Sensitivity of different organs and tissues as biomarkers of oxidative stress in juvenile tambaqui (Colossoma macropomum) submitted to fasting.

The present study was conducted to evaluate the effects of fasting on responses of oxidative biomarkers and antioxidant defenses using different organs and tissues of Colossoma macropomum. The fish were divided into two groups: fed (control) and fasting (7 days). After 7 days, the fish were sampled for assessment of oxidative stress biomarkers (MDA-lipid peroxidation and PCO-protein carbonyl) and antioxidant defenses (SOD-superoxide dismutase; CAT-catalase; GPX-glutathione peroxidase; and GST-glutathione-S -transferase) in the liver, intestine, gills, muscle, brain, and plasma. The results showed an increase in MDA, PCO, SOD, and GPX concentrations in the liver and intestine of fasting fish. In contrast, in the branchial tissue, there was a reduction in the activity of SOD and CAT enzymes in fasting fish. There was also a reduction in CAT activity in the muscle of fasting fish, while in the brain, there were no changes in oxidative stress biomarkers. Plasma showed a relatively low antioxidant response. In conclusion, our results confirm that a 7-day fasting period induced tissue-specific antioxidant responses, but the increase in antioxidant responses was only for the SOD and GPX enzymes of the liver and intestine. Additionally, the liver and intestine were the most responsive tissues, whereas the plasma was the least sensitive to oxidative stress.

Spirulina (Arthrospira platensis) supplementation: Impact on growth, metabolism, and antioxidant status in zebrafish.

Zebrafish (Danio rerio) is a valuable model for biomedical research because of its mammalian genetic similarities, rapid reproduction, and low maintenance costs. However, further investigation is required regarding their nutritional requirements and standardized laboratory diets. This study evaluated the metabolic and growth responses of zebrafish juveniles fed on diets supplemented with spirulina, Arthrospira platensis (SP) at different levels for 77 days. Six diets with SP inclusion levels of 0%, 2%, 4%, 6%, 8%, and 10% (SP0-SP10) were formulated. A total of 300 zebrafish juveniles with an average initial weight of 0.113 ± 0.10 g (mean ± SD) were randomly distributed across six groups, with five replicates per group, each containing 10 animals. After 77 days, the SP6 group demonstrated significantly enhanced growth performance compared with the other supplementation levels. The condition factor was markedly higher in the SP6 and SP8 groups than in the SP0 group. No significant effects on total cholesterol levels were observed, but the SP4, SP6, and SP10 diets decreased triglyceride levels. Lipase activity was higher in the SP6 and SP8 groups than in the control group, whereas amylase activity showed no significant differences between treatments. Catalase and superoxide dismutase activities were significantly higher in the SP8 and SP10 groups than in the SP0 and SP2 groups. Glutathione S-transferase activity was higher in the SP6, SP8, and SP10 groups than in the SP0 group. In addition, SP inclusion in zebrafish diets improved female gonadal development. In conclusion, this study indicates that SP supplementation has substantial potential as a growth promoter, positively influencing lipid metabolism and antioxidant enzyme activity without affecting zebrafish survival.

Hematological, biochemical and oxidative responses induced by thermal shock in juvenile Tambaqui (Colossoma macropomum) and its hybrid Tambatinga (Colossoma macropomum x Piaractus brachypomus).

