The effects of chronic morphine administration on spatial memory and microtubule dynamicity in male mice's brain.

The examination of the influence of morphine on behavioral processes, specifically learning and memory, holds significant importance. Additionally, microtubule proteins play a pivotal role in cellular functions, and the dynamics of microtubules contribute to neural network connectivity, information processing, and memory storage. however, the molecular mechanism of morphine on microtubule dynamics, learning, and memory remains uncovered. In the present study, we examined the effects of chronic morphine administration on memory formation impairment and the kinetic alterations in microtubule proteins induced by morphine in mice. Chronic morphine administration at doses of 5 and 10 mg/kg dose-dependently decreased subjects' performance in spatial memory tasks, such as the Morris Water Maze and Y-maze spontaneous alternation behavior. Furthermore, morphine was found to stabilize microtubule structure, and increase polymerization, and total polymer mass. However, it simultaneously impaired microtubule dynamicity, stemming from structural changes in tubulin dimer structure. These findings emphasize the need for careful consideration of different doses when using morphine, urging a more cautious approach in the administration of this opioid medication.

Crosstalk between tau protein autoproteolysis and amyloid fibril formation.

Tau cleavage has been shown to have a significant effect on protein aggregation. Tau truncation results in the formation of aggregation-prone fragments leading to toxic aggregates and also causes the formation of harmful fragments that do not aggregate. Thus, targeting proteolysis of tau would be beneficial for the development of therapeutics for Alzheimer's disease and related tauopathies. In this study, amino-terminal quantification and ThT fluorimetry were respectively used to analyze the kinetics of tau fragmentation and fibril formation. SDS-PAGE analysis of tau protein incubated with a disulfide-reducing agent demonstrated that the cysteines of tau have a crucial role in the fibrillation and autoproteolysis. However, the structures converted to amyloid fibrils were different with conformations that led to autoproteolysis. The quantification of the amino terminal indicated that the double-disulfide parallel structures formed in the presence of heparin did not have protease activity. The survey of possible tau disulfide-mediated dimer configurations suggested that the non-register single disulfide bound conformations were involved in the tau autoproteolysis process. Moreover, the inhibition of autoproteolysis resulted in the increment of aggregation rate; hence it seems that the tau auto-cleavage is the cellular defense mechanism against protein fibrillation.

Lysine ε-aminolysis and incorporation of sulfhydryl groups into human brain tau 4R/1N and 306VQIVYK311 enhances the formation of beta structures and toxicity.

In the present study, we investigated the effects of N-homocysteine thiolactone (tHcy) modification on expressed and purified tau protein and the synthesized VQIVYK target peptide. The modified constructs were subjected to comprehensive validation using various methodologies, including mass spectrometry. Subsequently, in vivo, in vitro, and in silico characterizations were performed under both reducing and non-reducing conditions, as well as in the presence and absence of heparin as a cofactor. Our results unequivocally confirmed that under reducing conditions and in the presence of heparin, the modified constructs exhibited a greater propensity for aggregation. This enhanced aggregative behavior can be attributed to the disruption of lysine positive charges and the subsequent influence of hydrophobic and p-stacking intermolecular forces. Notably, the modified oligomeric species induced apoptosis in the SH-SY5Y cell line, and this effect was further exacerbated with longer incubation times and higher concentrations of the modifier. These observations suggest a potential mechanism involving reactive oxygen species (ROS). To gain a deeper understanding of the molecular mechanisms underlying the neurotoxic effects, further investigations are warranted. Elucidating these mechanisms will contribute to the development of more effective strategies to counteract aggregation and mitigate neurodegeneration.

Comparable assessment of adolescent repeated physical or psychological stress effects on adult cardiac performance in female rats.

