Episymbiotic Saccharibacteria TM7x modulates the susceptibility of its host bacteria to phage infection and promotes their coexistence.

Bacteriophages (phages) play critical roles in modulating microbial ecology. Within the human microbiome, the factors influencing the long-term coexistence of phages and bacteria remain poorly investigated. Saccharibacteria (formerly TM7) are ubiquitous members of the human oral microbiome. These ultrasmall bacteria form episymbiotic relationships with their host bacteria and impact their physiology. Here, we showed that during surface-associated growth, a human oral Saccharibacteria isolate (named TM7x) protects its host bacterium, a Schaalia odontolytica strain (named XH001) against lytic phage LC001 predation. RNA-Sequencing analysis identified in XH001 a gene cluster with predicted functions involved in the biogenesis of cell wall polysaccharides (CWP), whose expression is significantly down-regulated when forming a symbiosis with TM7x. Through genetic work, we experimentally demonstrated the impact of the expression of this CWP gene cluster on bacterial-phage interaction by affecting phage binding. In vitro coevolution experiments further showed that the heterogeneous populations of TM7x-associated and TM7x-free XH001, which display differential susceptibility to LC001 predation, promote bacteria and phage coexistence. Our study highlights the tripartite interaction between the bacterium, episymbiont, and phage. More importantly, we present a mechanism, i.e., episymbiont-mediated modulation of gene expression in host bacteria, which impacts their susceptibility to phage predation and contributes to the formation of "source-sink" dynamics between phage and bacteria in biofilm, promoting their long-term coexistence within the human microbiome.

The Protective Roles of Self-Compassion and Parental Autonomy Support Against Depressive Symptoms in Peer-Victimized Chinese Adolescent Girls: A Longitudinal Study.

Peer victimization (PV) is a common and serious problem in school contexts, which hinders adolescents' emotional development and social adaptation. The present study aimed to test the longitudinal relationship between PV and the increase of depressive symptoms (DSs) among Chinese mid-late adolescents using a two-wave longitudinal design and examine the buffering effects of self-compassion (SC) and parental autonomy support (PAS) on this relationship. A relatively large sample of Chinese high school students (N = 722, 52.1% boys; age at Time 2 = 16.23 years old, SD = 0.79) were surveyed annually at two time points. The results showed that PV at Time 1 positively predicted DSs at Time 2 after controlling for the DSs at Time 1. In addition, SC and PAS moderated the longitudinal relationship between PV and the development of DSs, while such moderating effects only existed in girls but not in boys. Specifically, the positive relationship between PV and DSs was non-significant among girls with higher levels of SC or PAS. Our findings highlighted that SC and PAS might be important protective factors buffering against DSs for victimized girls.

Episymbiotic Saccharibacteria induce intracellular lipid droplet production in their host bacteria.

Saccharibacteria (formerly TM7) are a group of widespread and genetically diverse ultrasmall bacteria with highly reduced genomes that belong to Candidate Phyla Radiation, a large monophyletic lineage with poorly understood biology. Nanosynbacter lyticus type strain TM7x is the first Saccharibacteria member isolated from the human oral microbiome. With restrained metabolic capacities, TM7x lives on the surface of, and forms an obligate episymbiotic relationship with its bacterial host, Schaalia odontolytica strain XH001. The symbiosis allows TM7x to propagate but presents a burden to host bacteria by inducing stress response. Here, we employed super-resolution fluorescence imaging to investigate the physical association between TM7x and XH001. We showed that the binding with TM7x led to a substantial alteration in the membrane fluidity of XH001. We also revealed the formation of intracellular lipid droplets in XH001 when forming episymbiosis with TM7x, a feature that has not been reported in oral bacteria. The TM7x-induced lipid droplets accumulation in XH001 was confirmed by label-free Raman spectroscopy, which also unveiled additional phenotypical features when XH001 cells are physically associated with TM7x. Further exploration through culturing XH001 under various stress conditions showed that lipid droplets accumulation was a general response to stress. A survival assay demonstrated that the presence of lipid droplets plays a protective role in XH001, enhancing its survival under adverse conditions. In conclusion, our study sheds new light on the intricate interaction between Saccharibacteria and their host bacteria, highlighting the potential benefit conferred by TM7x to its host and further emphasizing the context-dependent nature of symbiotic relationships.

