Gut bacterial nutrient preferences quantified in vivo.

Great progress has been made in understanding gut microbiomes' products and their effects on health and disease. Less attention, however, has been given to the inputs that gut bacteria consume. Here, we quantitatively examine inputs and outputs of the mouse gut microbiome, using isotope tracing. The main input to microbial carbohydrate fermentation is dietary fiber and to branched-chain fatty acids and aromatic metabolites is dietary protein. In addition, circulating host lactate, 3-hydroxybutyrate, and urea (but not glucose or amino acids) feed the gut microbiome. To determine the nutrient preferences across bacteria, we traced into genus-specific bacterial protein sequences. We found systematic differences in nutrient use: most genera in the phylum Firmicutes prefer dietary protein, Bacteroides dietary fiber, and Akkermansia circulating host lactate. Such preferences correlate with microbiome composition changes in response to dietary modifications. Thus, diet shapes the microbiome by promoting the growth of bacteria that preferentially use the ingested nutrients.

Slow TCA flux and ATP production in primary solid tumours but not metastases.

Tissues derive ATP from two pathways-glycolysis and the tricarboxylic acid (TCA) cycle coupled to the electron transport chain. Most energy in mammals is produced via TCA metabolism1. In tumours, however, the absolute rates of these pathways remain unclear. Here we optimize tracer infusion approaches to measure the rates of glycolysis and the TCA cycle in healthy mouse tissues, Kras-mutant solid tumours, metastases and leukaemia. Then, given the rates of these two pathways, we calculate total ATP synthesis rates. We find that TCA cycle flux is suppressed in all five primary solid tumour models examined and is increased in lung metastases of breast cancer relative to primary orthotopic tumours. As expected, glycolysis flux is increased in tumours compared with healthy tissues (the Warburg effect2,3), but this increase is insufficient to compensate for low TCA flux in terms of ATP production. Thus, instead of being hypermetabolic, as commonly assumed, solid tumours generally produce ATP at a slower than normal rate. In mouse pancreatic cancer, this is accommodated by the downregulation of protein synthesis, one of this tissue's major energy costs. We propose that, as solid tumours develop, cancer cells shed energetically expensive tissue-specific functions, enabling uncontrolled growth despite a limited ability to produce ATP.

Spatially resolved isotope tracing reveals tissue metabolic activity.

Isotope tracing has helped to determine the metabolic activities of organs. Methods to probe metabolic heterogeneity within organs are less developed. We couple stable-isotope-labeled nutrient infusion to matrix-assisted laser desorption ionization imaging mass spectrometry (iso-imaging) to quantitate metabolic activity in mammalian tissues in a spatially resolved manner. In the kidney, we visualize gluconeogenic flux and glycolytic flux in the cortex and medulla, respectively. Tricarboxylic acid cycle substrate usage differs across kidney regions; glutamine and citrate are used preferentially in the cortex and fatty acids are used in the medulla. In the brain, we observe spatial gradations in carbon inputs to the tricarboxylic acid cycle and glutamate under a ketogenic diet. In a carbohydrate-rich diet, glucose predominates throughout but in a ketogenic diet, 3-hydroxybutyrate contributes most strongly in the hippocampus and least in the midbrain. Brain nitrogen sources also vary spatially; branched-chain amino acids contribute most in the midbrain, whereas ammonia contributes in the thalamus. Thus, iso-imaging can reveal the spatial organization of metabolic activity.

Simultaneous Detection of Adenosine-to-Inosine Editing and N6-Methyladenosine at Identical RNA Sites through Deamination-Assisted Reverse Transcription Stalling.

