Somatic cell nuclear transfer: failures, successes and the challenges ahead

Somatic cell nuclear transfer (SCNT) has a broad spectrum of potential applications, including rescue of endangered species, production of transgenic animals, drug production, and regenerative medicine. Unfortunately, the efficiency of SCNT is still disappointingly low. Many factors affecting cloning procedures have been described in several previous reviews; here we review the most effective improvements in SCNT, with a special emphasis on the effect of mitochondrial defects on SCNT embryo/ foetus development, an issue never touched upon before

Ultrastructural analysis reveals abnormal mitochondria in cloned blastocysts

Somatic cell nuclear transfer (SCNT) is a powerful technique, but still very inefficient despite 20 years passed by since the cloned mammal was born. We have recently shown that the major cause of abnormalities observed in cloned fetuses are mitochondrial dysfunctions in placenta collected from cloned sheep. Investigations on mitochondria in SCNT are limited to the mtDNA hetero/homoplasmy in cloned offspring, whereas no data is available for an eventual role of mitochondria dysfunction on the developmental failure of cloned animals. Here we wanted to know whether mitochondrial abnormalities are observed already in cloned blastocysts since mitochondrial replication does not occur after the hatched blastocysts stage. SCNT and in vitro processed (IVP) blastocysts were produced and analysed for mitochondrial structure and functionality. First, embryos were analysed using transmission electron microscope (TEM). Drastic differences in mitochondrial structure between SCNT and IVP blastocysts were observed. Decrease density of mature mitochondria, very high degree of cytoplasmic vacuolisation, numerous cytoplasmic vesicle and autophagosomes were observed in SCNT blastocysts. Moreover, statistically lower expression of major mitochondrial, autophagic and apoptotic proteins were observed in SCNT embryos. Obtained results clearly shown that mitochondrial abnormalities are already observed in blastocysts stage embryos. It is important to point out that activity of mitochondria are strictly control by nuclear signals, thus, obtained results may suggest that incomplete nuclear reprogramming in cloned nucleus might be responsible also for the impaired mitochondrial function in cloned embryos/fetuses

Dry storage of mammalian spermatozoa and cells: state-of-the-art and possible future directions

This review provides a snapshot of the current state-of-the-art of drying cells and spermatozoa. The major successes and pitfalls of the most relevant literature are described separately for spermatozoa and cells. Overall, the data published so far indicate that we are closer to success in spermatozoa, whereas the situation is far more complex with cells. Critical for success is the presence of xeroprotectants inside the spermatozoa and, even more so, inside cells to protect subcellular compartments, primarily DNA. We highlight workable strategies to endow gametes and cells with the right combination of xeroprotectants, mostly sugars, and late embryogenesis abundant (LEA) or similar ‘intrinsically disordered’ proteins to help them withstand reversible desiccation. We focus on the biological aspects of water stress, and in particular cellular and DNA damage, but also touch on other still unexplored issues, such as the choice of both dehydration and rehydration methods or approaches, because, in our view, they play a primary role in reducing desiccation damage. We conclude by highlighting the need to exhaustively explore desiccation strategies other than lyophilisation, such as air drying, spin drying or spray drying, ideally with new prototypes, other than the food and pharmaceutical drying strategies currently used, tailored for the unique needs of cells and spermatozoa

Agro-Industrial Waste from Cocoa Pod Husk (Theobroma cacao L.), as a Potential Raw Material for Preparation of Cellulose Nanocrystals

Obtaining cellulose from agro-industrial waste offers the possibility of generating added value to solid biomass that is currently deposited in sanitary landfills. This research performed the evaluation of a residue from cocoa husk pods (Theobroma cacao L.), from the agricultural industry. The cellulose fiber was obtained through chemical treatments with KOH at 5% w/v to remove non-cellulosic components and then the fiber was bleached with 3% v/v hydrogen peroxide. The changes in chemical structure were determined through Fourier transform infrared spectroscopy (FTIR). The FTIR analysis confirms the progressive decrease of lignin and hemicellulose after applying chemical treatment. The morphological changes in the surface of the fiber were characterized using the SEM technique. The mass percentage of cellulose increases up to 68 %. It is expected that the Nano Crystals (NCC) extracted from the biomass of the cocoa husk pods, present a high index of crystallinity and that they are also in suitable conditions to be useful as reinforcing agents in polymeric or mineral matrices, and may have potential application for technology transfer

Synthesis and Characterization of a Coagulating Agent from Plantain Peel Starch (Musa paradisiaca), as Coadjuvant in Water Treatment

Coagulation processes are widely used for water treatment, mainly with chemical coagulants. In this research, starch derived from a waste (unripe plantain peel, Musa paradisiaca) was used as a starting point for a chemical modifcation. Through acetylation, its chemical structure was modifed and characterized by infrared spectrophotometry, for its evaluation as a coadjuvant in coagulation operations to reduce the turbidity of raw water. Two experimental designs were developed to evaluate the incidence of modifed starch as the main coagulant, or in conjunction with a conventional coagulant (Al2(SO4)3), at diferent (Al2(SO4)3)/acetylated starch ratios, in jar-test experiments. In the frst experimental design, with the acetylated starch as the main coagulant, turbidity removal percentages reached 47.93% (average value, 41.18%). For the (Al2(SO4)3)/acetylated starch coagulation process, 98.91% turbidity removal was reached in the second experimental design (average value, 97.16%). The impact of starch chemical substitution degree and the (Al2(SO4)3)/acetylated starch ratio on the fnal turbidity obtained in the jar-tests was determined using ANOVA test. There was a great infuence of the chemical substitution degree and the concentration of acetylated starch utilized, when modifed starch was used as the main coagulant. For the second experimental design, the (Al2(SO4)3)/acetylated starch ratio had a greater incidence on the turbidity removal. Thus, modifed starch obtained from plantain peel waste is a promising coadjuvant material for water coagulation processes

