Applications of Data Characteristic AI-Assisted Raman Spectroscopy in Pathological Classification.

Raman spectroscopy has been widely used for label-free biomolecular analysis of cells and tissues for pathological diagnosis in vitro and in vivo. AI technology facilitates disease diagnosis based on Raman spectroscopy, including machine learning (PCA and SVM), manifold learning (UMAP), and deep learning (ResNet and AlexNet). However, it is not clear how to optimize the appropriate AI classification model for different types of Raman spectral data. Here, we selected five representative Raman spectral data sets, including endometrial carcinoma, hepatoma extracellular vesicles, bacteria, melanoma cell, diabetic skin, with different characteristics regarding sample size, spectral data size, Raman shift range, tissue sites, Kullback-Leibler (KL) divergence, and significant Raman shifts (i.e., wavenumbers with significant differences between groups), to explore the performance of different AI models (e.g., PCA-SVM, SVM, UMAP-SVM, ResNet or AlexNet). For data set of large spectral data size, Resnet performed better than PCA-SVM and UMAP. By building data characteristic-assisted AI classification model, we optimized the network parameters (e.g., principal components, activation function, and loss function) of AI model based on data size and KL divergence etc. The accuracy improved from 85.1 to 94.6% for endometrial carcinoma grading, from 77.1 to 90.7% for hepatoma extracellular vesicles detection, from 89.3 to 99.7% for melanoma cell detection, from 88.1 to 97.9% for bacterial identification, from 53.7 to 85.5% for diabetic skin screening, and mean time expense of 5 s.

Coupling of Perinuclear Actin Cap and Nuclear Mechanics in Regulating Flow-Induced Yap Spatiotemporal Nucleocytoplasmic Transport.

Mechanical forces, including flow shear stress, govern fundamental cellular processes by modulating nucleocytoplasmic transport of transcription factors like Yes-associated Protein (YAP). However, the underlying mechanical mechanism remains elusive. In this study, it is reported that unidirectional flow induces biphasic YAP transport with initial nuclear import, followed by nuclear export as actin cap formation and nuclear stiffening. Conversely, pathological oscillatory flow induces slight actin cap formation, nuclear softening, and sustained YAP nuclear localization. To elucidate the disparately YAP spatiotemporal distribution, a 3D mechanochemical model is developed, which integrates flow sensing, cytoskeleton organization, nucleus mechanotransduction, and YAP transport. The results unveiled that despite the significant localized nuclear stress imposed by the actin cap, its inherent stiffness counteracts the dispersed contractile stress exerted by conventional fibers on the nuclear membrane. Moreover, alterations in nuclear stiffness synergistically regulate nuclear deformation, thereby governing YAP transport. Furthermore, by expanding the single-cell model to a collective vertex framework, it is revealed that the irregularities in actin cap formation within individual cells have the potential to induce topological defects and spatially heterogeneous YAP distribution in the cellular monolayer. This work unveils a unified mechanism of flow-induced nucleocytoplasmic transport, providing a linkage between transcription factor localization and mechanical stimulation.

Rapid Antifungal Susceptibility Testing Based on Single-Cell Metabolism Analysis Using Stimulated Raman Scattering Imaging.

Rapid antifungal susceptibility testing (AFST) is urgently needed in clinics to treat invasive fungal infections with the appropriate antifungal drugs and to slow the emergence of antifungal resistance. However, current AFST methods are time-consuming (24-48 h) due to the slow growth of fungal cells and the methods not being able to work directly for clinical samples. Here, we demonstrate rapid AFST by measuring the metabolism in single fungal cells using stimulated Raman scattering imaging and deuterium probing. Distinct metabolic responses were observed in Candida albicans to different classes of antifungal drugs: while the metabolism was inhibited by amphotericin B, it was stimulated by azoles (fluconazole and voriconazole) and micafungin. Accordingly, we propose metabolism change as a biomarker for rapid AFST. The results were obtained in 4 h with 100% categorical agreement with the gold standard broth microdilution test. In addition, a protocol was developed for direct AFST from positive blood cultures. This method overcomes the limitation of slow growth in conventional methods and has the potential for the rapid diagnosis of candidemia and other clinical fungal infections.

Bio-inspired peptide-conjugated liposomes for enhanced planktonic bacteria killing and biofilm eradication.

