Interparticle interaction-dependent jamming in colloids: insights into glass transition and morphology modulation during rapid evaporation-induced assembly.

Understanding the role of interparticle interactions in jamming phenomena is essential for gaining insights into the intriguing glass transition behavior observed in atomic and molecular systems. In this study, we investigate the jamming behavior of colloids with tunable interparticle interactions during evaporation-induced assembly (EIA). By manipulating the interaction among charged colloids using cationic polyethyleneimine (PEI) through electro-sorption and subsequent free polymer induced repulsion, we observe distinct jamming behavior in silica colloids during EIA, depending on the interparticle interactions. Silica colloids with strong repulsive interactions exhibit a repulsive colloidal glass state with a volume fraction of silica colloids in supraparticle ϕ ∼ 0.70. On the other hand, PEI-mediated attractive interactions among silica colloids lead to an attractive colloidal glass phase with a significantly lower ϕ ∼ 0.43. Free polymer induced repulsion of colloids at higher PEI concentration once again results in a repulsive glassy state with ϕ ∼ 0.61. Furthermore, we revealed that interparticle interactions not only influence the jamming behavior but also play a significant role in shaping the morphology of self-assembled structures during EIA, and the assembled structure undergoes a morphological reentrant transition from a doughnut-like shape to a spherical form and again back to a doughnut-like configuration. Jamming-dependent evolution of micropores and dynamics of the confined PEI have been probed using positron annihilation lifetime spectroscopy (PALS) and broadband dielectric spectroscopy (BDS). PALS reveals distinct variations in the micropores of the supraparticles with different PEI loadings, confirming the impact of jamming on the evolution of the micropores within the supraparticles. BDS measurements uncover non-monotonic dynamics of PEI molecules confined in the evolved pore network. It is revealed that the reentrant jamming behavior of colloids, modulated by PEI, holds profound significance for the long-term stability of supraparticles.

A natural small molecule-mediated inhibition of alpha-synuclein aggregation leads to neuroprotection in Caenorhabditis elegans.

Small molecules are being explored intensively for their applications as therapeutic molecules in the management of metabolic and neurological disorders. The natural small molecules can inhibit protein aggregation and underlying cellular pathogenesis of neurodegenerative diseases involving multi-factorial mechanisms of action. Certain natural small molecular inhibitors of pathogenic protein aggregation are highly efficient and have shown promising therapeutic potential. In the present study, Shikonin (SHK), a natural plant-based naphthoquinone has been investigated for its aggregation inhibition activity against α-synuclein (α-syn) and the neuroprotective potential in Caenorhabditis elegans (C. elegans). SHK significantly inhibited aggregation of α-syn at sub-stochiometric concentrations, delayed the linear lag phase and growth kinetics of seeded and unseeded α-syn aggregation. The binding of SHK to the C-terminus of α-syn maintained α-helical and disordered secondary structures with reduced beta-sheet content and complexity of aggregates. Further, in C. elegans transgenic PD models, SHK significantly reduced α-syn aggregation, improved locomotor activity and prevented dopaminergic (DA) neuronal degeneration, indicating the neuroprotective role of SHK. The present study highlights the potential of natural small molecules in the prevention of protein aggregation that may further be explored for their therapeutic efficacy in the management of protein aggregation and neurodegenerative diseases.

Development and Clinical Validation of RT-LAMP-Based Lateral-Flow Devices and Electrochemical Sensor for Detecting Multigene Targets in SARS-CoV-2.

Consistently emerging variants and the life-threatening consequences of SARS-CoV-2 have prompted worldwide concern about human health, necessitating rapid and accurate point-of-care diagnostics to limit the spread of COVID-19. Still, However, the availability of such diagnostics for COVID-19 remains a major rate-limiting factor in containing the outbreaks. Apart from the conventional reverse transcription polymerase chain reaction, loop-mediated isothermal amplification-based (LAMP) assays have emerged as rapid and efficient systems to detect COVID-19. The present study aims to develop RT-LAMP-based assay system for detecting multiple targets in N, ORF1ab, E, and S genes of the SARS-CoV-2 genome, where the end-products were quantified using spectrophotometry, paper-based lateral-flow devices, and electrochemical sensors. The spectrophotometric method shows a LOD of 10 agµL-1 for N, ORF1ab, E genes and 100 agµL-1 for S gene in SARS-CoV-2. The developed lateral-flow devices showed an LOD of 10 agµL-1 for all four gene targets in SARS-CoV-2. An electrochemical sensor developed for N-gene showed an LOD and E-strip sensitivity of log 1.79 ± 0.427 pgµL-1 and log 0.067 µA/pg µL-1/mm2, respectively. The developed assay systems were validated with the clinical samples from COVID-19 outbreaks in 2020 and 2021. This multigene target approach can effectively detect emerging COVID-19 variants using combination of various analytical techniques at testing facilities and in point-of-care settings.

