Resting tremor is the characteristic motor sign in Parkinson’s disease (PD), predominantly affecting the upper extremity, unilaterally. The rest tremor can appear in other body regions at 4–6 Hz frequencies 2. It typOn the contrary to this steady rule of a distal-proximal gradient in parkinsonian resting tremor, we here to describe three patients with PD presented with unusual, isolated leg tremor starting from a thigh muscle without involving distal part of the leg, thereby extending our knowledge on the clinical features of parkinsonian resting tremor appearing in a leg.A 57-year-old man presented with a slowly progressive leg tremor developed two years ago. He felt tremor in the proximal part of the right leg only with resting state. Initially, the rest tremor selectively appeared when he lay down in a supine position. Then, it also appeared in his right thigh when he lay down with a semi-supine position. Video  or standA 59-year-old woman developed progressive left thigh tremor one month before the first clinic visit. She reported that the left thigh tremor specifically emerged only when she was lying on her back but with the knee flexed and did not experience leg tremors in other positions. On the initial neurological examination, there was mild bradykinesia in her left side. The left thigh tremor was explicitly observed while she was sitting with her legs crossed on the examination bed, and the tremor accentuated when she was distracted by doing repetitive finger tapping movement. Video . As parkA 71-year-old woman developed rest tremor in her left thigh one year ago. The thigh tremor appeared when she posed a semi-supine position and was sitting her legs crossed. There was no resting tremor in other body parts. Neurological examination revealed left-sided mild bradykinesia and rigidity. The resting tremor was provoked in her left thigh while she was sitting cross-legged Video . The MDS18F-fluorinated N-3-fluoropropyl-2-beta-carboxymethoxy-3-beta-(4-iodophenyl) nortropane positron emission tomography, which revealed asymmetric decrease in the posterior putaminal uptakes in all three cases . The response to dopaminergic medications of this thigh tremor was variable from excellent in the 2nd and 3rd cases might have been missed unless we appropriately examined them in a semi-supine or cross-legs position. Our atypical thigh tremor cases could extend our knowledge on the resting tremor appearing in the leg in humans. The dopaminergic loss in the dorsolateral striatum somatotopically corresponds to the lower extremities, but it can not precisely explain why the proximal leg involvement was more prominent than the distal part in these patients. When resting tremor restrictively involves a proximal thigh muscle, it is necessary to evaluate patients in more diverse positions. In particular, the resting thigh tremor while sitting with legs crossed can draw physicians’ attention due to a culturally distinct characteristic in Korean people who usually sit with this position in routine daily activities.Rest tremor examination is usually performed while patients sit, stand, walk, and lie down with commanding explicit maneuvers. The typical resting tremor in the legs is usually provoked in the distal part while the patient is seated with their feet free from the floor or lying down  with perIn conclusion, even if the patient showed a different pattern of the resting tremor compared with typical PD patients, the possibility of PD should be kept in mind with isolated unilateral resting tremor, especially in a thigh muscle.


Although DLBCL can be cured in more than half of all patients, up to 50% of patients become refractory to initial treatment or relapse after complete remission. We present a case of complete spontaneous remission of some tumors and concomitant newly developed tumors observed in a patient with relapsed DLBCL. Spontaneous remission of lymphoma without treatment is a rare phenomenon and can occur at baseline as well as in relapsed DLBCL. However, most patients who initially experience spontaneous remission later develop relapse. Thus, careful follow-up is required, and fluorine-18-fluorodeoxyglucose ( The sta


Electromyography (EMG)-driven musculoskeletal modeling relies on high-quality measurements of muscle electrical activity to estimate muscle forces. However, a critical challenge for practical deployment of this approach is missing EMG data from muscles that contribute substantially to joint moments. This situation may arise due to either the inability to measure deep muscles with surface electrodes or the lack of a sufficient number of EMG channels. Muscle synergy analysis (MSA) is a dimensionality reduction approach that decomposes a large number of muscle excitations into a small number of time-varying synergy excitations along with time-invariant synergy weights that define the contribution of each synergy excitation to all muscle excitations. This study evaluates how well missing muscle excitations can be predicted using synergy excitations extracted from muscles with available EMG data . The method was evaluated using a gait data set collected from a stroke survivor walking on an instrumented treadmill at self-selected and fastest-comfortable speeds. The evaluation process started with full calibration of a lower-body EMG-driven model using 16 measured EMG channels (collected using surface and fine wire electrodes) per leg. One fine wire EMG channel (either iliopsoas or adductor longus) was then treated as unmeasured. The synergy weights associated with the unmeasured muscle excitation were predicted by solving a nonlinear optimization problem where the errors between inverse dynamics and EMG-driven joint moments were minimized. The prediction process was performed for different synergy analysis algorithms , EMG normalization methods, and numbers of synergies. SynX performance was most influenced by the choice of synergy analysis algorithm and number of synergies. Principal component analysis with five or six synergies consistently predicted unmeasured muscle excitations the most accurately and with the greatest robustness to EMG normalization method. Furthermore, the associated joint moment matching accuracy was comparable to that produced by initial EMG-driven model calibration using all 16 EMG channels per leg. SynX may facilitate the assessment of human neuromuscular control and biomechanics when important EMG signals are missing. Knowledge of muscle forces could provide valuable insight into not only the neural control strategies employed by the central nervous system (CNS)  , physiological assumptions , and matrix decomposition algorithm   using cubic spline data interpolation. See Meyer et al.  was used to evaluate the SynX process  are the time-varying normalized muscle fiber length and velocity, respectively, and α is the pennation angle of the muscle. where p Zajac, .a) was calculated from muscle excitation (e) using a published model of muscle activation dynamics  to neural activation (μ) relationship and a nonlinear function (Equation 3) to define the neural activation (μ) to muscle activation (a) relationship:Muscle activation . For this model, every muscle was associated to an experimentally measured EMG signal collected using surface or fine wire electrodes. To perform full EMG-driven model calibration, we used experimental walking data over 10 gait cycles from the two walking speeds  . During this step, a sequence of optimizations was performed to identify the parameter values required by the activation dynamics model, Hill-type muscle-tendon model, and surrogate musculoskeletal geometric model  to extract a low-dimensional set of time-varying measured synergy excitations (Wm) and a corresponding time-invariant synergy vector containing weights (Hm) that defined how each synergy excitation contributed to the excitation of the measured muscles. Next, unmeasured muscle excitations (ex) were constructed using the measured synergy excitations (Wm) along with a trial-specific time-invariant synergy vector containing weights (Hx) associated with the unmeasured muscle. During this step, EMG-driven joint moments were estimated using a combination of both measured and unmeasured muscle excitations , and the unmeasured synergy vector weights (Hx) were identified iteratively through optimization by tracking experimental inverse dynamics joint moments (Equation 4). For NMF, the unmeasured synergy vector weights (Hx) were given a lower bound of zero, while they were unbounded for PCA. The unmeasured synergy vector weights (Hx) were initialized by the optimization using randomly chosen values between 0 and 1. The unmeasured muscle excitations predicted by SynX were constrained to be between 0 and 1.The second step in the evaluation process predicted unmeasured muscle excitations using SynX within the EMG-driven model calibrated in the first step . SpecifiWe implemented the second step of the evaluation process on the 10 calibration trials used in the first step as well as 10 other trials not used for calibration . Both steps of the evaluation process were performed with Matlab's built-in “fmincon” optimization algorithm using sequential quadratic programming. The structure of unmeasured synergy variables was slightly different between the matrix factorization algorithms, and more details are provided in the section below on methodological choices for synergy extrapolation.Muscle synergy analysis requires a number of methodological choices that can influence the results of the analysis  were represented as:To calculate measured muscle synergies, we used the two matrix factorization algorithms that render the most divergent MSA results: non-negative matrix factorization (NMF) and principal component analysis (PCA)  and “pca” were used to perform NMF and PCA, respectively.where ex) (either iliopsoas or adductor longus) was constructed from the measured synergy excitations (Wm) using the following relationships:Following MSA by either approach, the unmeasured muscle excitation  while keeping all musculotendon model parameters at their calibrated values.where Because EMG normalization method affects MSA results, this study explored five approaches for normalizing the magnitudes of processed EMG signals in preparation for MSA. EMG normalization was performed either within individual trials  or across all trials . Specifically, each processed muscle EMG signal was normalized using either: (1) maximum value over all trials (MaxOver), (2) maximum value per trial (MaxPer), (3) unit variance over all trials (VarOver), (4) unit variance per trial (VarPer), and (5) unit magnitude per trial (MagPer) , moderate (0.35 < r ≤ 0.67), strong (0.67 < r ≤ 0.9), or very strong (r ≥ 0.9)  was calculated to compare the ability of different methodological combinations to reconstruct measured muscle excitations  ANOVA tests on r and RMSE values between predicted and experimental unmeasured muscle excitations