Predicting hearing outcomes in idiopathic sudden sensorineural hearing loss (ISSNHL) is still challenging. We hypothesized that assessment of the semicircular canal (SCC) function via the video head impulse test (vHIT) might provide prognostic information. The medical records of patients diagnosed with ISSNHL from January 2015 to December 2018 were retrospectively reviewed. The prognostic values of the vHIT and other previously known factors in predicting hearing recovery were analyzed using a logistic regression model. A total of 148 patients with normal contra-lesional hearing were analyzed. Fifty-seven patients exhibited low gain (<0.7) on the vHIT in at least one SCC, more than the number of patients complaining of dizziness. Multivariable analysis revealed that non-recovery of normal hearing was associated with older age (OR 1.040), worse canal paresis on the caloric test (OR 1.023), worse initial hearing thresholds (OR 1.045) and abnormal vHIT result in the posterior SCC (OR 3.670). Low vHIT gain in the posterior SCC had specificity of 94.4% and positive predictive value of 85.7% in predicting non-recovery of normal hearing. In conclusion, abnormal vHIT gain in the posterior SCC appears to be a specific prognostic factor for incomplete hearing recovery in ISSNHL. In the majority of patients there is no specific identifiable cause of the hearing loss, and these cases are classified as idiopathic SSNHL (ISSNHL)1. Various etiologic backgrounds have been proposed, such as viral infection, vascular insufficiency and immunologic reaction4. ISSNHL has been treated with empirical therapies such as high dose steroid, antivirals, hyperbaric oxygen, etc5. Even if managed promptly, the outcomes of ISSNHL vary from non-recovery of previous hearing to complete recovery.Sudden sensorineural hearing loss (SSNHL) is defined as sensorineural hearing loss of 30 dB or more over at least three consecutive frequencies occurring within 72 hours1. The prevalence of dizziness or imbalance in SSNHL has been reported to be about 30%, and is related to poor prognosis4. Because vertigo is a subjective symptom, unlike the other indicators, a variety of studies have been performed to elucidate the clinical significance of vestibular function tests in SSNHL. Previous reports have focused on the value of the caloric test and vestibular evoked myogenic potentials (VEMP), which reflect the functioning of the horizontal semicircular canals (SCC) and otolith organs9. Theoretically, however, various types of vestibulocochlear involvement can be expected in ISSNHL. In terms of anatomic position, the closest vestibular organ from the cochlea is the saccule. The endolymphatic fluid spaces of cochlea and saccule are connected via ductus reunions, and their innervating nerve fibers are close together , bi-thermal caloric test and air-conducted cervical VEMP. The aim of the study was to evaluate the clinical significance of SCC involvement, as assessed by the vHIT, in predicting hearing outcomes of ISSNHL.Accordingly, we aimed to evaluate localized dysfunction of the vestibular organ in ISSNHL. We hypothesized that each of the components of the vestibular organ, especially the SCCs, can be involved variously in ISSNHL and might have distinct prognostic implications. Since subjective symptoms are not always correlated with objective vestibular function test results12. At the time of diagnosis, otoendoscopic examination, audiometry and vestibular function tests including the caloric test, vHIT and cVEMP, were routinely performed in all patients. Subjects with pre-existing hearing loss in the contra-lesional normal ear exceeding 25 dB HL were excluded to avoid possible debate about the assessment of outcomes. Those with retrocochlear pathology or other ear diseases - vestibular schwannoma, Meniere’s disease, inner ear anomaly, perilymphatic fistula, conductive hearing loss, or AICA infarction on MRI - were also excluded.The medical records of patients diagnosed with unilateral ISSNHL from January 2015 to December 2018 were retrospectively reviewed. The diagnosis of SSNHL was based on sudden hearing loss of more than 30 dB at a minimum of 3 consecutive frequencies over a period of 72 hours or lessAccording to the uniform treatment protocol, patients were initially treated with high dose oral steroid (prednisone 1 mg/kg daily for 7 days followed by 4 days of tapering). Thereafter, salvage intratympanic dexamethasone (5 mg/mL) injections (4 times/2 weeks) were performed in those whose hearing thresholds did not reach serviceable level (<40 dB HL) within a week.13. Complete recovery was defined as a final hearing threshold <25 dB HL. Partial recovery was defined as a final hearing threshold of 26–45 dB HL and >15 dB of hearing gain. Slight improvement referred to a final hearing threshold >46 dB HL and <than 15 dB of hearing gain, and no improvement meant a final hearing threshold >76 dB HL or <15 dB of hearing gain.Recovery of hearing was determined at 3 months from onset according to Siegel’s criterion, which is widely used to report hearing gain in SSNHLIn this study, the complete recovery group according to Siegel’s criterion, namely patients with final hearing threshold <25 dB HL, was the group of interest. The other groups were combined as the incomplete recovery group.This investigation was approved by the ethics review board of Hanyang University Guri Hospital (IRB #2019-11-016) and performed in accordance with the Declaration of Helsinki and good clinical practice guidelines. Informed consent was waived because of the retrospective nature of the study, and the analysis used anonymous clinical data after approval of ethics review board of Hanyang University Guri Hospital.14. Head impulses were given at least 10 times at the low amplitude of 10 degrees and a consistent peak velocity (100–300/second). Eye and head velocities were recorded. A calculated VOR gain of <0.7 was considered abnormal.The video head impulse test (vHIT) was performed at presentation to evaluate the high-frequency vestibulo-ocular reflex (VOR) in each SCC plane, using an ICS Impulse . To enhance test reliability, two experienced examiners performed the test, using the standard protocol proposed by Halmagy16. The induced nystagmus was recorded by video-nystagmography  until it had decayed to the null position. The maximum slow phase velocities of the corresponding stimuli were assessed and the asymmetry of vestibular function was calculated using Jongkees’ formula17. Canal paresis >25% was defined as horizontal semi-circular dysfunction18.The bi-thermal caloric test was performed using binaural alternative instillation of 8 liters of cold (24 °C) and warm (50 °C) air for 60 seconds20. Using a tone-burst sound stimulus of 500 Hz and 90 dB nHL via an EA-3 insert ear phone, the amplitudes of evoked potentials (P13-N23) on the same side of the sternocleidomastoid muscle were recorded20. cVEMP abnormality was defined as an asymmetry ratio of p13-n23 amplitude >35%, or no significant P13-N23 waveform21.The cVEMP test was performed with a Biologic Navigator Pro . The reference electrode, ground electrode and two active electrodes were placed at the sternoclavicular notch, center of the forehead and the middle third of each sternocleidomastoid muscle, respectivelyData were analyzed with IBM SPSS Statistics, version 24.0 for Windows . Descriptive data are expressed as means and standard deviations. A logistic regression model was used to evaluate the effects of prognostic factors on incomplete hearing recovery. Each parameter was assessed by univariable logistic regression analysis, and statistically significant factors were included in a subsequent multivariable analysis. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the vHIT for predicting non-recovery of normal hearing – incomplete recovery – were calculated. P values <0.05 were considered to indicate statistical significance. Figures were drawn with Microsoft Powerpoint 2016 MSO .A total of 148 consecutive patients (mean age 49.7 ± 13.9 years) with ISSNHL were analyzed. The average initial hearing threshold was 62.2 ± 29.5 dB HL  Table . Hearing4.5% TablUnivariable analyses showed that abnormal vHIT results in the horizontal SCC (P = 0.031) and posterior SCC (P < 0.001) were significantly related to poor recovery Table .In a multivariable analysis including the objective clinical factors that were statistically significant in the univariable analysis, significant predictive indicators for incomplete recovery were age , poorer initial hearing level , canal paresis on the caloric test  and abnormal vHIT gain in the posterior SCC   Various patterns of vHIT abnormality with SCC involvement were observed with or without subjective dizziness, (2) Multivariable analysis showed that incomplete recovery of hearing was significantly associated with decreased vHIT gain in the posterior SCC , worse initial hearing level at onset , age at onset , and canal paresis on the caloric test , (3) In our preliminary data, decreased vHIT gain in the posterior SCC had specificity of 94.4%, sensitivity of 31.2%, PPV of 85.7% and NPV of 56.7% in predicting incomplete recovery.22. The video method of examination, vHIT, is a noninvasive and quantitative method assessing each of the six SCCs individually14. In the vHIT, the adequacy of the VOR is usually measured by the gain, the ratio of the area under the eye velocity curve to the area under the head velocity curve during small, fast, passive unpredictable head impulses14. A VOR gain of <0.7 is usually considered as identifying a deficient SCC14. The vHIT has been widely applied to evaluate SCC function, especially in peripheral vestibular disorders and some central lesions28. In this study, we used it to assess vestibular involvement in ISSNHL.Since its introduction in 1988 by Halmagyi and Curthoys, the head impulse test has been used to evaluate the functioning of SCCs29. The well-known factors include age at onset, severity of initial hearing loss and time to initial treatment1. In our study, older age and poor initial hearing level were significantly associated with incomplete recovery of hearing  and saccule (cVEMP) was an important factor for hearing recovery, and that the extent of vestibular damage was related to the prognosis of hearing loss30. In addition, Fujimoto et al. reviewed the results of vestibular function tests in ISSNHL patients with vertigo to assess the extent of vestibular lesions7. They showed that the vestibular end organs close to the cochlea tended to be preferentially affected: there was involvement of saccule in 64%, of the horizontal SCC in 52% and of the utricle in 43% of the cases7. In our study, about 2/3 of the patients gave abnormal results in vestibular function tests. As in previous reports, the saccule was most often affected  followed by the horizontal SCC, based on abnormal caloric responses   Table . Among tIn terms of the inflammatory origin of ISSNHL, the involvement of adjacent vestibular organs can be explained by the flow of endolymphatic fluid within the inner ear, as well as the proximity of the vestibulocochlear nerve, and vestibular involvement can vary between individual patients. Empirical corticosteroid therapy may be expected to play a crucial role in such cases, as well as where viral infection and autoimmunity are implicated.9. Rambold et al. described a subgroup of patients with a distinct lesion pattern specifically involving the posterior SCC and cochlea9. In our study, twelve patients  showed this pattern (Fig. 32, the vulnerability of the cochlea to ischemia might offer a possible explanation for this outcome.It has also been suggested that certain patterns of vestibular involvement point to a vascular etiology for ISSNHL, as opposed to an inflammatory cause. In particular, compromise of the common cochlear artery can induce sudden hearing loss with isolated posterior SCC hypofunction Fig. 9. Rambolern Fig. . To compern Fig. , a post-In the present study, multivariable analysis showed that risk factors for incomplete recovery were older age, low initial hearing level, canal paresis on the caloric test and abnormal vHIT gain in the posterior SCC Table . PosteriTo the best of our knowledge this is the first study assessing the prognostic value of SCC involvement in ISSNHL using the vHIT. We assessed all ISSNHL patients, regardless of subjective dizziness. In addition, the inclusion criteria included normal contra-lesional hearing to alleviate possible debate about outcome determination. The evaluation of vestibular function, including each SCC, provided distinctive results pointing to a new prognostic indicator of hearing recovery. Unfortunately, oVEMP, which assesses utricular function, was not included in our analysis due to a lack of reliable data. Although we reviewed consecutive ISSNHL patients who were managed with a uniform treatment protocol during the enrollment period, there remains a possibility that biases may have arisen from the retrospective nature of the study. Hopefully, a future prospective study including all available vestibular function tests in a large number of patients will give further insight into the role of vestibular function in ISSNHL. Apart from providing prognostic information, we expect future studies to help develop individual treatment strategies to improve outcomes in ISSNHL.Abnormal vHIT gain in the posterior SCC is probably a specific prognostic factor indicating incomplete hearing recovery in ISSNHL.


