Hybrid modeling framework for process analytical technology: application to Bordetella pertussis cultures

In the process analytical technology (PAT) initiative, the application of sensors technology and modeling methods is promoted. The emphasis is on Quality by Design, online monitoring, and closed-loop control with the general aim of building in product quality into manufacturing operations. As a result, online high-throughput process analyzers find increasing application and therewith high amounts of highly correlated data become available online. In this study, an hybrid chemometric/mathematical modeling method is adopted for data analysis, which is shown to be advantageous over the commonly used chemometric techniques in PAT applications. This methodology was applied to the analysis of process data of Bordetella pertussis cultivations, namely online data of near-infrared, (NIR), pH, temperature and dissolved oxygen, and off-line data of biomass, glutamate, and lactate concentrations. The hybrid model structure consisted of macroscopic material balance equations in which the specific reactions rates are modeled by nonlinear partial least square (PLS). This methodology revealed a significant higher statistical confidence in comparison to PLSs, translated in a reduction of mean squared prediction errors (e.g., individual root mean squared prediction errors calibration/validation obtained through the hybrid model for the concentrations of lactate: 0.8699/0.7190 mmol/L; glutamate: 0.6057/0.2917 mmol/L; and biomass: 0.0520/0.0283 OD; and obtained through the PLS model for the concentrations of lactate: 1.3549/1.0087 mmol/L; glutamate: 0.7628/0.3504 mmol/L; and biomass: 0.0949/0.0412 OD). Moreover, the analysis of loadings and scores in the hybrid approach revealed that process features can, as for PLS, be extracted by the hybrid method.     

Identification of Chromobacterium violaceum genes with potential biotechnological application in environmental detoxification

Chromobacterium violaceum is a Gram-negative bacterium found in a wide variety of tropical and subtropical ecosystems. The complete genome sequence of C. violaceum ATCC 12472 is now available, and it has considerable biotechnological potential for various applications, such as environmental detoxification, as well as medical and agricultural use. We examined the biotechnological potential of C. violaceum for environmental detoxification. Three operons, comprising the ars operon, involved in arsenic resistance, the cyn operon, involved in cyanate detoxification, and the hcn operon, encoding a cyanase, responsible for biogenic production of cyanide, as well as an open reading frame, encoding an acid dehalogenase, were analyzed in detail. Probable catalytic mechanisms for the enzymes were determined, based on amino acid sequence comparisons and on published structural information for these types of proteins.     

Physical and chemical properties of primary defences in Tenebrio molitor

Prevention and reaction are the foundation for any defence system. In insects, the primary defences against pathogens and parasites limit invasion; the secondary ones (e.g. immune system) act when the cuticle and other primary defences fail. Because investment in both aspects of defence may be costly, they should be regulated in a plastic or variable way in accordance with the risk of infection. The mealworm beetle Tenebrio molitor L. changes cuticle colour and its resistance to fungal infection when subject to high population density, although such resistance is a result of the primary (cuticle) defences rather than the secondary (immunological) ones. The present study tests the hypothesis that the physical and chemical properties of the primary defences in T. molitor change with cuticular darkness. Beetles expressing black phenotypes (or with darker cuticle) have a thicker cuticle, with four well organized layers (epi‐, exo‐, endocuticle and formation zone) and more melanin than tan beetles. The cuticle properties investigated in the present study are likely to be the underlying mechanisms of pathogen resistance in black beetles, including the content of carbonylated proteins, which in black beetles was almost half that of tan beetles after exposure to ultraviolet radiation. It is proposed that, in polyphenic insects (such as mealworm beetles), primary and secondary defences are regulated pleiotropically, with the genes responsible for the expression of one defence having a positive effect on others, whereas, in polymorphic insects, there is no such link and so investment in one defence may impair others.     

Lysine and threonine biosynthesis in sorghum seeds: characterisation of aspartate kinase and homoserine dehydrogenase isoenzymes

