Background and Objectives: Metformin is currently the leading drug of choice for treating type 2 diabetes mellitus, being one of the most widely used drugs worldwide. The beneficial effects of Metformin, however, extend far beyond the reduction of blood glucose. Therefore, this study aimed to evaluate Metformin’s effects both in vitro and in ovo. Materials and Methods: Metformin has been tested in five different concentrations in human hepatocytes —HepaRG, in terms of cell viability, morphology, structure and number of nuclei and mitochondria, as well as the effect on cell migration. Through the application of HET-CAM, the biocompatibility and potential anti-irritant, as well as protective effects on the vascular plexus were also assessed. Results: According to the results obtained, Metformin increases cell viability without causing morphological changes to cells, mitochondria, or nuclei. Metformin displayed an anti-irritant activity rather than causing irritation at the level of the vascular plexus. Conclusions: In conclusion, Metformin enhances cell viability and proliferation and, has a protective effect on the vascular plexus. Nonetheless, more studies are required to clarify the mechanism of hepatoprotective effect of metformin. Type 2 diabetes mellitus is characterized by hyperglycemia and dyslipidemia as a result of the inability of islet β-cells to secrete insulin. This pathology has become a burden for modern society due to the explosion of the number of cases worldwide and multi-organ damage . ClinicaMetformin (Met) was introduced as a therapy in 1958 in the United Kingdom and has since become the most widely used drug for the treatment of type 2 diabetes. Met lowers blood glucose levels by decreasing plasma insulin levels and increasing insulin sensitivity and peripheral glucose uptake. In addition to these mechanisms of action, Met is involved in the process of gluconeogenesis by activating AMP protein kinase (AMPK) .The use of Met goes beyond its primary indication, as it is also used for other pathologies, such as obesity, metabolic syndrome, or polycystic ovary syndrome . Since MMet is attracting researchers’ attention because of the possibility that it may have antitumor activity as demonstrated in a retrospective report, indicating enhanced survival rates in cancer patients treated with Met . Met treMet reduces serum glucose levels by inhibiting hepatic gluconeogenesis and glycogenolysis in addition to enhancing striated muscle glucose uptake, inhibiting fatty acid oxidation, and reducing triglyceride levels. All these actions of Met cause a beneficial effect on the liver by reducing steatosis and improving liver enzymes ,11. The Moreover, drug-induced liver toxicity is also a major health concern. In many cases, the hepatotoxic potential is not detected during preclinical studies, due to the fact that hepatotoxicity may be idiosyncratic . Among tThe aim of the present study was to evaluate the in vitro and in ovo effect of Met using five concentrations . In in vitro studies, the effect exerted by Met on the hepatocyte cell line—HepaRG, was evaluated in terms of viability, cell morphology, influence on structures and number of nuclei and mitochondria and cell migration capacity. In ovo, the biocompatibility and irritant and anti-irritant potential of Met were evaluated by employing the chick chorioallantoic membrane (HET-CAM) assay.Metformin hydrochloride, trypsin-EDTA solution, phosphate saline buffer (PBS), dimethyl sulfoxide (DMSO), fetal calf serum (FCS), penicillin/streptomycin, insulin from bovine pancreas, Hydrocortisone 21-hemisuccinate sodium and MTT  reagents were purchased from Sigma Aldrich, Merck KgaA . The cell culture media, William’s E Medium  were purchased from Gibco, Waltham, MA, USA). All reagents were analytically pure and proper for cell culture use.2.The effect of Met on hepatocytes was studied in the HepaRG cell line provided by Gibco as frozen vials—HepaRG ™ Cells . The cell line was cultured in a culture-specific medium, represented by William’s E Medium containing 4 μg/mL Insulin from the bovine pancreas, 5 μM Hydrocortisone 21-hemisuccinate sodium, 10% FCS and 1% penicillin (100 U/mL)-streptomycin (100 µg/mL) mixture. During the experiment, the cells were maintained at a standard temperature (37 °C) and 5% CO4 cells / well. After reaching a confluence of approximately 90%, the cells were stimulated with five different concentrations of Met  for two time intervals of 24 h and 72 h. Test concentrations of Met hydrochloride were prepared in the culture medium. After these time intervals, the stimulation medium was removed and replaced with fresh medium (100 μL/well); 10 μL of reagent MTT was added to each well, and the plates were placed in an incubator at 37 °C for 3 h. After this time, the solubilization solution was added to a volume of 100 μL/well, and the plates were incubated for 30 min at room temperature, away from light. Subsequently, the measurement of absorbents at two wavelengths of 570 and 630 nM was performed using Cytation 5 .The MTT method was used to determine cell viability. For this purpose, the cells were cultured in 96-well plates in a number of 1 × 10In order to determine the possible effects induced by Met regarding the morphology and confluence of HepaRG cells, a microscopic evaluation was performed by photographing the cells under bright field illumination after the time intervals of 24 h and 72 h. The pictures were analyzed using the Gen5 ™ Microplate Data Collection and Analysis Software .5 cells / well, and after reaching a corresponding confluence of approximately 90%, they were stimulated for 72 h with Met . After 24 h, the cells were washed with ice-cold PBS and then fixed with paraformaldehyde 4% by maintaining room temperature for one hour. Cell permeabilization was performed with Triton X 2% in PBS. Then, 30% FBS solution in 0.01% Triton X was used for one hour to block Triton X 2% action. The cells were incubated at 4 °C overnight with Anti-COX IV antibody Mitochondrial marker (ab33985) at a dilution. of 1:500 for mitochondrial visualization. The next day, the primary antibody was washed with 0.01% Triton X solution in PBS and the secondary antibody specific for COX IV mitochondrial marker—Donkey Anti-Goat IgG H&L (Alexa Fluor® 488—ab150129) was added, and the plate was kept for 2 h at room temperature and protected from light. After this time, 4′, 6-diamidino-2-phenylindole (DAPI) staining was added to visualize the nuclei. The pictures were then taken with Cytation 1 and processed using Gen5 ™ Micro-plate Data Collection and Analysis Software .For the immunofluorescence visualization of the cellular components, HepaRG was cultured in plates of 12 wells, with 1 × 105 cells/ well) and an automatic scratch was made in the middle of each well using the AutoScratch ™ Wound Making Tool provided by BioTek® Instruments Inc., Winooski, VT, USA as recommended by the manufacturer. Cells were then stimulated with the five Met concentrations  for a 24-hour interval. Photographs (4 × magnification) were taken both at the beginning of the experiment (0 h) and after the stimulation was completed (24 h) using Cytation 1 and were processed using Gen5 ™ Microplate Data Collection and Analysis Software . The effect of Met on cell migration was quantified by calculating the migration rate, expressed as a percentage, using the formula described above [The wound healing (scratch) test was used to assess the effect of Met on cell migration. For this purpose, the cells were cultured in 24-well Corning plates  obtained from a local farmer were used to evaluate the effect of Met on the vascular plexus. They were disinfected with 70% alcohol (v/v), and after noting the date on them, they were placed in the incubator, under standard conditions of temperature (37 °C) and humidity in a horizontal position. The eggs were then prepared for the experiment as follows: (i) on the fourth day of incubation, a perforation was made at the level of the eggshell through which a volume of 7 mL of albumen was extracted in order to favor the detachment of the membrane from the inner shell of the egg; (ii) on the fifth day, a window was cut at the upper level of the egg, in order to allow the visualization of the chorioallantoic membrane. The cut-out window was then covered with duct tape, and eggs were placed in the incubator until the day the experiment began.Chicken eggs (2O and a positive control—1% sodium dodecylsulfate (SDS) were used. Both the test sample and the two controls were applied to the chorioallantoic membrane at a volume of 600 μL so that the entire surface of the membrane was covered. The changes observed at the vascular level were vascular lysis (L), coagulation (C) and hemorrhage (H). These changes were observed for a period of 5 min after application to the samples, using a stereomicroscope  and the images were taken  5 min before and after application to the samples. To quantify the potential irritant effect, the irritation score (IS) was calculated using the formula [H represents hemorrhage, L represents vascular lysis, and C represents intravascular coagulation.The Hen’s Egg Test (HET-CAM) method was applied to assess biocompatibility and possible irritant potential at the vascular level. The experiment was performed on the ninth day of incubation. For this purpose, Met was tested at 2 mM, at which the most beneficial in vitro effects were previously observed. In parallel, a negative control—H formula :IS=5×301IS = 0–0.9); (ii) Irritating (IS = 1–8.9) and (iii) Severe irritating (IS = 9–21) [Depending on the value of the irritation score, the substances can be classified into three categories, as follows: (i) Non-irritating , and the vascular plexus was examined for changes similar to those previously observed in the HET-CAM method . The changes were observed for a period of 5 min, with photographs being taken before and after application to the samples. The same parameters from the HET-CAM test  were followed, for calculating the following parameters:To determine the potential anti-irritant effect, the eggs were prepared as previously described, and the method applied was the one described above  and adapwww.graphpad.com, accessed on 27 April 2022). The statistically significant differences between data were labeled with * .All data are expressed as means ± SD, the differences being compared by applying the one-way ANOVA analysis followed by Dunnett’s multiple comparisons post-test. The used software was GraphPad Prism version 9.0.0 for Windows  a slight increase in viability can be observed, of approximately 