and cellsurvival ,13, and survival -16. Whilsurvival , CSC-asssurvival  leading Advances in next-generation sequencing technologies and bioinformatics tools have led to the identification of hundreds of thousands of RNA editing sites throughout the human transcriptome ,20, the To address these challenges, we developed and applied a straightforward, clinically amenable assay to validate and quantify RNA recoding in stem cell regulatory transcripts identified through whole transcriptome RNA-seq analysis of purified primary LSC . A site-+ cells  for subsequent FACS-purification of hematopoietic progenitor cells (CD34+CD38+Lin-) that represent the LSC fraction in BC CML [A large collection of leukemia patient samples and normal age-matched control bone marrow samples were obtained from consenting patients in accordance with Institutional Review Board approved protocols at UCSD and the University of Toronto  populations were isolated. Freshly-sorted cells were collected in lysis buffer  for RNA extraction followed by RNA-seq or qRT-PCR analyses as previously described [For primary patient-derived LSC purification, CD34-selected cells were stained with fluorescent antibodies against human CD34, CD38, lineage markers  and propidium iodide as previously described ,23,24. Fescribed .www.nucleobytes.com) and peak heights calculated using ImageJ (NIH). For RNA editing analysis of sequencing chromatograms, ratios of edited/WT peaks were calculated using the raw peak amplitude of each sequence trace.For PCR and targeted Sanger sequencing analysis, 1-2 μL of first-strand cDNA templates were prepared for PCR in 25-50 μL reaction volumes using the high-fidelity KOD Hot Start DNA Polymerase kit according to the manufacturer’s instructions . “Outer” primers  were maintained in complete medium containing DMEM , 10% fetal bovine serum (FBS), 1% Glutamax (Life Technologies), and 1% penicillin-streptomycin (Life Technologies). Parental cell lines and stably-transduced lines were authenticated as K562 by routine qRT-PCR analysis of BCR-ABL transcript levels . BrieflyWe have previously characterized lentiviral vectors (Thermo Scientific) for overexpression of human ADAR1 p150-IRES-GFP . PrimersFor preparation of stably-transduced K562 cell lines, 50,000 wild-type (wt) K562 cells were plated into 96-well U-bottom plates in complete culture medium and transduced with lentiviral vectors expressing GFP (ORF), ADAR1-GFP, or ADAR1m-GFP at multiplicities of infection (MOI) from 50-200. After transduction, cultures were expanded for at least 5 passages and then processed for FACS purification of GFP-positive cells to establish pure stably-transduced lines. Stable expression of lentivirus-enforced ADAR1 conferring increased transcript levels of human ADAR1 in K562-ADAR1 cells was confirmed at every 5 passages by qRT-PCR.For transduction of human normal HSC and CML progenitors, 50,000 CD34-selected  cells were plated in 96-well U-bottom plates in StemPro media (Life Technologies) supplemented with human cytokines  as previously described . Twenty-6 cells), washed in HBSS containing 2% FBS (staining media), and sorted using a FACS Aria II (Sanford Consortium Stem Cell Core Facility) for high GFP signal to purify the highly-transduced cell population. Purified cells were collected in complete media and maintained under routine culture conditions for K562 cells. The lentiviral-ORF and ADAR1 vectors include a blasticidin-resistance gene, but no significant change was observed in ADAR1 expression in stably-transduced cell lines following selection with blasticidin, and therefore no subsequent selection method was used after FACS purification. For all experiments, low-passage cells were thawed and maintained for no longer than two months in culture.For purification of stably-transduced K562 cell lines, K562 cells transduced with lentiviral-ADAR1 or ORF controls (MOI = 50-200) were collected  including an RNase A incubation step to digest any RNA present (Qiagen). RNA was stored at -80°C and gDNA stored at -20°C. Immediately prior to reverse transcription of RNA samples, nucleic acid concentrations were quantified on a NanoDrop 2000 spectrophotometer (Thermo Scientific), and purity was considered acceptable if A260/A280 values were ≥1.8. For standard qRT-PCR analysis of relative mRNA expression levels, DNA was synthesized using 50 ng - 1 μg of template RNA in 20 μL reaction volumes using the First-Strand SuperScript III Reverse Transcriptase Supermix (Life Technologies) followed by incubation with RNase H according to the manufacturer’s protocol and as described previously [Cell lines, lentivirus-transduced primary hematopoietic cells, or FACS-purified primary cells were harvested in lysis buffer (Qiagen). RNA was purified using RNeasy extraction kits with a DNase (Qiagen) incubation step to digest any trace genomic DNA (gDNA) present. For RNA extraction from cell line lysates, 1-2 × 10eviously . All cDNBecause RNA editing events often occur in pre-processed RNA species, for cDNA preparation, three different conditions were evaluated, including (1) reverse transcription with gene-specific primers, (2) random hexamer primers only, or (3) a supermix containing both random hexamers and oligo-dT primers. Using cDNA prepared with all three methods was suitable for detection of intronic regions in cDNA prepared from DNase-digested RNA extracts, and allowed detection of increased RNA editing in K562-ADAR1 cells. We therefore proceeded with the standard supermix reverse transcription method for RESSq-PCR, as this would provide the most versatility for use of valuable human tissue samples and would allow analysis of total mRNA expression of other genes in the same samples.-ddCt method. To ensure validity of results, only Ct values <35 were used in gene expression analyses. All primer sets were tested in a no-template control (NTC) reaction containing only water instead of cDNA, and all gave Ct values >35 in NTC reactions. Production of a single amplicon of the expected size was verified for each primer set by DNA gel electrophoresis on 2% agarose gels containing ethidium bromide. For all cell line experiments, assays were repeated at least three times using separate RNA extracts and cDNA preparations.We have made every effort to adhere to the Minimal Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines . PrimersIn order to implement a rapid, cost-effective and clinically amenable method to detect a CSC-specific RNA editing fingerprint of cancer progression, we devised an RNA editing site-specific primer design strategy that is compatible with SYBR green qRT-PCR protocols (RESSq-PCR). Since RNA-edited transcripts are predicted to differ from wild-type (WT) sequences at only one nucleotide position, detection of RNA editing by qRT-PCR requires highly sensitive and selective primer design strategies. We have previously developed qRT-PCR primers that specifically recognize a gene product with a single point mutation (JAK2 V617F ), and heCt Edit – Ct WT)-.qRT-PCR data were measured as a continuous outcome and each group was assessed for distribution. For normally distributed data, the Student’s Previous whole transcriptome analysis Figure A revealein vitro. ADAR1 expression was confirmed at regular intervals (approximately every 5 passages) by qRT-PCR ; lentiviral ADAR1 mutant (A5293C) that lacks deaminase activity   strategy was devised to detect aberrant RNA recoding in three different dsRNA contexts . An additional known editing site within exon 10 of the stem cell self-renewal factor GLI1 was included because ADAR-directed RNA editing of this site promotes transcriptional activity of GLI1 protein. The edited form of GLI1 is less susceptible to inhibition by the negative regulator of hedgehog signaling, suppressor of fused (SUFU) , promotiA modified secondary primer design strategy was used in some cases where it was not possible to design primers with similar characteristics directly upstream and downstream of the editing site. This strategy employs a similar principle, however the FW inner primer sequences detecting WT and edited variants are almost identical, with only the 3′ nucleotide position differing to discriminate A or G bases Figure B. An addRESSq-PCR primers specific for edited sites in MDM2, APOBEC3D, GLI1 and AZIN1 distinguished G(I) bases in cDNA from K562-ADAR1 cells, with no signal in gDNA  changes in MDM2, APOBEC3D and GLI1  or peripheral blood samples from CP CML patients. After 3-5 days in vitro, normal bone marrow cells transduced with lentiviral-ADAR1 displayed high levels of lentivirus-derived