Sulfuric acid is a mineral acid extensively utilized in various physical, chemical, and industrial applications. Undoubtedly, its concentration, particularly at lower levels, plays a pivotal role in these applications. Hence, there is an urgent demand for a highly accurate and sensitive tool to monitor even the slightest changes in its concentration, which is crucial for researchers. Herein, we presented a novel study on the optimization of the phononic crystal (PnC) sensor. The optimization process involves a comparative strategy between binary and ternary PnCs, utilizing a multilayer stack comprising 1D porous silicon (PSi) layers. Additionally, a second comparison is conducted between conventional Bragg and local resonant PnCs to demonstrate the design with the highest sensitivity. Moreover, we determine the optimum values for the materials’ thickness and number of periods. The results revealed that the ternary local resonant PnC design with the configuration of {silicone rubber/[PSi1/PSi2/PSi3]N/silicone rubber} is the optimal sensor design. The sensor provided a super sensitivity of 2.30 × 107 Hz for a concentration change of just 2%. This exceptional sensitivity is attributed to the presence of local resonant modes within the band gap of PnCs. The temperature effects on the local resonant modes and sensor performance have also been considered. Furthermore, additional sensor performance parameters such as quality factor, figure of merit, detection limit, and damping rate have been calculated to demonstrate the effectiveness of the proposed liquid sensor. The transfer matrix method was utilized to compute the transmission spectra of the PnC, and Hashin’s expression was employed to manipulate the porous silicon media filled with sulfuric acid at various concentrations. Lastly, the proposed sensor can serve as an efficient tool for detecting acidic rain, contaminating freshwater, and assessing food and liquid quality, as well as monitoring other pharmaceutical products.This research presented a comprehensive study of a one-dimensional (1D) porous silicon phononic crystal design as a novel fluidic sensor. The proposed sensor is designed to detect sulfuric acid (H The 1D ternary PnC structure is placed between two identical layers of silicon rubber, which are responsible for the emergence of the LR mode. Each unit cell of the designed 1D ternary PnC sensor comprises three layers of porous silicon with varying characteristics of porosity  can be simply introduced in the following manner 2. 1/PSi2]2/Silicone rubber. It is important to note that the pores of the PSi layers were filled with pure water (0% concentration). The transmission spectrum of [PSi1/PSi2]2, shown in 1 and PSi2 layers are close to each other. This means there is no significant acoustic mismatch between the two layers, resulting in no apparent band gaps appearing in the spectrum [1/PSi2]2 structure’s spectrum in the considered frequency ranges.Assume binary 1D-PSi-PnCs containing two-unit cells; each unit cell comprises two different PSi layers. The first material is PSispectrum . Additio1/PSi2]2 structure when two rubber layers are attached to its top and bottom sides. The resulting Silicone rubber/[PSi1/PSi2]2/Silicone rubber structure exhibits two complete PnBGs across the entire range of normalized frequencies. Within the band gap, the transmissivity of the incident acoustic waves is zero, resulting in a stopband where any acoustic waves cannot propagate through the structure and are effectively attenuated N/silicone rubber}. The studied sensor is presented for detecting a very important chemical acid represented by sulfuric acid (H2SO4) in a rare-used adopted range of concentrations (0–15%). In general, silicon can withstand the presence of H2So4 rather than other mechanical materials used in PnCs, such as epoxy and nylon, as sulfuric acid can affect these materials over the time, even with low concentrations. Additionally, silicon has a very high elastic constant to withstand sulfuric acid easily. On the other hand, silicone rubber is a highly chemically stable and low-cost material (with high melting temperature), and meanwhile is one of the preferred polymers that can generate local resonant modes easily. These modes are the main compass through the detection process. The modes shifted to new frequency positions by changing the sulfuric acid concentrations. Furthermore, we studied the optimum PnC sensor, including binary Bragg band gaps PnCs, ternary Bragg band gaps PnCs, binary local resonant PnCs, and ternary local resonant PnCs. In this regard, the sensor has provided a super sensitivity compared with many PnCs sensors with the value of 2.3 × In this paper, we presented a tunable porous silicon one-dimensional PnC structure as a liquid sensor. The sensor configuration is arranged as {silicone rubber /[PSiFurther, the other performance parameters are superb, such as a quality factor of 2.6 ×


Models of arterial injury in rodents have been invaluable to our current understanding of vessel restenosis and play a continuing role in the development of endovascular interventions for cardiovascular disease. Mechanical distention of the vessel wall and denudation of the vessel endothelium are the two major modes of vessel injury observed in most clinical pathologies and are critical to the reproducible modelling of progressive neointimal hyperplasia. The current models which have dominated this research area are the mouse wire carotid or femoral injury and the rat carotid balloon injury. While these elicit simultaneous distension of the vessel wall and denudation of the luminal endothelium, each model carries limitations that need to be addressed using a complementary injury model. Wire injuries in mice are highly technical and procedurally challenging due to small vessel diameters, while rat balloon injuries require permanent blood vessel ligation and disruption of native blood flow. Complementary models of vascular injury with reproducibility, convenience, and increased physiological relevance to the pathophysiology of endovascular injury would allow for improved studies of neointimal hyperplasia in both basic and translational research. In this study, we developed a new surgical model that elicits vessel distention and endothelial denudation injury using sequential steps using microforceps and a standard needle catheter inserted via arteriotomy into a rat common carotid artery, without requiring permanent ligation of branching arteries. After 2 weeks post-injury this model elicits highly reproducible neointimal hyperplasia and rates of re-endothelialisation similar to current wire and balloon injury models. Furthermore, evaluation of the smooth muscle cell phenotype profile, inflammatory response and extracellular matrix within the developing neointima, showed that our model replicated the vessel remodelling outcomes critical to restenosis and those becoming increasingly focused upon in the development of new anti-restenosis therapies. Minimally invasive endovascular device intervention has revolutionised the treatment of cardiovascular diseases, due to high acute success rates and low periprocedural complications ,2. DespiHigh pressure balloon catheter inflation characteristic of either stent or drug-eluting balloon delivery causes a significant vessel injury which leads to mechanical distention of the vessel wall and disruption of the native, protective endothelium . SubsequThe two predominantly used models of mechanically-induced NH are the wire denudation and balloon injury models. Wire denudation utilizes an angiocatheter guide wire that is repeatedly inserted into a mouse carotid or femoral artery to simultaneously denude the endothelium and distend the vessel wall. Despite rapid NH development within 2 to 4 weeks, the key limitations are its technical difficulty and corresponding reliance on operator skill, due to the small size of mouse arteries and demonstrated by the high variability in restenosis levels reported across the literature ,16. AlteDevelopment of a complementary injury model replicating the accessibility of the larger sized rat carotid vessel but using commonly available surgical equipment that allows for arterial injury without the permanent ligation of distal blood vessels would provide an invaluable expansion of current rodent models available for modelling NH. In this study, we report a novel carotid injury model using a surgical procedure the combines mechanical vessel distention and endothelial denudation into two distinct, sequential steps. This is achieved by combining the expansion of microforceps to a maximum width followed by the insertion of a needle catheter through an arteriotomy of the rat common carotid artery. Use of these tools allowed for greater control over the length of vessel injury requiring only 1.5 cm of the common carotid artery without additional permanent vessel ligation. Using male Sprague Dawley rats aged 7–10 weeks, we demonstrate that the microforceps and catheter injury is a simple microsurgical procedure that takes an operator roughly 20 minutes to complete from starting incision to final suture. Explanted rat carotids exhibit the classical vessel injury responses that include elevated M1 macrophage activation and pro-inflammatory cytokine secretion, SMC contractile-to-synthetic differentiation, and neointimal hyperplasia. Over multiple batches of surgeries, this model shows high reproducibility of these responses at a rate over 2 weeks that would allow deeper examination of pathological mechanisms and precise evaluation of restenosis therapies.Animal experiments were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purpose and issued under the Sydney Local Health District Animal Welfare Committee Ethics Protocol 2017/014. Male Sprague Dawley rats aged 7–10 weeks averaging 300 g were obtained from Animal Resources Centre .All surgical tools required in this procedure: fine pointed forceps, pointed forceps, spring scissors, and surgical scissors were sterilized using autoclave prior to surgery and maintained during surgery using a bead sterilizer . Rats weAnesthetised rats were placed in the supine position and using an electric shaver, hair was removed from the neck .Rat were placed in the supine position over a heat mat (at 37°C) on the operating table, betadine applied followed by surgical drapes, leaving only the neck exposed.Lignocaine anaesthetic  was given subcutaneously prior to using a No.22 scalpel blade to make a superior-to-inferior 5cm incision on the side of the neck, starting roughly 3 cm below the jaw and 3 cm to the side of the neck midline. (Syringe will be drawn back before injection to ensure a blood vessel has not been entered)Expose the underlying muscles (masseter and digastric), being careful with the trachea and gently push aside to expose the common carotid artery.Secure two arterial clamps  spaced around 1.5 cm apart at the proximal and distal ends to the exposed artery. Note: the top clamp is placed just below the bifurcation to the internal and external arteries, and the bottom clamp placed further proximal on the common carotid artery. This will occlude blood flow in the arterial segment which should last for only 15–20 minutes. Diagram included in Using spring scissors make a small entry incision at the proximal side of the carotid artery and flush with saline to remove the blood with sterile gauze from the artery.Insert microforceps (World Precision Instruments 500373-T) into the small entry incision until they reach the distal clamped end of the artery.Expand the microforceps to the maximum set width (1.55mm) and retract while expanded.Repeat steps 8–9 four additional times.Insert 22g catheter into the small entry incision until it reaches the distal clamped end of the artery, and pull the catheter out.Repeat steps 10–11 four additional times.Close the arteriotomy