across all calibration and evaluation trials, respectively. In addition, we performed paired t-tests on r and RMSE values to investigate whether matrix factorization algorithm  had a significant influence on SynX performance for the same number of synergies. Third, we performed a three-factor  ANOVA to compare MAE values characterizing the accuracy of joint moment tracking from different approaches. All statistical analyses were performed in Matlab, and significance levels were set at p < 0.05.Multiple statistical analyses were performed to assess whether the calculated metrics resulting from different SynX methodological choices were statistically different for each unmeasured muscle-leg combination. First, to assess whether reconstruction performance of measured muscle excitations was statistically different between the two matrix factorization algorithms and the five EMG normalization methods, we performed a two-factor ANOVA with a Tukey-Kramer p < 0.01) and EMG normalization method (p < 0.01) on the variance explained by factorization of measured muscle excitations. For five or fewer synergies, PCA generally had significantly higher VAF values than did NMF for the same number of synergies , VarPer (p = 0.028), and MagPer (p < 0.01), while MaxPer produced the lowest VAF amongst the five EMG normalization methods (p < 0.01). Moreover, no statistically significant interaction was observed between matrix factorization algorithm and EMG normalization method for mean VAF values.The two-way ANOVA for mean VAF values revealed main effects of matrix factorization algorithm , which was not consistently observed for the NMF results. Furthermore, RMSE values between the average predicted and actual unmeasured muscle excitations across all trials using PCA-based SynX were generally lower than those using NMF-based SynX. In addition, SynX performed using either PCA or NMF predicted more accurate unmeasured muscle excitations with less trial-to-trial variability for the left leg than for the right leg  and matrix factorization algorithm (p < 0.01) had a significant effect on both r and RMSE values for all unmeasured muscle-leg combinations, while EMG normalization method did not. Additionally, no statistically significant interaction was detected among the three factors for both r and RMSE values. For each unmeasured muscle-leg combination, as the number of synergies increased, PCA produced non-monotonic changes in r and RMSE values, with r values reaching a maximum and RMSE values a minimum at five or six synergies. Unlike PCA, r values for NMF initially rose with an increasing number of synergies and then remained high with further increases, while RMSE values initially dropped and then leveled off . The one exception was adductor longus in the left leg, where the only significant difference between PCA and NMF occurred for 3 synergies (p < 0.01). When the number of synergies increased above 6, NMF provided substantially higher r values for adductor longus in the left leg , five for iliopsoas in the right leg , five for adductors longus in the left leg , and five for adductors longus in the right leg . Additionally, for most trials, PCA required fewer synergies (between 3 and 8) than did NMF (between 4 and 10) to achieve the best SynX performance  but insensitive to EMG normalization method (p = 0.12) for all muscle-leg-joint combinations. Furthermore, no statistically significant interaction effects were observed among the three factors for MAE values. For the HipFE moment, MAE values for both PCA-based and NMF-based SynX decreased as the number of synergies increased (p < 0.01). Nonetheless, PCA produced more accurate joint moment matching, as indicated by smaller MAE values, than did NMF (p < 0.01). In contrast, for the HipAA moment, neither number of synergies nor matrix factorization algorithm had a significant influence on MAE values. The one exception was adductor longus in the left leg, where MAE values decreased with an increasing number of synergies for both algorithms, though PCA had significantly smaller MAE values than did NMF (p < 0.01)  .PCA-based and NMF-based SynX led to different levels of joint moment tracking accuracy compared to the full EMG-driven model calibration , 4. For r and RMSE values for muscle excitation matching were plotted as a function of MAE values for joint moment matching  stayed above or around the levels achieved by the full EMG-driven model, which caused SynX performance using NMF to level off. Therefore, even though the number of synergies that generated the best PCA-based SynX results varied across trials, muscles, and legs, PCA always needed fewer synergies than did NMF. This fact simplifies the implementation of our method and may improve computational efficiency when more variables need to be calibrated simultaneously.r and RMSE values across all five EMG normalization methods was considerably less for PCA than that for NMF using the same number of synergies  had higher reconstruction quality for both measured and unmeasured muscle excitations than did the paretic side (right leg) . This obOur study involved several important limitations that suggest areas for future investigation. First, we calibrated the EMG-driven model with a full set of EMG signals to obtain personalized model parameters , and SynX worked well when the calibrated model parameters were held constant. In the future, a challenge will be to develop an optimization problem formulation where design variables defining both unmeasured synergy vector weights and EMG-driven model parameters are found simultaneously while still predicting joint moments and unmeasured muscle excitations accurately. Second, the extraction of measured muscle synergies was performed before the optimization process. However, since changing some parameter values  can change the synergy analysis outcome, SynX may work better if MSA was performed iteratively within the optimization. Since PCA leads to unique and fast factorization solutions for synergy excitations while NMF does not  made before MSA affect the accuracy with which unmeasured muscle excitations can be predicted. The synergy vector weights associated with an unmeasured muscle excitation were optimized by minimizing errors between the EMG-driven model predicted and experimental joint moments. Our results highlighted that PCA was able to provide more accurate, reliable, and efficient estimates of unmeasured muscle excitations than was NMF. In general, PCA required five or six synergies to achieve the best prediction of unmeasured muscle excitations, and inclusion of additional synergies reduced SynX performance. Better SynX performance for PCA may be the result of the non-negativity restrictions imposed by NMF. Moreover, PCA was less sensitive to EMG normalization method than was NMF, which may reduce the need to identify the true maximum muscle excitations reliably for EMG normalization. SynX could be useful to address difficulties in collecting EMG signals from deep muscles inaccessible by surface electrodes, which is critical when predicting muscle forces with an EMG-driven musculoskeletal model. It could also be useful when using EMG systems with fewer channels than desired. Our proposed SynX method may eventually facilitate the assessment of human neuromuscular control and biomechanics after rehabilitation or surgical treatment when EMG data collection is limited.https://simtk.org/projects/synx.The experimental data, OpenSim model, and Matlab code used to perform this study are available at All experimental procedures were approved by the University of Florida Health Science Center Institutional Review Board (IRB-01), and the subject provided written informed consent prior to participation.CP and BF designed and performed the experiments. DA analyzed the data, prepared the figures, and drafted the manuscript. MS and BF supervised the research. DA, MS, CP, and BF revised the manuscript and approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The topical route is the most preferred one for administering drugs to eyes, skin and wounds for reaching enhanced efficacy and to improve patient compliance. Topical administration of drugs via conventional dosage forms such as solutions, creams and so forth to the eyes is associated with very low bioavailability (less than 5%) and hence, we cannot rely on these for delivering drugs to eyes more efficiently. An intravitreal injection is another popular drug delivery regime but is associated with complications like intravitreal hemorrhage, retinal detachment, endophthalmitis, and cataracts. The skin has a complex structure that serves as numerous physiological barriers to the entry of exogenous substances. Drug localization is an important aspect of some dermal diseases and requires directed delivery of the active substance to the diseased cells, which is challenging with current approaches. Existing therapies used for wound healing are costly, and they involve long-lasting treatments with 70% chance of recurrence of ulcers. Nanotechnology is a novel and highly potential technology for designing formulations that would improve the efficiency of delivering drugs via the topical route. This review involves a discussion about how nanotechnology-driven drug delivery systems have evolved, and their potential in overcoming the natural barriers for delivering drugs to eyes, skin and wounds. The oral route is the most preferred route of drug delivery for management of several diseases. When the oral route is used for treating topical infections of skin and eyes, drugs delivered systemically might cause adverse effects due to non-specific drug exposure . In contThe human eye is an anatomically complex structure, due to which delivering drugs to eyes remains a challenge . The drustratum corneum as a major barricade -N,N,N-trimethylammonium chloride (DOTMA)/Cholesterol (chol) liposomes significantly increased the pDNA ranging from 40 to 85 µg (p < 0.05). It was difficult to prepare the complex of pDNA with the DOTMA/Chol liposomal formulation having more than 60 µg pDNA. These liposomes  showed high transfection efficiency and luciferase activity in the cornea, aqueous humor, iris-ciliary body, lens, vitreous body and retina for 3 days, but at the same time showed high cytotoxicity and aggregation behavior with the retina, which decreases visual activity and causes blindness. Therefore, an anionic coating of natural polysaccharides or synthetic polymer is applied to reduce cytotoxicity as well as aggregation behaviors [Kawakami and co-workers prepared cationic liposomes with plasmid DNA and investigated the in vivo transfection efficiency in rabbits after intravitreal injection (100 µL). Plasmid DNA alone does not show significant transfection efficiency from 40 to 85 mg, however, pDNA complexed with ehaviors .Liu and coworkers formulated PEGylated liposome-protamine-hyaluronic acid nanoparticles (PEG-LPH-NP) loaded with siRNA (PEG-LPH-NP-S) for the treatment of choroidal neovascularization (CNV) on a laser-induced rat model. siRNA directed against vascular endothelial growth factor (VEGF) or VEGF receptors in CNV animal models have shown promising results. The PEG-LPH-NP-S were 132 nm in size with 20 mV cationic zeta potential, and depicted >95% encapsulation efficiency. It was found that PEG-LPH-NP could efficiently protect the siRNA load, facilitate the intracellular delivery of siRNA and the expression inhibition of VEGFR1. Apoptosis was detected by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick ending-labeling method (TUNEL). The TUNEL testing and morphologic observation showed low toxicity on the rat retina, and thus, these lioposomes were considered as a safe nanoformulation in the treatment of choroidal neovascularization .In another study, the delivery efficiency of diclofenac-loaded liposomes to the retina was compared with conventional diclofenac ophthalmic solution on rabbit eyes. It was reported that the liposomes modified with PVA or polyvinyl alcohol derivatives bearing a hydrophobic anchor (C16H33 S) at the terminal of the molecule (PVA-R) improved the physical stability of diclofenac-loaded liposomes. After administration to eye, PVA-R-liposomes took the non-corneal pathway to deliver diclofenac efficiently to the retina. These liposomes also displayed better physical stability and less aggregation .P. aeruginosa in rabbit eyes for 24 h when compared to the marketed preparation [It has been suggested that bioadhesive and biodegradable polymers can be used to prolong the residence time of liposomal formulations. Mehanna et al. demonstrated that chitosan-coated liposomes encapsulated with antibiotic ciprofloxacin HCl improved the ocular permeation of the drug. This formulation inhibited the growth of paration . Fernandparation . w/v. The prepared niosomal formulation provided prolonged and controlled release as compared to the marketed solution  of 24.3%  effects . Abdelbary and co-workers prepared an ophthalmic niosomal formulation for controlled delivery of gentamycin. Niosomal formulations were prepared using various surfactants , in the presence of cholesterol and a negative charge inducer dicetyl phosphate (DCP) in different molar ratios using a thin film hydration technique. An in vitro study suggested that niosomal formulation exhibited high drug retention in vesicles and showed sustained drug release as compared to the free drug solution. Prepared niosomal formulations were tested for ocular irritation and it was found that the prepared niosomal formulation did not produce irritation in albino rats .® (RS100 and RL100), hyaluronic acid and so forth [The first prototype of polymeric nanoparticles used for topical ocular delivery was made from poly- (PACA). Polymers that are commonly used for preparing nanoparticles are poly-ε-caprolactone (PCL), chitosan, poly-(lactic-co-glycolic acid) (PLGA), carbopol, gelatin, Eudragitso forth . Gold, sso forth . NanocapSimilarly to cationic liposomes, nanoparticles with positive surface charge demonstrate better corneal retention and permeability . VasconcChitosan-based nanoparticles loaded with ketorolac tromethamine were studied by Fathalla et al. for their enhanced delivery to the eyes. The size of the NPs was affected bychitosan/tripolyphosphate (CS/TPP) ratio where the diameter of the NPs ranged from 108.0 ± 2.4 nm to 257.2 ± 18.6 nm. The zeta potential was found to be in the range of 22.9 to 27.8 mV. The positive charge of NPs of different formulation was obtained due to the cationic nature of chitosan. The in vitro release study demonstrated a sustained release of the drug over a period of 6 h from the nanoparticles compared to a conventional solution, which was released within 3 h. The ex vivo studies conducted using porcine eye balls confirmed the ability of the prepared nanoparticles to retain the drug on the corneal surface for a longer time compared to the ketorolac tromethamine solution .Naringenin is being used for treating age-related macular degeneration (AMD), however, its use is limited due to the poor aqueous solubility. Hence, nanoparticular carrier systems have been examined for its ocular drug delivery. Naringenin (Nag)-loaded sulfobutyl ether-β-cyclodextrin/chitosan nanoparticles (Nag CD/CS-NPs) were formulated using an ionic gelation method of chitosan with butyl ether-β-cyclodextrin (SBE-β-CD). The Nag CD/CS-NPs with positive zeta potential (~22 mV) and size around 450 nm released the drug in a sustained manner. In vitro release studies were evaluated in simulated tear fluid (STF) using a dialysis tube with a molecular weight cutoff of 8000–14,000. After 5 h, Nag-CD gave 57% drug release whereas Nag CD/CS-NPs gave only 38% drug release. These results indicated that the coated nanoparticle complex provided a sustained release of drug as compared to the only cyclodextrin complex. The in vivo study was carried out on New Zealand white rabbits. The in vivo study showed that Nag CD/CS-NPs had a much higher ophthalmic bioavailability and prolonged residence time, which significantly increased Nag bioavailability in the aqueous humor compared to the suspension formulation and, consequently, reduced the frequency of administration .Polymeric micelles are also investigated for delivering drugs to both the anterior and posterior segments of eye, due to their ability to increase the solubility of hydrophobic drugs and to enhance drug absorption across biological barriers including the ones in the eye. Their ability to bind with cellular barriers and improve penetration of drugs through the cornea may be attributed to their similarity in size range to the membrane proteins, scleral pores (cornea), and other biomolecules. Polymeric micelles are a potential carrier to deliver hydrophobic drugs and proteins through biological membranes, to increase the chemical stability of unstable compounds, and to control the release of drugs ,45.® (350 ng CsA/g cornea) and cationic emulsions (750 ngCsA/g cornea). Despite the neutral charge of micelles, concentrations of drug at tissues levels were slightly higher as compared to cationic emulsion [Luschmann et al. prepared an in-situ forming nanosuspension made of liquid poly(ethylene glycols) (PEG) and a simple micellar solution containing nonionic surfactants that increase cyclosporine A solubility. The in vivo experiments suggested that the micellar solution is suitable for delivering drugs to the anterior segment of eye. The in-situ nanosuspension and the micellar solution deliver high levels of drug to eye, i.e., 1683 ± 430 ng CsA/g cornea and 826 ± 163 ng CsA/g cornea, respectively. Both formulations exceeded the drug tissue levels reported for Restasisemulsion .®. The Soluplus micelles were able to deliver an adequate amount of Cyclosporine A into the cornea for the treatment of immune-mediated corneal disease. The developed formulation also displayed a good ocular tolerance in rabbit eyes [In another similar study, cyclosporine A micelles were prepared using polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer (PVCL-PVA-PEG), also known as Soluplusbit eyes .Antioxidant α-lipoic acid (ALA) is used for treating diabetic keratopathy and retinopathy. Anterior eye segment structures are usually damaged in corneal neuropathy and keratopathy. Micelles prepared using Soluplus were investigated for their ability to solubilize ALA and the prepared formulation was compared with a marketed eye drop that was obtained using conventional micelles of sodium dioctylsulfo succinate surfactant. Prepared Soluplus-based nanoformulation was of around 70–80 nm in size and increased the solubility of ALA by around 10 times compared to the commercial eye drop. These micelles enhanced the flux of α-lipoic acid through cornea and were found to be less irritant .As an example of targeted delivery to the eyes, Boddu and co-workers developed doxorubicin entrapped local in-situ gel utilizing folic acid-PEG-PLGA copolymer micelles for ligand-mediated targeted delivery to retinoblastoma cells via intravitreal administration. After intravitreal administration, DOX micelles in PLGA-PEG-PLGA gel gave sustained release for up to two weeks. The uptake of DOX in micellar solution was four times greater than plain DOX solution .Dendrimers are “tree-like” nanostructured polymers that have been interesting in terms of ocular drug delivery. They are attractive systems for drug delivery due to their nanosize range, ability to display multiple surface groups that allows for targeting, and easy preparation and functionalization. Dendrimers demonstrate promising properties in topical delivery of drugs to the eyes. Holden et al. encapsulated the two anti-glaucoma drugs brimonidine and timolol maleate into dendrimer hydrogel (DH) made up of polyamidoamine (PAMAM) dendrimers tethered with multiple PEG chains and photoreactive acrylate groups. They investigated the intracellular uptake of these two encapsulated drugs on human corneal epithelial (HCE) cells and the transport across the bovine corneal endothelium. The prepared DH formulation showed mucoadhesive properties towards mucin particles and were non-toxic to human corneal epithelial cells, and depicted higher HCE uptake and significantly increased bovine corneal transport for both anti-glaucoma drugs, as compared to the conventional eye drop .Yavuz et al. investigated anionic and cationic PAMAM dendrimer complexes for delivering dexamethasone to the posterior segment of the eye (retina) for the treatment of diabetic retinopathy. An ex vivo transport study using rabbit cornea and sclera-choroid retina pigment epithelium (SCRPE) tissues demonstrated that anionic dendrimers delivered higher drug concentrations at the corneal and scleral tissues compared to the free drug solution and cationic dendrimers. From this finding, it was concluded that dexamethasone-PAMAM complex formulations enhanced ocular bioavailability of dexamethasone via topical route .Lancina et al. designed fast dissolving dendrimer-based nanofibers (DNF) as a topical delivery vehicle for brimonidine tartrate for glaucoma, where DNF showed better efficacy than pristine brimonidine tartrate solution . The watImplantable technology is not new and has been in use for many decades; however, the incorporation of micro- and nanotechnologies in implants enables the development of safe and more efficient drug delivery devices. Advancement in implants creates a huge impact on patient comfort and compliance, while treating chronic conditions by delivering drugs in a sustained manner to the posterior segment of eyes (posterior segment eye disease). These technologies are scalable technologies and offer localized drug delivery for a prolonged duration.