Molecular characterisation of hepatocellular carcinoma (HCC) is central to the development of novel therapeutic strategies for the disease. We have previously demonstrated mutagenic consequences of Long-Interspersed Nuclear Element-1 (LINE1s/L1) retrotransposition. However, the role of L1 in HCC, besides somatic mutagenesis, is not well understood.n = 372) and explored potential relationships between L1 expression and clinical features. The findings were confirmed by immunohistochemical (IHC) analysis of an independent human HCC cohort (n = 48) and functional mechanisms explored using in vitro and in vivo model systems.We analysed L1 expression in the TCGA-HCC RNAseq dataset . Experimental modulation of L1 ORF1p levels revealed an influence of L1 ORF1p on key hepatocarcinogenesis-related pathways. Reduction in cell migration and invasive capacity was observed upon L1 ORF1 knockdown, both in vitro and in vivo. In particular, L1 ORF1p increased PIN1 cytoplasmic localisation. Blocking PIN1 activity abrogated L1 ORF1p-induced NF-κB-mediated inflammatory response genes while further activated TGFβ-signalling confirming differential alteration of PIN1 activity in cellular compartments by L1 ORF1p.We observed positive associations between L1 and activated TGFβ-signalling, Our data demonstrate a causal link between L1 ORF1p and key oncogenic pathways mediated by PIN1, presenting a novel therapeutic avenue. CDKN2A silencing and CTNNB1 gene and TERT promoter mutations compared to iCuster2 and iCluster3. Overall, there was overexpression of proliferation marker genes and specific miRNA changes such as high expression of miR-181a and silencing of miR-122. In contrast, iCluster2 was associated with low-grade tumour and less microvascular invasion and exhibited enrichment for HNF1A mutation. iCluster3 was characterised by a higher degree of chromosomal instability with distinct 17p loss, high frequency of TP53 mutation and hypomethylation of multiple CpG sites. Notably, an immune subclass of HCC has also been defined by transcriptional profiling alongside immunohistochemical examination, potentially identifying patients responding to immune checkpoint inhibitors such as nivolumab [TGFB1, TGFB2, TGFBR1, SMAD3, SMAD4 and SPTBN1. Further transcriptomic analysis revealed, “activated”, “normal” and “inactivated” TGFβ signalling clusters in HCC cases, with patients in the inactivated cluster having poorest overall survival. However, non-disruption of the TGFβ pathway  appeared to be associated with better outcome than either activation or inactivation of the pathway, underscoring the importance of the balance of this pathway in hepatocarcinogenesis.Hepatocellular carcinoma (HCC) is the sixth most common type of cancer and the fourth most frequent cause of cancer-related death worldwide . HCC devivolumab . Approxic-MET gene has been reported [hTERT [Our previous work has demonstrated the activation and mutagenic consequences of long interspersed nuclear element  retrotransposition in HCC , 8. L1 breported . Likewisreported . The infreported –28. L1 Od [hTERT  and drugd [hTERT , 31 in td [hTERT .TP53 gene mutation [In gastrointestinal (GI) cancer patients, L1 retrotransposition and expression correlated with clinical factors, including age and survival . In addimutation , 17. ThiArchived diagnostic formalin-fixed paraffin-embedded (FFPE) biopsies with pathologically confirmed HCC were obtained from our own biobank, including patients diagnosed between 2002 and 2018, who consented to the use of their tissues surplus to diagnostic requirements for research purposes (project ID: 116370). Ethical approval was obtained for the use of FFPE HCC patient biopsies by the National Research Ethics Service (NRES) Committee North East (12/NE/0395). Clinical features were obtained from patient medical records. L1 ORF1p, pSMAD3 and PIN1 IHC was performed on a Ventana Discovery XT system. Details of antibodies are provided in Supplementary Table l-glutamine and penicillin–streptomycin and were incubated at 37 °C with 5% CO2. All the cell lines were authenticated by serial tandem repeat (STR) profiling  and tested to confirm the lack of mycoplasma infection routinely.All cell lines  were cultured in RPMI1640 medium supplemented with 10% foetal bovine serum, For L1 ORF1 knockdown, Huh7 cells were transduced with lentiviruses encoding L1 ORF1-targeting (CCGGAAATGAAGCGAGAAGGGAAGTCTCGAGACTTCCCTTCTCGCTTCATTTTTTTTG) or control (non-targeting) shRNA (CCGGCAACAAGATGAAGAGCACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG) followed by puromycin selection  as described previously .For conditional overexpression, L1 ORF1 was cloned under control of a DOX-ON (doxycycline-inducible) promoter in a piggybac vector containing a puromycin resistance cassette. Hep3B cells were co-transfected with PB-DOX-empty or PB-DOX-L1 ORF1 along with plasmid expressing Transposase in order to integrate the piggybac vector using Trans-LT1 transfection reagent  as per the manufacturer’s instructions. Cells with stable integration were selected by puromycin selection (1 µg/ml). The cells were then induced with doxycycline  for further experimentation.For chemical treatment experiments, cells were seeded into 12- or 6-well plates, and 24 h after seeding cells were treated with the indicated agents , SB525334 , DTM , BI605906 ) at indicated doses and time.www.svi.nl) software for co-localisation analysis [Cells were cultured into 12-well plates containing glass coverslips and fixed with 4% formaldehyde (10 min) for staining using standard ICC protocols. Details of antibodies are provided in Supplementary Table analysis .In total, 30,000 cells were seeded into 96-well plates to obtain confluence (at least five wells per cell line). Twenty-four hours later, a scratch wound was generated using an IncuCyte Wound Maker tool and plates were incubated and monitored in an IncuCyte time-lapse image capture system to visualise the initial wound and incremental wound closure at 6-h time intervals. Cell migration was then evaluated using an IncuCyte algorithm to measure wound confluence.6 cells/ml) was added at the top of the chamber in serum-free media. On the bottom, 500 µl complete media was added and the chambers were incubated at 37 °C and 5% CO2 for 72 h. Invaded cells were stained according to the manufacturer’s protocol. Phase-contrast images were taken (5–10 random fields) and quantified using ImageJ.Invasion assay was performed using QCM ECMatrix cell invasion assay kit . In short, 300 µl cell suspension  and Omega FLUOstar plate reader  with luminescence settings.Total RNA was extracted using an RNeasy Mini Kit . The RNA purity and concentration were estimated with an ND-1000 spectrophotometer . Complementary DNA was generated using the cDNA Reverse Transcription Kit  as per the manufacturer’s instructions. The qRT-PCR was carried out as described previously . PrimersTotal RNA was isolated for all the indicated cell lines from three biological replicates using an RNeasy kit. RNA integrity was confirmed by Agilent Bioanalyser . Illumina Tru-seq paired-end strand-specific sequencing was carried out on a NextSeq500 (Newcastle University Genomics Facility). Post-trimming quality control was performed with FastQC (version: 1.0.0). The resulting FastQ files were mapped on to human reference genome using RNAseq alignment tool (V1.1.1 for Huh7 and V2.0.2 for Hep3B) using Illumina BaseSpace software. A differential expression gene (DEG) list was obtained using DESeq2. Gene set enrichment analysis (GSEA) was carried out using broad institute’s GSEA software (V4.0.3), wherein “hallmark gene sets” from a molecular signatures database (MSigDB) were analysed by contrasting full gene lists of Huh7-L1KD versus Huh7-NT and Hep3B-DOX-Empty versus Hep3B-DOX-L1 ORF1 samples. Upstream regulators and interaction networks of DEGs were analysed using Qiagen Ingenuity Pathway Analysis  software. The data can be found in GEO database (GSE126615 and GSE194251).Human HCC RNAseq data was downloaded from the TCGA-LIHC project and mapped to the human L1-Ta sequence  by BLAT alignment using an in-house algorithm to obtain L1 counts . The couhttp://www.nc3rs.org.uk/page.asp?id=1357). Experiments were performed using 8–10-week-old male NSG mice obtained from Charles River. Mice under isoflurane general anaesthesia underwent laparotomy to expose the liver. Using an insulin syringe, 1 million cells  in a volume of 30 µl of 50% matrigel were injected directly into the left lobe of the liver. Whole-body bioluminescence images were acquired using an In Vivo Imaging System (IVIS spectrum: Perkin Elmer). Prior to in vivo imaging (10 min), mice were injected intraperitoneally with 10 mg/Kg of d-luciferin (Perkin Elmer). Following in vivo monitoring of tumour development, mice were humanly euthanised, organs were harvested and ex vivo imaging was performed. Both in vivo and ex vivo images were acquired using an open filter with auto-exposure. Images were analysed using Living Image . In vivo and ex vivo photon signals were quantified using the region of Interest method of analysis [All animal experiments were approved by the Newcastle University Ethical Review Committee and performed under a UK Home Office licence in accordance with the ARRIVE guidelines  or one- or two-way ANOVA with Tukey’s multiple comparison when required (three groups). Fold change (FC) data were analysed by one-sample t test using 1 as the hypothetical mean. P < 0.05 was taken as a cut-off for significance. Mean ±  standard errors or median lines are shown in figures where applicable. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.L1 transcript expression distribution amongst HCC subgroups (predefined clusters—information obtained from previous publications) and patients’ clinical features was analysed using the SPSS statistical package . Likewise, IHC data, along with patients’ clinical features, were analysed by the SPSS statistical package. Categorical and continuous datasets were compared using Pearson’s chi-square (Fishers Exact for groups with <5 cases) and Wilcoxson signed ranks  or Mann–Whitney (two groups) or Kruskal–Wallis (three groups) tests, respectively. Correlations between normalised L1 counts and a subset of genes were calculated by the Spearman correlation test. GraphPad Prism software (GraphPad 8.0) was used for cellular assays and analysed by Student’s TP53 mutation, and hypomethylation of multiple CpG sites [TP53 mutation . Besides TP53, no significant associations were observed with any other common HCC gene mutations such as CTNNB1 or TERT promoter. Similar to GI cancers [P < 0.001, Supplementary Fig. TP53 mutation and TGFβ activation, as they were for other subfamilies, with the exception of L1PA3 and TP53 mutation (data not shown).RNAseq data (TCGA-HCC samples) revealed a significant increase in L1 transcript levels in HCC compared to matched non-tumour (NT) tissues   consortium reported active retrotransposition in various cancer types, including a subset of TCGA-HCC samples . We combype Fig. .To analyse the relationship between L1 expression in HCC and other clinical characteristics of the patients in the TCGA cohort, we parsed the dataset with clinical characteristics including age, gender, ancestry, aetiology, pathological grade, patient AFP levels and tumour invasion. For this analysis, HCC samples were divided into two groups based on their L1 transcript expression level—L1-Low  liver tissue, L1 norm count ≤25) and L1-High (L1 expression higher than NT liver tissue). There were no significant correlations with age, gender, ancestry or aetiology   and data needs to be validated in a larger cohort  expression by IHC on formalin-fixed paraffin-embedded (FFPE) diagnostic biopsies from HCC patients in an independent cohort from our own biobank. The results were examined in relation to tumour grade, clinical features and patient outcome. Similar to the transcript data, L1 ORF1p expression was observed in HCC with minimal background expression in surrounding NT tissues or immune infiltrates Fig. . The L1 st) Fig. . There wFinally, we carried out IHC for