Efficacy in Predicting Negative Appendectomy Rates in Operated Acute Appendicitis Patients Using the Raja Isteri Pengiran Anak Saleha Appendicitis (RIPASA) Score Versus Modified Alvarado Score.

Introduction Acute appendicitis is the commonest abdominal surgical emergency globally. The most accepted management of acute appendicitis is surgical, either open or laparoscopic appendectomy. Overlapping clinical presentations with many genitourinary and gynecological conditions lead to difficulty in accurate diagnosis, making negative appendectomies an unwanted reality. With the advancement in technology, there have been constant efforts to minimize negative appendectomy rates (NAR) using imaging modalities like USG of the abdomen and the gold-standard imaging test, the contrast-enhanced computed tomography of the abdomen. Due to the cost incurred and the lesser availability of such imaging modalities and needed expertise in resource-poor settings, various clinical scoring systems were devised to accurately diagnose acute appendicitis and thereby decrease NAR. We conducted our study to determine the NAR between the Raja Isteri Pengiran Anak Saleha Appendicitis score (RIPASA) and the modified Alvarado (MA) scoring methods. Methods A prospective observational analytical study was conducted, including 50 patients presenting to our hospital with acute appendicitis and who underwent emergency open appendectomy. The need to operate was decided by the treating surgeon. Patients were stratified by both scores; the pre-operative scores were noted and were later compared with the histopathological diagnosis. Results A total of 50 clinically diagnosed patients with acute appendicitis were evaluated utilizing the RIPASA and the MA scores. The NAR was 2% using the RIPASA score vs 10% with the MA score. The sensitivity was 94.11% vs 70.58% (p<0.0001), the specificity was 93.75% vs 68.75% (p<0.0001), the positive predictive value (PPV) of 96.96% vs 82.75% (p<0.001), the negative predictive value (NPV) of 88.23% vs 52.38% (p<0.001), and NAR of 2% vs 10% (p<0.0001) in the RIPASA vs MA scoring method, respectively. Conclusions RIPASA score is highly efficacious and statistically significant in diagnosing acute appendicitis with higher PPV at higher scores and higher NPV with lower scores leading to decreased NAR compared with MA score.

Nucleophilic Radiofluorination Using Tri-tert-Butanol Ammonium as a Bifunctional Organocatalyst: Mechanism and Energetics.

We present a quantum chemical analysis of the 18F-fluorination of 1,3-ditosylpropane, promoted by a quaternary ammonium salt (tri-(tert-butanol)-methylammonium iodide (TBMA-I) with moderate to good radiochemical yields (RCYs), experimentally observed by Shinde et al. We obtained the mechanism of the SN2 process, focusing on the role of the -OH functional groups facilitating the reactions. We found that the counter-cation TBMA+ acts as a bifunctional promoter: the -OH groups function as a bidentate 'anchor' bridging the nucleophile [18F]F- and the -OTs leaving group or the third -OH. These electrostatic interactions cooperate for the formation of the transition states of a very compact configuration for facile SN2 18F-fluorination.

Sweetening Pharmaceutical Radiochemistry by 18F-Fluoroglycosylation: Recent Progress and Future Prospects.

In the field of 18F-chemistry for the development of radiopharmaceuticals for positron emission tomography (PET), various labeling strategies by the use of prosthetic groups have been implemented, including chemoselective 18F-labeling of biomolecules. Among those, chemoselective 18F-fluoroglycosylation methods focus on the sweetening of pharmaceutical radiochemistry by offering a highly valuable tool for the synthesis of 18F-glycoconjugates with suitable in vivo properties for PET imaging studies. A previous review covered the various 18F-fluoroglycosylation methods that were developed and applied as of 2014 (Maschauer and Prante, BioMed. Res. Int. 2014, 214748). This paper is an updated review, providing the recent progress in 18F-fluoroglycosylation reactions and the preclinical application of 18F-glycoconjugates, including small molecules, peptides, and high-molecular-weight proteins.