ADAR1 compared with ORF controls  cells transduced with lentiviral-ADAR1 and co-cultured with humanized mouse stromal cells that recapitulate the bone marrow microenvironment [In CP CML hematopoietic  , these cases were among the ones that showed highest editing rates in MDM2 , and higFuture studies evaluating larger cohorts of cancer patients, with comprehensive clinical annotation of patient treatment status and disease outcomes, will provide further insights into the relationship between aberrant RNA editing and risk for disease progression. Additionally, we hypothesize that RNA editing activity may be influenced by differing treatment status at the time of sample collection (e.g. hydroxyurea versus imatinib) and could also provide prognostic information related to risk for acquisition of drug resistance. Thus, assessment of sequential patient samples before and after treatment or disease progression will allow precise determination of the RNA editing levels that correlate with poorer prognosis in a variety of blood cancers and other CSC-driven malignancies.Recent evidence indicates that ADAR-directed RNA editing represents a novel mechanism of disease progression and a promising therapeutic target for diverse human cancers. Malignant RNA editing programs represent an unexplored resource for biomarker development, and the development of an RNA editing fingerprint assay demonstrates the application of novel findings from high-throughput sequencing and bioinformatics studies ,27 to va


Aptamers are short sequences of nucleic acid (DNA or RNA) or peptide molecules which adopt a conformation and bind cognate ligands with high affinity and specificity in a manner akin to antibody-antigen interactions. It has been globally acknowledged that aptamers promise a plethora of diagnostic and therapeutic applications. Although use of nucleic acid aptamers as targeted therapeutics or mediators of targeted drug delivery is a relatively new avenue of research, one aptamer-based drug “Macugen” is FDA approved and a series of aptamer-based drugs are in clinical pipelines. The present review discusses the aspects of design, unique properties, applications, and development of different aptamers to aid in cancer diagnosis, prevention, and/or treatment under defined conditions. Aptamers are relatively small biomolecules  whose three-dimensional structure confers on them the ability to bind their cognate ligands . The ter in vitro and can be selected to target virtually any protein even toxins or nonimmunogenic proteins within a relatively short period of time, whereas antibody generation is limited by the need to use live animals [13 to 1015 different variants of nucleic acid sequences, and involves the selection for oligonucleotides able to efficiently bind desired target molecules [ in vitro transcription of a random DNA oligonucleotide library using T7 RNA polymerase before starting the first round of RNA SELEX process. Target binding function of nucleic acid aptamers is mainly dependent on their unique three-dimensional folding. The secondary structures of aptamers mainly consist of short helical arms and single stranded loops, defined by intramolecular base complementarity, whereas tertiary structures of aptamers result from a combination of these secondary structures with pseudoknotting of segmental sequence complementarity of loops and bulges and allow aptamers to bind target by noncovalent interactions like Van der-Waals interactions, hydrogen bonding, topological compatibility, stacking of aromatic rings, and electrostatic interactions [Nucleic acid aptamers selected from a library of random sequences by systematic evolution of ligands by exponential enrichment (SELEX) bind to the chosen ligands with high specificity and affinity , 2. The  animals . In addi animals . Due to  animals . All the animals . SELEX solecules . For the20 oligonuclotides are technically feasible but are rarely used in practice [SELEX is started with a population of different random sequences flanked by defined sequences. The defined sequences are placed to ensure amplification of all different sequences in the selected population by polymerase chain reaction (PCR). The primers designed should anneal specifically to the template without forming “primer dimer” or secondary structures. Typically, up to 20-nucleotide long primers are used for PCR and can be synthesized with good yield. For selection of RNA aptamers, T7 RNA polymerase promoter sequence is required 5′ to the PCR template sequence within the primer design . In prinpractice . The majNA aptamer randomization depends upon sequence information of aptamer random sequence region. Three types of randomization are employed in designing aptamer random sequence region, that is, partial, segmental, and complete. Partially randomized (doped) library is used for selecting new functional molecules by taking in consideration the motifs whose structure and fuctions are known to bind with the target molecule . KnowledN nucleotides long, it will give a maximum of 4N possible sequence variants which means a theoretical maximum of 4N probable conformations. Short, randomized, nucleic acid segments of 20 to 50 nucleotides provide a vast sequence space for novel nucleic acid structures and functions [ in vitro selections of nucleic acids are typically limited to 430 (approx. 1020) different sequences.It is very important to choose the length of the random region while selecting for an aptamer. It is a wishful assumption that libraries with a longer random region may facilitate selection of more efficient aptamers because longer sequences are able to form a wider range of different three-dimensional structures . The lonunctions . If a ta In vitro selection can also make use of modified nucleotides (NTPs) which further confers the desired pharmacokinetic properties on the aptamers. The modified nucleotides can affect the chemistry, which in turn can bias the course and outcome of SELEX [Chemistry of the oligonucleotide pool also affects the selection method to be employed for the development of aptamers with affinity for chosen ligands. With increasing size of the DNA molecule, a larger mass would be needed to represent each possible variant in the pool. As the number of nucleotides increases, that is, the length of random nucleotide sequence, maximum number of variants per unit mass (mg) shows saturation and in fact declines for larger aptamers . So largof SELEX . The T7 of SELEX , 7. in vitro transcription, affinity binding and elution, and reverse transcription  or PCR (DNA aptamers),cloning and characterization of selected aptamers and further enrichment of high affinity aptamers by iterative rounds of selection.Negative selection for subtraction is a crucial step to minimize enrichment of nonspecifically bound oligonucleotides with the matrix absent of ligand [f ligand , 4. For f ligand . RNA aptAptamers are increasingly being used as a replacement option for antibody mainly because of the special features described in the following sections.The method for nucleic acid aptamer selection is quite simple and yields highly specific aptamers within a short time , 6. It hThere are no reports demonstrating toxicity or immunogenicity of aptamers. It is observed by clinical, cellular, or biochemical measures that high doses of aptamers (10 mg/kg daily for 90 days) are not toxic in rats or woodchucks . While tNucleic acid aptamers can be administered by either intravenous or subcutaneous injection, whereas most antibody therapeutics are administered via intravenous infusion. Due to low solubility, large volumes are necessary for therapeutic monoclonal antibodies. With good solubility (>150 mg/mL) and comparatively low molecular weight , aptamers are advantageous for tumor penetration and blood clearance. Aptamer bioavailability via subcutaneous administration is >80% in monkeys . Therefo in vivo half-life of the thrombin aptamer from 24 min to 6 h in rats, with little or no effect on thrombin binding affinity [For improving the stability of aptamers, pyrimidine as well as purine nucleotides can be modified at their 2′ positions and tested for their ability to accommodate stabilizing modifications like 2′-O-methyl groups . These maffinity . Howeveraffinity .Therapeutic aptamers are chemically synthesized and consequently can be readily scaled as needed to meet production demand. Therapeutic aptamers are chemically robust. They are intrinsically adapted to regain activity following exposure to heat, denaturants, and so forth and can be stored for extended periods (more than 1 