Hydrodynamic characterisation of macromolecules in cucurbits

This thesis comprises of the study performed on the extraction, isolation and structural characterisation of macromolecular components from the three members of the family Cucurbitaceae. In particular, the polysaccharides from C.moschata, C. maxima and C. pepo (butternut squash, zucchini and pumpkin, respectively) and oil bodies from the seeds of C. pepo are selected on the basis of their antidiabetic potential. The study centred around structural characterisation of the polysaccharides using hydrodynamical methods such as analytical ultracentrifugation (sedimentation velocity and sedimentation equilibrium), viscometry and dynamic light scattering followed by the use of gas chromatography and gas chromatography coupled with mass spectrophotometry for the assessment of monosaccharide composition. Bioactivity of these polysaccharides was also examined using complement fixation assay. Pumpkin seed oil bodies were extracted, isolated and characterised under various laboratory conditions to establish the zeta potential and size distribution of oil bodies in the solvent provided. Although the selection of the biomaterial for this study from the three species was based on their antidiabetic potential, other health benefits and practical applications are also associated with them. For example, the characterisation of these macromolecules could act as a stepping stone for the future investigation in therapeutics. These biomaterials can potentially be used in the pharmaceutical industry to act as a drug themselves or can be used as a part of any formulation or otherwise can be used as a nutraceutical compound

Hydrodynamic characterisation of macromolecules in cucurbits

This thesis comprises of the study performed on the extraction, isolation and structural characterisation of macromolecular components from the three members of the family Cucurbitaceae. In particular, the polysaccharides from C.moschata, C. maxima and C. pepo (butternut squash, zucchini and pumpkin, respectively) and oil bodies from the seeds of C. pepo are selected on the basis of their antidiabetic potential. The study centred around structural characterisation of the polysaccharides using hydrodynamical methods such as analytical ultracentrifugation (sedimentation velocity and sedimentation equilibrium), viscometry and dynamic light scattering followed by the use of gas chromatography and gas chromatography coupled with mass spectrophotometry for the assessment of monosaccharide composition. Bioactivity of these polysaccharides was also examined using complement fixation assay. Pumpkin seed oil bodies were extracted, isolated and characterised under various laboratory conditions to establish the zeta potential and size distribution of oil bodies in the solvent provided. Although the selection of the biomaterial for this study from the three species was based on their antidiabetic potential, other health benefits and practical applications are also associated with them. For example, the characterisation of these macromolecules could act as a stepping stone for the future investigation in therapeutics. These biomaterials can potentially be used in the pharmaceutical industry to act as a drug themselves or can be used as a part of any formulation or otherwise can be used as a nutraceutical compound

Management of Diabetes Patients across the Peri- Operative Pathway: A Systematic Review

Peri-operative environments are a hazardous setting for diabetes patients. A systematic review of literature regarding the management of diabetes patients across the peri-operative pathway has been undertaken to assess if the management of patients within this pathway is suitable and effective for patients. Methods A database search of Google Scholar, CINAHAL, Embase, OVID, Cochrane Library, Joanna Briggs institute and PUBMED was undertaken from 15 th of March 2019 to 30 th of March 2019. A total of 57 papers were found and reduced down to 11 final papers that answered the review question and met the inclusion and exclusion criteria. Inclusion criteria were: Full text, English language, human subjects, adult patients only and studies that focused on diabetes care in a section of the peri-operative pathway. Exclusion criteria: children or adults and children, studies that looked a one particular intervention or type of surgery. No date limit was set. PICO tool was used to frame the study question. Results Three main themes emerged from the literature. 1. Poor patient outcomes; 2. Longer length of stay (LOS); 3. Lack of adherence to guidance and or protocols and glycaemic control. Elective patients had advantageous outcomes compared to emergency surgical patients. Hyperglycaemia still remained a problem with an increase in other medical complications for diabetes patients. LOS in hospital was found to have increased due to medical complications. Adherence to protocols and guidance was found to be beneficial in monitoring and managing hyperglycaemia. However, this review found that best practice guidance and hospital protocol is not always adhered to. A liberal approach to glycaemic control is beneficial. Conclusion This systematic review investigated the management of diabetes patients across the peri-operative pathway. Three main themes emerged from the literature: poor patient outcomes; length of stay; and lack of adherence to guidance and or protocols and glycaemic control. We concluded the peri-operative environment is a hazardous setting for a diabetes patients. Elective patients had slightly more advantageous outcomes than emergency patients. Hyperglycaemia still remains a problem which leads to poor patient outcomes and longer LOS. Adherence to protocols and guidance was found to be beneficial in monitoring and managing hyperglycaemia

