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.

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.

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.

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.

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.

Antibiotic Susceptibility Determination within One Cell Cycle at Single-Bacterium Level by Stimulated Raman Metabolic Imaging.

The widespread use of antibiotics has significantly increased the number of resistant bacteria, which has also increased the urgency of rapid bacterial detection and profiling their antibiotic response. Current clinical methods for antibiotic susceptibility testing (AST) rely on culture and require at least 16 to 24 h to conduct. Therefore, there is an urgent need for a rapid method that can test the susceptibility of bacteria in a culture-free manner. Here we demonstrate a rapid AST method by monitoring the glucose metabolic activity of live bacteria at the single-cell level with hyperspectral stimulated Raman scattering (SRS) imaging. Using vancomycin-susceptible and -resistant enterococci E. faecalis as models, we demonstrate that the metabolic uptake of deuterated glucose in a single living bacterium can be quantitatively monitored via hyperspectral SRS imaging. Remarkably, the metabolic activity of susceptible bacteria responds differently to antibiotics from the resistant strain within only 0.5 h from the addition of antibiotics. Therefore, bacterial susceptibility and the minimum inhibitory concentration (MIC) of antibiotics can be determined within one cell cycle. Our metabolic imaging method is applicable to other bacteria species including E. coli, K. Pneumoniae, and S. aureus as well as different antibiotics, regardless of their mechanisms of inhibiting or killing bacteria.

Stimulated Raman Imaging Reveals Aberrant Lipogenesis as a Metabolic Marker for Azole-Resistant Candida albicans.

Candida albicans is the single most prevalent cause of fungal bloodstream infections worldwide causing significant mortality as high as 50 percent. This high mortality rate is, in part, due to the inability to initiate an effective antifungal therapy early in the disease process. Mortality rates significantly increase after 12 hours of delay in initiating the appropriate antifungal therapy following a positive blood culture. Early administration of appropriate antifungal therapy is hampered by the slow turnovers of the conventional antimicrobial testing techniques, which require days of incubation. To address this unmet need, we explored the potential of employing stimulated Raman scattering (SRS) imaging to probe for metabolic differences between fluconazole-susceptible and -resistant strains at a single cell level in search of a metabolic signature. Metabolism is integral to pathogenicity. Since only a few hours are needed to observe a full metabolic cycle in C. albicans, metabolic profiling provides an avenue for rapid antimicrobial susceptibility testing. C-H frequency (2850 cm-1) SRS imaging revealed a substantial difference in lipogenesis between the fluconazole-susceptible and -resistant C. albicans. Exposure to fluconazole, an antimicrobial drug that targets ergosterol biosynthesis, only affected the lipogenesis in the susceptible strain. These results show that single cell metabolic imaging via SRS microscopy can be used for rapid detection of antimicrobial susceptibility.

Evolution of Membrane Fouling Revealed by Label-Free Vibrational Spectroscopic Imaging.

Membrane fouling is the bottleneck that restricts the sustainability of membrane technology for environmental applications. Therefore, the development of novel analytical tools for characterizing membrane fouling processes is essential. In this work, we demonstrate a capability of probing the chemical structure of foulants and detecting their 3-dimentional spatial distribution on membranes based on stimulated Raman scattering (SRS) microscopy as a vibrational spectroscopic imaging approach. The adsorption process of foulants onto membrane surfaces and their aggregation process within membrane pores during the microfiltration of protein and polysaccharide solutions were clearly monitored. Pore constriction and cake layer formation were found to be the coupled membrane fouling mechanisms. This work establishes an ultrafast, highly sensitive, nondestructive and label-free imaging platform for the characterization of membrane fouling evolution. Furthermore, this work provides new insights into membrane fouling and offers a powerful tool for membrane-based process exploration.

The Effect of Temperature on Microtubule-Based Transport by Cytoplasmic Dynein and Kinesin-1 Motors.

