Generation of Anterior Segment of the Eye Cells from hiPSCs in Microfluidic Platforms.

Ophthalmic diseases affect many people, causing partial or total loss of vision and a reduced quality of life. The anterior segment of the eye accounts for nearly half of all visual impairment that can lead to blindness. Therefore, there is a growing demand for ocular research and regenerative medicine that specifically targets the anterior segment to improve vision quality. This study aims to generate a microfluidic platform for investigating the formation of the anterior segment of the eye derived from human induced pluripotent stem cells (hiPSC) under various spatial-mechanoresponsive conditions. Microfluidic platforms are developed to examine the effects of dynamic conditions on the generation of hiPSCs-derived ocular organoids. The differentiation protocol is validated, and mechanoresponsive genes are identified through transcriptomic analysis. Several culture strategies is implemented for the anterior segment of eye cells in a microfluidic chip. hiPSC-derived cells showed anterior eye cell characteristics in mRNA and protein expression levels under dynamic culture conditions. The expression levels of yes-associated protein and transcriptional coactivator PDZ binding motif (YAP/TAZ) and PIEZO1, varied depending on the differentiation and growth conditions of the cells, as well as the metabolomic profiles under dynamic culture conditions.

TRAIL promotes the polarization of human macrophages toward a proinflammatory M1 phenotype and is associated with increased survival in cancer patients with high tumor macrophage content.

Background: TNF-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that can either induce cell death or activate survival pathways after binding to death receptors (DRs) DR4 or DR5. TRAIL is investigated as a therapeutic agent in clinical trials due to its selective toxicity to transformed cells. Macrophages can be polarized into pro-inflammatory/tumor-fighting M1 macrophages or anti-inflammatory/tumor-supportive M2 macrophages and an imbalance between M1 and M2 macrophages can promote diseases. Therefore, identifying modulators that regulate macrophage polarization is important to design effective macrophage-targeted immunotherapies. The impact of TRAIL on macrophage polarization is not known. Methods: Primary human monocyte-derived macrophages were pre-treated with either TRAIL or with DR4 or DR5-specific ligands and then polarized into M1, M2a, or M2c phenotypes in vitro. The expression of M1 and M2 markers in macrophage subtypes was analyzed by RNA sequencing, qPCR, ELISA, and flow cytometry. Furthermore, the cytotoxicity of the macrophages against U937 AML tumor targets was assessed by flow cytometry. TCGA datasets were also analyzed to correlate TRAIL with M1/M2 markers, and the overall survival of cancer patients. Results: TRAIL increased the expression of M1 markers at both mRNA and protein levels while decreasing the expression of M2 markers at the mRNA level in human macrophages. TRAIL also shifted M2 macrophages towards an M1 phenotype. Our data showed that both DR4 and DR5 death receptors play a role in macrophage polarization. Furthermore, TRAIL enhanced the cytotoxicity of macrophages against the AML cancer cells in vitro. Finally, TRAIL expression was positively correlated with increased expression of M1 markers in the tumors from ovarian and sarcoma cancer patients and longer overall survival in cases with high, but not low, tumor macrophage content. Conclusions: TRAIL promotes the polarization of human macrophages toward a proinflammatory M1 phenotype via both DR4 and DR5. Our study defines TRAIL as a new regulator of macrophage polarization and suggests that targeting DRs can enhance the anti-tumorigenic response of macrophages in the tumor microenvironment by increasing M1 polarization.

Differential Occupancy and Regulatory Interactions of KDM6A in Bladder Cell Lines.

