Candida albicans. This study aimed to determine how much the resistance profile of non-biofilm microorganisms can change.Clinical strains of microorganisms, including pathogenic yeast-like fungi (YLF), are resistant to currently used antifungal agents. Thus, it is relevant to study the combinations of existing antimicrobial drugs and a medicinal extract of plant origin (farnesol). In previous studies, farnesol showed a relatively strong anti-biofilm effect against C. albicans and one reference strain were used to study the interaction of farnesol with the most used antimycotics. To determine the sensitivity of YLF to antimycotic drugs, such as nystatin (50 μg), amphotericin B (10 μg), ketoconazole (10 μg), clotrimazole (10 μg), voriconazole (10 μg), fluconazole (25 μg), miconazole (10 μg), and intraconazole (10 μg), the classic disk diffusion method was used. In the second stage, one of the six strains was used to simulate candidiasis of the gastrointestinal tract in an in vivo quail model. As an unusual experimental design, this study investigated the effects of pretreated C. albicans in quails, not the in vivo pathogenicity of C. albicans, after treatment with farnesol.Six clinical isolates of Candida strains did not improve with farnesol in all strains. All concentrations of farnesol  demonstrated a fungistatic effect  in 23 of 56 (7×8) cases (41%). The remaining 54% demonstrated no changes in the resistance to antifungal drugs or deterioration of the indicators in rare cases (5%). At 100 μM farnesol, sensitivity improved in 33 of 56 cases (59%). Candidiasis or the severity of clinical disease of the quail digestive tract developed to a lesser extent if fungi were treated with farnesol.The resistance profiles of in vivo model with biofilms, farnesol can be considered a new antimycotic drug or an additive to existing antimycotics.Farnesol does not always show a positive result on single cells without biofilm in the laboratory. However, in a biofilm or an  Candida albicans is a conditionally pathogenic yeast-like fungus (YLF) that threatens a human’s weakened immune system [C. albicans, and the main mechanism was probably associated with the suppression of hyphal development [e system ,2. Becauelopment -5.In biological communities, microorganisms use various mechanisms for their communication. Depending on the cell density, bacteria and YLF can produce quorum sensing (QS) signaling molecules  involved in regulating gene expression and coordinating collective behavior in their natural niche. These secondary metabolites play a major role in the adhesion and colonization of host tissues and surfaces, morphogenesis, and biofilm development ,7. In yeFarnesol is continuously produced in biofilms at temperatures from 23°C to 43°C and in quantities approximately proportional to colony-forming units per milliliter. Chemically, farnesol is acyclic sesquiterpene alcohol endogenously synthesized by the ergosterol pathway. Its synthesis depends on neither the type of carbon or nitrogen source nor the chemical nature of the nutrient medium, and it is a thermally stable molecule not exposed to extreme pH values .et al. [Escherichia coli. They found that phosphatidate phosphatase (PgpB) and undecaprenyl diphosphatase (YbjG), two integral membrane phosphatases of E. coli, can hydrolyze farnesyl diphosphate into farnesol [Farnesol is obtained in natural and synthetic ways. Farnesol can be isolated from linden-colored essential oils, musk grains, neroli, and pettigrain. In yeast, farnesol is a by-product of the ergosterol biosynthesis pathway formed due to the enzymatic dephosphorylation of farnesyl pyrophosphate. The enzymes of this pathway are encoded by ergosterol genes. In bacteria, phytoene/squalene synthase (YisP) acts as a phosphatase, catalyzing the formation of farnesyl diphosphate. Wang et al.  describefarnesol . Large-sCandida species. In these cases, studying whether farnesol affects changes in the sensitivity of clinical strains of Candida to antifungal drugs is especially interesting.The question is why study the QS farnesol molecule? The list of antifungal agents is huge, and new drugs appear every year. However, the sensitivity of microorganisms to new drugs is weak, variable, and unequal. In addition, tests for sensitivity to antimycotics, unfortunately, are not considered a routine procedure, not always available, and usually not considered a standard technique in patient management. As practice shows, resistance studies are prescribed by doctors only in cases of deep mycoses, mainly caused by non-albicans C. albicans pathogenicity in quail model experiments.This study aimed to prove that farnesol can increase the antifungal activity of some antimycotics and can reduce The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Peoples’ Friendship University of Russia (RUDN University) ethical committee ..The study was conducted from May to July 2021. The samples were processed at the Department of Microbiology and Virology, Medical Institute, Peoples’ Friendship University of Russia, Moscow, RussiaC. albicans isolates (C1-C6) were isolated from women with vulvovaginal candidiasis. The identification of microorganisms was carried out using the matrix-activated laser desorption/ionization technology “Bruker Daltonik MALDI Biotyper” . A score of >2000 was considered reliable. The reference strain (C7) was C. albicans ATCC 10231. Colonies of diurnal cultures of C. albicans from Saburo agar  were washed off with physiological solution . The YLF concentration was 0.5 according to McFarland, corresponding to 1.5×108 cells/mL [Clinical strains of six cells/mL -16.Farnesol (trans-farnesol) was purchased from Sigma-Aldrich (Germany): Molar mass 222.37 g/mol, mass of the substance 0.886 g/mL, Ns=The amount of the substance in moles = 0.886:222.37=0.004 M/mL or 4,000 μM/mL. Three concentrations of farnesol  were used, and PhS (pH 7.0) was used as the control.To determine the sensitivity of YLF to antimycotic drugs , nystatin , amphotericin B , ketoconazole , clotrimazole , voriconazole , fluconazole , miconazole , and intraconazole , the classic disk diffusion method was used.(a) 25 μL PhS+farnesol were applied to sterile paper disks, corresponding to a farnesol concentration of 100 μM; (b) 25 μL PhS was applied to sterile paper disks; (c) 25 μL PhS was applied to the disk with the antimycotic; (d) 25 μL farnesol (100 μM) was applied to the disk with an antimycotic; (e) 25 μL farnesol (50 μM) was applied to the disk with an antimycotic; and (f) 25 μL farnesol (25 μM) was applied to the disk with an antimycotic.The diameter of the growth suppression zone of the culture to the antimycotic was measured in accordance with the NCCLS M-44 protocol.ad libitum and were manipulated in accordance with the Local Ethics Committee for Animal Experimentation, Peoples’ Friendship University of Russia, Moscow, Russia .Female Texas white broiler quails , 21 days old, 30 heads, body weight 350-370 g, were used. Birds passed veterinary control and had all documentation. Quails were quarantined for 7 days before the experiment under the supervision of a veterinarian. All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals . Quails C. albicans to cause infection, the daily culture of YLF was washed 3 times with PhS, and the YLF concentration was 0.5 according to McFarland. Then, 25 μL of 100 μM farnesol was added to 1 mL microbial suspension (experiment), and 25 μL PhS (control) was added to another test tube. The tubes were incubated for 1 h at 37°C under constant shaking. After interaction with farnesol, YLF was washed three times with PhS [C. albicans+farnesol, and the control group was infected with C. albicans without farnesol.To identify the effects of farnesol on the ability of with PhS ,19. The C. albicans (1 mL) was given to quails through a digestive probe once daily for 5 days. The birds were killed every 5 days for five heads. The experiment lasted 20 days: 5 days for infection and 15 days for the course of the experiment. All animals injected with C. albicans were examined histopathologically [Statistical Package for the Social Sciences 20.0  was used for statistical analyses, and a significance level of 5% was adopted. Experiments with antimycotic disks were run with three replicates per condition. Data in the table on sensitivity to antimycotics are given without ± for better table perception, and ± did not exceed 0.4.in vitro. Especially, the clinical strain C1 showed resistance to most drugs. In the other six strains, the resistance levels differed among the groups of drugs and varied within the groups. The synergistic effect of farnesol with polyene antimycotics was as follows: farnesol (100 and 50 μM) increased sensitivity to NS in six strains, and farnesol 25 (μM) increased sensitivity to NS in four strains; farnesol (100 μM) increased sensitivity to AP (50 μM) in six strains, farnesol (50 μM) increased sensitivity to AP in four strains; and farnesol (100 μM) increased sensitivity to AP (25 μM) in six strains  were effective. The resistance of one clinical isolate was quite high, and farnesol even slightly worsened the indicators. Only in one strain of YLF, sensitivity to FU increased with farnesol at all three concentrations. Another culture increased sensitivity with farnesol (100 and 50 mM). In other cases, farnesol did not affect the sensitivity to FU. Sensitivity to MIC increased in three YLF cultures with farnesol . The growth retardation zone with IT in three of seven cases was higher than the control with the addition of farnesol at all three concentrations .th day after infection, curd overlays were more distinct, with rounded foci. On the 15th day before the slaughter, clinical signs of the disease were observed in the form of depression, drowsiness, and poor appetite. One of the five quails remaining died. On opening, the mucous membrane of the goiter was bumpier due to the different intensities of the overlays, which in sections took the form of a solid yellow-white film, and the serous membrane became folded. Delicate, loose, and yellow-white overlays were noted on the mucous membrane of the oral cavity and tongue, in which multiple budding and pseudomycelial forms of Candida were detected. On the 15th day, the remaining four quails killed presented with typical, well-marked candidiasis lesions in the anterior part of the digestive tract. In the histological sections, multiple filaments of the fungus were found growing perpendicular to the thickness of the mucous membrane.In the control group of birds killed within the first 5 days after infection, swelling, weak hyperemia of the goiter mucosa, thick viscous mucus and a delicate whitish plaque containing an abundance of budding cells, and pseudomycelia were observed. In the histological sections of the goiter, oral cavity, and esophagus, the start of fungal growth into the epithelial cover of the mucous membrane was detected. On the 10th day after infection on the mucous membranes of the digestive tract, a delicate whitish plaque containing a small number of budding cells was