Strain and Depot-specific Differences in Adipose Tissues of Obese BFMI Mice.

BACKGROUND/AIM: Obesity is associated with the structural and functional disorders related to the molecules of the tissues, cells, and membranes. This study aimed to examine the alterations in the secretion of inflammatory cytokines and metabolic factors and structural changes in inguinal (IF) and gonadal (GF) adipose tissues at the molecular level. MATERIALS AND METHODS: The IF and GF tissues of Berlin Fat Mouse Inbred (BFMI) lines namely BFMI852, BFMI856, BFMI860, BFMI861 obese and DBAJ control mouse lines were used for mRNA expression and Attenuated Total Reflection - Fourier Transform Infrared Spectroscopy (ATR-FTIR) studies. The mRNA levels of inflammatory cytokines including leptin, interleukin 6 (IL-6), tumor necrosis factor-alpha (Tnf-α), and insulin-like growth factor-1 (Igf-1), and peroxisome proliferator-activated receptor gamma 2 (Pparγ-2), were investigated using quantitative reverse transcriptase real-time PCR (qRT-PCR). Infrared spectroscopy does not provide information about specific proteins, instead, it gives information about overall (total) proteins, which is called global information. Therefore, in the current study, adequate information about secondary structures of adipose tissues proteins was obtained using artificial neural network (ANN) and secondary derivative-vector normalization methods based on the spectral profiles. RESULTS: According to the mRNA expression studies, high leptin resistance was found in all BFMI lines. Differences were observed in the levels of measured factors except for Igf-1 among BFMI lines. Protein secondary structure studies showed an increase in random coil contents, especially for BFMI860, which indicates denaturation of the proteins. CONCLUSION: Among the spontaneous obese BFMI mouse lines, the BFMI860 line is the most suitable for obesity studies. Obesity-induced effect on the adipose tissues varies considerably with location, type of adipose tissue, and animal line.

Rapid diagnosis of malignant pleural mesothelioma and its discrimination from lung cancer and benign exudative effusions using blood serum.

Malignant pleural mesothelioma (MPM), an aggressive cancer associated with exposure to fibrous minerals, can only be diagnosed in the advanced stage because its early symptoms are also connected with other respiratory diseases. Hence, understanding the molecular mechanism and the discrimination of MPM from other lung diseases at an early stage is important to apply effective treatment strategies and for the increase in survival rate. This study aims to develop a new approach for characterization and diagnosis of MPM among lung diseases from serum by Fourier transform infrared spectroscopy (FTIR) coupled with multivariate analysis. The detailed spectral characterization studies indicated the changes in lipid biosynthesis and nucleic acids levels in the malignant serum samples. Furthermore, the results showed that healthy, benign exudative effusion, lung cancer, and MPM groups were successfully separated from each other by applying principal component analysis (PCA), support vector machine (SVM), and especially linear discriminant analysis (LDA) to infrared spectra.

The structural effects of Vitamin A deficiency on biological macromolecules due to ethanol consumption and withdrawal: An FTIR study with chemometrics.

The structural effects of vitamin A-deficiency were examined on the molecular profiles of biomolecules of male rat hippocampus during prolonged ethanol intake/withdrawal using FT-IR spectroscopy coupled with chemometrics. Liquid ethanol diet with/without vitamin A was maintained to adult rats for 3-months. The rats were decapitated at different ethanol withdrawal times and FT-IR spectra were obtained. Ethanol consumption/withdrawal produced significant changes in proteins' conformations, while having insignificant structural effects on lipids. In vitamin A deficiency, ethanol produced structural changes in lipids by lipid ordering especially in the early-ethanol withdrawal. Furthermore, an increase in lipid and protein content, saturated/unsaturated lipid ratio, a decrease in nucleic acids content and decrease in membrane fluidity were observed. These changes were less severe in the presence of Vitamin A. This study is clinically important for individuals with vitamin A deficiency because they have to be more cautious when consuming alcohol to protect themselves from cognitive dysfunctions.

CoronaVac (Sinovac) COVID-19 vaccine-induced molecular changes in healthy human serum by infrared spectroscopy coupled with chemometrics.

