Recommendations for Method Development and Validation of qPCR and dPCR Assays in Support of Cell and Gene Therapy Drug Development.

The emerging use of qPCR and dPCR in regulated bioanalysis and absence of regulatory guidance on assay validations for these platforms has resulted in discussions on lack of harmonization on assay design and appropriate acceptance criteria for these assays. Both qPCR and dPCR are extensively used to answer bioanalytical questions for novel modalities such as cell and gene therapies. Following cross-industry conversations on the lack of information and guidelines for these assays, an American Association of Pharmaceutical Scientists working group was formed to address these gaps by bringing together 37 industry experts from 24 organizations to discuss best practices to gain a better understanding in the industry and facilitate filings to health authorities. Herein, this team provides considerations on assay design, development, and validation testing for PCR assays that are used in cell and gene therapies including (1) biodistribution; (2) transgene expression; (3) viral shedding; (4) and persistence or cellular kinetics of cell therapies.

The role of SR protein kinases in regulating lipid storage in the Drosophila fat body.

The survival of animals during periods of limited nutrients is dependent on the organism's ability to store lipids during times of nutrient abundance. However, the increased availability of food in modern western society has led to an excess storage of lipids resulting in metabolic diseases. To better understand the genes involved in regulating lipid storage, genome-wide RNAi screens were performed in cultured Drosophila cells and one group of genes identified includes mRNA splicing factor genes. Our lab has previously shown that a group of splicing factors important for intron/exon border recognition known as SR proteins are involved in controlling lipid storage in Drosophila; however, how these SR proteins are regulated to control lipid storage is not fully understood. Here, we focus on two SR protein kinases (SRPKs) in Drosophila: SRPK and SRPK79D. Decreasing the expression of these genes specifically in the adult fat body using RNAi resulted in lower levels of triglycerides and this is due to a decrease in the amount of fat stored per cell, despite having more fat cells, when compared to control flies. Decreasing SRPK and SRPK79D levels in the fat body leads to altered splicing of the ß-oxidation gene, carnitine palmitoyltransferase 1 (CPT1), resulting in increased production of a more active enzyme, which would increase lipid breakdown and be consistent with the lean phenotype observed in these flies. In addition, flies with decreased SRPK and SRPK79D levels in their fat bodies eat less, which may also contribute to the decreased triglyceride phenotype. Together, these findings provide evidence to support that lipid storage is controlled by the phosphorylation of factors involved in mRNA splicing.

A comparison of high-throughput SARS-CoV-2 sequencing methods from nasopharyngeal samples.

The COVID-19 pandemic caused by the new Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) continues to threaten public health and burden healthcare systems worldwide. Whole SARS-CoV-2 genome sequencing has become essential for epidemiological monitoring and identification of new variants, which could represent a risk of increased transmissibility, virulence, or resistance to vaccines or treatment. Different next-generation sequencing approaches are used in SARS-CoV-2 sequencing, although with different ability to provide whole genome coverage without gaps and to reliably detect new variants. In this study, we compared the performance of three target enrichment methods (two multiplex amplification methods and one hybridization capture) using nasopharyngeal swabs from infected individuals. We applied these target enrichment methods to the same set of nasopharyngeal samples (N = 93) in high-throughput mode. SARS-CoV-2 genome was obtained using short-read next-generation sequencing. We observed that each method has some advantages, such as high mapping rate (CleanPlex and COVIDSeq) or absence of systematic variant calling error (SureSelect) as well as their limitations such as suboptimal uniformity of coverage (CleanPlex), high cost (SureSelect) or supply shortages (COVIDSeq). Nevertheless, each of the three target enrichment kits tested in this study yielded acceptable results of whole SARS-CoV-2 genome sequencing and either of them can therefore be used in prospective programs of genomic surveillance of SARS-CoV-2. Genomic surveillance will be crucial to overcoming the ongoing pandemic of COVID-19, despite its successive waves and continually emerging variants.

Rapid protein evolution, organellar reductions, and invasive intronic elements in the marine aerobic parasite dinoflagellate Amoebophrya spp.