using a 9–0 silk suture using a single interrupted stitch and release distal and proximal clamps to restore blood flow.Repeated steps 4–12 on the other side of the carotid artery for bilateral injury .Close the neck surgical incision by 3–0 silk suture using a continuous stitch.To prevent infection, rats will be given an intramuscular injection of a prophylactic antibiotic; cephazolin (20 mg/kg) post-operatively prior to recovery from isoflurane.To further support the animals during recovery, Ringers fluid (warmed to 37°C) will be subcutaneously injected at 10 mL/kg.The animal will be allowed to recover on a heat mat (37°C) and monitored continuously until it has regained its righting reflexes.Rats were anaesthetised using the described pre-operative procedure (section 2.2.1) prior to being placed in the supine position over a heat mat (at 37°C) on the operating table.Using surgical scissors the neck incision was reopened and underlying muscles masseter and digastric moved aside and carotid artery exposed.Using the arteriotomy suture as a landmark, place a vessel clamp just below/proximal to the suture and another spaced 1.5 cm apart distally (the space between the clamps will lie over the common carotid artery where the injury was performed). Cut the vessel between the two clamps to remove the segment and flush with heparin-saline immediately to prevent the blood clot.Euthanise animal with a single I.V. bolus of pentobarbital (325mg/ml)Explanted arteries were fixed in paraformaldehyde (PFA) (4%) overnight and dehydrated through an ethanol gradient. Samples were then embedded in molten paraffin before being cooled at room temperature to allow the paraffin to solidify. The entire length of the artery was cut into 5 μm thick cross-sectional slices using an electronic rotary microtome  and mounted onto microscope slides with three sections per slide. Equidistant slides were selected from across the entire length of the carotid to represent proximal, middle, and distal segments . ParaffiAnalysis of histology and immunohistochemistry slides was done using ImageJ. For Haematoxylin and Eosin staining, neointima was quantified as percentage of total lumen area defined by the internal elastic lamina of the vessel wall. For PSR and Alcian Blue staining, proteoglycan and collagen deposition was quantified using the ‘Colour Threshold’ function in ImageJ to calculate amount of positive red or blue staining, respectively, present within the neointima and vessel wall. Fibrotic capsule was represented as total adventitial tissue area surrounding the graft. For SMCα, PCNA, CD68, MHC Class II, IL-1β, and TNF-α counts within the neointima was quantified using a constant threshold intensity and positive staining area measured. CD31 lumen coverage was quantified by first measuring the lumen circumference, followed by measuring the length of endothelium showing positive staining. Lumen coverage was then calculated as length of CD31+ staining divided by lumen circumference.Data are expressed as mean ± standard error of the mean (SEM) as calculated using GraphPad Prism 9  and statistically significant differences were determined by t-test with no corrections. Statistical analysis was performed on the averaged data comprising of all three vessel regions . The bar graphs in this paper represent the average amongst the vessels in each group with each vessel consisting of an average of its three regions . p < 0.0Under anaesthesia, the right common carotid artery was isolated and arteriotomy performed. Microforceps were inserted and expanded followed by catheter insertion, together repeated four times to mechanically distend the carotid vessel wall and subsequently denude the vessel endothelium . Across To determine the extent of endothelial denudation, vessels were explanted immediately after injury and 2 weeks post-injury. To visualise the injury to the endothelium en face- and cross-sections, respectively, were stained with the endothelial marker CD31. Enface staining showed an 87 ± 0.52% reduction of endothelial coverage across the length of the explanted vessel  indicatiThe most reported metric of all vessel injury models is neointima formation arising from the migration and over-proliferation of SMCs from the vessel wall, driven by a contractile-to-synthetic phenotype switch. To validate whether our model activated a similar response, vessels were explanted two weeks post-injury and stained with H&E to evaluate vessel patency and separately co-stained with PCNA and SMC-α to determine the ratio of contractile and synthetic phenotypes . Neointi+) and those polarised to the pro-inflammatory (M1) phenotype, using the MHCII+ and iNOS+ marker. In injured vessels 2 week-post injury, CD68+ was observed both within the vessel wall and the neointima highlighting the spatial recruitment of macrophages to the vessel injury  and Alcian Blue (AB) stains were used to visualise collagen and proteoglycan deposition, respectively. Collagen was found to be significantly upregulated within both the vessel wall and neointima, showing an overall total vessel increases of 157% . Using common surgical instruments readily available to any small animal surgery lab rather than custom-designed balloon catheters that require longer vessel lengths to reproducibly induce injury, our method enables a similar injury over half the required vessel length of rat balloon injury. Similar to the balloon injury, vessel distention and endothelial denudation can occur objectively between operators as the microforceps have maximum opening widths of 1.5mm and needle catheters are standardised to 22-gauge diameters. This combines into a single procedure the two modes of injury that have made mouse wire and rat balloon injury models a highly consistent means of evaluating new restenosis therapies.Our results firstly show that the model produces near complete endothelial denudation immediately after injury with substantial disruption of the endothelium sustained for at least 2 weeks with only approximately 30% recovery of the endothelium. This is more comparable to other balloon injury models in rats with reports of 15% recovery . Relativ2 throughout the literature, we back calculated our occlusion percentages to an average 0.228 ± 0.02 mm2 neointimal area after 2 weeks of injury. Compared to balloon injury models, which have reported neointimal areas of 0.255 ± 0.04 mm2 [2 [2 [The major reported metric of balloon injury models is neointimal growth and luminal patency, in our model we observed an average of 65% luminal occlusion. To make comparisons to neointimal areas which are often reported in mm0.04 mm2 , 0.218 ±m2 [2 [2 , our injm2 [2 [2 . This suDeveloping new therapeutic approaches to restenosis relies heavily on a solid understanding of neointimal development and its biological underpinnings. Hyperplasia arising within our model was consistent with high percentages of synthetic SMCs phenotype switching reflective of an unstable and growing neointima. Here we have used the proliferative marker PCNA to indicate synthetic SMC phenotypes, which allows us to determine the state of neointimal hyperplasia development. High levels of PCNA staining will indicate that the SMCs in the neointima are continuing to proliferate and therefore hyperplasia is likely to increase in size. Other studies have also used Ki67 as a proliferative marker as well as the apoptotic marker Caspase 3. The markers of choice reported in each study are largely dependent on the mechanism of action for therapy being evaluated. For example, proliferative markers, PCNA and Ki67, were appropriate for anti-proliferative drugs such as sirolimus which directly target the mTOR pathways . CytotoxIn addition to the crucial role of smooth muscle cells, mounting evidence points to the important role of vascular inflammation in the development of NH . Our injLastly, injured vessels within our model showed increased collagen deposition and proteoglycan synthesis both within the vessel wall and the developing neointima. While these are not commonly reported metrics in rodent-based injury models, both are critical vessel remodelling events that serve as precursors to more significant downstream vascular pathologies. In humans both balloon angioplasty and stenting are known to cause collagen accumulation in the arterial intima and media/adventitia layers. The processes underlying this occurrence are still not fully understood however, and numerous studies have acknowledged that both intact fibrillar collagen and its degradation are both integral to vascular repair . SimilarUsing a surgical technique that provides mechanical distention and endothelial denudation injury in two consecutive procedures, our new vascular injury model was able to reproduce the main outcomes of vascular injury, including neointimal hyperplasia and re-endothelialization, at levels comparable to the current established models of restenosis. This method involved a simple modification to current injury models yet may offers complementary improvements, most notably the access to the larger diameter rat vessels, shorter vessel lengths required for injury, and the lack of permanent ligation/disruption of distal blood vessels. This feature offers a main advantage as injury is only induced locally without permanent ligation or disruption of blood flow in collateral vessels. This could perhaps supplement restenosis injury research by providing a closer representation of the physiological response to vascular damage following endovascular injury. Despite the model’s procedural modifications, the traditional vascular damage responses, such as increased M1 macrophage activation, pro-inflammatory cytokine release, SMC contractile-to-synthetic differentiation, and proteoglycan and collagen deposition in the developing neointima are additional features of the model which can be accurately quantified and demonstrate remarkable consistency over 2 weeks across multiple batches of surgeries. The inclusion of this method into the current suite of rodent-based arterial injury models may help facilitate more in-depth analysis of pathogenic causes of restenosis and accurate evaluation of future anti-restenosis therapies.S1 Fig1) Surgical microscope 2) vessel clamps and 22G needle catheter 3) No.22 scalpel blade 4) sterilised gauze 5) surgical forceps and 6) microscissors. 7) Microforceps for performing vessel distention 8) sterilised gauze tips 9) 9–0 silk suture for arteriotomy closure and 10) 3–0 silk suture to close up neck incision.(TIF)Click here for additional data file.S2 FigA 1.5 cm segment of the carotid artery is isolated just proximal to the bifurcation of the internal and external carotid artery using vessel clamps. At the most proximal vessel clamp, an arteriotomy is performed to create an opening for the insertion of microforceps and needle catheter.(TIF)Click here for additional data file.S3 FigExplanted common carotid arterial segments were sectioned along their length in 5μm sections. Three sections were placed on each slide, and for each stain, three equidistant slides were selected at the proximal, middle, and distal ends. These slides were averaged to create a single data point for the sample. The total samples were then averaged and presented with ± as the standard error of the mean (SEM).(TIF)Click here for additional data file.S4 FigA) Representative co-staining using iNOS (M1) and CD68 (macrophages), scalebar = 300μm; inset: individual channels of iNOS and CD68, scalebar = 100μm. B-C) Secondary antibody (no primary) control images of AlexaFluor488 (AF488—green) and AlexaFluor594 (AF594) secondary antibodies showing negative staining in the neointima but auto-fluorescence of elastic laminae, scalebar = 100μm.(TIF)Click here for additional data file.S1 FilePDF file containing all raw data presented throughout the manuscript.(PDF)Click here for additional data file.


