In situ Reversible Aggregation of Extracellular Cellobiase in the Filamentous Fungus Termitomyces clypeatus

Samudra Prosad Banik,Swagata Pal,Shakuntala Ghorai,Sudeshna Chowdhury,Rajib Majumder,Soumya Mukherjee,Suman Khowala 한국생물공학회 2012 Biotechnology and Bioprocess Engineering Vol.17 No.5

Cellobiase (E.C. 3.2.1.21), is a widely exploited industrial glycosidase with a major role in biofuel industry. Its stability and shelf life are major bottlenecks in achieving a superior formulation for industry. In the filamentous fungus Termitomyces clypeatus, the enzyme is secreted in a co-aggregated form with sucrase; the separation of this co-aggregation results in substantial loss of the enzyme’s activity. The aim of the present study was to examine the mode of aggregation of the secreted cellobiase-sucrase coaggregate and its role in the stabilization of cellobiase. Transmission electron microscopy and dynamic light scattering of purified co-aggregates revealed reversible, concentration driven self-aggregation of the extracellular enzymes to form larger entities. However, the intracellular enzyme aggregates were rigid,non-interacting, and possessed a higher percentage of disulphide bonds. Circular dichroic spectra of the two coaggregates indicated no significant difference in secondary structures. Self-association increased the stability of extracellular aggregates towards heat by 1.5 fold, SDS by 4 ~ 7 fold, and chaotropic agents, by 1.5 ~ 2 fold, than the intracellular counterpart. The Km of extracellular aggregate varied between 0.29 and 0.45 mM as a result of spontaneous aggregation and disaggregation, whereas that of intracellular aggregate was 0.22 mM irrespective of its concentration status. In situ detection of cellobiase in native PAGE revealed two activity bands of the extracellular enzyme, which indicated a minimum of two active dissociated aggregate species, as compared to a single band for the intracellular enzyme. These studies are believed to improve the understanding of aggregation of the fungal glycosidases, which remains to be a blackbox, to increase the efficacy of these enzymes. Cellobiase (E.C. 3.2.1.21), is a widely exploited industrial glycosidase with a major role in biofuel industry. Its stability and shelf life are major bottlenecks in achieving a superior formulation for industry. In the filamentous fungus Termitomyces clypeatus, the enzyme is secreted in a co-aggregated form with sucrase; the separation of this co-aggregation results in substantial loss of the enzyme’s activity. The aim of the present study was to examine the mode of aggregation of the secreted cellobiase-sucrase coaggregate and its role in the stabilization of cellobiase. Transmission electron microscopy and dynamic light scattering of purified co-aggregates revealed reversible, concentration driven self-aggregation of the extracellular enzymes to form larger entities. However, the intracellular enzyme aggregates were rigid,non-interacting, and possessed a higher percentage of disulphide bonds. Circular dichroic spectra of the two coaggregates indicated no significant difference in secondary structures. Self-association increased the stability of extracellular aggregates towards heat by 1.5 fold, SDS by 4 ~ 7 fold, and chaotropic agents, by 1.5 ~ 2 fold, than the intracellular counterpart. The Km of extracellular aggregate varied between 0.29 and 0.45 mM as a result of spontaneous aggregation and disaggregation, whereas that of intracellular aggregate was 0.22 mM irrespective of its concentration status. In situ detection of cellobiase in native PAGE revealed two activity bands of the extracellular enzyme, which indicated a minimum of two active dissociated aggregate species, as compared to a single band for the intracellular enzyme. These studies are believed to improve the understanding of aggregation of the fungal glycosidases, which remains to be a blackbox, to increase the efficacy of these enzymes.