101% and 109%, respectively. The highest value of cell viability was recorded at a concentration of 2 mM (approximately 122%). As a result of the maximum concentration tested, 2.5 mM, a decrease in cell viability can be observed compared to the other concentrations tested, but not less than the control concentration . Thus, MAt the 72 h time interval, the same trend is observed in terms of influencing cell viability. Thus, at the first four concentrations tested  there is an increase in cell viability depending on the concentration, while at a concentration of 2.5 mM there is a decrease in cell viability compared to the first concentrations tested; however, the value of cell viability was higher than that of the control group, with the viability being approximately 100% in this case. However, the highest value of cell viability was recorded at a concentration of 2 mM, being about 171% .The effect of Met on the morphology and confluence of HepaRG cells was evaluated and monitored by photographing the cells after two time intervals, 24 h and 72 h.In addition to the results obtained from the previously conducted cell viability test, the morphology of the cells confirms the results obtained after stimulation for 24 h. In Regarding the time interval of 72 h, the results obtained are similar to those observed in the case of cell viability testing. Thus, at the first four concentrations tested, a dose-dependent increase in confluence and number of cells was observed, while at the highest concentration tested, 2.5 mM, there was a slight decrease in cell confluence. However, no change in cell morphology was observed in any of the cases, with the cells having morphological characteristics unaffected by Met treatment .To assess whether Met may exert effects on the morphology of the nucleus and mitochondria, the immunofluorescence method was applied. The tested concentrations showed no effect on cell morphology. Even in the case of the highest concentration tested, no significant changes were observed, such as condensation or a decrease in number. Similarly, in the case of mitochondria, treatment with Met does not cause changes in distribution and morphology. Thus, the mitochondria, in the case of the first four tested concentrations, show a uniform distribution, without signs of condensation or decrease in number. Instead, in the case of the highest concentration tested, a slight condensation of mitochondria around the nuclei is observed .Considering that Met may have a liver repair effect and may interfere with hepatocyte regeneration, the potential effect on cell migration was investigated by the Wound Healing method. For this purpose, the five previously tested concentrations  were tested. The effects observed in this case support the results obtained previously. There is an increase in the concentration-dependent cell migration rate in the case of the first four concentrations, with the highest approach rate being recorded in the case of the 2 mM concentration, followed by a slight decrease in cell migration in the case of the 2.5 mM concentration . Figure IS). For the best evaluation, two types of controls were used, a negative control (H20) and a positive control (SDS 1%), in parallel with testing the 2 mM Met solution.To determine the biocompatibility and evaluation of the irritant and toxic potential of Met in the vascular plexus, the HET-CAM method was applied, which is based on the calculation of the irritation score  being significantly lower than that obtained in the case of applying SDS without pretreatment. Within five minutes, the presence of small irritating effects at the vascular level was evident, manifested as slight vascular lysis, followed by intravascular coagulation and small areas of microhemorrhage (morrhage .Met belongs to the class of biguanides and was introduced in the 1950s for the treatment of type 2 diabetes mellitus (T2DM). However, many other therapeutic effects of Met have been observed over time, and these are not limited to the treatment of diabetes mellitus. Thus, clinical trials have suggested the usefulness of Met in the treatment of polycystic ovary syndrome, obesity, cardiovascular complications and, most importantly, in reducing the risk and mortality of cancer, especially pancreatic, liver and lung cancer . In addiThe association between T2DM and liver damage has been recognized for over 30 years . In patiBased on the premise that metformin may have a hepatoprotective effect, the present study aimed to evaluate the effect induced by Met on the hepatocyte cell line, HepaRG, in terms of viability, morphology and cell structures, such as nucleus and mitochondria. In addition, the effect of Met on the vascular plexus of the chorioallantoic membrane was evaluated, using an in ovo method, the HET-CAM assay, highlighting the biocompatibility and potential anti-irritant effect.An important factor to consider when evaluating the hepatic effect and toxicity of a compound is the choice of in vitro models that reflect the most relevant liver functions. As for isolated primary human hepatocytes, they rapidly lose their drug metabolism and transport characteristics, which greatly limits their use . An alteThus, for evaluating the effect on cell viability, the hepatocyte cell line—HepaRG—was selected, in which five concentrations of Met  were tested for two time intervals, of 24 and 72 h. Concentrations and their equivalents in mg: 0.173, 0.346, 0.519, 0.692 and 0.865 mg) were selected after extensive evaluation of the literature on in vitro testing of Metformin ,34,35,36Following stimulation for 24 h, an increase in cell viability was observed, at a maximum of approximately 122% in the case of a concentration of 2 mM. In the case of the highest tested concentration, 2.5 mM, cell viability was not significantly affected, with its value being similar to that of unstimulated control cells. After 72 h of stimulation, the effect of Met on cell viability was comparable to that observed after 24 h. In contrast, stimulation with Met for a period of 72 h led to a greater increase in cell viability than 24 h stimulation. Accordingly, the cell viability was approximately 171% at a 2 mM concentration. The highest concentration tested, 2.5 mM, did not influence the cell viability, the value, in this case, being about 108%. In addition, in terms of cell morphology, it did not show any changes, with the cells showing an increased confluence, in accordance with the values obtained from the cell viability test.To the best of our knowledge, no studies have been performed on the effect of metformin alone on liver cells in vitro. However, Met has been shown to be hepatoprotective in an insulin-resistant and leptin-deficient murine ob/ob model. In a study by Lin and colleagues, it was found that Met can improve fatty liver disease by reversing hepatomegaly, steatosis and aminotransferase abnormalities by inhibiting hepatic expression of tumoral necrosis factor (TNF) and TNF-inducing factors that promote the accumulation of liver lipids . One of Another study by Conde de la Rosa et al. evaluated the protective effect of Met against apoptosis induced by oxidative stress in primary hepatocytes isolated from rats. The study found that Met protects hepatocytes against oxidative stress-induced caspase activation, poly (ADP-ribose) polymerase (PARP) cleavage and apoptosis . Vilela The hepatoprotective effect of Met against drug-induced toxicity has also been evaluated in several in vitro and in vivo studies. Thus, Tripathi et al. evaluated the anti-inflammatory and protective effect of Met against acetaminophen-induced toxicity in the hepatic cell line HepG2. They pointed out that Met inhibits inflammatory responses and reduces liver damage . In vivoAlthough Met is widely used in the treatment of type 2 diabetes mellitus, the mechanism of action is not fully known. One of the possible mechanisms underlying the reduction of gluconeogenesis is the inhibition of the mitochondrial respiratory chain complex I or increased cytosolic redox status and decreased mitochondrial redox status due to the inhibition of mitochondrial glycerophosphate dehydrogenase . StartinCell migration is an important step in wound healing . In the For a more general picture of the effect induced by Met, the present study evaluated its action in blood vessels, both in terms of biocompatibility and anti-irritant potential, by using the chorioallantoic membrane of the hen’s egg. The obtained results showed that Met does not induce irritating effects at the level of the vascular plexus, having an irritation score of 0.7, which indicates that it is a substance without toxic effects. In addition, with respect to its potential protective role, pre-treatment with Met significantly decreased the irritant effects induced by SDS on the capillaries. Thus, if in the absence of pretreatment, the irritation score induced by SDS was 19.68, in the case of pre-treatment, it decreased to 7.29. These results support previous findings that Met increases cell viability and stimulates cell migration with a potential impact on wound healing. As far as we know, Met has not been previously tested for irritant and anti-irritant effects by the HET-CAM method. However, Met has previously been applied to the chorioallantoic membrane to assess its potential effect on angiogenesis. Thus, Wang and colleagues used the CAM method to highlight the antiangiogenic effect of Met in combination with Pemetrexed . Novel aMetformin has been used in clinical practice for over 50 years and is considered the therapy of choice for the treatment of type 2 diabetes. However, the effects of Met are not limited to the hypoglycemic effect. The results of the present study indicate that Met does not induce toxic effects in hepatocytes, but on the contrary, causes a stimulation of cell proliferation. In addition, by evaluating cell morphology and observing the structure, organization, and number of mitochondria and nuclei, it was confirmed that Met does not induce alterations in their characteristics. On the contrary, by applying the Scratch technique, it was found that Met stimulates cell migration, with a possibly beneficial effect on wound healing. Ovo studies complemented in vitro studies, supporting that Met is free of toxic and irritating effects, and has anti-inflammatory and protective effects against vascular irritation. In conclusion, Met presents a safety profile at the level of hepatocytes with possible hepatoprotective actions. However, further studies are needed to determine the possible biological mechanisms underlying this therapeutic action.