Escherichia coli), Gram-positive bacteria (Staphylococcus aureus), and yeast . The results demonstrated that the obtained composite materials were characterized by a homogenous porous structure and high water absorption capacity (more than 1000% w/w). These materials also possessed low degradation rates (<5% in simulated body fluid (SBF) at 37 °C) and considerable antimicrobial effect due to silver nanoparticles (10–70 nm) embedded in the polymer matrix. These properties could be finetuned by adjusting the content of AgNPs and the synthesis route. The samples prepared using the in situ route had a wider porosity range and better homogeneity.Hydroxyapatite (HAp) and bacterial cellulose (BC) composite materials represent a promising approach for tissue engineering due to their excellent biocompatibility and bioactivity. This paper presents the synthesis and characterization of two types of materials based on HAp and BC, with antibacterial properties provided by silver nanoparticles (AgNPs). The composite materials were obtained following two routes: (1) HAp was obtained in situ directly in the BC matrix containing different amounts of AgNPs by the coprecipitation method, and (2) HAp was first obtained separately using the coprecipitation method, then combined with BC containing different amounts of AgNPs by ultrasound exposure. The obtained materials were characterized by means of XRD, SEM, and FT-IR, while their antimicrobial effect was evaluated against Gram-negative bacteria ( One of the most significant advances in the field of tissue engineering is the development of a porous three-dimensional matrix . In ordeSuch characteristics allow cell penetration, vascularization, and adequate nutrient and oxygen diffusion to cells and to the unformed extracellular matrix, which ensures cells viability. The pore size is, in fact, a key element for material efficiency. The pores must be large enough to allow cells to enter and move into the framework of the scaffold, while a small dimension allows the attachment of essential cell number at the same level . DependiThe inorganic phase of the composites designed for bone replacement is usually hydroxyapatite (HAp) ,8. HAp iFor the organic phase of natural bone, replacement with bacterial cellulose (BC) has been attempted . Even thMany studies ,15,16,17Scaffolds embedded with antimicrobial agents, antibiotics, or several forms of silver nanoparticles, which are known antimicrobial agents, are attracting great interest in biomedical research. Metallic silver and silver nanoparticles (AgNPs) have been reported to provide a wide variety of antimicrobial activities ,26,27,28AgNPs are more toxic compared to bulk silver but they have a strong anti-inflammatory impact during tissue healing and can be integrated into composite materials to obtain antibacterial properties ,30,31. TThe purpose of this study was to develop a composite material based on hydroxyapatite, bacterial cellulose, and silver nanoparticles with biomedical applications. The material was obtained by the coprecipitation technique, which is a reliable, simple, economic, fairly rapid, and precise method that allows the synthesis of homogenous structures and favorable pore dimensions . Studies3)2•4H2O, >99%), ammonium phosphate dibasic ((NH4)2HPO4, 99%), ammonium hydroxide , sodium hydroxide , silver nitrate , sodium citrate , polyvinylpyrrolidone ((C6H9NO)n), and sodium borohydride , purchased from Sigma-Aldrich . The solvents were American Chemical Society  purity. Bacterial cellulose membrane was produced in the laboratory by Gluconacetobacter sp. strain isolated from traditionally fermented apple vinegar in the Microbiology Laboratory of the Chemical and Biochemical Engineering Department, University Politehnica of Bucharest, based on a protocol previously described ..3) was uGluconacetobacter sp.) was boiled at 80 °C in water alkalized with sodium hydroxide . After purification, BC was washed in distilled water until it reached neutral pH. Afterward, it was minced using a blender  and weighed according to the recipe. Previously, the amount of dry matter was determined on a quantity of BC by eliminating the humidity, and it was found that 0.25 g of dry BC can be obtained from 10.62 g of wet BC [The bacterial cellulose synthesized by the Gram-negative bacteria .Two synthesis methods were used to obtain the bacterial cellulose and HAp-based composites. The first method involved obtaining in situ hydroxyapatite nanoparticles directly on cellulose fibers and subsequently adding the AgNPs solution, followed by homogenization using an ultrasound probe  was calculated.2+ and PO43− precursors, Ca(NO3)2•4H2O, and (NH4)2HPO4 were solubilized in distilled water, and bacterial cellulose was added in the calcium nitrate solution  were performed using a Shimadzu DTG-TA-50H equipment  at 25–700 °C with a heating rate of 10 °C/min.Investigation of the crystallinity of the powders was performed by means of X-ray diffraction (XRD) technique using the PANalytrical Empyrean  equipment in Bragg–Brentano geometry equipped with a Cu anode (λCuKα = 1.541874 Å) X-ray tube. The spectra were acquired in the range of 10–80° 2θ angles (Bragg angle) with an acquisition step of 0.02° and an acquisition time of 100 s. The scanning electron microscopy (SEM) images were performed with a FEI Inspect F50 microscope coupled with an energy-dispersive spectrometer (EDS) . Both secondary electron and backscattered electron detectors were used at 30 kV accelerating voltage. The TEM images of AgNPs were obtained using the high-resolution transmission electron microscope TecnaiTM G2 F30 S-TWIN equipped with selected-area electron diffraction (SAED) detector, purchased from the company FEI. This microscope operates in transmission mode at a voltage of 300 kV with a resolution of 2 Å. Research conducted by Fourier transform infrared spectroscopy (FT-IR) involved the analysis of a small amount of samples using the Nicolet iS50R spectrometer . The measurements were performed at room temperature utilizing the total reflection attenuation module (ATR), and 32 scans of the samples between 4000 and 400 cmweM is the wet sample weight, dM is the dry sample weight, and wM is the sample weight in water.The open porosity of the freeze-dried composite materials was calculated with Equation (1) for each material prepared in order to observe the porosity level according to the chosen manufacturing method, while the water absorption was calculated with Equation (2):Mdays7 is the sample weight in SBF after 7 days of immersion in SBF, and initialM is the sample weight after immersion in SBF. All the weight values were obtained at room temperature using a hydrostatic analytic balance.To test their biodegradability, the samples were placed in a 12-well plate in which phosphate-buffered saline (PBS) and simulated body fluid (SBF) were added, similar to the processes involved in the human body. After immersion of the samples in fluid, their integrity was monitored for 7 days. The degradation rate was calculated with Equation (3) for each material:Staphylococcus aureus ATCC 23235), one Gram-negative bacteria (Escherichia coli ATCC 25922), and one yeast  laboratory strain were used. The standard work protocol for the adapted version of the disc diffusion method implies the preparation of microbial suspensions of 0.5 McFarland standard density (1.5 × 108 colony forming units (CFU)/mL), prepared in sterile buffered saline solution. The obtained microbial suspensions were afterward used to swab inoculate the entire surface of the nutrient agar Petri dishes. After inoculation, identical size samples of the sterile coatings were aseptically placed on the inoculated agar surface, and the plates were incubated at 37 °C for 24 h to allow the growth of bacteria. After incubation, the growth inhibition zone diameter (mm) was measured. A wider inhibition zone suggests a higher antimicrobial effect of the fibrous dressing, reflecting the ability of AgNPs contained into the composite material to diffuse within the agar.The antimicrobial behavior of the freeze-dried composite materials was qualitatively assessed by an adapted growth inhibition assay . To coveThe thermal analysis corresponding to the composite samples are presented in 1, 65% for BC4, 68% for BC5, and 70% for BC8. The composites obtained in situ had a lower weight loss, which suggests good loading of BC with calcium phosphate phases (HAp).We observed  significIn order to demonstrate the composition, hydroxyapatite was analyzed by XRD technique. The diffractograms are presented in 5–BC8 samples were made by direct mixing of bacterial cellulose with hydroxyapatite (ex situ), the composition of this sample was not expected to change; therefore, the XRD analysis was only performed for bacterial cellulose, simple hydroxyapatite, and BC3 and BC4 composites (in situ) [Due to the fact that BCin situ) ,45.3 and BC4 revealed peaks located at 2θ values of 15, 16, and 23°, which can be attributed to bacterial cellulose according to ICDD 00-056-1718.It was observed that, in all the analyzed samples, the existence of bacterial cellulose and HAp was obvious. In addition, the low-intensity peak around 2θ = 38°, which can be assigned to the (111) crystalline plane, indicated the presence of silver nanoparticles in the composite structure. Investigation of the composites BCAs the HAp peaks were poorly visible in XRD analysis, FT-IR analyses were used to better highlight hydroxyapatite formation. The results are presented in −1 (OH stretch vibrations) and 2958 cm−1 (CH2 and CH3 stretch vibrations). The wide band observed in the region of 3500–3200 cm−1, attributed to the hydroxyl groups within the bacterial cellulose, increased in absorbance with higher silver content. This behavior suggested that the presence of HAp crystals affected cellulose hydroxyl groups, probably by covering them at the surface. Furthermore, the change observed for the band attributed to intramolecular hydrogen bonding (~3500 cm−1) confirmed a strong interaction between the OH groups and calcium phosphate. The chemical interaction between HAp and BC stabilized the composite so that it could maintain its mechanical integrity, an aspect required for bone substituents [The vibrational frequencies characteristic of bacterial cellulose were observed at 3500–3200 cmtituents .−1 and 570–600 cm−1 were attributed to the vibrational modes of PO43−. Because the stretching vibration of CO32− also appeared (at 1418 cm−1), absorption of CO2 from the air is suggested [2). Carbonated hydroxyapatite contributes to the biomimetism increase of the obtained composites, which can promote the process of osteoregeneration. It was observed that the in situ method accelerated the nucleation of HAp crystals onto BC fibers instead of crystallization as higher absorbance values were registered for BC5–BC8 samples, which contained highly crystalized HAp. The bands observed at 1641 and 643 cm−1 correspond to the stretching and deformation vibrations of AgO, respectively, thus confirming the presence of silver in the obtained materials [The FT-IR bands observed at 1020 cmuggested . This isaterials . This reThrough the two analyses performed, it was possible to notice the elemental composition of materials (EDS) as well as the homogeneity of hydroxyapatite particle dispersion.2–d2) and 2–d2)).The SEM image highlighted the fibrous structure of BC (see green arrow), which were decorated with inorganic particles (see blue arrow). In the SEM images performed on the composites in which HAp was obtained in situ a–d, a beTransmission electron microscopy images showed the silver nanostructure a,b, withThe calculated open porosity for each prepared material is presented in As can be observed, when the silver nanoparticle concentration increased, the composite porosity decreased for susceptible types of composite, which resulted in lower absorption capacity. Even though high porosity, which is associated with increased absorption capacity, is an important structural parameter for bone substituents, the registered decrease due to Ag addition is not significant in this case as the water absorption was still greater than 1000–1500%.Visual inspection  is an efAfter seven days, no major visual changes were observed, and the degradation was below 5% see , a sign Due to the higher homogeneity and better interactions between the phosphate phase (HAp) and BC, the composites obtained in situ had a lower degradation compared to those obtained using ex situ methods.The antimicrobial effect of the obtained composite materials was different among the tested samples, being influenced by the synthesis approach, AgNP content, and microbial species. 