Thirty-six gilts were randomly assigned to fed control diet (CON), Na2SeO3 diet (0.3 mg Se/kg) or HMSeBA diet (0.3 mg Se/kg). The results showed that HMSeBA and Na2SeO3 supplementation both increased the total selenium content in liver and serum compared with control, while HMSeBA increased the total selenium content in liver compared with Na2SeO3 group. HMSeBA tended to increase the total selenium content in ovary compared with control. HMSeBA and Na2SeO3 supplementation both increased the weight of uteri in gilts at the third estrus. Moreover, HMSeBA supplementation down-regulated the gene expression of growth differentiation factor-9 (GDF-9) and bone morpho-genetic protein-15 (BMP-15) in cumulus-oocyte complexes (COCs). HMSeBA supplementation decreased malondialdehyde (MDA) content in serum, liver and ovary, increased activity of T-AOC in liver, TXNRD in ovary and GPX in serum, liver and ovary, while up-regulated the liver GPX2, SOD1 and TXNRD1, ovarian GPX1 gene expression. In vitro, HMSeBA treatment promoted GCs' proliferation and secretion of estradiol (E2). HMSeBA treatment increased the activity of T-AOC, T-SOD, GPX, TXNRD and decreased MDA content in GCs in vitro. Meanwhile, HMSeBA treatment up-regulated SOD2 and GPX1 gene expression in GCs in vitro. In conclusion, HMSeBA supplementation is more conducive to promoting follicle development by antioxidant pathway.Selenium (Se) is assumed to promote the follicle development by attenuating oxidative stress. The current study was developed to evaluate the effects of dietary 2-hydroxy-4-methylselenobutanoic acid (HMSeBA) supplementation on the follicle development  Model of proposed mechanism of 2-Hydroxy-4-methylselenobutanoic acid (HMSeBA) regulated follicle development by antioxidant pathway. E2 is one of the key ovarian hormones produced by the developing ovulatory follicle. E2 content reflect the differentiation of ovarian GCs. E2 is essential for female reproduction, as evidenced by severe fertility defects when its synthesis or action are suppressed (Ovarian follicular development is the most important event in reproductive performance in the gilt. The proliferation and differentiation of granulosa cells (GCs) determine ovarian follicular development . 17β-estppressed . With thSelenium (Se) is one of the essential trace elements for animals, which can effectively improve its reproductive performances –7. Typesin vitro were examined.Recently, one new organic selenium product named Selisseo® (SO) has been developed. Selenium in this product is present as 2-hydroxy-4-methylselenobutyric acid (HMSeBA). Studies have shown that HMSeBA is more bioavailable than selenium yeast or sodium selenite in poultry, growing pigs and weaned pigs , 13–15. 2SeO3) supplementation diet , (4) 2-hydroxy-4-methylselenobutyric acid supplementation diet . Each treatment had 12 gilts, six repetitions in each treatment and two gilts per repetition. CON diet was corn-soybean meal-based . Thirty-six female piglets  were randomly assigned to three dietary treatment groups: (1) control diet , (2) sodium selenite  of each treatment were randomly selected and slaughtered on the morning of the 19th day after the second estrus after fasting for 12 h. Before slaughter, 10 ml blood sample was collected from jugular vein. To obtaining the serum, blood samples were centrifuged at 3,000 × g, 4°C for 15 min, and then serum was stored at −20°C.One gilt from each repeatment . The number of corpora lutea was recorded, too. Then, the follicle contents of left ovary were aspirated from the follicles with > 5 mm in diameter by a 10-ml syringe. Cumulus-oocyte complexes (COCs) were recovered from the aspirate using a dissecting microscope (40 × magnification). Then the rest follicle contents were centrifuged at 2,000 rpm for 5 min. The follicular fluid (supernatant) was collected and stored at −20°C until analysis was conducted.in vitro experiments based on the high homology of swine and mice) at passage 3 or 4 at 2 × 105 cells  per well were seeded in 6-well tissue culture-treated plate in 2 ml DMEM supplemented with extra 1X antibiotic/antimicotic and 10% FBS. Added sodium selenite or HMSeBA working solution at 1 ng Se/ul according to the final concentration of 0, 2.5, 5, 10 ng Se /ml, respectively. Then the plate incubated in a humidified atmosphere of 5% CO2 at 37°C for 24 h. The cells should be around 40–50 % confluent, aspirated culture medium and washed attached cells once with 1* PBS. Added new culture medium with the final treatment concentration of selenium and incubated for 24 h (about 90 % confluent)  . Then thThe MTT  assay was applied to analysis the GCs' proliferation according to the manufacturer's protocol . Briefly, GCs were cultured with different concentration of sodium selenite or HMSeBA at 96-well tissue culture plates. After cultured for 44 h, the supernatant was removed and replaced by a solution composed of complete medium and MTT salt solution at ratio of 4:1. After 4 h of incubation, supernatant was removed, then added DMSO to each well and shake for 10 min to fully melt the crystals. Finally, the measurements were performed by a microplate reader  with 570 nm interference filters. Each experiment was carried out in triplicate.The concentrations of selenium in serum, tissue and feed samples were detected as previously described . Briefly2) and luteinizing hormone (LH) were detected by porcine enzyme-linked immunosorbent assay (ELISA) Kits . Optical density (OD) values were detected at 450 nm by a MuLtisKan MK3-Thermo Labsystems microplate reader . Serum three iodine thyroid (T3) and thyroxine (T4) were detected by porcine ELISA Kits  according to manufacturer's protocol.Serum and follicular fluid follicle stimulating hormone (FSH), estradiol  based on the manufacturer's protocol. Each experiment was carried out in triplicate for in vitro experiment.GCs were cultured with different concentration of Nain vitro experiment were quantified by the respective assay kits . Briefly, the activities of GPX, GR and SOD were assayed as described by Mou et al. (The activities of glutathione peroxidase (GPX), glutathione reductase (GR), thioredoxin reductase (TXNRD), total antioxidative capability (T-AOC), total superoxide dismutase (T-SOD) and malondialdehyde (MDA) of gilts' serum, liver, ovary, and supernatant and GCs of u et al. . MDA conin vitro experiment using the Trizol reagent . Agarose gel electrophoresis was applied to determine the integrity of RNA. By evaluating the OD260/OD280 ratio to determine RNA purity. Genomic DNA removal and reverse transcription (RT) were performed by PrimeScript RT reagent kit with gDNA eraser  based on the manufacturer's protocol. SYBR® Premix Ex TaqTM Kits  was appiled for real-time PCR analysis of mRNA expression. The primer sequences of the target genes were showed in Total RNA was isolated from gilt samples and GCs of P < 0.05. A tendency was assumed at P < 0.1.The repeatment was regarded as the experimental units for the animal growth performance. One gilt of a repeatment of three treatments had diarrhea or sick when younger, therefore, that repeatment was excluded from the present