A modified TILLING approach to detect induced mutations in tetraploid and hexaploid wheat

Background  

Wheat (Triticum ssp.) is an important food source for humans in many regions around the world. However, the ability to understand and modify gene function for crop improvement is hindered by the lack of available genomic resources. TILLING is a powerful reverse genetics approach that combines chemical mutagenesis with a high-throughput screen for mutations. Wheat is specially well-suited for TILLING due to the high mutation densities tolerated by polyploids, which allow for very efficient screens. Despite this, few TILLING populations are currently available. In addition, current TILLING screening protocols require high-throughput genotyping platforms, limiting their use.     

Mutational Profiling of Kinases in Human Tumours of Pancreatic Origin Identifies Candidate Cancer Genes in Ductal and Ampulla of Vater Carcinomas

Background

Protein kinases are key regulators of cellular processes (such as proliferation, apoptosis and invasion) that are often deregulated in human cancers. Accordingly, kinase genes have been the first to be systematically analyzed in human tumors leading to the discovery that many oncogenes correspond to mutated kinases. In most cases the genetic alterations translate in constitutively active kinase proteins, which are amenable of therapeutic targeting. Tumours of the pancreas are aggressive neoplasms for which no effective therapeutic strategy is currently available.

Methodology/Principal Findings

We conducted a DNA-sequence analysis of a selected set of 35 kinase genes in a panel of 52 pancreatic exocrine neoplasms, including 36 pancreatic ductal adenocarcinoma, and 16 ampulla of Vater cancer. Among other changes we found somatic mutations in ATM, EGFR, EPHA3, EPHB2, and KIT, none of which was previously described in cancers.

Conclusions/Significance

Although the alterations identified require further experimental evaluation, the localization within defined protein domains indicates functional relevance for most of them. Some of the mutated genes, including the tyrosine kinases EPHA3 and EPHB2, are clearly amenable to pharmacological intervention and could represent novel therapeutic targets for these incurable cancers.     

Lipid droplet hijacking by intracellular pathogens

Lipid droplets were long considered to be simple storage structures, but they have recently been shown to be dynamic organelles involved in diverse biological processes, including emerging roles in innate immunity. Various intracellular pathogens, including viruses, bacteria, and parasites, specifically target host lipid droplets during their life cycle. Viruses such as hepatitis C, dengue, and rotaviruses use lipid droplets as platforms for assembly. Bacteria, such as mycobacteria and Chlamydia, and parasites, such as trypanosomes, use host lipid droplets for nutritional purposes. The possible use of lipid droplets by intracellular pathogens, as part of an anti‐immunity strategy, is an intriguing question meriting further investigation in the near future.     

<Emphasis Type="Italic">Ochrobactrum</Emphasis> sp. MPV1 from a dump of roasted pyrites can be exploited as bacterial catalyst for the biogenesis of selenium and tellurium nanoparticles

Background

Bacteria have developed different mechanisms for the transformation of metalloid oxyanions to non-toxic chemical forms. A number of bacterial isolates so far obtained in axenic culture has shown the ability to bioreduce selenite and tellurite to the elemental state in different conditions along with the formation of nanoparticles—both inside and outside the cells—characterized by a variety of morphological features. This reductive process can be considered of major importance for two reasons: firstly, toxic and soluble (i.e. bioavailable) compounds such as selenite and tellurite are converted to a less toxic chemical forms (i.e. zero valent state); secondly, chalcogen nanoparticles have attracted great interest due to their photoelectric and semiconducting properties. In addition, their exploitation as antimicrobial agents is currently becoming an area of intensive research in medical sciences.

Results

In the present study, the bacterial strain Ochrobactrum sp. MPV1, isolated from a dump of roasted arsenopyrites as residues of a formerly sulfuric acid production near Scarlino (Tuscany, Italy) was analyzed for its capability of efficaciously bioreducing the chalcogen oxyanions selenite (SeO₃ ^2?) and tellurite (TeO₃ ^2?) to their respective elemental forms (Se⁰ and Te⁰) in aerobic conditions, with generation of Se- and Te-nanoparticles (Se- and TeNPs). The isolate could bioconvert 2 mM SeO₃ ^2? and 0.5 mM TeO₃ ^2? to the corresponding Se⁰ and Te⁰ in 48 and 120 h, respectively. The intracellular accumulation of nanomaterials was demonstrated through electron microscopy. Moreover, several analyses were performed to shed light on the mechanisms involved in SeO₃ ^2? and TeO₃ ^2? bioreduction to their elemental states. Results obtained suggested that these oxyanions are bioconverted through two different mechanisms in Ochrobactrum sp. MPV1. Glutathione (GSH) seemed to play a key role in SeO₃ ^2? bioreduction, while TeO₃ ^2? bioconversion could be ascribed to the catalytic activity of intracellular NADH-dependent oxidoreductases. The organic coating surrounding biogenic Se- and TeNPs was also characterized through Fourier-transform infrared spectroscopy. This analysis revealed interesting differences among the NPs produced by Ochrobactrum sp. MPV1 and suggested a possible different role of phospholipids and proteins in both biosynthesis and stabilization of such chalcogen-NPs.

Conclusions

In conclusion, Ochrobactrum sp. MPV1 has demonstrated to be an ideal candidate for the bioconversion of toxic oxyanions such as selenite and tellurite to their respective elemental forms, producing intracellular Se- and TeNPs possibly exploitable in biomedical and industrial applications.