year) at room temperature as lyophilized powders .The above-mentioned properties of aptamer specificity toward target ligands make them an ideal tool for diagnostics and therapeutics. Aptamers have been extensively used in analytical applications. Nucleic acid aptamers have wide applicability as exemplified in the following sections . Proteus mirabilis (causative agent of CAUTIs) blocks the flexible tube connecting body cavity, which disallows fluids to pass into or out of it by forming a crystalline biofilm [ Proteus mirabilis were selected using cell-SELEX in combination with in silico maturation (ISM). These aptamers would be utilized for biosensor development to get an insight into pathogen associated membrane proteins [The applicability of aptamer is not limited to targeted delivery to their cognate molecules, but they can function for prophylactic help as well. These molecules could either be intracellular , extracellular , or cell-surface targets . Nucleic acid aptamers have been projected as convenient molecular mimics of antibody to provide the basis for developing prophylactic idiotypes in much the same way as monoclonal antibodies have been used. Nowadays, increased chances of catheter associated urinary tract infections (CAUTIs) are imposing a big challenge . Proteus biofilm . Blockagproteins . In anotproteins . TCR-medproteins . cis-acting genetic control elements that regulate metabolic genes via structured sequences present in 5′-untranslated region of mRNA. Binding of metabolites to aptamer domain is independent of protein factors [Riboswitches are naturally occurring RNA sequences with specific structural recognition of cellular metabolites to modulate gene expression. The untranslated regions of many mRNAs are known to act as sensors of metabolic pool and reorganize themselves resulting in altered efficacy of translation . This he factors . Discove factors . It has  factors . These s factors . Guanine factors . Both be factors . A ribos factors . General factors . RiboswiClinical diagnosis of disease with the help of aptamers is attractive due to their small size, stable folding structure, and economy. There has been rapid advancement in application of such aptamers in disease diagnosis, imaging, and new biomarker discovery. Aptamers can detect very low amounts of diseased or tumor cells for which they are selected. For example, immobilized anti-EGFR RNA aptamer on a chemically modified glass surface can determine the presence and/or extent of GBM (glioblastoma) tumor cells . EGFR isBiomarkers are important and crucial in diagnosis and treatment of cancer . They ca botulinum toxin [ Staphylococcal enterotoxin B (SEB) [ Salmonella typhimurium with high affinity (Kd: 20 nM) and specificity [ Salmonella typhimurium. These may find use in prophylaxis or targeted delivery vehicles.Aptamer technology offers feasible alternative options for addressing the challenge of early diagnosis in human health . A singlum toxin . This DN B (SEB) . The cyt B (SEB) . RNA apt B (SEB) . Selecte B (SEB) . DNA apt B (SEB) . Biotoxi B (SEB) . Althougcificity . It did  in vitro-generated receptor for use as a biosensor for molecules used in biological warfare. Another sensory system (RNA aptamer) for opium alkaloid codeine, a class of benzylisoquinoline alkaloids (BIAs) and metabolite in opium alkaloid biosynthesis pathway, can precisely measure the codeine concentration. Two best codeine binding RNA aptamers (FC5 and FC45), with Kd 2.5 and 4.0 μM, respectively, are good enough to discriminate codeine from its structural analog [μM in human blood serum by a “split aptamer” [Aptamers are also used in biosensors due to the behaviour of ligand induced conformational changes. Such aptamers work as molecular beacons and may be used for the detection of environmental contaminant and monitoring carcinogen or to check the level of drugs in the blood . Over thl analog . Anotheraptamer” . The splaptamer” . Designiaptamer” .Aptamers can be used as chip based biosensor array by immobilizing fluorescent labelled nucleic acid aptamer on a glass slide where they can still rotate at a rate that corresponds to their apparent volume and mass . The bin in vitro and in vivo for its activity of ligand dependent splicing. Insertion of aptamers into the 5′-untranslated region of mRNA provides a handle for translational control of expression of specific genes in living cells. Translation of such aptamer RNA constructs can be regulated by reversible ligand dependent conformational change of aptamer domain [Chimeric RNA molecules have been constructed by fusion of aptamers with targeted ribozyme . Such chr domain . In vitro selected TCF RNA aptamer binds with T-cell factor and inhibits the tumorigenic function of beta-catenin in colon cancer cells by modulating beta-catenin target genes such as cyclin D1 and matrix metalloproteinase-7 [Cancer cells show altered expression of signaling pathways due to mutation in one or more than one gene. There are upregulation of oncogenes and downregulation of tumor suppressor genes. Cancer cells exhibit tendency for uncontrolled cell division and successfully escape from conventional apoptosis pathways. The Wnt (beta-catenin) signaling plays an important role in controlling human tumorigenesis. Beta-catenin serves as an effective anticancer drug target.einase-7 . CD28 iseinase-7 . This apeinase-7 .Due to their merits as cognate molecules, aptamers are quite attractive for therapeutic applications too. As therapeutic agent, RNA aptamers have advantages over other classes of RNA tool like short hairpin RNA (shRNA), small interfering RNA (siRNA), ribozyme, or antisense oligonucleotides (AS OGNs),  in terms165 is the dominant isoform, primarily responsible for pathological neovascularization in age-related macular degeneration (AMD) and diabetic macular edema (DME) [165 selectively, leaving other isoforms unaffected and modified them to increase their stability in serum by fluorination, methylation, and addition of a 5′-poly ethylene glycol moiety [The inhibition of viral invasion and replication using nucleic acid aptamer is another important area of research. An RNA aptamer “Macugen” is targeted against the angiogenic cytokine VEGF and its binding prevents choroidal neovascularization. In 2004, US Food and Drug Administration (FDA) approved the first aptamer-based drug Macugen or Pegaptanib sodium  . This wama (DME) . Researcl moiety , 80. Thiβ, and heregulin ligands to activate downstream signaling cascades [Similarly, EGFR protein family consists of four closely related receptor tyrosine kinases, namely, EGFR or ErbB-1 and its three homologs ErbB-2 or HER2/neu, ErbB-3 or HER3, and ErbB-4 or HER4 that can form homo- and heterodimers after receiving signals from EGF, TGF-cascades . Mutatiocascades . Furthercascades . HER2 ancascades . A30 RNAcascades . Further in vivo [Prostate specific membrane antigen (PSMA) is a unique marker for prostate cancer cells. PSMA specific RNA aptamers (xPSM-A9 and xPSM-A10) were isolated and used as intracellular delivery tools in conjugation with siRNAs or shRNAs for specifically targeting cancer cells instead of normal cells . In this in vivo , 92. Rad in vivo . This apThe surface glycoprotein gp120 of the human immunodeficiency virus interacts with host CD4 receptors leading to initiation of membrane fusion and delivery of viral RNA and enzymes to host cells. Aptamer siRNA chimeras have been developed to silence virus replication and propagation in T cells. In this chimera, siRNA is used against the HIV-1 tat/rev region . In combAptamers have wide applicability in pathway elucidation through their effect as specific inhibitors of intracellular signaling pathways. One example of such an inhibitory aptamer is the mitogen activated protein kinase (MAPK) RNA aptamer . MAPK paIn vitro selected nucleic acid aptamers serve as molecular mimics as well. Those aptamers can be selected employing simple chemistry of selection. Pathogen induced immune defense involves initiation of recruitment of inflammatory cells (leukocytes and platelets) to target sites  [ in vivo or inside the living cells. Similarly, Spinach aptamer fluoresces inside living cells in presence of provided fluorophore molecule, that is, 3, 5-difluoro-4-hydroxybenzylidene imidazolinone (DIHBI) [ in vivo transcript measurement and protein synthesis from the same mRNA simultaneously. Spinach aptamer sequences can serve as good tools to characterize transcription and translation inside cells. Such aptamer requires short RNA sequences with provided fluorophore [l cells) . In suchl