Nota Técnica. Variación longitudinal de densidad y peso específico básico en Pinus caribaea var. hondurensis de 25 años de edad, proveniente de las plantaciones de Uverito, (Monagas, Venezuela).

Se presenta el estudio de densidad (verde, seca al aire, seca al horno) y peso específico básico de Pinus caribaea var. hondurensis (Sénécl) W. Barret & Golfari (Pinaceae) procedente de plantaciones de 25 años de edad de la región de Uverito, estado Monagas (Venezuela) en tres niveles de altura: sección basal, media y apical para conocer su variación en sentido longitudinal. Se tomaron muestras en seis individuos y se prepararon siguiendo el procedimiento indicado por Hoheisel (1968). La densidad y peso específico mostraron los mayores valores en la sección basal, sin embargo, desde el punto de vista estadístico, no existieron diferencias significativas entre los tres niveles de altura y se puede indicar que las propiedades estudiadas tienden a mantenerse constantes en sentido longitudinal.

Establishment of an Efficient Protoplast Regeneration and Transfection Protocol for Field Cress (Lepidium campestre)

Field cress (Lepidium campestre) is a potential oilseed crop that has been under domestication in recent decades. CRISPR/Cas9 is a powerful tool for rapid trait improvement and gene characterization and for generating transgene-free mutants using protoplast transfection system. However, protoplast regeneration remains challenging for many plant species. Here we report an efficient protoplast regeneration and transfection protocol for field cress. Important factors such as type of basal media, type/combination of plant growth regulators, and culture duration on different media were optimized. Among the basal media tested, Nitsch was the best for protoplast growth in MI and MII media. For cell wall formation during the early stage of protoplast growth, relatively high auxin concentrations (0.5 mg L−1 NAA and 2,4-D), without addition of cytokinin was preferred for maintaining protoplast viability. After cell wall formation, 1.1 mg L−1 TDZ combined with either 0.05 mg L−1 NAA or 2,4-D was found to efficiently promote protoplast growth. On solid shoot induction medium, 1.1 mg L−1 TDZ without any auxin resulted in over 80% shoot generation frequency. A longer culture duration in MI medium would inhibit protoplast growth, while a longer culture duration in MII medium significantly delayed shoot formation. Using this optimized protoplast regeneration protocol, we have established an efficient PEG-mediated transfection protocol using a vector harboring the GFP gene, with transfection efficiencies of 50–80%. This efficient protoplast protocol would facilitate further genetic improvement of field cress via genome editing, and be beneficial to development of protoplast regeneration protocols for related plant species.

Screening of predominant microorganisms occurring on the concrete structures from selected locations

The aim of the present work is to provide an overview of the predominant microorganisms that effects structures of cementitious composites, which lead to biocorrosion, subsequently to the degradation of cementitious materials. Research is done by sampling of specimens taken from selected parts of the city of Košice. The sampling was mainly focused on insulated apartment panel buildings, part of the specimens was taken from uninsulated concrete and masonry plastered surfaces. Specimens were taken with sterile lab tubes, scalpels, and swabs by using dry, semi-dry and wet methods. The specimens were preserved in the cold place until their performance for the cultivation process. For cultivation purposes were important to prepare basal media for specimens of microorganisms and proceed isolation of cultures and their cultivation on appropriate media, identification of individual cultures. Data obtained after cultivation confirmed presence of predominant fungi genus such as Aspergillus, Botrytis, Cladosporium, Eutypella, Penicillium, Rhodotorula, Mucor, which create the ground for further research focused on the durability of cement paste based on secondary raw materials and their resistance against the predominant groups of microorganisms occurring in the objective location also by using extracts of several herbals as a prevention of microbial growth.

