The adult heart displays poor reparative capacities after injury. Cell transplantation and tissue engineering approaches have emerged as possible therapeutic options. Several stem cell populations have been largely used to treat the infarcted myocardium. Nevertheless, transplanted cells displayed limited ability to establish functional connections with the host cardiomyocytes. In this study, we provide a new experimental tool, named 3D eX vivo muscle engineered tissue (X-MET), to define the contribution of mechanical stimuli in triggering functional remodeling and to rescue cardiac ischemia. We revealed that mechanical stimuli trigger a functional remodeling of the 3D skeletal muscle system toward a cardiac muscle-like structure. This was supported by molecular and functional analyses, demonstrating that remodeled X-MET expresses relevant markers of functional cardiomyocytes, compared to unstimulated and to 2D- skeletal muscle culture system. Interestingly, transplanted remodeled X-MET preserved heart function in a murine model of chronic myocardial ischemia and increased survival of transplanted injured mice. X-MET implantation resulted in repression of pro-inflammatory cytokines, induction of anti-inflammatory cytokines, and reduction in collagen deposition. Altogether, our findings indicate that biomechanical stimulation induced a cardiac functional remodeling of X-MET, which showed promising seminal results as a therapeutic product for the development of novel strategies for regenerative medicine. Cell transplantation aims to replace compromised and scarred regions of the ischemic myocardium and represents a valid strategy to reduce ischemic area preserving cardiac activity after myocardial injury2. For this purpose, a variety of stem cells have been identified and tested for cardiac repair, an approach known as cellular ‘cardiomyoplasty’5. Among these, skeletal myoblasts have been widely investigated for their ability to generate a contractile phenotype6. The easy access to autologous muscle biopsies, the low risk of tumorigenicity and ethical concerns made muscle-derived cells transplantation a good alternative for treating myocardial ischemia and preventing loss of cell contractility7.Myocardial infarction (MI) represents one of the leading causes of death worldwide. Despite a rapid response would be required to repair myocardial damage, adult cardiomyocytes are terminally differentiated cells with limited regenerative capacities. Consequently, dead cardiomyocytes are replaced by non-functional fibrotic tissue that contributes to the development and progression of myocardial dysfunction and remodelling after an ischemic event8. Satellite cells were one of the first types of cells used in clinical trials of cardiology disease due to their promising features, including the relative resistance to the highly hypoxic environment that is found in ischemic tissue8. Evidence in animal models suggests that myoblast transplantation improves myocardial perfusion and cardiac contractility in the infarcted heart10. In addition, studies demonstrated that the autologous transplantation of skeletal myoblasts improved global heart function11 although they failed to define whether transplanted myoblasts can potentially counteract arrhythmogenic phenomena12. Indeed, clinical trials administrating muscle-derived cells displayed modest results with poor therapeutic benefits since these transplanted cells failed to establish a functional electrical syncytium with the hosting tissue14. This resulted in defective gap junction connection and arrhythmia15, a pathologic condition that has been overcome by increasing the levels of connexin-43 (Cx-43) in satellite cells, thus attenuating arrhythmia in an in vitro model of skeletal muscle transplantation16.Mature skeletal muscle hosts a pool of undifferentiated cells, called satellite cells, that can differentiate into muscle fibers and restore the injured tissue17.Tissue engineering (TE) represents an encouraging alternative approach to cell transplantation since it allows the rapid repair of the infarcted myocardial wall and may prevent the eventual rupture of the myocardium. This technology is based on the use of scaffolds together with specific culture conditions for creating in vitro engineered myocardial tissue. Of note, the scaffolds used for tissue engineering, while being supportive for myocardial regeneration, displayed critical features in terms of biocompatibility, biodegradability and cytotoxicity, triggering an inflammatory response that impairs implant efficacy18. Imposing a specific mechanical tension, able to promote a mechano-transduction-dependent cell differentiation19, we obtained a functional remodelling of the X-MET toward a cardiac-like phenotype. Interestingly, stretched X-MET displays the capacity to form gap junctions, guaranteeing electrical integration of the myotubes into a functional syncytium and preserving heart function in a murine model of chronic myocardial ischemia. These features make the X-MET an ideal clinically applicable cardiac-like patch.In our study, we aimed to overcome the potential limitations of using skeletal muscle cells and scaffolds by triggering a cardiac functional remodelling in a three-dimensional, scaffold-free, vascularized skeletal muscle engineered tissue (X-MET)21. To define whether these functional properties are linked to the imposed mechanical tension, we performed specific mechanical tests. We observed, performing a stretching test at constant velocity of 0.04 L0/s, that X-MET was able to develop its maximum passive force equal to about 200 μN when it reached the 66 ± 1.5% of its initial delamination length  and Cardiac ryanodine receptors (RyR 2)  with the aim to define the mechano-transduction related genes and pathways that allow intercellular communication. We observed a series of genes that were differentially expressed in the 3D stretched construct compared to the 2D culture system and to the unstretched X-MET 25. Real-time PCR analysis revealed a significant reduction of the miR-1 expression in the stretched X-MET structure compared to unstretched one . One end of the X-MET was labeled with a mixture of a low molecular weight (MW) fluorescent dye, namely Lucifer yellow (LY), and the rhodamine dextran (RD) high MW marker dye, unable to pass through gap junction. The transfer of the dye through gap junctions was monitored microscopically along the length of the X-MET. The time-lapse experiment demonstrated that LY was able to fully spread within the X-MET, while rhodamine dextran was restricted to the labelled site Fig. A, sugges27. The observed mean values of the Young's modulus, obtained through the Atomic Force Microscopy (AFM) curves analysis, were 7.5 ± 3.7 kPa and 25.0 ± 9.4 kPa for the unstretched and stretched X-MET, respectively , MI (infarcted mice), MI + X-MET 0% (infarcted mice that received unstretched X-MET implantation) and MI + X-MET 66% (infarcted mice that received X-MET 66% stretched implantation). Adverse ventricular remodelling after myocardial infarction represents the structural basis for ischemic heart failure (HF) allowing changes in left ventricle (LV) size, shape, function, and cellular composition. Forty days after MI we analysed, by Masson Trichrome staining, histological changes in infarcted hearts as compared to healthy and MI + X-MET transplanted hearts  in humans, followed by X-MET implantation immediately after the ischemic occlusionrts Fig. A. Animalrts Fig. A,C. Interts Fig. A,C. Baserts Fig. B, mice srts Fig. B.Figure 31.One of the first events associated with post-myocardial infarction is the activation of the inflammatory response. Nevertheless, impaired suppression of postinfarction inflammation leads to chronic inflammatory response that promotes fibrosis, causing adverse remodeling and contributing to the pathogenesis of heart failure. Among cytokines, Interleukin-6 (IL-6) and Interleukin 2 (IL-2) play key roles in the establishment of chronic inflammatory response and chronic heart failure32. In our context, we found an up-regulation of miR-29b correlated to an increase of collagen production in MI and MI + X-MET 0% compared to MI + X-MET 66% transplantation. This suggests that stretched X-MET promotes an efficient rescue of the damaged phenotype, avoiding excessive inflammatory processes, fibrosis, and abnormal cardiac remodeling.Real-time PCR analysis showed that forty days after ischemic damage proinflammatory IL-6 and IL-2 transcript levels significantly increased in infarcted hearts, whereas their expression was significantly decreased in MI + X-MET 66% transplanted hearts Fig. D. In conTo verify the ability of X-MET to also counteract the functional impairment of the infarcted myocardium, we performed an