The Philae lander reveals low-strength primitive ice inside cometary boulders.

On 12 November 2014, the Philae lander descended towards comet 67P/Churyumov-Gerasimenko, bounced twice off the surface, then arrived under an overhanging cliff in the Abydos region. The landing process provided insights into the properties of a cometary nucleus1-3. Here we report an investigation of the previously undiscovered site of the second touchdown, where Philae spent almost two minutes of its cross-comet journey, producing four distinct surface contacts on two adjoining cometary boulders. It exposed primitive water ice-that is, water ice from the time of the comet's formation 4.5 billion years ago-in their interiors while travelling through a crevice between the boulders. Our multi-instrument observations made 19 months later found that this water ice, mixed with ubiquitous dark organic-rich material, has a local dust/ice mass ratio of [Formula: see text], matching values previously observed in freshly exposed water ice from outbursts4 and water ice in shadow5,6. At the end of the crevice, Philae made a 0.25-metre-deep impression in the boulder ice, providing in situ measurements confirming that primitive ice has a very low compressive strength (less than 12 pascals, softer than freshly fallen light snow) and allowing a key estimation to be made of the porosity (75 ± 7 per cent) of the boulders' icy interiors. Our results provide constraints for cometary landers seeking access to a volatile-rich ice sample.

Identification of ß2 microglobulin, the product of B2M gene, as a Host Factor for Vaccinia Virus Infection by Genome-Wide CRISPR genetic screens.

Genome-wide genetic screens are powerful tools to identify genes that act as host factors of viruses. We have applied this technique to analyze the infection of HeLa cells by Vaccinia virus, in an attempt to find genes necessary for infection. Infection of cell populations harboring single gene inactivations resulted in no surviving cells, suggesting that no single gene knock-out was able to provide complete resistance to Vaccinia virus and thus allow cells to survive infection. In the absence of an absolute infection blockage, we explored if some gene inactivations could provide partial protection leading to a reduced probability of infection. Multiple experiments using modified screening procedures involving replication restricted viruses led to the identification of multiple genes whose inactivation potentially increase resistance to infection and therefore cell survival. As expected, significant gene hits were related to proteins known to act in virus entry, such as ITGB1 and AXL as well as genes belonging to their downstream related pathways. Additionally, we consistently found ß2-microglobulin, encoded by the B2M gene, among the screening top hits, a novel finding that was further explored. Inactivation of B2M resulted in 54% and 91% reduced VV infection efficiency in HeLa and HAP1 cell lines respectively. In the absence of B2M, while virus binding to the cells was unaffected, virus internalization and early gene expression were significantly diminished. These results point to ß2-microglobulin as a relevant factor in the Vaccinia virus entry process.

Phosphatidylinositol 3-kinase δ blockade increases genomic instability in B cells.

Activation-induced cytidine deaminase (AID) is a B-cell-specific enzyme that targets immunoglobulin genes to initiate class switch recombination and somatic hypermutation. In addition, through off-target activity, AID has a much broader effect on genomic instability by initiating oncogenic chromosomal translocations and mutations involved in the development and progression of lymphoma. AID expression is tightly regulated in B cells and its overexpression leads to enhanced genomic instability and lymphoma formation. The phosphatidylinositol 3-kinase δ (PI3Kδ) pathway regulates AID by suppressing its expression in B cells. Drugs for leukaemia or lymphoma therapy such as idelalisib, duvelisib and ibrutinib block PI3Kδ activity directly or indirectly, potentially affecting AID expression and, consequently, genomic stability in B cells. Here we show that treatment of primary mouse B cells with idelalisib or duvelisib, and to a lesser extent ibrutinib, enhanced the expression of AID and increased somatic hypermutation and chromosomal translocation frequency to the Igh locus and to several AID off-target sites. Both of these effects were completely abrogated in AID-deficient B cells. PI3Kδ inhibitors or ibrutinib increased the formation of AID-dependent tumours in pristane-treated mice. Consistently, PI3Kδ inhibitors enhanced AID expression and translocation frequency to IGH and AID off-target sites in human chronic lymphocytic leukaemia and mantle cell lymphoma cell lines, and patients treated with idelalisib, but not ibrutinib, showed increased somatic hypermutation in AID off-targets. In summary, we show that PI3Kδ or Bruton's tyrosine kinase inhibitors increase genomic instability in normal and neoplastic B cells by an AID-dependent mechanism. This effect should be carefully considered, as such inhibitors can be administered to patients for years.

