Mutation Silencing Research Articles

Phospholipid Signaling in Crop Plants: A Field to Explore.

In plant models such as Arabidopsis thaliana, phosphatidic acid (PA), a key molecule of lipid signaling, was shown not only to be involved in stress responses, but also in plant development and nutrition. In this article, we highlight lipid signaling existing in crop species. Based on open access databases, we update the list of sequences encoding phospholipases D, phosphoinositide-dependent phospholipases C, and diacylglycerol-kinases, enzymes that lead to the production of PA. We show that structural features of these enzymes from model plants are conserved in equivalent proteins from selected crop species. We then present an in-depth discussion of the structural characteristics of these proteins before focusing on PA binding proteins. For the purpose of this article, we consider RESPIRATORY BURST OXIDASE HOMOLOGUEs (RBOHs), the most documented PA target proteins. Finally, we present pioneering experiments that show, by different approaches such as monitoring of gene expression, use of pharmacological agents, ectopic over-expression of genes, and the creation of silenced mutants, that lipid signaling plays major roles in crop species. Finally, we present major open questions that require attention since we have only a perception of the peak of the iceberg when it comes to the exciting field of phospholipid signaling in plants.

Functional mechanism of hypoxia-like conditions mediating resistance to ferroptosis in cervical cancer cells by regulating KDM4A SUMOylation and the SLC7A11/GPX4 pathway.

The discovery of ferroptosis has unveiled new perspectives for cervical cancer (CC) management. We elucidated the functional mechanism of hypoxia-like conditions in CC cell ferroptosis resistance. CC cells were subjected to normoxia or hypoxia-like conditions, followed by erastin treatment to induce ferroptosis. The assessment of cell viability/ferroptosis resistance was performed by MTT assay/Fe2+, MDA, and glutathione measurement by colorimetry. KDM4A/SUMO1/Ubc9/SENP1 protein levels were determined by Western blot. Interaction and binding sites between KDM4A and SUMO1 were analyzed and predicted by immunofluorescence/co-immunoprecipitation and GPS-SUMO 1.0 software, with the target relationship verified by mutation experiment. SLC7A11/GPX4/H3K9me3 protein levels, and H3K9me3 level in the SLC7A11 gene promoter region were determined by RT-qPCR and Western blot/chromatin immunoprecipitation. H3H9me3/SLC7A11/GPX4 level alterations, and ferroptosis resistance after KDM4A silencing or KDM4A K471 mutation were assessed. Hypoxia-like conditions increased CC cell ferroptosis resistance and KDM4A, SUMO1, and Ubc9 protein levels, while it decreased SENP1 protein level. KDM4A and SUMO1 were co-localized in the nucleus, and hypoxia-like conditions promoted their interaction. Specifically, the K471 locus of KDM4A was the main locus for SUMO1ylation. Hypoxia-like conditions up-regulated SLC7A11 and GPX4 expression levels and decreased H3K9me3 protein level and H3K9me3 abundance in the SLC7A11 promoter region. KDM4A silencing or K471 locus mutation resulted in weakened interaction between KDM4A and SUMO1, elevated H3K9me3 levels, decreased SLC7A11 expression, ultimately, a reduced CC cell ferroptosis resistance. CoCl2-stimulated hypoxia-like conditions enhanced SUMO1 modification of KDM4A at the K471 locus specifically, repressed H3K9me3 levels, and up-regulated SLC7A11/GPX4 to enhance CC cell ferroptosis resistance.

Abstract LB059: Multimeric linker-exatecan-based ADC targeting guanylyl cyclase C (GCC) as novel therapeutic modality for treatment of colorectal cancer

