N-acetylcysteine absorption and its potential dual effect improve fitness and fruit yield in Xylella fastidiosa infected plants.

BACKGROUND: Xylella fastidiosa is a multi-host bacterium that can be detected in hundreds of plant species including several crops. Diseases caused by X. fastidiosa are considered a threat to global food production. The primary method for managing diseases caused by X. fastidiosa involves using insecticides to control the vector. Hence, it is necessary to adopt new and sustainable disease management technologies to control not only the insect but also the bacteria and plant health. We demonstrated that N-acetylcysteine (NAC), a low-cost cysteine analogue, is a sustainable molecule that can be used in agriculture to decrease the damage caused by X. fastidiosa and improve plant health. RESULTS: Using 15N-NAC we proved that this analogue was absorbed by the roots and transported to different parts of the plant. Inside the plant, NAC reduced the bacterial population by 60-fold and the number of xylem vessels blocked by bacterial biofilms. This reflected in a recovery of 0.28-fold of the daily sap flow compared to health plants. In addition, NAC-treated citrus variegated chlorosis (CVC) plants decreased the oxidative stress by improving the activity of detoxifying enzymes. Moreover, the use of NAC in field conditions positively contributed to the increase in fruit yield of CVC-diseased plants. CONCLUSION: Our research not only advances the understanding of NAC absorption in plants, but also indicates its dual effect as an antimicrobial and antioxidant molecule. This, in turn, negatively affects bacterial survival while improving plant health by decreasing oxidative stress. Overall, the positive field-based evidence supports the viability of NAC as a sustainable agricultural application. © 2024 Society of Chemical Industry.

The Midfoot Joint Complex (Foot Arch) Contributes to the Upper Body Position in Bipedal Walking and Coordinates With the Lower Limb Joints.

This study estimated the contribution of the midfoot joint complex (MJC) kinematics to the pelvis anterior-posterior positions during the stance phase of walking and investigated whether the MJC is functionally coordinated with the lower limb joints to maintain similar pelvic positions across steps. Hip, knee, ankle, and MJC sagittal angles were measured in 11 nondisabled participants during walking. The joints' contributions to pelvic positions were computed through equations derived from a link-segment model. Functional coordination across steps was identified when the MJC contribution to pelvic position varied and the summed contributions of other joints varied in the opposite direction (strong negative covariations [r ≤ -.7] in stance phase instants). We observed that the MJC plantarflexion (arch raising) during the midstance and late stance leads the pelvis backward, avoiding excessive forward displacement. The MJC was the second joint that contributed most to the pelvis positions (around 18% of all joints' contributions), after the ankle joint. The MJC and ankle were the joints that were most frequently coordinated with the other joints (≅70% of the stance phase duration). The findings suggest that the MJC is part of the kinematic chain that determines pelvis positions during walking and is functionally coordinated with the lower limb joints.

Persister Cells Form in the Plant Pathogen Xanthomonas citri subsp. citri under Different Stress Conditions.

Citrus canker disease, caused by the bacterium Xanthomonas citri subsp. citri is a constant threat to citrus-producing areas. Since it has no cure, agricultural practices to restrain its dissemination are essential to reduce the economic damage. Hence, increased knowledge of the basic aspects of X. citri biology could lead to more efficient management practices that can eliminate dormant bacteria in the field. The dormant cells, also referred to as persisters, are phenotypic variants with lowered metabolism, which in turn leads to tolerance to antimicrobials and undermines existing control approaches. We show here that X. citri forms persisters, identifying triggers for this phenotype, including antibiotics, high temperature, and metals (copper and zinc), which increase persistence rates by 10-100 times. The antioxidant N-acetylcysteine reduced copper and zinc-induced persisters, but not those induced by tetracycline, indicating that oxidative stress may be an important inducer of X. citri persistence. In addition, we found that metabolism-independent drugs like cisplatin and mitomycin C are able to eliminate X. citri persistent cells, as well as copper, at high concentrations. Specific amino acids like proline and isoleucine interfered with the physiological balance of the dormancy in X. citri, stimulating or preventing persister resuscitation. Taken together, we discover chemicals that can induce, wake, and kill X. citri persister cells; these results provide insights that should be considered for more efficient integrated control management in the field.