GABAergic projections from the nucleus accumbens core to the dorsolateral ventral pallidum are necessary for drug-conditioned cues to initiate relapse-like drug seeking. Astrocytes in the ventral pallidum are situated perisynaptically and regulate GABA transmission through expression of GABA uptake transporters, but whether they are involved in regulating drug seeking is unknown. To determine the contribution of ventral pallidal astrocytes to heroin seeking, we labeled astrocytes in male and female rats with a membrane-bound fluorescent tag and used confocal microscopy to quantify astroglial expression of the GABA transporter GAT-3 and astrocyte synaptic proximity after withdrawal from heroin self-administration and during 15 min of cued heroin seeking. We found that GAT-3 was upregulated in rats that had extinguished heroin seeking, but not in animals that were withdrawn from heroin without extinction training or in rats that extinguished sucrose seeking. When GAT-3 upregulation was reversed using a vivo-morpholino oligo, heroin seeking was restored in the extinguished context and extinction of cued heroin seeking was disrupted compared to control animals. Although astrocyte synaptic proximity was not altered overall after heroin withdrawal, examination of astrocyte proximity to accumbens D1- or D2-expressing afferents revealed a selective increase in astrocyte proximity with D1-expressing terminals during extinction of heroin self-administration. Experimentally-induced reduction of astrocyte synaptic proximity through knockdown of the astrocyte-selective actin-binding protein ezrin also markedly disrupted extinction of heroin seeking. Notably, GAT-3 or ezrin knockdown had no impact on context- or cue-induced seeking in sucrose-trained animals. These data show that astrocytes in the ventral pallidum undergo plasticity after extinction of heroin use that reduces seeking and highlight the importance of astrocyte-neuron interactions in shaping behaviors associated with opioid use disorder. Relapse vulnerability is a principal feature of opioid use disorder , 2 and aWhile much has been learned about how neural plasticity in the NAcore to dlVP circuit regulates drug seeking, the discovery process for drug-induced plasticity has largely ignored the critical regulation of synaptic signaling by astroglia. Astroglia promote synaptogenesis during brain development and after injury by secreting synaptogenic proteins, such as thrombospondin –19. AstrThe dlVP is a recipient of dense GABAergic innervation from both D1- and D2-MSNs in the NAcore  and dlVPAnimal procedures were approved by the Institutional Animal Care and Use Committee at the Medical University of South Carolina. Male and female Drd1a-iCre and Drd2-iCre (D1- and D2-Cre) rats  and their wild-type littermates were bred in house and maintained on a 12-h reverse light/dark cycle. Rats were caged with littermates until surgery, after which they were singly housed. When animals reached 200–250 g they were anesthetized with ketamine/xylazine and fitted with intrajugular catheters (Instech Laboratories) that were flushed daily with taurolidine-citrate catheter lock solution (Access Technologies) to maintain patency. Animals undergoing operant training with sucrose received no catheters. After recovery, animals were trained to self-administer heroin during 3 h sessions for 10 days and active lever presses resulted in i.v. heroin infusions  and light/tone cues for 5 s. Sucrose animals self-administered unflavored sucrose pellets  for oral consumption during 2 h daily sessions for 10 days. Yoked saline animals received i.v. saline along with cues when a paired rat self-administered heroin. Yoked controls for sucrose-trained animals received cues, but no pellet when a paired rat self-administered sucrose. All animals received 25 g chow/day throughout self-administration. Animals next underwent 10–11 days of extinction training (3 h/day for heroin-trained rats or 2 h/day for sucrose-trained rats) during which time active lever pressing had no consequence. Extinguished rats and yoked controls were sacrificed 48 h after the last extinction session. Rats in the abstinent group were sacrificed after a corresponding time of abstinence without extinction training. Reinstated rats were placed in the operant chamber a final time for 15 or 120 min and light/tone cues were restored to the active lever, but no reward was delivered.Following intrajugular catheter placement or 5 days prior to the beginning of operant sucrose training, AAV5.gfaABC1D.Lck-GFP (Addgene) was microinjected  bilaterally in the dlVP  before undergoing perfusion with 60 mL 1X phosphate buffer and 120 mL 4% paraformaldehyde (EM Sciences). Brains were extracted and submerged in 4% paraformaldehyde for 24 h at 4 °C before transfer into 1X phosphate buffer. Brains were sliced at 50 μm using a vibrating blade microtome (Leica) and slices were stored in a glycerol-based storage solution. For staining, sections containing the dlVP were permeabilized for 15 min at room temperature using 2% Triton X-100 in 1X phosphate-buffered saline. Tissue was transferred into block containing 0.2% Triton X-100 and 2% normal goat serum and incubated in primary antibodies  at 1:1000 in block for 24 h at 4 °C. The monoclonal anti-SYNJ1 antibody was previously validated by western blot and detects only one band in adult rat brain at ~145 kDa, the estimated molecular weight of SYNJ1 . GAT-3 iz-planes and z-stacks were deconvolved ten times (Auto-Quant). Animals were given alphanumerical designations following behavioral studies that were maintained throughout image analysis, so that all analyses were conducted by an investigator blind to animal treatment. Imaging parameters were kept constant for each marker analyzed and experimental groups were stained and imaged concurrently for each analysis. During imaging, distance from bregma was first determined by comparing the anterior commissure shape and position with atlas figures [z, but was instead used to normalize co-registered signal. Astrocyte synaptic proximity and GAT-3 expression were quantified by isolating SYNJ1 or GAT-3 signal that co-registered with the rendered astrocyte  and SYNJ1 intensity was quantified and normalized to quadrant size. For determination of astrocyte association with D1- or D2-MSN terminals, digitally rendered astroglia were used to isolate co-registered mCherry, from labeled NAcore neurons, and co-registered SYNJ1 immunoreactivity  that exhibited higher signal intensity due to antibody accumulation and thus poor signal:noise.Astroglia were imaged with a 63× oil immersion objective lens using a Leica SP5 confocal microscope. Z-series containing 3–8 astroglia were imaged at 12-bit resolution with a pinhole of 1 Airy unit, a frame size of 1024 × 1024, two-line averaging and a 1-μm step between  figures . The sub figures –39 and FBilateral cannulae (P1 Technologies) were placed 1.5 mm above the dlVP  5 days prior to the start of operant training and cannulae were kept patent by insertion of metal dummies  of the same length. Immediately following sessions 7–9 of extinction training, rats received bilateral infusions  of an ezrin-targeted vivo-morpholino oligo , a GAT-3-targeted vivo-morpholino oligo , or a standard control oligo with no expected binding target in the brain  under light isoflurane anesthesia . Intracranial delivery of vivo-morpholino oligos for 3–5 consecutive days has been used previously to obtain effective knockdown of ezrin and other astroglial proteins 4–7 days after the last oligo infusion , 40–43.Locomotion was analyzed in a subset of animals that underwent morpholino-mediated GAT-3 or ezrin knockdown in the dlVP as described above. Five to seven days after morpholino delivery, rats were placed in a novel open field environment (42 × 42 × 32 cm) and photocell beam breaks (VersaMax) were used to quantify distance traveled during the 2-h session.t-test, unless unequal variance was observed between groups. Astroglial measures were first tested for normality using D’Agostino-Pearson omnibus normality test  and are presented as scatter plots where possible, with cell and animal numbers clearly indicated. Statistical tests used for each analysis are stated in figure legends. Behavioral data are presented as mean ± SEM and were analyzed using one- or two-way ANOVA or Student’s Astroglia in the dlVP of male and female rats were transduced with AAV5.gfaABC1D.Lck.GFP Fig.  and traiN = 45–98/3–8 cells/animals), indicating that GAT-3 upregulation resulted from extinction training. Levels of GAT-3 on dlVP astroglia correlated negatively with lever pressing during 15-min of cue-induced heroin seeking   Fig. . In contls) Fig. . These dN = 36–41/5 cells/animals).To determine whether the reductions in GAT-3 expression and astrocyte synaptic proximity observed during cued heroin seeking in the rostral dlVP occurred during natural reward seeking, separate rats were trained to self-administer sucrose Fig. . We founN = 28–40/3–4 cells/animals). The GAT-3 oligo had no effect on GAT-1 expression, a GABA transporter expressed perisynaptically by both neurons and astroglia [Interestingly, the astroglial measurements of GAT-3 expression and SYNJ1 co-registration in rats undergoing cued reinstatement correlated negatively with active lever pressing Fig. , suggeststroglia  . Using this strategy, we compared active lever pressing in three treatment groups, rats that were infused bilaterally with a control oligomer and those that underwent either bilateral GAT-3 or ezrin knockdown. Compared to controls, knockdown of either GAT-3 or ezrin appeared to reverse extinction training and elevated heroin seeking in the extinguished context , illustrating that the increase in lever pressing in oligo-treated animals reflected motivation to acquire heroin and not nonspecific increases in lever pressing or motor activity. Together, these data show that increased GAT-3 expression and synaptic insulation by dlVP astroglia during extinction training suppress heroin seeking and are necessary for extinction of responding.Rats were trained to self-administer heroin and received bilateral oligo infusions in the dlVP immediately following sessions 7–9 of extinction training (ext Fig. . During ext Fig. . While cext Fig. . Remarkaext Fig. . ImportaExcitatory transmission onto D1- and D2-MSNs in the NAcore undergoes marked plasticity after drug use that regulates drug seeking in animal models of relapse and NAcore perisynaptic astroglia critically regulate glutamatergic transmission and cue-induced drug seeking . The dlVGAT-3 is a primary regulator of extracellular GABA concentrations . ThroughNeuronal activity in the VP is necessary to coordinate adaptive behavioral responses to motivationally salient information, including cues that reinstate drug seeking –50. The Since activation of neurons in the rostral VP is necessary for cued reinstatement of cocaine seeking , it is pAlthough the NAshell, which