Gastric cancer is one of the leading causes of cancer death in the world. Improving gastric cancer survival prediction can enhance patient prognostication and treatment planning.In this study, we performed gastric cancer survival prediction using machine learning and multi-modal data of 1061 patients, including 743 for model learning and 318 independent patients for evaluation. A Cox proportional-hazard model was trained to integrate clinical variables and CT imaging features (extracted by radiomics and deep learning) for overall and progression-free survival prediction. We further analyzed the prediction effects of clinical, radiomics, and deep learning features. Concordance index (c-index) was used as the model performance metric, and the predictive effects of multi-modal features were measured by hazard ratios (HRs) at pre- and post-operative settings.Among 318 patients in the independent testing group, the hazard predicted by Cox from multi-modal features is associated with their survival. The highest c-index was 0.783  and 0.770  for overall and progression-free survival prediction, respectively. The post-operative variables are significantly (p<0.001) more predictive than the pre-operative variables. Pathological tumor stage , pathological lymph node stage , carcinoembryonic antigen (CEA) , chemotherapy treatment , radiomics signature , and deep learning signature ) are significant survival predictors.Our study showed that CT radiomics and deep learning imaging features are significant pre-operative predictors, providing additional prognostic information to the pathological staging markers. Lower CEA levels and chemotherapy treatments also increase survival chances. These findings can enhance gastric cancer patient prognostication and inform treatment planning. Gastric cancer is one of the leading causes of death worldwide . AccuratPrevious studies have reported a variety of clinical factors indicative of gastric cancer prognosis, including serum tumor markers, lymphovascular invasion, perineural invasion, histological grade, etc. –10. ReceIt is expected that the combination of multi-modal data, such as demographic information, clinical variables, imaging data, histopathologic findings, lab measurements, therapeutic interventions, can empower survival analysis of gastric cancer . CurrentWe built a machine learning prognostic model Figure 1We performed a retrospective study that received approvals by a local ethics committee and an institutional review board with a waiver of written informed consent. Our study complies with the 1964 Helsinki declaration and its later amendments. Initially a total of 1,647 patients with pathologically confirmed gastric cancer during 2014 to 2018 were identified for the study. The exclusion criteria included: i) patients who failed to undergo radical surgery; ii) patients with diagnosis of other cancers in addition to gastric cancer; iii) patients with any intervention or therapy before surgery; iv) patients with poor imaging quality unacceptable for computational analysis; and v) patients without pre-operative CT imaging available. Finally, 1,061 patients were included for analysis, which were randomly split to two independent study groups: Group-A of 743 patients (70%) for model development and Group-B of 318 patients (30%) for independent evaluation. Patients were followed up every 3-6 months, starting from the time of gastrectomy and censored at the last alive contact or by the time of this study . For each patient, we collected various clinical data and a pre-operative contrast-enhanced computed tomography (CECT) scan.We collected a set of clinical data acquired before and after the gastrectomy operation. The pre-operative variables include lab tests , demographic variables, qualitative radiologic staging variables , tumor location assessed by radiographic imaging and endoscopy, histologic grades by endoscopic biopsy. The post-operative data includes chemotherapy treatment information as well as surgical pathology variables . See Quantitative radiomic features are extracted from the segmented 3D tumor volume in the CECT images. The gastric tumor was segmented slice-by-slice and semi-automatically by two radiologists (QL and QXF) using an in-house developed and validated software . QL first segmented the lesion for all cases; and one week later, QL repeated segmentation on 30 patients to evaluate intra-reader variability. To evaluate inter-observer variability, QXF performed lesion segmentation on a selected subset of 30 patients. The lesion segmentation was conducted over approximately two months. A total of 1,210 radiomic features, which describe the tumor characteristics in terms of intensity, shape, texture, etc., are extracted from the segmented gastric tumor volumes using an open-source Python package Pyradiomics . The robDeep learning was used to extract potentially different features from the approximate local regions around segmented tumor. To this end, we designed an attention-guided Variational AutoEncoder (attention-guided VAE) model , combination of the full set of pre- and post-operative data (Setting 2), and combination of only the variables that are shown in Setting 1 to be statistically significant (p<0.05) (Setting 3). In addition, we performed one more round of feature selection using the random survival forest method from the full set of data at Setting 1 and only the selected variables were combined for modeling (Setting 4).In the deep learning feature extraction, the 743 patients in Group-A were randomly split into a training set (669 patients) and a validation set (74 patients) for model learning. The axial view CECT image with the largest cross-sectional area of tumor was selected as the input of the attention-VAE model. We used open-source software libraries PyTorch  to impleThe model performance was measured on the independent Group-B of 318 patients using concordance index (c-index) . Hazard Out of the 16 variables listed in When the full set of the pre- and post-operative variables are combined (Setting 2), the respective c-index of overall and progression-free survival is 0.703 and 0.743, both outperforming (both p<0.001) the pre-operative variables alone but underperforming (both p<0.001) the post-operative variables alone. This implies that these variables may not be optimally integrated by the Cox model in Setting 2. When combining only the significant variables (those with p<0.05 at Setting 1), as shown at Setting 3, the c-index increases to 0.708 for overall survival prediction, slightly higher (p=0.19) than using all the variables (0.703) at Setting 2, while still significantly lower (p<0.001) than the post-operative variables (0.783) at Setting 1; meanwhile, the progression-free survival prediction shows a similar observation at Setting 3, where the c-index increases to 0.761, which is significantly higher (p<0.001) than using all the variables at Setting 2 (0.743), but again, significantly lower (p<0.001) than the post-operative variables (0.770) at Setting 1. At Setting 4, the c-index increases to 0.721 for overall survival prediction, which is still significantly (p<0.001) lower than the post-operative modeling (0.783) in Setting 1, but significantly (p<0.001) higher than the combined full set (0.703) at Setting 2. Likewise, at Setting 4, the performance pattern of the progression-free survival is similar to that of the overall survival. The comparisons of these results indicate the following: (I) when the full set of pre- and post-operative variables are all combined (Setting 2), the c-index values increase and become closer to, but are still lower than, just using the post-operative variables; and (II) regardless of using only the significant variables with p<0.05 (Setting 3) or using the variables selected by a second process of feature selection (Setting 4), the c-index is improved than using the full set at Setting 2. When comparing Setting 3 and Setting 4, the c-index for overall survival is higher (p<0.001) at Setting 4, while the c-index for progression-free survival is higher (p=0.02) at Setting 3. This indicates that the two methods of selecting subset variables for modeling  have respective advantages for the two different survival prediction tasks.After comparing the prediction model’s performance, here we analyze the prediction effects of individual variables in terms of their hazard ratios. Here we first look at the overall survival prediction. It shows CEA , deep learning signature , and radiomics signature  are significant variables for pre-operative prediction; for post-operative prediction, the significant variables are pT , pN , gross appearance , and chemotherapy . At Setting 2, the two imaging variables  remain significant with similar hazard ratios, along with the following new observations: CEA became marginal (p=0.06), pT became insignificant (p=0.88)), PNI became marginally significant (p=0.05), gross appearance became insignificant (p=0.64), and chemotherapy’s hazard ratio increased to 0.440 from 0.254. At Setting 3, those significant variables still remain significant except the pT and pN; it should be noted that in this case, the c-index (0.708) is much lower than the post-operative prediction (0.783), indicating very likely that the effects of pT and pN were lost in this setting. It is interesting to see that at Setting 4, rT and rN are selected in the models; however, as their p values are greater than 0.05 and the HRs are close to 1, the predictive values of rT and rN are limited when combined with other more significant variables. Comparing Setting 3 and Setting 4, CEA and gross appearance are significant in Setting 3, but they are not selected at Setting 4; in contrast, pN is marginally significant (p=0.05) at Setting 4 but is in-significant (p=0.17) at Setting 3; the two imaging signatures and chemotherapy treatment remain the significant predictors at both Setting 3 and Setting 4 for the overall survival prediction.Similarly, we now compare the effects of these variables for the progression-free survival prediction. Specifically, for pre-operative prediction at Setting 1, the significant variables are almost the same with the overall survival prediction, except here the biopsy histologic grade is also significant . For post-operative prediction at Setting 1, the significant variables are also almost the same with the overall survival prediction, except that gross appearance is not significant. Most significant variables at Setting 1 remain significant at Setting 2, except that biopsy histologic grade and pN became insignificant. Interestingly, when combining only the significant variables as shown at Setting 3, the significant variables are CEA, the two imaging signatures, pT, pN, and chemotherapy. Comparing Setting 3 and Setting 4, the significant predictors remain the same in the two settings.In all the four settings, the hazard ratios for chemotherapy treatment are lower than one, indicating the chemotherapy treatment reduces the risk of death . The two imaging signatures play a significant prediction role of survival in all the four settings. For pT and pN, at Setting 3, they are not significant for overall survival while significantly predictive of progression-free survival; at Setting 4, pN is a significant predictor for both overall and progression-free survival, while pT is only significantly predictive for progression-free survival.In this study, we evaluated the combination of various clinical variables and quantitative CECT imaging descriptors for overall and progression-free survival prediction on gastric cancer patients. We identified five primary