Structural-functional diversity of malaria parasite's PfHSP70-1 and PfHSP40 chaperone pair gives an edge over human orthologs in chaperone-assisted protein folding.

Plasmodium falciparum, the human malaria parasite harbors a metastable proteome which is vulnerable to proteotoxic stress conditions encountered during its lifecycle. How parasite's chaperone machinery is able to maintain its aggregation-prone proteome in functional state, is poorly understood. As HSP70-40 system forms the central hub in cellular proteostasis, we investigated the protein folding capacity of PfHSP70-1 and PfHSP40 chaperone pair and compared it with human orthologs (HSPA1A and DNAJA1). Despite the structural similarity, we observed that parasite chaperones and their human orthologs exhibit striking differences in conformational dynamics. Comprehensive biochemical investigations revealed that PfHSP70-1 and PfHSP40 chaperone pair has better protein folding, aggregation inhibition, oligomer remodeling and disaggregase activities than their human orthologs. Chaperone-swapping experiments suggest that PfHSP40 can also efficiently cooperate with human HSP70 to facilitate the folding of client-substrate. SPR-derived kinetic parameters reveal that PfHSP40 has higher binding affinity towards unfolded substrate than DNAJA1. Interestingly, the observed slow dissociation rate of PfHSP40-substrate interaction allows PfHSP40 to maintain the substrate in folding-competent state to minimize its misfolding. Structural investigation through small angle x-ray scattering gave insights into the conformational architecture of PfHSP70-1 (monomer), PfHSP40 (dimer) and their complex. Overall, our data suggest that the parasite has evolved functionally diverged and efficient chaperone machinery which allows the human malaria parasite to survive in hostile conditions. The distinct allosteric landscapes and interaction kinetics of plasmodial chaperones open avenues for the exploration of small-molecule based antimalarial interventions.

Expanding role of molecular chaperones in regulating α-synuclein misfolding; implications in Parkinson's disease.

Protein misfolding under stressful environmental conditions cause several cellular problems owing to the disturbed cellular protein homeostasis, which may further lead to neurological disorders like Parkinson's disease (PD), Alzheimer's disease (AD), Amyloid lateral sclerosis and Huntington disease (HD). The presence of cellular defense mechanisms like molecular chaperones and proteasomal degradation systems prevent protein misfolding and aggregation. Molecular chaperones plays primary role in preventing protein misfolding by mediating proper native folding, unfolding and refolding of the polypeptides along with vast number of cellular functions. In past few years, the understanding of molecular chaperone mechanisms has been expanded enormously although implementation to prevent protein aggregation diseases is still deficient. We in this review evaluated major classes of molecular chaperones and their mechanisms relevant for preventing protein aggregation, specific case of α-synuclein aggregation. We also evaluate the molecular chaperone function as a novel therapeutic approach and the chaperone inhibitors or activators as small molecular drug targets.

The Ti3 AlC2 MAX Phase as an Efficient Catalyst for Oxidative Dehydrogenation of n-Butane.

Dehydrogenation or oxidative dehydrogenation (ODH) of alkanes to produce alkenes directly from natural gas/shale gas is gaining in importance. Ti3 AlC2 , a MAX phase, which hitherto had not been used in catalysis, efficiently catalyzes the ODH of n-butane to butenes and butadiene, which are important intermediates for the synthesis of polymers and other compounds. The catalyst, which combines both metallic and ceramic properties, is stable for at least 30 h on stream, even at low O2 :butane ratios, without suffering from coking. This material has neither lattice oxygens nor noble metals, yet a unique combination of numerous defects and a thin surface Ti1-y Aly O2-y/2 layer that is rich in oxygen vacancies makes it an active catalyst. Given the large number of compositions available, MAX phases may find applications in several heterogeneously catalyzed reactions.

Assessment of autonomic symptoms in a medically complex, urban patient population.