Riboswitch theo/metE as a Transcription Regulation Tool for Xanthomonas citri subsp. citri.

: Xanthomonas citri subsp. citri (X. citri) is the causal agent of Asiatic Citrus Canker (ACC), a disease that affects citrus. ACC has no cure, and growers must rely on special agricultural practices to prevent bacterial spreading. Understanding X. citri basic biology is essential to foresee potential genetic targets to control ACC. Traditionally, microbial genetics use gene deletion/disruption to investigate gene function. However, essential genes are difficult to study this way. Techniques based on small-RNAs and antisense-RNAs are powerful for gene characterization, but not yet fully explored in prokaryotes. One alternative is riboswitches, which derive from bacteria, and can control transcription/translation. Riboswitches are non-coding RNAs able to modulate gene expression in the presence of specific ligands. Here we demonstrate that the riboswitch theo/metE decreases parB expression in X. citri in a platform responsive to theophylline. By monitoring cell respiration, we showed that higher concentrations of the ligand interfered with bacterial viability. Therefore, we determined the safe dose of theophylline to be used with X. citri. Finally, in downstream investigations of parB transcription modulation, we show evidence for the fact that ParB is stable, remains functional throughout the cell cycle, and is inherited by the daughter cells upon cell division.

A comparison between electrospinning and rotary-jet spinning to produce PCL fibers with low bacteria colonization.

One of the important components in tissue engineering is material structure, providing a model for fixing and the development of cells and tissues, which allows for the transport of nutrients and regulatory molecules to and from cells. The community claims the need for new materials with better properties for use in the clinic. Poly (ε-caprolactone) (PCL) is a biodegradable polymer, semi crystalline, with superior mechanical properties and has attracted an increasing interest due to its usefulness in various biomedical applications. Herein, two different methods (electrospinning versus rotary jet spinning) with different concentrations of PCL produced ultra thin-fibers each with particular characteristics, verified and analyzed by morphology, wettability, thermal and cytotoxicity features and for bacteria colonization. Different PCL scaffold morphologies were found to be dependent on the fabrication method used. All PCL scaffolds showed greater mammalian cell interactions. Most impressively, rotary-jet spun fibers showed that a special rough surface decreased bacteria colonization, emphasizing that no nanoparticle or antibiotic was used; maybe this effect is related with physical (scaffold) and/or biological mechanisms. Thus, this study showed that rotary jet spun fibers possess a special topography compared to electrospun fibers to reduce bacteria colonization and present no cytotoxicity when in contact with mammalian cells.

Persistence in Phytopathogenic Bacteria: Do We Know Enough?

Phytopathogenic bacteria affect a wide range of crops worldwide and have a negative impact in agriculture due to their associated economic losses and environmental impacts. Together with other biotic and abiotic stress factors, they pose a threat to global food production. Therefore, understanding bacterial survival strategies is an essential step toward the development of new strategies to control plant diseases. One mechanism used by bacteria to survive under stress conditions is the formation of persister cells. Persisters are a small fraction of phenotypic variants within an isogenic population that exhibits multidrug tolerance without undergoing genetic changes. They are dormant cells that survive treatment with antimicrobials by inactivating the metabolic functions that are disrupted by these compounds. They are thus responsible for the recalcitrance of many human diseases, and in the same way, they are thought to contribute to the survival of bacterial phytopathogens under a range of stresses they face in the environment. It is believed that persister cells of bacterial phytopathogens may lead to the reoccurrence of disease by recovering growth and recolonizing the host plant after the end of stress. However, compared to human pathogens, little is known about persister cells in phytopathogens, especially about their genetic regulation. In this review, we describe the overall knowledge on persister cells and their regulation in bacterial phytopathogens, focusing on their ability to survive stress conditions, to recover from dormancy and to maintain virulence.

