Publications by authors named "Katarzyna Szymanska"

61 Publications

The Multifaceted Regulation of SnRK2 Kinases.

Cells 2021 Aug 24;10(9). Epub 2021 Aug 24.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland.

SNF1-related kinases 2 (SnRK2s) are central regulators of plant responses to environmental cues simultaneously playing a pivotal role in the plant development and growth in favorable conditions. They are activated in response to osmotic stress and some of them also to abscisic acid (ABA), the latter being key in ABA signaling. The SnRK2s can be viewed as molecular switches between growth and stress response; therefore, their activity is tightly regulated; needed only for a short time to trigger the response, it has to be induced transiently and otherwise kept at a very low level. This implies a strict and multifaceted control of SnRK2s in plant cells. Despite emerging new information concerning the regulation of SnRK2s, especially those involved in ABA signaling, a lot remains to be uncovered, the regulation of SnRK2s in an ABA-independent manner being particularly understudied. Here, we present an overview of available data, discuss some controversial issues, and provide our perspective on SnRK2 regulation.
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http://dx.doi.org/10.3390/cells10092180DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8465348PMC
August 2021

Correction to: Preservation of connexin 43 and transzonal projections in isolated bovine pre-antral follicles before and following vitrification.

J Assist Reprod Genet 2021 Feb;38(2):493

Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Laboratory of Veterinary Physiology and Biochemistry, Gamete Research Centre, University of Antwerp, Universiteitsplein 1, U building, 2610, Wilrijk, Belgium.

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http://dx.doi.org/10.1007/s10815-020-02040-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884496PMC
February 2021

Targeting connexins with Gap27 during cold storage of the human donor uterus protects against cell death.

PLoS One 2020 10;15(12):e0243663. Epub 2020 Dec 10.

Department of Basic and Applied Medical Sciences, Ghent University, Ghent, Belgium.

Uterus transplantation is an experimental infertility treatment for women with uterine factor infertility. During donor uterus retrieval and subsequent storage, ischemia and other stressors are likely to occur, resulting in the delayed restoration of organ function and increased graft rejection. The uterus expresses connexin-based hemichannels, the opening of which can promote ischemic cell death, as well as gap junctions that may expand cell death by bystander signaling. We investigated if connexin channel inhibition with connexin channel inhibitor Gap27 could protect the uterus against cell death during the storage period. The study involved 9 female patients undergoing gender-change surgery. Before uterus removal, it was exposed to in situ warm ischemia with or without reperfusion. Uterus biopsies were taken before, during, and after ischemia, with or without reperfusion, and were subsequently stored under cold (4ᵒC) or warm (37ᵒC) conditions. TUNEL cell death assay was done at various time points along the combined in vivo/ex vivo experimental timeline. We found that Gap27 protected against storage-related cell death under cold but not warm conditions when the uterus had experienced in situ ischemia/reperfusion. For in situ brief ischemia without reperfusion, Gap27 reduction of cell death was delayed and significantly less, suggesting that protection critically depends on processes initiated when the organ was still in the donor. Thus, the inclusion of the connexin channel inhibitor Gap27 during cold storage protects the uterus against cell death, and the degree of protection depends on the history of exposure to warm ischemia. Gap27 protection may be indicated for uteri from deceased donors, in which ischemia is likely because life-saving organs have retrieval priority.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0243663PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7728185PMC
January 2021

Preservation of connexin 43 and transzonal projections in isolated bovine pre-antral follicles before and following vitrification.

J Assist Reprod Genet 2021 Feb 6;38(2):479-492. Epub 2020 Nov 6.

Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Laboratory of Veterinary Physiology and Biochemistry, Gamete Research Centre, University of Antwerp, Universiteitsplein 1, U building, 2610, Wilrijk, Belgium.

Purpose: Gap junctions and transzonal projections play a crucial role in intercellular communication between different follicular components and are necessary for follicle development. We aimed to demonstrate gap junction protein connexin 43 (Cx43) and transzonal projections (TZPs) in viable, category 1, isolated bovine pre-antral follicles (PAFs) during short-term culture and after vitrification and warming.

Methods: This study involved four experimental groups: fresh control, 2-day culture, 4-day culture, and vitrified secondary PAFs. Isolated PAFs were vitrified using a simple and efficient cryopreservation method by means of mini cell strainers.

Results: Cx43 and TZPs were detected in pre-antral follicles of all stages, as well as in every experimental group. The group fresh follicles showed a higher percentage of follicles that were positive for Cx43 (91.7%) than the follicles that were vitrified (77.4%). All follicles that were cultured for 2 days were Cx43-positive (100%). Follicles cultured for 4 days (65.8%) (P = 0.002) showed the lowest percentage of follicles that were Cx43-positive. The percentages of the presence or (partial) absence of the TZP network were shown to be very heterogeneous between follicles in different treatment groups.

Conclusions: These results suggest the maintenance of communication between the oocyte and the somatic companion cells after vitrification and warming. The varying percentages of the expression of the TZP network within groups suggests that it will be of interest to investigate whether this is truly due to variability in TZP integrity and follicle quality or due to methodological limitations.
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http://dx.doi.org/10.1007/s10815-020-01993-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7884540PMC
February 2021

Immobilization of the Highly Active UDP-Glucose Pyrophosphorylase From Provides a Highly Efficient Biocatalyst for the Production of UDP-Glucose.

Front Bioeng Biotechnol 2020 2;8:740. Epub 2020 Jul 2.

Department of Microbial Biotechnology, Faculty of Biology and Biotechnology, Ruhr-Universität Bochum, Bochum, Germany.

