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We and others have previously reported significant changes in chloride transport after cationic-lipid-mediated transfer of the cystic fibrosis transmembrane conductance regulator ( CFTR) gene to the nasal epithelium of patients with cystic fibrosis. We studied the safety and efficacy of this gene transfer to the lungs and nose of patients with cystic fibrosis in a double-blind placebo-controlled trial. Eight patients with cystic fibrosis were randomly assigned DNA-lipid complex (active) by nebulisation into the lungs followed 1 week later by administration to the nose. Eight control patients followed the same protocol but with the lipid alone (placebo). Safety was assessed clinically, by radiography, by pulmonary function, by induced sputum, and by histological analysis. Efficacy was assessed by analysis of vector-specific CFTR DNA and mRNA, in-vivo potential difference, epifluorescence assay of chloride efflux, and bacterial adherence. Seven of the eight patients receiving the active complex reported mild influenza-like symptoms that resolved within 36 h. Six of eight patients in both the active and placebo groups reported mild airway symptoms over a period of 12 h following pulmonary administration. No specific treatment was required for either event. Pulmonary administration resulted in a significant (p<0·05) degree of correction of the chloride abnormality in the patients receiving active treatment but not in those on placebo when assessed by in-vivo potential difference and chloride efflux. Bacterial adherence was also reduced. We detected no alterations in the sodium transport abnormality. A similar pattern occurred following nasal administration. Cationic-lipid-mediated CFTR gene transfer can significantly influence the underlying chloride defect in the lungs of patients with cystic fibrosis.

The diversity and complexity of the human brain are widely assumed to be encoded within a constant genome. Somatic gene recombination, which changes germline DNA sequences to increase molecular diversity, could theoretically alter this code but has not been documented in the brain, to our knowledge. Here we describe recombination of the Alzheimer’s disease-related gene APP , which encodes amyloid precursor protein, in human neurons, occurring mosaically as thousands of variant ‘genomic cDNAs’ (gencDNAs). gencDNAs lacked introns and ranged from full-length cDNA copies of expressed, brain-specific RNA splice variants to myriad smaller forms that contained intra-exonic junctions, insertions, deletions, and/or single nucleotide variations. DNA in situ hybridization identified gencDNAs within single neurons that were distinct from wild-type loci and absent from non-neuronal cells. Mechanistic studies supported neuronal ‘retro-insertion’ of RNA to produce gencDNAs; this process involved transcription, DNA breaks, reverse transcriptase activity, and age. Neurons from individuals with sporadic Alzheimer’s disease showed increased gencDNA diversity, including eleven mutations known to be associated with familial Alzheimer’s disease that were absent from healthy neurons. Neuronal gene recombination may allow ‘recording’ of neural activity for selective ‘playback’ of preferred gene variants whose expression bypasses splicing; this has implications for cellular diversity, learning and memory, plasticity, and diseases of the human brain. The gene for the amyloid precursor protein ( APP ) shows somatic gene recombination in neurons, and the abundance and diversity of APP variants is increased in neurons from individuals with Alzheimer’s disease.

Inactivated poliovirus vaccine (IPV) is believed to induce significantly lower mucosal immunity than oral poliovirus vaccine (OPV). Most of the data supporting this were generated before enhanced IPV (eIPV) was introduced. Excretion of poliovirus by OPV recipients can be used to assess intestinal immunity. We studied polymerase chain reaction amplification of viral complementary DNA from the stool of children vaccinated with either OPV alone or eIPV. Of first-time OPV recipients, 92% excreted virus after 1 week, and 81% excreted virus after 3 weeks. Prior vaccination with OPV reduced the number to 22% and shortened the duration of virus excretion (to 5% after 3 weeks). Two doses of IPV reduced the number of poliovirus-positive 1-week samples (to 76%), the duration of shedding (to 37% at 3 weeks), and the quantity of excreted virus. This suggests that IPV-vaccinated communities are partially protected from the spread of poliovirus. Further enhancement of IPV potency may lead to even higher levels of mucosal immunity

