Multifunctional calcium-based nanocarriers for synergistic treatment of triple-negative breast cancer.

Targeted breast cancer therapies hold the potential to improve the efficiency of drug delivery to the pathology site without impacting the viability and function of healthy cells. Herein, we developed multifunctional nanocarriers that target simultaneously several downstream signaling processes in triple negative breast cancer cells. The system comprises pH sensitive CaCO3 nanoparticles (NPs) as carriers of the anticancer drug doxorubicin (DOX). The NPs were coated in a layer-by-layer (LbL) fashion using poly-l-lysine and hyaluronic acid to target receptors overexpressed in breast cancer (e.g. CD44, RHAMM). Spheroids of the triple-negative Hs578T cell line were used as a 3D model to assess the therapeutic potential of this system. Our results showed that the NPs act via a synergistic mechanism that combines Ca2+ overload causing cell calcification and DNA damage by DOX. The LbL coating was crucial for the protection of the healthy cells, i.e. it provides NPs with targeting capacity. The overall data suggests that the LbL-coated NPs loaded with DOX hold great potential for the treatment of breast cancer.

Evaluation of bacterial growth, effects on albumin, and coagulation factors in canine fresh frozen plasma administered as continuous rate infusion exposed to room temperature for 12 hours.

OBJECTIVES: To determine the risk of bacterial growth and to analyze the stability of albumin and coagulation factors in canine fresh frozen plasma (FFP) units exposed to room temperature (24°C) administered as a continuous rate infusion (CRI) for 12 hours. DESIGN: Ex vivo study. SETTING: University teaching hospital and pet blood bank. ANIMALS: None. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: An FFP CRI was simulated to replicate the standard routine procedure used in dogs. Plasma samples were collected before starting the CRI (H0), after 4 hours (H4), and after 12 hours (H12). Bacterial culture of FFP was performed and albumin concentration and specific activity levels for factors V, VII, VIII, and IX were measured and compared. All plasma culture results were negative. There were no statistically significant differences at any time point in the factor VIII activity (median 105.5% [range, 75.6%-142.0%] at H0; median 107.8% [range, 75.0%-172.7%] at H4; and median 112.1% [range, 81.7%-171.0%] at H12); factor IX activity (median 119.3% [range, 89.1%-175.9%] at H0; median 123.1% [range, 72.5%-172.7%] at H4; and median 118.3% [range, 86.6%-177.5%] at H12); or albumin concentration (median 21.0 g/L [range, 17.0-23.0 g/L] at H0 and median 20.0 g/L [range, 17.0-24.0 g/L] at H12). A slight but significant increase in factor V activity was observed when comparing H0 (median 107.0% [range, 71.0%-159.0%]) to H4 (median 117.7% [range, 71.0%-176.7%]) (P = 0.002) or H12 (median 116.2% [range, 71.0%-191.6%]) (P = 0.001). A slight but significant increase in factor VII activity was observed when comparing H0 (median 115.4% [range, 70.6%-183.7%]) to H4 (median 118.2% [range, 82.7%-194.6%]) (P = 0.005); H0 to H12 (median 128.7% [range, 86.4%-200.0%]) (P < 0.001); and H4 to H12 (P = 0.002). CONCLUSIONS: FFP CRI at room temperature for 12 hours could be considered safe with regard to risk for bacterial growth and also effective by providing albumin and clotting factors.

Layer-by-layer coated calcium carbonate nanoparticles for targeting breast cancer cells.

