In vivo Labeling and Intravital Imaging of Bacterial Infection using a Near-infrared Fluorescent D-Amino Acid Probe.

Bacterial infections still pose a severe threat to public health, necessitating novel tools for real-time analysis of microbial behaviors in living organisms. While genetically engineered strains with fluorescent or luminescent reporters are commonly used in tracking bacteria, their in vivo uses are often limited. Here, we report a near-infrared fluorescent D-amino acid (FDAA) probe, Cy7ADA, for in situ labeling and intravital imaging of bacterial infections in mice. Cy7ADA probe effectively labels various bacteria in vitro and pathogenic Staphylococcus aureus in mice after intraperitoneal injection. Because of Cy7's high tissue penetration and the quick excretion of free probes via urine, real-time visualization of the pathogens in a liver abscess model via intravital confocal microscopy is achieved. The biodistributions, including their intracellular localization within Kupffer cells, are revealed. Monitoring bacterial responses to antibiotics also demonstrates Cy7ADA's capability to reflect the bacterial load dynamics within the host. Furthermore, Cy7ADA facilitates three-dimensional pathogen imaging in tissuecleared liver samples, showcasing its potential for studying the biogeography of microbes in different organs. Integrating nearinfrared FDAA probes with intravital microscopy holds promise for wide applications in studying bacterial infections in vivo.

Main/side chain asymmetric molecular design enhances charge transfer of two-dimensional conjugated polymer/g-C3N4 heterojunctions for high-efficiency photocatalytic sterilization and degradation.

Heterojunctions based on conjugated polymers (PHJs) are of promise as photocatalysts. Here, we fabricate the two-dimensional benzodithiophene (BDT) and thieno[2,3-f]benzofuran (TBF) based conjugated polymers/g-C3N4 PHJs creatively using the symmetry-breaking strategy. PD1 and PD3 with the asymmetric backbone TBF have better crystallinity. Moreover, PD3 utilizing fluorinated benzotriazole as the electron acceptor unit possesses more compact π - π stacking and higher charge mobility. The conjugated polymer PD5 with asymmetric side chains in the donor unit BDT guarantees more efficient charge transfer in the corresponding PD5/g-C3N4 PHJ while maintaining comparable light utilization rate. Consequently, PD5/g-C3N4 shows the champion performance with photocatalytic sterilization rates reaching 99.1% and 97.3% for S. aureus and E. coli. Notably, the reaction rate constant for Rhodamine B degradation of PD5/g-C3N4 is 8 times that of g-C3N4, a record high among conjugated polymers/g-C3N4. This study aims to reveal the structure - property correlation of asymmetric conjugated polymers/g-C3N4 for potential photocatalysis applications.

Visualizing germination of microbiota endospores in the mammalian gut.

Transmission of bacterial endospores between the environment and people and the following germination in vivo play critical roles in both the deadly infections of some bacterial pathogens and the stabilization of the commensal microbiotas in humans. Our knowledge about the germination process of different bacteria in the mammalian gut, however, is still very limited due to the lack of suitable tools to visually monitor this process. We proposed a two-step labeling strategy that can image and quantify the endospores' germination in the recipient's intestines. Endospores collected from donor's gut microbiota were first labeled with fluorescein isothiocyanate and transplanted to mice via gavage. The recipient mice were then administered with Cyanine5-tagged D-amino acid to label all the viable bacteria, including the germinated endospores, in their intestines in situ. The germinated donor endospores could be distinguished by presenting two types of fluorescent signals simultaneously. The integrative use of cell-sorting, 16S rDNA sequencing, and fluorescence in situ hybridization (FISH) staining of the two-colored bacteria unveiled the taxonomic information of the donor endospores that germinated in the recipient's gut. Using this strategy, we investigated effects of different germinants and pre-treatment interventions on their germination, and found that germination of different commensal bacterial genera was distinctly affected by various types of germinants. This two-color labeling strategy shows its potential as a versatile tool for visually monitoring endospore germination in the hosts and screening for new interventions to improve endospore-based therapeutics.

