Pf4CreRosa26mTmG). Using this microscopy tool, we discovered that fetal liver MKs provide higher thrombopoietic activity than yolk sac MKs. Mechanistically, fetal platelets were released from MKs either by membrane buds or the formation of proplatelets, with the former constituting the key process. In E14.5 c-Myb-deficient embryos that lack definitive hematopoiesis, MK and platelet numbers were similar to wild-type embryos, indicating the independence of embryonic thrombopoiesis from definitive hematopoiesis at this stage of development. In summary, our novel MP-IVM protocol allows the characterization of thrombopoiesis with high spatio-temporal resolution in the mouse embryo and has identified membrane budding as the main mechanism of fetal platelet production.Platelets are generated by specialized cells called megakaryocytes (MKs). However, MK’s origin and platelet release mode have remained incompletely understood. Here, we established direct visualization of embryonic thrombopoiesis in vivo by combining multiphoton intravital microscopy (MP-IVM) with a fluorescence switch reporter mouse model under control of the platelet factor 4 promoter ( Platelets play a crucial role in hemostasis in mammals . In addiIn contrast to platelet production in postnatal mammals, the mechanisms of embryonic thrombopoiesis have remained less well understood. The first platelets circulating in the fetus are provided by primitive megakaryocytes , which hAs such, the dynamics of MK’s development and their mode of embryonic platelet production have not been studied in detail by real-time in vivo imaging, which offers detailed insights into biologic processes . TherefoBlood formation relies on transcription factors driving hematopoietic differentiation. The transcription factor c-Myb promotes survival, proliferation, and differentiation of hematopoietic cells, and it is therefore essential for the establishment of definitive hematopoiesis ,19. EarlIn summary, we combined time-lapse MP-IVM with three-dimensional volumetric imaging (4D imaging) to visualize embryonic thrombopoiesis in hematopoietic organs  across different stages of embryonic development. By these means, we identified platelet budding as the major process in embryonic thrombopoiesis.Pf4Cre/wt (Stock No: 008535) [Rosa26mTmG/wt mice (Stock No: 007676) [−/− mice (Stock No: 004757) have previously been described [C57BL/6,  008535) , and Ros 007676)  were obtescribed . The micRosa26mTmG/wt × Pf4Cre/wt mice were mated with female C57BL/6 mice to generate heterozygous Rosa26mTmG/wt × Pf4Cre/wt embryos expressing the fluorescent marker mTmG. As controlled by activity of the Pf4 promoter, Cre activity leads to fluorescence switch from constitutive tomato (mT) to green fluorescent protein (mGFP) [Embryonic development was estimated as embryonic day 0.5 (E0.5) if a vaginal plug was present the following morning after mating. Pregnant females were used for subsequent experiments at indicated time points. Abdominal ultrasounds were carried out to monitor pregnancy. Embryos of E11.5 to E14.5 were adopted for flow cytometry analysis. Male n (mGFP) ,24. MP-I®, Norderstedt, Germany). A 1.5 cm skin incision was made on the lateral side of the abdomen, and subsequently, the underside portion of the peritoneal membrane was cut. A single uterine horn containing embryos was carefully exposed for dissection of the uterus to uncover the embryo. The uterine wall was cut with the spring scissor, revealing the individual amniotic sacs, each containing one fetus and the respective placenta. For YS imaging, this specific uterine area was placed and fixed on a holder that was custom-made by 3D printing. The membranes surrounding the YS were carefully removed and cleaned. Warm ultrasound gel was added around the embryo and YS to assure tissue hydration. A holder with coverslip was placed on top of the exposed YS. Then, the mouse preparation was moved to the microscope stage. For FL imaging, the YS and amniotic sac were cut to expose the fetus. The fetus was placed and fixed on the lower holder. The lateral abdominal skin of the embryo was removed to reduce light scattering and improve light penetration. Identically, the holder with coverslip was placed on the top of the exposed FL. During the whole preparation and following imaging process, warm saline or ultrasound gel  was continuously added to keep the hydration of the exposed uterus and embryo. The embryo was maintained in connection with the mother through the umbilical cord to preserve its circulation.Surgery of pregnant mice was performed under deep anesthesia by intraperitoneal injection of midazolam , medetomidine , and fentanyl . During the subsequent operation, surgical tolerance of anaesthetized mice was checked regularly. Mice were placed in supine position on surgical heating stage. The abdominal skin was disinfected with antiseptic solution , a biocompatible plastic resin (MiiCraft BV-007A), and a 3D printer .The coverslip holders were redesigned by 3D printing technology so that the holders and coverslip size could be adjusted according to the embryonic age and size. We have designed the different holder’s sizes using computer-aided design (CAD) software  before image scanning was carried out using the MP-IVM mode. Only Pf4 Cre-transgenic embryos expressed the GFP reporter in the megakaryocyte/platelet lineage. Cre-negative embryos were further used to confirm specificity of the fluorescence signal.z-axis) were recorded with 1.5 μm or 2 µm step size with the axial depth of 20–90 μm tissue volume. Sequential images were acquired as time lapse to allow observation of fetal blood flow.A multiphoton microscope  with an upright Olympus stand, all enclosed in a custom-built incubator maintaining 37 °C, was used for imaging. The microscope is equipped with a Chameleon Ti:Sapphire laser  with an output 900–1100 nm range pulsed laser for excitation and four photomultipliers (PMTs) with four simultaneous channel acquisitions. Images were acquired using a CFI75 LWD 16X objective (Nikon) with saline (for YS) or ultrasound gel (for FL) as immersion medium. Emitted fluorescent signals were collected by photomultipliers. Images were acquired using ImSpector software (Version ImspectorPro 275). Fluorescence excitation was provided by laser tuned to 1100 nm with an optical parametric oscillator (OPO) for simultaneous excitation of Tomato fluorophore. The 800 nm excitation wavelength was used for imaging GFP. The power of the laser and OPO were adjusted according to the thickness of the scan. The magnifications were described for each specific image. Image stacks  life drift correction software tool was employed to correct the tissue motion in real-time and maintain the region of interest in focus over the course of imaging ,26. Afte4 beads  were added before flow cytometry. Platelet counts were measured from 2 µL blood volume by flow cytometry. Peripheral platelets from embryonic and maternal blood as a control were gated with the same gating strategy  overnight at 4 °C. The aforementioned MK gating strategy in flow cytometry was used to identify MKs. Histograms were divided to approximate the different ploidy classes in terms of DNA content. We set boundaries of DNA with markers between peaks with geometric mean analysis [The fetal liver was isolated from surrounding tissue by blunt dissection and set aside in a solution of saline (NaCl 0.9%) on ice . With a analysis .Samples were analyzed on the flow cytometer . Acquired flow cytometry data sets were analyzed with a dedicated flow cytometry analysis software .3). Visualization and statistical analyses were performed with GraphPad Prism software Version 9 . In figures, all data are presented as mean ± SD. An unpaired student′s t-test was used for comparison between two groups. For comparison of multiple groups with one variable, one-way ANOVA testing was performed. A p value less than 0.05 was considered statistically significant, which was indicated by ‘*’ in figures (* p < 0.05).The fraction of MKs was calculated as the number of the respective MK type  divided by the total number of MKs observed in the volume. The concentration of bud-releasing MKs was calculated as follows: MK number was divided by observation time and volume scanned, which was expressed as MK/ than E11.5 . Still, the MK density was higher in FL than in YS. In FL, MK density at E13.5 was 1621 ± 798 cells per mm3 and at E14.5 1519 ± 655 cells per mm3. At E15.5, FL MK density decreased to 535 ± 303 cells per mm3. In YS, the diameter of MKs was 13.6 ± 1.6 µm at E10.5, which was similar to 14.8 ± 3.1 µm at E11.5. In FL at E13.5, MK diameter was 21.4 ± 0.9 µm. This was comparable to E14.5 with 21.5 ± 2.6 µm. At E15.5, the MK diameter increased to 24.4 ± 1.9 µm . In FL, the fraction of intravascular MKs was very low, from E13.5 to E14.5 (5.5 ± 6.9% and 3.6 ± 4.5%). At E15.5, the fraction of intravascular MKs was higher (14.6 ± 17.1%)  E left. A± 17.1%) E middle.± 17.1%) E right.We observed two distinct forms of potential platelet shedding from MKs by MP-IVM: (i) membrane budding and (ii) proplatelet formation. Detailed observation of MKs revealed a different form of platelet generation by expelling smaller particles without long proplatelet formation, which we termed the MK releasing bud . As repo3 at E10.5 and 238.9 ± 344.1 bud-forming MKs/h×mm3 at E11.5. Correspondingly, there were 118.8 ± 228.8 proplatelet-forming MKs/h×mm3 at E10.5 and 19.3 ± 61.1 proplatelet-forming MKs/h×mm3 at E11.5. In FL, there were 506.7 ± 228.8 bud-forming MKs/h×mm3 at E13.5 and 358.9 ± 206.8 bud-forming MKs/h×mm3 at E14.5. Correspondingly, there were 52.0 ± 85.6 proplatelet-forming MKs/h×mm3 at E13.5 and 76.1 ± 114.5 proplatelet-forming MKs/h×mm3 at E14.5. The same trend appeared at E15.5, as we quantified more bud-forming MKs (271.7 ± 118.8 MKs/h×mm3) than proplatelet-forming MKs (59.6 ± 87.8 MKs/h×mm3). In YS at E10.5, the fraction of bud-forming MKs was numerically higher than proplatelet-forming MKs  (p = 0.03). In FL, the fraction of bud-forming MKs was higher than the fraction of proplatelet-forming MKs at E13.5 , at E14.5 , and at E15.5 .“MK forming” bud means that we quantified all MKs’ assembling cytoplasmatic protrusions of less than 10 µm. In addition, we quantified those MKs that released buds, using the term “MK releasing buds”. We observed a significant number of MKs forming protrusions, including releasing events through buds and proplatelets C. In YS, = 0.26) D. Howeve3-scanned volume . This difference was also present at E14.5  and E15.5 . In both YS and FL, platelet release events were rare. This is in line with a former report [Next, we quantified how many MKs were releasing buds or proplatelets per hour per mmd volume E. In YS,pulation F. In YS,r report  pointing3/µL), with a sharp increase at E12.5 (83.2 ± 45.4 × 103/µL) and continued growth to E14.5 (165.3 ± 78.1 × 103/µL)  B. The pe 103/µL) .p < 0.0001). For the mother mice, the reticulated platelet fraction ranged from a minimum of 8.2 ± 2.9% at G13.5 to a maximum of 12.5 ± 2.2% at G11.5, although these differences over gestational age did not reach statistical significance   . These rn = 10–21 embryos and n = 3–5 mothers) were analyzed, showing an increased total cell number from 0.04 ± 0.05 × 106 cells at E11.5 to 19.7 ± 7.4 × 106 cells at E14.5 per FL .Using flow cytometry, we quantified MKs in FL B in diffThe maturation of fetal liver MKs was assessed with ploidy analysis using propidium iodide (PI) staining measured by flow cytometry across embryonic development D. The 2N−/− embryos lack definitive hematopoiesis and die around day E16.5 with severe anemia [Next, we evaluated the role of primitive and definitive hematopoiesis regulated by c-Myb in embryonic thrombopoiesis. MKs in embryos are believed to originate from both primitive hematopoiesis and definitive hematopoiesis . Homozyge anemia . We used−/− embryos were compared to heterozygous c-Myb+/−, and c-Myb+/+ embryos. Embryonic platelet count in c-Myb−/− embryos (190.0 ± 25.6 × 103/µL) was similar to c-Myb+/+ (173.3 ± 37.9 × 103/µL), but both numbers were lower than heterozygous c-Myb+/− (287.3 ± 16.1 × 103/μL) (We measured platelet counts of different c-Myb genotypes at E14.5: Homozygous c-Myb 103/μL) A.