echocardiography analysis 40 days after LAD Fig. A. End-di33. Analysis of trichrome-stained cross-sections of the heart , promotion of new viable tissue, promotion of angiogenesis, reduction of fibrosis, reduction of a pro-inflammatory milieu, up-regulation of different classes of cardiac functional-related genes. The more these goals are accomplished, the more a functional improvement of myocardial function will be achieved. A functional improvement of myocardial performance usually correlates also with an improvement in term of overall survival, in pre-clinical animal models as well as in humans. Interestingly, we demonstrated that stretched X-MET engraft within the infarcted myocardium, counteracting the morphological alterations of injured heart, reducing the infarct size, and significantly reducing mortality of infarcted mice. Indeed, we demonstrated that while the MI mice and MI + XMET 0% did not survive more than 40 days after infarction, the transplantation of X-MET stretched patch significantly reduced infarct size even after 100 days post-infarction and enhanced the survival of infarcted transplanted mice.While it is difficult to show a direct contribution of X-MET to cardiac contractility in vivo, all data provided in the manuscript suggest that X-MET implantation promotes physiological adaptation toward a beneficial adaptative remodelling. This positive remodelling is “clinically” corroborated by several in vivo echocardiographic functional analysis performed with a state-of-the-art echo-machine  40 days after the LAD ligature. In vivo functional analysis thus revealed a Fractional Shortening and End-diastolic/end systolic diameters similar to those of the sham mice. These data have non negligible implication since they replicate the post-acute MI trend observed in a real human clinical setting. In addition, while the MI mice and MI + XMET 0% did not survive more than 40 days after infarction, the transplantation of X-MET stretched patch significantly reduced infarct size even after 100 days post-infarction and improved ventricular function and long-term survival of transplanted mice.Of note, morphologic analysis revealed an increased ventricular wall 100 days after MI and X-MET implantation compared to sham operated mice. To define potential activation of pathological or physiological hypertrophic phenotype we analysed relevant markers of the hypertrophic phenotype, revealing a significant and selective upregulation of the ratio α-MHC/β-MHC gene expression but not of ANP and BNP expression, which represent well‐established markers of heart failure (HF). These data suggest the activation of a potential compensatory mechanism exerted by X-MET at a later stage of implantation.39. The compensatory phase can progress to a pathologic condition, characterized by contractile dysfunction, cell death, inflammation, and fibrosis.The heart has the capacity to adapt in response to several physio-pathologic stimuli, activating cardiomyocyte growth and concomitant ventricular hypertrophy, which normalizes wall stress and maintains cardiac output and contractile efficiencyInterestingly, echocardiography analysis revealed an improved ventricular function, modulation of inflammation, and reduced fibrosis in X-MET transplanted mice, associated also with long-term survival of infarcted mice. Moreover, X-MET implantation was associated with a down-modulation of relevant pro-inflammatory and pro-fibrotic cytokines, such as IL-6, and induction of anti-inflammatory cytokines, such as IL-4, preventing scar formation. Altogether these data suggest that X-MET implantation possibly promotes physiological adaptation.Although we were unable to conduct telemetric analyses to directly evaluate the impact of X-MET transplantation on the development of arrhythmias, we feel to exclude a significant effect in our model. In fact, we did not observe any increase in sudden death in the group treated with the patch, as it should occur in case of ventricular arrhythmia development. Actually, survival of mice treated with X-MET was significantly higher than control mice. Our biochemical analyses also support this notion, since X-MET transplantation induced the up-regulation of Cx-43, the downregulation of the arrhythmogenic miR-1 and the formation of gap junctions within the stretched X-MET. Interestingly, the down modulation of miR-29b expression in the infarcted heart transplanted mice with stretched X-MET further suggests that X-MET reduces overall cardiac dysfunction.In summary, biomechanical stimulation of X-MET induced a functional remodeling of a skeletal muscle cell structure toward a cardiac-like phenotype that showed promising seminal results for the development of novel strategies of myocardial patch transplantation and suggest a possible prominent role for this approach in cardiac regenerative therapy, providing a vascularized non-fibrotic patch that is viable and electrophysiologically stable after a long period implantation.Wild type and transgenic animals used to generate X-MET were housed in a temperature-controlled (22 °C) room with a 12-h light/dark cycle. All animal experiments were carried out in compliance with the guidelines of the Institutional Review Board of the animal facilities of DIEM and National Institute of Health-Italy  and reported in accordance with the ARRIVE guidelines.18. A heterogeneous population of cells was obtained by mechanic and enzymatic dissociation of hind limbs harvested from wild type mice (WT) or transgenic mice using the Skeletal Muscle Dissociation Kit by Miltenyi Biotech. Dissociated cells were filtered through a 70 μm cell strainer and centrifuged at 1200 rpm for 15 min. Cells were resuspended in growth medium (GM)  and plated for 30 min twice in a row to get an enrichment of myoblasts in the culture. Cells were resuspended and plated at a concentration of 40,000 cells/ml on a tissue culture dish of 35 mm diameter coated with type I collagen (Sigma) and incubated at 5% CO2 at 37 °C. After 5–6 days of culture, myoblasts were induced to differentiate using a differentiation medium . After 2–3 days of incubation with DM, a skeletal muscle primary cell monolayer is delaminated by gently moving a sterile tip around the peripheral area of the plate. The delaminated monolayer was then pinned on a silicone-coated dish  using 0.20 mm diameter stainless steel Minutiens pins (Austerlitz INSECT PINS®). The X-MET was tensioned at two different lengths: + 0%, which corresponds to the initial delamination length (unstretched X-MET), and 66 ± 1.5% (+ 66%) of initial delamination length (stretched X-MET). In these mechanical conditions X-MET was analysed at morphological, functional, and molecular levels after 15 days in culture. Generally, X-MET exhibits a self-organized cylindrical structure containing beating myotubes and shows, on average, a diameter of 200 ± 12 mm and a length (after stretching) of 2 ± 0.5 cm.Muscle primary culture and generation of X-MET were performed following the protocol detailed in Carosio et al.At first, the passive force generated by the tissue during stretch was measured using an actuator/transducer (Aurora Scientific Inc. 300B) for length control and a microforce transducer (Kronex AE801). A software developed in LabVIEW 2019 allowed the synchronization between the stretch signal and the force measurement as well as the settings of the test parameters. Starting from the initial delamination length of the tissue, the X-MET was stretched at 66 ± 1.5% with a constant velocity of 1 cm/min and simultaneously the passive force generated by the tissue was measured. Then, the X-MET spontaneous contraction force was measured 15 days after the construct delamination for both the unstretched and stretched X-MET conditions. In detail, one end of the tissue was maintained fixed by the pins and the other one was connected to a microforce transducer (Kronex AE801). Muscle contractile activity was acquired for 15 s through a National Instruments data acquisition board (DAQ NI-PCI 6251) and a software developed ad hoc in Labview 2019. The spontaneous contraction frequency was then computed through the Fast Fourier Transform (FFT). For the entire duration of the force measurement test, the X-MET was placed in a culture dish containing differentiation medium and was maintained at a temperature of 37 °C, by using a temperature control plate .Total RNA extraction was performed using TriReagentTM (SIGMA) and one microgram of each RNA sample was retrotranscribed using the QuantiTec Reverse Transcription kit (QIAGEN) to obtain double-stranded cDNA. Relative quantitative PCR was performed on ABI PRISM 7500 SDS , using premade 6-Carboxyfluorescein (FAM)-labeled TaqMan assay for Hprt, Cx-43, TNNT2, β-MHC, PDE1C, IL-6, IL-2, IL-4, IL-10, CCL2, COL3A1, CACNA 