Drosophila S2 cells. Contrary to the paradigm, the HLH subdomain actively suppressed the phosphotransferase activity of the enzyme, even in the absence of FKBP12. Unexpectedly, perturbation of the HLH subdomain elevated kinase activity at a distance, i.e., allosterically, at the ATP-binding and polypeptide-interacting active site cleft. Accessibility to polypeptide substrate  due to allosterically altered conformations of type I active sites within heterohexameric cytoplasmic signaling complexes—assembled noncanonically by activin-type II receptors extracellularly—is hypothesized to produce a gain of function of the R206H mutant protein responsible for episodic heterotopic ossification in FOP.Although structurally similar to type II counterparts, type I or activin receptor-like kinases (ALKs) are set apart by a metastable helix–loop–helix (HLH) element preceding the protein kinase domain that, according to a longstanding paradigm, serves passive albeit critical roles as an inhibitor-to-substrate-binding switch. A single recurrent mutation in the codon of the penultimate residue, directly adjacent the position of a constitutively activating substitution, causes milder activation of ACVR1/ALK2 leading to sporadic heterotopic bone deposition in patients presenting with fibrodysplasia ossificans progressiva, or FOP. To determine the protein structural–functional basis for the gain of function, R206H mutant, Q207D (aspartate-substituted caALK2) and HLH subdomain-truncated (208 Ntrunc) forms were compared to one another and the wild-type enzyme through in vitro kinase and protein–protein interaction analyses that were complemented by signaling read-out (p-Smad) in primary mouse embryonic fibroblasts and  The purpose of this study, commenced in 2006 with the identification of the recurrent R206H mutation causative of FOP , is to uUnfortunately, Smad protein substrate could not be analyzed, because phosphorylation in vitro cannot be detected. Assays of ALK protein kinase activities require surrogate substrates, dephosphorylated casein polypeptides, in descriptions of commercial sources and as reported by academic laboratories. That said, the pioneering work by Huse et al.  employedCell review article entitled “Conformational Plasticity of Protein Kinases” ATP  diluted into cold ATP (100 μM) and analyzed by Coomassie staining and by autoradiography of SDS-polyacrylamide gels air-dried between sheets of cellophane. Proteins were separated through 4% stacking/12% resolving acrylamide gels. Further specific method details are not available.Nondenaturing PAGE of native proteins for analysis of complex formation or autophosphorylation was performed as previously described . BrieflyRather than assessing radioactive ATP γ-phosphoryl group transfer, the ADP-Glo Kinase Assay  indirectly quantitated ADP produced by subsequent conversion into ATP, which is used to generate light in a luciferase reaction followed by white-light luminescence (RLU). Assays were performed per the instructions of the manufacturer. To ensure accuracy and reproducibility (reduction in scatter and noise), food colorings were added to components prior to mixing, which as a result was readily visualized as thorough and complete by elimination of input colors  and generation of a new one (yellow). Further specific method details are not available.MEFs were isolated from 13.5 dpc mouse embryos and used as described . Cells wFor signaling assays, MEFs were cultured in DMEM without serum for 2 h prior to adding BMP4  for positive control samples or FK506  at 0.001, 0.01, 0.1 and 1.0 µM for 1 h.For immunoblot detection, total cell protein was recovered using M-PER containing Halt Protease and Halt Phosphatase Inhibitor Cocktails and quantified using a Pierce BCA Protein Assay Kit . Proteins were electrophoresed through 10% SDS-polyacrylamide gels and transferred to nitrocellulose (Invitrogen). Membranes were blocked in 5% milk and incubated with primary antibodies against phosphorylated Smad1/5/8 (1:750) and β-actin (1:3000) , at 4 °C overnight. Bound antibodies were detected with anti-rabbit horseradish peroxidase-conjugated secondary antibody (1:6000)  at room temperature, 1 h. Detected proteins were imaged with Immobilon Chemiluminescent HRP Substrate  and quantified using ImageJ Software.ΔGS construct was generated using Quikchange Site-Directed Mutagenesis to remove the GS domain (T189–Q207) from wild-type ALK2. Following Sanger sequencing to confirm the deletion, the ALK2ΔGS was shuttled using LR reaction (Invitrogen) from the pDONR221 vector to the pAWF vector, which carries a C-terminal 3xFLAG tag and an actin 5C promoter for constitutive expression in cell culture. Drosophila S2 cells were cultured under standard conditions . For each transfection, 8 × 106 cells (at 4.0 × 106/mL) were used. Cells were cotransfected with 700 ng pAC FLAG-Mad  and 300 ng of one of the following constructs: pAWF ALK2ΔGS, pAWF ALK2 or pAWF ALK2R206H ATP and casein polypeptide substrates, in addition to autophosphorylation  casein is perhaps the most commonly employed substrate for assaying protein kinases in vitro. In actuality, caseins serve well as surrogate Smad substrates since the proline-containing C-terminal tails of Smads, the site of activating phosphorylation by ALK kinases, is loosely structured and composed of motifs of neighboring serine and or threonine residues.Casein, a major component of milk, is made up of four different polypeptides, αIn keeping with the relative transphosphorylation activities quantitated previously by the ADP-Glo assays A, R206H In conclusion, with respect to heterotopic ossification caused by ALK2 R206H, elevated basal activity appears responsible for the relatively mild gain of function of the mutant kinase, but not the robustly activated, aspartate-substituted caALK2 form, which must achieve a high level of activity due to a different mechanism of action.Because of the heterogeneous nature of the casein polypeptide substrates, we hypothesized that increased levels of phosphorylation by the HLH-perturbed forms were due to an increase in the number of sites modified. That is, the increase was not with respect to rate, but accessibility to more sites or clusters of dephosphorylated serine or threonine residues along the unstructured polypeptides. Toward that end, we assayed increasing amounts of wild-type and R206H mutant kinases to confirm that the reactions had reached completion. We also determined whether the caseins were heterogeneous not only structurally but functionally, that is to say, whether the compositions contained numerous sites of varied accessibility. The latter question was addressed by utilization of the dedicated cellular enzyme, Golgi Casein Kinase, which phosphorylates secreted phosphoproteins such as those found in milk .With respect to the former question, as can be seen in Drosophila S2 cells was performed, comparing the levels of Drosophila Smad (Mad) phosphorylation after transient transfection with wild-type, R206H mutant and HLH-deleted (ΔGS) ALK2 forms. As shown in Because truncation of the negatively acting HLH regulatory element dramatically increased the level of surrogate substrate phosphorylation , the queIn conclusion, basal kinase activities alone were not correlated with gain of function. Deletion of the second helix of the HLH element is predicted to eliminate Smad interaction; thus, the truncated form, though highly active in vitro with casein substrate, does not signal detectably. Similarly, caALK2 was less active than the mutant and marginally more so than the wild type with casein, although it signals robustly and constitutively in vivo. In other words, protein–protein interactions in vivo within the assembled heterohexameric  signaling complex appear necessary, while increased phosphotransferase activity alone, i.e., independent of complex formation, was insufficient.Native polyacrylamide gel electrophoresis as an assay for kinase activities by means of trans-autophosphorylation was a convenient method employed early on and throughout our studies. However, the nature of the modification proved to be unexpected. Because the established site of ALK phosphorylation by the upstream constitutively active type II receptor kinases in cellular assays was the cluster of serine and threonine residues in the glycine–serine-rich (GS) loop of the helix–loop–helix regulatory segment, we anticipated that the ALK forms would be multiply transphosphorylated within the same site.To our surprise, only a single residue was phosphorylated, as indicated by precursor–product relationships with respect to time with fixed amounts of kinase . Mass spIn the case of wild-type kinase, the more negatively charged, singly phosphorylated product migrated at a rate comparable to that of the unmodified mutant, which had a lower isoelectric point due to the loss of an arginine residue which would be positively charged at the pH (8.3) of electrophoresis A. SimilaInterestingly, neither form was phosphorylated to completion, perhaps only about one-third of the wild type and one-half of the mutant and in each case terminating by 30 min, the earliest time point sampled. Of further interest, we found that the fractions of product to precursor increased in each case reproducibly with increased age of preparations. That is, about one-half of aged  wild-type kinase was phosphorylated and nearly all of the mutant was  the ALK2 kinase.cf. Toward understanding the nature of the changes at the active site of the kinase forms triggered by perturbation of the HLH regulatory element, we compared the affinities for the ATP substrate by determining Km values for the four ALK2 forms . The valIn conclusion, the HLH element is hypothesized to perform allosteric regulation by acting as a choke, constricting the active site of the wild-type enzyme, opening up slightly in the recurrent mutant and flooding with water when ablated by deletion. The hyperphosphorylation of caseins by the HLH-truncated form becomes mechanistically clearer in this context, consistent with the hypothesis of polypeptide accessibility as the basis for the gain of function. Mildly increased perturbation of the HLH regulatory element by the R206H point mutation in FOP would slightly release the choke, which in vivo would allow for somewhat increased accessibility relative to the wild type to the serine hydroxyls of BMP Smad C-terminal tails within non-canonically (activin) assembled cytoplasmic signaling complexes.As described in the introduction above, a cytoplasmic BMPRII kinase–ALK2 kinase heterodimer model B was alsA zoomed view of a ternary complex D shows tNote that for a stable heterohexameric signaling complex to form, a pair of type II kinase–ALK2 heterodimers must be brought into proximity, which in FOP, results from ACVR2A/ActRIIa extracellular domains (ECDs) binding activin A, even without ALK2 ECD binding, which is essentially nonexistent . Such coThus, FOP due to the recurrent R206H mutation is an allosteric disorder, resulting from mild loss of an inhibitory choke at the active site, not diminished binding by an inhibitory protein. Unexpectedly, the second simplest basis postulated initially in 2007—increased affinity for or interaction with the immediate downstream effector, the BMP Smads—appears to be the case; however, this is not due to enhanced binding to the proximal L45 loop specificity determinant ,37 but oGiven the juxtaposition between the two sites of interaction, structure-based  homology modeling indicated that the recurrent FOP mutation led to either a loss of FKBP12 inhibitory protein binding or gain of Smad substrate recruitment . Analyse33P]ATP autoradiography was not detected in assays that yielded robust autophosphorylation or phosphotransfer to caseins, the surrogate substrates. Pre-incubation of ALK2 kinase forms with SUMO-BMPRII fusion protein, which was active with respect to binding by native PAGE, also failed to yield detectable Smad-MH2 phosphorylation. A similar result with ALK2 kinase was obtained by Sapkota and coworkers in their analysis of the differential effects of the inhibitor LDN-193189 through autoradiography [As mentioned above, altered interaction with the substrate of the kinase, the loosely structured C-terminal tails of BMP Smad-MH2 domains, was concluded to be causative of FOP due to the recurrent FOP mutation. Ironically, enhanced Smad binding at the L45 loop of the folded core proximal to the R206H mutation was ruled out early on as the protein structural—functional basis of the gain of function. Phosphorylation of Smad-MH2 by [γ-iography . SpecifiAlthough diminished inhibitory protein binding was ruled out as causative of classic FOP, and enhanced Smad interaction initially so, we did explicitly find that alteration of the helix–loop–helix regulatory element, due to the recurrent mutation, the activating aspartate substitution or by total truncation, elevated basal kinase activity as judged particularly by phosphorylation of a casein surrogate. Disruption of the conserved ion pair between the HLH element and the N-lobe of the kinase by the R206H mutation led to a several-fold increase in the basal phosphotransfer activity relative to the wild type. This increase appears not to be due to rearrangement of the active site to increase the catalytic rate, but instead due to increased accessibility of ATP and polypeptide substrates, the latter either surrogate in the case of caseins, or putatively C-terminal BMP Smad MH2 tails in ligand-mediated, cooperatively assembled heterohexameric signaling complexes in vivo.The proposed mild loss of an inhibitory choke at the active site is consistent with the pathology of FOP. That is, due to the relatively benign effects in utero and the episodic nature of flare-ups of soft tissue, the functional consequences of the mutation must be rather minimal, such as a slight elevation in the activity of the unphosphorylated, autoinhibited enzyme. The level for the mutant is likely sufficiently close to a threshold, which in certain cellular contexts, such as inflamed tissue, allows for localized BMP signaling to extents and for durations that trigger FOP. Since the recurrent mutation is heterozygous and phenotypically dominant, resulting in the dysregulation of half of the ALK2 receptor pool, the modest effect on activity observed in vitro might actually be even weaker in vivo. Thus, of note, the inhibitory activity of a therapeutic need not be overly high or a dosage strategy overly intense to dampen and maintain total basal activity at sub-threshold levels in tissues of FOP patients presenting with classic FOP due to the recurrent R206H mutation in the HLH element.


Bacillus coagulans after 90 days of storage at -18 °C.Various sectors of the food industry demand the enrichment of food with functional compounds. Probiotic products with valuable nutritional and therapeutic properties have attracted great attention in the fields of industry, nutrition and medicine. The aim of the present study is to investigate the sensory and physicochemical properties of probiotic ice cream containing fig syrup and to evaluate the survival of 9 CFU/g B. coagulans, ice cream containing 25 % fig syrup as sugar substitute and ice cream containing 25 % fig syrup as sugar substitute and 109 CFU/g B. coagulans. They were stored at -18 °C for 3 months. Texture, pH, acidity and viscosity were analysed and microbial counts were determined after 1, 30, 60 and 90 days of storage. The organoleptic evaluation was carried out on days 1 and 90.In this study, four experimental groups of ice cream were produced as follows: plain dairy ice cream (without additives), ice cream containing 10B. coagulans decreased from 109 to 107 CFU/g, without significant changes observed over the 90-day period. No significant changes were found in the sensory and textural properties of the samples either. Replacement of 25 % sugar with fig syrup reduced the pH, increased the acidity of the ice cream and improved their viscosity. In conclusion, the production of functional ice cream using fig syrup and B. coagulans is recommended for their health benefits.The results showed that during the initial freezing process and the transformation of the mixture into ice cream, the number of The results of this study can be used to prepare functional and healthy foods. Our results suggest that fig syrup has the potential to be used as a natural sweetener or sugar substitute in various products. Then they were homogenised with a blender  and filtered with cheesecloth. Using a refractometer , the Brix value of the fig syrup was determined to 16° Brix at room temperature. According to the results of the HPLC analysis , 100 mL fig syrup with Brix 16° contained 6.09 g fructose, 5.10 g glucose and 0.44 g sucrose. According to their sweetness coefficients , the sucrose equivalent of fig syrup (100 mL) was 12.8. To make 1 kg of ice cream with 25% sucrose replacement by fig syrup, 351.5 mL of fig syrup are needed to achieve the same sweetness as sucrose.To prepare fig syrup, dried white figs  supplied us with all the ingredients needed to make ice cream, including skimmed milk powder , hydrogenated vegetable oil , commercial sugar , fig syrup as sweetener, vanilla , carboxymethyl cellulose , panisol , cellulose gum (E 410), guar gum (E412), carrageenan (E 407), tocopherol-rich extract (E 306) and ascorbyl palmitate . The probiotic B. coagulans was obtained from the Pardis Roshd Mehregan Company, Shiraz, Iran. The bacteria were activated by inoculation of the nutrient yeast extract salt medium (NYSM) broth culture at 37 °C for 24 h. The probiotic cells were centrifuged at 6000×g  and then washed in sterile saline using the same centrifugation procedure. The probiotic bacteria were inoculated into the ice cream. The amount of the cells was adjusted to 109 CFU/mL. The ice cream mixture was frozen at −4 to −5 °C and stored at −20 °C to harden , the water was measured with a scaled cylinder, then the milk powder was added and heated to 40–45 °C. The other ingredients were then added to the reconstituted milk. After pasteurisation , the mixtures were stirred at 45 °C for 5 min using a simple mixer , cooled to room temperature and ripened overnight at 4 °C. They were then fortified with vanilla and a probiotic strain before freezing. The mixtures were whipped in an ice cream machine  for 20 min at 52 rpm and frozen. The final products at a temperature of -5 °C were packed in plastic cups, hardened at -30 °C for 2 h and stored at -18 °C (The overrun (%) of the samples was measured using the following formula .For the determination of total titratable acidity, the sample was titrated with 0.1 M sodium hydroxide (NaOH) and a phenolphthalein indicator. The results were expressed as a percentage of lactic acid  at 25 °C.The hardness, cohesiveness and adhesiveness of the samples were determined using a CT3 4500 texture analyser (Brookfield Engineering Laboratories) equipped with a stainless steel cylindrical probe . Each sample was compressed twice to 50 % of its original height at a test speed of 2 mm/s.B. coagulans number of probiotic samples was counted on days 1, 30, 60 and 90 of storage. Serial dilutions of the samples were plated on nutrient yeast extract salt agar  plates, which were then incubated at 37 °C for 24 h.The Sensory analysis was carried out by a panel of 30 individuals, 15 females and 15 males, ages 19 to 60, all non-smokers and regular consumers of dairy products. The sensory attributes of the samples, including flavour, texture, colour and mouthfeel, were rated on a five-point scale, where 0 and 4 meant ’unacceptable‘ and ’really like’, respectively . Analysis of variance (ANOVA) was performed for the comparison of physicochemical parameters and probiotic counts among the different experimental groups. The Duncan’s multiple range test was used to determine significant differences. Non-parametric comparisons, including the Kruskal–Wallis test and the Mann-Whitney Bacillus coagulans did not significantly change the pH and acidity of the different types of ice cream. On the other hand, the addition of fig syrup to ice cream lowered the pH and an increased the acidity of the ice cream, which is consistent with the results of Salama , corn syrup, sucralose and maltitol have also been reported to reduce overrun  of ice cream . In the The viability of probiotic bacteria can be affected by their initial count, temperature, type of food carrier and storage conditions. Other parameters that may influence probiotic viability include probiotic strain, pH and freezing and thawing conditions  were rated above three points in all treatments. Storing the samples in the freezer for 90 days did not lead to any organoleptic complaints. Crumbly, weak, greasy or sandy texture was not observed in any of the treatment groups.The results of the sensory analysis are shown in et al.  were developed. The presence of these functional ingredients did not show any adverse effects on the physicochemical, rheological and sensory properties of the ice cream. The microbial population of B. coagulans in the ice creams remained above the threshold of 106 CFU/g even after 90 days of storage at -18 °C. Due to the health benefits of fig syrup and B. coagulans probiotic bacteria, the production and consumption of functional ice cream are recommended.The development of ice cream enriched with fig syrup and a probiotic such as


