Biomarkers were selected via Least Absolute Shrinkage and Selection Operator (LASSO) regression modeling and machine learning analysis. A combination of five proteins from the proteomic layer was best at discriminating resolvers from non-resolvers from critical illness with an Area Under the Receiver Operating Characteristic curve (AUC) of 0.74, while 26 multi-omic features predicted 30-day survival with an AUC of 0.77. Patients with traumatic brain injury as part of their injury complex had a unique subset of features that predicted 30-day survival. Our findings indicate that multi-omic analyses can identify novel admission-based prognostic biomarkers for outcomes in trauma patients. Unique biomarker discovery also has the potential to provide biologic insights.Admission-based circulating biomarkers for the prediction of outcomes in trauma patients could be useful for clinical decision support. It is unknown which molecular classes of biomolecules can contribute biomarkers to predictive modeling. Here, we analyzed a large multi-omic database of over 8500 markers  to identify prognostic biomarkers in the circulating compartment for adverse outcomes, including mortality and slow recovery, in severely injured trauma patients. Admission plasma samples from patients ( Trauma is one of the leading causes of human morbidity and mortality worldwide, resulting in both short- and long-term health impairment and a significant economic burden . The cliAdvances in prehospital patient management and inpatient hospital care at designated trauma centers have increased 30-day survival in trauma patients. This improved survival is associated with a concomitant increase in the percentage of patients who remain critically ill after trauma ,6. GivenLarge-scale, admission-based multi-omic profiling has the potential to identify unique prognostic biomarkers and point to biologic processes associated with patient outcomes. A deep analysis of a high dimensional multi-omic dataset derived from a clinical trial  that demonstrated a survival benefit of prehospital thawed plasma was previously reported ,10, as wn = 112 for PAMPer and n = 93 for STAAMP) were obtained upon emergency department admission (0 h (h) timepoint). Patients transported by air ambulance were enrolled if they had tachycardia (defined as a heartrate > 108 beats per minute) and experienced at least one episode of hypotension (defined as systolic blood pressure < 90 mm Hg) or if they experienced severe hypotension (defined as systolic blood pressure < 70 mm Hg). Patients from the PAMPer dataset were used to train our models, which were validated for proteomics using patients from the STAAMP clinical trial. Further information on enrollment criteria for the PAMPer and STAAMP trials have been published [Human plasma samples .This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board (IRB) of the University of Pittsburgh . The PAMPer Trial received approval through the Emergency Exception from Informed Consent (EFIC) protocol from the Human Research Protection Office of the U.S. Army Medical Research and Material Command. Detailed information and study protocol for the PAMPer Trial are available through the following link: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4623322/#S3title (accessed on 1 June 2022).The Study of Tranexamic Acid During Air Medical and Ground Prehospital Transport (STAAMP) Trial enrolled adult trauma patients that had at least 1 episode of hypotension (systolic blood pressure < 90 mm Hg) or tachycardia (heart rate 110 bpm or greater). This study received approval through the EFIC protocol by the U.S. Food and Drug Administration as well as a community consultation process under the investigational new drug application (IND) protocol (IND # 121102). Detailed information and study protocol for the STAAMP Trial are available through the following link: Untargeted lipidomic and metabolomic assays (898 metabolites and 997 lipids) were conducted by Metabolon ; detailed information can be found in our previous analysis ,11. BrieAn aptamer-based, multiplexed method was used to measure protein levels  for 84 patients included in this study. Further details on the specific techniques are found in .https://data.mendeley.com/datasets/vt8nhp2y2t/1 (accessed on 1 June 2022).Missing data were less than 10% across all variables and the mean value for each variable was used to fill for any missing data points. Other methods of missing data imputation did not affect the data due to the low number of missing data. A prescreening of the biomarkers was conducted to reduce each layer  to the same number of variables based on median and quartiles to filter the most variable markers between patient groups. The bottom quartiles were removed based on abundance and the top 10% most variable markers between groups using R were kept. Each omic layer consisted of approximately 550 biomarkers after this prescreening, as shown in Several statistical and predictive models (Receiver Operating Characteristic (ROC) Curve, generalized linear model with Least Absolute Shrinkage and Selection Operator (LASSO) regression, support vector machine, random forest) were used to evaluate the predictive nature of biomarkers on either the individual layer scale or on the multi-omic platform for a binary outcome ). The R Caret package was used to train the data with a 10-fold cross validation. A Mann–Whitney U-test was incorporated to rank the significance and contribution of each biomarker during the analysis for both support vector machine and random forest methods of machine learning analysis along with a Benjamini–Hochberg correction with a false discovery rate of less than 5%.Linear regression with LASSO regularization was tuned by 6 different lambda  values ranging from 0.5 to 1 by 0.1 increments, making sure only balanced lambda coefficients were chosen to represent the model to avoid over- and under-fitting errors. These regularized data were used to train the support vector machine and random forest models.https://go.princeton.edu/cgi-bin/GOTermMapper) (accessed on 1 June 2022).GSEA was carried out using the proteins’ EntrezGeneID and the Princeton GO term site (The regularized data were used to train two different separate methods of machine learning (support vector machine and random forest). For each outcome tested , these models were repeated 20 times with 10-fold cross validation at each lambda value tested. The performance of each model was examined by calculating both the accuracy and Area Under the ROC Curve (AUC) of each model. The layers  were analyzed to determine which had the best predictive potential based on highest AUC and accuracy. For 30-day survival and TBI, the multi-omic predictive model functioned best with 84 patients and approximately 1650 biomarkers per run. For prediction of resolving status, the proteomic layer performed best with 84 patients and 550 biomarkers.To validate the accuracy of the models, proteomic data from the standard-of-care arm patient cohort derived from the STAAMP study  were usen = 112), a prospective trial that enrolled severely injured trauma patients transported to trauma centers via air ambulance [n = 93) [The admission circulating multi-omic data from patients in the standard-care arm of the PAMPer Trial  . The mul[n = 93) . Omic da[n = 93) , quantit[n = 93) .n = 25) vs. non-resolvers . The second clinical outcome was 30-day survival with 55 survivors and 57 non-survivors. Finally, we assessed 30-day survival in patients with TBI . Non-fasting healthy controls  were included and described in previous metabolomic and lipidomic studies [n = 29, ISS score of 1 or less) were from the STAAMP trial and were used for controls in the proteomic analysis.Patients in the primary cohort were segregated based on three outcomes as outlined in  studies ,11, whilFirst, multivariate machine learning analysis was used to determine which individual data layer provided the most predictive features for outcomes. This was then compared to a multivariate statistical model that combined the data layers of all three omics. LASSO-based feature selection was used to classify the down-selected features. To prevent over-fitting, L1-regularization and cross-validation were performed in every run. L1-regularization was used for this high-dimensional dataset as the number of features was higher than the number of sums. The performance of the models (using AUC) was measured in a k-fold cross-validation framework with permutation testing, as described previously ,18,19. TLASSO identified a minimal set of five proteins  that werWe next characterized the temporal patterns of the five protein biomarkers based on additional sampling time points of 24 and 72 h B. ActivaTo gain a more comprehensive understanding of the entire spectrum of features tracking with outcome, we constructed correlation networks, as described previously, around the LASSO-selected biomarkers ,18,19. TActivated protein C is an anti-coagulant that degrades the inactivated forms of coagulation factors V and VIII. Activated protein C also has anti-inflammatory and anti-apoptotic properties . Strong,Endophilin-B2 showed strong associations with ubiquitin carboxyl-terminal hydrolase 15, a key regulator of TGF-β receptor signaling, as well as serine/threonine-protein kinase that plays a role in cell survival via the caspase-3-dependent pathway . Associan = 46) than non-resolvers (n = 18).To validate our findings, we carried out a cross-prediction analysis using data from patients in the STAAMP clinical trial. These five proteins collectively predicted resolution of critical illness within 7 days in the STAAMP dataset with an AUC of 0.81 D. Of notp-value < 0.03.For 30-day survival, features identified from the combined multi-omic database were the most predictive of outcome with an AUC of 0.77 . LASSO iRelative values of all the selected features were projected for each layer in heatmaps C,D compaTo assess the prognostic capability of admission and multi-omic markers relative to clinical variables, we ran the same models but incorporated clinical variables into the model, including age, ISS, BMI, and other factors . ISS andn = 17) and low ISS controls over time (n = 29). For metabolomics and lipidomics, 27 survivors and 37 non-survivors were included, while 21 and 28 were included for proteomics, respectively. All the biomarkers were significantly higher (p < 0.05) in non-survivors except for PC.14:0/18:2  and protein SGT1 homolog (SUGT1), were correlated to two of the selected features, namely HEBP1 and Poly(rC)-binding protein 1 (PCBP1). IF1AY and SUGT1 both have functions related to protein biosynthesis and degradation ,29. PCBPTaken together, these analyses show that the addition of TBI to the injury complex impacts the features that predict survival.Gene set enrichment analysis (GSEA) was carried out between the outcome subgroups . p < 0.05) higher in resolvers, and coagulation factor VIII was highest in 30-day survivors. On the other hand, activated protein C (ApC) was significantly higher in both 30-day non-survivors and non-resolving patients, as shown by the tests of significance from the LASSO-selected features.The “hallmark coagulation” gene set shown in We also identified the “hallmark interferon-alpha response” gene set, comprised of 97 genes that are upregulated in response to alpha interferons  in 30-daThis study was undertaken to identify predictive biomarkers using high-dimensional multi-omic datasets derived from admission blood draws. By utilizing the standard-of-care arm patients from two recent interventional trials we were able to take advantage of patients enrolled across multiple institutions to better understand the features of severely injured humans. A multivariate machine learning model identified the combination of admission omics features that were most predictive for death within 30 days or resolution of critical illness. A consistent feature from both analyses was higher levels of activated protein C in patients destined for adverse outcomes. Gene enrichment analysis also strongly identified components of coagulation as correlative with patient outcomes. The inclusion of TBI in the injury complex altered the prognostic markers, indicating the polytrauma patients with TBI are likely to require unique models to predict outcomes.Trauma management has become standardized in order to have consistent and systematic care for patients by following guidelines such as the Advanced Trauma Life Support ,33. HoweThe GSEA and correlation network analysis yielded novel results with regards to biological pathways related to traumatic injury. The MYC target genes are reported to be involved in a large array of biological processes and have been studied in animals as regulators of energy metabolism in response to renal injury, particularly facilitating glycolysis . Our preIn patients with worse outcomes (non-survivors and non-resolvers), heme metabolism was enriched prominently compared to survivors and resolvers. The literature includes reports studying the relation between heme-related products and injury severity, especially in association with different arms of the immune system and brain recovery after stroke and TBI ,45,46,47The strengths of this study include a tightly controlled standard-care patient cohorts from the PAMPer clinical trial, along with available multi-omic databases. We included cross prediction and validation in an unrelated group of patients from another trauma clinical trial (STAAMP). The limitations include the inability to establish causality. The metabolomic layer uses qualitative ultra-performance liquid chromatography for measurement and the results should be validated with quantitative assays. Ultimately, the use of many of these biomarkers in the clinical setting would require validated assays. However, our findings can serve as a guide and resource to identify sets of biomarkers that combine predictive power and ease of measurement.