Single-cell whole-transcriptome analysis is a powerful tool for quantifying gene expression heterogeneity in populations of cells. Many techniques have, thus, been recently developed to perform transcriptome sequencing (RNA-Seq) on individual cells. To probe subtle biological variation between samples with limiting amounts of RNA, more precise and sensitive methods are still required. We adapted a previously developed strategy for single-cell RNA-Seq that has shown promise for superior sensitivity and implemented the chemistry in a microfluidic platform for single-cell whole-transcriptome analysis. In this approach, single cells are captured and lysed in a microfluidic device, where mRNAs with poly(A) tails are reverse-transcribed into cDNA. Double-stranded cDNA is then collected and sequenced using a next generation sequencing platform. We prepared 94 libraries consisting of single mouse embryonic cells and technical replicates of extracted RNA and thoroughly characterized the performance of this technology. Microfluidic implementation increased mRNA detection sensitivity as well as improved measurement precision compared with tube-based protocols. With 0.2 M reads per cell, we were able to reconstruct a majority of the bulk transcriptome with 10 single cells. We also quantified variation between and within different types of mouse embryonic cells and found that enhanced measurement precision, detection sensitivity, and experimental throughput aided the distinction between biological variability and technical noise. With this work, we validated the advantages of an early approach to single-cell RNA-Seq and showed that the benefits of combining microfluidic technology with high-throughput sequencing will be valuable for large-scale efforts in single-cell transcriptome analysis.

In parallel, whole chromosome tiling array interrogation of the RNA content of a variety of human tissues and cell lines revealed that, collectively, at least 93% of genomic bases are transcribed in one cell type or another [1],[10]-[13]. Since it is well established that highly expressed mRNAs dominate the non-ribosomal portion of the polyA+ transcriptome [7],[8],[10],[14]-[19], normalization approaches were used to reduce the quantity of highly expressed transcripts in these cDNA analyses [7],[8], and are implicit in tiling array approaches. [...]any estimate of the pervasiveness of transcription requires inclusion of all data sources, and less than exhaustive analyses can only provide lower bounds for transcriptional complexity.

Analyses of families affected by cold urticaria, immunodeficiency, and autoimmunity implicate mutations that activate phospholipase Cγ2 (PLCγ2), an enzyme pivotal to the translation of binding events at the cell surface to the intracellular milieu, as a cause of the disease. The genetic dissection of unique inflammatory phenotypes can identify and elucidate immunologic pathways and mechanisms. Such investigations have ultimately led to findings whose significance extends beyond the monogenic diseases harboring the mutations. Examples include the recognition that FOXP3 is essential for the differentiation of regulatory T cells in Scurfy mice and in patients with profound immune dysregulation, 1 – 4 the demonstration of a critical role for AIRE in thymic negative selection of T cells in patients with a specific autoimmune polyendocrinopathy, 5 and the identification of NLRP3 as a critical regulator of interleukin-1 in families with cold-induced inflammation. 6 Cold-induced urticaria is a . . .

Blood-based diagnostics tests, using individual or panels of biomarkers, may revolutionize disease diagnostics and enable minimally invasive therapy monitoring. However, selection of the most relevant biomarkers from liquid biosources remains an immense challenge. We recently presented the thromboSeq pipeline, which enables RNA sequencing and cancer classification via self-learning and swarm intelligence–enhanced bioinformatics algorithms using blood platelet RNA. Here, we provide the wet-lab protocol for the generation of platelet RNA-sequencing libraries and the dry-lab protocol for the development of swarm intelligence–enhanced machine-learning-based classification algorithms. The wet-lab protocol includes platelet RNA isolation, mRNA amplification, and preparation for next-generation sequencing. The dry-lab protocol describes the automated FASTQ file pre-processing to quantified gene counts, quality controls, data normalization and correction, and swarm intelligence–enhanced support vector machine (SVM) algorithm development. This protocol enables platelet RNA profiling from 500 pg of platelet RNA and allows automated and optimized biomarker panel selection. The wet-lab protocol can be performed in 5 d before sequencing, and the algorithm development can be completed in 2 d, depending on computational resources. The protocol requires basic molecular biology skills and a basic understanding of Linux and R. In all, with this protocol, we aim to enable the scientific community to test platelet RNA for diagnostic algorithm development. The particle-swarm optimization–enhanced thromboSeq pipeline enables RNA sequencing and cancer classification from blood platelet RNA, using self-learning and swarm intelligence–enhanced bioinformatics algorithms.