prognosis factors, including two pathological staging variables, the history of chemotherapy treatment, and two aggregated signatures from radiomics and deep learning. While multi-modal data have been increasingly used in machine learning modeling, our study provides a measurement on the quantitative effects of the examined multi-modal features for gastric cancer survival analysis. This can enhance gastric cancer patient prognostication.We found that in the models with the highest c-indices, the two pathological staging variables, pT and pN, are correlated with survival with highest hazard ratios. This suggests that the pathological staging data including both the depth of mural invasion and nodal involvement are closely indicative of patient survival. It is noted that when combined with pre-operative variables (including the imaging signatures), the effects of pT and pN are dismissed for overall survival prediction. This may have two important indications. First, because of the lower c-index at Settings 3 and 4, we suspect this may have to do with the modeling method in the Cox model, where variables are simply linearly concatenated and thus may not be optimal to capture more complicated non-linear interactions when the aggregated imaging signatures are incorporated in the model. Additional work on developing advanced modeling methods is therefore warranted. Second, at Settings 3 and 4 we found that the deep learning signature maintains high hazard ratios (like at Settings 1 and 2), while pT and pN are insignificant. This implies that the proposed deep learning model can extract quantitative imaging features that have overlapping information with pT and pN for overall survival prediction. This is a finding that highlights the important utility of pre-operative CECT imaging data coupled with the proposed deep learning modeling techniques. Interestingly, when looking at the progression-free survival at Setting 3, both the two imaging signatures and pT and pN are significant predictors with a similar magnitude of hazard ratios, which indicates that the information in the pre-operative CECT imaging signatures and the information in the pathological staging markers are complementary to each other for the progression-free survival prediction. Such complementary effects may align with the observation that CECT images can visualize the invasion of tumor into gastric wall (T stage) and the enlarged regional lymph nodes (N stage). Finally, it is not surprising to see that post-operative chemotherapy, with a hazard ratio consistently lower than one, can significantly increase survival.Radiomics are mathematically defined descriptors while deep learning features are less intuitive because of the complexity in deep neural networks. The two aggregated imaging signatures are identified as significant factors for both overall and progression-free survival prediction. These two signatures may convey distinct information on the high-dimensional CECT images. Radiomic features/signature quantify characteristics of the segmented intratumor regions. Tumor margins, or the peri-tumorous regions, may also carry active and predictive information related to patient outcomes . The deeThe focus of our study is to examine the effect and relationship of multi-modal features for gastric cancer survival prediction. Meanwhile, our model’s c-index values are in line with previously reported studies , 11. ForOur study has some limitations. While our study included more than one thousand patients with complete data to enable the performed analyses, additional evaluation using external datasets will further validate our findings. The Cox model is more explainable but may be less effective to integrate non-linear interactions among multi-modal features. This study indicates the needs of developing more advanced models in future work. In addition, indications to chemotherapy were not consistently applied to the enrolled patients according to the NCCN guideline, which reflects a limitation of retrospective analysis. Finally, the tumor segmentation is semi-automated, which may have introduced certain level of dependence to the data annotators. While showing a high intra- and inter-observer agreement on segmentations, we expect to use fully automated and robust tumor segmentation methods when they become available.We integrated multi-modal data for gastric cancer survival prediction and evaluated their individual and combined effects. Our study showed that quantitative radiomics and deep learning imaging features are significant pre-operative predictors of survival, providing additional prognostic information to the pathological staging markers. Lower CEA levels and chemotherapy treatments independently increase survival chances. Our findings provide quantitative effect measures on these markers in pre- and post-operative survival prediction, which will enhance gastric cancer patient prognostication and benefit treatment planningwus3@upmc.edu.The datasets presented in this article are not currently available to the public because of internal regulations and considerations of our research and publishing plans. Readers who are interested to the datasets may send a request to The studies involving human participants were reviewed and approved by an ethics committee of the First Affiliated Hospital with Nanjing Medical University (2018-SR-043) and the Institutional Review Board of University of Pittsburgh (STUDY19080135). The ethics committee waived the requirement of written informed consent for participation.SW and YZ jointly conceived the concept and supervised the study. DH, YZ, and SW designed the methodology. DH implemented the models. DH and QL performed major data analysis. QL, YZ, and XL collected and pre-processed data and provided clinical expertise. LQ, QXF, and QL performed imaging data annotation and/or clinical data review/re-interpretation. DA contributed to model evaluation. All authors contributed to data analysis and result interpretation. DH, QL, YZ, and SW drafted the manuscript. All authors contributed to the article and approved the submitted version.This work was supported in part by an Amazon Machine Learning Research Award.SW is a scientific consultant and stockholder of COGNISTX, Inc. SW has a research grant funded by Amazon.The remaining 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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In this study, we investigated the ultrastructural changes of the axon-myelin interface in the cuprizone model using serial block face scanning electron microscopy and immunohistochemistry. We show that non-inflammatory injury of oligodendrocytes by cuprizone intoxication results in myelin vacuole formation and axonal swellings, paralleled by early alterations of the node of Ranvier cytoarchitecture. This remarkable resemblance of ultrastructural myelin characteristics in multiple sclerosis and the cuprizone animal model suggests that the cuprizone model is a valuable tool to study early pathologies during lesion formation.Myelin damage is a histopathological hallmark of multiple sclerosis lesions. Results of  Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS), leading to irreversible neuronal damage and, in consequence, clinical disability. Different clinical disease courses are known. Most patients initially present with a relapsing-remitting disease course (RRMS) which is, on the clinical level, characterized by recurrent episodes of new or worsening symptoms (relapses) and periods of stability in between those relapses. It is believed that focal lesions of inflammatory demyelination result in the clinical deficits observed during the RRMS disease stage. On the histopathological level, such lesions are characterized by peripheral immune cell recruitment, destruction of oligodendrocytes and the myelin sheaths, glia cell activation (microglia and astrocytes) as well as acute axonal injury .The histopathological hallmark which distinguishes MS from other CNS disorders is the formation of large confluent plaques of primary demyelination. For this reason, any concept of MS pathogenesis has to provide an explanation for this highly specific destruction of the oligodendrocyte-myelin-axonal unit. The sequence of molecular events leading to oligodendrocyte loss and consequently demyelination are not fully understood. Different stressors are known which can induce oligodendrocyte degeneration including oxidative stress, mitochondrial dysfunction, nitric oxide, protein misfolding or inflammatory cytokine exposure . Of noteTo gain insight into the process of early demyelination, several studies on the ultrastructure of the myelin-axonal unit during demyelination have been conducted. We and others recently demonstrated that metabolic oligodendrocyte stress, induced by intoxication with the mitochondrial toxin cuprizone, results in myelin basic protein (MBP) citrullination which triggers autoimmune inflammatory demyelination . The ultN = 20) were used and the experiments were approved by the government of Upper bavaria/Regierung von Oberbayern (#55.2-154-2532-73-15). The experimental procedures were performed according to the Federation-of-European-Laboratory-Animal-Science-Associations recommendations. Cuprizone intoxication (0.25%) was induced as described previously  of 3 weeks cuprizone-intoxicated mice were processed for SBF-SEM as published (N = 3 each) were processed for toluidine blue stains and transmission electron microscopy (TEM) as published previously  was randomly placed within the first image of the z-stack. For reducing any bias, the 100th image (correlating with 8 μm in depth) of the z-stack was chosen as a starting point. In order to increase the number of evaluated axons images no. 300 (correlating with 24 μm depth) and no. 500 (correlating with 40 μm depth) were included in the analysis. A myelin detachment was counted only if an axon was clearly visible within the vacuoles.To quantify the relative frequency of myelin detachments in control and cuprizone-intoxicated mice, a virtual counting frame (area 4757 μm2 each) were randomly placed within the center of the z-stack, and the diameters of axons within the virtual counting frames were measured at six different z-stack positions. For each single axon, the results were averaged. In a next step, 15–25 virtual counting frames (areas 334 μm2 each) were randomly placed within the first third of the z-stack of cuprizone-intoxicated mice, and the 50 adjacent images (in both directions of the z-stack) were investigated for the presence of a myelin detachment. In cases where myelin detachments were detected within the virtual counting frames, respective axonal diameters were measured at six different z-stack positions distant to the myelin detachments. Again, for each single axon, the results were averaged.To assess whether small- or big-caliber axons are preferentially vulnerable to develop myelin detachments, in a first step we quantified axonal diameters (without the surrounding myelin sheath) in control mice. To this end, four virtual counting frames , mouse-anti-PLP  and rabbit-anti-Caspr . Appropriate negative controls (omission of primary antibody) were performed in parallel. ImageJ (version 1.52p) was used to evaluate the relative staining intensities using semi-automated densitometrical evaluation after automatic threshold setting  and to perform automated counting of Caspr+ or ankyrin G+ particles (particle size min. 4 pixel) after threshold settings were manually adjusted in a blinded manner to obtain the best signal to noise ratio (based on the threshold Rényi Entropy) , nodal