The impact of addition of pearl millet starch-germ complex in white bread on nutritional, textural, structural, and glycaemic response: Single blinded randomized controlled trial in healthy and pre-diabetic participants.

The rise of pre-diabetes at the global level has created a significant interest in developing low glycaemic index food products. The pearl millet is a cheaper source of starch and its germ contains significant amount of protein and fat. The complexing of pearl millet starch and germ by dry heat treatment (PMSGH) resulted an increase in the resistant starch content upto 45.09 % due to formation of amylose-glutelin-linoleic acid complex. The resulting pearl millet starch germ complex was incorporated into wheat bread at 20, 25, and 30 %. The PMSGH incorporated into bread at 30 % reduced the glycaemic index to 52.31. The PMSGH incorporated bread had significantly (p < 0.05)increased in the hardness with a reduction in springiness and cohesiveness. The structural attributes of the 30 % PMSGH incorporated bread revealed a significant (p < 0.05)increase in 1040/1020 cm-1 ratio and relative crystallinity. The consumption of functional bread incorporated with pearl millet starch germ complex reduced blood glucose levels and in vivo glycaemic index in healthy and pre-diabetic participants when compared to white bread. Hence, the study showed that the incorporation of pearl millet starch-germ complex into food products could be a potential new and healthier approach for improving dietary options in pre-diabetes care.

Green synthesis of chitosan nanoparticles using tea extract and its antimicrobial activity against economically important phytopathogens of rice.

The aim of the present work is to biosynthesize Chitosan nanoparticles (CTNp) using tea (Camellia sinensis) extract, with potent antimicrobial properties towards phytopathogens of rice. Preliminary chemical analysis of the extract showed that they contain carbohydrate as major compound and uronic acid indicating the nature of acidic polysaccharide. The structure of the isolated polysaccharide was analyzed through FTIR and 1H NMR. The CTNp was prepared by the addition of isolated tea polysaccharides to chitosan solution. The structure and size of the CTNp was determined through FTIR and DLS analyses. The surface morphology and size of the CTNp was analysed by SEM and HRTEM. The crystalinity nature of the synthesized nanoparticle was identified by XRD analysis. The CTNp exhibited the antimicrobial properties against the most devastating pathogens of rice viz., Pyricularia grisea, Xanthomonas oryzae under in vitro condition. CTNp also suppressed the blast and blight disease of rice under the detached leaf assay. These results suggest that the biosynthesized CTNp can be used to control the most devastating pathogens of rice.

DoseCal: a virtual calculator for dosage conversion between human and different animal species.

CONTEXT: The scientists as well as pharmacologists frequently convert the dose of drugs or extracts between human and different animal species. According to the literature, the extrapolation of the dose by simple conversion based only on body weight is not an accurate method. The miscalculation of dosage conversion may result in adverse effects due to overdose or reduced potency due to underdose. AIM: To develop a conversion calculator with factual factor like Km factor. METHOD: The virtual calculator was developed as a web-based application using hypertext pre-processor programming language. My SQL software was used to store the data related to DoseCal. RESULTS: Based on the Km factor, dose conversion calculator called DoseCal was developed. The DoseCal would provide the dosage for both per kg and actual weight of the animal taken for the experiment. CONCLUSION: The DoseCal would assist scientists in the dosage conversion easily and accurately between species.

Nutritional interventions for improving the endurance performance in athletes.

Endurance refers to the ability of skeletal muscles to perform continuously withstanding the hardships of exercise. Endurance exercises have three phases: pre-, during-, and post-workout phase. The nutritional requirements that drive these phases vary on intensity, type of workout, individual's body composition, training, weather conditions, etc. Generally, the pre-workout phase requires glycogen synthesis and spare glycogen breakdown. While workout phase, requires rapid absorption of exogenous glucose, insulin release to transport glucose into muscle cells, replenish the loss of electrolytes, promote fluid retention, etc. However, post-workout phase requires quick amino acid absorption, muscle protein synthesis, repair of damaged muscle fibres and tendon, ameliorate inflammation, oxidative stress, etc. Therefore, nutritional sources that can help these metabolic requirements is recommended. In this review, various dietary interventions including timing and amount of nutrient consumption that can promote the above metabolic requirements that in turn support in improving the endurance potential in athletes are discussed.HIGHLIGHTSReview article describes nutritional requirements of endurance exercises.It also describes nutritional interventions to enhance the endurance potential in athletes.