®, Retrisert®, Surodex™ and Ozurdex®). Biodegradable polymers including both synthetic (most commonly used for ocular implants), e.g., polylactic acid (PLA), polyglycolic acid (PGA), PLGA, PCL, polyanhydrides (PA) and polyortho-esters (POE), and natural polymers such as collagen, gelatin and chitosan have been utilized to fabricate nano-implants [Some nanocontrolled release systems that are implanted into the eyes to achieve drug delivery are already in clinical practices , which allows for drug release and an impermeable ethyl vinyl acetate polymer (EVA). The implant tablet contains 4.5 mg of drug and 0.015 mg magnesium stearate, which release from all sides of the tablet except the discontinuous EVA layer. The discontinuity in the EVA coating creates a diffusion port for the diffusion of ganciclovir from the implant into the eye. This technology releases active medicaments for 5–8 months and is devoid of systemic toxicity and thus cuts the costs of treatment. In the clinical studies, median time to disease progression of 6–8 months was observed in patients treated with a Vitrasert implant. The shelf life of the implant is 24 months and it is stored at a temperature between 15 and 25 °C [® is a United State of Food & Drug Administration (USFDA)-approved steroid implant manufactured by Bausch & Lomb, Rochester, NY, USA by incorporation of 0.59 mg of fluocinolone acetonide (FA) for controlled delivery into vitreous cavity (approximately 0.6 µg/day). It can release fluocinolone slowly for up to 3 years. In the long term, drug retention after release from the Retisert® effectively suppresses the inflammation, reduces the recurrence of uveitis and improves visual acuity in 80% of patients [Vitrasertnd 25 °C ,56. Retipatients .® are other examples of biodegradable implants that are widely used in clinical practice for intraocular delivery and sustained release of dexamethasone. Surodex™ is implanted into the atria to treat the post-operative inflammation of cataract patients and comprises 60 µg of dexamethasone into the PLGA hydroxypropyl methylcellulose polymer matrix. Ozurdex® is an intravitreal implant containing 0.7 mg of dexamethasone in PLGA polymer, which is capable of releasing the drug at a slow rate for up to 6 months. It is preloaded into a single-use drug delivery system (DDS) device for direct injection into the intravitreal cavity, administrated with the assistance of a healthcare expert. Clinical trial data suggest its potency in reducing vision loss. Data also suggest the improvement in vision acuity in eyes with macular edema associated with branch retinal vein occlusion (BRVO) or central retinal vein occlusion [Surodex™ and Ozurdexcclusion ,56.Skin has a complex structure that serves as a physical barrier to the entry of exogenous substances. Its detailed anatomical structure is presented in stratum corneum acts as a major barrier in delivering drugs to skin, especially for hydrophilic and larger drug molecules. The stratum corneum of skin consists of corneocytes and it is embedded in a lamellar structure. The unique arrangement of the corneocytes is the main reason for this layer to resist the permeation of molecules through it [stratum corneum maintains its hydration state of up to 80% relative humidity (RH), but an increase in hydration state due to humidity changes increases the diffusion coefficient. It has been observed that the penetration of lipophilic molecules increases with the increase in hydration of the stratum corneum. The chemical structure and physico-chemical characteristics of the drug molecule determine its permeability through the skin. Although the drugs with molecular weight of less than 500 Da are shown to have good permeation, there are a few exceptions, for instance heparin that has a molecular weight of 17,000 Da and still shows good penetration, which might be due to its inherent chemical nature [The rough it . Skin tel nature .The main objective of delivery drugs topically for skin diseases is to provide local and directed delivery to the diseased skin cells. The directed delivery of the drugs reduces the adverse effects that might otherwise occur when the drug is delivered systemically . TreatmeNovel drug delivery systems are being studied to solve the various challenges linked with conventional systems, mainly through improvement of the penetration of drugs through the skin. The aim with the novel systems is to provide controlled drug release, which reduces the frequent application of medicament and reduces the undesirable side effects, as well as protecting the degradation of encapsulated drugs. Nanotechnology-driven drug delivery systems aid in site-specific skin targeting, which might result in greater drug retention at the target site . The epistratum corneum and epidermis when compared with available commercial cream. Cell culture studies, trans-epidermal water loss (TEWL) measurements and histological observations confirmed that liposome in gel formulation was safe to be used in treating AD [Liposomes can deliver drugs to the skin efficiently due to their lipid composition that is similar to epidermal cells of the skin, which helps them penetrate the skin barrier better than other dosage forms. They can localize the drugs at the site of action, reduce systemic absorption, and therefore minimize drug side effects . Betametating AD .The elastic lipid-based vesicles (elastic liposomes) have recently gained interest in delivering drugs to skin. They are similar to conventional liposomes in morphology, but differ in function. They are highly deformable liposomes and are also called as transferosomes with better permeation through the skin barrier . They arstratum corneum and thereby the system increases the penetration, occlusion and accumulation of drug in the dermis region, which makes them the appropriate system for topical-targeted drug delivery. Pradhan et al. prepared fluocinolone acetonide SLN for treating psoriasis by emulsification-ultrasonication method. The authors prepared a gel-like formulation, which showed enhanced solubility of fluocinolone acetonide and helped the drug get deposited onto the epidermis, the location where psoriasis develops [Solid lipid nanoparticles (SLN) are novel carriers that are composed of a solid lipid core coated with surfactant. The lipids used are non-irritating and non-toxic to the skin, and they are also considered safe to be applied on damaged skin. The small size of the lipid particles is shown to have a close interaction with develops .Curcuma longa Linn. CUR also exhibits antioxidant activity, which is beneficial for several skin-related problems such as eczema, dermatitis, pigmentation, acne, psoriasis lesions and all the exfoliative skin diseases. Due to the presence of high amounts of epidermal lipids within the stratum corneum, SLNs seem to be a promising delivery system as compared to other nanoparticulate carriers. Considering this, Shrotriya et al. designed curcumin-loaded SLN (CUR-SLNs) incorporated into carbopol gel. Occlusive test, irritation test on skin, tyrosine enzyme inhibition activity and antioxidant activity using commercial gel and SLN-loaded gel was performed on skin model. It was demonstrated that CUR-SLN gel showed efficient occlusion properties, and enhanced skin deposition, inhibition of tyrosinase enzyme activity and improved antioxidant activity compared to that of a conventional CUR-plain gel. ICD was developed using dinitrochlorobenzene (DNCB) on the dorsal surface of ears of mice. In vivo study suggested that SLN-loaded gels are superior in suppression of ear swelling and reduction in skin water content in ICD disease compared to commercial gel [Irritant contact dermatitis (ICD), is one of the skin diseases manifested by edema, erythema, epidermal thickening, itching and scaling . Presentcial gel .Propionibacterium acnes. BPO has poor aqueous solubility, forms cluster and it gets crystallized in aqueous environment. These clusters are not able to penetrate through the follicles which is why conventional formulations like cream, lotion and gel have higher concentration of the drug (2.5–10%). Goyal et al. encapsulated BPO in niosome gel and found that the formulation resulted in better permeation of the drug through the skin and also reduced skin irritation. It was also suggested that BPO-loaded niosomal gel causes better reduction in colony forming unit (CFU) count of Propionibacterium acnes bacteria in mice ear than plain BPO [Meng et al. formulated celastrol-loaded niosomal hydrogel to treat psoriasis, which increased the permeation of celastrol in the skin and thus increased its anti-psoriasis activity in a mice model . Benzoyllain BPO .w/w) of ALA is applied on the lesion, left for 3–6 h for drug absorption, and removed before the irradiation of light at the lesion. The main limitation of this therapy was the hydrophilicity of ALA due to which permeation of ALA through stratum corneum was hindered. ALA-loaded noisome gel was prepared to improve the permeation of the drug across the stratum corneum. Ex vivo skin permeation studies performed on excised human skin showed that the ALA-loaded niosomal formulation showed better penetration across the skin when compared to the aqueous solution of ALA [Topical application of 5-aminolevulinic acid (ALA) has been employed in photodynamic therapy (PDT) treatment of superficial skin carcinoma. According to the PDT protocol, a concentrated aqueous solution  induced psoriasis-like mouse model was used to test the prepared formulation. Tumor necrosis factor (TNF) is one of the most potent inflammation activators that play a major role in signaling the classical nuclear factor kappa B (NF-B). The contribution of NF-B may be significant in IMQ-induced psoriasis-like inflammation. The results showed that TNF-α, NF-B, IL-6 expression was increased in IMQ-treated skin, but after the treatment with CUR-NPs-gel/clobetasol their expression was significantly decreased. Angiogenesis inhibition study was performed using the prepared nanoformulation gel by quantifying cluster of differentiation 31 (CD31) expression visually after skin staining with immunofluorescent dye. CD31 are biomarkers that are highly expressed during new blood vessel formation. Large amounts of new blood vessel formation were found in the control group, based on the large numbers of immunoflouroscent CD31 that were seen visually in the controlled group. It was found that a much smaller quantity of CD was shown in CUR-NPs-gel. It was concluded that curcumin-loaded nanoparticle gel inhibited the inflammatory cascade triggered by TNF-α and the inflammation associated angiogenesis stimulated in psoriasis, improving antioxidant activity. Curcumin nanoparticles were formed using a novel cationic amphiphilic polymer, an RRR-α-tocopheryl succinate-grafted ε-polylysine conjugate (VES-g-ε-PLL). These cationic nanoparticles were incorporated into 8% silk fibroin through electrostatic interaction. The increase in skin permeation of curcumin was responsible for an improved response in psoriatic plaque-like model, suggesting a promising potential of this drug delivery system to treat inflammatory skin disorders like psoriasis .