Tuberculosis (TB) is an infectious disease that can lead towards death if left untreated. TB detection involves extraction of complex TB manifestation features such as lung cavity, air space consolidation, endobronchial spread, and pleural effusions from chest x-rays (CXRs). Deep learning based approach named convolutional neural network (CNN) has the ability to learn complex features from CXR images. The main problem is that CNN does not consider uncertainty to classify CXRs using softmax layer. It lacks in presenting the true probability of CXRs by differentiating confusing cases during TB detection. This paper presents the solution for TB identification by using Bayesian-based convolutional neural network (B-CNN). It deals with the uncertain cases that have low discernibility among the TB and non-TB manifested CXRs. The proposed TB identification methodology based on B-CNN is evaluated on two TB benchmark datasets, i.e., Montgomery and Shenzhen. For training and testing of proposed scheme we have utilized Google Colab platform which provides NVidia Tesla K80 with 12 GB of VRAM, single core of 2.3 GHz Xeon Processor, 12 GB RAM and 320 GB of disk. B-CNN achieves 96.42% and 86.46% accuracy on both dataset, respectively as compared to the state-of-the-art machine learning and CNN approaches. Moreover, B-CNN validates its results by filtering the CXRs as confusion cases where the variance of B-CNN predicted outputs is more than a certain threshold. Results prove the supremacy of B-CNN for the identification of TB and non-TB sample CXRs as compared to counterparts in terms of accuracy, variance in the predicted probabilities and model uncertainty. Bayesian-based convolutional neural network (B-CNN) for tuberculosis identification: Input chest x-ray is given to N number of CNNs. Dropout is enabled at testing stage to get posterior probability distribution (PPD) of classes. I.Tuberculosis (TB) is a contagious disease that is designated among 10 highest causes of death and also the leading infectious disease above than human immunodeficiency Virus (HIV)/ acquired immune deficiency syndrome (AIDS) Researchers presented a number of mechanisms to differentiate between the CXRs of TB and non-TB patients. Machine learning (ML) and image analysis techniques are key components behind the recent developments in computer-aided image interpretation systems. Different researchers applied numerous techniques for feature-extraction including Gabor, histogram of oriented gradients (HOG) Deep learning (DL) is an emerging development in ML and image analysis that has been designated as one of the ten breakthrough technologies of this decade. DL is a resurgence of convolutional neural network (CNN) with the capability of learning higher levels of abstraction which is crucial for improving the performance of data analysis algorithms 1)Explored extensive literature to analyze radiograph screen schemes based on machine learning and deep learning algorithms. Through literature study we have identified the problem.2)Deep learning based TB diagnosis scheme is presented for effective mass screening of CXRs. In the proposed scheme three CNN architectures inspired by VGG-19 are presented to diagnose TB manifestations.3)Identification of the softmax function utilized at the end of CNNs can only classify the CXR as TB and non-TB with higher confidence which is error prone to the borderline TB cases.4)B-CNN based model is presented to identify TB by considering the uncertainty factor that effects results. It assigns a higher uncertainty/confusion for erroneous predictions to make better decisions and avoid false positive cases.The main problem is that uncertainty factor is not considered for TB diagnosis in most of the schemes including Rest of the paper is organized as follows: II.In this Section, we explore a number of TB diagnosis schemes based on machine-learning and deep learning. TB diagnosis relies primarily on radiological patterns. Early detection of TB can lead to its treatment and prevention of infection growth. However, lack of diagnostic resources such as skilled radiologists are major hindrance for effective and robust TB diagnostics. In comparison with human diagnoses, computer-aided tools can provide more significant results with less diagnostic errors and make it possible to perform efficient mass screening with fewer resources.A.et al.et al. presented a feature extraction and classification mechanism to compute local pixel characteristics. The method uses position features, texture features and features derived from the Hessian matrix. The hybrid active shape model pixel algorithm was used for classification. The receiver operator characteristic (ROC) based accuracy of 84.7% was achieved This section describes TB diagnosis schemes based on machine learning (ML) algorithms. The machine learning approaches rely on hand crafted features selection B.This section presents TB diagnostics methods based on Deep learning (DL). The DL-based methods achieved significant results for computer aided diagnostics. Lopes and Valiati et al.Alcantara et al. extracted features with GoogleNet and performed classification using SVM on their own collected dataset which comprised of 4701 images. It achieved 89.6% accuracy for binary classification of TB and 62.07% accuracy for multi-class classification of different TB manifestations. However, classes in dataset are uneven as Lymphadenopathy has 202 images whereas Infiltration comprises of 2252 images In our proposed work, we focus on recent model uncertainty integration tools with DL algorithm to cope with uncertain CXRs. In uncertain CXRs, it is difficult to identify whether TB manifestations are present or not in confusing cases. Softmax tends to misclassify TB from CXRs and represents model confidence either very high or very low which is not true. The next section explains the detailed technical background of DL approaches used for TB identification.III.DL techniques can learn the TB manifestation patterns and features automatically from raw CXR data used for classification and localization task. These DL techniques are capable of learning features at different abstraction levels by piling non-linear layers that allow the DL techniques to attain better simplification for difficult computer vision tasks such as image localization, segmentation, and classification.A.CNN is a DL based approach that became an emerging technique for image classification due to its significant achievements. The success achieved is because of the precise and robust assumptions of CNN for the natural image data including associations and pixel locality 1)The units of convolution layer are linked to their respective units of the local patch coming from the preceding layer with a filter. The units are activated by using the feature map, which is computed through applying Rectified Linear Unit (ReLU) 2)This layer works by merging features and patterns semantically into a solitary feature map. The pooling layer computes the maximum or average of input features from the previous layer and uses them as an output feature map.3)In the fully-connected layer, every unit is connected to previous layer units and making a mesh. Usually, before fully-connected layer two or three stacks of the convolution and pooling layers are placed to extract features.4)The purpose of the softmax layer is to converts the features into probabilities which belong to each output class. The total number of units in the softmax layer is equal to the total output classes. The softmax function is defined by ReLU can be used as an activation function for displaying non-linearity as well as achieving faster learning convergence B.To cope with low visibility between CXRs, a model is needed that should be proficient enough to represent the prediction uncertainty. The state-of-the-art techniques such as The inference in B-CNN model is implemented by using the initial stochastic regularization method such as dropout IV.In this Section, a robust TB diagnostic scheme based on Bayesian is presented. We have explored three baseline-CNN architectures that are trained on two benchmark datasets named Montgomery and Shenzhen datasets. Moreover, we have deployed Bayesian based CNN (B-CNN) architecture to overcome the softmax inference issue. We have calculated variance from prediction results of B-CNN on Shenzhen dataset to validate the robustness of proposed B-CNN. List of notations is presented in By applying B-CNN, TB identification task can be described as follows; consider a set of CXRs denoted as In Proposed CNN architecture for CXRs classification is shown in Moreover, it includes the computation of operations such as non-linear transformation, convolution, local normalization and max-pooling V.In this section, we explore experimental setup and B-CNN model based on baseline-CNN architectures. We analyze baseline-CNN architectures conversion into B-CNN and their prediction probability. In the last, we have discussed in detail important part of model uncertainty and its role to pronounce the model confidence. In this study, two benchmark datasets including Montgomery and Shenzhen A.et al.The identification accuracy is used to gauge the performance of proposed TB identification methodology based on B-CNN in comparison with other techniques from literature including: SVM To evaluate the robustness of our proposed scheme we have compared the results of Acr-2 B-CNN with other state-of-the-art schemes. From Furthermore, et al.The CXR in VI.TB identification is a challenging task due to the occurrence of complex and obscured patterns present in CXRs. Numerous TB identification approaches are proposed in the literature. However, the accuracy of TB identification is still low. Recently, DL based approaches have presented significant results for TB identification by discovering complex features from large datasets. However, conventional deep learning based models are not capable to suggest uncertainty for output class prediction. Proposed B-CNN exploits the model uncertainty and Bayesian confidence to improve the accuracy of TB identification as well as validation of the results. The dropout is utilized at the training and testing phase to acquire the posterior probability distribution of the classes. The variance in probabilities is utilized as uncertainty and the mean of the posterior probability distribution of the classes is used to make the final output classification decision. The proposed methodology based on B-CNN has experimented on two TB benchmark datasets: Montgomery and Shenzhen. The results have demonstrated that the proposed methodology achieved highly significant results in terms of TB identification accuracy, i.e., 96.42% and 86.46% for both datasets  in comparison with the state-of-the-art DL approaches in the literature. Furthermore, by utilizing the variance of proposed TB identification methodology based on B-CNN a threshold of 0.1 is applied to filter out the confusion cases of the Shenzhen dataset. The applied threshold helped to capture the 10.4% of false negative and the 4.5% of false positive as confusion cases. These confusion cases improve the classification in terms of validation up to 93.9% that is either the CXR belongs to TB or non-TB status or is a marginal confusion case that needs to be examined by an expert radiologist. This proposed work shows compelling results and provides an improved foundation for future work. However, the real challenge is to determine that, how such DL systems will accurately fit with the workflow for diagnostic of thorax diseases and clinical screening settings. In future, we shall focus on potential validation that will provide a robust approach to deal with multiple thorax diseases from CXRs. Furthermore, BCNN models shall be utilized to eliminate uncertainty factor in natural images classification.