Epigenetic deregulation is a critical theme which needs further investigation in bladder cancer research. One of the most highly mutated genes in bladder cancer is KDM6A, which functions as an H3K27 demethylase and is one of the MLL3/4 complexes. To decipher the role of KDM6A in normal versus tumor settings, we identified the genomic landscape of KDM6A in normal, immortalized, and cancerous bladder cells. Our results showed differential KDM6A occupancy in the genes involved in cell differentiation, chromatin organization, and Notch signaling depending on the cell type and the mutation status of KDM6A. Transcription factor motif analysis revealed HES1 to be enriched at KDM6A peaks identified in the T24 bladder cancer cell line; moreover, it has a truncating mutation in KDM6A and lacks a demethylase domain. Our co-immunoprecipitation experiments revealed TLE co-repressors and HES1 as potential truncated and wild-type KDM6A interactors. With the aid of structural modeling, we explored how truncated KDM6A could interact with TLE and HES1, as well as RUNX and HHEX transcription factors. These structures provide a solid means of studying the functions of KDM6A independently of its demethylase activity. Collectively, our work provides important contributions to the understanding of KDM6A malfunction in bladder cancer.

Transcriptome Dynamics of Human Neuronal Differentiation From iPSC.

The generation and use of induced pluripotent stem cells (iPSCs) in order to obtain all differentiated adult cell morphologies without requiring embryonic stem cells is one of the most important discoveries in molecular biology. Among the uses of iPSCs is the generation of neuron cells and organoids to study the biological cues underlying neuronal and brain development, in addition to neurological diseases. These iPSC-derived neuronal differentiation models allow us to examine the gene regulatory factors involved in such processes. Among these regulatory factors are long non-coding RNAs (lncRNAs), genes that are transcribed from the genome and have key biological functions in establishing phenotypes, but are frequently not included in studies focusing on protein coding genes. Here, we provide a comprehensive analysis and overview of the coding and non-coding transcriptome during multiple stages of the iPSC-derived neuronal differentiation process using RNA-seq. We identify previously unannotated lncRNAs via genome-guided de novo transcriptome assembly, and the distinct characteristics of the transcriptome during each stage, including differentially expressed and stage specific genes. We further identify key genes of the human neuronal differentiation network, representing novel candidates likely to have critical roles in neurogenesis using coexpression network analysis. Our findings provide a valuable resource for future studies on neuronal differentiation.

Assessment of bacterial diversity in Hyalomma aegyptium, H. marginatum and H. excavatum ticks through tag-encoded pyrosequencing.

Ticks are among the most significant human-biting ectoparasites and they play a major role in transmission of many pathogenic agents to humans. In the present study, three species of Hyalomma ticks, Hyalomma aegyptium, H. marginatum and H. excavatum, were examined for the presence of zoonotic bacteria, both male and female ticks alike. Examination of microbial diversity with tag-encoded pyrosequencing indicates that H. marginatum and H. excavatum were more diversity rich than H. aegyptium. Although numerous pathogenic and non-pathogenic bacterial genera were detected, including Acidovorax, Bacillus, Bacteroides, Bdellovibrio, Clostridium, Curvibacter, Escherichia, Flavobacterium, Limnohabitans, Paenibacillus, Ralstonia, Sarcina, Sediminibacterium, Segetibacter Stenotrophomonas and Variovorax, the predominant zoonotic bacteria represented in these ticks were genera Borrelia, Francisella, and Rickettsia. To the authors' knowledge, this work represents the first detection of Yersinia enterocolitica in the tick H. excavatum, raising questions regarding the vector competency of this tick, as well as associations of different disease representations perhaps through previously unforeseen routes of pathogen introduction. Likewise, similar questions are related to the presence of Legionella pneumophila in one H. excavatum sample.

Pyrosequencing based assessment of bacterial diversity in Turkish Rhipicephalus annulatus and Dermacentor marginatus ticks (Acari: Ixodidae).