Abstract Background: The approval of sacituzumabgovitecan (Trodelvy) and trastuzumab deruxtecan (Enhertu) demonstrated the potential of topoisomerase I inhibitors in cancer therapy. Both ADCs rely on payload conjugation to interchain cysteines of an unmodified wildtype antibody coming along with unfavorable properties like instability in circulation, premature payload release and unspecific uptake via Fc gamma receptors. Heidelberg Pharma uses a proprietary, site-specific conjugation to engineered cysteines andFcγR silencing mutations (LALA) to obtain a homogeneous and stable ADC void of Fc-mediated uptake. To achieve a DAR of 4, a multimeric linker comprisingexatecan and a solubility enhanceris used to obtain more than one payload per conjugation site. In the current study, in vitro and in vivo data of an ADC targeting GCC (guanylyl cyclase C) for the treatment of colorectal cancer with favorable potency and tolerability are presented. Material and methods: Cell lines: GCC+cell line: HEK293-GUCY2C mono2; GCC-cell line: HEK293 ADC: The cysteine-reactive multimeric linker-exatecan construct was synthesized at Heidelberg Pharma and conjugated site-specifically to engineered cysteine residues of a proprietary anti-GCC antibody yielding ADCs with a DAR of 4.0. Animal models: Female NOD SCID mice were subcutaneously inoculated with GCC-overexpressing HEK293-GUCY2C mono2 tumor cells. Animals were treated with anti GCC ADC at single or multiple doses. Tolerability was assessed in female NODSCID mice. Results: The anti-GCC ADC with multimeric linker-exatecan payload showed favorable in vitro cytotoxicity with nanomolar activity on the GCC+ cell line and no cytotoxic activity on target-negative cells. In mouse xenograft models, the anti-GCCADC caused dose-dependent tumor regression in HEK293-GUCY2C mono2 s.c. xenografts. Multiple dosing improved this anti-tumor efficacy even further without negative impact on toxicity. The MTD of the ADC in mice was determined to be ≥200 mg/kg. Conclusions: Targeted cytotoxic drug delivery to GCC positive cell lines was achieved by using an anti-GCC ADCwith multimeric linker-exatecan payload. The use of solubility enhancers within the structure of the multimeric linker facilitated site-specific coupling to cysteines resulting in a stable, potent, and well tolerated ADC. The use of anti-GCC ADCs with multimeric linker-exatecan payloads in the therapy of GCC positive colorectal cancer, represents a promising approach in cancer therapy. Citation Format: Sarah-Jane Neuberth, Christian Orlik, Aniko Palfi, Christoph Mueller, Hendrik Gruss, Torsten Hechler. Multimeric linker-exatecan-based ADC targeting guanylyl cyclase C (GCC) as novel therapeutic modality for treatment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB059.

Abstract 7040: Mutations in cytoskeleton genes SPTAN1/SPTBN1 serve as novel predictive markers for immunotherapy against bladder cancer

Abstract Cancer progression is often driven by somatic mutations. Despite several well-known driver mutations, more evidence has shown that genetic alterations in cytoskeleton organization enhance tumor aggressiveness and promote cancer progression, suggesting their roles as potential drivers. In this study, pan-cancer genetic analyses by using TCGA cohorts, more frequent mutation events were found in cytoskeleton-related genes as compared to total annotated genes, especially in cancers derived from hollow organs. High-dimensional analysis of molecular alterations in cancer (HD-MAC) suggested gene mutations in SPTAN1 and its partner SPTBN1 as potential prognostic biomarkers for clinical outcomes of patients with bladder cancer. Gene knockdown in either SPTAN1 or SPTBN1, mimicking silencing mutations in cancer tissues, caused cell morphology changes, DNA breaks, and alternations in cytokine profiles via activating cGAS-STING mediated interferon signaling. Xenograft tumor models also indicate accelerated tumor growth in SPTAN1- or SPTBN1-silenced cells, suggesting a more advanced cancer type. Based on immune staining in both human and mouse samples, increased CD8+ T cell infiltration was in tumor lesions with mutations or truncations in SPTAN1/SPTBN1 genes. In addition, several known substitution mutations were found as potent neo-antigens based on MHC II binding prediction, which could be utilized as targets for immune checkpoint therapy. This study provides a novel insight into how the cytoskeleton can contribute to cancer progression and find out predictive biological indicators with therapeutic values. Citation Format: Brian Yu-Ting Kuo. Mutations in cytoskeleton genes SPTAN1/SPTBN1 serve as novel predictive markers for immunotherapy against bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7040.

Inhibitory synaptic transmission is impaired in the Kölliker-Fuse of male, but not female, Rett syndrome mice.