Associations of CKIP-1 and LOX-1 polymorphisms with the risk of type 2 diabetes mellitus with hypertension among Chinese adults.

AIMS: Type 2 diabetes mellitus (T2DM) and hypertension are common high-incidence diseases, closely related, and have common pathogenic basis such as oxidative stress. Casein kinase 2 interacting protein-1 (CKIP-1) and low-density lipoprotein receptor (LOX-1) are considered to be important factors affect the level of oxidative stress in the body. The main purpose of this study was to explore the relationship between CKIP-1 (rs6693817 A > T, rs2306235 C > G) and LOX-1 (rs1050283 G > A, rs11053646 C > G) polymorphisms and the risk of hypertension and diabetes, and try to find new candidate genes for diabetes and diabetes with hypertension etiology in Chinese population. METHODS: 574 T2DM patients and 597 controls frequently matched by age and sex were selected for genotyping of CKIP-1 (rs6693817 A > T, rs2306235 C > G) and LOX-1 gene (rs1050283 G > A, rs11053646 C > G). Logistic regression was used to analyze the correlation between different genotypes and the risk of T2DM and T2DM with hypertension, and the results were expressed as odds ratio (OR) and 95% confidence interval (95% CI). RESULTS: We found that the risk of T2DM in the AA + AT genotype of rs6693817 was higher than that in the TT genotype in Chinese population (OR = 1.318, 95%CI: 1.011-1.717, P = 0.041), and the difference was still significant after adjustment (OR = 1.370, 95%CI: 1.043-1.799, Padjusted = 0.024), the difference of heterozygotes (AT vs TT: OR = 1.374, 95%CI: 1.026-1.840, Padjusted = 0.033) was statistically significant. But after Bonferroni correction, the significance of the above sites disappeared. And rs6693817 was associated with the risk of T2DM combined with hypertension before and after adjustment in dominant model (OR = 1.424, 95% CI: 1.038-1.954, P = 0.028; OR = 1.460, 95% CI: 1.057-2.015, Padjusted = 0.021, respectively) and in heterozygote model (OR = 1.499, 95% CI: 1.069-2.102, P = 0.019; OR = 1.562, 95% CI: 1.106-2.207, Padjusted = 0.011, respectively). However, only the statistical significance of the heterozygous model remained after Bonferroni correction. rs2306235, rs1050283 and rs11053646 were not significantly correlated with T2DM and T2DM combined with hypertension risk (P > 0.05). CONCLUSIONS: The results suggest that CKIP-1 rs6693817 is related to the susceptibility of Chinese people to T2DM with hypertension, providing a new genetic target for the treatment of diabetes with hypertension with in the future.

Chromophore-Targeting Precision Antimicrobial Phototherapy.

Phototherapy, encompassing the utilization of both natural and artificial light, has emerged as a dependable and non-invasive strategy for addressing a diverse range of illnesses, diseases, and infections. This therapeutic approach, primarily known for its efficacy in treating skin infections, such as herpes and acne lesions, involves the synergistic use of specific light wavelengths and photosensitizers, like methylene blue. Photodynamic therapy, as it is termed, relies on the generation of antimicrobial reactive oxygen species (ROS) through the interaction between light and externally applied photosensitizers. Recent research, however, has highlighted the intrinsic antimicrobial properties of light itself, marking a paradigm shift in focus from exogenous agents to the inherent photosensitivity of molecules found naturally within pathogens. Chemical analyses have identified specific organic molecular structures and systems, including protoporphyrins and conjugated C=C bonds, as pivotal components in molecular photosensitivity. Given the prevalence of these systems in organic life forms, there is an urgent need to investigate the potential impact of phototherapy on individual molecules expressed within pathogens and discern their contributions to the antimicrobial effects of light. This review delves into the recently unveiled key molecular targets of phototherapy, offering insights into their potential downstream implications and therapeutic applications. By shedding light on these fundamental molecular mechanisms, we aim to advance our understanding of phototherapy's broader therapeutic potential and contribute to the development of innovative treatments for a wide array of microbial infections and diseases.

Episymbiotic bacterium induces intracellular lipid droplet production in its host bacteria.