1 and BC5 did not contain antibacterial AgNPs, they were used as control samples for evaluation of the other samples.Because composites BCThe antimicrobial characteristic of the obtained composites was clearly influenced by the concentration of AgNPs, with samples with higher content of silver exhibiting the greatest microbial growth inhibition, regardless of the synthesis approach. It was observed that the materials obtained by the in situ method had a more pronounced overall antibacterial characteristic (growth inhibition zones ranging 5–11 mm) compared to the samples obtained by the ex situ method (diameter of inhibition zones ranging 5–9 mm).The different antimicrobial effects of the materials obtained by in situ and ex situ routes correlated with their physicochemical properties. Samples obtained by the in situ route showed a larger porosity, suggesting that the bioactive compounds  may be absorbed more efficiently in pores. Moreover, a higher porosity degree can be directly associated with an easier release of the bioactive agent, therefore inducing an increase in the antimicrobial activity of the final composite. This idea is supported by the results obtained with the control AgNPs utilized in equivalent amounts and added to sterile commercial filter paper, which demonstrated lower inhibition zones.C. albicans ATCC 10231, with the result being relevant for samples obtained by both in situ and ex situ methods. However, the composites obtained in situ also showed increased antimicrobial activity against the Gram-positive S. aureus strain. This result suggests that the obtained composite materials may act differently on microbial cells, depending on the particularities of their cellular wall. Such differences were observed before with silver nanoparticles [The most efficient growth inhibition was observed against the yeast strain articles ,51,52,534, which had a content of 5 wt % AgNPs. Due to the physicochemical structure, together with the already demonstrated great antimicrobial properties and low biodegradability of these materials, they have potential applications as successful candidates for biomedical applications, especially in hard tissue engineering. Their current limitation relates to the fact that further tests performed on osteoblast differentiation and mineralization  are needed.In this study, we report the synthesis of hydroxyapatite–bacterial cellulose silver nanocomposites obtained by two routes using coprecipitation, namely, in situ and ex situ assembly. These materials contained an organic part , an inorganic part (hydroxyapatite), and an antimicrobial agent (AgNPs) contained in various amounts, thereby conferring new bioactive properties on the composite materials. Physicochemical and antimicrobial studies demonstrated that the most efficient in terms of potential biomedical applications were the samples obtained by the in situ approach. The porosity range of the in situ materials was greater than the porosity of ex situ composites, while the best antimicrobial activity was observed for the material coded BC


Altogether, our results show for the first time that DBC1 regulates vascular response and function during hypertension and protects against vascular injury. This work also brings novel insights into the molecular mechanisms of the development of aortic dissections.Cardiovascular diseases are among the main causes of morbimortality in the adult population. Among them, hypertension is a leading cause for stroke, heart disease and kidney failure. Also, as a result of arterial wall weakness, hypertension can lead to the development of dissecting aortic aneurysms, a rare but often fatal condition if not readily treated. In this work, we investigated the role of DBC1 in the regulation of vascular function in an ANGII-induced hypertension mouse model. We found that WT and DBC1 KO mice developed hypertension in response to ANGII infusion. However, DBC1 KO mice showed increased susceptibility to develop aortic dissections. The effect was accompanied by upregulation of vascular remodeling factors, including MMP9 and also VEGF. Consistent with this, we found decreased collagen deposition and elastic fiber fragmentation, suggesting that increased expression of MMPs in DBC1 KO mice weakens the arterial wall, promoting the formation of aortic dissections during treatment with ANGII. Finally, DBC1 KO mice had reduced cell proliferation in the intima-media layer in response to ANGII, paralleled with an impairment to increase wall thickness in response to hypertension. Furthermore, VSMC purified from DBC1 KO mice showed impaired capacity to leave quiescence, confirming the  Sustained hypertension can lead to heart attack and stroke, kidney failure, aortic dissections and aneurysms3. In this regard, Angiotensin-II (ANGII), one of the key mediators of the renin-angiotensin system (RAS), is known to play a crucial role in the development of hypertension. ANGII, acting mainly through AT1 receptor, promotes a variety of cellular responses, including hypertrophy, fibrosis, reactive oxygen species (ROS) production and tissue remodeling4.Metabolic and aging diseases have become a main health concern worldwide. The increase in life expectancy, together with changes in lifestyle, mainly sedentarism, obesity and smoking have led to a dramatic increase in chronic non-infectious diseases, such as cardiovascular diseases . Altogether, these diseases constitute the main causes of morbimortality worldwide5. ANGII-mediated hypertension in mice by subcutaneous administration is by far the most widely used animal model for primary hypertension, accounting for nearly 50% of NIH-funded hypertension research5. This model has several distinctive advantages. First, it is possible to study the direct effect of ANGII on the vascular system. Second, it is useful to gain comprehensive insights into the molecular mechanisms of the RAS. Third, it is a very strong model in terms of reproducibility among species5. Also, animal models of ANGII-infusion are the best characterized and most widely used models of abdominal aortic aneurysms (AAA) or aortic dissections (AD)12.Animal models have proved to be extremely useful for understanding the pathogenesis of hypertension. There are several animal models available to study vascular and renal hypertension and they all have their advantages and limitations in terms of recapitulating the human disease13. Alterations in normal vessel remodeling during hypertension can lead to abnormal and life-threatening responses. One of them is the generation of aortic dissection (AD), a condition in which patients are highly vulnerable to life-threatening complications and death, particularly when the ascending aorta is involved. In humans, AD pathogenesis is mostly associated with a tear of the arterial wall, where blood dissects in intima-media, creating a “false lumen”. Based on their localization, the Stanford classification differentiate between type A and B aortic dissections. A-type dissections occur in the ascending aorta, while B-type ones localize in the descending portion of the aorta. AD is often fatal within the first hours to days after the onset (acute stage). Alternatively, it may evolve to a subacute stage (months since onset) and then to a chronic stage. The diversion of blood flow into the false lumen can cause hemodynamic alterations leading to ischemia, malperfusion, aortic valve insufficiency and tamponade (compression of the heart). These can lead to sudden aortic rupture, circulatory failure and death in the majority of patients without timely treatment15.In the vascular system, ANGII-mediated hypertension leads to remodeling of the arterial wall in order to cope with increased blood pressure. These changes involve, among others, extracellular matrix remodeling and increase in wall thickness. The latter is mainly caused due to proliferation and hypertrophy of vascular smooth muscle cells (VSMC)12. Despite reproducing several of the pathological features of human AAA such as elastin degradation, formation of intramural hematoma, aortic dissection, macrophage infiltration or thrombus formation, the ANGII-infused model of aneurism in mice seems triggered by different pathological mechanisms12. The most consistent lesion associated to ANGII-infusion models are the micro-ruptures of the vasa vasorum in the boundary of the tunica media with the adventitia, accompanied by a false channel formation with varying degrees of severity, and in some cases a lesion characterized by a dissected adventitia and an intramural hematoma (IMH). We therefore described these lesions as AD, a term that is currently used by other researchers12. Notably this pathogenic mechanism differs from the ones described for most of the AD seen in humans, where aortic dissection is characterized by development of an intimal flap caused by blood flowing into the media and forcing the intima and the adventitia apart (communicating AD). This intimal flap separates the true lumen  from a false lumen. It is worth noting, bleeding of the vasa vasorum with formation of IMH may evolve into non-communicating AD in 30–40% of patients with IMH17.It is important to notice that several lesions described previously as AAA, mainly in the model of ApoE-/- mice treated with ANGII, have been recently called into question as models of AD rather than AAA14. Importantly, the molecular mechanisms that operate in the development of AD are still not completely elucidated. Several studies have shown that dysregulation of Matrix Metalloproteinases (MMPs), as well as angiogenic factors like VEGF11, contribute to the development of AD18. In particular, MMP-9 has been shown to play an important role in this condition18. Considering that mice models of ANGII recapitulate AD, also, MMP12 seems to be involved in the AD progression9. In addition, it has been reported that MMP-9 and MMP-2 knockout mice were resistant to develop such lesions8. However, it is unclear which are the molecular pathways controlling MMPs and angiogenic factors expression during hypertension and how they lead to the formation of AD.Genetics, family history, age and gender, atherosclerosis and hypertension predispose to their development and progression19. DBC1 binds and regulates the biological activity of several transcription factors and epigenetic regulators, including SIRT122, HDAC323, p5324, BRCA125, AR26, ER28, Rev-erb-alpha29 and PARP130, among others.Deleted in Breast Cancer 1  is a nuclear protein that has many different functions in the regulation of metabolism34. We also showed that DBC1 KO mice have increased SIRT1 activity in vivo in liver and they are protected against non-alcoholic fatty liver disease22. In regards to cardiovascular diseases, we previously showed that DBC1 KO mice are protected against high-fat diet induced atherosclerosis35. However, our findings proved that protection against atherosclerosis was a consequence of increased lipid storage capacity in fat tissue rather than a local effect in blood vessels. Currently, there is no knowledge about the role of DBC1 in cardiovascular function.Many lines of research support that DBC1 is involved in the control of metabolism and metabolic diseasesin vivo and in vitro in VSMC, including MMP9, which has been linked to the development of AD. These changes were accompanied by decreased collagen levels and elastin fibers fragmentation, suggesting that DBC1 regulates extracellular matrix dynamics during hypertension. Finally, we also found that DBC1 KO mice failed to augment wall thickness in response to ANGII treatment, which was accompanied by decreased VSMC proliferation in vivo and in vitro. Altogether, our results constitute the first in vivo evidence that DBC1 is implicated in the tissue remodeling in response to ANGII, and also brings novel insights into the molecular mechanisms that regulate the development and progression of aortic dissections.In this work, we investigated the role of DBC1 in the regulation of vascular structure using a mouse model induced by ANGII infusion and hypertension. Both WT and DBC1 KO mice developed hypertension to a similar