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Great progress has been made in expanding the repertoire of genetically encoded fluorescent sensors for monitoring intracellular transition metals (TMs). This powerful toolkit permits dynamic and non-invasive detection of TMs with high spatial-temporal resolution, which enables us to better understand the roles of TM homeostasis in both physiological and pathological settings. Here we summarize the recent development of genetically encoded fluorescent sensors for intracellular detection of TMs such as zinc and copper, as well as heavy metals including lead, cadmium, mercury, and arsenic. [ABSTRACT FROM AUTHOR]

Multiple antibiotic resistance regulator (MarR) family proteins are widely conserved transcription factors that control bacterial resistance to antibiotics, environmental stresses, as well as the regulation of virulence determinants. Escherichia coli MarR, the prototype member of this family, has recently been shown to undergo copper(II)-catalyzed inter-dimer disulfide bond formation via a unique cysteine residue (Cys80) residing in its DNA-binding domain. However, despite extensive structural characterization of the MarR family proteins, the structural mechanism for DNA binding of this copper(II)-sensing MarR factor remains elusive. Here, we report the crystal structures of DNA-bound forms of MarR, which revealed a unique, concerted generation of two new helix-loop-helix motifs that facilitated MarR's DNA binding. Structural analysis and electrophoretic mobility shift assays (EMSA) show that the flexibility of Gly116 in the center of helix α5 and the extensive hydrogen-bonding interactions at the N-terminus of helix α1 together assist the reorientation of the wHTH domains and stabilize MarR's DNA-bound conformation. [ABSTRACT FROM AUTHOR]

Multiple antibiotic resistance regulator (MarR) family proteins are widely conserved transcription factors that are important for regulating bacterial resistance to various stresses. Escherichia coli MarR, the prototype member of this family, has recently been shown to undergo interdimer disulfide bond formation via a unique cysteine residue (Cys80) that ultimately triggered MarR's dissociation from its promoter DNA. However, these structural studies were performed with cysteine mutants while the structure of wild type MarR remains uncharacterized. Here we report the crystal structure of wild type MarR in the absence of DNA, which further revealed the roles of cysteine residues in MarR's transcriptional regulation. In addition, we developed a circularly permuted yellow fluorescent protein (cpYFP)-based environmental-sensitive fluorescent reporter for in situ detection of the DNA-binding induced conformational change on MarR, which verified the induction mechanism of this prototypical MarR family protein. This strategy might potentially be applicable for monitoring local conformational change within diverse protein structures. [ABSTRACT FROM AUTHOR]

The widely conserved multiple antibiotic resistance regulator (MarR) family of transcription factors modulates bacterial detoxification in response to diverse antibiotics, toxic chemicals or both. The natural inducer for Escherichia coli MarR, the prototypical transcription repressor within this family, remains unknown. Here we show that copper signaling potentiates MarR derepression in E. coli. Copper(II) oxidizes a cysteine residue (Cys80) on MarR to generate disulfide bonds between two MarR dimers, thereby inducing tetramer formation and the dissociation of MarR from its cognate promoter DNA. We further discovered that salicylate, a putative MarR inducer, and the clinically important bactericidal antibiotics norfloxacin and ampicillin all stimulate intracellular copper elevation, most likely through oxidative impairment of copper-dependent envelope proteins, including NADH dehydrogenase-2. This membrane-associated copper oxidation and liberation process derepresses MarR, causing increased bacterial antibiotic resistance. Our study reveals that this bacterial transcription regulator senses copper(II) as a natural signal to cope with stress caused by antibiotics or the environment. [ABSTRACT FROM AUTHOR]

A concise route was developed for the facile synthesis of a cyclic pyrrolysine analogue bearing an azide handle. Directed evolution enabled the encoding of this non-natural amino acid in both prokaryotic and eukaryotic cells, which offers a highly efficient approach for the site-specific protein labeling using click chemistry. [ABSTRACT FROM AUTHOR]