Ticks continue to be a threat to human and animal health in Turkey, as they are considered important vectors of human and animal diseases. The objectives of this investigation are to characterize the microbial communities of two tick species, Rhipicephalus annulatus and Dermacenter marginatus, analyze patterns of co-occurrence among microbial taxa, identify and compare pathogens contributing human diseases, and determine whether avirulent symbionts could exclude human pathogens from tick communities. Furthermore, this study explores a microbiome of the R. annulatus and D. marginatus via the bacterial 16S tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP) technique to describe their bacterial diversity. Pyrosequencing was performed on adult males and females isolated from humans from two high-risk Turkish provinces, Sivas and Amasya, during tick outbreaks in 2009. A total of 36,253 sequences were utilized for analyses of the 8 tick samples. Several pathogenic genera such as Francisella, Coxiella, Rickettsia, and Shigella were detected in the ticks tested. The most distinguishable difference between the two species of ticks was the lack of known human pathogen Rickettsia in R. annulatus and in samples 9 and 10 of D. marginatus. These samples had higher relative abundance of Flavobacterium sp., Curvibacter sp., Acidovorax sp., and Bacteroidaceae genera mostly representing symbionts which form a large component of normal tick microbiota. The outcome of this study is consistent with the predictions of the community ecological theory that diversity-rich bacteriomes are more resistant to bacterial invasion (and consequent pathogen dissemination) than diversity-deprived ones.

Molecular detection of spotted fever group rickettsiae in ticks removed from humans in Turkey.

A total of 229 attached hard ticks were collected from humans in Yozgat province (Turkey) in 2009. All ticks were individually examined for the presence of Spotted Fever Group (SFG) rickettsiae using the polymerase chain reaction (PCR) targeting rickettsial citrate synthase (gltA) and outer membrane protein (ompA) genes. Rickettsial DNA was detected in 24 (10.5%) tick samples belonging to Dermacentor marginatus (Sulzer) (9 males, 3 females), Hyalomma marginatum Koch (8 males, 2 females) and Haemaphysalis parva (Neumann) (2 males). Four SFG rickettsiae were identified: Rickettsia aeschlimannii in H. marginatum, Rickettsia raoultii and Rickettsia slovaca in D. marginatus; Rickettsia hoogstraalii in H. parva ticks. With the present study, the presence of SFG rickettsiae in ticks was reported in Yozgat province for the first time.

Ticks (Acari: Ixodida) parasitizing humans in Corum and Yozgat provinces, Turkey.

In order to identify ticks infesting humans in Corum and Yozgat provinces in Turkey, a total of 2110 ticks representing 14 species were collected on humans, between June and September 2009. Of those, 1551 (687♂, 450♀, 407 nymphs, 7 larvae) were collected from Corum and 559 (330♂, 180♀, 49 nymphs) were collected from Yozgat. The majority of ticks (n = 1121, 53.1 %) was Hyalomma marginatum. Other common ticks infesting humans were Dermacentor marginatus (n = 209, 9.9 %) and Rhipicephalus turanicus sensu lato (n = 145, 6.9 %) in the study area. In addition, a total of 386 immature Hyalomma were found on humans in Corum (335 nymphs, 7 larvae) and Yozgat (44 nymphs). Ixodes laguri and Haemaphysalis erinacei taurica were recorded for the first time in Corum. To the best of our knowledge, this study is the first detailed investigation on ticks infesting humans in Corum and Yozgat, except individual or incidental records. The present study provides useful information for those concerned with ticks and tick-borne diseases in Turkey.

A survey of ticks (Acari: Ixodida) infesting some wild animals from Sivas, Turkey.

In order to determine the species composition of infesting ticks, between 2011 and 2012 a total of 1118 wild animals were captured from various regions of Zara, Sivas province, Turkey. A total of 138 ticks were obtained from the 58 host animals. Ticks were identified as Dermacentor marginatus (Sulzer), Haemaphysalis erinacei taurica Pospelova-Shtrom, Haemaphysalis parva (Neumann), Haemaphysalis punctata Canestrini and Fanzago, Haemaphysalis sulcata Canestrini and Fanzago, Hyalomma marginatum Koch, Ixodes laguri Olenev, Ixodes ricinus (L.), Ixodes vespertilionis Koch and Rhipicephalus turanicus Pomerantzev. To the best of our knowledge, there are several new host records for D. marginatus, H. e. taurica and I. laguri. In addition, I. vespertilionis was recorded for the first time in the Central Anatolian Region in Turkey, whereas I. laguri and H. e. taurica are firstly reported in Sivas.

