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Gene therapy with LentiGlobin for sickle cell disease (bb1111, lovotibeglogene autotemcel) consists of autologous transplantation of a patient's hematopoietic stem cells transduced with the BB305 lentiviral vector that encodes the β

Serum levels of miR-194 have been reported to predict prostate cancer recurrence after surgery, but its functional contributions to this disease have not been studied. Herein, it is demonstrated that miR-194 is a driver of prostate cancer metastasis. Prostate tissue levels of miR-194 were associated with disease aggressiveness and poor outcome. Ectopic delivery of miR-194 stimulated migration, invasion, and epithelial–mesenchymal transition in human prostate cancer cell lines, and stable overexpression of miR-194 enhanced metastasis of intravenous and intraprostatic tumor xenografts. Conversely, inhibition of miR-194 activity suppressed the invasive capacity of prostate cancer cell lines in vitro and in vivo. Mechanistic investigations identified the ubiquitin ligase suppressor of cytokine signaling 2 (SOCS2) as a direct, biologically relevant target of miR-194 in prostate cancer. Low levels of SOCS2 correlated strongly with disease recurrence and metastasis in clinical specimens. SOCS2 downregulation recapitulated miR-194–driven metastatic phenotypes, whereas overexpression of a nontargetable SOCS2 reduced miR-194–stimulated invasion. Targeting of SOCS2 by miR-194 resulted in derepression of the oncogenic kinases FLT3 and JAK2, leading to enhanced ERK and STAT3 signaling. Pharmacologic inhibition of ERK and JAK/STAT pathways reversed miR-194–driven phenotypes. The GATA2 transcription factor was identified as an upstream regulator of miR-194, consistent with a strong concordance between GATA2 and miR-194 levels in clinical specimens. Overall, these results offer new insights into the molecular mechanisms of metastatic progression in prostate cancer. Cancer Res; 77(4); 1021–34. ©2016 AACR.

This files contains 1 Supplementary Table and 11 Supplementary Figures. The Supplementary Table shows Univariate and multivariate Cox proportional hazard analysis of prostate tumor levels of miR-194 and Gleason score/grade in relation to recurrence-free interval after radical prostatectomy in the TCGA cohort. The Supplementary Figures collectively include data that supports the key finding of this study; namely, that miR-194 is an important driver of prostate cancer metastasis.

Epithelial-mesenchymal transition (EMT) plays important roles in tumour progression and is orchestrated by dynamic changes in gene expression. While it is well established that post-transcriptional regulation plays a significant role in EMT, the extent of alternative polyadenylation (APA) during EMT has not yet been explored. Using 3’ end anchored RNA sequencing, we mapped the alternative polyadenylation landscape (APA) following TGF-β-mediated induction of EMT in human mammary epithelial cells and found APA generally causes 3’UTR lengthening during this cell state transition. Analysis of the RNA-binding protein Quaking (QKI), a splicing factor induced during EMT, revealed enrichment of its binding adjacent to cleavage and polyadenylation sites within 3’UTRs. Following QKI knockdown, APA of many transcripts are altered to produce predominantly shorter 3’UTRs associated with reduced gene expression. Among these, QKI binds to its own cleavage site to produce a transcript with a longer 3’UTR. These findings reveal extensive changes in APA occur during EMT and identify a novel function for QKI in this process.

Quantum states with long-lived coherence are essential for quantum computation, simulation and metrology. The nuclear spin states of ultracold molecules prepared in the singlet rovibrational ground state are an excellent candidate for encoding and storing quantum information. However, it is important to understand all sources of decoherence for these qubits, and then eliminate them, in order to reach the longest possible coherence times. Here, we fully characterise the dominant mechanisms for decoherence of a storage qubit in an optically trapped ultracold gas of RbCs molecules using high-resolution Ramsey spectroscopy. Guided by a detailed understanding of the hyperfine structure of the molecule, we tune the magnetic field to where a pair of hyperfine states have the same magnetic moment. These states form a qubit, which is insensitive to variations in magnetic field. Our experiments reveal an unexpected differential tensor light shift between the states, caused by weak mixing of rotational states. We demonstrate how this light shift can be eliminated by setting the angle between the linearly polarised trap light and the applied magnetic field to a magic angle of $\arccos{(1/\sqrt{3})}\approx55^{\circ}$. This leads to a coherence time exceeding 6.9 s (90% confidence level). Our results unlock the potential of ultracold molecules as a platform for quantum computation., Comment: 19 pages, 12 figures