Adenosine-to-inosine (A-to-I) editing and N6-methyladenosine (m6A) modifications are pivotal RNA modifications with widespread functional significance in physiological and pathological processes. Although significant effort has been dedicated to developing methodologies for identifying and quantifying these modifications, traditional approaches have often focused on each modification independently, neglecting the potential co-occurrence of A-to-I editing and m6A modifications at the same adenosine residues. This limitation has constrained our understanding of the intricate regulatory mechanisms governing RNA function and the interplay between different types of RNA modifications. To address this gap, we introduced an innovative technique called deamination-assisted reverse transcription stalling (DARTS), specifically designed for the simultaneous quantification of A-to-I editing and m6A at the same RNA sites. DARTS leverages the selective deamination activity of the engineered TadA-TadA8e protein, which converts adenosine residues to inosine, in combination with the unique property of Bst 2.0 DNA polymerase, which stalls when encountering inosine during reverse transcription. This approach enables the accurate quantification of A-to-I editing, m6A, and unmodified adenosine at identical RNA sites. The DARTS method is remarkable for its ability to directly quantify two distinct types of RNA modifications simultaneously, a capability that has remained largely unexplored in the field of RNA biology. By facilitating a comprehensive analysis of the co-occurrence and interaction between A-to-I editing and m6A modifications, DARTS opens new avenues for exploring the complex regulatory networks modulated by different RNA modifications.

Metabolite discovery through global annotation of untargeted metabolomics data.

Liquid chromatography-high-resolution mass spectrometry (LC-MS)-based metabolomics aims to identify and quantify all metabolites, but most LC-MS peaks remain unidentified. Here we present a global network optimization approach, NetID, to annotate untargeted LC-MS metabolomics data. The approach aims to generate, for all experimentally observed ion peaks, annotations that match the measured masses, retention times and (when available) tandem mass spectrometry fragmentation patterns. Peaks are connected based on mass differences reflecting adduction, fragmentation, isotopes, or feasible biochemical transformations. Global optimization generates a single network linking most observed ion peaks, enhances peak assignment accuracy, and produces chemically informative peak-peak relationships, including for peaks lacking tandem mass spectrometry spectra. Applying this approach to yeast and mouse data, we identified five previously unrecognized metabolites (thiamine derivatives and N-glucosyl-taurine). Isotope tracer studies indicate active flux through these metabolites. Thus, NetID applies existing metabolomic knowledge and global optimization to substantially improve annotation coverage and accuracy in untargeted metabolomics datasets, facilitating metabolite discovery.

Ultrafast control of the LnF+/LnO+ ratio from Ln(hfac)3.

The photo-induced dissociative ionization of lanthanide complexes Ln(hfac)3 (Ln = Pr, Er, Yb) is studied using ultrafast shaped laser pulses in a time-of-flight (TOF) mass spectrometry setup. Various fluorine and Ln-containing mass fragments were observed, which can be interpreted by the photo-fragmentation mechanistic pathway involving C-C bond rotation processes proposed previously. A set of experiments used pulse shaping guided by closed-loop feedback control to identify pulses that optimize the ratio of LnF+/LnO+. In agreement with previous studies in which very little LnO+ was observed, broad pulses were found to maximize the LnF+/LnO+ ratio, which involves metal-ligand bond-breaking followed by bond rotation and bond rearrangement. In contrast, a transform limited (TL) pulse favored the formation of LnO+. Finally, the recently developed experimental control pulse slicing (CPS) technique was applied to elucidate the dynamics induced by fields that either maximize or minimize the LnF+/LnO+ ratio, which also indicates that longer laser pulses facilitate LnF+ formation during the C-C bond rotation dissociative-ionization process.

A new 3,4-dinorsteroid from the marine sponge Cliona sp.

A new steroid, 2a-oxa-2-oxo-5ß-hydroxy-3,4-dinor-24-methylcholesta-22E-ene (1), together with 10 known ones (2-11), was isolated from the marine sponge Cliona sp. The structures of these compounds were determined by the spectroscopic methods (UV, IR, MS, and NMR) and X-ray diffraction analysis. Compound 1 was the third example of 3,4-dinorsteroid with a hemiketal at C-5 that was isolated from the natural source. In addition, the antibacterial activities of these compounds were also evaluated. However, none of them exhibited significant inhibition effects.

(+)- and (-)-Tedanine, a pair of new enantiomeric indolone alkaloids from the marine sponge Tedania sp.

(+)- and (-)-Tedanine [(+)-1 and (-)-1], a pair of new enantiomeric indolone alkaloids, along with nine compounds (2-10) were isolated from the marine sponge Tedania sp. The structures of (+)-1 and (-)-1 including absolute configurations were determined by spectroscopic analysis and quantum chemical calculation. Compounds (+)-1 and (-)-1 were the first examples of indolone alkaloids isolated from this genus. In addition, the cytotoxic and antibacterial activities of these compounds were also evaluated.

