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Campylobacter jejuni is the leading cause of food poisoning in the United States and Europe. The exterior cell surface of C. jejuni is coated with a capsular polysaccharide (CPS) that is essential for the maintenance and integrity of the bacterial cell wall and evasion of the host immune response. The identity and sequences of the monosaccharide components of the CPS are quite variable and dependent on the specific strain of C. jejuni. It is currently thought that the immediate precursor for the multiple variations found in the heptose moieties of the C. jejuni CPS is GDP-d-glycero-α-d-manno-heptose. In C. jejuni NCTC 11168, the heptose moiety is d-glycero-l-gluco-heptose. It has previously been shown that Cj1427 catalyzes the oxidation of GDP-d-glycero-α-d-manno-heptose to GDP-d-glycero-4-keto-α-d-lyxo-heptose using α-ketoglutarate as a cosubstrate. Cj1430 was now demonstrated to catalyze the double epimerization of this product at C3 and C5 to form GDP-d-glycero-4-keto-β-l-xylo-heptose. Cj1428 subsequently catalyzes the stereospecific reduction of this GDP-linked heptose by NADPH to form GDP-d-glycero-β-l-gluco-heptose. The three-dimensional crystal structure of Cj1430 was determined to a resolution of 1.85 A in the presence of bound GDP-d-glycero-β-l-gluco-heptose, a product analogue. The structure shows that it belongs to the cupin superfamily. The three-dimensional crystal structure of Cj1428 was solved in the presence of NADPH to a resolution of 1.50 A. Its fold places it into the short-chain dehydrogenase/reductase superfamily. Typically, members in this family display a characteristic signature sequence of YXXXK, with the conserved tyrosine serving a key role in catalysis. In Cj1428, this residue is a phenylalanine.

Host-cell cysteine proteases play an essential role in the processing of the viral spike protein of SARS coronaviruses. K777, an irreversible, covalent inactivator of cysteine proteases that has recently completed phase 1 clinical trials, reduced SARS-CoV-2 viral infectivity in several host cells: Vero E6 (EC50 10 μM. There was no toxicity to any of the host cell lines at 10-100 μM K777 concentration. Kinetic analysis confirmed that K777 was a potent inhibitor of human cathepsin L, whereas no inhibition of the SARS-CoV-2 cysteine proteases (papain-like and 3CL-like protease) was observed. Treatment of Vero E6 cells with a propargyl derivative of K777 as an activity-based probe identified human cathepsin B and cathepsin L as the intracellular targets of this molecule in both infected and uninfected Vero E6 cells. However, cleavage of the SARS-CoV-2 spike protein was only carried out by cathepsin L. This cleavage was blocked by K777 and occurred in the S1 domain of the SARS-CoV-2 spike protein, a different site from that previously observed for the SARS-CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of cathepsin L-mediated viral spike protein processing.

By repurposing DNICs designed for other medicinal purposes, the possibility of protease inhibition was investigated in silico using AutoDock 4.2.6 (AD4) and in vitro via a FRET protease assay. AD4 was validated as a predictive computational tool for coordinatively unsaturated DNIC binding using the only known crystal structure of a protein-bound DNIC, PDB- (calculation RMSD = 1.77). From the in silico data the dimeric DNICs TGTA-RRE, [(μ-S-TGTA)Fe(NO)2]2 (TGTA = 1-thio-β-d-glucose tetraacetate) and TG-RRE, [(μ-S-TG)Fe(NO)2]2 (TG = 1-thio-β-d-glucose) were identified as promising leads for inhibition via coordinative inhibition at Cys-145 of the SARS-CoV-2 Main Protease (SC2Mpro). In vitro studies indicate inhibition of protease activity upon DNIC treatment, with an IC50 of 38 ± 2 μM for TGTA-RRE and 33 ± 2 μM for TG-RRE. This study presents a simple computational method for predicting DNIC-protein interactions; the in vitro study is consistent with in silico leads.