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Thromboembolism after COVID-19 vaccine in patients with preexisting thrombocytopenia

While vaccination is the single most effective intervention to drastically reduce severe disease and death following SARS-CoV-2 infection, as shown in UK and Israel, some serious concerns have been raised for an unusual adverse drug reaction (ADR), including vaccine-induced immune thrombotic thrombocytopenia (VITT) with concurrent low platelets as well as capillary leak syndrome. In fact, the overall safety of the vaccine is highlighted by the low frequency of ADR considering that in UK, by the early June, 40 million first doses and 29 million second doses have been injected; nonetheless, 390 thrombotic events, including 71 fatal events have been reported. Interestingly, the cases reported low platelet counts with the presence of anti-platelet factor-4 (PF4) antibodies, indicating an abnormal clotting reaction. Here, out of three referred cases, we report a post-vaccine clinical case of fatal thrombosis with postmortem examination and whole exome sequencing (WES) analysis, whose pathogenesis appeared associated to a preexisting condition of thrombocytopenia due to myelodysplasia.Subject terms: Diseases, Medical research     

Human cytomegalovirus glycoprotein B genotyping from bronchoalveolar lavage specimens

The genes encoding glycoprotein complexes of human cytomegalovirus are often polymorphic; in particular, glycoprotein B (gB), which is essential for both in vivo and in vitro replication, is encoded by the highly polymorphic gene UL55. In this study, the distribution of gB genotypes was investigated in 44 bronchoalveolar lavage specimens from adult patients positive for human cytomegalovirus DNA by a multiplex nested fast PCR able to amplify 5 gB genotypes (gB1-gB5). The distribution of gB genotypes was as follows: 12 (27.3%) gB1, 11 (25%) gB2, 9 (20.4%) gB3, 4 (9.1%) gB4, 0 gB5, and 8 (18.2%) mixed genotypes. No difference in prevalence was found in relation to clinical features, including immunological status, non-transplant or transplant condition, and type of transplanted organ, or in follow-up specimens; while gB4 and gB3 were shown to be significantly more prevalent in patients with respiratory insufficiency, and gB4 and gB2 in those with pneumonia. The prevalence of gB genotypes in the lower respiratory tract was similar to that previously reported using other specimen types and patients, with gB1 found to be the most prevalent. The association of gB genotypes with specific clinical features should be further investigated.     

Yeasts from Macrobrachium amazonicum: a focus on antifungal susceptibility and virulence factors of Candida spp

In the present study, it was sought to compare yeast microbiota of wild and captive Macrobrachium amazonicum and evaluate the antifungal susceptibility and production of virulence factors by the recovered isolates of Candida spp. Additionally, cultivation water was monitored for the presence of fungi. Overall, 26 yeast isolates belonging to three genera and seven species were obtained, out of which 24 were Candida spp., with Candida famata as the most prevalent species for both wild and captive prawns. From cultivation water, 28 isolates of filamentous fungi were obtained, with Penicillium spp., Cladosporium spp. and Aspergillus spp. as the most frequent genera. Eight out of 24 Candida spp. isolates were resistant to azole derivatives, out of which four were recovered from wild-harvested prawns. As for production of virulence factors, three (12.5%) and eight (33.3%) isolates presented phospholipase and protease activity, respectively. This is the first comparative study between wild and captive prawns and the first report on yeast microbiota of M. amazonicum. The most relevant finding was the high percentage of resistant Candida spp., including from wild individuals, which suggests the occurrence of an environmental imbalance in the area where these prawns were captured.     

Dual nature of translational control by regulatory BC RNAs

In higher eukaryotes, increasing evidence suggests, gene expression is to a large degree controlled by RNA. Regulatory RNAs have been implicated in the management of neuronal function and plasticity in mammalian brains. However, much of the molecular-mechanistic framework that enables neuronal regulatory RNAs to control gene expression remains poorly understood. Here, we establish molecular mechanisms that underlie the regulatory capacity of neuronal BC RNAs in the translational control of gene expression. We report that regulatory BC RNAs employ a two-pronged approach in translational control. One of two distinct repression mechanisms is mediated by C-loop motifs in BC RNA 3' stem-loop domains. These C-loops bind to eIF4B and prevent the factor's interaction with 18S rRNA of the small ribosomal subunit. In the second mechanism, the central A-rich domains of BC RNAs target eIF4A, specifically inhibiting its RNA helicase activity. Thus, BC RNAs repress translation initiation in a bimodal mechanistic approach. As BC RNA functionality has evolved independently in rodent and primate lineages, our data suggest that BC RNA translational control was necessitated and implemented during mammalian phylogenetic development of complex neural systems.     

Glioblastoma cells: a heterogeneous and fatal tumor interacting with the parenchyma

Glioblastomas (GBMs) are considered to be one of the deadliest human cancers, characterized by a high proliferative rate, aggressive invasiveness and insensitivity to radio- and chemotherapy, as well as a short patient survival period. Moreover, GBMs are among the most vascularized and invasive cancers in humans. Angiogenesis in GBMs is correlated with the grade of malignancy and is inversely correlated with patient survival. One of the first steps in tumor invasions is migration. GBM cells have the ability to infiltrate and disrupt physical barriers such as basement membranes, extracellular matrix and cell junctions. The invasion process includes the overexpression of several members of a super-family of zinc-based proteinases, the Metzincin, in particular a sub-group, metalloproteinases. Another interesting aspect is that, inside the GBM tissue, there are up to 30% of microglia or macrophages. However, little is known about the immune performance and interactions of the microglia with GBMs. These singular properties of GBMs will be described here. A sub-population of cells with stem-like properties may be the source of tumors since, apparently, GBM stem cells (GSCs) are highly resistant to current cancer treatments. These cancer therapies, while killing the majority of tumor cells, ultimately fail in GBM treatment because they do not eliminate GSCs, which survive to regenerate new tumors. Finally, GBM patient prognostic has shown little improvement in decades. In this context, we will discuss how the membrane-acting toxins called cytolysins can be a potential new tool for GBM treatment.