Immunocompetent 3D Model of Human Upper Airway for Disease Modeling and In Vitro Drug Evaluation

The development of more complex in vitro models for the assessment of novel drugs and chemicals is needed because of the limited biological relevance of animal models to humans as well as ethical considerations. Although some human-cell-based assays exist, they are usually 2D, consist of single cell type, and have limited cellular and functional representation of the native tissue. In this study, we have used biomimetic porous electrospun scaffolds to develop an immunocompetent 3D model of the human respiratory tract comprised of three key cell types present in upper airway epithelium. The three cell types, namely, epithelial cells (providing a physical barrier), fibroblasts (extracellular matrix production), and dendritic cells (immune sensing), were initially grown on individual scaffolds and then assembled into the 3D multicell tissue model. The epithelial layer was cultured at the air–liquid interface for up to four weeks, leading to formation of a functional barrier as evidenced by an increase in transepithelial electrical resistance (TEER) and tight junction formation. The response of epithelial cells to allergen exposure was monitored by quantifying changes in TEER readings and by assessment of cellular tight junctions using immunostaining. It was found that epithelial cells cocultured with fibroblasts formed a functional epithelial barrier at a quicker rate than single cultures of epithelial cells and that the recovery from allergen exposure was also more rapid. Also, our data show that dendritic cells within this model remain viable and responsive to external stimulation as evidenced by their migration within the 3D construct in response to allergen challenge. This model provides an easy to assemble and physiologically relevant 3D model of human airway epithelium that can be used for studies aiming at better understanding lung biology, the cross-talk between immune cells, and airborne allergens and pathogens as well as drug delivery

Unusual congenital polydactyly in mini-pigs from the breeding group of the Institute of Cytology and Genetics (Novosibirsk, Russia)

The article describes a new phenomenon in the breeding group of mini-pigs at the Institute of Cytology and Genetics (ICG, Novosibirsk): polydactyly (extra digits), which is unusual because the additional digits are situated at the lateral surface of legs or at the lateral and medial ones. This anomaly was first found here in 2017 in adult animals intended for culling due to incorrect positioning of the legs caused by flexor tendon laxity and resulting in weight-bearing on the palmar surface of the proximal phalanges (“bear’s paw”). Therefore, the polydactyly of mini-pigs has a pronounced negative selection effect. A visual survey of the livestock was conducted, and a description of the detected anomaly was compiled. The polydactyly in mini-pigs is a stand-alone trait and is not part of any syndromes. Individuals with polydactyly may have extra digits either on pectoral or on pectoral and pelvic limbs. On thoracic limbs, there may be either one lateral digit or a lateral digit and a medially located rudimentary hooflet. On pelvic limbs, only lateral extra digits can occur. Anatomical and morphological analyses showed that the lateral extra digit is an anatomically complete (“mature”) structure, whereas the medial rudimentary digit consists of only a hooflet without other structures characteristic of normal digits. Cytological examination revealed no specific karyotypic features, except for Robertsonian translocation Rb 16;17 previously reported for the mini-pigs of the same livestock. Cytological findings indicated that the polydactyly and Robertsonian translocation are not linked genetically. Genealogical analysis and results of crosses are consistent with a working hypothesis of recessive inheritance of the trait. Overall, the study shows that this type of polydactyly is anatomically and morphologically unique and not typical of Sus scrofa. In this species, only polydactyly types with medial accessory toes have been described and are usually inherited as a dominant trait with incomplete penetrance. In our case, the results of test crosses indicate recessive inheritance of the trait with varying expression and incomplete penetrance, because of which poorly expressed phenotypes are not visually detectable