Ameliorative effect of enhanced Fischer ratio flaxseed protein hydrolysate in combination with antioxidant micronutrients on ethanol-induced hepatic damage in a rat model.

Alcohol abuse causes severe metabolic abnormalities inducing hepatic damage and malnutrition. Since higher Fischer ratio proteins have therapeutic value in liver diseases, an investigation was undertaken to study the ameliorative effect of the enhanced Fischer ratio flaxseed protein hydrolysate (EFR-FPH) alone and in combination with antioxidant micronutrients on ethanol-induced hepatotoxicity in a rat model. The EFR-FPH was prepared by dual enzymatic hydrolysis and charcoal treatment of flaxseed protein. The ratio of the branched-chain:aromatic amino acids (Fischer ratio) was found to be 7·08. The EFR-FPH, characterised using LC-MS/MS, showed the abundance of free leucine and isoleucine compared with phenylalanine and tyrosine. The matrix-assisted laser desorption/ionisation-time of flight MS analysis revealed the larger peptides present in EFR-FPH with mass 2·3 kDa. The EFR-FPH improved the nutritional status, liver function and antioxidant defense in the ethanol hepatotoxicity-induced rat model. The hepatoprotective effect of EFR-FPH was significantly enhanced when combined with selenium or vitamin E. Ethanol-induced changes in the liver tissue were effectively suppressed in the groups receiving EFR-FPH. Flaxseed-based hepatoprotective dietary supplement was formulated incorporating an optimum level of EFR-FPH (10 %) based on sensory acceptability and was fortified with selenium and vitamin E. The hepatoprotective formulation significantly lowered aspartate transaminase, alanine transaminase, alkaline phosphatase and bilirubin by 47, 61, 55 and 78 %, respectively, and improved the antioxidant defense in the ethanol hepatotoxicity-induced rat model. The current investigation suggests that EFR-FPH in synergy with antioxidant micronutrients is potent in ameliorating ethanol-induced hepatotoxicity and has a potential to form a hepatoprotective dietary supplement.

Application of proteins from different meat processing wastewater streams as a dietary protein source in animal feed.

The present study aimed to investigate the protein quality indices of meat processing wastewaters protein hydrolysate (FWPH) obtained by Pediococcus lolii fermentation using a rat bioassay. Different diets namely, SD (100% casein), F50 (50% FWPH+50% casein), F75 (75% FWPH+25% casein), and F100 (100% FWPH) were formulated and fed to 28 days old albino male Wistar rats (6 rats each group). SD and F50 group rats showed higher feed and protein intake among all diet group rats (P < 0.05). However, the highest body weight gain (142.16 ± 2.84 g) was noticed in the rats fed with the F50 diet (P < 0.05). F50 group rats presented better feed conversion ratio (FCR), feed efficiency ratio (FER), and protein efficiency ratio (PER) values as compared to other diet group counterparts (P < 0.05). Furthermore, no differences in values of urine, haematological, and serum biochemical parameters were observed in SD and F50 group rats (P > 0.05). However, haematological and serum biochemical parameters were slightly elevated in F100 group rats. F50 group rats presented the higher superoxide dismutase (SOD) and catalase (CAT) activities among all the diet groups (P < 0.05). Relative organ weights, serum glutamic pyruvic transaminase (SGPT), serum glutamic oxaloacetic transaminase (SGOT), and alkaline phosphatase (ALP) activities of F50 group rats were found similar to SD rats. Histopathological examination revealed that no incidence of organ toxicity is attributed to the F50 diet. Overall, F50 group rats exhibited good growth and development with no adverse effects in biochemical, haematological, and histopathology parameters. Therefore, FWPH at the 50% level could be used as a potential dietary protein source in feed for better growth performance of the animal.

A field study on the evaluation of day-of-hatch and in grow-out application of live infectious bursal disease virus vaccine in broiler chickens.

