Search

Aging is associated with functional decline and structural deteriorations of the myocardium and plays a crucial role in cardiovascular ailments. Decalepis hamiltonii (Dh) is well known medicinal plant used in traditional medicine and exhibits potent antioxidant properties. Therefore, the current study was proposed to assess the cardioprotective potential of Dh extract and its bioactive constituents [Ellagic acid (EA) and 4‑hydroxyisophthalic acid (4-HIA)] against isoproterenol (ISO)-induced region-specific myocardial damage in aging rats. Male albino Wistar rats aged 22 months were administered orally with Dh extract (100mg/kg BW), EA (50 mg/kg BW), and 4‑HIA (30 mg/kg BW) for one month before inducing MI using ISO (150 mg/kg BW) for the consecutive last two days. The ISO group exhibited a significant increase in hemodynamic parameters, cardiac marker enzymes, serum lipids, and oxidative stress markers. A significant reduction in region-specific antioxidant enzymes levels and histopathological alterations were observed in ISO-treated rats. Whereas, pretreatment of EA, 4-HIA, and Dh extract reduced the cardiac dysfunction biomarkers and mitigated oxidative stress by enhancing antioxidant enzymes and restoring histopathological changes in ISO-treated rats. The Overall findings give insights into the therapeutic applications of Dh extract and its compounds in ameliorating cardiovascular disorders through its antioxidant potential.

Glutamate dehydrogenase (GDH) is a salient metabolic enzyme which catalyzes the NAD+ - or NADP+ -dependent reversible conversion of α-ketoglutarate (AKG) to L-glutamate; and thereby connects the carbon and nitrogen metabolism cycles in all living organisms. The function of GDH is extensively regulated by both metabolites (citrate, succinate, etc.) and non-metabolites (ATP, NADH, etc.) but sufficient molecular evidences are lacking to rationalize the inhibitory effects by the metabolites. We have expressed and purified NADP+ -dependent Aspergillus terreus GDH (AtGDH) in recombinant form. Succinate, malonate, maleate, fumarate and tartrate independently inhibit the activity of AtGDH to different extents. The crystal structures of AtGDH complexed with the dicarboxylic acid metabolites and the coenzyme NADPH have been determined. Although AtGDH structures are not complexed with substrate; surprisingly, they acquire super closed conformation like previously reported for substrate and coenzyme bound catalytically competent Aspergillus niger GDH (AnGDH). These dicarboxylic acid metabolites partially occupy the same binding pocket as substrate; but interact with varying polar interactions and the coenzyme NADPH binds to the Domain-II of AtGDH. The low inhibition potential of tartrate as compared to other dicarboxylic acid metabolites is due to its weaker interactions of carboxylate groups with AtGDH. Our results suggest that the length of carbon skeleton and positioning of the carboxylate groups of inhibitors between two conserved lysine residues at the GDH active site might be the determinants of their inhibitory potency. Molecular details on the dicarboxylic acid metabolites bound AtGDH active site architecture presented here would be applicable to GDHs in general. This article is protected by copyright. All rights reserved.

Immune checkpoint inhibitors (ICIs) are a class of medications targeting mostly the PD-1/PD-L1 and CTLA-4 immune pathways in the treatment of many cancers. Despite the encouraging success of ICIs, they are associated with immune-related adverse events as well as exacerbation of underlying autoimmune conditions. The treatment of these conditions often involves discontinuation of ICI in addition to the utilization of immunomodulatory agents. In this report, we discuss a case in which a patient with metastatic renal cell carcinoma experienced exacerbation of underlying paraneoplastic dermatomyositis after treatment with ICI. He was successfully continued on ICI with the use of intravenous immunoglobulin. The patient experienced adequate control of his myositis but also experienced deepening of his antitumor response.

The focus of the metabolomic studies of mango fruit (Mangifera indica L.) so far has been mostly towards flavor and pulp quality metabolites and their biogenesis in domesticated cultivars. These studies are reliant on techniques, viz., gas chromatography and headspace solid-phase micro-extraction, and recently liquid chromatography–mass spectrometry. Here, we performed comparative untargeted metabolite analysis to understand flavor differentiation as fruit-specific changes in various biochemical pathways among Cv. Alphonso and two different natural mango populations from the Western Ghats, India. Significant stage-specific metabolome variations among these fruit sets were identified through principal component analysis. Furthermore, the multivariate analysis suggested quantitative expression variance in housekeeping and specialized metabolic pathways from the natural populations and Alphonso through network map for all the predominant biochemical pathways. The metabolites found in this study might be involved in growth and development processes, flavour, shelf-life, species interaction, and defences correlating with important general functions previously reported from mango and other fruits.

Ceropegia media is an endemic and endangered plant as its propagation through seeds is unreliable due to low germination, slow growth and seedling decay under natural conditions. Also, tubers of this plant are edible serving as carbohydrate source with medicinal values leading to severe population decline in the natural habitat. To provide a sustainable solution, an efficient in vitro propagation protocol along with phytochemical profiling was developed for C. media. Callus cultures were induced from seedling and wild leaf tissues using the most effective Murashige and Skoog’s (MS) medium with 2,4-dichlorophenoxyacetic acid (2,4-D; 2 µM) and sucrose (3%). Somatic embryos were acquired on MS medium with 1 µM 6-Benzylaminopurine (BAP) and 1 µM 2,4-D. Conversion into plantlets was attained only from tissue culture-derived seedling leaf (TCDSL) explant. Further, in vitro tuberization was achieved from TCDSL callus with BAP and Naphthalene acetic acid (NAA). AgNO3 as an elicitor had a positive effect on both fresh and dry weights of callus. Successful acclimatization (58%) was attained after two months resulting in normal phenotype in pots. Further, metabolite profiles of ten different tissues from wild and in vitro plants were compared. Total 82 compounds comprising alkaloids, fatty acids, fatty acid ester, steroids, terpenes and hydrocarbons were identified. Overall, results suggested enhanced production of selected metabolites with in vitro propagation and AgNO3, alleviating the problem of unavailability of planting materials. Thus, the current study might offer potential ways for the conservation of such RED enlisted species as C. media.

Based on detailed studies of the protologues, types, herbarium and comparison with live specimens, Caulokaempferia dinabandhuensis Biseshwori & Bipin, Globba teesta S. Nirola & A.P. Das and Zingibe...

Peritoneal carcinomatosis (PC) can occur as an advanced consequence of multiple primary malignancies. Surgical resection, radiation or systemic interventions alone have proven inadequate for this aggressive cancer presentation, since PC still has a poor survival profile. Photodynamic therapy (PDT), in which photosensitive drugs are exposed to light to generate cytotoxic reactive oxygen species, may be an ideal treatment for PC because of its ability to deliver treatment to a depth appropriate for peritoneal surface tumors. Additionally, epidermal growth factor receptor (EGFR) signaling plays a variety of roles in cancer progression and survival as well as PDT-mediated cytotoxicity, so EGFR inhibitors may be valuable in enhancing the therapeutic index of intraperitoneal PDT. This study examines escalating doses of benzoporphyrin derivative (BPD)-mediated intraperitoneal PDT combined with the EGFR-inhibitor cetuximab in a canine model. In the presence or absence of small bowel resection (SBR) and cetuximab, we observed a tolerable safety and toxicity profile related to the light dose received. Additionally, our findings that BPD levels are higher in the small bowel compared with other anatomical regions, and that the risk of anastomotic failure decreases at lower light doses will help to inform the design of similar PC treatments in humans.

