Carboxymethyl cellulase production optimization from Glutamicibacter arilaitensis strain ALA4 and its application in lignocellulosic waste biomass saccharification

Abstract

In this context, carboxymethyl cellulase (CMCase) production from Glutamicibacter arilaitensis strain ALA4 was initially optimized by one factor at a time (OFAT) method using goat dung as proficient feedstock. Two-level full factorial design (2⁵ factorial matrix) using first-order polynomial model revealed the significant (p?<?0.05) influence of pH, moisture, and peptone on CMCase activity. Central composite design at N?=?20 was further taken into account using a second-order polynomial equation, and thereby liberated maximum CMCase activity of 4925.56?±?31.61?U/g in the goat dung medium of pH 8.0 and 100% moisture containing 1% (w/w) peptone, which was approximately two fold increment with respect to OFAT method. Furthermore, the partially purified CMCase exhibited stability not only at high pH and temperature but also in the presence of varied metal ions, organic solvents, surfactants, and inhibitors with pronounced residual activities. The enzymatic hydrolysis using partially purified CMCase depicted the maximum liberation of fermentable sugars from alkali pretreated lignocellulosic wastes biomass in the order of paddy straw (13.8?±?0.15?mg/g)?>?pomegranate peel (9.1?±?0.18?mg/g)?>?sweet lime peel (8.37?±?0.16?mg/g), with saccharification efficiency of 62.1?±?0.8, 40.95?±?0.4, and 37.66?±?0.4%, respectively after 72?hr of treatment.     

‘Clustering’ SIRPα into the Plasma Membrane Lipid Microdomains Is Required for Activated Monocytes and Macrophages to Mediate Effective Cell Surface Interactions with CD47

SIRPα, an ITIMs-containing signaling receptor, negatively regulates leukocyte responses through extracellular interactions with CD47. However, the dynamics of SIRPα-CD47 interactions on the cell surface and the governing mechanisms remain unclear. Here we report that while the purified SIRPα binds to CD47 and that SIRPα is expressed on monocytes and monocytic THP-1 or U937, these SIRPα are ineffective to mediate cell binding to immobilized CD47. However, cell binding to CD47 is significantly enhanced when monocytes transmigrating across endothelia, or being differentiated into macrophages. Cell surface labeling reveals SIRPα to be diffused on naïve monocytes but highly clustered on transmigrated monocytes and macrophages. Protein crosslink and equilibrium centrifugation confirm that SIRPα in the latter cells forms oligomerized complexes resulting in increased avidity for CD47 binding. Furthermore, formation of SIRPα complexes/clusters requires the plasma membrane ‘lipid rafts’ and the activity of Src family kinase during macrophage differentiation. These results together suggest that ‘clustering’ SIRPα into plasma membrane microdomains is essential for activated monocytes and macrophages to effectively interact with CD47 and initiate intracellular signaling.     

Impact of VP1-Specific Protein Sequence Motifs on Adeno-Associated Virus Type 2 Intracellular Trafficking and Nuclear Entry

Adeno-associated virus type 2 (AAV2) has gained much interest as a gene delivery vector. A hallmark of AAV2-mediated gene transfer is an intracellular conformational change of the virus capsid, leading to the exposure of infection-relevant protein domains. These protein domains, which are located on the N-terminal portion of the structural proteins VP1 and VP2, include a catalytic phospholipase A(2) domain and three clusters of basic amino acids. We have identified additional protein sequence motifs located on the VP1/2 N terminus that also proved to be obligatory for virus infectivity. These motifs include signals that are known to be involved in protein interaction, endosomal sorting and signal transduction in eukaryotic cells. Among different AAV serotypes they are highly conserved and mutation of critical amino acids of the respective motifs led to a severe infection-deficient phenotype. In particular, mutation of a YXXQ-sequence motif significantly reduced accumulation of virus capsids around the nucleus in comparison to wild-type AAV2. Interestingly, intracellular trafficking of AAV2 was shown to be independent of PLA(2) activity. Moreover, mutation of three PDZ-binding motifs, which are located consecutively at the very tip of the VP1 N terminus, revealed a nuclear transport-defective phenotype, suggesting a role in nuclear uptake of the virus through an as-yet-unknown mechanism.     

A facile and rapid method for green synthesis of Achyranthes aspera stem extract-mediated silver nano-composites with cidal potential against Aedes aegypti L.

