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The European Green Deal proposes the reuse of any kind of waste that can be safely repurposed; sludge may also be reused as a biosolid. The present research aims to evaluate a biosolid made from sludge, originating from a wastewater treatment plant (WWTP) for its effect on tomato growth and enhancement of tolerance against the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici (Forl). Peat and/or two soil types were amended with biosolid (0, 80 and 160 tons/ha) to form substrates on which tomato plants were grown either under controlled conditions, in a growth chamber or outdoors, in a net protected area. Plant growth and disease parameters were recorded 4–7 weeks after application of Forl inoculum in the substrates. Results showed that biosolid addition enhanced plant growth and increased tolerance against tomato foot and root rot caused by Forl. Specifically, fresh weight, root weight, stem height and leaf number of tomato plants in Forl-inoculated soils that had received biosolid, were increased when compared to Forl-inoculated control soils. Forl resulted in higher disease severity on plants grown outdoors in biosolid plus clay soil than in biosolid plus sandy soil, while the opposite occurred under controlled conditions where higher disease index was recorded on plants grown in peat plus sandy soil than in peat plus clay soil. These findings strongly suggest that this biosolid may act as an organic fertilizer and possible stimulant of tomato tolerance against Forl, and the effect is soil type dependent. Therefore, this biosolid should be considered for its possible use in agriculture according to the principles of circular economy and waste minimization. • Biosolid addition enhances tomato growth in different substrates and conditions. • Disease is less severe on plants grown on soil mixed with biosolid. • In the outdoors trial biosolid renders higher disease tolerance in sandy soil. • Fusarium disease is less aggressive on tomato plants grown on biosolid mix. • Tolerance to disease is higher in sandy soil+biosolid than in clay soil+biosolid.

EIF2AK1 and EIF2AK2 encode members of the eukaryotic translation initiation factor 2 alpha kinase (EIF2AK) family that inhibits protein synthesis in response to physiologic stress conditions. EIF2AK2 is also involved in innate immune response and the regulation of signal transduction, apoptosis, cell proliferation, and differentiation. Despite these findings, human disorders associated with deleterious variants in EIF2AK1 and EIF2AK2 have not been reported. Here, we describe the identification of nine unrelated individuals with heterozygous de novo missense variants in EIF2AK1 (1/9) or EIF2AK2 (8/9). Features seen in these nine individuals include white matter alterations (9/9), developmental delay (9/9), impaired language (9/9), cognitive impairment (8/9), ataxia (6/9), dysarthria in probands with verbal ability (6/9), hypotonia (7/9), hypertonia (6/9), and involuntary movements (3/9). Individuals with EIF2AK2 variants also exhibit neurological regression in the setting of febrile illness or infection. We use mammalian cell lines and proband-derived fibroblasts to further confirm the pathogenicity of variants in these genes and found reduced kinase activity. EIF2AKs phosphorylate eukaryotic translation initiation factor 2 subunit 1 (EIF2S1, also known as EIF2α), which then inhibits EIF2B activity. Deleterious variants in genes encoding EIF2B proteins cause childhood ataxia with central nervous system hypomyelination/vanishing white matter (CACH/VWM), a leukodystrophy characterized by neurologic regression in the setting of febrile illness and other stressors. Our findings indicate that EIF2AK2 missense variants cause a neurodevelopmental syndrome that may share phenotypic and pathogenic mechanisms with CACH/VWM.

International audience; A small family composed of BRI1 KINASE INHIBITOR1 (BKI1) and MEMBRANE ASSOCIATED KINASE REGULATORs (MAKRs) has recently captured the attention of plant biologists, due to their involvement in developmental processes downstream of hormones and Receptor-Like Kinases (RLK) signaling. BKI1/MAKRs are intrinsically disordered proteins (so-called unstructured proteins) and as such lack specific domains. Instead, they are defined by the presence of two conserved linear motifs involved in the interaction with lipids and proteins, respectively. Here, we first relate the discovery of the MAKR gene family. Then, we review the individual function of characterized family members and discuss their shared and specific modes of action. Finally, we explore and summarize the structural, comparative, and functional genomics data available on this gene family. Together, this review aims at building a comprehensive reference about BKI1/MAKR protein function in plants.

Pseudomonas chlororaphis ToZa7 is a promising biocontrol agent possessing valuable characteristics and reducing disease severity caused by Fusarium oxysporum f. sp. radicis-lycopersici (Forl) in tomato. In this study, the strain's ability to induce three pathogenesis-related (PR) genes (PR-1a, GLUA, and CHI3) in tomato, was studied using quantitative reverse transcription PCR. The genes PR-1a and GLUA were up-regulated after 120 h exposure to P. chororaphis ToZa7 (15.22- and 13.11-fold, respectively), as compared to the untreated control, without challenge inoculation by the pathogen. To study the effects of individual or combined application of P. chororaphis ToZa7 and the compatible biocontrol fungus Clonostachys rosea IK726, challenged with the pathogen, the expression patterns of the above three PR genes were monitored, in tomato roots. Expression of PR1-a was noteworthy, especially 48 h after challenge inoculation, when C. rosea IK726 alone or in combination with P. chororaphis, ToZa7 was pre-inoculated on tomato roots (38.53-fold and 53.74-fold, respectively). Expression of PR1-a, 72 h after challenge inoculation, was the highest in P. chororaphis ToZa7, among biocontrol treatments. Expression of CHI3 was much lower, while up-regulation of GLUA was overall not observed. Confocal laser scanning microscopy of intact tomato roots and bacterial counts of superficially disinfected roots revealed, for the first time, that P. chororaphis ToZa7 colonizes the exterior as well as the internal tissues.

Optimal lysosome function requires maintenance of an acidic pH maintained by proton pumps in combination with a counterion transporter such as the Cl(−)/H(+) exchanger, CLCN7 (ClC-7), encoded by CLCN7. The role of ClC-7 in maintaining lysosomal pH has been controversial. In this paper, we performed clinical and genetic evaluations of two children of different ethnicities. Both children had delayed myelination and development, organomegaly, and hypopigmentation, but neither had osteopetrosis. Whole-exome and -genome sequencing revealed a de novo c.2144A>G variant in CLCN7 in both affected children. This p.Tyr715Cys variant, located in the C-terminal domain of ClC-7, resulted in increased outward currents when it was heterologously expressed in Xenopus oocytes. Fibroblasts from probands displayed a lysosomal pH approximately 0.2 units lower than that of control cells, and treatment with chloroquine normalized the pH. Primary fibroblasts from both probands also exhibited markedly enlarged intracellular vacuoles; this finding was recapitulated by the overexpression of human p.Tyr715Cys CLCN7 in control fibroblasts, reflecting the dominant, gain-of-function nature of the variant. A mouse harboring the knock-in Clcn7 variant exhibited hypopigmentation, hepatomegaly resulting from abnormal storage, and enlarged vacuoles in cultured fibroblasts. Our results show that p.Tyr715Cys is a gain-of-function CLCN7 variant associated with developmental delay, organomegaly, and hypopigmentation resulting from lysosomal hyperacidity, abnormal storage, and enlarged intracellular vacuoles. Our data supports the hypothesis that the ClC-7 antiporter plays a critical role in maintaining lysosomal pH.

Contains fulltext : 202800.pdf (Publisher’s version ) (Open Access) SMARCC2 (BAF170) is one of the invariable core subunits of the ATP-dependent chromatin remodeling BAF (BRG1-associated factor) complex and plays a crucial role in embryogenesis and corticogenesis. Pathogenic variants in genes encoding other components of the BAF complex have been associated with intellectual disability syndromes. Despite its significant biological role, variants in SMARCC2 have not been directly associated with human disease previously. Using whole-exome sequencing and a web-based gene-matching program, we identified 15 individuals with variable degrees of neurodevelopmental delay and growth retardation harboring one of 13 heterozygous variants in SMARCC2, most of them novel and proven de novo. The clinical presentation overlaps with intellectual disability syndromes associated with other BAF subunits, such as Coffin-Siris and Nicolaides-Baraitser syndromes and includes prominent speech impairment, hypotonia, feeding difficulties, behavioral abnormalities, and dysmorphic features such as hypertrichosis, thick eyebrows, thin upper lip vermilion, and upturned nose. Nine out of the fifteen individuals harbor variants in the highly conserved SMARCC2 DNA-interacting domains (SANT and SWIRM) and present with a more severe phenotype. Two of these individuals present cardiac abnormalities. Transcriptomic analysis of fibroblasts from affected individuals highlights a group of differentially expressed genes with possible roles in regulation of neuronal development and function, namely H19, SCRG1, RELN, and CACNB4. Our findings suggest a novel SMARCC2-related syndrome that overlaps with neurodevelopmental disorders associated with variants in BAF-complex subunits.