18F-Fluorination Using Tri-Tert-Butanol Ammonium Iodide as Phase-Transfer Catalyst: An Alternative Minimalist Approach.

The 18F syntheses of tracers for positron emission tomography (PET) typically require several steps, including extraction of [18F]fluoride from H2[18O]O, elution, and drying, prior to nucleophilic substitution reaction, being a laborious and time-consuming process. The elution of [18F]fluoride is commonly achieved by phase transfer catalysts (PTC) in aqueous solution, which makes azeotropic drying indispensable. The ideal PTC is characterized by a slightly basic nature, its capacity to elute [18F]fluoride with anhydrous solvents, and its efficient complex formation with [18F]fluoride during subsequent labeling. Herein, we developed tri-(tert-butanol)-methylammonium iodide (TBMA-I), a quaternary ammonium salt serving as the PTC for 18F-fluorination reactions. The favorable elution efficiency of [18F]fluoride using TBMA-I was demonstrated with aprotic and protic solvents, maintaining high 18F-recoveries of 96-99%. 18F-labeling reactions using TBMA-I as PTC were studied with aliphatic 1,3-ditosylpropane and aryl pinacol boronate esters as precursors, providing 18F-labeled products in moderate-to-high radiochemical yields. TBMA-I revealed adequate properties for application to 18F-fluorination reactions and could be used for elution of [18F]fluoride with MeOH, omitting an additional base and azeotropic drying prior to 18F-labeling. We speculate that the tert-alcohol functionality of TBMA-I promotes intermolecular hydrogen bonding, which enhances the elution efficiency and stability of [18F]fluoride during nucleophilic 18F-fluorination.

Metabolic engineering and synthetic biology for isoprenoid production in Escherichia coli and Saccharomyces cerevisiae.

Isoprenoids, often called terpenoids, are the most abundant and highly diverse family of natural organic compounds. In plants, they play a distinct role in the form of photosynthetic pigments, hormones, electron carrier, structural components of membrane, and defence. Many isoprenoids have useful applications in the pharmaceutical, nutraceutical, and chemical industries. They are synthesized by various isoprenoid synthase enzymes by several consecutive steps. Recent advancement in metabolic engineering and synthetic biology has enabled the production of these isoprenoids in the heterologous host systems like Escherichia coli and Saccharomyces cerevisiae. Both heterologous systems have been engineered for large-scale production of value-added isoprenoids. This review article will provide the detailed description of various approaches used for engineering of methyl-D-erythritol-4-phosphate (MEP) and mevalonate (MVA) pathway for synthesizing isoprene units (C5) and ultimate production of diverse isoprenoids. The review particularly highlighted the efforts taken for the production of C5-C20 isoprenoids by metabolic engineering techniques in E. coli and S. cerevisiae over a decade. The challenges and strategies are also discussed in detail for scale-up and engineering of isoprenoids in the heterologous host systems.Key points• Isoprenoids are beneficial and valuable natural products.• E. coli and S. cerevisiae are the promising host for isoprenoid biosynthesis.• Emerging techniques in synthetic biology enabled the improved production.• Need to expand the catalogue and scale-up of un-engineered isoprenoids. Metabolic engineering and synthetic biology for isoprenoid production in Escherichia coli and Saccharomyces cerevisiae.

Effect of tert-alcohol functional imidazolium salts on oligomerization and fibrillization of amyloid ß (1-42) peptide.