Acidic Methanol Treatment Facilitates Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry Imaging of Energy Metabolism.

Detection of small molecule metabolites (SMM), particularly those involved in energy metabolism using MALDI-mass spectrometry imaging (MSI), is challenging due to factors including ion suppression from other analytes present (e.g., proteins and lipids). One potential solution to enhance SMM detection is to remove analytes that cause ion suppression from tissue sections before matrix deposition through solvent washes. Here, we systematically investigated solvent treatment conditions to improve SMM signal and preserve metabolite localization. Washing with acidic methanol significantly enhances the detection of phosphate-containing metabolites involved in energy metabolism. The improved detection is due to removing lipids and highly polar metabolites that cause ion suppression and denaturing proteins that release bound phosphate-containing metabolites. Stable isotope infusions of [13C6]nicotinamide coupled to MALDI-MSI ("Iso-imaging") in the kidney reveal patterns that indicate blood vessels, medulla, outer stripe, and cortex. We also observed different ATP:ADP raw signals across mouse kidney regions, consistent with regional differences in glucose metabolism favoring either gluconeogenesis or glycolysis. In mouse muscle, Iso-imaging using [13C6]glucose shows high glycolytic flux from infused circulating glucose in type 1 and 2a fibers (soleus) and relatively lower glycolytic flux in type 2b fiber type (gastrocnemius). Thus, improved detection of phosphate-containing metabolites due to acidic methanol treatment combined with isotope tracing provides an improved way to probe energy metabolism with spatial resolution in vivo.

Two New Pairs of Enantiomeric Butenolides from the Marine Sponge Suberties sp.

Two new pairs of enantiomeric butenolides, (+)- and (-)-suberiteslide A, (+)- and (-)-subertieslide B had been obtained from the marine sponge Suberties sp. The structures with absolute configurations of these compounds were unequivocally determined by spectroscopic analyses and ECD (Electronic Circular Dichroism) method. It was the first separation of butenolides from the marine sponges of genus Suberites. Additionally, the anti-inflammatory, antibacterial and cytotoxic activities of these compounds were evaluated. The result indicated that only (-)-subertieslide B showed weak anti-inflammatory activity with the IC50 value of 40.8 µM.

The Design and Development of Instrumented Toys for the Assessment of Infant Cognitive Flexibility.

The first years of an infant's life represent a sensitive period for neurodevelopment where one can see the emergence of nascent forms of executive function (EF), which are required to support complex cognition. Few tests exist for measuring EF during infancy, and the available tests require painstaking manual coding of infant behaviour. In modern clinical and research practice, human coders collect data on EF performance by manually labelling video recordings of infant behaviour during toy or social interaction. Besides being extremely time-consuming, video annotation is known to be rater-dependent and subjective. To address these issues, starting from existing cognitive flexibility research protocols, we developed a set of instrumented toys to serve as a new type of task instrumentation and data collection tool suitable for infant use. A commercially available device comprising a barometer and an inertial measurement unit (IMU) embedded in a 3D-printed lattice structure was used to detect when and how the infant interacts with the toy. The data collected using the instrumented toys provided a rich dataset that described the sequence of toy interaction and individual toy interaction patterns, from which EF-relevant aspects of infant cognition can be inferred. Such a tool could provide an objective, reliable, and scalable method of collecting early developmental data in socially interactive contexts.

Regulation of circular RNAs act as ceRNA in a hypoxic pulmonary hypertension rat model.

To explore potential critical genes and identify circular RNAs (circRNAs) that act as the competitive endogenous RNA (ceRNA) in a hypoxic pulmonary hypertension (HPH) rat model. Constructed rat model, and a bioinformatics method was used to analyse differentially expressed (DE) genes and construct a circRNA-miRNA-mRNA ceRNA regulatory network. Then, qRT-PCR was used to verify. The significant DEcircRNAs/DEmiRNAs/DEmRNAs was showed, and a ceRNA network with 8 DEcircRNAs, 9 DEmiRNAs and 46 DEmRNAs were constructed. The functional enrichment suggested the inflammatory response, NF-κB signalling, MAPK cascade and Toll-like receptor were associated with HPH. Further assessment confirmed that circ_002723, circ_008021, circ_016925 and circ_020581 could have a potential ceRNA mechanism by sponging miR-23a or miR-21 to control downstream target gene and be involved in the pathophysiology of HPH. The qRT-PCR validation results were consistent with the RNA-Seq results. This study revealed potentially important genes, pathways and ceRNA regulatory networks in HPH.