In 2018, decline and necrosis were observed on olive tree cuttings (Olea europaea) in a nursery in northern Greece. The pathogen was identified as Alternaria alternata on the basis of conidia morphology and sequence analysis of the internal transcribed region (ITS) and part of the elongation factor 1a (EF-1a). The endopolygalacturonase (endoPG) and the Alternaria major allergen (Alta1) genes were also amplified. A pathogenicity test was performed and Koch's postulates were confirmed by re-isolation of the fungus A. alternata from artificially inoculated cuttings. This is the first report of A. alternata as the cause of decline and necrosis on olive tree cuttings in Greece.

Heterologous production of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) has been of interest for over 30 years, but implementation is sometimes difficult and can limit the scope of research. With the constant development of tools to improve recombinant protein production in Escherichia coli, we propose a method that takes characteristics of PhaZs from different bacterial strains into account. Recombinant His‐tagged versions of PhaZs (rPhaZ) from Comamonas testosteroni 31A, Cupriavidus sp. T1, Marinobacter algicola DG893, Pseudomonas stutzeri, and Ralstonia sp. were successfully produced with varying expression, solubility, and purity levels. PhaZs from C. testosteroni and P. stutzeri were more amenable to heterologous expression in all aspects; however, using the E. coli Rosetta‐gami B(DE3) expression strain and establishing optimal conditions for expression and purification (variation of IPTG concentration and use of size exclusion columns) helped circumvent low expression and purity for the other PhaZs. Degradation activity of the rPhaZs was compared using a simple PHB plate‐based method, adapted to test for various pH and temperatures. rPhaZ from M. algicola presented the highest activity at 15°C, and rPhaZs from Cupriavidus sp. T1 and Ralstonia sp. had the highest activity at pH 5.4. The methods proposed herein can be used to test the production of soluble recombinant PhaZs and to perform preliminary evaluation for applications that require PHB degradation., A rapid, efficient approach for the expression of active recombinant extracellular polyhydroxybutyrate (PHB) depolymerases originating from various bacterial strains. It enables the characterization of new PHB depolymerases and the direct comparison of activity between enzymes and conditions; as an example, the activity of five PHB depolymerases is evaluated and compared at different pH and temperatures. This approach can accelerate the investigation and development of applications based on the degradation of biopolymers.

Contains fulltext : 218305.pdf (Publisher’s version ) (Closed access) BACKGROUND: The X-chromosome gene USP9X encodes a deubiquitylating enzyme that has been associated with neurodevelopmental disorders primarily in female subjects. USP9X escapes X inactivation, and in female subjects de novo heterozygous copy number loss or truncating mutations cause haploinsufficiency culminating in a recognizable syndrome with intellectual disability and signature brain and congenital abnormalities. In contrast, the involvement of USP9X in male neurodevelopmental disorders remains tentative. METHODS: We used clinically recommended guidelines to collect and interrogate the pathogenicity of 44 USP9X variants associated with neurodevelopmental disorders in males. Functional studies in patient-derived cell lines and mice were used to determine mechanisms of pathology. RESULTS: Twelve missense variants showed strong evidence of pathogenicity. We define a characteristic phenotype of the central nervous system (white matter disturbances, thin corpus callosum, and widened ventricles); global delay with significant alteration of speech, language, and behavior; hypotonia; joint hypermobility; visual system defects; and other common congenital and dysmorphic features. Comparison of in silico and phenotypical features align additional variants of unknown significance with likely pathogenicity. In support of partial loss-of-function mechanisms, using patient-derived cell lines, we show loss of only specific USP9X substrates that regulate neurodevelopmental signaling pathways and a united defect in transforming growth factor beta signaling. In addition, we find correlates of the male phenotype in Usp9x brain-specific knockout mice, and further resolve loss of hippocampal-dependent learning and memory. CONCLUSIONS: Our data demonstrate the involvement of USP9X variants in a distinctive neurodevelopmental and behavioral syndrome in male subjects and identify plausible mechanisms of pathogenesis centered on disrupted transforming growth factor beta signaling and hippocampal function.

Purple spot caused by the fungus Stemphylium vesicarium is a disease that causes heavy damage in asparagus crops. It infects the leaves and stem and results in degradation of the quality in the harvested asparagus shoots, lowering their commercial value. Precautionary spraying with fungicides is practiced to prevent its appearance, posing a threat to consumers and the environment. The aim of this work was to evaluate the ability of visible and near-infrared field spectroscopy (UNISPEC-DC) to identify the appearance of the purple spot symptoms at the level of asparagus plants and the ability of very high resolution (Pleiades-1A) and wide area (Landsat 8) satellite images to map fungal distribution within asparagus fields and across the river plain, respectively. Field measurements were used as reference to select the optimum infection identification models. Results indicate that field spectroscopy and wide area remote sensing can be used to create sufficiently accurate quantification models of disease severity in asparagus plants (R2 = 0.83 and RMSE = 9.68%) or across asparagus fields (R2 = 0.85 and RMSE = 8.26%). The accuracy drops when using satellite data to quantify infection within field (R2 = 0.34 and RMSE = 13.13%). The contributing wavelengths, bands and indices can be utilized to provide timely information to farmers and agronomists in order to support precision plant protection applications.

The susceptible to Verticillium dahliae Kleb. tomato (Solanum lycopersicum L.) cv. ‘Early Pack’ was evaluated in 2004 and 2005, for tolerance to verticillium wilt, after grafted on the commercial rootstocks ‘He-man’, ‘Eldorado’ (48-S-548), ‘Beaufort’, ‘Primavera’, ‘Nova’, ‘Packmore’ and ‘Vigomax’. Self-grafted cv. ‘Early Pack’ was used as a control. The grafted plants, after artificial inoculation with a virulent isolate of V. dahliae during transplanting, were grown in a greenhouse and evaluated for disease tolerance, plant growth, yield, and fruit quality characteristics. In artificially inoculated plants, grafting on all rootstocks reduced disease severity significantly, in comparison to self-grafted cv. ‘Early Pack’, but effectiveness was repeated in both years only in ‘Beaufort’. In addition, in 2004, under high disease pressure, grafting on ‘Vigomax’, ‘Beaufort’, ‘Nova’, and ‘He-man’ increased root weight, while grafting on the first three increased also above ground fresh weight of tomato. In 2005, grafting on ‘Nova’, ‘Beaufort’ and ‘Vigomax’ increased total marketable yield, while ‘Beaufort’ increased early marketable yield also. Grafting on ‘Beaufort’, ‘Nova’, and ‘Vigomax’ decreased total soluble solids (oBrix) and dry matter of tomato fruits, in both years, as compared to self-grafted cv. ‘Early Pack’, however, values recorded are acceptable for tomato. None of the rootstocks had any significant effect on flesh firmness, pericarp thickness, number of locules, equatorial diameter, and polar diameter. These results strongly support that grafting on commercial rootstocks increases tomato tolerance to verticillium wilt, plant growth and productivity, without affecting any fruit quality characteristics.

Identification of a smut fungus with no visual signs on leaves using non-destructive spectroscopy.The method was applied in-situ on live plants using low cost tools.Three innovative classifiers were tested to evaluate their performance.An independent dataset was used for the validation of the results. Microbotryum silybum is a smut fungus infecting Silybum marianum (milk thistle) weed and is currently investigated as a means for its biological control. Although the fungus' detection is important for the evaluation of biological control effectiveness and decision making, in-situ diagnosis is not always possible. The presented approach describes the identification of systemically infected S. marianum plants by using field spectroscopy and hierarchical self-organizing maps. An experimental field that contained both healthy and artificially inoculated S. marianum plants was used to acquire leaf spectra using a handheld visible and near-infrared spectrometer (3101100nm). Three supervised hierarchical self-organizing models, including Supervised Kohonen Network (SKN), Counter propagation Artificial Neural Network (CP-ANN) and XY-Fusion network (XY-F) were utilized for the identification of the systemically infected S. marianum plants. As input features to the classifiers, the pre-processed spectral signatures were used. The pre-processing of the spectra included normalisation, second derivative and principal component extraction. The systemically infected S. marianum identification rates using SKN and CP-ANN reached high overall accuracy (up to 90%) and even higher using the XY-F (95.16%). The results demonstrate the potential for a high accuracy identification of systemically infected S. marianum plants during vegetative growth, with the assistance of hierarchical self-organizing maps.