−/− (56.5 ± 3.4%) and c-Myb+/+ (52.2 ± 3.8%) mice  mice B. These −/− embryos was smaller due to the lack of definitive hematopoiesis, FL cell counts were significantly lower in c-Myb−/−  than c-Myb+/+  embryos  (+/+ MKs (17.7 ± 1.5%) and highest in c-Myb−/− MKs (35.9 ± 31.0%). The 4N fraction was similar across all genotypes (between 44.8 ± 12.1% and 45.5 ± 1.3%). In contrast to 2N, the 8N fraction was lowest in c-Myb−/− MKs (16.8 ± 16.6%) and highest in c-Myb+/+ (31.8 ± 1.0%). The 16N, 32N and 64N fractions were very low in all genotypes.Since the size of the FL in c-Myb embryos C. When il liver) D. Ploidyl liver) E. The 2N+/+ embryos. These results confirmed that c-Myb had a strong impact on definitive haematopoiesis as FL cell numbers were very low. Additionally, c-Myb had no apparent impact on thrombopoiesis until E14.5. These findings could hint to at the potential of primitive thrombopoietic cells for maintaining platelet generation in the absence of definitive hematopoiesis.Even though c-Myb embryos had lower FL cell numbers, they presented a similar level of platelets and their reticulated forms in peripheral blood. This could be explained by their similar absolute FL MK numbers compared with c-MybUsing MP-IVM, we determined active thrombopoiesis in YS and FL. Comparing the different sites of thrombopoiesis in live embryos, we found higher density, increased ploidy, and a larger size of MKs in FL than in YS. This indicates higher differentiation and thrombopoietic activity in FL, reflecting a higher demand for circulating blood cells in the fast-growing embryo. Nevertheless, MK density decreases over time in both organs, likely due to the expansion of other hematopoietic lineages such as red blood cells. Regarding spatial distribution, the majority of MKs in YS were located in the intraluminal area of the vessels. On the contrary, in FL, a large fraction of MKs were positioned in the extravascular space. Whether this is due to a role of MKs in niche homeostasis as described for adult bone marrow, requires further investigations in future studies ,37,38.Using MP-IVM, we revealed the diverse MK and proplatelet/platelet populations present during embryonic development. Compared with the classic proplatelet formation and shedding pattern in adult bone marrow and lung ,10, we fThe potential mechanisms of budding vs. proplatelet shedding remain incompletely understood. Surely, the cytoskeleton and membrane re-organization play important roles, as regulators of this process are proteins of the Rho family , which are also required for many cellular functions beyond cell mobility and cytokinesis . These pp-selectin mobilization compared with adults [In this study, we revealed the physiological characteristics of thrombopoiesis in mouse embryos, which is very different from adult thrombopoiesis. Using flow cytometry, we quantified platelet and reticulated platelet numbers at different developmental ages. We found that the overall platelet count is lower in embryonic compared to adult mice, yet the fraction of reticulated platelets was particularly high in the embryo. Platelet counts were particularly low at E11.5, but expanded with development. As megakaryocytes and platelets are probably more mature towards later developmental ages, we also found a decrease in the reticulated platelet fraction over time. In a fetal thrombosis model, platelets presented hyporeactivity during early fetal development. Specifically, they showed less adhesion and spreading capacity as well as reduced h adults .We quantified the MK numbers and overall FL cell numbers and found that MKs are a small fraction among FL cells. However, absolute cell numbers of MKs also increase within the FL niche over time. As the embryo develops, the ploidy of MKs increases, and 8N/16N stages appear. The FL undergoes a complex developmental process, originating from the ventral foregut endoderm  and diffMKs can be generated from both primitive or definitive hematopoiesis . In c-MyWe established a new protocol for intravital, real-time three-dimensional imaging, MP-IVM, to visualize embryonic thrombopoiesis in hematopoietic organs  across different stages of embryonic development. This technique, combined with flow cytometry, gave us the possibility to classify that at least two patterns of platelet shedding from MKs exist, either by an important process termed membrane budding or by conventional proplatelet release. The former seems to be the predominant form, which might be independent of c-Myb. The cellular and molecular mechanisms for bud release require further research.


Hepatic ischemia/reperfusion (I/R) injury is one of the major pathological processes associated with various liver surgeries. However, there is still a lack of strategies to protect against hepatic I/R injury because of the unknown underlying mechanism. The present study aimed to identify a potential strategy and provide a fundamental experimental basis for treating hepatic I/R injury.A classic 70% ischemia/reperfusion injury was established. Immunoprecipitation was used to identify direct interactions between proteins. The expression of proteins from different subcellular localizations was detected by Western blotting. Cell translocation was directly observed by immunofluorescence. HE, TUNEL and ELISA were performed for function tests.We report that tripartite motif containing 37 (TRIM37) aggravates hepatic I/R injury through the reinforcement of IKK-induced inflammation following dual patterns. Mechanistically, TRIM37 directly interacts with tumor necrosis factor receptor-associated factor 6 (TRAF6), inducing K63 ubiquitination and eventually leading to the phosphorylation of IKKβ. TRIM37 enhances the translocation of IKKγ, a regulatory subunit of the IKK complex, from the nucleus to the cytoplasm, thereby stabilizing the cytoplasmic IKK complex and prolonging the duration of inflammation. Inhibition of IKK rescued the function of TRIM37 in vivo and in vitro.Collectively, the present study discloses some potential function of TRIM37 in hepatic I/R injury. Targeting TRIM37 might be potential for treatment against hepatic I/R injury.Targeting TRIM37 might be a potential treatment strategy against hepatic I/R injury.The online version contains supplementary material available at 10.1186/s10020-023-00653-2. Hepatic I/R injury is an inevitable pathological process associated with various liver operations  is a pivotal E3 ubiquitin ligase associated with innate immunity in solid organs  is composed of a ring finger, a B-Box-type zinc finger, a B-Box C-terminal domain, a TRAF domain and a polyacid domain and has recently been reported to participate in a variety of inflammatory immune responses . After replacing the medium, a normal incubator was used for reoxygenation for different times . The cell density was approximately 70% before hypoxia treatment. Total RNA, supernatant and proteins were isolated after H/R treatment.A standard hypoxia/reoxygenation (H/R) model was established according to our previous studies  injury was implemented based on our previous research  containing 10% cocktail protease inhibitor . Cytoplasmic proteins and nuclear proteins were extracted using the ExKine Nuclear and Cytoplasmic Protein Extraction Kit . β-Actin was used for the normalization of protein unless otherwise specified. Determination of TRIM37 , ASK1, p-ASK1, p-p38, p38, extracellular regulated protein kinases (ERK), p-ERK, p-JNK, JNK, c-caspase7, caspase7 , TRAF6, p-IKKα, p-IRAK1, IL-1β, IL-10, IKKγ, p-IKKγ , β-actin, p-IKK, IKK, IKKα, IKKβ, IL-6, TGF beta-activated kinase 1 (TAK1), p-TAK1, p-IκBα, IκBα, p-p65, p65, IRAK1, BAD, Bax, Bcl2, cleaved-caspase3 (c-caspase3), caspase3, Histone, α-tubulin , and p-IKKβ  was accomplished as described previously  staining based on our previous studies  to determine local or systemic inflammatory conditions. Liver enzymes in serum, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST), were detected by microplate kits  to determine liver function. The number of wells for each indicator was at least 3 and was specifically denoted in the relevant figure legend.−ΔΔCt method. The number of wells for each indicator was at least 3 and was specifically denoted in the relevant figure legend.TRIzol reagent (Beyotime) was used to isolate RNA from cells or tissues based on our previous research  48 h before treatment. All interference sequences are listed in Table 8 pfu, tail vein) 7 days before treatment. C25-140 was administered  at 24 h and 12 h before the treatment based on our previous research  dissolved in QuickBlock™ Blocking Buffer for Immunol Staining (Beyotime) for 20 min. The primary antibodies were as follows: F4/80 , p65 and p-IKKβ , TRIM37 , p-IKKγ , TRAF6 , and IKK . F4/80 was used as the marker of functional Kupffer cells. All primary antibodies were incubated for 8 h in a 4 °C room. Secondary antibodies conjugated with Cy3 or FITC were incubated at 37 °C for 30 min, followed by nuclear staining with DAPI (Beyotime). Cells were lysed after being treated using a specific lysis buffer containing HEPES (25 mmol/L), NaCl (150 mmol/L), EDTA (1 mmol/L), 2% glycerol and PMSF (1 mmol/L) based on previous studies  followed by Bonferroni’s post hoc test was used for comparisons among multiple groups. In vitro repetition was at least 3 and is described in detail in the corresponding figure legend. Sample sizes in vivo are shown in the corresponding figure legend. P < 0.05 was considered to be statistically significant. Statistical calculations were analyzed using SPSS (version 19.0).We identified the expression of TRIM37 and classical inflammatory signaling proteins in hepatic I/R injury in vivo and in vitro. H/R insult stimulated TRIM37 expression in Kupffer cells and increased the levels of p-ASK1 and TRAF6  (such as miR-125) can directly target TRAF6 and lead to its overall expression, as we verified (Huang et al. The functional diversity of TRIM37 includes its participation in disparate conditions (Avela et al. It has been shown that TRIM37 affects cell proliferation and is highly expressed during mitosis in other cells (Brigant et al. In the present study, we found that TRIM37 catalyzed the K63 chain and promoted the translocation of IKKγ by interacting with TRAF6, enhancing the activity and phase of the IKK complex in hepatic I/R injury. Our results might provide a potential target in hepatic I/R injury.Additional file 1: Figure S1. A-D, Suzuki score of liver after downregulating TRIM37 or TRAF6. E-F, transfection of si-TRAF2/4/6 in cells and tissues. G, transfection of adv-TRIM37 in cells and tissues. H, immunoflourence of p65.