phosphorylated SMAD family member 3 (pSMAD3-ser425) as a surrogate for TGFβ signalling activation status in the tumours in a subset of the initial L1-IHC cohort Fig. . Thus, tWestern blotting was performed on whole cell lysates of various liver cancer cell lines to evaluate the expression status of L1 ORF1p and to select cell lines for in vitro L1 manipulations. A range of L1 ORF1p expression was observed in these cell lines, as seen previously in primary HCC samples. L1 ORF1p expression was high in SNU475, SNU182 , SK-Hep1 (cholangiocarcinoma) and Huh7 , and lower in the other epithelial cell lines Huh1, PLC/PRF-5 and Hep3B while being almost undetectable in HepG2    and control cells expressing non-targeted shRNA (Huh7-NT) Fig. . A signi 4) Fig. g, h.Fig.Padj <0.05 was used, while 334 DEGs with a cut-off of log2 fold change 1, Padj < 0.05 . Moreover, Huh7-L1KD cells exhibited downregulation of SMAD3 at the protein level, when compared to Huh7-NT cells  [SMAD3 being a direct target of TGFβ signalling in these cells [RNAseq analysis of Huh7-L1KD versus Huh7-NT cells revealed 1512 differentially expressed genes (DEGs) when a cut-off of log2 fold change 0.5, .05 Fig. . In addills Fig. . The datng site)   cells showing the influence of TGFβ1 upstream of L1 . HoweverTo further explore the mechanism of influence of L1 on TGFβ signalling, we investigated common regulators/interacting partners of L1-encoded proteins (L1 ORF1p and L1 ORF2p) and TGFβ signalling. These included PIN1, which is a propyl isomerase that isomerises specific phosphorylated Ser/Thr-Pro proteins and is overexpressed in various cancer types . PIN1 biCo-staining for L1 ORF1p and PIN1 in Hep3B cells , which In human HCC biopsies studied by IHC, PIN1 positivity was observed in 15 out of 17 HCC, as either nuclear or both nuclear and cytoplasmic expression. The relationship between L1 ORF1p and PIN1 intensity, percentage positivity and localisation was assessed and scored by a liver pathologist in a blinded manner Fig. . This coTo evaluate the influence of L1 ORF1p on the cellular transcriptome, we generated a conditional (TET/DOX-ON) L1 ORF1 overexpression system using a piggybac vector in Hep3B cells  correlating positively with L1 ORF1, including those for KRAS upregulated signalling, Myc targets, inflammatory responses  and EMT. We concluded that L1 ORF1 overexpression influenced these tumorigenesis-associated pathways  were found in Hep3B-DOX-L1 ORF1 cells when compared to Hep3B-DOX-empty cells at a cut-off of log2 Fold change 0.5, ays Fig. .We went on to further interrogate the potential influence of PIN1 within these regulatory networks, using Ingenuity Pathway Analysis (IPA). An interaction network of PIN1 with the DEGs was generated, identifying 26 nodes and predicted PIN1 to be activated in Hep3B-DOX-L1 ORF1 compared to Hep3B-DOX-empty cells Fig. . BesidesPAI1 (SMAD3 target upon TGFβ stimulation) and CXCL10, IFIH1 and ICAM1 (inflammatory or NF-κB response genes) in the Hep3B-DOX-L1 ORF1p + Dox cells. The L1 ORF1p-induced upregulation of PAI1 was suppressed by treatment with SB525334, a specific inhibitor of the TGF-beta receptor I (ALK5) receptor. Upregulation of CXCL10, IFIH1 and ICAM1 was suppressed by treatment with BI605906, a specific inhibitor of IKKβ—a key activatory kinase of the NF-κB signalling pathway (Fig. RT-qPCR confirmed upregulation of way Fig. . These dway Fig. . Thus, iIn combination, we propose that in HCC, aberrant L1 ORF1p expression leads to higher expression of cytoplasmic PIN1, which contributes towards dysregulation of a number of oncogenic pathways—including upregulation of TGFβ and NF-κB signalling—that promote tumour invasion and metastasis.The development of personalised treatment for cancer requires the discovery of key drivers of tumour cell biology in diverse cancer types. The major contribution of activated L1 retrotransposons in cancer is attributed to their retrotransposition capability, leading to somatic mutagenesis and genomic rearrangements. The Influence of L1 on oncogenic pathways has been documented, but typically via insertional mutagenesis pathway , 19. OurTP53 mutation—it can promote the progression of tumours, mediating cancer metastasis by inducing EMT [TGFβ signalling has both tumour promoter and suppressor activities, with a complexity that makes it hard to target therapeutically . It is acing EMT , 58. Thucing EMT , 59, whiMoreover, targeting the interaction of L1 ORF1p with host proteins or downstream signalling may have therapeutic value worthy of further exploration. This novel approach to alter TGFβ or other inflammatory oncogenic pathways would have the potential advantage of being ‘cancer specific’. In contrast to our observations, Zhu et al. have reported direct interaction of L1 ORF1p with SMAD4 leading to the suppression of the translocation of SMAD4 from the cytoplasm to the nucleus elicited by TGFβ . This coOur observation of the upregulation of PIN1 in human HCC in situ, particularly in the cytoplasm in association with L1 ORF1p, is notable. In a number of other cancer types, cytoplasmic localisation of PIN1 has been associated with cancer aggressiveness and poor prognosis , 50. SimL1-encoded L1 ORF1p is specifically overexpressed in HCC compared to surrounding non-tumour liver tissue or immune infiltrates and influences oncogenic pathways by interacting with cellular proteins. In summary, we draw attention to the L1 ORF1p mediated cytoplasmic upregulation of PIN1 in human HCC, warranting its further exploration as a clinical predictive biomarker and novel anticancer drug development target for HCC.Supplementary Information


N = 41,209), we classified the content of existing AAT questions as invoking proximal versus distal details. We found better performance with proximal compared to distal questions, especially for low-achieving examinees. Studies 2 and 3 manipulated the distance of questions adapted from AATs and examined three moderators: overall AAT score, working-memory capacity, and presence of irrelevant information. In Study 2 (N = 129), proximity (versus distance) improved the performance of low-achieving participants. In Study 3 (N = 1744), a field study, among low-achieving examinees, proximity improved performance on questions that included irrelevant information. Together, these results suggest that the psychological distance that is invoked by the content of test questions has important consequences for performance in real-life high-stakes tests.Performance on standardized academic aptitude tests (AAT) can determine important life outcomes. However, it is not clear whether and which aspects of the content of test questions affect performance. We examined the effect of psychological distance embedded in test questions. In Study 1 ( Consider the following problem: Danielle prefers bananas over apples, and apples over strawberries. Considering her parents want to send her favorite fruit in the lunchbox, should they buy bananas or strawberries? This type of problem is often presented in academic aptitude tests (AAT), but can also emerge in real life, when, for example, we need to reason through a social situation , or come up with a novel, creative way to formalize a multifaceted problem.4. Specifically, CLT proposes that objects that are distal in time, space, or social perspective or are uncertain , are mentally represented  on a higher level of abstraction. Higher level, abstract mental representations extract the gist of informational input by retaining those features of input stimuli that the perceiver deems essential, whereas low-level construals include also incidental, contextual features of the input.The present paper examines how psychological distance affects reasoning with this type of questions. The psychological distance to an object is the extent to which a person feels that it is removed from “the reality of me, here and now” in either time (future or past), space, social perspective, or the realm of hypothetical worlds. Construal level theory (CLT) extensively studied the effects of psychological distance on the way information is represented and processedIn the present set of studies we examined whether it would help examinees to arrive at the correct solution in reasoning questions if the problems pertain to psychologically distal objects and events , or rather if the problems pertain to objects and events in the solver’s environment ? It is important to note that although there might be a potential utility of prior knowledge in problem-solving, the prior knowledge activated by proximity or distance is superfluous to solving the problem correctly. For example, the arithmetic problem “2 + 2” is not a-priori facilitated by knowledge of whether the items being counted are apples, bananas, persimmons, or pitaya; the physical characteristics of these fruits are extraneous to the arithmetic calculation. To perform the calculation, one does not rely on any known properties of the fruit. We designed our studies to investigate whether this extraneous aspect of the problem, specifically, whether the objects in the problem are psychologically proximal or distal, would affect performance.The literature seems to offer opposite answers. Some approaches suggest that distancing would make it easier for the problem solver to filter out irrelevant details , and thus represent the logical structure of the problem more clearly. In contrast, other approaches suggest that proximity would make the problem details more concrete and vivid and thus make it easier to represent the problem parameters and to ultimately solve it .The question of whether distance or rather proximity is more conducive to solving reasoning problems is obviously interesting for practical reasons, because knowing the answer would help people solve such problems, and design interventions to help others. But the question is also interesting for theoretical reasons, in view of the aforementioned disagreement. In what follows, we first review in more detail the opposing views on how distance might affect solving reasoning problems, and then report three studies that attempted to answer it empirically.5 found that physically distancing children from maps or models  helped them to solve problems that required the understanding of the relationship between the symbol and its referent . In contrast, increasing proximity to the model  impaired understanding of the mapping between the model and its referent and interfered with problem solving8.Many theoretical traditions seem to suggest that distancing a problem from oneself, here-and-now improves reasoning. For example, DeLoache9 and adults13. For example, in one of their studies, Polman and Emich12 showed that people solved a problem better when taking a socially distant perspective  compared to a proximal perspective (imagined themselves being immersed in the problem). As another example, studies on negotiation have shown that taking a distant perspective  improved the chances of the parties to reach integrative, mutually beneficial agreements about complex, multi-dimensional problems16.Distance has also been found to increase creative problem solving in both children17, psychological distance has been theorized and found to foster more abstract representations of objects. Hence, findings that show that more abstract (versus concrete) representations of problems facilitate their solution e.g.,20 are consistent with the possibility that psychological distancing would have a similar effect.Within CLT7. More specifically, abstraction engenders two distinct yet related processes : One process is filtering out of irrelevant information, such that distracting or irrelevant details are omitted, making it easier to concentrate on the critical parameters of the problem21. The second process is integration of information, such that objects are grouped together to form a broader category23. Both processes might assist mental operations that are critical for solving complex problems. Specifically, both filtering and grouping may reduce load on working memory25, a resource that is critical for solving complex