Vis-NIR spectroscopy based rapid and non-destructive method to quantitate microplastics: An emerging contaminant in farm soil.

Microplastics (MPs) is the second most important environmental issue and can potentially enter into food chain through farmland contamination and other means. There are no standardized extraction methods for quantification of MPs in soil. The embedded errors and biases generated serious problems regarding the comparability of different studies and leading to erroneous estimation. To address this gap, present study was formulated to develop an efficient method for MPs analysis suitable for a wide range of soil and organic matrices. A method based on Vis-NIR (Visible-Near Infra Red) spectroscopy is developed for four different soil belonging to Alfisol, Inceptisol, Mollisol and Vertisol and two organic matter matrices (FYM and Sludge). The developed method was found as rapid, reproducible, non-destructive and accurate method for estimation of all three-density groups of MPs (Low, Medium and High) with a prediction accuracy ranging from 1.9 g MPs/kg soil (Vertisol) to 3.7 g MPs/kg soil (Alfisol). Two different regression models [Partial Least Square Regression (PLSR) and Principal Component Regression (PCR)] were assessed and PLSR was found to provide better information in terms of prediction accuracy and minimum quantification limit (MQL). However, PCR performed better for organic matter matrices than PLSR. The method avoids any complicated sample preparation steps except drying and sieving thus saving time and acquisition of reflectance spectrum for single sample is possible within 18 s. Owing to have the minimum quantification limit ranging from 1.9-3.7 g/kg soil, the vis-NIR based method is perfectly suitable for estimation of MPs in soil samples collected from plastic pollution hotspots like landfill sites, regular based sludge amended farm soils. Additionally, the method can be adapted by small scale compost industries for assessing MPs load in product like city compost which are applied at agricultural fields and will be helpful in quantifying possible MPs at the sources itself.

Utility of peripheral blood monocyte subsets, circulating immune complexes and serum cytokines in assessment of SLE activity: an observational, cross-sectional study.

INTRODUCTION: SLE disease measurements by current standards are less than perfect. Monocytes and their subsets are part of innate immunity, and one of our objectives was to look at their role in SLE disease activity. We also looked at the common serum cytokines and the role of circulating immune complex (CIC) estimation in the assessment of disease activity. METHODS: We conducted a single-centre observational cross-sectional study of SLE patients with active and inactive disease as the comparison arms. Blood samples were collected for (a) peripheral blood monocyte separation and flowcytometric analysis of monocyte subsets based on CD14 and CD16 surface markers, and (b) ELISA for serum cytokines and CIC estimation. Results were analysed in terms of the difference in medians between the active and inactive disease groups using the Mann-Whitney U test (non-normally distributed data). RESULTS: The absolute monocyte count was lower in the active group than the inactive group (median (IQR) of 329 (228.5) vs. 628 (257)/microliter, p = 0.001). The frequency (%) of the intermediate monocyte subset showed a trend towards an increase in active disease (median (IQR) of 15.10% (9.65) vs. 11.85% (8.00), p = 0.09). It also had a significant positive correlation to the SLEDAI scores (r = 0.33, p = 0.046). The mean fluorescence intensity (MFI) of CD163, expressed primarily by intermediate subsets, was increased, and CD11c MFI was reduced in active disease. Serum TNF-a level was elevated in active disease (median (IQR) of 38 (48.5) pg/ml vs. 9 (48.5) pg/ml, p = 0.042). CIC ELISA at an optimal cut-off of 10 meq/ml provided an area under the curve (AUC) of 0.85 for detecting active SLE. CONCLUSION: Peripheral blood monocytes are depleted in active disease. The intermediate monocyte subset may have a role in disease activity. TNF-alpha correlated modestly with disease activity. CIC estimation by ELISA may be used in addition to or as an alternative to current standards of laboratory tests for the serological assessment of activity.

Health risk assessment of aerosol particles (PM2.5 and PM10) during winter crop at the agricultural site of Delhi, India.

