Evaluation of Results of Ponseti Technique in Idiopathic Clubfoot using Clinical Evaluation and Radiological Assessment.

Background: Congenital clubfoot (congenital talipes equinovarus) occurs in approximately one in 1000 live births and is one of the most common congenital birth defects. The Ponseti method is at present a well-established method of treatment for idiopathic clubfoot deformities. Aim: The aim of the present study was to evaluate the results of serial casting in clubfoot deformity with Ponseti method on the basis of Pirani's scoring and radiological findings before and after completion of treatment. Materials and Methods: A total of 30 patients were enrolled in the study and were treated with Ponseti's casting after grading the severity of deformity clinically by Pirani's scoring and radiological assessment by calculating the talo-first metatarsal angle in anteroposterior (AP) view and talocalcaneal angle in AP and lateral views. The same clinical and radiological assessment was done at the end of treatment before putting a patient on foot abduction orthosis (FAO). Results: The average number of casts applied before full correction was 5.56 (range: 5-8). The average duration of treatment was about 6.65 weeks before the patient was put on FAO. Pirani score significantly improved from an average of 5.50 (range: 4-6) on presentation to 0.24 (range: 0-2) after correction of deformity. Conclusion: The Ponseti method is an excellent method for the correction of all four deformities associated with congenital idiopathic clubfoot, and we found that the addition of radiographic to clinical evaluation helps in the better assessment of correction. It provides statistically significant results both clinically as measured by Pirani severity score and radiologically assessed by talocalcaneal and talo-first metatarsal angle.

To Analyze the Role of Intravenous Tranexamic Acid in Hip Fracture surgeries in Orthopedic Trauma.

INTRODUCTION: Hip fractures in orthopedic trauma cases are increasing. Majority of such patients undergoing surgery require blood transfusion of one or more units. Intravenous (I. V.) Tranexamic acid (TXA) may decrease loss of blood, decrease need of blood transfusion, and improve postoperative hemoglobin (Hb) along with lesser adverse effects. Risk of thromboembolic phenomena remains a concern. A study was done to analyze the role of I. V. TXA in hip fracture surgeries in trauma cases. MATERIALS AND METHODS: Sixty patients were included in the study; in two groups (37 males and 23 females), Group A in which two doses of I. V. TXA 15 mg/kg were given and Group B in which two doses of I. V. placebo were given. RESULTS: Total number of randomized hip arthroplasty cases was 22 (11 in Group A and 11 in Group B) whereas randomized osteosynthesis cases were 38 (19 in Group A and 19 in Group B). Mean preoperative Hb value in Group A was 10.8 gm% and in Group B was 10.7 gm% (P > 0.005. Mean postoperative Hb value in Group A was Hb 9.8 gm% and in Group B 9.5 gm% (difference of 3.061%). Mean duration of surgery in Group A was 64.2 min and in Group B was 66.3 min. Mean total blood loss (intraoperative and postoperative) in Group A was 384.6 ml and in Group B was 448.7 ml (14.29% less in Group A). A total of 14 patients in Group A (17 red blood cells [RBCs] units) and 17 patients (21 RBC units) in Group B required RBC transfusion. No major vascular event, severe bacterial infections, symptomatic deep vein thrombosis, pulmonary embolism, limb ischemia, acute coronary syndrome, or immediate postoperative mortality was noted in either group. CONCLUSION: I. V. TXA has the potential to decrease risk of blood transfusion, decrease total blood loss, and to maintain a higher postoperative Hb value with no significant adverse reactions. As the number of cases of hip fractures continues to increase along with increase in age, so the use of TXA in such cases may improve clinical outcomes, lessen number of inpatient days and hence decrease overall cost.

Whole-Genome Sequence Data Analysis of Anoxybacillus kamchatkensis NASTPD13 Isolated from Hot Spring of Myagdi, Nepal.