Bifidobacterium (fixed-effect beta (95% CI) = −0.22 , p = 3.90e-04) and Lachnospiraceae NK4A136 group (fixed-effect beta (95% CI) = −0.09 , p = 2.53e-06) were found to be the best predictors of anxiety and depressive symptoms, respectively, across our three cohorts and published literature taking into account demographic and lifestyle covariates, such as fibre intake. The association with anxiety was robust in accounting for heterogeneity between cohorts and supports previous observations of the potential prophylactic effect of Bifidobacterium against anxiety symptoms.The gut microbiome is a significant contributor to mental health, with growing evidence linking its composition to anxiety and depressive disorders. Gut microbiome composition is associated with signs of anxiety and depression both in clinically diagnosed mood disorders and subclinically in the general population and may be influenced by dietary fibre intake and the presence of chronic pain. We provide an update of current evidence on the role of gut microbiome composition in depressive and anxiety disorders or symptoms by reviewing available studies. Analysing data from three independent cohorts (osteoarthritis 1 (OA1); n = 46, osteoarthritis 2 (OA2); n = 58, and healthy controls (CON); n = 67), we identified microbial composition signatures of anxiety and depressive symptoms at genus level and cross-validated our findings performing meta-analyses of our results with results from previously published studies. The genera   •Symptoms of anxiety and depression are associated with the gut microbiota.•Five bacterial genera are associated with anxiety and depressive symptoms.•Associations were stronger in two chronic pain cohorts than in controls.•Bifidobacterium associated with lower anxiety symptoms.A meta-analysis found •Our results agree with observations of probiotic supplementation reducing anxiety. Depression and anxiety are mood disorders that affect 4.7% and 7.3% of the global population, respectively The impact of anxiety and depression on the gut-brain axis has been demonstrated in preclinical models A number of systematic reviews have explored the links between the gut microbiota and mood related clinical conditions Although several studies have reported an association between anxiety and gut microbiome composition Both anxiety and depression are more prevalent among individuals who experience chronic pain conditions than among the general population There is therefore a need to fill the gap of evidence for the links between gut microbes and anxiety and depressive symptoms in the general population which can provide a more comprehensive understanding of the complex relationship between the gut and the brain. In addition there is a need to understand whether the microbial features associated with anxiety or depressive symptoms in the general population have the same role in individuals with and without chronic pain. Establishing such links may aid in preventing the onset of full-fledged disease and lead to more personalised interventions for individuals with frequently coexisting health issues, such as chronic pain.The specific aims of the present study are, (i) to systematically search available evidence on gut microbiota composition in depressive and anxiety disorders and symptoms, providing a necessary update of the current literature; (ii) to identify microbial composition signatures of anxiety and depressive symptoms across three independent cohorts of individuals without mood disorders, two of osteoarthritis patients with chronic pain and a control cohort of similar age; (iii) to extend previous published studies by incorporating meta-analyses between anxiety/depressive symptom measures and the gut microbiota; and (iv) to cross-validate our findings combining our new data along with previously published reports, using a prediction-based approach, and taking into account dietary fibre intake and the presence of chronic pain.The study design is outlined in A systematic search of PubMed was conducted in April 2023 using the search terms: "Gastrointestinal Microbiome"[Mesh] OR Gastrointestinal[tiab] OR gut[tiab] OR intestinal[tiab] AND Microbiota[tiab] OR Microbiome[tiab] OR flora[tiab] AND "Anxiety"[Mesh] OR "Anxiety Disorders"[Mesh] OR "Phobia, Social"[Mesh] OR "Patient Health Questionnaire"[Mesh] OR Anxiety[tiab] OR stress[tiab] OR panic disorder[tiab] OR social anxiety disorder[tiab] OR generali* anxiety disorder[tiab] OR "Gastrointestinal Microbiome"[Mesh] OR Gastrointestinal[tiab] OR gut[tiab] OR intestinal[tiab] AND Microbiota[tiab] OR Microbiome[tiab] OR flora[tiab] AND "Depression"[Mesh] OR "Depressive Disorder"[Mesh] OR "Dysthymic Disorder"[Mesh] OR "Depressive Disorder, Major"[Mesh] OR Depression[tiab] OR Depressive disorder[tiab] OR Major Depression[tiab] OR Major Depressive Disorder[tiab] OR Dysthymic Disorder[tiab] OR Major Dysthymic Disorder[tiab] OR persistent depressive disorder[tiab]. The search yielded 1968 records. Studies were included if they: 1) assessed the gut microbiota composition in anxiety or depressive disorders, or 2) investigated associations between the gut microbiota and anxiety/depression symptom measures in healthy participants or relevant conditions . They were excluded if they: 1) examined the gut microbiota and anxiety/depression symptoms solely in another psychiatric disorder or disease, 2) assessed the effect of an intervention without reporting relevant baseline measurements. Studies were further excluded if they were: 3) in languages other than English, 4) letters, reports, or abstracts from congresses or symposia, 5) review, protocol and method studies, 6) animals and non-adult studies. The 73 included studies along with relevant findings are summarised in Data were taken from baseline visits of three independent study cohorts, iBEAT-OA (OA1), Molecular Pathways (OA2) and Omega-3/Fibre (CON). OA1 and OA2 are knee osteoarthritis cohorts of community dwelling individuals (Age: >45 y) recruited from the Nottinghamshire area All participants provided written informed consent. For OA1 and OA2, ethical approval was obtained from the East Midlands – Derby NHS Research Ethics Committee  and the Health Research Authority . The trials are registered under the clinicaltrials.gov database . For CON the trial was approved by the West Midlands Black Country NHS Research Ethics Committee (18/WM/0066) and is registered under the clinicaltrials.gov database (NCT03442348).In OA1 and CON, the Hospital Anxiety and Depression Scale Anxiety (HADS-A) and the HADS Depression (HADS-D) subscales were used to measure severity of anxiety and depression symptoms, respectively Dietary intake was measured in the OA1 cohort using the validated European Prospective Investigation into Cancer (EPIC) Norfolk Food Frequency Questionnaire (FFQ) version 6 (CAMB/PQ/6/1205) which contains a list of 130 commonly and less commonly consumed foods. Participants are asked to indicate their usual frequency of consumption ranging from “never or less than once/month” to “6 + times per day”. The servings are specified in terms of units or common portions  or household measures . Data from the EPIC-Norfolk FFQs were analysed using the FETA software which can be downloaded and used for free upon registration Faecal sample collection methods were the same in all cohort studies. Participants were given a stool collection kit and leaflet with detailed instructions on how to collect and post the sample to the assessment centre in the prepaid postbox provided at the end of their baseline visit. All samples were either immediately frozen at − 80 °C or at − 20 °C temporarily until they could be transported to − 80 °C freezers in the Clinical Science Building at City Hospital or at the Queen's Medical Centre following local standard operating procedures (SOPs) prior to analysis. SOPs were used at the collection sites and when transferring the samples which were handled by trained research personnel to ensure the high quality and reliability of the research data. Stool samples were outsourced to an external supplier for DNA extraction, quality control and preprocessing (see next section).The wet-lab procedure, i.e. the physical, hands-on work to process and prepare samples from stool for genetic analysis, followed by the Bioinformatic Genetic Lab, Department of Internal Medicine Erasmus Medical Center, Rotterdam was the same in all cohorts (i.e. the same amplicon generated and the same sequencing). In OA2 and CON cohorts, stool DNA extraction was carried out using the DNA isolation kit Invimag Stool DNA Kit by Stratec and 100 mg of the stool sample. In OA1 the MagMax Microbiome Ultra kit from Thermo Fisher was used. The laboratory has tested the effect of the DNA isolation kit on bacterial profiles extensively and has concluded that the Ultra kit does not have significant deviation from the Invimag kit. In addition, in a direct comparison between the Invimag Stool DNA Kit and the MagMax Microbiome Ultra kit with other DNA extraction kits, these two kits performed comparably Quality control and preprocessing, including demultiplexing using QIIME1 version 1.9.1 and primer trimming with TagCleaner v0.16 was performed by the Bioinformatic Genetic Lab. Reads were exported from the MiSeq as a run level FASTQ file, containing all reads generated above Q30, the average QC value threshold Illumina uses. Above Q30 in short means that each nucleotide in the read has at maximum a 1:1000 chance of being called wrong. In OA1 and OA2, the DADA2 pipeline in R version 4.0.0 was used for analysis of the sequencing data that turns sample-wise amplicon sequences into a feature count We carried out all statistical analyses in R v4.2.1. ASVs and OTUs with a relative abundance of < 0.01% in every sample were removed using a function adapted from Arumugam, Raes t-tests or Mann–Whitney U tests for continuous variables, and Chi-squared test for categorical variables and Fisher’s exact test when < 5 in a category. OA1 served as the Discovery cohort and OA2 and CON as the Replication cohorts. We implemented a predictive algorithm in the Discovery cohort to identify a model or microbial signature containing the minimum number of features with the maximum predictive power. This algorithm is part of the coda4microbiome package and is designed to identify all possible pairwise log-ratios between pairs of components and perform variable selection through penalized regression on all the log-ratio pairs Differences between the descriptive characteristics of the three cohorts were evaluated using one-way ANOVA, Student metafor and meta to perform both fixed-effects and Han-Eskin (H&E) random effect meta-analyses. The H&E random effect model was used in sensitivity analysis as it has higher statistical power than the fixed effects in the presence of heterogeneity (I2) and the Cochran's Q p-value. Publication bias was assessed visually using funnel plots and quantitatively with the Egger’s regression test and the trim- and fill-analysis We fitted linear regression models to identify which of the taxa comprising the microbial signature were statistically significant and survived correction for age, sex, body mass index (BMI) and fibre intake in the Discovery cohort. We confirmed consistent directionality of our results in the two independent Replication cohorts and published literature. Linear regression results from each participating cohort were standardised to allow for the different scales. We performed z-score normalisation .A