1C, RyR 2, α-MHC, ANP and BNP . The relative quantitative RT-PCR sample value was normalized for the expression of Hprt mRNA. To analyze miR-1 and miR-29b, extracted RNA was retrotranscribed using micro-RNA Reverse Transcription KIT (Applied Biosystem). Relative quantitative PCR was performed on ABI PRISM 7500 SDS , using premade 6-Carboxyfluorescein (FAM)-labeled TaqMan assay for miR1. The relative quantitative RT-PCR sample value was normalized for the expression of U6 snRNA.40;v2.1.1), quantified with HTSeq-count . Differential expression analysis was performed in R (v3.5.1) using DESeq2 . Counts data from all conditions were filtered based on their raw count, keeping only those whose sum of the counts for all samples was higher than 1. Principal Component Analysis (PCA) was based on the 42% most variant genes between the different samples. Genes were considered differentially expressed with Benjamini–Hochberg adjusted p-value (FDR) < 0.01.RNA was isolated with Trizol from stretched X-METs after 15 days in DM, unstretched X-METs and 2-dimensional primary cells. One microgram of RNA was sent to the Institute of Applied Genomics  for deep sequencing. cDNA libraries were processed accordingly with the standard Illumina protocol and sequenced with the HiSeq2500 . Reads were aligned to the UCSC mm10 version of the mouse genome using Tophat2  (Sigma-Aldrich), Connexin-43 (Cx-43) (Sigma-Aldrich) and Cardiac troponin (Troponin I) (RV-C2 Hybridoma Bank). Sections were then washed in PBS with 0.2% Triton X-100 and incubated with secondary antibody  45 min at room temperature. Nuclei were stained using Pibenzimol bisbenzimide H33342 (HOECHST). All analyses were performed using Zeiss Confocal software (Zen 3.0 Blue edition).2, washed with PBS for three times and then analysed with confocal microscopy. In order to verify the viability of X-MET and monitor the transferring of the dye, a time-lapse analysis was performed. Images were acquired with a Leica confocal microscope (laser scanning TCS SP2) equipped with Ar/ArKr and HeNe lasers, using a 10X objective. The laser line was 488 nm for the excitation of Lucifer yellow and 633 nm for the excitation of rhodamine dextran. Fluorescence was collected at 500/540 nm for Lucifer yellow and at 640/680 nm for rhodamine dextran. The fluorescence intensity was computed using Leica software.The X-MET was washed 2 times with calcium- and magnesium-free PBS. A mix of Lucifer yellow CH (0.2 mg/ml) (Molecular Probes) and rhodamine dextran (0.5 mg/ml) (Molecular Probes) diluted in differentiation medium was prepared. To allow the dyes to penetrate the cells, the construct was placed on a slide which was previously prepared to maintain the X-MET viable and create a ‘double incubation chamber’: in the chamber containing one end of the X-MET construct 50 µl of the above-detailed mix was added, while in the chamber containing the other end differentiation medium was added to keep the tissue wet. The X-MET was so incubated for 10 min at 37 °C, 5% CO2+]i Transient, X-MET unstretched (X-MET 0%) and X-MET stretched at 66% of the initial delamination length (X-MET 66%) were cultured on 35 mm dishes and incubated in culture medium containing 3.5 μmol/L 2-[6-[bis[2-[(Acetyloxy)methoxy]-2-oxoethyl]amino]-5-[2-[2-[bis[2-[(acetyloxy)methoxy]-2-oxoethyl]amino]-5-methylphenoxy]ethoxy]-2-benzofuranyl]-5-oxazolecarboxylic acid (acetyloxy)methyl ester  for 30 min at 37 °C. Then, the medium was rinsed with Hank’s balanced salt solution . Dishes were placed into a culture chamber on the support of an inverted fluorescence microscope , at 37 °C connected to a cooled charge-coupled devices camera . Random access monochromator was used to illuminate samples alternately at 340 and 380 nm  and the emission was detected using a 510 nm emission filter. Metafluor® software  was used to acquire images. At the end of each experiment, calibration was obtained by maximally increasing intracellular Ca2+-dependent FURA-2-AM fluorescence with 5 μmol/L ionomycin  followed by recording minimal fluorescence in a Ca2+-free medium42.FURA-2AM indicator_ To determinate the intracellular calcium [Ca2+/Mg2+ free PBS. Once reconstituted, it was protected from light and stored at − 20 °C to avoid freeze-thaws. The samples (X-METs 0% 15 DM and X-METs 66% 15 DM) were washed thrice in PBS. Then, INDO-1 AM was added slowly along the muscle constructs and the samples were incubated for 30 min at 37 °C. At the end, the X-METs were washed with PBS three times and were analysed with confocal microscopy. The detection was carried out considering the double emission of INDO-1 that shifts from 475 nm in Ca2+-free media to 400 nm when the dye is saturated with Ca2+43. All analyses were performed using Zeiss Confocal software (Zen 3.0 Blue edition)43.INDO-1AM indicator_INDO-1 AM (Invitrogen I1226) was reconstituted in high-quality freshly opened DMSO at a concentration of 1 mM and used at a final concentration of 100 μM in CaSamples were homogenized in lysis buffer .Equal amounts of protein (70 μg) from each lysate (previously quantified through Bradford assay) were separated in SDS polyacrylamide gel  and transferred into a nitrocellulose membrane  using Trans-Blot ® TurboTM Transfer System . The membrane was then stained with Ponceau (0.005% in 1% acetic acid) as an intermediate loading control, then blocked with 5% non-fat dry milk in TBS-1% Tween for 1 h at room temperature and then incubated overnight at 4 °C with a primary antibody for Cx-43 (Sigma-Aldrich). The membrane was thereafter washed four times for 5, 15, 15 and 5 min in TBS-1% Tween, then incubated with a specific peroxidase-conjugated secondary antibody for 45 min at room temperature. After three 10 min washes with TBS-1% Tween, the membrane was analysed by the enhanced chemiluminescence system  according to the manufacturer's indications. The acquired signal was quantified by scanning densitometry using a bio-image analysis system (Image LabTM Software). The results are expressed as relative integrated intensity compared to controls (GAPDH), after subtracting their respective backgrounds.44, before starting each experiment) that follows in the range 0.0065 ± 0.0005 N/m. Each sample has been measured by means of Force Volume maps of 16 × 16 force curves, at the speed of 3 force curve per second, in several different areas of 100 micron squared each. A prior manual selection (by visible inspection) of the force curves has been done by using the Bruker NanoScope Analysis software , in order to exclude from the analysis, the curves that presents a signal disturbed by the contraction of the sample. The stiffness calibration value  has been obtained from acquiring force curves on a pristine petri dish. After these preliminary steps, the force curves were analysed by means of the FC_analysis software, whose complete functioning is described in Dinarelli et al.45. The Young’s modulus value has been obtained by fitting the curves with an Hertian model by considering the Poisson ratio of the samples as 0.5 and a conical tip with an opening half angle of 35°. The total number of force curves included in the analysis are 700 and 1200 for the un-stretched X-MET and stretched 66% X-MET, respectively. The statistical and graphical elaborations have been carried out by using the software Origin .The elasticity measurements were conducted using the Bruker Dimension Icon Atomic Force Microscope  equipped with the probe holder to operate in fluid environment. The samples were maintained pinned into a petri dish covered with a layer of PDMS to hold the pins in position, during the entire measurements the samples were not adhered to the PDMS surface. The measurements were carried out in physiological buffer by using MLCT-BIO tips from Bruker, with elastic constants  artery was performed using a permanent 8–0 prolene suture , with silicon tubing (1 mm OD) placed on top of the LAD, 2 mm below the border between the left atrium and LV46. During the operation, mice were monitored with a rectal probe to keep body temperature between 36.8 and 37.2 °C using a heat pad. The chest was cut horizontally at the fourth intercostal space. Eventually, one end of the X-MET was fixed through a second ligation knot on site of damage at the same time of the LAD ligation surgery. The implantation was performed ensuring that the damage site was completely covered by the X-MET construct and the procedure was finished by carefully relocating the pericardium above the heart wall. Then, the chest was closed using 5–0 polypropylene suture. X-METs were obtained from C57BL/6J consanguineous mice and UBC/GFP mice excluding the use of immunosuppressive drugs. Forty and one hundred days after LAD, echocardiographic and histological analyses were performed to assess ischemia