Aspartate kinase (AK, EC 2.7.2.4) and homoserine dehydrogenase (HSDH, EC 1.1.1.3) have been partially purified and characterised from immature sorghum seeds. Two peaks of AK activity were eluted by anion‐exchange chromatography [diethylaminoethyl (DEAE)‐Sephacel] with 183 and 262 mM KCl, and both activities were inhibited by lysine. Similarly, two peaks of HSDH activity were eluted with 145 and 183 mM KCl; the enzyme activity in the first peak in elution order was shown to be resistant to threonine inhibition, whereas the second was sensitive to threonine inhibition. However, following gel filtration chromatography (Sephacryl S‐200), one peak of AK activity co‐eluted with HSDH and both activities were sensitive to threonine inhibition, suggesting the presence of a bifunctional threonine‐sensitive AK–HSDH isoenzyme with a molecular mass estimated as 167 kDa. The activities of AK and HSDH were studied in the presence of lysine, threonine, methionine, valine, calcium, ethylene glycol bis(2‐aminoethylether)‐N,N,N′N′‐tetraacetic acid, calmodulin, S‐adenosylmethionine (SAM), S‐2‐aminoethyl‐l ‐cysteine (AEC) and increasing concentrations of KCl. AK was shown to be inhibited by threonine and lysine, confirming the existence of two isoenzymes, one sensitive to threonine and the other sensitive to lysine, the latter being predominant in sorghum seeds. Methionine, SAM plus lysine and AEC also inhibited AK activity; however, increasing KCl concentrations and calcium did not produce any significant effect on AK activity, indicating that calcium does not play a role in AK regulation in sorghum seeds. HSDH also exhibited some inhibition by threonine, but the majority of the activity was not inhibited, thus indicating the existence of a threonine‐sensitive isoenzyme and a second predominant threonine‐insensitive isoenzyme. Valine and SAM plus threonine also inhibited HSDH; however, increasing concentrations of KCl and calcium had no inhibitory effect.     

Genetic transformation of Rangpur lime (Citrus limonia osbeck) with thebO (bacterio-opsin) genen and its initial evaluation forPhytophthora nicotianae resistance

Transgenic plants expressing the bacterio-opsin (bO) gene can spontaneously activate programmed cell death (ped) and may enhance broad-spectrum pathogen resistance by activating an intrinsic defense pathway in plant species such as tobacco and potato. In this work, we produced transgenic Rangpur lime plants with thebO gene, viaAgrobacterium tumefaciens-mediated transformation, and evaluated these plants forPhytophthora nicotianae resistance. Two transgenic lines were successfully regenerated and transformation was confirmed by GUS activity assay, PCR analysis, Southern, Northern and Western blot analyses, in addition to detecting the expressed bO protein by an immunological approach. Evaluation forPhytophthora nicotianae resistance was carried out by plant inoculations with the pathogen and quantification of the affected area. One of the two transgenic lines showed greater tolerance to the fungal pathogen as compared to the control, with significantly smaller stem lesions after pathogen challenge. This increase in pathogen tolerance is correlated with a significantly higher level of transgene expression in this line when compared with the other transgenic line. This is the first report of the introduction of a potentially important gene into Rangpur lime to provide novel pathogen tolerance.     

Ultrastructural aspects of the myxosporean Henneguya astyanax n. sp. (Myxozoa: Myxobolidae), a parasite of the Amazonian teleost Astyanax keithi (Characidae)

This study reports light and electron microscopical aspects of a myxosporean found in the gills of the freshwater teleost Astyanax keithi Géry, Planquete & Le Bail, 1996 (family Characidae), collected from the estuarine region of the Amazon River, near Belém, Brazil. The prevalence of infection was 23%. In interlamellar spaces of the gills, ellipsoidal whitish cyst-like plasmodia structures were present, which contained spores. The spores had a spermatozoa-like appearance (47.8 +/- 0.71 microm in total length) with a fusiform body (15.2 +/- 0.77 pm in length, 5.7 +/- 0.71 microm in width and 4.2 +/- 0.31 microm in thickness), and each of the 2 valves presented a tapering tail (32.6 +/- 1.11 microm in length). The valves surrounded a binucleate sporoplasm cell and 2 polar capsules (5.0 +/- 0.13 microm in length, 1.5 +/- 0.07 microm in width) that contained 8 to 9 coils of the polar filament. In the sporoplasm, several unique sporoplasmosomes were visible. A synoptic table of spore measurements of known Brazilian Henneguya species is presented. The spores differed from those of previously described species. Based on spore morphology, it is concluded that this species belongs to the family Myxobolidae, genus Henneguya, and that it constitutes a new species: H. astyanax n. sp.     