ankyrin-G or paranodal contactin-associated protein (Caspr), the following primary antibodies were used: mouse-anti-ankyrin G  . Please Compound action potentials (CAPs) across the corpus callosum were recorded in three animals per group as previously described . In briep ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001.All data are given as the arithmetic means ± SEM. Differences between groups were statistically tested using the software package GraphPad Prism 5 . All data sets were tested for normal distribution by Kolmogorov–Smirnov test . Further testing for significant differences between groups was conducted as stated in the figure legends with confidence intervals of 95%. The following symbols are used to indicate the level of significance: *To induce demyelination, mice were intoxicated with cuprizone for up to 5 weeks. Demyelination was visualized by immunohistochemical staining of the myelin protein PLP. In the medial corpus callosum, PLP immunoreactivity decreased over the course of cuprizone treatment indicating progressive demyelination . To inveOn close inspection of the anti-PLP stained sections, vacuole-like structures were evident especially after 3 weeks of cuprizone intoxication, and occasionally also found after 1 week of cuprizone intoxication , suggesting that not big-caliber but small-caliber axons are preferentially affected by the myelin detachments in this model.Next, we were interested whether small- or big-caliber axons are preferentially vulnerable to develop myelin detachments. Therefore, we first measured axonal diameters in randomly selected axons of control mice. In line with previous results  and as s4 ± 11.1 × 104 vacuoles/mm2 in control; 3.1 × 104 ± 2.9 × 104 vacuoles/mm2 in cuprizone), confirming the observations from anti-PLP immunohistochemical stainings  was decreasing during the course of cuprizone-induced demyelination  affects all calibers of axons to the same extent. Predominant myelin loss of large-caliber axons at early time points has not been observed by us or others but could introduce a bias to this analysis if existent. Additionally to myelin vacuolization, axonal swellings within vacuoles were demonstrated. Of note, similar myelinated axon morphologies were found in a model of focal, lysophosphatidylcholine (LPC)-induced demyelination . In thatIn this study, we observed a significant reduction of the expression of nodal ankyrin G and paranodal Caspr during the course of Cuprizone intoxication. This is in line with previous findings of reduction of nodal sodium channel expression  and reduIt is currently unclear, whether in MS such myelin abnormalities are induced by peripheral immune cells, can trigger or promote autoimmunity or promote the recruitment of peripheral immune cells. On the one hand, myelin vacuoles were found in the NAWM of MS patient material, where T cell densities are low , suggestThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The animal study was reviewed and approved by the Regierung von Oberbayern (#55.2-154-2532-73-15).MK and FS contributed to the conception and design of the study. SJ, FS, FP, CE, JK, and MF acquired and analyzed the data. MK and SJ drafted a significant portion of the manuscript and figures. All authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Dysregulation of transfer RNA (tRNA)-derived small noncoding RNA (tsRNA) signatures in human serum has been found in various diseases. Here, we determine whether the signatures of tsRNAs in serum can serve as biomarkers for diagnosis or prognosis of systemic lupus erythematosus (SLE).Initially, small RNA sequencing was employed for the screening serum tsRNAs obtained from SLE patients, followed by validation with TaqMan probe-based quantitative reverse transcription-PCR (RT-PCR) assay. Receiver operating characteristic (ROC) curve analysis was used to assess the diagnostic efficacy. The biological functions of tsRNAs were identified by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) assay.via exosome and can directly target signaling molecules that play crucial roles in regulating the immune system.We first analyzed tsRNA signatures in SLE serum and identified that tRF-His-GTG-1 was significantly upregulated in SLE serum. The combination of tRF-His-GTG-1 and anti-dsDNA could serve as biomarkers for diagnosing SLE with a high area under the curve (AUC) of 0.95 (95% CI = 0.92–0.99), sensitivity (83.72%), and specificity (94.19%). Importantly, the noninvasive serum tRF-His-GTG-1 could also be used to distinguish SLE with LN or SLE without LN with AUC of 0.81  and performance . Moreover, the serum tsRNA is mainly secreted In this study, it has been demonstrated for the first time that serum tsRNAs can be employed as noninvasive biomarkers for the efficient diagnosis and prediction of nephritis in SLE. Systemic lupus erythematosus (SLE) is a representative autoimmune disease leading to systemic autoimmunity resulting in various organ damage. SLE mainly occurs in women of childbearing age characterized with volcanic autoantibodies –4. Both The kidney, a vital organ in the human body, is severely affected by SLE . Lupus nSmall noncoding RNAs are a group of RNA molecules that are shorter than 200 nucleotides (nt) without coding potential, such as microRNA (miRNA), transfer RNA (tRNA)-derived small noncoding RNA (tsRNA), and PIWI-interacting RNAs (piRNAs) . Among tRecent studies from our group , 22 and In this study, we used RNA sequencing, qRT-PCR validation, and ROC curve analysis, leading to identifying SLE-associated tsRNA signatures in human serum. The serum tsRNA profile showed significant potential as a noninvasive biomarker for diagnosing and predicting LN in SLE.This study was divided into two stages Figure 1A total of 301 patients were divided into three groups; 76 patients with SLE without LN, 116 patients with SLE and LN, and 109 normal donors from Nanjing Drum Tower Hospital were enrolled in this study. The clinical characteristics of these patient samples are shown in The heavy decoration of tsRNAs by RNA modification is a main problem to deal with while constructing an sRNA sequence library. To construct a total RNA library, the following changes were made: 3’-aminoacyl was deacylated, 3’-cP  was removed from 3’-OH to facilitate 3’ adaptor ligation, and 5’-OH (hydroxyl group) was phosphorylated to 5’-P for ligating a 5’-adaptor. m1A and m3C were demethylated to facilitate reverse transcription.The integrity of RNA samples was validated using agarose gel electrophoresis. Quantification of the RNA samples was achieved by using NanoDrop ND-1000 instrument. The removal of the RNA modifications, the main issue during the construction of the RNA sequence library, was carried out by adopting the pretreatment strategy as priorly explained. Agilent 2100 Bioanalyzer was used to quantify the completed libraries. Based on the quantification outcomes, the libraries were mixed in equal quantities for sequencing operation.For sequencing, the single-stranded DNAs were needed. To achieve that, 0.1 M NaOH solution was used to denature the DNA fragments in well-mixed libraries. The single-stranded DNA with 1.8 pM concentration was loaded onto the reagent cartridge. NextSeq system using NextSeq 500/550 V2 kit  was used for sequencing operation following the guidelines provided by the production company. The sequencing operation included 50 cycles.The serum samples were centrifuged at 3,500 rpm for 10 min before being collected and stored at -80°C until further analysis. High-quality RNA extraction from serum was achieved by TRIzol  method. The quantity and quality of total RNA were measured using OneDrop-2000 (NanoDrop Technologies). For the cDNA synthesis, 500 ng of total RNA was reverse transcribed using the miRNA 1st Strand cDNA Synthesis Kit (Vazyme Biotech). Ultrasensitive detection of tsRNAs was performed using the miRNA Universal SYBR qPCR Master Mix (Vazyme Biotech). The expression of each gene was determined, and the corresponding standard curve was drawn. Gene-specific primers  are listed in Statistical analyses were performed using an SPSS software, version 20.0 , and GraphPad Prism software 7 . Data are presented as the means ± SEMs, and statistical validations were achieved by harnessing t-test or one-way ANOVA followed by Bonferroni’s multiple comparisons test. P < 0.05 was nominated to represent the statistically significant differences.Raw data generated by tsRNA sequencing have been deposited to Gene Expression Omnibus (GEO) under the accession code GSE90524. The main findings of the study are included in the article and supplementary materials. Other data supporting the study findings can be acquired upon reasonable request from the corresponding authors.). tsRNAs that satisfied two conditions showed significant differential expression: CPM >100 in serum by sequencing detection and fold change >6 between two groups. As shown in The presence of stable tsRNAs in human serum has been confirmed by recent studies . ExpressIn the training stage, we then used RT-qPCR assay to validate the sequencing results. The absolute quantification was used to validate the expression of the top 10 upregulated tsRNAs using individual serum samples from 32 SLE without LN and 32 healthy controls by RT-qPCR  and tRNA-derived fragments (tRFs). tiRNAs arise from the 5′ and 3′ tRNA halves consisting of about 30–40 nt. tRFs originate from mature and tRNA precursors by nucleases Dicer or angiogenin, etc. , 11, shoTo verify the value of tRF-His-GTG-1 for clinical practice, we used ROC curve analysis and assessed the sensitivity and specificity of prediction based on the risk scores. As shown in To further explore whether serum tRF-His-GTG-1 level is related to the progression of SLE, we then performed qRT-PCR to assay serum samples from 83 SLE with LN patients. The expression of the tRF-His-GTG-1 in serum was remarkably decreased in SLE with LN than in SLE patients without LN , Training Program of the Major Research Plan of the National Natural Science Foundation of China (No. 92049109), National Natural Science Foundation of China (Nos. 32000549 and 61971216), Key Research and Development Project of Jiangsu Province (Nos. BE2019603 and BE2020768), High-level Health Talent Project of Jiangsu Province (No. LGY2019001).