A quarter of prokaryotic Family II inorganic pyrophosphatases (PPases) contain a regulatory insert comprised of two cystathionine β-synthase (CBS) domains and one DRTGG domain in addition to the two catalytic domains that form canonical Family II PPases. The CBS domain-containing PPases (CBS-PPases) are allosterically activated or inhibited by adenine nucleotides that cooperatively bind to the CBS domains. Here we use chemical cross-linking and analytical ultracentrifugation to show that CBS-PPases from Desulfitobacterium hafniense and four other bacterial species are active as 200-250-kDa homotetramers, which seems unprecedented among the four PPase families. The tetrameric structure is stabilized by Co2+, the essential cofactor, pyrophosphate, the substrate, and adenine nucleotides, including diadenosine tetraphosphate. The deletion variants of dhPPase containing only catalytic or regulatory domains are dimeric. Co2+ depletion by incubation with EDTA converts CBS-PPase into inactive tetrameric and dimeric forms. Dissociation of tetrameric CBS-PPase and its catalytic part by dilution renders them inactive. The structure of CBS-PPase tetramer was modelled from the structures of dimeric catalytic and regulatory parts. These findings signify the role of the unique oligomeric structure of CBS-PPase in its multifaced regulation.Many antibacterial and antiparasitic drugs work by competitively inhibiting dihydrofolate reductase (DHFR), a vital enzyme in folate metabolism. The interactions between inhibitors and DHFR active site residues are known in many homologs but the contributions from distal residues are less understood. Identifying distal residues that aid in inhibitor binding can improve targeted drug development programs by accounting for distant influences that may be less conserved and subject to frequent resistance causing mutations. Previously, a novel, homology-based, computational approach that mines ligand inhibition data was used to predict residues involved in inhibitor selectivity in the DHFR family. Expectedly, some inhibitor selectivity determining residue positions were predicted to lie in the active site and coincide with experimentally known inhibitor selectivity determining positions. However, other residues that group spatially in clusters distal to the active site have not been previously investigated. In this study, the effect of introducing amino acid substitutions at one of these predicted clusters (His38-Ala39-Ile40) on the inhibitor selectivity profile in Bacillus stearothermophilus dihydrofolate reductase (Bs DHFR) was investigated. Mutations were introduced into these cluster positions to change sidechain chemistry and size. We determined kcat and KM values and measured KD values at equilibrium for two competitive DHFR inhibitors, trimethoprim (TMP) and pyrimethamine (PYR). Mutations in the His38-Ala39-Ile40 cluster significantly impacted inhibitor binding and TMP/PYR selectivity - seven out of nine mutations resulted in tighter binding to PYR when compared to TMP. These data suggest that the His38-Ala39-Ile40 cluster is a distal inhibitor selectivity determining region that favors PYR binding in Bs DHFR and, possibly, throughout the DHFR family. Adeno-associated virus (AAV) vectors have excellent properties as gene transfer vehicles. The recent development of AAV-PHP.eB, highly BBB-permeable capsid variant of AAV serotype 9, has opened up systemic application for whole brain transduction. To attain high transduction efficacy, much efforts have been paid to purify AAV vectors using gradient centrifugation or column chromatography. These methods are time-consuming, cost substantially and require expensive equipment. We propose a simple purification method for the production of systemically applicable AAV-PHP.eB targeting the brain. The new method, which we named minimal purification (MP) method, requires only 2 steps removal of cell debris using a syringe filter and concentration using a disposable ultrafiltration device. The MP method yielded 2 times more AAV-PHP.eB than the standard ultracentrifuge purification (UCP) method. Intravenous injection of AAV-PHP.eB prepared using the MP method caused robust whole brain transduction without overt toxicity on the liver and kidney. Moreover, we found almost no difference in cellular density and morphology of brain microglia between control mice and mice treated systemically with the MP viral solution, suggesting no influence of the viral injection on brain immunity. The new method, which requires only a benchtop centrifuge and takes only 2-4 h to obtain a ready-to-use viral solution, is much less expensive than the existing UCP method, and can avoid cumbersome and time-consuming purification processes. This simplified method further expands the use of AAV vectors in the neuroscience community.This simplified method further expands the use of AAV vectors in the neuroscience community. Rehabilitation robots integrated with brain-machine interaction (BMI) can facilitate stroke patients' recovery by closing the loop between motor intention and actual movement. The main challenge is to identify the patient's motor intention based on large training datasets with noise contamination in the Electroencephalogram (EEG) signal. To address this problem, this paper proposed a self-adaptively denoised Event-Related Desynchronization (ERD)-based motor intention recognition algorithm (DeERD) in order to enable BMI training with a small sample of calibration data. This study recruited 8 stroke patients. see more Each patient was required to execute paralyzed upper-limb motor attempt for 20 trials and remain in resting state for 20 trials randomly. ERD-based motor intention recognition algorithm, Common spatial filter algorithm (CSP) and Directed Transfer Function analysis (DTF) were used to extract features for classification respectively and compared with the proposed DeERD analysis. DeERD can filter the noise and extract the average lines as the principal trends. With denoising processing, Accuracy (ACC) was up to 70% for all 8 patients and they could be included in this BMI system effectively. The proposed DeERD model generated statistically significant increase in True Positive Rate (TPR) and in ACC than the DTF model. TPR and ACC standard deviation of DeERD was smaller than that of CSP. The proposed DeERD model can eliminate the principal noise and extract the principal trend of the time-frequency analysis. It provides a practical method to recruit more stroke patients into BMI training with fewer calibration trainings.The proposed DeERD model can eliminate the principal noise and extract the principal trend of the time-frequency analysis. It provides a practical method to recruit more stroke patients into BMI training with fewer calibration trainings.