study. The gilt was regarded as the experimental units for the animal study except for the animal growth performance. Before using parametric analyses, all data were checked for normality and homogeneity of variance with Shapio-Wilk W test and Levene's test, respectively. All data were analyzed by one-way ANOVA using SPSS Statistics 22  and GraphPad Prism 6.0 . Then multiple comparison by DUNCAN analysis  were used to determine statistical differences between treatment groups. The results were presented as mean with SE. Statistically significant difference was considered at 2SeO3 increased the total selenium content in serum and liver . Dietary supplementation with HMSeBA tended to increase the total selenium content in ovary compared with control treatment in gilts (P = 0.08).Dietary supplementation with HMSeBA and NaP < 0.05, Dietary supplementation with HMSeBA increased the average daily body weight gain (ADG)  and bone morphogenetic protein-15 (BMP-15). The relative gene expression of GDF-9 and BMP-15 in COCs was lower in HMSeBA treatment than that in control treatment  did not change the FSH, LH and estradiol concentration in serum and follicular fluid of gilts compared with control treatment  in HMSeBA treatment compared with Na2SeO3 treatment in the liver of gilts, while the relative gene expression of GPX1 increased in HMSeBA treatment compared with Na2SeO3 treatment in the ovary of gilts (SELENOW and SELENOO decreased (P < 0.05) in Na2SeO3 supplementation treatment compared with control in the ovary. Besides, HMSeBA supplementation significantly increased (P < 0.05) the expression of SOD1 in the liver compared with control and Na2SeO3 groups. The expression of TXN1 increased (P < 0.05) in HMSeBA treatment compared with Na2SeO3 group in the liver of gilts. In addition to the above genes, although the difference was not significant, it was observed a numerical improvement in gene expression with HMSeBA compared to Na2SeO3 for all of the selenoproteines in liver.The relative gene expression of of gilts . The expP < 0.05, 2SeO3 had tendency to increased cell proliferation at 2.5 ng/ml (P = 0.097). Estradiol secretion is an important manifestation of the physiological function of granular cells. HMSeBA significantly increased the E2 concentration compared with control treatment at 5 and 10 ng/ml  and growth differentiation factor-9 (GDF-9) are important oocyte-secreted factors which stimulate GCs proliferation and suppress progesterone production in the early stages of follicular development  and corpora lutea (33.0 vs. 23.2). Bone morphogenetic protein-15 . Fortier et al.  treatment on d −1 of peri-ovulatory and no difference among the treatments with other time of the peri-ovulatory. This response might be related to the different sampling time in vivo or the complexity of the in vivo test did not reflect the treatment effect. In our in vitro experiment, HMSeBA significantly increased the E2 concentration compared with control.In present study, consistent with previous study , we founed yeast . There ied yeast . In thisr et al.  found thin vitro experiment suggested that HMSeBA, a new organic selenium, played a regulatory role in GC proliferation and E2 synthesis. These findings are consistent with previous studies reporting that sodium selenite supplementation acts a regulatory role on E2 synthesis and the proliferation of bovine GCs  treatment increased the GR concentration, which was consistent with the results of gene expression. However, no difference in GR in the ovary at the transcription level and translation level in in vivo experiment. This may be due to HMSeBA regulates the expression of GR at the post-transcriptional level or too many cell types in the ovary mask the effect of HMSeBA on granulosa cells in vivo. However, this hypothesis requires more research to verify. The increasing of GR with the supplementation of HMSeBA indicated that the HMSeBA might be beneficial with GCs to increase oocyte viability and the fertility efficiency. In addition, this study found that HMSeBA (10 ng/ml) increased SOD2 mRNA expression in vitro, HMSeBA (5 ng/ml) and Na2SeO3  increased mRNA expression of GPX1 in vitro and HMSeBA group increased mRNA expression of GPX1 in the ovary in vivo. These results were agreed with previous researches, which found that Se might affect cell proliferation by regulating the activity of antioxidant enzymes in testis  is exclusively localized in the mitochondrial spaces, which been demonstrated that high expression in rodent steroidogenic , 43. Oxio et al.  found thsa cells . GPX1 issa cells . Mammalssa cells , 46, whisa cells  to allow75Se, the Se incorporated into TXNRD has been reported to increase with increasing selenium concentration in cancer cell culture medium (2SeO3 significantly increases the activity of TXNRD at the protein level in the GCs in vitro and in the ovary in vivo. These results indicated that HMSeBA might have much more effects on the TXN/TXNRD system to help cells to defense against oxidative stress.Thioredoxin reductase (TXNRD) in mammalian is a member of the selenium-containing pyridine nucleotide-disulphide oxidoreductases family. TXNRD, Thioredoxin (TXN) and NADPH are the components of the TXN/TXNRD system, which have been considered key antioxidant system against oxidative stress , 49. Usie medium . The mRNe medium . The stue medium , 52. In e medium . Similar2SeO3 and HMSeBA both decreased the concentration of MDA thus beneficial to the redox balance in vitro in granulosa cells and in the liver in vivo in gilts. At same time, HMSeBA decreased the concentration of MAD in the serum and ovary in vivo in gilts. Maintenance of redox balance is critical for effective immunity and health, and biomarkers of oxidative stress are linked to cellular function  secretion and enhanced antioxidant capacities in vitro. Therefore, this study proved that HMSeBA promoted follicle development by antioxidant pathway from the in vivo and in vitro data. In future, long-term effect of HMSeBA supplementation on farrowing performance in gilt and exact mechanism of HMSeBA will be further explored.Dietary HMSeBA supplementation increased the selenium deposition, antioxidant capacities and promoted the development of COCs in gilts. HMSeBA promoted follicle development by stimulated GCs' proliferation, estradiol .MK was employed by the company Adisseo France SAS. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Adrenocortical carcinoma (ACC) is an uncommon endocrine malignancy associated with poor clinical outcome. As a novel form of cell death, ferroptosis is reliant on the accumulation of iron and reactive oxygen species and is involved in the pathogenesis of various tumors, including ACC. Our study aimed to identify and characterize the prognostic ferroptosis-related lncRNA signature (FerRLSig) in ACC.Methods: A regulatory network of ferroptosis-related lncRNAs (FerRLs) and mRNAs was constructed based on The Cancer Genome Atlas (TCGA). Univariate and multivariate Cox regression assays were performed to construct the FerRLSig.Results: Twenty-four FerRLs were identified in the prognostic model, and the high-risk FerRLSig was related to the worse overall survival (OS) in ACC . The area under the curve (AUC) value of the FerRLSig was 0.936 according to the receiver operating characteristic (ROC) analyses, superior to other traditional clinicopathological features, further supported the utility in prognosis prediction of ACC. We further established a prognostic nomogram combining clinical factors with the FerRLSig, which showed favorable efficacy for survival prediction. Next, gene set enrichment analysis (GSEA) revealed that gene sets were involved in many immune regulatory biological processes related to malignancies. T-cell function of type II INF response and the immune checkpoints, including CD40, CD276, IDO2, NRP1, and CD80, were expressed with a significant difference between the low- and high-risk groups.Conclusion: This study offered new insights into the pathogenesis of ACC. The novel FerRLSig could be useful in predicting survival and may provide information of immunological research and treatment for ACC patients. Adrenocortical carcinoma (ACC) is an aggressive endocrine malignancy with a rare incidence but high mortality . AlthougPast decades have seen a growing number of research studies to investigate the function of ferroptosis in malignancies. As a novel form of cell death, ferroptosis is reliant on the accumulation of intracellular iron and reactive oxygen species, distinguished from other forms of cell death such as apoptosis and autophagy . ImbalanAs a subset of RNA molecules, a long noncoding RNA (lncRNA) has the length of over 200 nt and is involved in gene regulation . In addihttps://portal.gdc.cancer.gov/). https://www.gencodegenes.org/). We obtained 382 ferroptosis-related genes  from the FerrDb database, which is a data network with comprehensive and timely updates, covering the latest progress of ferroptosis-related genes along with their regulatory molecules and related diseases  + (coefficient of lncRNA2 × lncRNA2 expression) + + (coefficient of lncRNAn × lncRNAn expression). Taking the median value as the cut-off point, the lncRNAs were divided into two groups: high-risk group and low-risk group. Differentially expressed genes (DEGs) between the low- and high-risk groups were identified and statistically significant was considered at a false discovery rate (FDR) < 0.05 and |log2FC|≥1. A Kaplan–Meier survival analysis was then performed to evaluate the clinical correlation of risk stratification and patient prognosis. The FerRLSig was present as a risk plot, including the distribution of risk scores and survival status in ACC patients. We further calculated the area under the curve (AUC) value in the receiver operating characteristic (ROC) curve to evaluate the prediction accuracy of OS in ACC patients. Sensitivity and specificity of the FerRLSig were compared with other clinical and pathological factors via ROC curves and decision curve analysis (DCA)  . CytoscaFunctions of DEGs were compared between the low-risk group and the high-risk group. We evaluated the associated biological functions using gene ontology (GO), including biological processes (BPs), molecular functions (MFs), and cellular components (CCs). Based on the data in the Kyoto Encyclopedia of Genes and Genomes (KEGG), we further analyzed the biological pathways using the ggplot2 package in R 4.1.2.p-value <0.05 (GSEA 4.2.2). Statistical significance was expressed by the normalized enrichment score (NES) and FDR  to define the enriched terms in the KEGG. Significant gene sets were achieved at  and FDR . A nomoghttp://timer.comp-genomics.org/)  . The difcs.org/) .p < 0.05.R software (version 4.1.2), along with its appropriate packages, was used to perform the statistical analysis. The Wilcoxon test and unpaired Student’s t-test were used to analyze the non-normally and normally distributed variables, respectively. Differentially expressed genes between the low- and high-risk groups were identified using the Benjamini–Hochberg method. The association between FerRLs and clinicopathological characteristics of ACC patients was assessed by logistic regression analysis, presented in a cluster heatmap. Statistical significance was achieved at According to the Pearson correlation analysis, a total of 85 FerRLs were identified from the 382 ferroptosis-related genes in TCGA-ACC dataset . SubsequThe regulatory network of FerRLs, mRNAs, and pathways is shown in n = 437 for the high-risk group, Overall, 540 genes were identified to be differentially expressed between the low- and high-risk groups (n = 103 for the low-risk group and p < 0.001). In addition, the AUC value for the FerRLSig was 0.936 according to the ROC curve, significantly higher than that for traditional clinicopathological factors such as gender, age, and tumor stage  of the risk scores in the univariate analysis and multivariate Cox regression analysis were 1.975 (1.527–2.533) and 1.936 (1.484–2.526), respectively (p < 0.001), p53 signaling pathway , small-cell lung cancer , pancreatic cancer , homologous recombination , pyrimidine metabolism , pathogenic Escherichia coli infection , TGF-beta signaling pathway , primary bile acid biosynthesis , and drug metabolism cytochrome P450  signaling pathways . Most of the identified FerRLs were involved in tumor-related and immunological pathways including cell cycle (NES = 2.07; pathways . Taken tACC is an uncommon endocrine malignancy associated with high mortality . Early dvia the PRR11/PI3K/AKT pathway, predicting a poor prognosis in hepatocellular carcinoma (via the miR-135b-5p/CSF1 axis, indicating poor survival (Overall, 24 FerRLs were identified in the prognostic FerRLSig. In review of the literature, cell types that predominantly express these FerRLs along with the potential mechanisms are presented in arcinoma . In a rearcinoma . In addiarcinoma . Upregularcinoma . LINC015arcinoma . Wu et aarcinoma . Knockdosurvival . On the survival . In cervsurvival . HoweverNext, the present study identified 540 ferroptosis-related DEGs and stratified them by the novel FerRLSig. KEGG-based analyses further revealed that the genes of the high-risk group mainly participated in the Hippo signaling pathway, p53 signaling pathway, cancer-related pathway, ECM–receptor interaction, cellular senescence, and cell cycle. Recently, Lin and colleagues revealed that recurrent breast tumors are highly sensitive to ferroptosis, and upregulated expression of DDR2 could promote ferroptosis through the Hippo signaling pathway . Wen-HsuAnother significant contribution of the current study is the uncovering of the association between the FerRLSig and tumor immune microenvironment. Increasing evidence suggests that T-cells could promote tumor ferroptosis to enhance antitumor activity, which might be a potential therapeutic target for cancers combining with an immune checkpoint blockade . FunctioAs a novel form of cell death, ferroptosis provides a new way for tumor development and treatment . HoweverIn conclusion, a FerRLSig was established to predict the survival of ACC. Additionally, this model might be related to immune infiltration, providing potential immune targets for the control of ACC.