and myocardial remodelling.Three months old C57BL/6J mice were anesthetized with isoflurane (IsoFlo®) 1.35% + 2% OWhole hearts were fixed with 10% formalin and sectioned at 1 mm of intervals. Each slide had 10 sections, which started at the apex and ended at the suture ligation site (approximately 6 slides). Every slide was stained with Masson’s trichrome to identify areas of fibrosis or stained with Hematoxylin and Eosin (H&E). To evaluate the muscle fiber membrane integrity, mice were received the intravenous (i.v.) injection of Evans blue dye  in PBS at the dose of 0.1 mg/g of body weight. Each experimental mouse underwent 20 min continuous swimming after dye injection. Muscle samples were collected 24 h after injection. EBD bind to albumin and was observed under the fluorescent microscope.2 shaved and positioned on an electrically warmed surface. Ventricular wall thicknesses and diameters were studied by M-mode echocardiography, and fractional shortening was calculated. Mice body temperature was monitored using a rectal probe and heart rate was used as a validation parameter, excluding from the study bradycardic  mice. Once the functional characterization was completed, the anesthetized mouse was euthanized by cervical dislocation and tissue were harvested for histological and biochemical analysis.A high-frequency, high-resolution digital imaging platform with linear array technology and color Doppler mode for in vivo high-resolution micro-imaging was used for echocardiography . To assess the cardiovascular function of mice, a high-frequency transducer probe  was used by a skilled cardiologist under the supervision of a veterinarian. 4–5 weeks after surgery mice were anesthetized using (IsoFlo®) 1.35% + 2% OStatistical analysis was performed with GraphPad Prism Software. All data are expressed as mean ± SEM According to the different data analysed, the following statistical analyses were performed: nonparametric tests (Mann Whitney Rank Sum test) and 1-way ANOVA test . The differences were considered significant for p-value ≤ 0.05 .Supplementary Figures.


The cement industry and steel mills that produce reinforcing bars are among the largest and most emissions-intensive sectors emitting CO2 into the atmosphere. This article analyzes the possibility of achieving significant reductions in CO2 emissions by using basalt bars (BFRP) and glass bars (GFRP) in concrete structures, and—in the case of concrete—by using cement with the addition of metakaolinite and zeolite. There is a lack of literature reports on whether modifying concrete with the additions of metakaolinite and zeolite as substitutes for part of the cement affects the adhesion of FRP bars to concrete. It can be assumed, however, that improving the microstructure of concrete also improves the contact zone between the bar and the concrete. The aim of this research is to fill the aforementioned gap in the literature data by determining how the presence of metakaolinite and zeolite affects the adhesion of reinforcing bars to concrete and testing selected properties of hardened concrete. The test samples were prepared following the appropriate beam test procedure. The obtained results made it possible to perform a comparative analysis of reference samples and those with metakaolinite and zeolite additions. The research showed that introducing active pozzolanic additives in the form of metakaolinite and zeolite into concrete improved adhesion stress values by approximately 20% for glass GFRP bars and 15% for basalt BFRP bars, especially in the destruction phase.Today’s sustainable development policy in Europe, which is driven by concerns about the greenhouse effect and environmental protection, mandates a reduction in CO The construction materials market is characterized by a growing interest in the production, distribution, and application of composite rods as reinforcement in concrete elements. Their popularity has been increasing due to their numerous advantages, i.e., corrosion resistance, electrical non-conductivity, insensitivity to electromagnetic fields, and the relatively low weight of the rods. However, it should be noted that the low longitudinal elasticity coefficient and the absence of plastic properties limit their structural applications . VariousA proper interaction between the concrete and the reinforcement enables the transfer of the tensile or compressive stresses that occur in concrete to the reinforcing rods, which form the basis of reinforced concrete. Hosseini  identifiThe first of these mechanisms is friction, which is particularly significant in the transfer of forces between the bar and the concrete and depends mainly on the ribbing of the bar as the concrete mix can penetrate between the ribs and become wedged. This is related to another mechanism, i.e., mechanical interlocking. The final mechanism is chemical adhesion, i.e., chemical bonding. It generates shear strength in the steel-concrete connection, which can be broken even with minimal relative slip of the bar against the concrete. In addition to slip, adhesion can be compromised due to the formation of small cracks on the surface of the surrounding concrete. The complexity of the issue makes it impossible to unequivocally determine adhesion stresses, which are the result of the various factors described above .Concrete class (higher concrete classes result in greater adhesion).Friction between materials due to their interlocking.The surface condition of the reinforcement .The type of reinforcing bars—ribbed bars exhibit 1.5 times greater adhesion than smooth bars.Concrete age .Material adhesion—intermolecular attraction.Shear of concrete between the concrete ribs.Diameter, shape, as well as the arrangement and termination of the reinforcement.Placement and consolidation of the concrete mix (vibrating the concrete ensures thorough encasement of the reinforcing bars in the concrete mix).Proper curing of fresh concrete.Cooperation is primarily possible due to the phenomenon of adhesion that occurs between the materials in question, influenced by factors such as:At each segment of the reinforcing bar, longitudinal forces are balanced by tangential forces due to interactions with the concrete. These are adhesion forces or stresses, which are the greatest at the ends of the bars and occur as a result of the rapid changes in normal stresses along the length of the bar. In the case of long bars arranged in an uncracked tensile section, constant adhesion stress forces equal zero. On the other hand, significantly higher stresses occur in the reinforcement in cracks, compared to uncracked sections. Consequently, every crack induces adhesion forces.In the case of smooth steel, adhesion is provided by micron-sized irregularities present on the surface of the reinforcement, as well as by the cohesion and shrinkage of concrete. Unlike ribbed steel, smooth steel has a significantly lower yield strength. In ribbed steel, both adhesion and the resistance associated with the phenomenon of reinforcement slippage are provided by the bars’ ribs. Moreover, the adhesion of ribbed reinforcing steel also depends on the size and shape of the ribs present on the surface of the bars . Many studies have explored the way in which adhesive stress is transferred between FRP reinforcement and concrete ,14,15,16Numerous studies have been conducted to examine the interaction between FRP reinforcement and concrete, primarily using the direct pull-out test, the beam test, the splice test, or the ring pullout test. However, it is worth noting that the setups for the direct and ring pullout tests do not accurately replicate the actual bonding conditions in a reinforced concrete structure. Therefore, the most accurate assessment of the bonding behavior of reinforcement can be achieved through either the beam test or the splice test.Moreover, all previous studies demonstrate lower values of bond stresses in the case of FRP bars. For this reason, there is an urgent need to explore ways in which it would be possible to increase their bond strength.A relatively simple method for improving the adhesion of composite bars to concrete is the modification of the reinforcement-matrix cement contact zone by using active additives in the concrete. This enhances compressive and tensile strength of the concrete, friction between the materials, and material adhesion. What is more, the active additive can also partially replace cement.One of the valued additives with pozzolanic properties is metakaolin. It is produced from natural kaolin clay in the calcination process at temperatures ranging from 700 to 900 °C . The pro2 and Al2O3. Preliminary studies have confirmed the beneficial effect of zeolite used as a modifier on the increase in compressive and flexural strength, but