projects to the ventromedial VP, is often cited as a critical nucleus for extinction learning , our finIn our previous studies, we noted robust and transient astroglial plasticity in the NAcore associated with glutamatergic transmission during relapse . In the Research by others has uncovered sex differences in synaptic adjacency of astrocytes in the NAcore following cocaine self-administration and extinction . In the Consistent with the possible role for changes in astroglial GAT-3 and synaptic proximity selectively impacting extinction of drug seeking, sucrose self-administration followed by extinction training did not alter either measure. However, after 15 min of cue-reinstated sucrose seeking, both astroglial measurements were elevated, opposite to what occurred during heroin reinstatement, and would be expected to promote within-trial extinction of cue-induced sucrose seeking relative to heroin seeking. Indeed, we found that within-trial extinction during cued sucrose seeking occurred more rapidly than during heroin seeking (Supplementary Fig. In our animal model, heroin seeking behavior was extinguished after self-administration by removal of the reinforcer, a process that engaged plasticity in dlVP astroglia. In humans recovering from substance use disorder, cues and contexts previously associated with drug use can elicit strong craving and stimulate relapse –67 and tOpioid use disorder has reached epidemic proportions in the USA. We define critical astroglial adaptations in the dlVP that endogenously oppose drug-seeking behavior during withdrawal by supporting refraining from heroin seeking after extinction training. These adaptations are reversed during presentation of heroin cues, which contributes to more intense seeking in response to heroin cues compared with cues predicting a natural reward. These findings, combined with our previous studies in the NAcore, highlight that astroglia are uniquely impacted by withdrawal from drug use compared with natural rewards and that their morphological plasticity and expression of transporters are highly plastic during relapse in a manner that modulates drug seeking , 27. ThiSupplementary Figures and Legends


Alterations in the MET gene, including amplifications and exon 14 skipping mutations, have been identified as actionable oncogenic alterations. However, MET fusions are rarely detected in lung cancer, and their sensitivity to therapeutics has not been systematically analyzed.The data from 30876 lung cancer patients from the LAVA database and 7966 patients from cBioPortal database were screened. Basic demographic and clinical information for the patients harboring MET fusions were collected. A lung squamous cell cancer patient harboring a novel EML4-MET fusion was treated with crizotinib. Additionally, a literature review was performed to summarize the cases of patients harboring MET fusions and their treatment information.MET fusions were found in only 0.2% to 0.3% of lung cancer patients and appeared in almost all exons of the MET gene. Intragenic MET fusions were found in 52.6% (41/78) of the included patients. Crizotinib was effective for MET fusions, including a novel identified EML4-MET fusion, even after the failure of multiple lines of treatment. This result suggested that acquired MET fusions become more regionally selective, as they usually occurred in exons encoding the extracellular region. Interestingly, the MET-fused genes in primary MET fusions or acquired MET fusions were very different, which indicated the different functions and influences of the disease.MET fusions are rare, and half of the fusion types were intragenic fusions. Lung cancer patients harboring primary or acquired MET fusions could benefit from crizotinib. In addition, EML4-MET was first reported in this study as a novel MET fusion type.The online version contains supplementary material available at 10.1186/s12967-023-03999-7. Lung cancer, for which the 5-year overall survival (OS) is less than 20% in China, ranks first among all malignancies in cancer-related mortality  and is aThe MET gene is located on the 7th chromosome of the human genome and encodes the MET protein, which serves as the receptor of hepatocyte growth factor (HGF) that participates in the biological regulation of cell proliferation . On the We first collected the incidence data from a pan-cancer study, MSK MetTropism , of the cBioPortal database to analyze the incidences of MET gene alterations. No special pre-selection for patients in this dataset, such as tumor types, age, sex and race, in order to fully understood the incidences of MET fusions and the distribution of MET fusions among different cancer types. At the same time, we particularly screened lung cancer patients in the cBioPortal database, with a sample size of 7966 patients. As listed in Table Then, as of August 15th 2022, a total of 30,876 lung cancer patients from the Burning Rock® LAVA database  were screened via the system. The search term used was “lung cancer”, and no other pre-selection for the lung cancer patients. The LAVA database (system) is a targeted sequencing data cloud platform built by Burning Rock® Biotech, which can quickly query patients in the database through some simple functions, and can also see the overall data analysis of all patients sent by doctors, besides, the data is anonymous and protects patients’ privacy. Each piece of information in the database is real-world data, formed by Burning Rock® via collecting basic patients’ information, obtaining patients’ tissue sections for targeted sequencing, and uploading it to the database. The database contains patients from various hospitals, whose pathological sections were sent by clinicians, and the basic information of patients will be collected before genetic testing, such as: sex, age, pathological type and disease stage, etc., and genetic testing will be carried out only after all information is collected, and therefore no missing data in this database as well as in the process of database search in this study. All patients included in the database gave informed consent.DNA isolation and targeted sequencing were performed in Burning Rock® Biotech, a commercial clinical laboratory accredited by the College of American Pathologist (CAP) and certified by the Clinical Laboratory Improvement Amendments (CLIA), according to optimized protocols as described previously , 24. BriSequence data were mapped to the reference human genome (hg19) using Burrows-Wheeler Aligner version 0.7.10 . Local aIn this study, we collected the published cases of lung cancer patients harboring MET fusions. Basic information, including publication time, the types and appearance times of MET fusions, tumor stage, concomitant EGFR/ALK mutations, pathological types and treatment information, was collected, as displayed in Table https://www.proteinatlas.org/) database. The “GenVisR” package was used to generate the Oncoprint waterfall plots and the data were analyzed using the statistical software R, version 3.6.2. The Metascape [The fusion sites of the MET gene in patients from the LAVA database were detected, and the fusions in MET gene exons were then matched via the cBioPortal database for functional detection purposes. The locations of genes that fused with the MET gene were acquired from The Human Protein Atlas  to 0.3%  of all patients. From the point of view of MET gene structure, as shown in Fig. To elucidate the characteristics of lung cancer patients harboring MET fusions, we summarized the basic demographic and clinical information of the 78 selected patients in Table As described in the results above, MET fusions are rare in lung cancer patients. To determine what kind of therapeutics would benefit lung cancer patients harboring MET fusions, we performed a literature review of cases published by September 2022. As summarized in Table The case that we reported here is that of a unique LUSC patient harboring primary EML4-MET. The novel EML4-MET fusion type was not found in the 78 selected patients, nor was it reported in the published cases. The treatment timeline for the patient is displayed in Fig. In 56 selected lung cancer patients from the LAVA database harboring MET fusions, 38 patients had primary MET fusions and 18 patients acquired MET fusions after developing resistance to EGFR/ALK-TKIs, as shown in Table The rapid development of targeted sequencing has provided more opportunities for the treatment of lung cancer patients. Accordingly, patients harboring actionable genomic alterations respond differently to therapeutics. MET alterations, including MET mutations, amplifications and gene fusions, are relatively rare genomic alterations in NSCLC patients . SelectiMET fusions are rare in lung cancer patients, which has led to a lack of a systematic understanding of this kind of genomic alteration, both in terms of the genomic appearance and the treatment of patients. To the best of our knowledge, our study is the first to analyze the basic characteristics of lung cancer patients harboring MET fusions. Our analysis showed that MET fusions frequently appeared in LUAD patients, similar to other actionable genomic alterations in lung cancer –4. MET iMultiple concomitant genomic alterations were detected in lung cancer patients harboring MET fusions, but concomitant MET amplifications (35.7%) seemed to be the most common genomic alteration. There was no selectivity for the fusion sites for the MET gene, and nearly all of the exons could serve as the fusion sites, as well as introns. However, there were differences when comparing the fusion sites between primary MET fusions and acquired MET fusions in treatment-naive patients and EGFR/ALK-TKI-resistant patients. This result suggested that acquired MET fusions become more selective in fusion sites, which usually were in the extracellular region of MET. However, the underlying mechanism and the influence of the fusions on the function of MET remain unclear and need to be further explored. Interestingly, the MET-fused genes in primary MET fusions or acquired MET fusions were very different, which indicated the different functions and influences of the disease. Admittedly, the current study is not without limitations for confirming the role of MET fusions in the development of lung cancer resistance to EGFR/ALK-TKIs, and further exploration of these rare actionable genomic alterations is needed. However, our study first determined the basic characteristics of lung cancer patients harboring primary or acquired MET fusions, and the findings cast new light on their role in targeted therapy.MET fusions were rare in lung cancer and appeared in approximately 0.2%-0.3% of patients, most of whom had LUAD. Lung cancer patients harboring MET fusions could benefit from crizotinib treatment. In addition, EML4-MET was first reported in this study as a novel MET fusion type.Additional file 1: Figure S1. The framework for patients’ selection and study purposes.


