Multilevel Pain Assessment with Functional Near-Infrared Spectroscopy: Evaluating ΔHBO2 and ΔHHB Measures for Comprehensive Analysis.

Assessing pain in non-verbal patients is challenging, often depending on clinical judgment which can be unreliable due to fluctuations in vital signs caused by underlying medical conditions. To date, there is a notable absence of objective diagnostic tests to aid healthcare practitioners in pain assessment, especially affecting critically-ill or advanced dementia patients. Neurophysiological information, i.e., functional near-infrared spectroscopy (fNIRS) or electroencephalogram (EEG), unveils the brain's active regions and patterns, revealing the neural mechanisms behind the experience and processing of pain. This study focuses on assessing pain via the analysis of fNIRS signals combined with machine learning, utilising multiple fNIRS measures including oxygenated haemoglobin (ΔHBO2) and deoxygenated haemoglobin (ΔHHB). Initially, a channel selection process filters out highly contaminated channels with high-frequency and high-amplitude artifacts from the 24-channel fNIRS data. The remaining channels are then preprocessed by applying a low-pass filter and common average referencing to remove cardio-respiratory artifacts and common gain noise, respectively. Subsequently, the preprocessed channels are averaged to create a single time series vector for both ΔHBO2 and ΔHHB measures. From each measure, ten statistical features are extracted and fusion occurs at the feature level, resulting in a fused feature vector. The most relevant features, selected using the Minimum Redundancy Maximum Relevance method, are passed to a Support Vector Machines classifier. Using leave-one-subject-out cross validation, the system achieved an accuracy of 68.51%±9.02% in a multi-class task (No Pain, Low Pain, and High Pain) using a fusion of ΔHBO2 and ΔHHB. These two measures collectively demonstrated superior performance compared to when they were used independently. This study contributes to the pursuit of an objective pain assessment and proposes a potential biomarker for human pain using fNIRS.

Insights from a model based study on optimizing non invasive brain electrical stimulation for Parkinson's disease.

Parkinson's Disease (PD) is a disorder in the central nervous system which includes symptoms such as tremor, rigidity, and Bradykinesia. Deep brain stimulation (DBS) is the most effective method to treat PD motor symptoms especially when the patient is not responsive to other treatments. However, its invasiveness and high risk, involving electrode implantation in the Basal Ganglia (BG), prompt recent research to emphasize non-invasive Transcranial Electrical Stimulation (TES). TES proves to be effective in treating some PD symptoms with inherent safety and no associated risks. This study explores the potential of using TES, to modify the firing pattern of cells in BG that are responsible for motor symptoms in PD. The research employs a mathematical model of the BG to examine the impact of applying TES to the brain. This is conducted using a realistic head model incorporating the Finite Element Method (FEM). According to our findings, the firing pattern associated with Parkinson's disease shifted towards a healthier firing pattern through the use of tACS. Employing an adaptive algorithm that continually monitored the behavior of BG cells (specifically, Globus Pallidus Pars externa (GPe)), we determined the optimal electrode number and placement to concentrate the current within the intended region. This resulted in a peak induced electric field of 1.9 v/m at the BG area. Our mathematical modeling together with precise finite element simulation of the brain and BG suggests that proposed method effectively mitigates Parkinsonian behavior in the BG cells. Furthermore, this approach ensures an improvement in the condition while adhering to all safety constraints associated with the current injection into the brain.

Efficient Expression in the Prokaryotic Host System, Purification and Structural Analyses of the Recombinant Human ACE2 Catalytic Subunit as a Hybrid Protein with the B Subunit of Cholera Toxin (CTB-ACE2).