Aedes aegypti L. is the primary vector associated with transmission of globally concerned diseases; Zika, yellow fever, dengue and Chikungunya. Present study investigates an efficient, alternative and comparative approach for mosquito control which is safe to environment and non-target organisms. The silver nano-composites (AgNCs) were synthesized from the aqueous stem extract of Achyranthes aspera (AASE) using different concentration of aqueous silver nitrate (AgNO₃). The synthesis was tracked by UV-vis spectrophotometer and particle size analyser (DLS). The evaluation of their larvicidal potential against early fourth instars of Ae. aegypti showed significant potency, the toxicity increasing with the concentration of silver nitrate. The 24, 48 and 72 h bioassays resulted in respective LC₅₀ values of 26.693, 1.113 and 0.610 μg/mL (3 mM AASE-AgNO₃) 9.119, 0.420 and 0.407 μg/mL (4 mM AASE-AgNO₃) and that of 4.283, 0.3 and 0.248 μg/mL (5 mM AASE-AgNO₃). Keeping in view the significantly high larvicidal efficiency at lower concentration of silver nitrate, the 4 mM nano-composites were selected over 5 mM composites for further biophysical characterization carried out by X-ray Diffraction (XRD), Fourier transform infrared spectrometer (FTIR), Scanning electron microscopy (SEM), Energy dispersive X-ray (EDX) spectroscopy and Transmission electron microscopy (TEM). SEM and TEM confirmed the synthesis of spherical poly-dispersed AgNCs with average size ranging from 1–30 nm. Characterization through XRD showed the crystalline face-centered-cubic (fcc) structure of AgNCs with the highest intense peak obtained at 2θ value of 31.82°. FT-IR data suggests complex nature of AgNCs showing clearly defined peaks in different ranges. The present investigations recommend AgNCs of A. aspera stems as a low-cost and eco-friendly alternative to chemical insecticides for mosquito control.     

Analysis of the grasspea proteome and identification of stress-responsive proteins upon exposure to high salinity, low temperature, and abscisic acid treatment

Abiotic stress causes diverse biochemical and physiological changes in plants and limits crop productivity. Plants respond and adapt to such stress by altering their cellular metabolism and activating various defense machineries. To understand the molecular basis of stress tolerance in plants, we have developed differential proteomes in a hardy legume, grasspea (Lathyrus sativus L.). Five-week-old grasspea seedlings were subjected independently to high salinity, low temperature and abscisic acid treatment for duration of 36 h. The physiological changes of stressed seedlings were monitored, and correlated with the temporal changes of proteome using two-dimensional gel electrophoresis. Approximately, 400 protein spots were detected in each of the stress proteome with one-fourth showing more than 2-fold differences in expression values. Eighty such proteins were subjected to LC-tandem MS/MS analyses that led to the identification of 48 stress-responsive proteins (SRPs) presumably involved in a variety of functions, including metabolism, signal transduction, protein biogenesis and degradation, and cell defense and rescue. While 33 proteins were responsive to all three treatments, 15 proteins were expressed in stress-specific manner. Further, we explored the possible role of ROS in triggering the stress-induced degradation of large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase (Rubisco). These results might help in understanding the spectrum of stress-regulated proteins and the biological processes they control as well as having implications for strategies to improve stress adaptation in plants.     

Profiling the humoral immune response of acute and chronic Q fever by protein microarray

Antigen profiling using comprehensive protein microarrays is a powerful tool for characterizing the humoral immune response to infectious pathogens. Coxiella burnetii is a CDC category B bioterrorist infectious agent with worldwide distribution. In order to assess the antibody repertoire of acute and chronic Q fever patients we have constructed a protein microarray containing 93% of the proteome of Coxiella burnetii, the causative agent of Q fever. Here we report the profile of the IgG and IgM seroreactivity in 25 acute Q fever patients in longitudinal samples. We found that both early and late time points of infection have a very consistent repertoire of IgM and IgG response, with a limited number of proteins undergoing increasing or decreasing seroreactivity. We also probed a large collection of acute and chronic Q fever patient samples and identified serological markers that can differentiate between the two disease states. In this comparative analysis we confirmed the identity of numerous IgG biomarkers of acute infection, identified novel IgG biomarkers for acute and chronic infections, and profiled for the first time the IgM antibody repertoire for both acute and chronic Q fever. Using these results we were able to devise a test that can distinguish acute from chronic Q fever. These results also provide a unique perspective on isotype switch and demonstrate the utility of protein microarrays for simultaneously examining the dynamic humoral immune response against thousands of proteins from a large number of patients. The results presented here identify novel seroreactive antigens for the development of recombinant protein-based diagnostics and subunit vaccines, and provide insight into the development of the antibody response.     