Biocatalysis that produces economically interesting compounds can be carried out by using free enzymes or microbial cells. However, often the cell metabolism does not allow the overproduction or secretion of activated sugars and thus downstream processing of these sugars is complicated. Here enzyme immobilization comes into focus in order to stabilize the enzyme as well as to make the overall process economically feasible. Besides a robust immobilization method, a highly active and stable enzyme is needed to efficiently produce the product of choice. Herein, we report on the identification, gene expression, biochemical characterization as well as immobilization of the uridine-5'-diphosphate-glucose (UDP-glucose) pyrophosphorylase originating from the thermostable soil actinobacterium DSM 44070 (GalU). The enzyme immobilization was performed on organically modified mesostructured cellular foams (MCF) epoxy and amino group to provide a stable and active biocatalyst. The soluble and highly active GalU revealed a of 1698 U mg (uridine-5'-triphosphate, UTP) and a of 0.15 mM (UTP). The optimum reaction temperature was determined to be 50°C. GalU was stable at this temperature for up to 30 min with a maximum loss of activity of 65%. Interestingly, immobilized GalU was stable at 50°C for at least 120 min without a significant loss of activity, which makes this enzyme an interesting biocatalyst for the production of UDP-glucose.
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http://dx.doi.org/10.3389/fbioe.2020.00740DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343719PMC
July 2020

Unraveling the mechanisms of chemotherapy-induced damage to human primordial follicle reserve: road to developing therapeutics for fertility preservation and reversing ovarian aging.

Mol Hum Reprod 2020 08;26(8):553-566

Laboratory of Molecular Reproduction and Fertility Preservation, Department of Obstetrics, Gynecology and Reproductive Sciences, Yale University School of Medicine, New Haven, CT, USA.

Among the investigated mechanisms of chemotherapy-induced damage to human primordial follicle reserve are induction of DNA double-strand breaks (DSBs) and resultant apoptotic death, stromal-microvascular damage and follicle activation. Accumulating basic and translational evidence suggests that acute exposure to gonadotoxic chemotherapeutics, such as cyclophosphamide or doxorubicin, induces DNA DSBs and triggers apoptotic death of primordial follicle oocytes within 12-24 h, resulting in the massive loss of ovarian reserve. Evidence also indicates that chemotherapeutic agents can cause microvascular and stromal damage, induce hypoxia and indirectly affect ovarian reserve. While it is possible that the acute reduction of the primordial follicle reserve by massive apoptotic losses may result in delayed activation of some primordial follicles, this is unlikely to be a predominant mechanism of loss in humans. Here, we review these mechanisms of chemotherapy-induced ovarian reserve depletion and the potential reasons for the discrepancies among the studies. Based on the current literature, we propose an integrated hypothesis that explains both the acute and delayed chemotherapy-induced loss of primordial follicle reserve in the human ovary.
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http://dx.doi.org/10.1093/molehr/gaaa043DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7411370PMC
August 2020

Skin cancer in children after organ transplantation.

Postepy Dermatol Alergol 2019 Dec 30;36(6):649-654. Epub 2019 Dec 30.

Department of Nephrology, Transplantology and Internal Diseases, Medical University of Gdansk, Gdansk, Poland.

Skin cancer is the second most common complication of organ transplantation in children. The frequency of skin cancer incidence after organ transplantation is different in paediatric and adult populations. The post-transplant lymphoproliferative disease is the most common group of malignancies after organ transplantation in paediatric population. The majority of researchers who examined children with kidney, liver, heart or lungs grafts observed that the risk of skin cancer was three times higher than in the general population whereas in adults even200 times higher. The occurrence of skin cancer in children after transplantation is extremely rare during childhood. The risk increases in early adulthood. Malignancies occurring after solid organ transplantation result from many different factors. These include the immunological condition of the child, dose and time of immunosuppression, and oncogenic viruses. The increased risk of skin cancer following paediatric transplantation requires prevention and adequate education of children and their parents. These involve avoiding sun exposure and protection such as sunscreens and protective clothing. The early detection of cancer in transplant recipients is very important. Prevention of cancer includes regular dermatological examination.
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http://dx.doi.org/10.5114/ada.2019.82680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6986283PMC
December 2019

Leloir Glycosyltransferases in Applied Biocatalysis: A Multidisciplinary Approach.

Int J Mol Sci 2019 Oct 23;20(21). Epub 2019 Oct 23.

Department of Biotechnology, Delft University of Technology, Section Biocatalysis, Van der Maasweg 9, 2629 HZ Delft, The Netherlands.

Enzymes are nature's catalyst of choice for the highly selective and efficient coupling of carbohydrates. Enzymatic sugar coupling is a competitive technology for industrial glycosylation reactions, since chemical synthetic routes require extensive use of laborious protection group manipulations and often lack regio- and stereoselectivity. The application of Leloir glycosyltransferases has received considerable attention in recent years and offers excellent control over the reactivity and selectivity of glycosylation reactions with unprotected carbohydrates, paving the way for previously inaccessible synthetic routes. The development of nucleotide recycling cascades has allowed for the efficient production and reuse of nucleotide sugar donors in robust one-pot multi-enzyme glycosylation cascades. In this way, large glycans and glycoconjugates with complex stereochemistry can be constructed. With recent advances, LeLoir glycosyltransferases are close to being applied industrially in multi-enzyme, programmable cascade glycosylations.
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http://dx.doi.org/10.3390/ijms20215263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6861944PMC
October 2019

Exposing dairy bulls to high temperature-humidity index during spermatogenesis compromises subsequent embryo development in vitro.

Theriogenology 2020 Jan 29;141:16-25. Epub 2019 Aug 29.

Department of Reproduction, Obstetrics, and Herd Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium. Electronic address:

The possible impact of natural heat stress on animal fertility is currently a major concern for breeding companies. Here, we aimed to address this concern by determining the effects of natural heat stress on the fertility of Holstein bulls located in the Netherlands. Semen samples were collected from six bulls at two locations in March 2016 (low temperature-humidity index (THI) group; maximum THI of 51.8 and 55 at their respective locations) or August (high THI group; maximum THI of 77.9 and 80.5 during meiotic and spermiogenic stages of spermatogenesis, 42 to 14 days prior to semen collection). The effect of heat stress on semen quality was assessed by sperm morphology, motility, reactive oxygen species production, lipid peroxidation, viability, and DNA fragmentation. Moreover, we evaluated the development of embryos generated in vitro by low and high THI semen, and determined inner cell mass/trophectoderm ratio, apoptotic cell ratio, and embryonic gene expression in day-8 blastocysts. An increase in cell death (propidium iodide-positive cells; P = 0.039) was observed in the high THI group (31.5%) compared to the low THI group (27.6%). Moreover, a decrease (P < 0.001) was observed in the total blastocyst rates at day 7 post-insemination (15.3 vs 20.9%) and day 8 (23.2 vs 29.6%) in the high THI compared to the low THI group, respectively. There were no differences in the relative abundance of candidate transcripts examined. In conclusion, sperm samples from dairy bulls obtained during a period with higher THI had reduced viability and led to a decrease in blastocyst development and delayed hatching, compared to semen collected during a period with low THI.
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http://dx.doi.org/10.1016/j.theriogenology.2019.08.034DOI Listing
January 2020

Stable Immobilization of Enzymes in a Macro- and Mesoporous Silica Monolith.