Glucose repression in yeast leads to the sequential or diauxic utilization of mixed sugars and reduces the co-utilization of glucose and xylose from lignocellulosic biomasses. Study of the glucose sensing pathway helps to construct glucose repression-released yeast strains and enhance the utilization of lignocellulosic biomasses.Kluyveromyces marxianus which mainly consisted of KmSnf3, KmGrr1, KmMth1, and KmRgt1 was studied. The disruption of KmSNF3 led to a release of glucose repression, enhanced xylose consumption and did not result in deficient glucose utilization. Over-expression of glucose transporter gene restored the mild decrease of glucose utilization ability of Kmsnf3 strain to a similar level of the wildtype strain but did not restore glucose repression. Therefore, the repression on glucose transporter is parallel to glucose repression to xylose and other alternative carbon utilization. KmGRR1 disruption also released glucose repression and kept glucose utilization ability, although its xylose utilization ability was very weak with xylose as sole carbon source. The stable mutant of KmMth1-ΔT enabled the release of glucose repression irrespective that the genetic background was Kmsnf3, Kmmth1, or wildtype. Disruption of KmSNF1 in the Kmsnf3 strain or KmMTH1-ΔT overexpression in Kmsnf1 strain kept constitutive glucose repression, indicating that KmSNF1 was necessary to release the glucose repression in both SRR and Mig1-Hxk2 pathway. Finally, overexpression of KmMTH1-ΔT released the glucose repression to xylose utilization in S. cerevisiae.Herein, the glucose sensor/receptor repressor (SRR) pathway of K. marxianus strains constructed via a modified glucose SRR pathway did not lead to a deficiency in the utilization ability of sugar. The obtained thermotolerant, glucose repression-released, and xylose utilization-enhanced strains are good platforms for the construction of efficient lignocellulosic biomass utilization yeast strains.The glucose repression-released The online version contains supplementary material available at 10.1186/s12934-023-02138-7. Renewable and sustainable lignocellulosic biomasses have increasingly attracted attention as the consumption of fossil fuels not only leads to global warming, but the dwindling fossil resources impact the production of energy and chemicals. It is estimated that as much as 140 billion metric tons of biomass is generated annually from agriculture worldwide. Lignocellulosic biomasses, however, can partly substitute fossil resources, reduce greenhouse gas emissions, and provide renewable energy [The major components of lignocellulosic biomasses are cellulose (35–50%), hemicellulose 20–35%), and lignin (10–25%). The remaining fraction consists of proteins, oils, and ash [0–35%, anSaccharomyces cerevisiae, which has a weak xylose assimilation ability. Therefore, the study of glucose repression to xylose utilization is important in xylose-utilization yeasts, and the glucose repression mechanism may be different from that of S. cerevisiae.In most xylose assimilation yeasts, glucose repression causes sequential use of glucose and xylose. However, most information regarding glucose repression is obtained from S. cerevisiae, the main glucose-sensing and -repression pathways include the sensor/receptor repressor (SRR) pathway , Mig1 -Hxk2 pathway, the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway [S. cerevisiae has been proven to be related to glucose repression, while the mechanism of which remains obscure [In  pathway  and the  pathway , 6. In S pathway . The upt obscure . Stasyk SNF3 of S. cerevisiae leads to defects in glucose and fructose uptake [rgt2 snf3 double mutant of S. cerevisiae is growth-defective on 2% glucose [RGT2 and SNF3 leads to the repression of the HXT expression and reduces glucose uptake. Improving glucose uptake in S. cerevisiae restores glucose repression [S. cerevisiae.Disruption or deletion of e uptake  and the e uptake . The rgt glucose , 11. Thepression . TherefoS. cerevisiae and other xylose-using yeast species shows a sequential consumption of glucose and xylose due to the transport which has been directly verified by uptake assays for single transporter [Other than glucose sensing and repression, xylose transport is another limitation in co-utilization of glucose and xylose. The engineered nsporter . Transponsporter . To co-unsporter , 13.S. cerevisiae is weak, most studies on glucose repression do not include the repression to xylose utilization. The xylose utilization of S. cerevisiae is improved obviously through genetic engineering, but the informations obtained from the engineered strain currrently does not match the mechanism of glucose repression in xylose-assimilating yeasts. Therefore, special attention needs to be paid to the glucose repression of xylose utilization in such yeasts.As the xylose assimilation ability of K. marxianus is a microorganism “generally regarded as safe” and can assimilate various sugars including glucose, xylose, galactose, and inulin [K. marxianus is reported to be the fastest-growing known eukaryotic organism [K. marxianus strains can grow well at temperatures over 45 oC, and maintain a high growth rate at approximately 40 oC [K. marxianus allows for fermentation at elevated temperatures, which is convenient in high-temperature seasons and tropical environments. Additionally in industrial settings, fermentation at elevated temperatures reduces cooling costs, increases fermentation rate, and minimizes the risk of contamination [d inulin . K. marxorganism  that canly 40 oC . The themination , 18.K. marxianus can utilize xylose, it prefers glucose and causes sequential or diauxic fermentation, resulting in reduced volumetric productivity from sugar mixtures. Several studies on glucose repression in S. cerevisiae have been conducted [S. cerevisiae without genetic engineering is weak, and glucose effect studies mostly exclude xylose. The mechanism of glucose repression in S. cerevisiae is unlikely to apply to K. marxianus, particularly the repression of xylose utilization. In our previous study, we found that the glucose repression pathway for xylose is different from that of other sugars, such as galactose [K. marxianus (4912 open reading frames for NBRC1777) than those in the genome of S. cerevisiae. Therefore, it is unsurprisingly to find evidence of novel glucose-repression mechanisms or properties in K. marxianus [To efficiently utilize both glucose and xylose from lignocellulosic biomasses, glucose repression should be prevented. Although native onducted , 19–21; alactose . Furtherarxianus , 22.K. marxianus, glucose repression to xylose utilization was possibly not via Mig1 as to other alternative carbon source in the presence of glucose [K. marxianus. Herein, the glucose-sensing SRR pathway in K. marxianus were analyzed. A single gene-disruption strain  was constructed to evaluate effect of each gene on glucose repression , from our previous study [KmSNF1 was necessary to release glucose repression in both pathways. Furthermore, the results of overexpressing the glucose-transporter gene in KmSNF3 disrupted strain showed that improving glucose uptake did not restore the glucose repression  with appropriate supplements was used to screen transformants. Escherichia coli DH5α was used for gene cloning and was cultivated in lysogeny broth medium . For solid plates, 15 g/L of agar was added to each medium.D-glucose, D-xylose, 2-deoxy-D-glucose (2-DG), and yeast nitrogen base without amino acids (YNB) were purchased from Sangon Biotech Co., Ltd. . Yeast extract (LP0021), tryptone (LP0042), and peptone (LP0037) were purchased from Oxoid Ltd. . Restriction endonuclease and T4 DNA ligase were purchased from Takara Bio Co., Ltd. . NBRC1777 . YPD/YPGKmSNF3 (GenBank accession no. MT081965), KmGRR1 (GenBank accession no. BAP73380), KmMTH1 (GenBank accession no. BAP71026.1) and KmRGT1 (GenBank accession no. BAP73047.1) were amplified from the genomic DNA of K. marxianus NBRC 1777 with primer pairs KmSNF3F/KmSNF3R, KmGRR1F/KmGRR1R, KmMTH1F/KmMTH1R, and KmRGT1F/KmRGT1R . The strain that was obtained was YΔSNF3-U. Then, the KmSNF1disruption cassette was amplified from pKmSNF1-U [KmSNF1. After screening on an SD medium containing leucine and tryptophan, and colony-PCR confirmation, the Kmsnf, Kmsnf3 strain YΔSNF1-ΔSNF3 was obtained , glutamic acid (E), serine (S) and threonine (T) enriched (PEST) sequences and a phosphorylation site for YCK1 [KmPDC1-disrupted K. marxianus strain [KmMTH1-ΔT overexpression strains were constructed to evaluate whether this stable mutant could maintain or lead to the release of glucose repression. The ScURA3 in YΔSNF3 or YΔMTH1 was disrupted with the URA3 disruption cassette [KmMTH1-ΔT expression cassette. The obtained strain was YΔSNF3-KmMTH1-ΔT, YKmMTH1-ΔT or YHJ010-KmMTH1-ΔT.Stable Mth1 mutant (Mth1-ΔT) of for YCK1 . In our s strain . Here, scassette , and strcassette , which cKmMTH1-ΔT was also expressed in YΔSNF1 strain to evaluate the effect of the stable KmMTH1 mutant on glucose repression in KmSNF1-disrupted strain. The ScURA3 in YΔSNF1 [ScURA3 disruption cassette as described previously [n YΔSNF1  was disreviously . The obtKMAR_50343 or KMAR_50346, were constructed using K. marxianus YΔSNF3  [The putative sugar transporter genes, d Boles) , and YEBKmMTH1-ΔT-expressing S. cerevisiae was constructed to evaluate its effect on glucose repression. Plasmid pZB013 was transformed into S. cerevisiae W303-1 A and screened on an SD medium supplemented with leucine, tryptophan, histidine, and adenine, and the strain YW303-1 A-KmMTH1-ΔT was obtained  or YΔSNF3 was streaked on an SD plate medium containing 0.1% or 2% glucose, and the plates were cultivated at 37 oC for 2 days.As Snf3 is a glucose sensor of low-concentration glucose,  glucose . The groK. marxianus, 42 °C was used for the cultivation in liquid medium. Then, the cells from pre-cultivation were transferred into 250-mL Erlenmeyer flasks containing 50 mL of YPD (2% glucose), YPX (2% xylose), and YPDX (2% glucose + 2% xylose) media with an initial OD600 of 0.5 and cultivated at 42 °C with shaking at 250 rpm in an orbital shaker. Growth with 2% xylose, 2% glucose, and 2% xylose + 2% glucose was determined by monitoring the OD600. The sugar consumption during the growth was monitored by high-performance liquid chromatography (HPLC).As our study focused on glucose repression to xylose utilization, the glucose and xylose consumption of the constructed strains during culturing was analyzed. YWD005 (control), YΔSNF3, YΔGRR1, YΔMTH1, and YΔRGT1 cells were pre-cultivated in 5 mL of YPD medium at 42 °C overnight. Because elevated temperatures will be used in futural application, taking advantage of the thermotolerant nature of 600 of 0.5 and cultivated at 42 °C with shaking at 250 rpm in an orbital shaker. Growth was determined by measuring the OD600. The sugar consumption during growth was monitored by HPLC.YΔSNF3-ScTRP1, YΔSNF3-50343, YΔSNF3-50346, and YWD010  were pre-cultivated in 5 mL of YPD medium at 42 °C overnight. Then, the cells were transferred into 250-mL Erlenmeyer flasks containing 50 mL of YPDX  medium with an initial ODKmSNF3 disruption in the presence of glucose and xylose. K. marxianus YΔSNF3 and YWD005 were pre-cultivated in 5 mL of YPD medium at 42 °C overnight. Then, the cells were transferred into 250-mL Erlenmeyer flasks containing 30 mL of YPDX medium (2% glucose and 2% xylose) with an initial OD600 of 1.0 and cultivated at 42 °C with shaking at 250 rpm in an orbital shaker until the OD600 = 6.0 before the glucose was exhausted. Yeast cells were then recovered and stored at -80 °C for RNA isolation.RNA-seq was used to analyze the changes in gene expression before and after p-value < 0.05 and a greater than two-fold change in expression across libraries was observed.The total RNA of each sample was extracted and cDNA libraries were prepared as previously described . The resK. marxianus strains were overnight cultivated in YPD medium at 42 °C and with 250 rpm shaking, the cells were inoculated into YP medium containing 20 g/L glucose, 20 g/L glucose and 20 g/L alternative carbon source, 20 g/L alternative carbon source until the OD600 reached 6.0. The alternative carbon