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Background and Aims: Chronic hepatitis D (CHD) is the most severe form of chronic viral hepatitis, with a high risk of developing hepatocellular carcinoma (HCC) and liver-related mortality. Risk stratification is needed to guide HCC surveillance strategies and to prioritize treatment with antiviral agents., Methods: We conducted a multicenter retrospective cohort of anti-HDV positive individuals managed at sites in the Netherlands and the United Kingdom. We studied the 5-year cumulative incidences of HCC and liver-related events (first of HCC, liver transplantation and liver-related mortality), in the overall cohort and among relevant subgroups., Results: We analyzed 269 anti-HDV positive individuals with a median follow-up of 4.3 years in which 47 first events occurred. The 5-year cumulative incidences of HCC and liver-related events were 3.8% and 15.6% in the overall cohort. The 5-year cumulative incidence of HCC and liver-related events for individuals without cirrhosis was 0% and 0.9% compared to 12% and 41.3% for individuals with cirrhosis (p<0.001). The 5-year cumulative incidence of HCC and liver-related events was 0% and 2.1% among individuals with low PAGE-B scores, compared to 3.2% and 21.1% with intermediate and 25.4% and 45.5% with high risk scores (p<0.001). We found comparable results for the FIB-4 score. Findings were consistent regardless of cirrhosis or detectable HDV RNA (p<0.001)., Conclusion: Anti-HDV positive individuals are at high risk of adverse liver-related outcomes. The incidences of HCC was negligible among individuals without cirrhosis and among individuals with low baseline PAGE-B and/or FIB-4 scores. Therefore, these score can be used to guide HCC surveillance strategies and potentially also for treatment prioritization., (Copyright © 2024. Published by Elsevier Inc.)

Background: Pretreatment of HIV drug resistance among children living with HIV (CLHIV) can compromise antiretroviral therapy (ART) effectiveness. Resistance may be transmitted directly from mothers or acquired following exposure to antiretrovirals consumed through breastfeeding or administered as prophylaxis., Methods: We performed resistance testing in children aged <3 years, newly diagnosed with HIV in Western Cape, South Africa (2021-2022), who either (1) acquired HIV via possible breastfeeding transmission from mothers who received ART (any regimen) during pregnancy/postpartum and/or (2) were exposed to protease inhibitors or integrase strand transfer inhibitors (INSTIs) in utero. Possible breastfeeding transmission was defined as testing HIV-polymerase chain reaction positive at age >28 days, after previously testing negative. We used surveillance drug-resistance mutation lists to define mutations., Results: We included 135 CLHIV. Most mothers started ART prepregnancy (73%). Overall, 57% (77/135) of children had resistance mutations detected. Nonnucleoside reverse transcriptase inhibitor-associated, nucleoside reverse transcriptase inhibitor-associated, protease inhibitor-associated and INSTI-associated mutations were found in 55% (74/135), 10% (13/135), <1% (1/135) and <1% (1/122) of children tested, respectively. One child with breastfeeding transmission had high-level INSTI resistance detected at HIV diagnosis, aged 18 months (E138K and G118R mutations)., Conclusions: Although not clinically relevant, nonnucleoside reverse transcriptase inhibitor-associated mutations were common. Dolutegravir is currently the preferred first-line treatment for adults and CLHIV age ≥4 weeks, and although very low INSTI resistance levels have been observed in adults, limited data exist on genotyping the integrase region in children. Pretreatment INSTI resistance in children is likely to be unusual, but future surveillance, including longitudinal studies with paired mother-child resistance testing, is needed., Competing Interests: K.A., E.K. and M.-A.D. received funding from ViiV Healthcare for this project. The Provincial Health Data Centre was supported by grant U01AI069924 from National Institutes of Health (National Institute of Allergy and Infectious Diseases, National Institute of Child Health and Human Development, National Cancer Institute, National Institute on Drug Abuse and National Institute of Mental Health)—Principal Investigator: M. Egger and M.-A.D. E.K., A.B. and M.-A.D. were supported by grant R01HD080465 from National Institutes of Health (National Institute of Child Health and Human Development). The other authors have no conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Background: The increasing importance of artificial intelligence (AI) in health care has generated a growing need for health care professionals to possess a comprehensive understanding of AI technologies, requiring an adaptation in medical education., Objective: This paper explores stakeholder perceptions and expectations regarding AI in medicine and examines their potential impact on the medical curriculum. This study project aims to assess the AI experiences and awareness of different stakeholders and identify essential AI-related topics in medical education to define necessary competencies for students., Methods: The empirical data were collected as part of the TüKITZMed project between August 2022 and March 2023, using a semistructured qualitative interview. These interviews were administered to a diverse group of stakeholders to explore their experiences and perspectives of AI in medicine. A qualitative content analysis of the collected data was conducted using MAXQDA software., Results: Semistructured interviews were conducted with 38 participants (6 lecturers, 9 clinicians, 10 students, 6 AI experts, and 7 institutional stakeholders). The qualitative content analysis revealed 6 primary categories with a total of 24 subcategories to answer the research questions. The evaluation of the stakeholders' statements revealed several commonalities and differences regarding their understanding of AI. Crucial identified AI themes based on the main categories were as follows: possible curriculum contents, skills, and competencies; programming skills; curriculum scope; and curriculum structure., Conclusions: The analysis emphasizes integrating AI into medical curricula to ensure students' proficiency in clinical applications. Standardized AI comprehension is crucial for defining and teaching relevant content. Considering diverse perspectives in implementation is essential to comprehensively define AI in the medical context, addressing gaps and facilitating effective solutions for future AI use in medical studies. The results provide insights into potential curriculum content and structure, including aspects of AI in medicine., (© Julia-Astrid Moldt, Teresa Festl-Wietek, Wolfgang Fuhl, Susanne Zabel, Manfred Claassen, Samuel Wagner, Kay Nieselt, Anne Herrmann-Werner. Originally published in JMIR Medical Education (https://mededu.jmir.org).)

Different mechanisms driving a linear temperature dependence of the resistivity ρ ∼ T at van Hove singularities (VHSs) or metal-insulator transitions when doping a Mott insulator are being debated intensively with competing theoretical proposals. We experimentally investigate this using the exceptional tunability of twisted bilayer (TB) WSe 2 by tracking the parameter regions where linear-in- T resistivity is found in dependency of displacement fields, filling, and magnetic fields. We find that even when the VHSs are tuned rather far away from the half-filling point and the Mott insulating transition is absent, the T -linear resistivity persists at the VHSs. When doping away from the VHSs, the T -linear behavior quickly transitions into a Fermi liquid behavior with a T 2 relation. No apparent dependency of the linear-in- T resistivity, besides a rather strong change of prefactor, is found when applying displacement fields as long as the filling is tuned to the VHSs, including D ∼ 0.28 V/nm where a high-order VHS is expected. Intriguingly, such non-Fermi liquid linear-in- T resistivity persists even when magnetic fields break the spin-degeneracy of the VHSs at which point two linear in T regions emerge, for each of the split VHSs separately. This points to a mechanism of enhanced scattering at generic VHSs rather than only at high-order VHSs or by a quantum critical point during a Mott transition. Our findings provide insights into the many-body consequences arising out of VHSs, especially the non-Fermi liquid behavior found in moiré materials., Competing Interests: Competing interests statement:K.W. and T.T. are on multiple papers with reviewers due to their position as materials sources in this field. Because they provide hexagonal boron nitride to so many workers in the field, it is not feasible to find referees who are not co-authors with them.

This study presents a high-dimensional immunohistochemistry approach to assess human γδ T cell subsets in their native tissue microenvironments at spatial resolution, a hitherto unmet scientific goal due to the lack of established antibodies and required technology. We report an integrated approach based on multiplexed imaging and bioinformatic analysis to identify γδ T cells, characterize their phenotypes, and analyze the composition of their microenvironment. Twenty-eight γδ T cell microenvironments were identified in tissue samples from fresh frozen human colon and colorectal cancer where interaction partners of the immune system, but also cancer cells were discovered in close proximity to γδ T cells, visualizing their potential contributions to cancer immunosurveillance. While this proof-of-principle study demonstrates the potential of this cutting-edge technology to assess γδ T cell heterogeneity and to investigate their microenvironment, future comprehensive studies are warranted to associate phenotypes and microenvironment profiles with features such as relevant clinical characteristics., Competing Interests: Conflict of interest statement. M.W.L. reports personal fees from Boehringer Ingelheim for lectures/consultancy and is listed as a co-inventor on several patents owned by Immatics Biotechnologies, concerning peptides for use in immunotherapies., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Leukocyte Biology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

While the search for topological triplet-pairing superconductivity has remained a challenge, recent developments in optically stabilizing metastable superconducting states suggest a new route to realizing this elusive phase. Here, we devise a testable theory of competing superconducting orders that permits ultrafast switching to an opposite-parity superconducting phase in centrosymmetric crystals with strong spin-orbit coupling. Using both microscopic and phenomenological models, we show that dynamical inversion symmetry breaking with a tailored light pulse can induce odd-parity (spin triplet) order parameter oscillations in a conventional even-parity (spin singlet) superconductor, which when driven strongly can send the system to a competing minimum in its free energy landscape. Our results provide new guiding principles for engineering unconventional electronic phases using light, suggesting a fundamentally non-equilibrium route toward realizing topological superconductivity., (© 2024. The Author(s).)

