A Matrigel-based 3D construct of SH-SY5Y cells models the α-synuclein pathologies of Parkinson's disease

Parkinson's disease (PD) is featured with α-synuclein-based Lewy body pathology, which however was difficult to observe in conventional two-dimensional (2D) cell culture and even in animal models. We herein aimed to develop a three-dimensional (3D) cellular model of PD to recapitulate the α-synuclein pathologies. All-trans-retinoic acid-differentiated human SH-SY5Y cells and Matrigel were optimized for 3D construction. The 3D cultured cells displayed higher tyrosine hydroxylase expression and improved dopaminergic-like phenotypes than 2D cells as suggested by RNA-sequencing analyses. Multiple forms of α-synuclein, including monomer, low and high molecular weight oligomers, were differentially present in the 2D and 3D cells, but mostly remained unchanged upon the MPP+ or rotenone treatment. Phosphorylated α-synuclein was accumulated and detergent-insoluble α-synuclein fraction was observed in the neurotoxin-treated 3D cells. Importantly, Lewy body-like inclusions were captured in the 3D system, including proteinase K-resistant α-synuclein aggregates, ubiquitin aggregation, β-amyloid and β-sheet protein deposition. The study provides a unique and convenient 3D model of PD which recapitulates critical α-synuclein pathologies and should be useful in multiple PD-associated applications.

Cryo-EM demonstrates the in vitro proliferation of an ex vivo amyloid fibril morphology by seeding

Several studies showed that seeding of solutions of monomeric fibril proteins with ex vivo amyloid fibrils accelerated the kinetics of fibril formation in vitro but did not necessarily replicate the seed structure. In this research we use cryo-electron microscopy and other methods to analyze the ability of serum amyloid A (SAA)1.1-derived amyloid fibrils, purified from systemic AA amyloidosis tissue, to seed solutions of recombinant SAA1.1 protein. We show that 98% of the seeded fibrils remodel the full fibril structure of the main ex vivo fibril morphology, which we used for seeding, while they are notably different from unseeded in vitro fibrils. The seeded fibrils show a similar proteinase K resistance as ex vivo fibrils and are substantially more stable to proteolytic digestion than unseeded in vitro fibrils. Our data support the view that the fibril morphology contributes to determining proteolytic stability and that pathogenic amyloid fibrils arise from proteolytic selection.

Peptide stapling by late-stage Suzuki–Miyaura cross-coupling

The development of peptide stapling techniques to stabilise α-helical secondary structure motifs of peptides led to the design of modulators of protein–protein interactions, which had been considered undruggable for a long time. We disclose a novel approach towards peptide stapling utilising macrocyclisation by late-stage Suzuki–Miyaura cross-coupling of bromotryptophan-containing peptides of the catenin-binding domain of axin. Optimisation of the linker length in order to find a compromise between both sufficient linker rigidity and flexibility resulted in a peptide with an increased α-helicity and enhanced binding affinity to its native binding partner β-catenin. An increased proteolytic stability against proteinase K has been demonstrated.

Creation of a recombinant Komagataella phaffii strain, a producer of proteinase K from Tritirachium album