The molecular basis of the resistance of anaplastic lymphoma kinase (ALK)–driven lung cancer to the ALK inhibitor, crizotinib, was identified in a patient whose tumor initially responded to crizotinib treatment and then, after 5 months, progressed despite continued treatment. EML4-ALK is a fusion-type protein tyrosine kinase that is present in 4 to 5% of cases of non–small-cell lung cancer and is generated as a result of a small inversion within the short arm of human chromosome 2. 1 – 3 EML4-ALK undergoes constitutive dimerization through interaction between the coiled-coil domain within the EML4 region of each monomer, thereby activating ALK and generating oncogenic activity. In transgenic mice that express EML4-ALK specifically in lung epithelial cells, hundreds of adenocarcinoma nodules develop in both lungs soon after birth, and oral administration of a specific inhibitor of ALK tyrosine kinase activity rapidly eradicates such . . .

A simple, safe and cost-effective treatment protocol in ovarian stimulation is of great importance in IVF practice, especially in the case of previous unsuccessful attempts. hCG has been used as a substitute of LH because of the degree of homology between the two hormones. The main aim of this prospective randomized study was to determine, for the first time, whether low dose hCG added to rFSH for ovarian stimulation could produce better results compared to the addition of rLH in women entering IVF-ET, especially in those women that had previous IVF failures. An additional aim was to find an indicator that would allow us to follow-up ovarian stimulation and, possibly, modify it in order to achieve a better IVF outcome; and that indicator may be the cDNA copies of the LH/hCG receptor. Group A patients (n = 58) were administered hCG and Group B rLH (n = 56) in addition to rFSH in the first days of ovarian stimulation. The number of follicles and oocytes and, most importantly, implantation and pregnancy rates were shown to be statistically significantly higher in the hCG group. This study has also determined, for the first time to our best knowledge, m-RNA for LH/hCG receptors in the lymphocytes of peripheral blood 40 h before ovum pick-up. cDNA levels of the hCG receptor after ovarian stimulation were significantly higher among women receiving hCG compared to those receiving LH. In addition, higher levels were encountered among women with pregnancy compared to those without, although this was not statistically significant due to the small number of pregnancies. It seems that hCG permits a highly effective and more stable occupancy of rLH/hCG receptors and gives more follicles and more oocytes. The determination of cDNA copies could be, in the future, a marker during ovulation induction protocols and of course a predictor for the outcome of ART in the special subgroup of patients with previous failures.

Gene expression of microbes in anaerobic sediment from the Peru Margin at depths up to 159 metres below the sea floor is analysed: anaerobic metabolism of amino acids, carbohydrates and lipids are seen to be the dominant metabolic processes, and genes associated with cell division are found to be correlated with microbial cell concentration, suggesting that ongoing cell division contributes to biomass turnover. Microbial proliferation in sub-seafloor sediments Microbial metabolism in the marine subsurface environment is an important factor in various global biogeochemical cycles, yet our understanding of the types of metabolism occurring is limited. This study uses metatranscriptomics to investigate gene expression in sediments up to 159 metres below the seafloor off the coast of Peru. Anaerobic metabolism of amino acids, carbohydrates and lipids are the predominant metabolic processes in this 'deep biosphere' environment, and the distribution of transcripts associated with cell division suggests that microbial cell division is occurring, not just biomass turnover without division. Scientific ocean drilling has revealed a deep biosphere of widespread microbial life in sub-seafloor sediment. Microbial metabolism in the marine subsurface probably has an important role in global biogeochemical cycles 1 , 2 , 3 , but deep biosphere activities are not well understood 1 . Here we describe and analyse the first sub-seafloor metatranscriptomes from anaerobic Peru Margin sediment up to 159 metres below the sea floor, represented by over 1 billion complementary DNA (cDNA) sequence reads. Anaerobic metabolism of amino acids, carbohydrates and lipids seem to be the dominant metabolic processes, and profiles of dissimilatory sulfite reductase ( dsr ) transcripts are consistent with pore-water sulphate concentration profiles 1 . Moreover, transcripts involved in cell division increase as a function of microbial cell concentration, indicating that increases in sub-seafloor microbial abundance are a function of cell division across all three domains of life. These data support calculations 1 and models 4 of sub-seafloor microbial metabolism and represent the first holistic picture of deep biosphere activities.