Angiotensin-converting enzyme 2 (ACE2) has a specific interaction with the coronavirus spike protein, enabling its entry into human cells. This membrane enzyme converts angiotensin II into angiotensin 1-7, which has an essential role in protecting the heart and improving lung function. Many therapeutic properties have been attributed to the human recombinant ACE2 (hrACE2), especially in combating complications related to diabetes mellitus and hypertension, as well as, preventing the coronavirus from entering the target tissues. In the current study, we designed an appropriate gene construct for the hybrid protein containing the ACE2 catalytic subunit and the B subunit of cholera toxin (CTB-ACE2). This structural feature will probably help the recombinant hybrid protein enter the mucosal tissues, including the lung tissue. Optimization of this hybrid protein expression was investigated in BL21 bacterial host cells. Also, the hybrid protein was identified with an appropriate antibody using the ELISA method. A large amount of the hybrid protein (molecular weight of ~ 100 kDa) was expressed as the inclusion body when the induction was performed in the presence of 0.25 mM IPTG and 1% sucrose for 10 h. Finally, the protein structural features were assessed using several biophysical methods. The fluorescence emission intensity and oligomeric size distribution of the CTB-ACE2 suggested a temperature-dependent alteration. The ß-sheet and α-helix were also dominant in the hybrid protein structure, and this protein also displays acceptable chemical stability. In overall, according to our results, the efficient expression and successful purification of the CTB-ACE2 protein may pave the path for its therapeutic applications against diseases such as covid-19, diabetes mellitus and hypertension.

Toward a Multi-Modal Brain-Body Assessment in Parkinson's Disease: A Systematic Review in fNIRS.

Parkinson's disease (PD) causes impairments in cortical structures leading to motor and cognitive symptoms. While common disease management and treatment strategies mainly depend on the subjective assessment of clinical scales and patients' diaries, research in recent years has focused on advances in automatic and objective tools to help with diagnosing PD and determining its severity. Due to the link between brain structure deficits and physical symptoms in PD, objective brain activity and body motion assessment of patients have been studied in the literature. This study aimed to explore the relationship between brain activity and body motion measures of people with PD to look at the feasibility of diagnosis or assessment of PD using these measures. In this study, we summarised the findings of 24 selected papers from the complete literature review using the Scopus database. Selected studies used both brain activity recording using functional near-infrared spectroscopy (fNIRS) and motion assessment using sensors for people with PD in their experiments. Results include 1) the most common study protocol is a combination of single tasks. 2) Prefrontal cortex is mostly studied region of interest in the literature. 3) Oxygenated haemoglobin (HbO 2) concentration is the predominant metric utilised in fNIRS, compared to deoxygenated haemoglobin (HHb). 4) Motion assessment in people with PD is mostly done with inertial measurement units (IMUs) and electronic walkway. 5) The relationship between brain activity and body motion measures is an important factor that has been neglected in the literature.

Investigating the role of double mutations R12C/P20R, and R12C/R69C on structure, chaperone-like activity, and amyloidogenic properties of human αB-crystallin.

α-crystallin is a structurally essential small heat shock protein (sHSP) with a chaperone-like activity which maintains transparency of the lenticular tissues during a period of time that is as long as human life. α-crystallin is a multimeric protein consisting of αA and αB subunits, with 57 % homology. The CRYAB gene on chromosome 11 encodes human αB-crystallin (αB-Cry), which contains 175 amino acid residues. In the current study, the cataractogenic mutations R12C, P20R, R69C, and double mutations R12C/P20R and R12C/P20R were embedded into the human CRYAB gene. Following successful expression in the prokaryotic system and purification, a number of spectroscopic techniques, gel electrophoresis, dynamic light scattering (DLS), and transmission electron microscopy (TEM) were applied to assess the role of these mutations on the structure, amyloidogenicity, and biological function of human αB-Cry. The created mutations caused significant changes in the structure, and oligomeric state of human αB-Cry. These mutations, particularly R12C, R12C/P20R, and R12C/R69C, dramatically enhanced the tendency of this protein for the amyloid fibril formation and reduced its chaperone-like activity. Since double mutations R12C/P20R and R12C/P20R were able to intensely change the protein's structure and chaperone function, it can be suggested that they may play a destructive role in a cumulative manner. Our findings indicated that the simultaneous presence of two pathogenic mutations may have a cumulative destructive impacts on the structure and function of human αB-Cry and this observation is likely related to the disease severity of the mutated proteins.

An Analysis of Lower Limb Coordination Variability in Unilateral Tasks in Healthy Adults: A Possible Prognostic Tool.