Metal free benzothiadiazole-diketopyrrolopyrrole-based conjugated polymer/g-C3N4 photocatalyst for enhanced sterilization and degradation in visible to near-infrared region.

In recent years, photocatalytic technology has attracted wide attention in environmental treatment, exploring non-toxic and metal-free photocatalysts is imminent to meet sustainable development. However, semiconductors with wide spectral response are rarely studied and applied in the field of photocatalysis. Herein, a new narrow band-gap polymer PFBDT-DPP (P3) with wide absorption from 500 to 860 nm was synthesized and further constructed heterostructure with g-C3N4 for photocatalytic sterilization and degradation of organic pollutant Rhodamine B (RhB). The optimal antibacterial rate for Escherichia coli reached 99.8% after 190 min of light irradiation and for Staphylococcus aureus reached 96.8% after 120 min of irradiation, and the highest degradation efficiency of RhB by P3/g-C3N4 was 98.9% within 60 min light irradiation, while g-C3N4 displayed an unsatisfactory sterilization and photodegradation performance. This is mainly attributed to the broadened light absorption range and enhanced carrier separation efficiency of P3/g-C3N4. This work could provide a new strategy to fabricate metal-free photocatalysts with high utilization of sunlight and excellent photocatalytic performance.

Biodistributions of l,d-Transpeptidases in Gut Microbiota Revealed by In Vivo Labeling with Peptidoglycan Analogs.

By catalyzing a 3-3 cross-link in peptidoglycan, l,d-transpeptidases (Ldts) can cause resistance to ß-lactams in some pathogens in vitro. However, the prevalence of Ldt and Ldt-mediated responses to different ß-lactams in vivo have never been explored. Here, we apply an in vivo metabolic labeling strategy to study their biodistributions and Ldt-induced bacterial responses to ß-lactams in the mouse gut microbiota. A tetrapeptide-based fluorescent probe that functions as a substrate for Ldts in Gram-positive bacteria efficiently labels ∼18% of total gut bacteria after gavage, suggesting Ldts' high prevalence in gut microbiota. The cellular distributions of 3-3 cross-links on three gut bacterial species were then identified with the aid of fluorescence in situ hybridization to identify the bacterial taxa. After oral administration of two ß-lactams, ampicillin and meropenem, only the latter efficiently inhibits the tetrapeptide labeling, suggesting that Ldts may be able to cause resistance to some ß-lactams in the mammalian gut.

Imaging Commensal Microbiota and Pathogenic Bacteria in the Gut.

As a newly discovered organ, gut microbiota has been extensively studied in the last two decades, with their highly diverse and fundamental roles in the physiology of many organs and systems of the host being gradually revealed. However, most of the current research heavily relies on DNA sequencing-based methodologies. To truly understand the complex physiological and pathological functions demonstrated by commensal and pathogenic gut bacteria, we need more powerful methods and tools, among which imaging strategies suitable for approaching this ecosystem in different settings are one of the most desirable. Although the phrase gut "dark matter" is often used in referring to the unculturability of many gut bacteria, it is also applicable to describing the formidable difficulties in visualizing these microbes in the intestines. To develop suitable and versatile chemical and biological tools for imaging bacteria in the gut, great efforts have been devoted in the past several years.In this Account, we highlight the recent progress made by our group and other laboratories in the development of visualization strategies for commensal microbiota and pathogenic bacteria in the gut. First, we summarize our efforts toward the development of derivatized antibiotic staining probes that directly bind to specific bacterial surface structures for selective labeling of different groups of gut bacteria. Next, metabolic labeling-based imaging strategies, using unnatural amino acids, unnatural sugars, and stable isotopes, for imaging gut bacteria on various scales and in different settings are discussed in detail. We then introduce nucleic acid staining-based bacterial imaging, using either general nucleic acid-binding reagents or selective-labeling techniques (e.g., fluorescence in situ hybridization) to meet the diverse needs in gut microbiota research. This classical imaging strategy has witnessed a renaissance owing to a series of new technical advancements. Furthermore, despite the notorious difficulties of performing genetic manipulations in many commensal gut bacteria, great effort has been made recently in engineering gut bacteria with reporters like fluorescent proteins and acoustic response proteins.Our perspectives on the current limitations of the chemical tools and strategies and the future directions for improvement are also presented. We hope that this Account can offer valuable references to spark new ideas and invite new efforts to help decipher the complex biological and chemical interactions between commensal microbiota and pathogenic bacteria and the hosts.

