Subsequent Cultivation Research Articles

Efficiency of assisted hatching of the cryopreserved–melted embryos

Purpose . To measure outcomes of clinical research of efficiency of assisted hatching of cryopreserved embryos. Materials and methods. Patients who had un successful cycles IVF/ICSI with transfer of fresh embryos have been selected for participation in the research between 2014 and 2016 years. Patients were distributed in a random way for participation in the experiment and control groups. Results of embryos transfer of one or two cryopreserved and melted embryos were considered only. Embryos were cryopreserved at a stage of blastocyst, 5 days after extraction of oocytes by method of vitrification. Melting procedure was conducted in the morning of a day of embryos transfer following the instructions of the vitrification medium producer Cryotech (Japan). Assisted hatching was conducted with use of micropipettes of Holding Pipette Cook Medical (Australia) and Assisted Hatching/Zona Drilling Pipette Cook Medical (Australia). The treated embryos were cultivated up to a repeated estimation of morphology of embryos before transfer. Transfer of embryos has been conducted by a standard method with the use of catheter for non-invasive transfer of embryo Sydney IVF Cook Medical (Australia). The quantity of the transferred embryos varied from one to two. Results . 100 cryopreserved embryos were transferred which have been distributed in a random way either to the group with the assisted hatching or to the control group (without assisted hatching). A number of parameters of patients from both groups was analyzed, i.e. age of the patient at the time of melting of embryos, duration of infertility, causes of infertility, quantity of previous unsuccessful cycles IVF/ICSI. Any essential differences between patients within two groups based on the aforementioned parameters were not revealed. Also, there were no essential differences in number of the melted embryos, survival rate of embryos, quantity of the embryos transferred to patients. However, at the same time, parameters of positive results of tests on HCG (human chorionic gonadotropin) (42 % against 27 %) and quantity of clinical pregnancy (35 % against 24 %) were statistically higher in the group with assisted hatching comparatively to the control group (P < 0.004; P < 0.001 accordingly). Conclusions . Implantation of the transfered embryos and number of clinical pregnancies were statistically improved due to selection of the optimum freezing conditions and subsequent cultivation and conducting of procedure of mechanical incision of ZP.

Innovative Cryopreservation Process Using a Modified Core/Shell Cell-Printing with a Microfluidic System for Cell-Laden Scaffolds.

This work investigated the printability and applicability of a core/shell cell-printed scaffold for medium-term (for up to 20 days) cryopreservation and subsequent cultivation with acceptable cellular activities including cell viability. We developed an innovative cell-printing process supplemented with a microfluidic channel, a core/shell nozzle, and a low-temperature working stage to obtain a cell-laden 3D porous collagen scaffold for cryopreservation. The 3D porous biomedical scaffold consisted of core/shell struts with a cell-laden collagen-based bioink/dimethyl sulfoxide mixture in the core region and an alginate/poly(ethylene oxide) mixture in the shell region. Following 2 weeks of cryopreservation, the cells (osteoblast-like cells or human adipose stem cells) in the scaffold showed good viability (over 90%), steady growth, and mineralization similar to those of a control scaffold fabricated using a conventional cell-printing process without cryopreservation. We believe that these results are attributable to the optimized fabrication processes the cells underwent, including safe freezing/thawing processes. On the basis of these results, this fabrication process has great potential for obtaining core/shell cell-laden collagen scaffolds for cryopreservation, which have various tissue engineering applications.

Effects of land use change on soil physicochemical properties in selected areas in the North West region of Cameroon

BackgroundLand use changes from natural ecosystems into managed ecosystems may have deleterious effects on soil structure and quality. This study characterise the soils under, and assesses the effects of different land use systems on selected soil physicochemical properties in the North West region of Cameroon. Six land use systems including: natural forest, natural savanna, grazing land, afforested land, farmland and Eucalyptus plantation were identified. Ninety soil samples were collected from each at the 0–15 cm depth. Fifteen soil physicochemical properties were measured.ResultsThe conversion of natural forest or savanna to farmland reduces the silt contents, moisture content, organic matter, organic carbon, total nitrogen, available phosphorus, pH, cation exchange capacity and exchangeable bases, but increases bulk density, electrical conductivity, exchangeable acidity and sand content significantly (P < 0.05). The results revealed that deforestation and subsequent cultivation of soil had negative effects on the measured soil properties.ConclusionsLand use change has ruined soil quality in the North West region. To reverse soil degradation and promote restoration, emphases should be placed on promoting the use of sustainable land management practices within the savanna, grazing, agricultural and forest management systems.

