Natural polysaccharides regulate intestinal microbiota for inhibiting colorectal cancer.

The gastrointestinal tract is an important part of the human immune system. The gut microbiome, which constitutes a major component of the gastrointestinal tract, plays a crucial role in maintaining normal physiological functions and influences the development, diagnosis, and immunotherapy of colorectal cancer (CRC). Natural polysaccharides can be extracted from animals, plants, and traditional Chinese medicines. They serve as an essential energy source for the gut microbiome, promoting probiotic proliferation and regulating the intestinal microecological balance. Moreover, polysaccharides exhibit anti-tumor effects due to their immune regulatory functions and low toxicity. This review focuses on discussing these anti-tumor effects in CRC, along with improving gut microbiome dysbiosis and regulating the tumor immune microenvironment, providing evidence for effective therapeutic strategies against CRC.

Pt-S bond stabilized DNAzyme nanosensor with thiol-resistance enabling high-fidelity biosensing.

Gold nanoparticles (Au NPs) have been widely utilized in developing DNAzyme-functionalized nanosensors, most of which were engineered by attaching the thiolated DNAzymes to Au NPs via Au-S bonding. However, the Au NP-DNAzyme nanosensors always suffer from signal distortion when applied in complex environment with abundant thiols, which poses challenge for practical applications. Here, we focus on addressing the root cause of the issue and propose to decorate the Au NPs with a thin layer of platinum, thus facilitating the conjugation of DNAzymes through Pt-S bonding, a thiol-resistant cross-linking. The Pt-S bond stabilized DNAzyme nanosensor effectively minimized false positive signals when detecting l-histidine in infant formulas, as compared to the Au-S stabilized counterpart. This innovative strategy holds promise for high-fidelity biosensing, improving the practical applicability of Au NP-based DNAzyme nanosensor.

Lowering Entropic Barriers in Triplex DNA Switches Facilitating Biomedical Applications at Physiological pH.

Triplex DNA switches are attractive allosteric tools for engineering smart nanodevices, but their poor triplex-forming capacity at physiological conditions limited the practical applications. To address this challenge, we proposed a low-entropy barrier design to facilitate triplex formation by introducing a hairpin duplex linker into the triplex motif, and the resulting triplex switch was termed as CTNSds. Compared to the conventional clamp-like triplex switch, CTNSds increased the triplex-forming ratio from 30 % to 91 % at pH 7.4 and stabilized the triple-helix structure in FBS and cell lysate. CTNSds was also less sensitive to free-energy disturbances, such as lengthening linkers or mismatches in the triple-helix stem. The CTNSds design was utilized to reversibly isolate CTCs from whole blood, achieving high capture efficiencies (>86 %) at pH 7.4 and release efficiencies (>80 %) at pH 8.0. Our approach broadens the potential applications of DNA switches-based switchable nanodevices, showing great promise in biosensing and biomedicine.

DNA metabarcoding reveals ecological patterns and driving mechanisms of archaeal, bacterial, and eukaryotic communities in sediments of the Sansha Yongle Blue Hole.

The Sansha Yongle Blue Hole (SYBH) is the world's deepest marine blue hole with unique physicochemical characteristics. However, our knowledge of the biodiversity and community structure in SYBH sediments remains limited, as past studies have mostly focused on microbial communities in the water column. Here, we collected sediment samples from the aerobic zone (3.1 to 38.6 m) and the deep anaerobic zone (150 m, 300 m) of the SYBH and extracted DNA to characterize the archaeal, bacterial, and eukaryotic communities inhabiting these sediments. Our results showed that the archaeal and bacterial communities were dominated by Thaumarchaeota and Proteobacteria, respectively. The dominant taxa of eukaryotes in different sites varied greatly, mainly including Phaeophyceae, Annelida, Diatomea and Arthropoda. All three examined domains showed clear vertical distributions and significant differences in community composition between the aerobic and anaerobic zones. Sulfide played a prominent role in structuring the three domains, followed by salinity, nitrous oxide, pH, temperature and dissolved oxygen, all of which were positively correlated with the turnover component, the main contributor to beta diversity. Neutral community model revealed that stochastic processes contributed to more than half of the community variations across the three domains. Co-occurrence network showed an equal number of positive and negative interactions in the archaeal network, while positive interactions accounted for ~ 80% in the bacterial and eukaryotic networks. Our findings reveal the ecological features of prokaryotes and eukaryotes in SYBH sediments and shed new light on community dynamics and survival strategies in the special environment of marine blue holes.