Extensive evidence highlights a robust connection between various forms of chronic stress and cardiovascular disease (CVD). In today's fast-paced world, with chronic stressors abound, CVD has emerged as a leading global cause of mortality. The intricate interplay of physical and psychological stressors triggers distinct neural networks within the brain, culminating in diverse health challenges. This study aims to discern the unique impacts of chronic physical and psychological stress on the cardiovascular system, unveiling their varying potencies in precipitating CVD. Twenty-one adolescent female rats were methodically assigned to three groups: (1) control (n = 7), (2) physical stress (n = 7), and (3) psychological stress (n = 7). Employing a two-compartment enclosure, stressors were administered to the experimental rats over five consecutive days, each session lasting 10 min. After a 1.5-month recovery period post-stress exposure, a trio of complementary techniques characterized by high specificity or high sensitivity were employed to meticulously evaluate CVD. Echocardiography and single-photon emission computed tomography (SPECT) were harnessed to scrutinize left ventricular architecture and myocardial viability, respectively. Subsequently, the rats were ethically sacrificed to facilitate heart removal, followed by immunohistochemistry staining targeting glial fibrillary acidic protein (GFAP). Rats subjected to psychological stress showed a wider range of significant cardiac issues compared to control rats. This included left ventricular hypertrophy [IVSd: 0.1968 ± 0.0163 vs. 0.1520 ± 0.0076, P < 0.05; LVPWd: 0.2877 ± 0.0333 vs. 0.1689 ± 0.0057, P < 0.01; LVPWs: 0.3180 ± 0.0382 vs. 0.2226 ± 0.0121, P < 0.05; LV-mass: 1.283 ± 0.0836 vs. 1.000 ± 0.0241, P < 0.01], myocardial ischemia [21.30% vs. 32.97%, P < 0.001], and neuroinflammation. This outcome underscores the imperative of prioritizing psychological well-being during adolescence, presenting a compelling avenue to curtail the prevalence of CVD in adulthood. Furthermore, extending such considerations to individuals grappling with CVD might prospectively enhance their overall quality of life.

Beneficial effects of trehalose and gentiobiose on human sperm cryopreservation.

The protection of human sperm during cryopreservation is of great importance to infertility. Recent studies have shown that this area is still a long way from its ultimate aim of maintaining the maximum viability of sperm in cryopreservation. The present study used trehalose and gentiobiose to prepare the human sperm freezing medium during the freezing-thawing. The freezing medium of sperm was prepared with these sugars, and the sperm were then cryopreserved. The viable cells, sperm motility parameters, sperm morphology, membrane integrity, apoptosis, acrosome integrity, DNA fragmentation, mitochondrial membrane potential, reactive oxygen radicals, and malondialdehyde concentration was evaluated using standard protocols. A higher percentage of the total and progressive motility, rate of viable sperm, cell membrane integrity, DNA and acrosome integrity, and mitochondrial membrane potential were observed in the two frozen treatment groups compared to the frozen control. The cells had less abnormal morphology due to treatment with the new freezing medium than the frozen control. The higher malondialdehyde and DNA fragmentation were significantly observed in the two frozen treatment groups than in the frozen control. According to the results of this study, the use of trehalose and gentiobiose in the sperm freezing medium is a suitable strategy for sperm freezing to improve its motion and cellular parameters.

Inhibition Of Tau Protein Aggregation By a Chaperone-like ß-Boswellic Acid Conjugated To Gold Nanoparticles.

A potential therapeutic strategy to inhibit tau protein aggregation in neurons has substantial effects on preventing or controlling Alzheimer's disease (AD). In this work, we designed a covalent and noncovalent conjugation of ß-boswellic acid (BA) to gold nanoparticles (GNPs). We provided the opportunity to investigate the effect of the surface composition of BA-GNPs on the aggregation of the tau protein 1N/4R isoform in vitro. HR-TEM and FESEM micrographs revealed that GNPs were spherical and uniform, smaller than 25 nm. According to UV-visible and FTIR data, BA was successfully conjugated to GNPs. The finding illustrates the effect of the surface charge, size, and hydrophobicity of BA-GNPs on the kinetics of tau protein aggregation. The size and surface area of U-G-BA demonstrated that inhibited tau aggregation more effectively than covalently linked BA. The proposed method for preventing tau aggregation was monomer reduction. At the same time, a chaperone-like feature of GNP-BA while sustaining a tau native structure prevented the additional formation of fibrils. Overall, this study provides insight into the interaction of GNP-BAs with a monomer of tau protein and may suggest novel future therapies for AD.

Bivalent metal ions induce formation of α-synuclein fibril polymorphs with different cytotoxicities.