The inverted triangle configuration of the three cannulated screws is the classic fixation method most commonly performed for undisplaced femoral neck fractures in young and geriatric patients. However, the posterosuperior screw has a high incidence of cortical breach, known as an in-out-in (IOI) screw. In this study, we present a novel posterosuperior screw placement strategy to prevent the screw from becoming IOI.Using computed tomography data and image-processing software, 91 undisplaced femoral neck fractures were reconstructed. The anteroposterior (AP), lateral, and axial radiographs were simulated. To simulate the intraoperative screw placement process, participants used three screw insertion angles  to place the screw on the AP and lateral views of the radiograph according to the three established strategies. On the AP radiograph, a screw was placed abutting (strategy 1), 3.25 mm away from (strategy 2), or 6.5 mm away from (strategy 3) the superior border of the femoral neck. On the lateral radiograph, all the screws were placed abutting the posterior border of the femoral neck. Axial radiographs were used to evaluate the screw position.In strategy 1, all the placed screws were IOI regardless of the screw insertion angle. In strategy 2, 48.3% (44/91) of IOI screws occurred at a 0° screw insertion angle, 41.7% (38/91) of IOI screws occurred at a 10° screw insertion angle, and 42.9% (39/91) of IOI screws occurred at a 20° screw insertion angle situation. In strategy 3, no IOI screw occurred, and the screw insertion angles did not affect the safety and accuracy of screw placement.Screws placed according to strategy 3 are safe. The reliability of this screw placement strategy is unaffected by a screw insertion angle of less than 20 degrees. Percutaneous cannulated screw fixation is the most popular fixation method for femoral head preservation in young and geriatric patients with undisplaced femoral neck fractures. The inverted triangle configuration of the three cannulated screws is widely used and has been shown to have biomechanical advantages over other configurations . HoweverIn clinical practice, surgeons usually position and adjust the femoral neck screws under femoral AP and lateral C-arm fluoroscopy guidance. However, conventional standard anteroposterior (AP) and lateral radiographs are insensitive in assessing IOI screws . MultianFrom September 2018 to September 2021, pelvis computed tomography (CT) data of 91 consecutive patients with undisplaced femoral neck fractures treated in our hospital were collected. The baseline characteristics of the 91 patients are presented in First, pelvis CT data were imported into Mimics 20.0 software . To simulate a radiograph of the injured hip, the gray value in Mimics software's Volume Rendering module was adjusted. The intersection positioning line on the cross-section view was then rotated until the transverse line was parallel to the femur's head–neck axis. The standard AP simulated radiograph of the femur was obtained in this state . To simuIn the clinical setting, screws are typically inserted into the femur along but not exactly parallel to the head–neck axis. We simulated three screw insertion angles: 0°, 10°, and 20°. A 0° insertion angle is defined as the screw axis parallel to the head–neck axis . Screw aScrews were placed by two independent participants. Each participant was instructed to use three insertion angles  to place the screw on the AP and lateral views of the radiograph according to the three strategies listed below. All the three screw placement strategies were two-step placement strategies. Step one is to place the screw in the lateral radiograph, and step two is to adjust the screw position in the AP radiograph. On the lateral radiograph, the screw was all placed abutting the posterior border of the femoral neck in all three strategies . On the The circumscribed rectangle of the femoral neck safety zone was drawn on the axial view. The center of the circumscribed rectangle divided the femoral neck safety zone into four quadrants . The screw position was classified as excellent, acceptable, or poor based on the quadrant location and the cortical invasion. The simulated screw, which is located on the posterosuperior quadrant within 3 mm of the femoral neck cortex and has no cortex invasion, was classified as excellent . SimulatP value < 0.05 indicated statistical significance.Continuous variables are presented as the mean ± standard deviation. Categorical variables are presented as a total number plus percentage and were compared using the chi-squared test. All the screws were placed, and their corresponding positions were performed and evaluated by two independent reviewers. The assistance of a third reviewer judged the controversial categorical data. The interobserver reproducibility of screw placement strategies was assessed using Kappa coefficients. All screw placement strategies had Kappa coefficients greater than 0.75 . The repP = 0.1236)  of IOI screws occurring at a 0° screw insertion angle, 41.7% (38/91) of IOI screws occurring at a 10° screw insertion angle, and 42.9% (39/91) of IOI screws occurring at 20° screw insertion angle. There were no significant differences in screw placement among the three screw insertion angles under strategy 1 or 2. For screw placements at an insertion angle within 20°, strategy 3 is a reliable strategy for posterosuperior screw placement and for preventing it from being positioned IOI, with 95.6% of screws inserted at a 0° angle being excellent, 86.8% of screws inserted at a 10° angle being excellent, and 91.2% of screws inserted at a 20° angle being excellent. Under strategy 3, there were no significant differences in screw placement among the different screw insertion angles ( 0.1236) .Previous studies showed that the larger the cross-sectional area of the screw configuration, the better the stability of the internal fixation and the lower the risk of fracture nonunion , 7. ThusAlthough computed navigation techniques have advantages in accurate screw placement, conventional fluoroscopy is still the most common ancillary technique for percutaneous cannulated screw placement in femoral neck fracture –14. TradConcerning the AP and lateral radiographs that were insensitive to identifying the IOI screws, researchers are attempting to improve the safety assessment of the posterosuperior screw by using additional oblique radiographs. Terhune et al.  recommenThe limitation of this study was that patients with displaced femoral neck fractures with inadequate reduction were not evaluated. Posteromedial communications and malreduction also have an impact on screw placement accuracy. All of the CT data used in this study were obtained from Asians. Whether this screw placement strategy would be suitable for other ethnicities requires further investigation. In conclusion, to avoid the screw becoming IOI and to achieve cortical support, the posterosuperior screw should be placed more than 6.5 mm (diameter of the screw) away from the superior border of the femoral neck on the AP radiographs and abutting the posterior border of the femoral neck on the lateral view, and within a screw insertion angle of 20°, which rarely affected the accuracy of this strategy. Using our strategy, AP and lateral radiographs were sufficient in guiding and evaluating screw placement in patients with femoral neck fractures.


Conclusion: Dialkylcarbamoylchloride dressing is well-tolerated in paediatric and neonatal population. It represents a promising tool as a strategy for infection prevention.Dialkylcarbamoylchloride dressing is a fatty acid derivative that has been shown in vitro to bind a number of pathogenic microorganisms. The purpose of this prospective study was to evaluate the safety and the efficacy of this technology in the care of the exit site of central venous catheter in a paediatric and neonatal population. Methods: The study was conducted from September 2020 to December 2022 at the Infermi Hospital in Rimini. Central venous catheters were placed using the SIC bundle for insertion. Dialkylcarbamoylchloride dressing was placed below the subcutaneous anchoring at the time of CVC placement and at each dressing change. Data about the catheters and the exit site were recorded and then compared with an historical cohort. Results: 118 catheters were placed during the studied period. The dialkylcarbamoylchloride dressing was well-tolerated. No case of systemic or local infection was recorded. The comparison with the historical cohort showed a reduction in the rate of exit site infection ( Venous access is critical in the care of paediatric and neonatal patients. Venous access devices (VADs) can be divided into central devices  or peripheral devices (tip of the catheter in any other location). Central venous catheters (CVCs) should be distinguished according to the recent terminology suggested by the WoCoVA Foundation into PICCs = Peripherally Inserted Central Catheters, or CVCs inserted in the deep veins of the arm ; CICCs = Centrally Inserted Central Catheters, or CVCs inserted in the supraclavicular veins  or infra-clavicular ; and FICCs = Femoral Inserted Central Catheters . PICCs, CICCs and FICCs are indicated for the infusion of solutions incompatible with the peripheral route, for repeated and frequent blood sampling and for hemodynamic monitoring ,2.CVCs should be placed without any exception with the use of US guidance as recommended by current guidelines . SeveralIn the choice between PICC, CICC and FICC it is very important to consider the diameter of the vein throughout a preoperative ultrasound scan. The current international guidelines—such as the WoCoVA-GAVeCeLT-WINFOCUS consensus —suggest The main contraindications to the placement of a PICC are the presence of a chronic renal failure of grade 3b−4–5 or the bilateral presence of pathologies affecting the upper limbs. In newborns or in paediatric patients, when placement of a PICC is not feasible for the size of the available veins, CICC represents the best choice. However, independently from the type of VAD placed, the care of the exit site after the insertion procedure is the most important thing to be considered in order to minimize complications such as infection, catheter dislodgment and venous thrombosis.The main purposes in the care of the exit site are: (1) the prevention of extraluminal colonisation, which might lead to a systemic infection (Catheter-Related Blood Stream Infection (CRBSI)); (2) the maintenance of the skin integrity, thus preventing medical adhesive risk injury (MARSI) ; (3) theInfections may occur as infections at the exit site or infections of the tunnel or invasive infections (CRBSI). The latter is a severe and possibly life-threatening complication. The treatment of CRBSI depends on several factors, including the microorganism, the type of the vascular device itself, the availability of other vascular access devices for antibiotic administration and so on; however, the removal of the catheter in such cases is a quite often necessary ,13,14.Dialkylcarbamoylchloride (DACC) is a hydrophobic fatty acid that can be used to coat the material of a dressing, thus giving hydrophobic properties to the dressing itself. Microorganisms are, therefore, irreversibly bound to the surface of the dressing using hydrophobic interactions ,16. The Our