For the last few decades, air pollution in developing country like India is increasing, and it is a matter of huge concern due to its associated human health impacts. In this region, the burgeoning population, escalating urbanization and industrialization, has been cited as the major reason for such a high air pollution. The present study was carried out for health risk assessment of aerosol particles (PM10 and PM2.5) and its associated heavy metals of an agriculture farm site at Indian Agricultural Research Institute (IARI) considered to be green urban area in Delhi, India. The concentrations of both PM10 and PM2.5 varied significantly from 136 to 177 µg/m3 and 56 to 162 µg/m3, respectively at the site. In the present case, the highest PM10 and PM2.5 levels were reported in January, followed by December. The levels of ambient PM10 and PM2.5 are influenced by wind prevailing meteorology. These levels of PM10 and PM2.5 are more than the permissible limits of WHO guidelines of 15 and 5 µg/m3, respectively, thereby leading to high aerosol loadings specifically in winters. The PM concentration of the atmosphere was found to be negatively correlated with temperature during the sampling period. The concentrations of surface ozone O3 and NOx in the present study were observed to be high in February and March, respectively. The increasing air pollution in the city of Delhi poses a great risk to the human health, as the particulate matter loaded with heavy metals can enter humans via different pathways, viz., ingestion, inhalation, and absorption through skin. The mean hazard index for metals (Zn, Pb, Cd, As, Cr, and Ni) was observed within the acceptable limit (HI < 1), thereby indicating negligible non-carcinogenic effects to residing population. The carcinogenic risk assessment was conducted for Cd, Pb, and As only, as the concentrations for other metals were found to be quite low. The carcinogenic risk values were also within the limits of USEPA standards, indicating no carcinogenic risks to the health of children and adults residing near the site. This information about the PM pollution at the agricultural site and health risk assessment will serve as a baseline data in assessment of human health impacts due to air pollution at the local scale and can be used for development of mitigation strategies for tackling air pollution.

Characterization of immune responses to two and three doses of the adenoviral vectored vaccine ChAdOx1 nCov-19 and the whole virion inactivated vaccine BBV152 in a mix-and-match study in India.

Infections with SARS-CoV-2 variants and declining immunity after primary vaccination, encouraged the use of booster doses. Some countries changed their immunization programmes to boost with vaccines different from the ones in their original schedule, based on results from immunogenicity and effectiveness studies. This study reports immunological analysis of samples collected in a phase 4 randomized trial, where participants who had previously received two primary doses of ChAdOx1 nCov-19 (ChAd) or inactivated BBV152 vaccine were randomized to receive either ChAd or BBV152 booster and further categorized as: Group 1 (two primary doses of ChAd - ChAd booster), Group 2 (two primary doses of ChAd - BBV152 booster), Group 3 (two primary doses of BBV152 - ChAd booster), and Group 4 (two primary doses of BBV152 - BBV152 booster). SARS-CoV-2 specific cellular and humoral responses at day 0 (pre-boost samples 12-36 weeks after the second primary dose), and at day 28 post booster, were measured in a subset of participants (ChAd recipients, n = 37 and BBV152 recipients, n = 36). Additionally, on day180 post-booster humoral responses were assessed for the entire cohort (N = 378). Primary vaccination with 2 doses of BBV152 generated higher memory-B cells (median% 0.41 vs 0.35) and cytokine producing CD8-Tcells (median% 0.09 vs 0.04) while lower anti-spike IgG levels (medianAU/ml: 12,433 vs 27,074) as compared to ChAd. Irrespective of the primary vaccine received, ChAd boosted individuals generated higher memory-B cell frequencies and anti-spike IgG levels as compared to BBV152 booster. The percentage ACE-2 inhibition against Omicron and its sub-variants was higher in Group 3 (median > 60 %) as compared to other groups (median < 25 %). At day180 post booster the hierarchy of the antibody amounts was Group 1 âˆ¼ Group 2 âˆ¼ Group 3 > Group 4. Sustained humoral and robust cellular immune response to SARS-CoV-2 can be obtained with ChAd booster irrespective of the primary vaccination regimen. The trial is registered with ISRTCN (CTRI/2021/08/035648).

Lymphoepithelioma-Like Carcinoma of Urinary Bladder with Diagnostic Dilemma: Case Report of a Rare Variant of Urothelial Carcinoma.