problems, for review, see26.Indeed, abstraction has been proposed to mediate and explain the beneficial effects of psychological distance on problem solving e.g.,23. This, in turn, can help simplify mental organization and processing, further reducing the demands on working memory 27.The potential interaction between psychological distance, working memory capacity, and the presence of irrelevant information could manifest in a number of ways. For instance, when individuals are not immersed in the vivid details of a problem—i.e., they are psychologically distant from it—they may be less likely to be distracted by irrelevant information. Consequently, they may be able to focus more effectively on the problem’s critical aspects, thereby reducing the cognitive load on their working memory. Furthermore, psychological distance can facilitate categorization by enabling individuals to group related information into broader categories28, and hence, to the extent that problem solving relies on these processes, psychological proximity should assist it.Of course, solving complex problems does not rely solely on abstraction, but may also be assisted by a clear, vivid representation of the problem with all its relevant detail. It has been found that stimuli that are more proximal to me, here-and-now are processed with more attention, are represented in more detail, and are remembered better e.g.,29, were less prone to visual illusions30, exhibited improved letter recognition31, and faster discrimination between shapes32. Socially proximal information (about oneself or close others as opposed to about unfamiliar people) has also been shown to have similar advantages for cognitive processing: people remember information related to them and to objects they own better than information related to others and their objects33, an effect coined the self-reference effect in memory34, for a review and meta-analysis see37. Psychological proximity (compared to distance) also improved peoples’ performance on problems that required them to find missing parts in detailed pictures 38, Study 6.Several findings support this notion. For example, when objects were presented within physical reach, people segregated a figure from its background more accurately39, for a review, see40. It has also been suggested that proximal objects tend to attract more attention due to their relevance, that is, because when proximal, they can, for example, be consumed, manipulated, pose danger, or yield reward more than when distal41. Within the framework of CLT, proximity engenders more concrete, vivid and detailed representations. Thus, according to CLT, proximity should facilitate solving of problems that rely on these processes.Why are proximal objects processed better? Neuroscientific research suggests one reason according to which information presented in the space near the body  receives preferential processing42. A meta-analysis of 55 studies on students from kindergarten to college showed that the use of concrete objects (compared to abstract symbols) at math instruction facilitated problem solving, retention, and transfer of learned materials to new domains43.Consistent with the facilitating effects of proximity are findings that concrete, embodied processing of problems (compared to abstract processing) assist in solving them, for a review see44. Furthermore, the familiarity associated with psychologically proximal objects or events may decrease anxiety, which can free up working memory space45. Collectively, these processes may result in better problem-solving.Moreover, psychological proximity may enhance problem solving by influencing working memory. When an individual is psychologically close to a problem , they may be recruiting more memory traces from long-term memory to support information currently held in working memoryIn light of the aforementioned disagreement, our aim was to examine how psychological distance that is embedded in the problem parameters would affect performance on reasoning problems. We used verbal reasoning questions, because they require both an accurate representation of the specific parameters of the problem, and an accurate mapping of these parameters on an abstract logical structure. To better understand the effect of distance, we tested three moderators of its effect: overall AAT performance (Studies 1–3), working-memory capacity (Study 2), and presence of irrelevant details in the test question (Studies 2–3). All three moderators were expected to be related to performance . In addition, as noted above, different theoretical approaches predict interactions of these parameters with the effect of distance. In particular, if distance or higher working memory capacity facilitate performance by making it easier to filter-out irrelevant details, then their effect might be more pronounced in problems in which such details are present.In three studies, we examined performance on questions that included details that were either psychologically proximal or psychologically distal in time, space, and on the social dimension. Specifically, in the proximal condition, the questions were about objects and places in the participants’ culture and environment, about members of their ingroup and events in the recent past. In contrast, in the distal condition, questions were about objects and places from distant cultures, members of unfamiliar groups, and events in the distant past see Fig. .Fig. 1Sa46. This study’s design and its analyses were not preregistered. The methods were performed in accordance with relevant guidelines and regulations and approved by the Institutional Review Board of Tel Aviv University.The method section describes the sample, all data exclusions (if any), all manipulations, and all measures in the study. All data and analyses code are available at Open Science Repository (osf.io/2nvtq). Data were analyzed using R, version 4.1.2 and the package ggplot, version 3.3.547. Accuracy was coded as a binary variable . AAT scores were mean-centered48, and categorical variables were effect-coded . Type-III significance levels were calculated with the ‘car’ package version 3.1-0 49.In all studies we ran a generalized linear mixed model (GLMM) using the glmer function  in the lme4 package in R version 1.1-30Study 1N = 41,209). We analyzed accuracy on verbal reasoning questions that were distal versus proximal as coded by judges. We obtained information on performance on these questions from the organization that administered the test.Study 1 examined whether performance would be better with psychologically proximal  questions. We also included examinees’ final AAT scores as factor in the analysis. This enabled us to evaluate whether the effects of psychological distance vary according to ability level. We collected data from all publicly available AATs administered between the years 2014–2017 . An interaction between AAT and distance, B = 0.05, SE = 0.007, z = 7.64, p < 0.0001, indicated that the advantage of proximal questions over distal questions was more pronounced for low-achieving examinees (whose AAT scores were lower than the average), see Fig. We examined the effect of distance and AAT score on accuracy. As expected, accuracy was higher for participants with higher AAT scores, Study 2N = 129) aimed to examine the effects of distance on verbal reasoning while keeping the logical structure of the question constant. We used materials from a standardized AAT, and adjusted the content of the questions to refer to distant or proximal objects and situations (between participants), and to include only relevant or also irrelevant details (within participants). We also explored the possible moderating effects of AAT and of working-memory capacity on the effects of distance, relevance, and their interactions. We hypothesized that (a) AAT scores would positively correlate with performance, (b) working-memory capacity would positively correlate with performance, (c) adding irrelevant details would impair performance (compared to not adding them), and (d) replicating Study 1, proximity would enhance performance, especially for participants with low AAT scores.In study 1 we compared to each other questions that could have had different logical structure. Study 2  = 0.16, p = 0.087, possibly due to the restricted range of AAT scores which could have reflected the high admission threshold of students in the psychology program of the relevant university . We therefore included both AAT score and working-memory capacity (Kmax) in the same model. We present the regression coefficients in Table As both AAT scores and working-memory capacity reflect cognitive ability, they tend to be positively associated e.g.,Kmax and their interactions on accuracy. As expected, accuracy was higher for participants with higher AAT scores, B = 0.349, SE = 0.135, z = 2.59, p = 0.009, as well as for participants with higher working-memory capacity, Kmax, B = 0.297, SE = 0.147, z = 2.01, p = 0.044. An interaction between these scores, B = 0.315, SE = 0.150, z = 2.103, p = 0.035, indicated that their effects intensified each other, namely, with higher AAT scores, working-memory capacity had a larger effect on accuracy, and that with higher Kmax, AAT scores had a larger effect on accuracy , nor did relevance, B = −0.113, SE = 0.168, z = −0.67, p = 0.499, . The only effect that involved relevance was a marginally significant three-way interaction between relevance, distance and Kmax, B = 0.482, SE = 0.250, z = 1.93, p = 0.054. Inspection of the interaction revealed that adding irrelevant information impaired the performance only among participants with low Kmax in the distal condition, B = 0.284, SE = 0.135, z = 2.10, p = 0.035.Distance did not significantly affect accuracy, B = 0.536, SE = 0.235, z = 2.28, p = 0.022, indicated an advantage of proximal questions over distal questions among low-achieving participants but not among high-achieving participants. The effect of distance for participants with below-average AAT score was quite sizeable: accuracy with proximal questions (M = 0.50) was 28% higher than with distal questions (M = 0.39), although proximal and distal questions only differed in content and had the same logical structure , and the three-way interaction between AAT, distance and relevance (p = 0.21), were not significant.The interaction of AAT with relevance (Study 3N = 1744), aimed to replicate Study 2 in an ecological settings, with a large, representative sample, and a broader range of AAT scores. We used the context of a consequential high-stakes exam that would determine individuals’ admission chances to higher-education institutions. By looking at performance in this high-stakes, real life settings we aimed to increase the generalizability and reliability of the results by minimizing the extent to which the findings are attributable to low motivation or to a subset of participants who have been admitted to selective higher-education institutions. To that end, we collaborated with an organization that administers nation-wide academic aptitude tests, and incorporated the experimental questions into the exam administered as an entrance requirement for higher-education institutions. As in Study 2, we manipulated distance  between participants and relevance (relevant-only vs. irrelevant-added) within participants. Based on the results of Studies 1–2, we predicted that proximity would enhance performance, especially for participants with low AAT scores.Study 3 . Adding irrelevant information had a marginal negative effect on accuracy, B = 0.076, SE = 0.039, z = −1.92, p = 0.055, .We examined the effect of distance, relevance, AAT score, and their interactions on accuracy , distance (p = 0.890), or their interaction (p = 0.880), the performance of low-achieving examinees was impaired by adding irrelevant information B = −0.146, SE = 0.050, z = −2.91, p = 0.003. Also in this group, although distance did not affect performance as a main effect, (p = 0.390), it interacted with relevance, B = −0.087, SE = 0.040, z = −2.16, p = 0.031, such that accuracy was lower in distal-irrelevant