Anoxybacillus kamchatkensis NASTPD13 isolated from Paudwar hot spring of Myagdi, Nepal, upon morphological and biochemical analysis revealed to be Gram-positive, straight or slightly curved, rod-shaped, spore-forming, catalase, and oxidase-positive facultative anaerobes. It grows over a wide range of pH (5.0-11) and temperature (37-75°C), which showed growth in different reduced carbon sources such as starch raffinose, glucose, fructose, inositol, trehalose, sorbitol, mellobiose, and mannitol in aerobic conditions. Furthermore, the partial sequence obtained upon sequencing showed 99% sequence similarity in 16S rRNA gene sequence with A. kamchatkensis JW/VK-KG4 and was suggested to be Anoxybacillus kamchatkensis. Moreover, whole-genome analysis of NASTPD13 revealed 2,866,796 bp genome with a G+C content of 41.6%. Analysis of the genome revealed the presence of 102 RNA genes, which includes sequences coding for 19 rRNA and 79 tRNA genes. While the 16S rRNA gene sequence of strain NASTPD13 showed high similarity (>99%) to those of A. kamchatkensis JW/VK-KG4, RAST analysis of NASTPD13 genome suggested that A. kamchatkensis G10 is actually the closest neighbor in terms of sequence similarity. The genome annotation by RAST revealed various genes encoding glycoside hydrolases supporting that it can utilize several reduced carbon sources as observed and these genes could be important for carbohydrate-related industries. Xylanase pathway, particularly the genomic region encoding key enzymes for xylan depolymerization and xylose metabolism, further confirmed the presence of the complete gene in xylan metabolism. In addition, the complete xylose utilization gene locus analysis of NASTPD13 genome revealed all including D-xylose transport ATP-binding protein XylG and XylF, the xylose isomerase encoding gene XylA, and the gene XylB coding for a xylulokinase supported the fact that the isolate contains a complete set of genes related to xylan degradation, pentose transport, and metabolism. The results of the present study suggest that the isolated A. kamchatkensis NASTPD13 containing xylanase-producing genes could be useful in lignocellulosic biomass-utilizing industries where pentose polymers could also be utilized along with the hexose polymers.

FermFooDb: A database of bioactive peptides derived from fermented foods.

Globally fermented foods are in demands due to their functional and nutritional benefits. These foods are sources of probiotic organisms and bioactive peptides, various amino acids, enzymes etc. that provides numerous health benefits. FermFooDb (https://webs.iiitd.edu.in/raghava/fermfoodb/) is a manually curated database of bioactive peptides derived from wide range of foods that maintain comprehensive information about peptides and process of fermentation. This database comprises of 2205 entries with following major fields, peptide sequence, Mass and IC50, food source, functional activity, fermentation conditions, starter culture, testing conditions of sequences in vitro or in vivo, type of model and method of analysis. The bioactive peptides in our database have wide range of therapeutic potentials that includes antihypertensive, ACE-inhibitory, antioxidant, antimicrobial, immunomodulatory and cholesterol lowering peptides. These bioactive peptides were derived from different types of fermented foods that include milk, cheese, yogurt, wheat and rice. Numerous, web-based tools have been integrated to retrieve data, peptide mapping of proteins, similarity search and multiple-sequence alignment. This database will be useful for the food industry and researchers to explore full therapeutic potential of fermented foods from specific cultures.

Abundance and Diversity of Phages, Microbial Taxa, and Antibiotic Resistance Genes in the Sediments of the River Ganges Through Metagenomic Approach.

In this study, we have analyzed the metagenomic DNA from the pooled sediment sample of the river Ganges to explore the abundance and diversity of phages, microbial community, and antibiotic resistance genes (ARGs). Utilizing data from Illumina platform, 4,174 (∼0.0013%) reads were classified for the 285 different DNA viruses largely dominated by the group of 260 distinctive phages (3,602 reads, ∼86.3%). Among all, Microcystis (782 hits), Haemophilus (403), Synechococcus (386), Pseudomonas (279), Enterococcus (232), Bacillus (196), Rhodococcus (166), Caulobacter (163), Salmonella (146), Enterobacteria (143), Mycobacterium and (128) phages show the highest abundance and account for ∼90% of the total identified phages. In addition, we have also identified corresponding host pertaining to these phages. Mainly, Proteobacteria (∼69.3%) dominates the microbial population structure. Primarily, orders such as Caulobacterales (∼28%), Burkholderiales (∼13.9%), Actinomycetales (∼13.7%), and Pseudomonadales (∼7.5%) signify the core section. Furthermore, 21,869 (∼0.00695%) reads were classified in 20 ARG types (classes) and 240 ARGs (subtypes), among which 4 ARG types, namely multidrug resistance (12,041 reads, ∼55%), bacitracin (3,202 reads, ∼15%), macrolide-lincosamide-streptogramin (1,744 reads, ∼7.98%), and fosmidomycin (990 reads, ∼4.53%), have the highest abundance. Simultaneously, six resistance mechanisms were also recognized with the dominance of antibiotic efflux (72.8%, 15,919 reads). The results unveil the distribution of (pro)-phages; microbial community; and various ARGs in the Ganges river sediments.