hypothesis is that the use of DACC dressings for the management of the exit site allows for reduction of the bacterial load at each dressing change, since the microorganisms can be removed from the exit site along with the dressing itself. No antiseptic or antibiotics are involved in this process, therefore there is no theoretical risk of bacterial resistance .The aim of this study is to evaluate the feasibility, the safety and the skin tolerance of DACC dressings in the management of the exit site of tunnelled and non-tunnelled CVC in a paediatric and neonatal population. The secondary aim is to estimate the risk of infection compared with an historical cohort.p TAV) that is responsible for the placement and management of CVCs in the paediatric and neonatal population. All CVCs are inserted according to the SIC Ped protocol [Preprocedural ultrasound evaluation  ,12,30Real time ultrasound guided venepuncture Intra-procedural verification of central position of the tip by non-invasive methods (IC-ECG and RT-US) ,32,33Tunnelling according to RAVESTO (Rapid Assessment of Venous Exit Site and Tunnelling Options) Sutureless securement of the catheter or subcutaneous anchoring system ,36,37Protection of the exit site with glue ,39 and sThe study was conducted at the Infermi Hospital in Rimini from September 2020 to December 2022. In our hospital, there is a Neonatal/Paediatric Vascular Access Team  . Fully implantable catheters and central venous catheters stabilized only with tissue adhesive and other sutureless securement were excluded from the study. No antibiotic prophylaxis was used at the time of CVC placement.In our population, a DACC  dressing was placed around the exit site and below the subcutaneous anchoring system (SAS). The DACC dressing (sterile packaged) includes a four-layer sheet in pre-cut DACC of 4 × 2 cm to be placed in direct contact with the patient’s skin and a small non-woven gauze of the same size, also pre-cut to be placed immediately above the DACC dressing and under the subcutaneous anchoring system See .removal of the old dressing (transparent semipermeable membrane) and the DACC dressingcutaneous antisepsis around the exit site with chlorhexidine 2% gluconate in 70% isopropyl alcohol (PAH), using single-dose, disposable and sterile applicators application of a new DACC dressing and a transparent semipermeable membrane with high moist-vapour transmission rate The dressing change was then performed weekly according to international guidelines ,31. In pThe aforementioned four-layer DACC leaflet is green in colour and allows a constant indirect inspection of the exit site. In fact, if there are signs of initial infection of the exit site, the dressing changes colour, turning from green to dark brown and then to black. The non-woven gauze is mainly used as an anti-decubitus of the SAS, protecting the fragile skin of paediatric patients from the continuous and constant pressure of the SAS.Data recorded during the study period included the number of CVC placed, the number of catheter days, exit-site infections reported as a number per 1000 catheter days, CRBSIs reported as a number per 1000 catheter days, local skin injuries, number of dressing changes, reason for catheter removal, main catheter-related complications  and baseline characteristics of the population . All data were collected in the electronic database and then compared with a historical cohort of CVCs inserted from September 2018 to September 2020, before DACC dressing was introduced in our daily practice.https://www.medcalc.org; accessed on 26 March 2023). Continuous variables were analysed using Student’s t test or Mann–Whitney U test according to their distribution. Categorical variables were analysed using Fisher exact test. A p value < 0.05 was considered statistically significant.Statistical analysis was performed using MedCalc Statistical Software , 38 peripherally inserted central catheters (PICCs), 4 femoral inserted central catheters (FICCs). 30 CVCs were placed in the neonatal population (23 CICCs and 7 FICCs). Indications for CVC placement were oncologic condition (82%), parenteral nutrition (12%), prolonged antibiotic treatment (4%) and others (2%). Of the 118 CVCs placed, 92 catheters were tunnelled according to the SIC protocol. Mean age of the studied population was 6.8 years. Mean dwell time of the CVCs was 100 days, for a total of 11,151 catheter-days. 1721 dressing changes were performed during the studied period according to our protocol see . DACC drIn the historical cohort, 86 devices were placed and analysed. 39 CICCs, 25 PICCs and 3 FICCs were placed in the paediatric population, and 13 CICCs and 6 FICCs were placed in neonatal patients see . Of the A solid insertion bundle such as the one used in the present study, which includes, among others, the proper choice of the exit site, maximal barrier precautions, the use of 2% chlorexidine in 70% isoprohyl alcohol, the use of SAS, hand hygiene and cyanoacrylate glue at the time of CVC placementA proper policy of dressing changeCRBSI is the one of the most important complications related to VADs, since it is associated with high morbidity, loss of the device itself, failure of therapy administration, interruption in the care of patients and increased healthcare costs . The diaUse of needle-free connectors and port protectors, also known as “disinfecting caps”Hand hygieneProper protocol for changing of the administration setsProphylactic lock with taurolidine was also considered in children at high risk for CRBSI, especially if their venous patrimony is scarce and if the intravenous treatment is expected to last for a prolonged time In addition, the best available evidences to minimize intraluminal contamination have also been applied in the present study, including :Use of nHowever, not all catheter-related infections are necessarily systemic infections. Sometimes, local infection may occur, such as exit-site infection or tunnel infection, which are usually the results of an uncontrolled bacterial contamination of the skin around the VAD. These conditions might become relevant, since an exit-site infection of a non-tunnelled VAD could quite easily become a CRBSI. On the other hand, when a tunnel infection occurs the removal of the device is inevitable. Exit-site infection diagnosis can be performed by inspection and palpation: in fact, the presence of redness and tenderness of the exit site is highly indicative of a local infection . The treProper choice of the exit site. The exit site of a central VAD should be far from areas with high bacterial contamination, such as mouth, urethra, tracheostomy, ileostomy and so on. In addition, the stability of the area should be considered in the choice of the exit site, since an unstable area might be the cause of micro-movements of the device, which increases the risk of extraluminal contamination . The besProper hand hygiene. Every manipulation of the VADs should be preceded by proper hand hygiene.Skin antisepsis. During VAD insertion and at each dressing change, the skin should be disinfected using 2% chlorhexidine in 70% isopropyl alcohol with a single sterile applicator ,42,43.Maximal barrier precautions  as recommended by all guidelines including the recent SHEA published in 2022 .Cyanoacrylate glue. The use of Cyanoacrylate glue during CVC insertion is nowadays part of several insertion bundles ,39. The Securement using sutureless devices. In this regard, SAS must be applied at the time of insertion, and can stay in place as long as the catheter is needed without any routine replacements. Even though SASs are more expensive than other sutureless devices, they are cost-effective, especially when the risk of accidental dislodgment is estimated to be high; this is particularly true in the paediatric population. Recent clinical studies have demonstrated the high effectiveness of SASs in preventing dislodgment in neonates and children, as well as their safety since SASs are well-tolerated even with the fragile skin of preterm neonates ,36,37.A good, transparent, semipermeable membrane. The exit site must be covered and protected with a transparent semipermeable membrane ,12,42 whWeekly dressing change. Lastly, the exit site is protected throughout a weekly change of the exit site dressing.It is, therefore, clear that the prevention of exit-site infections is important to prevent CRBSI and is mainly based on strategies aiding the minimization of extraluminal contamination:It is clear that the effective management of the bacterial load at the exit site is particularly important as a strategy for infection prevention. Good care of the exit site might have an impact on the dwell time of the catheter itself and on patients’ comfort. The DACC technology is a complete novelty in this field, since it can bind bacteria rather than kill it in situ, thus preventing the release of potentially harmful substances (such as endotixin) that might damage the surrounding environment ,52,53,54p value 0.03).Our data suggest that the placement of a DACC dressing below the SAS is completely safe, even in the neonatal population. In the studied population, there was no case of local or systemic infection and no case of skin intolerance. The comparison with the historical cohort proves that the use of DACC dressings might have an impact as a strategy for infection prevention. In fact, in our population, there was a statistically significant reduction in the incidence of exit-site infections (The strengths of this work are: (1) the large population, (2) the solid insertion bundle that was applied through the whole study  and (3) the low rate of infection.The main weaknesses of the present work are: (1) this is not a randomized controlled trial  and (2) catheters stabilized without SAS were excluded.However, the preliminary results of the present paper might represent the basis for a larger randomized study aimed at evaluating the safety and efficacy of the dialkylcarbamoylchloride dressing compared to current, already standardized exit-site dressing techniques, such as cyanoacrylate glue ,51 and/oTotally safe in all patients (including newborns)Easy to be handled during dressing changesValid diagnostic tool for any change at the exit site levelBroad compatibility with other exit site management productsOur experience comes from the need to manage the exit site effectively, simply and cleanly. In fact, the DACC dressing comes pre-cut in sterile packaging in order to facilitate easy operation as much as possible. The DACC dressing might offer the following advantages:DACC dressings are easy to use and well-tolerated by paediatric and/or fragile skin patients and neonates. Their routine use could potentially have an advantage. both in terms of preventing CVC-related infections and in terms of reducing the healing time of any infections localized at the exit site. They represent a promising tool for infection prevention.


