A new isoform of the human estrogen receptor‐alpha (hER‐α) has been identified and characterized. This 46 kDa isoform (hERα46) lacks the N‐terminal 173 amino acids present in the previously ...characterized 66 kDa isoform (hERα66). hERα46 is encoded by a new class of hER‐α transcript that lacks the first coding exon (exon 1A) of the ER‐α gene. We demonstrated that these Δ1A hER‐α transcripts originate from the E and F hER‐α promoters and are produced by the splicing of exon 1E directly to exon 2. Functional analysis of hERα46 showed that, in a cell context sensitive to the transactivation function AF‐2, this receptor is an effective ligand‐inducible transcription factor. In contrast, hERα46 is a powerful inhibitor of hERα66 in a cell context where the transactivating function of AF‐1 predominates over AF‐2. The mechanisms by which the AF‐1 dominant‐negative action is exerted may involve heterodimeri zation of the two receptor isoforms and/or direct competition for the ER‐α DNA‐binding site. hERα66/hERα46 ratios change with the cell growth status of the breast carcinoma cell line MCF7, suggesting a role of hERα46 in cellular proliferation.
Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 ...presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.
Human brain cells generated by in vitro cell programming provide exciting prospects for disease modeling, drug discovery and cell therapy. These applications frequently require efficient and ...clinically compliant tools for genetic modification of the cells. Recombinant adeno-associated viruses (AAVs) fulfill these prerequisites for a number of reasons, including the availability of a myriad of AAV capsid variants with distinct cell type specificity (also called tropism). Here, we harnessed a customizable parallel screening approach to assess a panel of natural or synthetic AAV capsid variants for their efficacy in lineage-related human neural cell types. We identified common lead candidates suited for the transduction of directly converted, early-stage induced neural stem cells (iNSCs), induced pluripotent stem cell (iPSC)-derived later-stage, radial glia-like neural progenitors, as well as differentiated astrocytic and mixed neuroglial cultures. We then selected a subset of these candidates for functional validation in iNSCs and iPSC-derived astrocytes, using shRNA-induced downregulation of the citrate transporter SLC25A1 and overexpression of the transcription factor NGN2 for proofs-of-concept. Our study provides a comparative overview of the susceptibility of different human cell programming-derived brain cell types to AAV transduction and a critical discussion of the assets and limitations of this specific AAV capsid screening approach.
Respiratory viruses cause mild to severe diseases in humans every year, constituting a major public health problem. Characterizing the pathogenesis in physiologically relevant models is crucial for ...developing efficient vaccines and therapeutics. Here, we show that lung organoids derived from human primary or lung tumor tissue maintain the cellular composition and characteristics of the original tissue. Moreover, we show that these organoids sustain viral replication with particular infection foci formation, and they activate the expression of interferon-associated and proinflammatory genes responsible for mediating a robust innate immune response. All together, we show that three-dimensional (3D) lung organoids constitute a relevant platform to model diseases and enable the development of drug screenings.
Three-dimensional (3D) human lung organoids reflect the native cell composition of the lung as well as its physiological properties. Human 3D lung organoids offer ideal conditions, such as timely availability in large quantities and high physiological relevance for reassessment and prediction of disease outbreaks of respiratory pathogens and pathogens that use the lung as a primary entry portal. Human lung organoids can be used in basic research and diagnostic settings as early warning cell culture systems and also serve as a relevant platform for modeling infectious diseases and drug development. They can be used to characterize pathogens and analyze the influence of infection on, for example, immunological parameters, such as the expression of interferon-associated and proinflammatory genes in the context of cancer. In our study, we found that cancer-derived lung organoids were more sensitive to influenza A virus infection than those derived from healthy tissue and demonstrated a decreased innate immune response.
We present membrane-based steric exclusion chromatography (SXC) as a universal capture step for purification of adeno-associated virus (AAV) gene transfer vectors independent of their serotype and ...surface characteristics. SXC is performed by mixing an unpurified cell culture supernatant containing AAV particles with polyethylene glycol (PEG) and feeding the mixture onto a chromatography filter unit. The purified AAV particles are recovered by flushing the unit with a solution lacking PEG. SXC is an inexpensive single-use method that permits to concentrate, purify, and re-buffer AAV particles with yields >95% and >80% impurity clearance. SXC could theoretically be employed at industrial scales with units of nearly 20 m
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We have developed a novel technique which causes primary human hepatocytes to proliferate by transducing them with genes that upregulate their proliferation.
Upcyte® hepatocytes did not form colonies ...in soft agar and are not immortalised anchorage-independent cells. Confluent cultures expressed liver-specific proteins, produced urea and stored glycogen.
