A novel entity of massive multifocal osteolyses in the elderly.

Osteolyses are common findings in elderly patients and most frequently represent malignant or locally aggressive bone tumors, infection, inflammatory and endocrine disorders, histiocytoses, and rare diseases such as Gorham-Stout syndrome. We here report on a novel entity of massive multifocal osteolyses in both shoulders, the right hip and left knee joint and the dens of an 83-year-old patient not relatable to any previously known etiopathology of bone disorders. The soft tissue mass is of myxoid stroma with an unspecific granulomatous inflammatory process, aggressively destroying extensive cortical and cancellous bone segments and encroaching on articulating bones in diarthrodial large joints. Radiological, nuclear medical, serological, histological, and immunohistochemical analyses were incapable of further classifying the disease pattern within the existing scheme of pathology. Quantitative polymerase chain reaction and next generation sequencing revealed that mutations are not suggestive of any known hereditary or acquired bone disease. Possible treatment options include radionuclide therapy for pain palliation and percutaneous radiation to arrest bone resorption while surgical treatment is inevitable for pathological fractures. This case study shall increase the awareness of the musculoskeletal community and motivate to collect further information on this rare but mutilating disorder.

Experimental capture of miRNA targetomes: disease-specific 3'UTR library-based miRNA targetomics for Parkinson's disease.

The identification of targetomes remains a challenge given the pleiotropic effect of miRNAs, the limited effects of miRNAs on individual targets, and the sheer number of estimated miRNA-target gene interactions (MTIs), which is around 44,571,700. Currently, targetome identification for single miRNAs relies on computational evidence and functional studies covering smaller numbers of targets. To ensure that the targetome analysis could be experimentally verified by functional assays, we employed a systematic approach and explored the targetomes of four miRNAs (miR-129-5p, miR-129-1-3p, miR-133b, and miR-873-5p) by analyzing 410 predicted target genes, both of which were previously associated with Parkinson's disease (PD). After performing 13,536 transfections, we validated 442 of the 705 putative MTIs (62,7%) through dual luciferase reporter assays. These analyses increased the number of validated MTIs by at least 2.1-fold for miR-133b and by a maximum of 24.3-fold for miR-873-5p. Our study contributes to the experimental capture of miRNA targetomes by addressing i) the ratio of experimentally verified MTIs to predicted MTIs, ii) the sizes of disease-related miRNA targetomes, and iii) the density of MTI networks. A web service to support the analyses on the MTI level is available online ( https://ccb-web.cs.uni-saarland.de/utr-seremato ), and all the data have been added to the miRATBase database ( https://ccb-web.cs.uni-saarland.de/miratbase ).

DNA barcoding as new diagnostic tool to lethal plant poisoning in herbivorous mammals.

Reliable identification of plant species in the digestive tract of a deceased animal often represents the major key to diagnose a lethal intoxication with poisonous plants in veterinary pathology. In many cases, identification of the species is challenging or even impossible because the diagnostic morphological features have been degraded, and because the interpretation of such features requires a considerable expertise in plant anatomy and biodiversity. The use of DNA barcoding markers can support or even replace classical morphological assessment. While these markers have been widely used for plant taxonomy, their forensic application to clarify causes of animal poisoning is novel. In addition, we use specific single-nucleotide polymorphisms as fingerprints. This allows for a clear decision even in cases, where the conventionally used statistical e-values remain ambiguous. In the current work, we explore the feasibility of this strategy in a couple of exemplary cases, either in concert with anatomical diagnostics, or in cases where visual species identification is not possible, or where chemical toxin detection methods are not well established, complex, time consuming and expensive.

Dynamic and static circulating cancer microRNA biomarkers - a validation study.

For cancers and other pathologies, early diagnosis remains the most promising path to survival. Profiling of longitudinal cohorts facilitates insights into trajectories of biomarkers. We measured microRNA expression in 240 serum samples from patients with colon, lung, and breast cancer and from cancer-free controls. Each patient provided at least two serum samples, one prior to diagnosis and one following diagnosis. The median time interval between the samples was 11.6 years. Using computational models, we evaluated the circulating profiles of 21 microRNAs. The analysis yielded two sets of biomarkers, static ones that show an absolute difference between certain cancer types and controls and dynamic ones where the level over time provided higher diagnostic information content. In the first group, miR-99a-5p stands out for all three cancer types. In the second group, miR-155-5p allows to predict lung cancers and colon cancers. Classification in samples from cancer and non-cancer patients using gradient boosted trees reached an average accuracy of 79.9%. The results suggest that individual change over time or an absolute value at one time point may predict a disease with high specificity and sensitivity.