Background: The resorption of magnesium-based alloy bioabsorbable screws results in the release of hydrogen gas, which can mimic infection and enter the growth plate. The screw itself and the released gas may also affect image quality. Objective: The evaluation of magnetic resonance imaging (MRI) findings during the most active phase of screw resorption is the objective, with particular focus on the growth plate and to assess for the presence of metal-induced artifacts. Material and Methods: In total, 30 prospectively acquired MRIs from 17 pediatric patients with fractures treated with magnesium screws were assessed for the presence and distribution of intraosseous, extraosseous, and intra-articular gas; gas within the growth plate; osteolysis along the screw; joint effusion; bone marrow edema; periosteal reaction; soft tissue edema; and metal-induced artifacts. Results: Gas locules were found in the bone and soft tissues in 100% of the examinations, intra-articular in 40%, and in 37% of unfused growth plates. Osteolysis and the periosteal reaction were present in 87%, bone marrow edema in 100%, soft tissue edema in 100%, and joint effusion in 50% of examinations. Pile-up artifacts were present in 100%, and geometric distortion in 0% of examinations. Fat suppression was not significantly impaired in any examination. Conclusions: Gas and edema in the bone and soft tissues are normal findings during the resorption of magnesium screws and should not be misinterpreted as infection. Gas can also be detected within growth plates. MRI examinations can be performed without metal artifact reduction sequences. Standard fat suppression techniques are not significantly affected. Fractures are among the most common causes of presentation in pediatric emergency departments ,2. CertaA characteristic feature of magnesium-based alloy screws during resorption is the release of hydrogen gas into the bone and adjacent soft tissues. The gas can lead to alterations in the adjacent tissues that may be misinterpreted as soft tissue infection or osteomyelitis. In the long term, the gas may possibly also impair bone growth if it enters the growth plate. Currently, little is known about the appearance of bone and soft tissue alterations due to screw resorption on magnetic resonance imaging (MRI) in children ,22,23. IThe purpose of this single-center prospective study was to evaluate the screw resorption-induced alterations visible on MRI during the most active phase of resorption, i.e., during the first four months after implantation of the screws, whether gas can be detected in the growth plate, and to familiarize radiologists and clinicians with these findings in order to avoid inadvertently misdiagnosing a soft tissue infection or osteomyelitis .A prospective study was conducted at a single pediatric hospital over a 16-month period between November 2020 and February 2022. All patients included in the study sustained dislocated fractures of the elbow or tibial tuberosity. Patients and/or their parents could voluntarily choose between treatment with conventional titanium screws and resorbable magnesium-based alloy screws.Those who opted for magnesium screw treatment were invited to participate in this prospective radiological study. Ethics approval was obtained from the local ethics committee. Informed consent was obtained from the parents or patients (if 14 years of age and older). The ethics committee allowed a maximum of two MR examinations per patient as part of the radiological study and no MRI in patients younger than 7 years. Open fractures were not treated with magnesium screws. Demographic data, date of surgery, and clinical data to exclude signs of infection were obtained from the institutional patient database.® CBS or CS, Syntellix AG, Hannover, Germany) were used to fix the fractures. The diameter of all solid CBS screws was 3.5 mm and that of all hollow CS screws was 4.8 mm. Screw lengths were chosen according to bone size. The surgeons decided which type of screw to use. The alignment of the fracture fragments and drilling of the screw hole were performed with conventional metal tools.The surgeries were performed by two pediatric trauma surgeons with 20 years (T. K.) and 13 years (C. M.) of experience, respectively. Bioabsorbable magnesium-based compression screws alloyed with Yttrium  using a 4-channel flex coil. Sequences and parameters were adapted from a standard clinical routine elbow protocol. A T2-weighted true fast imaging with a steady-state free precession (TRUFI) 3D sequence with water excitation (we) fat suppression, performed in a transverse plane, aligned with the humerus/tibia, was additionally performed to improve gas detection due to the high spatial resolution of the sequence and due to gas causing blooming artifacts on the standard gradient echo technique. Detailed scan parameters are shown in The elbows were placed along the side of the patient, keeping the region of interest in the isocenter of the scanner. All elbows were in a flexed position, with some still in a cast. When the knee was examined, the knee was also placed in the isocenter of the scanner. No sedation was used. The sequences were oriented along the long axis of the humerus or tibia. No metal artifact reduction was performed. Radiological image files were stored in the hospital’s picture archiving and communication system .All MRI examinations were individually evaluated by two pediatric radiologists with an overall experience of 12 years (S. W.) and 7 years (S. M.), respectively. The examinations were assessed independently, blinded, and in a randomized order.Gas: The bubbly or grape-shaped gas components showed a signal void on all sequences ,27,28. TOsteolysis: Similar to previously published studies with titanium screws, a well-defined, hyperintense, linear signal in fluid-sensitive sequences directly adjacent to the screw shaft was interpreted as fluid collection/osteolysis . Its maxBone marrow edema: Bone marrow edema was defined as a hyperintense signal on fluid-sensitive sequences and an intermediate or low T1 signal compared to normal bone marrow ,30. A diPeriosteal reaction: A line of low signal intensity separated from the underlying cortex by a layer of high signal intensity fluid was interpreted as ossified periosteum ,32 FiguA,B.Soft tissue edema: Soft tissue edema was defined as a reticular, hyperintense signal on fluid-sensitive sequences and a hypointense signal on T1-weighted sequences in the subcutaneous fat and superficial fascia ,33,34. TJoint effusion: Joint effusion was defined as increased intra-articular fluid, with the distinction between a normal amount of joint fluid and a pathological effusion being subjective, or if the anterior or posterior fat pads were raised in the case of elbow fractures  Figure A,B.Metal-induced artifacts: MR images were evaluated for the presence of typical metal-induced artifacts generated by the screws . Signal pile-up is a curvilinear hyperintense signal that typically follows the contour of the screw and alternates with bands of signal loss, which is well seen in gradient-echo sequences . GeometrSignal pile-up and geometric distortion artifacts were assessed using a 4-point Likert scale: no artifacts, minimal artifacts, artifacts impairing diagnostic validity, and no interpretation of surrounding tissue possible. The 4-point Likert scale for fat suppression consisted of the following items: fat suppression not impaired, slightly impaired, heavily impaired but suppressed areas still present, and no fat suppression at all .If two screws were placed, then of all the parameters described above, the more pronounced finding  was included in the evaluation.Given the descriptive nature of this study, and the small number of replicates per patient, we treated each MRI examination as an independent observation. Whenever at least one reader scored a feature of interest to be present, it was recorded as such. Inter-reader agreement was expressed as the number of features for which both readers agreed/the total number of features (n/N), and additionally by Cohen’s kappa for categorical variables. For the only continuous variable of interest (osteolysis), we report the intraclass correlation coefficient (ICC) instead. Whenever the calculation of Cohen’s kappa was not possible (due to perfect agreement), or compromised (due to empty rows/columns in the confusion matrix), we had to fall-back on only reporting agreement in terms of n/N. All statistics and graphs were generated using the ‘pastecs’, ‘psych’, and ‘ggplot’ packages in R version 4.2.1 ,38,39,40In total, 30 MR examinations from seventeen pediatric patients  were available for this study. In the following, percentages have been rounded to the nearest whole number.Gas locules in the bone adjacent to the screw were found in thirty examinations (100%), and without contact to the screw in seven (30%) examinations A. The grOsteolysis adjacent to the screw thread was present in 26 (87%) examinations, with a mean width of 1.5 mm (range 0.60–3.40 mm).Bone marrow edema adjacent to the screw was found in thirty (100%) examinations, and not in continuity with the edema adjacent to the screw in three (10%) examinations A.A periosteal reaction was found in 26 (87%) examinations B.Soft tissue edema was found on the same side as the screw head in 30 (100%) examinations, and on the contralateral side of the screw head in 10 (33%) examinations C.Joint effusion was found in 15 (50%) examinations D.Pile-up artifacts were found in thirty (100%) examinations, of which twenty-six (87%) were minimal, and four (13%) were impairing diagnostic validity. Geometric distortion artifacts were found in zero (0%) examinations.Chemically selective saturation (CHESS) fat suppression was not impaired in twenty-nine (97%) examinations and was slightly impaired in one (3%) examination. STIR fat suppression was impaired in zero (0%) examinations. The DIXON fat suppression technique was not impaired in twenty-six (87%) examinations and was slightly impaired in four (13%) examinations. Water excitation fat suppression was not impaired in twenty-one (70%) examinations and slightly impaired in nine (30%) examinations.Inter-reader agreement for gas locules : Cohen’s kappa was moderate for gas locules in the growth plate (0.744), and intra-articular (0.545) and perfect for gas locules in the bone and in the soft tissues . An overIn this prospective, descriptive study, we have shown the findings typically seen on MRI in the early active stages of resorption of magnesium-based alloy bioabsorbable screws in a pediatric population. We believe that resorbable screws will be increasingly used in pediatric trauma in the future. This young population of patients may require additional imaging, for example, if they have persistent pain or a limited range of motion in the affected joint, and therefore will be examined by MRI due to its lack of radiation. Radiologists and clinicians should thus be familiar with the findings typically seen on MRI in patients with magnesium screws to avoid misdiagnosing pathologies  where there are none, which may ultimately lead to unnecessary revision surgery and intervention.In the growing skeleton, the osteosynthesis material is removed in a second surgery after the fracture has healed to avoid interference with growth. Resorbable magnesium screws eliminate the need for a second surgery. Resorption of the screw begins as soon as it comes into contact with chloride ions of the body fluids, which leads to the release of hydrogen gas. To ensure that resorption is not too rapid and excessive, magnesium is alloyed with rare-earth elements, such as yttrium (as in our case) or gadolinium ,47,48,49It has been shown that the radiographic findings of screw resorption may resemble those of osteomyelitis or soft tissue infection .Gas: It is known from radiographs in humans, μCT scans in animals, and a recent MR study in adults that hydrogen gas formation is particularly pronounced in the first few weeks and months and then decreases ,49,50,51Growth plate/growth disturbance: Gas within the growth plate was detected in 37% of the MR examinations with unfused growth plates. This is of particular concern in children, as it could potentially lead to growth disturbances. Kraus et al.  showed iOsteolysis: New bone is formed adjacent to the screw, but as shown in an animal study, lacunae are also formed ,56. ThesBone marrow edema: In our study, bone marrow edema was seen in 100% of the MR exams. The exact cause of bone marrow edema is not yet clear, but it may be a result of screw resorption. Hydrogen released during screw resorption may react with chloride ions in body fluids and thereby increase the pH value around the screw. The local increase in pH can result in a change of proton content, which in turn affects the MR signal ,57. ThisSoft tissue edema: Soft tissue edema adjacent to the screw head was visible in all examinations (100%) and in almost one-third (30%) on the contralateral side. We suspect the same causes as for the bone marrow edema.Periosteal reaction: A periosteal reaction is very common in children during fracture healing and is not a sign of infection or a finding caused by screw resorption . In our Artifacts: The magnetic properties of human tissue and metal screws differ significantly, resulting in local inhomogeneities of the magnetic field . The masSignal loss is limited to the screw itself and the screw edges are relatively sharply defined . In our The kappa values seemed to be poor for some features, despite quite good n/N values . This was due to the presence of empty rows/columns in the confusion matrix. Kappa attempts to correct for how often readers would agree by chance alone, and in order to do so, it must, among other things, calculate the row/column totals of the confusion matrix. If one of these is zero (or has very few observations), kappa’s estimates become very pessimistic. This is a general problem with all interobserver reliability statistics: their interpretation is very subjective, and easily compromised, especially in smaller datasets.Not all of the findings reported here are specific to alterations associated with magnesium screw resorption. Bone marrow edema, joint effusion, osteolysis, and periosteal reaction may also be seen in fractures that were not treated or were treated with conventional screws ,60,61,62This study has some limitations. Firstly, the number of patients and MR examinations was small, in part because it was difficult to motivate patients and their parents to partake in these optional MR examinations. Secondly, two different types of screws (hollow and solid) were used. Since we did not expect a significant difference in the findings between the screws and there were only very few cases with hollow screws, we did not separate the results by screw type. Thirdly, each MRI exam was considered individually. However, the majority of patients had two examinations, so the data are clustered. Fourthly, susceptibility artifacts may be caused not only by gas locules but also by metal abrasion, as is often seen in the bone and soft tissue adjacent to the surgical access . TherefoEdema and gas in the bone and soft tissue adjacent to resorbable magnesium screws are normal and should not be misinterpreted as infection. Gas may also be seen in growth plates. Its significance with respect to growth remains to be determined in future studies. With the exception of pile-up artifacts, resorbable magnesium screws do not cause significant artifacts. In patients with magnesium screws, MRI examinations can be performed without the need for special metal artifact reduction sequences. Standard fat suppression techniques are not significantly affected.