SI-ViT: Shuffle instance-based Vision Transformer for pancreatic cancer ROSE image classification.

BACKGROUND AND OBJECTIVE: The rapid on-site evaluation (ROSE) technique improves pancreatic cancer diagnosis by enabling immediate analysis of fast-stained cytopathological images. Automating ROSE classification could not only reduce the burden on pathologists but also broaden the application of this increasingly popular technique. However, this approach faces substantial challenges due to complex perturbations in color distribution, brightness, and contrast, which are influenced by various staining environments and devices. Additionally, the pronounced variability in cancerous patterns across samples further complicates classification, underscoring the difficulty in precisely identifying local cells and establishing their global relationships. METHODS: To address these challenges, we propose an instance-aware approach that enhances the Vision Transformer with a novel shuffle instance strategy (SI-ViT). Our approach presents a shuffle step to generate bags of shuffled instances and corresponding bag-level soft-labels, allowing the model to understand relationships and distributions beyond the limited original distributions. Simultaneously, combined with an un-shuffle step, the traditional ViT can model the relationships corresponding to the sample labels. This dual-step approach helps the model to focus on inner-sample and cross-sample instance relationships, making it potent in extracting diverse image patterns and reducing complicated perturbations. RESULTS: Compared to state-of-the-art methods, significant improvements in ROSE classification have been achieved. Aiming for interpretability, equipped with instance shuffling, SI-ViT yields precise attention regions that identifying cancer and normal cells in various scenarios. Additionally, the approach shows excellent potential in pathological image analysis through generalization validation on other datasets. CONCLUSIONS: By proposing instance relationship modeling through shuffling, we introduce a new insight in pathological image analysis. The significant improvements in ROSE classification leads to protential AI-on-site applications in pancreatic cancer diagnosis. The code and results are publicly available at https://github.com/sagizty/MIL-SI.

A Novel Feature Engineering Method Based on Latent Representation Learning for Radiomics: Application in NSCLC Subtype Classification.

Radiomics refers to the high-throughput extraction of quantitative features from medical images, and is widely used to construct machine learning models for the prediction of clinical outcomes, while feature engineering is the most important work in radiomics. However, current feature engineering methods fail to fully and effectively utilize the heterogeneity of features when dealing with different kinds of radiomics features. In this work, latent representation learning is first presented as a novel feature engineering approach to reconstruct a set of latent space features from original shape, intensity and texture features. This proposed method projects features into a subspace called latent space, in which the latent space features are obtained by minimizing a unique hybrid loss function including a clustering-like loss and a reconstruction loss. The former one ensures the separability among each class while the latter one narrows the gap between the original features and latent space features. Experiments were performed on a multi-center non-small cell lung cancer (NSCLC) subtype classification dataset from 8 international open databases. Results showed that compared with four traditional feature engineering methods (baseline, PCA, Lasso and L2,1-norm minimization), latent representation learning could significantly improve the classification performance of various machine learning classifiers on the independent test set (all p<0.001). Further on two additional test sets, latent representation learning also showed a significant improvement in generalization performance. Our research shows that latent representation learning is a more effective feature engineering method, which has the potential to be used as a general technology in a wide range of radiomics researches.

Weighted gene co-expression network analysis reveals key module and hub genes associated with the anthocyanin biosynthesis in maize pericarp.

Anthocyanins are the visual pigments that present most of the colors in plants. Its biosynthesis requires the coordinated expression of structural genes and regulatory genes. Pericarps are the rich sources of anthocyanins in maize seeds. In the experiment, the transcriptomes of transparent and anthocyanins-enriched pericarps at 15, 20, and 25 DAP were obtained. The results output 110.007 million raw reads and 51407 genes' expression matrix. Using data filtration in R language, 2057 genes were eventually identified for weighted gene co-expression network analysis. The results showed that 2057 genes were classified into ten modules. The cyan module containing 183 genes was confirmed to be the key module with the highest correlation value of 0.98 to the anthocyanins trait. Among 183 genes, seven structural genes were mapped the flavonoid biosynthesis pathway, and a transcription factor Lc gene was annotated as an anthocyanin regulatory gene. Cluster heatmap and gene network analysis further demonstrated that Naringenin, 2-oxoglutarate 3-dioxygenase (Zm00001d001960), Dihydroflavonol 4-reductase (Zm00001d044122), Leucoanthocyanidin dioxygenase (Zm00001d014914), anthocyanin regulatory Lc gene (Zm00001d026147), and Chalcone synthase C2 (Zm00001d052673) participated in the anthocyanins biosynthesis. And the transcription factor anthocyanin regulatory Lc gene Zm00001d026147 may act on the genes Chalcone synthase C2 (Zm00001d052673) and Dihydroflavonol 4-reductase (Zm00001d044122). The yeast one-hybrid assays confirmed that the Lc protein could combine with the promoter region of C2 and directly regulate the anthocyanin biosynthesis in the pericarp. These results may provide a new sight to uncover the module and hub genes related to anthocyanins biosynthesis in plants.

