Recently, the fabrication of MXenes thin films has attracted significant attention toward electronic devices and sensor applications. This review summarizes the exciting features of MXene thin film fabrication methods such as vacuum-assisted filtration (VAF), electrodeposition techniques, spin coating, spray coating, dip-coating methods, and other physical/chemical vapor deposition methods. Furthermore, a comparison between different methods available for synthesizing a variety of MXenes films was discussed in detail. This review further summarizes fundamental aspects and advances of MXenes thin films in solar cells, batteries, electromagnetic interference shielding, sensing, etc., to date. Finally, the challenges and opportunities in terms of future research, development, and applications of MXenes-based films are discussed. A comprehensive understanding of these competitive features and challenges shall provide guidelines and inspiration for further growth in MXenes-based functional thin films and contribute to the advances in MXenes technology. Two-dimensional MXenes possessed exceptional physiochemical properties such as high electrical conductivity (20,000 Scm T. T56]. Tan hours . Anotheran hours ,60.3C2 MXene nanosheets using the vacuum deposition method on Millipore filters . M. M3C2 MXapacitor . Furtherlectrons . VAF metapacitor . Other capacitor .3C2/PEDOT: PSS-based biomimetic thin films shield by using VAF . O. O75]. Oic value C.3C2 MXene thin films on silica substrate by using SCM for a memristive device  dispersant. The conductivity value for thicker and thinner V2CTx films was about 3300 ± 100 S/cm .ications . Multiplabsorber C,D 91]..3C2 MXene sensor E 96]. M. M3C2 MX3C2@montmorillonite MXene sheets were fabricated by dipping the substrate into piranha solution for one hour and then in poly(vinylpyrrolidone) solution for 15 min. Lastly, dipping this substrate into Ti3C2@montmorillonite solution caused the formation of bilayer sheets. After repeating these dipping cycles, it is possible to obtain the desired number of bilayers of sheets . . 3C2 MXeadiation . Anotherof 10 nm C 103]. . 3C2 MXen et al. B 104]..3C2 MXen3C2 or Mo1.33C MXene made a stable suspension with monomer . These monomers will lose electrons during the electrochemical polymerization process and form cationic radicals to form polymer chains. Negatively charged MXene will move toward the working electrode because of the electric field gradient and doped into the polymer chains to create a complex film  (PVA) and poly (sodium 4-styrene sulfonate) (PSS) . The tun2C and Nb2C MXene thin film fabrication by chemical etching of magnetron sputtered Ti2AlC and Nb2AlC . F. F3C2 MXto 9 nm) B 65]. S. S3C2 MXxibility C 88]. A. A3C2 MXhickness D 99]. . 3C2 MXe3C2 MXene thin films by using vacuum filtration and spray-coating methods . . −1) andin films C 147]..−1) and 3C2 MXene to obtain flexible and green EMI shielding material. The integrated electron migration in Ti3C2 MXene thin films causes the dissipation of EM waves in the form of heat ..−1) can 3O4 and NiCo2O4 with Ti3C2 MXene using three different approaches, including repeated filtration, spray coating, and wet chemical method. As prepared, flexible metal oxide and MXene-based films were utilized in Li-ion batteries without any binder. The free-standing hybrid films synthesized using spray coating have shown excellent electrochemical performance with a high reversible capacity of about 1330 mAh g−1 at 0.1 C  was obtained by VFM . . 2, and d by VFM . MXene m−1 pressure sensitivity at 0.45 MPa elastic modulus. Furthermore, this flexible sensor has shown a fast response time and monitors human heart motion . . 3C2Tx ther ions E.3C2Tx. The rigidity of Ti3C2Tx results in the formation of an ordered 2 nm channel, and this membrane is applied to separate dye molecules from water (MXene thin films, due to their extraordinary small thickness, have been explored to filter unrequired guests. Wang et al. utilized VFM to acquire regular and lamellar membrane of Tiom water F 172]. . 3C2Tx. om water G,H. The In recent years, along with diversity in the class of MXene thin films, a more comprehensive range of applicability has been observed in different fields, including sensors, photodetectors, motors, energy storage, catalysis, and separation. Few applications required specific thickness of the films, possibly obtained by utilizing different methods. MXene thin films have been widely explored in lab-based applications, but still, an improvement is required for industrial scale. Further, doping of MXene with other elements could enhance their applicability in energy storage, sensing, and biomedical field . Lastly,MXenes thin films have been explored widely because of their unique physical, chemical and electrical properties. This review explained MXenes thin film fabrication strategies, including vacuum filtration, electrodeposition, spin, spray, dip coatings, and physical deposition methods. Further, a comparative study on these methods explained well; modification or combination could further develop MXene thin film fabrication strategies. In addition, a suitable summary of progress in MXene thin film applications in electromagnetic interference shielding, solar cells, batteries, sensing, and separation is reviewed. Although MXenes thin films have been used in every aspect of science but still by improving their fabrication strategies, surface modifications, and doping chemistry, these thin films can be used in undiscovered applications, i.e., practical applications at the industrial level. By keeping these things in mind, a few things need to be considered in the future to develop the MXene thin films and their application in different fields.Almost 30 MXene materials have been discovered, and all have not been converted into thin films, which limits the applicability of these MXene materials. Considering fabrication methods of thin films of MXenes, it is well known that spray, spin, dip-coating methods, and VAF have been studied well. Still, less attention has been devoted to fully understanding the MXene thin film synthesis mechanisms involved in electrodeposition methods and chemical-physical vapor deposition. The thickness of MXene thin films fabricated by the aforementioned methods can be tuned from the micro to the nano level. Still, more development is required to fabricate tens of nanometers thin films.Another aspect is that MXene thin films can be easily oxidized in a humid environment, which affects the durability of the MXene films. By effectively using chemical grafting methods to modify the edges and defects of MXene thin films, oxidation can be reduced to a minimal level. In addition, lateral cross-linking of MXenes thin films and strategies to control the fabrication of vertically aligned pores could open new doors for development in electrochemical applications. In summary, the controllability, reproducibility, and robustness of the MXene-based thin film strategies need further development. Furthermore, MXene thin films used for separation and gas sensing should be studied well theoretically using density functional theory and other simulation software to obtain a clear insight into the mechanism. In recent years, along with diversity in the class of MXene thin films, a more comprehensive range of applicability has been observed in different fields, including sensors, photodetectors, motors, energy storage, catalysis, and separation. Few applications required specific thickness of the films, possibly obtained by utilizing different methods. MXene thin films have been widely explored in lab-based applications, but still, an improvement is required for industrial scale. These methods are being used at the lab scale for the fabrication of MXene films for a variety of applications, but to achieve industrial-scale applications still, more fabrication technologies need to be explored.Thin films based on other 2-D materials such as gold nanoparticles, graphene, carbon nanotubes, and black phosphorous gained tremendous attention but difficulty in thin film fabrication and unsatisfying stripping strategies limit their use for advanced applications. In contrast, solution-based procession and cost-effective, large-scale production of MXene-based thin films are favorable for a wide range of applications ,172,176.


The interest in astrocytes, the silent brain cells that accumulate polyamines (PAs), is growing. PAs exert anti-inflammatory, antioxidant, antidepressant, neuroprotective, and other beneficial effects, including increasing longevity in vivo. Unlike neurons, astrocytes are extensively coupled to others via connexin (Cx) gap junctions (GJs). Although there are striking modulatory effects of PAs on neuronal receptors and channels, PA regulation of the astrocytic GJs is not well understood. We studied GJ-propagation using molecules of different (i) electrical charge, (ii) structure, and (iii) molecular weight. Loading single astrocytes with patch pipettes containing membrane-impermeable dyes, we observed that (i) even small molecules do not easily permeate astrocytic GJs, (ii) the ratio of the charge to weight of these molecules is the key determinant of GJ permeation, (iii) the PA spermine (SPM) induced the propagation of negatively charged molecules via GJs, (iv) while no effects were observed on propagation of macromolecules with net-zero charge. The GJ uncoupler carbenoxolone (CBX) blocked such propagation. Taken together, these findings indicate that SPM is essential for astrocytic GJ communication and selectively facilitates intracellular propagation via GJs for negatively charged molecules through glial syncytium. In brain, neurons and glial cells are isolated one from another by the aquatic-intercellular space. Astrocytes are connected by large diameter connexin (Cx) gap junction pores (GJs) that facilitate astrocytic communication and function ,2,3,4. ORecently, PAs and their derivatives have been recognized as novel gliotransmitters ,18,24,34SPD/SPM are observed only in a few adult neurons and neurosecretory-neuronal- synaptic terminals  and the Recently, it was shown that glial cell-to-cell communication in brain , retina