Quadratic Soliton Frequency Comb at 4 µm from an OP-GaP-based Optical Parametric Oscillator

We report generation of quadratic solitons, i.e. temporal simultons, in an OP-GaP based halfharmonic optical parametric oscillator. We achieve 4-µm pulses with sech² spectrum of 790nm FWHM bandwidth, 197% slope efficiency, and 38% conversion efficiency

The Spatial Distribution of LGR5+ Cells Correlates With Gastric Cancer Progression

In this study we tested the prevalence, histoanatomical distribution and tumour biological significance of the Wnt target protein and cancer stem cell marker LGR5 in tumours of the human gastrointestinal tract. Differential expression of LGR5 was studied on transcriptional (real-time polymerase chain reaction) and translational level (immunohistochemistry) in malignant and corresponding non-malignant tissues of 127 patients comprising six different primary tumour sites, i.e. oesophagus, stomach, liver, pancreas, colon and rectum. The clinico-pathological significance of LGR5 expression was studied in 100 patients with gastric carcinoma (GC). Non-neoplastic tissue usually harboured only very few scattered LGR5+ cells. The corresponding carcinomas of the oesophagus, stomach, liver, pancreas, colon and rectum showed significantly more LGR5+ cells as well as significantly higher levels of LGR5-mRNA compared with the corresponding non-neoplastic tissue. Double staining experiments revealed a coexpression of LGR5 with the putative stem cell markers CD44, Musashi-1 and ADAM17. Next we tested the hypothesis that the sequential changes of gastric carcinogenesis, i.e. chronic atrophic gastritis, intestinal metaplasia and invasive carcinoma, are associated with a reallocation of the LGR5+ cells. Interestingly, the spatial distribution of LGR5 changed: in non-neoplastic stomach mucosa, LGR5+ cells were found predominantly in the mucous neck region; in intestinal metaplasia LGR5+ cells were localized at the crypt base, and in GC LGR5+ cells were present at the luminal surface, the tumour centre and the invasion front. The expression of LGR5 in the tumour centre and invasion front of GC correlated significantly with the local tumour growth (T-category) and the nodal spread (N-category). Furthermore, patients with LGR5+ GCs had a shorter median survival (28.0±8.6 months) than patients with LGR5− GCs (54.5±6.3 months). Our results show that LGR5 is differentially expressed in gastrointestinal cancers and that the spatial histoanatomical distribution of LGR5+ cells has to be considered when their tumour biological significance is sought

FSP1 is a glutathione-independent ferroptosis suppressor

Ferroptosis is an iron-dependent form of necrotic cell death marked by oxidative damage to phospholipids1,2. To date, ferroptosis has been believed to be controlled only by the phospholipid hydroperoxide-reducing enzyme glutathione peroxidase 4 (GPX4)3,4 and radical-trapping antioxidants5,6. However, elucidation of the factors that underlie the sensitivity of a given cell type to ferroptosis7 is critical to understand the pathophysiological role of ferroptosis and how it may be exploited for the treatment of cancer. Although metabolic constraints8 and phospholipid composition9,10 contribute to ferroptosis sensitivity, no cell-autonomous mechanisms have been identified that account for the resistance of cells to ferroptosis. Here we used an expression cloning approach to identify genes in human cancer cells that are able to complement the loss of GPX4. We found that the flavoprotein apoptosis-inducing factor mitochondria-associated 2 (AIFM2) is a previously unrecognized anti-ferroptotic gene. AIFM2, which we renamed ferroptosis suppressor protein 1 (FSP1) and which was initially described as a pro-apoptotic gene11, confers protection against ferroptosis elicited by GPX4 deletion. We further demonstrate that the suppression of ferroptosis by FSP1 is mediated by ubiquinone (also known as coenzyme Q10 (CoQ10)): the reduced form, ubiquinol, traps lipid peroxyl radicals that mediate lipid peroxidation, whereas FSP1 catalyses the regeneration of CoQ10 using NAD(P)H. Pharmacological targeting of FSP1 strongly synergizes with GPX4 inhibitors to trigger ferroptosis in a number of cancer entities. In conclusion, the FSP1–CoQ10–NAD(P)H pathway exists as a stand-alone parallel system, which co-operates with GPX4 and glutathione to suppress phospholipid peroxidation and ferroptosis

Garbage in, garbage out: how reliable training data improved a virtual screening approach against SARS-CoV-2 MPro