Dynamic Transcriptome-Based Weighted Gene Co-expression Network Analysis Reveals Key Modules and Hub Genes Associated With the Structure and Nutrient Formation of Endosperm for Wax Corn.

The endosperm of corn kernel consists of two components, a horny endosperm, and a floury endosperm. In the experiment, a kind of floury endosperm corn was identified. The result of phenotypic trait analysis and determination of amino acid content showed that the floury endosperm filled with the small, loose, and scattered irregular spherical shape starch granules and contained higher content of amino acid. The starch biochemical properties are similar between floury corns and regular flint corn. By using dynamically comparative transcriptome analysis of endosperm at 20, 25, and 30 DAP, a total of 113.42 million raw reads and 50.508 thousand genes were obtained. By using the weighted gene co-expression network analysis, 806 genes and six modules were identified. And the turquoise module with 459 genes was proved to be the key module closely related to the floury endosperm formation. Nine zein genes in turquoise module, including two zein-alpha A20 (Zm00001d019155 and Zm00001d019156), two zein-alpha A30 (Zm00001d048849 and Zm00001d048850), one 50 kDa gamma-zein (Zm00001d020591), one 22 kDa alpha-zein 14 (Zm00001d048817), one zein-alpha 19D1 (Zm00001d030855), one zein-alpha 19B1 (Zm00001d048848), and one FLOURY 2 (Zm00001d048808) were identified closely related the floury endosperm formation. Both zein-alpha 19B1 (Zm00001d048848) and zein-alpha A30 (Zm00001d048850) function as source genes with the highest expression level in floury endosperm. These results may provide the supplementary molecular mechanism of structure and nutrient formation for the floury endosperm of maize.

A label-free cyclic amplification strategy for microRNA detection by coupling graphene oxide-controlled adsorption with superlong poly(thymine)-hosted fluorescent copper nanoparticles.

Herein, based on a terminal deoxynucleotidyl transferase (TdT)-mediated superlong poly-T-templated-copper nanoparticles (poly T-CuNPs) strategy, a simple, universal and label-free fluorescent biosensor for the detection of miRNA was constructed by employing graphene oxide (GO) and DNase I. In this strategy, GO and DNase I were used as a switch and amplifier of the signal generation pathway, respectively, and the fluorescence of poly T-CuNPs was used as the signal output. In the presence of target miRNA, the DNA dissociated from the GO surface by forming a miRNA/DNA duplex and was degraded by DNase I. The short oligos with 3'-OH, the product of DNase I degradation, could be recognized by the TdT and added to a long poly-T tail. Finally, the fluorescence signal was output through the synthesis of poly T-CuNPs. As a proof of concept, let-7a was analyzed. The method showed good sensitivity and selectivity with a linear response in the 50 pM-10,000 pM let-7a concentration range and a 30 pM limit of detection (LOD = 30 pM, R2 = 0.9954, the relative standard deviation were 2.79%-5.30%). It was also successfully applied to the determination of miRNA in spiked human serum samples. It showed good linearity in the range of 500-10000 pM (R2 = 0.9969, the relative standard deviation were 1.61%-3.85%). Moreover, both the adsorption of GO and the degradation of DNase I are DNA sequence-independent; thus, this method can be applied to the detection of any miRNA simply by changing the assisted-DNA sequence.

A Polymeric Nanobeacon for Monitoring the Fluctuation of Hydrogen Polysulfides during Fertilization and Embryonic Development.