Intriguingly, the trafficking of glucose molecules with zero charge (fluorescent 2-NBDG and fluorescent 6-NBGD) via GJs was about two orders higher in the astrocytic syncytium, compared with a negatively charged phosphorylated glucose, such as 2-NBGD-6P (glucose-6-phosphate) ,59. TherAll procedures were carried out in accordance with the National Institute of Health guidelines for the humane treatment of laboratory animals and with the approval from the Universidad Central del Caribe Institutional Animal Care and Use Committee.2/95% O2 and slices were cut using a vibratome . The slices were then incubated for recovery in a standard ACSF solution containing (mM) 127 NaCl, 2.5 KCl, 1 MgCl2, 2 CaCl2, 1.25 NaH2PO4, 10 glucose, 26 NaHCO3, gassed with 5% CO2/95% O2, pH 7.4, (osmolarity 305 mOsm/L). The incubation was for 20 min at 35 °C and an additional 10 min at RT. After 30 min of total incubation, slices were placed in a recording chamber (0.5 mL volume) and superfused continuously with oxygenated ACSF at room temperature . Whole cell recording and fluorescent dye tracing studies were performed as described previously [To avoid age-dependent variation in Cx-based communication, we used brains of 25–30 postnatal-day-old Sprague-Dawley (P25–P30) rats. At this age, glial cells are mature and both Cx 43 gap junctions and Kir4.1 channels are fully developed ,67,68. Teviously ,65.2, 10 HEPES, (1 SPM-Cl was used in part of experiments), pH adjusted to 7.2 with KOH (osmolarity ~285 mOsm/L). After filling with ICS, the final micropipette resistance was close to 8 MΩ, which was optimized for astrocyte recordings to achieve seals of more than 3 GΩ on cell membranes. In slices, voltage clamping and current recording in the whole-cell patch-clamp mode from astrocytes were performed using MultiClamp700B . The pClamp 10 software package  was used for data acquisition and analysis. The traces were low-pass filtered at 3 kHz and digitized at 10 kHz (Axon DigiData 1440A interface) . The electrophysiological data were analyzed with the software, Clampfit 10.2 .MX7500R/L manipulators with MC-1000 drives  were used for positioning micropipettes for whole-cell voltage-clamp and current-clamp recordings. Astrocytes were clamped with patch pipettes made from borosilicate-glass tubing  pulled in four steps using a Flaming-Brown P-97 pipette puller . Pipettes were filled with intracellular solution (ICS) containing (mM) 117 K-gluconate, 13 KCl, 2 MgClAstrocytes were dialyzed in whole-cell configuration in current clamp (zero-current) mode. Each single astrocyte tested was loaded via a patch pipette with the physiological ICS containing different fluorescent dyes in the presence or absence of 1 mM SPM. This concentration of SPM was chosen because it is close to the cytoplasmic SPM concentration of about 800 µM in glia .Astrocyte recordings were accepted only if (i) the membrane potential was negative to ~−70 mV and (ii) if cells had a linear current-voltage relation (adult-passive astrocytes) and (iii) low input resistance (<20 MΩ). We used voltage-clamp mode to test current-to-voltage relationships and to characterize the type of cells. The holding potential was kept equal to the resting potential (to keep zero current), and cells were stimulated by short 60 ms voltage steps applied from the holding potential to −150, and then up to +150 mV in 10 mV increments, or by a “step-ramp” protocol using a step to −120 mV (for 100 ms), followed by a rising voltage ramp to +100 mV during 80 ms, and then a step back to resting voltage. We selected short protocols with a well-tolerated voltage range to help keep cells healthy while cell type was determined. Passive astrocytes, which are part of a large syncytium, produced linear I/V curves and were further utilized for GJ communication experiments. We avoided using astrocytes localized closer to the slice surface because those astrocytes may be artificially decoupled by the trauma obtained during cutting the slice. Instead, we recorded only from astrocytes situated at least 100 µm below the surface of the slice  with isoMorphologically and electrophysiologically identified astrocytes were used but not neurons, NG-2 cells, and oligodendrocytes. Astrocytes accumulating SPM/SPD, pyramidal neurons, and interneurons show different localizations  in stratWe specifically selected the CA1 area of hippocampus where (i) astrocytes are clearly separated from pyramidal neurons in specific areas  and (ii) the astrocyte size is about twice smaller than neurons . In addiWe visualized the dye-coupled astrocytes (loaded with membrane-impermeable dyes via the micropipette) and determined the size of the syncytium, the heterogeneous connectivity, and the extent of dye propagation in recipient astrocytes after 10 min of single-cell dialysis. Counting of coupled cells was carried out in a single X-Y plane (focused on the cell which was filled with fluorescent dye) and was equally applied for all the slices and procedures tested. In addition, for high-resolution imaging and for retention of the dyes after 10 min of single-cell dialysis, each slice was immersed in freshly prepared 4% paraformaldehyde in 0.1 M PBS solution, pH 7.4, in 5 mL tubes. After shaking for 5 min at room temperature, the slices were stored at 4 °C for 1 h. Freshly fixed slices were then processed via confocal microscopy to obtain fresh additional 3D images showing propagation of different dyes with and without cytoplasmic SPM in the glial syncytium using a confocal-scanning microscope .Brain slices (350 µm thick from 25–30 days old Sprague Dawley rats) were used for immunohistochemical studies. We used a total of 56 rats for the experiments presented in In this study, we used several different charged  molecules of fluorescent dyes. Alexa-488-biocytin, Alexa-568, and sulforhodamine-101 were purchased from Molecular Probes. Lucifer Yellow, sulforhodamine-B, 1,1-Diethyl-2,2-cyanine iodide  2-NBD-glucose, and 4-(4-(dimethylamino)-styryl)-N-methylpyridinium, (ASP+) were obtained from Sigma Chemical Co., Ltd. . For testing the compounds, one of these negatively or positively charged, or polar molecules, was added to the ICS at the following concentrations: 2 mg/mL (~3.6 µM) 2-NBD-Glucose ; 2 µM ASCarbenoxolone (200 µM CBX), a gap junction uncoupler, used in this study to block fluorescent dye propagation was purchased from Sigma Chemical Co., Ltd. .t-test. Statistical significance was expected if p < 0.05. Note: brain slices incubated for longer than 1 h were not used. In addition, all the data were tested for normality using the Shapiro-Wilk test .Data were analyzed using pCLAMP-10, version 10.4.0.36  or Origin 8 software, version 8.0725  and were reported as mean ± SEM. Significant differences between groups of data were evaluated using the paired Student’s To visualize astrocytes versus neurons, we used antibodies against the PAs SPD/SPM and the neuronal Kir2.4 channels. We found localization of PAs in astrocytes C. The KiThe next question was: which molecules may be used to analyze transfer via astrocytic GJs? For this, we need to distinguish between membrane-permeable versus membrane-impermeable dyes. To visualize the diffusion only through the gap-junctions, we carefully selected the dyes we would use in our experiments. We found that some dyes were crossing the astrocytic membrane and, therefore, were not suitable to study intracellular propagation through the astrocytic syncytium.Several dyes  were takIn addition, there are large pores, such as connexin ,99,100 aUsing the whole-cell configuration, we examined the propagation of various dyes through the astrocytic syncytium with and without SPM in the patch pipette. We used a concentration of SPM (1 mM) close to the physiological concentration  to estimTo obtain representative images  of dye pIn the absence of SPM  permeates via GJ syncytium much less than the larger molecular-weight dyes , indicating the opposite of what was predicted A. The avTherefore, we calculated the ratio of the charge to weight of the molecules, and this appears to be a key determinant for molecular propagation. Indeed, the ratio of net charge and molecular weight of the dye clearly correlated with the number of cells coupled C. Therefp < 0.01), and also increased the level of coupling of negatively charged Alexa 488-biocytin (p < 0.01). The most dramatic effect of SPM was seen with LY, control , compared to SPM-treated group  which was a 292% increase.It is known that Cx pores may be negatively charged and retain charged molecules, as has been shown for Cx40 and Cx43 ,113,114.biocytin C1, contrThese results suggest that SPM affects only permeation of negatively charged molecules, while having no effect on neutral ones. Indeed, as was expected, we observed a lack of correlation between dye coupling and the astrocytic membrane potential in the control group and in the SPM-treated group  to negatively charged dye molecules as was shown for acid proteins, ATP, and RNA, etc. in the cytoplasm [PA signaling through gap junctions may be a key to regulate cell proliferation , particuytoplasm . By bindytoplasm ,136,137;ytoplasm . So, an ytoplasm ) and resytoplasm ,26.4+ binds directly in the Cx43-pore and neutralizes the charge in this pore, and thus, making the pore wall electrically inert and enhancing the propagation of negatively charged molecules. Testing this mechanism in a separate large study is required. Therefore, PAs and their derivatives may serve many functions both within the astrocytic cytoplasm and upon being released from glial cells [An alternative, but only theoretical explanation, could be that positively charged SPMal cells ,11,14,15In conclusion, our findings suggest that we may predict the extent of coupling between astrocytes for novel, untested molecules or fluorescent dyes by knowing MW and charge, but most importantly, for biologically active molecules and drugs. By extension, this suggests that SPM will facilitate diffusion of negatively charged intracellular-organic and -inorganic anions, amino acids, polypeptides, and other signaling molecules through the astrocytic syncytium, and may therefore, have profound effects on glial-neuronal signaling.


