Visualizing Engrafted Human Cancer and Therapy Responses in Immunodeficient Zebrafish.

Xenograft cell transplantation into immunodeficient mice has become the gold standard for assessing pre-clinical efficacy of cancer drugs, yet direct visualization of single-cell phenotypes is difficult. Here, we report an optically-clear prkdc-/-, il2rga-/- zebrafish that lacks adaptive and natural killer immune cells, can engraft a wide array of human cancers at 37°C, and permits the dynamic visualization of single engrafted cells. For example, photoconversion cell-lineage tracing identified migratory and proliferative cell states in human rhabdomyosarcoma, a pediatric cancer of muscle. Additional experiments identified the preclinical efficacy of combination olaparib PARP inhibitor and temozolomide DNA-damaging agent as an effective therapy for rhabdomyosarcoma and visualized therapeutic responses using a four-color FUCCI cell-cycle fluorescent reporter. These experiments identified that combination treatment arrested rhabdomyosarcoma cells in the G2 cell cycle prior to induction of apoptosis. Finally, patient-derived xenografts could be engrafted into our model, opening new avenues for developing personalized therapeutic approaches in the future.

Tissue-specific Grb10/Ddc insulator drives allelic architecture for cardiac development.

Allele-specific expression of imprinted gene clusters is governed by gametic DNA methylation at master regulators called imprinting control regions (ICRs). Non-gametic or secondary differentially methylated regions (DMRs) at promoters and exonic regions reinforce monoallelic expression but do not control an entire cluster. Here, we unveil an unconventional secondary DMR that is indispensable for tissue-specific imprinting of two previously unlinked genes, Grb10 and Ddc. Using polymorphic mice, we mapped an intronic secondary DMR at Grb10 with paternal-specific CTCF binding (CBR2.3) that forms contacts with Ddc. Deletion of paternal CBR2.3 removed a critical insulator, resulting in substantial shifting of chromatin looping and ectopic enhancer-promoter contacts. Destabilized gene architecture precipitated abnormal Grb10-Ddc expression with developmental consequences in the heart and muscle. Thus, we redefine the Grb10-Ddc imprinting domain by uncovering an unconventional intronic secondary DMR that functions as an insulator to instruct the tissue-specific, monoallelic expression of multiple genes-a feature previously ICR exclusive.

Ultraviolet C intensity dependence of decontamination efficiency for pathogens as function of repacked metamaterials with screw channels.

A new method for repackaging optical metamaterials formed from quartz spheres (fibers) of various diameters is proposed for ultraviolet C disinfection of infected liquids by pathogens (viruses and bacteria). The main idea of the new equipment is connected with the rotation of a contaminated fluid by screw channels within a metamaterial matrix prepared from UVC fibers/spherical optics, to improve the decontamination efficiency. In demonstration of the viability of this approach, dynamic and static inactivation of Baker's yeast via Ultraviolet C radiation regimes are used in this paper to show the efficacy of decontamination within the screw channels.

Discovery of the parasite Marteilia cocosarum sp. nov. In common cockle (Cerastoderma edule) fisheries in Wales, UK and its comparison with Marteilia cochillia.

Diseases of bivalve molluscs caused by paramyxid parasites of the genus Marteilia have been linked to mass mortalities and the collapse of commercially important shellfish populations. Until recently, no Marteilia spp. have been detected in common cockle (Cerastoderma edule) populations in the British Isles. Molecular screening of cockles from ten sites on the Welsh coast indicates that a Marteilia parasite is widespread in Welsh C. edule populations, including major fisheries. Phylogenetic analysis of ribosomal DNA (rDNA) gene sequences from this parasite indicates that it is a closely related but different species to Marteilia cochillia, a parasite linked to mass mortality of C. edule fisheries in Spain, and that both are related to Marteilia octospora, for which we provide new rDNA sequence data. Preliminary light and transmission electron microscope (TEM) observations support this conclusion, indicating that the parasite from Wales is located primarily within areas of inflammation in the gills and the connective tissue of the digestive gland, whereas M. cochillia is found mainly within the epithelium of the digestive gland. The impact of infection by the new species, here described as Marteilia cocosarum n. sp., upon Welsh fisheries is currently unknown.

