bluuebunny/crossref_metadata_2025_split huggingface dataset:
Urine-HILIC: Automated sample preparation for bottom-up urinary proteome profiling in clinical proteomics
Authors: Govender Ireshyn , Mokoena Rethabile , Stoychev Stoyan , Naicker Previn 
Published: July 2023

AbstractUrine provides a diverse source of information related to a patient’s health status and is ideal for clinical proteomics because of its ease of collection. To date, there is no standard operating procedure for reproducible and robust urine sample preparation for mass spectrometry-based clinical proteomics. To this end, a novel workflow was developed based on an on-bead protein capture, clean up, and digestion without the requirement for processing steps such as precipitation or centrifugation. The workflow was applied to an acute kidney injury (AKI) pilot study. Urine from clinical samples and a pooled sample were subjected to automated sample preparation in a KingFisher™ Flex magnetic handling station using a novel urine-HILIC (uHLC) approach based on MagReSyn® HILIC microspheres. For benchmarking, the pooled sample was also prepared using a published protocol based on an on-membrane (OM) protein capture and digestion workflow. Peptides were analysed by LCMS in data independent acquisition (DIA) mode using a Dionex Ultimate 3000 UPLC coupled to a Sciex 5600 mass spectrometer. Data was searched in Spectronaut™ 17. Both workflows showed similar peptide and protein identifications in the pooled sample. The uHLC workflow was easier to set up and complete, having less hands-on time than the OM method, with fewer manual processing steps. Lower peptide and protein CV was observed in the uHLC technical replicates. Following statistical analysis, candidate protein markers were filtered, at ≥ 2-fold change in abundance, ≥ 2 unique peptides and ≤ 1% false discovery rate, and revealed many significant, differentially abundant kidney injury-associated urinary proteins. The pilot data derived using this novel workflow provides information on the urinary proteome of patients with AKI. Further exploration in a larger cohort using this novel high-throughput method is warranted.
Analyzing interaction of rhodacyanine inhibitor ‘MKT-077’ withPlasmodium falciparumHSP70 nucleotide binding domains
Authors: Nainani Chanchal , Upadhyay Vipul , Singh Bikramjit , Sandhu Komalpreet Kaur , Kaur Satinder , Hora Rachna , Mishra Prakash Chandra 
Published: July 2023

AbstractMKT-077 and its derivatives are rhodacyanine inhibitors that hold potential in treatment of cancer, neurodegenerative diseases and malaria. These allosteric drugs act by inhibiting the ATPase action of heat shock proteins of 70kDa (HSP70). MKT-077 accumulates in the mitochondria and displays differential activity against HSP70 homologs. The fourPlasmodium falciparumHSP70s (PfHSP70) are present at various subcellular locations to perform distinct functions. In the present study, we have used bioinformatics tools to understand interactions between MKT-077 and PfHSP70s. Nature of the identified interactions is primarily hydrophobic with different PfHSP70 homologs showing variable propensity to bind MKT-077. Our molecular docking approach has helped us to predict the binding pocket and specific residues on PfHSP70s that are involved in interacting with MKT-077. Information derived from this article may form the foundation for design and development of MKT-077 based drugs against malaria.
Mapping immunological and host receptor binding determinants of SARS-CoV spike protein utilizing the Qubevirus platform
Authors: Sanders Carrie , Dzelamonyuy Aristide , Ntemafack Augustin , Alatoom Nadia , Nchinda Godwin , Georgiadis Millie , Waffo Alain Bopda 
Published: July 2023