Interlimb coordination variability analysis can shed light into the dynamics of higher order coordination and motor control. However, it is not clear how the interlimb coordination of people with no known injuries change in similar activities with increasing difficulty. This study aimed to ascertain if the interlimb coordination variability range and patterns of healthy participants change in different unilateral functional tasks with increasing complexity and whether leg dominance affects the interlimb coordination variability. In this cross-sectional study fourteen younger participants with no known injuries completed three repeated unilateral sit-to-stands (UniSTS), step-ups (SUs), and continuous-hops (Hops). Using four inertial sensors mounted on the lower legs and thighs, angular rotation of thighs and shanks were recorded. Using Hilbert transform, the phase angle of each segment and then the continuous relative phase (CRP) of the two segments were measured. The CRP is indicative of the interlimb coordination. Finally, the linear and the nonlinear shank-thigh coordination variability of each participant in each task was calculated. The results show that the linear shank-thigh coordination variability was significantly smaller in the SUs compared to both UniSTS and Hops in both legs. There were no significant differences found between the latter two tests in their linear coordination variability. However, Hops were found to have significantly larger nonlinear shank-thigh coordination variability compared to the SUs and the UniSTS. This can be due to larger vertical and horizontal forces required for the task and can reveal inadequate motor control during the movement. The combination of nonlinear and linear interlimb coordination variability can provide more insight into human movement as they measure different aspects of coordination variability. It was also seen that leg dominance does not affect the lower limb coordination variability in participants with no known injuries. The results should be tested in participants recovering from lower limb injuries.

Chest and pelvis coordination during functional reach test: A possible indication of balance deficiency in older adults.

Falls in older adults represent the most common cause of injuries and a major cause of mortality in this vulnerable population. The morbidity and mortality rate of falls among older people makes balance analysis in older adults very important. Therefore, this study aims to explore different metrics that can potentially be used to identify early indications of balance loss and fall risk. To that end, the motion strategies and chest and pelvis coordination of a group of younger, a group of older non-faller and a group of older faller participants while conducting the functional reach test were investigated. To analyse the motion strategies of the different participant groups, four metrics of maximum angular rotation of chest, maximum angular rotation of pelvis, time warped chest and pelvis angular rotation difference, and the mean continuous relative phase of the chest and pelvis were assessed. In this study younger participants are found to have larger maximum chest rotation, maximum pelvis rotation, and time warped chest and pelvis angular rotation difference compared to older participants. However, these metrics were not significantly different in older non-fallers compared to older fallers. Meanwhile, the mean continuous relative phase of the chest and pelvis was the only metric found to be significantly different among all three participant groups. This metric is indicative of the chest and pelvis coordination which is associated with the ability to construct proper coordination and maintain balance. The mean continuous relative phase yielded the sensitivity of 92.3% and specificity of 73.7% in recognizing older fallers from older non-fallers. The results suggest that this metric might be useful in identifying the risk of falling in older population, thus, it should be further studied in a prospective study.

Increased chaperone activity of human α|B-crystallin with incomplete oxidation as a new defense mechanism against oxidative stress.

Previous research has shown that production of the high levels of oxidants overwhelms the body's antioxidant defense system during diabetes mellitus. Under this circumstance, ocular lens proteins are one of the main molecular targets for oxidative damage. In the present study, the individual effect of partial and extensive oxidation on the structure and function of human αB-crystallin was investigated using electrophoresis and various spectroscopic methods. The results of our study suggested that widespread oxidation causes loss of the chaperone activity of this protein, while partial oxidation significantly enhances this activity. Our studies also suggested that partial and extensive oxidation induces the formation of different structures in this protein. In fact, the chaperone-active and chaperone-inactive states of this protein are respectively associated with a minor and extensive structural alteration. Moreover, the oligomeric size distribution shows an inverse relationship with the chaperone activity of this protein. Increasing the chaperone activity of this protein during partial oxidation may be a natural defense mechanism to overcome the damages caused by oxidative stress, especially in diabetes and other pathological diseases.

Relationship between the Structure and Chaperone Activity of Human αA-Crystallin after Its Modification with Diabetes-Associated Oxidative Agents and Protective Role of Antioxidant Compounds.