Improved Annotation of Untargeted Metabolomics Data through Buffer Modifications That Shift Adduct Mass and Intensity.

Annotation of untargeted high-resolution full-scan LC-MS metabolomics data remains challenging due to individual metabolites generating multiple LC-MS peaks arising from isotopes, adducts, and fragments. Adduct annotation is a particular challenge, as the same mass difference between peaks can arise from adduct formation, fragmentation, or different biological species. To address this, here we describe a buffer modification workflow (BMW) in which the same sample is run by LC-MS in both liquid chromatography solvent with 14NH3-acetate buffer and in solvent with the buffer modified with 15NH3-formate. Buffer switching results in characteristic mass and signal intensity changes for adduct peaks, facilitating their annotation. This relatively simple and convenient chromatography modification annotated yeast metabolomics data with similar effectiveness to growing the yeast in isotope-labeled media. Application to mouse liver data annotated both known metabolite and known adduct peaks with 95% accuracy. Overall, it identified 26% of ∼27 000 liver LC-MS features as putative metabolites, of which ∼2600 showed HMDB or KEGG database formula match. This workflow is well suited to biological samples that cannot be readily isotope labeled, including plants, mammalian tissues, and tumors.

Autophagy in pulmonary hypertension: Emerging roles and therapeutic implications.

Autophagy is an important mechanism for cellular self-digestion and basal homeostasis. This gene- and modulator-regulated pathway is conserved in cells. Recently, several studies have shown that autophagic dysfunction is associated with pulmonary hypertension (PH). However, the relationship between autophagy and PH remains controversial. In this review, we mainly introduce the effects of autophagy-related genes and some regulatory molecules on PH and the relationship between autophagy and PH under the conditions of hypoxia, monocrotaline injection, thromboembolic stress, oxidative stress, and other drugs and toxins. The effects of other autophagy-related drugs, such as chloroquine, 3-methyladenine, rapamycin, and other potential therapeutic drugs and targets, in PH are also described.

Peak Annotation and Verification Engine for Untargeted LC-MS Metabolomics.

Untargeted metabolomics can detect more than 10 000 peaks in a single LC-MS run. The correspondence between these peaks and metabolites, however, remains unclear. Here, we introduce a Peak Annotation and Verification Engine (PAVE) for annotating untargeted microbial metabolomics data. The workflow involves growing cells in 13C and 15N isotope-labeled media to identify peaks from biological compounds and their carbon and nitrogen atom counts. Improved deisotoping and deadducting are enabled by algorithms that integrate positive mode, negative mode, and labeling data. To distinguish metabolites and their fragments, PAVE experimentally measures the response of each peak to weak in-source collision induced dissociation, which increases the peak intensity for fragments while decreasing it for their parent ions. The molecular formulas of the putative metabolites are then assigned based on database searching using both m/ z and C/N atom counts. Application of this procedure to Saccharomyces cerevisiae and Escherichia coli revealed that more than 80% of peaks do not label, i.e., are environmental contaminants. More than 70% of the biological peaks are isotopic variants, adducts, fragments, or mass spectrometry artifacts yielding ∼2000 apparent metabolites across the two organisms. About 650 match to a known metabolite formula based on m/ z and C/N atom counts, with 220 assigned structures based on MS/MS and/or retention time to match to authenticated standards. Thus, PAVE enables systematic annotation of LC-MS metabolomics data with only ∼4% of peaks annotated as apparent metabolites.

MicroRNA-214-3p Regulates Hypoxia-Mediated Pulmonary Artery Smooth Muscle Cell Proliferation and Migration by Targeting ARHGEF12.