Fertilization and early embryonic development as the beginning of a new life are key biological events. Hydrogen polysulfide (H2 Sn ) plays important roles during physiological regulation, such as antioxidation-protection. However, no report has studied in situ H2 Sn fluctuation during early embryonic development because of the low abundance of H2 Sn and inadequate sensitivity of probes. We herein construct a polymeric nanobeacon from a H2 Sn -responsive polymer and fluorophores, which is capable of detecting H2 Sn selectively and of signal amplification. Taking the zebrafish as a model, the polymeric nanobeacon revealed that the H2 Sn level was significantly elevated after fertilization due to the activation of cell multiplication, suppressed partially during embryonic development, and finally kept steady up to zebrafish emergence. This strategy is generally accessible for biomarkers by altering the responsive unit and significant for facilitating biological analysis during life development.

Biological Response of Planktic Foraminifera to Decline in Seawater pH.

Understanding the way in which a decline in ocean pH can affect calcareous organisms could enhance our ability to predict the impacts of the potential decline in seawater pH on marine ecosystems, and could help to reconstruct the paleoceanographic events over a geological time scale. Planktic foraminifera are among the most important biological proxies for these studies; however, the existing research on planktic foraminifera is almost exclusively based on their geochemical indices, without the inclusion of information on their biological development. Through a series of on-board experiments in the western tropical Pacific (134°33'54″ E, 12°32'47″ N), the present study showed that the symbiont-bearing calcifier Trilobatus sacculifer-a planktic foraminifer-responded rapidly to a decline in seawater pH, including losing symbionts, bleaching, etc. Several indices were established to quantify the relationships between these biological parameters and seawater pH, which could be used to reconstruct the paleoceanographic seawater pH. We further postulated that the loss of symbionts in planktic foraminifera acts as an adaptive response to the stress of low pH. Our results indicate that an ongoing decline in seawater pH may hinder the growth and calcification of planktic foraminifera by altering their biological processes. A reduction in carbonate deposition and predation could have profound effects on the carbon cycle and energy flow in the marine food web.

Bidirectional modulation of microRNA with a clamp-like triplex switch for enhanced and programmed gene therapy.

A clamp-like triplex switch (CTS) able to simultaneously downregulate an overexpressed onco-miRNA and replenish the lost tumor-suppressive miRNA in a controllable manner was developed for enhanced gene therapy. Compared to the "unidirectional" regulation approach, the CTS displayed improved anti-tumor efficacy in vitro and was harmless to healthy cells.

The fluorescence imaging and precise suppression of bacterial infections in chronic wounds by porphyrin-based metal-organic framework nanorods.

Nano-antibacterial agents can play a critical role in chronic wound management. However, the design of an intelligent nanosystem that can provide both a visual warning of infection and precise sterilization remains a hurdle. Herein, a rod-like porphyrin-based metal-organic framework theranostic nanosystem (Zn-TCPP nanorods) is fabricated via coordination chelation between tetrakis(4-carboxylphenyl)porphyrin and zinc ions. This system can show significant fluorescence activation in response to the local elevated pH shown by chronic wounds, a main indicator of wound infection. Meanwhile, under the guidance of fluorescence imaging, the highly spatiotemporally precise photodynamic inactivation of microorganisms can be carried out without the destruction of surrounding normal cells and nascent cells. The results demonstrated that the Zn-TCPP nanorods were a highly sensitive and reversible probe for sensing alkaline pH levels. Alterations in the fluorescence of the Zn-TCPP nanorods can accurately indicate the infection status and heterogeneity of infection within the wound bed. Under specific light irradiation, the Zn-TCPP nanorods can exterminate 97% of Staphylococcus aureus via the generation of reactive oxygen species (ROS). Assays of extensive wounds demonstrate that the precise fluorescence-imaging-guided suppression of bacterial infection can significantly reduce the mouse mortality rate and accelerate wound healing. This system provides the opportunity for "precision medicine" relating to chronic wounds and some large-area wounds.

Zn2+ -Coordination-Driven RNA Assembly with Retained Integrity and Biological Functions.