The effects of thermal shock on hematological, biochemical and antioxidant responses were evaluated in liver tissue of juvenile tambaqui (Colossoma macropomum) and tambatinga (♀ C. macropomum × â™‚ Piaractus brachypomus). Forty juveniles of tambaqui and 40 juveniles of tambatinga, of the same age and with an initial weight of 23.3 ± 6.7 g, were randomly distributed in eight 28L circular tanks. A tank (n = 10 fish) of tambaqui and a tank (n = 10 fish) of tambatinga were then used to obtain basal data. The other animals were subjected to thermal shock with sudden temperature reduction from 28 to 18 ºC. Blood and tissue were then collected after 1, 6 and 24 h from the onset of thermal shock. No mortality was observed during the experimental period. Thermal shock increased triglyceride levels after 24 h of stress for tambaqui and reduced values for tambatinga. There was an effect on plasma glucose only for fish group (P < 0.0001) and collection time (P < 0.0001) with a peak observed for the hybrid after 6 h. The interaction of factors for SOD indicated greater activity for tambatinga at the 6 h collection and lower at basal and 1 h collections. There was an interaction for CAT (P = 0.0020) with less activity for tambatinga at 1 h. However, thermal shock and hybridization did not influence GST and TBARS levels in liver tissue. Therefore, the results suggest that the hybrid, tambatinga, is more efficient at promoting adjustments of biochemical responses and antioxidant enzymes during thermal shock.

Functional Implications and Clinical Potential of MicroRNAs in Irritable Bowel Syndrome: A Concise Review.

MicroRNAs (miRNAs) are tiny (20-24 nucleotides long), non-coding, highly conserved RNA molecules that play a crucial role within the post-transcriptional regulation of gene expression via sequence-specific mechanisms. Since the miRNA transcriptome is involved in multiple molecular processes needed for cellular homeostasis, its altered expression can trigger the development and progression of several human pathologies. In this context, over the last few years, several relevant studies have demonstrated that dysregulated miRNAs affect a wide range of molecular mechanisms associated with irritable bowel syndrome (IBS), a common gastrointestinal disorder. For instance, abnormal miRNA expression in IBS patients is related to the alteration of intestinal permeability, visceral hyperalgesia, inflammatory pathways, and pain sensitivity. Besides, specific miRNAs are differentially expressed in the different subtypes of IBS, and therefore, they might be used as biomarkers for precise diagnosis of these pathological conditions. Accordingly, miRNAs have noteworthy potential as theragnostic targets for IBS. Hence, in this current review, we present an overview of the recent discoveries regarding the clinical relevance of miRNAs in IBS, which might be useful in the future for the development of miRNA-based drugs against this disorder.

Bacterial cell wall quantification by a modified low-volume Nelson-Somogyi method and its use with different sugars.

The study of peptidoglycan-binding proteins frequently requires in vitro binding assays, in which the isolated peptidoglycan used as a substrate must be carefully quantified. Here, we describe an easy and sensitive assay for peptidoglycan quantification based on a modified Nelson-Somogyi reducing sugar assay. We report the response of this assay to different common sugars and adapt its use to peptidoglycan samples subjected to acid hydrolysis. This method showed better sensitivity than the peptidoglycan quantification method based on the acid detection of diaminopimelic acid. The method described in this work, besides being valuable in the characterization of peptidoglycan-binding proteins, is also useful for the quantification of reducing monosaccharides or polysaccharides after acid or hydrolysis.

COVID-19-associated mucormycosis, diabetes and steroid therapy: Experience in a single centre in Western Mexico.

BACKGROUND: COVID-19-associated mucormycosis (CAM) has emerged as a challenging complication as the current pandemic has increased the population requiring treatment with corticosteroids. CAM has caused a massive outbreak in India, reported to be causing cases in Iran, Egypt and The Netherlands. OBJECTIVES: To describe CAM cases occurring in a single centre in Western Mexico. METHODS: Our group carried out a retrospective study from May 2020 to May 2021 to identify CAM cases in patients with previous COVID-19 diagnosis. RESULTS: Six CAM cases occurred in a single centre in Western Mexico during the study period, most of them with diabetes (n = 5/6) and all received corticosteroid therapy even when only three had severe COVID-19. After analysing local COVID-19 burden, it was estimated that in this region, CAM was 300 times more frequent among COVID individuals than the estimates for general population. CONCLUSION: Similar to large reports in India and other countries, CAM cases reported in this study were diagnosed in individuals with diabetes, hyperglycaemic status and with history of previous use of corticosteroids. Identifying these individuals at risk can help the early identification of CAM. In addition, strict glycaemic control and avoidance of unnecessary corticosteroid in non-severe COVID-19 cases could help in preventing this complicated fungal infection.