Effectiveness and Safety of Hyperbaric Oxygen Therapy (HBOT) in Treating Diabetic Foot Ulcers (DFUs)

Introduction: Diabetic Foot Ulcers (DFUs) are one of the more dangerous complications of diabetes, contributing to morbidity, mortality, and major financial strain, potentially affecting patients' quality of life. Therefore, an effective DFU treatment is needed to both heal and reduce severe consequences, such as amputation. Studies into effective multiple therapeutic interventions for DFUs indicate that Hyperbaric Oxygen Therapy (HBOT) may be a current alternative for treating patients presenting with DFUs

The effectiveness of lifestyle adaptation for the prevention of prediabetes in adults: a systematic review

Diabetes prevalence is increasing exceptionally worldwide and with this come associated healthcare costs. The primary outcome of this systematic review was to assess glycaemic control and incidence of Type 2 diabetes mellitus (T2DM) diagnosis after exercise and dietary intervention (measured with any validated scale). The secondary outcome assessed body mass index change, weight change, and physical exercise capacity after diet and exercise intervention (measured with any validated scale). 1,780 studies were identified from searching electronic databases. Relevant studies went through a selection process. The inclusion criteria for all studies were people with prediabetes diagnosed by either impaired glucose tolerance (IGT) or impaired fasting glucose (IFG). Lifestyle adaptation reduced the incidence of diabetes development more than standard treatment. Furthermore, better glycaemic control, improved physical exercise capacity, and increased weight reduction were observed with lifestyle intervention over standard treatment. Finally, improvements over the long term deteriorated, highlighting problems with long-term adherence to lifestyle changes. Overall, cumulative incidence of diabetes is drastically reduced in the intervention groups compared to control groups (standard care). Furthermore, glycaemic control was improved in the short term, with many participants reverting to normoglycaemia

Clinically Relevant Insulin Degludec and its Interaction with Polysaccharides: A Biophysical Examination.

Protein polysaccharide complexes have been widely studied for multiple industrial applications and are popular due to their biocompatibility. Insulin degludec, an analogue of human insulin, exists as di-hexamer in pharmaceutical formulations and has the potential to form long multi-hexamers in physiological environment, which dissociate into monomers to bind with receptors on the cell membrane. This study involved complexation of two negatively charged bio-polymers xanthan and alginate with clinically-relevant insulin degludec (PIC). The polymeric complexations and interactions were investigated using biophysical methods. Intrinsic viscosity [η] and particle size distribution (PSD) of PIC increased significantly with an increase in temperature, contrary to the individual components indicating possible interactions. [η] trend was X > XA > PIC > A > IDeg. PSD trend was X>A>IDeg>XA>PIC. Zeta (ζ)- potential (with general trend of IDeg < A < XA < X ≈ PIC) revealed stable interaction at lower temperature which gradually changed with an increase in temperature. Likewise, sedimentation velocity indicated stable complexation at lower temperature. With an increase in time and temperature, changes in the number of peaks and area under curve were observed for PIC. Conclusively, stable complexation occurred among the three polymers at 4 °C and 18 °C and the complex dissociated at 37 °C. Therefore, the complex has the potential to be used as a drug delivery vehicle

Characterisation of insulin analogues therapeutically available to patients

The structure and function of clinical dosage insulin and its analogues were assessed. This included ‘native insulins’ (human recombinant, bovine, porcine), ‘fast-acting analogues’ (aspart, glulisine, lispro) and ‘slow-acting analogues’ (glargine, detemir, degludec). Analytical ultracentrifugation, both sedimentation velocity and equilibrium experiments, were employed to yield distributions of both molar mass and sedimentation coefficient of all nine insulins. Size exclusion chromatography, coupled to multi-angle light scattering, was also used to explore the function of these analogues. On ultracentrifugation analysis, the insulins under investigation were found to be in numerous conformational states, however the majority of insulins were present in a primarily hexameric conformation. This was true for all native insulins and two fast-acting analogues. However, glargine was present as a dimer, detemir was a multi-hexameric system, degludec was a dodecamer (di-hexamer) and glulisine was present as a dimer-hexamer-dihexamer system. However, size-exclusion chromatography showed that the two hexameric fast-acting analogues (aspart and lispro) dissociated into monomers and dimers due to the lack of zinc in the mobile phase. This comprehensive study is the first time all nine insulins have been characterised in this way, the first time that insulin detemir have been studied using analytical ultracentrifugation and the first time that insulins aspart and glulisine have been studied using sedimentation equilibrium. The structure and function of these clinically administered insulins is of critical importance and this research adds novel data to an otherwise complex functional physiological protein