PURPOSE: Urban, minority communities are disproportionately affected by the chronic diseases associated with autonomic neuropathy; however validated measures of autonomic symptoms have not been studied in these complex populations. We sought to validate the Autonomic Symptom Profile (ASP) in a low income, medically complex, urban patient population. METHODS: Ninety-seven adults were recruited from the outpatient neurology clinic of an academic medical center serving the East Harlem neighborhood of New York City. Participants completed the ASP, and underwent a comprehensive neurologic examination, and a standardized battery of autonomic function tests (quantitative sweat testing, heart rate response to deep breathing (HRDB), Valsalva maneuver, and tilt table). Burden of chronic disease was summarized using the Charlson co-morbidity index (CCI), and detailed medication history was obtained. RESULTS: The ASP displayed good internal consistency (Cronbach's α = .88), even among lower literacy participants. In univariate analyses, the ASP was correlated with HRDB (r = -.301, p = .002), a marker of cardiac autonomic neuropathy, with the CCI (r = .37, p < .001), and with use of medications with autonomic effects [t(95) = -2.13, p = .036]. However, in multivariate analysis, only the CCI remained significant. CONCLUSIONS: In this urban, predominantly minority patient population, the symptoms captured by the ASP were more closely associated with burden of medical disease than with autonomic dysfunction. Due to this lack of specificity, it is essential that results from autonomic questionnaires be interpreted in the context of the neurologic history and exam, burden of co-morbid illness and medications, and most importantly autonomic function tests.

Insulin-degrading enzyme is activated by the C-terminus of α-synuclein.

The insulin-degrading enzyme (IDE) plays a key role in type-2 diabetes and typically degrades small peptides such as insulin, amyloid ß and islet amyloid polypeptide. We recently reported a novel non-proteolytical interaction in vitro between IDE and the Parkinson's disease 140-residue protein α-synuclein that resulted in dual effects: arrested α-synuclein oligomers and, simultaneously, increased IDE proteolysis activity. Here we demonstrate that these outcomes arise due to IDE interactions with the C-terminus of α-synuclein. Whereas a peptide containing the first 97 residues of α-synuclein did not improve IDE activity and its aggregation was not blocked by IDE, a peptide with the C-terminal 44 residues of α-synuclein increased IDE proteolysis to the same degree as full-length α-synuclein. Because the α-synuclein C-terminus is acidic, the interaction appears to involve electrostatic attraction with IDE's basic exosite, known to be involved in activation.

Novel, potent, orally bioavailable and selective mycobacterial ATP synthase inhibitors that demonstrated activity against both replicating and non-replicating M. tuberculosis.

The mycobacterial F0F1-ATP synthase (ATPase) is a validated target for the development of tuberculosis (TB) therapeutics. Therefore, a series of eighteen novel compounds has been designed, synthesized and evaluated against Mycobacterium smegmatis ATPase. The observed ATPase inhibitory activities (IC50) of these compounds range between 0.36 and 5.45µM. The lead compound 9d [N-(7-chloro-2-methylquinolin-4-yl)-N-(3-((diethylamino)methyl)-4-hydroxyphenyl)-2,3-dichlorobenzenesulfonamide] with null cytotoxicity (CC50>300µg/mL) and excellent anti-mycobacterial activity and selectivity (mycobacterium ATPase IC50=0.51µM, mammalian ATPase IC50>100µM, and selectivity >200) exhibited a complete growth inhibition of replicating Mycobacterium tuberculosis H37Rv at 3.12µg/mL. In addition, it also exhibited bactericidal effect (approximately 2.4log10 reductions in CFU) in the hypoxic culture of non-replicating M. tuberculosis at 100µg/mL (32-fold of its MIC) as compared to positive control isoniazid [approximately 0.2log10 reduction in CFU at 5µg/mL (50-fold of its MIC)]. The pharmacokinetics of 9d after p.o. and IV administration in male Sprague-Dawley rats indicated its quick absorption, distribution and slow elimination. It exhibited a high volume of distribution (Vss, 0.41L/kg), moderate clearance (0.06L/h/kg), long half-life (4.2h) and low absolute bioavailability (1.72%). In the murine model system of chronic TB, 9d showed 2.12log10 reductions in CFU in both lung and spleen at 173µmol/kg dose as compared to the growth of untreated control group of Balb/C male mice infected with replicating M. tuberculosis H37Rv. The in vivo efficacy of 9d is at least double of the control drug ethambutol. These results suggest 9d as a promising candidate molecule for further preclinical evaluation against resistant TB strains.

