Targeting low levels of MIF expression as a potential therapeutic strategy for ALS.

Mutations in SOD1 cause amyotrophic lateral sclerosis (ALS), a neurodegenerative disease characterized by motor neuron (MN) loss. We previously discovered that macrophage migration inhibitory factor (MIF), whose levels are extremely low in spinal MNs, inhibits mutant SOD1 misfolding and toxicity. In this study, we show that a single peripheral injection of adeno-associated virus (AAV) delivering MIF into adult SOD1G37R mice significantly improves their motor function, delays disease progression, and extends survival. Moreover, MIF treatment reduces neuroinflammation and misfolded SOD1 accumulation, rescues MNs, and corrects dysregulated pathways as observed by proteomics and transcriptomics. Furthermore, we reveal low MIF levels in human induced pluripotent stem cell-derived MNs from familial ALS patients with different genetic mutations, as well as in post mortem tissues of sporadic ALS patients. Our findings indicate that peripheral MIF administration may provide a potential therapeutic mechanism for modulating misfolded SOD1 in vivo and disease outcome in ALS patients.

Mind bomb 2 limits inflammatory dermatitis in Sharpin mutant mice independently of cell death.

Skin inflammation is a complex process implicated in various dermatological disorders. The chronic proliferative dermatitis (cpd) phenotype driven by the cpd mutation (cpdm) in the Sharpin gene is characterized by dermal inflammation and epidermal abnormalities. Tumour necrosis factor (TNF) and caspase-8-driven cell death causes the pathogenesis of Sharpincpdm mice; however, the role of mind bomb 2 (MIB2), a pro-survival E3 ubiquitin ligase involved in TNF signaling, in skin inflammation remains unknown. Here, we demonstrate that MIB2 antagonizes inflammatory dermatitis in the context of the cpd mutation. Surprisingly, the role of MIB2 in limiting skin inflammation is independent of its known pro-survival function and E3 ligase activity. Instead, MIB2 enhances the production of wound-healing molecules, granulocyte colony-stimulating factor, and Eotaxin, within the skin. This discovery advances our comprehension of inflammatory cytokines and chemokines associated with cpdm pathogenesis and highlights the significance of MIB2 in inflammatory skin disease that is independent of its ability to regulate TNF-induced cell death.

Osteopontin depletion in macrophages perturbs proteostasis via regulating UCHL1-UPS axis and mitochondria-mediated apoptosis.

Introduction: Osteopontin (OPN; also known as SPP1), an immunomodulatory cytokine highly expressed in bone marrow-derived macrophages (BMMΦ), is known to regulate diverse cellular and molecular immune responses. We previously revealed that glatiramer acetate (GA) stimulation of BMMΦ upregulates OPN expression, promoting an anti-inflammatory, pro-healing phenotype, whereas OPN inhibition triggers a pro-inflammatory phenotype. However, the precise role of OPN in macrophage activation state is unknown. Methods: Here, we applied global proteome profiling via mass spectrometry (MS) analysis to gain a mechanistic understanding of OPN suppression versus induction in primary macrophage cultures. We analyzed protein networks and immune-related functional pathways in BMMΦ either with OPN knockout (OPNKO) or GA-mediated OPN induction compared with wild type (WT) macrophages. The most significant differentially expressed proteins (DEPs) were validated using immunocytochemistry, western blot, and immunoprecipitation assays. Results and discussion: We identified 631 DEPs in OPNKO or GA-stimulated macrophages as compared to WT macrophages. The two topmost downregulated DEPs in OPNKO macrophages were ubiquitin C-terminal hydrolase L1 (UCHL1), a crucial component of the ubiquitin-proteasome system (UPS), and the anti-inflammatory Heme oxygenase 1 (HMOX-1), whereas GA stimulation upregulated their expression. We found that UCHL1, previously described as a neuron-specific protein, is expressed by BMMΦ and its regulation in macrophages was OPN-dependent. Moreover, UCHL1 interacted with OPN in a protein complex. The effects of GA activation on inducing UCHL1 and anti-inflammatory macrophage profiles were mediated by OPN. Functional pathway analyses revealed two inversely regulated pathways in OPN-deficient macrophages: activated oxidative stress and lysosome-mitochondria-mediated apoptosis (e.g., ROS, Lamp1-2, ATP-synthase subunits, cathepsins, and cytochrome C and B subunits) and inhibited translation and proteolytic pathways (e.g., 60S and 40S ribosomal subunits and UPS proteins). In agreement with the proteome-bioinformatics data, western blot and immunocytochemical analyses revealed that OPN deficiency perturbs protein homeostasis in macrophages-inhibiting translation and protein turnover and inducing apoptosis-whereas OPN induction by GA restores cellular proteostasis. Taken together, OPN is essential for macrophage homeostatic balance via the regulation of protein synthesis, UCHL1-UPS axis, and mitochondria-mediated apoptotic processes, indicating its potential application in immune-based therapies.