C) into infectious aggregates (PrPSc) causing fatal neurodegenerative diseases in humans and animals. Prions exist as strains, which are encoded by conformational variants of PrPSc. The transmissibility of prions depends on the PrPC sequence of the recipient host and on the incoming prion strain, so that some animal prion strains are more contagious than others or are transmissible to new species, including humans. Nor98/atypical scrapie (AS) is a prion disease of sheep and goats reported in several countries worldwide. At variance with classical scrapie (CS), AS is considered poorly contagious and is supposed to be spontaneous in origin. The zoonotic potential of AS, its strain variability and the relationships with the more contagious CS strains remain largely unknown. We characterized AS isolates from sheep and goats by transmission in ovinised transgenic mice (tg338) and in two genetic lines of bank voles, carrying either methionine (BvM) or isoleucine (BvI) at PrP residue 109. All AS isolates induced the same pathological phenotype in tg338 mice, thus proving that they encoded the same strain, irrespective of their geographical origin or source species. In bank voles, we found that the M109I polymorphism dictates the susceptibility to AS. BvI were susceptible and faithfully reproduced the AS strain, while the transmission in BvM was highly inefficient and was characterized by a conformational change towards a CS-like prion strain. Sub-passaging experiments revealed that the main strain component of AS is accompanied by minor CS-like strain components, which can be positively selected during replication in both AS-resistant or AS-susceptible animals. These findings add new clues for a better comprehension of strain selection dynamics in prion infections and have wider implications for understanding the origin of contagious prion strains, such as CS.Prions are infectious agents that replicate through the autocatalytic misfolding of the cellular prion protein , a sporadic prion disease of small ruminants. We found that AS was faithfully reproduced not only in a homologous context, i.e. ovinised transgenic mice, but also in an unrelated animal species, the bank vole. A natural polymorphism of the bank vole prion protein, coding for methionine (BvM) or for isoleucine (BvI) at codon 109, dictated the susceptibility of voles to AS, with BvI being highly susceptible to AS and BvM rather resistant. Most importantly, the M109I polymorphism mediated the emergence of AS-derived mutant prion strains resembling classical scrapie (CS), a contagious prion disease. Finally, by sub-passages in bank voles, we found that the main strain component of AS is accompanied by minor CS-like strain components, which can be positively selected during replication in both AS-resistant or AS-susceptible vole lines. These findings allow a better understanding of strain selection dynamics and suggest a link between sporadic and contagious prion diseases. Transmissible spongiform encephalopathies (TSEs), or prion diseases, are fatal neurodegenerative disorders of humans and animals. Although TSEs can occur as sporadic, acquired or inherited (genetic) forms, they have been historically defined based on their experimental transmissibility.In humans, most of TSEs have idiopathic  or genetic origin . However, those that have raised the most serious public concerns are the acquired forms, with zoonotic (variant Creutzfeldt Jakob disease) or iatrogenic origin (iatrogenic Creutzfeldt Jakob disease) .Animal TSEs present largely as acquired diseases: classical scrapie (CS) in small ruminants, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in cervids, and, likely, the recently discovered prion disease of dromedary camels , 3. HoweSc, the post-translational misfolded form of the host-encoded cellular prion protein (PrPC). In contrast to PrPC, PrPSc is partially resistant to protease digestion, insoluble in non-denaturant detergents and rich in β-sheet structure [C is fully hydrolysed while PrPSc is only partially cleaved at the amino terminal portion, leaving a PK-resistant core (PrPres), frequently characterized, in western blot analysis, by three bands corresponding to the unglycosylated, mono-glycosylated and di-glycosylated isoforms of PrPSc.Prion diseases are characterized by the accumulation in the central nervous system of PrPtructure . TypicalSc, while the phenomenon of prion strain variability, revealed by the existence of different phenotypes of the same disease, would depend on different PrPSc conformations [According to the protein-only hypothesis , the infrmations , 15.Sc and PrPC are of identical primary structure, as shown by several instances in which the barrier was overcome by replacing the PrP gene of the recipient with its counterpart from the donor [The transmission of prions between species is limited by the transmission barrier. Early studies argued that it resides in PrP primary structure differences between donor and recipient species. Many studies support the view that prion propagation proceeds most efficiently when the interacting PrPhe donor . Howeverhe donor , suggestIt has been proposed that prion populations are composed of a variety of conformational variants, or quasispecies; when the environment changes, the most efficiently replicating variants becomes the predominant components of the population, which then constitute a distinct sub-strain –20. In tSc.Although the gold standard for the characterization of prion strains is still based on bioassay in rodents, several studies demonstrated that prion strains can be partially distinguished based on different biochemical properties of PrPres [Sc [Sc during denaturation [The electrophoretic features of PrPres , 21, 22,res [Sc , 24, or turation –27 have res of around 27–30 kDa (PrPres 27–30), but atypical PrPres patterns have been described in both humans (Gerstmann-Sträussler-Scheinker disease and variably protease-sensitive prionopathy) and animals (AS) [Most prion diseases are characterized by PrPals (AS) –35.Sc deposition, the electrophoretic signature in western blot and the association with polymorphisms of the PrP gene [res from CS and AS cases implies that the PrPSc aggregates associated with these diseases have different conformations. Indeed, when treated with high concentration of PK, PrPSc from AS cases is cleaved at both N- and C-termini resulting in an atypical PK resistant core of ~8 kDa, in contrast to the classical, N-terminal cleaved PrPres of 27–30 kDa from CS cases [res from AS cases might also present as a multiband pattern, where the small N- and C-terminally truncated PrPres fragment is accompanied by at least two larger PrPres fragments [First recognized in Norwegian sheep in 1998 , AS has PrP gene , 41, 42.CS cases . By usinragments , 43.Reported mainly in aged sheep and goats, AS occurs with a sporadic distribution, compatible with a non-contagious, possibly spontaneous, origin , 44–46.Epidemiological evidences for natural transmission of AS are still lacking, although its transmissibility has been demonstrated experimentally in sheep  –50 and iMyodes glareolus), a wild type rodent model susceptible to a wide range of prion diseases [In the present study, we investigated the transmissibility of AS in bank voles  as previously described [Interestingly, the propagation of AS isolates in BvI was highly efficient, with 100% attack rate and apparent lack of transmission barrier, as showed by the absence of shortening of the survival times at the second and third passages . Furtherf ~8 kDa , cleavedf ~8 kDa , and by of 1.5 M . Thus, Bescribed , 34, 35.All AS isolates produced a similar distribution of vacuolar degeneration in the brain of BvI characterized by intense micro-vacuolar degeneration of the cerebellum, hippocampus, lateral septal nuclei, cerebral cortices , intense PrPeas Figs  and S2. eas Figs , stratumeas Figs , dorsal pocampus  and mediar layer . Intraceposition , 70, 71.res characteristics   , 72. In res accumulation within the life span of the voles  and PrPres 27–30 , which is in agreement with the presence of PrPres 27–30 at detectable levels in the mixed case, but not in bona fide AS. Overall, these findings suggest that the inefficient transmission of BvI-adapted bona fide AS to BvM depends on the inability of BvM to replicate AS, while the accompanying strain mutation reflects the presence of minor prion variants, along with bona fide AS, in AS-affected BvI, as also observed in sheep [How a single amino acid variation in bank vole PrP sequence could determine the susceptibility to AS prions was further investigated through the inoculation of BvI-adapted AS into BvM. The phenotype obtained was different from the AS strain observed in BvI, showing that BvM was not able to reproduce the original AS strain properties