summary of the relevant results (i.e. associations between anxiety/depressive symptoms and the gut microbiota or comparisons of gut microbiota between cases and controls) from the 73 included studies is provided in Fig. 1Bifidobacterium and Lactobacillus species Bifidobacterium and Lactobacillus counts Most studies employed 16 S rRNA gene sequencing to estimate gut microbiota composition (n = 64), two of which also included shotgun metagenomics. Six other studies used shotgun metagenomics to sequence all microbial genomes present in a sample. One study used oligonucleotide probes for The majority of studies published to date dealt with clinical diagnoses of depression/MDD or GAD (n = 58). Systematic reviews with quantitative and qualitative syntheses for these traits have already been published elsewhere e.g. . Only 15For the quantitative synthesis in this study we have only included studies that reported regression coefficients with anxiety or depression scales in participants without a clinical diagnosis of mood disorders. The selection strategy is depicted in The descriptive characteristics of the studied cohorts are shown in OA1 served as the Discovery cohort, while the OA2 and CON cohorts served as Replication cohorts.We first identified the microbial taxa that best predict anxiety and depressive symptoms in OA1 (Discovery cohort) using a prediction-based method . The preWe then conducted linear regressions with the aforementioned taxa, which are indicative of anxiety and depressive symptoms in the Discovery cohort, to examine their level of significance and determine whether they exhibit consistent directionality in the replication cohorts.Bifidobacterium, Clostridium sensu stricto 1, Coprococcus, Bacteroides, Blautia and Anaerostipes (positive) were in the same direction in all cohorts  had a positive association with higher anxiety symptom levels. As seen from Bifidobacterium has been negatively associated with anxiety in females only in Bacteroides abundance was lower in people with comorbid MDD and anxiety relative to people with MDD alone Bifidobacterium, Clostridium sensu stricto 1, and Coprococcus with anxiety symptoms were significant only in the two OA cohorts.For anxiety symptoms,  cohorts . BifidobLachnospiraceae NK4A136 group, Butyricicoccus and Roseburia were in the same direction in all cohorts  also found a negative association of Lachnospiraceae NK4A136 group and Roseburia taxa with higher depressive symptom levels Butyricicoccus. For example, Chen, et al. Butyricicoccus in controls relative to MDD, while Jiang and co-workers For depressive symptoms,  cohorts . LachnosFaecalibacterium taxon had a negative association with higher depressive symptom levels in OA1 but was found to be in the opposite direction in OA2 and CON. In the majority of studies Faecalibacterium taxon had lower abundance in depression . For instance, on e.g.,  consisteBacteroides and Blautia. Out of those, Bifidobacterium was the only genus previously found to be linked to anxiety symptoms in Taylor, Thompson Lachnospiraceae NK4A136 group remained significant in the Discovery cohort after adjusting for covariates and has been previously linked to depressive symptoms in Bosch, Nieuwdorp Bifidobacterium and Lachnospiraceae NK4A136 group taxa were negatively associated with anxiety and depressive symptom levels in all three cohorts but remained significant only in the two osteoarthritis cohorts.Linear regressions with the taxa that comprised the signatures predicting anxiety and depressive symptoms were adjusted for the a priori selected covariates  in the Discovery cohort. The taxa that remained statistically significant after adjustment for demographic and lifestyle covariates are highlighted in bold in Bifidobacterium and Lachnospiraceae NK4A136 group, respectively. We conducted meta-analyses with the data from our own cohorts and published studies to estimate the pooled effect of those genera in relation to anxiety and depressive symptoms . There was significant heterogeneity in the meta-analysis of Lachnospiraceae NK4A136 group taxon with depressive symptom scores (QEp < 0.05), which we followed up by conducting sensitivity analyses (see next section). The funnel plot for Bifidobacterium showed no evidence of publication bias , whereas the Lachnospiraceae NK4A136 group plot indicated potential publication bias as there was some asymmetry , indicating no evidence of publication bias. In addition, the trim- and fill- analysis produced almost identical results compared to our original meta-analyses , further supporting a lack of publication bias. A potential source of asymmetry in the funnel plot for the meta-analysis of Lachnospiraceae NK4A136 group with depressive symptom levels may be the presence of heterogeneity.The two genera that remained significant after adjustment for covariates and were previously reported in the literature to be associated with anxiety and depression symptoms were symptoms . Bifidobsymmetry . HoweverBifidobacterium and Lachnospiraceae NK4A136 group to remain statistically significant with negative associations with anxiety and depressive symptoms , respectively.We examined whether the results differed if OA2 was excluded from the meta-analysis due to the different instruments used to measure anxiety and depression symptoms in this cohort; the two validated items from the HADS as opposed to the full HADS used in OA1 and CON. Leave-one-out meta-analysis including only OA1 and CON resulted in the bacterial taxa Bifidobacterium and Lachnospiraceae NK4A136 taxa remained significantly negatively correlated with anxiety and depressive symptoms , respectively. Nevertheless, heterogeneity remained significant in the meta-analysis with Lachnospiraceae NK4A136 taxon.Considering the heterogeneity between the various cohorts, both ours and in the literature , we alsoBifidobacterium and Lachnospiraceae NK4A136 taxa associations in a small subset of the OA2 cohort for which we had microbiome data analysed with both pipelines (n = 33). Regardless of the pipeline, Bifidobacterium and Lachnospiraceae NK4A136 taxa were negatively associated with anxiety and depressive scores, respectively . Nevertheless, we observed differences in the magnitude of the effects between the two pipelines with DADA2 producing greater effects, especially for Lachnospiraceae NK4A136 taxon. This is consistent with the observation that some of the findings were only significant in the two osteoarthritis cohorts and not in the CON cohort, therefore indicating some level of indirect incompatibility between the two approaches. Despite this, our findings are valid as we could replicate them across our de novo data and published literature with the overall effect remaining significant, including in the random-effects models.Two different pipelines were used for microbiome analysis; DADA2 was used in the two osteoarthritis cohorts and MYcrobiota in the CON cohort. To check for potential incompatibilities between the two approaches, we compared the standardised beta estimates of the Bifidobacterium increased from − 0.45 to − 0.47 after adjusting for medication use whereas the effect size of Lachnospiraceae NK4A136 group decreased from − 0.38 to − 0.33 after adjustment. Neither NSAID nor opioid use was significantly associated with prevalence of anxiety or depressive symptoms in the two chronic pain cohorts tested.Finally, because our study used two chronic pain cohorts we also tested the potential effect of opioids and nonsteroidal anti-inflammatory inhibitors (NSAIDs) in our own datasets conducting a subanalysis of the linear regressions with anxiety and depressive symptom levels adjusting for use of pain medications (NSAIDs and opioids) in both OA cohorts. The results of this supplementary analysis, presented as Bifidobacterium genus and lower anxiety symptom levels. We also find a consistent association between Lachnospiraceae NK4A136 group and lower depressive symptom levels also reported in the HELIUS cohort by Bosch et al. Heterogeneity between cohorts was observed in the meta-analysis with Lachnospiraceae NK4A136 taxon, which suggests the need for further studies to confirm its actual effect.In this study we have for the first time carried out a meta-analysis of microbiome features associated with anxiety and depressive symptoms using both new data and data from the literature. We find consistent and robust associations between the presence of the No significant associations in richness/evenness measured with the Shannon diversity index were observed with either anxiety or depressive symptoms across the three cohorts. This is consistent with evidence from a meta-analysis in MDD Bifidobacterium, Clostridium sensu stricto 1 , Coprococcus and Lachnospiraceae NK4A136 group) and anxiety/depressive symptoms were stronger in the two osteoarthritis cohorts than in the CON group in our de novo data. While this effect could partly be driven by the different pipelines used for microbiome analysis as evident from the sensitivity analyses, we cannot disregard the possibility that some of the variation may be driven by the presence of chronic pain. Consistent with this notion are reports that Bifidobacterium and Clostridium genera are also reduced in fibromyalgia patients Bifidobacterium may therefore have a potential pleiotropic effect influencing the pathways shared by anxiety and chronic pain.One aim of the current study was to explore potential differences in microbial features previously associated with anxiety or depressive symptoms among individuals with or without chronic pain. A notable finding is that the magnitude and significance of the associations between several microbial taxa  in individuals with depression compared to controls Lachnospiraceae NK4A136 group in controls versus MDD Lachnospiraceae NK4A136 group in GAD [21]. Lachnospiraceae NK4A136 group therefore seems to exert beneficial effects on negative affect in general rather than having a specific association with either anxiety or depressive disorders and symptoms. This is in line with the idea that changes in the gut microbiome may be associated with overall changes in psychological health instead of showing taxonomic specificity, whereby specific taxa differentiate specific disorders.After full adjustment for covariates  in the Discovery cohort only the association between the presence of Bifidobacterium genus and lower levels of anxiety symptoms across our three cohorts and in the published literature withstood adjustments for heterogeneity and fibre intake both in our Discovery cohort and in Taylor, Thompson BifidobacteriumBifidobacterium strains are among the most widely used probiotics The consistent association between the presence of the Bifidobacterium genus reported here could be explained by its ability to produce acetate and lactate during carbohydrate fermentation, which in turn are converted into butyrate by butyrate-producing bacteria through cross-feeding interactions Bifidobacterium has been demonstrated in an animal model Bifidobacterium spp. have been shown to attenuate stress response in mice by modulating hippocampal neurogenesis and synaptic plasticity Bifidobacterium infantis species in particular was shown to reverse the stress response by inducing c-Fos activation in the paraventricular nucleus in germ-free mice Lachnospiraceae family are integral components of the gut microbiota and are prominent producers of short-chain fatty acids The beneficial effect of Bifidobacterium, personalized