only when the zeolite content is below 15% of the cement mass. Furthermore, the addition of zeolite enhances the durability of conventional concrete, not only by reducing concrete permeability but primarily by improving its resistance to the alkali-aggregate reaction.Zeolites are porous silicate minerals containing significant amounts of reactive SiOThere is a lack of literature reports on whether modifying concrete with additions of metakaolin and zeolite as substitutes for part of the cement affects the adhesion of FRP bars to concrete. It can be assumed that by improving the microstructure of concrete, the properties of the contact zone between the bar and the concrete will also improve. This study contributes to filling this gap in knowledge.The aim of this research was to determine how the presence of active pozzolanic additives affects the adhesion of steel, basalt, and glass bars to concrete, as well as its influence on selected properties of the tested concrete.In the study, both cement concrete and concrete in which 10% of the cement mass was replaced with either metakaolin or clinoptilolite zeolite were used. As reported by de Silva and Glosser, concrete strength is closely related to the reduction in microporosity caused by the presence of metakaolin and zeolite . EssentiThe influence of the additive on the adhesion of steel, basalt, and glass bars to the surrounding concrete was analyzed. The study was conducted in nine series, as presented in The composition of the concrete mixture is presented in 2). As a result, products in the form of hydrated calcium silicates are formed, which are compositionally and structurally similar to the products of Portland cement hydration. The material has a density of 2.61 g/cm3, and its specific surface area, determined using the BET method, is 14.6 m2/g. The pozzolanic activity index, tested after 28 days pursuant to EN 450-1:2012 ; The testing apparatus for the pullout test included two rotating roller bearings positioned 650 mm apart from each other. The sample was subjected to two-point loads applied symmetrically in relation to the center of span of the beam with a total length of 810 mm. During the test, the role of the steel joint was to transfer stresses from the compression zone, enabling deflection without causing stresses that would crush the concrete. During the test, the bar in the tensile zone was pulled out of the concrete. Measurements of the force–slip relationship of the bar were taken using sensors with a reading accuracy of 0.001 mm placed at the ends of the bar. To increase stresses σ in the bar, each sample was loaded in the following manner: 0-80-160-240 MPa. In accordance with , the totWhile it is possible to adopt smaller values of stress σ in the bar, it must be ensured that they increase stepwise by a constant amount.The force increment had to be achieved within 30 s and the load had to be maintained for long enough for the slip to stabilize, but for no longer than 2 min. The force increment, as per the recommendations, was gradual and continuous. The test was conducted until the adhesion of the bar was lost in both beams.σ is the stress of the tested bar [MPa]; Bond stress The bond stress was calculated for the following four measured slip values:ckf, tensile strength in bending ctmf, and longitudinal modulus of elasticity of concrete cmE. For the R series, reference concrete was used; for the M series, concrete with a 10% metakaolin addition was used; for the Z series, concrete with a 10% zeolite addition was used. The average strength was calculated based on the arithmetic mean of individual sample series.The mixture without the metakaolin additive showed a slightly lower slump height, although the difference was not significant, confirming the same construction class for both samples. The results of the consistency tests of the mixture of concrete with the zeolite additive showed that the mixture with zeolite reduced the flowability of the S4 class mixture (160–210 mm) down to the S1 class values (10–40 mm). This should be taken into account in future studies, i.e., a larger amount of superplasticizer would make it possible to achieve the desired consistency.Based on the results of the density tests of the concrete mixture, it was found that the density of the concrete with the zeolite additive was 8.22% lower than that of the reference concrete. The lack of a significant change in the composite density was due to the similar densities of metakaolin and sand, with the amount of sand adjusted according to the impermeability condition.Based on the obtained test results, it was found that concrete with 10% additions of metakaolin and zeolite was characterized by compressive strength values that were comparable to and 20% higher, respectively, than those of the reference concrete. A positive effect of metakaolin and zeolite additives on tensile strength in bending (a 10% increase) and longitudinal modulus of elasticity (a 5% increase) were also observed.For each series, measurements of diameters were taken at three different locations to compare the obtained results with the nominal representative diameter. The results are presented in Compared to steel bars, basalt BFRP bars exhibit 72% greater tensile strength, while glass bars exhibit 45% greater tensile strength. Unlike steel, composite materials do not have a plastic range, i.e., their state preceding failure is not signaled and failure occurs suddenly after the strain limits are exceeded.FRP bars are manufactured with various surface deformations to enhance their adhesion to concrete. The FRP bars used in this study were basalt BFRP and glass BFRP, along with reference steel bars. As they all differ in rib geometry, to help interpret the results, the surface characteristics of the bars are analyzed in the subsequent part of the study.s and Rr [2]; rR—relative rib area [–]; To account for the influence of bar geometry on bond behavior, two geometric ratios have been proposed in literature, i.e., as and Rr ,38,39..For basalt BFRP reinforcement, three phases can be distinguished in the force–displacement graphs. The first phase, up to half of the destructive force, is linear, followed by a phase of nonlinear growth, and the third phase of failure. The maximum adhesive force values obtained in the study were 21.9 kN for the reference concrete and 25.4 kN for the concrete with a metakaolin addition. This means that the addition of metakaolin to concrete increases the adhesive force by approx. 6%; the addition of zeolite increases this force by 11%.The graphs obtained for beams reinforced with GFRP bars show force–displacement characteristics that are similar to the series with BFRP reinforcement. This is due to the material characteristics of these bars, which are similar to basalt BFRP bars. In this case, however, the maximum values of force proved to be the highest for concrete with a metakaolin addition at 25.7 kN and for concrete with a zeolite addition at 26.7 kN.In the series with composite bars, an increasing incline of the curve can be observed from the second phase onwards, until failure occurs. A plastic plateau and plastic material strengthening were formed, as indicated by the nonlinear character of the graph, which also points to gradual damage at the interface between the concrete and the bar due to mechanical locking and wedging effects.However, the different behavior of steel bars under load, compared to basalt BFRP and glass GFRP bars, must be confirmed unequivocally. In the conducted tests, it could be observed that composite bars, due to their different types of wrap and their composition consisting of resin and glass, as well as basalt roving, exhibit different mechanical and chemical properties of adhesion between the concrete and the bar. The use of metakaolin resulted in an increase in the compressive and tensile strength of concrete, which is also one of the components responsible for adhesion.2].The results for the tensile stresses in the bar measured at slip show that stresses increase with increasing slippage. In the R-GFRP and M-GFRP series, in which metakaolin was added to the concrete along with glass bars, the highest increase in the values of tensile stresses was observed at all stages, i.e., from 0.01 to failure. In the R-BFRP and M-BFRP series, in which metakaolin was added to the concrete along with basalt bars, a 25% increase in tensile stress was noticed at failure. In the Z series, a remarkable increase in the applied forces at slip, i.e., up to 64%, was observed for glass bars (GFRP). On average, compared to the reference concrete, the increase was approx. 