From the beginning of the COVID-19 coronavirus pandemic in December of 2019, the disease has infected millions of people worldwide and caused hundreds of thousands of deaths. Since then, several vaccines have been developed. One of those vaccines is inactivated CoronaVac-Sinovac COVID-19 vaccine. In this proof of concept study, we first aimed to determine CoronaVac-induced biomolecular changes in healthy human serum using infrared spectroscopy. Our second aim was to see whether the vaccinated group can be separated or not from the non-vaccinated group by applying chemometric techniques to spectral data. The results revealed that the vaccine administration induced significant changes in some functional groups belonging to lipids, proteins and nucleic acids. In addition, the non-vaccinated and vaccinated groups were successfully separated from each other by principal component analysis (PCA) and linear discriminant analysis (LDA). This proof-of-concept study will encourage future studies on CoronaVac as well as other vaccines and will lead to make a comparison between different vaccines to establish a better understanding of the vaccination outcomes on serum biomolecules.

Discrimination of heavy metal acclimated environmental strains by chemometric analysis of FTIR spectra.

Heavy metal acclimated bacteria are profoundly the preferred choice for bioremediation studies. Bacteria get acclimated to toxic concentrations of heavy metals by induction of specific enzymes and genetic selection favoring new metabolic abilities leading to activation of one or several of resistance mechanisms creating bacterial populations with differences in resistance profile and/or level. Therefore, to use in bioremediation processes, it is important to discriminate acclimated bacterial populations and choose a more resistant strain. In this study, we discriminated heavy metal acclimated bacteria by using Attenuated Total Reflectance Fourier Transform Infrared (ATR-FTIR) spectroscopy and multivariate analysis methods namely Hierarchical Cluster Analysis (HCA), Principal Component Analysis (PCA) and Soft Independent Modeling of Class Analogy (SIMCA). Two acclimation methods, acute and gradual, were used which cause differences in molecular changes resulting in bacterial populations with different molecular and resistance profiles. Brevundimonas sp., Gordonia sp., and Microbacterium oxydans were exposed to the toxic concentrations of Cd (30 µg/ml) or Pb (90 µg/ml) by using broth medium as a growth media. Our results revealed that PCA and HCA clearly discriminated the acute-acclimated, gradual-acclimated, and control bacteria from each other in protein, carbohydrate, and whole spectral regions. Furthermore, we classified acclimated (acute and gradual) and control bacteria more accurately by using SIMCA with 99.9% confidence. This study demonstrated that heavy metal acclimated and control group bacteria can be discriminated by using chemometric analysis of FTIR spectra in a powerful, cost-effective, and handy way. In addition to the determination of the most appropriate acclimation procedure, this approach can be used in the detection of the most resistant bacterial strains to be used in bioremediation studies.

Diagnosis of malignant pleural mesothelioma from pleural fluid by Fourier transform-infrared spectroscopy coupled with chemometrics.

This study was conducted to differentiate malignant pleural mesothelioma (MPM) from lung cancer (LC) and benign pleural effusion (BPE) from pleural fluids using the diagnostic power of Fourier transform-infrared spectroscopy with attenuated total reflectance mode coupled with chemometrics. Infrared spectra of MPM (n = 24), LC (n = 20), and BPE (n = 25) were collected, and hierarchical cluster analysis (HCA) and principal component analysis (PCA) were applied to their spectra. HCA results indicated that MPM was differentiated from LC with 100% sensitivity and 100% specificity and from BPE, with 100% sensitivity and 88% specificity, which were also confirmed by PCA score plots. PCA loading plots indicated that these separations originated mainly from lipids, proteins, and nucleic acids-related spectral bands. There was significantly higher lipid, protein, nucleic acid, and glucose contents in the MPM and LC. However, the significant changes in triglyceride and cholesterol ester content, protein and nucleic acid structure, a lower membrane fluidity, and higher membrane order were only observed in the MPM. To check the classification success of some test samples/each group, soft independent modeling of class analogies was performed and 96.2% overall classification success was obtained. This approach can provide a rapid and inexpensive methodology for the efficient differentiation of MPM from other pleural effusions.

Structural and functional damages of whole body ionizing radiation on rat brain homogenate membranes and protective effect of amifostine.