The objects of the study were recombinant clones of Komagataella phaffii K51 carrying the heterologous proteinase K (PK-w) gene from Tritirachium album integrated into their genome as well as samples of recombinant proteinase K isolated from these clones. The aims of this work were i) to determine whether it is possible to create recombinant K. phaffii K51 clones overexpressing functionally active proteinase K from T. album and ii) to analyze the enzymatic activity of the resulting recombinant enzyme. The following methods were used: computational analysis of primary structure of the proteinase K gene, molecular biological methods (PCR, electrophoresis of DNA in an agarose gel, electrophoresis of proteins in an SDS polyacrylamide gel under denaturing conditions, spectrophotometry, and quantitative assays of protease activity), and genetic engineering techniques (cloning and selection of genes in bacterial cells Escherichia coli TOP10 and in the methylotrophic yeast K. phaffii K51). The gene encoding natural proteinase K (PK-w) was designed and optimized for expression in K. phaffii K51. The proteinase K gene was synthesized and cloned within the plasmid pPICZα-A vector in E. coli TOP10 cells. The proteinase K gene was inserted into pPICZα-A in such a way that – at a subsequent stage of transfection into yeast cells – it was efficiently expressed under the control of the promoter and terminator of the AOX1 gene, and the product of the exogenous gene contained the signal peptide of the Saccharomyces cerevisiae a-factor to ensure the protein’s secretion into the culture medium. The resultant recombinant plasmid (pPICZα-A/PK-w) was transfected into K. phaffii K51 cells. A recombinant K. phaffii K51 clone was obtained that carried the synthetic proteinase K gene and ensured its effective expression and secretion into the culture medium. An approximate productivity of the yeast recombinant clones for recombinant proteinase K was 25 μg/ mL after 4 days of cultivation. The resulting recombinant protease has a high specific proteolytic activity: ~5000 U/mg.

Optimization of enzymatic hydrolysis of Pleurotus ostreatus derived proteins through RSM and evaluation of nutritional and functional qualities of mushroom protein hydrolysates

Pleurotus ostreatus (Jacq.) P. Kumm., the second most widely cultivated oyster mushroom was grown on paddy straw, which is cheap and readily available waste material. After harvesting and drying, nutritional, and antinutritional composition of P. ostreatus were estimated using the standard assay methods. Tannin and phytic acid were present in very negligible amount (0.095 ± 0.027 mg/g and 0.150 ± 0.083 mg/g, respectively), whereas oxalate and cyanide were absent in whole mushroom. In fact, P. ostreatus was hydrolysed with commercially available proteinase K, pepsin and trypsin with different concentrations of the enzymes (0.05%, 0.10% and 0.15%), at different temperatures (30 °C, 40 °C and 50 °C) for different time periods (60, 90 and 120 min) to get the mushroom protein hydrolysates. Degree of hydrolysis and protein content varied from 4.29 ± 1.12% to 99.42 ± 0.02% and from 0.25 ± 0.07 mg/mL to 3.22 ± 0.12 mg/mL, respectively. Maximum degree of hydrolysis and the highest protein content of protein hydrolysate was obtained when using 0.15% proteinase K, at 50 °C for 120 minutes. Mushroom protein hydrolysates thus obtained exhibited improved functional characteristics such as foaming capacity, foaming stability and emulsifying property than the unhydrolysed mushroom. Based on the result of the present study, the mushroom protein hydrolysates could be served as useful ingredient for food and nutraceutical applications.

Method versatility in RNA extraction-free PCR detection of SARS-CoV-2 in saliva samples

Early in the pandemic, a simple, open-source, RNA extraction-free RT-qPCR protocol for SARS-CoV-2 detection in saliva was developed and made widely available. This simplified approach (SalivaDirect) requires only sample treatment with proteinase K prior to PCR testing. However, feedback from clinical laboratories highlighted a need for a flexible workflow that can be seamlessly integrated into their current health and safety requirements for the receiving and handling of potentially infectious samples. To address these varying needs, we explored additional pre-PCR workflows. We built upon the original SalivaDirect workflow to include an initial incubation step (95°C for 30 minutes, 95°C for 5 minutes or 65°C for 15 minutes) with or without addition of proteinase K. The limit of detection for the workflows tested did not significantly differ from that of the original SalivaDirect workflow. When tested on de-identified saliva samples from confirmed COVID-19 individuals, these workflows also produced comparable virus detection and assay sensitivities, as determined by RT-qPCR analysis. Exclusion of proteinase K did not negatively affect the sensitivity of the assay. The addition of multiple heat pretreatment options to the SalivaDirect protocol increases the accessibility of this cost-effective SARS-CoV-2 test as it gives diagnostic laboratories the flexibility to implement the workflow which best suits their safety protocols.

