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Whole‐organ transcriptomic analyses have emerged as a common method for characterizing developmental transitions in mammalian organs. However, it is unclear if all cell types in an organ follow the whole‐organ defined developmental trajectory. Recently, a postnatal two‐stage developmental process was described for the mouse stomach. Here, using laser capture microdissection to obtain in situ transcriptomic data, we show that mouse gastric pit cells exhibit four postnatal developmental stages. Interestingly, early stages are characterized by the up‐regulation of genes associated with metabolism, a functionality not typically associated with pit cells. Hence, beyond revealing that not all constituent cells develop according to the whole‐organ determined pathway, these results broaden our understanding of the pit cell phenotypic landscape during stomach development. [ABSTRACT FROM AUTHOR]

The activation of monocytes and their trans-differentiation into macrophages are critical processes of the immune response. Prior work has characterized the differences in the expression between monocytes and macrophages, but the transitional process between these cells is poorly detailed. Here, we analyzed the temporal changes of the transcriptome during trans-differentiation of primary human monocytes into M0 macrophages. We find changes with many transcription factors throughout the process, the vast majority of which exhibit a maximally different expression at the intermediate stages. A few factors, including AP-1, were previously known to play a role in immunological transitions, but most were not. Thus, these findings indicate that this trans-differentiation requires the dynamic expression of many transcription factors not previously discussed in immunology, and provide a foundation for the delineation of the molecular mechanisms associated with healthy or pathological responses that involve this transition. [ABSTRACT FROM AUTHOR]

Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) can profile genome-wide epigenetic marks associated with regulatory genomic elements. However, conventional ChIP-seq is challenging when examining limited numbers of cells. Here, we developed a new technique by supplementing carrier materials of both chemically modified mimics with epigenetic marks and dUTP-containing DNA fragments during conventional ChIP procedures (hereafter referred to as 2cChIP-seq), thus dramatically improving immunoprecipitation efficiency and reducing DNA loss of low-input ChIP-seq samples. Using this strategy, we generated high-quality epigenomic profiles of histone modifications or DNA methylation in 10–1000 cells. By introducing Tn5 transposase-assisted fragmentation, 2cChIP-seq reliably captured genomic regions with histone modification at the single-cell level in about 100 cells. Moreover, we characterized the methylome of 100 differentiated female germline stem cells (FGSCs) and observed a particular DNA methylation signature potentially involved in the differentiation of mouse germline stem cells. Hence, we provided a reliable and robust epigenomic profiling approach for small cell numbers and single cells. [ABSTRACT FROM AUTHOR]

Mechanistic understanding of how living systems sense, transduce, and respond to mechanical cues has important implications in development, physiology, and therapy. Here, the authors use an integrated atomic force microscope (AFM) and brightfield/epifluorescent microscope platform to precisely simulate living single cells or groups of cells under physiological conditions, in real time, concomitantly measuring the single‐cell autophagic response and its transmission to neighboring cells. Dual‐color fluorescence monitoring of the cellular autophagic response reveals the dynamics of autophagosome formation, degradation, and induction in neighboring contacting and noncontacting cells. Autophagosome formation is dependent on both the applied force and contact area of the AFM tip. More importantly, the enhancement of the autophagic responses in neighboring cells via a gap junction‐dependent mechanism is observed. This AFM‐based nanoacupuncture platform can serve as a tool for elucidating the primary mechanism underlying mechanical stimulation of living systems and other biomechanical therapeutics. [ABSTRACT FROM AUTHOR]

Immortalized cell lines have long been used as model systems to systematically investigate biological processes under controlled and reproducible conditions, providing insights that have greatly advanced cellular biology and medical sciences. Recently, the widely used monocytic leukemia cell line, THP-1, was comprehensively examined to understand mechanistic relationships between the 3D chromatin structure and transcription during the trans-differentiation of monocytes to macrophages. To corroborate these observations in primary cells, we analyze in situ Hi-C and RNA-seq data of human primary monocytes and their differentiated macrophages in comparison to that obtained from the monocytic/macrophagic THP-1 cells. Surprisingly, we find significant differences between the primary cells and the THP-1 cells at all levels of chromatin structure, from loops to topologically associated domains to compartments. Importantly, the compartment-level differences correlate significantly with transcription: those genes that are in A-compartments in the primary cells but are in B-compartments in the THP-1 cells exhibit a higher level of expression in the primary cells than in the THP-1 cells, and vice versa. Overall, the genes in these different compartments are enriched for a wide range of pathways, and, at least in the case of the monocytic cells, their altered expression in certain pathways in the THP-1 cells argues for a less immune cell-like phenotype, suggesting that immortalization or prolonged culturing of THP-1 caused a divergence of these cells from their primary counterparts. It is thus essential to reexamine phenotypic details observed in cell lines with their primary counterparts so as to ensure a proper understanding of functional cell states in vivo. [ABSTRACT FROM AUTHOR]

Objective: To alleviate the severe limitation of the prohibitively long process of immune-fluorescence labeling on the routine applications of revolutionary intact tissue clearing techniques in diverse biomedical arenas. Methods: We proposed an easily adaptable approach, electro-enhanced rapid staining (EERS), for highly efficient and fast immuno-labeling of thick clarified tissues. In EERS, an optimized and precisely controlled weak external electric field is engineered into a compact device to enable efficient and uniform transport of antibodies into clarified tissues while minimizing the detrimental effect of macromolecular crowding at the tissue-solution interface. Results and Conclusions: The experimental results show that, with EERS, a current density of only ∼0.2 mA mm−2 is sufficient to achieve uniform labeling of clarified tissues of several millimeters thick in a few hours without detectable tissue damage. In addition, the amount of antibodies required is also several-fold lower than conventional immuno-labeling assays under comparable conditions. Significance: It is expected that the implementation of EERS in most laboratories should significantly expedite the application of tissue clearing in a broad range of research explorations, both basic and clinical. [ABSTRACT FROM AUTHOR]