PURPOSE: To investigate the effects of whole body ionizing radiation at a sublethal dose on rat brain homogenate membranes and the protective effects of amifostine on these systems at molecular level. MATERIALS AND METHODS: Sprague-Dawley rats, in the absence and presence of amifostine, were whole-body irradiated at a single dose of 8 Gy and decapitated after 24 h. The brain homogenate membranes of these rats were analyzed using Fourier Transform Infrared (FTIR) spectroscopy. RESULTS: Ionizing radiation caused a significant increase in the lipid to protein ratio and significant decreases in the ratios of olefinic = CH/lipid, CH2/lipid, carbonyl ester/lipid and CH3/lipid suggesting, respectively, a more excessive decrease in the protein content and the degradation of lipids as a result of lipid peroxidation. In addition, radiation changed the secondary structure of proteins and the status of packing of membrane lipid head groups. Furthermore, it caused a decrease in lipid order and an increase in membrane fluidity. The administration of amifostine before ionizing radiation inhibited all the radiation-induced alterations in brain homogenate membranes. CONCLUSIONS: The results revealed that whole body ionizing radiation at a sublethal dose causes significant alterations in the structure, composition and dynamics of brain homogenate membranes and amifostine has a protective effect on these membranes.

Structural effects of simvastatin on liver rat [corrected] tissue: Fourier transform infrared and Raman microspectroscopic studies.

Simvastatin is one of the most frequently prescribed statins because of its efficacy in the treatment of hypercholesterolemia, reducing cardiovascular risk and related mortality. Determination of its side effects on different tissues is mandatory to improve safe use of this drug. In the present study, the effects of simvastatin on molecular composition and structure of healthy rat livers were investigated by Fourier transform infrared and Raman imaging. Simvastatin-treated groups received 50 mg/kg/day simvastatin for 30 days. The ratio of the area and/or intensity of the bands assigned to lipids, proteins, and nucleic acids were calculated to get information about the drug-induced changes in tissues. Loss of unsaturation, accumulation of end products of lipid peroxidation, and alterations in lipid-to-protein ratio were observed in the treated group. Protein secondary structure studies revealed significant decrease in α-helix and increase in random coil, while native ß-sheet decreases and aggregated ß-sheet increases in treated group implying simvastatin-induced protein denaturation. Moreover, groups were successfully discriminated using principal component analysis. Consequently, high-dose simvastatin treatment induces hepatic lipid peroxidation and changes in molecular content and protein secondary structure, implying the risk of liver disorders in drug therapy.

Effect of thermal treatment on secondary structure and conformational change of mushroom polyphenol oxidase (PPO) as food quality related enzyme: A FTIR study.

In order to understand the conformational changes of polyphenol oxidase (PPO), which is a food quality related enzyme, after thermal treatment, secondary structure changes of the enzyme were analyzed by using Fourier Transform Infrared (FTIR) spectroscopy and compared with the change in enzyme activity in the temperature range of 25-80 °C. Fourier self-deconvolution, neural network (NN) and curve-fitting analysis were applied to the amide I band of FTIR spectra for detail analysis of secondary structure elements. FTIR analysis indicated that PPO is an α-helix dominating enzyme. Detail analysis revealed that, as temperature increased, α-helix and ß-sheet decreased, but aggregated ß-sheet, turns and random coil increased. The marked changes were noted at 40 °C with the occurrence of new bands due to aggregated ß-sheet structures, all of which indicate protein denaturation. These aggregation bands were still observed when the temperature was reduced back to 25 °C, from 70 °C, demonstrating an irreversible change in the structure.

Ionizing radiation induces structural and functional damage on the molecules of rat brain homogenate membranes: a Fourier transform infrared (FT-IR) spectroscopic study.

Humans can be exposed to ionizing radiation, due to various reasons, whose structural effects on biological membranes are not well defined. The current study aims to understand the ionizing radiation-induced structural and functional alterations in biomolecules of brain membranes using Fourier transform infrared (FT-IR) spectroscopy using rat animal models. For this purpose, 1000 cGy of ionizing radiation was specifically directed to the head of Sprague Dawley rats. The rats were decapitated after 24 h. The results revealed that the lipid-to-protein ratio decreased and that irradiation caused lipid peroxidation and increases in the amounts of olefinic =CH, carbonyl, and methylene groups of lipids. In addition, ionizing radiation induced a decrease in membrane fluidity, disordering of membrane lipids, strengthening of the hydrogen bonding of the phosphate groups of lipid head-groups, and weakening in the hydrogen bonding of the interfacial carbonyl groups of lipids. Radiation further caused significant decrements in the α-helix and turns, and significant increments in the ß-sheet and random coil contents in the protein structure. Hierarchical cluster analyses, performed in the whole region (3030-1000 cm(-1)), lipid (3030-2800 cm(-1)), and protein (1700-1600 cm(-1)) regions separately, successfully differentiated the control and irradiated groups of rat brain membranes and showed that proteins in the membranes are affected more than lipids from the damages induced with ionizing radiation. As a result, the current study showed that FT-IR spectroscopy can be used successfully as a novel method to monitor radiation-induced alterations on biological membranes.

