Structure of Tau filaments in Prion protein amyloidoses

In human neurodegenerative diseases associated with the intracellular aggregation of Tau protein, the ordered cores of Tau filaments adopt distinct folds. Here, we analyze Tau filaments isolated from the brain of individuals affected by Prion-Protein cerebral amyloid angiopathy (PrP-CAA) with a nonsense mutation in the PRNP gene that leads to early termination of translation of PrP (Q160Ter or Q160X), and Gerstmann-Sträussler-Scheinker (GSS) disease, with a missense mutation in the PRNP gene that leads to an amino acid substitution at residue 198 (F198S) of PrP. The clinical and neuropathologic phenotypes associated with these two mutations in PRNP are different; however, the neuropathologic analyses of these two genetic variants have consistently shown the presence of numerous neurofibrillary tangles (NFTs) made of filamentous Tau aggregates in neurons. We report that Tau filaments in PrP-CAA (Q160X) and GSS (F198S) are composed of 3-repeat and 4-repeat Tau isoforms, having a striking similarity to NFTs in Alzheimer disease (AD). In PrP-CAA (Q160X), Tau filaments are made of both paired helical filaments (PHFs) and straight filaments (SFs), while in GSS (F198S), only PHFs were found. Mass spectrometry analyses of Tau filaments extracted from PrP-CAA (Q160X) and GSS (F198S) brains show the presence of post-translational modifications that are comparable to those seen in Tau aggregates from AD. Cryo-EM analysis reveals that the atomic models of the Tau filaments obtained from PrP-CAA (Q160X) and GSS (F198S) are identical to those of the Tau filaments from AD, and are therefore distinct from those of Pick disease, chronic traumatic encephalopathy, and corticobasal degeneration. Our data support the hypothesis that in the presence of extracellular amyloid deposits and regardless of the primary amino acid sequence of the amyloid protein, similar molecular mechanisms are at play in the formation of identical Tau filaments

American Society of Transplantation and Cellular Therapy, Center of International Blood and Marrow Transplant Research, and European Society for Blood and Marrow Transplantation Clinical Practice Recommendations for Transplantation and Cellular Therapies in Mantle Cell Lymphoma

Autologous (auto-) and allogeneic (allo-) hematopoietic cell transplantation (HCT) are accepted treatment modalities in contemporary treatment algorithms for mantle cell lymphoma (MCL). Chimeric antigen receptor (CAR) T cell therapy recently received approval for MCL; however, its exact place and sequence in relation to HCT remain unclear. The American Society of Transplantation and Cellular Therapy, Center of International Blood and Marrow Transplant Research, and the European Society for Blood and Marrow Transplantation jointly convened an expert panel to formulate consensus recommendations for role, timing, and sequencing of auto-HCT, allo-HCT, and CAR T cell therapy for patients with newly diagnosed and relapsed/refractory (R/R) MCL. The RAND-modified Delphi method was used to generate consensus statements. Seventeen consensus statements were generated, with a few key statements as follows: in the first line setting, auto-HCT consolidation represents standard of care in eligible patients, whereas there is no clear role of allo-HCT or CAR T cell therapy outside of clinical trials. In the R/R setting, the preferential option is CAR T cell therapy, especially in patients with MCL failing or intolerant to at least one Bruton's tyrosine kinase inhibitor, while allo-HCT is recommended if CAR T cell therapy fails or is infeasible. Several recommendations were based on expert opinion, where the panel developed consensus statements for important real-world clinical scenarios to guide clinical practice. In the absence of contemporary evidence-based data, the panel found RAND-modified Delphi methodology effective in providing a formal framework for developing consensus recommendations for the timing and sequence of cellular therapies for MCL

Ubiquitination and degradation of SUMO1 by small-molecule degraders extends survival of mice with patient-derived tumors

Discovery of small-molecule degraders that activate ubiquitin ligase–mediated ubiquitination and degradation of targeted oncoproteins in cancer cells has been an elusive therapeutic strategy. Here, we report a cancer cell–based drug screen of the NCI drug-like compounds library that enabled identification of small-molecule degraders of the small ubiquitin-related modifier 1 (SUMO1). Structure-activity relationship studies of analogs of the hit compound CPD1 led to identification of a lead compound HB007 with improved properties and anticancer potency in vitro and in vivo. A genome-scale CRISPR-Cas9 knockout screen identified the substrate receptor F-box protein 42 (FBXO42) of cullin 1 (CUL1) E3 ubiquitin ligase as required for HB007 activity. Using HB007 pull-down proteomics assays, we pinpointed HB007’s binding protein as the cytoplasmic activation/proliferation-associated protein 1 (CAPRIN1). Biolayer interferometry and compound competitive immunoblot assays confirmed the selectivity of HB007’s binding to CAPRIN1. When bound to CAPRIN1, HB007 induced the interaction of CAPRIN1 with FBXO42. FBXO42 then recruited SUMO1 to the CAPRIN1-CUL1-FBXO42 ubiquitin ligase complex, where SUMO1 was ubiquitinated in several of human cancer cells. HB007 selectively degraded SUMO1 in patient tumor–derived xenografts implanted into mice. Systemic administration of HB007 inhibited the progression of patient-derived brain, breast, colon, and lung cancers in mice and increased survival of the animals. This cancer cell–based screening approach enabled discovery of a small-molecule degrader of SUMO1 and may be useful for identifying other small-molecule degraders of oncoproteins