questions, B = −0.137, SE = 0.063, z = −2.17, p = 0.030. For participants with below-average AAT score, performance on proximal questions (M = 0.52) was higher compared to distal questions (M = 0.48) by 7%, but only when the questions were made more difficult by including irrelevant details.Although the interaction between distance and AAT score was not significant, Across three studies - in a corpus of large-scale real-life data attained from an academic aptitude test , in the lab (Study 2), and in the field (Study 3), we examined how performance on verbal reasoning questions is affected by the examinee’s psychological distance from the content of those questions. In all studies, individuals completed verbal reasoning questions from standardized AATs. The questions were about objects that were either psychologically distal or psychologically proximal to the examinees in time, space, and social distance. In Study 1, questions included only information necessary for solving the problem, whereas in Studies 2 and 3 some of the questions also included additional, irrelevant details.M = 0.43), and in Study 2 (M = 40), and for low-achieving examinees in the irrelevant-added problems in Study 3 (M = 0.42) was similar. In this way, the differences between the results obtained in the three studies may in fact point to a consistent pattern - whereby proximity is facilitative  when difficulty is high. Importantly, these effects emerged not only in the lab, but also when participants took a consequential, high-stakes exam that could determine their admission chances to higher-education institutions, and possibly affect their future career paths.In Study 1 we found that people scored higher on questions that presented proximal content compared to questions that presented distal content. Notably, the facilitative effect of proximity was more pronounced among low-achieving examinees than among high-achieving examinees. In Study 2, we found the same pattern: proximity (compared to distance) enhanced performance for participants who attained low scores on the AAT. In Study 3, we predicted that proximity would improve the performance of low-achieving examinees (as in Studies 1–2), but we found this to be true only in questions that were made more difficult by including irrelevant details. Our results, therefore, suggest that proximity is especially advantageous  for low-achieving individuals and when difficulty is high. In Studies 1 and 2 proximity facilitated correct responding among examinees who attained below-average AAT scores, and in Study 3 proximity facilitated correct responding on irrelevant-added problems among examinees who attained below-average AAT scores. Indeed, the average accuracy rate for low-achieving examinees (those with 1 SD below the mean) in Study 1  to be at some distance – to occur in a certain proximal or distal point in time and space, to be familiar to the examinee or their social group, or, conversely to denote a socially distal group.These findings are in line with previous research which documented that proximity enhanced cognitive performance, for a review, seePsychological distance to test items is idiosyncratic: It may depend on the level of familiarity, knowledge and exposure to various objects, people and situations that are mentioned in the item. However, some differences in a person’s distance to test items may also depend on social group. Culture-specific entities such as public figures, symbols, historical events or heritage sites would be typically regarded as close and familiar for certain people while distant and alien for others. Our results suggest that the extent to which an examinee feels proximal to the content of a question can affect performance. For example, the extent to which an examinee feels close to the British philosopher David Hume can determine how well they do on a question that invokes this historical intellectual figure. We thus suggest that test developers take into account the demographic and socioeconomic characteristics of examinees, and try to construct questions that do not create or amplify differences in level of achievement that are based on these differences. This could be achieved by pre-testing the materials and tailoring the content to avoid making it psychologically closer to some examinees than to others. Our findings thus identify a new source of potential cultural bias in AAT questions. The finding that low-scoring examinees are more affected by distance is also informative in this regard, as it suggests that this cultural bias would impact this segment in particular.52, possibly because familiar objects rely on long-term memory processes that shield representations from interference44. Moreover, people tend to physically hold, manipulate, and interact with proximal objects more often than with distal objects and thus would tend to have a more embodied representation of them55. According to embodied cognition theory, cognition has evolved to facilitate action, and as such, cognitive processes that support action are more accurate and efficient. It is possible, then, that reasoning is better with proximal objects because they are more easily embodied. Also, a meta-analysis of 19 neuroimaging studies56 showed that the verbal system is involved to a greater extent with the processing of abstract rather than concrete concepts, while the perceptual system is involved to a greater extent with processing of concrete rather than abstract concepts. To the extent that mental imagery or other perceptual simulation facilitate reasoning about a problem, concrete (rather than abstract) representation might be more conducive for performance. Future research, however, is needed to determine what are the conditions in which vivid mental imagery would assist problem solving.Why does proximity improve reasoning? Some of the features that characterize proximal stimuli have been found to facilitate cognitive performance. For example, people are usually more familiar with proximal objects than with distal objects, and studies have found that familiar objects are remembered better than unfamiliar ones57. These processes have been shown to reduce working memory capacity and negatively affect cognitive performance59.Social psychology suggests another potential mediator for the effect of psychological distance, namely, the possibility that distal content might communicate to some examinees that they do not belong to the relevant social environment. For example, it is possible that questions about David Hume, about distal planets and even about a brioche might suggest that “anybody who is not familiar with these things does not belong here”. Studies on stereotype threat have found that performance is undermined when (and to the extent that) difficulty suggests to students that they do not belong to the current social environment60, aptitude tests , cognitive functioning tests 61; and even skills assessment tests in job interviews. Accordingly, it is imperative to understand how performance is affected by seemingly neutral aspects of test questions, such as whether they pertain to distal or proximal objects.From a practical perspective, our findings can inform users and developers of cognitive tests. Tasks akin to the one used in our studies are integral to many cognitive assessment tests, such as intelligence tests Study 1Participants were 41,209 examinees taking the AAT. Overall, we collected data from 10 exams, see Table r = 0.88) as either proximal or distal, with a total of 38 unique questions .Examinees underwent the AAT, a test administered nationally, in which each examinee received a test form, comprised of three sections in the domains of quantitative reasoning, verbal reasoning, and English. Examinees also complete a writing assignment. Each of the three sections has 20–23 multiple-choice questions, which the examinee has 20 minutes to complete. We selected questions from the verbal reasoning section which were coded by two judges  All of M. Petreu’s books are children’s books; (2) M. Petreu published his first book of poetry in 1953; (3) All of M. Petreu’s books are books of poetry; (4) M. Petreu published nothing after 1940.”An example of a proximal question is: “In recent years a new, mechanized method for separating the seeds of the corn from its pulp was developed. This led to a reduction in the price of corn seeds and their sales rose significantly. As a result, the number of corns grown in the US increased greatly. However, a problem arose: the amount of waste – the corn pulp – that accumulated in the factories separating the seeds grew tremendously. A new study found that adding corn pulp to the fodder for sheep and cows greatly improved their health and the quality and quantity of the milk they produced. Which of the following sentences best describes the connection between the problem presented in the first paragraph and the study described in the second paragraph? (1) The study findings explain how the problem arose; (2) The study examines how the problem impacts the new method; (3) The study shows that the advantage of the new method exceeds the damage caused by the problem; (4) The solution to the problem is incorporated in the study findings.” The SOM present more examples of proximal and distal questions.Scores on the AAT are composed from a score in the writing assignment and scores in the three multiple-choice sections . Each correct answer on the multiple-choice sections is worth one point, there is no penalty for incorrect answers. The raw score for these sections is equal to the number of correct answers. To compute the final AAT score, the administering institute assigns twice as much weight to the verbal reasoning and quantitative reasoning scores as it does to the English score. This policy of the institute reflects considerations of optimizing prediction of academic success. The raw scores of the writing assignment and the score in the multiple-choice sections are then converted to a uniform scale ranging from 200–800.Study 2Mage = 24.20, SD = 2.94) took part in the study and were paid 30 NIS (around US$8) for participation. All participants were native Hebrew speakers. Six participants did not complete the working memory task, and 10 participants did not report their AAT scores. Overall, 112 participants provided both measures. We did not have an estimate of the effect, but planned to be able to detect an effect (main effects of distance and relevance and their interaction) of medium size with a probability of 0.80 at a 5% level of significance. An a-priori power analysis using the G*Power calculator62 indicated that a sample size of at least 132 was required. Aiming to meet this goal, data collection continued until the end of the semester. All participants provided informed consent. The study was approved by the institutional review board (IRB) of Tel Aviv University.One hundred twenty-eight undergraduate students from a large Israeli university , 18 questions  were presented, and participants rated the extent to which each question was about “a distal  person, time, or place” on two separate scales . The correlation between the two scales was negative and high (r = −0.92). From the 18 pretest questions, we chose 12.Questions were selected from an online pool of previous academic aptitude tests in Hebrew. We created a proximal and a distal version for each question and added irrelevant information to each version . These two values were then averaged to form a single parameter with a standard formula64, Kmax = S(H-F), where S is the size of the array, H is the observed hit rate , and F is the observed false alarm rate . Higher Kmax scores indicate higher capacity .Visual working memory capacity was measured via the standard change detection taskParticipants were randomly assigned to either a proximal or a distal condition. Participants were seated individually in a quiet room and were provided with a digital stopwatch and an answer booklet, conditions that resembled a standard AAT. Participants had 12 minutes to complete the verbal reasoning section, and were instructed