Imaging surface structure and premelting of ice Ih with atomic resolution.

Ice surfaces are closely relevant to many physical and chemical properties, such as melting, freezing, friction, gas uptake and atmospheric reaction1-8. Despite extensive experimental and theoretical investigations9-17, the exact atomic structures of ice interfaces remain elusive owing to the vulnerable hydrogen-bonding network and the complicated premelting process. Here we realize atomic-resolution imaging of the basal (0001) surface structure of hexagonal water ice (ice Ih) by using qPlus-based cryogenic atomic force microscopy with a carbon monoxide-functionalized tip. We find that the crystalline ice-Ih surface consists of mixed Ih- and cubic (Ic)-stacking nanodomains, forming 19 × 19 periodic superstructures. Density functional theory reveals that this reconstructed surface is stabilized over the ideal ice surface mainly by minimizing the electrostatic repulsion between dangling OH bonds. Moreover, we observe that the ice surface gradually becomes disordered with increasing temperature (above 120 Kelvin), indicating the onset of the premelting process. The surface premelting occurs from the defective boundaries between the Ih and Ic domains and can be promoted by the formation of a planar local structure. These results put an end to the longstanding debate on ice surface structures and shed light on the molecular origin of ice premelting, which may lead to a paradigm shift in the understanding of ice physics and chemistry.

Heat shock potentiates aminoglycosides against gram-negative bacteria by enhancing antibiotic uptake, protein aggregation, and ROS.

The potentiation of antibiotics is a promising strategy for combatting antibiotic-resistant/tolerant bacteria. Herein, we report that a 5-min sublethal heat shock enhances the bactericidal actions of aminoglycoside antibiotics by six orders of magnitude against both exponential- and stationary-phase Escherichia coli. This combined treatment also effectively kills various E. coli persisters, E. coli clinical isolates, and numerous gram-negative but not gram-positive bacteria and enables aminoglycosides at 5% of minimum inhibitory concentrations to eradicate multidrug-resistant pathogens Acinetobacter baumannii and Klebsiella pneumoniae. Mechanistically, the potentiation is achieved comprehensively by heat shock-enhanced proton motive force that thus promotes the bacterial uptake of aminoglycosides, as well as by increasing irreversible protein aggregation and reactive oxygen species that further augment the downstream lethality of aminoglycosides. Consistently, protonophores, chemical chaperones, antioxidants, and anaerobic culturing abolish heat shock-enhanced aminoglycoside lethality. We also demonstrate as a proof of concept that infrared irradiation- or photothermal nanosphere-induced thermal treatments potentiate aminoglycoside killing of Pseudomonas aeruginosa in a mouse acute skin wound model. Our study advances the understanding of the mechanism of actions of aminoglycosides and demonstrates a high potential for thermal ablation in curing bacterial infections when combined with aminoglycosides.

Advances in miniature CRISPR-Cas proteins and their applications in gene editing.

The CRISPR-Cas system consists of Cas proteins and single-stranded RNAs that recruit Cas proteins and specifically target the nucleic acid. Some Cas proteins can accurately cleave the target nucleic acid under the guidance of the single-stranded RNAs. Due to its exceptionally high specificity, the CRISPR-Cas system is now widely used in various fields such as gene editing, transcription regulation, and molecular diagnosis. However, the huge size of the most frequently utilized Cas proteins (Cas9, Cas12a, and Cas13, which contain 950-1,400 amino acids) can limit their applicability, especially in eukaryotic gene editing, where larger Cas proteins are difficult to deliver into the target cells. Recently discovered miniature CRISPR-Cas proteins, consisting of only 400 to 800 amino acids, offer the possibility of overcoming this limitation. This article systematically reviews the latest research progress of several miniature CRISPR-Cas proteins (Cas12f, Cas12j, Cas12k, and Cas12m) and their practical applications in the field of gene editing.

The effect of surface hydrophobicity and hydrophilicity on ion-ion interactions at water-solid interfaces.

