Metallocenyl 7-ACA Conjugates: Antibacterial Activity Studies and Atomic-Resolution X-ray Crystal St
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Lewandowski, Eric M.↗
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Simple Analysis of Primary and Secondary Bile Salt Hydrolysis in Mouse and Human Gut Microbiome Samples by Using Fluorogenic Substrates
Animals produce bile to maximize absorption of lipophilic nutrients in the gut. The physical properties of bile are largely dictated by amphipathic bile salt molecules, which also act as signaling molecules that bind host receptors and modulate physiological processes. Upon excretion of bile salts into the gut, the gut microbiome can create metabolites that have modified signaling capabilities. The category and magnitude of bile salt metabolism can affect the host either positively or negatively. A key modification is bile salt hydrolysis, which is a prerequisite step for all additional microbial modification. Here, we have synthesized five different fluorogenic bile salts that report on hydrolysis activity using a simple, continuous assay. We profile activity in both murine and human fecal samples. Our data demonstrate that most gut microbiomes have the highest capacity for hydrolysis of host-produced bile salts, but some microbially modified bile salts also display significant turnover.
59 BASIC BIOLOGICAL SCIENCES↗
A Hybrid Structural Method for Investigating Low Molecular Weight Oligomeric Structures of Amyloid Beta
Spurred in part by the failure of recent therapeutics targeting amyloid β plaques in Alzheimer's Disease (AD), attention is increasingly turning to the oligomeric forms of this peptide that form early in the aggregation process. However, while numerous amyloid β fibril structures have been characterized, primarily by NMR spectroscopy and cryo-EM, obtaining structural information on the low molecular weight forms of amyloid β that presumably precede and/or seed fibril formation has proved challenging. Furthermore, these transient forms are heterogeneous, and depend heavily on experimental conditions such as buffer, temperature, concentration, and degree of quiescence during measurement. Here, we present the concept for a new approach to delineating structural features of early-stage low molecular weight amyloid β oligomers, using a solvent accessibility assay in conjunction with simultaneous fluorescence measurements.
60 APPLIED LIFE SCIENCES↗
Development of the Safe and Broad‐Spectrum Aldehyde and Ketoamide Mpro inhibitors Derived from the Constrained α, γ‐AA Peptide Scaffold
Abstract SARS‐CoV‐2 is still wreaking havoc all over the world with surging morbidity and high mortality. The main protease (M pro ) is essential in the replication of SARS‐CoV‐2, enabling itself an active target for antiviral development. Herein, we reported the design and synthesis of a new class of peptidomimetics‐constrained α, γ‐AA peptides, based on which a series of aldehyde and ketoamide inhibitors of the M pro of SARS‐CoV‐2 were prepared. The lead compounds showed excellent inhibitory activity in the FRET‐based M pro enzymatic assay not only for the M pro of SARS‐CoV‐2 but also for SARS‐CoV and MERS‐CoV, along with HCoVs like HCoV‐OC43, HCoV‐229E, HCoV‐NL63 and HKU1. The X‐ray crystallographic results demonstrated that our compounds form a covalent bond with the catalytic Cys145. They also demonstrated effective antiviral activity against live SARS‐CoV‐2. Overall, the results suggest that α, γ‐AA peptide could be a promising molecular scaffold in designing novel M pro inhibitors of SARS‐CoV‐2 and other coronaviruses.
Chemistry↗
CO 2 Activation with Manganese Tricarbonyl Complexes through an H–Atom Responsive Benzimidazole Ligand
Herein, we report the synthesis and characterization of two manganese tricarbonyl complexes, Mn I (HL)(CO) 3 Br (1 a-Br) and Mn I (MeL)(CO) 3 Br (1 b-Br) (where HL=2-(2’-pyridyl)benzimidazole; MeL=1-methyl-2-(2’-pyridy)benzimidazole) and assayed their electrocatalytic properties for CO 2 reduction. A redox-active pyridine benzimidazole ancillary ligand in complex 1 a-Br displayed unique hydrogen atom transfer ability to facilitate electrocatalytic CO 2 conversion at a markedly lower reduction potential than that observed for 1 b-Br. Notably, a one-electron reduction of 1 a-Br yields a structurally characterized H-bonded binuclear Mn(I) adduct (2 a’) rather than the typically observed Mn(0)-Mn(0) dimer, suggesting a novel method for CO 2 activation. Furthermore, combining advanced electrochemical, spectroscopic, and single crystal X-ray diffraction techniques, we demonstrate the use of an H-atom responsive ligand may reveal an alternative, low-energy pathway for CO 2 activation by an earth-abundant metal complex catalyst.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
A Quick Route to Multiple Highly Potent SARS-CoV-2 Main Protease Inhibitors**
The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2M Pro ) to digest two of its translated long polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replicating in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1M Pro ), we have designed and synthesized a series of SC2M Pro inhibitors that contain β-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2M Pro active-site cysteine C145. All inhibitors display high potency with K i values at or below 100 nM. The most potent compound, MPI3, has as a K i value of 8.3 nM. Crystallographic analyses of SC2M Pro bound to seven inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549/ACE2 cells. Two inhibitors, MPI5 and MPI8, completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 μM and A549/ACE2 cells at 0.16-0.31 μM. Their virus inhibition potency is much higher than that of some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2M Pro inhibitors with ultra-high antiviral potency.