observed. On the 15th day before slaughter, clinical signs of the disease in the form of depression, drowsiness, and poor appetite were not observed. Not a single quail died. On autopsy, the mucous membrane of the goiter was covered with a delicate white biofilm, in which mainly C. albicans budding cells were found.In the experimental group of quails, where YLF was treated with farnesol, hyperemia of the goiter mucosa was not noticed in the first 5 days after infection. In the histological sections of the goiter, oral cavity, and esophagus, the start of fungal growth into the epithelial cover of the mucous membrane was detected but to a lesser extent than in control. On the 10C. albicans whether in the form of a biofilm or individual cells not connected by a matrix. In the previous experiments, where Candida was in the form of a biofilm, farnesol worked perfectly. In living models, microorganisms also form biofilms. Therefore, farnesol reduces the pathogenicity of Candida, and the infection does not develop so rapidly and strongly. Thus, this experiment showed that C. albicans treated with farnesol becomes less pathogenic for animals. YLF are less able to form a biofilm under the influence of farnesol.This study demonstrated a significant difference in Candida spp. is a justification for the preliminary determination of their sensitivity to antifungal drugs and continuous mycological monitoring of the spread of these strains. Identifying such resistant strains significantly limits chemotherapy possibilities for candidiasis [The increasing dominance of polyresistant strains of didiasis ,6,21. TrCandida, including filamentation, biofilm formation, drug susceptibility, and apoptosis [C. albicans, Candida dubliniensis, Candida tropicalis, Candida parapsilosis, Candida guilliermondii, Candida kefyr, Candida krusei, and Candida glabrata. Whether farnesol can affect the resistance of the strain to modern drugs remains an open question. Because this process of stability or sensitivity will always be dynamic for different isolates and change over time, it will always be relevant.As a QS molecule, farnesol participates in regulating various physiological processes in poptosis ,4,16,22.poptosis . The secCandida biofilm and the concentration used. However, the effect of farnesol on its synergy or antagonism with known antifungal drugs has been studied to a lesser extent. This study showed that farnesol certainly affects the degree of YLF resistance, indicating its potential role as an antimicrobial agent [Candida strains are not improved by farnesol in all strains. All concentrations of farnesol  demonstrated a fungistatic effect  in 23 of 56 (7×8) cases (41%). The remaining 54% demonstrated no changes in the resistance to antifungal drugs or deterioration of the indicators in rare cases (5%). At 100 μM farnesol, sensitivity improved in 33 of 56 cases (59%).Farnesol and its derivatives exhibit antibiofilm, anticancer, antitumor, and fungicidal properties ,18,24. Tal agent ,4,25,26.in vivo model. Candidiasis of the digestive tract developed to a lesser extent if fungi were treated with farnesol. In combination with increased sensitivity to existing drugs, the effect of reducing the pathogenicity and reducing biofilm formation is a good result. We believe that the effect of farnesol on the sensitivity of C. albicans to antifungal drugs is successful. Our plans are to check the change in sensitivity to other drugs for this purpose. Of course, this experience will be useful for a deeper understanding of the potential antifungal mechanism of QS molecules and a continuing search for new drugs against Candida and other YLF.In the previous studies ,4, farneThe supplementary data can be available from the authors on reasonable request.AK, NS and EV: Conception and designed the study. NS, AS, and OK: Collected the samples and data analysis. IP, AI, NZ, and MM: Drafted the manuscript. All authors read and approved the final manuscript.


The in utero microbiome hypothesis has been long debated. This hypothesis will change our comprehension of the pioneer human microbiome if proved correct. In 60 uncomplicated pregnancies, we profiled the microbiome of chorionic villi (CV) and amniotic fluids (AF) in relation to maternal saliva, rectum, and vagina and the soluble cytokines cascade in the vagina, CV and AF. In our series, 12/37 (32%) AF and 10/23 (44%) CV tested positive for bacterial DNA. CV and AF harbored bacterial DNA of Streptococcus and Lactobacillus, overlapping that of the matched oral and vaginal niches, which showed a dysbiotic microbiome. In these pregnant women, the immune profiling revealed an immune hyporesponsiveness in the vagina and a high intraamniotic concentration of inflammatory cytokines. To understand the eventual role of bacterial colonization of the CV and AF and the associated immune response in the pregnancy outcome, further appropriate studies are needed. In this context, further studies should highlight if the hematogenous route could justify the spread of bacterial DNA from the oral microbiome to the placenta and if vaginal dysbiosis could favor the likelihood of identifying CV and AF positive for bacterial DNA. The host–microbiome interrelationship is considered a mutualistic symbiosis in which the human body provides sustenance and an adequate environment for the microbial growth, while the microbes accomplish essential functions, such as helping the immune system develop, metabolic functions, and defense against infections .The in utero microbiome has been debated for almost 150 years ,3. AccorIf this “in utero colonization hypothesis” proves correct, our comprehension of the establishment of the pioneer human microbiome and its relationship with environmental, lifestyle, and clinical factors will change. Indeed, prenatal microbial colonization is expected to exert a significant influence on the developing fetus, e.g., through the production of short chain fatty acids (SCFAs), positively affecting the immune system’s development  or, convThus, hypothesizing that the microbial footprint and its metabolic and immunological priming of the newborn happen already in utero during the first phase of pregnancy, it is important to understand how the microbial transfer occurs. Although the vaginal microbiome plays a potential role in successful fertilization and healthy pregnancies , the preIn order to investigate the origin of the fetal-placental microbiome during the early phases of pregnancy, we have investigated the bacterial composition of chorionic villi and amniotic fluids in relation to maternal saliva, the rectum, and the vagina during the first and second trimesters of gestation. Next, in order to explore how the maternal-fetal microbiome transfer may impact immune tolerance, we dosed the soluble cytokine cascade in vaginal, chorionic villi, and amniotic fluid samples.In the present study, 64 Caucasian women carrying singleton pregnancies were included, with a mean age of 38 ± 4 years. The indications for invasive procedures were fetal malformation , advanced maternal age , high-risk screening test , anamnestic risk , combined advanced maternal age and high-risk screening test , combined advanced maternal age and fetal malformation , and combined advanced maternal age and anamnestic risk . Before further analysis, four women were excluded from the study as the chorionic villus/amniotic fluid sampling was not sufficient for downstream analysis.A total of 240 biological samples were sequenced, including chorionic villi or amniotic fluid, vaginal swabs, rectal swabs, and saliva samples from 60 pregnant women.After the DADA2 filtering, the sequencing of the V3 region of the 16S rRNA gene produced a total of 9,916,147 reads, identifying 15,207 features. The 24 no template controls produced a total of 59,290 reads, identifying 1560 features.For the analysis, we grouped the samples as follows: chorionic villi samples  and the matched samples, including vaginal swabs (Vag.CVS), rectal swabs (Rect.CVS), and saliva samples . The same sample grouping was performed for the amniotic fluid samples  and matched samples, including vaginal swabs (Vag.AF), rectal swabs (Rect.AF), and saliva samples .Ralstonia pickettii, Escherichia coli, Bacillus pseudofirmus, Cutibacterium acnes, and Pseudomonas fulva sequences. There were no shared features when considering more than 30% of the no template controls. We computed the core microbiome of the no template controls, identifying features present in at least 30% of these samples, including p value = 0.008)  A. When c= 0.008) C,D.After that, we performed a beta diversity analysis. Anaerococcus (W = 551), Corynebacterium (W = 560), Dialister (W = 561), Gemella (W = 554), Haemophilus (W = 556), Lactobacillus (W = 561), Mobiluncus (W = 520), Peptoniphilus (W = 552), Porphyromonas (W = 561), Prevotella (W = 561), Streptococcus (W = 560), Staphylococcus (W = 543), and Veillonella (W = 558).At this point, we performed the differential abundance testing using the ANCOM test. The bacteria that significantly changed between the compared groups were Lactobacillus (n = 5), which was shared with the vaginal samples, and Streptococcus (n = 5), which was shared with the saliva samples (Lactobacillus (n = 7), shared with vaginal samples, and Streptococcus (n = 6), shared with vaginal and saliva samples  compared to those matched to negative CVS/AF samples. Concerning the rectal samples, those matched to the positive CVS and AF samples showed a higher relative abundance of Campylobacter, Peptoniphilus, and Lactobacillus. In the vaginal samples matched to positive CVS, an increase in lactobacilli was observed compared to the vaginal samples matched to negative CVS, whereas the opposite trend was observed for the vaginal samples matched to positive AF samples  were significantly upregulated in AF samples positive for bacterial DNA compared with the AF samples negative for bacterial DNA A.When accounting for the immune-soluble factors dosed in the vaginal swabs, we did not observe significant differences in the vaginal swabs matching the CVS. On the contrary, we observed significantly modulated factors in vaginal swabs matched to the AF samples positive for the bacterial DNA compared to vaginal swabs matched to the AF samples negative for the bacterial DNA. Namely, IL-4, IL-10, IFN-γ, and IL-1ra were significantly down-regulated in vaginal swabs matched to the AF samples positive for the bacterial DNA B.In the present study, we examine in utero colonization by analyzing the microbiome of chorionic villi and the amniotic fluids of 60 singleton pregnancies. In addition, in order to identify the possible origin of the identified bacterial DNA, vaginal, rectal, and saliva samples collected at the same time as CVS or AF sampling were tested.Lactobacillus spp. alongside several other bacteria [In the CVS and AF samples positive for bacterial DNA, a heterogeneous microbiome was observed, similar to that of the matched vaginal, rectal, and saliva samples . In partbacteria .Apart from the heterogeneity of the microbial community, positive