regardless of whether the inoculum derived from natural hosts or BvI. The transmissions of BvI-adapted bona fide AS (i.e. without detectable phenotype shift) and of the mixed case to BvM led to the emergence of similar, although not identical, mutant PrPs 284 vs  dpi, whiin sheep .Sc with atypical features similar to AS [Sc conformations characterised by N- and C-terminal cleaved PrPres types. In AS natural hosts, the key role of single amino acid residues in this PrP region is highlighted by the strong association of the disease with specific sheep and goat PrP polymorphisms, such as F141 and H154 [Bank voles are the first wild type rodents showing susceptibility to AS. The peculiar susceptibility of bank voles to a wide range of prion sources has been mainly ascribed to features of vole PrP rather than to the vole genetic background , 81–83. ar to AS , such asar to AS , 67, 84.and H154 . Interesand H154 –89.Sc conformations, with a main component characterized by 8 kDa PrPres and minor components by PrPres 27–30. Although it is a minor component, PrPres 27–30 can still play an important role as it might be endowed with the contagiousness that is typical of CS and can be positively selected under specific circumstances, as we have shown here in BvI. Recent data showing that classical BSE prions are present in AS isolates led to suggest that AS cases might be a possible source of classical BSE prions [In conclusion, we show that BvI are uniquely susceptible to AS and are the first available wild type rodent model for experimental studies with this prion strain. The resistance of BvM to AS and the emergence of CS-like strains in this bank vole genetic line can be explained in the context of the conformational selection model, supporting evidences that AS isolates are composed of an ensemble of prion strain components, i.e. PrPE prions . Thus, tExperiments involving animals adhered to the guidelines contained in the Italian Legislative Decree 116/92, which transposed the European Directive 86/609/EEC on Laboratory Animal Protection, and then in the Legislative Decree 26/2014, which transposed the European Directive 2010/63/UE on Laboratory Animal Protection. The experimental protocols were approved and supervised by the Service for Biotechnology and Animal Welfare of the Istituto Superiore di Sanità, and were authorized by the Italian Ministry of Health (decree numbers 84/12.B and 1119/2015-PR).Bank voles carrying methionine (BvM) or isoleucine (BvI) at the polymorphic PRNP codon 109 and tg338 transgenic mice that overexpress the VRQ allele of ovine PrP were obtained from the breeding colony of Istituto Superiore di Sanità (ISS).Sc biochemical properties [Nor98/atypical scrapie (AS) isolates from sheep and goat were identified through active or passive surveillance programs in Italy and Norway. Most of the AS isolates were previously characterized for their PrPoperties .DNA extraction, PCR amplification and sequencing were carried out as previously reported . BrieflySc and half fixed in formol-saline for histological analysis. The attack rate was calculated as the number of animals scoring positive at post-mortem/number inoculated. For primary transmissions, animals found dead or culled for intercurrent disease before 200 dpi and scoring negative at postmortem were excluded from analyses. The survival time for animals scoring positive at post-mortem was calculated as the time from inoculation to culling or death.For the preparation of the inocula, brain tissues from TSE affected animals were homogenized at 10% (w/v) in phosphate buffered saline (PBS) and stored at -80°C. Groups of six-to-eight week old rodents were inoculated intracerebrally with 20μl of homogenate into the left cerebral hemisphere, under ketamine anaesthesia (ketamine 0.1μg/g). All animals were individually identified by a passive integrated transponder. Animals were examined twice a week until neurological signs appeared, after which they were examined daily. Animals were culled with carbon dioxide before neurological impairment was such as to compromise their welfare, in particular their ability to drink and feed adequately. At post-mortem, brains from inoculated mice were removed and divided sagittally. Half brain was frozen for WB analysis of PrPBrain homogenates (20% w/v) were prepared as previously described .After adding an equal volume of 100 mM Tris-HCl containing 4% sarkosyl, the homogenates were incubated for 30min at 37°C with gentle shaking. Proteinase K (Sigma-Aldrich) was added at a final concentration of 50 μg/ml and then the samples were incubated for 1 h at 55°C with gentle shaking. Protease treatment was stopped with 3mM PMSF (Sigma-Aldrich). Aliquots of samples were added with an equal volume of isopropanol/butanol (1:1 v/v) and centrifuged at 20,000 g for 5min. Supernatants were discarded and the pellets were resuspended in denaturing sample buffer  and heated for 10 min at 90°C.After electrophoresis on 12% bis-Tris polyacrylamide gels (Invitrogen) and WB on polyvinylidene fluoride membranes using the Trans-Blot Turbo Transfer System (Bio-Rad), the blots were processed with anti-PrP mAbs by using the SNAP i.d. 2.0 system (Millipore). Membranes were probed with mAbs SAF84 , 9A2 (aa 102–104) or 12B2 (aa 93–97). The PrP was visualized by enhanced chemiluminescent substrate  and the ChemiDoc imaging system (Bio-Rad). The chemiluminescent signal was quantified by Image Lab software (Bio-Rad).Sc in brain homogenates from bank voles was analysed by CSSA as previously described [Sc (GdnHCl1/2). Individual denaturation curves were analyzed and best-fitted by plotting the fraction of PrPSc remaining in the pellet as a function of GdnHCl concentration, and using a four parameter logistic equation (GraphPad Prism).The conformational stability of PrPescribed . BrieflyHistopathology and immunohistochemistry were carried out on formalin-fixed tissues as previously described . BrieflyS1 Figres from brains of BvI inoculated with classical (CS) or atypical scrapie (AS). Replica blots were probed with 12B2, 9A2 and SAF84 mAbs. The positions of MW markers are indicated on the left of the blot .Representative blots of PrP(TIF)Click here for additional data file.S2 FigSc deposition patterns observed in hippocampus and red nucleus of BvI and BvM inoculated with AS and with AS-passaged in BvM and BvI, as indicated. Note that AS in BvI showed a fine punctate pattern in the alveus and stratum lacunosum-moleculare (Slm) of CA1 and no labeling in the red nucleus, while AS in BvM was characterized by intraglial and intraneuronal PrPSc depositions in the red nucleus, accompanied by diffuse and punctate immunolabeling in the neuropil. PrPSc deposits in BvM inoculated with BvI-passaged AS were mainly intraglial and intraneuronal in the hippocampus and red nucleus, accompanied by punctate, granular and plaque-like deposits in the red nucleus. Finally, PrPSc deposits in BvI inoculated with BvM-passaged AS overlapped with those observed in BvM inoculated with AS, mainly characterized by by intraglial and intraneuronal PrPSc depositions.PrP(TIF)Click here for additional data file.S3 Figres from brains of tg338 infected with AS from sheep (AS-Sh) or with BvI-adapted AS (AS-BvI). (C) Lesion profiles in groups of BvI infected with AS natural isolates or with tg338-adapted AS (Sh-I2 tg338).(A) Lesion profiles in groups of tg338 mice infected with AS natural isolates and BvI-passaged AS (Sh-N3 BvI). (B) Representative western blot showing PrP(TIF)Click here for additional data file.S4 FigSc from brains of BvM infected with AS isolates Sh-N1 and Sh-N2. As with the others AS isolates, most BvM were negative. The positive BvM shown is one of the two only BvM, both inoculated with Sh-N2, resulting in positive transmission. Note that PrPSc in BvM inoculated with AS is characterized by PrPres 27–30, similar to that in BvM inoculated with CS (control lanes) and different from AS in small ruminants and BvI. Membrane was probed with SAF84 mAb. The positions of MW markers are indicated on the left of the blot .Representative western blot showing PK-resistant PrP(TIF)Click here for additional data file.S5 Figres from the brain of individual BvI inoculated with Sh-N3 (second passage), Sh-N3 (third passage) or heathy controls, as indicated. The BvI from the second passage of Sh-N3 shows the classical 27–30 PrPres accompanied by small amount of the 8 kDa PrPres (indicated with the arrow in long exposure blot on the right). Membrane was probed with 12B2 mAb. (B) Immunohistochemical analysis of the mixed case highlights the simultaneous presence of intracellular PrPSc deposits in CA1, characteristic of BvM inoculated with AS, and fine punctate deposits in the alveus, characteristic of BvI inoculated with AS.(A) Western blot of PrP(TIF)Click here for additional data file.