Pattern of Syringomyelia in Presumed Idiopathic and Congenital Scoliosis

Mohanty Simanchal Prosad,Kanhangad Madhava Pai,Saifuddin Sibin,Narayana Kurup Jayakrishnan K. 대한척추외과학회 2021 Asian Spine Journal Vol.15 No.6

Study Design: Retrospective study. Purpose: This study was undertaken to compare the patterns of syringomyelia in patients with presumed idiopathic and congenital scoliosis. Overview of Literature: The incidence of neuraxial anomalies presenting as idiopathic scoliosis ranges from 2% to 14%; the common ones are idiopathic syringomyelia (IS) and Chiari malformation type 1 (CM1) with syringomyelia. Some authors have speculated that scoliosis is caused by the asymmetrical compression of anterior horn cells by a syrinx, which causes an imbalance of the trunk musculature. In congenital scoliosis, syringomyelia is the second commonest cord anomaly, and the deformity progression depends upon the underlying vertebral abnormality, the location of the abnormality, and the age of patient, and is independent of the intraspinal anomaly. Methods: We analyzed the radiological records of 44 consecutive patients with scoliosis and syringomyelia. Of these 44 patients, 13 had IS, 12 had CM1, and 19 had congenital scoliosis. The radiographs were evaluated to determine the curve magnitude, sagittal alignment, side of convexity, and type of vertebral anomaly, if any. T1- and T2-weighted magnetic resonance imaging of the whole spine was analyzed to determine the presence of craniovertebral anomalies, syrinx length, syrinx diameter, and syrinx-cord ratio (SCR). Results: The frequency of left convex curves was 26.1%, with no significant differences across the three groups. The mean length of the syrinx was 7.2±4.9 vertebral levels, and the mean SCR was 0.39±0.2. The mean syrinx length was significantly higher in patients with CM1 and IS, compared to patients with congenital scoliosis. The mean SCR was highest in patients with CM1. In congenital scoliosis, syringomyelia was seen most frequently in patients with a failure of formation, and 63.2% had concomitant cord anomalies. Conclusions: Syrinxes were smaller in size and length in patients with congenital scoliosis, and are distinct from those seen in IS and CM1.

Evidence of an Alternative Route of Cellobiase Secretion in the Presence of Brefeldin A in the Filamentous Fungus Termitomyces clypeatus

( Banik Samudra Prosad ),( Swagata Pal ),( Sudeshna Chowdhury ),( Shakuntala Ghorai ),( Suman Khowala ) 한국미생물 · 생명공학회 2011 Journal of microbiology and biotechnology Vol.21 No.4

Secretion of cellobiase occurred in a brefeldin A (BFA) uninhibited manner in the filamentous fungus Termitomyces clypeatus. Fluorescence confocal microscopy revealed that application of the drug at a concentration of 50 μg/ml caused arrest of Spitzenkorper assembly at the hyphal tip. This resulted in greater than 30% inhibition of total protein secretion in the culture medium. However, the cellobiase titer increased by 17%, and an additional 13% was localized in the vacuolar fraction en route secretion. The secretory vacuoles formed in the presence of the drug were also found to be bigger (68 nm) than those in the control cultures (40 nm). The enzyme secreted in the presence and absence of BFA revealed a single activity band in both cases in native PAGE and had similar molecular masses (approx. 120 kDa) in SDS-PAGE. The BFA enzyme retained 72% of native glycosylation. It also exhibited a higher stability and retained 98% activity at 50oC, 93.3% activity at pH 9, 63.64% activity in the presence of 1M guanidium hydrochloride, and 50% activity at a glucose concentration of 10 mg/ml in comparison to 68% activity, 75% activity, 36% activity, and 19% activity for the control enzyme, respectively. The observations collectively aimed at the operation of an alternative secretory pathway, distinct from the target of brefeldin A, which bypassed the Golgi apparatus, but still was able to deliver the cargo to the vacuoles for secretion. This can be utilized in selectively enhancing the yield and stability of glycosidases for a successful industrial recipe.

Zygapophyseal Joint Orientation and Facet Tropism and Their Association with Lumbar Disc Prolapse

Madhava Pai Kanhangad,Simanchal Prosad Mohanty,Siddarth Kamath,Asha Kamath 대한척추외과학회 2018 Asian Spine Journal Vol.12 No.5