Cytoplasmic dynein and kinesin are both microtubule-based molecular motors but are structurally and evolutionarily unrelated. Under standard conditions, both move with comparable unloaded velocities toward either the microtubule minus (dynein) or plus (most kinesins) end. This similarity is important because it is often implicitly incorporated into models that examine the balance of cargo fluxes in cells and into models of the bidirectional motility of individual cargos. We examined whether this similarity is a robust feature, and specifically whether it persists across the biologically relevant temperature range. The velocity of mammalian cytoplasmic dynein, but not of mammalian kinesin-1, exhibited a break from simple Arrhenius behavior below 15°C-just above the restrictive temperature of mammalian fast axonal transport. In contrast, the velocity of yeast cytoplasmic dynein showed a break from Arrhenius behavior at a lower temperature (∼8°C). Our studies implicate cytoplasmic dynein as a more thermally tunable motor and therefore a potential thermal regulator of microtubule-based transport. Our theoretical analysis further suggests that motor velocity changes can lead to qualitative changes in individual cargo motion and hence net intracellular cargo fluxes. We propose that temperature can potentially be used as a noninvasive probe of intracellular transport.

Coherent anti-Stokes Raman scattering imaging under ambient light.

We demonstrate an ambient light coherent anti-Stokes Raman scattering microscope that allows CARS imaging to be operated under environmental light for field use. The CARS signal is modulated at megahertz frequency and detected by a photodiode equipped with a lab-built resonant amplifier, then extracted through a lock-in amplifier. The filters in both the spectral domain and the frequency domain effectively blocked the room light contamination of the CARS image. In situ hyperspectral CARS imaging of tumor tissue under ambient light is demonstrated.

Single-molecule inhibition of human kinesin by adociasulfate-13 and -14 from the sponge Cladocroce aculeata.

Two merotriterpenoid hydroquinone sulfates designated adociasulfate-13 (1) and adociasulfate-14 (2) were purified from Cladocroce aculeata (Chalinidae) along with adociasulfate-8. All three compounds were found to inhibit microtubule-stimulated ATPase activity of kinesin at 15 µM by blocking both the binding of microtubules and the processive motion of kinesin along microtubules. These findings directly show that substitution of the 5'-sulfate in 1 for a glycolic acid moiety in 2 maintains kinesin inhibition. Nomarski imaging and bead diffusion assays in the presence of adociasulfates showed no signs of either free-floating or bead-bound adociasulfate aggregates. Single-molecule biophysical experiments also suggest that inhibition of kinesin activity does not involve adociasulfate aggregation. Furthermore, both mitotic and nonmitotic kinesins are inhibited by adociasulfates to a significantly different extent. We also report evidence that microtubule binding of nonkinesin microtubule binding domains may be affected by adociasulfates.

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.

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.

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.

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.

[Stratified analysis of the relationship between traditional Chinese medicine constitutional types and health status in the general population based on data of 8,448 cases].

OBJECTIVE: To examine the relationship between traditional Chinese medicine (TCM) constitutional types and health status among groups of different age or gender in the general population of China. METHODS: Data of 8 448 cases were randomly sampled from a database of 21 948 cases of a cross-sectional survey on the TCM constitutional types and health status which was carried out in 9 provinces or municipalities of China (Jiangsu, Anhui, Gansu, Qinghai, Fujian, Beijing, Jilin, Jiangxi and Henan) according to gender and age structure of the Chinese population in 2005. Scores of health-related quality of life scale--the Medical Outcomes Study 36-Item Short-Form Health Survey (MOS SF-36)--were analyzed by Nemenyi test to compare the health status of individuals with different constitutional types. RESULTS: Compared with the gentleness type, the MOS SF-36 scores of the 8 types of pathological constitution were significantly low (P<0.05) among groups of different age or gender. The MOS SF-36 score was the lowest in men of the qi-deficiency, qi-depression and blood-stasis types, while it was the lowest in women of the phlegm-dampness, qi-depression and qi-deficiency types. For the age group of 15 to 34, the special diathesis, qi-depression and blood-stasis types had the lowest MOS SF-36 scores; for the age group of 35 to 59, the qi-deficiency, qi-depression and blood-stasis types had the lowest MOS SF-36 scores; for the age group of over 60, the qi-deficiency, qi-depression and phlegm-dampness types had the lowest MOS SF-36 scores. CONCLUSION: In groups of different gender or age, the MOS SF-36 scores of the 8 types of pathological constitution were significantly lower than that of the gentleness type, indicating a deficient health status. The health status of different types of constitution showed different characteristics in groups of different gender or age.

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.