Two-step Pathway Research Articles

Engineering Escherichia coli via introduction of the isopentenol utilization pathway to effectively produce geranyllinalool

BackgroundGeranyllinalool, a natural diterpenoid found in plants, has a floral and woody aroma, making it valuable in flavors and fragrances. Currently, its synthesis primarily depends on chemical methods, which are environmentally harmful and economically unsustainable. Microbial synthesis through metabolic engineering has shown potential for producing geranyllinalool. However, achieving efficient synthesis remains challenging owing to the limited availability of terpenoid precursors in microorganisms. Thus, an artificial isopentenol utilization pathway (IUP) was constructed and introduced in Escherichia coli to enhance precursor availability and further improve terpenoid synthesis.ResultsWe first constructed an artificial IUP in E. coli to enhance the supply of precursor geranylgeranyl diphosphate (GGPP) and then screened geranyllinalool synthases from plants to achieve efficient synthesis of geranyllinalool (274.78 ± 2.48 mg/L). To further improve geranyllinalool synthesis, we optimized various cultivation factors, including carbon source, IPTG concentration, and prenol addition and obtained 447.51 ± 6.92 mg/L of geranyllinalool after 72 h of shaken flask fermentation. Moreover, a scaled-up production in a 5-L fermenter was investigated to give 2.06 g/L of geranyllinalool through fed-batch fermentation. To the best of our knowledge, this is the highest reported titer so far.ConclusionsEfficient synthesis of geranyllinalool in E. coli can be achieved through a two-step pathway and optimization of culture conditions. The findings of this study provide valuable insights into the production of other terpenoids in E. coli.

52 The bag is not always bad: Implementing a two-step method for urine testing on the inpatient paediatric units

Abstract Background Urinary tract infections (UTIs) are a common source of infection in children. Best practice for diagnosis includes interpreting a urinalysis (UA) and urine culture. Urine cultures obtained through bladder catheterization are invasive and time consuming. A two-step approach with noninvasive UA screen, followed by urine culture if positive has previously demonstrated a reduction in unnecessary catheterizations and urine cultures in children 6-24 months with suspected UTIs. Pre-intervention, there was inconsistent use of screening point-of-care urinalysis prior to bladder catheterization for culture. Objectives To decrease the number of bladder catheterizations in children 6-24 months with a negative screen on Point of Care Test (POCT) urinalysis by 25% over 1 year on paediatrics wards at a paediatric tertiary care centre. Outcome measures: 1. Proportion of bladder catheterizations with negative or no UA screen prior 2. Proportion of UA completed as POCT rather than lab sample Process measures: 1. Number of urinary catheterizations performed for UTI screen 2. Number of times pathway is followed Balancing measure: 1. Missed UTIs Design/Methods Baseline data was collected from the paediatric inpatient units at a tertiary paediatric hospital from April 2020 – April 2021. A two-step pathway for collecting urine samples was adopted, including POCT UA screen on a bag sample, followed by bladder catheterization for culture if positive. The pathway was implemented using the Model for Improvement and multiple PDSA cycles. Intervention focused on education, interdisciplinary collaboration, and process standardization. Results Baseline data demonstrated a high rate of urinary catheter cultures despite a negative UA or no UA prior (69%). After pathway implementation, this rate decreased to 23%, with a reduction in initial UA collected by catheter from 14% to 5% (figure 1). The number of POCT UA performed has increased from a median of 64% pre-intervention to 80% post-intervention, resulting in fewer samples processed in the lab (figure 2). Conclusion Variability in practice for specimen choice for collection and investigation contributed to unnecessary catheterizations. Process standardization successfully improved patient-centered care and efficiency. Next steps include broadening pathway inclusion criteria to the 3–6-month age group. Potential competing interests This work was partially supported by the UMC (Utilization Management Committee) Resource Stewardship Grant. The authors declare no additional sources of funding or conflicts of interest.

Two-dimensional silk.

Despite the promise of silk-based devices, the inherent disorder of native silk limits performance. Here, we report highly ordered two-dimensional silk fibroin (SF) films grown epitaxially on van der Waals (vdW) substrates. Using atomic force microscopy, nano-Fourier transform infrared spectroscopy, and molecular dynamics, we show that the films consist of lamellae of SF molecules that exhibit the same secondary structure as the nanocrystallites of native silk. Increasing the SF concentration results in multilayers that grow either by direct assembly of SF molecules into the lamellae or, at high concentrations, along a two-step pathway beginning with a disordered monolayer that then crystallizes. Scanning Kelvin probe measurements show that these films substantially alter the surface potential; thus, they provide a platform for silk-based electronics on vdW solids.

