High resolution imaging of human development: shedding light on contrast agents.

BACKGROUND: Visualizing (micro)vascular structures remains challenging for researchers and clinicians due to limitations in traditional radiological imaging methods. Exploring the role of vascular development in craniofacial malformations in experimental settings can enhance understanding of these processes, with the effectiveness of high-resolution imaging techniques being crucial for successful research in this field. Micro-CT imaging offers 3D microstructural insights, but requires contrast-enhancing staining agents (CESAs) for visualizing (micro)-vascular tissues, known as contrast-enhanced micro-CT (CECT). As effective contrast agents are crucial for optimal visualization, this review focuses on comparative studies investigating such agents for micro-vascular tissue imaging using micro-CT. Furthermore, we demonstrate the utilization of B-Lugol solution as a promising contrast agent for acquiring high-quality micro-CT images of (micro)vascular structures in human embryonic samples. METHOD: This scoping review followed Preferred Reporting Items for Systematic Reviews and Meta-analysis Protocols. PubMed database provided relevant articles, screened initially by title and abstract. Inclusion and exclusion criteria defined outcomes of interest. RESULTS: From an initial search, 273 records were identified, narrowed down to 9 articles after applying our criteria. Additionally, two articles were added through citation searching. This, a total of 11 articles were incorporated in this study. CONCLUSION: This micro-CT contrast agent review underscores the need for tailored choices based on research goals. Both Barium sulfate and Iodine-based agents showing excellent results, providing high resolution (micro) vascular content, especially in ex-vivo specimens. However, careful consideration of protocols and tissue characteristics remains imperative for optimizing the effectiveness of micro-CT imaging for the study of cranio-facial vascular development.

Venetoclax resistance in acute lymphoblastic leukemia is characterized by increased mitochondrial activity and can be overcome by co-targeting oxidative phosphorylation.

Deregulated apoptosis signaling is characteristic for many cancers and contributes to leukemogenesis and treatment failure in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Apoptosis is controlled by different pro- and anti-apoptotic molecules. Inhibition of anti-apoptotic molecules like B-cell lymphoma 2 (BCL-2) has been developed as therapeutic strategy. Venetoclax (VEN), a selective BCL-2 inhibitor has shown clinical activity in different lymphoid malignancies and is currently evaluated in first clinical trials in BCP-ALL. However, insensitivity to VEN has been described constituting a major clinical concern. Here, we addressed and modeled VEN-resistance in BCP-ALL, investigated the underlying mechanisms in cell lines and patient-derived xenograft (PDX) samples and identified potential strategies to overcome VEN-insensitivity. Leukemia lines with VEN-specific resistance were generated in vitro and further characterized using RNA-seq analysis. Interestingly, gene sets annotated to the citric/tricarboxylic acid cycle and the respiratory electron transport chain were significantly enriched and upregulated, indicating increased mitochondrial metabolism in VEN-resistant ALL. Metabolic profiling showed sustained high mitochondrial metabolism in VEN-resistant lines as compared to control lines. Accordingly, primary PDX-ALL samples with intrinsic VEN-insensitivity showed higher oxygen consumption and ATP production rates, further highlighting that increased mitochondrial activity is a characteristic feature of VEN-resistant ALL. VEN-resistant PDX-ALL showed significant higher mitochondrial DNA content and differed in mitochondria morphology with significantly larger and elongated structures, further corroborating our finding of augmented mitochondrial metabolism upon VEN-resistance. Using Oligomycin, an inhibitor of the complex V/ATPase subunit, we found synergistic activity and apoptosis induction in VEN-resistant BCP-ALL cell lines and PDX samples, demonstrating that acquired and intrinsic VEN-insensitivity can be overcome by co-targeting BCL-2 and the OxPhos pathway. These findings of reprogrammed, high mitochondrial metabolism in VEN-resistance and synergistic activity upon co-targeting BCL-2 and oxidative phosphorylation strongly suggest further preclinical and potential clinical evaluation in VEN-resistant BCP-ALL.

Highly confined epsilon-near-zero and surface phonon polaritons in SrTiO3 membranes.