The study was aimed to evaluate the impact of peroxynitrite (PON, oxidative stress agent in diabetes), methylglyoxal (MGO, diabetes-associated reactive carbonyl compound), and their simultaneous application on the structural and functional features of human αA-crystallin (αA-Cry) using various spectroscopy techniques. Additionally, the surface tension and oligomer size distribution of the treated and untreated protein were tested using tensiometric analysis and dynamic light scattering, respectively. Our results indicated that the reaction of PON and MGO with human αA-Cry leads to the formation of new chromophores, alterations in the secondary to quaternary protein structure, reduction in the size of protein oligomers, and significant enhancement in the chaperone activity of αA-Cry. To reverse the effects of the tested compounds, ascorbic acid and glutathione (main components of lens antioxidant defense system) were applied. As expected, the two antioxidant compounds significantly prevented formation of high molecular weight aggregates of αA-Cry (according to SDS-PAGE). Our results suggest that the lens antioxidant defense system, in particular, glutathione, may provide a strong protection against rapid incidence and progression of diabetic cataract by preventing the destructive reactions of highly reactive DM-associated metabolites.

Myopathy-associated G154S mutation causes important changes in the conformational stability, amyloidogenic properties, and chaperone-like activity of human αB-crystallin.

Glycine to serine substitution at position 154 of human αB-crystallin (αB-Cry) is behind the development of cardiomyopathy and late-onset distal myopathy. The current study was conducted with the aim to investigate the structural and functional features of the G154S mutant αB-Cry using various spectroscopic techniques and microscopic analyses. The secondary and tertiary structures of human αB-Cry were preserved mainly in the presence of G154S mutation, but the mutant protein indicated a reduced chaperone-like activity when γ-Cry as its natural partner in eye lenses was the substrate protein. Moreover, a significant reduction in the enzyme refolding ability and in vivo chaperone activity of the mutant protein were observed. Also, the mutant protein displayed reduced conformational stability upon urea-induced denaturation. Both fluorescence and electron microscopic analyses suggested that G154S mutant protein has an increased susceptibility for amyloid fibril formation. Therefore, the pathomechanism of G154S mutation can be explained by its attenuated chaperone function, decreased conformational stability, and increased amyloidogenic propensity. Some of these important changes may also alter the correct interaction of the mutated αB-Cry with its target proteins in myopathy.

Structural and functional studies of D109A human αB-crystallin contributing to the development of cataract and cardiomyopathy diseases.

αB-crystallin (heat shock protein ß5/HSPB5) is a member of the family of small heat shock proteins that is expressed in various organs of the human body including eye lenses and muscles. Therefore, mutations in the gene of this protein (CRYAB) might have many pathological consequences. A new mutation has recently been discovered in the α-crystallin domain of this chaperone protein which replaces aspartate 109 with alanine (D109A). This mutation can cause myofibrillar myopathy (MFM), cataracts, and cardiomyopathy. In the current study, several spectroscopic and microscopic analyses, as well as gel electrophoresis assessment were applied to elucidate the pathogenic contribution of human αB-crystallin bearing D109A mutation in development of eye lens cataract and myopathies. The protein oligomerization, chaperone-like activity and chemical/thermal stabilities of the mutant and wild-type protein were also investigated in the comparative assessments. Our results suggested that the D109A mutation has a significant impact on the important features of human αB-crystallin, including its structure, size of the protein oligomers, tendency to form amyloid fibrils, stability, and chaperone-like activity. Given the importance of aspartate 109 in maintaining the proper structure of the α-crystallin domain, its role in the dimerization and chaperone-like activity, as well as preserving protein stability through the formation of salt bridges; mutation at this important site might have critical consequences and can explain the genesis of myopathy and cataract disorders. Also, the formation of large light-scattering aggregates and disruption of the chaperone-like activity by D109A mutation might be considered as important contributing factors in development of the eye lens opacity.

The biochemical association between R157H mutation in human αB-crystallin and development of cardiomyopathy: Structural and functional analyses of the mutant protein.

In human αB-crystallin or HspB5, the substitution of arginine residue at position 157 with histidine has been reported to cause cardiomyopathy. In this study, the impact of R157H mutation on the structure, stability and functional properties of human αB-crystallin was investigated using a variety of spectroscopic techniques and microscopic analyses. Our spectroscopic analyses revealed that this mutation has a negligible impact on the secondary and tertiary structures of HspB5 but its quaternary structure underwent fundamental changes. Although the chemical stability of the mutant protein remained largely unchanged, the differential scanning calorimetry (DSC) measurement suggested that its thermal stability was reduced. As examined with transmission electron microscopy, αB-crystallin and its mutant indicated a similar tendency for the amyloid fibril formation under thermochemical stress. Dynamic light scattering (DLS) analysis suggested important changes in the quaternary (oligomeric) structures of the mutant protein as compared with the native protein counterpart. Also, the mutant protein indicated an improved chaperone-like activity under in vitro assessment. In a pH-dependent manner, the side chains of arginine and histidine have different capabilities for establishing hydrogen bonds and electrostatic interaction (salt bridge) and this variation may be sufficient to produce the larger changes that ultimately alter the interaction of this protein with other target proteins. Overall, the pathogenic contribution of this mutation in cardiomyopathy can be explained by its role in quaternary structure/stability alteration of the mutated protein.