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The individual placement and support (IPS) model of supported employment is a leading evidence-based practice in community mental health services. In Japan, individualized supported employment that is highly informed by the philosophy of the IPS model has been implemented. While there is a body of evidence demonstrating the association between program fidelity and the proportion of participants gaining competitive employment, the association between fidelity and a wider set of vocational and individual outcomes has received limited investigation. This study aimed to assess whether high-fidelity individualized supported employment programs were superior to low-fidelity programs in terms of vocational outcomes, preferred job acquisition, and patient-reported outcome measures (PROMs).A prospective longitudinal study with 24-month follow-up analyzed 16 individualized supported employment programs. The Japanese version of the individualized Supported Employment Fidelity scale (JiSEF) was used to assess the structural quality of supported employment programs . Job acquisition, work tenure, work earnings, job preference matching , and PROMs such as the INSPIRE and WHO-Five Well-being index were compared between groups.There were 75 and 127 participants in the low-fidelity group (k = 6) and high-fidelity group (k = 10), respectively. The high-fidelity group demonstrated better vocational outcomes than the low-fidelity group, i.e., higher competitive job acquisition  = 3.6, p = 0.002), longer work tenure , and better match for illness disclosure preference . However, we found no differences between groups in other preference matches or PROM outcomes.High-fidelity individualized supported employment programs resulted in good vocational outcomes in a real-world setting. However, enhancing service quality to increase desired job acquisition and improve PROMs will be important in the future.UMIN000025648The online version contains supplementary material available at 10.1007/s10488-021-01161-3. Employment is as important for many people with a diagnosis of mental illness as it is for much of the general population. The individual placement and support (IPS) model of supported employment has been a leading evidence-based practice in vocational rehabilitation and community mental health services over the past three decades  and implementation studies have consistently reported that IPS programs worldwide are superior to other services in terms of improving vocational outcomes  have reported a significant association between the quality of program-level structural services and program-level employment rates  was the fidelity score of each individualized supported employment program. We assessed participants’ characteristics and job preferences at baseline, as well as vocational outcomes and PROMs over the study period. The primary outcome was competitive job acquisition.The study was designed based on the STROBE reporting guidelines  score APA,  was obtaData for each participant’s competitive employment status over the 24-month follow-up period were obtained from their service records and employment agreement documents. Competitive employment was operationally defined as working at least 1 day a month during the follow up period at a minimum wage or higher, as determined by Japanese law. We calculated the employment rate, percentage of participants who maintained their employment for 6 or 12 months after starting their job, length of time to find the first job, work tenure, and total earnings during the study period. Using the employment contract documents, the employment specialists in each program obtained data on salary and hours worked per week, and assessed commute time and illness disclosure based on participant interviews. Data collection was completed in December 2019.Two PROMs were used in this study. To assess the overall well-being of each individual, the Japanese version of the WHO-Five Well-being Index (WHO-5) was administered at baseline and at 12- and 24-month follow-up assessments. The WHO-5 was initially introduced in a European research project and has been internationally used  for continuous data. Sensitivity analyses for vocational outcomes and PROMs were conducted to control for demographic variables at baseline. Specifically, based on past systematic reviews, adjustments were made for potential covariates such as age, diagnosis, education level, past job experience, past hospitalization, and GAF score . Multilevel mixed-effects logistic regression models were used to compare binary outcome data between the groups, and multi-level mixed-effects generalized linear models were used to compare continuous outcome data and INSPIRE scores. Each model included adjustment for agency as a cluster-level variable. There was also adjustment for sex as a fixed-effect variable, since a significant difference in this variable was identified between groups. A multilevel, mixed-effects, generalized linear repeated measures model was performed for the WHO-5, and included the variables of group, time, group and time interaction, baseline score, and sex. These analyses reported the adjusted odds ratio (aOR) with 95% confidence interval (95%CI) for binary data, and the adjusted mean difference (aMD) with 95%CI and effect size . One author (TS) blinded to group allocation conducted all analyses using Stata version 16.Although the particular sample size was not calculated, the statistical power of this study was estimated for the primary outcome. A previous Japanese fidelity study found a 30% difference in the employment rate of participants in low- and high-fidelity individualized supported employment programs  were enrolled . Four participants withdrew their consent during the 24-month follow-up period. The ITT analysis ultimately included 75 and 127 participants in the low-fidelity group (k = 6) and high-fidelity group (k = 10), respectively. Of these, 130 (59.4%) consented to complete the WHO-5 at the baseline assessment and were included in the analysis of the WHO-5, and 95 (43.4%) completed the INSPIRE at the 12-month follow-up assessment and were included in the analysis of the INSPIRE.2 = 5.446, p = 0.020). No significant between-group differences were found in other variables at baseline. An analysis of only the participants who completed the PROMs also showed no significant differences between groups in any baseline variables (Online Supplementary Table 2). The results of job preference assessment are shown in Online Supplementary Tables 3 and 4. In each domain, participants in the high-fidelity group were more likely to identify their job preferences than those in the low-fidelity group.At baseline, the mean age was approximately 35 years and the most frequent diagnosis in both groups was schizophrenia, based on the ICD-10 . In addition, the commute times for first jobs were shorter for participants in the high-fidelity group than for those in the low-fidelity group . For participants who were employed at least once, Fig. There were no significant group differences in the WHO-5 at any assessment point Table . FurtherThis longitudinal study examined the predictive association between individualized supported employment program quality, as measured by the JiSEF, and multiple outcomes in a real-world setting. High-fidelity programs yielded better vocational outcomes than low-fidelity programs, whereas we obtained mixed results regarding job preference matching and PROMs. We discuss these results in comparison with previous findings of both the IPS and individualized supported employment.This study demonstrated an association between program fidelity and several vocational outcomes, confirming the results of past program-level fidelity research on the IPS and individualized supported employment models Supplementary file2 (PPTX 49 KB)Below is the link to the electronic supplementary material.


Due to the unique structural characteristics allowing for facile modification of targeting ligands and radionuclides, dendrimers can be served as a versatile scaffold to build up various PET imaging agents, and significant breakthroughs have been made in this field over the past decades. This review focuses on the recent advances in dendrimer-based contrast agents for PET imaging of cancer, cardiovascular and other diseases. In particular, radiolabeling strategies for different PET isotopes are described in detail. Several challenges involved in clinical translation of radiolabeled dendrimers are also discussed.Positron emission tomography (PET) imaging offers physiological and biological information through the  By replacing a nonradioactive 12 C atom, 11 C can be easily labeled with biological molecules, including but not limited to amino acids, nucleic acids, choline and dopamine , while the addition of 18 F to complex molecules is still challenging. Usually, the 18 F radiolabeling is carried out through the nucleophilic substitution of some excellent leaving groups in precursors such as mesylate, tosylate or triflate. Despite many 18 F-labeling methods have been proposed, most of them suffer from harsh reaction conditions, multistep protocols and low radiochemical yields, which is also a major obstacle of 18 F-labeled NPs  are indispensable, which forms stable complexes with these radiometals. As the well-established coordination chemistry, a wide range of BFCs have been designed and synthesized, which greatly facilitates the development of various radiometal-labeled NPs for PET-imaging applications  and optimal positron emission energy (0.635 MeV). This means a short positron linear range in tissue that enables high-resolution PET imaging  for tumor imaging , which has been used to label antibody and small molecules for PET imaging  forming protective shells to prevent dehalogenation in vivo. Radiolabeling was achieved using Chloramine-T method and the 76Br-labeled dendrimers displayed excellent stability in PBS and mouse serum within 48 h. Moreover, the pharmacokinetics could be modulated through appropriate level of dendritic branching and PEO length. RGD peptides were modified with lysine and could be further installed at the ends of the PEO chains. The targeted nanoprobes exhibited a 50-fold enhancement in binding affinity over the mono-RGD peptide and 6-fold increase in αvβ3 receptor-mediated endocytosis compared with nontargeted nanoprobe. Highly specific accumulation of 76Br-labeled dendritic nanoprobe was found in a murine hindlimb ischemia model and the potential of dendritic nanoprobe as a PET-imaging agent of angiogenesis was verified in vivo. Remarkably, the design of protective shells in this study could be potentially used to improve radiostability of radioiodine-labeled nanoprobes that are more vulnerable to dehalogenation in vivo.ogenesis   and attractive decay characteristics   and 1,4,8,11-tetraazacyclotetradecane-N,N′,N″,N″″-tetraacetic acid (TETA) are additional macrocyclic chelators 64Cu nuclear reaction in cyclotron accelerator, 64CuCl2 is provided in a hydrochloric acid solution, and then mixed with the NPs dissolved in nearly neutral or weakly acidic buffer solution. The mixture is incubated at room temperature or heated to higher temperatures for one half to several hours. The crude products can be purified by adding another chelating agent (e.g. DTPA), gel filtration chromatography  or centrifuged filtration to remove free 64Cu.2 Affibody-based dual imaging probe using PAMAM generation 0 (PAMAM G0) as a platform to assemble 64Cu and Cy5.5 for dual-modality imaging of ovarian cancer  cyclohexane-1-carboxylate (Sulfo-SMCC), which could react with the amine terminal groups of G0 and coupled with the cysteine in anti-HER2 Affibody. The developed dendrimer conjugates could effectively chelate with 64Cu, but the 64Cu-labeled complexes were not very stable in vivo. The main reason of instability is considered to be the reduction of Cu(II) to Cu(I) in the 64Cu-DOTA moiety, while DOTA is unsuitable for the chelation of Cu(I), generating transchelation between 64Cu-DOTA and some proteins such as serum albumin and superoxide dismutase. Nevertheless, both NIRF and PET imaging exhibited high tumor uptake with obvious contrast effects at 1 h post injection, and excellent tumor imaging results were observed within 20 h, which attributed to favorable pharmacokinetic properties. Interestingly, tumor fluorescence signals gradually increased during the period investigated, whereas a radioactivity peak from PET were found at 4 h after injection. This difference might be explained by the fact that NIRF and PET followed the fate of Cy5.5 and 64Cu moieties, respectively. Biodistribution studies showed that this dendrimer-based dual-modality imaging probe is accumulated prominently in liver and kidneys, suggesting the excretion through both hepatobiliary and kidney systems.Wang et al. developed an anti-HERn cancer   for MR imaging . Similar to the fluorescence property, the telodendrimers also possessed the ability of photodynamic transduction. After laser irradiation, telodendrimers could convert energy in the form of heat in PBS, while fluorescence and singlet oxygen generation with the addition of SDS. Furthermore, chemotherapeutic drugs could be efficiently encapsulated inside the telodendrimers as programmable releasing nanocarriers for drug delivery. These properties enabled telodendrimers as theranostic agents for NIRF imaging, PTT, PDT and chemotherapy, which had been demonstrated in both ovarian cancer xenograft model and murine transgenic breast cancer model in vivo.In another study, Li et al. developed smart and versatile telodendrimers consisting of various imaging and therapeutic functions such as NIRF, PET and MR imaging, photothermal therapy (PTT), photodynamic therapy (PDT), as well as image-guided drug delivery , adamantane-grafted polyamidoamine (Ad-PAMAM) and Ad-grafted polyethylene glycol (Ad-PEG). The 64Cu labeling of TCO⊂SNPs was designed via Diels-Alder reaction between TCO and tetrazine-DOTA-64Cu (64Cu-Tz). 