Recent theoretical studies have suggested that transition metal perovskite oxide membranes can enable surface phonon polaritons in the infrared range with low loss and much stronger subwavelength confinement than bulk crystals. Such modes, however, have not been experimentally observed so far. Here, using a combination of far-field Fourier-transform infrared (FTIR) spectroscopy and near-field synchrotron infrared nanospectroscopy (SINS) imaging, we study the phonon polaritons in a 100 nm thick freestanding crystalline membrane of SrTiO3 transferred on metallic and dielectric substrates. We observe a symmetric-antisymmetric mode splitting giving rise to epsilon-near-zero and Berreman modes as well as highly confined (by a factor of 10) propagating phonon polaritons, both of which result from the deep-subwavelength thickness of the membranes. Theoretical modeling based on the analytical finite-dipole model and numerical finite-difference methods fully corroborate the experimental results. Our work reveals the potential of oxide membranes as a promising platform for infrared photonics and polaritonics.

The evolution of exceptional diversity in parental care and fertilization modes in ray-finned fishes.

Among vertebrates, ray-finned fishes (Actinopterygii) display the highest diversity in parental care, and their diversification has been hypothesized to be related to phylogenetic changes in fertilization modes. Using the most comprehensive, sex-specific data from 7600 species of 62 extant orders of ray-finned fishes, we inferred ancestral states and transitions among care types and caring episodes (i.e. the stage of offspring development). Our work has uncovered three novel findings. First, transitions among different care types (i.e. male-only care, female-only care, biparental care and no care) are common, and the frequencies of these transitions show unusually diverse patterns concerning fertilization modes (external, or internal via oviduct, mouth or brood pouch). Second, both oviduct and mouth fertilization select for female-biased care, whereas fertilization in a brood pouch selects for male-biased care. Importantly, internal fertilization without parental care is extremely unstable phylogenetically. Third, we show that egg care in both sexes is associated with nest building (which is male-biased) and fry care (which is female-biased). Taken together, the aquatic environment, which supports considerable flexibility in care, facilitated the diversification of parenting behavior, creating the evolutionary bases for more comprehensive parenting to protect offspring in semiterrestrial or terrestrial environments.

Prediction of resistance to bevacizumab plus FOLFOX in metastatic colorectal cancer-Results of the prospective multicenter PERMAD trial.

BACKGROUND: Anti-vascular endothelial growth factor (VEGF) monoclonal antibodies (mAbs) are widely used for tumor treatment, including metastatic colorectal cancer (mCRC). So far, there are no biomarkers that reliably predict resistance to anti-VEGF mAbs like bevacizumab. A biomarker-guided strategy for early and accurate assessment of resistance could avoid the use of non-effective treatment and improve patient outcomes. We hypothesized that repeated analysis of multiple cytokines and angiogenic growth factors (CAFs) before and during treatment using machine learning could provide an accurate and earlier, i.e., 100 days before conventional radiologic staging, prediction of resistance to first-line mCRC treatment with FOLFOX plus bevacizumab. PATIENTS AND METHODS: 15 German and Austrian centers prospectively recruited 50 mCRC patients receiving FOLFOX plus bevacizumab as first-line treatment. Plasma samples were collected every two weeks until radiologic progression (RECIST 1.1) as determined by CT scans performed every 2 months. 102 pre-selected CAFs were centrally analyzed using a cytokine multiplex assay (Luminex, Myriad RBM). RESULTS: Using random forests, we developed a predictive machine learning model that discriminated between the situations of "no progress within 100 days before radiological progress" and "progress within 100 days before radiological progress". We could further identify a combination of ten out of the 102 CAF markers, which fulfilled this task with 78.2% accuracy, 71.8% sensitivity, and 82.5% specificity. CONCLUSIONS: We identified a CAF marker combination that indicates treatment resistance to FOLFOX plus bevacizumab in patients with mCRC within 100 days prior to radiologic progress.

Extinction training suppresses activity of fear memory ensembles across the hippocampus and alters transcriptomes of fear-encoding cells.

Contextual fear conditioning has been shown to activate a set of "fear ensemble" cells in the hippocampal dentate gyrus (DG) whose reactivation is necessary and sufficient for expression of contextual fear. We previously demonstrated that extinction learning suppresses reactivation of these fear ensemble cells and activates a competing set of DG cells-the "extinction ensemble." Here, we tested whether extinction was sufficient to suppress reactivation in other regions and used single nucleus RNA sequencing (snRNA-seq) of cells in the dorsal dentate gyrus to examine how extinction affects the transcriptomic activity of fear ensemble and fear recall-activated cells. Our results confirm the suppressive effects of extinction in the dorsal and ventral dentate gyrus and demonstrate that this same effect extends to fear ensemble cells located in the dorsal CA1. Interestingly, the extinction-induced suppression of fear ensemble activity was not detected in ventral CA1. Our snRNA-seq analysis demonstrates that extinction training markedly changes transcription patterns in fear ensemble cells and that cells activated during recall of fear and recall of extinction have distinct transcriptomic profiles. Together, our results indicate that extinction training suppresses a broad portion of the fear ensemble in the hippocampus, and this suppression is accompanied by changes in the transcriptomes of fear ensemble cells and the emergence of a transcriptionally unique extinction ensemble.