Rapid and dynamic arginylation of the leading edge ß-actin is required for cell migration.

ß-actin plays key roles in cell migration. Our previous work demonstrated that ß-actin in migratory non-muscle cells is N-terminally arginylated and that this arginylation is required for normal lamellipodia extension. Here, we examined the function of ß-actin arginylation in cell migration. We found that arginylated ß-actin is concentrated at the leading edge of lamellipodia and that this enrichment is abolished after serum starvation as well as in contact-inhibited cells in confluent cultures, suggesting that arginylated ß-actin at the cell leading edge is coupled to active migration. Arginylated actin levels exhibit dynamic changes in response to cell stimuli, lowered after serum starvation and dramatically elevating within minutes after cell stimulation by readdition of serum or lysophosphatidic acid. These dynamic changes require active translation and are not seen in confluent contact-inhibited cell cultures. Microinjection of arginylated actin antibodies into cells severely and specifically inhibits their migration rates. Together, these data strongly suggest that arginylation of ß-actin is a tightly regulated dynamic process that occurs at the leading edge of locomoting cells in response to stimuli and is integral to the signaling network that regulates cell migration.

Reduced passive force in skeletal muscles lacking protein arginylation.

Arginylation is a posttranslational modification that plays a global role in mammals. Mice lacking the enzyme arginyltransferase in skeletal muscles exhibit reduced contractile forces that have been linked to a reduction in myosin cross-bridge formation. The role of arginylation in passive skeletal myofibril forces has never been investigated. In this study, we used single sarcomere and myofibril measurements and observed that lack of arginylation leads to a pronounced reduction in passive forces in skeletal muscles. Mass spectrometry indicated that skeletal muscle titin, the protein primarily linked to passive force generation, is arginylated on five sites located within the A band, an important area for protein-protein interactions. We propose a mechanism for passive force regulation by arginylation through modulation of protein-protein binding between the titin molecule and the thick filament. Key points are as follows: 1) active and passive forces were decreased in myofibrils and single sarcomeres isolated from muscles lacking arginyl-tRNA-protein transferase (ATE1). 2) Mass spectrometry revealed five sites for arginylation within titin molecules. All sites are located within the A-band portion of titin, an important region for protein-protein interactions. 3) Our data suggest that arginylation of titin is required for proper passive force development in skeletal muscles.

High risk for seizures following subarachnoid hemorrhage regardless of referral bias.

BACKGROUND: To investigate the frequency, predictors, and clinical impact of electrographic seizures in patients with high clinical or radiologic grade non-traumatic subarachnoid hemorrhage (SAH), independent of referral bias. METHODS: We compared rates of electrographic seizures and associated clinical variables and outcomes in patients with high clinical or radiologic grade non-traumatic SAH. Rates of electrographic seizure detection before and after institution of a guideline which made continuous EEG monitoring routine in this population were compared. RESULTS: Electrographic seizures occurred in 17.6 % of patients monitored expressly because of clinically suspected subclinical seizures. In unselected patients, seizures still occurred in 9.6 % of all cases, and in 8.6 % of cases in which there was no a priori suspicion of seizures. The first seizure detected occurred 5.4 (IQR 2.9-7.3) days after onset of subarachnoid hemorrhage with three of eight patients (37.5 %) having the first recorded seizure more than 48 h following EEG initiation, and 2/8 (25 %) at more than 72 h following EEG initiation. High clinical grade was associated with poor outcome at time of hospital discharge; electrographic seizures were not associated with poor outcome. CONCLUSIONS: Electrographic seizures occur at a relatively high rate in patients with non-traumatic SAH even after accounting for referral bias. The prolonged time to the first detected seizure in this cohort may reflect dynamic clinical features unique to the SAH population.

Topological Avenue for Efficient Decontamination of Large Volumes of Fluids via UVC Irradiation of Packed Metamaterials.

