Single-cell deconvolution reveals high lineage- and location-dependent heterogeneity in mesenchymal multivisceral stage 4 colorectal cancer.

Metastasized colorectal cancer (CRC) is associated with a poor prognosis and rapid disease progression. Besides hepatic metastasis, peritoneal carcinomatosis is the major cause of death in Union for International Cancer Control (UICC) stage IV CRC patients. Insights into differential site-specific reconstitution of tumor cells and the corresponding tumor microenvironment are still missing. Here, we analyzed the transcriptome of single cells derived from murine multivisceral CRC and delineated the intermetastatic cellular heterogeneity regarding tumor epithelium, stroma, and immune cells. Interestingly, we found an intercellular site-specific network of cancer-associated fibroblasts and tumor epithelium during peritoneal metastasis as well as an autologous feed-forward loop in cancer stem cells. We furthermore deciphered a metastatic dysfunctional adaptive immunity by a loss of B cell-dependent antigen presentation and consecutive effector T cell exhaustion. Furthermore, we demonstrated major similarities of this murine metastatic CRC model with human disease and - based on the results of our analysis - provided an auspicious site-specific immunomodulatory treatment approach for stage IV CRC by intraperitoneal checkpoint inhibition.

Magainin 2 and PGLa in bacterial membrane mimics IV: Membrane curvature and partitioning.

We previously reported that the synergistically enhanced antimicrobial activity of magainin 2 (MG2a) and PGLa is related to membrane adhesion and fusion. Here, we demonstrate that equimolar mixtures of MG2a and L18W-PGLa induce positive monolayer curvature stress and sense, at the same time, positive mean and Gaussian bilayer curvatures already at low amounts of bound peptide. The combination of both abilities-membrane curvature sensing and inducing-is most likely the base for the synergistically enhanced peptide activity. In addition, our coarse-grained simulations suggest that fusion stalks are promoted by decreasing the free-energy barrier for their formation rather than by stabilizing their shape. We also interrogated peptide partitioning as a function of lipid and peptide concentration using tryptophan fluorescence spectroscopy and peptide-induced leakage of dyes from lipid vesicles. In agreement with a previous report, we find increased membrane partitioning of L18W-PGLa in the presence of MG2a. However, this effect does not prevail to lipid concentrations higher than 1 mM, above which all peptides associate with the lipid bilayers. This implies that synergistic effects of MG2a and L18W-PGLa in previously reported experiments with lipid concentrations >1 mM are due to peptide-induced membrane remodeling and not their specific membrane partitioning.

Lactoferricins impair the cytosolic membrane of Escherichia coli within a few seconds and accumulate inside the cell.

We report the real-time response of Escherichia coli to lactoferricin-derived antimicrobial peptides (AMPs) on length scales bridging microscopic cell sizes to nanoscopic lipid packing using millisecond time-resolved synchrotron small-angle X-ray scattering. Coupling a multiscale scattering data analysis to biophysical assays for peptide partitioning revealed that the AMPs rapidly permeabilize the cytosolic membrane within less than 3 s-much faster than previously considered. Final intracellular AMP concentrations of ∼80-100 mM suggest an efficient obstruction of physiologically important processes as the primary cause of bacterial killing. On the other hand, damage of the cell envelope and leakage occurred also at sublethal peptide concentrations, thus emerging as a collateral effect of AMP activity that does not kill the bacteria. This implies that the impairment of the membrane barrier is a necessary but not sufficient condition for microbial killing by lactoferricins. The most efficient AMP studied exceeds others in both speed of permeabilizing membranes and lowest intracellular peptide concentration needed to inhibit bacterial growth.

Magainin 2 and PGLa in bacterial membrane mimics III: Membrane fusion and disruption.

We previously speculated that the synergistically enhanced antimicrobial activity of Magainin 2 and PGLa is related to membrane adhesion, fusion, and further membrane remodeling. Here we combined computer simulations with time-resolved in vitro fluorescence microscopy, cryoelectron microscopy, and small-angle X-ray scattering to interrogate such morphological and topological changes of vesicles at nanoscopic and microscopic length scales in real time. Coarse-grained simulations revealed formation of an elongated and bent fusion zone between vesicles in the presence of equimolar peptide mixtures. Vesicle adhesion and fusion were observed to occur within a few seconds by cryoelectron microscopy and corroborated by small-angle X-ray scattering measurements. The latter experiments indicated continued and time-extended structural remodeling for individual peptides or chemically linked peptide heterodimers but with different kinetics. Fluorescence microscopy further captured peptide-dependent adhesion, fusion, and occasional bursting of giant unilamellar vesicles a few seconds after peptide addition. The synergistic interactions between the peptides shorten the time response of vesicles and enhance membrane fusogenic and disruption properties of the equimolar mixture compared with the individual peptides.