Ceria-boosted Ni/Al2O3 catalysts for enhanced H2 production via acetic acid dry reforming

Acetic acid dry reforming (ADR) is a promising route for sustainable H2 generation. However, coke inhibition during ADR is the main challenge and not resolved by using suitable promoted catalysts. In this work, Ce promotion on 10%Ni/Al2O3 catalysts with 1-5 wt%Ce was evaluated for ADR at varied temperatures of 923–998 K and stoichiometric feed in a fixed-bed rig. CeO2 addition of 1–3% enhanced metal dispersion, and surface area whilst basic CeO2 character significantly boosted the concentration and density of basic sites on catalysts. Particularly, the CO2 uptake of promoted catalysts was about 2.49–3.73 times greater than that of counterpart sample. CH3COOH and CO2 conversions were enhanced with rising Ce loading and the highest reactant conversions were observed at 3 wt%Ce. The improved adsorption of acidic CH3COOH and CO2 molecules due to increasing amount of basic sites as well as redox attributes of CeO2 promoter could be responsible for the enhancement in ADR activity and yield of H2 and CO. The mechanistic two-step pathway for coke suppression induced by CeO2 promotion was elaborated in this work. Generally, carbonaceous species formation on 3%Ce–10%Ni/Al2O3 was considerably reduced about 1.6–2.0 times. H2/CO ratio varied from 0.59 to 0.65 relying on ADR temperature over 3%Ce–10%Ni/Al2O3. These H2/CO values, two times higher than theoretical H2/CO ratio in ADR, are compatible for downstream gas-to-liquid processes to selectively yield high molecular weight olefins. Water formation rate increased from 8.67 × 10−6 to 4.71 × 10−5molH2O gcat−1 s−1 with rising temperature within 923–998 K on 3%Ce–10%Ni/Al2O3.

A BN-Benzvalene.

The synthesis and crystallographic characterization of BN-benzvalene, the first second-row heteroatom-containing benzvalene, is described. BN-benzvalenes are produced via photoexcitation of C5-aryl-substituted 1,2-azaborines under flow conditions. Mechanistic studies support a boron-specific, two-step photoisomerization pathway involving a BN-Dewar benzene intermediate, which is distinct from the photoisomerization pathway proposed in benzene and phospha- and silabenzenes for the formation of their respective benzvalene analogues.

Dissecting diazirine photo-reaction mechanism for protein residue-specific cross-linking and distance mapping

While photo-cross-linking (PXL) with alkyl diazirines can provide stringent distance restraints and offer insights into protein structures, unambiguous identification of cross-linked residues hinders data interpretation to the same level that has been achieved with chemical cross-linking (CXL). We address this challenge by developing an in-line system with systematic modulation of light intensity and irradiation time, which allows for a quantitative evaluation of diazirine photolysis and photo-reaction mechanism. Our results reveal a two-step pathway with mainly sequential generation of diazo and carbene intermediates. Diazo intermediate preferentially targets buried polar residues, many of which are inaccessible with known CXL probes for their limited reactivity. Moreover, we demonstrate that tuning light intensity and duration enhances selectivity towards polar residues by biasing diazo-mediated cross-linking reactions over carbene ones. This mechanistic dissection unlocks the full potential of PXL, paving the way for accurate distance mapping against protein structures and ultimately, unveiling protein dynamic behaviors.

Photoinduced Cu(II)-Mediated Decarboxylative Thianthrenation of Aryl and Heteroaryl Carboxylic Acids.