CVS showed a partial overlap in terms of bacterial identity with the matched vaginal and rectal samples. We can speculate that only some bacterial species are shared between these body districts. The same observation was confirmed for the AF-positive samples, which clustered near vaginal and rectal samples .When accounting for the significantly modulated bacteria, several bacteria were identified. The identified bacteria in the CVS and AF-positive samples belonged to commensal and opportunistic pathogens of the reproductive tract and the oral cavity ,19 that Fusobacterium, Prevotella, Streptococcus, and Veillonella increased compared to the saliva samples matched to the negative CVS/AF samples. During pregnancy, an increase in these opportunistic pathogens [Streptococcus salivarius, have been previously observed [Streptococcus salivarius decreased in saliva samples matched to positive CVS/AF samples in concomitance with the increase in keystone low-abundance microbial pathogens, remodeling a normally eubiotic microbiota into a dysbiotic one [Streptococcus salivarius strain has been suggested as a preemptive treatment to limit vaginal colonization from pathogens during pregnancy and thereby prevent neonatal transmission [We observed that in saliva samples matched to the positive CVS and, to a minor extent, in the oral microbiome of the colonized AF samples, athogens ,22,23 anobserved ,25 In ouotic one ,27. Althsmission ,29. BaseL. crispatus in the samples matched to the positive CVS and AF samples compared to the samples matched to the negative CVS/AF samples. To note, the association of L. crispatus with the stability of the vaginal microbiota has been observed, especially during pregnancy [S. salivarius, L. crispatus has long been studied for its role in protecting the vaginal epithelium from pathogen colonization, such as Streptococcus agalactiae, which predisposes to a number of adverse pregnancy outcomes, including stillbirth, preterm birth, and neonatal invasive disease [In vaginal samples, the most evident result was the absence and decrease, respectively, of regnancy ,31  or amniocentesis (second trimester of pregnancy) were enrolled. The study was proposed to patients who decided to perform an invasive diagnostic procedure for clinical reasons, such as advanced age, previous medical history (genetic pathology), or a high-risk screening test for major aneuploidies. Women with sexually transmitted infections, hormonal or antibiotic/probiotic therapy in the previous 6 months to the enrollment, history of chronic or infectious diseases, history of recurrent vaginal and urinary infections, or documented risk factors  were excluded. All women were asymptomatic at the time of the invasive procedure. All enrolled patients had to give their consent to use excessive material for the purposes of the study.In this study, 60 pregnant women were enrolled. Chorionic villi or amniotic fluid were collected from each pregnant woman. In addition, swabs were performed in vaginal, oral, and rectal maternal body districts.Chorionic villus sampling is an invasive procedure performed in our center in the first trimester (11–14 weeks) trans-abdominally, after preparation of a sterile field, and involves the insertion of a guide needle of 18 G inside the placenta. Subsequently, the second 20-G needle is introduced, with which the villi are aspirated. The quantification of the sampled villi is performed only at the end of the procedure. Approximately 15 mg of villi are sufficient for cytogenetic analysis. Cytogenetic analysis was privileged only in the case in which there was an excess quantity of the collected villi; these were destined for the study and were deposited in a sterile container with 1 mL of sterile physiological solution and immediately sent to the laboratory of Advanced Microbiological Translational Diagnostics.Amniocentesis is an invasive procedure performed in our center starting from the 15th week of gestation and is carried out trans-abdominally. After the preparation of a sterile field, amniocentesis involves the insertion of a single 21-G needle. Usually, 16–20 cc of liquid is withdrawn for cytogenetic analysis, depending on gestational age. For the purpose of the study, we used 1–2 cc of amniotic fluid that was deposited in a sterile container and immediately sent to the laboratory of Advanced Microbiological Translational Diagnostics.The saliva samples were collected in an empty sterile tube and harvested at least 1 h after the last meal and after subsequent oral hygiene. The rectal swabs  were obtained by inserting 1–3 cm to ensure sampling of the distal rectum. The vaginal swabs  were obtained by a single gentle 360° rotation at the vaginal wall under speculum examination. Amniotic fluid and chorionic villi samples were stored at −80 °C until the time of analysis. Regarding the swabs, they were suspended in 1.5 mL of sterile saline solution and stored at −80 °C.After being thawed, samples were vortexed, and then total DNA was extracted from 300 µL of each sample in a final elution volume of 50 µL by the automatic extractor Maxwell CSC DNA Blood Kit , according to the manufacturer’s instructions.® dye , the Kapa 2G HiFi Hotstart ready mix 2X , 0.5 µM of each primer, and 400 ng/µL of Bovine Serum Albumin (BSA), in a final volume of 10 µL. The temperature cycling conditions were: 5 min at 95 °C, 30 s at 95 °C, 30 s at 59°/57 °C, 45 s at 72 °C and a final elongation step at 72 °C for 10 min. No template controls, processed in parallel with clinical samples starting from the pre-analytic phase, were used as negative controls. The correct size of the amplicons (560 bp for the first PCR and 260 bp for semi-nested PCR) was assessed on a 2% agarose gel. The amount of dsDNA in each sample after the semi-nested PCR was quantified with a Qubit® 2.0 Fluorimeter  using the Qubit® dsDNA BR Assay Kit , and an equal amount of each sample (100 ng) was mixed into a single batch to generate a pooled library at a final concentration of 100 pM, according to the manufacturer’s instructions. Template preparation was performed by emulsion PCR using the Ion OneTouch™ 2 System , with the Ion PGM Hi-Q View OT2 200 kit , and subsequent quality control was carried out on a Qubit® 2.0 Fluorimeter. Sequencing was performed with the Ion PGM™ System technology by using the Ion PGM Hi-Q View sequencing kit .The bacterial composition of the samples was performed by sequencing region V3 of the 16S rRNA gene. Briefly, a qPCR targeting the V1-V3 region of the 16S rRNA gene (500 bp) was performed by employing the U534R primer and the degenerated primer 27FYM. Subsequently, a semi-nested PCR was carried out with the primers B338F_P1-adaptor and U534R_A_adaptor_barcode, targeting the V3 region (200 bp) of the 16S rRNA gene, with a different barcode for each sample linked to the reverse primer . The PCRg, the undiluted samples (50 μL) were mixed with biomagnetic beads in 96-well flat-bottom plates with the addition of 0.5% of BSA. The amniotic fluid and chorionic villi samples were centrifugated at 1000× g, and diluted 1:4 using Bioplex Sample Diluent before analysis. Then, 50 μL of diluted samples were mixed with biomagnetic beads in 96-well flat-bottom plates. After incubation for 30 min at room temperature, followed by a washing plate with Bio-Plex wash buffer, 25 μL of the antibody–biotin reporter was added. After the addition of 50 μL of streptavidin–phycoerythrin and following incubation for 10 min, the concentrations of the cytokines were determined using the Bio-Plex-200 system  and Bio-Plex Manager software . The data were expressed as median fluorescence intensity (MFI) and concentration (pg/mL).The soluble concentration of 27 cytokines, chemokines, and growth factors was assessed in duplicate in all 60 vaginal swabs, amniotic fluid, and chorionic villi samples using magnetic bead-based multiplex immunoassays , according to the pre-optimized protocol . RegardiFor the big data analysis, using QIIME 2.22.2, evenness  and observed ASVs  metrics were calculated to assess the alpha diversity (microbiome diversity within a community) and compared by means of the Kruskal–Wallis test. The Bray–Curtis dissimilarity index was calculated to assess the beta diversity, which measures the similarity or dissimilarity of the analyzed groups, visualized by the principal coordinate analysis (PcoA), and compared by the PERMANOVA test. To highlight the differences in the microbial composition, we performed differential abundance testing using the ANCOM test. Using MicrobiomeAnalyst, we applied the LEfSE test to identify microbiological biomarkers .p-value was observed (p < 0.05), a multiple comparison test was used to determine which groups were different.To test the differences in the immune-soluble factors, GraphPad Prism  was used. Specifically, the Kruskal–Wallis one-way analysis of variance was used for comparisons between groups. When a significant bona fide researchers.Due to the sensitive nature of the data, information created during and/or analyzed during the current study is available from the corresponding author upon reasonable request to All the participants signed an informed consent, and the experiment was carried out according to the principles of the Declaration of Helsinki. Ethical approval for this study was obtained from the ethical committee board .Taken together, our results support the presence of bacterial DNA in CVS and AF samples. These results are relevant as the exposure of the fetus to the maternal microbiome could impact the proper development of the fetal immune system but also play a vital role in predisposing the newborn to pathologies such as asthma, allergies, obesity, neurodevelopmental disorders, and liver steatosis ,49.Further studies on a wider cohort are needed to understand the possible role of bacterial colonization of amniotic fluid and chorionic villi, the associated immune response in the pregnancy outcome, and the route of colonization. In this regard, studies should highlight if a hematogenous spread could justify the spread of bacterial DNA belonging to oral microbes to the placenta. If, in the vaginal microbiome, the dysbiosis could contribute to the likelihood of retrieving CVS and AF samples positive for bacterial DNA. If, despite the absence of an ongoing infection, bacterial translocation could be elicited or accompanied by the upregulation of inflammatory cytokines in the amniotic fluid coupled with an immune hyporesponsiveness in the vaginal milieu.On one side, we had the possibility to collect CVS to study a microbial composition that is less explored compared to AF samples and placentas of at-term pregnancies. In addition, we simultaneously collected samples from the other maternal body sites in order to identify the possible origin of the DNA identified in the CVS and AF samples. On the other side, we acknowledge that our cohort is small, and we cannot argue for the clinical relevance of our findings. Furthermore, we cannot demonstrate the route of colonization of CVS and AF samples from maternal body districts.