Differentiation of Anatolian honey samples from different botanical origins by ATR-FTIR spectroscopy using multivariate analysis.

Botanical origin of the nectar predominantly affects the chemical composition of honey. Analytical techniques used for reliable honey authentication are mostly time consuming and expensive. Additionally, they cannot provide 100% efficiency in accurate authentication. Therefore, alternatives for the determination of floral origin of honey need to be developed. This study aims to discriminate characteristic Anatolian honey samples from different botanical origins based on the differences in their molecular content, rather than giving numerical information about the constituents of samples. Another scope of the study is to differentiate inauthentic honey samples from the natural ones precisely. All samples were tested via unsupervised pattern recognition procedures like hierarchical clustering and Principal Component Analysis (PCA). Discrimination of sample groups was achieved successfully with hierarchical clustering over the spectral range of 1800-750 cm(-1) which suggests a good predictive capability of Fourier Transform Infrared (FTIR) spectroscopy and chemometry for the determination of honey floral source.

Vitamin A deficiency induces structural and functional alterations in the molecular constituents of the rat hippocampus.

To date, no structural study has been carried out on the effects of vitamin A deficiency (VAD) on hippocampal macromolecules. Therefore, in the present study, the effect of dietary VAD on the structure, content and function of rat hippocampal molecules was investigated using Fourier transform IF spectroscopy. Male Wistar rats were randomly divided into three groups: an experimental group maintained on a vitamin A-deficient liquid diet (VAD, n 7); a control group maintained on a vitamin A-supplemented liquid diet (CON, n 9); a pure control group maintained on standard solid laboratory chow (PC, n 7). The PC group was included in the study to ensure that the usage of liquid diet did not influence the outcomes of VAD. Both the CON and PC groups were successfully discriminated from the VAD group by principal component analysis and hierarchical cluster analysis. The spectral analysis indicated a significant decrease in the contents of saturated and unsaturated lipids, cholesteryl esters, TAG and nucleic acids in the VAD group when compared with the CON group (P≤ 0·05). In addition, a significant decrease in membrane fluidity and a significant increase in lipid order (e.g. acyl chain flexibility) were observed in the VAD group (P≤ 0·001). The results of the artificial neural network analysis revealed a significant decrease in the α-helix structure content and a significant increase in the turn and random coil structure contents, indicating protein denaturation, in the VAD group when compared with the CON and PC groups (P≤ 0·05). Dietary exclusion of vitamin A for 3 months apparently had an adverse impact on compositional, structural and dynamical parameters. These changes can be due to increased oxidative stress, confirming the antioxidant protection provided by vitamin A when used as a dietary supplement at low-to-moderate doses.

Epileptic seizures induce structural and functional alterations on brain tissue membranes.

Epilepsy is characterized by disruption of balance between cerebral excitation and inhibition, leading to recurrent and unprovoked convulsions. Studies are still underway to understand mechanisms lying epileptic seizures with the aim of improving treatment strategies. In this context, the research on brain tissue membranes gains importance for generation of epileptic activities. In order to provide additional information for this field, we have investigated the effects of pentylenetetrazol-induced and audiogenetically susceptible epileptic seizures on structure, content and function of rat brain membrane components using Fourier transform infrared (FT-IR) spectroscopy. The findings have shown that both two types of epileptic seizures stimulate the variations in the molecular organization of membrane lipids, which have potential to influence the structures in connection with functions of membrane proteins. Moreover, less fluid lipid structure and a decline in content of lipids obtained from the ratio of CH3 asym/lipid, CH2 asym/lipid, CO/lipid, and olefinicCH/lipid and the areas of the PO2 symmetric and asymmetric modes were observed. Moreover, based on IR data the changes in the conformation of proteins were predicted by neural network (NN) analysis, and displayed as an increase in random coil despite a decrease in beta sheet. Depending on spectral parameters, we have successfully differentiated treated samples from the control by principal component analysis (PCA) and cluster analysis. In summary, FT-IR spectroscopy may offer promising attempt to identify compositional, structural and functional alterations in brain tissue membranes resulting from epileptic activities.