The role of length of oral resection margin and survival in esophageal cancer surgery after neoadjuvant therapy: A retrospective propensity score-matched study.

BACKGROUND: Multimodal therapy regimens became the standard of care for patients with esophageal cancer, whereas surgical resection remains at the center of curative treatment modalities. Current guidelines provide no recommendations on the extent of the oral resection margin, especially in the era of neoadjuvant therapy. Therefore, this study aimed to evaluate the relationship between the oral tumor-free resection margin and overall survival. METHODS: Retrospective study with 382 1:1 propensity-matched patients out of 660 patients, operated between 2013 and 2019, with an Ivor-Lewis-esophagectomy for adenocarcinoma and squamous cell carcinoma of the esophagus or esophagogastric junction after neoadjuvant therapy. Independent pathologists measured the oral resection margin after formalin fixation. RESULTS: The mean oral tumor-free resection margin was 37.2 ± 0.6 mm. The ideal cut-off for survival differences was determined for 33 mm. Patients with an oral resection margin of more than 33 mm had a better median overall survival (≤33 mm: 45.0 months, 95% confidence interval: 22.4-67.6 months, >33 mm: not reached, P = .005). An oral resection margin of more than 33 mm proved to be an independent favorable prognostic factor for patients' overall survival in multivariate Cox regression analyses (P = .049). CONCLUSION: This study analyzed a patient cohort retrospectively after curative intended Ivor-Lewis-esophagectomy after neoadjuvant therapy. An oral resection margin of more than 33 mm is a factor for improved overall survival. Therefore, a minimum resection margin of 34 mm after fixation could be suggested.

Surgical and multimodal treatment of metastatic oesophageal cancer: retrospective cohort study.

BACKGROUND: In contrast to the well-established multimodal therapy for localized oesophageal cancer, the metastatic stage is commonly treated only with systemic therapy as current international guidelines recommend. However, evidence suggesting that multimodal therapy including surgery could benefit selected patients with metastasized oesophageal cancer is increasing. The aim of this study was to investigate the survival of patients diagnosed with metastatic oesophageal cancer after different treatment regimens. METHODS: This was a retrospective single-centre study of patients with adenocarcinoma or squamous cell carcinoma of the oesophagus with synchronous or metachronous metastases who underwent Ivor Lewis oesophagectomy between 2010 and 2021. Each patient received an individual treatment for their metastatic burden based on an interdisciplinary tumour board conference. Survival differences between different treatments were assessed using the Kaplan-Meier method, as well as univariable and multivariable Cox regression models. RESULTS: Out of 1791 patients undergoing Ivor Lewis oesophagectomy, 235 patients diagnosed with metastases were included. Of all of the included patients, 42 (17.9%) only underwent surgical resection of their metastatic disease, 37 (15.7%) underwent multimodal therapy including surgery, 78 (33.2%) received chemotherapy alone, 49 (20.9%) received other therapies, and 29 (12.3%) received best supportive care. Patients who underwent resection or multimodal therapy including surgery of their metastatic burden showed superior overall survival compared with chemotherapy alone (median overall survival of 19.0, 18.0, and 11.0 months respectively) (P < 0.001). This was confirmed in subcohorts of patients with metachronous solid-organ metastases and with a single metastasis. In multivariable analyses, resection with or without multimodal therapy was an independent factor for favourable survival. CONCLUSION: Surgical resection could be a feasible treatment option for metastasized oesophageal cancer, improving survival in selected patients. Further prospective randomized studies are needed to confirm these findings and define reliable selection criteria.

Embracing digital health: German otolaryngology patients' usage and prospects of digital information and communication technologies for cross-sectoral care.