Background:Clostridioides difficile infection (CDI) is the leading cause of healthcare-associated diarrhea. Significant risk factors for CDI include antibiotic use and healthcare exposure. Antibiotics are often administered before, during and/or after surgery to prevent postsurgical infection. The contribution of surgery-related antibiotics to the overall CDI burden has not been well described, and assessment of the appropriateness of surgical antibiotic use is complicated by complex clinical guidelines. We have described surgical antibiotic prophylaxis history among adult with CDI in Minnesota in 2018. Method: The Minnesota Department of Health (MDH) performs 5-county active population- and laboratory-based CDI surveillance as a CDC Emerging Infections Program site. Incident CDI was defined as stool positive for C. difficile by toxin or molecular assay from a person aged ≥18 years with no positive test in the preceding 8 weeks. History of CDI was defined as having had a previous CDI episode in the 2009–2018 surveillance data set. Medical records were reviewed for 12 weeks prior to incident CDI test date to identify antibiotic prescriptions. Antibiotics with documented indication for surgical-site infection prevention or surgical prophylaxis were classified as “surgical antibiotic prophylaxis” (SPPX). SPPX type , appropriateness of SPPX, and clinical guideline adherence were not assessed. Results: During 2018, 812 incident CDIs were reported to MDH among 736 patients. SPPX preceded 84 (10.3%) cases, non-SPPX antibiotic use preceded 465 cases (57.3%), and 263 cases (32.4%) had no documented prior antibiotic use. The median age of incident CDIs with preceding SPPX was 68 years . In 25 incident CDI cases with preceding SPPX (29.8%), there were no other antibiotic exposures. Among incident CDIs with preceding SPPX, 11 (13.1%) had >1 surgery event with SPPX. Prior CDI was identified for 13 (15.7%) with SPPX. Among 99 procedures with preceding SPPX, orthopedic surgeries , gastrointestinal surgeries , and cardiovascular surgeries  were most common. In total 18 SPPX prescriptions (18.2%) originated in outpatient settings. SPPX drugs included cefazolin , ceftriaxone , ertapenem , and clindamycin . Median SPPX duration was 1 day , and the median number days between surgery and specimen collection date was 19 . Conclusions: Antibiotic stewardship programs should assess surgical prescribing, including in outpatient centers. Even short antibiotic duration for surgery could put patients at risk for CDI. More data are needed to evaluate the appropriateness of SPPX prescribing and to describe the impact of SPPX on CDI.Funding: NoneDisclosures: None


Pure adhesive served as control. The physicochemical (degree of conversion (DC), linear shrinkage (LS), shear and complex viscosity, water sorption (WS), sol fraction (SF)), biological  and bioactive  properties were investigated. DC and LS remained unchanged. The combination of BAG-Bi/MA-POSS resulted in a significantly increased WS and SF compared to control. In addition, the combination of CAP/MA-POSS slightly increased the shear viscosity of the adhesive. The addition of the nanoparticles did not influence the antimicrobial effects compared to the pure adhesive. Improved mineral inducing capacity could be detected in all nanoparticle combinations. The combination of bioactive and/or antibacterial nanoparticles showed improved mineral inducing capacity, but no antibacterial properties. The material properties were not or only slightly affected.The aim of the study was to examine the applicability of bioactive and antibacterial nanoparticles to an experimental adhesive. The adhesive  was mixed with combinations of 5 wt% methacryl-functionalized polyhedral oligomeric silsesquioxane (MA-POSS) and one kind of bioactive/antibacterial nanoparticles: 1 wt% core-shell silica-silver nanoparticle (SiO In teeth with deep carious lesions and a symptom-free pulp, the so-called selective caries excavation method allows to keep the microorganisms under a filling to avoid root canal treatment. A compact adhesive composite restoration is then intended to hinder caries progression . HoweverA well-known and very effective antibacterial agent is silver, which has been used in medical applications before ,6. By reBeside the bacterial activity in the carious lesions, the parallel degradation of the hybrid layer worsens the prognosis and is still a significant problem despite the continuous improvement of the adhesive systems ,15. AnotIn this study, an experimental adhesive was filled by the core-shell MA-POSS nanoparticles combined with the ion-releasing (bioactive) or antibacterial particles to obtain bioactive and antibacterial properties without compromising the applicability of such adhesive. The null hypothesis was that there are no significant differences in the physicochemical properties, but an improved mineral inducing capacity and improved antibacterial effect in the adhesives filled with the nanoparticles.(a)2@Ag core-shell 5 wt% MA-POSS and 1 wt% SiO(b)5 wt% MA-POSS and 1 wt% BAG-Bi (c)5 wt% MA-POSS and 1 wt% CAP .An experimental adhesive consisting of 60 wt% BisGMA, 15 wt% TEGDMA and 25 wt% HEMA  was prepared and magnetically mixed in dark for 10 min as described previously . As photThe unfilled adhesive served as a control group.−1 and 4000 cm−1 with a resolution of 4 cm−1. One test consisted of 26 measurements with an interval of 30 s. For this purpose, one drop of each adhesive group was applied on the diamond crystal to cover the sampling area and polymerized after four initial measurements (120 s) for 60 s . A background scan was carried out between each test. The benzyl groups were taken as reference and the DC was estimated from the height of the vinyl group peaks before and after polymerization, as described elsewhere [The DC was investigated by ATR-FTIR (n = 3). The tests were carried out at a wavelength range between 700 cmlsewhere .The viscosity was studied by a rheometer  using a cone plate geometry (40 mm diameter and 2° cone angle). To avoid polymerization, all test samples were prepared without an initiator. Firstly, the linearity was confirmed by an amplitude sweep experiment (γ = 5 to 20% and ꞷ = 20 rad/s). The complex viscosity was measured at the constant deformation of 15% and the frequency range (ꞷ = 10 to 100 rad/s and ꞷ = 100 to 5 rad/s). Two following frequency sweep tests were used to ensure no internal structure or possible polymerization affecting the results. Complex viscosity was estimated at the region where the most systems exhibited a constant viscosity (ꞷ = 30 rad/s). Following, the shear viscosity was estimated through two steady shear tests  and the data were fitted by cross-fit model (Newtonian).2, Bluephase, Ivoclar Vivadent GmbH, Ellwangen, Germany). Afterwards, the specimens were dried in a desiccator and weighted each two days until the difference between the two following measurements was below or equal 0.1 mg (m0).Five specimens  from each adhesive were prepared in a teflon form and light cured for 60 s from both sides (>1000 mW/cmmV0). Then the specimens were stored in distilled water at 37 °C and weighted every second day. The cycle was repeated until the difference between two measurements was again below or equal 0.1 mg (m1). Next, the specimens were dried in a desiccator and weighted again every second day until they reached a steady value (m2). Based on the determined values, WS and SF could be calculated by the following equations:After drying, the diameter and the thickness of the specimens were estimated to determine the volume (The measurements of LS (each group n = 5) were performed by a linometer at room temperature . Twenty 2), 24 h before the test. Each test was performed in triplicate. The specimens were shortly cleaned with ethanol and sterile distilled water before embedding in silicone in a 24-well plate  and storage at 4 °C.To prepare tester plates for bacterial adhesion measurement, adhesives (each group n = 6) were applied onto autoclaved glass specimens  with a microbrush  and polymerized for 60 s  were prepared from overnight  grown agar cultures  in Phosphate-buffered saline (PBS) adjusted to an OD550 of 1.0 with a photometer . 1 mL of the bacterial suspension was then applied directly on each test specimen prepared in a well-plate. Bacteria in PBS on a glass specimen was used as negative control. After two hours at 37 °C on a rocking incubator, the bacterial suspensions were aspirated with a pipette and plates were washed with PBS to remove non-adherent cells. To determine the number of living cells an ATP-bioluminescence-assay  was used according to the manufacturer’s specifications.Inocula of 2, Bluephase, Ivoclar Vivadent GmbH, Ellwangen, Germany). Artificial dentinal fluid was prepared with accordance to Jungbluth et al. [2PO4, 0.807 g Na2HPO4, and 0.617 g CaCl2·2H2O. Artificial saliva was prepared pursuant to Klimek et al. [2PO4, 0.34 g Na2HPO4, 0.225 g CaCl2·2H2O, 0.16 g NaSCN, 0.16 g NH4Cl, 0.2 g urea, 0.03 g glucose, and 0.002 g ascorbic acid mixed into 1000 mL distilled water. Specimens were stored in vertical position for four weeks either in artificial dentinal fluid or in artificial saliva at 37 °C while the medium was changed every second to third day (each group n = 2). Afterwards, the specimens were removed from the media, carefully washed with distilled water and left in a desiccator for drying phase. Then, the surface of the test specimens was sputtered with carbon and examined under the scanning electron microscope  at 10 kV. The same specimens were then analyzed by energy-dispersive X-ray spectroscopy  at 15 kV to estimate the Ca/P ratio.Cylindrical specimens  were prepared in a teflon form and polymerized for 60 s from both sides .All statistical analyses were performed using the software IBM SPSS Statistics for Macintosh . Statistical analysis was performed using Kruskal-Wallis-tests followed by multiple pairwise comparisons adjusted to Bonferroni. Pearson correlation was applied to assess the association between oscillatory and steady shear viscosity  could be observed (p < 0.001), the complex and steady shear viscosity were associated in a linear way and thus the Cox-Merz rule applies for the studied resins and oscillatory and shear parameters used [As shown by Pearson correlation  and led to a significant increase in shear viscosity only by the addition of CAP/MA-POSS compared to the pure adhesive (p = 0.013). Still, a tendency to viscosity (complex and shear) enhancement, even though not significant, can be seen in all particle-filled groups (The different combinations of the nanoparticles had a significant effect on shear viscosity (d groups .p = 0.007) and sol fraction (p = 0.005). No significant