Parp3 promotes long-range end joining in murine cells.

Chromosomal rearrangements, including translocations, are early and essential events in the formation of many tumors. Previous studies that defined the genetic requirements for rearrangement formation have identified differences between murine and human cells, most notably in the role of classic and alternative nonhomologous end-joining (NHEJ) factors. We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. We show here that in contrast to classic (c-NHEJ) factors, Parp3 also promotes rearrangements in murine cells, including translocations in murine embryonic stem cells (mESCs), class-switch recombination in primary B cells, and inversions in tail fibroblasts that generate Eml4-Alk fusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing of Eml4-Alk junctions in tail fibroblasts and is consistent with a role for Parp3 in promoting the processing of DNA double-strand breaks. We confirmed a previous report that Parp1 also promotes rearrangement formation. In contrast with Parp3, rearrangement junctions in the absence of Parp1 had longer deletion lengths, suggesting that Parp1 may suppress double-strand break processing. Together, these data indicate that Parp3 and Parp1 promote rearrangements with distinct phenotypes.

The CRISPR/Cas9 System as a Tool to Engineer Chromosomal Translocation In Vivo.

The CRISPR/Cas9 system has emerged as a powerful tool to edit the genome. Among many applications, the system generates the exciting possibility of engineering small and large portions of chromosomes to induce a variety of structural alterations such as deletions, inversions, insertions and inter-chromosomal translocations. Furthermore, the availability of viral vectors that express Cas9 has been critical to deliver the CRISPR/Cas9 system directly in vivo to induce chromosomal rearrangements. This review provides an overview of the state-of-the-art CRISPR/Cas9 technology to model a variety of rearrangements in vivo in animal models.

A Cyanide-Induced 3-Cyanoalanine Nitrilase in the Cyanide-Assimilating Bacterium Pseudomonas pseudoalcaligenes Strain CECT 5344.

Pseudomonas pseudoalcaligenes CECT 5344 is a bacterium able to assimilate cyanide as a sole nitrogen source. Under this growth condition, a 3-cyanoalanine nitrilase enzymatic activity was induced. This activity was encoded by nit4, one of the four nitrilase genes detected in the genome of this bacterium, and its expression in Escherichia coli enabled the recombinant strain to fully assimilate 3-cyanoalanine. P. pseudoalcaligenes CECT 5344 showed a weak growth level with 3-cyanoalanine as the N source, unless KCN was also added. Moreover, a nit4 knockout mutant of P. pseudoalcaligenes CECT 5344 became severely impaired in its ability to grow with 3-cyanoalanine and cyanide as nitrogen sources. The native enzyme expressed in E. coli was purified up to electrophoretic homogeneity and biochemically characterized. Nit4 seems to be specific for 3-cyanoalanine, and the amount of ammonium derived from the enzymatic activity doubled in the presence of exogenously added asparaginase activity, which demonstrated that the Nit4 enzyme had both 3-cyanoalanine nitrilase and hydratase activities. The nit4 gene is located downstream of the cyanide resistance transcriptional unit containing cio1 genes, whose expression levels are under the positive control of cyanide. Real-time PCR experiments revealed that nit4 expression was also positively regulated by cyanide in both minimal and LB media. These results suggest that this gene cluster including cio1 and nit4 could be involved both in cyanide resistance and in its assimilation by P. pseudoalcaligenes CECT 5344.IMPORTANCE Cyanide is a highly toxic molecule present in some industrial wastes due to its application in several manufacturing processes, such as gold mining and the electroplating industry. The biodegradation of cyanide from contaminated wastes could be an attractive alternative to physicochemical treatment. P. pseudoalcaligenes CECT 5344 is a bacterial strain able to assimilate cyanide under alkaline conditions, thus avoiding its volatilization as HCN. This paper describes and characterizes an enzyme (Nit4) induced by cyanide that is probably involved in cyanide assimilation. The biochemical characterization of Nit4 provides a segment for building a cyanide assimilation pathway in P. pseudoalcaligenes This information could be useful for understanding, and hopefully improving, the mechanisms involved in bacterial cyanide biodegradation and its application in the treatment of cyanide-containing wastes.