Nowadays, metamaterials application enjoys notoriety in fluid decontamination and pathogen annihilation, which are frequently present in polluted fluids (e.g., water, blood, blood plasma, air or other gases). The depollution effect is largely enhanced by UVC irradiation. The novelty of this contribution comes from the significant increase by packing of the total surface of metamaterials in contact with contaminated fluids. Packed metamaterial samples are subjected to UVC irradiation, with expected advantages for implant sterilization and long-term prevention of nosocomial infections over large clinical areas. The novel aspect of the investigation consists of a combination of big and small elements of the metamaterial to optimize the above effects connected with fluids and irradiation. The big elements allow the radiation to penetrate deep inside the fluid, and the small elements optimally disperse this radiation toward deeper regions of the metamaterial. A packing scheme of smaller, in-between large metamaterial spheres and fibres is proposed for promoting enhanced depollution against pathogen agents. It is demonstrated that the total surface of metamaterials in contact with contaminated fluids/surface is significantly increased as a result of packing. This opens, in our opinion, new auspicious perspectives in the construction of novel equipment with high sensibility in the detection and decontamination of microorganisms.

TET1 Catalytic Activity is Required for Reprogramming of Imprinting Control Regions and Patterning of Sperm-Specific Hypomethylated Regions.

DNA methylation erasure is required for mammalian primordial germ cell reprogramming. TET enzymes iteratively oxidize 5-methylcytosine to generate 5-hyroxymethylcytosine (5hmC), 5-formylcytosine, and 5-carboxycytosine to facilitate active genome demethylation. Whether these bases are required to promote replication-coupled dilution or activate base excision repair during germline reprogramming remains unresolved due to the lack of genetic models that decouple TET activities. Here, we generated two mouse lines expressing catalytically inactive TET1 ( Tet1-HxD ) and TET1 that stalls oxidation at 5hmC ( Tet1-V ). Tet1 -/- , Tet1 V/V , and Tet1 HxD/HxD sperm methylomes show that TET1 V and TET1 HxD rescue most Tet1 -/- hypermethylated regions, demonstrating the importance of TET1’s extra-catalytic functions. Imprinted regions, in contrast, require iterative oxidation. We further reveal a broader class of hypermethylated regions in sperm of Tet1 mutant mice that are excluded from de novo methylation during male germline development and depend on TET oxidation for reprogramming. Our study underscores the link between TET1-mediated demethylation during reprogramming and sperm methylome patterning.

Aggressive primary scalp lymphoma mimicking an acute epidural hematoma: Case report and review of the literature.

BACKGROUND: Cutaneous Central Follicular Lymphoma (CCFL) is a type B cutaneous lymphoma with a usually indolent course. Scalp localization of CCFL is extremely rare, we report a new case mimicking an epidural hematoma, and showing a rapid progression with aggressive infiltration of skin, calvaria, dura and brain parenchyma. CASE REPORT: A 58-year-old patient with an unlabeled polymalformative syndrome was admitted to the Emergency department following a head injury secondary to a self-resolving tonic-clonic epileptic seizure. The initial CT-scan was interpreted as a minor subcutaneous and epidural hematoma initially deemed for conservative management. Within 4 days, the patient showed a progressive neurological deterioration culminating into a stuporous status which prompted a constrast-enhanced brain MRI. The scan revealed a multilayered solid lesion, extending from the subgaleal compartment to the subdural space, threatening the integrity of overlying skin and causing infiltration of the brain parenchyma. Following emergency neurosurgical excision a definitive histology diagnosis of central follicular lymphoma was made. A focused chemotherapy with high-dose Methotrexate with R-CHOP protocol led to disease control until the latest follow up at 2 years. CONCLUSION: To our knowledge, this case represents the first CCFL invading the brain parenchyma and the second extending to the dura. Although such tumor is usually indolent the aggressive behavior herein reported extend the differential diagnosis to high-grade meningiomas, sarcomas, and metastases. Prognostication and appropriate adjuvant treatment require prompt surgical excision and histological confirmation.

UVC radiation intensity dependence of pathogen decontamination rate: semiclassical theory and experiment.