Lymphoepithelioma-like carcinoma of the urinary bladder is a rare variant of urothelial carcinoma that was first described by Zukerberg in 1991 and confirmed as a type of urothelial carcinoma by the WHO classification of the urinary system tumors. A 63-year-old man presented with the chief complaint of gross hematuria for 2 months. Sections from transurethral resection of the bladder revealed urothelial carcinoma. A radical cystoprostatectomy was performed and a final histopathological examination revealed high-grade muscle-invasive urothelial carcinoma, lymphoepithelial variant. The patient has been receiving adjuvant chemotherapy with no recurrence reported to date. This is the third case reported in India to date to the best of our knowledge. Owing to its rare presentation, no definite treatment guidelines have been established for this entity. Various studies from the English literature suggest a conservative approach.

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.

Comparison of Human Neonatal and Adult Blood Leukocyte Subset Composition Phenotypes.

The human peripheral leukocyte subset composition depends on genotype variation and pre-natal and post-natal environmental influence diversity. We quantified this composition in adults and neonates, and compared the median values and dispersal ranges of various subsets in them. We confirmed higher frequencies of monocytes and regulatory T cells (Tregs), similar frequencies of neutrophils, and lower frequencies of CD8 T cells, NKT cells, B1 B cells and gamma-delta T cells in neonatal umbilical cord blood. Unlike previous reports, we found higher frequencies of eosinophils and B cells, higher CD4:CD8 ratios, lower frequencies of T cells and iNKT cells, and similar frequencies of CD4 T cells and NK cells in neonates. We characterized monocyte subsets and dendritic cell (DC) subsets in far greater detail than previously reported, using recently described surface markers and gating strategies and observed that neonates had lower frequencies of patrolling monocytes and lower myeloid dendritic cell (mDC):plasmacytoid DC (pDC) ratios. Our data contribute to South Asian reference values for these parameters. We found that dispersal ranges differ between different leukocyte subsets, suggesting differential determination of variation. Further, some subsets were more dispersed in adults than in neonates suggesting influences of postnatal sources of variation, while some show the opposite pattern suggesting influences of developmental process variation. Together, these data and analyses provide interesting biological possibilities for future exploration.

Therapeutic treatment with a novel hypoxia-inducible factor hydroxylase inhibitor (TRC160334) ameliorates murine colitis.

BACKGROUND AND AIM: Mucosal healing in inflammatory bowel disease (IBD) can be achieved by improvement of intestinal barrier protection. Activation of hypoxia-inducible factor (HIF) has been identified as a critical factor for barrier protection during mucosal insult and is linked with improvement in symptoms of colitis. Although prophylactic efficacy of HIF hydroxylase inhibitors in murine colitis have been established, its therapeutic efficacy in clinically relevant therapeutic settings have not been established. In the present study we aim to establish therapeutic efficacy of TRC160334, a novel HIF hydroxylase inhibitor, in animal models of colitis. METHODS: The efficacy of TRC160334 was evaluated in two different mouse models of colitis by oral route. A prophylactic efficacy study was performed in a 2,4,6-trinitrobenzene sulfonic acid-induced mouse model of colitis representing human Crohn's disease pathology. Additionally, a therapeutic efficacy study was performed in a dextran sulfate sodium-induced mouse model of colitis, a model simulating human ulcerative colitis. RESULTS: TRC160334 treatment resulted in significant improvement in disease end points in both models of colitis. TRC160334 treatment resulted into cytoprotective heatshock protein 70 induction in inflamed colon. TRC160334 successfully attenuated the rate of fall in body weight, disease activity index, and macroscopic and microscopic scores of colonic damage leading to overall improvement in study outcome. CONCLUSION: Our findings are the first to demonstrate that therapeutic intervention with a HIF hydroxylase inhibitor ameliorates IBD in disease models. These findings highlight the potential of TRC160334 for its clinical application in the treatment of IBD.

Modulation of nutritional and antioxidant potential of seeds and pericarp of pea pods treated with microbial consortium.

Microbial populations have diverse roles within rhizosphere where interactions among distinct microorganisms along with the host may lead to mutualistic associations. The present study aimed to investigate the nutritional and antioxidant qualities of seeds and pericarp of pea raised from seeds treated with beneficial microbes namely Bacillus subtilis BHHU100, Trichoderma harzianum TNHU27 and Pseudomonas aeruginosa PJHU15 either singly and/or in consortia. A significant increase in total phenolic, flavonoid, ascorbic acid and protein contents, free radical scavenging activity, hydroxyl radical scavenging activity, iron chelation and reducing power were observed in the seeds and pericarp of pods treated with a consortium of microbes in comparison to control pods. Also, the differential accumulation of phenolic compounds, namely, shikimic acid, gallic acid, tannic acid, syringic acid, p-coumaric acid, quercetin and kaempferol, was observed from the HPLC chromatogram of the seed extracts of different treatments. We especially emphasized on dietary importance of the pod pericarp, other than seeds, along with their modulation by microbial consortium. The study also highlights the role of beneficial microbes in improving nutritional value by providing protection against oxidative stress.