Development of Site-Specific PEGylated Granulocyte Colony Stimulating Factor With Prolonged Biological Activity.

Currently, amino-terminal PEGylated human granulocyte colony stimulating factor (huG-CSF) is used to prevent and treat neutropenia. Although huG-CSF has been used as a drug for more than 20 years, it has three significant drawbacks: (i) it relies on PEG aldehyde for PEGylation of the alpha-amino group of the first amino acid, and this leads to non-specific PEGylation of the epsilon amino group of lysine residues within the G-CSF; (ii) longer-acting G-CSF variants are desirable to reduce the risk of chemotherapy-associated neutropenia; and (iii) G-CSF cannot be administered on the day of chemotherapy. In an attempt to overcome the above drawbacks, we engineered cysteine variants of G-CSF to facilitate the maleimide PEG-based site-specific PEGylation that leads to a highly homogenous PEGylated product. Importantly, we have demonstrated that 20 kDa thiol-reactive PEG conjugated by maleimide chemistry to the Cys2 G-CSF variant exhibits leukocyte proliferative activity similar to that of the commercially available G-CSF conjugated with aldehyde PEG in a neutropenia mice model. Moreover, we have demonstrated that PEGylation of the cysteine variant of huG-CSF with higher molecular weight PEGs, such as 30 kDa PEG and 40 kDa PEG, leads to significantly prolonged leukocyte proliferation activity compared to the variant conjugated with 20 kDa PEG. Importantly, even a half-dose of the engineered variant conjugated with 40 kDa PEG exhibited significantly longer biological activity than the commercially available 20 kDa PEGylated huG-CSF. Finally, we have demonstrated that administration of the engineered variant conjugated with 40 kDa PEG on the day of administration of cyclophosphamide for inducing neutropenia in mice can alleviate neutropenia through leukocyte proliferation. In summary, this study provides the design of site-specific PEGylated huG-CSF variants with improved therapeutic potential. It opens the possibility of long-acting and same-day prophylactic administration of G-CSF after chemotherapy drug regimens. These results may pave the way for the development of potential G-CSF derivatives possessing longer half-lives and favorable clinical attributes.

Recombinant expression, purification and PEGylation of Paneth cell peptide (cryptdin-2) with value added attributes against Staphylococcus aureus.

Cryptdins are disulfide-rich cationic antimicrobial peptides secreted by mouse Paneth cells and are known to exhibit potent antimicrobial activity against various deadly pathogens. Keeping in view the extremely low yield obtained from mouse Paneth cells and high cost of synthetic peptide(s), herein, we have attempted to produce cryptdin-2 in Escherichia coli using recombinant technology. To avoid lethal effects of peptide on the host cells, cryptdin-2 was expressed as a fusion protein with thioredoxin as fusion partner which yielded 40 mg/L protein in the soluble fraction. Subsequently, mature cryptdin-2 was cleaved from the fusion partner and purified by cation exchange chromatography. Since conjugation of poly(ethylene) glycol (PEG) has been known to improve the biological properties of biomolecules, therefore, we further attempted to prepare PEG-conjugated variant of cryptdin-2 using thiol specific PEGylation. Though the antimicrobial activity of PEGylated cryptdin-2 was compromised to some extent, but it was found to have enhanced serum stability for longer duration as compared to its un-modified forms. Also, it was found to exhibit reduced toxicity to the host cells. Further, its synergism with gentamicin suggests that PEGylated cryptdin-2 can be used with conventional antibiotics, thereby indicating its possibility to be used as an adjunct therapy.

Kringles of substrate plasminogen provide a 'catalytic switch' in plasminogen to plasmin turnover by Streptokinase.

