Standard Cultivation Research Articles

Isolation and Diagnosis of Methicillin-Resistant Staphylococcus aureus in Meat and Butcher Tool Surfaces in Baghdad

Due to the medical and epidemiological importance of the spread of Methicillin-Resistant Staphylococcus aureus, this study was conducted for the purpose of isolating and diagnosing these bacteria from local sheep meat and butcher's tools in Baghdad. Methods. 200 samples were collected. Mannitol salt agar and Staph.110 medium were used to isolate the bacteria. The isolates were identified using standard cultivation methods, biochemical tests, the GP24 diagnostic system, and an integrated Vitek 2 device. The isolates were tested for sensitivity to methicillin by the disk diff usion method. DNA was extracted and the mecA gene was detected in the isolates that showed methicillin resistance by polymerase chain reaction. Results. S. aureus was diagnosed in 83 (41.5%) of the samples. Of them, 35 (42.2%) were methicillin-resistant. Out of these, 24 (68.6%) were found to have the mecA gene. Conclusions. Methicillin-Resistant S. aureus strains were detected with a high prevalence due to the underdeveloped reality of slaughter places.

Rapid detection of φX-174virus based on synchronous fluorescence of tryptophan.

The development of rapid methods for the detection of virus particles based on their intrinsic fluorescence (the native auto-fluorescence that originates from the non-labeled analyte) is challenging. Pure viruses may be detected in filtered solutions, based on the strong fluorescence of the amino acid tryptophan (Trp) in their proteins. Nevertheless, Trp also exists in high quantities in the hosts and host cultivation media. In this work, we developed a new method for the detection of the naked φX-174virus. We show that a separation of φX-174from its Escherichia coli host (grown on the standard cultivation medium nutrient agar) by simple extraction and filtration is not sufficient for its detection based on the intrinsic fluorescence since ~ 70% of the Trp fluorescence is derived from impurities. We formulate a new cultivation medium with a very low Trp concentration. We apply synchronous fluorescence measurements to show that no Trp fluorescence is detected in the extract solution upon incubation of this medium substrate with ammonium acetate extraction buffer. Finally, we apply synchronous fluorescence to detect φX-174based on the spectral fingerprint of its native Trp content. Such a method is more rapid than usual traditional separation and detection methods which can take several hours and does not require any addition of labeling agents such as fluorescent dyes or antibodies for the detection. As other virus species contain Trp as one of the amino acids presents in their proteins, this method has the potential to apply to the detection of other viral species.

Influence of soil cultivation method and fertilization system on the yield of sunflower (Helianthus L.) when growing in conditions of Central Polissia of Ukraine

The paper presents three-year field and laboratory research on studying the sunflower hybrids of different cultivation technologies, types of ripeness groups with yield indices depending on using different fertilisers and different types of soil cultivation. The study was conducted when using new hybrids of domestic and foreign selection. The hybrids Grand Admiral and Pegas were selected according to their biological characteristics that are more typical for the zone of Polissia when using standard cultivation technology. It was noted that on sod-podzolic soils with a light mechanical composition, the use of disk tillage tools (BDVP-4.2) to a depth of 10–12 cm was not significantly inferior to the deep loosener (GR-4.5) to a depth of 40–45 cm, which was a shortfall grain yield from disking in both experiments was 0.08 t/ha (37.7%) and 0.21 t/ha (23%). The above-mentioned type of soil is naturally poor in terms of its physical and chemical composition, namely the content of humus (1.0–1.3%), availability of exchangeable potassium and mobile phosphorus (32–52 mg/kg), which creates the need to use scientific approach to the reproduction of soil fertility with the use of organic and mineral fertilizers. It has been proven that when using fertilizers N16P16K16 + N46 (nitroammophosca, 200 kg + urea fertilizer, 75 kg) an increase in sunflower seed yield equals 1.66–1.72 t/hа (281–322%), when using the fertilizers N10P26K26 + N46 (diammophos, 200 kg + urea fertilizer, 75 kg) the increase in yield equals — (287–328%) and when using P5K55 + N46 (phosphorus-potassium fertilizer, 20 kg + urea fertilizer, 75 kg) the increase in yield is the highest and equals 1.85–1.97 t/ha depending on the experiment variant. It is noted that the Pegasus sunflower hybrid was inferior to the Grand Admiral hybrid in all indicators of the experiment, from the form of mineral fertilizers to soil cultivation, and is: when applying N16P16K16 + N46 — 0.66 t/ha; N10P26K26 + N46 — 0.39 t/ha; P5K55 + N46 — 0.38 t/ha.