30%.Basalt bars (BFRP) and glass bars (GFRP) exhibited lower tensile stress values than steel bars. Comparing both types of composite reinforcement used, it can be concluded that glass bars were characterized by higher tensile stress values in the bar.In the case of BFRP bars, despite the greater spacing in the wrap compared to the ribs in steel bars, the adhesion values were higher than those obtained for steel bars. The loss of adhesion in both GFRP and BFRP bars was caused by complete deformation of the wrap’s ribs. Damage was initiated at the base of the wrap and then propagated outward. Ultimately, in both GFRP and BFRP bars, the shearing surface appeared at the interface between the concrete and the wrap. The adhesion mechanism was primarily enacted by the mechanical interlocking caused by the wrap of the composite bar. Hence, it can be stated that in the case of the adhesion mechanism, in addition to the aforementioned influence of the concrete composition, the type of wrap deformation also plays an equally important role.According to EN2 annex C  and RILECompared to steel bars, the average adhesion stresses in all the series with composite reinforcement were characterized by lower adhesion. However, the introduction of an active pozzolanic additive into the concrete, in the form of metakaolin and zeolite, resulted in adhesion stress values improved by approximately 20% for glass bars (GFRP) and approximately 15% for basalt bars (BFRP), especially in the failure phase . In the In all the tested series, the loss of adhesion in both steel and composite bars was observed in the final stage of loading. According to the EN2 standard  and RILECompared to the flexural tensile strength of the reference concrete or concrete with metakaolin The diverse adhesion force values inspired the authors to conduct detailed studies of the contact zone between the bars and the concrete with the use of microscopic analysis, as presented in The microscopic image of the concrete structure presented in In the case of concrete with a metakaolin or zeolite addition, the sealing of the concrete structure can be observed (as confirmed by the higher results of the strength test of the concrete). It can be assumed that metakaolin and zeolite act as sealing fillers, reducing the total porosity of the surface, as confirmed in . Frias rThe addition of metakaolin and zeolite to concrete had a positive effect on the adhesion of reinforcing bars to concrete;The force–displacement relationship showed a consistent pattern in all the tested series;The maximum force values achieved during the tests of the adhesion of basalt bars to concrete were 21.9 kN for reference concrete, 25.4 kN for concrete with metakaolin, and 26.7 kN for concrete with zeolite;The GFRP bars in beams exhibited similar force–displacement curves to the basalt reinforcement series, but the maximum force value was the highest, reaching 26.7 kN;The use of metakaolin improved adhesion by 8% for basalt BFRP bars and 28% for glass GFRP bars;The use of zeolite improved adhesion by 11% for basalt BFRP bars and 33% for glass GFRP bars;The addition of active pozzolanic additives in the form of metakaolin to concrete improved the adhesion stress values (Adhesion stresses at slip met the normative conditions in all the series.The aim of this study was to perform comparative research on the adhesion of steel, basalt, and glass bars to reference concrete and to concrete with active pozzolanic additives in the form of metakaolin or zeolite. The strength of the concrete was tested and the mechanical properties of steel and of the composite bars were analyzed. Based on the obtained results, it can be concluded that:The limitations of this research and the methodologies apply only to the two specific types of composite bars.In future research, the authors plan to conduct detailed SEM analyses of the contact zone between the FRP bar and concrete. This will help explain the beneficial influence of active pozzolanic additives. Furthermore, crucial considerations will also focus on a numerical analyses of bond zones, which will make it possible to design FRP structures with metakaolin and zeolite without the need for additional scientific research.


Introduction: Visualizing small individual biomolecules at subcellular resolution in live cells and tissues can provide valuable insights into metabolic activity in heterogeneous cells, but is challenging.Methods: Here, we used stimulated Raman scattering (SRS) microscopy to image deuterated methionine (d-Met) incorporated into Drosophila tissues in vivo.Results: Our results demonstrate that SRS can detect a range of previously uncharacterized cell-to-cell differences in d-Met distribution within a tissue at the subcellular level.Discussion: These results demonstrate the potential of SRS microscopy for metabolic imaging of less abundant but important amino acids such as methionine in tissue. Small biomolecules, such as amino acids, are essential for life and play a crucial role in controlling cell fate through their participation in metabolic reactions. The metabolic profile of an individual can provide valuable information about their physiological status. However, while our understanding of the molecular details of these processes has improved, we still lack knowledge of their finer spatial details, which are important for understanding cell-to-cell differences. In cancer, such differences can lead to differential sensitivity to treatment and the emergence of resistant cells . To bettin vivo imaging, but does not have the ability to resolve subcellular details. Mass spectrometry techniques   is applichniques  provide chniques , but is hemistry  has morehemistry  and may Drosophila have further demonstrated the non-invasive nature of deuterium for systemic labelling in vivo and long-term imaging, highlighting its potential as a universal metabolic probe (8-Met) and demonstrated the ability to detect the CD stretching peak of d8-Met by careful subtraction of background signal (Stimulated Raman scattering (SRS) microscopy is an imaging technique that can visualize the vibrational resonances of small biomolecules at subcellular resolution . Its lowic probe . Howeveric probe , and Metic probe . In our d signal .8-Met, in multiple dissected Drosophila tissues, incorporated systemically in vivo. By using our integrated SRS and fluorescence microscope  instead of Met, for up to 48 h prior to dissection unless otherwise stated. Third instar larvae or one-week-old adult Drosophila were placed in phosphate buffered saline (PBS) (4°C) and dissected using forceps  under a dissecting microscope. n = 3 or greater and carried out on different days using at least two different populations. Tissues were mounted on glass coverslips ; ) with SecureSeal imaging spacers , in PBS, PBS with 4% paraformaldehyde (adult gut only), or PBS with LysoTracker Deep Red , 50 μM.Flies were transferred to a modified version of holidic medium  suppleme−1) or CH stretching  regions, respectively. The light is focused on the sample by an objective lens  and the transmitted light is collected by another objective of the same type. After confirming the position of the spectral peak for d8-Met, measurements were taken for four spectral points each with 1,500 frames: 2,107, 2,127, 2,130, and 2,150 cm−1. SRS signal of d8-Met was obtained as th spectral image  gene, which is known to be expressed in cells including ISCs and EBs. In the case of the adult gut, we fixed the dissected tissue to avoid any movement during the time required for imaging. d8-Met uptake in Drosophila adult gut shows a heterogeneous distribution , a kinase that is a central regulator of cell metabolism , integraDrosophila  may be affected by the additional five deuterium atoms present in other parts of d8-Met. Additionally, while the CD3 group of deuterated methionine provides a strong signal (3 may have alternative fates aside from methylation. On the other hand, deuterated Met may also be useful for studying other aspects of Met metabolism. A recent study using SRS imaging and deuterated water as a probe found that manipulating the level of L-Met together with insulin can alter de novo lipogenesis, the process of synthesizing new lipids, in cancer cells (It is known that SAM is a key regulator of epigenetic control during normal development and disease . It has g signal , it is per cells . This rain vivo has been carried out by other groups but has been limited to accessible tissues or embryos (in vivo, should their size permit. Still, the limitation of tissue accessibility for SRS imaging in vivo may require miniaturization and an introduction of flexible components or remote technologies. Alternatively, from a systems perspective SRS imaging’s capability to track molecular fate appears to offer high potential for diagnostics using more accessible tissues.Also, while all our experiments thus far have been carried out on dissected tissue, SRS imaging on tissue  embryos . SRS ima8-Met in several dissected Drosophila tissues and demonstrated the ability to overcome the challenges that have previously limited long-term uptake of small biomolecules in live cells and tissue at subcellular resolution. In agreement with the known role of mTOR signaling in regulating Met uptake, we provided direct, real-time visualization of mTOR function controlling d8-Met uptake and distribution in the fat body. The ability to visualize the range of cell-to-cell differences of d8-Met distribution within tissue using SRS has significant potential for basic research on metabolic imaging of less abundant but important amino acids such as methionine and may also have potential for medical applications.In this study, we have generated SRS images of d