BACKGROUND miR-214-3p has been found to inhibit proliferation and migration in cancer cells. The objective of this study was to determine whether ARHGEF12 is involved in miR-214-3p-mediated suppression of proliferation and migration of pulmonary artery smooth muscle cells (PASMCs). MATERIAL AND METHODS PASMCs were cultured under normoxia or hypoxia. miR-214-3p mimics or inhibitors were transiently transfected into PASMCs. Proliferation, apoptosis, and migration of PASMCs were evaluated using MTT assay, flow cytometry, and Boyden chamber apparatus. Western blot analysis was used to examine expression of Rho guanine nucleotide exchange factor 12 (ARHGEF12), c-fos, c-jun, and caspase-3. Luciferase reporter assay was used to test the direct regulation of miR-214-3p on the 3'-untranslated region (UTR) of ARHGEF12. RESULTS miR-214-3p was significantly upregulated in hypoxia-treated PASMCs. Knockdown of miR-214-3p significantly attenuated hypoxia-induced proliferation and migration in PASMCs and promoted apoptosis, whereas this effect was aggravated by overexpression of miR-214-3p. In addition, dual-luciferase reporter assay demonstrated that ARHGEF12 is a direct target gene of miR-214-3p. The protein levels of ARHGEF12 were downregulated after knockdown of miR-214-3p in PASMCs. Rescue experiment results indicated that decreased proliferation of PASMCs resulted from knockdown of miR-214-3p were partially reversed by silencing of ARHGEF12 by siRNA. Furthermore, knockdown of miR-214-3p reduced expression of c-jun and c-fos, but increased expression of caspase-3 in PASMCs under hypoxia. CONCLUSIONS In conclusion, these results indicate that miR-214-3p acts as a novel regulator of hypoxia-induced proliferation and migration by directly targeting ARHGEF12 and dysregulating c-jun and c-fos in PASMCs, and may be a potential therapeutic target for treating pulmonary hypertension.

Gaining Mechanistic Insight with Control Pulse Slicing: Application to the Dissociative Ionization of CH2BrI.

In quantum control experiments with shaped femtosecond laser pulses, adaptive feedback control is often used to identify pulse shapes that can optimally steer the quantum system toward the desired outcome. However, gaining mechanistic information can pose a challenge due to the varied structural features of the control pulses and/or the often complex nature of the associated simulations of the experiments. In this article, we introduce control pulse slicing (CPS) as an easy-to-implement experimental analysis tool that can be employed directly in the laboratory without the need for modeling, to gain mechanistic insights about control experiments, regardless of whether the pulse is optimal or chosen by other means. As an illustration, we apply CPS to dissociative ionization of CH2BrI with mass spectral detection, where two pulses with similar intensities are investigated, with each capable of distinctively controlling the ratio of Br+/CH2Br+. These two control pulses were, respectively, first identified with closed loop and open loop procedures, and then the multispecies experimental data was analyzed with CPS. By comparing the dynamical evolution of the observed multiple fragment ion yields upon slicing scans of the two distinct pulses, we were able to reveal insights about the control mechanism for manipulating the objective ratio. In addition, we also identified the relationship between the temporal structures of the control pulses and the associated key reaction pathways involved in ionic as well as neutral electronic states, in spite of the signals only directly being from the ionic species. The CPS technique is not limited to controlled fragmentation mass spectrometry, and it may be applied to gain mechanistic insights in any control experiment, reflected in the nature of the recorded signal.

A highly sensitive fluorescence assay for methyltransferase activity by exonuclease-aided signal amplification.

DNA methylation, catalyzed by methyltransferases, plays critical roles in various biological processes in both prokaryotes and eukaryotes. Bacterial DNA adenine methyltransferases (DAM) are associated with bacterial pathogenesis and essential for bacterial virulence and viability. Since mammals do not methylate DNA at adenine, bacterial DAM is considered to be a great candidate target for developing new therapeutics for diseases. In the current study, we developed a simple, rapid and highly sensitive fluorescence method for the detection of DAM based on exonuclease-aided signal amplification. In the proposed strategy, a liberated amplifier upon DAM methylation and Dpn I digestion of the substrate can hybridize with a reporter (FT) that contains a quencher (TAMRA) at the second base of the 3' end and a fluorophore (FAM) at the fifth base. Upon hybridization, exonuclease III degrades the reporter in the formed duplex DNA from the 3' end successively, releasing the fluorophore from the quencher and resulting in an intensive appearance of the fluorescent signal. The amplifier will hybridize with another reporter and enter a new cycle, which therefore can amplify the signal and dramatically increase the detection sensitivity even with an extremely low amount of amplifier. Using this strategy, the detection limit down to 0.0025 U mL(-1) of DAM was achieved within a short assay time of 30 min. Furthermore, the assay was applied to evaluate endogenous DAM activity in E. coli cell at different growth stages as well as the effects of inhibitors on DAM activity. Given the attractive analytical performance, the sensing strategy may find many important applications in biomedical research and clinical diagnosis.

