Preclinical evaluation of general toxicity and safety pharmacology of a receptor-binding domain-based COVID-19 subunit vaccine in various animal models.

The coronavirus disease 2019 pandemic has resulted in the introduction of several naïve methods of vaccine development, which have been used to prepare novel viral vectors and mRNA-based vaccines. However, reluctance to receive vaccines owing to the uncertainty regarding their safety is prevalent. Therefore, rigorous safety evaluation of vaccines through preclinical toxicity studies is critical to determine the safety profiles of vaccine candidates. This study aimed to evaluate the toxicity profile of HuVac-19, a subunit vaccine of SARS-CoV-2 utilizing the receptor-binding domain as an antigen, in rats, rabbits, and dogs using single- and repeat-dose study designs. Repeat-dose toxicity studies in rats and rabbits showed transient changes in hematological and serum biochemical parameters in the adjuvant and/or vaccine groups; however, these changes were reversed or potentially reversible after the recovery period. Moreover, temporary reversible changes in absolute and relative organ weights were observed in the prostate of rats and the thymus of rabbits. Gross examination of the injection sites in rats and rabbits treated with the adjuvant- and HuVac-19 showed discoloration and foci, whereas histopathological examination showed granulomatous inflammation, inflammatory cell infiltration, and myofiber degeneration/necrosis. This inflammatory response was local, unassociated with other toxicological changes, and resolved. In a pharmacological safety study, no toxicological or physiological changes associated with HuVac-19 administration were observed. In conclusion, HuVac-19 was not associated with any major systemic adverse effects in the general toxicity and safety pharmacology evaluation, demonstrating that HuVac-19 is a vaccine candidate with sufficient capacity to be used in human clinical trials.

Toxic Misfolded Transthyretin Oligomers with Different Molecular Conformations Formed through Distinct Oligomerization Pathways.

Protein aggregation is initiated by structural changes from native polypeptides to cytotoxic oligomers, which form cross-ß structured amyloid. Identification and characterization of oligomeric intermediates are critically important for understanding not only the molecular mechanism of aggregation but also the cytotoxic nature of amyloid oligomers. Preparation of misfolded oligomers for structural characterization is, however, challenging because of their transient, heterogeneous nature. Here, we report two distinct misfolded transthyretin (TTR) oligomers formed through different oligomerization pathways. A pathogenic TTR variant with a strong aggregation propensity (L55P) was used to prepare misfolded oligomers at physiological pH. Our mechanistic studies showed that the full-length TTR initially forms small oligomers, which self-assemble into short protofibrils at later stages. Enzymatic cleavage of the CD loop was also used to induce the formation of N-terminally truncated oligomers, which was detected in ex vivo cardiac TTR aggregates extracted from the tissues of patients. Structural characterization of the oligomers using solid-state nuclear magnetic resonance and circular dichroism revealed that the two TTR misfolded oligomers have distinct molecular conformations. In addition, the proteolytically cleaved TTR oligomers exhibit a higher surface hydrophobicity, suggesting the presence of distinct oligomerization pathways for TTR oligomer formation. Cytotoxicity assays also revealed that the cytotoxicity of cleaved oligomers is stronger than that of the full-length TTR oligomers, indicating that hydrophobicity might be an important property of toxic oligomers. These comparative biophysical analyses suggest that the toxic cleaved TTR oligomers formed through a different misfoling pathway may adopt distinct structural features that produce higher surface hydrophobicity, leading to the stronger cytotoxic activities.

Untwisted α-Synuclein Filaments Formed in the Presence of Lipid Vesicles.

Accumulation of filamentous aggregates of α-synuclein is a pathological hallmark of several neurodegenerative diseases, including Parkinson's disease (PD). The interaction between α-synuclein and phospholipids has been shown to play a critical role in the aggregation of α-synuclein. Most structural studies have, however, been focused on α-synuclein filaments formed in the absence of lipids. Here, we report the structural investigation of α-synuclein filaments assembled under the quiescent condition in the presence of anionic lipid vesicles using electron microscopy (EM), including cryogenic electron microscopy (cryo-EM). Our transmission electron microscopy (TEM) analyses reveal that α-synuclein forms curly protofilaments at an early stage of aggregation. The flexible protofilaments were then converted to long filaments after a longer incubation of 30 days. More detailed structural analyses using cryo-EM reveal that the long filaments adopt untwisted structures with different diameters, which have not been observed in previous α-synuclein fibrils formed in vitro. The untwisted filaments are rather similar to straight filaments with no observable twist that are extracted from patients with dementia with Lewy bodies. Our structural studies highlight the conformational diversity of α-synuclein filaments, requiring additional structural investigation of not only more ex vivo α-synuclein filaments but also in vitro α-synuclein filaments formed in the presence of diverse cofactors to better understand the molecular basis of diverse molecular conformations of α-synuclein filaments.