AbstractThe motifs involved in tropism and immunological interactions of SARS-CoV spike (S) protein were investigated utilizing the Qubevirus platform. We showed that separately, 14 overlapping peptide fragments representing the S protein (F1-14 of 100 residues each) could be inserted into the C-terminus of A1 on recombinant Qubevirus without affecting its viability. Additionally, recombinant phage expression resulted in the surface exposure of different engineered fragments in an accessible manner. The F6 from S425-525, was found to contain the binding determinant of the recombinant human angiotensin converting enzyme 2 (rhACE2), with the shortest active binding motif situated between residues S437-492. Upstream, another fragment, F7, containing an overlapping portion of F6 would not bind to rhACE2, confirming not just only that residues were linear but equally also the appropriate structural orientation of F6 upon the Qubevirus. The F6 (S441-460) and other inserts, including F7/F8 (S601-620) and F10 (S781-800), were demonstrated to contain important immunological determinants through recognition and binding of S protein specific (anti-S) antibodies. An engineered chimeric insert bearing the fusion of all three anti-S reactive epitopes, improved substantially the recognition and binding to their cognate antibodies. These results provide insights into humoral immune relevant epitopes and tropism characteristics of the S protein with implications for the development of subunit vaccines or other biologics against SARS-CoV.SignificanceMapping epitopes within the receptor binding domains of viruses which are essential for viral tropism is critical for developing antiviral agents and subunit vaccines. In this study we have engineered the surface of Qubevirus to display a peptide library derived from the SARS-CoV S protein. In biopanning with S protein antibodies, we have identified three peptide fragments (EP1, EP2 and EP3) which reacted selectively with antibodies specific to the S protein. We demonstrated that all recombinant phage displayed peptide fragments both individually and as chimera exposed important immunological epitopes to their cognate antibodies. A peptide fragment F6 situated at S425-525, was found containing the binding determinant of the recombinant human angiotensin converting enzyme 2 (rhACE2), with the shortest active binding motif situated between residues S437-492. The platform is rapidly to identify epitopes and receptor binding sites within viral receptors found in target host cell. Thus, this platform holds great significance.
Functional and anatomical connectivity predict brain stimulation’s mnemonic effects
Authors: Ezzyat Youssef , Kragel James E. , Solomon Ethan A. , Lega Bradley C. , Aronson Joshua P. , Jobst Barbara C. , Gross Robert E. , Sperling Michael R. , Worrell Gregory A. , Sheth Sameer A. , Wanda Paul A. , Rizzuto Daniel S. , Kahana Michael J. 
Published: July 2023

AbstractClosed-loop direct brain stimulation is a promising tool for modulating neural activity and behavior. However, it remains unclear how to optimally target stimulation to modulate brain activity in particular brain networks that underlie particular cognitive functions. Here, we test the hypothesis that stimulation’s behavioral and physiological effects depend on the stimulation target’s anatomical and functional network properties. We delivered closed-loop stimulation as 47 neurosurgical patients studied and recalled word lists. Multivariate classifiers, trained to predict momentary lapses in memory function, triggered stimulation of the lateral temporal cortex (LTC) during the study phase of the task. We found that LTC stimulation specifically improved memory when delivered to targets near white matter pathways. Memory improvement was largest for targets near white matter that also showed high functional connectivity to the brain’s memory network. These targets also reduced low-frequency activity in this network, an established marker of successful memory encoding. These data reveal how anatomical and functional networks mediate stimulation’s behavioral and physiological effects, provide further evidence that closed-loop LTC stimulation can improve episodic memory, and suggest a method for optimizing neuromodulation through improved stimulation targeting.
Targeting BMI-1 to deplete antibody-secreting cells in autoimmunity
Authors: Polmear Jack , Hailes Lauren , Olshansky Moshe , Rischmueller Maureen , L’Estrange-Stranieri Elan , Fletcher Anne L , Hibbs Margaret L , Bryant Vanessa L , Good-Jacobson Kim L 
Published: July 2023

ABSTRACTObjectivesB cells drive the production of autoreactive antibody-secreting cells (ASCs) in autoimmune diseases such as Systemic Lupus Erythematosus (SLE) and Sjögren’s syndrome, causing long-term organ damage. Current treatments for antibody-mediated autoimmune diseases target B cells or broadly suppress the immune system. However, pre-existing long-lived ASCs are often refractory to treatment, leaving a reservoir of autoreactive cells that continue to produce antibody. Therefore, the development of novel treatment methods targeting ASCs is vital to improve patient outcomes. Our objective was to test whether targeting the epigenetic regulator BMI-1 could deplete ASCs in autoimmune conditionsin vivoandin vitro.MethodsUse of a BMI-1 inhibitor in both mouse and human autoimmune settings was investigated.Lyn-/-mice, a model of SLE, were treated with the BMI-1 small molecule inhibitor PTC-028, before assessment of ASCs, serum antibody and immune complexes. To examine human ASC survival, a novel human fibroblast-based assay was established, and the impact of PTC-028 on ASCs derived from Sjögren’s syndrome patients evaluated.ResultsBMI-1 inhibition significantly decreased splenic and bone marrow ASCs inLyn-/-mice. The decline in ASCs was linked to aberrant cell cycle gene expression and led to a significant decrease in serum IgG3, immune complexes and anti-DNA IgG. PTC-028 was also efficacious in reducingex vivoplasma cell survival from both Sjögren’s syndrome patients and age-matched healthy donors.ConclusionThese data provide evidence that inhibiting BMI-1 can deplete ASC in a variety of contexts and thus BMI-1 is a viable therapeutic target for antibody-mediated autoimmune diseases.
The fate of bacterial secondary metabolites in the rhizosphere:Streptomycesdegrades and feeds on cyclic lipopeptides produced by competitors
Authors: Rigolet Augustin , Argüelles Arias Anthony , Anckaert Adrien , Quinton Loïc , Rigali Sébastien , Tellatin Deborah , Burguet Pierre , Ongena Marc 
Published: July 2023