Five biologics, targeting different effectors in the type 2 inflammatory pathway, are approved for asthma treatment. However, biologics have not been compared against each other or add-on inhaled therapies in head-to-head clinical trials. As a result, their positioning versus that of current and anticipated small-molecule strategies is largely unknown. Furthermore, with the emergence of biomarkers for predicting response to biologics, a more personalized treatment approach—currently lacking with inhaled therapies—may be possible. To gain perspective, we reviewed recent advances in asthma pathophysiology, phenotypes, and biomarkers; the place of biologics in the management and personalized treatment of severe asthma; and the future of biologics and small-molecule drugs. We propose an algorithm for the stepwise treatment of severe asthma based on recommendations in the GINA strategy document that accounts for the broad range of phenotypes targeted by inhaled therapies and the specificity of biologics. In the future, both biologics and small molecules will continue to play key roles in the individualized treatment of severe asthma. However, as targeted therapies, their application will continue to be focused on patients with certain phenotypes who meet the specific criteria for use as identified in pivotal clinical trials.Globally, a small proportion (5–12%) of asthma patients are estimated to have severe disease. However, severe asthma accounts for disproportionately high healthcare resource utilization. The Global Initiative for Asthma (GINA) management committee recommends treating patients with asthma with inhaled corticosteroids plus long-acting βThe online version contains supplementary material available at 10.1186/s13223-022-00676-0. The Global Initiative for Asthma (GINA)  cells), or innate lymphoid cells type 2 (ILC2s), which are also a major source of type 2 cytokines , 12. ILC2 (PGD2) pathway is also involved in T2 inflammation ), asthma control, and asthma-related QoL to varying degrees, although only reslizumab and dupilumab were found to significantly reduce asthma exacerbation rates [Head-to-head clinical trials between biologics are lacking and indirect comparison of biologics has provided conflicting results, presumably because patient populations are not comparable across studies with respect to demographics and clinical characteristics, such as age, lung function, and eosinophil counts. In a network meta-analysis, benralizumab, dupilumab, mepolizumab, and reslizumab were associated with improvements in lung function  should be considered as a rescue medication in adults with severe asthma despite medical therapy at GINA Step 5 . With brIL-25 and IL-33 hold potential as upstream targets for the treatment of asthma. Although no biologics under investigation directly target IL-25, two anti-IL-33 antibodies, i.e., REGN3500 and ANB020 or etokimab, are under investigation , 91.2 [2 are involved in allergic inflammation [Various small molecules targeting specific inflammatory pathways (T2 or non-T2) are also being evaluated  . NotablyIf all recommended inhaled and oral therapies are ineffective, phenotyping is recommended, and biomarker screening tests should be considered to determine the most appropriate step-up therapy using a shared decision-making approach with the patient. In the absence of direct clinical comparisons of biologics, the choice of biologic may be determined by the physician based on specific biomarkers underlying patient asthma phenotypes. Bronchial thermoplasty should be considered in appropriate patients as well .Asthma is a chronic disease comprising multiple clinical and inflammatory phenotypes. Besides medium- or high-dose ICS + LABA, tiotropium and several biologics, tailored toward specific inflammatory phenotypes, are approved as add-on therapies for treatment of severe asthma. Before considering the use of biologics, add-on inhaled therapies, such as LAMAs, may provide scope for improvement in asthma control owing to their comparatively low cost. Notably, the on-going development of new biologics and small molecules may pave the way for targeted treatments of patients with severe asthma with an appropriate phenotype.Additional file 1: Video S1. Overview of the pathophysiology of asthma, mechanism of action of available inhaled therapies and biologics, and the proposed treatment algorithm for severe asthma.Additional file 2: Table S1. Small molecules in asthma under investigation.


I2 = 73·9%). The risk of stillbirth was significantly lower in the vaccinated cohort by 15% . There was no evidence of a higher risk of adverse outcomes including miscarriage, earlier gestation at birth, placental abruption, pulmonary embolism, postpartum haemorrhage, maternal death, intensive care unit admission, lower birthweight Z-score, or neonatal intensive care unit admission . COVID-19 mRNA vaccination in pregnancy appears to be safe and is associated with a reduction in stillbirth.Safety and effectiveness of COVID-19 vaccines during pregnancy is a particular concern affecting vaccination uptake by this vulnerable group. Here we evaluated evidence from 23 studies including 117,552 COVID-19 vaccinated pregnant people, almost exclusively with mRNA vaccines. We show that the effectiveness of mRNA vaccination against RT-PCR confirmed SARS-CoV-2 infection 7 days after second dose was 89·5% (95% CI 69·0-96·4%, 18,828 vaccinated pregnant people,  Pregnant women have been disproportionately under-vaccinated against COVID-19, partly because they were excluded from initial trials. This systematic review and meta-analysis supports efficacy of vaccination in pregnancy, and finds no evidence of adverse maternal or perinatal outcomes. One under-vaccinated group is pregnant people. Exclusion of pregnant people from initial COVID-19 vaccine trials, lack of experience with mRNA vaccine platforms outside research settings in this group, and resultant variable and ambiguous guidance on vaccination from official and professional bodies, as well as antivaccine disinformation, contributed to vaccine hesitancy among pregnant people8.With the first COVID-19 vaccination trials reporting the effectiveness of vaccination against COVID-19 in December 202010. In the UK, the rate of hospital and ICU admission and the associated co-morbidities has increased with each wave11. Data from the UK Obstetric Surveillance System (UKOSS) indicate that the overwhelming majority of pregnant people who required hospitalization or ICU care for COVID-19 during the delta wave were unvaccinated12. With the emergence of the Omicron variant, the pandemic appears to be far from over and the urgent need for vaccination of pregnant people cannot be overemphasised.SARS-CoV-2 infection in pregnancy can have devastating effects, with evidence showing increased rates of admission to hospital and intensive care units (ICU), maternal death, stillbirth, pre-eclampsia and preterm birth17. Clinical trials are underway to investigate the outstanding questions about COVID-19 vaccination in pregnancy, including the optimal dosing schedule, and the duration and efficacy of antibodies transferred to the neonate transplacentally and in breastmilk18. There is an immediate need for high-quality robust information to support pregnant people considering COVID-19 vaccination, pending additional updates from large national registries and the results of ongoing trials. We conducted a systematic review and meta-analysis of published data on the effects of COVID-19 vaccination in pregnancy, and on vaccine effectiveness in pregnancy.Emerging data from individual observational studies and large case series are consistent with pre-clinical studies that suggested that COVID-19 vaccines have no adverse effects on pregnancy or neonatal outcomes34 and 5 randomised trials37, reporting on 117,552 people vaccinated during pregnancy . Dagan et al. 23 and Goldshtein et al. 24 both used national-level data in Israel; the sampling period coincided with a pandemic peak for Goldshtein et al. (December 2020–February 2021), while Dagan et al. also captured the time period following the end of the same peak (December 2020–June 2021). Butt et al. 22 included a population in Qatar and the sampling period included a peak and a low-case period preceding it (December 2020–May 2021). Both Israel and Qatar recommend a 21-day dosing interval for mRNA vaccines, and follow-up in the included studies started one week after the second dose and ranged from 42 to 50 days.Two of the three included studies matched for demographic and clinical characteristicsI2 = 73.9%)  Table , Fig. 2.33. Twelve studies were observational34 and three were governmental reports33. We did not include two studies reporting vaccination side-effects26 and a single study25 reporting antibody levels, as there was no pregnancy control group. Three studies were at overall serious risk of bias32, with the remainder at moderate risk34 (Supplementary Tables 3 and 4). No study was deemed to be at risk of bias for classification of interventions or deviation from intended interventions domains. Problematic missing outcome data were observed in one study19.Of 18 observational studies of the impact of COVID-19 vaccination (vs. no vaccination) on pregnancy outcomes, the vast majority were of mRNA vaccines, with very few of viral vector vaccinesTable P = 0.035, I2 = 93.9%, Fig. 31. The stillbirth rates reported by Morgan et al. 30 and Goldshtein et al. 24 were unusually low (<1:1000) and may have overestimated the protective effect due to zero and one events observed in the vaccinated group, respectively. The study by Rottenstreich et al. 31 was at risk of confounding bias as vaccinated pregnant people were older, had higher rates of previous miscarriage, fertility treatments and diabetes.A 15% decrease in the odds of stillbirth was associated with vaccination (vs. no vaccination) in pregnancy .Hypoxic brain injury, labelled as ‘hypoxic ischaemic encephalopathy’ and ‘birth asphyxia’ (each undefined further), was reduced in odds by 71% in association with vaccination (vs. no vaccination) in pregnancy 28 and Blakeway et al. 8 reported hazard ratio estimates after adjusting for confounding effects. Blakeway et al. 8 originally reported a hazard ratio censored after 39 weeks’ gestation and we re-estimated the hazard ratio for 37 weeks using the original raw data, so it can be comparable to Lipkind et al. 28 The cumulative effect shows a non-statistically significant 10% reduction in preterm birth before 37 weeks’ gestation following COVID-19 vaccination . No study distinguished spontaneous from iatrogenic preterm birth.There was no significant impact of vaccination (vs. no vaccination) on the odds of preterm birth as reported, before 37 weeks’ gestation  and the vaccine types varied , quantitative synthesis was not undertaken.Although miscarriage rate was reported in multiple studiesThis systematic review and meta-analysis summarise current data on COVID-19 vaccine effectiveness and safety during pregnancy. The mRNA vaccines appear highly effective against SARS-CoV-2 in pregnancy. The incidence of adverse pregnancy outcomes was not increased among vaccinated