Variability in case fatality rate risk due to Covid-19 according to health services provider in Mexico City hospitals.

OBJECTIVE: To describe differences in Case Fatality Rate (CFR) for Covid-19 among healthcare subsystems in Mexico City between March and December 2020. MATERIALS AND METHODS: This is a retrospective secondary data analysis from the National Epidemiological Surveillance System data of Covid-19 cases. Information about health provider institutions was retrieved from the Catalogue of Health Establishments (CLUES). Logistic regressions were fitted to determine the association between health subsystems and mortality associated to Covid-19. The analyses were divided between hospitalized and ambulatory patients. RESULTS: The probability of dying from Covid-19 was higher among those treated at Instituto Mexicano del Seguro Social (IMSS) (Hospitalized:OR=5.11, Ambulatory:OR=36.57), Instituto de Se-guridad y Servicios Sociales de los Trabajadores del Estado (ISSSTE) (Hospitalized:OR=2.10, Ambulatory:OR=9.19), Secretaría de Salud (SS) (Hospitalized:OR=1.94, Ambulatory:OR=5.29) or other public institutions (Hospitalized: OR=1.70, Ambulatory:OR=9.56) than in those treated in private in-stitutions. CONCLUSIONS: Differences in healthcare quality and access between health subsystems are profound. It is imperative to increase the capacity and quality of the different health subsystems to improve health outcomes.

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Disturbances of brain cholesterol metabolism: A new excitotoxic process associated with status epilepticus.

The understanding of the excitotoxic processes associated with a severe status epilepticus (SE) is of major importance. Changes of brain cholesterol homeostasis is an emerging candidate for excitotoxicity. We conducted an overall analysis of the cholesterol homeostasis both (i) in fluids and tissues from patients with SE: blood (n = 63, n = 87 controls), CSF (n = 32, n = 60 controls), and post-mortem brain tissues (n = 8, n = 8 controls) and (ii) in a mouse model of SE induced by an intrahippocampal injection of kainic acid. 24-hydroxycholesterol levels were decreased in kainic acid mouse hippocampus and in human plasma and post-mortem brain tissues of patients with SE when compared with controls. The decrease of 24-hydroxycholesterol levels was followed by increased cholesterol levels and by an increase of the cholesterol synthesis. Desmosterol levels were higher in human CSF and in mice and human hippocampus after SE. Lanosterol and dihydrolanosterol levels were higher in plasma from SE patients. Our results suggest that a CYP46A1 inhibition could occur after SE and is followed by a brain cholesterol accumulation. The excess of cholesterol is known to be excitotoxic for neuronal cells and may participate to neurological sequelae observed after SE. This study highlights a new pathophysiological pathway involved in SE excitotoxicity.

Phase I study to assess safety, biodistribution and radiation dosimetry for 89Zr-girentuximab in patients with renal cell carcinoma.

PURPOSE: In this phase I study, we evaluated the safety, biodistribution and dosimetry of [89Zr]Zr-DFO-girentuximab (89Zr-girentuximab) PET/CT imaging in patients with suspicion of clear cell renal cell carcinoma (ccRCC). METHODS: Ten eligible patients received an intravenous administration of 37 MBq (± 10%) of 89Zr-girentuximab at mass doses of 5 mg or 10 mg. Safety was evaluated according to the NCI CTCAE (version 4.03). Biodistribution and normal organ dosimetry was performed based on PET/CT images acquired at 0.5, 4, 24, 72 and 168 h post-administration. Additionally, tumour dosimetry was performed in patients with confirmed ccRCC and visible tumour uptake on PET/CT imaging. RESULTS: 89Zr-girentuximab was administered in ten patients as per protocol. No treatment-related adverse events ≥ grade 3 were reported. 89Zr-girentuximab imaging allowed successful differentiation between ccRCC and non-ccRCC lesions in all patients, as confirmed with histological data. Dosimetry analysis using OLINDA/EXM 2.1 showed that the organs receiving the highest doses (mean ± SD) were the liver (1.86 ± 0.40 mGy/MBq), the kidneys (1.50 ± 0.22 mGy/MBq) and the heart wall (1.45 ± 0.19 mGy/MBq), with a mean whole body effective dose of 0.57 ± 0.08 mSv/MBq. Tumour dosimetry was performed in the 6 patients with histologically confirmed ccRCC resulting in a median tumour-absorbed dose of 4.03 mGy/MBq (range 1.90-11.6 mGy/MBq). CONCLUSIONS: This study demonstrates that 89Zr-girentuximab is safe and well tolerated for the administered activities and mass doses and allows quantitative assessment of 89Zr-girentuximab PET/CT imaging in patients with suspicion of ccRCC. TRIAL REGISTRATION: NCT03556046-14th of June, 2018.