The search for the “Holy Grail” in clinical diagnostic microbiology—a reliable, accurate, low-cost, real-time, easy-to-use method—has brought up several methods with the potential to meet these criteria. One is Raman spectroscopy, an optical, nondestructive method based on the inelastic scattering of monochromatic light. The current study focuses on the possible use of Raman spectroscopy for identifying microbes causing severe, often life-threatening bloodstream infections. We included 305 microbial strains of 28 species acting as causative agents of bloodstream infections. Raman spectroscopy identified the strains from grown colonies, with 2.8% and 7% incorrectly identified strains using the support vector machine algorithm based on centered and uncentred principal-component analyses, respectively. We combined Raman spectroscopy with optical tweezers to speed up the process and captured and analyzed microbes directly from spiked human serum. The pilot study suggests that it is possible to capture individual microbial cells from human serum and characterize them by Raman spectroscopy with notable differences among different species.IMPORTANCE Bloodstream infections are among the most common causes of hospitalizations and are often life-threatening. To establish an effective therapy for a patient, the timely identification of the causative agent and characterization of its antimicrobial susceptibility and resistance profiles are essential. Therefore, our multidisciplinary team of microbiologists and physicists presents a method that reliably, rapidly, and inexpensively identifies pathogens causing bloodstream infections—Raman spectroscopy. We believe that it might become a valuable diagnostic tool in the future. Combined with optical trapping, it offers a new approach where the microorganisms are individually trapped in a noncontact way by optical tweezers and investigated by Raman spectroscopy directly in a liquid sample. Together with the automatic processing of measured Raman spectra and comparison with a database of microorganisms, it makes the whole identification process almost real time. Even in the modern world, bloodstream infections are associated with high morbidity and mortality rates. Together with myocardial infarction, they are among the most common causes of hospitalizations. Patients with an increased risk of bloodstream infections include immunocompromised patients, patients with chronic infections, and elderly patients. Bloodstream infections frequently have a nosocomial origin: they are often associated with intravenous catheters. Another clinically significant and common type of bloodstream infection is infectious endocarditis, which occurs mainly in patients with preexisting heart disease, immunocompromised patients, or patients with artificial heart valves , 2.Staphylococcus aureus). However, the number of Gram-negative bacteria has been increasing, with Escherichia coli at the top. Also, other members of the family Enterobacteriaceae play a role. Other common causative agents of bloodstream infections include enterococci and yeasts of the genus Candida  (–Numerous bloodstream infections are caused by staphylococci (both coagulase-negative staphylococci and lbicans) –5. Due tbicans) –.Bloodstream infections often lead to sepsis, a life-threatening organ dysfunction caused by the deregulated response of a host organism to infection . Therefoin situ hybridization- or DNA microarray-based methods, the Accelerate Pheno system, and the Verigene system, which significantly shorten the time necessary for identification; however, they still require subcultivation. A breakthrough in this diagnostic process came with matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS), an effective, quick, and relatively inexpensive method based on protein fingerprinting of microbes  , 10. In microbes , 12. In microbes –18. Seveicrobes –, but eitRaman spectroscopy belongs to optical methods with multiple applications in clinical medicine, such as measurements of inflammatory markers, including C-reactive protein (CRP) , 21; mea23–27–39–42–This study presents the possibility of differentiating microbes causing bloodstream infections by Raman spectroscopy. Furthermore, it describes a pilot study aiming to identify microbes directly from human serum. It compares the results obtained by Raman spectroscopy to the results obtained by MALDI-TOF MS.We acquired Raman spectra from 305 strains belonging to 28 microbial species from colonies grown on Mueller-Hinton agar plates. Pseudomonas aeruginosa’s peaks associated with pyoverdine include vibrations at 715, 830, 1,355, 1,488, and 1,611 cm−1. In staphylococcal species, we can detect carotenoid pigments visible at wavenumbers of 1,110, 1,160, and 1,525 cm−1, corresponding to C-C-(CH3), =C-C=, and -C=C vibrations  and support vector machine (SVM). Although the two classification methods have different principles, both approaches lead to similar results, with an accuracy of 93.5 to 93.8% for both 1NN and SVM using both centered and uncentered principal-component analyses (PCAs). The accuracy values were obtained by 5-fold cross-validation applied to the testing data. The results of cross-validation may be presented in the form of a confusion matrix , where tSubsequently, using the “semaphore” , we independently evaluated our method’s performance by classifying 71 testing strains with five possible outcomes. The green and orange species were either correctly or incorrectly identified as the true class identification , with indistinguishable species being the fifth outcome. The results comparing the 1NN and SVM methods, which use features extracted by centered or uncentered PCAs, are summarized in Candida and Staphylococcus). The remaining incorrectly classified strain is the one strain of Klebsiella oxytoca, which was identified as Escherichia coli.Finally, Rows (true class) correspond to identifications by MALDI-TOF mass spectroscopy plus biochemical methods; columns (predicted class) correspond to identifications predicted by the SVM method. Numbers in the cells show correctly identified spectra on a green background  and incorrectly identified spectra on a red background . The colors of the numbers represent the confidence of the identification based on the semaphore scheme, with the indistinguishable samples (red semaphore) placed into the rightmost column. Moreover, gray rows depict the bacterial strains used in the training process, but no samples were present in the testing data set. Squares with a light-blue background depict bacterial species of the same genus.The cultivation step significantly slows the identification of pathogens in clinical diagnostic processes. Especially for life-threatening infections, the time to proper treatment is an essential component of treatment success. To shorten the time necessary for the identification of the causative agent, we performed a pilot study on spiked human sera using Raman tweezers, aiming to skip the cultivation step. The goal of this experiment was to test the feasibility of this application. In this case, optical tweezers attract microbes from the illuminated volume of the serum, catching them in an optical trap . Since the laser beam is tightly focused, microbial cells cover most of the volume from which Raman spectra are acquired. In this