Gene expression analyses of immune responses in Atlantic salmon during early stages of infection by salmon louse (Lepeophtheirus salmonis) revealed bi-phasic responses coinciding with the copepod-chalimus transition

The salmon louse (Lepeophtheirus salmonis Krøyer), an ectoparasitic copepod with a complex life cycle causes significant losses in salmon aquaculture. Pesticide treatments against the parasite raise environmental concerns and their efficacy is gradually decreasing. Improvement of fish resistance to lice, through biological control methods, needs better understanding of the protective mechanisms. We used a 21 k oligonucleotide microarray and RT-qPCR to examine the time-course of immune gene expression changes in salmon skin, spleen, and head kidney during the first 15 days after challenge, which encompassed the copepod and chalimus stages of lice development. Results Large scale and highly complex transcriptome responses were found already one day after infection (dpi). Many genes showed bi-phasic expression profiles with abrupt changes between 5 and 10 dpi (the copepod-chalimus transitions); the greatest fluctuations (up- and down-regulation) were seen in a large group of secretory splenic proteases with unknown roles. Rapid sensing was witnessed with induction of genes involved in innate immunity including lectins and enzymes of eicosanoid metabolism in skin and acute phase proteins in spleen. Transient (1-5 dpi) increase of T-cell receptor alpha, CD4-1, and possible regulators of lymphocyte differentiation suggested recruitment of T-cells of unidentified lineage to the skin. After 5 dpi the magnitude of transcriptomic responses decreased markedly in skin. Up-regulation of matrix metalloproteinases in all studied organs suggested establishment of a chronic inflammatory status. Up-regulation of putative lymphocyte G0/G1 switch proteins in spleen at 5 dpi, immunoglobulins at 15 dpi; and increase of IgM and IgT transcripts in skin indicated an onset of adaptive humoral immune responses, whereas MHCI appeared to be down-regulated. Conclusions Atlantic salmon develops rapid local and systemic reactions to L. salmonis, which, however, do not result in substantial level of protection. The dramatic changes observed after 5 dpi can be associated with metamorphosis of copepod, immune modulation by the parasite, or transition from innate to adaptive immune responses

Applying genetic technologies to combat infectious diseases in aquaculture

Disease and parasitism cause major welfare, environmental and economic concerns for global aquaculture. In this review, we examine the status and potential of technologies that exploit genetic variation in host resistance to tackle this problem. We argue that there is an urgent need to improve understanding of the genetic mechanisms involved, leading to the development of tools that can be applied to boost host resistance and reduce the disease burden. We draw on two pressing global disease problems as case studies—sea lice infestations in salmonids and white spot syndrome in shrimp. We review how the latest genetic technologies can be capitalised upon to determine the mechanisms underlying inter- and intra-species variation in pathogen/ parasite resistance, and how the derived knowledge could be applied to boost disease resistance using selective breeding, gene editing and/or with targeted feed treatments and vaccines. Gene editing brings novel opportunities, but also implementation and dissemination challenges, and necessitates new protocols to integrate the technology into aquaculture breeding programmes. There is also an ongoing need to minimise risks of disease agents evolving to overcome genetic improvements to host resistance, and insights from epidemiological and evolutionary models of pathogen infestation in wild and cultured host populations are explored. Ethical issues around the different approaches for achieving genetic resistance are discussed. Application of genetic technologies and approaches has potential to improve fundamental knowledge of mechanisms affecting genetic resistance and provide effective pathways for implementation that could lead to more resistant aquaculture stocks, transforming global aquaculture.publishedVersio

Orthogonally photocontrolled non-autonomous DNA walker.

There is considerable interest in developing progressively moving devices on the nanoscale, with the aim of using them as parts of programmable therapeutics, smart materials and nano factories. We present an entirely light‐induced DNA walker based on orthogonal photo‐control. Implementing two azobenzene derivatives, S‐DM‐Azo and DM‐Azo, enabled precise coordination of strand displacement reactions that powered a biped walker and guided it along a defined track in a non‐autonomous way. This unprecedented type of molecular walker design offers high precision control over the movement in back‐and‐forth directions as desired, regulated solely by the sequence of the irradiation wavelengths. This concept may open new avenues for advancing non‐autonomous progressive molecular motors, ultimately facilitating their application on the nanoscale

Design, assembly, characterization, and operation of double-stranded interlocked DNA nanostructures

Mechanically interlocked DNA nanostructures are useful as flexible entities for operating DNA-based nanomachines. Interlocked structures made of double-stranded (ds) DNA components can be constructed by irreversibly threading them through one another to mechanically link them. The interlocked components thus remain bound to one another while still permitting large-amplitude motion about the mechanical bond. The construction of interlocked dsDNA architectures is challenging because it usually involves the synthesis and modification of small dsDNA nanocircles of various sizes, dependent on intrinsically curved DNA. Here we describe the design, generation, purification, and characterization of interlocked dsDNA structures such as catenanes, rotaxanes, and daisy-chain rotaxanes (DCRs). Their construction requires precise control of threading and hybridization of the interlocking components at each step during the assembly process. The protocol details the characterization of these nanostructures with gel electrophoresis and atomic force microscopy (AFM), including acquisition of high-resolution AFM images obtained in intermittent contact mode in liquid. Additional functionality can be conferred on the DNA architectures by incorporating proteins, molecular switches such as photo-switchable azobenzene derivatives, or fluorophores for studying their mechanical behavior by fluorescence quenching or fluorescent resonance energy transfer experiments. These modified interlocked DNA architectures provide access to more complex mechanical devices and nanomachines that can perform a variety of desired functions and operations. The assembly of catenanes can be completed in 2 d, and that of rotaxanes in 3 d. Addition of azobenzene functionality, fluorophores, anchor groups, or the site-specific linkage of proteins to the nanostructure can extend the time line