Precipitation Stripping of V(V) as a Novel Approach for the Preparation of Two-Dimensional Transition Metal Vanadates.

Cobalt, nickel, manganese and zinc vanadates were synthesized by a hydrometallurgical two-phase method. The extraction of vanadium (V) ions from alkaline solution using Aliquat® 336 was followed by the production of metal vanadates through precipitation stripping. Precipitation stripping was carried out using solutions of the corresponding metal ions (Ni (II), Co (II), Mn (II) and Zn (II), 0.05 mol/L in 4 mol/L NaCl), and the addition time of the strip solution was varied (0, 1 and 2 h). The time-dependent experiments showed a notable influence on the composition, structure, morphology and crystallinity of the two-dimensional vanadate products. Inspired by these findings, we selected two metallic vanadate products and studied their properties as alternative cathode materials for nonaqueous sodium and lithium metal batteries.

A Temporary Pause in the Replication Licensing Restriction Leads to Rereplication during Early Human Cell Differentiation.

Gene amplifications in amphibians and flies are known to occur during development and have been well characterized, unlike in mammalian cells, where they are predominantly investigated as an attribute of tumors. Recently, we first described gene amplifications in human and mouse neural stem cells, myoblasts, and mesenchymal stem cells during differentiation. The mechanism leading to gene amplifications in amphibians and flies depends on endocycles and multiple origin-firings. So far, there is no knowledge about a comparable mechanism in normal human cells. Here, we describe rereplication during the early myotube differentiation of human skeletal myoblast cells, using fiber combing and pulse-treatment with EdU (5'-Ethynyl-2'-deoxyuridine)/CldU (5-Chlor-2'-deoxyuridine) and IdU (5-Iodo-2'-deoxyuridine)/CldU. We found rereplication during a restricted time window between 2 h and 8 h after differentiation induction. Rereplication was detected in cells simultaneously with the amplification of the MDM2 gene. Our findings support rereplication as a mechanism enabling gene amplification in normal human cells.

Gene Amplification in Tumor Cells: Developed De Novo or Adopted from Stem Cells.

Gene amplifications have been known for several decades as physiological processes in amphibian and flies, e.g., during eggshell development in Drosophila and as part of pathological processes in humans, specifically in tumors and drug-resistant cells. The long-held belief that a physiological gene amplification does not occur in humans was, however, fundamental questioned by findings that showed gene amplification in human stem cells. We hypothesis that the physiological and the pathological, i.e., tumor associated processes of gene amplification share at their beginning the same underlying mechanism. Re-replication was reported both in the context of tumor related genome instability and during restricted time windows in Drosophila development causing the known developmental gene amplification in Drosophila. There is also growing evidence that gene amplification and re-replication were present in human stem cells. It appears likely that stem cells utilize a re-replication mechanism that has been developed early in evolution as a powerful tool to increase gene copy numbers very efficiently. Here, we show that, several decades ago, there was already evidence of gene amplification in non-tumor mammalian cells, but that was not recognized at the time and interpreted accordingly. We give an overview on gene amplifications during normal mammalian development, the possible mechanism that enable gene amplification and hypothesize how tumors adopted this capability for gene amplification.

Characterization of micro-RNA in women with different ovarian reserve.

Women undergoing infertility treatment are routinely subjected to one or more tests of ovarian reserve. Therefore, an adequate assessment of the ovarian reserve is necessary for the treatment. In this study, we aimed to characterize the potential role of microRNAs (miRNAs) as biomarkers for women with different ovarian reserves. A total of 159 women were recruited in the study and classified according to their anti-Müllerian hormone (AMH) level into three groups: (1) low ovarian reserve (LAMH, n = 39), (2) normal ovarian reserve (NAMH, n = 80), and (3) high ovarian reserve (HAMH, n = 40). SurePrint Human miRNA array screening and reverse transcription-quantitative PCR (RT-qPCR) were respectively employed to screen and validate the miRNA abundance level in the three tested groups. Compared with NAMH, the abundance level of 34 and 98 miRNAs was found to be significantly altered in LAMH and HAMH, respectively. The abundance level of miRNAs was further validated by RT-qPCR in both, the screening samples as well as in an independent set of validation samples. The abundance levels of the validated miRNAs were significantly correlated with the AMH level. The best AUC value for the prediction of the increase and decrease in the AMH level was obtained for the miR-100-5p and miR-21-5p, respectively. The level of miRNAs abundance correlates with the level of AMH, which may serve as a tool for identifying women with a different ovarian reserve and may help to lay the ground for the development of novel diagnostic approaches.