CHASERR (AC013394.2 or LINC01578) that could regulate target genes co-transcriptionally via interaction with a nascent transcript by directing CHD2 helicase. The obtained results suggested that this nuclear lncRNA may be able to activate expression of the target genes in trans by base-pairing with the nascent transcripts and directing the CHD2 helicase to the regulated promoters leading to open the chromatin and active transcription. Our study highlights the possible importance of base-pairing between nuclear lncRNAs and nascent transcripts for the regulation of gene expression.Although thousands of mammalian long non-coding RNAs (lncRNAs) have been reported in the last decade, their functional annotation remains limited. A wet-lab approach to detect functions of a novel lncRNA usually includes its knockdown followed by RNA sequencing and identification of the deferentially expressed genes. However, identification of the molecular mechanism(s) used by the lncRNA to regulate its targets frequently becomes a challenge. Previously, we developed the ASSA algorithm that detects statistically significant inter-molecular RNA-RNA interactions. Here we designed a workflow that uses ASSA predictions to estimate the ability of an lncRNA to function via direct base pairing with the target transcripts . The workflow was applied to 300+ lncRNA knockdown experiments from the FANTOM6 pilot project producing statistically significant predictions for 71 unique lncRNAs (104 knockdowns). Surprisingly, the majority of these lncRNAs were likely to function co-transcriptionally, i.e., hybridize with the nascent transcripts of the target genes. Moreover, a number of the obtained predictions were supported by independent iMARGI experimental data on co-localization of lncRNA and chromatin. We detected an evolutionarily conserved lncRNA  Long non-coding RNAs (lncRNAs)—transcripts of at least 200 nucleotides long lacking protein-coding potential—are the most populated class of transcripts in the mammalian genomes. They are expressed from more than 50,000 loci in the human genome , which iUp to date, several genomic screening approaches to functional annotation of lncRNA have been developed. Based on lncRNA knockdown (knockout) followed by cellular or molecular phenotyping, these approaches characterize a fair share of lncRNAs in one series of experiments ,13,14,15Complementary approaches to lncRNA annotation aim to search for their targets via detecting interactions between lncRNAs and chromatin or other RNA molecules. LncRNAs use different molecular mechanisms to bind to the chromatin, including RNA-DNA binding mediated by protein complexes, direct RNA-DNA hybridization via triplexes, RNA binding to single-stranded DNA regions (known as R-loops) and co-transcriptional RNA-RNA interactions via duplexes with nascent RNA transcripts (reviewed in ). ExperiIn this work, we focus on RNA:RNA interactions. We investigated two possible scenarios— lncRNA bound co-transcriptionally to the nascent RNAs via duplexes and lncRNA bound to mature transcripts—by applying the ASSA RNA-RNA interaction prediction tool  to the dhttps://github.com/vanya-antonov/assa) tool was used in this study to predict inter-molecular RNA:RNA interactions [The AntiSense Search Approach  with the first 1kb of the target gene . We included the introns assuming that lncRNA-RNA binding may precede splicing.By contrast, post-transcriptional lncRNA:RNA hybridizations occur with mature spliced target transcripts in the cytoplasm or the nucleus . We hypothesized that this type of interaction results in post-transcriptional regulation of gene expression. It should be noted that CAGE-seq technology which has been used in the FANTOM6 project measures expression changes at the promoter level. Thus, to predict post-transcriptional interactions we considered the longest isoform that can be expressed from the corresponding promoter only.In the present work ASSA was run with default settings in both the co- and post-transcriptional modes.p-values To estimate the correspondence between the iMARGI peaks and ASSA predictions for each lncRNA we only considered differentially expressed genes (DEGs) that had predicted antisense interactions with the corresponding lncRNA  were cophyper R function). We used 45480 as the total number of the human genes that was computed as the sum of the protein-coding (19954), long (17957) and small (7569) non-coding RNA genes as annotated in the GENCODE version 35. The human orthologs of the DEGs Chaserr-/- mEFs [getLDS function of the biomaRt R package. The expression of the CHD2 gene was obtained from the FANTOM5 SSTAR online tool (https://fantom.gsc.riken.jp/5/sstar/EntrezGene:1106) for the “Fibroblast-skin normal, donor2 (nuclear fraction).CNhs12582.14302-155B9” sample.The statistical significance of the gene overlaps was estimated using the hypergeometric distribution  taking into account the lengths and the GC contents of both transcripts.We used the AntiSense Search Approach (ASSA) tool  to identWe analyzed all the lncRNAs knockdown experiments available from the recent pilot FANTOM6 project . In brieCHASERR and the promoters that were differentially expressed upon its knockdown and detected strong ASSA hits in the co-transcriptional mode [p-value < 0.01) was observed for 21 of those ambiguous 41 ASO knockdowns . This indicated the CHASERR functional significance and reproducibility of the results.Two knockdown experiments for the AC013394.2 lncRNA  demonstrate coordinated and strong enrichment of the ASSA predicted RNA:RNA duplexes in DEG D. The prCHASERR is mainly localized in the nucleus where it is bound to the chromodomain helicase DNA binding protein 2 (Chd2) [CHASERR orthologs are highly similar to each other (Chaserr-/- mouse embryonic fibroblases (mEFs) (see Methods) and compared them to the lists of the DEGs from the two FANTOM6 knockdowns. We observed a statistically significant overlap between the three sets of genes  supporting conservation of the CHASERR target genes (It has recently been shown that the mouse ortholog of 2 (Chd2) . The chr2 (Chd2) . Thus, C2 (Chd2) . Importach other . To checet genes A.CHASERR orthologs as well as the fact that the CHD2 helicase is also expressed in the human fibroblasts (FANTOM5 expression = 113.25), we assumed that CHASERR lncRNA and CHD2 helicase interact in the nucleus of human fibroblasts as well. Our computational analysis suggested that CHASERR may directly interact with the beginning . It should also be noted that ASSA predicted statistically significant interactions between CHASERR and transcripts of these two genes . A more detailed analysis of the


Ovarian cancer is a primary gynecological malignancy with a global 5-year survival rate of 44%. The majority of patients present with advanced disease at initial diagnosis because of the lack of an effective early detection screening test. Circular RNAs (circRNAs) within exosomes in the circulatory system are effective diagnostic and therapeutic biomarkers for many diseases, especially tumors. In this study, we used microarrays to identify 6 circRNAs that were upregulated and 37 circRNAs that were downregulated in exosomes from ovarian cancer patients as compared to healthy volunteers. We validated the accumulation trends for the 6 upregulated circRNAs in the training set using qRT-PCR and found that circ-0001068 was significantly higher in the serum exosomes from the ovarian cancer patients as than healthy volunteers. Circ-0001068 was next evaluated further in a larger cohort. As with the training set, results from the larger cohort revealed that levels of circ-0001068 in the exosomes were significantly higher in ovarian cancer patients than healthy volunteers. Circ-0001068 was also delivered into T cells and induced PD1 expression by acting as a competing endogenous RNA (ceRNA) for miR-28-5p through the exosomes. Ovarian cancer (OC) is recognized as a primary gynecological malignancy with a global 5-year survival rate of 44% . CurrentLiquid biopsy is a novel and minimally invasive blood-based technique that can be used for the early diagnosis of and population screening for OC , 3. CurrCircular RNAs are a novel class of endogenous noncoding RNAs that are characterized by back splicing that results in covalently closed loop structures without 5′ caps and 3′ poly tails . CircRNAThe aim of the present study was to compare differences in the expression of circRNAs in the serum exosomes between OC patients and healthy volunteers using microarray analyses in order to identify biomarkers for the diagnosis and prognosis of OC. Our results showed that circ-0001068 was significantly increased in the serum exosomes of the OC patients as compared with that in the healthy controls. Additionally, OC cells delivered circ-0001068 into T cells to promote tumor immune invasion.High-throughput human circRNA microarray was performed using serum exosomes from 4 OC patients and 4 healthy volunteers to assess circRNA expression profiles. The heat map showed that the profiles of exosomal circRNA were different between OC patients and healthy volunteers . Specifip < 0.05) in OC patients as compared with those in healthy volunteers were used as biomarkers. Consequently, 6 upregulated