Predictive value of α-synuclein expression in peripheral blood of multiple sclerosis patients: A two-dimensional assessment of a selected biomarker.

INTRODUCTION: Our study aimed to evaluate whether assessing α-synuclein expression levels in blood samples could provide a reliable and straightforward alternative to existing diagnostic and prognostic methods for neurodegenerative disorders, including multiple sclerosis (MS). We specifically investigated if α-synuclein and IL-6 expression levels from serum and peripheral blood mononuclear cells (PBMCs) could accurately predict MS severity in patients using a two-dimensional approach. METHODS: We designed a case-control study to analyze the expression of α-synuclein and IL-6 in the peripheral blood of an MS patient group (n = 51) and a control group (n = 51). We statistically evaluated the PBMCs and serum profiles of α-synuclein and IL-6 in MS patients, along with their age of onset, disease duration, tobacco exposure, and Expanded Disability Status Scale (EDSS) score, using SPSS V22.0 software and GraphPad Prism V9.0. RESULTS: Our findings indicate that α-synuclein production was significantly downregulated in MS patients. Principal component analysis also revealed distinct profiles between MS patients and controls. PBMCs and serum profiles of α-synuclein correlated with the EDSS score, suggesting that disease severity can be predicted using α-synuclein profiles. Moreover, α-synuclein showed a significant correlation with IL-6 and age of onset. Lastly, receiver operating characteristic curves of PBMCs and serum activity of α-synuclein profiles displayed discrimination with area under the curve values of 0.856 and 0.705, respectively. CONCLUSION: Our results imply that measuring α-synuclein levels in both serum and PBMCs could be a valuable method for diagnosing and predicting MS severity, potentially serving as a non-invasive biomarker for the disease.

Faulty Metabolism: A Potential Instigator of an Aggressive Phenotype in Cdk5-dependent Medullary Thyroid Carcinoma.

Mechanistic modeling of cancers such as Medullary Thyroid Carcinoma (MTC) to emulate patient-specific phenotypes is challenging. The discovery of potential diagnostic markers and druggable targets in MTC urgently requires clinically relevant animal models. Here we established orthotopic mouse models of MTC driven by aberrantly active Cdk5 using cell-specific promoters. Each of the two models elicits distinct growth differences that recapitulate the less or more aggressive forms of human tumors. The comparative mutational and transcriptomic landscape of tumors revealed significant alterations in mitotic cell cycle processes coupled with the slow-growing tumor phenotype. Conversely, perturbation in metabolic pathways emerged as critical for aggressive tumor growth. Moreover, an overlapping mutational profile was identified between mouse and human tumors. Gene prioritization revealed putative downstream effectors of Cdk5 which may contribute to the slow and aggressive growth in the mouse MTC models. In addition, Cdk5/p25 phosphorylation sites identified as biomarkers for Cdk5-driven neuroendocrine tumors (NETs) were detected in both slow and rapid onset models and were also histologically present in human MTC. Thus, this study directly relates mouse and human MTC models and uncovers vulnerable pathways potentially responsible for differential tumor growth rates. Functional validation of our findings may lead to better prediction of patient-specific personalized combinational therapies.

Protein-Protein Interaction Network Exploration Using Cytoscape.

As the protein-protein interaction (PPI) data increase exponentially, the development and usage of computational methods to analyze these datasets have become a new research horizon in systems biology. The PPI network analysis and visualization can help identify functional modules of the network, pathway genes involved in common cellular functions, and functional annotations of novel genes. Currently, a variety of tools are available for network graph visualization and analysis. Cytoscape, an open-source software tool, is one of them. It provides an interactive visualization interface along with other core features to import, navigate, filter, cluster, search, and export networks. It comes with hundreds of in-built Apps in App Manager to resolve research questions related to network visualization and integration. This chapter aims to illustrate the Cytoscape application to visualize and analyze the PPI network using Arabidopsis interactome-1 main (AI-1MAIN) PPI network dataset from Plant Interactome Database.