sources used were lactose, sucrose, xylose, raffinose or glycerol. The cells were collected by centrifugation at 5000×g for 5 min and total RNA was extracted using the Yeast Total RNA Isolation Kit . The genomic DNA in the sample was removed by 4×DN Master Mix with gDNA Remover. The cDNA was reverse-transcribed using the ReverTra Ace® qPCR RT Master Mix with gDNA Remover . The expression levels of KmXYL1, KmXYL2, KmXYL3, KmLAC4, KmINU1, KmGUT1, KmGUT2, and KmHXK1 (hexokinase 1) were analyzed in the presence of glucose, alternative carbon sources or a glucose and alternative carbon source mixture. The primers used for RT-PCR are listed in Supplementary Table Quantitative real-time PCR (qRT-PCR) was used to evaluate the expression of the key genes for alternative carbon source utilization in the presence of glucose. After S. cerevisiae EBY.VW4000, YEBYVW4000-50343, YEBYVW4000-50345, and YEBYVW4000-10531  grew better than the non-disrupted control (YWD005) and accumulated more biomass in the liquid medium with glucose, with a final OD600 of 21.83 and 18.74, respectively  to 0.34 g/(L·h)) [Kmhxk1 strain releases glucose repression with a significantly decreased glucose-assimilating ability [KmGLK improves glucose assimilation ability and maintains the release of glucose repression, but the strain loses fructose utilization ability [In other reports, the glucose utilization ability of glucose repression-released ecreased , 32. The ability . Express ability , 27. TheThe ethanol accumulations of YΔSNF3 and YWD005 during the cultivation were also determined. YWD005 produced 6.82 g/L ethanol at 8th h with glucose, whereas, YΔSNF3 produced less ethanol (6.14 g/L) with longer time (12 h) . In the case of xylose as carbon source, only very small amount of ethanol accumulated  . When glucose and xylose mixture was used as carbon source, Y ΔSNF3 produced 5.63 g/L ethanol at 12th h and YWD005 produced 5.54 g/L ethanol at 8th h. The ethanol accumulation of Y ΔSNF3 was slower than that of YWD005, and was consistent with the slower sugar consumption of Y ΔSNF3 than YWD005.Although a spot assay in the presence of 2-DG is a traditional method for glucose-repression determination, the changes in gene expression still need to be verified. YΔSNF3 and YWD005 were cultivated with mixture of glucose and xylose, RNA-seq was used to analyze the transcriptional changes of KmSNF3-disruption in response to the glucose repression with YWD005 as control strain. In GO enrichment analysis, among those significantly enriched pathways, single-organism process, small molecule metabolic process, single-organism biosynthetic process, oxoacid metabolic process, carboxylic acid metabolic process, and organic acid metabolic process were among the top 40 GO terms with significant enrichment .As to KEGG enrichment analysis, among those significantly enriched pathways, citrate cycle (TCA cycle), pyruvate metabolism, glycolysis/gluconeogenesis, and many amino acids metabolism and biosynthesis such as alanine, aspartate, glutamate, phenylalanine, tyrosine, tryptophan, and arginine etc., were in the top 40 pathways with significant enrichment. Galactose metabolism, fructose and mannose metabolism were also in this top 40 pathways , xylitol dehydrogenase (KmXYL2), and xylulokinase (KmXYL3), improved by Log2FC values of 3.71, 4.14, 1.94, respectively  , which is downstream of xylose catabolism, the expression of genes involved in PPP was also analyzed. There was no obvious change, except for the upregulated expression of glucose-6-phosphate 1-dehydrogenase (KmBGL1) and inulinase (KmINU1) genes that correspond to cellobiose or inulin  utilization, respectively, did not change significantly. This was possibly due to the absence of an inducer (carbon source) in the medium.The expression of genes encoding the enzymes involved in alternative carbon source utilization was also analyzed. The expressions of the β-glucosidase (KmGALK), lactose regulatory protein LAC9 (KmGAL40), and galactose/lactose metabolism regulatory protein (KmGAL80), was upregulated ; however, the expression of β- galactosidase (KmLAC4) was not changed.The expression of genes related to lactose utilization, such as galactokinase  were found in the genome. KmAdh1 and KmAdh2 are cytosolic enzymes, whereas KmAdh3 and KmAdh4 are mitochondrial ADHs [KmADH1 and KmADH3 were upregulated (Log2FC 6.10 and 2.99) and KmADH2 was downregulated (Log2FC -3.82), whereas there was no significant difference of KmADH4 in YΔSNF3. This is possible because KmADH3 and KmADH4 cannot catalyze the conversion of acetaldehyde to ethanol, and they are the enzymes involved in ethanol consumption. Although the Kcat/Km of KmAdh1 (4.3 × 103 min− 1 mM− 1) and KmAdh2 (2.7 × 103 min− 1 mM− 1) with acetaldehyde as the substrate is relatively high, KmAdh1 has a low Kcat/Km (5.2 min− 1 mM− 1) with ethanol as the substrate, in contrast to KmAdh2 (2.1 × 102 min− 1 mM− 1). Therefore, KmAdh2 is a respondent to ethanol production [2FC: -3.82) and increased expressions of KmAdh1 (Log2FC: 6.10) and Adh3 (Log2FC: 2.99) indicated that the expression of genes related to ethanol production decreased and those related to ethanol utilization increased in YΔSNF3.In ial ADHs . In the oduction . The decKmHXK1 and KmMIG1 in YΔSNF3 was downregulated with a Log2FC value of -2.20 or -3.90, respectively, compared to those in YWD005 . In the SRR pathway, the expression of KmRGT1 were upregulated (Log2FC: 1.58), while KmMTH1 was downregulated (Log2FC: -1.59) in YΔSNF3 ) and changed significantly , the relative expression (fold-change) of KmXYL1 in glucose, glucose-xylose, and xylose was 1:0.72:2.55 , the relative expression of KmINU1 in the above carbon sources was 1:1.06:2.06. This result indicates that KmINU1 is an inducible gene and is repressed by glucose in YWD005 , the relative expression of KmLAC4 on the above carbon sources was 1:1.41:7.87. This result shows that KmLAC41 is an inducible gene and is repressed by glucose in YWD005 , the relative expressions of KmGUT1 and KmGUT2 on the above carbon sources were 1:1.23:1.99 and 1:1.02:1.29, respectively  [KMAR_50343 and KMAR_50346, respectively, grew well on a synthetic dropout (SD) solid medium with glucose as the carbon source, whereas YEBYVW4000-10531, which expressed KMAR_10531, did not grow  have been studied [K. marxianus [The function of d Boles) . YEBYVW4 studied , and Donarxianus .S. cerevisiae is released, which allows Rgt1 to bind the promoters of several HXT genes and represses their expression [KMAR_50343 and 50,346 which transports glucose probably led to a decreased glucose uptake which was consistent with the reduced glucose utilization ability of K. marxianus YΔSNF3  of YΔSNF3-50343 and YΔSNF3-50346 increased but was still lower than that of YΔSNF3-TRP1 . YΔSNF3-TRP1, YΔSNF3-50343, and YΔSNF3-50346 consumed 13.58, 14.12, and 12.67 g/L xylose, respectively, in 60 h, in contrast to YWD010, which consumed 7.73 g/L in 60 h. The xylose consumption rates of YWD010, Y△SNF3-TRP1, Y△SNF3-50343, and Y△SNF3-50346 were 0.25 g/(L·h), 0.39 g/(L·h), 0.66 g/(L·h), and 0.33 g/(L·h), respectively. Moreover, YΔSNF3-50343 consumed 10.66 g/L xylose in 12 h with a xylose consumption rate of 0.87 g/(L·h)  partially released glucose repression. YΔGRR1 grew better on all of the alternative carbon source plates in the presence of 2-DG than the control strain, YWD005, but worse than YΔSNF3 . The xylose-utilization ability of YΔGRR1 improved when the glucose-xylose mixture was used as the carbon source. YΔGRR1 consumed 12.05 g/L of xylose in 24 h, which was faster than that by YWD005  on all plates was similar to YWD005. Therefore, the disruption of KmMTH1 or KmRGT1 did not release glucose repression. The growth of YΔMTH1 and YΔRGT1 in the presence of glucose in liquid medium was similar to that of YWD005  in xylose, and 12.82 in the mixed carbon source. The growth of YΔRGT1 in xylose was slower than that of YWD005 initially and finally accumulated more biomass (OD600 = 21.62), whereas the growth of YΔRGT1 and YWD005 was similar in the glucose-xylose mixture . These results were expectable as KmMTH1 and KmRGT1 are required to the release of glucose repression [As shown in Fig. 005 Fig. B. The gl005 Fig. C. The gr005 Fig. D-H. The S. cerevisiae, in the presence of glucose, Mth1 and Std1 are phosphorylated and targeted for degradation via the ubiquitin-proteasome pathway by Grr1. Therefore, the disruption of GRR1 may result in stabilized Mth1 and Std1. In K. marxianus, STD1 homolog was not found in the genome. We postulated that Kmgrr1 disruption led to reduced degradation of KmMth1 and KmRgt1; thereafter, the signal transmitted downstream of KmMth1 and KmRgt1was no- or low-glucose in the medium. The partial release of glucose repression by YΔGRR1 was achieved  was constructed. Its growth on the alternative carbon source plate in the presence of 2-DG was repressed and similar to the growth of YΔRGT1 and the control (YWD005)  which is possibly stable.KmMTH1-ΔT was overexpressed in strains YΔSNF3 and YΔMTH1. The growth of YΔSNF3-KmMTH1-ΔT in glucose or alternative carbon sources with or without 2-DG was similar to that of YΔSNF3  in the presence of glucose  releases glucose repression of K. marxianus, although the glucose utilization ability decreased markedly [KmSNF1 (YΔSNF1) led to a defective alternative carbon source utilization ability [KmSNF1 in the YΔSNF3 glucose-repression released strain was disrupted. As shown in Fig. Kmsnf1 strain (YΔSNF1) . Therefore, double disruption strain was more sensitive to 2-DG.In our previous study, we investigated the Mig1-Hxk2 pathway and proved that the disruption of the sole hexose kinase gene  Fig. A. In S. sporters . The disKmMTH1-ΔT was expressed in strain YΔSNF1 to evaluate the crosstalk between the two pathways  of KmHXK1 in YΔSNF3 was the lowest and approximately 0.16, 0.13, and 0.06, respectively, in contrast to the 1.00 of YWD005  on 0.1% or 2% glucose medium. Fig. S2 The ethanol accumulations of YΔSNF3 with glucose (A), xylose (B) or mixture of glucose and xylose as carbon source (C). Fig. S3 GO and KEEG enrichment analyses of the RNA-seq results. Fig. S4 The growth of S. cerevisiae strains which expressed the transporter genes. Fig. S5 Evaluation of the effect of KmRGT1 disruption on glucose repression. Table S1 plasmids used in this study. Table S2 Primers used in this study. Table S3 RNA-seq results.Supplementary Material 1:


TLR9 gene is known to be a major element in an innate immune system. The relation between TLR9 and diabetes is connected via chronic inflammation, which affects the β-cell function. Previous studies on diabetes were with rs187084, rs352140, and rs5743836 SNPs in the TLR9 gene, and there are no direct studies. The prevalence of T2DM was expanding in Saudi Arabia. One of the foot ulcer infections in diabetic individuals is linked to the relationship between infection and diabetes. Individuals who had a COVID-19 infection are at an increased risk of developing chronic diseases, such as diabetes; this has been observed in Saudi Arabia. We design this study between rs187084, rs352140, and rs5743836 SNPs in the TLR9 gene and T2DM in the Saudi population. The current study results confirmed rs187084 and rs5743836 SNPs were associated in the Saudi population. This indicates that they play a role in the Saudi population. The TLR9 gene in different forms of diabetes. As a result, the purpose of this study was to investigate the relationship between rs187084, rs352140, and rs5743836 SNPs in the TLR9 gene among T2DM patients in the Saudi population. This was a case-control study that included 100 T2DM cases and 100 control subjects. The three SNPs were identified in the study population (n = 200) using polymerase chain reaction (PCR), restriction enzymes for rs352140, and Sanger sequencing for rs187084 and rs5783836. Next, statistical analyses were performed using various software to determine the association between the SNPs and T2DM. rs187084 and rs5743836 were associated with an increased risk of T2DM development. rs187084 and rs5743836 allelic frequencies were associated with a 3.2 times increased risk of T2DM development (p < 0.05). DBP was associated with T2DM (p = 0.02). rs187084 was associated with TC and HDLc; rs352140 was associated with DBP, HbA1c, and HDLc; rs5743836 was associated with waist (p < 0.05). The CGT haplotype was strongly associated with T2DM (p < 0.003). Gene–gene interaction, graphical presentation, and dendrogram showed the strong association with T2DM patients (p < 0.05). This study concluded that rs187084 and rs5743836 were strongly associated with T2DM in Saudi Arabian patients. This study provides further evidence that SNPs in the TLR9 gene play a significant role in T2DM development in a Saudi community.Diabetes mellitus is a complex disease with a wide range of manifestations. Diabetes, notably type 2 diabetes mellitus (T2DM), is becoming more common in Saudi Arabia as a result of obesity and an aging population. T2DM is classified as a noncommunicable disease, and its incidence in the Saudi population continues to grow as a consequence of socioeconomic changes. Toll-like receptors (TLRs) are innate immune receptors that mediate the inflammatory response in diabetes mellitus. Previous studies have documented the relationship between different SNPs in the  Globally, diabetes mellitus (DM) is one of the most common chronic diseases, and its prevalence has increased significantly over the last three decades . At presType 2 diabetes mellitus (T2DM) is characterized by hyperglycemia and is caused by a combination of deficient insulin secretion, inadequate insulin resistance, and misleading glucagon secretion . Since 1KB pathway ; p = 0.0003) and the dominant model . However, other genetic models  showed negative associations.The quality of genomic DNA was found to be between 1.7 and 2.0 with an estimated OD260/OD280 ratio. p = 0.01 and AA vs. GG: OR—0.61 [95% CI: 0.27–1.37]; p = 0.23); however, there was no association between different genetic models . There was a positive association in the co-dominant model, i.e., GG + AA vs. GG: OR—1.91 [95% CI: 1.08–3.34]; p = 0.02. GA and AA genotypes were high in control as well as T2DM patients. This indicates that the rs352140 SNP has a protective effect in T2DM patients.The rs352140 SNP, presented as GG, GA, and AA genotypes, was found in the T2DM group  and the control group . Statistical analysis confirmed a positive association between genotypes  and the dominant model . However, other genetic models  showed negative associations.The rs5743836 SNP, present as TT, TC, and CC genotypes, was found in the T2DM group  and the control group . Heterozygous (TC) and homozygous variants (TC) were highly prevalent in the T2DM group. Genotype analysis confirmed positive association between genotypes . The rs352140 SNP A and G alleles were present in A: 46.5%/G: 53.5% and A: 52.5%/G: 47.5% of T2DM and control patients, respectively. We did not find a significant association between the rs352140 SNP present in G and A alleles . The rs5743836 C and T alleles were found in C: 18.5%/T: 81.5% and C: 6.5%/T: 93.5% of the T2DM and control patients, respectively. There was a significant association between alleles .The differences between the three SNPs present in the p = 0.02) was associated (p > 0.05) with the SNPs in T2DM covariates.p = 0.03) and HDLC (p = 0.04) were significantly associated (p < 0.05). ANOVA analysis of the rs352140 SNP revealed increased weight (80.17 ± 17.44), waist (101.65 ± 22.58), hip (105.79 ± 13.77), FBG (13.72 ± 3.34), TC (2.67 ± 2.95), and TG (5.81 ± 1.01) in GG genotype. In the GA genotype, age (63.53 ± 7.65), BMI (31.73 ± 5.30), and SBP (127.70 ± 11.75) were high. In the AA genotype, DBP (81.77 ± 5.97), HbA1c (7.51 ± 0.86), and HDLC (1.57 ± 1.13) were elevated. ANOVA analysis of the rs352140 SNP showed an association between GG, GA, and AA genotypes and DBP (p = 0.0003), HbA1c (p = 0.03), and HDLC (p = 0.04). In the rs5743836 TT genotype, weight (80.74 ± 13.41), waist (99.38 ± 14.81), and FBG (13.41 ± 4.52) were elevated. In the TC genotype, BMI (31.59 ± 5.96), hip (104.67 ± 11.95), DBP (80.20 ± 6.29), HbA1c (7.21 ± 1.01), TG (2.39 ± 2.18), and HDLC (1.62 ± 1.74) were elevated. In the CC genotype, age (63.82 ± 7.52), SBP (123.50 ± 12.91), TC (5.95 ± 0.87), and LDLC (3.99 ± 0.51) were elevated. The ANOVA analysis confirmed that waist was significantly associated (p = 0.02) with the three genotypes.One-way ANOVA analysis was performed between the SNPs and the 13 covariates present in T2DM subjects . In the p < 0.003). These results indicate that the C-G-T haplotype confers risk towards T2DM. In addition, analysis of T2DM in combination with three SNPs in the TLR9 gene showed that the C-G-C, C-A-T haplotypes were found to offer T2DM protection .In the present study, seven haplotypes based on the three SNPs were constructed and analyzed to determine association with T2DM . The C-GTLR9 gene) was determined for both T2DM and control groups. No linkage disequilibrium was observed between the three SNPs, as shown in Linkage disequilibrium analysis (LD), defined by the delta coefficient (D), of the three SNPs , with the combination of testing accuracy among S1 (T = 0.645), S1/S3 (T = 0.7), and S1/S2/S3 (T = 0.705). In addition, the MDR analysis  involvement, in addition to environmental factors.Gene–gene interaction was studied in 100 T2DM cases, and it was carried out in the SNPs  in the LR9 gene . The preanalysis  ensures TLR9 gene and β-cells is considered to be the specialized cells in the pancreas, which is produced via insulin and also plays a major role in elevating blood glucose levels. However, this is not directly connected with immune cells. The connection between TLR9 and the innate immune system may be influenced by β-cell function and diabetes [Toll-like receptors are found on cells of the innate immune system and contribute to the diagnosis of T2DM by identifying damage-associated molecular patterns. Upon TLR recognition, innate immune cells perform a number of actions, such as phagocytosis, cytokine synthesis, co-stimulatory molecule expression, and adhesion molecule expression . TLRs ardiabetes ,25. One diabetes .p = 0.02), TC (p = 0.03), and LDLC (p = 0.04) were significantly associated with rs187084. DBP (p = 0.0003), Hb1Ac (p = 0.03), and HDLC (p = 0.04) were significantly associated with rs352140, and waist (p = 0.02) was significantly associated with rs5743836. The C-G-T haplotype increased the risk for developing T2DM (p < 0.003) by approximately four-fold, and the combination of C-G-C/C-A-T haplotypes showed protective role towards T2DM. LD analysis showed a negative association, and both MDR/GMDR analyses showed strong association of gene–gene interaction, dendrogram, and graphical representation (p < 0.05) with T2DM cases. Overall, we confirmed that T2DM development is associated with SNPs present in the TLR9 gene in the Saudi population.We conducted a case-control study among Saudi Arabian T2DM patients and determined the prevalence of three SNPs  in the study population. We confirmed a strong association between T2DM and rs187084 and rs5743836 SNPs. In addition, a negligible association was observed between T2DM and the rs352140 SNP. Additionally, DBP levels (TLR9 gene. rs187084 is present at the promoter region [p = 0.02), with 1.9 times increased risk.TLRs are widely expressed in the human body cells, and 12 SNPs are present in the r region . In thisp = 0.01) and diabetes foot (DF) (p = 0.02). In T2DM patients, 73.3% and 6.7% of the heterozygous genotypes were observed. In DF patients, TT and TC genotypes were present (50% each), and the CC genotype was not observed. The genotype frequencies observed in our study were different and could be due to a number of factors. First, the Egyptian study contained a small sample size. In addition, differences could be due to the ethnic diversity between the populations [A previous study in an Egyptian population has confirmed a significant role of rs5743836 in T2DM . These results were confirmed with multiple linear regression; a positive association was observed between DBP and T2DM (p = 0.02). The association between the rs352140 SNP and DBP was statistically significant (p = 0.0003). The elevated levels of DBP were found in the rs352140 AA genotype with 81.77 ± 5.97, which indicates there is a relation between HTN and the TLR9 gene. The relationship between the TLR9 gene and HTN has been previously documented [TLR9 gene plays a role in T2DM and HTN development in the Saudi Arabian population.HTN  is defined as a chronic condition marked by expanded SBP and DBP levels. In our study, we have found an association between DBP and the cumented ,34. OverTLR9 gene. Additional statistics was another strength.The major limitation of this study was small population size . The other limitations of this study could be not matching with gender and anthropometric characteristics in T2DM cases and control subjects. However, the number of participants included in this study was based on a sample size calculation. Only three SNPs were screened in this study. Another limitation of this study was not selecting T2DM patients with any of the infectious diseases. The final limitation of this study was missing of functional studies in TLR9. The strength of this study was enrolling T2DM patients with a family history of diabetes. By selecting the patients with complete family history, we were able to determine the molecular role of the TLR9 gene in T2DM. Previously, different SNPs in the TLR9 gene were well-established with infectious diseases and cancers. Presently, a link has been established between infectious diseases and T2DM [TLR9 gene in HTN development.In this study, we aimed to understand the molecular role of rs187084, rs352140, and rs5743836 SNPs in the


Vibrio harveyi infection from mRNA and protein level, respectively. We concluded that: a) at the mRNA level, IL-15Rα was significantly upregulated in each tissue after V. harveyi infection of grouper, as well as in HKL cells after stimulated by pathogenic stimuli. b) at the protein level, IL-15Rα can increase the cytokine expression level and promote the proliferation of HKL cells, and can also improve the survival of groupers after V. harveyi infection. Our study contributes to a better understanding of the immune responses of Ec-IL15Rα (Epinephelus coioides IL15Rα) in preventing Vibrio infection in groupers.In recent years, grouper industry has been severely devastated by vibriosis. The present study explored the function of IL-15Rα (α Chain of Interleukin-15 Receptor) response to Epinephelus coioides (Ec-IL15Rα), a natural regulator of IL15, was identified, and immune response functions of fish were determined and characterized. Ec-IL15Rα contains a 720 bp open reading frame that encodes 239 amino acids, including four typical conserved cysteine residues with a highly conserved sushi domain. Ec-IL15Rα is closely related to Epinephelus lanceolatus and is the most clustered with teleost. Subcellular localization studies showed that Ec-IL15Rα was situated in the cytoplasm and cell membrane. Ec-IL15Rα was detected in 11 tissues, with the highest expression in the liver and blood. Meanwhile, the Ec-IL15Rα transcriptional levels substantially increased in nine tissues after Vibrio harveyi infection. Ec-IL15Rα was significantly up-regulated in HKLs by ConA, PHA, LPS and poly I:C stimulation. In vitro analysis, the recombinant protein of rEc-IL15Rα stimulates HKL proliferation and IL1R, IL6R, IL10, and IL16 expression. Challenge experiments revealed that IL15Rα protein showed an increase of 6.67–10% survival protection rate after V. harveyi infection. This study provides a better understanding of the immune protection of IL15Rα in vertebrate fish. Interleukin-15 (IL15) is a proinflammatory cytokine that could induce the production of inflammatory cytokines. In this study, the α chain of the IL15 receptor of  Interleukin-15 (IL15) is a pro-inflammatory cytokine produced mainly by activated monocytes, macrophages and dendritic cells, and it can activate immune cells ,3. The 1h IL15Rα ,5,6,7,8.h IL15Rα ,10. IL15h IL15Rα ,12. The h IL15Rα . Differeh IL15Rα .Misgurnus anguillicaudatus [Oncorhynchus mykiss [Oplegnathus fasciatus [Edwardsiella tarda and Streptococcus iniae, Rb-IL15Ra of Oplegnathus fasciatus increased in the trunk kidney, head kidney and spleens [Flavobacterium columnare G4, Ichthyophthirius multifiliis and