Rett syndrome (RTT) is a severe neurodevelopmental disorder that mainly affects females due to silencing mutations in the X-linked MECP2 gene. One of the most troubling symptoms of RTT is breathing irregularity, including apneas, breath-holds, and hyperventilation. Mice with silencing mutations in Mecp2 exhibit breathing abnormalities similar to human patients and serve as useful models for studying mechanisms underlying breathing problems in RTT. Previous work implicated the pontine, respiratory-controlling Kölliker-Fuse (KF) in the breathing problems in RTT. The goal of this study was to test the hypothesis that inhibitory synaptic transmission is deficient in KF neurons from symptomatic male and female RTT mice. We performed whole cell voltage-clamp recordings from KF neurons in acute brain slices to examine spontaneous and electrically evoked inhibitory post-synaptic currents (IPSCs) in RTT mice and age- and sex-matched wild-type mice. The frequency of spontaneous IPSCs was reduced in KF neurons from male RTT mice but surprisingly not in female RTT mice. In addition, electrically evoked IPSCs were less reliable in KF neurons from male, but not female, RTT mice, which was positively correlated with paired-pulse facilitation, indicating decreased probability of release. KF neurons from male RTT mice were also more excitable and exhibited shorter-duration action potentials. Increased excitability of KF neurons from male mice was not explained by changes in axon initial segment length. These findings indicate impaired inhibitory neurotransmission and increased excitability of KF neurons in male but not female RTT mice and suggest that sex-dependent mechanisms contribute to breathing problems in RTT.NEW & NOTEWORTHY Kölliker-Fuse (KF) neurons in acute brain slices from male Rett syndrome (RTT) mice receive reduced inhibitory synaptic inputs compared with wild-type littermates. In female RTT mice, inhibitory transmission was not different in KF neurons compared with controls. The results from this study show that sex-specific alterations in synaptic transmission occur in the KF of RTT mice.

Construction of RNA silencing system of Penicillium brevicompactum and genetic manipulation of the regulator pbpcz in mycophenolic acid production

Penicillium brevicompactum is a critical industrial strain for the production of mycophenolic acid (MPA). However, the genetic background of Penicillium brevicompactum is unclear, and there are few tools available for genetic manipulation. To investigate its gene function, we first verified the feasibility of a pair of citrate synthase promoter (Pcit) and terminator (Tcit) from P. brevicompactum by constructing a fluorescent expression cassette. Based on this, an RNAi vector was designed and constructed with reverse promoters. This study focused on the functional investigation of the pbpcz gene in P. brevicompactum, a regulator belonging to the Zn(II)2Cys6 family. RNAi was used to silence the pbpcz gene, providing a valuable tool for genetic studies in P. brevicompactum. After seven days, we observed differences in the number of spores between different phenotypes strains of pbpcz gene. Compared to the wild-type strain (WT), the spore yield of the pbpcz gene silencing mutant (M2) was only 51.4 %, while that of the pbpcz gene overexpressed mutant (SE4) was increased by 50 %. Expression levels of the three genes (brlA, abaA, and wetA) comprising conidia's central regulatory pathway were significantly reduced in the pbpcz gene silencing mutant, while fluorescence localization showed that PbPCZ protein was mainly distributed in spores. The results indicated that the pbpcz gene is critical for conidia and asexual development of P. brevicompactum. In addition, overexpressing the pbpcz gene resulted in a 30.3 % increase in MPA production compared to the wild type, with a final yield of 3.57 g/L. These results provide evidence that PbPCZ acts as a positive regulator in P. brevicompactum, controlling MPA production and regulating conidia and asexual development.

CREB-H is a stress-regulator of hepcidin gene expression during early postnatal development.

Hepcidin, the hepatic iron hormone, is the central regulator of iron homeostasis. Cyclic AMP-Responsive Element-Binding protein 3-like 3 (CREB3L3/CREB-H) is a liver homeostatic regulator of essential nutrients (i.e. glucose and lipids) and has been previously involved in hepcidin response to pathologic stress signals. Here, we asked whether CREB-H has also a physiologic role in iron homeostasis through hepcidin. To this end, we analyzed hepcidin gene expression and regulation in the liver of wild type and Creb3l3 knockout mice during early postnatal development, as a model of "physiologic" stressful condition. The effect of iron challenge in vivo and BMP6 stimulation in vitro have been also addressed. In addition, we investigated the BMP signaling pathway and hepcidin promoter activity following CREB3L3 silencing and hepcidin promoter mutation in HepG2 cells. Creb3l3 knockout suckling and young-adult mice showed a prominent serum and hepatic iron accumulation, respectively, due to impaired hepcidin mRNA expression which progressively returned to normal level in adult mice. Interestingly, upon iron challenge, while the upstream BMP/SMAD signaling pathway controlling hepcidin was equally responsive in both strains, hepcidin gene expression was impaired in knockout mice and more iron accumulated in the liver. Accordingly, hepcidin gene response to BMP6 was blunted in primary CREB-H knockout hepatocytes and in HepG2 cells transfected with CREB-H siRNA or carrying a hepcidin promoter mutated in the CREB-H binding site. In conclusion, CREB-H has a role in maintaining the homeostatic balance of iron traffic through hepcidin during the critical postnatal period and in response to iron challenge. KEY MESSAGES: CREB-H KO mice develop liver iron overload shortly after weaning that normalizes in adulthood. CHEB-H is involved in hepcidin gene response to oral iron in vivo. CREB-H loss hampers hepcidin promoter response to BMP6. CREB-H is a key stress-sensor controlling hepcidin gene transcription in physiologic and pathophysiologic states.