State-of-the-art REDOR and TEDOR methods for elucidation of protein structure and interactions

Protein structure determination is vital for elucidating their function, folding or misfolding pathways, and their interaction with other biomolecules, small molecules and membranes. In the field of neurodegenerative disorders like Parkinson’s Disease and Alzheimer’s Disease, amyloid fibrils that constitute the aggregates in the diseased brain are formed due to misfolding of proteins like a-synuclein (a-syn) and amyloid-b. Understanding the structure of these amyloid fibrils will be beneficial for probing amyloid- ligand interactions, amyloid-membrane interactions and even their misfolding pathways. This dissertation thesis introduces some state-of-the-art solid-state nuclear magnetic resonance (SSNMR) techniques to investigate protein structures and their interactions, which will facilitate the study of amyloid fibrils formed in the neurodegenerative disorders. One of the major biophysical techniques to probe biomolecular structure and their interactions is magic angle spinning (MAS) SSNMR spectroscopy due to its ability to examine insoluble systems and with no inherent restriction on the size of the molecule. For structure determination and refinement, we employ famous and robust techniques like rotational echo double resonance (REDOR) and transferred echo double resonance (TEDOR) that recouples the dipolar couplings between two selective heteronuclear spins present in the biomolecule. The dipolar coupling is directly proportional to the gyromagnetic ratio (g) of the spins and indirectly proportional to the cube of the distance between them. Hence, fitting the dipolar dephasing curves to Bessel function of first kind provide us quantitative, precise and unambiguous distance restraints within or between molecules and these distance restraints will assist the structure calculations or docking of small molecules or other biomolecules to the proteins. Structure or ligand binding mode determination with REDOR or TEDOR has been performed extensively on all types of biomolecules, small molecules and inorganic compounds in the past couple of decades. However, due to utilization of lower g nuclei in the spin pair and restrictions in the pulse sequence, the sensitivity, resolution, the distance detection range, and the types of sample on which REDOR and TEDOR can be applied have been limited. In my thesis, I have focused on advancing REDOR and TEDOR methodology such that we can obtain quantitative and precise distances up to 1.5-2 nm. These are the longest quantitative distances obtained by SSNMR so far. This has been made possible by utilizing high g spins, like 1H and 19F as REDOR or TEDOR spins. The novel methods are multidimensional in nature, can be applied to uniformly 13C or 2H labeled proteins, highly sensitive and will be performed in reduced experimental time due to 1H-detection. 1H-detection is feasible under fast MAS and in perdeuterated proteins. Perdeuteration of protein leads to enhanced 1H spin-spin relaxation time (T2), which is a huge advantage for performing REDOR dephasing or TEDOR build-up for longer times and hence increasing the distance detection range. The REDOR or TEDOR techniques introduced in this thesis have been mainly applied on uniformly-13C, 2H, 15N (U-CDN) labeled proteins, like GB1 crystalline protein and a-syn fibrils and their mutants, back-exchanged to 10-30% 1H. 1H-detected REDOR or TEDOR techniques developed here are unique in measuring distances between the side chain 13C atoms and backbone 1H atoms in the proteins, thereby providing accurate restraints for side chain orientations and not only backbone restraints. Performing REDOR with 19F atom is advantageous because of the usefulness and popularity of 19F atom in the drug or diagnostic agent development industry. In our studies, we have also successfully demonstrated the incorporation of 19F atom in a-syn fibrils through mutagenesis and chemical modification. Thereafter, we performed 13C-19F REDOR to obtain distances of up to 10 Å and with our newly developed 1H-detected REDOR pulse sequence, we obtained 1H-19F unambiguous REDOR distances of up to 17 Å in a-syn fibrils. Taken together, this thesis lays a foundation towards shaping REDOR and TEDOR spectroscopy to be feasible for — (1) structure determination and refinement of uniformly 13C labeled and perdeuterated proteins, and (2) binding mode determination of fluorinated imaging agents or drugs, and lipid membranes to amyloid fibrils like a-syn, or non-fluorinated small molecules to 19F- labeled proteins

State-of-the-art REDOR and TEDOR methods for elucidation of protein structure and interactions