Introduction: The identification of chemical compounds that interfere with SARS-CoV-2 replication continues to be a priority in several academic and pharmaceutical laboratories. Computational tools and approaches have the power to integrate, process and analyze multiple data in a short time. However, these initiatives may yield unrealistic results if the applied models are not inferred from reliable data and the resulting predictions are not confirmed by experimental evidence.Methods: We undertook a drug discovery campaign against the essential major protease (MPro) from SARS-CoV-2, which relied on an in silico search strategy –performed in a large and diverse chemolibrary– complemented by experimental validation. The computational method comprises a recently reported ligand-based approach developed upon refinement/learning cycles, and structure-based approximations. Search models were applied to both retrospective (in silico) and prospective (experimentally confirmed) screening.Results: The first generation of ligand-based models were fed by data, which to a great extent, had not been published in peer-reviewed articles. The first screening campaign performed with 188 compounds (46 in silico hits and 100 analogues, and 40 unrelated compounds: flavonols and pyrazoles) yielded three hits against MPro (IC50 ≤ 25 μM): two analogues of in silico hits (one glycoside and one benzo-thiazol) and one flavonol. A second generation of ligand-based models was developed based on this negative information and newly published peer-reviewed data for MPro inhibitors. This led to 43 new hit candidates belonging to different chemical families. From 45 compounds (28 in silico hits and 17 related analogues) tested in the second screening campaign, eight inhibited MPro with IC50 = 0.12–20 μM and five of them also impaired the proliferation of SARS-CoV-2 in Vero cells (EC50 7–45 μM).Discussion: Our study provides an example of a virtuous loop between computational and experimental approaches applied to target-focused drug discovery against a major and global pathogen, reaffirming the well-known “garbage in, garbage out” machine learning principle

0.5-W Few-Cycle Frequency Comb at 4 μm from an Efficient Simulton-based Optical Parametric Oscillator

We report generation of three-cycle pulses at 4.18 μm with 565-mW average power, 900-nm instantaneous FWHM bandwidth, 350% slope efficiency, and 44% conversion efficiency, based on a half-harmonic optical parametric oscillator operating in simulton regime

<i>Ko</i>^<i>G</i> mice are viable, but fail to gain weight after weaning.

<p>(A) Scheme of the skin. Keratinocytes of the epidermis and the hair follicles/sebaceous glands, which are affected by the knockout, are shown in blue. Mice heterozygous for the <i>K5Cre</i> transgene and homozygous for the floxed <i>Gclc</i> allele (ho-Gclc^floxed) lack Gclc in keratinocytes (<i>ko</i>^<i>G</i> mice) (B) qRT-PCR of <i>Gclc</i> relative to <i>Rps29</i> using RNA from primary keratinocytes (1°KC; N = 6) or epidermis (3W N = 5; 2M N = 7). (C) Total GSH/GSSG levels (GSH_T) in 1°KC (N = 6/4) and epidermis (3W N = 5; 2M N = 3). (D) Western blot with lysates from epidermis (pooled from 2 mice per genotype), total skin, and liver for Gclc and Gapdh (loading control). (E) Macroscopic appearance of <i>ko</i>^<i>G</i> and <i>ctrl</i> mice at 3W and 2M. (F) Body weight of <i>ctrl</i> and <i>ko</i>^<i>G</i> mice surviving until 2M. Arrow indicates **<i>P</i> ≤ 0.01 for each time point after P23. N = 7/5. (G) Kaplan-Meier survival curves of <i>ko</i>^<i>G</i> versus <i>ctrl</i> mice. N = 14. Significance was analyzed using Log-rank test. *<i>P</i> ≤ 0.05. (H) Hematoxylin/eosin (H&E) staining of stomach sections from mice at 2M. Scale bar: 1 mm. Squares indicate the area shown at higher magnification on the right side. Note the hyperkeratosis in the forestomach of <i>ko</i>^<i>G</i> mice. (I) Picture of the stomach from <i>ko</i>^<i>G</i> and <i>ctrl</i> mice at 2M. Scale bar: 0.5 cm. Scatter plots show the median with interquartile range.</p

Cell damage and apoptosis in the hyperproliferative wound epithelium of Gclc-deficient mice.