We explore coherent multi-photon processes in $^{87}$Rb$^{133}$Cs molecules using 3-level lambda and ladder configurations of rotational and hyperfine states, and discuss their relevance to future applications in quantum computation and quantum simulation. In the lambda configuration, we demonstrate the driving of population between two hyperfine levels of the rotational ground state via a two-photon Raman transition. Such pairs of states may be used in the future as a quantum memory, and we measure a Ramsey coherence time for a superposition of these states of 58(9) ms. In the ladder configuration, we show that we can generate and coherently populate microwave dressed states via the observation of an Autler-Townes doublet. We demonstrate that we can control the strength of this dressing by varying the intensity of the microwave coupling field. Finally, we perform spectroscopy of the rotational states of $^{87}$Rb$^{133}$Cs up to $N=6$, highlighting the potential of ultracold molecules for quantum simulation in synthetic dimensions. By fitting the measured transition frequencies we determine a new value of the centrifugal distortion coefficient $D_v=h\times207.3(2)~$Hz.

Refereed/Peer-reviewed The RNA-binding protein Quaking (QKI) has emerged as a potent regulator of cellular differentiation in developmental and pathological processes. The QKI gene is itself alternatively spliced to produce three major isoforms, QKI-5, QKI-6, and QKI-7, that possess very distinct functions. Here, we highlight roles of the different QKI isoforms in neuronal, vascular, muscle, and monocyte cell differentiation, and during epithelial-mesenchymal transition in cancer progression. QKI isoforms control cell differentiation through regulating alternative splicing, mRNA stability and translation, with activities in gene transcription now also becoming evident. These diverse functions of the QKI isoforms contribute to their broad influences on RNA metabolism and cellular differentiation. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications; RNA Processing > Splicing Regulation/Alternative Splicing; RNA in Disease and Development > RNA in Development

Simple Summary Prostate cancer is the second most common cancer and the fifth leading cause of cancer death in men worldwide. The current diagnosis methods for prostate cancer are invasive and costly. In particular, digital rectal examination (DRE) or prostate massage adds considerable discomfort to patients, reduces compliance to cancer screening schedules, and raises the cost of the diagnostic procedure. New technologies are urgently needed for the effective and yet noninvasive detection of these conditions. This manuscript describes streamlined biotechnology for the noninvasive detection of prostate cancer from malignant cells shed in urine. For the first time, a whole-cell immunocapture approach combined with photodynamic diagnostic principles is used in a device to detect whole cancer cells from unprocessed patient urine samples collected without prior DRE. Abstract Urine-based biomarkers have shown suitable diagnostic potential for prostate cancer (PCa) detection. Yet, until now, prostatic massage remains required prior to urine sampling. Here, we test a potential diagnostic approach using voided urine collected without prior digital rectal examination (DRE). In this study, we evaluated the diagnostic performance of a microfluidic-based platform that combines the principle of photodynamic diagnostic with immunocapture for the detection of PCa cells. The functionality and sensitivity of this platform were validated using both cultured cells and PCa patient urine samples. Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) demonstrated this platform had a detection limit of fewer than 10 cells per 60 µL and successfully validated the presence of a PCa biomarker in the urine of cancer patients without prior DRE. This biosensing platform exhibits a sensitivity of 72.4% and a specificity of 71.4%, in suitable agreement with qRT-PCR data. The results of this study constitute a stepping stone in the future development of noninvasive prostate cancer diagnostic technologies that do not require DRE.

Huntington’s disease (HD) is a dominantly inherited neurodegenerative disorder caused by a CAG trinucleotide expansion in the huntingtin gene (HTT), which codes for the pathologic mutant HTT (mHTT) protein. Since normal HTT is thought to be important for brain function, we engineered zinc finger protein transcription factors (ZFP-TFs) to target the pathogenic CAG repeat and selectively lower mHTT as a therapeutic strategy. Using patient-derived fibroblasts and neurons, we demonstrate that ZFP-TFs selectively repress >99% of HD-causing alleles over a wide dose range while preserving expression of >86% of normal alleles. Other CAG-containing genes are minimally affected, and virally delivered ZFP-TFs are active and well tolerated in HD neurons beyond 100 days in culture and for at least nine months in the mouse brain. Using three HD mouse models, we demonstrate improvements in a range of molecular, histopathological, electrophysiological and functional endpoints. Our findings support the continued development of an allele-selective ZFP-TF for the treatment of HD. Zinc finger protein transcription factors are developed for the selective silencing of the mutant huntingtin gene in human neurons in vitro and multiple animal models of Huntington’s disease in vivo while preserving expression of the wild-type allele.

