The third set of annotation conditions, where the user obtains a chemical structure of a metabolite for which the biosynthesis/biodegradation pathway is unknown, has also been tackled using RDM patterns (Oh et al., 2007), as an extension of the E-zyme approach. We recently developed a new web-based server named PathPred (Moriya et al., 2010)

for predicting the metabolic fate of a given chemical compound, based on the conserved RCLASS depending on the types of pathways. This server provides plausible reactions and transformed compounds, and displays all predicted reaction pathways in a tree-shaped graph (Figure 5a). The suggested pathway includes the steps see more with the plausible EC numbers, which are predicted by E-zyme (Figure 5b). The user can choose the type of pathway according to their purpose, the biodegradation of xenobiotics in bacteria and the biosynthesis of secondary metabolites in plants, which utilizes different characteristic

subsets of the RDM patterns. In the first step, the query compound structure is compared with those in the selected metabolic category. In the second step, possible RDM patterns on the query compound are selected from the RDM pattern library based on the structurally similar compounds containing the corresponding RDM DAPT research buy patterns with the use of the SIMCOMP program (Hattori et al., 2003, 9). The third step is to obtain the plausible products according to the selected RDM patterns. The generated products become the next query compound and the prediction is iterated if possible. Optionally, if already known, the final compound in the biodegradation or the initial compound in the biosynthesis can be specified, (bi-directional prediction). As an expansion of our study to reconstruct metabolic pathway based on chemical structures, we have been trying to predict accompanying genes for predicted reactions based on the relationships between metabolite chemical structures and protein sequences. The key to archive this is the classification of enzymes from both genomic and metabolomic points of view. There

are many ways to classify enzymes. Enzymes in the IUBMB׳s Enzyme List are systematically classified according Docetaxel clinical trial to the chemical structures of their substrates and products, and co-factors, as well as reaction selectivity and substrate specificity, which are inalienably related to Enzyme Nomenclature. Enzymes can also be classified based on enzyme proteins, such as the amino acid sequences and the 3D structure of proteins. Other factors that can group enzymes include the location in the pathway (i.e., biological functions), and the location of the cells. Enzymes are classified into membrane-bound enzymes and soluble enzymes. The membrane-bound enzymes can be further classified into buried type (such as receptor proteins), transmembrane type (such as channel, transporter, ATP syntheses) and membrane adhesion type (such as hydrogenases).

We analyzed 4 glands in this manner. MAGs were homogenized in 0.2 M HEPES buffer, pH7, 0.2% Triton X-100 (15 pairs of glands in 150 μl). Aliquots (10 μl) were incubated with 1.25 nmoles of peptide at 25 °C. Reactions were stopped by addition of 260 μl of 0.1% TFA and the GSK1120212 research buy amount of parent peptide remaining was quantified by reversed phase HPLC [17]. Injections of either Aea-HP-1 or SP

into the abdomen of virgin D. melanogaster females were performed as described previously [42]. After injection, females were transferred to individual food vials and were tested after 5 h for receptivity with a naive Canton S male. Ligand-mediated receptor activation was measured using an established expression system employing CHO-K1 cells expressing the Ca2+ reporter aequorin [42]. The construction of the expression constructs for D. melanogaster sex peptide receptor (SPR) and A. aegypti SPR and the measurement of the luminescent signals have been reported previously [19]. Peptides were extracted from MAGs plus SVs for click here analysis by MALDI/TOF-MS. The spectrum (m/z 800–4000) revealed two prominent monoisotopic peaks, one at m/z, 1227.8 and a less intense peak at m/z, 1211.8 ( Fig. 1a). The mass difference of 16 Da between these peaks suggested they might be

related, with a difference in oxidation state between them. The molecular ion (m/z, 1227.8) was subjected to post-source decay analysis and the fragmentation spectra generated revealed the amino acid sequence of the parent peptide as pERPhPSLKTRFamide (pE, pyro-glutamic acid, hP, 4-hydroxyproline; amide, amidated C-terminus; Fig. 2). The sequence was identical to a neuropeptide previously isolated from A. aegypti heads collected from a mixed sex population and known as the head peptide or before Aea-HP-1 [30]. Aea-HP-1 is modified post-translationally in three places, including hydroxylation of one proline residue. The fully modified mature peptide has a theoretical molecular mass ion m/z of 1227.7, which agrees closely with the m/z peaks observed in our spectra. The