Objective: Liver metastases are often resistant to immune checkpoint inhibitor therapy (ICI) and portend a worse prognosis compared with metastases to other locations. Regulatory T cells (Tregs) are one of several immunosuppressive cells implicated in ICI resistance of liver tumours, but the role played by Tregs residing within the liver surrounding a tumour is unknown., Design: Flow cytometry and single-cell RNA sequencing were used to characterise hepatic Tregs before and after ICI therapy., Results: We found that the murine liver houses a Treg population that, unlike those found in other organs, is both highly proliferative and apoptotic at baseline. On administration of αPD-1, αPD-L1 or αCTLA4, the liver Treg population doubled regardless of the presence of an intrahepatic tumour. Remarkably, this change was not due to the preferential expansion of the subpopulation of Tregs that express PD-1. Instead, a subpopulation of CD29 + ( Itgb1 , integrin β1) Tregs, that were highly proliferative at baseline, doubled its size in response to αPD-1. Partial and full depletion of Tregs identified CD29 + Tregs as the prominent niche-filling subpopulation in the liver, and CD29 + Tregs demonstrated enhanced suppression in vitro when derived from the liver but not the spleen. We identified IL2 as a critical modulator of both CD29 + and CD29 - hepatic Tregs, but expansion of the liver Treg population with αPD-1 driven by CD29 + Tregs was in part IL2-independent., Conclusion: We propose that CD29 + Tregs constitute a unique subpopulation of hepatic Tregs that are primed to respond to ICI agents and mediate resistance., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Background & Aim(s): Current guidelines suggest that nucleos(t)ide analogues (NA) can be discontinued before HBsAg loss in a selected group of chronic hepatitis B (CHB) patients. We aimed to study the safety and off-treatment response after NA cessation., Methods: This is a prospective, multicentre, cohort study in which eligible patients discontinued NA therapy. Adult patients, with a CHB mono-infection, HBeAg-negative, without a (history of) liver cirrhosis, who had achieved long-term viral suppression were eligible. Follow-up visits were planned at week 2-4-8-12-24-36-48-72-96. Re-treatment criteria included severe hepatitis (ALT >10x ULN), signs of imminent liver failure (bilirubin >1.5x ULN or INR >1.5), or at the physician's own discretion., Results: In total, 33 patients were enrolled. Patients were predominantly Caucasian (45.5%) and had genotype A/B/C/D/unknown in 3/4/6/10/10 (9.1/12.1/18.2/30.3/30.3%). At week 48, 15 patients (45.5%) achieved a sustained response (HBV DNA <2,000 IU/mL). At week 96, 13 patients (39.4%) achieved a sustained response, 4 (12.1%) achieved HBsAg loss, and 12 (36.4%) were re-treated. Severe hepatitis was the main reason for re-treatment (n=7, 21.2%). One patient with severe hepatitis developed jaundice, without signs of hepatic decompensation. Re-treatment was successful in all patients., Conclusion: NA therapy can be ceased in a highly selected group of CHB patients if close follow-up can be guaranteed. Treatment cessation may increase the chance of HBsAg loss in selected patients, which is counterbalanced by a significant risk of severe hepatitis., Competing Interests: Declaration of Competing Interest AB has received research grants from GSK, Gilead Sciences, Fujirebio, and Janssen. RdK has received honoraria for consulting/speaking from Gilead, Janssen, Echosens, AbbVie, and Norgine and received research grants form Gilead, GSK and Janssen. MS has received speaker's fees and research support from Roche, Innogenetics, BMS, Gilead and Fujirebio. The other authors report no disclosures., (Copyright © 2023. Published by Elsevier Masson SAS.)

Introduction: Children with underlying comorbidities and infants are most severely affected by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection, including in low- and middle-income countries with a high prevalence of HIV and TB. We describe the clinical presentation of SARS-CoV-2 infection in children during the Omicron wave, in Cape Town, South Africa., Methods: We analysed routine care data from a prospective cohort of children aged 0-13 years, with a positive SARS-CoV-2 real-time reverse-transcription polymerase chain reaction (rRT-PCR) or SARS-CoV-2 antigen test, admitted to Tygerberg Hospital between 1 November 2021 until 1 March 2022. Risk factors for severity of disease were assessed., Results: Ninety-five children tested positive for SARS-CoV-2, of whom 87 (91.6%) were symptomatic. Clinical data were available for 86 children. The median age was 11 months (IQR 3.0-60.0), 37 (43.0%) were females, 21 (24.7%) were HIV-exposed and 7 (8.1%) were living with HIV (CLHIV). In total, 44 (51.2%) children had at least one underlying comorbidity. TB co-infection was seen in 11 children, 6 children were newly diagnosed and 5 children were already on TB treatment at the time of admission., Conclusion: There was no evidence of more severe disease in children living with HIV or TB., (© 2024 The Authors.)

Introduction: Most childhood-onset SLE patients (cSLE) develop lupus nephritis (cLN), but only a small proportion achieve complete response to current therapies. The prognosis of children with LN and end-stage renal disease is particularly dire. Mortality rates within the first five years of renal replacement therapy may reach 22%. Thus, there is urgent need to decipher and target immune mechanisms that drive cLN. Despite the clear role of autoantibody production in SLE, targeted B cell therapies such as rituximab (anti-CD20) and belimumab (anti-BAFF) have shown only modest efficacy in cLN. While many studies have linked dysregulation of germinal center formation to SLE pathogenesis, other work supports a role for extrafollicular B cell activation in generation of pathogenic antibody secreting cells. However, whether extrafollicular B cell subsets and their T cell collaborators play a role in specific organ involvement in cLN and/or track with disease activity remains unknown., Methods: We analyzed high-dimensional mass cytometry and gene expression data from 24 treatment naïve cSLE patients at the time of diagnosis and longitudinally, applying novel computational tools to identify abnormalities associated with clinical manifestations (cLN) and disease activity (SLEDAI)., Results: cSLE patients have an extrafollicular B cell expansion signature, with increased frequency of i) DN2, ii) Bnd2, iii) plasmablasts, and iv) peripheral T helper cells. Most importantly, we discovered that this extrafollicular signature correlates with disease activity in cLN, supporting extrafollicular T/B interactions as a mechanism underlying pediatric renal pathogenesis., Discussion: This study integrates established and emerging themes of extrafollicular B cell involvement in SLE by providing evidence for extrafollicular B and peripheral T helper cell expansion, along with elevated type 1 IFN activation, in a homogeneous cohort of treatment-naïve cSLE patients, a point at which they should display the most extreme state of their immune dysregulation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Baxter, Wang, Garcia-Perez, Kong, Coleman, Larchenko, Schuyler, Jackson, Ghosh, Rudra, Paul, Claassen, Rochford, Cambier, Ghosh, Cooper, Smith and Hsieh.)

Laser-induced ultrafast demagnetization is a phenomenon of utmost interest and attracts significant attention because it enables potential applications in ultrafast optoelectronics and spintronics. As a spin-orbit coupling assisted magnetic insulator, α-RuCl 3 provides an attractive platform to explore the physics of electronic correlations and unconventional magnetism. Using time-dependent density functional theory, we explore the ultrafast laser-induced dynamics of the electronic and magnetic structures in α-RuCl 3 . Our study unveils that laser pulses can introduce ultrafast demagnetizations, accompanied by an out-of-equilibrium insulator-to-metal transition in a few tens of femtoseconds. The spin response significantly depends on the laser wavelength and polarization on account of the electron correlations, band renormalizations, and charge redistributions. These findings provide physical insights into the coupling between the electronic and magnetic degrees of freedom in α-RuCl 3 and shed light on suppressing the long-range magnetic orders and reaching a proximate spin liquid phase for two-dimensional magnets on an ultrafast time scale.