Understanding ultracold collisions involving molecules is of fundamental importance for current experiments, where inelastic collisions typically limit the lifetime of molecular ensembles in optical traps. Here we present a broad study of optically trapped ultracold RbCs molecules in collisions with one another, in reactive collisions with Rb atoms, and in nonreactive collisions with Cs atoms. For experiments with RbCs alone, we show that by modulating the intensity of the optical trap, such that the molecules spend 75% of each modulation cycle in the dark, we partially suppress collisional loss of the molecules. This is evidence for optical excitation of molecule pairs mediated via sticky collisions. We find that the suppression is less effective for molecules not prepared in the spin-stretched hyperfine ground state. This may be due either to longer lifetimes for complexes or to laser-free decay pathways. For atom-molecule mixtures, RbCs+Rb and RbCs+Cs, we demonstrate that the rate of collisional loss of molecules scales linearly with the density of atoms. This indicates that, in both cases, the loss of molecules is rate-limited by two-body atom-molecule processes. For both mixtures, we measure loss rates that are below the thermally averaged universal limit., Comment: 21 pages, 9 figures

Neuroendocrine prostate cancer is an aggressive disease subtype associated with poor patient outcome. Fernandes et al. demonstrate that a microRNA, miR-194, promotes the emergence of neuroendocrine features in prostate cancer cells by targeting genes that regulate epithelial-neuroendocrine plasticity. Inhibiting miR-194 suppresses the growth of neuroendocrine prostate cancer models. © 2020 The Author(s)Potent therapeutic inhibition of the androgen receptor (AR) in prostate adenocarcinoma can lead to the emergence of neuroendocrine prostate cancer (NEPC), a phenomenon associated with enhanced cell plasticity. Here, we show that microRNA-194 (miR-194) is a regulator of epithelial-neuroendocrine transdifferentiation. In clinical prostate cancer samples, miR-194 expression and activity were elevated in NEPC and inversely correlated with AR signaling. miR-194 facilitated the emergence of neuroendocrine features in prostate cancer cells, a process mediated by its ability to directly target a suite of genes involved in cell plasticity. One such target was FOXA1, which encodes a transcription factor with a vital role in maintaining the prostate epithelial lineage. Importantly, a miR-194 inhibitor blocked epithelial-neuroendocrine transdifferentiation and inhibited the growth of cell lines and patient-derived organoids possessing neuroendocrine features. Overall, our study reveals a post-transcriptional mechanism regulating the plasticity of prostate cancer cells and provides a rationale for targeting miR-194 in NEPC. Refereed/Peer-reviewed

The attachment of unique molecular identifiers (UMIs) to RNA molecules prior to PCR amplification and sequencing, makes it possible to amplify libraries to a level that is sufficient to identify rare molecules, whilst simultaneously eliminating PCR bias through the identification of duplicated reads. Accurate de-duplication is dependent upon a sufficiently complex pool of UMIs to allow unique labelling. In applications dealing with complex libraries, such as total RNA-seq, only a limited variety of UMIs are required as the variation in molecules to be sequenced is enormous. However, when sequencing a less complex library, such as small RNAs for which there is a more limited range of possible sequences, we find increased variation in UMIs are required, even beyond that provided in a commercial kit specifically designed for the preparation of small RNA libraries for sequencing. We show that a pool of UMIs randomly varying across eight nucleotides is not of sufficient depth to uniquely tag the microRNAs to be sequenced. This results in over de-duplication of reads and the marked under-estimation of expression of the more abundant microRNAs. Whilst still arguing for the utility of UMIs, this work demonstrates the importance of their considered design to avoid errors in the estimation of gene expression in libraries derived from select regions of the transcriptome or small genomes.

We investigate the effects of static electric and magnetic fields on the differential ac Stark shifts for microwave transitions in ultracold bosonic $^{87}$Rb$^{133}$Cs molecules, for light of wavelength $\lambda = 1064~\mathrm{nm}$. Near this wavelength we observe unexpected two-photon transitions that may cause trap loss. We measure the ac Stark effect in external magnetic and electric fields, using microwave spectroscopy of the first rotational transition. We quantify the isotropic and anisotropic parts of the molecular polarizability at this wavelength. We demonstrate that a modest electric field can decouple the nuclear spins from the rotational angular momentum, greatly simplifying the ac Stark effect. We use this simplification to control the ac Stark shift using the polarization angle of the trapping laser.