second peak at m/z, approx. 1211.7 most likely represents a version of the peptide known as Aea-HP-3 [39], in which the proline at position four was not hydroxylated. An almost identical fragment ion spectrum was generated when synthetic Aea-HP-1 was analyzed in the same manner (not shown). An extract from a total of 70 pairs of MAGs and SVs was fractionated using RP-HPLC and MALDI/TOF-MS analysis of collected fractions established that the retention times of the natural and synthetic Aea-HP-1 were identical. MAGs and SVs were also analyzed separately by directly placing tissues from individual insects onto the MALDI plate. The prominent ions previously observed in the acidified methanol extract were also the major peaks (m/z, 12211.6 and 1227.6, Fig. 1b) in the spectra obtained directly from pairs of MAGs and were consistently seen in tissues from seven individual mosquitoes.

Major beaches in the Polish part of the lagoon are Stepnica, Trzebież, Czarnocin, Lake Nowowarpieńskie and Wolin. The municipalities differ considerably in terms of population and income. The municipalities with the highest income are the tourism resorts located on the Baltic Sea coast. Municipalities around the lagoon have an income below the national average mainly from farming, light industry and commerce. However, tourism is still growing and of increasing importance. Especially the fast development of marinas in the lagoon with about 2 400 mooring spaces is one indicator (10 marinas on the Polish side

with altogether about 600 mooring spaces as well as 14 marinas in the German part of the lagoon) (Steingrube et al., 2004). The regional plan by the Marshal of Zachodniopomorskie Voivodship Ceritinib in vitro suggests the creation of a West Pomeranian Sailing Route covering Lenvatinib cost the lagoon and the Baltic. It includes new sport boat harbours and the modernization of existing ones. For a further development of tourism around the lagoon a good water quality is imperative. During recent years, the Oder/Odra estuary faced many problems with microorganisms. A Salmonella pollution event in the sea-side resort Miedzyzdroje caused a beach closing for more than 4 weeks during high-season in August 2008. High concentrations of V. vulnificus were frequently found in Karlshagen, Island of Usedom

and in Lubmin, Greifswald Bodden. In 2009, the maximum was above 1 million germs per litre in Lubmin. In 2003, LY294002 2010 bathers died after a vibrion infection and in 2006, three people fell ill but and were saved only by fast application of antibiotics (LAGUS pers. com). However, most common are problems due to high concentrations of coliform, E. coli and Enterococci bacteria. In the past, coliform bacteria often caused a closing of beaches according to EU Bathing Water Quality Directive (76/160/EEC), e.g. in Stepnica (from 08.08.2006 for 25 days; from 19.07.2006 for 15 days), in Trzebież (from 01.08.2006 for 42 days; from 20.07.2007 for 42 days; from 24.07.2008

for an unknown period) and in Czarnocin (from 27.07.2006 for 50 days; from 10.07.2007 for 35 days and from 01.08. 2008 for an unknown number of days). Data on bathing water quality in Poland are publicly provided by the Chief Sanitary Inspectorate. Data on local bathing water quality are also available on the websites of public health services. Insufficiently treated sewage water is the most important reason for microbial problems and caused serious water quality problems in the lagoon during the last decades. Today the situation is improving because 288 million Euros have recently been invested in sewage treatment plants around the city of Szczecin, which is the major centre and located at the Odra river, north of the lagoon.

Vaccine-strain viruses are expanded by culture through a eukaryotic cell line. Cell culture may be derived from primary cells (including eggs

and primary monkey kidney cells), diploid cells (such as MRC-5 [a diploid line from normal human lung fibroblasts], WI-38 [Wistar Institute; a diploid line from normal human lung fibroblasts] and selleck chemicals FRhL-2 [a cell line from foetal rhesus monkey lung]), yeast and other continuous cell lines (CCLs). Once the cell cultures are established, the vaccine-strain virus is seeded and cultivated. In the case of microorganisms that are not able to grow in vitro, recombinant antigens have been produced through expression systems (eg yeast or baculovirus/insect cells) used to generate the protein antigen from gene sequences inserted into the expression system and under the control of a promoter sequence (see Chapter 3 – Vaccine antigens). Recovery of antigens from the culture media involves a combination of optimised processes, including microfiltration,

purification, homogenisation and batch clarification using ion-exchange resins. Vaccines based on whole living organisms/pathogens can be made using a genetically altered (thus attenuated) organism grown in the culture system or by attenuating the viral pathogen itself. Attenuation can be achieved by repeatedly propagating the microbial pathogen in human and/or non-human cell lines grown in culture,