ACS Omega 2019 Apr 29;4(4):7795-7806. Epub 2019 Apr 29.

Department of Materials (D-MATL), ETH Zürich, Vladimir-Prelog-Weg 5, 8093 Zürich, Switzerland.

Horseradish peroxidase isoenzyme C (HRP) and proteinase K (proK) were immobilized inside macro- and mesoporous silica monoliths. Stable immobilization was achieved through simple noncovalent adsorption of conjugates, which were prepared from a polycationic, water-soluble second generation dendronized polymer (denpol) and the enzymes. Conjugates prepared from three denpols with the same type of repeating unit (r.u.), but different average lengths were compared. It was shown that there is no obvious advantage of using denpols with very long chains. Excellent results were achieved with denpols having on average 750 or 1000 r.u. The enzyme-loaded monoliths were tested as flow reactors. Comparison was made with microscopy glass coverslips onto which the conjugates were immobilized and with glass micropipettes containing adsorbed conjugates. High enzyme loading was achieved using the monoliths. Monoliths containing immobilized denpol-HRP conjugates exhibited good operational stability at 25 °C (for at least several hours), and good storage stability at 4 °C (at least for weeks) was demonstrated. Such HRP-containing monoliths were applied as continuous flow reactors for the quantitative determination of hydrogen peroxide in aqueous solution between 1 μM (34 ng/mL) and 50 μM (1.7 μg/mL). Although many methods for immobilizing enzymes on silica surfaces exist, there are only a few approaches with porous silica materials for the development of flow reactors. The work presented is a promising contribution to this field of research toward bioanalytical and biosynthetic applications.
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http://dx.doi.org/10.1021/acsomega.9b00286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6648689PMC
April 2019

CiliaCarta: An integrated and validated compendium of ciliary genes.

PLoS One 2019 16;14(5):e0216705. Epub 2019 May 16.

Medical Proteome Center, Institute for Ophthalmic Research, University of Tuebingen, Tuebingen, Germany.

The cilium is an essential organelle at the surface of mammalian cells whose dysfunction causes a wide range of genetic diseases collectively called ciliopathies. The current rate at which new ciliopathy genes are identified suggests that many ciliary components remain undiscovered. We generated and rigorously analyzed genomic, proteomic, transcriptomic and evolutionary data and systematically integrated these using Bayesian statistics into a predictive score for ciliary function. This resulted in 285 candidate ciliary genes. We generated independent experimental evidence of ciliary associations for 24 out of 36 analyzed candidate proteins using multiple cell and animal model systems (mouse, zebrafish and nematode) and techniques. For example, we show that OSCP1, which has previously been implicated in two distinct non-ciliary processes, causes ciliogenic and ciliopathy-associated tissue phenotypes when depleted in zebrafish. The candidate list forms the basis of CiliaCarta, a comprehensive ciliary compendium covering 956 genes. The resource can be used to objectively prioritize candidate genes in whole exome or genome sequencing of ciliopathy patients and can be accessed at http://bioinformatics.bio.uu.nl/john/syscilia/ciliacarta/.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0216705PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522010PMC
January 2020

Expression of maize calcium-dependent protein kinase (ZmCPK11) improves salt tolerance in transgenic Arabidopsis plants by regulating sodium and potassium homeostasis and stabilizing photosystem II.

Physiol Plant 2020 Jan 27;168(1):38-57. Epub 2019 Feb 27.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.

In plants, CALCIUM-DEPENDENT PROTEIN KINASES (CDPKs/CPKs) are involved in calcium signaling in response to endogenous and environmental stimuli. Here, we report that ZmCPK11, one of maize CDPKs, participates in salt stress response and tolerance. Salt stress induced expression and upregulated the activity of ZmCPK11 in maize roots and leaves. Activation of ZmCPK11 upon salt stress was also observed in roots and leaves of transgenic Arabidopsis plants expressing ZmCPK11. The transgenic plants showed a long-root phenotype under control conditions and a short-root phenotype under NaCl, abscisic acid (ABA) or jasmonic acid (JA) treatment. Analysis of ABA and JA content in roots indicated that ZmCPK11 can mediate root growth by regulating the levels of these phytohormones. Moreover, 4-week-old transgenic plants were more tolerant to salinity than the wild-type plants. Their leaves were less chlorotic and showed weaker symptoms of senescence accompanied by higher chlorophyll content and higher quantum efficiency of photosystem II. The expression of Na /K transporters (HKT1, SOS1 and NHX1) and transcription factors (CBF1, CBF2, CBF3, ZAT6 and ZAT10) with known links to salinity tolerance was upregulated in roots of the transgenic plants upon salt stress. Furthermore, the transgenic plants accumulated less Na in roots and leaves under salinity, and showed a higher K /Na ratio in leaves. These results show that the improved salt tolerance in ZmCPK11-transgenic plants could be due to an upregulation of genes involved in the maintenance of intracellular Na and K homeostasis and a protection of photosystem II against damage.
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http://dx.doi.org/10.1111/ppl.12938DOI Listing
January 2020

SNF1-Related Protein Kinases SnRK2.4 and SnRK2.10 Modulate ROS Homeostasis in Plant Response to Salt Stress.

Int J Mol Sci 2019 Jan 2;20(1). Epub 2019 Jan 2.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5a, 02-106 Warsaw, Poland.

