ATP-free in vitro biotransformation of starch-derived maltodextrin into poly-3-hydroxybutyrate via acetyl-CoA.

In vitro biotransformation (ivBT) facilitated by in vitro synthetic enzymatic biosystems (ivSEBs) has emerged as a highly promising biosynthetic platform. Several ivSEBs have been constructed to produce poly-3-hydroxybutyrate (PHB) via acetyl-coenzyme A (acetyl-CoA). However, some systems are hindered by their reliance on costly ATP, limiting their practicality. This study presents the design of an ATP-free ivSEB for one-pot PHB biosynthesis via acetyl-CoA utilizing starch-derived maltodextrin as the sole substrate. Stoichiometric analysis indicates this ivSEB can self-maintain NADP+/NADPH balance and achieve a theoretical molar yield of 133.3%. Leveraging simple one-pot reactions, our ivSEBs achieved a near-theoretical molar yield of 125.5%, the highest PHB titer (208.3 mM, approximately 17.9 g/L) and the fastest PHB production rate (9.4 mM/h, approximately 0.8 g/L/h) among all the reported ivSEBs to date, and demonstrated easy scalability. This study unveils the promising potential of ivBT for the industrial-scale production of PHB and other acetyl-CoA-derived chemicals from starch.

Basic Properties of Coordinated Neuronal Ensembles in the Auditory Thalamus.

Coordinated neuronal activity has been identified to play an important role in information processing and transmission in the brain. However, current research predominantly focuses on understanding the properties and functions of neuronal coordination in hippocampal and cortical areas, leaving subcortical regions relatively unexplored. In this study, we use single-unit recordings in female Sprague Dawley rats to investigate the properties and functions of groups of neurons exhibiting coordinated activity in the auditory thalamus-the medial geniculate body (MGB). We reliably identify coordinated neuronal ensembles (cNEs), which are groups of neurons that fire synchronously, in the MGB. cNEs are shown not to be the result of false-positive detections or by-products of slow-state oscillations in anesthetized animals. We demonstrate that cNEs in the MGB have enhanced information-encoding properties over individual neurons. Their neuronal composition is stable between spontaneous and evoked activity, suggesting limited stimulus-induced ensemble dynamics. These MGB cNE properties are similar to what is observed in cNEs in the primary auditory cortex (A1), suggesting that ensembles serve as a ubiquitous mechanism for organizing local networks and play a fundamental role in sensory processing within the brain.

Differentiated responses of the phyllosphere bacterial community of the yellowhorn tree to precipitation and temperature regimes across Northern China.

Introduction: As an ephemeral and oligotrophic environment, the phyllosphere harbors many highly diverse microorganisms. Importantly, it is known that their colonization of plant leaf surfaces is considerably influenced by a few abiotic factors related to climatic conditions. Yet how the dynamics of phyllosphere bacterial community assembly are shaped by detailed climatological elements, such as various bioclimatic variables, remains poorly understood. Methods: Using high-throughput 16S rRNA gene amplicon sequencing technology, we analyzed the bacterial communities inhabiting the leaf surfaces of an oilseed tree, yellowhorn (Xanthoceras sorbifolium), grown at four sites (Yinchuan, Otogqianqi, Tongliao, and Zhangwu) whose climatic status differs in northern China. Results and Discussion: We found that the yellowhorn phyllosphere's bacterial community was generally dominated by four phyla: Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. Nevertheless, bacterial community composition differed significantly among the four sampled site regions, indicating the possible impact of climatological factors upon the phyllosphere microbiome. Interestingly, we also noted that the α-diversities of phyllosphere microbiota showed strong positive or negative correlation with 13 bioclimatic factors (including 7 precipitation factors and 6 temperature factors). Furthermore, the relative abundances of 55 amplicon sequence variants (ASVs), including three ASVs representing two keystone taxa (the genera Curtobacterium and Streptomyces), exhibited significant yet contrary responses to the precipitation and temperature climatic variables. That pattern was consistent with all ASVs' trends of possessing opposite correlations to those two parameter classes. In addition, the total number of links and nodes, which conveys community network complexity, increased with rising values of most temperature variables. Besides that, remarkably positive relevance was found between average clustering coefficient and most precipitation variables. Altogether, these results suggest the yellowhorn phyllosphere bacterial community is capable of responding to variation in rainfall and temperature regimes in distinctive ways.