3C-like protease↗
Design, Synthesis and X-Ray Structural Studies of Potent HIV-1 Protease Inhibitors Containing C-4 Su
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Ghosh, Arun K.↗
Programmable Gene Knockdown in Diverse Bacteria Using Mobile‐CRISPRi
Abstract Facile bacterial genome sequencing has unlocked a veritable treasure trove of novel genes awaiting functional exploration. To make the most of this opportunity requires powerful genetic tools that can target all genes in diverse bacteria. CRISPR interference (CRISPRi) is a programmable gene‐knockdown tool that uses an RNA‐protein complex comprised of a single guide RNA (sgRNA) and a catalytically inactive Cas9 nuclease (dCas9) to sterically block transcription of target genes. We previously developed a suite of modular CRISPRi systems that transfer by conjugation and integrate into the genomes of diverse bacteria, which we call Mobile‐CRISPRi. Here, we provide detailed protocols for the modification and transfer of Mobile‐CRISPRi vectors for the purpose of knocking down target genes in bacteria of interest. We further discuss strategies for optimizing Mobile‐CRISPRi knockdown, transfer, and integration. We cover the following basic protocols: sgRNA design, cloning new sgRNA spacers into Mobile‐CRISPRi vectors, Tn 7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria, and ICE Bs1 transfer of Mobile‐CRISPRi to Bacillales. © 2020 The Authors. Basic Protocol 1 : sgRNA design Basic Protocol 2 : Cloning of new sgRNA spacers into Mobile‐CRISPRi vectors Basic Protocol 3 : Tn 7 transfer of Mobile‐CRISPRi to Gram‐negative bacteria Basic Protocol 4 : ICE Bs1 transfer of Mobile‐CRISPRi to Bacillales Support Protocol 1 : Quantification of CRISPRi repression using fluorescent reporters Support Protocol 2 : Testing for gene essentiality using CRISPRi spot assays on plates Support Protocol 3 : Transformation of E. coli by electroporation Support Protocol 4 : Transformation of CaCl 2 ‐competent E. coli
59 BASIC BIOLOGICAL SCIENCES↗
Selective Enrichment Coupled with Proteomics to Identify S-Acylated Plasma Membrane Proteins in Arabidopsis
Protein S-acylation, predominately in the form of palmitoylation, is a reversible lipid post-translational modification (PTM) on cysteines that plays important roles in protein localization, trafficking, activity, and complex assembly. The functions and regulatory mechanisms of Sacylation have been extensively studied in mammals, owing to remarkable development of the high-resolution proteomics and the discovery of the S-acylation-related enzymes. However, the advance of S-acylation studies in plants lags farther behind than that in mammals, mainly due to the lack of knowledge about proteins responsible for this process, such as protein acyltransferases (PATs) and their substrates. In this article, a systematic protocol to study global S-acylation in Arabidopsis seedlings is described. The procedures are presented in detail, including preparation of Arabidopsis seedlings, enrichment of plasma membrane (PM) proteins, followed by enrichment of S-acylated proteins/peptides based on the acyl-biotin exchange (ABE) method, and large-scale identification of S-acylated proteins/peptides via mass spectrometry. This approach enables researchers to study S-acylation of PM proteins in plants in a systematic and straightforward way. Basic protocol 1: Preparation of Arabidopsis seedling materials Basic protocol 2: Isolation and enrichment of plasma membrane proteins Support protocol 1: Determination of protein concentration using BCA assay Basic Protocol 3: Enrichment of S-acylated proteins by ABE method Support protocol 2: Protein precipitation by methanol/chloroform method Basic Protocol 4: Trypsin digestion and proteomic analysis Alternate protocol 1: Pre-resin digestion and peptide level enrichment.