Developing new antimicrobial agents has become an urgent task to address the increasing prevalence of multidrug-resistant pathogens and the emergence of biofilms. Cationic antimicrobial peptides (AMPs) have been regarded as promising candidates due to their unique non-specific membrane rupture mechanism. However, a series of problems with the peptides hindered their practical application due to their high toxicity and low bioactivity and stability. Here, inspired by broadening the application of cell-penetrating peptides (CPPs), we selected five different sequences of cationic peptides which are considered as both CPPs and AMPs, and developed a biomimetic strategy to construct cationic peptide-conjugated liposomes with the virus-like structure for both enhancements of antibacterial efficacy and biosafety. The correlation between available peptide density/peptide variety and antimicrobial capabilities was evaluated from quantitative perspectives. Computational simulation and experimental investigations assisted to identify the optimal peptide-conjugated liposomes and revealed that the designed system provides high charge density for enhanced anionic bacterial membrane binding capability without compromised cytotoxicity, being capable of enhanced antibacterial efficacy of bacteria/biofilm of clinically important pathogens. The bio-inspired design has shown enhanced therapeutic efficiency of peptides and may promote the development of next-generation antimicrobials.

RIIß-PKA in GABAergic Neurons of Dorsal Median Hypothalamus Governs White Adipose Browning.

The RIIß subunit of  cAMP-dependent protein kinase A (PKA) is expressed in the brain and adipose tissue. RIIß-knockout mice show leanness and increased UCP1 in brown adipose tissue. The authors have previously reported that RIIß reexpression in hypothalamic GABAergic neurons rescues the leanness. However, whether white adipose tissue (WAT) browning contributes to the leanness and whether RIIß-PKA in these neurons governs WAT browning are unknown. Here, this work reports that RIIß-KO mice exhibit a robust WAT browning. RIIß reexpression in dorsal median hypothalamic GABAergic neurons (DMH GABAergic neurons) abrogates WAT browning. Single-cell sequencing, transcriptome sequencing, and electrophysiological studies show increased GABAergic activity in DMH GABAergic neurons of RIIß-KO mice. Activation of DMH GABAergic neurons or inhibition of PKA in these neurons elicits WAT browning and thus lowers body weight. These findings reveal that RIIß-PKA in DMH GABAergic neurons regulates WAT browning. Targeting RIIß-PKA in DMH GABAergic neurons may offer a clinically useful way to promote WAT browning for treating obesity and other metabolic disorders.

Compound Raman microscopy for rapid diagnosis and antimicrobial susceptibility testing of pathogenic bacteria in urine.

Rapid identification and antimicrobial susceptibility testing (AST) of bacteria are key interventions to curb the spread and emergence of antimicrobial resistance. The current gold standard identification and AST methods provide comprehensive diagnostic information but often take 3 to 5 days. Here, a compound Raman microscopy (CRM), which integrates Raman spectroscopy and stimulated Raman scattering microscopy in one system, is presented and demonstrated for rapid identification and AST of pathogens in urine. We generated an extensive bacterial Raman spectral dataset and applied deep learning to identify common clinical bacterial pathogens. In addition, we employed stimulated Raman scattering microscopy to quantify bacterial metabolic activity to determine their antimicrobial susceptibility. For proof-of-concept, we demonstrated an integrated assay to diagnose urinary tract infection pathogens, S. aureus and E. coli. Notably, the CRM system has the unique ability to provide Gram-staining classification and AST results within ~3 h directly from urine samples and shows great potential for clinical applications.

Cytosolic peptides encoding CaV1 C-termini downregulate the calcium channel activity-neuritogenesis coupling.

L-type Ca2+ (CaV1) channels transduce channel activities into nuclear signals critical to neuritogenesis. Also, standalone peptides encoded by CaV1 DCT (distal carboxyl-terminus) act as nuclear transcription factors reportedly promoting neuritogenesis. Here, by focusing on exemplary CaV1.3 and cortical neurons under basal conditions, we discover that cytosolic DCT peptides downregulate neurite outgrowth by the interactions with CaV1's apo-calmodulin binding motif. Distinct from nuclear DCT, various cytosolic peptides exert a gradient of inhibitory effects on Ca2+ influx via CaV1 channels and neurite extension and arborization, and also the intermediate events including CREB activation and c-Fos expression. The inhibition efficacies of DCT are quantitatively correlated with its binding affinities. Meanwhile, cytosolic inhibition tends to facilitate neuritogenesis indirectly by favoring Ca2+-sensitive nuclear retention of DCT. In summary, DCT peptides as a class of CaV1 inhibitors specifically regulate the channel activity-neuritogenesis coupling in a variant-, affinity-, and localization-dependent manner.

