Formation of a barrier capable of protecting tissue from external damage, chemical factors, and pathogens is one of the main functions of the epidermis. Furthermore, upon development and during aging, mechanoprotective epidermal functions change dramatically. However, comparative studies between embryonic and adult skin in comparison to skin equivalents are still scarce which is especially due to the lack of appropriate measurement systems with sufficient accuracy and long-term tissue compatibility. Our studies fill this gap by developing a combined bioreactor and tensile testing machine for biomechanical analysis of living epithelia. Based on this tissue stretcher, our data clearly show that viscoelastic and plastic deformation behavior of embryonic and adult skin differ significantly. Tissue responses to static strain compared to cyclic strain also show a clear dependence on differentiation stage. Multilayered unkeratinized epidermis equivalents, on the other hand, respond very similar to mechanical stretch as adult tissue. This mechanical similarity is even more evident after a single cycle of mechanical preconditioning. Our studies therefore suggest that skin equivalents are well suited model systems to analyze cellular interactions of epidermal cells in natural tissues. Epithelia separate organs and tissues from one another and from the environment. Hereby they act as physical barriers with protective and regulatory functions. The skin, in particular, is permanently affected by mechanical stress and had to develop structures and mechanisms to preserve integrity . Mature From an engineering point of view, mature skin is a highly non-linear, anisotropic and viscoelastic material ,6. WhileAdditionally, despite intense research, the complex mechanical response of mature full-thickness skin under physiological conditions is poorly understood. This holds especially true at comparatively small strains. In general, in vivo measurements are performed in this small strain regime but they are often difficult to reproduce whereas measurement devices for in vitro mechanical testing almost invariably perform much better in the high strain and high force regime ,17,18. TAs an alternative for ex vivo skin samples, many biomedical studies use isolated cells or epithelial cell monolayers to investigate mutations accounting for a variety of diseases like epidermolysis bullosa ,25. To iFor this reason, we developed a highly sensitive, environmental-controlled tensile testing machine. This instrument, which we call “tissue stretcher” for simplicity reasons, allowed, in a first direct approach, to evaluate and optimally compare the mechanical properties of a cell culture-based, multilayered epidermis model system with native tissue. Besides full-thickness adult rat skin, we used skin samples of 18-day-old rat embryos.In this study, a custom-made stretching device for long-term, strain-controlled measurements of vital and even delicate tissue samples was developed and used. Adapted from our previously published stretcher for elastic silicone chambers ,30, the To calibrate the force measurement of our tissue stretcher, cuboidal elastomer samples of the polydimethylsiloxane two-component formulation Sylgard 184 , a previously well-characterized material ,31, wereAfter system equilibration for at least 30 min, the holding grip was lowered and raised in buffer without any sample attached. Upper and lower amplitude were held for 1 min each; the full cycle was repeated four times. Force values were sampled at a rate of 10 Hz. For each sequence, histograms of all forces measured in the lower position and in the upper position, respectively, were taken. The cumulative normal distribution was fitted to these histograms. Noise value reported is the mean of all standard deviations from these fits. Amplitudes from these fits were used to measure buoyancy of the grip holder.2+ (50 µM) as described previously [2+ medium (1.8 mM) to induce cell-cell contact formation. After 18–24 h, Ca2+ was depleted with EGTA  in PBS for 1 min and cells were trypsinized for 7 min . Subsequently, cells were replated in high density (4 × 105 keratinocytes per cm2) in a total volume of 800 µL on fibronectin  coated silicone elastomer chambers  as free floating sheets in the tissue stretcher, one sidewall of the chamber was removed and the multilayered sheet was gently detached from the silicone using fine forceps. Subsequently, the SEE was folded 2 times and transferred on a C-shaped PVDF-membrane frame, which served as carrier and distance piece. Together they were mounted in grip and fixture of the holding frame. The lateral ligament of the PVDF frame allowed accurate length maintenance of the specimen. After mounting, the frame was cut and the prepared sample placed in the medium-filled incubation chamber. After connecting the motor-driven load cell with the moving grip, the needle was unblocked for subsequent analysis.2 and decapitated according to local regulations; 18-day-old fetuses were directly removed and decapitated before the full-thickness back skin was carefully excised and placed in cold Hank’s balanced salt solution . To prevent the tissue from excessive mechanical stress during preparation, a removal of underlying hypodermal tissue was explicitly avoided. The explants were cut into rectangular pieces of 16 × 5 mm with its long axis in cranial-caudal orientation and stored in DMEM/F-12 medium supplemented with HEPES , penicillin (100 U mL−1), streptomycin  and FBS  at 37 °C until further use in stretching experiments within 8 h after isolation. For this, the fetal rat skin explants were transferred on a PVDF-membrane frame and mounted as described for SEEs above.The tissue used in this study was isolated from animals prepared for other projects  and no additional animals were harmed. A pregnant rat  was sacrificed by COThe adult skin samples were also isolated from the animal’s back. Therefore, a region of approximately 3 × 3 cm next to the spine was depilated and the used dimensions of 5 × 10 mm with long axis in cranial-caudal orientation were marked with waterproof ink. The skin was generously excised and placed in cold HBSS. Subsequently, tissue samples were cut to exact size and stored in medium at 4 °C until further use. All experiments with adult explants were equally performed at 37 °C after placing in the medium-filled chamber unit as described for fetal skin and SEEs. All measurements were started within 24 h after tissue isolation.Tissue explants and SEEs were fixed in formaldehyde  overnight at 4 °C (rat skins) or for 30 min at 37 °C (multilayer models). After brief washing in PBS, the adult rat skin was manually cut into thin slices with a sharp razor blade and placed laterally on a glass slide with some drops of buffer. Images of the slices and a calibrating scale were taken on a binocular microscope  equipped with a camera . The fetal rat skin was transferred in dissolved agarose  at 60 °C, filled in cryomolds  and cured at room temperature. Subsequently, the agarose blocks were cut in 50 µm slices at the VT1000 S vibratome , collected on glass slides and imaged with a light microscope . Since the multilayered SEEs were too thin for proper sectioning, the fixed cell sheets were permeabilized with Triton-X 100 in PBS  for 15 min at room temperature, washed with PBS and stained with Phallodin-Atto633  for 4 h at room temperature. Cell nuclei were stained with NucBlue in PBS  for 1 h at room temperature. After washing with PBS, the samples were mounted in Fluoromount and covered with a cover glass. Confocal z-stacks of the full thickness layer were taken at a laser scanning microscope . Thicknesses were determined as mean of several independent measurements.After transfer of the samples in the tissue stretcher and complete cut of the PVDF transfer frames, the starting sample length was set to 10 mm using a single-lens reflex camera  equipped with a macro objective . After equilibration for approximately 30 min, the respective protocol was directly started. Data points were recorded every 0.1 s. Photos of the tissues were manually taken during static stretch protocol at certain time points for every cycle. The normalization of the initial force relaxation for each strain cycle was related to each cycle’s peak force. Accordingly, the mean and standard deviation of at least 3 different samples for every data point were calculated. Furthermore, the tissue force recovery was defined as the mean peak force of a certain cycle divided by the mean peak force of the first cycle. In order to apply cyclic tensile forces, we changed the straining protocol to a trapezoidal approximation of a sine wave for some experiments. Here, the tissues were cyclically loaded and unloaded with identical velocity compared to static stretch. The dwell time at loaded and unloaded position was set to 0.4 s to result in a stretch frequency of 0.3 Hz. For further analysis and visualization, only peak forces of every cycle were plotted.−1, F7378, Sigma, USA) and ethidium bromide (10 µg mL−1) for 15 min at 37 °C. A positive control was kept in medium supplemented with 1% sodium azide for 2 h prior to staining. Imaging was directly performed after a brief washing step with fresh medium at the LSM 880 confocal microscope  with a 20× plan apochromatic objective and appropriate filter settings. The SEEs were transferred in trypsin-EDTA solution  and incubated for 10 min. Once the cell sheets started to break into pieces by smooth agitation, the SEEs were mechanically dissolved by pipetting up and down. The cells were then centrifuged and the pellet either resuspended in PBS with FBS  or in PBS with EtOH (35%) as positive control. The following viability assay was performed with the LIVE/DEAD™ Fixable Cell Stain Kit  according to the manufacturer’s protocol and analyzed by flow cytometry .For viability analysis, fetal rat skin explants were stretched and subsequently transferred in medium with fluorescein diacetate . Microscopic images of tissue sections were evaluated using Fiji ImageJ software . SchemeIn order to enable precise and sensitive tensile test measurements in a cell culture environment, we developed a new tensile test instrument for long-term, strain-controlled experiments . Here, t−1.We characterized the predicted high temporal resolution and sensitivity for force measurements with elastomer test specimens of known, well-defined Young’s moduli. The stepwise straining of such elastomer samples resulted in a linear force increase, followed by a fast stress relaxation that approached a plateau within minutes A. For reIn this study, epidermal model systems of different complexity, structure and composition were mechanically characterized and compared. The full-thickness back skin of adult rats is composed of a fully developed and sparsely cellularized dermal layer with attached hypodermal fatty tissue and a considerably thinner epidermal layer. Since the initial and non-preconditioned mechanical response should be measured in this study, mechanical stress was avoided as much as possible and adipose tissue therefore not entirely removed. We also performed some tensile tests on skin in which the subcutaneous tissue was previously separated and did not find major differences in curve progression and force levels. For this reason, we used the dermo-epidermal thickness for tissue stress estimation and neglected the attached fatty layer. The mean dermo-epidermal thickness was determined to be around 1550 µm (SD 130 µm) with the cornified epidermis of approximately 50 µm A. Even tTo characterize the mechanical properties of all skin models, we used a static stretch protocol of four repetitive cycles of 1 h strain followed by 1 h rest at zero position A. Here, Interestingly, strained tissue also recovered to its initial length during the following rest period with fastest response during the first seconds E. This cTo evaluate the influence of repetitive cycles on the force relaxation behavior of our different model systems, we normalized each stretch cycle to its initial peak force A. SurpriThe absolute force decay of repetitive static stretch cycles revealed further similarities and differences between the mechanical properties of the tested tissues. Here, fetal rat skin showed not only the most striking force decrease under stretch but also the lowest force recovery after a 1 h rest period B,D. EspeTo gain a deeper understanding of mechanical properties, we additionally applied cyclic stretch and higher strain amplitudes to subsequently analyze force relaxation A. In casDue to their inherent fragility and often low reaction forces, biomechanical testing of delicate tissue samples or cell culture tissue models under physiological conditions can be quite challenging. The instrument developed here closes the gap between classic tensile test devices known from material sciences and very sensitive experimental approaches to measure mechanical properties on a molecular or cellular level ,36,37.Our instrument exhibited a noise level of 22 µN at a sampling rate of 10 Hz. This noise in forces corresponds for simplified skin equivalents to a standard deviation in stresses of 100 Pa; for fetal skin, the corresponding value is 7.6 Pa. As can be seen, for example, in In this study, the device enabled, for the first time, a direct comparison of mature full-thickness skin, fetal skin and an ECM-free epidermal model tissue  . Please Another important aspect in which our measurements on adult skin are set apart from the bulk of the literature is the strain regime. Whereas most measurements on adult skin were performed in the high strain linear regime, where collagen fibrils are almost uniformly aligned and intra- and interfibrillary sliding are the most important mechanisms of elasticity ,10,11, wIn our experiments, we focused on physiological conditions and therefore stress levels below 30 kPa, which were largely ignored in previous studies A–D. In fAs our samples were not preconditioned, the first force relaxation curve of adult skin differed from later ones C,D. WhilInterestingly, tissue length is not equally affected and revealed an impressive recovery. Indeed, fetal skin samples reproducibly regain their initial length within the rest periods of the test protocol E. This tIn the response to applied forces, a fundamental difference between mature connective tissue and living cells emerged when we applied comparatively fast cyclic stretch instead of a constant strain. While in SEEs, higher amplitudes did not affect force relaxation, we observed a surprisingly faster relaxation when the specimens were cyclically instead of statically stretched D. HoweveIn summary, we present here a direct mechanical comparison of simplified epidermal equivalents, fetal, and adult rat skin. Despite the structural differences between these samples, mechanical behavior exhibited surprising similarities that highlight the high value of epidermal equivalents as in vitro model systems for basic research in dermatology and epidermal cell biology.