extent. However, we found a higher incidence of AD in DBC1 KO mice in response to ANGII infusion. Absence of DBC1 led to up-regulation of MMPs All general reagents and chemicals were purchased from Sigma-Aldrich, including angiotensin II , unless otherwise specified. Lipofectamine RNAiMax, Bradford protein assay reagent, Trizol and SuperScript II RT were bought from Invitrogen. SiRNAs oligos were purchased from Ambion  or Invitrogen (DBC1 MSS211964 and SIRT1 MSS234959). Antibodies were purchased from Bethyl , Abcam , or Cell Signaling . DNase I and Fast SYBR Green were purchased from Roche.ad libitum.All mice used in this study were maintained at the Institut Pasteur de Montevideo Animal facility (UATE). The experimental protocol was approved by the Institutional Animal Care and Use Committee of the Institut Pasteur de Montevideo . All the studies described were performed according to the methods approved in the protocol and following all international guidelines and legal regulations. WT and whole-body DBC1 KO mice were in a C57BL6/J pure background. DBC1 KO mice were backcrossed into C57BL/6 J for more than 10 generations in order to ensure genetic purity. Mice received standard chow and water Alzet mini-osmotic pumps  were subcutaneously implanted in mice under isoflurane anesthesia. Prior to the implant, pumps were filled either with ANGII (1 mg/kg/day) or vehicle (NaCl 0.9%) and left for 7 and 28 days. All the results are representative of at least 3 independent experiments. Each group contained at least 5 mice per group. A total of 101 mice were infused with either ANGII or vehicle (50 WT and 51 DBC1 KO).Blood pressure (BP) in mice was measured by a non-invasive CODA system , using a Volume Pressure Recording (VPR) sensor. This methodology consists of a cuff placed on the tail to occlude the blood flow. Upon deflation, the VPR sensor uses a specially designed differential pressure transducer to measure the blood volume in the tail. Before recording, animals were allowed to acclimate (1 h at 30 °C). BP was registered in the afternoon, twice a week for two consecutive days during 6 weeks (2 weeks before the pumps implantation and during the 4 weeks of treatment). BP values for each week correspond to the mean value of both days (15 recordings per session). When Systolic BP values were obtained with a tail blood volume below 15 μl they were discarded.Mice were placed in individual metabolic cages for 48 h at the fourth week of treatment. The first day was for acclimatization and the following 24 h were the ones considered for the analysis of urine and water intake. Urine production was measured and centrifuged and the supernatant was frozen for subsequent procedures.5 Bromo-2′-deoxyuridine (BrdU) was injected into mice  at the first, second and third week of treatment. At the first or fourth week, mice were sacrificed and a segment of descending thoracic aorta was fixed in 4% paraformaldehyde (PFA) for 24 h for immunohistochemistry.2O2 for 20 minutes to block non-specific peroxidase signals. Briefly, samples were incubated with 2 M HCl for 15 minutes at 37 °C, blocked with 2% BSA-0.03% Triton x-100 during 1 h, incubated with BrdU antibody and revealed using an anti-rat -HRP-diaminobenzidine. Hematoxylin was used as counterstaining.Aortic sections were obtained from paraffin-embedded tissue and BrdU immunolabel were performed according to standard procedure. The paraffin sections (5 µM) were deparaffinized and rehydrated, incubated with 2% Hin situ by macroscopic analysis of the whole aorta (ascending and descending). Once identified, AD was diagnosed under stereoscopic microscopy, as a blood clot surrounded by greatly expanded adventitial tissue and neovasculature on the outer surface, that made the artery difficult to remove. In all cases, the nature of the lesion was confirmed by histological analysis. Aorta scheme is illustrated to show different portions used for analysis (Supplementary methods). A portion of thoracic aorta was used to immunohistochemistry and staining techniques: Hematoxylin & Eosin (H&E) and Verhoeff (VF). In the cases when AD was observed macroscopically, tissue was processed to histological analysis stained with H&E and VF. Finally, a section of abdominal aorta below AD was used for molecular biology processing.Mice were anesthetized with intraperitoneal ketamine and xylazine . Depth of anesthesia was assessed by toe-pinch procedure and absence of muscular tone. Aortas were isolated after the first or fourth week of ANGII infusion, and either frozen in liquid nitrogen for subsequent molecular biology methods or fixed in 4% PFA. The presence of AD was evidenced 36. VSMCs were cultured in full medium containing DMEM supplemented with 10% fetal bovine serum (FBS), 2 mmol/L glutamine, 100 U/mL penicillin, 100 mg/mL streptomycin. Cells were cultured in a water-jacketed incubator at 37 °C and 5% CO2.Vascular smooth muscle cells (VSMCs) were obtained by outgrowth from abdominal aorta explants from WT or DBC1 KO male mice as previously described by othersFor siRNA experiments, cells were plated in six well plates in medium used for VSMCs. When cultures reached 80% confluence, cells were transfected with 30 nM siRNA oligos  were lysed using NETN buffer  or RIPA buffer , respectively. Protein concentrations were determined using the Bradford protein assay reagent. Proteins were separated in SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes, which were then blocked in Tris-buffered saline containing 0.2% Tween 20 and 5% nonfat milk. Membranes were then incubated overnight with anti DBC1, Cyclin D1, PCNA, P27 or tubulin. After that, secondary antibodies were incubated 1 h, washed and detected using chemiluminescence. Densitometry of bands was analyzed using Image J.Paraffin sections (5 µm) were stained with H&E, Picrosirius red (PIC) for collagen and Verhoeff (VF) for elastic fibers stain. The specimens were observed under a light microscope . In order to measure the wall thickness of the aortic rings, we used a custom designed plugin for Image J. The procedure starts by clicking in the center of the aorta lumen. Then, 4 lines passing at that point are drawn, marking the aorta wall at 8 different places. Those are the 8 places where the wall is measured by clicking at the intima and at the outer limit of the media without considering the adventitia layer. The wall thickness is automatically obtained as the average of those 8 measurements. Collagen deposition was evaluated in intima-media aortic ring using the Wake Segmentation plugin from Image J, reporting results as area (%) occupied by collagen.VSMCs were counted, plated in 10 cm plate and cultured to 60% confluence. After that, FBS was removed for 48 h to induce cellular quiescence. Later, FBS was restored and cells collected at different time points. Total viable cells were selected by propidium iodide exclusion and counted by flow cytometry using BD Accuri C6 cytometer. Cell proliferation state under described experimental conditions was further confirmed by quantification of Ki67-positive cells, using immunofluorescence.37.Cells were grown until 90% confluence and then incubated with 0.1% FBS for 48 h. After that, cells were treated with 100 nm ANGII (or vehicle) for 12 h as previously described by othersCells and tissues, were homogenized in Trizol for RNA extraction according to manufacturer’s protocol. DNase I treatment was used to eliminate genomic DNA contamination. Reverse transcription was done using SuperScript II RT and quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed using Fast SYBR Green Mix. Relative quantification of changes in gene expression were expressed in relation to a housekeeping gene. Expression was calculated as fold increase with respect to control condition. Primers containing the following sequences were synthesized by Integrated DNA Technology (IDT) and listed in Table P < 0.05 was considered to be significant. Mann-Whitney U test was used to compare two independent samples (groups) in cases where sample distribution was not normal. For comparisons of proportions, Fisher’s exact test was used.Results are expressed as mean ± SEM unless otherwise specified. Shapiro Wilk Analysis were performed to confirm normal distributions. Unpaired t-test was used to compare two independent groups. In multiple comparisons, analysis of variance (ANOVA) followed by Bonferroni’s post hoc test, In order to investigate the role of DBC1 on vascular function, we used the model of ANGII-induced vascular hypertension. WT and DBC1 KO mice were exposed to subcutaneous infusion of ANGII (1 mg/kg/day) for 4 weeks. Under these conditions, we found that both WT and DBC1 KO mice developed hypertension in a similar manner  and in vivo in a model of liver regeneration48. In order to evaluate if failure to leave quiescence could explain decreased in vivo proliferation of VSMC, we cultured VSMC form WT and DBC1 KO mice and measured cell cycle progression. WT and DBC1 KO VSMC were synchronized into quiescence by serum deprivation48 and later allowed to re-enter cell cycle by restoring normal serum. Serum restoration in WT cells was accompanied by up-regulation of the proliferation marker Ki67  triggered by ANGII. Mechanistically, our results show that absence of DBC1 increases the expression of vascular remodeling factors, including MMPs, specially MMP9, that are involved in the development of AD both in mice models and humans35. DBC1 regulates SIRT1 activity in the liver and adipose tissue during different metabolic states31. Furthermore, we also showed that DBC1 KO mice are protected against vascular injury and atherosclerosis, due to increased fat adipose tissue buffer capacity during obesity35. In this work we found that ANGII promotes hypertension to a similar extent in WT and DBC1 KO mice, although the latter were partially protected against cardiac hypertrophy. This result is consistent with several reports which have shown that SIRT1 activation protects against cardiac hypertrophy51. To our surprise, we found that DBC1 KO mice had a higher incidence of AD triggered by the treatment. Our results suggest that DBC1 affects different aspects of vascular response during hypertension, which are discussed below.We and others have extensively shown that DBC1 plays major roles in metabolism and metabolic diseases52. However, others have suggested that ANGII-induced AD develop even in the absence of hypertension53. The fact that AD can develop before the onset of hypertension (1 week of ANGII infusion) and also that in DBC1 KO mice they still develop at lower doses of ANGII, suggest that AD and hypertension may be considered as independent events. However, further studies will be needed in order to determine if AD formation in DBC1 KO is independent from blood pressure.Our results show that DBC1 KO mice exhibit increased incidence of AD in response to ANGII. This effect was as early as 1 week after ANGII infusion  conditions, while this was not detected in vivo in untreated WT and DBC1 KO mice. We cannot provide a definite explanation for this discrepancy. One possibility is that the expression of MMPs by VSMC in vivo is too low to detect differences among genotypes. Another possibility is that other cell types rather than VSMC are the main driving force for the observed phenotype. In this regard, it has been shown that macrophages57 and neutrophils58 that infiltrate vascular tissue during hypertension are an important source of MMPs.It is important to notice that only DBC1 KO mice showed a clear up-regulation of MMP9 mRNA after one week of ANGII treatment .Many of the biological effects of DBC1 are mediated by regulation of SIRT1 activity, and in fact it was recently shown that SIRT1 activity protects against AD in the context of caloric restriction (CR). The authors showed that specific knock-down of SIRT1 in VSMC 59. Indeed, p53 regulates VSMC function60. Since DBC1 is a direct regulator of p5324, it is possible that the DBC1-p53 axis is involved in our results. Another possible downstream effector of DBC1 in AD may be the long non-coding RNA (lncRNA) MALAT1. This lncRNA was recently involved in the regulation of aneurysmatic lesions by regulating VSMC function61. MALAT1 also regulates MMPs expression and cell invasiveness in cancer cells63. MALAT1 is directly regulated by DBC164, suggesting that dysregulation of this lncRNA could explain the DBC1-dependent effect on AD in response