differences were observed between the other groups.In comparison with the pure adhesive, the sample filled with BAG-Bi/MA-POSS exhibited a significantly higher water sorption (p = 0.002). Compared to control, no significant differences to CAP/MA-POSS (p = 0.256), BAG-Bi/MA-POSS (p = 1.000) and SiO2@Ag/MA-POSS (p = 0.149) could be observed (2@Ag/MA-POSS group (p = 0.009) and CAP/MA-POSS (p = 0.019).The Kruskal-Wallis test showed a significant effect of the different combinations of the nanoparticles on linear shrinkage  or 0.4 and 1.0 , respectively.The experimental adhesive used in this study was based on the study by Kreutz et al. , which aThe hypothesis that there is no effect on the physicochemical properties by adding the nanoparticles can be partially accepted. The linear shrinkage was not changed by the addition of any particle combination. The shear viscosity was significantly increased only with the CAP/MA-POSS fillers, solely the BAG-Bi/MA-POSS combination showed a significantly increased water sorption and sol fraction. A low sol fraction has a beneficial effect on biological properties. In the oral environment, monomers and nanoparticles can be released through the saliva and thus enter the body’s circulation. Due to the nanometer size, nanoparticles can be absorbed by cells and lead to inflammation . SimilarThe increase in water sorption in BAG-Bi group in this study is probably due to hydrophilic and ion-releasing nature of BAG-Bi, as similar results have been observed ,23. SincThe study results indicate nanoparticles having no significant effect on the degree of conversion. This finding is comparable to those of previous studies presenting no influence on the degree of conversion due to BAG-Bi, MA-POSS or CAP nanoparticles ,23,26,392@Ag group from all filled adhesives indicates again a better particle distribution due to the silica shell. The complex and shear viscosity of all tested groups was unaffected or only increased to an extent that does not hinder the applicability for dentistry [The degree of conversion is strongly associated with the viscosity of the resin blend. Low viscosity typically results in a higher degree of conversion, as the diffusion of the reactive groups is enhanced. Low viscosity is also advantageous for adhesives improving the infiltration into the acid etched collagen matrix. In this study, contrasting the pure adhesive, solely the addition of CAP/MA-POSS significantly influenced the shear viscosity. This may be an indication for a poor particle distribution and a stronger aggregation within the tested adhesive ,42. Neveentistry ,43. Slig2@Ag/MA-POSS showed a significantly higher linear shrinkage than the BAG-Bi/MA-POSS combination. This result goes parallel to the observations on WS, SF and DC and supports the assumption about a slightly worsen network density at the BAG-Bi/MA-POSS group in comparison to other filled resins. Similar correlation between DC and polymerization shrinkage was observed on the BAG-filled composite resin by Par et al. [The polymerization shrinkage is also related to the degree of conversion . There wr et al.  and Jäger et al. . The difAn additional mechanical stabilization of the hybrid layer is to be achieved by the addition of nanoparticles that can induce mineral precipitation. After storage in artificial saliva  mineral There are several factors known to influence bioactivity. In this regard, there are many factors with a crucial role: pH and (super)saturation of the immersion medium and immersion time ,50, sizeThe initial dissolution of ion-releasing particles within an immersion medium is the first step of the Ca/P crystal precipitation. This leads to an increase of the medium’s  supersaturation, especially around the surface. Followed by the actual precipitation and ion exchange .The precipitates mainly exhibited a cauliflower- or plate-like structure in all particle-filled groups. Such crystals have also been found in other studies ,55,56 an2@Ag was shown elsewhere [2@Ag particles were embedded in a polymer matrix and thus had a small area of contact with bacteria. As a result, the SiO2@Ag particles were not sufficiently exposed to the solution and could not be effective. However, a later release of the particles would be beneficial for clinical success, so that studies should be conducted to observe the antibacterial potential over a longer period of time. It was noted that the combination of SiO2@Ag and MA-POSS did not have a negative influence on material properties, as observed in other studies for silver particles [In order to consider new standards of minimally invasive caries therapies, the antibacterial effect was also investigated in this study. No antibacterial effect could be observed in any particle-combination. Although an antibacterial efficacy of SiOlsewhere ,57, the articles . As indiarticles ,58,59,60This study showed that while the concentration and combinations of the particles were sufficient for the mineral precipitation inhibition, no antimicrobial effects could be reached in such compositions. Therefore, despite no antimicrobial effect, the studied particle combinations are promising in dental adhesives for the inhibition of a remineralization process.The combination of 1 wt% mineral releasing or silica-silver core-shell nanoparticles with 5 wt% MA-POSS in an experimental adhesive showed an improved bioactive potential. However, no additional antibacterial effect was observed for any particle combination. Regarding the studied concentration of the particles, there only was a modest effect on material properties. Therefore, such nanoparticle-combinations were shown to be promising for the application in dentistry.


We aimed to evaluate the effect of procalcitonin (PCT) guided therapy on antibiotic exposure in pediatric patients with infectious disease.We performed an updated systematic review and meta-analysis of randomized controlled trials (RCTs) identified in systematic searches of MEDLINE, Embase, the Cochrane Database, Google Scholar, and SinoMed (through July 2021). The primary outcome was the length of the antibiotic therapy. Required information size (RIS) was calculated using trial sequential analysis (TSA).Four RCTs with 1,313 patients with infectious disease were included. Overall, after a mean 22-day follow-up, PCT-guided antibiotic therapy was associated with a significantly shorter length of antibiotic therapy compared with the control group  and a decreased rate of antibiotic adverse events . However, the length of hospital stay , rates of antibiotic prescription , hospital readmission  and mortality  were comparable between the PCT-guided antibiotic and control groups. TSA showed that the RIS was 2,340, indicating a statistically significantly shorter length of antibiotic therapy between PCT-guided antibiotic and control groups (P <0.05).PCT-guided management seems to be able to decrease antibiotic exposure in patients with infectious disease. However, much larger prospective clinical studies are warranted to confirm these findings. It is not affected by hormone levels in the body. Under physiological conditions, thyroid C cells are the main cellular source of PCT  is a new inflammatory indicator discovered by Assicot et al. in 1993. PCT is a more specific infection-related biological marker index for diagnosing bacterial infection than CRP . In recee of PCT . Under pe of PCT . PCT leve of PCT .In addition to early, timely, and accurate diagnosis of systemic bacterial infection in children, active control of infection and correct selection of antibiotics are the keys to improving the success rate of treatment. In routine clinical practice, when the exact pathogenic bacteria and drug susceptibility results are unknown, the antibiotic therapy regimen is often empirical. Many conditions, such as the local and hospital microbiological data and antibiotic susceptibility, the severity of the disease of the child, the antibiotic susceptibility of common pathogens, and the risk of drug-resistant bacterial infections . Initialvs. 10%, P = 0.037), a 2.4-day reduction in antibiotic exposure time , and antibiotic-related adverse events   . TherefoHowever, no RCT published after 2016 demonstrated that PCT-guided management was associated with decreased antibiotic exposure compared with empirical care, which might be associated with the recent progress in antibiotic stewardship or access to infectious disease in the empirical care group . MeanwhiTherefore, as the evidence gathered has recently increased, we have performed a meta-analysis and a trial sequential analysis (TSA) to evaluate the effect of PCT-guided therapy on antibiotic exposure in pediatrics with infectious disease.https://inplasy.com/.We searched MEDLINE , Embase (2005 to July 2021), the Cochrane Controlled Clinical Trials Register Database (to July 2021), Google Scholar (to July 2021), SinoMed (to July 2021), and the ClinicalTrials.gov website (to July 2021) using the terms “infection,” “pneumonia,” “sepsis,” “procalcitonin,” “pediatric,” “children,” “length of antibiotic therapy,” and “randomized trial.” Manual reference checking of the bibliographies of all relevant articles was performed. No restrictions were applied. The review was registered at We first conducted an initial screening of titles and abstracts; the second evaluation was based on a full-text review. Trials were considered eligible if they met these criteria: 1) pediatric patients with infection ; 2) pediatrics in the PCT group were given PCT-guided antibiotic therapy regimen; 3) pediatrics in the control group were treated with empirical antibiotic regimen; 4) primary outcome of interest was the length of antibiotic therapy; and 5) the study was an RCT. Exclusion criteria were (1) neonate, adult patients; (2) complicated with immune deficiency , or infection requiring prolonged antibiotic therapy ; (3) single-arm study; (4) without primary outcome; (5) cohort study, retrospective study, animal study, case report, or review; and (6) duplicated data.Two reviewers extracted data concerning patient characteristics, the PCT-guided and empirical antibiotic therapy regimens used, study quality, and clinical outcomes using a standard data-collection form. Disagreements are resolved by discussion.The primary outcome was the length of the antibiotic therapy. Secondary outcomes were the length of hospital stay, rates of antibiotic prescription, hospital readmission, mortality, and antibiotic adverse events.The Preferred Reporting Items for Systemic Reviews and Meta-Analyses (PRISMA) statement  was follResults were analyzed quantitatively with STATA 14.0 software  using the fixed-effects model . We calc2 statistic and the chi-square test. A value of I2 >50% was considered a substantial level of heterogeneity .In the meta-analyses, trial sequential analysis (TSA) was used to reduce the risk of reaching a false-negative conclusion . When thWe initially identified 219 potentially relevant articles. Seventy studies were considered of interest and were retrieved for full-text review. Seventeen articles were excluded owing to duplication (n = 6), reviews (n = 5), incorrect comparisons (n = 4), and no clinical outcomes (n = 3). Therefore, four randomized trials were finally included in the analysis. Four published RCTs with a total of 1,313 patients were included, and all have completed and reported the clinical outcomes . One RCTAmong the pediatrics enrolled, the average age was in the range of 2.3 to 4.7 years. The total number of patients in each study was in the range of 270–337, and most were male. About 67.5% of pediatrics had pneumonia, and 14.7% had non-CAP lower respiratory tract infections. The average levels of PCT were 0.97 ug/L, C-reactive protein was 51.0 mg/L, and leukocytes were 13.7 cells/ul. Most pediatrics were followed up for less than 1 month, and the average follow-up period was 22 days Table 2.Four trials randomized the participants and reported the details about the means of generation of random sequences . Two stu2 = 89.0%). However, sensitivity analysis was performed by removing each of the trials individually, which did not detect any influence on the length of antibiotic therapy.The length of antibiotic therapy: All four trials  provided2 = 79.2%).Length of hospital stay: Length of hospital stay was shown in all four trials . Overall2 = 0).Antibiotics prescription: The rate of antibiotic prescription was shown in four trials . Overall2 = 4.7%).Hospital readmission: All four studies  reportedMortality: Only one study  reported2 = 92.8%).Antibiotic adverse event: The rate of antibiotic adverse events was reported in four studies , and wasThe TSA analysis showed that, assuming a mean difference of 2.0 days between PCT-guided antibiotic therapy and control groups in the length of antibiotic therapy, the RIS required 2,340 participants. The cumulative Z curve crossed trial sequential boundaries, indicating statistically significant differences in the length of antibiotic therapy between groups Figure 8This meta-analysis, including all the available evidence, showed that the application of PCT can guide the antibiotic management of pediatrics with infective disease, not only shorten the length of antibiotic therapy , but also decrease the risk of antibiotic adverse events .vs. 9.3 day). Meanwhile, 28-day (20% vs. 25%) and 1-year mortality (36% vs. 43%) were significantly lower in the PCT-guided group  (vs. 15.3%), 28-day mortality (15.2% vs. 28.2%), length of antibiotic therapy (5 days vs. 10 days) and cost of hospitalization (P <0.001) than the standard of care guided by guidelines (vs. 8.1 days) and a reduction in antibiotic-related side-effects   Whe= 0.008) . Recentlidelines . Among 1idelines . Howeveridelines , which midelines . In a pa57–0.82) . Among t57–0.82) . Therefovs. 6.3 days) (vs. 10.96 days). In another study of 396 pediatrics with LRTIs, there were 198 subjects in each group. Dai et al. found that the length of antibiotic therapy was significantly decreased in the PCT-guided group (4 days vs. 6 days). Meanwhile, in neonates with sepsis, PCT-guided therapy was also associated with markedly reduced duration of antibiotic therapy (P <0.001) (vs. 7.6 days). Similar to previous studies, our study confirmed that PCT-guided antibiotic therapy was associated with a significantly shorter length of antibiotic therapy of about 2.22 days compared to the control group .In contrast to the numerous published trials enrolling adult patients, there are only four pediatric  and two .3 days) . In anot <0.001) . HoweverClinical guidelines for antibiotic management vary in their recommendations on PCT-guided management for pediatrics with pneumonia, reflecting the conflicting evidence in this area of research . Our stuNevertheless, the evidence for this effect is not yet robust. In a recently published study, Sekmen et al. included 488 children (<18 years) with pneumonia. PCT was tested at enrollment . They found that PCT values were not significantly associated with higher rates of antibiotic initiation  or empirical antibiotic selection  . TherefoThere are several limitations. (1) Our meta-analyses are based on study-level data with the flaws of the original study. (2) There is a risk of geographical variations. All four studies showed slight differences in the characteristics of the patient, conditions, PCT-guided treatment strategies, and following period. (3) The sample size is small. The TSA analysis showed that, assuming a 20% difference between groups in the length of antibiotic therapy, the RIS required 2,340 participants, and the accrued information size was 1,313.On the basis of our pooled analysis, PCT-guided therapy appears to reduce antibiotic therapy duration and adverse event risks in pediatric patients with infectious diseases. However, the sample size is still small, and there is insufficient evidence to reach a definitive conclusion on the final effectiveness.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical review and approval was not required for the study on human participants in accordance with the local legislation and institutional requirements. Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.PL wrote the main manuscript text. PL and JiaL analyzed the data. JiaL designed this work. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Most antibiotic prescribing for upper respiratory tract infections (URTIs) and acute bronchitis is inappropriate. Substantive and sustained reductions in prescribing are needed to reduce antibiotic resistance. Prescribing habits develop early in clinicians’ careers. Hence, general practice (GP) trainees are an important group to target.We aimed to establish temporal trends in antibiotic prescribing for URTIs and acute bronchitis/bronchiolitis by Australian GP trainees (registrars).A longitudinal analysis, 2010–2019, of the Registrars Clinical Encounters in Training (ReCEnT) dataset. In ReCEnT, registrars record clinical and educational content of 60 consecutive consultations, on 3 occasions, 6 monthly. Analyses were of new diagnoses of URTI and acute bronchitis/bronchiolitis, with the outcome variable a systemic antibiotic being prescribed. The independent variable of interest was year of prescribing (modelled as a continuous variable).28,372 diagnoses of URTI and 5,289 diagnoses of acute bronchitis/bronchiolitis were recorded by 2,839 registrars. Antibiotic prescribing for URTI decreased from 24% in 2010 to 12% in 2019. Prescribing for acute bronchitis/bronchiolitis decreased from 84% to 72%. “Year” was significantly, negatively associated with antibiotic prescribing for both URTI  and acute bronchitis/bronchiolitis  on multivariable analysis, with estimates representing the mean annual change.GP registrars’ prescribing for URTI and acute bronchitis/bronchiolitis declined over the 10-year period. Prescribing for acute bronchitis/bronchiolitis, however, remains higher than recommended benchmarks. Continued education and programme-level antibiotic stewardship interventions are required to further reduce registrars’ antibiotic prescribing for acute bronchitis/bronchiolitis to appropriate levels. GP registrars’ antibiotic prescribing for URTI and acute bronchitis has reduced.Registrar URTI/acute bronchitis antibiotics prescribing is less than other GPs.Registrars’ antibiotic prescribing for acute bronchitis (72%) remains high.1 The number of deaths is expected to rise if efforts to reduce antibiotic prescribing are not made.2 The majority of antibiotics are prescribed in primary care. Inappropriate antibiotic prescribing contributes to this crisis.3 Inappropriate antibiotic prescribing refers to prescribing for conditions for which antibiotics provide little-to-no benefit, including self-limiting respiratory diseases such as acute bronchitis/bronchiolitis (hereafter referred to as acute bronchitis) and upper respiratory tract infections (URTIs).Antibiotic resistance is a global health threat, with an attributable mortality of 1.27 million deaths in 2019.3 Although overall antibiotic use in Australia is trending downwards, greater reductions are needed.3 Guidelines state that antibiotics are not indicated for these conditions, due to compelling evidence for lack of substantive benefit.5In Australian general practice, antibiotics are prescribed in 81.5% of diagnoses of acute bronchitis, and in 35.8% of cases of URTI in 2019.6 and comprised approximately 10% of the GP workforce in Australia by headcount in 2020.7 Although registrars have lower antibiotic prescribing rates than their established GP colleagues, their prescribing is still high.8 However, their antibiotic prescribing habits have been shown to be amenable to education interventions.10 The effect of these interventions could have a long-term impact as antibiotic prescribing habits established early in a clinical career may tend to persist.11 Previous research in an Australian GP registrar population found no reduction in individual registrars’ antibiotic prescribing for acute bronchitis or URTI as they progressed through training.8 However, these findings, considered together with those of an earlier cross-sectional analysis in this registrar population, suggest that there may have been some reduction over time in overall (training programme level) antibiotic prescribing for URTI.12General practitioner (GP) registrars  are of particular interest regarding appropriate antibiotic prescribing,In this study, we aimed to establish trends (from 2010 to 2019) in Australian GP registrars’ antibiotic prescribing for acute bronchitis and URTI in Australia.13 Before 2016, 5 of Australia’s 17 Regional Training Providers (RTPs) participated in ReCEnT. In 2016, there was a major reorganization of Australia’s GP vocational training programme, with 3 of Australia’s 9 Regional Training Organisation (RTOs)  subsequently participating in ReCEnT. In ReCEnT, registrars record 60 consecutive consultations  3 times during training . Only in-practice consultations are recorded (not home visits or nursing home visits) and not “single issue” clinics (such as influenza immunization clinics). Registrars also complete a cross-sectional questionnaire at each 6-month period. The questionnaire elicits characteristics of the GP registrar and their current practice. The methods of the ReCEnT study have been described elsewhere.15This was a longitudinal analysis of data from the Registrars Clinical Encounters in Training (ReCEnT) from 2010 to 2019, recording content of 413,306 consultations. ReCEnT is a multicentre, ongoing