To understand the role of substrate plasminogen kringles in its differential catalytic processing by the streptokinase - human plasmin (SK-HPN) activator enzyme, Fluorescence Resonance Energy Transfer (FRET) model was generated between the donor labeled activator enzyme and the acceptor labeled substrate plasminogen (for both kringle rich Lys plasminogen - LysPG, and kringle less microplasminogen - µPG as substrates). Different steps of plasminogen to plasmin catalysis i.e. substrate plasminogen docking to scissile peptide bond cleavage, chemical transformation into proteolytically active product, and the decoupling of the nascent product from the SK-HPN activator enzyme were segregated selectively using (1) FRET signal as a proximity sensor to score the interactions between the substrate and the activator during the cycle of catalysis, (2) active site titration studies and (3) kinetics of peptide bond cleavage in the substrate. Remarkably, active site titration studies and the kinetics of peptide bond cleavage have shown that post docking chemical transformation of the substrate into the product is independent of kringles adjacent to the catalytic domain (CD). Stopped-flow based rapid mixing experiments for kringle rich and kringle less substrate plasminogen derivatives under substrate saturating and single cycle turnover conditions have shown that the presence of kringle domains adjacent to the CD in the macromolecular substrate contributes by selectively speeding up the final step, namely the product release/expulsion step of catalysis by the streptokinase-plasmin(ogen) activator enzyme.

Design of a thrombin inhibitory staphylokinase based plasminogen activator with anti-reocclusion potential.

In the quest of a therapeutically improved thrombolytic drug, we designed and expressed a Staphylokinase based, recombinant fusion protein with fibrin specific clot lysis and anti-thrombin activities derived from human thrombomodulin, a transmembrane glycoprotein with anticoagulant properties, known to form a complex with thrombin and then activating Protein C. The fusion construct was expressed in the yeast Pichia pastoris for cost effective and facile production. The purified construct had comparable plasminogen activation and clot lysis capability as compared to native SAK in that it showed plasmin dependent Plasminogen activation, but exhibited significantly lower fibrinogenolysis (an indicator of fibrin-specific action) even at much higher doses than SAK. In addition, its successful activation of the thrombin-mediated Protein C anticoagulant pathway was reflected in increased in vitro plasma clotting time, as well as inhibition of clot bound thrombin, which led to nearly 15-25% lesser re-formation of fibrin clots after initial successful clot lysis in a specially designed in vitro assay for re-occlusion. Thus, this novel chimeric protein could be of high therapeutic potential as a thrombolytic drug with enhanced fibrin specific clot dissolving properties along with lower bleeding and re-thrombosis complications- a dire need today, especially in the treatment of thrombotic strokes.

Cloning and purification of an anti-thrombotic, chimeric Staphylokinase in Pichia pastoris.

There has been an increasing prevalence of cardiovascular diseases such as myocardial infarction and stroke in modern societies because of multiple lifestyle related issues like sedentariness and obesity, alcohol consumption and many more "life-style"factors. The FDA-approved thrombolytics such as Tissue Plasminogen Activator, Streptokinase etc. are used to lyse the clots in thrombotic disorders such as myocardial infarction, stroke etc. but re-occlusion and bleeding that are co-incident to their clinical usage are not addressed. Hence, there is need to develop thrombolytics having properties like increased fibrin clot specificity and thrombin inhibition capability to prevent re-occlusion. In the present work, a fusion protein construct containing two components i.e. Staphylokinase (SAK) and Epidermal Growth Factor (EGF) 4, 5, 6-like domains of human thrombomodulin (THBD) was expressed in Pichia pastoris after genetic optimization. SAK isolated from Staphylococcus aureus is a fibrin-specific plasminogen activator while EGF 4, 5, 6-like domains are reported to be responsible for imparting thrombin inhibition to human thrombomodulin, and therefore, expected could help prevent re-occlusion in the novel construct - SAK_EGF, which is a 43 kDa protein. After expression, it was purified (approx. 13-fold) using two-step purification protocol involving ion-exchange followed by Gel Filtration Chromatography (GFC). The functional characterization including plasminogen activation and thrombin inhibition showed that both the fusion partners viz. SAK and 4,5,6 EGF-like domains retained their respective activities after fusion, confirming it to be a bio-active construct. Thus, this engineered protein could be clinically promising due to the combinatorial effect of fibrin-specific thrombus lysis and prevention of re-occulusion.

Site-specific PEGylation of micro-plasmin for improved thrombolytic therapy through engineering enhanced resistance against serpin mediated inhibition.