Three-Dimensional Quantitative Imaging of Native Microbiota Distribution in the Gut.

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.

Revealing the in vivo growth and division patterns of mouse gut bacteria.

Current techniques for studying gut microbiota are unable to answer some important microbiology questions, like how different bacteria grow and divide in the gut. We propose a method that integrates the use of sequential d-amino acid-based in vivo metabolic labeling with fluorescence in situ hybridization (FISH), for characterizing the growth and division patterns of gut bacteria. After sequentially administering two d-amino acid-based probes containing different fluorophores to mice by gavage, the resulting dual-labeled peptidoglycans provide temporal information on cell wall synthesis of gut bacteria. Following taxonomic identification with FISH probes, the growth and division patterns of the corresponding bacterial taxa, including species that cannot be cultured separately in vitro, are revealed. Our method offers a facile yet powerful tool for investigating the in vivo growth dynamics of the bacterial gut microbiota, which will advance our understanding of bacterial cytology and facilitate elucidation of the basic microbiology of this gut "dark matter."

Highly Sensitive Minimal Residual Disease Detection by Biomimetic Multivalent Aptamer Nanoclimber Functionalized Microfluidic Chip.

Minimal residual disease (MRD) offers a highly independent prognostic factor for leukemia patients. However, challenges confronting conventional MRD assays are high invasiveness, as well as limited detection sensitivity and clinical applicability. Inspired by the self-adaptive skeleton and multiple suckers or tendrils of climbing plants, a biomimetic Multivalent Aptamer Nanoclimber (MANC)-functionalized microfluidic chip (MANC-Chip) is reported for minimally invasive, highly sensitive and clinically applicable MRD detection in the peripheral blood of T-cell acute lymphoblastic leukemia patients. The MANCs are synthesized by a simple co-polymerization reaction. Due to their flexible structure and cooperative multivalent effect, MANCs dramatically enhance the binding affinity of aptamers targeting leukemia cells. A deterministic lateral displacement-patterned microfluidic chip is designed to further increase the collision probability between MANCs and leukemia cells. Benefiting from the synergistic effect of multivalent binding and enhanced collision, a high capture efficiency of 92.2% for leukemia cells is achieved. Moreover, the captured leukemia cells can be released with high efficiency of 88.9% and high viability of 93.8% via nuclease treatment prior to downstream analysis. Overall, the excellent features of MANC-Chip make it very useful for precise detection of MRD and better understanding of leukemia.

Quantification of Bacterial Metabolic Activities in the Gut by d-Amino Acid-Based In†Vivo Labeling.

Herein, we propose a metabolic d-amino acid-based labeling and in situ hybridization-facilitated (MeDabLISH) strategy for the quantitative analysis of the indigenous metabolic status of gut bacteria. The fluorescent d-amino acid (FDAA)-based labeling intensities of bacteria were found to highly correlate with their temporal and steady-state metabolic status. Then, after taxonomic identification of bacterial genera in the in vivo FDAA-labeled mouse gut microbiota, by corresponding fluorescence in situ hybridization (FISH) probes, the metabolic activities of different gut bacterial genera are quantified by flow cytometry, using FISH signals to differentiate genera and FDAA signals to indicate their basal metabolic levels. It was found that Gram-negative genera in the mouse microbiota have stronger metabolic activities during the daytime, and Gram-positive genera have higher activities at the night. Our strategy will be instrumental in deepening our understanding of the highly complex microbiota.