Background: Numerous vancomycin population pharmacokinetic models in neonates have been published; however, their predictive performances remain unknown. This study aims to evaluate their external predictability and explore the factors that might affect model performance.Methods: Published population pharmacokinetic models in neonates were identified from the literature and evaluated using datasets from two clinical centers, including 171 neonates with a total of 319 measurements of vancomycin levels. Predictive performance was assessed by prediction- and simulation-based diagnostics and Bayesian forecasting. Furthermore, the effect of model structure and a number of identified covariates was also investigated.Results: Eighteen published pharmacokinetic models of vancomycin were identified after a systematic literature search. Using prediction-based diagnostics, no model had a median prediction error of ≤ ± 15%, a median absolute prediction error of ≤30%, and a percentage of prediction error that fell within ±30% of >50%. A simulation-based visual predictive check of most models showed there were large deviations between observations and simulations. After Bayesian forecasting with one or two prior observations, the predicted performance improved significantly. Weight, age, and serum creatinine were identified as the most important covariates. Moreover, employing a maturation model based on weight and age as well as nonlinear model to incorporate serum creatinine level significantly improved predictive performance.Conclusion: The predictability of the pharmacokinetic models for vancomycin is closely related to the approach used for modeling covariates. Bayesian forecasting can significantly improve the predictive performance of models. Staphylococcus aureus , the administration target for vancomycin is an area under the concentration-time curve (AUC)/minimum inhibitory concentration of ≥400 h in neonates and infants . AlthougChoosing the appropriate population PK model to estimate the initial and maintenance dosage for vancomycin is essential in clinical practice; however, the performance of most of the published pharmacokinetic models is still unknown. Our research aimed to systematically evaluate the published population pharmacokinetic models of vancomycin in neonates, using data from independent cohorts collected from two clinical centers. Moreover, factors that may influence model predictability were also investigated, such as structural model selection and covariate screening approaches, to provide informed guidance for future studies.The PubMed, Scopus, and Web of Science databases were systematically searched for population pharmacokinetic analyses of vancomycin published up to October 2020. The key words “vancomycin,” “pharmacokinetic” or “pharmacokinetics” or “model” or “nonlinear mixed effect model” were used in the search strategy. The publications were included if 1) the study was a population pharmacokinetic analysis of vancomycin in neonates and 2) the article was written in English.The publications were excluded if 1) the model was not created using a nonlinear mixed-effects modeling approach, or 2) the model could not be recreated using the published information, or 3) the modeling populations overlapped or the articles were duplicated.Datasets were derived from published population PK studies conducted in neonates who received vancomycin at Shanghai Children’s Hospital between January 2013 and December 2016 , and SuzThe following information was collected in each study: gestational age (GA), PMA, PNA, current weight (WT), birth weight, dosing records, measurements of vancomycin levels, and SCR level.The doses of vancomycin ranged from 10 to 15 mg/kg, administered every 8 h or every 12 h with a 1 h or 2 h infusion duration. Peak samples were collected 1 h after completion of drug infusion, and trough samples were collected half an hour before vancomycin administration in each neonate. Trough and peak levels were determined after at least four repeated doses.Vancomycin levels were determined using a fluorescence polarization immunoassay with an Architect i2000SR . The limit of detection was 1 mg/L, and the calibration range was 3–50 mg/L. The intra-day and inter-day coefficients of variation were <20%.SCR assays were performed at the Shanghai Children’s Hospital using the enzymatic method and were analyzed with a 7,180 automatic analyzer . The calibration range was from 3 to 100 mg/L. SCR assays were performed at the Suzhou Hospital Affiliated to Nanjing Medical University using the enzymatic method and were analyzed with a 7,600 Automatic Analyzer . The calibration range was 0.08–100 mg/L.Creatinine clearance was calculated using the Schwartz formula as in †) was employed in the external model by SCR was standardized to the enzymatic method (SCR*) if the Jeff method . Statistical analysis and graphing were performed using R  and the xpose package.Data were analyzed using a nonlinear mixed-effects modeling program  compiled with gFortran  were estimated and compared with the corresponding observations (OBS) by estimating the relative prediction error (PE%) using 20 and F30 were also calculated as combination indexes of both accuracy and precision, and indicate the percentage of PE that fell within the ±20% and ±30% ranges, respectively. When the standards of MDPE ≤ ± 15%, MAPE ≤30%, F20 > 35%, and F30 > 50% were reached, the model could be determined as satisfactory and clinically acceptable.The median prediction error (MDPE) was used to evaluate predictive accuracy, whereas the median absolute prediction error (MAPE) was used to evaluate predictive precision. FA prediction- and variability-corrected VPC (pvcVPC)  was condMaximum a posteriori Bayesian (MAPB) forecasting was conducted to assess the influence of prior observations on model predictability. Patients with ≥1, 2, three observations were included in the analysis for Bayesian forecasting using zero, one and two previous observations, respectively. For a patient, the individual prediction (IPRED) of the third observation was predicted using the first and second observations, the second observation was predicted using the first observation, and then compared with the corresponding observations. The relative differences denoted by the individual prediction error (IPE%) were calculated using 20 > 35%, and an IF30 > 50% were used for MAPB forecasting.To evaluate the predictability of the candidate models when prior information is increased, the standards of an IPE% (MDIPE) ≤ ± 15%, an IPE% (MAIPE) ≤ 30%, an IFDifferent modeling strategies were used in previous studies, which may affect the predictive performance of the model. To explore the impact of these different modeling strategies, we evaluated the predictability of various structural models and covariate models employed in previous studies. The assessment methods include the aforementioned prediction-based diagnostics and Bayesian forecasting methods.After a systematic literature search, 18 neonatal vancomycin models  were incMost studies employed sparse sampling strategies. Six models were established with a two-compartment model (2CMT), whereas 12 models were established with a one-compartment (1CMT) model.Weight, age, and renal function were the most important covariates for clearance identified in the previous studies. Maturation models were employed in 11 studies and could be described by 50 is the value of PMA when clearance maturation reaches 50% of adults; Hill is the slope parameter for the sigmoid Emax maturation model.Weight (current weight and birth weight) and age  were regarded as the main factors for body size and maturation, respectively. The current weight was used in most of the reported studies. PMA was chosen over GA and PNA as it presented the most parsimonious way to account for both antenatal and postnatal maturation, which can be incorporated as a sigmoid Emax model and asymptotic exponential model, as shown in Renal function was often presented by SCR in reported studies and was included in nonlinear manner.The characteristics of each study are summarized in The study population consisted of 171 neonates in whom there were 319 assessments of vancomycin levels. Of these, 80 neonates with 165 vancomycin levels were from Shanghai Children’s Hospital (SH), and 91 neonates with 154 vancomycin levels were from Suzhou Hospital Affiliated to Nanjing Medical University (SZ). The proportion of preterm neonates was larger in the SZ group than in the SH group. The patient demographics of both cohorts are shown in 20 > 35%, and F30 > 50%. Nine models showed good predictive accuracy, with an MDPE of less than ±15%. However, MAPE was more than 30% in all models, indicating a poor predictive precision for all models. Of note, the model reported by 20 > 30% and F30 > 45%, showing better accuracy and precision of predictability than other models. The boxplot for PE% for each model is shown in There were large differences in predictability of the different models. As can be seen from the results of the prediction-based diagnostics shown in In the case of simulation-based diagnosis, the pvcVPC showed significant differences between observations and model simulations in all reported studies . A clear20, and F30 compared favorably with the prediction-based diagnostics, indicating that the predictive performances had improved, as shown in 20 > 35%, and an IF30 > 50% after Bayesian forecasting with one or two prior observations. The box plots for predictability are shown in In total, 171 neonates and 171 observations, 112 neonates and 224 observations, 24 neonates and 72 observations with zero, one, two previous samples, respectively, were included in the Bayesian forecasting. After Bayesian forecasting with one or two prior observations for all models, the mean values of MDPE, MAPE, FThe structural model employed in the previous studies included the 1CMT model or 2CMT model. We evaluated the predictive performance of these two models by establishing 1CMT and 2CMT base models using the external data. Because trough concentrations could not fully describe a two-compartment model, the volume of distribution of the central compartment was fixed at 1.27 L, and the inter-compartmental clearance was fixed at 1.161 L/h in the 2CMT model, according to the study by Song et al. . The MDPTo assess the predictive performance of the various covariate models, we developed models with the identified covariates  and corresponding formulas  based on the 1CMT or 2CMT structural models. The maturation model was used by 20 and F30 were improved significantly compared to the base 1CMT model (F20: 27.65% vs. 14.12% and F30: 44.71% vs. 19.41%), or that of the 2CMT model (F20: 30.0% vs. 14.12% and F30: 43.53% vs. 19.41%). Moreover, the predictive performance of the model with WT and SCR included in a nonlinear fashion was much better than in the model where they were included in a linear fashion , and immunosuppressive agents .20 and F30 improved by 30%–50% compared with the linear model.Body size is a pivotal index for the CL and V of vancomycin in neonates. Comparing different covariate modeling approaches, nonlinear models, especially the maturation model, showed much better predictability than the linear model. When the maturation model was adopted, FFor drugs with narrow therapeutic windows, weight-based dosing is most commonly used for neonates because it is easy to perform. However, some studies have reported that there are adverse drug reactions related to weight-based dosage regimens for children, especially for drugs with narrow therapeutic ranges, leading to ineffective treatment outcomes and even fatalities in some cases .As body weight does not fully describe organ maturity, the maturation model could better explain the physiological status of early, slower growth and subsequent faster growth in neonates . It alsoRenal function is also a very important factor affecting the pharmacokinetics of vancomycin, since vancomycin is mainly eliminated via the kidney. Creatinine clearance is used as index to describe renal function in adult patients; however, in neonates, SCR levels are a more reliable indicator of renal function, which is consistent with the findings of previous population PK studies. It has previously been shown that incorporating SCR in CL in a nonlinear fashion is better than incorporating it in a linear fashion. This finding also supports the fact that renal function matures in a nonlinear manner in neonates.Our study has several limitations. As mentioned previously, the creatinine determination method (Jeff and enzymatic method) has been shown to have a large impact on external predictability . In thisBased on our study, the published models performed poorly in prediction-based and simulation-based diagnostics. The maturation model based on weight, age, and nonlinear incorporation of SCR had better predictability than other modeling approaches. Moreover, the Bayesian method significantly improved the predictive performance of the published models, and could thus play an important role in vancomycin dosing recommendations and guiding clinical practice.


