PrbP modulates biofilm formation in Liberibacter crescens

In Liberibacter asiaticus, PrbP is a transcriptional regulatory protein involved in survival and persistence during host infection. Tolfenamic acid was previously found to inhibit interactions between PrbP and the promotor region of rplK, resulting in reduced survival of L. asiaticus in the citrus host. In this study, we performed transcriptome analyses to elucidate the PrbP regulon in L. crescens, as it is phylogenetically the closest related species to L. asiaticus that can be grown in laboratory conditions. Chemical inhibition of PrbP with tolfenamic acid revealed that PrbP is involved in the regulation of diverse cellular processes, including stress response, cell motility, cell cycle and biofilm formation. In vitro DNA binding and bacterial two-hybrid assays also suggested that PrbP is a global regulator of multiple transcription factors (RpoH, VisN, PleD, MucR, MocR and CtrA) at both transcriptional and/or post-transcriptional levels. Sub-lethal concentrations of tolfenamic acid significantly reduced the attachment of L. crescens during biofilm formation and decreased long-term persistence in biofilm structures. Overall, our findings show the importance of PrbP in regulating diverse biological processes through direct and indirect interactions with other transcriptional regulators in L. crescens.Fil: Pan, Lei. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Gardner, Christopher L.. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Beliakoff, Reagan. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Da Silva, Danilo. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Zuo, Ran. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Pagliai, Fernando A.. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Padgett Pagliai, Kaylie A.. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Merli, Marcelo Luciano. University of Florida. Department of Microbiology and Cell Science; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Bahadiroglu, Erol. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Gonzalez, Claudio F.. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Lorca, Graciela L.. University of Florida. Department of Microbiology and Cell Science; Estados Unido

An IMU and USBL-aided buoy for underwater localization

Autonomous underwater navigation remains, as of today, a challenging task. The marine environment limits the number of sensors available for precise localization, hence Au- tonomous Underwater Vehicles (AUVs) usually rely on inertial and velocity sensors to obtain an estimate of their position either through dead reckoning or by means of more sophisticated navigation filters (such as Kalman filters and its extensions [1]). On the other hand, acoustic localization makes possible the determination of a reliable vehicles pose estimate exploiting suit- able acoustic modems [3]; such estimate can even be integrated within the navigation filter of the vehicle in order to increase its accuracy. In this paper, the authors discuss the development and the performance of an Ultra-Short BaseLine (USBL)-aided buoy to improve the localization of underwater vehicles. At first, the components and the physical realization of the buoy will be discussed; then, the procedure to compute the position of the target will be analyzed. The following part of the paper will be focused on the development of a recursive state estimation algorithm to process the measurements computed by the buoy; specifically, Extended Kalman Filter [4] has been adopted to deal with the nonlinearities of the sensors housed on the buoy. A validation of the measurement filtering through experimental tests is also proposed

An IMU and USBL-aided buoy for underwater localization

Autonomous underwater navigation remains, as of today, a challenging task. The marine environment limits the number of sensors available for precise localization, hence Au- tonomous Underwater Vehicles (AUVs) usually rely on inertial and velocity sensors to obtain an estimate of their position either through dead reckoning or by means of more sophisticated navigation filters (such as Kalman filters and its extensions [1]). On the other hand, acoustic localization makes possible the determination of a reliable vehicles pose estimate exploiting suit- able acoustic modems [3]; such estimate can even be integrated within the navigation filter of the vehicle in order to increase its accuracy. In this paper, the authors discuss the development and the performance of an Ultra-Short BaseLine (USBL)-aided buoy to improve the localization of underwater vehicles. At first, the components and the physical realization of the buoy will be discussed; then, the procedure to compute the position of the target will be analyzed. The following part of the paper will be focused on the development of a recursive state estimation algorithm to process the measurements computed by the buoy; specifically, Extended Kalman Filter [4] has been adopted to deal with the nonlinearities of the sensors housed on the buoy. A validation of the measurement filtering through experimental tests is also proposed