Growth hormone transgenesis affects osmoregulation and energy metabolism in zebrafish (Danio rerio)

Growth hormone (GH) transgenic fish are at a critical step for possible approval for commercialization. Since this hormone is related to salinity tolerance in fish, our main goal was to verify whether the osmoregulatory capacity of the stenohaline zebrafish (Danio rerio) would be modified by GH-transgenesis. For this, we transferred GH-transgenic zebrafish (T) from freshwater to 11 ppt salinity and analyzed survival as well as relative changes in gene expression. Results show an increased mortality in T versus non-transgenic (NT) fish, suggesting an impaired mechanism of osmotic acclimation in T. The salinity effect on expression of genes related to osmoregulation, the somatotropic axis and energy metabolism was evaluated in gills and liver of T and NT. Genes coding for Na⁺, K⁺-ATPase, H⁺-ATPase, plasma carbonic anhydrase and cytosolic carbonic anhydrase were up-regulated in gills of transgenics in freshwater. The growth hormone receptor gene was down-regulated in gills and liver of both NT and T exposed to 11 ppt salinity, while insulin-like growth factor-1 was down-regulated in liver of NT and in gills of T exposed to 11 ppt salinity. In transgenics, all osmoregulation-related genes and the citrate synthase gene were down-regulated in gills of fish exposed to 11 ppt salinity, while lactate dehydrogenase expression was up-regulated in liver. Na⁺, K⁺-ATPase activity was higher in gills of T exposed to 11 ppt salinity as well as the whole body content of Na⁺. Increased ATP content was observed in gills of both NT and T exposed to 11 ppt salinity, being statistically higher in T than NT. Taking altogether, these findings support the hypothesis that GH-transgenesis increases Na⁺ import capacity and energetic demand, promoting an unfavorable osmotic and energetic physiological status and making this transgenic fish intolerant of hyperosmotic environments.     

24-Epibrassinolide Mechanisms Regulating Blossom-End Rot Development in Tomato Fruit

Blossom-end rot (BER) is a physiological disorder believed to be triggered by low Ca²⁺ content in the distal fruit tissue. However, many other factors can also determine fruit susceptibility to BER. It is possible that during fruit growth, Ca²⁺ imbalance can increase membrane leakiness, which may trigger the accumulation of reactive oxygen species, leading to cell death. Brassinosteroids are a class of plant hormones involved in stress defenses, specially increasing the activity of antioxidant enzymes and the accumulation of antioxidant compounds, such as ascorbic acid. The objective of this study was to understand the mechanisms by which 24-epibrassinolide (EBL) reduces fruit susceptibility to BER. Tomato plants ‘BRS Montese’ were cultivated in a greenhouse and were weekly sprayed with water (control) or EBL (0.01 µM) after full bloom. Plants and fruits were evaluated at 15 days after pollination (DAP). According to the results, EBL treatment inhibited BER development, increased fruit diameter, length, and fresh weight. EBL-treated fruit showed higher concentrations of soluble Ca²⁺ and lower concentrations of cell wall-bound Ca²⁺. EBL-treated fruit also had higher concentrations of ascorbic acid and lower concentrations of hydrogen peroxide, compared to water-treated fruit. EBL treatment increased the activity of the three main antioxidant enzymes known as ascorbate peroxidase, catalase, and superoxide dismutase. According to the results, EBL treatment maintained higher soluble Ca²⁺ and antioxidant capacity, reducing fruit susceptibility to BER.

     

Biospeckle activity in coffee seeds is associated non‐destructively with seedling quality

Seeds age during storage, resulting in a decline in germination and seedling quality. Seed quality tests are important to monitor this decline. However, such tests are usually destructive and require large seed numbers and long time. For coffee seeds the standard germination test and assessment of seedling quality takes 30 days. Biospeckle has been used previously as a non‐destructive optical tool to monitor biological activity in a range of tissues. Biospeckle was applied 3–6 days after imbibition (DAI) to investigate an association with coffee seedling quality after 30 days. Two distinct areas of biospeckle activity were demonstrated, concurring with the locations of the embryonic axis and the cotyledons in the apical and central seed parts, respectively. Moisture content analysis revealed that embryos of imbibed seeds contained more water than endosperm. Different areas within the endosperm did not differ in moisture content, while the moisture content of the axis was higher than that of the cotyledons, and this did not change from 4 DAI. Therefore, it was concluded that high biospeckle activity was not the result of increased water content in any seed part, but more likely of growth and metabolism in the axis and cotyledons, which had been described previously. A threshold biospeckle ratio apical : central of 1.02 after 6 days distinguished between seeds that produced dead and viable seedlings after 30 days and provided similar results as a tetrazolium test, a widely acknowledged but destructive test for seed quality. Thus, biospeckle data provided a non‐destructive early parameter for seedling quality, based on embryo growth during germination.