We show that the lifetime of ultracold ground-state ^{87}Rb^{133}Cs molecules in an optical trap is limited by fast optical excitation of long-lived two-body collision complexes. We partially suppress this loss mechanism by applying square-wave modulation to the trap intensity, such that the molecules spend 75% of each modulation cycle in the dark. By varying the modulation frequency, we show that the lifetime of the collision complex is 0.53±0.06 ms in the dark. We find that the rate of optical excitation of the collision complex is 3_{-2}^{+4}×10^{3} W^{-1} cm^{2} s^{-1} for λ=1550 nm, leading to a lifetime of

We show that the lifetime of ultracold ground-state ^{87}Rb^{133}Cs molecules in an optical trap is limited by fast optical excitation of long-lived two-body collision complexes. We partially suppress this loss mechanism by applying square-wave modulation to the trap intensity, such that the molecules spend 75% of each modulation cycle in the dark. By varying the modulation frequency, we show that the lifetime of the collision complex is 0.53±0.06 ms in the dark. We find that the rate of optical excitation of the collision complex is 3_{-2}^{+4}×10^{3} W^{-1} cm^{2} s^{-1} for λ=1550 nm, leading to a lifetime of100 ns for typical trap intensities. These results explain the two-body loss observed in experiments on nonreactive bialkali molecules.

PurposeTo compare the effects of morphine and methadone on length of hospital stay (LOS) or treatment (LOT) and adverse effects in infants with neonatal abstinence syndrome (NAS).DesignSystematic review.SamplePubMed, Google Scholar, Cochrane library, CINAHL, IPA, American Academy of Pediatrics, and clinicaltrials.gov were systematically searched to identify randomized controlled trials (RCTs) and observational studies. comparing morphine and methadone for NAS.OutcomesLOS, LOT, adverse effects.ResultsOne RCT, two cohort studies, and two chart reviews met inclusion criteria. Each had a low risk of bias. LOS ranged from 12.08 to 36 days with morphine and 21 to 44.23 days with methadone. LOT ranged from 7.46 to 22.9 days (morphine) and 13.9 to 38.08 days (methadone). Adverse effects were not reported. Clinical evidence comparing morphine to methadone for NAS treatment is limited and conflicting. A recommendation for one over the other cannot be made based on these outcomes.

Lentiviral vector (LVV)-mediated transduction of human CD34+ hematopoietic stem and progenitor cells (HSPCs) holds tremendous promise for the treatment of monogenic hematological diseases. This approach requires the generation of a sufficient proportion of gene-modified cells. We identified staurosporine, a serine/threonine kinase inhibitor, as a small molecule that could be added to the transduction process to increase the proportion of genetically modified HSPCs by overcoming a LVV entry barrier. Staurosporine increased vector copy number (VCN) approximately 2-fold when added to mobilized peripheral blood (mPB) CD34+ cells prior to transduction. Limited staurosporine treatment did not affect viability of cells post-transduction, and there was no difference in in vitro colony formation compared to vehicle-treated cells. Xenotransplantation studies identified a statistically significant increase in VCN in engrafted human cells in mouse bone marrow at 4 months post-transplantation compared to vehicle-treated cells. Prostaglandin E2 (PGE2) is known to increase transduction efficiency of HSPCs through a different mechanism. Combining staurosporine and PGE2 resulted in further enhancement of transduction efficiency, particularly in short-term HSPCs. The combinatorial use of small molecules, such as staurosporine and PGE2, to enhance LVV transduction of human CD34+ cells is a promising method to improve transduction efficiency and subsequent potential therapeutic benefit of gene therapy drug products. Keywords: lentiviral, HSPC, transduction

As a powerful regulator of cellular homeostasis and metabolism, adenosine is involved in diverse neurological processes including pain, cognition, and memory. Altered adenosine homeostasis has also been associated with several diseases such as depression, schizophrenia, or epilepsy. Based on its protective properties, adenosine has been considered as a potential therapeutic agent for various brain disorders. Since systemic application of adenosine is hampered by serious side effects such as vasodilatation and cardiac suppression, recent studies aim at improving local delivery by depots, pumps, or cell-based applications. Here, we report on the characterization of adenosine-releasing human embryonic stem cell-derived neuroepithelial stem cells (long-term self-renewing neuroepithelial stem [lt-NES] cells) generated by zinc finger nuclease (ZFN)-mediated knockout of the adenosine kinase (ADK) gene. ADK-deficient lt-NES cells and their differentiated neuronal and astroglial progeny exhibit substantially elevated release of adenosine compared to control cells. Importantly, extensive adenosine release could be triggered by excitation of differentiated neuronal cultures, suggesting a potential activity-dependent regulation of adenosine supply. Thus, ZFN-modified neural stem cells might serve as a useful vehicle for the activity-dependent local therapeutic delivery of adenosine into the central nervous system.