Given that (hetero)aryl carboxylic acids are inexpensive materials available in a great variety from commercial and natural resources or synthesis, the strategies enabling their use as starting materials for preparing fine chemicals are highly sought after. Here we report a photoinduced Cu(II)-mediated protocol converting (hetero)aryl carboxylic acids into (hetero)aryl thianthrenium salts, high value-added building blocks that can undergo various subsequent transformations, creating an attractive two-step pathway for the divergent functionalization of these ubiquitous starting materials. The excellent compatibility of the method is shown by preparing a broad range of sterically and electronically varied (hetero)aryl thianthrenium salts, including derivatives of pharmaceuticals, such as ataluren, celecoxib, flavoxate, probenecid, repaglinide, and tamibarotene. The syntheses of 13 C-labeled probenecid and bioisosteres of ataluren as well as the unconventional modifications of celecoxib and flavoxate, illustrate the synthetic potential of the strategy. Mechanistic studies are in line with a reaction occurring through a photoinduced ligand-to-metal charge transfer (LMCT) of Cu(II)-arylcarboxylates, enabling radical decarboxylative carbometallation to form arylcopper(II) intermediates that in turn react with thianthrene to form the product. Noteworthy, the susceptibility of aryl thianthrenium salts to photodegradation is overcome by a Cu(I)-driven salvage loop, which continuously intercepts the transiently formed radicals and regenerates the products.

Chloroimidazolium Deoxyfluorination Reagent with H2F3- Anion as a Sole Fluoride Source.

In the study, we introduce an air-stable NHC-based deoxyfluorination reagent ImCl[H2F3], offering a promising avenue for deoxyfluorination across various substrates. Reagent efficiently fluorinates benzyl alcohols, carboxylic acids, and P(V) compounds without external fluoride sources. A mechanistic study reveals a two-step process involving benzyl chloride as an intermediate, shedding light on the two-step reaction pathway. The Hammet plot provides insights into reaction mechanisms with different substrates, enhancing our understanding of this versatile deoxyfluorination method.

Ex vivo metabolism kinetics of primary to secondary bile acids via a physiologically relevant human faecal microbiota model

Bile acids (BA) are synthesized in the human liver and undergo metabolism by host gut bacteria. In diseased states, gut microbial dysbiosis may lead to high primary unconjugated BA concentrations and significant perturbations to secondary BA. Hence, it is important to understand the microbial-mediated formation kinetics of secondary bile acids using physiologically relevant ex vivo human faecal microbiota models. Here, we optimized an ex vivo human faecal microbiota model to recapitulate the metabolic kinetics of primary unconjugated BA and applied it to investigate the formation kinetics of novel secondary BA metabolites and their sequential pathways. We demonstrated (1) first-order depletion of primary BA, cholic acid (CA) and chenodeoxycholic acid (CDCA), under non-saturable conditions and (2) saturable Michaelis-Menten kinetics for secondary BA metabolite formation with increasing substrate concentration. Notably, relatively lower Michaelis constants (Km) were associated with the formation of deoxycholic acid (DCA, 14.3 μM) and lithocholic acid (LCA, 140 μM) versus 3-oxo CA (>1000 μM), 7-keto DCA (443 μM) and 7-keto LCA (>1000 μM), thereby recapitulating clinically observed saturation of 7α-dehydroxylation relative to oxidation of primary BA. Congruently, metagenomics revealed higher relative abundance of functional genes related to the oxidation pathway as compared to the 7α-dehydroxylation pathway. In addition, we demonstrated gut microbial-mediated hyocholic acid (HCA) and hyodeoxycholic acid (HDCA) formation from CDCA. In conclusion, we optimized a physiologically relevant ex vivo human faecal microbiota model to investigate gut microbial-mediated metabolism of primary BA and present a novel gut microbial-catalysed two-step pathway from CDCA to HCA and, subsequently, HDCA.

Molecular basis of one-step methyl anthranilate biosynthesis in grapes, sweet orange, and maize.

Plants synthesize an array of volatile compounds, many of which serve ecological roles in attracting pollinators, deterring herbivores, and communicating with their surroundings. Methyl anthranilate (MeAA) is an anti-herbivory defensive volatile responsible for grape aroma that is emitted by several agriculturally relevant plants, including citrus, grapes, and maize. Unlike maize, which uses a one-step anthranilate methyltransferase (AAMT), grapes have been thought to use a two-step pathway for MeAA biosynthesis. By mining available transcriptomics data, we identified two AAMTs in Vitis vinifera (wine grape), as well as one ortholog in "Concord" grape. Many angiosperms methylate the plant hormone salicylic acid (SA) to produce methyl salicylate, which acts as a plant-to-plant communication molecule. Because the Citrus sinensis (sweet orange) SA methyltransferase can methylate both anthranilate (AA) and SA, we used this enzyme to examine the molecular basis of AA activity by introducing rational mutations, which identified several active site residues that increase activity with AA. Reversing this approach, we introduced mutations that imparted activity with SA in the maize AAMT, which uncovered different active site residues from those in the citrus enzyme. Sequence and phylogenetic analysis revealed that one of the Vitis AAMTs shares an ancestor with jasmonic acid methyltransferases, similar to the AAMT from strawberry (Frageria sp.). Collectively, these data demonstrate the molecular mechanisms underpinning AA activity across methyltransferases and identify one-step enzymes by which grapes synthesize MeAA.

