Chem-CRISPR/dCas9FCPF: a platform for chemically induced epigenome editing.

Epigenetic aberration is one of the major driving factors in human cancer, often leading to acquired resistance to chemotherapies. Various small molecule epigenetic modulators have been reported. Nonetheless, outcomes from animal models and clinical trials have underscored the substantial setbacks attributed to pronounced on- and off-target toxicities. To address these challenges, CRISPR/dCas9 technology is emerging as a potent tool for precise modulation of epigenetic mechanism. However, this technology involves co-expressing exogenous epigenetic modulator proteins, which presents technical challenges in preparation and delivery with potential undesirable side effects. Recently, our research demonstrated that Cas9 tagged with the Phe-Cys-Pro-Phe (FCPF)-peptide motif can be specifically targeted by perfluorobiphenyl (PFB) derivatives. Here, we integrated the FCPF-tag into dCas9 and established a chemically inducible platform for epigenome editing, called Chem-CRISPR/dCas9FCPF. We designed a series of chemical inhibitor-PFB conjugates targeting various epigenetic modulator proteins. Focusing on JQ1, a panBET inhibitor, we demonstrate that c-MYC-sgRNA-guided JQ1-PFB specifically inhibits BRD4 in close proximity to the c-MYC promoter/enhancer, thereby effectively repressing the intricate transcription networks orchestrated by c-MYC as compared with JQ1 alone. In conclusion, our Chem-CRISPR/dCas9FCPF platform significantly increased target specificity of chemical epigenetic inhibitors, offering a viable alternative to conventional fusion protein systems for epigenome editing.

Post-COVID breathlessness: a mathematical model of respiratory processing in the brain.

Breathlessness is among the most common post-COVID symptoms. In a considerable number of patients, severe breathlessness cannot be explained by peripheral organ impairment. Recent concepts have described how such persistent breathlessness could arise from dysfunctional processing of respiratory information in the brain. In this paper, we present a first quantitative and testable mathematical model of how processing of respiratory-related signals could lead to breathlessness perception. The model is based on recent theories that the brain holds an adaptive and dynamic internal representation of a respiratory state that is based on previous experiences and comprises gas exchange between environment, lung and tissue cells. Perceived breathlessness reflects the brain's estimate of this respiratory state signaling a potentially hazardous disequilibrium in gas exchange. The internal respiratory state evolves from the respiratory state of the last breath, is updated by a sensory measurement of CO2 concentration, and is dependent on the current activity context. To evaluate our model and thus test the assumed mechanism, we used data from an ongoing rebreathing experiment investigating breathlessness in patients with post-COVID without peripheral organ dysfunction (N = 5) and healthy control participants without complaints after COVID-19 (N = 5). Although the observed breathlessness patterns varied extensively between individual participants in the rebreathing experiment, our model shows good performance in replicating these individual, heterogeneous time courses. The model assumes the same underlying processes in the central nervous system in all individuals, i.e., also between patients and healthy control participants, and we hypothesize that differences in breathlessness are explained by different weighting and thus influence of these processes on the final percept. Our model could thus be applied in future studies to provide insight into where in the processing cascade of respiratory signals a deficit is located that leads to (post-COVID) breathlessness. A potential clinical application could be, e.g., the monitoring of effects of pulmonary rehabilitation on respiratory processing in the brain to improve the therapeutic strategies.

[Walking aids seen from a cultural historical perspective : Functional and semantic diversity of assistive systems facilitating locomotion in old age]. / Gehhilfen aus kulturgeschichtlicher Perspektive : Funktionelle und semantische Vielfalt von Assistenzsystemen für die Fortbewegung im Alter.