Increasing soybean productivity by plant equidistant arrangements and modification of technology levels of cropping systems

The arrangement of soybean plants defines their ability to recognize the environment and improve their abiotic and biotic interactions with it. This study aimed to evaluate the effect of planting arrangements associated to two distinct crop systems (high technological level or conventional crop level) for the soybean varieties 8473 RSF and AS 3730, to evaluate the plant performance and productivity in the field. A double factorial scheme was used with two varieties and six spatial arrangements. Two experiments were conducted during the 2017–2018 crop season: the first was based on standard cultivation of the Brazilian Cerrado (without irrigation systems and adequate fertilizer), whereas the second utilized high technology (irrigation systems and increased fertilizer). The morphophysiological parameters, normalized difference vegetation index (NDVI), leaf pigment content and crop yield parameters were evaluated. Our study pointed out the positive responses for both cultivated varieties and two technological levels adopted cultivated under equidistant arrangement and high density. The leaf area index, leaf pigment content, NDVI, and crop production showed substantial responses under different equidistant arrangements. Therefore, this procedure requires adjustments in the level of cultivation technology and identification of the most suitable soybean variety.

SELECTION OF APPROPRIATE NUTRIENT MEDIUM FOR CULTIVATION OF PYRENOPHORA TERES DRECHSLER

Net blotch (Pyrenophora teres Drechsler) is the dominant pathogenic complex of barley leaf diseases worldwide. The study of various methods of controlling hemibiotrophic fungus requires careful preparation and development of a sufficient amount of pathogenic inoculum. The aim of our research was to select appropriate nutrient medium for obtaining P. teres inoculum with maximum aggressiveness parameters (colony growth rate, sporulation, short latency period) and ergonomics in preparation. Three nutrient media were used: carrot-beet agar (CBA), potato-glucose agar (PGA), Czapek's medium for standard methods and cultivation conditions. The selection consisted of 10 isolates for each nutrient medium and had statistically significant differences among the experimental variants. It was found that appropriate combination of parametres such as growth rate (5.3±0.5 mm/day), colony diameter (53.4±1.1 mm), latent period (4 days), sporulation intensity (4100 conidia in 1 ml) was observed in case of cultivation of P. teres isolates on carrot-beet agar on the 6th day. When cultivating the fungus on potato-glucose agar, rather high growth rates (5.2±0.8 mm/day), colony diameter (52.0±2.9 mm), sporulation intensity (3700 conidia per 1 ml) were revealed, the latent period was the longest among the studied variants, the first sporulation was detected on the 6th day. In case of usage of the standard Czapek medium, the minimum aggressiveness of P. teres isolates was revealed (growth rate - 3.2 ± 0.3 mm/day; colony diameter - 31.6 ± 1.9 mm; latent period - spores were revealed on the 4th day in 1 cup; the intensity of sporulation was 500 conidia per 1 ml). It was noted that there is a larger range of morphological types of colonies on carrot-beet agar, including morphotypes of isolates which grow on other media. Almost all experimental variants cultivated on CBA medium had a clear structured mycelium with a dark color, which indicates a sufficient availability of nutrients in the culture medium. Thus, it is reasonable to use carrot-beet agar in order to obtain P. teres inoculum with maximum aggressiveness parameters and low labor costs when using standard cultivation methods.

MediaDive: the expert-curated cultivation media database.

We present MediaDive (https://mediadive.dsmz.de), a comprehensive and expert-curated cultivation media database, which comprises recipes, instructions and molecular compositions of >3200 standardized cultivation media for >40 000 microbial strains from all domains of life. MediaDive is designed to enable broad range applications from every-day-use in research and diagnostic laboratories to knowledge-driven support of new media design and artificial intelligence-driven data mining. It offers a number of intuitive search functions and comparison tools, for example to identify media for related taxonomic groups and to integrate strain-specific modifications. Besides classical PDF archiving and printing, the state-of-the-art website allows paperless use of media recipes on mobile devices for convenient wet-lab use. In addition, data can be retrieved using a RESTful web service for large-scale data analyses. An internal editor interface ensures continuous extension and curation of media by cultivation experts from the Leibniz Institute DSMZ, which is interlinked with the growing microbial collections at DSMZ. External user engagement is covered by a dedicated media builder tool. The standardized and programmatically accessible data will foster new approaches for the design of cultivation media to target the vast majority of uncultured microorganisms.