Refereed/Peer-reviewed

Gene therapy currently in development for hemoglobinopathies utilizes ex vivo lentiviral transduction of CD34+ hematopoietic stem and progenitor cells (HSPCs). A small-molecule screen identified prostaglandin E2 (PGE2) as a positive mediator of lentiviral transduction of CD34+ cells. Supplementation with PGE2 increased lentiviral vector (LVV) transduction of CD34+ cells approximately 2-fold compared to control transduction methods with no effect on cell viability. Transduction efficiency was consistently increased in primary CD34+ cells from multiple normal human donors and from patients with β-thalassemia or sickle cell disease. Notably, PGE2 increased transduction of repopulating human HSPCs in an immune-deficient (nonobese diabetic/severe combined immunodeficiency/interleukin-2 gamma receptor null [NSG]) xenotransplantation mouse model without evidence of in vivo toxicity, lineage bias, or a de novo bias of lentiviral integration sites. These data suggest that PGE2 improves lentiviral transduction and increases vector copy number, therefore resulting in increased transgene expression. As a result, PGE2 may be useful in clinical gene therapy applications using lentivirally modified HSPCs., Heffner et al. performed a small-molecule screen and identified prostaglandin E2 (PGE2) as a positive mediator of lentiviral transduction of CD34+ cells. Their data suggest that PGE2-mediated improvements in transduction efficiency may aid in clinical gene therapy applications using lentivirally modified HSPCs.

Recent evidence has implicated the transmembrane co-receptor neuropilin-1 (NRP1) in cancer progression. Primarily known as a regulator of neuronal guidance and angiogenesis, NRP1 is also expressed in multiple human malignancies, where it promotes tumor angiogenesis. However, non-angiogenic roles of NRP1 in tumor progression remain poorly characterized. In this study, we define NRP1 as an androgen-repressed gene whose expression is elevated during the adaptation of prostate tumors to androgen-targeted therapies (ATTs), and subsequent progression to metastatic castration-resistant prostate cancer (mCRPC). Using short hairpin RNA (shRNA)-mediated suppression of NRP1, we demonstrate that NRP1 regulates the mesenchymal phenotype of mCRPC cell models and the invasive and metastatic dissemination of tumor cells in vivo. In patients, immunohistochemical staining of tissue microarrays and mRNA expression analyses revealed a positive association between NRP1 expression and increasing Gleason grade, pathological T score, positive lymph node status and primary therapy failure. Furthermore, multivariate analysis of several large clinical prostate cancer (PCa) cohorts identified NRP1 expression at radical prostatectomy as an independent prognostic biomarker of biochemical recurrence after radiation therapy, metastasis and cancer-specific mortality. This study identifies NRP1 for the first time as a novel androgen-suppressed gene upregulated during the adaptive response of prostate tumors to ATTs and a prognostic biomarker of clinical metastasis and lethal PCa. Refereed/Peer-reviewed

MicroRNA-194 (miR-194) promotes prostate cancer metastasis, but the precise molecular mechanisms by which it achieves this are unknown. Here, by integrating Argonaute high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (Ago-HITS-CLIP) with RNA sequencing and exon-intron split analysis, we defined a 163-gene miR-194 “targetome” in prostate cancer. These target genes were predominantly down-regulated through canonical 3’UTR recognition sites and were enriched within pathways involved in cytoskeletal organisation and cell movement. In clinical prostate cancer samples, miR-194 activity was inversely correlated with the androgen receptor (AR) signalling axis. At a mechanistic level, this inverse correlation was explained by down-regulation of miR-194 expression by AR. Accordingly, miR-194 expression and activity was significantly elevated in neuroendocrine prostate cancer (NEPC), an aggressive AR-independent disease subtype. MiR-194 enhanced the transdifferentiation of prostate adenocarcinoma cells to a neuroendocrine-like state, at least in part by targeting FOXA1, a transcription factor with a key role in maintaining the prostate epithelial lineage. Importantly, a miR-194 inhibitor effectively inhibited the growth of cell lines and patient-derived organoids with neuroendocrine features. Overall, our study reveals a novel post-transcriptional mechanism regulating the plasticity of prostate cancer cells and provides a rationale for targeting miR-194 in this NEPC.