Condensation and arrangement of ions at water-solid interfaces are of great importance in the formation of electrical double layers (EDL) and the transport of ions under a confined geometry. So far, the microscopic understanding of interfacial ion configurations is still far from complete, especially when the local ion concentration is high and ion-ion interactions become prominent. In this study, we directly visualized alkali metal cations within the hydrogen-bonding network of water on graphite and Cu(111)-supported graphene surfaces, using qPlus-based noncontact atomic force microscopy (NC-AFM). We found that the codeposition of the alkali cations and water molecules on the hydrophobic graphite surface leads to the formation of an ion-doped bilayer hexagonal ice (BHI) structure, where the ions are repelled from each other and scattered in a disordered distribution. In contrast, the hydrated alkali cations aggregate in one dimension on the more hydrophilic graphene/Cu(111) surface, forming a nematic state with a long-range order. Such a nematic state arises from the delicate interplay between water-ion and water-water interactions under surface confinement. These results reveal the high sensitivity of ion-ion interactions and ionic ordering to the surface hydrophobicity and hydrophilicity.

Mechanisms involved in the antinociceptive and anti-inflammatory effects of xanthotoxin.

Xanthotoxin (XAT) is a natural furanocoumarin clinically used in the treatment of skin diseases such as vitiligo and psoriasis. Recent studies have also investigated its effects on anti-inflammatory, anti-cognitive dysfunction, and anti-amnesia as a guideline for clinic application. However, little is known about its effects on pain relief. Here, we tested the analgesic effects of XAT in serious acute pain and chronic pain models. For acute pain, we used hot-, capsaicin- and formalin-induced paw licking. Nociceptive threshold was measured by mechanical stimuli with von Frey filaments. For chronic pain, we injected complete Freund's adjuvant (CFA) into the mice's plantar surface of the hind paw to induce inflammatory pain. Heat and mechanical hyperalgesia were evaluated by radiant heat and von Frey filament tests, respectively. To investigate the mechanisms underlying the analgesic effect of XAT, we used calcium imaging and western blot to assess transient receptor potential vanilloid 1 (TRPV1) activity and expression in isolated L4-L6 dorsal root ganglion (DRG) neurons. Haematoxylin and eosin (HE) staining, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay (ELISA) were used to examine immune cell recruitment and proinflammatory factor release from skin tissue from paw injection sites. Our results demonstrated that XAT not only reduced acute pain behaviors generated by hot, capsaicin, and formalin but also attenuated CFA-induced heat and mechanical hyperalgesia. The analgesic activity of XAT may be achieved by controlling peripheral inflammation, lowering immune cell infiltration at the site of inflammatory tissue, reducing inflammatory factor production, and therefore inhibiting TRPV1 channel sensitization and expression.

Toxicity Characterization of Environment-Related Pollutants Using a Biospectroscopy-Bioreporter-Coupling Approach: Potential for Real-World Toxicity Determination and Source Apportionment of Multiple Pollutants.

Exposure to environmental pollutants occurs ubiquitously and poses many risks to human health and the ecosystem. Although many analytical methods have been developed to assess such jeopardies, the circumstances applying these means are restricted to linking the toxicities to compositions in the pollutant mixtures. The present study proposes a novel analytical approach, namely, biospectroscopy-bioreporter-coupling (BBC), to quantify and apportion the toxicities of metal ions and organic pollutants. Using a toxicity bioreporter ADPWH_recA and Raman spectroscopy, both bioluminescent signals and spectral alterations had similar dosage- and time-response behavior to the toxic compounds, validating the possibility of coupling these two methods from practical aspects. Raman spectral alterations successfully distinguished the biomarkers for different toxicity mechanisms of individual pollutants, such as ring breathing mode of DNA/RNA bases (1373 cm-1) by Cr, reactive oxygen species-induced peaks of proteins (1243 cm-1), collagen (813 cm-1), and lipids (1255 cm-1) by most metal ions, and indicative fingerprints of organic toxins. The support vector machine model had a satisfactory performance in distinguishing and apportioning toxicities of individual toxins from all input data, achieving a sensitivity of 88.54% and a specificity of 97.80%. This work set a preliminary database for Raman spectral alterations of whole-cell bioreporter response to multiple pollutants. It proved the state-of-the-art concept that the BBC approach is feasible to rapidly quantify and precisely apportion toxicities of numerous pollutant mixtures.

Ultrasensitive SERS Analysis of Liquid and Gaseous Putrescine and Cadaverine by a 3D-Rosettelike Nanostructure-Decorated Flexible Porous Substrate.

