Efficient Clones Research Articles

Identification of monoclonal antibodies from naive antibody phage-display libraries for protein detection in formalin-fixed paraffin-embedded tissues

Most antibodies used in immunohistochemistry (IHC) have been developed by animal immunization. We wanted to explore naive antibody repertoires displayed on filamentous phages as a source of full-length antibodies for IHC on Formalin-Fixed and Paraffin-Embedded (FFPE) tissues. We used two isogenic mouse fibroblast cell lines that express or not human HER2 to generate positive and negative FFPE pseudo-tissue respectively. Using these pseudo-tissues and previously described approaches based on differential panning, we isolated very efficient antibody clones, but not against HER2. To optimize HER2 targeting and tissue specificity, we first performed 3–4 rounds of in vitro panning using recombinant HER2 extracellular domain (ECD) to enrich the phage library in HER2 binders, followed by one panning round using the two FFPE pseudo-tissues to retain binders for IHC conditions. We then analyzed the bound phages using next-generation sequencing to identify antibody sequences specifically associated with the HER2-positive pseudo-tissue. Using this approach, the top-ranked clone identified by sequencing was specific to the HER2-positive pseudo-tissue and showed a staining pattern similar to that of the antibody used for the clinical diagnosis of HER2-positive breast cancer. However, we could not optimize staining on other tissues, showing that specificity was restricted to the tissue used for selection and screening. Therefore, future optimized protocols must consider tissue diversity early during the selection by panning using a wide collection of tissue types.

Trends on Human Norovirus Virus-like Particles (HuNoV-VLPs) and Strategies for the Construction of Infectious Viral Clones toward In Vitro Replication.

Most acute gastroenteritis (AGE) outbreaks and sporadic cases in developing countries are attributable to infection by human norovirus (HuNoV), the enteric virus mainly transmitted via fecal-contaminated water. However, it has been challenging to study HuNoV due to the lack of suitable systems to cultivate and replicate the virus, hindering the development of treatments and vaccines. Researchers have been using virus-like particles (VLPs) and infectious viral clones to overcome this challenge as alternatives to fresh virus isolates in various in vitro and ex vivo models. VLPs are multiprotein structures that mimic the wild-type virus but cannot replicate in host cells due to the lack of genetic materials for replication, limiting downstream analysis of the virus life cycle and pathogenesis. The development of in vitro cloning systems has shown promise for HuNoV replication studies. This review discusses the approaches for constructing HuNoV-VLPs and infectious viral clones, the techniques involved, and the challenges faced. It also highlights the relationship between viral genes and their protein products and provides a perspective on technical considerations for producing efficient HuNoV-VLPs and infectious viral clones, which could substitute for native human noroviruses in future studies.

Physiological marker of phosphorus nutritional status in potatoPhysiological marker of phosphorus nutritional status in potato

The development of more efficient plants in the use of nutrients will be fundamental for agriculture in the coming years. The objective of this work was to define a physiological marker of the nutritional status of P for potato clones cultivated off-soil and in vitro, in order to facilitate the selection process of more efficient clones in the use of P. For this, two experiments were carried out with seven potato clones (SMIC 148-A, Dakota Rose, SMINIA 793101-3, SMIB 106-7, SMIF 212-3, SMIJ 319-1 and P 150) grown at two levels of P (low and high) in the culture systems in vitro (1,935 and 19,346mg P L-1) and in off-soil (2.32 and 23.2mg P L-1) using sand as substrate. The inorganic P content and the total accumulation of P in shoot and roots seem to be good indicators of the nutritional status of P in potato plants, both in off-soil and in vitro cultivation. The activity of the acidic phosphatase enzymes (APases) in the tissues of the potato depends very much on the culture system, the clone and the organ of the analyzed plant, making it difficult to choose it as a physiological marker for restriction of P in potatoes. The dry mass of the potato plant may be a good indicator of the nutritional status of P in clones grown off-soil.