Selection of a suitable viral DNA extraction method for Sheeppox virus in cell culture

A suitable method for the extraction of nucleic acids should be efficient, sensitive, rapid and simple. Moreover, ideally, good method should yield pure nucleic acid-free from any contaminant inhibitors. Several methods have been reported for viral deoxyribonucleic acid (DNA) isolation but limited information is available on quick and simple isolation of Sheeppox virus (SPPV) genomic DNA in cell culture. In this study, the healthy Vero cells and primary lamb testis cells were infected with SPPV strains such as SPPV-Jaipur, SPPV-Ranipet and SPPV-Roumanian Fanar (RF) and harvested when it exhibited clear cytopathic effect (CPE) in culture. Four different DNA extraction methods i.e., (i) Phenol/chloroform/Isoamyl alcohol method, (ii) Cell lysis buffer method, (iii) Proteinase-k method, and (iv) commercial nucleic acid extraction kit was used to extract optimum yield of viral genomic DNA from clarified culture supernatant of harvested SPPV virus. The DNA sample was characterized using the Nanodrop spectrophotometer and agarose gel electrophoresis. Significantly (p<0.05) higher yield of SPPV genomic DNA was obtained in proteinase-k method which was about 3-5 times more than other methods. Among these methods, proteinase-k protocol was found to be comparatively very effective method in terms of yield of viral genomic DNA, and was free from PCR inhibitors.

Distribution of phosphorylated alpha-synuclein in non-diseased brain implicates olfactory bulb mitral cells in synucleinopathy pathogenesis

Synucleinopathies including Parkinsons disease and dementia with Lewy bodies are neurodegenerative diseases characterized by the intracellular accumulation of the protein alpha-synuclein called Lewy pathology. Alpha-synuclein within Lewy pathology is aggregated into protease resistant filamentous structures and is predominantly phosphorylated at serine 129 (PSER129). Lewy pathology has been hypothesized to spread throughout the nervous system as the disease progresses. Cross-sectional studies have shown the olfactory bulb and olfactory tract consistently bare LP for common synucleinopathies, making these structures likely starting points for the spreading process, and thus disease. Here we examined the distribution of PSER129 in non-diseased brain. To do this we used a sensitive tyramide signal amplification (TSA) technique to detect low abundance endogenous PSER129 under ideal antibody binding conditions. In wild-type non-diseased mice, PSER129 was detected in the olfactory bulb and several brain regions of the olfactory cortex across the neuroaxis (i.e., olfactory bulb to brain stem). PSER129 was particularly apparent in the mitral cell layer and the outer plexiform layer of the olfactory bulb where it was observed as cytosolic/nuclear puncta or fibers, respectively. PSER129 immunoreactivity in the healthy olfactory bulb was abolished by pretreatment of the tissue with proteinase K, pre-absorption of the primary antibody against the purified PSER129 peptide fragment, or the omission of the PSER129 antibody. Furthermore, PSER129 immunoreactivity was not observed in any brain region of alpha-synuclein knockout mice. Dual labeling for the PSER129 and the mitral cell marker TBX21 showed that PSER129 positive structures of the healthy OB were found in mitral cells. We found evidence of the same PSER129 positive structures in the olfactory bulb of non-diseased rats, non-human primates, healthy humans, but not individuals diagnosed with PD. Results suggest biological pathways responsible for alpha-synuclein phosphorylation are constitutively active in OB mitral cells and alpha-synuclein in these cells may be predisposed to pathological aggregation. Pathological seeds originating in mitral cells may act as a source for alpha-synuclein spread competent assemblies that spreads throughout the brain via fibers of the olfactory tract. Future studies should investigate the normal function of alpha-synuclein in the mitral cells of the olfactory bulb, which may give insight into synucleinopathy disease origins.