The effects of temperature and donor piglet age on the transcriptomic profile and energy metabolism of myoblasts.

Rapid climate change is associated with frequent extreme heat events and the resulting thermal stress has consequences for the health, welfare, and growth of farm animals. The aim of this study was to characterize the transcriptional changes and the effects on energy metabolism in proliferating porcine myoblasts derived from piglets of different ages, representing differences in thermoregulatory abilities, and cultivated below (35°C) and above (39°C, 41°C) the standard cultivation temperature (37°C). Satellite cells originating from Musculus rhomboideus of piglets isolated on days 5 (P5, thermolabile) and 20 (P20, thermostable) of age were used. Our expression analyses highlighted differentially expressed genes in porcine myoblasts cultures under heat or cold induced stress. These gene sets showed enrichment for biological processes and pathways related to organelle fission, cell cycle, chromosome organization, and DNA replication. Culture at 35°C resulted in increased metabolic flux as well as a greater abundance of transcripts of the cold shock protein-encoding gene RBM3 and those of genes related to biological processes and signaling pathways, especially those involving the immune system (cytokine–cytokine receptor interaction, TNF and IL-17 signaling pathways). For cultivation at 39°C, differences in the expression of genes related to DNA replication and cell growth were identified. The highest glutathione index ratio was also found under 39°C. Meanwhile, cultivation at 41°C induced a heat stress response, including the upregulation of HSP70 expression and the downregulation of many biological processes and signaling pathways related to proliferative ability. Our analysis also identified differentially expressed genes between cells of donors with a not yet (P5) and already fully developed (P20) capacity for thermoregulation at different cultivation temperatures. When comparing P5 and P20, most of the changes in gene expression were detected at 37°C. At this optimal temperature, muscle cells can develop to their full capacity. Therefore, the most diverse molecular signaling pathways, including PI3K-Akt signaling, Wnt signaling, and EGFR tyrosine kinase inhibitor, were found and are more pronounced in muscle cells from 20-day-old piglets. These results contribute to a better understanding of the mechanisms underlying the adaptation of skeletal muscle cells to temperature stress in terms of their thermoregulatory ability.

A droplet-based microfluidic platform enables high-throughput combinatorial optimization of cyanobacterial cultivation

Cyanobacteria are fast-growing, genetically accessible, photoautotrophs. Therefore, they have attracted interest as sustainable production platforms. However, the lack of techniques to systematically optimize cultivation parameters in a high-throughput manner is holding back progress towards industrialization. To overcome this bottleneck, here we introduce a droplet-based microfluidic platform capable of one- (1D) and two-dimension (2D) screening of key parameters in cyanobacterial cultivation. We successfully grew three different unicellular, biotechnologically relevant, cyanobacteria: Synechocystis sp. PCC 6803, Synechococcus elongatus UTEX 2973 and Synechococcus sp. UTEX 3154. This was followed by a highly-resolved 1D screening of nitrate, phosphate, carbonate, and salt concentrations. The 1D screening results suggested that nitrate and/or phosphate may be limiting nutrients in standard cultivation media. Finally, we use 2D screening to determine the optimal N:P ratio of BG-11. Application of the improved medium composition in a high-density cultivation setup led to an increase in biomass yield of up to 15.7%. This study demonstrates that droplet-based microfluidics can decrease the volume required for cyanobacterial cultivation and screening up to a thousand times while significantly increasing the multiplexing capacity. Going forward, microfluidics have the potential to play a significant role in the industrial exploitation of cyanobacteria.