Glutamate is the major donor of nitrogen for anabolic reactions. The Gram-positive soil bacterium Bacillus subtilis either utilizes exogenously provided glutamate or synthesizes it using the gltAB-encoded glutamate synthase (GOGAT). In the absence of glutamate, the transcription factor GltC activates expression of the GOGAT genes for glutamate production. Consequently, a gltC mutant strain is auxotrophic for glutamate. Using a genetic selection and screening system, we could isolate and differentiate between gltC suppressor mutants in one step. All mutants had acquired the ability to synthesize glutamate, independent of GltC. We identified (i) gain-of-function mutations in the gltR gene, encoding the transcription factor GltR, (ii) mutations in the promoter of the gltAB operon and (iii) massive amplification of the genomic locus containing the gltAB operon. The mutants belonging to the first two classes constitutively expressed the gltAB genes and produced sufficient glutamate for growth. By contrast, mutants that belong to the third class appeared most frequently and solved glutamate limitation by increasing the copy number of the poorly expressed gltAB genes. Thus, glutamate auxotrophy of a B. subtilis gltC mutant can be relieved in multiple ways. Moreover, recombination-dependent amplification of the gltAB genes is the predominant mutational event indicating a hierarchy of mutations.

Whole-exome sequencing (WES) has increasingly enabled new pathogenic gene variant identification for undiagnosed neurodevelopmental disorders and provided insights into both gene function and disease biology. Here, we describe seven children with a neurodevelopmental disorder characterized by microcephaly, profound developmental delays and/or intellectual disability, cataracts, severe epilepsy including infantile spasms, irritability, failure to thrive, and stereotypic hand movements. Brain imaging in these individuals reveals delay in myelination and cerebral atrophy. We observe an identical recurrent de novo heterozygous c.892C>T (p.Arg298Trp) variant in the nucleus accumbens associated 1 ( NACC1 ) gene in seven affected individuals. One of the seven individuals is mosaic for this variant. NACC1 encodes a transcriptional repressor implicated in gene expression and has not previously been associated with germline disorders. The probability of finding the same missense NACC1 variant by chance in 7 out of 17,228 individuals who underwent WES for diagnoses of neurodevelopmental phenotypes is extremely small and achieves genome-wide significance (p = 1.25 × 10 −14 ). Selective constraint against missense variants in NACC1 makes this excess of an identical missense variant in all seven individuals more remarkable. Our findings are consistent with a germline recurrent mutational hotspot associated with an allele-specific neurodevelopmental phenotype in NACC1 .

Early B cell factor 3 (EBF3) is a member of the highly evolutionarily conserved Collier/Olf/EBF (COE) family of transcription factors. Prior studies on invertebrate and vertebrate animals have shown that EBF3 homologs are essential for survival and that loss-of-function mutations are associated with a range of nervous system developmental defects, including perturbation of neuronal development and migration. Interestingly, aristaless-related homeobox (ARX), a homeobox-containing transcription factor critical for the regulation of nervous system development, transcriptionally represses EBF3 expression. However, human neurodevelopmental disorders related to EBF3 have not been reported. Here, we describe three individuals who are affected by global developmental delay, intellectual disability, and expressive speech disorder and carry de novo variants in EBF3. Associated features seen in these individuals include congenital hypotonia, structural CNS malformations, ataxia, and genitourinary abnormalities. The de novo variants affect a single conserved residue in a zinc finger motif crucial for DNA binding and are deleterious in a fly model. Our findings indicate that mutations in EBF3 cause a genetic neurodevelopmental syndrome and suggest that loss of EBF3 function might mediate a subset of neurologic phenotypes shared by ARX-related disorders, including intellectual disability, abnormal genitalia, and structural CNS malformations.

International audience; Cadherins constitute a family of transmembrane proteins that mediate calcium-dependent cell-cell adhesion. The extracellular domain of cadherins consists of extracellular cadherin (EC) domains, separated by calcium binding sites. The EC interacts with other cadherin molecules in cis and in trans to mechanically hold apposing cell surfaces together. CDH2 encodes N-cadherin, whose essential roles in neural development include neuronal migration and axon pathfinding. However, CDH2 has not yet been linked to a Mendelian neurodevelopmental disorder. Here, we report de novo heterozygous pathogenic variants (seven missense, two frameshift) in CDH2 in nine individuals with a syndromic neurodevelopmental disorder characterized by global developmental delay and/or intellectual disability, variable axon pathfinding defects (corpus callosum agenesis or hypoplasia, mirror movements, Duane anomaly), and ocular, cardiac, and genital anomalies. All seven missense variants (c.1057G>A [p.Asp353Asn]; c.1789G>A [p.Asp597Asn]; c.1789G>T [p.Asp597Tyr]; c.1802A>C [p.Asn601Thr]; c.1839C>G [p.Cys613Trp]; c.1880A>G [p.Asp627Gly]; c.2027A>G [p.Tyr676Cys]) result in substitution of highly conserved residues, and six of seven cluster within EC domains 4 and 5. Four of the substitutions affect the calcium-binding site in the EC4-EC5 interdomain. We show that cells expressing these variants in the EC4-EC5 domains have a defect in cell-cell adhesion; this defect includes impaired binding in trans with N-cadherin-WT expressed on apposing cells. The two frameshift variants (c.2563_2564delCT [p.Leu855Valfs∗4]; c.2564_2567dupTGTT [p.Leu856Phefs∗5]) are predicted to lead to a truncated cytoplasmic domain. Our study demonstrates that de novo heterozygous variants in CDH2 impair the adhesive activity of N-cadherin, resulting in a multisystemic developmental disorder, that could be named ACOG syndrome (agenesis of corpus callosum, axon pathfinding, cardiac, ocular, and genital defects).

Carbocyclic nucleosides have long played a role in antiviral, antiparasitic, and antibacterial therapies. Recent results from our laboratories from two structurally related scaffolds have shown promising activity against both Mycobacterium tuberculosis and several parasitic strains. As a result, a small structure activity relationship study was designed to further probe their activity and potential. Their synthesis and the results of the subsequent biological activity are reported herein.

WD40 repeat-containing proteins form a large family of proteins present in all eukaryotes. Here, we identified five pediatric probands with de novo variants in WDR37, which encodes a member of the WD40 repeat protein family. Two probands shared one variant and the others have variants in nearby amino acids outside the WD40 repeats. The probands exhibited shared phenotypes of epilepsy, colobomas, facial dysmorphology reminiscent of CHARGE syndrome, developmental delay and intellectual disability, and cerebellar hypoplasia. The WDR37 protein is highly conserved in vertebrate and invertebrate model organisms and is currently not associated with a human disease. We generated a null allele of the single Drosophila ortholog to gain functional insights and replaced the coding region of the fly gene CG12333/wdr37 with GAL4. These flies are homozygous viable but display severe bang sensitivity, a phenotype associated with seizures in flies. Additionally, the mutant flies fall when climbing the walls of the vials, suggesting a defect in grip strength, and repeat the cycle of climbing and falling. Similar to wall clinging defect, mutant males often lose grip of the female abdomen during copulation. These phenotypes are rescued by using the GAL4 in the CG12333/wdr37 locus to drive the UAS-human reference WDR37 cDNA. The two variants found in three human subjects failed to rescue these phenotypes, suggesting that these alleles severely affect the function of this protein. Taken together, our data suggest that variants in WDR37 underlie a novel syndromic neurological disorder.