Imidazolium based IL's has gained vast interest in developing biological applications. Oligomerization and fibrillization of amyloid ß (1-42) peptide are mainly responsible for the extra-neuronal deposition of amyloid fibrils in neurodegenerative disorders like Alzheimer's disease (AD). Here, we report an effect of tert-BuOH-functional imidazolium ILs on oligomerization and fibrillization of amyloid ß (1-42) Peptide in vitro. In this study, a series of these [alkyl-tOHim][OMs] ILs with methyl sulphonate counter anion by varying alkyl chains were used. Among the seven protic ILs, four showed strong binding and inhibition activity for the formation of amyloid ß (1-42) aggregation by using Thioflavin T fluorescence binding assay. The secondary structural analysis of the peptide, pre-incubated with active ILs shows the loss of ordered ß-sheet amyloid structure. The longer alkyl chain ILs showed that an increased in amyloid binding and hence an inhibition effect on amyloid aggregation was enhanced. Thus, we propose that ILs could be presented as potential candidates for therapeutic intervention against Alzheimer's disease (AD).

Retraction: Synthesis of deuterated isopentyl pyrophosphates for chemo-enzymatic labelling methods: GC-EI-MS based 1,2-hydride shift in epicedrol biosynthesis.

[This retracts the article DOI: 10.1039/C9RA00163H.].

Retracted Article: Synthesis of deuterated isopentyl pyrophosphates for chemo-enzymatic labelling methods: GC-EI-MS based 1,2-hydride shift in epicedrol biosynthesis.

A sesquiterpene epicedrol cyclase mechanism was elucidated based on the gas chromatography coupled to electron impact mass spectrometry fragmentation data of deuterated (2H) epicedrol analogues. The chemo-enzymatic method was applied for the specific synthesis of 8-position labelled farnesyl pyrophosphate and epicedrol. EI-MS fragmentation ions compared with non-labelled and isotopic mass shift fragments suggest that the 2H of C6 migrates to the C7 position during the cyclization mechanism.

Enhancing epi-cedrol production in Escherichia coli by fusion expression of farnesyl pyrophosphate synthase and epi-cedrol synthase.

Terpene synthase catalyses acyclic diphosphate farnesyl diphosphate into desired sesquiterpenes. In this study, a fusion enzyme was constructed by linking Santalum album farnesyl pyrophosphate synthase (SaFPPS) individually with terpene synthase and Artemisia annua Epi-cedrol synthase (AaECS). The stop codon at the N-terminus of SaFPPS was removed and replaced by a short peptide (GSGGS) to introduce a linker between the two open reading frames. This fusion clone was expressed in Escherichia coli Rosseta DE3 cells. The fusion enzyme FPPS-ECS produced sesquiterpene 8-epi-cedrol from substrates isopentenyl pyrophosphate and dimethylallyl pyrophosphate through sequential reactions. The K m values for FPPS-ECS for isopentyl diphosphate was 4.71 µM. The fusion enzyme carried out the efficient conversion of IPP to epi-cedrol, in comparison to single enzymes SaFPPS and AaECS when combined together in enzyme assay over time. Further, the recombinant E. coli BL21 strain harbouring fusion plasmid successfully produced epi-cedrol in fermentation medium. The strain having fusion plasmid (pET32a-FPPS-ECS) produced 1.084 ± 0.09 mg/L epi-cedrol, while the strain harbouring mixed plasmid (pRSETB-FPPS and pET28a-ECS) showed 1.002 ± 0.07 mg/L titre in fermentation medium by overexpression and MEP pathway utilization. Structural analysis was done by I-TASSER server and docking was done by AutoDock Vina software, which suggested that secondary structure of the N- C terminal domain and their relative positions to functional domains of the fusion enzyme was greatly significant to the catalytic properties of the fusion enzymatic complex than individual enzymes.

Enzymatic Formation of a Skipped Methyl-Substituted Octaprenyl Side Chain of Longestin (KS-505a): Involvement of Homo-IPP as a Common Extender Unit.

