Bone Morphogenetic Protein 4 (BMP4) Enhances the Differentiation of Human Induced Pluripotent Stem Cells into Limbal Progenitor Cells

Corneal epithelium maintains visual acuity and is regenerated by the proliferation and differentiation of limbal progenitor cells. Transplantation of human limbal progenitor cells could restore the integrity and functionality of the corneal surface in patients with limbal stem cell deficiency. However, multiple protocols are employed to differentiate human induced pluripotent stem (iPS) cells into corneal epithelium or limbal progenitor cells. The aim of this study was to optimize a protocol that uses bone morphogenetic protein 4 (BMP4) and limbal cell-specific medium. Human dermal fibroblast-derived iPS cells were differentiated into limbal progenitor cells using limbal cell-specific (PI) medium and varying doses (1, 10, and 50 ng/mL) and durations (1, 3, and 10 days) of BMP4 treatment. Differentiated human iPS cells were analyzed by real-time polymerase chain reaction (RT-PCR), Western blotting, and immunocytochemical studies at 2 or 4 weeks after BMP4 treatment. Culturing human dermal fibroblast-derived iPS cells in limbal cell-specific medium and BMP4 gave rise to limbal progenitor and corneal epithelial-like cells. The optimal protocol of 10 ng/mL and three days of BMP4 treatment elicited significantly higher limbal progenitor marker (ABCG2, ∆Np63α) expression and less corneal epithelial cell marker (CK3, CK12) expression than the other combinations of BMP4 dose and duration. In conclusion, this study identified a successful reprogramming strategy to induce limbal progenitor cells from human iPS cells using limbal cell-specific medium and BMP4. Additionally, our experiments indicate that the optimal BMP4 dose and duration favor limbal progenitor cell differentiation over corneal epithelial cells and maintain the phenotype of limbal stem cells. These findings contribute to the development of therapies for limbal stem cell deficiency disorders.

Photobook phenomenon: an interview with Moritz Neumüller

In this interview, Moritz Neumüller reflects on the nature of the photobook as a specific medium, and examines its place in contemporary photography. The interview was made by João Queiroz and Ana Luiza Fernandes in February and March 2021.

R U ready 4 new subtitles? Investigating the potential of social translation practices and creative spellings

In this paper I investigate novel and creative linguistic features used in non-conventional subtitling settings such as fansubbing, arguing that they can be advantageously used in professional subtitling practices for a specific medium, such as the Internet. The integration of txt lingo in subtitling is supported by the recent explosion of social translation practices as a response to an ever-growing audience fragmentation as well as changes in technology which make the integration of several customised subtitling tracks possible. In an attempt to provide empirical evidence to support this argument I present the initial results of a pilot eye-tracker-based experiment to elicit data on the reception of “unregimented” subtitling when offered as an alternative to conventional subtitling from consumers in selected new subtitling contexts.

Assessment of Miniature Piezoelectric Travelling-Wave Beam and Plate Robots

Different up-to-date utilizations have found several benefits in condensing the size of autonomous robots. Miniature traveling wave piezoelectric robots have proven to be appropriate for many of these applications. The principles of locomotion embraced in these robots are mainly inspired by natural biological locomotion and could be categorized by their movement through a specific medium. In this article, after having highlighted the amplifying effect of piezoelectric actuators generating the locomotion necessary for robotic requests, we will review the different types of such locomotion. Next, we will discuss the traveling wave piezoelectric resonant robots. Succeeding, we will look at the operation and usages of piezoelectric beam and plate robots. Finally, we will discuss the modeling aspects implicated in these robots and more generally, the modeling of piezoelectric patches stuck on thin structures. Keywords: piezoelectric, miniature, travelling-wave, locomotion, beam and plate robots

Media Matters, Examining Historical and Modern Streptococcus pneumoniae Growth Media and the Experiments They Affect