compared with unvaccinated pregnancies. In fact, the incidence of stillbirths and possibly, preterm births, was lower among the vaccinated pregnant population. Importantly, there was no increased risk of miscarriage following COVID-19 vaccination, based on data from large national registries and reports of inadvertent exposures in early pregnancy during RCTs.The strengths of this study include its robust methodology and comprehensive literature search, including incorporation of data from the ‘grey’ literature, such as governmental reports, and data from trials in which pregnant people were inadvertently exposed to the vaccine in early pregnancy. Also, we have included the broadest possible range of reported outcomes, to provide a comprehensive summary of relevant information to support informed and shared decision-making by pregnant people and maternity care providers.38. Where aggregate meta-analysis was feasible, the conclusions were further challenged by low numbers in the exposed (vaccinated) groups and by the low rates of adverse obstetric outcomes in the countries where published data have emerged. Moreover, even when included studies collected data from pregnant people who received viral vector vaccines, outcome data were not reported according to vaccine type, preventing relevant subgroup analyses. Booster shots gained prominence, especially with the advent of Omicron variant, and most countries allow a mix-and-match approach for booster doses. This increases the importance of safety data on other vaccine types and as well as the effectiveness of booster shots in pregnancy—both significantly lacking in the literature at the time of this review39. Finally, we were unable to study the impact of vaccination on spontaneous preterm birth, or vaccine effectiveness in infection-naïve and previously infected subgroups of pregnant people, or according to the COVID-19 variants of concern.This study, like most systematic reviews, was limited by the literature on which it was based. Very few studies reported maternal and neonatal outcomes after each dose of the vaccine and according to trimester at vaccination, and there was variation in the outcomes reported, so planned sensitivity analyses could not be performed40 and are increasingly available to pregnant people, particularly in low-and-middle-income countries. Lack of reports from these settings and for these vaccine types represent an important knowledge gap about the safety and efficacy of COVID-19 vaccines in pregnancy.All studies included data from high-income countries, but vaccines are now readily available to pregnant people in many low-and-middle-income countries. An increasing number of inactivated and viral vector vaccines  have been endorsed by the World Health Organization41. The mRNA vaccine causes both antibody and cellular immune responses; given the importance of T-cell suppression in placental development and fetal wellbeing42, concern has been expressed that the vaccine may increase miscarriage risk. Social media has been full of reports that have fuelled this concern, and many pregnant people have cited this fear as their primary reason for vaccine hesitancy. Our data do not support such concerns, based on both observational data29  and inadvertent exposure in early pregnancy in vaccine trial participants37.One question of particular interest was the risk of miscarriage following COVID-19 vaccination in early pregnancy. This is particularly important as up to 40% of pregnancies are unintended and may go unrecognised until 4–8 weeks’ gestation or beyond, so that inadvertent vaccination in early pregnancy is likely to be common43. A recent report of population-level data from Scotland found that following SARS-CoV-2 infection in unvaccinated pregnant people, the perinatal death rate was 22.6 per 1000 births, while in contrast, no vaccinated pregnant people with breakthrough infection suffered a perinatal death44. Taken together with our findings, these data point to the severity of COVID-19 as the mediator of the COVID-19 and stillbirth relationship. However, the observational nature of the original studies, significant statistical heterogeneity observed in the results and other probable confounders should caution not interpret these results as causal.Our finding of a lower incidence of stillbirths in the vaccinated cohort is important, given that COVID-19 in pregnancy is associated with an increased risk of stillbirth, particularly in the period of Delta dominance28 and Blakeway et al. 8) which adjust for time-varying and other possible confounders were combined, we found a non-statistically significant reduction in preterm birth by 10%. This finding is consistent with multiple studies linking COVID-19 infection to preterm birth.Although we noted no significant difference in the crude preterm birth rates, the data do not account for time-varying confounding. For instance, the crude rates from the recent CDC data and UKHSA data are conflicting to the extent that the former suggests a 32% reduction in preterm birth, while the latter suggests a 9% increase. However, the reduced rate in the CDC study is likely explained by the fact that most (~60%) vaccinated people were in their third trimester at the time of vaccination, so many were too late to have a preterm birth. On the other hand, the UK data did not account for the probable higher baseline preterm birth risk of vaccinated people and may have overestimated the effect. When two estimates of hazard ratio  but not all countries to withhold this vaccine from people under 40 years of age. These anecdotal reports of very rare complications should not deter the scientific community, healthcare workers and policy makers from disseminating information about the clear benefits of COVID-19 vaccination in pregnancy to both mother and baby.Much of the literature reviewed  was assessed as being at some risk of bias and with substantial between-study differences in some outcomes. Nevertheless, the data reviewed should prove very reassuring to pregnant people and healthcare providers who must make decisions about COVID-19 vaccination in pregnancy now. Further clinical trials will report in 2022. In the interim, prospective registries and active post-marketing surveillance should continue, to build more data about the effectiveness and safety of COVID-19 vaccination in pregnancy, including any rare side-effects that would not be expected to be seen in vaccine trials. In Brazil earlier this year, there was a report of a maternal death after Astra Zeneca vaccination, but we are unaware of other cases in the published literature48, and occur more commonly in association with COVID-19 infection49. We are unaware of such cases in pregnancy.More recently, reports have emerged of rare post-mRNA vaccination myocarditis, estimated to occur in 2 per million females and 10 per million males aged 18–40 years. Such reactions are typically mild and rapidly self-limiting52. This is likely due to limited evidence on vaccine safety in pregnancy early in the pandemic, and conflicting and changing advice given to pregnant people as the pandemic has evolved54. Our review corroborates the CDC V-Safe registry and MHRA reports, and further emphasizes that COVID-19 vaccination is highly effective in pregnancy to prevent COVID-19, without increasing the risk of adverse pregnancy outcomes. This provides further evidence that the risks of COVID-19 outweigh the rare risks of vaccination in pregnancy, and pregnant people should be encouraged to pursue vaccination, even in the first trimester. However, the lack of high-quality  studies with uniform reporting of clinically important outcomes, as well as under reporting of other vaccines types used in low-to-middle-income countries, are problematic. Studies of other vaccine types are important for improving vaccine coverage in underserved areas. Moreover, a core outcome set for reporting newly developed therapeutics would facilitate uniform high-quality evidence accumulation.Worldwide, there has been significant hesitancy among pregnant people to accept COVID-19 vaccination, with one study in the UK finding uptake of only 28.5%—significantly lower than in their non-pregnant peers56 advising COVID-19 vaccination in pregnancy, as it provides maternal and fetal benefit, without increasing perinatal risk. Ongoing post-marketing surveillance, ideally prospective in nature and controlling for bias, is needed to grow the evidence base related to vaccine type, timing in pregnancy, and vaccine effectiveness against newer evolving variants, like Omicron.The evidence supports recommendationsThis systematic review and meta-analysis on the effectiveness and safety of COVID-19 in pregnancy was registered with PROSPERO 2021 (CRD42021274016) and reported according to PRISMA guidelines.We electronically searched the COVID-19 Research, MEDLINE and Embase databases from 1 December 2020 to 8 September 2021. We updated our search on 13 November 2021 and again on 9 January 2022. For safety reports and briefings, we searched preprint servers including medRxiv; bioRxiv; and national regulatory and advisory body websites, including the Centers for Disease Prevention and Control (CDC), Medicines and Healthcare products Regulatory Agency (MHRA), United Kingdom Health Security Agency , Federal Drug Administration (FDA), European Medicines Agency (EMA), and Health Canada. The search included relevant medical subject heading terms, keywords, and word variants for pregnancy outcomes, perinatal outcomes, neonatal outcomes, stillbirth, preterm birth, obstetric complications, COVID-19 vaccination and immunization. No language restrictions were applied, and only human studies were included. The search strategy can be found in the supplementary material (Supplementary Table 5). Abstracts and potentially relevant full texts were reviewed independently by two authors (H.B. and S.P.) with any conflicts resolved by consulting a third senior author (A.K.).We included randomised trials, case-control studies, cohort studies, and data from national registries comparing outcomes between vaccinated (during pregnancy) and unvaccinated pregnancies. Additionally, we retrieved from trial safety reports and briefings, data on outcomes of inadvertent pregnancies occurring during clinical trials. We excluded narrative literature reviews  and reports without contemporary control groups of unvaccinated pregnant people.The intervention of interest was COVID-19 vaccination in pregnancy, inclusive of all vaccine platforms . Data were extracted with use of Covidence systematic review software  on a pre-designed spreadsheet. The following data were extracted where available: author’s name, publication date, study design, country, reported outcome categories, sample size of exposed (vaccinated) and non-exposed (unvaccinated) control population, data collection period, type of vaccine, number of doses received, and timing of administration (trimester of pregnancy). The total number of vaccinated pregnant people and the sum of adverse events in each group were extracted for categorical outcomes. Mean, standard deviation, and the total number of vaccinated pregnant people