Infectious bursal disease (IBD) is an acute, highly contagious, economically important disease of young chickens caused by Avibirnavirus, the infectious bursal disease virus (IBDV). The causative virus is highly resilient in poultry environments and vaccination is the most effective measure for IBDV control. However, the susceptibility of highly attenuated IBDV vaccine strains to neutralization by maternally derived antibodies (MDA) and overwhelming virulence of partly attenuated strains have limited the application of conventional live IBDV vaccines in pre- and posthatch chicks. Nevertheless, preliminary data have raised questions about the validity of this prevailing dogma. India is an IBD endemic country and the disease causes sizeable economic losses in the sector. To evaluate the feasibility of application of live IBDV vaccine strain, the IBDV MB-1, to the maternally immunized day-of-hatch chicks in Indian production environment, 4 large-scale field trials have been conducted. The 4 trials have measured the relative safety, IBDV immunization parameters, and production performances of MB-1 vs. the established live and immune complex IBDV vaccines in a variety of commercial broiler systems. The overall health and production performances in all 4 trials have been better in the MB-1 groups. The results challenge the prevailing notion that live IBDV strains may be neutralized or break through maternal immunity and induce permanent damage to the young broiler chick's immune response. A delayed replication phenomenon following parenteral administration of the live IBDV vaccine strain has been observed, while the delayed replication mechanism remains to be elucidated. This study warrants further research on the molecular mechanism of live IBDV vaccine strain, MB-1, and its interaction with the chicken immune system.

A Rare Case of Fractured Posterior Facet of the Talus in Association With Open Medial Subtalar Dislocation.

Subtalar or peritalar dislocation is the loss of contact between the articular surface of the talus distally and the calcaneum and navicular. In this paper, a case of open medial type of subtalar dislocation associated with fractured posterior facet of the talus in a 27-year-old man with a history of road traffic accident was reported. Immediate wound irrigation and open reduction under general anesthesia at the emergency room operation theater was successful followed by cast immobilization. At one-year follow-up, the patient was walking and carrying out his daily activities with mild restriction of inversion and eversion movements. Extensive wound debridement followed by immediate reduction and, when required, stabilization are the principal features of management. Open subtalar dislocation is an extremely rare injury and often poses a treatment dilemma. Early debridement and open reduction of the dislocation like in our case can give good functional outcome for an open medial subtalar dislocation at one-year follow-up. Temporary stabilization of dislocation in the form of Kirschner wires maybe needed in some cases only.

Is the Acetabulum Retroverted in SCFE? A Study of Acetabular Morphology in Indian Children with SCFE.

BACKGROUND: Acetabular morphology has always been an area of interest in patients with slipped capital femoral epiphysis (SCFE). Acetabular retroversion in SCFE is proposed as a pre-disposing factor and also can predispose the hip to pincer impingement. But there is controversial data in literature regarding the acetabular morphology. All available data are from the West and we present the first study on acetabular morphology in Indian children with SCFE. METHODS: CT scans of 29 patients with SCFE were collected from our database and anteversion was measured as described by Dandachli et al. Lateral central edge angle (LCEA) was measured in the standard AP radiographs of the pelvis. There were 20 boys and 9 girls with a mean age of 13.5. Seven patients had right side SCFE, eighteen had left side SCFE, and four had bilateral involvement. Measurements were done in 33 affected hips and compared with 25 normal hips. RESULTS: The mean AVsup and AVcen of affected hips were 6.59° and 13.51°, respectively, and that in normal hips were 8.36° and 14.04° (p > 0.05). The mean LCEA was 23.05° on the affected hips and 25.45° on the normal hips (p > 0.05). The AVsup showed retroversion in 24.24% (8/33) of the affected hips and 20% (5/25) of the normal hips. Though the mean version was less in SCFE hips, it was not statistically significant. CONCLUSION: Our study documents that 25% of hips with SCFE had acetabular retroversion. However, this was not significantly high compared to the contralateral hips.

High-Throughput Process Development: II-Membrane Chromatography.

Membrane chromatography is gradually emerging as an alternative to conventional column chromatography. It alleviates some of the major disadvantages associated with the latter, including high-pressure drop across the column bed and dependence on intraparticle diffusion for the transport of solute molecules to their binding sites within the pores of separation media. In the last decade, it has emerged as a method of choice for final polishing of biopharmaceuticals, in particular, monoclonal antibody products. The relevance of such a platform is high in view of the constraints with respect to time and resources that the biopharma industry faces today.This protocol describes the steps involved in performing HTPD of a membrane chromatography step. It describes the operation of a commercially available device (AcroPrep™ Advance filter plate with Mustang S membrane from Pall Corporation). This device is available in 96-well format with a 7 µL membrane in each well. We will discuss the challenges that one faces when performing such experiments as well as possible solutions to alleviate them. Besides describing the operation of the device, the protocol also presents an approach for statistical analysis of the data that are gathered from such a platform. A case study involving the use of the protocol for examining ion-exchange chromatography of the Granulocyte Colony Stimulating Factor (GCSF), a therapeutic product, is briefly discussed. This is intended to demonstrate the usefulness of this protocol in generating data that are representative of the data obtained at the traditional lab scale. The agreement in the data is indeed very significant (regression coefficient 0.9866). We think that this protocol will be of significant value to those involved in performing high-throughput process development of membrane chromatography.