Saccharibacteria (formerly TM7) Nanosynbacter lyticus type strain TM7x exhibits a remarkably compact genome and an extraordinarily small cell size. This obligate epibiotic parasite forms a symbiotic relationship with its bacterial host, Schaalia odontolytica, strain XH001 (formerly Actinomyces odontolyticus strain XH001). Due to its limited genome size, TM7x possesses restrained metabolic capacities, predominantly living on the surface of its bacterial host to sustain this symbiotic lifestyle. To comprehend this intriguing, yet understudied interspecies interaction, a thorough understanding of the physical interaction between TM7x and XH001 is imperative. In this study, we employed super-resolution fluorescence imaging to investigate the physical association between TM7x and XH001. We found that the binding with TM7x led to a substantial alteration in the membrane fluidity of the host bacterium XH001. Unexpectedly, we revealed the formation of intracellular lipid droplets in XH001 when forming episymbiosis with TM7x, a feature not commonly observed in oral bacteria cells. The TM7x-induced LD accumulation in XH001 was further confirmed by label-free non-invasive Raman spectroscopy, which also unveiled additional phenotypical features when XH001 cells are physically associated with TM7x. Further exploration through culturing host bacterium XH001 alone under various stress conditions showed that LD accumulation was a general response to stress. Intriguingly, a survival assay demonstrated that the presence of LDs likely plays a protective role in XH001, enhancing its overall survival under adverse conditions. In conclusion, our study sheds new light on the intricate interaction between Saccharibacteria and its host bacterium, highlighting the potential benefit conferred by TM7x to its host, and further emphasizing the context-dependent nature of symbiotic relationships.

Development of a mammalian cell-based ZZ display system for IgG quantification.

BACKGROUND: Biological laboratories and companies involved in antibody development need convenient and versatile methods to detect highly active antibodies. METHODS: To develop a mammalian cell-based ZZ display system for antibody quantification, the eukaryotic ZZ-displayed plasmid was constructed and transfected into CHO cells. After screening by flow cytometric sorting, the stable ZZ display cells were incubated with reference IgG and samples with unknown IgG content for 40 min at 4℃, the relative fluorescence intensity of cells was analyzed and the concentration of IgG was calculated. RESULTS: By investigating the effects of different display-associated genetic elements, a eukaryotic ZZ-displaying plasmid with the highest display efficiency were constructed. After transfection and screening, almost 100% of the cells were able to display the ZZ peptide (designated CHO-ZZ cells). These stable CHO-ZZ cells were able to capture a variety of IgG, including human, rabbit, donkey and even mouse and goat. CHO-ZZ cells could be used to quantify human IgG in the range of approximately 12.5-1000 ng/mL, and to identify high-yielding engineered monoclonal cell lines. CONCLUSIONS: We have established a highly efficient CHO-ZZ display system in this study, which enables the quantification of IgG from various species under physiological conditions. This system offers the advantage of eliminating the need for antibody purification and will contribute to antibody development.

Fingerprint Stimulated Raman Scattering Imaging Unveils Ergosteryl Ester as a Metabolic Signature of Azole-Resistant Candida albicans.

Candida albicans (C. albicans), a major fungal pathogen, causes life-threatening infections in immunocompromised individuals. Fluconazole (FLC) is recommended as first-line therapy for treatment of invasive fungal infections. However, the widespread use of FLC has resulted in increased antifungal resistance among different strains of Candida, especially C. albicans, which is a leading source of hospital-acquired infections. Here, by hyperspectral stimulated Raman scattering imaging of single fungal cells in the fingerprint window and pixel-wise spectral unmixing, we report aberrant ergosteryl ester accumulation in azole-resistant C. albicans compared to azole-susceptible species. This accumulation was a consequence of de novo lipogenesis. Lipid profiling by mass spectroscopy identified ergosterol oleate to be the major species stored in azole-resistant C. albicans. Blocking ergosterol esterification by oleate and suppressing sterol synthesis by FLC synergistically suppressed the viability of C. albicans in vitro and limited the growth of biofilm on mouse skin in vivo. Our findings highlight a metabolic marker and a new therapeutic strategy for targeting azole-resistant C. albicans by interrupting the esterified ergosterol biosynthetic pathway.

Targeting Fusobacterium nucleatum through chemical modifications of host-derived transfer RNA fragments.