Evaluation of the effectiveness of RNA silencing in the filamentous fungus Aspergillus niger using a binary vector integrated with Gateway recombination cloning technology

The filamentous fungus Aspergillus niger is widely employed in the production of citric acid and some industrial enzymes such as phytase, glucoamylase, and glucose oxidase. Currently, several genetic modification tools have been developed and applied to this fungal species. However, the RNA silencing technique is less used for A. niger. In the present study, an RNA silencing binary vector integrated with the Gateway recombination cloning technology was evaluated in A. niger. The integration of Gateway recombination cloning technology in binary vectors for rapid generation of hairpin RNA structure allowed the assessment of inhibition of target gene expression through mRNA degradation. The RNA silencing constructs were successfully transferred into A. nigervia Agrobacterium tumefaciens. Results showed that the RNA silencing vectors were effective in downregulating the expression of the DsRed fluorescent reporter gene and the stuA regulatory gene. Interestingly, the silenced mutants of stuA exhibited a significant reduction in fungal sporulation. This finding revealed that the RNA silencing technique with the examined Gateway binary vector represents a potential genetic tool for functional studies of target genes in A. niger.

Caveolae and Bin1 form ring-shaped platforms for T-tubule initiation.

Excitation-contraction coupling requires a highly specialized membrane structure, the triad, composed of a plasma membrane invagination, the T-tubule, surrounded by two sarcoplasmic reticulum terminal cisternae. Although the precise mechanisms governing T-tubule biogenesis and triad formation remain largely unknown, studies have shown that caveolae participate in T-tubule formation and mutations of several of their constituents induce muscle weakness and myopathies. Here, we demonstrate that, at the plasma membrane, Bin1 and caveolae composed of caveolin-3 assemble into ring-like structures from which emerge tubes enriched in the dihydropyridine receptor. Bin1 expression lead to the formation of both rings and tubes and we show that Bin1 forms scaffolds on which caveolae accumulate to form the initial T-tubule. Cav3 deficiency caused by either gene silencing or pathogenic mutations results in defective ring formation and perturbed Bin1-mediated tubulation that may explain defective T-tubule organization in mature muscles. Our results uncover new pathophysiological mechanisms that may prove relevant to myopathies caused by Cav3 or Bin1 dysfunction.

Insights into the Evolution of P. aeruginosa Antimicrobial Resistance in a Patient Undergoing Intensive Therapy

Whole genome sequencing (WGS) provides insights into the evolution of antimicrobial resistance, an urgent global health threat. Using WGS, we observe evolutionary adaptation of a Pseudomonas aeruginosa strain within an immunocompromised patient undergoing antibiotic therapy. Two blood isolates (EA-86 and EA-87) from the patient evolved separate adaptations for antibiotic resistance, while sharing common adaptive mutations for host immune evasion. In EA-86, a silencing mutation in the antibiotic efflux pump repressor, NfxB, increased antibiotic resistance, while in EA-87, a similar mutation was seen in the antibiotic efflux pump repressor mexR. The number of genomic variants between the two isolates give a divergence time estimate of the order of 1000 generations. This time is sufficient for a bacterial lineage to have evolved an SNP in every position in the genome and been fixed if advantageous. This demonstrates the evolutionary adaptive power accessible to bacteria and the timescale for a brute-force functional survey of the SNP fitness landscape.