Expanding the Toolbox of Photoswitches for DNA Nanotechnology Using Arylazopyrazoles

Photoregulation is among the most promising tools for development of dynamic DNA nanosystems, due to its high spatiotemporal precision, biocompatibility, and ease of use. So far, azobenzene and its derivatives have shown high potential in photocontrolling DNA duplex hybridization by light-dependent photoisomerization. Despite many recent advances, obtaining sufficiently high photoswitching efficiency under conditions more suitable for work with DNA nanostructures are challenging. Here we introduce a pair of arylazopyrazoles as new photoswitches for efficient and reversible control of DNA hybridization achieved even at room temperature with a low number of required modifications. Their photophysical properties in the native state and in DNA strands result in near-quantitative isomerization rates by irradiation with UV and orange light. To demonstrate the applicability of these photoswitches, we have successfully applied one of them to open and close a DNA hairpin by light at room temperature

Skeletal Age as a Determinant of Bone Mass in Preadolescent Females

Objective. To evaluate the association between chronological age, skeletal age, pubertal stage, and basic anthropometry with bone mass of the total body, forearm, and second metacarpal bone in 456 healthy Caucasian females, aged 8–13 years. Design. Total body and forearm bone measurements were performed by dual X-ray absorptiometry, while bone mass of the second metacarpal was assessed by radiogrammetry. Skeletal age (SA) was assessed by the FELS method and pubertal stage was self-determined by selecting corresponding illustrations of breast and pubic hair development. The Cp criterion was used to select the best multiple regression model containing the subset of independent variables with the least bias and best predictive ability for each of the measured bone mass variables. Results. Of all the independent variables, weight, stature, and SA emerged as the most significant predictors for almost all the bone mass variables. Multiple regression models were created based on the Cp criterion with the resulting R2 (adjusted) for bone mineral content of total body, proximal forearm, ultradistal forearm, length of second metacarpal, as well as of total, medullary, and cortical areas: 0.793, 0.523, 0.390, 0.602, 0.232, 0.073, and 0.264, respectively. The measured bone variables were also regressed on SA using either quadratic or linear equations, depending on the shape of the cubic splines used for the best curve fitting. Significant positive association (p\u3c0.0001) of SA and each of the bone variables was noted, the highest being with bone mineral density and content of total body (R2=0.176, 0.338) and proximal and ultradistal forearm (R2=0.216, 0.203, 0.106, 0.201), respectively, as well as with the length of the second metacarpal bone (R2=0.339). Chronological age and pubertal stage did not have statistically significant predictive abilities for bone mass variables in the multiple regression models. Conclusions. We conclude that skeletal age is a powerful determinant of bone mass in children. It can be used as the criterion for the selection of a biologically homogeneous population with regard to bone mass. This may be important for the design of intervention studies targeting bone mass of children and adolescents

Skeletal Age as a Determinant of Bone Mass in Preadolescent Females

Objective. To evaluate the association between chronological age, skeletal age, pubertal stage, and basic anthropometry with bone mass of the total body, forearm, and second metacarpal bone in 456 healthy Caucasian females, aged 8–13 years. Design. Total body and forearm bone measurements were performed by dual X-ray absorptiometry, while bone mass of the second metacarpal was assessed by radiogrammetry. Skeletal age (SA) was assessed by the FELS method and pubertal stage was self-determined by selecting corresponding illustrations of breast and pubic hair development. The Cp criterion was used to select the best multiple regression model containing the subset of independent variables with the least bias and best predictive ability for each of the measured bone mass variables. Results. Of all the independent variables, weight, stature, and SA emerged as the most significant predictors for almost all the bone mass variables. Multiple regression models were created based on the Cp criterion with the resulting R2 (adjusted) for bone mineral content of total body, proximal forearm, ultradistal forearm, length of second metacarpal, as well as of total, medullary, and cortical areas: 0.793, 0.523, 0.390, 0.602, 0.232, 0.073, and 0.264, respectively. The measured bone variables were also regressed on SA using either quadratic or linear equations, depending on the shape of the cubic splines used for the best curve fitting. Significant positive association (p\u3c0.0001) of SA and each of the bone variables was noted, the highest being with bone mineral density and content of total body (R2=0.176, 0.338) and proximal and ultradistal forearm (R2=0.216, 0.203, 0.106, 0.201), respectively, as well as with the length of the second metacarpal bone (R2=0.339). Chronological age and pubertal stage did not have statistically significant predictive abilities for bone mass variables in the multiple regression models. Conclusions. We conclude that skeletal age is a powerful determinant of bone mass in children. It can be used as the criterion for the selection of a biologically homogeneous population with regard to bone mass. This may be important for the design of intervention studies targeting bone mass of children and adolescents