Drug repurposing: tolfenamic acid inactivates PrbP, a transcriptional accessory protein in liberibacter asiaticus

CLIBASIA_01510, PrbP, is a predicted RNA polymerase binding protein in Liberibacter asiaticus. PrbP was found to regulate expression of a small subset of ribosomal genes through interactions with the β-subunit of the RNA polymerase and a short, specific sequence on the promoter region. Molecular screening assays were performed to identify small molecules that interact with PrbP in vitro. Chemical hits were analyzed for therapeutic efficacy against L. asiaticus via an infected leaf assay, where the transcriptional activity of L. asiaticus was found to decrease significantly after exposure to tolfenamic acid. Similarly, tolfenamic acid was found to inhibit L. asiaticus infection in highly symptomatic citrus seedlings. Our results indicate that PrbP is an important transcriptional regulator for survival of L. asiaticus in planta, and the chemicals identified by molecular screening assays could be used as a therapeutic treatment for huanglongbing disease.Fil: Gardner, Christopher L.. University of Florida; Estados UnidosFil: Pagliai, Fernando A.. University of Florida; Estados UnidosFil: Pan, Lei. University of Florida; Estados UnidosFil: Bojilova, Lora. University of Florida; Estados UnidosFil: Torino, Maria Ines. University of Florida; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Tucumán. Centro de Referencia para Lactobacilos; ArgentinaFil: Lorca, Graciela L.. University of Florida; Estados UnidosFil: Gonzalez, Claudio F.. University of Florida; Estados Unido

‘Candidatus Liberibacter asiaticus’ Multimeric LotP Mediates Citrus sinensis Defense Response Activation

‘Candidatus Liberibacter asiaticus’ is known as the most pathogenic organism associated with citrus greening disease. Since its publicized emergence in Florida in 2005, ‘Ca. L. asiaticus’ remains unculturable. Currently, a limited number of potential disease effectors have been identified through in silico analysis. Therefore, these potential effectors remain poorly characterized and do not fully explain the complexity of symptoms observed in citrus trees infected with ‘Ca. L. asiaticus.’ LotP has been identified as a potential effector and have been partially characterized. This protein retains structural homology to the substrate binding domain of the Lon protease. LotP interacts with chaperones like GroEL, Hsp40, DnaJ, and ClpX and may exercise its biological role through interactions with different proteins involved in proteostasis networks. Here, we evaluate the interactome of LotP—revealing a new protein–protein interaction target (Lon-serine protease) and its effect on citrus plant tissue integrity. We found that via protein–protein interactions, LotP can enhance Lon protease activity, increasing the degradation rate of its specific targets. Infiltration of purified LotP strained citrus plant tissue causing photoinhibition and chlorosis after several days. Proteomics analysis of LotP tissues recovering after the infiltration revealed a large abundance of plant proteins associated with the stabilization and processing of mRNA transcripts, a subset of important transcription factors; and pathways associated with innate plant defense were highly expressed. Furthermore, interactions and substrate binding module of LotP suggest potential interactions with plant proteins, most likely proteases.Fil: Merli, Marcelo Luciano. University of Florida. Department of Microbiology and Cell Science; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Padgett Pagliai, Kaylie A.. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Cuaycal, Alexandra E.. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: García, Lucila. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Marano, María Rosa. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Lorca, Graciela L.. University of Florida. Department of Microbiology and Cell Science; Estados UnidosFil: Gonzalez, Claudio F.. University of Florida. Department of Microbiology and Cell Science; Estados Unido

Raman and Computational Study on the Adsorption of Xanthine on Silver Nanocolloids