Heterologous production of extracellular polyhydroxybutyrate (PHB) depolymerases (PhaZs) has been of interest for over 30 years, but implementation is sometimes difficult and can limit the scope of research. With the constant development of tools to improve recombinant protein production inEscherichia coli, we propose a method that takes characteristics of PhaZs from different bacterial strains into account. Recombinant His-tagged versions of PhaZs (rPhaZ) fromComamonas testosteroni31A,Cupriavidussp.,Marinobacter algicolaDG893,Pseudomonas stutzeri, andRalstoniasp. were successfully produced with varying expression, solubility, and purity levels. PhaZs fromC. testosteroniandP. stutzeriwere more amenable to heterologous expression in all aspects; however, strategies were developed to circumvent low expression and purity for the other PhaZs. Degradation activity of the rPhaZs was compared using a simple PHB plate-based method, adapted to test for various pH and temperatures. rPhaZ fromM. algicolapresented the highest activity at 15 °C, and rPhaZs fromCupriavidussp. andRalstoniasp. had the highest activity at pH 5.4. The methods proposed herein can be used to test the production of soluble recombinant PhaZs, and to perform preliminary evaluation for applications that require PHB degradation.

OBJECTIVE: To characterize the phenotypic spectrum associated with GNAO1 variants and establish genotype-protein structure-phenotype relationships. METHODS: We evaluated the phenotypes of 14 patients with GNAO1 variants, analyzed their variants for potential pathogenicity, and mapped them, along with those in the literature, on a three-dimensional structural protein model. RESULTS: The 14 patients in our cohort, including one sibling pair, had 13 distinct, heterozygous GNAO1 variants classified as pathogenic or likely pathogenic. We attributed the same variant in two siblings to parental mosaicism. Patients initially presented with seizures beginning in the first 3 months of life (8/14), developmental delay (4/14), hypotonia (1/14), or movement disorder (1/14). All patients had hypotonia and developmental delay ranging from mild to severe. Nine had epilepsy, and nine had movement disorders, including dystonia, ataxia, chorea, and dyskinesia. The 13 GNAO1 variants in our patients are predicted to result in amino acid substitutions or deletions in the GNAO1 guanosine triphosphate (GTP)-binding region, analogous to those in previous publications. Patients with variants affecting amino acids 207-221 had only movement disorder and hypotonia. Patients with variants affecting the C-terminal region had the mildest phenotypes. SIGNIFICANCE: GNAO1 encephalopathy most frequently presents with seizures beginning in the first 3 months of life. Concurrent movement disorders are also a prominent feature in the spectrum of GNAO1 encephalopathy. All variants affected the GTP-binding domain of GNAO1, highlighting the importance of this region for G-protein signaling and neurodevelopment. ispartof: EPILEPSIA vol:60 issue:3 pages:406-418 ispartof: location:United States status: published

The ubiquitin system regulates diverse biological processes, many involved in cancer pathogenesis, by altering the ubiquitination state of protein substrates. This is accomplished by ubiquitin ligases and deubiquitinases (DUBs), which respectively add or remove ubiquitin from substrates to alter their stability, activity, localization and interactions. While lack of catalytic activity makes therapeutic targeting of ubiquitin ligases difficult, DUB inhibitors represent an active area of research and the identification of cancer-associated DUBs may lead to the development of novel therapeutics. A growing body of literature demonstrates that the DUB Otubain 1 (OTUB1) regulates many cancer-associated signaling pathways including MAPK, ERa, epithelial-mesenchymal transition (EMT), RHOa, mTORC1, FOXM1 and P53 to promote tumor cell survival, proliferation, invasiveness and therapeutic resistance. In addition, clinical studies have associated elevated OTUB1 expression with high grade, invasiveness and metastasis in several tumor types including lung, breast, ovarian, glioma, colon and gastric. Interestingly, in addition to catalytic DUB activity, OTUB1 displays a catalytic-independent, non-canonical activity where it inhibits the transfer of ubiquitin onto protein substrates by sequestration of E2 ubiquitin-conjugating enzymes. The aim of this review is to describe the canonical and non-canonical activities of OTUB1, summarize roles for OTUB1 in cancer-associated pathways and discuss its potential therapeutic targeting.

Silybum marianum L. Gaertn weed has the tendency to grow in patches. In order to perform site-specific weed management, determining the spatial distribution of weeds is important for its eradication. Remote sensing has been used to perform species discrimination and it offers promising techniques for operational weed mapping. In the present study, the feasibility of high-resolution imaging for S. marianum detection and mapping is reported. A multispectral camera green–red–near-infrared mounted on a fixed wing unmanned aerial vehicle UAV was used for the acquisition of high-resolution images with pixel size of 0.1 m. The maximum likelihood ML classifier was used to classify the S. marianum among other weed species present in a field, with Avena sterilisL. being predominant. As input to the classifier, the three spectral bands and the texture were used. The scale of the mapping was varied by degrading the image resolution to evaluate classification performance, with 1 m resolution providing the highest classification accuracy. The classification rates obtained using ML reached an overall accuracy of 87.04% with a K-hat statistic of 74%. The results prove the feasibility of operational S. marianum mapping using UAV and multispectral imaging.

Purpose Radiation-induced pulmonary fibrosis (RIPF) is a late toxicity of therapeutic radiation. Signaling of the mammalian target of rapamycin drives several processes implicated in RIPF, including inflammatory cytokine production, fibroblast proliferation, and epithelial senescence. We sought to determine if mammalian target of rapamycin inhibition with rapamycin would mitigate RIPF. Methods and Materials C57BL/6NCr mice received a diet formulated with rapamycin (14 mg/kg food) or a control diet 2 days before and continuing for 16 weeks after exposure to 5 daily fractions of 6 Gy of thoracic irradiation. Fibrosis was assessed with Masson trichrome staining and hydroxyproline assay. Cytokine expression was evaluated by quantitative real-time polymerase chain reaction. Senescence was assessed by staining for β-galactosidase activity. Results Administration of rapamycin extended the median survival of irradiated mice compared with the control diet from 116 days to 156 days ( P =.006, log-rank test). Treatment with rapamycin reduced hydroxyproline content compared with the control diet (irradiation plus vehicle, 45.9 ± 11.8 μg per lung; irradiation plus rapamycin, 21.4 ± 6.0 μg per lung; P =.001) and reduced visible fibrotic foci. Rapamycin treatment attenuated interleukin 1β and transforming growth factor β induction in irradiated lungs compared with the control diet. Type II pneumocyte senescence after irradiation was reduced with rapamycin treatment at 16 weeks (3-fold reduction at 16 weeks, P Conclusions Rapamycin protected against RIPF in a murine model. Rapamycin treatment reduced inflammatory cytokine expression, extracellular matrix production, and senescence in type II pneumocytes.

Comparison of tumors from The Cancer Genome Atlas (TCGA) reveals that head and neck squamous cell carcinomas (HNSCC) harbor the most frequent genomic amplifications of Fas-associated death domain (FADD), with or without Baculovirus inhibitor of apoptosis repeat containing BIRC2 (cIAP1), affecting about 30% of patients in association with worse prognosis. Here, we identified HNSCC cell lines harboring FADD/BIRC2 amplifications and overexpression by exome sequencing, RT-PCR, and Western blotting. In vitro, FADD or BIRC2 siRNA knockdown inhibited HNSCC displaying amplification and increased expression of these genes, supporting their functional importance in promoting proliferation. Birinapant, a novel SMAC mimetic, sensitized multiple HNSCC lines to cell death by agonists TNFα or TRAIL and inhibited cIAP1>XIAP>IAP2. Combination of birinapant and TNFα induced sub-G0 DNA fragmentation in sensitive lines and birinapant alone also induced significant G2–M cell-cycle arrest and cell death in UM-SCC-46 cells. Gene transfer and expression of FADD sensitized resistant UM-SCC-38 cells lacking FADD amplification to birinapant and TNFα, supporting a role for FADD in sensitization to IAP inhibitor and death ligands. HNSCC varied in mechanisms of cell death, as indicated by reversal by inhibitors or protein markers of caspase-dependent apoptosis and/or RIPK1/MLKL-mediated necroptosis. In vivo, birinapant inhibited tumor growth and enhanced radiation-induced TNFα, tumor responses, and host survival in UM-SCC-46 and -11B xenograft models displaying amplification and overexpression of FADD+/− BIRC2. These findings suggest that combination of SMAC mimetics such as birinapant plus radiation may be particularly active in HNSCC, which harbor frequent FADD/BIRC2 genomic alterations. Cancer Res; 76(18); 5442–54. ©2016 AACR.

