New Safety Aspects in Corneal Donation-Studies on SARS-CoV-2-Positive Corneal Donors.

In the tissue donation field, to prevent pathogen transmission, all donors are screened by postmortem swabs for SARS-CoV-2 using qRT-PCR. Corneas from donors who tested positive for SARS-CoV-2 were subjected to further investigations. Corneal transplants and culture medium from positive donors were cultured under appropriate safety conditions for further analyses. Cornea tissue samples, including sclera/limbus/cornea, and culture media were taken at different time points for testing for SARS-CoV-2 using qRT-PCR, immunohistochemistry (IHC) and subgenomic RNA (sgRNA) analysis. Between January and May 2021, in four donors with initial negative premortem rapid tests, SARS-CoV-2 was detected post-mortem using qRT-PCR. In these cases, SARS-CoV-2 was observed at the beginning of cultivation in both tissue and culture medium using qRT-PCR and IHC. The virus was mainly localized in the limbus epithelial cells, with a stable detection level. Premortem rapid tests are potentially insufficient to exclude SARS-CoV-2 infection in corneal donors. While, for SARS-CoV-2, the risk of infection via transplants is considered low, a residual risk remains for presymptomatic new infections. However, our investigations provide the first indications that, with organ cultures, the risk of virus transmission is minimized due to the longer minimum culture period.

Cell-type-specific metabolic labeling of nascent proteomes in vivo.

Although advances in protein labeling methods have made it possible to measure the proteome of mixed cell populations, it has not been possible to isolate cell-type-specific proteomes in vivo. This is because the existing methods for metabolic protein labeling in vivo access all cell types. We report the development of a transgenic mouse line where Cre-recombinase-induced expression of a mutant methionyl-tRNA synthetase (L274G) enables the cell-type-specific labeling of nascent proteins with a non-canonical amino-acid and click chemistry. Using immunoblotting, imaging and mass spectrometry, we use our transgenic mouse to label and analyze proteins in excitatory principal neurons and Purkinje neurons in vitro (brain slices) and in vivo. We discover more than 200 proteins that are differentially regulated in hippocampal excitatory neurons by exposing mice to an environment with enriched sensory cues. Our approach can be used to isolate, analyze and quantitate cell-type-specific proteomes and their dynamics in healthy and diseased tissues.

NONO couples the circadian clock to the cell cycle.

Mammalian circadian clocks restrict cell proliferation to defined time windows, but the mechanism and consequences of this interrelationship are not fully understood. Previously we identified the multifunctional nuclear protein NONO as a partner of circadian PERIOD (PER) proteins. Here we show that it also conveys circadian gating to the cell cycle, a connection surprisingly important for wound healing in mice. Specifically, although fibroblasts from NONO-deficient mice showed approximately normal circadian cycles, they displayed elevated cell doubling and lower cellular senescence. At a molecular level, NONO bound to the p16-Ink4A cell cycle checkpoint gene and potentiated its circadian activation in a PER protein-dependent fashion. Loss of either NONO or PER abolished this activation and circadian expression of p16-Ink4A and eliminated circadian cell cycle gating. In vivo, lack of NONO resulted in defective wound repair. Because wound healing defects were also seen in multiple circadian clock-deficient mouse lines, our results therefore suggest that coupling of the cell cycle to the circadian clock via NONO may be useful to segregate in temporal fashion cell proliferation from tissue organization.

Screening for propagation suitability in vitro of different Cyclamen species / Revisión para la adecuación de propagación in vitro de diferentes especies de Cyclamen

In the present study we examined the possibility of propagating different Cyclamen species (C. africanum Boiss. and Reut., C. cilicium Boiss. and Heldr., C. coum Mill., C. hederifolium Ait., C. persicum Mill., C. purpurascens Mill.) including some of their subspecies and cultivars in vitro using explants of adult plants. For this purpose two protocols have been applied to eleven genotypes combined with mostly four explant types (placentas with ovules, leaves, petioles and peduncles). The use of these protocols has given rise to either somatic embryo-like structures and/or adventitious shoots in all genotypes. This way it was possible to propagate each of the examined genotypes in vitro using explants of adult plants in a time less than one year. These results may be used in breeding and propagation of Cyclamen as an ornamental plant and as a medicinal plant.

Bacillary angiomatosis.

An infection with Bartonella henselae transmitted from domestic cats to humans by scratching normally leads to cat-scratch disease. When the human host has severe immunosuppression or HIV infection, the potentially life-threatening disease bacillary angiomatosis can develop. A 79-year-old man presented with livid-erythematous, angioma-like skin lesions. We considered a cutaneous infiltrate from his known chronic lymphocytic leukemia, Merkel cell carcinoma, cutaneous metastases of internal tumors, cutaneous sarcoidosis, mycobacterial infection and even atypical herpes simplex infection. The correct diagnosis was proven histologically and by PCR. Because of increasing numbers of immunosuppressed and HIV-positive patients, as well as an infection rate of 13% for B. henselae in domestic cats in Germany, one must be alert to the presence of bacillary angiomatosis.

Human leukocyte antigen-DR expression in peripheral blood mononuclear cells from healthy donors influenced by the sera of injured patients prone to severe sepsis.

OBJECTIVE: To study the influence of sera from severely injured patients on the human leukocyte antigen (HLA)-DR expression of normal peripheral blood mononuclear cells (PBMC). DESIGN: In vitro study. SETTING: University hospital. PATIENTS AND PARTICIPANTS: Sera from 34 patients were obtained within 8 h after trauma. Seventeen of these patients developed posttraumatic sepsis (sepsis group) and 17 recovered without infectious complications. Sera from ten healthy individuals served as controls. Phytohemagglutinin (PHA)-activated PBMC from 44 healthy donors were used to study the effects of a patient's serum. MEASUREMENTS AND RESULTS: Medium containing 5% of serum from the sepsis group significantly ( p<0.05) reduced the HLA-DR expression (channels, mean +/- standard error of the mean) on monocytes (patients 883+/-22, controls 962+/-15), B (patients 922+/-14, controls 972+/-7) and T cells (patients 932+/-13, controls 968+/-5) of PHA-activated PBMC. Significantly increased accumulation of TNFalpha on (1.8+/-0.4% of PBMC) and within T cells (0.98+/-0.26% of PBMC) was observed by flow cytometry after incubation with medium containing sera of the sepsis group compared with controls (on 0.5+/-0.1%, within 0.27+/-0.05% of PBMC). A significant negative correlation between relative cell counts of intracellular TNFalpha-positive T cells with HLA-DR expression was observed for monocytes ( r= -0.61), B cells ( r= -0.57) and proliferation ( r= -0.68) as estimated by (3)H-thymidine uptake [patients 139971+/-12844 counts per minute (cpm), controls 198973+/-19347 cpm, p<0.05] in the presence of sera from the sepsis group. CONCLUSIONS: Reduced cellular immunity and, therefore, immunodeficiency after trauma appears to be caused by soluble factors influencing T cell function in particular.