Visible light-driven molecular oxygen activation by lead-zinc smelting slag and tartaric acid for efficient organic pollutant degradation and Cr(VI) reduction

The remediation of heavy metal and organic-contaminated wastewater remains a significant concern in environmental science, necessitating the development of cost-effective and efficient treatment systems. This study focuses on the setup of a highly effective molecular oxygen (O2) activation system using lead–zinc smelting slag (LZSS) and tartaric acid (TA) under visible light. The LZSS-TA complex exhibited rapid photochemical reactions and demonstrated a remarkable capacity for O2 activation, resulting in the in situ generation of hydrogen peroxide (H2O2) and 99 % degradation of para-chloroaniline (p-CA). The formation of H2O2 occurred through a two-step single electron transfer pathway, whereby C-centered radicals (CHOHCHOHCOO−) generated within the system initiated the reduction of O2 to form O2−, as a crucial intermediate. The produced O2− is then reduced by Fe(II) to generate H2O2 via proton-coupled electron transfer (PCET) processes. Light-induced ligand-to-metal charge transfer (LMCT) process is the key to the production of C-centered radicals and Fe(II), which determines the production efficiency of H2O2 and OH. Additionally, this system enabled simultaneous oxidation of p-CA by 98 % and reduction of Cr(VI) by 100 % within 60 min. More importantly, O2 activation by TA-assisted LZSS under natural sunlight/solar irradiation could efficiently degrade p-CA in both ultrapure water (94 %) and reservoir water (92.5 %) within 210 min. This study significantly contributes to the understanding of light-driven O2 activation and lays the foundation for the design of cost-effective and efficient pollution remediation systems.

Probing the energy barriers and stages of membrane protein unfolding using solid-state NMR spectroscopy.

Understanding how the amino acid sequence dictates protein structure and defines its stability is a fundamental problem in molecular biology. It is especially challenging for membrane proteins that reside in the complex environment of a lipid bilayer. Here, we obtain an atomic-level picture of the thermally induced unfolding of a membrane-embedded α-helical protein, human aquaporin 1, using solid-state nuclear magnetic resonance spectroscopy. Our data reveal the hierarchical two-step pathway that begins with unfolding of a structured extracellular loop and proceeds to an intermediate state with a native-like helical packing. In the second step, the transmembrane domain unravels as a single unit, resulting in a heterogeneous misfolded state with high helical content but with nonnative helical packing. Our results show the importance of loops for the kinetic stabilization of the whole membrane protein structure and support the three-stage membrane protein folding model.

Engineering of methionine-auxotroph Escherichia coli via parallel evolution of two enzymes from Corynebacterium glutamicum's direct-sulfurylation pathway enables its recovery in minimal medium