Longestin (KS-505a), a specific inhibitor of phosphodiesterase, is a meroterpenoid that consists of a unique octacyclic terpene skeleton with branched methyl groups at unusual positions (C1 and C12). Biochemical analysis of Lon23, a methyltransferase involved in the biosynthesis of longestin, demonstrated that it methylates homoisopentenyl diphosphate (homo-IPP) to afford (3Z)-3-methyl IPP. This compound, along with IPP, is selectively accepted as extender units by Lon22, a geranylgeranyl diphosphate (GGPP) synthase homologue, to yield dimethylated GGPP (dmGGPP). The absolute configuration of dmGGPP was determined to be (4R,12R) by degradation and chiral GC analysis. These findings allowed us to propose an enzymatic sequence for key steps of the biosynthetic pathway of the unusual homoterpenoid longestin.

Sucrose capped gold nanoparticles as a plasmonic chemical sensor based on non-covalent interactions: Application for selective detection of vitamins B1 and B6 in brown and white rice food samples.

We report simple and selective method for detection of vitamins B1 and B6 in brown and white rice samples using localized surface plasmon resonance (LSPR) of sucrose capped gold nanoparticles (AuNPs) as a chemical sensor colorimetrically. Here, detection is based on the color change of AuNPs from pink to blue followed by a red shift of LSPR absorption band in UV-vis region with the addition of vitamins B1 and B6 into the NPs solution. A good linear range was observed in the range of 25-1000 ngmL-1 with detection limit of 8 ngmL-1 for B1 and 50-1000 ngmL-1 with detection limit of 15 ngmL-1 for vitamins B6. The employment of AuNPs for detection of B1 and B6 vitamins in rice food samples showed remarkable abilities in terms of the simplicity, low cost, stability, reproducibility and sensitivity.

Removal of endrin and dieldrin isomeric pesticides through stereoselective adsorption behavior on the graphene oxide-magnetic nanoparticles.

A novel stereoselective removal behavior of isomeric endrin and dieldrin pesticides from sample solution is demonstrated using nanocomposite of graphene oxide (GO) and iron oxide (Fe3O4) magnetic nanoparticles (MNPs). The removal efficiency of endrin and dieldrin was found higher when GO-MNPs was used as a separating probe than the individual use of GO and MNPs. The removal efficiency of both the pesticides was found to be more favorable when the dosage amount of GO-MNPs was 30 mg for 30-min contact time with pH 4.0 at room temperature. The good correlation of determination (R 2) with 0.975 and 0.973 values obtained for endrin and dieldrin, respectively demonstrated a well fitting of Langmuir adsorption isotherm model. The higher removal percentage (86.0%) and higher slope value of Langmuir adsorption isotherm were estimated for endrin compared to dieldrin (74.0%). The reason for higher adsorption percentage of endrin is due to the endo-position of oxygen atom in molecule favors more interaction of molecules with GO-MNPs compared to the exo-position of oxygen present in dieldrin. In addition, the higher value of R 2 for endrin and dieldrin demonstrated better suitability of pseudo-first-order and pseudo-second-order kinetic models, respectively. The advantages of the present method are use of simple UV-vis spectrophotometry for monitoring and low-cost use of GO-MNPs nanomaterial for the removal of pesticides from sample solution.

Cyclization mechanism of phomopsene synthase: mass spectrometry based analysis of various site-specifically labeled terpenes.

Elucidation of the cyclization mechanism catalyzed by terpene synthases is important for the rational engineering of terpene cyclases. We developed a chemoenzymatic method for the synthesis of systematically deuterium-labeled geranylgeranyl diphosphate (GGPP), starting from site-specifically deuterium-labeled isopentenyl diphosphates (IPPs) using IPP isomerase and three prenyltransferases. We examined the cyclization mechanism of tetracyclic diterpene phomopsene with phomopsene synthase. A detailed EI-MS analysis of phomopsene labeled at various positions allowed us to propose the structures corresponding to the most intense peaks, and thus elucidate a cyclization mechanism involving double 1,2-alkyl shifts and a 1,2-hydride shift via a dolabelladien-15-yl cation. Our study demonstrated that this newly developed method is highly sensitive and provides sufficient information for a reliable assignment of the structures of fragmented ions.