Nicotiana tabacum) root affects the yield and quality of tobacco leaves. To gain insight into the responses of the tobacco root system to different soil types, we integrated morphological characteristics, the physiological index, the metabolic pathways of the root system, and the aboveground biomass of tobacco cultivated in limestone soil (LS), paddy soil (PS), and red soil (RS). Compared with plants growing in LS and PS, the chemical composition of tobacco leaves in RS tended to be coordinated. Red soil facilitated the accumulation of aboveground and belowground biomass of flue-cured tobacco and had the most significant effect on the dry matter quality of the roots. In addition, it promoted an increased root length, root surface area (RSA), root volume, and a higher number of root forks and improved root vigor and nitrate reductase (NR) activity; however, the activities of superoxide dismutase (SOD) and peroxidase (POD) were decreased. We studied differentially the abundant proteins (DAPs) of the flue-cured tobacco roots cultivated in different soil types by isobaric tags for the relative and absolute quantification (iTRAQ) of the proteomic profiles of cultivar. In total, 699, 650, and 569 differentially abundant proteins (DAPs) were identified from limestone soil (LS) vs. PS, LS vs. RS, and PS vs. RS, respectively, including 412/287, 291/359, and 323/246 up-/downregulated proteins, respectively. These DAPs were mainly involved in starch and sucrose metabolism, phenylalanine metabolism, the biosynthesis of secondary metabolites, microbial metabolism in different environments, and ribosomes. The parallel reaction monitoring (PRM) and quantitative reverse transcription PCR (qRT-PCR) analysis showed that the results of the iTRAQ proteomics were reliable. Overall, our study facilitates a new understanding of the responses of tobacco roots to different soil types at the protein level.Tobacco ( The root system in an important vegetative organ of plants, providing support, water and nutrient absorption storage, and the secretion of metabolites , making The root system of flue-cured tobacco is the main place where tobacco plants secrete secondary metabolites, such as nicotine and plant hormones . The amoFlue-cured tobacco can produce numerous adventitious roots after transplanting, which are younger than the main and lateral roots and are the main sites of absorption and synthesis. When tobacco plants are subjected to nutritional stress, adventitious roots will compensate or over-compensate nutrient losses .The effect of soil type on the root growth of flue-cured tobacco is greater than that of soil moisture and temperature. In another study, different nitrogen forms had different effects on microorganisms in the yellow cinnamon soil and fluvo-aquic soil, thereby differing in their impacts on the growth and development of roots and the absorption and use of nitrogen . Yellow Appropriate field water conditions can promote root growth, whereas drought or waterlogging will seriously affect the root growth. Tobacco soil acidity and alkalinity can also impact the growth and development of flue-cured tobacco roots, consequently affecting mineral absorption and nicotine synthesis . Xu et aThe term proteome was originally put forward by the Australian scholars Isotope-labeled relative and absolute quantitative isobaric tags for relative and absolute quantitation (iTRAQ)/tandem mass tag (TMT) is a new quantitative technique for the high-throughput screening of proteins. The tags used in the analysis can be divided into reflection group, equilibrium group, and reporter group; they are used for labeling 8–10 samples at the same time, and almost all proteins in the sample can be labeled . The undRecently, tobacco has been intensively studied, and as a new method, the proteomics technology has achieved important results. For example, In this context, the purpose of this study is to understand the genetic regulation mechanism of tobacco roots to different soil types at the protein level. This work will provide new insights into the response of tobacco roots to soil types and into the key proteins, with the overall aim to improve the tobacco yield and quality.2 (4.15 m × 5.76 m), and the different planting areas were divided by concrete pouring. A random block arrangement was used, with three replicates per treatment. The pest control and other field management measures were identical for each treatment and carried out in accordance with the guidelines for the production and management of high-quality tobacco leaves in Yunnan Province. After topping, three tobacco plants with uniform growth were randomly selected from each plot as the biological replicates; the roots were rinsed under running water, the root tip was preserved in liquid nitrogen (3–5 cm), and the samples were stored in a refrigerator at −80°C for the determination of various indices.In this study, the flue-cured tobacco variety K326  was used. The experiment was conducted at the Yanhe Experimental Base, Hongta District, Yuxi City, Yunnan Province, China, from April to August 2019, at an elevation of 1,635 m. In the dry shed experiment, different soil types widely distributed in Yunnan were selected, namely, limestone soil (LS), paddy soil (PS), and red soil (RS) . Table 1The flue-cured tobacco roots were cleaned with deionized water, scanned with a root scanner , and the image was stored on the computer. When scanning, 10–15 mm distilled water was injected into the transparent tray to facilitate the expansion of flue-cured tobacco roots. The characteristic parameters of tobacco roots, such as root length, root surface area (RSA), root volume, root average diameter, root tip number, and root fork number, were measured using the root analysis software WinRHIZO . Each process was repeated three times.Root vigor was determined according to 2PO4 (pH 7.5), 2% propanol, and 30 mmol/L KNO3. The samples were vacuumed for 5 min and incubated in the dark for 30 min in an oscillating water bath at 25°C. After incubation, 1 ml from each sample was transferred to a new test tube, and 1 ml of sulfonamide reagent, 0.02% naphthalene diamine, and 50 μl of acetonitrile were added. After incubation at room temperature for 30 min, absorbance at 540 nm was measured using a spectrophotometer. Each process was performed in triplicate.The determination of NR activity was performed as described in g at 4°C for 15 min, and the supernatant was used to determine the activity of SOD. Subsequently, 250 μmol/L nitroblue tetrazolium chloride (NBT), 2.8 mmol/L N, N, N′, N′-tetramethylethylenediamine (TEMED), 22 μmol/L riboflavin, and 3 ml crude enzyme solution were added, and the mixture was incubated in the dark for 25 min. After this, the tubes were placed in a box lined with aluminum foil and irradiated at room temperature with two 20-W light-emitting diode (LED) illuminators until the SOD active band was visible. After the reaction, the absorbance values of the blank and the reaction solution were determined spectrophotometrically at 560 nm; each process was performed in triplicate.The activity of superoxide dismutase (SOD) was determined according to g, and 0.1 ml of the supernatant was added to a new test tube and spiked with 1 ml of 0.05 mol/L guaiacol and 1 ml of 2% H2O2. The final volume was 5 ml, and absorbance was recorded at 470 nm; each process was carried out in triplicate.The activity of peroxidase (POD) was determined according to g for 20 min at 4°C, and the obtained supernatant was transferred to a new centrifuge tube. After the addition of four times the volume of the supernatant of cold acetone, the mixture was left overnight in a dark chamber at −20°C and subsequently centrifuged at 13,000 g for 20 min; the precipitated protein pellet was collected. The reduction reaction was carried out with 8 mol/L urea and 100 mmol/L tetraethylammonium borohydride (TEAB) (pH 8.0) in a water bath at 56°C for 30 min. Finally, iodacetamide (IAM) with a final concentration of 55 mmol/L was added, and the protein concentration was determined by the Bradford method  was added. After mixing and incubating for 5 min on ice, a dithiothreitol (DTT) solution with a final concentration of 10 mmol/L was added. The solution was subjected to ultrasound for, centrifuged at 13,000 d method . Each prThe peptides were dissolved in 0.5 mol/L TEAB and labeled with iTRAQ , according to the instructions of the iTRAQ-8 standard kit. The root samples of flue-cured tobacco grown in PS, RS, and LS were labeled with iTRAQ 114, 116, and 118, respectively, at three replications. After labeling and mixing, the samples were separated by an SCX column in triplicate.2O) and buffer B , and finally freeze-dried. The mass-charge ratio ranges of primary and secondary scanning were 350–1,500 and 100–1,500 m/z, respectively. Each process was performed in triplicate.Triple TOF 5600  + liquid chromatography-mass spectrometry  was used for mass spectrometry (MS) data acquisition, and the samples were analyzed using a Triple TOF 5600 plus mass spectrometer coupled with an Eksigent nanoLC system . The polypeptide sample was dissolved in 2% acetonitrile and 1% formic acid and eluted with a time gradient of 90 min and a flow rate of 300 nl/min. The two mobile phases were buffer A . The trusted protein contains at least one unique peptide before it can be used for protein quantification.For this, the data were retrieved from the Protein-PilotTM (v4.5) search engine and compared with the tobacco protein database. By further filtering the identification results of Protein-Pilot, we assumed that further quantitative identification can be carried out only at a credibility level above 95% (unused score ≥ 1.3) and a false discovery rate (FDR) below 5%. In the pairwise comparisons of flue-cured tobacco roots under three different soil types, the filter criterion of differential proteins was set as a maximum 1 and their pathways were analyzed using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database.2The differentially abundant proteins were functionally classified and annotated by Gene Ontology (GO),The protein expression level and the protein function obtained by iTRAQ analysis were verified using the parallel reaction monitoring (PRM) technique. For this, the protein extract and trypsin digestion solution were prepared according to the iTRAQ technology; subsequently, the trypsin peptides were loaded on a C18 capture column for desalination, and PRM verification was carried out using a TripleTOF 5600 LC-MS/MS system . Collections MS1 and MS2 were analyzed in the range of 350–1,500 and 100–1,500 m/z. Some trypsin peptides were mixed and detected by MS data-dependent (DD) acquisition. Protein quality was determined by the Protein-Pilot software, and the MS data were processed using the Skyline software (spectrum library). The target peptide m/z was added to the inclusion list, the PRM collection method was established, and the PRM data collection of mixed samples was optimized and adjusted. Using the optimized PRM MS collection method, the data of each sample and PRM spectral files were collected, and the quantitative information of the proteins was obtained by analysis.