KMT9 Controls Stemness and Growth of Colorectal Cancer.

Colorectal cancer is among the leading causes of cancer-associated deaths worldwide. Treatment failure and tumor recurrence due to survival of therapy-resistant cancer stem/initiating cells represent major clinical issues to overcome. In this study, we identified lysine methyltransferase 9 (KMT9), an obligate heterodimer composed of KMT9α and KMT9ß that monomethylates histone H4 at lysine 12 (H4K12me1), as an important regulator in colorectal tumorigenesis. KMT9α and KMT9ß were overexpressed in colorectal cancer and colocalized with H4K12me1 at promoters of target genes involved in the regulation of proliferation. Ablation of KMT9α drastically reduced colorectal tumorigenesis in mice and prevented the growth of murine as well as human patient-derived tumor organoids. Moreover, loss of KMT9α impaired the maintenance and function of colorectal cancer stem/initiating cells and induced apoptosis specifically in this cellular compartment. Together, these data suggest that KMT9 is an important regulator of colorectal carcinogenesis, identifying KMT9 as a promising therapeutic target for the treatment of colorectal cancer. SIGNIFICANCE: The H4K12 methyltransferase KMT9 regulates tumor cell proliferation and stemness in colorectal cancer, indicating that targeting KMT9 could be a useful approach for preventing and treating this disease.

Antimicrobial peptide activity in asymmetric bacterial membrane mimics.

We report on the response of asymmetric lipid membranes composed of palmitoyl oleoyl phosphatidylethanolamine and palmitoyl oleoyl phosphatidylglycerol, to interactions with the frog peptides L18W-PGLa and magainin 2 (MG2a), as well as the lactoferricin derivative LF11-215. In particular we determined the peptide-induced lipid flip-flop, as well as membrane partitioning of L18W-PGLa and LF11-215, and vesicle dye-leakage induced by L18W-PGLa. The ability of L18W-PGLa and MG2a to translocate through the membrane appears to correlate with the observed lipid flip-flop, which occurred at the fastest rate for L18W-PGLa. The higher structural flexibility of LF11-215 in turn allows this peptide to insert into the bilayers without detectable changes of membrane asymmetry. The increased vulnerability of asymmetric membranes to L18W-PGLa in terms of permeability, appears to be a consequence of tension differences between the compositionally distinct leaflets, but not due to increased peptide partitioning.

Evolution of the analytical scattering model of live Escherichia coli.

A previously reported multi-scale model for (ultra-)small-angle X-ray (USAXS/SAXS) and (very) small-angle neutron scattering (VSANS/SANS) of live Escherichia coli was revised on the basis of compositional/metabolomic and ultrastructural constraints. The cellular body is modeled, as previously described, by an ellipsoid with multiple shells. However, scattering originating from flagella was replaced by a term accounting for the oligosaccharide cores of the lipopolysaccharide leaflet of the outer membrane including its cross-term with the cellular body. This was mainly motivated by (U)SAXS experiments showing indistinguishable scattering for bacteria in the presence and absence of flagella or fimbrae. The revised model succeeded in fitting USAXS/SAXS and differently contrasted VSANS/SANS data of E. coli ATCC 25922 over four orders of magnitude in length scale. Specifically, this approach provides detailed insight into structural features of the cellular envelope, including the distance of the inner and outer membranes, as well as the scattering length densities of all bacterial compartments. The model was also successfully applied to E. coli K12, used for the authors' original modeling, as well as for two other E. coli strains. Significant differences were detected between the different strains in terms of bacterial size, intermembrane distance and its positional fluctuations. These findings corroborate the general applicability of the approach outlined here to quantitatively study the effect of bactericidal compounds on ultrastructural features of Gram-negative bacteria without the need to resort to any invasive staining or labeling agents.

Bridging the Antimicrobial Activity of Two Lactoferricin Derivatives in E. coli and Lipid-Only Membranes.

