Detection of early proteomic alterations in 5xFAD Alzheimer's disease neonatal mouse model via MALDI-MSI.

Alzheimer's disease (AD) is a debilitating neurodegenerative disorder, characterized by memory deficit and dementia. AD is considered a multifactorial disorder where multiple processes like amyloid-beta and tau accumulation, axonal degeneration, synaptic plasticity, and autophagic processes plays an important role. In this study, the spatial proteomic differences in the neonatal 5xFAD brain tissue were investigated using MALDI-MSI coupled to LC-MS/MS, and the statistically significantly altered proteins were associated with AD. Thirty-five differentially expressed proteins (DEPs) between the brain tissues of neonatal 5xFAD and their littermate mice were detected via MALDI-MSI technique. Among the 35 proteins identified, 26 of them were directly associated with AD. Our results indicated a remarkable resemblance in the protein expression profiles of neonatal 5xFAD brain when compared to AD patient specimens or AD mouse models. These findings showed that the molecular alterations in the AD brain existed even at birth and that some proteins are neurodegenerative presages in neonatal AD brain. HIGHLIGHTS: Spatial proteomic alterations in the 5xFAD mouse brain compared to the littermate. 26 out of 35 differentially expressed proteins associated with Alzheimer's disease (AD). Molecular alterations and neurodegenerative presages in neonatal AD brain. Alterations in the synaptic function an early and common neurobiological thread.

Comparison of protein expression profile of limb regeneration between neotenic and metamorphic axolotl.

The axolotl (Ambystoma mexicanum) salamander, a urodele amphibian, has an exceptional regenerative capacity to fully restore an amputated limb throughout the life-long lasting neoteny. By contrast, when axolotls are experimentally induced to metamorphosis, attenuation of the limb's regenerative competence is noticeable. Here, we sought to discern the proteomic profiles of the early stages of blastema formation of neotenic and metamorphic axolotls after limb amputation by employing LC-MS/MS technology. We quantified a total of 714 proteins and qRT-PCR for selected genes was performed to validate the proteomics results and provide evidence for the putative link between immune system activity and regenerative potential. This study provides new insights for examination of common and distinct molecular mechanisms in regeneration-permissive neotenic and regeneration-deficient metamorphic stages at the proteome level.

Clinical applications of MALDI imaging technologies in cancer and neurodegenerative diseases.

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) imaging mass spectrometry (IMS) enables localization of analytes of interest along with histology. More specifically, MALDI-IMS identifies the distributions of proteins, peptides, small molecules, lipids, and drugs and their metabolites in tissues, with high spatial resolution. This unique capacity to directly analyze tissue samples without the need for lengthy sample preparation reduces technical variability and renders MALDI-IMS ideal for the identification of potential diagnostic and prognostic biomarkers and disease gradation. MALDI-IMS has evolved rapidly over the last decade and has been successfully used in both medical and basic research by scientists worldwide. In this review, we explore the clinical applications of MALDI-IMS, focusing on the major cancer types and neurodegenerative diseases. In particular, we re-emphasize the diagnostic potential of IMS and the challenges that must be confronted when conducting MALDI-IMS in clinical settings. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.

Hydroquinidine displays a significant anticarcinogenic activity in breast and ovarian cancer cells via inhibiting cell-cycle and stimulating apoptosis.

Breast and ovarian cancers are women's most commonly diagnosed cancers. Seeking an efficient anticarcinogenic compound is still a top priority regarding the aggressiveness of these cancers and the limited benefit of current therapies. Hydroquinidine (HQ) is a natural alkaloid used in arrhythmia and Brugada syndrome. As an ion channel blocker, HQ exhibits its activity by altering ion gradient and membrane potential. Considering the growing evidence of ion channel blockers' antineoplastic potential, we were prompted to test HQ's effect on breast and ovarian cancers. MCF-7 and SKOV-3 cell lines were used to inspect how HQ acts on survival, clonogenicity, migration, tumorigenicity, proliferation, and apoptosis. The molecular basis for the remarkable antiproliferative and proapoptotic effect of HQ in these cells was dissected by proteomics. CDK1, PSMB5, PSMC2, MCM2, MCM7, YWHAH, YWHAQ, and YWHAB proteins in HQ-treated MCF-7 cells, and RRM2, PSMD2, PSME2, COX2, COX4l1, and CDK6 proteins in HQ-treated SKOV-3 cells were found as low-abundant, which was noteworthy. Based on the in-depth analysis, upon HQ treatment, several cell cycle-related processes were found as suppressed, whereas apoptosis and ferroptosis pathways were found to be activated. The observed proteome alteration in cancer cells may provide mechanistic explanations for the growth-limiting effects of HQ at the cellular level.