in each outcome group were extracted for outcomes reported on a continuous scale. We sought to study the following outcomes: (i) confirmed SARS-CoV-2 infection; (ii) maternal outcomes of hypertensive disorders of pregnancy (HDP), pre-eclampsia, placental abruption, pulmonary embolism, postpartum haemorrhage, intensive care unit admission, and maternal death; and (iii) fetal/newborn outcomes of miscarriage, fetal anomalies, stillbirth, gestational age at birth, preterm birth , birthweight, status at birth, and neonatal intensive care unit admission. For this review, we did not study non-pregnancy-related safety outcomes, reactogenicity or immunogenicity of the candidate COVID-19 vaccines.Each study was scored according to Cochrane Risk of Bias Tool 2 (randomised) and ROBINS-I , independently by two assessors , and disagreements were resolved by referral to a third reviewer (A.K.).57. When the event rate prohibited model convergence, approximate likelihood was used to facilitate model convergence. When the number of studies for an outcome was less than three or likelihood-based approaches did not converge, random-effects Mantel–Haenszel or Peto method were preferred depending on event rate. When data extraction for re-estimation of the effect was not possible, study reported estimates  and variances were combined directly using generic inverse variance meta-analysis with a restricted maximum-likelihood estimator for between-study variance. Continuous outcome measures were summarised using a random-effects model with restricted maximum likelihood estimation of between study variance58. The degree of between study heterogeneity that could not be ascribed to sampling error was explored using the I2 statistic59, and was interpreted as low (I2: <25%), low to moderate (I2: 25–50%), moderate to substantial (I2: 50–75%), or substantial (I2: >75%). Potential publication bias was assessed using Egger’s test and the creation of funnel plots for visual inspection when a sufficient number of studies (N > 10) was available. Sensitivity analyses were planned for studies reporting outcomes for single or double dose of vaccination, type of vaccine and trimester at first vaccination. Analyses were conducted using R for statistical computing software (v.4.0.3) and meta package .Results were reported as summary odds ratios, hazard ratios or mean differences with 95% confidence intervals (CI). Dichotomous outcomes were summarised with a hypergeometric-normal model based on maximum-likelihood estimatorsFurther information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


AG appeared to be superior to DT and ST, as it was the only condition that resulted in significant improvements in both single-task and dual-task walking abilities (p < 0.001). Implementing movement analogies could be a feasible and useful gait rehabilitation strategy for fall prevention and wellbeing promotion among older adults.It has been suggested that implicit motor learning via dual-task or analogy training during gait rehabilitation may yield better outcomes in older adults by reducing the propensity for the conscious processing of movements (movement-specific reinvestment). The current study investigated the immediate effects of single-task, dual-task, and analogy training on reinvestment propensity and fall-related rehabilitation outcomes among older adults at risk of falling. Seventy-one older adults were randomly allocated to the single-task (ST), dual-task (DT), or analogy (AG) training conditions and received 12 training sessions. We assessed the reinvestment propensity, functional gait and balance, functional mobility, balance ability, single-task and dual-task walking abilities, and fear of falling at baseline (before training) and immediately after training. Our findings revealed a lack of training effect on reinvestment propensity for all groups. However, all groups displayed significant improvements in functional gait and balance ( Falls constitute a concern that can undermine the wellbeing of the older population. Falls can lead to serious physical injuries, such as hip fractures, hospitalization, and death as well aHealthy older adults generally walk with automaticity . HoweverTherefore, research has emerged to explore methods that aim to reduce the propensity for movement-specific reinvestment to generate better outcomes  in the context of gait rehabilitation. Potential training approaches to mitigate movement-specific reinvestment propensity have been developed from the concept of implicit motor learning. In these approaches, the accrual of explicit rules and the cognitive involvement of working memory are deliberately suppressed . ImpliciOne type of implicit motor learning is via dual-task training, wherein a concurrent cognitive secondary task is performed during a primary motor task ,16. DualHowever, to the best of our knowledge, research has yet to investigate (and compare) the training effects of different implicit motor learning methodologies on movement-specific reinvestment propensity and fall-related rehabilitation outcomes among community-dwelling older adults. Such knowledge serves as critical evidence for physiotherapists in geriatric rehabilitation who aim to restore, maintain, or improve daily locomotion and independent function in older adults . ExploriThe current study aimed to examine the immediate effects of dual-task and analogy training on movement-specific reinvestment propensity and fall-related outcomes in the context of gait rehabilitation among older adults at risk of falling. We hypothesized that at-risk older adults who complete dual-task or analogy training will display a greater reduction in movement-specific reinvestment propensity and greater improvements in fall-related rehabilitation outcomes than those who complete single-task training (active control group). The outcomes include functional gait and balance, balance ability, functional mobility, single-task and dual-task walking abilities, and reduced fear of falling.This study consists of a three-arm, parallel group, randomized controlled trial (RCT). Participants were randomly allocated into a single-task (ST) (active control), a dual-task (DT), or an analogy (AG) walking training group. The study protocol was published  and prerParticipants were recruited from nine elderly community centers in Hong Kong. Community-dwelling older adults were recruited by convenience sampling based on the following inclusion criteria: (1) age 65 or above; (2) able to walk independently indoors for a minimum of 10 m; (3) a total score of 24 or higher out of 30 in the Chinese version of the Mini-Mental State Examination (MMSE-C) ; (4) no The training groups were differentiated by the walking instructions that were provided. An expert group of physiotherapists that specialized in geriatric rehabilitation designed the instructions. To ensure that participants were able to learn and understand the instructions comprehensively, all participants took part in their own group’s pre-training session , where they were clearly briefed about the specific walking instruction assigned to them.“please walk with bigger steps and even weight-bearing on your two feet”). The instructions, as a common practice, were dependent on participants’ walking ability. In the DT, specific dual-task instruction was utilized to instruct participants to walk with a concurrent cognitive task . In the AG, a specific analogy instruction was utilized to instruct participants during walking .In the ST, participants were instructed using typical therapeutic walking instructions . A battery of outcome assessments was subsequently performed to assess the participants’ cognitive and physical abilities at baseline before training (T0) and just after completion of training (T1). All participants took part in three 45 min training sessions per week for four consecutive weeks, resulting in a total of 12 training sessions that were held at local community centers. These training sessions were the only treatment that we provided to the participants. The training sessions were arranged in small groups of five participants and each consisted of (i) 5 min of warm-up and cool down; (ii) 5 min of balance training; (iii) 5 min of body transport training; (iv) 5 min of body transport training with hand manipulation; and (v) 20 min of walking training with various levels of difficulties on a 10 m walkway  under specific instructions as stated by their assigned groups. All training sessions were conducted by registered physiotherapists with experience in geriatric exercise training.The primary outcome was movement-specific reinvestment propensity, which was assessed by the Chinese version of the Movement-Specific Reinvestment Scale (MSRS-C) . The 10-m/s) was represented by (i) comfortable walking speed for 10 m and (ii) fast walking speed for 10 m [m/s) was represented by (i) comfortable walking speed for 10 m with concurrent verbal secondary task  [The secondary outcomes were functional gait and balance, functional mobility, balance ability, walking ability, and fear of falling. Functional gait and balance were evaluated by POMA , a widelfor 10 m . Dual-taop task) ; (ii) faop task) ; (iii) cop task) ; and (ivop task) . Fear ofop task) . A higheop task) .n = 84) was calculated to provide sufficient power based on an effect size of 0.31 for the primary outcome of movement-specific reinvestment propensity from a pilot study. After screening 179 potential participants, we found 71 who met the inclusion criteria. We note that we were unable to conduct further recruitment due to the local COVID-19 situation that led to prolonged suspensions of community activities for older adults who were deemed vulnerable to COVID-19.A sample size of 28 participants per group , DT (n = 27), or AG (n = 25) using concealed block randomization  by an independent researcher with an opaque and sealed envelope. An allocation schedule was generated by a computerized random-number generator.Recruited participants were randomly assigned to either ST . Means (with standard deviation) and numbers (with percentage) were used to describe continuous and categorical outcome variables, respectively. One-way analysis of variance (ANOVA) and chi-square tests were performed to detect between-group differences  in the dependent variables (primary and secondary outcomes) at baseline (T0).p < 0.05.The effects of walking training  on the primary (movement-specific reinvestment propensity) and secondary  outcomes across two time points (T0 and T1) were examined using 3  × 2  mixed-model ANOVA with Bonferroni adjusted post hoc tests. The level of significance for all statistical tests was set at n = 46, 82.14%) and had a history of falls . No significant differences among the three groups were observed in any variables at baseline (T0).p = 0.99, ηp2 < 0.001) on movement-specific reinvestment propensity. There were no main effects of group  or time . MSRS-C scores did not differ across groups and time. There was no significant group × time interaction effect . There were also no significant main effects of group . There were significant main effects of time, as the POMA, BBS, and TUG scores