Putrescine and cadaverine are toxic biogenic amines in spoiled food, which poses a serious threat to food security. In this work, we reported a highly sensitive three-dimensional (3D)-rosettelike surface-enhanced Raman spectroscopy (SERS) substrate functionalized with a p-mercaptobenzoic acid (p-MBA) monolayer to detect liquid and gaseous putrescine and cadaverine in pork samples. The SERS substrate was made by a combination of the merit of the 3D morphology of ZnO nanorod arrays on a flexible porous poly(vinylidene fluoride) (PVDF) membrane and the in situ chemical growth of Au nanoparticle seeds on Au film-coated ZnO nanorods, which produced a 3D-rosettelike BigAuNP/Au/ZnO/P heterostructure with abundant SERS-active hot spots that significantly enhanced the localized surface plasmonic resonance (LSPR) effect and charge-transfer (CT) effect of Raman enhancement. This SERS substrate showed high sensitivity, reproducibility, stability, and uniformity. With the p-MBA molecular monolayer as the sensing interface, our SERS substrate realized the highly sensitive and quantitative detection of liquid putrescine and cadaverine within 10 min, with a limit of detection (LOD) of 3.2 × 10-16 and 1.6 × 10-13 M, respectively. Additionally, the sensor showed efficient SERS responses to gaseous amine molecules at low concentrations (putrescine: 1.26 × 10-9 M, cadaverine: 2.5 × 10-9 M). Further, the sensor was successfully applied to determine the total content of putrescine and cadaverine. Moreover, the practicability of this SERS sensor was verified by the measurement of liquid and gaseous amines in pork samples, and it showed great potential applications for sensitive detection of food spoilage.

Development of a Fast Raman-Assisted Antibiotic Susceptibility Test (FRAST) for the Antibiotic Resistance Analysis of Clinical Urine and Blood Samples.

Human health is at great risk due to the spreading of antimicrobial resistance (AMR). The lengthy procedure of conventional antimicrobial susceptibility testing (AST) usually requires a few days. We developed a fast Raman-assisted antibiotic susceptibility test (FRAST), which detects single bacterial metabolic activity in the presence of antibiotics, using Raman single-cell spectroscopy. It was found that single-cell Raman spectra (SCRS) would show a clear and distinguishable Raman band at the "silent zone" (2000-2300 cm-1), due to the active incorporation of deuterium from heavy water (D2O) by antibiotic-resistant bacteria. This pilot study has compared the FRAST and the conventional AST for six clinical standard quality controls (four Gram-negative and two Gram-positive bacteria strains) in response to 38 antibiotics. In total, 3200 treatments have been carried out and approximately 64 000 SCRS have been acquired for FRAST analysis. The result showed an overall agreement of 88.0% between the FRAST and the conventional AST assay. The gram-staining classification based on the linear discriminant analysis (LDA) model of SCRS was developed, seamlessly coupling with the FRAST to further reduce the turnaround time. We applied the FRAST to real clinical analysis for nine urinary infectious samples and three sepsis samples. The results were consistent with MALDI-TOF identification and the conventional AST. Under the optimal conditions, the "sample to report" of the FRAST could be reduced to 3 h for urine samples and 21 h for sepsis samples. The FRAST provides fast and reliable susceptibility tests, which could speed up microbiological analysis for clinical practice and facilitate antibiotic stewardship.

Proteorhodopsin Overproduction Enhances the Long-Term Viability of Escherichia coli.