BACKGROUND: Assistance systems serving the locomotion of older people interact in many ways with the culture of a society. Since early modern times at the latest, walking aids were tantamount to human frailty; however, the cane also symbolized governmental power or reputation. Nowadays, the cane, the wheelchair, and the rollator have not only a functional significance in terms of a better mobility, they also enable people to take an active part in social life. OBJECTIVE: This study aimes at tracing back these provisional insights into the history of civilization and thereby analyze the roots, new forms and pictures of the handling and metaphors of these assistance systems. The goal in the context of this special issue is to decipher a central textual and pictorial symbol of old age, comparing it with more recent symbols of assistance in old age. MATERIAL AND METHODS: Methodologically, the text combines approaches of philology and history of medicine with those of the history of art. It analyzes (after a brief retrospection of ancient times and the Middle Ages) by means of textual and pictorial sources from the sixteenth to the twenty-first centuries the historical development of these aids of locomotion for older people. Additionally, it explores the cultural relevancy of these assistance systems. RESULTS AND DISCUSSION: In history the medical profession paid relatively late and then only minor attention to the assistive systems analyzed here. Its semantic diversity is closely related to the age roles and stereotypes of age prevailing in certain epochs. The more the respective assistive tool is used by old people, the more suitable it is as a symbol of old age and the more biased and negative the semantic connotation seems to be. The development of a symbol of age connoting frailty, at present symbolized most clearly by the rollator, tends to refer to a pejorative image of age in a society. The cultural historical analysis suggests that a contrasting development will only be possible when the assistive systems will again fulfil a diversity of alternative functions and semantics.

Interaction of Flavin-Dependent Fructose Dehydrogenase with Cytochrome c as Basis for the Construction of Biomacromolecular Architectures on Electrodes.

The creation of electron transfer (ET) chains based on the defined arrangement of enzymes and redox proteins on electrode surfaces represents an interesting approach within the field of bioelectrocatalysis. In this study, we investigated the ET reaction of the flavin-dependent enzyme fructose dehydrogenase (FDH) with the redox protein cytochrome c (cyt c). Two different pH optima were found for the reaction in acidic and neutral solutions. When cyt c was adsorbed on an electrode surface while the enzyme remained in solution, ET proceeded efficiently in media of neutral pH. Interprotein ET was also observed in acidic media; however, it appeared to be less efficient. These findings suggest that two different ET pathways between the enzyme and cyt c may occur. Moreover, cyt c and FDH were immobilized in multiple layers on an electrode surface by means of another biomacromolecule: DNA (double stranded) using the layer-by-layer technique. The biprotein multilayer architecture showed a catalytic response in dependence on the fructose concentration, indicating that the ET reaction between both proteins is feasible even in the immobilized state. The electrode showed a defined response to fructose and a good storage stability. Our results contribute to the better understanding of the ET reaction between FDH and cyt c and provide the basis for the creation of all-biomolecule based fructose sensors the sensitivity of which can be controlled by the layer preparation.

Metformin-Derived Growth Inhibition in Renal Cell Carcinoma Depends on miR-21-Mediated PTEN Expression.

PURPOSE: Metformin (MF) acts as a tumour-suppressor in renal cell carcinoma (RCC) by inhibiting the AKT/mTOR pathway via AMPK activation. Here, we explore the influence of miR-21 and its target gene PTEN on MF effects in CAKI-1 and CAKI-2 cells. METHODS: Proliferation assays (MTS) and qRT-PCR after transient transfection with pre- and anti-miR-21 and MF treatment were conducted. AMPK-dependency was assessed via transfection of siAMPK. The expression of PTEN, AKT and miR-21 after transient pre-miR-21 transfection and MF treatment was analysed. RESULTS: We demonstrate that CAKI-1 cells, which were found to be less sensitive towards MF, showed a significant higher miR-21 and lower PTEN expression than CAKI-2. This was confirmed in a primary RCC collective (n = 28): miR-21 and PTEN expression correlated negatively. MF treatment lowered miR-21 AMPK-dependently and increased PTEN expression in the cell lines. Ectopic miR-21 regulation modulated MF sensitivity. Western blot analysis showed that pre-miR-21 transfection and MF treatment regulated PTEN expression with impact on pAKT levels in the cells. CONCLUSIONS: We show that differing MF sensitivity in RCC cells is associated with and mediated through the regulation of miR-21/PTEN expression with an impact on subsequent AKT signalling. This provides imaginable clinical implications regarding MF therapy of RCC patients for the future.