nutritional therapies can be developed to target specific bacteria and their interactions with the gut-brain axis. Miyaoka et al. have observed that the combination of antidepressants and probiotics is more effective to treat drug-resistant depression By identifying the key bacterial candidates associated with anxiety and MDD and stress resilience-promoting microorganisms like Strengths of the current study include an updated and extensive systematic literature search both for depression and anxiety and the inclusion of dietary fibre as covariate in the Discovery cohort. The present analysis on the microbial signatures was based specifically on model prediction rather than differential abundance in the Discovery cohort. Another strength is the cross-validation using three databases and performing meta-analysis with previous published literature and the use of both chronic pain and non-chronic pain cohorts which has enabled us to observe potential differences in microbiome associations with anxiety and depression in these two groups. The results from our de novo data analysis are based on adults without clinical depression, anxiety, or stress disorders, and therefore can be extended to the general population that experiences negative mood states at a subclinical level.Lachnospiraceae NK4A136 group, Roseburia, and Faecalibacterium, have been identified in the literature  that are highly correlated with clinical screening terature  to be asBifidobacterium and Lachnospiraceae NK4A136 group, respectively, has only minor effects in the standardized effect sizes in either cohort . Therefore, we do not expect these minimal gender differences to impact our results.Another potential limitation may be seen in the marginal gender differences between the OA2 and CON groups. The higher proportion of women in the control group result of this having started as a female only cohort although it is widely recognised as representative of the general female UK population Bifidobacterium and Lachnospiraceae NK4A136 group are reproducibly associated with lower anxiety and depressive symptoms, respectively. Heterogeneity between cohorts was observed only for the association with Lachnospiraceae NK4A136 group, suggesting that more meta-analyses are needed to elucidate its effect. The association with Bifidobacterium was not affected by heterogeneity and supports previous observations of its potential prophylactic effect against anxiety symptoms.In conclusion, we report that two bacterial genera, NIHR Nottingham Biomedical Research Centre, by Versus Arthritis Project grants 22473 and 23139, and by 10.13039/100014013UKRI grant MR/W026813/1. The control cohort was funded by the 10.13039/100011721Chronic Disease Research Foundation. TwinsUK receives funding from the 10.13039/100004440Wellcome Trust, the 10.13039/501100000780European Commission H2020 grants SYSCID (contract #733100); the 10.13039/501100000272National Institute for Health Research (NIHR) Clinical Research Facility and the 10.13039/100014461Biomedical Research Centre based at Guy's and St Thomas' NHS Foundation Trust in partnership with King's College London, the 10.13039/100011721Chronic Disease Research Foundation, the UKRI Medical Research Council (MRC)/British Heart Foundation Ancestry and Biological Informative Markers for Stratification of Hypertension , and Zoe Limited. This work was also supported by UKRI grant MR/W026813/1 to AMV and CM is supported by the 10.13039/100011721Chronic Disease Research Foundation.This study was supported by grant the Afroditi Kouraki: Conceptualization, Methodology, Software, Formal analysis, Data curation, Writing − original draft, Writing − review & editing, Visualization. Anthony Kelly: Investigation, Resources, Data curation, Writing − review & editing, Project administration. Amrita Vijay: Investigation, Resources, Data curation, Writing − review & editing, Project administration. Sameer Gohir: Investigation, Resources, Data curation, Writing − review & editing, Project administration. Stuart Astbury: Software, Formal analysis, Writing − review & editing. Vasileios Georgopoulos: Investigation, Writing − review & editing. Bonnie Millar: Project administration, Data curation, Writing − review & editing. David Andrew Walsh: Resources, Funding acquisition, Writing − review & editing. Eamonn Ferguson: Methodology, Writing − review & editing. Cristina Menni: Methodology, Resources, Writing − review & editing, Funding acquisition. Ana M Valdes: Conceptualization, Methodology, Supervision, Writing − original draft, Writing − review & editing, Funding acquisition.AMV and AV are members of the Chuckling Goat (a nutrition company) Scientific Advisory Board. AMV is a member of the CP Kelco SAB. AMV and CM are consultants for Zoe Ltd. DAW has undertaken consultancy through University of Nottingham for Contura International A/S, Glaxo SmithKline, AKL Research and Development Ltd, Pfizer Ltd, Abbvie Ltd, Ely Lilly & Co.Ltd, Galapagos Ltd., and Reckitt Benckiser Health Ltd and is responsible for research grants paid to University of Nottingham from Versus Arthritis, UKRI, NIHR, Pfizer Ltd, Ely Lilly & Co Ltd, UCB Pharma, and Orion Pharma .


Volumetric muscle loss overwhelms skeletal muscle’s ordinarily capable regenerative machinery, resulting in severe functional deficits that have defied clinical repair strategies. In this manuscript we pair the early in vivo functional response induced by differing volumetric muscle loss tissue engineering repair strategies that are broadly representative of those explored by the field  to the transcriptomic response induced by each intervention. We demonstrate that an implant strategy comprising allogeneic decellularized skeletal muscle scaffolds seeded with autologous minced muscle cellular paste (scaffold + cells) mediates a pattern of increased expression for several genes known to play roles in axon guidance and peripheral neuroregeneration, as well as several other key genes related to inflammation, phagocytosis, and extracellular matrix regulation. The upregulation of several key genes in the presence of both implant components suggests a unique synergy between scaffolding and cells in the early period following intervention that is not seen when either scaffolds or cells are used in isolation; a finding that invites further exploration of the interactions that could have a positive impact on the treatment of volumetric muscle loss.The online version contains supplementary material available at 10.1186/s12891-023-06401-1. Skeletal muscle exhibits an innate ability to regenerate small amounts of functional tissue following minor traumas including strains and contusions .The immeAlternatively, in the case of volumetric muscle loss (VML), satellite cell recruitment and differentiation are overwhelmed by inflammatory signaling and damage to the basal lamina, with ECM-deposition featuring prominently in the weeks following injury . With thExploratory biomaterials for treatment of VML generally attempt to restore the architectural and compositional cues lost to injury and thereby provide a pro-regenerative substrate for endogenous progenitor cell migration. The more promising scaffolding materials appear to be those derived from native tissues, which can be remodeled by the body’s own wound healing machinery. This includes scaffolds prepared using decellularized skeletal muscle (DSM), porcine small intestinal, and bladder tissue. However, the use of tissue derived scaffolds on their own has yielded mixed results, with little evidence of de-novo muscle fiber regeneration –13. To eThe mechanisms by which these combinatorial therapies (scaffold + cells) may enhance muscle recovery when compared to either strategy used in isolation remains unclear, particularly during the early weeks following injury. The lower level of recovery occurring with the use of scaffolds or cells alone emphasizes the importance of a multifactor approach to VML treatment, and suggests a biomolecular synergy exists between delivered cells and scaffolds which to date has not been explored in depth. Here we extend the body of VML knowledge by profiling the transcriptome-wide responses of VML-injured tissue to scaffolding and cell delivery strategies employed in isolation and in combination at 3 to 14 days post injury (DPI) to better assess early regulatory changes potentially mediating the significant differences observed in long-term functional outcome between these distinct tissue engineering schemes. Clarifying the effects of these therapies on the complex inflammatory, fibrotic, and regenerative networks in the early period following injury contributes to a foundational understanding of VML pathobiology upon which to further refine VML repair strategies for the clinic.ad libitum. All animal procedures were approved by and performed in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Arkansas.Sprague Dawley rats  were used in this study . The 3 and 14 day timepoints are supported by findings by Corona in which they observed a rise in many key regenerative markers during the first two weeks post VML injury and repair . BuprenoVML injuries were created in rats using an established VML model . BrieflyPeak tetanic contractile force of injured and uninjured contralateral limbs was measured at three days and fourteen days post-injury as previously described . BrieflySamples of TA muscles (n = 3 / group / timepoint) encapsulating the interface between the defect and defect-adjacent regions were harvested from euthanized rats at three days and fourteen days post-injury using a 2 mm biopsy punch, snap frozen on liquid nitrogen, and stored at -80 °C pending RNA isolation. Tissues were thawed and homogenized for 15 s at room temperature. Total RNA was isolated using the Purelink RNA Mini Kit (ThermoFisher). RNA concentration was determined by nanodrop spectrophotometry, with RNA quality  confirmed using a Tapestation (Agilent Technologies). cDNA libraries were sequenced on the BGISeq-500 platform to a mean depth of 20,000,000 reads per library.Rattus norvegicus genome (RGSC build 6.0) using STAR [RNA sequencing reads were mapped to the ing STAR . Reads wing STAR , followeing STAR . Differeing STAR . Enrichming STAR . Pathwaying STAR .2) and the significance of the relationship was examined using ANOVA. Significance was accepted at P ≤ 0.05 (*) P ≤ 0.01 (**), P ≤ 0.001 (***), and P ≤ 0.0001 (****). Quantitative data are displayed as mean ± standard deviation.Statistical analyses for assessment of muscle force and mass were performed using Prism 9 , using repeated measures ANOVA with post-hoc Dunnett’s test. Assessment of significance for differential gene expression was performed using EdgeR with TMM normalization of read counts and adjustment of p-values for multiple comparisons by the Benjamini-Hochberg procedure. Linear regression using the least squares method was used to model the relationship of Reln and Robo1 gene expression to peak contractile muscle torque for all treatment groups at 14 DPI. The quality of the relationship was evaluated with the Coefficient of Determination (Rhttps://www.ncbi.nlm.nih.gov/geo/) under accession number GSE125896. Supplementary Figures are publicly available at a GitHub repository (http://github.com/RobertsEng/VML-RNA-seq).All RNA-sequencing FASTQ files and processed transcipts expression files were deposited in the Gene Expression Omnibus  resulted in defects clearly visible at 3 and 14 days post injury (DPI) Fig. A. While ). No significant differences