Stochastic techno-economic analysis of the production of aviation biofuel from oilseeds.

BACKGROUND: The economic viability of hydrodeoxygenation process using Camelina, Carinata and Jatropha feedstocks for aviation biofuel production was evaluated for two product profiles: (i) maximum diesel production and (ii) maximum jet fuel production (HRJ). RESULTS: Deterministic analysis of Camelina and Carinata diesel facilities returned positive NPVs and IRRs of 25 and 18%, respectively. Stochastic analysis suggested that the probabilities of positive NPVs were 75, 59 and 15%, respectively, for Camelina, Carinata and Jatropha diesel plants. Jet fuel facilities presented probabilities of loss of 98, 99 and 100% for Camelina, Carinata and Jatropha scenarios, respectively. Sensitivity analysis determined that financial performance was majorly influenced by feedstock and fuel prices. Categories of subsidies to enhance the attractiveness of the projects were studied. CONCLUSIONS: Camelina, Carinata and Jatropha plants targeting HRJ required incentives of 0.31, 0.39 and 0.61 US$/L of biofuel produced, respectively, to reduce the probabilities of loss to approximately 30%.

New Vaccine Formulations Containing a Modified Version of the Amastigote 2 Antigen and the Non-Virulent Trypanosoma cruzi CL-14 Strain Are Highly Antigenic and Protective against Leishmania infantum Challenge.

Visceral leishmaniasis (VL) is a major public health issue reported as the second illness in mortality among all tropical diseases. Clinical trials have shown that protection against VL is associated with robust T cell responses, especially those producing IFN-γ. The Leishmania amastigote 2 (A2) protein has been repeatedly described as immunogenic and protective against VL in different animal models; it is recognized by human T cells, and it is also commercially available in a vaccine formulation containing saponin against canine VL. Moving toward a more appropriate formulation for human vaccination, here, we tested a new optimized version of the recombinant protein (rA2), designed for Escherichia coli expression, in combination with adjuvants that have been approved for human use. Moreover, aiming at improving the cellular immune response triggered by rA2, we generated a recombinant live vaccine vector using Trypanosoma cruzi CL-14 non-virulent strain, named CL-14 A2. Mice immunized with respective rA2, adsorbed in Alum/CpG B297, a TLR9 agonist recognized by mice and human homologs, or with the recombinant CL-14 A2 parasites through homologous prime-boost protocol, were evaluated for antigen-specific immune responses and protection against Leishmania infantum promastigote challenge. Immunization with the new rA2/Alum/CpG formulations and CL-14 A2 transgenic vectors elicited stronger cellular immune responses than control groups, as shown by increased levels of IFN-γ, conferring protection against L. infantum challenge. Interestingly, the use of the wild-type CL-14 alone was enough to boost immunity and confer protection, confirming the previously reported immunogenic potential of this strain. Together, these results support the success of both the newly designed rA2 antigen and the ability of T. cruzi CL-14 to induce strong T cell-mediated immune responses against VL in animal models when used as a live vaccine vector. In conclusion, the vaccination strategies explored here reveal promising alternatives for the development of new rA2 vaccine formulations to be translated human clinical trials.

Evaluation of cell proliferation and endometrial thickness of bitches in different periods of diestrus

ABSTRACT This study aimed to determine the histological features of the endometrium of bitches, as well as the cell proliferation at specific moments of diestrus, 10, 20, 30, 40, 50 and 60 days post ovulation, correlating the endometrial thickness with the uterine cell proliferation and the metabolic state (weight, blood glucose and plasma cholesterol) of the animals. Therefore, the right and left uterine horns of 26 clinically healthy bitches submitted to ovariohysterectomy were histologically analyzed 10, 20, 30, 40, 50 and 60 days post ovulation. The hematoxylin-eosin and AgNOR staining techniques were performed. All parameters were evaluated by ANOVA and post-hoc Tukey test (p<0.05). The correlation between endometrial thickness and uterine cell proliferation, weight, blood glucose and plasma cholesterol of animals was observed using the Pearson method (p<0.05). In the present study, it is concluded that endometrial thickness does not differ at any of the moments analyzed in diestrus. The endometrial thickness is not influenced by hormones, weight, blood glucose or serum cholesterol of bitches in this phase of the estrous cycle. However, there is greater cell proliferation in the endometrium at day 40 compared to day 60 post ovulation under the influence of the endocrine profile.