The serine/threonine kinase 33 is present and expressed in palaeognath birds but has become a unitary pseudogene in neognaths about 100 million years ago.

BACKGROUND: Serine/threonine kinase 33 (STK33) has been shown to be conserved across all major vertebrate classes including reptiles, mammals, amphibians and fish, suggesting its importance within vertebrates. It has been shown to phosphorylate vimentin and might play a role in spermatogenesis and organ ontogenesis. In this study we analyzed the genomic locus and expression of stk33 in the class Aves, using a combination of large scale next generation sequencing data analysis and traditional PCR. RESULTS: Within the subclass Palaeognathae we analyzed the white-throated tinamou (Tinamus guttatus), the African ostrich (Struthio camelus) and the emu (Dromaius novaehollandiae). For the African ostrich we were able to generate a 62,778 bp long genomic contig and an mRNA sequence that encodes a protein showing highly significant similarity to STK33 proteins from other vertebrates. The emu has been shown to encode and transcribe a functional STK33 as well. For the white-throated tinamou we were able to identify 13 exons by sequence comparison encoding a protein similar to STK33 as well. In contrast, in all 28 neognath birds analyzed, we could not find evidence for the existence of a functional copy of stk33 or its expression. In the genomes of these 28 bird species, we found only remnants of the stk33 locus carrying several large genomic deletions, leading to the loss of multiple exons. The remaining exons have acquired various indels and premature stop codons. CONCLUSIONS: We were able to elucidate and describe the genomic structure and the transcription of a functional stk33 gene within the subclass Palaeognathae, but we could only find degenerate remnants of stk33 in all neognath birds analyzed. This led us to the conclusion that stk33 became a unitary pseudogene in the evolutionary history of the class Aves at the paleognath-neognath branch point during the late cretaceous period about 100 million years ago. We hypothesize that the pseudogenization of stk33 might have become fixed in neognaths due to either genetic redundancy or a non-orthologous gene displacement and present potential candidate genes for such an incident.

Cytoplasmic replication of Staphylococcus aureus upon phagosomal escape triggered by phenol-soluble modulin α.

Staphylococcus aureus is a Gram-positive human pathogen that is readily internalized by professional phagocytes such as macrophages and neutrophils but also by non-professional phagocytes such as epithelial or endothelial cells. Intracellular bacteria have been proposed to play a role in evasion of the innate immune system and may also lead to dissemination within migrating phagocytes. Further, S. aureus efficiently lyses host cells with a battery of cytolytic toxins. Recently, phenol-soluble modulins (PSM) have been identified to comprise a genus-specific family of cytolytic peptides. Of these the PSMα peptides have been implicated in killing polymorphonuclear leucocytes after phagocytosis. We questioned if the peptides were active in destroying endosomal membranes to avoid lysosomal killing of the pathogen and monitored integrity of infected host cell endosomes by measuring the acidity of the intracellular bacterial microenvironment via flow cytometry and by a reporter recruitment technique. Isogenic mutants of the methicillin-resistant S. aureus (MRSA) strains USA300 LAC, USA400 MW2 as well as the strongly cytolytic methicillin-sensitive strain 6850 were compared with their respective wild type strains. In all three genetic backgrounds, PSMα mutants were unable to escape from phagosomes in non-professional (293, HeLa, EAhy.926) and professional phagocytes (THP-1), whereas mutants in PSMß and δ-toxin as well as ß-toxin, phosphatidyl inositol-dependent phospholipase C and Panton Valentine leucotoxin escaped with efficiencies of the parental strains. S. aureus replicated intracellularly only in presence of a functional PSMα operon thereby illustrating that bacteria grow in the host cell cytoplasm upon phagosomal escape.

Higher order asymptotics for negative binomial regression inferences from RNA-sequencing data.