way, the signal of the surrounding serum is suppressed , 48. TheOur study shows that Raman spectroscopy can differentiate among pathogens causing bloodstream infections. We achieved approximately 94% correctly identified strains within the training set. Using the SVM classification method on the testing set and the “semaphore” approach, ~3% and 7% of the strains were incorrectly classified, with 86% and 88.7% correctly identified strains for centered and uncentered PCA feature extractions, respectively. These results closely correspond to those of previous studies using Raman spectroscopy to identify microbes , 38, 48.Proteus vulgaris, the spectra of which were repeatedly classified as Escherichia coli, suggesting a high level of similarity in their Raman profiles. Unsatisfactory results were also obtained for Candida tropicalis (identified as other Candida species in some instances), Enterococcus faecium , Staphylococcus warneri and Staphylococcus lugdunensis , and Streptococcus oralis . This led to a relatively high false discovery rate for Enterococcus faecalis (29%). Although these isolated problems occurred in the training set, all strains belonging to the species mentioned above from the testing set were identified correctly.When training the artificial intelligence (AI) classifier, the most problematic species was P. aeruginosa, Micrococcus luteus, S. aureus, and S. lugdunensis, the problems occurred with only one strain of P. aeruginosa and several individual spectra of S. lugdunensis. The set of S. aureus strains included 13 nonpigmented strains, 9 slightly pigmented strains, and 38 pigmented strains. Although the peaks associated with carotenoids are very intense, the machine learning algorithms did not consider them as the base for identifying S. aureus, so we can presume that this variability is suppressed by the rest of the characteristic spectral features.For the species with considerable observed variance in pigment production reflected in Raman spectra, namely, 2 slides and aluminum, etc.). Sample preparation for Raman tweezers depends on the sample; in the case of complex samples, preprocessing steps might be necessary. However, when the object of interest is in a liquid medium without other particulate matter or the particular matter is well defined, the objects can be directly captured and analyzed. Based on the principle of Raman tweezers, objects in the liquid medium are attracted by Raman tweezers and captured and can be subsequently analyzed. This allows the analysis of microbes in very low concentrations: the analysis is performed with one to four cells captured at once, so when the concentration is low, it takes more time to capture the object from the sample. The capture of an object is visible by the change in the laser beam appearance.We can conclude that despite some problematic strains, Raman spectroscopy offers an elegant, rapid approach to identifying microbes. It is noninvasive and nondestructive. Sample preparation is easy and does not require expensive consumables. When analyzing microbial colonies, it is sufficient to use a grown culture directly on a suitable culture medium . Alternatively, the colonies/microbial pellets after centrifugation and similar samples can be transferred to any Raman-inactive substrate or Raman-grade substrate with a defined low-interference signal  and acquires their Raman spectra —it might, after repeated measurements, give some indication of a possible mixed infection . Moreover, recent studies by Bernatová et al. , Pilát eAltogether, this novel approach could significantly accelerate the identification of pathogens and their antimicrobial susceptibility/resistance profiles, leading to the timely and accurate treatment of a patient. Upon this pilot testing, we plan to perform a more extensive study to include more strains/species as well as more microbial species in the testing of the method.Identifying and characterizing pathogens from tiny sample volumes rapidly and reliably are the crucial first steps in the diagnosis of microbial infections. Thus, we can envision the following wishful scenario in clinical practice. A sick person enters a hospital emergency ward with what appears to be an infection. Next, the doctor on duty collects a specimen  from them. Consequently, in an ideal case, the doctor could quickly and simply analyze the sample in the examination room. This analysis should give the doctor enough information for quantitative and conclusive pathogen identification. In this scenario, a point-of-care (POC) instrument that quickly identifies the pathogens in minutes is required. Thus, the clinician could prescribe tailored antibiotics, which improves the prognosis for a patient, shortens the time to proper treatment, and decreases problems with ever-increasing drug resistance. Since Raman spectroscopy and Raman tweezers have many possible applications in clinical medicine (as described above), they can provide a clinician with complementary information like the CRP level, the presence of inflammatory markers, and/or the presence of other conditions, including cancer. It could make the device suitable for complex clinical diagnostics.Currently, the main limitations of Raman spectroscopy include the relatively high input costs necessary for purchasing the highly optimized instrumentation and the need for staff specifically trained in the acquisition of Raman spectra. Since microbial samples are heterogeneous, and variability can be expected, manual measurements are necessary at the current stage. They involve finding the object of interest , focusing on the sample, and analysis (preferably more measurements/strain). The training necessary for personnel should include mostly the finding and focusing phases, as they are essential for the outcome. The analysis can be performed with the same (or very similar) settings for most of the microbial samples of the same type , with some basic adjustments for specific situations. Therefore, we believe that the necessary training would not be more complicated than the training required for MALDI-TOF MS analyses.Specially engineered portable systems could overcome these limitations of POC diagnostics in the future. Also, the absence of a large-scale database of microbial Raman fingerprints poses a problem for potential future clinical use. This is partially covered by our current (305 strains/28 species) and previous , 28, 48 34–64–Beyond the limitations mentioned above, Raman spectroscopy has numerous advantages, proving its high potential to become a clinical diagnostic tool. It is noninvasive and nondestructive, so the samples (cells) can be used for further/complementary testing. It has a broad spectrum of applications across scientific fields to be used for numerous purposes in clinical diagnostics. It does not require any expensive consumables. Sample preparation is easy and quick. Therefore, we believe that Raman spectroscopy could become a valuable diagnostic tool in the future, significantly improving the management of infections and helping to reduce the speed of the ever-growing propagation of antimicrobial resistance. This would save the lives of patients as well as save costs for the management of infectious diseases.The current study on 305 microbial strains belonging to 28 microbial species shows that Raman spectroscopy can reliably differentiate among microbes causing bloodstream infections, with only 2.8 to 7% incorrectly identified strains depending on the approach used. Moreover, the pilot study based on Raman tweezers suggests that it is possible to locate microbes directly in human serum; their Raman fingerprints are distinguishable among each other and the serum itself. Therefore, we believe that Raman spectroscopy and Raman-based techniques (Raman tweezers) could significantly contribute to the development of rapid approaches for the diagnosis and management of infectious diseases.