circRNAs were selected for further validation in two independent cohorts (53 HV and 95 OC) using RT-qPCR assays  . HoweverExosomes are 30–150 nm nanoscale extracellular vesicles that are shed by almost all kinds of tumor cells and play multiple roles in tumor progression, including cell proliferation , 17, druOverall, we revealed that circ-0001068 was significantly upregulated in the exosomes of serum from OC patients as compared with that in healthy volunteers. We also confirmed circ-0001068 that was secreted by OC cells was delivered into T cells and sponged miR-28-5p, which resulted in the increased expression of PD1 and T cell exhaustion. Our results demonstrate that circ-0001068 may serve as novel noninvasive biomarker and target for the diagnosis and treatment OC.Twenty pairs of OC tissues and adjacent non-cancerous tissues were collected from patients who were diagnosed with OC at the Tongde Hospital of Zhejiang Province. The serum samples from 95 OC patients who were not receiving any treatments and 53 matched healthy volunteers were collected at the Tongde Hospital of Zhejiang Province. The patient characteristics and clinical features are summarized in Caenorhabditis elegans miRNA cel-miR-39 (5’-UCACCGGGUGUAAAUCAGCUUG-3’),  was used for normalization [The Total Exosome Isolation Reagent (from serum)  and Total Exosome Isolation Reagent (from cell culture media)  were used to isolate exosomes from the serum and cell culture media according to the manufacturer’s protocol. Total RNAs were extracted from exosomes using the mirVana PARIS Kit  according to the manufacturer's protocol. Synthetic lization . FinallyT method; cel-miR-39 was used as the exogenous control.A hydrolysis probe–based RT-qPCR assay was performed for exosomal circRNAs and miRNAs according to the manufacturer's protocol. The primers used for circRNAs were synthesized by GenScript  and are listed in Jurkat T cells were seeded onto 12-well dishes, and 500 μg of exosomes were added into each well. After incubation for 24 h, the Jurkat T cells were collected for qRT-PCR or flow cytometry analysis. The Jurkat T cells were stained with the PD1 antibody , and the expression of PD1 was determined by flow cytometry. Data were analyzed with FlowJo-10 software.https://circinteractome.nia.nih.gov) was used to predict the miRNAs that interacted with circ-0001068 and miRNAs [The Circular RNA Interactome database  analysis was performed to estimate the diagnostic value of circRNAs in the exosomes.Supplementary FiguresSupplementary Table 1


C. elegans to human. These domains are thought to mediate interactions between proteins but also between proteins and RNA in complex networks associated with mRNA splicing, chromatin structure, transcription, cell cycle and cell structure. A precise and tight regulation of their function is achieved through phosphorylation of a number of serine residues within the SR/RS motifs by the Serine-Arginine protein kinases (SRPKs) that lead to delicate structural alterations. Given that coronavirus N proteins also contain SR/RS domains, we formulate the hypothesis that the viruses exploit the properties of these motifs to promote unpacking of viral RNA and virion assembly.SR/RS domains are found in almost all eukaryotic genomes from  Coronaviruses (CoVs) have a single-stranded positive-sense RNA genome covered by an enveloped structure. The order of genes (5′–3′) contained in their RNA genome is as follows: replicase ORF1a/b, spike (S), envelope (E), membrane (M), and nucleocapsid (N) . The twoOf the four structural proteins, the nucleocapsid protein N is the one that is responsible for packaging the viral RNA . Many N The N protein of coronaviruses is structurally organized into two domains, the N-terminal domain (NTD) and C-terminal domain (CTD), which are separated by a flexible linker . Both NTThe flexible linkers of the different coronaviruses share very low sequence homology, yet they share some common properties: they are intrinsically disordered regions, they all contain a serine-arginine (SR)-rich domain  and theyViruses hijack cellular machineries in many ways to cause infection and to force the host cell to produce copies of the virus. In the context of this hijacking procedure the viruses take advantage of short linear motifs that have the capacity to encode a functional interaction interface and are found in intrinsically disordered regions of both cellular and viral proteins . These sFollowing entry, the coronavirus genome needs to be translated by the cellular translational machinery to yield initially polyproteins pp1a and pp1ab and then through proteolytic processing the non-structural proteins that will drive viral genome replication and subgenomic mRNA synthesis . For traCoronavirus RNA-dependent RNA synthesis includes two distinct processes: genome replication, during which the coronavirus replicase synthesizes full-length negative “antigenomic” RNA which in turn serves as a template for the synthesis of new genomic RNA and transcription during which the replicase-transciptase complex synthesizes a set of negative sub-genomic RNAs which are then used as templates for the synthesis of positive-sense sub-genomic RNAs encoding the viral structural and accessory proteins . Both thN protein is the most abundantly expressed protein during viral infection  and is sVirus assembly occurs in the ER-Golgi intermediate compartment (ERGIC) and is orchestrated by the M protein , 2019. AThe nucleocapsid is then incorporated into a viral particle. Finally, coronavirus particles budded into the ERGIC are transported in smooth-wall vesicles and trafficked via the secretory pathway for release by exocytosis .Although the N proteins of different coronaviruses share low sequence homology, they all contain an SR/RS domain. A SARS-CoV genetic construct lacking the SR motif from the N gene showed defective replication and significantly reduced levels of infectious virions , highligAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Viral RNA exported from the coronavirus replication organelle is likely to be mediated by a crown-shaped molecular pore. et al. used cryo–electron microscopy to identify a molecular pore that spans the double membrane . Six copies of a large coronavirus transmembrane protein formed the core of this structure, which may constitute a viral RNA export channel and provide a target for future antiviral interventions.Coronaviruses transform host cell membranes into peculiar double-membrane vesicles that have long been thought to accommodate viral genome replication. However, because these compartments appeared to be completely sealed, it has remained unknown how the newly made viral RNA could be exported to the cytosol for translation and packaging into new virions. Wolff Science, this issue p. 1395; see also p. 1306 Coronavirus genome replication is associated with virus-induced cytosolic double-membrane vesicles, which may provide a tailored microenvironment for viral RNA synthesis in the infected cell. However, it is unclear how newly synthesized genomes and messenger RNAs can travel from these sealed replication compartments to the cytosol to ensure their translation and the assembly of progeny virions. In this study, we used cellular cryo–electron microscopy to visualize a molecular pore complex that spans both membranes of the double-membrane vesicle and would allow export of RNA to the cytosol. A hexameric assembly of a large viral transmembrane protein was found to form the core of the crown-shaped complex. This coronavirus-specific structure likely plays a key role in coronavirus replication and thus constitutes a potential drug target. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the third and most impactful example of a potentially lethal coronavirus infection in humans within the past 20 years , Middle East respiratory syndrome coronavirus (MERS-CoV), and SARS-CoV-2. One advantage of MHV over these class 3 agents is the absence of serious biosafety constraints, thus making MHV suitable for in situ cryo-EM studies. We performed electron tomography (ET) on cryo-lamellae prepared by focused ion beam milling of cells in the middle stage of MHV infection. The tomograms revealed abundant perinuclear DMVs with an average diameter of 257 ± 63 nm (±SD), occasionally interconnected or connected to the ER as part of the reticulovesicular network described in previous work  to analyze the structure of coronavirus-induced ROs in their native host cellular environment. The murine hepatitis coronavirus (MHV) is a well-studied model for the genus Reoviridae  , and engTo investigate whether nsp3 is a component of the DMV molecular pore, we imaged cells infected with a well-characterized engineered MHV expressing nsp3 with an enhanced green fluorescent protein (EGFP) moiety fused to the Ubl1 domain [MHV-Δ2-GFP3  revealedThe molecular pores frequently appeared to interact with other macromolecules on both the cytosolic and DMV luminal sides (fig. S8). In the subtomogram averages, these appeared as largely blurred out densities , which sThe interaction partners of the cytosolic nsp3 prong ranged from chain-like masses to larger assemblies . The subdomains of the long N-terminal nsp3 domain engage in a range of viral and virus-host interactions (Our findings suggest a pathway for newly made viral genomic RNA from the DMV interior, via the channel of the pore, to the cytosolic sites of encapsidation. In our model, specific replicase subunits may associate with the pore complex to guide the newly synthesized RNA toward it . As propThe double-membrane–spanning molecular pore revealed here may constitute the exit pathway for coronaviral RNA products from the DMV’s interior toward the cytosol, with the large and multifunctional nsp3 being its central component. Although the exact mode of function of this molecular pore remains to be elucidated, it seems to be a key structure in the viral replication cycle that is likely conserved among coronaviruses and thus may offer a coronavirus-specific drug target.