Spatial proteomic alterations detected via MALDI-MS imaging implicate neuronal loss in a Huntington's disease mouse (YAC128) brain.

Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder that occurs with the increase of CAG trinucleotide repeats in the huntingtin gene. To understand the mechanisms of HD, powerful proteomics techniques, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) were employed. However, one major drawback of these methods is loss of the region-specific quantitative information of the proteins due to analysis of total tissue lysates. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a MS-based label-free technique that works directly on tissue sections and gathers m/z values with their respective regional information. In this study, we established a data processing protocol that includes several software programs and methods to determine spatial protein alterations between the brain samples of a 12 month-old YAC128 HD mouse model and their non-transgenic littermates. 22 differentially expressed proteins were revealed with their respective regional information, and possible relationships of several proteins were discussed. As a validation of the MALDI-MSI analysis, a differentially expressed protein (GFAP) was verified using immunohistochemical staining. Furthermore, since several proteins detected in this study have previously been associated with neuronal loss, neuronal loss in the cortical region was demonstrated using an anti-NeuN immunohistochemical staining method. In conclusion, the findings of this research have provided insights into the spatial proteomic changes between HD transgenic and non-transgenic littermates and therefore, we suggest that MALDI-MSI is a powerful technique to determine spatial proteomic alterations between biological samples, and the data processing that we present here can be employed as a complementary tool for the data analysis.

Molecular Effects of Pteryxin and Scopoletin in the 5xFAD Alzheimer's Disease Mouse Model.

BACKGROUND: Alzheimer's disease (AD) is one of the most prevalent diseases with rapidly increasing numbers, but there is still no medication to treat or stop the disease. Previous data on coumarins suggests that scopoletin may have potential benefits in AD. OBJECTIVE: Evaluate the therapeutic potential of the coumarins with natural origin - scopoletin and pteryxin- in a 5xFAD mouse model of AD. METHODS: Both compounds were administered at two doses to 12-month-old mice, which represent severe AD pathology. The effects of coumarins were assessed on cognition in mouse experiments. Changes in the overall brain proteome were evaluated using LCMS/ MS analyses. RESULTS: The Morris water maze test implicated that a higher dose of pteryxin (16 mg/kg) significantly improved learning, and the proteome analysis showed pronounced changes of specific proteins upon pteryxin administration. The amyloid-ß precursor protein, glial fibrillary acid protein, and apolipoprotein E protein which are highly associated with AD, were among the differentially expressed proteins at the higher dose of the pteryxin. CONCLUSION: Overall, pteryxin may be evaluated further as a disease-modifying agent in AD pathology in the late stages of AD.

Kappa/Lambda light-chain typing in Alzheimer's Disease.

BACKGROUND: Alzheimer's disease is a progressive neurodegenerative disorder characterized by memory loss and cognitive impairment. The diagnosis of Alzheimer's disease according to symptomatic events is still a puzzling task. Developing a biomarker-based, low-cost, and high-throughput test, readily applicable in clinical laboratories, dramatically impacts the rapid and reliable detection of the disease. OBJECTIVE: This study aimed to develop an accurate, sensitive, and reliable screening tool for diagnosing Alzheimer's disease, which can significantly reduce the cost and time of existing methods. METHODS: We have employed a MALDI-TOF-MS-based methodology combined with a microaffinity chromatography enrichment approach using affinity capture resins to determine serum kappa (κ) and lambda (λ) light chain levels in control and patients with AD. RESULTS: We observed a statistically significant difference in the kappa light chain over lambda light chain (κLC/λLC) ratios between patients with AD and controls (mean difference -0,409; % 95 CI:- 0.547 to -0.269; p<0.001). Our method demonstrated higher sensitivity (100.00%) and specificity (71.43%) for discrimination between AD and controls. CONCLUSION: We have developed a high-throughput screening test with a novel sample enrichment method for determining κLC/λLC ratios associated with AD diagnosis. Following further validation, we believe our test has the potential for clinical laboratories.