Host mucosal barriers possess an arsenal of defense molecules to maintain host-microbe homeostasis such as antimicrobial peptides and immunoglobulins. In addition to these well-established defense molecules, we recently reported small RNAs (sRNAs)-mediated interactions between human oral keratinocytes and Fusobacterium nucleatum (Fn), an oral pathobiont with increasing implications in extra-oral diseases. Specifically, upon Fn infection, oral keratinocytes released Fn-targeting tRNA-derived sRNAs (tsRNAs), an emerging class of noncoding sRNAs with gene regulatory functions. To explore potential antimicrobial activities of tsRNAs, we chemically modify the nucleotides of the Fn-targeting tsRNAs and demonstrate that the resultant tsRNA derivatives, termed MOD-tsRNAs, exhibit growth inhibitory effect against various Fn type strains and clinical tumor isolates without any delivery vehicle in the nanomolar concentration range. In contrast, the same MOD-tsRNAs do not inhibit other representative oral bacteria. Further mechanistic studies uncover the ribosome-targeting functions of MOD-tsRNAs in inhibiting Fn. Taken together, our work provides an engineering approach to targeting pathobionts through co-opting host-derived extracellular tsRNAs.

Antagonistic interaction between two key endodontic pathogens Enterococcus faecalis and Fusobacterium nucleatum.

Background: Endodontic infections are known to be caused by pathogenic bacteria. Numerous previous studies found that both Fusobacterium nucleatum and Enterococcus faecalis are associated with endodontic infections, with Fusobacterium nucleatum more abundant in primary infection while Enterococcus faecalis more abundant in secondary infection. Little is known about the potential interactions between different endodontic pathogens. Objective: This study aims to investigate the potential interaction between F. nucleatum and E. faecalis via phenotypical and genetic approaches. Methods: Physical and physiological interactions of F. nucleatum and E. faecalis under both planktonic and biofilm conditions were measured with co-aggregation and competition assays. The mechanisms behind these interactions were revealed with genetic screening and biochemical measurements. Results: E. faecalis was found to physically bind to F. nucleatum under both in vitro planktonic and biofilm conditions, and this interaction requires F. nucleatum fap2, a galactose-inhibitable adhesin-encoding gene. Under our experimental conditions, E. faecalis exhibits a strong killing ability against F. nucleatum by generating an acidic micro-environment and producing hydrogen peroxide (H2O2). Finally, the binding and killing capacities of E. faecalis were found to be necessary to invade and dominate a pre-established in vitro F. nucleatum biofilm. Conclusions: This study reveals multifaceted mechanisms underlying the physical binding and antagonistic interaction between F. nucleatum and E. faecalis, which could play a potential role in the shift of microbial composition in primary and secondary endodontic infections.

Analysis of influencing factors for prognosis of patients with ventricular septal perforation: A single-center retrospective study.

Background: Ventricular septal rupture (VSR) is a type of cardiac rupture, usually complicated by acute myocardial infarction (AMI), with a high mortality rate and often poor prognosis. The aim of our study was to investigate the factors influencing the long-term prognosis of patients with VSR from different aspects, comparing the evaluation performance of the Gensini score, Sequential Organ Failure Assessment (SOFA) score and European Heart Surgery Risk Assessment System II (EuroSCORE II) score systems. Methods: This study retrospectively enrolled 188 patients with VSR between Dec 9, 2011 and Nov 21, 2021at the First Affiliated Hospital of Zhengzhou University. All patients were followed up until Jan 27, 2022 for clinical data, angiographic characteristics, echocardiogram outcomes, intraoperative, postoperative characteristics and major adverse cardiac events (MACEs) (30-day mortality, cardiac readmission). Cox proportional hazard regression analysis was used to explore the predictors of long-term mortality. Results: The median age of 188 VSR patients was 66.2 ± 9.1 years and 97 (51.6%) were males, and there were 103 (54.8%) patients in the medication group, 34 (18.1%) patients in the percutaneous transcatheter closure (TCC) group, and 51 (27.1%) patients in the surgical repair group. The average follow-up time was 857.4 days. The long-term mortality of the medically managed group, the percutaneous TCC group, and the surgical repair group was 94.2, 32.4, and 35.3%, respectively. Whether combined with cardiogenic shock (OR 0.023, 95% CI 0.001-0.054, P = 0.019), NT-pro BNP level (OR 0.027, 95% CI 0.002-0.34, P = 0.005), EuroSCORE II (OR 0.530, 95% CI 0.305-0.918, P = 0.024) and therapy group (OR 3.518, 95% CI 1.079-11.463, P = 0.037) were independently associated with long-term mortality in patients with VSR, and this seems to be independent of the therapy group. The mortality rate of surgical repair after 2 weeks of VSR was much lower than within 2 weeks (P = 0.025). The cut-off point of EuroSCORE II was determined to be 14, and there were statistically significant differences between the EuroSCORE II < 14 group and EuroSCORE II≥14 group (HR = 0.2596, 95%CI: 0.1800-0.3744, Logrank P < 0.001). Conclusion: Patients with AMI combined with VSR have a poor prognosis if not treated surgically, surgical repair after 2 weeks of VSR is a better time. In addition, EuroSCORE II can be used as a scoring system to assess the prognosis of patients with VSR.