While some bacteria can thrive for generations in minerals and salts, many require lavish nutrition and specific chemicals to survive to the point where they can be observed and researched. Although researchers once boiled and rendered animal flesh and bones to obtain a media that facilitated bacterial growth, we now have a plethora of formulations and manufacturers to provide dehydrated flavors of historical, modified, and modern media. The purpose of media has evolved from simple isolation to more measured study. However, in some instances, media formulated to aid the metabolic, nutritional, or physical properties of microbes may not be best suited for studying pathogen behavior or resilience as a function of host interactions. While there have been comparative studies on handfuls of these media in Streptococcus pneumoniae, this review focuses on describing both the historical and modern composition of common complex (Todd Hewitt and M17), semi-defined (Adams and Roe), and defined pneumococcal media (RPMI and Van de Rijn and Kessler), key components discovered/needed for cultivation/growth enhancement, and effects these different media have on bacterial phenotypes and experimental outcomes. While many researchers find the best conditions to grow and experiment on their bacteria of choice, the reasons for some researchers to use a specific medium is at best, not discussed, and at worst, arbitrary. As such, the goal of this review is to highlight the differences in pneumococcal media to encourage investigators to challenge their decisions on why they use a given medium, discuss the recipe, and explain their reasoning.

Hybrid Collagen Hydrogel/Chondroitin-4-Sulphate Fortified with Dermal Fibroblast Conditioned Medium for Skin Therapeutic Application

The current strategy for rapid wound healing treatment involves combining a biomaterial and cell-secreted proteins or biomolecules. This study was aimed at characterizing 3-dimensional (3D) collagen hydrogels fortified with dermal fibroblast-conditioned medium (DFCM) as a readily available acellular skin substitute. Confluent fibroblasts were cultured with serum-free keratinocyte-specific medium (KM1 and KM2) and fibroblast-specific medium (FM) to obtain DFCM. Subsequently, the DFCM was mixed with collagen (Col) hydrogel and chondroitin-4-sulphate (C4S) to fabricate 3D constructs termed Col/C4S/DFCM-KM1, Col/C4S/DFCM-KM2, and Col/C4S/DFCM-FM. The constructs successfully formed soft, semi-solid and translucent hydrogels within 1 h of incubation at 37 °C with strength of <2.5 Newton (N). The Col/C4S/DFCM demonstrated significantly lower turbidity compared to the control groups. The Col/C4S/DFCM also showed a lower percentage of porosity (KM1: 35.15 ± 9.76%; KM2: 6.85 ± 1.60%; FM: 14.14 ± 7.65%) compared to the Col (105.14 ± 11.87%) and Col/C4S (143.44 ± 27.72%) constructs. There were no changes in both swelling and degradation among all constructs. Fourier transform infrared spectrometry showed that all groups consisted of oxygen–hydrogen bonds (O-H) and amide I, II, and III. In conclusion, the Col/C4S/DFCM constructs maintain the characteristics of native collagen and can synergistically deliver essential biomolecules for future use in skin therapeutic applications.

Publishing Patterns in Greek Media Websites

The concept of different publishing patterns during a day has been employed for many decades in the broadcasting industry. These patterns are close related with dayparts, which are defined as sequential time blocks on comparable days during which the audience size is homogeneous, as is the group depiction using the specific medium. During the first decade of the World Wide Web, Internet media strategy was focused on total web reach, demographics and affinity of content without particular attention to how the nature of the audience changes by time of day. This paper studies the variation of publishing patterns of the top 22 Greek media websites. More than 550 thousand articles were indexed in a period of four and a half months. The study identified distinct WWW time periods that exhibit specific publishing characteristics. Specifically, different categories of news articles present different publishing patterns during weekdays and weekends. The results appear to be in agreement with findings of previous studies.