Silverleaf nightshade (Solanum elaeagnifolium, Cav.) is one of the important invasive plant species in Mediterranean Basin countries. Over the last 60 years, this plant has gone from a few accidental introductions to near monospecific populations in many areas of the Mediterranean Basin (in particular Greece and Morocco). Recent findings from Lebanon show that the invasion is ongoing. Solanum elaeagnifolium has a negative impact on crops, causing up to 75% yield loss, as well as an indirect impact by harbouring plant pests and diseases. Solanum elaeagnifolium is toxic to livestock and has a negative affect on the quality of life for humans. Losses of potential resource or revenue caused by its invasion include: decreased forage quality on grazing lands; decreased cropping land and amenity values of public space; increased water loss; increased water conveyancing costs; and increased forest restoration costs. Available control techniques need to be strengthened to reduce the impact of S. elaeagnifolium and prevent its spread. More attention needs to be devoted to biological control, which could provide regional management of this invasive alien plant. Sustainable management of S. elaeagnifolium will require coordination, education and support across the affected countries. Governments must have the means to detect, manage and control S. elaeagnifolium. © 2016 The Authors.

Graphical abstract, A method for measurement of dimensions of platy particles of exfoliating, or delaminating, materials, such as clays, in aqueous suspensions in situ is proposed. Equivalent spherical diameter (esd), measured by many common methods, depends more on the major (lateral) dimension of a particle, while it is less sensitive to changes of the particle thickness. Addition of the second method, results of which are a function of the particle diameter and thickness too, would provide more accurate determination of the particle dimensions. Previously, a combination of low-temperature nitrogen adsorption (BET) and dynamic light scattering (DLS) methods for determination of specific surface area of dry powder of platy particles and their esd in suspension was suggested. While such combination was suitable for measurement of particle size for non-exfoliating materials, it gave incorrect results for exfoliating materials, which dramatically change their surface area when dispersed in liquid. We modify this method by substituting BET method with NMR relaxometry, which allows to measure wetted surface area of the dispersed material directly in suspension. The advantages of this method are:•More accurate determination of diameter and thickness of platy, particularly exfoliating, materials directly in suspension.•Possibility of routine monitoring of particle size changes during the dispersing process.

Ecological communities in forests have been shown to be strongly affected by forest management but a detailed understanding of how different components of management affect insect communities directly and indirectly via environmental variables, how management influences functional trait diversity and composition, and whether these results can be transferred to other functional groups besides insects (e.g. bacteria or nematodes) is still missing. To address these questions we used water-filled tree holes, which provide habitats for insect larvae and other aquatic organisms in forests, as a model system. We mapped all water-filled tree holes in 75 forest plots (1 ha) under different management intensity in three regions of Germany. We measured structural and climatic conditions at different spatial scales, sampled insect communities in 123 tree holes and bacterial and nematode communities in a subset of these. We found that forest management in terms of harvesting intensity and the proportion of non-natural tree species (species not part of the natural vegetation at the sites) negatively affected tree-hole abundance. An increased proportion of non-natural tree species had a positive direct effect on insect richness and functional diversity in the tree holes. However, a structural equation model showed that increasing management intensity had negative indirect effects on insect abundance and richness, operating via environmental variables at stand and tree-hole scale. Functional diversity and trait composition of insect communities similarly responded to changes in management-related variables. In contrast to insects, bacterial and nematode richness were not directly impacted by forest management but by other environmental variables. Our results suggest that forest management may strongly alter insect communities of tree holes, while nematodes and bacteria seem less affected. Most effects in our study were indirect and negative, indicating that management has often complex consequences for forest communities that should be taken into account in forest management schemes.

Interferon regulatory factor 2 binding protein-like (IRF2BPL) encodes a member of the IRF2BP family of transcriptional regulators. Currently the biological function of this gene is obscure, and the gene has not been associated with a Mendelian disease. Here we describe seven individuals who carry damaging heterozygous variants in IRF2BPL and are affected with neurological symptoms. Five individuals who carry IRF2BPL nonsense variants resulting in a premature stop codon display severe neurodevelopmental regression, hypotonia, progressive ataxia, seizures, and a lack of coordination. Two additional individuals, both with missense variants, display global developmental delay and seizures and a relatively milder phenotype than those with nonsense alleles. The IRF2BPL bioinformatics signature based on population genomics is consistent with a gene that is intolerant to variation. We show that the fruit-fly IRF2BPL ortholog, called pits (protein interacting with Ttk69 and Sin3A), is broadly detected, including in the nervous system. Complete loss of pits is lethal early in development, whereas partial knockdown with RNA interference in neurons leads to neurodegeneration, revealing a requirement for this gene in proper neuronal function and maintenance. The identified IRF2BPL nonsense variants behave as severe loss-of-function alleles in this model organism, and ectopic expression of the missense variants leads to a range of phenotypes. Taken together, our results show that IRF2BPL and pits are required in the nervous system in humans and flies, and their loss leads to a range of neurological phenotypes in both species.

xiii Preface xiv Rationale xiv Dissertation organization xiv Authors’ roles xiv Chapter 1. General Introduction 1 Malnutrition 1 The problem of malnutrition 1 Potential solutions to malnutrition 1 Biofortification 3 Iron deficiency and improvements in dietary iron 4 Iron deficiency and absorption 4 Inhibitors and enhancers of iron bioavailability 5 Iron forticants 6 Biofortification of iron 8 Hemoglobin 9 Plant hemoglobin 9 Protein deficiency and seed storage proteins in maize 11 Protein deficiency 11 Dietary requirements for protein 12

Microbotryum silybum, a smut fungus, is studied as an agent for the biological control of Silybum marianum (milk thistle) weed. Confirmation of the systemic infection is essential in order to assess the effectiveness of the biological control application and assist decision-making. Nonetheless, in situ diagnosis is challenging. The presently demonstrated research illustrates the identification process of systemically infected S. marianum plants by means of field spectroscopy and the multilayer perceptron/automatic relevance determination (MLP-ARD) network. Leaf spectral signatures were obtained from both healthy and infected S. marianum plants using a portable visible and near-infrared spectrometer (310&ndash, 1100 nm). The MLP-ARD algorithm was applied for the recognition of the infected S. marianum plants. Pre-processed spectral signatures served as input features. The spectra pre-processing consisted of normalization, and second derivative and principal component extraction. MLP-ARD reached a high overall accuracy (90.32%) in the identification process. The research results establish the capacity of MLP-ARD to precisely identify systemically infected S. marianum weeds during their vegetative growth stage.

The biodegradation of polyhydroxybutyrate (PHB) has been broadly investigated, but studies typically focus on a single strain or enzyme and little attention has been paid to comparing the interaction of different PHB depolymerase (PhaZ)-producing strains with this biopolymer. In this work, we selected nine bacterial strains-five with demonstrated and four with predicted PhaZ activity-to compare their effectiveness at degrading PHB film provided as sole carbon source. Each of the strains with demonstrated activity were able to use the PHB film (maximum mass losses ranging from 12% after 2 days for Paucimonas lemoignei to 90% after 4 days for Cupriavidus sp.), and to a lower extent Marinobacter algicola DG893 (with a predicted PhaZ) achieved PHB film mass loss of 11% after 2 weeks of exposure. Among the strains with proven PhaZ activity, Ralstonia sp. showed the highest specific activity since less biomass was required to degrade the polymer in comparison to the other strains. In the case of Ralstonia sp., PHB continued to be degraded at pH values as low as pH 3.3-3.7. In addition, analysis of the extracellular fractions of the strains with demonstrated activity showed that Comamonas testosteroni, Cupriavidus sp., and Ralstonia sp. readily degraded both PHB film and PHB particles in agar suspensions. This study highlights that whole cell cultures and enzymatic (extracellular) fractions display different levels of activity, an important factor in the development of PHB-based applications and in understanding the fate of PHB and other PHAs released in the environment. Furthermore, predictions of PhaZ functionality from genome sequencing analyses remain to be validated by experimental results; PHB-degrading ability could not be proven for three of four investigated species predicted by the polyhydroxyalkanoates (PHA) depolymerase engineering database.

