These double knockout (DKO) mice showed compromised sarcolemmal membrane integrity compared to mdx mice, as measured by immunoglobulin G staining and ex vivo muscle laser microscopy wounding assays. We also found a significant decrease in muscle ex vivo contractile function as compared to mdx mice at both 6 weeks and 1.5 years of age. As the DKO mice aged, they developed more extensive fibrosis in skeletal muscles compared to mdx. Our findings indicate that TRIM72/MG53-mediated membrane repair can partially compensate for the sarcolemmal fragility associated with DMD and that the loss of membrane repair results in increased pathology in the DKO mice.Duchenne muscular dystrophy (DMD) is a common X-linked degenerative muscle disorder that involves mutations in the DMD gene that frequently reduce the expression of the dystrophin protein, compromising the structural integrity of the sarcolemmal membrane and leaving it vulnerable to injury during cycles of muscle contraction and relaxation. This results in an increased frequency of sarcolemma disruptions that can compromise the barrier function of the membrane and lead to death of the myocyte. Sarcolemmal membrane repair processes can potentially compensate for increased membrane disruptions in DMD myocytes. Previous studies demonstrated that TRIM72, a muscle-enriched tripartite motif (TRIM) family protein also known as mitsugumin 53 (MG53), is a component of the cell membrane repair machinery in striated muscle. To test the importance of membrane repair in striated muscle in compensating for the membrane fragility in DMD, we crossed TRIM72/MG53 knockout mice into the  Plasma membrane disruptions occur in many cell types in a variety of tissues as the result of cellular injury, such as physical damage or oxidative stress. Once the plasma membrane is physically disrupted, the cell uses a specialized membrane repair process to reseal the injury and restore the barrier function of the membrane to allow for survival of the cell. This process is triggered by the influx of extracellular calcium into the cell through membrane disruptions; this leads to the recruitment of intracellular vesicles and associated proteins that create a membrane repair patch to reseal the injury ,2,3,4. AMembrane repair is particularly essential in striated muscle myocytes, as the contractile nature of these cells exposes the sarcolemmal membrane to increased mechanical stress ,12. SeveTRIM72/MG53 regulates vesicle trafficking and exocytosis , which lTRIM72/MG53 and other proteins of the membrane repair machinery are thought to support tissue homeostasis, both by maintaining cellular function and preventing energetically demanding replacement following cell death. During normal physiology, membrane repair can play a limited role in some tissues; however, it becomes more important during stress. When cell membrane disruptions are more prevalent, including during injuries induced by permeabilizing toxins ,44, ischmdx mouse model, in which treatment with recombinant human MG53 protein (rhMG53) [Muscular dystrophies are a group of disorders characterized by loss of muscle mass and muscle weakness. Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy, with a prevalence in the United States and Europe of approximately 6 per 100,000 individuals ,51. DMD rhMG53)  or polox  or polo(rhMG53)  to incre(rhMG53) ,57,58,59mdx mouse background. When dysferlin knockout mice were crossed with dystrophin-deficient mice, the pathology was increased in the double knockout mouse in an age-dependent manner [mdx mouse. Given that TRIM72/MG53 binds dysferlin [mdx mouse. To do so, we crossed the TRIM72/MG53 knockout mouse into the mdx mouse model of DMD to create a double knockout (DKO) mouse for TRIM72/MG53 and dystrophin. This mouse model shows reduced sarcolemmal membrane repair capacity and age-dependent increases in the hallmarks of DMD, including decreased force production by the extensor digitorum longus (EDL) muscle and increased fibrosis in multiple skeletal muscles.Previous studies have attempted to address this question by knocking out the expression of specific membrane repair proteins in the t manner . This stysferlin ,61 and rTRIM72 knockout mice (−/−TRIM72) were previously generated [mdx mice (Jackson Laboratory). Three generations of crosses of male progeny with mdx female mice produced littermates −/−TRIM72/mdx double knockout (DKO), or +/+TRIM72/mdx (mdx). Lack of MG53 and dystrophin expression was confirmed by Western blotting using standard techniques as described below.Mice were housed and bred at standard conditions with a temperature of 22 ± 2C and a 12-h/12-h light cycle. Mice were provided standard mouse chow and drinking water ad libitum. All animal care and experimental procedures were preapproved by The Ohio State University Institutional Animal Care and Use Committee. 2 asphyxiation and cervical dislocation. Hind limb muscles were dissected and fixed for 24 h with 10% phosphate-buffered formalin, followed by a 70% ethanol incubation for 24 h. Tissue was then processed and embedded in paraffin  and 12 μm sections were collected on SuperFrost Plus slides . Slides were deparaffinized with two xylene incubations and rehydrated with a series of ethanol incubations at the following concentrations: 100%, 100%, 95%, 70%, 50%. The slides were then stained for hematoxylin/eosin  or Masson’s trichrome  for immunohistochemical staining and analysis.Male mice at various ages (indicated where appropriate) were euthanized via COMyocyte cross-sectional area (CSA) was determined from sections of EDL, soleus, and TA muscles stained with hematoxylin/eosin. Image files were blinded before analysis to preclude any bias. Myocyte CSA was determined by outlining individual myocytes using ImageJ. Myocyte CSA was analyzed for their frequency distribution (200 µm bins), and graphed based on the percentage of myocytes corresponding to the binned myocyte size.Centrally located nuclei were counted from myocytes measured for CSA. Myocytes were counted as positive for centrally located nuclei if nuclei were located inside of the perimeter of the outlined myocyte. Image files were blinded before analysis to preclude any bias.First, 12 μm paraffin-embedded sections of EDL, soleus, and TA muscles were deparaffinized with xylene incubations and rehydrated with ethanol incubations, followed by antigen retrieval with Citra Plus Solution . Tissue sections were blocked with 2.5% bovine serum albumin  for 1 h at room temperature, and incubated overnight at 4C with goat anti-mouse IgG antibody conjugated with Alexa Fluor 488 . ImageJ was utilized to analyze the IgG positivity within the muscle tissue. To eliminate dark background without tissue present and staining artifacts (including areas with tissue folding or separation due to processing and heavily fibrotic or necrotic areas where distinct fibers with intact membranes could not be identified), the outline of the tissue was traced to only include analysis of the skeletal muscle tissue. The IgG-positive area was divided by the total area to determine the percent area of IgG-positive skeletal muscle per image. To preclude bias in the selection of areas, all experiments were performed in a blinded fashion.Analysis of the percent area of fibrosis was conducted using ImageJ  on Masson’s trichrome-stained sections. First, 20× images were stitched together, encompassing the entire muscle, using the EVOS FL Auto 2 inverted microscope imaging system . ImageJ was utilized to identify the skeletal muscle area within each stitched image. To exclude histological artifacts (including areas with tissue folding or separation due to processing), the ImageJ plug-in color deconvolution was used to separate the blue channel and an additional threshold corresponding to areas of the tissue positive for fibrosis was measured. The percent area of fibrosis in the tissue section was determined by dividing the fibrosis-positive tissue area by the total tissue area of each image representing whole tissue. To preclude bias in the selection of areas, all experiments were performed in a blinded fashion.Isolated mouse muscle contractility was measured as described previously . BrieflySkeletal muscle tissue was dissected from mice and protein lysate was isolated using Radioimmunoprecipitation Assay buffer . Protein concentrations were determined by the standard Bradford Assay using bovine serum albumin (BSA) standards ranging from 0 to 1 mg/mL. Then, 20 μg protein samples were separated by SDS-PAGE at room temperature on 4–15% gradient gels  and were transferred on ice to 0.45 μm nitrocellulose membranes . Membranes were stained with Ponceau S stain  and then photographed for determination of protein loading levels. Membranes were probed for TRIM72/MG53 with a custom polyclonal antibody , dysferlin , caveolin-3 , anti-mouse horseradish peroxidase (HRP)-conjugated secondary antibodies, and anti-rabbit HRP-conjugated secondary antibodies . The blots were developed using enhanced chemiluminescence (ECL) substrate . An Azure Biosystems imager was used to visualize chemiluminescent blots. Quantification of immunoreactive bands was performed using ImageJ’s integrated density measurement and normalized to total protein levels using Ponceau S staining. Creatine kinase (CK) levels were measured in mouse serum using a mouse CK enzyme-linked immunoassay (ELISA) kit  per manufacturer’s directions.2+ was added to the dish to induce membrane damage. The Olympus FV1000 multi-photon laser scanning confocal system was used to irradiate the sarcolemma to assess membrane repair. Membrane injury was induced in the presence of 2.5 μM FM4-64 fluorescent lipophilic dye . A circular area was selected along the edge of the cell membrane and irradiated at 20–30% laser power for 5 s (s). Pre- and post-damage images were captured every 3 s, continuing for 57 s. The ImageJ software  was used to analyze cell membrane repair kinetics by measuring the fluorescence intensity, encompassing the site of damage and the background dye fluorescence. ΔF/F0 values were calculated with the following equation at each timepoint: (injury fluorescence-background fluorescence)/background fluorescence. To preclude any potential for bias, all experiments were performed in a blinded fashion.Whole flexor digitorum brevis (FDB) muscles were dissected from mice and mounted on a 35 mm glass-bottom imaging dish with commercially available liquid bandage. Physiologic Tyrode’s solution containing 2.0 mM Cat-test assuming unequal variances, and with Welch’s t-test for unbalanced designs. Other appropriate statistical tests, including Mann–Whitney tests, were used on specific data sets as indicated in the text.Graphical representation and statistical analysis of data was performed using Prism version 8 . All results are presented as mean ± SEM. Cross-sectional area measurements were grouped based on frequency distribution in bins of 200 µm. Measurements were analyzed by a two-way ANOVA with a Bonferroni post-hoc test. Central nuclei, F-max, laser injury AUC, percent fibrosis, CK, and protein expression measurements were analyzed by unpaired two-tailed mdx mouse [mdx mouse lacks functional dystrophin, the integrity of the plasma membrane is compromised, potentially leading to myocytes becoming more reliant on membrane repair to prevent the death of myocytes. To further investigate this reliance on membrane repair, we crossed the TRIM72/MG53 knockout mice into the mdx mouse model of DMD. These double knockout (DKO) mice were viable and, when sacrificed at 6 weeks of age, did not show differences in the wet weight of the heart, diaphragm, or skeletal muscle as compared to mdx mice at this age, even when normalized to body weight . We then assessed the extent of changes in sarcolemmal membrane permeability by immunostaining the EDL and tibialis anterior (TA) muscles from 6-week-old mdx and DKO mice to quantify IgG antibodies trapped in myocytes after membrane injury. DKO TA muscles showed a significant increase in IgG-positive myocytes as compared to mdx muscles . Thus, the DKO model provides an opportunity to evaluate the impact of membrane repair in compensating for the membrane fragility in DMD muscle and altering the pathologic hallmarks of this disease.Membrane repair has been shown to be a crucial process to maintain the integrity of skeletal muscle, and targeting membrane repair has shown potential as a therapeutic approach in the dx mouse . Since t muscles A. To dir muscles B. These mdx background, it is not clear if there is compensation by increased expression of other membrane repair proteins. In fact, in both the EDL and soleus, we observed an increase in caveolin-3 protein. Interestingly, we only resolved increased dysferlin expression in the soleus after normalizing to Ponceau S staining of the membrane as a loading control . These differences in body mass could result from metabolic differences in the TRIM72/MG53 knockout mouse background [mdx and DKO mice when the muscles were normalized to their