S-acylation, palmitoylation, plasma membrane prote↗
Quantitative Proteomic Analysis of the Senescence-Associated Secretory Phenotype by Data-Independent Acquisition
Cellular senescence is a complex stress response that induces an essentially permanent cell cycle arrest and a complex secretory phenotype termed the senescence-associated secretory phenotype (SASP), which drives numerous aging pathologies. Characterization of the SASP can provide insights into aging and disease mechanisms, aging biomarker candidates, and targets for counteracting the deleterious effects of senescent cells. Here we describe a mass spectrometry (MS)-compatible protocol to (1) generate senescent cells using different stimuli, (2) collect conditioned medium containing proteins secreted by senescent cells (i.e., SASP), and (3) prepare the SASP for quantitative proteomic analysis using data-independent acquisition (DIA) MS. Basic Protocol 1: Generating ionizing radiation-induced senescent and control cells Alternate Protocol 1: Generating doxorubicin-induced senescent and control cells Alternate Protocol 2: Generating oncogenic RAS-induced senescent and control cells Alternate Protocol 3: Generating mitochondrial dysfunction-induced senescent and control cells Alternate Protocol 4: Generating atazanavir/ritonavir-induced senescent and control cells Support Protocol: A multiple-assay approach to confirm the phenotype of senescent cells Basic Protocol 2: Generating conditioned medium from senescent cells cultured in low serum and quiescent control cells Alternate Protocol 5: Generating conditioned medium from senescent cells cultured in complete medium and quiescent control cells Basic Protocol 3: Quantitative proteomic analysis of the SASP.
60 APPLIED LIFE SCIENCES↗
No evidence of top–down effects by ants on litter decomposition in a temperate grassland
Ants play multiple roles in ecosystems, but their ability to affect decomposition processes in temperate grasslands is relatively unknown. We investigated whether the suppression of ant populations influenced litter decomposition in grasslands via predation of some decomposers (e.g., mites and springtails) and/or microbial activity and composition. We performed two successful ant suppression treatments (seven weeks, 37% suppression, year 1, 10 weeks, 70% suppression, year 2) over the course of a 59-week experiment. We then assayed the effects of ant suppression using coarse- and fine-mesh litterbags and evaluated litter chemistry, microbial and arthropod communities, and microbial enzyme activity. Ant suppression efforts reduced ant abundance and altered ant, arthropod decomposer, and non-ant predator community composition. However, ant suppression did not affect decomposer arthropod abundance, litter mass loss, microbial composition, or enzyme activity in litterbags. Litterbag mesh size did not alter microbial composition, perhaps due to a failure to exclude decomposers, as mites and springtails were more or equally abundant in fine-mesh bags. Nevertheless, mesh size did change litter chemistry, suggesting that mesh size-mediated microenvironments affect decomposition environment regardless of invertebrate exclusion. Coarse-mesh litterbags had higher concentrations of microbial sugars, lignin, and N and lower concentrations of litter C and crystalline cellulose than fine-mesh litterbags. Litterbag mesh size may alter decomposition processes irrespective of invertebrate abundance. We found no evidence that ant predation was an important driver of decomposer populations or decomposition in these systems, and we suspect redundancy at the top of the detrital food web dilutes the role of ants.
54 ENVIRONMENTAL SCIENCES↗
Deprotection of N ‐ tert ‐Butoxycarbonyl (Boc) Protected Functionalized Heteroarenes via Addition–Elimination with 3‐Methoxypropylamine
Continued pursuit of functionalized soft‐N‐donor complexant scaffolds with favorable solubility and kinetics profiles applicable for the separation of the trivalent minor actinides from the lanthanides has attracted significant interest over the last three decades. Recent work from this laboratory resulted in the production of various N ‐Boc protected [1,2,4]triazinyl‐pyridin‐2‐yl indole Lewis basic procomplexants which necessitated the removal of the indole N ‐Boc protecting group prior to evaluation of complexant efficacy in separations assays. Traditional deprotection strategies involving trifluoroacetic and other protic and Lewis acids proved unsuccessful in removal of the recalcitrant indole‐ N ‐Boc protecting group necessitating the development of a new strategy for deprotection of this complexant class. A serendipitous result facilitated utilization of 3‐methoxypropylamine as a mild deprotecting agent for various N ‐Boc protected heteroarenes via a proposed addition–elimination mechanism. Method development, application to various heteroarenes including indoles, 1,2‐indazoles, 1,2‐pyrazoles, and related derivatives, a ten‐fold scale‐up reaction, and experimental evaluation of a preliminary mechanistic hypothesis are reported herein.