Saprolegnia parasitica infection of dojo loaches [IL15Rα was first identified in human and mouse as a specific receptor for IL15. IL15Rα gene gaps of human and mouse were found in chromosome 10 and chromosome 2, with the encoding protein sharing 54% homology ,15. In tcaudatus , Oncorhys mykiss  and Opleasciatus , althoug spleens . Ma-IL15 loaches . However loaches . CompareVibrio harveyi, a kind of Gram-negative opportunistic pathogen [Litopenaeus vannamei [Larimichthys crocea [Epinephelus coioides [V. harveyi had arisen in many areas of cultured grouper, devastating grouper breeding industries and reducing the production of groupers. Although antibiotics used to control vibriosis in grouper, they also caused severe consequences, including drug resistance and drug residual issues [E. coioides was first demonstrated, and the roles of Ec-IL15Rα in regulating immune response against V. harveyi infection were also investigated. The present study contributes to a better understanding of the role of Ec-IL15Rα in preventing vibrio infection in groupers.The orange-spotted grouper is an important agricultural product in southern China and Southeast Asia . Howeverpathogen , which cvannamei , Larimics crocea , Epinephcoioides  etc. V. l issues . UnderstHealthy orange-spotted groupers with body weight 65.0 ± 5.0 g and body length 10.0 ± 2.0 cm were purchased from a grouper breeding base in Dapeng New District, Shenzhen City, Guangdong Province, China. Groupers were cultured in sea water at 26.0 ± 2.0 °C. The sea water parameters were as follows: salinity was 30.0 ± 2.0‰, dissolved oxygen was 5.0 ± 1.0 mg/L, pH was 8.0 ± 0.2, and total alkalinity was 100.0 ± 5.0 mg/L. Groupers were fed about 2~3% of their body weight in feed a day for 2 weeks. Before injection, fish were anesthetized with 100 mg/mL tricaine methane sulfonate (MS-222). V. harveyi (PRJNA165825) was stored in our laboratory. This bacterium was isolated from grouper Epinephelus coioides. The bacterium was cultured in TSB at 28 °C overnight, washed in phosphate-buffered saline (PBS), and adjusted to the concentration of 1 × 109 CFU/mL [The strain ZJ0603 of 9 CFU/mL .® One-Step gDNA Removal and cDNA Synthesis SuperMix , respectively. Based on the E. coioides transcriptome database, we designed primers for IL15Rα, and listed them in The total RNA was extracted and first-strand cDNA was synthesized from the spleen of healthy groupers using TransZol Up Plus RNA Kit  and EasyScriptV. harveyi  was intraperitoneally injected into 18 groupers. The total RNA was extracted and cDNA was amplified using same methods of ® Green qPCR SuperMix . V. harveyi-stimulated tissues including liver, blood, intestine, head kidney, spleen, skin, gills, thymus, muscle, brain and heart were obtained from injected groupers (3 fish per time point) at 0, 6, 12, 24, 48 and 72 h time points, with 0 h as control. Healthy tissues including liver, blood, intestine, head kidney, spleen, skin, gills, thymus, muscle, brain and heart were obtained from healthy groupers. Fold change of IL15Rα in tissues was relative to the expression in the heart as a control. Primers are listed in 6 cell/mL) were added in 48-well plates and divided into 5 groups. Then, 250 μL per well of 4 different pathogenic stimuli, including conA type A IV , lipopolysaccharides , phytohemagglutinin PHA-P  and polyinosinic-polycytidylic acid , with a concentration of 10 μg/mL, were mixed with HKLs; PBS was used as the control group. The mixtures were incubated and collected at six time points  after the addition, respectively.HKLs were prepared as previously described with minor modifications . HKLs (12) for 12 h. Then, empty pEGFP-N1 and pEGFP-IL15Rα were transfected into 293T cells. The cells were washed with PBS, fixed with 4% paraformaldehyde, and stained with DAPI (1 g/mL) 24 h after transfection. Finally, confocal fluorescence microscope  was used to observe the cells after they had been rinsed with PBS and mounted with 50% glycerol.According to the Lipofectamine™ 3000 Transfection Reagent kit , the following instructions were followed for transfection experiments. HEK-293T cells were added and cultured in 24-well plates at 37 °C in Dulbecco’s modified Eagle’s medium  (IL15Rα was ligated with pGEX-4T-1 vector. The recombinant vector was transformed into Escherichia coli BL21 (DE3) . The positive clone was cultured in Luria–Bertani (LB) added 100 mg/mL Ampicillin. After OD600 reached 0.4–0.6, 0.5 mmol/L, isopropyl-β-D-thiogalactopyranoside (IPTG) was added. The bacteria were induced for 6 h, then collected and washed using PBS. The protein was purified and analyzed by SDS-PAGE and Western blot. The method of protein expression and purification was consistent with what Wang described . The prime (ORF) . EcIL15RThe Ec-IL15Rα recombinant protein (rEc-IL15Rα) was used to test cytokine production and proliferation of HKLs in vitro. The regulation of rEc-IL15Rα to the expression level changes in 4 cytokines, including pro-inflammatory factors IL1R, IL6R, and IL16, and anti-inflammatory factor IL-10, were detected in HKLs. 500 μL HKLs with the concentration of 2000 cells/μL were added into 24-well plates and divided into 3 groups: GST, PBS and rEc-IL15Rα group. Then, GST, PBS and rEc-IL15Rα with the concentration of 10 μg/mL were added into 24-well plates, respectively. HKLs were collected after 0, 3, 6, 9, 12 and 24 h. RNA was extracted and cDNA was reverse transcribed. β-actin was used to normalize the mRNA level.450. An amount of 100 μL HKLs with the concentration of 50 cells/μL were added into 96-well plates and divided into 4 groups: PHA, PBS, GST and rEc-IL15Rα group. Then, 1 μL/per well  of PHA, GST, PBS and rEc-IL15Rα were added into 96-well plates, respectively. An amount of 10 μL CCK-8  was added to wells and incubated for 4 h. Finally, microplate reader  was used to detect ODV. harveyi and the survival rate (SR) was detected from 1 to 7 d. Bacterium was re-isolated from the liver of dead fish and incubated on TSA plates. 16s rDNA was used to identify V. harveyi infection in fish. The SR was calculated as SR = (surviving fish ÷ 30) × 100%. The experiment was repeated three times.A total of 180 fish were divided into 6 groups (30 fish/group), with VH + GST(L), VH + GST(H), VH + Ec-IL15Rα(L) and VH + Ec-IL15Rα(H) as the experimental groups, and the PBS group and VH groups as the negative control . To testAdobe Photoshop CC  and Adobe Illustrator  were used to create the drawings and final panels. SPSS 26.0 software with Duncan’s new multiple range test was used to analyze the data using one-way analysis of variance (ANOVA).IL15Rα contains 720 bp ORF that encodes the putative protein of 239 amino acids, including a 26 amino acid signal peptide domain and a 23 amino acid transmembrane domain. SMART domain architecture analysis predicted that Ec-IL15Rα contains a CCP (sushi) domain that was from 31 to 94 amino acids (E. lanceolatus and mainly clustered with teleost (IL15Rα to form the CCP (sushi) domain (no acids A. Ec-IL1 teleost B. Multip) domain C.Ec-IL15Rα mRNA levels in tissues of healthy orange-spotted groupers were measured  and VH+Ec-IL15Rα(H) groups started decreasing from 2 d post-injection and finally decreased to 10% and 6.67% at 7 d, respectively. The results showed that compared with PBS, VH+GST(L) and VH+GST(H) groups with the SR of 0%, IL15Rα protein showed an increase of 6.67–10% in relative survival rate against nfection F.Ec-IL15Rα in 11 tissues and under four stimuli, as well as tested the immune-protective effects of Ec-IL15Rα protein in vivo and in vitro. These results provide insight for understanding the function and immune mechanism of IL15Rα in fish against bacterial infection.IL15Rα, as a receptor of IL15, was well characterized in avians, mammals and teleosts. However, the functions of orange-spotted grouper IL15Rα are still unclear. In order to interpret the function of IL15Rα, our study identified and described the molecular characterizations of IL15Rα from orange-spotted grouper . We alsoThe human and mouse IL15R complex is stable, and makes it extremely difficult for the free form of IL15 to be detected in humans and mice . Vice veIL15Rα was proved to be widely expressed in a variety of cell types and tissues, and mainly expressed in immune-relevant tissues that could be activated by immune response to adjust bacterium challenge. Ec-IL15Rα was obviously up-regulated within 48 h in tissues after V. harveyi infection. The mRNA levels of Ec-IL15Rα were obviously positively regulated and maximized at 6 h post-infection in gill, spleen and head kidney, at 12 h in the liver, heart, brain and muscles, at 48 h in thymus, and at 72 h in the intestines. Although different expression patterns were detected in different fish, IL15Rα was widely distributed and could certainly be induced by bacterial infection to play roles in immune defense. A similar conclusion was drawn in several studies: that the IL15Rα in different tissues can be enhanced after M. anguillicaudatus challenge against F. columnare [E. tarda and S. iniae [IL15Rα was highly expressed in the blood, brain, skin and gill of M. anguillicaudatus [O. fasciatus [Ec-IL15Rα was detected in 11 tissues, with the highest levels in the liver and blood. Ec-IL15Rα was highly expressed in immune-related tissues in both bacteria-infected and healthy groupers, indicating that IL15Rα may play key roles throughout the immune process of the body. Subsequently, HKLs were isolated and stimulated by ConA, PHA, LPS, and poly I:C, respectively. Results revealed that IL15Rα was significantly up-regulated by four pathogenic stimuli, which showed that IL15Rα could stimulate immune defense. The above results indicate that Ec-IL15Rα could be expressed in both immune and non-immune organs to activate immune responses in fish. The same results were shown in Ec-IL15 [Ec-IL15Rα cooperated with Ec-IL15 as a constitutive expression pattern to mediate signal transduction in the form of trans-presentation, to better activate the immune-relevant signaling pathways.olumnare , and rocS. iniae . In telecaudatus , while dasciatus . In our  Ec-IL15 . Until nIn this study, IL15Rα contains 720 bp ORF that encodes the putative protein of 239 amino acids, including a 26-amino-acid signal peptide domain and a 23-amino-acid transmembrane domain . In HEK-IL1R, IL6R, IL10 and IL16 could be activated by Ec-IL15Rα. IL1 and IL10, as inflammatory cytokines, could provide innate inflammation that is required for nonspecific host defense as well as acquired immunity [+ T lymphocytes, monocytes and eosinophils, but also initiate CD4+ T cells and induce T lymphocytes to express IL2 receptor [In order to investigate whether Ec-IL15Rα proteins can activate downstream signaling molecules in HKLs, the function of Ec-IL15Rα on the cytokine production and proliferation of HKLs was detected in vitro. As the results suggest, cytokines and cytokine receptors including immunity . IL6R asimmunity . As a mureceptor . In addireceptor . As a reDanio rerio [+ T cells, etc., promoting immune response and anti-infection [V. harveyi infection. Suggesting that Ec-IL15Rα may play roles in enhancing the binding and assisting Ec-IL15, to co-express and operate together against pathogen infection.In vivo, most reports showed that IL15 or IL15/IL15Rα complex could effectively improve immune protection to the host, including female BALB/c mice , Danio rio rerio , and femio rerio . IL15 ornfection ,39,40,41nfection . CompareEc-IL15Rα were expressed in all 11 tested tissues, with the highest expression in liver. Ec-IL15Rα could be stimulated by ConA, PHA, LPS and poly I:C stimuli, and take part in the immune response against infective V. harveyi. A subcellular localization study showed that Ec-IL15Rα is distributed in both cytoplasm and cell membrane. Although Ec-IL15Rα only improved 6.67%-10% of SR in vivo, but it could stimulate activate IL1R, IL6R, IL10 and IL16 expression and proliferation of HKL in vitro. This study establishes the groundwork for future research into the mechanism through which IL15R defends fish from infections.In conclusion, IL15Rα from orange-spotted grouper was cloned, and the amino acid sequence and structure of Ec-IL15Rα showed high similarity with other teleost species.