TRC210258, a novel TGR5 agonist, reduces glycemic and dyslipidemic cardiovascular risk in animal models of diabesity.

BACKGROUND: Patients with diabesity have a significantly increased risk of developing cardiovascular disease. Therefore, therapy addressing the multiple metabolic abnormalities linked with diabesity and leading to further reduction of cardiovascular risk is highly desirable. Activation of the TGR5 receptor holds therapeutic potential for diabesity. In the present study, we evaluated the efficacy of TRC210258, a novel TGR5 agonist, in clinically relevant animal models of diabesity. METHODS: A novel small molecule, TRC210258 (N-(4-chlorophenyl)-2-(4-fluorophenoxy)-N-methylimidazo (1, 2-a) pyrimidine-3-carboxamide), was synthesized. The in vitro TGR5 receptor activation potential of TRC210258 was assessed by cyclic adenosine monophosphate (cAMP) assay and cAMP-responsive element reporter assay using cells overexpressing the human TGR5 receptor. The effect of TRC210258 on glucagon-like peptide-1 release was evaluated in vitro using a human enteroendocrine cell line. The effect of TRC210258 on energy expenditure and glycemic control was evaluated in high-fat diet-induced obese mice. Additionally, the effect of TRC210258 on dyslipidemic parameters was determined in high fat-fed hamsters. RESULTS: TRC210258 demonstrated potent TGR5 agonist activity, with enhanced glucagon-like peptide-1 release and energy expenditure. Treatment with TRC210258 resulted in better glycemic control and improved parameters of dyslipidemia such as plasma triglyceride, low-density lipoprotein cholesterol, and non-high-density lipoprotein cholesterol levels. Treatment with TRC210258 also improved emerging dyslipidemic cardiovascular risk parameters, including remnant cholesterol and triglyceride clearance. CONCLUSION: This study highlights the potential of TRC210258, a novel TGR5 agonist, to improve dyslipidemic cardiovascular risk beyond glycemic control in patients with type 2 diabetes.

Treatment with a novel hypoxia-inducible factor hydroxylase inhibitor (TRC160334) ameliorates ischemic acute kidney injury.

BACKGROUND: Hypoxia-inducible factor (HIF) transcriptional system plays a central role in cellular adaptation to low oxygen levels. Preconditional activation of HIF and/or expression of its individual target gene products leading to cytoprotection have been well established in hypoxic/ischemic renal injury. Increasing evidence indicate HIF activation is involved in hypoxic/ischemic postconditioning of heart, brain and kidney. Very few studies evaluated the potential benefits of postischemia HIF activation in renal injury employing a pharmacological agent. We hypothesized that postischemia augmentation of HIF activation with a pharmacological agent would protect renal ischemia/reperfusion injury. For this, TRC160334, a novel HIF hydroxylase inhibitor, was used. METHODS: TRC160334, a novel HIF hydroxylase inhibitor, was synthesized. Ability of TRC160334 for stabilization of HIF-α and consequent HIF activation was evaluated in Hep3B cells. Efficacy of TRC160334 was evaluated in a rat model of ischemia/reperfusion-induced AKI. Two different treatment protocols were employed, one involved treatment with TRC160334 before onset of ischemia, the other involved treatment after the reperfusion of kidneys. RESULTS: TRC160334 treatment results in stabilization of HIF-α leading to HIF activation in Hep3B cells. Significant reduction in renal injury was observed by both treatment protocols and remarkable reduction in serum creatinine (23 and 71% at 24 and 48 h, respectively, p < 0.01) was observed with TRC160334 treatment applied after reperfusion. Urine output was significantly improved up to 24 h by both treatment protocols. CONCLUSION: The data presented here provide pharmacologic evidence for postischemia augmentation of HIF activation by TRC160334 as a promising and clinically feasible strategy for the treatment of renal ischemia/reperfusion injury.