Integrated microRNA and mRNA Expression Profiling Identifies Novel Targets and Networks Associated with Ebstein's Anomaly.

Little is known about abundance level changes of circulating microRNAs (miRNAs) and messenger RNAs (mRNA) in patients with Ebstein's anomaly (EA). Here, we performed an integrated analysis to identify the differentially abundant miRNAs and mRNA targets and to identify the potential therapeutic targets that might be involved in the mechanisms underlying EA. A large panel of human miRNA and mRNA microarrays were conducted to determine the genome-wide expression profiles in the blood of 16 EA patients and 16 age and gender-matched healthy control volunteers (HVs). Differential abundance level of single miRNA and mRNA was validated by Real-Time quantitative PCR (RT-qPCR). Enrichment analyses of altered miRNA and mRNA abundance levels were identified using bioinformatics tools. Altered miRNA and mRNA abundance levels were observed between EA patients and HVs. Among the deregulated miRNAs and mRNAs, 76 miRNAs (49 lower abundance and 27 higher abundance, fold-change of ≥2) and 29 mRNAs (25 higher abundance and 4 lower abundance, fold-change of ≥1.5) were identified in EA patients compared to HVs. Bioinformatics analysis identified 37 pairs of putative miRNA-mRNA interactions. The majority of the correlations were detected between the lower abundance level of miRNA and higher abundance level of mRNA, except for let-7b-5p, which showed a higher abundance level and their target gene, SCRN3, showed a lower abundance level. Pathway enrichment analysis of the deregulated mRNAs identified 35 significant pathways that are mostly involved in signal transduction and cellular interaction pathways. Our findings provide new insights into a potential molecular biomarker(s) for the EA that may guide the development of novel targeting therapies.

MicroRNA-targeting in spermatogenesis: Over-expressions of microRNA-23a/b-3p and its affected targeting of the genes ODF2 and UBQLN3 in spermatozoa of patients with oligoasthenozoospermia.

BACKGROUND: Male infertility is a multifactorial syndrome with diverse phenotypic representations. MicroRNAs (miRNAs) are small, non-coding RNAs that are involved in the post-transcriptional regulation of gene expression. Altered abundance levels of ODF2 and UBQLN3 have been reported in patients with different spermatogenic impairments. However, the transcriptional regulation of these two genes by miR-23a/b-3p is still unclear. OBJECTIVES: To investigate experimentally whether miR-23a/b-3p targets the genes ODF2 and UBQLN3 and whether this targeting impacts abundance levels of ODF2 and UBQLN3 in patients with oligoasthenozoospermia. MATERIALS AND METHODS: A total of 92 men attending a fertility clinic were included in the study, including 46 oligoasthenozoospermic men and 46 age-matched normozoospermic volunteers who served as controls. Reverse transcription-quantitative PCR (RT-qPCR), Western blot, and dual-luciferase (Firefly-Renilla) assays were used to validate the miRNAs and their target genes. RESULTS: RT-qPCR revealed that miR-23a/b-3p was more abundant and ODF2 and UBQLN3 targets were less abundant in men with impaired spermatogenesis. Besides, Western blot shows that ODF2 and UBQLN3 protein levels were reduced in men with impaired spermatogenesis. In silico prediction and dual-luciferase assays revealed that potential links exist between the higher abundance level of miR-23a/b-3p and the lower abundance level of ODF2 and UBQLN3 targets. Mutations in the miR-23a/b-3p-binding site within the 3'UTRs (3'untranslated regions) of ODF2 and UBQLN3 genes resulted in abrogated responsiveness to miR-23a/b-3p. Correlation analysis showed that sperm count, motility, and morphology were negatively correlated with miR-23a/b-3p and positively correlated with the lower abundance level of UBQLN3, while ODF lower abundance level was positively correlated with sperm motility. CONCLUSION: Findings indicate that the higher abundance level of miR-23a/b-3p and the lower abundance level of ODF2 and UBQLN3 targets are associated with oligoasthenozoospermia and male subfertility.

MicroRNAs in combined spent culture media and sperm are associated with embryo quality and pregnancy outcome.