N 6-Methyldeoxyadenosine (6mA) has recently been shown to exist and play regulatory roles in eukaryotic genomic DNA (gDNA). However, the biological functions of 6mA in mammals have yet to be adequately explored, largely due to its low abundance in most mammalian genomes. Here, we report that mammalian mitochondrial DNA (mtDNA) is enriched for 6mA. The level of 6mA in HepG2 mtDNA is at least 1,300-fold higher than that in gDNA under normal growth conditions, corresponding to approximately four 6mA modifications on each mtDNA molecule. METTL4, a putative mammalian methyltransferase, can mediate mtDNA 6mA methylation, which contributes to attenuated mtDNA transcription and a reduced mtDNA copy number. Mechanistically, the presence of 6mA could repress DNA binding and bending by mitochondrial transcription factor (TFAM). Under hypoxia, the 6mA level in mtDNA could be further elevated, suggesting regulatory roles for 6mA in mitochondrial stress response. Our study reveals DNA 6mA as a regulatory mark in mammalian mtDNA. • N 6-methyldeoxyadenosine (6mA) is enriched in human mitochondria DNA (mtDNA) • METTL4 can mediate mammalian mtDNA 6mA methylation • mtDNA 6mA affects mitochondrial transcription, replication, and activity • The 6mA level in mtDNA is significantly elevated under hypoxic stress Hao et al. show that N 6-methyldeoxyadenosine (6mA) modification is notably enriched in human mitochondria DNA and regulates mitochondrial transcription, replication, and activity. [ABSTRACT FROM AUTHOR]

Protein-based hydrogels are considered ideal biomaterials due to their high biocompatibility, diverse structure, and their improved bioactivity and biodegradability. However, it remains challenging to mimic the native extracellular matrices that can dynamically respond to environmental stimuli. The combination of stimuli-responsive functionalities with engineered protein hydrogels has facilitated the development of new smart hydrogels with tunable biomechanics and biological properties that are triggered by cyto-compatible stimuli. This review summarizes the recent advancements of responsive hydrogels prepared from engineered proteins and integrated with physical, chemical or biological responsive moieties. We underscore the design principles and fabrication approaches of responsive protein hydrogels, and their biomedical applications in disease treatment, drug delivery, and tissue engineering are briefly discussed. Finally, the current challenges and future perspectives in this field are highlighted. [ABSTRACT FROM AUTHOR]

Transforming growth factor (TGF)-β1, an angiogenic factor in the maternal circulation, has been suggested to be related to preeclampsia. However, the findings from previous studies were inconsistent. Thus, we conducted a meta-analysis to assess the difference in circulating TGF-β1 levels between women with preeclampsia and normal pregnancies. Twenty-four studies including 1748 women with PE and 1404 women with normal pregnancy were included in our study. The results showed that circulating TGF-β1 levels were not different before the time of active disease (standardized mean differences, − 0.46 [95% CI, − 0.16 to 0.15]; P = 0.000). At the time of active disease, women with preeclampsia (n = 1207) had higher circulating TGF-β1 levels than normotensive controls (n = 912; standardized mean differences, 0.94 [95% CI, 0.52 to 1.35]; P = 0.000). Circulating TGF-β1 levels were higher in both early-onset/severe and late-onset/mild types of preeclampsia. No publication biases were found. We conclude that preeclamptic women have higher circulating TGF-β1 than those with normal pregnancy at the time of preeclampsia diagnosis. [ABSTRACT FROM AUTHOR]

Functional studies of the RNA N6-methyladenosine (m6A) modification have been limited by an inability to map individual m6A-modified sites in whole transcriptomes. To enable such studies, here, we introduce m6A-selective allyl chemical labeling and sequencing (m6A-SAC-seq), a method for quantitative, whole-transcriptome mapping of m6A at single-nucleotide resolution. The method requires only ~30 ng of poly(A) or rRNA-depleted RNA. We mapped m6A modification stoichiometries in RNA from cell lines and during in vitro monocytopoiesis from human hematopoietic stem and progenitor cells (HSPCs). We identified numerous cell-state-specific m6A sites whose methylation status was highly dynamic during cell differentiation. We observed changes of m6A stoichiometry as well as expression levels of transcripts encoding or regulated by key transcriptional factors (TFs) critical for HSPC differentiation. m6A-SAC-seq is a quantitative method to dissect the dynamics and functional roles of m6A sites in diverse biological processes using limited input RNA.m6A-SAC-seq uses chemical labeling to quantify m6A at single-base resolution in the mammalian transcriptome. [ABSTRACT FROM AUTHOR]