decreased body weight at this age (data not shown). Histologically, there was also no difference in cross-sectional area or central nuclei counts in the EDL, soleus, and TA muscles (mdx mouse while the soleus performed the same in each mouse line, just as we observed in the 6-week-old mice (Since there was no major increase in structural defects in the young DKO mouse muscle, we aged these animals to determine if the accumulation of membrane injuries over time would lead to a difference in pathology between the nt mouse . Additiont mouse . We tests muscle . We obseckground ,67. Howe muscles A–C. We dold mice D.mdx mouse undergoes repeated cycles of muscle damage and regeneration, which results in increased fibrotic muscle tissue as the mice age [mdx mouse (The repeated damage to muscle that occurs during the progression of muscular dystrophy eventually results in the extensive deposition of fibrous tissue, which impairs muscle function and myocyte regeneration, increasing susceptibility to re-injury . The mdxmice age , and thidx mouse A–C.mdx mice by IgG staining (mdx model. These results indicate that loss of TRIM72/MG53-mediated membrane repair, although not debilitating in young mice, is relevant for the maintenance of myocytes as they undergo repeated bouts of damage as the mice age.Aged muscle undergoes various molecular and physical changes in response to the stresses of aging. This is particularly relevant to dystrophic muscle due to repeated bouts of injury and regeneration. Similar to the fibrosis analysis, the DKO EDL, soleus, and TA muscles all showed breakdown of membrane integrity when compared to aged staining A–C. The staining D. These mdx dystrophic muscles are dependent on the TRIM72/MG53-mediated membrane repair to survive repeated injury to the sarcolemmal membrane. At six weeks of age, we observed minimal histological differences between the DKO and mdx mouse; however, we found that the EDL muscle of the DKO mouse was functionally weaker than that of the mdx age-matched control muscles. These changes at a young age also include increased protein expression levels of two TRIM72/MG53 binding partners, dysferlin and caveolin-3, potentially in an attempt to compensate for the absence of TRIM72/MG53. This could occur through clearance of damaged portions of the membrane, reducing inflammatory signaling and limiting collagen deposition [DMD myocytes are dependent on the membrane repair process due to the lack of the dystrophin protein that provides a force-transferring link from the cytoskeleton to the extracellular matrix. Our studies show that myocytes from position ,69. mdx mice. Since the mdx mouse shows high levels of muscle pathology at baseline, the decreased membrane repair may not produce a major elevation in pathological measurements, which are already quite high in some muscles. Additionally, various age-related changes could contribute to the observed differences between the young and the old mice. We observed that TRIM72/MG53 protein levels decreased with age in the EDL muscle, while the soleus muscle showed no change (In response to aging, DKO mice develop significantly more fibrotic tissue, a hallmark of muscular dystrophy, in all skeletal muscles examined. We interpret these findings to indicate that the absence of TRIM72/MG53 exacerbates certain hallmarks of DMD, with these effects being somewhat blunted by the upregulation of other membrane repair proteins or because the absence of TRIM72/MG53 may alter other aspects of muscle function . Howevero change , which cmdx model also leads to increased myocyte necrosis and muscle weakness. This mouse model was observed to have increased muscle damage at 6 months of age when compared to the mdx mouse [mdx mice due to its susceptibility to membrane damage and is likely a cause of differences between these double knockout models. The combination of these results indicates that the loss of any one of these proteins can compromise membrane repair and accelerate the dystrophic phenotype. Future studies assessing the combinatorial loss of membrane repair proteins in dystrophic muscle will help to establish the relative contribution of each protein and how compensatory changes in the expression of membrane repair proteins can contribute to the phenotype seen in the mdx mouse.These results indicate that membrane repair is an important process for DMD myocytes to avoid death due to membrane fragility and support that targeting membrane repair could be an effective therapeutic approach to treat DMD. Our findings point to the value of membrane repair in compensating for the membrane fragility observed in DMD, supporting previous studies that illustrated the importance of membrane repair in dystrophic skeletal muscle . Deletiodx mouse . Althougdx mouse , whereasdx mouse . The formdx age-matched controls. Previous studies have shown that the EDL of mdx mice is particularly susceptible to contraction-related membrane injuries [mdx EDL to contraction-related membrane injuries [Our results also indicate that TRIM72/MG53 is important for the function of the EDL muscle of dystrophic mice. EDL muscles from 6-week- and 1.5-year-old DKO mice generate less maximal contractile force when compared to Decreased force production in the EDL muscle also argues against a role for increased caveolin-3 and dysferlin fully compensating for the loss of TRIM72/MG53 expression. Our previous work indicates that TRIM72/MG53 plays more of a role in the function of dysferlin  than it mdx mouse, where the diaphragm is heavily affected while the heart is relatively spared. Different anatomical muscles show varying levels of fibrosis in the mdx mouse (There are also certain limitations to our studies. In certain experimental circumstances, we could not collect all endpoints from certain muscle types. For example, it is challenging to collect contractility measurements of the tibialis anterior muscle using our ex vivo approach. In other experiments, biochemical or histological measurements were performed on muscles that were not used for contractility measurements to avoid confounding changes that could occur to muscles during ex vivo contractility assessments. It is not uncommon for specific anatomical muscles to be more affected than others in patients with certain neuromuscular diseases. This is the case in DMD patients and the dx mouse  and divedx mouse . This sumdx mouse model of DMD. We knocked out the membrane repair protein TRIM72/MG53 to elicit a membrane repair defect in these mice. Minimal histological changes were observed in 6-week-old mice; however, robust fibrosis and reduced membrane integrity were observed in aged mice. This suggests that the dystrophic muscle can compensate for membrane repair deficits early in life; however, in the absence of membrane repair, the continuous bouts of injury that lead to increased death of the fragile dystrophic myocytes tax the regenerative capacity of muscle, leading to increased fibrosis and loss of muscle function. This model demonstrates the value of membrane repair in repeated injury and supports a role for TRIM72/MG53 in maintaining myocyte survival in dystrophic muscle.Our current study focused on the value of membrane repair in the


Successful walking requires the execution of the pre-swing biomechanical tasks of body propulsion and leg swing initiation, which are often impaired post-stroke. While excess rectus femoris activity during swing is often associated with low knee flexion, previous work has suggested that deficits in propulsion and leg swing initiation may also contribute. The purpose of this study was to determine underlying causes of propulsion, leg swing initiation and knee flexion deficits in pre-swing and their link to stiff knee gait in individuals post-stroke.Musculoskeletal models and forward dynamic simulations were developed for individuals post-stroke (n = 15) and healthy participants (n = 5). Linear regressions were used to evaluate the relationships between peak knee flexion, braking and propulsion symmetry, and individual muscle contributions to braking, propulsion, knee flexion in pre-swing, and leg swing initiation.Four out of fifteen of individuals post-stroke had higher plantarflexor contributions to propulsion and seven out of fifteen had higher vasti contributions to braking on their paretic leg relative to their nonparetic leg. Higher gastrocnemius contributions to propulsion predicted paretic propulsion symmetry (p = 0.005) while soleus contributions did not. Higher vasti contributions to braking in pre-swing predicted lower knee flexion (p = 0.022). The rectus femoris had minimal contributions to lower knee flexion acceleration in pre-swing compared to contributions from the vasti. However, for some individuals with low knee flexion, during pre-swing the rectus femoris absorbed more power and the iliopsoas contributed less power to the paretic leg. Total musculotendon work done on the paretic leg in pre-swing did not predict knee flexion during swing.These results emphasize the multiple causes of propulsion asymmetry in individuals post-stroke, including low plantarflexor contributions to propulsion, increased vasti contributions to braking and reliance on compensatory mechanisms. The results also show that the rectus femoris is not a major contributor to knee flexion in pre-swing, but absorbs more power from the paretic leg in pre-swing in some individuals with stiff knee gait. These results highlight the need to identify individual causes of propulsion and knee flexion deficits to design more effective rehabilitation strategies.The online version contains supplementary material available at 10.1186/s12984-022-01029-z. Over 795,000 people in the United States experience a stroke each year and over half of individuals post-stroke over age 65 have reduced mobility . RegainiFor example, modeling studies having identified knee flexion velocity at toe-off as the primary contributor to peak knee flexion during swing  and low Braking and propulsion deficits are also common in individuals post-stroke , and in Previous work has established the importance of pre-swing conditions to achieving adequate swing phase knee flexion. However, actual muscle contributions to propulsion, knee velocity and leg-swing initiation in individuals post-stroke and their relationship to swing-phase knee flexion has not been established. Thus, the objectives of this study were to determine the underlying causes of propulsion and braking deficits and identify muscle contributors to pre-swing knee flexion acceleration and leg swing initiation in individuals post-stroke with and without stiff knee gait. We hypothesized that (1) braking and propulsion asymmetries would be caused by both low plantarflexor contributions to propulsion and high vasti contributions to braking, (2) vasti and plantarflexor contributions to propulsion and braking in pre-swing would predict swing phase knee flexion, (3) the rectus femoris would be a major contributor to knee extension in pre-swing in individuals with stiff knee gait, and (4) total musculotendon power delivered to the leg in pre-swing would predict knee flexion during swing. The outcomes of this work will highlight specific deficits in propulsion and leg swing initiation post-stroke and their implications for swing phase knee flexion, which will provide a basis for developing targeted rehabilitation strategies.Kinematic, kinetic and electromyography data were collected from 15 individuals post-stroke  and 5 age-similar control subjects  (Table A representative paretic leg gait cycle (left gait cycle for control subjects) for each participant was chosen for further analysis using the functional medial distance depth method . Using OBody propulsion and braking were calculated from the integral of the anterior and posterior GRF, respectively. Propulsion and braking asymmetries were defined as the percentage of paretic propulsion (PP) and paretic braking (PB), i.e., the paretic propulsive or braking impulse divided by the sum of paretic and nonparetic propulsive or braking impulses . Heel strike and toe-off were identified from the vertical GRF using a threshold of 20 N. Knee flexion velocity at toe-off and peak knee flexion during swing were identified using joint kinematics from RRA. Pre-swing was defined as the double support phase between nonparetic (right) heel strike and paretic (left) toe-off. Individuals were classified as having stiff knee gait if their peak paretic knee flexion during swing was at least 15° less than their peak nonparetic knee flexion , 35.Induced acceleration and segmental power analyses  were useTo test the hypotheses that greater vasti contributions to braking and lower plantarflexor contributions to propulsion would predict braking and propulsion asymmetries, linear regression models were created with PP and PB as the dependent measures and either soleus, gastrocnemius or vasti contributions to AP COM acceleration over stance as the independent measure. To test the hypothesis that pre-swing braking