Assessing base-resolution DNA mechanics on the genome scale.

Intrinsic DNA properties including bending play a crucial role in diverse biological systems. A recent advance in a high-throughput technology called loop-seq makes it possible to determine the bendability of hundred thousand 50-bp DNA duplexes in one experiment. However, it's still challenging to assess base-resolution sequence bendability in large genomes such as human, which requires thousands of such experiments. Here, we introduce 'BendNet'-a deep neural network to predict the intrinsic DNA bending at base-resolution by using loop-seq results in yeast as training data. BendNet can predict the DNA bendability of any given sequence from different species with high accuracy. To explore the utility of BendNet, we applied it to the human genome and observed DNA bendability is associated with chromatin features and disease risk regions involving transcription/enhancer regulation, DNA replication, transcription factor binding and extrachromosomal circular DNA generation. These findings expand our understanding on DNA mechanics and its association with transcription regulation in mammals. Lastly, we built a comprehensive resource of genomic DNA bendability profiles for 307 species by applying BendNet, and provided an online tool to assess the bendability of user-specified DNA sequences (http://www.dnabendnet.com/).

The intratumor mycobiome promotes lung cancer progression via myeloid-derived suppressor cells.

Although polymorphic microbiomes have emerged as hallmarks of cancer, far less is known about the role of the intratumor mycobiome as living microorganisms in cancer progression. Here, using fungi-enriched DNA extraction and deep shotgun metagenomic sequencing, we have identified enriched tumor-resident Aspergillus sydowii in patients with lung adenocarcinoma (LUAD). By three different syngeneic lung cancer mice models, we find that A. sydowii promotes lung tumor progression via IL-1ß-mediated expansion and activation of MDSCs, resulting in suppressed activity of cytotoxic T lymphocyte cells and accumulation of PD-1+ CD8+ T cells. This is mediated by IL-1ß secretion via ß-glucan/Dectin-1/CARD9 pathway. Analysis of human samples confirms that enriched A. sydowii is associated with immunosuppression and poor patient outcome. Our findings suggest that intratumor mycobiome, albeit at low biomass, promotes lung cancer progression and could be targeted at the strain level to improve patients with LUAD outcome.

A self-cascade system based on Ag nanoparticle/single-walled carbon nanotube nanocomposites as an enzyme mimic for ultrasensitive detection of L-cysteine.

L-Cysteine, widely used in medicine and the food industry, is of great essentiality to organisms and the food quality. Given that current detection approaches require exacting lab conditions and tedious sample treatment, there is a pressing demand for developing a method that possesses advantages of user friendliness, prominent performance, and cost-effectiveness. Herein, a self-cascade system was developed for the fluorescence detection of L-cysteine based on the ingenious performance of Ag nanoparticle/single-walled carbon nanotube nanocomposites (AgNP/SWCNTs) and DNA-templated Ag nanoclusters (DNA-AgNCs). The fluorescence of DNA-AgNCs could be quenched on account of the adsorption of DNA-AgNCs on AgNP/SWCNTs by π-π stacking. With the cooperation of Fe2+, AgNP/SWCNTs with oxidase and peroxidase-like activities could catalyze the oxidation of L-cysteine to produce cystine and hydrogen peroxide (H2O2) and then break the O-O bond of H2O2 to generate a hydroxyl radical (·OH), which could cleave the DNA strand into different sequence fragments which subsequently peeled off from the AgNP/SWCNTs, resulting in a "turn-on" fluorescence response. In this paper, AgNP/SWCNTs with multi-enzyme activities was synthesized enabling the reaction to proceed in just one step. The successful preliminary applications for the L-cysteine detection in pharmaceutical, juice beverage, and serum samples indicated that the developed method exhibited great potential in medical diagnosis, food monitoring, and the biochemical field, which also broadened the horizon for follow-up research.

A thermophilic phosphatase from Methanothermobacter marburgensis and its application to in vitro biosynthesis.