α-Synuclein (α-Syn) aggregates are key components of intracellular inclusion bodies characteristic of Parkinson's disease (PD) and other synucleinopathies. Metal ions have been considered as the important etiological factors in PD since their interactions with α-Syn alter the kinetics of fibrillation. In the present study, we have systematically explored the effects of Zn2+, Cu2+, Ca2+, and Mg2+ cations on α-Syn fibril formation. Specifically, we determined fibrillation kinetics, size, morphology, and secondary structure of the fibrils and their cytotoxic activity. While all cations accelerate fibrillation, we observed distinct effects of the different ions. For example, Zn2+ induced fibrillation by lower tlag and higher kapp and formation of shorter fibrils, while Ca2+ ions lead to formation of longer fibrils, as evidenced by dynamic light scattering and atomic force microscopy studies. Additionally, the morphology of formed fibrils was different. Circular dichroism and attenuated total reflection-Fourier transform infrared spectroscopies revealed higher contents of ß-sheets in fibrils. Interestingly, cell viability studies indicated nontoxicity of α-Syn fibrils formed in the presence of Zn2+ ions, while the fibrils formed in the presence of Cu2+, Ca2+, and Mg2+ were cytotoxic. Our results revealed that α-Syn fibrils formed in the presence of different divalent cations have distinct structural and cytotoxic features.

Unravelling the Novel Effects of Three Volatile Compounds in Preventing Fibril Formation of Disease Related Tau and α-Synuclein Proteins- Towards Identifying Candidate Aromatic Substances for Treating Neurodegenerative Diseases.

Background: The aggregation of tau and α-synuclein into fibrillary assemblies in nerve cells is the molecular hallmark of Alzheimer's and Parkinson's diseases, respectively. In our previous studies, we investigated the anti-amyloidogenic effects of three different aroma-producing (volatile) compounds including cinnamaldehyde, phenyl ethyl alcohol, and TEMED on the fibrillation process of HEWL, as a model protein. Our previous results showed that while TEMED was able to completely stop the process of fibril formation, cinnamaldehyde and phenyl ethyl alcohol gave rise to oligomeric/protofibrillar forms and were involved in the entrapment of intermediate species of HEWL. In this study, we investigated the anti-amyloidogenic effect of the same three volatile compounds on recombinantly produced tau and α-synuclein proteins. Methods: The thioflavin T fluorescence assay, circular dichroism, SDS-PAGE/native-PAGE, dynamic light scattering, and atomic force microscopy were used, where necessary, to further our understanding of the inhibitory effects of the three volatile compounds on the fibril formation of tau and α-synuclein proteins and allow for a comparison with previous data obtained for HEWL. Results: Our results revealed that contrary to the results obtained for HEWL (a globular protein), the volatile compound TEMED was no longer able to prevent fibril formation in either of the natively unstructured tau or α-synuclein proteins, and instead, cinnamaldehye and phenyl ethyl alcohol, in particular, had the role of preventing fibril formation of tau or α-synuclein. Conclusion: The results of this study further emphasized the exclusion of HEWL as a model protein for fibrillation studies and highlighted the importance of studying brain-related proteins such as tau or α-synuclein and the need to assess the effects of volatile compounds such as cinnamaldehye and phenyl ethyl alcohol as potential substances in the treatment of neurodegenerative diseases.

Supplementation of freezing medium with encapsulated or free glutathione during cryopreservation of bull sperm.