This unique ambidextrous optical reflection arises from left-handed chiral photonic architectures with lateral gradient photonic bandgaps and nematic-like defects at the film-substrate interface and between left-handed photonic bandgap layers acting as a half-wavelength retarder. We demonstrate that the tilted angle self-assembly method provides a feasible step toward color patterning of CNC-based photonic films capable of ambidextrous optical reflection.Artificial photonic materials displaying ordered reflected color patterns are desirable in the field of photonic technologies, however, it is challenging to realize. Here we present that self-assembly of cellulose nanocrystals (CNC) in a tilted cuvette leads to the formation of rainbow color CNC films. We show that the self-organized CNC films enable simultaneous reflection of left-handed circularly polarized (LCP) and right-handed circularly polarized (RCP) light with lateral gradient transmittance ratio (LCP/RCP: 8.7–0.9) and the maximum reflectance value up to  Plusiotis resplendens displaying optically ambidextrous reflection in a left-handed chiral nematic organization and the cuticle of beetle Chrysina gloriosa enabling strong polarization-insensitive reflection  offer ways to manipulate light, which are of significance for applications including circular polarizers , photoniflection . This efflection . These sflection . The lefflection . The wavnavg is the average refractive index, P is the helical pitch and θ is the incident angle from nematic surface  and the acid/cotton pulp mass ratio was kept at 10:1. The hydrolysis reaction took place at 45°C for 90 min under vigorous stirring, and the reaction was terminated using deionized water. The solid product was centrifuged  several times and dialyzed against deionized water until pH = 7. The suspension was diluted to desired concentrations for keeping.Two milliliter of CNC suspensions  without macroscopic phase separation was allowed to assemble in a tilted quartz cuvette (25°C) as illustrated in CNC-30 film showed colors in order of red, orange, green and blue from the higher to the lower end of the cuvette against a black background when viewed in normal white light . Notablyca. 38 and 8% at 655 and 660 nm, respectively, consistent with the optical microscopy studies . High-magnification SEM images of the film cross-section revealed a chiral nematic-nematic bilayer architecture , where tL to the propagating light following the Eq. 2,When rotated around a plane perpendicular to the crossed polarizers in a transmission polarizing optical microscopy, the zone 1 showed birefringent patterns and a change in the transmitted intensity of some color appearances from minimum to maximum at a 45° rotation, confirming the presence of nematic-like phase . These nn is the net birefringence, ne and no are the extraordinary and ordinary refractive indices of birefringent medium, respectively  by evaporation-induced self-assembly method and by vertical deposition method , respectca. 72%. It allows lateral gradient change in assembly duration and local CNC concentrations and can be manipulated by a change in tilt angle. The formation of nematic-like phase at the film-substrate interface is owing to the combined effect of kinetic stabilization of transient phase and seeded growth. The self-organization of rainbow color CNC films in a tilted cuvette is demonstrated as a step toward synthetic construction of one-dimensional chiral photonic materials displaying optical patterning and ambidextrous reflection with lateral gradient chiroptical properties for photonic applications.We have presented experimental evidences that tilt angle self-assembly produces chiral photonic CNC films displaying rainbow colors and lateral gradient ambidextrous optical reflection that can be tuned by a change in the tilt angle. We demonstrate how the circularly polarized light reflection of both handedness can be manipulated to produce chiral photonic CNC films with changing ambidextrous reflectivity and maximum reflectance values of The original contributions presented in the study are included in the article/JT wrote this manuscript, designed, and carried out the whole experiments. JL and XY contributed to cellulose nanocrystals preparation and partial data collection. LW contributed to SEM imaging collection. YX supervised the work, contributed to data analysis, and manuscript revision. All authors discussed the results and contributed to the completion of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Studies have suggested that embryo-endometrial developmental asynchrony caused by slow-growing embryos can be corrected by freezing the embryo and transferring it back in a subsequent cycle. Therefore, we hypothesized that live birth rates (LBR) would be higher in frozen embryo transfer (FET) compared with fresh embryo transfers.To compare LBR between fresh and FET cycles.A cross-sectional analysis of 10,744 single autologous embryo transfer cycles that used a single cleavage stage embryo was performed. Multivariate analysis was performed to compare LBR between FET and fresh cycles, after correcting for various confounding factors. Sub-analysis was also performed in cycles using slow embryos.Both LBR (19.13% vs 14.13%) and clinical pregnancy (22.48% vs 16.25%) rates (CPR) were higher in the fresh cycle group (p This study shows a lower LBR in FET cycles when compared to fresh cycles. Our results suggest that any potential gains in LBR due to improved embryo-endometrial synchrony following FET are lost, presumably due to freeze-thaw process-related embryo damage. Prerequisites for successful implantation are an embryo with implantation competency, a receptive endometrium, and synchronous development between the embryo and the endometrium. (1-4). The relative contributions of reduced embryo viability, a non-receptive endometrium, or embryo-endometrial asynchronous to implantation failure following IVF are unknown. There have been statistical analyses that suggest both embryo viability and uterine receptivity play significant roles . Another approach to investigating these issues is to compare pregnancy outcomes following fresh versus frozen embryo transfers (FETs).It is widely believed that controlled ovarian hyperstimulation (COH) used in IVF cycles has detrimental effects on the endometrium, as well as potentially disrupting normal synchronous development between the endometrium and the embryo (7-17). It has been shown that pregnancy is more likely when there are fewer endometrial histological alterations after COH (18). No pregnancy was reported if development of the endometrium was greater than 3 days more advanced than the embryos .Evidence that synchronous development between the embryo and the endometrium is important for successful implantation comes from work comparing normal-growing and slow-growing embryos on implantation rates and pregnancy rates (PR) between fresh autologous and FET cycles. As expected, the clinical PR was higher for normal-growing than the slow-growing embryos in fresh cycles (51% vs. 33.3%). However, if the slower blastocyst growth rates are compensated for by transferring on developmental age (day 5) rather than chronological age (day 6), then there was no significant difference in The PR between the normal- and slow-growing cryopreserved blastocysts following FET cycles (63.6% vs. 58.9%). Slow embryos were also associated with a significantly greater PR in FET cycles than in fresh autologous cycles (58.9% vs. 33.3%). (20) This study supports the hypothesis that embryo-endometrial developmental asynchrony caused by slow-growing embryos can be corrected by freezing the embryo and transferring it back a day earlier in a subsequent cycle.Further support for FET giving improved results to fresh transfers comes from a recent randomized multicenter trial involving women with polycystic ovarian syndrome (PCOS) (21). This study demonstrated a higher live birth rate  with FET than fresh embryo transfer cycles.The primary aim of this retrospective study of 10,744 single embryo transfers from the Melbourne IVF (MIVF) database was to compare pregnancy outcomes between fresh versus frozen autologous transfer cycles. We hypothesized that live birth and clinical pregnancy rates (CPR) would be higher in FET compared with fresh embryo transfers due to a combination of improved endometrial receptivity and improved embryo-endometrial synchrony in the FET