Owing to the challenges to acquire detailed spatial information of gut bacteria in situ, three‐dimensional (3D) microbiota distributions in the gut remain largely uncharted. Here, we propose a tissue clearing‐based and D‐amino acid labeling‐facilitated (TiDaL) strategy that combines a novel microbiota in vivo labeling protocol, CUBIC‐based tissue clearing and whole‐mount tissue imaging, to achieve 3D imaging of indigenous gut microbiota. We demonstrate high‐resolution 3D acquisition of their biogeography in different gut sections, and present quantitative spatial details in relation to the host epithelium. We unexpectedly observe microbiota in the small intestine crypts, which were thought to be bacteria‐free. Significant bacterial overgrowth in the first two‐thirds of the small intestine is detected in an enteritis model. We expect that this quantitative 3D imaging strategy for native gut microbiota will provide insightful information into the host–microbiota interactions. [ABSTRACT FROM AUTHOR]

One of the most widely used approaches to characterize transmembrane ion transport through nanoscale synthetic or biological channels is a straightforward, liposome-based assay that monitors changes in ionic flux across the vesicle membrane using pH- or ion-sensitive dyes. However, failure to account for the precise experimental conditions, in particular the complete ionic composition on either side of the membrane and the inherent permeability of ions through the lipid bilayer itself, can prevent quantifications and lead to fundamentally incorrect conclusions. Here we present a quantitative model for this assay based on the Goldman–Hodgkin–Katz flux theory, which enables accurate measurements and identification of optimal conditions for the determination of ion channel permeability and selectivity. Based on our model, the detection sensitivity of channel permeability is improved by two orders of magnitude over the commonly used experimental conditions. Further, rather than obtaining qualitative preferences of ion selectivity as is typical, we determine quantitative values of these parameters under rigorously controlled conditions even when the experimental results would otherwise imply (without our model) incorrect behavior. We anticipate that this simply employed ultrasensitive assay will find wide application in the quantitative characterization of synthetic or biological ion channels. Image 1 • Popular liposome-based assay only provides qualitative properties of ion channels. • First theoretical description of this assay enables optimization and quantification. • The sensitivity to detect channel permeability improves ∼100-fold. • Misleading experimental results of ion selectivity can be distinguished and corrected. [ABSTRACT FROM AUTHOR]

Background. Lymph node (LN) metastasis was an independent risk factor for stomach cancer recurrence, and the presence of LN metastasis has great influence on the overall survival of stomach cancer patients. Thus, accurate prediction of the presence of lymph node metastasis can provide guarantee of credible prognosis evaluation of stomach cancer patients. Recently, increasing evidence demonstrated that the aberrant DNA methylation first appears before symptoms of the disease become clinically apparent. Objective. Selecting key biomarkers for LN metastasis presence prediction for stomach cancer using clinical DNA methylation based on a machine learning method. Methods. To reduce the overfitting risk of prediction task, we applied a three-step feature selection method according to the property of DNA methylation data. Results. The feature selection procedure extracted several cancer-related and lymph node metastasis-related genes, such as TP73, PDX1, FUT8, HOXD1, NMT1, and SEMA3E. The prediction performance was evaluated on the public DNA methylation dataset. The results showed that the three-step feature procedure can largely improve the prediction performance and implied the reliability of the biomarkers selected. Conclusions. With the selected biomarkers, the prediction method can achieve higher accuracy in detecting LN metastasis and the results also proved the reliability of the selected biomarkers indirectly. [ABSTRACT FROM AUTHOR]

As a naturally-abundant biopolymer, chitosan (CS) exhibit pH-sensitive structural transformation within a narrow pH range. Integrating hydrophobic groups to CS molecules gives modified CS polymers with more adjustable pH responsiveness. In this paper, near-infrared (NIR) photoluminescent Ag 2 S QDs capped by long-chain carboxylic acid were synthesized and then conjugated with CS via esterification reaction. The anticancer drug doxorubicin (DOX) has an affinity for the hydrophobic oleoyl groups and was entrapped by them to produce Ag 2 S(DOX)@CS nanospheres. A variety of experiments were performed to characterize the nanospheres. In vitro and in vivo experiments showed that the nanospheres can release DOX at lowered pH in tumor cells and have high antitumor efficacy. In addition, the strong NIR signal derived from the encapsulated Ag 2 S QDs makes real-time monitoring of the nanosphere distribution in a body possible. This study provides a new CS-based nanocomposite drug carrier for efficient cancer therapy. [ABSTRACT FROM AUTHOR]

The need for smart materials in the area of biotechnology has accelerated the development of stimuli-responsive copolymer micelles. Here, we reported a novel dual-stimuli-responsive block copolymer PEG-DMTK-SS-PLA with both dimethyl thioketal (DMTK) and disulfide linkage incorporated into the backbone, capable of triggering fast drug release properties upon both oxidative (H 2 O 2 ) and reductive (GSH) environment. The CMC values of these copolymer micelles ranging from 0.051 to 0.087 mg mL −1 , the average diameters are from 34 nm to 55 nm and 65 nm to 198 nm for blank and DOX-loaded micelles respectively. MTT assay conducted in NIH 3T3 cells showed the low cytotoxicity of these micelles even when the concentration reached up to 500 μg/mL. Considering tumor microenvironment’s diverse in kinds of tumor cells, fluorescence microscopy, flow cytometry and MTT activity analysis were conducted in several cancer cells (e.g., cervix, lung, gastric, and colon cancer cells), and further confirmed that the dual responsive PEG-DMTK-SS-PLA micelles were degraded much faster than that of non-responsive PEG-PLA and single responsive PEG-SS-PLA and PEG-DMTK-PLA micelles. These results indicate that the dual-responsive micelles are promising for efficient anticancer drug delivery. [ABSTRACT FROM AUTHOR]