significantly improved across all groups at T1 compared to T0 .The results showed no significant group × time interaction effects on the functional gait and balance, functional mobility, and balance ability  and dual-task walking abilities . Post hoc comparisons revealed that, for single-task walking abilities, ST and AG showed significant improvements in comfortable  and fast speed  at T1 compared to T0 (p < 0.001) and fast speed (t[p < 0.001) and visual-spatial dual-task with comfortable speed (t[p < 0.001) at T1 compared to T0 (p > 0.05).There were significant group × time interaction effects on single-task walking abilities , main effect of group , or main effect of time  on the fear of falling, as the FES−13 scores did not differ across groups and time.There was no significant group × time interaction effect . Our theoretical assumption was that strategies that promote implicit motor learning could reduce the propensity for movement-specific reinvestment.Our findings only partially support our hypotheses; we did not find any expected reductions (or increases) in movement-specific reinvestment propensity, as revealed by the MSRS-C scores for all three groups  after training. We speculate that the MSRS-C, which quantifies the propensity for trait movement-specific reinvestment, might not be sufficiently sensitive to reflect potentially reduced levels of real-time conscious processing of movements in older adults, particularly in the context of level-ground walking. Although one might argue the possibility that implicit motor learning indeed has minimal effects on the propensity for real-time movement-specific reinvestment, our argument is consistently supported by recent evidence demonstrating the lack of significant association between the MSRS-C score (indicative of trait reinvestment) and EEG T3-Fz coherence  in community-dwelling older adults during gait tasks ,36. TherDespite the lack of significant changes in the propensity for trait movement-specific reinvestment, all three groups showed significant but comparable improvements in functional gait and balance, functional mobility, and balance ability after 12 sessions of walking training. The results suggest that both DT and AG might have similar functional benefits to ST in older adults who are at risk of falling. While all three types of training with expertly designed instructions were equally effective at improving functional performance, our findings showed evidence of the advantage of training with an analogy instruction in walking ability among older adults at risk of falling. The AG was found to be superior to the DT and ST in improving walking, as it was the only group that significantly improved both single-task and dual-task walking abilities immediately after training. Analogy is a form of instruction that facilitates skill acquisition by associating it with a fundamentally similar concept ,42,43. AWhen considering the outcomes of walking ability through other training groups, ST only showed significant improvements in single-task walking speeds, while DT did not show any improvements in all measures of walking ability. While it was reasonable to expect improvements in (single-task) walking speeds after a 4-week group-based training targeting solely walking (ST), our findings might hint at the notion that walking training under single-task contexts might not be able to generalize to locomotor control during dual-task conditions . For duaSeveral limitations should be taken into account when interpreting the results. First, the study was severely impacted by public health measures in response to COVID-19 in Hong Kong. Hence, we were not able to meet our target sample size, and there was a high drop-out number for the ST group, leading to an imbalance in sample sizes among the three groups . Unequal sample sizes might potentially affect statistical power and the risk of type I error. Second, one cannot neglect the possibility that older adults were using previous locomotor experience and/or adaptive learning strategies rather than solely relying on the instructions provided by us during training. Another limitation was the use of gait speed alone to quantify the performance of walking abilities under single-task and dual-task conditions. Gait speed has been shown to be a good predictor for falls ,58, physTo the best of our knowledge, this is the first RCT that compared the immediate effects of single-task, dual-task, and analogy training on movement-specific reinvestment propensity and fall-related rehabilitation outcomes in older adults. Our findings, while tentative, have potential implications for the design and development of psychomotor interventions that aim to enhance the wellbeing of older adults through fall prevention. Implementing the concept of analogy learning could be a feasible and effective intervention in geriatric rehabilitation settings. Physiotherapists could consider using analogies as a way of modifying instructions for older adults at risk of falling. Further research is recommended to strengthen the evidence and support implementation in practice.The present study revealed that walking training through single-task, dual-task, and analogy strategies significantly improved the functional outcomes  immediately after training. There were no significant effects on movement-specific reinvestment propensity by any training strategy. Using analogy training appeared to yield significant improvements in both single-task and dual-task walking abilities. We provide evidence for the notion that walking training with analogy instruction is a promising approach that has the potential to improve functional walking outcomes in older adults at risk of falling. For geriatric rehabilitation, our findings might be considered for service provision, where analogy instructions during gait training could potentially contribute to the prevention of falls and the promotion of wellbeing.


Lung cancer is the most common solid tumor in the worldwide. Targeted therapy and immunotherapy are important treatment options in advanced non-small cell lung cancer (NSCLC). The association of PTEN mutation and tumor immunotherapy is less established for patients with NSCLC. We present the case of an Asian woman diagnosed with stage IV lung adenocarcinoma harboring an ERBB2 mutation. She received Nivolumab treatment when her disease progresses after previous chemotherapy and Afatinib treatment. However, the patient did not response to Nivolumab. PTEN mutation was detected by next-generation sequencing (NGS) after treatment with Nivolumab. PTEN, a secondary mutation, may be served as a biomarker of resistance to anti-PD-1 immunotherapy in lung adenocarcinoma. The relationship between PTEN mutation and immunotherapy is complex and needs further study. Non-small cell lung cancer (NSCLC) is the most common type of lung cancer accounting for approximately 85% of all lung cancers .Targeted therapy is the standard treatment for advanced NSCLC with driver genetic alterations. The common driver genes changes in lung adenocarcinoma include EGER, ALK, ROS1. For advanced NSCLC patients without driver genetic alterations, immune checkpoint inhibitor (ICI) therapy is an alternative treatment option. Currently, the cell programmed death receptor 1 (PD-1) and its ligand (PD-L1) inhibitors are approved by FDA for the treatment of metastatic NSCLC after failed platinum-containing chemotherapy. Based on the data from CheckMate 057 trial , NivolumPTEN is a known tumor suppressor whose mutation rate is only second to p53 in human tumors. PTEN facilitates fundamental anti-oncogenic tasks and its encoded PTEN protein can promote cell cycle arrest in G1 phase and apoptosis, thereby inhibiting tumor cell growth. PTEN can also inhibit cell invasion and metastasis . HoweverA 40-year-old Chinese woman who had never smoked presented to our center due to right shoulder pain in September 2016. Physical examination revealed an enlarged lymph node about 1 × 1 cm in size placed on the left supraclavicular fossa. A chest computed tomography (CT) scan indicated a space-occupying lesion in the left lower lobe with miliary distribution of multiple tiny nodules in both lungs, and local bone destruction at the right scapula . Biopsy The patient received six cycles of Pemetrexed and Cisplatin which result in partial response (PR) . Then shIn July 2018, the patient has been enrolled in a clinical trial to receive anti-PD-1 treatment with the 240 mg q2w regimen of Nivolumab. Unfortunately, she does not benefit from immunotherapy. After 4 cycles of Nivolumab, she developed further PD in the lung. Compared with the CT images before immunotherapy, it appeared pleural effusion . At thisThis report of an advanced NSCLC patient who has received comprehensive treatment, including chemotherapy, radiotherapy, targeted therapy and immunotherapy. Checkpoint inhibitors can activate the immune system, enhance anti-tumor activity and prolong the survival of patients with NSCLC . During PTEN is an antitumor gene, and its inactivation mutation can lead to continuous activation of mTOR pathway, so that tumor growth cannot be controlled . PreviouLoss of PTEN can activate the PI3K pathway, leading to decreased responserates to immunotherapy . WhetherNotably, genetic alterations in this patient‘s cancer included not only PTEN mutation, but also ERBB2 and TP53 mutations. Then, what are the influence of ERBB2 or TP53mutations on immunotherapy? BT Li et al. reported seven patients with HER2 mutant lung adenocarcinomas who received anti-PD1 immune checkpoint inhibitors, and none responded . Recent In summary, the case we reported indicate that the effect of immunotherapy is related to genetic abnormality. PTEN mutation may be used as biomarker to predict resistance to anti-PD1 immune checkpoint inhibitors in lung adenocarcinoma. However, larger samples are required for validation. There is still a long way to go to understand the role and regulatory mechanism of PTEN in immunology. It is hope that in-depth research on these aspects in the future will contribute to the development of PTEN-related therapies for human cancer. The exploration of combination therapy in patients with drug resistance has the potential to improve the response rate of NSCLC with PTEN mutation to anti-PD-1 treatment. Afatinib, as a non-reversible tyrosine kinase inhibitor of HER2, is a potential treatment option for patients with advanced lung adenocarcinoma with ERBB2 20 exon insertion. In addition, repeated NGS testing in the process of tumor treatment has not only guided targeted therapy, but also contributed to determine the efficacy of immunotherapy and understand the resistance mechanism of immunotherapy. NGS detection can track find more genetic mutations, but now the high cost, less cost, we repeat biopsy in the treatment process, blood samples at differ-ent times, puncture, the results of multiple genes, tracking guidance significance. In a-ddition, patients with advanced lung adenocarcinoma patients, early attention to treat-ment effect, respect the wishes of the patients with late treatment, in order to improve the quality of life, Prolonging life is the main purpose.