NeuroLINCS Proteomics: Defining human-derived iPSC proteomes and protein signatures of pluripotency.

The National Institute of Health (NIH) Library of integrated network-based cellular signatures (LINCS) program is premised on the generation of a publicly available data resource of cell-based biochemical responses or "signatures" to genetic or environmental perturbations. NeuroLINCS uses human inducible pluripotent stem cells (hiPSCs), derived from patients and healthy controls, and differentiated into motor neuron cell cultures. This multi-laboratory effort strives to establish i) robust multi-omic workflows for hiPSC and differentiated neuronal cultures, ii) public annotated data sets and iii) relevant and targetable biological pathways of spinal muscular atrophy (SMA) and amyotrophic lateral sclerosis (ALS). Here, we focus on the proteomics and the quality of the developed workflow of hiPSC lines from 6 individuals, though epigenomics and transcriptomics data are also publicly available. Known and commonly used markers representing 73 proteins were reproducibly quantified with consistent expression levels across all hiPSC lines. Data quality assessments, data levels and metadata of all 6 genetically diverse human iPSCs analysed by DIA-MS are parsable and available as a high-quality resource to the public.

Answer ALS, a large-scale resource for sporadic and familial ALS combining clinical and multi-omics data from induced pluripotent cell lines.

Answer ALS is a biological and clinical resource of patient-derived, induced pluripotent stem (iPS) cell lines, multi-omic data derived from iPS neurons and longitudinal clinical and smartphone data from over 1,000 patients with ALS. This resource provides population-level biological and clinical data that may be employed to identify clinical-molecular-biochemical subtypes of amyotrophic lateral sclerosis (ALS). A unique smartphone-based system was employed to collect deep clinical data, including fine motor activity, speech, breathing and linguistics/cognition. The iPS spinal neurons were blood derived from each patient and these cells underwent multi-omic analytics including whole-genome sequencing, RNA transcriptomics, ATAC-sequencing and proteomics. The intent of these data is for the generation of integrated clinical and biological signatures using bioinformatics, statistics and computational biology to establish patterns that may lead to a better understanding of the underlying mechanisms of disease, including subgroup identification. A web portal for open-source sharing of all data was developed for widespread community-based data analytics.

Potential early clinical stage colorectal cancer diagnosis using a proteomics blood test panel.

BACKGROUND: One of the most significant challenges in colorectal cancer (CRC) management is the use of compliant early stage population-based diagnostic tests as adjuncts to confirmatory colonoscopy. Despite the near curative nature of early clinical stage surgical resection, mortality remains unacceptably high-as the majority of patients diagnosed by faecal haemoglobin followed by colonoscopy occur at latter stages. Additionally, current population-based screens reliant on fecal occult blood test (FOBT) have low compliance (~ 40%) and tests suffer low sensitivities. Therefore, blood-based diagnostic tests offer survival benefits from their higher compliance (≥ 97%), if they can at least match the sensitivity and specificity of FOBTs. However, discovery of low abundance plasma biomarkers is difficult due to occupancy of a high percentage of proteomic discovery space by many high abundance plasma proteins (e.g., human serum albumin). METHODS: A combination of high abundance protein ultradepletion (e.g., MARS-14 and an in-house IgY depletion columns) strategies, extensive peptide fractionation methods (SCX, SAX, High pH and SEC) and SWATH-MS were utilized to uncover protein biomarkers from a cohort of 100 plasma samples (i.e., pools of 20 healthy and 20 stages I-IV CRC plasmas). The differentially expressed proteins were analyzed using ANOVA and pairwise t-tests (p < 0.05; fold-change > 1.5), and further examined with a neural network classification method using in silico augmented 5000 patient datasets. RESULTS: Ultradepletion combined with peptide fractionation allowed for the identification of a total of 513 plasma proteins, 8 of which had not been previously reported in human plasma (based on PeptideAtlas database). SWATH-MS analysis revealed 37 protein biomarker candidates that exhibited differential expression across CRC stages compared to healthy controls. Of those, 7 candidates (CST3, GPX3, CFD, MRC1, COMP, PON1 and ADAMDEC1) were validated using Western blotting and/or ELISA. The neural network classification narrowed down candidate biomarkers to 5 proteins (SAA2, APCS, APOA4, F2 and AMBP) that had maintained accuracy which could discern early (I/II) from late (III/IV) stage CRC. CONCLUSION: MS-based proteomics in combination with ultradepletion strategies have an immense potential of identifying diagnostic protein biosignature.