Evaluation of the Therapeutic Effect of Lycoramine on Alzheimer's Disease in Mouse Model.

BACKGROUND: Alzheimer's disease is one of the leading health problems characterized by the accumulation of Aß and hyperphosphorylated tau that account for the senile plaque formations causing extensive cognitive decline. Many of the clinical diagnoses of Alzheimer's disease are made in the late stages, when the pathological changes have already progressed. OBJECTIVE: The objective of this study is to evaluate the promising therapeutic effects of a natural compound, lycoramine, which has been shown to have therapeutic potential in several studies and to understand its mechanism of action on the molecular level via differential protein expression analyses. METHODS: Lycoramine and galantamine, an FDA approved drug used in the treatment of mild to moderate AD, were administered to 12 month-old 5xFAD mice. Effects of the compounds were investigated by Morris water maze, immunohistochemistry and label- free differential protein expression analyses. RESULTS: Here we demonstrated the reversal of cognitive decline via behavioral testing and the clearance of Aß plaques. Proteomics analysis provided in-depth information on the statistically significant protein perturbations in the cortex, hippocampus and cerebellum sections to hypothesize the possible clearance mechanisms of the plaque formation and the molecular mechanism of the reversal of cognitive decline in a transgenic mouse model. Bioinformatics analyses showed altered molecular pathways that can be linked with the reversal of cognitive decline observed after lycoramine administration but not with galantamine. CONCLUSION: Lycoramine shows therapeutic potential to halt and reverse cognitive decline at the late stages of disease progression, and holds great promise for the treatment of Alzheimer's disease.

Proteins associated with neutrophil degranulation are upregulated in nasopharyngeal swabs from SARS-CoV-2 patients.

COVID-19 or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appeared throughout the World and currently affected more than 9 million people and caused the death of around 470,000 patients. The novel strain of the coronavirus disease is transmittable at a devastating rate with a high rate of severe hospitalization even more so for the elderly population. Naso-oro-pharyngeal swab samples as the first step towards detecting suspected infection of SARS-CoV-2 provides a non-invasive method for PCR testing at a high confidence rate. Furthermore, proteomics analysis of PCR positive and negative naso-oropharyngeal samples provides information on the molecular level which highlights disease pathology. Samples from 15 PCR positive cases and 15 PCR negative cases were analyzed with nanoLC-MS/MS to identify the differentially expressed proteins. Proteomic analyses identified 207 proteins across the sample set and 17 of them were statistically significant. Protein-protein interaction analyses emphasized pathways like Neutrophil degranulation, Innate Immune System, Antimicrobial Peptides. Neutrophil Elastase (ELANE), Azurocidin (AZU1), Myeloperoxidase (MPO), Myeloblastin (PRTN3), Cathepsin G (CTSG) and Transcobalamine-1 (TCN1) were found to be significantly altered in naso-oropharyngeal samples of SARS-CoV-2 patients. The identified proteins are linked to alteration in the innate immune system specifically via neutrophil degranulation and NETosis.

Stereotype-based priming without stereotype activation: A tale of two priming tasks.

An extensive literature has demonstrated stereotype-based priming effects. What this work has only recently considered, however, is the extent to which priming is moderated by the adoption of different sequential-priming tasks and the attendant implications for theoretical treatments of person perception. In addition, the processes through which priming arises (i.e., stimulus and/or response biases) remain largely unspecified. Accordingly, here we explored the emergence and origin of stereotype-based priming using both semantic- and response-priming tasks. Corroborating previous research, a stereotype-based priming effect only emerged when a response-priming (vs. semantic-priming) task was used. A further hierarchical drift diffusion model analysis revealed that this effect was underpinned by differences in the evidential requirements of response generation (i.e., a response bias), such that less evidence was needed when generating stereotype-consistent compared with stereotype-inconsistent responses. Crucially, information uptake (i.e., stimulus bias, efficiency of target processing) was faster for stereotype-inconsistent than stereotype-consistent targets. This reveals that stereotype-based priming originated in a response bias rather than the automatic activation of stereotypes. The theoretical implications of these findings are considered.

Proteomics Analysis of CA1 Region of the Hippocampus in Pre-, Progression and Pathological Stages in a Mouse Model of the Alzheimer's Disease.