The objective was to compare effects of encapsulated or free glutathione (GSH) on the quality of frozen-thawed bull sperm. Ejaculates were collected via artificial vagina from six mature Holstein bulls once weekly for 6 weeks. All ejaculates had motility ≥70%, sperm concentration ≥1.0 × 109 /ml and ≤15% morphologically abnormal sperm. Each week, semen was pooled and diluted with lecithin-based extenders containing various concentrations of encapsulated (E0, E1, E2.5 and E5 mM) or free (F0, F1, F2.5 and F5 mM) GSH, with total glutathione content determined before and after cryopreservation. Total GSH in fresh semen was (mean+SEM) 4.8 ± 0.2 nmol/108 sperm, whereas in frozen-thawed semen of group F0 (control), it decreased to 1.4 ± 0.2 nmol/108 sperm, a 70.8% reduction (p < .05). In addition, total GSH in frozen-thawed semen from groups E2.5, E5 and F5 were 2.4 ± 0.2, 2.8 ± 0.2 and 1.8 ± 0.2 nmol/108 sperm, respectively (E5 versus. F0, p < .05). Compared to group F0, frozen-thawed sperm from group E2.5 had greater (p < .05) percentages of sperm that were viable (Annexin-V) (61.1 ± 1.8 versus. 71.1 ± 1.8) and that had cell membrane integrity (eosin-nigrosin) (64.5 ± 3.1 versus. 80.0 ± 3.1). Furthermore, frozen-thawed sperm from group E2.5 had the numerically highest total and progressive motility (CASA) and cell membrane functionality (HOS) and the lowest percentage of early apoptotic sperm (Annexin-V). However, acrosome membrane integrity (PSA) of E5 had the lowest mean (p < .05), whereas E2.5 caused a small nonsignificant decrease (69.1 ± 1.4%) compared to E0 and F0. In conclusion, 2.5 mM encapsulated GSH in semen extender significantly improved the quality of frozen-thawed bull sperm.

Cannabidiol Inhibits Tau Aggregation In Vitro.

A hallmark of Alzheimer's disease (AD) is the accumulation of tau protein in the brain. Compelling evidence indicates that the presence of tau aggregates causes irreversible neuronal destruction, eventually leading to synaptic loss. So far, the inhibition of tau aggregation has been recognized as one of the most effective therapeutic strategies. Cannabidiol (CBD), a major component found in Cannabis sativa L., has antioxidant activities as well as numerous neuroprotective features. Therefore, we hypothesize that CBD may serve as a potent substance to hamper tau aggregation in AD. In this study, we aim to investigate the CBD effect on the aggregation of recombinant human tau protein 1N/4R isoform using biochemical methods in vitro and in silico. Using Thioflavin T (ThT) assay, circular dichroism (CD), and atomic force microscopy (AFM), we demonstrated that CBD can suppress tau fibrils formation. Moreover, by quenching assay, docking, and job's plot, we further demonstrated that one molecule of CBD interacts with one molecule of tau protein through a spontaneous binding. Experiments performed by quenching assay, docking, and Thioflavin T assay further established that the main forces are hydrogen Van der Waals and some non-negligible hydrophobic forces, affecting the lag phase of tau protein kinetics. Taken together, this study provides new insights about a natural substance, CBD, for tau therapy which may offer new hope for the treatment of AD.

Astaxanthin Decreases Spatial Memory and Glutamate Transport Impairment Induced by Fluoride.

Excessive exposure to the sources of fluoride in drinking water, oral care products, and food is a widespread problem. Fluoride is associated with impairment in child intelligence development. It causes DNA damage, oxidative stress, and mitochondrial dysfunction, mainly due to the production of reactive oxygen species (ROS). It has been postulated that the use of antioxidants such as astaxanthin, may alleviate fluoride's adverse effects. This study assessed the effects of fluoride on cellular ROS content and rat's learning and memory ability and investigated the protective potency of astaxanthin with emphasis on the role of glutamate using the Morris Water Maze test, glutamate concentration determination, and western blot techniques. The fluoride treatment of cells results in an increment of cellular ROS, whereas astaxanthin inhibits lipid peroxidation. Fluoride significantly decreases the cellular glutamate uptake and glutamate transporter, protein level, possibly due to the disruption of mitochondrial energy metabolism and defect of the transporter recycle, respectively. The in-vivo study indicated that the treatment of rats with fluoride led to a loss of learning, while astaxanthin improved memory dysfunction. Measurement of ROS and glutamate levels of rat brain hippocampus showed that fluoride increased the ROS but decreased the glutamate. On the other hand, the utilization of astaxanthin decreased the brain ROS content and increased the glutamate level. It seems that fluoride disrupts the normal function of neurons via increment of ROS production and decrement of glutamate level, whereas astaxanthin has neuroprotective potency due to the ROS scavenging ability.

Interplay of isoform 1N4R tau protein and amyloid-ß peptide fragment 25-35 in reducing and non-reducing conditions.