ST-246 is a key antiviral to inhibit the viral F13L phospholipase, one of the essential proteins for orthopoxvirus wrapping.

OBJECTIVES: ST-246 is one of the key antivirals being developed to fight orthopoxvirus (OPV) infections. Its exact mode of action is not completely understood, but it has been reported to interfere with the wrapping of infectious virions, for which F13L (peripheral membrane protein) and B5R (type I glycoprotein) are required. Here we monitored the appearance of ST-246 resistance to identify its molecular target. METHODS: Vaccinia virus (VACV), cowpox virus (CPXV) and camelpox virus (CMLV) with reduced susceptibility to ST-246 were selected in cell culture and further characterized by antiviral assays and immunofluorescence. A panel of recombinant OPVs was engineered and a putative 3D model of F13L coupled with molecular docking was used to visualize drug-target interaction. The F13L gene of 65 CPXVs was sequenced to investigate F13L amino acid heterogeneity. RESULTS: Amino acid substitutions or insertions were found in the F13L gene of six drug-resistant OPVs and production of four F13L-recombinant viruses confirmed their role(s) in the occurrence of ST-246 resistance. F13L, but not B5R, knockout OPVs showed resistance to ST-246. ST-246 treatment of WT OPVs delocalized F13L- and B5R-encoded proteins and blocked virus wrapping. Putative modelling of F13L and ST-246 revealed a probable pocket into which ST-246 penetrates. None of the identified amino acid changes occurred naturally among newly sequenced or NCBI-derived OPV F13L sequences. CONCLUSIONS: Besides demonstrating that F13L is a direct target of ST-246, we also identified novel F13L residues involved in the interaction with ST-246. These findings are important for ST-246 use in the clinic and crucial for future drug-resistance surveillance programmes.

Draft whole genome sequence of the cyanide-degrading bacterium Pseudomonas pseudoalcaligenes CECT5344.

Pseudomonas pseudoalcaligenes CECT5344 is a Gram-negative bacterium able to tolerate cyanide and to use it as the sole nitrogen source. We report here the first draft of the whole genome sequence of a P. pseudoalcaligenes strain that assimilates cyanide. Three aspects are specially emphasized in this manuscript. First, some generalities of the genome are shown and discussed in the context of other Pseudomonadaceae genomes, including genome size, G + C content, core genome and singletons among other features. Second, the genome is analysed in the context of cyanide metabolism, describing genes probably involved in cyanide assimilation, like those encoding nitrilases, and genes related to cyanide resistance, like the cio genes encoding the cyanide insensitive oxidases. Finally, the presence of genes probably involved in other processes with a great biotechnological potential like production of bioplastics and biodegradation of pollutants also is discussed.

Characterization of hepatitis E virus recombinant ORF2 proteins expressed by vaccinia viruses.

Hepatitis E virus (HEV), an enterically transmitted pathogen, is one of the major causes of acute hepatitis in humans worldwide, being responsible for outbreaks and epidemics in regions with suboptimal sanitary conditions, in many of which it is endemic. In industrialized countries, hepatitis E is rarely reported, but recent studies have revealed quite high human seroprevalence rates and the possibility of porcine zoonotic transmission. There is currently no specific therapy or licensed vaccine against HEV infection, and little is known about its intracellular growth cycle, as until very recently no efficient cell culture system has been available. In the present study, vaccinia viruses have been used to express recombinant HEV ORF2 proteins, allowing the study of their glycosylation patterns and subcellular localization. Furthermore, the expressed proteins have been shown to be good antigens for diagnostic purposes and to elicit high and long-lasting specific anti-HEV titers of antibodies in mice that are passively transferred to the offspring by both transplacental and lactation routes.