Influence of visceral obesity on the secretion of adipokines with epicardial adipocytes in patients with coronary heart disease.

To study adipokine-cytokine profile of epicardial adipocytes (EAT) and subcutaneous adipose tissue (SAT) in conjunction with the area of visceral adipose tissue (VAT), biochemical and clinical characteristics of patients with coronary heart disease. Examined 84 patients (70 men and 14 women) with coronary artery disease. In fact the presence of visceral obesity (VO) the patients were divided into two groups. Patients VO the sampling of adipocytes of EAT and SAT, with subsequent cultivation and evaluation of adipokine and provospalitelna activity. Carried out the determination of carbohydrate and lipid metabolism, adipokine and pro-inflammatory status in the blood serum. It was found that adipokine-cytokine profile of adipocytes of EAT and SAT differ. Adipocytes art of the disease on the background characterized by an increase IL-1, TNF-α, leptin-adiponectin relationships and a decrease in the content of protective factors: adiponectin and anti-inflammatory cytokine IL-10. While the SAT adipocytes was characterized by a decrease in the concentration of soluble receptor for leptin and the more pronounced leptinresistance, and the increase in proinflammatory cytokines was offset by the increase in the concentration of IL-10. The presence associated with multi-vessel coronary bed lesion, multifocal atherosclerosis, insulin resistance, atherogenic dyslipidemia, an imbalance of adipokines and markers of inflammation. So the value of the square VAT determined higher concentrations of leptin, TNF-α in adipocytes and serum, lipid and carbohydrate metabolism and a lower content of soluble receptor for leptin. Thus, the disease on the background of the status of the adipocytes of EAT characterized as a &quot;metabolic inflammation&quot;, and may indicate the direct involvement of adipocytes in the pathogenesis of coronary artery disease, due to the formation of adipokine imbalance and the activation of proinflammatory reactions.

Design and Production of Rotary Type Machine for Chop Up Organic Waste Plantation

Agricultural organic waste chopping machine are tools that can be used to chop leaves, twigs and could be used to help farmers in processing of the agricultural disposal products such as stalks and corn stalks, cassava stems, rice stalks in order to accelerate farmers in processing of those disposal in order that the plantation land can be prepared immediately for subsequent cultivation, as well as the chopped products can be reused for various purposes such as animal feeds and composts fertilizer. The research of this engineering method is carried out with the following stages: (1) study of the characteristics of several organic wastes to be enumerated, (2) analysis of the Chopping machine elements, consists of Chopping Blades, the house of the chopper blades, hopper, frame and transmission system. (3) A Prototype chop up mechanism manufacturing. And (4) Functional testing machine. The Copping Machine design resume as follows: Production capacity of enumeration 220 kg / hour. The cutting mechanism uses 2 static blades and 6 rotary blades. Transmission system using Belt-Pulley mechanism. The 24 Hp Diesel engine is used.

Effect of light spectrum on isolation of microalgae from urban wastewater and growth characteristics of subsequent cultivation of the isolated species

Light quality plays an important role in regulation of microalgae growth. The aim of this work was to investigate the role of light spectrum in isolation of different strains of microalgae from urban wastewater. Screening and isolation under three light spectrums of white, red, and blue resulted in three different strains of Chlorella sorokiniana IG-W-96, Chlorella vulgaris IG-R-96, and Chlorella sp. IG-B-96, respectively. The strains were identified and named based on morphological characterization under light and confocal microscopy followed by molecular characterization. It was revealed that the light spectrum had significant influence on microalgae cell size. The largest and smallest cells were observed under blue and red light, respectively. The isolated strain under blue light when subsequently cultured under blue light in suspension mode showed the highest biomass density of 1.21±0.00gl−1, as well as lipid density and productivity of 0.29±0.02gl−1 and 40.24±4.02mglipidl−1day−1, respectively. Due to its shorter growth time, however, maximum biomass productivity was obtained for the isolated and cultured strain under red lights. The proposed illumination methodology in isolation and cultivation improved growth characteristics, in such a way that similar light spectra in both steps led to the highest amounts of biomass, lipid, and productivities for each strain.