By exploring the manufacturing methods of historic objects, cultural heritage studies can yield new insights into the history of technology. The required tunability of processing steps, however, is often unknown and the underlying physics and chemistry that provide insight into the object’s properties may also be lacking. A case in point is Böttger luster, a purple overglaze decoration famous for its distinctive iridescence, produced in the Meissen Manufactory from its earliest Böttger period (1710–1719) to around 1735. The iridescence of Böttger luster distinguishes it from contemporaneous purple glazes, motivating the exploration of what compositional and structural features give rise to this optical phenomenon. In this study, historic samples of Böttger luster and Purple of Cassius from Meissen are characterized and compared microscopically. The composition of both overglaze enamels is presented, including results from scanning transmission electron microscopy (STEM)-energy-dispersive X-ray spectroscopy (EDX) analyses. It was found that the iridescence and purple color of Böttger luster is due to the presence of gold nanoparticles in the glaze of the porcelain. Of specific interest is how the underlying physics of scattering and interference of the nanoparticle array is responsible for the iridescence that distinguishes Böttger luster from other gold-based purple colorants, including Purple of Cassius. Coupling these results with prior findings of Meissen porcelains, the glazes were recreated and characterized using scanning electron microscopy (SEM)-EDX, X-ray diffraction (XRD), and ultraviolet-visible spectroscopy (UV-Vis), offering insights into compositional requirements to produce purple luster.

The leading cause of bacterial gastroenteritis, Campylobacter jejuni, is a Gram-negative pathogen that contains a unique O-methyl phosphoramidate (MeOPN) on its capsular polysaccharide. Previously, MeOPN has been linked to the evasion of host immune responses and serum resistance. Despite the involvement of MeOPN in pathogenicity, the complete biosynthesis of this modification is unknown; however, the first four enzymatic steps have been elucidated. The second enzyme in this pathway, Cj1416, is a CTP:phosphoglutamine cytididylyltransferase that catalyzes the displacement of pyrophosphate from MgCTP by L-glutamine phosphate to form CDP-L-glutamine. Initially, Cj1416 was predicted to use phosphoramidate to form cytidine diphosphoramidate but no activity was detected with MgATP as a substrate. However, in the presence of MnCTP, Cj1416 can directly catalyze the formation of cytidine diphosphoramidate from phosphoramidate and MnCTP. Here we characterize the manganese-induced promiscuity of Cj1416. In the presence of Mn(2+), Cj1416 catalyzes the formation of 12 different reaction products using L-glutamine phosphate, phosphoramidate, methyl phosphate, methyl phosphonate, phosphate, arsenate, ethanolamine phosphate, glycerol-1-phosphate, glycerol-2-phosphate, serinol phosphate, L-serine phosphate or 3-phospho-D-glycerate as the nucleophile to displace pyrophosphate from CTP.

Three-dimensional (3D) multicomponent metal oxides with complex architectures could enable previously impossible energy storage devices, particularly lithium-ion battery (LIB) electrodes with fully controllable form factors. Existing additive manufacturing approaches for fabricating 3D multicomponent metal oxides rely on particle-based or organic-inorganic binders, which are limited in their resolution and chemical composition, respectively. In this work, aqueous metal salt solutions are used as metal precursors to circumvent these limitations, and provide a platform for 3D printing multicomponent metal oxides. As a proof-of-concept, architected lithium cobalt oxide (LCO) structures are fabricated by first synthesizing a homogenous lithium and cobalt nitrate aqueous photoresin, and then using it with digital light processing printing to obtain lithium and cobalt ion containing hydrogels. The 3D hydrogels are calcined to obtain micro-porous self-similar LCO architectures with a resolution of ~100μm. These free-standing, binder- and conductive additive-free LCO structures are integrated as cathodes into LIBs, and exhibit electrochemical capacity retention of 76% over 100 cycles at C/10. This facile approach to fabricating 3D LCO structures can be extended to other materials by tailoring the identity and stoichiometry of the metal salt solutions used, providing a versatile method for the fabrication of multicomponent metal oxides with complex 3D architectures.

K777 is a di-peptide analog that contains an electrophilic vinyl-sulfone moiety and is a potent, covalent inactivator of cathepsins. Vero E6, HeLa/ACE2, Caco-2, A549/ACE2, and Calu-3, cells were exposed to SARS-CoV-2, and then treated with K777. K777 reduced viral infectivity with EC50 values of inhibition of viral infection of: 74 nM for Vero E6, 50 values in the low micromolar range. No toxicity of K777 was observed for any of the host cells at 10-100 μM inhibitor. K777 did not inhibit activity of the papain-like cysteine protease and 3CL cysteine protease, encoded by SARS-CoV-2 at concentrations of ≤ 100 μM. These results suggested that K777 exerts its potent anti-viral activity by inactivation of mammalian cysteine proteases which are essential to viral infectivity. Using a propargyl derivative of K777 as an activity-based probe, K777 selectively targeted cathepsin B and cathepsin L in Vero E6 cells. However only cathepsin L cleaved the SARS-CoV-2 spike protein and K777 blocked this proteolysis. The site of spike protein cleavage by cathepsin L was in the S1 domain of SARS-CoV-2, differing from the cleavage site observed in the SARS CoV-1 spike protein. These data support the hypothesis that the antiviral activity of K777 is mediated through inhibition of the activity of host cathepsin L and subsequent loss of viral spike protein processing.SIGNIFICANCEThe virus causing COVID-19 is highly infectious and has resulted in a global pandemic. We confirm that a cysteine protease inhibitor, approved by the FDA as a clinical-stage compound, inhibits SARS-CoV-2 infection of several human and monkey cell lines with notable(nanomolar) efficacy. The mechanism of action of this inhibitor is identified as a specific inhibition of host cell cathepsin L. This in turn inhibits host cell processing of the coronaviral spike protein, a step required for cell entry. Neither of the coronaviral proteases are inhibited, and the cleavage site of spike protein processing is different from that reported in other coronaviruses. Hypotheses to explain the differential activity of the inhibitor with different cell types are discussed.

