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Spatial transcriptomics expression prediction from histopathology based on cross-modal mask reconstruction and contrastive learning
No full textSpatial transcriptomics is a technology that captures gene expression at different spatial locations, widely used in tumor microenvironment analysis and molecular profiling of histopathology, providing valuable insights into resolving gene expression and clinical diagnosis of cancer. Due to the high cost of data acquisition, large-scale spatial transcriptomics data remain challenging to obtain. In this study, we develop a contrastive learning-based deep learning method to predict spatially resolved gene expression from the whole-slide images (WSIs). Unlike existing end-to-end prediction frameworks, our method leverages multi-modal contrastive learning to establish a correspondence between histopathological morphology and spatial gene expression in the feature space. By computing cross-modal feature similarity, our method generates spatially resolved gene expression directly from WSIs. Furthermore, to enhance the standard contrastive learning paradigm, a cross-modal masked reconstruction is designed as a pretext task, enabling feature-level fusion between modalities. Notably, our method does not rely on large-scale pretraining datasets or abstract semantic representations from either modality, making it particularly effective for scenarios with limited spatial transcriptomics data. Evaluation across six different disease datasets demonstrates that, compared to existing studies, our method improves Pearson Correlation Coefficient (PCC) in the prediction of highly expressed genes, highly variable genes, and marker genes by 6.27 %, 6.11 %, and 11.26 % respectively. Further analysis indicates that our method preserves gene-gene correlations and applies to datasets with limited samples. Additionally, our method exhibits potential in cancer tissue localization based on biomarker expression. The code repository for this work is available at https://github.com/ngfufdrdh/CMRCNet
How accurate is probabilistic tractography when used to predict the “sweet spot” in deep brain stimulation? Mind the gap!
No full textBackground
Tractography has been used in various studies with respect to the improvement of patient-specific DBS targeting. Nevertheless, methodological influences of the chosen parameters and associated errors are often neglected. The aim of this study was to estimate concrete errors associated with specific image processing steps when using measurements of distances to specific subcortical fiber tracts to predict optimal stimulation sites for DBS targeting.
Method
Probabilistic tractography of the crossing and non-decussating part of the dentato-rubro-thalamic-tract (c-/ nd-DRTT) was performed using FSL 6.0.3 in 40 PD- and ET-patients having received bilateral DBS surgery. DBS-electrodes were reconstructed using LeadDBS. The influence of (1) the choice of threshold for binarization of fiber tracts, (2) manual measurements compared to measurements using automized distance maps and (3) normalization into the MNI standard space on measured distances were investigated.
Results
Different thresholds for binarization resulted in non-linear and unpredictable variations of measured distances up to 1.72 ± 1.49 mm (mean value ± standard deviation). Manual measurements on the axial slice of the electrode contact showed a mean error of 0.91 ± 1.36 mm (maximum 14.9 mm) compared to automated measurements. Regarding normalization, a mean error of 0.82 ± 0.50 mm (maximum 2.34 mm) was found compared to measurements in native space.
Conclusion
Measured maximum errors reach up to several millimeters, which might have significant impact on clinical targeting in DBS. Researchers should be aware of these errors and define individual standards for specific studies
Stabilität oder Sensitivität: Makroökonomische Einflussfaktoren auf Mittelzu- und -abflüsse in deutschen offenen Immobilienfonds
No full textEffect of Ropivacain and Bupivacain on Calcium‐Related and G‐Protein Coupled Processes in PMNs: A Human In‐Vitro Study
No full textBackground and Aims:
We investigated the impact of altered intracellular calcium levels and G-protein-coupled receptor (GPCRs) signaling inhibition on migration and NETosis of polymorphonuclear leukocytes (PMNs) under influence of bupivacaine and ropivacaine.
Methods:
PMNs were isolated from whole blood of healthy volunteers by centrifugation. In vitro µSlide chemotaxis assays were conducted, where PMNs migrated along a formyl-methionyl-leucyl-phenylalanine (fMLP) chemotactic gradient through a type I collagen matrix, tracked over 6 h using fluorescence microscopy. Bupivacaine and ropivacaine were added, along with the calcium chelator BAPTA AM, GPCR inhibitor gallein and phospholipase C (PLC) inhibitor U-73122.
Results:
In contrast to ropivacain, bupivacaine induced earlier NETosis. Both local anesthetics caused an earlier cessation of PMN migration. Chelation of intracellular calcium demonstrated a concentration-dependent effect on migration. The addition of Gallein and U-73122 resulted in earlier NETosis and an increase in maximum intracellular calcium concentration.