Exploitation of a Bacterium-Encoded Lytic Transglycosylase by a Human Oral Lytic Phage To Facilitate Infection.

Bacteriophages (phages) are an integral part of the human oral microbiome. Their roles in modulating bacterial physiology and shaping microbial communities have been discussed but remain understudied due to limited isolation and characterization of oral phage. Here, we report the isolation of LC001, a lytic phage targeting human oral Schaalia odontolytica (formerly known as Actinomyces odontolyticus) strain XH001. We showed that LC001 attached to and infected surface-grown, but not planktonic, XH001 cells, and it displayed remarkable host specificity at the strain level. Whole-genome sequencing of spontaneous LC001-resistant, surface-grown XH001 mutants revealed that the majority of the mutants carry nonsense or frameshift mutations in XH001 gene APY09_05145 (renamed ltg-1), which encodes a putative lytic transglycosylase (LT). The mutants are defective in LC001 binding, as revealed by direct visualization of the significantly reduced attachment of phage particles to the XH001 spontaneous mutants compared that to the wild type. Meanwhile, targeted deletion of ltg-1 produced a mutant that is defective in LC001 binding and resistant to LC001 infection even as surface-grown cells, while complementation of ltg-1 in the mutant background restored the LC001-sensitive phenotype. Intriguingly, similar expression levels of ltg-1 were observed in surface-grown and planktonic XH001, which displayed LC001-binding and nonbinding phenotypes, respectively. Furthermore, the overexpression of ltg-1 failed to confer an LC001-binding and -sensitive phenotype to planktonic XH001. Thus, our data suggested that rather than directly serving as a phage receptor, ltg-1-encoded LT may increase the accessibility of phage receptor, possibly via its enzymatic activity, by cleaving the peptidoglycan structure for better receptor exposure during peptidoglycan remodeling, a function that can be exploited by LC001 to facilitate infection. IMPORTANCE The evidence for the presence of a diverse and abundant phage population in the host-associated oral microbiome came largely from metagenomic analysis or the observation of virus-like particles within saliva/plaque samples, while the isolation of oral phage and investigation of their interaction with bacterial hosts are limited. Here, we report the isolation of LC001, the first lytic phage targeting oral Schaalia odontolytica. Our study suggested that LC001 may exploit the host bacterium-encoded lytic transglycosylase function to gain access to the receptor, thus facilitating its infection.

Photoinactivation of catalase sensitizes a wide range of bacteria to ROS-producing agents and immune cells.

Bacteria have evolved to cope with the detrimental effects of ROS using their essential molecular components. Catalase, a heme-containing tetramer protein expressed universally in most aerobic bacteria, plays an indispensable role in scavenging excess hydrogen peroxide (H2O2). Here, through use of wild-type and catalase-deficient mutants, we identified catalase as an endogenous therapeutic target of 400-420 nm blue light. Catalase residing inside bacteria could be effectively inactivated by blue light, subsequently rendering the pathogens extremely vulnerable to H2O2 and H2O2-producing agents. As a result, photoinactivation of catalase and H2O2 synergistically eliminated a wide range of catalase-positive planktonic bacteria and P. aeruginosa inside biofilms. In addition, photoinactivation of catalase was shown to facilitate macrophage defense against intracellular pathogens. The antimicrobial efficacy of catalase photoinactivation was validated using a Pseudomonas aeruginosa-induced mouse abrasion model. Taken together, our findings offer a catalase-targeting phototherapy approach against multidrug-resistant bacterial infections.

Photoinactivation of Catalase Sensitizes Candida albicans and Candida auris to ROS-Producing Agents and Immune Cells.