Objective: The usage of digital information and communication technologies in European healthcare is growing. Unlike numerous technological possibilities, the present use of these technologies and perspectives towards them in relation to otolaryngology care have so far been of less interest. This study evaluates the utilisation of and attitudes towards digital information and communication technologies in cross-sectoral otolaryngology care among German patients. Methods: A structured interview-based study was conducted at the outpatient facility of a tertiary hospital in Germany. It focused on chief complaints, current use of digital technologies, estimated benefits of increased digital technology use in otolaryngology care, and sociodemographic data. The detailed statistical analysis employed Chi-squared tests and multivariate logistic regression. Results: A total of 208 otolaryngology patients completed the interview. Digital communication technologies exhibited a high penetration rate (91.8%) and were regularly used in daily life (78.7%) and for health reasons (73.3%). Younger age (p ≤ 0.003) and higher education levels (p ≤ 0.008) were significantly correlated with the increased digital communication technology use. The overall potential of eHealth technologies was rated significantly higher by younger patients (p ≤ 0.001). The patients' chief complaints showed no significant influence on the current and potential use of these technologies for cross-sectoral otolaryngology care. Conclusion: Regardless of their chief complaints, German otolaryngology patients regularly use digital information and communication technologies for health reasons and express interest in their further use for cross-sectoral care. To enhance digital patient communication in otolaryngology, attention should be given to treatment quality, usability, data security and availability and financial remuneration for service providers.

Repeat Radiosurgery for Sporadic Vestibular Schwannoma After Primary Radiosurgical Failure: An International Multi-institutional Investigation.

OBJECTIVE: To describe outcomes of patients with sporadic vestibular schwannoma (VS) who underwent repeat stereotactic radiosurgery (SRS) after primary SRS failure. STUDY DESIGN: Multi-institutional historical cohort study. SETTING: Five tertiary care referral centers. PATIENTS: Adults ≥18 years old with sporadic VS. INTERVENTION: Primary and repeat treatment with SRS. MAIN OUTCOME MEASURE: Microsurgery-free survival after repeat SRS. RESULTS: Across institutions, 32 patients underwent repeat SRS after primary SRS. Most patients (74%) had tumors with cerebellopontine angle extension at primary SRS (median size, 13.5 mm [interquartile range, 7.5-18.8] mm). After primary SRS, patients underwent repeat SRS at a median of 4.8 years (interquartile range, 3.2-5.7 yr). For treatment modality, 30 (94%) patients received gamma knife for primary treatment and 31 (97%) patients received gamma knife as their repeat treatment. Median tumor volume increased from 0.970 cm3 at primary SRS to 2.200 cm3 at repeat SRS. Facial nerve function worsened in two patients after primary SRS and in two patients after repeat SRS. There were no instances of intracranial complications after repeat SRS. Microsurgery-free survival rates (95% confidence interval; number still at risk) at 1, 3, and 5 years after repeat SRS were 97% (90-100%, 24), 84% (71-100%, 13), and 68% (48-96%, 6), respectively. There was one occurrence of malignancy diagnosed after repeat radiosurgery. CONCLUSION: Overall, repeat SRS for sporadic VS has comparable risk profile, but lower rates of tumor control, compared with primary SRS.

Transition-state theory reexamined.

Two quantum systems, each described as a random-matrix ensemble, are coupled to each other via a number of transition states. Each system is strongly coupled to a large number of channels. The average transmission probability is the product of three factors describing, respectively, formation of the first system from the entrance channel, decay of the second system through the exit channel, and transport through the transition states. Each of the transition states contributes a Breit-Wigner resonance. In general, the resonances overlap.

Segmentation-based cardiomegaly detection based on semi-supervised estimation of cardiothoracic ratio.

The successful integration of neural networks in a clinical setting is still uncommon despite major successes achieved by artificial intelligence in other domains. This is mainly due to the black box characteristic of most optimized models and the undetermined generalization ability of the trained architectures. The current work tackles both issues in the radiology domain by focusing on developing an effective and interpretable cardiomegaly detection architecture based on segmentation models. The architecture consists of two distinct neural networks performing the segmentation of both cardiac and thoracic areas of a radiograph. The respective segmentation outputs are subsequently used to estimate the cardiothoracic ratio, and the corresponding radiograph is classified as a case of cardiomegaly based on a given threshold. Due to the scarcity of pixel-level labeled chest radiographs, both segmentation models are optimized in a semi-supervised manner. This results in a significant reduction in the costs of manual annotation. The resulting segmentation outputs significantly improve the interpretability of the architecture's final classification results. The generalization ability of the architecture is assessed in a cross-domain setting. The assessment shows the effectiveness of the semi-supervised optimization of the segmentation models and the robustness of the ensuing classification architecture.