In response to salinity and various other environmental stresses, plants accumulate reactive oxygen species (ROS). The ROS produced at very early stages of the stress response act as signaling molecules activating defense mechanisms, whereas those produced at later stages in an uncontrolled way are detrimental to plant cells by damaging lipids, DNA, and proteins. Multiple systems are involved in ROS generation and also in ROS scavenging. Their level and activity are tightly controlled to ensure ROS homeostasis and protect the plant against the negative effects of the environment. The signaling pathways responsible for maintaining ROS homeostasis in abiotic stress conditions remain largely unknown. Here, we show that in , two abscisic acid- (ABA)-non-activated SNF1-releted protein kinases 2 (SnRK2) kinases, SnRK2.4 and SnRK2.10, are involved in the regulation of ROS homeostasis in response to salinity. They regulate the expression of several genes responsible for ROS generation at early stages of the stress response as well as those responsible for their removal. Moreover, the SnRK2.4 regulate catalase levels and its activity and the level of ascorbate in seedlings exposed to salt stress.
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http://dx.doi.org/10.3390/ijms20010143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6337402PMC
January 2019

Tuning the Magnetic Properties of Columnar Benzo[e][1,2,4]triazin-4-yls with the Molecular Shape.

Chemphyschem 2019 02 1;20(4):636-644. Epub 2019 Feb 1.

Faculty of Chemistry, University of Łódź, Tamka 12, 91-403, Łódź, Poland.

A homologous series of disc-like 1,3,6-trisubstituted benzo[e][1,2,4]triazin-4-yls 1[n] was synthesized and their structural, thermal, optical, magnetic, and electric properties were investigated. The results demonstrate that all members of the series display a Col phase with clearing temperatures depending on the length of the alkoxy chains at the N(1) position, hence the shape of the disc. Powder XRD and magnetic data indicate a gradual change in the column diameter and magnetic behavior in the series in transition from half-disc in 1[0] (antiferromagnetic interactions) to full-disc geometry in the 1[12] homologue (ferromagnetic interactions with J/k =+7.5 K). Studies of binary systems revealed that a 1 : 1 mixture of 1[0] and 1[12] exhibits modest stabilization of the Col phase with an expanded range, and magnetic behavior typical for 1[0] in the rigid phase obtained from the melt. Electric measurements demonstrated hole mobility of ∼10  cm  V  s and dark conductivity of ∼10  Scm in the mixture and individual compounds. The latter is enhanced up to 4 times by simultaneous illumination with UV light.
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http://dx.doi.org/10.1002/cphc.201800965DOI Listing
February 2019

Isolation and Characterization of Functionally Active Extracellular Vesicles from Culture Medium Conditioned by Bovine Embryos In Vitro.

Int J Mol Sci 2018 Dec 21;20(1). Epub 2018 Dec 21.

Department of Reproduction, Obstetrics and Herd Health, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133, B-9820 Merelbeke, Belgium.

Extracellular vesicles (EVs) play a possible role in cell⁻cell communication and are found in various body fluids and cell conditioned culture media. The aim of this study was to isolate and characterize EVs in culture medium conditioned by bovine embryos in group and to verify if these EVs are functionally active. Initially, ultracentrifuged bovine serum albumin (BSA) containing medium was selected as suitable EV-free embryo culture medium. Next, EVs were isolated from embryo conditioned culture medium by OptiPrep density gradient ultracentrifugation. Isolated EVs were characterized by nanoparticle tracking analysis, western blotting, transmission, and immunoelectron microscopy. Bovine embryo-derived EVs were sizing between 25⁻230 nm with an average concentration of 236.5 ± 1.27 × 10⁸ particles/mL. Moreover, PKH67 EV pre-labeling showed that embryo-secreted EVs were uptaken by zona-intact bovine embryos. Since BSA did not appear to be a contaminating EV source in culture medium, EV functionality was tested in BSA containing medium. Individual embryo culture in BSA medium enriched with EVs derived from conditioned embryo culture medium showed significantly higher blastocyst rates at day 7 and 8 together with a significantly lower apoptotic cell ratio. In conclusion, our study shows that EVs play an important role in inter embryo communication during bovine embryo culture in group.
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http://dx.doi.org/10.3390/ijms20010038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6337605PMC
December 2018

Connexin hemichannels and cell death as measures of bovine COC vitrification success.

Reproduction 2019 01;157(1):87-99

Physiology Group, Department of Basic Medical Sciences, Ghent University, Ghent, Belgium.

Vitrification of immature germinal vesicle-stage oocytes is a promising method in assisted reproduction but is associated with reduced developmental potential and low birth rates. Cumulus-oocyte complexes (COCs) express several connexins that form hexameric hemichannels, which interact head to head to create a gap junction or exist as unopposed free hemichannels. The latter are normally closed but open under stress conditions and may exert detrimental effects. We determined whether minimizing hemichannel opening and cell death during vitrification could improve COC quality. Bovine immature COCs underwent vitrification, storage and warming, followed by dye uptake to assess hemichannel opening and TUNEL staining to detect cell death. Based on these scores, we optimized the procedure by tuning the equilibration time, temperature, cryoprotectant concentration and extracellular Ca2+ concentration and assessed its impact on maturation, cleavage and blastocyst formation after parthenogenetic activation. We found that the major stressor resides in the cooling/warming phase of the vitrification procedure and observed that hemichannel opening and cell death in cumulus cells measure different aspects of cell stress. Optimization of the hemichannel and cell death readouts demonstrated that combined minimal hemichannel opening/cell death gave the highest cleavage rates but had no effect on maturation and blastocyst formation. Neither hemichannel nor cell death optimization performed better than the non-optimized protocol, leading to the conclusion that cell stress factors other than those detected by hemichannel dye uptake or TUNEL positivity are involved.
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http://dx.doi.org/10.1530/REP-18-0387DOI Listing
January 2019

Phosphoproteomic analysis reveals that dehydrins ERD10 and ERD14 are phosphorylated by SNF1-related protein kinase 2.10 in response to osmotic stress.

Plant Cell Environ 2019 03 10;42(3):931-946. Epub 2018 Dec 10.

Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.