and propulsion would predict swing-phase knee flexion, linear regression models were created with peak knee flexion as the dependent measure and either total pre-swing AP GRF impulse , soleus, gastrocnemius or vasti contributions to AP COM acceleration in pre-swing  as the independent measures. Similarly, to test the hypothesis that individuals with low knee flexion would have less paretic musculotendon work performed on the paretic leg, a linear regression was created with peak knee flexion as the dependent measure and net musculotendon work performed on the paretic leg as the independent measure. Linear regressions were created with one predictor and one outcome measure at a time using the linear regression model tool in MATLAB . Significance was defined as α = 0.05.2 = 0.47, β = 0.43). Vasti, gastrocnemius and soleus contributions to AP GRFs did not predict braking asymmetry . Lower vasti contributions to braking in pre-swing relative to walking speed also predicted greater peak knee flexion during swing  while soleus and gastrocnemius contributions to propulsion in pre-swing did not . The low knee flexion group had lower total muscle contributions to paretic leg swing on average, but with high variability between participants  plantarflexor contributions to propulsion and 2) vasti contributions to braking would predict propulsion asymmetry. These hypotheses were partially supported. Gastrocnemius contributions to AP GRF predicted propulsion symmetry. However, soleus and vasti contributions to AP GRF impulses did not predict braking or propulsion symmetry  due to prolonged braking from the vasti and rectus femoris despite producing more propulsion with the paretic plantarflexors (Fig. We hypothesized that vasti and plantarflexor contributions to propulsion and braking in pre-swing would predict swing phase knee flexion. This hypothesis was partially supported. Lower pre-swing AP GRF impulse and greater vasti contributions to braking predicted less knee flexion in swing, but plantarflexor contributions did not Fig. . SimilarWe hypothesized that the rectus femoris would be a major contributor to knee extension in pre-swing in individuals with stiff knee gait. This hypothesis was not supported. Rectus femoris contributions to knee extension were minimal in all groups Fig. , while tThere is evidence that the rectus femoris does contribute to stiff knee gait in some but not all cases, as rectus femoris Botox injections for individuals post-stroke improved knee flexion for some individuals but not for those with more severe knee flexion deficits . Thus, tWe hypothesized that greater net musculotendon power delivered to the leg in pre-swing would predict greater knee flexion, but this hypothesis was not supported. While on average the low knee flexion group had less power delivered to the paretic leg in pre-swing compared to the typical knee flexion and control groups, there was significant variability between participants Fig. .While the rectus femoris was not a primary contributor to knee extension in individuals with low knee flexion, it may still limit leg swing by absorbing power from the leg. Muscles act to either generate, absorb or transfer power between body segments , 45. In The iliopsoas contributed less to leg swing initiation in both stroke groups on average compared to healthy controls, but especially the low knee flexion group Fig. . These rA potential limitation of this study is that individuals post-stroke often have high levels of muscle co-contraction , 47, whiWe observed that some participants had paretic propulsion deficits due to low plantarflexor contributions to propulsion and/or excess vasti contributions to braking. Others appeared to produce sufficient or high paretic propulsion, but accomplished that propulsion via compensatory mechanisms such as a reliance on the hamstrings rather than appropriate plantarflexor activity. Greater vasti contributions to braking in pre-swing predicted lower knee flexion. The rectus femoris and iliopsoas did not directly contribute to lower knee flexion in pre-swing. However, in a number of cases the rectus femoris absorbed more power and the iliopsoas contributed less power from the paretic leg in pre-swing in individuals with low knee flexion. These results highlight the need to identify the underlying causes of propulsion and knee flexion deficits in each individual in order to design more effective rehabilitation strategies.Additional file 1: Table S3. Propulsion and braking symmetry, muscle contributions to propulsion and braking, and knee kinematics. Values for participants in the low knee flexion group are bolded.


They have a distinctive bipolar morphology, characterized by the presence of a single axon that bifurcates into two branches with distinct projections and functions1 . Electri6 The scaffolding protein ankyrin-G, considered the master organizer of the AIS, orchestrates its formation and maintenance in combination with voltage-gated ion channels, membrane proteins, and a specialized cytoskeletal network.7 Detecting these components has revealed the presence of an AIS in DRG neurons in culture,8 but evidence for DRG AISs in vivo has remained elusive. Given the AIS’s key role in spike initiation, if the structure were found to exist in cells of the intact DRG it would be a prime candidate for generating the spontaneous activity that contributes to neuropathic pain and could become a promising therapeutic target.The AIS is located within the proximal axon and, in most neurons of the mammalian CNS, is the site of action potential initiation.5 began by providing convincing anatomical evidence that DRG neurons in the intact adult mouse can indeed have a bone fide AIS. Using immunohistochemistry in ex vivo DRG tissue, they identified ankyrin-G-positive elongated structures in the proximal axon of medium and large—but not small—DRG cells, cell types which also possess myelinated axons and nodes of Ranvier. The AISs of DRG neurons have an unusual curly trajectory, perhaps explaining why they have remained undetected for so long. Nevertheless, they contain classical AIS components such as βIV-spectrin, as well as a high density of voltage-gated sodium channels (Nav) and the proximal axonal protein TRIM46. Notably, the authors also observed that the nociception-associated channel Nav1.7 is concentrated at DRG AISs. This is the first time this particular sodium channel has been found at the AIS, and its discovery raises some interesting cell biological questions about how it accumulates there.Here, Nascimento and colleaguespost hoc immunolabelling for Nav1.7. This revealed that, while only a small percentage of DRG neurons showed spontaneous activity, over 90% of those with spontaneous activity had an AIS. In contrast, only ∼60% of non-spontaneously active cells were AIS-positive. No differences in AIS length or position were found between AIS-positive neurons with and without spontaneous activity, but cells showing spontaneous activity had significantly higher levels of AIS Nav1.7 fluorescence, suggesting that this channel contributes to pathological activity levels. Indeed, Nav1.7 intensity was significantly and positively correlated with the frequency of spontaneous activity at the cell-to-cell level.To assess the potential for these AISs to contribute to pathological neuronal activity, the authors used a chronic constriction injury (CCI) model of neuropathic pain that produced aberrant spontaneous activity in DRG neurons. In an elegant approach to assessing structure and function in individual cells, they combined functional imaging experiments using the calcium indicator GCaMP6s in an acute dissected preparation with v conductance and action potential initiation: in the absence of higher Nav levels at the AIS, only higher threshold, somatically initiated action potentials could be fired. These data suggest that the DRG AIS plays a major role in the generation of pain-associated aberrant firing, although the fact that not all neurons generating such activity had a detectable AIS indicates that it may not be absolutely required for spontaneous activity in all cells.A computational model of DRGs also showed a clear relationship between AIS Naet al.5 selectively removed AISs from DRG neurons before assessing the effects on spontaneous activity generation and behavioural measures of pain. They triggered AIS disassembly in adult DRG neurons by crossing the sensory ganglia-specific Advillin-CreERT2 inducible conditional line with mice floxed for the ankyrin-G gene Ank3 (Avil-Ank3fl/fl). Knockout generation via tamoxifen injection resulted in a progressive decrease in the percentage of medium and large DRG neurons with an ankyrin-G-positive AIS. Since ankyrin-G depletion is known to also disassemble nodes of Ranvier,9 the authors had to be extremely careful with the timing of their experiments. Deficits in node integrity and axonal conduction velocity were indeed seen in Avil-Ank3fl/fl mice at 6 weeks post-tamoxifen injection (6 wpt). However, they were not evident at an earlier 5 wpt time point when AISs were already severely compromised. Studying Avil-Ank3fl/fl mice at 5 wpt therefore allowed the authors to specifically assess the role of DRG AISs—and not other axonal compartments—in generating pain-associated aberrant activity.Finally, to provide causal evidence for the role of the DRG in producing pain-associated neuronal activity, Nascimento et al.5 used this approach to ask two key questions. First, what happens to spontaneous activity when DRGs lack an AIS? Calcium imaging after CCI showed that, compared to wild-type controls, the percentage of neurons with spontaneous activity in Avil-Ank3fl/fl mice was significantly reduced and calcium transient rates were decreased. Only a few remaining AIS-negative neurons still had some spontaneous activity. These effects were restricted to medium and large DRG neurons, in line with the exclusive presence of AISs in these cell types. Thus, the AIS is key to generating spontaneous activity in DRG neurons in a model of neuropathic pain.Nascimento 5 carried out behavioural tests for mechanical allodynia, where pain is experienced after exposure to harmless tactile stimuli.10 Before CCI induction, no differences were found between wild type and Avil-Ank3fl/fl tamoxifen-injected mice in the allodynia measure of paw withdrawal threshold. However, one week after CCI, Avil-Ank3fl/fl mice had a significantly higher threshold, with the majority of AIS-knockout animals showing no difference at all compared to baseline, pre-CCI levels. Given the specific depletion of DRG AISs in the inducible Avil-Ank3fl/fl line at the time point tested, these experiments show that the AIS in DRG neurons is required for both aberrant electrical activity and mechanical allodynia in the CCI model.The second, crucial question was therefore: when DRGs lack an AIS, what happens to neuropathic pain itself? To address this, Nascimento and colleaguesin situ, but also presents correlative and causal evidence that those AISs generate the pathological spontaneous activity underpinning neuropathic pain (5 have built a high-quality study with several strong points, including careful description of basic anatomy, combinations of structural and functional readouts in the same neurons, and a highly specific in vivo AIS manipulation. We watch with interest to see whether rapidly expanding knowledge of AIS composition and function can now be harnessed to develop therapeutic interventions specifically targeted to this DRG substructure.Altogether, this study not only provides the first description of AISs in DRG neurons hic pain . Nascimeex vivo, ultrastructural studies using electron microscopy will be essential to complete the morphological picture. In light of the results showing that spontaneous activity is not completely gone in the absence of DRG AISs, it will be important to assess what else is contributing to spontaneous activity generation. In vivo functional imaging is also required to assess whether pain-associated spontaneous activity is present, and associated with DRG AISs, in the intact animal.The study also raises a wide range of interesting questions. To continue the AIS characterization of DRG neurons Finally, while the DRG AIS is clearly important in generating pathological activity, we wonder what its role might be in the healthy nervous system. Presumably this complex structure has not evolved purely to generate debilitating sensations of pain! Given the DRG neuron’s unusual morphology, perhaps an additional highly excitable domain is necessary in larger cells to boost action potential propagation towards the spinal cord, or to shape the properties of spikes as they move into the CNS. Alternatively, or additionally, the AIS’s well characterized role in the maintenance of neuronal polarity might mean that it is essential for upkeep of the very morphology that makes DRG neurons so distinctive. Work to understand the role of the AIS in these fascinating cells is only just beginning.


