Bone scintigraphy predicts bisphosphonate-induced osteonecrosis of the jaw (BRONJ) in patients with metastatic castration-resistant prostate cancer (mCRPC).

OBJECTIVES: The aim of this study was to evaluate the role of bone scintigraphy (BS) in early prediction of clinically asymptomatic bisphosphonate (BP)-related osteonecrosis of the jaws (BRONJ) in patients with metastatic castration-resistant prostate cancer (mCRPC). MATERIAL AND METHODS: BS of mCRPC patients treated with BP was evaluated for pathologic tracer uptake of the jaws in BS suspicious for BRONJ. Results were compared to development of clinically evident BRONJ. Sensitivity, specificity and predictive values of BS for the detection of BRONJ as well as time from beginning of BP therapy to pathologic tracer uptake in BS and time from pathologic tracer uptake in BS to clinically evident BRONJ were determined. RESULTS: Thirty BP-treated patients were included. Nine patients (30%) had pathologic BS lesions of the jaws. Six patients (20%) developed BRONJ. Sensitivity and specificity of BS for BRONJ prediction were 67 and 79%. Median time from the start of BP treatment to pathologic tracer uptake in BS was 28 months (range 10-33) and from pathologic tracer uptake in BS to clinically evident BRONJ 6.5 months (range 2-19). Pathologic tracer uptake in BS was significantly more often observed in patients who developed BRONJ compared to patients who did not (p = 0.049; OR 7.6). CONCLUSIONS: Patients with pathologic tracer uptake in the jaws in BS significantly more often develop BRONJ. An unsuspicious BS is predictive for absence of BRONJ in the future. CLINICAL RELEVANCE: We conclude that when BS has been performed, it should not only be used to assess tumour stage and treatment response but also to check for pathologic tracer uptake in the jaws in BS to detect BRONJ at an early stage in mCRPC patients receiving bisphosphonates.

Anks3 interacts with nephronophthisis proteins and is required for normal renal development.

Nephronophthisis (NPH) is a heterogenetic autosomal recessive disorder associated with kidney cysts and multiple extrarenal manifestations. The disease-associated gene products (NPHPs) typically contain domains involved in protein-protein interactions, and appear to exert their tissue-specific functions in large protein complexes. Most NPHPs localize to the cilium and/or basal body; however, their precise molecular functions remain largely unknown. We have recently identified the SAM-domain containing protein Anks3 as a potential ANKS6/NPHP16-interacting protein, and report now that Anks3 interacts with several NPHPs as well as with Bicc1 and the oxygen-sensitive asparaginyl hydroxylase HIF1AN. Knockdown of anks3 in zebrafish embryos was associated with NPH-typical manifestations, including ciliary abnormalities, cyst formation, and laterality defects. In multi-ciliated epidermal cells, GFP-tagged Anks3 localizes to the cilium, but forms large aggregates in the absence of NPHP1, indicating that the negatively charged NPHP1 curtails the polymerization of Anks3. Collectively, these findings suggest that Anks3 is a cilia-associated molecule that partners with the ANKS6- and via NPHP1 to the NPHP1-4-8 module. Thus, developmental defects associated with Anks3 depletion in zebrafish suggest that ANKS3 mutations may cause NPH or NPH-like disease in humans.

Anks3 alters the sub-cellular localization of the Nek7 kinase.