Assessing the impact of freshwater discharge on the fluid chemistry in the Svalbard fjords.

Changes in the cryosphere extent (e.g., glacier, ice sheet, permafrost, and snow) have been speculated to impact (bio)geochemical interactions and element budgets of seawater and pore fluids in Arctic regions. However, this process has rarely been documented in Arctic fjords, which leads to a poor systematic understanding of land-ocean interactions in such a warming-susceptible region. Here, we present the chemical and isotopic (δ18O, δD, δ11B, and 87Sr/86Sr) compositions of seawater and pore fluids from five fjords in the Svalbard archipelago. Compared to bottom seawater, the low Cl- concentrations and depleted water isotopic signatures (δ18O and δD) of surface seawater and pore fluids delineate freshwater discharge originating from precipitation and/or meltwater of the cryosphere (i.e., glacier, snow, and permafrost). In contrast, the high Cl- concentrations with light water isotopic values in pore fluids from Dicksonfjorden indicate a brine probably resulted from submarine permafrost formation during the late Holocene, a timing supported by the numerical simulation of dissolved Cl- concentration. The freshwater is influenced by the local diagenetic processes such as ion exchanges indicated by δ11B signatures as well as interactions with bedrock during fluid migration inferred from pore fluid 87Sr/86Sr ratios. The interactions with bedrock significantly alter the hydrogeochemical properties of pore fluids in each fjord, yielding spatiotemporal variations. Consequently, land-ocean interactions in combination with the hydrosphere-cryosphere-lithosphere are critical factors for understanding and predicting the hydrology and elemental cycling during global climate change periods in the past, present, and future of the Svalbard archipelago.

A rapid procedure for bacterial identification and antimicrobial susceptibility testing directly from positive blood cultures.

There is an urgent need to develop a rapid procedure that can rapidly identify and obtain antimicrobial susceptibility testing (AST) results directly from positive blood cultures. Here, we report a semi-automatic bacterial diagnosis procedure, which includes (1) a bacterial concentration process to isolate bacteria from a positive blood culture bottle (PBCB), (2) an identification process using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and (3) a rapid AST process based on stimulated Raman scattering imaging of deuterium oxide (D2O) incorporation in bacteria. A total of 105 samples were tested for bacterial identification, and a bacterial identification accuracy of 92.3% was achieved. AST takes about 2.5 h after identification. This semi-automatic procedure only takes 3.5 h, which is demonstrated to be the fastest process to obtain identification and AST results starting from PBCBs.

Distinct methane-dependent biogeochemical states in Arctic seafloor gas hydrate mounds.

Archaea mediating anaerobic methane oxidation are key in preventing methane produced in marine sediments from reaching the hydrosphere; however, a complete understanding of how microbial communities in natural settings respond to changes in the flux of methane remains largely uncharacterized. We investigate microbial communities in gas hydrate-bearing seafloor mounds at Storfjordrenna, offshore Svalbard in the high Arctic, where we identify distinct methane concentration profiles that include steady-state, recently-increasing subsurface diffusive flux, and active gas seepage. Populations of anaerobic methanotrophs and sulfate-reducing bacteria were highest at the seep site, while decreased community diversity was associated with a recent increase in methane influx. Despite high methane fluxes and methanotroph doubling times estimated at 5-9 months, microbial community responses were largely synchronous with the advancement of methane into shallower sediment horizons. Together, these provide a framework for interpreting subseafloor microbial responses to methane escape in a warming Arctic Ocean.

Rapid antimicrobial susceptibility testing by stimulated Raman scattering metabolic imaging and morphological deformation of bacteria.

Methods for rapid antimicrobial susceptibility testing (AST) are urgently needed to address the emergence and spread of antimicrobial resistance. Here, we report a new method based on stimulated Raman scattering (SRS) microscopy, which measures both the metabolic activity and the morphological deformation of bacteria to determine the antimicrobial susceptibility of ß-lactam antibiotics rapidly. In this approach, we quantify single bacteria's metabolic activity by the carbon-deuterium (C-D) bond concentrations in bacteria after D2O incubation. In the meantime, bacterial morphological deformation caused by ß-lactam antibiotics is also measured. With these two quantifiable markers, we develop an evaluation method to perform AST of cefotaxime on 103 E. coli strains. Our method achieved a 93.2% categorical agreement and a 93.2% essential agreement with the standard reference method.