Several recent viraloutbreaks, culminating in the COVID-19 pandemic,have illustrated the need for comprehensive improvement in the detection,control, and treatment of emerging viruses that exhibit the potentialto cause epidemics. Nanotechnology approaches have the potential tomake major contributions in all these areas. This perspective is intendedto outline how nanotechnology can be employed to improve upon respiratorydisease detection and containment measures, and therapeutics, witha particular emphasis on applications that can address key areas,including home diagnostics, contact tracing, and the evaluation ofdurability of vaccine protection over time and against future variants.Nanotechnology offers potent tools to address these needs, but furtherresearch is required to validate these applications to address needsof future epidemics. However, the use of bivalentvaccine boosters that also contain sequences from the SARS-CoV-2 OmicronVOC is also under evaluation.3 Future SARS-CoV-2 vaccinesmay require seasonal updates in response to predictions made usingepidemiologic data concerning emerging VOCs,4 similar to the influenza vaccine production. However, there is significantinterest for multivalent vaccines that display spike proteins froma pool of VOCs to enhance potential protective coverage of a predictivevaccine, and the development of vaccines that can target conservedregions that can offer protection against a broad array of potentialVOCs. Broad spectrum therapeutics are also of considerable interest,and studies examining the efficacy of oral antiviral drugs to reducesevere illness by SARS-CoV-2 variants when taken soon after symptomonset and to prevent or attenuate long COVID pathology are now underway.Further developments on all these topics are required to permit theworld to “live with SARS-CoV-2” and deal with futurepathogens that have the potential to cause pandemics. This perspectivewill employ SARS-CoV-2 as an example to discuss what measures maybe useful in addressing these issues.Global efforts to eradicatesevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) haveled to acceptance that this virus has or will become endemic in theglobal population. Learning mistakes made during early SARS-CoV-2diagnosis and containment efforts is critical to prevent or containfuture outbreaks of SARS-CoV-2 variants of concern or emerging pathogenscapable of producing pandemics. One of these key lessons had beenthe critical need for simple, rapid, and inexpensive point-of-carediagnostics that can accurately detect forthcoming cases to preventlarge outbreaks. Vaccines and antibody therapeutics can provide amore long-term solution given their development and validation time,although their effectiveness can be diminished by variants that ariseduring ongoing transmission and by the progressive decline in protectiveimmunity following vaccination. Broad spectrum vaccines that targetmultiple SARS-CoV-2 variants of concern (VOCs) are therefore of greatinterest for their potential to produce enduring protection againstemerging variants. Most current vaccines target the spike proteinof WuhanHu-1 strain,What lies ahead? Countriesaround the world are easing restrictionsand stepping toward economic recovery, but while this is inevitableat some point, it risks another wave of infection that may furtherdamage already strained economic and healthcare infrastructure. Tominimize the potential for and extent of such events, additional worldwideeffort should be focused on the development of smart strategies toallow the safe resumption of daily activities by developing more advancedmeans to rapidly detect future outbreaks and variants and better meansof treatment. Specifically, nanotechnology approaches provide multipleopportunities to improve critical means of infection prevention anddetection, including personal protection measures, environmental sanitationapproaches, and rapid diagnostic tests, among others 1. For ex5 Measures employed to reduce respiratory transmission, such as facemasks,have played a key role in reducing transmission, but extended useof facemasks in day-to-day life can have drawbacks, including physicaldiscomfort, that can reduce compliance even in individuals willingto adhere to their use. Facemasks intended for protection againstrespiratory pathogens should have excellent filtration efficacy andbe comfortable when worn for extended time periods. However, currentN95 and surgical facemasks lack self-sterilizing properties and arethus prone to accumulate pathogens during the course of prolongeduse, leading to increased risk of exposure. One recent study foundthat facemasks coated with a graphene nanolayer could block interactionswith small droplets, which tended to bounce off this superhydrophobicnanolayer.6 Further, exposing this graphenenanolayer surface to direct sunlight for 1 min could raise its temperatureto >80 °C, allowing its photothermal sterilization to permitextended and repeated mask usage without the need for harsh chemicalsterilization procedures that could damage their filtration performance.This study also found that conjugation of silver nanoparticles withinthe graphene layers improved the disinfectant characteristics of thefacemasks. The addition of thin copper nanowire metal–organicframework core–shell structures7 or copper oxide nanoparticles into facemask designs was also foundto be a promising means for incorporating antiviral activity.9 Another key limitation of the traditional facemask is the thermaldiscomfort or skin damage caused by constant use. The thermal characteristicsof a facemask primarily depend on fiber thickness, which influencesair filtration and permeability. Studies indicate that facemasks thatuse nanofibers can reduce the respiratory effort required during normalair filtration, while still protecting protect against small particles(<50 nm).10 Nanoporous polyethylenenanofibers can provide filtration efficiencies superior to those oftraditional facemasks material, while their infrared transparencyallows improved radiative cooling to reduce thermal discomfort.11 Thus, nanomaterials have potential to improve the performance oftraditional mask designs by incorporating self-sanitizing and antiviral/antimicrobialactivities, reducing their insulating properties to increase comfort,and improving their particulate filtration properties. Several externalparameters can affect the filtration efficacy and comfort of a facemask,including the environmental temperature and humidity, which can promoteoverheating and condensation to promote the growth and survival ofmicroorganisms on its filtration materials, and elevation, which canfurther restrict air intake. Facemask design and composition can alsoaffect the trade-off between filtration efficiency and air intake,but the use of nanomaterials can improve the filtration propertiesof current facemask designs. For example, nanomaterials can be incorporatedinto textiles to modify the fibers used in mask construction and improvetheir filtration efficiency by altering their electrostatic propertiesor other parameters. Several nanomaterials with antiviral activities,including those that employ silver, copper oxide, and carbon nanoparticles,have been successfully embedded into textiles used in facemasks.12 Additional nanomaterials properties, such astheir relative hygroscopic and thermal properties, may also permitthe design changes to produce more comfortable masks that still exhibitexcellent filtration properties.SARS-CoV-2 transmission predominantlyoccurs by inhalation of aerosolsand droplets generated by coughing, sneezing, or talking, with surfacetransmission appearing to play a modest role.13 Another study conducted between January 2018and December 2020 by the U.S. Poison Control Center identified 299adverse events caused by abundant exposure to hand sanitizers, primarilyheadache and dizziness attributed to vapors from these products.13 Disposable medical gloves are an alternativefor those who cannot tolerate hand sanitizers but cannot be readilysterilized and can thus transfer contamination, can be difficult towear for extended periods, must be removed carefully to avoid handcontamination, are intended for single use and can be nonbiodegradable(vinyl or nitrile gloves) and thus present a disposal problem, orcan have risks of contact dermatitis and even more severe allergicreactions in some individuals (latex gloves). These issues could,however, potentially be addressed by the use of self-sanitizing biodegradablegloves. For example, nanofibrous mesh layers that contain metal nanoparticles with antiviral properties could be incorporatedinto the outer surface of a biodegradable glove design to reduce theirsurface contamination, reducing the potential for wearer exposureand secondary transfer of this contamination to other surfaces. Thenanofibrous mesh structures of these gloves could also provide ventilationto reduce sweat accumulation inside the gloves, a common issue forlatex and plastic gloves used in bioprotective applications.Hand sanitization is anotheressential personal protective measurethat has become an inseparable part of personal safety in public settingswhere it is often not feasible to wash one’s hands with soapand water after exposures to high contact surfaces, such as publictransport, shopping venues, and large public events. However, effectiveuse of hand sanitizers requires either contactless application orsanitization of individuals containers during use to prevent possiblerecontamination from their surfaces. The Centers for Disease Controland Prevention recommends that such hand sanitizers contain at least60% alcohol, but widespread and frequent use of such hand sanitizerscan entail risks. For example, the Food and Drug Administration found50 adverse events attributed to the use of alcohol-based sanitizersshortly after the start of the COVID-19 in March 2020.14 Virus viability on a contaminated surface dependson multiple factors, including the surface type (porous or nonporous),virus structure (enveloped or nonenveloped), and temperature and humidityconditions. Recent studies indicate that SARS-CoV-2 viability is greateron plastic or stainless-steel surfaces (up to 72 h) than copper orcardboard surfaces (3–4 h),15 withgreater virus viability and stability detected on nonporous versusporous surfaces.16 Many researchers haveinvestigated viability by virus type and stain and found that surfacesurvival times for enveloped viruses, including influenza virus, parainfluenzavirus, and corona virus can vary from hours to days, whereas nonenvelopedviruses, such as astroviruses and rotaviruses, can survive on surfacesfor up to a few months.14Many viruses, including SARS-CoV-2, can surviveon surfaces forextended periods under favorable conditions, raising the potentialfor “supply chain contagion” where rapid national andinternational transport of contaminated packages could serve as avector for distant outbreaks. Surface-based transmission events playa crucial role in the spread of several human viruses, including porcineepidemic diarrhea virus, the middle east respiratory syndrome (MERS)coronavirus, and endemic human coronaviruses 229E and OC43.17 Frequent usage of concentratedalcohol solutions used in disinfectants also can damage many commonsurfaces, including plastics, rubber, and