Biodistribution and dosimetry of a single dose of albumin-binding ligand [177Lu]Lu-PSMA-ALB-56 in patients with mCRPC.

INTRODUCTION: PSMA-targeted radionuclide therapy with lutetium-177 has emerged as an effective treatment option for metastatic, castration-resistant prostate cancer (mCRPC). Recently, the concept of modifying PSMA radioligands with an albumin-binding entity was demonstrated as a promising measure to increase the tumor uptake in preclinical experiments. The aim of this study was to translate the concept to a clinical setting and evaluate the safety and dosimetry of [177Lu]Lu-PSMA-ALB-56, a novel PSMA radioligand with albumin-binding properties. METHODS: Ten patients (71.8 ± 8.2 years) with mCRPC received an activity of 3360 ± 393 MBq (120-160 µg) [177Lu]Lu-PSMA-ALB-56 followed by whole-body SPECT/CT imaging over 7 days. Volumes of interest were defined on the SPECT/CT images for dosimetric evaluation for healthy tissue and tumor lesions. General safety and therapeutic efficacy were assessed by measuring blood biomarkers. RESULTS: [177Lu]Lu-PSMA-ALB-56 was well tolerated, and no severe adverse events were observed. SPECT images revealed longer circulation of [177Lu]Lu-PSMA-ALB-56 in the blood with the highest uptake in tumor lesions at 48 h post injection. Compared with published data for other therapeutic PSMA radioligands (e.g. PSMA-617 and PSMA I&T), normalized absorbed doses of [177Lu]Lu-PSMA-ALB-56 were up to 2.3-fold higher in tumor lesions (6.64 ± 6.92 Gy/GBq) and similar in salivary glands (0.87 ± 0.43 Gy/GBq). Doses to the kidneys and red marrow (2.54 ± 0.94 Gy/GBq and 0.29 ± 0.07 Gy/GBq, respectively) were increased. CONCLUSION: Our data demonstrated that the concept of albumin-binding PSMA-radioligands is feasible and leads to increased tumor doses. After further optimization of the ligand design, the therapeutic outcomes may be improved for patients with prostate cancer.

Bee pollen as a dietary supplement for fish: Effect on the reproductive performance of zebrafish and the immunological response of their offspring.

Bee pollen, a natural resource collected by bees, is rich in many nutrients, therefore it may represent a useful dietary supplement. Different uses of bee pollen are proposed due to its beneficial health properties, which includes the capacity to improve animal performance and promote immunostimulation. Animal nutrition can directly affect adults and their offspring, and larval stage is a critical moment for fish due to high mortality related to immune challenges. Thus, the present study attempted to evaluate the effects of adding bee pollen to a zebrafish diet, specifically, analyzing the effects on reproduction and immunity transference to descendants. Zebrafish adults received control diets based on commercial flakes and live food Artemia sp. nauplii or bee pollen-supplemented diets, administered three times a day, at the same time. The animals received the diets over 60 d, and throughout this period, they were tested for: egg production per female, total number of eggs, embryo viability rate, larval survival rate after exposure to spring viremia of carp virus and to Salmonella enterica serovar Typhimurium, and larval neutrophil recruitment after tail wounding. Bee pollen supplementation failed to improve egg production and embryo viability, and was unable to substitute flakes in zebrafish breeders. Instead, the offspring of breeders fed with bee pollen supplemented diets showed longer survival upon virus exposure and higher neutrophil migration to wounds. These results indicate that bee pollen can influence vertical immunity through important mechanisms related to offspring immunity in the early stages, when larval immune system is not fully developed.