Evaluation of cell proliferation and endometrial thickness of bitches in different periods of diestrus.

This study aimed to determine the histological features of the endometrium of bitches, as well as the cell proliferation at specific moments of diestrus, 10, 20, 30, 40, 50 and 60 days post ovulation, correlating the endometrial thickness with the uterine cell proliferation and the metabolic state (weight, blood glucose and plasma cholesterol) of the animals. Therefore, the right and left uterine horns of 26 clinically healthy bitches submitted to ovariohysterectomy were histologically analyzed 10, 20, 30, 40, 50 and 60 days post ovulation. The hematoxylin-eosin and AgNOR staining techniques were performed. All parameters were evaluated by ANOVA and post-hoc Tukey test (p<0.05). The correlation between endometrial thickness and uterine cell proliferation, weight, blood glucose and plasma cholesterol of animals was observed using the Pearson method (p<0.05). In the present study, it is concluded that endometrial thickness does not differ at any of the moments analyzed in diestrus. The endometrial thickness is not influenced by hormones, weight, blood glucose or serum cholesterol of bitches in this phase of the estrous cycle. However, there is greater cell proliferation in the endometrium at day 40 compared to day 60 post ovulation under the influence of the endocrine profile.

Protein depletion using the arabinose promoter in Xanthomonas citri subsp. citri.

Xanthomonas citri subsp. citri (X. citri) is a plant pathogen and the etiological agent of citrus canker, a severe disease that affects all the commercially important citrus varieties, and has worldwide distribution. Citrus canker cannot be healed, and the best method known to control the spread of X. citri in the orchards is the eradication of symptomatic and asymptomatic plants in the field. However, in the state of São Paulo, Brazil, the main orange producing area in the world, control is evolving to an integrated management system (IMS) in which growers have to use less susceptible plants, windshields to prevent bacterial spread out and sprays of cupric bactericidal formulations. Our group has recently proposed alternative methods to control citrus canker, which are based on the use of chemical compounds able to disrupt vital cellular processes of X. citri. An important step in this approach is the genetic and biochemical characterization of genes/proteins that are the possible targets to be perturbed, a task not always simple when the gene/protein under investigation is essential for the organism. Here, we describe vectors carrying the arabinose promoter that enable controllable protein expression in X. citri. These vectors were used as complementation tools for the clean deletion of parB in X. citri, a widespread and conserved gene involved in the essential process of bacterial chromosome segregation. Overexpression or depletion of ParB led to increased cell size, which is probably a resultant of delayed chromosome segregation with subsequent retard of cell division. However, ParB is not essential in X. citri, and in its absence the bacterium was fully competent to colonize the host citrus and cause disease. The arabinose expression vectors described here are valuable tools for protein expression, and especially, to assist in the deletion of essential genes in X. citri.

PAMPs, PRRs, effectors and R-genes associated with citrus-pathogen interactions.

BACKGROUND: Recent application of molecular-based technologies has considerably advanced our understanding of complex processes in plant-pathogen interactions and their key components such as PAMPs, PRRs, effectors and R-genes. To develop novel control strategies for disease prevention in citrus, it is essential to expand and consolidate our knowledge of the molecular interaction of citrus plants with their pathogens. SCOPE: This review provides an overview of our understanding of citrus plant immunity, focusing on the molecular mechanisms involved in the interactions with viruses, bacteria, fungi, oomycetes and vectors related to the following diseases: tristeza, psorosis, citrus variegated chlorosis, citrus canker, huanglongbing, brown spot, post-bloom, anthracnose, gummosis and citrus root rot.