КРУПНОПЛОДНОСТЬ МИНИ-СВИНЕЙ ИЦиГ СО РАН: ПОТЕНЦИАЛ НЕРЕАЛИЗУЕМЫХ ВОЗМОЖНОСТЕЙ

This publication presents the results of the analysis of the dynamics of changes in the largefruited indicators of the breeding group of minipigs of the ICG SB RAS. The analysis showed that the four large-copious indicators are divided into two pairs. The first pair is made up of sample values of the characteristic: average and maximum. These indicators are characterized by stability throughout the studied period. The second pair includes the sample minimum values and standard deviations of the trait. These two indicators are dynamic: the sample minimum values are characterized by a decrease, and the sample standard deviations are characterized by a uniform increase, described by linear regression equations. It is shown that the dynamic characteristics are related to each other. It is determined that in this complex, the leader is the minimum value, and the follower is the standard deviation. This is explained by the fact that an increase in the standard deviation is associated with a decrease in the minimum value and the stability of the maximum in the studied period of time. The result of this process is the growth of the genetic potential in the breeding group, which is responsible for the high weight of the newborn individual. However, due to the small size of sows in comparison with commercial breeds (60-70 kg), this potential cannot be realized. Nevertheless, its redundancy ensures the stabilization of the maximum and average values of the trait - the mass of a newborn individual in minipigs of the ICG SB RAS. A possible way to increase the realization of the potential of large-copious breeding group is to reduce the multiple fertility of sows, which is quite solvable, but hardly advisable. Thus there is natural selection directed against individuals with a low birth weight in the herd. Natural and artificial selection for live weight of piglets at birth of 700 g or more, both help to stabilize the average value of the trait at the level optimal for the broodstock.Настоящая публикация представляет результаты анализа динамик изменения показателей крупноплодности селекционной группы мини-свиней ИЦиГ СО РАН. Анализ показал, что четыре показателя крупноплодности делятся на две пары. Первую пару составляют выборочные значения признака: среднее и максимальное. Для этих показателей характерна стабильность на протяжении изучаемого периода. Во вторую пару вошли выборочные минимальные значения и стандартные отклонения признака. Эти два показателя являются динамическими: для выборочных минимальных значений характерно снижение, а для выборочных стандартных отклонений равномерное повышение, описываемое уравнениями линейной регрессии. Показано, что динамические характеристики связаны друг с другом. Определено, что в данном комплексе ведущим является минимальное значение, а ведомым – стандартное отклонение. Объясняется это тем, что увеличение стандартного отклонения сопряжено с уменьшением минимального значения и стабильностью максимального в изучаемый промежуток времени. Результатом этого процесса является рост в селекционной группе генетического потенциала, отвечающего за высокую массу новорожденной особи. Однако из-за малых по сравнению с коммерческими породами размеров свиноматок (60–70 кг) этот потенциал не может быть реализован. Тем не менее его избыточность обеспечивает стабилизацию максимального и среднего значения признака – массы новорождённой особи у мини-свиней ИЦиГ СО РАН. Возможный путь повышения реализации потенциала крупноплодности селекционной группы – это снижение многоплодия свиноматок, что вполне решаемо, но вряд ли целесообразно. Таким образом, в стаде присутствует естественный отбор, направленный против особей с малой массой при рождении, который в совокупности с искусственным отбором на живую массу поросят при рождении 700 г и более способствует стабилизации среднего значения признака на оптимальном для маточного поголовья уровне

Multiscale multifactorial approaches for engineering tendon substitutes

The physiology of tendons and the continuous strains experienced daily make tendons very prone to injury. Excessive and prolonged loading forces and aging also contribute to the onset and progression of tendon injuries, and conventional treatments have limited efficacy in restoring tendon biomechanics. Tissue engineering and regenerative medicine (TERM) approaches hold the promise to provide therapeutic solutions for injured or damaged tendons despite the challenging cues of tendon niche and the lack of tendon-specific factors to guide cellular responses and tackle regeneration. The roots of engineering tendon substitutes lay in multifactorial approaches from adequate stem cells sources and environmental stimuli to the construction of multiscale 3D scaffolding systems. To achieve such advanced tendon substitutes, incremental strategies have been pursued to more closely recreate the native tendon requirements providing structural as well as physical and chemical cues combined with biochemical and mechanical stimuli to instruct cell behavior in 3D architectures, pursuing mechanically competent constructs with adequate maturation before implantation.Authors acknowledge the project “Accelerating tissue engineering and personalized medicine discoveries by the integration of key enabling nanotechnologies, marinederived biomaterials and stem cells,” supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). Authors acknowledge the H2020 Achilles Twinning Project No. 810850, and also the European Research Council CoG MagTendon No. 772817, and the FCT Project MagTT PTDC/CTM-CTM/ 29930/2017 (POCI-01-0145-FEDER-29930

Investigating NF-kappa B signaling in lung fibroblasts in 2D and 3D culture systems

BACKGROUND: Inflammatory respiratory diseases are amongst major global health challenges. Lung fibroblasts have been shown to play a key role in lung inflammatory responses. However, their exact role in initiation and maintenance of lung diseases has remained elusive partly due to the limited availability of physiologically relevant in vitro models. Therefore, developing new tools that enable investigating the molecular pathways (e.g. nuclear factor-kappa B (NF-κB) activation) that underpin inflammatory responses in fibroblasts could be a valuable resource for scientists working in this area of research. RESULTS: In order to investigate NF-κB activation in response to pro-inflammatory stimuli in real-time, we first developed two detection systems based on nuclear localization of NF-κB by immunostaining and luciferase reporter assay system. Furthermore using electrospun porous scaffolds, with similar geometry to human lung extracellular matrix, we developed 3D cultures of lung fibroblasts allowing comparing NF-κB activation in response to pro-inflammatory stimuli (i.e. TNF-α) in 2D and 3D. Our data clearly show that the magnitude of NF-κB activation in 2D cultures is substantially higher than 3D cultures. However, unlike 2D cultures, cells in the 3D model remained responsive to TNF-α at higher concentrations. The more subdued and wider dynamic range of NF-κB responses in 3D culture system was associated with a different expression pattern for TNF receptor I in 3D versus 2D cultures collectively reflecting a more in vivo like TNF receptor I expression and NF-κB activation pattern in the 3D system. CONCLUSION: Our data suggest that lung fibroblasts are actively involved in the pathogenesis of lung inflammation by activation of NF-κB signaling pathway. The 3D culture detection system provides a sensitive and biologically relevant tool for investigating different pro-inflammatory events involving lung fibroblasts