Previous epidemiological studies have demonstrated that the lower serum concentration of vitamin D was associated with elevated risk of open-angle glaucoma (OAG). However, few studies have examined the association between aqueous humor vitamin D concentrations and OAG. Hence, we investigated the relationship between 25-hydroxyvitamin D (25(OH)D) concentrations in aqueous humor and OAG. We measured 25(OH)D concentrations in aqueous humor and serum of 126 patients who underwent cataract surgery. 36 were patients with OAG and 90 were control patients. The 25(OH)D concentrations were measured using Elecsys Vitamin D Total Kits with the Cobas e602 module , an electrochemiluminescence assay. Multiple linear regression analysis was performed to investigate factors associated with serum and aqueous humor 25(OH)D concentrations. Patients with OAG had significantly lower 25(OH)D concentrations in aqueous humor than control patients. Serum 25(OH)D concentrations were higher in patients with OAG than in the control, but this was not statistically significant. 25(OH)D concentrations in aqueous humor of patients with OAG were significantly associated with axial length but not with glaucoma severity, which was determined by the retinal nerve fiber layer thickness or mean deviation. Vitamin D concentrations in aqueous humor of patients with OAG were significantly lower than those in patients without OAG. The core event in glaucoma is irreversible damage of retinal ganglion cell axons, which carry visual information from the eye to the brain, due to elevated intraocular pressure (IOP)2. Depending on the mechanism of increasing IOP, glaucoma could be classified into two categories: angle-closure glaucoma (ACG) or open-angle glaucoma (OAG). Unlike ACG, which has a narrow iridocorneal angle, OAG is characterized by increased resistance in the trabecular meshwork (TM), which causes an elevation of IOP. Several studies have suggested that changes in the concentration of various molecules in aqueous humor, such as vitamin C3, hyaluronic acid4, transforming growth factor β (TGF-β)5, and endothelin-16, increase TM resistance.Glaucoma is a leading cause of irreversible blindness worldwide, contributing to approximately 10% of cases of legal blindness registered in the United States8. The vitamin D status is usually evaluated by measuring 25(OH)D concentrations. Its deficiency affects bone and mineral metabolism; however, recent studies have reported that vitamin D insufficiency is associated with various systemic diseases. Adequate vitamin D intake can prevent diseases, such as myocardial infarction, stroke, diabetes mellitus types 1 and 2, infectious or chronic respiratory diseases, and autoimmune diseases12. Furthermore, the serum concentrations of vitamin D has been linked to the prevalence or occurrence of eye disorders, such as diabetic retinopathy, age-related macular degeneration, myopia, and dry eye syndrome16. In particular, several studies have demonstrated that the serum concentration of vitamin D is associated with OAG22.Vitamin D does not exhibit biological activity until two-step hydroxylation occurs. Following the hydroxylation of vitamin D into 25-hydroxy vitamin D [25(OH)D] in the liver, the vitamin D metabolite is transported to kidneys where it is converted to 1α, 25-dihydroxyvitamin D , which is an active form of vitamin DHowever, most of these studies were only epidemiological investigations of the association between serum vitamin D concentrations and eye diseases; few studies have examined the association between aqueous humor vitamin D concentrations and eye diseases. To the best of our knowledge, no studies have reported on the relationship between the aqueous humor vitamin D concentration and glaucoma. In the present study, we measured 25(OH)D concentrations in aqueous humor of patients with cataract with or without OAG. We then performed a comparative analysis of the 25(OH)D concentrations in the two groups to determine whether there was a correlation with the presence of OAG.p =  < 0.01), and CCT was significantly lower (p = 0.03) in the OAG group than in the control group. In contrast, there was no significant difference between the two groups in terms of IOP, ECC, and ACD D concentrations were 21.22 ± 12.18 ng/mL in the OAG group and 18.12 ± 9.55 ng/mL in the control group, which was slightly but not significantly higher in the OAG group , whereas the concentration became statistically lower as the AL increased  D concentrations in aqueous humor was conducted for all patients, and the results showed that the presence of OAG had a significant effect on the 25(OH)D concentration in aqueous humor D concentration in aqueous humor of patients with cataract with glaucoma was significantly lower than that in patients with cataract without glaucoma, while the presence of OAG had no significant correlation with the serum 25(OH)D concentration. The 25(OH)D concentration in aqueous humor of patients with OAG exhibited a significant correlation with AL. However, no statistical significance between the aqueous 25(OH)D concentration and glaucoma severity  was detected.. reported that reduced serum vitamin D concentrations increased the risk for developing primary OAG17. However, no other study has investigated the relationship between glaucoma and aqueous vitamin D concentration. Vitamin D concentrations in aqueous humor may have a stronger correlation with glaucoma than its concentrations in serum. First, aqueous vitamin D can have a direct effect on tissues related to the pathophysiology of glaucoma, such as the ciliary epithelium, iris, and TM. Second, there is a blood–aqueous barrier in the eye, which makes it difficult for substances present in the serum to pass directly into aqueous humor; as a result, the serum concentration does not completely reflect the concentration of aqueous humor. Reviews of previous studies on 25(OH)D indicate a lack of any significant correlation between the serum and aqueous humor vitamin D concentrations. Cho et al. investigated differences in the concentrations of 25(OH)D in aqueous humor and serum of senile and diabetic patients with cataract23. Thus, this study could be used as basic reference data for studies on the relationship between glaucoma and aqueous humor vitamin D concentrations.The main purpose of this study was to determine whether the aqueous humor vitamin D concentration differs depending on the presence of glaucoma; further investigation was made into whether this difference was related to the duration of glaucoma, medications, or severity of glaucoma. Previous studies on the association between vitamin D and glaucoma generally investigated the correlation between serum concentrations and the incidence or progression of glaucoma. Among the previous studies, Yoo et al. examined the relationship between diabetic macular edema and vitamin D concentrations in aqueous humor. They reported that diabetic macular edema was associated with high concentrations of vitamin D in aqueous humor and that the aqueous vitamin D concentration might be another indicator of diabetic macular edema severity24. In the present study, the 25(OH)D concentration in aqueous humor of patients with cataract with OAG was significantly lower than that of control patients. However, in this study, there was no correlation between the 25(OH)D concentration in aqueous humor and indirect measures of glaucoma severity, i.e., RNFL thickness and MD values.There are few studies on the relationship between aqueous vitamin D concentration and several eye diseases. Recently, Kim et al26. In particular, when the concentrations of oxygen radicals in aqueous humor increase, the TGF-β-SMAD3 pathway is activated in TM cells, which results in structural changes that increase the levels of ECM proteins, such as laminin, fibronectin, and myocilin. Such changes cause fibrosis of TM cells and increase the resistance of aqueous outflow, which then lead to increased IOP. However, when 1,25-(OH)2D3 is added after treating the oxygen radicals of TM cells, VDRs are activated and block the activation of the TGF-β-SMAD3 pathway, which then prevents fibrosis in TM cells27. Although structural changes in TM cells could be prevented when the concentrations of vitamin D in aqueous humor are high, at low concentrations, these changes in TM cells may increase the risk for glaucoma. However, to the best of our knowledge, no studies have measured vitamin D concentrations in aqueous humor of patients with glaucoma. Therefore, the findings of the present study may support this hypothesis. In addition, a previous study reported a decrease in IOP when topical 1,25-(OH)2D3 or its analog was injected in an animal experiment, and another study demonstrated an increased concentration in aqueous humor after the oral injection of vitamin D in rabbits. These findings indicate that vitamin D may be a potential candidate for developing a novel treatment for glaucoma.Several experimental studies have provided evidence of the correlation between decreased vitamin D concentrations in aqueous humor and glaucoma development. The changes occurring in TM cells, such as apoptosis, compound accumulation in extracellular matrix (ECM), and cytoskeletal disruption, are primary mechanisms involved in OAG developmentIn this study, the glaucoma group was 36 subjects, so the precision or power of the results may be low due to the small number of subjects. Further study with larger sample size might be required for more precision.As all patients had cataract, it was difficult to eliminate the effect of cataract on the changes in vitamin D concentrations. In this study, the glaucoma group included only stable glaucoma patients who had no history of laser treatment or glaucoma surgery and who did not progress only with medications. If the study included patients with progressive visual field defect, or if the aqueous humor was collected from patients undergoing glaucoma surgery, the concentration of 25(OH)D could be measured lower than the results of this study. In addition, the correlation between the concentration of 25(OH)D in the aqueous humor and the MD values or RNFL thickness may have been statistically significant. Based on the results of this study, we plan to conduct a study by collecting a aqueous humor from patients undergoing glaucoma surgery for progressing glaucoma despite maximal antiglaucoma medication and measuring the 25(OH)D concentration. Furthermore, the effect of vitamin D transport on the various antiglaucoma topical medications used by patients with OAG could not be eliminated; In this study, OAG and control group showed a statistically significant difference in axial length values and the multivariate linear regression analysis revealed that there was a significant association between axial length and vitamin D concentration in aqueous humor. Previous studies demonstrated a strong relationship between vitamin D and myopia, so the potential role of longer axial length in determining lower concentrations of vitamin D in OAG group cannot be completely excluded in our study. The previous investigations that reported an association between OAG and serum vitamin D concentrations were generally large-scale studies, whereas the present study included a small number of participants. Therefore, it is challenging to make accurate judgments about serum