to answer as accurately as they can as many questions as they can. They were also instructed to guess in case time was over. Each participant was presented with only one version of each question. Relevance was manipulated within participants, such that each participant had six questions with only relevant details, and six questions that included also irrelevant details, presented pseudo-randomly. Participants were informed that the three best performers in the test would receive a 100 NIS cash bonus (around $27). At the end of the experiment, participants provided demographic information and reported their highest attained AAT score. One hundred twenty-three participants (95% of the sample) also completed the change-detection task for another study that took place on the same day, prior to the current study.Study 3Mage = 22.30, SD = 2.95) were sampled. In the second wave, which took place in the summer of 2019, 873 examinees  were sampled. While we aimed to reach as many examinees, the organization administering the exam allocated the experimental version on the test according to their operational constraints. Participants signed informed consent as part of the enrollment process to the test.Participants were 1744 examinees, randomly sampled from all the examinees who took the AAT, in two waves. In the first wave, which took place in the summer of 2018, 870 examinees (469 women; The test was comprised of several chapters that are used for calculating the AAT score and several pilot chapters that do not count toward the final test score but are rather used for operational purposes. Examinees do not know which chapters are the pilot chapters. The experimental questions were incorporated into one of these pilot chapters. We used the same method as in Study 2 with several changes. First, the order of questions in each verbal reasoning section was fixed. In Wave 1 we were able to administer nine new questions, and in Wave 2 we administered 10 of the questions from Study 2. Questions were different in the two waves because the rules of the administering agency do not allow repeating questions across tests.Participants were randomly assigned to either a proximal or a distal condition. Each examinee received a test form, comprised of several chapters. Each chapter contained 20–23 multiple choice questions with 20 min allotted to complete the chapter. The experimental questions were incorporated into the pilot chapter which did not count toward the examinees’ final AAT score.Further information on research design is available in the Supplementary MaterialReporting Summary


Furthermore, the AP3.0G1.5 sample (the 3D-printed composite containing 3.0 wt.% of PANI nanofibers and 1.5 wt.% of graphene flakes) exhibited 2.63 times (22.23 MPa) higher tensile strength, 1.47 times (553.8 MPa) higher Young’s modulus, and 5.07 times (25.83%) higher elongation at break values compared to the pristine ABS-like resin with a tensile strength of 8.46 MPa, a Young’s modulus of 376.6 MPa, and an elongation at break of 5.09%. Our work suggests the potential use of highly conductive and mechanically robust ABS-like resin composites in the 3D printing industry. This article not only provides optimized DLP 3D printing conditions for the ABS-like resin, which has both the advantages of the ABS resin and the advantages of a thermoplastic elastomer (TPE), but also presents the effective manufacturing process of ABS-like resin composites with significantly improved conductivity and mechanical properties.Three-dimensional printing is regarded as a future-oriented additive manufacturing technology that is making significant contributions to the field of polymer processing. Among the 3D printing methods, the DLP  technique has attracted great interest because it requires a short printing time and enables high-quality printing through selective light curing of polymeric materials. In this study, we report a fabrication method for ABS-like resin composites containing polyaniline (PANI) nanofibers and graphene flakes suitable for DLP 3D printing. As-prepared ABS-like resin composite inks employing PANI nanofibers and graphene flakes as co-fillers were successfully printed, obtaining highly conductive and mechanically robust products with the desired shapes and different sizes through DLP 3D printing. The sheet resistance of the 3D-printed composites was reduced from 2.50 × 10 Z-axis, requires a short printing time, and ensures excellent printing quality and selective light curing [co-poly(ethylene glycol methyl ether methacrylate) (PHEMA-co-PEGMEMA) for use as a shape memory polymer (SMP) stabilized by hydrogen bonding interactions [Among various polymer processing methods, 3D printing is becoming a future-oriented polymer processing technology because it can produce customized polymer products of the desired design, shape, and size. 3D-printed polymer products have been used in various applications such as automotive and dental applications, machinery, jewelry, electronics, building materials, and tissue engineering ,2. In adt curing ,13,14,15t curing ,13,14,15t curing , polyuret curing ,8,9,10, t curing ,12, isopractions . The ure2) or zinc oxide (ZnO) nanoparticles for use as in antibacterial applications; the ABS-like resin composites employing 1 wt.% of TiO2 nanoparticles and 1 wt.% of ZnO nanoparticles demonstrated the maximum tensile strengths of 29.533 and 33.696 MPa, respectively [In particular, the ABS-like resin is very suitable for DLP 3D printing because it retains the intrinsic properties of a thermoplastic elastomer (TPE) and provides the unique advantages of ABS, such as processability, adhesion, and mechanical strength. Recently, Liu et al. reported the DLP 3D printing of ABS-like resin composites containing titanium dioxide (TiOectively . Accordiectively . DespiteConducting polymers (CPs) not only retain the inherent properties of polymer materials but also provide unique advantages of tunable electrical conductivity and electrochemical activity, which make them very suitable as fillers that can form conducting networks inside 3D printable resins ,18,19,20sp2-hybridized carbons, which has attractive properties such as high theoretical surface area (2630 m2/g), excellent electrical conductivity, high mobility of charge carriers, and high flexibility [Graphene is a two-dimensional (2D) carbon nanomaterial composed of xibility ,21,22. Sxibility . TherefoIn this work, PANI nanofibers and graphene flakes were chosen as co-fillers to enhance the mechanical and electrical properties of 3D printable ABS-like resin composites, and the DLP 3D printing technology was applied to achieve improved printing quality and reduced printing time in the 3D printing process of ABS-like resin composite materials. A field emission scanning electron microscope (FE-SEM) was used to investigate the presence and dispersion of PANI nanofibers and graphene flakes within the 3D-printed ABS-like resin composites. Fourier-transform infrared (FT−IR) spectroscopy identified the effects of the PANI/graphene co-fillers on the bonding structures of the 3D-printed composites. The sheet resistance values of the 3D-printed composites showed the reinforcing efficiency of various contents of the PANI/graphene co-fillers in relation to the electrical properties of ABS-like resin composites. Furthermore, the reinforcing effects of the PANI/graphene co-fillers on the mechanical properties of the ABS-like resin composites were studied by determining the stress–strain curves, tensile strength, Young’s modulus, and elongation at break values of the 3D-printed composites.3. Then, 55 mmol of aniline was added to the 1M HCl/CHCl3  mixture, followed by vigorous stirring for 1 h. Then, 26.5 mmol of APS was added to the reaction medium and reacted at 3 °C for 9 h to obtain the PANI precipitates. The as-prepared PANI precipitates were washed with water, ethanol, and acetone.Ammonium persulfate  and aniline (99%) were obtained from Sigma-Aldrich . Water-dispersible graphene flakes were purchased from MExplorer Co., Ltd. . The graphene flake paste had a density of 25 mg/mL, while the average thickness and lateral size of the graphene sheets were about <5 nm and 2–3 μm, respectively. The ABS-like resin solution (Carima ABS) was purchased from Carima . Hydrochloric acid , ethanol (95%), acetone (99%), and isopropyl alcohol  were purchased from Daejung Chemical & Metals Co., Ltd. . The PANI nanofibers used in this experiment were synthesized by a modified interfacial polymerization method ,18,19,20The 3D printer used in this work was a DLP-type 3D printing system , and a 405 nm laser was used as a light source for photo-crosslinking inside the ABS-like resin composites. The ABS-like resin composites containing different amounts of PANI nanofibers and graphene flakes were 3D-printed, and the printability of each sample was evaluated. The maximum printable concentration of PANI was confirmed to be 5.3 wt.% with respect to the resin composite ink, and no lamination occurred when the content of PANI exceeded 5.3 wt.%. The maximum printable concentration of graphene flakes was 2.0 wt.% with respect to the resin composite ink, and no lamination occurred when the content of graphene flakes exceeded 2.0 wt.%. A FE-SEM  was used to identify the morphologies inside the ABS-like resin composites. A FT−IR spectrometer  was used to investigate the chemical bonds inside the ABS-like resin composites. Ultraviolet–visible (UV–visible) spectra of the ABS-like resin composites were recorded on a UV–visible spectrometer . The sheet resistance values of the 3D-printed composites were measured using a 4-point probe conductivity meter  equipped with a current source meter  .In order to evaluate the mechanical properties of the 3D-printed composites, tensile tests were performed according to the ASTM standard D638 method using a universal testing machine  with a crosshead speed of 10 mm/min and a gauge length of 50 mm at room temperature under a relative humidity (RH) of 30% . Each sp−1  to 1.95 × 108, 9.35 × 107, and 2.84 × 107 ohm/sq by adding 1.0, 2.0, and 3.0 wt.% of PANI nanofibers, respectively  to 1.06 × 107, 3.99 × 106, and 1.61 × 106 ohm/sq by adding 1.0, 1.3, and 1.5 wt.% of graphene flakes, respectively. When the contents of the graphene flakes were larger than 1.5 wt.%, the reinforcing efficiency of the graphene flakes decreased, as shown for the AP3.0G1.7 and AP3.0G2.0 samples. Accordingly, the AP3.0G1.5 sample demonstrated the lowest surface resistance. This indicated that the synergistic effect of the PANI nanofibers and graphene flakes enabled a better-reinforcing od the electrical properties of the ABS-like resin composites. Considering these results, the appropriate amounts of PANI nanofibers and graphene flakes providing effective conductive pathways within the ABS-like resin composites were confirmed to be 3.0 and 1.5 wt.%, respectively [ectively a. This sectively ,21,22.5 to 106 times higher than that of a neat polymer matrix  compared to the pristine ABS-like resin. Furthermore, the mechanical properties of the 3D-printed composites were found to be significantly improved by adding 3.0 wt.% of PANI nanofibers and 1.5 wt.% of graphene flakes into the ABS-like resin matrix. The enhanced electrical and mechanical properties could be attributed to the successful formation of a conductive network and the improved dispersion of the filler within the ABS-like resin matrix due to the synergistic effect of the PANI nanofibers and graphene flakes. The novelty of our study is that it provides manufacturing conditions and successful 3D printing conditions for composites that can improve the conductivity and mechanical properties of ABS-like resins, a TPE material with the advantages of the ABS resin. Although specific applications of the ABS-like resin composites were not presented in this article, such highly conductive and mechanically robust ABS-like resin composites could be widely used as essential materials for electronic devices, automotive parts, thermal pads, thermoelectric devices, sensors, and energy storage devices in the future.