The sit to stand to sit postural transition variability in the five time sit to stand test in older people with different fall histories.

BACKGROUND: It has been proved that increased motion variability can be indicative of lack of stability. Despite the extensive literature on single sit to stand/stand to sit postural transitions analysis in older people, no studies to this date have assessed the sit to stand to sit (STSTS) transition variability in older adults. RESEARCH QUESTION: To investigate the variability in STSTS transition during the five times sit to stand (FTSS) test in older people with different fall histories. METHODS: Seventy-five older (80.5 ± 7.5) and twenty-five younger (27.7 ± 6.5) subjects participated in the study. The older participants were categorized into three groups of non-fallers, once-fallers, and multiple-fallers based on their fall histories. Subjects were fitted with an IMU at their lower backs and asked to fully stand up and then sit down again five times in a row. The angular rotation of the trunk in the sagittal plane was recorded. Using the DTW method, the first STSTS transition of each subject was compared to the last transition and the variability was measured. The correlation between STSTS variability and older participants' Berg balance scale (BBS) was investigated. RESULTS: The STSTS variability results were significantly different in older fallers (multiple-fallers and once-fallers) compared to both younger participants and older non-fallers. The results yielded a sensitivity of 85.4 % and a specificity of 83.3 % in recognizing older fallers from older non-fallers and a sensitivity and specificity of 86.7 % and 85.7 % respectively in recognizing older multiple-fallers from other older participants. The STSTS variability was found to be significantly correlated with BBS. SIGNIFICANCE: The findings demonstrated a strong indication of variability in the STSTS transition in older fallers and a significant correlation between STSTS variability and BBS. The results suggest that variability analysis of the STSTS transition has the potential to be used for fall risk analysis in older adults.

Analysis of the data on titration of native and peroxynitrite modified αA- and αB-crystallins by Cu2+-ions.

The interaction of αA- and αB-crystallins with Cu2+ ion modulates their structure and chaperone-like activity which is important for lens transparency. Theoretical analysis of the dependences of fluorescence intensity of native αA- and αB-crystallins and αA- and αB-crystallins modified by peroxynitrite on concentration of Cu2+ ions has been carried out. It has been shown that one subunit of native αA-crystallin contains two equivalent Cu2+-binding sites. The microscopic dissociation constant for Cu2+-αA-crystallin complex (K diss) was found to be equal to 9.7 µM. For peroxynitrite modified αA-crystallin the K diss value is equal to 17 µM. One subunit of native αB-crystallin contains two non-equivalent Cu2+-binding sites. The corresponding microscopic dissociation constants for Cu2+-αB-crystallin complexes (K 1 and K 2) were found to be equal to 0.94 and 36 µM. For peroxynitrite modified αB-crystallin the K 1 and K 2 values are equal to 4.3 and 70 µM, respectively.

The congenital cataract-causing mutations P20R and A171T are associated with important changes in the amyloidogenic feature, structure and chaperone-like activity of human αB-crystallin.

Cataract is the major reason for human blindness worldwide. α-Crystallin, as a key chaperone of eye lenses, keeps the lenticular tissues in its transparent state over time. In this study, cataract-causing familial mutations, P20R and A171T, were introduced in CRYАB gene. After successful expression in Escherichia coli and subsequent purification, the recombinant proteins were subjected to extensive structural and functional analyses using various spectroscopic techniques, gel electrophoresis, and electron microscopy. The results of fluorescence and Raman assessments suggest important but discreet conformational changes in human αB-Cry upon these cataractogenic mutations. Furthermore, the mutant proteins exhibited significant secondary structural alteration as revealed by FTIR and Raman spectroscopy. An increase in conformational stability was seen in the human αB-Cry bearing these congenital cataractogenic mutations. The oligomeric size distribution and chaperone-like activity of human αB-Cry were significantly altered by these mutations. The P20R mutant protein was observed to loose most of the chaperone-like activity. Finally, these cataractogenic mutant proteins exhibited an increased propensity to form the amyloid fibrils when incubated under environmental stress. Overall, the structural and functional changes in mutated human αB-Cry proteins can shed light on the pathogenic development of congenital cataracts.