Amyloid-ß (Aß) peptide and tau protein are two hallmark proteins in Alzheimer's disease (AD); however, the parameters, which mediate the abnormal aggregation of Aß and tau, have not been fully discovered. Here, we have provided an optimum method to purify tau protein isoform 1N4R by using nickel-nitrilotriacetic acid agarose chromatography under denaturing condition. The biochemical and biophysical properties of the purified protein were further characterized using in vitro tau filament assembly, tubulin polymerization assay, circular dichroism (CD) spectroscopy and atomic force microscopy. Afterwards, we investigated the effect of tau protein on aggregation of Aß (25-35) peptide using microscopic imaging and cell viability assay. Incubation of tau at physiologic and supra-physiologic concentrations with Aß25-35 for 40 days under reducing and non-reducing conditions revealed formation of two types of aggregates with distinct morphologies and dimensions. In non-reducing condition, the co-incubated sample showed granular aggregates, while in reducing condition, they formed annular protofibrils. Results from cell viability assay revealed the increased cell viability for the co-incubated sample. Therefore, the disassembling action shown by tau protein on Aß25-35 suggests the possibility that tau may have a protective role in preventing Aß peptide from acquiring the cytotoxic, aggregated form against oxidative stress damages.

Peptide LIQ Promotes Cell Protection against Zinc-Induced Cytotoxicity through Microtubule Stabilization.

Stability of the microtubule protein (MTP) network required for its physiological functions is disrupted in the course of neurodegenerative disorders. Thus, the design of novel therapeutic approaches for microtubule stabilization is a focus of intensive study. Dynamin-related protein-1 (Drp1) is a guanosine triphosphatase (GTPase), which plays a prevailing role in mitochondrial fission. Several isoforms of Drp1 have been identified, of which one of these isoforms (Drp1-x01) has been previously described with MTP stabilizing activity. Here, we synthesized peptide LIQ, an 11-amino-acid peptide derived from the Drp1-x01 isoform, and reported that LIQ could induce tubulin assembly in vitro. Using a Stern-Volmer plot and continuous variation method, we proposed one binding site on tubulin for this peptide. Interestingly, FRET experiment and docking studies showed that LIQ binds the taxol-binding site on ß-tubulin. Furthermore, circular dichroism (CD) spectroscopy and 8-anilino-1-naphthalenesulfonic acid (ANS) assay provided data on tubulin structural changes upon LIQ binding that result in formation of more stable tubulin dimers. Flow cytometry analysis and fluorescence microscopy displayed that cellular internalization of 5-FAM-labeled LIQ is attributed to a mechanism that mostly involves endocytosis. In addition, LIQ promoted polymerization of tubulin and stabilized MTP in primary astroglia cells and also protected these cells against zinc toxicity. This excellent feature of cellular neuroprotection by LIQ provides a promising therapeutic approach for neurodegenerative diseases.

Anti-metastatic properties of a potent herbal combination in cell and mice models of triple negative breast cancer.

AIM: To determine the anti-metastatic potential of combinations of two bioactive carotenoids of saffron, crocin and crocetin, on 4T1 breast cancer and on a mice model of TNBC, and assess the effect of the most potent combination on the Wnt/ß-catenin pathway. MAIN METHODS: The effects of the carotenoid combinations on the viability of 4T1 cells were determined by MTT assay. The effects of the nontoxic doses on migration, mobility, invasion and adhesion to ECM were examined by scratch assay, Transwell/Matrigel-coated Transwell chamber and adhesion assay respectively. Tumors were inoculated by injecting mice with 4T1 cells. The weights and survival rates of the mice and tumor sizes were monitored. Histological analysis of the tissues was conducted. The expression levels of Wnt/ß-catenin pathway genes were measured by Real-time PCR and western blotting. KEY FINDINGS: Treatment of 4T1 cells with combination doses inhibited viability in a dose-dependent manner. The nontoxic combinations significantly inhibited migration, cell mobility and invasion, also attenuating adhesion to ECM. The combination therapy mice possessed more weight, higher survival rates and smaller tumors. Histological examination detected remarkably fewer metastatic foci in their livers and lungs. It was also demonstrated that the combinations exerted anti-metastatic effects by disturbing the Wnt/ß-catenin target genes in the liver and tumors. SIGNIFICANCE: Our findings propose a carotenoid combination as an alternative potent herbal treatment for TNBC, which lacks the adverse effects associated with either chemotherapeutic agents or herb-chemotherapeutic drugs.