SNF1-related protein kinases 2 (SnRK2s) regulate the plant responses to abiotic stresses, especially water deficits. They are activated in plants subjected to osmotic stress, and some of them are additionally activated in response to enhanced concentrations of abscisic acid (ABA) in plant cells. The SnRK2s that are activated in response to ABA are key elements of ABA signalling that regulate plant acclimation to environmental stresses and ABA-dependent development. Much less is known about the SnRK2s that are not activated by ABA, albeit several studies have shown that these kinases are also involved in response to osmotic stress. Here, we show that one of the Arabidopsis thaliana ABA-non-activated SnRK2s, SnRK2.10, regulates not only the response to salinity but also the plant sensitivity to dehydration. Several potential SnRK2.10 targets phosphorylated in response to stress were identified by a phosphoproteomic approach, including the dehydrins ERD10 and ERD14. Their phosphorylation by SnRK2.10 was confirmed in vitro. Our data suggest that the phosphorylation of ERD14 within the S-segment is involved in the regulation of dehydrin subcellular localization in response to stress.
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http://dx.doi.org/10.1111/pce.13465DOI Listing
March 2019

Disrupted alternative splicing for genes implicated in splicing and ciliogenesis causes PRPF31 retinitis pigmentosa.

Nat Commun 2018 10 12;9(1):4234. Epub 2018 Oct 12.

Leeds Institute of Medical Research, University of Leeds, St James's University Hospital, Beckett Street, Leeds, LS9 7TF, UK.

Mutations in pre-mRNA processing factors (PRPFs) cause autosomal-dominant retinitis pigmentosa (RP), but it is unclear why mutations in ubiquitously expressed genes cause non-syndromic retinal disease. Here, we generate transcriptome profiles from RP11 (PRPF31-mutated) patient-derived retinal organoids and retinal pigment epithelium (RPE), as well as Prpf31 mouse tissues, which revealed that disrupted alternative splicing occurred for specific splicing programmes. Mis-splicing of genes encoding pre-mRNA splicing proteins was limited to patient-specific retinal cells and Prpf31 mouse retinae and RPE. Mis-splicing of genes implicated in ciliogenesis and cellular adhesion was associated with severe RPE defects that include disrupted apical - basal polarity, reduced trans-epithelial resistance and phagocytic capacity, and decreased cilia length and incidence. Disrupted cilia morphology also occurred in patient-derived photoreceptors, associated with progressive degeneration and cellular stress. In situ gene editing of a pathogenic mutation rescued protein expression and key cellular phenotypes in RPE and photoreceptors, providing proof of concept for future therapeutic strategies.
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http://dx.doi.org/10.1038/s41467-018-06448-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6185938PMC
October 2018

Blocking connexin channels during vitrification of immature cat oocytes improves maturation capacity after warming.

Theriogenology 2018 Dec 14;122:144-149. Epub 2018 Sep 14.

Faculty of Veterinary Medicine, Department of Reproduction, Obstetrics and Herd Health, Ghent University, Merelbeke, Belgium.

In the domestic cat, nuclear maturation and embryo development after vitrification of immature oocytes have been obtained but developmental competence after warming remains low. It has been reported that during folliculogenesis, the association and communication between the oocyte and the surrounding cumulus cells through connexin-based gap junctions is essential for normal oocyte and follicular development. Gap junctions result from the head-to-head interaction of two hemichannels; however, there is always a population of hemichannels not incorporated into gap junctions. These unopposed hemichannels are normally closed but may open under certain stress conditions, potentially also during vitrification and warming, turning them into toxic pores inducing cell injury and cell death. The aim of our study was to test whether inhibiting connexin 37 (Cx37) and connexin 43 (Cx43) channels with the connexin-targeting peptide Gap26 during vitrification and warming of cat immature cumulus-oocyte-complexes (COCs) could improve oocyte maturation and competence of resultant blastocysts derived by parthenogenetic activation. In the first experiment, our immunostainings confirmed the presence of Cx43 protein in the cytoplasm of immature cat oocytes and in the plasma membranes of cumulus cells. In the second experiment, COCs were randomly divided in three different groups: a control group (control), a group vitrified without Gap26 (vitrified) and a group vitrified with Gap26 (vitrified-peptide). The maturation rate was checked and oocytes from all three different experimental groups were parthenogenetically activated and cultured in vitro until day 8. After vitrification and warming, 49% of the oocytes in the control group matured, while this was 8% and 19% in the vitrified and vitrified-peptide groups, respectively. Compared to the vitrified group, oocytes in the vitrified-peptide group had significantly larger maturation rates. No blastocysts were detected at day 8 in the vitrified group, while 2% and 13% of the oocytes further developed to blastocyst at day 8 in the vitrified-peptide and control non-vitrified group, respectively. We conclude that the use of Gap26 in vitrification and warming media to vitrify immature cat oocytes improves maturation success and allows such oocytes to reach the blastocyst stage (2%) at day 8 after parthenogenetic activation.
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http://dx.doi.org/10.1016/j.theriogenology.2018.09.011DOI Listing
December 2018

Analysis of desferrioxamine-like siderophores and their capability to selectively bind metals and metalloids: development of a robust analytical RP-HPLC method.

Res Microbiol 2018 Dec 20;169(10):598-607. Epub 2018 Aug 20.

Institute of Biological Sciences, Environmental Microbiology Group, TU Bergakademie Freiberg, Leipziger Straße 29, 09599, Freiberg, Germany; Microbial Biotechnology, Ruhr University Bochum, Universitätsstr. 150, 44801, Bochum, Germany. Electronic address:

The Actinobacterium Gordonia rubripertincta CWB2 (DSM 46758) produces hydroxamate-type siderophores (188 mg L) under iron limitation. Analytical reversed-phase HPLC allowed determining a single peak of ferric iron chelating compounds from culture broth which was confirmed by the Fe-CAS assay. Elution profile and its absorbance spectrum were similar to those of commercial (des)ferrioxamine B which was used as reference compound. This confirms previously made assumptions and shows for the first time that the genus Gordonia produces desferrioxamine-like siderophores. The reversed-phase HPLC protocol was optimized to separate metal-free and -loaded oxamines. This allowed to determine siderophore concentrations in solutions as well as metal affinity. The metal loading of oxamines was confirmed by ICP-MS. As a result, it was demonstrated that desferrioxamine prefers trivalent metal ions (Fe > Ga > V > Al) over divalent ones. In addition, we aimed to show the applicability of the newly established reversed-phase HPLC protocol and to increase the re-usability of desferrioxamines as metal chelators by immobilization on mesocellular silica foam carriers. The siderophores obtained from strain CWB2 and commercial desferrioxamine B were successfully linked to the carrier with a high yield (up to 95%) which was verified by the HPLC method. Metal binding studies demonstrated that metals can be bound to non-immobilized and to the covalently linked desferrioxamines, but also to the carrier material itself. The latter was found to be unspecific and, therefore, the effect of the carrier material remains a field of future research. By means of a reversed CAS assay for various elements (Nd, Gd, La, Er, Al, Ga, V, Au, Fe, As) it was possible to demonstrate improved Ga- and Nd-binding to desferrioxamine loaded mesoporous silica carriers. The combination of the robust reversed-phase HPLC method and various CAS assays provides new avenues to screen for siderophore producing strains, and to control purification and immobilization of siderophores.
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http://dx.doi.org/10.1016/j.resmic.2018.08.002DOI Listing
December 2018

Lamp1 Increases the Efficiency of Lassa Virus Infection by Promoting Fusion in Less Acidic Endosomal Compartments.

mBio 2018 01 2;9(1). Epub 2018 Jan 2.

Department of Microbiology, University of Virginia, Charlottesville, Virginia, USA

Lassa virus (LASV) is an arenavirus whose entry into host cells is mediated by a glycoprotein complex (GPC) comprised of a receptor binding subunit, GP1, a fusogenic transmembrane subunit, GP2, and a stable signal peptide. After receptor-mediated internalization, arenaviruses converge in the endocytic pathway, where they are thought to undergo low-pH-triggered, GPC-mediated fusion with a late endosome membrane. A unique feature of LASV entry is a pH-dependent switch from a primary cell surface receptor (α-dystroglycan) to an endosomal receptor, lysosomal-associated membrane protein (Lamp1). Despite evidence that the interaction between LASV GP1 and Lamp1 is critical, the function of Lamp1 in promoting LASV infection remains poorly characterized. Here we used wild-type (WT) and Lamp1 knockout (KO) cells to show that Lamp1 increases the efficiency of, but is not absolutely required for, LASV entry and infection. We then used cell-cell and pseudovirus-cell surface fusion assays to demonstrate that LASV GPC-mediated fusion occurs at a significantly higher pH when Lamp1 is present compared to when Lamp1 is missing. Correspondingly, we found that LASV entry occurs through less acidic endosomes in WT (Lamp1-positive) versus Lamp1 KO cells. We propose that, by elevating the pH threshold for fusion, Lamp1 allows LASV particles to exit the endocytic pathway before they encounter an increasingly acidic and harsh proteolytic environment, which could inactivate a significant percentage of incoming viruses. In this manner Lamp1 increases the overall efficiency of LASV entry and infection. Lassa virus is the most clinically important member of the , a family that includes six additional biosafety level 4 (BSL4) hemorrhagic fever viruses. The lack of specific antiviral therapies for Lassa fever drives an urgent need to identify druggable targets, and interventions that block infection at the entry stage are particularly attractive. Lassa virus is only the second virus known to employ an intracellular receptor, the first being Ebola virus. Here we show that interaction with its intracellular receptor, Lamp1, enhances and upwardly shifts the pH dependence of fusion and consistently permits Lassa virus entry into cells through less acidic endosomes. We propose that in this manner, Lamp1 increases the overall efficiency of Lassa virus infection.
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http://dx.doi.org/10.1128/mBio.01818-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5750398PMC
January 2018

DNAAF1 links heart laterality with the AAA+ ATPase RUVBL1 and ciliary intraflagellar transport.

Hum Mol Genet 2018 02;27(3):529-545

Leeds Institute of Biomedical and Clinical Sciences, Faculty of Medicine & Health, University of Leeds, Leeds LS9 7TF, UK.

DNAAF1 (LRRC50) is a cytoplasmic protein required for dynein heavy chain assembly and cilia motility, and DNAAF1 mutations cause primary ciliary dyskinesia (PCD; MIM 613193). We describe four families with DNAAF1 mutations and complex congenital heart disease (CHD). In three families, all affected individuals have typical PCD phenotypes. However, an additional family demonstrates isolated CHD (heterotaxy) in two affected siblings, but no clinical evidence of PCD. We identified a homozygous DNAAF1 missense mutation, p.Leu191Phe, as causative for heterotaxy in this family. Genetic complementation in dnaaf1-null zebrafish embryos demonstrated the rescue of normal heart looping with wild-type human DNAAF1, but not the p.Leu191Phe variant, supporting the conserved pathogenicity of this DNAAF1 missense mutation. This observation points to a phenotypic continuum between CHD and PCD, providing new insights into the pathogenesis of isolated CHD. In further investigations of the function of DNAAF1 in dynein arm assembly, we identified interactions with members of a putative dynein arm assembly complex. These include the ciliary intraflagellar transport protein IFT88 and the AAA+ (ATPases Associated with various cellular Activities) family proteins RUVBL1 (Pontin) and RUVBL2 (Reptin). Co-localization studies support these findings, with the loss of RUVBL1 perturbing the co-localization of DNAAF1 with IFT88. We show that RUVBL1 orthologues have an asymmetric left-sided distribution at both the mouse embryonic node and the Kupffer's vesicle in zebrafish embryos, with the latter asymmetry dependent on DNAAF1. These results suggest that DNAAF1-RUVBL1 biochemical and genetic interactions have a novel functional role in symmetry breaking and cardiac development.
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http://dx.doi.org/10.1093/hmg/ddx422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5886296PMC
February 2018

Meckel-Gruber Syndrome: An Update on Diagnosis, Clinical Management, and Research Advances.

Front Pediatr 2017 20;5:244. Epub 2017 Nov 20.

Leeds Institute of Biomedical and Clinical Sciences, University of Leeds, Leeds, United Kingdom.