Application of cisplatin (DDP) for treating lung cancer is restricted due to its toxicity and lung cancer’s drug resistance. In this study, we examined the effect of Jinfukang (JFK), an effective herbal medicine against lung cancer, on DDP-induced cytotoxicity in lung cancer cells. Morphologically, we observed that JFK increases DDP-induced pro-apoptosis in A549 cells in a synergistic manner. Transcriptome profiling analysis indicated that the combination of JFK and DDP regulates genes involved in apoptosis-related signaling pathways. Moreover, we found that the combination of JFK and DDP produces synergistic pro-apoptosis effect in other lung cancer cell lines, such as NCI-H1975, NCI-H1650, and NCI-H2228. Particularly, we demonstrated that AIFM2 is activated by the combined treatment of JFK and DDP and partially mediates the synergistic pro-apoptosis effect. Collectively, this study not only offered the first evidence that JFK promotes DDP-induced cytotoxicity, and activation of AIFM2 enhances apoptosis of human lung cancer cells undergoing toxicological stress, but also provided a novel insight for improving cytotoxicity by combining JFK with DDP to treat lung cancer cells. [ABSTRACT FROM AUTHOR]

Redox-responsive micelles self-assembled from triblock copolymers of poly( l -lactic acid)-poly(ethylene glycol)-poly( l -lactic acid) ( PLA-PEG-PLA) with double-disulfide linkage in the backbone were synthesized and characterized by proton nuclear magnetic resonance ( 1 H NMR) and size exclusion chromatography (SEC), in which both PEG ( M n = 1000, 2000 and 4000 g mol −1 ) and PLA ( M n = 1600 g mol −1 ) have different molecular weights respectively. The triblock copolymers PLA 3000 -PEG 2000 -PLA 3000 and PLA 3000 -PEG 4000 -PLA 3000 can self-assemble into flower-like micelles in aqueous media with average diameters 110 nm and 43 nm and lower critical micelle concentrations (CMC) 0.017 and 0.014 mg mL −1 respectively compared with that of diblock copolymers. Moreover, in vitro drug release analyses indicated that reductive environment can result in triggered drug release profiles. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl- 2-H-tetrazolium bromide (MTT) assay in vitro showed no significant cytotoxicity as NIH 3T3 cells incubated in the micelles even when the concentrations up to 1000 μg/mL. Additionally fluorescence microscopy measurements and MTT assay demonstrated that the micelles exhibited a faster drug release and higher cellular proliferation inhibition due to the effect of intracellular reduction responsiveness compared with that of diblock copolymers. The above results suggest that the reduction-responsive, biodegradable and biocompatibility micelles could provide a platform to construct potential drug delivery systems for cancer therapy. [ABSTRACT FROM AUTHOR]

Traditional Chinese medicine Jinfukang (JFK) has been clinically used for treating lung cancer. To examine whether epigenetic modifications are involved in its anticancer activity, we performed a global profiling analysis of H3K4Me3, an epigenomic marker associated with active gene expression, in JFK-treated lung cancer cells. We identified 11,670 genes with significantly altered status of H3K4Me3 modification following JFK treatment (P<0.05). Gene Ontology analysis indicates that these genes are involved in tumor-related pathways, including pathway in cancer, basal cell carcinoma, apoptosis, induction of programmed cell death, regulation of transcription (DNA-templated), intracellular signal transduction, and regulation of peptidase activity. In particular, we found that the levels of H3K4Me3 at the promoters of SUSD2, CCND2, BCL2A1, and TMEM158 are significantly altered in A549, NCI-H1975, NCI-H1650, and NCI-H2228 cells, when treated with JFK. Collectively, these findings provide the first evidence that the anticancer activity of JFK involves modulation of histone modification at many cancer-related gene loci. [ABSTRACT FROM AUTHOR]

An acid-cleavable linker based on a dimethylketal moiety was synthesized and used to connect a nucleotide with a fluorophore to produce a 3′-OH unblocked nucleotide analogue as an excellent reversible terminator for DNA sequencing by synthesis. [ABSTRACT FROM AUTHOR]

A cleavable azo linker was synthesized and reacted with 5-(6)-carboxytetramethyl rhodamine succinimidyl ester, followed by further reactions with di(N-succinimidyl) carbonate and 5-(3-amino-1-propynyl)-2′-deoxyuridine 5′-triphosphate [dUTP(AP3)] to obtain the terminal product dUTP-azo linker-TAMRA as a potential reversible terminator for DNA sequencing by synthesis with no need for 3′-OH blocking. [ABSTRACT FROM AUTHOR]

[Display omitted] • The polyMOF (CuPP-PELA) composite was prepared by in situ ROP reaction first time. • The in situ formed PELA could accelerate the degradation of MOF (CuPP). • The burst release of Curcumin under NIR sensitize the photothermal sterilization. • Curcumin gradually released without NIR remodulate the immune microenvironment. Chronic wounds, especially those caused by drug-resistant bacteria, remain mostly intractable to existing treatments. Photothermal therapy, apart from the additional inflammation it may cause, exhibits potent ability to efficiently destroy drug-resistant bacteria. A system capable of thermal therapy together with active regulation of the repairing process could be a potentially effective treatment for these wounds. Here we developed a highly integrated hydrogel made of curcumin loaded copper-tetrakis(4-carboxyphenyl)porphyrin metal–organic framework and poly (ethylene glycol)-poly(ε-caprolactone-co-lactide) hybrid by in situ ring-opening polymerization. It combines photothermal effect, immune modulatory, hemostasis and temperature-sensitive sol–gel transition capacity. This multifunctional polymer-metal–organic framework (polyMOF) hydrogel dressing showed the ability to completely heal drug-resistant bacteria infected wounds within 14 days and could be an effective and biocompatible material for chemo-photothermal therapy against chronic wounds. Moreover, our strategy to prepare the polyMOF hybrid may provide a universal approach for the development of multifunctional gels with great significance in clinical application. [ABSTRACT FROM AUTHOR]