Microbes have developed their own specific strategies to cope with reactive oxygen species (ROS). Catalase, a heme-containing tetramer expressed in a broad range of aerobic fungi, shows remarkable efficiency in degrading hydrogen peroxide (H2 O2 ) for fungal survival and host invasion. Here, it is demonstrated that catalase inactivation by blue light renders fungal cells highly susceptible to ROS attack. To confirm catalase as a major molecular target of blue light, wild type Candida albicans are systematically compared with a catalase-deficient mutant strain regarding their susceptibility to ROS through 410 nm treatment. Upon testing a wide range of fungal species, it is found that intracellular catalase can be effectively and universally inactivated by 410 nm blue light. It is also found that photoinactivation of catalase in combination with ROS-generating agents is highly effective in total eradication of various fungal species, including multiple Candida auris strains, the causative agent of the global fungal epidemic. In addition, photoinactivation of catalase is shown to facilitate macrophage killing of intracellular Candida albicans. The antifungal efficacy of catalase photoinactivation is further validated using a C. albicans-induced mouse model of skin abrasion. Taken together, the findings offer a novel catalase-photoinactivation approach to address multidrug-resistant Candida infections.

Acquisition of the arginine deiminase system benefits epiparasitic Saccharibacteria and their host bacteria in a mammalian niche environment.

Saccharibacteria are a group of widespread and genetically diverse ultrasmall bacteria with highly reduced genomes that belong to the Candidate Phyla Radiation. Comparative genomic analyses suggest convergent evolution of key functions enabling the adaptation of environmental Saccharibacteria to mammalian microbiomes. Currently, our understanding of this environment-to-mammal niche transition within Saccharibacteria and their obligate episymbiotic association with host bacteria is limited. Here, we identified a complete arginine deiminase system (ADS), found in further genome streamlined mammal-associated Saccharibacteria but missing in their environmental counterparts, suggesting acquisition during environment-to-mammal niche transition. Using TM7x, the first cultured Saccharibacteria strain from the human oral microbiome and its host bacterium Actinomyces odontolyticus, we experimentally tested the function and impact of the ADS. We demonstrated that by catabolizing arginine and generating adenosine triphosphate, the ADS allows metabolically restrained TM7x to maintain higher viability and infectivity when disassociated from the host bacterium. Furthermore, the ADS protects TM7x and its host bacterium from acid stress, a condition frequently encountered within the human oral cavity due to bacterial metabolism of dietary carbohydrates. Intriguingly, with a restricted host range, TM7x forms obligate associations with Actinomyces spp. lacking the ADS but not those carrying the ADS, suggesting the acquired ADS may also contribute to partner selection for cooperative episymbiosis within a mammalian microbiome. These data present experimental characterization of a mutualistic interaction between TM7x and their host bacteria, and illustrate the benefits of acquiring a novel pathway in the transition of Saccharibacteria to mammalian microbiomes.

Single cell imaging reveals cisplatin regulating interactions between transcription (co)factors and DNA.

Cisplatin is an extremely successful anticancer drug, and is commonly thought to target DNA. However, the way in which cisplatin-induced DNA lesions regulate interactions between transcription factors/cofactors and genomic DNA remains unclear. Herein, we developed a dual-modal microscopy imaging strategy to investigate, in situ, the formation of ternary binding complexes of the transcription cofactor HMGB1 and transcription factor Smad3 with cisplatin crosslinked DNA in single cells. We utilized confocal microscopy imaging to map EYFP-fused HMGB1 and fluorescent dye-stained DNA in single cells, followed by the visualization of cisplatin using high spatial resolution (200-350 nm) time of flight secondary ion mass spectrometry (ToF-SIMS) imaging of the same cells. The superposition of the fluorescence and the mass spectrometry (MS) signals indicate the formation of HMGB1-Pt-DNA ternary complexes in the cells. More significantly, for the first time, similar integrated imaging revealed that the cisplatin lesions at Smad-binding elements, for example GGC(GC)/(CG) and AGAC, disrupted the interactions of Smad3 with DNA, which was evidenced by the remarkable reduction in the expression of Smad-specific luciferase reporters subjected to cisplatin treatment. This finding suggests that Smad3 and its related signalling pathway are most likely involved in the intracellular response to cisplatin induced DNA damage.

Ultrasensitive Vibrational Imaging of Retinoids by Visible Preresonance Stimulated Raman Scattering Microscopy.