GOT2  is essential for regulating cellular energy production and biosynthesis, linking multiple pathways. Nevertheless, the expression profile and prognostic significance of GOT2 in KIRC remain unclear. This study comprehensively analyzed the transcriptional levels, epigenetic regulation, correlation with immune infiltration, and prognosis of GOT2 in KIRC using rigorous bioinformatics analysis. We discovered that the expression levels of both mRNA and protein of GOT2 were remarkably decreased in KIRC tissues in comparison with normal tissues and were also significantly related to the clinical features and prognosis of KIRC. Remarkably, low GOT2 expression was positively associated with poorer overall survival (OS) and disease-free survival (DFS). Further analysis revealed that GOT2 downregulation is driven by DNA methylation in the promoter-related CpG islands. Finally, we also shed light on the influence of GOT2 expression in immune cell infiltration, suggesting that GOT2 may be a potential prognostic marker and therapeutic target for KIRC patients.Clear cell renal cell carcinoma (KIRC) is the most common and highly malignant pathological type of kidney cancer, characterized by a profound metabolism dysregulation. As part of aspartate biosynthesis, mitochondrial  Renal cell carcinoma (RCC) is one of the most common malignancies of the genitourinary system, representing 3% of all malignancies in adults . In the VHL) gene. VLH inactivation results in constitutive activation of hypoxia-inducible factors (HIF-1α and HIF-2α), thereby altering many genes involved in angiogenesis, metabolism, chromatin remodeling, extracellular matrix (ECM) and DNA repair [PBRM1, SETD2, BAP1, KDM5C, and MTOR) have been continuously identified to contribute to the pathogenesis (used to classify tumors into subgroups) and metabolic remodeling process of KIRC [Current shreds of evidence have demonstrated that KIRC is a metabolic disease ,16,17,18A repair ,19,20,21 of KIRC ,23.MDH2 and glutamic-oxaloacetic transaminase 2 (GOT2) and then transported to the cytosol for use by GOT1 and other enzymes [The metabolic shift in KIRC tumors covers different pathways and specific intermediates  ,24, allo enzymes .GOT2, situated on chromosome 16q21, is a crucial enzyme for cancer cell metabolism, (i) mediating the reversible interconversion of oxaloacetate and glutamate into aspartate and α-ketoglutarate, providing energy for tumor cells (Krebs cycle) [GOT2 expression significantly influences tumor growth and the prognosis of several human neoplasms [GOT2 in the development and prognosis of KIRC has not been reported. To address these issues, this study aims to evaluate the expression levels of GOT2 in KIRC and determine its epigenetic modulation, prognostic value, and correlation with tumor-infiltrating immune cells in KIRC patients through multiple databases.s cycle) ; (ii) bes cycle) , maintais cycle) . Increaseoplasms ,36,37,38GOT2 transcription levels was performed via the TIMER2.0 database  [p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001).Initially, pan-cancer analysis of er 2021) , using thttp://gepia.cancer-pku.cn/index.html, accessed on 1 December 2021) [http://www.gtexportal.org/home/index.html, accessed on 1 December 2021) (100 samples). The differential threshold of log2FC was 1 and the value cutoff of 0.05.To keep the focus of our analyses on KIRC tumors, the GEPIA2 database  (er 2021)  was usedhttp://ualcan.path.uab.edu/index.html, accessed on 1 December 2021) [GOT2 expression in the high-throughput mass spectrometry data, obtained from the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of normal kidney tissues (n = 84) and primary KIRC tumors (N = 110) [At the protein level, we used the UALCAN platform (er 2021) ,42 to miN = 110) . IntegraN = 110) ,45. Briehttps://www.proteinatlas.org/, accessed on 1 December 2021) [https://www.proteinatlas.org/about/assays+annotation#ihk, accessed on 1 December 2021).In this study, we checked the expression of GOT2 in the protein expression module of the HPA database  ,47,48, aGOT2 were analyzed using the UALCAN  [Associations between clinicopathological parameters and mRNA expression of er 2021) . For theer 2021) , nodal mhttp://kmplot.com/analysis/, accessed on 5 December 2021) [http://gepia.cancer-pku.cn/index.html, accessed on 1 December 2021) [http://www.proteinatlas.org/, accessed on 5 December 2021) [GOT2, and then these two groups were compared in terms of relapse-free survival. Moreover, the hazard ratio (HR) with a 95% confidence interval (CI) and the p-value of the log-rank test were obtained. For all survival analyses, p < 0.05 was considered statistically significant.The prognosis analysis was estimated by Kaplan–Meier (KM) survival curves generated by the Kaplan Meier (KM) Plotter (er 2021) , GEPIA2 er 2021)  and HPA er 2021) ,47,48. Ihttps://biit.cs.ut.ee/methsurv/, accessed on 10 December 2021) [http://www.bioinfo-zs.com/smartapp/, accessed on 10 December 2021) [GOT2 probe and Spearman’s correlation between methylation level  and mRNA level . CpG-aggregated methylation values were determined by mean .To explore the DNA methylation level of all CpG islands located in GOT2 of KIRC-TCGA samples, we used the MethSurv database (er 2021) . Next, Ser 2021)  was usedGOT2 gene expression contributed by different immune cell types in kidney samples  by the GEPIA2021  [+ T cells, resting memory CD4+ T cells, naïve CD4+ T cells, CD8+ T cells, follicular helper T cells (Tfh), regulatory T cells (Tregs), and gamma/delta T cells, activated dendritic cells (DC), resting dendritic cells, eosinophils, macrophages (M0–M2), activated mast cells, resting mast cells, monocytes, resting NK cells, activated NK cells, neutrophils, and plasma cells. ANOVA  was used for quantitative comparison. Sidak’s multiple comparisons test was used for the post-test, and p < 0.05 was considered significant.We calculated and compared the er 2021) . For eachttp://tisch.comp-genomics.org/home/, accessed on 1 April 2022) is an online single-cell RNA-seq database focused on the tumor microenvironment (TME) [GOT2 average expression at the single-cell level. The expression of GOT2 was collapsed by the mean value. The gene expression level displayed using UMAP and violin plots was quantified by the normalized values.The Tumor Immune Single-cell Hub  . In our nt (TME) ,56 were GOT2 expression correlates with cancer, we surveyed GOT2 expression in multiple cancer types and adjacent normal tissues through the TIMER database. As shown in GOT2 expression was higher in BLCA , CESC , COAD (Colon adenocarcinoma), KICH (Kidney Chromophobe), ESCA , LUAD (Lung adenocarcinoma), LUSC (Lung squamous cell carcinoma), STAD (Stomach adenocarcinoma) and UCEC . Conversely, GOT2 had markedly lower expression in CHOL (Cholangiocarcinoma), GBM (Glioblastoma), KIRC , LIHC (Liver hepatocellular carcinoma), PRAD (Prostate adenocarcinoma) and THCA (Thyroid carcinoma). All these data indicated that the dysregulation of this glutamic-oxaloacetic transaminase was common across several tumors, including KIRC.To determine whether GOT2 performing a single-gene differential analysis using RNA-seq data from the TCGA database (KIRC-TCGA), compared with non-tumor tissues from the GTEx database by GEPIA2. Our results showed that the mRNA expression levels of GOT2 in KIRC tissues (n = 523) were significantly lower than in adjacent normal tissues (n = 100)  A. Corresn = 100) B, consisn = 100) C. This fGOT2 expression in KIRC. Data showed that GOT2 expression levels were significantly associated with stage, gender, grade, KIRC subtypes, and nodal metastasis status  and disease-free survival (DFS). As shown in GOT2 were significantly shorter than those with high expression. At the same time, we also noticed that the low level of GOT2 protein was significantly related to the worse OS (p = 0.023) , grade 3 , and low mutation burden   C. Additi × 10−2) . Here, tGOT2 underexpression, we analyzed the methylation level of seventeen probes covering the island (promoter region), N Shelf, S Shore, and Open Sea regions of GOT2, chosen through the UCSC Genome Browser (GOT2 lay on probes at the promoter (island). At the same time, most hypermethylated sites fell in the open sea, N Shelf, and S Shore regions   (GOT2 in KIRC patients.To further explore the epigenetic mechanism underlying  Browser . Notably regions . Given t samples . Interes0.00022) A. Furthe 0.0071) B, thus iGOT2 could potentially impact immune cell infiltration in KIRC, we first examined the differences in GOT2 expression across the six immune subtypes proposed by Thorsson et al. [GOT2 expression was highest in patients harboring the C5 subtype  and lowest in patients exhibiting the C2 subtype (IFN-gamma dominant), indicating that GOT2 can be used as a marker for immunophenotyping of patients with clear-cell renal cell carcinoma  cells, M1 and M2 Macrophages in KIRC-TCGA tumors than in normal tissues , plasmacytoid dendritic cells (pDCs), exhausted CD8+ T Cells (CD8Tex), Treg cells and conventional dendritic cells 2 (cDC2)  that also co-expressed some immune checkpoint inhibitors   A,B. Thesnd CD96) , thus stGOT2 generates aspartate from oxaloacetate and glutamate [GOT2 in KIRC. Herein, we elucidated the most comprehensive insights into understanding the epigenetic regulation and the potential association of GOT2 with the clinical and immunity of KIRC.KIRC is characterized by profound metabolic reprogramming that involves multiple pathways ,15. Currlutamate . AdditioGOT2 is differentially expressed in 18 tumor types, thus potentially being a therapeutic target. Further interrogating KIRC, we showed that the GOT2 mRNA and protein levels were markedly decreased in KIRC patients than in normal tissues. Besides, we observed that this transaminase was markedly lower as the pathological stage increased and was also strongly impacted by other clinicopathological characteristics, which conferred a worse outcome. Our findings are consistent with Zhao et al. [GOT2 in hepatocellular carcinoma (HCC). The results from a recent study support a scenario in which in VHL-deficient KIRC, but not in non-clear renal cell carcinomas (NCRCC), the simultaneous suppression of GOT1 and GOT2 is HIF1α-dependent, which impairs oxidative and reductive aspartate biogenesis [GOT2 in KIRC patients harboring VHL, PBRM1, and SETD2 mutations seen in our study, thus suggesting that GOT2 repression represents a specific metabolic feature of KIRC.Based on a pan-cancer perspective, we initially demonstrated that o et al. , who alsogenesis . Hence, ogenesis , glutamiogenesis . This coGOT2 expression in KIRC. Here, for the first time, we provided evidence that the methylation of the GOT2 promoter was increased in KIRC patients compared to normal samples. Additionally, the correlation analysis results revealed that promoter methylation was negatively correlated with the regulation of gene expression. According to these results, it can be speculated that the DNA hypermethylation in the promoter-associated CpG islands may be one of the mechanisms leading to GOT2 down-expression in KIRC. However, additional efforts are necessary to determine the potential impact of additional events, such as chromatin structural modifications, miRNAs, and the influence of metabolites on patients exhibiting GOT2 promoter hypermethylation.DNA methylation of specific CpG sites in the promoter region is tightly linked with transcription repression. In the last few years, its role in carcinogenesis has been of considerable interest ,68,69. IGOT2 expression and various tumor-infiltrating immune cells in