Selleck Natural Product Library which reduces their efficiency at replicating in human Phloretin cells or alters other virulence properties. Whole pathogen antigens can be inactivated by methods including chemical or heat treatment to produce whole killed formulations. Pathogens can be split or fractionated to produce subunit antigens, which may be subsequently purified to retain highly selected antigenic components. Vaccine polysaccharide antigens may also be conjugated to protein carriers once they have been isolated, to produce vaccines for diseases caused by encapsulated bacteria such as Meningococci (see Chapter 2 – Vaccine immunology and Chapter 3 – Vaccine antigens). Finishing operations include sterilising/clarifying filtration, freezing, freeze drying, glassifying (drying vaccines in the presence of sugars or other stabilisers) after formulation with adjuvants, stabilisers, preservatives (if required), filling syringes or vials, and labelling and packaging the products. All procedures need to be conducted according to strict cGMP regulations. QA and QC are performed at every step of the vaccine manufacturing process. Production of a vaccine, whether by fermentation, cultivation, isolation or synthesis, usually starts with raw materials. Subsequent steps of the procedure involve preparation, characterisation and purification of intermediates eventually resulting in the bulk material.

Protocol II was used only for P. brasiliensis, in which the incubation time was 12 h and the methodology was adapted

from Travassos and collaborators [42]. The peptides P1, P2, P3, and P4 were serially diluted from 16 to 500 μg ml−1 in phosphate buffer saline (PBS, pH 7.2). A 2-fold dilution series (100 μl) were added to 100 μl of 2 × 104 viable cells of the P. brasiliensis in 500 μl plastic tubes. The tubes were incubated at 36 °C in rotatory shaker (100 rpm) during 12 h. After this period, 100 μl of each tube were plated in solid medium Brain Heart Infusion (BHI, Acumedia®, USA) supplemented with 4% (v/v) horse serum (Gibco, USA), 5% (v/v) supernatant of the culture filtrate of the isolate Pb192 and 40 mg l−1 gentamycin (Schering-Plow, USA). The filtrate was prepared according to methodology described previously [42]. The growth of colony-forming units was observed for INCB024360 clinical trial 21 days. The lowest concentration of peptide that completely inhibited growth of the fungi was defined as the minimal inhibitory concentration. The MICs were calculated by the average Osimertinib values obtained in triplicates on

three independent measurements [36]. The experimental controls used in both protocols were amphotericin B (Sigma–Aldrich, USA) and for protocol II the killer peptide (KP) as control was also used. The antibacterial activity was evaluated against human pathogenic bacteria Escherichia coli ATCC8739 and Staphylococcus aureus ATCC25923, both obtained from the American Type Culture Collection (ATCC). Briefly, the bacterial cultures were grown in Lysogeny Broth (LB) medium, pH 7.0, at 37 °C until they reached the exponential phase. The method used to study the antibacterial activity of the peptides was based on the broth microdilution assay. The culture for the assay was prepared by diluting 1:11 the bacteria obtained on the exponential phase. The peptides P1, P2, P3, and P4 were serially diluted from 2 to 256 μg ml−1 in LB medium. A 2-fold dilution series (100 μl) were added to 10 μl of approximately 5 × 106 CFU of bacteria

in each well of a 96-well polypropylene plate. The plates were incubated for 4 h at 37 °C and the peptides antibacterial activities were Adenosine observed in every 30 min by measuring the absorbance in a plate reader (Bio-Rad 680 Microplate Reader) at 595 nm. The controls utilized were distilled water and chloramphenicol 60 μg ml−1. Primaries sequences were obtained from initial selection previously described in this section. All of them being of synthetic peptide amidate. PSI-BLAST was used for templates data mining [48]. For P1 and P2 peptides models, it was possible to obtain templates by homology method (pdb: 2jx6 and 1id3), showing 55 and 88% of identity respectively [45] and [48]. Fifty models for each peptide were constructed by using Modeller v9.8.