Recent Development of Rapid Antimicrobial Susceptibility Testing Methods through Metabolic Profiling of Bacteria.

Due to the inappropriate use and overuse of antibiotics, the emergence and spread of antibiotic-resistant bacteria are increasing and have become a major threat to human health. A key factor in the treatment of bacterial infections and slowing down the emergence of antibiotic resistance is to perform antimicrobial susceptibility testing (AST) of infecting bacteria rapidly to prescribe appropriate drugs and reduce the use of broad-spectrum antibiotics. Current phenotypic AST methods based on the detection of bacterial growth are generally reliable but are too slow. There is an urgent need for new methods that can perform AST rapidly. Bacterial metabolism is a fast process, as bacterial cells double about every 20 to 30 min for fast-growing species. Moreover, bacterial metabolism has shown to be related to drug resistance, so a comparison of differences in microbial metabolic processes in the presence or absence of antimicrobials provides an alternative approach to traditional culture for faster AST. In this review, we summarize recent developments in rapid AST methods through metabolic profiling of bacteria under antibiotic treatment.

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.

Diagnosis of severe scrub typhus infection by next-generation sequencing:a case report.

BACKGROUND: Scrub typhus is an acute febrile illness, which was caused by Orientia tsutsugamushi and transmitted through the bite of chiggers. The diagnosis of scrub typhus could be missed diagnosis due to the absence of the pathognomonic eschar. CASE PRESENTATION: A 76-year-old man was hospitalized with fever and kidney injury and was diagnosed of hemorrhagic fever with renal syndrome first. However, the situation of the illness deteriorated into refractory septic shock and multiple organ dysfunction rapidly,although the treatment of anti-sepsis was used in 3rd-5th day. Orientia tsutsugamushi was determined to be the causative pathogen by Next-generation sequencing of his plasma sample in 6th day. Then, the patient was treated with doxycycline and azithromycin and recovered quickly. CONCLUSIONS: Next-generation sequencing was a new diagnostic technology and could identify scrub typhus in accurately and fast without the pathognomonic eschar.

Discharge of deeply rooted fluids from submarine mud volcanism in the Taiwan accretionary prism.

Qualitative and quantitative assessments of fluid cycling are essential to address the role and transport of deeply sourced fluids in subduction systems. In this study, sediment cores distributed across a submarine mud volcano (SMV) offshore southwestern Taiwan were investigated to determine the characteristics of fluids generated through the convergence between the Eurasian and Phillippine Sea Plates. The low dissolved chloride concentration combined with the enrichment of 18O, and depletion of 2H of pore fluids suggest the discharge of deep freshwater formed by smectite dehydration at an equilibrium temperature of 100 to 150 °C. The upward fluid velocities, decreasing from 2.0 to 5.0 cm yr-1 at the center to a negligible value at margin sites, varied with the rate and efficiency of anaerobic methanotrophy, demonstrating the impact of fluid migration on biogeochemical processes and carbon cycling. By extrapolating the velocity pattern, the flux of fluids exported from 13 SMVs into seawater amounted up to 1.3-2.5 × 107 kg yr-1, a quantity accounting for 1.1-28.6% of the smectite-bound water originally stored in the incoming sediments. Our results imply that SMVs could act as a conduit to channel the fluids produced from great depth/temperature into seafloor environments in a subduction system of the western Pacific Ocean.

The effects of an enteral nutrition feeding protocol on critically ill patients: A prospective multi-center, before-after study.

PURPOSE: The aim of this study was to explore the effects of an enteral nutrition (EN) feeding protocol in critically ill patients. METHODS: This was a prospective multi-center before-after study. We compared energy related and prognostic indicators between the control group (pre-implementation stage) and intervention group (post-implementation stage). The primary endpoint was the percentage of patients receiving EN within 7 days after ICU admission. RESULTS: 209 patients in the control group and 230 patients in the intervention group were enrolled. The implementation of the EN protocol increased the percentage of target energy reached from day 3 to day 7, and the difference between two groups reached statistical significance in day 6 (P = .01) and day 7 (P = .002). But it had no effects on proportion of patient receiving EN (P = .65) and start time of EN (P = .90). The protocol application might be associated with better hospital survival (89.1% vs 82.8%, P = .055) and reduce the incidence of EN related adverse (P = .004). There was no difference in ICU length of stay, duration of mechanical ventilation and ICU cost. CONCLUSION: The implementation of the enteral feeding protocol is associated with improved energy intake and a decreased incidence of enteral nutrition related adverse events for critically ill patients, but it had no statistically beneficial effects on reducing the hospital mortality rate. Trial registration ClinicalTrials.gov, NCT02976155. Registered November 29, 2016- Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT02976155.