Kinetic data analysis of chaperone-like activity of Wt, R69C and D109H αB-crystallins.

The α-Crystallin (α-Cry) functions as a molecular chaperone, preventing the formation of stress-induced protein aggregation which is important for maintenance of lens transparency. The kinetic data of Wt, R69C and D109H αB-Crys chaperone-like activity were obtained by UV-Vis spectroscopy in both thermal- and chemical-induced aggregation methods. The data were analyzed using physical parameters describing the aggregation process including t* (the characteristic of the stage of nucleation), and t 0.5 (the characteristic of the stage of aggregate growth) and I lim (the limiting value of the light scattering intensity). Parameter t* is duration of the lag phase and the lower t* value is associated with the higher rate of the nucleation stage. Also, the lower values of t 0.5 indicated the higher rate of aggregate growth stage. The change in parameter I lim in the presence of chaperones can be connected with the change in the size of protein aggregates. These data are related to the research article entitled "Structural and functional characterization of D109H and R69C mutant versions of human αB-crystallin: the biochemical pathomechanism underlying cataract and myopathy development" [1].

Structural and functional characterization of D109H and R69C mutant versions of human αB-crystallin: The biochemical pathomechanism underlying cataract and myopathy development.

In human αB-crystallin (αB-Cry), the highly conserved residues arginine 69 (R69) and aspartate 109 (D109) are located within a critical motif of α-crystallin domain (ACD), contributing to the subunit interactions and oligomeric assembly. Recently, two missense mutations (R69C and D109H) in human αB-Cry have been reported to cause congenital cataract and myopathy disorders. We used various spectroscopic techniques, dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), gel electrophoresis and transmission electron microscopy (TEM) to show how these mutations cause significant changes in structure, amyloidogenic feature and biological function of human αB-Cry. These pathogenic mutations resulted in the important alterations of the secondary, tertiary and oligomeric (quaternary) structures of human αB-Cry. The missense mutations were also capable to significantly increase the amyloidogenic propensity of human αB-Cry and to diminish the chaperone-like activity of this protein. The above mentioned changes were observed more noticeably after D109H mutation. The detrimental effects of D109H mutation may be due to the loss of salt bridge with R120 in the dimeric interface, flagging the anti-aggregation ability of αB-Cry chaperone. In conclusion, the R69C and D109H mutations displayed a significant damaging effect on the structure and chaperone function of human αB-Cry which could be considered as their biochemical pathomechanisms in development of congenital cataract and myopathy disorders.

Body postural sway analysis in older people with different fall histories.

A cross-sectional study of postural sway analysis in older non-fallers, once-fallers and multiple-fallers using five common standing tests was conducted. Eighty-six older subjects with an average age of 80.4 years (SD ± 7.9) participated in the study. The angular rotation and velocity of the trunk of the participants in the roll (lateral) and pitch (sagittal) planes were recorded using an inertial sensor mounted on their lower backs. The Gaussian Mixture Models (GMM), Expectation-Maximisation (EM) and the Minimum Message Length (MML) algorithms were applied to the acquired data to obtain an index indicative of the body sway. The standing with feet together and standing with one foot in front, sway index distinguished older fallers from non-fallers with specificity of 75.7% and 77.7%, respectively, and sensitivity of 78.6% and 82.1%, respectively. This compares favourably with the Berg Balance Scales (BBS) with specificity of 70.5% and sensitivity of 75.3%. The results suggest that the proposed method has potential as a protocol to diagnose balance disorder in older people. Graphical abstract.

Structural and functional alteration of human αA-crystallin after exposure to full spectrum solar radiation and preventive role of lens antioxidants.