Breast cancer is resistant to conventional treatments due to the specific tumour microenvironment, the associated acidic pH and the overexpression of receptors that enhance cells tumorigenicity. Herein, we optimized the synthesis of acidic resorbable calcium carbonate (CaCO3) nanoparticles and the encapsulation of a low molecular weight model molecule (Rhodamine). The addition of ethylene glycol during the synthetic process resulted in a particle size decrease: we obtained homogeneous CaCO3 particles with an average size of 564 nm. Their negative charge enabled the assembly of layer-by-layer (LbL) coatings with surface-exposed hyaluronic acid (HA), a ligand of tumour-associated receptor CD44. The coating decreased Rhodamine release by two-fold compared to uncoated nanoparticles. We demonstrated the effect of nanoparticles on two breast cancer cell lines with different aggressiveness - SK-BR-3 and the more aggressive MDA-MB-231 - and compared them with the normal breast cell line MCF10A. CaCO3 nanoparticles (coated and uncoated) significantly decreased the metabolic activity of the breast cancer cells. The interactions between LbL-coated nanoparticles and cells depended on HA expression on the cell surface: more particles were observed on the surface of MDA-MB-231 cells, which had the thickest endogenous HA coating. We concluded that CaCO3 nanoparticles are potential candidates to carry low molecular weight chemotherapeutics and deliver them to aggressive breast cancer sites with an HA-abundant pericellular matrix.

Risk Assessment of Nine Coccidiostats in Commercial and Home-Raised Eggs.

The poultry industry, in order to prevent and control coccidiosis caused by Eimeria spp., widely uses coccidiostats as feed additives. The main objective of this study was to evaluate the presence of nine coccidiostats in 62 egg samples by UHPLC-MS/MS. Overall, detection frequency and average concentration were 90.3% (56/62) and 106.3 µg kg-1, respectively. Only diclazuril and nicarbazin were detected. Diclazuril, only found in home-raised eggs, showed an overall detection frequency of 8.1% (5/62), with average and maximum concentrations of 0.46 ± 1.90 µg kg-1 and 13.6 µg kg-1, respectively. Nicarbazin presented an overall higher frequency, 88.7% (55/62), with levels up to 744.8 µg kg-1. Additionally, four samples (6.5%) presented both nicarbazin and diclazuril. Home-raised egg samples (n = 28) showed a detection frequency of 89.3%, with nicarbazin found in more samples (85.7% vs. 17.9%) and at higher levels (266.3 ± 169.4 µg kg-1 vs. 0.91 ± 2.78 µg kg-1) when compared to diclazuril. In supermarket samples (n = 34), only nicarbazin was detected in 31 samples (91.1%), with an average of 167.6 ± 62.2 µg kg-1. Considering the average contamination scenario, consumers' health should not be adversely affected by egg consumption. In every scenario considered, children were the most vulnerable population group.

Coccidiostats and Poultry: A Comprehensive Review and Current Legislation.

Coccidiosis remains one of the major problems of the poultry industry. Caused by Eimeria species, Coccidiosis is a contagious parasitic disease affecting poultry with great economic significance. Currently, in order to prevent health problems caused by this disease, broiler farmers make extensive use of coccidiostats in poultry feed, maintaining animal health and, in some cases, enhancing feed conversion. The presence of unauthorized substances, residues of veterinary products and chemical contaminants in the food industry is of concern, since they may pose a risk to public health. As the use of coccidiostats has been increasing without any requirements for veterinary prescription, research and surveillance of coccidiostat residues in poultry meat is becoming imperative. This review presents an up-to-date comprehensive discussion of the state of the art regarding coccidiosis, the most used anticoccidials in poultry production, their mode of action, their prophylactic use, occurrence and the European Union (EU) applicable legislation.

Identification of a Cryptic t(8;20;21)(q22;p13;q22) Resulting in RUNX1T1/RUNX1 Fusion in a Patient with Newly Diagnosed Acute Myeloid Leukemia.

The detection of recurrent genetic abnormalities in acute myeloid leukemia (AML), including RUNX1T1/RUNX1 gene fusion, is critical for optimal medical management. Herein, we report a 45 year old woman with newly diagnosed AML and conventional chromosome studies that revealed an apparently balanced t(8;20)(q22;p13) in all 20 metaphases analyzed. A RUNX1T1/RUNX1 dual-color dual-fusion fluorescence in situ hybridization (FISH) probe set was subsequently performed and revealed a RUNX1T1/RUNX1 gene fusion. Metaphase FISH studies performed on abnormal metaphases revealed a cryptic, complex translocation resulting in RUNX1T1/RUNX1 fusion, t(8;20;21)(q22;p13;q22). This case study shows the importance of performing FISH studies or other high-resolution genetic testing concurrently with conventional chromosome studies for the detection of cryptic recurrent gene fusions in AML, particularly a focused genetic evaluation such as RUNX1T1/RUNX1 gene fusion, when specific abnormalities involving 8q22 are identified.