Exploration of temperature and shelf-life dependency of the therapeutically available Insulin Detemir

© 2020 Elsevier B.V. Purpose: Insulin, in typical use, undergoes multiple changes in temperature; from refrigerator, to room temperature, to body temperature. Although long-term storage temperature has been well-studied, the short term changes to insulin are yet to be determined. Insulin detemir (IDet) is a clinically available, slow-acting, synthetic analogue characterised by the conjugation of a C14 fatty acid. The function of this modification is to cause the insulin to form multi-hexameric species, thus retarding the pharmacokinetic rate of action. In this investigation, the temperature dependence properties of this synthetic analogue is probed, as well as expiration. Methods: Dynamic light scattering (DLS) and viscometry were employed to assess the effect of temperature upon IDet. Mass spectrometry was also used to probe the impact of shelf-life and the presence of certain excipients. Results: IDet was compared with eight other insulins, including human recombinant, three fast-acting analogues and two other slow-acting analogues. Of all nine insulins, IDet was the only analogue to show temperature dependent behaviour, between 20 °C and 37 °C, when probed with non-invasive backscatter dynamic light scattering. Upon further investigation, IDet observed significant changes in size related to temperature, direction of temperature (heated/cooled) and expiration with cross-correlation observed amongst all 4 parameters. Conclusions: These findings are critical to our understanding of the behaviour of this particular clinically relevant drug, as it will allow the development of future generations of peptide-based therapies with greater clinical efficacy

Analysis of insulin glulisine at the molecular level by X-ray crystallography and biophysical techniques

© 2021, The Author(s). This study concerns glulisine, a rapid-acting insulin analogue that plays a fundamental role in diabetes management. We have applied a combination of methods namely X-ray crystallography, and biophysical characterisation to provide a detailed insight into the structure and function of glulisine. X-ray data provided structural information to a resolution of 1.26Å. Crystals belonged to the H3 space group with hexagonal (centred trigonal) cell dimensions a = b = 82.44 and c = 33.65Å with two molecules in the asymmetric unit. A unique position of D21Glu, not present in other fast-acting analogues, pointing inwards rather than to the outside surface was observed. This reduces interactions with neighbouring molecules thereby increasing preference of the dimer form. Sedimentation velocity/equilibrium studies revealed a trinary system of dimers and hexamers/dihexamers in dynamic equilibrium. This new information may lead to better understanding of the pharmacokinetic and pharmacodynamic behaviour of glulisine which might aid in improving formulation regarding its fast-acting role and reducing side effects of this drug

Clinically Relevant Insulin Degludec and Its Interaction with Polysaccharides: A Biophysical Examination

Protein polysaccharide complexes have been widely studied for multiple industrial applications and are popular due to their biocompatibility. Insulin degludec, an analogue of human insulin, exists as di-hexamer in pharmaceutical formulations and has the potential to form long multi-hexamers in physiological environment, which dissociate into monomers to bind with receptors on the cell membrane. This study involved complexation of two negatively charged bio-polymers xanthan and alginate with clinically-relevant insulin degludec (PIC). The polymeric complexations and interactions were investigated using biophysical methods. Intrinsic viscosity [&eta;] and particle size distribution (PSD) of PIC increased significantly with an increase in temperature, contrary to the individual components indicating possible interactions. [&eta;] trend was X &gt; XA &gt; PIC &gt; A &gt; IDeg. PSD trend was X &gt; A &gt; IDeg &gt; XA &gt; PIC. Zeta (&zeta;)- potential (with general trend of IDeg &lt; A &lt; XA &lt; X &asymp; PIC) revealed stable interaction at lower temperature which gradually changed with an increase in temperature. Likewise, sedimentation velocity indicated stable complexation at lower temperature. With an increase in time and temperature, changes in the number of peaks and area under curve were observed for PIC. Conclusively, stable complexation occurred among the three polymers at 4 &deg;C and 18 &deg;C and the complex dissociated at 37 &deg;C. Therefore, the complex has the potential to be used as a drug delivery vehicle