Xanthine is a nucleobase, deriving from adenine and guanine by deamination and oxidation processes, which may deposit in the human body causing diseases, similar to uric acid. Here, we have investigated the adsorption of xanthine on silver colloidal nanoparticles by means of surface-enhanced Raman scattering (SERS) with an exciting radiation in the near-infrared spectral region, where interference due to fluorescence does not occur, along with density functional theory calculations of molecule/metal model systems. By adopting a combined experimental and computational approach, we have identified the "marker" SERS bands of xanthine and the tautomer that preferentially binds the silver particles, as well as the molecular group involved in the interaction with metal. This investigation allows using the FT-SERS spectroscopy for biosensory and diagnostic purposes in body fluids, detecting abnormal levels of xanthine, and preventing metabolic diseases

A serralysin-like protein of Candidatus Liberibacter asiaticus modulates components of the bacterial extracellular matrix

Huanglongbing (HLB), the current major threat for Citrus species, is caused by intracellular alphaproteobacteria of the genus Candidatus Liberibacter (CaL), with CaL asiaticus (CLas) being the most prevalent species. This bacterium inhabits phloem cells and is transmitted by the psyllid Diaphorina citri. A gene encoding a putative serralysin-like metalloprotease (CLIBASIA_01345) was identified in the CLas genome. The expression levels of this gene were found to be higher in citrus leaves than in psyllids, suggesting a function for this protease in adaptation to the plant environment. Here, we study the putative role of CLas-serralysin (Las1345) as virulence factor. We first assayed whether Las1345 could be secreted by two different surrogate bacteria, Rhizobium leguminosarum bv. viciae A34 (A34) and Serratia marcescens. The protein was detected only in the cellular fraction of A34 and S. marcescens expressing Las1345, and increased protease activity of those bacteria by 2.55 and 4.25- fold, respectively. In contrast, Las1345 expressed in Nicotiana benthamiana leaves did not show protease activity nor alterations in the cell membrane, suggesting that Las1345 do not function as a protease in the plant cell. Las1345 expression negatively regulated cell motility, exopolysaccharide production, and biofilm formation in Xanthomonas campestris pv. campestris (Xcc). This bacterial phenotype was correlated with reduced growth and survival on leaf surfaces as well as reduced disease symptoms in N. benthamiana and Arabidopsis. These results support a model where Las1345 could modify extracellular components to adapt bacterial shape and appendages to the phloem environment, thus contributing to virulence

The treatment of diffuse cutaneous systemic sclerosis with autologous hemopoietic stem cells transplantation (HSCT): our experience on 2 cases

Objectives: Autologous hematopoietic stem cell transplantation (HSCT) is a treatment option which may be considered for severe diffuse cutaneous systemic sclerosis (dcSSc) patients not responding to cyclophophamide (CY). We present two cases of dcSSc not responding to CY >10 g who were successfully treated with HSCT. Patients and methods: Two dcSSc patients were unresponsive to monthly i.v. pulse of CYC (0.75 g m2). Both patients had significant reduction of DLCO and mild-moderate pulmonary hypertension and HSCT was considered due to the rapid progression of the disease. Following informed consent and ethics committee approval, HSCT was performed. Mobilisation was performed with CY 4g/m2 and recombinant human granulocyte colony stimulating factor (rHu GCSF) followed by a successful apheresis (CD34+ cells, >7X106). Conditioning regimens were: CY 100mg/kg body weight plus thiotepa 10 mg/ kg in the first patient and CY 200 mg/kg in the second. Both graft products were CD34 selected. No arrythmias occurred during the procedure and no other severe side effects were observed during hospitalisation. Results: Follow up: Patients underwent a monthly follow up with physical examination, pulmonary function tests and echocardiography every 3 months. Chest CT has been performed 6 months post transplantation. The following was observed: skin score (from 40 to 10 for the first patient and from 38 to 12 for the second one), LVEF and pulmonary function remained stable, PAP decreased from 45 mmHg to 35 mmHg and from 40 to 32 mmHg. No late complications or cardiac toxicity was observed. Conclusion: These two dcSSc cases demonstrate that HSCT may be successfully performed without serious side effects in cases in whom despite a cumulative CY dose was ineffective. This suggests an “immunological threshold” effect which may be exploited in other severe, therapy refractory autoimmune cases