in contractile kinematics were detected between groups during the first two weeks of recovery.Unrepaired, repaired, and uninjured contralateral limbs were assessed for peak tetanic force output at both time points. At 3 DPI, MM and DSM + MM -treated groups exhibited significantly lower force output (N/kg body weight) than uninjured contralateral muscles   suggests an activation of CREB signaling which persists in the DSM and DSM + MM groups at 14 DPI and is known to promote survival of macrophages [Analysis of RNA-seq data in EdgeR revealed a cumulative total of 5174 differentially expressed genes (DEGs) across all treatment groups relative to uninjured muscle Fig. A. A broarophages . This toAssessment of all groups for the classic markers of precursor cell myogenesis indicate a general lack of coordinated myogenesis signaling at 14 DPI. Notable exceptions include the upregulation of MYOG in all 3 DPI groups, the observation of upregulated MYF5 within the DSM group relative to uninjured controls at 14 DPI (p = 0.007), and significant (p < 0.05) increases in MYMK across most groups relative to control, though between-treatment differences were insubstantial. , with IPA identifying significant upregulation within the hepatic fibrosis canonical pathway for each . Multiple common pro-inflammatory cytokines and chemokines were significantly upregulated in all implant groups at 3 DPI as well as the DSM group at 14 DPI, with DSM + MM at 14 DPI also exhibiting increased transcription of CCL2 and CXCL10 . Upregulation of the Fcy receptor-mediated phagocytosis pathway was further identified  with upregulation of transcripts for phagocytosis-activating FC receptors as well as SRC and SYK family kinases [). The Cell Movement category of transcripts was found by Ingenuity Pathway Analysis (IPA) to be significantly upregulated for all groups at 3 DPI and the DSM (p = 1.73E-91 with 525 DEGs) and DSM + MM (p = 2.34E-109 with 736 DEGs) groups at 14 DPI. While all 3 DPI groups exhibited patterns of transcription identified by IPA as consistent with immune cell trafficking, at 14 DPI only DSM and DSM + MM treatment continued to show such patterns . Of particular note, at 3 DPI, expression of the IL-10 receptor subunits IL10ra / IL10rb were significantly upregulated across all groups, however by 14 DPI expression had largely decreased and remained upregulated in the DSM + MM group only . Among these genes, both Reln  and Robo1  expression were found to correlate moderately with limb contractile force .IPA identified substantial changes in the Neuroinflammation and Axonal Guidance Signaling canonical pathways in all 3 DPI groups, with VML, MM, DSM, and DSM + MM each exhibiting ~ 80 DEGs for neuroinflammation and ~ 100 DEGs for axonal guidance. Notably, DSM and DSM + MM at 14 DPI exhibit 60 and 109 DEGs associated with axon guidance signaling, respectively, while VML and MM at 14 DPI did not exhibit such patterns; further, pathway analysis of the 299 upregulated DEGs unique to the DSM + MM group at 14 DPI indicates that 66 of the 299 are known to have roles in nervous system development and function, including neurotransmission (20 DEGs) and motor function (6 DEGs). Querying the Gene Ontology Cellular Component database for the 299 unique DEGs additionally revealed that 37 DEGs are known to localize to the projections of neurons. Querying the Gene Ontology Biological Process database indicated that 20 of the 299 DSM + MM14 unique genes have roles in synaptic signaling, and that 25 of the human homologs of the 299 DEGs have similar roles in humans. Expression levels of several genes known to have important roles in axon guidance and peripheral neuroregeneration  were found to be significantly upregulated in the DSM + MM group but not others at 14 DPI . A standard of care for VML remains elusive; MM implantation can promote some degree of repair but requires prohibitively large amounts of donor tissue for extensive VML wounds. An emerging alternative is the use of extracellular matrix scaffolds; however, many of those piloted in the literature thus far have mixed impacts on functional outcome. In our previous work the application of MM pastes to DSM prior to implantation restored half of the peak contractile force lost to VML (81% of uninjured peak contractile force vs. 62%), while other minced muscle or ECM grafts result in comparatively modest improvement to force outcomes . In thisWhile upregulation of some myogenesis-related transcripts relative to uninjured muscle was observed in some groups, between-treatment differences were insubstantial, in agreement with the findings of Aguilar et al. in their transcriptomic profiling of VML and MM repair . The shode novo myogenesis [Implantation of cellular constructs following VML has shown the ability to promote muscle innervation as soon as a week post-repair , and canogenesis . Therefoogenesis  from theogenesis . The bidogenesis . While wOur observation of inflammatory and phagocytosis related transcription -including several chemokines- is likely a reflection of the diverse population of macrophages and other key immune cells known to infiltrate VML defects in the early period following injury. While the recruitment of macrophages as part of the acute wound response and foreign body response to implants is well described, direct participation of these cells in a variety of regenerative processes is only recently becoming better appreciated. Intriguingly, Stratton et al. demonstrated that macrophages regulate Schwann cell function during regeneration, with ablation of macrophages associated with the injury site resulting in marked reductions in conduction velocity and remyelination mediated by the absence of macrophage-derived GAS6 . Cattin We detected elevated transcription of IL-10 receptor subunits in all groups except VML and MM at 14 days post injury, yet the transcription of IL-10 only achieved significance in the DSM and DSM + MM groups at the 3-day timepoint. IL-10 is a well-established wound healing cytokine with known anti-inflammatory effects , and itsWe find that the results of this transcriptomic study suggest that DSM + MM treatment promotes a pattern of transcription consistent with peripheral neuroregenerative signaling at two weeks following injury Fig. . FurtherIn sum, our data suggest the involvement of several key immunomodulation, neuritogenesis, axonogenesis, and axon guidance transcripts in VML repair in the early period following injury; these changes were particalular notable in the scaffold + cell treatment (DSM + MM) group. Interrogation of these and other factors influencing nerve regeneration and immune-assisted repair within injured skeletal muscle is relatively unexplored and may yield fruitful targets exploitable by a combination of drug and biomaterials-based therapies. Ultimately, VML injuries exhibit significant heterogeneity. Therefore, a range of clinical strategies incorporating appropriate biomaterials, drugs, and physical rehabilitation will be required to best address the complications of this challenging condition. As the expense of RNA-sequencing technologies decreases and the ease of downstream data analysis increases, whole transcriptome expression profiling will be an invaluable tool in guiding the development of promising therapies for the treatment of VML.Below is the link to the electronic supplementary material.Supplementary Material 1


Introduction: Human body models (HBMs) play a key role in improving modern vehicle safety systems to protect broad populations. However, their geometry is commonly derived from single individuals chosen to meet global anthropometric targets, thus their internal anatomy may not fully represent the HBM’s target demographic. Past studies show sixth rib cross-sectional geometry differences between HBM ribs and population-derived ribs, and corrections to HBM ribs based on these data have improved HBM’s abilities to predict rib fracture locations.Methods: We measure and report average and standard deviations (SDs) in rib cross-sectional geometric properties derived from live subject CT scans of 240 adults aged 18–90. Male and female results are given as functions of rib number and rib lengthwise position for ribs 2 through 11. Population means/SDs are reported for measures of rib total area, rib cortical bone area, and rib endosteal area, as well as inertial moment properties of these rib sections. These population corridors are compared between males and females, and against the baseline rib geometries defined in six current HBMs.Results: Total cross-sectional area results found average males ribs to be larger than those of females by between approximately 1–2 SDs depending on rib number and position, and larger in cortical bone cross-sectional area by between 0–1 SDs. Inertial moment ratios showed female ribs being between approximately 0–1 SDs more elongated than male ribs, dependent again on rib number and position. Rib cross-sectional areas from 5 of the 6 HBMs were found to be overly large along substantial portions of most ribs when compared to average population corridors. Similarly, rib aspect ratios in HBMs deviated from average population data by up to 3 SDs in regions towards sternal rib ends.Discussion: Overall, while most HBMs capture overall trends such as reductions in cross-section along shaft lengths, many also exhibit local variation that deviates from population trends. This study’s results provide the first reference values for assessing the cross-sectional geometry of human ribs across a wide range of rib levels. Results also further provide clear guidelines to improve rib geometry definitions present in current HBMs in order to better represent their target demographic. The thorax remains the most commonly injured body region of vehicle occupants in motor vehicle crashes, and rib fractures are the most common type of thoracic injury . Finite Current HBMs have typically been developed to represent population-based percentiles of male or female in terms of mass and stature . As such, their specific geometry is commonly derived from a single individual meeting target population measures of anthropometry , but further local and internal anatomy  is not strictly controlled for, and thus may not accurately represent that of the HBM’s target demographic. The biofidelity of the ribs in these HBMs needs to be evaluated and improved.Experimental studies have shown that the cross-sectional shapes and sizes of ribs, measured local to the eventual fracture sites, have strong correlations with overall rib stiffness and force required to reach fracture . These fIn order to build HBMs with rib geometry that represents their target demographic it is necessary to first quantify those geometric targets. With appropriate image analysis tools, medical imaging is an abundant source from which to derive these geometric targets. Previously, we used CT scans of 33 cadaveric sixth ribs to understand local bone thickness distributions and also reported average cross-sectional geometry properties within that population . These cSince that time, we have improved upon CT image analysis techniques to report rib cortical bone thickness from a wider range of live subject CT scans, and from rib levels 2 through 11 . To dateRibs from a total of 240 chest CT scans were used for this study under IRB HUM00041441 as described in detail previously in . The popIn previous work , 3D curvTt.Ar), the cortical bone area (Ct.Ar), the endosteal area (Es.Ar), and the cortical