Callus Induction from Root Fragments of Falcataria moluccana Plantlets

Callus induction of F. moluccana (sengon) was still an obstacle to indirect organogenesis regeneration. The purpose of the study was to determine the callus induction formation from root fragments of F. moluccana plantlets. Primary explants (fragments of roots) were cultivated on MS induction basal media and three concentration combination of PGRs (BAP and NAA): 0.5 ml/l BAP; 0,1 ml/l NAA (single PGR); and combination of 0.5 ml/l BAP + 0.1 ml/l NAA (double PGR). When roots were used as explants, high formation rates of callus (more than 70%) were obtained. Highest formation rates of callus were in NAA added at all clones (12 clones), then BAP added (7 clones) and BAP + NAA added (5 clones). The results indicated that BAP and NAA concentrations used in the media were influence the producing callus and affect the amount of callus produced from roots of sengon. The addition of NAA also gives higher callus proliferation results than the addition of BAP or the addition of a combination of the two hormones. The results indicated that BAP and NAA concentrations used in the media were influence the producing callus and affect the amount of callus produced from roots of sengon.

P–174 Globulin-rich protein supplements improve blastulation efficiency in culture and promote implantation in vitro

Study question Can globulin-rich compared to albumin (HSA) supplements improve blastulation and support embryo development towards post implantation? Summary answer Yes, globulin supplements with clinical-grade quality increase blastulation efficiency by 20% (50% in older mothers) and support the transition of embryos towards post-implantation development. What is known already During embryonic development at the morula stage there is a metabolic transition towards glycolysis as demand from outsourced energy increases. Therefore as cleavage proceeds, the demand for nutrients in the embryo increases accordingly. With few exceptions, HSA from human plasma or recombinant origin has been the main an only protein supplement used in almost all IVF-procedures. Globulin rich supplements are available but their use is not widespread and little is known about their efficiency in post-implantation development. Study design, size, duration We have cultured more than 600 mouse embryos in continuous media containing a protein supplement#1 (PS#1), from 1-cell up to blastocyst stage. At blastocyst stage embryos were replaced into fresh media containing protein supplement#2 (PS#2). The embryos were allowed to hatch naturally and then transferred into a proprietary matrix for further development and implantation for an additional 48h. Participants/materials, setting, methods: The blastulation rate, measured for HSA-supplemented embryo cohort was compared with embryos cultured in PS#1. Hatching efficiency was reported for embryos cultured in transfer media including PS#2. Once embedded in the matrix, advanced label-free imaging techniques and custom algorithms to measure matrix implantation strength were used. Key molecular markers (i.e. OCT4, CDX2) for correct post-implantation lineage patterning were documented by conventional 3D confocal immunofluorescence imaging. Main results and the role of chance Embryos supplemented with PS#1 reached blastocyst with overall 21% higher efficiency than embryos supplemented by HSA. When separated by age cohorts, embryos obtained from older females (ex-colony breeders, >14 weeks old) reached blastocyst stage with 55% higher efficiency than the same type of embryos cultured in the presence of HSA. Embryos obtained from females at optimal reproductive age reached blastocyst stage 10% more efficiently under PS#1 supplementation than with HSA. Hatching efficiency was 45% higher for embryos cultured with PS#2 than embryos supplemented with HSA. For every variable tested (e.g.% of arrested or degenerated embryos) or condition implemented (e.g. mouse basal media, human basal media from different brands, etc.) PS#1 and PS#2 outperformed, without exception, the supplementation with HSA. When embedded in the implantation matrix, the embryos cultured with PS#1 (cleavage) and transferred to PS#2 at blastocyst stage showed a remarkable implantation ability as measured by trophoblast outgrowth and matrix deformations. The embryos in PS#2 medium exerted stronger force into the matrix and also survived longer times than the embryos in HSA. PS#2 supported the transition of blastocyst towards post-implantation stages of development showing the correct lineage patterning of embryonic and extraembryonic molecular markers, including Oct4, CDx2, EOMES or GATA4. Limitations, reasons for caution This is a study based on an animal model. These observations need to be confirmed by ongoing experiments with human embryos. Wider implications of the findings: This work constitutes a proof-of-concept for the use of globulin-rich supplements as higher performance substitute of albumin in the culture of IVF embryos, both as (i) a standard protein source for culture media and (ii) as a supplement for transfer media to capacitate the embryo for implantation. Trial registration number Not applicable