The composition and remodelling of the extracellular matrix (ECM) are important factors in the development and progression of cancers, and the ECM is implicated in promoting tumour growth and restricting anti-tumour therapies through multiple mechanisms. The characterisation of differences in ECM composition between normal and diseased tissues may aid in identifying novel diagnostic markers, prognostic indicators and therapeutic targets for drug development.Using tissue from non-small cell lung cancer (NSCLC) patients undergoing curative intent surgery, we characterised quantitative tumour-specific ECM proteome signatures by mass spectrometry.PXDN and ADAMTS16 gene expression was associated with shorter survival of lung adenocarcinoma and squamous cell carcinoma patients, respectively.We identified 161 matrisome proteins differentially regulated between tumour tissue and nearby non-malignant lung tissue, and we defined a collagen hydroxylation functional protein network that is enriched in the lung tumour microenvironment. We validated two novel putative extracellular markers of NSCLC, the collagen cross-linking enzyme peroxidasin and a disintegrin and metalloproteinase with thrombospondin motifs 16 (ADAMTS16), for discrimination of malignant and non-malignant lung tissue. These proteins were up-regulated in lung tumour samples, and high These data chart extensive remodelling of the lung extracellular niche and reveal tumour matrisome signatures in human NSCLC. Lung cancer, including non-small cell lung cancer (NSCLC), is a common cancer and the leading cause of cancer-related deaths . Lung caThe extracellular matrix (ECM) proteome, or matrisome, is composed of core ECM proteins, including collagens, glycoproteins and proteoglycans which form the structure of the ECM, and ECM-associated proteins, such as mucins, enzymes that can modify the ECM and secreted factors such as cytokines . MatrisoThis study aimed to detect changes in matrisome proteins between human NSCLC tissues and patient-matched non-cancerous lung using a quantitative proteomic approach to curate an extensive resource of lung tumour matrisome proteins. We analysed tissue from 34 patients undergoing curative intent resections for NSCLC, coupling MS-based proteomics to fractionation of tissue samples to enrich for matrisome proteins. We quantified the differential abundance of proteins between tumour tissue and non-cancerous lung, characterising tumour matrisome signatures in NSCLC. Functional network analysis identified a collagen hydroxylation module enriched in tumour tissue, and we validated two novel putative extracellular markers, peroxidasin and a disintegrin and metalloproteinase with thrombospondin motifs 16 (ADAMTS16), as being up-regulated within tumours in separate patient cohorts.th edition), one patient had a T1, 17 had T2, 10 had T3 and six had T4 tumours   and cathepsin S (1.7-fold) , MXRA5 (14.4-fold), peroxidasin (2.5-fold), thrombospondin-1 (2.5-fold), SPARC (2.3-fold), FRAS1 (2.3-fold) and tenascin-C (2.1-fold)  and ADAMTS16 were significantly up-regulated in adenocarcinoma tumours compared to matched adjacent normal lung tissue   = 1.57; 95% confidence interval (CI), 1.05–2.37; P = 0.028, log-rank test), whereas that of ADAMTS16 was not   or squamous cell carcinoma (ADAMTS16) patients undergoing curative intent surgery.We next assessed whether the protein expression levels of peroxidasin and ADAMTS16 were predictors of survival in the TMA cohort  is one of several collagen-modifying enzymes that we detected as up-regulated in NSCLC tumour tissue in this study, alongside a concomitant increase in collagen lysine hydroxylation. Lysyl hydroxylase 2 is secreted by lung cancer cells in culture, and its hydroxylation of collagen telopeptidyl lysine residues leads to the formation of stable hydroxylysine aldehyde-derived collagen cross-links that are up-regulated in lung cancer tissue and generate stiffer tumour tissue . All samples were assigned an anonymised code, and researchers were blinded to patient details for experiments. The TMA dataset was approved by Lothian NRS Bioresource, REC number 15/ES/0094 (reference SR1208), and approved by the NHS Lothian Caldicott Guardian (reference CRD19031).Tissues samples used for mass spectrometry (MS) were collected from patients with NSCLC undergoing curative-intent surgical procedures. Following resection, samples were handled by an experienced thoracic pathologist, and samples of tumour and non-cancerous lung  were dissected and provided for MS analysis. Samples were snap frozen and stored at −70°C until required. Anonymised patient details were recorded, including age, gender, smoking history, histopathological diagnosis of tumour and non-tumour tissues, degree of differentiation of the tumour tissue, tumour stage and lymph node stage (determined by TNM status), survival and PET tracer uptake.2+ or Mg2+), containing 1% (v/v) protease inhibitor cocktail (Sigma-Aldrich), using a Precellys 24 tissue homogeniser (Bertin Instruments) at 6,500 rpm for 50 s. Samples were incubated for 5 min on ice between homogenisation cycles. The homogenate was centrifuged at 14,000 × g for 10 min at 4°C. The supernatant was removed (fraction 1), and the remaining pellet was resuspended and incubated in 10 mM Tris-HCl, pH 8, 150 mM NaCl, 25 mM EDTA, 1% (v/v) Triton X-100, 1% (v/v) protease inhibitor cocktail for 30 min on ice. The lysate was centrifuged at 14,000 × g for 10 min at 4°C. The supernatant was removed (fraction 2), and the remaining pellet was resuspended and incubated in 20 mM NH4OH containing 0.5% (v/v) Triton X-100 in PBS (without Ca2+ or Mg2+). The lysate was centrifuged at 14,000 × g for 10 min at 4°C. The supernatant was removed (fraction 3), and the remaining pellet was incubated with 10 µg/ml DNase I in PBS (with Ca2+ and Mg2+) for 30 min on ice. Samples were centrifuged at 14,000 × g for 10 min at 4°C, and the supernatant was removed (fraction 4). The remaining pellet was washed in ice-cold PBS and centrifuged at 14,000 × g for 10 min at 4°C three times. The final insoluble pellets enriched for ECM proteins were then stored at −70°C until further use.Tissue samples were enriched for predominately insoluble matrisome proteins by depleting soluble intracellular proteins . Tissue 4HCO3, pH 8, 10 mM dithiothereitol for 30 min at 37°C. Samples were resolved by SDS-PAGE using 4–20% Tris-glycine gels. Proteins were transferred to PVDF membranes using an iBlot 2 dry blotting system (Thermo Fisher Scientific) according to manufacturer’s instructions. Membranes were blocked using milk blocking buffer (5% (w/v) non-fat skimmed milk powder (Marvel) in 1× Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBS-T)) for 1 h at room temperature with shaking. Following blocking, membranes were incubated with primary antibodies, diluted 1:1,000 in milk blocking buffer, overnight at 4°C with rolling. Antibodies used were rabbit polyclonal anti-fibronectin , mouse monoclonal anti-lamin A/C  and rabbit monoclonal anti-GAPDH . Membranes were washed three times in TBS-T and then incubated with anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies, diluted 1:10,000 or 1:5,000, respectively, in milk blocking buffer, for 45 min at room temperature with rolling. Membranes were washed three times in TBS-T. Membranes was developed using Pierce ECL western blotting substrate (Thermo Fisher Scientific) according to the manufacturer’s instructions. Membranes were imaged using an Odyssey Fc imaging system (LI-COR Biosciences).To confirm matrisome proteins were enriched in the final protein pellet prior to proteomics experiments, the supernatants from serially extracted fractions from a subset of samples were probed by SDS-PAGE and western blotting. Three percent of the supernatant from each fraction was used for western blotting to allow comparison between fractions. Fractions 1–4 were incubated in 1× Laemmli buffer containing 50 mM dithiothereitol for 10 min at 95°C. The final ECM pellet was precipitated using TCA–acetone (see below), homogenised and incubated in 8 M urea, 100 mM NHg for 30 min at 4°C. The supernatant was discarded, and the ECM-enriched pellet was resuspended in ice-cold acetone, using vortexing and sonication, and incubated for 20 min at −20°C. The sample was centrifuged at 16,000 × g for 30 min at 4°C, and the supernatant was discarded. The acetone wash step was then repeated. Samples were air dried at room temperature until residual solvent had evaporated. Precipitated protein pellets were stored at −70°C until further use.Ice-cold TCA (10% (v/v) final concentration) was added to final ECM-enriched fractions for 20 min at 4°C. The sample was centrifuged at 16,000 × 4HCO3, pH 8, 10 mM dithiothreitol). Samples were sonicated on ice using a probe sonicator and then incubated for 30 min at 37°C. After cooling to room temperature, sample pH in the range pH 8–9 was verified using pH indicator paper. To 50 µl sample, 8.3 μl of 175 mM iodoacetamide in 100 mM NH4HCO3, pH 8, was added  and incubated for 30 min at room temperature in the dark to block thiol groups of cysteine residues. Excess iodoacetamide was quenched by the addition of 3 µl of 100 mM dithiothreitol in 100 mM NH4HCO3, pH 8. For protein digestion, urea was diluted from 8 M to 2 M using 100 mM NH4HCO3, pH 8. Samples were incubated with 833 units of PNGase F for 2 h at 37°C with shaking (900 rpm), then with 800 ng of Lys-C for 2 h at 37°C, then with 1.6 µg of MS-grade trypsin for 16 h at 37°C with shaking . Samples were then incubated with an additional 0.8 µg of trypsin for 2 h at 37°C with shaking . Samples were acidified using trifluoroacetic acid (TFA) to obtain sample pH in the range pH 3–4, and samples were clarified by centrifugation at 18,000 × g for 15 min.Precipitated protein pellets were resuspended in 300 μl solubilisation buffer  TFA and centrifuged at 500 × g for 5 min. Samples estimated to contain 10 µg of acidified peptide (based on Nanodrop readings) were then added to Stage tips, and tips were centrifuged at 500 × g for 5 min. Stage tips with bound protein were stored at −20°C until elution.Peptide concentrations in the digested samples were estimated using a Nanodrop spectrophotometer measuring absorbance at 280 nm. Stop-and-go extraction (Stage) tips were made in-house, using a method adapted from Rappsilber et al. . Brieflyg for 5 min. Acetonitrile was evaporated using a vacuum centrifuge. Samples were adjusted to 15 μl volume with 0.1% (v/v) TFA, and peptide concentration was re-measured using a Nanodrop spectrophotometer measuring absorbance at 280 nm.Peptides were eluted from C18-containing Stage tips with 40 µl of 80% (v/v) acetonitrile, 0.1% (v/v) TFA, and tips were centrifuged at 200 × ‘Bottom-up’ liquid chromatography-coupled tandem MS (LC-MS/MS) was used to elucidate the structure of isolated peptides and to detect post-translational modifications. LC-MS/MS analysis was carried out using an Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific) coupled to an UltiMate 3000 UHPLC Nano (Thermo Fisher Scientific), Aurora C18 column (IonOpticks), column oven  and Proxeon Nanospray ion source (Thermo Fisher Scientific). Peptides (1 µg) were injected onto an Aurora C18 column in buffer A (2% (v/v) acetonitrile, 0.5% (v/v) acetic acid) and eluted with a linear 120-min gradient of 2%–45% (v/v) buffer B (80% (v/v) acetonitrile, 0.5% (v/v) acetic acid). Eluting peptides were ionised in positive-ion mode before data-dependent analysis. A dynamic exclusion window of 30 s was enabled and lockmass was not used.2 transformed, and proteins quantified in less than one-third of samples were removed. Missing values were imputed from a width-compressed, down-shifted Gaussian distribution using Perseus (version 1.6.2.3)  . MaxQuan1.6.2.3) .Gene Ontology (GO) over-representation analysis was performed using DAVID 2021  . The funin-silico and in-vivo data from the Matrisome Project  set to 1 using Perseus. Statistical data were visualised using Prism . For differentially expressed matrisome proteins, potential confounding variables were also considered. These variables included necrotic tumour samples versus non-necrotic tumour samples, adenocarcinoma versus squamous cell carcinoma, well and moderately differentiated tumours versus poorly differentiated tumours, tumours with local lymph node spread versus no known lymph node metastasis and normal non-tumour samples versus non-tumour samples with underlying pathology. Necrosis was crudely assessed based on the composition of the tumour samples when dissected and minced. PET standardised uptake value was scored as mild, moderate or marked. In some instances, absolute values were provided in reports rather than categorical values. These numbers were reclassified as no uptake for 0.6–0.8, low uptake for 1.0–2.0, moderate uptake for 1.5–2.0 and high uptake for >2.5. These variables were compared using multiple Student’s t-tests .Identified proteins were defined as belonging to the matrisome if they exist in searchable databases of matrisome proteins based on data from 17 studies of the ECM, MatrisomeDB , and in- Project . Label-f2-transformed label-free quantification intensities were standardised by row-wise (protein-wise) Z-scoring. Differentially expressed matrisome proteins were hierarchically clustered on the basis of Euclidean distance, computed using average linkage, using Cluster 3.0   . The folon 1.54) .Composite functional association networks were constructed using GeneMANIA   in CytosThe TMA was constructed for sequential patients undergoing surgical resection for lung cancer over a 2-year period at a regional thoracic surgery centre. Formalin-fixed paraffin pathological blocks were annotated by an experienced pathologist, and 1-mm cores taken from tumours and non-cancerous lung were embedded into new blocks and, subsequently, 4-µm sections were cut onto glass slides. Slides were deparaffinised and rehydrated, and antigen retrieval was undertaken with citrate buffer  twice for 5 min in a microwave. Slides were processed with a commercial DAB cell and tissue staining kit . Primary antibody immunostaining was optimised for polyclonal rabbit anti-human peroxidasin  and polyclonal rabbit anti-human ADAMTS16  . Secondary antibodies were incubated at room temperature for 30 min and DAB was developed. Slides were counterstained with haematoxylin and mounted, and images were acquired on an Axioscan microscope slide scanner (Zeiss).For peroxidasin immunostaining, there were 151 non-cancerous lung cores that were available for evaluation (149 interpretable following staining) and 138 tumour cores, of which 119 were paired samples . All samples were assigned an anonymised code, and researchers were blinded to patient details for experiments. The TMA dataset was approved by Lothian NRS Bioresource, REC number 15/ES/0094 (reference SR1208), and approved by the NHS Lothian Caldicott Guardian (reference CRD19031).HT, AK, JW, RO’C, AB and AA undertook experimentation and data generation. HT, AK, JW, RO’C, KD, MF, AB and AA interpretated the data and conceptualised the manuscript. AA, SP, DD constructed the TMA, retrieved the clinical data and provided samples for ECM extraction. HT, AB and AA wrote the manuscript. AB and AA jointly supervised the work. All authors contributed to the article and approved the submitted version.