Metal-coordination-directed biomolecule crosslinking in nature has been used for synthesizing various biopolymers, including DNA, peptides, proteins, and polysaccharides. However, the RNA biopolymer has been avoided so far, as due to the poor stability of the RNA molecules, the formation of a biopolymer may alter the biological function of the molecules. Herein, for the first time, we report Zn2+ -driven RNA self-assembly forming spherical nanoparticles while retaining the integrity and biological function of RNA. Various functional RNAs of different compositions, shapes, and lengths from 20 to nearly 1000 nucleotides were used, highlighting the versatility of this approach. The assembled nanospheres possess a superior RNA-loading efficiency, pharmacokinetics, and bioavailability. In-vitro and in-vivo evaluation demonstrated mRNA delivery for expressing GFP proteins, and microRNA delivery to triple-negative breast cancer. This coordination-directed self-assembly behavior amplifies the horizons of RNA coordination chemistry and the application scope of RNA-based therapeutics.

A Glucose-Powered Activatable Nanozyme Breaking pH and H2 O2 Limitations for Treating Diabetic Infections.

The peroxidase-like activity of nanozymes is promising for chemodynamic therapy by catalyzing H2 O2 into . OH. However, for most nanozymes, this activity is optimal just in acidic solutions, while the pH of most physiological systems is beyond 7.0 (even >8.0 in chronic wounds) with inadequate H2 O2 . We herein communicate an activatable nanozyme with targeting capability to simultaneously break the local pH and H2 O2 limitations under physiological conditions. As a proof of concept, aptamer-functionalized nanozymes, glucose oxidase, and hyaluronic acid constitute an activatable nanocapsule "APGH", which can be activated by bacteria-secreted hyaluronidase in infected wounds. Nanozymes bind onto bacteria through aptamer recognition, and glucose oxidation tunes the local pH down and supplements H2 O2 for the in-situ generation of . OH on bacteria surfaces. The activity switching and enhanced antibacterial effect of the nanocapsule were verified in vitro and in diabetic wounds. This strategy for directly regulating local microenvironment is generally accessible for nanozymes, and significant for facilitating biological applications of nanozymes.

In Situ Modulating DNAzyme Activity and Internalization Behavior with Acid-Initiated Reconfigurable DNA Nanodevice for Activatable Theranostic.

DNAzyme-mediated gene silencing was still challenged by off-target toxicity. In this study, we developed a split DNAzyme-based nanodevice (sDz-ND) that leveraged acidic tumor microenvironments to drive in situ assembly, thus modulating internalization behavior and silencing activity of DNAzymes. sDz-ND consisted of two different modules, which functionalized with split DNAzyme fragments, respectively. At psychological pH (∼7.4), the two modules were monodispersed, showing cleavage anergy and quenched fluorescence. At pH 6.3, the separated modules could cross-link with each other to form integrated sDz-ND, resulting activation of theranostic function. Meanwhile, the increased particle size and acquired multivalent effect favored 2.1-fold enhanced binding ability, which further facilitated rapid endocytosis of sDz-ND into target cancer cells, then allowing DNAzyme mediated gene silencing. The strategy provides a promising and general concept for precise tumor imaging and gene therapy.

Thiol-suppressed I2-etching of AuNRs: acetylcholinesterase-mediated colorimetric detection of organophosphorus pesticides.

For the first time it is demonstrated that sulfhydryl compounds can suppress longitudinal etching of gold nanorods via consuming oxidizers, which provides a new signaling mechanism for colorimetric sensing. As a proof of concept, a colorimetric assay is developed for detecting organophosphorus pesticides, which are most widely used in modern agriculture to improve food production but with high toxicity to animals and the ecological environment. Triazophos was selected as a model organophosphorus pesticide. In the absence of triazophos, the active acetylcholinesterase can catalyze the conversion of acetylthiocholine iodide to thiocholine whose thiol group can suppress the I2-induced etching of gold nanorods. When triazophos is present, the activity of AchE is inhibited, and I2-induced etching of gold nanorods results in triazophos concentration-dependent color change from brown to blue, pink, and red. The aspect ratio of gold nanorods reduced with gradually blue-shifted longitudinal absorption. There was a linear detection range from 0 to 117 nM (R2 = 0.9908), the detection limit was 4.69 nM, and a good application potential was demonstrated by the assay of real water samples. This method will not only contribute to public monitoring of organophosphorus pesticides but also has verified a new signaling mechanism which will open up a new path to develop colorimetric detection methods. It has been first found that sulfhydryl compounds can suppress longitudinal etching of gold nanorods (AuNRs) via consuming oxidizers, which provides a new signaling mechanism for colorimetric sensing. As a proof of concept, a colorimetric assay is developed for sensitively detecting organophosphorus pesticides (OPs). It will not only contribute to public monitoring of OPs but also has verified a new signaling mechanism which will open up a new path to develop multicolor colorimetric methods.