Sperm characterization and cryopreservation of the endangered freshwater fish Chirostoma estor (Atheriniformes).

The knowledge of the physiology of sperm of an endangered species allows the implantation of reproductive biotechnologies that aim at conservation. The aim of this study was to characterize fresh sperm and evaluate different cryopreservation solutions for sperm in Chirostoma estor. The characterization of Chirostoma estor fresh sperm (n = 22 males) was performed through analyzes of sperm concentration, membrane integrity, sperm morphology, motility rate, motility quality score, and motility duration. For cryopreservation (n = 42 males), 3 extenders (BTS™, MIII™, or Androstar Plus™) in combination with 2 permeable cryoprotectants (dimethyl sulfoxide (DMSO) or methyl glycol (Methyl)) were used. Analyzes of post-thaw sperm were performed as described for fresh sperm and additionally the fertilization rate analysis was performed. Fresh sperm presented a sperm concentration of 29.2 × 109 spermatozoa/mL, membrane integrity of 82.4%, and morphologically normal cells of 53%. After glucose activation (150 mM) a motility rate of 87.5%, sperm quality score of 5.0, and a duration of motility of 285 s were observed. For post-thaw sperm, MIII + Methyl and Androstar + Methyl solutions resulted in the highest motility rates of 40-48%. No differences were observed for motility duration, membrane integrity, and sperm morphology. Samples cryopreserved in Methyl (12-20%) showed a higher fertilization rate than DMSO, independently of the extender. In conclusion, the fresh sperm collected artificially from Chirostoma estor presents a compatible quality to carry out fertilization and can be cryopreserved in the commercial extenders MIII™ and Androstar Plus™ together with the cryoprotectant Methyl glycol.

[Opportunity cost approximation of underage caregivers in Mexico]. / Aproximación al costo de oportunidad de los cuidadores menores de edad en México.

OBJECTIVE: To calculate the cost assumed by society for having underaged individuals who do not study and provide care to dependent older adults. MATERIALS AND METHODS: The Mexican Health and Ageing Study (MHAS) 2015 was used to calculate the number of underage caregivers who do not attend school. The Household Income and Expenditure Survey (ENIGH) 2018 was used to estimate wage differences between populations of working age with complete elementary and complete high school education. Those differences were then converted to present value, added, and multiplied by the number of underage caregivers. RESULTS: The total annual cost of having underage caregivers who drop out of school adds to 1 112 179 210 pesos. CONCLUSIONS: The topic concerning underage caregivers has not received enough attention from specialized literature, and there is still a large research gap in this area.

Synergism and Subadditivity of Verbascoside-Lignans and -Iridoids Binary Mixtures Isolated from Castilleja tenuiflora Benth. on NF-κB/AP-1 Inhibition Activity.

Pharmacodynamic interactions between plant isolated compounds are important to understand the mode of action of an herbal extract to formulate or create better standardized extracts, phytomedicines, or phytopharmaceuticals. In this work, we propose binary mixtures using a leader compound to found pharmacodynamic interactions in inhibition of the NF-κB/AP-1 pathway using RAW-Blue™ cells. Eight compounds were isolated from Castilleja tenuiflora, four were new furofuran-type lignans for the species magnolin, eudesmin, sesamin, and kobusin. Magnolin (60.97%) was the most effective lignan inhibiting the NF-κB/AP-1 pathway, followed by eudesmin (56.82%), tenuifloroside (52.91%), sesamin (52.63%), and kobusin (45.45%). Verbascoside, a major compound contained in wild C. tenuiflora showed an inhibitory effect on NF-κB/AP-1. This polyphenol was chosen as a leader compound for binary mixtures. Verbacoside-aucubin and verbascoside-kobusin produced synergism, while verbascoside-tenuifloroside had subadditivity in all concentrations. Verbascoside-kobusin is a promising mixture to use on NF-κB/AP-1 related diseases and anti-inflammatory C. tenuiflora-based phytomedicines.