YIELD AND NUT QUALITY OF 29 CASHEW MOTHER TREES (ANACARDIUM OCCIDENTALE L) ESTABLISHED AT THE GERMPLASM OF OUOGHI IN CENTRAL REGION OF BENIN

Description of subject: One of the main constraints to cashew crop development in Benin and Africa is the unavailability of efficient planting material for the plantations establishment. This constraint is largely responsible for the low level of nut yieldsobtained in cashew plantations. Objective: This study aimed to assess the agronomic performances of 29 different cashew mother trees established in the germplasm of the Central Agricultural Research Center (CRA-Centre) of the National Agricultural Research Institute of Benin located in Save, Central region, in sightof selecting high-performance clones to be distributed in rural areas. Materials and Methods: Nuts production, unit nuts weight and the kernelrate data were collected on 29 cashew mother trees during the harvest seasons from 2017 to 2020. These data were submitted to the analysis of variance and comparison of the means tests with software R. Results: The average nut yields of the 29 cashew mother trees varied from 0.85 to 11.98 kg / tree with a general average of 3.57 kg / tree at the age of 7 years old. Average unit nut weights ranged from 4.61 to 8.28 g with an overall average of 6.40 g. The average kernel ratio of nuts ranged from 24.08% to 32.46% with an overall average of 27.18%. The average weight of akernel varied from 1.43 to 2.15g with an average of 1.71g. At the end of the fouryears assessment, five of the 29 cashew mother trees with a minimum nut yield of 5 kg / tree were preselected as the first efficient cashew clones from Benin. Conclusion: These preliminary results are very important for the cashew breeding program in Benin. Furtherstudieswill plan to assess the cashew mother trees performance at 10, 15, 20 and 25 years old.

Intraspecific variation in resource use is not explained by population persistence or seasonality.

Populations of generalist grazers often contain genotypes with "powerful" and "efficient" strategies. Powerful genotypes grow rapidly on rich-quality resources, but slowly on poorer-quality ones, while efficient genotypes grow relatively better on poorer resources but cannot exploit richer resources as well. Via a "power-efficiency" trade-off, variation in resource quality could maintain genetic diversity. To evaluate this mechanism, we sampled six populations of the freshwater cladoceran Daphnia pulicaria. In persisting (year-round) populations, Daphnia consume resources that vary in quality, whereas in non-persisting (spring-only) populations, Daphnia primarily encounter rich-quality resources. We hypothesized that non-persisting populations harbor no efficient clones (hence should show lower growth on poor-quality resources). Although individuals from non-persisting populations remained smaller than individuals from persisting populations, no evidence arose for a trade-off between powerful and efficient strategies. In fact, growth rates on the two diets were positively correlated (instead of negatively, as predicted). Furthermore, in the persisting populations, we predicted that clonal selection from spring to summer should shift the distribution of genotypes from powerful (specialists on richer spring resources) to efficient (poorer, summer resources). Genetic composition of populations shifted from spring to summer, but not toward more efficient genotypes. Therefore, in these lakes, maintenance of variation among genotypes must stem from more complicated factors than population persistence patterns or seasonal shifts in resource quality alone.

Isolation, Propagation, and Prion Protein Expression During Neuronal Differentiation of Human Dental Pulp Stem Cells.

Bioethical issues related to the manipulation of embryonic stem cells have hindered advances in the field of medical research. For this reason, it is very important to obtain adult stem cells from different tissues such as adipose, umbilical cord, bone marrow and blood. Among the possible sources, dental pulp is particularly interesting because it is easy to obtain in respect of bioethical considerations. Indeed, human Dental Pulp Stem Cells (hDPSCs) are a type of adult stem cells able to differentiate in neuronal-like cells and can be obtained from the third molar of healthy patients (13-19 ages). In particular, the dental pulp was removed with an excavator, cut into small slices, treated with collagenase IV and cultured in a flask. To induce the neuronal differentiation, hDPSCs were stimulated with EGF/bFGF for 2 weeks. Previously, we have demonstrated that during the differentiation process the content of cellular prion Protein (PrPC) in hDPSCs increased. The cytofluorimetric analysis showed an early expression of PrPC that increased after neuronal differentiation process. Ablation of PrPC by siRNA PrP prevented neuronal differentiation induced by EGF/bFGF. In this paper, we illustrate that as we enhanced the isolation, separation and in vitro cultivation methods of hDPSCs with several easy procedures, more efficient cell clones were obtained and large-scale expansion of the mesenchymal stem cells (MSCs) was observed. We also show how the hDPSCs, obtained with methods detailed in the protocol, are an excellent experimental model to study the neuronal differentiation process of MSCs and subsequent cellular and molecular processes.