Genes encoding the photoreactive protein proteorhodopsin (PR) have been found in a wide range of marine bacterial species, reflecting the significant contribution that PR makes to energy flux and carbon cycling in ocean ecosystems. PR can also confer advantages to enhance the ability of marine bacteria to survive periods of starvation. Here, we investigate the effect of heterologously produced PR on the viability of Escherichia coli Quantitative mass spectrometry shows that E. coli, exogenously supplied with the retinal cofactor, assembles as many as 187,000 holo-PR molecules per cell, accounting for approximately 47% of the membrane area; even cells with no retinal synthesize ∼148,000 apo-PR molecules per cell. We show that populations of E. coli cells containing PR exhibit significantly extended viability over many weeks, and we use single-cell Raman spectroscopy (SCRS) to detect holo-PR in 9-month-old cells. SCRS shows that such cells, even incubated in the dark and therefore with inactive PR, maintain cellular levels of DNA and RNA and avoid deterioration of the cytoplasmic membrane, a likely basis for extended viability. The substantial proportion of the E. coli membrane required to accommodate high levels of PR likely fosters extensive intermolecular contacts, suggested to physically stabilize the cell membrane and impart a long-term benefit manifested as extended viability in the dark. We propose that marine bacteria could benefit similarly from a high PR content, with a stabilized cell membrane extending survival when those bacteria experience periods of severe nutrient or light limitation in the oceans.IMPORTANCE Proteorhodopsin (PR) is part of a diverse, abundant, and widespread superfamily of photoreactive proteins, the microbial rhodopsins. PR, a light-driven proton pump, enhances the ability of the marine bacterium Vibrio strain AND4 to survive and recover from periods of starvation, and heterologously produced PR extends the viability of nutrient-limited Shewanella oneidensis We show that heterologously produced PR enhances the viability of E. coli cultures over long periods of several weeks and use single-cell Raman spectroscopy (SCRS) to detect PR in 9-month-old cells. We identify a densely packed and consequently stabilized cell membrane as the likely basis for extended viability. Similar considerations are suggested to apply to marine bacteria, for which high PR levels represent a significant investment in scarce metabolic resources. PR-stabilized cell membranes in marine bacteria are proposed to keep a population viable during extended periods of light or nutrient limitation, until conditions improve.

Selenium enhanced phytoremediation of diesel contaminated soil by Alternanthera philoxeroides.

Using a 60-day pot culture experiment, we investigated the effect of selenium on phytoremediation of soil containing high-level diesel by Alternanthera philoxeroides (alligator weed). Diesel (20 g kg-1) decreased the growth of A. philoxeroides and induced oxidative stress, as indicated by tissue levels of hydrogen peroxide (H2O2) and malondialdehyde (MDA). Adding Se (0.5 or 1.5 mg kg-1) to diesel-treated soil alleviated oxidative stress and improved biomass production, and the low dose was as effective as the high dose. After 60 days, the reduction in rhizospheric soil diesel was 20.1 ±â€¯0.55% without Se and 35.2 ±â€¯3.6% with Se, showing a significant increase in efficiency. Again, the low Se dose was as effective as the high dose. These findings advance the field phytoremediation by demonstrating that Se, at 0.5 mg kg-1, enhances removal and increases plant tolerance to petroleum hydrocarbons.

Raman-activated cell sorting and metagenomic sequencing revealing carbon-fixing bacteria in the ocean.

It is of great significance to understand CO2 fixation in the oceans. Using single cell Raman spectra (SCRS) as biochemical profiles, Raman activated cell ejection (RACE) was able to link phenotypes and genotypes of cells. Here, we show that mini-metagenomic sequences from RACE can be used as a reference to reconstruct nearly complete genomes of key functional bacteria by binning shotgun metagenomic sequencing data. By applying this approach to 13 C bicarbonate spiked seawater from euphotic zone of the Yellow Sea of China, the dominant bacteria Synechococcus spp. and Pelagibacter spp. were revealed and both of them contain carotenoid and were able to incorporate 13 C into the cells at the same time. Genetic analysis of the reconstructed genomes suggests that both Synechococcus spp. and Pelagibacter spp. contained all genes necessary for carotenoid synthesis, light energy harvesting and CO2 fixation. Interestingly, the reconstructed genome indicates that Pelagibacter spp. harbored intact sets of genes for ß-carotene (precursor of retional), proteorhodopsin synthesis and anaplerotic CO2 fixation. This novel approach shines light on the role of marine 'microbial dark matter' in global carbon cycling, by linking yet-to-be-cultured Synechococcus spp. and Pelagibacter spp. to carbon fixation and flow activities in situ.

Effect of Laser Irradiation on Cell Function and Its Implications in Raman Spectroscopy.