Exploiting Polyelectrolyte Complexation for the Development of Adhesive and Bioactive Membranes Envisaging Guided Tissue Regeneration.

Mussels secrete protein-based byssal threads to tether to rocks, ships, and other organisms underwater. The secreted marine mussel adhesive proteins (MAPs) contain the peculiar amino acid L-3,4-dihydroxyphenylalanine (DOPA), whose catechol group content contributes greatly to their outstanding adhesive properties. Inspired by such mussel bioadhesion, we demonstrate that catechol-modified polysaccharides can be used to obtain adhesive membranes using the compaction of polyelectrolyte complexes (CoPEC) method. It is a simple and versatile approach that uses polyelectrolyte complexes as building blocks that coalesce and dry as membrane constructs simply as a result of sedimentation and mild temperature. We used two natural and biocompatible polymers: chitosan (CHI) as a polycation and hyaluronic acid (HA) as a polyanion. The CoPEC technique also allowed the entrapment of ternary bioactive glass nanoparticles to stimulate mineralization. Moreover, combinations of these polymers modified with catechol groups were made to enhance the adhesive properties of the assembled membranes. Extensive physico-chemical characterization was performed to investigate the successful production of composite CoPEC membranes in terms of surface morphology, wettability, stability, mechanical performance, in vitro bioactivity, and cellular behavior. Considering the promising properties exhibited by the obtained membranes, new adhesives suitable for the regeneration of hard tissues can be envisaged.

Risk Assessment of Nine Coccidiostats in Commercial and Home Raised Poultry.

For the first time, this paper aimed to evaluate nine ionophore and synthetic coccidiostat residues in poultry muscle samples, obtained from different production types, by solid-liquid extraction followed by liquid chromatography with tandem mass spectrometry (LC-MS/MS). The fully validated methodology was successfully applied to a total of 101 chicken and turkey samples obtained from canteens, supermarkets, and home productions in Portugal. Halofuginone, diclazuril, decoquinate, narasin, lasalocid, and salinomycin were detected in 20.8% of the samples. Home raised samples showed a greater frequency, 47.1%. The synthetic coccidiostats halofuginone, diclazuril, and decoquinate were found in averages of 0.7 µg kg-1,2.9 µg kg-1, and 3.7 µg kg-1, respectively, while averages of 1.2 µg kg-1, 1.6 µg kg-1, and 1.3 µg kg-1 were found regarding the ionophores narasin, lasalocid, and salinomycin. As for the risk assessment, values lower than 8.06% of the acceptable daily intake (ADI) were observed, indicating that exposure to coccidiostats through consumption of poultry meat does not represent risk to consumers.

Liposomes embedded in layer by layer constructs as simplistic extracellular vesicles transfer model.

Extracellular vesicles (EVs) are particles originating from the exfoliation of the cellular membrane. They are involved in cell-to-cell and cell-to-matrix signaling, exchange of bioactive molecules, tumorigenesis and metastasis, among others. To mitigate the limited understanding of EVs transfer phenomena, we developed a simplistic model that mimics EVs and their interactions with cells and the extracellular matrix. The proposed model is a layer by layer (LbL) film built from the polycationic poly-l-lysine (PLL) and the glycosaminoglycan hyaluronic acid (HA) to provide ECM mimicry. Positively charged 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and N1,N1,N14,N14-tetramethyl-N1,N14-ditetradecyltetradecane-1,14-diaminium dibromide (GS14) liposomes were embedded in this construct to act as EVs analogs. To simulate EVs carrying substances, Nile Red was loaded as a model of lipophilic cargo molecules. The integration of each component was followed by quartz crystal microbalance measurements, which confirmed the immobilization of intact liposomes on the underlying (PLL/HA)3 soft film. The release of Nile Red from liposomes either embedded in the LbL construct or exposed at its surface revealed a fast first order release. This system was validated as a model for EV/cell interactions by incubation with breast cancer cells MDA-MB-231. We observed higher internalization for embedded liposomes when compared with surface-exposed ones, showcasing that the ECM mimic layers do not constitute a barrier to liposome/cell interactions but favor them.