Site Directed Disulfide PEGylation of Interferon-ß-1b with Fork Peptide Linker.

The attachment of PEG to biopharmaceuticals has been applied for enhancement of bioavailability and improved stability. The PEG polymer is highly hydrated; thus effective attachment to inaccessible sites could be hindered. We have devised a scheme to address this issue by introducing a considerable distance between PEG and protein by addition of a linear peptide, appended to long chained reactive linkers. Second, the position of PEG conjugation directly affects biological activity. Accordingly, a disulfide bond could be considered as an ideal choice for site directed PEGylation; but reactivity of both thiol moieties to bridging reagent is critical for maintenance of protein structure. In our design, a forked structure with two arms provides essential flexibility to account for dissociation of reduced cysteines. An efficient yield for disulfide PEGylation of IFN-ß1b was attained and specificity, biophysical characterization, biological activity, and pharmacokinetics were surveyed.

Aggregate Forms of Recombinant Human Erythropoietin With Different Charge Profile Substantially Impact Biological Activities.

Recombinant human erythropoietin (rHuEPO) as a glycoprotein growth factor has been considered a biological drug for treatment of anemic patients with chronic renal failure or who receive cancer chemotherapy. Biological activity and circulation time are 2 parameters that are important to achieve EPO's efficacy. Previous efforts for increasing EPO's efficacy have focused on glycosylation modification via adding more sialic acid antenna and generates more negative charged protein. Evidences cleared that EPO's activity increased by numbers of N-glycan moieties with presence of sialic acids at their terminus. Correlation between bioactivity and glycosylation with terminal sialylation is theoretically achieved using the calculation of the amount of charge profile of the EPO variants called "I-number." Here, we studied and compared the relationship between bioactivities of different EPOs that contained various I-numbers and the effect of their secondary and tertiary protein structures on measured in vivo efficacy. Eight recombinant EPOs batches were produced under the same condition. I-numbers found out by EPO's charge profiles determination using capillary electrophoresis and activities were studied upon erythroid precursor cell stimulation in mice. Analyzing the bioactivity, I-number, and structural studies revealed that in spite of I-number, conformational changes in protein structure and presence of aggregated species impact bioactivity substantially.

Ac-SDKP ameliorates the progression of experimental autoimmune encephalomyelitis via inhibition of ER stress and oxidative stress in the hippocampus of C57BL/6 mice.

Despite the attention given to the treatment of multiple sclerosis (MS), still no certain cure is available. The main purpose of MS drugs is acting against neuroinflammation which underlies the pathology of MS. Neuroinflammation is associated with endoplasmic reticulum (ER) stress that mediates neural apoptosis. In the present study, we hypothesized that the tetrapeptide N-acetyl-ser-asp-lys-pro (Ac-SDKP) with the previously described anti-fibrotic effects might have anti-inflammatory, anti-oxidative and anti-ER stress roles in the hippocampus. We used myelin oligodendrocyte glycoprotein (MOG) to induce experimental autoimmune encephalomyelitis (EAE), a widely-accepted animal model of MS, in C57BL/6 mice. The protein levels of ER stress-related molecules including caspase-12, C/EBP homologous protein (CHOP), and protein disulfide isomerase (PDI) in the hippocampus were examined by immunoblotting. Hence, reactive oxygen species (ROS) production, lipid peroxidation and antioxidant capacity of the hippocampus were studied. Moreover, hippocampal morphology changes, leukocytes infiltration, and the levels of IL-6 and IL-1ß pro-inflammatory cytokines were evaluated. Our results displayed that Ac-SDKP down regulates caspase-12 and CHOP expression in the hippocampus-resident oligodendrocytes of EAE mice. Further, treatment with Ac-SDKP decreased oxidative stress markers and caspase-3 activation in the hippocampus of EAE mice. According to our findings, Ac-SDKP showed beneficial effects against ER stress and oxidative stress in addition to inflammation in the hippocampus of EAE mice. The present study provides the basis for further research on the therapeutic applications of Ac-SDKP to reduce ER stress and oxidative stress-induced apoptosis in neurodegenerative disorders.