CLASSIFICAÇÃO CLIMÁTICA PARA O SUL DO BRASIL UTILIZANDO O SISTEMA DE HOLDRIDGE (1967) / CLIMATIC CLASSIFICATION FOR SOUTHERN BRAZIL USING HOLDRIDGE (1967) SYSTEM

O objetivo deste trabalho é classificar o clima da região Sul do Brasil utilizando o sistema de classificação climática por zonas de vidas estabelecido por Holdridge (1967). Foram utilizados um sequenciamento de 30 anos (1989-2019) de dados climáticos para região Sul do Brasil obtidos através da plataforma National Aeronautics and Space Administration/Prediction of Worldwide Energy Resources – (NASA/POWER). A evapotranspiração potencial (ETP) foi estimada utilizando o método definido por Camargo (1971). Através da combinação entre a precipitação anual, biotemperatura média anual, biotemperatura basal média anual, relação da evapotranspiração, delimitou-se as zonas de vidas para o sistema de Holdridge (1967). A temperatura e precipitação apresentaram médias anuais de 18,58 (± 1,57) °C e 1777 (± 239) mm respectivamente. Foram encontradas doze zonas de vidas para a região Sul, sendo as mais predominantes: Floresta subtropical de montana baixa úmida (31,37%), e Floresta subtropical premontana úmida (32,11%).

Optimization of Media to Enhance the Growth of Tissue Engineered Cartilage

Unlike other self-repairing tissues, cartilage has a very low regenerative capacity, thus, giving reason to examine different approaches to potential reparative therapies such as tissue engineering. Although once chondrocytes are placed in vitro they start to synthesize less cartilaginous extracellular matrix (ECM). A promising method used to upregulate the synthesis of ECM constituents is new media formulations. Thus, the objective of this study was to explore different media formulations to upregulate the accumulation of cartilaginous extracellular matrix (specifically, proteoglycans and collagen) by providing the cells with different availability of nutrients (e.g. glucose, glutamine) as well as examining the influence of different basal media formulations. The accumulation of GAG and collagen had two different media formulations which showed a significant increase in upregulation of each constituent in the ECM; highlighting the importance of having new media formulations specifically geared to each constituent.

Optimization of Media to Enhance the Growth of Tissue Engineered Cartilage

Unlike other self-repairing tissues, cartilage has a very low regenerative capacity, thus, giving reason to examine different approaches to potential reparative therapies such as tissue engineering. Although once chondrocytes are placed in vitro they start to synthesize less cartilaginous extracellular matrix (ECM). A promising method used to upregulate the synthesis of ECM constituents is new media formulations. Thus, the objective of this study was to explore different media formulations to upregulate the accumulation of cartilaginous extracellular matrix (specifically, proteoglycans and collagen) by providing the cells with different availability of nutrients (e.g. glucose, glutamine) as well as examining the influence of different basal media formulations. The accumulation of GAG and collagen had two different media formulations which showed a significant increase in upregulation of each constituent in the ECM; highlighting the importance of having new media formulations specifically geared to each constituent.

Similarity assessment by multivariate statistics method based on the distance between biosimilar and originator

The development of biosimilar products or follow-on biologics has been flourishing in recent years because of their lower price than the originators. In this study, a multivariate data analysis method based on JMP software was proposed to assess the glycosylation pattern similarity of antibody candidates from different conditions in optimization experiments with a reference. A specific distance was generated by this method and indicated the glycoform similarity between the biosimilar and the reference. This method can be applied to analyze the similarity of other physicochemical and functional characteristics between follow-on biologics and originators. Then, the design of experimental methods can be realized to optimize the conditions of cell culture to attain similar antibody candidates. A higher concentration of GlcNAc added to the basal media made the glycan of the antibody more similar to the glycan of the reference in this study.