Identification of In Vitro Inhibitors of Monkeypox Replication.

Monkeypox virus (MPXV) infections in humans have historically been restricted to regions of endemicity in Africa. However, in 2022, an alarming number of MPXV cases were reported globally, with evidence of person-to-person transmission. Because of this, the World Health Organization (WHO) declared the MPXV outbreak a public health emergency of international concern. The supply of MPXV vaccines is limited, and only two antivirals, tecovirimat and brincidofovir, approved by the U.S. Food and Drug Administration (FDA) for the treatment of smallpox, are currently available for the treatment of MPXV infection. Here, we evaluated 19 compounds previously shown to inhibit different RNA viruses for their ability to inhibit orthopoxvirus infections. We first used recombinant vaccinia virus (rVACV) expressing fluorescence (mScarlet or green fluorescent protein [GFP]) and luciferase (Nluc) reporter genes to identify compounds with antiorthopoxvirus activity. Seven compounds from the ReFRAME library (antimycin A, mycophenolic acid, AVN-944, pyrazofurin, mycophenolate mofetil, azaribine, and brequinar) and six compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) showed inhibitory activity against rVACV. Notably, the anti-VACV activity of some of the compounds in the ReFRAME library (antimycin A, mycophenolic acid, AVN-944, mycophenolate mofetil, and brequinar) and all the compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) were confirmed with MPXV, demonstrating their inhibitory activity in vitro against two orthopoxviruses. IMPORTANCE Despite the eradication of smallpox, some orthopoxviruses remain important human pathogens, as exemplified by the recent 2022 monkeypox virus (MPXV) outbreak. Although smallpox vaccines are effective against MPXV, access to those vaccines is limited. In addition, current antiviral treatment against MPXV infections is limited to the use of the FDA-approved drugs tecovirimat and brincidofovir. Thus, there is an urgent need to identify novel antivirals for the treatment of MPXV infection and other potentially zoonotic orthopoxvirus infections. Here, we show that 13 compounds, derived from two different libraries, previously found to inhibit several RNA viruses, also inhibit VACV. Notably, 11 compounds also displayed inhibitory activity against MPXV.

Antivirals against monkeypox infections.

Monkeypox virus (MPXV) infection in humans are historically restricted to endemic regions in Africa. However, in 2022, an alarming number of MPXV cases have been reported globally with evidence of person-to-person transmission. Because of this, the World Health Organization (WHO) declared the MPXV outbreak a public health emergency of international concern. MPXV vaccines are limited and only two antivirals, tecovirimat and brincidofovir, approved by the United States (US) Food and Drug Administration (FDA) for the treatment of smallpox, are currently available for the treatment of MPXV infection. Here, we evaluated 19 compounds previously shown to inhibit different RNA viruses for their ability to inhibit Orthopoxvirus infections. We first used recombinant vaccinia virus (rVACV) expressing fluorescence (Scarlet or GFP) and luciferase (Nluc) reporter genes to identify compounds with anti-Orthopoxvirus activity. Seven compounds from the ReFRAME library (antimycin A, mycophenolic acid, AVN- 944, pyrazofurin, mycophenolate mofetil, azaribine, and brequinar) and six compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) showed antiviral activity against rVACV. Notably, the anti-VACV activity of some of the compounds in the ReFRAME library (antimycin A, mycophenolic acid, AVN- 944, mycophenolate mofetil, and brequinar) and all the compounds from the NPC library (buparvaquone, valinomycin, narasin, monensin, rotenone, and mubritinib) were confirmed with MPXV, demonstrating the broad-spectrum antiviral activity against Orthopoxviruses and their potential to be used for the antiviral treatment of MPXV, or other Orthopoxvirus, infections. IMPORTANCE: Despite the eradication of smallpox, some Orthopoxviruses remain important human pathogens, as exemplified by the recent 2022 monkeypox virus (MPXV) outbreak. Although smallpox vaccines are effective against MPXV, there is presently limited access to those vaccines. In addition, current antiviral treatment against MPXV infections is limited to the use of the FDA-approved drugs tecovirimat and brincidofovir. Thus, there is an urgent need to identify novel antivirals for the treatment of MPXV, and other potentially zoonotic Orthopoxvirus infections. Here, we show that thirteen compounds, derived from two different libraries, previously found to inhibit several RNA viruses, exhibit also antiviral activity against VACV. Notably, eleven compounds also displayed antiviral activity against MPXV, demonstrating their potential to be incorporated into the therapeutic armamentarium to combat Orthopoxvirus infections.