many wood finishes. Newmaterials with broader virucidal activities and fewer downsides arethus highly desirable. Recent studies indicate that single-walledcarbon nanotubes can be used in a spray disinfectant, since theirphotothermal activity could increase the temperature of treated surfacesto 90 °C when exposed to direct solar illumination for effectivevirucidal activity.18 Spraying these single-walledcarbon nanotubes onto a polypropylene surface also formed nanospikestructures that exhibited super hydrophobicity when exposed to variousbiological fluids to reduce surface contamination. A spray containingthe trifunctional polymer poly(DMA-PEGMA-QA) has also been reportedto provide at least 24 h of protection against viral contaminationby forming a nanoscale coating layer when applied to the fabric ofpersonal protective equipment.19 Biocompatibilitytests found that poly(DMA-PEGMA-QA) did not induce any discernibletoxicity in vitro or in vivo. Studies have also reported that lowtoxicity alginate-based nanocomposites can inhibit a wide spectrumof viruses, including the human immunodeficiency virus and the hepatitisA, B, and C, herpes, rabies, rubella, and polio viruses.20 Metal oxide nanomaterials, including a zincoxide nanospray, are also effective as antiviral surface disinfectantagents,21 although care may need to betaken to balance the virucidal activity against potential cytotoxiceffects. More research is therefore warranted to evaluate the potentialutility of nanomaterial-based disinfectant sprays against the spectrumof virus types known to cause human disease. This potential for surfacetransmission raised questions about the safety of transported goodsat the consumer level and changed the consumer-merchandise landscapeearly in the SARS-CoV-2 pandemic.23 Surface contaminationof packages has not proven to be an issue for SARS-CoV-2 or otherpathogens to date, but the incorporation of antiviral coatings orfilms on packaging materials could be used to reduce the potentialfor viral contamination, particularly when handling materials thatmay be more prone to such contamination. Hybrid nanocomposites containingcopper,25 zinc, graphene,26 and silver28 have antiviralactivities and could be incorporated into packaging materials or othermaterials used on high contact surfaces. Such nanocomposites can alsobe added to paints and varnishes to produce antiviral coatings capableof continuously decontaminating walls and other potential contractsurfaces in public spaces, including airports, bus and train stations,public vehicles, and grocery stores, shopping malls, theaters, andhealthcare units. Large-scale deployment of such antiviral coatingsin paint and other materials thus represents another key approachfor nonspecific reduction of viral surface contamination, which requiresadditional research to determine its best applications and potentialutility to attenuate viral infections, including current and emergingviruses capable of major disease outbreaks.Ideal disinfectantsshould cover the entire antimicrobial spectrum,exhibit rapid activity without toxic effects on the environment orhuman health, be cost-effective, biodegradable, and compatible witha broad array of potential decontamination surface types. Alcohol-baseddisinfectants played a key role during the COVID-19 pandemic, butthe virucidal efficacy of alcohol disinfectants varies with theiralcohol concentration, contact time, and the targeted virus type.For example, isopropyl alcohol is effective only for enveloped viruses and lackseffective virucidal activity against nonenveloped viruses, includingadenoviruses and rhinoviruses that are responsible for common humaninfections.29 There isthus a need for less hazardous disinfectants, which has led to researchon the development of biocompatible disinfectants, including solutionsthat contain biocompatible metal nanoparticles, which could providea safe alternative to disinfectant chemicals in current use. Nanomaterialsare now in broad use in different biological applications, but concernshould still be applied for their use in human health applicationsand the potential impact of such applications on the environment.Nanomaterial toxicity effects are mediated by their size, charge,and composition. Studies are still underway to investigate how physicochemicalfeatures of different nanomaterials influence toxicity.30 More research is therefore required to studytoxic nanomaterial effects that could result from changes in theirdimension, structure, surface area, functionality, constituent, source,and exposure dose before deploying them as a disinfectant spray.Disinfectant spraysare commonly used to sanitize high contactsurfaces in public areas, including public transport, healthcare facilities,as well as households. However, routine and/or excessive use of commondisinfectant chemicals  may have adverse effects on humanhealth and the environment.The demand for and rapid expansion of decentralizedand home-basedtesting technologies has been a dramatic effect of the COVID-19 pandemic.This demand has led to the development of simple diagnostic assaysthat use inexpensive portable or disposable sample-in-result-out readoutdevices. Such devices can eliminate the need to transport clinicalsamples to a centralized lab and eliminate the need for trained personneland specialized equipment that delay results and can increase costs.This development has produced both advantages and challenges, butgreatly increased test capacity and sample-to-answer times at thecost of reduced testing accuracy and the loss of epidemiologic datathat would normally be reported by testing laboratories. Nanomaterialapplications can be employed to address the first of these drawbacks,and it should be possible to address the second with smartphone-basedreporting apps or other approaches, although these still entail potentialdifficulties with user compliance and data security.35 An epidemiological study conducted early in the pandemic estimatedthat 80% of SARS-CoV-2 in the analyzed population could be attributedto virus transmission by an undiagnosed SARS-CoV-2 infected population.36 Thus, early detection measures can play a crucialrole in disease control by rapidly identifying these infected individualsand implementing measures to reduce their potential for virus transmission.Reverse transcription polymerase chain reaction (RT-PCR) is the goldstandard for SARS-CoV-2 RNA detection, but can be time-consuming sinceassays are usually collected and analyzed in batches in clinical laboratoriesthat have the equipment and trained personnel required to performthese assays. It can thus require more than 24 h to obtain a testresult after sample collection, providing a significant window forSARS-CoV-2 transmission from individuals with asymptomatic infections.Nanomaterial solutions have the potential to address this testingbottleneck to allow assays to be performed at sites lacking theseresources to increase testing capacity and thus avoid shipping andprocessing bottlenecks that increase sample-to-answer times. For example,a plasmonic nanomaterial that rapidly and efficiently converts lightto heat has been used to develop a simple photonic PCR device thatpermits ultrarapid template amplification and real-time analysis (40PCR cycles in ∼5 min) with ∼90% yield, using an on-chipformat ideal for point-of-care diagnosis at resource limited settings.37 Nanomaterial devices can also simplify the extractionof nucleic acids from biofluid, which is typically required from mostconventional PCR assays, but can be a complex process that requirestrained personnel and reagents and equipment that may be difficultto obtain at sites with limited resources. For example, single-walledcarbon nanotubes can permit direct recovery of nucleic acid from complexbiological samples using a single-tube isolation procedure that doesnot require multiple steps, expensive supplies or equipment, or substantialtechnical expertise.38 Nanodroplets loadedwith target-specific Cas13a/CRISPR RNA complexes and quenched fluorescentprobes have also been used to detect target RNAs in a digital dropletassay format, avoiding the need for RT-PCR,39 although this approach is not suitable for high-throughput analysesor use in resource limited settings. Nanomaterials have also madesignificant contributions to rapid serodiagnosis approaches. For example,a lateral flow immunoassay that uses fluorescent dye-loaded nanoparticlesis reported to detect SARS-CoV-2-specific immunoglobulin G in mostof the tested subjects within 1 day after symptoms onset40 and thus has a strong potential for rapid point-of-carediagnosis. Nanoparticles functionalized with virus-specific epitopescan also be used in seroprevalence surveys to identify individualswith late-stage or past SARS-CoV-2 infections, where positive casesare detected by a colorimetric change induced by nanoparticle aggregationupon cross-linking by these specific antibodies.41 A similar approach has also been employed for direct detectionof SARS-CoV-2 RNA from processed patient specimens to permit rapidvisual detection of positive samples.42 In both cases, this approach eliminates the need for sophisticateddetection instruments to facilitate their use in point-of-care settings.Such nanomaterial sensors should also be readily incorporated intolab-on-a-chip devices employed in at-home testing kits. Future sensorsshould ideally also evaluate alternate sample types since relianceon upper respiratory tract specimens may miss respiratory virus infectionsin the lower respiratory tract that are not detected by these specimensdue to limited or transient infection of these regions during an activeinfection. Diagnosis of these infections may require more invasivespecimens  or expensive computed tomography scans that can have poor diagnosticspecificity. Lab-on-a-chip designs that couple the optical and chemicalproperties of nanomaterials to biological receptors for target detectionhave already been reported for diverse biosensing applications.48 However, even with advances in sensitivity and specificity, severalconcerns still need to be addressed before such devices can be usedin large-scale applications. One such concern is that disposable readoutdevices and batteries would generate a substantial amount of electronicwaste, although this could be offset if these devices could be mademodular to accept small test chips and employ rechargeable batteries.Ideally, these devices should also be able to communicate their resultsin a format that permits their simple and secure transmission to acentral epidemiologic data repository that can be used to monitorpotential outbreaks. These concerns, and battery weight, also applyto wearable sensors and may be particularly relevant in poor in remoteor resource limited settings. More attention thus needs to be paidto develop sensor platforms with small replaceable sensor chips andrechargeable batteries or other power supplies. Power generation approachesthat employ small photovoltaic panels, generators that capture mechanicalenergy from small body movements, or other forms of renewable powercould serve this purpose.Severalstudies have evaluated nanomaterials for electroactivefunctional properties required to obtain the signal amplificationrequired to sensitively detect the target virus across a wide