shell’s maximal  and minimal (or secondary) area inertial moments (Imax and Imin). A derived ratio of these moments (the log transform of Imax divided by Imin), Irat, is also created.In the current study we measure and report the cross-sectional area and inertial moment properties from the shapes created by these cortical bone borders. The measurements consist of the total sub-periosteal area , and average and standard deviations in these property distributions are reported.The same cross-sectional rib measurements were obtained from all ribs of 6 human body models that are currently use within the automotive safety community, listed in Sample cross-sectional geometry from ribs 3 and 8 of the VIVA + M50, GHBMC M50, and THUMS M50 HBMs are shown in Tt.Ar, Ct. Ar, and Es.Ar) of ribs 2 through 11 are summarized by rib number and position along the rib in Tt.Ar and Es.Ar) and 1 SDs (Ct.Ar) as compared to females. These differences are consistently seen across all rib levels and regions with the exception of the sternal ends of ribs 2-4, whereby the difference in Ct.Ar diminishes towards parity at between 70% and 90% of the rib’s length.Male and female average cross-sectional area measurements (Imax) and secondary inertial moments (Imin) than females. This difference is found across all ribs and all rib locations, and ranges between approximately 1 and 2 standard deviations at different regions across the rib cage. Female ribs are also seen to have higher Irat ratios  by a margin of less than 1 SD across most rib regions.Comparisons of area inertial moment measures between males and females are presented in Tt.Ar and Es.Ar measures) than the male population average. Correspondences between the HBM rib sizes and the population corridors vary greatly by rib and by local region. Rib geometry measurements taken from the three male HBMs  are shown superimposed over the average male corridors for those same measures in Tt.Ar than average male ribs (over 75% of these ribs’ length beyond 1SD above population averages), with lower ribs 7 through 11 falling near to or within 1 SD of male average values. Measured cortical bone content (Ct.Ar) matches population values more closely, falling within 1SD of expected average values along approximately 90% of all GHBMC M50 ribs.Comparing to population average values, the GHBMC M50 model ribs 2 through 5 are generally larger in Tt.Ar from average male values of over 4 SDs for portions of ribs 9 through 11. With their uniform bone thickness, the THUMS M50’s Ct.Ar measures are relatively uniform across all ribs, leading to largest discrepancies with population Ct.Ar data towards the sternal ends of ribs 4–11.The THUMS M50 model ribs are uniformly larger in cross-section than average male ribs, with greatest discrepancy in Tt.Ar than average male data by approximately 1.5SDs across most ribs, and track population averages in Ct.Ar more closely on most rib regions. The greatest discrepancies in Ct.Ar tend to be seen at the sternal ends of all ribs.The VIVA + M50 model ribs are larger in Imax, Imin) being larger than expected for the THUMS M50 model across most ribs. This is true also for Imax in the VIVA + M50 model across ribs 2 through 9, but Imin values fall much closer to population averages across all ribs. Inertial moment discrepancies are most prominent when considering Irat, which reaches a ratio of over 10-to-1 towards the sternal end of average adult ribs, whereas corresponding regions of the HBMs reach only ratios of 4-to-1 in similar regions.Comparisons of the area inertial moments of these male HBM ribs to average male population data are shown in Tt.Ar) than average female ribs. With uniform cortical bone thickness definitions of 0.7 mm, these models also tend to have rib regions with greater Ct. Ar than a female population average, particularly in central regions of ribs 2-4 and towards the sternal ends of ribs 5–9. The GHBMC F05 model, on the other hand, is generally smaller in both Tt. Ar and Ct. Ar than an average female cohort by approximately 1SD for most ribs.Cross-sectional area comparisons between female HBM ribs and female population average measurements are shown in Imax and Imin inertial measures being larger than average female ribs for THUMS F05 and VIVA + F50 models, and Imax being smaller than average ribs for the GHBMC F05 model.Area inertial moment comparisons between average population data and female HBMs are shown in Irat) is generally lower than average population values for the THUMS F05 and GHBMC F05 models. Like for the male models, this discrepancy is particularly prominent towards the sternal end of ribs 2-4 which is highly elongated in females up to an average ratio of 18-to-1. Notably the VIVA + F50 model does stay primarily within a 1SD corridor across all rib regions, and reaches an Irat ratio of 10-to-1 at these elongated regions of ribs 2-4.Taken together, the ratio of inertial moments  of the two inertial moments, Imax and Imin. Population results here found a distinct elongation of ribs 2 through 5 towards their sternal ends producing a peak in Irat at 90% along the rib’s length of up to logn15 in males and logn18 in females . Most HBMs failed to reproduce this characteristic rib shape, with only the VIVA + models matched this aspect ratio along parts of their upper ribs, albeit with their Irat magnitudes increasing somewhat linearly along the whole rib’s shaft rather than following the exact change in ratio that was expected from population data. Taken together, these differences in Tt.Ar and Irat indicate that most HBM ribs can be overly large and overly round towards their sternal ends - a region in which rib fractures commonly occur  whereas the data presented here is average across the full male or female sampled population. This choice was made for a number of reasons. Firstly, true population variance in these specific measures is useful to quantify, and this would be lost if reporting only variance within the smaller subset. Secondly, inspection of measurements obtained from only subjects meeting average height and weight criteria found that their average values were highly similar to whole population averages. And finally, the inclusion of all subjects reduces the influence that any given outlier individual may have on overall average values.Despite providing cross-sectional data from the largest number of ribs and population available to date, the current study does not include data from rib 1 or rib 12 since each of these were excluded from the original cortical bone thickness study. The first rib has greater mechanical significance in most blunt trauma scenarios and efforts should be taken to characterize its shape and cross-sectional properties. Furthermore, the included population is sourced from a Midwestern United States adult population thus care should be taken when applying results to subjects from differing regions.This study has derived sex-specific corridors of cross-sectional geometric measurements along lengths of ribs 2 through 11 from an adult population of 240 subjects and over 4,000 ribs. Through comparisons to current HBMs we have identified key geometric differences that are likely to affect each models’ mechanical behavior. The reference data provided within this study can be used to alter these HBM ribs and improve their fracture prediction capabilities.


Considerable reductions in antimicrobial stewardship efforts have been attributed to the COVID-19 pandemic, especially among low-resourced healthcare facilities, such as long-term care settings. Our study objectives were to (1) assess the appropriateness of antibiotic prescriptions for urinary (UTI) and respiratory tract infections (RTI) using standardized infection definitions, known as the McGeer criteria, and (2) to determine whether the prescribing practices differed prior to and during the COVID-19 pandemic.We employed a HIPPA-compliant REDCap data collection tool to abstract Electronic Medical Record data from an Arizona-based, Skilled Nursing Facility between March 2019 and January 2022. Clinical and microbiologic infection characteristics were abstracted to determine whether indications for antibiotic prescriptions met the McGeer criteria for UTI and RTI. The frequency and proportion of antibiotic prescriptions for suspected infections that did not meet McGeer criteria were compared for the time period prior to and during the COVID-19 pandemic.388 total antibiotic events prescribed for UTI and RTI infections between March 2019 – January 2022 were analyzed . 61% (n=14) and 78% (n=154) of UTI prescriptions did not meet McGeer criteria prior to and during the COVID-19 pandemic, respectively. 70% (n=14) of RTI antibiotic prescriptions prior to the pandemic and 60% (n=89) of antibiotic events during the pandemic were not in accordance with McGeer criteria (Table 1) .Flow diagram to demonstrate Electronic Medical Record abstraction analysis inclusion criteriaNote: a suspected infection was defined as a laboratory order , or an antibiotic prescriptionFrequency and percent of UTI and RTI antibiotic prescriptions that did not meet McGeer criteria, prior to and during the COVID-19 pandemicNote: suspected infections from March 01, 2019 - January 25, 2020 are considered “pre-pandemic,” and January 26, 2020 – January 23, 2022 are considered “during pandemic” *The McGeer criteria differentiate infection definitions based on the presence or absence of an indwelling catheterAntibiotic prescription accordance with McGeer criteria from March 2019 – January 2022Despite consorted efforts to improve antimicrobial stewardship infrastructure, the majority of antibiotic prescribing for UTIs and RTIs prior to the pandemic did not meet McGeer criteria. This trend also persisted during the pandemic, indicating a need for increased stewardship practices that can operate concurrently with future public health threats.All Authors: No reported disclosures