Treatment of local recurrence of esophageal cancer following Ivor-Lewis esophagectomy-Experiences of a high-volume center.

PURPOSE: Patients with local recurrence of esophageal cancer have a highly decreased overall survival. There is currently no standardized treatment algorithm for this group. This retrospective cohort study aimed to evaluate the survival of patients with local recurrence, despite receiving individualized treatment options. METHODS: 241 of 1791 patients were diagnosed with a local recurrence following Ivor-Lewis esophagectomy at the University Hospital of Cologne. 59 patients, who were diagnosed only with a local recurrence of adeno- or squamous cell carcinoma and received their individualized therapy regimes at our high-volume center, were included. RESULTS: The study included 52 patients with adenocarcinoma and 7 with squamous cell carcinoma. Among these, 6 patients underwent resection, 19 received solely chemotherapy, 29 received chemoradiotherapy, and 5 were provided with best supportive care. Patients who underwent resection showed a better survival outcome compared to patients without resection (median OS: not reached vs. 15.1 months, p = 0.012). Best supportive care and palliative care were found to be independent risk factors for shorter overall survival compared to curative intended treatment options like local resection or chemoradiotherapy. CONCLUSION: In this study, different treatment strategies for patients with local recurrence of esophageal cancer were depicted. Resection as well as chemoradiotherapy could play a role in selected patients. Further prospective studies are needed to improve the selection of eligible patients.

Myriad Mapping of nanoscale minerals reveals calcium carbonate hemihydrate in forming nacre and coral biominerals.

Calcium carbonate (CaCO3) is abundant on Earth, is a major component of marine biominerals and thus of sedimentary and metamorphic rocks and it plays a major role in the global carbon cycle by storing atmospheric CO2 into solid biominerals. Six crystalline polymorphs of CaCO3 are known-3 anhydrous: calcite, aragonite, vaterite, and 3 hydrated: ikaite (CaCO3·6H2O), monohydrocalcite (CaCO3·1H2O, MHC), and calcium carbonate hemihydrate (CaCO3·½H2O, CCHH). CCHH was recently discovered and characterized, but exclusively as a synthetic material, not as a naturally occurring mineral. Here, analyzing 200 million spectra with Myriad Mapping (MM) of nanoscale mineral phases, we find CCHH and MHC, along with amorphous precursors, on freshly deposited coral skeleton and nacre surfaces, but not on sea urchin spines. Thus, biomineralization pathways are more complex and diverse than previously understood, opening new questions on isotopes and climate. Crystalline precursors are more accessible than amorphous ones to other spectroscopies and diffraction, in natural and bio-inspired materials.

Salvage Microsurgery Following Failed Primary Radiosurgery in Sporadic Vestibular Schwannoma.

Importance: Management of sporadic vestibular schwannoma with radiosurgery is becoming increasingly common globally; however, limited data currently characterize patient outcomes in the setting of microsurgical salvage for radiosurgical failure. Objective: To describe the clinical outcomes of salvage microsurgery following failed primary stereotactic radiosurgery (SRS) or fractionated stereotactic radiotherapy (FSRT) among patients with sporadic vestibular schwannoma. Design, Setting, and Participants: This was a cohort study of adults (≥18 years old) with sporadic vestibular schwannoma who underwent salvage microsurgery following failed primary SRS/FSRT in 7 vestibular schwannoma treatment centers across the US and Norway. Data collection was performed between July 2022 and January 2023, with data analysis performed between January and July 2023. Exposure: Salvage microsurgical tumor resection. Main Outcomes and Measures: Composite outcome of undergoing less than gross total resection (GTR) or experiencing long-term facial paresis. Results: Among 126 patients, the median (IQR) age at time of salvage microsurgery was 62 (53-70) years, 69 (55%) were female, and 113 of 117 (97%) had tumors that extended into the cerebellopontine angle at time of salvage. Of 125 patients, 96 (76%) underwent primary gamma knife SRS, while 24 (19%) underwent linear accelerator-based SRS; the remaining patients underwent FSRT using other modalities. Postoperative cerebrospinal fluid leak was seen in 15 of 126 patients (12%), hydrocephalus in 8 (6%), symptomatic stroke in 7 (6%), and meningitis in 2 (2%). Each 1-mm increase in cerebellopontine angle tumor size was associated with a 13% increased likelihood of foregoing GTR (64 of 102 patients [63%]) or long-term postoperative House-Brackmann grade higher than I (48 of 102 patients [47%]) (odds ratio, 1.13; 95% CI, 1.04-1.23). Following salvage microsurgery, tumor growth-free survival rates at 1, 3, and 5 years were 97% (95% CI, 94%-100%), 93% (95% CI, 87%-99%), and 91% (95% CI, 84%-98%), respectively. Conclusions: In this cohort study, more than half of patients who received salvage microsurgery following primary SRS/FSRT underwent less than GTR or experienced some degree of facial paresis long term. These data suggest that the cumulative risk of developing facial paresis following primary SRS/FSRT by the end of the patient's journey with treatment approximates 2.5% to 7.5% when using published primary SRS/FSRT long-term tumor control rates.