(i) Sample preparation. We measured 3,127 Raman spectra originating from 305 microbial strains of 28 species of 14 genera  (Czech Republic) and 1 reference strain from the National Collection of Yeast Cultures (NCYC) (United Kingdom) . Three SThe strains were identified by biochemical methods and MALDI-TOF MS using the extended direct transfer method according to the manufacturer’s instructions  or blood agar with 5% sheep erythrocytes (streptococci), which do not have a significant influence on Raman microbial fingerprints .(ii) Experimental setup. We acquired Raman spectra from colonies grown on Mueller-Hinton agar plates after 24 h of cultivation at 37°C using a commercial Raman spectrometer  equipped with a Leica N Plan EPI microscope objective . Spectral acquisition was performed using a 785-nm single-mode diode laser. The spectra were acquired in the range of 614 to 1,724 cm−1  for 10 s.We conducted at least 10 measurements of each microbial strain from 3 different colonies at a minimum, aiming the laser beam on the colonial surface. Before each spectral acquisition, the laser was refocused onto a colony surface, ensuring that the collected signal originated within the focal depth of the laser excitation and imaging optics. Considering the above-described geometry ensuring the collection of the Raman signal over an axial range of about 8 μm, we can neglect the contribution of the cultivation medium to the Raman spectra .Measurements and sample preparations were performed in the same way on all measurement days. The high reproducibility of microbial Raman fingerprints acquired in this way was validated in our previous work .(iii) Data analysis. We processed all acquired spectra using custom-written routines implemented in MATLAB software  (Acinetobacter ursingii and Citrobacter koseri), all spectra were assigned to the training data set. MA, USA) , 28, 48.−1) . The high fluorescence background present in microbial spectra was removed by a rolling-circle filter (RCF)  . The speFor the extraction of the main spectral features, an aid for the identification of microbial strains, we employed centered and uncentered principal-component analysis (PCA) variants. The extracted features (PCA scores) were used as the inputs for the one-nearest-neighbor (1NN) and support vector machine (SVM) classification methods. The 1NN method is based on identifying the closest data point of the known class , and theHowever, for testing the classification performance, we used the testing data set consisting of 741 spectra belonging to 71 strains that were not used for training our classifier. As each sample consists of multiple spectra, the classifiers are trained to provide results based on one spectrum only; we introduce a “semaphore” scheme to evaluate the testing results. The “semaphore-like” colors green, orange, and red represent the confidences of species classifications of high-confidence-level, low-confidence-level, and indistinguishable species. For this, we introduce the following set of rules: (i) at least 50% of the spectra must be classified as a single species, or the sample is indistinguishable; (ii) if the number of spectra belonging to the most probable species is at least three times the number of spectra of the second most probable species, the result is obtained with a high confidence level, i.e., green; (iii) if the number of spectra belonging to the most probable species is at least two times the number of spectra of the second most probable species, the result is obtained with a lower confidence level, i.e., orange; and (iv) otherwise, the sample is indistinguishable (red).For example, let us assume that we take 10 spectra to classify one sample. We will obtain results with a high confidence level if 8 to 10 spectra belong to one species but also if the most populous species contains 6 or 7 spectra, assuming that the second most populous species has just 2 spectra, and also if 5 spectra are classified as a single species while the remaining 5 spectra are split, each of which gives a different species. On the other hand, we will obtain a low level of confidence of the classification if spectra are divided into groups according to 7-3, 6-3-1, and 5-2-2-1. The whole process is summarized in Fig. S1 in the supplemental material.(i) Sample preparation. We used pooled anonymized serum samples from the Department of Microbiology, St. Anne’s University Hospital, Brno, Czech Republic. First, aliquots of the serum samples (1 mL) were spiked with S. aureus CCM 3953, Staphylococcus epidermidis CCM 4418, E. coli CCM 3954, and Candida albicans CCM 8261  and mixed thoroughly for at least 1 min using a vortex mixer. Ten microliters of the suspension was transferred to a glass chamber (one coverslip separated by a 120-μm spacer from a 200-μm thin CaF2 slide). This setup allowed us to obtain approximately 50 to 70 cells within the microscope field of view (approximately 100 μm by 100 μm).(ii) Experimental setup. Freely moving microbial cells in spiked human serum were trapped by using a custom-built experimental Raman tweezers system: a combination of a Raman microspectrometer and optical tweezers using the same laser for trapping and Raman scattering . This experimental setup achieves a full lateral width of the excitation region of ~0.8 μm. Therefore, only a few cells can be trapped and analyzed simultaneously (one to four cells), and the background (serum) signal is almost suppressed. Trapped cells were exposed to the laser beam only during spectral acquisition (50 s), and the laser was then blocked. Afterward, different cells were trapped in a new location within the sample, and we acquired their Raman spectra. Further details on the experimental setup and the procedure can be found in recent articles by Bernatová et al.  Data analysis. In the case of Raman tweezers experiments, the strong Raman signal of the medium (human serum) was suppressed according to the essential principle of optical tweezers: confining microbial cells in a focal volume of a laser beam, leading to the filling of almost the whole Raman scattering area by microbial cells. The acquired Raman spectra were processed using the Savitzky-Golay filter  and an advanced rolling-filter background removal routine . The data were further analyzed using custom-written routines in MATLAB software  using an uncentred PCA. PCA was also used to present results from Raman tweezers-based experiments. Ellipses around principal components were plotted using a Mahalanobis distance of 2.15 .https://osf.io/6t5r4/?view_only=3ec09e05ff5145639e1079ee772450a5 .Raw data are available at


