Saima Zafar
Georg-August-Universität Göttingen, Neurologie, Faculty Member
- Dr. Saima Zafar directs a large interdisciplinary neuro-proteomics research projects primarily centered on understand... moreDr. Saima Zafar directs a large interdisciplinary neuro-proteomics research projects primarily centered on understanding the patho-physiology of rapid progressive brain disorders such as Prion, Alzheimer’s, and Parkinson's disease as well as the mechanisms underlying the early disease biomarkers and therapeutic targets of deep brain stimulation. Much of my work focuses on the ultimate translation of basic laboratory research discoveries into clinical treatment options for affected patients in order to reduce symptoms, minimize side effects and enhance function and quality of life.My impending research plans are to develop early neurological disease biomarkers and detection of therapeutic targets and enhance new discoveries and their meaningful translation from “bench to bedside.”edit
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Recent advances in understanding of the molecular biology of prion diseases and improved clinical diagnostic techniques might allow researchers to think about therapeutic trials in Creutzfeldt-Jakob disease (CJD) patients. Some attempts... more
Recent advances in understanding of the molecular biology of prion diseases and improved clinical diagnostic techniques might allow researchers to think about therapeutic trials in Creutzfeldt-Jakob disease (CJD) patients. Some attempts have been made in the past and various compounds have been tested in single case reports and patient series. Controlled trials are rare. However, in the past few years, it has been demonstrated that clinical trials are feasible. The clinicians might face several specific problems when evaluating the efficacy of the drug in CJD, such as rareness of the disease, lack of appropriate preclinical tests and heterogeneous clinical presentation in humans. These problems have to be carefully addressed in future.
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β-glucocerebrosidase (GBA)-associated mutations are a significant risk factor for Parkinson’s disease (PD) that aggravate the disease pathology by upregulating the deposition of α-Synuclein (α-Syn). The resultant clinical profile varies... more
β-glucocerebrosidase (GBA)-associated mutations are a significant risk factor for Parkinson’s disease (PD) that aggravate the disease pathology by upregulating the deposition of α-Synuclein (α-Syn). The resultant clinical profile varies for PD patients without GBA mutations. The current study aimed to identify the proteomic targets involved in the pathogenic pathways leading to the differential clinical presentation of GBA-associated PD. CSF samples (n = 32) were obtained from PD patients with GBA mutations (n = 22), PD patients without GBA mutations (n = 7), and healthy controls that were carriers of GBA mutations (n = 3). All samples were subjected to in-gel tryptic digestion followed by the construction of the spectral library and quantitative SWATH-based analysis. CSF α-Syn levels were reduced in both PDIdiopathic and PDGBA cases. Our SWATH-based mass spectrometric analysis detected 363 proteins involved in immune response, stress response, and cell signaling in various groups. ...
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Additional file 6. The spreadsheet contains the raw proteomics data (unique peptide counts data) of HDFs, and subsequent normalized data along with the results of pairwise statistical testing (Welch's t-test).
Cathepsin S and GAPDH levels in lysosomal and cytoplasmic enriched fractions derived from prion protein peptide treatment. (PPTX 88 kb)
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Calpain substrates levels in sCJD. (A) qPCR analysis of CAPNS1/CAPN4 in the frontal cortex and cerebellum of controls and sCJD MM and VV2 cases. (B) Western-blot and densitometry analysis of Neurofilament Light (NF-L) and γ-Tubulin in the... more
Calpain substrates levels in sCJD. (A) qPCR analysis of CAPNS1/CAPN4 in the frontal cortex and cerebellum of controls and sCJD MM and VV2 cases. (B) Western-blot and densitometry analysis of Neurofilament Light (NF-L) and γ-Tubulin in the frontal cortex and cerebellum of control, sCJD MM1 and sCJD VV2 cases. ANOVA test followed by post-test Tukey's Multiple Comparison Test was used to compare the values from different groups. P values for the comparisons of the three groups are indicated in the figure:*p