Agricultural waste as a source for the production of silica nanoparticles.

The major interest of the paper deals with the extraction of silica from four natural sources such as rice husk, bamboo leaves, sugarcane bagasse and groundnut shell. These waste materials in large quantities can create a serious environmental problem. Hence, there is a need to adopt proper strategy to reduce the waste. In the present investigation, all the waste materials are subjected to moisture removal in a hot plate and sintered at 900°C for 7 h. The sintered powder was treated with 1 M NaOH to form sodium silicate and then with 6M H2SO4 to precipitate silica. The prepared silica powders were characterized by FT-IR, XRD and SEM-EDAX analysis. The silica recovered from different sources was found to vary between 52% and 78%. Magnesium substituted silica was formed from the groundnut waste and further treatment is required to precipitate silica.

Serum proteome changes in dengue virus-infected patients from a dengue-endemic area of India: towards new molecular targets?

The global burden of dengue continues to worsen, specifically in tropical and subtropical countries, and has evolved as a major public health problem. We investigated the changes in serum proteome in dengue fever (DF) patients from a dengue-endemic area of India to obtain mechanistic insights about the disease pathogenesis, the host immune response, and identification of potential serum protein biomarkers of this infectious disease. In this study, serum samples from DF patients, healthy subjects, and patients with falciparum malaria (an infectious disease control) were investigated by 2D-DIGE in combination with MALDI-TOF/TOF MS. The findings were validated with Western blotting. Functional clustering of the identified proteins was performed using PANTHER and DAVID tools. Compared to the healthy controls, we found significant changes in the expression levels of 48 protein spots corresponding to 18 unique proteins (7 downregulated and 11 upregulated) in DF patients (p<0.05). Among these differentially-expressed proteins, 11 candidates exhibited different trends in dengue fever compared to falciparum malaria. Importantly, our results suggest that dengue virus infection leads to alterations in expression levels of multiple serum proteins involved in diverse and vital physiological pathways, including acute phase response signaling, complement cascades, hemostasis, and blood coagulation. For the first time we report here that the serum levels of hemopexin, haptoglobin, serum amyloid P, and kininogen precursor, are altered in DF. This study informs the pathogenesis and host immune response to dengue virus infection, as well as the current search for new diagnostic and molecular drug targets.

Serum profiling of leptospirosis patients to investigate proteomic alterations.

Leptospirosis is a zoonotic infectious disease of tropical, subtropical and temperate zones, which is caused by the pathogenic spirochetes of genus Leptospira. Although this zoonosis is generally not considered as fatal, the pathogen can eventually cause severe infection with septic shock, multi-organ failure and lethal pulmonary hemorrhages leading to mortality. In this study, we have performed a proteomic analysis of serum samples from leptospirosis patients (n=6), febrile controls (falciparum malaria) (n=8) and healthy subjects (n=18) to obtain an insight about disease pathogenesis and host immune responses in leptospiral infections. 2DE and 2D-DIGE analysis in combination with MALDI-TOF/TOF MS revealed differential expression of 22 serum proteins in leptospirosis patients compared to the healthy controls. Among the identified differentially expressed proteins, 8 candidates exhibited different trends compared to the febrile controls. Functional analysis suggested the involvement of differentially expressed proteins in vital physiological pathways, including acute phase response, complement and coagulation cascades and hemostasis. This is the first report of analysis of human serum proteome alterations in leptospirosis patients, which revealed several differentially expressed proteins, including α-1-antitrypsin, vitronectin, ceruloplasmin, G-protein signaling regulator, apolipoprotein A-IV, which have not been reported in context of leptospirosis previously. This study will enhance our understanding about leptospirosis pathogenesis and provide a glimpse of host immunological responses. Additionally, a few differentially expressed proteins identified in this study may further be investigated as diagnostic or prognostic serum biomarkers for leptospirosis. This article is part of a Special Issue entitled: Integrated omics.