A colocação de cecostomia percutânea permite a realização de enemas anterógrados por meio de uma sonda e, como tal, evita as complicações associadas ao estoma. Inicialmente descrita como um procedimento colocado com auxílio da fluoroscopia4 and 5, o recurso à endoscopia permite uma visualização direta do cego, evitando que a colocação da sonda seja feita noutros locais que não esse, ao mesmo tempo que o doente não é exposto a radiação prolongada. É tecnicamente

simples e fácil de realizar. Também o tempo de procedimento é curto6, sendo menos moroso que a realização de apendicostomia/cecostomia e que a colocação sob controlo radioscópico. Como desvantagens apresenta-se a presença de uma sonda permanente que atravessa a pele e o tecido subcutâneo terminando Akt targets no cego e a possibilidade de ocorrência de complicações relacionadas com a sonda (sua remoção acidental, rotura4 e migração). No caso que se apresenta ocorreu a migração da sonda inicial, tendo esta complicação sido facilmente resolvida com colonoscopia e substituição por sonda com

balão. Estão ainda descritas outras possíveis complicações: peritonite, celulite e hemorragia7. No caso particular das crianças com derivações ventriculoperitoneais, a colocação de CEP parece ser uma opção segura, na medida em não tem sido associada a risco maior de infeção do líquido céfalo-raquidiano2 and 4. A CEP é recomendada nos casos de Glutathione peroxidase incontinência fecal EPZ015666 solubility dmso associada a espinha bífida, lesão medular, malformação anorretal e alterações neurológicas. Na doença de Hirschsprung

com enterocolite recorrente e pseudo-obstrução cólica pode ser útil para irrigação e descompressão do cólon. A sua aplicabilidade poderá ser alargada também para aos adultos para descompressão em casos de obstrução maligna do cólon esquerdo2. Não terá de ser necessariamente realizado sob anestesia geral, podendo ser um procedimento de ambulatório com sedação e anestesia local, desde que a colonoscopia seja bem tolerada. Longe de ser a forma de abordagem ideal da incontinência fecal, a CEP revela-se como uma boa opção terapêutica. Apesar de ainda não ser frequentemente utilizada e divulgada na literatura e apesar da ausência de estudos comparativos com as opções mais amplamente usadas como a apendicostomia/cecostomia e os enemas retrógrados, alia as vantagens da realização de enemas anterógrados sem a presença de estoma e colocado de uma forma rápida e segura. O doente adquire autonomia e independência na realização dos enemas e alcança o controlo da continência fecal melhorando significativamente a sua qualidade de vida e adaptação social, aspetos fundamentais na vida de um adolescente. Os autores declaram não haver conflito de interesses.

In this work, the improvement of the performance of StAP3 was achieved by means of a covalent modification with PEG. The separation of a mono-PEGylated StAP3 fraction could easily be performed by gel filtration chromatography. The mono-PEGylated StAP3 fraction was studied in terms of in vitro antimicrobial activity,

exhibiting higher antimicrobial activity against Fusarium solani spores and Bacillus selleck chemical cereus. In addition, PEGylation did not affect the selective cytotoxicity of StAP3, since no hemolytic activity was observed. Succinimidyl valerate monomethoxy polyethylene glycol (mPEG-SVA, 5 kDa) was purchased from Laysan Bio Inc. (Arab, AL, USA). Sodium dodecyl sulphate (SDS) and dithiothreitol (DTT) were supplied by Sigma (St. Louis, MO, USA). All the reagents were purchased in the highest purity and used without further purification. F. solani f. sp. eumartii, isolate 3122 (EEA-INTA, Balcarce, Argentina) was grown at 25 °C on potato dextrose

agar (PDA) plates supplemented with 100 μg/ml ampicillin. Spores were collected from 8-day-old cultures by suspension in sterile water. B. cereus and Escherichia coli were provided by the American Type Culture Collection (ATCC) and were grown in Luria–Bertani medium at 37 °C with continuous shaking. Bacterial growth was quantified by measuring absorbance at 600 nm. Potato leaves were detached and placed at 18 °C in a moist chamber. StAP3 was purified from leaves using the protocol previously described by Guevara et al. [53]. A solution of purified StAP3 (5 ml, 0.6 mg/ml) in 50 mM Tris–HCl Pirfenidone price pH 8, was added to a 40-fold molar excess of mPEG-SVA. The mixture was incubated at 25 °C with stirring at 500 rpm, and the reaction was quenched after 6 h by addition of 2 ml 1 M glycine solution. The mixture was then concentrated to 230 μl using Vivaspin 15R (MW cut-off 5 kDa) (VIVASCIENCE, Germany), and 0.4% SDS (w/v) and 0.2 mM DTT were added. PEG-StAP3