Bioprospecting thermophiles for cellulase production: a review

Most of the potential bioprospecting is currently related to the study of the extremophiles and their potential use in industrial processes. Recently microbial cellulases find applications in various industries and constitute a major group of industrial enzymes. Considerable amount of work has been done on microbial cellulases, especially with resurgence of interest in biomass ethanol production employing cellulases and use of cellulases in textile and paper industry. Most efficient method of lignocellulosic biomass hydrolysis is through enzymatic saccharification using cellulases. Significant information has also been gained about the physiology of thermophilic cellulases producers and process development for enzyme production and biomass saccharification. The review discusses the current knowledge on cellulase producing thermophilic microorganisms, their physiological adaptations and control of cellulase gene expression. It discusses the industrial applications of thermophilic cellulases, their cost of production and challenges in cellulase research especially in the area of improving process economics of enzyme production.

Optimization of fermentation conditions for cellulases production by Bacillus licheniformis MVS1 and Bacillus sp. MVS3 isolated from Indian hot spring

The aim of this work was to study the effect of some nutritional and environmental factors on the production of cellulases, in particular endoglucanase (CMCase) and exoglucanases (FPase) from Bacillus licheniformis MVS1 and Bacillus sp. MVS3 isolated from an Indian hot spring. The characterization study indicated that the optimum pH and temperature value was 6.5 to 7.0 and 50-55°C, respectively. Maximum cellulases production by both the isolates was detected after 60 h incubation period using wheat and rice straw. The combination of inorganic and organic nitrogen source was suitable for cellulases production. Overall, FPase production was much higher than CMCase production by both of the strains. Between the two thermophiles, the cellulolytic activity was more in B.licheniformis MVS1 than Bacillus sp. MVS3 in varying environmental and nutritional conditions.

Impact of transgenic cotton varieties on activity of enzymes in their rhizosphere.

The impact of five Bacillus thuringiensis (Bt) cotton varieties and their respective isogenic non-Bt(NBt) isolines (ANKUR-2534, MECH-6304, RCH-317, ANKUR-651 and MECH-6301) was assessed on the key soil enzymes i.e., dehydrogenase, alkaline phosphatase and urease in their rhizosphere at four growth stages of the crop, namely vegetative, flowering, bolling and harvesting. These varieties were grown on farmer's field in villages 22 miles and 24 miles of Ganganagar District of Rajasthan State in India. Results showed that dehydrogenase, alkaline phosphatase and urease activities were higher in rhizosphere of Bt isolines as compared to NBt isolines of all the varieties. Except phosphatase, differences in dehydrogenase and urease activities in rhizosphere of Bt and NBt isolines of all five varieties were significant (P < 0.05). Maximum enhancement in the three enzymes activities was observed in MECH-6304 Bt isoline rhizosphere. Maximum and minimum activities of dehydrogenase and urease were observed in MECH-6304 and RCH-317 Bt isolines, respectively, whereas phosphatase activity was maximum and minimum in MECH-6304 and ANKUR-651 Bt isolines, respectively. Maximum dehydrogenase and urease activities were observed at boll formation and minimum at flowering and harvesting stage, respectively, while maximum phosphatase activity was observed at vegetative stage and minimum at harvesting stage. In conclusion, all the studied Bt isolines of cotton varieties showed no adverse effect on dehydrogenase, alkaline phosphatase and urease activities in the rhizosphere.

Bioprospecting thermophiles for cellulase production: a review.

Most of the potential bioprospecting is currently related to the study of the extremophiles and their potential use in industrial processes. Recently microbial cellulases find applications in various industries and constitute a major group of industrial enzymes. Considerable amount of work has been done on microbial cellulases, especially with resurgence of interest in biomass ethanol production employing cellulases and use of cellulases in textile and paper industry. Most efficient method of lignocellulosic biomass hydrolysis is through enzymatic saccharification using cellulases. Significant information has also been gained about the physiology of thermophilic cellulases producers and process development for enzyme production and biomass saccharification. The review discusses the current knowledge on cellulase producing thermophilic microorganisms, their physiological adaptations and control of cellulase gene expression. It discusses the industrial applications of thermophilic cellulases, their cost of production and challenges in cellulase research especially in the area of improving process economics of enzyme production.