Copper Environment in Artificial Metalloproteins Probed by Electron Paramagnetic Resonance Spectroscopy.

The design of binding sites for divalent metals in artificial proteins is a productive platform for examining the characteristics of metal-ligand interactions. In this report, we investigate the spectroscopic properties of small peptides and four-helix bundles that bind Cu(II). Three small peptides, consisting of 15 amino acid residues, were designed to have two arms, each containing a metal-binding site comprised of different combinations of imidazole and carboxylate side chains. Two four-helix bundles each had a binding site for a central dinuclear metal cofactor, with one design incorporating additional potential metal ligands at two identical sites. The small peptides displayed pH-dependent, metal-induced changes in the circular dichroism spectra, consistent with large changes in the secondary structure upon metal binding, while the spectra of the four-helix bundles showed a predominant α-helix content but only small structural changes upon metal binding. Electron paramagnetic resonance spectra were measured at X-band revealing classic Cu(II) axial patterns with hyperfine coupling peaks for the small peptides and four-helix bundles exhibiting a range of values that were related to the specific chemical natures of the ligands. The variety of electronic structures allow us to define the distinctive environment of each metal-binding site in these artificial systems, including the designed additional binding sites in one of the four-helix bundles.

One molecule of ionic liquid and tert-alcohol on a polystyrene-support as catalysts for efficient nucleophilic substitution including fluorination.

The tert-alcohol and ionic liquid solvents in one molecule [mim-(t)OH][OMs] was immobilized on polystyrene and reported to be a highly efficient catalyst in aliphatic nucleophilic substitution using alkali metal salts. Herein, we investigated the catalytic activity of a new structurally modified polymer-supported tert-alcohol functionalized imidazolium salt catalyst in nucleophilic substitution of 2-(3-methanesulfonyloxypropyoxy)naphthalene as a model substrate with various metal nucleophiles. The tert-alcohol moiety of the ionic liquid with a hexyl chain distance from polystyrene had a better catalytic activity compared to the other resin which lacked an alkyl linker and tert-alcohol moiety. We found that the maximum [mim-(t)OH][OMs] loading had the best catalytic efficacy among the tested polystyrene-based ionic liquids (PSILs) in nucleophilic fluorination. The catalytic efficiency of the PS[him-(t)OH][OMs] as a phase transfer catalyst (PTC) was determined by carrying out various nucleophilic substitutions using the corresponding alkali metal salts from the third to sixth periodic in CH3CN or tert-BuOH media. The scope of this protocol with primary and secondary polar substrates containing many heteroatoms is also reported. This PS[him-(t)OH][OMs] catalyst not only enhances the reactivity of alkali metal salts and reduces the formation of by-products but also affords high yield with easy isolation.

A designed buried salt bridge modulates heterodimerization of a membrane peptide.

Specific helix-helix interactions underpin the correct assembly of multipass membrane proteins. Here, we show that a designed buried salt bridge mediates heterodimer formation of model transmembrane helical peptides in a pH-dependent manner. The model peptides bear side chains functionalized with either a carboxylic acid or a primary amine within a hydrophobic segment. The association behavior was monitored by Förster resonance energy transfer, revealing that heterodimer formation is maximized at a pH close to neutrality (pH 6.5), at which each peptide is found in a charged state. In contrast, heterodimerization is disfavored at low and high values of pH, because either the carboxylic acid or the primary amine is present in its neutral state, thus preventing the formation of a salt bridge. These findings provide a blueprint for the design and modulation of protein-protein interactions in membrane proteins.

Cyclic N-terminal loop of amylin forms non amyloid fibers.