ПЕРСПЕКТИВНАЯ МЯГКОСЕМЯННАЯ СТОЛОВАЯ ФОРМА ВИНОГРАДА

The paper is devoted to varietal study and evaluation of grapevine hybrid form 4-6-15, selected for the elite as promising in terms: of high yield, good taste and commercial qualities, and increased disease resistance. The soft-seeded elite seedling of grapevine was obtained by crossing varieties Talisman and Kishmish Luchisty in All-Russian Re-search Ya.I. Potapenko Institute for Viticul-ture and Winemaking – Branch of Federal State Budget Scientific Institution «FRARC». An ampelographic description of the seedling was compiled for leading fea-tures. The vineyards are non-irrigated, graft-ed, covered, fan-shaped, planting scheme 3×1.5 m. In the variety study, methods gen-erally accepted in viticulture and standard vineyard cultivation technology were used. The hybrid form 4-6-15 is characterized by a very early ripening period (106 days from bud break to full ripeness of berries), large berries for the seedless variety (5.3 g), seed-less class IV (weight of rudiments 16,7 mg), very large loose clusters (average weight 705 g), attractive color of berries, increased re-sistance to fungal diseases (mildew 3.0‒3.5 points, oidium 3.5 points, botrytis 2.0‒2.5 points). Frost resistance is average, covering for winter is recommended. The yield is high, 11.5 t/ha, gives a second crop on lateral shoots. We believe that, based on the combination of economically valuable traits, the seedling 4-6-15 can be recom-mended for further use in grapevine breed-ing for seedlessness, the quality of berries in combination with high yields, as well as for backyards viticulture.

Listeria monocytogenes genes supporting growth under standard laboratory cultivation conditions and during macrophage infection.

The Gram-positive bacterium Listeria monocytogenes occurs widespread in the environment and infects humans when ingested along with contaminated food. Such infections are particularly dangerous for risk group patients, for whom they represent a life-threatening disease. To invent novel strategies to control contamination and disease, it is important to identify those cellular processes that maintain pathogen growth inside and outside the host. Here, we have applied transposon insertion sequencing (Tn-Seq) to L. monocytogenes for the identification of such processes on a genome-wide scale. Our approach identified 394 open reading frames that are required for growth under standard laboratory conditions and 42 further genes, which become necessary during intracellular growth in macrophages. Most of these genes encode components of the translation machinery and act in chromosome-related processes, cell division, and biosynthesis of the cellular envelope. Several cofactor biosynthesis pathways and 29 genes with unknown functions are also required for growth, suggesting novel options for the development of antilisterial drugs. Among the genes specifically required during intracellular growth are known virulence factors, genes compensating intracellular auxotrophies, and several cell division genes. Our experiments also highlight the importance of PASTA kinase signaling for general viability and of glycine metabolism and chromosome segregation for efficient intracellular growth of L. monocytogenes.

Cultivation of previously uncultured sponge-associated bacteria using advanced cultivation techniques: A perspective on possible key mechanisms

Most of the microbes from natural habitats cannot be cultivated with standard cultivation in laboratory, and sponge-associated microbes are no exception. We used two advanced methods based on a continuous-flow bioreactor (CF) and in situ cultivation (I-tip) to isolate previously uncultivated marine sponge-associated bacteria. We also characterized the physiological properties of the isolates from each method and attempted to clarify the mechanisms operating in each cultivation method. A greater number of novel bacteria were isolated using CF and in situ cultivation compared to standard direct plating (SDP) cultivation. Most isolates from CF cultivation were poor growers (with lower specific growth rates and saturated cell densities than those of isolates from SDP cultivation), demonstrating that it is effective to carry out pre-enrichment cultivation targeting bacteria that are less competitive on conventional cultivation, especially K-strategists and bacterial types inhibited by their own growth. Isolates from in situ cultivation showed a positive influence on cell recovery stimulated by chemical compounds in the extract of sponge tissue, indicating that some of the bacteria require a “growth initiation factor” that is present in the natural environment. Each advanced cultivation method has its own distinct key mechanisms allowing cultivation of physiologically and phylogenetically different fastidious bacteria for cultivation compared with conventional methods.

Effects of Plant Growth Regulators on Plum (Prunus domestica L.) Grown on Two Rootstocks at Harvest and at the Postharvest Period