Synthego's Engineered Cells Allow Scientists to "Cut Out" CRISPR Optimization [SPONSORED

Synthego's Engineered Cells Allow Scientists to "Cut Out" CRISPR Optimization [SPONSORED

Consequences of moderate drought stress on the net photosynthesis, water-use efficiency and biomass production of three poplar clones

The breeding of efficient but drought-tolerant poplar clones is very important for short-rotation planting because of frequent periods with low precipitation in Central Europe. Three clones exhibiting very different shoot–root ratios under well-watered conditions were investigated: (1) the aspen clone Grosdubrau No. 1 (Populus tremula); (2) the newly bred aspen clone L316 × L9 No. 21 Thermo (P. tremula × P. tremula); (3) and the poplar clone Max2 (P. maximowiczii × P. trichocarpa). All three clones were exposed to moderate drought stress. Photosynthesis and transpiration were measured. The influences of drought on the biometrical parameters of the plants were evaluated. The intrinsic water-use efficiency (WUEintrinsic) was calculated. Height growth was decreased in stressed plants. Photosynthesis and water conductivity were significantly decreased, which is why the WUEintrinsic exhibited a greater increase in Max2 than in the aspen clones. The poplar clone Max2 showed low WUE after sufficient watering, but this parameter exhibited a greater increase under drought stress compared with the aspen clones. Max2 was characterised by intensive root growth that was diminished under stress. In contrast, both aspen clones were less adaptive to moderate drought stress. The capability to change the WUEintrinsic under different water availabilities can be considered a possible selection criterion for breeding.

HTLV-1 gene expression by individual infected clones determines susceptibility to lysis by cytotoxic T lymphocytes specific for Tax and HBZ

Host cytotoxic T-lymphocyte (CTL) responses are critical in limiting expansion of HTLV-1-infected CD4+T-cells in vivo, and most individuals generate abundant Tax-specific CTL. Although Tax is immunodominant, the ability to efficiently present peptides from HBZ on MHC class 1 is associated with a lower proviral load and a reduced frequency of HAM/TSP. HBZ mRNA is expressed in vivo, directing proliferation of infected cells. However, HBZ-specific CTL were detectable in fresh PBMCs in only 25% of chronically infected individuals. We hypothesised that HBZ has evolved to evade the generation of effective HBZ-specific CTLs. To evaluate the selective capacity of a potent HBZ-specific CTL response, we assayed the ability of equally efficient Tax-and HBZ-specific CTL clones to kill unstimulated, naturally infected cells from 16 HLA-A*02+HTLV-1+ individuals. Infected cells which expressed Tax during the course of the assay upregulated surface expression of HLA-A*02, and were eliminated efficiently by Tax-specific CTL. HBZ-specific CTL killed Tax+ cells less efficiently, preferentially killing cells with high levels of HLA-A*02. As a proportion of infected cells do not express Tax owing to silencing, mutation or viral integration site (IS) location, we tested whether HBZ-specific CTLs could kill Tax- infected cells, using high-throughput sequencing to monitor survival of infected clones after CTL selection. We are now validating our findings using patient-derived HBZ-specific CTL, and mapping HBZ epitopes recognised in vivo. In conclusion, the efficacy of HBZ-specific CTLs appears to be limited by the level of antigen presentation, but may confer the ability to target infected cells which escape Tax-specific CTL.