We coupled the antimicrobial activity of two well-studied lactoferricin derivatives, LF11-215 and LF11-324, in Escherichia coli and different lipid-only mimics of its cytoplasmic membrane using a common thermodynamic framework for peptide partitioning. In particular, we combined an improved analysis of microdilution assays with ζ-potential measurements, which allowed us to discriminate between the maximum number of surface-adsorbed peptides and peptides fully partitioned into the bacteria. At the same time, we measured the partitioning of the peptides into vesicles composed of phosphatidylethanolamine (PE), phosphatidylgylcerol (PG), and cardiolipin (CL) mixtures using tryptophan fluorescence and determined their membrane activity using a dye leakage assay and small-angle X-ray scattering. We found that the vast majority of LF11-215 and LF11-324 readily enter inner bacterial compartments, whereas only 1-5% remain surface bound. We observed comparable membrane binding of both peptides in membrane mimics containing PE and different molar ratios of PG and CL. The peptides' activity caused a concentration-dependent dye leakage in all studied membrane mimics; however, it also led to the formation of large aggregates, part of which contained collapsed multibilayers with sandwiched peptides in the interstitial space between membranes. This effect was least pronounced in pure PG vesicles, requiring also the highest peptide concentration to induce membrane permeabilization. In PE-containing systems, we additionally observed an effective shielding of the fluorescent dyes from leakage even at highest peptide concentrations, suggesting a coupling of the peptide activity to vesicle fusion, being mediated by the intrinsic lipid curvatures of PE and CL. Our results thus show that LF11-215 and LF11-324 effectively target inner bacterial components, while the stored elastic stress makes membranes more vulnerable to peptide translocation.

Increasing complexity in small-angle X-ray and neutron scattering experiments: from biological membrane mimics to live cells.

Small-angle X-ray and neutron scattering are well-established, non-invasive experimental techniques to interrogate global structural properties of biological membrane mimicking systems under physiologically relevant conditions. Recent developments, both in bottom-up sample preparation techniques for increasingly complex model systems, and in data analysis techniques have opened the path toward addressing long standing issues of biological membrane remodelling processes. These efforts also include emerging quantitative scattering studies on live cells, thus enabling a bridging of molecular to cellular length scales. Here, we review recent progress in devising compositional models for joint small-angle X-ray and neutron scattering studies on diverse membrane mimics - with a specific focus on membrane structural coupling to amphiphatic peptides and integral proteins - and live Escherichia coli. In particular, we outline the present state-of-the-art in small-angle scattering methods applied to complex membrane systems, highlighting how increasing system complexity must be followed by an advance in compositional modelling and data-analysis tools.

Experimental concepts for linking the biological activities of antimicrobial peptides to their molecular modes of action.

The search for novel compounds to combat multi-resistant bacterial infections includes exploring the potency of antimicrobial peptides and derivatives thereof. Complementary to high-throughput screening techniques, biophysical and biochemical studies of the biological activity of these compounds enable deep insight, which can be exploited in designing antimicrobial peptides with improved efficacy. This approach requires the combination of several techniques to study the effect of such peptides on both bacterial cells and simple mimics of their cell envelope, such as lipid-only vesicles. These efforts carry the challenge of bridging results across techniques and sample systems, including the proper choice of membrane mimics. This review describes some important concepts toward the development of potent antimicrobial peptides and how they translate to frequently applied experimental techniques, along with an outline of the biophysics pertaining to the killing mechanism of antimicrobial peptides.

Chemoenzymatic Approaches to the Synthesis of the Calcimimetic Agent Cinacalcet Employing Transaminases and Ketoreductases.

Several chemoenzymatic routes have been explored for the preparation of cinacalcet, a calcimimetic agent. Transaminases (TAs) and ketoreductases (KREDs) turned out to be useful biocatalysts for the preparation of key optically active precursors. Thus, the asymmetric amination of 1-acetonaphthone yielded an enantiopure (R)-amine, which can be alkylated in one step to yield cinacalcet. Alternatively, the bioreduction of the same ketone resulted in an enantiopure (S)-alcohol, which was easily converted into the previous (R)-amine. In addition, the reduction was efficiently performed with the KRED and its cofactor co-immobilized on the same porous surface. This self-sufficient heterogeneous biocatalyst presented an accumulated total turnover number (TTN) for the cofactor of 675 after 5 consecutive operational cycles. Finally, in a preparative scale synthesis the TA-based approach was performed in aqueous medium and led to enantiopure cinacalcet in two steps and 50% overall yield.