Plant growth regulators (PGRs), such as cytokinins (6-benzyladenine; BA) and gibberellins (GAs), are widely used in fruit production. This study focused on the plum cultivar “Čačanska rodna” (P. domestica L.) grafted on vegetative rootstock “WaVit” and generative rootstock Prunus cerasifera, with interstock Prunus spinosa. PGR treatments included 50 mg L−1 and 100 mg L−1 of BA and 200 mg L−1 of gibberellin A3 (GA3) and non-treated control. Pomological characteristics of plum fruits were examined at harvest, while physicochemical properties were analyzed at harvest and after 28 days of cold storage and 4 days of shelf life. GA3 and BA application changed the fruit morphological traits, improved skin strength, and increased carotenoid, anthocyanin and sucrose content while decreasing the titratable acidity at harvest. The beneficial effects of higher sucrose, anthocyanin and carotenoid levels persisted in all PGR-treated fruits after cold storage and shelf life. GA3-treated fruits had firmer flesh, stronger skin and higher total soluble solids (TSS) content, while in BA-treated plums, these effects were rootstock-dependent. The physical properties and chemical composition of plum fruit in the postharvest period suggest beneficial effects of the applied PGR treatments. Moreover, these chemical treatments might have prolonged the beneficial impact on fruit storability, nutritional profile and sensory properties. Based on our results, GA3 preharvest treatment can be included in standard cultivation practices within contemporary production systems of European plums not only to improve fruit quality at harvest but also to improve the storage potential and nutritional value, regardless of the rootstock used.

The Penicillium brasilianum Histone Deacetylase Clr3 Regulates Secondary Metabolite Production and Tolerance to Oxidative Stress.

Most of the biosynthetic gene clusters (BGCs) found in microbes are silent under standard laboratory cultivation conditions due to the lack of expression triggering stimuli, representing a considerable drawback in drug discovery. To access the full biosynthetic potential, studies towards the activation of cryptic BGCs are essential. Histone acetylation status is an important regulator of chromatin structure, which impacts cell physiology and the expression of BGCs. In this study, clr3, a gene encoding a histone deacetylase in Penicillium brasilianum LaBioMMi 136, is deleted and associated phenotypic and metabolic changes are evaluated. The results indicate reduced growth under oxidative stress conditions in the ∆clr3 strain, higher intracellular reactive oxygen species (ROS) levels, and a different transcriptional profile of 13 ROS-related genes of both strains under basal and ROS-induced conditions. Moreover, the production of 14 secondary metabolites, including austin-related meroterpenoids, brasiliamides, verruculogen, penicillic acid, and cyclodepsipeptides was evaluated in the ∆clr3 strain, most of them being reduced. Accordingly, the addition of epigenetic modulators responsible for HDAC inhibition into P. brasilianum’s growth media also culminated in the reduction in secondary metabolite production. The results suggest that Clr3 plays an essential role in secondary metabolite biosynthesis in P. brasilianum, thus offering new strategies for the regulation of natural product synthesis by assessing chromatin modification.

Improving Microalgal Biomass Productivity Using Weather-Forecast-Informed Operations.

The operation of microalgal cultivation systems, such as culture dilution associated with harvests, affects biomass productivity. However, the constantly changing incident light and ambient temperature in the outdoor environment make it difficult to determine the operational parameters that result in optimal biomass growth. To address this problem, we present a pond operation optimization tool that predicts biomass growth based on future weather conditions to identify the optimal dilution rate that maximizes biomass productivity. The concept was tested by comparing the biomass productivities of three dilution scenarios: standard batch cultivation (no dilution), fixed-rate dilution (harvest 60% of the culture every three days), and weather-forecast-informed dilution. In the weather-forecast-informed case, the culture was diluted daily, and the dilution ratio was optimized by the operation optimization tool according to the future 24 h weather condition. The results show that the weather-forecast-informed dilution improved the biomass productivity by 47% over the standard batch cultivation and 20% over the fixed-rate dilution case. These results demonstrate that the pond operation optimization tool could help pond operators to make decisions that maximize biomass growth in the field under ever-changing weather conditions.

Environmental impact assessment of discharge of treated wastewater effluent in Upper Iskar sub-catchment

The upper Iskar sub-catchment is one of Bulgaria’s most important economic and socially significant water sources because of its role in supplying Sofia with drinking water. Among the critical factors that carry potential high-risk levels for water quality in this hydrosystem are the discharge from the Samokov Wastewater Treatment Plant (WWTP), diffuse pollution from agriculture, and the percolation of untreated sewage from the small villages. In this study, we assessed the effect of treated wastewater effluent on water quality, and on the ecological state and microbial communities in the river sector of Samokov’s WWTP discharge area. The assessment was based on the complex use of chemical and microbiological indicators and biological quality elements. The concentrations of organics, nutrients and microcomponents were determined with results confirming the expected increase for parameters associated with the discharge of urban wastewater. The ecological state, according to macrozoobenthos indicators, was “good” throughout the river sector but local deterioration was registered in a proximal location downstream of the WWTP outfall. The analysis of stream water and bed sediment microbial communities by a fluorescent technique showed the high metabolic activity and intensive transformation processes in addition to high abundance registered with standard cultivation methods. The importance of the studied sub-catchment for the functioning of the urban water cycle, and for the quality of Sofia’s drinking water, underlines the need to extend an existing monitoring program with a more detailed assessment of the environmental impact of wastewater discharge.