3D-QSAR and molecular modeling studies on 2,3-dideoxy hexenopyranosid-4-uloses as anti-tubercular agents targeting alpha-mannosidase.

Ligand-based and structure-based methods were applied in combination to exploit the physicochemical properties of 2,3-dideoxy hex-2-enopyranosid-4-uloses against Mycobacterium tuberculosis H37Rv. Statistically valid 3D-QSAR models with good correlation and predictive power were obtained with CoMFA steric and electrostatic fields (r(2) = 0.797, q(2) = 0.589) and CoMSIA with combined steric, electrostatic, hydrophobic and hydrogen bond acceptor fields (r(2) = 0.867, q(2) = 0.570) based on training set of 33 molecules with predictive r(2) of 0.808 and 0.890 for CoMFA and CoMSIA respectively. The results illustrate the requirement of optimal alkyl chain length at C-1 position and acceptor groups along hydroxy methyl substituent of C-6 to enhance the anti-tubercular activity of the 2,3-dideoxy hex-2-enopyranosid-4-uloses while any substitution at C-3 position exert diminishing effect on anti-tubercular activity of these enulosides. Further, homology modeling of M. tuberculosis alpha-mannosidase followed by molecular docking and molecular dynamics simulations on co-complexed models were performed to gain insight into the rationale for binding affinity of selected inhibitors with the target of interest. The comprehensive information obtained from this study will help to better understand the structural basis of biological activity of this class of molecules and guide further design of more potent analogues as anti-tubercular agents.

Hsp110 is a bona fide chaperone using ATP to unfold stable misfolded polypeptides and reciprocally collaborate with Hsp70 to solubilize protein aggregates.

Structurally and sequence-wise, the Hsp110s belong to a subfamily of the Hsp70 chaperones. Like the classical Hsp70s, members of the Hsp110 subfamily can bind misfolding polypeptides and hydrolyze ATP. However, they apparently act as a mere subordinate nucleotide exchange factors, regulating the ability of Hsp70 to hydrolyze ATP and convert stable protein aggregates into native proteins. Using stably misfolded and aggregated polypeptides as substrates in optimized in vitro chaperone assays, we show that the human cytosolic Hsp110s (HSPH1 and HSPH2) are bona fide chaperones on their own that collaborate with Hsp40 (DNAJA1 and DNAJB1) to hydrolyze ATP and unfold and thus convert stable misfolded polypeptides into natively refolded proteins. Moreover, equimolar Hsp70 (HSPA1A) and Hsp110 (HSPH1) formed a powerful molecular machinery that optimally reactivated stable luciferase aggregates in an ATP- and DNAJA1-dependent manner, in a disaggregation mechanism whereby the two paralogous chaperones alternatively activate the release of bound unfolded polypeptide substrates from one another, leading to native protein refolding.

Deoxysugars as antituberculars and alpha-mannosidase inhibitors.

A promising modified sugar molecule was identified which was active against multidrug-resistant (MDR) strains of Mycobacterium tuberculosis, suggesting involvement of a new target. The compound was demonstrated to be bactericidal, inhibited the growth of M. tuberculosis in mice, and targeted alpha-mannosidase as a competitive inhibitor with a Ki value of 353.9 µM.

Arsenite interferes with protein folding and triggers formation of protein aggregates in yeast.

Several metals and metalloids profoundly affect biological systems, but their impact on the proteome and mechanisms of toxicity are not fully understood. Here, we demonstrate that arsenite causes protein aggregation in Saccharomyces cerevisiae. Various molecular chaperones were found to be associated with arsenite-induced aggregates indicating that this metalloid promotes protein misfolding. Using in vivo and in vitro assays, we show that proteins in the process of synthesis/folding are particularly sensitive to arsenite-induced aggregation, that arsenite interferes with protein folding by acting on unfolded polypeptides, and that arsenite directly inhibits chaperone activity. Thus, folding inhibition contributes to arsenite toxicity in two ways: by aggregate formation and by chaperone inhibition. Importantly, arsenite-induced protein aggregates can act as seeds committing other, labile proteins to misfold and aggregate. Our findings describe a novel mechanism of toxicity that may explain the suggested role of this metalloid in the etiology and pathogenesis of protein folding disorders associated with arsenic poisoning.

A paper-based chromogenic strip and electrochemical sensor for the detection of 4-(dimethylamino)azobenzene.