High-quality cardiopulmonary resuscitation (CPR) is a key element in the rescue of cardiac arrest patients but is difficult to achieve in circumstances involving aerosol transmission, such as the COVID-19 pandemic.  This prospective randomized crossover trial included 30 experienced health care providers to evaluate the impact of personal protective equipment (PPE) on CPR quality and rescuer safety. Participants were asked to perform continuous CPR for 5 minutes on a manikin with three types of PPE: level D-PPE, level C-PPE, and PAPR. The primary outcome was effective chest compression per minute. Secondary outcomes were the fit factor by PortaCount, vital signs and fatigue scores before and after CPR, and perceptions related to wearing PPE. Repeated-measures ANOVA was used, and a two-tailed test value of 0.05 was considered statistically significant. p = 0.584). The fit-factor test values of level C-PPE and PAPRs were 182.9 ± 39.9 vs. 198.9 ± 9.2 (p < 0.001). The differences in vital signs before and after CPR were not significantly different among the groups. In addition, the fatigue and total perception scores of wearing PPE were significantly higher for level C-PPE than PAPRs: 3.8 ± 1.6 vs. 3.0 ± 1.6 (p < 0.001) and 27.9 ± 5.4 vs. 26.0 ± 5.3 (p < 0.001), respectively.  The rates of effective chest compressions for 5 minutes with level D-PPE, level C-PPE, and PAPRs were 82.0 ± 0.2%, 78.4 ± 0.2%, and 78.0 ± 0.2%, respectively (NCT04802109. PAPRs are recommended when performing CPR in situations where aerosol transmission is suspected. When PAPRs are in short supply, individual fit-tested N95 masks are an alternative. This trial is registered with  High-quality cardiopulmonary resuscitation (CPR) is a key element in rescuing patients from sudden cardiac death. According to the 2015 American Heart Association guidelines, high-quality CPR includes chest compression that requires a depth between 5 and 6 cm, a rate between 100 and 120 compressions per minute, complete chest recoil, and correct hand position , chemicaIn the past, the Ebola virus epidemic in West Africa caused many infections and deaths in medical personnel. In these circumstances, experts have especially emphasized the importance of personal protective equipment (PPE) . There wHowever, regarding the impact of PPE on CPR quality, the advantages and disadvantages of different types of PPE on rescuer safety remain unclear and the number of studies in this area is limited –17. HencThis simulation study was designed as a prospective randomized crossover trial performed from 1 April 2021 to 30 June 2021 at Annan Hospital, China Medical University, Tainan, Taiwan. The participants were required to perform five minutes of continuous CPR on a Laerdal manikin QCPR  in the kneeling position while using three types of PPE separated by 120 minutes. The AHA recommends changing rescuers every two minutes during CPR to prevent fatigue [The decision to refrain from employing the standing position at the bedside to assess CPR quality was based on well-established evidence indicating that this posture had a significant impact on CPR quality . This efParticipants who met all the following criteria were eligible for inclusion: (1) doctors or nurses in An Nan Hospital; (2) working experience of more than 1 year; and (3) those who had certified advanced cardiovascular life support or basic life support. The exclusion criteria were as follows: (1) those who had a history of spine surgery, sciatica, coronary artery disease, or lung disease such as asthma or chronic obstructive pulmonary disease or were pregnant and (2) those who could not tolerate 2 minutes of CPR. The participants had to sign informed consent forms before joining the study.The researchers used the modified ambient aerosol condensation nuclei counter quantitative fit testing protocol (M-QNFT) to test two types of N95 masks before the study: fold-type N95 masks  and cup-type N95 masks  Figures . The M-QThe three types of PPE were level D PPE, includes a surgical mask, face shield, hair cover, nitrile gloves, isolation gown, and foot cover (D-PPE); level C PPE, includes an N95 mask, face shield, hair cover, protective clothing , nitrile gloves, foot cover, and isolation gown (C-PPE); and PAPR, including level C PPE plus a PAPR  with a loose-fitting hood (S-433 L-5), breathing tube (BT-20 L), P3 particulate filter, 8-hour battery , with an N95 mask inside the hood Figures .With three types of PPE, six different sequences can be drawn for randomization (from A to F) . To ensuThe study was conducted in the pediatric room in the emergency department in this hospital, in which temperature and humidity were controlled at approximately 20∼22°C and 60∼80%, respectively. The primary outcome was the percentage of effective chest compression, which was defined as each chest compression that fit all requirements of the AHA basic life support guidelines . The sect-tests were also applied to measure differences between the two groups, and one-way analysis of variance (ANOVA) was used to measure differences between three or more groups, followed by post hoc Bonferroni tests for pairwise comparisons if the ANOVA results were significant. This study applied the McNemar test for repeated measurements between categorical variables. All statistical tests were two-tailed, the level of significance was 0.05, and the analysis was performed using SPSS for Windows, version 17.0 .Based on a pilot study using the “5 min effective chest compressions” factor, the authors estimated that with a significance level of 0.05, a power of 80%, and an effect size of 0.899, a sample size of 12 participants with repeated measurements would be adequate for evaluating the primary outcome. Additionally, we recruited 30 participants with an actual power of 0.91 (91%). Categorical variables are presented as numbers and percentages, and continuous variables are presented as the mean ± standard deviation. Independent Thirty-two participants were enrolled in the study, two of whom were excluded due to an inability to complete at least one minute of CPR during the study period. Among the remaining participants, there were 7 physicians and 23 nurses; 16 were male and 14 were female; 80% (24/30) of the participants worked in the emergency department, while the remaining participants worked in the intensive care unit and COVID-19-dedicated ward. The mean, standard deviation, and range of the participants' information were as follows: age, 34.2 ± 5.8 years (25–45 years); working experience, 10.9 ± 5.7 years (2–25 years); height, 168.5 ± 8.5 cm (150–184 cm); weight, 69.7 ± 14.4 kilograms (47–108 kilograms); and body mass index, 24.4 ± 3.8 (17.9–36.5).p < 0.001, talking was 185.8 ± 44.9 vs. 110.2 ± 78.3, p < 0.001, head side-to-side was 177.3 ± 47.6 vs. 116.9 ± 72.8, p < 0.001 and head up-and-down was 178.2 ± 50.7 vs. 109.3 ± 74.5, p < 0.001, respectively. The overall M-QNFT scores were 175.4 ± 51.5 vs. 100.2 ± 75.8, p < 0.001. For all participants who wore the face mask in the first round, the pass rate with the Champak F550 was higher than that with the 3 M 1860, 25/30 (83.3%) vs. 14/30 (46.7%), respectively, p=0.064 (McNemar test). After the intervention for teaching participants how to wear the mask, the pass rate with the Champak F550 increased to 30/30 (100%), while that with 3M1860 increased only to 18/30 (60%), p < 0.001 (McNemar test). Therefore, the researchers chose the Champak F550 as a standard N95 mask in this study.This study required the M-QNFT to decide which face mask to choose. The M-QNFT scores were significantly higher with the Champak F550 than with the 3 M 1860: bending was 182.6 ± 41.7 vs. 91.8 ± 76.6, p=0.716). For the entire five-minute evaluation, D-PPE was 82.1 ± 0.2%, C-PPE was 78.4 ± 0.2%, and PAPR was 78.0 ± 0.2%, and these differences in performance still did not reach statistical significance (p=0.584)  .p=0.011) for the initial two minutes and 183.0 ± 39.9 vs. 198.9 ± 9.2 (p < 0.001) for the overall five-minute comparison. However, if an FF ≥ 100 was considered a pass, the C-PPE still met the standard for 96.7% (29/30) in the first two minutes and 86.7% (26/30) in the overall five minutes.C-PPE was worse than PAPR in terms of FF at any particular minute across the five-minute continuous monitoring analysis . The FF After 5 minutes of CPR, the participants' systolic blood pressure, diastolic blood pressure, heart rate, and subjective visual analog fatigue score were all increased compared with those before CPR, while the pulse oximeter measured in room air conditions slightly decreased . Among tp < 0.001). In addition, C-PPE scores were significantly higher than PAPR scores for difficulty breathing, feeling hot while wearing, and difficulty communicating, and PAPR scores were significantly higher than C-PPE scores for donning. For other information, please refer to Regarding the total scores across the ten items, C-PPE was associated with the worst perception, PAPR was in the middle, and D-PPE was considered the best, and there were significant differences among them  , acting However, our study also has many advantages; the most important is that it was a very rigorous study design and was quite comprehensive in terms of prognostic parameters. CPR quality, FF, fatigue scores, vital signs, and various aspects of the perception of wearing PPE have not yet been reported in other studies.PAPR is recommended when performing CPR in situations where aerosol transmission is suspected. If PAPR cannot be fully implemented, N95 masks with an acceptable FF are recommended for use. Additionally, when using N95 masks, rescuers should avoid speaking loudly during CPR to minimize the risk of mask leakage.