Sviluppi di Ricevitori e di Componentistica per Banda 3 mm ad INAF-OA Cagliari

L'INAF-OA Cagliari (OACa) sta sviluppando un ricevitore criogenico a basso rumore basato su un mixer SSB (Single Side Band) a superconduttore SIS (Superconductor-Insulator-Superconductor) per la banda 3 mm. Il ricevitore, acquistato da IRAM, è stato fortemente modificato per essere adattato al fuoco Gregoriano di SRT (Sardinia Radio Telescope). Lo strumento è caratterizzato da una nuova criogenia a ciclo chiuso 4 K (per evitare l'uso di elio liquido in antenna), da un nuovo oscillatore locale (di tipo ALMA Banda 3) e da un nuovo sistema di controllo e di monitoraggio basato su schede Raspberry ed Arduino sviluppato ad OACa. Verranno presentati i recenti sviluppi sul ricevitore, inclusi i risultati preliminari della misura della temperatura di rumore, che raggiunge un valore pari a Trec=66 K alla frequenza di 86 GHz, nonostante la criogenia non sia ancora ottimizzata. L'INAF-OACa è coinvolto nel progetto AETHRA (Advanced European Technologies for Heterodyne Receivers for Astronomy) nel quadro del programma Radionet/Horizon2020 per il quale sta contribuendo al WP1 (Work Package 1). Lo scopo del WP1 è di sviluppare e costruire un dimostratore di un array di ricevitori a doppia polarizzazione per la banda 3 mm basato su amplificatori criogenici a basso rumore (LNA) in tecnologia a semiconduttore MMIC. Nell'ambito del WP1 l'OACa ha in carico il progetto di un OrthomodeTransducer (OMT) in guida d'onda o in tecnologia planare per la banda 72-116 GHz che sia integrabile con amplificatori MMICs ed adatto all'integrazione in un array da installare nel piano focale di un radiotelescopio. Verranno presentati i design preliminari degli OMT per AETHRA, che sono basati su prototipi sviluppati in passato da OACa

Effect of shoot parameters on cracking in vegetated soil

The relationship of shoot parameters, which play a major role in transpiration, with the cracking of soil has rarely been investigated. Such relation helps to analyse water use efficiency accurately. This study investigated the effect of vegetation (cowpea) age on crack formation and explored any correlation between age and cracking. The age of vegetation was expressed in the form of shoot parameters (shoot length (SL) and leaf area index (LAI)). Crack formation was expressed in the form of crack intensity factor (CIF). Ten experimental test pots were used to observe crack formation on vegetated and bare soil in a greenhouse. Image analysis in the experimental pots revealed that under drying–wetting cycles, the CIF of vegetated soil increased compared with that of bare soil. There was an evident increase in CIF with SL growth, up to a threshold length (400 mm), where lateral branch growth starts forming. There was no observable increase in CIF, with further SL growth (with negligible lateral branch formation). CIF increased with LAI up to a certain threshold value (0·56), after which the CIF was relatively the same. Two correlations have been identified for shoot parameters (SL, LAI) with the CIF for the selected species

The RNA-binding protein hnRNP K mediates the effect of BDNF on dendritic mRNA metabolism and regulates synaptic NMDA receptors in hippocampal neurons

Brain-derived neurotrophic factor (BDNF) is an important mediator of long-term synaptic potentiation (LTP) in the hippocampus. The local effects of BDNF depend on the activation of translation activity, which requires the delivery of transcripts to the synapse. In this work, we found that neuronal activity regulates the dendritic localization of the RNA-binding protein heterogeneous nuclear ribonucleoprotein K (hnRNP K) in cultured rat hippocampal neurons by stimulating BDNF-Trk signaling. Microarray experiments identified a large number of transcripts that are coimmunoprecipitated with hnRNP K, and about 60% of these transcripts are dissociated from the protein upon stimulation of rat hippocampal neurons with BDNF. In vivo studies also showed a role for TrkB signaling in the dissociation of transcripts from hnRNP K upon high-frequency stimulation (HFS) of medial perforant path-granule cell synapses of male rat dentate gyrus (DG). Furthermore, treatment of rat hippocampal synaptoneurosomes with BDNF decreased the coimmunoprecipitation of hnRNP K with mRNAs coding for glutamate receptor subunits, Ca2+- and calmodulin-dependent protein kinase IIβ (CaMKIIβ) and BDNF. Downregulation of hnRNP K impaired the BDNF-induced enhancement of NMDA receptor (NMDAR)-mediated mEPSC, and similar results were obtained upon inhibition of protein synthesis with cycloheximide. The results demonstrate that BDNF regulates specific populations of hnRNP-associated mRNAs in neuronal dendrites and suggests an important role of hnRNP K in BDNF-dependent forms of synaptic plasticity.publishe

Freeze-Dried Somatic Cells Direct Embryonic Development after Nuclear Transfer

The natural capacity of simple organisms to survive in a dehydrated state has long been exploited by man, with lyophylization the method of choice for the long term storage of bacterial and yeast cells. More recently, attempts have been made to apply this procedure to the long term storage of blood cells. However, despite significant progress, practical application in a clinical setting is still some way off. Conversely, to date there are no reports of attempts to lyophilize nucleated somatic cells for possible downstream applications. Here we demonstrate that lyophilised somatic cells stored for 3 years at room temperature are able to direct embryonic development following injection into enucleated oocytes. These remarkable results demonstrate that alternative systems for the long-term storage of cell lines are now possible, and open unprecedented opportunities in the fields of biomedicine and for conservation strategies