cells) . P- or Ll cells) . Such apl cells) . Furtherl cells) . Anotherl cells) . Certainl cells) . General (DIHBI) . A repororophore .The importance of the applicability of aptamers has significantly increased with recent advancement in nanotechnology, microfluidics, microarray, and other technologies for application in the clinical field. Aptamers have been proven as multifunctional molecules having tremendous paramedical and medical applications. A number of such aptamers are in various stages of clinical trials. Recent advancement in aptamers selection and development can potentially deal with targeted drug delivery, targeted inhibition of protein function, and regulation of gene expression. Aptamers can also help in early cancer detection and alleviate detection complications. Aptamers compete with antibodies and offer promising tools in different applications. Targeted delivery, target specificity, and high affinity sum up into a higher application index in favor of aptamers, although, some limitations also exist with aptamers tools such as the stability of aptamers with modified nucleotides, intake, and its retention time inside the living system. Despite the enormous possibilities, nucleic acid aptamers suffer from certain limitations. These include a typical molecular mass being in excess of 10 kD, relative unstability of unmodified RNA aptamers, and possible off-target effects. However, further studies can confirm and validate outcomes of this selective approach by profiling the targeting specificity of the chosen ligand. Aptamers binding with transition state analogues of biochemical reaction may be expected to catalyze the respective biochemical reactions. An aptamer called “Spinach” mimics green fluorescent protein and promises use as tool for characterization of gene expression. This could be used in other applications such as RNA imaging. Variants of such aptamers offer great potential applications in synthetic biology.


This review highlights results leading to a novel concept, stating that substrate specificity, but also transport kinetics and transporter turnover, are determined by subtle intramolecular interactions between a major substrate binding site and independent outward- or cytoplasmically-facing gating domains, analogous to those present in channels. This concept is supported by recent structural evidence from several, phylogenetically and functionally distinct transporter families. The significance of this concept is discussed in relationship to the role and potential exploitation of transporters in drug action.Transporters are ubiquitous proteins mediating the translocation of solutes across cell membranes, a biological process involved in nutrition, signaling, neurotransmission, cell communication and drug uptake or efflux. Similarly to enzymes, most transporters have a single substrate binding-site and thus their activity follows Michaelis-Menten kinetics. Substrate binding elicits a series of structural changes, which produce a transporter conformer open toward the side opposite to the one from where the substrate was originally bound. This mechanism, involving alternate outward- and inward-facing transporter conformers, has gained significant support from structural, genetic, biochemical and biophysical approaches. Most transporters are specific for a given substrate or a group of substrates with similar chemical structure, but substrate specificity and/or affinity can vary dramatically, even among members of a transporter family that show high overall amino acid sequence and structural similarity. The current view is that transporter substrate affinity or specificity is determined by a small number of interactions a given solute can make within a specific binding site. However, genetic, biochemical and  They comprise a single major substrate-binding site interacting specifically with a single substrate molecule in each transport cycle. Consequently they are characterized, in most cases, with Michaelis-Menten kinetics, with measurable substrate affinity, inhibition or dissociation constants  and transport capacities or rates (V or mV). Substrate binding elicits conformational changes, which eventually produce a transporter conformer open to the opposite side from the one from where the substrate originally bound. In other words, transporters do not contain a continuous open pore accessible from both sides of a membrane. This “rocking-switch” mechanism  transmembrane, mostly α-helical, segments (TMS), well packed between hydrophilic cytoplasmic or extracellular termini. The TMS, which are linked by hydrophilic loops of different lengths depending on the transporter family, host the amino acid residues that interact with substrates or catalyze intra-helical interactions associated with the dynamic translocation of substrates. The hydrophilic loops linking TMS can also play a role in substrate recognition and transport, while in eukaryotes the hydrophilic termini have major roles in the post-translational regulation of transporter trafficking, endocytosis and vacuolar turnover (see later). Transporters can function as facilitators (substrate transport down a concentration gradient), active transporters , or exchangers (antiporters) of different substrates translocated in opposite directions. Substrates of transporters can be all kinds of natural metabolites, including lipids, xenobiotics, antibiotics or drugs. In fact, very few natural molecules  can efficiently cross the plasma membrane without the need of a transporter. Thus, it becomes apparent that antibiotic and drug action is absolutely dependent on relevant transporters, which can mediate efficient uptake or efflux of these chemicals. However, while we know much on the action of transporter pumping out drugs and leading to multidrug or pleiotropic drug resistance in cancer cells and resistant microbial pathogens  are polytopic transmembrane proteins that function as topological enzymes, that is, they catalyze the translocation of substrates from one side . Thus, channels function only as facilitators, are much faster (up to 108 ions/s) than transporters, and may lead to the generation of electric currents when translocating charged ions that alter the cellular electrostatic status.Channels (or pores) translocate mostly ions or some small solutes  and are considered to have a very different mechanism of action compared to transporters, also reflected in a different structure. Similarly to transporters, most channels are made by several, mostly α-helical, TMSs , but in most cases their functional unit is a homo-oligomer made by association of 2 or more subunits. Channels potentially form a continuous pore, commonly in the interphase of different subunits, but in most cases this pore is restricted by so called selectivity filters and controlled by gating domains or http://www.membranetransport.org/). Among the champions of transporters are most free-living bacteria, fungi, and plants, whereas channels are more abundant in metazoa. The importance of transport proteins is also reflected in more than 60 human genetic diseases caused by the malfunctioning of transporters and channels  (http://www.tcdb.org/disease_explore.php). As already mentioned above, transporters, unlike channels, are also key mediators of drug and antibiotic action. The present review has as its main goal to highlight novel findings, somehow neglected in the field of pharmacology, on the mechanism of transporter functioning, which I believe are essential to make a systematic and rational use of transporters as unique tools to develop novel pharmacological approaches.Transporters and channels are ubiquitously involved in cell nutrition, homeostasis, detoxification, stress response, but also in signaling and neurotransmission. Their biological importance is reflected by the observation that all known genomes include at least 5–15% genes coding for transport proteins  represent more than one-quarter of drug targets, currently approved inhibitors of transporters are ten times less . In addition, even in cases where the host cells and a microbial pathogen possess similar transport activities, these are often characterized by distinct kinetics and specificity profiles. Thus, in principle, transporters of microbial pathogens can well be exploited as specific gateways of antimicrobials. Compatible with this idea is that today, with the easiness and efficiency of genome sequencing and the development of powerful reverse genetics approaches, the systematic characterization of the complete complement of transporters in the most important