The spatial organization of various cell types within the tissue microenvironment is a key element for the formation of physiological and pathological processes, including cancer and autoimmune diseases. Here, we present S 3 -CIMA, a weakly supervised convolutional neural network model that enables the detection of disease-specific microenvironment compositions from high-dimensional proteomic imaging data. We demonstrate the utility of this approach by determining cancer outcome- and cellular-signaling-specific spatial cell-state compositions in highly multiplexed fluorescence microscopy data of the tumor microenvironment in colorectal cancer. Moreover, we use S 3 -CIMA to identify disease-onset-specific changes of the pancreatic tissue microenvironment in type 1 diabetes using imaging mass-cytometry data. We evaluated S 3 -CIMA as a powerful tool to discover novel disease-associated spatial cellular interactions from currently available and future spatial biology datasets., Competing Interests: M.C. is a co-founder of, is a scientific advisor to, and holds stock in Scailyte AG. This work is independent of this status. C.M.S. is a scientific advisor to, has stock options in, and has received research funding from Enable Medicine, Inc., all outside of this work., (© 2023 The Author(s).)

Mucosal-associated invariant T (MAIT) cells represent an abundant innate-like T cell subtype in the human liver. MAIT cells are assigned crucial roles in regulating immunity and inflammation, yet their role in liver cancer remains elusive. Here, we present a MAIT cell-centered profiling of hepatocellular carcinoma (HCC) using scRNA-seq, flow cytometry, and co-detection by indexing (CODEX) imaging of paired patient samples. These analyses highlight the heterogeneity and dysfunctionality of MAIT cells in HCC and their defective capacity to infiltrate liver tumors. Machine-learning tools were used to dissect the spatial cellular interaction network within the MAIT cell neighborhood. Co-localization in the adjacent liver and interaction between niche-occupying CSF1R + PD-L1 + tumor-associated macrophages (TAMs) and MAIT cells was identified as a key regulatory element of MAIT cell dysfunction. Perturbation of this cell-cell interaction in ex vivo co-culture studies using patient samples and murine models reinvigorated MAIT cell cytotoxicity. These studies suggest that aPD-1/aPD-L1 therapies target MAIT cells in HCC patients., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Background & Aims: The progression of non-alcoholic steatohepatitis (NASH) to fibrosis and hepatocellular carcinoma (HCC) is aggravated by auto-aggressive T cells. The gut-liver axis contributes to NASH, but the mechanisms involved and the consequences for NASH-induced fibrosis and liver cancer remain unknown. We investigated the role of gastrointestinal B cells in the development of NASH, fibrosis and NASH-induced HCC., Methods: C57BL/6J wild-type (WT), B cell-deficient and different immunoglobulin-deficient or transgenic mice were fed distinct NASH-inducing diets or standard chow for 6 or 12 months, whereafter NASH, fibrosis, and NASH-induced HCC were assessed and analysed. Specific pathogen-free/germ-free WT and μMT mice (containing B cells only in the gastrointestinal tract) were fed a choline-deficient high-fat diet, and treated with an anti-CD20 antibody, whereafter NASH and fibrosis were assessed. Tissue biopsy samples from patients with simple steatosis, NASH and cirrhosis were analysed to correlate the secretion of immunoglobulins to clinicopathological features. Flow cytometry, immunohistochemistry and single-cell RNA-sequencing analysis were performed in liver and gastrointestinal tissue to characterise immune cells in mice and humans., Results: Activated intestinal B cells were increased in mouse and human NASH samples and licensed metabolic T-cell activation to induce NASH independently of antigen specificity and gut microbiota. Genetic or therapeutic depletion of systemic or gastrointestinal B cells prevented or reverted NASH and liver fibrosis. IgA secretion was necessary for fibrosis induction by activating CD11b+CCR2+F4/80+CD11c-FCGR1+ hepatic myeloid cells through an IgA-FcR signalling axis. Similarly, patients with NASH had increased numbers of activated intestinal B cells; additionally, we observed a positive correlation between IgA levels and activated FcRg+ hepatic myeloid cells, as well the extent of liver fibrosis., Conclusions: Intestinal B cells and the IgA-FcR signalling axis represent potential therapeutic targets for the treatment of NASH., Impact and Implications: There is currently no effective treatment for non-alcoholic steatohepatitis (NASH), which is associated with a substantial healthcare burden and is a growing risk factor for hepatocellular carcinoma (HCC). We have previously shown that NASH is an auto-aggressive condition aggravated, amongst others, by T cells. Therefore, we hypothesized that B cells might have a role in disease induction and progression. Our present work highlights that B cells have a dual role in NASH pathogenesis, being implicated in the activation of auto-aggressive T cells and the development of fibrosis via activation of monocyte-derived macrophages by secreted immunoglobulins (e.g., IgA). Furthermore, we show that the absence of B cells prevented HCC development. B cell-intrinsic signalling pathways, secreted immunoglobulins, and interactions of B cells with other immune cells are potential targets for combinatorial NASH therapies against inflammation and fibrosis., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Data from low- and middle-income countries (LMICs) show higher morbidity and mortality in children with acute respiratory illness (ARI) from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). However, whether SARS-CoV-2 infection is distinct from other causes of ARI in this regard is unclear. We describe clinical characteristics and outcomes of South African children with SARS-CoV-2 and non-SARS-CoV-2 ARIs., Methods: We performed a cross-sectional study including 0-13 years old children admitted to Tygerberg Hospital between May and December 2020 with an ARI. Routine clinical data were collected by the attending clinicians. All children underwent SARS-CoV-2 polymerase chain reaction testing. For severity of disease, the need for respiratory support and duration of support was considered. Multivariable logistic regression models were built to determine the factors associated with SARS-CoV-2 infection and severity., Results: Data for 176 children were available, 38 (22%) children were SARS-CoV-2 polymerase chain reaction positive and 138 (78%) were negative. SARS-CoV-2 positive children were more likely to be female (OR: 2.68, 95% CI: 1.18-6.07), had lower weight-for-age Z score (OR: 0.76, 95% CI: 0.63-0.93), presented more frequently with fever (OR: 3.56, 95% CI: 1.54-8.24) and less often with cough (OR: 0.27, 95% CI: 0.11-0.66). SARS-CoV-2 infection was associated with significantly longer duration of oxygen treatment (median 8 vs. 3 days; OR: 1.1, 95% CI: 1.01-1.20). Overall, 66% of children had viral coinfection, with no significant difference between the groups. In total, 18% of SARS-CoV-2 positive children were readmitted within 3 months for a respiratory reason, compared with 15% SARS-CoV-2 negative children ( P = 0.64)., Conclusions: Our data show that ARIs from SARS-CoV-2 cannot be easily differentiated, but were associated with a higher morbidity compared with ARIs from other causes. Overall outcomes were good. The long-term implications of severe SARS-CoV-2 pneumonia in young children in low- and middle-income countries require further study., Competing Interests: This work is part of the VERDI project (101045989) which is funded by the European Union. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union or the European Health and Digital Executive Agency. Neither the European Union nor the granting authority can be held responsible for them. M.V.D.Z. was supported by a career development grant from the EDCTP2 program supported by the European Union (grant number TMA2019SFP-2836 TB-Lung FACT2), by the Fogarty International Center of the National Institutes of Health under Award Number K43TW011028 and funding from a SIR grant from the SAMRC. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. L.V. is supported by a Radboud University Medical Center Hypatia Tenure Track Grant. The data collection was supported by an unrestricted SEED funding from Stellenbosch University special vice-rector fund for COVID research. The other authors have no conflict of interest to disclose., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc.)