Pedicle screw fixation (PSF) has been the standard therapy for the treatment of various spinal diseases. Although complications are identified regularly, iatrogenic vascular injury is one of the rare but life-threatening complications. In this literature, we describe the first case of inferior vena cava (IVC) injury during pedicle screw removal.A 31-year-old man was treated by percutaneous pedicle screw fixation for an L1 compression fracture. After a year, the fracture healed well and hardware removal surgery was performed. During the procedure, the hardware on the right was removed unremarkably except for the L2 pedicle screw which slipped into the retroperitoneum because of the improper technique. The CT angiogram revealed the screw had breached the anterior cortex of the L2 vertebral body and penetrated the IVC. After multidisciplinary cooperation, the defect of IVC was reconstructed and the L2 screw was removed from the posterior approach in the end.The patient recovered well and was discharged after 3 weeks without further events. The removal of the contralateral implants was unremarkable at 7 months postoperatively. At the 3-year follow-up, the patient returned to his normal daily activity without any complaints.Although pedicle screw removal is a rather simple procedure, severe complications may have occurred from this procedure. Surgeons should keep vigilant to avoid the complication noted in this case. The pedicle screw fixation (PSF) has been the standard therapy for the treatment of various spine diseases . HoweverHardware removal is the most commonly performed orthopedic surgery, the procedure carries risks of unexpected complications , 7. AccoHere, we present a case of inferior vena cava (IVC) injury secondary to the removal of the pedicle screw following percutaneous PSF in treating a thoracolumbar fracture.A 31-year-old man was treated by percutaneous PSF for an L1 compression fracture. The implant was required to remove one year after the initial surgery without sign of hardware failure or loosening, and bone healing was achieved according to radiography .Hardware removal surgery was performed in a prone position. During surgery, the caps on the right side was firstly loosened using a remover, and the caps and the rod were removed sequentially, then the pedicle screws of the T12 and L1 were removed unremarkably using the proper screwdriver. However, the removal of the right L2 pedicle screw was unsuccessful. With a limited view of minimal invasive incision, the screwdriver has never been able to fully fit the tail of the screw. After multiple attempts, the screwdriver seemed to fit. To enhance the sense of fit, the surgeon put forwarding pressure on the screw and rotated the screwdriver in a clockwise direction(in a wrong manner). Even worse, he suddenly felt that the screw was pushed into the retroperitoneum.Intraoperative fluoroscopy was taken and confirmed that the screw migrated anteriorly to the vertebral body . An endoThe patient was hemodynamically stabilized by fluid resuscitation. Subsequently, a laparotomy was performed in a supine position by a vascular surgeon. The circumference of the IVC wall was evaluated. It was identified that the IVC was traversed, and the bilateral renal veins were also contorted by the screw. After carefully separating the blood vessels from screw, it was found that the IVC was completely punctured and there was a severe crush injury at the transition between the renal vein and the IVC. Vascular control above and below the level of injury was performed, and the screw was slowly backed out . FinallyImmediately following the abdomen incision was closed, the L2 screw was then removed from the posterior approach . ConsideHe recovered well and was discharged from our institution after 3 weeks without further event. At 7 months postoperatively visit, the removal of the contralateral implants was done with unremarkable. At the 3-year follow-up, the patient returned to his normal daily activity without any complaints. The diagnosis and treatment process of patient were showed in PSF has developed into the standard therapy for the treatment of spine diseases . After tHardware removal remains a controversial topic in spine surgery after a successful fusion . Smits eAlthough whether to remove the pedicle screw was controversial, the procedure was still frequently performed. Its associated unexpected complications were occasionally reported. Crasto et al. reported a case of pneumothorax secondary to the broken bolt cutter jaw entering the pulmonary cavity during removal of hardware performed on the thoracolumbar spine, which resulted in the patient death . VanichkIatrogenic IVC injury is a rare but life-threatening complication. To our knowledge, there was only three pedicle screw related IVC injuries reported in the literature. Wang et al. reported a case of IVC tear which occurred during posterior spinal fusion surgery . The patin situ without removal is also an alternative option in certain circumstances. lin et al. reported a case in which the vascular replacement surgery was performed without removing the screw for the stability of the spine (Injury of the vessel was associated with a mortality rate high of 50% due to its catastrophic hemorrhage occurred . CTA or he spine . A similhe spine . In addihe spine .Although numerous cases of large vascular injury by implant have been listed, sparse literatures regarding the systematic management tactics for this extremely condition , 22–24. This present case suggested that although pedicle screw removal was a rather simple procedure, serious complications may arise from this procedure. Surgeons should keep vigilant to avoid the fatal complication noted in this case. Once it occurred during operation, an emergent diagnostic imaging scanning and an appropriate management approach are mandatory to make the best clinical decisions.


We employ the Tight Binding Fishbone-Wire Model to study the electronic structure and coherent transfer of a hole (the absence of an electron created by oxidation) in all possible ideal B-DNA dimers as well as in homopolymers . The sites considered are the base pairs and the deoxyriboses, with no backbone disorder. For the time-independent problem, we calculate the eigenspectra and the density of states. For the time-dependent problem after oxidation , we calculate the mean-over-time probabilities to find the hole at each site and establish the frequency content of coherent carrier transfer by computing the Weighted Mean Frequency at each site and the Total Weighted Mean Frequency of a dimer or polymer. We also evaluate the main oscillation frequencies of the dipole moment along the macromolecule axis and the relevant amplitudes. Finally, we focus on the mean transfer rates from an initial site to all others. We study the dependence of these quantities on the number of monomers that are used to construct the polymer. Since the value of the interaction integral between base pairs and deoxyriboses is not well-established, we treat it as a variable and examine its influence on the calculated quantities. The purpose of this work is to establish the main features of the Fishbone-Wire Tight Binding (TB) model for B-DNA dimers and homopolymers. The Fishbone-Wire Model (FWM) and the Fishbone-Ladder Model for DNA were introduced a few years ago now. In Ref. , the autFishbone-like models are named as such because the backbone sites are not connected to each other, yielding a fishbone shape see . FishbonAdditionally, a fishbone-like model was used in Ref.  to studyhopping integral or transfer integral or even transport integral or parameter are some of the terms used for quantities of the form interaction integral or parameter—this is the term we are going to use throughout the text.The Even though the purine (guanine or adenine)–deoxyribose connection is not expected to be identical with the pyrimidine (cytosine or thymine)–deoxyribose connection, in this article, we make the simplification that the interaction integral between the base pairs and deoxyriboses, The article is organized as follows: In A depiction of our FWM for DNA is provided in We study all possible dimers and homopolymers. This work is a fishbone-wire extension of the wire model of Refs. ,18, in tH. For, example, the Hamiltonian matrix of a dimer has the formThe Hamiltonian describing the Highest Occupied Molecular Orbital (HOMO) or the Lowest Unoccupied Molecular Orbital (LUMO) regime of a given DNA polymer within the FWM isi by i and j by For base pairs, we denote the on-site energy of base pair  in Ref. .The state, Plugging Equation  into theMultiplying Equation  by 〈λ1| Given that, within the TB approximation for the FWM, Similarly, multiplying Equation  by 〈λ2| Given that Finally, multiplying Equation  by 〈λ3| Given that For example, for a dimer  an anλ=1,2)quations , we obtaH is the Hamiltonian matrix and The time-independent problem expressed in Equations , 10), a, a10), aIn other words, we have to solve an eigenvalue–eigenvector problem.E, k is a collective generic eigenenergy index. In our FWM, for N monomers, we have The DOS, N, discrete energy levels occur. However, as N increases, the eigenenergies are gathered into subbands. The DOS diagrams demonstrated in N (=2000). We notice that the persistence length of DNA is ≈150 monomers, and so our quasi one-dimensional model is not really adequate for longer distances. Additionally, we are dealing with coherent phenomena, which are expected to dominate at small distances. The main reason for using large N for DOS diagrams is to obtain smooth images. The number of subbands is equal to the number of sites within the repetition unit. For homopolymers, a monomer  is composed of three sites; hence, three subbands are obtained. The states are distributed in energy regions close to the on-site energies of base pairs (A–T or G–C) as well as to the deoxyribose on-site energy.For a small number of monomers, The state, Plugging Equation  into theMultiplying Equation  by 〈λ1| Similarly, multiplying Equation  by 〈λ2| Finally, multiplying Equation  by 〈λ3| The time-dependent problem expressed in Equations , 23), a, a23), aH is the Hamiltonian matrix. Equation , then it can be shown that the vector matrix Equation  can be sEquation  leads toEquation , i.e., uEquation  is(28)CLet us now suppose that, initially, the extra carrier is placed at site In other words, the coefficients From Equation , it follm, then the number of different m would be N degenerate eigenvalues equal to cyclic homopolymers within the wire model  or numex- and y- axes are defined so that y-center is always at x-axis); due to the symmetry of our FWM, no dipole moment occurs across the molecule, cf. also y-axis); there is also some activity along the macromolecule, but the carrier basically remains at the negative x- region, i.e., mainly at x corresponds to the first half of the homopolymer, cf. The character of these oscillations is better revealed in N for five values of N increases, i.e., as the fishbone chord becomes longer. Moreover, when