Access to the Enantiopure Axially Chiral Cyclophane Isoplagiochin†D through Atropo-diastereoselective Heck Coupling.

Macrocyclization is typically the key step in the syntheses of cyclophane-type natural products. Considering cyclophanes with axially chiral biaryl moieties, the control of atroposelectivity is essential with biological activity and is synthetically challenging. We report an atroposelective approach involving Heck cyclization, which for the first time enables the total synthesis of an enantiopure macrocyclic bis(bibenzyl), namely isoplagiochin D. An enantiopure sulfinyl auxiliary in the ortho position of a biaryl axis (still flexible) was used to induce an atropo-diastereoselective Heck coupling (up to 98 % de). The traceless character of the sulfinyl auxiliary enables the introduction of a hydroxy group to give the target molecule with 98 % ee as well.

Anti-inflammatory Effects of Fungal Metabolites in Mouse Intestine as Revealed by In vitro Models.

Inflammatory bowel diseases (IBD), which include Crohn's disease and ulcerative colitis, are chronic inflammatory disorders that can affect the whole gastrointestinal tract or the colonic mucosal layer. Current therapies aiming to suppress the exaggerated immune response in IBD largely rely on compounds with non-satisfying effects or side-effects. Therefore, new therapeutical options are needed. In the present study, we investigated the anti-inflammatory effects of the fungal metabolites, galiellalactone, and dehydrocurvularin in both an in vitro intestinal inflammation model, as well as in isolated myenteric plexus and enterocyte cells. Administration of a pro-inflammatory cytokine mix through the mesenteric artery of intestinal segments caused an up-regulation of inflammatory marker genes. Treatment of the murine intestinal segments with galiellalactone or dehydrocurvularin by application through the mesenteric artery significantly prevented the expression of pro-inflammatory marker genes on the mRNA and the protein level. Comparable to the results in the perfused intestine model, treatment of primary enteric nervous system (ENS) cells from the murine intestine with the fungal compounds reduced expression of cytokines such as IL-6, TNF-α, IL-1ß, and inflammatory enzymes such as COX-2 and iNOS on mRNA and protein levels. Similar anti-inflammatory effects of the fungal metabolites were observed in the human colorectal adenocarcinoma cell line DLD-1 after stimulation with IFN-γ (10 ng/ml), TNF-α (10 ng/ml), and IL-1ß (5 ng/ml). Our results show that the mesenterially perfused intestine model provides a reliable tool for the screening of new therapeutics with limited amounts of test compounds. Furthermore, we could characterize the anti-inflammatory effects of two novel active compounds, galiellalactone, and dehydrocurvularin which are interesting candidates for studies with chronic animal models of IBD.

The German Version of the Gaze Anxiety Rating Scale (GARS): Reliability and Validity.

OBJECTIVE: Fear of eye gaze and avoidance of eye contact are core features of social anxiety disorders (SAD). To measure self-reported fear and avoidance of eye gaze, the Gaze Anxiety Rating Scale (GARS) has been developed and validated in recent years in its English version. The main objectives of the present study were to psychometrically evaluate the German translation of the GARS concerning its reliability, factorial structure, and validity. METHODS: Three samples of participants were enrolled in the study. (1) A non-patient sample (n = 353) completed the GARS and a set of trait questionnaires to assess internal consistency, test-retest reliability, factorial structure, and concurrent and divergent validity. (2) A sample of patients with SAD (n = 33) was compared to a healthy control group (n = 30) regarding their scores on the GARS and the trait measures. RESULTS: The German GARS fear and avoidance scales exhibited excellent internal consistency and high stability over 2 and 4 months, as did the original version. The English version's factorial structure was replicated, yielding two categories of situations: (1) everyday situations and (2) situations involving high evaluative threat. GARS fear and avoidance displayed convergent validity with trait measures of social anxiety and were markedly higher in patients with GSAD than in healthy controls. Fear and avoidance of eye contact in situations involving high levels of evaluative threat related more closely to social anxiety than to gaze anxiety in everyday situations. CONCLUSIONS: The German version of the GARS has demonstrated reliability and validity similar to the original version, and is thus well suited to capture fear and avoidance of eye contact in different social situations as a valid self-report measure of social anxiety and related disorders in the social domain for use in both clinical practice and research.