Two-step Pathway Research Articles

Single-molecule mechanical folding and unfolding kinetics of armless mitochondrial tRNAArg from Romanomermis culicivorax * *Project supported by the Natural Science Foundation of Guangdong Province, China (Grant No. 2017A030310085) and the Science and Technology Planning Project of Guangdong Province, China (Grant No. 2018A050506034).

The mechanical stability of tRNAs contributes to their biological activities. The mitochondrial tRNAArg from Romanomermis culicivorax is the shortest tRNA ever known. This tRNA lacks D- and T-arms, represents a stem–bulge–stem architecture but still folds into a stable tertiary structure. Although its structure had been reported, studies on its mechanical folding and unfolding kinetic characteristics are lacking. Here, we directly measured the single-molecule mechanical folding and unfolding kinetics of the armless mt tRNAArg by using optical tweezers in different solution conditions. We revealed a two-step reversible unfolding pathway: the first and large transition corresponds to the unfolding of acceptor stem and bulge below 11 pN, and the second and small transition corresponds to the unfolding of anticodon arm at 12 pN–14 pN. Moreover, the free energy landscapes of the unfolding pathways were reconstructed. We also demonstrated that amino acid-chelated Mg2+(aaCM), which mimics the intracellular solution condition, stabilizes the bulge of mitochondrial tRNAArg possibly by reducing the topological constraints or stabilizing the possible local non-canonical base pairings within the bulge region. Our study revealed the solution-dependent mechanical stability of an armless mt tRNA, which may shed light on future mt tRNA studies.

Temperature-dependent kinetic pathways of heterogeneous ice nucleation competing between classical and non-classical nucleation

Ice nucleation on the surface plays a vital role in diverse areas, ranging from physics and cryobiology to atmospheric science. Compared to ice nucleation in the bulk, the water-surface interactions present in heterogeneous ice nucleation complicate the nucleation process, making heterogeneous ice nucleation less comprehended, especially the relationship between the kinetics and the structures of the critical ice nucleus. Here we combine Markov State Models and transition path theory to elucidate the ensemble pathways of heterogeneous ice nucleation. Our Markov State Models reveal that the classical one-step and non-classical two-step nucleation pathways can surprisingly co-exist with comparable fluxes at T = 230 K. Interestingly, we find that the disordered mixing of rhombic and hexagonal ice leads to a favorable configurational entropy that stabilizes the critical nucleus, facilitating the non-classical pathway. In contrast, the favorable energetics promotes the formation of hexagonal ice, resulting in the classical pathway. Furthermore, we discover that, at elevated temperatures, the nucleation process prefers to proceed via the classical pathway, as opposed to the non-classical pathway, since the potential energy contributions override the configurational entropy compensation. This study provides insights into the mechanisms of heterogeneous ice nucleation and sheds light on the rational designs to control crystallization processes.

Comparative Study on the Hydrogenation of Naphthalene over Both Al2O3-Supported Pd and NiMo Catalysts against a Novel LDH-Derived Ni-MMO-Supported Mo Catalyst.

Naphthalene hydrogenation was studied over a novel Ni–Al-layered double hydroxide-derived Mo-doped mixed metal oxide (Mo-MMO), contrasted against bifunctional NiMo/Al2O3, and Pd-doped Al2O3 catalysts, the latter of which with Pd loadings of 1, 2, and 5 wt %. Reaction rate constants were derived from a pseudo-first-order kinetic pathway describing a two-step hydrogenation pathway to tetralin (k1) and decalin (k2). The Mo-MMO catalyst achieved comparable reaction rates to Pd2%/Al2O3 at double concentration. When using Pd5%/Al2O3, tetralin hydrogenation was favored over naphthalene hydrogenation culminating in a k2 value of 0.224 compared to a k1 value of 0.069. Ni- and Mo-based catalysts produced the most significant cis-decalin production, with Mo-MMO culminating at a cis/trans ratio of 0.62 as well as providing enhanced activity in naphthalene hydrogenation compared to NiMo/Al2O3. Consequently, Mo-MMO presents an opportunity to generate more alkyl naphthenes in subsequent hydrodecyclization reactions and therefore a higher cetane number in transport fuels. This is contrasted by a preferential production of trans-decalin observed when using all of the Al2O3-supported Pd catalysts, as a result of octalin intermediate orientations on the catalyst surface as a function of the electronic properties of Pd catalysts.

