Developmental- and stress-mediated expression analysis of cinnamoyl-CoA reductase 1 (CCR1) from Hibiscus cannabinus

Cinnamoyl-CoA reductase (CCR, EC 1.2.1.44) is an important enzyme responsible for lignin biosynthesis in plants that belongs to the family of oxidoreductases. We analyzed developmental, tissue specific, and stress-mediated expression of the HcCCR1 (HM151381) gene from Hibiscus cannabinus. Gene expression analysis revealed that HcCCR1 was highly upregulated in mature leaves of 16-week-old plants. The maximum downregulation and upregulation of HcCCR1 was caused by cold and MeJA treatment, respectively. Sequence analysis demonstrated that HcCCR1 protein (ADK24219) contains a conserved NWYCYGK catalytic domain, while bioinformatics prediction indicated the presence of a palmitoylation site in the HcCCR1 protein. Phylogenetic analysis showed that HcCCR1 is more closely related to HcCCR2 (AGJ84130) and AtCCR proteins than CCR-like proteins. Comparative sequence analysis showed presence of significant differences between HcCCR1 and HcCCR2, which are homologs of H. cannabinus. Expression analysis demonstrated that the HcCCR1 gene is modulated by different external stresses.     

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessaryprior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.     

Teasing apart plant community responses to N enrichment: the roles of resource limitation,competition and soil microbes

Although ecologists have documented the effects of nitrogen enrichment on productivity, diversity and species composition, we know little about the relative importance of the mechanisms driving these effects. We propose that distinct aspects of environmental change associated with N enrichment (resource limitation, asymmetric competition, and interactions with soil microbes) drive different aspects of plant response. We test this in greenhouse mesocosms, experimentally manipulating each factor across three ecosystems: tallgrass prairie, alpine tundra and desert grassland. We found that resource limitation controlled productivity responses to N enrichment in all systems. Asymmetric competition was responsible for diversity declines in two systems. Plant community composition was impacted by both asymmetric competition and altered soil microbes, with some contributions from resource limitation. Results suggest there may be generality in the mechanisms of plant community change with N enrichment. Understanding these links can help us better predict N response across a wide range of ecosystems.     

Roles of actin binding proteins in mammalian oocyte maturation and beyond

Actin nucleation factors, which promote the formation of new actin filaments, have emerged in the last decade as key regulatory factors controlling asymmetric division in mammalian oocytes. Actin nucleators such as formin-2, spire, and the ARP2/3 complex have been found to be important regulators of actin remodeling during oocyte maturation. Another class of actin-binding proteins including cofilin, tropomyosin, myosin motors, capping proteins, tropomodulin, and Ezrin-Radixin-Moesin proteins are thought to control actin cytoskeleton dynamics at various steps of oocyte maturation. In addition, actin dynamics controlling asymmetric-symmetric transitions after fertilization is a new area of investigation. Taken together, defining the mechanisms by which actin-binding proteins regulate actin cytoskeletons is crucial for understanding the basic biology of mammalian gamete formation and pre-implantation development.     

Structural insights into the catalytic mechanism of 5-methylthioribose 1-phosphate isomerase

5-Methylthioribose 1-phosphate isomerase (M1Pi) is a crucial enzyme involved in the universally conserved methionine salvage pathway (MSP) where it is known to catalyze the conversion of 5-methylthioribose 1-phosphate (MTR-1-P) to 5-methylthioribulose 1-phosphate (MTRu-1-P) via a mechanism which remains unspecified till date. Furthermore, although M1Pi has a discrete function, it surprisingly shares high structural similarity with two functionally non-related proteins such as ribose-1,5-bisphosphate isomerase (R15Pi) and the regulatory subunits of eukaryotic translation initiation factor 2B (eIF2B). To identify the distinct structural features that lead to divergent functional obligations of M1Pi as well as to understand the mechanism of enzyme catalysis, the crystal structure of M1Pi from a hyperthermophilic archaeon Pyrococcus horikoshii OT3 was determined. A meticulous structural investigation of the dimeric M1Pi revealed the presence of an N-terminal extension and a hydrophobic patch absent in R15Pi and the regulatory α-subunit of eIF2B. Furthermore, unlike R15Pi in which a kink formation is observed in one of the helices, the domain movement of M1Pi is distinguished by a forward shift in a loop covering the active-site pocket. All these structural attributes contribute towards a hydrophobic microenvironment in the vicinity of the active site of the enzyme making it favorable for the reaction mechanism to commence. Thus, a hydrophobic active-site microenvironment in addition to the availability of optimal amino-acid residues surrounding the catalytic residues in M1Pi led us to propose its probable reaction mechanism via a cis-phosphoenolate intermediate formation.     

Biophysical factors in the regulation of asymmetric division of stem cells

Stem cells are a promising cell source for regenerative medicine due to their characteristics of self‐renewal and differentiation. The intricate balance between these two cell fates is maintained by precisely controlled symmetric and asymmetric cell divisions. Asymmetric division has a fundamental importance in maintaining tissue homeostasis and in the development of multi‐cellular organisms. For example, during development, asymmetric cell divisions are responsible for the formation of the body axis. Mechanistically, mitotic spindle dynamics determine the assembly and separation of chromosomes and regulate the orientation of cell division. Interestingly, symmetric and asymmetric cell division is not mutually exclusive and a range of factors are involved in such cell‐fate decisions, the measurement of which can provide efficient and reliable information on the regenerative potential of a cell. The balance between self‐renewal and differentiation in stem cells is controlled by various biophysical and biochemical cues. Although the role of biochemical factors in asymmetric stem cell division has been widely studied, the effect of biophysical cues in stem‐cell self‐renewal is not comprehensively understood. Herein, we review the biological relevance of stem‐cell asymmetric division to regenerative medicine and discuss the influences of various intrinsic and extrinsic biophysical cues in stem‐cell self‐renewal. This review particularly aims to inform the clinical translation of efforts to control the self‐renewal ability of stem cells through the tuning of various biophysical cues.     

Modifying bio-catalytic properties of enzymes for efficient biocatalysis: a review from immobilization strategies viewpoint

Enzyme-based catalysis has become one of the most important disciplines in organic synthesis and plays a noteworthy role in the establishment of many chemical industries, e.g. fine chemicals, food or energy, textiles, agricultural, cosmeceutical, medicinal and pharmaceutical industries. However, pristine enzymes fail to demonstrate requisite functionalities for an industrial setting where extremely specific and stable catalysts are required. Immobilization enhances the catalytic stability and activity of enzymes and trims the overall cost burden of the enzyme. Therefore, it widely endeavours for proficient, sustainable, and environmentally responsive catalytic processes. Amongst several immobilization strategies, e.g. (1) supports-assisted, i.e. physical or covalent coupling and (2) supports-free techniques, i.e. cross-linked enzyme crystals (CLECs) or aggregates are the most promising ones and widely pursued for enzyme immobilization purposes. This perspective review focuses on up-to-date developments in the area of enzyme immobilization and presents their potentialities to upgrade and/or modify enzyme properties. Both types of immobilization strategies, i.e. supports-assisted and supports-free techniques are discussed with particular reference to CLECs or aggregates and protein-coated microcrystals. Also, several useful traits achieved after immobilization are also discussed in the second half of the review.     

A Surface-Induced Asymmetric Program Promotes Tissue Colonization by Pseudomonas aeruginosa

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