Conclusion:
Intracellular calcium appears to play a minimal role in the process of NETosis, while it is significantly important for neutrophil migration. Inhibition of the Gβγ subunit using gallein and PLC using U-73122 led to an earlier onset of NETosis and an increase in the maximum intracellular calcium. An additional effect of ropivacaine on the GPCR signaling pathway was not detectable
Lactic acid promotes an MDSC-like phenotype via HIF1α stabilization with impact on prognosis in renal cell carcinoma
No full textParadoxically, immune cell infiltration correlates with worse prognosis in renal cell carcinoma (RCC) patients, with tumor-associated myeloid cells playing a key role in tumor progression. However, little is known about factors driving their polarization. Here, we investigated the link between RCC-related glycolysis, hypoxia-inducible factor (HIF)1α-associated myeloid inflammation, and patient prognosis.
TCGA data revealed a strong correlation between the expression of monocarboxylate transporter 4 (MCT4), the myeloid marker CD14 and patient survival in ccRCC patients. scRNAseq data confirmed high MCT4 expression in both tumor and myeloid cells, suggesting lactate transport. In vitro analyses proved lactate uptake by CD14+ monocytes, which stabilized HIF1α and induced an MDSC-like, HLA-DR low phenotype. In line, the HIF1α-stabilizing drug Roxadustat increased the number of CD14+ HLA-DR low cells. Lactate uptake also increased protein lactylation.
To further investigate the interplay between RCC tumor and myeloid cells, we established a 3D spheroid co-culture model and analyzed the effects of MCT inhibitors. This 3D model reflected tumor-myeloid cell interactions, as spheroid-infiltrating myeloid cells exhibited spontaneous IL-6 secretion comparable to patient-derived RCC cultures. Inhibition of lactate secretion reduced lactate and IL-6 secretion while increasing CD14+ HLA-DR+ cells. These findings were validated in patient-derived RCC cultures treated with anti-glycolytic drugs.
Our data dissect the intratumoral network of RCC and show that tumor-derived lactate promotes a pro-tumorigenic myeloid phenotype with low MHC-II but high immune-checkpoint, LOX-1 and S100A8/9 expression. Blocking MCT disrupts this interplay, offering a promising strategy to re-educate tumor-associated myeloid cells and enhance tumor immune surveillance
On the non-neutrality of socially responsible investing in the presence of a greenium
No full textThe neutrality of SRI in the AD-GE model by Arnold (2023) ceases to hold once the law of one price is violated for an asset that sufficiently many individuals (a single one may suffice) are not indifferent towards. The introduction of a green bond priced at a premium leads to an illusory gain, that is, a pure utility gain accompanied by a reduction of consumption, of green investors. Their financial losses are allocated to those that were sufficiently un-green to not buy too many green bonds themselves. To profit financially this way, an individual needs to start out as (partial) owner of a firm that “turns out” to be a green bond issuer. Impact investing still does not generate environmental impact in this model
Fertility correlates with queen size and sperm quality in an ant
No full textFitness can vary between individuals of the same population for reasons related to nutrient acquisition during development, but also to the quality of the mating partners. We focus on ant queens of Cardiocondyla obscurior to investigate the effects of female and male morphology and sperm characteristics on productivity. We monitored queens for 12 weeks after egg production started as a proxy for lifetime productivity, and found that larger queens are more productive, as commonly found in other insects, producing more workers and winged males. Sperm viability, but not sperm length, was positively correlated with female-biased sex ratios, pointing to a better insemination rate. The high correlation between sperm viability, male body size, and mandible size suggests strong selection for larger and stronger males. However, male competition for access to unmated queens in the maternal colony may result in a trade-off between male size and developmental time in this species
Characterization of Monomers and Heteromers of the Dopamine and Histamine Receptor Families Using Bioluminescence- and Radioactivity-Based Techniques
No full textIn the last decades various test systems were established to characterize pharmacological tools like radioligands and fluorescent ligands, as well as, evaluating binding affinities and selectivity of novel ligands. Furthermore, they are capable to investigate receptor monomers and heteromers and their functional properties.