A Simplified Model for Reversible Solid Oxide Fuel Cells

A simplified reversible steady-state solid oxide cell model is formulated and solved analytically with only a few calibration parameters to match experimental performance data. A Butler-Volmer type model is used for the hydrogen electrode while a two-step single pathway reduced-order oxygen reduction reaction model is applied in the oxygen electrode. It is shown that this simple model is capable of accurately predicting the trends of V-I data produced experimentally at various gas concentrations in both fuel cell and electrolysis modes.

Clinically Relevant Mutations of Mycobacterial GatCAB Inform Regulation of Translational Fidelity.

ABSTRACTMost bacteria employ a two-step indirect tRNA aminoacylation pathway for the synthesis of aminoacylated tRNAGln and tRNAAsn. The heterotrimeric enzyme GatCAB performs a critical amidotransferase reaction in the second step of this pathway. We have previously demonstrated in mycobacteria that this two-step pathway is error prone and translational errors contribute to adaptive phenotypes such as antibiotic tolerance. Furthermore, we identified clinical isolates of the globally important pathogen Mycobacterium tuberculosis with partial loss-of-function mutations in gatA, and demonstrated that these mutations result in high, specific rates of translational error and increased rifampin tolerance. However, the mechanisms by which these clinically derived mutations in gatA impact GatCAB function were unknown. Here, we describe biochemical and biophysical characterization of M. tuberculosis GatCAB, containing either wild-type gatA or one of two gatA mutants from clinical strains. We show that these mutations have minimal impact on enzymatic activity of GatCAB; however, they result in destabilization of the GatCAB complex as well as that of the ternary asparaginyl-transamidosome. Stabilizing complex formation with the solute trehalose increases specific translational fidelity of not only the mutant strains but also of wild-type mycobacteria. Therefore, our data suggest that alteration of GatCAB stability may be a mechanism for modulation of translational fidelity.

Molecular-level insights into the immobilization of vapor-phase mercury on Fe/Co/Ni-doped hierarchical molybdenum selenide

A novel and efficient adsorbent (TM-MoSe2, TM = Fe, Co, Ni) for mercury removal was developed and studied. The adsorption of mercury species (Hg0, HgCl, and HgCl2) and the oxidation of Hg0 by HCl on TM-MoSe2 (001) surface were explored at molecular level by density functional theory (DFT). The results shown that the Hg0 adsorption capacity of MoSe2 was improved by the doping of Fe/Co/Ni, which was also confirmed by experiments. The initial Hg0 removal efficiency of MoSe2-based adsorbents reached 96.4–100.0%. In addition, HgCl was mainly adsorbed on TM-MoSe2 (001) surface in the form of dissociation. The escape of Hg atom from HgCl resulted in the release of Hg0 again. However, HgCl2 could be fixed well on the surface of adsorbent through molecular adsorption or dissociative adsorption. For the oxidation process of Hg0 by HCl, it abided with the Langmuir–Hinshelwood mechanism. In comparison with direct oxidation (Hg → HgCl2), two-step pathway (Hg → HgCl → HgCl2) was an achievable reaction route with lower energy. Furthermore, the Hg → HgCl process was the rate-limiting step of the two-step pathway. The proposed adsorption and oxidation mechanism of mercury species on TM-MoSe2 (001) provide advanced strategies on the development of adsorbents for industrial mercury removal.

Chemoselective transfer hydrogenation of nitriles to secondary amines with nickel(II) catalysts

• Chemoselective nickel-mediated nitrile hydrogenation is reported. • The catalyst system displays the highest TON values for chemoselective nitrile hydrogenation. • The catalyst system mediates ammonia borane dehydrogenation near-quantitatively. • Evidence is provided for a two-step mechanistic pathway. Herein we report the selective transfer hydrogenation (TH) of nitriles to secondary (2°) amines with simple Ni(II)-catalysts using ammonia borane (AB) as a source of hydrogen (H 2 ). A bis(pyrazolylmethyl)pyridine ( L1 ) or ethylenediamine ( L4 ) ligated Ni(II) pre-catalyst, created in situ, could hydrogenate several aromatic- and aliphatic nitriles in full conversions and isolated yields of up to 88% under ambient temperature and in very short reaction times. Deuterium labelling experiments illustrated the incorporation of a proton on the nitrogen and hydride on the α-carbon of dibenzylamine. Using α-picoline borane, containing no dissociable protons, assisted with the postulation of a two-step TH mechanism of benzonitrile. AB was subjected to dehydrogenation and it was observed that a maximum of 2.96 equivalents of H 2 gas could be generated from NiCl 2 •6H 2 O/ L1 .