The relatively rapid inhibition of microplasmin by α2-AP leads to short functional half-life of the molecule in vivo, causing inefficient clot dissolution, even after site-specific, local catheter-based delivery. Here, we describe a PEGylation approach for improving the therapeutic potential via improving the survival of microplasmin in presence of its cognate inhibitor, α2-AP, wherein a series of strategically designed cysteine analogs of micro-plasminogen were prepared and expressed in E. coli, and further modified by covalent grafting in vitro with PEG groups of different molecular sizes so as to select single or double PEG chains that increase the molecular weight and hydrodynamic radii of the conjugates, but with a minimal discernible effect on intrinsic plasmin activity and structural framework, as explored by amidolytic activity and CD-spectroscopy, respectively. Interestingly, some of the purified PEG-coupled proteins after conversion to their corresponding proteolytically active forms were found to exhibit significantly reduced inhibition rates (up to 2-fold) by α2-AP relative to that observed with wild-type microplasmin. These results indicate an interesting, and not often observed, effect of PEG groups through reduced/altered dynamics between protease and inhibitor, likely through a steric hindrance mechanism. Thus, the present study successfully identifies single- and double-site PEGylated muteins of microplasmin with significantly enhanced functional half-life through enhanced resistance to inactivation by its in vivo plasma inhibitor. Such an increased survival of bioactivity in situ, holds unmistakable potential for therapeutic exploitation, especially in ischemic strokes where a direct, catheter-based deposition within the cranium has been shown to be promising, but is currently limited by the very short in vivo bioactive half-life of the fibrin dissolving agent/s.

Production, Purification, and Characterization of Thermostable Alkaline Xylanase From Anoxybacillus kamchatkensis NASTPD13.

Anoxybacillus kamchatkensis NASTPD13 used herein as a source for thermostable alkaline xylanase were isolated from Paudwar Hot Springs, Nepal. NASTPD13 cultured at 60°C, pH 7 and in presence of inorganic (ammonium sulfate) or organic (yeast extract) nitrogen sources, produced maximum xylanase enzyme. Xylanase production in the cultures was monitored by following the ability of culture media to hydrolyze beech wood xylan producing xylooligosaccharide and xylose by thin layer chromatography (TLC). The extracellular xylanase was isolated from optimized A. kamchatkensis NASTPD13 cultures by ammonium sulfate (80%) precipitation; the enriched xylanase preparation was dialyzed and purified using Sephadex G100 column chromatography. The purified xylanaseshowed 11-fold enrichment with a specific activity of 33 U/mg and molecular weight were37 kDa based on SDS-PAGE and PAGE-Zymography. The optimum pH and temperature of purified xylanase was 9.0 and 65°C respectively retainingmore than 50% of its maximal activity over a broad range of pH (6-9) and temperature (30-65°C). With beech wood xylan, the enzyme showed Km 0.7 mg/ml and Vmax 66.64 µM/min/mg The xylanase described herein is a secretory enzyme produced in large quantities by NASTPD13 and is a novel thermostable, alkaline xylanase with potential biotechnological applications.

Production, purification and characterization of raw starch hydrolyzing thermostable acidic α-amylase from hot springs, India.

Alpha-amylase is an important hydrolytic enzyme used for various industrial processes. In the present study, Geobacillus bacterium (K1C), producing a thermostable α-amylase was isolated from Manikaran hot springs, India. We have purified and characterized the biochemical properties of α-amylase. The optimum temperature and pH for α-amylase activity was 80 °C and pH 6.0 respectively. The far-UV CD spectra of the enzyme indicated the presence of random coil conformation and showed an intermediate phase during temperature-induced unfolding. In the presence of substrate, thermostability of the α-amylase was increased as 50% initial activity was retained at 70 °C for 6 h and at 80 °C for 2 h. Moreover, the enzyme also showed remarkable pH stability as 90% of the initial activity was retained even after 48 h of incubation at pH 5.0, 6.0 and 7.0. Interestingly, amylase activity of the purified enzyme was Ca2+independent, whereas the complete inhibition of activity was observed in the presence of Cu2+, Pb2+, and Hg2+. The purified α-amylase was stable in the presence of detergents, organic solvents and Proteinase K. Furthermore, it exhibited the ability to hydrolyze raw starches (e.g. rice, wheat, corn, potato) efficiently; thus this enzyme has the potential to be used for industrial applications.

