Flat Γ Moiré Bands in Twisted Bilayer WSe_{2}.

The recent observation of correlated phases in transition metal dichalcogenide moiré systems at integer and fractional filling promises new insight into metal-insulator transitions and the unusual states of matter that can emerge near such transitions. Here, we combine real- and momentum-space mapping techniques to study moiré superlattice effects in 57.4° twisted WSe_{2} (tWSe_{2}). Our data reveal a split-off flat band that derives from the monolayer Γ states. Using advanced data analysis, we directly quantify the moiré potential from our data. We further demonstrate that the global valence band maximum in tWSe_{2} is close in energy to this flat band but derives from the monolayer K states which show weaker superlattice effects. These results constrain theoretical models and open the perspective that Γ-valley flat bands might be involved in the correlated physics of twisted WSe_{2}.

Altered lipid metabolism in synovial fibroblasts of individuals at risk of developing rheumatoid arthritis.

OBJECTIVE: Fibroblast-like synoviocytes (FLS) can augment the inflammatory process observed in synovium of patients with rheumatoid arthritis (RA). A recent transcriptomic study in synovial biopsies revealed changes in metabolic pathways before disease onset in absence of synovial tissue inflammation. This raises the question whether alterations in cellular metabolism in tissue resident FLS underlie disease pathogenesis. MATERIALS AND METHODS: To study this, we compared the metabolic profile of FLS isolated from synovial biopsies from individuals with arthralgia who were autoantibody positive but without any evidence of arthritis (RA-risk individuals, n = 6) with FLS from patients with RA (n = 6), osteoarthritis (OA, n = 6) and seronegative controls (n = 6). After synovial digestion, FLS were cultured in vitro and cellular metabolism was assessed using quantitative PCR, flow cytometry, XFe96 Seahorse Analyzer and tritium-labelled oleate oxidation assays. RESULTS: Real-time metabolic profiling revealed that basal (p < 0.0001) and maximum mitochondrial respiration (p = 0.0024) were significantly lower in RA FLS compared with control FLS. In all donors, basal respiration was largely dependent on fatty acid oxidation while glucose was only highly used by FLS from RA patients. Moreover, we showed that RA-risk and RA FLS are less metabolically flexible. Strikingly, mitochondrial fatty acid ß-oxidation was significantly impaired in RA-risk (p = 0.001) and RA FLS (p < 0.0001) compared with control FLS. CONCLUSION: Overall, this study showed several metabolic alterations in FLS even in absence of synovial inflammation, suggesting that these alterations already start before clinical manifestation of disease and may drive disease pathogenesis.

Quantitative analysis of spectroscopic low energy electron microscopy data: High-dynamic range imaging, drift correction and cluster analysis.

For many complex materials systems, low-energy electron microscopy (LEEM) offers detailed insights into morphology and crystallography by naturally combining real-space and reciprocal-space information. Its unique strength, however, is that all measurements can easily be performed energy-dependently. Consequently, one should treat LEEM measurements as multi-dimensional, spectroscopic datasets rather than as images to fully harvest this potential. Here we describe a measurement and data analysis approach to obtain such quantitative spectroscopic LEEM datasets with high lateral resolution. The employed detector correction and adjustment techniques enable measurement of true reflectivity values over four orders of magnitudes of intensity. Moreover, we show a drift correction algorithm, tailored for LEEM datasets with inverting contrast, that yields sub-pixel accuracy without special computational demands. Finally, we apply dimension reduction techniques to summarize the key spectroscopic features of datasets with hundreds of images into two single images that can easily be presented and interpreted intuitively. We use cluster analysis to automatically identify different materials within the field of view and to calculate average spectra per material. We demonstrate these methods by analyzing bright-field and dark-field datasets of few-layer graphene grown on silicon carbide and provide a high-performance Python implementation.

Inhibition of rat colon tumors by sulindac and sulindac sulfone is independent of K-ras (codon 12) mutation.