First record of Ixodes arboricola (Ixodida: Ixodidae) from Turkey with presence of Candidatus Rickettsia vini (Rickettsiales: Rickettsiaceae).

Birds are the specific hosts of many tick species and may contribute to the dissemination of ticks and tick-borne pathogens throughout the world. Determination of ticks infesting birds and their pathogens are important for the detection of natural foci of human pathogens. Unfortunately, there is very limited information about the occurrence of ticks on birds and associated pathogens in Turkey. We performed a tick survey on three passerine bird species; Parus major, Sylvia atricapilla, and Turdus merula. Ticks collected from these birds were identified to species and tested for the presence of Borrelia, Ehrlichia, and Rickettsia species. Ixodes arboricola Schulze & Schlottke, Ixodes frontalis Panzer, and Ixodes ricinus L. were found on the birds. This is the first study reporting the presence of I. frontalis and I. arboricola on S. atricapilla and P. major, respectively, in Turkey. In addition, the results of polymerase chain reaction (PCR) with primers specific for gltA and ompA genes and DNA sequence analysis of positive PCR products indicated the presence of Candidatus Rickettsia vini in I. arboricola ticks. In conclusion, this is the first record of both I. arboricola and Candidatus Rickettsia vini in Turkey. Therefore, future studies needed to be conducted on the ticks infesting birds and their pathogens to elucidate the role of birds in the dispersal of tick species and tick-borne pathogens in Turkey.

Parasitism of immature stages of Haemaphysalis sulcata (Acari: Ixodidae) on some reptiles in Turkey.

Reptiles may contribute to maintaining tick populations by feeding larvae, nymphs, and adults. The life cycles and tick-host associations of many Turkish ticks are still poorly known, and only 3 ixodid tick species have been reported on 7 reptile species in Turkey. In this study, we performed a tick survey on reptiles in the Southeastern Anatolia Region of Turkey. In 2005, 57 reptiles (52 lizards and 5 snakes) comprising 10 species from 5 families were captured and examined for tick infestation. A total of 427 ticks was collected. The majority of ticks found on lizards was the immature stages of Haemaphysalis sulcata, 420 larvae and 4 nymphs. The only adult ticks recorded on the agamid lizard, Laudakia stellio, were Hyalomma aegyptium (1 ♂, 2 ♀). The highest tick infestation rate was recorded on specimens of Timon princeps. This study is the first detailed investigation on ticks infesting reptiles in Turkey. To the best of our knowledge, these tick-host associations have never been documented in the literature.

Ticks (Acari: Ixodida) infesting humans in the provinces of Kelkit Valley, a Crimean-Congo Hemorrhagic Fever endemic region in Turkey.

Ticks are mandatory blood feeding ectoparasites leading transmission of various tick-borne pathogens to human and animals. Since 2002, thousands of human tick bites and numerous Crimean-Congo Hemorrhagic Fever cases have been reported in several provinces in the Kelkit Valley region in Turkey. Despite increased cases of tick bites and tick-borne diseases, no taxonomic information is available about the tick species infesting humans in the region. In the present study, a tick survey on humans was performed to determine the species composition of ticks infesting humans in several provinces of Kelkit Valley. In the survey, 1,460 ticks (721 males, 516 females and 223 nymphs) were collected from tick-infested humans. A total of 19 tick species have been found on humans in the region, including 7 Hyalomma, 2 Argas, 2 Haemaphysalis, 2 Ixodes, Dermacentor and 3 Rhipicephalus species. Infestation of Dermacentor reticulatus on humans was documented for the first time in Turkey.

A case of gynandromorphism in Hyalomma marginatum Koch, 1844 (Acari: Ixodidae).