Phosphatases catalyze the irreversible dephosphorylation of phosphate-containing compounds, and hence can be applied as the final enzymatic step for the synthesis of various biochemicals. However, the extensive substrate spectrums of phosphatases impose a great challenge for efficient biomanufacturing. Characterization of phosphatases is therefore of extreme importance. In this study, MmPase, a putative HAD phosphatase from Methanothermobacter marburgensis, was expressed, purified, and characterized. Recombinant MmPase was readily expressed in Escherichia coli, and required metal ions such as Mn2+ or Mg2+ to function. MmPase worked optimally at 50 °C, pH 6.5, and exhibited a half-life of 6.5 h under this condition. Among all substrates tested, MmPase established the highest dephosphorylation activity against D-tagatose 6-phosphate, and was relatively specific for this substrate than for D-glucose 1-phosphate, D-glucose 6-phosphate, and D-fructose 6-phosphate. Therefore, MmPase was integrated into an in vitro synthetic enzymatic biosystem for the one-pot production of D-tagatose from maltodextrin, and achieved a product yield of 37.6%. Our studies of MmPase provided a promising strategy for the economic and efficient production of D-tagatose in the future.

A ratiometric fluorescence strategy based on copper nanoclusters/carbon dots for sensitive detection of doxorubicin.

Sensitive detection of doxorubicin (DOX) is critical for clinical theranostics. A novel ratiometric fluorescence strategy based on the inner filter effect (IFE) has been established for the sensitive detection of DOX by designing a ratiometric fluorescence probe. In the presence of DOX, the fluorescence intensity of copper nanoclusters (CuNCs) at 485 nm decreases, and the fluorescence intensity of carbon dots at 560 nm increases. Therefore, DOX can be quantitatively detected by measuring the ratio of the fluorescence intensities at 560 and 485 nm (F560 /F485 ). The F560 /F485 ratio exhibits a linear correlation with the DOX concentration in the range from 1.0 × 10-8 M to 1.0 × 10-4 M with the detection limit of 3.7 nM. Furthermore, this method was also successfully applied to the analysis of DOX in human plasma samples, affording an effective platform for drug safety management.

A novel liquid crystal sensing platform for highly selective UO22+ detection based on a UO22+-specific DNAzyme.

A label-free and selective sensor was established for uranyl ion (UO22+) detection based on a UO22+-dependent DNAzyme and liquid crystals (LCs). In the presence of UO22+, the substrate chains can be cleaved at the rA site by the DNAzyme strands. The cleaved products released from the DNAzyme strand will hybridize with the capture probes that are fixed on the LC sensing substrate to form double strands. The formation of double strands would disturb the original orientation and induce the rearrangement of liquid crystal molecules, resulting in the polarization images changing from uniform black to bright. Attributed to the specificity of the DNAzyme and the optical signal of the LC, a highly selective and label-free method was established with a detection limit of 25 nM. This approach showed satisfactory analytical performance and offered an inspiring platform for detecting other radioactive elements.

The Value of Brain Resting-State Functional Magnetic Resonance Imaging on Image Registration Algorithm in Analyzing Abnormal Changes of Neuronal Activity in Patients with Type 2 Diabetes.

The aim of this paper was to analyze the application value of resting-state functional magnetic resonance imaging (FMRI) parameters and rigid transformation algorithm in patients with type 2 diabetes (T2DM), which could provide a theoretical basis for the registration application of FMRI. 107 patients confirmed pathologically as T2DM and 51 community medical healthy volunteers were selected and divided into an experimental group and a control group, respectively. Besides, all the subjects were scanned with FMRI. Then, the rigid transformation-principal axis algorithm (RT-PAA), Levenberg-Marquardt iterative closest point (LMICP), and Demons algorithm were applied to magnetic resonance image registration. It was found that RT-PAA was superior to LMICP and Demons in image registration. The amplitude of low-frequency fluctuation (ALFF) values of the left middle temporal gyrus, right middle temporal gyrus, left fusiform gyrus, right inferior occipital gyrus, and left middle occipital gyrus in patients from the experimental group were lower than those of the control group (P < 0.05). The Montreal cognitive assessment (MoCA) score was extremely negatively correlated with the ALFF of the left middle temporal gyrus (r = -0.451 and P < 0.001) and highly positively associated with the ALFF of the right posterior cerebellar lobe (r = -0.484 and P < 0.001). In addition, the MoCA score of patients had a dramatically negative correlation with the ALFF of the left middle temporal gyrus (r = -0.602 and P < 0.001) and had a greatly positive correlation with the ALFF of the right posterior cerebellar lobe (r = -0.516 and P < 0.001). The results showed that RT-PAA based on rigid transformation in this study had a good registration effect on magnetic resonance images. Compared with healthy volunteers, the left middle temporal gyrus, right middle temporal gyrus, left fusiform gyrus, right inferior occipital gyrus, and left middle occipital gyrus in patients with T2DM showed abnormal neuronal changes and reduced cognitive function.