BACKGROUND: CA1 subregion of the hippocampal formation is one of the primarily affected structures in AD, yet not much is known about proteome alterations in the extracellular milieu of this region. OBJECTIVE: In this study, we aimed to identify the protein expression alterations throughout the pre-pathological, progression and pathological stages of AD mouse model. METHODS: The CA1 region perfusates were collected by in-vivo intracerebral push-pull perfusion from transgenic 5XFAD mice and their non-transgenic littermates at 3, 6 and 12 wereßmonths of age. Morris water maze test and immunohistochemistry staining of A performed to determine the stages of the disease in this mouse model. The protein expression differences were analyzed by label-free shotgun proteomics analysis. RESULTS: A total of 251, 213 and 238 proteins were identified in samples obtained from CA1 regions of mice at 3, 6 and 12 months of age, respectively. Of these, 68, 41 and 33 proteins showed statistical significance. Pathway analysis based on the unique and common proteins within the groups revealed that several pathways are dysregulated during different stages of AD. The alterations in glucose and lipid metabolisms respectively in pre-pathologic and progression stages of the disease, lead to imbalances in ROS production via diminished SOD level and impairment of neuronal integrity. CONCLUSION: We conclude that CA1 region-specific proteomic analysis of hippocampal degeneration may be useful in identifying the earliest as well as progressional changes that are associated with Alzheimer's disease.

Neonatal Neurodegeneration in Alzheimer's Disease Transgenic Mouse Model.

Alzheimer's disease (AD) is a progressive disorder characterized by a variety of molecular pathologies causing cortical dementia with a prominent memory deficit. Formation of the pathology, which begins decades before the diagnosis of the disease, is highly correlated with the clinical symptoms. Several proteomics studies were performed using animal models to monitor the alterations of the brain tissue proteome at different stages of AD. However, proteome changes in the brain regions of newborn transgenic mouse model have not been investigated yet. To this end, we analyzed protein expression alterations in cortex, hippocampus and cerebellum of transgenic mice carrying five familial AD mutations (5XFAD) at neonatal day-1. Our results indicate a remarkable difference in protein expression profile of newborn 5XFAD brain with region specific variations. Additionally, the proteins, which show similar expression alteration pattern in postmortem human AD brains, were determined. Bioinformatics analysis showed that the molecular alterations were mostly related to the cell morphology, cellular assembly and organization, and neuroinflammation. Moreover, morphological analysis revealed that there is an increase in neurite number of 5XFAD mouse neurons in vitro. We suggest that, molecular alterations in the AD brain exist even at birth, and perhaps the disease is silenced until older ages when the brain becomes vulnerable.

Early Stage Alterations in CA1 Extracellular Region Proteins Indicate Dysregulation of IL6 and Iron Homeostasis in the 5XFAD Alzheimer's Disease Mouse Model.

In recent years, an increasing number of research papers revealed that the compositional and volumetric alterations in the extracellular matrix are the consequences of aging and may be related to Alzheimer's disease (AD). In this study, we aimed to demonstrate the alterations in hippocampal extracellular fluid proteins in vivo using the 5XFAD mouse model. Samples were obtained from hippocampi of 5XFAD mice (n = 6) and their non-transgenic littermates by intracerebral push-pull perfusion technique at 3 months of age, representing the pre-pathological stage of the AD. Proteins in the hippocampal perfusates were analyzed by Ultra Performance Liquid Chromatography-Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry (UPLC-ESI-qTOF-MS/MS). 178 proteins were identified and 19 proteins of them were found to be statistically significantly altered (p≤0.05, fold change ≥40%, unique peptide count ≥3) in the hippocampal CA1 extracellular fluid of the 5XFAD mouse model. Ingenuity pathway analysis of the protein expression results identified IL6 as an upstream regulator. The upregulation of IL6 was validated by immunohistochemical staining of the hippocampus and cortex of the 5XFAD mice prior to Aß plaque formation. Furthermore, the iron level in the hippocampus was measured by inductively coupled plasma-mass spectrometry as IL6 is mentioned in several studies to take part in iron homeostasis and inflammation and found to be increased in 5XFAD mice hippocampus. Alterations in extracellular matrix proteins in addition to increasing amount of hippocampal IL6 and iron in the early stages of AD may reveal inflammation-mediated iron dyshomeostasis in the early stages of neurodegeneration.

Comparison of the effects of radiofrequency tissue ablation, CO2 laser ablation, and partial turbinectomy applications on nasal mucociliary functions.