Multipoint Optimal Minimum Entropy Deconvolution Adjusted (MOMEDA) is an advanced deconvolution method, which can effectively inhibit the interference of background noise and distinguish the fault period by calculating the multipoint kurtosis values. However, multipoint kurtosis (MKurt) could lead to misjudgment since it is sensitive to spurious noise spikes. Considering that L-kurtosis has good robustness with noise, this paper proposes a multipoint envelope L-kurtosis (MELkurt) method for establishing the temporal features. Then, an enhanced image representation method of vibration signals is proposed by employing the Gramian Angular Difference Field (GADF) method to convert the MELkurt series into images. Furthermore, to effectively learn and extract the features of GADF images, this paper develops a deep learning method named Conditional Super Token Transformer (CSTT) by incorporating the Super Token Transformer block, Super Token Mixer module, and Conditional Positional Encoding mechanism into Vision Transformer appropriately. Transfer learning is introduced to enhance the diagnostic accuracy and generalization capability of the designed CSTT. Consequently, a novel bearing fault diagnosis framework is established based on the presented enhanced image representation and CSTT. The proposed method is compared with Vision Transformer and some CNN-based models to verify the recognition effect by two experimental datasets. The results show that MELkurt significantly improves the fault feature enhancement ability with superior noise robustness to kurtosis, and the proposed CSTT achieves the highest diagnostic accuracy and stability. [ABSTRACT FROM AUTHOR]

Rolling bearings play a critical role in wind turbine generators, operating continuously in an electromagnetic excitation environment. Their fault vibration characteristics are affected by unbalanced magnetic pull (UMP), which has received little attention in previous studies. Moreover, torsional vibration can provide advantages in bearing fault diagnosis, since it has a good signal-to-noise ratio (SNR) and it can be easily measured by the encoder. However, torsional vibration is rarely considered in the existing model of wind turbine generator. Accordingly, this paper proposes a dynamic model with 8 degrees of freedom to analyze the bearing outer ring fault characteristics of wind turbine generator under the influence of UMP, while also taking into account the torsional vibration in the model. The validity of the model is verified through experiments conducted on a test rig. The results displays modulation of the ball pass frequency outer (BPFO) harmonics with both UMP frequency and rotation frequency in lateral vibration, and this phenomenon can also be observed in instantaneous angular speed (IAS) and instantaneous angular acceleration (IAA) signals that represent torsional vibration. Besides, the lateral vibration spectrum reveals two distinct resonance bands, and simulation analysis suggested that the rotor and bearings give rise to low-frequency and high-frequency resonance, respectively. This study provides a reference for bearing faults diagnosis in wind turbine generator. • A dynamic model of generator bearing outer ring fault is studied considering UMP. • The modulation phenomenon of fault characteristic frequency and UMP is revealed. • The double resonance phenomenon of rotor-bearing system of generator is revealed. [ABSTRACT FROM AUTHOR]

The varying speed can cause the significant data distribution shift of bearings, making it difficult for deep learning-based bearing fault diagnosis models to ensure good generalization. Domain adaptation methods have been developed to address the domain shifts, while they struggle with the class-invariant features extraction under variable speed. Accordingly, a time–frequency supervised contrastive learning framework (TF-SupCon) is proposed for unsupervised cross-speed fault diagnosis of bearing. TF-SupCon adopts a pre-training-downstream task framework that aims to extract speed immune class-invariant features. During the pre-training phase, the physical consistency of the time-domain information and the frequency-domain information of bearing signal, a general feature applicable to different speed conditions, is learned in a targeted manner through supervised contrastive learning. Additionally, a K-Nearest Neighbor (KNN) algorithm based on cosine distance is designed to assign pseudo-labels to unlabeled target domain data, enabling cross-domain supervised contrastive pre-training. In the downstream task, unsupervised cross-domain fault diagnosis is performed using a KNN classifier and the speed immune time–frequency features by the trained encoders. It is worth noting that the same metric is maintained throughout both the pre-training phase and the downstream task, ensuring organic connection between the two stages. The superiority of TF-SupCon was demonstrated through a variety of fault diagnosis experiments conducted on both public and self-collected datasets. Lastly, the distances between time-domain and frequency-domain features of different categories were studied to verify the physical consistency between the features. [ABSTRACT FROM AUTHOR]