OBJECTIVE: To identify differentially abundant miRNAs in sperm samples and spent culture media (SCM) of embryos of different grade toward a prediction of pregnancy outcome. DESIGN: Array-based reverse-transcription quantitative polymerase chain reaction profiling and validation. SETTING: University research institute and in vitro fertilization center. PATIENT(S): Couples (n = 61) undergoing infertility treatment with the use of intracytoplasmic sperm injection. INTERVENTIONS(S): None. MAIN OUTCOME MEASURE(S): Abundance levels of miRNAs in combined SCM of embryos of different quality and in sperm samples associated with pregnancy outcome. RESULT(S): Out of 372 screened miRNAs, miR-19b-3p and let-7a-5p were detected consistently in all SCM and sperm samples. The abundance levels of miRNAs were significantly altered between SCM of embryos with different quality (G1, G2, and G3 grades). Specifically, miR-320a and miR-15a-5p were differentially abundant in G1 vs. G2, miR-21-5p in G1 vs. G3, and miR-20a-5p in G2 vs. G3. The abundance levels of combined SCM and sperm derived miRNAs were also significantly altered between different pregnancy outcomes. MiR-19b-3p showed the highest area under the receiver operating characteristic curve values between positive and negative outcomes, with lower abundance levels in both combined SCM and sperm samples associated with a positive pregnancy outcome. MiR-320a, miR-15a-5p, miR-21-5p, and miR-20a-5p showed similar results in combined SCM samples. CONCLUSION(S): miRNA abundance levels in combined SCM and sperm differed significantly depending on embryo quality and pregnancy outcome. MiR-19b-3p may serve as a potential biomarker to predict pregnancy outcome.

Low miR-150-5p and miR-320b Expression Predicts Reduced Survival of COPD Patients.

Chronic obstructive pulmonary disease (COPD) is associated with an increased risk of death, reducing life expectancy on average between 5 and 7 years. The survival time after diagnosis, however, varies considerably as a result of the heterogeneity of COPD. Therefore, markers that predict individual survival of COPD patients are of great value. We analyzed baseline molecular profiles and collected 54 months of follow-up data of the cohort study "COPD and SYstemic consequences-COmorbidities NETwork" (COSYCONET). Genome-wide microRNA signatures from whole blood collected at time of the inclusion in the study were generated for 533 COPD patients including patients that deceased during the 54-month follow-up period (n = 53) and patients that survived this period (n = 480). We identified two blood-born microRNAs (miR-150-5p and miR-320b) that were highly predictive for survival of COPD patients. The expression change was then confirmed by RT-qPCR in 245 individuals. Ninety percent of patients with highest expression of miR-150-5p survived the 54-month period in contrast to only 50% of patients with lowest expression intensity. Moreover, the abundance of the oncogenic miR-150-5p in blood of COPD patients was predictive for the development of cancer. Thus, molecular profiles measured at the time of a COPD diagnosis have a high predictive power for the survival of patients.

Differential expression of miR-23a/b-3p and its target genes in male patients with subfertility.

OBJECTIVE: To elucidate the potential regulatory function of miR-23a/b-3p on spermatogenesis-specific genes. DESIGN: Reverse transcription quantitative polymerase chain reaction (RT-qPCR) validation, Northern blot, dual luciferase assay, and Western blot confirmation. SETTING: University research and clinical institutes. PATIENT(S): A total of 115 men presenting at an infertility clinic. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Significant higher abundance levels of miR-23a/b-3p and lower abundance levels of PFKFB4, HMMR, SPATA6, and TEX15 in oligoasthenozoospermic men compared with those in normozoospermic men. RESULT(S): In oligoasthenozoospermic men, the abundance levels of miR-23a/b-3p were significantly higher when compared with controls as determined by RT-qPCR. After in silico prediction of potential targets of miR-23a/b-3p, PFKFB4, HMMR, SPATA6, and TEX15 have been identified as direct targets by dual luciferase assays. Mutations in the miR-23a/b-3p binding site within the 3'UTRs resulted in abrogated responsiveness to miR-23a/b-3p. PFKFB4, HMMR, SPATA6, and TEX15 mRNA and HMMR and SPATA6 protein levels were significantly lower in oligoasthenozoospermic men compared with in normozoospermic men. Correlation analysis showed that the sperm count, motility, and morphology were negatively correlated with miR-23a/b-3p and positively correlated with PFKFB4, HMMR, SPATA6, and TEX15 abundance levels (lower ΔCt, the higher abundance levels). CONCLUSION(S): This study establishes a link between up-regulation of miR-23a/b-3p and the coincident down-regulation of four expressed genes in the sperm of men with oligoasthenozoospermia, compared with men with normozoospermia. This study provides a novel insight into some of the mechanisms leading to male subfertility, offering a possible therapeutic target for treatment, or even for male contraception.