Nephronophthisis (NPH) is an autosomal recessive cystic kidney disease, and a frequent cause of end-stage renal failure in children. To date, 17 NPH-associated gene products (NPHPs) have been identified. Most NPHPs participate in large multi-protein complexes that localize to the cilium and/or basal body; however, the precise composition of these complexes and their biological function remain largely unknown. We recently observed that the ankyrin repeat protein Anks3 interacts with the NPH family member Anks6. Both Anks3 and Anks6 form complexes with multiple other NPHPs, suggesting that both proteins function in similar or overlapping signaling pathways. Here, we show that Anks3, but not Anks6 interacted with the NIMA-related kinase Nek7, and was heavily modified in the presence of Nek7, resulting in an approximately 20 kD increase in molecular weight. Although mass spectrometry revealed increased serine and threonine phosphorylation of Anks3 primarily within the N-terminal ankyrin repeats also required for Nek7 interaction, the molecular weight increase occurred even in the presence of a kinase-dead Nek7 mutant, indicating that this modification was not caused by Nek7-dependent Anks3 phosphorylation. Furthermore, the Anks3 modification was specific for Nek7, and did not occur in the presence of Nek8. Importantly, Anks3 retained Nek7 in the cytoplasm, suggesting that, Nek7 triggers the modification of Anks3, which in turn prevents the nuclear localization of Nek7.

The nephronophthisis gene product NPHP2/Inversin interacts with Aurora A and interferes with HDAC6-mediated cilia disassembly.

BACKGROUND: Nephronophthisis (NPH) is a rare recessive disease caused by several different gene mutations. Most gene products localize to the cilium, and thus, the various NPH manifestations including kidney cysts and situs inversus have been linked to ciliary defects. RESULTS: Here, we describe that targeted knockdown of NPHP2 significantly reduced the number of cilia on polarized MDCK cells. As one of the underlying molecular mechanisms, we identified a direct interaction between NPHP2 and Aurora A, a cell cycle kinase that promotes ciliary disassembly after activation by Hef1. NPHP2 inhibited the phosphorylation and activation of Aurora A, and reduced its kinase activity in vitro. Aurora A and histone deacetylase inhibitors ameliorated the ciliogenesis defect in NPHP2-deficient MDCK cells, supporting our hypothesis that NPHP2 is involved in the control of ciliary disassembly. Furthermore, we observed that nephrocystin (NPHP1), an interaction partner of NPHP2, also binds Aurora A, exerting very similar inhibitory effects on Hef1-mediated Aurora A activation. CONCLUSIONS: Taken together, these findings suggest that NPHP gene products can interfere with ciliary disassembly through interaction with the Hef1/Aurora A module, thereby modulating cell cycle control and cell proliferation.

Cell stress is related to re-localization of Argonaute 2 and to decreased RNA interference in human cells.

Various kinds of stress on human cells induce the formation of endogenous stress granules (SGs). Human Argonaute 2 (hAgo2), the catalytic core component of the RNA-induced silencing complex (RISC), can be recruited to SGs as well as P-bodies (PBs) indicating that the dynamic intracellular distribution of hAgo2 in SGs, in PBs or at other sub-cellular sites could be related to the efficiency of the RNA interference (RNAi) machinery. Here, we studied the influence of heat shock, sodium arsenite (NaAsO2), cycloheximide (CHX) and Lipofectamine 2000-mediated transfection of phosphorothioate (PS)-modified oligonucleotides (ON) on the intracellular localization of hAgo2 and the efficiency of RNAi. Fluorescence microscopy and sedimentation analysis of cell fractions indicate stress-induced accumulation of hAgo2 in SGs and the loss of distinctly composed complexes containing hAgo2 or their sub-cellular context. Transfection of cells with PS-ON induces cell stress that is phenotypically similar to the established inducers heat shock and NaAsO2. The intracellular re-distribution of hAgo2 is related to its increased metabolic stability and to decreased RNAi directed by microRNA or by short interfering RNA. Here, we propose a functional model of the relationship between cell stress, translocation of hAgo2 to SGs providing a depot function, and loss of RNAi activity.

PRKCSH/80K-H, the protein mutated in polycystic liver disease, protects polycystin-2/TRPP2 against HERP-mediated degradation.