Protein-Protein Interaction Network Analysis Using NetworkX.

In recent years, extracting information from biological data has become a particularly valuable way of gaining knowledge. Molecular interaction networks provide a framework for visualizing cellular processes, but their complexity frequently makes their interpretation difficult. Proteins are one of the primary determinants of biological function. Indeed, most biological activities in the living cells are functionally regulated by protein-protein interactions (PPIs). Thus, studying protein interactions is critical for understanding their roles within the cell. Exploring the PPI networks can open new avenues for future experimental studies and offer interspecies predictions for effective interaction mapping. In this chapter we will demonstrate how to construct, visualize, and analyze a protein-protein interaction network using NetworkX.

Building Protein-Protein Interaction Graph Database Using Neo4j.

A cell's various components interact with each other in a coordinated manner to respond to environmental cues and intracellular signals. Compared to the other biological networks, the protein-protein interaction (PPI) is mostly responsible for maintaining signaling pathways. Increasing numbers of experimentally verified and predicted PPIs in plants demand a scalable platform to deal with large and complex datasets. Network/graph data can be organized and analyzed using different tools. This chapter uses Neo4j, a graph database management system, to store and analyze plant PPI networks. To make the graph database and analyze network centrality, we used Arabidopsis interactome-1 main (AI-1MAIN) PPI network.

Mechanism underlying follicular hyperproliferation and oncogenesis in hidradenitis suppurativa.

Hidradenitis suppurativa (HS) is a skin disorder that causes chronic painful inflammation and hyperproliferation, often with the comorbidity of invasive keratoacanthoma (KA). Our research, employing high-resolution immunofluorescence and data science approaches together with confirmatory molecular analysis, has identified that the 5'-cap-dependent protein translation regulatory complex eIF4F is a key factor in the development of HS and is responsible for regulating follicular hyperproliferation. Specifically, eIF4F translational targets, Cyclin D1 and c-MYC, orchestrate the development of HS-associated KA. Although eIF4F and p-eIF4E are contiguous throughout HS lesions, Cyclin D1 and c-MYC have unique spatial localization and functions. The keratin-filled crater of KA is formed by nuclear c-MYC-induced differentiation of epithelial cells, whereas the co-localization of c-MYC and Cyclin D1 provides oncogenic transformation by activating RAS, PI3K, and ERK pathways. In sum, we have revealed a novel mechanism underlying HS pathogenesis of follicular hyperproliferation and the development of HS-associated invasive KA.

The analysis of dynamic gene expression patterns in peripheral blood of multiple sclerosis patients indicates possible diagnostic and prognostic biomarkers.

INTRODUCTION: Among numerous invasive procedures for the research of biomarkers, blood-based indicators are regarded as marginally non-invasive procedures in the diagnosis and prognosis of demyelinating disorders, including multiple sclerosis (MS). In this study, we looked into the blood-derived gene expression profiles of patients with multiple sclerosis to investigate their clinical traits and linked them with dysregulated gene expressions to establish diagnostic and prognostic indicators. METHODS: We included 51 patients with relapsing-remitting MS (RRMS, n = 31), clinically isolated syndrome (CIS, n = 12), primary progressive MS (PPMS, n = 8) and a control group (n = 51). Using correlational analysis, the transcriptional patterns of chosen gene panels were examined and subsequently related with disease duration and the expanded disease disability score (EDSS). In addition, principal component analysis, univariate regression, and logistic regression analysis were employed to highlight distinct profiles of genes and prognosticate the excellent biomarkers of this illness. RESULTS: Our findings demonstrated that neurofilament light (NEFL), tumor necrosis factor α (TNF-α), Tau, and clusterin (CLU) were revealed to be increased in recruited patients, whereas the presenilin-1 (PSEN1) and cell-surface glycoprotein-44 (CD44) were downregulated. Principal Component Analysis revealed distinct patterns between the MS and control groups. Correlation analysis indicated co-dependent dysregulated genes and their differential expression with clinical findings. Furthermore, logistic regression demonstrated that Clusterin (AUC=0.940), NEFL (AUC=0.775), TNF-α (AUC=0.817), Tau (AUC=0.749), PSEN1 (AUC=0.6913), and CD44 (AUC=0.832) had diagnostic relevance. Following the univariate linear regression, a significant regression equation was found between EDSS and IGF-1 (R2 adj = 0.10844; p= 0.0060), APP (R2 adj = 0.1107; p= 0.0098), and PSEN1 (R2 adj = 0.1266; p=0.0102). CONCLUSION: This study exhibits dynamic gene expression patterns that represent the significance of specified genes that are prospective diagnostic and prognostic biomarkers for multiple sclerosis.