Synthesis, characterization and anti-inflammatory properties of karanjin (Pongamia pinnata seed) and its derivatives.

Karanja (Pongamia pinnata) is a medicinal tree used in the Indian traditional ayurvedic system for treating several ailments. The seeds contain a unique furano-flavonoid karanjin, which has shown to possess many medicinal properties. Its usage at the clinical level is affected due to poor solubility and absorption. In the present investigation, molecular modifications of karanjin were attempted and evaluated their effect on anti-inflammatory activity. Firstly, Karanja ketone was obtained from karanjin by hydrolysis, and it was converted into karanja ketone oxime. The oxime undergoes Beckmann rearrangement and cyclized to yield furano benzoxazole (karanja oxazole). The new derivatives were purified with >95% purity (HPLC) and spectrally characterized (HR-MS, FTIR, and NMR). Among the test compounds, karanja ketone oxime exhibited higher antioxidant activity with an IC50 value of 360 µg/ml (DPPH). Soy lipoxygenase-1 (LOX-1) inhibitory activity of oxime was higher (IC50 = 65.4 µM) than other compounds. Fluorescence studies showed that oxime had higher quenching capacity with a Qmax of 76.3% and a binding constant of 0.9 × 105 M-1 for soy LOX-1. In-silico interaction studies showed that karanja ketone oxime had the least binding energy of -5.76 kcal/mol with LOX-1 by forming two hydrogen bonds with hydrophobic amino acids Leu 390 and Gly 392. The compounds were evaluated for their acute anti-inflammatory activity by the paw and ear edema in the rat model. Karanjin inhibits paw edema and ear edema by 34.13% and 51.13%, respectively, whereas the derivatives inhibited by 45-57 % and 70-76.8%. This study reports a rational approach to synthesize karanjin derivatives with considerable anti-inflammatory properties, both in-vitro and in-vivo.

Preeclampsia: Pathophysiology and management.

Preeclampsia is a pregnancy-related multisystem disorder, frequently encountered pregnancy-related medical complications next to gestational diabetes mellitus. It is the onset of hypertension during pregnancy. The preeclampsia can be of two types, placental or maternal preeclampsia. Among these two types former, i.e., placental preeclampsia is more severe than the latter. According to the recent survey by National Health Portal of India, the incidence of preeclampsia is about 8-10 % among pregnant women. Though our understanding of preeclampsia has improved in recent years, the development and interpretation of the clinical tests remain difficult for preeclampsia. Hence, we have made an attempt to understand the pathophysiology, associated conditions/consequences, treatment and management/prevention of the condition in this review.

Chitosan guar nanoparticle preparation and its in vitro antimicrobial activity towards phytopathogens of rice.

The aim of the present study was to prepare chitosan guar nanoparticle (CGNP) with high antimicrobial activity to use as a bioprotectant against rice phytopathogens. Nanoparticles were prepared using sodium tripolyphosphate by the ionic gelation method. The physico-chemical properties of nanoparticles were characterized through DLS, FTIR, TEM, SEM, AFM and XRD. The application of CGNP to rice seeds stimulated seed germination and seedling growth. CGNP showed growth inhibition towards rice pathogens P. grisea and X. oryzae under in-vitro condition. Excised rice leaves treated with CGNP and challenged with P. grisea showed no blast disease symptom whereas control leaves showed very high blast disease symptom. The results of this study indicate that CGNP can be used as an antimicrobial agent to control blast, blight disease of rice.

Rare Case Report of Closed Traumatic Dislocation of Second to Fifth Metatarsophalangeal Joints.