Probe-Free Identification of RNA Virus Variants with Point Mutations by Surface-Enhanced Raman Spectroscopy.

As observed in the COVID-19 pandemic, RNA viruses continue to rapidly evolve through mutations. In the absence of effective therapeutics, early detection of new severely pathogenic viruses and quarantine of infected people are critical for reducing the spread of the viral infections. However, conventional detection methods require a substantial amount of time to develop probes specific to new viruses, thereby impeding immediate response to the emergence of viral pathogens. In this study, we identified multiple types of viruses by obtaining the spectral fingerprint of their surface proteins with probe-free surface-enhanced Raman scattering (SERS). In addition, the SERS-based method can remarkably distinguish influenza virus variants with several surface protein point mutations from their parental strain. Principal component analysis (PCA) of the SERS spectra systematically captured the key Raman bands to distinguish the variants. Our results show that the combination of SERS and PCA can be a promising tool for rapid detection of newly emerging mutant viruses without a virus-specific probe.

Is there a relationship between intensity of occupational therapy and functional outcomes in hospitalised older patients? A prospective cohort study.

INTRODUCTION: Delivering high-intensity occupational therapy can improve functional outcomes for patients and reduce length of stay. However, there is little published evidence of this in the aged rehabilitation setting. This study aims to explore the association between intensity of occupational therapy interventions and functional outcomes in geriatric rehabilitation inpatients. METHODS: A prospective cohort study was conducted with adult inpatients admitted to a geriatric rehabilitation program. The intervention was the intensity of occupational therapy measured as high (≥30 minutes per day) versus low (<30 minutes per day). The primary outcome measured was change in functional performance, defined as a minimum of half a point improvement in the Katz Index of Activities of Daily Living (ADL) and/or the Lawton and Brody Scale of Instrumental ADL (IADL) at admission to rehabilitation, discharge and 3months post-discharge. RESULTS: A total of 693 patients were included in the analysis. The mean age was 82.2 years (standard deviation [SD] = 7.9), 57% were females, and 64% had cognitive impairment. Patients (n = 210) who received greater than or equal to 30 minutes of occupational therapy daily were more likely to have clinically relevant functional improvements.; for both ADL (odds ratio [OR] = 1.87, 95% confidence interval [CI]: 1.24-2.83) and IADL (OR = 3.00, 95% CI: 1.96-4.61), after adjusting for age, sex, severity of function (ADL ≤ 2) at admission, frailty and cognitive impairment. Improvements in ADL and IADL were maintained for at least 3 months following discharge. CONCLUSION: This study found that geriatric rehabilitation inpatients who received higher intensity of occupational therapy interventions were more likely to functionally improve than those who received lower intensity. Further research is required to determine if other factors, such as therapy type, influence functional outcomes.

Tau induces formation of α-synuclein filaments with distinct molecular conformations.

Recent structural investigation of amyloid filaments extracted from human patients demonstrated that the ex vivo filaments associated with different disease phenotypes adopt diverse molecular conformations, which are different from those of in vitro amyloid filaments. A very recent cryo-EM structural study also revealed that ex vivo α-synuclein filaments extracted from multiple system atrophy patients adopt distinct molecular structures from those of in vitro α-synuclein filaments, suggesting the presence of co-factors for α-synuclein aggregation in vivo. Here, we report structural characterizations of α-synuclein filaments formed in the presence of a potential co-factor, tau, using cryo-EM and solid-state NMR. Our cryo-EM structure of the tau-promoted α-synuclein filaments reveals some similarities to one of the previously reported polymorphs of in vitro α-synuclein filaments in the core region, while illustrating distinct conformations in the N- and C-terminal regions. The structural study highlights the conformational plasticity of α-synuclein filaments and the importance of the co-factors, requiring additional structural investigation of not only more ex vivo α-synuclein filaments, but also in vitro α-synuclein filaments formed in the presence of diverse co-factors. The comparative structural analyses will help better understand molecular basis of diverse structures of α-synuclein filaments and possible relevance of each structure to the disease phenotype.