Photostimulation of sequential degradation and assimilation of recalcitrant carbonaceous organics in Scenedesmus quadricauda

Abstract This study investigated the photostimulation of organic degradation with Scenedesmus quadricauda microalga, which is capable of assimilating organic carbon as a carbon source under mixo– and hetero–trophic growth conditions. The assimilability of carbonaceous organics in the microalga was significantly inhibited for polymeric organic compounds (a mixture of humic substances and polysaccharides) due to their recalcitrant characteristics when compared to using glucose. However, continuous illumination (150 μmol photons m−2 s−1) under non-aerated conditions stimulated the microalga to increase the degradability of the organic mixture. The subsequent algal cultivation in the light using aeration with 10% (v/v) CO2 achieved a significant assimilation of the carbonaceous organics along with nitrate, which was comparable to that observed when using glucose as a carbon source. This is the first time that the photostimulation of S. quadricauda has been shown to induce the degradation of humic-like substances that are typically resistant to microbial decomposition.

Degradation of Mollisols quality after deforestation and cultivation on a transect with Mediterranean condition

In order to assess the impact of deforestation—cultivation on soil quality attributes of Mollisols under native forest, 8 soil pedons and 32 soil cores were described and sampled from four different soil types of forest and adjacent cultivated soil on a transect. Deforestation and subsequent cultivation have resulted in a remarkable change in the morphological and physicochemical soil properties, thereby contributing to extensive land degradation through nutrient depletion and poor physical conditions. Without significant variation, 14–20% higher bulk density and 10–22% lower porosity were observed in the cultivated soils compared to those of the forest lands which have, in turn, contributed to the structural deterioration of the cultivated soil. A pronounced depletion has occurred at the values of organic carbon (60–85%), total N (67–88%), cation exchange capacity (9–18%), and exchangeable cations (4–60%) after deforestation—cultivation showing a degrading and declining effect of deforestation on soil fertility indices. Changes in particle-size distribution and mainly organic matter of soils due to deforestation were responsible for a significant increase in the values of soil erodibility factor or K-factor (a rise of 10–270%). The natural forest promoted remarkably the expansion of smectite (peak of 18.2 A) toward illite (peak of 10 A) in a K incorporation context compared to the cultivated soil based on X-ray patterns. Soil quality index, evaluated based on 14 soil attributes, revealed that deforestation led to a negative change in the index for all soil types ranging from 18 to 43%.

Apoptosis and the processes of DNA fragmentation in native and cryopreserved human sperm cells with normo- and pathosperma

Cryopreserved human sperm is widely applied in the treatment of infertility using methods of assisted reproductive technologies. Comprehensive studies of the sperm condition after cryopreservation and subsequent cultivation will update the efficiency of the programs based on assisted reproductive technologies. The aim of this study was to investigate the effect of cryopreservation factors on the state of DNA and apoptotic processes in isolated human spermatozoa fractions in normo- and pathospermia. The analysis of DNA fragmentation and the assessment of apoptotic processes (human spermatozoa necrosis) were performed using the sperm chromatin dispersion test and the flow cytometry method. According to our study, sperm cryopreservation has not affected the DNA fragmentation index in normozoospermia and significantly increased it in oligoastenoteratozoospermia. The postcultivation increase of apoptotic and necrotic processes in oligoastenoteratozoospermia gives reason for choosing an alternative technique for oocyte fertilization— intracytoplasmic sperm injection.

Case-control study: Determination of potential risk factors for the colonization of healthy volunteers with Streptococcus gallolyticus subsp. gallolyticus.