An ATP-free in vitro synthetic enzymatic biosystem facilitating one-pot stoichiometric conversion of starch to mannitol.

D-Mannitol (hereinafter as mannitol) is a six-carbon sugar alcohol with diverse applications in food and pharmaceutical industries. To overcome the drawbacks of the chemical hydrogenation method commonly used for mannitol production at present, there is a need to search for novel prospective mannitol production strategies that are of high yield and low cost. In this study, we present a novel approach for the stoichiometric synthesis of mannitol via an in vitro synthetic enzymatic biosystem using the low-cost starch as substrate. By dividing the overall reaction pathway into three modules which could be executed sequentially in one pot, our design aimed at the stoichiometric conversion of starch-based materials into mannitol in an ATP-independent and cofactor-balanced manner. At optimized conditions, high product yields of around 95-98% were achieved using both 10 g/L and 50 g/L maltodextrin as substrate, indicating the potential of our designed system for industrial applications. This study not only provides a high-efficient strategy for the synthesis of mannitol but also expands the product scope of sugar alcohols by the in vitro synthetic enzymatic biosystems using low-cost starch-based materials as the input. KEY POINTS : • We described a design-build-test-learn pipeline to construct in vitro biosystems. • The designed system comprised six key enzymes and another three enzymes. • The system converted maltodextrin stoichiometrically to mannitol in one pot.

l-Cysteine modified silver nanoparticles-based colorimetric sensing for the sensitive determination of Hg2+ in aqueous solutions.

A simple and sensitive colorimetric sensing method was constructed for detection of Hg2+ in aqueous solutions and based on silver nanoparticles functionalized with l-cysteine (l-Cys-Ag NPs). In this method, adenosine triphosphate (ATP) induced aggregation of l-Cys-Ag NPs. Simultaneously, the solution colour changed from bright yellow to brown. In the presence of Hg2+ , Hg2+ chelated ATP to form a complex and reduce the degree of aggregation of l-Cys-Ag NPs and was accompanied by a colour change from brown to bright yellow. The changing values of absorbance at 390 nm were linearly correlated with concentration of Hg2+ over the 4.00 × 10-8 to 1.04 × 10-6 mol·L-1 range, with a detection limit of 8 nM. This method was used successfully for detection of Hg2+ in real water samples and performed good selectivity and sensitivity. The recovery range was 91.5-109.1%, indicating that the method has vast application potential for determination of Hg2+ in the environment.

Colorimetric detection uranyl ions based on the enhanced peroxidase-like activity by GO adsorption.

Based on the fact that uranyl ions (UO22+) adsorbed on GO can enhanced the peroxidase-like activity of graphene oxide (GO), a novel colorimetric strategy for visualizing quantitative determination of uranyl ions was established. The peroxidase-like activity of GO-UO22+ nanocomposites was assessed by catalyzing H2O2 oxidation of TMB to produce a distinct color reaction. A good linearity between the UO22+ concentration and absorption at 652 nm was acquired in the range of 5.90 × 10-6 to 9.43 × 10-4 M with a detection limit of 4.70 µM. This strategy was also successfully applied to determination of uranyl ions in environmental water samples.

Biomimetic synthesis of hyperolactones.

Through a biomimetic pathway, hyperolactone D, 4-hydroxyhyperolactone D, and hyperolactone C were synthesized from methyl acetoacetate via Weiler's dianion method, asymmetric allylic alkylation, biomimetic lactonization, oxidation, and cyclization. The stereochemistry of the quaternary carbon was controlled efficiently by Palladium-catalyzed asymmetric allylic alkylation. This strategy was also used for the synthesis of hyperolactone B.