INHBA is Enriched in HPV-negative Oropharyngeal Squamous Cell Carcinoma and Promotes Cancer Progression.

Patients with oropharyngeal squamous cell carcinoma (OPSCC) caused by human papilloma virus (HPV) exhibit a better prognosis than those with HPV-negative OPSCC. This study investigated the distinct molecular pathways that delineate HPV-negative from HPV-positive OPSCC to identify biologically relevant therapeutic targets. Bulk mRNA from 23 HPV-negative and 39 HPV-positive OPSCC tumors (n = 62) was sequenced to uncover the transcriptomic profiles. Differential expression followed by gene set enrichment analysis was performed to outline the top enriched biological process in the HPV-negative compared with HPV-positive entity. INHBA, the highest overexpressed gene in the HPV-negative tumor, was knocked down. Functional assays (migration, proliferation, cell death, stemness) were conducted to confirm the target's oncogenic role. Correlation analyses to reveal its impact on the tumor microenvironment were performed. We revealed that epithelial-to-mesenchymal transition (EMT) is the most enriched process in HPV-negative compared with HPV-positive OPSCC, with INHBA (inhibin beta A subunit) being the top upregulated gene. INHBA knockdown downregulated the expression of EMT transcription factors and attenuated migration, proliferation, stemness, and cell death resistance of OPSCC cells. We uncovered that INHBA associates with a pro-tumor microenvironment by negatively correlating with antitumor CD8+ T and B cells while positively correlating with pro-tumor M1 macrophages. We identified three miRNAs that are putatively involved in repressing INHBA expression. Our results indicate that the upregulation of INHBA is tumor-promoting. We propose INHBA as an attractive therapeutic target for the treatment of INHBA-enriched tumors in patients with HPV-negative OPSCC to ameliorate prognosis. SIGNIFICANCE: Patients with HPV-negative OPSCC have a poorer prognosis due to distinct molecular pathways. This study reveals significant transcriptomic differences between HPV-negative and HPV-positive OPSCC, identifying INHBA as a key upregulated gene in HPV-negative OPSCC's oncogenic pathways. INHBA is crucial in promoting EMT, cell proliferation, and an immunosuppressive tumor environment, suggesting its potential as a therapeutic target for HPV-negative OPSCC.

Saccharomyces cerevisiae CellWall Remodeling in the Absence of Knr4 and Kre6 Revealed by Nano-FourierTransform Infrared Spectroscopy.

The cell wall integrity (CWI) signaling pathway regulates yeast cell wall biosynthesis, cell division, and responses to external stress. The cell wall, comprised of a dense network of chitin, ß-1,3- and ß-1,6- glucans, and mannoproteins, is very thin, <100 nm. Alterations in cell wall composition may activate the CWI pathway. Saccharomyces cerevisiae, a model yeast, was used to study the role of individual wall components in altering the structure and biophysical properties of the yeast cell wall. Near-field Fourier transform infrared spectroscopy (nano-FT-IR) was used for the first direct, spectrochemical identification of cell wall composition in a background (wild-type) strain and two deletion mutants from the yeast knock-out collection: kre6Δ and knr4Δ. Killer toxin resistant 6 (Kre6) is an integral membrane protein required for biosynthesis of ß-1,6-glucan, while Knr4 is a cell signaling protein involved in the control of cell wall biosynthesis, in particular, biosynthesis and deposition of chitin. Complementary spectral data were obtained with far-field (FF)-FT-IR, in transmission, and with attenuated total reflectance (ATR) spectromicroscopy with 3-10 µm wavelength-dependent spatial resolution. The FF-FT-IR spectra of cells and spectra of isolated cell wall components showed that components of the cell body dominated transmission spectra and were still evident in ATR spectra. In contrast, the nano-FT-IR at ∼25 nm spatial resolution could be used to characterize the yeast wall chemical structure. Our results show that the ß-1,6-glucan content is decreased in kre6Δ, while all glucan content is decreased in the knr4Δ cell wall. The latter may be thinner than in wild type, since not only are mannan and chitin detectable by nano-FT-IR, but also lipid membranes and protein, indicative of cell interior.