Obesity induction in adult zebrafish leads to negative reproduction and offspring effects.

Obesity is a transgenerational epigenetic metabolic disturbance. Although the diet-induced obese (DIO) zebrafish model is well established, reproductive parameters and changes in offspring have not yet been evaluated. Thus, the aim of this study was to evaluate possible changes in reproductive parameters, embryos and offspring (F1) generated by the reproduction of diet-induced obese males and females. The adult zebrafish were divided into two groups: one group receiving a balanced diet (control group) and the other group was overfed (DIO group) . The dietary protocol was maintained for 8 weeks. During this period, males and females in the same group were stimulated through a weekly reproduction protocol. To verify parental obesity, body weight, blood glucose, triglyceride, the hepatosomatic and gonadosomatic index and adipose tissue morphometry evaluations were carried out. Reproductive parameters were evaluated through ovarian and oocyte maturation stage, total spawning, fertility and fertilization index. To verify possible changes caused by parenteral obesity, all offspring were kept in separate groups in correspondence with their parents and were fed a control diet. Plasma glucose, triglycerides, mortality rate, hatching, and deformities were determined. After 8 weeks under the diet protocol, the DIO group exhibited characteristic obesity alterations, displaying significant increases in body mass and hepatosomatic and gonadosomatic indices, hyperglycemia and visceral and subcutaneous adipocyte hypertrophy. In addition, high mortality rates, morphologic deformities and high plasmatic glucose and triglyceride levels, with 100% mortality at 60 dpf, were observed for the offspring. Therefore, obesity induction in adults led to negative effects on their offspring, with a high occurrence of deformities and mortality.

Effect of melatonin supplementation to a cytoprotective medium on post-thawed Brycon orbignyanus sperm quality preserved during different freezing times.

The aim of the present study is to verify the viability of frozen B. orbignyanus sperm cells after freezing with dilution media containing different concentrations of the melatonin and after different freezing times. Semen from 15 males was collected and pooled as five pools from three random animals. Oocytes (100) from three females were separately used for fertilization. There were three treatments: (C) Control Medium: 90% of extender Beltsville thawing solution (5% concentration) + 10% methylglycol (MG); (M1) Control Medium + 1 mM melatonin; and (M2) Control Medium + 2 mM melatonin. Sperm samples were diluted in media at a final proportion of 1:4 [125 µl sperm (25% V/V) + 337.5 µl BTS (65% V/V) + 37.5 µl MG (10% V/V)]. Melatonin was added at final solution. Three Dry shipper freezing times were used: T1 (15 min), T2 (12 h) and T3 (24 h). The samples were transferred, stored in a cryobank and thawed in a water bath at 60 °C for 5 s and evaluated concerning viability, morphology and fertilization rate. B. orbignyanus semen frozen in M2 presented the highest fertilization rate (8.40 ± 2.54%). The highest vitality (85.2 ± 2.8%), motility (64.63 ± 8.3%), motility duration (84.22 ± 11.4 s) and progressive motility (17.01 ± 1.2%) rates were maintained for M2. The highest number of altered cells was observed in C (57.4 ± 5.9%). Melatonin at 2 mmol L-1 associated with the cryoprotectant methylglycol in cryopreservation could be used to improve a cryobank for endangered Brycon orbignyanus populations.

[Particularities of coronary syndrome in women]. / Particularités du syndrome coronarien chez les femmes.