to ANGII. Which of these pathways, or any other still to be known, are involved in the DBC1-dependent AD incidence needs to be further investigated.As it was mentioned in the introduction, DBC1 has many molecular targets, which makes it very laborious to single out a molecular effector of its function. In this case, based on the available data, we can propose two possible DBC1-dependent pathways implicated in the development of AD. The first one involves the regulation of the p53 protein. It has been recently shown that loss of p53 sensitizes to ANGII-induced AD during caloric restrictionin vivo in response to ANGII treatment. This was evidenced by a decreased rate of BrdU incorporation into DNA and paralleled by a failure to increase the thickness of tunica-media in response to hypertension. Several reports have shown that VSMC proliferation capacity is compromised in AD47 and that failure of VSMC could be a cause for aortic wall weakening and aneurysm formation. Recently, we showed that deletion of DBC1 delays cell proliferation in previously quiescent cells, both in vitro and in vivo during liver regeneration48. In fact, when DBC1 KO VSMC were made quiescent by serum deprivation, they failed to re-enter cell cycle upon serum restoration. This was evidenced both by the expression cell cycle progression markers and by cell count. Quiescent VSMC from WT mice showed the appearance of the putative quiescence marker DN-DBC148. Serum restoration triggered the down-regulation of DN-DBC1 and up-regulation of cell cycle progression markers. DBC1 KO VSMC were unable to up-regulate these proteins and failed to progress through cell cycle. These results suggest that, similar to the liver, the ratio between DBC1 and DN-DBC1 plays a role in the control of cell cycle progression of VSMC. Thus, this may affect the response of VSMC to vascular injury in vivo. Interestingly, it has been shown that SIRT1 overactivation in VSMC inhibits proliferation after vascular injury65. We propose that SIRT1 overactivation might impair the proliferation of VSMC in DBC1 KO mice in response to ANGII. Also, we speculate that under basal conditions the expression of DN-DBC1, which does not bind to SIRT148, contributes to maintaining the quiescent state of VSMC by increasing SIRT1 activity. Upon vascular injury, DN-DBC1 might be fully replaced by DBC1, promoting cell proliferation in intima-media thickening.Besides showing altered expression of vascular remodeling factors, the cells belonging to the intima and media layers from DBC1 KO mice showed impaired proliferation Based on all the results presented here, we generated a working model for DBC1 role in the control of vascular response during ANGII-mediated hypertension Fig. . We propThis work constitutes the evidence that DBC1 is directly involved in the control of vascular response to injury by regulating the expression of vascular remodeling factors and also the proliferative response of VSMC. Furthermore, it strengthens the emerging role of DBC1 as a key regulator of non-transformed cells and their transition from quiescent to proliferative states. Finally, our findings could contribute to the understanding of the molecular mechanisms that underlie the development of AD.Supplementary Information.


Silver nanoparticles (AgNPs) are constituents of many consumer products, but the future of their production depends on ensuring safety. The stability of AgNPs in various physiological solutions and aging in storage may affect the accuracy of predicted nanoparticle toxicity. The goal of this study was to simulate the transformation of AgNPs in different media representatives to the life cycle in the environment and to identify their toxicity to Hepa1c1c7 cells in a long-term aging process. AgNPs coated with citrate, polyethylene glycol (PEG), polyvinylpyrrolidone (PVP), and branched polyethyleneimine (BPEI) were studied. Our results show that the exposure media had a significant impact on the transformation of AgNPs. Citrate-coated AgNPs showed significant aggregation in phosphate-buffered saline. The aging of AgNPs in optimal storage showed that the charge-stabilized particles (citrate) were more unstable, with significant aggregation and shape changes, than sterically stabilized particles . The BPEI AgNPs showed the highest dissolution of AgNPs, which induced significantly increased toxicity to Hepa1c1c7 cells. Overall, our findings showed that storage and media of AgNPs influenced the transformation of AgNPs and that the resulting changes in the AgNPs’ physicochemical properties influenced their toxicity. Our study contributes to the understanding of AgNPs’ transformations under realistic exposure scenarios and increasing the predictability of risk assessments. Silver nanoparticles (AgNPs) are one of the most commonly used engineered nanomaterials in many applications , which results in their significant release into the environment and induces potential risks to ecological receptors and humans ,2. WhileThe high surface to volume ratio of AgNPs makes them highly dynamic in natural systems and significantly changes their physical and chemical properties in the environment, including their ability to oxidize and sulfidize. Sulfidation of the nanoparticles can change their aggregation state, surface chemistry, and charge, as well as their ability to release toxic Ag ions. In turn, this modifies their persistence and toxicity ,4. AdditMoreover, complex nano–bio interactions on the surface of AgNPs with different coatings can also cause toxicity. Different surface coatings are used to develop faster electronics, brighter displays, and more sensitive diagnostic agents for medical imaging. Previous studies have demonstrated that the toxicity of nanomaterials’ particle size, composition, surface charge, shape, and aggregation drive the toxicity of nanomaterials ,9,10,11.To address these knowledge gaps, we investigated the transformation of different surface-coated AgNPs in different media and identified the toxicity of transformed AgNPs to Hepa1c1c7 cells in a long-term aging process. The Hepa1c1c7 test is applied as a tool to gain an insight into the relative development of toxicity during the different treatments. Four types of AgNPs coated with citrate, polyethylene glycol, polyvinylpyrrolidone, and branched polyethyleneimine  were tested in the study.3 (CAS NO. 7761880). The Kinetic Turbidity Assay (NCL Method STE-1.2) was used to test endotoxin concentration in four AgNP stock suspensions. The diameter of AgNPs was determined by transmission electron microscopy . The measurement of mass concentration and corresponding number concentration of the Ag in stock solutions was measured by Inductively Coupled Plasma Mass Spectrometry . We assumed a 5 nm thick layer of polymeric capping ligand  on each particle and a 0.5 nm thick layer of molecular citrate for estimating the total mass of capping ligand in each solution. A UV-Visible Spectrophotometer  was used to investigate the spectral properties of materials. Zetasizer Nano ZS  was used to measure the hydrodynamic diameter and zeta potential of nanoparticles.Citrate, polyethylene glycol, polyvinylpyrrolidone, and branched polyethyleneimine  were used as coatings for AgNPs. These materials were purchased from nanoComposix . Sigma-Aldrich  supplied AgNO2HPO4: 14.4 g/L, and KH2PO4: 2.4 g/L, pH 7.4), serum , and α-MEM . After shaking by hand for about half a minute, the samples were analyzed by TEM and dynamic light scattering (DLS).To identify media impact on AgNPs’ physicochemical properties, Citrate AgNPs (1.14 mg/mL) were added to 1 mL of the different media, respectively. The media were deionized water, saline , PBS  and ICP-MS. The toxicity of AgNPs to Hepa1c1c7 cell lines was analyzed before and after storage in the fridge.2 in an incubator. Cells were cultured in α-MEM  with 10% FBS  and antibiotics.The Department of Environmental Toxicology, University of California, Davis provided Hepa1c1c7 cells. They were maintained at 37 °C with a humidified atmosphere of 5% CO® luminescent assay  was used to measure cell viability. Detergents were added to the reagent to break cell membranes. This resulted in the release of ATPase inhibitors to media and their stabilization. ATPases are a group of enzymes that catalyze the hydrolysis of a phosphate bond in adenosine triphosphate (ATP) to form adenosine diphosphate (ADP). The assay is based on the conversion of beetle luciferin to oxyluciferin by a recombinant luciferase in the presence of ATP. The quantity of ATP in cells is proportional to the observed luminescence. The observed luminescence was measured by a microplate reader machine . White, opaque, walled, 96-well plates  were used for experiments. The ATP assay was conducted by platin, 5 × 103 cells per well. Different concentrations  of Citrate AgNPs, PVP AgNPs, PEG AgNPs, BPEI AgNPs, and AgNO3, for 24 h, were used for treatment.CellTiter-Glop < 0.05. Levene’s test was used to check homogeneity of variances. SPSS version 16 was used for the analyses.Statistical analyses for the cell viability were done using analysis of variance (ANOVA) and Tukey’s test with significance level at All studied AgNPs were monodispersed with similar primary size (around 30 nm), as shown by TEM images A–D. ζ poThe stock suspensions were stable in Milli-Q water media, as evidenced by the hydrodynamic size of all AgNPs. To identify the media impact on citrate AgNPs, we studied physicochemical properties of Citrate AgNPs in different media b–f. Our Our aging studies show that the changes in physicochemical properties of AgNPs after one year relates to their surface coatings. The TEM images showed significant aggregation of Citrate AgNPs, and slight aggregation of PEG AgNPs and PVP AgNPs during one-year storage , Table 150 of PEG AgNPs and BPEI AgNPs to Hepa1c1c7 cells decreased four and five times after one year of aging, respectively (Cell viability studies show higher toxicity of AgNPs stored for one year to Hepa1c1c7 cells as compared to particles tested without aging . The obsectively C.− in PBS and dissolved Ag of Citrate AgNPs. Our results are consistent with others who conclude that silver ions are mainly precipitated as AgCl in the presence of chloride. Because phosphate is mostly protonated to hydrogen phosphate and dihydrogen phosphate in a moderate pH (around 7), silver phosphate was never detected [− (8 g/L) in saline than the Cl− (80g/L) in PBS. This result is consistent with the aggregation kinetics of the electrostatically stabilized AgNPs . The ionic strength correlates with aggregation increases up to a certain ion concentration in the medium [Our results showed that the media impact on Citrate AgNPs is lowest in deionized water, highest in PBS and Serum, and α-MEM is in between. We found that Citrate AgNPs aggregated significantly in 10× PBS, where the color changed from yellow to gray d,D. The detected . We did e medium .Nanomaterials can interact with proteins, peptides, and lipids (nanocorona) on their surface once the nanomaterials enter into biological systems ,17. The Our results showed that citrate coating plays an important role in both AgNP stability and antibacterial activity. We found that the presence of salt (NaCl) increases the aggregation of Citrate AgNPs but the presence of serum decreases their aggregation in different media. Previous studies showed that the interaction of AgNPs with serum albumin had a significant effect on their antibacterial activity. Citrate AgNPs exhibited antibacterial properties due to minimized interactions with serum proteins . HoweverInteraction of NPs with the environment significantly changes their physical and chemical properties because the high surface to volume ratio and reactivity of NPs makes them highly dynamic in environmental systems . HoweverThe TEM images showed that Citrate AgNPs had substantial aggregation resulting in increased size, and change in shape from spherical particles to triangular or irregular shapes. Such shape transformations have been found in other studies ,29,30,31There were no observable changes in size and shapes of PVP and PEG-coated AgNPs, but a slight aggregation of PVP and PEG-coated AgNPs were observed after one year of storage. The relative stability of PEG and PVP-coated AgNPs in comparison to Citrate AgNPs may be because PEG and PVP are sterically stabilized AgNPs, which are strongly bound to the core and potentially