increasing N and In N for initial carrier placement at sites N. The decrease in k to sites of the last monomer is more acute.In On the left panels (initial oxidation at the first base pair, N and Similarly, although less pronounced, all rates involving the last monomer, i.e., k values of the triplet of each monomer are closer than for In r is the traveled distance, or N monomers, the number of steps is In experiments, it is common to fit transfer rates using the past ,20,21. HIn the experiments of Ref. , the autIn For all dimer types, for initial oxidation at the first base pair (re model . As tS iers cf., , initialRegarding the frequency content of hole transfer, increasing ers cf., , for iniOn the contrary, for initial placement at a deoxyribose, all rates increase with We studied the electronic structure and the hole-transfer properties of DNA homopolymers and of all types of dimers within the fishbone-wire variant of the TB method, using known HOMO on-site energies for the base pairs and deoxyriboses as well as interaction integrals between successive base pairs. We treated the interaction integral between base pairs and deoxyriboses (N degenerate eigenenergies (at N eigenenergies each, above and below N to clarify their form. As Studying the time-independent problem for homopolymers, we compared the eigenenergies for different sequence lengths and for different values of se pairs  plus isoWe also studied the time-dependent problem for homopolymers and discussed quantities related to hole transfer. Examining the mean-over-time probability to find the hole at each site for the initial placement at the first base pair, we showed that it can readily be transferred to the remaining base pairs. Edge monomers are clearly preferred, and the probabilities display palindromicity.within each monomer, not between monomers; the latter is solely dependent on N. We also showed that, for given N and N for a hole to be transferred from the first monomer, had it been initially placed at one of its deoxyriboses.When increasing the interaction between base pairs and deoxyriboses, increased transfer to deoxyriboses occurs. On the contrary, for initial placement at one of the deoxyriboses of the first monomer, the deoxyriboses of the first monomer are clearly preferred. When increasing Additionally, dipole moment components can clearly show the type of carrier movement: for initial placement at the first base pair, the dipole moment oscillates solely along the macromolecule, while, for initial placement at a deoxyribose site, it mainly oscillates across the macromolecule. We also examined the frequency content of carrier movement, using analytical and numerical Fourier transforms and obtained the WMF of all sites and the TWMF of the polymer. These quantities can also be used to characterize the readiness of coherent charge transfer within the polymer. The TWMF decreases with polymer size, increases with N, as expected. The increase in N, the coherent mean transfer rates involving end-to-end transfer are larger for initial hole placement at the base pair of the first monomer compared to initial hole placement at a deoxyribose of the first monomer, in accordance with what happens with the mean probabilities. Similar conclusions can be reached when studying hole transfer in all categories of DNA dimers, i.e., made up of identical monomers with purine on purine, made up of identical monomers with purine on pyrimidine, and made up of different monomers.The mean transfer rate between a site of the first monomer and a site of the last monomer decreases with


Streptococcus downii is a recently reported bacterial species of oral origin, with inhibitory capacity against Streptococcus mutans, Actinomyces naeslundii, Veillonella parvula and Aggregatibacter actinomycetemcomitans, which confers upon it the potential of being an oral probiotic. The aim of the present study was to identify the potential mechanisms by which S. downii exerts its inhibitory effect on S. mutans. To this end, the study assessed the consumption of glucose and proteins available in the culture medium, the modification of the pH, the production of short-chain fatty acids, the changes in the protein panel of the inhibition halo, the production of hydrogen peroxide and the effect of proteinase K. There were no differences in the glucose values or in the protein content of the medium, but there was a reduction in pH (with no effect on the growth of S. mutans). Significant increases were detected in the levels of lactic and formic acid (with no effect on the growth of S. mutans), as well as changes in the peptide panel (with no effect on the growth of S. mutans). The inhibitory effect was maintained in the presence of peroxidase but disappeared after adding proteinase K. Based on these results, it is suggested that the main mechanism of inhibition of S. downii against S. mutans is the production of bacteriocins. Analysis of oral microbiota using next-generation sequencing techniques has enabled the identification of differing clusters of microbial communities that are dependent on oral health, thereby suggesting that good oral health corresponds to a combination of innocuous commensal species that make up a stable ecosystem and hinder the growth of pathogenic bacteria ,2,3. AnyProbiotics are living microorganisms that, when administered in the appropriate quantities, are able to produce a health benefit in the host . The useStreptococcus downii is a new bacterial species initially detected as part of the oral microbiome in a teenager with Down syndrome with no history of dental caries. S. downii has shown inhibitory capacity against Streptococcus mutans, Actinomyces naeslundii, Veillonella parvula and Aggregatibacter actinomycetemcomitans. In a biofilm model, S. downii showed an antibiofilm effect against S. mutans and a significant reduction in S. mutans and Aggregatibacter actinomycetemcomitans counts and significantly reduced the growth of P. gingivalis and V. parvula in well-structured biofilms [biofilms ,15.S. mutans is the main bacterial species involved in the onset and progress of dental caries [S. downii exerts its inhibitory effect on S. mutans.Given that l caries , the aimS. mutans ATCC (American Type Culture Collection) 25175T prior to culturing S. downii and those obtained from the inhibition halo generated by S. downii after culture with S. mutans ATCC 25175T showed that the mean protein contents were 6.363 ± 0.110 and 6.640 ± 0.100 mg/mL (p = 0.1), respectively, with a mean glucose content of 6.377 ± 0.070 and 7.620 ± 0.080 g/L (p = 0.1), respectively.A comparison of solubilized agar samples obtained from a plate of S. mutans ATCC 25175T plate with no S. downii and the medium for the inhibition halo generated by S. downii after culture in the presence of S. mutans ATCC 25175T, with mean pH values of 6.667 ± 0.321 and 5.500 ± 0.200 (p = 0.1), respectively.The pH levels detected in distinct growth environments varied between the medium for the S. mutans ATCC 25175T, the growth ability of this bacterial strain was studied at a pH of 5.5, finding that the decrease in pH was not responsible for the inhibition.To determine whether this acidification was responsible for the growth inhibition of S. mutans ATCC 25175T plate before culture with S. downii and those obtained from the inhibition halo generated by S. downii after culture with S. mutans ATCC 25175T.S. downii than in those obtained prior to culture with S. downii, although these results did not reach statistical significance. In light of these findings, the direct inhibitory effect of both acids on S. mutans ATCC 25175T was investigated by diluting the acids to various concentrations . A combination of 0.24 mg/mL of formic acid and 1.6 mg/mL of lactic acid was also tested. After adding 5 µL of each acid to S. mutans ATCC 25175T and incubating for 17 h, no inhibition was observed at any concentration or after adding the combination of both organic acids. Consequently, the production of short-chain organic acids by S. downii was ruled out as being responsible for the inhibition of S. mutans ATCC 25175T.Higher levels of lactic (27-fold) and formic acid (10-fold) were detected in the samples obtained from the inhibition halo of S. downii and from the inhibition halo generated after culture thereof with S. mutans ATCC 25175T were analyzed, fractionating the protein content by applying cut-offs with a molecular size of 3 and 10 kDa. The fractionated protein content < 3 kDa was 3.190 ± 0.165 and 2.107 ± 0.200 (p = 0.1), respectively, for both sample types, while the content < 10 kDa was 5.017 ± 0.101 and 3.733 ± 0.208 (p = 0.1), respectively.Solubilized agar samples collected before culture with S. downii caused changes in the peptide profile, the inhibitory effect was not reproduced when inoculating these fractions directly onto S. mutans ATCC 25175T. Consequently, the presence of peptide compounds in the <3 kDa and <10 kDa fractions produced by S. downii with a potential ability to inhibit S. mutans ATCC 25175T was ruled out.Although the growth of S. downii on S. mutans ATCC 25175T. This inhibitory effect is maintained in the presence of peroxidase but disappears after adding proteinase K, suggesting that inhibition might be caused by a molecule of a peptide-type nature.S. downii supernatant produces an inhibitory effect against the growth of S. mutans compared with the growth curve obtained when adding the concentrated brain heart infusion (BHI) medium.S. mutans). Significant increases were detected in the levels of lactic and formic acid (with no effect on the growth of S. mutans), as well as changes in the peptide panel (with no effect on the growth of S. mutans). The inhibitory effect was maintained in the presence of peroxidase but disappeared after adding proteinase K, suggesting the presence of the inhibitory molecules in the S. downii supernatant. S. downii inhibition of S. mutans as well as the results.In summary, there were no differences in the glucose values or in the protein content of the medium, but there was a reduction in pH  from lactic acid, thereby minimizing the decrease in pH. H2O2 inhibits the growth of S. mutans and numerous other oral pathogens at concentrations that do not markedly affect the producing strains [2O2, low molecular weight antimicrobial peptides, bacteriocins and adhesion inhibitors [Lactobacillus acidophilus might inhibit the growth of other bacteria. Specifically, Lactobacilli from the strain GG produce organic acids, H2O2, carbon dioxide, diacetyl, low molecular weight antimicrobial peptides, bacteriocins and adhesion inhibitors against Streptococcus spp. and can therefore be considered a potent probiotic [S. sanguinis, which is a H2O2 producer, has been found to suppress the growth of A. actinomycetemcomitans in vitro and to antagonize the colonization thereof in gnotobiotic rats [S. oligofermentans inhibits the growth of S. mutans by producing H2O2, both in suspension and in biofilm studies [Streptococcus A12 also inhibits growth and intercellular signaling in S. mutans and can exert a