Methionine biosynthesis relies on the sequential catalysis of multiple enzymes. Escherichia coli, the main bacteria used in research and industry for protein production and engineering, utilizes the three-step trans-sulfurylation pathway catalyzed by L-homoserine O-succinyl transferase, cystathionine gamma synthase and cystathionine beta lyase to convert L-homoserine to L-homocysteine. However, most bacteria employ the two-step direct-sulfurylation pathway involving L-homoserine O-acetyltransferases and O-acetyl homoserine sulfhydrylase. We previously showed that a methionine-auxotroph Escherichiacoli strain (MG1655) with deletion of metA, encoding for L-homoserine O-succinyl transferase, and metB, encoding for cystathionine gamma synthase, could be complemented by introducing the genes metX, encoding for L-homoserine O-acetyltransferases and metY, encoding for O-acetyl homoserine sulfhydrylase, from various sources, thus altering the Escherichia coli methionine biosynthesis metabolic pathway to direct-sulfurylation. However, introducing metX and metY from Corynebacterium glutamicum failed to complement methionine auxotrophy. Herein, we generated a randomized genetic library based on the metX and metY of Corynebacterium glutamicum and transformed it into a methionine-auxotrophic Escherichia coli strain lacking the metA and metB genes. Through multiple enrichment cycles, we successfully isolated active clones capable of growing in M9 minimal media. The dominant metX mutations in the evolved methionine-autotrophs Escherichia coli were L315P and H46R. Interestingly, we found that a metY gene encoding only the N-terminus 106 out of 438 amino acids of the wild-type MetY enzyme is functional and supports the growth of the methionine auxotroph. Recloning the new genes into the original plasmid and transforming them to methionine auxotroph Escherichia coli validated their functionality. These results show that directed enzyme-evolution enables fast and simultaneous engineering of new active variants within the Escherichia coli methionine direct-sulfurylation pathway, leading to efficient complementation.

Understanding the stability of a plastic-degrading Rieske iron oxidoreductase system.

Rieske oxygenases (ROs) are a diverse metalloenzyme class with growing potential in bioconversion and synthetic applications. We postulated that ROs are nonetheless underutilized because they are unstable. Terephthalate dioxygenase (TPADO PDB ID 7Q05)is a structurally characterized heterohexameric α3β3 RO that, with its cognate reductase (TPARED), catalyzes the first intracellular step of bacterial polyethylene terephthalate plastic bioconversion. Here, we showed that the heterologously expressed TPADO/TPARED system exhibits only ~300 total turnovers at its optimal pH and temperature. We investigated the thermal stability of the system and the unfolding pathway of TPADO through a combination of biochemical and biophysical approaches. The system's activity is thermally limited by a melting temperature (Tm) of 39.9°C for the monomeric TPARED, while the independent Tm of TPADO is 50.8°C. Differential scanning calorimetry revealed a two-step thermal decomposition pathway for TPADO with Tm values of 47.6 and 58.0°C (ΔH = 210 and 509 kcal mol-1, respectively) for each step. Temperature-dependent small-angle x-ray scattering and dynamic light scattering both detected heat-induced dissociation of TPADO subunits at 53.8°C, followed by higher-temperature loss of tertiary structure that coincided with protein aggregation. The computed enthalpies of dissociation for the monomer interfaces were most congruent with a decomposition pathway initiated by β-β interface dissociation, a pattern predicted to be widespread in ROs. As a strategy for enhancing TPADO stability, we propose prioritizing the re-engineering of the β subunit interfaces, with subsequent targeted improvements of the subunits.

Simulations predict preferred Mg2+ coordination in a nonenzymatic primer-extension reaction center

The mechanism by which genetic information was copied prior to the evolution of ribozymes is of great interest because of its importance to the origin of life. The most effective known process for the nonenzymatic copying of an RNA template is primer extension by a two-step pathway in which 2-aminoimidazole-activated nucleotides first react with each other to form an imidazolium-bridged intermediate that subsequently reacts with the primer. Reaction kinetics, structure-activity relationships, and X-ray crystallography have provided insight into the overall reaction mechanism, but many puzzles remain. In particular, high concentrations of Mg2+ are required for efficient primer extension, but the mechanism by which Mg2+ accelerates primer extension remains unknown. By analogy with the mechanism of DNA and RNA polymerases, a role for Mg2+ in facilitating the deprotonation of the primer 3′-hydroxyl is often assumed, but no catalytic metal ion is seen in crystal structures of the primer-extension complex. To explore the potential effects of Mg2+ binding in the reaction center, we performed atomistic molecular dynamics simulations of a series of modeled complexes in which a Mg2+ ion was placed in the reaction center with inner-sphere coordination with different sets of functional groups. Our simulations suggest that coordination of a Mg2+ ion with both O3′ of the terminal primer nucleotide and the pro-Sp nonbridging oxygen of the reactive phosphate of an imidazolium-bridged dinucleotide would help to pre-organize the structure of the primer/template substrate complex to favor the primer-extension reaction. Our results suggest that the catalytic metal ion may play an important role in overcoming electrostatic repulsion between a deprotonated O3′ and the reactive phosphate of the bridged dinucleotide and lead to testable predictions of the mode of Mg2+ binding that is most relevant to catalysis of primer extension.