YcjR from Escherichia coli K-12 MG1655 catalyzes the manganese-dependent reversible epimerization of 3-keto-α-D-gulosides to the corresponding 3-keto-α-D-glucosides as a part of a proposed catabolic pathway for the transformation of D-gulosides to D-glucosides. The three-dimensional structure of the manganese-bound enzyme was determined by X-ray crystallography. The divalent manganese ion is coordinated to the enzyme by ligation to Glu-146, Asp-179, His-205, and Glu-240. When either of the two active site glutamate residues are mutated to glutamine the enzyme loses all catalytic activity for the epimerization of α-methyl-3-keto-D-glucoside at C4. However, the E240Q mutant is able to catalyze hydrogen/deuterium exchange of the proton at C4 of α-methyl-3-keto-D-glucoside in solvent D(2)O. The E146Q mutant does not catalyze this exchange reaction. These results indicate that YcjR catalyzes the isomerization of 3-keto-D-glucosides via proton abstraction at C4 by Glu-146 to form a cis-enediolate intermediate that is subsequently protonated on the opposite face by Glu-240 to generate the corresponding 3-keto-D-guloside. This conclusion is supported by docking of the cis-enediolate intermediate into the active site of YcjR based on the known binding orientation of D-fructose and D-psicose in the active site of D-psicose-3-epimerase. It is proposed that YcjR be named 3-keto-D-glucoside-4-epimerase.

Campylobacter jejuni, a leading cause of gastroenteritis world wide, has a unique O-methyl phosphoramidate (MeOPN) moiety attached to its capsular polysaccharide. Investigations into the biological role of MeOPN have revealed that it contributes to the pathogenicity of C. jejuni, and this modification is important for the colonization of C. jejuni. Previously, the reactions catalyzed by four enzymes (Cj1418-Cj1415) from C. jejuni that are required for the biosynthesis of the phosphoramidate modification have been elucidated. Cj1418 (L-glutamine kinase) catalyzes the formation of the initial phosphoramidate bond with the ATP-dependent phosphorylation of the amide nitrogen of L-glutamine. Here we show that Cj1418 catalyzes the phosphorylation of L-glutamine through a three-step reaction mechanism via the formation of covalent pyrophosphorylated (Enz-X-P(β)-P(γ)) and phosphorylated (Enz-X-P(β)) intermediates. In the absence of L-glutamine, the enzyme was shown to catalyze a positional isotope exchange (PIX) reaction within β-[(18)O(4)]-ATP in support of the formation of the Enz-X-P(β)-P(γ) intermediate. In the absence of ATP, the enzyme was shown to catalyze a molecular isotope exchange (MIX) reaction between L-glutamine phosphate and [(15)N-amide]-L-glutamine in direct support of the Enz-X-P(β) intermediate. The active site nucleophile has been identified as His-737 based on the lack of activity of the H737N mutant and amino acid sequence comparisons. The enzyme was shown to also catalyze the phosphorylation of D-glutamine, γ-L-glutamyl hydroxamate, γ-L-glutamyl hydrazide and β-L-aspartyl hydroxamate, in addition to L-glutamine.

Optimal procedures for reliable anti-cancer treatments involve the systematic delivery of zinc oxide nanoparticles, which spread through the circulatory system. The success of these procedures may largely depend on the NPs&rsquo, ability of self-adapting their physicochemical properties to overcome the different challenges facing at each stage on its way to the interior of a cancerous cell. In this article, we combine a multiscale approach, a unique nanoparticle model, and available experimental data to characterize the behavior of zinc oxide nanoparticles under different vessels rheology, pH levels, and biological environments. We investigate their ability to prevent aggregation, allow prolonged circulation time in the bloodstream, avoid clearance, conduct themselves through the capillarity system to reach damaged tissues, and selectively approach to target cancerous cells. Our results show that non-functionalized spherical zinc oxide nanoparticles with surface density N = 5.89 &times, 10&minus, 6 mol/m2, protonation and deprotonation rates pKa = 10.9 and pKb = &minus, 5.5, and NP size in the range of 20&ndash, 50 nm are the most effective, smart anti-cancer agents for biomedical treatments.