Determination of urokinase-type plasminogen activator serum levels in healthy and oncologic cats.

The urokinase plasminogen activator system (uPAS) has been poorly investigated in veterinary oncology. The aim of this study was to determine uPA serum concentrations in healthy and oncologic cats to understand the potential value of uPA as a cancer biomarker. Serum samples were collected from 19 healthy cats and 18 cats with spontaneous malignant neoplasms and uPA was measured through a specific enzyme-linked immunosorbent assay kit. The differences between uPA values and their relation with intrinsic factors and clinicopathological parameters were analyzed using an analysis of variance (ANOVA) and independent t-test. The average serum concentration of uPA in cancerous cats (0.54 ± 0.22 ng/mL) differed from that of healthy cats (1.10 ± 1.16 ng/mL) but was not significantly influenced by cats' clinicopathological parameters or by the presence of metastases. This study describes, for the first time, the serum concentrations of uPA in cats and proposes directions for future studies to uncover the relevance of uPAS in feline carcinogenesis.

Le système activateur de plasminogène de type urokinase (uPAS) a été peu étudié en oncologie vétérinaire. L'objectif de la présente étude était de déterminer les concentrations sériques d'uPA chez des chats en santé et oncologiques afin de comprendre la valeur potentielle d'uPA comme marqueur de cancer. Des échantillons de sérum furent prélevés de 19 chats en santé et de 18 chats avec des néoplasmes malins spontanés et l'uPA fut mesuré à l'aide d'une trousse immuno-enzymatique. Les différences entre les valeurs d'uPA et leur relation avec des facteurs intrinsèques et des paramètres clinico-pathologiques furent analysées par analyse de variance (ANOVA) et test de t indépendant. La concentration moyenne d'uPA chez les chats avec cancer (0,54 ± 0,22 ng/mL) différait de celle des chats en santé (1,10 ± 1,16 ng/mL) mais n'était pas influencée de manière significative par les paramètres clinico-pathologiques des chats ou la présence de métastases. Cette étude décrit, pour la première fois, les concentrations sériques d'uPA chez les chats et propose des orientations pour des études ultérieures afin de révéler la pertinence d'uPAS dans la carcinogénèse chez les chats.(Traduit par Docteur Serge Messier).

Immunotherapy- (Blinatumomab-) Related Lineage Switch of KMT2A/AFF1 Rearranged B-Lymphoblastic Leukemia into Acute Myeloid Leukemia/Myeloid Sarcoma and Subsequently into B/Myeloid Mixed Phenotype Acute Leukemia.

The presence of KMT2A/AFF1 rearrangement in B-lymphoblastic leukemia (B-ALL) is an independent poor prognostic factor and has been associated with higher rate of treatment failure and higher risk of linage switch under therapy. Blinatumomab has shown promising therapeutic results in refractory or relapsed B-ALL; however, it has potential risk of inducing lineage switch, especially in KMT2A/AFF1 rearranged B-ALL into acute myeloid leukemia and/or myeloid sarcoma. We report a 40-year-old female with KMT2A/AFF1-rearranged B-ALL that was refractory to conventional chemotherapy. Following administration of blinatumomab, she developed a breast mass proven to be myeloid sarcoma, in addition to bone marrow involvement by AML. Approximately six weeks after cessation of blinatumomab, a repeat bone marrow examination revealed B/myeloid MPAL.

Characterization of a rarely reported STAT5B/RARA gene fusion in a young adult with newly diagnosed acute promyelocytic leukemia with resistance to ATRA therapy.