Electromagnetic field in human sperm cryopreservation improves fertilizing potential of thawed sperm through physicochemical modification of water molecules in freezing medium.

Physicochemical properties of water molecules as the main compositions of the freezing media can be affected by the electromagnetic fled. The purpose of this study was to apply extremely low repetition rate electromagnetic fields (ELEFs) to change the molecular network of water molecules existing in freezing media used for human sperm cryopreservation. First, different time periods and pulsed electromagnetic fields were used to evaluate the physiochemical properties of water. The lowest rate of cluster size, surface tension, viscosity, and density was observed for water samples exposed to 1000 Hz ELEF for 60 min (P < 0.05) that could be results in small ice crystal formation. Therefore, this treatment was selected for further evaluations in human sperm freezing because there was minimal probability of amorphous ice crystallization in this group. To assess fertilizing potential, human semen samples were subjected to ELEF (1000 Hz) water-made freezing medium and cryopreserved. The highest percentage of total motility, progressive motility, viability, membrane integrity, mitochondrial membrane potential, DNA integrity, and TAC were obtained in frozen ELEF as compared to other groups. The percentage of viable spermatozoa (Annexin V-/PI-) in frozen ELEF was significantly higher than in frozen control. The level of ROS was significantly lower in frozen ELEF when compared to frozen control. It can be concluded that the modification of physicochemical properties of water existing in cryopreservation media by ELEF is a suitable strategy to improve the outcome of cryopreservation.

Comparison of polymerization and structural behavior of microtubules in rat brain and sperm affected by the extremely low-frequency electromagnetic field.

BACKGROUND: Microtubule proteins are able to produce electromagnetic fields and have an important role in memory formation, and learning. Therefore, microtubules have the potential to be affected by exogenous electromagnetic fields. This study aimed to examine the comparison of microtubule polymerization and its structural behavior in brain and sperm affected by 50 Hz extremely low-frequency electromagnetic field (ELEF). RESULTS: Twenties adult male rats were randomly and equally divided into control and experimental groups, to evaluate the effect of 50 Hz ELEF on the sperm and brain functions. Plus-maze, serum testosterone and corticosterone, and sperm evaluation were performed. Next, the semen and brain samples were obtained, and they were divided into four experimental groups for investigation of microtubule polymerization. There was no significant difference in testosterone and, corticosterone levels, anxiety behaviors, and sperm morphology between control and ELEF-exposure groups. The sperm viability, total and progressive motility were significantly higher in the ELEF-exposed group than that of the control group. The microtubule polymerization in sperm ELEF was significantly higher than in other groups. The secondary and tertiary structures of tubulins were significantly affected in the brain, and sperm ELEF groups. CONCLUSION: It seems that the polymerization of microtubules and conformational changes of tubulin dimers are improved by ELEF application.

Microtubule network as a potential candidate for targeting by gold nanoparticle-assisted photothermal therapy.

Photothermal therapy is achieving ever-increasing attention as a promising method for killing cancer cells. Although, gold nanoparticles are regarded as one of the most effective photothermal therapy agents, the mechanisms underlying their action have to be addressed. Moreover, studies have showed that gold nanoparticles induce apoptosis in treated cultures. Hence, in this study, we investigated the interaction of folic acid functionalized gold nanoparticles and gold-shelled Fe3O4 nanoparticles with microtubule and microtubule associated protein tau in order to introduce intracellular targets of these nanoparticles and provide a holistic view about the mechanism of action of gold nanoparticles used in photothermal therapy. Various spectroscopic methods were used to find gold nanoparticles interaction with Tubulin and Tau. Our results indicated that these gold nanoparticles interact with both Tau and Tubulin and their affinity increases as temperature rises. Also, the results illustrated that quenching mechanism for gold nanoparticles interaction with Tubulin and Tau was static. The hydrophobic interaction was determined as driving force for gold nanoparticles binding to Tubulin and Tau. Moreover, it was showed that both type of gold nanoparticles stabilize microtubule polymers. These results suggest Tau and Tubulin as intracellular target of gold nanoparticles and propose that microtubule network is at the heart of apoptosis mechanisms initiated by photothermal therapy.