Lasers are instrumental in advanced bioimaging and Raman spectroscopy. However, they are also well known for their destructive effects on living organisms, leading to concerns about the adverse effects of laser technologies. To implement Raman spectroscopy for cell analysis and manipulation, such as Raman-activated cell sorting, it is crucial to identify nondestructive conditions for living cells. Here, we evaluated quantitatively the effect of 532-nm laser irradiation on bacterial cell fate and growth at the single-cell level. Using a purpose-built microfluidic platform, we were able to quantify the growth characteristics, i.e., specific growth rates and lag times of individual cells, as well as the survival rate of a population in conjunction with Raman spectroscopy. Representative Gram-negative and Gram-positive species show similar trends in response to a laser irradiation dose. Laser irradiation could compromise the physiological function of cells, and the degree of destruction is both dose and strain dependent, ranging from reduced cell growth to a complete loss of cell metabolic activity and finally to physical disintegration. Gram-positive bacterial cells are more susceptible than Gram-negative bacterial strains to irradiation-induced damage. By directly correlating Raman acquisition with single-cell growth characteristics, we provide evidence of nondestructive characteristics of Raman spectroscopy on individual bacterial cells. However, while strong Raman signals can be obtained without causing cell death, the variety of responses from different strains and from individual cells justifies careful evaluation of Raman acquisition conditions if cell viability is critical.IMPORTANCE In Raman spectroscopy, the use of powerful monochromatic light in laser-based systems facilitates the detection of inherently weak signals. This allows environmentally and clinically relevant microorganisms to be measured at the single-cell level. The significance of being able to perform Raman measurement is that, unlike label-based fluorescence techniques, it provides a "fingerprint" that is specific to the identity and state of any (unlabeled) sample. Thus, it has emerged as a powerful method for studying living cells under physiological and environmental conditions. However, the laser's high power also has the potential to kill bacteria, which leads to concerns. The research presented here is a quantitative evaluation that provides a generic platform and methodology to evaluate the effects of laser irradiation on individual bacterial cells. Furthermore, it illustrates this by determining the conditions required to nondestructively measure the spectra of representative bacteria from several different groups.

Dynamic SAP102 expression in the hippocampal subregions of rats and APP/PS1 mice of various ages.

The hippocampus is a structurally and functionally complex brain area that plays important and diverse roles in higher brain functions, such as learning and memory, and mounting evidence indicates that different hippocampal subregions play distinctive roles. The hippocampus is also one of the first regions in the brain to suffer damage in Alzheimer's disease (AD). Synaptic dysfunction in the hippocampus, rather than neuronal loss per se, is paralleled by behavioural and functional deficits in AD. The membrane-associated guanylate kinase (MAGUK) family of proteins, including SAP102, PSD-95, PSD-93 and SAP97, have long been recognized as essential components of the postsynaptic density (PSD) at excitatory synapses. Hippocampal spines are the predominant synaptic transmission sites of excitatory glutamatergic synapses. During postnatal brain development, individual MAGUK members show distinct expression patterns. Although SAP102 has been confirmed as the dominant scaffold protein in neonatal synapses, its expression profiles in adult and ageing rodent hippocampi are discrepant. Furthermore, in AD brains, significantly reduced SAP102 protein levels have been found, suggesting that SAP102 may be related to AD progression; however, the precise mechanism underlying this result remains unclear. Herein, we observed distinct SAP102 expression profiles in the hippocampal CA1, CA3 and DG subregions of rats and APPswe/PS1dE9 (APP/PS1) mice at various ages using immunofluorescence. In Wistar rats, SAP102 was not only highly expressed in the hippocampal subregions of neonatal rats but also maintained relatively high expression levels in adult hippocampi and displayed no obvious decreases in the CA1 and DG subregions of aged rats. Surprisingly, we observed abnormally high SAP102 expression levels in the CA1 stratum moleculare and CA3 stratum polymorphum subregions of 2-month-old APP/PS1 mice, but low SAP102 levels in the DG and CA3 subregions of 7-month-old APP/PS1 mice, reflecting the subregion-specific reactivity and vulnerability of AD mouse models in different disease stages. Our findings provide fundamental data to support the functional differences of SAP102 in different hippocampal subregions during postnatal periods and may serve as the basis for additional functional studies on SAP102 in normal physiological conditions and different stages of AD.

Se enhanced phytoremediation of diesel in soil by Trifolium repens.