pathogens is only a matter of dedicated effort. In such an effort, and especially to carry out sophisticated reverse genetic approaches and rigorous biochemical assays, the use of model bacteria, fungi and protozoa, will constitute a major step. This is necessary as one of the major problems studying the function and specificity of a given transporter is the existence of a multitude of transporters of overlapping specificities in most organisms. Thus, it is practically impossible to rigorously characterize with simple uptake assays the kinetics and specificity of a transporter if other similar transporters are also present. This problem can only be overcome by studying a single transporter in an appropriate genetic background lacking any other functionally related transporter. Such strains can be easily constructed and tested by combining multiple gene knock-out mutations in model microbes such as Escherichia coli, Aspergillus nidulans or Saccharomyces cerevisiae. Transporters from pathogenic microbes can also be isolated, expressed and functionally characterized in appropriate strains of model relatives, as for example has been the case for a number of A. fumigatus and Candida albicans purine transporters studied in A. nidulans or S. cerevisiae , named BBB22 and BBB23. This is supported by the observation that B. burgdorferi lacking bbb22-23 was non-infectious in mice  and a handful of other transporters, the majority of which were of bacterial origin . Among those, less than 20 concern ATP-dependent ABC primary active transporters and the rest are cation symporters, facilitators, antiporters or exchangers. The great majority of transporters consist of 10–14 α-helical transmembrane segments (TMS) connected via intracellular and extracellular loops, and cytoplasmic N- and C-terminal regions. These transporters seem to function as monomers, although some also form dimers or oligomers. Shorter transporters made of 3–6 TMS seem to form homo- or hetero-oligomeric functional complexes. Surprisingly, transporters of functionally and evolutionary distinct protein families with no primary amino acid sequence similarity and different substrate specificities seem to exhibit similar folds.In recent years since 2003), the availability of 2D and 3D structures obtained by EM crystallography and X-ray, as well as contributions from computational and theoretical approaches, have greatly enhanced our understanding of transporter structure-function relationships. The first crystal structure of a secondary active transporter at atomic resolution was obtained in 2002  type  of outer molecular gates to hinder substrate diffusion. The gate closure is facilitated by the substrate-induced rearrangement of single amino acid side chains or by the bending of single α-helices, parts of helices or helical hairpins. The transport cycle then proceeds by a substantial conformational change from the occluded (closed) outward-facing to the occluded inward-facing conformation. During this structural switch the transporter passes through the intermediate occluded form, where the substrates are inaccessibly buried within the protein. This is followed by the opening of the inner molecular gates thus enabling the release of the substrates from the transporter protein into the cytosol. The transport cycle is completed when the transporter switches from the empty internal form, back to the empty external conformation, enabling the protein to start a new cycle , including the drugs allopurinol or oxypurinol  the development of rapid uptake assays using germinated conidiospores of and transporting hypoxanthine, or any other purine that is not uric acid and xanthine, was a surprise. Hypoxanthine is smaller than xanthine and very similar in structure, so how does UapA select between these two purines? Or how can UapA distinguish between allopurinol and hypoxanthine in favor of the former , or gate and major binding site mutations , led to UapA versions with distinct transport activities and specificities, supporting the functional independence of the two presumed gates and the major substrate binding site  none of which is part of the presumed substrate binding pocket, which is made by residues in TMS3, TMS8, and TMS10. All these specificity mutations enlarged the capacity of UapA to transport purines, pyrimidines and bulky analogs of xanthine. Most critically, none of the specificity mutations affected significantly the A. nidulans transporter  seems to function as a substrate filter rather than a selectivity gate.What is the lesson from the classical genetic approaches used in UapA? No rational design would have predicted the role of the presumed gate residues which affect substrate specificity as these, unlike residues in the major binding site, are not well conserved. Why are these residues not well conserved? In fact, in the case of the NAT (Nucleobase Ascorbate Transporters) family where UapA belongs, there is no genetic evidence supporting the existence of residues critical for substrate specificity in analogous domains of the bacterial homologs. For example, in XanQ (xanthine permease), mutations in Asn430 and Ile432, which correspond to residues Thr526 and Phe528 in UapA, do not confer the ability for transport of purines other than xanthine, in sharp contrast with the case of the Overall, it seems that the evolution of substrate selectivity gates is a functional novelty that might have arisen independently in different transporter families or in different members of the same family. The reason for that might be connected with a specific selective advantage related to the need for more dynamic control of substrate binding and transport in eukaryotic cells . The variability in the evolution of gating mechanisms is most strongly supported by recent structural reports that show that the closure and opening of gates seems to be elicited by different mechanisms and distinct molecular elements in different transporters (see below). Finally, the evolution of gates seems to have also affected how transporters are regulated at the level of cellular expression, sorting, endocytosis and turnover, as will be discussed later.open and occluded conformations of transporters , biochemical  and homology modeling approaches, supports directly the existence of transiently es Table . The ind+ or H+. Binding of these cations, probably at the extracellular gate or deeper into the major substrate binding pocket, stabilizes the outward-facing open state, creating a high-affinity substrate-binding site. Following substrate and potentially additional ion binding, the extracellular gate closes and this closure promotes a transition to the intermediate or inward-facing occluded state. In contrast to the local changes associated opening and closing of gates, the isomerization between the outward- and inward-facing states involves larger-scale conformational changes spread throughout the transporter. This structural transition from outward- to inward-facing does not seem to involve the same domain movements in all transporters and further experimental and computational studies will be required to understand the movements associated with the transport cycle in each transporter studied. In the inward-facing state, the initial event triggering substrate release appears to be the release of Na+ or H+ ions. Following release of the symported cations, the open state of the intracellular gate is stabilized, leading to release of further ions and substrate. The empty inward-facing open transporter can then transit back outward-facing state and, thereby, complete the transport cycle. This model is predominantly based on the study of secondary active transporters and whether it is applicable to passive facilitators is still disputable  family, have suggested that there is an additional secondary binding site between the primary site and the bulk extracellular solution  and sorted to the endocytic/vacuolar pathway when cell grow in the presence of relevant substrates, but remain stable in the PM for many hours in the absence of external substrates. This phenomenon has been well studied in several fungal amino acid or purine transporters .The primary signal for substrate-elicited endocytic turnover of transporters is generally believed to be the over-accumulation of substrates at potential toxic levels. Transporter down-regulation is thought to provide a feedback control mechanism against cell poisoning form excess substrate. In this case, cellular levels of substrates would control the steady state levels of relevant transporter A. nidulans  