A NodD-like protein activates transcription of genes involved with naringenin degradation in a flavonoid-dependent manner in Herbaspirillum seropedicae.

Herbaspirillum seropedicae is an associative, endophytic non-nodulating diazotrophic bacterium that colonises several grasses. An ORF encoding a LysR-type transcriptional regulator, very similar to NodD proteins of rhizobia, was identified in its genome. This nodD-like gene, named fdeR, is divergently transcribed from an operon encoding enzymes involved in flavonoid degradation (fde operon). Apigenin, chrysin, luteolin and naringenin strongly induce transcription of the fde operon, but not that of the fdeR, in an FdeR-dependent manner. The intergenic region between fdeR and fdeA contains several generic LysR consensus sequences (T-N11 -A) and we propose a binding site for FdeR, which is conserved in other bacteria. DNase I foot-printing revealed that the interaction with the FdeR binding site is modified by the four flavonoids that stimulate transcription of the fde operon. Moreover, FdeR binds naringenin and chrysin as shown by isothermal titration calorimetry. Interestingly, FdeR also binds in vitro to the nod-box from the nodABC operon of Rhizobium sp. NGR234 and is able to activate its transcription in vivo. These results show that FdeR exhibits two features of rhizobial NodD proteins: nod-box recognition and flavonoid-dependent transcription activation, but its role in H. seropedicae and related organisms seems to have evolved to control flavonoid metabolism.

Recombinant expression of Ixolaris, a Kunitz-type inhibitor from the tick salivary gland, for NMR studies.

Ixolaris is an anticoagulant protein identified in the tick saliva of Ixodes scapularis. Ixolaris contains 2 Kunitz like domains and binds to Factor Xa or Factor X as a scaffold for inhibition of the Tissue Factor (TF)/Factor VIIa (FVIIa). In contrast to tissue factor pathway inhibitor (TFPI), however, Ixolaris does not bind to the active site cleft of FXa. Instead, complex formation is mediated by the FXa heparin-binding exosite. Due to its potent and long-lasting antithrombotic activity, Ixolaris is a promising agent for anticoagulant therapy. Although numerous functional studies of Ixolaris exist, three-dimensional structure of Ixolaris has not been obtained at atomic resolution. Using the pET32 vector, we successfully expressed a TRX-His6-Ixolaris fusion protein. By combining Ni-NTA chromatography, enterokinase protease cleavage, and reverse phase HPLC (RP-HPLC), we purified isotopically labeled Ixolaris for NMR studies. 1D 1H and 2D 15N-1H NMR analysis yielded high quality 2D 15N-1H HSQC spectra revealing that the recombinant protein is folded. These studies represent the first steps in obtaining high-resolution structural information by NMR for Ixolaris enabling the investigation of the molecular basis for Ixolaris-coagulation factors interactions.

On the origins and genetic diversity of South American chickens: one step closer.

Local chicken populations are a major source of food in the rural areas of South America. However, very little is known about their genetic composition and diversity. Here, we analyzed five populations from South America to investigate their maternal genetic origin and diversity, hoping to mitigate the lack of information on local chicken populations from this region. We also included three populations of chicken from the Iberian Peninsula and one from Easter Island, which are potential sources of the first chickens introduced in South America. The obtained sequencing data from South American chickens indicate the presence of four haplogroups (A, B, E and D) that can be further subdivided into nine sub-haplogroups. Of these, four (B1, D1a, E1a(b), E1b) were absent from local Iberian Peninsula chickens and one (D1a) was present only on Easter Island. The presence of the sub-haplogroups A1a(b) and E1a(b) in South America, previously only observed in Eastern Asia, and the significant population differentiation between Iberian Peninsula and South American populations, suggest a second maternal source of the extant genetic pool in South American chickens.

Flow characteristics of Luer and non-Luer spinal needles.