Abstract: Developing smart nanocarriers for drug delivery system is advantageous for many kinds of successful biomedicinal therapy. In this study, we designed an amphiphilic block copolymers containing pH-responsive tetrahydropyran (THP) and tetrahydrofuran (THF) linkage. Their structures were confirmed by 1H NMR and gel permeation chromatography (GPC). The release rate of encapsulated drugs depends upon the pH value and pH sensitive linkage in the backbone of copolymers. For PLA–THP–PEG micelles the cumulative release amount of doxorubicin (DOX) was 62% at pH 5.0, which is about four times higher than that at pH 7.4. Under the same conditions the release rate for PLA–THF–PEG micelles is a little faster than that of the PLA–THP–PEG micelles. Cellular uptake study demonstrates that DOX-loaded micelles can easily enter the cells and produce the desired pharmacological action and minimizing the side effect of free DOX. These findings indicate that THP and THF linked diblock copolymer micelles is a promising candidate for drug carrier. [Copyright &y& Elsevier]

Highlights: [•] The paper describes the way of a mercury arc lamp as a key component in localization-based super-resolution microscope. [•] The achievable sub-diffraction resolution is comparable to other more complicated system. [•] The setup only requires simple modification on conventional fluorescence microscope. [ABSTRACT FROM AUTHOR]

Abstract: In the budding yeast, cohesin is loaded onto the chromosome during the late G1 phase, establishes sister chromatid cohesion concomitant with DNA replication, and dissociates by the telophase. Here, using oligonucleotide tiling arrays, we show that, at the anaphase, nearly all of the cohesin binding sites contain nucleosome-free regions. The majority of these sites remain nucleosome-free throughout the cell cycle, consistent with the suggestion of a DNA-binding anchoring protein present at these sites, although such a region could also serve as part of a marker for the binding of cohesin in the next cell cycle. However, a third of these sites are remodeled in the G1 phase, being reoccupied by nucleosomes by the G1/S boundary, though their subsequent removal in the S phase appears to be independent of DNA replication. Whether this difference is a result of other functions of cohesin or of the chromatin remains to be elucidated. [Copyright &y& Elsevier]

Sentinel lymph node (SLN) imaging and biopsy has been advocated as an important technique to evaluate the metastatic status of regional lymph nodes and determine subsequent surgical procedure for many cancers, yet there is no reliable means to provide accurate and rapid diagnosis of metastatic SLN during surgery. Here we develop a new approach, named "Ratiometric Raman dual-nanotag strategy", that using folic acid functionalized targeted and nontargeted gap-enhanced Raman tags (FA-GERTs and Nt-GERTs) to detect metastatic SLN based on Raman imaging combined with classical least square data processing methods. By using this strategy, with built-in self-calibration for signal correction, rather than absolute intensity-dependent signal readout, we realize the visualization and prompt intraoperative diagnosis of metastatic SLN with a high accuracy of 87.5 % when the cut-off value of ratio (FA-GERTs/Nt-GERTs) set at 1.255. This approach may outperform the existing histopathological assessment in diagnosing SLN metastasis and is promising for guiding surgical procedure in the future. [ABSTRACT FROM AUTHOR]

Atomic Force Microscopy (AFM) is a powerful technique with widespread applications in various scientific fields, including biology. It operates by precisely detecting the interaction between a sharp tip and a sample surface, providing high-resolution topographical information and mechanical properties at a nanoscale. Through the years, a deeper understanding of this tip-sample interaction and the mechanisms by which it can be more precisely regulated have invariably led to improvements in AFM imaging. Additionally, AFM can serve not only as a sensor but also as a tool for actively manipulating the mechanical properties of biological systems. By applying controlled forces to the sample surface, AFM allows for a deeper understanding of mechanotransduction pathways, the intricate signaling cascades that convert physical cues into biochemical responses. This review, is an extensive overview of the current status of AFM working either as a mechanosensor or a mechanotransducer to probe biological systems across diverse scales, from individual molecules to entire tissues is presented. Challenges are discussed and potential future research directions are elaborated., (© 2024 Wiley‐VCH GmbH.)

Visualizing the whole vascular network system is crucial for understanding the pathogenesis of specific diseases and devising targeted therapeutic interventions. Although the combination of light sheet microscopy and tissue-clearing methods has emerged as a promising approach for investigating the blood vascular network, leveraging the spatial resolution down to the capillary level and the ability to image centimeter-scale samples remains difficult. Especially, as the resolution improves, the issue of photobleaching outside the field of view poses a challenge to image the whole vascular network of adult mice at capillary resolution. Here, we devise a fluorescent microsphere vascular perfusion method to enable labeling of the whole vascular network in adult mice, which overcomes the photobleaching limit during the imaging of large samples. Moreover, by combining the utilization of a large-scale light-sheet microscope and tissue clearing protocols for whole-mouse samples, we achieve the capillary-level imaging resolution (3.2 × 3.2 × 6.5 μm) of the whole vascular network with dimensions of 45 × 15 × 82 mm in adult mice. This method thus holds great potential to deliver mesoscopic resolution images of various tissue organs for whole-animal imaging.