The commonly used diffuse fraction models are generally expressed as a single-value function of clearness index (K t). Whereas in fact, the same K t occurring at different times of the day may correspond to different diffuse fraction (K f). Using a 10-year dataset from 2005 to 2014, this study intended to model hourly solar diffuse fraction based on sky conditions. Five cities in China, including Golmud, Kashgar, Sanya, Wuhan, and Wenjiang, were selected as representatives of five solar climatic zones. The measured radiation data on a horizontal surface were used for k-means clustering of the sky conditions with two factors of K f and K t. The results show that the sky conditions could be divided into two groups according to time dimension: the morning (6:00–12:00) and the afternoon (13:00–18:00) segments. Subsequently, a morning-afternoon segmented K f -K t model for each zone was constructed based on quartic polynomial regression, with the smallest error rate at a range of 10.4%–28.5%. Comparison with the existing models further validates that the respective models for the morning and afternoon periods can assist in improving the modeling of the hourly solar diffuse fraction on a horizontal surface in variable solar climatic zones. • Same clearness index at various times may correspond to different diffuse fraction. • Sky conditions can be divided into morning and afternoon periods by time dimension. • Using quartic polynomial to regress hourly diffuse fraction performs well in China. • Constructing morning-afternoon segmented K f -K t model at each solar zone in China. [ABSTRACT FROM AUTHOR]

A controllable antisense oligonucleotides delivery carrier based on Frame-Guided Assembly strategy, which can inhibit Bcl-2 gene expression. [Display omitted] • A controllable antisense oligonucleotides delivery carrier based on Frame-Guided Assembly strategy. • Thermo sensitivity and redox responsiveness of Frame-Guided Assembly vesicles. • Antisense oligonucleotides vesicles revealed an obviously decreased cell viability of 59.8% after 3 days treatment. Using nanoparticles to deliver antisense oligonucleotides is a fabulous way which can solve the difficulty of penetrating cell membrane on account of high molecular weight and high negative charges. Herein, we have reported a frame-guided assembly strategy to prepare drug delivery system for anti-sense DNA. The in vitro experiments demonstrated that the anti-sense oligonucleotides vesicles perform good cytotoxicity to the cancer cells by downregulation of the expression of anti-apoptotic Bcl-2 gene, finally resulting in the apoptosis. It is feasible that taking advantage of the programmable assembly process and good stability of the frame-guided assembly (FGA) systems, this strategy can deliver a wide variety of drugs. [ABSTRACT FROM AUTHOR]

Background: Directed DNA methylation on N6-adenine (6mA), N4-cytosine (4mC), and C5-cytosine (5mC) can potentially increase DNA coding capacity and regulate a variety of biological functions. These modifications are relatively abundant in bacteria, occurring in about a percent of all bases of most bacteria. Until recently, 5mC and its oxidized derivatives were thought to be the only directed DNA methylation events in metazoa. New and more sensitive detection techniques (ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-ms/ms) and single molecule real-time sequencing (SMRTseq)) have suggested that 6mA and 4mC modifications could be present in a variety of metazoa. Results: Here, we find that both of these techniques are prone to inaccuracies, which overestimate DNA methylation concentrations in metazoan genomic DNA. Artifacts can arise from methylated bacterial DNA contamination of enzyme preparations used to digest DNA and contaminating bacterial DNA in eukaryotic DNA preparations. Moreover, DNA sonication introduces a novel modified base from 5mC that has a retention time near 4mC that can be confused with 4mC. Our analyses also suggest that SMRTseq systematically overestimates 4mC in prokaryotic and eukaryotic DNA and 6mA in DNA samples in which it is rare. Using UHPLC-ms/ms designed to minimize and subtract artifacts, we find low to undetectable levels of 4mC and 6mA in genomes of representative worms, insects, amphibians, birds, rodents and primates under normal growth conditions. We also find that mammalian cells incorporate exogenous methylated nucleosides into their genome, suggesting that a portion of 6mA modifications could derive from incorporation of nucleosides from bacteria in food or microbiota. However, gDNA samples from gnotobiotic mouse tissues found rare (0.9–3.7 ppm) 6mA modifications above background. Conclusions: Altogether these data demonstrate that 6mA and 4mC are rarer in metazoa than previously reported, and highlight the importance of careful sample preparation and measurement, and need for more accurate sequencing techniques. [ABSTRACT FROM AUTHOR]

Summary tRNA is a central component of protein synthesis and the cell signaling network. One salient feature of tRNA is its heavily modified status, which can critically impact its function. Here, we show that mammalian ALKBH1 is a tRNA demethylase. It mediates the demethylation of N 1 -methyladenosine (m 1 A) in tRNAs. The ALKBH1-catalyzed demethylation of the target tRNAs results in attenuated translation initiation and decreased usage of tRNAs in protein synthesis. This process is dynamic and responds to glucose availability to affect translation. Our results uncover reversible methylation of tRNA as a new mechanism of post-transcriptional gene expression regulation. [ABSTRACT FROM AUTHOR]