concentrationrange in complex biological specimens including saliva and nasal tissuespecimens employed by current SARS-CoV-2 diagnostics.51 Flexible electronics that usebiocompatible nanomesh structures derived from polymeric nanomaterialscould be adopted to develop flexible and gas permeable skin-contactsensors suitable for extended use in these applications. Integratingartificial intelligence (AI) into an Internet of medical things (IOMT)lab-on-a-chip device would be an incredibly powerful strategy to developan extensive health surveillance framework. Such devices could allowreal-time monitoring for changes in heart rate, blood oxygenationlevel, resting body temperature, or other parameters that could potentiallybe used as a suggestive indicator for asymptomatic, presymptomatic,or mild infections and provide a wealth of valuable epidemiologicinformation. Similarly, other wearable devices designed to constantlymonitor for changes in blood oxygen levels, respiratory effort, changesin voice pitch, coughing, or other respiratory parameters in sickindividuals and/or those at-risk for severe disease could be usefulearly indicators to seek rapid medical care. IOMT diagnostic devicescould be used to detect new outbreaks, identify individuals who shouldbe tested for infection, and assist in contact tracing and quarantineconfirmation by transmitting real-time remote data and location signalsremotely in several locations. Thus, AI-IOMT-assisted lab-on-a-chipdevices appear likely to be one important pillar of next-generationdiagnostic devices needed to avoid or reduce the extent and durationof future lockdowns or guide the reopening of business and socialactivities in the wake of such events.Wearable lab-on-a-chip devices couldhave significant clinicalutility if they can rapidly detect early signs of infection to preventthe spread of infection and improve contact tracing and/or identifysymptoms associated with the development of severe disease to allowrapid intervention that could minimize disease pathology and improvepatient outcomes. Such wearable sensors may need direct physical contactwith the skin through electroactive thin film sensors to monitor somekey biometric parameters.52 Nanotechnology approaches havethe potential to streamline current B- and T-cell response assays,which are not suitable for high-throughput use to monitor protectiveimmunity at scale, to identify the decline in vaccine efficacy againstfuture variants or to better evaluate the need for specific populationsor individuals to receive a vaccine booster dose to achieve protectiveimmunity.The SARS-CoV-2 pandemichas also illustrated the need for assaysthat can accurately measure specific immune responses generated inresponse to vaccination or infection to predict the likelihood ofprotection from infection, severe infection, or death upon subsequentexposure. A better understanding of how these immune responses changeover time and respond to future variants will be essential in allowingpublic health officials to make rapid and informed decisions on vaccineefficacy and dosing strategies and other policy measures. However,current methods have limited utility for monitoring the adaptive immuneresponses regulated by effector and memory B-cells and T-cells thatare responsible for durable specific immunity after vaccination orexposure to a viral pathogen and can remain protective against SARS-CoV-2infection after protective neutralizing antibodies decline below protectivelevels.53 Another SARS-CoV-2antiviral drug developed by Pfizer, paxlovid that contains nirmatrelvirand ritonavir. Nirmatrelvir functions as a peptidomimetic to inhibitthe activity of the main SARS-CoV-2 protease nsp5 and has antiviralactivity against coronaviruses known to infect humans.54 Ritonavir, a strong cytochrome P450 3A4 inhibitor,must be administered with nirmatrelvir to permit systemic nirmatrelvirconcentrations to reach a therapeutic range, since cytochrome P4503A4 is highly expressed in the liver and intestines. Due to its requiredinclusion of ritonavir, paxlovid is thus prone to interact with othercoadministered drugs and may not be beneficial if a patient is receivingother medications for comorbid conditions.55 However, these detrimental effects could be minimized or avoidedfor current and future antiviral drugs—and other drug types—byemploying targeted delivery approaches to selectively increase drugbioavailability only at the selected sites or tissues. One study hasexamined a nanoparticle-mediated strategy for targeted delivery ofremdesivir, a nucleotide prodrug that promotes premature terminationof viral RNA transcription56 and can reducethe risk of hospitalization and death in nonhospitalized patientsat high risk for progression to severe disease.57 This study evaluated the potential for aerosol deliveryof remdesivir-loaded liposome nanoparticles to enable direct drugdelivery to affected respiratory tissue and avoid off-target effectsthat could arise during its standard, three-day intravenous systemicdelivery protocol.58 Nanotechnology offersseveral nanocarrier platforms to permit selective and efficient drugdelivery to targeted sites,61 which could increase drug bioavailabilityat therapeutic concentrations at sites of viral infections while reducingtheir systemic levels to decrease their systemic levels and associatedoff-target effects. Such nanocarriers can be employed to permit thedelivery of multiple drugs to a target site to allow targeted deliveryof synergistic drugs for more effective treatment.Nanomaterials may also haveutility in three distinct types oftherapeutic applications: nanocarriers that can permit targeted drugdelivery, nanodecoys that can adsorb virus particles, or pro-inflammatoryfactors to attenuate infection or its resulting pathology, and nanovaccinesthat can enable alter vaccine delivery approaches and targeted delivery1 and 2. 63 Further, due to their small size and other properties, these nanoparticlescan readily penetrate most biological barriers, including the blood–brainbarrier. This may be of significant importance since data now indicatethat several established and emerging viral pathogens  exhibit neurovirulence, neuroinvasiveness, orneurotropism associated with neural pathology and thus the abilityof drugs to cross the blood–brain barrier may be critical intreating the neuropathology of these infections. Notably, severalnanomaterials  demonstrate great potential as nanocarriersfor drug delivery across the blood–brain barrier,67 including functionalized nanoparticles that can be targeted fortissue- or cell-specific drug delivery.68 One caveat for this approach is that such nanoparticles are subjectto clearance by the reticuloendothelial system and thus should beformulated to reduce their clearance by this mechanism.Nanocarriers can be engineered to display factors that permit theirtargeted uptake by specific tissues or cell types that express thecorresponding receptors, ligands, or other interacting factor.70 This might also be useful early in infection or shortly after anexposure event if these approaches could reduce the burden of theactive virus to attenuate virus replication and reduce the potentialfor virus transmission. For instance, SARS-CoV-2 employs angiotensin-convertingenzyme 2 (ACE2), which is highly expressed in nasal and oral tissue,as its primary receptor for cell entry,72 and researchhas focused on the ability of nanodecoys that display ACE2 or cellmembranes that highly express ACE2 to attenuate in vivo infectionsresulting from exposure to SARS-CoV-2 or an engineered SARS-CoV-2pseudovirus.70 Severe respiratory virusinfections, including severe COVID-19 cases, can produce dysregulatedcytokine expression resulting in a cytokine storm and inducing acutelung injury that can progress to acute respiratory distress syndrome(ARDS) and multiple organ failure. Early intervention to reduce inflammationcould reduce this progression to reduce ARDS morbidity and mortalityresulting from severe respiratory virus infections, but care mustbe taken to avoid the deleterious effects of systemic immunosuppression.Targeted delivery of nanodecoys to the upper and lower respiratorytract using aerosol delivery approaches could, however, address thisissue by restricting the immunosuppressive effect to the primary infectionsites. Notably, one recent study employing a nanodecoy that carriedhigh levels of ACE2 and multiple cytokine receptors found that thisparticle could both attenuate SARS-CoV-2 infection and independentlyreduce lung cytokine levels and injury stimulated by a nonviral stimulus.69 Studies have shown that extracellular vesiclesderived from lung tissues and cells, and which carry viral receptors,are promising candidates for nanodecoys that can bind viruses to restricttheir uptake by host cells.73 However,further work needs to be done to evaluate optimal approaches, doses,intervention times, and efficacy of such interventions. For instance,oral vaccine doses required to produce a robust immune response canbe 100-fold higher than required in standard subcutaneous injectionapproaches,74 although this requirementmight be attenuated by modifying the targeting, adjuvant, and otherproperties of these nanocarriers. Nasal delivery of nanocarrier-basedvaccines could also reduce vaccine degradation and enhance its interactionswith immune cells to decrease the required dosage while also promotingthe development of a protective mucosal immune response.Nanoparticlesmay also have effective therapeutic activity as nanodecoysthat express receptors, ligands, or other materials that are specificallyrecognized by the target virus or factors that can have pathologiceffects when expressed in excess during the response to infection.Respiratory virus exposure primarily occurs through direct contactwith target receptors expressed by cells present on the epithelialsurface of the nasal, oral, and tracheal mucosae. It may thereforebe possible to employ biocompatible nanomaterials as virucidal agentsor decoys in nasal or oral sprays to disrupt virion structure or blockvirus interaction with its target receptor to limit infection.The recent history of multiple outbreaks by emerging coronaviruses(MERS-CoV and SAR-CoV) that culminated in the SARS-CoV-2-induced COVID-19pandemic, and the constant threat of virus crossover events, has emphasizedthe need for multilevel approaches to combat future respiratory diseaseoutbreaks capable of producing epidemics and pandemics. Current andfuture nanomaterial applications appear to hold great potential toprevent, detect, and treat existing and emerging respiratory infectionsto reduce their social and economic impact and avert future pandemics.One notable example of this potential was the rapid deployment oftwo major SARS-CoV-2 mRNA vaccines that used lipid nanoparticles astheir delivery vehicles on the basis of previous studies. Researchconducted during the COVID-19 pandemic has highlighted the potentialof these approaches and the need for multidisciplinary research requiredto develop and validate future applications in key areas, includingpersonal protection, environmental sanitation, and virus diagnosisand treatment. This perspective suggests areas where nanomaterialcould contribute to improved applications, but it is to be expectedthat future research could greatly expand this list.