Hybrid immunity to SARS-CoV-2 in patients with chronic lymphocytic leukemia.

OBJECTIVE: Preventing severe COVID-19 remains a priority globally, particularly in the immunocompromised population. As shown in healthy individuals, immunity against SARS-CoV-2 can be yielded by previous infection, vaccination, or both (hybrid immunity). The objective of this observation study was to investigate hybrid immunity in patients with chronic lymphocytic leukemia (CLL). METHODS/RESULTS: Blood samples of six patients with CLL were collected 55 days after fourth COVID-19 vaccination. All patients had a SARS-CoV-2 infection within 12 months before the second booster (fourth vaccination). SARS-CoV-2 spike receptor binding domain (RBD)-specific IgG antibodies were detectable in 6/6 (100.0%) CLL patients after four compared to 4/6 (66.7%) after three vaccinations. The median number of SARS-CoV-2 spike-specific T cells after repeated booster vaccination plus infection was 166 spot-forming cells (SFC) per million peripheral blood mononuclear cells. Overall, 5/5 (100%) studied patients showed a detectable increase in T cell activity. CONCLUSION: Our data reveal an increase of cellular and humoral immune response in CLL patients after fourth COVID-19 vaccination combined with SARS-CoV-2 infection, even in those undergoing B cell-depleting treatment. Patients with prior vaccination failure now show a specific IgG response. Future research should explore the duration and effectiveness of hybrid immunity considering various factors like past infection and vaccination rates, types and numbers of doses, and emerging variants.

Iterative design of training data to control intricate enzymatic reaction networks.

Kinetic modeling of in vitro enzymatic reaction networks is vital to understand and control the complex behaviors emerging from the nonlinear interactions inside. However, modeling is severely hampered by the lack of training data. Here, we introduce a methodology that combines an active learning-like approach and flow chemistry to efficiently create optimized datasets for a highly interconnected enzymatic reactions network with multiple sub-pathways. The optimal experimental design (OED) algorithm designs a sequence of out-of-equilibrium perturbations to maximize the information about the reaction kinetics, yielding a descriptive model that allows control of the output of the network towards any cost function. We experimentally validate the model by forcing the network to produce different product ratios while maintaining a minimum level of overall conversion efficiency. Our workflow scales with the complexity of the system and enables the optimization of previously unobtainable network outputs.

Extinction Training Suppresses Activity of Fear Memory Ensembles Across the Hippocampus and Alters Transcriptomes of Fear-Encoding Cells.

Contextual fear conditioning has been shown to activate a set of "fear ensemble" cells in the hippocampal dentate gyrus (DG) whose reactivation is necessary and sufficient for expression of contextual fear. We previously demonstrated that extinction learning suppresses reactivation of these fear ensemble cells and activates a competing set of DG cells - the "extinction ensemble." Here, we tested whether extinction was sufficient to suppress reactivation in other regions and used single nucleus RNA sequencing (snRNA-seq) of cells in the dorsal dentate gyrus to examine how extinction affects the transcriptomic activity of fear ensemble and fear recall-activated cells. Our results confirm the suppressive effects of extinction in the dorsal and ventral dentate gyrus and demonstrate that this same effect extends to fear ensemble cells located in the dorsal CA1. Interestingly, the extinction-induced suppression of fear ensemble activity was not detected in ventral CA1. Our snRNA-seq analysis demonstrates that extinction training markedly changes transcription patterns in fear ensemble cells and that cells activated during recall of fear and recall of extinction have distinct transcriptomic profiles. Together, our results indicate that extinction training suppresses a broad portion of the fear ensemble in the hippocampus, and this suppression is accompanied by changes in the transcriptomes of fear ensemble cells and the emergence of a transcriptionally unique extinction ensemble.