RNA sequencing (RNA-Seq) is the current method of choice for characterizing transcriptomes and quantifying gene expression changes. This next generation sequencing-based method provides unprecedented depth and resolution. The negative binomial (NB) probability distribution has been shown to be a useful model for frequencies of mapped RNA-Seq reads and consequently provides a basis for statistical analysis of gene expression. Negative binomial exact tests are available for two-group comparisons but do not extend to negative binomial regression analysis, which is important for examining gene expression as a function of explanatory variables and for adjusted group comparisons accounting for other factors. We address the adequacy of available large-sample tests for the small sample sizes typically available from RNA-Seq studies and consider a higher-order asymptotic (HOA) adjustment to likelihood ratio tests. We demonstrate that 1) the HOA-adjusted likelihood ratio test is practically indistinguishable from the exact test in situations where the exact test is available, 2) the type I error of the HOA test matches the nominal specification in regression settings we examined via simulation, and 3) the power of the likelihood ratio test does not appear to be affected by the HOA adjustment. This work helps clarify the accuracy of the unadjusted likelihood ratio test and the degree of improvement available with the HOA adjustment. Furthermore, the HOA test may be preferable even when the exact test is available because it does not require ad hoc library size adjustments.

[Feaver Concepts in the Early Work of Poeta Medicus Friedrich Schiller]. / Fieberkrankheiten im frühen Werk des Poeta medicus Friedrich Schiller.

Friedrich Schiller wrote a Latin "Prüfschrift" (thesis) on fever diseases in 1780 as part of his medical studies in Stuttgart. In it, he accuses nature of aggravating inflammation through excessive resistance. This concept of fever interacts with Schiller's early literary texts: In his first drama, "Die Räuber" (1781), the two protagonists embody the two main types of fever. Accordingly, the descriptions of the two main types of fever in the "Prüfschrift" do not turn out to be "objective" either but contain positive and negative connotations: In a sense, personified heroes and hypocrites are medically juxtaposed and pathologised. In another early poem about the plague (1782), Schiller also interpreted and used fever as an expression of human vitality and natural power, as an anthropological sign for the interconnectedness of soul and body: soul forces are revealed in fever, and the drama of human existence becomes particularly clear in the struggle between nature and disease.

Culture expansion of CAR T cells results in aberrant DNA methylation that is associated with adverse clinical outcome.

Chimeric antigen receptor (CAR) T cells provide new perspectives for treatment of hematological malignancies. Manufacturing of these cellular products includes culture expansion procedures, which may affect cellular integrity and therapeutic outcome. In this study, we investigated culture-associated epigenetic changes in CAR T cells and found continuous gain of DNAm, particularly within genes that are relevant for T cell function. Hypermethylation in many genes, such as TCF7, RUNX1, and TOX, was reflected by transcriptional downregulation. 332 CG dinucleotides (CpGs) showed an almost linear gain in methylation with cell culture time, albeit neighboring CpGs were not coherently regulated on the same DNA strands. An epigenetic signature based on 14 of these culture-associated CpGs predicted cell culture time across various culture conditions. Notably, even in CAR T cell products of similar culture time higher DNAm levels at these CpGs were associated with significantly reduced long-term survival post transfusion. Our data demonstrate that cell culture expansion of CAR T cells evokes DNA hypermethylation at specific sites in the genome and the signature may also reflect loss of potential in CAR T cell products. Hence, reduced cultivation periods are beneficial to avoid dysfunctional methylation programs that seem to be associated with worse therapeutic outcome.

Are Old Men Impotent? On a Sparse Discourse of Early Modern Medicine and Its Forensic Implications in Paolo Zacchia's Quaestiones medico-legales.

In early modern medical literature, there are increasing references to sterility and impotence in older men. This is especially true of the Quaestiones medico-legales by the Roman physician Paolo Zacchia (1584-1659). In several books of this systematically structured manual, its author discusses medical and legal arguments on the one hand. On the other hand, in the 10th, only posthumously published volume, a total of five cases of impotence in old men are described on the basis of court decisions of the Rota Romana and expert opinions of the author. The paper examines these cases with regard to central statements on male impotence in old age, which are placed in the medical as well as the social and legal-historical context of the time. It becomes clear that old-age impotence and sterility were less a medical than a legal problem in the 17th century. However, the physician Zacchia emphasises the concept of biological age instead of historically transmitted numerical age limits. In this respect, his expert opinions already show the first signs of medical empiricism.