The detection of PML/RARA or variant RARA rearrangements is critical for the diagnosis and treatment of patients with newly diagnosed acute promyelocytic leukemia (APL). While most cases of APL harboring the PML/RARA fusion respond to all-trans retinoic acid (ATRA), some variant RARA rearrangements are ATRA insensitive. Herein, we report a 27-year-old male with newly diagnosed, rapidly progressive APL and a rarely described STAT5B/RARA fusion with known resistance to ATRA therapy. While the PML/RARA dual-color dual-fusion fluorescence in situ hybridization (FISH) probe study was negative, the RARA break-apart probe study revealed an atypical RARA rearrangement in 95% of nuclei. A next generation sequencing assay, mate-pair sequencing, was subsequently performed to further characterize the RARA rearrangement and identified the RARA gene fusion partner STAT5B.

Bioinspired baroplastic glycosaminoglycan sealants for soft tissues.

We describe biomimetic adhesives inspired by the marine glues fabricated by the sandcastle worm. The formation of stable polyelectrolyte complexes between poly-L-lysine (PLL) and glycosaminoglycans (GAGs) with different sulfation degree - heparin (HEP), chondroitin sulfate (CS) and hyaluronic acid (HA) - is optimized by zeta-potential titrations. These PLL/GAG complexes are transformed into compact polyelectrolyte complexes (coPECs) with controlled water contents and densities via baroplastic processing. Rotational shear tests demonstrate that coPECs containing sulfated GAGs (HEP or CS) have solid-like properties, whereas HA-based complexes form highly hydrated viscous-like networks. The adhesiveness of the generated coPECs (normalized lap shear strength) is tested in dry and wet states using polystyrene and rabbit skin, respectively. In dry state, the adhesives exhibit lap shear strengths in the order of hundreds of kPa, with coPLL/HEP and coPLL/CS being about 1.5 times stronger than coPLL/HA. In wet state, all coPECs seal rabbit skin and recover over 60% of the elongation capacity of intact skin with coPLL/HA providing the sturdiest adhesion (∼85% elongation recovery). We demonstrate that this is due to the higher water fraction that improves the bonding between the wet specimens, showcasing the potential superior mechanical recovery on injured tissues. STATEMENT OF SIGNIFICANCE: The development of medical sealants with sufficient adhesive strength in the presence of water and moist remains a huge challenge. We present glycosaminoglycans (GAGs) as biomaterials for the assembly of baroplastics with strong adhesive strength to soft tissues at physiological conditions. Baroplastics with tacky properties were generated by a mild assembly process based on polyelectrolyte complexation and compaction. These materials behave as versatile sealants: their adhesiveness can be adjusted to either dry or wet specimens because of the different sulfation degree of GAGs. These sealants were noncytotoxic towards L929 cells and allowed the damaged skin to recover a great deal of its native elasticity: they preserved the J-shaped stress/strain mechanical response that is typical of biological soft tissues.

In vitro hemolysis of stored units of canine packed red blood cells.

BACKGROUND: Hemolysis is an important quality parameter of packed red blood cells (pRBCs) that is used to assess the cellular integrity of stored blood units. According to human standards, hemolysis at the end of storage must not exceed 1%, as otherwise it may be responsible for decreased transfusion effectiveness and acute life-threatening reactions. OBJECTIVES: This prospective study was designed to evaluate the hemolysis of canine pRBCs stored in an additive solution containing adenine, dextrose, mannitol, and sodium chloride, and to assess its associations with storage time, duration of the collection process, collection disturbances, and with the final volume and PCV of the pRBCs units. METHODS: One hundred eighty pRBCs units were collected from canine donors. Hemolysis of the pRBCs units was determined immediately after processing (t = 0). The units were then stored and retested (t = 1) either before administration (during weeks 2, 3, 4, 5, or 6 of storage) or at the end of the storage period (42 d) if not used. RESULTS: Mean hemolysis at t = 0 was 0.09% (SD 0.06) and increased during storage, at a more pronounced rate from the 5th (mean values of 0.52%, SD 0.29) to the 6th week (1.2%, SD 0.72). Almost 51% of the units with 36-42 days of shelf-life showed more than 1% hemolysis. Disturbances in the collection process, the volume of the whole blood units, and the volume of stored pRBCs units or their PCV were not related to pRBCs hemolysis. CONCLUSIONS: According to human blood bank recommendations regarding acceptable hemolysis, canine pRBCs stored for more than 35 days should be tested to ensure <1% hemolysis prior to administration.