AbstractCyclic lipopeptides are key bioactive secondary metabolites produced by some plant beneficial rhizobacteria such asPseudomonasandBacillus. They exhibit antimicrobial properties, promote induced systemic resistance in plants and support key developmental traits including motility, biofilm formation and root colonization. However, our knowledge about the fate of lipopeptides once released in the environment and especially upon contact with neighboring rhizobacteria remains limited. Here, we investigated the enzymatic degradation ofBacillusandPseudomonascyclic lipopeptides byStreptomyces venezuelae. We observed thatStreptomycesis able to degrade the three lipopeptides surfactin, iturin and fengycin upon confrontation with ofB. velezensis in vitroandin plantaaccording to specific mechanisms.S. venezuelaewas also able to degrade the structurally diverse sessilin, tolaasin, orfamide, xantholisin and putisolvin-type lipopeptides produced byPseudomonas, indicating that this trait is likely engage in the interaction with various competitors.Furthermore, the degradation of CLPs is associated with the release of free amino and fatty acids and was found to enhanceStreptomycesgrowth, indicating a possible nutritional utilization. Thereby, this work stresses on how the enzymatic arsenal ofS. venezuelaemay contribute to its adaptation to BSMs-driven interactions with microbial competitors. The ability ofStreptomycesto degrade exogenous lipopeptides and feed on them adds a new facet to the implications of the degradation of those compounds byStreptomyces, where linearization of surfactin was previously reported as a detoxification mechanism. Additionally, we hypothesize that lipopeptide-producing rhizobacteria and their biocontrol potential are impacted by the degradation of their lipopeptides as observed with the polarized motility ofB. velezensis, avoiding the confrontation zone withStreptomycesand the loss of antifungal properties of degraded iturin. This work illustrates how CLPs, once released in the environment, may rapidly be remodeled or degraded by members of the bacterial community, with potential impacts on CLP-producing rhizobacteria and the biocontrol products derived from them.
Measurable ResidualIDH1before Allogeneic Transplant for Acute Myeloid Leukemia
Authors: Gui Gege , Dillon Laura W. , Ravindra Niveditha , Hegde Pranay S. , Andrew Georgia , Mukherjee Devdeep , Wong Zoë , Auletta Jeffery , Chaer Firas El , Chen Evan , Chen Yi-Bin , Corner Adam , Devine Steven M. , Iyer Sunil , Jimenez Jimenez Antonio Martin , De Lima Marcos J.G. , Litzow Mark R. , Kebriaei Partow , Spellman Stephen R , Zeger Scott L. , Page Kristin M. , Hourigan Christopher S. 
Published: August 2023

AbstractMeasurable residual disease (MRD) in adults with acute myeloid leukemia (AML) in complete remission is an important prognostic marker, but detection methodology requires optimization. The persistence of mutatedNPM1orFLT3-ITD in the blood of adult patients with AML in first complete remission (CR1) prior to allogeneic hematopoetic cell transplant (alloHCT) has been established as associated with increased relapse and death after transplant. The prognostic implications of persistence of other common AML-associated mutations, such asIDH1, at this treatment landmark however remains incompletely defined. We performed testing for residualIDH1variants (IDH1m) in pre-transplant CR1 blood of 148 adult patients undergoing alloHCT forIDH1-mutated AML at a CIBMTR site between 2013-2019. No post-transplant differences were observed between those testingIDH1m positive (n=53, 36%) and negative pre-transplant (overall survival: p = 0.4; relapse: p = 0.5). For patients withIDH1mutated AML co-mutated withNPM1and/orFLT3-ITD, only detection of persistent mutatedNPM1and/orFLT3-ITD was associated with significantly higher rates of relapse (p = 0.01). These data, from the largest study to date, do not support the detection ofIDH1mutation in CR1 blood prior to alloHCT as evidence of AML MRD or increased post-transplant relapse risk.
Enhancing gene transfer to renal tubules and podocytes by context-dependent selection of AAV capsids
Authors: Furusho Taisuke , Adachi Kei , Galbraith-Liss Mia , Sairavi Anusha , Das Ranjan , Nakai Hiroyuki 
Published: July 2023