KIRC. Previous studies have found that T cells and macrophages represented the dominant populations in most KIRC cases [GOT2 expression was more likely to affect the tumor infiltration of subtypes of T cells, especially CD8+ T cells and follicular helper CD4+ T (Tfh) cells, and M1 and M2 macrophages compared to normal renal tissue. Our deep-dissection of individual cell subsets from scRNA-seq data revealed that GOT2 was broadly expressed within exhausted CD8+ T Cells (CD8Tex) and in the proliferative T cells (Tprolif). Unlike all solid tumors, high tumor-infiltrating CD8+ T-cells predicted poor overall survival and inferior therapeutic responsiveness in patients with KIRC [+ T-cells exhibited four distinct groups that may represent transcriptional states upon tumor infiltration with distinct prognostic significance: two of them were associated with a PD-1+TIM-3+ exhausted subcluster, one with a proliferative subcluster, and a fourth with the higher levels of cytokine signaling [+T-cells) and TAM_3 (macrophages) showed a better prognosis. In another study, a first-in-class CAR T-cell therapy co-expressing GOT2 enhanced T-cell metabolic function for treating GPC3-positive solid tumors, supporting the progress of a future first-in-human trial in subjects with GPC3-positive tumors [GOT2 is likely to play distinct roles at different stages of T-cell exhaustion and might potentially be modulated by the spectrum of changes in TME conditions of KIRC patients, including tumor metabolism, hypoxia, nutrient restriction, and exhaustion driven by chronic stimulation, thus strengthening the potential application of synergic modulation of the GOT2 and T cell exhaustion markers in non-responsive KIRC patients to boost antitumor and immune responses.In addition, another innovative aspect of this study clarified the significant correlations between RC cases ,74, consith KIRC ,76,77. Hignaling . Moreovee tumors . ConsideGOT2 protein were significantly decreased in KIRC patients compared to normal ones. This low expression was positively associated with clinicopathological features, culminating in poor clinical outcomes for KIRC patients. Notably, we provide the first mechanism insights into the epigenetic-mediated regulation of GOT2, which is driven by the DNA methylation in the promoter-related CpG islands. Finally, we also shed light on the influence of GOT2 expression in immune cell infiltration, suggesting that GOT2 may be a potential prognostic marker and therapeutic target for KIRC patients.In conclusion, using a series of rigorous bioinformatics analyses, we showed that the mRNA and the expression levels of the


Klebsiella pneumoniae is a notorious human pathogen involved in healthcare-associated infections. The worldwide expansion of infections induced by colistin-resistant and carbapenemase-producing Enterobacterales (CPE) isolates has been increasingly reported. This study aims to analyze the phenotypic and molecular profiles of 10 colistin-resistant (CR) isolates and 2 pairs of colistin-heteroresistant (ChR)  isolates of K. pneumoniae CPE sourced from two hospitals. The phenotypes of strains in the selected collection had been previously characterized. Antimicrobial susceptibility testing was performed using a Vitek 2 Compact system , the disc diffusion method, and broth microdilution (BMD) for colistin. Whole-genome sequencing (WGS) did not uncover evidence of any mobile colistin resistance (mcr) genes, although the mgrB gene of seven isolates appeared to be disrupted by insertion sequences (ISKpn25 or ISKpn26). Possible deleterious missense mutations were found in phoP (L4F), phoQ , pmrB , and crrB (P151S) genes. The identified isolates belonged to the following clonal lineages: ST101 (n = 6), ST147 (n = 5), ST258 (n = 2), and ST307 (n = 1). All strains harbored IncF plasmids. OXA-48 producers carried IncL and IncR plasmids, while one blaNDM-1 genome was found to harbor IncC plasmids. Ceftazidime–avibactam remains a therapeutic option for KPC-2 and OXA-48 producers. Resistance to meropenem–vaborbactam has emerged in some blakPC-2-carrying isolates. Our study demonstrates that the results of WGS can provide essential evidence for the surveillance of antimicrobial resistance. Klebsiella pneumoniae being the principal pathogen associated with significant mortality [Recently, it has been reported that carbapenemase-producing Enterobacterales (CPE) isolates are expanding at an alarming rate around the globe ,2. Theseortality ,4.K. pneumoniae strains continues to be problematic [Antimicrobial resistance of blematic . Resistablematic ,5,6. Aroblematic . K. pneumoniae carbapenemase (KPC), metallo-β-lactamases (MBL), and oxacillinase-48-like (OXA-48-like), are principally encoded on plasmids and linked with diverse mobile genetic elements [The main carbapenem-hydrolyzing enzymes, K. pneumoniae multilocus sequence type ST258/512 is a recognized “high-risk international clone” [K. pneumoniae ST258 and the special relationship with epidemic resistance plasmids of incompatibility group F (IncF), which have diverse replicon types  [K. pneumoniae ST258 and have not been determined to contribute to international dominance [blaOXA-48-like genes, and IncA/C and IncN types with blaNDM genes that encode New Delhi metallo-β-lactamases (NDMs) [The global propagation of carbapenemases is the result of a continuous interaction between the clonal dispersion of some efficient CPE lineages and the horizontal transfer of their resistance genes . KPC-prol clone” . In Eurol clone” , offerinl clone” . The issand FII) ,12. The and FII) ,12. The ominance . IncL/M s (NDMs) ,11.K. pneumoniae strain ST23, which integrates both hypervirulence and resistance to carbapenems, with dramatic clinical consequences [peg-344, iro , iuc (aerobactin synthesis), rmpA (regulator of mucoid phenotype), and rmpA2 are highly predictive of strains as hypervirulent [The situation has evolved with the emergence of equences ,13. Biomvirulent . phoPQ and pmrAB and the feedback regulator mgrB gene and the colistin resistance regulation (crrAB) operon, respectively [mgrB gene mediates the inactivation and disruption of this regulator gene, conferring colistin resistance [mcr) genes, mcr-1 to mcr-10, with diverse variants [The worldwide escalation of colistin-resistant CPE isolates is of significant concern . Colistiectively ,18. Transistance ,20,21. Asistance . Howeversistance . Of partsistance ,15,16,18variants  that encvariants .Despite continuous research activity, there is still an incomplete understanding of the molecular mechanisms of colistin resistance and heteroresistance ,24, alonK. pneumoniae CPE, collected from two Romanian hospitals in 2017 and 2021, toward delineating the variety of molecular substrates of colistin resistance and heteroresistance, identifying the genetic determinants of resistance to other classes of antibiotics, confirming the presence of circulating high-risk clones and dominant plasmids, and exploring potential therapeutic options, including novel β-lactam/β-lactamase inhibitor combinations.Consequently, the aim of the study is to perform a detailed phenotypic and genomic characterization of 10 colistin-resistant (CR) isolates and 2 pairs of colistin-heteroresistant (ChR)  isolates of All isolates presented resistance to multiple classes of antimicrobial agents, specifically meropenem, ertapenem, doripenem, aztreonam, cephalosporins, old generations of β-lactam/β-lactamase inhibitors, ciprofloxacin, and levofloxacin. The phenotypic results of antibacterial drug testing are given in Of the three aminoglycosides tested, gentamicin was the most active in vitro (n = 8 susceptible isolates), while total resistance was noted for tobramycin . Most ofMinimum inhibitory concentrations (MICs) of colistin obtained by Vitek 2 Compact software are given in Colistin susceptibility test results obtained with other phenotypic methods and reference broth microdilution (BMD) are given in SPAdes assembly metrics for the sequenced genomes are presented in All genomes belonged to MDR strains, with several defined resistance genes against different categories of antibiotics, along with other antibiotic resistance determinants, such as insertions, point mutations, or substitutions that lead to the disruption of genes, thus leading to resistance to at least six antibiotic classes.(A) Molecular mechanism of resistance to various antibiotic classes, except colistinblaNDM-1-positive BC7_BM and BC1_TM presented the highest numbers of genetic determinants , while the strain BC2_BM OXA-48 producer had the lowest (n = 12) (The highest heterogeneity of resistance-conferring elements was noted for β-lactams (n = 13), aminoglycosides (n = 11), and fluoroquinolones (n = 8) . Isolate(n = 12) .In agreement with the phenotypic results, the genotypic analysis confirmed the presence of carbapenem-hydrolyzing enzymes responsible for carbapenem resistance.blaCTX-M-15 (n = 12), followed by blaSHV-12 (n = 2). All blaOXA-48 genes were accompanied by blaCTX-M-15 genes. The NDM-1 producer isolate BC1_TM co-presented blaCTX-M-15 and blaSHV-12 genes. In the genome of isolate BC7_BM, blaNDM-1, blaCTX-M-15, and blaCMY-16 genes were found  were the molecular elements most frequently detected, represented by re found .aac(6′)-Ib gene being the most commonly encountered (n = 12 strains)  (aac(6′)-Ib (n = 12), aac(6′)-Ib-cr5 (n = 2), aph(3′)-VI (n = 2), and rmtC (n = 1) (aac(3)-IIe gene (n = 3), followed by the aac(3)-IId gene (n = 1) (aac(6′)-Ib gene (n = 6) and the rmtC gene (n = 1) were responsible for tobramycin resistance.Genes encoding the aminoglycoside-modifying enzyme (AME) were the main elements conferring resistance to aminoglycosides in all strains, with the strains) . All isose (RMT) . Amikaci (n = 1) . Gentami (n = 1) . The aacoqxAB efflux pump genes and various chromosomal mutations in the quinolone resistance-determining region (QRDR) of gyrA and parC genes contributed to fluoroquinolone resistance in all the analyzed strains. Six strains were observed with the genotype profile of gyrA D87N and parC S80I mutations  genes were noted: aac(6′)-Ib-cr5 (n = 2) and qnrS1 (n = 1) (The utations . All the (n = 1) .ompK36 and ompK37, which potentially confer resistance to cephalosporins and carbapenems, identified by ResFinder, are summarized in A number of alterations in the principal outer membrane of porin-encoding genes ompK36 was visualized in five strains . The insertion of ISKpn26 (IS5) upstream of ompK36 (position -48 versus the start of ompK36 gene), which leads to ompK36 inactivation, was visualized in WGS data and further confirmed by Sanger sequencing (data not shown) in the BC3_TM and BC8_BM isolates. The BC1_TM strain possessed an N186K mutation, while BC7_BM carried several single nucleotide polymorphisms (SNPs), along with deletions, insertions, and substitutions. Similarly, the isolates BC3_TM, BC7_BM, and BC8_BM presented SNPs and substitutions in ompK37, while an insertion in ompK35 that produced a frame shift (FS) at amino acid 166 was noted in isolate BC6_BM. ClustalW (BioEdit) alignment of ompK35, ompK36, and ompK37 genes is shown in Similarly, using BioEdit for sequence alignment, the glycine–aspartic acid duplication (GD) (position 134–135 duplicated in 136–137) in (B) Molecular determinants of colistin resistancemcr genes.The genomic results indicate the absence of plasmidial mgrB gene or in the two-component regulatory systems phoPQ, pmrAB, and crrAB , phoQ , and pmrB genes   with the highest colistin MIC value by BMD, 32 mg/L, two distinct classes of insertion sequences, ISL3 (ISKpn25) and IS5 (ISKpn26), were identified