Metabolic Labeling of Peptidoglycan with NIR-II Dye Enables In Vivo Imaging of Gut Microbiota.

Deepening our understanding of mammalian gut microbiota has been greatly hampered by the lack of a facile, real-time, and in vivo bacterial imaging method. To address this unmet need in microbial visualization, we herein report the development of a second near-infrared (NIR-II)-based method for in vivo imaging of gut bacteria. Using d-propargylglycine in gavage and then click reaction with an azide-containing NIR-II dye, gut microbiota of a donor mouse was strongly labeled with NIR-II fluorescence on their peptidoglycan. The bacteria could be readily visualized in recipient mouse gut with high spatial resolution and deep tissue penetration under NIR irradiation. The NIR-II-based metabolic labeling strategy reported herein, provides, to the best of our knowledge, the first protocol for facile in vivo visualization of gut microbiota within deep tissues, and offers an instrumental tool for deciphering the complex biology of these gut "dark matters".

Bacterial Extracellular Electron Transfer Occurs in Mammalian Gut.

As a well-studied biochemical reduction process in environmental microbiology, extracellular electron transfer (EET) was recently discovered in bacteria closely related to human health, and orthologues of a flavin-based EET gene were found in the genomes of many species across Firmicutes, a major phylum in mammalian gut microbiota. However, EET has not yet been confirmed to occur in mammalian gut, the presence of which may have broad physiological influences. Toward this end, here we first confirmed the occurrence of EET in mouse gut microbiotas cultured in vitro. Cyclic voltammetry analysis was then performed by directly inserting electrodes into the mouse cecum under anaerobic conditions, and a characteristic catalytic wave was observed in the gut of conventional but not germ-free mouse, proving the existence of in vivo bacterial EET. We also detected similar catalytic waves in the cecal microbiotas of rat and guinea pig in vivo, suggesting EET's high prevalence in mammalian intestines. Our finding on the bacterial electron production in mammalian guts offers a new bioelectrochemical scope for deciphering the complex microbiology of gut bacteria and its effects on host physiology.

Assessing the viability of transplanted gut microbiota by sequential tagging with D-amino acid-based metabolic probes.

Currently, there are more than 200 fecal microbiota transplantation (FMT) clinical trials worldwide. However, our knowledge of this microbial therapy is still limited. Here we develop a strategy using sequential tagging with D-amino acid-based metabolic probes (STAMP) for assessing the viabilities of transplanted microbiotas. A fluorescent D-amino acid (FDAA) is first administered to donor mice to metabolically label the gut microbiotas in vivo. The labeled microbiotas are transplanted to recipient mice, which receive a second FDAA with a different color. The surviving transplants should incorporate both FDAAs and can be readily distinguished by presenting two colors simultaneously. Isolation of surviving bacteria and 16S rDNA sequencing identify several enriched genera, suggesting the importance of specific bacteria in FMT. In addition, using STAMP, we evaluate the effects on transplant survival of pre-treating recipients using different antibiotics. We propose STAMP as a versatile tool for deciphering the complex biology of FMT, and potentially improving its treatment efficacy.

Superelastic Few-Layer Carbon Foam Made from Natural Cotton for All-Solid-State Electrochemical Capacitors.

Flexible/stretchable devices for energy storage are essential for future wearable and flexible electronics. Electrochemical capacitors (ECs) are an important technology for supplement batteries in the energy storage and harvesting field, but they are limited by relatively low energy density. Herein, we report a superelastic foam consisting of few-layer carbon nanowalls made from natural cotton as a good scaffold to growth conductive polymer polyaniline for stretchable, lightweight, and flexible all-solid-state ECs. As-prepared superelastic bulk tubular carbon foam (surface area ∼950 m(2)/g) can withstand >90% repeated compression cycling and support >45,000 times its own weight but no damage. The flexible device has a high specific capacitance of 510 F g(-1), a specific energy of 25.5 Wh kg(-1) and a power density of 28.5 kW kg(-1) in weight of the total electrode materials and withstands 5,000 charging/discharging cycles.