cycles. Given that this is the largest such dataset ever published, we also hypothesized that the analysis would also provide important insights into factors that influence implantation rates in IVF and FET cycles.We performed a cross-sectional analysis using data obtained from the MIVF patient database. Transfer of cleavage-stage embryos (day 2 embryos) was a standard practice at the MIVF at the time of the data collection. We, and the majority of IVF centers, now transfer and freeze blastocysts; however, given the unique size of the dataset, we anticipated that we would gain new insights into factors influencing embryo implantation.According to the MIVF laboratory protocol, each embryo was evaluated twice before transfer. The first evaluation was performed 23-24 hr post-insemination/ICSI; referred to henceforth as the syngamy check. During this evaluation, embryos were assessed for the presence and number of cells (early cleavage (EC), nuclear envelope breakdown (NEBD), or 2 pronuclei (2PN)). The second evaluation was done on the morning of fresh embryo transfer (or before cryopreservation) on day 2 post-insemination/ICSI. Number of cells, degree of fragmentation, and multinucleation were assessed at the day 2 check. All embryos were cryopreserved using the slow freeze method (routine practice at the time of study) (22). The MIVF freeze-thaw protocols use post-thaw embryo culture to confirm resumed embryo development. Embryos for transfer in FET cycles are thawed the afternoon before the day of embryo transfer. These embryos are again evaluated twice; immediately after thawing for the number of surviving cells and a second time just prior to transfer in order to assess the resumption of mitosis and the total number of cells. Embryos are deemed suitable for transfer only if they survive the freeze-thaw process, defined as survival of Information about embryo transfer cycles using cleavage-stage embryos was retrieved from the MIVF database. Given the large size of the database, we were able to enforce strict inclusion and exclusion criteria, but still retained a large dataset to analyze. A maximum of two stimulated cycles  were included in the analysis for each patient. Loss of a blastomere that is evident immediately post thaw is known to be associated with reduced embryo implantation potential (23). Therefore, we excluded those cycles using embryos where cell loss occurred during post-thaw embryo culture and evaluation. We also excluded cycles involving transfer of more than one embryo, use of donor gametes, or embryos with pre-implantation genetic testing. Women who had been pregnant from a previous IVF treatment were also excluded from the analysis.We compared the clinical outcomes between fresh embryo transfer and FET cycles. The main outcome was LBR. A `live birth' is defined by the World Health Organization to be `the complete expulsion or extraction from its mother of a baby, irrespective of the duration of the pregnancy, which, after such separation, breathes or shows any other evidence of life'. The secondary outcome of this study was CPR. Clinical pregnancy is defined as the presence of fetal heart beat at first viability ultrasound .Stratification of cycles into fast- and slow-growing embryos was also performed. Slow cleavage-stage embryos were defined as those embryos that had 2PN during the syngamy check and were still at the 2-cell stage at the day 2 check. For the FET cycles, all embryos were at the 2-cells stage when frozen, and both cells survived the thawing process. Due to the post-thaw embryo culture protocol at MIVF for FET cycles, extra time allowed for development of frozen embryos, these embryos were half a day more advanced chronologically than their counterparts in fresh cycles. With the assumption that endometrial development is slightly advanced by COH, transferring frozen-thawed embryos that are half a day more advanced chronologically should improve embryo-endometrial developmental synchrony, leading to better CPR and LBR outcomes.This study was approved by the Royal Women's Hospital (RWH) Research Committee and RWH Human Research Ethics Committee (Project AQA19/15).t test, while binary variables were initially examined using the Chi-square test. Multivariate analysis was undertaken using logistic regression. For the purpose of model building, variables that reached p-value of Data were analyzed using the STATA 9.2  statistical and data analysis program. Continuous variables were examined in relation to relevant outcomes using the students A total 10,744 cycles involving transfer of a single cleavage-stage embryo were identified between July 2009 and April 2015, comprising 7,014 fresh cycles and 3,730 FET cycles. Table I shows the main outcomes and potential confounders in the two groups. Surprisingly, both LBR and CPR were higher in the fresh cycle group compared to the FET group . Controlling for these confounding factors confirmed the finding of lower LBR in FET cycles. These results do not support our hypothesis that LBR and CPR would be higher in FET compared with fresh embryo transfers due to a combination of improved endometrial receptivity and improved embryo-endometrial synchrony in the FET cycles. Rather, they suggest that any potential gains in LBR due to improved endometrial receptivity and improved embryo-endometrial synchrony are lost with FET, presumably due to embryo damage caused during the freeze-thaw process.A secondary finding from this study was a sub-analysis of cycles with slow-growing embryos. We hypothesized that embryo-endometrial developmental synchrony would be improved in FET compared to fresh cycles using slow embryos due to the post-thaw embryo culture protocol. In our analysis of 1,154 cycles using slow embryos, there was no statistical difference in LBR or CPR between the two groups. We interpret this result as showing that the reduction in LBR due to freeze-thawing damage to the embryo seen in fast-growing embryos is fully compensated for by the improved synchrony in the slow-growing embryos. Slower-growing embryos typically result in lower PR in fresh cycles, potentially due to reduced embryo viability and increased embryo-endometrial asynchrony. Taken together, the results from this study confirm the positive influence of younger maternal age, better embryo quality, faster embryo development and improved embryo-endometrial developmental synchrony on LBR, while also demonstrating that embryo freezing has a negative impact on LBR.There have been a limited number of published studies involving smaller sample sizes comparing clinical outcomes between fresh and FET cycles. While each of these provides some insight into the relative contributions of embryo viability, uterine receptivity, and embryo-endometrial synchrony to LBR, the individual studies are not all directly comparable and their findings not in complete agreement.The study published by Shapiro and colleagues mentioned earlier in this paper, found increased CPR in the FET cycle cohorts. It is important to note that the best blastocysts of the cohort were transferred in fresh cycles, while only good-quality embryos (expanded supernumerary blastocysts) were selected for cryopreservation and subsequent use in FET cycles. Embryo selection was a potential confounder in this study, which was acknowledged by the authors in their article (20). Two subsequent prospective randomized studies by the same group comparing fresh blastocyst transfer and oocyte cryopreservation (with subsequent blastocyst transfer grown from the frozen-thawed oocytes) has shown a significantly greater CPR in the oocyte cryopreservation group in normal responders, but no statistical difference in high responders . Overall, these studies support the hypothesis that FET improves CPR, presumably through improving uterine receptivity and/or embryo-endometrial synchrony. There is no evidence from these studies for a negative effect from freeze-thawing on embryo viability, possibly due to factors such as reduced blastomere size in the blastocyst compared to a cleavage-stage embryo. An alternative explanation is that by selecting embryos that have reached the expanded blastocyst stage, it is possible to eliminate all the nonviable embryos that appeared “normal” at the cleavage stage.In two prospective cohort studies involving use of cleavage-stage embryos and large number of cycles, LBR was lower in FET cycles when compared to fresh cycles . Four other randomized controlled trials using cleavage-stage embryos have also found no difference in LBR between fresh and FET cycles (28-31). The first trial involved 2,157 young (20-35 year old) women undergoing IVF/ICSI treatment due to tubal and/or male factors infertility (28). In the second trial, 782 infertile women were randomly assigned to fresh transfer or frozen embryos on day 3. In the FET group, only good-quality embryos (grade 1 or 2) were used (29). The last two studies involved women at risk of OHSS . All studies involved the transfer of multiple embryos (28-31). Differences between the above studies and our study include patient population, number of embryos transferred, and embryo freezing method. Any or all of these factors could have contributed to the different clinical outcomes between