Tinospora cordifolia (Guduchi/Giloy) is a relatively common herbal supplement whose use has recently become prominent in Southeast Asia. It was promoted to the public in India as an immunity booster, especially against the novel COVID-19. There have been reports, mostly from India, of an association between Guduchi/Giloy and liver injury. We present a 50-year-old female with a history of Hashimoto thyroiditis, who presented with abdominal discomfort and nausea of two weeks duration, which coincided with starting HistaEzeTM supplement containing Tinospora cordifolia. The vital signs upon presentation showed no significant abnormalities. Labs were significant for severely elevated transaminases; however, viral panels, autoimmune serologies, and imaging studies were unremarkable. Roussel Uclaf causality assessment method (RUCAM) score was at 6, which was indicative of probable drug/herb-induced liver injury. HistaEzeTM was discontinued, and the patient took a three-day course of oral steroids with significant interval improvement in clinical status, as evidenced by progressive normalization of the transaminases level. The transaminases decreased by greater than 50% within two weeks of discontinuation and trended back to baseline within three months. This case highlights the worldwide availability and use of Tinospora cordifolia, which can cause liver injury that appears to be idiosyncratic and possibly immune-mediated. Further research on the precise mechanism of its hepatotoxicity is warranted. Tinospora cordifolia, commonly described as heart-leaved moonseed , is an Ayurvedic herbal supplement used as an immune booster mostly in India, especially during the COVID-19 pandemic [Tinospora cordifolia-induced liver injury in the United States.Dietary and herbal supplements are known to be significant contributors to acute liver injury in the United States, accounting for about 15.5% of drug-induced liver injury (DILI), and are also a major cause of fulminant hepatitis in the Western world . It’s knpandemic . Here, wTM, which contains 900 mg of Tinospora cordifolia extract (stem)/Giloy supplement for immune support against COVID-19 infection. Other than the reported symptoms above, the patient denied weight changes, regurgitation, heartburn, jaundice, skin rash, pruritus, change in stool or urine color, recent travel, exposure to blood or body fluids, needle stick injuries, or change in bowel habits. The patient reported a family history of autoimmune diseases; however, she was unsure of the specific type. She denied a history of smoking or alcohol intake and had no recreational drug use. Active medications at presentation include levothyroxine sodium, progesterone, estradiol patch, and HistaEzeTM supplement. A 50-year-old female with a past medical history of Hashimoto thyroiditis on levothyroxine replacement therapy presented with mild abdominal discomfort of two weeks duration. Abdominal pain was constant, graded 3-4/10 in severity, located in the right upper quadrant and epigastrium region, non-radiating, and had no known aggravating or relieving factors. The pain was associated with generalized fatigue, loss of appetite, nausea, and rare vomiting. Four to five weeks prior to presentation, the patient started taking 1 tablet of HistaEzeOn presentation to the gastrointestinal/liver clinic, her vitals were unremarkable. Physical examination noted mild discomfort to palpation in the right upper quadrant without rebound tenderness or guarding. Labs were notable for elevated alanine transaminase (ALT) 1336 IU/L and aspartate aminotransferase (AST) 375 IU/L . Alkaline phosphatase (ALP) was 57 IU/L (reference range: 45-117 IU/L), which was normal. Likewise, albumin, bilirubin, thyroid function tests, and the coagulation panel were all within normal limits. The hepatotropic and non-hepatotropic viral panels, autoimmune serologies, and imaging were also unremarkable. The Roussel Uclaf causality assessment model (RUCAM) score was 6, indicating probable drug-induced liver injury. TM, and counseled on liberal oral fluid intake. The patient received a three-day course of prednisone 40 mg daily during the first week of the presentation, with interval improvement in symptoms noted. Table The patient was managed as an outpatient in the gastroenterology clinic, advised to stop taking HistaEzeHerbal supplements, although used quite frequently for improving health, have been associated with damage to the body, especially the liver. However, many patients do not fully volunteer this information as they feel it is harmless and can be misdiagnosed as acute viral hepatitis, autoimmune hepatitis, or alcoholic liver disease . Its effTM contains quercetin 600 mg, stinging nettle (Urtica dioica) 600 mg, and sodium bicarbonate in addition to Tinospora cordifolia extract (stem). There are no case reports of liver injury from quercetin or Urtica dioica which have both been widely used all over the world for many years. Hence, we suspect that the liver injury is from Tinospora cordifolia extract [HistaEze extract . Tinospora cordifolia-induced liver injury remains unclear, its mechanism of toxicity appears to be an idiosyncratic liver injury, or immunomodulatory mechanism, or the unveiling of subtle autoimmune hepatitis. Autoimmunity has been thought to play a role as liver histology shows some similarities with atypical autoimmune features like hepatic plasma-lymphocytic infiltrations but without explicit features of autoimmune hepatitis and negative anti-smooth antibodies [Although the precise pathophysiology of tibodies . Hence, Tinospora cordifolia to symptom onset is about 46 days [Herb-induced liver injury shares similarities with DILI regarding manifestations, which may depend on frequency, dosage, duration, and associated risk factors like autoimmunity and chronic liver disease. The median time from intake of  46 days . The pat 46 days ,7. The s 46 days . Before diagnosing HILI, it is imperative to thoroughly rule out other causes of liver injury by obtaining a liver and coagulation panel, viral hepatitis panel, autoimmune serologies, and imaging. The RUCAM score, which is used as a grading system for clinical diagnosis of DILI, could be utilized for HILI as well: <1 excluded; 1-3 unlikely; 3-5 possible; 6-8 probable, and >8 highly probable . A liverTreatment entails discontinuing the offending agent and monitoring liver enzymes weekly or biweekly for a gradual decline in transaminase levels. Steroid therapy may be considered as there are some features of drug-induced autoimmune hepatitis with this herbal supplement and reports of faster improvement of liver labs and spontaneous resolution . The proTinospora cordifolia-induced liver injury, whose use has become prevalent since the COVID-19 pandemic. A high level of suspicion is needed due to its close similarities to other forms of liver injury. Early identification and discontinuation of the offending medication provide critical diagnostic and prognostic value. Further studies are needed to define the prognosis of patients with underlying liver disease and co-morbidities. There is also a critical need to raise public awareness of the dangers of herbal supplements, which are not well-regulated, unlike allopathic medications.This case highlights a case of