Autosomal dominant polycystic liver disease (PCLD) is caused by mutations of either PRKCSH or Sec63, two proteins associated with the endoplasmic reticulum (ER). Both proteins are involved in carbohydrate processing, folding and translocation of newly synthesized glycoproteins. It is postulated that defective quality control of proteins initiates endoplasmic reticulum-associated degradation (ERAD), which disrupts hepatic homeostasis in patients with PRKCSH or Sec63 mutations. However, the precise molecular mechanisms are not known. Here, we show that over-expression or depletion of PRKCSH in zebrafish embryos leads to pronephric cysts, abnormal body curvature and situs inversus. Identical phenotypic changes are induced by depletion or over-expression of TRPP2. Increased PRKCSH levels ameliorate developmental abnormalities caused by over-expressed TRPP2, whereas excess TRPP2 can compensate the loss PRKCSH, indicating that the proteins share a common signaling pathway. PRKCSH binds the C-terminal domain of TRPP2, and both proteins co-localize within the ER. Furthermore, PRKCSH interacts with Herp, and inhibits Herp-mediated ubiquitination of TRPP2. Our findings suggest that PRKCSH functions as a chaperone-like molecule, which prevents ERAD of TRPP2. Dysequilibrium between TRPP2 and PRKCSH may lead to cyst formation in PCLD patients with PRKCSH mutations, and thereby account for the overlapping manifestations observed in PCLD and autosomal dominant polycystic kidney disease.

Antisense tools for functional studies of human Argonaute proteins.

The Argonaute proteins play essential roles in development and cellular metabolism in many organisms, including plants, flies, worms, and mammals. Whereas in organisms such as Caenorhabditis elegans and Arabidopsis thaliana, creation of Argonaute mutant strains allowed the study of their biological functions, in mammals the application of this approach is limited by its difficulty and in the specific case of Ago2 gene, by the lethality of such mutation. Hence, in human cells, functional studies of Ago proteins relied on phenotypic suppression using small interfering RNA (siRNA) which involves Ago proteins and the RNA interference mechanism. This bears the danger of undesired or unknown interference effects which may lead to misleading results. Thus, alternative methods acting by different regulatory mechanisms would be advantageous in order to exclude unspecific effects. The knockdown may be achieved by using specific antisense oligonucleotides (asONs) which act via an RNase H-dependent mechanism, not thought to interfere with processes in which Agos are involved. Different functional observations in the use of siRNA versus asONs indicate the relevance of this assumption. We developed asONs specific for the four human Agos (hAgos) and compared their activities with those obtained by siRNA. We confirm that hAgo2 is involved in microRNA (miRNA)- and in siRNA-mediated silencing pathways, while the other hAgos play a role only in miRNA-based gene regulation. Using combinations of asONs we found that the simultaneous down-regulation of hAgo1, hAgo2, and hAgo4 led to the strongest decrease in miRNA activity, indicating a main role of these proteins.

Scribble participates in Hippo signaling and is required for normal zebrafish pronephros development.

Spatial organization of cells and their appendages is controlled by the planar cell polarity pathway, a signaling cascade initiated by the protocadherin Fat in Drosophila. Vertebrates express 4 Fat molecules, Fat1-4. We found that depletion of Fat1 caused cyst formation in the zebrafish pronephros. Knockdown of the PDZ domain containing the adaptor protein Scribble intensified the cyst-promoting phenotype of Fat1 depletion, suggesting that Fat1 and Scribble act in overlapping signaling cascades during zebrafish pronephros development. Supporting the genetic interaction with Fat1, Scribble recognized the PDZ-binding site of Fat1. Depletion of Yes-associated protein 1 (YAP1), a transcriptional co-activator inhibited by Hippo signaling, ameliorated the cyst formation in Fat1-deficient zebrafish, whereas Scribble inhibited the YAP1-induced cyst formation. Thus, reduced Hippo signaling and subsequent YAP1 disinhibition seem to play a role in the development of pronephric cysts after depletion of Fat1 or Scribble. We hypothesize that Hippo signaling is required for normal pronephros development in zebrafish and that Scribble is a candidate link between Fat and the Hippo signaling cascade in vertebrates.

Microridge-like structures anchor motile cilia.