64Cu-Tz could be prepared with high radiochemical yield and its stability was up to 95% with 8 h both in PBS and serum. TCO groups were encapsulated into supramolecular NPs to be protected from potential degradation in vivo. When preferential accumulation of TCO⊂SNPs in tumor through EPR effect occurred, TCO/CD-PEI could be released from the inside of TCO⊂SNPs and reacted with 64Cu-Tz to retain radioactivity in tumor. After the quick clearance of the unreacted 64Cu-Tz from the body, high-contrast tumor PET imaging were achieved. In contrast to traditional nanoparticle-based imaging platforms with faint tumor uptake and excessive liver distribution, the pretargeted approach showed approximately equivalent uptake in tumor and liver.The development of PET nanoprobes is vulnerable to be restricted by the contradiction between intrinsic pharmacokinetics (PKs) of NPs and limited half-lives of positron-emitting isotopes. To this issue, the pretargeted imaging strategy may be one of the promising solutions . The 64Cu-labeled dendritic peptide showed significantly enhanced accumulation in atherosclerotic plaque and higher aorta/blood ratio as compared with both the monomer and control peptide through in vivo PET imaging. In another study, Pant et al. exploited 64Cu-labeled dendritic polyglycerol sulfates (dPGS) as inflammation-specific agents for PET imaging  shows significant superiority over 64Cu  to gain improved image quality in theory for PET imaging and more suitable half-life of 67.8 min for clinical applications Gal linkage in glycoclusters played an important role in the circulatory residence of N-glycans and varied remarkably in the clearance pathway from those of glycoclusters of 16-mer-b and 16-mer-c, which were cleared through a biofiltration pathway in the kidney.Tanaka et al. reported PET imaging of dendrimer-type asparagine-linked oligosaccharide (N-glycan) clusters to visualize their dynamics and biodistributions vβ3 integrin receptors has been widely used in the fields of cancer and cardiovascular diseases  peptide that can target αin vivo stability and reasonable tumor uptake, PESIN peptide is regard as one of the most promising ligands to gastrin releasing peptide receptor (GRPR), which is overexpressed on several tumor types. Lindner et al. synthesized a series of PESIN monomers, dimers, tetramers and octamers comprising PEG linkers of different lengths, followed by conjugation with PAMAM dendrimers and efficient radiolabeling of 68Ga. The labeled dendrimers could be obtained in high radiochemical yields and purities of 96–99% with excellent stability. The shortest linker within each group (monomers to octamers) resulting in minimal distances between the peptide moieties showed the highest binding affinities in vitro. However, the effect of binding affinities increasing proportionally to the number of peptide moieties was not observed in the case of these PESIN multimers. The dimers presented the optimized binding affinity, namely a 2.5-fold avidity enhancement compared to the monomeric peptides, which was further validated by the result that PET study of 68Ga-labeled dimers displayed a twice higher tumor uptake in tumor-bearing mice. Interestingly, benefiting from the much faster blood clearance, the dimer also showed twofold high tumor-to-background ratios than the respective monomers.This synthesis approach using dendritic structures as scaffolds for multimerization of bioactive molecules was broadened in a following work  or EPR effect. The unique structural characteristics of dendrimers have offered opportunities to incorporate various molecular imaging modalities in a single system for multimodal imaging, such as PET/MR and PET/NIRF imaging. More importantly, therapeutic capabilities are able to be introduced into multifunctional dendrimers as well for theranostic applications. On the other hand, appropriate isotopes and efficient radiolabeling strategies must be carefully considered in the construction of dendrimer-based contrast agents for PET imaging. Interestingly, pretargeted strategy can provide a great deal of flexibility of imaging time, especially for the short-lived isotopes. Several radiolabeling approaches for different PET isotopes have been developed with satisfactory results.in vivo. In addition, the physical properties of NPs, such as size, shape and surface charge, significantly determine their in vivo biodistribution behavior; therefore, dendrimers with optimal surface modifications can prolong circulation time, decrease MPS uptake and have the potential to increase the target-to-background ratio as compared to the uncoated counterparts. Further, the construction of smart and versatile dendrimers for molecular imaging still remains an open area of investigation, for instance, exploring new synthetic techniques specific to dendrimer-based molecular imaging contrast agents. Thus, in order to fulfill PET applications, novel radiolabeling strategies with sufficient radiochemical yields and in vivo stabilities must be developed. It should be noted that click chemistry in recent years has shown significant benefits over traditional synthetic methods for the clean, high yielding and rapid preparation of imaging agents labeled with PET radionuclides such as 18 F, 64Cu and 68Ga. However, the application of click techniques to radiolabeled dendrimer-based NPs is still in an early stage and much more effort is needed in this field. In the aspect of safety, the long-term toxicity of dendrimer-based contrast agents is still confusing, especially the large systems with lack of complete clearance from the body. To resolve this issue, the biodegradable dendrimers will be a better choice. Lastly, due to that fact that dendrimers are able to be loaded with various drugs, genes and therapeutic radionuclides, more types of dendrimer-based theranostic systems could be developed in order to expand the scope of molecular imaging applications, in particular PET image-guided drug delivery (Chakravarty et al., Although positron isotope-labeled dendrimers have shown a great promise in the field of molecular imaging, quite a number of problems need to be solved in their clinical translation (Stylianopoulos & Jain,


Current tuberculosis treatments leave patients with clinically significant lung injury and increased all-cause mortality post-cure. Adjunctive host-directed therapies could protect the lungs, improve long-term survival, and shorten treatment duration; however, few have been tested clinically. Therefore, we aimed to assess the safety and preliminary efficacy of four host-directed therapies for tuberculosis.Mycobacterium tuberculosis, a sputum Xpert cycle threshold of less than 20, and moderately advanced or far advanced disease on chest radiography. By use of numbers generated in blocks of ten and stratification by site, eligible patients were randomly assigned (1:1:1:1:1) to receive one of the four oral host-directed treatments plus standard tuberculosis treatment or standard treatment alone (the control group). Host-directed treatments were: CC-11050 ; everolimus ; auranofin ; and ergocalciferol . All study participants received oral rifabutin-substituted standard tuberculosis treatment for 180 days. Patients and clinicians were not masked to treatment assignment. Spirometry and sputum culture with solid and liquid media were done at baseline and up to 180 days at specified intervals throughout treatment. The primary endpoint was safety and tolerability up to day 210. Secondary preliminary efficacy endpoints were treatment effects on sputum microbiology (culture status at day 56 and the hazard ratio for stable culture conversion up to day 180) and lung function  measured by spirometry at day 56, day 180, and day 540. Safety was analysed in the intention-to-treat population and preliminary efficacy primarily in the per-protocol population. The trial is registered at ClinicalTrials.gov, NCT02968927. Post-treatment follow-up was completed in 2020.In this prospective, open-label, phase 2, randomised controlled trial, patients with pulmonary tuberculosis were recruited at three clinical sites in South Africa. Eligible patients were aged 18–65 years, HIV-1-negative, and had rifampicin-susceptible 1 in the control group was 61·7% of predicted (95% CI 56·3–67·1) at baseline and 69·1% (62·3–75·8) at day 180. Patients treated with CC-11050 and everolimus had increased recovery of FEV1 at day 180 relative to the control group , whereas auranofin and ergocalciferol recipients did not. None of the treatments had an effect on FVC during 180 days of follow-up or on measures of sputum culture status over the course of the study.Between Nov 18, 2016, and Sept 27, 2018, 200 patients were screened and randomly assigned to different treatment groups . 11 treatment-emergent serious adverse events occurred either during treatment or within 30 days after treatment discontinuation, of which three were attributable to a host-directed treatment. Life-threatening thrombocytopenia occurred in an auranofin recipient; apparent intra-abdominal sepsis leading to death occurred in another auranofin recipient and was classified as a suspected unexpected serious adverse reaction. Tuberculous spondylitis occurred as an apparent paradoxical reaction in a patient receiving ergocalciferol. Two patients in the control group had life-threatening, treatment-attributable liver injury. No treatment-emergent, treatment-attributable serious adverse events occurred in patients receiving CC-11050 or everolimus. Mean FEV1, a key measure of lung function and predictor of all-cause mortality. Further studies of these candidates are warranted.CC-11050 and everolimus were safe and reasonably well tolerated as adjunctive therapies for tuberculosis, and analysis of preliminary efficacy suggests they might also enhance the recovery of FEVThe Bill & Melinda Gates Foundation and the South African Medical Research Council. Evidence before this study1 is an independent, generalisable predictor of all-cause mortality. Tuberculosis leaves most patients with permanent impairment of lung function, including of FEV1. There is growing recognition that long-term all-cause mortality risks are increased after tuberculosis, to a degree consistent with impairment of FEV1. We searched PubMed for articles published in English between database inception and Dec 15, 2019, using the search terms “tuberculosis”, “clinical trial”, and “spirometry”. The search did not identify any studies of interventions to protect the lung done more recently than the 