vitamin D concentrations. If a large number of participants were included in this study, different results may have been obtained for serum vitamin D concentrations. Moreover, we did not survey patients regarding their dietary intake of vitamin D, which could have had a significant influence on the test results.In conclusion, vitamin D concentrations in aqueous humor of patients with cataract with glaucoma were significantly lower than those in patients with cataract without glaucoma. These findings suggest that future studies should explore the use of vitamin D as a candidate therapy for glaucoma treatment.This prospective study included 126 patients who underwent uneventful cataract surgery at Gyeongsang National University Hospital between January 2017 and December 2019. This study was approved by the Gyeongsang National University Hospital Institutional Review Board (2017-01-011), and adhered to the principals of the Declaration of Helsinki. Informed consents were received from all enrolled patients. The OAG group included patients who were diagnosed with primary open angle glaucoma (POAG) or normal tension glaucoma (NTG). The inclusion criteria for subjects with OAG were the characteristic visual field loss, glaucomatous optic neuropathy, and IOP > 21 mmHg. The inclusion criteria for subjects with NTG were visual field loss, glaucomatous optic neuropathy, and IOP ≦21 mmHg. Eyes with glaucomatous visual field defects were defined as (1) outside of the normal limits on the glaucoma hemifield test; (2) a pattern of standard deviation of < 5%; or (3) a cluster of 3 points with probabilities of < 5% in at least 1 hemifield on the pattern deviation map, including at least 1 point with a probability of < 1% or a cluster of 2 points with a probability of < 1%, as confirmed by at least 2 reliable visual field examinations. In these patients with glaucoma, glaucomatous visual field defects did not progress and IOP was stably controlled with antiglaucoma medications. The patients were also provided questionnaires to assess their occupation, regular daily activity (average time the patient was exposed to sunlight during the day), and use of vitamin D supplements. Of the patients who had undergone cataract surgery and ophthalmic examinations, we examined the medical records of patients who were receiving treatment for OAG.Patients who had diseases related to vitamin D, such as bone metabolism disorders or hyperparathyroidism, and patients using vitamin D supplementation were excluded. In addition, we excluded patients who had other ophthalmic diseases , as well as patients who underwent ophthalmological surgeries or laser treatments. In addition, patients with brunescent or hypermature cataract were excluded. Finally, cases in which the test was impossible to perform because the amount of collected aqueous humor was too low were excluded from this study.23, we targeted only senile cataract patients, excluding those with diabetic cataract or complicated cataract.In addition, based on a previous report that the vitamin D concentration in aqueous humor could be affected by the type of cataract23. All participants provided complete medical histories and underwent ophthalmologic examination, including measurements of best-corrected visual acuity and IOP, as well as slit-lamp biomicroscopy and fundus examinations. Axial anterior chamber depth (ACD) and central corneal thickness (CCT) were measured using the Pentacam system . Ocular biometry and axial length (AL) measurements were performed using the IOL Master system . The number of corneal endothelial cells was determined using CellChek XL . RNFL and macular thickness were measured using Spectralis Optical coherence tomography .All procedures were conducted as described previouslyA single surgeon (S.J.K.) performed all cataract surgeries. After irrigation with 5% povidone and a balanced salt solution, approximately 0.15 mL of aqueous humor was collected using a 30-gauge needle through the clear cornea near the limbus. A 2.2-mm clear corneal incision was created, and two side ports were formed. A continuous curvilinear capsulorrhexis was made using a capsulorrhexis forceps. After hydrodissection, the nucleus was phacoemulsified and the residual cortex was aspirated with an Infiniti emulsifier . After filling the anterior chamber with viscoelastics, a single-piece hydrophobic acrylic IOL was inserted into the capsular bag.23. Immediately after collection, all aqueous humor samples were transferred to the laboratory, where their 25(OH)D concentrations were measured. In contrast, all serum samples were collected and stored at − 70 °C until analysis. The 25(OH)D concentrations were measured using Elecsys Vitamin D Total Kits with the Cobas e602 module , which is an electrochemiluminescence assay that includes the ruthenium-labeled vitamin D-binding protein, biotin-labeled vitamin D, and streptavidin-coated microparticles. The coefficients of variation for four concentrations  of intra- and interassay were 1.7–7.8% and 2.2–10.7%, respectively.Laboratory analyses were conducted as described previouslyt-test or Fisher’s exact test .Clinical characteristics are presented as mean and standard deviation when they were continuous variables. Categorical variables were presented as number and proportions. The characteristics of patients with cataract and OAG were compared using independent To identify factors related to the vitamin D concentration in aqueous humor, univariate linear regression analysis was performed for each variable, and significant variables were included in the multivariate linear regression model and analyzed. However, the serum vitamin D concentrations were corrected by including them in the model regardless of their significance. Regression analysis was performed in all subjects . A two-tailed All procedures performed in studies involving human participants were in accordance with the ethical standards of the Gyeongsang National University Hospital Institutional Review Board (2017-01-011), and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.


Desmognathus) currently comprise only 22 described, extant species. However, recent mitochondrial and nuclear estimates indicate the presence of up to 49 candidate species based on ecogeographic sampling. Previous studies also suggest a complex history of hybridization between these lineages. Studies in other groups suggest that disregarding admixture may affect both phylogenetic inference and clustering‐based species delimitation. With a dataset comprising 233 Anchored Hybrid Enrichment (AHE) loci sequenced for 896 Desmognathus specimens from all 49 candidate species, we test three hypotheses regarding (i) species‐level diversity, (ii) hybridization and admixture, and (iii) misleading phylogenetic inference. Using phylogenetic and population‐clustering analyses considering gene flow, we find support for at least 47 candidate species in the phylogenomic dataset, some of which are newly characterized here while others represent combinations of previously named lineages that are collapsed in the current dataset. Within these, we observe significant phylogeographic structure, with up to 64 total geographic genetic lineages, many of which hybridize either narrowly at contact zones or extensively across ecological gradients. We find strong support for both recent admixture between terminal lineages and ancient hybridization across internal branches. This signal appears to distort concatenated phylogenetic inference, wherein more heavily admixed terminal specimens occupy apparently artifactual early‐diverging topological positions, occasionally to the extent of forming false clades of intermediate hybrids. Additional geographic and genetic sampling and more robust computational approaches will be needed to clarify taxonomy, and to reconstruct a network topology to display evolutionary relationships in a manner that is consistent with their complex history of reticulation.Dusky Salamanders  contain only 22 described species, but previous phylogenetic research suggests up to 49 candidate species. With a genome‐scale dataset, we find support for at least 47 species‐level lineages with a complex history of hybridization and admixture. These reticulations continue to confound attempts to untangle historical evolutionary relationships. Consequently, we strongly suspect that many relationships in the group are characterized by non‐level‐1 networks, and therefore cannot be estimated by current methods  model assuming incomplete lineage‐sorting (ILS) as the primary driver of gene‐tree discordance, in the program ASTRAL‐III v5.7.7  as well as determining the optimal value of K minimizing median cross‐entropy across 100 replicates, if these differed. In a few cases where an elbow did not form, we selected the lowest value of K representing a significant improvement in cross‐entropy using the ‘notch’ test of the boxplots .Our final aim is to estimate the prevalence and location of hybrid individuals, and the overall degree and spatial extent of genomic admixture between candidate species. We approached this with both individual‐ and taxon‐based approaches. First, we used the ‘sNMF’ algorithm in the R package ‘LEA’ to estimate individual ancestry coefficients for each specimen . Accurately identifying such models is still challenging, and similar distributions of allele frequencies can be generated by a variety of processes, such as variation in substitution rates  was related to the amount of hybridization within that clade. We measured imbalance as Colless’ I, normalized proportional to distinguishable arrangements, rather than a Yule process which is likely inappropriate at the phylogeographic level  will cluster together, which would preserve the expected pattern. Second, we tested whether the imbalance of the subtrees for each of the 47 mito‐nuclear candidate species  suggests that numerous hybrid individuals occupy intermediate positions as an artifactual consequence of their admixed ancestry, which cannot be resolved adequately by concatenation or species‐tree analysis. We estimate that 169 of the 894 specimens have >20% ancestry from a secondary phylogeographic lineage or candidate species Figure . Chief ai Figure  clades  between clade level admixture  and Colless’ I normalized by the PDA model  and a restricted lineage in the southern Nantahala mountains. We refer to these as wrighti A1 & A2, respectively. A small amount of admixture is estimated both between wrighti A1 & A2 and between organi and wrighti A1. Whether the latter represents ILS or possible genetic contact across the Asheville Basin as recently as the LGM is unclear  with two phylogeographic lineages. The sNMF analysis estimated the quadramaculatus A1 & A2 lineages east and west of the Little Tennessee River valley as in our previous study  ancestry with roughly equal genomic contributions from the other six lineages, though a small plurality from marmoratus C (~30%). We observe significant admixture  between quadramaculatus C & E, C & G, D & E, and E & G; between marmoratus E/H & quadramaculatus E; between marmoratus E/H & quadramaculatus G; and between marmoratus C & quadramaculatus C. Second, sNMF estimates two phylogeographic lineages quadramaculatus E1/E2, distributed approximately east and west of the boundary between the Upper French Broad/Nolichucky River