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Partial UPR activation in the frontal cortex of sCJD cases. (A) Western-blot and densitometric analysis of UPR proteins CHOP, ATF4, P-IRE-1, IRE-1, XBP1 and ATF6 in the frontal cortex of control and sCJD MM1 cases. (B) Immunohistochemical... more
Partial UPR activation in the frontal cortex of sCJD cases. (A) Western-blot and densitometric analysis of UPR proteins CHOP, ATF4, P-IRE-1, IRE-1, XBP1 and ATF6 in the frontal cortex of control and sCJD MM1 cases. (B) Immunohistochemical detection of CHOP in the frontal cortex of control and sCJD MM1 cases. (B) Immunohistochemical detection of CHOP in the cortex of control and sCJD MM1 inoculated tg340-PRNP129MM mice. Brain slices were counterstained with DAPI. (PPTX 4 kb)
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Alzheimer’s disease is the most prevalent neurodegenerative disorder leading to progressive cognitive decline. Despite decades of research, understanding AD progression at the molecular level, especially at its early stages, remains... more
Alzheimer’s disease is the most prevalent neurodegenerative disorder leading to progressive cognitive decline. Despite decades of research, understanding AD progression at the molecular level, especially at its early stages, remains elusive. Here, we identified several presymptomatic AD markers by investigating brain proteome changes over the course of neurodegeneration in a transgenic mouse model of AD (3×Tg-AD). We show that one of these markers, heme-binding protein 1 (Hebp1), is elevated in the brains of both 3×Tg-AD mice and patients affected by rapidly-progressing forms of AD. Hebp1, predominantly expressed in neurons, interacts with the mitochondrial contact site complex (MICOS) and exhibits a perimitochondrial localization. Strikingly, wildtype, but not Hebp1-deficient, neurons showed elevated cytotoxicity in response to heme-induced apoptosis. Increased survivability in Hebp1-deficient neurons is conferred by blocking the activation of the mitochondrial-associated caspase s...
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Cerebellar damage and granular and Purkinje cell loss in sporadic Creutzfeldt-Jakob disease (sCJD) highlight a critical involvement of the cerebellum during symptomatic progression of the disease. In this project, global proteomic... more
Cerebellar damage and granular and Purkinje cell loss in sporadic Creutzfeldt-Jakob disease (sCJD) highlight a critical involvement of the cerebellum during symptomatic progression of the disease. In this project, global proteomic alterations in the cerebellum of brain from the two most prevalent subtypes (MM1 and VV2) of sCJD were studied. Two-dimensional gel electrophoresis (2DE) coupled mass spectrometric identification revealed 40 proteins in MM1 and 43 proteins in VV2 subtype to be differentially expressed. Of those, 12 proteins showed common differential expression in their expression between two subtypes. Differentially expressed proteins mainly belonged to (i) cell cycle, gene expression and cell death; (ii) cellular stress response/oxidative stress (OS) and (iii) signal transduction and synaptic functions, related molecular functions. We verified 10 differentially expressed proteins at transcriptional and translational level as well. Interestingly, protein deglycase DJ-1 (a...
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Cerebrospinal fluid (CSF) biomarkers are routinely used for the differential diagnosis of rapidly progressive dementia, but are also affected by patients' characteristics. To assess if stratification by age, sex, and genetic risk... more
Cerebrospinal fluid (CSF) biomarkers are routinely used for the differential diagnosis of rapidly progressive dementia, but are also affected by patients' characteristics. To assess if stratification by age, sex, and genetic risk factors improves the accuracy of cerebrospinal fluid (CSF) biomarkers in patients with rapidly progressive dementia. 1,538 individuals with sporadic Creutzfeldt-Jakob disease (CJD), 173 with classic Alzheimer's disease (cAD), 37 with rapidly progressive Alzheimer's disease (rpAD), and 589 without signs of dementia were included in this retrospective diagnostic study. The effect of age, sex, PRNP codon 129, and APOE genotype on CSF levels of tau, p-tau, Aβ1-42, and Aβ1-40 values measured at time of diagnostic work-up was assessed. Tau was a better marker for the differentiation of CJD and rpAD in older (AUC:0.97; 95% CI:0.96-1.00) than in younger (AUC:0.91; 95% CI:0.87-0.94) patients as tau levels increased with age in CJD patients, but not in rp...