1960s. Several clinical trials of vitamin D have been done in patients with tuberculosis. Only one found that vitamin D enhanced sputum culture conversion at week 8 compared with standard treatment alone; none examined lung function. Studies of the type 4 phosphodiesterase inhibitor CC-11050 in experimental mouse and rabbit models of tuberculosis found that it improved the resolution of lung pathology and the clearance of Mycobacterium tuberculosis infection. One previous study of a non-specific phosphodiesterase inhibitor (pentoxifylline) found no benefit on short-term or long-term outcomes but did not examine effects on lung function.FEVAdded value of this study1. Interventions to enhance the recovery of FEV1 in patients with tuberculosis might help to reduce the increased long-term mortality risks observed after treatment.This phase 2 study found that adjunctive treatment with CC-11050 or everolimus in adults with pulmonary tuberculosis and baseline predictors of poor outcome was safe and well tolerated, and might have resulted in improved recovery of FEVImplications of all the available evidenceMore studies of CC-11050 and everolimus in patients with tuberculosis are warranted to further investigate their effects on lung function and assess their impact on long-term health outcomes.Tuberculosis is a leading cause of morbidity and mortality globally.3There is growing interest in the potential of adjunctive host-directed therapies to improve treatment options for patients with tuberculosis. As modulators of the host immune response, these treatments have the potential to protect the lungs, improve long-term survival, and shorten treatment duration by reducing lung inflammation, improving lesional drug penetration, and inducing antimicrobial activity in phagocytic cells.Mycobacterium tuberculosis infection.M tuberculosis.M tuberculosis.Therefore, we aimed to assess the safety and preliminary efficacy of four adjunctive host-directed therapies in patients with hard-to-treat tuberculosis: CC-11050, everolimus, auranofin, and vitamin D . CC-11050 is a type 4 phosphodiesterase inhibitor with anti-inflammatory properties and has shown efficacy in preclinical models of M tuberculosis/rifampin  test results were asked to consent for further screening to enter the study.In this prospective, open-label, phase 2, randomised controlled trial, patients with pulmonary tuberculosis were recruited at three clinical sites in Greater Johannesburg and Pretoria, South Africa: the Tembisa Clinical Research Centre, Tembisa; the Clinical HIV Research Unit, Johannesburg; and the Setshaba Research Centre, Soshanguve. Patients attending neighbouring public health clinics were asked to give permission for a review of laboratory records for possible study eligibility. Those with positive sputum Cepheid Xpert M tuberculosis and one or more probes with a cycle threshold less than 20. Cycle threshold indicates the number of cycles of DNA amplification required to result in detection of the target and is inversely related to the log of the target copy number. Patients also had moderately advanced or far advanced pulmonary tuberculosis on chest radiography , the London School of Hygiene & Tropical Medicine (reference number 10645), the South African Medicines Control Council (reference number 20160506), is registered as study 4297 in the South African Human Research Electronic Application System, and can be found online.By use of numbers computer-generated by the study statistician (SG) in blocks of ten and with stratification by site, patients were randomly assigned (1:1:1:1:1) to receive CC-11050, everolimus, auranofin, or ergocalciferol in addition to standard tuberculosis therapy, or standard tuberculosis therapy alone (control). At each study site, the individuals responsible for assigning patients to treatment groups were not involved in evaluating treatment outcomes. Envelopes containing allocation information were provided to each site by SG. Patients and clinicians were not masked to treatment assignment. However, microbiology and spirometry laboratory personnel and senior study leadership (RSW and GC) were masked to treatment assignment. Summaries of study progress prepared by SG that described outcomes by treatment group were available only to members of the study's data and safety monitoring committee.NCT01300208) and erythema nodosum leprosum (NCT03807362). The dose of everolimus was selected as the lowest showing human biological activity.Participants randomly allocated to adjunctive host-directed tuberculosis therapies received CC-11050 , everolimus , auranofin , or ergocalciferol  orally. Adjunctive CC-11050, everolimus, or auranofin were given from day 1 to day 112 as follows: 200 mg of CC-11050 twice a day with food; 0·5 mg/day of everolimus; or 3 mg/day of auranofin for the first week, which was then increased to 6 mg/day. Ergocalciferol was given as a 5 mg dose on day 1, then 2·5 mg on days 28 and 56. CC-11050 was given at the same dose used by Celgene in two phase 2 studies in patients with cutaneous lupus erythematosus  and liquid media  on days 0, 1, 7, 14, 21, 28, 35, 42, 56, 84, 112, 140, and 180, with particular attention to culture status on day 56. Patients were repeatedly encouraged to produce adequate sputum specimens, including through video instructional materials. Sputum induction was not done. M tuberculosis molecular testing, culture, and drug susceptibility testing, and a single commercial laboratory  for safety studies.Spirometry was done at the study sites by use of EasyOne Pro instruments  beginning on study day 1. The protocol originally specified that spirometry be done on days 1, 14, 28, 56, 84, 112, 140, and 180, with particular attention to lung function on days 56 and 180. A protocol amendment later specified that spirometry should additionally be done on day 540. All sites used a single microbiology laboratory  for The primary endpoint of the study was the safety and tolerability of the study treatments, in terms of treatment-emergent serious adverse events for CC-11050 and suspected unexpected serious adverse reactions for the other treatments up to day 210, and was centrally assessed. Secondary safety endpoints included treatment-emergent adverse events other than serious adverse events, categorised according to severity, drug-relatedness, and whether they lead to early withdrawal, and the proportion of patients with disease exacerbation (assessed at each scheduled visit) due to study treatments. Safety events were assessed by site personnel at each study visit and subsequently reviewed by the data and safety monitoring committee. Clinicians at each site provided initial assessments of severity (using adverse event grading tables from the Division of Allergy and Infectious Diseases) and treatment-relatedness. These assessments could be upgraded following central review.M tuberculosis (as indicated by sputum culture status) and on lung function. The proportion of patients with positive sputum culture status was assessed at day 56 using solid culture medium, because previous trial data suggest culture status at this timepoint is a predictor of tuberculosis recurrence.M tuberculosis was detected in more than one specimen after day 112. For the entire treatment period, stable culture conversion was defined as occurring on the day of the first of two consecutive sputum specimens obtained at least 2 weeks apart without growth of M tuberculosis. Specimens with liquid or solid cultures showing growth of M tuberculosis were considered positive. Instances in which patients were unable to provide a sputum specimen despite encouragement were considered negative. Cultures that were unevaluable because of contamination or other causes were considered missing. To assess lung function, FEV1 and forced vital capacity (FVC) were measured by spirometry, and change from control was assessed at numerous timepoints, with particular attention to days 56, 180, and 540. Spirometry findings were interpreted according to guidelines from the American Thoracic Society and the European Respiratory Society,1 was expressed as a percentage of the predicted value based on age, sex, height, and race (FEV1%).Secondary preliminary efficacy endpoints were key measures of the effects of host-directed therapies on the clearance of 18F-fluorodeoxyglucose-PET, CT imaging, and serum markers of inflammation, will be undertaken and reported separately.Analyses of other secondary microbiology and lung function outcomes and of early biomarkers of treatment response, including changes in NCT01300208) included only 48 patients with cutaneous lupus erythematosus. We also considered microbiological outcomes in choosing sample size. Near-complete (≤1%) culture conversion at 2 months, measured by use of solid medium, has been proposed as a target for new 4-month regimens.With 40 patients per group, this study was predicted to have a 77% power, at a two-sided 0·05 significance level, of detecting a significant difference in the proportion of serious adverse events between the control and experimental treatments, assuming proportions of 0·05 (n=2) in the control group and 0·3 (n=12) in the experimental groups. This sample size was considered appropriate to detect treatment-emergent serious adverse events attributable to CC-11050 in an exploratory trial in patients with tuberculosis because the sole previous phase 2 study . The secondary efficacy outcomes for microbiology and lung function were analysed in the per-protocol population, which included patients in the modified intention-to-treat population, but excluded those who did not complete treatment or were found to have inadequate adherence . The secondary efficacy analyses were repeated using the modified intention-to-treat population.1 and FVC at day 56, day 180, and day 540 were compared by ANCOVA. We did not adjust for multiple comparisons, but instead independently compared each candidate host-directed therapy with the control group, as each might be advanced independently on the basis of this analysis. Analyses were adjusted for baseline covariates. We considered adjusting for multiple baseline factors in each analysis, but found that the effect of additional factors was small. Missing data were excluded from analyses and not otherwise imputed or estimated.Safety data are presented descriptively, with particular attention to severity, treatment-relatedness, and action on study drug for serious adverse events and suspected unexpected serious adverse reactions. Logistic regression was used to assess associations of treatment group with culture-positive status measured after 56 days of treatment. We report unadjusted and adjusted odds ratios (ORs) and 95% CIs for culture status on solid medium, with the adjusted values controlling for baseline differences in time to positivity in automated liquid cultures. We individually compared experimental groups with the control group to calculate the hazard ratio (HR) for stable culture conversion up to 180 days using Cox proportional hazards regression. FEV1 were done to investigate adjustment for study site in the per-protocol population and to examine the use of more stringent spirometry criteria at day 180 in the per-protocol population (grades A–D were considered acceptable). An additional post-hoc analysis fit a random effects model for the repeated measurements of FEV1% on a study day until day 180 using the modified intention-to-treat population and included an interaction between day and study group, and a random intercept for repeated measurement of FEV1%. Further post-hoc analyses separately examined