buffer effect by modifying the pH via the arginolytic pathway [Another mechanism exhibited by oral commensal streptococci and that affects the ecosystem of the oral biofilm due to its inhibitory effect on certain microorganisms is the production of hydrogen peroxide  that produces a bacteriocin, namely mutacin 1140, which inhibits the growth of a wide range of bacteria, including Streptococcus spp., Actinomyces spp. and Lactobacillus spp. in vitro, has been isolated [S. salivarius has been shown to contain the biosynthetic loci for the bacteriocins salivaricin A3 and E, streptin, and streptococcin SA-FF22; this strain also produces the bacteriocin zoocin A, which, in combination with the preparation of dextranase, exhibited potent anti-S. mutans activity [S. salivarius K12, in turn, produces salivaricin, which is mainly responsible for its potential as an oral probiotic [S. salivarius M18 produced bacteriocins that antagonize the acidogenic activity of S. mutans. When this strain colonizes the human oral mucosa, it produces dextranase and urease, which can counteract plaque formation and the acidity of saliva, respectively [S. dentisani, in turn, metabolizes arginine and produces bacteriocins to exert its oral probiotic effect [However, the percentage contribution of lactic acid and other organic acids to the antibacterial activity of lactic acid bacteria has been estimated at less than 50% . Terai ebstances . Indeed,livarius , reuteri reuteri  or plantlantarum . Reuteriucleatum . A straiisolated ,40,41. Pactivity . The strrobiotic . Similarectively . S. dentc effect ,46.S. downii exerts its probiotic action: competition for the binding site to oral tissues and/or nutrients, the production of antimicrobial agents and/or bacteriocins and modification of the host’s immune response. Given that the experiments in this study were performed in vitro, we were unable to demonstrate a role for immunomodulation in the inhibitory effect observed.In light of the above, we can consider at least three mechanisms by which S. mutans ATCC 25175T plate prior to culture with S. downii and those obtained from the inhibition halo generated by S. downii after culture with S. mutans ATCC 25175T. It therefore appears unlikely that S. mutans inhibition is the result of competition for the substrate or a depletion of nutrients. Although differences in terms of pH were observed after culture with S. downii, the effect thereof on the growth of S. mutans was ruled out in subsequent studies. A marked increase in the levels of lactic acid and formic acid was detected in the samples obtained from the inhibition halo generated by S. downii after culture with S. mutans ATCC 25175T compared with the solubilized agar samples obtained from the S. mutans ATCC 25175T plate prior to culture with S. downii. However, neither of these two acids exhibited a direct inhibitory effect on S. mutans. H2O2 production was also ruled out as a mechanism for bacterial antagonism, given that the inhibitory effect remained unchanged after adding peroxidase.No major differences in nutrient content were detected for the solubilized agar samples obtained from the S. downii either produces a metabolite that diffuses through the agar or modifies the medium, thereby favoring S. mutans inhibition. Consequently, the most likely option to explain the inhibitory effect observed is the production of antimicrobial substances of a peptide-type nature, which can diffuse freely through the agar, thereby inhibiting the growth of susceptible strains [Given that inhibition in the in vitro tests occurred after pre-growth of the strain,  strains . The lik strains .Streptococcus spp. isolates were shown to be bacteriocin-producing [Streptococcus pyogenes [Streptococcus MIA (M 18) [S. dentisani [S. downii has revealed the presence of putative genes of the family of bacteriocin IIb lactobinA/cerein 7B [S. downii to detect the presence of these genes related to the production of bacteriocins would confirm the study’s findings. Moreover, it is essential to isolate the bacteriocin through the concentrated supernatant, similar to the study by Conrads et al. with S. dentisani [Based on the results and given that a significant number of roducing , class Ipyogenes , StreptoA (M 18)  and S. dentisani , among oerein 7B . A compaentisani .S. mutans ATCC 25175T was prepared on BHI plates to obtain a carpet-type growth. A 10-µL aliquot of S. downii was deposited on each plate in the form of a button after completing growth in pure culture for 24 h. S. mitis ATCC 49456T and S. salivarius ATCC 7073T were used as negative controls, and S. dentisani CECT 7747T was used as a positive control. The plates were incubated at 35–37 °C in an aerobic atmosphere for 24 h. The inhibitory capacity versus S. mutans ATCC 25175T was confirmed at 48 h by the presence of inhibition haloes. The test was performed in triplicate.A 0.3 McFarland suspension of S. downii in 50 mL of brain heart infusion broth  incubating at 37 °C for 48 h in an aerobic atmosphere. After incubation, the mixture was centrifuged at 4000 rpm for 10 min, discarding the pellets. The supernatant was passed through filters (pore size of 0.2 μm) (Millipore) and concentrated in a Rotavapor (rotary evaporator) 10 times. The resulting 5 mL was once again passed through filters (pore size of 0.2 μm) (Millipore) and stored at −20 °C until its use.To obtain the concentrated supernatant, we inoculated several colonies of S. mutans with BHI medium until an optical density of 0.1 was achieved. We then mixed 160 μL of the S. mutans suspension with 40 μL of the concentrated S. downii supernatant. As a negative control, we mixed 160 μL of the S. mutans suspension with 40 μL of concentrated BHI 10 times. The experiment was repeated 3 times.The supernatant’s inhibitory activity was determined by measuring the absorption at 610 nm in an automated microplate reader, incubating the sample at 37 °C for 14 h and recording the absorption every 30 min. To this end, we diluted S. mutans ATCC 25175T did not occur as a result of the consumption of glucose or proteins present in the medium by S. downii, agar samples  were collected, solubilized and analyzed. Glucose was analyzed using the biochemical kit K-GLUHK . Proteins were analyzed using a Pierce BCA Protein Assay Kit .To confirm that the inhibition of S. mutans plaque before culture with S. downii and from the inhibition halo generated by the co-culture of both species. Each fragment of agar medium was added to 1.5 mL of double-distilled sterile water and melted in a bath at 85–90 °C for 3 min (the agar’s melting temperature). We then measured the resulting volume to be able to adjust the calculations according to the dilution factor obtained in each case. We used as copies equidistant fragments of the S. mutans plaque before culture with S. downii and those obtained from the inhibition halo generated by the co-culture of both species.The agar solubilization protocol was performed as described previously with minor modifications . Using aS. downii affected the growth of S. mutans ATCC 25175T, agar samples were taken, solubilized in a similar manner to those used in the nutrient-consumption experiments at various growth points and in the inhibition zone generated, and analyzed to detect any differences in terms of pH.To study whether pH modification of the medium caused by the growth of S. mutans ATCC 25175T. To analyze the differential production of these organic acids, two samples of solubilized agar  were taken. The organic acids present therein were analyzed by high-performance liquid chromatography coupled to a refractive index detector [S. mutans ATCC 25175T at supposedly inhibitory concentrations was also studied. To that end, the acids were diluted in phosphate-buffered saline (PBS) and a 5-µL aliquot inoculated to S. mutans ATCC 25175T simultaneously.Certain short-chain organic acids, such as succinic, lactic, formic, acetic, propionic, isobutyric, butyric, isovaleric, valeric and caproic, might inhibit the growth of detector . The dirS. downii, the protein content of two solubilized agar samples was analyzed. One sample lacked S. downii, and the other was obtained from the inhibition halo generated after the culture of S. downii together with S. mutans ATCC 25175T. Samples were ultrafiltered on an exclusion column  of 3 and 10 kDa (molecular weight cut-offs), and the protein content was quantified using the Pierce BCA Protein Assay Kit . Both protein fractions were also tested directly, adding 5 µL of each while simultaneously inoculating S. mutans ATCC 25175T.To evaluate the possible changes in the protein (peptide) profile after the culture of 2O2 by S. downii, after incubation in a solid medium for 24 h, 10 μL of peroxidase (64 μg/mL) was added to one side of the colony, and 10 μL of PBS was added to the other. Subsequently, 5 μL of S. mutans ATCC 25175T was inoculated to the right and left sides, as reported previously by other authors [S. sanguinis DMSZ (German Collection of Microorganims and Cell Cultures) 20567T was used as a positive control [S. mutans ATCC 25175T was used as a negative control. If S. downii is a H₂O₂-producer, slight effervescence is observed immediately, and no inhibition haloes are observed after 24 h.To evaluate the production of Hroxidase 4 μg/mL w authors . S. sangS. downii, after incubation in a solid medium for 24 h, 10 μL of proteinase K  was added to one side of the colony, and 10 μL of PBS was added to the other. Subsequently, 5 μL of S. mutans ATCC 25175T was inoculated to the right and left sides, as reported previously by other authors [S. mutans ATCC 25175T [S. sanguinis DMSZ 20567T was used as a negative control. The plates were visualized after incubation at 37 °C for 24 h, and the absence of haloes indicated that inhibition was caused by molecules of a peptide-type nature.To determine whether bacteriocins might be responsible for the inhibition by  authors ,18. MutaC 25175T  was usedp ≤ 0.05. All experiments have been repeated 3 times, and we agree that the statistical power of 3 samples is very weak, and therefore these results should be interpreted with extreme caution.For the statistical analysis of the experimental data, we employed the statistical package SPSS . Due to the non-normal distribution, the differences between the mean values were assessed using the nonparametric Mann–Whitney U test. Significance was established for S. downii exerts its inhibitory effect on S. mutans is not regulated by the consumption of glucose and proteins available in the culture medium, the change in pH, the production of short-chain fatty acids, the changes in the inhibition halo’s protein panel or the production of hydrogen peroxide. Consequently, and given that the inhibitory effect disappeared after adding proteinase K, these results allow us to speculate that the most likely mechanism by which S. downii exerts its inhibitory action on certain oral pathogenic bacteria is the production of antimicrobial substances of a peptide nature.The results of this study show that the mechanism of action by which