Quantitative detection of various molecules at very low concentrations in complex mixtures has been the main objective in many fields of science and engineering, from the detection of cancer-causing mutagens and early disease markers to environmental pollutants and bioterror agents 1-5 . Moreover, technologies that can detect these analytes without external labels or modifications are extremely valuable and often preferred 6 . In this regard, surface-enhanced Raman spectroscopy can detect molecular species in complex mixtures on the basis only of their intrinsic and unique vibrational signatures 7 . However, the development of surface-enhanced Raman spectroscopy for this purpose has been challenging so far because of uncontrollable signal heterogeneity and poor reproducibility at low analyte concentrations 8 . Here, as a proof of concept, we show that, using digital (nano)colloid-enhanced Raman spectroscopy, reproducible quantification of a broad range of target molecules at very low concentrations can be routinely achieved with single-molecule counting, limited only by the Poisson noise of the measurement process. As metallic colloidal nanoparticles that enhance these vibrational signatures, including hydroxylamine-reduced-silver colloids, can be fabricated at large scale under routine conditions, we anticipate that digital (nano)colloid-enhanced Raman spectroscopy will become the technology of choice for the reliable and ultrasensitive detection of various analytes, including those of great importance for human health., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

The faithful transmission of a cell's identity and functionality to its daughters during mitosis requires the proper assembly of mitotic chromosomes from interphase chromatin in a process that involves significant changes in the genome-bound material, including the RNA. However, our understanding of the RNA that is associated with the mitotic chromosome is presently limited. Here, we present complete and quantitative characterizations of the full-length mitotic chromosome-associated RNAs (mCARs) for 3 human cell lines, a monkey cell line, and a mouse cell line derived from high-depth RNA sequencing (3 replicates, 47 M mapped read pairs for each replicate). Overall, we identify, on average, more than 20,400 mCAR species per cell-type (including isoforms), more than 5,200 of which are enriched on the chromosome. Notably, overall, more than 2,700 of these mCARs were previously unknown, which thus also expands the annotated genome of these species. We anticipate that these datasets will provide an essential resource for future studies to better understand the functioning of mCARs on the mitotic chromosome and in the cell., (© 2024. The Author(s).)

The histone variant macroH2A has been found to play important regulatory roles in genomic processes, especially in regulating transcriptomes. However, whether macroH2A nucleosomes are retained on mitotic chromosomes to enable maintenance of cell-specific transcriptomes is not known. Here, examining mouse embryonic fibroblast cells (NIH-3T3) with native chromatin immunoprecipitation and sequencing (nChIP-seq), we show that the overwhelming majority (~90%) of macroH2A1 domains identified at the G1/S stage are indeed stably retained on mitotic chromosomes. Unexpectedly though, we also find that there are a number of macroH2A domains that are specific for either mitotic or G1/S cells. Notably, more than 7,000 interphase expressed genes flanked by macroH2A1 domains are loaded with macroH2A1 nucleosomes on the mitotic chromosome to form extended domains. Overall, these results reveal that, while the majority of macroH2A1 domains are indeed faithfully transmitted through the mitotic chromosomes, there is a previously unknown cell-cycle dependent exchange of macroH2A1 nucleosomes at numerous genomic loci, indicating the existence of molecular machineries for this dynamically regulated process. We anticipate that these findings will prove to be essential for the integrity of mitotic progression and the maintenance of cellular identity.

The convergent positioning of functional groups in biomacromolecules leads to good binding, catalytic and transport capabilities. Synthetic frameworks capable of convergently locking functional groups with minimized conformational uncertainty-leading to similar properties-are highly desirable but rare. Here we report C5-symmetric aromatic pentaamide macrocycles synthesized in one pot from the corresponding monomers. Their crystal structures reveal a star-shaped, fully constrained backbone that causes ten alternating NH/CH hydrogen-bond donors and five large amide dipoles to orient towards the centre of the macrocycle. With a highly electropositive cavity in a high-energy unbound state, the macrocycles bind anions in a 1:1 stoichiometry in solution, with high affinity for halides and very high affinity for oxoanions. We demonstrate that such macrocycles are able to transport anions across lipid bilayers with a high chloride selectivity and restore the depleted airway surface liquid of cystic fibrosis airway cell cultures., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

The stoichiometry of molecular components within supramolecular biological complexes is often an important property to understand their biological functioning, particularly within their native environment. While there are well established methods to determine stoichiometry in vitro , it is presently challenging to precisely quantify this property in vivo, especially with single molecule resolution that is needed for the characterization stoichiometry heterogeneity. Previous work has shown that optical microscopy can provide some information to this end, but it can be challenging to obtain highly precise measurements at higher densities of fluorophores. Here we provide a simple approach using already established procedures in single-molecule localization microscopy (SMLM) to enable precise quantification of stoichiometry within individual complexes regardless of the density of fluorophores. We show that by focusing on the number of fluorophore detections accumulated during the quasi equilibrium-state of this process, this method yields a 50-fold improvement in precision over values obtained from images with higher densities of active fluorophores. Further, we show that our method yields more correct estimates of stoichiometry with nuclear pore complexes and is easily adaptable to quantify the DNA content with nanodomains of chromatin within individual chromosomes inside cells. Thus, we envision that this straightforward method may become a common approach by which SMLM can be routinely employed for the accurate quantification of subunit stoichiometry within individual complexes within cells., (© 2023 IOP Publishing Ltd.)

With improvements in the survival rate of patients with cancer, fertility maintenance has become a major concern in terms of cancer treatment for women of reproductive age. Thus, it is important to examine the impact on fertility of anticancer drugs that are used clinically or are undergoing trials. The HuR small-molecule inhibitor MS-444 has been used in many cancer treatment studies, but its reproductive toxicity in females is unknown. Here, we reported that MS-444 blocked the nucleocytoplasmic transport of Agbl2 mRNA by inhibiting HuR dimerization, resulting in the developmental arrest of 2-cell stage embryos in mouse. Combining analysis of low-input RNA-seq for MS-444-treated 2-cell embryos and mapping binding sites of RNA-binding protein, Agbl2 was predicted to be the target gene of MS-444. For further confirmation, RNAi experiment in wild-type zygotes showed that Agbl2 knockdown reduced the proportion of embryos successfully developed to the blastocyst stage: from 71% in controls to 23%. Furthermore, RNA-FISH and luciferase reporter analyses showed that MS-444 blocked the nucleocytoplasmic transport of Agbl2 mRNA and reduced its stability by inhibiting HuR dimerization. In addition, optimized stochastic optical reconstruction microscopy (STORM) imaging showed that MS-444 significantly reduced the HuR dimerization, and HuR mainly existed in cluster form in 2-cell stage embryos. In conclusion, this study provides clinical guidance for maintaining fertility during the treatment of cancer with MS-444 in women of reproductive age. And also, our research provides guidance for the application of STORM in nanometer scale studies of embryonic cells. HuR inhibitor MS-444 arrested embryonic development at 2-cell stage. Low-input RNA-seq revealed that Agbl2 was the target gene of MS-444. MS-444 blocked the nucleocytoplasmic transport of Agbl2 mRNA by inhibiting HuR dimerization and reduced the stability of Agbl2 mRNA. STORM with our optimized protocol showed that HuR tended to form elliptical and dense clusters in 2-cell stage embryos., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