ALK inhibitors increase ALK expression and sensitize neuroblastoma cells to ALK.CAR-T cells.

Selection of the best tumor antigen is critical for the therapeutic success of chimeric antigen receptor (CAR) T cells in hematologic malignancies and solid tumors. The anaplastic lymphoma kinase (ALK) receptor is expressed by most neuroblastomas while virtually absent in most normal tissues. ALK is an oncogenic driver in neuroblastoma and ALK inhibitors show promising clinical activity. Here, we describe the development of ALK.CAR-T cells that show potent efficacy in monotherapy against neuroblastoma with high ALK expression without toxicity. For neuroblastoma with low ALK expression, combination with ALK inhibitors specifically potentiates ALK.CAR-T cells but not GD2.CAR-T cells. Mechanistically, ALK inhibitors impair tumor growth and upregulate the expression of ALK, thereby facilitating the activity of ALK.CAR-T cells against neuroblastoma. Thus, while neither ALK inhibitors nor ALK.CAR-T cells will likely be sufficient as monotherapy in neuroblastoma with low ALK density, their combination specifically enhances therapeutic efficacy.

Vaccinia Virus Strain MVA Expressing a Prefusion-Stabilized SARS-CoV-2 Spike Glycoprotein Induces Robust Protection and Prevents Brain Infection in Mouse and Hamster Models.

The COVID-19 pandemic has underscored the importance of swift responses and the necessity of dependable technologies for vaccine development. Our team previously developed a fast cloning system for the modified vaccinia virus Ankara (MVA) vaccine platform. In this study, we reported on the construction and preclinical testing of a recombinant MVA vaccine obtained using this system. We obtained recombinant MVA expressing the unmodified full-length SARS-CoV-2 spike (S) protein containing the D614G amino-acid substitution (MVA-Sdg) and a version expressing a modified S protein containing amino-acid substitutions designed to stabilize the protein a in a pre-fusion conformation (MVA-Spf). S protein expressed by MVA-Sdg was found to be expressed and was correctly processed and transported to the cell surface, where it efficiently produced cell-cell fusion. Version Spf, however, was not proteolytically processed, and despite being transported to the plasma membrane, it failed to induce cell-cell fusion. We assessed both vaccine candidates in prime-boost regimens in the susceptible transgenic K18-human angiotensin-converting enzyme 2 (K18-hACE2) in mice and in golden Syrian hamsters. Robust immunity and protection from disease was induced with either vaccine in both animal models. Remarkably, the MVA-Spf vaccine candidate produced higher levels of antibodies, a stronger T cell response, and a higher degree of protection from challenge. In addition, the level of SARS-CoV-2 in the brain of MVA-Spf inoculated mice was decreased to undetectable levels. Those results add to our current experience and range of vaccine vectors and technologies for developing a safe and effective COVID-19 vaccine.

ALK peptide vaccination restores the immunogenicity of ALK-rearranged non-small cell lung cancer.

Anaplastic lymphoma kinase (ALK)-rearranged non-small cell lung cancer (NSCLC) is treated with ALK tyrosine kinase inhibitors (TKIs), but the lack of activity of immune checkpoint inhibitors (ICIs) is poorly understood. Here, we identified immunogenic ALK peptides to show that ICIs induced rejection of ALK+ tumors in the flank but not in the lung. A single-peptide vaccination restored priming of ALK-specific CD8+ T cells, eradicated lung tumors in combination with ALK TKIs and prevented metastatic dissemination of tumors to the brain. The poor response of ALK+ NSCLC to ICIs was due to ineffective CD8+ T cell priming against ALK antigens and is circumvented through specific vaccination. Finally, we identified human ALK peptides displayed by HLA-A*02:01 and HLA-B*07:02 molecules. These peptides were immunogenic in HLA-transgenic mice and were recognized by CD8+ T cells from individuals with NSCLC, paving the way for the development of a clinical vaccine to treat ALK+ NSCLC.