Role of AtYap1 in the reactive oxygen species regulation of lovastatin production in Aspergillus terreus

Lovastatin has great medical and economic importance, and its production in Aspergillus terreus is positively regulated at transcriptional level, by reactive oxygen species (ROS) generated during idiophase. To investigate the role of the transcription factor Yap1 in the regulation of lovastatin biosynthesis by ROS, an orthologue of yap1 was identified in A. terreus TUB F-514 and knocked down (silenced) by RNAi. Results confirmed that the selected knockdown strain (Siyap1) showed decreased yap1 expression in both culture systems (submerged and solid-state fermentation). Transformants showed higher sensitivity to oxidative stress. Interestingly, knockdown mutant showed higher ROS levels in idiophase and an important increase in lovastatin production in submerged and solid-state fermentations: 60 and 70% increase, respectively. Furthermore, sporulation also increased by 600%. This suggested that AtYap1 was functioning as a negative regulator of the biosynthetic genes, and that lack of AtYap1 in the mutants would be derepressing these genes and could explain increased production. However, we have shown that lovastatin production is proportional to ROS levels, so ROS increase in the mutants alone could also be the cause of production increase. In this work, when ROS levels were decreased with antioxidant, to the levels shown by the parental strain, the lovastatin production and kinetics were similar to the ones of the parental strain. This means that AtYap1 does not regulate lovastatin biosynthetic genes, and that production increase observed in the knockdown strain was an indirect effect caused by ROS increase. This conclusion is compared with studies on other secondary metabolites produced by other fungal species. KEY POINTS: • ROS regulates lovastatin biosynthesis at transcriptional level, in solid-state, and in submerged fermentations. • ATyap1 knockdown mutants showed important lovastatin production increases (60 and 70%) and higher ROS levels. • When ROS were decreased in the silenced mutant to the parental strain's level, lovastatin kinetics were identical to the parental strain's.

Silencing of ROT2, the Encoding Gene of the Endoplasmic Reticulum Glucosidase II, Affects the Cell Wall and the Sporothrix schenckii-Host Interaction.

Sporothrix schenckii is a member of the Sporothrix pathogenic clade and one of the most common etiological agents of sporotrichosis, a subcutaneous fungal infection that affects both animal and human beings. Like other fungal pathogens, the Sporothrix cell wall is composed of structural polysaccharides and glycoproteins that are covalently modified with both N-linked and O-linked glycans. Thus far, little is known about the N-linked glycosylation pathway in this organism or its contribution to cell wall composition and interaction with the host. Here, we silenced ROT2, which encodes the catalytic subunit of the endoplasmic reticulum α-glucosidase II, a processing enzyme key for the N-linked glycan core processing. Silencing of ROT2 led to the accumulation of the Glc2Man9GlcNAC2 glycan core at the cell wall and a reduction in the total content of N-linked glycans found in the wall. However, the highly silenced mutants showed a compensatory mechanism with increased content of cell wall O-linked glycans. The phenotype of mutants with intermediate levels of ROT2 silencing was more informative, as they showed changes in the cell wall composition and exposure of β-1.3-glucans and chitin at the cell surface. Furthermore, the ability to stimulate cytokine production by human mononuclear cells was affected, along with the phagocytosis by human monocyte-derived macrophages, in a mannose receptor-, complement receptor 3-, and TLR4-dependent stimulation. In an insect model of experimental sporotrichosis, these mutant cells showed virulence attenuation. In conclusion, S. schenckii ROT2 is required for proper N-linked glycosylation, cell wall organization and composition, and interaction with the host.

Abstract 298: Targeting KRAS mutant non-small cell lung cancer with EGFR aptamer displaying 3WJ pRNA nanoparticles

Abstract Background and Purpose: KRAS mutations are one of the most common oncogenic mutations in human cancers, including non-small cell lung cancer (NSCLC), and have established roles in cancer pathogenesis and therapeutic resistance. Development of effective inhibitors of mutant KRAS represent a significant challenge. The field of three-way junction (3WJ) based RNA nanoparticles has advanced rapidly and shows promise as an effective in vivo siRNA delivery system. This aim of this study is to explore the potential of 3WJ-pRNA nanoparticles for specific and efficient delivery of KRASG12C siRNA to NSCLC cells harboring a KRASG12C mutation to effectively silence KRAS oncogenic activity. Experimental design: Multifunctional EGFRapt-3WJ-siKRASG12C nanoparticles were prepared using bottom-up self-assembly approach, and characterized by agarose gel shift assay, dynamic light scattering (DLS), and temperature gradient gel electrophoresis system (TGGE). Three NSCLC cell lines, H2122 and H2030 carrying KRASG12C, and H1299 with wild type KRAS were used in this study. Binding specificity and delivery efficiency of EGFRapt-3WJ-siKRASG12C nanoparticles in NSCLC cells were evaluated in vitro and in vivo. Biological effects of KRAS mutant silencing were assessed by in vitro functional assays, and in vivo tumor xenograft experiments. Results: Characterization of EGFRapt-3WJ-siKRASG12C by DLS showed 7.2 ± 0.5 nm average size, and TGGE indicated a melting temperature (Tm) of 51.77°C. Flow cytometry, IVIS lumina imaging assay, real-time quantitative PCR, western blot, and KRAS activity assay confirmed that EGFRapt-3WJ-siKRASG12C potently and selectively depleted cellular KRAS expression, resulting in suppression of downstream MAPK pathway signaling in KRASG12C NSCLC cells and in heterotopic tumor models. In addition, in vitro cell proliferation, migration/invasion, and chemotherapy and radiotherapy sensitivity assays, demonstrated that KRAS silencing by EGFRapt-3WJ-siKRASG12C suppressed NSCLC cell growth, migration/invasion ability, and sensitized NSCLC cells to chemotherapy or radiotherapy. In vivo, these nanoparticles led to regression of tumor growth in heterotopic models. Conclusions: Together, this study suggests that 3WJ pRNA based platform has clinical potential to suppress mutant KRAS activity for the treatment of KRAS driven human cancers, and warrants further development. Citation Format: Linlin Yang, Zhefeng Li, Peixuan Guo, Terence M. Williams. Targeting KRAS mutant non-small cell lung cancer with EGFR aptamer displaying 3WJ pRNA nanoparticles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 298.