Closed traumatic dislocation of multiple metatarsophalangeal joints is a rare injury. Until now only one case of simultaneous dislocation of all five metatarsophalangeal joints has been reported in peer-reviewed studies. The complex anatomy of the metatarsophalangeal joints prevents the relocation of the joints in a closed manner in maximum cases. We are reporting a case of dorsal dislocation of the second to fifth metatarsophalangeal joints in the left foot after road traffic accident. Bony prominence over the plantar aspect and increased web space between toes on presentation, then incongruity of metatarsophalangeal joints has to be thoroughly checked on radiograph. Since closed reduction attempts failed open reduction was done through dorsal approach using two incisions. Button holing of the capsule with interposition of capsule and plantar plate was noted. Dorsal approach avoids damage to the plantar plate and surrounding soft tissues.

Experimental models of polycystic ovary syndrome: An update.

Polycystic ovary syndrome (PCOS) is one of the major endocrine disorders in women. PCOS is a disorder with alterations in the structure as well as functions of female reproductive organs and is also associated with metabolic disorders. Studies on humans have limitations due to ethical issues, hence animal models are used to understand the different aspects of PCOS. Animal models of PCOS aids in studying various facts beginning from etiology to the treatment, hence, several animal models have been developed. Despite of the establishment of several models and a number of studies on PCOS, lacunae exist. This may be due to the failure in selecting a suitable animal model, as all animal models may not exhibit all the key features of the human PCOS condition or may exhibit traits similar to other diseased conditions in addition to the PCOS which should be excluded. Therefore, in this review, we have discussed the different animal models, features they exhibit, their merits and limitations which may aid in the selection of the relevant animal model of PCOS based upon the investigation's focus. In addition, a few nonmammalian models as an alternative to mammalian models have also been discussed which is to be validated further.

The potential of the iron concentrated germinated wheat in wheat flour fortification: an alternative to the conventional expensive iron fortification.

Investigation of the effect of the Fe abiotic stress on the germination rate, iron accumulation, root and shoot elongation of wheat (Triticum aestivum) was carried out. The grains were exposed to different experimental concentrations of ferrous sulfate (FeSO4) (0-15 mM). The effect of the treatment on the shoot and root elongation of the seeds were reported. There is a linear relationship between the treatment and the inhibition of shoot elongation. The half-inhibition dose (ID50) of FeSO4 on inhibition of shoot elongation was 7.3 mM. Each treatment groups (3-15 mM) were used to fortify the wheat flour at 0.1 mg Fe/g. The effect of fortification on rheology (farinograph, extensograph, and amylograph), quality of pasta and iron bioavailability was studied. The pasta cooking properties, texture and sensory properties of 12 and 15 mM composite pasta were equally acceptable as wheat without fortification, or NaFeEDTA fortified pasta. The iron dialysability of 3-15 mM composite pasta was similar to the NaFeEDTA fortified pasta. The iron bioavailability (in vivo) of 15 mM group based pasta was evaluated in the anemic rats. The pasta at 4% (Fe 0.026 mg/g) in iron-deficient diet fed to anemic rats for 2 weeks showed percentage iron absorption (PIA) and hemoglobin regeneration efficiency (HRE) of 85.3% and 44.4% respectively which is higher than the NaFeEDTA. In conclusion, iron-fortified pasta is the promising food fortificant with more iron bioavailability in the prevention of iron deficiency anemia.

A Prospective Comparative Study on Improvement of Hyperthyroid Cardiovascular Dysfunction in Patients Undergoing Total Thyroidectomy Versus Medical Management.

INTRODUCTION: Cardiovascular dysfunction (CVD) is a well-recognized complication in patients with hyperthyroidism and is the major cause of mortality. Very few studies have compared the outcome of CVD following different treatment modalities. In this study we intended to compare treatment modalities (antithyroid drugs vs surgery) for reversal of CVD. MATERIALS AND METHODS: Patients with newly detected hyperthyroidism were grouped into, Group I [n = 123, age <60 years, undergoing total thyroidectomy], Group II [n = 42, age <60 years, treated with antithyroid medications] were evaluated with 2D echocardiography, serum N terminal pro brain natriuretic peptide (NT-pro-BNP) at the time of diagnosis (Point A), after achieving euthyroidism (Point B) with antithyroid drugs and 6 months after surgery/continuation of antithyroid medications (Point C). Forty patients (Group III), age < 60 years, undergoing total thyroidectomy for nontoxic benign thyroid nodules served as controls. RESULTS: All groups were age and sex matched. At Point A, CVD was evident in 80/123 (65%) in Group I and 28/42 (66.7%) in Group II. At Point B improvement in CVD occurred in 84/123 (68.3%) in Group and 29/42 (69.04%) in Group II. At Point C dramatic improvement in CVD occurred in 118/123 (95.9%) in Group I, whereas only 33/42 (78.5%) improved in Group II. CVD were comparable between Groups I and II at Point A and Point B (p > 0.05). At Point C there was a significant decrease in all the diastolic dysfunction parameters in Group I, whereas the same was not observed in Group II patients. Systolic dysfunction between Groups II and II had no statistical significance at Point C. CONCLUSION: Total thyroidectomy seems to be the definitive treatment of choice for hyperthyroid cardiac dysfunction with diastolic dysfunction completely reversing at 6 months after TT.

