STAR syndrome-associated CDK10/Cyclin M regulates actin network architecture and ciliogenesis

CDK10/CycM is a protein kinase deficient in STAR (toe Syndactyly, Telecanthus and Anogenital and Renal malformations) syndrome, which results from mutations in the X-linked FAM58A gene encoding Cyclin M. The biological functions of CDK10/CycM and etiology of STAR syndrome are poorly understood. Here, we report that deficiency of CDK10/Cyclin M promotes assembly and elongation of primary cilia. We establish that this reflects a key role for CDK10/Cyclin M in regulation of actin network organization, which is known to govern ciliogenesis. In an unbiased screen, we identified the RhoA-associated kinase PKN2 as a CDK10/CycM phosphorylation substrate. We establish that PKN2 is a bone fide regulator of ciliogenesis, acting in a similar manner to CDK10/CycM. We discovered that CDK10/Cyclin M binds and phosphorylates PKN2 on threonines 121 and 124, within PKN2′s core RhoA-binding domain. Furthermore, we demonstrate that deficiencies in CDK10/CycM or PKN2, or expression of a non-phosphorylatable version of PKN2, destabilize both the RhoA protein and the actin network architecture. Importantly, we established that ectopic expression of RhoA is sufficient to override the induction of ciliogenesis resulting from CDK10/CycM knockdown, indicating that RhoA regulation is critical for CDK10/CycM's negative effect on ciliogenesis. Finally, we show that kidney sections from a STAR patient display dilated renal tubules and abnormal, elongated cilia. Altogether, these results reveal CDK10/CycM as a key regulator of actin dynamics and a suppressor of ciliogenesis through phosphorylation of PKN2 and promotion of RhoA signaling. Moreover, they suggest that STAR syndrome is a ciliopathy.     

Estimating the global distribution of field size using crowdsourcing

There is an increasing evidence that smallholder farms contribute substantially to food production globally, yet spatially explicit data on agricultural field sizes are currently lacking. Automated field size delineation using remote sensing or the estimation of average farm size at subnational level using census data are two approaches that have been used. However, both have limitations, for example, automatic field size delineation using remote sensing has not yet been implemented at a global scale while the spatial resolution is very coarse when using census data. This paper demonstrates a unique approach to quantifying and mapping agricultural field size globally using crowdsourcing. A campaign was run in June 2017, where participants were asked to visually interpret very high resolution satellite imagery from Google Maps and Bing using the Geo‐Wiki application. During the campaign, participants collected field size data for 130 K unique locations around the globe. Using this sample, we have produced the most accurate global field size map to date and estimated the percentage of different field sizes, ranging from very small to very large, in agricultural areas at global, continental, and national levels. The results show that smallholder farms occupy up to 40% of agricultural areas globally, which means that, potentially, there are many more smallholder farms in comparison with the two different current global estimates of 12% and 24%. The global field size map and the crowdsourced data set are openly available and can be used for integrated assessment modeling, comparative studies of agricultural dynamics across different contexts, for training and validation of remote sensing field size delineation, and potential contributions to the Sustainable Development Goal of Ending hunger, achieve food security and improved nutrition and promote sustainable agriculture.     

Can morphing methods predict intermediate structures?

Movement is crucial to the biological function of many proteins, yet crystallographic structures of proteins can give us only a static snapshot. The protein dynamics that are important to biological function often happen on a timescale that is unattainable through detailed simulation methods such as molecular dynamics as they often involve crossing high-energy barriers. To address this coarse-grained motion, several methods have been implemented as web servers in which a set of coordinates is usually linearly interpolated from an initial crystallographic structure to a final crystallographic structure. We present a new morphing method that does not extrapolate linearly and can therefore go around high-energy barriers and which can produce different trajectories between the same two starting points. In this work, we evaluate our method and other established coarse-grained methods according to an objective measure: how close a coarse-grained dynamics method comes to a crystallographically determined intermediate structure when calculating a trajectory between the initial and final crystal protein structure. We test this with a set of five proteins with at least three crystallographically determined on-pathway high-resolution intermediate structures from the Protein Data Bank. For simple hinging motions involving a small conformational change, segmentation of the protein into two rigid sections outperforms other more computationally involved methods. However, large-scale conformational change is best addressed using a nonlinear approach and we suggest that there is merit in further developing such methods.     

Gene dosage of CuZnSOD and Down''s syndrome: diminished prostaglandin synthesis in human trisomy 21, transfected cells and transgenic mice.

Patients with Down's syndrome (DS) exhibit elevated activity of copper zinc superoxide dismutase (CuZnSOD) caused by the trisomy 21 state. To investigate the possible involvement of CuZnSOD gene dosage in perturbation of prostaglandin biosynthesis we analyzed transfected cells and transgenic mice that express elevated levels of human CuZnSOD. It was found that the synthesis of prostaglandin E2 (PGE2) was diminished in transfected PC12-CuZnSOD cells as well as in fibroblasts from DS patients. Primary cells derived from transgenic CuZnSOD mice showed similar reduction. Impaired biosynthesis of prostaglandins was not confined to cells grown in culture since secretion of PGE2 and PGD2 by kidney and cerebellum of transgenic CuZnSOD was significantly lower than in non-transgenic littermate mice. These findings strongly suggest that overexpression of the CuZnSOD gene induces a demotion in PGE2 and PGD2 formation and establish a connection between alteration of prostaglandin biosynthesis in trisomy 21 cells and gene dosage of CuZnSOD.     