GABAergic and Glycinergic Synaptic Transmission in Respiratory‐Controlling Kolliker‐Fuse Neurons in Female and Male Rett Syndrome Mice

Rett Syndrome (RTT) is a neurodevelopmental disorder characterized by severe motor behavior disturbances including breathing problems. RTT is caused by silencing mutations in the methyl‐CpG binding protein 2 (MeCP2) gene located on the X chromosome. Accordingly, girls and women account for the majority of RTT patients. The genetic nature of RTT lends itself to study in mice with loss of function mutations in the MeCP2 gene. Importantly, RTT mice display breathing irregularities similar to human patients. Insufficient inhibitory neurotransmission in the brainstem respiratory network is thought to contribute to the breathing problems in RTT. In the pontine Kölliker‐Fuse (KF) area of RTT mice, there are fewer GABAergic axon terminals and injection of a GABA reuptake antagonist alleviates breathing problems in vivo. However, no one has examined the physiology of GABA or glycine synapses in the KF of RTT mice. In the present work, we tested the hypothesis that GABAergic and glycinergic synapses are deficient in the KF of RTT mice. We performed whole‐cell voltage clamp recordings from KF neurons contained in brain slices of MeCP2‐deficient RTT mice and wild type littermates. The frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in the KF neurons of male RTT mice was reduced compared to wild type (WT) littermates. In contrast, female RTT mice showed no change in sIPSC frequency in the KF compared to control, despite having breathing abnormalities. The amplitude of sIPSCs in the KF of female mice were smaller compared to WT littermates, whereas male RTT mice showed no change in amplitude compared to WT littermates. These results spotlight the importance of studying respiratory controlling KF neurons in both male and female RTT mice, and suggest that reductions in IPSC frequency and amplitude are not sufficient to explain the breathing irregularities observed in RTT in females and males, respectively.

The ectomycorrhizal basidiomycete Laccaria bicolor releases a GH28 polygalacturonase that plays a key role in symbiosis establishment.

In ectomycorrhiza, root penetration and colonization of the intercellular space by symbiotic hyphae is thought to rely on the mechanical force that results from hyphal tip growth, enhanced by the activity of secreted cell-wall-degrading enzymes. Here, we characterize the biochemical properties of the symbiosis-induced polygalacturonase LbGH28A from the ectomycorrhizal fungus Laccaria bicolor. The transcriptional regulation of LbGH28A was measured by quantitative PCR (qPCR). The biological relevance ofLbGH28A was confirmed by generating RNA interference (RNAi)-silenced LbGH28A mutants. We localized the LbGH28A protein by immunofluorescence confocal and immunogold cytochemical microscopy in poplar ectomycorrhizal roots. Quantitative PCR confirmed the induced expression of LbGH28A during ectomycorrhiza formation. Laccaria bicolor RNAi mutants have a lower ability to establish ectomycorrhiza, confirming the key role of this enzyme in symbiosis. The purified recombinant LbGH28A has its highest activity towards pectin and polygalacturonic acid. In situ localization of LbGH28A indicates that this endopolygalacturonase is located in both fungal and plant cell walls at the symbiotic hyphal front. These findings suggest that the symbiosis-induced pectinase LbGH28A is involved in the Hartig net formation and is an important determinant for successful symbiotic colonization.