Research Interests: Cognitive Science, Dementia, Biomarkers, Medicine, Cerebrospinal Fluid, and 15 moreHumans, Internal Medicine, Female, Male, Differential Diagnosis, Apolipoprotein E, Clinical Sciences, Aged, Middle Aged, Genotype, Retrospective Studies, Disease Progression, Alzheimer Disease, Neurosciences, and PRNP
In vitro amplification assays, such as real-time quaking-induced conversion (RT-QuIC) are used to detect aggregation activity of misfolded prion protein (PrP) in brain, cerebrospinal fluid (CSF) and urine samples from patients with a... more
In vitro amplification assays, such as real-time quaking-induced conversion (RT-QuIC) are used to detect aggregation activity of misfolded prion protein (PrP) in brain, cerebrospinal fluid (CSF) and urine samples from patients with a prion disease. We believe that the method also has a much broader application spectrum. In the present study, we applied RT-QuIC as a pre-screening test for substances that potentially inhibit the aggregation process of the cellular PrP (PrPC) to proteinase (PK)-resistant PrPres. We chose doxycycline as the test substance as it has been tested successfully in animal models and proposed in clinical studies as a therapeutic for prion diseases. The RT-QuIC-reaction was seeded with brain tissue or CSF from sCJD patients and doxycycline was then added in different concentrations as well as at different time points. In both experiments, we observed a dose- and time-dependent inhibition of the RT-QuIC seeding response and a decrease of PK resistant PrPres when...
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There is an increasing demand for the understanding of pathophysiology on neurodegeneration diseases at early stages. Changes in endocytic machinery and the cytoskeleton-associated response are the first alterations observed in... more
There is an increasing demand for the understanding of pathophysiology on neurodegeneration diseases at early stages. Changes in endocytic machinery and the cytoskeleton-associated response are the first alterations observed in Creutzfeldt-Jakob disease (CJD) and Alzheimer's disease AD brain. In this study, we performed a targeted search for endocytic pathway proteins in the different regions of the brain. We found late endosome marker Rab7a which was significantly upregulated in the frontal cortex region in the rapid progressive CJD form (MM1) and rapid progressive AD (rpAD) forms. However, Rab9 expression was significantly downregulated only in CJD-MM1 brain frontal cortex region. In the cerebellum, Rab7a expression showed significant upregulation in both subtype MM1 and VV2 CJD forms, in contrast to Rab9 which showed significant downregulation in both subtype MM1 and VV2 CJD forms at terminal stage of the disease. To check regulatory response at pre-symptomatic stage of the d...
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Although a physiological function of the cellular prion protein (PrP(c)) is still not fully clarified a PrP(c)-mediated neuroprotection against hypoxic/ischemic insult is intriguing. After ischemic stroke prion knockout mice (Prnp(0/0))... more
Although a physiological function of the cellular prion protein (PrP(c)) is still not fully clarified a PrP(c)-mediated neuroprotection against hypoxic/ischemic insult is intriguing. After ischemic stroke prion knockout mice (Prnp(0/0)) display significantly greater lesions as compared to wild-type (WT) mice. Earlier reports suggested an interaction between the glycolytic enzyme lactate dehydrogenase (LDH) and PrP(c). Since hypoxic environment enhances LDH expression levels and compels neurons to rely on lactate as an additional oxidative substrate for energy metabolism, we examined possible differences in LDH protein expression in WT and Prnp(0/0) knockout models under normoxic/hypoxic conditions in vitro and in vivo, as well as in a HEK293 cell line. While no differences are observed under normoxic conditions, LDH expression is markedly increased after 60-min and 90-min of hypoxia in WT vs. Prnp(0/0) primary cortical neurons with concurrent less hypoxia-induced damage in the forme...
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Regulatory enzymes, which facilitate the transferring of phosphate groups to the specific substrates, are called kinases. Protein kinases are the numerous groups of kinases, which involve in the regulation of cell function and further... more
Regulatory enzymes, which facilitate the transferring of phosphate groups to the specific substrates, are called kinases. Protein kinases are the numerous groups of kinases, which involve in the regulation of cell function and further modification. In prokaryotes and eukaryotes, several protein kinases exert specific and reversible control on protein phosphorylation. Protein kinases are classified both by the type of amino acid they phosphorylate in the protein target and by their location in the cell. Biochemically, phosphorylation distinguishes five different groups, that is, histidine, serine, threonine or tyrosine, and dual-specificity kinases. The majority of eukaryotic kinases are serine/threonine-specific protein kinases. Tyrosine-specific protein kinases are used in signal transduction processes in eukaryotes and a low level of activity is detectable in yeast. Protein kinases are established in cytosol and in the plasma membrane. Ser/Thr protein kinases and the dual-specificity kinases are most often cytosolic. Most tyrosine kinases are found on the plasma membrane. Protein kinases have profound effects on a cell with highly regulated activity by turning on and off and by binding of proteins with advantage as regulatory strategy. Regulation of protein kinases is ubiquitous in eukaryotic development, physiology, and metabolism and deregulation is a common cause of disease, particularly cancer.