Uveal melanoma driver mutations in GNAQ/11 yield numerous changes in melanocyte biology

Uveal melanoma (UM) is the most common primary intraocular cancer and has a high incidence of metastasis, which lacks any effective treatment. Here, we present zebrafish models of UM, which are driven by melanocyte‐specific expression of activating GNAQ or GNA11 alleles, GNAQ/11^Q209L, the predominant initiating mutations for human UM. When combined with mutant tp53, GNAQ/11^Q209L transgenics develop various melanocytic tumors, including UM, with near complete penetrance. These tumors display nuclear YAP localization and thus phenocopy human UM. We show that GNAQ/11^Q209L expression induces profound melanocyte defects independent of tp53 mutation, which are apparent within 3 days of development. First, increases in melanocyte number, melanin content, and subcellular melanin distribution result in hyperpigmentation. Additionally, altered melanocyte migration, survival properties, and evasion of normal boundary cues lead to aberrant melanocyte localization and stripe patterning. Collectively, these data show that GNAQ/11^Q209L is sufficient to induce numerous protumorigenic changes within melanocytes.     

Canine Hereditary Ataxia in Old English Sheepdogs and Gordon Setters Is Associated with a Defect in the Autophagy Gene Encoding RAB24

Old English Sheepdogs and Gordon Setters suffer from a juvenile onset, autosomal recessive form of canine hereditary ataxia primarily affecting the Purkinje neuron of the cerebellar cortex. The clinical and histological characteristics are analogous to hereditary ataxias in humans. Linkage and genome-wide association studies on a cohort of related Old English Sheepdogs identified a region on CFA4 strongly associated with the disease phenotype. Targeted sequence capture and next generation sequencing of the region identified an A to C single nucleotide polymorphism (SNP) located at position 113 in exon 1 of an autophagy gene, RAB24, that segregated with the phenotype. Genotyping of six additional breeds of dogs affected with hereditary ataxia identified the same polymorphism in affected Gordon Setters that segregated perfectly with phenotype. The other breeds tested did not have the polymorphism. Genome-wide SNP genotyping of Gordon Setters identified a 1.9 MB region with an identical haplotype to affected Old English Sheepdogs. Histopathology, immunohistochemistry and ultrastructural evaluation of the brains of affected dogs from both breeds identified dramatic Purkinje neuron loss with axonal spheroids, accumulation of autophagosomes, ubiquitin positive inclusions and a diffuse increase in cytoplasmic neuronal ubiquitin staining. These findings recapitulate the changes reported in mice with induced neuron-specific autophagy defects. Taken together, our results suggest that a defect in RAB24, a gene associated with autophagy, is highly associated with and may contribute to canine hereditary ataxia in Old English Sheepdogs and Gordon Setters. This finding suggests that detailed investigation of autophagy pathways should be undertaken in human hereditary ataxia.     

Synthesis,characterization and antineoplastic activity of bis-aziridinyl dimeric naphthoquinone – A novel class of compounds with potent activity against acute myeloid leukemia cells

The synthesis, characterization and antileukemic activity of rationally designed amino dimeric naphthoquinone (BiQ) possessing aziridine as alkylating moiety is described. Bis-aziridinyl BiQ decreased proliferation of acute myeloid leukemia (AML) cell lines and primary cells from patients, and exhibited potent (nanomolar) inhibition of colony formation and overall cell survival in AML cells. Effective production of reactive oxygen species (ROS) and double stranded DNA breaks (DSB) induced by bis-aziridinyl BiQ is reported. Bis-dimethylamine BiQ, as the isostere of bis-aziridinyl BiQ but without the alkylating moiety did not show as potent anti-AML activity. Systemic administration of bis-aziridinyl BiQ was well tolerated in NSG mice.     

In situ copolyesters containing poly(L-lactide) and poly(hydroxyalkanoate) units

In situ copolyesters containing polylactide (PLA) and polyhydroxyalkanoate (PHA) segments were obtained via ring-opening polymerization of L-lactide using PHA as a macroinitiator with stannous octoate as catalyst. Incorporation of PHA (20 wt %) into PLA affords a novel copolymer with Mn values ranging from 25 to 50 KDa and low polydispersities of 1.8-2.3. DSC analysis of the copolymer indicates well-defined crystallization and melting transitions different from the homopolymers and corresponding blend. The polymers were characterized by FT-IR, GPC, DSC, optical microscopy, NMR, and TGA. The results show successful reactivity of PHA as a macroinitiator for the ring-opening polymerization of lactide.     

Vesicular stomatitis virus as an oncolytic agent against pancreatic ductal adenocarcinoma

Vesicular stomatitis virus (VSV) is a promising oncolytic agent against a variety of cancers. However, it has never been tested in any pancreatic cancer model. Pancreatic ductal adenocarcinoma (PDA) is the most common and aggressive form of pancreatic cancer. In this study, the oncolytic potentials of several VSV variants were analyzed in a panel of 13 clinically relevant human PDA cell lines and compared to conditionally replicative adenoviruses (CRAds), Sendai virus and respiratory syncytial virus. VSV variants showed oncolytic abilities superior to those of other viruses, and some cell lines that exhibited resistance to other viruses were successfully killed by VSV. However, PDA cells were highly heterogeneous in their susceptibility to virus-induced oncolysis, and several cell lines were resistant to all tested viruses. Resistant cells showed low levels of very early VSV RNA synthesis, indicating possible defects at initial stages of infection. In addition, unlike permissive PDA cell lines, most of the resistant cell lines were able to both produce and respond to interferon, suggesting that intact type I interferon responses contributed to their resistance phenotype. Four cell lines that varied in their permissiveness to VSV-ΔM51 and CRAd dl1520 were tested in mice, and the in vivo results closely mimicked those in vitro. While our results demonstrate that VSV is a promising oncolytic agent against PDA, further studies are needed to better understand the molecular mechanisms of resistance of some PDAs to oncolytic virotherapy.     

NDNF interneurons in layer 1 gain-modulate whole cortical columns according to an animal’s behavioral state

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