Cardiovascular disease, including myocardial infarction, has been less studied in the female population, leading to higher mortality among women. Differences between the sexes exist in terms of clinical presentation, pathophysiology and even management. This article is a non-systematic review of literature of recent articles addressing this theme. A broader understanding of the peculiarities of coronary syndrome in women improves their management and reduces the inequality of prognosis for women.

Les maladies cardiovasculaires, dont l'infarctus du myocarde, ont été moins étudiées dans la population féminine. Des différences entre les sexes existent en termes de présentation clinique, de physiopathologie et même de prise en charge. Cet article est une revue non systématique de la littérature des articles récents abordant ce thème. Une meilleure compréhension des particularités du syndrome coronarien chez les femmes peut contribuer à améliorer leur prise en charge et réduire l'inégalité de pronostic actuellement en leur défaveur.

Establishment of a Protein Concentration Gradient in the Outer Membrane Requires Two Diffusion-Limiting Mechanisms.

OmpA-like proteins are involved in the stabilization of the outer membrane, resistance to osmotic stress, and pathogenesis. In Caulobacter crescentus, OmpA2 forms a physiologically relevant concentration gradient that forms by an uncharacterized mechanism, in which the gradient orientation depends on the position of the gene locus. This suggests that OmpA2 is synthesized and translocated to the periplasm close to the position of the gene and that the gradient forms by diffusion of the protein from this point. To further understand how the OmpA2 gradient is established, we determined the localization and mobility of the full protein and of its two structural domains. We show that OmpA2 does not diffuse and that both domains are required for gradient formation. The C-terminal domain binds tightly to the cell wall and the immobility of the full protein depends on the binding of this domain to the peptidoglycan; in contrast, the N-terminal membrane ß-barrel diffuses slowly. Our results support a model in which once OmpA2 is translocated to the periplasm, the N-terminal membrane ß-barrel is required for an initial fast restriction of diffusion until the position of the protein is stabilized by the binding of the C-terminal domain to the cell wall. The implications of these results on outer membrane protein diffusion and organization are discussed.IMPORTANCE Protein concentration gradients play a relevant role in the organization of the bacterial cell. The Caulobacter crescentus protein OmpA2 forms an outer membrane polar concentration gradient. To understand the molecular mechanism that determines the formation of this gradient, we characterized the mobility and localization of the full protein and of its two structural domains an integral outer membrane ß-barrel and a periplasmic peptidoglycan binding domain. Each domain has a different role in the formation of the OmpA2 gradient, which occurs in two steps. We also show that the OmpA2 outer membrane ß-barrel can diffuse, which is in contrast to what has been reported previously for several integral outer membrane proteins in Escherichia coli, suggesting a different organization of the outer membrane proteins.

Evolution of bacteria seen through their essential genes: the case of Pseudomonas aeruginosa and Azotobacter vinelandii.

Pseudomonas aeruginosa is a metabolically versatile bacterium and also an important opportunistic pathogen. It has a remarkable genomic structure since the genetic information encoding its pathogenicity-related traits belongs to its core-genome while both environmental and clinical isolates are part of the same population with a highly conserved genomic sequence. Unexpectedly, considering the high level of sequence identity and homologue gene number shared between different P. aeruginosa isolates, the presence of specific essential genes of the two type strains PAO1 and PA14 has been reported to be highly variable. Here we report the detailed bioinformatics analysis of the essential genes of P. aeruginosa PAO1 and PA14 that have been previously experimentally identified and show that the reported gene variability was owed to sequencing and annotation inconsistencies, but that in fact they are highly conserved. This bioinformatics analysis led us to the definition of 348 P. aeruginosa general essential genes. In addition we show that 342 of these 348 essential genes are conserved in Azotobacter vinelandii, a nitrogen-fixing, cyst-forming, soil bacterium. These results support the hypothesis of A. vinelandii having a polyphyletic origin with a Pseudomonads genomic backbone, and are a challenge to the accepted theory of bacterial evolution.