Mutagenesis of the palmitoylation site in vaccinia virus envelope glycoprotein B5.

The outer envelope of vaccinia virus extracellular virions is derived from intracellular membranes that, at late times in infection, are enriched in several virus-encoded proteins. Although palmitoylation is common in vaccinia virus envelope proteins, little is known about the role of palmitoylation in the biogenesis of the enveloped virus. We have studied the palmitoylation of B5, a 42 kDa type I transmembrane glycoprotein comprising a large ectodomain and a short (17 aa) cytoplasmic tail. Mutation of two cysteine residues located in the cytoplasmic tail in close proximity to the transmembrane domain abrogated palmitoylation of the protein. Virus mutants expressing non-palmitoylated versions of B5 and/or lacking most of the cytoplasmic tail were isolated and characterized. Cell-to-cell virus transmission and extracellular virus formation were only slightly affected by those mutations. Notably, B5 versions lacking palmitate showed decreased interactions with proteins A33 and F13, but were still incorporated into the virus envelope. Expression of mutated B5 by transfection into uninfected cells showed that both the cytoplasmic tail and palmitate have a role in the intracellular transport of B5. These results indicate that the C-terminal portion of protein B5, while involved in protein transport and in protein-protein interactions, is broadly dispensable for the formation and egress of infectious extracellular virus and for virus transmission.

Vaccinia Virus Attenuation by Codon Deoptimization of the A24R Gene for Vaccine Development.

Poxviruses have large DNA genomes, and they are able to infect multiple vertebrate and invertebrate animals, including humans. Despite the eradication of smallpox, poxvirus infections still remain a significant public health concern. Vaccinia virus (VV) is the prototypic member in the poxviridae family and it has been used extensively for different prophylactic applications, including the generation of vaccines against multiple infectious diseases and/or for oncolytic treatment. Many attempts have been pursued to develop novel attenuated forms of VV with improved safety profiles for their implementation as vaccines and/or vaccines vectors. We and others have previously demonstrated how RNA viruses encoding codon-deoptimized viral genes are attenuated, immunogenic and able to protect, upon a single administration, against challenge with parental viruses. In this study, we employed the same experimental approach based on the use of misrepresented codons for the generation of a recombinant (r)VV encoding a codon-deoptimized A24R gene, which is a key component of the viral RNA polymerase. Similar to our previous studies with RNA viruses, the A24R codon-deoptimized rVV (v-A24cd) was highly attenuated in vivo but able to protect, after a single intranasal dose administration, against an otherwise lethal challenge with parental VV. These results indicate that poxviruses can be effectively attenuated by synonymous codon deoptimization and open the possibility of using this methodology alone or in combination with other experimental approaches for the development of attenuated vaccines for the treatment of poxvirus infection, or to generate improved VV-based vectors. Moreover, this approach could be applied to other DNA viruses. IMPORTANCE The family poxviridae includes multiple viruses of medical and veterinary relevance, being vaccinia virus (VV) the prototypic member in the family. VV was used during the smallpox vaccination campaign to eradicate variola virus (VARV), which is considered a credible bioterrorism threat. Because of novel innovations in genetic engineering and vaccine technology, VV has gained popularity as a viral vector for the development of vaccines against several infectious diseases. Several approaches have been used to generate attenuated VV for its implementation as vaccine and/or vaccine vector. Here, we generated a rVV containing a codon-deoptimized A24R gene (v-A24cd), which encodes a key component of the viral RNA polymerase. v-A24cd was stable in culture cells and highly attenuated in vivo but able to protect against a subsequent lethal challenge with parental VV. Our findings support the use of this approach for the development of safe, stable, and protective live-attenuated VV and/or vaccine vectors.

Genomic and biological study of fusion genes as resistance mechanisms to EGFR inhibitors.