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Proteinopathy refers to a group of disorders defined by depositions of amyloids within living tissue. Neurodegenerative proteinopathies, including Alzheimer’s disease, Parkinson’s disease, Creutzfeldt–Jakob disease, and others, constitute... more
Proteinopathy refers to a group of disorders defined by depositions of amyloids within living tissue. Neurodegenerative proteinopathies, including Alzheimer’s disease, Parkinson’s disease, Creutzfeldt–Jakob disease, and others, constitute a large fraction of these disorders. Amyloids are highly insoluble, ordered, stable, beta-sheet rich proteins. The emerging theory about the pathophysiology of neurodegenerative proteinopathies suggests that the primary amyloid-forming proteins, also known as the prion-like proteins, may exist as multiple proteoforms that contribute differentially towards the disease prognosis. It is therefore necessary to resolve these disorders on the level of proteoforms rather than the proteome. The transient and hydrophobic nature of amyloid-forming proteins and the minor post-translational alterations that lead to the formation of proteoforms require the use of highly sensitive and specialized techniques. Several conventional techniques, like gel electrophore...
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The molecular determinants of atypical clinical variants of Alzheimer’s disease, including the recently discovered rapidly progressive Alzheimer’s disease (rpAD), are unknown to date. Fibrilization of the amyloid-β (Aβ) peptide is the... more
The molecular determinants of atypical clinical variants of Alzheimer’s disease, including the recently discovered rapidly progressive Alzheimer’s disease (rpAD), are unknown to date. Fibrilization of the amyloid-β (Aβ) peptide is the most frequently studied candidate in this context. The Aβ peptide can exist as multiple proteoforms that vary in their post-translational processing, amyloidogenesis, and toxicity. The current study was designed to identify these variations in Alzheimer’s disease patients exhibiting classical (sAD) and rapid progression, with the primary aim of establishing if these variants may constitute strains that underlie the phenotypic variability of Alzheimer’s disease. We employed two-dimensional polyacrylamide gel electrophoresis and MALDI-ToF mass spectrometry to validate and identify the Aβ proteoforms extracted from targeted brain tissues. The biophysical analysis was conducted using RT-QuIC assay, confocal microscopy, and atomic force microscopy. Interact...
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Background High-density oligomers of the prion protein (HDPs) have previously been identified in brain tissues of patients with rapidly progressive Alzheimer’s disease (rpAD). The current investigation aims at identifying interacting... more
Background High-density oligomers of the prion protein (HDPs) have previously been identified in brain tissues of patients with rapidly progressive Alzheimer’s disease (rpAD). The current investigation aims at identifying interacting partners of HDPs in the rpAD brains to unravel the pathological involvement of HDPs in the rapid progression. Methods HDPs from the frontal cortex tissues of rpAD brains were isolated using sucrose density gradient centrifugation. Proteins interacting with HDPs were identified by co-immunoprecipitation coupled with mass spectrometry. Further verifications were carried out using proteomic tools, immunoblotting, and confocal laser scanning microscopy. Results We identified rpAD-specific HDP-interactors, including the growth arrest specific 2-like 2 protein (G2L2). Intriguingly, rpAD-specific disturbances were found in the localization of G2L2 and its associated proteins i.e., the end binding protein 1, α-tubulin, and β-actin. Discussion The results show t...
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Internet of Things (IoT) is expected to play a major role in our lives through pervasive systems of sensor networks encompassing our environment. These systems are designed to monitor vital physical phenomena generating data which can be... more
Internet of Things (IoT) is expected to play a major role in our lives through pervasive systems of sensor networks encompassing our environment. These systems are designed to monitor vital physical phenomena generating data which can be transmitted and saved at cloud from where this information can be accessed through applications and further actions can be taken. This paper presents the implementation and results of an environmental monitoring system which employs sensors for temperature and humidity of the surrounding area. This data can be used to trigger short term actions such as remotely controlling heating or cooling devices or long term statistics. The sensed data is uploaded to cloud storage and an Android application accesses the cloud and presents the results to the end users. The system employs Arduino UNO board, DHT11 sensor, ESP8266 Wi-Fi module, which transmits data to open IoT API service ThingSpeak where it is analyzed and stored. An Android application is develope...