®). It was reported that the crystallinity of adapalene was decreased and the TyroSpheres got accumulated in the hair follicles, due to which the topical delivery of adapalene was improved as compared to the commercial product. Drug distribution in the hair follicles was visualized using adapalene inherent fluorescence and Nile red loaded TyroSpheres with the aid of a fluorescence microscope  loaded tyrosine-derived polymeric nanoparticles . The formulation was evaluated ex vivo using human cadaver and porcine ear skin and compared with the marketed adapalene formulation  for topical skin delivery to treat psoriasis. The skin distribution studies showed that vitamin D3-TyroSpheres with 15 wt% loading were efficient in delivering the drug to the epidermis and dermis. It was also observed that the exposure time of skin with to micellar formulation (from 6 to 12 h) increased vitamin D3 diffusion significantly to the epidermis. On the other hand, increasing the exposure time of commercially available vitamin D3 Transcutolpidermis .n = 27 nm) polymeric micelles was reported using block co-polymer methoxyPEG-hexyl-substituted PLA (mPEG-hexPLA) for the treatment of skin cancer. IMQ micelles (0.05%) were then incorporated in gel using carboxy methyl cellulose sodium (CMC) and tested on porcine and human skin to evaluate their efficiency. It was found that prepared gel is more efficient in delivering IMQ in the different layers as compared with the Aldara™ cream [Fabrication of IMQ-loaded nanosized  molecule production by resident cells in the skin. Proliferation involves re-epithelialization, angiogenesis and granulation. The expansion of keratinocytes epithelial cells, stem cells and fibroblasts is known as re-epithelialization. Activation of endothelial cells and the creation of a site in which these cells can proliferate in the wound is called angiogenesis. Granulation is defined as the promotion of tissue granulation by fibroblasts, granulocytes and macrophages. Finally, remodeling involves the production of collagen- and elastin-including fibroblasts leading to myofibroblasts in the presence of T cells and macrophages . MonocytNumerous strategies based on nanotechnology are available for wound healing and are summarized in It has been reported that deficiency of epidermal growth factor (EGF) is one of the causes of diabetic foot ulcer (DFU). The effective concentration of exogenous recombinant human EGF (rhEGF) in wound for the treatment of DFU could not be achieved after local administration due to the short biological half-life of rhEGF, rapid dilution by tissue fluid, leakage from the wound surface, and degradation by enzymes. In order to overcome these challenges, Chu et al. designed rhEGF nanoparticles using PLGA polymer. These nanoparticles showed a controlled release of rhEGF for up to 24 h. These nanoparticles possessed better wound healing effects than those of pristine rhEGF . LL37, a2) and silicon dioxide (SiO2) play important roles in the production of reactive oxygen species, which stimulate the proliferation of fibroblasts and wound healing [Inorganic nanoparticles, especially metal oxide nanoparticles (MONPs), are also studied for the speedy recovery of wounds. MONPs like zinc oxide (ZnO), titanium dioxide  engineered with titanium dioxide nanoparticles was applied topically on the wound for improving the therapeutic efficacy of the extract. The results suggested significant wound healing activity in Albino rats, established by measuring wound closure, histopathology and protein expression profiling [In a study, rofiling . Bacterirofiling .Solid lipid nanoparticles (SLN) and nanostructured lipid carriers (NLC) are efficient and non-toxic drug carriers, which can provide high drug concentration at the site of wound. SLNs consist of solid lipids or blending of solid lipids whereas NLCs are a mixture of solid lipid with liquid lipids. In NLC, imperfection is more as compared to the SLN, hence, it avoids expulsion of drugs from the matrix as compared to the SLNs which have fewer imperfections due to their high order structure. There is close contact between these nanoparticles and the skin due to their small size and lipid content. NLCs are very suitable for dermal administration because they can remain on the skin and release drugs in a sustained manner and are also known to improve skin hydration . Many grGainza et al. encapsulated recombinant human epidermal growth factor (rhEGF) into SLN and NLC. The in vitro evaluation involved determining the bioactivity of rhEGF using fibroblasts and keratinocytes. The in vivo experiments concluded that 10 and 20 μg of rhEGF-loaded SLN and NLC were more effective in the wound healing process compared with the same number of intralesional doses of 75 μg of free rhEGF nanoparticles, and as effective as a single dose of 75 μg MS-rhEGF in genetically diabetic db/db mice (diabetic) .2 affinity to increase tissue perfusion, suppress inflammatory cytokines and promote faster skin wound healing. The results suggest that the above-mentioned parameters were significantly improved in diabetic dB/dB mice and are comparable to normal mice when treated with liposomal formulation [Stromal cell-derived factor-1 (SDF-1) is a chemokine known to increase the rate of healing of excisional skin wounds in mice. Olekson et al. formulated a cellular dermal scaffold loaded with SDF-1 liposomes and evaluated in a diabetic mouse excisional wound model with free SDF-1. The SDF-1 liposomes maintained and promoted sustained proliferation of SDF-1 in the wound that led to a positive effect on wound closure . Since tmulation .2 is believed to have the ability to inhibit these reactive oxygen species. Smith et al. modified a titanium implant surface via TiO2 nanotubes to increase the TiO2 surface area and eventually the catalytic degradation of NO species. It was observed that the TiO2 nanotubes reduced the levels of the pro-inflammatory signaling molecule NO compared with an implant having a planar TiO2 surface [Implants placed in any soft tissue induce a cell-mediated inflammatory response immediately following implantation. The inflammatory response at the site of the wound involves inflammatory cells, reactive oxygen species, and nitrogen oxygen (NO) species; TiO surface . Similar surface . Nanosur surface .Toxicity is a prime concern for any topical formulation and the establishment of a toxicity profile is a mandatory requirement for regulatory approval. Nanoparticles have the potential to penetrate the cells and can produce free radicals, which leads to oxidative stress and collagen depletion. This depletion induces keratinization, atrophy of the dermis and skin wrinkling ,125. In o/w formulation comprising 5% hydroquinone (HQ) and a different base. The major challenge associated with HQ is its poor permeability and physicochemical stability, which leads to the use of p-benzoquinone (pBQ) in the formulation which is known as carcinogenic [The various critical quality attributes including the polymer concentration, drug characteristic and critical process parameters of the topical formulation can lead to remarkable differences in terms of stability, safety, efficacy and toxicity. In a study, the authors investigated the signs of toxicity after exposure of inogenic ,128,129.o/w formulation with 5% HQ after 6 h of exposure [2) nanoparticles in vitro and in vivo using isolated porcine skin. The results suggest that the pathological lesions are likely to occur probably due to oxidative stress induced by the deposition of TiO2 nanoparticles. They also reported that with a prolonged exposure time, this topically applied nano-TiO2 can induce skin aging and may pose health risk issues to humans after long dermal exposure [The high HQ concentration was found in skin from formulation 1 containing Beeler’s based plus antioxidants, followed by formulation 2, which contains Beeler’s base and dimethyl isosorbide as solubilizer. This interaction might be between the cationic proteins of the skin and the anionic nature of Beeler’s base (sodium lauryl sulfate). Some signs of toxicity were also observed on the skin with all topical semisolid exposure .Wu and cexposure .Trop and co-workers prepared silver nanoparticles for the treatment of wound healing. Silver coated dressing acticoat caused liver enzymes and argyria-like symptoms in acute burn patients (30% burns). The silver levels in urine and plasma were monitored regularly to avoid the toxic effect of silver in burn patients. After 1 week of local treatment, hepatotoxicity and argyria-like symptoms and grayish discoloration of the patient’s face was also reported . NumerouConventional drug delivery systems for topical delivery possess several challenges, including repetitive dosing administration and rapid release, which impact product performance in healthcare sectors. Nowadays, nanotechnology-driven topical complex formulations such as polymeric micelles, liposomes, niosomes, nanoparticles, and implantable devices are gaining significant attention in the pharmaceutical sector for topical applications.Nanotechnology has provided minimally invasive ocular drug delivery systems. The invasive nature of intravitreal and periocular injection has spurred researchers to work on topical delivery systems that offer higher patient compliance. The nanotechnology-driven topical drug delivery systems to eyes possess properties such as enhanced residence time in the pre-corneal area, better permeation ability through the ocular barriers, sustained delivery of drugs at targeted sites, ultimately improving therapeutic efficacy. Although the in vivo studies have shown that the nanotechnology-based drug delivery systems did not produce any irritation and inflammatory response, their long-term effect must be properly analyzed. The existing topical formulations to treat skin diseases face challenges including a smaller amount of drug penetrating the skin barrier, systemic absorption leading to unwanted side effects and, if administered orally, adverse effects and poorly targeted therapy. Psoriasis, skin cancer, fungal infections and bacterial infections are a few of the most common skin diseases that require topical treatment. Most of the in vivo studies performed have shown better skin permeability of the nanoformulations, their ability to reach the diseased site, being free from irritation and inflammatory responses. However, extensive investigation has to be carried out to translate the novel formulations for clinical use. Nano-based systems certainly exemplify an improvement in safety and efficacy for topical drug delivery and hold great potential for efficient treatment leading to improved quality of the patient’s life.Nanotechnology is still a promising and emerging field, presently focusing on encapsulating drugs which require a low dose for treatment. Several products encompass technical challenges, e.g., (i) nanoparticles have a low loading capacity, (ii) dendrimers show toxicity problems, and (iii) liposomes have stability challenges. Scale-up, batch-to-batch reproducibility, stability and clinical performance can be regarded as the major challenges in the clinical translation from industry perspectives. The number of nanotechnology-based products going into the market is smaller compared to the efforts that have been put at the research level. We need to focus on the technology transfer, scale-up and validation of these products in R&D laboratories for clinical translation. Amalgamation of nanotechnology with other delivery systems such as implants, nanofibers, hydrogels, etc., may be further investigated for improved results in drug delivery by topical route. The main challenges that researchers need to focus on for clinical translation are retention time, vision interferences, nano-bio interactions, drug permeation and accumulation.