Presenting symptoms of COVID-19 and clinical outcomes in hospitalised older adults.

BACKGROUND: In July 2020, a COVID-19 outbreak was recognised in the geriatric wards at a subacute campus of the Royal Melbourne Hospital affecting patients and staff. Patients were also admitted to this site after diagnosis in residential care. AIMS: To describe the early symptoms and the outcomes of COVID-19 in older adults. METHODS: Patients diagnosed with COVID-19 at the facility in July or August 2020 were identified and their medical records were examined to identify symptoms present before and after their diagnosis and to determine their outcomes. RESULTS: Overall, 106 patients were identified as having COVID-19, with median age of 84.3 years (range 41-104 years); 64 were diagnosed as hospital inpatients after a median length of stay of 49 days, 31 were transferred from residential aged care facilities with a known diagnosis and 11 were diagnosed after discharge. There were 95 patients included in an analysis of symptom type and timing onset. Overall, 61 (64.2%) were asymptomatic at the time of diagnosis of COVID-19, having been diagnosed through screening initiated on site. Of these, 88.6% developed symptoms of COVID-19 within 14 days. The most common initial symptom type was respiratory, but there was wide variation in presentation, including fever, gastrointestinal and neurological symptoms, many initially not recognised as being due to COVID-19. Of 104 patients, 32 died within 30 days of diagnosis. CONCLUSIONS: COVID-19 diagnosis is challenging due to the variance in symptoms. In the context of an outbreak, asymptomatic screening can identify affected patients early in the disease course.

Structural Characterization of Cardiac Ex Vivo Transthyretin Amyloid: Insight into the Transthyretin Misfolding Pathway In Vivo.

Structural characterization of misfolded protein aggregates is essential to understanding the molecular mechanism of protein aggregation associated with various protein misfolding disorders. Here, we report structural analyses of ex vivo transthyretin aggregates extracted from human cardiac tissue. Comparative structural analyses of in vitro and ex vivo transthyretin aggregates using various biophysical techniques revealed that cardiac transthyretin amyloid has structural features similar to those of in vitro transthyretin amyloid. Our solid-state nuclear magnetic resonance studies showed that in vitro amyloid contains extensive nativelike ß-sheet structures, while other loop regions including helical structures are disrupted in the amyloid state. These results suggest that transthyretin undergoes a common misfolding and aggregation transition to nativelike aggregation-prone monomers that self-assemble into amyloid precipitates in vitro and in vivo.

Disruption of the CD Loop by Enzymatic Cleavage Promotes the Formation of Toxic Transthyretin Oligomers through a Common Transthyretin Misfolding Pathway.

Amyloid formation of full-length TTR involves dissociation of the native tetramers into misfolded monomers that self-assemble into amyloid. In addition to the full-length TTR, C-terminal fragments including residues 49-127 were also observed in vivo, implying the presence of additional misfolding pathways. It was previously proposed that a proteolytic cleavage might lead to the formation of the C-terminal fragment TTR amyloid. Here, we report mechanistic studies of misfolding and aggregation of a TTR variant (G53A) in the absence and presence of a serine protease. A proteolytic cleavage of G53A in the CD loop (K48 and T49) with agitation promoted TTR misfolding and aggregation, suggesting that the proteolytic cleavage may lead to the aggregation of the C-terminal fragment (residues 49-127). To gain more detailed insights into TTR misfolding promoted by proteolytic cleavage, we investigated structural changes in G53A TTR in the presence and absence of trypsin. Our combined biophysical analyses revealed that the proteolytic cleavage accelerated the formation of spherical small oligomers, which exhibited cytotoxic activities. However, the truncated TTR appeared to maintain native-like structures, rather than the C-terminal fragment (residues 49-127) being released and unfolded from the native state. In addition, our solid-state nuclear magnetic resonance and Fourier transform infrared structural studies showed that the two aggregates derived from the full-length and cleaved TTR exhibited nearly identical molecular structural features, suggesting that the proteolytic cleavage in the CD loop destabilizes the native tetrameric structure and accelerates oligomer formation through a common TTR misfolding and aggregation mechanism rather than through a distinct molecular mechanism.