Streptococcus gallolyticus subsp. gallolyticus was identified in humans and animals as commensal of the gut and can act as a causative agent of endocarditis and septicemia. A case-control study was performed to identify yet unknown risk factors for the transmission of this facultative pathogen. The prevalence in the gut of 99 healthy volunteers was determined using real-time polymerase chain reaction resulting in 62.5% S. gallolyticus subsp. gallolyticus positive excrements. Subsequent cultivation offered three isolates and epidemiological analysis based on MLST revealed sequence type (ST) 3 and ST 7, previously detected from bovine and endocarditis patients. These results support the hypotheses of the zoonotic potential of this bacterium. Participant questionnaires were evaluated concerning personal characteristics, nutritional habits and animal contact. Specifically, closer contact between participants and animals influenced the colonization of the human gut significantly and was further affected if volunteers used excrement for the fertilization of plants.

Forest clearance and land use by early farmers in Europe: Insights from north Greek oral history

European prehistorians have long debated whether Neolithic farmers, especially in temperate central and northern Europe, exploited cleared woodland for short-term ‘slash-and-burn’ crop husbandry or for cultivation of ‘permanent’ gardens/fields. Because empirical evidence was scarce and rather indirect, circumstantial arguments based on the relative costs and benefits of the two rival regimes have hitherto dominated this debate, but more direct archaeobotanical proxies for Neolithic crop husbandry (based on weed ecology and crop isotopes) now favour the predominance of intensively cultivated permanent plots. At the same time, it is argued from ongoing experimental cultivation at Forchtenberg in southern Germany that short-term slash-and-burn husbandry offers much better returns on both land and labour, at least on poor soils marginal for Neolithic cereals. This paper presents oral historical evidence for mid-twentieth century clearance and subsequent long-term cultivation of mixed deciduous woodland in the Pieria region of northern Greece. These data extend the sparse European ethnographic and historical record of manual clearance for crop husbandry to deciduous woodland of the northern Mediterranean, while also providing comparative material against which to evaluate experimental results. Pierian farmers achieved good cereal yields for many years on cleared woodland, with very limited use of fire, demonstrating the viability of a permanent-cultivation regime for early farmers in the north Mediterranean, but suitable land was scarce in Pieria and informants had no experience of the alternative slash-and-burn regime. Conversely, the Forchtenberg experiments have effectively tested only the slash-and-burn regime under soil pH conditions hostile to successful long-term cultivation. Evaluation of these experiments in the light of the Pierian data suggests that long-term cultivation of cleared woodland would have offered Neolithic famers better returns than slash and burn under good or neutral soil conditions. Comparison of the likely returns of the two rival regimes thus offers no grounds for questioning archaeobotanical indications of Neolithic permanent cultivation. The Pierian and Forchtenberg data agree, however, in suggesting that the time elapsed between clearance and reliable grain harvests, under either regime, may have been long enough to pose a significant challenge to the survival of pioneer Neolithic farmers, especially if these were colonists settling beyond ‘commuting’ distance from their previous homes.

Maximum Plant Uptakes for Water, Nutrients, and Oxygen Are Not Always Met by Irrigation Rate and Distribution in Water-based Cultivation Systems.