Comparison of BCYE and BMPA media on recovery rate of Legionella pneumophila

Latar belakang: Legionella pneumophila (L. pneumophila) telah banyak diketahui sebagai penyebab legionellosis; habitat nya di berbagai sumber air, lingkungan lembab dan hangat. Metode kultur menggunakan media spesifik masih menjadi baku emas untuk identifikasi L. pneumophila. Penelitian ini bertujuan membandingkan recovery rate L. pneumophila ATCC®33823 pada medium spesifik BCYE, dan medium selektif BMPA. Metode: Dilakukan dilusi serial pada suspensi 0,5 McFarland L. pneumophila ATCC®33823; 100 ul dari tiap tingkat dilusi diinokulasi pada medium Buffered Charcoal Yeast Extract (BCYE) dan medium BMPA (medium BCYE yang ditambahkan suplemen BMPA-α), secara duplo. Konsentrasi suspensi dihitung menggunakan metode Angka Lempeng Total (ALT) dari Standar Nasional Indonesia No. 01-2332.3-2006. Persentase recovery rate dihitung dan dianalisis secara statistik menggunakan SPSS versi 23,0. Hasil: Jumlah koloni L. pneumophila yang tumbuh pada medium BMPA jauh lebih tinggi daripada yang tumbuh di medium BCYE; konsentrasi tertinggi yang diperoleh adalah pada medium BMPA sebesar 1,45 x 107 CFU/ml. Persentase recovery rate pada medium BMPA adalah 96,67%, dan 60,67% pada medium BCYE. Kesimpulan: Recovery rate medium BMPA untuk pertumbuhan koloni L. pneumophila ATCC®33823 jauh lebih tinggi daripada media BCYE, karena itu medium BMPA dapat direkomendasikan untuk kultivasi L. pneumophila, khususnya pada program surveilans berbagai sumber air dengan biaya lebih terjangkau. Kata kunci: Legionella pneumophila, medium spesifik, BCYE , BMPA, recovery rate Abstract Background: Legionella pneumophila (L. pneumophila) has been known as the etiology of legionellosis; they live in aquatic environment, warm and moist. Culture method using specific medium remains as the gold standard in the identification of L. pneumophila. This study aimed to compare the recovery rate of L. pneumophila ATCC® 33823 on the specific medium BCYE for the cultivation of Legionella, and BMPA, the selective medium. Methods: Suspension of L. pneumophila ATCC® 33823 of 0.5 McFarland was diluted to 10 fold serial dilution; 100 ul of each dilution was inoculated on Buffered Charcoal Yeast Extract (BCYE) medium, and BMPA (BCYE supplemented with BMPA-α) in duplicate manner. The concentration was calculated using Total Plate Count standard as of Indonesian Nasional Standard number 01-2332.3-2006. The percentage of recovery rate was calculated, and the statistical analysis was performed using SPSS version 23.0. Results: Numbers of colonies of L. pneumophila grew on BMPA was much higher than on BCYE medium; the highest concentration was yielded on BMPA medium i.e. 1.45x107 CFU/ml. The recovery rates were 96.67% and 60.67% on BMPA medium and BCYE subsequently. Conclusion: The recovery rate of the BMPA medium on the colony growth of L. pneumophila ATCC®33823 was markedly higher than the BCYE, therefore BMPA medium can be suggested to be used in the cultivation of L. pneumophila especially in the routine surveillance program for water sources with less cost. Keywords: Legionella pneumophila, specific medium, BCYE , BMPA, recovery rate

A multi-organ-chip co-culture of liver and testis equivalents: a first step toward a systemic male reprotoxicity model