or internally accumulated substrates due to genetic mutations , do not elicit endocytosis. Finally, UapA or Can1 mutant versions with modified specificity undergo endocytosis in the presence of all “novel” substrates. For example, a UapA version (Gln408Glu/Thr526Leu) transporting efficiently hypoxanthine, in addition its natural substrates uric acid or xanthine, is endocytosed by all three purines  specificity mutant Phe528Ala, which transports with increased rate all purines, does not undergo substrate-elicited endocytosis. Similarly, in Can1, the hyperactive mutant Thr456Ser also did not undergo substrate-elicited endocytosis. In addition, in Can1, the inactivation of transport activity due to a mutation in a residue critical for proton-coupling (Glu184Gln) did not abolish substrate-elicited endocytosis. Uncoupling of transport-activity from endocytosis was also shown in Gap1 using different substrates or substrate analogs  H+ symporters and exhibit a rather broad specificity for most purines, pyrimidines and several analogs  which acts as substrate and non-competitive inhibitor of xanthine oxidase/dehydrogenase (XO/XDH), a key enzyme for purine catabolism (Lewis et al., The atypical phenomenon underlying allopurinol transport by UapA was rationalized by the existence of substrate-specific alternative mechanisms and partially overlapping translocation trajectories in a single transporter. Interestingly, the existence of alternative and partially overlapping substrate trajectories has also been shown in the mammalian glucose transporter GLUT1 (Jiang et al., bona fidae transporter crystal structures are known, of which less than 50 concern secondary active transporters, facilitators or antiporters, and only 4 of them are of eukaryotic origin. Bacterial transporter structures are used quite successfully to model human homologs, but this approach risks erroneous or overstated conclusions. This is so because bacterial transporters are usually smaller in length due to much shorter N- or C- terminal regions, or internal hydrophilic loops, which in eukaryotes have been shown to be extremely critical for transporter function, specificity, cellular sorting and turnover. In addition, several of the known prokaryotic structures correspond to transporters of unknown physiological function, specificity and regulation, simply because these proteins proved more stable for crystallography.Transporters, despite their proven role in drug uptake and efflux, have been little exploited toward this direction, obviously due to the significant lack of knowledge concerning their function, specificity and regulation of expression. Currently nearly 70 The lack of knowledge on transporters, especially those of eukaryotes, is obviously due to general technical difficulties associated with their transmembrane nature, but also due to limited use of appropriate genetic or other alternative approaches to structural biology. In this review, I selected to present some paradigmatic genetic studies on fungal transporters, which led and continue to lead to novel knowledge on transporter function and regulation. In my opinion, one of the most prominent recent discoveries on transporter function is the existence of channel-like gates. Their existence was indeed predicted using rigorous genetic and biochemical approaches, before crystals became available and confirmed their presence. A second novel finding is related to substrate-elicited endocytosis and in particular to the idea that eukaryotic cells directly sense transporter function and accordingly regulate their turnover in order to achieve cell homeostasis to specific physiological, developmental or stress conditions. Impressively, what has been found on fungal transporter substrate-elicited endocytosis seems to hold true for mammalian transporters involved in neurotransmission. Finally, I gave an example, using a fungal transporter, but also discussing a similar case in a human transporter, on how transporters can, in some cases, show atypical kinetic behavior.in vivo microscopy, rational and random genetic screens, in addition to crystallography and other biophysical approaches. Knowledge of the different states in the transporter cycles at a molecular level will eventually assist in developing new highly targeted drugs.In summary, I think we need to learn much more on transporter function and regulation before we use them as tools in drug action. Recent crystal structures of mostly prokaryotic transporters and recent functional and computational studies of eukaryotic homologs have generated hypotheses on how transporters accomplish solute uptake. However, not all states in the transporter cycles have yet been identified in transporters, and only 4 crystal structures are available for eukaryotic transporters. More crystal structures might help in solving these controversies. In addition, many of the states proposed from existing crystal structures still need to be verified by other methods. To obtain new knowledge we definitely need to use appropriate eukaryotic model systems and interdisciplinary approaches, including functional genomics, reverse genetics, easy transport assays, The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Whole transcriptome sequencing (RNA-seq) represents a powerful approach for whole transcriptome gene expression analysis. However, RNA-seq carries a few limitations, e.g., the requirement of a significant amount of input RNA and complications led by non-specific mapping of short reads. The Ion AmpliSeq™ Transcriptome Human Gene Expression Kit (AmpliSeq) was recently introduced by Life Technologies as a whole-transcriptome, targeted gene quantification kit to overcome these limitations of RNA-seq. To assess the performance of this new methodology, we performed a comprehensive comparison of AmpliSeq with RNA-seq using two well-established next-generation sequencing platforms (Illumina HiSeq and Ion Torrent Proton). We analyzed standard reference RNA samples and RNA samples obtained from human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs).r = 0.92) and Ion Torrent Proton (Pearson’s r = 0.92). We used ROC, Matthew’s correlation coefficient and RMSD to determine the overall performance characteristics. All three statistical methods demonstrate AmpliSeq as a highly accurate method for differential gene expression analysis. Additionally, for genes with high abundance, AmpliSeq outperforms the two RNA-seq methods. When analyzing four closely related hiPSC-CM lines, we show that both AmpliSeq and RNA-seq capture similar global gene expression patterns consistent with known sources of variations.Using published data from two standard RNA reference samples, we observed a strong concordance of log2 fold change for all genes when comparing AmpliSeq to Illumina HiSeq  contains supplementary material, which is available to authorized users. Transcriptome analysis has enhanced our understanding of the molecular constituents of cells and tissues. As one of the most widely used tools for transcriptome profiling, quantification of differential gene expression has played a pivotal role in the identification of pathway and gene-network interactions –3. In thWhole-transcriptome sequencing (RNA-seq) technology has emerged as a revolutionary platform for genome-wide quantification of mRNA transcripts. This technique enables the sequencing of all RNA molecules in a high-throughput manner. Short sequence reads are generated by either single-end or paired-end sequencing. The number of sequencing reads that map to each transcript is used to infer the abundance of mRNA molecules. RNA-seq offers several advantages over microarrays or RT-qPCR for detecting differentially expressed genes (DEGs). These include a wide coverage of transcripts, high sensitivity, the ability to detect allele-specific differential expression and the identification of novel transcripts –14. Howehttp://www.illumina.com/products/truseq-targeted-rna-expression-kits.html). Similarly, Thermo Fisher Scientitic developed AmpliSeq-RNA panels for the study of a small number of pre-defined gene sets (between 150 to 900 genes). Based on a number of studies that used a targeted sequencing approach, there is strong evidence that gene expression quantification using targeted sequencing method is reliable and accurate [TM technology). This kit is designed for targeted amplification of over 20,000 distinct human RNA targets simultaneously in a single primer pool. A short amplicon (~150 bp) is amplified for each targeted gene [Taking advantage of high-throughput sequencing technology