Type II Toxin-Antitoxin Distribution and Adaptive Aspects on Xanthomonas Genomes: Focus on Xanthomonas citri.

Prokaryotic toxin-antitoxin (TA) systems were first described as being designed to prevent plasmid loss in bacteria. However, with the increase in prokaryotic genome sequencing, recently many TAs have been found in bacterial chromosomes, having other biological functions, such as environmental stress response. To date, only few studies have focused on TA systems in phytopathogens, and their possible impact on the bacterial fitness. This may be especially important for pathogens like Xanthomonas spp., which live epiphytically before entering the host. In this study, we looked for TA systems in the genomes of 10 Xanthomonas strains. We verified that citrus-infecting pathovars have, on average, 50% more TAs than other Xanthomonas spp. and no genome harbors classical toxins such as MqsR, RelB, and HicA. Only one TA system (PIN_VapC-FitB-like/SpoVT_AbrB) was conserved among the Xanthomonas genomes, suggesting adaptive aspects concerning its broad occurrence. We also detected a trend of toxin gene loss in this genus, while the antitoxin gene was preferably maintained. This study discovers the quantitative and qualitative differences among the type II TA systems present in Xanthomonas spp., especially concerning the citrus-infecting strains. In addition, the antitoxin retention in the genomes is possibly related with the resistance mechanism of further TA infections as an anti-addiction system or might also be involved in regulation of certain specific genes.

A protein expression system for tandem affinity purification in Xanthomonas citri subsp. citri

Abstract Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (Xac), is one of the most devastating diseases to affect citrus crops. There is no treatment for citrus canker; effective control against the spread of Xac is usually achieved by the elimination of affected plants along with that of asymptomatic neighbors. An in depth understanding of the pathogen is the keystone for understanding of the disease; to this effect we are committed to the development of strategies to ease the study of Xac. Genome sequencing and annotation of Xac revealed that ∼37% of the genome is composed of hypothetical ORFs. To start a systematic characterization of novel factors encoded by Xac, we constructed integrative-vectors for protein expression specific to this bacterium. The vectors allow for the production of TAP-tagged proteins in Xac under the regulation of the xylose promoter. In this study, we show that a TAP-expression vector, integrated into the amy locus of Xac, does not compromise its virulence. Furthermore, our results also demonstrate that the polypeptide TAP can be overproduced in Xac and purified from the soluble phase of cell extracts. Our results substantiate the use of our vectors for protein expression in Xac thus contributing a novel tool for the characterization of proteins and protein complexes generated by this bacterium in vivo.

A protein expression system for tandem affinity purification in Xanthomonas citri subsp. citri.

Citrus canker, caused by the Gram-negative bacterium Xanthomonas citri subsp. citri (Xac), is one of the most devastating diseases to affect citrus crops. There is no treatment for citrus canker; effective control against the spread of Xac is usually achieved by the elimination of affected plants along with that of asymptomatic neighbors. An in depth understanding of the pathogen is the keystone for understanding of the disease; to this effect we are committed to the development of strategies to ease the study of Xac. Genome sequencing and annotation of Xac revealed that ∼37% of the genome is composed of hypothetical ORFs. To start a systematic characterization of novel factors encoded by Xac, we constructed integrative-vectors for protein expression specific to this bacterium. The vectors allow for the production of TAP-tagged proteins in Xac under the regulation of the xylose promoter. In this study, we show that a TAP-expression vector, integrated into the amy locus of Xac, does not compromise its virulence. Furthermore, our results also demonstrate that the polypeptide TAP can be overproduced in Xac and purified from the soluble phase of cell extracts. Our results substantiate the use of our vectors for protein expression in Xac thus contributing a novel tool for the characterization of proteins and protein complexes generated by this bacterium in vivo.

Temperature stress promotes cell division arrest in Xanthomonas citri subsp. citri.