Nonsteroidal anti-inflammatory drug (NSAID) use reduces the risk of colorectal cancer by 40-50%. Previous studies suggest that effective inhibition of colorectal cancer by NSAIDs may be dependent on the presence or absence of a K-ras mutation. This study was aimed at determining the relationship between inhibition of colorectal cancer by sulindac and sulindac sulfone and the presence of activating K-ras mutations in the 1,2-dimethylhydrazine dihydrochloride rat model. Sulindac (20 mg x kg(-1) x day(-1)), sulindac sulfone (40 mg x kg(-1) x day(-1)), or vehicle was administered orally to male Sprague-Dawley rats for a 4-wk period beginning 20 wk after tumor induction. Tumor number and volume were measured before treatment by laparotomy and colonoscopy and again after treatment. Sulindac and sulindac sulfone treatment significantly reduced the number and volume of colorectal tumors compared with control rats. For K-ras (codon 12) mutation detection, frozen tumor tissue was collected at the endpoint. We found K-ras codon 12 mutations in 11 of 21 (52%) control tumors. The proportion of tumors with K-ras mutations in the sulindac-treated group [5 of 8 (62%); odds ratio = 1.51 (95% confidence interval = 0.29, 8.33)] and the proportion of sulindac sulfone-treated tumors [9 of 14 (64%); odds ratio = 1.63 (95% confidence interval = 0.41, 6.66)] were not significantly different from controls. Tumor inhibition did not correlate with K-ras (codon 12) mutation status, which suggests that the mechanism of inhibition of rat colorectal cancer by sulindac and sulindac sulfone is independent of K-ras mutation.

A cytosolic granzyme B inhibitor related to the viral apoptotic regulator cytokine response modifier A is present in cytotoxic lymphocytes.

Using a polymerase chain reaction strategy we identified a serine proteinase inhibitor (serpin) in human bone marrow that is related to the cellular serpin proteinase inhibitor 6 (PI-6) and the viral serpin cytokine response modifier A (CrmA). This serpin, proteinase inhibitor 9 (PI-9), has an unusual reactive center P1(Glu)-P1'(Cys), which suggests that it inhibits serine proteinases that cleave after acidic residues. The only known serine proteinase with this specificity is granzyme B, a granule cytotoxin produced by cytotoxic lymphocytes. To test the interaction of PI-9 with granzyme B we prepared recombinant hexa-histidine tagged PI-9 in a yeast expression system. Addition of the recombinant protein to native granzyme B resulted in an SDS-resistant complex typical of serpin-serine proteinase interactions. Further analysis showed that complex formation followed bimolecular kinetics with a second order rate constant of 1.7 +/- 0.3 x 10(6) M-1 s-1, which is in the range for a physiologically significant serpin-proteinase interaction. Recombinant PI-9 also completely abrogated granzyme B and perforin-mediated cytotoxicity in vitro. Examination of PI-9 mRNA distribution demonstrated that it is expressed in immune tissue, primarily in lymphocytes. The highest levels of PI-9 mRNA and protein were observed in natural killer cell leukemia cell lines and in interleukin-2 stimulated peripheral blood mononuclear cells, which also produce granzyme B. Like PI-6, PI-9 was shown to be a cytosolic protein that is not secreted. Fractionation of natural killer cells and stimulated peripheral blood mononuclear cells demonstrated that PI-9 is in a separate subcellular compartment to granzyme B. These results suggest that PI-9 serves to inactivate misdirected granzyme B following cytotoxic cell degranulation. This may explain why cytotoxic cells are not damaged by their own granzyme B during destruction of abnormal cells.

Immunolocalization of interleukin-4 in eosinophils in the bronchial mucosa of atopic asthmatics.