Dysfunctional RNA-binding proteins (RBPs) have been implicated in several neurodegenerative disorders. Recently, this paradigm of RBPs has been extended to pathophysiology of Alzheimer’s disease (AD). Here, we identified disease subtype... more
Dysfunctional RNA-binding proteins (RBPs) have been implicated in several neurodegenerative disorders. Recently, this paradigm of RBPs has been extended to pathophysiology of Alzheimer’s disease (AD). Here, we identified disease subtype specific variations in the RNA-binding proteome (RBPome) of sporadic AD (spAD), rapidly progressive AD (rpAD), and sporadic Creutzfeldt Jakob disease (sCJD), as well as control cases using RNA pull-down assay in combination with proteomics. We show that one of these identified proteins, splicing factor proline and glutamine rich (SFPQ), is downregulated in the post-mortem brains of rapidly progressive AD patients, sCJD patients and 3xTg mice brain at terminal stage of the disease. In contrast, the expression of SFPQ was elevated at early stage of the disease in the 3xTg mice, and in vitro after oxidative stress stimuli. Strikingly, in rpAD patients’ brains SFPQ showed a significant dislocation from the nucleus and cytoplasmic colocalization with TIA-...
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Background α-Synuclein is a small soluble protein, whose physiological function in the healthy brain is poorly understood. Intracellular inclusions of α-synuclein, referred to as Lewy bodies (LBs), are pathological hallmarks of... more
Background α-Synuclein is a small soluble protein, whose physiological function in the healthy brain is poorly understood. Intracellular inclusions of α-synuclein, referred to as Lewy bodies (LBs), are pathological hallmarks of α-synucleinopathies, such as Parkinson’s disease (PD) or dementia with Lewy bodies (DLB). Main body Understanding of the molecular basis as well as the factors or conditions promoting α-synuclein misfolding and aggregation is an important step towards the comprehension of pathological mechanism of α-synucleinopathies and for the development of efficient therapeutic strategies. Based on the conversion and aggregation mechanism of α-synuclein, novel diagnostic tests, such as protein misfolding seeded conversion assays, e.g. the real-time quaking-induced conversion (RT-QuIC), had been developed. In diagnostics, α-synuclein RT-QuIC exhibits a specificity between 82 and 100% while the sensitivity varies between 70 and 100% among different laboratories. In addition...
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Background: Thyroid is a highly vascular gland and hemostasis is a key limiting factor in morbidity and mortality in thyroid surgery. Hemostasis has always been a challenge and various techniques have been used. Vessel sealers like... more
Background: Thyroid is a highly vascular gland and hemostasis is a key limiting factor in morbidity and mortality in thyroid surgery. Hemostasis has always been a challenge and various techniques have been used. Vessel sealers like Ligasure are now gaining popularity. Objectives: The objective of this study is to compare thyroidectomy by Ligasure with conventional technique in terms of mean operative time and post-operative blood loss. Study Design: It is a Randomized Control Trial. Setting: Surgical Department of Allied Hospital Faisalabad. Period: Six months (March 2017 to August 2017). Material & Methods: After approval of study from ethical review committee and informed consent, the patients were subjected to complete history and clinical examination in addition to baseline investigations. Total 135 patients of multinodular goiter (MNG) and euthyroid were divided in two groups by simple random sampling. All surgeries were performed under general anesthesia with orotracheal intub...