conjugates were analyzed by size exclusion chromatography on an equilibrated Superose 12 HR (10/30) column (Pharmacia, Uppsala, Sweden), connected to a fast-protein liquid ZD1839 order chromatography system, at a constant flow rate of 0.4 ml/min at room temperature. The column was calibrated using a mixture of four proteins of known molecular mass, i.e. pyruvate kinase (230 kDa), native StAP3 (45 kDa), glyceraldehyde-3P-dehydrogenase (36 kDa), and lysozyme (14.3 kDa). The column was equilibrated and eluted with 20 mM Tris–HCl pH 8, 0.4% SDS (w/v), and 0.2 mM DTT. Fractions of 0.4 ml were collected and the elution was monitored at 280 nm. Fractions from the size exclusion chromatography corresponding to different peaks were pooled and then analyzed by SDS-PAGE using 12% acrylamide. Gel was stained with Coomassie Brilliant Blue R250 coloidal [54].

(2004), from one half of a filter, representing 50 ml of water samples. The DNA was quantified with a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA) and yielded 10–50 ng genomic DNA per 100 ml water sample. A terminal-restriction fragment length polymorphism (T-RFLP) analysis was performed following the protocol of Hahnke et al. (2013). In short: the fluorescently labelled general bacterial primers 27F (FAM, 5′-AGA GTT TGA TCC TGG CTC AG-3′) and 907R (HEX, 5′-CCG TCA ATT CCT TTR AGT TT-3′) were used to amplify the partial 16S rRNA gene (Muyzer et al. 1995). Approximately 25 ng of purified PCR products were digested with 5 U of the restriction

enzyme AluI. The terminal restriction fragments (TRFs) were detected on an ABI Prism LEE011 mw 3130 XL Genetic Analyzer (Applied Biosystems, California), equipped with an 80 cm capillary, a POP-7 polymer and the filter set D (Filter DS-30). The ROX-labelled MapMarker 1000 (Eurogentec, Belgium) served as a size standard between 50 bp and 1000 bp. Forward TRFs were analysed only because of the higher variability at the beginning of the 16S rRNA gene ( Hahnke et al. 2013). The T-RFLP patterns were analysed following the protocol of Hahnke et al. (2013). In short: TRFs between 50 and 1000 bp were identified and sized with the Genetic Analyser

3.7 (Applied Biosystems, California, USA) software, click here using a fluorescence intensity threshold of 20 U. The individual patterns were processed, applying the interactive binner (Ramette 2009) in R (http://www.r-project.org, version 2.3.1). The binning size was one nucleotide and the binning shift 0.1 nucleotides. The TRFs were named by subtracting 0.1 bases from the TRF length. The resulting pattern with normalised relative fluorescence intensities

(RFI) were visualised in rank versus cumulated abundance curves with the k-dominance plot in PRIMER (v.6, PRIMER-E, Plymouth Marine Laboratory, UK) (Clarke 1993), in order to identify and remove outlying samples within the triplicates (one from station E53 and one from station E54) and identify the final T-RFLP data set. Fragments smaller than 100 nt were not included. There was a shift between closely situated intensive fluorescence peaks, which Wilson disease protein impaired data interpretation. Fragments of 230–232 nt were therefore excluded from analysis. Visual comparisons between bacterial communities at each station were explored by ordination using non-metric multidimensional scaling (nMDS) and applying the isoMDS function of the MASS package (Venables & Ripley 2002) with 100 random restarts, Bray-Curtis dissimilarity and 999 iterations. The environmental parameters were fitted into the nMDS plot by applying the function envfit of the R package VEGAN (v.1.8–3, Dixon 2003) with 1000 permutations, Euclidian distances and P-values smaller than 0.001.