Nitric oxide synthases (NOS) are important mediators of progrowth signaling in tumor cells, as they regulate angiogenesis, immune response, and immune-mediated wound healing. Ionizing radiation (IR) is also an immune modulator and inducer of wound response. We hypothesized that radiation therapeutic efficacy could be improved by targeting NOS following tumor irradiation. Herein, we show enhanced radiation-induced (10 Gy) tumor growth delay in a syngeneic model (C3H) but not immunosuppressed (Nu/Nu) squamous cell carcinoma tumor-bearing mice treated post-IR with the constitutive NOS inhibitor NG-nitro-l-arginine methyl ester (L-NAME). These results suggest a requirement of T cells for improved radiation tumor response. In support of this observation, tumor irradiation induced a rapid increase in the immunosuppressive Th2 cytokine IL10, which was abated by post-IR administration of L-NAME. In vivo suppression of IL10 using an antisense IL10 morpholino also extended the tumor growth delay induced by radiation in a manner similar to L-NAME. Further examination of this mechanism in cultured Jurkat T cells revealed L-NAME suppression of IR-induced IL10 expression, which reaccumulated in the presence of exogenous NO donor. In addition to L-NAME, the guanylyl cyclase inhibitors ODQ and thrombospondin-1 also abated IR-induced IL10 expression in Jurkat T cells and ANA-1 macrophages, which further suggests that the immunosuppressive effects involve eNOS. Moreover, cytotoxic Th1 cytokines, including IL2, IL12p40, and IFNγ, as well as activated CD8+ T cells were elevated in tumors receiving post-IR L-NAME. Together, these results suggest that post-IR NOS inhibition improves radiation tumor response via Th1 immune polarization within the tumor microenvironment. Cancer Res; 75(14); 2788–99. ©2015 AACR.

Canada thistle (Cirsium arvense) is one of the worst weeds in temperate areas of the world. A rust fungus, Puccinia punctiformis, was first proposed as a biological control agent for C. arvense in 1893. The rust causes systemic disease which ultimately kills C. arvense plants. In 2013 it was demonstrated in four countries, that inoculation of C. arvense rosettes in the fall with ground telia-bearing leaves can initiate epidemics of systemic rust disease with an average of 28% of inoculated rosettes producing a systemically diseased shoot the following spring. Other rosettes that emerged near inoculation points in spring were stunted and appeared diseased. To determine whether other rosettes were diseased, a chemiluminescence western slot blot test, applying polyclonal antibodies raised against P. punctiformis antigens, was developed to detect the fungus in roots. Rosettes were inoculated with telia-bearing leaves in the fall in Maryland, USA and Veroia, Greece. Roots of asymptomatic rosettes that emerged adjacent to inoculation points the following spring were tested for the presence of the fungus with the slot blot test. Rosettes that had diseased shoots were recorded. Based on the slot blot tests, 50–60% of the asymptomatic rosettes adjacent to inoculation points were positive for presence of the rust and likely to be systemically diseased. To demonstrate that systemic disease leads to C. arvense decline, C. arvense shoot densities were measured annually at 10 sites, in three countries, that had been inoculated with telia-bearing leaves in the fall between 2008 and 2012. Changes in C. arvense shoot densities over time were calculated. Average reductions in C. arvense density across the 10 sites were 43.1 ± 10.0% at 18 months after inoculation, 63.8 ± 8.0% at 30 months after inoculation, and 80.9 ± 16.5% at 42 months after inoculation, and 72.9 ± 27.2% at 54 months after inoculation; the 54 month reduction was, however, based on only two sites.

Purpose: Radiation remains a mainstay for the treatment of nonmetastatic head and neck squamous cell carcinoma (HNSCC), a malignancy characterized by a high rate of PI3K/mTOR signaling axis activation. We investigated the ATP-competitive dual PI3K/mTOR inhibitor, PF-05212384, as a radiosensitizer in preclinical HNSCC models. Experimental Design: Extent of radiation enhancement of two HNSCC cell lines (UMSCC1-wtP53 and UMSCC46-mtP53) and normal human fibroblast (1522) was assessed by in vitro clonogenic assay with appropriate target inhibition verified by immunoblotting. Radiation-induced DNA damage repair was evaluated by γH2AX Western blots with the mechanism of DNA double-strand break repair abrogation investigated by cell cycle analysis, immunoblotting, and RT-PCR. PF-05212384 efficacy in vivo was assessed by UMSCC1 xenograft tumor regrowth delay, xenograft lysate immunoblotting, and tissue section immunohistochemistry. Results: PF-05212384 effectively inhibited PI3K and mTOR, resulting in significant radiosensitization of exponentially growing and plateau-phase cells with 24-hour treatment following irradiation, and variable radiation enhancement with 24-hour treatment before irradiation. Tumor cells radiosensitized to a greater extent than normal human fibroblasts. Postirradiation PF-05212384 treatment delays γH2AX foci resolution. PF-05212384 24-hour exposure resulted in an evident G1–S phase block in p53-competent cells. Fractionated radiation plus i.v. PF-05212384 synergistically delayed nude mice bearing UMSCC1 xenograft regrowth, with potential drug efficacy biomarkers identified, including pS6, pAkt, p4EBP1, and Ki67. Conclusions: Taken together, our results of significant radiosensitization both in vitro and in vivo validate the PI3K/mTOR axis as a radiation modification target and PF-05212384 as a potential clinical radiation modifier of nonmetastatic HNSCC. Clin Cancer Res; 21(12); 2792–801. ©2015 AACR.

Local beneficial rhizobacteria were selected based upon their ability to control the fungus Fusarium oxysporum f. sp. radicis-lycopersici which causes crown and root rot of tomato. Seven out of 384 strains prevailed in multiple and dual cultures and were identified as Pseudomonas chlororaphis (one strain), Bacillus cereus (one strain), Serratia marcescens (three strains) and Serratia rubidaea (two strains), by sequencing the 16S rRNA or the 16S and 23S rRNA inter-spacer region. The seven selected rhizobacteria were tested for their biocontrol and growth-promoting effects in planta, and their antifungal properties in vitro. All strains significantly reduced disease severity under controlled conditions, in a gnotobiotic system and in pots. Moreover, one P. chlororaphis and one S. marcescens strain significantly decreased disease severity to the level of the healthy control under natural conditions in pots experiments. The inhibitory activity of bacterial liquid cultures' metabolites on the fungus was demons...

PurposeTo describe examples of missed pathogenic variants on whole-exome sequencing (WES) and the importance of deep phenotyping for further diagnostic testing.MethodsGuided by phenotypic information, three children with negative WES underwent targeted single-gene testing.ResultsIndividual 1 had a clinical diagnosis consistent with infantile systemic hyalinosis, although WES and a next-generation sequencing (NGS)-based ANTXR2 test were negative. Sanger sequencing of ANTXR2 revealed a homozygous single base pair insertion, previously missed by the WES variant caller software. Individual 2 had neurodevelopmental regression and cerebellar atrophy, with no diagnosis on WES. New clinical findings prompted Sanger sequencing and copy number testing of PLA2G6. A novel homozygous deletion of the noncoding exon 1 (not included in the WES capture kit) was detected, with extension into the promoter, confirming the clinical suspicion of infantile neuroaxonal dystrophy. Individual 3 had progressive ataxia, spasticity, and magnetic resonance image changes of vanishing white matter leukoencephalopathy. An NGS leukodystrophy gene panel and WES showed a heterozygous pathogenic variant in EIF2B5; no deletions/duplications were detected. Sanger sequencing of EIF2B5 showed a frameshift indel, probably missed owing to failure of alignment.ConclusionThese cases illustrate potential pitfalls of WES/NGS testing and the importance of phenotype-guided molecular testing in yielding diagnoses.

HIV and M. tuberculosis are two intersecting epidemics making the search for new dual action drugs against both pathogens extremely important. Here, we report on the synthesis and suppressive activities of five dual-targeted HIV/TB compounds. These compounds constitute heterodimers of AZT, as anti-HIV molecules, and 5-arylaminouracil derivatives or 2′-deoxyuridine monophosphates analogs, as anti-TB molecules. We found that these compounds inhibit the growth of M. tuberculosis and suppress the replication of HIV in human cell cultures and human lymphoid tissues ex vivo. We identified one particular heterodimer that inhibited most potently both HIV and the drug-resistant TB strain MS −115. This compound demonstrated low toxicity and had no cytostatic effect on cells in culture. This is a leading candidate for future development and further in vivo testing.