Evaluation of a new two-step frailty assessment of head and neck patients in a prospective cohort

PurposeAssessing frailty, in head and neck cancer (HNC) patients is key when choosing appropriate treatment. Optimal screening is challenging, as it should be feasible and should avoid over-referral for comprehensive geriatric assessment (CGA) This study aims to evaluate the association between geriatric assessment using a new two-step care pathway, referral to geriatrician and adverse outcomes.MethodsThis institutional retrospective analysis on a prospective cohort analysed the multimodal geriatric assessment (GA) of newly diagnosed HNC patients. Uni- and multivariable logistic regression was performed to study the association between the screening tests, and referral to the geriatrician for complete geriatric screening, and adverse outcomes.ResultsThis study included 539 patients, of whom 276 were screened. Patients who underwent the GA, were significantly older and more often had advanced tumour stages compared to non-screened patients. Referral to the geriatrician was done for 30.8% of patients. Of the 130 patients who underwent surgery, 26/130 (20%) experienced clinically relevant postoperative complications. Of the 184 patients who underwent (radio)chemotherapy, 50/184 (27.2%) had clinically relevant treatment-related toxicity. Age, treatment intensity, polypharmacy and cognitive deficits, were independently associated with referral to geriatrician. A medium to high risk of malnutrition was independently associated with acute radiation induced toxicity and adverse outcomes in general.ConclusionThe current study showed a 30.8% referral rate for CGA by a geriatrician. Age, treatment intensity, cognitive deficits and polypharmacy were associated with higher rates of referral. Furthermore, nutritional status was found to be an important negative factor for adverse treatment outcomes, that requires attention.

Process intensification for production of dispersants via integration of reaction and separation in a horizontal thin film evaporator

Process intensification can be achieved in specialty chemicals manufacturing via transition from batch-to-continuous processing. However, the absence of headspace in a continuous tubular reactor limits the ability to remove volatile byproducts from the reacting liquid flow. Previous studies showed that the production of succinimide dispersants follows a two-step amidation-dehydration pathway in which water is formed as a by-product. The inability to remove this water imposed a thermodynamic equilibrium limitation on the dehydration step, and hence necessitated a downstream drying step to obtain a dehydrated dispersant product within commercial specifications. In the present study, the use of an agitated, horizontal thin film evaporator (TFE) was investigated as a continuously operated reactive separator which combines reaction and dehydration into a single operating unit, reducing the overall cost and physical footprint of the process. The continuous TFE unit was first experimentally investigated as a stand-alone, integrated reactor-separator. Using this configuration, the obtained imide yield consistently exceeded the maximum thermodynamic yield at each operating temperature, confirming the efficiency of the integrated reaction and separation steps in the TFE. Next, the unit was modeled by integrating reaction kinetics with mass and energy balance equations, using appropriate correlations for heat and mass transfer from the literature which were verified for the experimental set-up. This model provided insights into the system behavior across different operating conditions, including the role of forward and reverse reactions along the TFE unit length. The model was then used to confirm the feasibility of replacing the two-step tubular reactor-evaporator process with a single TFE unit as an intensified reactive separator, further reducing the physical footprint of the process by enabling compact modular process designs.

An energy-conserving reaction in amino acid metabolism catalyzed by arginine synthetase

All forms of life are presumed to synthesize arginine from citrulline via a two-step pathway consisting of argininosuccinate synthetase and argininosuccinate lyase using citrulline, adenosine 5'-triphosphate (ATP), and aspartate as substrates. Conversion of arginine to citrulline predominantly proceeds via hydrolysis. Here, from the hyperthermophilic archaeon Thermococcus kodakarensis, we identified an enzyme which we designate "arginine synthetase". In arginine synthesis, the enzyme converts citrulline, ATP, and free ammonia to arginine, adenosine 5'-diphosphate (ADP), and phosphate. In the reverse direction, arginine synthetase conserves the energy of arginine deimination and generates ATP from ADP and phosphate while releasing ammonia. The equilibrium constant of this reaction at pH 7.0 is [Cit][ATP][NH3]/[Arg][ADP][Pi] = 10.1 ± 0.7 at 80 °C, corresponding to a ΔG°' of -6.8 ± 0.2 kJ mol-1. Growth of the gene disruption strain was compared to the host strain in medium composed of amino acids. The results suggested that arginine synthetase is necessary in providing ornithine, the precursor for proline biosynthesis, as well as in generating ATP. Growth in medium supplemented with citrulline indicated that arginine synthetase can function in the direction of arginine synthesis. The enzyme is widespread in nature, including bacteria and eukaryotes, and catalyzes a long-overlooked energy-conserving reaction in microbial amino acid metabolism. Along with ornithine transcarbamoylase and carbamate kinase, the pathway identified here is designated the arginine synthetase pathway.