drainages, with extensive hybridization between them. This region is also the contact zone between carolinensis and orestes  from several other lineages that stack on the end of the barplot or occur together as a single cluster of slices in the pie chart. We generally do not interpret this further as evidence of admixture but note that it may have several distinct causes. First, it may represent actual evidence of small amounts of individual ancestry from those estimated clades. Second, it may represent noise in the algorithm or result from sequencing error, missing data, or ILS. Third, it may represent real individual ancestry from lineages not included in the comparison, such as other candidate species or ghost admixture from extinct lineages  by the admixture analyses in sNMF  of individual ancestry from abditus, but we do not consider this significant evidence of admixture between them at present. While we treat both candidate species together in this analysis, they are not sister lineages in the concatenated phylogenetic estimate presented here, but rather successive divergences  are distributed primarily in the Piedmont and Atlantic Coastal Plain south and east of the Blue Ridge Mountains. Within these five candidate species as previously defined, all are reciprocally monophyletic in the concatenated phylogeny, with auriculatus A forming the sister lineage to the remaining groups on a relatively long branch , one primarily in the southern Blue Ridge foothills of western North Carolina and the Piedmont and Coastal Plain of South Carolina (C3), and one primarily in the Piedmont and Coastal Plain of northern North Carolina and southern Virginia (C1).The second clade of  Figures , 15, 16.fuscus C contains ~40% ancestry from auriculatus B/C, while another early‐diverging sample has ~30%. Significant admixture is also observed between fuscus C & D and between auriculatus B/C and both fuscus C and D. Finally, one specimen of auriculatus A is estimated to have ~14% ancestry from fuscus C. While this does not meet our 20% threshold for significance, it is remarkable in potentially corroborating a previous finding of a sister‐group relationship between auriculatus A and fuscus C in a previous phylogenetic network analysis . Additional sampling of populations and individuals is desirable to evaluate possible gene flow between valentinei and valentinei B and possibly between either and brimleyorum.As in recent concatenated, species‐tree, and network analyses, 3.2.10ocoee species group estimated here includes apalachicolae, monticola A/C & B, and ocoee D, E, and F/G/H, as in most previous mitochondrial (excluding ocoee D) and nuclear concatenated, species tree, and network analyses  was not sampled in our previous work , and several have streaks.The admixture analyses in sNMF estimate two phylogeographic lineages k et al. ’s “ocoeemonticola A/C & B are both reciprocally monophyletic sister lineages here, with a few admixed specimens at a narrow contact zone in northern Georgia at the transition from the Piedmont to the Blue Ridge mountains. Several monticola B specimens have streaks of ~20% non‐monticola ancestry. Whether this represents admixture from the other ocoee lineages, unaccounted for introgression from a non‐ocoee species, noise in the SNP data, or uncertainty from the admixture algorithm remains unclear.The previously defined candidate species apalachicolae and ocoee E & F/G/H  represents a difficult proposition for delimiting candidate species. First, ocoee F/G/H is the monophyletic sister lineage to the remaining candidate species, and none of the sampled specimens have significant ancestry from any other single lineage. As with the monticola B and ocoee D specimens described above, one ocoee F/G/H has a ~40% “streak” of non‐F/G/H ancestry. The geographically expanded apalachicolae  is not monophyletic and exhibits extensive admixture with ocoee E2 (see below) which it contacts parapatrically. The southern form  is monophyletic and nested within apalachicolae A2, and does not exhibit significant  admixture with any other lineage. A possible exception is the two earliest diverging specimens which are estimated to have ~10% ancestry from apalachicolae A2.The clade comprising ocoee E contains two phylogeographic lineages, ocoee E1 & E2, in the southern Nantahala mountains of the Blue Ridge. The first, ocoee E1, is the more northerly and contacts ocoee F/G/H parapatrically, while ocoee E2 occurs near the Georgia/North Carolina border, parapatrically contacting ocoee E1 to the north, apalachicolae A2 to the south, and ocoee D1 to the east. Additional sampling is needed to clarify which lineages occur to the west of ocoee E2. Both ocoee E1 & E2 exhibit extensive admixture with each other across their contact zone. While several early‐diverging specimens of ocoee E1 exhibit estimated individual ancestry from ocoee F/G/H and apalachicolae A2, they do not meet our 20% threshold for further interpretation. In contrast, one specimen of ocoee E2 has ~20% ancestry from apalachicolae A2, and one specimen of ocoee E1 contains ~30% ancestry from ocoee D2. Taken at face value, these patterns suggest extensive genetic contact between these lineages over large distances.Finally, 3.2.11ocoee A & B is estimated here as the sister lineage of the conanti species group  without any evidence of significant admixture . While we do not directly compare those candidate species here to estimate individual ancestry coefficients, the Dsuite results suggest an additional facet of complex history for this group to be examined in the future.The enigmatic clade of p Figure , as in pp Figure . This ise Figure . However3.2.12conanti species group  represents perhaps the most complex and challenging set of candidate species and phylogeographic lineages. This group was first addressed in detail, in part, by Tilley et al.  individual ancestry from any other lineage, but the two earliest diverging specimens have ~10% from santeetlah and conanti F, respectively. A series of conanti A2 specimens also exhibit “streaks” of mixed ancestry along with admixture from conanti A1.First, santeetlah form a monophyletic group, with most of those from the core range of the species centered on the Great Smoky Mountains exhibiting exclusive santeetlah ancestry. In contrast, the four earliest diverging specimens exhibit significant admixture with ‘beta,’ carolinensis, conanti F, and conanti A at the contact zones between those lineages and santeetlah. Accordingly, another deeply nested specimen exhibits significant individual ancestry from ‘gamma’ at the contact zone between the two lineages. The sampled specimens of the geographically distinct candidate species conanti E found exclusively in lowlands west of the Mississippi River are monophyletic and do not exhibit significant admixture from any other lineage, though the two individuals close to the Mississippi exhibit “streaks” comprising ~15%–25% of their total ancestry.Similarly, the sampled specimens assigned to conanti B/C/D form reciprocally monophyletic sister candidate species. As previously defined based on more limited sampling  are more closely related to carolinensis in the concatenated phylogeny. These individuals have significant genomic ancestry from conanti F, carolinensis, ‘beta,’ and conanti A, and notable amounts (~10%) from conanti E. Similarly, the earliest diverging specimen of carolinensis contains notable admixture (~10%) from ‘beta’ and conanti F, respectively, while several other carolinensis individuals also possess ~10%–20% individual ancestry from ‘beta.’ These admixed individuals occur around the geographic contact zones between these lineages (except the distant conanti E), suggesting complex hybridization or other genomic admixture dynamics in southwestern North Carolina. In contrast, at least one specimen with ~100% conanti F ancestry occurs sympatrically with specimens exhibiting ~100% conanti A2 ancestry, suggesting that these lineages can nonetheless maintain genetic distinctiveness in close geographic proximity.Finally, the sampled individuals of ‘beta’ and the primary monophyletic group of sampled 3.3Desmognathus, and present additional insight for future targeted analyses. The matrix of fb values can be read to indicate the proportion of alleles shared between a donor species in the columns and the recipient branch in the rows in excess of that predicted by the MSC model  being supported most strongly, but with conanti B/C/D, ‘gamma,’ santeetlah, and conanti A also being implicated.First is the complex interplay within and between candidate species from conanti species group from the Balsam clade (ocoee A & B), most strongly from ocoee B. Given the partial symmetricity of the matrix, the reverse scenario is also possible, with ocoee B receiving alleles from one or more conanti‐group lineages. This is likely related to the ghost admixture scenario estimated in a previous network analysis in TreeMix  are all implicated in contributing alleles to species in the Balsam and conanti clades. As this result is not significantly symmetric, the directionality may be more meaningful. This partially corroborates previous network analyses in SNAq that estimated conanti B/C/D arising as the result of a hybrid speciation event between ocoee F/G/H and ‘gamma’ , which is strongly corroborated in the admixture analyses  for clustering analysis yielded 3126 variable sites. However, the two specimens (USNM 468094–5) only overlapped at a few sites, because successful reads for each specimen generally mapped to different loci or different parts of the same locus. We, therefore, divided the dataset into fully sampled SNP matrices for each specimen, yielding 1176 SNPs from 56 loci for USNM 468094 and 1605 from 79 for USNM 468095. The phylogenetic and clustering analyses both strongly estimated membership of these specimens in auriculatus A .As noted above, extraction and library preparation for the two fluid‐preserved specimens was modestly successful but yielded sufficient coverage for only 55% of the loci (129/233) and just 5% of the total alignment . Pruning this reduced alignment to the relevant clades (fuscus C ancestry in USNM 468094 (16%) and 468095 (25%), the existence of which was hypothesized in our previous study  by attempting to sequence more loci to greater depth while accounting for DNA degradation  loci. This study had three primary aims: (i) to objectively evaluate the distinctiveness of the 49 previously delimited mito‐nuclear candidate species, (ii) to estimate the presence of phylogeographic lineages therein and the prevalence of admixture both within and between candidate species, and (iii) to estimate the possible impact of both deep‐time reticulation and recent gene flow on topological estimates within and among clades. We estimate 47 candidate species supported by monophyly in the concatenated ML topology of the AHE dataset, all of which coincide with estimates of distinct genomic ancestry and partial to complete genealogical exclusivity in the admixture analyses. However, many of the 47 candidate species contain significant geographic genetic structure, with up to 30 phylogeographic lineages within 13 of those 47 taxa, and therefore as many as 64 geographically and genetically distinct population segments within Desmognathus , and appear to have clear genetic and geographic