AbstractDespite recent remarkable advancements in adeno-associated virus (AAV) vector technologies, effective gene delivery to the kidney remains a significant challenge. Here we show that AAV vector transduction in proximal tubules and podocytes, the crucial targets for renal gene therapy, can be enhanced remarkably through a meticulous selection of both AAV capsids and route of administration, tailored to the condition of the kidney. In this study, we performed a side-by-side comparison of 47 AAV capsids using AAV Barcode-Seq and identified six AAV capsids, including AAV-KP1, that exhibit remarkable enhancement of renal transduction in mice when delivered locally via the renal vein or the renal pelvis. Individual capsid validation analyses revealed that local delivery of AAV-KP1, but not AAV9, enables remarkably enhanced proximal tubule transduction while minimizing off-target liver transduction. In a mouse model of chronic kidney disease, intravenous administration of AAV9, not AAV-KP1, showed efficient renal tubule and podocyte transduction, which was not observed in the control wild-type mice. We also provide evidence that these contrasting observations between AAV-KP1 and AAV9 are attributed to their distinct pharmacokinetic profiles. Thus, this study highlights the importance of context-dependent capsid selection and engineering for successful renal gene therapy.
Nepicastat, a dopamine-beta-hydroxylase inhibitor decreases blood pressure and induces the infiltration of macrophages and B cells in the heart of spontaneous hypertensive rats
Authors: Chandan Shivanshu , Kosher Ganesh 
Published: July 2023

AbstractNepicastat is a potent dopamine-beta-hydroxylase inhibitor that modulates the sympathetic nervous system by inhibiting the synthesis of norepinephrine. Nepicastat is a potential drug for the treatment of congestive heart failure. We sought to investigate the mechanistic role of Nepicastsat in the heart of Spontaneous Hypertensive Rats (SHR) rats. Here, we investigated if Nepicastat at both acute (7 days) and chronic administration (14 days) decrease blood pressure and echocardiography parameters in SHR rats. SHR 3-4 months male rats were administered either Nepicastsat (30mg/kg, orally), Enalapril (10 mg/kg, orally), or vehicle for 7 days or 14 days. Blood pressure and echocardiography parameters were recorded on day 0, day 3, day 7, and day 14 of drug administration. The animals were sacrificed, and tissues are collected for histology, qRTPCR, and flow cytometry analysis. At both acute and chronic administration, Nepicastat decreased systolic blood pressure and intraventricular septal thickness of SHR rats compared to vehicle groups. The decrease in blood pressure was comparable to Enalapril treated rats. Interestingly, Nepicastat also decreased the infiltrating macrophages and B cells in the hearts of SHR rats. In conclusion, Nepicastsat consistently decreased the systolic blood pressure but increased the macrophages and B cell infiltration in the heart of SHR rats.
Adapting antibacterial display to identify serum active macrocyclic peptide antibiotics
Authors: Randall Justin R. , Groover Kyra E. , O’Donnell Angela C. , Garza Joseph M. , Cole T. Jeffrey , Davies Bryan W. 
Published: July 2023

AbstractThe lack of available treatments for many antimicrobial resistant infections highlights the critical need for antibiotic discovery innovation. Peptides are an underappreciated antibiotic scaffold because they often suffer from proteolytic instability and toxicity towards human cells, makingin vivouse challenging. To investigate sequence factors related to serum activity, we adapt an antibacterial display technology to screen a library of peptide macrocycles for antibacterial potential directly in human serum. We identify dozens of new macrocyclic peptide antibiotic sequences and find that serum activity within our library is influenced by peptide length, cationic charge, and the number of disulfide bonds present. Interestingly, an optimized version of our most active lead peptide permeates the outer membrane of gram-negative bacteria without strong inner membrane disruption and kills bacteria slowly while causing cell elongation. This contrasts with traditional cationic antimicrobial peptides, which kill rapidly via lysis of both bacterial membranes. Notably, this optimized variant is not toxic to mammalian cells and retains its functionin vivo, suggesting therapeutic promise. Our results support the use of more physiologically relevant conditions when screening peptides for antimicrobial activity which retainin vivofunctionality.SignificanceTraditional methods of natural antibiotic discovery are low throughput and cannot keep pace with the development of antimicrobial resistance. Synthetic peptide display technologies offer a high-throughput means of screening drug candidates, but rarely consider functionality beyond simple target binding and do not consider retention of functionin vivo. Here, we adapt a function-based, antibacterial display technology to screen a large library of peptide macrocycles directly for bacterial growth inhibition in human serum. This screen identifies an optimized non-toxic macrocyclic peptide antibiotic retainingin vivofunction, suggesting this advancement could increase clinical antibiotic discovery efficiency.
Changes in coding and efficiency through modular modifications to a One Pot PURE system for in vitro transcription & translation
Authors: Ngo Phuoc H. T. , Ishida Satoshi , Busogi Bianca B. , Do Hannah , Ledesma Maximiliano A. , Kar Shaunak , Ellington Andrew 
Published: July 2023