–ΔΔCt method, using three biological replicates.After extracting the total RNA from tobacco root samples with TRTzol reagent , cDNA was reverse-transcribed from 1 μg total RNA with ReverTra Ace qPCR RT kit (TOYOBO) according to the manufacturer’s instructions, using cDNA as a template for three replications. Real-time fluorescence quantitative PCR (RT-qPCR) internal reference gene actin primers were used for q-PCR amplification to verify cDNA quality, using the following conditions: denaturation at 95°C for 1 min, 95°C for 15 s, 40 cycles, followed by annealing and extension at 60°C for 30 s. The expression level of each gene was calculated and analyzed as described in For data analyses, SPPS18.0  Duncan’s multiple test (Duncan’s multiple range test) and the logarithmic data processing and analysis software Origin8.0  were used.As shown in p < 0.05), following the order RS-grown plants > PS-grown plants > LS-grown plants. The root vigor of RS-grown plants was 35.23 and 21.02% higher than that of LS- and PS-grown plants, respectively. The NR activity of RS-grown plants was 51.43 and 25.71% higher than that of LS- and PS-grown plants, respectively. The SOD activity of RS-grown plants was 34.09 and 20.45% higher than that of LS- and PS-grown plants, respectively, whereas the POD activity of RS-grown plants was 37.29 and 21.19% higher than that of LS- and PS-grown plants, respectively.As shown in p) as the ordinate. In this study, 699 differentially abundant proteins were identified in PS vs. LS, such as 412 upregulated and 287 downregulated proteins . Among the 1,282 differential proteins in the three comparison groups, 102 proteins occurred in the three comparison groups . In the PS vs. RS comparison group, 398 differential proteins were annotated to 101 pathways and significantly enriched in 15 metabolic pathways.To further understand the biological functions of differential proteins in flue-cured tobacco roots in different soil types, we investigated the different pathways. In the PS vs. LS comparison group, 520 differential proteins were annotated to 103 pathways and significantly enriched in 15 metabolic pathways, based on KEGG enrichment analysis. In the LS vs. RS comparison group, 444 differential proteins were annotated to 104 pathways and significantly enriched in 18 metabolic pathways  to provide accurate data for studying the molecular mechanisms of tobacco root responses to different soil types. These seven proteins or genes are involved in the defense mechanism; the qRT-PCR data of the seven genes were consistent with the results of the iTRAQ .Root morphological characteristics  play an important role in determining nutrient and water absorption efficiencies and in assessing the adaptability of a plant to stress . The rooBased on previous studies, soil physical and chemical properties, temperature, nutrient content, moisture, air permeability, organic matter content, poPODity, and other factors affect the root growth of flue-cured tobacco. LS has a high sand content, light texture, fast fertilizer effect, poor ability of water and fertilizer conservation, low organic matter content, and large temperature changes; however, its air permeability and water permeability are good . PS, as Affected by various factors in the soil, the root morphology of flue-cured tobacco also differs with the soil type . It is gIn this study, the RSA and root volume of RS flue-cured tobacco were larger than those of LS and PS plants, suggesting that RS promoted an increase in RSA and root volume. According to a previous study, higher RSA and root volume values facilitate the drought resistance of plants . GeneralRoot vigor is a physiological index reflecting the metabolic level of the root system and the synthesis of various compounds. It directly affects the growth and development of plants, with a significant correlation between root vigor and the chemical composition of tobacco leaves. The higher the root vigor, the stronger the stress resistance of flue-cured tobacco plants. According to Nitrate reductase is a key enzyme in plant nitrogen metabolism. Under the action of NR, glutamine synthetase, and glutamate synthetase, nitrate nitrogen is reduced to nitrite nitrogen in roots, which is transformed into amino acids and then participates in nitrogen metabolism. The NR activity therefore reflects the level of nitrogen metabolism. Cell metabolism can produce reactive oxygen species (ROS), which can harm the biological system; the activity of antioxidant enzymes is therefore a key factor in active oxygen detoxification. SOD plays an important role in plant oxygen metabolism  and is tAs defense molecules, secondary metabolites can protect plants under various adverse conditions. Numerous plants can produce secondary metabolites after external stimulation, and the regulation mechanism of these metabolites involved in plant defense responses is highly complex. A variety of defense signal pathways form a defense network, which can effectively resist various biological stresses to a certain extent . Lei et Phenylpropane compounds are secondary metabolites of plants and play an important role in the interaction between plant growth and the environment. Studies have shown that phenylpropanoids can prevent the invasion of pathogens by increasing the thickness of the secondary cell walls. The metabolism of phenylpropanoids provides plants with a large number of important phenolic compounds . In our In this study, 21 proteins related to defense mechanisms were screened from 102 common differential proteins in three comparison groups in which peroxidase could inactivate toxic substances and protect plants from the toxic effects of high oxygen concentrations. Glutathione S-transferase (GST) plays an important role by catalyzing the binding reaction of nucleophilic glutathione with various foreign chemicals, thereby facilitating detoxification. GST is the key enzyme of the glutathione binding reaction and catalyzes the initial step of this reaction .Fructokinase has also been found to be involved in the stress response of plant cells. The abundance of these proteins decreased in the comparison group PS vs. LS and increased in the LS vs. RS and PS vs. RS groups. The pathways of 13 differential proteins involved in stress defense were analyzed; among them, 8 differential proteins were involved in secondary metabolism, 2 in glucose metabolism, 2 in glutathione synthesis, and 1 in methionine synthesis. Among the 14 differential proteins related to defense mechanisms, 10 were upregulated in LS vs. RS and/or PS vs. RS, and 3 proteins were downregulated. Based on these results, the defense mechanism of flue-cured tobacco roots in different soil types is highly complex, with an even more complex regulatory network. The above results lead us to infer that compared with LS and PS, the resistance of tobacco roots to stress was higher in RS, in addition to the well-developed root system in RS, providing enhanced resistance.Carbohydrate and energy metabolism is one of the most important biological metabolisms of flue-cured tobacco roots in response to different soil types. The pathways related to carbohydrate and energy metabolism include the glycolysis gluconeogenesis pathway, the starch and sucrose metabolism pathway, the TCA cycle, and the pentose phosphate metabolism pathway, among others. In plants, sucrose synthase is a key enzyme in sucrose metabolism and can catalyze the decomposition and synthesis of sucrose; it also plays an important role in starch synthesis, stress resistance, and plant growth . In our Based on a previous study, the TCA cycle is the main way for plants to obtain energy, providing the carbon skeleton for plants to synthesize various substances . SeveralIn this study, we selected 10 proteins related to carbohydrate and energy metabolism, out of 102 common differential proteins. For example, glucose-6-phosphate and glucose-1-phosphate adenylate transferase are involved in carbohydrate transport and metabolism, aconitine hydratase is involved in the TCA cycle, and other proteins are involved in carbohydrate transport and metabolism. Therefore, the biological processes and pathways related to sugar and energy metabolism may be important factors regulating the adaptation of flue-cured tobacco roots to different soil types.Since proteins are the direct executors of various biological processes, protein synthesis is directly related to the general activities of plants . RibosomBased on determining the morphological characteristics and physiological indices of flue-cured tobacco roots, an iTRAQ technique was used to study the differential changes in root proteins under different soil types . The results showed that root length, RSA, and root volume were higher in RS than in LS and PS, indicating that RS was most suitable for tobacco growth. The soil type significantly changed the proteomic profiles of tobacco roots. The stress resistance, carbohydrate and energy metabolism, and protein synthesis of RS flue-cured tobacco roots were higher than those in LS and PS. Based on PRM and qRT-PCR analysis, the results of iTRAQ proteomics were correct and reliable. The response mechanism of flue-cured tobacco roots to different soil types could be revealed at the protein level.IPX0004001000.The data presented in the study are deposited in the Integrated Proteome Resources repository, accession number Written informed consent for participation in this study was obtained from the participants.RY and YC: conceptualization. RY: data curation, methodology, and writing–original draft. YC: funding acquisition, project administration, supervision, and validation. JlL: writing–review and editing. All authors contributed to the article and approved the submitted version.RY was employed by Shiyan Branch of Hubei Tobacco Company. SS and YL was employed by Research Center of Hubei Tobacco Industrial Co., Ltd. XL was employed by Hunan Zhangjiajie Municipal Tobacco Co.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.