Pseudomonas fluvialis sp. nov., a novel member of the genus Pseudomonas isolated from the river Ganges, India.

A bacterial strain, designated ASS-1T, was isolated and identified from a sediment sample of the river Ganges, Allahabad, India. The strain was Gram-stain-negative, formed straw-yellow pigmented colonies, was strictly aerobic, motile with a single polar flagellum, and positive for oxidase and catalase. The major fatty acids were C16 : 1ω7c/ 16 : 1 C16 : 1ω6c, C18 : 1ω7c and C16 : 0. Sequence analysis based on the 16S rRNA gene revealed that strain ASS-1T showed high similarity to Pseudomonas guguanensis CC-G9AT (98.2 %), Pseudomonas alcaligenes ATCC 14909T (98.2 %), Pseudomonas oleovorans DSM 1045T (98.1 %), Pseudomonas indolxydans IPL-1T (98.1 %) and Pseudomonas toyotomiensis HT-3T (98.0 %). Analysis of its rpoB and rpoD housekeeping genes confirmed its phylogenetic affiliation and showed identities lower than 93 % with respect to the closest relatives. Phylogenetic analysis based on the 16S rRNA, rpoB, rpoD genes and the whole genome assigned it to the genus Pseudomonas. The results of digital DNA-DNA hybridization based on the genome-to-genome distance calculator and average nucleotide identity revealed low genome relatedness to its close phylogenetic neighbours (below the recommended thresholds of 70 and 95 %, respectively, for species delineation). Strain ASS-1T also differed from the related strains by some phenotypic characteristics, i.e. growth at pH 5.0 and 42 °C, starch and casein hydrolysis, and citrate utilization. Therefore, based on data obtained from phenotypic and genotypic analysis, it is evident that strain ASS-1T should be regarded as a novel species within the genus Pseudomonas, for which the name Pseudomonasfluvialis sp. nov. is proposed. The type strain is ASS-1T (=KCTC 52437T=CCM 8778T).

Mechanism of plasmin generation by S100A10.

Plasminogen (Pg) is cleaved to form plasmin by the action of specific plasminogen activators such as the tissue plasminogen activator (tPA). Although the interaction of tPA and Pg with the surface of the fibrin clot has been well characterised, their interaction with cell surface Pg receptors is poorly understood. S100A10 is a cell surface Pg receptor that plays a key role in cellular plasmin generation. In the present report, we have utilised domain-switched/deleted variants of tPA, truncated plasminogen variants and S100A10 site-directed mutant proteins to define the regions responsible for S100A10-dependent plasmin generation. In contrast to the established role of the finger domain of tPA in fibrin-stimulated plasmin generation, we show that the kringle-2 domain of tPA plays a key role in S100A10-dependent plasmin generation. The kringle-1 domain of plasminogen, indispensable for fibrin-binding, is also critical for S100A10-dependent plasmin generation. S100A10 retains activity after substitution or deletion of the carboxyl-terminal lysine suggesting that internal lysine residues contribute to its plasmin generating activity. These studies define a new paradigm for plasminogen activation by the plasminogen receptor, S100A10.

Biochemical and structural characterization of a novel halotolerant cellulase from soil metagenome.

Cellulase catalyzes the hydrolysis of ß-1,4-linkages of cellulose to produce industrially relevant monomeric subunits. Cellulases find their applications in pulp and paper, laundry, food and feed, textile, brewing industry and in biofuel production. These industries always have great demand for cellulases that can work efficiently even in harsh conditions such as high salt, heat, and acidic environments. While, cellulases with high thermal and acidic stability are already in use, existence of a high halotolerant cellulase is still elusive. Here, we report a novel cellulase Cel5R, obtained from soil metagenome that shows high halotolerance and thermal stability. The biochemical and functional characterization of Cel5R revealed its endoglucanase activity and high halostability. In addition, the crystal structure of Cel5R determined at 2.2 Å resolution reveals a large number of acidic residues on the surface of the protein that contribute to the halophilic nature of this enzyme. Moreover, we demonstrate that the four free and non-conserved cysteine residues (C65, C90, C231 and C273) contributes to the thermal stability of Cel5R by alanine scanning experiments. Thus, the newly identified endoglucanase Cel5R is a promising candidate for various industrial applications.