Investigation of compositional, structural, and dynamical changes of pentylenetetrazol-induced seizures on a rat brain by FT-IR spectroscopy.

To accomplish the appropriate treatment strategies of epilepsy action mechanisms underlying epileptic seizures should be lightened. The identification of epileptic seizure-induced alterations on the brain related to their pathologies may provide information for its action mechanism. Therefore, the current study determined molecular consequences of seizures induced by pentylenetetrazol (PTZ), which is a widely used convulsant agent, on rat brain. The rats were administered subconvulsant (25 mg/kg) and convulsant (60 mg/kg) doses of PTZ during a week, and brain tissues were studied by Fourier transform infrared (FT-IR) spectroscopy. Results revealed a decrease in lipid fluidity and lipid and protein content and also the differences in membrane packing by changing the nature of hydrogen bonding as indicated by the C═O, the PO(-)2 symmetric, and asymmetric bands. Monitoring of the olefinic band elicited seizure-induced lipid peroxidation further confirmed by the thiobarbituric acid (TBAR) assay. Additionally, PTZ-induced convulsions led to alterations in protein structures obtained by neural network (NN) predictions like an increase in random coils. On the basis of the spectral changes, treated samples could be successfully differentiated from the controls by cluster analysis. Consequently, the convulsive dose of PTZ caused more significant molecular variations compared to the subconvulsive one. All findings might have an important role in understanding the molecular mechanisms underlying epileptic activities.

Structural alterations in rat liver proteins due to streptozotocin-induced diabetes and the recovery effect of selenium: fourier transform infrared microspectroscopy and neural network study.

The relation between protein structural alterations and tissue dysfunction is a major concern as protein fibrillation and/or aggregation due to structural alterations has been reported in many disease states. In the current study, Fourier transform infrared microspectroscopic imaging has been used to investigate diabetes-induced changes on protein secondary structure and macromolecular content in streptozotocin-induced diabetic rat liver. Protein secondary structural alterations were predicted using neural network approach utilizing the amide I region. Moreover, the role of selenium in the recovery of diabetes-induced alterations on macromolecular content and protein secondary structure was also studied. The results revealed that diabetes induced a decrease in lipid to protein and glycogen to protein ratios in diabetic livers. Significant alterations in protein secondary structure were observed with a decrease in α-helical and an increase in ß-sheet content. Both doses of selenium restored diabetes-induced changes in lipid to protein and glycogen to protein ratios. However, low-dose selenium supplementation was not sufficient to recover the effects of diabetes on protein secondary structure, while a higher dose of selenium fully restored diabetes-induced alterations in protein structure.

Biophysical and microbiological study of high hydrostatic pressure inactivation of Bovine Viral Diarrheavirus type 1 on serum.

The effect of high hydrostatic pressure application on fetal bovine serum components and the model microorganism (Bovine Viral Diarrheavirus type 1 NADL strain) was studied at 132 and 220 MPa pressure for 5 min at 25°C. Protein secondary structures were found to be unaffected by an artificial neural network application on the amide I region for both untreated and HHP treated samples. FTIR spectroscopy study of both the HHP-treated and control samples revealed changes in the intensity of some bands in the finger-print region (1500-900 cm(-1)) originating mainly from lipids which are thought to result from changes in the lipoprotein structure. The virus strain lost its infectivity completely after 220 MPa HHP treatments. These results indicate that HHP can be successfully used for inactivation of pestiviruses while leaving structural and functional properties of serum and serum products unaffected.

The effects of short-term chronic ethanol intoxication and ethanol withdrawal on the molecular composition of the rat hippocampus by FT-IR spectroscopy.