Carbocyclic nucleoside analogues have a distinguished history as anti-infectious agents, including key antiviral agents. Toxicity was initially a concern but this was reduced by the introduction of 5′-nor variants. Here, we report the result of our preliminary screening of a series of 5′-norcarbocyclic uridine analogues against protozoan parasites, specifically the major pathogens Leishmania mexicana and Trypanosoma brucei. The series displayed antiparasite activity in the low to mid-micromolar range and establishes a preliminary structure-activity relationship, with the 4′,N3-di-(3,5-dimethylbenzoyl)-substituted analogues showing the most prominent activity. Utilizing an array of specially adapted cell lines, it was established that this series of analogues likely act through a common target. Moreover, the strong correlation between the trypanocidal and anti-leishmanial activities indicates that this mechanism is likely shared between the two species. EC50 values were unaffected by the disabling of pyrimidine biosynthesis in T. brucei, showing that these uridine analogues do not act directly on the enzymes of pyrimidine nucleotide metabolism. The lack of cross-resistance with 5-fluorouracil, also establishes that the carbocyclic analogues are not imported through the known uracil transporters, thus offering forth new insights for this class of nucleosides. The lack of cross-resistance with current trypanocides makes this compound class interesting for further exploration.

One major challenge encountered with interpreting human genetic variants is the limited understanding of the functional impact of genetic alterations on biological processes. Furthermore, there remains an unmet demand for an efficient survey of the wealth of information on human homologs in model organisms across numerous databases. To efficiently assess the large volume of publically available information, it is important to provide a concise summary of the most relevant information in a rapid user-friendly format. To this end, we created MARRVEL (model organism aggregated resources for rare variant exploration). MARRVEL is a publicly available website that integrates information from six human genetic databases and seven model organism databases. For any given variant or gene, MARRVEL displays information from OMIM, ExAC, ClinVar, Geno2MP, DGV, and DECIPHER. Importantly, it curates model organism-specific databases to concurrently display a concise summary regarding the human gene homologs in budding and fission yeast, worm, fly, fish, mouse, and rat on a single webpage. Experiment-based information on tissue expression, protein subcellular localization, biological process, and molecular function for the human gene and homologs in the seven model organisms are arranged into a concise output. Hence, rather than visiting multiple separate databases for variant and gene analysis, users can obtain important information by searching once through MARRVEL. Altogether, MARRVEL dramatically improves efficiency and accessibility to data collection and facilitates analysis of human genes and variants by cross-disciplinary integration of 18 million records available in public databases to facilitate clinical diagnosis and basic research.

Russian thistle ( Salsola tragus , tumbleweed, RT) is a problematic invasive weed in the United States (U.S.) and is a target of biological control efforts. The facultative saprophytic fungus Colletotrichum salsolae (CS) kills RT plants in greenhouse tests and is specific to Salsola spp., which are not native in the U.S. However, the effectiveness of CS in controlling RT has not been previously demonstrated. The objectives of this study were to determine in field tests: (1) disease progress of CS in time; (2) the relationship of disease progress to rainfall and temperature; (3) the effect of CS on RT plant density. Field tests were established in Serres and Kozani, Greece and Taman and Tuzla, Russia with isolates of the pathogen collected in the respective countries. Solid inoculum was prepared by asceptically inoculating sterile mixtures of grain and grain hulls with axenic cultures of CS. Spore suspensions used in Russia were prepared by blending pure sporulating cultures of CS with distilled water and diluting the suspension to 10 6 conidia per ml. Six field plots, each subdivided into 36 subplots, of an RT infested field in Serres, Greece were inoculated on October 23, 2006 by placing about 300 g of solid inoculum in the center of each plot. Four field plots, similarly subdivided, in each of two fields at Kozani, Greece were inoculated in the same way on October 1, 2010. RT density was counted and recorded in each sub-plot prior to inoculation and in September in each of 2 years following inoculation. Disease incidence and/or severity in each sub-plot were recorded at about 7-days intervals after inoculation. Rainfall and temperature data, from inoculation until 40–55 days after inoculation, were collected and recorded at Serres and at the Kozani airport meteorological station. Disease progressed rapidly at both sites and was correlated with cumulative rainfall. By 2 years after inoculation, RT had been eliminated from the Serres site and one field in Kozani. In the other Kozani field, RT density declined to 0–25% from original densities of about 80% in large areas of the field. RT plants in Taman and Tuzla, Russia were inoculated either with 250 g of grain inoculum or with a suspension of 10 6 conidia sprayed onto each plant until runoff. The proportion of diseased tissue reached 1.0 by 55 days after inoculation in both sites. Non-inoculated plants that were near inoculated plants became diseased quickly and reached the same disease severity as inoculated plants. Disease severity was correlated with cumulative rainfall but not temperature. This pathogen and inoculation procedure offers a low-cost solution to RT infestations. Since CS is specific to Salsola spp., this effective biological control is also environmentally friendly.

Chaperone Hsp70 can activate adaptive immunity suggesting its possible application as an antitumor vaccine. To assess the therapeutic capacity of Hsp70 we administered purified chaperone into a C6 glioblastoma brain tumor and explored the viability and tumor size as well as interferon gamma (IFNγ) production and cytotoxicity of lymphocytes in the treated animals. Targeted intratumoral injection of Hsp70 resulted in its distribution within the area of glioblastoma, and caused significant inhibition of tumor progression as confirmed by magnetic resonance imaging. The delay in tumor growth corresponded to the prolonged survival of tumor-bearing animals of up to 31 days versus 20 days in control. Continuous administration of Hsp70 with an osmotic pump increased survival even further (39 days). Therapeutic efficacy was associated with infiltration to glioblastoma of NK cells (Ly-6c+) and T lymphocytes (CD3+, CD4+ and CD8+) as well as with an increase in the activity of NK cells (granzyme B production) and CD8+ T lymphocytes as shown by IFNγ ELISPOT assay. Furthermore, we found that Hsp70 treatment caused concomitantly, with a tenfold elevated IFNγ production, an increase in anti-C6 tumor cytotoxicity of lymphocytes. In conclusion, continuous intratumoral delivery of Hsp70 demonstrates high therapeutic potential and therefore could be applied in the treatment of glioblastoma.

Summary Tissue engineering using cardiomyocytes derived from human pluripotent stem cells holds a promise to revolutionize drug discovery, but only if limitations related to cardiac chamber specification and platform versatility can be overcome. We describe here a scalable tissue-cultivation platform that is cell source agnostic and enables drug testing under electrical pacing. The plastic platform enabled on-line noninvasive recording of passive tension, active force, contractile dynamics, and Ca2+ transients, as well as endpoint assessments of action potentials and conduction velocity. By combining directed cell differentiation with electrical field conditioning, we engineered electrophysiologically distinct atrial and ventricular tissues with chamber-specific drug responses and gene expression. We report, for the first time, engineering of heteropolar cardiac tissues containing distinct atrial and ventricular ends, and we demonstrate their spatially confined responses to serotonin and ranolazine. Uniquely, electrical conditioning for up to 8 months enabled modeling of polygenic left ventricular hypertrophy starting from patient cells.