in combination with multiplexed enrichment or amplification methods, a few targeted RNA expression kits have recently been developed. For example, Illumina developed TruSeq targeted gene expression kit. This kit enables the testing of 10–100 assays for targeted genes . In comparison to the reference samples, these lines exhibit a spectrum of expression patterns, which can be expected in typical expression studies. Notably, hiPSC-based cell lines are gaining popularity as significant tools for disease modeling and comprehensive functional analysis, e.g., the analysis of gene expression influenced by human genome variation and validation of functional impact of a causal variant. In addition to correlating the fold-change in expression, we also used clustering and principal component analysis to compare the overall performance of AmpliSeq to RNA-seq in identifying distinct global expression patterns according to known source of variations. AmpliSeq offers comparable resolution to RNA-seq by clustering cell lines based on their origin, phenotypic and disease status. Our study strongly suggests that AmpliSeq is a highly reliable tool for gene expression quantification as demonstrated by the analysis of both reference libraries and real life samples such as iPSC disease models.For the three gene expression quantification methods we compared in this study, three library preparation methods are used: poly-A enrichment for Illumina RNA-seq, ribosomal RNA depletion for Proton RNA-seq, and genome-wide, targeted amplicon amplification for AmpliSeq. For the library preparation, both Illumina and Proton RNA-seq are similar: RNA is subjected to fragmentation prior to random amplification and down-stream high-throughput next-generation sequencing. Both methods have a certain percentage of reads mapped to intronic regions .The major difference between AmpliSeq and the two whole transcriptome RNA sequencing methods is that AmpliSeq is designed to profile over 20,000 distinct human RNA targets using a highly multiplexed amplification method. Each amplicon represents a unique targeted gene. The average size of each amplicon is ~150 bp. Because of the targeted nature and small amplicon size, the total number of raw reads needed for DEG analysis for each library prepared with AmpliSeq is much smaller than typical whole-transcriptome RNA sequencing. For library preparation, a barcoded cDNA library is first generated with SuperScript® VILO™ cDNA Synthesis kit from 10 ng of total RNA. Then cDNA is amplified using Ion AmpliSeq™ technology to accurately maintain expression levels of all targeted genes. Amplified cDNA Libraries were evaluated for quality and quantified using Agilent Bioanalyzer High sensitivity chip. Libraries were then diluted to 100pM and pooled equally, with eight individual samples per pool. Pooled libraries were amplified using emulsion PCR on Ion Torrent OneTouch2 instruments (OT2) and enriched following manufactures instructions. Templated libraries were then sequenced on Ion Torrent Proton™ sequencing system, using Ion PI kit and chip V2.Two established next-generation sequencing platforms were involved in this study: Illumina RNA-seq and Proton RNA-seq. Both the Proton RNA-seq and AmpliSeq rely on Ion Proton for the sequencing step, though each uses different RNA library preparation protocols (as mentioned above). Illumina RNA-seq and Proton RNA-seq use two next-generation sequencing technologies. Illumina RNA-seq uses the technology of sequencing by synthesis, which requires fluorescence and signal scanning. For this method, prepared libraries are denatured to single strands by linearization. The four nucleotides (GCAT) are coupled to a cleavable fluorescent dye and a removable blocking group, which complements the template one base at a time, yielding a signal to be captured by a charge-coupled device . Proton ; and the Ambion FirstChoice® Human Brain Reference Total RNA (HBRR) was generated from multiple brain regions from 23 donors [For initial performance analysis, we used two commercially available reference RNA samples. The Agilent Universal Human Reference RNA (UHRR) (reference rna cat. #: #740000) was a pooled sample from 10 cancer cell lines3 donors  .We performed AmpliSeq on both UHRR and HBRR samples. For quality control, ten External RNA Controls Consortium (ERCC)  referencWe assessed the performance of AmpliSeq in comparison to Proton RNA-Seq using four biologically related RNA samples. We selected RNA from two patient-specific hiPSC-CM lines. These two lines are selected from participants in the NHLBI HyperGen cohort , 25. ThiNPPB. For this study, we performed both AmpliSeq and RNA-seq on the Ion Proton platform for both stimulated and unstimulated hiPSC-CMs.These two cell lines differ genetically as well as phenotypically in various echocardiographic measurements such as left ventricular mass and ejection fraction. The hiPSC lines were generated from white blood cells using episomal reprogramming . hiPSC-CRaw reads from all whole transcriptome RNA-seq libraries were aligned using a two-step alignment approach. First, TopHat (v.2.0.3)  was usedPrimary analysis for AmpliSeq sequencing data of all samples was performed using the ampliSeqRNA plugin available for Ion Torrent™ sequencing platforms. This plugin uses the Torrent Mapping Alignment Program (TMAP). TMAP is optimized for Ion Torrent™ sequencing data for aligning the raw sequencing reads against a custom reference sequence set containing all transcripts targeted by the AmpliSeq kit. To maintain specificity and sensitivity, TMAP implements a two-stage mapping approach. First, four alignment algorithms, BWA-short , BWA-lonp-value and the log2 fold change (log2FC) by DESeq2.DEG analysis was performed using R/Bioconductor package DESeq2  with rawT value of each target was normalized by subtracting the average CT of endogenous control from the CT of each RT-qPCR target.Two published RT-qPCR datasets for DEGs between the UHRR and HBRR library were used as gold standard to assess the overall performance of each method. One dataset is comprised of TaqMan assays for 843 genes from the MAQC-I study (MAQC) (GSE5350) . The othThe overall concordance between each of the interested method and RT-qPCR was measured by calculating the Pearson’s r on individual gene’s log2FC values. To measure the similarity of each method in ordering the expression of genes, we calculated Spearman’s ranked r between each method using log2 transformed read counts. Three statistical analyses were used to compare transcript FC detected by each of the three methods to the gold-standard qPCR data: root-mean-square deviation (RMSD) for genes with at least two FC in expression, Receiver Operator Characteristic curves (ROC curve) with their associated Area Under the Curve (AUC) value, and Matthew’s correlation coefficient (MCC).Using the ABRF gene set as the gold-standard, we calculated the RMSD for genes with at least two FC in expression. This statistical analysis provided a quantitative measure of the difference in the identified FC of each method to RT-qPCR standard . We set p-value cutoffs as described by Li et al. (2014), transcripts were then grouped by p-values at 0.05, 0.01 and 0.001. Only transcripts with at least two-fold change were included in the analysis.We used AUC and MCC to measure the overall specificity and sensitivity of each method. Higher AUC values indicate better overall accuracy. It is perceivable that transcript abundance influences the ROC characteristics. Therefore, we assessed the impact of transcript abundance by categorizing transcript levels into quartiles based on Illumina RNA-seq data. ROC analysis was performed in comparison to the ABRF PrimePCR dataset. Similar to the study of Li et al. , we calcIn addition to the standard reference libraries, we also analyzed four RNA samples taken from two patient-specific, hiPSC-derived CM lines. Both cell lines were stimulated with ET-1 to induce a hypertrophic phenotype . The stiWhen preparing the libraries for AmpliSeq, ten ERCC transcripts with known concentrations were spiked in for each technical run before library preparation of UHRR and HBRR samples. These transcripts have a nearly 2000 fold difference regarding transcript abundance. The detected abundance of ERCC transcripts helped us evaluate the quality of the sequenced UHRR and HBRR libraries. Values of Pearson’s r were calculated for the following: (1) The overall concordance between detected read-counts and known concentrations of each ERCC transcript using log2 transfromed data; and (2) the overall concordance