Vacuolar targeting of r‐proteins in sugarcane leads to higher levels of purifiable commercially equivalent recombinant proteins in cane juice

Sugarcane is an ideal candidate for biofarming applications because of its large biomass, rapid growth rate, efficient carbon fixation pathway and a well‐developed storage tissue system. Vacuoles occupy a large proportion of the storage parenchyma cells in the sugarcane stem, and the stored products can be harvested as juice by crushing the cane. Hence, for the production of any high‐value protein, it could be targeted to the lytic vacuoles so as to extract and purify the protein of interest from the juice. There is no consensus vacuolar‐targeting sequence so far to target any heterologous proteins to sugarcane vacuole. Hence, in this study, we identified an N‐terminal 78‐bp‐long putative vacuolar‐targeting sequence from the N‐terminal domain of unknown function (DUF) in Triticum aestivum 6‐SFT (sucrose: fructan 6‐fructosyl transferase). In this study, we have generated sugarcane transgenics with gene coding for the green fluorescent protein (GFP) fused with the vacuolar‐targeting determinants at the N‐terminal driven by a strong constitutive promoter (Port ubi882) and demonstrated the targeting of GFP to the vacuoles. In addition, we have also generated transgenics with His‐tagged β‐glucuronidase (GUS) and aprotinin targeted to the lytic vacuole, and these two proteins were isolated and purified from the transgenic sugarcane and compared with commercially available protein samples. Our studies have demonstrated that the novel vacuolar‐targeting determinant could localize recombinant proteins (r‐proteins) to the vacuole in high concentrations and such targeted r‐proteins can be purified from the juice with a few simple steps.     

Population connectivity and genetic structure of Asian green mussel,Perna viridis along Indian waters assessed using mitochondrial markers

Molecular Biology Reports - Perna viridis (Linnaeus, 1758), the Asian green mussel, belonging to the family Mytilidae is widely distributed along the Indian coast. The species is majorly found in...     

Drug repositioning for orphan diseases

The need and opportunity to discover therapeutics for rare or orphan diseases are enormous. Due to limited prevalence and/or commercial potential, of the approximately 6000 orphan diseases (defined by the FDA Orphan Drug Act as <200 000 US prevalence), only a small fraction (5%) is of interest to the biopharmaceutical industry. The fact that drug development is complicated, time-consuming and expensive with extremely low success rates only adds to the low rate of therapeutics available for orphan diseases. An alternative and efficient strategy to boost the discovery of orphan disease therapeutics is to find connections between an existing drug product and orphan disease. Drug Repositioning or Drug Repurposing--finding a new indication for a drug--is one way to maximize the potential of a drug. The advantages of this approach are manifold, but rational drug repositioning for orphan diseases is not trivial and poses several formidable challenges--pharmacologically and computationally. Most of the repositioned drugs currently in the market are the result of serendipity. One reason the connection between drug candidates and their potential new applications are not identified in an earlier or more systematic fashion is that the underlying mechanism 'connecting' them is either very intricate and unknown or indirect or dispersed and buried in an ever-increasing sea of information, much of which is emerging only recently and therefore is not well organized. In this study, we will review some of these issues and the current methodologies adopted or proposed to overcome them and translate chemical and biological discoveries into safe and effective orphan disease therapeutics.     

Electroencephalographic Variation during End Maintenance and Emergence from Surgical Anesthesia

The re-establishment of conscious awareness after discontinuing general anesthesia has often been assumed to be the inverse of loss of consciousness. This is despite the obvious asymmetry in the initiation and termination of natural sleep. In order to characterize the restoration of consciousness after surgery, we recorded frontal electroencephalograph (EEG) from 100 patients in the operating room during maintenance and emergence from general anesthesia. We have defined, for the first time, 4 steady-state patterns of anesthetic maintenance based on the relative EEG power in the slow-wave (<14 Hz) frequency bands that dominate sleep and anesthesia. Unlike single-drug experiments performed in healthy volunteers, we found that surgical patients exhibited greater electroencephalographic heterogeneity while re-establishing conscious awareness after drug discontinuation. Moreover, these emergence patterns could be broadly grouped according to the duration and rapidity of transitions amongst these slow-wave dominated brain states that precede awakening. Most patients progressed gradually from a pattern characterized by strong peaks of delta (0.5–4 Hz) and alpha/spindle (8–14 Hz) power (‘Slow-Wave Anesthesia’) to a state marked by low delta-spindle power (‘Non Slow-Wave Anesthesia’) before awakening. However, 31% of patients transitioned abruptly from Slow-Wave Anesthesia to waking; they were also more likely to express pain in the post-operative period. Our results, based on sleep-staging classification, provide the first systematized nomenclature for tracking brain states under general anesthesia from maintenance to emergence, and suggest that these transitions may correlate with post-operative outcomes such as pain.     

Double Deception: Ant-Mimicking Spiders Elude Both Visually- and Chemically-Oriented Predators

Biological mimicry is often multimodal, in that a mimic reinforces its resemblance to another organism via different kinds of signals that can be perceived by a specific target audience. In this paper we describe a novel scenario, in which a mimic deceives at least two distinct audiences, each of which relies primarily on a different sensory modality for decision-making. We have previously shown that Peckhamia picata, a myrmecomorphic spider that morphologically and behaviorally resembles the ant Camponotus nearcticus, experiences reduced predation by visually-oriented jumping spiders. Here we report that Peckhamia also faces reduced aggression from spider-hunting sphecid wasps as well as from its model ant, both of which use chemical cues to identify prey. We also report that Peckhamia does not chemically resemble its model ants, and that its total cuticular hydrocarbons are significantly lower than those of the ants and non-mimic spiders. Although further studies are needed to clarify the basis of Peckhamia''s chemically-mediated protection, to our knowledge, such ‘double deception,’ in which a single organism sends misleading visual cues to one set of predators while chemically misleading another set, has not been reported; however, it is likely to be common among what have until now been considered purely visual mimics.     