to disrupt the nctional . Four ot, V352E) .pmrB gene, but with a supplementary presumptive intolerant L224Q SNP in the phoQ gene. Notably, only the resistant mutants BC12_TM_B_m and BC14_TM_C_m presented deleterious Q317K SNPs in the phoQ gene and P151S in the crrB gene  in the same nucleotide position and an R256G SNP in the rrB gene .pmrA and crrA genes resulting in functional amino acid substitutions were identified in any isolates; only changes encoding neutral amino acid substitutions were identified  scores for amino acid changes with a potentially harmful influence on the biological function of the analogous proteins are given in (C) Association between phenotypic antimicrobial susceptibility test results and genotypic predictionaac(6′)-Ib-cr5 and dfrA14 were detected. Two isolates, BC7_BM and BC9_TM, were categorized as susceptible to tigecycline, but tet(A) and tet(D) genes were identified. Two other strains (BC4_BM and BC10_TM) that showed intermediate susceptibility to tigecycline did not carry tetracycline resistance markers. Conversely, isolate BC14_TM_C_m was interpreted as resistant to chloramphenicol based on both testing methods, but no resistance genes were found upon genetic analysis  and 98.21% (330/336 × 100), respectively.Molecular serotyping determined that six strains were KL17;O1v1, five were KL112;O2v2, two were KL106;O2v2, and one isolate belonged to serotype KL102;O2v2 . No geneblaNDM-1 genome was associated with the IncC plasmid  were assigned by multilocus sequence typing (MLST): ST101 (n = 6), ST147 (n = 5), ST258 (n = 2), and ST307 (n = 1) . The colK. pneumoniae database of the cgMLST.org Nomenclature Server.Nine complex types were identified by core genome MLST (cgMLST). Five of them  were considered new founders by SeqSphere and were included in the Overall, ST101 isolates presented the highest number of distinct plasmid replicon types (n = 7 types). KL17 and KL112 serotypes were exclusively associated with ST101 and ST147, respectively. blaKPC-2 producers: two pairs of ChR with their corresponding resistant mutants (from Târgu Mureș) and BC6_BM (from Baia Mare), collected in the period 2019–2020.MST cluster 1, comprising blaOXA-48 strains, BC2_BM, BC4_BM, and BC10_TM (two from Baia Mare and one from Târgu Mureș), collected in the period 2018–2019.MST cluster 2, consisting of three blaKPC-2 strains, BC3_TM and BC8_BM , collected in the period 2017–2018.MST cluster 3, comprising two The minimum spanning tree of the 14 CR and ChR strains revealed three clusters of closely related strains :MST cluK. pneumoniae CPE, collected from two Romanian hospitals between 2017 and 2021. This information is of critical epidemiological importance in the context of the continuous global expansion of high-risk MDR K. pneumoniae clones [K. pneumoniae CPE isolates at Romanian hospitals, from 32.3% in 2019 to 48.3% in 2020 [In this study, we analyzed the phenotypic features, resistome, virulome, and plasmid content of 10 CR and 2 pairs of ChR  isolates of e clones ,10,27. A in 2020 ,28. The K. pneumoniae CPE isolates. The results may also contribute to mitigating the spread of antimicrobial resistance in Romanian hospitals.To the best of our knowledge, this is the first Romanian study that focuses on evaluating molecular determinants of resistance in CR and ChR blaOXA-48-carrying isolates. In contrast, the resistance to meropenem–vaborbactam in three out of the eight KPC producers represents an unwelcome occurrence in the context of patients who have not previously been exposed to this promising novel antimicrobial compound. In line with previous reports ,3,5, ourK. pneumoniae isolates with the MDR or XDR phenotype [K. pneumoniae by offering an additional reliable basis for resistance [The resistome analysis confirms the existence of an extensive repertoire of antibiotic resistance genes, along with insertions or point mutations, and all our isolates possessed determinants of resistance to β-lactams, amikacin, tobramycin, fosfomycin, fluoroquinolones, colistin, and phenicol. Similarly, several previous reports described a great variety of resistance genes in henotype ,30,31,32henotype . Furthersistance . In the current evaluation, concordance of more than 96% between phenotypic and genomic predictions of antimicrobial resistance was observed. Similarly, Ruppe et al., obtained more than 96% concordance in a study that included 187 Enterobacterales strains tested phenotypically using the disc diffusion method . Howeveraac(6′)-Ib-cr5 gene but without phenotypic expression of amikacin resistance. Vaziri et al. underscored the fundamental role of the qnrB gene in the K. pneumoniae genome, which, in association with the aac(6′)-Ib-cr5 gene, contributes to increased resistance to aminoglycosides, fluoroquinolones, and cephalosporins [Of note, some discrepancies were identified in this study. Two isolates, BC3_TM and BC8_BM, were categorized as resistant to gentamicin by Vitek 2 Compact and susceptible by disc diffusion, but no AME or RMT genes were detected. However, inadequate correspondence between genotypes and inference of the presence of substrates with resistance to aminoglycosides have been extensively reported based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) clinical breakpoints in association with expert rules . Insteadosporins , a conclosporins . In addiosporins . catA1 acetyltransferase and the nonenzymatic cmlA5 genes, encoding resistance to chloramphenicol, were detected in most of our strains. However, discrepant results for chloramphenicol were noted in the case of four strains , showing resistance based on Vitek 2 Compact and susceptibility based on the Kirby–Bauer method, while no resistance genes were detected by WGS. The contribution of another mechanism responsible for chloramphenicol resistance (high expression of efflux systems) should not be underestimated [The stimated , given trmtC gene in NDM-1-positive isolates belonging to the order Enterobacterales [rmtC gene along with blaNDM-1, blaCTX-M-15, and blaCMY-16 resistance genes and express pan-aminoglycoside resistance. Similarly, the co-expression of NDM-1 and rmtC genes, along with at least one ESBL-encoding gene in K. pneumoniae isolates, has previously been documented in Kenya, Turkey, and Saudi Arabia [armA) in K. pneumoniae in 2003 [rmtA to rmtH) and N1-A1408 methyltransferase (MTase) (npmA) have emerged in Gram-negative pathogens in various parts of the world [K. pneumoniae isolates harboring the rmtC gene; the most frequently identified was the armA gene [armA, rmtB, and rmtC genes are distributed worldwide and confer high-level, broad-range aminoglycoside resistance, including against plazomicin, a newly approved aminoglycoside compound that can evade virtually all clinically relevant AMEs, including acetyltransferase (aac), phosphotransferase (aph), and adenylyltransferase (aad or ant) [blaNDM, blaKPC, and mcr genes is a multifaceted topic because of the possible involvement in the expansive dissemination of extensively pandrug-resistant organisms [Previous studies in Romania have reported on the worrisome emergence of the cterales ,40. Thisi Arabia ,42,43. S in 2003 , eight ohe world . In contrmA gene . Among t or ant) . The assrganisms . blaKPC-2-carrying isolates resistant to meropenem–vaborbactam , detailed molecular analysis verified their affiliation with the international high-risk lineages ST258 and ST101, with evidence of either GD134-135 duplication in the ompK36 gene or insertion of ISKpn26 (IS5) upstream of ompK36 in association with SNPs and substitutions in the ompK37 porin gene. Furthermore, even though BC6_BM showed susceptibility to meropenem–vaborbactam, an FS mutation at amino acid 166 in ompK35 was recorded. These mutations are responsible for alterations in permeability that contribute to reduced susceptibility to meropenem–vaborbactam, as previously reported [K. pneumoniae mainly through ompK35 and ompK36 porins, but vaborbactam prefers the latter, which has a narrower inner channel [Among our three reported ,48,49. M channel . However channel , and a h channel . mcr genes were identified. In agreement with other reports from diverse geographical regions ,31,32, cphoPQ signaling, but the expression of this TCRS regulator is upregulated in the presence of a modified mgrB gene, and it remodels LPS through the addition of cationic L-Ara4N, which prevents colistin molecules attaching to the LPS membrane [mgrB gene was the basis for resistance most frequently encountered in our study  and was induced by either ISKpn25 (n = 6), ISKpn26 (n = 1), or a missense mutation M27K (n = 4), predicted as deleterious by bioinformatics tools. Several recent studies have highlighted the pivotal role of ISs in contributing to the emergence of colistin resistance by disrupting the mgrB gene [mgrB gene was also reported to mediate colistin resistance in a previous study [mgrB has also been shown to promote virulence in K. pneumoniae isolates by suppressing the initiation of host defense reactions and limiting the action of multiple antimicrobial peptides [MgrB, a small negative regulatory transmembrane protein, represses membrane ,18,51. MgrB gene ,30,31,32grB gene . The M27us study . In our us study . Inactivpeptides . phoP and phoQ genes, which potentially mediate colistin resistance and heteroresistance. We observed an alteration in the phoP gene induced by a novel deleterious L4F substitution in the BC2_BM isolate, while several new intolerant SNPs , as anticipated by bioinformatics tools, were detected in phoQ in both CR and ChR strains. Meanwhile, other researchers have mentioned the deleterious L26Q substitution in the phoP gene [phoQ gene  [phoP gene (V3F) [In comparison with prior studies, our analysis indicates the occurrence of five previously unreported point mutations in hoP gene . Furthertutions) ,57,58 anphoP can also stimulate the production of PmrA protein, which belongs to the pmrAB TCRS, either directly or indirectly through the adaptor PmrD protein, leading to the addition of cationic pEtN and L-Ara4N moieties to LPS [pmrB, as part of the pmrAB TCRS, was subject to diverse mutations . The R256G substitution has been widely reported to contribute to colistin resistance [Within the complex chromosomal cascade mechanism that confers colistin resistance, s to LPS ,51,59. Isistance ,31,32,53sistance ,58, suggsistance , and thisistance . In agresistance , our KPCsistance , and thesistance .mgrB gene disruption by ISs. Consequently, in the first case, a combination of mutations in mgrB (M27K), phoP (L4F), and phoQ (Q426L) genes was noted, while in the second case, an L26Q substitution in the phoQ gene was associated with R256G mutation in the pmrB gene. However, several reports have mentioned that Vitek 2 Compact is inappropriate for colistin susceptibility testing [Notably, our BC2_BM and BC3_TM isolates, with colistin-susceptible results by Vitek 2 Compact AST N222 cards but an MIC value of ≥8 mg/L colistin by BMD, did not present any  testing ,64,65, e testing . The man testing , at leascrrAB TCRS can activate Pmr A [crrB alone can activate the expression of genes leading to colistin resistance without any contribution from the pmrAB TCRS [crrB gene only in the resistant mutant BC14_TM_C_m isolate, which, in contrast to its susceptible parental strain BC13_TM_C_hR, expressed an MIC value of >64 mg/L colistin. The P151S substitution detected in the putative histidine kinase domain has been validated to induce elevated resistance to colistin [crrB, as conferring colistin resistance [Moreover, the te Pmr A ,59, and colistin . Similarsistance , while Psistance .mgrB alteration caused by an ISKpn25 element insertion at the same position (with the deletion of nucleotides 1–5) and an R256G substitution in pmrB. An identical molecular profile was observed with the BC6_BM strain, which, unlike the two parental ChR strains, had an MIC value of 32 mg/L colistin indicated by BMD. In particular, the two pairs of isolates were supplementarily accompanied by a new L224Q substitution in phoQ, and the two mutants presented either an additional amino acid change P151S in crrB or Q317K in phoQ. Despite the presence of a disrupted mgrB gene in the two parental ChR strains, a possible explanation for the results of colistin susceptibility according to BMD might be the potential suppressor effect of the L224Q mutation in phoQ. In addition, BMD has been considered an unreliable method for the detection of colistin heteroresistance [K. pneumoniae isolates exclusively carrying an ISKpn26-like element exhibit an MIC of ≥64 mg/L colistin, while the introduction of a mutated phoP (P47L or A95S) or phoQ (N253T or V446G) gene into an IS mgrB-altered strain led to a decrease in the MIC [phoQ, phoP, and pmrD expression [phoPQ, pmrAB, and mgrB regulatory systems or in lpxM and yciM alleles are involved [mcr-1 gene is not connected with this phenomenon based on PCR results [Remarkably, another significant aspect of our study is the molecular basis of the two pairs of ChR  isolates, which are reported for the first time in Romania . These tsistance ,69. Pitt the MIC . These mpression . Howeverpression ,51,59. Minvolved ,71,72 an results ,71.blaKPC genes [blaOXA-48, blaKPC-2, and blaNDM-1 genes, consistent with Palmieri et al. [K. pneumoniae genotype ST101 has been associated with resistance to carbapenem, colistin, aminoglycosides, fluoroquinolones, and fosfomycin, with the potential to become a “perfect storm” clone, considering the similarity with the genetic profiles of hypervirulent strains [K. pneumoniae ST147 and ST307 have been associated with pandrug resistance and have been reported from endemic regions as well as in global nosocomial outbreaks, with proven links to KPC, NDM, OXA-48-like, and Verona integron-encoded MBL (VIM) [blaCTX-M-15 gene along with gyrA S83I and parC S80I mutations. Contrary to a recent report on an outbreak in northeast Germany induced by K. pneumoniae ST307 co-harboring blaNDM-1 and blaOXA-48 genes and colistin resistance [blaOXA-48 gene was not detected in our ST307 isolate, nor were any virulence genes or elements conferring susceptibility to chloramphenicol detected.The strains included in our collection were concentrated in either established  or emerging (ST307) international clonal lineages ,27,73,74i et al. . K. pneu strains . In addi strains . All of  strains ,79. K. pmgrB, phoQ, pmrB, and crrB alterations, and were recovered from the same intensive care unit (ICU) approximately 3 weeks apart from different patients, all of which are indicative of silent clonal expansion and intraward dissemination. There is also concern regarding the close relationship of these two ChR isolates from Târgu Mureș with the BC6_BM strain isolated in Baia Mare, suggesting the interregional propagation of ST147. Furthermore, the OXA-48-producer cluster ST101 revealed both interward and interregional spread, while the KPC-2-producer cluster ST258 showed interregional transmission. The constituent isolates of each cluster possessed similar or almost similar plasmids, implying plasmid-mediated propagation of blaOXA-48 and blaKPC-2 genes.It is noteworthy that the two parental strains, BC11_TM_B_hR and BC13_TM_C_hR, showed the same phenotypic and genomic profiles belonged to the successful ST147 clone, carried the same K. pneumoniae NDM-1-positive isolates [blaNDM plasmid in this geographical area, which was previously named pKOX_NDM1-like by Phan et al. [The interdependence between KPC-2-producing isolates with FII(K) and IncX3 plasmids and between OXA-48 producers with IncL and IncR plasmids concurs with previous observations made by Becker et al. in Germany . In contisolates , our twon et al. . Addition et al. . These en et al. ,83, whicn et al. ,38.The present investigation has some limitations. The retrospective nature of the study did not allow us to provide a reliable picture of all significant clinical and therapeutic aspects or previous hospitalizations, which was beyond the scope of this manuscript. However, the data on previous colistin therapy in patients diagnosed with ChR strains may be found elsewhere . BMD wasFuture studies should be oriented toward the reliable detection of both existing and novel mutations, combined with functional analysis, to establish their real contribution to colistin resistance and heteroresistance. Additional research should be conducted to expand our knowledge of the complex mechanisms underlying colistin resistance and heteroresistance; for the last phenomenon to establish clinical relevance, we should formulate a harmonized international definition and develop a standardized detection methodology. Furthermore, investigations using long-read sequencing technology will offer an adequate resolution for exploring plasmid structures.K. pneumoniae CPE strains were analyzed: 5 selected from patients admitted to Dr. Constantin Opriș County Emergency Hospital, Baia Mare, Romania, between January 2017 and April 2021 and 5 from patients at Târgu Mureș County Emergency Clinical Hospital, Romania, between January 2017 and April 2019. Additionally, 2 ChR K. pneumoniae strains obtained from Târgu Mureș in March 2019 and their corresponding colistin-resistant mutants were included in the study. The collection comprising the 10 CR and 2 ChR isolates has been phenotypically characterized in an earlier study [A total of 10 unique clinical CR er study . The first medical center is a public 920-bed general acute care nonteaching hospital in the northwest region of Romania, and the second is a large 1089-bed teaching hospital located in Transylvania, in the central region; they are located approximately 200 km apart.K. pneumoniae CPE isolates were randomly selected based on (i) MIC values greater than 2 mg/L colistin (categorized as resistance) [CR istance) , as detePathogens were mostly isolated from patients in the ICU (n = 6) and were obtained from different anatomical sites, as summarized in Data from electronic medical records available in the 2 laboratories are presented in All strains were identified at the species level using standard techniques and a Vitek 2 Compact system . AST-XN05 and AST-N233 pair testing cards, starting from the same inoculum, were used with the Vitek 2 Compact system, and AST-N222 cards were added for particular strains. Vitek 2 Compact version 9.02 software was used. The Kirby–Bauer disc diffusion method was used in all cases. Meropenem–vaborbactam (30 µg), imipenem–relebactam (35 µg), ceftazidime–avibactam (14 µg), and doripenem (10 µg) were tested exclusively by disc diffusion.K. pneumoniae CPE strains were additionally assessed for colistin resistance by BMD and using the following 5 phenotypic methods, as described elsewhere [All lsewhere : Micronalsewhere .The EUCAST breakpoint  was applThe modified carbapenem inactivation method (mCIM) ,87 and tConcordance between phenotypic and genotypic susceptibility test results was calculated individually for Vitek 2 Compact  and the disc diffusion method (14 isolates × 24 agents = 336 combinations) as the proportion of concordant results in the total combinations tested by each method. In this study, results of susceptibility or susceptibility to increased exposure were considered to be discrepant if a resistance molecular marker was noted or resistance results were not linked with genetic elements.Escherichia coli ATCC 25922, E. coli NCTC 13846, E. coli ATCC 35218, K. pneumoniae ATCC 700603, and K. pneumoniae BAA 2814. Other details regarding quality control for all methods used can be found elsewhere [Routine and extended quality control were performed according to EUCAST  with thelsewhere .The isolates were frozen at −70 °C and subcultured twice on solid medium before additional testing.https://www.ebi.ac.uk/ena/browser/home, accessed on 11 August 2022). The 14 genomes of CR, ChR, and corresponding resistant mutants of CPE strains were sequenced using the Ion Torrent PGM platform  according to the 400 bp protocol for library preparation, which includes enzymatic shearing, Ion OneTouch2 emulsion PCR, enrichment, and Hi-Q View sequencing kits (Thermo Fisher Scientific). The sequences obtained were subjected to de novo assembly into contigs using the Assembler SPAdes plugin version 5.12   installeWGS data were analyzed using both commercial and free online bioinformatics tools.http://www.genomicepidemiology.org/services/, accessed on 20 April 2022) , the Basic Local Alignment Search Tool (BLAST) search against reference sequences from the National Center for Biotechnology Information (NCBI) nucleotide repository, and BioEdit software version 7.0.5.3. [For antibiotic resistance characterization, we used AMRFinderPlus version 1.1  through 7.0.5.3. .mcr genes, the presence and integrity of TCRS genes , and the regulatory transmembrane protein-coding mgrB gene were investigated to identify colistin resistance determinants. ISfinder was used to depict the insertion sequence (IS) types that led to gene disruption [K. pneumoniae NC_009648.1 were used as references.Plasmid-mediated sruption . In ordesruption , with a sruption , with deIn order to depict the allelic profile of the genomes, MLST, ST , and cgMhttps://kaptive-web.erc.monash.edu/, accessed on 16 June 2022) [Capsular serotyping was conducted using the Kaptive web interface (ne 2022) . The Virne 2022) , includeblaNDM plasmids circulating during the period 2014–2015 in the town of Târgu Mureș [blaNDM-1-positive strains (BC1_TM and BC7_BM) to 3 reference plasmid sequences from 2014–2015 for comparison, namely, K. pneumoniae strain 1TM plasmid pNDM_1TM (MF042353.1), K. pneumoniae strain 6TM plasmid pNDM_6TM (MF042354.1), and K. pneumoniae strain 18ES plasmid pNDM_18ES (MF042350.1).Plasmid replicons were detected using the web-based PlasmidFinder version 2.1  on the Cgu Mureș . Since tmgrB, phoPQ, pmrAB, or crrAB gene, predicted to confer colistin resistance. We report on a novel and rare potential suppressor mutation in phoQ, L224Q, with possible involvement in heteroresistance to colistin, which in the presence of an altered mgrB, modified by IS, leads to low MIC values for colistin, as determined using BMD and other phenotypic methods aside from the Rapid Polymyxin NP test and PAP assays. Evidence for the silent intrahospital dissemination of heteroresistant mutant ST147 clones is thus provided. This study highlights the significant challenges involved in the phenotypic and molecular diagnoses of colistin resistance and heteroresistance. Each of our isolates presented at least one mutation in the K. pneumoniae MDR isolates, in addition to being an innovative and promising strategy for continuous surveillance of antimicrobial resistance in order to limit worldwide transmission. This research underlines the importance of WGS in identifying a catalog of molecular markers of various classes of antibiotics coupled with descriptions of circulating plasmids and clones, data that can be useful when investigating the dynamics of the dispersion of Ceftazidime-avibactam, imipenem/cilastatin-relebactam, meropenem-vaborbactam, tigecycline, gentamicin, and trimethoprim-sulfamethoxazole are still potential in vitro agents with activity against some of the studied pathogens.


