Recombinant adeno-associated viruses (rAAVs) are the predominant gene therapy vector. Several rAAV vectored therapies have achieved regulatory approval, but production of sufficient rAAV quantities ...remains difficult. The AAV Rep proteins, which are essential for genome replication and packaging, represent a promising engineering target for improvement of rAAV production but remain underexplored. To gain a comprehensive understanding of the Rep proteins and their mutational landscape, we assayed the effects of all 39,297 possible single-codon mutations to the AAV2
gene on AAV2 production. Most beneficial variants are not observed in nature, indicating that improved production may require synthetic mutations. Additionally, the effects of AAV2
mutations were largely consistent across capsid serotypes, suggesting that production benefits are capsid independent. Our results provide a detailed sequence-to-function map that enhances our understanding of Rep protein function and lays the groundwork for Rep engineering and enhancement of large-scale gene therapy production.
Business-to-business (B2B) firms regularly engage in collaborative selling where an outside sales (OS) representative (rep) interfaces with customers and an inside sales (IS) rep supports the OS reps ...through remote selling. While anecdotal evidence abounds, there is little empirical research examining factors driving successful IS-OS co-selling, as evidenced by objective sales performance. The authors use an organizational behavior lens and theories of shared team experiences and member knowledge diversity to posit that the collaboration experience (length and intensity) and product knowledge diversity affect customer sales outcomes. Further, they unpack how the efficacy of dyadic attributes should be contingent on the length of the customer–firm relationship and the customer’s product need complexity. Using field data and identification strategies suitable to their setting, the authors confirm that an IS–OS dyad’s collaboration experience and product knowledge diversity have a positive effect on customer-level sales outcome. However, they find a nuanced interplay of IS-OS dyadic attributes with customer characteristics. For example, customers with more complex product needs buy more when dyadic collaboration is long or intensive but not when the dyad possesses diverse product knowledge. The conceptual framework and empirical results together enable sales managers to match IS-OS dyads to customers they can serve profitably.
The blaNDM-1 gene and its variants encode metallo-beta-lactamases that confer resistance to almost all beta-lactam antibiotics. Genes encoding blaNDM-1 and its variants can be found in several ...Acinetobacter species, and they are usually linked to two different plasmid clades. The plasmids in one of these clades contain a gene encoding a Rep protein of the Rep_3 superfamily. The other clade consists of medium-sized plasmids in which the gene (s) involved in plasmid replication initiation (rep)have not yet been identified. In the present study, we identified the minimal replication region of a blaNDM-1-carrying plasmid of Acinetobacter haemolyticus AN54 (pAhaeAN54e), a member of this second clade. This region of 834 paired bases encodes three small peptides, all of which have roles in plasmid maintenance. The plasmids containing this minimal replication region are closely related; almost all contain blaNDM genes, and they are found in multiple Acinetobacter species, including A. baumannii. None of these plasmids contain an annotated Rep gene, suggesting that their replication relies on the minimal replication region that they share with the plasmid pAhaeAN54e. These observations suggest that this plasmid lineage plays a crucial role in the dissemination of the blaNDM-1 gene and its variants.
The metabolic versatility of
, a heterofermentative lactic acid bacterium, could benefit environmentally compatible and low salt cucumber fermentation. The biodiversity of
autochthonous to cucumber ...fermentation was studied using genotypic and phenotypic analyses to identify unique adjunct cultures. A group of 131 isolates autochthonous to industrial fermentations was screened using rep-PCR-(GTG)
and a fermentation ability assay under varied combinations of salt (0 or 6%), initial pH (4.0 or 5.2), and temperature (15 or 30°C). No apparent similarities were observed among the seven and nine clusters in the genotypic and phenotypic dendrograms, respectively. A total of 14 isolates representing the observed biodiversity were subjected to comparative genome analysis. The autochthonous
clustered apart from allochthonous isolates, as their genomes lack templates for citrate lyase, several putative hypothetical proteins, and some plasmid- and phage-associated proteins. Four and two representative autochthonous and allochthonous
, respectively, were subjected to phenotype microarray analysis using an Omnilog. Growth of all
strains was supported to various levels by glucose, fructose, gentiobiose, 1,2-propanediol, and propionic acid, whereas the allochthonous isolate ATCC14890 was unique in utilizing citric acid. All the
genomes encode for 1,2-propanediol utilization microcompartments. This study identified a unique
strain, autochthonous to cucumber, as a potential functional adjunct culture for commercial fermentation that is distinct in metabolic activities from allochthonous isolates of the same species.
A new index derived from existing indices - an index-based built-up index (IBI) - is proposed for the rapid extraction of built-up land features in satellite imagery. The IBI is distinguished from ...conventional indices by its first-time use of thematic index-derived bands to construct an index rather than by using original image bands. The three thematic indices used in constructing the IBI are the soil adjusted vegetation index (SAVI), the modified normalized difference water index (MNDWI) and the normalized difference built-up index (NDBI). Respectively, these represent the three major urban components of vegetation, water and built-up land. The new index has been verified using the Landsat ETM+ image of Fuzhou City in southeastern China. The result shows that the IBI can significantly enhance the built-up land feature while effectively suppressing background noise. A statistical analysis indicates that the IBI possesses a positive correlation with land surface temperature, but negative correlations with the NDVI and the MNDWI.