Since its introduction into the field of oncology, deep learning (DL) has impacted clinical discoveries and biomarker predictions. DL-driven discoveries and predictions in oncology are based on a variety of biological data such as genomics, proteomics, and imaging data. DL-based computational frameworks can predict genetic variant effects on gene expression, as well as protein structures based on amino acid sequences. Furthermore, DL algorithms can capture valuable mechanistic biological information from several spatial "omics" technologies, such as spatial transcriptomics and spatial proteomics. Here, we review the impact that the combination of artificial intelligence (AI) with spatial omics technologies has had on oncology, focusing on DL and its applications in biomedical image analysis, encompassing cell segmentation, cell phenotype identification, cancer prognostication, and therapy prediction. We highlight the advantages of using highly multiplexed images (spatial proteomics data) compared to single-stained, conventional histopathological ("simple") images, as the former can provide deep mechanistic insights that cannot be obtained by the latter, even with the aid of explainable AI. Furthermore, we provide the reader with the advantages/disadvantages of DL-based pipelines used in preprocessing highly multiplexed images (cell segmentation, cell type annotation). Therefore, this review also guides the reader to choose the DL-based pipeline that best fits their data. In conclusion, DL continues to be established as an essential tool in discovering novel biological mechanisms when combined with technologies such as highly multiplexed tissue imaging data. In balance with conventional medical data, its role in clinical routine will become more important, supporting diagnosis and prognosis in oncology, enhancing clinical decision-making, and improving the quality of care for patients. Since its introduction into the field of oncology, deep learning (DL) has impacted clinical discoveries and biomarker predictions. DL-driven discoveries and predictions in oncology are based on a variety of biological data such as genomics, proteomics, and imaging data. DL-based computational frameworks can predict genetic variant effects on gene expression, as well as protein structures based on amino acid sequences. Furthermore, DL algorithms can capture valuable mechanistic biological information from several spatial "omics" technologies, such as spatial transcriptomics and spatial proteomics. Here, we review the impact that the combination of artificial intelligence (AI) with spatial omics technologies has had on oncology, focusing on DL and its applications in biomedical image analysis, encompassing cell segmentation, cell phenotype identification, cancer prognostication, and therapy prediction. We highlight the advantages of using highly multiplexed images (spatial proteomics data) compared to single-stained, conventional histopathological ("simple") images, as the former can provide deep mechanistic insights that cannot be obtained by the latter, even with the aid of explainable AI. Furthermore, we provide the reader with the advantages/disadvantages of the DL-based pipelines used in preprocessing the highly multiplexed images (cell segmentation, cell type annotation). Therefore, this review also guides the reader to choose the DL-based pipeline that best fits their data. In conclusion, DL continues to be established as an essential tool in discovering novel biological mechanisms when combined with technologies such as highly multiplexed tissue imaging data. In balance with conventional medical data, its role in clinical routine will become more important, supporting diagnosis and prognosis in oncology, enhancing clinical decision-making, and improving the quality of care for patients., Competing Interests: CMS is a scientific advisor to AstraZeneca plc, and is on the scientific advisory board of, has stock options in, and has received research funding from Enable Medicine, Inc., all outside the current work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zidane, Makky, Bruhns, Rochwarger, Babaei, Claassen and Schürch.)

Monitoring of HIV drug resistance (HIVDR) remains critical for ensuring countries attain and sustain the global goals for ending HIV as a public health threat by 2030. On an individual patient level, drug resistance results assist in ensuring unnecessary treatment switches are avoided and subsequent regimens are tailored on a case-by-case basis, should resistance be detected. Although there is a disparity in access to HIVDR testing in high-income countries compared to low- and middle-income countries (LMICS), more LMICs have now included HIVDR testing for individual patient management in some groups of patients. In this review, we describe different strategies for surveillance as well as where HIVDR testing can be implemented for individual patient management. In addition, we briefly review available technologies for HIVDR testing in LMICs, including Sanger sequencing, next-generation sequencing, and some point-of-care options. Finally, we describe how South Africa has implemented HIVDR testing in the public sector.

Background: The need for reliable clinical biomarkers to predict which patients with melanoma will benefit from immune checkpoint blockade (ICB) remains unmet. Several different parameters have been considered in the past, including routine differential blood counts, T cell subset distribution patterns and quantification of peripheral myeloid-derived suppressor cells (MDSC), but none has yet achieved sufficient accuracy for clinical utility., Methods: Here, we investigated potential cellular biomarkers from clinical routine blood counts as well as several myeloid and T cell subsets, using flow cytometry, in two independent cohorts of a total of 141 patients with stage IV M1c melanoma before and during ICB., Results: Elevated baseline frequencies of monocytic MDSCs (M-MDSC) in the blood were confirmed to predict shorter overall survival (OS) (HR 2.086, p=0.030) and progression-free survival (HR 2.425, p=0.001) in the whole patient cohort. However, we identified a subgroup of patients with highly elevated baseline M-MDSC frequencies that fell below a defined cut-off during therapy and found that these patients had a longer OS that was similar to that of patients with low baseline M-MDSC frequencies. Importantly, patients with high M-MDSC frequencies exhibited a skewed baseline distribution of certain other immune cells but these did not influence patient survival, illustrating the paramount utility of MDSC assessment., Conclusion: We confirmed that in general, highly elevated frequencies of peripheral M-MDSC are associated with poorer outcomes of ICB in metastatic melanoma. However, one reason for an imperfect correlation between high baseline MDSCs and outcome for individual patients may be the subgroup of patients identified here, with rapidly decreasing M-MDSCs on therapy, in whom the negative effect of high M-MDSC frequencies was lost. These findings might contribute to developing more reliable predictors of late-stage melanoma response to ICB at the individual patient level. A multifactorial model seeking such markers yielded only MDSC behavior and serum lactate dehydrogenase as predictors of treatment outcome., Competing Interests: Competing interests: TA reports institutional grants from SkylineDx, institutional grants and personal fees from Novartis, institutional grants from NeraCare, personal fees from BMS, institutional grants from Sanofi, personal fees from CeCaVa, personal fees from Pierre Fabre, outside the submitted work. NW reports an advisory role for Pierre Fabre and Sanofi, consultant's honoraria from Novartis, and has received travel support from AbbVie and Amgen outside the submitted work. FM reports receiving commercial research grants from Novartis and Roche; and has received travel support and/or speaker’s fees and/or advisor’s honoraria by Novartis, Roche, Bristol-Myers Squibb, Merck Sharp & Dohme and Pierre Fabre. PT has received travel support and/or speaker’s fees and/or advisor’s honoraria by Almirall, Biofrontera, Bristol-Myers Squibb, Curevac, Kyowa Kirin, Merck, Merck Sharp & Dohme, Novartis, Pierre Fabre, Roche, Sanofi and 4SC. CG reports receiving commercial research grants from Bristol-Myers Squibb, Novartis and Roche; and is a consultant/advisory board member for Amgen, Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis and Roche. GP has received speaker’s honoraria from Novartis, Roche, Pfizer, GlaxoSmithKline and Astellas. TE has received travel support and/or speaker’s fees and/or advisor’s honoraria by Sanofi, Novartis, Bristol-Myers Squibb, Merck Sharp & Dohme, Almiral Hermal and Pierre Fabre. BW reports receiving commercial research grants from, is a consultant/advisory board member for and reports receiving travel reimbursement from Bristol-Myers Squibb and Merck Sharp & Dohme. KW-H received commercial research grants from CatalYm GmbH and travel support from Society for Immunotherapy of Cancer. No potential conflicts of interest were disclosed by the other authors., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Adult-born cells, arriving daily into the rodent olfactory bulb, either integrate into the neural circuitry or get eliminated. However, whether these two populations differ in their morphological or functional properties remains unclear. Using longitudinal in vivo two-photon imaging, we monitored dendritic morphogenesis, odor-evoked responsiveness, ongoing Ca 2+ signaling, and survival/death of adult-born juxtaglomerular neurons (abJGNs). We found that the maturation of abJGNs is accompanied by a significant reduction in dendritic complexity, with surviving and subsequently eliminated cells showing similar degrees of dendritic remodeling. Surprisingly, ∼63% of eliminated abJGNs acquired odor responsiveness before death, with amplitudes and time courses of odor-evoked responses similar to those recorded in surviving cells. However, the subsequently eliminated cell population exhibited significantly higher ongoing Ca 2+ signals, with a difference visible even 10 days before death. Quantitative supervised machine learning analysis revealed a relationship between the abJGNs' activity and survival probability, with low neuronal activity being supportive for survival., Competing Interests: Conflict of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Healing wounds and cancers present remarkable cellular and molecular parallels, but the specific roles of the healing phases are largely unknown. We developed a bioinformatics pipeline to identify genes and pathways that define distinct phases across the time-course of healing. Their comparison to cancer transcriptomes revealed that a resolution phase wound signature is associated with increased severity in skin cancer and enriches for extracellular matrix-related pathways. Comparisons of transcriptomes of early- and late-phase wound fibroblasts vs skin cancer-associated fibroblasts (CAFs) identified an "early wound" CAF subtype, which localizes to the inner tumor stroma and expresses collagen-related genes that are controlled by the RUNX2 transcription factor. A "late wound" CAF subtype localizes to the outer tumor stroma and expresses elastin-related genes. Matrix imaging of primary melanoma tissue microarrays validated these matrix signatures and identified collagen- vs elastin-rich niches within the tumor microenvironment, whose spatial organization predicts survival and recurrence. These results identify wound-regulated genes and matrix patterns with prognostic potential in skin cancer., Competing Interests: Declaration of Competing Interest RD has intermittent, project focused consulting and/or advisory relationships with Novartis, Merck Sharp & Dohme (MSD), Bristol-Myers Squibb (BMS), Roche, Amgen, Takeda, Pierre Fabre, Sun Pharma, Sanofi, Catalym, Second Genome, Regeneron, Alligator, T3 Pharma, MaxiVAX SA, Pfizer and touchIME, all outside of the submitted work. No other authors have potential competing interests to declare., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Cellular decision making often builds on ultrasensitive MAPK pathways. The phosphorylation mechanism of MAP kinase has so far been described as either distributive or processive, with distributive mechanisms generating ultrasensitivity in theoretical analyses. However, the in vivo mechanism of MAP kinase phosphorylation and its activation dynamics remain unclear. Here, we characterize the regulation of the MAP kinase Hog1 in Saccharomyces cerevisiae via topologically different ODE models, parameterized on multimodal activation data. Interestingly, our best fitting model switches between distributive and processive phosphorylation behavior regulated via a positive feedback loop composed of an affinity and a catalytic component targeting the MAP kinase-kinase Pbs2. Indeed, we show that Hog1 directly phosphorylates Pbs2 on serine 248 (S248), that cells expressing a non-phosphorylatable (S248A) or phosphomimetic (S248E) mutant show behavior that is consistent with simulations of disrupted or constitutively active affinity feedback and that Pbs2-S248E shows significantly increased affinity to Hog1 in vitro. Simulations further suggest that this mixed Hog1 activation mechanism is required for full sensitivity to stimuli and to ensure robustness to different perturbations., (© 2023. The Author(s).)