MACSima imaging cyclic staining (MICS) technology reveals combinatorial target pairs for CAR T cell treatment of solid tumors.

Many critical advances in research utilize techniques that combine high-resolution with high-content characterization at the single cell level. We introduce the MICS (MACSima Imaging Cyclic Staining) technology, which enables the immunofluorescent imaging of hundreds of protein targets across a single specimen at subcellular resolution. MICS is based on cycles of staining, imaging, and erasure, using photobleaching of fluorescent labels of recombinant antibodies (REAfinity Antibodies), or release of antibodies (REAlease Antibodies) or their labels (REAdye_lease Antibodies). Multimarker analysis can identify potential targets for immune therapy against solid tumors. With MICS we analysed human glioblastoma, ovarian and pancreatic carcinoma, and 16 healthy tissues, identifying the pair EPCAM/THY1 as a potential target for chimeric antigen receptor (CAR) T cell therapy for ovarian carcinoma. Using an Adapter CAR T cell approach, we show selective killing of cells only if both markers are expressed. MICS represents a new high-content microscopy methodology widely applicable for personalized medicine.

Anti-CD19 CARs displayed at the surface of lentiviral vector particles promote transduction of target-expressing cells.

Recently, a rare type of relapse was reported upon treating a B cell acute lymphoblastic leukemia (B-ALL) patient with anti-CD19 chimeric antigen receptor (CAR)-T cells caused by unintentional transduction of residual malignant B cells (CAR-B cells). We show that anti-CD19 and anti-CD20 CARs are presented on the surface of lentiviral vectors (LVs), inducing specific binding to the respective antigen. Binding of anti-CD19 CAR-encoding LVs containing supernatant was reduced by CD19-specific blocking antibodies in a dose-dependent manner, and binding was absent for unspecific LV containing supernatant. This suggests that LVs bind via displayed CAR molecules to CAR antigen-expressing cells. The relevance for CAR-T cell manufacturing was evaluated when PBMCs and B-ALL malignant B cells were mixed and transduced with anti-CD19 or anti-CD20 CAR-displaying LVs in clinically relevant doses to mimic transduction conditions of unpurified patient leukapheresis samples. Malignant B cells were transduced at higher levels with LVs displaying anti-CD19 CARs compared to LVs displaying non-binding control constructs. Stability of gene transfer was confirmed by applying a potent LV inhibitor and long-term cultures for 10 days. Our findings provide a potential explanation for the emergence of CAR-B cells pointing to safer manufacturing procedures with reduced risk of this rare type of relapse in the future.

Identification of CD318, TSPAN8 and CD66c as target candidates for CAR T cell based immunotherapy of pancreatic adenocarcinoma.

A major roadblock prohibiting effective cellular immunotherapy of pancreatic ductal adenocarcinoma (PDAC) is the lack of suitable tumor-specific antigens. To address this challenge, here we combine flow cytometry screenings, bioinformatic expression analyses and a cyclic immunofluorescence platform. We identify CLA, CD66c, CD318 and TSPAN8 as target candidates among 371 antigens and generate 32 CARs specific for these molecules. CAR T cell activity is evaluated in vitro based on target cell lysis, T cell activation and cytokine release. Promising constructs are evaluated in vivo. CAR T cells specific for CD66c, CD318 and TSPAN8 demonstrate efficacies ranging from stabilized disease to complete tumor eradication with CD318 followed by TSPAN8 being the most promising candidates for clinical translation based on functionality and predicted safety profiles. This study reveals potential target candidates for CAR T cell based immunotherapy of PDAC together with a functional set of CAR constructs specific for these molecules.

Phagolysosomal integrity is generally maintained after Staphylococcus aureus invasion of nonprofessional phagocytes but is modulated by strain 6850.