PEGylation of Truncated Streptokinase Leads to Formulation of a Useful Drug with Ameliorated Attributes.

Streptokinase (SK) remains a favored thrombolytic agent in the developing world as compared to the nearly 10-fold more expensive human tissue-plasminogen activator (tPA) for the dissolution of pathological fibrin clots in myocardial infarction. However, unlike the latter, SK induces systemic activation of plasmin which results in a greater risk of hemorrhage. Being of bacterial origin, it elicits generation of unwanted antibody and has a relatively short half-life in vivo that needs to be addressed to make it more efficacious clinically. In order to address these lacunae, in the present study we have incorporated cysteine residues specifically at the N- and C-termini of partially truncated SK and these were then PEGylated successfully. Some of the obtained derivatives displayed enhanced plasmin resistance, longer half-life (upto several hours), improved fibrin clot-specificity and reduced immune-reactivity as compared to the native SK (nSK). This paves the way for devising next-generation SK-based thrombolytic agent/s that besides being fibrin clot-specific are endowed with an improved efficacy by virtue of an extended in vivo half-life.

Amino-Terminal Fusion of Epidermal Growth Factor 4,5,6 Domains of Human Thrombomodulin on Streptokinase Confers Anti-Reocclusion Characteristics along with Plasmin-Mediated Clot Specificity.

Streptokinase (SK) is a potent clot dissolver but lacks fibrin clot specificity as it activates human plasminogen (HPG) into human plasmin (HPN) throughout the system leading to increased risk of bleeding. Another major drawback associated with all thrombolytics, including tissue plasminogen activator, is the generation of transient thrombin and release of clot-bound thrombin that promotes reformation of clots. In order to obtain anti-thrombotic as well as clot-specificity properties in SK, cDNAs encoding the EGF 4,5,6 domains of human thrombomodulin were fused with that of streptokinase, either at its N- or C-termini, and expressed these in Pichia pastoris followed by purification and structural-functional characterization, including plasminogen activation, thrombin inhibition, and Protein C activation characteristics. Interestingly, the N-terminal EGF fusion construct (EGF-SK) showed plasmin-mediated plasminogen activation, whereas the C-terminal (SK-EGF) fusion construct exhibited 'spontaneous' plasminogen activation which is quite similar to SK i.e. direct activation of systemic HPG in absence of free HPN. Since HPN is normally absent in free circulation due to rapid serpin-based inactivation (such as alpha-2-antiplasmin and alpha-2-Macroglobin), but selectively present in clots, a plasmin-dependent mode of HPG activation is expected to lead to a desirable fibrin clot-specific response by the thrombolytic. Both the N- and C-terminal fusion constructs showed strong thrombin inhibition and Protein C activation properties as well, and significantly prevented re-occlusion in a specially designed assay. The EGF-SK construct exhibited fibrin clot dissolution properties with much-lowered levels of fibrinogenolysis, suggesting unmistakable promise in clot dissolver therapy with reduced hemorrhage and re-occlusion risks.

Site-Specific Thiol-mediated PEGylation of Streptokinase Leads to Improved Properties with Clinical Potential.

Streptokinase (SK) is an efficient thrombolytic agent that dissolves fibrin blood clots with clinical efficiency comparable to the high priced drug, tissue plasminogen activator (tPA). However, being of bacterial origin, its major drawbacks are its potentially high antigenicity, and relatively short circulating half-life (approximately 10-15 min). In the present investigation, an attempt has been made to address both these shortcomings by site-specific pegylation, and to obtain longer lasting thrombolytics, which are consistent with clinical requirements. Therefore, we employed available three-dimensional structural information on SK to carry out site-specific cysteine incorporation at 'optimal' surfaceexposed sites within all the three domains in streptokinase followed by pegylation with 20KDa PEG groups, and screening for biologically active variants. Interestingly, some of these SK PEG-conjugates exhibited considerably subdued immunereactivity along with enhanced in-vitro proteolytic stability profiles and extended circulating in-vivo half-lives (2 to 20-fold compared to that of native unconjugated SK) depending upon location and number of PEG-groups per molecule obtained in homogeneous form. The obtained results are a promising approach for favorably modulating immune-reactivity and half-life by cysteine- specific PEGylation of SK to achieve therapeutic attributes desirable for the treatment of different circulatory disorders, such as ischemic stroke, myocardial infarction and pulmonary embolism.