The Indian subcontinent has experienced a major shift in climatic regime from a wet tropical regime to increased seasonal rainfall, since the late Miocene. This shift has been attributed to the intensification of monsoons, which led to opening up of dry habitats in humid forests and formation of deciduous forests. We explored the role of this climatic shift in the origin and diversification of dry-adapted plant genera Ceropegia and Brachystelma (Ceropegiae, Asclepiadoideae, Apocynaceae). We sampled Ceropegia and Brachystelma from across India and used five markers (two nuclear and three plastid regions) to reconstruct a global phylogeny of this group. Indian members of the tribe Ceropegiae were derived from Africa through at least four independent dispersal events. All dispersal events occurred in late Miocene after establishment of a monsoon climate. One of these early dispersing lineages underwent rapid radiation in peninsular India, giving rise to around 50 species. Thus, both dispersal and diversification events coincided with the intensification of monsoons and concomitant aridification. The role of environment in the evolution of floral characteristics and root type in the Indian radiation is also discussed. This is one of the first reports on a dry-adapted endemic radiation of plants in India. © 2020 Institute of Botany, Chinese Academy of Sciences

Treatment with epidermal growth factor receptor monoclonal antibodies extends life for patients with advanced colorectal cancers (CRCs) whose tumors exhibit wild-type KRAS, but KRAS testing may be underused. We studied the role of socioeconomic factors in the application of KRAS testing.We identified subjects with stage IV colorectal adenocarcinoma diagnosed 2010-2015 in the Surveillance, Epidemiology, and End Results (SEER) database. We used multivariable logistic regression models to evaluate associations between clinical/demographic factors and the rate of KRAS testing. We used multivariable-adjusted Cox proportional hazards models to assess survival.We identified 37,676 patients with stage IV CRC, 31.1% of whom were tested for KRAS mutations, of those who had documented KRAS testing, 44% were KRAS mutant. Patients were more likely to be tested if they were younger (odds ratio [OR]=5.10 for age 20 to 29 vs. 80+, 95% confidence interval [CI]: 3.99-6.54, P0.01), diagnosed more recently (OR=1.92 for 2015 vs. 2010, 95% CI: 1.77-2.08, P0.01), or lived in an area of high median household income (OR=1.24 for median household income of$69,311 vs.$49,265, 95% CI: 1.14-1.35, P0.01). Patients were less likely to be tested if they had Medicaid (OR=0.83, 95% CI: 0.77-0.88, P0.01) or were unmarried (OR=0.78, 95% CI: 0.75-0.82, P0.0001). The risk of death was decreased in patients who received KRAS testing (hazard ratio=0.77, 95% CI: 0.75-0.80, P0.01).We found a low rate of KRAS testing in CRC patients with those living in low-income areas less likely to be tested, even after controlling for Medicaid insurance. Our study suggests that socioeconomic disparities persist despite Medicaid insurance.

Glutamate dehydrogenase (GDH) is a salient metabolic enzyme which catalyzes the NAD+ - or NADP+-dependent reversible conversion of α-ketoglutarate (AKG) to L-glutamate; and thereby connects the carbon and nitrogen metabolism cycles in all living organisms. The function of GDH is extensively regulated by both metabolites (citrate, succinate, etc.) and non-metabolites (ATP, NADH, etc.) but sufficient molecular evidences are lacking to rationalize the inhibitory effects by the metabolites. We have expressed and purified NADP+-dependent Aspergillus terreus GDH (AtGDH) in recombinant form. Succinate, malonate, maleate, fumarate and tartrate independently inhibit the activity of AtGDH to different extents. The crystal structures of AtGDH complexed with the dicarboxylic acid metabolites and the coenzyme NADPH have been determined. Although AtGDH structures are not complexed with substrate; surprisingly, they acquire super closed conformation like previously reported for substrate and coenzyme bound catalytically competent Aspergillus niger GDH (AnGDH). These dicarboxylic acid metabolites partially occupy the same binding pocket as substrate; but interact with varying polar interactions and the coenzyme NADPH binds to the Domain-II of AtGDH. The low inhibition potential of tartrate as compared to other dicarboxylic acid metabolites is due to its weaker interactions of carboxylate groups with AtGDH. Our results suggest that the length of carbon skeleton and positioning of the carboxylate groups of inhibitors between two conserved lysine residues at the GDH active site might be the determinants of their inhibitory potency. Molecular details on the dicarboxylic acid metabolites bound AtGDH active site architecture presented here would be applicable to GDHs in general.

The carboxylesterases (EC 3.1.1.x) are widely distributed and form an important yet diverse group of hydrolases catalysing the ester bond cleavage in a variety of substrates. Besides acting on plant cell wall components like cutin, tannin and feruloyl esters, they are often the first line of defence to metabolize drugs, xenobiotics, pesticides, insecticides and plastic. But for the promiscuity of some carboxylesterases and cutinases, very few enzymes act exclusively on aromatic carboxylic acid esters. Infrequent occurrence of aromatic carboxylesterases suggests that aromatic carboxylesters are inherently more difficult to hydrolyse than the regular carboxylesters because of both steric and polar effects. Naturally occurring aromatic carboxylesters were rare before the anthropogenic activity augmented their environmental presence and diversity. An appraisal of the literature shows that the hydrolysis of aromatic carboxylic esters is a uniquely difficult endeavour and hence deserves special attention. Enzymes to hydrolyse such esters are evolving rapidly in nature. Very few such enzymes are known and they often display much lower catalytic efficiencies. Obviously, the esters of aromatic carboxylic acids, including polyethylene terephthalate waste, pose an environmental challenge. In this review, we highlight the uniqueness of aromatic carboxylesters and then underscore the importance of relevant carboxylesterases.

NADP-glutamate dehydrogenase from Aspergillus niger (AnGDH) exhibits sigmoidal 2-oxoglutarate saturation. Despite sharing 88% amino acid identity, the homologous enzyme from Aspergillus terreus (AtGDH) shows hyperbolic 2-oxoglutarate saturation. In order to address the structural origins of this phenomenon, six AnGDH-AtGDH chimeras were constructed and characterized. The C-terminal sequence (residues 315-460, named the D-segment) was implicated in the AnGDH cooperativity. The D-segment residues largely contribute to the monomer-monomer interface of each trimer in the native hexamer and are far removed from the enzyme active site. The D-segment appears to be a part of the allosteric network responsible for 2-oxoglutarate homotropic interactions in AnGDH. AnGDH and its C415S mutant, but not AtGDH, also showed atypical, biphasic ammonium saturation, particularly at sub-saturating 2-oxoglutarate concentrations. We found that the sigmoidal 2-oxoglutarate saturation and the biphasic ammonium response are tightly coupled; the analysis of AnGDH-AtGDH chimeras ascribes the two features to the AnGDH D-segment. The two non-Michaelis-Menten substrate saturations of AnGDH were influenced by ionic strength. Increase in ionic strength reduced the nH of 2-oxoglutarate saturation as well as abolished the biphasic response, suggesting that polar/ionic interactions determine the allosteric, inter-subunit communications. The biochemical analysis in the context of available structural data implicates the D-segment of AnGDH in the allosteric feature of this enzyme. The coupling of sigmoidal 2-oxoglutarate saturation and the biphasic ammonium response could possibly confer growth advantage to A. niger experiencing carbon and/or nitrogen limitation.

Wilhelmine Meeraus,1 Robert Wood,2 Rafal Jakubanis,2 Tim Holbrook,2 Geoffray Bizouard,3 Johanna Despres,3 Camille Correia Da Silva,4 Gaelle Nachbaur,4 Sarah H Landis,1 Yogesh Punekar,5 Bernard Aguilaniu,6 Afisi S Ismaila7,8 1GlaxoSmithKline, Stockley Park West, Uxbridge, UK; 2Adelphi Real World, Bollington, Cheshire, UK; 3IQVIA, Paris, France; 4GlaxoSmithKline, Rueil-Malmaison, France; 5ViiV Healthcare, Brentford, Middlesex, UK; 6Faculty of Medicine, University Grenoble-Alpes, Grenoble, France; 7GlaxoSmithKline, Collegeville, PA, USA; 8Department of Health Research Methods, Evidence and Impact, McMaster University, Hamilton, ON, Canada Background: Increasing availability of therapeutic options for COPD may drive new treatment pathways. This study describes COPD treatment in France, focusing on identifying initial treatment modifications in patients with COPD who either initiated long-acting bronchodilator (LABD)-based therapy or escalated to triple therapy (long-acting muscarinic antagonist [LAMA] + long-acting β2-agonist [LABA] + inhaled corticosteroid [ICS]). Methods: This retrospective analysis of patients with COPD in a large general practitioner database (IQVIA Longitudinal Patient Database) in France included two cohorts: Cohort 1 – new initiators of LABD-based therapy (LAMA, LABA, LAMA + LABA, LAMA + ICS, LABA + ICS or LAMA + LABA + ICS); Cohort 2 – patients escalating to triple therapy from mono- or dual-bronchodilator-based maintenance treatment. Both cohorts were indexed on the date of initiation/escalation (January 2008–December 2013), and the first treatment modification (at class level) within the 18-month post-index observational period was described. Five mutually exclusive outcomes were defined: continuous use (no modification), discontinuation (permanent [≥91 days with no restart] or temporary [≥91 days with subsequent restart]), switch, and augmentation (Cohort 1 only). Exploratory analysis of Cohort 1 explored potential drivers of treatment initiation. Results: Overall, 5,065 patients initiated LABD-based therapy (Cohort 1), and 501 escalated to triple therapy (Cohort 2). In Cohort 1, 7.0% of patients were continuous users, 46.5% discontinued permanently, 28.5% discontinued temporarily, 2.8% augmented (added LAMA and/or LABA and/or ICS), and 15.2% switched therapy. In Cohort 2, 18.2% of patients were continuous users, 7.2% discontinued permanently, 27.9% discontinued temporarily, and 46.7% switched therapy. Exploratory analyses showed that time since COPD diagnosis was first recorded, pre-index exacerbation events, and concomitant medical conditions were potential drivers of initial maintenance treatment choices. Conclusion: Discontinuation among new initiators of LABD-based therapy was high in France, whereas few switched or augmented treatment. In comparison, permanent discontinuation within 18 months was low in patients escalating to triple therapy. Keywords: triple therapy, France, treatment pathways, treatment modification, maintenance therapy