We investigated the flow rates of 25-G and 27-G spinal needles, of 90-mm and 120-mm lengths, from Vygon, BD, B. Braun and Pajunk; the needles had either a Luer connector, or a Surety® or UniVia® non-Luer connector. We used a bench-top model of entering the spinal space, pressurised to 35 cmH2 O to simulate cerebrospinal fluid pressure in the sitting position. We examined the time to first appearance of simulated cerebrospinal fluid in the needle hub, as well as the amount of fluid collected over 120 s after the needle was introduced. The mean (SD) times to first appearance of fluid in the needle hub of Luer spinal needles varied from 0.36 (0.22) s for the 25-G 90-mm BD to 3.14 (0.72) s for the 27-G 120-mm B. Braun, and in the non-Luer spinal needles from 0.22 (0.17) s for the 25-G 90-mm B. Braun to 2.99 (0.71) s for the 27-G 120-mm Pajunk. There was a significant difference in the time to first appearance of fluid in the needle hub between Luer and non-Luer needles of the same type for seven of 14 comparisons made, of which four showed slower appearance of fluid in the non-Luer version. In some of these cases, the time to appearance of fluid was nearly twice as long with the non-Luer counterpart. The mean (SD) weight of fluid collected in 120 s using the Luer spinal needles varied from 0.21 (0.05) g for the 27-G 120-mm Pajunk to 1.21 (0.18) g for the 25-G 90-mm Vygon, and using the non-Luer spinal needles from 0.25 (0.05) g for the 27-G 120-mm Pajunk to 1.55 (0.05) g for the 25-G 90-mm B. Braun. All of the needle types showed a greater weight of fluid collected using the non-Luer compared with the Luer version, with six of the 14 needle types showing a significant difference. Significant variations in flow were also seen between the same needle type from different manufacturers. We conclude that changing from Luer to non-Luer versions of spinal needles does not merely change the hub design and connection, but may introduce important differences in function.

Strength of commonly used spinal needles: the ability to deform and resist deformation.

We investigated the strength of commonly used spinal needles in relation to the amount of deformation, and registered forces during standardised testing. We investigated differences between manufacturers for the same length and gauge of Luer and non-Luer needles, and examined the effect of the internal stylet in terms of needle strength. A specialised rig was designed to perform the testing in both the horizontal and axial plane, reflecting common industrial tests and clinical use. Needles from four commonly used manufacturers were used (Vygon, Becton Dickinson, B Braun, and Pajunk). Needles of 25 G and 27 G were tested in 90-mm and 120-mm lengths. We found significant differences in terms of the size of final deformation and 'toughness'/resistance to deformation between needles of different brands. There were also significant differences between horizontal tests conducted as an industry standard and our own axial test. This may have bearing on clinical use in terms of the incidence of bending and breakage. The presence of the internal stylet resulted in significantly greater toughness in many needles, but had little effect on the degree of deformation. Comparison of Luer and non-Luer needles of the same brand and size showed few significant differences in strength. This result is reassuring, given the imminent change from Luer to non-Luer needles that is to occur in the UK.

RNA-seq transcriptional profiling of Herbaspirillum seropedicae colonizing wheat (Triticum aestivum) roots.

Herbaspirillum seropedicae is a diazotrophic and endophytic bacterium that associates with economically important grasses promoting plant growth and increasing productivity. To identify genes related to bacterial ability to colonize plants, wheat seedlings growing hydroponically in Hoagland's medium were inoculated with H. seropedicae and incubated for 3 days. Total mRNA from the bacteria present in the root surface and in the plant medium were purified, depleted from rRNA and used for RNA-seq profiling. RT-qPCR analyses were conducted to confirm regulation of selected genes. Comparison of RNA profile of root attached and planktonic bacteria revealed extensive metabolic adaptations to the epiphytic life style. These adaptations include expression of specific adhesins and cell wall re-modeling to attach to the root. Additionally, the metabolism was adapted to the microxic environment and nitrogen-fixation genes were expressed. Polyhydroxybutyrate (PHB) synthesis was activated, and PHB granules were stored as observed by microscopy. Genes related to plant growth promotion, such as auxin production were expressed. Many ABC transporter genes were regulated in the bacteria attached to the roots. The results provide new insights into the adaptation of H. seropedicae to the interaction with the plant.

Toll-like receptor 3 expression and function in childhood idiopathic nephrotic syndrome.

The efficacy of steroids and immunosuppressive treatments in idiopathic nephrotic syndrome (INS) hints at the implication of immune cells in the pathophysiology of the disease. Toll-like receptor (TLR) dysfunctions are involved in many kidney diseases of immune origin, but remain little described in INS. We investigated the expression and function of TLRs in peripheral blood mononuclear cells (PBMC) of INS children, including 28 in relapse, 23 in remission and 40 controls. No child had any sign of infection, but a higher Epstein-Barr virus viral load was measured in the PBMC of relapsing patients. TLR-3 expression was increased in B cells only during INS remission. There was a negative correlation between proteinuria and TLR-3 expression in total and the main subsets of PBMC from INS patients. The expression of TLR-8 was also increased in both CD4(+) T cells and B cells in INS remission. There was a negative correlation between proteinuria and TLR-8 expression in total PBMC, CD4(+) T cells and B cells of INS patients. Nevertheless, TLR-3 and TLR-8 expression was normalized in all PBMC subsets in an additional group of 15 INS patients in remission with B cell repletion after rituximab therapy. Paradoxically, interferon (IFN) regulatory factor 3 transactivation was increased in PBMC of all INS patients. In-vitro secretion of IFN-α and interleukin 6 were increased spontaneously in PBMC of INS remission patients, whereas PBMC from all INS patients displayed an impaired IFN-α secretion after TLR-3 stimulation. Thus, TLR-3 pathway dysfunctions may be closely involved in INS pathogenesis.