Supplementation with vitamin D has become increasingly popular over the past decade, and numerous organizations have developed recommendations for the appropriate intake of vitamin D. Vitamin D supplements come in a variety of formulations and strengths and vary in their directions for use. This study was designed to compare vitamin D label dosing information with the recommendations in North American guidelines. A systematic search was conducted to identify 62 single-ingredient vitamin D products of which 1000 IU was the most common strength. Assessment of North American guidelines found recommended vitamin D dosing to range from 400 to 1000 IU daily, depending on age. Twenty-four (39%) of the products recommended a maximum dose within the range of 400 to 1000 IU daily. Thirty-eight (61%) and 19 (31%) products recommended maximum doses more than 1000 IU daily and 2000 IU daily, respectively. Labeled dosing recommendations of commercially available vitamin D supplements are largely inconsistent with North American recommendations.

Chimeric antigen receptor (CAR) T cell therapies have achieved promising outcomes in several cancers, however more challenging oncology indications may necessitate advanced antigen receptor designs and functions. Here we describe a bipartite receptor system comprised of separate antigen targeting and signal transduction polypeptides, each containing an extracellular dimerization domain. We demonstrate that T cell activation remains antigen dependent but can only be achieved in the presence of a dimerizing drug, rapamycin. Studies performed in vitro and in xenograft mouse models illustrate equivalent to superior anti-tumor potency compared to currently used CAR designs, and at rapamycin concentrations well below immunosuppressive levels. We further show that the extracellular positioning of the dimerization domains enables the administration of recombinant re-targeting modules, potentially extending antigen targeting. Overall, this novel regulatable CAR design has exquisite drug sensitivity, provides robust anti-tumor responses, and is uniquely flexible for multiplex antigen targeting or retargeting, which may further assist the development of safe, potent and durable T cell therapeutics.

Background Altering the biochemical makeup of chromatin by the incorporation of histone variants during development represents a key mechanism in regulating gene expression. The histone variant H2A.B, H2A.B.3 in mice, appeared late in evolution and is most highly expressed in the testis. In the mouse, it is encoded by three different genes. H2A.B expression is spatially and temporally regulated during spermatogenesis being most highly expressed in the haploid round spermatid stage. Active genes gain H2A.B where it directly interacts with polymerase II and RNA processing factors within splicing speckles. However, the importance of H2A.B for gene expression and fertility are unknown. Results Here, we report the first mouse knockout of this histone variant and its effects on fertility, nuclear organization, and gene expression. In view of the controversy related to the generation of off-target mutations by gene editing approaches, we test the specificity of TALENs by disrupting the H2A.B multi-copy gene family using only one pair of TALENs. We show that TALENs do display a high level of specificity since no off-target mutations are detected by bioinformatics analyses of exome sequences obtained from three consecutive generations of knockout mice and by Sanger DNA sequencing. Male H2A.B.3 knockout mice are subfertile and display an increase in the proportion of abnormal sperm and clogged seminiferous tubules. Significantly, a loss of proper RNA Pol II targeting to distinct transcription–splicing territories and changes to pre-mRNA splicing are observed. Conclusion We have produced the first H2A.B knockout mouse using the TALEN approach. Electronic supplementary material The online version of this article (10.1186/s13059-019-1633-3) contains supplementary material, which is available to authorized users.

Epithelial–mesenchymal transition (EMT) has been a subject of intense scrutiny as it facilitates metastasis and alters drug sensitivity. Although EMT-regulatory roles for numerous miRNAs and transcription factors are known, their functions can be difficult to disentangle, in part due to the difficulty in identifying direct miRNA targets from complex datasets and in deciding how to incorporate ‘indirect’ miRNA effects that may, or may not, represent biologically relevant information. To better understand how miRNAs exert effects throughout the transcriptome during EMT, we employed Exon–Intron Split Analysis (EISA), a bioinformatic technique that separates transcriptional and post-transcriptional effects through the separate analysis of RNA-Seq reads mapping to exons and introns. We find that in response to the manipulation of miRNAs, a major effect on gene expression is transcriptional. We also find extensive co-ordination of transcriptional and post-transcriptional regulatory mechanisms during both EMT and mesenchymal to epithelial transition (MET) in response to TGF-β or miR-200c respectively. The prominent transcriptional influence of miRNAs was also observed in other datasets where miRNA levels were perturbed. This work cautions against a narrow approach that is limited to the analysis of direct targets, and demonstrates the utility of EISA to examine complex regulatory networks involving both transcriptional and post-transcriptional mechanisms.

We present the production of ultracold 87 RbCs molecules in the electronic, rovibrational and hyperfine ground state, using stimulated Raman adiabatic passage to transfer the molecules from a weakly bound Feshbach state. We measure one-way transfer efficiencies of 92(1)% and fully characterise the strengths and linewidths of the transitions used. We model the transfer, including a Monte Carlo simulation of the laser noise, and find this matches well with both the transfer efficiency and our previous measurements of the laser linewidth and frequency drifts.