Tekupalli Ravikiran,1 Santosh Anand,2 Mohammad Azam Ansari,3 Mohammad N Alomary,4 Sami AlYahya,4 Sowbhagya Ramachandregowda,1 Saad Alghamdi,5 Anupama Sindhghatta Kariyappa,1 Bhagyalakshmi Dundaiah,1 Mamatha Madhugiri Gopinath,1 Sumreen Sultana,1 Shital Manohar Punekar,1 Thimappa Ramachandrappa Lakshmeesha1 1Department of Biotechnology, Bangalore University, Bangalore, India; 2Department of Biotechnology, School of Applied Sciences, Reva University, Bengaluru, India; 3Department of Epidemic Disease Research, Institutes for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, Dammam, 31441, Saudi Arabia; 4National Center for Biotechnology, Life Science and Environmental Research Institute, King Abdulaziz City for Science and Technology, Riyadh, Saudi Arabia; 5Laboratory Medicine Department, Faculty of Applied Medical Sciences, Umm Al-Qura University, Makkah, Saudi ArabiaCorrespondence: Thimappa Ramachandrappa LakshmeeshaDepartment of Biotechnology, Bangalore University, Bangalore, 560056, Karnataka, IndiaTel +91 8710937464Email lakshmeeshat6@gmail.comMohammad Azam AnsariDepartment of Epidemic Disease Research, Institutes for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, Dammam, 31441, Saudi ArabiaTel +966 580306835Email maansari@iau.edu.saPurpose: 4-Hydroxyisophthalic acid (4-HIA) is a bioactive compound present in the roots of Decalepis hamiltonii, which has attracted considerable attention in attenuating oxidative stress-related neurodegenerative diseases. However, its efficacy is limited because of its low solubility and bioavailability. Therefore, the present study aimed to encapsulate 4-HIA using biocompatible copolymer polylactide-co-glycolide (PLGA) and evaluate its antioxidant and neuroprotective potential.Methods: The nanoparticles (NPs) were fabricated by solid/oil/water (s/o/w) emulsion technique and characterized using XRD, SEM, HR-TEM, and FTIR spectroscopy. Antioxidant assays such as 1,1 diphenyl-2-picrylhydrazyl (DPPH), superoxide, and hydroxyl radical scavenging ability were performed to assess the antioxidant potential of the fabricated NPs.Results: The bioactive component, 4-HIA, was efficiently encapsulated by the PLGA polymer and was found to be spherical and smooth with a size < 10nm. 4-HIA showed better scavenging capability in DPPH and superoxide assays as compared to 4-HIA encapsulated PLGA and butylated hydroxytoluene (BHT). In contrast, 4-HIA encapsulated PLGA NPs exhibited a significant hydroxyl radical scavenging activity than 4-HIA and BHT alone. Further, the encapsulated NPs efficiently curtailed hydrogen peroxide (H2O2)-induced cytotoxicity in PC12 cells.Conclusion: Our findings indicate that 4-HIA encapsulated PLGA NPs might be a therapeutic intervention towards the effective management of oxidative stress as it has exhibited efficient neuroprotective potential against H2O2-induced oxidative stress in PC12 cells.Keywords: 4-Hydroxyisophthalic acid, polylactic-co-glycolic acid, nanoencapsulation, PC12 cells, cytotoxicity

In the search for optimal platforms for protein expression and secretion, filamentous fungi in principle provide some of the best microbial cell factories. They are inherently endowed with the ability to secrete proteins. Fungi belonging to Aspergillus and Trichoderma species are well-studied for industrial production of proteins and enzymes. Our understanding of these organisms at the level of transcription, translation, post-translational processing and the secretory pathways has improved significantly in recent years. Despite this, the ability of these fungal secretion platforms has not yet been able to match their intrinsic secretion capacity to produce foreign proteins. Details of the molecular mechanisms of the secretory pathways in filamentous fungi are emerging. This knowledge can be gainfully employed to enhance protein production in filamentous fungi, particularly in the secretion of heterologous proteins of value.

Penicillin binding proteins (PBPs) catalyzing transpeptidation reactions that stabilize the peptidoglycan component of the bacterial cell wall are the targets of β-lactams, the most clinically successful antibiotics to date. However, PBP-transpeptidation enzymology has evaded detailed analysis, because of the historical unavailability of kinetically competent assays with physiologically relevant substrates and the previously unappreciated contribution of protein cofactors to PBP activity. By re-engineering peptidoglycan synthesis, we have constructed a continuous spectrophotometric assay for transpeptidation of native or near native peptidoglycan precursors and fragments by Escherichia coli PBP1B, allowing us to (a) identify recognition elements of transpeptidase substrates, (b) reveal a novel mechanism of stereochemical editing within peptidoglycan transpeptidation, (c) assess the impact of peptidoglycan substrates on β-lactam targeting of transpeptidation, and (d) demonstrate that both substrates have to be bound before transpeptidation occurs. The results allow characterization of high molecular weight PBPs as enzymes and not merely the targets of β-lactam acylation.

Summary We identify the Plasmodium falciparum acetyl-coenzyme A synthetase (PfAcAS) as a druggable target, using genetic and chemical validation. In vitro evolution of resistance with two antiplasmodial drug-like compounds (MMV019721 and MMV084978) selects for mutations in PfAcAS. Metabolic profiling of compound-treated parasites reveals changes in acetyl-CoA levels for both compounds. Genome editing confirms that mutations in PfAcAS are sufficient to confer resistance. Knockdown studies demonstrate that PfAcAS is essential for asexual growth, and partial knockdown induces hypersensitivity to both compounds. In vitro biochemical assays using recombinantly expressed PfAcAS validates that MMV019721 and MMV084978 directly inhibit the enzyme by preventing CoA and acetate binding, respectively. Immunolocalization studies reveal that PfAcAS is primarily localized to the nucleus. Functional studies demonstrate inhibition of histone acetylation in compound-treated wild-type, but not in resistant parasites. Our findings identify and validate PfAcAS as an essential, druggable target involved in the epigenetic regulation of gene expression.

Geophytic Euphorbia species are distributed across four sections in subgenus Euphorbia of genus Euphorbia. At least nine Indian geophytic Euphorbia species have been identified, but delimitation of...

The Cretaceous-Paleogene boundary (KPB) mass extinction (~ 66.02 Ma) and the Paleocene-Eocene Thermal Maximum (PETM) (~ 55.8 Ma) are two remarkable climatic and faunal events in Earth's history that have implications for the current Anthropocene global warming and rapid diversity loss. Here we evaluate these two events at the stratotype localities in Tunisia and Egypt based on climate warming and environmental responses recorded in faunal and geochemical proxies. The KPB mass extinction is commonly attributed to the Chicxulub impact, but Deccan volcanism appears as a major culprit. New mercury analysis reveals that major Deccan eruptions accelerated during the last 10 ky and reached the tipping point leading up to the mass extinction. During the PETM, climate warmed rapidly by ~ 5 °C, which is mainly attributed to methane degassing from seafloor sediments during global warming linked to the North Atlantic Igneous Province (NAIP). Biological effects were transient, marked by temporary absence of most planktic foraminifera due to ocean acidification followed by the return of the pre-PETM fauna and diversification. In contrast, the current rapid rise in atmospheric CO_2 and climate warming are magnitudes faster than at the KPB or PETM events leading to predictions of a PETM-like response as best case scenario and rapidly approaching sixth mass extinction as worst-case scenario.