Using lasso regression, 18 microRNAs substantially linked with the prognosis of HNSCC patients were identified and included in risk scores. The ROC curves demonstrate that risk scores derived from microRNAs involved in fatty acid metabolism can accurately predict the prognosis of HNSCC patients at 1, 3, and 5 years. Moreover, we discovered that 11 microRNAs included in the risk score properly distinguished the prognosis of HNSCC patients. This paper indicated that microRNAs involved with fatty acid metabolism are strongly linked to the prognosis of HNSCC patients. It also indicated that reprogramming of fatty acid metabolism in tumor tissues may play an important role in HNSCC cancer.Head and neck squamous cell carcinoma (HNSCC) is the sixth most frequent cancer in humans globally. In addition to smoking and drinking, genetic and epigenetic changes also play a big role in how HNSCC starts and grows. MicroRNAs are short, non-coding RNAs that control cell differentiation and apoptosis by interfering with gene expression. In addition, microRNAs in HNSCC have been shown to affect the clinical behaviors of HNSCC in amazing ways. Moreover, metabolic reprogramming is a key part of cancer and is needed for cancer to turn into a tumor and grow. But it is still not clear what effect microRNAs related to fatty acid metabolism have on the prognosis of HNSCC patients. We downloaded the data of HNSCC patients from the TCGA database and obtained the genes associated with fatty acid metabolism according to the GSEA database. Then, the microRNAs associated with fatty acid metabolism genes were matched. Finally, fatty acid metabolism gene-associated microRNAs for calculating risk scores and then building multifactorial Cox regression models in patients with HNSCC. Heatmap analysis showed that microRNAs involved in fatty acid metabolism were significantly different in HNSCC patients than in healthy controls. A total of 27 microRNAs associated with fatty acid metabolism were screened by univariate Cox analysis ( Head and neck squamous cell carcinoma (HNSCC), one of the most prevalent malignancies in the world, is a cancer of the squamous epithelium that lines the head and neck organs, such as the oral cavity, larynx, hypopharynx, oropharynx, nasopharynx, paranasal sinuses, nasal cavity, and salivary glands with poor outcomes and survival rates . HoweverIn the last 10 years, it has been shown that microRNAs play an important role in the development of HNSCC cancer and its spread . This maLipid metabolism, particularly fatty acid synthesis, is a critical physiological process that turns nutrients into metabolic intermediates for membrane formation, energy storage, and the creation of signaling molecules. A major metabolic characteristic of cancer cells is altered lipid metabolism . Furtherhttps://tcga-data.nci.nih.gov/tcga/). Ninety-two fatty acid metabolism-related genes were downloaded from the GeneCards (https://www.genecards.org/), including ABCD1, ABCD2, ACAA2, ACAD9, ACADL, ACADM, ACADS, ACADVL, ACAT1, ACSL1, ACSL3, ACSL4, ACSL5, ACSL6, ACSM1, ACSM2A, ACSM2B, ACSM3, ACSM4, ACSS1, ACSS2, ACSS3, CPT1A, CPT1B, CPT2, CRAT, CROT, ECHS1, ETFA, ETFB, ETFDH, HADH, HADHA, HADHB, HSD17B10, PEX11G, PEX13, PEX14, SLC25A20, ACAT2, ACAA1, EHHADH, ACOX3, ACOX1, ACADSB, GCDH, ACSBG1, ACSBG2, CPT1C, ECI1, ECI2, CYP4A11, CYP4A22, ADH1A, ADH1B, ADH1C, ADH7, ADH4, ADH5, ADH6, ALDH2, ALDH3A2, ALDH1B1, ALDH7A1, ALDH9A1, ACACA, ACACB, ACLY, FASN, MCAT, OLAH, OXSM, MECR, PPT1, PPT2, ELOVL1, ELOVL2, ELOVL3, ELOVL4, ELOVL5, ELOVL6, ELOVL7, HSD17B12, HACD2, HACD1, HACD4, HACD3, TECR, ACOT4, ACOT2, ACOT1, ACOT7 (collect and download from GeneCards). MicroRNAs related to these 92 genes were retrieved for further investigation.The RNA-sequencing transcriptome data and clinical data on patients with HNSCC and a matched normal group were extracted from the TCGA database . The ratios for all fatty acid metabolism-related microRNAs were computed between samples to measure the fold-change (FC) in expression between groups. DEG was determined using the following criteria: A false discovery rate (FDR) < 0.05 and a |log2FC| value greater than 1.0 are necessary. The expression of these fatty acid metabolism-related microRNAs was then visualized using vioplot and heatmap.The expression of fatty acid metabolism-related microRNAs in tumor tissues and matched normal tissues was compared to detect DEGs using the R package  in R (p < 0.05). Using 10-fold cross-validation and a p-value of 0.05, Least Absolute Shrinkage and Selection Operator (LASSO) regression was conducted. After identifying possible prognostic genes, the risk score was determined using the risk score formula. By using the median risk score as the dividing line, we classified the patients as either low-risk or high-risk. Using the Kaplan-Meier technique and the log-rank test, the overall survival (OS) between the two groups was determined. Risk scores are represented graphically after separating high and low risk categories. Risk scores are represented graphically. Using the “survival,” “survminer,” and “timeROC” functions of the R package, the 1-, 3-, and 5-year time-dependent receiver operating characteristics (ROC) curves were shown. Using microRNAs associated with fatty acid metabolism that were filtered by lasso regression, multivariate Cox regression models were built, and forest plots were generated. The univariate Cox regression and multivariable Cox regression were used to explore whether the risk score based on fatty acid metabolism-related microRNAs and characteristics of patient and tumor  were independent risk factors and to construct prognostic Norman diagrams based on the above characteristics. The log-rank test of the Kaplan–Meier analysis was used to generate a survival curve to compare subgroup survival. Using the immunoscore website (https://bioinformatics.mdanderson.org/estimate/disease.html), we immunoscore all genes, filter for differential genes, and locate intersections with differential genes based on risk score. Analysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG), Disease Ontology (DO), and Gene Ontology (GO) for functional annotation of DEGs based on the immunoscore and risk score. These DEGs were also used for analysis of the correlation between genes and immune cells and immune function.The univariate Cox regression analysis was used to screen for fatty acid metabolism-related microRNAs associated with HNSCC patients’ outcome (https://string-db.org/), a protein-protein interaction (PPI) network of DEGs was constructed to reveal the interactions of the 92 metabolism-related genes  were filtered . The risThe results of univariate Cox regression analysis indicated age, stage  and risk score are risk factors for HNSCC patients . MoreoveAnalysis of the Kyoto Encyclopedia of Genes and Genomes (KEGG), the Disease Ontology (DO), and the Gene Ontology (GO) for functional annotation of DEGs using intersection genes between immunoscore and risk score. According to the KEGG analysis, the intersection genes were mostly enriched in “Primary immunodeficiency,” “Neuroactive ligand-receptor interaction,” “Intestinal immune network for IgA production,” “Hematopoietic cell lineage,” “Salivary secretion,” “Malaria,” “Fat digestion and absorption,” and “Staphylococcus aureus infection” . AccordiThe reprogrammed metabolism of tumor cells results in changed metabolite levels in the tumor ecotone. Current research focuses on the effect of these metabolic modifications on the activity of immune cell compartments in the microenvironment. Consequently, it is essential to study and identify the multiple potential metabolic targets that impact tumor development. Thus, metabolic imbalance and transformation are regarded as hallmarks of cancer. Cancer cells have a very dynamic metabolism and continually alter how they use nutrients as fuel in and around the tumor . ReprogrCurrent research focuses on the effect of these metabolic modifications on the activity of immune cell compartments in the microenvironment. Immune cell metabolic reprogramming is a novel cancer characteristic that alters immune cell function by interfering with critical transcriptional and post-transcriptional activation mechanisms . NumerouFatty acid synthesis is an important cellular process that makes lipids, which can then be used to make membranes or ATP. However, lipid metabolism is a sign that a disease is getting worse, and it may be very important for some types of cancer to get worse. Increased quantities of fatty acids generated and accumulated by several solid tumors have been documented to provide a tumor microenvironment rich in fatty acids. In malignancies of the prostate, colon, ovary, liver, and lung, genes involved in adipogenesis have been identified as being activated . There ip < 0.05). We utilized lasso regression to identify microRNAs that have great strongly linked with the prognosis of HNSCC patients and used them to create risk scores. The results of ROC and survival curve data demonstrated that the risk score has a better prognosis distinction for HNSCC patients. Furthermore, survival analysis of these microRNAs revealed that 11 of them  discriminated effectively between HNSCC patient risk groups. However, the function of these genes in HNSCC remains unclear. A recent study found that PSMC2 promotes gastric cancer progression by targeting hsa-let-7c-3p to increase the level of RPS15A, which then activates the mTOR pathway (Previous study has shown a potential relationship between fatty acid metabolism and tumor development and patient prognosis. However, fatty acid metabolism-related microRNAs in HNSCC and how they affect a patient’s prognosis is not clear. The results of this paper revealed that microRNAs involved in fatty acid metabolism were considerably altered in HNSCC tumor tissues compared to healthy controls. Through univariate Cox regression analysis, we found that 27 of these differentially expressed microRNAs are linked with HNSCC patient prognosis ( pathway . This in pathway . Althoug pathway . Since a pathway , its rol pathway . Our res pathway . Overexp pathway . In addi pathway . Previou pathway . Our finIn conclusion, acid metabolism-related microRNAs may play a role in the progression of HNSCC. Although there has been much research on HNSCC related microRNAs, there has been little on its reprogramming of fatty acid metabolism and microRNAs that regulate fatty acid metabolism. Moreover, the enrichment analysis of KEGG, GO, and DO revealed that interconnection genes were predominantly in fatty acid metabolism-related pathways, including fat digestion and absorption, obesity, and overnutrition, as well as immune-related pathways, including B cell proliferation, B cell differentiation, B cell activation, cell killing, killing of other organisms’ cells, regulation of B cell proliferation, lymphocyte proliferation, leukocyte proliferation, mononuclear cell proliferation, and regulation of B cell proliferation. So, in the future, more emphasis should be directed to HNSCC metabolic reprogramming, particularly changes in fatty acid metabolism and the accompanying microRNA regulatory network, which may reveal new immunotherapeutic targets for the treatment of HNSCC.