Antigen-Specific Gut Inflammation and Systemic Immune Responses Induced by Prolonging Wheat Gluten Sensitization in BALB/c Murine Model.

Gluten-related diseases such as wheat allergy, celiac disease, and gluten intolerance are widespread around the globe to genetically predisposed individuals. The present study aims to develop a wheat-gluten induced BALB/c murine model for addressing wheat-gluten related disorders by sensitizing the wheat gluten through the route of intraperitoneal and oral challenge in prolonged days. During the sensitization, the sera were collected for specific antigliadin antibodies response and proinflammatory markers quantification. Ex vivo primary cells and organs were collected for subsequent analysis of inflammatory profile. Prolonging sensitization of gluten can moderate the antigen-specific inflammatory markers such as IL-1ß, IL-4, IL-15, IL-6, IFN-γ and TNF-α levels in mice sera. However, ex vivo primary cells of splenocytes (SPLs) and intestinal epithelial lymphocytes (IELs) significantly increased the IL-6, IL-15, IL-1ß, and IL-4 levels in G+ (gliadin and gluten) treated cells. Histopathology staining of jejunum sections indicates enterocyte degeneration in the apical part of villi and damage of tight junctions in G+ (gliadin and gluten) sensitized murine model. Immunohistochemistry of embedded jejunum sections showed significant expression of positive cells of IL-15, tTG and IL-4 in G+ sensitized murine model. In contrast, all markers of gluten-related disorders are expressed exclusively such as tTG, ZO-1, IL-15, IL-6, IL-4, and intestinal inflammation was mediated by iNOS, COX-2, TLR-4 and NF-kBp50 signaling mechanism in G+ sensitized mice.

Characterization of nutrients, amino acids, polyphenols and antioxidant activity of Ridge gourd (Luffa acutangula) peel.

Ridge gourd (Luffa acutangula) is consumed as a vegetable after peeling off the skin which is a domestic waste. Luffa acutangula peel (LAP) was observed to be a good source of fiber (20.6 %) and minerals (7.7 %). Amino acid analysis revealed presence of the highest content of Carnosine followed by aspartic acid and aminoadipic acid. Antioxidant activity of different extracts showed that ethyl acetate extract was more potent when compared to other solvent extractions. It exhibited a significant amount of phenolic acids like p-coumaric acid (68.64 mg/100 g of dry weight) followed by gallic acid (34.98 mg/100 g of dry weight), protocatechuic acid (30.52 mg/100 g of dry weight) in free form and ferulic acid (13.04 mg/100 g of dry weight) in bound form.

Analytical characterization of in vitro refolding in the quality by design paradigm: Refolding of recombinant human granulocyte colony stimulating factor.

Protein based therapeutics dominate most pharmaceutical pipelines today. For a therapeutic product to be effective, it is important that it is in its native form as slight modifications have been known to result in significantly different performance in the clinic. When expressed in hosts such as Escherichia coli, formation of inactive insoluble aggregates of proteins popularly known as inclusion bodies occurs in most cases. This necessitates the need for in vitro refolding to generate the native (and active) form of the therapeutic protein. This paper aims to provide an approach to generate a deeper understanding of refolding of a therapeutic protein and then to use it for its optimal production commercially. Recombinant human granulocyte colony stimulating factor has been chosen as the model protein. Seven orthogonal analytical tools have been used to elucidate the refolding process. By strategically using these tools protein refolding has been segregated into a series of well-defined sequence of events, starting from the unfolded random coil and ending with the uniquely folded metastable state. The study also suggests the choice of tools that can be used to monitor each event. We believe that this paper successfully demonstrates an approach to generate deeper understanding of the protein refolding process as per the expectations laid out in the Quality by Design paradigm.