Volden and Conzen present

a complementary review of the influence buy LDK378 of glucocorticoid signaling on tumor progression through cell context-specific transcriptional networks (Volden and Conzen, 2012 1045). In the clinical context, disruption of HPA rhythms, as indicated by diurnal cortisol slopes, predicted early metastatic breast cancer mortality (Sephton et al., 2000). Sephton and colleagues, as reported in this volume, replicate those findings in a small sample of lung cancer patients followed for a median of 4 years from date of diagnosis (Sephton et al., 2012). Volden and Conzen foreshadow emerging interest in stress regulation of epithelial cancer biology through metabolic pathways and energy regulators such as insulin, leptin, ghrelin, and adiponectin (Cao and During, 2012 and Williams et al., 2009). Convergence of animal models and human correlative studies led Neeman and Ben-Eliyahu to identify catecholamine and prostaglandin-mediated immunosuppression as a perioperative risk factor for cancer recurrence and metastasis (Neeman and Ben-Eliyahu, 2012). The authors advance a theoretical model that captures the cumulative

risk and review mechanistic support for the use of pharmacological blockade of key mediators during the perioperative period. Sheridan and colleagues review the utility of a mouse find protocol model of repeated social defeat to elucidate neural-immune mechanisms in cancer (Powell et al., 2012). This review highlights the role of myeloid-derived cells in stress-primed inflammation, in tissue remodeling in non-immune and immune organs, and in support of behavioral states experienced as cancer-associated

sickness behaviors (see reviews in this volume by Bower and Lamkin, 2012, Costanzo et al., 2012 and Irwin et Rho al., 2012). The empirical paper by Madden et al. examines the impact of social isolation on breast cancer pathogenesis in adult severe combined immunodeficiency mice using a human breast cancer cell line known to express β-ARs (Madden et al., 2012). The results raise implications of mild vs. chronic stress exposure, timing of exposure during the life span of experimental animals, and the need to capture transient shifts in target cell populations. Further, the study supports the importance of myeloid-derived suppressor cells and stress-associated leukocyte recruitment as indicated by changes in macrophage populations in tumor and spleen, similar to that observed with social disruption (SDR) stress paradigms (Engler et al., 2004 and Powell et al., 2012). Bower and Lamkin identify two questions that direct contemporary research on cancer-related fatigue, i.e.

a.s.l. From planting to harvesting, mean rainfall and temperature range were respectively 1121 mm and 16.7–28.7 °C at Namulonge, 1095 mm and 17.3–29.2 °C at Jinja, and 424 mm and 18.5–29.4 °C at Nakasongola. Twelve genotypes (Table 1) were sourced from farmers’ fields and from the National Cassava Breeding Programme (NCBP) at the National Crops Resources Research Institute, Namulonge. Genotypes from farmers’ compound screening assay fields were landraces, while genotypes from the NCBP were introductions from the International Institute of Tropical Agriculture (IITA) and genotypes developed

by crossing cassava lines from the International Centre for Tropical Agriculture (CIAT) with lines from Uganda. Selection of the genotypes was based on their performance for storage root yield, early bulking and relative degrees of field resistance to two diseases prevalent in Uganda: cassava brown streak disease (CBSD) and cassava mosaic disease (CMD). The trial at each location

was laid out in a randomised complete block design with three replications. Healthy stem cuttings each 25 cm in length were horizontally planted in a flat seedbed at a spacing of 1 m × 1 m giving a population density of 10,000 plants ha− 1. Each plot measured 2 m × 6 m, comprising 3 rows of 6 plants each. The first and last rows and the first and last plant within the middle row of each plot were considered as border plants. The plots and blocks were separated by 2.0 m and 2.5 m find more alleys, to reduce inter-plot and inter-block plant competition, respectively. The trials were conducted without supplemental irrigation and weeded regularly. Data for the following traits were collected from a net plot of four randomly selected and hand-uprooted plants of each genotype: storage root number (SRN); storage root mass (SRM); FSRY and cassava brown streak disease root necrosis (CBSD-RN). Cassava mosaic disease severity (CMD-S) was assessed during the crop growth at 6 MAP on an increasing scale of 1–5, where: 1 = no symptoms;

and 5 = severe mosaic symptoms [16]. Storage roots of the four plants were bulked, counted and weighed selleck to obtain SRN and SRM (kg), respectively. The FSRY (t ha− 1) per genotype was then estimated from the SRM of the four-plant bulk of storage roots as: FSRY=(SRM×10,000)/(4×1000).FSRY=SRM×10,000/4×1000. Storage root necrosis due to CBSD (CBSD-RN) was scored on an increasing scale of 1 to 5 where 1 = no visible necrosis, and 5 = severe necrosis [17]. The data for each location were first analysed independently and then the error variances for the environments were tested for homogeneity using Hartley’s Fmax test [18]. The differences were non-significant, and accordingly an unweighted combined AMMI analysis of variance was conducted across the locations. Correlations among various plant parameters were calculated as Spearman correlation coefficients [19].