The Crucial p53-Dependent Oncogenic Role of JAB1 in Osteosarcoma in vivo

Osteosarcoma (OS) is the most common primary bone cancer and ranks amongst the leading causes of cancer mortality in young adults. Jun activation domain binding protein 1 (JAB1) is overexpressed in many cancers and has recently emerged as a novel target for cancer treatment. However, the role of JAB1 in osteosarcoma was virtually unknown. In this study, we demonstrate that JAB1-knockdown in malignant osteosarcoma cell lines significantly reduced their oncogenic properties, including proliferation, colony formation, and motility. We also performed RNA-sequencing analysis in JAB1-knockdown OS cells and identified 4110 genes that are significantly differentially expressed. This demonstrated for the first time that JAB1 regulates a large and specific transcriptome in cancer. We also found that JAB1 is overexpressed in human OS and correlates with a poor prognosis. Moreover, we generated a novel mouse model that overexpresses Jab1 specifically in osteoblasts upon a TP53 heterozygous sensitizing background. Interestingly, by 13 months of age, a significant proportion of these mice spontaneously developed conventional OS. Finally, we demonstrate that a novel, highly specific small molecule inhibitor of JAB1, CSN5i-3, reduces osteosarcoma cell viability and has specific effects on the ubiquitin-proteasome system in OS. Thus, we show for the first time that the overexpression of JAB1 in vivo can result in accelerated spontaneous tumor formation in a p53-dependent manner. In summary, JAB1 might be a unique target for the treatment of osteosarcoma and other cancers

LARGE-COPIOUS MINIPIGS OF ICG SB RAS: POTENTIAL OF UNREALIZABLE OPPORTUNITIES

This publication presents the results of the analysis of the dynamics of changes in the largefruited indicators of the breeding group of minipigs of the ICG SB RAS. The analysis showed that the four large-copious indicators are divided into two pairs. The first pair is made up of sample values of the characteristic: average and maximum. These indicators are characterized by stability throughout the studied period. The second pair includes the sample minimum values and standard deviations of the trait. These two indicators are dynamic: the sample minimum values are characterized by a decrease, and the sample standard deviations are characterized by a uniform increase, described by linear regression equations. It is shown that the dynamic characteristics are related to each other. It is determined that in this complex, the leader is the minimum value, and the follower is the standard deviation. This is explained by the fact that an increase in the standard deviation is associated with a decrease in the minimum value and the stability of the maximum in the studied period of time. The result of this process is the growth of the genetic potential in the breeding group, which is responsible for the high weight of the newborn individual. However, due to the small size of sows in comparison with commercial breeds (60-70 kg), this potential cannot be realized. Nevertheless, its redundancy ensures the stabilization of the maximum and average values of the trait - the mass of a newborn individual in minipigs of the ICG SB RAS. A possible way to increase the realization of the potential of large-copious breeding group is to reduce the multiple fertility of sows, which is quite solvable, but hardly advisable. Thus there is natural selection directed against individuals with a low birth weight in the herd. Natural and artificial selection for live weight of piglets at birth of 700 g or more, both help to stabilize the average value of the trait at the level optimal for the broodstock.</jats:p

Unusual congenital polydactyly in mini-pigs from the breeding group of the Institute of Cytology and Genetics (Novosibirsk, Russia)

The article describes a new phenomenon in the breeding group of mini-pigs at the Institute of Cytology and Genetics (ICG, Novosibirsk): polydactyly (extra digits), which is unusual because the additional digits are situated at the lateral surface of legs or at the lateral and medial ones. This anomaly was first found here in 2017 in adult animals intended for culling due to incorrect positioning of the legs caused by flexor tendon laxity and resulting in weight-bearing on the palmar surface of the proximal phalanges (“bear’s paw”). Therefore, the polydactyly of mini-pigs has a pronounced negative selection effect. A visual survey of the livestock was conducted, and a description of the detected anomaly was compiled. The polydactyly in mini-pigs is a stand-alone trait and is not part of any syndromes. Individuals with polydactyly may have extra digits either on pectoral or on pectoral and pelvic limbs. On thoracic limbs, there may be either one lateral digit or a lateral digit and a medially located rudimentary hooflet. On pelvic limbs, only lateral extra digits can occur. Anatomical and morphological analyses showed that the lateral extra digit is an anatomically complete (“mature”) structure, whereas the medial rudimentary digit consists of only a hooflet without other structures characteristic of normal digits. Cytological examination revealed no specific karyotypic features, except for Robertsonian translocation Rb 16;17 previously reported for the mini-pigs of the same livestock. Cytological findings indicated that the polydactyly and Robertsonian translocation are not linked genetically. Genealogical analysis and results of crosses are consistent with a working hypothesis of recessive inheritance of the trait. Overall, the study shows that this type of polydactyly is anatomically and morphologically unique and not typical of Sus scrofa. In this species, only polydactyly types with medial accessory toes have been described and are usually inherited as a dominant trait with incomplete penetrance. In our case, the results of test crosses indicate recessive inheritance of the trait with varying expression and incomplete penetrance, because of which poorly expressed phenotypes are not visually detectable.</jats:p