Staphylococcus aureus is a major cause of a variety of both local and systemic infections. It can invade human host cells, a process that may account for disseminated and recurrent infections. S. aureus postinvasion events in nonprofessional phagocytes are only partially understood. While morphological data suggest a phagosomal escape, there is a lack of corroborating functional data. Using a combination of pH determination and morphological techniques, we have tested the integrity of Staphylococcus-containing phagosomes in 293 (HEK-293), HeLa, and EA.hy926 cells over time. Rapid acidification of S. aureus-containing phagosomes occurred and was sustained for up to 24 h. All S. aureus strains tested displayed equally sustained intraphagosomal pH levels without exhibiting any correlation with pH level and hemolytic activity. The membrane morphology of the phagosomal compartment was heterogeneous, even under conditions where acidic pH was fully maintained, an observation incompatible with phagolysosomal membrane destruction. As an exception, S. aureus strain 6850 showed a reduced phagosomal acidification signal 6 h after invasion. Additionally, only strain 6850 failed to localize to LAMP-1-positive vesicles in HeLa cells, although this was observed only rarely. Several other strongly beta-hemolytic strains did not modulate phagolysosomal pH, suggesting that S. aureus alpha-toxin and beta-toxin are not sufficient for this process. Taken together, our data suggest that S. aureus-containing phagolysosomes generally remain functionally intact in nonprofessional phagocytes, thereby contrasting with transmission electron micrographic results.

Rational design of thiolated polyenes as trifunctional Raman reporter molecules in surface-enhanced Raman scattering nanotags for cytokine detection in a lateral flow assay.

The characteristic vibrational spectroscopic fingerprint of Raman reporter molecules adsorbed on noble metal nanoparticles is employed for the identification of target proteins by the corresponding surface-enhanced Raman scattering (SERS) nanotag-labeled antibodies. Here, we present the modular synthesis of thiolated polyenes with two to five C═C double bonds introduced via stepwise Wittig reactions. The experimental characterization of their electronic and vibrational properties is complemented by density functional theory calculations. Highly SERS-active nanotags are generated by using the thiolated polyenes as Raman reporter molecules in Au/Au core/satellite supraparticles with multiple hot spots. The cytokines IL-1ß and IFN-γ are detected in a duplex SERS-based lateral flow assay on a nitrocellulose test strip by Raman microscopy. The thiolated polyenes are suitable for use in immuno-SERS applications such as point-of-care testing as well as cellular and tissue imaging.

Privatization of Biofilm Matrix in Structurally Heterogeneous Biofilms.

The self-produced biofilm provides beneficial protection for the enclosed cells, but the costly production of matrix components makes producer cells susceptible to cheating by nonproducing individuals. Despite detrimental effects of nonproducers, biofilms can be heterogeneous, with isogenic nonproducers being a natural consequence of phenotypic differentiation processes. For instance, in Bacillus subtilis biofilm cells differ in production of the two major matrix components, the amyloid fiber protein TasA and exopolysaccharides (EPS), demonstrating different expression levels of corresponding matrix genes. This raises questions regarding matrix gene expression dynamics during biofilm development and the impact of phenotypic nonproducers on biofilm robustness. Here, we show that biofilms are structurally heterogeneous and can be separated into strongly and weakly associated clusters. We reveal that spatiotemporal changes in structural heterogeneity correlate with matrix gene expression, with TasA playing a key role in biofilm integrity and timing of development. We show that the matrix remains partially privatized by the producer subpopulation, where cells tightly stick together even when exposed to shear stress. Our results support previous findings on the existence of "weak points" in seemingly robust biofilms as well as on the key role of linkage proteins in biofilm formation. Furthermore, we provide a starting point for investigating the privatization of common goods within isogenic populations.IMPORTANCE Biofilms are communities of bacteria protected by a self-produced extracellular matrix. The detrimental effects of nonproducing individuals on biofilm development raise questions about the dynamics between community members, especially when isogenic nonproducers exist within wild-type populations. We asked ourselves whether phenotypic nonproducers impact biofilm robustness, and where and when this heterogeneity of matrix gene expression occurs. Based on our results, we propose that the matrix remains partly privatized by the producing subpopulation, since producing cells stick together when exposed to shear stress. The important role of linkage proteins in robustness and development of the structurally heterogeneous biofilm provides an entry into studying the privatization of common goods within isogenic populations.