The Gut Microbiota in Parkinson Disease: Interactions with Drugs and Potential for Therapeutic Applications.

The concept of a 'microbiota-gut-brain axis' has recently emerged as an important player in the pathophysiology of Parkinson disease (PD), not least because of the reciprocal interaction between gut bacteria and medications. The gut microbiota can influence levodopa kinetics, and conversely, drugs administered for PD can influence gut microbiota composition. Through a two-step enzymatic pathway, gut microbes can decarboxylate levodopa to dopamine in the small intestine and then dehydroxylate it to m-tyramine, thus reducing availability. Inhibition of bacterial decarboxylation pathways could therefore represent a strategy to increase levodopa absorption. Other bacterial perturbations common in PD, such as small intestinal bacterial overgrowth and Helicobacter pylori infection, can also modulate levodopa metabolism, and eradication therapies may improve levodopa absorption. Interventions targeting the gut microbiota offer a novel opportunity to manage disabling motor complications and dopa-unresponsive symptoms. Mediterranean diet-induced changes in gut microbiota composition might improve a range of non-motor symptoms. Prebiotics can increase levels of short-chain fatty acid-producing bacteria and decrease pro-inflammatory species, with positive effects on clinical symptoms and levodopa kinetics. Different formulations of probiotics showed beneficial outcomes on constipation, with some of them improving dopamine levels; however, the most effective dosage and duration and long-term effects of these treatments remain unknown. Data from faecal microbiota transplantation studies are preliminary, but show encouraging trends towards improvement in both motor and non-motor outcomes.This article summarises the most up-to-date knowledge in pharmacomicrobiomics in PD, and discusses how the manipulation of gut microbiota represents a potential new therapeutic avenue for PD.

Fundamentals of NO reduction by liquid wastes: The first application in hazardous waste incineration

To meet increasingly stringent standards of Nitrogen oxide (NOx), it is highly desirable to develop high-efficiency denitration technology during hazardous waste incineration. Therefore, a state-of-art decoupling combustion technology of liquid hazardous wastes (LHW) has been proposed to achieve low NOx emission. In this technology, LHW is introduced into a low-NOx reactor to reduce NOx generated from solid hazardous waste (SHW) incineration prior to its complete combustion. The present article is devoted to clarifying the optimal conditions for denitration reactions by LHW, so the capabilities of nitric oxide (NO) reduction by reduction by LHW model compounds, namely methanol and methylbenzene, were evaluated in fixed-bed and fluidized-bed reactors. The highest denitration efficiency is 78.9% at 900 °C for methanol and 70.0% at 1000 °C for methylbenzene in the test temperature range. The NO reduction activities were closely associated with cracking characteristics of LHW. The presence of oxygen (O2) exhibited different effects on denitration: it promoted NO reduction at low temperatures due to the generation of more denitration-favored agents but suppressed NO reduction at high temperatures due to the oxidation of denitration-favored agents into CO2 and H2O by excessive O2. Moreover, the fluidized-bed reactor, characterized by good heat transfer, exhibited an approximately 17% higher NO reducing efficiency compared to the fixed-bed reactor at low temperatures, but both reactors demonstrated similar NO reduction efficiencies of around 83% at high temperatures. In addition, methanol and methylbenzene showed higher NO reduction efficiencies than their cracking-produced reducing gases, with the reducing gases accounting for approximately 50% of the overall NO reduction efficiency at 1000 °C. According to the above results, a two-step denitration reaction pathway was proposed in which both cracking-formed free radicals and reducing gases contribute to the overall NO reduction. The results of this study can provide valuable insights for NO reduction in the hazardous waste disposal.