Several studies have shown that human eosinophils can synthesize and release a number of cytokines. The aim of this study was to investigate whether eosinophils contain interleukin (IL)-4 protein. We examined the ultrastructural localization of IL-4 in eosinophils in bronchial mucosal biopsies of 29 nonsmoking atopic asthmatics and 7 controls. No eosinophils were detected in the bronchial mucosa of controls. In the eosinophils (n = 42) of the asthmatics, IL-4 was localized to the electron-dense crystalloid core compartment of 85% of the secondary or specific eosinophil granules (n = 468). Other structures in the eosinophils were unlabeled. Control sections, incubated with an irrelevant primary antibody, were negative. This study demonstrates that pre-formed IL-4 is stored in the secondary eosinophil granules. These results were extended by light microscopic immunodouble-staining for IL-4 protein and eosinophil cationic protein, which showed that a subpopulation of activated eosinophils express IL-4 immunoreactivity in bronchial mucosal biopsies of asthmatics as well as controls. These data indicate that eosinophils may be an important source of IL-4 in allergic inflammation.

Ultrastructural immunogold localization of interleukin 5 to the crystalloid core compartment of eosinophil secondary granules in patients with atopic asthma.

Bronchial biopsies from two patients with atopic asthma were analyzed by immunogold labeling to detect the ultrastructural location of interleukin 5 (IL-5). In eosinophils, IL-5 was localized to the electron-dense crystalloid core compartment of the secondary or specific eosinophil granules. Other structures in the eosinophils were unlabeled. No IL-5 was detected in mast cells. Control sections incubated with an irrelevant primary antibody were negative. This study demonstrates that pre-formed IL-5 is stored within the major population of secondary granules in human eosinophils.

Expression of cytokeratin 8 in basal cell carcinoma: a comparative immunohistochemical and immunoelectron microscopy study.

The comparative study reported here was undertaken in order to resolve the discrepancies in the detection of cytokeratin (Ck) 8 reported in previous studies. The expression of Ck 8 was compared in 6 basal cell carcinomas (BCCs) using immunohistochemical and immunoelectron microscopic techniques and a panel of 4 different commercially available monoclonal antibodies (MoAbs). The results of this comparative study demonstrated not only that the consistent expression of Ck 8 using one of the MoAbs in immunohistochemistry was confirmed by immunoelectron microscopy, but that the inconsistent expression of Ck 8 observed using two other MoAbs was also confirmed. One of the MoAbs did not show any staining at all. The inability of this MoAb to detect the expression of Ck 8 using either of the techniques also indicated that this MoAb may be directed against an epitope of Ck8 that is not detectable in BCC in situ.

Neurofibromatosis type 2 protein co-localizes with elements of the cytoskeleton.

The product of the neurofibromatosis type 2 (NF2) tumor suppressor gene is a 595-amino-acid protein bearing resemblance to a family of band-4.1-related proteins. These proteins, including ezrin, radixin, and moesin, probably function as molecular linking proteins, connecting the cytoskeleton to the cell membrane. On the grounds of the homology to the ezrin, radixin, and moesin proteins and on the basis of its predicted secondary structure, the NF2 protein is also thought to act as a cytoskeleton-cell membrane linking protein. Using monoclonal antibodies to amino- and carboxyl-terminal synthetic NF2 peptides we demonstrate the co-localization of the NF2 protein with elements of the cytoskeleton in a COS cell model system and in cultured human cells. Furthermore, the presence of the NF2 protein in tissue sections is shown. The monoclonal antibodies specifically stain smooth muscle cells and the stratum granulosum of the human epidermis. In cultured smooth muscle cells the NF2 protein co-localizes with actin stress fibers. Immunoelectron microscopy demonstrates the presence of the NF2 protein associated with keratohyalin granules and to a lesser extent with intermediate filaments in the human epidermis. We conclude that the NF2 protein is indeed associated with multiple elements of the cytoskeleton.

Genome type analysis of adenovirus 37 isolates.

Seventy-six strains of adenovirus type 37 (AV37), isolated during a period of 10 years from 73 patients on three continents, were analysed with eight DNA restriction endonucleases. Strains identical with the prototype (56 strains) prevailed; four deviating genome types were found, which differed only slightly from the prototype, with one exception. Half of the strains were tested in detail by neutralisation and haemagglutination inhibition. Minor differences in neutralisation were not reflected by differences in DNA restriction patterns. In comparison to genome types of other subgenera, field strains of adenovirus 37 showed only a moderate genetic variability.