Conversion from Heterometallic to Homometallic Metal-Organic Frameworks.

Two new heterometallic metal-organic frameworks (MOFs), LnZnTPO 1 and 2, and two homometallic MOFs, LnTPO 3 and 4 (Ln=Eu for 1 and 3, and Tb for 2 and 4; H3 TPO=tris(4-carboxyphenyl)phosphine oxide) were synthesized, and their structures and properties were analyzed. They were prepared by solvothermal reaction of the C3 -symmetric ligand H3 TPO with the corresponding metal ion(s) (a mixture of Ln3+ and Zn2+ for 1 and 2, and Ln3+ alone for 3 and 4). Single-crystal XRD (SXRD) analysis revealed that 1 and 3 are isostructural to 2 and 4, respectively. TGA showed that the framework is thermally stable up to about 400 °C for 1 and 2, and about 450 °C for 3 and 4. PXRD analysis showed their pore-structure distortions without noticeable framework-structure changes during drying processes. The shapes of gas sorption isotherms for 1 and 3 are almost identical to those for 2 and 4, respectively. Solvothermal immersion of 1 and 2 in Tb3+ and Eu3+ solutions resulted in the framework metal-ion exchange affording 4 and 3, respectively, as confirmed by photoluminescence (PL), PXRD, IR, inductively coupled plasma atomic emission spectroscopy (ICP-AES), and energy-dispersive X-ray (EDX) analyses.

Alteration of gammaretroviral vector integration patterns by insertion of histone and leucine zipper into integrase.

Retroviral vectors show long-term gene expression in gene therapy through the integration of transgenes into the human cell genome. Murine leukemia virus (MLV), a well-studied gammaretrovirus, has been often used as a representative retroviral vector. However, frequent integrations of MLV-based vectors into transcriptional start sites (TSSs) could lead to the activation of oncogenes by enhancer effects of the genetic components within the vectors. Therefore, the MLV integration preference for TSSs limits its wider use in clinical applications. To reduce the integration preference of MLV-based vectors, we attempted to perturb the structure of the viral integrase that plays a key role in determining integration sites. For this goal, we inserted histones and leucine zippers, having DNA-binding property, into internal sites of MLV integrase. This integrase engineering yielded multiple mutant vectors that showed significantly different integration patterns compared with that of wild-type vector. Some mutant vectors did not prefer the key regulatory genomic domains of human cells, TSSs. Moreover, a couple of engineered vectors did not integrate into the genomic sites near the TSSs of oncogenes. Overall, this study suggests that structural perturbation of integrase is a simple way to develop safer MLV-based retroviral vectors for use in clinical applications.

Tau Interacts with the C-Terminal Region of α-Synuclein, Promoting Formation of Toxic Aggregates with Distinct Molecular Conformations.

An increasing body of evidence suggests that aggregation-prone proteins associated with various neurodegenerative diseases synergistically promote their mutual aggregation, leading to the co-occurrence of multiple neurodegenerative diseases in the same patient. Here we investigated teh molecular basis of synergistic interactions between the two pathological proteins, tau and α-synuclein, using various biophysical techniques including transmission electron microscopy (TEM), circular dichroism (CD), and solution and solid-state NMR. Our biophysical analyses of α-synuclein aggregation in the absence and presence of tau reveal that tau monomers promote the formation of α-synuclein oligomers and subsequently fibril formation. Solution NMR results also indicate that monomeric forms of tau selectively interact with the C-terminal region of the α-synuclein monomer, accelerating α-synuclein aggregation. In addition, a combined use of TEM and solid-state NMR spectroscopy reveals that the synergistic interactions lead to the formation of toxic α-synuclein aggregates with a distinct morphology and molecular conformation. The filamentous α-synuclein aggregates as well as α-synuclein monomers were also able to induce tau aggregation.

Identification of Newly Emerging Influenza Viruses by Detecting the Virally Infected Cells Based on Surface Enhanced Raman Spectroscopy and Principal Component Analysis.