Interaction with the CCT chaperonin complex limits APOBEC3A cytidine deaminase cytotoxicity.

The APOBEC3 cytidine deaminases are implicated as the cause of a prevalent somatic mutation pattern found in cancer genomes. The APOBEC3 enzymes act as viral restriction factors by mutating viral genomes. Mutation of the cellular genome is presumed to be an off-target activity of the enzymes, although the regulatory measures for APOBEC3 expression and activity remain undefined. It is therefore difficult to predict circumstances that enable APOBEC3 interaction with cellular DNA that leads to mutagenesis. The APOBEC3A (A3A) enzyme is the most potent deaminase of the family. Using proteomics, we evaluate protein interactors of A3A to identify potential regulators. We find that A3A interacts with the chaperonin-containing TCP-1 (CCT) complex, a cellular machine that assists in protein folding and function. Importantly, depletion of CCT results in A3A-induced DNA damage and cytotoxicity. Evaluation of cancer genomes demonstrates an enrichment of A3A mutational signatures in cancers with silencing mutations in CCT subunit genes. Together, these data suggest that the CCT complex interacts with A3A, and that disruption of CCT function results in increased A3A mutational activity.

Disruption of protein rhamnosylation affects the Sporothrix schenckii-host interaction

Sporotrichosis is a fungal disease caused by the members of the Sporothrix pathogenic clade, and one of the etiological agents is Sporothrix schenckii. The cell wall of this organism has been previously analyzed and thus far is known to contain an inner layer composed of chitin and β -glucans, and an outer layer of glycoproteins, which are decorated with mannose and rhamnose-containing oligosaccharides. The L-rhamnose biosynthesis pathway is common in bacteria but rare in members of the Fungi kingdom. Therefore, in this study, we aimed to disrupt this metabolic route to assess the contribution of rhamnose during the S.schenckii-host interaction. We identified and silenced in S. schenckii a functional ortholog of the bacterial rmlD gene, which encodes for an essential reductase for the synthesis of nucleotide-activated L-rhamnose. RmlD silencing did not affect fungal growth or morphology but decreased cell wall rhamnose content. Compensatory, the β-1,3-glucan levels increased and were more exposed at the cell surface. Moreover, when incubated with human peripheral blood mononuclear cells, the RmlD silenced mutants differentially stimulated cytokine production when compared with the wild-type strain, reducing TNFα and IL-6 levels and increasing IL-1 β and IL-10 production. Upon incubation with human monocyte-derived macrophages, the silenced strains were more efficiently phagocytosed than the wild-type strain. In both cases, our data suggest that rhamnose-based oligosaccharides are ligands that interact with TLR4. Finally, our findings showed that cell wall rhamnose is required for the S. schenckii virulence in the G. mellonella model of infection.

Contribution of soluble ORF2 viral protein to viral replication and host immune response during acute Hepatitis E virus infection

Abstract The hepatitis E virus (HEV) is a small, positive-stranded RNA virus that is a major cause of acute viral hepatitis globally. Acute HEV infection is typically asymptomatic and resolves within 8–10 weeks. HEV encodes 2 forms of capsid protein. A cytoplasmic form (ORF2c) is essential for virion structure. A secreted glycosylated form (ORF2s) accumulates at high titer in serum and can mask anti-ORF2 neutralizing antibodies. Here, we explored the contribution of ORF2s to HEV replicative fitness in vivo, and its role in generating anti-ORF2 antibodies (Abs). Rhesus Macaques (RM) were challenged by direct hepatic injection of infectious ORF2s+ and ORF2s− RNA. The replication of an HEV mutant lacking ORF2s expression was delayed by ~2 weeks when compared with wildtype virus and peak titers were nearly 10-fold lower for ORF2s−. No reversion of the 3 ORF2s silencing mutations was detected in the ORF2s− genomes, indicating genetic stability. The delay in replication and lower peak titer was unexpected as the viruses replicate similarly in cell culture. In addition, our data demonstrated that ORF2s has a significant and unexpected impact on generation of antibodies. Specifically, serum anti-ORF2 antibodies were only transiently detected in ORF2s− infected RM. As expected, anti-ORF2 titers were high and sustained in ORF2s+ infected RM. Furthermore, anti-ORF2 Ab response primed by ORF2s− infection differed in protection against reinfection when compared to ORF2s+. The ORF2s− challenged animals were re-infected upon second exposure to HEV infection. These findings indicate ORF2s may be dispensable for viral replication in vivo but is required for long-lived antibody response to mediate protection against re-exposure.