Graphene-induced energy transfer (GIET) is a recentlydevelopedfluorescence-spectroscopic technique that achieves subnanometric opticallocalization of fluorophores along the optical axis of a microscope.GIET is based on the near-field energy transfer from an opticallyexcited fluorescent molecule to a single sheet of graphene. It hasbeen successfully used for estimating interleaflet distances of singlelipid bilayers and for investigating the membrane organization ofliving mitochondria. In this study, we use GIET to measure the cholesterol-inducedsubtle changes of membrane thickness at the nanoscale. We quantifymembrane thickness variations in supported lipid bilayers (SLBs) asa function of lipid composition and increasing cholesterol content.Our findings demonstrate that GIET is an extremely sensitive toolfor investigating nanometric structural changes in biomembranes. In particular, MD simulations predictedthat for bilayers constituted of unsaturated lipids , the addition of Cholfirst increases membrane thickness but then decreases it again beyonda Chol concentration of around 35 mol %.23 SANS did not show any difference in thickness for pure DOPC bilayerswithout and with 44 mol % Chol,18 whileAFM did not find any systematic trend in the thickness to Chol concentrationrelationship.19 In contrast, X-ray studies17 saw a monotonic increases of bilayer thickness with increasing Cholconcentration for multiple stacks of DOPC bilayers. It should be notedthat biological plasma membranes contain predominantly highly unsaturatedlipids,24 so the influence of Chol on theproperties of such membranes is particularly important. Complementarily,fluorescence spectroscopy has been widely used to study membrane dynamics,25 but it is not easy to see with structural variationson the length scale of membrane thickness (∼5 nm). For thatpurpose, one has to use Förster resonance energy transfer (FRET)spectroscopy,26 which requires labelingwith two different fluorescent probes, but even then, measuring membranethickness values with angstrom resolution remains challenging. Onereason is the difficulty of making FRET measurement of length valuesquantitatively accurate by taking into account all the complexityof a FRET experiment such as possible direct excitation of acceptor,bleed-through of emission from donor, relative sensitivity of detectionat different wavelengths, and in particular the usually unknown relativeorientation of donor and acceptor molecules with respect to each otherand the studied system.Lipid membranes are one of thefundamental building blocks of cells and form the division betweena cell’s interior and its environment and between differentsubcellular compartments . They are composed of lipid bilayers that are decorated withtransmembrane and peripheral proteins which are involved in a myriadof different processes and functions . The lipidbilayers themselves are made of amphipathic phospholipid molecules.Among them, cholesterol (Chol) is most abundant (∼30 mol %)and plays a crucial role in defining a membrane’s architectureand dynamics.28 imaging/spectroscopy. This method is based on the strongly distance-dependentquenching of a fluorescent molecule by a single planar sheet of graphene.This leads to a strong modulation of a fluorophore’s intensityand fluorescence lifetime as a function of its distance from the graphene,with full quenching directly on the surface of the graphene and nearlyfull recovery of bulk fluorescence properties at around 25 nm awayfrom the graphene. Thus, over a range of ca. 25 nm, one can use thegraphene-induced quenching for measuring a distance of a fluorophoreto the graphene with extreme accuracy by simply measuring its fluorescencelifetime and converting this value into a distance 27 and by mappingthe activity-dependent organization of mitochondrial membranes.31 In the current work, we use GIET for quantifyingthickness variations in SLBs as a function of Chol content with subnanometerresolution, which is impossible to do with other fluorescence-spectroscopictechniques.An alternative for measuring distancesalong one direction withangstrom resolution is graphene-induced energy transfer (GIET) and ref ). GIET w2) using chemical vapordeposition (see “Substrate preparation” in the Supporting Information). SLBs with few fluorescentlylabeled lipids (Atto655 headgroup labeling) are prepared using vesiclefusion (see the Supporting Information “Samplepreparation”). From the solution of Maxwell’s equations,one can then calculate the total emission power of the emitter whichis assumed to be proportional to the radiative transitionrate form the dye’s excited state to its ground state. Knowingalso the fluorescence lifetime τ0 and quantum yieldϕ of the dye in free space (with no GIET substrate or sample)allows then to compute the final GIET calibration curve (for detailssee the section “Working principle of GIET” in the Supporting Information). One additional detailthat has to be taken into account is that fluorescent dyes have broademission spectra and that the dielectric properties of graphene  are wavelength-dependent, whichis taken into account by computing the emission rate as a functionof wavelength and then averaging the result with the emission spectrumas weight function. Finally, the result depends also on the relativeorientation of the dye with respect to the GIET substrate, so thatthis orientation has also to be known a priori andthen used in the calculations. For the Atto655 dye used in this study,we have determined all these parameters in separate measurements andfound the values τ0 = 2.6 ns and ϕ = 0.36 aswell as a fluorophore orientation parallel to the membrane surface.27 The emission spectrum of Atto655 and the graphenedispersion curve required for the calculations are shown in not affect the GIET calibrationcurve for dyes above the SLB—these curves are indistinguishablefrom the GIET calibration curve in the absence of a SLB .These lifetime values were then converted into distance values usingthe precalculated GIET calibration curves.For determining values of bilayer thickness, fluorescencedecaycurves of fluorescently labeled SLBs on GIET substrates were recordedat 20 °C by scanning areas of 5 × 5 μmlong, correspondingto the dyes labeling lipid head groups in the top leaflet, were convertedinto height values ztop by using the “nolipid/lipid below” curve shown in short, corresponding to dyes in the bottomleaflet, into height values zbottom, wehad to use an iterative procedure. In that case, the GIET calibrationcurve zbottom = gbottom that relatesa measured lifetime τshort to the height value zbottom depends on the SLB thickness d itself, which is calculated as the difference betweenthe height value ztop of dyes above theSLB minus the height value zbottom ofdyes below the SLB. The height value ztop is already known from the conversion of the long lifetime componentτlong. Thus, zbottom isfound by solving the implicit equation zbottom = gbottom with d = ztop – zbottom. This is done by iterativelyevaluating zbottom = gbottom startingwith d = 0 and then updating the value of d with any new value of zbottom. After five iterations, the results for zbottom and d do not change anymore. The resulting heighthistograms for the lifetime data shown in The long lifetime components τsn-glycero-3-phosphocholine  and 1,2-diphytanoyl-sn-glycero-3-phosphocholine , and the thirdSLB was prepared with the monounsaturated fatty acid DOPC. For allthree SLBs, we performed measurements for increasing Chol contentof 0, 15, 30, and 44 mol %. DPhPC consists of saturated branched chainhydrocarbons and is mainly found in archaebacterial membranes. DPhPCis known to readily form bilayers32 andhas been used for preparing model membranes in previous studies.34For studying the dependence of SLBthickness on Chol concentration,we chose three different lipids for preparing SLBs. The first twoSLBs were made with the saturated fatty acids 1,2-dilauroyl-Supporting Information. The obtained thickness values areshown in We determined the height and resulting thickness values forthe12 SLBs prepared from the three lipids DOPC, DLPC, and DPhPC and forfour Chol concentrations of 0, 15, 30, and 44 mol %. The determinedheight values are displayed in An important result of our GIET-based thicknessmeasurements isthat we consistently achieve a measurement accuracy of ca. 0.5 nm(as estimated from the standard deviations). The exception is theDPhPC SLB with 44 mol % Chol showing a standard deviation of 1 nm.We attribute this to SLB heterogeneity which cannot be spatially resolvedby the diffraction-limited lateral resolution of our confocal scanningmicroscope but which leads to variations in the determined fluorescencelifetime values.35 Moreover, literature values doalso suggest a nonlinear increase in bilayer thickness of DLPC SLBwith increasing molar fractions of Chol,18 in accordance with our current findings. It should be mentionedthat the length of the linker between dye and lipid headgroup as wellas the size of the dye itself increases the apparent bilayer thicknessas measured by our method, so that our values are systematically largerby ca. 5 Å than those determined by SAXS and SANS, which measurethe direct lipid headgroup to headgroup distance. Literature valuesof bilayer thickness for Chol-free DPhPC SLBs range from 3.6 to 4.4nm,38 in good agreement with our value of 4.3 ± 0.4nm. However, no literature reports for Chol-containing DPhPC SLBsare available. The most interesting behavior shows DOPC SLBs, withan increase of bilayer thickness for small Chol concentrations anda decrease at large Chol concentrations. These results are in goodagreement with previously reported computational studies,23 showing a similar trend in thickness variation of DOPC SLB as afunction of Chol content . Furthermore, the thickness values for Chol-freeDOPC SLBs agree well with literature values obtained by using atomicforce microscopy39 and with our previousmeasurements using GIET.27Our obtained thickness value for a Chol-freeDLPC SLB is in excellentagreement with previous findings from X-ray scattering and independentGIET measurements.d) phase, where the hydrocarbon tailsof lipids are loosely packed and randomly oriented. Adding Chol toan SLB induces the formation of a liquid-ordered (Lo) phasethat will grow in size with increasing Chol concentration (“condensingeffect”).6 In the Lo phase, hydrocarbontails are extended and more closely packed, and Chol condensing leadsto a decrease of area per lipid that is considerably more pronouncedthan one would expect for the case of ideal mixing, where the areaper molecule would be a weighted average of Chol and lipid areas.Abovethe phase-transition temperature, SLBs are supposed to exhibita liquid-disordered . For SLBs containing unsaturated lipids, a recentstudy using neutron spin-echo (NSE) spectroscopy, solid-state nuclearmagnetic resonance (NMR) spectroscopy, and molecular dynamics (MD)simulations has shown that Chol stiffens the membrane by increasingits lipid packing.7 Another atomistic MDsimulation of DOPC membranes reported an initial increase of membranethickness with increasing Chol concentration, followed by a decreasefor Chol concentrations above 35 mol %.22 The explanation is that in DOPC membranes acyl chains are much lessordered than in membranes containing high concentrations of saturatedlipids.44 When adding Chol, thisleads first to a reduction of the lipids’ tilt angle for accommodatingmore space to incorporate Chol,22 whichis later counteracted by reducing the order of lipid tails at highcholesterol concentrations.23 Thus, onefirst observes an increase of bilayer thickness at moderate Chol concentrations,followed by thickness reduction beyond a critical Chol concentration(thinning effect). Our results show that the transition from thickeningto thinning occurs between 30 and 44 mol % Chol concentration. Furthermore,our results may answer the question why almost no Chol-related thicknesschanges are observed for the plasma membranes (PM) of cells45 because these membranes contain predominantlyhighly unsaturated lipids,24 so that thePM thickness under physiological conditions (Chol ∼25–43mol %) is the same as after Chol depletion.Another effect of Chol on membrane organization is that it reducesthe tilt angle of lipid backbones (with respect to the direction perpendicularto the membrane) and thus increases the interleaflet distance betweenlipid headgroups.To summarize, GIETis a powerful fluorescence-spectroscopic toolfor membrane biophysics. We demonstrated this here by studying theimpact of Chol on membrane thickness, showing that GIET can easilyresolve changes in membrane thickness with an accuracy of ca. 5 Å.An important advantage of GIET is its experimental simplicity, requiringonly a conventional fluorescence lifetime imaging microscope (FLIM).Potential further applications of GIET in membrane biophysics canbe the precise localization of membrane-associated proteins (or otherbiomolecules) with respect to a membrane or the study of membranedynamics by combining GIET with fluorescence correlation spectroscopy.