Growing on rooting media other than soils in situ -i.e., substrate-based growing- allows for higher yields than soil-based growing as transport rates of water, nutrients, and oxygen in substrate surpass those in soil. Possibly water-based growing allows for even higher yields as transport rates of water and nutrients in water surpass those in substrate, even though the transport of oxygen may be more complex. Transport rates can only limit growth when they are below a rate corresponding to maximum plant uptake. Our first objective was to compare Chrysanthemum growth performance for three water-based growing systems with different irrigation. We compared; multi-point irrigation into a pond (DeepFlow); one-point irrigation resulting in a thin film of running water (NutrientFlow) and multi-point irrigation as droplets through air (Aeroponic). Second objective was to compare press pots as propagation medium with nutrient solution as propagation medium. The comparison included DeepFlow water-rooted cuttings with either the stem 1 cm into the nutrient solution or with the stem 1 cm above the nutrient solution. Measurements included fresh weight, dry weight, length, water supply, nutrient supply, and oxygen levels. To account for differences in radiation sum received, crop performance was evaluated with Radiation Use Efficiency (RUE) expressed as dry weight over sum of Photosynthetically Active Radiation. The reference, DeepFlow with substrate-based propagation, showed the highest RUE, even while the oxygen supply provided by irrigation was potentially growth limiting. DeepFlow with water-based propagation showed 15–17% lower RUEs than the reference. NutrientFlow showed 8% lower RUE than the reference, in combination with potentially limiting irrigation supply of nutrients and oxygen. Aeroponic showed RUE levels similar to the reference and Aeroponic had non-limiting irrigation supply of water, nutrients, and oxygen. Water-based propagation affected the subsequent cultivation in the DeepFlow negatively compared to substrate-based propagation. Water-based propagation resulted in frequent transient discolorations after transplanting in all cultivation systems, indicating a factor, other than irrigation supply of water, nutrients, and oxygen, influencing plant uptake. Plant uptake rates for water, nutrients, and oxygen are offered as a more fundamental way to compare and improve growing systems.

Propagation of Brazilian Zika virus strains in static and suspension cultures using Vero and BHK cells

The recent spread of Zika virus (ZIKV) in the Americas and the Pacific has reached alarming levels in more than 60 countries. However, relatively little is known about the disease on a virological and epidemiological level and its consequences for humans. Accordingly, a large demand for in vitro derived Brazilian ZIKV material to support in vitro and in vivo studies has arisen. However, a prompt supply of ZIKV and ZIKV antigens cannot be guaranteed as the production of this virus typically using Vero or C6/36 cell lines remains challenging.Here we present a production platform based on BHK-21 suspension (BHK-21SUS) cells to propagate Brazilian ZIKV at larger quantities in perfusion bioreactors. Scouting experiments performed in tissue culture flasks using adherent BHK-21 and Vero cells have demonstrated similar permissivity and virus yields for four different Brazilian ZIKV isolates. The cell-specific yield of infectious virus particles varied between respective virus strains (1–48PFU/cell), and the ZIKV isolate from the Brazilian state Pernambuco (ZIKVPE) showed to be a best performing isolate for both cell lines. However, infection studies of BHK-21SUS cells with ZIKVPE in shake flasks resulted in poor virus replication, with a maximum titer of 8.9×103PFU/mL. Additional RT-qPCR measurements of intracellular and extracellular viral RNA levels revealed high viral copy numbers within the cell, but poor virus release. Subsequent cultivation in a perfusion bioreactor using an alternating tangential flow filtration system (ATF) under controlled process conditions enabled cell concentrations of about 1.2×107cells/mL, and virus titers of 3.9×107PFU/mL. However, while the total number of infectious virus particles was increased, the cell-specific yield (3.3PFU/cell) remained lower than determined in adherent cell lines. Nevertheless, the established perfusion process allows to provide large amounts of ZIKV material for research and is a first step towards process development for manufacturing inactivated or live-attenuated ZIKV vaccines.

Benzene Degradation by a Variovorax Species within a Coal Tar-Contaminated Groundwater Microbial Community.

Benzene is a human carcinogen whose presence in contaminated groundwater drives environmental cleanup efforts. Although the aerobic biodegradation of benzene has long been established, knowledge of the identity of the microorganisms in complex naturally occurring microbial communities responsible for benzene biodegradation has evaded scientific inquiry for many decades. Here, we applied a molecular biology technique known as stable isotope probing (SIP) to the microbial communities residing in contaminated groundwater samples to identify the community members active in benzene biodegradation. We complemented this approach by isolating and growing in the laboratory a bacterium representative of the bacteria found using SIP. Further characterization of the isolated bacterium enabled us to track the expression of a key gene that attacks benzene both in pure cultures of the bacterium and in the naturally occurring groundwater microbial community. This work advances information regarding the documentation of microbial processes, especially the populations and genes that contribute to bioremediation.