Several tissues contain cells with multiple motile cilia that generate a fluid or particle flow to support development and organ functions; defective motility causes human disease. Developmental cues orient motile cilia, but how cilia are locked into their final position to maintain a directional flow is not understood. Here we find that the actin cytoskeleton is highly dynamic during early development of multiciliated cells (MCCs). While apical actin bundles become increasingly more static, subapical actin filaments are nucleated from the distal tip of ciliary rootlets. Anchorage of these subapical actin filaments requires the presence of microridge-like structures formed during MCC development, and the activity of Nonmuscle Myosin II. Optogenetic manipulation of Ezrin, a core component of the microridge actin-anchoring complex, or inhibition of Myosin Light Chain Kinase interfere with rootlet anchorage and orientation. These observations identify microridge-like structures as an essential component of basal body rootlet anchoring in MCCs.

Quantification of microaerobic growth of Geobacter sulfurreducens.

Geobacter sulfurreducens was originally considered a strict anaerobe. However, this bacterium was later shown to not only tolerate exposure to oxygen but also to use it as terminal electron acceptor. Research performed has so far only revealed the general ability of G. sulfurreducens to reduce oxygen, but the oxygen uptake rate has not been quantified yet, nor has evidence been provided as to how the bacterium achieves oxygen reduction. Therefore, microaerobic growth of G. sulfurreducens was investigated here with better defined operating conditions as previously performed and a transcriptome analysis was performed to elucidate possible metabolic mechanisms important for oxygen reduction in G. sulfurreducens. The investigations revealed that cell growth with oxygen is possible to the same extent as with fumarate if the maximum specific oxygen uptake rate (sOUR) of 95 mgO2 gCDW-1 h-1 is not surpassed. Hereby, the entire amount of introduced oxygen is reduced. When oxygen concentrations are too high, cell growth is completely inhibited and there is no partial oxygen consumption. Transcriptome analysis suggests a menaquinol oxidase to be the enzyme responsible for oxygen reduction. Transcriptome analysis has further revealed three different survival strategies, depending on the oxygen concentration present. When prompted with small amounts of oxygen, G. sulfurreducens will try to escape the microaerobic area; if oxygen concentrations are higher, cells will focus on rapid and complete oxygen reduction coupled to cell growth; and ultimately cells will form protective layers if a complete reduction becomes impossible. The results presented here have important implications for understanding how G. sulfurreducens survives exposure to oxygen.

Nephronophthisis gene products display RNA-binding properties and are recruited to stress granules.

Mutations of cilia-associated molecules cause multiple developmental defects that are collectively termed ciliopathies. However, several ciliary proteins, involved in gating access to the cilium, also assume localizations at other cellular sites including the nucleus, where they participate in DNA damage responses to maintain tissue integrity. Molecular insight into how these molecules execute such diverse functions remains limited. A mass spectrometry screen for ANKS6-interacting proteins suggested an involvement of ANKS6 in RNA processing and/or binding. Comparing the RNA-binding properties of the known RNA-binding protein BICC1 with the three ankyrin-repeat proteins ANKS3, ANKS6 (NPHP16) and INVERSIN (NPHP2) confirmed that certain nephronophthisis (NPH) family members can interact with RNA molecules. We also observed that BICC1 and INVERSIN associate with stress granules in response to translational inhibition. Furthermore, BICC1 recruits ANKS3 and ANKS6 into TIA-1-positive stress granules after exposure to hippuristanol. Our findings uncover a novel function of NPH family members, and provide further evidence that NPH family members together with BICC1 are involved in stress responses to maintain tissue and organ integrity.

Long-Term Behavior of Defined Mixed Cultures of Geobacter sulfurreducens and Shewanella oneidensis in Bioelectrochemical Systems.