The folding of interphase chromatin into highly compact mitotic chromosomes is one of the most recognizable changes during the cell cycle. However, the structural organization underlying this drastic compaction remains elusive. Here, we combine several super resolution methods, including structured illumination microscopy (SIM), binding-activated localization microscopy (BALM), and atomic force microscopy (AFM), to examine the structural details of the DNA within the mitotic chromosome, both in the native state and after up to 30-fold extension using single-molecule micromanipulation. Images of native chromosomes reveal widespread ∼125 nm compact granules (CGs) throughout the metaphase chromosome. However, at maximal extensions, we find exclusively ∼90 nm domains (mitotic nanodomains, MNDs) that are unexpectedly resistant to extensive forces of tens of nanonewtons. The DNA content of the MNDs is estimated to be predominantly ∼80 kb, which is comparable to the size of the inner loops predicted by a recent nested loop model of the mitotic chromosome. With this DNA content, the total volume expected of the human genome assuming closely packed MNDs is nearly identical to what is observed. Thus, altogether, these results suggest that these mechanically stable MNDs, and their higher-order assembly into CGs, are the dominant higher-level structures that underlie the compaction of chromatin from interphase to metaphase.

The functioning of tissues is fundamentally dependent upon not only the phenotypes of the constituent cells but also their spatial organization in the tissue, as local interactions precipitate intra-cellular events that often lead to changes in expression. However, our understanding of these processes in tissues, whether healthy or diseased, is limited at present owing to the difficulty in acquiring comprehensive transcriptional programs of spatially- and phenotypically-defined cells in situ . Here we present a robust method based on immunofluorescence-guided laser capture microdissection (immuno-LCM-RNAseq) to acquire finely resolved transcriptional programs with as few as tens of cells from snap-frozen or RNAlater-treated clinical tissues sufficient to resolve even isoforms. The protocol is optimized to protect the RNA with a small molecule inhibitor, the ribonucleoside vanadyl complex (RVC), which thereby enables the typical time-consuming immunostaining and laser capture steps of this procedure during which RNA is usually severely degraded in existing approaches. The efficacy of this approach is exemplified by the characterization of differentially expressed genes between the mouse small intestine lacteal cells at the tip versus the main capillary body, including those that function in sensing and responding to local environmental cues to stimulate intra-cellular signalling. With the extensive repertoire of specific antibodies that are presently available, our method provides an unprecedented capability for the analysis of transcriptional networks and signalling pathways during development, pathogenesis, and aging of specific cell types within native tissues., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhang, Hu, Huang, Wei, Li, Hu, Li, Wu, Czajkowsky, Guo and Shao.)

Recent studies have characterized the genomic structures of many eukaryotic cells, often focusing on their relation to gene expression. However, these studies have largely investigated cells grown in 2D cultures, although the transcriptomes of 3D-cultured cells are generally closer to their in vivo phenotypes. To examine the effects of spatial constraints on chromosome conformation, we investigated the genomic architecture of mouse hepatocytes grown in 2D and 3D cultures using in situ Hi-C. Our results reveal significant differences in higher-order genomic interactions, notably in compartment identity and strength as well as in topologically associating domain (TAD)-TAD interactions, but only minor differences are found at the TAD level. Our RNA-seq analysis reveals an up-regulated expression of genes involved in physiological hepatocyte functions in the 3D-cultured cells. These genes are associated with a subset of structural changes, suggesting that differences in genomic structure are critically important for transcriptional regulation. However, there are also many structural differences that are not directly associated with changes in gene expression, whose cause remains to be determined. Overall, our results indicate that growth in 3D significantly alters higher-order genomic interactions, which may be consequential for a subset of genes that are important for the physiological functioning of the cell., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

There is presently intense interest in the development of synthetic nanopores that recapitulate the functional properties of biological water channels for a wide range of applications. To date, all known synthetic water channels have a hydrophobic lumen, and while many exhibit a comparable rate of water transport as biological water channels, there is presently no rationally designed system with the ability to regulate water transport, a critical property of many natural water channels. Here, we describe a self-assembling nanopore consisting of stacked macrocyclic molecules with a hybrid hydrophilic/hydrophobic lumen exhibiting water transport that can be regulated by alkali metal ions. Stopped-flow kinetic assays reveal a non-monotonic-dependence of transport on cation size as well as a strikingly broad range of water flow, from essentially none in the presence of the sodium ion to as high a flow as that of the biological water channel, aquaporin 1, in the absence of the cations. All-atom molecular dynamics simulations show that the mechanism underlying the observed sensitivity is the binding of cations to defined sites within this hybrid pore, which perturbs water flow through the channel. Thus, beyond revealing insights into factors that can modulate a high-flux water transport through sub-nm pores, the obtained results provide a proof-of-concept for the rational design of next-generation, controllable synthetic water channels., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

