Optimizing performance of semi‐continuous cell culture in an ambr15™ microbioreactor using dynamic flux balance modeling

The ambr bioreactors are single‐use microbioreactors for cell line development and process optimization. With operating conditions for large‐scale biopharmaceutical production properly scaled down, microbioreactors such as the ambr15? can potentially be used to predict the effect of process changes such as modified media or different cell lines. While there have been some recent studies evaluating the ambr15? technology as a scale‐down model for fed‐batch operations, little has been reported for semi‐continuous or continuous operation. Gassing rates and dilution rates in the ambr15? were varied in this study to attempt to replicate performance of a perfusion process at the 5 L scale. At both scales, changes to metabolite production and consumption, and cell growth rate and therapeutic protein production were measured. Conditions were identified in the ambr15? bioreactor that produced metabolic shifts and specific metabolic and protein production rates that are characteristic of the corresponding 5 L perfusion process. A dynamic flux balance (DFB) model was employed to understand and predict the metabolic changes observed. The DFB model predicted trends observed experimentally, including lower specific glucose consumption and a switch from lactate production to consumption when dissolved CO₂ was maintained at higher levels in the broth. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:420–431, 2018     

Suicide in Nepal: Qualitative Findings from a Modified Case-Series Psychological Autopsy Investigation of Suicide Deaths

South Asia accounts for the majority of the world’s suicide deaths, but typical psychiatric or surveillance-based research approaches are limited due to incomplete vital surveillance. Despite rich anthropological scholarship in the region, such work has not been used to address public health gaps in surveillance and nor inform prevention programs designed based on surveillance data. Our goal was to leverage useful strategies from both public health and anthropological approaches to provide rich narrative reconstructions of suicide events, told by family members or loved ones of the deceased, to further contextualize the circumstances of suicide. Specifically, we sought to untangle socio-cultural and structural patterns in suicide cases to better inform systems-level surveillance strategies and salient community-level suicide prevention opportunities. Using a mixed-methods psychological autopsy approach for cross-cultural research (MPAC) in both urban and rural Nepal, 39 suicide deaths were examined. MPAC was used to document antecedent events, characteristics of persons completing suicide, and perceived drivers of each suicide. Patterns across suicide cases include (1) lack of education (72% of cases); (2) life stressors such as poverty (54%), violence (61.1%), migrant labor (33% of men), and family disputes often resulting in isolation or shame (56.4%); (3) family histories of suicidal behavior (62%), with the majority involving an immediate family member; (4) gender differences: female suicides were attributed to hopeless situations, such as spousal abuse, with high degrees of social stigma. In contrast, male suicides were most commonly associated with drinking and resulted from internalized stigma, such as financial failure or an inability to provide for their family; (5) justifications for suicide were attributions to ‘fate’ and personality characteristics such as ‘stubbornness’ and ‘egoism’; (5) power dynamics and available agency precluded some families from disputing the death as a suicide and also had implications for the condemnation or justification of particular suicides. Importantly, only 1 out of 3 men and 1 out of 6 women had any communication to family members about suicidal ideation prior to completion. Findings illustrate the importance of MPAC methods for capturing cultural narratives evoked after completed suicides, recognizing culturally salient warning signs, and identifying potential barriers to disclosure and justice seeking by families. These findings elucidate how suicide narratives are structured by family members and reveal public health opportunities for creating or supplementing mortality surveillance, intervening in higher risk populations such as survivors of suicide, and encouraging disclosure.     