The chronically exposure of eye lenses to ultra violet and visible light of the solar radiation is an important risk factor for development of the senile cataract diseases. Various photosensitizer molecules including riboflavin (RF) play a significant role in photo-oxidative damages of lens proteins underlying development of opacity in the lenticular tissues. In the current study, RF-mediated photo-oxidation of human αA-crystallin (αA-Cry) was assessed using SDS-PAGE analysis, dynamic light scattering and other spectroscopic assessments. The RF-photosensitized reactions led to non-disulfide covalent cross-linking, oligomerization and significant structural changes in αA-Cry. The photo-damaging of αA-Cry under solar radiation was also accompanied by the reduction in both Trp and Tyr fluorescence intensities which followed by the formation of new photosensitizer chromophores. The solvent exposed hydrophobic patches, secondary structures and chaperone-like activity of αA-Cry were significantly altered after exposure to the solar radiation in the presence of RF. Although glutathione and ascorbate were capable to partially protect the photo-induced structural damages of human αA-Cry, they also disrupted its chaperone function when co-exposed with this protein to the solar radiation. Also, the most promising data were obtained with cysteine which its availability in the lenticular tissues is a rate limiting factor for the biosynthesis of glutathione. Overall our results suggest that glutathione and ascorbate, as the major anti-oxidant compounds within lenticular tissues, demonstrate controversial effect on structure and chaperone-like activity of human αA-Cry. Elucidation of this effect may demand further experiments.

A Comparative Study of the Impact of Calcium Ion on Structure, Aggregation and Chaperone Function of Human αA-crystallin and its Cataract- Causing R12C Mutant.

BACKGROUND: The chaperone activity of α-crystallin (α-Cry) plays an important role in maintenance of eye lens transparency. Various mutations in the α-Cry genes have been indicated to cause cataract diseases in human. Also, the calcium imbalance has been shown to induce aggregation in α-Cry. We investigated the impact of calcium ion on structure, chaperone activity of the recombinant wild-type and mutant R12C αA-Cry. We suggested that the raise of calcium level in eye lens is an additional contributory factor accelerating the development of cataract diseases in patients with R12C mutation. OBJECTIVES: The main objective of this study was to investigate the impact of calcium ion on structure, chaperone activity and amyloidogenic properties of the recombinant wild-type and mutant R12C αA-Cry, in a comparative study. METHODS: The mutagenesis was performed on confirmed αA-Cry cDNA in pET-28b (+) which applied as a template to generate R12C mutant, using polymerase chain reaction (PCR) and a Quick Change Lightning Multi Site-Directed Mutagenesis kit (Stratgene). Both wild-type and mutant plasmids were chemically transformed into E.coli BL21 (DE3) and the respective recombinant proteins over-expressed in LB broth. The protein purification was done using Q-Sepharose anion exchange and Sephacryl S-300 gel filtration chromatography. The purified αA-Cry samples were incubated with different concentrations of calcium ion (0-40 mM) at 37 °C for 1 week. The secondary and tertiary structural analyses of each protein were performed by far-UV CD and Try/Trp and ANS fluorescence assessments, respectively. The assessment of chaperone activity was done spectrophotometrically in both thermal and chemical-induced aggregation systems using γ-Cry and bovine pancreatic insulin as the substrate proteins, respectively. Also, the amyloidogenic properties of proteins was investigated by CR absorption and ThT fluorescence measurements. RESULTS: The results of fluorescence and CD assessments suggested the significant secondary and tertiary structural alterations upon R12C mutation. R12C mutant αA-Cry demonstrated preserved secondary and tertiary structures in the presence of calcium. The chaperone activity of wild-type and mutant R12C αA-Cry was reduced in the presence of calcium. Also, the extent of chaperone activity reduction was significantly higher for R12C αA-Cry. Both wild-type and mutant R12C αA-Cry revealed slight amount of aggregation when incubated with different calcium concentrations for 1 week, at 37 °C. However, the susceptibility of both proteins for aggregation was significantly increased in the presence of 40 mM calcium, at the elevated temperature (60 °C). Also, the mutant protein exhibited extensive disulfide bridge cross-linking as indicated by gel electrophoresis. Moreover, the mutant R12C αA-Cry significantly resists against amyloid fibril formation in the presence of calcium ion compared to the wild-type protein as indicated by CR and ThT assessments. CONCLUSION: Our data suggested that αA-Cry conformational changes occurring upon R12C mutation and further functional damages induced by calcium may play an important role in the pathomechanism of the cataract development by this mutant protein.