Colostrum and milk samples from Month 0 (M0), 2 (M2), 4 (M4) and 6 (M6) were analysed using whole-genome shotgun-sequencing. The microbial diversity from antibiotic-treated groups was different and higher than that of the non-antibiotic group. This difference was more evident in milk compared to colostrum, with increasing diversity seen only in antibiotic-treated groups. The microbiome of antibiotic-treated groups clustered separately from the non-antibiotic group at M2-, M4- and M6 milk samples, showing the effect of antibiotic treatment on between-group (beta) diversity. The non-antibiotic group did not show a high relative abundance of mastitis-causing pathogens during early lactation and was more associated with genera such as Psychrobacter, Serratia, Gordonibacter and Brevibacterium. A high relative abundance of antibiotic resistance genes (ARGs) was observed in the milk of antibiotic-treated groups with the Cephaguard group showing a significantly high abundance of genes conferring resistance to cephalosporin, aminoglycoside and penam classes. The data support the use of non-antibiotic alternatives for drying off in cows.This study investigated the longitudinal impact of methods for the drying off of cows with and without dry cow therapy (DCT) on the microbiota and resistome profile in colostrum and milk samples from cows. Three groups of healthy dairy cows ( The first milk produced after the non-lactating period, colostrum, is highly nutrient-rich, provides the calf with both nutritional and immune benefits, and shapes the calf gut microbiota . It helpIn cows, before the milking begins, there is a period called the dry period (six to eight weeks long), which allows the teats to dry up. This can be performed by allowing the drying to occur naturally, using teat sealants. Alternatively, drying of the teats can be performed by using medications and antibiotics to ensure the teats are free of any infection, called dry cow therapy (DCT). The objectives of DCT are to eliminate infections presenting from previous lactation and to avoid new udder infections during the dry period—when mammary glands are highly susceptible to new infections . The useThe use of antimicrobials for DCT is common globally ,12,13,14Milk from cows treated with prophylactic antibiotics usually contains significant antibiotic residues ,18,19. STM (CEF)  and Ubro red (UBRO)  group during DCT will result in varying abundance and composition of microbiota and resistance genes as compared to the non-antibiotic group (NOAB). Thus, in this study, shotgun metagenomics sequencing was used to investigate the longitudinal microbial composition and resistome profiles of colostrum and milk samples obtained from healthy cows throughout lactation, which were previously treated with and without DCT.Moreover, the presence of ARGs is not limited to pathogenic bacteria but may also occur in commensals. Mostly, the antibiotic resistance of pathogens in bovine milk has been studied in cases of mastitis ,34,35, bp-value = 0.026, Chao1 p-value = 0.011; for CEF: Shannon p-value = 0.00016, Chao1 p-value = 0.003; Wilcoxon test), M0 and M4 , and M0 and M6  in both Cephaguard (CEF)- and UBRO red (UBRO)-treated groups (p = 0.0030) and NOAB vs. UBRO (p = 0.0045) at M2, NOAB vs. CEF (p = 0.0015) and NOAB vs. UBRO (p = 0.0015) at M4, and NOAB vs. CEF (p = 0.0015) and NOAB vs. UBRO at M6 (p = 0.0015). Furthermore, discrete clustering of samples was observed based on time points, with samples at M0 clustering separately from all later time points . Both Shannon and Chao1 diversity indices did not show a variation between time points within the no-antibiotic group . Howeverd groups . Additiod groups . Concernd groups . The betd groups C,D, incle points D.2 = 0.15 and R2 = 0.11, reciprocally). To test the interactions between antibiotics and farms, we performed a PERMANOVA on the two most explanatory grouping variables. The results showed that grouping based on antibiotic exposure explains a higher variance compared to farms . These results considering the effect of farm variables must be interpreted cautiously due to the low subject size for each farm.Similar to distinct clustering of the NOAB group from antibiotic-treated groups overall, we also observed clustering based on farms, where farm “C”  clusters separately from the other antibiotic-treated farms . Furthermore, samples from nulliparous cows, which corresponded to the NOAB group, showed a similar clustering pattern because of an overlapping between the antibiotics groups and parity . Based oDifferences in the relative abundance of the microbial composition of milk between the three groups at all time points were observed using plots at phyla and genera levels . OverallAcinetobacter, Brevibacterium, Corynebacterium, Lactobacillus, Lactococcus, Microbacterium, Pseudomonas, Propionibacterium, Kocuria and Staphylococcus . The relative abundance of Corynebacterium was higher in the NOAB group than the CEF group at M0, and higher than both antibiotic-treated groups at M2  did not show any pattern, highlighting a stronger association of any genera to a particular antibiotic group.Relative associations of genera to each group were estimated with Songbird  using thRGI-bwt was used to predict antibiotic resistance genes in filtered and quality-controlled shotgun sequencing reads. ARGs belonging to 189 AMR gene families conferring resistance to 43 different classes of antibiotics were found. Of these, several genes conferred resistance to more than one class and genes conferring resistance to three or more classes were termed multi-drug-resistance (MDR) genes A. FurtheThe alpha diversity of ARGs over time was not different in milk samples from all groups (Shannon index), while the CEF and NOAB groups showed significant differences (Chao1 index), pointing towards higher richness in the CEF group. No significant differences were observed between groups or between time points in individual groups A. Beta dThe CEF group showed the highest ARG abundance, with both antibiotic-treated groups showing a higher ARG abundance compared to the NOAB group . The abuStreptococcus spp.  and Staphylococcus aureus, which are coagulase-negative staphylococci for Cephaguard, while UBRO red targets Staphylococcus spp., Streptococcus spp., Corynebacterium spp., Escherichia spp., Klebsiella and Pseudomonas spp. The European Medicine Agency (EMA) classifies cefquinome as a category B (restricted use) antibiotic, while Framycetin is to be used with caution [In this study, we compared the microbial and resistome profiles of colostrum and milk from healthy cows throughout lactation, separated into three groups treated with differing dry cow therapies—two antibiotic-treated groups and one non-antibiotic-treated group using shotgun metagenomics. Studying the bovine milk microbiome is considered challenging as it is a low-biomass sample, difficult to collect without contamination, along with the possibility of kit contaminants, with an inter-individual variation in microbiota , differe caution .Escherichia spp., Streptococcus spp. and Staphylococcus spp. in the groups, especially in the NOAB group. Similar to other studies [This study demonstrated significant differences in the diversity of microbial genera within the antibiotic-treated groups over the time points (Shannon diversity) similar to . Similar studies ,42,43,44Acinetobacter, Brevibacterium, Corynebacterium, Lactobacillus, Lactococcus, Microbacterium, Pseudomonas, Propionibacterium, Kocuria and Staphylococcus . However, Acinetobacter and Pseudomonas are also psychrotrophic and observed in stored bulk milk samples [The microbial diversity in milk from cows is affected by several factors. For instance,  demonstr the cow  and seas the cow  can impa the cow ,48,53,63 the cow  observed samples  and are As reported earlier, we observed the presence of ARGs in cow colostrum; however, changes in ARG diversity over time were