Role of stromal cells-mediated Notch-1 in the invasion of T-ALL cells.

Extra-medullary infiltration is still one of the main causes of recurrence and treatment failure of T-cell acute lymphoblastic leukemia (T-ALL). Intensive studies revealed that Notch pathway plays an important role in the invasion of tumor cells. Notch pathway can be triggered by binding of Notch receptors on T-ALL cells to their ligands on bone marrow stromal cells (BMSCs), which contributes to the development of T-ALL. However, the effect and molecular mechanisms of BMSCs in invasion of T-ALL cells remain unclear. To explore the effect of Notch-1 on the invasiveness of T-ALL cells, we co-cultured T-ALL cells with BMSCs (from healthy donors)/BMSCs(⁎) (from newly diagnosed T-ALL patients). The results demonstrated that BMSCs/BMSCs(⁎) promoted invasion of T-ALL cells through activating Notch-1 signaling. In particular, T-ALL cells showed a higher invasive potential in the presence of BMSCs(⁎) than BMSCs. Knockdown of Notch-1 prevented the positive effect of stromal cells-mediated invasion. Our study also showed that BMSCs/BMSCs(⁎)-induced Notch-1 activation increased the expression of matrix metalloprote inase-2 (MMP-2) and matrix metalloprote inase-9 (MMP-9), which increased invasiveness. These results provided theoretical and laboratory basis for the prevention and treatment of extra-medullary infiltration of T-ALL cells.

SMG1 acts as a novel potential tumor suppressor with epigenetic inactivation in acute myeloid leukemia.

Suppressor with morphogenetic effect on genitalia family member (SMG1) belongs to a family of phosphoinositide 3-kinase-related kinases and is the main kinase involved in nonsense-mediated mRNA decay. Recently, SMG1 was suggested as a novel potential tumor suppressor gene, particularly in hypoxic tumors. To investigate the function of SMG1 in acute myeloid leukemia (AML), we performed methylation-specific polymerase chain reaction and found that SMG1 was hypermethylated in the promoter region. SMG1 hypermethylation was found in 66% (33/50) of AML samples compared with none (0/14) of the normal controls. SMG1 mRNA was down-regulated in AML patients with hypermethylation status whereas it was readily expressed in patients without methylation. Moreover, treatment of AML cells with demethylating agent 5-aza-2'-deoxycytidine (decitabine) inhibited AML cell growth and induced apoptosis by reversing SMG1 methylation status and restoring SMG1 expression. On the other hand, knockdown of SMG1 by RNA interference inhibited apoptosis. We also found that mTOR expression level was negatively correlated to SMG1 expression in AML patients which indicated that SMG1 and mTOR maybe act antagonistically to regulate AML cell growth. In conclusion, our results indicate that SMG1 acts as a potential tumor suppressor with epigenetic regulation in AML.

Dysregulation of hydrogen sulphide metabolism impairs oviductal transport of embryos.

Embryo retention in the fallopian tube is thought to lead to ectopic pregnancy, which is a significant cause of morbidity. Hydrogen sulphide (H2S) is a gaseotransmitter produced mainly by cystathionine-γ-lyase and cystathionine-ß-synthase. Here we show that cystathionine-γ-lyase and cystathionine -ß-synthase are ubiquitously distributed in human fallopian tube epithelium and that H2S signalling relaxes the spontaneous contraction of the human oviduct. Furthermore, an aberration in H2S signalling, either silenced or enhanced activity induced by pharmacologic or genetic methods, causes embryo retention and developmental delay in the mouse oviduct, which is partly reversed by administration of either GYY4137, a slow-releasing H2S donor, or NaHS. Our findings reveal a new regulatory mechanism for oviductal embryo transport.

miR-181b increases drug sensitivity in acute myeloid leukemia via targeting HMGB1 and Mcl-1.