Inference of Gene Regulatory Network from Single-Cell Transcriptomic Data Using pySCENIC.

With the advent of recent next-generation sequencing (NGS) technologies in genomics, transcriptomics, and epigenomics, profiling single-cell sequencing became possible. The single-cell RNA sequencing (scRNA-seq) is widely used to characterize diverse cell populations and ascertain cell type-specific regulatory mechanisms. The gene regulatory network (GRN) mainly consists of genes and their regulators-transcription factors (TF). Here, we describe the lightning-fast Python implementation of the SCENIC (Single-Cell reEgulatory Network Inference and Clustering) pipeline called pySCENIC. Using single-cell RNA-seq data, it maps TFs onto gene regulatory networks and integrates various cell types to infer cell-specific GRNs. There are two fast and efficient GRN inference algorithms, GRNBoost2 and GENIE3, optionally available with pySCENIC. The pipeline has three steps: (1) identification of potential TF targets based on co-expression; (2) TF-motif enrichment analysis to identify the direct targets (regulons); and (3) scoring the activity of regulons (or other gene sets) on single cell types.

Fixed Dose Single Tablet Formulation with Differential Release of Amlodipine Besylate and Simvastatin and Its Pharmacokinetic Profile: QbD and Risk Assessment Approach.

PURPOSE: A differential release fixed dose matrix tablet of amlodipine besylate (AML-B) and simvastatin (SIM) was formulated to enhance patient compliance. MATERIAL AND METHOD: In the first phase, release controlling parameters of AML-B and SIM granules were identified and in the second phase a fixed dose AML-B and SIM tablet formulation was prepared and optimized for a differential release of the drugs using a quality by design (QbD) and risk assessment approach. A validated HPLC method was employed for simultaneous determination of AML-B and SIM for FDC formulation. A pharmacokinetics of the above drugs was studied in healthy dogs in the third phase. RESULTS: In QbD-based optimized formulation, Eudragit® RSPO-dicalcium phosphate (DCP) blend controlled the release of AML-B over 8 h, though this diffusion-controlled release assumed first order kinetics. DCP and Eudragit® RS 100 also retarded release of SIM causing SIM release over 8 h after AML-B release from the optimized FDC tablet formulation. The HPLC retention times of AML-B and SIM were 2.10 and 15.52 min, respectively. Linearity for AML-B was 5.0-50 ng/mL and 0.01-2.0 µg/mL for SIM with percent recoveries of 92.85-101.53% and 94.51-117.75% for AML-B and SIM. AUC0-∞ of AML-B was increased 3 fold, while AUC0-∞ of SIM was decreased 2 fold. The tmax values for AML-B and SIM were 12 and 6 h, respectively. AML-B was absorbed without any lag time (tlag) while tlag was 6.33 ± 0.81 h for SIM, thus met the study objective. CONCLUSION: The pharmacokinetic study showed an immediate absorption of AML-B while that of SIM was withheld for 6 h, close to the desired delay time of 8 h.