The CD47/PD‐L1 antibodies combination exhibits durable antitumor immunity but also elicits excessive immune‐related adverse events (IRAEs) caused by the on‐target off‐tumor immunotoxicity, hindering their clinical benefits greatly. Here, a microfluidics‐enabled nanovesicle using ultra‐pH‐sensitive polymer mannose‐poly(carboxybetaine methacrylate)‐poly(hydroxyethyl piperidine methacrylate) (Man‐PCB‐PHEP) is developed to deliver CD47/PD‐L1 antibodies (NCPA) for tumor‐acidity‐activated immunotherapy. The NCPA can specifically release antibodies in acidic environment, thereby stimulating the phagocytosis of bone marrow‐derived macrophages. In mice bearing Lewis lung carcinoma, NCPA shows significantly improved intratumoral CD47/PD‐L1 antibodies accumulation, promoted tumor‐associated macrophages remodeling to antitumoral status, and increased infiltration of dendritic cells and cytotoxic T lymphocytes, resulting in more favorable treatment effect compared to those of free antibodies. Additionally, NCPA also shows less IRAEs, including anemia, pneumonia, hepatitis, and small intestinal inflammation in vivo. Altogether, a potent dual checkpoint blockade immunotherapy utilizing NCPA with enhanced antitumor immunity and reduced IRAEs is demonstrated. A microfluidics‐enabled nanovesicle using ultra‐pH‐sensitive polymer mannose‐poly(carboxybetaine methacrylate)‐poly(hydroxyethyl piperidine methacrylate) (Man‐PCB‐PHEP) is developed to deliver CD47/PD‐L1 antibodies (NCPA) for tumor‐acidity‐activated immunotherapy. The NCPA can specifically release antibodies in acidic tumor microenvironment, resulting in more favorable treatment effect compared to those of free antibodies. Moreover, NCPA shows less IRAEs, including anemia, pneumonia, hepatitis, and small intestinal inflammation in vivo. Moreover, multiple clinical trials of dual blockade of CD47/PD‐L1 by antibody combinations, bispecific antibodies, or fusion proteins are ongoing, revealing the great potential of this strategy. It is noteworthy that CD47/PD‐L1 coexpression predicts long‐term survival and suggests the potential of dual‐targeting immunotherapy in non‐small cell lung cancer. However, although accumulated data has shown that the CD47 antibody (aCD47), which blocks the SIRPα/CD47 innate immune checkpoint, shows great potential for eliminating tumors by enhancing phagocytic macrophages to phagocytize tumor cells and promote antigen presentation, durable antitumor responses to CD47 blockade requires adaptive immune stimulation by a therapeutic antibody such as PD‐L1 antibody (aPD‐L1). Nevertheless, the clinical benefit of this strategy is unsatisfactory due to on‐target off‐tumor immunotoxicity and the inevitable increased dosage of antibodies also leads to excessive immune‐related adverse events (IRAEs). Although immunosuppressive drugs, including glucocorticoids, are currently in routine clinical use to manage the toxicity of IRAEs, they induce deleterious outcomes in optimal immunotherapy.Checkpoint blockade antibody therapy is the hotspot strategy in cancer treatment.1 Rece These strategies remain limited clinical transformable so far due to uncontrollable alteration of affinity for antigen binding, uncertain pharmacokinetics, unacceptable stability, and unaffordable costs. Thus, to optimize the clinical outcome of dual checkpoint blockade immunotherapy, there is an urgent need to develop a strategy for improving intratumor antibody delivery with reduced IRAEs.To tackle these problems, many substantial attempts have been employed, including biomaterial‐delivery platforms for antibody delivery by chemical or biological modification.10 The However, the microfluidic synthesis of droplets usually intersects water with organic reagents, such as ethanol and oil, which are harmful to the stability and activity of biomacromolecules and increase tedious purification processes. Hence, numerous improvements are needed to enable the production of a delivery platform for biomacromolecules using microfluidic devices.Recently, microfluidics‐enabled synthesis can produce monodisperse particles on a large scale in a straightforward procedure with high controllability and reproducibility, which has been used to produce lipid nanoparticles for the delivery of biomacromolecules, including mRNA and proteins.12 HowSchemeIn the present study, we demonstrated a microfluidics‐enabled tumor acidity‐responsive nanovesicle to deliver and specifically on‐site release CD47/PD‐L1 antibodies (NCPA) in an acidic tumor microenvironment (TME), thereby assuring dual CD47/PD‐L1 checkpoint blockade and avoiding IRAEs induced by the on‐target off‐tumor immunotoxicity. The NCPA was developed by the organic solvent‐free microfluidic synthesis in one step using ultra‐pH‐sensitive zwitterion polymer Man‐PCB‐PHEP Scheme1, maxi22.11H nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). The characteristic peaks of the synthesized monomers and polymers were labeled in the 1H NMR spectrum ‐poly(hydroxyethyl piperidine methacrylate) (Man‐PCB‐PHEP), by copper‐free organocatalyzed atom transfer radical polymerization of carboxybetaine methacrylate and hydroxyethyl piperidine methacrylate using brominated mannose as the initiator  and aPD‐L1 (1.51±0.10 vs 0.28±0.16 h) due to zwitterion polymeric nanovesicle shielding protection , aPD‐L1 (100 µg), and CPA or NCPA at low (50 µg aCD47+50 µg aPD‐L1), medium (100 µg aCD47+100 µg aPD‐L1), and high (200 µg aCD47+200 µg aPD‐L1) dosages via tail vein injection  of the mice due to CD47 overexpression on the red blood cell membrane , enabling good laboratory practice (GLP) toxicology studies to confirm its safety before application in human research.4−1. Then, the antibody solution (10 mg mL−1) was introduced into the chip through the center inlet 1 at 0.2 mL h−1. And inlet 2 introduced polymer hydrochloric acid solution (4 mg mL−1) at 4 mL h−1, and inlet 3 introduced a base sodium hydroxide solution (pH 8) at 40 mL h−1. At last, the collected solution from the outlet was filtrated by a 220 nm syringe filter and stored at 4 °C for further usage.To avoid using organic solvents, which may be harmful to the bioactivity of antibodies, the primitive aqueous solution was introduced by syringe pumps . First, Man‐PCB‐PHEP was dissolved in a hydrochloric acid solution of pH 4 at a concentration of 4 mg mLFirst, BSA as the substitute for antibody, labeled with CY5 if needed, was encapsulated into NCPA using the microfluidic chip. Then, the collected solution was treated with PBS at pH 7.4 and 6.5 for 0.5, 1, 2, 4, 8, and 24 h and ultracentrifugated by an Amicon centrifugation unit . Finally, the fluorescent signal was recorded by fluorescence spectrometer and the protein concentration was quantified by Pierce BCA Protein Assay Kit (Thermo Scientific) as manufacture instructions.5 cells per well and cultured with DMEM overnight. Then, the BMDMs were stained with CD45‐APC‐CY7 and cultured with RPMI‐1640. The LLC cells were treated with IgG, aCD47, aPD‐L1, CPA, and NCPA (pH 7.4 and 6.5) for 1 h and then stained with CFSE by 10 min incubation at room temperature. After repeating freeze‐thaw cycles 3 times with liquid nitrogen and washing with PBS 3 times, the CFSE labeled LLC cells were cocultured with BMDMs in 6‐well plates at a concentration of 1×105 cells per well for 2 h. After aspirating the dissociative LLC cells, the nuclei of reserved cells were stained with DAPI after fixing by 4% paraformaldehyde and captured the images by confocal laser scanning microscopy. Similarly, the coculture cells were collected without aspirated and analyzed by BD FACSCanto Clinical Flow Cytometry System.The BMDMs were seeded to a 6‐well plate at a concentration of 4×10Animal experiments were approved by the Second Clinical Medicine College of Jinan University (AUP‐220302‐SZW‐029‐01). Mouse transportation, housing, and breeding were conducted according to the recommendations of “The use of nonhuman animals in research.” Mice were killed by cervical dislocation to prevent suffering. All mouse experiments were conducted by the ethical standards and guidelines of the Asian Federation of Laboratory Animal Science Associations.5 cells suspended in PBS were subcutaneous injected into the right flank of 4 weeks old C57BL/6J male mice. When the tumor volume reached 50–100 mm3 after about 1 week of inoculation, the mice were randomly divided for designed experiments.To construct the xenografted LLC mouse tumor model, a total of 5×10LLC‐bearing mice were administrated with CPA or NCPA at an antibody concentration of 200 µg per mouse, while the aCD47 was stained with CY3 and aPD‐L1 was stained with CY5. The in vivo animal fluorescence imaging was conducted at predetermined time intervals  using the IVIS imaging system, and the ex vivo imaging was performed after removing organs and tumors from mice. Moreover, frozen sections of tumor tissues were captured by CLSM.n = 5) and administrated with IgG, aCD47 (100 µg), aPD‐L1 (100 µg), and CPA or NCPA at low (50 µg aCD47+50 µg aPD‐L1), medium (100 µg aCD47+100 µg aPD‐L1), and high (200 µg aCD47+200 µg aPD‐L1) dosages via tail vein injection at day 6, 9, and 12, respectively. The body weight and tumor volume were recorded every 3 days. The tumor volume was obtained by the formula tumor volume = (width2 × Length)/2 for caliper measurements. On day 15 or 21, blood, organs, and tumors were collected for other experiments.The LLC‐bearing mice were randomly divided into 9 groups . Statistical differences were performed using a one‐way analysis of variance (ANOVA) followed by Tukey's test with GraphPad Prism 9.0 software . Tukey's post hoc tests and one‐way analysis of variance (ANOVA) were performed for multiple comparisons. The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Sarcoidosis is a multisystem disease characterized by non-caseating epithelioid cell granulomas. The diagnosis of sarcoidosis is based on compatible clinical presentation, non-necrotizing granulomatous inflammation in one or more tissue samples, and the exclusion of alternative causes of granulomatous disease . With thThis Research Topic consists of one case report, three reviews, and six original research articles focusing on advances in diagnosis and treatment, together with findings in genetics and transcriptomics that might be helpful in the early diagnosis of sarcoidosis.Xiong et al. identify sex-dependent genetic variations in two clinical sarcoidosis phenotypes, namely Löfgren's syndrome (LS) and non-Löfgren's syndrome (non-LS), in multiethnic cohorts from Sweden, Germany, and the US. The authors confirm that differences in genetic findings between the sex groups in LS are explicitly located in the extended Major Histocompatibility Complex, while genetic differences between the sex groups in non-LS are primarily located in the MHC class II subregion and ANXA11. Transcriptome-wide expression studies have been conducted to reveal the mechanisms of sarcoidosis. Jiang et al. performed a systematic database search of the Gene Expression Omnibus and utilized transcriptomic data from blood and sarcoidosis-affected tissues in a meta-analysis to identify a cross-tissue, cross-platform signature. They identify 29 robustly sarcoidosis-associated genes, including the top genes LINC01278, GBP5, and PSMB9. They report that pathway enrichment analysis revealed activation of IFN-γ, IL-1 and IL-18, autophagy, and viral infection response. This study provides a cross-tissue meta-analysis for expression profiles and identifies a potential non-invasive diagnostic classifier for sarcoidosis. Similar efforts have been made by Duo et al., who report on a study in which transcriptomes from eleven independent sarcoidosis cohorts comprising 313 patients and 400 healthy controls were analyzed and machine learning was employed to fit a diagnostic model. A ten-gene sarcoidosis diagnosis signature consisting of GBP1, LEF1, IFIT3, LRRN3, IFI44, LHFPL2, RTP4, CD27, EPHX2, and CXCL10 was constructed in the training cohorts; this signature performed well in the four independent cohorts and has been further validated in seven independent publicly available gene expression datasets. Transcriptional signatures, developed through bioinformatics analysis, could improve the accuracy of early diagnosis of sarcoidosis.Multiple factors, such