Obesity is a risk factor for severe airway obstruction and hypoxemia. High-flow nasal cannula (HFNC) is considered as a novel method for oxygen therapy, but the efficacy of HFNC for obese patients is controversial. This meta-analysis aimed to assess the efficacy of HFNC compared with conventional oxygen therapy (COT) in obese patients during the perioperative period. 2, the need for additional respiratory support, and the hospital length of stay (LOS).  We searched the PubMed, Embase, Web of Science, the Cochrane Library, and Google scholar databases for randomized controlled trials (RCTs) that compared the efficacy of HFNC with COT in obese patients during the perioperative period. The primary outcome was the incidence of hypoxemia, while the secondary outcomes included the lowest SpOP=0.002; I2 = 24%; 8 RCTs; n = 458), increased the lowest SpO2 , decreased the need for additional respiratory support , and shortened the hospital LOS .  Twelve trials with 798 obese patients during the perioperative period were included. Compared with COT, HFNC reduced the incidence of hypoxemia (RR, 0.60; 95% CI, 0.43 to 0.83; 2, decrease the need for additional respiratory support, and shorten the hospital LOS in obese patients during the perioperative period. Well-organized trials with large sample size should be conducted to support our findings. This meta-analysis showed that compared with COT, the use of HFNC was able to reduce the incidence of hypoxemia, increase the lowest SpO The definition for hypoxemia was varied in included trials, such as SpO2 < 90%, SpO2 < 92%, and SpO2 < 95%. The additional respiratory support was defined as an escalation in oxygen support therapy, including the intermittent positive-pressure ventilation, continuous positive airway pressure (CPAP), NIV, HFNC, or reintubation.The primary outcome was the incidence of hypoxemia, while the secondary outcomes included the lowest SpOTwo authors (R. Z. and H. T. W.) examined the titles and abstracts of the retrieved trials independently, and reviewed the full texts of the potentially eligible trials based on the inclusion and exclusion criteria. Any disagreement was resolved by a third author (W. G.).2, the need for additional respiratory support, and the hospital LOS. Disagreements were adjudicated by a third author.Two authors independently extracted the trial characteristics, which were summarized in a standardized Excel file. The following information were retrieved from each trial: first author, year of publication, location, population, clinical setting, sample size, interventional time point, intervention and control details, the incidence of hypoxemia, the lowest SpOThe methodological quality of each trial was assessed by two authors through the Cochrane Risk of Bias Tool , and theI2tatistica, and the I2 ver 50% indicated significant heterogeneity. The intention-to-treat principle was used for performing the analyses. We adopted a priori random-effects model to pool the outcome data, on the assumption of heterogeneity across the included trials. Differences of the outcomes were graphically displayed with a forest plot, and a P value of <0.05 was considered to be statistically significant. To assess the potential impact of the findings from a single trial on the overall meta-analytical outcome, we adopted sensitivity analysis with a leave-one-out approach. The statistical analyses were performed using Review Manager, Version 5.3.Dichotomous outcomes were presented as the relative risks (RRs), and continuous outcomes were presented as the mean differences (MDs), both with corresponding 95% confidence intervals (CIs). For continuous outcomes presented as the median (25th and 75th percentile), we converted inter-quartile ranges to standard deviation using formula conversion suggested by the Cochrane Collaboration . StatistThe process of trial selection is shown in 2 < 90% [2 < 92% [2 < 95% [The basic characteristics of the included trials are summarized in 2 < 90% , 19, 21,[2 < 92% , 22, 23,[2 < 95% .The risks of bias of individual trials are summarized in Figures n = 228 vs. COT group, n = 230) provided data on the incidence of hypoxemia [P=0.002; I2 = 24%; 8 RCTs; n = 458; Incidence of hypoxemia. Eight trials involving a total of 458 patients  The modest amount of positive end-expiratory pressure generated by HFNC could greatly flush potential nasopharyngeal dead space, reduce the carbon dioxide levels, as well as improve ventilation and perfusion matching for the obese patients . (2) HFN2, additional respiratory support, and the hospital LOS rather than some physiologic outcomes. Finally, we used the intention-to-treat principle and a random-effects model for a more conservative estimate accounting for clinical heterogeneity.The strengths of our study lie in the population included in all obese patients during the perioperative period. Second, we only focused on the efficacy of HFNC versus COT in the obese patients, and excluded NIV, CPAP, and other oxygen treatments. Third, we prioritized the patient-centered outcomes including hypoxemia, the lowest SpOOur study also has limitations. First, the patient characteristics , and clinical characteristics  all these might affect the pooled results. We did not perform the subgroup analysis, due to the limited trials. Second, because of few included trials, the conclusions for some outcomes may not have clinical significance. Third, formula conversion was used for the data that were not represented by the meanula , which m2, and shorten the hospital LOS in obese patients during the perioperative period. Therefore, HFNC may be superior to COT for reducing hypoxemia or enhancing oxygenation in obese patients during the perioperative period, but further large-scale and well-organized clinical trials are required to confirm the efficacy of HFNC in the obese patients. Although in our study outcomes did not demonstrate statistical heterogeneity, the definition of hypoxemia and the intervention time point were varied in included trials. Therefore, for the further research, it is important for to keep consistent in the intervention time point, obese degree, the surgery type, the definition of hypoxemia, as well as the exact threshold for additional respiratory support.In our meta-analysis, compared with COT, the use of HFNC was able to reduce the incidence of hypoxemia, increase the lowest SpO


Background: Carboplatin, the key drug used in treating gynaecological cancer, has an approximately 12–16% risk of hypersensitivity reactions. We aimed to investigate the efficacy and adverse effects of carboplatin desensitisation therapy for gynaecological cancer. Methods: The desensitisation protocol was standardised as a four-step, 4-h, carboplatin administration in the hospital. A retrospective medical record review was conducted on 15 patients who underwent carboplatin desensitisation for gynaecological malignancies at our hospital. Patients’ data were analysed to evaluate the treatment success rate, therapeutic effect of desensitisation, adverse events, and treatment. Results: Of 91 carboplatin desensitisation cycles scheduled; the completion rate was 93.4% (85/91). Adverse events occurred in 23 of these 91 (25.3%). In four (4.4%) of the 23 cycles, hypersensitivity reactions could be treated only by discontinuing the infusion and slowing the administration, while in the remaining 19 (20.9%), medication was administered intravenously after discontinuing the infusion to manage hypersensitivity reactions. No treatment-related deaths occurred. Overall, 23 series of anti-cancer agent regimens, including carboplatin desensitisation, were administered to the 15 patients. The therapeutic response rate was 82.6% and the disease control rate was 95.7%. Conclusions: Carboplatin desensitisation was beneficial in patients with a history of carboplatin-induced hypersensitivity reactions. Platinum agents are some of the most effective drugs for the treatment of gynaecological malignancies including ovarian, cervical, and endometrial cancers. Carboplatin, one of the platinum agents, is used most frequently. Compared to cisplatin, carboplatin demonstrates the same therapeutic efficacy with fewer adverse events  ,2. PlatiThe clinical symptoms of platinum HSRs range from mild to severe and death can also occur ,12,13. MHSRs to key drugs are problematic in some cases because of the lack of alternative therapies. Management options include enhanced premedication with antihistamines and corticosteroids, the discontinuation of carboplatin and switching to another drug, and carboplatin desensitisation (CD).The goal of desensitisation is for patients to react less strongly to chemotherapeutic agents. In some reports, CD was successful in patients who experienced platinum-related HSRs ,19,20,21The purpose of this study was to summarise the clinical features, toxicity, and effects of CD therapy on therapeutic efficacy, and outline possible appropriate prophylaxis and management strategies for treating the adverse events.We retrospectively evaluated the experience of CD in patients with gynaecological malignancies who experienced HSRs with previous chemotherapy. Our study was conducted at a teaching hospital attached to a university medical school, a general hospital with approximately 900 beds. This study was approved by the Institutional Review Board of Nippon Medical School (No. 30-01-1067). We reviewed all patients who underwent CD between January 2015 and December 2019. In this retrospective review study, only patients who underwent CD following careful consultation were included. That is, among all gynaecological cancer patients, only those who had developed HSRs because of carboplatin administration and who consented to the risk of undergoing desensitisation were included. Patients who developed HSRs to carboplatin and who consented to CD but for whom detailed information on HSRs was not available, and patients who had not completed an anti-cancer agent treatment cycle, including CD at our hospital, were excluded. Due to the nature of the disease, all subjects were female. Initial HSR symptoms varied and included, cutaneous symptoms ; respiratory symptoms ; cardiovascular symptoms ; gastrointestinal symptoms ; and atypical symptoms (limb dysesthesia and discomfort). Patients with adverse events associated with carboplatin administration were examined by oncologists. If the oncologist determined that their symptoms were carboplatin-induced HSRs, they were offered treatment alternatives, including CD therapy, another anti-cancer agent therapy, or best supportive care only. All patients understood the risks, including death, as well as the benefits of re-medication from the description of CD therapy, and they signed written informed consent for desensitization therapy. Consent to use the participants’ clinical information was obtained in writing and consent limited to this study was obtained by an opt-out method. After applying the inclusion and exclusion criteria, 15 patients with gynaecological malignancies  subsequently opted for CD at our hospital. None of the patients underwent treatment after developing HSRs at another hospital or underwent an intradermal reaction test for carboplatin.The desensitisation protocol followed at our institution was standardised as a four-step, 4-h, carboplatin administration based on the report by Takase et al.  