Development of purification process for dual-function recombinant human heavy-chain ferritin by the investigation of genetic modification impact on conformation.

Ferritin is a promising drug delivery platform and has been functionalized through genetic modifications. This work has designed and expressed a dual‐functional engineered human heavy‐chain ferritin (HFn) with the inserted functional peptide PAS and RGDK to extend half‐life and improve tumor targeted drug delivery. A facile and cost‐effective two‐step purification pathway for recombinant HFn was developed. The genetic modification was found to affect HFn conformation, and therefore varied the purification performance. Heat‐acid precipitation followed by butyl fast flow hydrophobic interaction chromatography (HIC) has been developed to purify HFn and modified HFns. Nucleic acid removal reached above 99.8% for HFn and modified HFns. However, HFn purity reached above 95% and recovery yield (overall) above 90%, compared with modified HFns purity above 82% and recovery yield (overall) above 58%. It is interesting to find that the inserted functional peptides significantly changed the molecule conformation, where a putative turnover of the E‐helix with the inserted functional peptides formed a “flop” conformation, in contrast with the “flip” conformation of HFn. It could be the cause of fragile stability of modified HFns, and therefore less tolerant to heat and acid condition, observed by the lower recovery yield in heat‐acid precipitation.

Interaction mechanism between cadmium species and SiO2 of municipal solid waste incineration fly ash: Effect of HCl

Cadmium (Cd) pollutants from the incineration of municipal solid waste (MSW) has drawn considerable attention due to their high volatility and biological toxicity. Current experimental results have revealed the strong interaction between SiO2 and Cd species, which provides an insight for the removal of Cd species, but the detailed mechanism is still unclear. Thus, with the density functional theory (DFT) calculations, the interaction mechanisms of Cd species over the SiO2(001) surface were investigated, considering the effect of HCl in MSW incineration flue gas. It is found that the adsorption manners of Cd0 and CdO are chemisorption over the SiO2 surface and the active sites are O sites, while the interaction between CdCl2 and the surface is dominated by weak chemisorption. HCl plays a dual role in the interaction of Cd0 and CdO with the surface. The introduction of HCl can promote the fixation of CdO and Cd0, but also promote their chlorination into CdCl2 whose adsorption on SiO2 is too weak to be captured. The chlorination of Cd0 can be achieved by two-step and one-step routes, both following the Eley-Rideal mechanism. Whereas, CdO can only be chloridized through a two-step pathway, in which the first step follows the Eley-Rideal mechanism and the second step follows the Langmuir-Hinshelwood mechanism. Compared with Cd0, the chlorination of CdO has a higher reaction energy barrier and is more difficult to proceed. Therefore, CdO is the most effortless cadmium pollutant for SiO2 to control.

Comparative Life Cycle Assessment of CO2 Electrosynthesis to Fuels and Feedstocks

Development of electrochemical pathways to convert CO2 into fuels and feedstock is rapidly progressing over the past decade. Here we present a comparative cradle-to-gate life cycle assessment (LCA) of one and two-step electrochemical conversion of CO2 to eight major value-added products; wherein we consider CO2 capture, conversion and product separation in our process model. We measure the carbon intensity (i.e., global warming impact) of one and two-step electrochemical routes with its counterparts – thermochemical CO2 utilization and fossil-fuel based incumbent synthesis routes for those products. Due to inevitable carbonate formation or CO2 crossover in one-step CO2 electrolysis using neutral pH membrane electrode assembly, the two-step electrosynthesis pathways (CO2 to CO in solid oxide electrolysis cell followed by CO electroreduction in alkaline flow cell) would be distinctively compelling through the lens of climate benefits. This analysis further reveals that the carbon intensity of electrosynthesis products is due to significant energy requirement for conversion (75–85% of total energy consumption for gas products) and product separation (30–85% of total energy consumption for liquid products) phases. Electrochemical route is highly sensitive to the electricity emission intensity and is compelling only when coupled with low emission intensity (<0.25 kg CO2e per kWh). As the technology advances, we identify near-term compelling products that would provide climate benefits over incumbent routes, including syngas, ethylene and n-propanol. We further identify technological goals required for electrochemical route to be competitive, notably achieving liquid product concentration >20 wt%. It is our hope that this analysis will guide the CO2 electrosynthesis community to target achieving these technological goals, such that when coupled with low-carbon electricity, electrochemical route would bring climate benefits in near future.