Multidrug resistance (MDR) remains the major cause of disease relapse and poor prognosis in adults with acute myeloid leukemia (AML). Emerging evidence shows that drug resistance not only exists against conventional chemotherapeutic drugs, but also limits the efficacy of new biological agents. Therefore, it is important to elucidate the mechanisms through which AML patients develop drug resistance. MicroRNAs have been shown to play an important role in regulating the chemotherapy resistance in AML. A detailed understanding of the mechanisms of microRNA that are clinically relevant in AML may enhance our ability to predict and overcome drug resistance. Here, we demonstrated, for the first time, that miR-181b was decreased significantly in human multidrug-resistant leukemia cells and relapsed/refractory AML patient samples. Overexpression of miR-181b increased the sensitivity of leukemia cells to cytotoxic chemotherapeutic agents and promoted drug-induced apoptosis. Moreover, miR-181b inhibited HMGB1 and Mcl-1 expression by direct binding to their 3'-untranslated regions. In addition, HMGB1 was expressed at high levels in relapsed/refractory AML patients and suppression of HMGB1 via RNA interference sensitized multidrug-resistant leukemia cells to chemotherapy and induced apoptosis. In conclusion, these results provide a strong rationale for the development of miR-181b-based therapeutic strategies for the enhancement of efficacy in AML treatment.

Gene expression profiling of the DNMT3A R882 mutation in acute leukemia.

DNA methyltransferase 3A (DNMT3A) is one of two human de novo DNA methyltransferases essential for the regulation of gene expression. DNMT3A mutations and deletions have been previously observed in acute myeloid leukemia (AML), myelodysplastic sydromes and myeloproliferative neoplasms. However, the involvement of DNMT3A in acute lymphoblastic leukemia (ALL) has rarely been reported. In the present study, PCR and direct sequencing was performed to analyze mutations of DNMT3A amino acid residue 882 in 99 acute leukemia patients, including 57 AML patients, 41 ALL patients and a single biphenotypic acute leukemia (BAL) patient. DNMT3A expression was detected in mono-nuclear cells of the bone marrow in these patients and in normal individuals using real-time quantitative polymerase chain reaction, and 17.5% (10/57) of AML patients were found to exhibit DNMT3A mutations. Four missense mutations were observed in the DNMT3A-mutated AML patients, including R882 mutations and a novel single nucleotide polymorphism resulting in the M880V amino acid substitution. However, the ALL and BAL patients were not found to exhibit DNMT3A mutations. The DNMT3A expression levels in the AML patients were significantly higher compared with those of the ALL patients or normal controls. The reduced expression levels of DNMT3A were associated with a significantly lower complete remission rate in the AML patients. However, in the ALL patients, no statistical significance was identified. The results of the present study indicate that DNMT3A may play varying roles in the regulation of DNA methylation in AML and ALL.

Simultaneous and Direct Determination of Vancomycin and Cephalexin in Human Plasma by Using HPLC-DAD Coupled with Second-Order Calibration Algorithms.

A simple, rapid, and sensitive method for the simultaneous determination of vancomycin and cephalexin in human plasma was developed by using HPLC-DAD with second-order calibration algorithms. Instead of a completely chromatographic separation, mathematical separation was performed by using two trilinear decomposition algorithms, that is, PARAFAC-alternative least squares (PARAFAC-ALSs) and self-weight-alternative-trilinear-decomposition- (SWATLD-) coupled high-performance liquid chromatography with DAD detection. The average recoveries attained from PARAFAC-ALS and SWATLD with the factor number of 4 (N = 4) were 101 ± 5% and 102 ± 4% for vancomycin, and 96 ± 3% and 97 ± 3% for cephalexininde in real human samples, respectively. The statistical comparison between PARAFAC-ALS and SWATLD is demonstrated to be similar. The results indicated that the combination of HPLC-DAD detection with second-order calibration algorithms is a powerful tool to quantify the analytes of interest from overlapped chromatographic profiles for complex analysis of drugs in plasma.