Background and Objectives: Marfan syndrome (MS) is a genetic disorder with autosomal dominant inheritance that affects the connective tissue and consequently many organ systems. The cardiovascular manifestations of MS are notorious and include aortic root dilatation or acute aortic dissection, which can cause morbidity and early mortality. However, surgical treatment of aortic pathology may be complicated by musculoskeletal deformity of the chest wall, as in pectus excavatum. In this regard, single-stage combined Bentall and Ravitch surgery is an extreme rarity that has also been scarcely reported in the literature. Patients and Methods: We present the medical history and single-stage Bentall and modified Ravitch surgical treatment of an 18-year-old male MS patient with symptomatic and severe pectus excavatum (PEX) in conjunction with a pear-shaped aortic root aneurysm. To discuss our case in the context of a synopsis of similar published cases, we present a systematic review of combined Bentall surgical aortic aneurysm repair and Ravitch correction of PEX. Results: A total of four studies (one case series and three case reports) and a case from our institution describing a single-stage combined Bentall and Ravitch operation were included. Patients were 22 ± 5.9 years of age (median = 22.5 years) and predominantly male (60%). All cases reported a midline vertical skin incision over the sternum. The most common surgical approach was midsternotomy (80%). In all cases metal struts were used to reinforce the corrected chest wall. Postoperative mortality was zero. Conclusions: Single-stage combined Bentall and Ravitch surgery is an underutilized surgical approach. Its use in MS patients with concomitant PEX and ascending aortic aneurysm that require surgical treatment warrants further investigation. Midsternotomy seems to be a viable access route that provides sufficient exposure in the single-stage surgical setting. Although operative time is long, the intraoperative and postoperative risks appear to be low and manageable. FBN1, which encodes the extracellular matrix glycoprotein fibrillin-1, has been identified as the cause of MS [Marfan syndrome (MS) is a hereditary connective tissue disorder that encompasses multiple organ systems. Pathogenetic alteration in se of MS ,3. Hencese of MS . Pectus se of MS  that is se of MS . Inward se of MS ,8,9. As se of MS . Moreovese of MS ,12. CardFBN1 mutation, and congenital PEX presented with acute atypical retrosternal chest pain that radiated to the back and differed in character from his chronic chest pain. Electrocardiography (ECG) and laboratory testing ruled out acute myocardial infarction. One month earlier, the patient had been referred to our institution because an aortic root dilatation had increased by 6 to 8 mm over 6 months to a diameter of 53 mm on his most recent transthoracic echocardiography (TTE) and magnetic resonance angiography. The same studies showed a 47 mm fusiform aneurysm of the ascending aorta. At this point, a surgical therapy option was discussed with the patient due to the rapid size-progression of the aneurysm, and an elective inpatient admission was scheduled. ECG-gated computed tomography angiography (CTA) of the aorta excluded acute aortic dissection, confirming the previous findings , language other than English (n = 5), PEX-correction technique other than modified Ravitch procedure (n = 5), valve-sparing ascending aortic replacement (n = 3), no correction of PEX (n = 3), and staged surgery (n = 1). The remaining six articles were adjusted for duplicates, resulting in a total of four eligible papers, three of which were case reports and one of which enclosed a patient of a case series (n = 4); one article reported left-sided costotomy. Only a single article reported the cannulation technique for connection to CPB  [The systematic search yielded a total of 29 peer-reviewed publications; 23 did not meet the selection criteria and were excluded. Reasons for exclusion were as follows: cardiac procedure performed in addition to Bentall procedure  ; aorto-rulation) . In the ulation) ,22. Likeulation) . The totulation) ; mean anCardiovascular lesions and thoracic skeletal deformities are frequent complications in patients with MS . The neeThe main question is whether and how patients with concomitant pectus deformity and cardiovascular disease should be treated surgically in one step. Currently, there is no consensus. A few publications advocate staged repair because of concerns about serious complications such as limited surgical exposure, severe bleeding, and prolonged operative time ,23,24,25Once the decision has been made to proceed with combined surgery, the key question of thoracic access technique arises. In addition to median sternotomy with transverse sternal split and median sternotomy with staged bilateral costochondral osteotomy both before and after the surgical procedure, parasternal approaches and sternal turnover techniques have been described. Thus, controversy persists regarding the best surgical approach. However, median sternotomy was the most commonly used surgical approach in previous publications. Adequate surgical-field exposure was reported in all cases.Another point of contention is whether a vertical or horizontal skin incision should be performed for a satisfactory cosmetic result, as some authors consider a vertical incision in the keloid triangle to be cosmetically questionable. Nevertheless, our review showed that a longitudinal midline skin incision was used in all patients who underwent single-stage Bentall and modified Ravitch surgery. Postoperative keloid formation was not reported in any case.Since its initial description in 1949, the Ravitch sternochondroplasty has undergone numerous changes and modifications over the years to allow simultaneous surgical treatment of cardiac lesions. In this respect, controversy remains over whether the use of a metal strut is essential to reinforce the pectus in its surgically corrected shape. Some authors have expressed concern that a metal strut may be associated with complications such as migration or dislocation, which may cause intrathoracic organ injury . Our revSingle-stage combined Bentall and modified Ravitch surgery is an underutilized surgical approach. Its use in MS patients with concomitant PEX and ascending aortic aneurysm that require surgical treatment warrants further investigation. Midsternotomy seems to be a viable access route that provides sufficient exposure in the single-stage surgical setting. Although operative time is long, the intraoperative and postoperative risks appear to be low and manageable.


Helmet noninvasive support may provide advantages over other noninvasive oxygenation strategies in the management of acute hypoxemic respiratory failure. In this narrative review based on a systematic search of the literature, we summarize the rationale, mechanism of action and technicalities for helmet support in hypoxemic patients.2/FiO2 ≤ 200 mmHg), possibly because higher positive end-expiratory pressure (10–15 cmH2O) can be applied for prolonged periods with good tolerability. This improves oxygenation, limits ventilator inhomogeneities, and may attenuate the potential harm of lung and diaphragm injury caused by vigorous inspiratory effort. The potential superiority of helmet support for reducing the risk of intubation has been hypothesized in small, pilot randomized trials and in a network metanalysis.In hypoxemic patients, helmet can facilitate noninvasive application of continuous positive-airway pressure or pressure-support ventilation via a hood interface that seals at the neck and is secured by straps under the arms. Helmet use requires specific settings. Continuous positive-airway pressure is delivered through a high-flow generator or a Venturi system connected to the inspiratory port of the interface, and a positive end-expiratory pressure valve place at the expiratory port of the helmet;  alternatively, pressure-support ventilation is delivered by connecting the helmet to a mechanical ventilator through a bi-tube circuit. The helmet interface allows continuous treatments with high positive end-expiratory pressure with good patient comfort. Preliminary data suggest that helmet noninvasive ventilation (NIV) may provide physiological benefits compared to other noninvasive oxygenation strategies  in non-hypercapnic patients with moderate-to-severe hypoxemia (PaOHelmet noninvasive support represents a promising tool for the initial management of patients with severe hypoxemic respiratory failure. Currently, the lack of confidence with this and technique and the absence of conclusive data regarding its efficacy render helmet use limited to specific settings, with expert and trained personnel. As per other noninvasive oxygenation strategies, careful clinical and physiological monitoring during the treatment is essential to early identify treatment failure and avoid delays in intubation. The role of non-invasive respiratory support in the management of acute hypoxemic respiratory failure (AHRF) is unclear, but evolving. Avoidance of intubation through noninvasive support improves patient outcomes by preventing the complications of invasive mechanical ventilation –3. HowevThe optimal balance between benefits and harms of preserving spontaneous breathing in patients with AHRF and/or acute respiratory distress syndrome (ARDS) is not fully understood . For theNIV can be delivered through different interfaces, namely, oro-nasal masks, full-face masks, and helmets . Most stIn this narrative review, we discuss the physiological rationale for the use of helmet support as first-line treatment of AHRF/ARDS, and we describe the technicalities for its safe application in hypoxemic patients.This narrative review was based on a systematic search of the medical literature, which was performed according to a protocol published in PROSPERO (CRD42020201563). We performed a computerized search of MEDLINE, PubMed, Embase and the Cochrane Central Register of Controlled Trials (CENTRAL) database for relevant English-language studies from inception to June 2021. Most relevant studies published up to August 2022 were subsequently included. Study inclusion for our review included any observational study, interventional trial or reviews on adults with AHRF treated with helmet NIV or describing the physiological effects of spontaneous breathing during hypoxemic respiratory failure. We included studies describing (1) how to set up helmet support, (2) its physiological effects, (3) ventilator settings capable of limiting lung injury during spontaneous breathing and (4) clinical outcomes of patients receiving helmet support, with or without a comparison to other noninvasive oxygenation strategies. Two independent reviewers performed an initial screening of all retrieved papers by title and abstract. Then, full-text screening was performed. At any stage, when discussion was unable to reach a definitive conclusion, disagreements were solved by a third reviewer.Among 510 citations, a total of 100 studies, including 8 randomized trials and three meta-analyses, were included.In patients with AHRF in intensive care unit (ICU), maintenance of spontaneous breathing avoids sedation and passive ventilation, thereby limiting diaphragm dysfunction and delirium, facilitating mobilization, and reducing the risk of ventilator-associated complications  –23. MorePreserving spontaneous breathing with noninvasive support may yield, however, risks related to delays in endotracheal intubation, with detrimental effects on mortality , 28, 29.2), and higher CO2 production by respiratory muscles [2 clearance [In critically ill patients with AHRF, respiratory drive and inspiratory effort are increased by lung injury, high alveolar dead space, reduced pulmonary compliance, increased neural ventilatory response to carbon dioxide  and the application of high PEEP levels may mitigate the risk of lung injury due to dysregulated inspiratory effort.2O) favours a more homogeneous distribution of inspiratory pressure across the lung tissue, thus reducing pendelluft  [2O are usually applied [2O) for prolonged treatments with good tolerability and without significant leaks.In summary, application of moderate-to-high PEEP may be essential to minimize the risk of self-inflicted lung injury in spontaneously breathing AHRF and ARDS, especially in case of moderate-to-severe hypoxemia (PaO00 mmHg) . During  applied , while h applied . By contThe Helmet is a transparent hood that covers the entire head of the patient with soft neck collar that allows the system to seal at the patient’s neck. The interface is further secured by straps under the arms. At least 2 ports are present, which are connected to separate tubes for inhaled and exhaled gas, respectively. All commonly used helmets are latex-free and available in multiple sizes.2O), while NIV mostly benefits patients with high inspiratory effort (> 10 cmH2O) [Helmet interface may be used to deliver either continuous positive airway pressure  or NIV in pressure support mode (PSV). For the same PEEP level, the major difference between CPAP and NIV in the capability of the latter to best reduce inspiratory effort. From a theoretical standpoint, in hypoxemic patients, CPAP could be preferred in case the inspiratory effort before treatment start is low (< 10 cmH0 cmH2O) .Given the unique characteristics of the interface, specific settings are required to optimize the treatment: these are described in Table 2 rebreathing [2O) represent the safest set-up to deliver helmet CPAP. In this setting, the application of a heated humidifier is needed, since fresh gas flows > 40 L/min would otherwise lead to under-humidification inside the helmet [Theoretically, helmet CPAP can be delivered through a mechanical ventilator or by an adjustable continuous flow-generator in combination with a PEEP valve . Ventilareathing –57. For e helmet –60.The main helmet-specific PSV settings are , 61–64:2 rebreathing.Double-limb ventilators should be used to provide helmet NIV. Both modern high-performance turbine ventilators and gas-compressed ventilators can be used, with the latter being preferable. A double-tube circuit should be preferred over a Y-piece circuit, in terms of ventilator pressurization performance, patient-ventilator interaction and avoidance of COIncreasing PEEP reduces interface compliance, thus minimizing the amount of pressure support wasted to pressurize the interface and reducing airway pressurization time. Importantly, increasing PEEP contributes to reduce air leaks by abutting the helmet against the patient's shoulders.2 rebreathing.Increasing pressure support further reduces helmet compliance reducing the amount of pressure wasted to pressurize the interface and ensuring adequate inspiratory support to unload the respiratory muscles. Moreover, a higher-pressure support generates a higher washout flow, which is crucial to avoid COThis aims at minimizing the under-assistance of respiratory muscles during the peak inspiratory effort. Vargas and colleagues demonstrated that increasing PEEP and pressure support by 50% and use of the fastest pressurization rate significantly improved the unloading of respiratory muscles.2O/L is recommended during facemask NIV [Gas conditioning by either heated humidifiers or heat and moisture exchangers to reach a minimum absolute humidity of 15 mgHmask NIV –68. Howemask NIV .2 rebreathing [2 concentration inside the helmet depends on the balance between the patient’s CO2 elimination and the system’s washout flow. Consequently, high fresh gas flows are needed to avoid rebreathing (flow rates of at least 30–50 L/min have been shown to be necessary for this purpose) during CPAP [2O is usually efficient to avoid the risk of clinically relevant CO2 rebreathing during NIV [2 washout by ventilator expiratory flow-by, that can reach 15 L/min in modern gas-compressed mechanical ventilators provided with a NIV-dedicated module.The internal volume of the helmet is much larger than any other NIV interface (around 18 L) and it behaves as a semi-closed mixing chamber. As such, some of the patient’s exhaled gas is not eliminated from the helmet and instead mixes with fresh gas coming from the inspiratory limb of the circuit, possibly resulting in COreathing , 70–72. ing CPAP , and prering NIV . During During NIV, inspiratory pressurization is slower than with mask interfaces due to significant trigger delays (0.1–0.5 s) and because part of the pressure is dissipated to distend the interface. Similarly, pressure decay after cycling off is slower and delayed, often leading to patient’s expiration against a positive pressure which is higher than the set PEEP  . Inspira2, comfort, or transpulmonary pressures. Patients with lower inspiratory effort during high-flow nasal oxygen can develop increased transpulmonary pressures on helmet NIV, while patients with higher effort during high-flow nasal oxygen show the most relevant reduction in transpulmonary pressure swings on helmet NIV.As compared to high-flow nasal oxygen, helmet NIV improves oxygenation and reduces inspiratory effort and dyspnoea without changes in PaCOTaken together, these data indicate that helmet NIV might have advantages over high-flow nasal oxygen in most severely hypoxemic patients, especially among those exhibiting intense inspiratory effort, perhaps because of the higher achievable PEEP levels with their attendant benefits in terms of alveolar recruitment and reducing inspiratory effort. Conversely, helmet NIV may increase transpulmonary pressures in patients with low inspiratory effort, since the increase in delivered pressure support is not offset by a clinically relevant decrease in negative swings of pleural pressure. In this latter subgroup, the use of a lower level of pressure support or CPAP may help mitigate the increase in transpulmonary pressure.As per any other noninvasive oxygenation strategy, helmet support should be used under strict clinical and physiological monitoring. Careful monitoring is needed to promptly identify treatment failure, and not to delay endotracheal intubation and the institution of protective ventilation –77.Clinically, worsening hypoxemia, increased respiratory rate, lack of dyspnea relief are all factors that should prompt the decision to intubate patients , 78–80.0.1) may not be reliable [2O during NIV, both delivered with facemask and helmet, is strongly associated with the subsequent need for endotracheal intubation, suggesting that inspiratory effort monitoring may play a crucial role in assessing the risk of self-inflicted lung injury during helmet NIV [Given the mechanical characteristics of the helmet interface, such as the inability to transiently occlude the airway, some of the non-invasive validated methods to assess inspiratory effort and drive  and the HACOR scale  have been used to early predict failure during high-flow nasal oxygen and facemask NIV, respectively [Patients with high severity of illness (Simplified Acute Physiology Score II > 34), older age, or those who fail to improve PaO failure . Validatectively , 85. TheWith standard equipment, tidal and minute ventilation cannot be reliably monitored during helmet support, since a substantial portion of the tidal volume inflates the helmet and does not reach the patient. During PSV, minute ventilation displayed by the ventilator represents the system’s washout flow.Aside from the physiological benefits, the helmet interface offers several practical advantages over other interfaces. First, it allows the patient to see, read, interact with the environment, it facilitates coughing, improves overall comfort, and can facilitate early mobilization and physiotherapy. It also allows the patient to drink though a specific straw independently. High tolerability allows continuous treatment, reducing or eliminating the need for intermittent sessions, that are unavoidable during facemask NIV . It can A summary of the clinical studies comparing helmet support with other techniques is provided in Table In a small trial, helmet CPAP reduced intubation rate (15% vs. 63%) and mortality  in patients with community-acquired pneumonia, compared to conventional oxygen therapy .2/FiO2ratio < 200 mmHg), whereas it remained significant for helmet NIV across all degrees of hypoxemia.In a recent meta-analysis by Ferreyro et al. hypothesized the superiority of helmet support over standard oxygen therapy in AHRF: helmet support showed the most significant improvements in mortality  and intubation rate  [4–0.63, a2O), anatomical dead space clearance, and an inspiratory flow capable of matching the peak inspiratory flow of a hypoxemic patient, an important advantage over conventional low-flow oxygen therapy devices [In recent years, there has been significant interest in high flow nasal oxygen as an alternative method to noninvasively manage AHRF. High-flow nasal oxygen provides small, variable amounts of PEEP  with high-flow nasal oxygen [In a physiologic, helmet NIV was shown to improve oxygenation and lower inspiratory effort, compared to high-flow nasal oxygen. The most beneficial effects by helmet NIV was observed among most severely hypoxemic patients and those exhibiting intense inspiratory effort , with no significant effect on mortality. Treatment with helmet NIV was associated with an increased number of days free of invasive ventilation at 28 days from randomization. Patients in the helmet NIV group experienced less dyspnea, improved gas exchange values, with increased discomfort as compared with high-flow nasal oxygen. The most significant clinical benefit of helmet NIV over high-flow nasal oxygen was observed in patients exhibiting hypocapnia before treatment start, which may identify the sub-population with the most dysregulated inspiratory effort [Recently, a multicenter, randomized trial compared early continuous treatment with helmet NIV followed by high-flow nasal oxygen vs. high-flow nasal oxygen on days free of respiratory support in patients with COVID-19 and moderate to severe hypoxemic respiratory failure . This fiy effort .In a matched-control pilot trial in early 2000s, helmet was as effective as the conventional facemask NIV in improving oxygenation and avoiding intubation with better patient comfort and fewer complications  .More recently, a retrospective observational study to assess the differences between patients who succeeded or failed noninvasive respiratory support showed that the use of helmet CPAP was an independent predictor of noninvasive respiratory support success and lower intubation rate when compared with facemask NIV .The most rigorous head-to-head comparison of helmet and facemask NIV comes from a randomized trial by Patel and colleagues : patientA recent non-randomized study confirmed the possible superiority of helmet over facemasks for delivering CPAP in the specific population of COVID-19 patients: use of helmet allowed prolonged treatments with higher PEEP, and was associated with lower rate of intubation and improved survival .Three meta-analyses including studies comparing helmet with facemask NIV in patients with acute respiratory failure confirmed a possible clinical benefit by helmet support , 16, 96.In the network meta-analysis by Ferreyro and colleagues , helmet Theoretically, avoidance of intubation is particularly important in immunocompromised patients, for whom respiratory complications are a predominant cause of morbidity and mortality. Squadrone et al. showed that early helmet CPAP in immunosuppressed patients, when compared to standard oxygen, may prevent evolution to ARDS requiring ventilatory support and ICU admission . These rNoninvasive respiratory support is playing an increasingly important role in the management of patients with severe AHRF. Helmet support may enhance tolerability with greater physiological effectiveness than other noninvasive oxygenation strategies in patients with moderate-to-severe hypoxemia. This is attributable to its ability to deliver higher levels of PEEP for prolonged periods of time with good comfort, which may improve outcomes by improving oxygenation, relieving dyspnea and preventing self-inflicted lung injury and diaphragm injury.Clinically, helmet support appears to be an effective tool to manage AHRF, especially in patients with the most severe oxygenation impairment. In these patients, helmet NIV could even reduce need for endotracheal intubation, but further research is warranted to confirm findings from preliminary randomized studies and to discriminate the effect of helmet CPAP and NIV. Currently, the lack of confidence with this and technique and the absence of conclusive data regarding its efficacy render helmet use limited to specific settings, with expert and trained personnel. As per any other noninvasive oxygenation strategy, careful monitoring of the patient remains paramount to avoid delays in intubation and institution of protective ventilation.