Rapid diagnosis and quarantine of influenza virus mutant-infected people is critical to contain the fatal viral infection spread because effective antiviral drugs are normally not available. Conventional methods, however, cannot be used for the diagnosis because these methods need predefined labels, likely also unavailable for just emerging viruses. Here, we propose label-free identification of cells infected with different influenza viruses based on surface-enhanced Raman spectroscopy (SERS) and principal component analysis (PCA). Viral envelope proteins that are displayed on the surface of cells after infection of influenza viruses were targeted for this identification. Cells that expressed the envelope proteins of A/WSN/33 H1N1 or A/California/04/2009 H1N1 influenza viruses produced distinct SERS signals. Cells that displayed combinations of the envelope proteins from these two viral variants, an indication of emergence of a new virus, also generated characteristic SERS patterns. However, the cell's own surface proteins often hindered the identification of virally infected cells by producing SERS peaks similar to viral ones. PCA of the obtained SERS patterns could effectively capture the virus-specific signal components from the jumbled SERS peaks. Our study demonstrates a potential of combination of SERS and PCA to identify newly emerging influenza viruses through sensing the cells infected with the viruses.

Single-molecule DNA visualization using AT-specific red and non-specific green DNA-binding fluorescent proteins.

The recent advances in the single cell genome analysis are generating a considerable amount of novel insights into complex biological systems. However, there are still technical challenges because each cell has a single copy of DNA to be amplified in most single cell genome analytical methods. In this paper, we present a novel approach to directly visualize a genomic map on a large DNA molecule instantly stained with red and green DNA-binding fluorescent proteins without DNA amplification. For this visualization, we constructed a few types of fluorescent protein-fused DNA-binding proteins: H-NS (histone-like nucleoid-structuring protein), DNA-binding domain of BRCA1 (breast cancer 1), high mobility group-1 (HMG), and lysine tryptophan (KW) repeat motif. Because H-NS and HMG preferentially bind A/T-rich regions, we combined A/T specific binder (H-NS-mCherry and HMG-mCherry as red color) and a non-specific complementary DNA binder (BRCA1-eGFP and 2(KW)2-eGFP repeat as green color) to produce a sequence-specific two-color DNA physical map for efficient optical identification of single DNA molecules.

Attomolar detection of virus by liquid coplanar-gate graphene transistor on plastic.

The direct method of detecting a virus with extremely low concentration is recommended for the diagnosis of viral disease. In this study, coplanar-gate graphene field-effect transistors (GFETs) were built on flexible polyethylene terephthalate substrates for the attomolar detection of a virus. The GFETs exhibited a very low detection limit of 47.8 aM with relatively low source/drain voltage due to aqueous dielectric media which stabilizes viruses and antibodies for specific bonding. The antibody as a probe molecule was decorated on a graphene surface using 1-pyrenebutanoic acid succinimidyl ester that had previously been immobilized on a graphene surface. The Dirac point voltage shifted downward after dropping the virus solution, due to the electrostatic gating effect of graphene in the antigen (namely, virus)-antibody complex. The virus detection platform used in this study is expected to be beneficial for direct diagnosis in saline environments, since the performances of GFETs were not significantly affected by the presence of Na+ and Cl-. Furthermore, since our flexible and transparent virus sensors can be used in a wearable device, they provide a simple and fast method for diagnosing viruses.

Molecular-Level Interactions between Engineered Materials and Cells.

Various recent experimental observations indicate that growing cells on engineered materials can alter their physiology, function, and fate. This finding suggests that better molecular-level understanding of the interactions between cells and materials may guide the design and construction of sophisticated artificial substrates, potentially enabling control of cells for use in various biomedical applications. In this review, we introduce recent research results that shed light on molecular events and mechanisms involved in the interactions between cells and materials. We discuss the development of materials with distinct physical, chemical, and biological features, cellular sensing of the engineered materials, transfer of the sensing information to the cell nucleus, subsequent changes in physical and chemical states of genomic DNA, and finally the resulting cellular behavior changes. Ongoing efforts to advance materials engineering and the cell-material interface will eventually expand the cell-based applications in therapies and tissue regenerations.

Recent Advances in Mitochondria-Targeted Gene Delivery.