A semiclassical (light classical and molecule quantum) model describing the dependence of DNA/RNA dimerization rate as function of the ultraviolet C (UVC) radiation's intensity is proposed. Particularly, a nonlinear model is developed based on the Raman-like processes in quantum optics. The main result of the theory shows that the process of dimerization in the DNA/RNA depends strongly on the UVC light's intensity, thus proving a possible quantum microscopical mechanism of the interaction of UV light with the DNA. To corroborate the theoretical findings, we realize some experiments, by which want to investigate how the inactivation rate of the yeast colonies depends on the intensity of the UVC irradiation. The experimental results evidence a nonlinear decreasing of the residual yeast colonies as a function of the intensity in the irradiation process. The possibilities to optimize the intensity of UVC radiation in the considered decontamination equipment by using metamaterials are studied. The application of such equipment in disinfection of fluids (air, water, droplets, etc.), as well for the SARS-CoV-2-infected aerosols, is discussed.

Histopathological features in fatal COVID-19 acute respiratory distress syndrome.

Background: COVID-19 acute respiratory distress syndrome (ARDS) shares the common histological hallmarks with other forms of ARDS. However, the chronology of the histological lesions has not been well established. Objective: To describe the chronological histopathological alterations in the lungs of patients with COVID-19 related ARDS. Design: A prospective cohort study was carried out. Setting: Intensive Care Unit of a tertiary hospital. Patients: The first 22 consecutive COVID-19 deaths. Measurements: Lung biopsies and histopathological analyses were performed in deceased patients with COVID-19 related ARDS. Clinical data and patient course were evaluated. Results: The median patient age was 66 [63-74] years; 73% were males. The median duration of mechanical ventilation was 17 [8-24] days. COVID-19 induced pulmonary injury was characterized by an exudative phase in the first week of the disease, followed by a proliferative/organizing phase in the second and third weeks, and finally an end-stage fibrosis phase after the third week. Viral RNA and proteins were detected in pneumocytes and macrophages in a very early stage of the disease, and were no longer detected after the second week. Limitation: Limited sample size. Conclusions: The chronological evolution of COVID-19 lung histopathological lesions seems to be similar to that seen in other forms of ARDS. In particular, lung lesions consistent with potentially corticosteroid-sensitive lesions are seen.

Antecedentes: El síndrome de dificultad respiratoria aguda (SDRA) asociado a la COVID-19 comparte características histológicas con otros tipos de SDRA. Sin embargo, no se ha establecido adecuadamente la cronología de las lesiones histológicas. Objetivo: Describir las alteraciones histopatológicas cronológicas en los pulmones de los pacientes con síndrome de dificultad respiratoria aguda asociado a COVID-19. Diseño: Estudio prospectivo de cohortes. Ámbito: Unidad de cuidados intensivos de un hospital terciario. Pacientes: Las primeras 22 muertes consecutivas por COVID-19. Intervenciones: Se llevaron a cabo biopsias pulmonares y análisis histopatológicos en pacientes fallecidos por SDRA asociado a COVID-19. Se evaluaron los datos clínicos y la evolución médica. Resultados: La mediana de edad de los pacientes fue de 66 (63-74) años y el 73% eran varones. La mediana de la duración de la ventilación mecánica fue de 17 (8-24) días. La lesión pulmonar inducida por COVID-19 se caracterizó por una fase exudativa durante la primera semana de la enfermedad, seguida de una fase proliferativa/organizativa en la segunda y tercera semana y, por último, una fase de fibrosis en fase terminal tras la tercera semana de evolución. Se detectaron proteínas y ARN vírico en neumocitos y macrófagos en una fase muy temprana de la enfermedad, pero estos ya no se volvieron a detectar a partir de la segunda semana. Limitación: Tamaño limitado de la muestra. Conclusión: La evolución cronológica de las lesiones histopatológicas pulmonares asociadas a la COVID-19 parece ser similar a la de otras formas de SDRA. En particular, se observan daños pulmonares coherentes con las lesiones potencialmente sensibles a los corticosteroides.

Histopathological features in fatal COVID-19 acute respiratory distress syndrome.