Prospect and challenge of detecting dynamic gene copy number increases in stem cells by whole genome sequencing.

Gene amplification is an evolutionarily well-conserved and highly efficient mechanism to increase the amount of specific proteins. In humans, gene amplification is a hallmark of cancer and has recently been found during stem cell differentiation. Amplifications in stem cells are restricted to specific tissue areas and time windows, rendering their detection difficult. Here, we report on the performance of deep WGS sequencing (average 82-fold depth of coverage) on the BGISEQ with nanoball technology to detect amplifications in human mesenchymal and neural stem cells. As reference technology, we applied array-based comparative genomic hybridization (aCGH), fluorescence in situ hybridization (FISH), and qPCR. Using different in silico strategies for amplification detection, we analyzed the potential of WGS for amplification detection. Our results provide evidence that WGS accurately identifies changes of the copy number profiles in human stem cell differentiation. However, the identified changes are not in all cases consistent between WGS and aCGH. The results between WGS and the validation by qPCR were concordant in 83.3% of all tested 36 cases. In sum, both genome-wide techniques, aCGH and WGS, have unique advantages and specific challenges, calling for locus-specific confirmation by the low-throughput approaches qPCR or FISH. KEY MESSAGES: WGS allows for the identification of dynamic copy number changes in human stem cells. Less stringent threshold setting is crucial for detection of copy number increase. Broad knowledge of dynamic copy number is pivotal to estimate stem cell capabilities.

An estimate of the total number of true human miRNAs.

While the number of human miRNA candidates continuously increases, only a few of them are completely characterized and experimentally validated. Toward determining the total number of true miRNAs, we employed a combined in silico high- and experimental low-throughput validation strategy. We collected 28 866 human small RNA sequencing data sets containing 363.7 billion sequencing reads and excluded falsely annotated and low quality data. Our high-throughput analysis identified 65% of 24 127 mature miRNA candidates as likely false-positives. Using northern blotting, we experimentally validated miRBase entries and novel miRNA candidates. By exogenous overexpression of 108 precursors that encode 205 mature miRNAs, we confirmed 68.5% of the miRBase entries with the confirmation rate going up to 94.4% for the high-confidence entries and 18.3% of the novel miRNA candidates. Analyzing endogenous miRNAs, we verified the expression of 8 miRNAs in 12 different human cell lines. In total, we extrapolated 2300 true human mature miRNAs, 1115 of which are currently annotated in miRBase V22. The experimentally validated miRNAs will contribute to revising targetomes hypothesized by utilizing falsely annotated miRNAs.

Large-scale validation of miRNAs by disease association, evolutionary conservation and pathway activity.

The validation of microRNAs (miRNAs) identified by next generation sequencing involves amplification-free and hybridization-based detection of transcripts as criteria for confirming valid miRNAs. Since respective validation is frequently not performed, miRNA repositories likely still contain a substantial fraction of false positive candidates while true miRNAs are not stored in the repositories yet. Especially if downstream analyses are performed with these candidates (e.g. target or pathway prediction), the results may be misleading. In the present study, we evaluated 558 mature miRNAs from miRBase and 1,709 miRNA candidates from next generation sequencing experiments by amplification-free hybridization and investigated their distributions in patients with various disease conditions. Notably, the most significant miRNAs in diseases are often not contained in the miRBase. However, these candidates are evolutionary highly conserved. From the expression patterns, target gene and pathway analyses and evolutionary conservation analyses, we were able to shed light on the complexity of miRNAs in humans. Our data also highlight that a more thorough validation of miRNAs identified by next generation sequencing is required. The results are available in miRCarta ( https://mircarta.cs.uni-saarland.de ).

Gene amplification in mesenchymal stem cells and during differentiation towards adipocytes or osteoblasts.