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High levels of total α-synuclein (t-α-synuclein) in the cerebrospinal fluid (CSF) were reported in sporadic Creutzfeldt-Jakob disease (sCJD). The potential use of t-α-synuclein in the discrimination of Lewy body dementias (i.e.,... more
High levels of total α-synuclein (t-α-synuclein) in the cerebrospinal fluid (CSF) were reported in sporadic Creutzfeldt-Jakob disease (sCJD). The potential use of t-α-synuclein in the discrimination of Lewy body dementias (i.e., Parkinson's disease dementia (PDD) and dementia with Lewy bodies (DLB)) is still under investigation. In addition, phospho-serine-129 α-synuclein (p-α-synuclein) has been described to be slightly increased in the CSF of synucleinopathies. Here, we analyzed t-α-synuclein and p-α-synuclein concentrations and their ratio in the context of differential diagnosis of neurodegenerative diseases. We quantified the levels of CSF t-α-synuclein and p-α-synuclein in a cohort of samples composed of neurological controls (NC), sCJD, PDD, and DLB by means of newly developed specific enzyme-linked immunosorbent assays. T-α-synuclein and p-α-synuclein were specifically elevated in sCJD compared to other disease groups. The area under the curve (AUC) values for t-α-synucl...
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Cerebrospinal fluid (CSF) contains a dynamic and complex mixture of proteins, which reflects physiologic or pathologic states of the central nervous system. Changes in CSF proteome have been described in various neurodegenerative... more
Cerebrospinal fluid (CSF) contains a dynamic and complex mixture of proteins, which reflects physiologic or pathologic states of the central nervous system. Changes in CSF proteome have been described in various neurodegenerative disorders. Earliest publications came from the field of prion disease. Two major approaches have been followed aiming to detect the pathologic form of prion protein (PrPSc) in various peripheral tissues on one hand, but also looking for surrogate parameters as a consequence of the underlying neurodegenerative process. First observations were made using two-dimensional gel electrophoresis for proteins named p130/131, identified as belonging to the 14-3-3 protein family group. This protein became known as the first "wet" biomarker part of clinical diagnostic criteria. Other proteins were identified; most of the work in addition to 14-3-3 has been done on tau/p-tau. The development of PrPSc-based biomarkers was hampered by technical problems and dete...
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Increasing evidence indicates that microRNAs (miRNAs) are contributing factors to neurodegeneration. Alterations in miRNA signatures have been reported in several neurodegenerative dementias, but data in prion diseases are restricted to... more
Increasing evidence indicates that microRNAs (miRNAs) are contributing factors to neurodegeneration. Alterations in miRNA signatures have been reported in several neurodegenerative dementias, but data in prion diseases are restricted to ex vivo and animal models. The present study identified significant miRNA expression pattern alterations in the frontal cortex and cerebellum of sporadic Creutzfeldt-Jakob disease (sCJD) patients. These changes display a highly regional and disease subtype-dependent regulation that correlates with brain pathology. We demonstrate that selected miRNAs are enriched in sCJD isolated Argonaute(Ago)-binding complexes in disease, indicating their incorporation into RNA-induced silencing complexes, and further suggesting their contribution to disease-associated gene expression changes. Alterations in the miRNA-mRNA regulatory machinery and perturbed levels of miRNA biogenesis key components in sCJD brain samples reported here further implicate miRNAs in sCJD...
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This study examines the bioremediation potential and cadmium-induced cellular response on a molecular level in Candida tropicalis 3Aer. Spectroscopic analysis clearly illustrated the involvement of yeast cell wall components in... more
This study examines the bioremediation potential and cadmium-induced cellular response on a molecular level in Candida tropicalis 3Aer. Spectroscopic analysis clearly illustrated the involvement of yeast cell wall components in biosorption. Cadmium bioaccumulation was confirmed by TEM, SEM, and EDX examination. TEM images revealed extracellular as well as cytoplasmic and vacuolar cadmium nanoparticle formation, further validated by presence of ycf1 gene and increased biosynthesis of GSH under cadmium stress. Fourteen proteins exhibited differential expression and during cellular redox homeostasis are found to involve in nitrogen metabolism, nucleotide biosynthesis, and carbohydrate catabolism. Interestingly, C. tropicalis 3Aer is equipped with nitrile hydratase enzyme, rarely been reported in yeast. It has the potential to remove nitriles from the environment. The Cd(+2) toxicity not only caused growth stasis but also upregulated the cysteine biosynthesis, protein folding and cytoplasmic detoxification response elements. The present study suggests that C. tropicalis 3Aer is a potential candidate for bioremediating environmental pollution by Cd(+2).