treatment effects on culture conversion in liquid cultures in the per-protocol population using Cox proportional hazards methods.Post-hoc analyses of FEVNCT02968927 by ClinicalTrials.gov.All analyses were done by use of Stata (version 14). Study progress was monitored at regular intervals by an independent data and safety monitoring committee. This trial is registered as study 4297 in the South African Human Research Electronic Application System, as DOH-27–0616–5297 by the South African National Clinical Trial Registration, and as The Bill & Melinda Gates Foundation provided funding for this trial and contributed to the selection of host-directed therapy candidates and discussions of trial design. The South African Medical Research Council provided additional funding for administrative aspects of the study. Celgene provided CC-11050. The Aurum Institute served as a study sponsor. The funders of the study had no role in determining other aspects of study design, data collection, data analysis, data interpretation, or writing of the report.M tuberculosis burden  at baseline and 69·1% (62·3–75·8) at 180 days, with much of the apparent increase occurring early during standard treatment. All groups except for the ergocalciferol group showed an apparent increase in mean FEV1 from baseline to day 14, although none of these increases reached statistical significance .1 can be impaired by the inability to fully inhale or rapidly exhale, which can be attributed to two predominant post-tuberculosis pathological mechanisms: fibrosis and bronchiectasis. The finding that enhanced recovery of FEV1 occurred relatively late (at day 180 but not day 56) during treatment is of particular interest. Inflammation due to recurring viral or bacterial lung infection is thought to drive progressive FEV1loss in COPD by promoting airway remodelling, a process that results in structural changes to the airways by depositing collagen in the extracellular matrix, altering the cellular composition of the epithelial barrier, and narrowing small airways.In analyses of preliminary efficacy, both CC-11050 and everolimus appeared to enhance the recovery of FEV1 is an independent, generalisable predictor of all-cause mortality, even in individuals with only mild to moderate impairment .1 declines to 70% of predicted (moderately impaired), and doubles again as FEV1 further declines to 50% of predicted (severely impaired).1. Multiple studies have confirmed that mortality risk is increased after tuberculosis.FEVM tuberculosis colony-forming unit counts to a greater extent than did isoniazid alone.M tuberculosis-infected rabbits, CC-11050 additionally reduced the expression of multiple matrix metalloproteinase genes (particularly MMP12) and reduced the extracellular deposition of collagen in the lung.30CC-11050, a type 4 phosphodiesterase inhibitor, was previously a backup compound for apremilast , sharing similar chemical structures and biological activities. Apremilast is now approved for multiple anti-inflammatory indications. CC-11050 decreases the production of pro-inflammatory cytokines, including tumour necrosis factor, by preventing the degradation of cyclic AMP. In preclinical models of active tuberculosis in the mouse and rabbit, treatment with CC-11050 plus isoniazid reduced the number and size of lung lesions, ameliorated lung pathology, and reduced lung 1 values at baseline in this study tended to be somewhat higher in everolimus recipients than in control recipients, the difference from controls at 180 days persisted after adjusting for this potential confounding factor. This clinical trial is, to our knowledge, the first of an mTOR inhibitor in tuberculosis.Everolimus, an inhibitor of mTOR, is used clinically at high doses for the treatment of some cancers, and at intermediate doses as an immunosuppressive agent for solid organ transplantation. Anti-inflammatory and anti-fibrotic effects of mTOR inhibitors have also been described.M tuberculosis, HIV, and severe acute respiratory syndrome coronavirus 2.Auranofin is an orally bioavailable gold salt introduced in the 1980s as a disease-modifying treatment for rheumatoid arthritis. In addition to their anti-inflammatory properties, gold salts show broad-spectrum antibacterial and antiviral activities in vitro, including against diverse intracellular pathogens such as Vitamin D is essential for host defences against tuberculosis.1 on day 14 is a true early treatment response or merely a learned improvement in test taking. Larger studies with longer follow-up will be necessary to directly assess effects on mortality.The findings of this trial are best considered in the context of its experimental medicine design. The study's main shortcomings are a small sample size and low statistical power. Experimental groups were compared with the control without adjustment for multiple comparisons. Patients and clinicians could not readily be masked to treatment assignment because of the multiplicity of experimental treatments, which potentially introduced bias. As an exploratory study, its key findings will require verification in future trials. Further studies will be necessary to assess effects in patients with common comorbidities such as HIV-1 infection and diabetes, in patients with rifampicin-resistant disease, and in greater numbers of female patients. Repeated spirometry on day 2 in future studies would help to elucidate whether the apparent improvement in FEV1, a key measure of lung function and predictor of long-term all-cause mortality. Further definitive studies of CC-11050 and everolimus in patients with pulmonary tuberculosis are warranted.In summary, two adjunctive host-directed therapies for—CC-11050 and everolimus—showed adequate safety and tolerability in patients beginning treatment for pulmonary tuberculosis. An analysis of preliminary efficacy suggests that these therapies might preserve FEVstudy protocol see https://bit.ly/2MA6bJLFor the rwallis@auruminstitute.org.Data collected for the study, including deidentified individual participant data, a data dictionary defining each field in the set, and the statistical analysis plan will be made available to other researchers on request from the time of publication of this manuscript. Research proposals can be submitted to the corresponding author (RSW) at


Overall, weekly smoking remained stable (±11.5%), weekly binge drinking decreased (from 27.8% to 13.9%) and weekly cannabis use increased (from 6.7% to 8.6%). Male gender, not living with parents, being a bachelor student, having less financial resources and less adherence to the COVID-19 measures were found to increase the risk of substance use (before/during the first COVID-19 lockdown). Additionally, male gender, not living with parents, being a bachelor student, not being born in the Netherlands and having a student loan contributed to the likelihood of increased substance use during COVID-19. Patterns of characteristics contributing to the likelihood of decreased weekly substance use during COVID-19 were less clear. The risk factors male gender, not living with parents and being a bachelor student do not only contribute to the likelihood of using substances but also contribute to the likelihood of increased use during a lockdown. Prevention and intervention programs should especially target these risk groups.Tobacco, alcohol and cannabis are commonly used among university students. However, student lives and their substance use have changed dramatically since the start of the COVID-19 pandemic. This study investigated the impact of COVID-19 on (trends in) weekly smoking, weekly binge drinking and weekly cannabis use in Dutch university students and investigated associated student-, study- and COVID-19-related characteristics. Between April and June 2020, several Dutch higher educational institutes invited their students to participate in an online survey. Data of 9967 students (M Before the COVID-19 pandemic, the prevalence of tobacco smoking, binge drinking and cannabis use among university students were relatively high compared to peers who are not attending higher education ,3,4. In Since the start of the COVID-19 pandemic (March 2020), student lives have changed dramatically worldwide. Governments implemented measures to prevent the spread of the virus, e.g., social distancing, the closing of public spaces and the catering industry, and restrictions regarding indoor and team sports ,12. In mThe impact of the first COVID-19 lockdown on students’ smoking, binge drinking and cannabis use was already studied in several international (university) student samples. Smoking did not change in German and Italian students ,16. In cInsights from before the COVID-19 pandemic showed that several student- and study-related factors play a role in students’ smoking, binge drinking and/or cannabis use. Being male, being a gender minority, being of white ethnicity, mental health problems, participation in extracurricular activities , college residence (not living with parents) and being in their first year of college were associated with higher substance use ,24. AddiAlthough these studies provide valuable insights, the amount of evidence regarding student- and study-related factors associated with substance use during the COVID-19 pandemic is limited. Up-to-date insights regarding the impact of the first COVID-19 lockdown on students’ substance use and associated characteristics are essential to provide adequate interventions and targeted services to (specific subgroups of) students and prepare for future lockdown situations.According to our knowledge, no study exists that extensively investigated a wide variety of student- and study-related factors associated with (trends in) smoking, binge drinking and/or cannabis use in students in higher education, comparing the period before and during the first COVID-19 lockdown in the Netherlands (March 2020 to June 2020). In the current study, we aimed to (1) investigate changes in the prevalence of weekly smoking, weekly binge drinking and weekly cannabis use before and during the first COVID-19 lockdown; (2) explore which student-, study- and/or COVID-19-related characteristics are associated with weekly smoking, weekly binge drinking and weekly cannabis use both before and during the first COVID-19 lockdown; and (3) explore which pre-COVID-19 student-, and study-related characteristics contribute to changes in weekly smoking, weekly binge drinking or weekly cannabis use during the first COVID-19 lockdown in (a) non-users before COVID-19 and (b) weekly users before COVID-19.The methods and data analyses were pre-registered in the Open Science Framework (OSF) .This study is part of the COVID-19 International Student Well-Being Study  and applied university  students, referred to as university students. Students who had not finished the survey (N = 3462), international students who moved back to their country of residence before or at the start of the COVID-19 pandemic , participants aged ≥ 31 years old  and participants who did not identify themselves as male or female were excluded from further analyses . In total, data of 9967 students were available for the main analyses (model A). The set of analyses described under model B was conducted in subgroups of non-users before COVID-19 for all three substances. The set of analyses described under model C was conducted in subgroups of weekly users before COVID-19 for all three substances.Validated survey instruments were selected from previous international (student) surveys when available, and new survey items were developed when existing instruments were not suitable