Single-cell chromatin studies provide insights into how chromatin structure relates to functions of individual cells. However, balancing high-resolution and genome wide-coverage remains challenging. We describe a computational method for the reconstruction of large 3D-ensembles of single-cell (sc) chromatin conformations from population Hi-C that we apply to study embryogenesis in Drosophila. With minimal assumptions of physical properties and without adjustable parameters, our method generates large ensembles of chromatin conformations via deep-sampling. Our method identifies specific interactions, which constitute 5-6% of Hi-C frequencies, but surprisingly are sufficient to drive chromatin folding, giving rise to the observed Hi-C patterns. Modeled sc-chromatins quantify chromatin heterogeneity, revealing significant changes during embryogenesis. Furthermore, >50% of modeled sc-chromatin maintain topologically associating domains (TADs) in early embryos, when no population TADs are perceptible. Domain boundaries become fixated during development, with strong preference at binding-sites of insulator-complexes upon the midblastula transition. Overall, high-resolution 3D-ensembles of sc-chromatin conformations enable further in-depth interpretation of population Hi-C, improving understanding of the structure-function relationship of genome organization.

Background: Spasmolytic polypeptide-expressing metaplasia (SPEM) is present in more than 90% of resected gastric cancer tissues. However, although widely regarded as a pre-cancerous tissue, its genetic characteristics have not been well studied., Methods: Immunohistochemistry using Trefoil factor 2 (TFF2) antibodies was used to identify TFF2-positive SPEM cells within SPEM glands in the stomach of Helicobacter felis (H. felis) -infected mice and human clinical samples. Laser microdissection was used to isolate specific cells from both the infected mice and the human samples. The genetic instability in these cells was examined by measuring the lengths of microsatellite (MS) markers using capillary electrophoresis. Also, genome-wide genetic variations in the SPEM cells from the clinical sample was examined using deep whole-exome sequencing., Results: SPEM cells indeed exhibit not only heightened MS instability (MSI), but also genetic instabilities that extend genome-wide. Furthermore, surprisingly, we found that morphologically normal, TFF2-negative cells also contain a comparable degree of genomic instabilities as the co-resident SPEM cells within the SPEM glands., Conclusion: These results, for the first time, clearly establish elevated genetic instability as a critical property of SPEM glands, which may provide a greater possibility for malignant clone selection. More importantly, these results indicate that SPEM cells may not be the sole origin of carcinogenesis in the stomach and strongly suggest the common progenitor of these cells, the stem cells, as the source of these genetic instabilities, and thus, potential key players in carcinogenesis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Monocyte-to-macrophage trans-differentiation has long been studied to better understand this immunological response and aspects of developmental processes more generally. A key question is the nature of the corresponding changes in chromatin conformation and its relationship to the transcriptome during this process. This question is especially intriguing since this trans-differentiation is not associated with progression through mitosis, often considered a necessary step for gross changes in chromosomal structure. Here, we characterized the transcriptional and genomic structural changes during macrophage development of primary human monocytes using RNA-seq and in situ Hi-C. We found that, during this transition, the genome architecture undergoes a massive remodeling to a degree not observed before between structured genomes, with changes in ~90% of the topologically associating domains (TADs). These changes in the TADs are associated with changed expression of immunological genes. These structural changes, however, differ extensively from those described recently in a study of the leukemia cell line, THP-1. Furthermore, up-regulation of the AP-1 family of genes that effected functionally important changes in the genomic structure during the differentiation of the THP-1 cells was not corroborated with the primary cells. Taken together, our results provide a comprehensive characterization of the changes in genomic structure during the monocyte-to-macrophage transition, establish a framework for the elucidation of processes underlying differentiation without proliferation, and demonstrate the importance of verifying with primary cells the mechanisms discovered with cultured cells., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Nucleosomal profiling is an effective method to determine the positioning and occupancy of nucleosomes, which is essential to understand their roles in genomic processes. However, the positional randomness across the genome and its relationship with nucleosome occupancy remains poorly understood. Here we present a computational method that segments the profile into nucleosomal domains and quantifies their randomness and relative occupancy level. Applying this method to published data, we find on average ~ 3-fold differences in the degree of positional randomness between regions typically considered "well-ordered", as well as an unexpected predominance of only two types of domains of positional randomness in yeast cells. Further, we find that occupancy levels between domains actually differ maximally by ~ 2-3-fold in both cells, which has not been described before. We also developed a procedure by which one can estimate the sequencing depth that is required to identify nucleosomal positions even when regional positional randomness is high. Overall, we have developed a pipeline to quantitatively characterize domain-level features of nucleosome randomness and occupancy genome-wide, enabling the identification of otherwise unknown features in nucleosomal organization.

Atomic force microscopy-based single-molecule-force spectroscopy is limited by low throughput. We introduce addressable DNA origami to study multiple target molecules. Six target DNAs that differed by only a single base-pair mismatch were clearly differentiated a rupture force of only 4 pN.

Background: In mammals, fine-tuned regulation of gene expression leads to transcription initiation from diverse transcription start sites (TSSs) and multiple core promoters. Although polysome association is a critical step in translation, whether polysome selectively uses TSSs and core promoters and how this could impact translation remains elusive., Results: In this study, we used CAGE followed by deep sequencing to globally profile the transcript 5' isoforms in the translatome and transcriptome of human HEK293 cells at single-nucleotide resolution. By comparing the two profiles, we identified the 5' isoforms preferentially used in translatome and revealed a widespread selective usage of TSSs (32.0%) and core promoters (48.7%) by polysome. We discovered the transcription initiation patterns and the sequence characteristics that were highly correlated with polysome selection. We further identified 5804 genes significantly enriched or depleted in translatome and showed that polysome selection was an important contributing factor to the abundance of related gene products. Moreover, after comparison with public transcriptome CAGE data from 180 human tissues and primary cells, we raised a question on whether it is a widely adopted mechanism to regulate translation efficiency by changing the transcription initiation sites on the transcription level in cells of different conditions., Conclusions: Using HEK293 cells as a model, we delineated an indirect selection toward TSSs and core promoters by the translation machinery. Our findings lend additional evidence for a much closer coordination between transcription and translation, warranting future translatome studies in more cell types and conditions to develop a more intricate regulatory model for gene expression.