In this thesis three different bioluminescent test systems comprising the spilt NanoLuc-based miniG recruitment method, the further downstream CAMYEN BRET-based biosensor and a G-case sensor derived from the TRUPATH system were applied to the five (human) dopamine receptors. Therefore, in case of the miniG recruitment assay the small bit of the split NanoLuc was fused to the C-terminus of the respective (human) dopamine receptor, while the large bit was introduced N-terminally to the canonical miniG protein. For the CAMYEN BRET assay the whole NanoLuc was cloned to the C-terminus of an Epac cAMP binding domain, and mCitrine to the N-terminus. In case of the G-case sensor the whole NanoLuc is cloned into the Gα subunit of a G protein trimer, and the cpVenus protein is fused to the N-terminus of the corresponding Gγ subunit. With the combination of these different techniques the five (human) dopamine receptors were characterized with respect to ligand induced efficacy, different G protein recruitment with involving bias signaling and comparison of close-receptor-signaling or further downstream signal trafficking. Those techniques enable to investigate a pharmacological profile of the dopamine receptor family and the characterization of G protein coupling, ligand efficacies and potencies, as well as the differentiation between agonist, antagonists and inverse agonists.
Moreover, a radioligand as a useful tool for radioligand binding experiments for the H3R was characterized by overcoming drawbacks, of agonistic ligands such as receptor internalization in cell-based systems or selectivity issues within the histamine receptor family. The chosen pharmacophore was a propionylated JNJ-5207852 scaffold, which was tritium labeled. In radioligand saturation binding experiments the radioligand revealed curves in a saturable manner with a binding affinity in the sub nanomolar range and obtained a high H3R selectivity compared to the other members of the histamine receptor family. In kinetic studies the fast association and complete dissociation was verified. Furthermore, in competition binding experiments in the presence of the radioligand [3H]UR-MN259 several H3R standard agonists and antagonist were tested and the resulted pKi values were comparable with literature data. Therefore, a selective and high affinity radioligand for the H3R was developed and characterized as a novel tool for the investigation of new ligands in robust radioligand binding studies.
The investigation of the pharmacological properties of heterodimers of the class A family is necessary due to their involvement in e.g., neurodegenerative diseases like Parkinson’s disease or Alzheimer’s disease. Furthermore, to address these drug targets novel bivalent ligands were synthesized in-house and needed to be evaluated in their binding affinities, efficacies and confirmed as true bivalent ligands. Therefore, a radioligand binding assay was developed for the reported D1R-H3R and D2lR-H3R heteromers.
For the D1R-H3R the suggested stoichiometric receptor ratio in vitro of 1:2, leading to functional heterodimers, was obtained and biphasic curves for the bivalent ligand NR330 were achieved. The resulting pKi,high value was not higher as at the monomer receptor. Further experiments with the bivalent ligand NR330 in the presence of an additional competitor were performed, in order to test whether the biphasic curve can be transformed to a monophasic curve by blocking one protomer of the D1-H3R heteromer. For the H3R mode the D1R antagonist SCH-23390 (c = 10 µM) and in the D1R mode the antagonistic H3R ligand JNJ-5207852 (c =10 µM) were used. The resulting monophasic curve of NR330 in the presence of either the D1R or H3R competitor was right shifted compared to the curve on the mono-expressed receptor and showed an additional decrease in the specific binding of both radioligands (D1R protomer: [3H]SCH-23390, H3R protomer: [3H]UR-MN259). We hypothesized a cross binding affinity of the D1R competitor to the H3R protomer and vice versa. Thus, the respective ligands were investigated on the respective D1R and H3R mono-expressing cell lines. The bivalent ligand NR330 resulted a monophasic curve in all three chosen buffers (sodium-free or sodium-containing) at the D1R monomer, as expected, but different binding affinities which were negatively affected in the presence of sodium. At the H3R monomer the expected monovalent curve was observed in sodium-free BB and BB supplemented with 100 mM NaCl. In contrast, in both buffers containing 140 mM NaCl (Leibovitz’s L15 media and BB with 140 mM NaCl) no statistically correct binding mode was validated and both modes (monophasic and biphasic curves) were specified. With respect to the amount of the specific binding of the radioligand [3H]SCH-23390 the observed plateau of NR330 at the H3R was different compared to the resulted one of NR330 on D1R-H3R co-expressing HEK239T cells. Therefore, we assume a true bivalent binding mode of NR330 on D1R-H3R co-expressing HEK239T cells with a stoichiometric receptor ratio of 1:2.