A pot-culture experiment was conducted to assess the effects of selenium (Se) (0.5 mg kg-1) on Trifolium repens exposed to various levels of diesel (0, 15, 20, 25 g kg-1) for 30 days and 60 days. Exposure to diesel for 60 day led to concentration-dependent decreases in root morphogenesis, chlorophyll content and CAT activity, and to dose-dependent increases in MDA content and SOD activity. The residual diesel concentration in soil increased and the removal efficiency decreased with soil diesel concentration. The chlorophyll content and residual diesel concentration after were slightly higher at 30 days than at 60days. Application of Se to soil increased Trifolium repens tolerance to diesel and significantly increased the phytoremediation effect at 60 days, with a removal rate of 36 ±â€¯8%, compared to 28 ±â€¯7% in the control. These results contribute to the ongoing effort to develop an effective phytoremediation system for soils highly contaminated by diesel.

Selenium alleviates phytotoxicity of phenanthrene and pyrene in Alternanthera Philoxeroides.

To investigate if selenium can alleviate phytotoxicity of phenanthrene and pyrene, two high molecular weight (HMW) PAHs (polycyclic aromatic hydrocarbons) in Alternanthera philoxeroides are considered. A 60-day pot-culture experiment was carried out to assess the effects of selenium (0.5 mg Se·kg-1 soil) on A. philoxeroides exposed to two PAH pollutants, pyrene (PYR) and phenanthrene (PHE), at levels of 10, 100, and 1000 mg·kg-1. The test index included growth, chlorophyl, gas exchange and chlorophyl fluorescence parameters, and indicators of oxidative stress (H2O2 and malondialdehyde MDA). The response of plants to PAH exposure was concentration dependent; indicators of plant health declined, while indicators of plant stress rose. The maximum values of H2O2 and MDA were recorded at 1000 mg·kg-1 PYR, followed by 1000 mg·kg-1 PHE. However, application of Se (0.5 mg·kg-1) to the soil significantly decreased the phytotoxic response to PAH exposure. This study demonstrated that Se increases the tolerance of A. philoxeroides to PYR and PHE, improving the feasibility of phytoremediating high level PAH contamination and expediting ecological restoration.

Raman Deuterium Isotope Probing Reveals Microbial Metabolism at the Single-Cell Level.

We illustrate that single-cell Raman microspectroscopy, coupled with deuterium isotope probing (Raman-DIP), provides a culture-independent and nondestructive approach to probe metabolic pathways of carbon substrates at the single-cell level. We found a distinguishable C-D vibration band at 2070-2300 cm-1 in single-cell Raman spectra (SCRS) when Escherichia coli used deuterated glucose and Pseudomonas sp. used deuterated naphthalene as sole carbon sources. The intensity of the C-D band is proportional to the extent of deuteration in the carbon source, and as little as 5% deuteration can be distinguished by analysis of SCRS. It suggests that Raman-DIP could be used to semiquantitatively and sensitively indicate the metabolism of deuterated carbon source in microbes. A lower lipid conversion rate of deuterated naphthalene compared to that of deuterated glucose was observed, presumably owing to different anabolic pathways and membrane alteration. Apart from the C-D band shift from C-H, SCRS also reveal several isotopic shifts of the phenylalanine band, of which the positions correlate well with a computational model. A reduction in phenylalanine deuteration in Pseudomonas sp. compared to that in E. coli is due to the dilution effect of different pathways of phenylalanine biosynthesis in Pseudomonas sp. Collectively, we demonstrate that Raman-DIP can not only indicate metabolic activity using deuterated carbon sources but also reveal different metabolic pathways by analyzing SCRS. By harnessing such low-cost and versatile deuterated substrates, Raman-DIP has the potential to probe a wide range of metabolic pathways and functions at the single-cell level.

Use of selenium to alleviate naphthalene induced oxidative stress in Trifolium repens L.

Polycyclic aromatic hydrocarbons (PAHs) are among the most dangerous of environmental contaminants, due to their toxicity, carcinogenicity and mutagenicity. This study investigated the use of selenium (Se) to protect plants from the toxic effects of naphthalene (NPH). Exposing Trifolium repens L. (white clover) to a high concentration of NPH (soil spiked to 500mgkg-1) for 60 d significantly decreased biomass, CO2 assimilation rate (Pn), stomatal conductance (Gs) and intercellular CO2 concentration (Ci), while inducing production of H2O2 and malondialdehyde (MDA). Application of Se (soil spiked to 0.5mgkg-1) to plants exposed to NPH clearly protected the plants; biomass, Pn, Gs and Ci were significantly higher and contents of MDA and H2O2 decreased. The protection provided to Trifolium repens L. by Se is attributed primarily to an increase in photosynthesis and a decrease in oxidative stress. This study demonstrates that a low concentration of Se protects plants against oxidative stress induced by NPH and can provide a means for improving phytoremediation in PAHs contaminated soils.