as environmental and genetic predisposition, have been implicated in the pathogenesis of sarcoidosis. The search for gene candidates in sarcoidosis can be conducted on the basis of gene expression data and protein profile data from genomic, transcriptomic, or proteomic studies. Strambu). The most challenging issue is the suspicion of CS with initial cardiac manifestations in a patient with no previous diagnosis of sarcoidosis, and diagnosis can be delayed. Another difficulty in diagnosis is the absence of a gold standard for diagnosis, because biopsy of the endomyocardial tissue has low sensitivity given the risk level of the procedure. The increasing use of newer imaging modalities such as cardiac magnetic resonance (CMR) and positron emission tomography (PET) may provide valuable information for accurate diagnosis and assessment in CS patients. Brazile et al. review a set of medical records for diagnostic features of CS, including late gadolinium enhancement (LGE) patterns, increased signal on T2-weighted imaging, and non-caseating granulomas. They confirm that CMR is an important tool in the non-invasive diagnosis of CS, and the presence of LGE on CMR in a pattern consistent with CS has been shown to be a predictor of mortality.Cardiac sarcoidosis (CS) remains diagnostically challenging as the sensitivity and specificity of the diagnostic modalities are limited. Strambu reviews current knowledge of the diagnosis and decision to treat cardiac sarcoidosis, and illustrates the information with a case presentation  in patients with and without sarcoidosis. They found that 31% (5/16) of CINS patients had associated sarcoidosis. CINS symptoms, biological and CSF features, and treatments were similar for CINS patients with and without sarcoidosis, except regarding CD4 cell percentage and CD4/CD8 ratio, which were higher in blood from those with sarcoidosis. Ji et al. present a case report of pleural sarcoidosis with pleural nodules and effusion, along with a review of the literature on this rare manifestation. Another study describes the natural course and prognostic value of cancer-associated sarcoid-like reaction (SLR) . During follow-up, progression of cancer-related SLR to overt sarcoidosis was not observed. Development of SLR was also not associated with overall survival or disease-free survival in patients with non-small cell lung cancer.Three manuscripts published in this Research Topic relate to rare clinical features associated with or mimicking sarcoidosis. Valeyre et al. the authors establish optimal differential diagnosis strategies tailored to each of several situations in order to help non-sarcoidosis experts. They emphasize that alternative diagnoses must be ruled out before a diagnosis of sarcoidosis can be made. First, epidemiological factors must be clarified. Subsequently, a detailed medical history and physical examination are also crucial. Chest CT is also helpful to characterize the findings as typical or atypical for sarcoidosis. Ultimately, the Sarcoidosis Diagnostic Score  can be very helpful to assess sarcoidosis diagnosis before and after evidence of granuloma. Recent therapeutic drug trials and treatment approaches are reviewed by Obi et al.. Up to now, only two medications (prednisone and repository corticotropin injection) have been approved in the treatment of sarcoidosis by the United States Food and Drug Administration. This past decade has seen a renewed interest in developing new drugs and exploring novel therapeutic pathways for the treatment of sarcoidosis. The next challenge will lie in funding and in evaluating the effectiveness and safety of treatments.Two reviews of differential diagnosis and treatment approaches are presented in our Research Topic. In the review by For accurate diagnosis and effective treatment, further efforts should be focused on early detection, new diagnostic technologies, and multicenter clinical trials of new drugs. Moreover, the identification of new biomarkers is warranted for differential diagnosis and for individualized treatment. This will provide a powerful decision-making tool for doctors that can be used to better manage sarcoidosis patients.The draft was written by YZ and edited by FJ and RB. All authors reviewed the final document.


The manufacturing process for ointments typically involves a series of heating, cooling, and mixing steps. Precise control of the level of mixing through homogenization and the cooling rate, as well as temperature at different stages, is important in delivering ointments with the desired quality attributes, stability, and performance. In this work, we investigated the influence of typical plant processing conditions on the microstructure, stability, and sensorial properties of a model ointment system through a Design of Experiments (DoE) approach. Homogenization speed at the cooling stage after the addition of the solvent  was found to be the critical processing parameter that affects stability and the rheological and sensorial properties of the ointment. A lower PG addition temperature was also found to be beneficial. The stabilization of the ointment at a lower PG addition temperature was hypothesized to be due to more effective encapsulation by crystallizing mono- and diglycerides at the lower temperature. The in vitro release profiles were found to be not influenced by the processing parameters, suggesting that for the ointment platform studied, processing affects the microstructure, but the effects do not translate into the release profile, a key performance indicator. Our systematic study represents a Quality-by-Design (QbD) approach to the design of a robust manufacturing process for delivering stable ointments with the desired performance attributes and properties. The typical ointment formulation is an oil-based formulation with a semi-solid texture comprising a solvent phase dispersed in an oil phase stabilized by emulsifying agents. A drug or an active ingredient can be dissolved in the solvent phase and dispersed in the external oil phase. The typical oil phase of ointment comprises petrolatum and stiffening agents and/or emulsifying agents such as glyceride derivatives that provide the desired stiffness/strength to keep the solvent phase dispersed. In addition, to provide emollience for skin, mineral oil is typically used in ointments .The manufacturing process for ointments involves heating to melt the petrolatum and waxes, followed by addition of other liquid components, such as polar solvents, with mixing/homogenization for uniform dispersion. The ointment is then cooled to room temperature before filling into containers such as jars and tubes. It is anticipated that homogenization should affect the distribution or dispersion of the various components at the different stages, and both heating and cooling, along with homogenization, affect the distribution and crystallization of emulsifying wax and stiffening agents around the solvent droplets and the oil phase. Although the term microstructure has been recently used to characterize semi-solid topical products such as ointments, its definition is not clearly articulated and agreed upon . The micBesides influencing the active release and skin permeation behavior, the microstructural properties—including droplet size, structure, and distribution of the stiffening and emulsifying agents around the oil droplet—also affect the sensorial properties as the product is applied to skin. In addition, congealing of petrolatum and distribution of the waxes and stiffening agents as they crystallize would generate a viscoelastic system with different rheological properties. The rheological properties would influence the phase separation of dispersed solvent due to various de-mixing phenomena such as coalescence, sedimentation, and/or creaming, and potentially impact the bio-performance, i.e., in vitro release and skin permeation. While the in vitro release and permeation profile of a drug (flux) influences drug delivery and hence efficacy, sensorial properties are also an important concern, as they affect consumer perception and acceptance of the product.The objective of this study was, therefore, to study the influence of typical plant processing conditions of various unit processes on the microstructure, stability, and sensorial properties of a topical ointment formulation through a systematic DoE approach. The focus of the current study is the effect of processing on the microstructure of the ointment formulation. However, if significant impacts of process conditions on microstructure are observed, the second study would involve an assessment of the performance attributes, such as API release.Both the white petrolatum and paraffin wax used in this study were Ph. Eur grades manufactured by H&R Gruppe  and VWR International (Singapore), respectively. Mono-and-Diglycerides (Geleol™) was purchased from Gattefosse . Propylene glycol (Ph. Eur grade) was purchased from Thermo Fisher Scientific . The active pharmaceutical ingredient, lidocaine, was purchased from Sigma Aldrich (Singapore). All the ingredients were used directly as supplied for ointment preparation.High-speed mixing emulsifying equipment  consisting of a glass-jacketed vessel equipped with a high-speed shearing homo-mixer and a scraper/paddle mixer was used as the representative manufacturing container for ointments. The equipment was capable of manufacturing semi-solid products under vacuum. The temperature was controlled by a water circulator  with an attached thermocouple (Julabo Pt100). The ointment was prepared in two stages, namely compounding and post compounding, as shown in the schematic in Step 1:Charge white petrolatum, paraffin wax and Mono-and-Diglycerides into the jacketed mixing vessel and heat to 75 ± 5 °C while mixing until all materials are fully melted.Step 2:Transfer melted waxes into the main vessel and mix (90 rpm) at 75 ± 5 °C for at least 10 min.Compounding1] to [F4] represent set points of various processing parameters/factors investigated for their effect on ointment properties with a statistically appropriate DoE study.Step 3:1] to cool the contents of the main vessel while mixing at 90 rpm. When the temperature reaches 50 ± 3 °C, homogenize at [F2] for 5 min while continuing mixing at 90 rpm until the temperature reaches [F3]. [F1], [F2], and [F3] are critical process parameters investigated for the effect on drug product quality attributes.The cooling rate is set at [FStep 4:3]. After the addition is completed, homogenize at [F2] for 20 min at every 30 min interval. Homogenization is repeated twice for a total mixing and homogenization duration of 60 min.Dissolve lidocaine in propylene glycol. Charge propylene glycol into main vessel while mixing (90 rpm) at [FStep 5:4] is applied for 5 min when the temperature reaches 36 ± 3 °C.The cooling rate is set at 0.5 ± 0.1 °C/min to cool the contents to 32 ± 3 °C while mixing (50 rpm). Homogenization at  was varied from 2500 to 8000 rpm while the cooling rate [F1] was set at 0.8 °C/min, and the compounding shear temperature [F3] was fixed at 43 °C. The maximum homogenization speed at the final cooling stage [F4] was limited to 5000 rpm because the mixture became highly viscous at that stage, and higher homogenization would result in a significant temperature rise in the mixture. No significant temperature rise was detected in the mixture inside the main vessel, even at the highest homogenization speed and longest duration applied in this study.Runs OT1, OT2, and OT3  technology, and its instability index was determined via SEPView 6 software. Oscillatory rheology measurements were performed to study the viscoelastic behavior of the ointments. Storage modulus (G′) and loss modulus (G″) were calculated from the small angle oscillatory shear (SAOS) measurements. The complex shear modulus (G*), which is an indication of the material stiffness as a function of strain, was then computed by the equation below:cross), the ointment lost its structural integrity, and the viscous characteristics dominated over the elastic response. The viscoelastic parameters obtained for OT1-3 are shown in 4] at 5000 rpm to investigate if varying the cooling rate [F1] and homogenization speed [F2] will shift the formulation properties to the desirable range. The