Gene amplifications are an attribute of tumor cells and have for long time been overlooked in normal cells. A growing number of investigations describe gene amplifications in normal mammalian cells during development and differentiation. Possibly, tumor cells have rescued the gene amplification mechanism as a physiological attribute of stem cells. Here, we investigated human mesenchymal stem cells (hMSCs) for gene amplification using array-CGH, single cell fluorescence in situ hybridization and qPCR. Gene amplifications were detected in mesenchymal stem cells and in mesenchymal stem cells during differentiation towards adipocytes and osteoblasts. Undifferentiated hMSCs harbor 12 amplified chromosomal regions, hMSCs that differentiated towards adipocytes 18 amplified chromosome regions, and hMSCs that differentiate towards osteoblasts 19 amplified regions. Specifically, hMSCs that differentiated towards adipocytes or osteoblasts harbor CDK4 and MDM2 amplifications both of which frequently occur in osteosarcoma and liposarcoma that are both of same cell origin. Beside the amplifications, we identified 36 under-replicated regions in undifferentiated and in differentiating hMSC cells.

A high-resolution map of the human small non-coding transcriptome.

Motivation: Although the amount of small non-coding RNA-sequencing data is continuously increasing, it is still unclear to which extent small RNAs are represented in the human genome. Results: In this study we analyzed 303 billion sequencing reads from nearly 25 000 datasets to answer this question. We determined that 0.8% of the human genome are reliably covered by 874 123 regions with an average length of 31 nt. On the basis of these regions, we found that among the known small non-coding RNA classes, microRNAs were the most prevalent. In subsequent steps, we characterized variations of miRNAs and performed a staged validation of 11 877 candidate miRNAs. Of these, many were actually expressed and significantly dysregulated in lung cancer. Selected candidates were finally validated by northern blots. Although isolated miRNAs could still be present in the human genome, our presented set likely contains the largest fraction of human miRNAs. Contact: c.backes@mx.uni-saarland.de or andreas.keller@ccb.uni-saarland.de. Supplementary information: Supplementary data are available at Bioinformatics online.

Specific amplifications and copy number decreases during human neural stem cells differentiation towards astrocytes, neurons and oligodendrocytes.

There is growing evidence that gene amplifications are an attribute of normal cells during development and differentiation. During neural progenitor cell differentiation half of the genome is involved in amplification process. To answer the question how specific amplifications occur at different stages and in different lineages of differentiation we analyzed the genes CDK4, MDM2, EGFR, GINS2, GFAP, TP53, DDB1 and MDM4 in human neural stem cells that were induced to differentiate towards astrocytes, neurons and oligodendrocytes. We found specific amplification pattern for each of the eight analyzed genes both in undifferentiated neural stem and progenitor cells and in cells that were induced for differentiation. Different amplification patterns were also found between adherently grown neural stem cells and cells that were grown as spheres. The most frequently amplified genes were MDM2 and CDK4 with the latter amplified in all three lineages at all analyzed stages. Amplification of the analyzed genes was also found in four glioma stem-like cells. The combined amplification data of stem cells and of tumor stem cells can help to define cell populations at the origin of the tumor. Furthermore, we detected a decrease of gene copies at specific differentiation stages most frequently for MDM4. This study shows specific amplification pattern in defined stem cell populations within specific time windows during differentiation processes indicating that amplifications occur in an orderly sequence during the differentiation of human neural stem and progenitor cells.

Human alveolar epithelial cells expressing tight junctions to model the air-blood barrier.

This paper describes a new human alveolar epithelial cell line (hAELVi - human Alveolar Epithelial Lentivirus immortalized) with type I-like characteristics and functional tight junctions, suitable to model the air-blood barrier of the peripheral lung. Primary human alveolar epithelial cells were immortalized by a novel regimen, grown as monolayers on permeable filter supports and characterized morphologically, biochemically and biophysically. hAELVi cells maintain the capacity to form tight intercellular junctions, with high trans-epithelial electrical resistance (> 1000 Ω*cm²). The cells could be kept in culture over several days, up to passage 75, under liquid-liquid as well as air-liquid conditions. Ultrastructural analysis and real time PCR revealed type I-like cell properties, such as the presence of caveolae, expression of caveolin-1, and absence of surfactant protein C. Accounting for the barrier properties, inter-digitations sealed with tight junctions and desmosomes were also observed. Low permeability of the hydrophilic marker sodium fluorescein confirmed the suitability of hAELVi cells for in vitro transport studies across the alveolar epithelium. These results suggest that hAELVi cells reflect the essential features of the air-blood barrier, as needed for an alternative to animal testing to study absorption and toxicity of inhaled drugs, chemicals and nanomaterials.