permeable to solutes and solvents.BPEI AgNPs showed only slightly decreased size and aggregation after one year of storage. BPEI AgNPs are electrosterically stabilized and have abundant amine motifs that can form stable complexes with Ag atoms, which can explain their relative stability.The DLS analysis did not show significant hydrodynamic size changes for any AgNPs in media after one year of storage. The findings of the TEM images analysis discussed above contradict these results. Because DLS provides information on the hydrodynamic radii of particles in solution, an increase in AgNPs’ size from DLS over TEM measurements is expected. It can indicate a successful capping of AgNPs with coatings . Our resThe surface coating was found to be important for the dissolution of AgNPs based on the analysis by ICP-MS and ultracentrifugation. The positively charged BPEI AgNPs showed the highest dissolution (371.1 µg/L) but the negatively charged Citrate AgNPs, PEG AgNPs, and PVP AgNPs showed lower dissolution of 3.12 µg/L, 67.36 µg/L, and 130.60 µg/L, respectively . NP surfWe found that one-year storage significantly changes the physicochemical properties of AgNPs. The 24-h toxicity of AgNPs aged for one year to Hepa1c1c7 cells is higher than the toxicity of pristine AgNPs . The obs+) can increase the ionic strength in media, which induced significant aggregation of Citrate AgNPs in PBS. However, Citrate AgNPs in saline did not show aggregation, which indicated that the kinetics of aggregation depends on the ionic composition of media. The decreased zeta potential of Citrate AgNPs in serum demonstrates that the diversity of the proteins forming the protein corona may be affected, reducing the aggregation of AgNPs. The aging of AgNPs with different coatings shows that the observed changes in AgNPs’ toxicity are related to different processes such as dissolution, agglomeration, and capping agent degradation. Our study confirms the strong impact of aging and media properties on increased toxicity of AgNPs. These processes need to be explicitly modeled to predict the fate of silver in the environment and humans.Our study showed that media had a significant impact on the physicochemical properties of Citrate AgNPs. The electrolyte solution containing positive ions (e.g., Na


Eccentric resistance training has been shown to improve performance outcomes in a range of populations, making it a popular choice for practitioners. Evidence suggests that neuromuscular adaptations resulting from eccentric overload (EO) and accentuated eccentric loading (AEL) methods could benefit athletic populations competing in team sports. The purpose of this review was to determine the effects of eccentric resistance training on performance qualities in trained male team sport athletes. A systematic review was conducted using electronic databases PubMed, SPORTDiscus and Web of Science in May 2019. The literature search resulted in 1402 initial articles, with 14 included in the final analysis. Variables related to strength, speed, power and change of direction ability were extracted and effect sizes were calculated with a correction for small sample size. Trivial, moderate and large effect sizes were reported for strength (−0.17 to 1.67), speed (−0.08 to 1.06), power (0.27 to 1.63) and change of direction (0.48 to 1.46) outcomes. Eccentric resistance training appears to be an effective stimulus for developing neuromuscular qualities in trained male team sport athletes. However, the range of effect sizes, testing protocols and training interventions suggest that more research is needed to better implement this type of training in athletic populations. There is growing evidence in the literature that suggests eccentric resistance training is an effective stimulus for enhancing physical performance . During One reviewer conducted the literature search according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines for systematic reviews . The elewww.zotero.org). The titles and abstracts of the remaining records were then screened. Articles not meeting the inclusion/exclusion criteria were removed, and the remaining records were assessed. Those full-text studies meeting the eligibility criteria were then assessed for inclusion in the review. An additional search was carried out using the reference lists of articles; those records identified through the additional search were then subjected to the same systematic process. Finally, all of the studies deemed to meet the criteria were assessed for methodological quality and included in the review. Once the initial search had been conducted, the articles were stored in reference manager software . Duplicate articles were manually reviewed and merged using the included “Duplicate Items” function. From the company’s website, “Zotero assesses records for duplicates based on the title, DOI, and ISBN fields to determine duplicates. If these fields match (or are absent), Zotero also compares the years of publication (if they are within a year of each other) and author/creator lists  to determine duplicates”  and were between 17 and 35 years of age.The sports included in the review following the screening process were basketball, soccer, handball, and rugby union.Studies investigated the effects of longitudinal (≥three weeks) EO training interventions. Eccentric training load  needed to be quantified.Data on at least one of the following outcome measures were reported: strength , maximum sprint times , power , and change of direction .Studies meeting the following inclusion criteria were included in the review:Participants were individual sport athletes  or untrained (students or with less than six months training experience). Studies not listing the training experience/sport status of participants were also excluded.Studies investigating male and female athletes were excluded if the results were not reported separately.The training intervention included injured participants.Supplements or ergogenic aids were used in the intervention.Studies with the following exclusion criteria were not included in the review:n = 5).The eligibility assessment was performed in an unblinded manner by a single reviewer. The study selection process used is visually represented by n = 8). The score for each criterion was as follows: 0 = “clearly no”, 1 = “maybe”, and 2 = “clearly yes”.The remaining 14 articles were evaluated using a 10 item scale designed for exercise training studies [Inclusion criteria were clearly stated;Subjects were randomly allocated to groups;Intervention was clearly defined;Groups were tested for similarity at baseline;Use of a control group;Outcome variables were clearly defined;Assessments were practically useful;Duration of intervention was practically useful;Between-group statistical analysis was appropriate;Point measures of variability.The items included: 1) and post-test (SD2) standard deviation and pre- and post-test sample size pairs  . A majorple bias .(1)CoheValues were interpreted as trivial 0.00 < trivial < 0.20, 0.20 ≤ small < 0.60, 0.60 ≤ moderate < 1.20, 1.20 ≤ large < 2.00, 2.00 ≤ very large < 4.00 .n = 20). Background variables were provided in all studies except one [Data for participant and training intervention characteristics are reported as mean ± standard deviation, unless otherwise stated. A total of 14 studies met the inclusion criteria and were included in the review, with a summary of the participant characteristics provided in cept one . Particicept one ,22,23. Acept one ,26,27,28Training programs lasted from 3 to 10 weeks (8.1 ± 2.6), including 7 to 18 training sessions (13.3 ± 3.1), with the exception of Suarez-Arrones et al. , whose iThe prescription method of exercise intensity used in the experimental groups was quantified in only three studies. These authors prescribed intensity based on percentage of one repetition maximum (1RM) , percentStrength outcomes were assessed in 9 of the 14 studies included in the literature review . Five ofNine of the fourteen studies reported outcomes measures of speed : these mExplosive movement was measured using a variety of tests (counter-movement jump (CMJ), triple jump, leg press and throwing) in 7 of the 14 studies included in this review . CMJ wasOnly three studies investigated the effects of eccentric training on change of direction performance . The invThe goal of this systematic review was to identify and evaluate the existing literature surrounding EO training interventions and their effects on performance measures in trained team sport athletes. The current evidence is in support of the inclusion of eccentric training in training programs to improve performance measures of strength, speed, power and change of direction. However, inconsistencies exist within the literature with regard to methodologies and variables of interest that need careful consideration before the results can be extrapolated to other athletes or populations. The quantification and prescription of EO intensity in well-trained athletes was only reported in four studies focused on performance outcomes ,25,30,31Maximum strength, as measured by a single maximal voluntary contraction, is influenced by both neurological and morphological factors that can be influenced through EO . Studies−1) had a greater degree of transfer than slow velocity EO training (30°·s−1) during isokinetic testing. Furthermore, one review [The velocity of EO training appears to play an important role in determining subsequent strength adaptation following the intervention. For example, Roig et al.  reviewede review  challengStudies in the current review reported effect sizes from −0.17  to 1.67 Three studies investigating NORD and strength outcomes found small to moderate improvements in trained athletes ,22,25. TThe effect and time course of training duration and number of sessions on strength outcomes following EO training is unclear, as both shorter (3 weeks) and longer 10 weeks) interventions and smaller (7)  and larg0 weeks iThe effect of EO training on measures of linear sprint ability are thought to involve numerous components . BrieflySeveral articles identified throughout the literature search were in agreement with the apparent positive effect of EO on sprint performance. Each of the nine studies included in the review reported trivial to moderate improvements in speed measures. Investigations involving short sprints (<10 m) showed trivial to moderate effect sizes. Krommes et al.  reported−1 to 210°·s−1. Askling et al. [−1) used in training interventions for speed outcomes, which resulted in a moderate effect (0.73) in trained athletes. Based on previous research investigating high- and low-velocity training [−1 may represent a relatively low training velocity. Limited availability of EO literature examining trained populations makes it difficult to draw generalisations; however, it is speculated that distinct sprint qualities may be differentially affected by EO training parameters such as velocity.Contraction velocity appears to be a contributing factor to subsequent adaptations in EO research ,6,16, wig et al.  were thetraining ,58, 60°·The storage and reutilisation of elastic potential energy during the eccentric phase of the SSC is thought to contribute to jump performance ,61,62,63However, investigations using EO have also reported no change in squat jump, counter-movement jump or rate of force development . AdditioDespite recent evidence ,70,71 onThese findings are in agreement with existing literature that has reported improvements in change of direction ability following EO training interventions. Gonzalo-Skok et al.  reportedEO appears to be an effective training strategy for athletes and sports practitioners looking to improve measures of strength, speed, power and change of direction. The review highlights evidence suggesting EO can improve performance qualities even in experienced athletes. The exact neurological and morphological mechanisms underlying these changes have been the focus of a growing body of research. Due to a large degree of variation in the existing research, a dose-response relationship for a specific method and its intended adaptation has yet to be determined in trained team sport athletes. Future research should explore the quantification of eccentric ability, the prescription of eccentric training variables and the relationship between EO-induced adaptation and performance qualities.