That is,Under this desensitisation protocol, four different dilutions of carboplatin solutions were administered to patients over 1 h each. The total dose of carboplatin (AUC5-6 according to the regimen) was calculated using the Calvert formula. First, the total dose of carboplatin was dissolved in 250 mL of 5% glucose. Next, 25 mL of the solution were removed from the first solution and diluted with 225 mL of 5% glucose to obtain the second solution. This operation was repeated in sequence to prepare 1/10, 1/100, and 1/1000 diluted solutions. In other words, 250 mL of 0.1% solution, 225 mL of 1% solution, 225 mL of 10% solution, and 225 mL of 100% solution were prepared. Each solution was infused over 1 h, and all infusions were completed over 4 h. Prior to desensitisation, all patients were intravenously treated with glucocorticoids (dexamethasone 19.8 mg), H2 antagonists (famotidine 20 mg), H1 antagonists (chlorpheniramine 5 mg), and 5-HT3 antagonists  .If HSRs re-emerged during desensitisation, carboplatin administration was immediately discontinued, and the patient was examined by the attending doctor. After confirming the improvement of the symptoms, the patient was allowed to resume the treatment 30 min later. When the administration was resumed, it was re-initiated at half the infusion rate at the time of discontinuation, and if no abnormalities were observed for 30 min, the infusion rate was restored to the original rate. If a mild reaction occurred, careful observation of the symptoms was performed without medication. When a moderate reaction occurred, an H1 antagonist (chlorpheniramine 5 mg), an H2 antagonist (famotidine 20 mg), and glucocorticoid (hydrocortisone 100 mg or methylprednisolone 125 mg) were administered intravenously. In addition, a phosphodiesterase inhibitor (aminophylline 250 mg) was used to treat respiratory symptoms. For severe reactions or anaphylaxis, an additional 0.3 mg of adrenaline was administered in addition to the glucocorticoid. The gynaecological oncologist in charge decided whether to continue or discontinue the desensitisation depending on the symptoms of CHRs. If treatment continuation was an option, the patient was verbally consulted, and consent obtained. Even if the doctor-in-charge determined that continuous administration was possible, the administration was discontinued according to the patient wishes. Desensitisation was discontinued in all cases of severe hypersensitivity and anaphylaxis.The clinical-stage was evaluated based on the staging criteria of the International Federation of Gynaecology and Obstetrics. Therapeutic effects were evaluated according to the Revised Response Evaluation Criteria in Solid Tumours guidelines (version 1.1) . AdverseThe primary endpoint was the success rate of CD administration, which was calculated as the percentage of patients who completed all planned CDs among all patients and the percentage of completed CD cycles among the total CD cycles. The secondary endpoint was the response rate of the anti-cancer agent therapy, including CD. The treatment response rate was calculated as the percentage of consecutive treatments in which tumour shrinkage was observed in all consecutive treatments, including CD. An analysis using descriptive statistics was performed. The survey results were described and evaluated in terms of range, median, percentage, and so forth.2 . The initial HSR grades ranged from 1 to 3 .Patient characteristics are shown in n = 20), paclitaxel + carboplatin + bevacizumab (n = 30), docetaxel + carboplatin (n = 6), docetaxel + carboplatin + bevacizumab (n = 8), gemcitabine + carboplatin (n = 19), and gemcitabine + carboplatin + bevacizumab (n = 8).The chemotherapy regimens used for desensitisation were paclitaxel + carboplatin  were completed without adverse events. In 23 (25.3%) of the CD treatments, patients developed some CHRs, but symptoms were relieved by a temporary suspension of administration or additional medication; among those, 17 CD cycles were completed as planned. As a result, 85 CD cycles (93.4%) were completed as originally planned.Seven patients (46.7%) did not develop CHRs. Eight patients (53.3%) developed CHRs during the CD. Among them, three patients (20.0%) completed the scheduled treatment with only temporary interruptions or some changes in medication, while five patients (33.3%) discontinued the CD after consulting with their doctor. Treatment was not discontinued due to adverse events other than CHR or tumour exacerbation during the course of the treatment.After completing the series of regimens including CD, the therapeutic effect was determined by comparing tumour image measurements before and after treatment. A total of 23 series of anti-cancer agent regimens, including CD, were administered to the 15 patients. Results were evaluated according to the Response Evaluation Criteria in Solid Tumours (version 1.1) as complete response (CR) 15 series, partial response (PR) 4 series, stable disease (SD) 3 series, and progressive disease (PD) 1 series. The response rate [(CR + PR)/(CR + PR + SD + PD)] was 82.6%, and the disease control rate [(CR + PR + SD)/(CR + PR + SD + PD)] was 95.7%. The average platinum-free interval before the start of the anti-cancer treatment series, including the CD regimen, was 17.8 months  in 22 series, excluding one series of adjuvant therapy. Comparing the median platinum-free interval by treatment result, 14 series of CR was 19.5 (6–33) months, 4 series of PR was 14.5 (10–29) months, 3 series of SD was 10.0 (7–12) months, and 1 series of PD was 24 months.Adverse events were observed in 23 of the 91 total CD cycles (25.3%). In 4 (4.4%) of these cycles, CHRs could be treated only by discontinuing the infusion and slowing down the administration. In the remaining 19 cycles (20.9%), medication was administered intravenously after the discontinuation of the infusion to manage CHRs.Cutaneous adverse events were most common. Skin rash and itching were observed in seven patients; they were alleviated by discontinuation of CD or administration of additional drugs (grade 1 or grade 3). The next most commonly observed adverse events were those affecting the gastrointestinal system, such as stomach-aches and diarrhoea. Two patients developed symptoms and were treated with CD discontinuation and administration of H1 or H2 blockers (grade 1 or grade 3). One patient experienced grade 3 anaphylaxis (hypotension) but recovered quickly after discontinuing the carboplatin infusion, without requiring adrenaline and with no sequelae. Another patient experienced grade 3 anaphylaxis (bronchospasm and hypoxia) but quickly recovered with the addition of medications containing adrenaline. No treatment-related deaths occurred.We reported that carboplatin could be re-administered to patients who experienced CHRs, using the CD protocol. The therapeutic effect was satisfactory, and the recurrence of adverse events was in an acceptable range. This result is of great importance for patients; it stresses that prolonging their lives through additional carboplatin is feasible.Platinum agents, such as carboplatin, are essential for the treatment of gynaecological malignancies, especially ovarian cancer. However, multiple doses of carboplatin can cause HSRs, and abandoning subsequent platinum administration and switching to platinum-free anti-cancer therapy is of great detriment to a patient’s prognosis. CHRs usually appear in 8–19% of patients receiving repeated doses of six cycles or more (eight times on average) . CHRs arPreviously, unsuccessful attempts to re-administer the drug that caused HSR using premedication alone have been reported . On the In this study, we adopted a four-step, 4-h CD protocol in the hospital. The advantages of this protocol include its simplicity, short infusion times, and acceptable HSR rates . The CD The majority of our patient cohort completed the CD without experiencing serious adverse events. In our study, HSRs occurred in 25.3% of the CD cycles, which is close to the incidence of 25.0% reported in the largest desensitisation study by Altwerger et al. . OverallReviewing our results, discontinuation of CD was limited to 6 CD cycles, with 93.4% of cycles ending successfully. However, five patients did not complete the targeted number of CD cycles. Of the five dropouts, three were discontinued at the patient’s request. Strict adherence to our protocol rules meant that two patients failed CD, for a success rate of 86.7%. These two patients had initial CHRs with respiratory and circulatory symptoms and were at risk of developing severe adverse events during CD. As mentioned previously, we speculate that more stringent eligibility criteria for CD and exclusion of these two patients would have resulted in a higher success rate of desensitization, consistent with that reported by Li et al. .The discontinuation criteria in this study were not clear, and the decision to discontinue the CD depended on the judgment of the attending doctor, which could obscure the results of the analysis. In our study, two patients with CHR that affected their hemodynamic status and respiratory function discontinued CD and did not resume the subsequent CD cycle because of the strong expectation of more serious life-threatening adverse events. On the other hand, the three patients with milder symptoms also discontinued the subsequent CD cycle after consulting with their doctors, considering that the purpose of treatment for recurrent cancer was not curative. Lee et al. and Castells et al. allowed resumption of subsequent CD, even in patients who required adrenaline, corticosteroids, or oxygen, but Takase et al. did not ,19,22. TApart from the ambiguity of patient eligibility criteria and treatment discontinuation criteria mentioned above, the limitations of our study are its small sample size and retrospective nature. Because this was a retrospective study, patient characteristics were uncontrolled and varied in disease, stage, and the number of treatments. Therefore, in the future, it will be necessary to carefully consider the adaptation and discontinuation criteria, develop and comply with a protocol with unified standards, and conduct a large-scale investigation. In addition, the fact that male patients are not included in this study limited to gynaecologic cancers, will limit the versatility of this protocol. Although there are successful reports of CD on other types of cancer patients, that included males, using a protocol similar to ours, the findings derived from our study are limited to females ,42. As aIn conclusion, our findings suggest that CD is a good option for patients with a history of platinum-sensitive gynaecological cancer and CHR. CD carries some risk, but strict adaptation and discontinuation criteria may contribute to risk reduction. In addition, our improved protocol, which secures a day dedicated to carboplatin administration only, and which strictly regulates a slow administration, as a re-administration method after CHR, made it possible to administer CD more safely than the conventional protocols.