<p>(A,B) Immunofluorescence-stained sections of 2-, 3- and 5-day wounds and quantification of (A) cleaved caspase-3 (arrowheads; arrows: positive cells in the adjacent epidermis), and (B) γH2AX positive cells per area HE. White dotted lines show the area of HE in which positive cells were counted. Each picture includes a magnification of the indicated square to show positive cells (if existing). Scale bar: 100 μm. For (A): 2dw N = 6; 3dw N = 6; 5dw N = 10/8. For (B): 2dw N = 4; 3dw N = 7/8; 5dw N = 5. (C) OxyBlot of lysates (pooled from 2 wounds of individual mice) from 3-day wounds. Equal loading was confirmed by Ponceau S staining of the membrane. (D) H&E staining on sections from 3-day wounds. Scale bar: 200 μm. D: dermis Es: eschar, G: granulation tissue, HF: hair follicle, HE: hyperproliferative epithelium. (E-G) Morphometric analysis of (E) percentage wound closure, (F) length HE and (G) area HE of 3-day wounds of <i>ko</i>^<i>G/N</i> and control mice. For (E): N = 6/7; for (F): N = 7/8; for (G): N = 7/8. (H-J) Quantification of (H) Ki-67 (N = 4), (I) cleaved caspase-3 (N = 3/4), and (J) γH2AX (N = 6/5) positive cells per area HE. Scatter plots show the median with interquartile range. *<i>P</i> ≤ 0.05. n.d.: not detectable.</p

Nrf2 is activated in <i>ko</i>^<i>G</i> mice, but does not compensate for the loss of Gclc.

<p>(A,B) qRT-PCR of <i>Nqo1</i>, <i>Srxn1</i>, <i>Slpi</i> and <i>Sprr2d</i> relative to <i>Rps29</i> using RNA from (A) the epidermis at 3W (N = 5) and from (B) primary keratinocytes (N = 11/9). (C) Scheme of the skin from mice of all four genotypes used for the analysis. Keratinocytes affected by the knockout are shown in the respective color. (D) qRT-PCR of <i>Gclc</i> and <i>Nrf2</i> relative to <i>Rps29</i> using RNA from epidermis at 3W (left panel; N = 8/8/7/8) or primary keratinocytes of control and <i>ko</i>^<i>G/N</i> mice (right panel; N = 8). (E,F) qRT-PCR of <i>Nqo1</i>, <i>Srxn1</i>, <i>Slpi</i>, and <i>Sprr2d</i> relative to <i>Rps29</i> using RNA from the epidermis at 3W (N = 7-8/7-8/5-6/7-8) and of <i>Nqo1</i>, <i>Srxn1</i>, <i>Slpi</i> relative to <i>Rps29</i> using RNA from primary keratinocytes (N = 15/8/7). (G) Macroscopic appearance of mice of all four genotypes at 3W. (H) H&E staining of longitudinal skin sections from mice at 3W and immunofluorescence staining for epidermal differentiation markers. Nuclei were counterstained with Hoechst. Scale bars: 40 μm. (I) Immunofluorescence staining of skin sections for Ki-67 and quantification of positive cells per length epidermis at 3W (N = 22/11/6/8). Scale bar: 20 μm. White dotted lines indicate the basement membrane. (J) TEWL of mice at 3W (N = 25/11/7/8). (K) Body weight of mice at 3W (N = 26/11/5/11). (L) H&E staining of skin from <i>ko</i>^<i>G/N</i> mice at 3W demonstrating malformed hair follicles and cyst formation. The number of abnormal hair follicles per length epidermis is shown in the bar graph (N = 9/9/6/8; n.d. = not detectable). Scale bar: 40 μm. Scatter plots show the median with interquartile range. *<i>P</i> ≤ 0.05, **<i>P</i> ≤ 0.01, ***<i>P</i> ≤ 0.001.</p

Mild wound healing abnormalities in <i>ko</i>^<i>G</i> mice.

<p>(A) Schematic representation of a 3–5 day wound showing the parameters for morphometric analysis: percentage of wound closure = (I+III/I+II+III)*100; length HE = average length I+III; area HE = average area IV+V. D: dermis Es: eschar, G: granulation tissue, HF: hair follicle, HE: hyperproliferative epithelium. (B) Immunofluorescence staining of K14 (green) and Hoechst counterstaining (blue) on sections from 3- and 5-day wounds. Scale bar: 100 μm. (C-E) Morphometric analysis of (C) percentage of wound closure, (D) length HE, and (E) area HE of 2-, 3- and 5-day wounds. For (C): 2dw N = 4/7; 3dw N = 6/5; 5dw N = 9/12. For (D): 2dw N = 5/7; 3dw N = 6/5; 5dw N = 11/14. For (E): 2dw N = 5/7; 3dw N = 6/5; 5dw N = 11/14. (F) Schematic representation of the HE with the region of the cells that were analyzed in (G). (G) Immunofluorescence-stained sections of 2-, 3- and 5-day wounds were used for quantification of Ki-67 positive cells per area HE. White dotted lines show the area of HE in which positive cells were counted. Each picture includes a magnification of the indicated square to show positive cells. Scale bar = 100 μm. 2dw N = 4; 3dw N = 7/8; 5dw N = 5. Scatter plots show the median with interquartile range. **<i>P</i> ≤ 0.01.</p