Tuning the Stiffness of Surfaces by Assembling Genetically Engineered Polypeptides with Tailored Amino Acid Sequence.

We introduce elastin-like recombinamers (ELRs) as polypeptides with precise amino acid positioning to generate polypeptide coatings with tunable rigidity. Two ELRs are used: V84-ELR, a hydrophobic monoblock, and EI-ELR, an amphiphilic diblock. Both were modified with the amine-reactive tetrakis (hydroxymethyl) phosphonium chloride compound. We evaluated the affinity, conformation, and dissipative behavior of ELRs assembled on alkanethiol self-assembled coatings by quartz crystal microbalance with dissipation monitoring, multiparametric surface plasmon resonance, and atomic force microscopy. The thickness of the polypeptide coatings showcases the preferential affinity of ELRs to NH2- and CH3-terminated surfaces. We demonstrate that V84-ELR strongly bonded to the substrate and reorganizes into an extended and more hydrated layer as the adsorbed amount increases, whereas EI-ELR has a less dissipative behavior. The results suggest that ELR adsorption depends on the amino acid sequence and the substrate chemistry, ultimately influencing the stiffness of the polypeptide coatings.

Bioinformatics analysis of common differential genes of coronary artery disease and ischemic cardiomyopathy.

OBJECTIVE: This paper aims at screening the common differential genes of coronary atherosclerotic heart disease (CAD) and ischemic cardiomyopathy (ICM), and to conduct pathway analysis and protein-protein interaction (PPI) network analysis for the differential genes. MATERIALS AND METHODS: The CAD and ICM datasets were collected from the Gene Expression Omnibus (GEO) database for human tumors to extract data components of peripheral blood RNA of patients and normal people in GSE71226 and GSE9128 chips; "limma" package of "R" software was used to screen the differential genes, and "pheatmap" package was applied to construct heat maps for the differential genes; Cytoscape, Database for Annotation, Visualization and Integration Discovery (DAVID) and String platforms were utilized for PPI network analysis, Genome Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis on the selected differential genes. RESULTS: A total of 575 differential genes were screened from GSE71226, including 350 genes with up-regulated expression and 225 with down-regulated expression, which was statistically significant (p<0.05, fold change >1). 75 differential genes were screened from GSE9128, including 47 genes with up-regulated expression and 28 with down-regulated expression. By virtue of String, DAVID and Cytoscape software, the PPI network diagram was constructed, and GO and KEGG analyses were performed successfully. CONCLUSIONS: A total of 8 common differential genes are screened, and functional annotation and pathway analysis are conducted, which is conducive to further studying the interactions between the differentially expressed genes.

Development of Inhalable Superparamagnetic Iron Oxide Nanoparticles (SPIONs) in Microparticulate System for Antituberculosis Drug Delivery.

Tuberculosis (TB) is an infectious disease which affects millions of people worldwide. Inhalable polymeric dry powders are promising alternatives as anti-TB drug carriers to the alveoli milieu and infected macrophages, with potential to significantly improve the therapeutics efficiency. Here, the development of a magnetically responsive microparticulate system for pulmonary delivery of an anti-TB drug candidate (P3) is reported. Microparticles (MPs) are developed based on a cast method using calcium carbonate sacrificial templates and incorporate superparamagnetic iron oxide nanoparticles to concentrate MPs in alveoli and enable drug on demand release upon actuation of an external alternate magnetic field (AMF). The MPs are shown to be suitable for P3 delivery to the lower airways and for alveolar macrophage phagocytosis. The developed MPs reveal unique and promising features to be used as an inhalable dry powder allowing the AMF control over dosage and frequency of drug delivery anticipating improved TB treatments.

In vitro quality control analysis after processing and during storage of feline packed red blood cells units.