The incidence of intramedullary infection is increasing with increased use of intramedullary fixation for long bone fractures. However, appropriate treatment for infection after intramedullary nailing is unclear. The purpose of this study was to report the results of our treatment protocol for infection after intramedullary nailing: intramedullary nail removal, local debridement, reaming and irrigation, and antibiotic-loaded calcium sulfate implantation with or without segmental bone resection and distraction osteogenesis.We retrospectively reviewed the records of patients with an infection after intramedullary nailing treated from 2014 to 2017 at our center. Patients with follow-up of less than 24 months, received other treatment methods, or those with serious medical conditions were excluded from the analysis. Patients met the criteria were treated as described above, followed by distraction osteogenesis in 9 cases to repair bone defect. The infection remission rate, infection recurrence rate, and post-operative complication rates were assessed.A total of 19 patients were included in the analysis. All of patients had satisfactory outcomes with an average follow-up of 38.1 ± 9.4 months . Eighteen patients (94.7%) achieved infection remission; 1 patient (5.3%) developed a reinfection that resolved after repeat debridement. Nine patients with bone defects  were treated with bone transport which successfully restored the length of involved limb. The mean bone transport duration was 10.7 ± 4.0 months . The majority of patients achieved full weight bearing and became pain free during the follow-up period. Postoperative complications mainly included prolonged aseptic drainage , re-fracture  and joint stiffness, which were successfully managed by regular dressing changes and re-fixation, respectively.Intramedullary nail removal, canal reaming and irrigation, and antibiotic-loaded calcium sulfate implantation (with or without distraction osteogenesis) is effective for treating infections after intramedullary nailing. Infection after intramedullary nailing is uncommon, with a reported rate of 0.9 to 3.8% , 2. In a. However, opinions on the best treatments for Stage II and III infections vary greatly [The management of this type of infection remains controversial , 6. Makr greatly , 7, 8, e greatly , 9. ThusIn our experience, an intramedullary nail as a foreign fixator lacks an adequate blood supply, which means systemic antibiotics cannot reach the interface of the nail to eradicate the biofilm bacteria effectively . FurtherAfter debridement, there may be remaining bacterial residue in the marrow canal or the surrounding soft tissue, and it only takes about 72 h for a bacterial biofilm to develop and become mature . Due to Based on literature data and our experience, we have developed a protocol for the treatment of infections after intramedullary nailing: intramedullary nail removal, local debridement, medullary canal reaming and irrigation, and antibiotic-loaded calcium sulfate implantation, with or without secondary osteotomy and distraction osteogenesis. The purpose of this study is to describe this technique and report our results using the protocol for treating infections after intramedullary nailing.The records of patients diagnosed with an infection after intramedullary nail fixation and treated at our center from 2014 to 2017 were retrospectively analyzed. The inclusion criteria for study were: 1) Infection after initial intramedullary nail fixation and the intramedullary nail was in place when admitted to the hospital; 2) Treated as per our protocol described above; 3) Minimum follow-up of 24 months. The exclusion criteria were: 1) Patients with severe liver or kidney dysfunction, cardiovascular disease, or diabetes with uncontrolled blood glucose; 2) Patient was not treated with our described method; 3) Follow-up < 24 months. Diagnosis of infection after intramedullary nailing was based on the clinical criteria described by Metsemakers et al. [Before surgical interventions, patients were informed with the details of treatment protocols, and the informed consents were signed by patients themselves. Our study was approved by the Ethical Committee of Guangdong Second Provincial General Hospital and Nanfang Hospital.The intramedullary nail was removed first, and subsequent debridement procedures were based on whether or not the fracture had healed.In the case of healed fractures, the entry point of the intramedullary nail was enlarged. Since the reamer-irrigator-aspirator (RIA) system is not available in our country, irrigation fluid cannot be aspirated directly. Therefore, distal diaphysis fenestration was performed to allow drainage of the irrigation fluid and necrotic tissues. The medullary canal was reamed repeatedly with a larger-diameter reamer head  to completely debride necrotic tissue. Local debridement was also performed which consisted of removal of all infected bone, soft tissue, and any sinuses. Segmental bone resection was not performed in cases with healed fractures, since none of cases presented with diffuse osteomyelitis according to the Cierny-Mader classification .For cases without bone union, after intramedullary nail removal the fracture site was segmentally resected to reduce the possibility of infection recurrence . Bony de3. An appropriate amount of saline was added to the mixture, and the calcium sulfate preparation was injected into the medullary canal and the segmental bone defect. Considering the economic situations of patients and the potential cytotoxic effect of calcium sulfate, the total volume of the calcium sulfate preparation used per patient was no more than 50 ml. Vancomycin and gentamicin were both used in the preparation, in order to cover both Gram-positive and -negative bacteria.Surrounding soft tissues were also debrided. In all patients, samples were sent for culture and pathological examination. After radical debridement, the medullary canal was irrigated with saline using an impulsive irrigation gun. Elimination of the dead space caused by removal of the intramedullary nail and reaming was directly filled with antibiotic-loaded calcium sulfate . The antibiotic-loaded calcium sulfate was prepared with a ratio of vancomycin 0.5 g + gentamycin 2 ml + calcium sulfate 5 cmAfter implantation, an external fixator was used in 13 cases for avoiding debridement-related fracture (4 cases) or secondary distraction osteogenesis in 9 cases with bone defects.Intravenous antibiotics were empirically administrated until culture results were available, and then adjusted based on the results. Due to the application of antibiotic-loaded calcium sulfate, the total course of systemic antibiotics was less than 2 weeks. Patients with bony defects received an osteotomy in a secondary surgery, and bone transport was carried out 1 week after the surgery, with an initial rate of 1 mm/day, and then regulated according to the rate of bone formation and patient feedback. During follow-up, patients received testing of white blood cell (WBC) count, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), and standard anteroposterior (AP) and lateral radiographs to determine the effectiveness of infection elimination and bone formation.External fixators were removed once bones were determined to be strong enough for weight bearing. Walking-aids were recommended for the first several weeks after the operation and 1 month after external fixation removal for patients who received segmental bone resection and bone transport. For patients without bone transport, however, early rehabilitation training without any assistance was encouraged.Infection Remission was defined as the absence of any positive markers of infection, no evidence of infection on radiographs or physical examination, and a completely healed wound. Bone Union was assessed by the formation of new bone on radiographs. Infection Recurrence was defined by the presence of positive clinical symptoms, radiographic findings, and elevated levels of inflammatory markers. Continuous drainage without signs of a local infection for more than 1 month was defined as prolonged aseptic drainage.9/L. Patient preoperative data are summarized in Table A total of 19 patients who met the inclusion criteria were included in the analysis. There were 15 men and 4 women with a mean age of 39.0 ± 10.1 years . The femur was involved in 7 patients and tibia in 12 patients, and all fractures were due to some form of trauma . Nine patients suffered from infection on right extremity while 10 patients on left extremity. All patients presented with a sinus at their first physical examination, and preoperative radiographs indicated that 10 patients had healed fractures and 9 patients had unhealed fractures. The mean preoperative ESR was 49.4 ± 34.2 mm/h, mean CRP level was 33.1 ± 23.8 mg/L, and the mean WBC count was 12.75 ± 6.63 × 10Thirteen patients required external fixation, and 9 of these patients received segmental bone resection and bone transport after the infection markers became normal, and started bone transport 1 week later Fig.  aiming tStaphylococcus  was the most commonly isolated pathogen, followed by methicillin-resistant Staphylococcus aureus (MRSA) , methicillin-sensitive Staphylococcus aureus (MSSA) , and Escherichia coli . Polymicrobial infections were identified in 3 patients . The distribution of bacterial culture results are shown in Fig. A total of 20 strains of bacteria were isolated from 17 cases, with a positive culture rate of 89.5% (17/19). Coagulase-negative With a mean follow-up of 38.1 ± 9.4 months , 18 (94.7%) patients achieved infection remission after the first surgical treatment, while 1 patient (5.3%) developed infection recurrence 3 months after surgery and underwent segmental resection and bone transport. The representative cases were presented in Figs. Infection after intramedullary nailing is defined as a confined or diffuse infection of medullary cavity caused by the invasion of pathogens during intramedullary nailing. Although uncommon, it often leads to serious consequences if not treated timely and appropriately. Today’s treatment protocols of such disease vary widely according to the severity of infection and doctor’s experience, and a wide-accepted method remains unclear. In our study, we hold a more aggressive treatment method on the management of intramedullary infection since we put the infection controlling on a higher priority.After surgical debridement and antibiotic-loaded calcium sulfate implantation, 94.7% of patients achieved infection remission after surgical treatment, while only 1 patient developed a recurrence of infection and required a second surgery. We speculate that the high remission rate is attributed to radical debridement and the use of a local antibiotic delivery system. This method thoroughly removes infected tissues and eliminates residual bacteria due to a high local antibiotic concentration and long treatment duration. Such remarkable result was similar to the previous reports. Kanakaris et al.  performeNine of our 19 patients were treated with additional segmental bone resection and bone transport, and all of patients achieved infection remission, and the lengths of the involved limbs were well-restored. Although our outcomes were satisfactory, the best protocol for the management of non-healing fractures is not clear and treatments are primarily based on the experiences of individual surgeons. Those who prefer to retain the nails believe that fracture healing is more important, and that management of intramedullary infection can be postponed until after bone union. On the other hand, some surgeons believe infection control should take priority. Thus, based on different concepts treatment protocols mainly include 1) local debridement and antibiotic administration and retaining the nail until after bone union, 2) nail removal, re-reaming, and replacement of a larger diameter intramedullary nail or a resorbable antibiotic coated nail , 23, andLocal antibiotic-carriers were proposed in 1970s , 34, andProlonged aseptic drainage was the most frequent complication in our study, with a relatively high rate of 36.8%, with prior studies reporting rates ranging from 4.2 to 33% –40. In oThere are limitations of this study that need to be considered. Firstly, our outcomes were not compared with those of other surgical methods. In addition, some detailed patient data were not available because of the retrospective nature of the study, and this might influence the understanding of outcomes. Finally, our patients were heterogeneous, with different sites of infection (tibia or femur), infection with bone union or non-union, and receiving bone transport or not, all of which inevitably can lead to more complex outcomes. However, we have to point out that the emphasis of our study was to introduce an effective method to eliminate infection after intramedullary nailing, and from this perspective all patients received the same management and overall the outcomes were good.Intramedullary nail removal, medullary reaming and irrigation, and antibiotic-loaded calcium sulfate implantation seems effective in the treatment of infection after intramedullary nailing. Additional prospective studies with larger case numbers are necessary to confirm our findings.