Pollination by flies (Diptera) has been important to the diversification and ecology of the flowering plants, but is poorly understood in contrast to pollination by other groups such as bees, butterflies and birds. Within the Apocynaceae the genera Ceropegia and Riocreuxia temporarily trap flies, releasing them after a fixed, species-specific period of time, during which pollination and/or pollen removal occurs. This “trap flower” pollination system shows convergent evolution with unrelated species in other families and fascinated Stefan Vogel for much of his career, leading to ground-breaking work on floral function in Ceropegia (Apocynaceae). In this new study we extend the work of the latest broad analysis published by some of the authors (Ollerton et al., 2009 − Annals of Botany). This incorporates previously unpublished data from India and Africa, as well as recently published information, on the diversity of po llinators exploited by Ceropegia. The analyses are based on a more accurate phylogenetic understanding of the relationships between the major groups, and significantly widens the biogeographic scope of our understanding of fly pollination within Ceropegia. Information about the pollinators of 69 taxa (species, subspecies and natural varieties) of Ceropegia is now available. Twenty five families of Diptera are known to visit the flowers of Ceropegia, of which sixteen are confirmed as pollinators. Most taxa are pollinated by species from a single family. Overall, there were no major biogeographic differences in the types of Diptera that were used in particular regions, though some subtle differences were apparent. Likewise there were no differences between the two major clades of Ceropegia, but clear differences when comparing the range of Diptera exploited by Ceropegia with that of the stapeliads. This clade, one of the largest in the Asclepiadoideae, is a fascinating example of a species radiation driven by an apparently relatively uniform set of pollinators.

Ceropegia karulensis is an endemic and critically endangered plant of the Western Ghats from India. Exploitation of the tubers and poor regeneration from seed has narrowed distribution and propagation of the species. There is a need to develop in vitro propagation methods for C. karulensis to alleviate these problems. Here, we optimized callus induction, somatic embryogenesis and microtuberization from different seedling explants viz. cotyledonary leaf and root. The environmental scanning electron microscopy was used to observe somatic embryonic origin and their developmental stages. Highest callus proliferation was recorded with 2 µM 6-benzylaminopurine and 1 µM 2,4-dichlorophenoxyacetic acid. Somatic embryos derived from cotyledonary leaf explants were more proliferative than root explants. The combination of 2 µM 6-benzylaminopurine, 2 µM naphthalene acetic acid and 7% sucrose in MS media resulted in highest microtuberization. Further, gas chromatography-mass spectrometry based metabolic profiling was carried out from native wild plants and in vitro callus tissues which identified various phytochemicals such as alkaloids, fatty acids, esters alcohols, etc. Multivariate analysis revealed the chemical disparities, where considerable variations were observed between native wild type and in vitro tissues, but no significant differences were found among in vitro callus from both root and cotyledonary explants. Overall, our results suggested that the production of various secondary metabolites found in C. karulensis was not affected by in vitro propagation and could be utilized in the conservation strategies for this plant.

Planktic foraminiferal analysis, including species populations, diversity trends, high-stress indices and stable isotopes of the latest Campanian through Maastrichtian in the South Atlantic, Tethys and Indian oceans reveal four major climate and faunal events that ended with the Cretaceous-Paleogene (K/Pg), formerly Cretaceous-Tertiary (K/T), mass extinction. The prelude to these events is the late Campanian cooling that reached minimum temperatures in the earliest Maastrichtian (base C31r) correlative with low primary productivity and species diversity. Event-1 begins during the persistent cool climate of the early Maastrichtian (lower C31r) when primary productivity rapidly increased accompanied by rapid species originations, attributed to increased nutrient influx from increased upwelling, erosion during the sea-level fall ~ 70.6 Ma, and Ninety East Ridge volcanism. During Event-2 (upper C31r to lower C30n), climate rapidly warmed by 2–3 °C in deep waters and peaked at 22 °C on land, primary productivity remained high and diversification reached maximum for the entire Cretaceous. We attribute this climate warming to intense Ninety East Ridge volcanic activity beginning ~ 69.5 Ma, accompanied by rapid reorganization of intermediate oceanic circulation. Enhanced greenhouse conditions due to the eruption of Deccan Phase-1 in India resulted in detrimental conditions for planktic foraminifera marking the end of diversification. Global cooling resumed in Event-3 (C30n), species diversity declined gradually accompanied by dwarfing, decreased large specialized species, increased small ecologically tolerant taxa, and ocean acidification. Event-3 is mainly the result of enhanced weathering and volcanogenic CO2 adsorption by the oceans during the preceding warm Event-2 that led to cooling and lower pH in the surface ocean. Event-4 marks the last 250 kyr of the Maastrichtian (C29r), which began with the largest Deccan eruptions (Phase-2) that caused rapid climate warming of 4 °C in deep waters and 8 °C on land, acid rain and ocean acidification leading to a major carbonate crisis preceding the K/T mass extinction.

The main objectives of this study, based on a large cohort of German COPD patients, were to assess the level of non-persistence (NP) and non-adherence (NA) with long-acting COPD inhaler treatment and to describe factors that may be associated with NP and NA.This was a retrospective cohort analysis based on claims data provided by a German statutory health insurance fund (years 2010-2012). NP was analyzed for treatment-naïve patients only; it was defined as a gap of90 days in medication availability. With regard to NA, first the overall yearly medication possession ratio (MPR) was analyzed, NA was defined as MPR80%. Secondly, adherence was explored only for the period in which a patient continued therapy with a long-acting COPD agent (no gap90 days).45,937 COPD patients who received at least one prescription of any long-acting COPD agent were identified (mean age 71.4 years; 45.2% female). Among these, 22,276 (42.4%) were classified as newly treated. The percentage of NP patients after 12 months was 65.3% on an overall patient level. Agent-specific NP rates were: 58.5% for LABA, 47.9% for LAMA, 78.0% for ICS, and 69.4% for single-device LABA/ICS combination treatment. The overall 12-month MPR across all agent classes on a patient level was 57.9% (70.0% of patients classified as non-adherent). During periods of general treatment continuation, the mean MPR/NA rates were 85.0%/30.1% (patient level across all agents), 89.3%/28.2% (LABA), 92.1%/16.2% (LAMA), 84.2%/43.8% (ICS) and 84.1%/42.8% (LABA/ICS combination). In the Cox regression analyses, several factors like female gender, higher CCI or lower number of specialist' visits were associated with earlier discontinuation of therapy. In comparison to LABA therapy, LAMA therapy was less likely to be associated with early NP, whereas patients who initiated ICS therapy or a single-device LABA/ICS combination therapy faced a higher NP risk.In German COPD patients, persistence and adherence with respect to long-acting bronchodilator therapy is poor. Approximately two thirds of patients fail to continue treatment after 12 months. In addition, about one third implement their treatment poorly during periods of general therapy continuation.

Exploration with high-throughput transcriptomics and metabolomics of two varieties of Ceropegia bulbosa identifies candidate genes, crucial metabolites and a potential cerpegin biosynthetic pathway. Ceropegia bulbosa is an important medicinal plant, used in the treatment of various ailments including diarrhea, dysentery, and syphilis. This is primarily attributed to the presence of pharmaceutically active secondary metabolites, especially cerpegin. As this plant belongs to an endemic threatened category, genomic resources are not available hampering exploration on the molecular basis of cerpegin accumulation till now. Therefore, we undertook high-throughput metabolomic and transcriptomic analyses using different tissues from two varieties namely, C. bulbosa var. bulbosa and C. bulbosa var. lushii. Metabolomic analysis revealed spatial and differential accumulation of various metabolites. We chemically synthesized and characterized the cerpegin and its derivatives by liquid chromatography tandem–mass spectrometry (LC–MS/MS). Importantly, these comparisons suggested the presence of cerpegin and 5-allyl cerpegin in all C. bulbosa tissues. Further, de novo transcriptome analysis indicated the presence of significant transcripts for secondary metabolic pathways through the Kyoto encyclopedia of genes and genomes database. Tissue-specific profiling of transcripts and metabolites showed a significant correlation, suggesting the intricate mechanism of cerpegin biosynthesis. The expression of potential candidate genes from the proposed cerpegin biosynthetic pathway was further validated by qRT-PCR and NanoString nCounter. Overall, our findings propose a potential route of cerpegin biosynthesis. Identified transcripts and metabolites have built a foundation as new molecular resources that could facilitate future research on biosynthesis, regulation, and engineering of cerpegin or other important metabolites in such non-model plants.