This minireview focuses on the current lung organoid systems that recapitulate developmental events occurring before and during branching morphogenesis. In addition, we also discuss their limitations and future directions.Three-dimensional (3D) organoid culture systems have rapidly emerged as powerful tools to study organ development and disease. The lung is a complex and highly specialized organ that comprises more than 40 cell types that offer several region-specific roles. During organogenesis, the lung goes through sequential and morphologically distinctive stages to assume its mature form, both structurally and functionally. As branching takes place, multipotent epithelial progenitors at the distal tips of the growing/bifurcating epithelial tubes progressively become lineage-restricted, giving rise to more differentiated and specialized cell types. Although many cellular and molecular mechanisms leading to branching morphogenesis have been explored, deeper understanding of biological processes governing cell-fate decisions and lung patterning is still needed. Given that these distinct processes cannot be easily analyzed  These buds comprise three cellular layers: an inner epithelial layer enclosed by mesenchyme and a thin external mesothelial layer  and SFTPC+ alveolar epithelial cells type 2 (AECII)  and SFTPC+ alveolar epithelial cells type 2 (AECII) obtained from hPSCs developed into organoids expressing EpCAM and SFTPC when co-cultured with mouse fibroblasts  and early alveolar structures (AECII and immature AECI) displaying proximodistal specification  (PLG) scaffold niche and then transplanted into the fat pad of immunocompromised mice  and bud tip-like domains   were co-cultured with primary mesenchymal cells  and lung resident mesenchymal cells (rMC) (CD45−CD31−EpCAM− SCA-1+) isolated from adult mouse lungs gave rise to mainly bronchioalveolar lung organoids (BALO) that exhibit distinct bronchiolar-like structures containing basal, club, goblet, and ciliated cells and alveolar-like structures comprising differentiated AECI and AECII that resembled the proximodistal pattering of the lung (highαSMA+) and lipofibroblasts (PDGFRαlowLipidTOX+) that were shown to be indispensable for proximodistal specification, differentiation, and branching formation. Accordingly, this model could be used to study epithelial-mesenchymal crosstalk mechanisms and the role of these subsets within the niche during lung branching. In addition, following treatment of BALO with morpholino oligos against miR-142-3p, a miRNA known to activate the canonical WNT β-catenin pathway, BALO mimicked developmental defects observed in ex vivo embryonic lung implants  and β-catenin destruction complex activation. Of note, BALO model could be supplemented with tissue-resident alveolar macrophages by direct cell microinjection adding another level of complexity to the system and opening new avenues to study the interactions between immune cells and various cellular components ex vivo. A significant drawback of BASC-derived organoid models is that BASCs have not been identified in humans; therefore, equivalent human organoid models are still missing. Nevertheless, further BASC genome profiling may reveal specific cell markers that might enable the identification of a human counterpart.The canalicular phase of lung development is delineated by further branching, elongation and broadening of alveolar airspaces Burri, . Particuthe lung  (Vazquezthe lung . The BALWithin the last few years, significant progress has been made on organoid technology; nevertheless, major research challenges remain to be addressed in order to rely on 3D organoid platforms to study lung developmental biology. For instance, current human lung organoid models lack terminal cellular maturity and structural complexity, particularly, maturation of the alveolar lineages and progressive formation of branches in hPSC-derived organoids. Nevertheless, there are diverse directions that could be taken to achieve these key features of branching morphogenesis.First, cell microinjection of relevant cell types into lung organoids to monitor the cellular interactions happening during branching formation. In this regard, lipofibroblasts are a type of lung-resident mesenchymal cell that are found in close proximity to AECII and have been shown to be involved in mesenchymal-epithelial crosstalk during alveolarization (Rehan and Torday, Second, engineering of a lung-specific hydrogel scaffold to guide branching formation. One interesting approach would be to engineer culture environments resembling the organ native niche by use of scaffold materials that could facilitate the generation of lung organoids that closely recapitulate lung structures and functions. Such methodology has been recently successfully applied to intestinal organoids by placing human stem cells inside a microfluidic chip that was laser-sculpted to resemble the gut (Nikolaev et al., in vitro. Vascularization of lung organoids has so far only been established by transplantation of organoids into host animals to promote cell differentiation and maturation (Dye et al., in vitro after hPSC-derived human mesodermal progenitors were introduced into the culture (Wörsdörfer et al., in vitro. For instance, the use of hPSC-derived mesodermal and lung progenitors may hold the key for the establishment of a vascular system within human lung organoids that could allow analysis of all the stages of branching morphogenesis, including septation and alveolar formation.Third, establishment of a vascularized lung organoid model to facilitate cell differentiation and branching morphogenesis In summary, although there are several valuable lung organoid models available to study different features of lung development, the ultimate goal would be the development of multicellular, highly differentiated, branched organoid systems that more closely resemble the lung architecture. Such complex branching organoid models in combination with the latest technology such as single-cell RNA-sequencing and genome editing tools could prompt the identification of novel molecular cues and cellular interactions occurring during lung bud elongation, branching and alveolarization, providing exciting new insights into branching morphogenesis.AV-A designed and wrote the manuscript. SH and AV-A contributed to the interpretation and discussion, read, and approved the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Dermatitis artefacta is a rare psychological disorder in which patients self-inflict cutaneous lesions to satisfy an emotional need. Due to the nature of this disease, patients can present with a wide array of sometimes very severe skin lesions. Here, we describe a case of dermatitis artefacta initially misdiagnosed as pyoderma gangrenosum and treated as such for eight years. The patient reported a long history of cutaneous ulcers on her extremities and trunk, with resultant extensive scarring. Upon presentation, she displayed rapidly progressing necrotizing skin lesions on her bilateral distal lower extremities. Both the skin manifestations and histologic sections were extremely atypical for pyoderma gangrenosum leading to extensive medical records review and subsequent diagnosis of dermatitis artefacta. This case represents the importance of the timely recognition and treatment of dermatitis artifacta to prevent its progression to severe harm and even death. Dermatitis artefacta (DA) represents a type of factitious disorder within the broad Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) category of “somatic symptom and related disorders”. Patients inflict cutaneous lesions in order to satisfy a psychological or emotional need of which they are usually not consciously aware . Skin leA 36-year-old female was transferred to the burn unit with rapidly progressing necrotizing skin lesions on her bilateral distal lower extremities. She reported noting small red-brown lesions on her right lower leg that rapidly progressed within 24 hours into black necrotic plaques, circumferential around both of her lower extremities Figure .Examination revealed discrete hypoesthetic eschar plaques on the bilateral lower distal extremities. Some plaques showed sharp well-demarcated borders and some with branching edges. No retiform purpura, palpable purpura, crepitus, or livedo was noted. Right lower extremity showed a background of atrophic shiny plaques coupled with scars at sites of reported prior PG lesions. She was afebrile with a leukocytosis of 20, C-reactive protein (CRP) of 80 (up from 23 during recent admission), and comprehensive metabolic panel (CMP) within normal limits.Histologic sections from wedge biopsies taken from the edge of the lesions showed extensive necrosis involving the epidermis, dermis, and subcutaneous tissue with vascular thrombosis and acute inflammation Figure . Due to Although the patient’s self-reported history was significant for PG, the extremely atypical presentation led to consideration of a broader differential diagnosis. PG is a rare, inflammatory dermatosis often associated with systemic disease and is a diagnosis of exclusion. The classic presentation of PG begins as painful erythematous pustules or nodules most often arising on the lower extremities or truck. The lesions then rapidly progress to cutaneous ulcerations with a well-defined, raised inflammatory border and mucopurulent base. Ulcers are exquisitely tender and may expand quickly in one direction and slow in another, leading to a serpiginous pattern . The hypIn dermatologic practice, it is important to take note of patients with persistent chronic conditions that do not respond to treatment. Conditions that have atypical presentations should be questioned even further. In this case specifically, the presence of chronic ulcers that were resistant to treatment coupled with a complicated medical history and no confirmed diagnosis should prompt medical record investigation to decrease misdiagnosis of DA and prevent severe self-harm.