Investigations on the artificial synthesis of amphidiploids of Brassica tournefortii Gouan with the other elementary species of Brassica. I. Genomic relationships

With the object of studying the genomic relationships of Brassica tournefortii Gouan with the other elementary species of Brassica viz. B. campestris (2n=20, A genome), B. oleracea (2n=18, C genome) and B. nigra (2n=16, B genome), it has been hybridized with them. The percentage of F₁ hybrids formed, their morphology and meiotic behaviour have been described. Based upon crossability relationships and meiotic pairing in the F₁ hybrids, it is inferred that the D genome of B. tournefortii is more closely related to the A genome than to the B and C genomes. It may have been derived from the A genome which likewise shows a strong genetic isolation from B and C. The species has developed a strong genetic barrier in the course of its evolution and shows little crossability, high hybrid sterility and no gene flow with any of the other elementary species. The fact that it has not formed any natural amphidiploids with the elementary species which otherwise are formed in all combinations, is more evidence that it originated more recently than the A genome. It is presumed that B. tournefortii, being more distantly related to B. nigra than to other elementary species, may form stable artificial aphid resistant amphidiploids with the former.     

Seed traits,fatty acid profile and genetic diversity assessment in <Emphasis Type="Italic">Pongamia pinnata</Emphasis> (L.) Pierre germplasm

Phenotypic variation of important seed traits like seed length, seed breadth, seed thickness, 100 seed weight and seed oil content were recorded in a total of 157 collected accessions of Pongamia. Out of these, fatty acid profiles of 38 accessions selected based on their high and low oil content was analyzed. Fatty acid profile revealed high variability in stearic, oleic and linoleic acid which varied from 0.42 to 10.61 %, 34.34 to 74.58 %, and 7.00 to 31.28 % respectively. Variations in palmitic and linolenic acid were small. Iodine value, saponification number and cetane number (CN) of fatty acid methyl esters (FAME) of seed oil ranges from 186.99 to 201.25, 81.13 to 108.19 and 46.16 to 56.47 respectively. Fatty acid compositions, degree of unsaturation and CN are the important parameters, which are used to determine quality of FAME were used as biodiesel. Some of the Pongamia accessions identified were higher in oil content while some accessions showed higher degree of unsaturation and a few of them had CN values higher than 55. Genetic diversity analysis with six TE-AFLP primers generated a total of 334 bands out of which 174 (52.10 %) were polymorphic. The genetic similarity ranged from 0.11 to 0.47. These findings clearly showed high level of genetic diversity and all economically desirable traits were not present in a single genotype of Pongamia. All these traits could be selected from these CPTs and transfer to a single elite variety through selection and breeding programme and could be utilized for large scale multiplication and plantation to produce high quantity and quality biodiesel in future.     

Analysis of genetic diversity and population structure in <Emphasis Type="Italic">Capsicum</Emphasis> landraces from North Eastern India using TE-AFLP markers

North eastern (NE) India harbours a precious germplasm repository of Capsicum in the form of various landraces. The present study was undertaken to characterise the extent of genetic variation present in different Capsicum landraces from north eastern India. A set of 171 Capsicum accessions were characterised using three-endonuclease amplified fragment length polymorphism (AFLP) markers. Out of 416 bands obtained from six primer combinations, 254 (61 %) were polymorphic. The pairwise genetic dissimilarity among accessions ranged from 0.03 to 0.97. Cluster analysis based on neighbour joining showed two major clusters. Cluster I contained most of the bhut jolokia accessions whereas cluster II contained all of the Capsicum annuum genotypes. Similar grouping was observed with population STRUCTURE analysis as well as principle coordinate analysis. Analysis of molecular variance (AMOVA) revealed 45 and 54 % variation among and within populations, respectively. This information on population structure analysis and molecular characterisation will be helpful for effective utilisation of this germplasm in Capsicum improvement programs.     

A structural basis for the allosteric regulation of non-hydrolysing UDP-GlcNAc 2-epimerases

The non-hydrolysing bacterial UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc 2-epimerase) catalyses the conversion of UDP-GlcNAc into UDP-N-acetylmannosamine, an intermediate in the biosynthesis of several cell-surface polysaccharides. This enzyme is allosterically regulated by its substrate UDP-GlcNAc. The structure of the ternary complex between the Bacillus anthracis UDP-GlcNAc 2-epimerase, its substrate UDP-GlcNAc and the reaction intermediate UDP, showed direct interactions between UDP and its substrate, and between the complex and highly conserved enzyme residues, identifying the allosteric site of the enzyme. The binding of UDP-GlcNAc is associated with conformational changes in the active site of the enzyme. Kinetic data and mutagenesis of the highly conserved UDP-GlcNAc-interacting residues confirm their importance in the substrate binding and catalysis of the enzyme. This constitutes the first example to our knowledge, of an enzymatic allosteric activation by direct interaction between the substrate and the allosteric activator.     