boundaries as candidate species, albeit permeable ones , but with marmoratus G having perhaps the strongest evidence for being of hybrid origin, and therefore potentially being considered conspecific with marmoratus C. However, the difficulty of treating multiple morphologically similar but distantly related candidate species that hybridize in parapatry without any apparent geographic, ecological, or microhabitat partitioning  is a thorny conceptual challenge.The first is the Pisgah clade of ocoee clade, with respect to apalachicolae and ocoee D, E, and F/G/H. These candidate species and their constituent phylogeographic lineages each exhibit signatures of geographically distinct genomic ancestry, while simultaneously exhibiting admixture across broad parapatric contact zones in the southern Blue Ridge mountains. While some peripheral, Piedmont, and Coastal Plains populations exhibit more distinctive phenotypes , or possibly an additional distinct candidate species endemic to the foothills of the Blue Ridge in Georgia.A similar situation arises in the conanti species group  all exhibit extensive admixture at their contact zones in parapatry, including a region in southwestern North Carolina characterized by 4‐ or 5‐way hybrids in our admixture analyses. Interaction between this group and the ocoee A & B and F/G/H lineages and fuscus C is also indicated by our Dsuite analyses and previous network estimates , a larger “dusky” morphology with a laterally compressed tail , and the distinctively colored santeetlah  that have yet to be estimated in any network or admixture analyses. There are numerous other putative instances of hybridization between distantly related but sympatric or parapatric species pairs  of hybrid ancestry from some s Figure . Our pres Figure  using Phs Figure  actuallyauriculatus A populations (such as the former fuscus subspecies “carri”) may have limited our ability to recover this signal  examine these putative taxa for additional phylogeographic lineages and the existence and extent of hybrid zones, and (iii) assess the impact of introgression on the reconstruction of bifurcating phylogenetic topologies. We find that previous estimates have converged on a roughly stable estimate of species‐level diversity in the genus and corroborate the existence of 47 candidate species. Many of these candidate species exhibit extensive admixture with each other along their geographic margins, and in many cases with non‐sister or even distantly related clades. Similarly, many candidate species contain significant geographic genetic structuring, with multiple phylogeographic lineages exhibiting broad hybrid zones. This extensive gene flow across species boundaries even at great phylogenetic distance apparently exerts a strong influence on topological reconstructions, both the placement of terminal specimens and entire clades. Concatenated, species‐tree, and network analyses have yet to conclusively resolve the placement and relationships of groups, such as abditus, carolinensis, ocoee A & B, and brimleyorum + valentinei. Similarly, a satisfactory model of species limits in the conanti, ocoee, and Pisgah clades in particular will require additional genomic and geographic sampling, along with more computationally sophisticated and biologically realistic models. Our results here provide a comprehensive if basic evaluation of the landscape of genetic diversity in Desmognathus and should support these future studies in targeting further comparisons.Our aim in this study was to (i) evaluate the distinctiveness of the 49 previously defined mito‐nuclear candidate species of The authors declare that they have no conflict of interest.R. Alexander Pyron: Conceptualization ; Data curation ; Formal analysis ; Funding acquisition ; Investigation ; Methodology ; Project administration . Kyle A. O’Connell: Conceptualization ; Data curation ; Formal analysis ; Investigation ; Methodology ; Project administration . Emily Moriarty Lemmon: Data curation . Alan R. Lemmon: Data curation . Dave A. Beamer: Conceptualization ; Data curation ; Formal analysis ; Funding acquisition ; Investigation ; Methodology ; Project administration .


Breviarium romanum ad usumFratrum Minorum S.P. (1738), demonstrating the promisingpotential of the suggested approach. Amperometric measurements, carriedout with a smart electrochemical tool developed in our laboratory,demonstrate the reversibility of the proposed treatment by removalof the nanocrystalline cellulose from the paper surface with a suitablecleaning hydrogel. This completely new feature of the consolidationtreatment proposed here satisfies a pivotal requisite in culturalheritage conservation because the methodological requirement for the ″reversibility″of any conservation measure is a fundamental goal for restorers. Apaper artifact, in fact, is subject to a number of natural and man-madehazards, inducing continuous degradation. With time, monitoring andconsolidation actions need to be often performed to ensure conservation,and this tends to modify the status quo and compromise the artifactintegrity. Removable treatments can potentially avoid erosion of theartifact integrity.An innovative consolidationstrategy for degraded paper is presentedbased on the reversible application of cellulose nanocrystals as sustainablefillers to reinforce mechanical properties and resistance to furtherdegradation. The compatibility and efficacy of the proposed consolidationtreatment are assessed first on pure cellulose paper, used as a model,by reliable techniques such as field emission scanning electron microscopy,atomic force microscopy, tensile tests, X-ray powder diffraction,and Fourier transform infrared spectroscopy, evidencing the influenceof the surface functionalization of nanocellulose on the consolidationand protection effects. Then, the consolidation technique is appliedto real aged paper samples from  The crystalline nanocelluloses are the idealmaterials for restoring the original properties of paper. In a fewattempts reported in the literature, bacterial nanocellulose (BNC)or cellulose nanofiber (CNF) thin films were applied by conventionallining  on the deteriorated paperwith encouraging results that well compare to the ones given by themore traditional Japanese paper.10Precious and degradedpaper artworks necessitate restoration treatmentsto preserve their unmeasurable artistic and historical value and toslow down irreversible degradation processes. In this context, theconsolidation of very fragile artworks often represents a necessary,but delicate step. Typically, the strengthening of fragile papersis performed by the use of linings. This method could have severaldisadvantages because it involves the use of: (i) an external (otherthan artwork itself) paper material with different properties withrespect to the original one and (ii) a glue. With aging, some of theseadditional materials undergo structural transformations that couldcause a loss in compactness, yellowing, and an acidity increase, acceleratingthe degradation processes of the artwork itself.However, some issues ofthe above-described treatments with CNFsand BNC are still open, mainly pertinent to the durability of thetreatment and demonstration of its reversibility.13 These nanomaterialsdisplay exceptional mechanical16 and thermal17 properties, optical transparency,18 and high crystallinity19 and have extensiveapplication in many fields of chemistry and chemicalengineering.27 Cellulose nanocrystals 28 are often used as fillers andreinforcing agents in nanocomposites29 andas paper pulp modifier to increase its resistance or control its waterabsorption.30 CNCs are highly efficientnanocomposite formers: for example, they can be dispersed at highconcentration in water and applied on a canvas surface. In this specificapplication, nanocelluloses differing in dimension and chemical functionalizationmanifest diverse consolidation effects, with the highest reinforcementper equivalent number of coatings yielded by CNC treatment with respectto nanofibers with a higher aspect ratio.6 Moreover,they form the most homogenous film which best protects canvases frommechanical changes upon relative humidity variations.6 CNCs mixed to silver nanoparticles can protect Whatmanpaper from microbial attacks.31 Finally,an interesting study from the University of Florence on the applicationof oleic acid-treated CNCs combined with calcium carbonate nanoparticlesfor consolidation of artificially aged filter paper as a model ofancient paper artworks has further highlighted the potential of nanocellulosecoating for paper preservation.32Nanocrystallinecelluloses are abundant and nontoxic.37 in the present work, the use of neutral and sulfatedCNCs in paper artwork conservation treatments is presented, usingpure cellulose paper samples (Whatman paper) as a test. The nanocrystalsused in our study differ in their surface functionalization: whileN_CNCs are composed of pure cellulose, S_CNCs present surface derivatizationwith sulfate groups, which may influence their behavior once theyare applied on paper. Therefore, in our work, we offer an importanthighlight on the influence of the surface functionalization of CNCson their behavior as a paper consolidant. Given the positive outcomesin terms of paper characteristics, we extend the study to a writtenreal aged paper sample from the XVIII century (BreviariumRomanum ad usum fratrum minorum), a rag pulp book previouslycharacterized in our laboratories.40 Finally, we proposehere a solution to a pivotal issue of restoration processes,41 demonstrating for the first time that the treatmentwith CNCs is reversible by their easy and safe removal via gellangel application.38 Indeed, degraded artworks needthat measures are taken often to ensure the conservation, and in somecases, removal of previous consolidating materials can erode artifact’sintegrity. Therefore, herein, we demonstrate that they can be safelyremoved by paper treatment with a gel.Given the above-described scenario and in connection with our previousstudies,In order to offer a thoroughand quantitative evaluation of theconsolidation method, experiments using different techniques havebeen performed on paper samples before and after the treatment withCNCs: field emission scanning electron microscopy (FE-SEM), atomicforce microscopy (AFM) topographies, elemental analysis, tensile tests,colorimetry, pH measurements, and Fourier transform infrared spectroscopy(FTIR). Each of them gives complementary information on the goodnessof CNCs as reinforcement materials for paper artworks.Cellulase , D (+)-glucose monohydrate, glucoseoxidase, Avicel PH 10.1, and sulfuric and hydrochloric acid were obtainedfrom Merck . All reagentswere of analytical grade and used without further purification. Inthe preparation, solutions of bidistilled water  was used. The book “Breviarium Romanumad usum fratrum minorum”  of 1738 is from a private collection.Pure cellulose paper samples(Whatman filterpaper N° 1) were obtained from Whatman (Maidstone UK). Gellangum was a KELCOGELCG-LA product obtained by CP Kelco . 