Patched1 haploinsufficiency severely impacts intermediary metabolism in the skin of Ptch1+/-/ODC transgenic mice.

The study of dominantly heritable cancers has provided insights about tumor development. Gorlin syndrome (GS) is an autosomal dominant disorder wherein affected individuals develop multiple basal cell carcinomas (BCCs) of the skin. We developed a murine model of Ptch1 haploinsufficiency on an ornithine decarboxylase (ODC) transgenic background (Ptch1+/-/ODCt/C57BL/6) that is more sensitive to BCCs growth as compared with Ptch1+/+/ODCt/C57BL/6 littermates. Ptch1+/-/ODCt/C57BL/6 mice show an altered metabolic landscape in the phenotypically normal skin, including restricted glucose availability, restricted ribose/deoxyribose flow and NADPH production, an accumulation of α-ketoglutarate, aconitate, and citrate that is associated with reversal of the tricarboxylic acid cycle, coupled with increased ketogenic/lipogenic activity via acetyl-CoA, 3-hydroybutyrate, and cholesterol metabolites. Also apparent was an increased content/acetylation of amino-acids, glutamine and glutamate, in particular. Accordingly, metabolic alterations due to a single copy loss of Ptch1 in Ptch1+/-/ODCt/C57BL/6 heterozygous mice may provide insights about the cancer prone phenotype of BCCs in GS patients, including biomarkers/targets for early intervention.

Frequency of non-motor symptoms in Parkinson's disease presenting to tertiary care centre in Pakistan: an observational, cross-sectional study.

OBJECTIVE: To determine the frequency of non-motor symptoms (NMS) in patients of Parkinson's disease (PD) presenting to a movement disorder clinic at a tertiary care centre in Pakistan, and how frequency of NMS is different in male and female patients. STUDY DESIGN: Observational, cross-sectional study. SETTING: Tertiary care centre. PARTICIPANTS: Out of 102 patients, 85 were included. Inclusion criteria were patients with PD diagnosed according to the UK Parkinson's Disease Society Brain Bank Clinical Diagnostic Criteria, age ≥18 years, able to give consent and have no difficulty in answering questions. Exclusion criteria were diseases that resemble PD, stroke, dementia, patients unable to provide information and history of antipsychotic use. RESULTS: The NMSQuest revealed a mean of nearly seven different NMS per patient. Autonomic problems such as constipation (56%) and nocturia (49%) were the most common NMS, while urinary urgency was reported by 35% of patients. Low mood and feeling sad were reported by 47%, whereas feeling anxious/panicky was reported by 36%. Problem with memory was reported by 45% of patients. Feeling of light-headedness and dizziness was reported by 40% of patients. Problems with sexual relationship were reported by 30% of patients. The most common sleep problem was difficulty falling sleep (29%). Pain not related to the musculoskeletal system was reported by 30% of patients. Loss or change in the ability to taste or smell was reported by 29% of patients. The rest of NMS were less than 25% in frequency. Feeling sad or blue, feeling light-headed/dizzy, unexplained pain, unpleasant sensations in the legs, difficulty in swallowing and faecal incontinence were more common in female participants, while problems with sex were more common in male participants. CONCLUSION: NMS are quite prevalent in PD in our population. Certain NMS are more common in women as compared with men. There is a need for a large-scale study to look for the association of different NMS with sex.

A novel mutation in ALS2 associated with severe and progressive infantile onset of spastic paralysis.

Infantile onset ascending spastic paralysis (IAHSP) is a type of recessively inherited spastic paraplegia. We investigated the clinical and genetic cause of a recessively inherited disorder in two siblings manifesting severe spasticity in the lower limbs which hindered their gait. A novel homozygous nonsense mutation c.1918 C > T (p.Arg640*) was identified after whole-exome sequencing within ALS2 in the DNA of both patients. The obligate carriers were heterozygous for the mutation and other unaffected members were homozygous for the wild type allele. The variant was absent from 100 control chromosomes and all public databases. This report extends the allelic heterogeneity of ALS2 mutations and emphasizes the importance of genetic testing for diagnosis of pediatric disorders.