Campylobacter jejuni , a leading cause of gastroenteritis, produces a capsular polysaccharide that is derivatized with a unique O-methyl phosphoramidate (MeOPN) modification. This modification contributes to serum resistance and invasion of epithelial cells. Previously, the first three biosynthetic steps for the formation of MeOPN have been elucidated. The first step is catalyzed by a novel glutamine kinase (Cj1418), which catalyzes the ATP-dependent phosphorylation of the amide nitrogen of L-glutamine. L-Glutamine phosphate is used by CTP:phosphoglutamine cytidylyltransferase (Cj1416) to displace pyrophosphate from CTP to generate CDP-L-glutamine, which is then hydrolyzed by γ-glutamyl-CDP-amidate hydrolase (Cj1417) to form cytidine diphosphoramidate (CDP-NH(2)). Here we show that Cj1415 catalyzes the ATP-dependent phosphorylation of CDP-NH(2) to form 3′-phospho-cytidine-5′-diphosphoramidate. Cj1415 will also catalyze the phosphorylation of adenosine diphosphoramidate (ADP-NH(2)) and uridine diphosphoamidate (UDP-NH(2)) but at significantly reduced rates. It is proposed that Cj1415 be named cytidine diphosphoramidate kinase.

Campylobacter jejuni is a pathogenic Gram-negative bacterium and a leading cause of food-borne gastroenteritis. C. jejuni produces a capsular polysaccharide (CPS) that contains a unique O-methyl phosphoramidate modification (MeOPN). Recently, the first step in the biosynthetic pathway for the assembly of the MeOPN modification to the CPS was elucidated. It was shown that the enzyme Cj1418 catalyzes the phosphorylation of the amide nitrogen of L-glutamine to form L-glutamine phosphate. In this investigation the metabolic fate of L-glutamine phosphate was determined. The enzyme Cj1416 catalyzes the displacement of pyrophosphate from MgCTP by L-glutamine phosphate to form CDP-L-glutamine. The enzyme Cj1417 subsequently catalyzes the hydrolysis of CDP-L-glutamine to generate cytidine diphosphoramidate and L-glutamate. The structures of the two novel intermediates, CDP-L-glutamine and cytidine diphosphoramidate, were confirmed by 31P NMR spectroscopy and mass spectrometry. It is proposed that the enzyme Cj1416 be named CTP: phosphoglutamine cytidylyltransferase and that the enzyme Cj1417 be named γ-glutamyl-CDP-amidate hydrolase.

Bacterial capsular polysaccharides (CPS) are complex carbohydrate structures that play a role in the overall fitness of the organism. Campylobacter jejuni, known for being a major cause of bacterial gastroenteritis worldwide, produces a CPS with a unique O-methyl phosphoramidate (MeOPN) modification on specific sugar residues. The formation of P–N bonds in nature is relatively rare, and the pathway for the assembly of the phosphoramidate moiety in the CPS of C. jejuni is unknown. In this investigation we discovered that the initial transformation in the biosynthetic pathway for the MeOPN modification of the CPS involves the direct phosphorylation of the amide nitrogen of l-glutamine with ATP by the catalytic activity of Cj1418. The other two products are AMP and inorganic phosphate. The l-glutamine-phosphate product was characterized using 31P NMR spectroscopy and mass spectrometry. We suggest that this newly discovered enzyme be named l-glutamine kinase.

A guided inquiry laboratory experiment for teaching liquid/liquid extractions to first semester undergraduate organic chemistry students is described. This laboratory is particularly useful for introductory students as the analytes that are separated are highly colored dye molecules. This allows students to track into which phase each analyte partitions and allows them to determine if they have successfully separated a mixture of two dyes. During the first part of this experiment, students determine how the partitioning of the various analytes changes as a function of the pH of the aqueous layer. They use these data to determine appropriate solvents for separating mixtures of dyes. After students have learned about the theory and practice of liquid/liquid extractions using dyes, they then separate and identify an unknown simulated cutting agent from a simulated drug. This experiment provides students with a better understanding of the theory and practice of liquid/liquid extractions, and introduces them t...