Respiratory tract infections such as the ongoing coronavirus disease 2019 (COVID-19) has seriously threatened public health in the last decades. The experience of fighting against the epidemic highlights the importance of user-friendly and accessible point-of-care systems for nucleic acid (NA) detection. To realize low-cost and multiplexed point-of-care NA detection, a swing-assisted multiplexed analyzer for point-of-care respiratory tract infection testing (SMART) was proposed to detect multiple respiratory tract pathogens using visible loop-mediated isothermal amplification. By performing hand-swing movements to generate acceleration force to distribute samples into reaction chambers, the design of the SMART system was greatly simplified. By using different format of chips and integrating into a suitcase, this system can be applied to on-site multitarget and multi-sample testing. Three targets including the N and Orf genes of SARS-CoV-2 and the internal control were simultaneously analyzed . Twenty-three clinical samples with eight types of respiratory bacteria and twelve COVID-19 clinical samples were successfully detected. These results indicate that the SMART system has the potential to be further developed as a versatile tool in the diagnosis of respiratory tract infection. Respiratory tract infections pose a major global health challenge to the public worldwide, and are associated with high morbidity and mortality . The annMany well-established commercial POC products have been further developed to detect SARS-CoV-2 and have obtained emergency use authorizations (EUAs) by the Food and Drug Administration (FDA) in the USA . Among tStaphylococcus aureus, Streptococcus pneumoniae, Klebsiella pneumoniae, Haemophilus influenzae, Acinetobacter baumannii, Pseudomonas aeruginosa, Stenotrophomonas maltophilia and Legionella pneumophila, and consistent results were obtained for 23 clinical samples compared to the results detected by a commercial LAMP-based bacterial detection kit. To further facilitate field-deployable detection of pathogens, a suitcase with six portable analyzers installed inside was constructed. This suitcase can analyze 36 SARS-CoV-2 samples in parallel in a convenient and flexible manner, and thus possesses potential as a tool to fight against pandemics.Inspired by our previous studies ,29 that ®7759, Beijing, China).SARS-CoV-2 pseudovirus was commercially purchased from Fubio Biological Technology Corporation . The pseudovirus was quantified using digital droplet PCR . The sample releasing reagent was obtained from Sansure Biotech . A total of 20 ng human genomic DNA  was added as a standard IC of each LAMP reaction. Disposable swabs were purchased from MaiRuiKeLin Technology . For SARS-CoV-2 RNA extraction, a self-developed lysis-binding buffer from our group [2+ free) , 6.25 mM MgSO4 , 1.175 mM dNTP mixture , 0.8 μL Bst Enzyme Mix (UDG plus) , 0.12 mM EBT , and LAMP primer mixture . The primers were synthesized by Sangon , and their sequences are listed in ®Dye was ordered from Biotium . In-tube RT-LAMP was performed on a 7500 Fast Real-Time PCR System . For on-chip LAMP, the RT-LAMP buffer was lyophilized by SP Scientific Lyophilizer . The RT-LAMP reaction for respiratory bacteria detection with a commercial kit was performed on an RTisochipTM—A Analyzer . The DL 2000 DNA marker and GeneGreen dye were purchased from Tiangen Biotech . Agarose gel images were obtained and processed using a gel imager .RT-LAMP was performed in a 20 μL reaction mixture containing 1.57 μL Isothermo buffer  were carefully placed into the reaction chambers under low humidity and electrostatic elimination conditions . Finally2) was placed behind the chip for contact heating. A polyimide-heating membrane  was adhered to the back of the copper plate with an NTC temperature sensor , which was inserted to measure the temperature. Two flat springs were fixed on this copper plate to ensure that the contact between the chip and the copper was tight when the chip was inserted into the analyzer  for 10 min to lyse the viral particles at room temperature. Then, the released viral NAs and human genomic DNA were diluted to the known concentrations and detected with LAMP reagents to determine the limit of detection (LoD) for SARS-CoV-2. After that, throat swabs from healthy donors were dipped in 1 mL of rinsing solution (lysis and binding buffer of the kit) to simulate negative clinical samples, while serial dilutions of the SARS-CoV-2 pseudoviruses were immediately added into the rinsing solutions of the swabs to mimic positive clinical samples. All throat swabs were collected more than 30 min after eating or drinking, and the throats of donors were rinsed with water before scraping.Nine SARS-CoV-2 positive and three negative clinical samples were provided by the CapitalBio MedLab. All clinical samples were collected by clinical nasopharyngeal swabs and stored and transported in VTM following the standard guidelines of the National Health Commission (NHC) of China. To extract the NAs from SARS-CoV-2 mock and clinical samples, all procedures were performed as described in detail in our previous paper . In brieg for 5 min for bacterial collection. The pellet was then resuspended in 1 mL of washing buffer for washing, and after another centrifugation at 20,000× g for 5 min, the supernatant was discarded. Then, 200 μL eluent was added to the tube and resuspended. Next, the sample was transferred to a tube with zirconium beads, and the tube was placed in a 95 °C-metal bath for incubation at 100× g for 10 min to mechanically lyse the bacteria. Finally, after the impurities were precipitated by instantaneous centrifugation, the supernatant with bacterial NA was taken as the template for testing. For the commercial RTisochipTM—A system test, 20 μL of the thawed amplification reagent at room temperature was mixed well with 34.5 μL of extracted NA sample in a 200 μL tube. Next, 50 μL of the mixture was added to the centrifugal microfluidic chip in the kit, and then the centrifugal chip was placed on the RTisochipTM—A system for LAMP amplification and result interpretation. For our SMART system test, a ten-target hand-swing chip was fabricated and used. DEPC water was added to the 100 μL purified NA sample to increase the volume to 250 μL, and then the solution was loaded into our hand-swing chip as the template, and also to dissolve the lyophilized RT-LAMP reagents for NA amplification.To detect the respiratory bacteria, 23 sputum samples with respiratory tract infections were collected. It was necessary that the white blood cell count was greater than 25/low power field, the epithelial cells were less than 10/low power field to be qualified sputum samples, and the sample volume was not less than 0.6 mL in the sample collection tube. Clinical samples were collected with the patients’ informed consent in the Beijing Tsinghua Changgung Hospital in 2021. This research was approved by the Institution Review Board of Tsinghua University . NA extractions from these clinical sputum samples were performed by following the protocol as recommended by the respiratory bacterial NA detection kit. In brief, the viscous sputum sample was first liquefied with an equal volume of 4% NaOH solution, and then centrifuged at 20,000× As shown in For multitarget detection, the distribution of samples into isolated reaction chambers with respective primers is an important prerequisite. Centrifugation-assisted liquid distribution is a commonly used strategy for multiplexed RPA ,33, LAMPTo achieve this goal, we fabricated microfluidic chips  by folloAlthough there was no observed liquid connection in the sample-infusing channel c, the poSensitivity is one of the most important indicators for a POC system. Here, we tested the detection limit of our SMART system using serial dilutions of RNA templates of known concentrations released from SARS-CoV-2 pseudoviruses. As shown in NA extraction and purification steps are still necessary and effective for robust and sensitive detection. Therefore, the detection limit of the extracted samples from mock swab samples was evaluated using our system. Based on an optimized extraction method proposed in our previous study , two tarAlthough NA extraction allows sensitive detection, it is time-consuming and sometimes labor-intensive. Direct amplification of samples for POC applications has been frequently reported ,30,31, sIt is challenging to perform multitarget and multi-sample detections with the current POC systems. For instance, the cartridge from GeneXpert only has one reaction chamber for multiplexed PCR , thus thAs shown in 3, weight: 370 g). The systems in the suitcase work with an external power supply or an internal battery, and thus greatly promote the on-site use for fighting epidemics, such as COVID-19.To further facilitate field-deployable application, a suitcase was constructed to support the work of six SMART analyzers introduced above . This suPseudomonas aeruginosa, Stenotrophomonas maltophilia and Klebsiella pneumoniae, which are commonly found pathogens in the sputum of long-stay patients [Streptococcus pneumoniae and Legionella pneumophila were not detected in these samples. Nine COVID-19 positive samples and three negative samples were also successfully detected (We collected 23 sputum samples from patients infected with respiratory pathogens, 9 nasopharyngeal swab samples from patients infected with SARS-CoV-2, and 3 negative swab samples to test our SMART system and to demonstrate the ability of our SMART system to analyze real clinical samples . The respatients , were dedetected c. The abIn this study, we proposed an easy-to-use and low-cost SMART system to detect multiple pathogens with EBT-based visible LAMP in a multiplexed manner. An essential step for distributing the sample into reaction chambers was achieved by a hand-swinging process; thus, the structure of the chip and its supporting analyzer is quite simple, making low-cost and flexible detection of multitarget and multiple samples possible . This SM