these studies (no difference in LBR between fresh and FET cycles) and ours (higher LBR in fresh cycles).While studies involving blastocyst transfer have suggested a better LBR with FET cycles , our results and other studies using cleavage-stage embryos have not found the same (28-30). A major difference between protocols is that cleavage-stage embryos are cryopreserved according to their chronological age (day 2), while blastocysts are only cryopreserved if they reached the desired developmental stage of expanded blastocyst. The developmental stage of embryos is an important factor when considering embryo-endometrial synchrony. The cleavage-stage embryo freezing protocol has not been designed to correct for any potential asynchrony between developmental stage for the embryo and endometrium. There may be clinical benefit in the future to considering developmental stage rather than chronological age when thawing cleavage-stage embryos for transfer.In conclusion, in this retrospective study of 10,744 IVF cycles, we were not able to support our hypothesis that LBR and CPR would be higher in FET compared with fresh embryo transfers. Multivariate analysis with correction for five significant variables showed a significantly higher LBR rate in cycles using fresh embryos compared to frozen-thawed embryos. In our sub-analysis of cycles involving slow-growing embryos, we interpreted the fact that there was no statistical difference in LBR between the fresh and FET groups as evidence for improved embryo-endometrial synchrony in the FET cycles. The large size of our dataset coupled with the strict inclusion and exclusion criteria have allowed us to identify six variables that significantly impact LBR, in addition to the results from our sub-analysis on slow-growing embryos which provides data to support the view that embryo-endometrial developmental synchrony is important. While most IVF units have moved to blastocysts transfer and freezing, the findings from this study on two-day old embryos remain highly relevant for understanding and optimizing LBR in today's IVF clinic.None declared.


T cells constituting one of the arms of adaptive immune responses provide cell-mediated immunity against offending pathogens. Thymus is the maturation site for T cells that have been shown to be involved in cell-mediated immunity and humoral immune response in 1961–1962. It took another couple of decades to identify heterodimeric T cell receptor, which is crucial for the T cell activation, differentiation, and functions . As the appreciation of T helper subset plasticity increases, it becomes even more important to characterize them. Distinctly opposite T helper subsets can express the same receptor, secrete a common cytokine and be regulated by the same transcription factor. Study by Huang et al., urge caution in using LAG3/CD49b co-expression as standalone markers for Tr1 cell identification as they can also be expressed by Foxp3+ Tregs and CD8+ T cells. Further studies would dissect the physiological relevance of the expression of these markers in different T cell subsets. Several studies have indicated the participation of distinct T helper subsets in the pathophysiology of inflammatory disorders . B cells regulated by Tfh cells could move to the CNS and undergo class switching that correlated with disease severity .Differentiation and regulation of CD4+ T cells depend on a plethora of factors including strength of antigen-antibody interaction, amount of co-stimulation, cytokines present in the milieu, expression of transcription factors and their interaction with histone modifiers. During the development of thymocytes CD154 co-stimulation plays prominent role to the TCR repertoire diversity. CD154 deficiency attenuates the sharing of TCRβ clone compared to the wild-type in T-cell dependent immune responses, leading to incorrect editing of T-cell clonotypes during the negative thymic selection . 25-OHC can negatively regulate Tr1 cells for the production of IL-10. T cell metabolism has been targeted for efficient cancer immunotherapy and altered effector T cell functions [. Additional metabolites including lipids, ATP, nitric oxide, NAD also play crucial role in the differentiation of CD4+ T cells. Both mTOR and AMPK are sensors that regulate the metabolic checkpoints of T cell differentiation. mTOR inhibitors can attenuate glycolysis to induce memory T cell differentiation, while AMPK inhibitors decrease metabolism of fatty acids that in turn promote the differentiation of Th1 and Th17 cells. In contrast, activation of AMPK pathway can impart an analgesic effect in inflammatory pain by attenuating Il1β expression and blocking NF-κB activation (Significant amount of information has been generated by researchers on the outcome of CD4+ T cells in various inflammatory disorders in both mice and humans. Behavior of CD4+ T cells, are regulated by internal metabolic properties. Lipid metabolites can act as regulators of immune responses. Alteration of steroids pathways can affect inflammation and be responsible for the pathophysiology of various diseases. The enzyme cholesterol 25-hydroxylase, which synthesizes 25-OHC, can enhance IL-27-induced Ttivation . In naïvtivation . The zinCollectively, the articles published within the Research Topic highlight the emerging roles and underlying mechanisms of T cell differentiation and functions in tissue inflammation and their impact in the pathogenesis of inflammatory diseases. Based on the published work under this topic, it is further required to understand the functional dynamics of Th cell plasticity that lead to the ultimate outcome of immune-pathogenesis of diseases and lead to advancing our understanding for the immunological basis of diseases. The acquired immunological-based knowledge from the published articles will contribute further refined and novel immune strategies for inflammatory conditions.RG and AA conceived, designed, and wrote the manuscript. All the authors read and approved the final manuscript for publication.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The polymer self-assemblies were convenient to encapsulate arbutin (ARB) as the selected active substances. Moreover, the conjugates of PEG graft copolymers with ferulic acid (FA) or lipoic acid (LA) were also investigated. The permeability of released active substances through a membrane mimicking skin was evaluated by conducting transdermal tests in Franz diffusion cells. The biological response to new carriers with active substances was tested across cell lines, including normal human dermal fibroblasts (NHDF), human epidermal keratinocyte (HaCaT), as well as cancer melanoma (Me45) and metastatic human melanoma (451-Lu), for comparison. These polymer systems were safe and non-cytotoxic at the tested concentrations for healthy skin cell lines according to the MTT test. Cytometric evaluation of cell cycles as well as cell death defined by Annexin-V apoptosis assays and senescence tests showed no significant changes under action of the delivery systems, as compared to the control cells. In vitro tests confirmed the biochemical potential of these antioxidant carriers as beneficial components in cosmetic products, especially applied in the form of masks and eye pads.The in vitro biochemical evaluation of the applicability of polymers carrying active substances (micelles and conjugates) was carried out. Previously designed amphiphilic graft copolymers with retinol or 4- Carriers of active substances are a topic often examined due to the high demand for medical and cosmetic products that ensure a controlled course of delivery and release of substances. Many biologically active substances are unstable and sensitive to temperature, pH, light, and oxidation; therefore, they require carriers that protect them against undesirable degradation . DesirabAn important aspect for the determination of newly prepared transdermal systems is the ability to penetrate and diffuse through the skin and/or diffusion of the released active substance, including drug. In vitro tests of absorption through the skin have been applied for parabens , ketoproDepending on the target application, the cytotoxicity may be desirable activity (cancer therapy) or is a feature that causes disqualification of the bioactive substance/carrier (cosmetics) . Comprehn-butylresorcinol as the terminal or center unit in the main chain, as it was also postulated for linear polymethacrylates b-PEG-b-preported  systems.I/DLCII = 99%/77%). This means, in the case of skin melanoma cells, the resulting carriers will not be therapeutic. A similar relationship was detected for the conjugate with FA (III) or LA (IV). They presented the effect of increasing cell viability, especially for the Me45 cell line (cell proliferation 123–143%). However, from the point of view of the target application of the tested carriers with active cosmetic substances, their therapeutic effect on skin melanoma cells was rather unexpected, but it was tested comparatively due to a possible positive therapeutic “side effect”, which in our case did not exist.Additional MTT tests were conducted on two cancer skin cell lines . The appExposure of NHDF and HaCaT cell lines to carriers caused a significant increase