BACKGROUND: Dinoflagellates are aquatic protists particularly widespread in the oceans worldwide. Some are responsible for toxic blooms while others live in symbiotic relationships, either as mutualistic symbionts in corals or as parasites infecting other protists and animals. Dinoflagellates harbor atypically large genomes (~ 3 to 250 Gb), with gene organization and gene expression patterns very different from closely related apicomplexan parasites. Here we sequenced and analyzed the genomes of two early-diverging and co-occurring parasitic dinoflagellate Amoebophrya strains, to shed light on the emergence of such atypical genomic features, dinoflagellate evolution, and host specialization. RESULTS: We sequenced, assembled, and annotated high-quality genomes for two Amoebophrya strains (A25 and A120), using a combination of Illumina paired-end short-read and Oxford Nanopore Technology (ONT) MinION long-read sequencing approaches. We found a small number of transposable elements, along with short introns and intergenic regions, and a limited number of gene families, together contribute to the compactness of the Amoebophrya genomes, a feature potentially linked with parasitism. While the majority of Amoebophrya proteins (63.7% of A25 and 59.3% of A120) had no functional assignment, we found many orthologs shared with Dinophyceae. Our analyses revealed a strong tendency for genes encoded by unidirectional clusters and high levels of synteny conservation between the two genomes despite low interspecific protein sequence similarity, suggesting rapid protein evolution. Most strikingly, we identified a large portion of non-canonical introns, including repeated introns, displaying a broad variability of associated splicing motifs never observed among eukaryotes. Those introner elements appear to have the capacity to spread over their respective genomes in a manner similar to transposable elements. Finally, we confirmed the reduction of organelles observed in Amoebophrya spp., i.e., loss of the plastid, potential loss of a mitochondrial genome and functions. CONCLUSION: These results expand the range of atypical genome features found in basal dinoflagellates and raise questions regarding speciation and the evolutionary mechanisms at play while parastitism was selected for in this particular unicellular lineage.

GROOLS: reactive graph reasoning for genome annotation through biological processes.

BACKGROUND: High quality functional annotation is essential for understanding the phenotypic consequences encoded in a genome. Despite improvements in bioinformatics methods, millions of sequences in databanks are not assigned reliable functions. The curation of protein functions in the context of biological processes is a way to evaluate and improve their annotation. RESULTS: We developed an expert system using paraconsistent logic, named GROOLS (Genomic Rule Object-Oriented Logic System), that evaluates the completeness and the consistency of predicted functions through biological processes like metabolic pathways. Using a generic and hierarchical representation of knowledge, biological processes are modeled in a graph from which observations (i.e. predictions and expectations) are propagated by rules. At the end of the reasoning, conclusions are assigned to biological process components and highlight uncertainties and inconsistencies. Results on 14 microbial organisms are presented. CONCLUSIONS: GROOLS software is designed to evaluate the overall accuracy of functional unit and pathway predictions according to organism experimental data like growth phenotypes. It assists biocurators in the functional annotation of proteins by focusing on missing or contradictory observations.

Two genomes of highly polyphagous lepidopteran pests (Spodoptera frugiperda, Noctuidae) with different host-plant ranges.

Emergence of polyphagous herbivorous insects entails significant adaptation to recognize, detoxify and digest a variety of host-plants. Despite of its biological and practical importance - since insects eat 20% of crops - no exhaustive analysis of gene repertoires required for adaptations in generalist insect herbivores has previously been performed. The noctuid moth Spodoptera frugiperda ranks as one of the world's worst agricultural pests. This insect is polyphagous while the majority of other lepidopteran herbivores are specialist. It consists of two morphologically indistinguishable strains ("C" and "R") that have different host plant ranges. To describe the evolutionary mechanisms that both enable the emergence of polyphagous herbivory and lead to the shift in the host preference, we analyzed whole genome sequences from laboratory and natural populations of both strains. We observed huge expansions of genes associated with chemosensation and detoxification compared with specialist Lepidoptera. These expansions are largely due to tandem duplication, a possible adaptation mechanism enabling polyphagy. Individuals from natural C and R populations show significant genomic differentiation. We found signatures of positive selection in genes involved in chemoreception, detoxification and digestion, and copy number variation in the two latter gene families, suggesting an adaptive role for structural variation.

Online and Mobile Interventions for Problem Gambling, Alcohol, and Drugs: A Systematic Review.