Gynandromorphism is a rare, abnormal phenomenon in which both female and male characteristics are simultaneously displayed in an organism. It has been described in many arthropods, including ticks. This unique occurrence is known within several species of Amblyomma, Dermacentor, Haemaphysalis, Hyalomma, Ixodes, and Rhipicephalus. Bipartite protogynander is the most common form of gynandromorphism, whereas gynander intriqué is the rarest type among the ticks. Here, we report the first case of a gynandromorph of Hyalomma marginatum Koch, 1844 collected from a naturally infested human during a tick survey in the Tokat Province of Turkey in 2006. It is an interesting gynander intriqué, with features of a protogynander. The tick described here displays abnormal characters such as an alloscutum with a male consucutum in dorsal view, male and female spiracular plates, female genital aperture under the male genital flap, and adanal plates located on the both side of the anus, whereas accessory plates are on the left side only in ventral view.

A review of the ticks (Acari: Ixodida) of Turkey: species diversity, hosts and geographical distribution.

Ticks are important ectoparasites, causing a variety of serious infectious diseases in humans and domestic animals. There is very limited taxonomic information about the tick species of Turkey in the literature, even though Turkey has very suitable climate and vegetation for ticks. In the current study, species diversity, hosts and geographical distribution of the ticks present in Turkey are reviewed based on taxonomic data in the literature from 1915 to 2011 and our recent observations. The names of tick species are arranged according to the most recent check lists. The taxonomic records in the literature and our studies on actual tick samples indicated that the tick fauna of Turkey consists of 46 species; 38 species from Ixodidae and 8 species from Argasidae.

Crimean-Congo hemorrhagic fever virus in various ixodid tick species from a highly endemic area.

Ticks are major vectors of numerous diseases affecting animals and humans. Presence of various tick-borne pathogens such as Crimean-Congo hemorrhagic fever virus (CCHFV) in various tick species was documented. CCHF is a severe tick-borne illness caused by the CCHFV which is a member of the Nairovirus genus (family: Bunyaviridae). Presence of CCHFV was shown in the most prevalent ixodid tick species such as Hyalomma marginatum and Rhiphicephalus bursa in Turkey. In the present study, prevalence and species diversity of ixodid ticks carrying CCHFV in Tokat province where CCHF is highly endemic were determined by using real-time reverse transcription-polymerase chain reaction (RRT-PCR). As a result, 15 out 745 ticks from various hosts (2%) were found to be CCHFV positive. The CCHFV positive ticks were Haemaphysalis concinna, Hyalomma anatolicum, Hyalomma detritum, Hyalomma marginatum, Hyalomma turanicum, Rhipicephalus bursa, and Rhiphicephalus turanicus indicating that multiple ixodid tick species may contribute to transmission of CCHFV to humans and animals in Turkey.

Ixodid ticks (Acari: Ixodidae) infesting humans in Tokat Province of Turkey: species diversity and seasonal activity.

Ixodid ticks (Acari: Ixodidae) are the major vectors of pathogens threatening animal and human health. Tokat Province, Turkey, is a suitable habitat for extended tick activity with its moderate climate and vegetation. In the present study, we surveyed humans visiting health centers to determine the species diversity, geographical distribution, and seasonal activity of ixodid ticks infesting them. Out of 5,999 adult ticks collected from humans from April to September, 2008, 800 ticks were identified to species, while the remaining were identified to genus according to their distinct morphological characteristics. Hyalomma marginatum, Hyalomma detritum, Rhipicephalus bursa, Rhipicephalus (Boophilus) annulatus, Haemaphysalis punctata, Dermacentor marginatus, and Ixodes laguri were the most prevalent tick species among 24 ixodid tick species infesting humans in the region. One of these tick species, Hyalomma isacii was identified as a new record for the ixodid tick fauna of Turkey. Hyalomma species were the most abundant in summer, while Dermacentor and Ixodes species displayed the lowest frequency. Hyalomma aegyptium infestation was very common on humans in the province. Results indicated that a variety of ixodid tick species infest humans depending on the season in the target area. It is possible that a variety of ixodid tick species may contribute to the spread of tick-borne diseases such as Crimean-Congo haemorrhagic fever virus (CCHFV), which is endemic in the region.