OBJECTIVES: One of the major causes of chronic nasal airway obstruction is disease of the inferior turbinate. However, there is no agreement on how to deal with this problem. Comparison was made of the nasal functions after treatment by radiofrequency tissue ablation, laser ablation, and partial turbinectomy using subjective symptom scores and objective tests. STUDY DESIGN: Prospective, randomized clinical trial. METHODS: The study was conducted on three groups of 45 adult volunteer patients with symptoms and signs of nasal obstruction and stuffiness related to enlarged turbinates. In group A, laser ablation was applied to the inferior turbinate on one side and partial turbinectomy to the inferior turbinate on the other side. In group B, radiofrequency tissue ablation was applied to the inferior turbinate on one side and partial turbinectomy to the inferior turbinate on the other side. In group C, patients who were not treated by any surgical techniques were the control subjects. Clinical examinations, visual analogue scales, rhinomanometry, and isotopic study of nasal mucociliary transport time were used to assess treatment outcomes. RESULTS: At 12 weeks after surgery, the nasal mucociliary transport time results were compared in the same patients. The average time was 25.60 minutes on the side where laser ablation was applied and 11.40 minutes on the side where partial turbinectomy (PT) was applied. In the patients on whom radiofrequency tissue ablation and partial turbinectomy were applied, the average nasal mucociliary transport time was 10.33 minutes on the radiofrequency tissue ablation side, whereas it was 11.33 minutes on the partial turbinectomy side. Rhinomanometric measurements demonstrated a significant decrease in nasal resistances at 12 weeks in both sides in groups A and B. CONCLUSIONS: In the study it was demonstrated that radiofrequency tissue ablation to the turbinate is effective in improving nasal obstruction objectively and in preserving nasal mucociliary function. Laser ablation of the turbinate is effective in improving the nasal obstruction; however, it disturbs the mucociliary function significantly. With the partial turbinectomy technique, results obtained were similar to the results with the radiofrequency tissue ablation technique.

[Results of radiofrequency tissue ablation]. / Radyofrekans cerrahisi sonuçlarimiz.

OBJECTIVES: We evaluated the effectiveness of radiofrequency tissue ablation in respiratory obstruction due to snoring and turbinate hypertrophy. PATIENTS AND METHODS: Thirty-three patients (9 females, 24 males; mean age 35.3 years; range 19 to 55 years) underwent 38 submucosal probe applications in the inferior turbinate (n = 15), soft palate (n = 13), and both (n = 5). Applications were performed in four regions of the soft palate, and in the anterior, medial, or posterior regions of the inferior turbinate depending on the hypertrophic site. Evaluations were made prior to, in the second month, and at the end of the first year of treatment. Snoring was evaluated using the Epworth Sleepiness Scale and subjective assessment. Respiration was evaluated by a visual analog scale. Maximum follow-up period was one year. RESULTS: Subjective evaluations made at the end of the first year showed that snoring decreased in 44.5% and disappeared in 50%. No change was observed in 5.5%. According to the patients' subjective evaluations and clinical examination findings complaints of obstruction decreased by 65.4% and 45.61%, respectively. No significant complications occurred. CONCLUSION: Radiofrequency tissue ablation may prove to be useful in appropriate patients. It is minimally invasive, requires low power and heat, and no general anesthesia, and is associated with low complication risks.

Adaptive optics with pupil tracking for high resolution retinal imaging.

Adaptive optics, when integrated into retinal imaging systems, compensates for rapidly changing ocular aberrations in real time and results in improved high resolution images that reveal the photoreceptor mosaic. Imaging the retina at high resolution has numerous potential medical applications, and yet for the development of commercial products that can be used in the clinic, the complexity and high cost of the present research systems have to be addressed. We present a new method to control the deformable mirror in real time based on pupil tracking measurements which uses the default camera for the alignment of the eye in the retinal imaging system and requires no extra cost or hardware. We also present the first experiments done with a compact adaptive optics flood illumination fundus camera where it was possible to compensate for the higher order aberrations of a moving model eye and in vivo in real time based on pupil tracking measurements, without the real time contribution of a wavefront sensor. As an outcome of this research, we showed that pupil tracking can be effectively used as a low cost and practical adaptive optics tool for high resolution retinal imaging because eye movements constitute an important part of the ocular wavefront dynamics.