DNA methylation plays a critical role in tumorigenesis through regulating oncogene activation and tumor suppressor gene silencing. Although extensively analyzed, the implication of DNA methylation in gene regulatory network is less characterized. To address this issue, in this study we performed an integrative analysis on the alteration of DNA methylation patterns and the dynamics of gene regulatory network topology across distinct stages of stomach cancer. We found the global DNA methylation patterns in different stages are generally conserved, whereas some significantly differentially methylated genes were exclusively observed in the early stage of stomach cancer. Integrative analysis of DNA methylation and network topology alteration yielded several genes which have been reported to be involved in the progression of stomach cancer, such as IGF2 , ERBB2 , GSTP1 , MYH11 , TMEM59 , and SST . Finally, we demonstrated that inhibition of SST promotes cell proliferation, suggesting that DNA methylation-associated SST suppression possibly contributes to the gastric cancer progression. Taken together, our study suggests the DNA methylation-associated regulatory network analysis could be used for identifying cancer-related genes. This strategy can facilitate the understanding of gene regulatory network in cancer biology and provide a new insight into the study of DNA methylation at system level.

The cell cortex is a layer of cytoskeletal networks underneath the plasma membrane, formed by filamentous actin (F-actin) and cortex proteins including spectrin, adducin, and myosin. It provides cells with proper stiffness, elasticity, and surface tension to allow morphogenesis, division, and migration. Although its architecture and formation have been widely studied in red blood cells, they are poorly understood in non-erythrocytes due to structural complexity and versatile functions. In this study, we identify the actin-binding protein abLIM1 as a novel non-erythroid cell-specific cortex organizer. Endogenous abLIM1 colocalized with cortical βII spectrin but upon overexpression redistributed to thick cortical actin bundles. abLIM1 associated with major cortex proteins such as spectrins and adducin in vivo. Depletion of abLIM1 by RNAi induced prominent blebbing during membrane protrusions of spreading or migrating RPE1 cells and impaired migration efficiency. Reducing cortical tensions by culturing the cells to confluency or inhibiting myosin activity repressed the blebbing phenotype. abLIM1-depleted RPE1 or U2OS cells lacked the dense interwoven cortical actin meshwork observed in control cells but were abundant in long cortical actin bundles along the long axis of the cells. In-vitro assays indicated that abLIM1 was able to crosslink and bundle F-actin to induce dense F-actin network formation. Therefore, abLIM1 governs the formation of dense interconnected cortical actin meshwork in non-erythroid cells to prevent mechanical tension-induced blebbing during cellular activities such as spreading and migration., Competing Interests: The authors declare that they have no conflict of interest.

It is widely recognized that the higher-order spatial organization of the genome, beyond the nucleosome, plays an important role in many biological processes. However, to date, direct information on even such fundamental structural details as the typical sizes and DNA content of these higher-order structures in situ is poorly characterized. Here, we examine the nanoscopic DNA organization within human nuclei using super-resolution direct stochastic optical reconstruction microscopy (dSTORM) imaging and 5-ethynyl-2'-deoxyuridine click chemistry, studying single fully labeled chromosomes within an otherwise unlabeled nuclei to improve the attainable resolution. We find that, regardless of nuclear position, individual subchromosomal regions consist of three different levels of DNA compaction: (i) dispersed chromatin; (ii) nanodomains of sizes ranging tens of nanometers containing a few kilobases (kb) of DNA; and (iii) clusters of nanodomains. Interestingly, the sizes and DNA content of the nanodomains are approximately the same at the nuclear periphery, nucleolar proximity, and nuclear interior, suggesting that these nanodomains share a roughly common higher-order architecture. Overall, these results suggest that DNA compaction within the eukaryote nucleus occurs via the condensation of DNA into few-kb nanodomains of approximately similar structure, with further compaction occurring via the clustering of nanodomains.

LIF-mediated STAT3 signaling is critically involved in stem cells and development. However, its function in mouse female germline cells (FGSCs) remains elusive. In this study, we demonstrated that LIF-induced STAT3 activation contributes to the proliferation and undifferentiation maintenance of mouse FGSCs. Characterization of the STAT3-mediated transcriptional network by intersecting ChIP-seq and RNA-seq datasets revealed 405 direct target genes of STAT3, which are primarily involved in proliferation and germline development. In particular, we observed that STAT3 exhibits a FGSC-specific binding pattern when compared with mouse embryonic stem cells. Taken together, our study reported that the LIF-mediated STAT3 activation is actively involved in FGSCs and functions through a distinctive binding pattern across the FGSC genome. This cell-type specific binding preference provides an insight into understanding the genetic base for STAT3-driven cellular functions in germline stem cells.

One of the most successful applications of atomic force microscopy (AFM) in biology involves monitoring the effect of force on single biological molecules, often referred to as force spectroscopy. Such studies generally entail the application of pulling forces of different magnitudes and velocities upon individual molecules to resolve individualistic unfolding/separation pathways and the quantification of the force-dependent rate constants. However, a less recognized variation of this method, the application of compressive force, actually pre-dates many of these "tensile" force spectroscopic studies. Further, beyond being limited to the study of single molecules, these compressive force spectroscopic investigations have spanned samples as large as living cells to smaller, multi-molecular complexes such as viruses down to single protein molecules. Correspondingly, these studies have enabled the detailed characterization of individual cell states, subtle differences between seemingly identical viral structures, as well as the quantification of rate constants of functionally important, structural transitions in single proteins. Here, we briefly review some of the recent achievements that have been obtained with compressive force spectroscopy using AFM and highlight exciting areas of its future development., Competing Interests: The authors declare no conflict of interest.