Cancer cells are known to have distinct metabolic characteristics compared with normal cells, given the catabolic and anabolic demands of increased cell growth and proliferation. This altered metabolism in cancer cells imbues differential dependencies, and substantial effort has been invested in developing therapeutic strategies to exploit these potential vulnerabilities. Parallel to these efforts has been a growing appreciation for the presence of notable intratumoral metabolic heterogeneity. Although many novel agents are showing some promising results in targeting specific metabolic processes, the challenge moving forward will be to develop combination strategies to address the aforementioned metabolic heterogeneity and its interplay with both epigenetic and immune factors in the tumor microenvironment. In this review, we discuss recent developments in targeting tumor catabolism, lipid biosynthesis, glycolysis, and the citric acid cycle as well as efforts to combine these approaches with immunotherapy.

Arginase is the only fungal ureohydrolase that is well documented in the literature. More recently, a novel route for agmatine catabolism in Aspergillus niger involving another ureohydrolase, 4-guanidinobutyrase (GBase), was reported. We present here a detailed characterization of A. niger GBase - the first fungal (and eukaryotic) enzyme to be studied in detail. A. niger GBase is a homohexamer with a native molecular weight of 336 kDa and an optimal pH of 7.5. Unlike arginase, the Mn2+ enzyme from the same fungus, purified GBase protein is associated with Zn2+ ions. A sensitive fluorescence assay was used to determine its kinetic parameters. GBase acted 25 times more efficiently on 4-guanidinobutyrate (GB) than 3-guanidinopropionic acid (GP). The Km for GB was 2.7±0.4 mM, whereas for GP it was 53.7±0.8 mM. While GB was an efficient nitrogen source, A. niger grew very poorly on GP. Constitutive expression of GBase favoured fungal growth on GP, indicating that GP catabolism is limited by intracellular GBase levels in A. niger. The absence of a specific GPase and the inability of GP to induce GBase expression confine the fungal growth on GP. That GP is a poor substrate for GBase and a very poor nitrogen source for A. niger offers an opportunity to select GBase specificity mutations. Further, it is now possible to compare two distinct ureohydrolases, namely arginase and GBase, from the same organism.

Made available in DSpace on 2019-10-06T16:57:32Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-01-23 British Ecological Society Consejo Nacional de Investigaciones Científicas y Técnicas Background and Aims: Large clades of angiosperms are often characterized by diverse interactions with pollinators, but how these pollination systems are structured phylogenetically and biogeographically is still uncertain for most families. Apocynaceae is a clade of >5300 species with a worldwide distribution. A database representing >10 % of species in the family was used to explore the diversity of pollinators and evolutionary shifts in pollination systems across major clades and regions. Methods: The database was compiled from published and unpublished reports. Plants were categorized into broad pollination systems and then subdivided to include bimodal systems. These were mapped against the five major divisions of the family, and against the smaller clades. Finally, pollination systems were mapped onto a phylogenetic reconstruction that included those species for which sequence data are available, and transition rates between pollination systems were calculated. Key Results: Most Apocynaceae are insect pollinated with few records of bird pollination. Almost three-quarters of species are pollinated by a single higher taxon (e.g. flies or moths); 7 % have bimodal pollination systems, whilst the remaining approx. 20 % are insect generalists. The less phenotypically specialized flowers of the Rauvolfioids are pollinated by a more restricted set of pollinators than are more complex flowers within the Apocynoids + Periplocoideae + Secamonoideae + Asclepiadoideae (APSA) clade. Certain combinations of bimodal pollination systems are more common than others. Some pollination systems are missing from particular regions, whilst others are over-represented. Conclusions: Within Apocynaceae, interactions with pollinators are highly structured both phylogenetically and biogeographically. Variation in transition rates between pollination systems suggest constraints on their evolution, whereas regional differences point to environmental effects such as filtering of certain pollinators from habitats. This is the most extensive analysis of its type so far attempted and gives important insights into the diversity and evolution of pollination systems in large clades. Faculty of Arts Science and Technology University of Northampton, Newton Building, Avenue Campus Lehrstuhl für Pflanzensystematik Universität Bayreuth Department of Systematic and Evolutionary Botany University of Zurich, Zollikerstrasse 107 Universidade Federal dos Vales do Jequitinhonha e Mucuri (UFVJM) Curso de Licenciatura em Educação do Campo - LEC Campus JK - Diamantina School of Life Sciences University of KwaZulu-Natal, Private Bag X01 Instituto de Botánica del Nordeste UNNE-CONICET Department of Plant Biology Ecology and Evolution, 301 Physical Sciences Laboratorio de Plantas Vasculares Departamento de Biología Comparada Facultad de Ciencias UNAM Laboratório de Ecologia da Polinização e Interações - LEPI Departamento de Botânica Instituto de Biociências Universidade Estadual Paulista 'Júlio de Mesquita Filho'- Unesp Saint Louis University Department of Biology Biology Extension Building, 1008 S. Spring Ave. Mehmet Akif Ersoy Mah. 269. Cad. Urankent Prestij Konutlarý Department of Biological Sciences University of Cape Town, P/Bag X3 Ecosystem Management Group ETH Zurich EDB UMR 5174 Université de Toulouse UPS, 118 route de Narbonne Laboratorio de Ecología Evolutiva - Biología Floral IMBIV (UNC-CONICET) Departamento de Botânica Museu Nacional Universidade Federal do Rio de Janeiro Quinta da Boa Vista Department of Plant Sciences Faculty of Natural and Agricultural Sciences University of the Free State, PO Box 339 Laboratório de Ciências Ambientais Centro de Biociências e Biotecnologia Universidade Estadual do Norte Fluminense Darcy Ribeiro Departamento de Botânica - CB Laboratório de Biologia Floral e Reprodutiva - POLINIZAR Universidade Federal de Pernambuco Universidade Federal de São Carlos - UFSCar Centro de Ciências Agrárias Depto. Ciências da Natureza Matemática e Educação, Rod. Anhanguera Km 174 Jardim Botânico do Rio de Janeiro, Rua Pacheco Leão 915 Facultad de Ciencias Exactas Fisicas y Naturales Universidad Nacional de Córdoba (UNC) IMBIV (CONICET-UNC) Herbarium - Royal Botanic Gardens Laboratorio de Investigaciones Botánicas (LABIBO) Facultad de Ciencias Naturales Universidad Nacional de Salta-CONICET, Av. Bolivia 5150 Department of Biosciences University of Salzburg Biology Department Franklin and Marshall College Natural History Museum Georgia College, 231 W. Hancock Street Centre for Ecology and Conservation University of Exeter Penryn Campus Department of Biology Willamette University Department of Botany Faculty of Science Chulalongkorn University, Phayathai Road Department of Biodiversity Earth and Environmental Sciences Academy of Natural Sciences Drexel University Center for Ecological Research Kyoto University, Hirano 2-509-3 Universidade Estadual Paulista UNESP Instituto de Biociências Departamento de Botânica Laboratório de Fenologia Tanzania Wildlife Research Institute (TAWIRI), PO Box 661 School of Biological Sciences Royal Holloway University of London Graduate School of Arts and Sciences University of Tokyo, 3-8-1 Komaba Universidade do Estado da Bahia, Campus IX. Rodovia BR 242, km 4, s/n. Flamengo Tropical Biology Association David Attenborough Building, Pembroke Street Instituto de Biologia - UFU, Campus Umuarama Bloco 2D Department of Botany Rhodes University, PO Box 94 Biospheres Eshwari, 52/403, Nanasaheb Peshva Marg, Near Ramna Ganpati Department of Evolution Ecology and Organismal Biology University of California Riverside, 900 University Avenue Departamento de Biologia Universidade Estadual de Feira de Santana, Av. Universitária s/n, Novo Horizonte Key Laboratory for Plant Diversity and Biogeography of East Asia Kunming Institute of Botany Chinese Academy of Sciences, 132 Lanhei Road Laboratorio de Ecología UBIPRO FES-Iztacala Universidad Nacional Autónoma de México Tlalnepantla de Baz Escuela de Ciencias Biológicas Universidad Pedagógica y Tecnológica de Colombia Departamento de Biologia Vegetal Instituto de Biologia Universidade Estadual de Campinas, Caixa Postal 6109 Department of Plant Sciences Natural and Agricultural Sciences University of the Free State, Qwaqwa campus, Kestell Road Penn State University, 542 ASI Building Department of Chemistry and Bioscience Aalborg University, Fredrik Bajers Vej 7H Graduate School of Human Development and Environment Kobe University, 3-11 Tsurukabuto Departamento de Biologia Vegetal Universidade Federal de VIçosa (UFV) Museo Botánico Córdoba y Cátedra de Morfologia Vegetal (IMBIVUNC-CONICET) Graduate School of Technology Industrial and Social Science Tokushima University, 2-1 Minamijyosanjima Centro Acadêmico de Vitória Universidade Federal de Pernambuco Departamento de Educação Universidade Federal da Paraiba Departamento de Engenharia e Meio Ambiente Universidade Federal da Paraiba Laboratório de Ecologia da Polinização e Interações - LEPI Departamento de Botânica Instituto de Biociências Universidade Estadual Paulista 'Júlio de Mesquita Filho'- Unesp Universidade Estadual Paulista UNESP Instituto de Biociências Departamento de Botânica Laboratório de Fenologia