Protein structure determination is vital for elucidating their function, folding or misfolding pathways, and their interaction with other biomolecules, small molecules and membranes. In the field of neurodegenerative disorders like Parkinson’s Disease and Alzheimer’s Disease, amyloid fibrils that constitute the aggregates in the diseased brain are formed due to misfolding of proteins like a-synuclein (a-syn) and amyloid-b. Understanding the structure of these amyloid fibrils will be beneficial for probing amyloid- ligand interactions, amyloid-membrane interactions and even their misfolding pathways. This dissertation thesis introduces some state-of-the-art solid-state nuclear magnetic resonance (SSNMR) techniques to investigate protein structures and their interactions, which will facilitate the study of amyloid fibrils formed in the neurodegenerative disorders. One of the major biophysical techniques to probe biomolecular structure and their interactions is magic angle spinning (MAS) SSNMR spectroscopy due to its ability to examine insoluble systems and with no inherent restriction on the size of the molecule. For structure determination and refinement, we employ famous and robust techniques like rotational echo double resonance (REDOR) and transferred echo double resonance (TEDOR) that recouples the dipolar couplings between two selective heteronuclear spins present in the biomolecule. The dipolar coupling is directly proportional to the gyromagnetic ratio (g) of the spins and indirectly proportional to the cube of the distance between them. Hence, fitting the dipolar dephasing curves to Bessel function of first kind provide us quantitative, precise and unambiguous distance restraints within or between molecules and these distance restraints will assist the structure calculations or docking of small molecules or other biomolecules to the proteins. Structure or ligand binding mode determination with REDOR or TEDOR has been performed extensively on all types of biomolecules, small molecules and inorganic compounds in the past couple of decades. However, due to utilization of lower g nuclei in the spin pair and restrictions in the pulse sequence, the sensitivity, resolution, the distance detection range, and the types of sample on which REDOR and TEDOR can be applied have been limited. In my thesis, I have focused on advancing REDOR and TEDOR methodology such that we can obtain quantitative and precise distances up to 1.5-2 nm. These are the longest quantitative distances obtained by SSNMR so far. This has been made possible by utilizing high g spins, like 1H and 19F as REDOR or TEDOR spins. The novel methods are multidimensional in nature, can be applied to uniformly 13C or 2H labeled proteins, highly sensitive and will be performed in reduced experimental time due to 1H-detection. 1H-detection is feasible under fast MAS and in perdeuterated proteins. Perdeuteration of protein leads to enhanced 1H spin-spin relaxation time (T2), which is a huge advantage for performing REDOR dephasing or TEDOR build-up for longer times and hence increasing the distance detection range. The REDOR or TEDOR techniques introduced in this thesis have been mainly applied on uniformly-13C, 2H, 15N (U-CDN) labeled proteins, like GB1 crystalline protein and a-syn fibrils and their mutants, back-exchanged to 10-30% 1H. 1H-detected REDOR or TEDOR techniques developed here are unique in measuring distances between the side chain 13C atoms and backbone 1H atoms in the proteins, thereby providing accurate restraints for side chain orientations and not only backbone restraints. Performing REDOR with 19F atom is advantageous because of the usefulness and popularity of 19F atom in the drug or diagnostic agent development industry. In our studies, we have also successfully demonstrated the incorporation of 19F atom in a-syn fibrils through mutagenesis and chemical modification. Thereafter, we performed 13C-19F REDOR to obtain distances of up to 10 Å and with our newly developed 1H-detected REDOR pulse sequence, we obtained 1H-19F unambiguous REDOR distances of up to 17 Å in a-syn fibrils. Taken together, this thesis lays a foundation towards shaping REDOR and TEDOR spectroscopy to be feasible for — (1) structure determination and refinement of uniformly 13C labeled and perdeuterated proteins, and (2) binding mode determination of fluorinated imaging agents or drugs, and lipid membranes to amyloid fibrils like a-syn, or non-fluorinated small molecules to 19F- labeled proteins.U of I OnlyAuthor requested U of Illinois access only (OA after 2yrs) in Vireo ETD syste

¹H‑Detected REDOR with Fast Magic-Angle Spinning of a Deuterated Protein

Rotational echo double resonance (REDOR) is a highly successful method for heteronuclear distance determination in biological solid-state NMR, and ¹H detection methods have emerged in recent years as a powerful approach to improving sensitivity and resolution for small sample quantities by utilizing fast magic-angle spinning (>30 kHz) and deuteration strategies. In theory, involving ¹H as one of the spins for measuring REDOR effects can greatly increase the distance measurement range, but few experiments of this type have been reported. Here we introduce a pulse sequence that combines frequency-selective REDOR (FSR) with ¹H detection. We demonstrate this method with applications to samples of uniformly ¹³C,¹⁵N,²H-labeled alanine and uniformly ¹³C,²H,¹⁵N-labeled GB1 protein, back-exchanged with 30% H₂O and 70% D₂O, employing a variety of frequency-selective ¹³C pulses to highlight unique spectral features. The resulting, robust REDOR effects provide (1) tools for resonance assignment, (2) restraints of secondary structure, (3) probes of tertiary structure, and (4) approaches to determine the preferred orientation of aromatic rings in the protein core