BACKGROUND: COVID-19 acute respiratory distress syndrome (ARDS) shares the common histological hallmarks with other forms of ARDS. However, the chronology of the histological lesions has not been well established. OBJECTIVE: To describe the chronological histopathological alterations in the lungs of patients with COVID-19 related ARDS. DESIGN: A prospective cohort study was carried out. SETTING: Intensive Care Unit of a tertiary hospital. PATIENTS: The first 22 consecutive COVID-19 deaths. MEASUREMENTS: Lung biopsies and histopathological analyses were performed in deceased patients with COVID-19 related ARDS. Clinical data and patient course were evaluated. RESULTS: The median patient age was 66 [63-74] years; 73% were males. The median duration of mechanical ventilation was 17 [8-24] days. COVID-19 induced pulmonary injury was characterized by an exudative phase in the first week of the disease, followed by a proliferative/organizing phase in the second and third weeks, and finally an end-stage fibrosis phase after the third week. Viral RNA and proteins were detected in pneumocytes and macrophages in a very early stage of the disease, and were no longer detected after the second week. LIMITATION: Limited sample size. CONCLUSIONS: The chronological evolution of COVID-19 lung histopathological lesions seems to be similar to that seen in other forms of ARDS. In particular, lung lesions consistent with potentially corticosteroid-sensitive lesions are seen.

Unknown: A woman with an erythematous nodule on the scalp.

A 77-year-old healthy woman presented with an isolated exophytic lesion of the scalp for 4 years, which had grown progressively. On exam, there was a 4 cm, firm erythematous and lobulated tumor with telangiectasias on the scalp. The rest of her cutaneous exam was normal. No lymphadenopathy was appreciated. A solitary cylindroma was diagnosed by biopsy.

A new Elf5CreERT2-GFP BAC transgenic mouse model for tracing Elf5 cell lineages in adult tissues.

Elf5 is a transcription factor known to regulate critical developmental processes and has been shown to act as a tumour suppressor in multiple cancers. Elf5 knockout mice are embryonically lethal, limiting in vivo studies pertaining to its function. Moreover, haploinsufficiency of Elf5 limits the use of current mouse models to investigate adult tissue distribution of Elf5. Here, we successfully generated Elf5CreERT2-GFP bacterial artificial chromosome (BAC) transgenic mice and show that Elf5+ cells are present in several adult tissues, where its expression was previously not known. Our study demonstrates the unique distribution of Elf5+ cells in multiple adult organs, which will facilitate future studies investigating the function of Elf5 in these tissues during homeostasis, repair and cancer.

Conventional radiology and right heart catheterization in estimating primary pulmonary hypertension and pulmonary hypertension secondary to left-sided valvular disease.

AIM: to estimate the comparative value of conventional radiology and cardiac catheterization in establishing the diagnosis and severity of primary and secondary pulmonary hypertension. We also tried to achieve some correlations between the radiological and invasive parameters. METHODS: we performed a retrospective study over an 11-years time period, analyzing data from a group of 14 patients diagnosed with primary pulmonary hypertension compared to a matched group of 20 patients suffering from pulmonary hypertension secondary to mitral and aortic valvular disease. All the patients had undergone conventional radiology (chest X-ray) and catheterization of the right heart cavities and pulmonary artery. RESULTS: we detected significantly elevated pulmonary artery pressures and resistances in the primary pulmonary hypertension group compared to secondary hypertension patients. Cardiac output values were much lower in the primary pulmonary hypertension individuals compared with secondary pulmonary hypertension in left-sided valvular disease. The pulmonary artery arch diameter, the diameter of the right descending pulmonary artery and the value of the arterio-bronchial ratio were similar in the two groups. CONCLUSION: in both groups, we found a statistically significant positive correlation between the values of the pulmonary artery vascular resistance and the diameter of the right descending pulmonary artery, which are very specific parameters in the diagnosis of pulmonary hypertension. In the primary pulmonary hypertension group we identified a significant inverse correlation between the diameter of the right descending pulmonary artery and the values of cardiac output. This finding was not confirmed in the secondary group.