of read-counts of all genes between replicates using log10 transformed raw read-counts.The values of Pearson’ r between the log2 transformed known concentration and detected read-counts are > = 0.98 Fig. , indicatFor the published RNA-seq data, 40 million reads per sample were obtained for both Illumina RNA-seq and Proton RNA-seq. Due to its targeted nature and short length of each targeted amplicon, an average of nine million reads was obtained for each sample with each AmpliSeq run (GEO accession #: GSE74760). Vast majority of the reads (> = 93.2 %) were on target for each run  from Illumina RNA-Seq, we observe a nearly equal distribution in each quartile of transcript abundance for genes with at least two FC in expression . Using Gencode gtf as the reference, the mapping rate for each sample is between 93.2 % and 94.6 %    Fig. . To furtAnalysis using UHRR and HBRR provided valuable insights about the performance of AmpliSeq. However, UHRR and HBRR are pooled samples from multiple sources. Therefore, the differential gene expression profile may not represent the typical RNA samples in a research or clinical setting. Consequently, we extended our analysis and analyzed four biologically relevant samples. These samples were obtained from two different hiPSC-CM lines allowing us to further assess the performance of AmpliSeq in comparison to the Proton RNA-Seq. Since the two individuals, from which the hiPSC-CMs were derived, differ in genetic as well as phenotypic features, we expect to detect different, cell-line specific gene expression profiles.r = 0.96 for genes with FC > =2; and Pearson’r > =0.90 for genes with FC > =1.5). Thus, AmpliSeq has very consistent performance compared to Proton RNA-seq in identifying differentially expressed genes when closely related RNA samples were used.As expected, different set of DEGs were identified for samples 1156 and 1104. 67 % of all DEGs identified in sample 1156 were shared by sample 1104, and 24 % of DEGs identified in sample 1104 were shared by sample 1156. Though a largely different set of genes showed significant differential expression for each sample, we observed strong correlation of the log2FC between AmpliSeq and Proton RNA-seq for both CM lines  differences due to the genetic makeup of hiPSC donors; (2) differences based on hypertrophy phenotypes, non-hypertrophic (unstimulated) vs. hypertrophic (stimulated) cells, and (3) differences associated with the library preparation methods: targeted amplification for AmpliSeq and random amplification of ribo-depleted RNA library for Proton RNA-seq. As can be seen in the dendrogram in Fig. de novo assembly. However, for research focused on studying differential expression of known genes, sequence information at single-base level may not be required, thus motivating the development of alternative methods for high-throughput, yet highly accurate quantification of gene expression. Further, there are few well-recognized limitations for the application of whole-transcriptome RNA-seq. At the time of our analysis, whole-transcriptome sequencing methods require a significant amount of input material. To ensure sufficient statistical power, studies of functional consequences of differential gene expression generally entail large scale of cohorts and multiple replicates of each sample. Such requirement can easily lead to formidable sequencing cost and significant efforts for downstream analysis. After sequencing, conventional work-flow of RNA-seq involves mapping short reads to genomic reference, which unavoidably involves the mapping of non-specific in highly repetitive regions [Whole transcriptome sequencing has emerged as a new standard for high-throughput transcriptomic studies , 21, 40. regions . Such noAmpliSeq, as a whole-transcriptome, targeted gene quantification kit, seems to excel in all these limiting areas of RNA-seq. With 10 ng of total RNA, minimal prep time and the requirement of fewer sequencing reads compared to RNA-seq, AmpliSeq displayed very robust performance without losing specificity and sensitivity. First, results from ERCC spike-in controls in our AmpliSeq run of UHRR and HBRR indicated that AmpliSeq is highly consistent and yields high-quality transcript quantification. Despite the wide range in defined transcript abundance (in a range of 2000 fold) for the ERCC spike-in controls, detected read-counts and known concentrations of each ERCC transcript are highly correlated, reflecting great sequencing quality of AmpliSeq. By comparing the raw read-counts of all genes between replicates, we observed a very consistent performance between technical replicates. Second, our analysis using the DEGs between two reference libraries indicated that the performance of AmpliSeq closely resembles established RNA-seq methods in terms of overall specificity and sensitivity as indicated by the values of RMSD, AUC and MCC. Notably, AmpliSeq displayed excellent accuracy for genes in bottom two quartiles and outperforms the two RNA-seq methods for genes with high abundance. Third, even though AmpliSeq does not have capacity in isoform-level quantification or resolution at single-base level, sequencing-based, targeted quantification gives AmpliSeq methodology an advantage in handling off-target amplification. By only considering reads matching to defined target regions, AmpliSeq reduces the complexity and could avoid issues related to non-specific mapping [As a new method for sequence-based, genome-scale gene expression quantification, AmpliSeq stands as a very versatile and cost-effective approach for large-scale gene expression analysis with high accuracy. These merits enable AmpliSeq as a highly attractive method for very large-scale studies including replication analysis or other studies that require a very large number of samples to increase statistical power.One concern of using the standard RNA samples for quality assessment from various platforms for DEG analysis has been the distinct gene expression profile between the two reference RNA samples. Both UHRR and HBRR samples are pooled samples either from different cancer cell lines or from several regions of the brain from multiple donors. Additionally, more than 50 % the genes in human brain have brain-specific expression, which makes HBRR drastically different from UHRR , 42, 43.In order to gain insights into the performance of AmpliSeq to an established RNA-seq method, we used samples which represent expression patterns more commonly observed in a typical experiment. We performed AmpliSeq on four RNA samples obtained from an in vitro model of cardiac hypertrophy . The hiPComparing Proton RNA-seq and AmpliSeq results for the four RNA samples, we observe nearly the same clustering pattern between the two platforms. Out of the three levels of variations predefined by our experiments, we expect hiPSC-donors to be the largest variation, followed by hypertrophy phenotype and with library preparation methods being the least variation that contribute to global gene expression pattern. As expected, AmpliSeq, just like RNA-seq, is able to capture these variations shown in the clustering analysis. Both methods first differentiate the patient-specific cell lines, and then subsequently separate them based on their hypertrophy phenotype. Most importantly, AmpliSeq achieves the same resolution as RNA-seq with much less number of reads. An independent method, PCA, also revealed the same conclusion. Combined with our findings from UHRR and HBRR samples, our results strongly support that AmpliSeq is a very sensitive and competent approach for very large-scale mRNA-marker screening in hiPSC-based cellular models.Our understanding of the complexity and diversity of the human transcriptome is far from being comprehensive. Hence, there is a clear need to develop more complex RNA samples with well-validated RNA content that are from a wide range of physiological conditions. Create and analyze a very comprehensive RNA dataset requires major effort like MAQC III , 45 and AmpliSeq, as a whole-transcriptome, targeted gene quantification method, clearly stands as a highly robust approach for large-scale, genome-wide differential gene expression analysis. Further, AmpliSeq is a very sensitive and competent approach for very large-scale mRNA-marker screening in cellular models.The data sets supporting the results of this article are available in the NCBI Gene Expression Omnibus (GEO) repository, GSE74760. This includes AmpliSeq raw read counts on both RNA reference samples and hiPSC-CM samples, and Proton RNA-seq raw read counts on hiPSC-CM samples.