IL10RA deficiency.Umbilical cord blood transplantation (UCBT) is associated with a relatively high rate of engraftment failure. This study aimed at exploring whether any fecal microbiota could be associated with engraftment failure following UCBT in Crohn’s disease patients with Thirteen patients were recruited and their 230 fecal samples were collected longitudinally from immediately before conditioning chemotherapy to 8 weeks post the UCBT. The V3-V4 regions of the bacterial 16S rRNA gene were amplified by PCR and sequenced, followed by bioinformatics analyses.P = 0.0176) and less abundance of Bifidobacterium longum, Bifidobacterium pseudolongum, Enterobacteriaceae_538000, and one taxon of Lachnospiraceae family was detected in the F group, accompanied by significantly higher abundances of four taxa including Lautropia, Pseudomonas, and species Microvirgula aerodenitrificans during the chemotherapy period as well as UCBT. The abundances of thirty OTUs were correlated significantly with clinical indices.Following the UCBT, 7 out of 13 patients achieved neutrophil and platelet engraftment with a median of 21 and 28 days, respectively (S group), while 6 patients failed to achieve engraftment (F group). In comparison with that in the S group, significantly lower Shannon diversity values on the UCBT day (Bifidobacterium longum, for engraftment failure would require validation. These indicators may help for the risk stratification in patients with IL10RA deficiency undergoing UCBT.Microbial indicators of reduced diversity of microbiota and signatures of specific bacterial abundances, such as a lower abundance of  IL10RA gene, the most common genetic factor of IBD in China (Inflammatory bowel diseases (IBD) are chronic relapsing inflammatory disorders in the gastrointestinal tract, and classified into Crohn’s disease (CD), ulcerative colitis (UC), and IBD-undefined. IBD can affect different ages of subjects, including infants, who usually have genetic mutations, such as the in China . Pediatrin China . Patholoin China .IL10RA-associated immune deficiency, making it a potential treatment for IBD  transplantation is emerging as an alternative therapeutic approach for the treatment of IBD . Current for IBD . Unfortupatients . Howeverpatients  and littIL10RA deficiency.It is well known that many factors, such as high number of HLA mismatches and low doses of cord blood cells, are associated with inferior transplant outcomes . HoweverIL10RA deficiency after the UCBT. In this study, we monitored the dynamic changes in engraftment and the intestinal microbiome longitudinally in 13 CD patients with IL10RA deficiency throughout the UCBT course to establish the preliminary association between intestinal microbial profiles and engraftment failure in these patients.Given that altered microbiota affect hematopoiesis and immune reconstitution in allo-HSCT recipients, modulation of intestinal microbiota may promote healthy post-transplant immunity. Accordingly, it is critical to define the unique intestinal bacterial profiles associated with engraftment failure to predict and prevent engraftment failure in IBD patients with This study was approved by the Ethical Committee of Children’s Hospital, Fudan University . Oral or written informed consent was obtained from individual parents or their guardians prior to sample collection.IL10RA deficient patients (age: 0–18 years) with CD and receiving UCBT were enrolled at the Children’s Hospital of Fudan University (China) between October 1, 2017 and December 31, 2018. Individual patients with CD were diagnosed, according to the revised Porto criteria for the diagnosis of PIBD  regimens, including fludarabine, busulfan and cyclophosphamide beginning on day -9. They received pre-medications and hydration prior to UCB infusion (day 0), as described previously . Those p9/L, and the first 7 consecutive days with a platelet count of ≥20 × 109/L without platelet transfusion , and then weekly for three times and biweekly for two times post the UCBT. All fecal samples were stored at -80°C immediately after collection.Total DNA of individual fecal samples was extracted using the standardized IHMS protocol Q recommended by the International Human Microbiome Consortium (IHMS_SOP_06_V1). Briefly, total DNA in individual fecal samples was extracted using the QIAamp DNA Stool Mini Kit  and bead-beating with 0.5 g of autoclaved zirconium beads  in a Scientz‐48 High-throughput tissue grinder . The extracted DNA was used as the templates for in triplicate amplification of the V3-V4 variable regions of the bacterial 16S rRNA gene using barcoded primers by PCR. The PCR products were purified using AMPure XP beads , and equal amount of products from each sample was pooled to form a library. After quantification and qualification using Agilent Bioanalyzer 2100 , the DNA samples were sequenced on a Novaseq 6000 platform  using the 2 × 250 bp paired end protocol.join_paired_ends.py command. The merged sequences were demultiplexed using split_libraries_fastq.py command at Phred ≥ Q20. The unique sequences were clustered into operational taxonomic units (OTUs) using pick_closed_reference_otus.py against the Greengenes database (version 13.8) at 97% identity  was the Shannon diversity index divided by the maximum diversity The raw sequencing data were processed using QIIME1 (version 1.9) . The paiidentity . The OTUidentity . Alpha d BugBase .IL10RA-deficient CD patients undergoing UCBT were assessed, 13 patients were included and their 230 stool samples were collected (13–22 samples per patient). Four patients were excluded due to ostomy (N = 2) or inadequate stool samples (N = 2). Following the UCBT, 7 out of 13 patients achieved neutrophil and platelet engraftment with a median of 21 and 28 days, respectively (S group), while the remaining 6 patients failed to achieve engraftment (F group). There was no significant difference in age, gender, body weights, BMI, disease duration, disease severity, laboratory results and medications at their admission for the UCBT (P = 0.0003) and blood hemoglobin levels (P = 0.0004) were significantly higher in the S group during the RIC chemotherapy and after the transfusion, respectively. In contrast, there was no significant difference in the median platelet counts and GVHD rates between these two groups during the observation period. While there was no patient death in the S group, five out of six patients in the F group died of the UCBT-related severe complications, such as respiratory failure (N = 2) and sepsis (N = 3). The extensive details of individual patients are shown in To determine the potential association of gut microbiota with the outcomes of UCBT in CD, 17 P > 0.05). Although the average Shannon diversity values between these two groups were similar, a hallmark of relatively stable during the chemotherapy and UCBT, the average alpha diversity on the transplant day (T0d) in the F group was significantly lower than in the S group (P = 0.0176).Analysis of the gut microbiota profiles with 16S rRNA gene sequencing revealed considerable variations of the gut microbiota alpha diversity (a wide interquartile range) in the F group during the chemotherapy period , and one taxon of the Lachnospiraceae family in the S group during chemotherapy throughout the UCBT course  with four clinical indices   . Third, IL10RA deficiency after the UCBT. Reduced diversity of microbiota and low abundance of Bifidobacterium longum whose abundance had significant positive correlation with blood hemoglobin levels might be good candidates of microbial biomarkers. If validated, these findings may be valuable for predicting and preventing engraftment failure in IBD patients following the allo-HSCT, particularly for those with deficiency in XIAP and FOXP3. We are interested in further investigating the precise biological mechanism and the clinical impact of these microbiota using new technologies. Potentially, these findings may develop new biomarkers in precociously predicting HSCT complications and guiding clinical intervention.In summary, we provided new insights into the early microbial biomarkers for predicting an engraftment failure in pediatric CD patients with The data generated during the current study are available from the corresponding author on reasonable request.This study was approved by the Ethical Committee of Children’s Hospital, Fudan University . Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.YH was responsible for study design and supervision, drafting and critical revision of the manuscript. AX contributed to study design, acquisition and analysis of clinical data, and drafting and revision of manuscript. XQ and XG contributed to data collection and analysis and drafting the article. PW, LW, CZ, ZH, WH, and JS contributed to acquisition of samples and analysis of data. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.