BTRFS

BTRFS is a Linux filesystem that has been adopted as the default filesystem in some popular versions of Linux. It is based on copy-on-write, allowing for efficient snapshots and clones. It uses B-trees as its main on-disk data structure. The design goal is to work well for many use cases and workloads. To this end, much effort has been directed to maintaining even performance as the filesystem ages, rather than trying to support a particular narrow benchmark use-case. Linux filesystems are installed on smartphones as well as enterprise servers. This entails challenges on many different fronts. --- Scalability . The filesystem must scale in many dimensions: disk space, memory, and CPUs. --- Data integrity . Losing data is not an option, and much effort is expended to safeguard the content. This includes checksums, metadata duplication, and RAID support built into the filesystem. --- Disk diversity . The system should work well with SSDs and hard disks. It is also expected to be able to use an array of different sized disks, which poses challenges to the RAID and striping mechanisms. This article describes the core ideas, data structures, and algorithms of this filesystem. It sheds light on the challenges posed by defragmentation in the presence of snapshots, and the tradeoffs required to maintain even performance in the face of a wide spectrum of workloads.

A power–efficiency trade‐off in resource use alters epidemiological relationships

Trade-offs play pivotal roles in the ecology and evolution of natural populations. However, trade-offs are probably not static, invariant relationships. Instead, ecological factors can shift, alter, or reverse the relationships underlying trade-offs and create critical genotype x environment (G x E) interactions. But which ecological factors alter trade-offs or create G x E interactions, and why (mechanistically) do they do this? We tackle these questions using resource quality as the central ecological factor and a case study of disease in the plankton. We show that clonal genotypes of a zooplankton host (Daphnia dentifera) exhibit a "power-efficiency" trade-off in resource use, where powerful (fast-feeding) host clones perform well on richer algal resources, but more efficient (slow-feeding) clones perform relatively well on poorer resources. This resource-based trade-off then influences epidemiological relationships due to fundamental connections between resources and fecundity, transmission rate (an index of resistance), and replication of a virulent fungal parasite (Metschnikowia bicuspidata) within hosts. For instance, using experiments and dynamic energy budget models, we show that the power-efficiency trade-off overturned a previously detected trade-off between fecundity and transmission risk of hosts to this parasite. When poorer resources were eaten, transmission risk and fecundity were negatively, not positively, correlated. Additionally, poor resource quality changed positive relationships between yield of infectious stages (spores) and host fecundity: those fecundity-spore relationships with poor food became negative or nonsignificant. Finally, the power-efficiency trade-off set up an interaction between host clone and resource quality for yield of fungal spores: powerful clones yielded relatively more spores on the better resource, while efficient clones yielded relatively more on the poorer resource. Thus, the physiological ecology of resource use can offer potent, mechanistic insight linking environmental factors to epidemiological relationships.

Induction of Pluripotent Stem Cells from Human Third Molar Mesenchymal Stromal Cells*[S

The expression of four transcription factors (OCT3/4, SOX2, KLF4, and MYC) can reprogram mouse as well as human somatic cells to induced pluripotent stem (iPS) cells. We generated iPS cells from mesenchymal stromal cells (MSCs) derived from human third molars (wisdom teeth) by retroviral transduction of OCT3/4, SOX2, and KLF4 without MYC, which is considered as oncogene. Interestingly, some of the clonally expanded MSCs could be used for iPS cell generation with 30-100-fold higher efficiency when compared with that of other clonally expanded MSCs and human dermal fibroblasts. Global gene expression profiles demonstrated some up-regulated genes regarding DNA repair/histone conformational change in the efficient clones, suggesting that the processes of chromatin remodeling have important roles in the cascade of iPS cells generation. The generated iPS cells resembled human embryonic stem (ES) cells in many aspects, including morphology, ES marker expression, global gene expression, epigenetic states, and the ability to differentiate into the three germ layers in vitro and in vivo. Because human third molars are discarded as clinical waste, our data indicate that clonally expanded MSCs derived from human third molars are a valuable cell source for the generation of iPS cells.

Novel Efficient Primary Human Peripheral Blood Progenitor Cell-Targeted Recombinant Adeno-Associated Viral Vectors.