Gulledge, Zachary Z.↗
Transcriptomics-Based Points of Departure for Daphnia magna Exposed to 18 Per- and Polyfluoroalkyl Substances
Here, per- and polyfluoroalkyl substances (PFAS) represent a large group of contaminants of concern based on their widespread use, environmental persistence, and potential toxicity. Many traditional models for estimating toxicity, bioaccumulation, and other toxicological properties are not well suited for PFAS. Consequently, there is a need to generate hazard information for PFAS in an efficient and cost-effective manner. In the present study, Daphnia magna were exposed to multiple concentrations of 22 different PFAS for 24 h, in 96-well plate format. Following exposure, whole body RNA was extracted and extracts, each representing five exposed individuals, were subjected to RNA sequencing. Following analytical measurements to verify PFAS exposure concentrations, and quality control on processed cDNA libraries for sequencing, concentration-response modeling was applied to the data sets for 18 of the tested compounds, and the concentration at which a concerted molecular response occurred (transcriptomic point of departure; tPOD) was calculated. The tPODs, based on measured concentrations of PFAS, generally ranged from 0.03-0.58 µM (9.9-350 µg/L; interquartile range). In most cases, these concentrations were two orders of magnitude lower than similarly calculated tPODs for human cell lines exposed to PFAS. They were also lower than apical effect concentrations reported for seven PFAS for which some crustacean or invertebrate toxicity data were available, although there were a few exceptions. Despite being lower than most other available hazard benchmarks, Daphnia magna tPODs were, on average, four orders of magnitude greater than the maximum aqueous concentrations of PFAS measured in Great Lakes tributaries. Overall, this high throughput transcriptomics assay with Daphnia magna holds promise as a component of a tiered hazard evaluation strategy employing new approach methodologies.
59 BASIC BIOLOGICAL SCIENCES↗
Amount and reactivity of dissolved organic matter export are affected by land cover change from old‐growth to second‐growth forests in headwater ecosystems
Abstract Headwater forest ecosystems of the western USA generate a large portion of the dissolved organic matter (DOM) transported in streams across North America. Land cover changes that alter forest structure and species composition affect the quantity and composition of DOM transferred to aquatic ecosystems. Clear‐cut harvesting affects ~1% of the forest area of North America annually, leaving most western forests in varying stages of regrowth and the total area of old‐growth forest is decreasing. The consequences of this widespread management practice on watershed carbon cycling remain unknown. We investigated the role of land cover change, because of clear‐cut harvesting, from mixed‐species old‐growth to lodgepole pine‐dominated second‐growth forest on the character and reactivity of hillslope DOM exports. We evaluated inputs of DOM from litter leachates and export of DOM collected at the base of trenched hillslopes during a 3‐year period (2016–2018) at the Fraser Experimental Forest in north‐central Colorado, USA. Dissolved organic carbon and total dissolved nitrogen were higher in lateral subsurface flow draining old‐ versus second‐growth forest. Fluorescence spectroscopy showed that the DOM exported from the old‐growth forest was more heterogeneous and aromatic and that proteinaceous, microbially processed DOM components were more prevalent in the second‐growth forest. Biological oxygen demand assays revealed much lower microbial metabolism of DOM in litter leachate and subsurface exports from the old‐growth forest relative to second growth. Old‐growth and second‐growth forests are co‐mingled in managed ecosystems, and our findings demonstrate that land cover change from a mixture of conifer species to lodgepole pine dominance influences DOM inputs that can increase the reactivity of DOM transferred from terrestrial to aquatic ecosystems.
Fegel, Timothy S.↗
Quantitative assessment of distant recurrence risk in early stage breast cancer using a nonlinear combination of pathological, clinical and imaging variables
Abstract Use of genomic assays to determine distant recurrence risk in patients with early stage breast cancer has expanded and is now included in the American Joint Committee on Cancer staging manual. Algorithmic alternatives using standard clinical and pathology information may provide equivalent benefit in settings where genomic tests, such as OncotypeDx, are unavailable. We developed an artificial neural network (ANN) model to nonlinearly estimate risk of distant cancer recurrence. In addition to clinical and pathological variables, we enhanced our model using intraoperatively determined global mammographic breast density (MBD) and local breast density (LBD). LBD was measured with optical spectral imaging capable of sensing regional concentrations of tissue constituents. A cohort of 56 ER+ patients with an OncotypeDx score was evaluated. We demonstrated that combining MBD/LBD measurements with clinical and pathological variables improves distant recurrence risk prediction accuracy, with high correlation ( r = 0.98) to the OncotypeDx recurrence score.