ABSTRACTThe incorporation of unnatural amino acids are attractive methods for improving or bringing new and novel functions in peptides and proteins. Cell-free protein synthesis using the Protein Synthesis Using Recombinant Elements (PURE) system is an attractive platform for efficient unnatural amino acid incorporation. In this work, we further adapted and modified the One Pot PURE for a robust and modular system of enzymatic single site-specific of unnatural amino acid. We demonstrated the flexibility of this system through the introduction of two orthogonal aminoacyl tRNA synthetases and the suppression of two distinctive stop codons.
Genetic and functional diversity of β-N-acetylgalactosamine residue-targeting glycosidases expanded by deep-sea metagenome
Authors: Sumida Tomomi , Hiraoka Satoshi , Usui Keiko , Ishiwata Akihiro , Sengoku Toru , Stubbs Keith A , Tanaka Katsunori , Deguchi Shigeru , Fushinobu Shinya , Nunoura Takuro 
Published: July 2023

Abstractβ-N-Acetylgalactosamine-containing glycans play essential roles in several biological processes, including cell adhesion, signal transduction, and immune responses. β-N-Acetylgalactosaminidases hydrolyze β-N-acetylgalactosamine linkages of various glycoconjugates. However, their biological significance remains ambiguous, primarily because only one type of enzyme, exo-β-N-acetylgalactosaminidases that specifically act on β-N-acetylgalactosamine residues, has been documented so far. In this study, we identified three novel glycoside hydrolase families distributed among all three domains of life and characterized eight novel β-N-acetylgalactosaminidases and β-N-acetylhexosaminidase through sequence-based screening of deep-sea metagenomes and subsequent searching of public protein databases. Despite low sequence similarity, the crystal structures of these enzymes demonstrate that all enzymes share a prototype structure and diversify their substrate specificities (endo-, dual-endo/exo-, and exo-) through the accumulation of mutations and insertional amino acid sequences. The diverse β-N-acetylgalactosaminidases reported in this study could facilitate the comprehension of their structures and functions and present novel evolutionary pathways for expanding their substrate specificity.
Functional and topological analysis of PSENEN, the fourth subunit of the γ-secretase complex
Authors: Serneels Lutgarde , Bammens Leen , Zwijsen An , Tolia Alexandra , Chávez-Gutiérrez Lucía , De Strooper Bart 
Published: July 2023

AbstractThe γ-secretase complexes are intramembrane cleaving proteases involved in the generation of the Aβ peptides in Alzheimer’s Disease. The complex consists of four subunits, with Presenilin, harboring the catalytic site. Here, we study the role of the smallest subunit, PSENEN or Presenilin enhancer 2 (PEN-2), encoded by the genePsenen,in vivoandin vitro. We find a profound Notch-deficiency phenotype inPsenen−/−embryos confirming the essential role of PSENEN in the γ-secretase complex. We usedPsenen−/−fibroblasts to explore the structure-function of PSENEN by the Scanning Cysteine Accessibility Method. Glycine22 and Proline27 which border the membrane domains 1 and 2 of PSENEN are involved in complex formation and stabilization of γ-secretase. The hairpin structured hydrophobic membrane domains 1 and 2 are exposed to a water-containing cavity in the complex, while transmembrane domain 3 is not water exposed. We finally demonstrate the essential role of PSENEN for the cleavage activity of the complex. PSENEN is more than a structural component of the γ-secretase complex and might contribute to the catalytic mechanism of the enzyme.