Lead-free cesium tin halide nanocrystals for light-emitting diodes and color down conversion.

Organometal halide perovskites are attracting a great deal of attention because of their long carrier diffusion lengths, wide wavelength tunability, and narrow-band emission. However, the toxicity of lead has caused considerable environmental and health concerns. In this work, lead-free cesium tin halide nanocrystals are synthesized and investigated. CsSnBr3 and CsSnI3 nanocrystals, 25 and 7 nm in size, are synthesized by a facile hot injection method. Absorption spectroscopy, photoluminescence spectroscopy, and X-ray diffraction were used to understand their structural and optical properties. CsSnBr3 and CsSnI3 nanocrystals show emission peaks at 683 and 938 nm, respectively. These nanocrystals show shelf stability for a few months. Temperature-dependent photoluminescence is utilized to know more about fundamental physical parameters, such as exciton binding energy, charge carrier-phonon interactions and band gap. Light-emitting diodes and color down-conversion films are also demonstrated using these lead free perovskite nanocrystals.

Microbial communities from Arctic marine sediments respond slowly to methane addition during ex situ enrichments.

Anaerobic methanotrophic archaea (ANME) consume methane in marine sediments, limiting its release to the water column, but their responses to changes in methane and sulfate supplies remain poorly constrained. To address how methane exposure may affect microbial communities and methane- and sulfur-cycling gene abundances in Arctic marine sediments, we collected sediments from offshore Svalbard that represent geochemical horizons where anaerobic methanotrophy is expected to be active, previously active, and long-inactive based on reaction-transport biogeochemical modelling of porewater sulfate profiles. Sediment slurries were incubated at in situ temperature and pressure with different added methane concentrations. Sediments from an active area of seepage began to reduce sulfate in a methane-dependent manner within months, preceding increased relative abundances of anaerobic methanotrophs ANME-1 within communities. In previously active and long-inactive sediments, sulfur-cycling Deltaproteobacteria became more dominant after 30 days, though these communities showed no evidence of methanotrophy after nearly 8 months of enrichment. Overall, enrichment conditions, but not methane, broadly altered microbial community structure across different enrichment times and sediment types. These results suggest that active ANME populations may require years to develop, and consequently microbial community composition may affect methanotrophic responses to potential large-scale seafloor methane releases in ways that provide insight for future modelling studies.

Image based quantitative comparisons indicate heightened megabenthos diversity and abundance at a site of weak hydrocarbon seepage in the southwestern Barents Sea.

BACKGROUND: High primary productivity in the midst of high toxicity defines hydrocarbon seeps; this feature usually results in significantly higher biomass, but in lower diversity communities at seeps rather than in the surrounding non-seep benthos. Qualitative estimates indicate that this dichotomy does not necessarily hold true in high latitude regions with respect to megafauna. Instead, high latitude seeps appear to function as local hotspots of both megafaunal diversity and abundance, although quantitative studies do not exist. In this study, we tested this hypothesis quantitatively by comparing georeferenced seafloor mosaics of a seep in the southwestern Barents Sea with the adjacent non-seep seafloor. METHODS: Seafloor images of the Svanefjell seep site and the adjacent non seep-influenced background seabed in the southwestern Barents Sea were used to construct georeferenced mosaics. All megafauna were enumerated and mapped on these mosaics and comparisons of the communities at the seep site and the non-seep background site were compared. Sediment push cores were taken in order to assess the sediment geochemical environment. RESULTS: Taxonomic richness and abundance were both considerably higher at the seep site than the non-seep location. However, taxa were fewer at the seep site compared to other seeps in the Barents Sea or the Arctic, which is likely due to the Svanefjell seep site exhibiting relatively low seepage rates (and correspondingly less chemosynthesis based primary production). Crusts of seep carbonates account for the higher diversity of the seep site compared to the background site, since most animals were either colonizing crust surfaces or using them for shelter or coverage. Our results indicate that seeps in northern latitudes can enhance local benthic diversity and this effect can take place even with weak seepage. Since crusts of seep carbonates account for most of the aggregating effect of sites experiencing moderate/weak seepage such as the study site, this means that the ability of seep sites to attract benthic species extends well beyond the life cycle of the seep itself, which has important implications for the larger marine ecosystem and its management policies.