Background: Despite a high paediatric tuberculosis (TB) burden globally, sensitive and specific diagnostic tools are lacking. In addition, no data exist on the impact of pulmonary TB on long-term child lung health in low- and middle-income countries. The prospective observational UMOYA study aims (1) to build a state-of-the-art clinical, radiological, and biological repository of well-characterised children with presumptive pulmonary TB as a platform for future studies to explore new emerging diagnostic tools and biomarkers for early diagnosis and treatment response; and (2) to investigate the short and long-term impact of pulmonary TB on lung health and quality of life in children., Methods: We will recruit up to 600 children (0-13 years) with presumptive pulmonary TB and 100 healthy controls. Recruitment started in November 2017 and is expected to continue until May 2023. Sputum and non-sputum-based samples are collected at enrolment and during follow-up in TB cases and symptomatic controls. TB treatment is started by routine care services. Intensive follow-up for 6 months will allow for TB cases to retrospectively be classified according to international consensus clinical case definitions for TB. Long-term follow-up, including imaging, comprehensive assessment of lung function and quality of life questionnaires, are done yearly up to 4 years after recruitment., Discussion: The UMOYA study will provide a unique platform to evaluate new emerging diagnostic tools and biomarkers for early diagnosis and treatment response and to investigate long-term outcomes of pulmonary TB and other respiratory events on lung health in children., (© 2023. The Author(s).)

Many-body entanglement in condensed matter systems can be diagnosed from equilibrium response functions through the use of entanglement witnesses and operator-specific quantum bounds. Here, we investigate the applicability of this approach for detecting entangled states in quantum systems driven out of equilibrium. We use a multipartite entanglement witness, the quantum Fisher information, to study the dynamics of a paradigmatic fermion chain undergoing a time-dependent change of the Coulomb interaction. Our results show that the quantum Fisher information is able to witness distinct signatures of multipartite entanglement both near and far from equilibrium that are robust against decoherence. We discuss implications of these findings for probing entanglement in light-driven quantum materials with time-resolved optical and x-ray scattering methods.

Objectives: Low-capital-layout sequencing options from Oxford Nanopore Technologies (ONT) could assist in expanding HIV drug resistance testing to resource-limited settings. HIV drug resistance mutations often occur as mixtures, but current ONT pipelines provide a consensus sequence only. Moreover, there is no integrated pipeline that provides a drug resistance report from an ONT sequence file without intervention from skilled bioinformaticists. We therefore investigated Nano-RECall, which provides seamless drug resistance interpretation while requiring low-read coverage ONT sequence data from affordable Flongle or MinION flow cells and which provides mutation mixtures similar to Sanger Sequencing., Methods: We compared Sanger sequencing to ONT sequencing of the same HIV-1 subtype C polymerase chain reaction (PCR) amplicons, respectively using RECall and the novel Nano-RECall bioinformatics pipelines. Amplicons were from separate assays: (a) Applied Biosystems HIV-1 Genotyping Kit (ThermoFisher) spanning protease (PR) to reverse transcriptase (RT) (PR-RT) (n = 46) and (b) homebrew integrase (IN) (n = 21). The agreement between Sanger sequences and ONT sequences was assessed at nucleotide level, and at codon level for Stanford HIV drug resistance database mutations at an optimal ONT read depth of 400 reads only., Results: The average sequence similarity between ONT and Sanger sequences was 99.3% (95% CI: 99.1%-99.4%) for PR-RT and 99.6% (95% CI: 99.4%-99.7%) for INT. Drug resistance mutations did not differ for 21 IN specimens; 8 mutations were detected by both ONT- and Sanger sequencing. For the 46 PR and RT specimens, 245 mutations were detected by either ONT or Sanger, of these 238 (97.1%) were detected by both., Conclusions: The Nano-RECall pipeline, freely available as a downloadable application on a Windows computer, provides Sanger-equivalent HIV drug resistance interpretation. This novel pipeline combined with a simple workflow and multiplexing samples on ONT flow-cells would contribute to making HIV drug resistance sequencing feasible for resource-limited settings., (© 2023 John Wiley & Sons Ltd.)

We report Cytopath, a method for trajectory inference that takes advantage of transcriptional activity information from the RNA velocity of single cells to perform trajectory inference. Cytopath performs this task by defining a Markov chain model, simulating an ensemble of possible differentiation trajectories, and constructing a consensus trajectory. We show that Cytopath can recapitulate the topological and molecular characteristics of the differentiation process under study. In our analysis, we include differentiation trajectories with varying bifurcated, circular, convergent, and mixed topologies studied in single-snapshot as well as time-series single-cell RNA sequencing experiments. We demonstrate the capability to reconstruct differentiation trajectories, assess the association of RNA velocity-based pseudotime with actually elapsed process time, and identify drawbacks in current state-of-the art trajectory inference approaches., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Background: Identification of SARS-CoV-2 infected individuals is imperative to prevent hospital transmission, but symptom-based screening may fail to identify asymptomatic/mildly symptomatic infectious children and their caregivers., Methods: A COVID-19 period prevalence study was conducted between 13 and 26 August 2020 at Tygerberg Hospital, testing all children and their accompanying asymptomatic caregivers after initial symptom screening. One nasopharyngeal swab was submitted for SARS-CoV-2 using real-time reverse-transcription polymerase chain reaction (rRT-PCR). An additional Respiratory Viral 16-multiplex rRT-PCR test was simultaneously done in children presenting with symptoms compatible with possible SARS-CoV-2 infection., Results: SARS-Co-V 2 RT-PCR tests from 196 children and 116 caregivers were included in the analysis. The SARS-CoV-2 period prevalence in children was 5.6% (11/196) versus 15.5% (18/116) in asymptomatic caregivers ( p <0.01). Presenting symptoms did not correlate with SARS-CoV-2 test positivity; children without typical symptoms of SARS-CoV-2 were more likely to be positive than those with typical symptoms (10.2% [10/99] vs 1% [1/97]; p <0.01). Children with typical symptoms (97/196; 49.5%) mainly presented with acute respiratory (68/97; 70.1%), fever (17/97; 17.5%), or gastro-intestinal complaints (12/97; 12.4%); Human Rhinovirus (23/81; 28.4%) and Respiratory Syncytial Virus (18/81; 22.2%) were frequently identified in this group. Children-caregiver pairs' SARS-CoV-2 tests were discordant in 83.3%; 15/18 infected caregivers' children tested negative. Symptom-based COVID-19 screening alone would have missed 90% of the positive children and 100% of asymptomatic but positive caregivers., Conclusion: Given the poor correlation between SARS-CoV-2 symptoms and RT-PCR test positivity, universal testing of children and their accompanying caregivers should be considered for emergency and inpatient paediatric admissions during high COVID-19 community transmission periods. Universal PPE and optimising ventilation is likely the most effective way to control transmission of respiratory viral infections, including SARS-CoV-2, where universal testing is not feasible. In these settings, repeated point prevalence studies may be useful to inform local testing and cohorting strategies., Competing Interests: The authors declared no conflict of interest., (© 2022 Published by Elsevier B.V. on behalf of African Federation for Emergency Medicine.)