Many patients who take antiretroviral drugs also take alternative therapies including dietary supplements. Some drug–supplement combinations may result in clinically meaningful interactions. We aimed to investigate the evidence for dietary supplement interactions with antiretrovirals. A systematic review was conducted using multiple resources including PubMed, Natural Medicine Comprehensive Database, The Review of Natural Products, and Google Scholar. All human studies or case reports evaluating an interaction between a dietary supplement and an antiretroviral were selected for inclusion. Twenty-eight pharmacokinetic studies and case-series/case reports were selected for inclusion. Calcium carbonate, ferrous fumarate, some forms of ginkgo, some forms of garlic, some forms of milk thistle, St. John's wort, vitamin C, zinc sulfate, and multivitamins were all found to significantly decrease the levels of selected antiretrovirals and should be avoided in patients taking these antiretrovirals. Cat's claw and evening primrose oil were found to significantly increase the levels of antiretrovirals and patients should be monitored for adverse effects while taking these dietary supplements with antiretrovirals. This systematic review shows the importance of screening all human immunodeficiency virus patients for dietary supplement use to prevent treatment failure or adverse effects related to an interaction.

Purpose: To characterize requests received through an academic drug information consultation service related to complementary and alternative medicines. Methods: A retrospective review and descriptive analysis of drug information consultations was conducted. Results: A total of 195 consultations related to complementary and alternative medicine were evaluated. All consultation requests involved questions about dietary supplements. The most common request types were related to safety and tolerability (39%), effectiveness (38%), and therapeutic use (34%). Sixty-eight percent of the requests were from pharmacists. The most frequent consultation requests from pharmacists were questions related to drug interactions (37%), therapeutic use (37%), or stability/compatibility/storage (34%). Nearly 60% of complementary and alternative medicine-related consultation requests were able to be completely addressed using available resources. Among review sources, Natural Medicines Comprehensive Database, Clinical Pharmacology, Micromedex, and Pharmacist’s Letter were the most common resources used to address consultations. Conclusion: Utilization of a drug information service may be a viable option for health care professionals to help answer a complementary and alternative medicine-related question. Additionally, pharmacists and other health care professionals may consider acquiring resources identified to consistently answering these questions.

Genome editing in hematopoietic stem and progenitor cells (HSPCs) is a promising novel technology for the treatment of many human diseases. Here, we evaluated whether the disruption of the C-C chemokine receptor 5 (CCR5) locus in pigtailed macaque HSPCs by zinc finger nucleases (ZFNs) was feasible. We show that macaque-specific CCR5 ZFNs efficiently induce CCR5 disruption at levels of up to 64% ex vivo, 40% in vivo early posttransplant, and 3% to 5% in long-term repopulating cells over 6 months following HSPC transplant. These genome-edited HSPCs support multilineage engraftment and generate progeny capable of trafficking to secondary tissues including the gut. Using deep sequencing technology, we show that these ZFNs are highly specific for the CCR5 locus in primary cells. Further, we have adapted our clonal tracking methodology to follow individual CCR5 mutant cells over time in vivo, reinforcing that CCR5 gene-edited HSPCs are capable of long-term engraftment. Together, these data demonstrate that genome-edited HSPCs engraft, and contribute to multilineage repopulation after autologous transplantation in a clinically relevant large animal model, an important step toward the development of stem cell-based genome-editing therapies for HIV and potentially other diseases as well.

Objective:To evaluate whether clinical data support the safety and efficacy of probiotics for the management of infantile colic.Background:Probiotics have been suggested as a potential strategy for infantile colic, and the specific species that have been studied in healthy infants are considered to be safe.Methodology:A systematic review was conducted to identify randomized controlled trials (RCTs) evaluating the use of probiotic supplementation in infants with colic. RCTs with a primary end point assessing crying or fussing time were selected. A meta-analysis comparing “responders” to “nonresponders” in infants receiving probiotic versus control was conducted. The quality of trials selected was assessed.Results:Five RCTs assessing 2 different strains of the probiotic Lactobacillus reuteri in mostly breastfed infants were identified. Analysis of response rates showed that infants receiving probiotics had a 2.3-fold greater chance of having a 50% or greater decrease in crying/fussing time compared to controls ( P = .01). Probiotic supplementation was not associated with any adverse events.Conclusion:Supplementation with the probiotic L. reuteri in breastfed infants appears to be safe and effective for the management of infantile colic. Further research is needed to determine the role of probiotics in infants who are formula-fed.

The Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency caused by mutations in the WAS gene and characterized by severe thrombocytopenia. Although the role of WASp in terminally differentiated lymphocytes and myeloid cells is well characterized, its role in early hematopoietic differentiation and in platelets (Plts) biology is poorly understood. In the present manuscript, we have used zinc finger nucleases targeted to the WAS locus for the development of two isogenic WAS knockout (WASKO) human embryonic stem cell lines (hESCs). Upon hematopoietic differentiation, hESCs-WASKO generated increased ratios of CD34(+)CD45(+) progenitors with altered responses to stem cell factor compared to hESCs-WT. When differentiated toward the megakaryocytic linage, hESCs-WASKO produced increased numbers of CD34(+)CD41(+) progenitors, megakaryocytes (MKs), and Plts. hESCs-WASKO-derived MKs and Plts showed altered phenotype as well as defective responses to agonist, mimicking WAS patients MKs and Plts defects. Interestingly, the defects were more evident in WASp-deficient MKs than in WASp-deficient Plts. Importantly, ectopic WAS expression using lentiviral vectors restored normal Plts development and MKs responses. These data validate the AND-1_WASKO cell lines as a human cellular model for basic research and for preclinical studies for WAS.

Genome editing is the introduction of directed modifications in the genome, a process boosted to therapeutic levels by designer nucleases. Building on the experience of ex vivo gene therapy for severe combined immunodeficiencies, it is likely that genome editing of haematopoietic stem/progenitor cells (HSPC) for correction of inherited blood diseases will be an early clinical application. We show molecular evidence of gene correction in a mouse model of primary immunodeficiency. In vitro experiments in DNA-dependent protein kinase catalytic subunit severe combined immunodeficiency (Prkdc scid) fibroblasts using designed zinc finger nucleases (ZFN) and a repair template demonstrated molecular and functional correction of the defect. Following transplantation of ex vivo gene-edited Prkdc scid HSPC, some of the recipient animals carried the expected genomic signature of ZFN-driven gene correction. In some primary and secondary transplant recipients we detected double-positive CD4/CD8 T-cells in thymus and single-positive T-cells in blood, but no other evidence of immune reconstitution. However, the leakiness of this model is a confounding factor for the interpretation of the possible T-cell reconstitution. Our results provide support for the feasibility of rescuing inherited blood disease by ex vivo genome editing followed by transplantation, and highlight some of the challenges.

Members of the miR-200 family are critical gatekeepers of the epithelial state, restraining expression of pro-mesenchymal genes that drive epithelial-mesenchymal transition (EMT) and contribute to metastatic cancer progression. Here, we show that miR-200cand another epithelial-enriched miRNA, miR-375, exert wide spread control of alternative splicing in cancer cells by suppressing the RNA-binding protein Quaking (QKI). During EMT, QKI-5 directly binds to and regulates hundreds of alternative splicing targets and exerts pleiotropic effects, such as increasing cell migration and invasion and restraining tumour growth, without appreciably affecting mRNA levels. QKI-5 is both necessary and sufficient to direct EMT-associated alternative splicing changes, and this splicing signature is broadly conserved across many epithelial-derived cancer types. Importantly, several actin cytoskeleton-associated genes are directly targeted by both QKI and miR-200c, revealing coordinated control of alternative splicing and mRNA abundance during EMT. These findings demonstrate the existence of a miR200/miR-375/QKI axis that impacts cancer-associated epithelial cell plasticity through widespread control of alternative splicing. Refereed/Peer-reviewed

MicroRNAs (miRNAs) are important post-transcriptional regulators of gene expression, functioning in part by facilitating the degradation of target mRNAs. They have an established role in controlling epithelial-mesenchymal transition (EMT), a reversible phenotypic program underlying normal and pathological processes. Many studies demonstrate the role of individual miRNAs using over expression at levels greatly exceeding physiological abundance.This can influence transcripts with relatively poor targeting and may in part explain why over 130 different miRNAs are directly implicated as EMT regulators.Analyzing a human mammary cell model of EMT we found evidence that a set of miRNAs, including the miR-200 and miR-182/183 family members, cooperatein post-transcriptional regulation, both reinforcing and buffering transcriptional output.Investigating this, we demonstrate that combinatorial treatment altered cellular phenotype with miRNA concentrations much closer to endogenous levels and with less off-target effects. This suggests that co-operative targeting by miRNAs is important for their physiological function and future work classifying miRNAs should consider such combinatorial effects. Refereed/Peer-reviewed