109 - Precision thoracic oncology in the UK: a two-step NGS pathway for extended molecular profiling in NSCLC

109 - Precision thoracic oncology in the UK: a two-step NGS pathway for extended molecular profiling in NSCLC

An environmental approach for the photodegradation of toxic pollutants from wastewater using silver nanoparticles decorated titania-reduced graphene oxide

Light-induced demineralization of organic pollutants is a recent development in the field of waste treatment. The present study focuses on TiO2-rGO-Ag hybrid nanocomposite through a two-step simultaneous synthesis pathway. The methods primarily involving facile solvothermal treatment (TiO2-rGO nanocomposite) in a water-ethanol mixture, followed by microwave irradiation method for deposition of silver (Ag) nanoparticles over the Binary nanocomposite synthesized. The resulting hybrid photocatalyst was well, and in-detail characterized using analytical techniques such as High-Resolution X-ray Diffractometer (HR-XRD), Transmission Electron Microscopy and High-Resolution Transmission Electron Microscopy (TEM/HR-TEM), High-Resolution Scanning Electron Microscopy (HR-SEM), Fourier-Transform Infra-Red Spectroscopy (FT-IR), Energy Dispersive X-Ray Spectroscopy (EDX), Raman Spectroscopy, Solid-State UV–Vis Spectroscopy and X-Ray Photoelectron spectroscopy (XPS). The nanomaterial was examined to determine photocatalytic property by conducting a photodegradation experiment of an aqueous solution of Methylene Blue (MB) dye and monitoring the changes. The photocatalytic efficiency of the nano-hybrid synthesized was also analyzed in detail for the degradation of compounds that active components of petrochemical pollutants such as Benzene, Toluene, and Phenol the Visible region of radiation in a photochemical reactor under ambient reaction conditions. The set of experiments suggested that the photocatalytic efficiency of ternary nanocomposite synthesized was quite noticeable. The variation in the catalyst's photoactivity with the change in the functional group was monitored, and the rate of reaction has been correlated with attached substituents.

The use of Lactobacillus plantarum esterase genes: a biotechnological strategy to increase the bioavailability of dietary phenolic compounds in lactic acid bacteria

In Lactobacillus plantarum the metabolism of hydroxybenzoic and hydroxycinnamic acid derivatives follows a similar two-step pathway, an esterase action followed by a decarboxylation. The L. plantarum esterase genes involved in these reactions have been cloned into pNZ8048 or pT1NX plasmids and transformed into technologically relevant lactic acid bacteria. None of the strains assayed can hydrolyse methyl gallate, a hydroxybenzoic ester. The presence of the L. plantarum tannase encoding genes (tanALp or tanBLp ) on these bacteria conferred their detectable esterase (tannase) activity. Similarly, on hydroxycinnamic compounds, esterase activity for the hydrolysis of ferulic acid was acquired by lactic acid bacteria when L. plantarum esterase (JDM1_1092) was present. This study showed that the heterologous expression of L. plantarum esterase genes involved in the metabolism of phenolic acids allowed the production of healthy compounds which increase the bioavailability of these dietary compounds in food relevant lactic acid bacteria.

Resolving atomic-scale phase transformation and oxygen loss mechanism in ultrahigh-nickel layered cathodes for cobalt-free lithium-ion batteries

Summary Doped LiNiO2 has recently become one of the most promising cathode materials for its high specific energy, long cycle life, and reduced cobalt content. Despite this, the degradation mechanism of LiNiO2 and its derivatives still remains elusive. Here, by combining in situ electron microscopy and first-principles calculations, we elucidate the atomic-level chemomechanical degradation pathway of LiNiO2-derived cathodes. We uncover that the O1 phase formed at high voltages acts as a preferential site for rock-salt transformation via a two-step pathway involving cation mixing and shear along (003) planes. Moreover, electron tomography reveals that planar cracks nucleated simultaneously from particle interior and surface propagate along the [100] direction on (003) planes, accompanied by concurrent structural degradation in a discrete manner. Our results provide an in-depth understanding of the degradation mechanism of LiNiO2-derived cathodes, pointing out the concept that suppressing the O1 phase and oxygen loss is key to stabilizing LiNiO2 for developing next-generation high-energy cathode materials.