The antagonistic H3R ligands JNJ-5207852 and MN259 showed no binding affinity to the D1R monomer, although a negative cooperativity was observed in combination with the D1R standard antagonist SCH-23390. In case of the H3R monomer the affinity of the D1R antagonist SCH-23390 was dependent on the used buffers. In sodium containing buffer supplemented 140 mM NaCl, the binding affinity was in micromolar range, while in BB supplemented with 100 mM NaCl, the affinity was increased to the nanomolar range. In contrast, in BB without supplements a biphasic curve for SCH-23390 was observed. The observed two binding modes in sodium-free buffer were thus changed to a monovalent binding mode in sodium containing buffer, which implies a sodium-dependent effect. Furthermore, the combination of the H3R ligands JNJ-5207852 or MN259 with 10 µM SCH-23390 led to right-shifted curves and a drop in signal of the specific binding of the radioligand [3H]UR-MN259 in all buffers. Therefore, we hypothesized a possible allosteric modulation of SCH-23390 to the H3R ligands. Unfortunately, we could not confirm this hypothesis completely and further investigations are necessary.
For the D2lR-H3R heteromer a radioligand binding assay was likewise developed. The observed curves of the bivalent ligands MN079 and MN240 were primarily monophasic in different receptor ratios (1:1, 1:2.4 and 1:3.7), while MN240 achieved a biphasic curve only in the D2lR-mode on co-expressing cells with a stoichiometric receptor ratio of 1:1.8. The assumed receptor ratio for the formation of functional heteromers of the class A GPCRs is 1:2 in vitro, which could not be achieved during this PhD.
However, to characterize the efficacies of bivalent ligands a functional miniG protein recruitment assay was applied to the D2lR-H3R heteromer. The small bit of the split Nano luciferase was fused to the C-terminus of the H3R, whereas the large bit was cloned between the D2lR and the miniGsi protein, tethered through flexible linkers. The recruitment of the miniGsi could be observed in real-time after the activation by histamine and gave hints to the existence of the formation of the D2lR-H3R heteromer, compared to the observed traces on the H3R monomer. All analyzed H3R standard ligands gave robust concentration-response-curves, while in contrast no activation for the D2-like agonist dopamine and pramipexole was achieved. Moreover, we were able to observe a possible cross-interaction between the D2lR-H3R heteromer. The affinity of histamine in the presence of 100 µM dopamine was right-shifted to nanomolar range and a drop in the signal was observed compared to histamine without additional competitor. A further possible explanation is the asymmetric recruitment of the miniGsi protein by one protomer, due to an excess limitation. Thus, further experiments are necessary to confirm the negative cooperativity by introducing a point mutation in the G protein binding pocket of the D2lR7 and changing of the linker length to the miniG protein to evaluate a possible impact, as well as, the fusion of the small bit to the D2lR and the NLucN-miniGsi construct to the H3R.
In summary, it was not possible to develop a radioligand binding assay for both heteromers to characterize the respective novel bivalent ligands with sufficient replicates. However, the results are promising to confirm the ligands as true bivalent ligands, but further investigations are necessary. Thus, for competition binding experiments with an additional competitor the competitors should be changed to (+)-butaclamol in the H3R mode and to GSK33449 in case of the D1R mode to obtain monophasic curves of the bivalent ligand and to obtain monophasic curves without a decreased signal-to-noise ratio on co-expressing cells. Additionally, the end-capped ligands as further controls, should be included. For all following studies on the D1R-H3R heteromer, the buffer needs to be changed to a sodium-containing buffer, like Leibovitz’s L-15 media, to alleviate the sodium impact on the binding affinities and properties. Therefore, all previously obtained experiments with the bivalent ligand with or without an additional competitor need to be performed in this buffer. For the D2lR-H3R heteromer the correct stoichiometric receptor ratio in vitro needs to be evaluated, followed by competition binding experiments in the presence of suitable competitors in both modes, for the complete characterization of the novel bivalent ligands.
The last project of this thesis was the development of a NanoBRET-based assay for the five (human) dopamine receptors to investigate the binding affinities of novel ligands with a reliable and robust test system like in radioligand binding experiments. The NanoLuc as BRET donor was N-terminally fused to the respective (human) dopamine receptor where a fluorescent ligand can bind and the non-radioactive energy transfer takes place, referred as BRET. The investigated fluorescent ligands of either the D1-like or D2-like dopamine receptors consist of a selective pharmacophore (D1-like: SCH-23390 analogue, D2-like: spiperone analogue) connected through alkyl or PEG linker with the TAMRA or DY-549P1 dye.