Fecal microbiota transplantation (FMT) has been proposed as a potential treatment for irritable bowel syndrome (IBS); however, the consensus regarding its efficacy and safety is limited.We performed a systematic search of the literature using PubMed, EMBASE, Ovid MEDLINE, and Cochrane. Meta-analyses were conducted in relative risk (RR) or standard mean difference (SMD) using 95% confidence intervals (CI). Cochrane risk-of-bias 2 tool (RoB2) was employed to evaluate the study quality.Of 2,589 potential records, 7 studies with 9 cohorts involving 505 participants were included. Meta-analyses showed no significant difference in the short-term (12 weeks) and long-term (12 months) global improvement of IBS symptoms of FMT vs. placebo . There were statistically significant differences of short-term IBS-SSS improvement  and short-term IBS-QoL improvement . Eight from 9 studies (88.9%) had a low risk of bias. The subgroup analysis revealed the short-term global symptoms improvement in studies with low-risk of bias , studies with well-defined donors , and studies with FMT using colonoscopy . Major FMT adverse events are transient and rapidly self-limiting.FMT significantly improved IBS-SSS and IBS-QoL in the short-term period in IBS patients. However, global symptom improvement showed no significance. Well-defined donors and appropriate fecal administration routes appear to be important factors for the successful outcomes of FMT in IBS.www.crd.york.ac.uk/prospero], identifier [CRD42021246101].[ Irritable bowel syndrome (IBS) is a clinical-based functional bowel disorder characterized by abdominal pain and altered bowel habits in the absence of structural abnormalities , 2. The Recent studies have highlighted an important role of the gut microbiota in patients with IBS, which diminishes in microbial biodiversity compared with healthy controls –13. Manivia invasive routes significantly improved global IBS symptoms. However, neither the characteristics of stool donors nor the significance of stool donor inclusion were discussed in detail. Moreover, although the microbiome profile was mentioned, the association between the microbiome profile and global symptoms were unclear. Therefore, this study aimed to conduct a systematic review and meta-analyses of RCTs to estimate the efficacy and safety of FMT for the treatment of IBS, with subgroup analyses by route of FMT administration, type of feces used in FMT, donors’ microbiome profiles, patients’ microbiome profiles, and quality of stool donors.Recent randomized studies of fecal microbiota transplantation (FMT) in IBS have shown significant results on relieving symptoms compared to placebo , 18. DesWe conducted a systematic review and meta-analysis following the recommendations of the Preferred Reporting Items for Systematic Review and Meta-Analysis Protocols (PRISMA-P) 2015 statement . We prosWe performed a systematic search of the literature using PubMed, EMBASE, Ovid MEDLINE, and Cochrane. The term of the search strategy is presented in detail in The inclusion criteria were randomized-controlled trials of patients with IBS defined by Rome I, Rome II, Rome III, or Rome IV compared FMT with placebo. Both adult and pediatric studies were allowed. The exclusion criteria were: (1) studies failing to report our outcomes of interest (2) type of study designs which are review article, protocol, letter, comment, guidelines, case-control, or cohort studies. (3) Not yet published as full manuscript or not peer-reviewed, and (4) not in the English language.The primary outcome was the global improvement in IBS symptoms after FMT. Global improvement was defined as dichotomous assessment in the form of either an assessment of global symptom cure or improvement, or abdominal pain cure or improvement, after completion of therapy. It was also defined as a dichotomous response determined from the IBS Severity Scoring System instrument (IBS-SSS) score or Gastrointestinal Symptom Rating Scale for IBS (GSRS-IBS) total score using a predefined cut point of response and non-response. The secondary outcomes were the improvement of IBS-SSS (reduction in IBS-SSS score), the improvement of QoL (score increase on IBS-QoL), and adverse events (AEs). Subgroup analyses of the primary outcome were conducted by study characteristics including the risk of bias, characteristic of donors , route of FMT administration , type of feces used in FMT (fresh or frozen), and microbiome profiles. We gathered data for two time periods: the short term and the long term. Eight to 12 weeks’ outcomes were classified as “short-term” and 6–12 months as “long-term” , 27. We Two independent reviewers  screened articles for eligible studies and then extracted data from eligible published articles of FMT in patients with IBS. Discrepancies between two reviewers were re-checked and discussed to reach a consensus. If the authors were unable to reach a consensus, the third-person GI specialist (R.P.) reached the judgment.The data collection included: (1) study characteristics including authors, study type, country, (2) patients characteristics including mean age, sex, IBS criteria, IBS subtype, the number of patients, IBS severity, the year with IBS, current medication, and naïve or refractory to standard treatment, (3) stool donor including the number of donors, inclusion and exclusion criteria, stool preparation, (4) placebo preparation, (5) primary and secondary outcomes, (6) details of FMT methods, including preparation for FMT, the FMT route, the frequency and duration of FMT, (7) the duration of follow-up after FMT, and (8) FMT-related adverse events. We did contact the corresponding authors to request incompletely reported data on the outcomes of interest. If we could not get the response by 14 days, the analyses would be conducted by using available data.We applied the Cochrane risk-of-bias 2 (RoB2) tool for evaluating the quality of each eligible study in terms of randomization process, allocation concealment, blinding of participants, personnel and outcome assessment, complete outcome data addressed, selective outcome reporting, and other sources of bias.P-value < 0.05 was considered statistically significant. Heterogeneity was determined using the Cochran’s Q-test [a p-value of 0.10 indicated heterogeneity] and the Higgins’ test [I2]   were calculated using random-effects models. Meta-analyses were conducted using the relative risk (RR) method for dichotomous outcomes and the standard mean difference (SMD) method for continuous outcomes. han 75%] . If a puMicrobiome subgroup analyses of the primary outcome were planned to be performed by microbiome profiles, including recipients’ baseline microbiota, donors’ baseline microbiota, and the difference of baseline microbiota between donors and recipients. In addition, the association of the change in recipients’ microbiome profiles after FMT, the difference of dysbiosis index of recipients after FMT, as well as other gut microbiome profiles  of recipients after FMT and the primary outcome would be scrutinized using meta-analysis.Stool donor subgroup analyses of the primary outcome were planned to be performed by classifying the donors into “well-defined donors,” “relatively well-defined donors,” and “unclearly defined donors” using the following 3 main factors. In the first factor, we utilized the standard donor selection and collection criteria for FMT in clinical practice to reduce the risk of disease transmission –32 to en(1) The donor selection and collection process.; (a) The protocol defined by Kim and Gluck ((b) one of the following international guidelines: European consensus conference on FMT in clinical practice  onepractice , A jointpractice , Internapractice , A jointpractice , Australpractice .(2) The donor microbiome profile.via direct and indirect assessment. The direct assessment was determined by at least one of the following conditions; (a) high microbiome diversity or microbiome richness  The stability of the donor microbiome composition.via direct and indirect assessment. The direct assessment was determined by the stability of microbiome composition throughout the duration of the study measured by gut microbiome diversity , 2 editorials, 1 review article, and 1 comment article. Therefore, 7 articles with 9 studies were eligible for the data synthesis , 38, 39 via oral capsules.We performed a meta-analysis of 7 studies with 9 randomized control trials involving FMT in patients with IBS. All patients in these studies were adults. Five studies , 38, 39 There were 7 , 38, 39 I2 81%)  .I2 69%)  .I2 85%)  , 19, 39 I2 85%) . The meaI2 54%)  .I2 21%) (I2 34%) (Four studies (5 cohorts) , 18, 39 I2 21%) . Two stuI2 21%) , 39 demo(I2 34%) .I2 74%) (I2 69%) (Among 6 studies (8 cohorts) , 38, 39 I2 74%) . However(I2 69%) .Six studies (7 cohorts) , 38, 39 I2 85%) (Among 2 studies (3 cohorts) , 18 withI2 85%) . The chaI2 80%)  . The chaI2 45%) (Among 2 studies (3 cohorts) , 21 withI2 45%) . The chaI2 0%) (I2 69%)  . However(I2 69%) .I2 88%) (Four studies (6 cohorts) , 20, 21 I2 88%) .via capsule evaluated only a short-term but not a long-term global symptom improvement. There was no significant difference in the short-term global symptom improvement compared between the FMT and placebo  (Two studies (3 cohorts) , 20 in w I2 74%) .I2 86%) (p = 0.03). Johnsen et al. (Five studies (7 cohorts) , 38, 39,I2 86%) . LahtineI2 86%)  revealedn et al.  used botSix studies (8 cohorts) , 38, 39 I2 91%) (Six studies (8 cohorts) , 38, 39 I2 91%) . One stuI2 91%)  did not Two studies (3 cohorts) , 20 evalFive studies (7 cohorts) , 38, 39 Three studies (4 cohorts) , 20, 38 Six studies with 8 cohorts , 38, 39 I2-value show that the distributions of the included studies were heterogeneity. The Egger regression test did not revealed data asymmetry . The funnel plot was shown in According to Cochrane guidelines, one study was considered having an uncertain risk of bias because it did not adequately explain allocation concealment . A funnevia colonoscopy significantly improved global IBS symptoms. However, our meta-analysis provides additional results that the prior meta-analysis did not. First, our meta-analysis is the first to find a significant improvement in IBS-SSS after FMT compared to placebo after 12 weeks. The result was different from previous meta-analyses since the El-Salhy et al. study  , 21 via  studies , 39 were 1 study  via gaste et al.  conducteparation , 62. Addvia colonoscopy can improve global IBS symptoms may be only partially correct. This could be because pre-FMT lavage and a higher volume of stool suspension infusion per FMT contribute to the FMT’s effectiveness. Fifth, only a few studies reported on the association between global symptom improvement and donor microbiome profile, the patient microbiome profile at baseline, and the patient microbiome profile following FMT. Therefore, the results in this meta-analysis about microbiome profile and global symptom improvement were limited. Sixth, the results of this meta-analysis should not be applied to pediatric patients, as only studies in adults were available for this meta-analysis. Seventh, Additional factors that may limit the generalizability of the results include the fact that all stool donors are men or N/A. In addition, all of the included studies was conducted in either Europe or the United States. Finally, statistical heterogeneity was high in this meta-analysis. The factors that contributed to the heterogeneity may be route of administration and risk of bias, respectively. When we performed subgroup analysis on these factors, heterogeneity was reduced. In particular, the I2 for the colonoscopy subgroup is 0. Therefore, additional RCTs with colonoscopy as the route of administration and a low risk of bias are required to reduce statistical heterogeneity in prospective meta-analyses. Also, high-quality RCTs with large sample sizes, well-defined donors, appropriate route of administration and well-studied gut microbiome profiles of donors & recipients are needed to confirm the efficacy of FMT in IBS.There are limitations to this systematic review and meta-analysis. First, as the number of identified studies was fewer than 10, the Egger’s regression asymmetry test and funnel plots did not effectively indicate only publication bias but a variety of possible reasons such as heterogeneity, chance, and publication bias . Second,In conclusion, this meta-analysis of FMT in IBS demonstrated that FMT had a significant positive impact on the IBS-SSS and IBS-QoL in the short term, although its long-term efficacy was unclear. The improvements in clinical outcome with FMT for IBS may be attributed to the difference in route of administration, the donor selection criteria, and donor microbiome profile.The original contributions presented in this study are included in the article/CS, RP, TP, and SG involved in the conception and design the study. CS, PK, and RP extracted the data and involved in the methodology. RP, TP, and SG involved in supervision and validation and critically revised the manuscript. CS and PK wrote the manuscript. All authors contributed to the article and approved the submitted version.