Pathways to mental health consultations: A study from a tertiary care setting in India

Research indicates that help seeking for mental health is low and often delayed. Understanding pathways to care is crucial to facilitate mental health referrals and reduce the time to consultation. In the present study, 63 individuals were assessed on illness severity, attitudes towards help-seeking and pathways-to-care. Multiple pathways for therapy were noted, a delayed-pathway, two-step referral pathway and a direct-pathway. Most prominent pathway was the delayed-pathway. The direct-pathway had least treatment delay, contributed by timely recognition of symptoms by the patient. As first point of contact, patients preferred psychiatrists and popularly sought information about treatment via media. There are multiple pathways to consultations, often leading to treatment delay in care received. Timely recognition of symptoms was associated a direct pathway and the least delay. These findings have implications for strengthening routes to mental health specialists at early stages and increasing awareness about treatment available.

Microscopic Kinetics Pathway of Salt Crystallization in Graphene Nanocapillaries.

The fundamental understanding of crystallization, in terms of microscopic kinetic and thermodynamic details, remains a key challenge in the physical sciences. Here, by using insitu graphene liquid cell transmission electron microscopy, we reveal the atomistic mechanism of NaCl crystallization from solutions confined within graphene cells. We find that rock salt NaCl forms with a peculiar hexagonal morphology. We also see the emergence of a transitory graphitelike phase, which may act as an intermediate in a two-step pathway. With the aid of density functional theory calculations, we propose that these observations result from a delicate balance between the substrate-solute interaction and thermodynamics under confinement. Our results highlight the impact of confinement on both the kinetics and thermodynamics of crystallization, offering new insights into heterogeneous crystallization theory and a potential avenue for materials design.

Oxidation of trimethylamine to trimethylamine N-oxide facilitates high hydrostatic pressure tolerance in a generalist bacterial lineage.

High hydrostatic pressure (HHP) is a characteristic environmental factor of the deep ocean. However, it remains unclear how piezotolerant bacteria adapt to HHP. Here, we identify a two-step metabolic pathway to cope with HHP stress in a piezotolerant bacterium. Myroides profundi D25T, obtained from a deep-sea sediment, can take up trimethylamine (TMA) through a previously unidentified TMA transporter, TmaT, and oxidize intracellular TMA into trimethylamine N-oxide (TMAO) by a TMA monooxygenase, MpTmm. The produced TMAO is accumulated in the cell, functioning as a piezolyte, improving both growth and survival at HHP. The function of the TmaT-MpTmm pathway was further confirmed by introducing it into Escherichia coli and Bacillus subtilis Encoded TmaT-like and MpTmm-like sequences extensively exist in marine metagenomes, and other marine Bacteroidetes bacteria containing genes encoding TmaT-like and MpTmm-like proteins also have improved HHP tolerance in the presence of TMA, implying the universality of this HHP tolerance strategy in marine Bacteroidetes.

Application of Cell-Free Protein Synthesis System for the Biosynthesis of l-Theanine.