42 where40 mL of deionized water was introduced in a 250 mL three-necked round-bottomedflask equipped with a water condenser and a mechanical stirrer. Then,the flask was cooled in an ice bath, and 40 mL of concentrated H2SO4 was added. After that, 4 g of Avicel PH-101was added, and the suspension was warmed to 50 °C for 80 min.The system was cooled to room temperature, and the mixture was transferredto polypropylene centrifugation tubes. Centrifugation at 4000 rpmwas repeated, replacing the supernatant liquid with fresh deionizedwater until the pH was approximately 1. Then, the precipitate wassuspended in deionized water with the aid of a Branson sonifier 250 equipped with an ultrasonic horn with a 3.5 mm diameter(micro-tip) operated in pulsed mode, with a power of 40 W, 0.6 s pulsesfor 10 min, and dialyzed against distilled water until neutralityusing a cellulose nitrate membrane with a molecular weight cut-offof 12,400 Da. The resulting suspension was transferred to polypropylenecentrifugation tubes and centrifuged at 4000 rpm for 20 min. The supernatantsuspension was kept, and water was removed under reduced pressure,yielding 905 mg of CNCs with an average length of 280 ± 70 nm.This procedure was adaptedfrom the work by Operamolla et al.Fifty milliliters of deionizedwater was introduced in a 250 mL three-necked round-bottomed flaskequipped with a water condenser and a mechanical stirrer. Then, theflask was cooled in an ice bath and 50 mL of concentrated HCl wasadded. After that, 5.00 g of Avicel PH-101 was added and the suspensionwas warmed to 105 °C for 6 h. The system was cooled to room temperatureand diluted with 50 mL of distilled water and the mixture was transferredto polypropylene centrifugation tubes. Centrifugation at 3000 rpmwas repeated four times, replacing the supernatant liquid with freshdeionized water until the pH was approximately 3. Then, the precipitatewas dialyzed against distilled water until neutrality using a cellulosenitrate membrane with a molecular weight cut-off of 12,400 Da. Theresulting suspension was recovered, and water was removed by distillationunder reduced pressure, yielding 4.71 g of CNCs.Breviarium paper samples of 8.0 cm2 dimensions. After drying, theresulting weight increase (%) of treated paper is in accordance withthe amount of CNCs added .The results obtained for these CNC-treated samples were compared,Whatman and Breviarium paper treated with only pure water, appliedwith a soft brush .We have decided to compare the data of CNC-treated paper samples withthose treated with pure water because all the specimens have undergonetreatment with water (with or without CNCs), thus rendering the resultsclearer and making the effect of CNCs more evident.Sonication of water suspensionswas carried out with a Branson Sonifier 250 used at 40 W power inthe case of S_CNC and at 80 W power for N_CNC. A total of 1.5 mL of1.5% of sonicated suspension of N_CNC or of 2% suspension of S_CNCwas applied by a soft brush on stripes of Whatman or 43 and Calvini et al.:44 paper samples wereimmersed for 1 h in a solution of 0.1 M HCl to catalyze hydrolysisand then aged for 1 week at 80 °C and RH = 65%.Paper agingon untreated and treated paper samples was performed, following theprocedures reported by Lojewska et al.–1 nominal force constant, and 10 nm guaranteed tipcurvature radius. Surface areas were sampled with a scan rate of 1Hz. The topographies were analyzed using the software XEI . For the AFM analyses of CNCs, 15 mg ofsulfated or neutral samples was dispersed in 5 mL of distilled waterwith the aid of a tip sonicator , and the resulting suspension was cast on mica and allowedto dry in ambient conditions.AFM topographies were carriedout using a Park XE-100 SPM system microscope. Images were acquiredin the tapping mode using tips (Type PPP-NCHR) on a cantilever of125 μm length, about 330 kHz resonance frequency, 42 N m2 quartzglasses or silicon slabs were used as substrates for FE-SEM measurements.All samples were dried in vacuum before the analysis.The morphology of the surface of papersamples was analyzed by FE-SEM on a field emission scanning electronmicroscope ZEISS Merlin equipped with a GEMINI IIs column and Beam-Booster,with acceleration voltages between 0.05 and 30 kV and 0.8 nm as thebest resolution, four optional detectors for SE and BSE, charge compensation,and an in situ sample cleaning system. 1 × 1 cm45 according to the followingequationI200 represents the maximum intensity of the peak with Miller’sindexes 200 (centered at 22.6° in our spectra), while intensityof the amorphous peak is calculated at the maximum, which dependson the typology of cellulose and is centered at 18° for celluloseI and 16° for cellulose II.The surface-sensitivestructural analysis was performed by using an X-ray diffraction (XRD)system  equipped with a parallel beam mirror(incident optic) coupled with a diffracted beam collimator . In a typical measurement, the spectra arerecorded by maintaining the incident angle ω (angle betweenthe incident beam and sample surface) fixed at 1.5° and movingthe X-ray detector and post-sample collimator along the goniometercircle in the 2θ range between 5 and 45° with a step sizeof 0.02°. Where necessary, the crystallinity index (C.I.) ofcellulose was calculated from the XRD spectra by the method reportedby Segal,38 CIELab is a three-dimensional space, and the distance  between two color coordinatescan be calculated by applying the Pythagorean Theorem. This valueis useful to interpret the meaning of the psychrometric differencesin color (only if ΔE > 3 color differencecanbe detected by human eyes).46 Variationof the colorimetric values are calculated with respect to those obtainedusing the pristine paper sample.Colorimetric measurementswere performed using a Minolta operating in the D65/10 mode. Resultswere expressed in CIELab color space , L* for the lightnessfrom black (0) to white (100), a* from green (−) to red (+),and b* from blue (−) to yellow (+). An average of six measurementswas performed for each colorimetric value.Measurements of pH were carriedout on the paper surface by using an Amel Instrument 334-B pH meterwith a combined glass electrode Ag/AgCl and a porous PTFE diaphragm; relative standard deviation was 5%, calculatedon three measurements of the same sample.–1 region. A total of 64 scans were collected at a resolution of 4cm–1 for each sample. ATR-FTIR spectra were recordedin triplicate.Spectra were acquired on a ThermoScientific(mod. Is50) instrument, equipped with an attenuated total reflectance(ATR) diamond cell for measurement in the 4000–525 cm–1 S_CNC dispersion in 1 mM aqueousKCl at pH ∼ 7.0 without addition of any surfactant.Theζ-potentialof CNC in water could only be determined for the colloidally stableS_CNC dispersion. The measurements were performed with a Brookhaven90 Plus Dynamic Light Scattering particle size analyzer equipped withZetaPlus software and electrophoretic light scattering cell for determinationof the ζ-potential. The measurements were performed on a sonicated10 mg·LElemental analyses were performedon a Carlo Erba EA 1108 CHNS Elemental analyzer or on an ElementarVario Micro Cube analyzer. Sample preparation: paper samples werecut into different pieces, dried in vacuum, and weighed on a precisionbalance, placed inside tin caps, and analyzed immediately afterward.Samples weight was maintained between 2.0 and 3.3 mg, except for analysesof S_CNCs and N_CNCs, for which the sample weight was ∼5 mgto ensure detection of small amounts of elements (S or N).), followingthe UNI EN ISO 1924-2:2009 standard. A gauge length of 125 mm hasbeen set. Eight repetitions for each sample have been performed.Tensile test on paper specimens, withdimensions 165 × 15 mm, have been performed by means of a universaltesting machine  is composed ofa flow sampling plate in contact with a hydrogel (Gellan gel). Theflow sampling plate is connected to an electrochemical biosensor,sensitive to d-glucose. The biosensor and the preparationof Gellan gel and its application on the paper surface have been alreadydescribed.38 Briefly, theflow sampling system is applied directly on the Gellan gel on theopposite side with respect to the paper surface and is constitutedby a plate in Perspex with a serpentine with 12 channels. Throughthe serpentine, the buffer flows continuously , lapping the gel applied on papersamples . All the material removedby the gel from the paper sample is taken and dragged away, throughthe flow tubes, to a bioreactor where the enzymatic degradation ofnanocellulose (see below) occurs. Then, the material is dragged intoan electrochemical thin layer cell, where a screen-printed biosensoris integrated. The thin layer cell is a liquid chromatography–electrochemistrycell obtained from Bio-Analytical Systems ,adapted for screen-printed electrodes (SPEs). The working and carrierbuffer was 0.05 M phosphate buffer +0.1 M KCl, pH 6.8. The bufferflow (0.1 mL/min) was regulated by a peristaltic pump, connected directlyto the plate. Amperometric measurements were carried out using a PalmSenspotentiostat  connected to alaptop computer.The tool  [potassium iron(III) hexacyanoferrate(II)] priorto enzyme immobilization. The procedure of the working electrode surfacemodification protocol was reported in a previous work.34SPEs were printed in the Laboratory of AnalyticalChemistry of the University of Rome Tor Vergata. The diameter of theworking electrode was 0.3 cm, resulting in a geometric area of 0.07cm2, witha thickness of 150 μm. A total of 13 μL of enzyme solution[200 IU ml–1Cellulase ] in 0.1 Mphosphate buffer (pH 8.0) was added on both membrane faces and incubatedfor 1 h at room temperature. The gel was applied on paper samplesfor 1 h. This electrochemical device is able to produce a currentproportional to the amount of glucose present in the solution flow,yielding a real-time efficient monitor tool.The bioreactor for nanocellulose analysiswas a hand-made cylindricalreactor in Plexiglass in which an enzymatic ABC Immunodyne membrane was inserted into the flow system. Themembrane had an immobilization surface of 300 cm48 at controlled reaction conditions. Sulfuric acid hydrolysis yieldedeasily suspendable negatively charged (at pH > 1) CNCs,42 named S_CNC (sulfated CNC), due to a parallelprocess of surface sulfation, randomly involving the primary hydroxylgroups on the surface glucopyranose units.49 S_CNCs formed stable colloidal suspensions in water at 2% (w/w)concentration,52 thanks to surface charge repulsion. Conversely, hydrolysisconducted with hydrochloric acid produced uncharged nanocrystals,named N_CNC , which displayed higher aggregation tendency,and were suspended in distilled water at 1.5% (w/w) final concentration.53CNCs wereprepared by hydrolysis with strong mineral acidsFigure S3) demonstrate the good filming abilityof both, the FE-SEM investigation revealed average lengths of 149± 32 nm and 151 ± 17 nm for S_CNCs and N_CNCs, respectively.In both cases, the thickness of the nanocrystals was detected by AFMto be 10 ± 2 nm. These data are collected in –1. These nanocrystals showed the tendencyto form small clusters connecting to each other by the long side.Panel (b) shows N_CNCs deposited on the same substrate from dimethylsulfoxide (DMSO) at a concentration of 1 mg L–1.Apparently, N_CNCs looked very similar in morphology to S_CNCs, buttheir dispersing ability in water was much poorer, as we could notdetect separate N_CNCs deposited from water but only clusters or films.The quite analogous length and thickness displayed by individual nanocrystalsof S_CNCs and N_CNCs should be related, more than the kind of acidused for the digestion, to