BACKGROUND: During the storage of packed red blood cells (pRBC), packed cell volume (PCV), bacterial contamination and percentage of haemolysis [percentage of free haemoglobin (HGB) in relation to the total HGB] are important quality parameters. Both PCV and haemolysis are indicators of the cellular integrity of stored units. There are no published experimental studies that evaluated these parameters during storage of feline pRBC using SAGM (adenine, dextrose, mannitol and sodium chloride) as the additive solution. The present study aims to (1) evaluate the quality of feline pRBCs stored in SAGM; (2) test for the semi-closed system's suitability for use and risk of bacterial contamination; (3) establish the maximum storage time that may be appropriate to meet the criteria established by the United States Food and Drug Administration (US-FDA) guidelines for human blood banking; and (4) evaluate the need to calculate the percentage of haemolysis prior to the administration of units stored for more than 4 weeks. Four hundred eighty nine feline pRBC units were analyzed. Bacterial culture, PCV and percentage of haemolysis were determined within 6 h after processing (t0). One hundred and eighty units were re-tested for haemolysis and PCV after 29-35 days of storage (t1) and 118 units after 36-42 days (t2). RESULTS: Bacterial contamination was not detected in any pRBC unit. Mean PCV at t0 was 52.25% (SD: ±5.27) and decreased significantly (p < 0.001) during storage to 48.15% (SD: ±3.79) at t1 and to 49.34% (SD: ±4.45) at t2. Mean percentage of haemolysis at t0 was 0.07% (SD: ±0.06) and increased significantly (p < 0.001) to 0.69% (SD: ±0.40) at t1 and to 0.81% (SD: ±0.47) at t2. In addition, 13.88% and 19.49% of pRBC units exceeded 1% haemolysis at t1 and t2, respectively. CONCLUSIONS: According to the US-FDA guidelines for human blood banking that recommend a maximum of 1% haemolysis, the results of this study show that all feline pRBC units with less than 24 h of shelf life have low levels of haemolysis. However, units preserved up to 28 days can only be administered if tested for haemolysis before use, since 13.88% units exceeded the 1% limit. The semi-closed system was considered safe for use as bacterial contamination was not detected in any pRBC unit.

Assessing sufficiency and necessity of enhancer activities for gene expression and the mechanisms of transcription activation.

Enhancers are important genomic regulatory elements directing cell type-specific transcription. They assume a key role during development and disease, and their identification and functional characterization have long been the focus of scientific interest. The advent of next-generation sequencing and clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9-based genome editing has revolutionized the means by which we study enhancer biology. In this review, we cover recent developments in the prediction of enhancers based on chromatin characteristics and their identification by functional reporter assays and endogenous DNA perturbations. We discuss that the two latter approaches provide different and complementary insights, especially in assessing enhancer sufficiency and necessity for transcription activation. Furthermore, we discuss recent insights into mechanistic aspects of enhancer function, including findings about cofactor requirements and the role of post-translational histone modifications such as monomethylation of histone H3 Lys4 (H3K4me1). Finally, we survey how these approaches advance our understanding of transcription regulation with respect to promoter specificity and transcriptional bursting and provide an outlook covering open questions and promising developments.

Resolving systematic errors in widely used enhancer activity assays in human cells.

The identification of transcriptional enhancers in the human genome is a prime goal in biology. Enhancers are typically predicted via chromatin marks, yet their function is primarily assessed with plasmid-based reporter assays. Here, we show that such assays are rendered unreliable by two previously reported phenomena relating to plasmid transfection into human cells: (i) the bacterial plasmid origin of replication (ORI) functions as a conflicting core promoter and (ii) a type I interferon (IFN-I) response is activated. These cause confounding false positives and negatives in luciferase assays and STARR-seq screens. We overcome both problems by employing the ORI as core promoter and by inhibiting two IFN-I-inducing kinases, enabling genome-wide STARR-seq screens in human cells. In HeLa-S3 cells, we uncover strong enhancers, IFN-I-induced enhancers, and enhancers endogenously silenced at the chromatin level. Our findings apply to all episomal enhancer activity assays in mammalian cells and are key to the characterization of human enhancers.