Recognition-Driven Remodeling of Dual-Split Aptamer Triggering In Situ Hybridization Chain Reaction for Activatable and Autonomous Identification of Cancer Cells.

Proximity-dependent hybridization chain reaction (HCR) has shown great potential in sensing biomolecules on the cell surface. However, the requirement of two adjacent bioevents occurring simultaneously limits its application. To solve the problem, split aptamers with target binding ability were introduced to combine with split triggers for initiating HCR, thus producing a novel dual-split aptamer probe (DSAP). By employing cancer-related receptors as models, in situ HCR on a cancer cell surface induced by recognition-driven remodeling of the DSAP was demonstrated. The DSAP consisted of two sequences. Each contained two segments; one derived from split aptamers and the other originated in split triggers. In the presence of target cells, split aptamers reassembled on the cell surface under the "induced-fit effect", thus forcing two split triggers close to each other. The remodeled DSAP worked as an intact trigger, which opened the H1 hairpin probe and then hybridized with the H2 hairpin probe, thus initiating HCR to produce an activated fluorescence signal. As a proof of concept, human liver cancer SMMC-7721 cells and their split ZY11 aptamer were used to construct the DSAP. Results indicated that the DSAP realized sensitive analysis of target cells, permitting the actual detection of 20 cells in the buffer. Moreover, the specific identification of target cells in mixed cell samples and the quantitative analysis of target cells in serum were also achieved. The DSAP strategy is facile and universal, which not only would expand the application range of HCR but also might be developed as a multitarget detection technique for bioanalysis.

Enzyme-free amplified detection of miRNA based on target-catalyzed hairpin assembly and DNA-stabilized fluorescent silver nanoclusters.

MicroRNAs (miRNAs) have been shown to be promising biomarkers for disease diagnostics and therapeutics. However, the rapid, low-cost, sensitive, and selective detection of miRNAs remains a challenge because of their characters of small size, vulnerability to degradation, low abundance, and sequence similarity. Herein, we describe an enzyme-free amplification platform, consisting of a catalytic hairpin assembly (CHA) and DNA-templated silver nanoclusters (DNA/AgNCs), for miRNA analysis. In this work, two DNA hairpins (H1 and H2) were first designed for target miR-21-induced CHA, and then the fluorescence of DNA/AgNCs was quenched by BHQ1 to construct an activatable probe (AP). In the presence of target miR-21, hairpin H1 was opened by miR-21 through a hybridization reaction, and hairpin H2 was then opened by H1. During this process, miR-21 was released from H1 and participated in the next round of hybridization, triggering the CHA cycle reaction. The obtained H1-H2 products with sticky ends could react with the AP, forcing BHQ1 away from the DNA/AgNCs and thus causing the fluorescence recovery of the DNA/AgNCs. The assay for miR-21 detection demonstrated an excellent linear response to concentrations varying from 200 pM to 20 nM with the detection limit of 200 pM. The simple and cost-effective strategy holds great potential for application in biomedical research and clinical diagnostics.

Diversity hotspot and unique community structure of foraminifera in the world's deepest marine blue hole - Sansha Yongle Blue Hole.

Marine blue holes are precious geological heritages with high scientific research values. Their physical and chemical characteristics are unique because of the steep-walled structure and isolated water column which create isolated ecosystems in geographically restricted areas. The Sansha Yongle Blue Hole (SYBH) is the world's deepest marine blue hole. Here, we generated the first DNA metabarcoding dataset from SYBH sediment focusing on foraminifera, a group of protists that have colonized various marine environments. We collected sediment samples from SYBH along a depth gradient to characterize the foraminiferal diversity and compared them with the foraminiferal diversity of the costal Jiaozhou Bay (JZB) and the abyssal Northwest Pacific Ocean (NWP). We amplified the SSU rDNA of foraminifera and sequenced them with high-throughput sequencing. The results showed that the foraminiferal assemblages in SYBH were vertically structured in response to the abiotic gradients and diversity was higher than in JZB and NWP. This study illustrates the capacity of foraminifera to colonize hostile environments and shows that blue holes are natural laboratories to explore physiological innovation associated with anoxia.