Naringenin degradation by the endophytic diazotroph Herbaspirillum seropedicae SmR1.

Several bacteria are able to degrade flavonoids either to use them as carbon sources or as a detoxification mechanism. Degradation pathways have been proposed for several bacteria, but the genes responsible are not known. We identified in the genome of the endophyte Herbaspirillum seropedicae SmR1 an operon potentially associated with the degradation of aromatic compounds. We show that this operon is involved in naringenin degradation and that its expression is induced by naringenin and chrysin, two closely related flavonoids. Mutation of fdeA, the first gene of the operon, and fdeR, its transcriptional activator, abolished the ability of H. seropedicae to degrade naringenin.

Antimicrobial resistance in faecal enterococci and Escherichia coli isolates recovered from Iberian wolf.

The aim of this study was to report the antimicrobial resistance, the molecular mechanisms associated and the detection of virulence determinants within faecal Enterococcus spp. and Escherichia coli isolates of Iberian wolf. Enterococci (n = 227) and E. coli (n = 195) isolates were obtained from faecal samples of Iberian wolf (Canis lupus signatus). High rates of resistance were detected for tetracycline and erythromycin among the enterococci isolates, and most of resistant isolates harboured the tet(M) and/or tet(L) and erm(B) genes, respectively. The blaTEM, tet(A) and/or tet(B), and aadA or strA-strB genes were detected among most ampicillin-, tetracycline- or streptomycin-resistant E. coli isolates, respectively. E. coli isolates were ascribed to phylogroups A (n = 56), B1 (91), B2 (13) and D (35). The occurrence of resistant enterococci and E. coli isolates in the faecal flora of Iberian wolf, including the presence of resistant genes in integrons, and virulence determinants was showed in this study. Iberian wolf might act as reservoir of certain resistance genes that could be spread throughout the environment.

Culture-independent analysis of endophytic bacterial communities associated with Brazilian sugarcane.

Sugarcane is an economically important culture in Brazil. Endophytic bacteria live inside plants, and can provide many benefits to the plant host. We analyzed the bacterial diversity of sugarcane cultivar RB-72454 by cultivation-independent techniques. Total DNA from sugarcane stems from a commercial plantation located in Paraná State was extracted. Partial 16S rRNA genes were amplified and sequenced for library construction. Of 152 sequences obtained, 52% were similar to 16S rRNA from Pseudomonas sp, and 35.5% to Enterobacter sp. The genera Pantoea, Serratia, Citrobacter, and Klebsiella were also represented. The endophytic communities in these sugarcane samples were dominated by the families Enterobacteriaceae and Pseudomonadaceae (class Gammaproteobacteria).

Assigning F-words as ingredients of interventions for children with cerebral palsy functioning at GMFCS IV and V: A scoping review protocol.

Introduction: Children with Cerebral Palsy (CP) functioning at Gross Motor Function Classification System (GMFCS) levels IV and V require "on time" identification and intervention. Interventions offered continue to be a challenge, in high-, but even more so in middle-, and low-income countries. Aim: To describe the methods developed to explore the ingredients of published studies on early interventions in young children with cerebral palsy (CP) at highest risk of being non-ambulant based on the "F-words for child development framework" and the design of a scoping review exploring these ingredients. Method: An operational procedure was developed through expert panels to identify ingredients of published interventions and related F-words. After sufficient agreement among researchers was reached, a scoping review was designed. The review is registered in the Open Science Framework database. The "Population, Concept and Context" framework was used. Population: young children (0-5 years with CP and at highest risk for being non-ambulant (GMFCS levels IV or V); Concept: non-surgical and non-pharmacological early intervention services measuring outcomes from any ICF domain; Context: studies published from 2001 to 2021. After duplicated screening and selection, data will be extracted and quality will be assessed with the American Academy for Cerebral Palsy and Developmental Medicine (AACPDM) and Mixed Methods Appraisal (MMAT) tools. Results: We present the protocol to identify the explicit (directly measured outcomes and respective ICF domains) and implicit (intervention features not explicitly intended or measured) ingredients. Conclusion: Findings will support the implementation of the F-words in interventions for young children with non-ambulant CP.

11ß-Hydroxysteroid dehydrogenase type 1: relevance of its modulation in the pathophysiology of obesity, the metabolic syndrome and type 2 diabetes mellitus.