Cultivation of microalgae in adjusted wastewater to enhance biofuel production and reduce environmental impact: Pyrolysis performances and life cycle assessment

The interest in microalgae as a renewable and sustainable feedstock for biofuels production has inspired a new focus in biorefinery. Current innovations in microalgae technology include the use of wastewater as the cultivation medium towards nutrients recovery, renewable energy generation, as well as wastewater treatment. Though recent studies have favoured the competitiveness of such an approach, how to maintain a high-quality microalgae-derived biofuel production in real wastewater with fluctuations in nutrient contents is still a challenge. This study investigated a strategy of adjusting the nutrient composition of the feeding wastewater (i.e. anaerobic digestion effluent) for microalgae cultivation (Desmodesmus sp.) and biofuel production. The addition of an appropriate amount of nutrients, including magnesium, iron and phosphorus, significantly enhanced the microalgal biomass production (0.78 g L−1) compared with the original wastewater (0.35 g L−1) and the standard BG11 microalgae cultivation medium (0.54 g L−1). In terms of the potential biofuel quality, the use of adjusted wastewater led to a higher content of valuable products (aliphatic hydrocarbon and fatty acids were accounted for 23.98% and 42.33% of the whole biomass, respectively) along with a reduction in potentially toxic substances (nitrogen-containing compounds and polycyclic aromatic hydrocarbons were accounted for 7.96% and 7.09% of the whole biomass, respectively) compared with the other cultivation groups. Moreover, the lower optimal temperature of pyrolysis engendered by the adjusted wastewater was significant for reduction of process energy consumption, which in turn led to overall lowered environmental impacts (838.52 mPET2000, applying life cycle assessment) than did the original wastewater (1165.67 mPET2000) and standard cultivation medium (1347.63 mPET2000). This study demonstrated that the adjustment of wastewater can act as a potential approach for the improvement in the quality of microalgal biofuel production, with consequent reduced environmental impacts.

HLA-G and CD152 Expression Levels Encourage the Use of Umbilical Cord Tissue-Derived Mesenchymal Stromal Cells as an Alternative for Immunosuppressive Therapy.

Mesenchymal stromal cells (MSCs) have been used in immunosuppressive therapy due to their therapeutic effects, with the HLA-G molecule seeming to play a fundamental role. This work evaluated alternative MSC sources to bone marrow (BM), namely, umbilical cord tissue (UC), adipose tissue (AD) and dental pulp tissue (DP), and the influence of interferon-γ (IFN-γ) and hypoxia on the cultivation of these cells for use in immunosuppression therapies. Expression of costimulatory markers CD40, CD80 and CD86 and immunosuppressive molecules CD152 and HLA-G was analyzed. Lymphocyte inhibition assays were also performed. Sequencing of the HLA-G gene from exons 1 to 5 was performed using next-generation sequencing to determine the presence of alleles. UC-derived MSCs (UCMSCs) expressed higher CD152 and HLA-G1 under standard cultivation. UCMSCs and DP-derived MSCs (DPSCs) secreted similar levels of HLA-G5. All MSC sources inhibited the proliferation of peripheral blood mononuclear cells (PBMCs); growth under regular versus hypoxic conditions resulted in similar levels of inhibition. When IFN-γ was added, PBMC growth was inhibited to a lesser extent by UCMSCs. The HLA-G*01:04:01:01 allele appears to generate a more efficient MSC response in inhibiting lymphocyte proliferation. However, the strength of this conclusion was limited by the small sample size. UCMSCs are an excellent alternative to BM in immunosuppressive therapy: they express high concentrations of inhibitory molecules and can be cultivated without stimuli, which minimizes cost.