Introduction: G-CSF mobilized peripheral blood progenitor cells (PBPC) are an excellent target for the cure of acquired and inherited disorders of the hematopoietic system. AAV-based vectors have an advantageous safety profile, due to their low immunogenicity, the lack of any associated human disease and its episomal localization. Unfortunately, currently standard rAAV2-based vectors lack the required efficiency for gene transfer into human blood progenitor cells. To increase the gene transfer efficiency on primary hematopoietic cells we applied an AAV random peptide library on primary human CD34+ PBPC.Materials and Methods: The AAV random peptide library was applied on primary human CD34+ PBPC samples (3 selection rounds). Several mutant blood progenitor-targeted rAAV2 capsid clones were obtained and sequenced. Five new rAAV vectors with the most efficient clones were produced. Titers were verified using real time PCR-based titration. To determine efficiency and specificity, a panel of leukemia (CML and AML) and non-leukemia cell lines, as well as primary human CD34+ PBPC were transduced. GFP expression was determined against uninfected control cells.Results: On a panel of leukemia cell lines at least one capsid mutant was significantly more efficient (%GFP+; p<0.01) than standard rAAV2 vectors. On solid tumor cell lines rAAV2 was more efficient, suggesting the increased specificity of our capsid mutants for hematopoietic cells. Significantly higher gene transfer efficiency (up to 16% GFP+ cells) could be obtained with the newly generated vectors compared to standard rAAV2 (up to 9-fold higher; p<0.01). Using a different approach, up to 25% gene transfer into CD34+ PBPC was observed.Conclusion: These novel vectors may finally enable efficient (>20%) gene transfer using rAAV-based vectors into primary human PBPC and may offer a safe alternative to other vector systems which is applicable in vivo.

Clonal variation in nutrient content in woody biomass of hybrid aspen (Populus tremula L. x P. tremuloides Michx.)

Differences in the nutrient concentrations and nutrient amounts of stems and branches amongst clones of hybrid aspen (Populus tremula L. x P. tremuloides Michx.) were investigated. Seven clones with superior and seven with medium growth rates were selected from a test of 119 clones in southern Sweden. Four trees per clone were randomly identified and harvested in dormant conditions. Sample discs from the stems and branches were collected and analysed for N, K, P, Ca, Mg, and S concentrations, as well as wood density. The analyses revealed significant genetic differences in wood density, K, P, and Mg concentrations in the stems. There were weak (non-significant) and negative genetic correlations between stem volume and concentrations of all the nutrients, except potassium, suggesting that nutrient-efficient clones could be selected without significantly sacrificing genetic gain for growth. In the branches K, Ca, and Mg concentrations differed significantly among clones. After selecting more nutrient efficient clones, the potential savings of nutrients compared with current hybrid aspen material was estimated to be around 5%, which seems fairly low, at least in a short-term perspective. However, the use of clones with different nutrient storage strategies may be regarded as a possible way in the long run to save nutrients in hybrid aspen ecosystems, or of removing them when sludge is applied.

VARIABILITY AMONG APHID CLONES OF RHOPALOSIPHUM PADI L. AND SITOBION AVENAE FABR. (HOMOPTERA: APHIDIDAE) IN TRANSMISSION OF THREE PAV ISOLATES OF BARLEY YELLOW DWARF VIRUSES

AbstractVariability in vectoring efficiency among six clones of Rhopalosiphum padi L. and five clones of Sitobion avenae Fabr. in transmission of three French PAV isolates (PAV-RG, PAV-2t, and PAV-13t) of barley yellow dwarf viruses (BYDVs) on seedlings of barley (Hordeum vulgare L.) cv. Plaisant was determined. All the clones could transmit these three isolates, but their vectoring efficiency was significantly different: 91% and 56% transmission efficiencies were obtained from the most efficient clones (Rp-M and Sa-R1) but only 21% and 9% from the least efficient clones (Rp-R26 and Sa-V) with 5-day acquisition and inoculation access periods (AAP, IAP). A significant difference in overall transmission between apterous adults and winged aphids of the tested clones was also found. In most cases, apterous adults were more efficient than were winged aphids. The overall transmission efficiency of apterous adults was 1.5 and 1.7 times that of winged ones for R. padi and S. avenae, respectively. The transmissibility of PAV-RG and PAV-2t isolates was similar within each of the tested aphid clones, but that of the PAV-13t isolate differed, particularly for the poorly efficient vector clones of S. avenae. Temperature could significantly influence the vectoring efficiency of the tested clones of R. padi, but the influence was greater on Rp-R26. High temperature (25 °C) facilitated more transmission than did low temperature (14 °C), and high temperature for AAP played a more important role than it did for IAP.