Histopathological features in fatal COVID-19 acute respiratory distress syndrome

Background COVID-19 acute respiratory distress syndrome (ARDS) shares the common histological hallmarks with other forms of ARDS. However, the chronology of the histological lesions has not been well established. Objective To describe the chronological histopathological alterations in the lungs of patients with COVID-19 related ARDS. Design A prospective cohort study was carried out. Setting Intensive Care Unit of a tertiary hospital. Patients The first 22 consecutive COVID-19 deaths. Measurements Lung biopsies and histopathological analyses were performed in deceased patients with COVID-19 related ARDS. Clinical data and patient course were evaluated.Results The median patient age was 66 [63–74] years; 73% were males. The median duration of mechanical ventilation was 17 [8–24] days. COVID-19 induced pulmonary injury was characterized by an exudative phase in the first week of the disease, followed by a proliferative/organizing phase in the second and third weeks, and finally an end-stage fibrosis phase after the third week. Viral RNA and proteins were detected in pneumocytes and macrophages in a very early stage of the disease, and were no longer detected after the second week. Limitation Limited sample size. Conclusions The chronological evolution of COVID-19 lung histopathological lesions seems to be similar to that seen in other forms of ARDS. In particular, lung lesions consistent with potentially corticosteroid-sensitive lesions are seen. (AU)

Antecedentes El síndrome de dificultad respiratoria aguda (SDRA) asociado a la COVID-19 comparte características histológicas con otros tipos de SDRA. Sin embargo, no se ha establecido adecuadamente la cronología de las lesiones histológicas. Objetivo Describir las alteraciones histopatológicas cronológicas en los pulmones de los pacientes con síndrome de dificultad respiratoria aguda asociado a COVID-19. Diseño Estudio prospectivo de cohortes. Ámbito Unidad de cuidados intensivos de un hospital terciario. Pacientes Las primeras 22 muertes consecutivas por COVID-19. Intervenciones Se llevaron a cabo biopsias pulmonares y análisis histopatológicos en pacientes fallecidos por SDRA asociado a COVID-19. Se evaluaron los datos clínicos y la evolución médica. Resultados La mediana de edad de los pacientes fue de 66 (63-74) años y el 73% eran varones. La mediana de la duración de la ventilación mecánica fue de 17 (8-24) días. La lesión pulmonar inducida por COVID-19 se caracterizó por una fase exudativa durante la primera semana de la enfermedad, seguida de una fase proliferativa/organizativa en la segunda y tercera semana y, por último, una fase de fibrosis en fase terminal tras la tercera semana de evolución. Se detectaron proteínas y ARN vírico en neumocitos y macrófagos en una fase muy temprana de la enfermedad, pero estos ya no se volvieron a detectar a partir de la segunda semana. Limitación Tamaño limitado de la muestra. Conclusión La evolución cronológica de las lesiones histopatológicas pulmonares asociadas a la COVID-19 parece ser similar a la de otras formas de SDRA. En particular, se observan daños pulmonares coherentes con las lesiones potencialmente sensibles a los corticosteroides. (AU)

Enhancing a Wnt-Telomere Feedback Loop Restores Intestinal Stem Cell Function in a Human Organotypic Model of Dyskeratosis Congenita.

Patients with dyskeratosis congenita (DC) suffer from stem cell failure in highly proliferative tissues, including the intestinal epithelium. Few therapeutic options exist for this disorder, and patients are treated primarily with bone marrow transplantation to restore hematopoietic function. Here, we generate isogenic DC patient and disease allele-corrected intestinal tissue using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated gene correction in induced pluripotent stem cells and directed differentiation. We show that DC tissue has suboptimal Wnt pathway activity causing intestinal stem cell failure and that enhanced expression of the telomere-capping protein TRF2, a Wnt target gene, can alleviate DC phenotypes. Treatment with the clinically relevant Wnt agonists LiCl or CHIR99021 restored TRF2 expression and reversed gastrointestinal DC phenotypes, including organoid formation in vitro, and maturation of intestinal tissue and xenografted organoids in vivo. Thus, the isogenic DC cell model provides a platform for therapeutic discovery and identifies Wnt modulation as a potential strategy for treatment of DC patients.