Shenfu injection alleviates intestine epithelial damage in septic rats.

BACKGROUND: Shenfu injection (SFI) promotes tissue microcirculation and oxygen metabolism. We aimed to assess its effects on intestinal epithelial damage in septic rats. METHODS: Fifty Sprague-Dawley rats were randomly divided into sham operation (Sham), sepsis (cecal ligation and puncture [CLP]), and SFI (low-dose, middle-dose, high-dose) groups (n = 10). For Sham animals, the abdominal cavity was opened and closed. For other groups, severe sepsis was induced by CLP. After surgery, saline (Sham and CLP rats) and SFI (treatment groups) were administered intraperitoneally. Samples were collected 12 hours after injection. Serum tumor necrosis factor α, diamineoxidase, and d-lactate levels and ileal mucosal damage and ultrastructural change, as well as protein and messenger RNA expression of tight junction markers, including Claudin-3 and zonula occludens protein-1 in ileal mucosa's epithelial cells, were assessed. All animal experiments were carried out under aseptic conditions. RESULTS: Compared with Sham animals, serum tumor necrosis factor α, DAO, and d-lactic acid levels in CLP animals were significantly higher; the ileal mucosal damage was more severe; and the expression levels of tight junction markers were significantly decreased. These indexes were significantly improved in SFI groups, in a concentration-dependent manner, compared with CLP rats. Sham animals displayed orderly arranged ileal mucosal villi, continuous tight junctions between epithelial cells, intact organelles, and microvilli. Compared with CLP animals (with obvious damage in these structures), an overt improvement was observed in SFI groups, especially in the high-dose SFI group, with tight junctions clearly visible between epithelial cells. CONCLUSIONS: Shenfu injection significantly alleviates intestinal epithelial damage in septic rats, in a dose-dependent manner.

Effect of Shenfu injection on intestinal mucosal barrier in a rat model of sepsis.

PURPOSE: The effects of Shenfu injection on protecting the intestinal mucosal barrier were investigated in rats with sepsis. METHODS: Severe sepsis was established by cecal ligation and puncture (CLP) in 30 healthy Sprague-Dawley rats. Twelve rats that received sham surgery received 10 mL/kg of normal saline. Rats with CLP were randomized to receive 10 mL/kg of normal saline (n = 12) and 5 mL/kg Shenfu (n = 12), and 10 received 10 mL/kg Shenfu injection (n = 12) by tail intravenous injection. Rats were killed after 8 hours. Serum levels of tumor necrosis factor α and interleukin-10, and ileal malondialdehyde and superoxide dismutase activity were measured by enzyme-linked immunosorbent assay. Ileum tissue structures and pathological score were observed by microscopy. Ileal mucosal epithelial cell apoptosis index was calculated by TUNEL assay. Ileal proapoptotic protein Bax, antiapoptotic protein Bcl-2, and tight junction transmembrane protein occludin were measured by immunohistochemistry and immunoblot. RESULTS: The level of tumor necrosis factor α, the ileal malondialdehyde level, ileum pathological score, apoptosis index of ileal mucosal epithelial cells, and Bax protein level were significantly higher, and serum level of interleukin-10, the ileal superoxide dismutase activity, Bcl-2 protein level, Bcl-2/Bax ratio, and occludin protein level were significantly lower in the CLP group than in the sham group (P < .01 or P < .05). Both low- and high-dose Shenfu significantly ameliorated these changes (P < .01 or P < .05), but high-dose injection achieved more significant improvements than did the low-dose injection (P < .01 or P < .05). CONCLUSIONS: Shenfu injection might ameliorate the mucosal barrier function in a model of sepsis in rats in a dose-dependent manner.