Citrus canker is an economically important disease that affects orange production in some of the most important producing areas around the world. It represents a great threat to the Brazilian and North American citriculture, particularly to the states of São Paulo and Florida, which together correspond to the biggest orange juice producers in the world. The etiological agent of this disease is the Gram-negative bacterium Xanthomonas citri subsp. citri (Xcc), which grows optimally in laboratory cultures at ~30 °C. To investigate how temperatures differing from 30 °C influence the development of Xcc, we subjected the bacterium to thermal stresses, and afterward scored its recovery capability. In addition, we analyzed cell morphology and some markers of essential cellular processes that could indicate the extent of the heat-induced damage. We found that the exposure of Xcc to 37 °C for a period of 6 h led to a cell cycle arrest at the division stage. Thermal stress might have also interfered with the DNA replication and/or the chromosome segregation apparatuses, since cells displayed an increased number of sister origins side-by-side within rods. Additionally, Xcc treated at 37 °C was still able to induce citrus canker symptoms, showing that thermal stress did not affect the ability of Xcc to colonize the host citrus. At 40-42 °C, Xcc lost viability and became unable to induce disease symptoms in citrus. Our results provide evidence about essential cellular mechanisms perturbed by temperature, and can be potentially explored as a new method for Xanthomonas citri synchronization in cell cycle studies, as well as for the sanitation of plant material.

Asymmetric chromosome segregation in Xanthomonas citri ssp. citri.

This study was intended to characterize the chromosome segregation process of Xanthomonas citri ssp. citri (Xac) by investigating the functionality of the ParB factor encoded on its chromosome, and its requirement for cell viability and virulence. Using TAP tagging we show that ParB is expressed in Xac. Disruption of parB increased the cell doubling time and precluded the ability of Xac to colonize the host citrus. Moreover, Xac mutant cells expressing only truncated forms of ParB exhibited the classical phenotype of aberrant chromosome organization, and seemed affected in cell division judged by their reduced growth rate and the propensity to form filaments. The ParB-GFP localization pattern in Xac was suggestive of an asymmetric mode of replicon partitioning, which together with the filamentation phenotype support the idea that Xac may control septum placement using mechanisms probably analogous to Caulobacter crescentus, and perhaps Vibrio cholerae, and Corynebacterium glutamicum. Xac exhibits asymmetric chromosome segregation, and the perturbation of this process leads to an inability to colonize the host plant.

Low-cost and low maintenance preservation of Agaricus brasiliensis cultures.

Agaricus brasiliensis cultures quickly lose viability when stored at cool temperatures, even for a short period of time. We evaluated several low-cost preservation methods using varied substrates, preservation solutions, and storage temperatures. Agaricus brasiliensis was intolerant to freezing temperatures, making liquid nitrogen use and deep-freezing methods impossible for its preservation. The best preservation conditions for the A. brasiliensis CS1 strain tested in this study were obtained by using rice as substrate and water as preservation solution, with storage at room temperature or when using soil, mushroom cultivation compost, or rice and stored at 10 °C without preservation solution. Those cultures that were reactivated showed the same productivity attributes as the control. In addition, no effect on productivity or biological efficiency was observed through successive subculturing of the strain (CS1). Parboiled rice was successfully used for other A. brasiliensis strains (CS2, CS5, CS7, CS9, and CS10), and also for Pleurotus ostreatus, P. sajor-caju, and Lentinula edodes.

Treatment of mesenteric angina in patients with Takayasu's arteritis.

Takayasu's arteritis (TA) is an idiopathic chronic inflammatory disease of the connective tissue that affects mainly the aorta and its branches. Treatment is mainly based on corticosteroids and immunosuppressants. We report the case of a 33-year-old female complaining of malaise, fever, myalgia, severe pulsing holocranial headache resistant to analgesics, systemic arterial hypertension hard to control, right lower limb claudication, and severe abdominal pain that worsened after the meals. Angiotomography revealed aneurysm of the ascending aorta, and stenosis of the following vessels: right common iliac artery, renal arteries, and superior mesenteric artery. Those findings supported the diagnosis of mesenteric angina and the interventional approach by use of percutaneous transluminal angioplasty with stent placement.