Carbaryl is the most widely used carbamate family pesticide, and its persistent nature causes it to pollute both soil and water ecosystems. Microbes maintain the Earth’s biogeochemical cycles by metabolizing various compounds present in the matter, including xenobiotics, as a sole source of carbon, nitrogen, and energy. Soil isolate Pseudomonas sp. strain C5pp metabolizes carbaryl efficiently as the carbon source. Periplasmic carbaryl hydrolase catalyzes the conversion of carbaryl to 1-naphthol and methylamine. 1-Naphthol was further used as a carbon source via gentisate, whereas the metabolic fate of methylamine is not known. Here, we demonstrate that strain C5pp showed efficient growth on carbaryl when supplied as a carbon and nitrogen source, suggesting that the methylamine generated was used as the nitrogen source. Genes involved in the methylamine metabolism were annotated and characterized at the biochemical and molecular level. Transcriptional and enzyme activity studies corroborate that the γ-glutamylmethylamide/N-methylglutamate (GMA/NMG) pathway is involved in the metabolism of carbaryl and methylamine as a nitrogen source. Compared to carbaryl, methylamine was found to be an effective inducer for the metabolic and transporter genes. Strain C5pp also harbored genes involved in sarcosine metabolism that were cotranscribed and induced by sarcosine. The presence of inducible pathways for metabolism of carbaryl as a nitrogen and carbon source helps in complete and efficient mineralization of carbaryl by strain C5pp, thereby maintaining the biogeochemical cycles. IMPORTANCE The degradation of xenobiotics plays a significant role in the environment to maintain ecological systems as well as to prevent the imbalance of biogeochemical cycles via carbon-nitrogen cycling. Carbaryl is the most widely used pesticide from the carbamate family. Pseudomonas sp. strain C5pp, capable of utilizing carbaryl as a carbon and nitrogen source for its growth, subsequently helps in complete remediation of carbaryl. Thus, it maintains the ecosystem by balancing the biogeochemical cycles. The metabolic versatility and genetic diversity of strain C5pp for the transformation of contaminants like carbaryl and 1-naphthol into less harmful products make it a suitable candidate from the perspective of bioremediation.

This retrospective cohort study aimed to assess treatment patterns over 24 months amongst patients with chronic obstructive pulmonary disease (COPD), initiating a new COPD maintenance treatment, and to understand clinical indicators of treatment change. Patients included in the study initiated a long-acting β2-agonist (LABA), a long-acting muscarinic antagonist (LAMA), or a combination of LABA and an inhaled corticosteroid (ICS/LABA) between January 1, 2009, and November 30, 2013, as recorded in the United Kingdom Clinical Practice Research Datalink (UK CPRD). Treatment modifications (switching or adding maintenance treatments) over 24 months were assessed, and patient characteristics, disease burden, medication and healthcare resource use during the 30 days before treatment modification were evaluated. The cohort comprised 17,258 patients [LABA (8%), LAMA (39%) and ICS/LABA (54%)] with similar age, body mass index and dyspnoea distribution. LABA users were more likely than LAMA users to add a maintenance therapy. Distinct patterns of treatment augmentations were noted, whereby LABA users typically received dual therapy before moving to triple therapy, while LAMA users moved to triple therapy by directly adding an ICS/LABA. Exacerbation events immediately prior to treatment change were not frequently recorded; however, the need for rescue short-acting medication and assessment of dyspnoea in the 30 days prior to the treatment change suggest that dyspnoea is a remaining unmet need driving therapy change.

Isoprenoids and phenylpropanoids are the major secondary metabolite constituents in Ocimum genus. Though enzymes from phenylpropanoid pathway have been characterized from few plants, limited information exists on how they modulate levels of secondary metabolites. Here, we performed phenylpropanoid profiling in different tissues from five Ocimum species, which revealed significant variations in secondary metabolites including eugenol, eugenol methyl ether, estragole and methyl cinnamate levels. Expression analysis of eugenol synthase (EGS) gene showed higher transcript levels especially in young leaves and inflorescence; and were positively correlated with eugenol contents. Additionally, transcript levels of coniferyl alcohol acyl transferase, a key enzyme diverting pool of substrate to phenylpropanoids, were in accordance with their abundance in respective species. In particular, eugenol methyl transferase expression positively correlated with higher levels of eugenol methyl ether in Ocimum tenuiflorum. Further, EGSs were functionally characterized from four Ocimum species varying in their eugenol contents. Kinetic and expression analyses indicated, higher enzyme turnover and transcripts levels, in species accumulating more eugenol. Moreover, biochemical and bioinformatics studies demonstrated that coniferyl acetate was the preferred substrate over coumaryl acetate when used, individually or together, in the enzyme assay. Overall, this study revealed the preliminary evidence for varied accumulation of eugenol and its abundance over chavicol in these Ocimum species. Current findings could potentially provide novel insights for metabolic modulations in medicinal and aromatic plants.

Mass extinctions generally involve a complex array of interrelated causes and are best evaluated by a multi-proxy approach as applied here for the end-Cretaceous mass extinction. This study documents and compares the planktic foraminiferal records, carbonate dissolution effects, stable isotopes, and magnetic susceptibility in France (Bidart), Austria (Gamsbach) and Tunisia (Elles) in order to explore the environmental conditions during the uppermost Maastrichtian Plummerita hantkeninoides zone CF1 leading up to the mass extinction. Planktic foraminiferal assemblages at Bidart and Gamsbach appear to be more diverse than those at Elles, with unusually high abundance (20–30%) and diversity (~ 15 species) of globotruncanids in the two deep-water sections but lower abundance (

The genus Ocimum is a boutique of a diverse set of specialized metabolites such as terpenoids and phenylpropanoids. Each Ocimum species and its cultivars represent a characteristic chemical profile. The present study explored the two interspecific Ocimum hybrids originating through a serendipitous natural cross between O. kilimandscharicum and O. basilicum. These two novel Ocimum hybrids exhibited intermediate morphological features of two parental species. Inter simple sequence repeats (ISSR) analysis and DNA barcoding with the plastid non-coding trnH-psbA intergenic spacer region reaffirmed unambiguous parental identification and differentiation of these natural hybrids from other available Ocimum species. Consequently, gas chromatography-mass spectrometry-based metabolite profiling of two hybrids identified them as specific chemotypes with the presence of a unique blend of specialized metabolites from the parental species, which are either rich in terpenes or phenylpropanoids. Additionally, expression analysis of key genes from terpenoid and phenylpropanoid pathways corroborated with differential metabolite accumulation. Thus, these two Ocimum hybrids represented the novel chemotypes, which could be useful in commercial cultivation to produce novel essential oil and bioactive constituents. Further, the resulting metabolite diversity could have potential industrial applications in the areas of healthcare, bioremediation, and crop protection.