Carex gayana, and then assessed whether genetic variation of candidate climate-selected loci better predicted their genome-wide responses than genetic variation of non-candidate loci. Contrary to this expectation, genomic responses of climate-linked loci only related significantly to environmental variables and genetic diversity of non-candidate loci. The effects of genome-wide genetic diversity detected in this study may be a result of either the combined influence of small effect variants or neutral and demographic factors altering the adaptive evolutionary potential of C. gayana populations. Regardless of the processes involved, our results redeem genome-wide genetic diversity as a potentially useful indicator of population adaptive evolutionary potential.Assessing population evolutionary potential has become a central tenet of conservation biology. Since adaptive responses require allelic variation at functional genes, consensus has grown that genetic variation at genes under selection is a better surrogate for adaptive evolutionary potential than neutral genetic diversity. Although consistent with prevailing theory, this argument lacks empirical support and ignores recent theoretical advances questioning the very concept of neutral genetic diversity. In this study, we quantified genome-wide responses of single nucleotide polymorphism loci linked to climatic factors over a strong latitudinal gradient in natural populations of the high Andean wetland plant,  Because genetic variation defines opportunities for adaptive evolution6, it is universally recognized as a crucial population characteristic indicative of adaptive potential9. Nevertheless, there is some debate as to whether traditional estimates of genetic variation are suitable for this purpose10. Many studies have resorted to neutral markers for their convenience8. However, neutral genetic diversity has increasingly been dismissed as a useful proxy for adaptive potential13, since it is not directly targeted by selection and covaries only weakly with quantitative genetic variation14.In the current context of unprecedented biodiversity loss16. For instance, successful adaptations to new selective pressures are more likely in large populations than in small ones; this is because large populations hold more absolute copies of potentially favorable alleles, have a lower risk of losing those alleles by drift, and have greater potential for mutation, including beneficial de novo mutations5. Gene flow can also either constrain or favor adaptation by causing fitness declines resulting from the introduction of maladapted individuals18 or, in contrast, by introducing new beneficial genetic variants20. Thus, in populations where neutral and demographic processes significantly alter adaptive evolutionary capacity, neutral genetic diversity could provide useful insights into adaptive potential. On the other hand, growing evidence indicates that neutral genetic diversity can be influenced by selection at linked loci23 and that a significant proportion of genetic variants within genomes, originally thought to be neutral, could in fact be under selection and functional as a result of the combined genome-wide influence of small effect variants26. Thus, genome-wide genetic diversity could also reflect adaptive processes and, as such, be a useful predictor of population adaptive potential.Dismissing traditional neutral genetic diversity estimates as useful indicators of adaptive potential both negates the possibility that neutral and demographic processes play a role in populations’ capacity to respond to selective pressures and assumes that neutral genetic diversity is well characterized and unimpacted by adaptive processes. Yet, neither of these assumptions is likely to be universally true. While adaptive variation is necessary for populations to adapt to new environmental conditions, there is no one-to-one relationship between adaptive genetic diversity and responses. Both neutral and demographic factors, such as population size and gene flow, can actually alter this relationshipCarex gayana as a model. A key challenge in these types of studies is how to quantify adaptive responses. Ideally, these should be assessed directly from genes under selection27, but such genes are in most cases unknown28. While this issue has been partially resolved thanks to large-scale next-generation sequencing, which allows candidate-selected loci29 with generally strong effects to be identified, tracking changes in allele frequencies driven by environmental factors is often impossible, particularly in natural populations. Here, we used multivariate analyses  of single nucleotide polymorphism (SNP) loci datasets to quantify divergence between candidate climate-selected loci and genome-wide non-candidate loci datasets. By applying this approach, we demonstrate that genome-wide genetic diversity of non-candidate loci is a more powerful predictor of evolutionary responses of climate-linked loci in C. gayana than genetic diversity of the loci used to measure these responses.We investigated this by analyzing the extent to which genomic responses of climate-linked loci relate to the genetic diversity of candidate climate-selected loci as well as genome-wide genetic diversity of non-candidate selected loci over a strong latitudinal gradient, using the high Andean wetland plant 30, we identified candidate climate-selected loci from the WorldClim database based on redundancy analysis (RDA), a genotype-environment association method31 linking genotypes at each locus to variables likely to exert selective pressures. The final RDA model used for this purpose (Supplementary Note on variable selection and detection of candidate climate-selected loci) included annual precipitation (BIO12), annual mean temperature (BIO01) and mean diurnal range (BIO02) as predictor variables. The model explained 14% of the total genetic variance  and identified three significant RDA axes  comprising 90 putatively adaptive loci notable for their outlier scores  of these loci did not correlate with those of the non-candidate loci .Using an original database of 1709 SNPs genotyped for 158 specimens by Pfeiffer et al.32 (CoIA), we projected the SNP profiles of the genotyped individuals for the candidate and non-candidate selected loci onto a common multivariate space. For these analyses, we used a dataset composed of 90 loci randomly selected from the full non-candidate loci dataset  to ensure a matching number of loci between the non-candidate and candidate selected loci datasets. The CoIA, which captured 80.2% of the total inertia of the non-candidate and candidate selected SNP matrices on its first three axes, characterized spatial genetic structure, showing that individuals from the same wetland, and sites from the same basin, tended to be grouped . This indicates that a large fraction of the genetic variation of the candidate and non-candidate selected loci did not co-vary. To ensure that this pattern was truly specific to these two datasets, we compared the observed RV correlation to a null distribution simulated by a bootstrap procedure from two datasets of 90 loci randomly selected from the full non-candidate loci dataset. None of the 10,000 bootstrap iterations produced an RV coefficient as low as that of the candidate and non-candidate selected SNP datasets . Candidate climate-selected loci data diverged less strongly from non-candidate ones in the Huasco, Elqui and Limarí basins relative to the northernmost (Copiapó) and southernmost (Choapa) basins . Effects were entirely independent, with mean annual precipitation explaining slightly more variation in population divergence than non-candidate loci genetic diversity  and genetic diversity of the candidate and non-candidate selected loci. The regression model revealed a significant effect of mean annual precipitation and of genetic diversity of the non-candidate loci. In contrast, no such effects were found with respect to genetic diversity of the candidate climate-selected loci Fig. c–e. Altoity Fig. .Figure 4C. gayana genomic responses of climate-linked loci than genetic variation at the loci used to measure this response. This suggests that genome-wide genetic variation may be an important indicator of population adaptive potential, which is also supported by a recent laboratory study demonstrating that it favored evolutionary responses of fitness-related traits in the vinegar fly22.Our results demonstrate that genome-wide genetic diversity was more informative in predicting 26. Indeed, a growing body of evidence indicates that natural selection can impact genetic diversity through its effects on linked neutral diversity34. In addition, it has recently been suggested that genetic variants with small individual effects along the genome, originally considered to be selectively neutral, may in fact be functional as a result of their combined effects26. Traits important in plant responses to climate change are often controlled by genetic regulatory networks involving complex signaling pathways and the effects of several genes 36, which suggests that many genetic variants with small effects on adaptive responses to climatic variables could be spread along the genome of C. gayana. Since genetic-environmental methods only detect loci with major effects on adaptation37, there is a high likelihood that a large number of variants with small individual effects on climate-selected traits were included in the non-candidate climate-loci dataset, and could have contributed to genomic responses of climate-linked loci. Nevertheless, this does not explain why these responses were better predicted by genome-wide genetic diversity than genetic variation at the loci used to measure them, given that an equal number of loci between the two datasets was used.The effects of genome-wide genetic diversity detected in this study may have resulted from the persistence of loci relevant