We predict that twisted bilayers of 1T-ZrS 2 realize a novel and tunable platform to engineer two-dimensional topological quantum phases dominated by strong spin-orbit interactions. At small twist angles, ZrS 2 heterostructures give rise to an emergent and twist-controlled moiré Kagome lattice, combining geometric frustration and strong spin-orbit coupling to give rise to a moiré quantum spin Hall insulator with highly controllable and nearly-dispersionless bands. We devise a generic pseudo-spin theory for group-IV transition metal dichalcogenides that relies on the two-component character of the valence band maximum of the 1T structure at Γ, and study the emergence of a robust quantum anomalous Hall phase as well as possible fractional Chern insulating states from strong Coulomb repulsion at fractional fillings of the topological moiré Kagome bands. Our results establish group-IV transition metal dichalcogenide bilayers as a novel moiré platform to realize strongly-correlated topological phases in a twist-tunable setting., (© 2022. The Author(s).)

Dimethyl fumarate (DMF) is an immunomodulatory treatment for multiple sclerosis (MS). Despite its wide clinical use, the mechanisms underlying clinical response are not understood. This study aimed to reveal immune markers of therapeutic response to DMF treatment in MS. For this purpose, we prospectively collected peripheral blood mononuclear cells (PBMCs) from a highly characterized cohort of 44 individuals with MS before and at 12 and 48 wk of DMF treatment. Single cells were profiled using high-dimensional mass cytometry. To capture the heterogeneity of different immune subsets, we adopted a bioinformatic multipanel approach that allowed cell population-cluster assignment of more than 50 different parameters, including lineage and activation markers as well as chemokine receptors and cytokines. Data were further analyzed in a semiunbiased fashion implementing a supervised representation learning approach to capture subtle longitudinal immune changes characteristic for therapy response. With this approach, we identified a population of memory T helper cells expressing high levels of neuroinflammatory cytokines (granulocyte-macrophage colony-stimulating factor [GM-CSF], interferon γ [IFNγ]) as well as CXCR3, whose abundance correlated with treatment response. Using spectral flow cytometry, we confirmed these findings in a second cohort of patients. Serum neurofilament light-chain levels confirmed the correlation of this immune cell signature with axonal damage. The identified cell population is expanded in peripheral blood under natalizumab treatment, substantiating a specific role in treatment response. We propose that depletion of GM-CSF-, IFNγ-, and CXCR3-expressing T helper cells is the main mechanism of action of DMF and allows monitoring of treatment response.

Background: Comorbidities are risk factors for development of severe coronavirus disease 2019 (COVID-19). However, the extent to which an underlying comorbidity influences the immune response to severe acute respiratory syndrome coronavirus 2 remains unknown., Objective: Our aim was to investigate the complex interrelations of comorbidities, the immune response, and patient outcome in COVID-19., Methods: We used high-throughput, high-dimensional, single-cell mapping of peripheral blood leukocytes and algorithm-guided analysis., Results: We discovered characteristic immune signatures associated not only with severe COVID-19 but also with the underlying medical condition. Different factors of the metabolic syndrome (obesity, hypertension, and diabetes) affected distinct immune populations, thereby additively increasing the immunodysregulatory effect when present in a single patient. Patients with disorders affecting the lung or heart, together with factors of metabolic syndrome, were clustered together, whereas immune disorder and chronic kidney disease displayed a distinct immune profile in COVID-19. In particular, severe acute respiratory syndrome coronavirus 2-infected patients with preexisting chronic kidney disease were characterized by the highest number of altered immune signatures of both lymphoid and myeloid immune branches. This overall major immune dysregulation could be the underlying mechanism for the estimated odds ratio of 16.3 for development of severe COVID-19 in this burdened cohort., Conclusion: The combinatorial systematic analysis of the immune signatures, comorbidities, and outcomes of patients with COVID-19 has provided the mechanistic immunologic underpinnings of comorbidity-driven patient risk and uncovered comorbidity-driven immune signatures., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Immune checkpoint blockade (ICB) is standard-of-care for patients with metastatic melanoma. It may re-invigorate T cells recognizing tumors, and several tumor antigens have been identified as potential targets. However, little is known about the dynamics of tumor antigen-specific T cells in the circulation, which might provide valuable information on ICB responses in a minimally invasive manner. Here, we investigated individual signatures composed of up to 167 different melanoma-associated epitope (MAE)-specific CD8+ T cells in the blood of stage IV melanoma patients before and during anti-PD-1 treatment, using a peptide-loaded multimer-based high-throughput approach. Additionally, checkpoint receptor expression patterns on T cell subsets and frequencies of myeloid-derived suppressor cells and regulatory T cells were quantified by flow cytometry. Regression analysis using the MAE-specific CD8+ T cell populations was applied to identify those that correlated with overall survival (OS). The abundance of MAE-specific CD8+ T cell populations, as well as their dynamics under therapy, varied between patients. Those with a dominant increase of these T cell populations during PD-1 ICB had a longer OS and progression-free survival than those with decreasing or balanced signatures. Patients with a dominantly increased MAE-specific CD8+ T cell signature also exhibited an increase in TIM-3+ and LAG-3+ T cells. From these results, we created a model predicting improved/reduced OS by combining data on dynamics of the three most informative MAE-specific CD8+ T cell populations. Our results provide insights into the dynamics of circulating MAE-specific CD8+ T cell populations during ICB, and should contribute to a better understanding of biomarkers of response and anti-cancer mechanisms., Competing Interests: CG reports receiving commercial research grants from Bristol-Myers Squibb, Novartis, and Roche, and is a consultant/advisory board member for Amgen, Bristol-Myers Squibb, Merck Sharp & Dohme, Novartis, and Roche. BW reports receiving commercial research grants from, is a consultant/advisory board member for, and reports receiving travel reimbursement from Bristol-Myers Squibb and Merck Sharp & Dohme. FM reports receiving commercial research grants from Novartis and Roche, and has received travel support or/and speaker´s fees or/and advisor´s honoraria by Novartis, Roche, Bristol-Myers Squibb, Merck Sharp & Dohme, and Pierre Fabre. NW reports an advisory role for Pierre-Fabre and Sanofi, consultant’s honoraria from Novartis, and has received travel support from AbbVie and Amgen outside the submitted work. GP has received speaker´s honoraria from Novartis, Roche, Pfizer, GlaxoSmithKline and Astellas. KW-H received commercial research grants from the CatalYm GmbH and travel support from SITC (Society for Immunotherapy of Cancer). SH is the cofounder of Immumap, Tetramer-shop and PokeAcell and is the co-inventor of the patents WO2015185067 and WO2015188839 for the barcoded MHC technology which is licensed to Immudex. The data presented in this study is not directly involved in these activities. TE has received travel support or/and speaker´s fees or/and advisor´s honoraria by Sanofi, Novartis, Bristol-Myers Squibb, Merck Sharp & Dohme, Almiral Hermal, and Pierre Fabre. TA reports institutional grants from SkylineDx, institutional grants and personal fees from Novartis, institutional grants from NeraCare, personal fees from BMS, institutional grants from Sanofi, personal fees from CeCaVa, personal fees from Pierre Fabre, outside the submitted work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Gaißler, Meldgaard, Heeke, Babaei, Tvingsholm, Bochem, Spreuer, Amaral, Wagner, Klein, Meier, Garbe, Eigentler, Pawelec, Claassen, Weide, Hadrup and Wistuba-Hamprecht.)

Motivation: Improvements in single-cell RNA-seq technologies mean that studies measuring multiple experimental conditions, such as time series, have become more common. At present, few computational methods exist to infer time series-specific transcriptome changes, and such studies have therefore typically used unsupervised pseudotime methods. While these methods identify cell subpopulations and the transitions between them, they are not appropriate for identifying the genes that vary coherently along the time series. In addition, the orderings they estimate are based only on the major sources of variation in the data, which may not correspond to the processes related to the time labels., Results: We introduce psupertime, a supervised pseudotime approach based on a regression model, which explicitly uses time-series labels as input. It identifies genes that vary coherently along a time series, in addition to pseudotime values for individual cells, and a classifier that can be used to estimate labels for new data with unknown or differing labels. We show that psupertime outperforms benchmark classifiers in terms of identifying time-varying genes and provides better individual cell orderings than popular unsupervised pseudotime techniques. psupertime is applicable to any single-cell RNA-seq dataset with sequential labels (e.g. principally time series but also drug dosage and disease progression), derived from either experimental design and provides a fast, interpretable tool for targeted identification of genes varying along with specific biological processes., Availability and Implementation: R package available at github.com/wmacnair/psupertime and code for results reproduction at github.com/wmacnair/psupplementary., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2022. Published by Oxford University Press.)