This work aims to investigate the long-term behavior of interactions of electrochemically active bacteria in bioelectrochemical systems. The electrochemical performance and biofilm characteristics of pure cultures of Geobacter sulfurreducens and Shewanella oneidensis are being compared to a defined mixed culture of both organisms. While S. oneidensis pure cultures did not form cohesive and stable biofilms on graphite anodes and only yielded 0.034 ± 0.011 mA/cm2 as maximum current density by feeding of each 5 mM lactate and acetate, G. sulfurreducens pure cultures formed 69 µm thick, area-wide biofilms with 10 mM acetate as initial substrate concentration and yielded a current of 0.39 ± 0.09 mA/cm2. Compared to the latter, a defined mixed culture of both species was able to yield 38% higher maximum current densities of 0.54 ± 0.07 mA/cm2 with each 5 mM lactate and acetate. This increase in current density was associated with a likewise increased thickness of the anodic biofilm to approximately 93 µm. It was further investigated whether a sessile incorporation of S. oneidensis into the mixed culture biofilm, which has been reported previously for short-term experiments, is long-term stable. The results demonstrate that S. oneidensis was not stably incorporated into the biofilm; rather, the planktonic presence of S. oneidensis has a positive effect on the biofilm growth of G. sulfurreducens and thus on current production.

RIG-I Resists Hypoxia-Induced Immunosuppression and Dedifferentiation.

A hypoxic tumor microenvironment is linked to poor prognosis. It promotes tumor cell dedifferentiation and metastasis and desensitizes tumor cells to type-I IFN, chemotherapy, and irradiation. The cytoplasmic immunoreceptor retinoic acid-inducible gene-I (RIG-I) is ubiquitously expressed in tumor cells and upon activation by 5'-triphosphate RNA (3pRNA) drives the induction of type I IFN and immunogenic cell death. Here, we analyzed the impact of hypoxia on the expression of RIG-I in various human and murine tumor and nonmalignant cell types and further investigated its function in hypoxic murine melanoma. 3pRNA-inducible RIG-I-expression was reduced in hypoxic melanoma cells compared with normoxic controls, a phenomenon that depended on the hypoxia-associated transcription factor HIF1α. Still, RIG-I functionality was conserved in hypoxic melanoma cells, whereas responsiveness to recombinant type-I IFN was abolished, due to hypoxia-induced loss of type I IFN receptor expression. Likewise, RIG-I activation in hypoxic melanoma cells, but not exposure to recombinant IFNα, provoked melanocyte antigen-specific CD8+ T-cell and NK-cell attack. Scavenging of hypoxia-induced reactive oxygen species by vitamin C restored the inducible expression of RIG-I under hypoxia in vitro, boosted in vitro anti-melanoma NK- and CD8+ T-cell attack, and augmented 3pRNA antitumor efficacy in vivo These results demonstrate that RIG-I remains operational under hypoxia and that RIG-I function is largely insensitive to lower cell surface expression of the IFNα receptor. RIG-I function could be fortified under hypoxia by the combined use of 3pRNA with antioxidants. Cancer Immunol Res; 5(6); 455-67. ©2017 AACR.

The C175R mutation alters nuclear localization and transcriptional activity of the nephronophthisis NPHP7 gene product.

Nephronophthisis (NPH) is a rare autosomal ciliopathy, but the leading cause for hereditary end-stage renal disease in children. Most NPH family members form large protein networks, which appear to participate in structural elements of the cilium and/or function to restrict access of molecules to the ciliary compartment. The zinc-finger protein GLIS2/NPHP7 represents an exception as it has been implicated in transcriptional regulation; only two families with GLIS2/NPHP7 mutations and typical NPH manifestations have been identified so far. We describe here that the recently identified GLIS2/NPHP7(C175R) point mutation abolished the nuclear localization of GLIS2/NPHP7. Forced nuclear import did not rescue the transcriptional defects of GLIS2/NPHP7(C175R), indicating additional defects as DNA-binding protein. We further observed that wild type, but not GLIS2/NPHP7(C175R), prevented the cyst formation caused by depletion of nphp7 in zebrafish embryos. Taken together, our findings indicate that the C175R mutation affects both localization and function of GLIS2/NPHP7, supporting a role of this mutation in NPH, but questioning the direct involvement of GLIS2/NPHP7 in ciliary functions.