Acinetobacter baumannii
is an emergent bacterial pathogen that provokes many types of infections in hospitals around the world. The genome of this organism consists of a chromosome and plasmids. ...These plasmids vary over a wide size range and many of them have been linked to the acquisition of antibiotic-resistance genes. Our bioinformatic analyses indicate that
A. baumannii
plasmids belong to a small number of plasmid lineages. The general structure of these lineages seems to be very stable and consists not only of genes involved in plasmid maintenance functions but of gene sets encoding poorly characterized proteins, not obviously linked to survival in the hospital setting, and opening the possibility that they improve the parasitic properties of plasmids. An analysis of genes involved in replication, suggests that members of the same plasmid lineage are part of the same plasmid incompatibility group. The same analysis showed the necessity of classifying the Rep proteins in ten new groups, under the scheme proposed by
Bertini et al. (2010)
. Also, we show that some plasmid lineages have the potential capacity to replicate in many bacterial genera including those embracing human pathogen species, while others seem to replicate only within the limits of the
Acinetobacter
genus. Moreover, some plasmid lineages are widely distributed along the
A. baumannii
phylogenetic tree. Despite this, a number of them lack genes involved in conjugation or mobilization functions. Interestingly, only 34.6% of the plasmids analyzed here possess antibiotic resistance genes and most of them belong to fourteen plasmid lineages of the twenty one described here. Gene flux between plasmid lineages appears primarily limited to transposable elements, which sometimes carry antibiotic resistance genes. In most plasmid lineages transposable elements and antibiotic resistance genes are secondary acquisitions. Finally, broad host-range plasmids appear to have played a crucial role.
Black rot, caused by Xanthomonas campestris pv. campestris (Xcc) is a disease of crucifer crops. The objective of this study was to characterize races of Xcc, their distribution and genetic diversity ...in India. Two hundred and seventeen isolates of bacteria were obtained from 12 different black rot‐infected crucifer crops from 19 states of India; these were identified as Xcc based on morphology, hrpF gene and 16S rRNA gene based molecular markers and pathogenicity tests. Characterization of races was performed by using a set of seven differential crucifer hosts, comprising two cultivars of turnip (Brassica rapa var. rapa) and cultivars of Indian mustard (B. juncea), Ethiopian mustard (B. carinata), rapeseed mustard (B. napus), cauliflower (B. oleracea) and Savoy cabbage (B. oleracea var. sabauda). Races 1, 4 and 6 of Xcc were identified and, among these races, race 1 followed by race 4 dominated most of the states of India. Genetic diversity of the Indian isolates of Xcc was analysed using repetitive sequence‐based PCR (rep‐PCR) including primers for REP (repetitive extragenic palindromic), ERIC (enterobacterial repetitive intergenic consensus) and BOX (amplifying with BOX A1 R primer) repetitive elements. This method of fingerprinting grouped the isolates into 56 different DNA types (clusters) with a 75% similarity coefficient. Among these clusters, DNA types 22 and 53 contained two different races 1 and 4, whereas DNA type 12 contained races 1, 4 and 6. However, no clear relationship was observed between fingerprints and races, hosts or geographical origin.
The article clarifies the issues of modeling robotic systems in the V-REP program and provides skills in modeling the process of robotic and mechatronic complexes operation taking into account the ...laws of physics. The aim of the research paper is to investigate a 3D robotic simulator based on a distributed control architecture: control programs (or scripts) can be directly attached to objects in the scene and executed simultaneously in a streaming or non-streaming mode. V-REP can be used for remote monitoring, for hardware control, for rapid prototyping and verification, for rapid algorithm development/parameter tuning, for safety retesting, for robotics education, factory automation simulation, etc.
Artykuł wyjaśnia kwestie modelowania systemów zrobotyzowanych w programie V-REP i zapewnia umiejętności modelowania procesu działania robotów i zespołów mechatronicznych z uwzględnieniem praw fizyki. Celem artykułu jest zbadanie symulatora robotycznego 3D opartego na rozproszonej architekturze sterowania: programy sterujące (lub skrypty) mogą być bezpośrednio dołączane do obiektów na scenie i wykonywane jednocześnie w trybie strumieniowym lub niestrumieniowym. V-REP może być wykorzystywany do zdalnego monitorowania, kontroli sprzętu, szybkiego prototypowania i weryfikacji, szybkiego opracowywania algorytmów / dostrajania parametrów, ponownego testowania bezpieczeństwa, edukacji robotyki, symulacji automatyzacji fabryki itp.
Cytosine methylation is an epigenetic mark that promotes gene silencing and plays an important role in genome defence against transposons and invading DNA viruses. Previous data showed that the ...largest family of single-stranded DNA viruses, Geminiviridae, prevents methylation-mediated transcriptional gene silencing (TGS) by interfering with the proper functioning of the plant methylation cycle.
Here, we describe a novel counter-defence strategy used by geminiviruses, which reduces the expression of the plant maintenance DNA methyltransferases, METHYLTRANSFERASE 1 (MET1) and CHROMOMETHYLASE 3 (CMT3), in both locally and systemically infected tissues.
We demonstrated that the virus-mediated repression of these two maintenance DNA methyltransferases is widespread among geminivirus species. Additionally, we identified Rep (Replication associated protein) as the geminiviral protein responsible for the repression of MET1 and CMT3, and another viral protein, C4, as an ancillary player in MET1 down-regulation. The presence of Rep suppressed TGS of an Arabidopsis thaliana transgene and of host loci whose expression was strongly controlled by CG methylation. Bisulfite sequencing analyses showed that the expression of Rep caused a substantial reduction in the levels of DNA methylation at CG sites.
Our findings suggest that Rep, the only viral protein essential for replication, displays TGS suppressor activity through a mechanism distinct from that thus far described for geminiviruses.
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