cohort study of the in-consultation clinical experiences of Australian GP registrars.16 In ReCEnT, prescription (ATC) is directly linked to the problem/diagnosis  for which the prescription is made.The outcome factor was whether a systemic antibiotic was prescribed. Antibiotics were defined as J01 (systemic antibiotics) according to the Anatomical Therapeutic Chemical (ATC) classification system.The independent variable of interest was year (2010–2019), modelled as a continuous variable.These included patient-, registrar-, practice-, and consultation-level variables:Registrar variables: age, gender, full time/part time status, Australian or non-Australian medical degree, training stage .Patient: age, gender, Aboriginal and/or Torres Strait Island status, non-English speaking background, new to practice, new to registrar.Practice: rurality, socio-economic index for areas (SEIFA), bulk-billing (consultations are government funded with no out-of-pocket cost to patient) or non-bulk-billing, size of practice, region.Consultation: pathology/imaging ordered, duration, number of issues addressed, whether or not the registrar sought information advice , referral ordered, follow-up ordered, learning goals generated).17 We excluded subsection R74-6 from the URTI classification due to its ambiguity of “sore throat” and its potential to include misclassified conditions such as pharyngitis or tonsilitis. All analyses were at the level of problem/diagnosis (rather than consultation). The proportion of problems managed that were URTIs and acute bronchitis were each calculated with 95% confidence intervals (CIs). Proportions of acute bronchitis problems/diagnoses and of URTI problems/diagnoses that were treated with antibiotics were also calculated with 95% CIs. Descriptive analysis of the type of antibiotic prescribed for acute bronchitis and for URTI was also performed.Two separate analyses were performed, limited to diagnoses classified as new cases of acute bronchitis  and URTI (R74), respectively.Descriptive statistics included frequencies for categorical variables and mean with SD for continuous variables.A mixed-effects logistic regression model was used for the longitudinal analyses, to account for repeated measures on registrars over time. The model included a random intercept for registrar to account for nonindependence of prescribing practice within registrars. A fixed effect for time was included, representing the calendar year of the consultation, assuming linearity in the log-odds of the response. The exponentiated parameter estimate represents the estimated multiplicative change per year in the odds of prescribing an antibiotic, as a combination of between- and within-registrar effects.P value less than 0.20 were considered for inclusion in the multiple regression model. Once the model with all significant covariates was fitted, model reduction was assessed. Covariates that were no longer significant (at P < 0.2) in the multivariable model were tested for removal from the model. If the removal of the covariate did not substantively change the resulting model, the covariate was removed from the final model. A substantive change to the model was defined as any covariate in the model having a change in the effect size  of greater than 10%. The regressions modelled the log-odds that an antibiotic was prescribed for a new diagnosis of acute bronchitis/bronchiolitis. Goodness of fit was assessed by checking linearity of the effect of time, checking residuals, performing the Hosmer–Lemeshow (H–L) test, and checking for the presence of influential observations.All other measured variables were modelled as fixed effects. Covariates were selected for inclusion in the multivariable model by screening each covariate for association with the outcome, using a univariate model, adjusted for year of consultation. Covariates with a univariate Analyses were programmed using STATA 16.0 and SAS V9.4. The study was approved by the Human Research Ethics Committee of the University of Newcastle: approval number H-2009-0323.A total of 2,839 registrars recorded 413,306 consultations with a response rate of 96.1%. See From 2010 to 2019, there were 28,372 new diagnoses of URTI and 5,289 new diagnoses of acute bronchitis/bronchiolitis. Antibiotics were prescribed in 13.5% (95% CI: 13.2%–14.0%) of URTI cases, and 74.6% (95% CI: 73.4%–75.8%) of acute bronchitis cases. The proportions of individual antibiotics prescribed for URTI and acute bronchitis are presented in P ≤ 0.001) and acute bronchitis . See Prescribing for both conditions decreased in the 10-year period, from 24%  to 12%  for URTI and 84%  to 72%  for acute bronchitis. The characteristics associated with prescribing of antibiotics for acute bronchitis and URTI are presented in χ2 = 20.5, P = 0.30) and acute bronchitis  and there were no influential observations.Regression diagnostics found reasonable linearity of the time effect. The model had a good fit for both URTI  and 84% to 72% for acute bronchitis. The time trend was statistically significant following adjustment for potential confounding. The most prescribed antibiotic for both conditions was amoxicillin.3 The AURA report found that overall volume of systemic antibiotics fell by 3.5% in the past 5 years, with a 11.2% reduction in proportion of patients with URTI prescribed antibiotics and a 9% reduction for patients with acute bronchitis. Comparison of our findings with those of AURA suggests registrars consistently prescribe less antibiotics than the wider population of established GPs; the GP registrars had a 23% lower rate for URTI and a 10% lower antibiotic prescribing rate for acute bronchitis.5Our findings are consistent with those from the Antimicrobial Use and Resistance in Australia (AURA) report, which collects deidentified data extracted from GP clinical records.8 This suggests that, over the course of an individual registrar’s training, prescribing rates for these conditions are stable. Our current analysis demonstrates that there is “within-programme” temporal change (as opposed to “within-registrar” change) in antibiotic prescribing. This is consistent with other findings from the ReCEnT study examining benzodiazepine prescribing habits, i.e. within-registrar benzodiazepine prescribing patterns do not change through training, but within-programme benzodiazepine prescribing reduces significantly.18A previous analysis of registrar antibiotic prescribing for URTI and acute bronchitis (covering the period 2010–2015) found no significant change in individual registrars’ antibiotic prescribing rate during training .3 The high response rate of the ReCEnT study is a major strength.19 Like clinical notes, ReCEnT data are requested to be recorded at the time of the consultation, therefore recall bias is minimized. Our analyses accounted for a large number of potential confounding variables such as patient age or socioeconomic status. However, a limitation was that we do not have data on patient comorbidities .The ReCEnT study is a unique database that describes the in-consultation clinical and educational experiences and behaviours of GPs in the earliest stages of their general practice careers. It allows in-depth analysis of prescribing habits, specifically linked to the problem/diagnosis for which prescriptions are made. Other data sources utilize national prescribing databases, which although having very high volume of information/participants, do not have the clear linkage of prescription and indication present in ReCEnT. This is a limitation of the AURA report data where GPs can voluntarily fill in “reason for prescription” which is only documented in 36.4% of consultations.Utilizing the ICPC-2 system to link diagnoses to prescribing has limitations, as it does not indicate if the patient is experiencing more complex symptoms. Symptom severity, however, is not an indication for the prescription of antibiotics if the diagnosis is URTI or acute bronchitis. Another potential limitation is that data collection was noncontinuous (it was obtained every 6 months); this may not represent the patterns of prescribing for respiratory tract infections over an entire year . The timing of data collection, however, was at approximately the same times each year and is unlikely to have biased our estimates of the annual temporal trend.20 We do not know the temporal trend in delayed versus immediate prescribing, but any change would plausibly be an increase in delayed prescribing. Any consequent potential increase in overall prescribing over time would make our findings of reduced prescribing more robust.Differentiation of delayed and immediate prescribing is not included in this particular analysis due to lack of data on this variable in earlier data collection periods, but it has been included in ReCEnT data in later collection periods. Delayed prescribing accounts for approximately 9% of URTI prescribing, and 11% of acute bronchitis in registrars’ practice.10 Registrar prescribing for acute bronchitis, however, remains inappropriately high at 72%. This represents a continuing high burden of unnecessary antibiotic use. Other countries have achieved much lower prescribing rates, and have low rates of antibiotic resistance.22 National antibiotic prescribing levels need to reduce further in order to have the desired impact on antimicrobial resistance, and this may be achieved through targeting the next generation of GPs.23 Previous evidence suggests that focussed education of registrars can reduce antibiotic prescribing for acute bronchitis.10 Wider implementation of similar educational strategies is essential.GP registrars prescribe considerably fewer antibiotics for both URTI and acute bronchitis compared with established GPs and prescribing rates have declined in the past 10 years. URTI antibiotic prescribing is relatively low, more in-line with current clinical guidelines. An intervention to reduce antibiotic prescribing which found no significant change in prescribing for URTI in this registrar population may, thus, have been subject to a “ceiling” effect.We have found significant reductions in registrars’ prescribing of antibiotics for URTI and acute bronchitis over the period 2010–2019. Prescribing for acute bronchitis, though, remains high and should be the subject of further educational interventions and antimicrobial stewardship interventions.cmac052_suppl_Supplementary_Appendix_Table_1Click here for additional data file.cmac052_suppl_Supplementary_Appendix_Table_2Click here for additional data file.cmac052_suppl_Supplementary_Appendix_Table_3Click here for additional data file.cmac052_suppl_Supplementary_Appendix_Table_4Click here for additional data file.cmac052_suppl_Supplementary_ChecklistClick here for additional data file.