Motivation: Single-cell RNA sequencing (scRNA-seq) allows studying the development of cells in unprecedented detail. Given that many cellular differentiation processes are hierarchical, their scRNA-seq data are expected to be approximately tree-shaped in gene expression space. Inference and representation of this tree structure in two dimensions is highly desirable for biological interpretation and exploratory analysis., Results: Our two contributions are an approach for identifying a meaningful tree structure from high-dimensional scRNA-seq data, and a visualization method respecting the tree structure. We extract the tree structure by means of a density-based maximum spanning tree on a vector quantization of the data and show that it captures biological information well. We then introduce density-tree biased autoencoder (DTAE), a tree-biased autoencoder that emphasizes the tree structure of the data in low dimensional space. We compare to other dimension reduction methods and demonstrate the success of our method both qualitatively and quantitatively on real and toy data., Availability and Implementation: Our implementation relying on PyTorch and Higra is available at github.com/hci-unihd/DTAE., Supplementary Information: Supplementary data are available at Bioinformatics online., (© The Author(s) 2022. Published by Oxford University Press.)

Treatment with dimethyl fumarate (DMF) leads to lymphopenia and infectious complications in a subset of patients with multiple sclerosis (MS). Here, we aimed to reveal immune markers of DMF-associated lymphopenia. This prospective observational study longitudinally assessed 31 individuals with MS by single-cell mass cytometry before and after 12 and 48 weeks of DMF therapy. Employing a neural network-based representation learning approach, we identified a CCR4-expressing T helper cell population negatively associated with relevant lymphopenia. CCR4-expressing T helper cells represent a candidate prognostic biomarker for the development of relevant lymphopenia in patients undergoing DMF treatment. ANN NEUROL 2022;91:676-681., (© 2022 The Authors. Annals of Neurology published by Wiley Periodicals LLC on behalf of American Neurological Association.)

A 38-year-old male patient presented to the emergency department with fever and dyspnea. Hospitalization was warranted and soon coronavirus disease 2019 (COVID-19) was diagnosed based on a positive SARS-CoV-2-PCR. Over the following weeks his condition gradually worsened, leading to admission at the intensive care unit. Because of unexplained weight loss before admission, a HIV screening was performed. HIV was confirmed and additional tests showed an undetectable CD4+ T cell count, alongside a number of co-infections. Convalescent plasma therapy, which has been shown to be effective in severe humoral immunodeficiency was tried, but was not effective. One week after the HIV diagnosis, antiretroviral therapy was started and finally, 3 months after the initial positive test and after partial recovery of cellular immunity, the COVID-19 virus was cleared. In the end, the patient made a full recovery. Our case demonstrates a prolonged COVID-19 infection in a patient with undiagnosed HIV with severely impaired cellular immunity.

Training accurate and robust machine learning models requires a large amount of data that is usually scattered across data silos. Sharing or centralizing the data of different healthcare institutions is, however, unfeasible or prohibitively difficult due to privacy regulations. In this work, we address this problem by using a privacy-preserving federated learning-based approach, PriCell , for complex models such as convolutional neural networks. PriCell relies on multiparty homomorphic encryption and enables the collaborative training of encrypted neural networks with multiple healthcare institutions. We preserve the confidentiality of each institutions' input data, of any intermediate values, and of the trained model parameters. We efficiently replicate the training of a published state-of-the-art convolutional neural network architecture in a decentralized and privacy-preserving manner. Our solution achieves an accuracy comparable with the one obtained with the centralized non-secure solution. PriCell guarantees patient privacy and ensures data utility for efficient multi-center studies involving complex healthcare data., Competing Interests: J.R.T.-P. and J.-P.H. are co-founders of the start-up Tune Insight SA (https://tuneinsight.com). The other authors declare no competing interests. The methods presented in this work are partially covered by the PCT patent applications under publication nos. WO/2022/04284848 and WO/2021/223873.49, (© 2022 The Authors.)

Ample evidence pinpoints the phenotypic diversity of blood vessels (BVs) and site-specific functions of their lining endothelial cells (ECs). We harnessed single-cell RNA sequencing (scRNA-seq) to dissect the molecular heterogeneity of blood vascular endothelial cells (BECs) in healthy adult human skin and identified six different subpopulations, signifying arterioles, post-arterial capillaries, pre-venular capillaries, post-capillary venules, venules and collecting venules. Individual BEC subtypes exhibited distinctive transcriptomic landscapes associated with diverse biological pathways. These functionally distinct dermal BV segments were characterized by their unique compositions of conventional and novel markers (e.g., arteriole marker GJA5; arteriole capillary markers ASS1 and S100A4; pre-venular capillary markers SOX17 and PLAUR; venular markers EGR2 and LRG1), many of which have been implicated in vascular remodeling upon inflammatory responses. Immunofluorescence staining of human skin sections and whole-mount skin blocks confirmed the discrete expression of these markers along the blood vascular tree in situ, further corroborating BEC heterogeneity in human skin. Overall, our study molecularly refines individual BV compartments, whilst the identification of novel subtype-specific signatures provides more insights for future studies dissecting the responses of distinct vessel segments under pathological conditions.

Single-cell RNA sequencing (scRNA-seq) approaches have transformed our ability to resolve cellular properties across systems, but are currently tailored toward large cell inputs (>1,000 cells). This renders them inefficient and costly when processing small, individual tissue samples, a problem that tends to be resolved by loading bulk samples, yielding confounded mosaic cell population read-outs. Here, we developed a deterministic, mRNA-capture bead and cell co-encapsulation dropleting system, DisCo, aimed at processing low-input samples (<500 cells). We demonstrate that DisCo enables precise particle and cell positioning and droplet sorting control through combined machine-vision and multilayer microfluidics, enabling continuous processing of low-input single-cell suspensions at high capture efficiency (>70%) and at speeds up to 350 cells per hour. To underscore DisCo's unique capabilities, we analyzed 31 individual intestinal organoids at varying developmental stages. This revealed extensive organoid heterogeneity, identifying distinct subtypes including a regenerative fetal-like Ly6a + stem cell population that persists as symmetrical cysts, or spheroids, even under differentiation conditions, and an uncharacterized 'gobloid' subtype consisting predominantly of precursor and mature (Muc2 + ) goblet cells. To complement this dataset and to demonstrate DisCo's capacity to process low-input, in vivo-derived tissues, we also analyzed individual mouse intestinal crypts. This revealed the existence of crypts with a compositional similarity to spheroids, which consisted predominantly of regenerative stem cells, suggesting the existence of regenerating crypts in the homeostatic intestine. These findings demonstrate the unique power of DisCo in providing high-resolution snapshots of cellular heterogeneity in small, individual tissues., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Objectives: To determine the prevalence and types of viral pathogens in the myocardium of patients presenting with clinically suspected myocarditis in South Africa., Method: This is a prospective cross-sectional study. Consecutive adults presenting to a single tertiary centre in South Africa between August 2017 and January 2021 who fulfilled the European Society of Cardiology's diagnostic criteria for clinically suspected myocarditis and who had undergone the appropriate investigations, including cardiac MRI (CMR) and endomyocardial biopsy (EMB), were included., Results: One hundred and two patients with clinically suspected myocarditis were enrolled. Acute myocarditis (AM) was confirmed by CMR or EMB in 82 (80.39%) patients. Viral genomes were detected by PCR in EMB specimens of 50 patients with AM. Parvovirus B19 (PVB19) was the most frequently detected virus, in 37 as monoinfection and 4 as coinfection. This was followed by Epstein-Barr virus (n=6), human herpesvirus 6 (n=2) and human bocavirus (n=1). PVB19 was also detected in 9 patients with no evidence of AM on CMR or EMB., Conclusion: Viral myocarditis is the most common form of myocarditis in South Africa. Local viral prevalence appears to be similar those of the developed world. The clinical significance and pathogenic role of PVB19 remains questioned, and its local background prevalence will have to be further investigated., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

The application of machine learning approaches to imaging flow cytometry (IFC) data has the potential to transform the diagnosis of hematological diseases. However, the need for manually labeled single-cell images for machine learning model training has severely limited its clinical application. To address this, we present iCellCnn, a weakly supervised deep learning approach for label-free IFC-based blood diagnostics. We demonstrate the capability of iCellCnn to achieve diagnosis of Sézary syndrome (SS) from patient samples on the basis of bright-field IFC images of T cells obtained after fluorescence-activated cell sorting of human peripheral blood mononuclear cell specimens. With a sample size of four healthy donors and five SS patients, iCellCnn achieved a 100% classification accuracy. As iCellCnn is not restricted to the diagnosis of SS, we expect such weakly supervised approaches to tap the diagnostic potential of IFC by providing automatic data-driven diagnosis of diseases with so-far unknown morphological manifestations., Competing Interests: The authors declare no competing interests., (© 2021 The Authors.)