in cell confluence after a 72 h incubation as compared to control cells CTR, . The typAn in vitro evaluation of NHDF, HaCaT, and Me45 model cell lines showed an almost non-toxic effect on human dermal fibroblasts and epidermis, as well as melanoma cells. A cytometric evaluation revealed the effect of the tested carriers with active substances (micelles and conjugates) did not significantly alter the cell cycle phase distribution compared to the untreated control populations of NHDF a,b or HaAnalysis of the sub-G1 cell fraction indicated the presence of a small number of damaged cells containing fragmented DNA but little additional information. Therefore, an apoptosis test was conducted to characterize the type of cell death induced by a carrier solution with active substances. Studies on necrotic (A−/PI+) and apoptotic  cell changes provide important information on the cytotoxicity of tested carriers with active substances. The test was based on the use of Annexin-V (that detects early apoptosis) in combination with propidium iodide (PI), which penetrates cells being in necrosis. The cell lines were treated with carriers in two doses: 3 µg/mL and 100 μg/mL.100, II100). The slight increase in necrotic (A−/PI+ = 5–6%) and apoptotic (A+/PI- and A+/PI+ = 5–6%) cells detected in samples treated with 100 μg/mL conjugate solutions  was small enough that there was no coverage in previously obtained cell cycles or cytotoxicity results. Additionally, there was no significant effect of the carrier or its concentration on apoptosis and necrosis for HaCaT cells noticed in comparison with the control , the possibility of uncontrolled cell death as a result of mechanical damage was lower, and instead, these cells entered the apoptosis pathway.The results for the NHDF cells  showed l control . As in t control , where aThe last stage for determining the safe use of the tested carriers in cosmetic products was realized by cell senescence tests. Cell aging is associated with the loss of the ability of cells to divide, changes in cell morphology, shape, physical appearance, and gene expression patterns. The assay performed was based on a histochemical stain for β-galactosidase as the most widely used biomarker of cellular senescence activated at pH = 6. Under these conditions, β-galactosidase catalyzes the hydrolysis of the chromogenic substrate, i.e., galactose linked to a substituted indole, which after cleavage gives blue staining easily detectable in senescent cells, but undetectable in quiescent or immortal cells. The senescent cells were observed as blue-stained cells . The shagraft-PEG)-co-MMA) and P(HEMA-graft-PCL)-co-MPEGMA), as well as the graft copolymer based conjugates with covalently attached antioxidants P(HEMA-click-FA)-co-MPEGMA) and P((HEMA-click-LA)-co-MPEGMA) were designed for delivery of substances applicable in cosmetology, including dermatological problems. The biochemical evaluation of both types of polymer carriers (micelles and conjugates) indicated diffusion of the released active substance into the solution in Franz cells, but its significant part was remaining in the membrane working as the artificial skin. The permeability properties were dependent on the carrier particle size and strongly related to the hydrophilic-lipophilic balance in the copolymer. The application of a higher concentration of the carrier resulted in slightly higher viability of the healthy skin cell lines . The exposure of healthy cell lines to carriers caused a significant increase in cell confluence, especially for the NHDF cell line treated with the conjugate with FA. The carriers did not significantly alter the cell cycle phase distribution, the type of cell death of tested cell lines and did not induce cell senescence. The evaluated PEG graft copolymers are promising safe carriers for the transdermal delivery of active substances, e.g., antioxidants, including skin therapy in cosmetology. In vitro biological studies are an excellent starting point for the next phase of mandatory ex-vivo tests on human skin in lab conditions, dermatological studies with a contact test on hypoallergenic tape, and finally in vivo studies on volunteers with different skin types.Polymeric micelles with ARB loaded by amphiphilic graft copolymers P((HEMA-


Allergen-specific immunotherapy (AIT) is a highly economic, effective and disease-modifying form of allergy treatment but requires accurate prescription and monitoring. New molecular approaches are currently under development to improve AIT by reducing treatment-related side effects, cumbersome protocols and patients’ compliance. We review the current advances regarding refined diagnosis for prescription and monitoring of AIT and the development of novel molecular vaccines for AIT. Finally, we discuss prophylactic application of AIT.There is evidence that molecular allergy diagnosis not only assists in the prescription and monitoring of AIT but also allows a refined selection of patients to increase the likelihood of treatment success. New data regarding the effects of AIT treatment with traditional allergen extracts by alternative routes have become available. Experimental approaches for AIT, such as virus-like particles and cell-based treatments have been described. New results from clinical trials performed with recombinant hypoallergens and passive immunization with allergen-specific antibodies highlight the importance of allergen-specific IgG antibodies for the effect of AIT and indicate opportunities for preventive allergen-specific vaccination.Molecular allergy diagnosis is useful for the prescription and monitoring of AIT and may improve the success of AIT. Results with molecular allergy vaccines and by passive immunization with allergen-specific IgG antibodies indicate the importance of allergen-specific IgG capable of blocking allergen recognition by IgE and IgE-mediated allergic inflammation as important mechanism for the success of AIT. New molecular vaccines may pave the road towards prophylactic allergen-specific vaccination. AIT is the only treatment that instructs the immune system of the patients for protection by therapeutic vaccination and can prevent the progression of the severity of allergic disease . Using allergen microarrays containing small amounts of immobilized allergens, it was found that the development of blocking IgG antibodies is reflected by a decrease of allergen-specific IgE binding through competition of IgG with IgE for the binding sites on the allergens. This decrease of the IgE binding also serves as surrogate marker for effective AIT  suggesting that clinical outcomes might be improved by selecting AIT extracts fitting the patient's sensitization profile . Another interesting diagnostic option are chips-containing micro-arrayed peptides derived from human rhinovirus-strains, which together with allergen chips can be used for the differential diagnosis of allergen-induced and rhinovirus-induced asthma . The latter study reflects well the magnitude of effect of SLIT with tablets observed in earlier large AIT trials   studies showed that ILIT is clinically effective but the treatment-induced IgG antibody levels were not higher than in subcutaneous AIT (SCIT) studies and in one study no effective boost of antibody levels by the booster injection was observed ▪▪,36▪▪  studies performed with peanut allergen extracts provide evidence for clinical effects ▪▪,38▪▪  is mainly conducted with digestion-resistant food allergens  but does45▪] Table . Palforzet al. .Despite the use of allergen peptides, which were thought to be hypoallergenic severe side effects were observed in the course of a randomized, multicenter DBPC trial . There win vitro[▪▪]  of the molecules and at the same time to maintain allergen-specific T-cell epitopes  Fig. 2)2). The fin vitro. In the in vitro  [A few preclinical results describing recombinant vaccine constructs may also be mentioned. For example, basophil activation experiments demonstrated that a hybrid consisting of major allergens from two different aeroallergen sources, Bet v 1 and Phl p 5 induced a much reduced activation, compared with the monomeric forms of the proteins . SimilarDer p 7) . Mice imDer p 7) .Allergen-derived synthetic peptides containing T-cell epitopes and lacking IgE reactivity have been considered for AIT for almost 30 years. This approach was thought to induce T-cell tolerance with the hope that it would reduce allergen-specific IgE production and induce protective allergen-specific IgG responses. The AIT studies performed in experimental animal models and in clinical trials in humans provide indeed evidence that peptide-based AIT may induce signs of immunological tolerance Fig. . In a re4 antibodies up to two pollen seasons after treatment [An induction of allergen-specific IgG antibodies was only observed with longer, immunogenic peptides as reported in a phase IIb trial performed with contiguous overlapping peptides from Bet v 1 ▪  and by the Country of Lower Austria.R.V. has received research grants from the Austrian Science Fund (FWF), from Biomay AG, Vienna, Austria and from Viravaxx, Vienna, Austria. He serves as a consultant for Viravaxx, Vienna, Austria. The other authors have no conflict of interest to declare.