Natural and synthetic dyes are added to different food commodities to enhance their appearance and acceptance by consumers. Acute and chronic exposure owing to the consumption of non-permitted dyes may lead to health concerns such as allergic reactions, eczema, and asthma. 4-(Dimethylamino)azobenzene (4-DMAAB) is a non-permitted dye that has been reported in adulterated mustard oil. Consumption of 4-DMAAB poses severe risks due to its mutagenic and carcinogenic properties. Several sensitive methods such as FT-NIR, FT-MIR and SERS are available for the detection of 4-DMAAB. Here, a spectrophotometric method was developed for the detection of 4-DMAAB. The developed method was translated to a point-of-test paper-based, chromogenic strip which showed a detection limit of 0.025 mM for 4-DMAAB. Also, an electrochemical sensor was developed by electro-depositing the test solution on a screen-printed electrode. The electrochemical sensor showed an LOD of 0.027 ± 0.008 mM with recovery in the range of 91-107% of 4-DMAAB. Oil samples collected from the market were processed by liquid-liquid extraction and the content of 4-DMAAB was assessed. The developed point-of-use sensors for the detection of 4-DMAAB have potential for use by the consumers, food industry and regulatory agencies for on-site analysis and assuring the quality of edible oils.

A polylactic acid-carbon nanofiber-based electro-conductive sensing material and paper-based colorimetric sensor for detection of nitrates.

Plastics are ubiquitous in today's lifestyle, and their indiscriminate use has led to the accumulation of plastic waste in landfills and oceans. The waste accumulates and breaks into micro-particles that enter the food chain, causing severe threats to human health, wildlife, and the ecosystem. Environment-friendly and bio-based degradable materials offer a sustainable alternative to the vastly used synthetic materials. Here, a polylactic acid and carbon nanofiber-based membrane and a paper-based colorimetric sensor have been developed. The membrane had a surface area of 3.02 m2 g-1 and a pore size of 18.77 nm. The pores were evenly distributed with a pore volume of 0.0137 cm3 g-1. The membrane was evaluated in accordance with OECD guidelines and was found to be safe for tested aquatic and terrestrial models. The activated PLA-CNF membrane was further used as a bio-based electrode for the electrochemical detection of nitrates (NO3-) in water samples with a detection limit of 0.046 ppm and sensitivity of 1.69 × 10-4 A ppm-1 mm-2, whereas the developed paper-based colorimetric sensor had a detection limit of 156 ppm for NO3-. This study presents an environment-friendly, low-carbon footprint disposable material for sensing applications as a sustainable alternative to plastics.

Revealing the nano-level molecular packing in chitosan-NiO nanocomposite by using positron annihilation spectroscopy and small-angle X-ray scattering.

Chitosan-NiO nanocomposite (CNC) is shown to be a potential dielectric material with promising properties. CNCs containing NiO nanoparticles (0.2, 0.6, 1, 2, 5 wt %) are prepared through chemical methods. The inclusion of NiO nanoparticles in the chitosan matrix is confirmed by scanning electron microscopy (SEM) and X-ray diffraction. The morphology of the NiO nanoparticles and the nanocomposites is investigated by transmission electron microscopy and SEM, respectively. Positron annihilation lifetime spectroscopy (PALS) and the coincidence Doppler broadening (CDB) technique are used to quantify the free volume and molecular packing in the nanocomposites. The triplet-state positronium lifetime and the corresponding intensity show the changes in nanohole size, density, and size distribution as a function of NiO loading. Small-angle X-ray scattering indicates that the NiO aggregates are identical in all the CNCs. The momentum density distribution obtained from CDB measurements excludes the possibility of a contribution of vacant spaces (pores) available in NiO aggregates to the free volume of nanocomposites upon determination by using PALS. The results show systematic variation in free-volume properties and nano-level molecular packing as a function of NiO loading, which is presumed to play a vital role in determining the various properties of the nanocomposites.

A QR code-integrated chromogenic paper strip for detection of hydrogen peroxide in aqueous samples.

Hydrogen peroxide (H2O2) is commonly used as a preservative, disinfectant, bleach, and oxidizing agent. The prolonged consumption of H2O2 adulterated milk is harmful to human health when consumed in the diet. Exposure to H2O2 can lead to oxidative stress, cell damage and tissue injury. Due to the potential adverse effects, the use of hydrogen peroxide is regulated in certain applications, such as in food, water treatment plants and medical products. Several methods are available for the detection of H2O2 in various matrices. Here, a method and QR code-integrated chromogenic paper strip for the detection of H2O2 in aqueous samples has been developed. The spectrophotometric method showed an LOD and LOQ of 0.00087 ± 8.70 ×10-5% (v/v) and 0.0037 ± 0.0003% (v/v), respectively. The paper-based chromogenic strip prepared by immobilizing recognition solution onto a QR code was able to detect 0.0005% v/v of H2O2 in aqueous samples. The QR integrated chromogenic paper strip sensors can serve as a useful tool for consumers, regulatory agencies, and the food industry to assess food quality and authenticity.