English

The study examined the impact of deforestation and subsequent cultivation on soil fertility and acidity conditions under varying soil depths. Soil profiles were opened in two adjacent land units, namely forestland and arable land and samples were collected from genetic horizons. Deterioration of soil fertility was observed after deforestation and traditional cultivation. The main aim of deforestation was agricultural expansion. Soil pH consistently decreased with depth in both land units and it was relatively lowest in arable land perhaps due to depletion of organic matter (OM) and decrease in buffering capacity of the soil. The OM and total nitrogen (N) ranged from 0.78 and 0.06% in the 75 to160 cm layer of arable land to 15 and 0.61% in the 0 to10 cm layer of forestland, respectively. Total N was strongly and positively correlated with soil OM (r = 0.99). Exchangeable Al was poorly and negatively correlated with available phosphorous (r = -0.41). Conversion of forestland to arable land reduced the mean available phosphorus (P) from 4.04 ppm to 1.95 ppm most probably due to decline in OM, soil acidification and erosion. Deforestation and subsequent continuous cultivation over the past 25 years apparently amplified the mean exchangeable acids from 0.83 cmol (+) kg-1 to 5.96 cmol (+) kg-1. Soil acidification and related problems were the major challenges of continuous cultivation in the study area. The study indicated that land use change and management practices have had a considerable negative effect on soil physical and chemical properties. Key words: Deforestation, continuous cultivation, land use change, soil properties, soil fertility, soil acidity, Western Ethiopia.

Aerobic denitrification by a heterotrophic nitrifying-aerobic denitrifying (HN-AD) culture enriched activated sludge

Heterotrophic nitrifying bacteria capable of denitrification under aerobic conditions, which are known as HN-AD species, were investigated. Instead of the isolation and subsequent cultivation of a pure HN-AD strain, the activated sludge obtained from the domestic wastewater treatment plant was enriched to enhance the fraction and activity of the indigenous HN-AD species by adding ammonium and organic carbon. The enriched culture showed both rapid ammonium removal at a rate of 13.3 mg-N/L/hr and nitrate removal at a rate of 12.3 mg-N/L/hr, which was higher than those for the pure HN-AD bacteria reported in the literature. On the contrary, the activated sludge that was not enriched showed insignificant nitrification and aerobic denitrification. These results implied that the selective enrichment procedure improved the fraction and/or activity of the HN-AD culture in the mixed biomass. Consequently, the enriched HN-AD culture from activated sludge could be a feasible alternative over the conventional BNR process.

Proper period and temperature for transportation of cucumber and tomato transplants

With an increased demand for high-quality transplants, vegetable growers have begun to purchase grafted transplants from specialized propagators. These are often shipped over long distances, but improper transport conditions can deteriorate transplant quality. To determine the appropriate period and temperature for transporting grafted transplants, we stored cucumber and tomato transplants in darkness at 6, 9, 12, 15, and 18 o C for 3, 6, 9, 12, and 15 days, and subsequently cultivated these in a greenhouse for 15 days. We investigated the changes in plant height and SPAD value during storage, as well as the early growth of the stored transplants during subsequent cultivation. Increasing temperature during storage promoted stem elongation in both transplant species, with cucumber transplants showing greater stem elongation than tomato transplants. SPAD values of cucumber and tomato transplants tended to decrease with increasing storage temperature, declining even further with extended storage period. The cucumber transplants stored for 3 days and the tomato transplants stored for 6 days, showed no decrease in percent survival and no delay of growth after transplanting compared with non-stored transplants. From these results, we suggest that the appropriate temperature for transporting cucumber and tomato transplants in darkness is 6°C, and the appropriate period of storage in darkness are 3 and 6 days for cucumber and tomato transplants, respectively. Controlling transportation conditions can avoid the deterioration of transplant quality during transport and the delay of early growth after transplanting.

The boosted lipid accumulation in microalga Chlorella vulgaris by a heterotrophy and nutrition-limitation transition cultivation regime.