Identification of Norway Spruce MYB-bHLH-WDR Transcription Factor Complex Members Linked to Regulation of the Flavonoid Pathway.

Transcription factors (TFs) forming MYB-bHLH-WDR complexes are known to regulate the biosynthesis of specialized metabolites in angiosperms through an intricate network. These specialized metabolites participate in a wide range of biological processes including plant growth, development, reproduction as well as in plant immunity. Studying the regulation of their biosynthesis is thus essential. While MYB (TFs) have been previously shown to control specialized metabolism (SM) in gymnosperms, the identity of their partners, in particular bHLH or WDR members, has not yet been revealed. To gain knowledge about MYB-bHLH-WDR transcription factor complexes in gymnosperms and their regulation of SW, we identified two bHLH homologs of AtTT8, six homologs of the MYB transcription factor AtTT2 and one WDR ortholog of AtTTG1 in Norway spruce. We investigated the expression levels of these genes in diverse tissues and upon treatments with various stimuli including methyl-salicylate, methyl-jasmonate, wounding or fungal inoculation. In addition, we also identified protein-protein interactions among different homologs of MYB, bHLH and WDR. Finally, we generated transgenic spruce cell lines overexpressing four of the Norway spruce AtTT2 homologs and observed differential regulation of genes in the flavonoid pathway and flavonoid contents.

Aneurysmal Subarachnoid Hemorrhage: Outcome of Aneurysm Clipping Versus Coiling in Anterior Circulation Aneurysm.

OBJECTIVE: To compare the neurological outcome of microsurgical clipping versus coiling in patients with anterior circulation aneurysm. STUDY DESIGN: Comparative study. PLACE AND DURATION OF STUDY: Department of Neurosurgery, Lahore General Hospital, Lahore, from January 2010 to December 2013. METHODOLOGY: Patients aged 14 - 60 years, with ruptured cerebral aneurysm of anterior circulation and World Federation of Neurosurgical Society (WFNS) grades 1, 2 and 3 were included. Patients more than 60 years, medically unfit patient and posterior circulation aneurysms and WFNS grades 4 and 5 were excluded. Aneurysm sac obliteration was done in randomized manner with microsurgical clipping or coiling. Postoperatively, the patients were assessed and followed-up upto one year for outcome parameters on the bases of WFNS grade and Modified Ranking Scale (mRS) as favourable (mRS ≤2 ) and unfavourable (mRS > 2). RESULTS: Among 140 subjects selected for study, 70 were included in group A, i.e. coiling and other 70 were in group B, i.e. clipping. The median age of patients in group A was 52.5 ± 10 years and in group B was 51.00 ± 10 years. Overall, 56 (40%) males, 28 (60%) males in each group; and 84 (60%) females, 42 (60%) in each group were included. The male to female ratio in this study was 1:1.5. In group A, i.e. coiling, 27 (38.6%) patients had no disability (grades 1 and 2), 25 (35.7%) were slightly disabled (grade 3) and 18 (25.7%) had moderate disability (grade 4); whereas in group B, i.e. clipping group 23 (32.9%) patients had no disability (grades 1 and 2), 23 (32.9%) were slightly disabled (grade 3) and 24 (34.3%) had moderate disability (grade 4). At one year follow-up, in group A, favourable outcome was achieved in 56 (80%) of patients compared to 48 (68.6%) in group B; whilst, 14 (20%) patients in group Aand 22 (33.1%) in group B showed unfavourable outcome. Although mortality rate was higher in clipping (n=3, 4.3%) as compared to coiling (n=1, 1.4%), but was not statistically significant (p = 0.310). CONCLUSION: Endovascular coiling of anterior circulation aneurysms is safe and as effective and successful as aneurysm clipping and is less invasive also.