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The development of in vitro protein misfolding amplification assays for the detection and analysis of abnormally folded proteins, such as proteinase K resistant prion protein (PrP(res)) was a major innovation in the prion field. In prion... more
The development of in vitro protein misfolding amplification assays for the detection and analysis of abnormally folded proteins, such as proteinase K resistant prion protein (PrP(res)) was a major innovation in the prion field. In prion diseases, these types of assays imitate the pathological conversion of the cellular PrP (PrP(C)) into a proteinase resistant associated conformer or amyloid, called PrP(res). Areas covered: The most prominent protein misfolding amplification assays are the protein misfolding cyclic amplification (PMCA), which is based on sonication and the real-time quaking-induced conversion (RT-QuIC) technique based on shaking. The more recently established RT-QuIC is fully automatic and enables the monitoring of misfolded protein aggregates in real-time by using a fluorescent dye. Expert Commentary: RT-QuIC is a very robust and highly reproducible test system which is applicable in diagnosis, prion strain-typing, drug pre-screening and other amyloidopathies.
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Anti-apoptotic properties of physiological and elevated levels of the cellular prion protein (PrP(c)) under stress conditions are well documented. Yet, detrimental effects of elevated PrP(c) levels under stress conditions, such as... more
Anti-apoptotic properties of physiological and elevated levels of the cellular prion protein (PrP(c)) under stress conditions are well documented. Yet, detrimental effects of elevated PrP(c) levels under stress conditions, such as exposure to staurosporine (STS) have also been described. In the present study, we focused on discerning early apoptotic STS-induced proteome and phospho-proteome changes in SH-SY5Y human neuroblastoma cells stably transfected either with an empty or PRNP-containing vector, expressing physiological or supraphysiological levels of PrP(c), respectively. PrP(c)-overexpression per se appears to stress the cells under STS-free conditions as indicated by diminished cell viability of PrP(c)-overexpressing versus control cells. However, PrP(c)-overexpression becomes advantageous following exposure to STS. Thus, only a short exposure (2 h) to 1 μM STS results in lower survival rates and significantly higher caspase-3 activity in control versus PrP(c)-overexpressing...
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A high priority in the prion field is to identify pre-symptomatic events and associated profile of molecular changes. In this study, we demonstrate the pre-symptomatic dysregulation of cytoskeleton assembly and its associated cofilin-1... more
A high priority in the prion field is to identify pre-symptomatic events and associated profile of molecular changes. In this study, we demonstrate the pre-symptomatic dysregulation of cytoskeleton assembly and its associated cofilin-1 pathway in strain and brain region-specific manners in MM1 and VV2 subtype-specific Creutzfeldt-Jakob disease at clinical and pre-clinical stage. At physiological level, PrP(C) interaction with cofilin-1 and phosphorylated form of cofilin (p-cofilin(Ser3)) was investigated in primary cultures of mouse cortex neurons (PCNs) of PrP(C) wild-type and knockout mice (PrP(-/-)). Short-interfering RNA downregulation of active form of cofilin-1 resulted in the redistribution/downregulation of PrP(C), increase of activated form of microglia, accumulation of dense form of F-actin, and upregulation of p-cofilin(Ser3). This upregulated p-cofilin(Ser3) showed redistribution of expression predominantly in the activated form of microglia in PCNs. At pathological leve...
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The analysis of cerebrospinal fluid (CSF) biomarkers gains importance in the differential diagnosis of prion diseases. However, no single diagnostic tool or combination of them can unequivocally confirm prion disease diagnosis.... more
The analysis of cerebrospinal fluid (CSF) biomarkers gains importance in the differential diagnosis of prion diseases. However, no single diagnostic tool or combination of them can unequivocally confirm prion disease diagnosis. Electrochemiluminescence (ECL)-based immunoassays have demonstrated to achieve high diagnostic accuracy in a variety of sample types due to their high sensitivity and dynamic range. Quantification of CSF α-synuclein (a-syn) by an in-house ECL-based ELISA assay has been recently reported as an excellent approach for the diagnosis of sporadic Creutzfeldt-Jakob disease (sCJD), the most prevalent form of human prion disease. In the present study, we validated a commercially available ECL-based a-syn ELISA platform as a diagnostic test for correct classification of sCJD cases. CSF a-syn was analysed in 203 sCJD cases with definite diagnosis and in 445 non-CJD cases. We investigated reproducibility and stability of CSF a-syn and made recommendations for its analysi...