Mitochondria are the energy-producing organelles of cells. Mitochondrial dysfunctions link to various syndromes and diseases including myoclonic epilepsy and ragged-red fiber disease (MERRF), Leigh syndrome (LS), and Leber hereditary optic neuropathy (LHON). Primary mitochondrial diseases often result from mutations of mitochondrial genomes and nuclear genes that encode the mitochondrial components. However, complete intracellular correction of the mutated genetic parts relevant to mitochondrial structures and functions is technically challenging. Instead, there have been diverse attempts to provide corrected genetic materials with cells. In this review, we discuss recent novel physical, chemical and biological strategies, and methods to introduce genetic cargos into mitochondria of eukaryotic cells. Effective mitochondria-targeting gene delivery systems can reverse multiple mitochondrial disorders by enabling cells to produce functional mitochondrial components.

Pathogenic Mutations Induce Partial Structural Changes in the Native ß-Sheet Structure of Transthyretin and Accelerate Aggregation.

Amyloid formation of natively folded proteins involves global and/or local unfolding of the native state to form aggregation-prone intermediates. Here we report solid-state nuclear magnetic resonance (NMR) structural studies of amyloid derived from wild-type (WT) and more aggressive mutant forms of transthyretin (TTR) to investigate the structural changes associated with effective TTR aggregation. We employed selective 13C labeling schemes to investigate structural features of ß-structured core regions in amyloid states of WT and two mutant forms (V30M and L55P) of TTR. Analyses of the 13C-13C correlation solid-state NMR spectra revealed that WT TTR aggregates contain an amyloid core consisting of nativelike CBEF and DAGH ß-sheet structures, and the mutant TTR amyloids adopt a similar amyloid core structure with nativelike CBEF and AGH ß-structures. However, the V30M mutant amyloid was shown to have a different DA ß-structure. In addition, strand D is more disordered even in the native state of L55P TTR, indicating that the pathogenic mutations affect the DA ß-structure, leading to more effective amyloid formation. The NMR results are consistent with our mass spectrometry-based thermodynamic analyses that showed the amyloidogenic precursor states of WT and mutant TTRs adopt folded structures but the mutant precursor states are less stable than that of WT TTR. Analyses of the oxidation rate of the methionine side chain also revealed that the side chain of residue Met-30 pointing between strands D and A is not protected from oxidation in the V30M mutant, while protected in the native state, supporting the possibility that the DA ß-structure might be disrupted in the V30M mutant amyloid.

Cell surface-engineering to embed targeting ligands or tracking agents on the cell membrane.

The key challenge to improve the efficacy of cell therapy is how to efficiently modify cells with a specific molecule or compound that can guide the cells to the target tissue. To address this, we have developed a cell surface engineering technology to non-invasively modify the cell surface. This technology can embed a wide variety of bioactive molecules on any cell surface and allow for the targeting of a wide range of tissues in a variety of disease states. Using our cell surface engineering technology, mesenchymal stem cells (MSC)s were modified with: 1) a homing peptide or a recombinant protein to facilitate the migration of the cells toward a specific molecular target; or 2) magnetic resonance imaging (MRI) contrast agents to allow for in vivo tracking of the cells. The incorporation of a homing peptide or a targeting ligand on MSCs facilitated the migration of the cells toward their molecular target. MRI contrast agents were successfully embedded on the cell surfaces without adverse effects to the cells and the contrast agent-labeled cells were detectable by MRI. Our technology is a promising method of cell surface engineering that is applicable to a broad range of cell therapies.

Luminescent Metal-Organic Framework Sensor: Exceptional Cd2+ Turn-On Detection and First In Situ Visualization of Cd2+ Ion Diffusion into a Crystal.

With regard to fluorescence quenching commonly observed during metal-ion detection, "turn-on" chemical sensing has been rarely reported, but could be extremely important because it facilitates the selective recognition of target objects of interest against a dark background. A metal-organic framework (MOF) chemosensor has been prepared that serves as an efficient platform for the selective detection of Cu2+ and Cd2+ ions over other metal ions. In particular, this framework shows the highest fluorescence enhancement (≈60-fold relative to Cd-free MOF) for the hazardous metal ion Cd2+ among luminescent MOFs and displays excellent reusability in repeated cycles. The direct diffusion of Cd2+ into the crystal pores has also been visualized for the first time.