Online interventions for gambling, alcohol, and illegal drug related problems have been developing at a fast pace over the past decade. Yet, little is known about the content and efficacy of interventions provided entirely online for reducing drug/alcohol use and gambling, or about the characteristics of those who use these interventions. This systematic review aims to describe the characteristics of online interventions, their efficacy, and the profile of their clientele. Documentation was mainly obtained through four scientific databases in psychology, technology, and medical research (PsychINFO, MedLine, Francis, and INSPEC) using three keywords (substances or gambling, intervention, Internet). Of the 4,708 documents initially identified, 18 studies meeting admissibility criteria were retained and analyzed after exclusion of duplicates and non-relevant documents. No study in the review related to problem gambling. The majority of interventions were based upon motivational or cognitive-behavioral theoretical approaches and called upon well-established therapeutic components in the field of addictions. The participants in these studies were generally adults between 30 and 46 years old with a high school education and presenting a high risk or problematic use. More than three quarters of the studies showed a short-term decrease in use that was maintained 6 months later, but only two studies included a 12 months follow-up. Online interventions seem promising and appear to meet the needs of participants who are in the workforce and seeking help for the first time. Long-term efficacy studies should nonetheless be conducted.

MicroScope in 2017: an expanding and evolving integrated resource for community expertise of microbial genomes.

The annotation of genomes from NGS platforms needs to be automated and fully integrated. However, maintaining consistency and accuracy in genome annotation is a challenging problem because millions of protein database entries are not assigned reliable functions. This shortcoming limits the knowledge that can be extracted from genomes and metabolic models. Launched in 2005, the MicroScope platform (http://www.genoscope.cns.fr/agc/microscope) is an integrative resource that supports systematic and efficient revision of microbial genome annotation, data management and comparative analysis. Effective comparative analysis requires a consistent and complete view of biological data, and therefore, support for reviewing the quality of functional annotation is critical. MicroScope allows users to analyze microbial (meta)genomes together with post-genomic experiment results if any (i.e. transcriptomics, re-sequencing of evolved strains, mutant collections, phenotype data). It combines tools and graphical interfaces to analyze genomes and to perform the expert curation of gene functions in a comparative context. Starting with a short overview of the MicroScope system, this paper focuses on some major improvements of the Web interface, mainly for the submission of genomic data and on original tools and pipelines that have been developed and integrated in the platform: computation of pan-genomes and prediction of biosynthetic gene clusters. Today the resource contains data for more than 6000 microbial genomes, and among the 2700 personal accounts (65% of which are now from foreign countries), 14% of the users are performing expert annotations, on at least a weekly basis, contributing to improve the quality of microbial genome annotations.

Decoding the oak genome: public release of sequence data, assembly, annotation and publication strategies.

The 1.5 Gbp/2C genome of pedunculate oak (Quercus robur) has been sequenced. A strategy was established for dealing with the challenges imposed by the sequencing of such a large, complex and highly heterozygous genome by a whole-genome shotgun (WGS) approach, without the use of costly and time-consuming methods, such as fosmid or BAC clone-based hierarchical sequencing methods. The sequencing strategy combined short and long reads. Over 49 million reads provided by Roche 454 GS-FLX technology were assembled into contigs and combined with shorter Illumina sequence reads from paired-end and mate-pair libraries of different insert sizes, to build scaffolds. Errors were corrected and gaps filled with Illumina paired-end reads and contaminants detected, resulting in a total of 17,910 scaffolds (>2 kb) corresponding to 1.34 Gb. Fifty per cent of the assembly was accounted for by 1468 scaffolds (N50 of 260 kb). Initial comparison with the phylogenetically related Prunus persica gene model indicated that genes for 84.6% of the proteins present in peach (mean protein coverage of 90.5%) were present in our assembly. The second and third steps in this project are genome annotation and the assignment of scaffolds to the oak genetic linkage map. In accordance with the Bermuda and Fort Lauderdale agreements and the more recent Toronto Statement, the oak genome data have been released into public sequence repositories in advance of publication. In this presubmission paper, the oak genome consortium describes its principal lines of work and future directions for analyses of the nature, function and evolution of the oak genome.