for questioning the COVID-19 pandemic. Details on survey topics are described in the study protocol . Items iFor a substantial amount of the survey items, participants were asked to describe the situation before and during the COVID-19 regulations. “Before COVID-19” referred to the average situation during the month prior to the implementation of the first COVID-19 regulations. “During COVID-19” referred to the week prior to filling out the survey .In order to investigate weekly substance use before and during COVID-19, six outcome variables were created: weekly smoking before COVID-19 (1) and during COVID-19 (2); weekly binge drinking before COVID-19 (3) and during COVID-19 (4); weekly cannabis use before COVID-19 (5) and during COVID-19 (6). These variables were created based on questions regarding the frequency of tobacco smoking, binge drinking (drinking ≥6 glasses on a single occasion) and cannabis use before and during COVID-19. Response categories were dichotomised into “non-user” (0) (including (1)  never, (2) less than once a week) versus “weekly user” (1) (including (3) once a week, (4) more than once a week and (5)  daily). Participants who answered (6) “prefer not to say” either before or during COVID-19 were disregarded for the analysis of that particular substance.In order to investigate increases in weekly smoking, weekly binge drinking and weekly cannabis use in students who were non-users before COVID-19, dichotomous outcome variables were created for all three substances: “stable low” (0) versus “increase” (1), where “stable low” referred to being a non-user before and during COVID-19 and “increase” referred to moving from being a non-user before COVID-19 to weekly user during COVID-19.In order to investigate decreases in weekly smoking, weekly binge drinking and weekly cannabis use in students who were weekly users before COVID-19, dichotomous outcome variables were created for all three substances: “stable high” (0) versus “decrease” (1), where “stable high” referred to being a weekly user before and during COVID-19 and “decrease” referred to moving from being a weekly user before COVID-19 to non-user during COVID-19.Student-, study- and COVID-19-related characteristics were added in blocks: (1) demographics , (2) study-related information before and/or during COVID-19 , (3) study attitude factors during COVID-19 , (4) study stress and well-being during COVID-19 (study-related concerns and depressive symptoms) and (5) other factors related to the COVID-19 situation . Detailed information on coding and scales is shown in Prevalence rates of weekly smoking, weekly binge drinking and weekly cannabis use before and during the first COVID-19 lockdown were analysed by using Chi-square tests (to test gender differences) and McNemar tests for paired observations  over time).p < 0.05) for the next step in the model. In the final model, only variables with p < 0.0083 were deemed statistically significant . The OR, p-value and explained variance (Nagelkerke R2) were reported. Additionally, to investigate the effect of combining multiple independent variables on the obtained associations, univariate logistic regression analyses (including gender and age as covariates) were performed to explore the individual relationships between the mean outcome variables and all separate independent variables  for the next step in the model. In the final models, only variables with p < 0.0167 were deemed statistically significant . OR, p-values and NagelKerke R2 were reported.Model B and C—Two multivariate logistic regression analyses were used to investigate whether pre-COVID-19 independent variables were associated with trends in weekly smoking, weekly binge drinking and weekly cannabis use during the first COVID-19 lockdown see . In bothThe majority of the students were female, born in the Netherlands, had highly educated parents, enrolled in bachelor programs and were not first-year students . A subsep < 0.001) while the number of weekly cannabis users increased (p < 0.001). Both before and during the first COVID-19 lockdown, there were more males than females who were weekly smokers, weekly binge drinkers or weekly cannabis users (p < 0.001).No difference in the number of weekly smokers was observed when comparing before and during the first COVID-19 lockdown . The numOverall, male gender , not living with parents (vs. living with parents), being a bachelor student (vs. master student) and having less financial resources to cover monthly costs were associated with higher chances of weekly smoking, weekly binge drinking and weekly cannabis use (before/during COVID-19), see For all three substances, the majority of the students were non-users both before and during the first COVID-19 lockdown (stable low) . In the In the group of non-smokers, non-binge drinkers and non-cannabis users before COVID-19, male gender, not living with parents, bachelor programs, not being born in the Netherlands and having a student loan were associated with increased likelihood of becoming a weekly user during the first COVID-19 lockdown  . Older aIn these analyses, many of the variables were not significant and therefore excluded from the final models . Sample This study investigated the impact of the first COVID-19 lockdown on weekly substance use and associated student-, study- and COVID-19-related characteristics in Dutch university students.The findings indicate clear risk factors for substance use among all three substances: male gender, not living with parents, being a bachelor student, having less financial resources and less adherence to the COVID-19 regulations. Other risk factors such as not being born in the Netherlands, not paying tuition themselves or parents paying intuition and depressive symptoms were associated with increased risk of using either one or two of the investigated substances. Additionally, clear overlap was observed in factors contributing to substance use before and during COVID-19. This indicates that characteristics contributing to substance use in university students are stable over time and are not so much influenced by unexpected stressful events such as the first lockdown. Additionally, a large overlap in identified risk factors  was observed in the cross-sectional analyses regarding substance use before and during COVID-19 (Model A) and in the analyses investigating changes in substance use during the first lockdown compared to before (Model B and C). This indicates that the identified characteristics are not only contributing to the likelihood of substance use but also contribute to changes in substance use. Striking findings both in line and in contrast with the previous literature are discussed below.In line with other studies, we did not observe a change in smoking prevalence during the first COVID-19 lockdown ,16. ThisInsights regarding the role of gender in changes in substance use during the COVID-19 pandemic in students are inconsistent, possibly due to different methodological approaches, study samples or implemented COVID-19 measures. Observed changes in substance use suggested positive and negative effects for males on binge drinking and cannabis use , positivIn line with our findings, White et al.  showed tPrevious studies conducted before COVID-19 showed that mental health or mood problems often co-occur with substance use (disorders) ,33,34,35To the best of our knowledge, this is the first study to examine the association between (trends in) substance use and student-, study- and COVID-19-related characteristics in Dutch university students, investigating a comprehensive set of factors both cross-sectionally and over time (retrospective). This broader perspective is important to understand the mechanisms of students’ substance use (and the relative importance of contributing factors).p < 0.05. It appeared that after adding variables from additional blocks, some variables from earlier added blocks did not remain significant in the final model. Therefore, the order of adding the variables influenced the obtained results.Several limitations need to be considered. The study sample consists of different subsamples of Dutch (applied) university students. Although this makes it possible to generalise the results to different Dutch student populations, it limits the generalisability to international student populations due to differences in the education system and COVID-19 regulations. Selection bias might be playing a role if students with extremely high levels of substance use or poor well-being did not participate in the study. However, a major impact is not expected, as prevalence rates of smoking, binge drinking and cannabis use align with earlier research in Dutch university students ,36. Next(1) Follow-up research should pay attention to high substance use risk groups  and how substance use within these groups will further develop during the current continuation of the pandemic. (2) Despite the general not so alarming trends in prevalence rates, there are subgroups of students who (re)started using substances or who further increased their weekly use during the COVID-19 pandemic. Extra support and further research are needed for these groups. (3) In this study, some risk factors were identified who contribute to the use of all three investigated substances, while also risk factors were identified who only contribute to the use of one or two substances only. Future research should focus on the unique mechanisms and involved characteristics per substance. (4) Polysubstance use should be taken into account since it is known that the use of one substance may increase the likelihood of using another . AdditioThis study provides valuable insights for public authorities and higher education institutes, especially since the COVID-19 pandemic is still ongoing, and students, higher education institutes and policymakers will also have to cope with its consequences in the near future. Public authorities might want to pay extra attention to high-risk groups of substance use when developing/improving interventions and policies addressing substance use among students in higher education. Additionally, public authorities should ensure that higher education institutes have adequate resources to train their staff and provide effective and accessible services to help students adopt healthy lifestyles. Higher education institutes should ensure that support services  are available and that these services are accessible and user-friendly. Communication towards students about available services and practicalities should be provided in a clear and accessible way. Lastly, higher education institutes should be aware that there are different subgroups of students who are at elevated risk of using substances and that these students might need to be actively offered extra support. This is essential, not only during the COVID-19 pandemic but also when regulations are loosened, as supporting these students will contribute to the prevention or decrease in hazardous substance use (and related consequences) in the future.This study makes a substantial contribution to the knowledge on the underlying mechanisms of substance use and the identification of substance use high-risk groups both before and during the first COVID-19 lockdown. These valuable new insights on how students cope during a global health crisis can directly be applied by public authorities and higher education institutes by enhancing implemented policies, interventions and support services. This is of utmost relevance as the COVID-19 pandemic is still ongoing, and new lockdowns/restrictions are still in place.