compounding shear temperature [F3] was set at 40 °C, which is the lower limit set by the manufacturer. During PG addition, homogenization was applied twice for 20 min duration, with 30 min between each application. The operating conditions of OT4 to OT9 are shown in cross . Most importantly, OT4 to OT9 exhibited similar stability as assessed by the instability index measured by the LUMiSizer. From these findings, we believe that the homogenization speed of 5000 rpm or above at the final cooling step during the compounding stage may have damaged the microstructure built up in the previous steps, thus masking the effect of the other three operating parameters on the stability of the ointment. Hence, lower homogenization speeds should be employed at step 5 in subsequent experiments.From the results of OT1-OT3, the operating conditions at OT3 yielded the most drastic changes to the properties of the ointment compared to OT1 and OT2. Therefore, the subsequent set of experiments (OT4-OT9) was designed based on OT3 by fixing [F4] was capped at 2500 rpm in the final DoE. The cooling rate [F1] did not significantly affect the properties of the ointment, so the cooling rate [F1] was fixed at 0.8 °C/min, which is more practically achievable in the plant than the faster rate of 1.5 °C/min. Homogenization speed at steps 3 and 4 [F2] was varied from 2500 to 8000 rpm. During PG addition, the homogenization was applied twice for 20 min duration, with 30 min between each application. Propylene glycol addition temperature [F3] was assessed at two levels, 40 °C and 46 °C, which are the lower and upper limits of the manufacturing process. The operating conditions or factors of the final DoE are shown in Based on the preliminary runs OT1-OT9, the homogenization speed at step 5 [F3], the stability of the ointment increases (instability index decreases) when the homogenization speed at step 5 [F4] was lowered. An exception was when the homogenization speed at the previous steps [F2] was at 8000 rpm, [F4] no longer had any influence on the stability. This means that if the mixture is subjected to a high level of shear before or during the addition of propylene glycol, either the formation process of the microstructure is being hindered or the microstructures already formed are destroyed before reaching step 5; therefore, the damage cannot be reversed, even if no homogenization is applied at step 5. Even though our results suggest that no homogenization at step 5 is beneficial to the stability of the ointment, this is not practical in a manufacturing plant. Some degree of gentle homogenization is still required to ensure homogeneity within the mixing vessel and the final ointment product.The instability indices of the ointments produced in the final DoE are shown in 3] was at 46 °C, the homogenization speed at step 5 [F4] did not affect the stability of the ointment since the instability indices were almost the same . Similar to the observation when [F3] was set at 40 °C, at the same homogenization speed at step 5, the instability index increased significantly when the homogenization speed [F2] was increased from 2500 to 8000 rpm. In addition, the mixing duration of the PG addition step also influenced the stability of the ointment. The stability of the ointment was improved when the mixing duration was reduced from 60 min to 30 min.When the propylene glycol addition temperature . This can be better visualized graphically in cross decrease with increasing homogenization speed [F2], regardless of the homogenization speed at step 5. This shows that shear at the steps before step 5 also plays a part in controlling the rheological properties of the ointment. The same can also be observed for samples OS-7 to OS-12.From the rheological properties listed in −1 and then reduced back to 0 using a stepwise shear ramp. −1 are also shown in To understand why step 5 heavily influences the rheological properties and stability of the ointment, thixotropy tests were performed on petrolatum, which is the dominant component in the formulation. The tests were conducted using a rough parallel plate geometry with a 1 mm gap between plates. The shear rate was increased from 0 to 100 sThe effect of PG addition mixing duration at 46 °C on complex shear modulus and crossover stress is shown in 4]  is the same as that observed in textural properties of the ointments. This similarity is because textural properties are largely dependent on the bulk rheological properties of the ointments.The sensorial attributes measured for ointments in the final DoE are shown in 4] . Going fperature , which a4], PG additional temperature [F3], and PG addition mixing duration on the CoF are illustrated graphically in The coefficient of friction (CoF) as a function of probe displacement on the bioskin plate without the application of ointment is shown in v/v ethanol:water solution was chosen as the receptor medium to ensure that the solubility of lidocaine is high enough to provide sink condition for the release studies. In vitro release studies were performed using the most stable (OS5-4) and least stable (OS5-8) ointments in the final DoE to investigate if the stability of the ointment would affect the drug release performance. The assumption is that the less stable formulation is due to inadequate microstructure and not appropriate rheological properties. Lidocaine, a local anaesthetic agent, was chosen as the model drug for this study. It was predissolved in propylene glycol and incorporated into the ointment during PG addition at step 4 of the compounding stage  are negatively correlated with linear rheological response and the measured textural properties. This suggests that lower homogenization speeds should result in ointments that are more rigid and less susceptible to shear damage (higher complex shear modulus and crossover stress) while giving a skin feel of being more adhesive, firmer, and more cohesive and spreadable. It is clear from the biplot that the textural properties are correlated positively with the linear rheological response , as their vectors form acute angles. Among the textural properties, adhesive force is most highly correlated with complex shear modulus and crossover stress.Given the large number of measured properties and operating conditions, PCA analysis was performed to understand how the various operating parameters are correlated to the measured properties. The scree plot shown as the inset in 3] is also positively correlated with the instability index, although not as highly correlated as the homogenization speed, since the angle between the instability index and PG addition temperature is larger than the angles between the instability index and homogenization speeds. Unlike homogenization speed, PG addition temperature correlates highly with the coefficient of friction. Hence, if PG is added at a lower temperature, a more stable and lubricating ointment can be achieved, although an ointment with a lower coefficient of friction may also be perceived as greasy during application on skin. PG addition temperature does not have much influence on the rheological properties and the pick-up characteristics , although it correlates negatively, to a certain extent, with the rubout characteristics (cohesion strength and spreadability).PG addition temperature  can also be explained by our proposed hypothesis. In step 5, the MDG crystals begin to crystallize and encapsulate the PG droplets. Homogenization, subsequently, may disrupt the formation process of the microstructure or destroy the microstructure already formed. Therefore, we observed that a lower step 5 homogenization speed resulted in more stable ointment.The effect of step 5 homogenization speed [FThis study demonstrated the significant effects that selected processing parameters have on the critical quality attribute of ointment drug product. However, not all processing parameters are equal and have an equivalent significant effect on all or some critical quality attributes, including performance, stability, and sensorial properties. It appears that the temperature of addition of solvent and homogenization speed during cooling at the compounding stage had the most significant effect on rheological properties, stability, and sensorial properties. These were attributed to the complex dynamics of stabilizing agents (MDG) crystallizing at different temperatures during/after addition of solvent to enable droplet encapsulation and stabilization. Once the optimal microstructure is formed, additional homogenization (speed and time) may disrupt the structure to cause instability. Rheological properties and instability, as measured by the LUMiSizer, appear to correlate well and provide insight into the formed microstructure of the ointment DP. However, based upon similar in vitro release profiles for the suboptimal DP compared to the most stable DP, it appears that the variations in microstructure in the ranges affected by changes in the processing parameters studied may have no influence on delivery performance. This suggests that it is difficult to obtain an ideal product profile from key quality attributes, including performance, stability, and sensorial properties, by modifications of process alone. From a manufacturing scale-up perspective, we have demonstrated how a typical semi-solid manufacturing process can be studied and optimized to achieve the target product profile from a quality and performance attributes perspective.


Pseudomonas aeruginosa; however, real-world comparative-effectiveness data are lacking.Ceftolozane-tazobactam (CT) and ceftazidime-avibactam (CZA) are front-line agents for treatment of multidrug-resistant (MDR) P. aeruginosa. CT and CZA patients were matched 1:1 within each study site by the presence/absence of septic shock/severe sepsis, infection site, and time to treatment initiation. The primary outcome was clinical success at day 30 defined as survival, resolution of signs/symptoms with the intended treatment course, and absence of recurrent infections. Patients with cystic fibrosis or COVID-19 infection within 90 days were excluded.CACTUS is a retrospective, matched, multicenter study to compare the efficacy of CT and CZA among patients with bacteremia or pneumonia due to MDR Table 1). The overall median age was 61 years, 61% were male, and the median Charlson score was 5. At study drug initiation, 77% of patients were in the ICU, 67% received mechanical ventilation and the median SOFA score was 7. 79% of patients were treated for pneumonia; 72% of which occurred in ventilated patients. The median time from index culture to treatment initiation was 72 hours in both groups; CT patients were more likely to receive a prolonged infusion of ≥3 hours . Clinical success occurred in 62% and 55% of patients receiving CT and CZA, respectively . Corresponding rates of success for pneumonia were 63% and 52%, respectively . All-cause, 30-day mortality rate was 20% and 19%, respectively. Microbiologic failures, recurrent infections, and development of resistance within 90 days were similar between groups. Time to a composite endpoint of recurrent infection or death within 90 days was similar between groups in the overall analysis and the subgroup of patients with pneumonia .234 patients were included from 20 sites. Patient demographics, severity of illness, infection types, and treatment durations were similar for patients treated with CT or CZA , Merck: Advisor/Consultant Erin K. McCreary, PharmD, Abbvie: Advisor/Consultant|Ferring: Advisor/Consultant|GSK: Honoraria|La Jolla (Entasis): Advisor/Consultant|LabSimply: Advisor/Consultant|Merck: Advisor/Consultant|Shionogi: Advisor/Consultant|Shionogi: Honoraria Christopher Polk, MD, ViiVHealthcare: Job change to work for ViiV as Medical Director Michael J. Satlin, MD, AbbVie: IDMC member|Biomerieux: Grant/Research Support|Merck: Grant/Research Support|SNIPRBiome: Grant/Research Support Michael Veve, PharmD, MPH, National Institutes of Health: Grant/Research Support|Paratek Pharmaceuticals: Grant/Research Support jason M. Pogue, PharmD, AbbVie: Advisor/Consultant|Entasis: Advisor/Consultant|Ferring: Advisor/Consultant|GSK: Advisor/Consultant|Merck: Advisor/Consultant|Merck: Grant/Research Support|Qpex: Advisor/Consultant|Shionogi: Advisor/Consultant