The final ~50 ky of the Maastrichtian leading up to the Cretaceous-Paleogene (K-Pg) boundary mass extinction at Bidart (France) and Gamsbach (Austria) record poor carbonate preservation indicative of ocean acidification preceding the mass extinction. Further evidence includes enhanced planktic foraminifera test fragmentation, anomalously low bulk-rock magnetic susceptibility (acidification) and peak mercury content (toxicity) related to peak Deccan volcanism in India. Here we present new experimental data that validates the Fragmentation Index of planktic foraminifera as a reliable proxy for the end-Cretaceous acidification. Pristine Cretaceous planktic foraminiferal shells (from DSDP 525A, palaeodepth ~1000 m) were exposed to buffers of pH 8.0, 7.5, 7.0 and 6.5 for 15 days each, and their preservation state was quantified as a function of time. The critical variables affecting test taphonomy are morphology, pH and time of exposure. Thin-walled fragile biserial species constitute an average ~60% of typical Late Maastrichtian assemblages (e.g Heterohelix globulosa and H. planata) and are most susceptible to dissolution, followed by simple coiled forms such as Rugoglobigerina sp. (~19% of the assemblage) and Hedbergella sp. (~6% of the assemblage). The globotruncanids (Globotruncana, Globotruncanita, ~12%) are least susceptible to chemical and physical damage. Lower pH conditions render tests more vulnerable to physical fragmentation. Caution is needed to assess taphonomic inflation of morphologically robust yet environmentally sensitive Cretaceous species (e.g. globotruncanids), that may result in an underestimation of the degree/nature of the faunal crisis and tempo of extinctions in the pre-extinction acidification interval.

Ocimum species produces a varied mix of different metabolites that imparts immense medicinal properties. To explore this chemo-diversity, we initially carried out metabolite profiling of different tissues of five Ocimum species and identified the major terpenes. This analysis broadly classified these five Ocimum species into two distinct chemotypes namely, phenylpropanoid-rich and terpene-rich. In particular, β-caryophyllene, myrcene, limonene, camphor, borneol and selinene were major terpenes present in these Ocimum species. Subsequently, transcriptomic analysis of pooled RNA samples from different tissues of Ocimum gratissimum, O. tenuiflorum and O. kilimandscharicum identified 38 unique transcripts of terpene synthase (TPS) gene family. Full-length gene cloning, followed by sequencing and phylogenetic analysis of three TPS transcripts were carried out along with their expression in various tissues. Terpenoid metabolite and expression profiling of candidate TPS genes in various tissues of Ocimum species revealed spatial variances. Further, putative TPS contig 19414 (TPS1) was selected to corroborate its role in terpene biosynthesis. Agrobacterium-mediated transient over-expression assay of TPS1 in the leaves of O. kilimandscharicum and subsequent metabolic and gene expression analyses indicated it as a cis-β-terpineol synthase. Overall, present study provided deeper understanding of terpene diversity in Ocimum species and might help in the enhancement of their terpene content through advanced biotechnological approaches. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12298-018-0612-6) contains supplementary material, which is available to authorized users.

All reactions relevant to biology belong to one of the two groups: those with a kinetic barrier and those with a thermodynamic barrier. The kinetic barrier can be overcome by employing a catalyst. Reactions with a thermodynamic barrier (uphill, endergonic reactions) require provision of energy (such as ATP hydrolysis) in addition to a catalyst. Regardless of the nature of barriers faced, greater than 99% of all the reactions occurring in biological systems are catalyzed. With minor exception of a few RNA catalysts (ribozymes), all the enzymes are built from a protein scaffold. These catalysts par excellence are at the very foundation of life. The three hallmark features of these biocatalysts are catalysis, specificity, and regulation.

Acids and bases are enormously important in enzyme chemistry. A thorough knowledge of acid–base chemistry is crucial to understand reaction mechanism and catalysis. According to the Bronsted (1923) definition of acids and bases, any substance (or a functional group) that has a tendency to lose a proton is an acid. Correspondingly, a base will then be a proton (H+) acceptor. This definition, as we will see below, eminently suits our understanding of the role of acid–base groups at the enzyme active site. However, Lewis provided a broader definition of acids and bases. Accordingly, a Lewis acid is a substance that accepts an electron pair from a base while a Lewis base is a substance that donates an electron pair to an acid. Lewis acids are involved in enzyme-catalyzed processes as cofactors. Metal cations such as Mg2+, Mn2+, Zn2+, and iron–sulfur clusters are Lewis acids. One way of visualizing this acidity is to consider water molecule coordinated to Zn2+, for instance. Because a lone pair of oxygen is donated to Zn2+ (the Lewis acid), the O-H bond of bound water is better polarized and therefore more readily loses a proton (i.e., H+). While the definition of base is practically same in the two definitions, the concept of Lewis acids is much broader and goes beyond just the H+ donors.

We have so far noted that a substrate molecule normally forms a productive complex at the enzyme active site. However there are cases where substrate also interacts with the enzyme (or the ES complex) in a nonproductive fashion. If this interaction is kinetically silent, it will not show up in routine steady-state kinetic analysis. Other methods (like equilibrium dialysis, fluorescence difference spectroscopy, or MALDI-TOF) may however be able to detect such binding phenomena. Most often nonproductive interactions of substrate are not considered at all – except when they also interact with the same enzyme either as activators or as inhibitors.

One marvels at the intricate design of living systems, and we cannot but wonder how life originated on this planet. Whether first biological structures emerged as the self-reproducing genetic templates (genetics-first origin of life) or the metabolic universality preceded the genome and eventually integrated it (metabolism-first origin of life) is still a matter of hot scientific debate. There is growing acceptance that the RNA world came first – as RNA molecules can perform both the functions of information storage and catalysis. Regardless of which view eventually gains acceptance, emergence of catalytic phenomena is at the core of biology. The last century has seen an explosive growth in our understanding of biological systems. The progression has involved successive emphasis on taxonomy → physiology → biochemistry → molecular biology → genetic engineering and finally the large-scale study of genomes. The field of molecular biology became largely synonymous with the study of DNA – the genetic material. Molecular biology however had its beginnings in the understanding of biomolecular structure and function. Appreciation of proteins, catalytic phenomena, and the function of enzymes had a large role to play in the progress of modern biology.

There can be no doubt that study of enzymes will continue to occupy the prime position in modern biology (and chemistry!). This much is amply obvious from each and every context and examples that we have come across in the preceding chapters. The study of enzymes in isolation, most often in purified form, has occupied much of the time in this field. However, as we have seen in the last chapter, the importance of understanding enzyme function in vivo is very much appreciated now; this will form one of the frontiers in enzymology. Present emphasis on systems biology is a pointer in this direction. Enzymes in sequence, in combination with other enzymes and other cellular components, bring in interesting features often not manifested by an enzyme in isolation – coupled reactions, regulatory networks, and distributed control of metabolism are some of them.

While enzymes can be studied from the viewpoint of (a) the active site environment, (b) kinetic mechanism, and (c) chemical mechanism, the enzyme is a unit, and all these approaches tell valid but partial tales. A complete understanding of enzyme mechanism requires a correlation of these and other information, like enzyme structure and its dynamics. Although chemical mechanisms have been elucidated for many enzymes, how they achieve a catalytically competent state has become approachable only recently through experiments and computation. Synergy between structure and plasticity results in the unique power of enzymes. Structural enzymology aims to address these catalytic motions in detail.

Side chains of many amino acid residues are known to participate in nucleophilic catalysis by forming covalent enzyme–substrate intermediates. Enzymes have a choice of many nucleophilic groups but have little to offer in terms of good electrophiles. Therefore a number of small molecules (cofactors and prosthetic groups) are recruited by nature to complement an apoenzyme – resulting in a functional holoenzyme. These small molecules act as temporary electron sinks during catalysis, by forming covalent adducts with substrates. Much of enzyme chemistry is carbanion chemistry. Abstraction of a proton or decarboxylation from a sp3 carbon leaves behind a carbanion that is not so stable. Developing carbanions may be stabilized – hence effecting rate accelerations – by suitably placing temporary electron sinks. Coenzymes like pyridoxal phosphate and thiamine pyrophosphate function to stabilize them via their electrophilic adducts. We already listed more commonly encountered electrophilic reagents in Table 31.1. Decarboxylations involving carboxylic acids (other than amino acids) have already been dealt with previously (Chap. 34). Amino acid transformations including decarboxylations offer a different chemical challenge and are described in this chapter.