Atorvastatin is a commonly used statin for the treatment of hypercholesterolemia in people at high risk for coronary, cerebrovascular, and peripheral artery disease. However, fatal liver failure due to atorvastatin treatment has been rarely reported, especially during the very short incubation period.A 63-year-old male patient was admitted due to unexplained chest pain. After admission, his liver function had decreased < 24 hours after taking 20 mg tablets of atorvastatin due to lacunar infarction, which was improved after drug withdrawal. The treatment regimen was restarted 15 days later, but within 16 hours, the patient developed multiple organ failure, including liver failure and renal failure.The pathological results after 7 days indicated focal inflammatory necrosis, virus and autoimmune correlation tests were negative, which did not rule out drug-induced liver injury. Interventions: receiving artificial liver therapy, continuous renal replacement therapy and other organ support treatment.The patient died 18 days after admission.Statin idiosyncratic liver injury is very rare, but the consequences can be serious. A large proportion of commonly used medications, in addition to herbal and dietary supplements, can cause liver injury. The most important drug groups are antibiotics, antituberculous medications, nonsteroidal anti-inflammatory drugs and immunosuppressants. Because many elderly and frail patients have basic cardiovascular and cerebrovascular diseases, severe infection is more common and more often complicated by other conditions requiring medication treatment. Once a drug-induced liver injury occurs, it is sometimes difficult to determine which drug is responsible for the injury. Statins are commonly used to treat hyperlipidemia, coronary artery disease and other atherosclerotic diseases because of their ability to lower cholesterol. Side effects limit the wide application of these drugs to a certain extent. Common side effects include neurological and neurocognitive effects, hepatotoxicity, renal toxicity, gastrointestinal, urogenital, reproductive, etc. A DILI is a relatively important side effect, with an incidence of approximately3%. A statin-induced liver injury is generally dose-and time-dependent and can be relieved after drug withdrawal, thus the probability of chronic hepatitis is very low. Although an idiosyncratic liver injury has a 75% risk of death or need for liver transplantation, itis not generally dose-dependent. Therefore, clinicians should be aware of and suspect fatal liver failure induced by atorvastatin.A drug-induced liver injury (DILI) is an important but rare adverse event that can lead tomild liver enzyme elevations, liver failure, the need for transplantation or even death.A 63-year-old male farmer was admitted for “chest pain for 8 hours”. More than 2 years ago, the patient underwent thoracolaparoscopic radical resection of an esophageal carcinoma under general anesthesia, was treated with 2 courses of gimeracil and oteracil potassium capsules and followed up regularly. Eight hours before admission, the patient reported he experienced chest pain without obvious inducement, which was aggravated with respiratory movement and relieved after rest. No chest distress, shortness of breath, nausea, cough, expectoration, abdominal pain, or abdominal distension was reported nor observed at admission. Physical examination showed the following: body temperature, 38.0°C; pulse, 101 beats/minutes; respiration rate, 18 breaths/minutes; blood pressure,123/67 mm Hg; NRS score for left shoulder and back pain, 2 points; stable breathing; no icteric skin or sclera or bilateral supraclavicular lymph node enlargement; old surgical scars on the neck and chest, clear breath sounds in both lungs, with no obvious rales; flat and soft abdomen, no tenderness; and the liver and spleen were not palpable under the ribs. Chest CT showed the following: Postoperative changes in the esophagus; Arcuate gas shadow in front of the heart (suspected pneumomediastinum); A small amount of pericardial effusion, and; A patchy shadow in the anterior segment of the right upper lobe , which has been rarely reported, with this being the second report to my knowledge. Early Li et al reported a case of mild-to-moderately impaired liver function 12 hours after the application of fluvastatin drugs, and the prognosis was good after drug withdrawal. The mechanism of statin-induced liver injury is unknown, and the possible cause may be autoimmune hepatitis induced by this class of drugs, but we examined autoimmune-related parameters and found no abnormalities. Thus, hepatic impairment due to this cause was tentatively excluded. It has also been suggested that a statin liver injury is associated with oxidative stress. However, because we lacked previous knowledge of this aspect, no relevant examinations were performed. A liver injury occurs very early in the course of treatment, is apparently not dose-dependent and is individual specific, similar to amoxicillin and clavulanate potassium. Therefore, a clinically significant statin-induced liver injury may have idiosyncratic and immune allergic mechanisms. The specific mechanism of statin-induced liver failure in this patient is still unclear. It is likely that individual susceptibility to statins, genetics and polymorphisms play an important role in the hepatotoxicity of statins.A liver injury occurs very early in the course of treatment, is apparently not dose-dependent and is individual specific. Therefore, although statins are considered safe medications, it is still necessary to be aware of a potentially fatal liver injury based on this case and previous literature reports, especially for patients with acute liver disease, decompensated liver disease, or a history of suspected drug liver damage, for whom the use of these drugs should be avoided as much as possible. There is currently a lack of evidence supporting the reinitiation of a statin regimen or a reduced dosage regimen, and some data even suggest that it is relatively safer to replace statins with other drugs than to continue a statin regimen, but this hypothesis requires validation through more observational studies. However, due to insufficient attention in the early stage of treatment, a statin-induced liver injury was once mistaken for an infection-related liver injury, so the severity of a heterogeneous liver injury caused by atorvastatin was not fully recognized. In this patient, the later stage of liver failure progressed rapidly, with abnormal coagulation function and unstable vital signs, and liver transplantation was unfortunately not performed.As the proportion of aging adults within the general population increases, the incidence of cardiovascular and cerebrovascular diseases is increasing yearly. Statins have been confirmed to significantly reduce serum cholesterol and triglyceride levels, reduce lipids in atheromatous plaques, reduce inflammation and improve endothelial function to effectively reduce the incidence and mortality of cardiovascular and cerebrovascular diseases, despite unknown side effects. They are considered to be relatively safe drugs and have become one of the most prescribed drugs in developed countries.In conclusion, statin idiosyncratic liver injury is very rare, but the consequences can be serious.We thank the staff and patients for their contributions and participation in the study.Formal analysis: Ye Fu.Investigation: Shiyi Liu, Chenjie Zhou.Methodology: Shiyi Liu.Writing – review & editing: Huajun Wang.