We report for the first time, to our knowledge, that the N-terminal loop (N_loop) of amylin (islet amyloid polypeptide (IAPP) residues 1-8) forms extremely long and stable non-ß-sheet fibers in solution under the same conditions in which human amylin (hIAPP) forms amyloid fibers. This observation applies to the cyclic, oxidized form of the N_loop but not to the linear, reduced form, which does not form fibers. Our findings indicate a potential role of direct N_loop-N_loop interactions in hIAPP aggregation, which has not been previously explored, with important implications for the mechanism of hIAPP amyloid fiber formation, the inhibitory action of IAPP variants, and the competition between ordered and disordered aggregation in peptides of the calcitonin peptide family.

PNA-peptide assembly in a 3D DNA nanocage at room temperature.

Proteins and peptides fold into dynamic structures that access a broad functional landscape; however, designing artificial polypeptide systems is still a great challenge. Conversely, DNA engineering is now routinely used to build a wide variety of 2D and 3D nanostructures from hybridization based rules, and their functional diversity can be significantly expanded through site specific incorporation of the appropriate guest molecules. Here we demonstrate a new approach to rationally design 3D nucleic acid-amino acid complexes using peptide nucleic acid (PNA) to assemble peptides inside a 3D DNA nanocage. The PNA-peptides were found to bind to the preassembled DNA nanocage in 5-10 min at room temperature, and assembly could be performed in a stepwise fashion. Biophysical characterization of the DNA-PNA-peptide complex was performed using gel electrophoresis as well as steady state and time-resolved fluorescence spectroscopy. Based on these results we have developed a model for the arrangement of the PNA-peptides inside the DNA nanocage. This work demonstrates a flexible new approach to leverage rationally designed nucleic acid (DNA-PNA) nanoscaffolds to guide polypeptide engineering.

Structure analysis of geranyl pyrophosphate methyltransferase and the proposed reaction mechanism of SAM-dependent C-methylation.

In the typical isoprenoid-biosynthesis pathway, condensation of the universal C(5)-unit precursors isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) occurs via the common intermediates prenyl pyrophosphates (C(10)-C(20)). The diversity of isoprenoids reflects differences in chain length, cyclization and further additional modification after cyclization. In contrast, the biosynthesis of 2-methylisonorneol (2-MIB), which is responsible for taste and odour problems in drinking water, is unique in that it primes the enzymatic methylation of geranyl pyrophosphate (GPP) before cyclization, which is catalyzed by an S-adenosyl-L-methionine-dependent methyltransferase (GPPMT). The substrate of GPPMT contains a nonconjugated olefin and the reaction mechanism is expected to be similar to that of the steroid methyltransferase (SMT) family. Here, structural analysis of GPPMT in complex with its cofactor and substrate revealed the mechanisms of substrate recognition and possible enzymatic reaction. Using the structures of these complexes, methyl-group transfer and the subsequent proton-abstraction mechanism are discussed. GPPMT and SMTs contain a conserved glutamate residue that is likely to play a role as a general base. Comparison with the reaction mechanism of the mycolic acid cyclopropane synthase (MACS) family also supports this result. This enzyme represented here is the first model of the enzymatic C-methylation of a nonconjugated olefin in the isoprenoid-biosynthesis pathway. In addition, an elaborate system to avoid methylation of incorrect substrates is proposed.

Sequential enzymatic epoxidation involved in polyether lasalocid biosynthesis.

Enantioselective epoxidation followed by regioselective epoxide opening reaction are the key processes in construction of the polyether skeleton. Recent genetic analysis of ionophore polyether biosynthetic gene clusters suggested that flavin-containing monooxygenases (FMOs) could be involved in the oxidation steps. In vivo and in vitro analyses of Lsd18, an FMO involved in the biosynthesis of polyether lasalocid, using simple olefin or truncated diene of a putative substrate as substrate mimics demonstrated that enantioselective epoxidation affords natural type mono- or bis-epoxide in a stepwise manner. These findings allow us to figure out enzymatic polyether construction in lasalocid biosynthesis.