the cellulose source (Avicel) and purificationprotocol applied. The quite different dispersing properties, shownby S_CNCs and N_CNCs in water, were attributed to the different surfacecharges possessed by the two nanocellulose samples: ζ-potentialmeasurements were performed on nanocrystals suspensions, revealingfor S_CNCs an observed ζ-potential value of −28.3 ±1.3 mV at pH 7.0. Such negative surface charge endows the aqueousdispersions of sulfated CNCs54 with a faircolloidal stability as opposed to the case of N_CNCs. We also performedelemental analyses on both S_CNCs and N_CNCs for detecting the degreeof substitution with sulfate groups on S_CNCs. As expected, N_CNCsrevealed the absence of sulfur. Conversely, S_CNC revealed the followingcomposition: C% 40.35, H% 5.89, S% 0.88, and O% 52.88. For the calculationof the cellulose degree of substitution , we elaborated the resultsof elemental analyses, as described in the Supporting Information. S_CNCs were composed of pure cellulose with asubstitution degree of sulfate groups of ∼0.05. The water contentcalculated from elemental analyses, as well as the value of pH foundfor the relevant S_CNC suspensions  of 3.94, would suggest that sulfate groups andH3O+ ions were co-present in the freeze-driedsample.The two nanocellulose forms were investigatedby AFM and FE-SEM to detect their morphology and dimension. WhileAFM topographies of thin films of S_CNCs and N_CNCs  lower thermal stability forsulfated nanocellulose with respect to the corresponding less dispersibleneutral form, which is a relevant property, considering the conditionsthat were used in our artificial aging experiments on paper samples(see below). The reported lower decomposition temperatures were ascribedto the easier elimination reaction of sulfate groups from the glucopyranoseunits, with respect to the elimination of a molecule of water, whichrequires more energy. These findings, combined with an observed increaseof char content, hinted at a fire-retardant role of the sulfate moieties.The behavior was observed even at very low degrees of functionalizationwith sulfate groups. This is the reason why both nanocellulose formsand not just the most common sulfated one were judged worth investigating:in this way, we aimed at providing a benchmark study on consolidationtreatments performed with the nanocelluloses, including useful newinformation about the effect of nanocellulose surface functionalizationon the restoration success. Indeed, aging experiments performed ontreated and untreated paper helped, in our case, to understand therole of sulfate groups on the preservation of paper artworks.Other different properties could be expected on thebasis of otherliterature reports,Figure S2. The spectra revealed thepresence of much purer cellulose I phase in N_CNCs. Conversely, S_CNCsdisplayed the still predominant presence of cellulose I phase, butwith higher contamination from amorphous or cellulose II phases, asshown by the characteristic diffraction peaks in the region between19 and 21 θ, indicating the influence of the sulfuric acid hydrolysisreaction on the phase composition of the starting crystalline cellulose.XRD spectra acquired on pure N_CNCs and S_CNCs thin films are shownin Sulfate and neutral CNC aqueous dispersions were appliedon new and artificially aged Whatman paper samples. Paper aging onpaper samples was performed by a modified literature procedure, asdetailed in the FE-SEM micrographsof pristine Whatman paper and the same paper treated with S_CNCs orN_CNCs are given in 2 scanning areas allowedus to acquire surface morphologies, where paper fibers and CNCs couldbe easily distinguished. We preliminarily investigated the followingsamples: (a) pristine Whatman paper; (b) Whatman paper treated withS_CNCs; and (c) Whatman paper treated with N_CNCs. The pristine paper obtainable from the analysis of the FTIR spectra, so that it does not perturb significantly the overallcrystallinity properties of paper. Macroscopically, the possible perturbationon the optical quality of paper after application of CNCs was evaluatedby performing colorimetric experiments. These experiments are morereliable than a simple human eye inspection, which is insensitiveto yellowing or darkening of paper if the colorimetric change ΔE*  is lower than 3.46 These parameters are reportedin a* and b* values. Moreover,pH, a fundamental parameter in evaluating the health state of a papermaterial,46 did not change substantially upon treatingthe samples with CNCs. Conversely, the mechanical properties of thesamples coated with CNCs slightly improved, suggesting that CNCs increase,even if slightly, the tensile strength of paper. This result is incontrast with other literature reports, where the negative influenceof the mechanical coating with nanocellulose water suspensions onpaper properties was observed due to the forced penetration of waterinto the fibrous structure of paper;59 itis in agreement with the results reported by Bergamonti et al.,31 indicating the positive effect of the manualapplication with a brush on paper properties, which will not forcewater to penetrate deeply among paper fibers.This finding was supported by the invariance in theC.I. , accelerates the decrease ofpH, showing the worsening effect on pH of the oxidation of C6 to carboxylgroups, but the point remains open to further investigation.The morphological stability of the coating was demonstratedbycomparing FE-SEM micrographs acquired on the samples restored withboth typologies of CNCs after the aging treatment 2, while Finally, it should be noted that the effect of this preservationtreatment on the mechanical properties of aged paper samples is verypromising. In spite of the very low nanocellulose content (about 0.3%by weight), tensile strength values, reported in Table S3), performedon the pristine and treated samples, evidenced in pristine Whatmanpaper the presence of humidity traces, as witnessed by the lower carboncontent with respect to the theoretical carbon percentage expectedfrom pure cellulose . An increasein carbon content was observed with the aging process for all samples(from 41.88% for Whatman paper to 42.48% for Whatman paper after aging).The higher percentage of carbon was retained after treatment withS_CNCs and lowered after N_CNC treatment. We took this observationas a confirmation of a different behavior of neutral or charged CNCs,probably connected to loss of sulfur due to desulfation processesoccurring on the S_CNC with aging. However, elemental analyses couldnot give us any information on the amount of eliminated sulfur becauseits overall content was too low when compared to the detection limitof the technique.Elemental analyses  cleaning task. When applied on CNC-treatedsamples, Gellan gel acts also as a CNC remover and carrier, deliveringthem through the flow system applied on it, to a suitable electrochemicaldetector .The interesting aspect and true novelty of therestoration of paperperformed by the application of a protecting coating with CNCs isthe reversibility of the treatment, a capability that we are ableto demonstrate. To ensure this reversibility, we used a cleaning method,based on the application of a Gellan hydrogel on the paper surface;Figure S6) supported this result as, after cleaning with gel, the coatingon paper fibers due to CNCs completely disappeared, restoring themorphology of pristine paper. In particular, as shown by the AFM resultsacquired after cleaning and CNC removal process reported in As shown in Breviarium romanum ad usumfratrum minorum (1738), called, in the following, Breviarium. We had completely characterized that in ourprevious studies.40Breviarum page (left image), a page treated with S_CNCs (middle), and a pagetreated with N_CNCs (right). The surface morphology appears more compactin samples treated with both, with S_CNCs coating showing a betterfilm-forming ability, confirming what was observed on the Whatmansamples and the coating properties of CNCs, while control water-treatedsamples presented pores and exposed fibers, which is less observedin the case of CNC-treated paper samples. Again, the smoother surfaceyielded by S_CNCs may indicate the higher penetration ability of N_CNCsamong paper fibers, as hypothesized previously.The last step of this study wasthe application of the CNC treatmenton samples from a real aged book, Breviarium pages treated with the two typologies of CNCs . However,on longer aging times, the pH decreases induced by the surface sulfationof S_CNCs could increase the risk of cellulose hydrolysis, decreasingthe polymerization degree of paper fibers and degrading the mechanicalproperties. Conversely, the strong reinforcement yielded by the N_CNCstreatment, with up to 50% enhancement of maximum stress and straincompared to the values of the pristine Breviarium paper, may be ascribedagain to the penetration of the N_CNCs and effective coating of theindividual fibers, resulting in improved bulk (or at least not limitedto the paper surface layer) interfiber adhesion at the microscopiclevel and increased cohesivity at the macroscopic level. Furthermore,no pH variation was produced by the application of an N_CNCs protectivetreatment.XRD analysis, the results of which are reported in Figure S7; it supported the reversibilityof nanocellulose coating.As for the Whatman paper, also in the case of Breviarium, the removalof CNCs was followed by coupling of the electrochemical monitoringsystem with Gellan hydrogel. In this work, an innovative procedure torestore and reinforce,in a sustainable way, paper artworks is presented. The outstandingresults reported here demonstrate that CNC application is compatiblewith paper materials, and, when applied on real aged samples, theycan improve optical quality and mechanical properties. However, careshould be taken in choosing the chemical surface derivatization methodof CNCs, which could severely influence the effectiveness of the consolidationmethod. Indeed, the presence of surface sulfation suggests differentaggregation properties in S_CNCs, when compared to N_CNC. Moreover,the functional group determines a difference in the consequence ofaging. The pH of paper treated with S_CNCs decreases under acceleratedaging, which means that it will negatively affect the conservationof a paper artifact treated with S_CNCs in the long term. This suggeststhat the use of S_CNCs for water protection would require a deacidifyingadditive to neutralize the excess acidity due to the sulfate functionalization.Conversely, N_CNCs are safer for paper conservation treatment becausethey do not compromise its pH and mechanical properties with aging.Moreover, for the first time, we demonstrated the reversibility ofthis restoration strategy by an easy and straightforward removal ofthe conservation treatment with Gellan hydrogel. The removal can beeasily followed by interfacing of an efficient real-time monitoringelectrochemical tool to the hydrogel, which allows us to remove anddetect the removal of the nanocrystals at approximately 400 s afterthe application of the gel. This work represents a fundamental studyon the interaction of CNCs with aged and non-aged paper materials.The manuscript was written through contributionsof all authors.All authors have given approval to the final version of the manuscript.L.M. and C.M. equally contributed to this work as corresponding authors.