Sensor-integrated biocomposite membrane for food quality assessment.

The use of biopolymers is gaining momentum owing to their compatibility with various food commodities and acceptability by the industry and consumers. Biopolymers integrated with natural pigments can be used for assessing the quality of perishable packaged food products. Here, a biodegradable composite membrane of starch and chitosan, integrated with anthocyanin has been developed. The chromogenic response of the developed biocomposite membrane was used to assess spoilage in milk. The surface roughness recorded using atomic force microscopy and scanning electron microscopy showed smooth membrane surface, and a pore size 4.21 nm was determined by Brunauer-Emmett-Teller analysis. The water vapour transmissibility of the membrane was 4.616E-09 and 1.147E-08 g/h Pa.mm and water solubility was 5.6 and 5.8 % at 25 and 37 ℃, respectively, indicating high water resistance and low vapour transmission rate. The developed biocomposite membrane offers an environment friendly substrate for biosensing with a promising potential in smart food-packaging applications.

Band gap and defect engineering of bismuth vanadate using La, Ce, Zr dopants to obtain a photoelectrochemical system for ultra-sensitive detection of glucose in blood serum.

Bismuth vanadate (BiVO4) is a promising photoactive material for the design of photoelectrochemical (PEC) analytical devices for the non-enzymatic detection of glucose. In this work, un-doped and La/Ce/Zr doped BiVO4 photo anodes were developed by spray pyrolysis coating to generate unique 2D hierarchical architectures using the facile ultrasonic spray coating technique without any complex pre or post-treatment. The influence of different dopants on the morphology and photoelectrochemical activity of BiVO4 coatings was investigated. X-ray diffraction, scanning electron microscopy, UV-vis optical absorbance, and positron annihilation techniques were used to evaluate the structure, defects, and optical properties of BiVO4 films. DFT simulation confirmed the Zr doping induced band gap reduction in the BiVO4 lattice. The Zr doping on the Bi site in BiVO4 lattice provided significantly low Bi and V-based defect density and a higher bulk diffusion length of charge pairs (4 times that of pristine) as well as charge transfer efficiency and this led to the foremost photocurrent for water splitting. The Zr-doped BiVO4 photo anode showed remarkable sensitivity in glucose sensing. The sensitivity and limit of detection of the Zr-doped BiVO4 PEC device towards glucose were 0.14 mA cm-2 mM-1 and 1.22 µM, respectively, in the concentration range of 1-7 mM. The system showed sensitive detection of glucose in blood serum. This is the first time that a 2D morphology electrode design consisting of Zr-doped BiVO4, which leads to exceptionally high sensitivity for glucose sensing, has been reported.

Implications of In Vitro Multi-Serine Phosphorylation of Alpha-Synuclein in Aggregation and Cytotoxicity.

Post-translational modifications guide the functional diversity and identity of proteins. Phosphorylation is one such post-translational modification that has been reported in pathological proteins related to various neurodegenerative disorders such as α-synuclein (α-syn) phosphorylation in Parkinson's disease and other synucleinopathies. In α-syn, the phosphorylation has mostly been observed at S129; however, the occurrence of other serine modifications at S9, S42, and S87 is partially explored. In pathogenic conditions, where α-syn is phosphorylated by complex kinase pathways, multi-site modifications may happen and alter the mechanism of α-syn aggregation. Here, using Polo-like kinase 2 and G-protein coupled receptor kinase 4, the in vitro phosphorylation of α-syn was performed, which revealed multi-serine phosphorylation. Mass spectrometry with customized proteolytic digestion showed prominent phosphorylation at S129 and modifications at S87 and S42 with PLK2 and S87 with GRK4. The phosphorylation at the identified serine residues was further validated with NMR and western blotting. Multi-serine phosphorylation aggravates the aggregation potential of monomeric α-syn, seeding capacity, and cytotoxicity in the SH-SY5Y cell line. This study proposes evidence for in vitro multi-site phosphorylation and its significance in α-syn aggregation, toxicity, and related pathogenesis.