STUDY QUESTION Is it possible to co-culture and functionally link human liver and testis equivalents in the combined medium circuit of a multi-organ chip? SUMMARY ANSWER Multi-organ-chip co-cultures of human liver and testis equivalents were maintained at a steady-state for at least 1 week and the co-cultures reproduced specific natural and drug-induced liver–testis systemic interactions. WHAT IS KNOWN ALREADY Current benchtop reprotoxicity models typically do not include hepatic metabolism and interactions of the liver–testis axis. However, these are important to study the biotransformation of substances. STUDY DESIGN, SIZE, DURATION Testicular organoids derived from primary adult testicular cells and liver spheroids consisting of cultured HepaRG cells and hepatic stellate cells were loaded into separate culture compartments of each multi-organ-chip circuit for co-culture in liver spheroid-specific medium, testicular organoid-specific medium or a combined medium over a week. Additional multi-organ-chips (single) and well plates (static) were loaded only with testicular organoids or liver spheroids for comparison. Subsequently, the selected type of medium was supplemented with cyclophosphamide, an alkylating anti-neoplastic prodrug that has demonstrated germ cell toxicity after its bioactivation in the liver, and added to chip-based co-cultures to replicate a human liver–testis systemic interaction in vitro. Single chip-based testicular organoids were used as a control. Experiments were performed with three biological replicates unless otherwise stated. PARTICIPANTS/MATERIALS, SETTING, METHODS The metabolic activity was determined as glucose consumption and lactate production. The cell viability was measured as lactate dehydrogenase activity in the medium. Additionally, immunohistochemical and real-time quantitative PCR end-point analyses were performed for apoptosis, proliferation and cell-specific phenotypical and functional markers. The functionality of Sertoli and Leydig cells in testicular spheroids was specifically evaluated by measuring daily inhibin B and testosterone release, respectively. MAIN RESULTS AND THE ROLE OF CHANCE Co-culture in multi-organ chips with liver spheroid-specific medium better supported the metabolic activity of the cultured tissues compared to other media tested. The liver spheroids did not show significantly different behaviour during co-culture compared to that in single culture on multi-organ-chips. The testicular organoids also developed accordingly and produced higher inhibin B but lower testosterone levels than the static culture in plates with testicular organoid-specific medium. By comparison, testosterone secretion by testicular organoids cultured individually on multi-organ-chips reached a similar level as the static culture at Day 7. This suggests that the liver spheroids have metabolised the steroids in the co-cultures, a naturally occurring phenomenon. The addition of cyclophosphamide led to upregulation of specific cytochromes in liver spheroids and loss of germ cells in testicular organoids in the multi-organ-chip co-cultures but not in single-testis culture. LARGE-SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION The number of biological replicates included in this study was relatively small due to the limited availability of individual donor testes and the labour-intensive nature of multi-organ-chip co-cultures. Moreover, testicular organoids and liver spheroids are miniaturised organ equivalents that capture key features, but are still simplified versions of the native tissues. Also, it should be noted that only the prodrug cyclophosphamide was administered. The final concentration of the active metabolite was not measured. WIDER IMPLICATIONS OF THE FINDINGS This co-culture model responds to the request of setting up a specific tool that enables the testing of candidate reprotoxic substances with the possibility of human biotransformation. It further allows the inclusion of other human tissue equivalents for chemical risk assessment on the systemic level. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by research grants from the Scientific Research Foundation Flanders (FWO), Universitair Ziekenhuis Brussel (scientific fund Willy Gepts) and the Vrije Universiteit Brussel. Y.B. is a postdoctoral fellow of the FWO. U.M. is founder, shareholder and CEO of TissUse GmbH, Berlin, Germany, a company commercializing the Multi-Organ-Chip platform systems used in the study. The other authors have no conflict of interest to declare.

Evidence of in utero infection by Mycobacterium avium subsp. paratuberculosis using Multiple-Locus Variable-number tandem-repeat Analysis:

A pregnant heifer with an advanced clinical stage of paratuberculosis was reported in a herd in Argentina. Thus, the animal was euthanized and samples of organs of the cow and its fetus was taken and cultured for bacteriology in specific medium. Tissues were analyzed by histopathology (hematoxylin-eosin and Ziehl-Neelsen staining). Histopathological analysis of the cow’s samples revealed the presence of lesions consistent with paratuberculosis, and Ziehl-Neelsen staining revealed the presence of acid-fast bacilli, whereas the fetal tissues showed absence of lesions but the presence of acid-fast bacilli by Ziehl-Neelsen staining. After growing in specific medium, colonies in tissues from both cow and fetus were positive for IS900-PCR, confirming the presence of Mycobacterium avium subsp. paratuberculosis (MAP). Finally, the isolates were typed by Multiple-Locus Variable-number tandem-repeat Analysis (MLVA), which confirmedthe epidemiological link between them. This study is the first in Argentina to report the detection of MAP that shares an identical MLVA type in a pregnant cow and its fetus. The results of this study are consistent with previous reports and highlight the intra-uterine transmission of MAP as an important source of infection within herds.