Recently, the twist angle between adjacent sheets of stacked van der Waals materials emerged as a new knob to engineer correlated states of matter in two-dimensional heterostructures in a controlled manner, giving rise to emergent phenomena such as superconductivity or correlated insulating states. Here, we use an ab initio based approach to characterize the electronic properties of twisted bilayer MoS 2 . We report that, in marked contrast to twisted bilayer graphene, slightly hole-doped MoS 2 realizes a strongly asymmetric p x -p y Hubbard model on the honeycomb lattice, with two almost entirely dispersionless bands emerging due to destructive interference. The origin of these dispersionless bands, is similar to that of the flat bands in the prototypical Lieb or Kagome lattices and co-exists with the general band flattening at small twist angle due to the moiré interference. We study the collective behavior of twisted bilayer MoS 2 in the presence of interactions, and characterize an array of different magnetic and orbitally-ordered correlated phases, which may be susceptible to quantum fluctuations giving rise to exotic, purely quantum, states of matter., (© 2021. The Author(s).)

Twisting two adjacent layers of van der Waals materials with respect to each other can lead to flat two-dimensional electronic bands which enables a wealth of physical phenomena. Here, we generalize this concept of so-called moiré flat bands to engineer flat bands in all three spatial dimensions controlled by the twist angle. The basic concept is to stack the material such that the large spatial moiré interference patterns are spatially shifted from one twisted layer to the next. We exemplify the general concept by considering graphitic systems, boron nitride, and WSe 2 , but the approach is applicable to any two-dimensional van der Waals material. For hexagonal boron nitride, we develop an ab initio fitted tight binding model that captures the corresponding three-dimensional low-energy electronic structure. We outline that interesting three-dimensional correlated phases of matter can be induced and controlled following this route, including quantum magnets and unconventional superconducting states.

Intrahepatic cholangiocarcinoma (iCCA) has emerged as a promising candidate for precision medicine, especially in the case of activating FGFR2 gene fusions. In addition to fusions, a considerable fraction of iCCA patients reveals FGFR2 mutations, which might lead to uncontrolled activation of the FGFR2 pathway but are mostly of unknown functional significance. A current challenge for molecular tumor boards (MTB) is to predict the functional consequences of such FGFR2 alterations to guide potential treatment decisions. We report two iCCA patients with extracellular and juxtamembrane FGFR2 mutations. After in silico investigation of the alterations and identification of activated FGFR2 downstream targets in tumor specimens by immunohistochemistry and transcriptome analysis, the MTB recommended treatment with an FGFR-inhibiting tyrosine kinase inhibitor. Both patients developed a rapidly detectable and prolonged partial response to treatment. These two cases suggest an approach to characterize further detected FGFR2 mutations in iCCA to enable patients´ selection for a successful application of the FGFR -inhibiting drugs., (© 2021. The Author(s).)

Immune profiling of COVID-19 patients has identified numerous alterations in both innate and adaptive immunity. However, whether those changes are specific to SARS-CoV-2 or driven by a general inflammatory response shared across severely ill pneumonia patients remains unknown. Here, we compared the immune profile of severe COVID-19 with non-SARS-CoV-2 pneumonia ICU patients using longitudinal, high-dimensional single-cell spectral cytometry and algorithm-guided analysis. COVID-19 and non-SARS-CoV-2 pneumonia both showed increased emergency myelopoiesis and displayed features of adaptive immune paralysis. However, pathological immune signatures suggestive of T cell exhaustion were exclusive to COVID-19. The integration of single-cell profiling with a predicted binding capacity of SARS-CoV-2 peptides to the patients' HLA profile further linked the COVID-19 immunopathology to impaired virus recognition. Toward clinical translation, circulating NKT cell frequency was identified as a predictive biomarker for patient outcome. Our comparative immune map serves to delineate treatment strategies to interfere with the immunopathologic cascade exclusive to severe COVID-19., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Women living with HIV (WLWH) experience high rates of anal cancer. Screening using anal cytology, high-resolution anoscopy (HRA) with biopsies, can histologically diagnose anal cancer precursors called high-grade squamous intraepithelial lesions (HSIL). The low specificity of screening using anal cytology results in HRA referral for many WLWH without HSIL. Screening using high-risk human papillomavirus (HR-HPV) may improve specificity., Methods: Two hundred seven WLWH (63% non-Hispanic black) were screened for anal histologic HSIL (hHSIL) using cytology, HRA-guided biopsies, and Xpert HPV. Xpert performance for predicting anal hHSIL was compared with that of cytology. Usng Xpert 5 HPV genotypic results and accompanying cycle thresholds, receiver operator characteristic curve and recursive partitioning analyses were used to create predictive models for hHSIL., Results: The performance of Xpert to predict hHSIL was not different from that of cytology with a sensitivity (Sn) of 89% and specificity (Sp) of 49%. Interpretation of Xpert was modified using genotypic results and receiver operator characteristic curve analysis, which produced a screen with an Sn and Sp of 75% and 84% for hHSIL, respectively. Another reinterpretation of Xpert was created using recursive partitioning and cycle thresholds, which predicted hHSIL with an Sn and Sp of 75% and 86%, respectively. The detection of HPV-16 was highly predictive of hHSIL in all analyses. These modified screening tests would reduce HRA referral in this population by almost half compared with anal cytology., Conclusions: Xpert HPV is an alternative to anal cytology to screen for anal HSIL and can be optimized to reduce the number of unnecessary HRAs performed in WLWH., Competing Interests: G.B.E. was supported by T32 AI007613 (Division of Infectious Weill Cornell Medicine, R. Gulick) and UL1 TR002384 (Weill Cornell Medicine Clinical and Translational Science Center). Funding for this work came from UM1 CA121947 (AIDS Malignancy Consortium, R. Mitsuyasu) and R01 CA163103 (E.Y.C.). N.J. has received honoraria from Antiva and is on Merck and Coadvisory board. R.D.C. has received personal fees from UpToDate, outside the submitted work. S.E.G. has received personal fees from Merck and Co, grants from Antiva and Inovio, and other support from THD America. T.D. has received nonfinancial support from Hologic and personal fees from Roche, BD, Antiva, and TheVax. M.H.E. has advised or participated in educational speaking activities but does not receive honoraria from any companies; his employers have received payment for his time spent for these activities from Papivax, Cynvec, Altum Pharma, Photocure, Becton Dickinson, and PDS Biotechnologies. Rutgers has received grant funding for research-related costs of clinical trials on which M.H.E. has been the overall or local principal investigator from Johnson & Johnson, Pfizer, AstraZeneca, Advaxis, and Inovio; he has also received other support from Photocure, Papivax, Cynvec, PDS, Altum Pharma, and Becton Dickinson, outside the submitted work. E.A.S. has received nonfinancial support from Qiagen and Hologic. J.M.B.-L. has received personal fees from Antiva. J.M.P. has received grants and nonfinancial support from Merck and Co; grants, personal fees, and other support from Vir Biotechnologies, Ubiome, and Antiva Biosciences; personal fees from Janssen Pharmaceuticals, Novan, and Vaccitech; and nonfinancial support from Virion Therapeutics. T.J.W. has received grants and personal fees from GlaxoSmithKline/ViiV Healthcare, outside the submitted work. The remaining authors have no conflicts of interest to disclose., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Clustering high-dimensional data, such as images or biological measurements, is a long-standing problem and has been studied extensively. Recently, Deep Clustering has gained popularity due to its flexibility in fitting the specific peculiarities of complex data. Here we introduce the Mixture-of-Experts Similarity Variational Autoencoder (MoE-Sim-VAE), a novel generative clustering model. The model can learn multi-modal distributions of high-dimensional data and use these to generate realistic data with high efficacy and efficiency. MoE-Sim-VAE is based on a Variational Autoencoder (VAE), where the decoder consists of a Mixture-of-Experts (MoE) architecture. This specific architecture allows for various modes of the data to be automatically learned by means of the experts. Additionally, we encourage the lower dimensional latent representation of our model to follow a Gaussian mixture distribution and to accurately represent the similarities between the data points. We assess the performance of our model on the MNIST benchmark data set and challenging real-world tasks of clustering mouse organs from single-cell RNA-sequencing measurements and defining cell subpopulations from mass cytometry (CyTOF) measurements on hundreds of different datasets. MoE-Sim-VAE exhibits superior clustering performance on all these tasks in comparison to the baselines as well as competitor methods., Competing Interests: The authors have declared that no competing interests exist.