BACKGROUND: The numerous adverse effects of ethanol abuse and ethanol withdrawal on biological systems are well documented. Conversely, the understanding of the molecular mechanisms underlying these pathological effects is still incomplete. This study was undertaken to investigate the effects of short-term chronic ethanol administration and ethanol withdrawal on the molecular structure and function of hippocampal tissue, a brain region important for mnemonic processes and known to be highly susceptible to ethanol intoxication. METHODS: Ethanol was administered to adult Wistar rats by intragastric intubation for 15 days with a stepwise increase in the daily dose from 6 to 12 g/kg body weight, with the highest dose delivered for the last 2 days only. The total daily dose of ethanol was divided into 3 equal portions administered 4 hours apart. Animals were sacrificed by decapitation at 4, 24, and 72 hours after the last ethanol administration to examine potential effects of ethanol intoxication and ethanol withdrawal. Ethanol-related molecular changes were monitored by Fourier transform infrared (FT-IR) spectroscopy. RESULTS: Significant changes in the hippocampal content, structure, and function of lipids, proteins, and nucleic acids were recorded under ethanol intoxication. Seventy-two hours after the cessation of ethanol administration, during the late phase of withdrawal, alterations in the macromolecules' content and conformational changes in protein and nucleic acid structure ameliorated, while the changes in macromolecular ratios, lipid order, and dynamics aggravated. CONCLUSIONS: Our results suggest that 15 days of binge-like drinking resulting in the high blood alcohol concentration (varying in the dose-dependent manner between 253 and 606 mg/dl) produced a strong physical dependence manifested mainly by the changes in lipid profiles pointing toward withdrawal-induced oxidative stress. These results show that ethanol withdrawal may cause equal to or even more severe brain damage than the ethanol itself, which should be considered when designing antialcohol therapies.

Screening of protective effect of amifostine on radiation-induced structural and functional variations in rat liver microsomal membranes by FT-IR spectroscopy.

In this study, the protective effect of amifostine, which is the only FDA-approved radioprotective agent, was investigated against the deleterious effects of ionizing radiation on rat liver microsomal membranes at molecular level. Sprague-Dawley rats, which were either administered amifostine or not, were whole-body irradiated with a single dose of 800 cGy and decapitated after 24 h. The microsomal membranes isolated from the livers of these rats were investigated using FT-IR spectroscopy. The results revealed that radiation caused a significant decrease in the lipid-to-protein ratio and the degradation of lipids into smaller fragments that contain less CH(2) and more carbonyl esters, olefinic═CH and CH(3) groups, which could be interpreted as a result of lipid peroxidation. Radiation altered the secondary structure of proteins by inducing a decrease in the ß-sheet structures and an increase in the turns and random coil structures. Moreover, a dramatic increase in lipid order and a significant decrease in the membrane dynamics were observed in the irradiated group. The administration of amifostine before ionizing radiation inhibited all the radiation induced compositional, structural, and functional damages. In addition, these results suggest that FT-IR spectroscopy provides a novel approach to monitoring radiation-induced damage on biological membranes.

Evaluation and discrimination of simvastatin-induced structural alterations in proteins of different rat tissues by FTIR spectroscopy and neural network analysis.

Statins are commonly used to control hypercholesterolemia and to prevent cardiovascular diseases. Among the statins, Simvastatin is one of the most frequently prescribed statins because of its efficacy in reducing LDL lipoprotein cholesterol levels, its tolerability, and its reduction of cardiovascular risk and mortality. Conflicting results have been reported with regard to benefits (pleiotropic effects) as well as risks (adverse effects) of simvastatin on different soft and hard tissues. In the current study, Attenuated Total Reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy was used to obtain detailed information about protein conformational changes due to simvastatin therapy of soft tissues namely liver, testis, sciatic nerve and hard tissues such as femur and tibia. Protein secondary structural changes were predicted by intensity calculations from second derivative spectra and neural network (NN) analysis, using the amide I band (1700-1600 cm(-1)) of FTIR spectra. Moreover, based on protein secondary structural differences, hierarchical cluster analysis was carried out in the 1700-1600 cm(-1) region. The results of our study in liver, testis and sciatic nerve tissues revealed that simvastatin treatment significantly decreased alpha helix structure and beta sheet structure at 1638 cm(-1), while increased the anti-parallel and aggregated beta sheet and random coil structures implying a simvastatin-induced protein denaturation in treated groups. Different to soft tissues, the results of hard tissue studies on femur and tibia bones revealed increased alpha helix structure and decreased anti-parallel beta sheet, aggregated beta sheet and random coil structures implying more strengthened bone tissues in simvastatin-treated groups. Finally, the simvastatin-treated and control groups for all soft and bone tissues were successfully differentiated using cluster analysis. According to the heterogeneity values in the cluster analysis of these tissues, the sciatic nerve tissue was found to be the most affected tissue from simvastatin treatment among the studied soft tissues. In addition, the high heterogeneity value implied high secondary structural difference between control and simvastatin-treated groups in tibia bone tissues. These findings reveal that FTIR spectroscopy with bioinformatic analyses such as neural network and hierarchical clustering, allowed us to determine the simvastatin-induced protein conformational changes as adverse and pleiotropic effects of the drug on different soft and hard tissues.