Clonal variation in apical growth and content in vegetative storage proteins in Populus

Apical shoot growth and storage protein content in various poplar species and clones were followed in trees growing in the field and in micropropagated plants cultivated in the growth chamber under a controlled environment. In autumn a 32 kD and a 36 kD vegetative storage protein accumulate in wood, bark and roots of poplar and comprise together about 25% of the soluble proteins. In spring, at the time of dormancy break, the storage proteins are degraded and 3 weeks after budburst these proteins are no longer immunologically detectable. As in autumn, short day exposure of black cottonwood plants (Populus trichocarpa Torr. and Gray) induces cessation of apical growth and accumulation of the 32 kD and 36 kD vegetative storage proteins in all clones studied. In order to simulate spring conditions, short day induced plants were transferred back to long days. Like the situation in spring, budburst and storage protein degradation occurred considerably earlier in clone 9/60 than in clone Muhle Larsen. The latter clone accumulates both in winter and after short day exposure more storage proteins than the former. Furthermore two P. trichocarpa clones differ qualitatively in storage protein content: they possess an additional 34 kD polypeptide which cross-reacts with the anti-32 kD antibody. In conclusion, apical shoot growth and the capacity to synthesize storage proteins can be easily followed in micropropagated poplar cultivated in the growth chamber under inducing photoperiods. This offers the major advantage of independence from the annual growth cycle. Within one species considerable clonal variance in storage protein content and in the induction times needed for dormancy and dormancy break were observed. The suitability of storage protein content and apical growth as early selection traits in breeding programs focusing on nitrogen efficient poplar and clones adapted to specific latitudes will be discussed.

Stomatal control of water use efficiency in poplar clones and hybrids

Water use efficiency (WUE, the ratio of dry matter produced to water used in transpiration) was studied in 17 poplar clones and hybrids. Although WUE could not be predicted from the poplar group alone, water efficiency was found to vary in different poplar genotypes. For example, a balsam poplar, Populus maximowiczii M-4 (section Tacamahaca), and a white poplar clone, P. alba A-499 (section Leuce), had twice the dry matter production per unit of water transpired compared with another clone of P. maximowiczii (M-13) and a Cottonwood clone, P. nigra N-80 (section Aegeiros), which showed low WUE. The reduced transpiration in water-efficient clones per unit of dry matter produced was associated with a higher stomatal resistance on the abaxial leaf surface. However, the physiological and morphological basis of WUE varied in different clones. Water-efficient clones exhibited one or more of the following adaptations restricting water loss: (i) conspicuous cuticular ledges or hairs above the pore openings of stomata, (ii) earlier partial stomatal opening in the morning, and (iii) smaller stomata and a lower stomatal frequency on the adaxial surface of the upper leaves. Poplar clones of low WUE exhibited less stomatal control of transpiration as a result of lower stomatal resistances and they lacked the above adaptations. Relative ranking of genotypes could not be predicted from either dry matter productivity or transpiration rate alone although the most water efficient clones generally exhibited lower transpiration rates compared with less efficient clones.

Water‐Use Efficiency and Its Association with Several Characteristics Blue Panicgrass (Panicum antidotale Retz.) Clones1

Water‐use efficiency and its relationship to physiological and morphological characteristics was investigated using blue panicgrass (Panicum antidotale Retz.). Four clones which had been characterized previously as being seedling drouth tolerant and four as being seedling drouth susceptible were subjected to three levels of soil‐moisture stress. A program‐controlled environment was used to simulate climatic conditions of the arid southwestern U.S. Clones differed significantly in water‐use efficiency. As soil‐moisture stress increased water‐use efficiency and dry matter production decreased. Dry weight, fresh weight, and total protein were significantly associated with water‐use efficiency. The clones most efficient in water use had more vascular bundles in the leaves when compared with the least efficient clones. The amino acids (micromoles/mg) lysine, histidine, arginine, glycine, and alanine were significantly associated with water‐use efficiency. A non‐significant association was found between seedling drouth tolerance and water‐use efficiency for the clones studied. Additional clones are being studied to elucidate further the relationships between these characteristics.