Construction and validation of an N6-methyladenosine-associated prognostic signature in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is among the most common types of cancer that threat the public health worldwide. N6-methyladenosine (m6A) RNA methylation is associated with cancer initiation and progression, and is dynamically regulated by m6A RNA methylation-associated genes. However, little is known about the expression status and the prognostic value of m6A-associated genes in HCC. The present study aimed to identify the expression profiling pattern and clinical significance of m6A-associated genes in HCC. Consensus clustering analysis was performed to identify the clusters of HCC with different clinical outcomes. A prognostic signature built by the least absolute shrinkage and selection operator Cox regression model was utilized to discover subtypes associated with different clinical outcomes of patients with HCC in the discovery cohort from The Cancer Genome Atlas. The differences between subgroups were characterized in terms of epigenetic dysregulation and somatic mutation frequencies. The International Cancer Genome Consortium cohort and two independent cohorts from the meta-Gene Expression Omnibus database were used for external validation. Most of the m6A-associated genes were upregulated and involved in the prognosis and malignancy of HCC. A four-gene prognostic signature revealed two HCC subtypes (namely, high- and low-risk group) that was associated with different clinical outcomes. Patients in the high-risk group were accompanied with increased epigenetic silencing and significant mutations in TP53 and FLG, while ALB was frequently mutated in the low-risk group. In conclusion, an m6A-based signature was constructed to predict the prognosis of patients with HCC, which may provide a tool for reliable prognosis assessment for clinicians, and aid clinical treatment decision-making.

Mutant Kras as a Biomarker Plays a Favorable Role in FL118-Induced Apoptosis, Reactive Oxygen Species (ROS) Production and Modulation of Survivin, Mcl-1 and XIAP in Human Bladder Cancer.

Simple SummaryFL118 is a novel orally available small molecule anticancer drug. We found that bladder cancer cells with a mutant Kras is highly sensitive to FL118-induced cell growth inhibition and cell death induction through inhibiting the anti-cancer cell death and drug resistance factors (survivin, Mcl-1, XIAP). In the Kras-mutation bladder cancer cells, FL118 can stimulate the reactive oxygen species (ROS) over-production for killing bladder cancer cells and inhibiting bladder cancer cell-established tumor growth. Elimination of mutant Kras by Kras-specific shRNA technology in mutant Kras-containing bladder cancer cell-established tumor decreased FL118 effectiveness to inhibit bladder cancer tumor growth. In this regard, mutant Kras is a potential favorable biomarker for FL118. This finding is significant because mutant Kras is known to be a formidable challenge treatment resistant factor in various types of cancer. Thus, FL118 could use mutant Kras as favorable biomarker for patient selection to carry out precision medicine.Tumor heterogeneity in key gene mutations in bladder cancer (BC) is a major hurdle for the development of effective treatments. Using molecular, cellular, proteomics and animal models, we demonstrated that FL118, an innovative small molecule, is highly effective at killing T24 and UMUC3 high-grade BC cells, which have Hras and Kras mutations, respectively. In contrast, HT1376 BC cells with wild-type Ras are insensitive to FL118. This concept was further demonstrated in additional BC and colorectal cancer cells with mutant Kras versus those with wild-type Kras. FL118 strongly induced PARP cleavage (apoptosis hallmark) and inhibited survivin, XIAP and/or Mcl-1 in both T24 and UMUC3 cells, but not in the HT1376 cells. Silencing mutant Kras reduced both FL118-induced PARP cleavage and downregulation of survivin, XIAP and Mcl-1 in UMUC3 cells, suggesting mutant Kras is required for FL118 to exhibit higher anticancer efficacy. FL118 increased reactive oxygen species (ROS) production in T24 and UMUC3 cells, but not in HT1376 cells. Silencing mutant Kras in UMUC3 cells reduced FL118-mediated ROS generation. Proteomics analysis revealed that a profound and opposing Kras-relevant signaling protein is changed in UMUC3 cells and not in HT1376 cells. Consistently, in vivo studies indicated that UMUC3 tumors are highly sensitive to FL118 treatment, while HT1376 tumors are highly resistant to this agent. Silencing mutant Kras in UMUC3 cell-derived tumors decreases UMUC3 tumor sensitivity to FL118 treatment. Together, our studies revealed that mutant Kras is a favorable biomarker for FL118 targeted treatment.