NMR characterization of conformational fluctuations and noncovalent interactions of SUMO protein from Drosophila melanogaster (dSmt3)

Structural heterogeneity in the native-state ensemble of dSmt3, the only small ubiquitin-like modifier (SUMO) in Drosophila melanogaster, was investigated and compared with its human homologue SUMO1. Temperature dependence of amide proton's chemical shift was studied to identify amino acids possessing alternative structural conformations in the native state. Effect of small concentration of denaturant (1M urea) on this population was also monitored to assess the ruggedness of near-native energy landscape. Owing to presence of many such amino acids, especially in the β₂-loop-α region, the native state of dSmt3 seems more flexible in comparison to SUMO1. Information about backbone dynamics in ns-ps timescale was quantified from the measurement of ¹⁵N-relaxation experiments. Furthermore, the noncovalent interaction of dSmt3 and SUMO1 with Daxx12 (Daxx⁷²⁹DPEEIIVLSDSD⁷⁴⁰), a [V/I]-X-[V/I]-[V/I]-based SUMO interaction motif, was characterized using Bio-layer Interferometery and NMR spectroscopy. Daxx12 fits itself in the groove formed by β₂-loop-α structural region in both dSmt3 and SUMO1, but the binding is stronger with the former. Flexibility of β₂-loop-α region in dSmt3 is suspected to assist its interaction with Daxx12. Our results highlight the role of native-state flexibility in assisting noncovalent interactions of SUMO proteins especially in organisms where a single SUMO isoform has to tackle multiple substrates single handedly.     

Enhanced phosphorus nutrition in monocots and dicots over-expressing a phosphorus-responsive type I H+-pyrophosphatase

Plants challenged by limited phosphorus undergo dramatic morphological and architectural changes in their root systems in order to increase their absorptive surface area. In this paper, it is shown that phosphorus deficiency results in increased expression of the type I H⁺-pyrophosphatase AVP1 (AVP, Arabidopsis vacuolar pyrophosphatase), subsequent increased P-type adenosine triphosphatase (P-ATPase)-mediated rhizosphere acidification and root proliferation. Molecular genetic manipulation of AVP1 expression in Arabidopsis , tomato and rice results in plants that outperform controls when challenged with limited phosphorus. However, AVP1 over-expression and the resulting rhizosphere acidification do not result in increased sensitivity to AlPO₄, apparently because of the enhancement of potassium uptake and the release of organic acids. Thus, the over-expression of type I H⁺-pyrophosphatases appears to be a generally applicable technology to help alleviate agricultural losses in low-phosphorus tropical/subtropical soils and to reduce phosphorus runoff pollution of aquatic and marine environments resulting from fertilizer application.     

DNA Damage Regulates Translation through β-TRCP Targeting of CReP

The Skp1-Cul1-F box complex (SCF) associates with any one of a number of F box proteins, which serve as substrate binding adaptors. The human F box protein βTRCP directs the conjugation of ubiquitin to a variety of substrate proteins, leading to the destruction of the substrate by the proteasome. To identify βTRCP substrates, we employed a recently-developed technique, called Ligase Trapping, wherein a ubiquitin ligase is fused to a ubiquitin-binding domain to “trap” ubiquitinated substrates. 88% of the candidate substrates that we examined were bona fide substrates, comprising twelve previously validated substrates, eleven new substrates and three false positives. One βTRCP substrate, CReP, is a Protein Phosphatase 1 (PP1) specificity subunit that targets the translation initiation factor eIF2α to promote the removal of a stress-induced inhibitory phosphorylation and increase cap-dependent translation. We found that CReP is targeted by βTRCP for degradation upon DNA damage. Using a stable CReP allele, we show that depletion of CReP is required for the full induction of eIF2α phosphorylation upon DNA damage, and contributes to keeping the levels of translation low as cells recover from DNA damage.     

Cloning and characterization of two neuropeptide genes from cereal cyst nematode, Heterodera avanae from India

The cereal cyst nematode, Heterodera avenae (Wollenweber, 1924) is one of the most important plant parasitic nematodes of cereals. It is an obligate sedentary endo parasite causing considerable crop losses in wheat, barley and oats worldwide. FMRFamide-like peptides (FLPs) play critical role as neurotransmitters or neuromodulators in the nervous system and proposed as one of the important targets for the plant parasitic nematode management. Therefore, for the first time we have cloned and characterized two neuropeptide genes (flp-12 and flp-16) from the cDNA library of feeding female of H. avenae. Sequence analysis of FLPs revealed that both the neuropeptides are closely related with the parasitic as well as free-living nematodes. The flp-12 contains putative 22 residue long signal peptide at N-terminal suggesting its association with extra-cellular functions, while flp-16 does not contain signal peptide. Besides this, we have found highly conserved motif KFEFIRF in flp-12 and RFGK motif in flp-16. These two flp genes could be interesting and potential targets for functional validation to explore their utility for designing management strategies.