The clinical significance of gene fusions detected by DNA-based next generation sequencing remains unclear as resistance mechanisms to EGFR tyrosine kinase inhibitors in EGFR mutant non-small cell lung cancer. By studying EGFR inhibitor-resistant patients treated with a combination of an EGFR inhibitor and a drug targeting the putative resistance-causing fusion oncogene, we identify patients who benefit and those who do not from this treatment approach. Through evaluation including RNA-seq of potential drug resistance-imparting fusion oncogenes in 504 patients with EGFR mutant lung cancer, we identify only a minority of them as functional, potentially capable of imparting EGFR inhibitor resistance. We further functionally validate fusion oncogenes in vitro using CRISPR-based editing of EGFR mutant cell lines and use these models to identify known and unknown drug resistance mechanisms to combination therapies. Collectively, our results partially reveal the complex nature of fusion oncogenes as potential drug resistance mechanisms and highlight approaches that can be undertaken to determine their functional significance.

Cyanide degradation by Pseudomonas pseudoalcaligenes CECT5344 involves a malate:quinone oxidoreductase and an associated cyanide-insensitive electron transfer chain.

The alkaliphilic bacterium Pseudomonas pseudoalcaligenes CECT5344 is able to grow with cyanide as the sole nitrogen source. Membrane fractions from cells grown under cyanotrophic conditions catalysed the production of oxaloacetate from L-malate. Several enzymic activities of the tricarboxylic acid and glyoxylate cycles in association with the cyanide-insensitive respiratory pathway seem to be responsible for the oxaloacetate formation in vivo. Thus, in cyanide-grown cells, citrate synthase and isocitrate lyase activities were significantly higher than those observed with other nitrogen sources. Malate dehydrogenase activity was undetectable, but a malate:quinone oxidoreductase activity coupled to the cyanide-insensitive alternative oxidase was found in membrane fractions from cyanide-grown cells. Therefore, oxaloacetate production was linked to the cyanide-insensitive respiration in P. pseudoalcaligenes CECT5344. Cyanide and oxaloacetate reacted chemically inside the cells to produce a cyanohydrin (2-hydroxynitrile), which was further converted to ammonium. In addition to cyanide, strain CECT5344 was able to grow with several cyano derivatives, such as 2- and 3-hydroxynitriles. The specific system required for uptake and metabolization of cyanohydrins was induced by cyanide and by 2-hydroxynitriles, such as the cyanohydrins of oxaloacetate and 2-oxoglutarate.

Role of Fur on cyanide tolerance of Pseudomonas pseudoalcaligenes CECT5344.

Pseudomonas pseudoalcaligenes CECT5344 can be used in cyanide bioremediation processes because it grows at pH 9.5 using 2.0 mM cyanide at the sole nitrogen source. Cyanide strongly binds to metals creating iron-deprivation conditions. The bacterium responds to the presence of cyanide by inducing several processes such as siderophore synthesis for iron capture, cyanide-insensitive respiration system and defence mechanisms against oxidative stress. Since high concentrations of cyanide cause iron deficiency and because iron is an essential nutrient, bacterial growth in the presence of cyanide requires an efficient iron uptake. Fur is a global transcription factor that regulates a diversity of biological processes such as iron homoeostasis, TCA (tricarboxylic acid) cycle metabolism and oxidative stress response. Fur's regulation of iron uptake and storage genes should play a significant role in the lives of these bacteria. In the present review, current knowledge of Fur is summarized.

Metabolic adaptation of Pseudomonas pseudoalcaligenes CECT5344 to cyanide: role of malate-quinone oxidoreductases, aconitase and fumarase isoenzymes.

In general, the biodegradation of a toxic compound by a micro-organism requires the concurrence of, at least, two features in the biological system: first, the capability of the micro-organism to metabolize the toxic compound, and secondly, the capacity to resist its toxic effect. Pseudomonas pseudoalcaligenes CECT5344 is a bacterium used in the biodegradation of cyanide because it is capable to use it as a nitrogen source. The present review is mainly focused on the putative role of iron-containing enzymes of the tricarboxylic acid cycle in cyanide resistance by P. pseudoalcaligenes CECT5344.