CYP activities were low but similar to that in 5-day cultures of primary human hepatocytes. CYP1A2 and CYP3A4 were inducible; moreover, upcyte® hepatocytes predicted the in vivo induction potencies of known CYP3A4 inducers using the "relative induction score" prediction model. Placing cells into 3D culture increased their basal CYP2B6 and CYP3A4 basal activities and induction responses.
Phase 2 activities (UGTs, SULTs and GSTs) were comparable to activities in freshly isolated hepatocytes.
Upcyte® hepatocytes were markedly more sensitive to the hepatotoxin, α-amanitin, than HepG2 cells, indicating functional OATP1B3 uptake. The cytotoxicity of aflatoxin B1, was decreased in upcyte® hepatocytes by co-incubation with the CYP3A4 inhibitor, ketoconazole. Upcyte® hepatocytes also differentiated between ten hepatotoxic and eight non-hepatotoxic compounds.
In conclusion, upcyte® hepatocyte cultures have a differentiated phenotype and exhibit functional phase 1 and 2 activities. These data support the use of upcyte® hepatocytes for CYP induction and cytotoxicity screening.
The isolation and characterization of several new
human estrogen receptor-α (hERα) mRNAs are described. Together with
those previously identified, they give rise to a total of six hERα
mRNA isoforms ...(A–F hERα mRNAs). Produced from a single hERα gene
by multiple promoter usage, all these transcripts encode a common
protein but differ in their 5′-untranslated region as a consequence of
alternative splicing of five upstream exons (1B–1F). RT-PCR and S1
nuclease mapping analysis of these different hERα mRNA isoforms
revealed a differential pattern of expression of the hERα gene in
human tissues and cell types. The A hERα mRNA is the main isoform
detected in mammary glands or in the tumor cell lines derived from this
tissue. In endometrium, the predominant forms are the A and C hERα
mRNA isoforms, whereas the C and F hERα mRNA isoforms are the major
forms detected in ovary. Finally, high levels of the E hERα mRNA
isoform are restricted to the liver with an increased expression in
females. Taken together, our results demonstrate that the hERα gene
is a complex genomic unit exhibiting alternative splicing and promoter
usage in a tissue-specific manner.
The existence of two forms of the chicken estrogen
receptor-α protein (ER-α) in chicken tissues is demonstrated: the
previously reported receptor (cER-α form I), which has a size of 66
kDa, and a new ...form (cER-α form II), which lacks the N-terminal 41
amino acids present in form I and thus gives rise to a protein of 61
kDa. Whereas the 66-kDa protein is the translation product of several
cER-α mRNAs (A1–D), the cER-α protein isoform II is encoded
by a new cER-α mRNA (A2), which is transcribed in vivo
from a specific promoter that is located in the region of the
previously assigned translation start site of the cER-α gene. SI
nuclease mapping analysis reveals that cER-α mRNA A2 is liver
enriched. The resulting cER-α forms I and II differ in their ability
to modulate estrogen target gene expression in a promoter- and cell
type-specific manner. Whereas cER-α form I activates or represses in
a strictly E2-dependent manner, the truncated
form is characterized by a partial transactivating or repressing
activity in the absence of its ligand. Comparison of the N-terminal
coding regions of different vertebrate ER-α reveal a conservation of
the translation start methionine of the protein ER-α form II in other
oviparous species but not in mammals. The expression of two classes of
ER-α transcripts encoding the two ER-α receptor forms in the liver
of Xenopus laevis and rainbow trout is demonstrated.
Therefore, the existence of two functionally different protein isoforms
produced from the ER-α gene is probably a common and specific feature
in oviparous species.
The adenovirus E1A gene products are nuclear phosphoproteins that can transactivate the other adenovirus early genes as well as several cellular genes, and can transform primary rodent cells in ...culture. Transformation and transactivation by E1A proteins is most likely to be mediated through binding to several cellular proteins, including the retinoblastoma gene product pRb, the pRb‐related p107 and p130, and the TATA box binding protein TBP. We report here the cloning of BS69, a novel protein that specifically interacts with adenovirus 5 E1A. BS69 has no significant homology to known proteins and requires the region that is unique to the large (289R) E1A protein for high affinity binding. BS69 and E1A proteins coimmunoprecipitate in adenovirus‐transformed 293 cells, indicating that these proteins also interact in vivo. BS69 specifically inhibits transactivation by the 289R E1A protein, but not by the 243R E1A protein. BS69 also suppressed the E1A‐stimulated transcription of the retinoic acid receptor in COS cells, but did not affect the cellular E1A‐like activity that is present in embryonic carcinoma cells. Our data indicate that BS69 is a novel and specific suppressor of E1A‐activated transcription.
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