For the D1-like family the most promising fluorescent ligand NR435 consists of the SCH-23390 pharmacophore, a short linker and the TAMRA fluorophore and yielded an affinity in the nanomolar range. For the D2-like family the fluorescent ligand MN212 with a spiperone analogue, the shortest linker and the TAMRA dye was characterized for the D2lR and obtained likewise the highest affinity. Due to the high homology between the D2-like receptors MN212 was tested at the D3R with an eight-fold lower affinity compared to the D2lR. For both, competition binding experiments with different literature described D2-like standard ligands were performed, with comparable pKi values to published data. In this study, we could observe a negative modulation of the binding affinity by introducing the DY-549P1 dye to the fluorescent ligands. In previous work the respective linker length, properties like hydrophilic or lipophilic entities and the fluorophore are considered to affect the binding properties of the linker.
Due to a change of the device (from Tecan Infinite® Lumi plate reader to CLARIOStarPlus plate reader) all observed results were not reproducible at the CLARIOStarPlus under the same conditions. We suggest, due to a device specific signal-to-noise ratio, that the emission of the fluorescent ligand was not detectable anymore with the CLAIOStarPlus, although we were able to detect the blue light emission of the NanoLuc (λ = 480 nM). It is known that there is no direct correlation between the receptor expression and the observed BRET signal. The optimal amount of NanoLuc-tagged receptor can be verified by transiently performed experiments. Due to the limitation in time kinetic studies, outstanding saturation or competition binding experiments for all dopamine receptor and the respective fluorescent ligands could not be performed
The Glutamine Synthetase in lymphocytic leukemia cells and non-malignant T lymphocytes
No full textT cells exhibit similar metabolic characteristics to tumor cells during their activation phase. Rapidly growing and dividing cells need to adapt their metabolism, with glutamine playing a significant role as a partially essential amino acid, especially for cells with high energy demands.
First, we investigated the effects of glutamine deprivation on the proliferation, survival, and metabolism of tumor cells. We chose the two cell lines T-ALL CCRF-CEM-C7H2 (C7H2) and Jurkat-FHCRC as model cells. It was found that both cell lines proliferated less under glutamine deprivation, with the deprivation having a greater impact on C7H2 cells. While Jurkat cells grew more slowly under glutamine deprivation, they continued their growth and division.
To gain insights into the underlying mechanisms, we analyzed the effects of glutamine deficiency on mitochondrial respiration in both cell lines. It was found that cellular respiration in C7H2 cells was significantly reduced, whereas respiration in Jurkat cells was only slightly affected.
Next, we examined the ability for endogenous glutamine synthesis and found that both C7H2 and Jurkat cells expressed glutamine synthetase under glutamine deprivation. To rule out differences in enzymatic activity, we conducted metabolite tracing experiments. We demonstrated the activity of glutamine synthetase in both cell lines under glutamine deprivation in the tracing analysis using labeled 13C-glutamate.
Finally, we tested whether the addition of glutamate could compensate for glutamine deprivation. We found that high doses of glutamate significantly improved the survival of C7H2 cells.
In the second part of the study, we investigated how glutamine deprivation affected human CD8+ T cells. It is known that these T cells do not proliferate, are not activated, and have reduced cytokine production under glutamine deprivation. High concentrations of glutamate in cell culture were able to partially compensate for glutamine deprivation in T cells, resulting in slightly increased proliferation and cytokine production compared to the group with no added glutamate or glutamine.
Further studies are necessary, particularly with regard to the tumor microenvironment where immune cells encounter the tumor. Since certain amino acids, such as glutamine, are present in very low concentrations in the tumor environment, tumor cells might have a potential survival advantage there. Future studies should continue to focus on the tumor microenvironment
Data integration with Fusion Searchlight: Classifying brain states from resting-state fMRI
No full textResting-state fMRI captures spontaneous neural activity characterized by complex spatiotemporal dynamics. Various metrics, such as local and global brain connectivity and low-frequency amplitude fluctuations, quantify distinct aspects of these dynamics. However, these measures are typically analyzed independently, overlooking their interrelations and potentially limiting analytical sensitivity. Here, we introduce the Fusion Searchlight (FuSL) framework, which integrates complementary information from multiple resting-state fMRI metrics. We demonstrate that combining these metrics enhances the accuracy of pharmacological treatment prediction from rs-fMRI data, enabling the identification of additional brain regions affected by sedation with alprazolam. Furthermore, we leverage explainable AI to delineate the differential contributions of each metric, which additionally improves spatial specificity of the searchlight analysis. Moreover, this framework can be adapted to combine information across imaging modalities or experimental conditions, providing a versatile and interpretable tool for data fusion in neuroimaging