Canada thistle ( Cirsium arvense , CT) is one of the worst weeds in temperate areas of the world. The rust fungus Puccinia punctiformis was first proposed as a biological control agent for CT in 1893. The rust causes systemic disease, is specific to CT, and is in all countries where CT is found. Despite a 120-year lapse since biological control with the rust was proposed, establishment of epiphytotics of the rust have previously been unsuccessful due to incomplete understanding of the disease cycle. In this study, newly-emerging rosettes in the fall are proposed as the physical and temporal infection courts for basidiospores, from germinating teliospores, to systemically infect CT and give rise to systemically diseased shoots the following spring. To test this hypothesis, rosettes of CT were inoculated in the fall with either telia-bearing leaves collected in mid-summer or with greenhouse-produced teliospores. Field sites were located near Kozani, Greece, Moscow, Russia, Christchurch, New Zealand, and Ft. Detrick, Maryland, USA. Telia-bearing leaves, which were used as inoculum in 12 of 13 field sites, were collected near each field site from CT shoots in close proximity to systemically diseased CT shoots producing aeciospores in the spring. Aeciospore infections of the leaves of these nearby shoots gave rise to uredinia which turned to telia in mid- to late-summer. Temperature and dew conditions at inoculation in the fall at each site were very favorable for teliospore germination. Rosettes inoculated in the fall were marked with flags, and systemically diseased shoots emerging near these flags the following spring were recorded. In 11 of the sites in these countries, individual rosettes were inoculated 2, 4, 6, or 8 times with telia-bearing leaves. Proportions of rosettes giving rise to systemically diseased shoots, out of the number of rosettes inoculated, were analyzed. Inoculations in all 13 sites produced systemically diseased shoots. A separate study on the phenology of CT showed that the maximum rate of leaf abscission occurred at the time of maximum emergence of new CT rosettes in the fall. This period coincided with an annually occurring period of sustained dew and favorable temperatures for teliospore germination. In nature, abscising telia-bearing leaves likely come into contact with a receptive rosette during favorable conditions for teliospore germination in the fall. This study demonstrates that epiphytotics of systemic rust disease of CT can be routinely established, by mimicking the natural disease cycle.

Several 5-aminouracil derivatives that have previously been shown to inhibit Mycobacterium tuberculosis growth at concentrations of 5-40 g/mL are demonstrated to act also as noncompetitive non-nucleoside inhibitors of HIV-1 reverse transcriptase without causing toxicity in vitro (МT-4 cells) and ex vivo (human tonsillar tissue).

Maize ( Zea mays ) is an important staple crop in many parts of the world but has low iron bioavailability, in part due to its high phytate content. Hemoglobin is a form of iron that is highly bioavailable, and its bioavailability is not inhibited by phytate. It was hypothesized that maize hemoglobin is a highly bioavailable iron source and that biofortification of maize with iron can be accomplished by overexpression of maize globin in the endosperm. Maize was transformed with a gene construct encoding a translational fusion of maize globin and green fluorescent protein under transcriptional control of the maize 27 kDa γ-zein promoter. Iron bioavailability of maize hemoglobin produced in Escherichia coli and of stably transformed seeds expressing the maize globin-GFP fusion was determined using an in vitro Caco-2 cell culture model. Maize flour fortified with maize hemoglobin was found to have iron bioavailability that is not significantly different from that of flour fortified with ferrous sulfate or bovine hemoglobin but is significantly higher than unfortified flour. Transformed maize grain expressing maize globin was found to have iron bioavailability similar to that of untransformed seeds. These results suggest that maize globin produced in E. coli may be an effective iron fortificant, but overexpressing maize globin in maize endosperm may require a different strategy to increase bioavailable iron content in maize.

Benzophenone-uracil (BPU) scaffold-derived candidate compounds are efficient non-nucleoside reverse transcriptase inhibitors (NNRTI) with extremely low solubility in water. We proposed to use hydrophobic core (methoxypolyethylene glycol-polylysine) graft copolymer (HC-PGC) technology for stabilizing nanoparticle-based formulations of BPU NNRTI in water. Co-lyophilization of NNRTI/HC-PGC mixtures resulted in dry powders that could be easily reconstituted with the formation of 150–250 nm stable nanoparticles (NP). The NP and HC-PGC were non-toxic in experiments with TZM-bl reporter cells. Nanoparticles containing selected efficient candidate Z107 NNRTI preserved the ability to inhibit HIV-1 reverse transcriptase polymerase activities with no appreciable change of EC50. The formulation with HC-PGC bearing residues of oleic acid resulted in nanoparticles that were nearly identical in anti-HIV-1 potency when compared to Z107 solutions in DMSO (EC50 = 7.5 ± 3.8 vs. 8.2 ± 5.1 nM). Therefore, hydrophobic core macromolecular stabilizers form nanoparticles with insoluble NNRTI while preserving the antiviral activity of the drug cargo.

Anticancer chemotherapeutics often rely on induction of apoptosis in rapidly dividing cells. While these treatment strategies are generally effective in debulking the primary tumor, post-therapeutic recurrence and metastasis are pervasive concerns with potentially devastating consequences. We demonstrate that the amiloride derivative 5-(N,N-hexamethylene) amiloride (HMA) harbors cytotoxic properties particularly attractive for a novel class of therapeutic agent. HMA is potently and specifically cytotoxic toward breast cancer cells, with remarkable selectivity for transformed cells relative to non-transformed or primary cells. Nonetheless, HMA is similarly cytotoxic to breast cancer cells irrespective of their molecular profile, proliferative status, or species of origin, suggesting that it engages a cell death mechanism common to all breast tumor subtypes. We observed that HMA induces a novel form of caspase- and autophagy-independent programmed necrosis relying on the orchestration of mitochondrial and lysosomal pro-death mechanisms, where its cytotoxicity was attenuated with ROS-scavengers or lysosomal cathepsin inhibition. Overall, our findings suggest HMA may efficiently target the heterogeneous populations of cancer cells known to reside within a single breast tumor by induction of a ROS- and lysosome-mediated form of programmed necrosis.

A rapid, efficient technique for the attachment of bacteriophages (phages) onto polyhydroxyalkanoate (PHA) surfaces has been developed and compared to three reported methods for phage immobilization. Polymer surfaces were modified to facilitate phage attachment using (1) plasma treatment alone, (2) plasma treatment followed by activation by 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride (EDC) and N-hydroxysulfosuccinimide (sulfo-NHS), (3) plasma-initiated acrylic acid grafting, or (4) plasma-initiated acrylic acid grafting with activation by EDC and sulfo-NHS. The impact of each method on the surface chemistry of PHA was investigated using contact angle analysis and X-ray photoelectron spectroscopy. Each of the four treatments was shown to result in both increased hydrophilicity and in the modification of the surface functional groups. Modified surfaces were immersed in suspensions of phage T4 for immobilization. The highest level of phage binding was observed for the surfaces modified by plasma treatment alone. The change in chemical bond states observed for surfaces that underwent plasma treatment is suspected to be the cause of the increased binding of active phages. Plasma-treated surfaces were further analyzed through phage-staining and fluorescence microscopy to assess the surface density of immobilized phages and their capacity to capture hosts. The infective capability of attached phages was confirmed by exposing the phage-immobilized surfaces to the host bacteria Escherichia coli in both plaque and infection dynamic assays. Plasma-treated surfaces with immobilized phages displayed higher infectivity than surfaces treated with other methods; in fact, the equivalent initial multiplicity of infection was 2 orders of magnitude greater than with other methods. Control samples - prepared by immersing polymer surfaces in phage suspensions (without prior plasma treatment) - did not show any bacterial growth inhibition, suggesting they did not bind phages from the suspension.

Item does not contain fulltext The ASXL genes (ASXL1, ASXL2, and ASXL3) participate in body patterning during embryogenesis and encode proteins involved in epigenetic regulation and assembly of transcription factors to specific genomic loci. Germline de novo truncating variants in ASXL1 and ASXL3 have been respectively implicated in causing Bohring-Opitz and Bainbridge-Ropers syndromes, which result in overlapping features of severe intellectual disability and dysmorphic features. ASXL2 has not yet been associated with a human Mendelian disorder. In this study, we performed whole-exome sequencing in six unrelated probands with developmental delay, macrocephaly, and dysmorphic features. All six had de novo truncating variants in ASXL2. A careful review enabled the recognition of a specific phenotype consisting of macrocephaly, prominent eyes, arched eyebrows, hypertelorism, a glabellar nevus flammeus, neonatal feeding difficulties, hypotonia, and developmental disabilities. Although overlapping features with Bohring-Opitz and Bainbridge-Ropers syndromes exist, features that distinguish the ASXL2-associated condition from ASXL1- and ASXL3-related disorders are macrocephaly, absence of growth retardation, and more variability in the degree of intellectual disabilities. We were also able to demonstrate with mRNA studies that these variants are likely to exert a dominant-negative effect, given that both alleles are expressed in blood and the mutated ASXL2 transcripts escape nonsense-mediated decay. In conclusion, de novo truncating variants in ASXL2 underlie a neurodevelopmental syndrome with a clinically recognizable phenotype. This report expands the germline disorders that are linked to the ASXL genes.