Reverse and Multiple Stable Isotope Probing to Study Bacterial Metabolism and Interactions at the Single Cell Level.

The interactions between microorganisms driven by substrate metabolism and energy flow are important to shape diversity, abundance, and structure of a microbial community. Single cell technologies are useful tools for dissecting the functions of individual members and their interactions in microbial communities. Here, we developed a novel Raman stable isotope probing (Raman-SIP), which uses Raman microspectroscopy coupled with reverse and D2O colabeling to study metabolic interactions in a two-species community consisting of Acinetobacter baylyi ADP1 and Escherichia coli DH5α-GFP. This Raman-SIP approach is able to detect carbon assimilation and general metabolic activity simultaneously. Taking advantage of Raman shift of single cell Raman spectra (SCRS) mediated by incorporation of stable-isotopic substrates, Raman-SIP with reverse labeling has been applied to detect initially 13C-labeled bands of ADP1 SCRS reverting back to 12C positions in the presence of 12C citrate. Raman-SIP with D2O labeling has been employed to probe metabolic activity of single cells without the need of cell replication. Our results show that E. coli alone in minimal medium with citrate as the sole carbon source had no metabolic activity, but became metabolically active in the presence of ADP1. Mass spectrometry-based metabolite footprint analysis suggests that putrescine and phenylalanine excreted by ADP1 cells may support the metabolic activity of E. coli. This study demonstrates that Raman-SIP with reverse labeling would be a useful tool to probe metabolism of any carbon substrate, overcoming limitations when stable isotopic substrates are not readily available. It is also found that Raman-SIP with D2O labeling is a sensitive and reliable approach to distinguish metabolically active cells but not quiescent cells. This novel approach extends the application of Raman-SIP and demonstrates its potential application as a valuable strategic approach for probing cellular metabolism, metabolic activity, and interactions in microbial communities at the single cell level.

Use of a whole-cell bioreporter, Acinetobacter baylyi, to estimate the genotoxicity and bioavailability of chromium(VI)-contaminated soils.

A whole-cell bioreporter, Acinetobacter baylyi ADPWH_recA, was used to estimate the genotoxicity and bioavailability of chromium (VI) [Cr(VI)] in contaminated soils. Upon direct exposure to pre-sonicated soil samples, ADPWH_recA gave the highest response to the genotoxicity of Cr(VI) within 5 h with a detection limit of 2 µM Cr(VI). Investigations on sites contaminated with Cr(VI) revealed that soil-associated Cr(VI) was bioavailable to the bioreporter although it could not be extracted into the aqueous phase. The physical and chemical properties of soil might influence the bioavailability of Cr(VI), and higher genotoxicity was found in soils with a lower pH. This whole cell bioreporter approach makes it feasible to evaluate the bioavailability and genotoxicity of Cr(VI)-contaminated soils to uncover their potential impact on human health.

Raman Flow Cytometry and Its Biomedical Applications.

Raman flow cytometry (RFC) uniquely integrates the "label-free" capability of Raman spectroscopy with the "high-throughput" attribute of traditional flow cytometry (FCM), offering exceptional performance in cell characterization and sorting. Unlike conventional FCM, RFC stands out for its elimination of the dependency on fluorescent labels, thereby reducing interference with the natural state of cells. Furthermore, it significantly enhances the detection information, providing a more comprehensive chemical fingerprint of cells. This review thoroughly discusses the fundamental principles and technological advantages of RFC and elaborates on its various applications in the biomedical field, from identifying and characterizing cancer cells for in vivo cancer detection and surveillance to sorting stem cells, paving the way for cell therapy, and identifying metabolic products of microbial cells, enabling the differentiation of microbial subgroups. Moreover, we delve into the current challenges and future directions regarding the improvement in sensitivity and throughput. This holds significant implications for the field of cell analysis, especially for the advancement of metabolomics.