to climate adaptation in the non-candidate loci datasets. Recent theoretical and empirical advances clearly question the definition of neutral genetic diversity, and point towards a larger role of natural selection in genome-wide genetic diversity levelsC. gayana could have limited genomic responses of climate-linked loci and could therefore also have contributed to the apparent association of the latter with genome-wide genetic diversity. Since they fuel all genetic variation  and can cause its loss4, neutral and demographic processes determine the availability of and population capacity to produce new genetic material for selection to act upon. In addition, they can also limit evolutionary adaptive responses even in the presence of substantial adaptive genetic variation38, which may occur in the event of demographic constraints38 linked, for instance, to small population size, or where gene flow is extensive and outweighs selection effects40. In discrete ecosystems such as high Andean wetlands, neutral processes can strongly impact species and genetic diversity due to greater potential for drift, demographic stochasticity and limited opportunity for dispersal41. The importance of neutral processes on genome-wide diversity of C. gayana is supported by previous evidence demonstrating strong effects of wetland isolation on this genetic component42. Here, we found that high and low precipitation levels were driving genomic responses of climate-linked loci of relatively similar amplitude in the north and south of the study region. Yet, genomic responses of candidate climate-selected loci were more restricted in northernmost wetlands where C. gayana populations displayed lower levels of genome-wide genetic diversity. This suggests that demographic constraints could be limiting adaptive potential of this species in the northern sites, which tend to be both smaller and more isolated than the southernmost wetlands42.Constraints imposed by neutral processes on the evolutionary adaptive potential of C. gayana populations at climate-selected loci are not solely conditioned by the amount of adaptive genetic variation at loci used to measure this response. As mentioned earlier, there is a high probability that a large number of variants contributing to genomic responses of climate-linked loci  were absent from the candidate climate-loci dataset. In addition, adaptive genetic diversity may not be very informative when de novo mutations make up a substantial part of the evolutionary response, as may be the case in later phases of the adaptive process16. Finally, even when adaptation from standing genetic variation dominates, high genetic variation at adaptive loci does not guarantee that variation does exist in the direction of selection and thus may not necessarily facilitate evolutionary adaptive responses. In sum, adaptive genetic diversity may also fail to capture the adaptive potential of populations. Of course, our results do not rule out the utility of adaptive genetic diversity to predict the ability for populations to adapt in situ, nor did they demonstrate the superiority of genome-wide genetic diversity to do so. However, they indicate that this diversity component could be relevant and should not to be discarded. Far more research is needed to understand the circumstances under which genome-wide genetic diversity can be expected to be a reliable predictor of a populations’ adaptive potential.The poor performance of genetic diversity at candidate climate-selected loci in predicting genomic responses may be attributable to several factors. Firstly, genetic diversity estimates from contemporary samples may inadequately represent levels of genetic variation available at the climate-selected loci prior to selection. Alternatively, this result may indicate that adaptive responses of 30, produced by genotyping-by-sequencing43 and consisting of 1709 SNPs filtered and genotyped for 158 specimens of the plant C. gayana (Cyperaceae) distributed among 17 high Andean wetlands in Chile´s Norte Chico region . This region is characterized by a large precipitation gradient increasing with latitude, with climatic conditions varying from Mediterranean to hyperarid. Sample size per wetland ranged from 4 to 10 C. gayana individuals. Detailed information regarding the study area, study sites, species biology and sampling, DNA extraction and SNP data production can be found elsewhere44. A map of the study area is provided in Supplementary Fig. We used the same SNP dataset used by Pfeiffer et al.https://cran.r-project.org). We performed redundancy analyses (RDA) to detect putatively selected loci based on the correlation between individual genotypes and climatic variables potentially acting as selective factors. Prior to this analysis, we used a clustering of variables around latent variables (CLV)45 to reduce the initial set of 21 predictor variables, composed of longitude, latitude and 19 standard WorldClim bioclimatic variables (BIO1-19) at 30 s spatial resolution, downloaded from WorldClim version 246 (available at https://worldclim.org/version2). We followed the recommendations of Vigneau et al.47 in determining the number of clusters, then selected the variable most representative of each  to check for multicollinearity problems considering a maximum value of 10. Significance of the RDA model and each individual RDA axis was tested using ANOVA-like permutation tests with 9999 randomizations. We identified outlier loci on each significant axis with a cut-off of 2.5 SD around the mean. Based on these results, we separated the SNPs into candidate climate-selected and non-candidate loci datasets, composed of outlier loci associated with climate variables (i.e. 90) and loci not identified as outliers according to multiple detection methods (i.e. 1421), respectively. In order to remove as many loci with potentially large effects as possible, the non-candidate loci dataset was constructed by eliminating the 229 outliers detected by Pfeiffer et al.30, 17 monomorphic loci, and 42 of the 90 climate-selected loci that were unique to the present study, from the 1709 original loci. However, to ensure adequate comparison of variation patterns of candidate climate-selected and non-candidate loci using a matching number of loci, we created a non-candidate dataset by randomly selecting 90 loci out of the 1421 non-candidate loci. This dataset was used in all subsequent analyses. Note that all analyses were also performed using the full non-candidate loci dataset. Patterns detected with the 90 and 1421 non-candidate selected loci were always consistent  from two data matrices, in this instance the non-candidate and candidate climate-selected loci datasets, along successive canonical axes. The CoIA axes are calculated such that congruence between the two tables is maximized, approximated by their squared covariance (i.e. inertia). CoIA first requires separate ordinations of the two matrices, which we performed by subjecting the non-candidate and candidate climate-selected datasets to principal component analyses. The number of principal components that we retained accounted for 90% of the total variation of each dataset. To test the significance of the correlation between the genetic variation of the non-candidate and candidate climate-selected loci datasets, we calculated the RV coefficient, a multivariate generalization of the Pearson correlation coefficient33, and tested its significance based on 9999 permutations. Both the principal component and CoIA analyses were performed using the ade4 package49. The projection of the genetic profiles of the individuals onto the co-inertia space allows common genetic patterns between the non-candidate and the candidate climate-selected data to be visualized. The distance between the non-candidate and candidate climate-selected dataset in the CoIA space reflects the effects of processes causing the candidate and non-candidate selected loci datasets to diverge. Thus, to quantify the genome-wide signature of these processes, we computed the Euclidean distances between their CoIA scores on the first seven canonical axes, which together represented 94.7% of the co-inertia. We analyzed whether the divergence levels varied geographically by applying a linear model to test for the effects of basins and sites nested within basins.Genetic differentiation between wetlands was assessed through various traditional approaches (Supplementary Note on population genetic structure). In addition, we investigated the genetic structure common to both the non-candidate and candidate climate-selected SNP data by carrying out co-inertia analyses (CoIA)50 as the expected heterozygosity (He) calculated for each SNP dataset (non-candidate and candidate selected loci, Supplementary Table 30. We used the leaps package51 to identify the optimal subset of bioclimatic variables for use in detecting candidate climate-selected loci. We considered that these variables can have both linear as well as quadratic effects. The best model was selected based on a performance analysis returned by leaps using the AICcmodavg package52. To estimate the contribution of each of the predictors found to be significant, we performed a variation partition using vegan’s varpart function48. Before running these analyses, we ensured that there was no effect of the number of genotyped individuals, as this may act as a possible confounding factor.We searched for factors linked to population divergence between the non-candidate and candidate climate-selected loci datasets using linear models. To assess divergence between the two datasets at the population level, we averaged divergence estimates of all individuals belonging to the same wetland. The set of explanatory variables included climatic variables potentially acting as selective factors and genetic diversity of candidate and non-candidate selected loci, estimated using gstudioSupplementary information.