A model of heterotrophy and nutrition-limitation transition cultivation for efficient algal biomass and lipid production was proposed in this study, wherein sufficient robust heterotrophic-seed cells submitted into nitrogen-starvation induction for boosted lipid accumulation. The results demonstrated that heterotrophic-seed (HS) achieved specific growth rate of 1.35day-1 and biomass productivity of 1.93mg/L/d, representing 6.42- and 32.16-fold, 2.01- and 2.75-fold more than that of photoautotrophic-seed (PS) and mixtrophic-seed (MS). Even though subsequent nutrition-limitation cultivation repressed the growth of HS, the overall lipid productivity caused by nitrogen-starvation was not offset by biomass loss. The most favorable lipid productivity (465.61mg/L/d) of HS was 3.25 and 52.31 times higher than that of MS and PS. The high content of monounsaturated fatty acids (50.13%) over saturated and polyunsaturated fatty acids (totally 47.39%) in HS cells could provide superior oxidation stability and lower viscosity for biofuels generated from algal biomass feedstock. These findings suggested the feasibility of using heterotrophy and nutrition-limitation transition cultivation for enhancing the overall lipid productivity. Further, several critical enzymes (i.e. G3PDH, ME, and ACAD) were highly related to lipid accumulation and showed especially pronounced up-regulation or down-regulation expression in HS, which provide indications for shedding light on the molecular mechanisms of lipid accumulation and a prospective metabolic engineering for lipid production.

A Catalog of Regulatory Sequences for Trait Gene for the Genome Editing of Wheat.

Wheat has been cultivated for 10000 years and ever since the origin of hexaploid wheat it has been exempt from natural selection. Instead, it was under the constant selective pressure of human agriculture from harvest to sowing during every year, producing a vast array of varieties. Wheat has been adopted globally, accumulating variation for genes involved in yield traits, environmental adaptation and resistance. However, one small but important part of the wheat genome has hardly changed: the regulatory regions of both the x- and y-type high molecular weight glutenin subunit (HMW-GS) genes, which are alone responsible for approximately 12% of the grain protein content. The phylogeny of the HMW-GS regulatory regions of the Triticeae demonstrates that a genetic bottleneck may have led to its decreased diversity during domestication and the subsequent cultivation. It has also highlighted the fact that the wild relatives of wheat may offer an unexploited genetic resource for the regulatory region of these genes. Significant research efforts have been made in the public sector and by international agencies, using wild crosses to exploit the available genetic variation, and as a result synthetic hexaploids are now being utilized by a number of breeding companies. However, a newly emerging tool of genome editing provides significantly improved efficiency in exploiting the natural variation in HMW-GS genes and incorporating this into elite cultivars and breeding lines. Recent advancement in the understanding of the regulation of these genes underlines the needs for an overview of the regulatory elements for genome editing purposes.

Trehalose, an easy, safe and efficient cryoprotectant for the parasitic protozoan Trypanosoma brucei

Trehalose, a non-permeating cryoprotective agent (CPA), has been documented as less toxic and highly efficient at cryopreserving different kinds of cells or organisms. In the present study, trehalose was evaluated for its application in cryopreservation of both Trypanosoma brucei procyclic and bloodstream form cells. The cryopreservation efficiency was determined by the motility of trypanosomes after thawing, as well as a subsequent recovery and infectivity assessment. The viability of trypanosomes from cultivation that were frozen in a serial concentrations of trehalose showed similar results to classical CPAs of glycerol and DMSO. Nevertheless, trypanosomes cryopreserved in 0.2M trehalose showed the best growth characteristic during subsequent cultivation. In addition, CPA cocktails with trehalose and permeating CPA glycerol or DMSO were developed and evaluated. Interestingly, trypanosomes in host (mouse) blood cryopreserved in 0.4M trehalose plus 5% glycerol showed higher infectivity than those preserved in trehalose/DMSO cocktails as well as individually. Further investigations showed that, in comparison with slow freezing at −80°C, flash freezing in liquid nitrogen provided better cryopreservation for bloodstream form cells than slow freezing. In conclusion, trehalose is an easy, safe and efficient CPA for cryopreservation of T. brucei and potentially for other protozoan species and cells.