Tuning a MAb glycan profile in cell culture: Supplementing N-acetylglucosamine to favour G0 glycans without compromising productivity and cell growth.

Glycosylation is a critical quality attribute of many therapeutic proteins, particularly monoclonal antibodies (MAbs). Nucleotide-sugar precursors supplemented to growth medium to affect the substrate supply chain of glycosylation has yielded promising but varied results for affecting glycosylation. Glucosamine (GlcN), a precursor for N-acetylglucosamine (GlcNAc), is a major component of mammalian glycans. The supplementation of GlcN to CHO cells stably-expressing a chimeric heavy-chain monoclonal antibody, EG2-hFc, reduces the complexity of glycans to favour G0 glycoforms, while also negatively impacting cell growth. Although several researchers have examined the supplementation of glucosamine, no clear explanation of its impact on cell growth has been forthcoming. In this work, the glucosamine metabolism is examined. We identified the acetylation of GlcN to produce GlcNAc to be the most likely cause for the negative impact on growth due to the depletion of intracellular acetyl-CoA pools in the cytosol. By supplementing GlcNAc in lieu of GlcN to CHO cells producing EG2-hFc, we achieve the same shift in glycan complexity with marginal impacts on the cell growth and protein production.

The polarity protein Inturned links NPHP4 to Daam1 to control the subapical actin network in multiciliated cells.

Motile cilia polarization requires intracellular anchorage to the cytoskeleton; however, the molecular machinery that supports this process remains elusive. We report that Inturned plays a central role in coordinating the interaction between cilia-associated proteins and actin-nucleation factors. We observed that knockdown of nphp4 in multiciliated cells of the Xenopus laevis epidermis compromised ciliogenesis and directional fluid flow. Depletion of nphp4 disrupted the subapical actin layer. Comparison to the structural defects caused by inturned depletion revealed striking similarities. Furthermore, coimmunoprecipitation assays demonstrated that the two proteins interact with each other and that Inturned mediates the formation of ternary protein complexes between NPHP4 and DAAM1. Knockdown of daam1, but not formin-2, resulted in similar disruption of the subapical actin web, whereas nphp4 depletion prevented the association of Inturned with the basal bodies. Thus, Inturned appears to function as an adaptor protein that couples cilia-associated molecules to actin-modifying proteins to rearrange the local actin cytoskeleton.

A complex of BBS1 and NPHP7 is required for cilia motility in zebrafish.

Bardet-Biedl syndrome (BBS) and nephronophthisis (NPH) are hereditary autosomal recessive disorders, encoded by two families of diverse genes. BBS and NPH display several overlapping phenotypes including cystic kidney disease, retinitis pigmentosa, liver fibrosis, situs inversus and cerebellar defects. Since most of the BBS and NPH proteins localize to cilia and/or their appendages, BBS and NPH are considered ciliopathies. In this study, we characterized the function of the transcription factor Nphp7 in zebrafish, and addressed the molecular connection between BBS and NPH. The knockdown of zebrafish bbs1 and nphp7.2 caused similar phenotypic changes including convergent extension defects, curvature of the body axis, hydrocephalus, abnormal heart looping and cystic pronephros, all consistent with an altered ciliary function. Immunoprecipitation assays revealed a physical interaction between BBS1 and NPHP7, and the simultaneous knockdown of zbbs1 and znphp7.2 enhanced the cystic pronephros phenotype synergistically, suggesting a genetic interaction between zbbs1 and znphp7.2 in vivo. Deletion of zBbs1 or zNphp7.2 did not compromise cilia formation, but disrupted cilia motility. Although NPHP7 has been shown to act as transcriptional repressor, our studies suggest a crosstalk between BBS1 and NPHP7 in regulating normal function of the cilium.