High-sensitivity chemical imaging offers a window to decipher the molecular orchestra inside a living system. Based on vibrational fingerprint signatures, coherent Raman scattering microscopy provides a label-free approach to map biomolecules and drug molecules inside a cell. Yet, by near-infrared (NIR) pulse excitation, the sensitivity is limited to millimolar concentration for endogenous biomolecules. Here, the imaging sensitivity of stimulated Raman scattering (SRS) is significantly boosted for retinoid molecules to 34 micromolar via electronic preresonance in the visible wavelength regime. Retinoids play critical roles in development, immunity, stem cell differentiation, and lipid metabolism. By visible preresonance SRS (VP-SRS) imaging, retinoid distribution in single embryonic neurons and mouse brain tissues is mapped, retinoid storage in chemoresistant pancreatic and ovarian cancers is revealed, and retinoids stored in protein network and lipid droplets of Caenorahbditis elegans are identified. These results demonstrate VP-SRS microscopy as an ultrasensitive label-free chemical imaging tool and collectively open new opportunities of understanding the function of retinoids in biological systems.

Polarization-sensitive stimulated Raman scattering imaging resolves amphotericin B orientation in Candida membrane.

Ergosterol-targeting amphotericin B (AmB) is the first line of defense for life-threatening fungal infections. Two models have been proposed to illustrate AmB assembly in the cell membrane; one is the classical ion channel model in which AmB vertically forms transmembrane tunnel and the other is a recently proposed sterol sponge model where AmB is laterally adsorbed onto the membrane surface. To address this controversy, we use polarization-sensitive stimulated Raman scattering from fingerprint C═C stretching vibration to visualize AmB, ergosterol, and lipid in single fungal cells. Intracellular lipid droplet accumulation in response to AmB treatment is found. AmB is located in membrane and intracellular droplets. In the 16 strains studied, AmB residing inside cell membrane was highly ordered, and its orientation is primarily parallel to phospholipid acyl chains, supporting the ion channel model. Label-free imaging of AmB and chemical contents offers an analytical platform for developing low-toxicity, resistance-refractory antifungal agents.

Granadaene Photobleaching Reduces the Virulence and Increases Antimicrobial Susceptibility of Streptococcus agalactiae.

Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is increasingly recognized as a major cause of soft tissue and invasive diseases in the elderly and diabetic populations. Antibiotics like penicillin are used with great frequency to treat these infections, although antimicrobial resistance is increasing among GBS strains and underlines a need for alternative methods not reliant on traditional antibiotics. GBS granadaene pigment is related to the hemolysin/cytolysin of GBS, which is critical for the pathogenesis of GBS diseases. Here, we show that photobleaching granadaene dampens the hemolytic activity of GBS. Furthermore, photobleaching of this antioxidant was found to increase GBS susceptibility to killing by reactive oxygen species like hydrogen peroxide. Treatment with light was also shown to affect GBS membrane permeability and contribute to increased susceptibility to the cell membrane-targeting antibiotic daptomycin. Overall, our study demonstrates dual effects of photobleaching on the virulence and antimicrobial susceptibility of GBS and suggests a novel approach for the treatment of GBS infection.

Rapid Determination of Antimicrobial Susceptibility by Stimulated Raman Scattering Imaging of D2O Metabolic Incorporation in a Single Bacterium.

Rapid antimicrobial susceptibility testing (AST) is urgently needed for treating infections with appropriate antibiotics and slowing down the emergence of antibiotic-resistant bacteria. Here, a phenotypic platform that rapidly produces AST results by femtosecond stimulated Raman scattering imaging of deuterium oxide (D2O) metabolism is reported. Metabolic incorporation of D2O into biomass in a single bacterium and the metabolic response to antibiotics are probed in as short as 10 min after culture in 70% D2O medium, the fastest among current technologies. Single-cell metabolism inactivation concentration (SC-MIC) is obtained in less than 2.5 h from colony to results. The SC-MIC results of 37 sets of bacterial isolate samples, which include 8 major bacterial species and 14 different antibiotics often encountered in clinic, are validated by standard minimal inhibitory concentration blindly measured via broth microdilution. Toward clinical translation, stimulated Raman scattering imaging of D2O metabolic incorporation and SC-MIC determination after 1 h antibiotic treatment and 30 min mixture of D2O and antibiotics incubation of bacteria in urine or whole blood is demonstrated.