Focus on frailty status has become increasingly important when determining care plans within and across health care sectors. A standardized frailty measure applicable for both primary and secondary health care sectors is needed to provide a common reference point. The aim of this study was to translate the Clinical Frailty Scale (CFS) into Danish (CFS-DK) and test inter-rater reliability for key health care professionals in the primary and secondary sectors using the CFS-DK.n = 10), community nurses (n = 10), hospital doctors from internal medicine (n = 10) and intensive care (n = 10). Inter-rater reliability was assessed using intraclass correlation coefficients (ICC), and sensitivity analysis was performed using multilevel random effects linear regression.The Clinical Frailty Scale was translated into Danish using the ISPOR principles for translation and cultural adaptation that included forward and back translation, review by the original developer, and cognitive debriefing. For the validation exercise, 40 participants were asked to rate 15 clinical case vignettes using the CFS-DK. The raters were distributed across several health care professions: primary care physicians (The Clinical Frailty Scale was translated and culturally adapted into Danish and is presented in this paper in its final form. Inter-rater reliability in the four professional groups ranged from ICC 0.81 to 0.90. Sensitivity analysis showed no significant impact of professional group or length of clinical experience. The health care professionals considered the CFS-DK to be relevant for their own area of work and for cross-sectoral collaboration.The Clinical Frailty Scale was translated and culturally adapted into Danish. The inter-rater reliability was high in all four groups of health care professionals involved in cross-sectoral collaborations. However, the use of case vignettes may reduce the generalizability of the reliability findings to real-life settings. The CFS has the potential to serve as a common reference tool when treating and rehabilitating older patients.The online version contains supplementary material available at 10.1186/s12877-020-01850-y. It is a global concern that even highly effective health care systems will struggle to meet the demands of the increasing share of aged and frail populations . FrailtyMultiple scales and instruments for measuring frailty exist and have been tested in various settings , 8. The The CFS was developed in Canada in 2005 and was validated for diagnostic accuracy of frailty in people aged 65 years and over in the primary care sector . The oriThe aim of the current study was first to translate the CFS into Danish (CFS-DK) using standard methodology and then to test inter-rater reliability for key health care professionals in the primary and secondary sectors using the CFS-DK.The study was conducted from 16th March to 8th May 2020.To ensure cultural and conceptual compliance with the source instrument , 11, we Health care professionals from the primary and secondary health care sectors were recruited to validate the CFS-DK and to support potential future use in a cross-sectoral context. Forty raters assessed 15 written clinical case vignettes using the CFS-DK. The raters were 10 community nurses, 10 general practitioners, and 20 hospital doctors  and were recruited as a convenience sample from the authors’ professional network.Cases consisted of a short text and a picture of each case-patient and were selected to collectively represent all nine levels of the CFS. Cases provided essential information on 1) symptoms of diseases, 2) dependency on others, 3) cognitive function, and 4) physical condition. Each case was presented alongside a picture of the CFS-DK for reference  , 19, an Inter-rater reliability was assessed using the intraclass correlation coefficient (ICC) with 95% confidence intervals (CI) , 21 thatThe sensitivity of the inter-rater reliability was examined using a random effect linear regression  with CFSP < 0.05. Figures were made using “R” software (Version 3.6.1) ” and “[declined palliative therapy]”, which require health care professionals recognizing the implied consequences .Calibration of the CFS scale is likely dependent on frailty incidence , and mayThe responsibility for care in the frail population varies across European countries, and this has implications for the future use of the CFS. In the Scandinavian countries and the Netherlands, there is societal consensus for a welfare system in which the public sector is responsible for providing good and equal health and long-term social care –32. Howen = 15). However, the use of case vignettes allowed a high number of observations of 600 , a quadrupling compared to previous investigations of inter-rater reliability following translation of the CFS [This study used clinical case vignettes, and although the case vignettes were clinically appropriate and built to imitate real-life patients, they could also be regarded as hypothetical. Case vignettes provide the opportunity to let raters assess the exact same information, with the same structure and nearly identical setting at the same time point, allowing equal comparisons between professional groups. The cases were designed to encompass all the CFS levels. Ratings given to the 15 cases clearly reflect these different degrees of severity. Conversely, case vignettes introduce a risk of inflated inter-rater reliability and ICC as raters assess all cases at once, which does not reflect clinical practice.Another limitation to this study is the relatively small number of cases ( the CFS .Finally, raters were recruited as a convenience sample, inherently risking inflated reliability measures and sensitivity analyses.This study has several strengths. First, the translation process was completed using a rigorous procedure that followed the ISPOR guidelines. Poorly translated instruments threaten the validity of the data, and quality is dependent on methodology . Second,The Clinical Frailty Scale was translated and culturally adapted into Danish following a careful and well-established standard process. The inter-rater reliability was high in all four groups of health care professionals involved in cross-sectoral collaborations. However, the use of case vignettes may reduce the generalizability of the reliability findings to real-life settings. The CFS has the potential to serve as a common reference tool when treating and rehabilitating older patients.Additional file 1.


Only vertebrates and Caenorhabditis genomes carry two uggt gene copies (uggt–1 and uggt–2) and phylogenetic inference showed that uggt genes went through independent duplications in Caenorhabditis and vertebrates. UGGT-1 retained canonical UGGT activity both in vertebrates and Caenorhabditis and vertebrate UGGT-2 underwent a specialization process. In Caenorhabditis, uggt-2 evolved by means of a putative neofunctionalization process in a non-redundant paralog and its biological function is still unknown . Hence, UGGT-1 is the only protein engaged in monitoring the folding state of every glycoprotein in Caenorhabditis ER. To determine C. elegans UGGT-1’s body pattern expression we used fosmid recombineering technology  to generate the Puggt-1::sl2::nls::gfp::unc-54 3’UTR transcriptional fusion reporter and established worm lines expressing this construct. UGGT-1 is expressed in the head, both in the pharynx,  and in the pharyngeal intestinal valve. In the same image its expression is detected in the hypodermis and in the secretory gland (B and C). The somatic cells of the spermatheca express UGGT-1, but not the germline (D). Consistent with our previous findings  UGGT-1 is widely expressed in the nervous system, both in ventral and dorsal nerve cords (E and F), as well as in the muscle cells as shown in (E-F) and in the anal depressor muscle (G). In the tail expression is also observed both in the rectal gland cell and the intestine.The endoplasmic reticulum (ER) uses an elaborate system called the ER quality control (QC) to monitor the proper folding of newly synthesized glycoproteins. The QC allows cells to differentiate between properly folded and misfolded proteins, allowing only those proteins which have acquired their native conformations to exit the ER and reach their final destinations. Alternatively, misfolded glycoproteins or incompletely formed glycoprotein complexes are translocated to the cytosol where they are finally degraded by proteasomes (Caramelo and Parodi 2007). The key element of this mechanism is the UDP-Glc: glycoprotein glucosyltransferase (UGGT) that functions as a folding sensor as it glucosylates exclusively those glycoproteins that have not acquired their native structures . Images were acquired with LSM software (Carl Zeiss) using a 20 x plan apochromat objective.Worms of stable transgenic lines carrying the