Meckel-Gruber syndrome (MKS) is a lethal autosomal recessive congenital anomaly syndrome caused by mutations in genes encoding proteins that are structural or functional components of the primary cilium. Conditions that are caused by mutations in ciliary genes are collectively termed the ciliopathies, and MKS represents the most severe condition in this group of disorders. The primary cilium is a microtubule-based organelle, projecting from the apical surface of vertebrate cells. It acts as an "antenna" that receives and transduces chemosensory and mechanosensory signals, but also regulates diverse signaling pathways, such as Wnt and Shh, that have important roles during embryonic development. Most MKS proteins localize to a distinct ciliary compartment called the transition zone (TZ) that regulates the trafficking of cargo proteins or lipids. In this review, we provide an up-to-date summary of MKS clinical features, molecular genetics, and clinical diagnosis. MKS has a highly variable phenotype, extreme genetic heterogeneity, and displays allelism with other related ciliopathies such as Joubert syndrome, presenting significant challenges to diagnosis. Recent advances in genetic technology, with the widespread use of multi-gene panels for molecular testing, have significantly improved diagnosis, genetic counseling, and the clinical management of MKS families. These include the description of some limited genotype-phenotype correlations. We discuss recent insights into the molecular basis of disease in MKS, since the functions of some of the relevant ciliary proteins have now been determined. A common molecular etiology appears to be disruption of ciliary TZ structure and function, affecting essential developmental signaling and the regulation of secondary messengers.
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http://dx.doi.org/10.3389/fped.2017.00244DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5701918PMC
November 2017

Application of 3-D Fluorescence: Characterization of Natural Organic Matter in Natural Water and Water Purification Systems.

J Fluoresc 2017 Nov 21;27(6):2069-2094. Epub 2017 Aug 21.

School of Civil and Environmental Engineering, Graduate School, Harbin Institute of Technology Shenzhen, Shenzhen, 518055, China.

Natural organic matter (NOM) found in water sources is broadly defined as a mixture of polyfunctional organic molecules, characterized by its complex structure and paramount influence on water quality. Because the inevitable release of pollutants into aquatic environments due to an ineffective control of industrial and agricultural pollution, the evaluation of the interaction of NOM with heavy metals, nanoparticles, organic pollutants and other pollutants in the aquatic environment, has greatly increased. Three-dimensional (3-D) fluorescence has the potential to reveal the interaction mechanisms between NOM and pollutants as well as the source of NOM pollution. In water purification engineering system, the 3-D fluorescence can indicate the variations of NOM composition and gives an effective prediction of water quality as well as the underline water purification mechanisms. Inadequately treated NOM is a cause of precursors of disinfection byproducts (DBPs), posing a potential threat to human health. Effective control and measurement/evaluation of NOM have long been an important factors in the prevention of water pollution. Overall, 3-D fluorescence allows for a rapid identification of organic components thus indicating possible sources of water pollution, mechanisms of pollutant interactions, and possible DBPs formed during conventional treatment of this water. This article reviews the 3-D fluorescence characteristics of NOM in natural water and typical water purification systems. The 3-D fluorescence was effective for indicating the variabilities in NOM composition and chemistry thus providing a better understanding of NOM in natural water system and water engineering system.
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http://dx.doi.org/10.1007/s10895-017-2146-7DOI Listing
November 2017

Blocking connexin channels improves embryo development of vitrified bovine blastocysts.

Biol Reprod 2017 02;96(2):288-301

Physiology group, Department of Basic Medical Sciences, Ghent University, Ghent, Belgium.

Connexins (Cxs) are required for normal embryo development and implantation. They form gap junctions (GJs) connecting the cytoplasm of adjacent cells and hemichannels (HCs), which are normally closed but open in response to stress conditions. Excessive HC opening is detrimental for cell function and may lead to cell death. We found that hatching of in vitro-produced bovine embryos, matured in serum-containing conditions, was significantly improved when vitrification/warming was done in the presence of Gap26 that targets GJA1 (Cx43) and GJA4 (Cx37). Further work showed that HCs from blastocysts produced after oocyte maturation in the presence of serum were open shortly after vitrification/warming, and this was prevented by Gap26. Gap26, applied for the exposure times used, inhibited Cx43 and Cx37 HCs while it did not have an effect on GJs. Interestingly, Gap26 had no effect on blastocyst degeneration or cell death. We conclude that blocking HCs protects embryos during vitrification and warming by a functional effect not linked to cell death.
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http://dx.doi.org/10.1095/biolreprod.116.144121DOI Listing
February 2017

Characterizing the morbid genome of ciliopathies.

Genome Biol 2016 11 28;17(1):242. Epub 2016 Nov 28.

Division of Experimental Ophthalmology and Medical Bioanalytics, Center for Ophthalmology, Eberhard-Karls University Tübingen, 72076, Tübingen, Germany.

Background: Ciliopathies are clinically diverse disorders of the primary cilium. Remarkable progress has been made in understanding the molecular basis of these genetically heterogeneous conditions; however, our knowledge of their morbid genome, pleiotropy, and variable expressivity remains incomplete.

Results: We applied genomic approaches on a large patient cohort of 371 affected individuals from 265 families, with phenotypes that span the entire ciliopathy spectrum. Likely causal mutations in previously described ciliopathy genes were identified in 85% (225/265) of the families, adding 32 novel alleles. Consistent with a fully penetrant model for these genes, we found no significant difference in their "mutation load" beyond the causal variants between our ciliopathy cohort and a control non-ciliopathy cohort. Genomic analysis of our cohort further identified mutations in a novel morbid gene TXNDC15, encoding a thiol isomerase, based on independent loss of function mutations in individuals with a consistent ciliopathy phenotype (Meckel-Gruber syndrome) and a functional effect of its deficiency on ciliary signaling. Our study also highlighted seven novel candidate genes (TRAPPC3, EXOC3L2, FAM98C, C17orf61, LRRCC1, NEK4, and CELSR2) some of which have established links to ciliogenesis. Finally, we show that the morbid genome of ciliopathies encompasses many founder mutations, the combined carrier frequency of which accounts for a high disease burden in the study population.