Molecular effects of lithium exposure during mouse and chick gastrulation and subsequent valve dysmorphogenesis

BACKGROUND: Lithium (Li) has been associated with cardiac teratogenicity in the developing fetus. We took advantage of the association of therapeutic administration of Li with an increase in heart defects to gain insight into both normal and pathological heart and valve development with GSK‐3 inhibition. The objective of this study was to define whether Li mimicry of canonical Wnt/β‐catenin signaling induces cardiac valve defects. METHODS: Li was administered by a single intraperitoneal injection to the pregnant mouse on embryonic day E6.75, much earlier than heretofore analyzed. On E15.5 developing heart defects were defined by Doppler ultrasound. The embryonic hearts were analyzed for changes in patterning of active canonical Wnt expression and nuclear factor of the activated T cells‐c1 (NFATc1), both key regulators of valve development. Li‐exposed chick embryos were used to define the early cell populations during gastrulation that are susceptible to GSK‐3 inhibition and may relate to valve formation. RESULTS: Li exposure during gastrulation decreased the number of prechordal plate (PP) cells that reached the anterior intestinal portal, a region associated with valve development. Li decreased expression of Hex, an endoderm cardiac inducing molecule, normally also expressed by the PP cells, and of Sox 4 at the anterior intestinal portal and NFAT, critical factors in valvulogenesis. CONCLUSIONS: Cells existing already during gastrulation are associated with valve formation days later. The Wnt/β‐catenin signaling in PP cells is normally repressed by Wnt antagonists and Hex is up‐regulated. The antagonism occurring at the receptor level is bypassed by Li exposure by its intracellular inactivation of GSK‐3 directly to augment Wnt signaling. Birth Defects Research (Part A), 2008. © 2008 Wiley‐Liss, Inc.     

Genotypic differences in water status,membrane integrity,ionic content,N2-fixing efficiency and dry matter of mungbean nodules under saline irrigation

A pot experiment was conducted under natural conditions of screen house to evaluate the effect of saline irrigation given at flowering stage (30–35 DAS) on nodule functioning and their tolerance in two mungbean genotypes viz. Asha and Muskan based on various physiological traits. The pots containing sandy soil (Typic Torrispamments) were saturated with Cl-dominated saline irrigation to maintain ECe of 2.5, 5.0, 7.5 dS m⁻¹ as compared to control. In both the genotypes osmotic potential (Ψs) and relative water content (RWC %) of nodules decreased significantly, while a sharp rise in proline and total soluble sugars contents were observed with the increasing level of saline irrigation after 10 and 20 days of treatment. A marked increase in hydrogen peroxide (H₂O₂), lipid peroxidation (MDA content) and relative stress injury (RSI %) was noticed in nodules which were much higher in Muskan. The decrease in Ψs of nodules was more pronounced in Asha than in Muskan, while reverse was true for RWC and proline accumulation. A sharp decline in acetylene reduction assay (ARA) for N₂-fixation, leghemoglobin content and dry matter of the nodules was observed, but was more in Muskan than in Asha. Nitrogen (N) content declined while Na⁺/K⁺ ratio and Cl⁻ content increased significantly. The genotype Asha maintained better N₂-fixing efficiency but lower Na⁺/K⁺ ratio and Cl⁻ content in nodules than Muskan. Though the nodule functioning was further deteriorated at 20 DAT in both the genotypes yet the tolerance capacity of nodules in Asha was better than in Muskan under saline conditions which is correlated with the compensatory mechanism i.e. osmoregulation in nodules.Key words: Leghemoglobin, Lipid peroxidation, Minerals, N₂-ase activity, Vigna radiata, Water relations