This study performed a prediction and risk factor analysis of diuretic resistance (DR) in patients with decompensated heart failure during hospitalization.The data of patients with decompensated heart failure treated in 2010–2018 with DR  or without DR  were retrospectively collected from Chinese PLA General Hospital medical records. Statistical analysis of baseline was performed on two groups of people, and the risk factor of DR was analyzed through logic regression. Six machine learning models were built accordingly, and the adjustment of model super parameters was performed by using Bayesian optimization method. Finally, the optimal algorithm was selected according to prediction efficiency.2); serum albumin, 33 g/L; hematocrit, 0.32% and 0.56%; red blood cell volume distribution width, 13; and age, 59 years. The optimal area under the curve was 0.9512. The ranked features derived from the model were age, abnormal sodium level, pro-BNP level, serum albumin level, D-dimer level, direct bilirubin level, and eGFR.The preliminary analysis of variance showed significant differences in the incidence of DR among patients with lung infection, hyperlipidemia, type 2 diabetes, and kidney disease. There were significant differences in estimated glomerular filtration rate (eGFR) (P < 0.001). In addition, some physical indicators like BMI were different, the laboratory results like mean red blood cell volume or C-reactive protein assay were also significantly different. The optimal classification model indicated that the best cutoff points for risk factors were vein carbon dioxide, 21 mmol/L and 29 mmol/L; total protein, 64 g/L; pro-brain natriuretic peptide (pro-BNP), 7,600 pg/mL; eGFR, 50 mL/(min ∙ 1.73 mThe DR risk prediction model based on a gradient boosting decision tree created here identified its important risk factors. The model made very accurate predictions using simple indicators and simultaneously calculated cutoff values to help doctors predict the occurrence of DR. Heart failure, a complex syndrome that develops in the terminal stage of cardiovascular disease, seriously threatens patient life and health. More than 26 million people worldwide are hospitalized annually for acute heart failure 23, with 24Congestion, the most frequent 70%) clinical manifestation is characterized by volume overload and insufficient cardiac output 7. Its re0% clinic9101113+) output sufficiently to relieve volume overload, edema, or congestion, despite escalating doses of a loop diuretic to a ceiling level. It is a major cause of recurrent hospitalizations in patients with chronic heart failure and predicts death but is difficult to diagnose unequivocally  = 186 × serum creatinine (mg/dL) – 1.154 × age – 0.203[The patients’ baseline characteristics were the first valid recorded values after hospital admission. All the indicators used for prediction were the baseline indicators of the patient at the time of admission, and the ion fluctuations were also the data measured before the diagnosis of DR. Hypertension, hyperlipidemia, kidney disease, pulmonary infection, and tumor were obtained from the history diagnosis results in the electronic medical record system. Body mass index (BMI) was calculated as follows: BMI = weight (kg)/height .t-test for continuous variables. Normality tests were performed using the Shapiro–Wilk test. Normally distributed continuous variables, non-normally distributed continuous variables, and categorical variables are expressed as mean ± standard deviation, quartiles, and count or percentage, respectively; differences were detected using the two-sample independent t-test, rank sum test, and chi-square test, respectively. The difference between eGFR and categorical variables in the occurrence of DR was analyzed using binary logistic regression, and the odds ratio (OR) and 95% confidence interval (CI) was calculated. SPSS software for Windows  was used for the statistical analyses.The demographic and baseline characteristics of the study population were compared using the Pearson chi-square test for categorical variables and Student’s https://shap.readthedocs.io/en/latest/api.html.We randomly split the data of DR and non-DR patients by a 7:2:1 ratio for training, internal validation, and testing, respectively, and 10-fold cross validation was adopted. Different machine-learning classification algorithms were used for the classification analysis. The six algorithms were gradient boosting decision tree (GBDT), extreme gradient boosting, random forest, light gradient boosted machine, decision tree, and support vector machine. For model evaluation, we added five evaluation indexes including precision, recall, F1-score, accuracy and AUC. As for parametric tuning, we used Bayesian optimization method to automatically adjust the model hyperparameters. The predictive capabilities of the different machine learning algorithms through the 5 evaluation index enabled the more reliable calculation of the precise cutoff values of the indicators. Shapley additive explanation (SHAP) value analysis graphs were used to show the specific values of the turning points of the important biological detection indicators to provide better guidance and advice for clinical judgment . The SHATable 1. Of the population, 17.97% developed DR. Women had a higher DR rate than men. More DR patients had hypokalemia, hyponatremia, pulmonary infection, type 2 diabetes mellitus, and kidney disease, whereas a smaller proportion suffered from hyperlipidemia, and there were no significant differences in the incidence of tumor and hypertension. Patients with DR had a lower blood pressure and BMI. Pro-brain natriuretic peptide (BNP), troponin T, and international normalized (IN) ratio levels were very high in patients with DR, indicating that these patients had advanced heart failure. Concurrently, the mean direct bilirubin level, which reflects congested liver function, was higher in the DR group. Elevated creatinine and reduced eGFR also indicated poor kidney function in patients with DR. According to the four CFR staging derived from eGFR values (eGFR below 15 was excluded), the proportion of patients with diuretic resistance was significantly lower in stageI–II and significantly higher in stage III–IV. Fewer DR patients had undergone percutaneous coronary intervention. The all-cause mortality rate was 20.1%, while that of the non-DR group was only 3.7%.The baseline characteristics of the total population and the groups stratified by the occurrence of DR are presented in Figure 2, abnormal sodium and potassium levels increased the risk of DR . Pulmonary infection and kidney disease also increased the risk of DR ; P < 0.001, respectively). The population was divided into four age groups: ≤45, 45–65, 65–75, and ≥75 years, showed as generation; each additional unit increased the OR to 5.289. eGFR was negatively proportional to DR, the smaller the eGFR value, the more likely DR occurred.Binary logistic regression modeling was performed for the risk factor analysis. As shown in Figure 3, six machine learning algorithm models were implemented to classify the patients into DR and non-DR groups. According to the values of comprehensive indexes F1-score and AUC, GBDT is the model with the best performance . According to the optimal model, the best cutoff point for the risk factors was calculated . The optimal classification model indicated that the best cutoff points for risk factors were: vein carbon dioxide, 21 and 29 mmol/L; total protein, 64 g/L; pro-BNP, 7,600 pg/mL; eGFR, 50 mL/(min × 1.73 m2); serum albumin, 33 g/L; hematocrit (HCT), 0.32% and 0.56%; blood cell volume distribution width, 13; and age, 59 years .As shown in Figure 5A,B). The SHAP value is used to calculate the marginal contribution of a feature when it is added to the model, and the different marginal contributions of the feature in the case of all feature sequences [Figure 5A represents the SHAP value of one feature of an instance. The position on the y-axis was determined by the feature, while the position on the x-axis was determined by the SHAP value. The color represents the feature’s value from small to large, which allows understanding of the distribution of the SHAP values for each feature. Figure 5B presents the feature importance ranking, showing that age; sodium abnormality; pro-BNP, serum albumin, D-dimer, and direct bilirubin levels; and eGFR are more important. Figure 5C shows that we used the SHAP to explain the prediction of a single sample. Each feature value is a force that increases or decreases the DR likelihood. The prediction starts from the baseline, which is the average of all predictions, and each SHAP value is an arrow that increases  or decreases  the prediction. Figure 5D shows the probability of DR after a patient’s indicators are entered in the model and the impact of each indicator on the model’s results. The random input of a patient’s indicators shows that the probability of this patient suffering from DR is 97%. We verified that DR indeed occurred, indicating the model’s reliability.We calculated the importance of each feature according to the SHAP values .The main finding of this study is that the application of machine learning to predict DR prediction is more efficient than traditional statistical analysis and can find the best cutoff value that will provide doctors with more advisable information. Interestingly, the optimal model accurately predicted the probability of a person developing DR and provided the relevant indicators’ importance. A sub-analysis of the Organized Program to Initiate Lifesaving Treatment in Hospitalized Patients with Heart Failure (OPTIMIZE-HF) illustrated that 20% of patients had hyponatremia at the time of admission , a findi30Predicting whether a patient will develop DR at an early stage of admission is important since it facilitates the adjustment of drugs, relieves dyspnea sooner, shortens the length of hospital stay, and reduces the risk of readmission. Valente et al. previously reported the association of a poor diuretic response with more advanced heart failure, renal impairment, diabetes, and atherosclerotic disease . Many clIn our research, SHAP was used to explain the model to obtain the influence index of this result. By inputting the observed indicators of one person into the model, we were able to quickly predict the probability of DR at the initial admission. This could help in the early treatment of patients with DR, which might improve their prognosis.In conclusion, here we constructed a DR risk prediction model based on a GBDT that can identify important risk factors. The model can make very accurate predictions for individuals based on simple indicators. Simultaneously, the model can calculate cutoff values to help doctors judge the occurrence of DR.Since this is a retrospective data analysis, the drug used by patients with heart failure is very complicated, and our research has not been included in the drug analysis, which is a limitation. Additionally, this was a single-center observational study and not a randomized controlled trial; as such, although the total dataset was large, unforeseen confounding factors could have affected the results. We currently only predicted the occurrence of DR in hospitals; in the future, we will continue to study and analyze the applicability of diuretic drugs for avoiding adverse events.Since the data used in this study is the medical data of the military hospital, involving the management of the army, so our data cannot be copied and disclosed.