The order of addition of substrates to and release of products from the enzyme active site, along with establishment of relative rates of various events, defines the kinetic mechanism. These mechanisms fall into two broad groups – those where the full complement of substrates have to assemble on the enzyme active site before the reaction occurs are termed “sequential mechanisms.” In the other category, a product(s) is released between additions of two substrates and are called “ping–pong mechanisms.” In this category, substitution on the enzyme active site groups occurs and hence is also known as double displacement mechanism. Study of these mechanisms is best approached by rigorous experimental design where data is collected by systematically varying one parameter at a time. This method of reduction is in full display in enzyme kinetics. In fact, elucidating enzyme kinetic mechanisms offers the best example of how scientific hypotheses are tested. Elements of scientific method in sequence include problem recognition → collation of available information → hypothesis building → experimentation → reasoning and deduction → and refining the hypothesis. These steps are iterated in arriving at an enzyme mechanism. Finally when experiments and measurements agree with the theory, truth is secured. In practical enzyme kinetics, this exercise translates into following steps (Table 18.1) for the elucidation of mechanisms.

Most organisms, however evolutionarily distant, contain a set of common metabolites. But their intracellular concentrations are unique to each individual species. This metabolic identity of an organism is the consequence of quantitative differences in relevant enzyme properties and their associated regulation. Features of metabolic regulation are unique to each organism – often within a closely related group of organisms. It is becoming increasingly clear that the concept of unity in biochemistry does not always extend to the metabolic pathway control and enzyme regulation. Historically, metabolic regulation and control of enzyme activity have developed as closely related phenomena. What then is the justification to place regulation of enzyme activity here in “Frontiers in Enzymology”? Over the years, molecular developments in biology have outshone the progress made in physiological and system-level understanding of organisms. While the basic principles of enzyme/metabolic regulation may have been uncovered, novel modes of regulation continue to be discovered. Nature continues to surprise us with original ways of regulating enzyme activity. The novelty may be in the conceptual mechanism or the regulatory ligands involved. For instance, fructose-2,6-bisphosphate as a regulator of glycolysis (at the phosphofructokinase step) was discovered much later (in the early 1980s) – many decades after the complete description of glycolytic enzymes. In this sense the topic of regulation of enzyme activity will always be at the frontiers of enzymology.

Understanding enzyme function is an exciting research because diverse and often unpredictable solutions are developed to perform seemingly impossible tasks. Enzymes are such powerful catalysts as they lower the height of the activation energy barrier. How do enzymes bring about this decrease in ΔG≠? Many excellent attempts to dissect this into discrete enthalpic and entropic factors have been made. Rate accelerations are favored when enzyme specifically binds and assembles substrates at the active site and then provides optimal arrangement of catalytic groups. This is clearly an entropic (ΔS≠) contribution. Stabilization of transition state through enthalpic (ΔH≠) factors like select hydrogen bonds, salt links, acid–base groups, covalent interactions, etc., is another feature. Various tricks that enzymes employ in achieving catalytic prowess are shown in Fig. 6.1. These components are best understood through case studies, and we will do this through representative examples for each.

The word enzyme (eνζυμη meaning in yeast in Greek), first used by Kuhne in 1877, is now well accepted to describe a biological catalyst. Majority of enzyme names today carry the suffix “-ase” as recommended for all enzyme names by Duclaux in 1898. Proteolytic enzymes are a significant exception to this generally accepted norm. Some of them have retained the older tradition of usually ending with “-in,” for example, trypsin, chymotrypsin, papain, and subtilisin.

Much before enzymes were identified as discrete biochemical entities, they found favor through their useful properties. Early applications included use of enzyme preparations in meat tenderizing and starch hydrolysis. From the very beginning, commercial enzyme applications have largely belonged to a group of hydrolytic reactions. But a few oxidative enzymes were also exploited. While this trend holds even today, examples of designer enzymes and catalysts for more complex chemical processes are being developed. The first application of diastase (α-amylase) was by Jokichi Takamine. His 1894 patent (US Patent No. 525823) describes a process to make Taka-diastase from Aspergillus oryzae. This α-amylase was useful as a digestive aid, in eliminating starchy material from textiles and laundry. In a short but succinct paper, E.F. Leuchs (in 1931) described “the action of saliva on starch.” The possible practical utility of such activity was clearly anticipated by him. The last line of his report reads “it will be possible to use saliva and gastric juice of killed animals very successfully in cases of defective digestion.”

Enzymes are the only biomolecules that combine three hallmark features, namely, catalysis, specificity, and regulation. No wonder that study of enzyme catalysis continues to occupy prime position in modern biology (and chemistry!). Up until the fashion of omics took over studying one enzyme at a time formed the thrust of enzymology. Importance of enzyme function in vivo will form one of the key frontiers with the present emphasis on systems biology as a pointer in this direction. Enzymes in sequence and in combination with other enzymes and other cellular components often manifest interesting features like coupled reactions, regulatory networks, and distributed control of metabolism. While single enzyme studies have taken the back seat, the knowledge gleaned from such research is important for a systems biology approach. After all any biologist who follows their research interest to the finest level of detail will become an enzymologist (quote by Perry Frey).

Enzymology is a quantitative and exact science. Therefore it is important to understand how enzyme activity is measured and presented. A robust and reliable measure of the progress of an enzyme-catalyzed reaction is first and foremost requirement. Like with any other chemical reaction, progress of an enzyme-catalyzed reaction can be monitored either by the product formed (d[P]/dt) or by the substrate consumed (−d[A]/dt). The two rates are of course related by the reaction stoichiometry. It is desirable and often safe to follow the formation of product – a substance is better estimated when it is formed in a background where very little (or none) of it exists. On the other hand, to measure a decrease in the concentration of a reactant as it disappears – a small change in a large background – becomes daunting. In practice, a small decrease in substrate is relatively more difficult to observe than to follow a buildup of product from nothing. This is particularly relevant when we wish to record the initial rate (rate during very early time after the reaction is initiated, abbreviated as “v”), which is given by d[A]/dt when [P] ≈ 0. This is the rate at the beginning of the reaction or the instantaneous rate extrapolated to time zero.

This is more so in enzymology, as it is a quantitative and exact branch of biology. It is necessary to understand how enzyme activity is measured, calculated, and presented.

Glutamate dehydrogenase (GDH) is a key enzyme connecting carbon and nitrogen metabolism in all living organisms. Despite extensive studies on GDHs from both prokaryotic and eukaryotic organisms in the last 40 years, the structural basis of the catalytic features of this enzyme remains incomplete. This study reports the structural basis of the GDH catalytic mechanism and allosteric behavior. We determined the first high-resolution crystal structures of glutamate dehydrogenase from the fungus Aspergillus niger (AnGDH), a unique NADP(+)-dependent allosteric enzyme that is forward-inhibited by the formation of mixed disulfide. We determined the structures of the active enzyme in its apo form and in binary/ternary complexes with bound substrate (α-ketoglutarate), inhibitor (isophthalate), coenzyme (NADPH), or two reaction intermediates (α-iminoglutarate and 2-amino-2-hydroxyglutarate). The structure of the forward-inhibited enzyme (fiAnGDH) was also determined. The hexameric AnGDH had three open subunits at one side and three partially closed protomers at the other, a configuration not previously reported. The AnGDH hexamers having subunits with different conformations indicated that its α-ketoglutarate–dependent homotropic cooperativity follows the Monod–Wyman–Changeux (MWC) model. Moreover, the position of the water attached to Asp-154 and Gly-153 defined the previously unresolved ammonium ion-binding pocket, and the binding site for the 2′-phosphate group of the coenzyme was also better defined by our structural data. Additional structural and mutagenesis experiments identified the residues essential for coenzyme recognition. This study reveals the structural features responsible for positioning α-ketoglutarate, NADPH, ammonium ion, and the reaction intermediates in the GDH active site.