Conclusions: Our study increases our understanding of the morbid genome of ciliopathies. We also provide the strongest evidence, to date, in support of the classical Mendelian inheritance of Bardet-Biedl syndrome and other ciliopathies.
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http://dx.doi.org/10.1186/s13059-016-1099-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5126998PMC
November 2016

Covalently immobilized lipase on aminoalkyl-, carboxy- and hydroxy-multi-wall carbon nanotubes in the enantioselective synthesis of Solketal esters.

Enzyme Microb Technol 2016 Jun 2;87-88:61-9. Epub 2016 Mar 2.

Department of Organic Chemistry, Bioorganic Chemistry and Biotechnology, Silesian University of Technology, Krzywoustego 4, Gliwice 44-100, Poland. Electronic address:

Aiming at the preparation of efficient, stable on storage and recyclable nanobiocatalysts for enantioselective transesterification, alkaline lipase from Pseudomonas fluorescens was covalently immobilized (up to 8.5wt.%) on functionalized multi-wall carbon nanotubes (f-MWCNTs). f-MWCNTs were synthesized via: (a) (2+1)-cycloaddition of a nitrene to the C-sp(2) nanotube walls (3.2mmolg(-1), a novel synthetic approach) and, (b) oxidative treatments, i.e. Fenton reagent (3.5mmolg(-1)) and nitrating mixture (2.5mmolg(-1)), yielding aminoalkyl-, hydroxyl- and carboxyl-MWCNTs, respectively. Amino- and epoxy- functionalized mesoporous silica (f-SBA-15) were used as the reference supports. Transesterification of vinyl n-butyrate by racemic Solketal with a chromatographically (GC) traced kinetics was selected as the model reaction. The studies revealed that different chemical functionalization of morphologically identical nanotube supports led to various enzyme loadings, catalytic activities and enantioselectivities. MWCNT-NH2-based nanobiocatalyst was found to be the most active composite among all of the tested systems (yield 20%, t=0.5h, 1321Ug(-1)), i.e. 12 times more active than the native enzyme. In turn, lipase immobilized on MWCNT-COOH emerged as the most enantioselective system (ex aequo with SBA-NH2) (eeR=74%, t=0.5h at yield of 3-5%). The activity of the MWCNT-NH2-based nanobiocatalyst after 8 cycles of transesterification dropped to 60% of its initial value, whereas for SBA-NH2-based composite remained unchanged. Importantly, stability on storage was fully maintained for all MWCNT-based nanobiocatalysts or even 'extra-enhanced' for MWCNT-OH.
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http://dx.doi.org/10.1016/j.enzmictec.2016.02.015DOI Listing
June 2016

TMEM107 recruits ciliopathy proteins to subdomains of the ciliary transition zone and causes Joubert syndrome.

Nat Cell Biol 2016 Jan 23;18(1):122-31. Epub 2015 Nov 23.

School of Biomolecular and Biomedical Science, UCD Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland.

The transition zone (TZ) ciliary subcompartment is thought to control cilium composition and signalling by facilitating a protein diffusion barrier at the ciliary base. TZ defects cause ciliopathies such as Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP) and Joubert syndrome (JBTS). However, the molecular composition and mechanisms underpinning TZ organization and barrier regulation are poorly understood. To uncover candidate TZ genes, we employed bioinformatics (coexpression and co-evolution) and identified TMEM107 as a TZ protein mutated in oral-facial-digital syndrome and JBTS patients. Mechanistic studies in Caenorhabditis elegans showed that TMEM-107 controls ciliary composition and functions redundantly with NPHP-4 to regulate cilium integrity, TZ docking and assembly of membrane to microtubule Y-link connectors. Furthermore, nematode TMEM-107 occupies an intermediate layer of the TZ-localized MKS module by organizing recruitment of the ciliopathy proteins MKS-1, TMEM-231 (JBTS20) and JBTS-14 (TMEM237). Finally, MKS module membrane proteins are immobile and super-resolution microscopy in worms and mammalian cells reveals periodic localizations within the TZ. This work expands the MKS module of ciliopathy-causing TZ proteins associated with diffusion barrier formation and provides insight into TZ subdomain architecture.
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http://dx.doi.org/10.1038/ncb3273DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5580800PMC
January 2016

Screen-based identification and validation of four new ion channels as regulators of renal ciliogenesis.

J Cell Sci 2015 Dec 6;128(24):4550-9. Epub 2015 Nov 6.

Department of Nephrology and Hypertension, University Medical Center Utrecht, Utrecht 3584 CX, The Netherlands

To investigate the contribution of ion channels to ciliogenesis, we carried out a small interfering RNA (siRNA)-based reverse genetics screen of all ion channels in the mouse genome in murine inner medullary collecting duct kidney cells. This screen revealed four candidate ion channel genes: Kcnq1, Kcnj10, Kcnf1 and Clcn4. We show that these four ion channels localize to renal tubules, specifically to the base of primary cilia. We report that human KCNQ1 Long QT syndrome disease alleles regulate renal ciliogenesis; KCNQ1-p.R518X, -p.A178T and -p.K362R could not rescue ciliogenesis after Kcnq1-siRNA-mediated depletion in contrast to wild-type KCNQ1 and benign KCNQ1-p.R518Q, suggesting that the ion channel function of KCNQ1 regulates ciliogenesis. In contrast, we demonstrate that the ion channel function of KCNJ10 is independent of its effect on ciliogenesis. Our data suggest that these four ion channels regulate renal ciliogenesis through the periciliary diffusion barrier or the ciliary pocket, with potential implication as genetic contributors to ciliopathy pathophysiology. The new functional roles of a subset of ion channels provide new insights into the disease pathogenesis of channelopathies, which might suggest future therapeutic approaches.
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http://dx.doi.org/10.1242/jcs.176065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4696498PMC
December 2015

An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes.

Nat Cell Biol 2015 Aug 13;17(8):1074-1087. Epub 2015 Jul 13.

Institute of Human Genetics, Friedrich-Alexander-Universität Erlangen-Nürnberg, 91054 Erlangen, Germany.

Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.
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http://dx.doi.org/10.1038/ncb3201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4536769PMC
August 2015
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