l-Theanine, as an active component of the leaves of the tea plant, possesses many health benefits and broad applications. Chemical synthesis of l-theanine is possible; however, this method generates chiral compounds and needs further isolation of the pure l-isoform. Heterologous biosynthesis is an alternative strategy, but one main limitation is the toxicity of the substrate ethylamine on microbial host cells. In this study, we introduced a cell-free protein synthesis (CFPS) system for l-theanine production. The CFPS expressed l-theanine synthetase 2 from Camellia sinensis (CsTS2) could produce l-theanine at a concentration of 11.31 μM after 32 h of the synthesis reaction. In addition, three isozymes from microorganisms were expressed in CFPS for l-theanine biosynthesis. The γ-glutamylcysteine synthetase from Escherichia coli could produce l-theanine at the highest concentration of 302.96 μM after 24 h of reaction. Furthermore, CFPS was used to validate a hypothetical two-step l-theanine biosynthetic pathway consisting of the l-alanine decarboxylase from C. sinensis (CsAD) and multiple l-theanine synthases. Among them, the combination of CsAD and the l-glutamine synthetase from Pseudomonas taetrolens (PtGS) could synthesize l-theanine at the highest concentration of 13.42 μM. Then, we constructed an engineered E. coli strain overexpressed CsAD and PtGS to further confirm the l-theanine biosynthesis ability in living cells. This engineered E. coli strain could convert l-alanine and l-glutamate in the medium to l-theanine at a concentration of 3.82 mM after 72 h of fermentation. Taken together, these results demonstrated that the CFPS system can be used to produce the l-theanine through the two-step l-theanine biosynthesis pathway, indicating the potential application of CFPS for the biosynthesis of other active compounds.

Role of integrated noise in pathway-specific signal propagation in feed-forward loops

Cells impose optimal noise control mechanism in diverse situations to cope with distinct environmental cues. Sometimes, it is desirable for the cell to utilize fluctuations for noise-driven processes. In other cases, noise can be harmful to the cell to show optimal fitness. It is, therefore, important to unravel the noise propagation mechanism inside the cell. Such noise controlling mechanism is accomplished by using gene transcription regulatory networks. One such gene regulatory network is feed-forward loop, having three regulatory nodes S, X and Y. Here, we consider the most abundant type 1 of coherent and incoherent feed-forward loops with both OR and AND logic functions, forming four different architectures. In OR logic function, the functions representing S and X act additively for the regulation of Y, while in AND logic function, the same functions (S and X) act multiplicatively for the regulation of Y. Measurement of susceptibility of the signal at output Y is done using elasticity of each regulation in FFLs. Using susceptibility, we demonstrate the nature of pathway integration by which one-step and two-step pathways get overlapped. The integration type is competitive for motifs having OR gate, while it is noncompetitive for the same with AND gate. The pathway integration property explains the output noise behavior of the motifs properly but cannot infer about the mechanism by which the upstream noise propagates to output. To account this, the total output noise is decomposed, which results in integrated noise as an additional noise source along with pathway-specific noise components. The integrated noise is found to appear as a consequence of integration between the pathways and has different functional characteristics explaining noise amplification and noise attenuation property of coherent and incoherent feed-forward loops, respectively. The noise decomposition also quantifies the contribution of different noise sources toward total noise. Finally, the noise propagation is being tuned as a function of input signal noise and its time scale of fluctuations, which shows considerable intrinsic noise strength and relatively slow relaxation time scale causes a higher degree of noise propagation in FFLs.

Interexciton nonradiative relaxation pathways in the peridinin-chlorophyll protein

Excitation energy transfer can be unusually efficient and structurally robust when mediated by molecular excitons, which arise in a photosynthetic light-harvesting protein when delocalized excitations of the electronic chromophores are created upon absorption of light. Here we report results from two-dimensional electronic spectroscopy and electronic structure calculations, revealing that excitation energy is transferred between the peridinin and chlorophyll excitons in the peridinin-chlorophyll protein from marine dinoflagellates over a delocalized, two-step pathway. Upon absorption of light by the peridinins in the strong, mid-visible absorption band, excitation energy is trapped by the chlorophylls in less than 50 fs at room temperature. The overall process is slowed only by a few fs upon replacement of chlorophyll a with chlorophyll b. The key step in the pathway transfers excitation from higher-energy peridinin excitons with more extensive delocalization to the lowest-energy peridinin exciton, which is delocalized only over peridinin 614 and the neighboring chlorophyll. A two-step pathway transfers energy from peridinin excitons to chlorophylls Excitation energy is trapped by chlorophyll acceptors in less than 50 fs Delocalization collapses as excitation progresses irreversibly down the pathway Transfer rates are robust when chlorophyll a is replaced with chlorophyll b Tilluck et al. reveal how excitation energy transfer in the peridinin-chlorophyll protein from marine dinoflagellates involves quantum coherent mixing of the excited states of the clustered chromophores in each protein domain. The results suggest that solar photons might be captured efficiently in photocatalytic devices and solar cells by delocalized nanomaterials.