Parameter Optimization of the Harvest Method in the Standardized Hedge Cultivation Mode of Lycium barbarum Using Response Surface Methodology

In a previously published study, to optimize the vibrating and comb-brushing harvesting, the main factors and their parameter values were obtained based on the FEM and RSM. However, the study was based on the extensive cultivation mode which need to be improve. To realize the mechanization of the harvesting of Lycium barbarum L., as well as to face the standardized hedge cultivation mode, a vibrating and comb-brushing harvester machine was designed, which was primarily composed of an execution system, a motion system, and a control system. The mathematical model between the harvest effect index and the operation parameters was established based on response surface methodology (RSM). The effects of various parameters on the harvest index were analyzed, and the best parameter combination was determined: a vibration frequency of 38.73 Hz, a brush speed of 14.21 mm/s, and an insertion depth of 26.07 mm. The field experiment showed that the harvesting rate of ripe fruit was 83.65%, the harvesting rate of unripe fruit was 7.22%, the damage rate of the ripe fruit was 11.49%, and the comprehensive picking index was 87.85. The findings provided a reference for the development of L. barbarum harvesting mechanization in a standardized hedge cultivation mode.

Venous catheters in horses - early ultrasonographic venous abnormalities and evidence of biofilms

Complications associated with indwelling intravenous catheters vary from minor to severe. Changes in tissue architecture and vein structure may be detectable prior to clinical alterations. The aim of the study was to characterize and compare changes in a catheterized vein and surrounding tissue by clinical and ultrasonographic examination. Microbiological infestation of catheter specimens was assessed. In this prospective, observational, clinical study 55 horses with an indwelling intravenous catheter have been included. Subsequent to catheter placement, vein and surrounding tissue were daily examined clinically and by ultrasonographic examination at predetermined localizations. After sterile removal of the catheters, specimens underwent microbiological testing and scanning electron microscopy. Obtained data were analyzed via descriptive statistics. Thickness of the venous wall was compared at predetermined localizations and time points for several parameters with the help of non-parametric tests (level of significance at p < 0.05, post-hoc Bonferroni correction). Overall, in 41.8 % of the horses transient alterations (swelling) of the surrounding tissue occurred during catheterization. Median catheterization was 69.5 hours (19 hour-10 days). With ultrasonography, venous valves and collateral vessels could be detected reliably. Significant alterations in wall thickness during catheterization were associated with various factors (location site [p ≤ 0.001], season [p = 0.006], anesthesia [p ≤ 0.001]).Microbiological analysis revealed a positive result in 23.5 %, 12 of the 51 samples; raster electron microscopy showed presence of bacteria in 25.0 %, 4 of the 16 investigated catheter samples. Local tissue changes at the insertion site of the catheter are commonly associated with catheterization, and are easily detectable with ultrasonography. Despite the easy performance in a clinical setting, the benefit of routine ultrasonographic monitoring of catheterized veins might be questionable with regard to early identification and prediction of catheter-associated venous disease. Bacteria might be detectable morphologically on the catheter but frequently lack a positive result from standard bacterial cultivation.

Molecular genetic approaches to decrease the uncontrolled misincorporation of non-canonical branched chain amino acids into recombinant mini-proinsulin expressed in Escherichia coli

The uncontrolled incorporation of non-canonical branched chain amino acids (ncBCAAs) such as norleucine, norvaline and β-methylnorleucine into recombinant proteins in E. coli production processes is a crucial problem in the pharmaceutical industry, since it can lead to the production of altered proteins with non-optimal characteristics. Despite various solutions, to date there are no engineered strains that exhibit a reduced accumulation of these ncBAAs. In this study, novel E. coli K-12 BW25113 strains with exogenous tunable expression of target genes of the BCAA biosynthetic pathway were developed. For this purpose, single gene knock-outs for thrA, ilvA, leuA, ilvIH, ilvBN, ilvGM and ilvC were complemented with plasmids containing the respective genes under control of an arabinose promoter. These clones were screened in a mL-bioreactor system in fed-batch mode under both standard cultivation conditions and with pyruvate pulses, and induction of a min-proinsulin. Screening was performed by comparing the impurity profile of the recombinant mini-proinsulin expressed of each clone with the E. coli BW25113 WT strain, and the most promising clones were cultivated in a 15L Screening showed that up-regulation of ilvC, ilvIH and ilvGM, and downregulation of leuA and ilvBN trigger a reduction of norvaline and norleucine accumulation and misincorporation into mini-proinsulin. The stirred tank bioreactor cultivations confirmed that up-regulation of ilvIH and ilvGM were most effective to reduce the ncBCAA misincorporation. This novel approach for a reduced ncBCAA misincorporation may be solution to this old challenging problem in the large-scale production of human therapeutics.