not observed in any group in this study. There are certain antibiotic resistance genes (ARGs) found in the NOAB group, while other ARGs that do not belong to commonly used antibiotic classes might be attributed to past antibiotic exposure or environmental factors such as contaminated water or manure, as well as horizontal gene transfer within the herd. Further, a high abundance of ARGs in the antibiotic-treated groups was observed. Certain genes conferring resistance to classes like beta-lactams, penams and cephalosporin and MDR genes were observed. The presence of ARGs (those that confer resistance to the antibiotic in question) in the antibiotic-treated groups can be justified due to the use of antibiotics, while many other resistance genes conferring resistance to drug classes like tetracycline, intercalating dye, disinfecting agents, triclosan and glycopeptide antibiotics could be due to contaminating bacteria from environmental sources such as irrigation , groundwThe limitations of this study include the relatively small number of animals included per group, although we would emphasise that these animals were all followed longitudinally for six months of lactation. Another limitation of the study was the long sample intervals, which spans various seasonal and feeding variations; however, all cows belonged to the same geographic location; thus, the effect of these variables, if any, was similar throughout the dataset. We suggest that this study demonstrates the value of conducting temporal microbial and resistome studies on bovine colostrum and milk microbiota to examine the effect of DCT and antibiotic use. More studies with larger cohort sizes and shorter between-sample intervals are needed to understand the longitudinal effect of antibiotics on bovine colostrum and milk samples. This will help reduce antibiotic use in livestock and decrease the generation and transfer of ARGs.n = 9, CEF: n = 8, UBRO: n = 7), with four samples collected longitudinally from each cow throughout the first six months of lactation. In terms of the farm-wise distribution of the cows included in our study, all NOAB cows were from a single farm, Farm C (n = 9), while farm D and P constituted the CEF group and had n = 4 and n = 4, respectively. Farm L and T formed the UBRO group and had n = 5 and n = 2, respectively. Farms D and P reported routinely using Cephaguard while farms L and T routinely used Ubro red therapy. Cows showing signs of mastitis or any other infections were excluded from the study and milk samples were collected only from healthy cows. Cows were not subjected to antibiotic treatment during the time interval of sample collection.Colostrum and subsequent milk samples at month 2 (M2), month 4 (M4) and month 6 (M6) were collected from healthy cows from five different Irish farms between February and September 2020 as detailed below. All cows were housed in different herds in farms located in County Cork, Ireland. The farms included in our study were labelled farm D, farm P, farm L, farm T and farm C. Cows from each farm were selected randomly and divided into three groups based on the DCT treatment. Cows on all farms were treated with a teat sealant—Boviseal—in addition to antibiotics as described during the drying-off period. NOAB did not use any antibiotics during the drying-off period  and was assigned as the control group. The UBRO group was treated with blanket DCT using Ubro Red Dry Cow Intramammary Suspension at drying off. The CEF group was treated with blanket DCT using Cephaguard DC 150 mg intramammary ointment at drying off. Twenty-four cows divided into three groups were included in the study  with modifications as follows: Briefly, the cell pellets were washed with PBS   and centrifuged at 13,000× g for one minute at 20 °C to discard the supernatants. This process was repeated until the supernatant was no longer cloudy. The pellet was then re-suspended in PBS and treated with 90 µL of 50 mg/mL lysozyme  and 50 µL of 5 KU/mL mutanolysin (Sigma Aldrich) followed by 15 min incubation at 55 °C with vortexing at intervals of 5 min. Further, samples were treated with Proteinase k , incubated at 55 °C for 15 min and then treated according to the manufacturer’s instructions using the PowerFood microbial DNA Isolation Kit protocol. The extracted DNA was stored at −30 °C until further use.Milk samples were thawed by placing them at 4 °C the night prior to DNA extractions. The samples were homogenised by inverting the tubes and centrifuged at 4000× The quantification of DNA was performed using the Qubit double-stranded DNA (dsDNA) high-sensitivity assay kit (Invitrogen). Some samples had less than 0.2 ng/µL of DNA; thus, a modified low-input Nextera XP protocol was used for shotgun sequencing library preparation as described previously . BrieflyTrim galore (v0.6.1) and Bowtie2 (v 2.4.4) using the Bos taurus reference genome database from NCBI (Genome assembly ARS-UCD1.3) to remove any host contaminant reads. Reads obtained post-host removal step were considered microbial and were used for further analysis. The filtered and trimmed reads were then used for taxonomic assignment with Kraken2 (v2.1.1) and kraken-biom (https://github.com/smdabdoub/kraken-biom). Further, antibiotic resistance analysis was performed using RGI (Resistance Gene Identifier) (RGI-bwt) (v3.1.4) and CARD (Comprehensive Antibiotic Resistance Database) [−4 reads per taxa, and those belonging to viruses, Euryarchaeota and Archaea, and not classified at phylum level were removed. Initially, 5194 taxa were obtained, which were filtered down to 235 taxa. Stringent filtering parameters were applied to make sure that no taxa arising as contaminants/singletons remain in downstream analysis. Further, decontam package (v1.14.0) in R was used to look for contaminants in samples and remove them. Using the default parameters, no PCR blank reads were observed in any of the study samples. The negative control sample (with less than 500 reads) was thus excluded from further analysis and the phyloseq object with all study samples was used for further analysis. Downstream analysis was performed in R (v4.0.2) using packages phyloseq (v1.38.0), microbiome (v1.16.0) and ggplot2 (v3.3.2). Shannon and Chao1 diversity indices were used to calculate the alpha diversity of the microbiota within the groups. The Shannon diversity index provides details about richness and evenness while the Chao1 index estimates total richness only. The Wilcoxon test was performed to test if the difference between any two groups or time points was significant. Beta diversity analysis was performed using the Bray–Curtis dissimilarity matrix to determine compositional dissimilarities between the samples based on group, time, farm and parity variables in the study. The Bray–Curtis distance matrix takes into account abundance/occurrence data and does not rely solely on the presence or absence of data. The impact of various study variables on the microbiota diversity was examined using permutational multivariate analysis of variance (PERMANOVA). Statistical analysis was also performed in R using the Wilcoxon test and Dunn test with the function stat_compare_means and stat_pwc from package ggpubr (v0.4.0) and Permanova using the pairwiseAdonis (v0.4) package. p-values were adjusted using the false discovery rate (fdr) method in R unless specified otherwise.Raw metagenomics shotgun reads received post sequencing were quality-checked using FastQC (0.11.8) and MultiQC (v1.9). The reads were then filtered and trimmed using atabase) . The RGIatabase) , using tatabase) . The difatabase)  formed fAntibiotic treatment during DCT was associated with high abundances of ARGs in milk compared to the non-antibiotic control group, and altered microbial composition throughout lactation. Using teat sealant without antibiotics did not lead to the presence of a high abundance of mastitis-causing pathogens. Furthermore, the microbial diversity  in antibiotic-treated groups increased over time, which was not observed in the non-antibiotic group. The reason for this is unknown; however, it could be due to effect of antibiotics on highly abundant bacteria, leaving a higher diversity of unaffected species. The presence of ARGs at high abundance in cow colostrum and milk is alarming because of the threat of passage of ARGs to the calf and to humans through the food chain. Both previous and current antibiotic use may result in the selection of bacteria that carry resistance genes, thus increasing the overall resistome profile diversity of the microbiota. It is challenging to trace the source of bacteria and thus the resistance genes in question; thus, preventive and protective approaches to reduce the use of antibiotics on farms and associated resources is crucial.