Preparation and characterization of macroporous pure alumina capillary membrane using boehmite as binder for filtration application

Macro-porous pure alumina membrane was prepared from boehmite sol having a pH of 3.6 as a binder with alumina powder in different concentrations in weight percentage (2.5, 4 and 7 %) to make extrudable ceramic paste. The pastes were extruded by a plunger type extruder to 3/2 mm (outer/inner diameter) capillary tube. The capillaries were dried in a roller drier and subjected to retarded sintering at 1350 A degrees C for 2.5 h. Prepared membrane were characterized and compared with each other and membrane prepared with Methocel as organic binder for pore size, porosity, mechanical strength and clean water flux. The pore size of 7 % boehmite sol based membrane was found to be 400 nm when compared to 1.3 mu m for membrane prepared with Methocel. The boehmite act as sintering agent and carry out the retarded sintering of alumina at 1350 A degrees C in place of 1700 A degrees C to form porous alumina membrane. Boehmite membrane was used for treatment of contaminated tannery wastewater and compared with membrane prepared from conventional organic binder. About 99 % removal in total organic carbon was obtained in case of boehmite membrane and chemical oxygen demand (COD) was reduced to 100 mg/L from an initial value of 6600 mg/L whereas in case of Methocel membrane COD was only reduced to 390 mg/L

Motijheel Lake - Victim of Cultural Eutrophication

Destruction of natural water bodies due to cultural eutrophication is a predominant problem in India. Motijheel Lake of Murshidabad district is an environmentally, economically and historically significant water body. However, Anthropogenic activities including unplanned settlements around this lake and its over exploitation have deteriorated its water quality to a great extent. Motijheel Lake acts as a sink for domestic sewage, human and animal excreta. Surface runoffs are discharged into the lake which further adds to the list of pollutants. High phosphate, nitrate and nitrite-Nitrogen and chlorophyll content of the lake categorizes it as hypereutrophic one. Also, the amount of phosphate, nitrite and iron present in Motijheel Lake exceed the permissible limit in drinking water, as prescribed by US Environmental Protection Agency and Bureau of Indian Standards. When the Below Poverty Line residents of the surrounding area consume such water, they become susceptible to various fatal diseases. The low level of Dissolved Oxygen in the lake water signifies huge amount of organic matter deposited in the lake and indicates the lake water to be poor in quality. The high load of coliform bacteria in the lake water further corroborates the deposition of domestic, human and animal wastes. If water with such high concentration of faecal coliform is consumed, it could lead to fatal gastrointestinal and enteric diseases

Antimicrobial nano-zinc oxide-2S albumin protein formulation significantly inhibits growth of "Candidatus Liberibacter asiaticus" in planta.

Huanglongbing (HLB, also known as citrus greening) is considered to be the most devastating disease that has significantly damaged the citrus industry globally. HLB is caused by the Candidatus Liberibacter asiaticus (CLas), the fastidious phloem-restricted gram-negative bacterium, vectored by the asian citrus psyllid. To date, there is no effective control available against CLas. To alleviate the effects of HLB on the industry and protect citrus farmers, there is an urgent need to identify or develop inhibitor molecules to suppress or eradicate CLas from infected citrus plant. In this paper, we demonstrate for the first time an in planta efficacy of two antimicrobial compounds against CLas viz. 2S albumin (a plant based protein; ~12.5 kDa), Nano-Zinc Oxide (Nano-ZnO; ~ 4.0 nm diameter) and their combinations. Aqueous formulations of these compounds were trunk-injected to HLB affected Mosambi plants (Citrus sinensis) grafted on 3-year old rough lemon (C. jambhiri) rootstock with known CLas titer maintained inside an insect-free screen house. The effective concentration of 2S albumin (330 ppm) coupled with the Nano-ZnO (330 ppm) at 1:1 ratio was used. The dynamics of CLas pathogen load of treated Mosambi plants was assessed using TaqMan-qPCR assay every 30 days after treatment (DAT) and monitored till 120 days. We observed that 2S albumin-Nano-ZnO formulation performed the best among all the treatments decreasing CLas population by 96.2%, 97.6%, 95.6%, and 97% of the initial bacterial load (per 12.5 ng of genomic DNA) at 30, 60, 90, and 120 DAT, respectively. Our studies demonstrated the potency of 2S albumin-Nano-ZnO formulation as an antimicrobial treatment for suppressing CLas in planta and could potentially be developed as a novel anti CLas therapeutics to mitigate the HLB severity affecting the citrus industry worldwide