Parvoviruses Cause Nuclear Envelope Breakdown by Activating Key Enzymes of Mitosis

Disassembly of the nuclear lamina is essential in mitosis and apoptosis requiring multiple coordinated enzymatic activities in nucleus and cytoplasm. Activation and coordination of the different activities is poorly understood and moreover complicated as some factors translocate between cytoplasm and nucleus in preparatory phases. Here we used the ability of parvoviruses to induce nuclear membrane breakdown to understand the triggers of key mitotic enzymes. Nuclear envelope disintegration was shown upon infection, microinjection but also upon their application to permeabilized cells. The latter technique also showed that nuclear envelope disintegration was independent upon soluble cytoplasmic factors. Using time-lapse microscopy, we observed that nuclear disassembly exhibited mitosis-like kinetics and occurred suddenly, implying a catastrophic event irrespective of cell- or type of parvovirus used. Analyzing the order of the processes allowed us to propose a model starting with direct binding of parvoviruses to distinct proteins of the nuclear pore causing structural rearrangement of the parvoviruses. The resulting exposure of domains comprising amphipathic helices was required for nuclear envelope disintegration, which comprised disruption of inner and outer nuclear membrane as shown by electron microscopy. Consistent with Ca⁺⁺ efflux from the lumen between inner and outer nuclear membrane we found that Ca⁺⁺ was essential for nuclear disassembly by activating PKC. PKC activation then triggered activation of cdk-2, which became further activated by caspase-3. Collectively our study shows a unique interaction of a virus with the nuclear envelope, provides evidence that a nuclear pool of executing enzymes is sufficient for nuclear disassembly in quiescent cells, and demonstrates that nuclear disassembly can be uncoupled from initial phases of mitosis.     

Identification of Optimum Sequencing Depth Especially for De Novo Genome Assembly of Small Genomes Using Next Generation Sequencing Data

Next Generation Sequencing (NGS) is a disruptive technology that has found widespread acceptance in the life sciences research community. The high throughput and low cost of sequencing has encouraged researchers to undertake ambitious genomic projects, especially in de novo genome sequencing. Currently, NGS systems generate sequence data as short reads and de novo genome assembly using these short reads is computationally very intensive. Due to lower cost of sequencing and higher throughput, NGS systems now provide the ability to sequence genomes at high depth. However, currently no report is available highlighting the impact of high sequence depth on genome assembly using real data sets and multiple assembly algorithms. Recently, some studies have evaluated the impact of sequence coverage, error rate and average read length on genome assembly using multiple assembly algorithms, however, these evaluations were performed using simulated datasets. One limitation of using simulated datasets is that variables such as error rates, read length and coverage which are known to impact genome assembly are carefully controlled. Hence, this study was undertaken to identify the minimum depth of sequencing required for de novo assembly for different sized genomes using graph based assembly algorithms and real datasets. Illumina reads for E.coli (4.6 MB) S.kudriavzevii (11.18 MB) and C.elegans (100 MB) were assembled using SOAPdenovo, Velvet, ABySS, Meraculous and IDBA-UD. Our analysis shows that 50X is the optimum read depth for assembling these genomes using all assemblers except Meraculous which requires 100X read depth. Moreover, our analysis shows that de novo assembly from 50X read data requires only 6–40 GB RAM depending on the genome size and assembly algorithm used. We believe that this information can be extremely valuable for researchers in designing experiments and multiplexing which will enable optimum utilization of sequencing as well as analysis resources.     

The zebrafish as a tool to identify novel therapies for human cardiovascular disease

Over the past decade, the zebrafish has become an increasingly popular animal model for the study of human cardiovascular disease. Because zebrafish embryos are transparent and their genetic manipulation is straightforward, the zebrafish has been used to recapitulate a number of cardiovascular disease processes ranging from congenital heart defects to arrhythmia to cardiomyopathy. The use of fluorescent reporters has been essential to identify two discrete phases of cardiomyocyte differentiation necessary for normal cardiac development in the zebrafish. These phases are analogous to the differentiation of the two progenitor heart cell populations in mammals, termed the first and second heart fields. The small size of zebrafish embryos has enabled high-throughput chemical screening to identify small-molecule suppressors of fundamental pathways in vasculogenesis, such as the BMP axis, as well as of common vascular defects, such as aortic coarctation. The optical clarity of zebrafish has facilitated studies of valvulogenesis as well as detailed electrophysiological mapping to characterize the early cardiac conduction system. One unique aspect of zebrafish larvae is their ability to oxygenate through diffusion alone, permitting the study of mutations that cause severe cardiomyopathy phenotypes such as silent heart and pickwick^m171, which mimic titin mutations observed in human dilated cardiomyopathy. Above all, the regenerative capacity of zebrafish presents a particularly exciting opportunity to discover new therapies for cardiac injury, including scar formation following myocardial infarction. This Review will summarize the current state of the field and describe future directions to advance our understanding of human cardiovascular disease.KEY WORDS: Cardiovascular, Drug discovery, Zebrafish