Nichols, Brandon S.↗
Mitigating the allergenicity of peanut allergen Ara h 1 by cold atmospheric pressure argon plasma jet
Abstract BACKGROUND Peanut allergy is recognized as a major food allergy that triggers severe and even fatal symptoms. Avoidance of peanuts in the diet is the main option for current safety management. Processing techniques reducing peanut allergenicity are required to develop other options. Cold plasma is currently considered as a novel non‐thermal approach to alter protein structure and has the potential to alleviate immunoreactivity of protein allergen. RESULTS The application of a cold argon plasma jet to peanut protein extract could reduce the amount of a 64 kDa protein band corresponding to a major peanut allergen Ara h 1 using sodium dodecyl sulfate–polyacrylamide gel electrophoresis, but the overall protein size distribution did not change significantly. A decrease in peanut protein solubility was a possible cause that led to the loss of protein content in the soluble fraction. Immunoblotting and enzyme‐linked immunosorbent assay elucidated that the immunoreactivity of Ara h 1 was significantly decreased with the time treated with plasma. Ara h 1 antigenicity reduced by 38% after five scans (approximately 3 min) of cold argon plasma jet treatment, and the reduction was up to 66% after approximately 15 min of treatment. CONCLUSION The results indicate that cold argon plasma jet treatment could be a suitable platform for alleviating the immunoreactivity of peanut protein. This work demonstrates an efficient, compact, and rapid platform for mitigating the allergenicity of peanuts, and shows great potential for the plasma platform as a non‐thermal technique in the food industry. © 2023 Society of Chemical Industry.
Hsu, Fu‐Chiun↗
Detection of a multi–disease biomarker in saliva with graphene field effect transistors
Human carbonic anhydrase 1 (CA1) has been suggested as a biomarker for identification of several diseases including cancers, pancreatitis, diabetes, and Sjogren’s syndrome. However, the lack of a rapid, cheap, accurate, and easy-to-use quantification technique has prevented widespread utilization of CA1 for practical clinical applications. To this end, we present a label-free electronic biosensor for detection of CA1 utilizing highly sensitive graphene field effect transistors (G-FETs) as a transducer and specific RNA aptamers as a probe. The binding of CA1 with aptamers resulted in a positive shift in Dirac voltage V D of the G-FETs, the magnitude of which depended on target concentration. These aptameric G-FET biosensors showed the binding affinity (K D ) of ~2.3 ng/ml (70 pM), which is four orders lower than that reported using a gel shift assay. This lower value of K D enabled us to achieve a detection range (10 pg/ml - 100 ng/ml) which is well in line with the clinically relevant range. These highly sensitive devices allowed us to further prove their clinical relevance by successfully detecting the presence of CA1 in human saliva samples. In conclusion, the utilization of this label-free biosensor could facilitate the early stage identification of various diseases associated with changes in concentration of CAs.
77 NANOSCIENCE AND NANOTECHNOLOGY↗
Phenotypic effects of changes in the FTVTxK region of an Arabidopsis secondary wall cellulose synthase compared with results from analogous mutations in other isoforms
Abstract Understanding protein structure and function relationships in cellulose synthase (CesA), including divergent isomers, is an important goal. Here, we report results from mutant complementation assays that tested the ability of sequence variants of AtCesA7, a secondary wall CesA of Arabidopsis thaliana , to rescue the collapsed vessels, short stems, and low cellulose content of the irx3‐1 AtCesA7 null mutant. We tested a catalytic null mutation and seven missense or small domain changes in and near the AtCesA7 FTVTSK motif, which lies near the catalytic domain and may, analogously to bacterial CesA, exist within a substrate “gating loop.” A low‐to‐high gradient of rescue occurred, and even inactive AtCesA7 had a small positive effect on stem cellulose content but not stem elongation. Overall, secondary wall cellulose content and stem length were moderately correlated, but the results were consistent with threshold amounts of cellulose supporting particular developmental processes. Vibrational sum frequency generation microscopy allowed tissue‐specific analysis of cellulose content in stem xylem and interfascicular fibers, revealing subtle differences between selected genotypes that correlated with the extent of rescue of the collapsing xylem phenotype. Similar tests on PpCesA5 from the moss Physcomitrium (formerly Physcomitrella ) patens helped us to synergize the AtCesA7 results with prior results on AtCesA1 and PpCesA5. The cumulative results show that the FTVTxK region is important for the function of an angiosperm secondary wall CesA as well as widely divergent primary wall CesAs, while differences in complementation results between isomers may reflect functional differences that can be explored in further work.
Burris, Jason N.↗