Study Design: Cross-sectional study. Purpose: To evaluate the association between zygapophyseal joint angle (ZJA), facet tropism (FT), and lumbar intervertebral disc prolapse (IVDP). Overview of Literature: Several studies have shown that FT increases the risk of IVDP and have postulated that a more sagittally oriented zygapophyseal joint provides less mechanical resistance to axial torque, thereby exerting excessive rotational strain on the intervertebral disc, resulting in an annular tear. In contrast, other studies have found no definitive association between FT and IVDP. Therefore, conclusive evidence regarding the role of FT in the pathogenesis of disc prolapse is currently lacking. Methods: Magnetic resonance imaging scans of 426 patients with single-level lumbar IVDP were analyzed. Right and left ZJAs of the lumbar segments were measured on axial sections. The frequency and severity of FT were determined by calculating the absolute difference between the right and left ZJAs. Patients without IVDP at L4–L5 and L5–S1 served as controls for those with IVDP at L4–L5 and L5–S1, respectively. Chi-square test and t -test were used to compare the severity and frequency of FT between patients with and without IVDP. The receiver operating characteristic analysis was performed to determine the critical FT values that were predictive of IVDP. Results: Patients with IVDP exhibited a higher frequency (L4–L5: 47% vs. 15.08%; L5–S1: 39.62% vs. 22.69%; p =0.001) and severity (L4–L5: 7.85°±3.5° vs. 4.05°±2.62°; L5–S1: 7.30°±3.07° vs. 4.82°±3.29°; p <0.001) of FT than those without IVDP. Critical FT values of 5.7° at L4–L5 and 6° at L5–S1 increased the likelihood of IVDP by a factor of 2.89 and 1.75, respectively. Conclusions: Our results confirm the existence of a significant association between lumbar IVDP and FT; however, a causal relationship could not be ascertained.

Fight Detection in Hockey Videos using Deep Network

Mukherjee, Subham,Saini, Rajkumar,Kumar, Pradeep,Roy, Partha Pratim,Dogra, Debi Prosad,Kim, Byung-Gyu Korea Multimedia Society 2017 The journal of multimedia information system Vol.4 No.4

Understanding actions in videos is an important task. It helps in finding the anomalies present in videos such as fights. Detection of fights becomes more crucial when it comes to sports. This paper focuses on finding fight scenes in Hockey sport videos using blur & radon transform and convolutional neural networks (CNNs). First, the local motion within the video frames has been extracted using blur information. Next, fast fourier and radon transform have been applied on the local motion. The video frames with fight scene have been identified using transfer learning with the help of pre-trained deep learning model VGG-Net. Finally, a comparison of the methodology has been performed using feed forward neural networks. Accuracies of 56.00% and 75.00% have been achieved using feed forward neural network and VGG16-Net, respectively.

Plant Disease Identification using Deep Neural Networks

Mukherjee, Subham,Kumar, Pradeep,Saini, Rajkumar,Roy, Partha Pratim,Dogra, Debi Prosad,Kim, Byung-Gyu Korea Multimedia Society 2017 The journal of multimedia information system Vol.4 No.4

Automatic identification of disease in plants from their leaves is one of the most challenging task to researchers. Diseases among plants degrade their performance and results into a huge reduction of agricultural products. Therefore, early and accurate diagnosis of such disease is of the utmost importance. The advancement in deep Convolutional Neural Network (CNN) has change the way of processing images as compared to traditional image processing techniques. Deep learning architectures are composed of multiple processing layers that learn the representations of data with multiple levels of abstraction. Therefore, proved highly effective in comparison to many state-of-the-art works. In this paper, we present a plant disease identification methodology from their leaves using deep CNNs. For this, we have adopted GoogLeNet that is considered a powerful architecture of deep learning to identify the disease types. Transfer learning has been used to fine tune the pre-trained model. An accuracy of 85.04% has been recorded in the identification of four disease class in Apple plant leaves. Finally, a comparison with other models has been performed to show the effectiveness of the approach.

A Survey of Real-time Road Detection Techniques Using Visual Color Sensor

Hong, Gwang-Soo,Kim, Byung-Gyu,Dogra, Debi Prosad,Roy, Partha Pratim Korea Multimedia Society 2018 The journal of multimedia information system Vol.5 No.1

A road recognition system or Lane departure warning system is an early stage technology that has been commercialized as early as 10 years but can be optional and used as an expensive premium vehicle, with a very small number of users. Since the system installed on a vehicle should not be error prone and operate reliably, the introduction of robust feature extraction and tracking techniques requires the development of algorithms that can provide reliable information. In this paper, we investigate and analyze various real-time road detection algorithms based on color information. Through these analyses, we would like to suggest the algorithms that are actually applicable.