Bacterial and archaeal phylogenetic diversity of a cold sulfur-rich spring on the shoreline of Lake Erie, Michigan.

Studies of sulfidic springs have provided new insights into microbial metabolism, groundwater biogeochemistry, and geologic processes. We investigated Great Sulphur Spring on the western shore of Lake Erie and evaluated the phylogenetic affiliations of 189 bacterial and 77 archaeal 16S rRNA gene sequences from three habitats: the spring origin (11-m depth), bacterial-algal mats on the spring pond surface, and whitish filamentous materials from the spring drain. Water from the spring origin water was cold, pH 6.3, and anoxic (H(2), 5.4 nM; CH(4), 2.70 microM) with concentrations of S(2-) (0.03 mM), SO(4)(2-) (14.8 mM), Ca(2+) (15.7 mM), and HCO(3)(-) (4.1 mM) similar to those in groundwater from the local aquifer. No archaeal and few bacterial sequences were >95% similar to sequences of cultivated organisms. Bacterial sequences were largely affiliated with sulfur-metabolizing or chemolithotrophic taxa in Beta-, Gamma-, Delta-, and Epsilonproteobacteria. Epsilonproteobacteria sequences similar to those obtained from other sulfidic environments and a new clade of Cyanobacteria sequences were particularly abundant (16% and 40%, respectively) in the spring origin clone library. Crenarchaeota sequences associated with archaeal-bacterial consortia in whitish filaments at a German sulfidic spring were detected only in a similar habitat at Great Sulphur Spring. This study expands the geographic distribution of many uncultured Archaea and Bacteria sequences to the Laurentian Great Lakes, indicates possible roles for epsilonproteobacteria in local aquifer chemistry and karst formation, documents new oscillatorioid Cyanobacteria lineages, and shows that uncultured, cold-adapted Crenarchaeota sequences may comprise a significant part of the microbial community of some sulfidic environments.

Insular carcinoma of thyroid: A case report.

A histological distinct subset of thyroid carcinoma, which falls between well differentiated and anaplastic carcinoma with respect to cell differentiation and clinical behaviour. It has been categorized as poorly differentiated or insular carcinoma based on its characteristic cell groupings. Insular carcinoma warrants aggressive management with total thyroidectomy followed by radioactive iodine ablation of the residual deposit. We present a case of insular carcinoma, which was diagnosed post operatively based on the histopathological enzyme study.

Domain truncation studies reveal that the streptokinase-plasmin activator complex utilizes long range protein-protein interactions with macromolecular substrate to maximize catalytic turnover.

To explore the interdomain co-operativity during human plasminogen (HPG) activation by streptokinase (SK), we expressed the cDNAs corresponding to each SK domain individually (alpha, beta, and gamma), and also their two-domain combinations, viz. alphabeta and betagamma in Escherichia coli. After purification, alpha and beta showed activator activities of approximately 0.4 and 0.05%, respectively, as compared with that of native SK, measured in the presence of human plasmin, but the bi-domain constructs alphabeta and betagamma showed much higher co-factor activities (3.5 and 0.7% of native SK, respectively). Resonant Mirror-based binding studies showed that the single-domain constructs had significantly lower affinities for "partner" HPG, whereas the affinities of the two-domain constructs were remarkably native-like with regards to both binary-mode as well as ternary mode ("substrate") binding with HPG, suggesting that the vast difference in co-factor activity between the two- and three-domain structures did not arise merely from affinity differences between activator species and HPG. Remarkably, when the co-factor activities of the various constructs were measured with microplasminogen, the nearly 50-fold difference in the co-factor activity between the two- and three-domain SK constructs observed with full-length HPG as substrate was found to be dramatically attenuated, with all three types of constructs now exhibiting a low activity of approximately 1-2% compared to that of SK.HPN and HPG. Thus, the docking of substrate through the catalytic domain at the active site of SK-plasmin(ogen) is capable of engendering, at best, only a minimal level of co-factor activity in SK.HPN. Therefore, apart from conferring additional substrate affinity through kringle-mediated interactions, reported earlier (Dhar et al., 2002; J. Biol. Chem. 277, 13257), selective interactions between all three domains of SK and the kringle domains of substrate vastly accelerate the plasminogen activation reaction to near native levels.