Recent evidence strongly argues for a pathogenic role of glucocorticoids and 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) in obesity and the metabolic syndrome, a cluster of risk factors for atherosclerotic cardiovascular disease and type 2 diabetes mellitus (T2DM) that includes insulin resistance (IR), dyslipidaemia, hypertension and visceral obesity. This has been partially prompted not only by the striking clinical resemblances between the metabolic syndrome and Cushing's syndrome (a state characterized by hypercortisolism that associates with metabolic syndrome components) but also from monogenic rodent models for the metabolic syndrome (e.g. the leptin-deficient ob/ob mouse or the leptin-resistant Zucker rat) that display overall increased secretion of glucocorticoids. However, systemic circulating glucocorticoids are not elevated in obese patients and/or patients with metabolic syndrome. The study of the role of 11ß-HSD system shed light on this conundrum, showing that local glucocorticoids are finely regulated in a tissue-specific manner at the pre-receptor level. The system comprises two microsomal enzymes that either activate cortisone to cortisol (11ß-HSD1) or inactivate cortisol to cortisone (11ß-HSD2). Transgenic rodent models, knockout (KO) for HSD11B1 or with HSD11B1 or HSD11B2 overexpression, specifically targeted to the liver or adipose tissue, have been developed and helped unravel the currently undisputable role of the enzymes in metabolic syndrome pathophysiology, in each of its isolated components and in their prevention. In the transgenic HSD11B1 overexpressing models, different features of the metabolic syndrome and obesity are replicated. HSD11B1 gene deficiency or HSD11B2 gene overexpression associates with improvements in the metabolic profile. In face of these demonstrations, research efforts are now being turned both into the inhibition of 11ß-HSD1 as a possible pharmacological target and into the role of dietary habits on the establishment or the prevention of the metabolic syndrome, obesity and T2DM through 11ß-HSD1 modulation. We intend to review and discuss 11ß-HSD1 and obesity, the metabolic syndrome and T2DM and to highlight the potential of its inhibition for therapeutic or prophylactic approaches in those metabolic diseases.

In vitro suppression of drug-induced methaemoglobin formation by Intralipid(®) in whole human blood: observations relevant to the 'lipid sink theory'.

To provide further evidence for the lipid sink theory, we have developed an in vitro model to assess the effect of Intralipid® 20% on methaemoglobin formation by drugs of varying lipid solubility. Progressively increasing Intralipid concentrations from 4 to 24 mg.ml⁻¹ suppressed methaemoglobin formation by the lipid soluble drug glyceryl trinitrate in a dose-dependent manner (p < 0.001). Both dose and timing of administration of Intralipid to blood previously incubated with glyceryl trinitrate for 10 and 40 min resulted in significant suppression of methaemoglobin formation (p < 0.0001 and p < 0.05, respectively). Mathematical modelling demonstrated that the entire process of methaemoglobin formation by glyceryl trinitrate was slowed down in the presence of Intralipid. Intralipid did not significantly suppress methaemoglobin formation induced by 2-amino-5-hydroxytoluene (partially lipid soluble) or sodium nitrite (lipid insoluble; both p > 0.5). This work may assist determination of the suitability of drugs taken in overdose for which Intralipid might be deployed.

Influence of antimicrobial photodynamic therapy with different pre-irradiation times on children's dental biofilm: randomized clinical trial.

PURPOSE: Photodynamic therapy (PDT) is effective in reducing pathogenic microorganisms in the oral cavity and in preventing dental diseases. This study evaluated the pre-irradiation time using PDT (diode laser associated with 0.01% methylene blue) to decrease the number of microorganisms in the visible plaque in permanent teeth. METHODS: This randomized clinical trial included 108 homologous lower permanent first molars (36 and 46) with biofilm from 54 children aged six to 12 years. PDT was performed (0.01% methylene blue photosensitizer/therapeutic laser-InGaAIP), according to the following protocols: Group 1, biofilm collection of the distal area of the lingual surface of 36 µm before PDT; group 2, mesial area of the lingual surface of 36 µm 1 min after PDT; group 3, area of the lingual surface of 46 µm before PDT; and group 4, mesial area of the lingual surface of 46 µm 5 min after PDT. RESULTS: After statistical analysis, significant differences were observed between the groups (p = 0.000). In groups 2 and 4, the number of bacteria tended to decrease, with a more evident bacterial reduction in group 4. CONCLUSIONS: Pre-irradiation reduced the number of colony-forming units of mature bacterial biofilms in vivo. A time of 5 min resulted in a greater reduction in the number of colony-forming units. CLINICAL TRIAL REGISTRATION: ReBEC Identifier: RBR-6bqfp3; Date of Register: March 2nd, 2020. Retrospectively Registered.

Naringenin regulates expression of genes involved in cell wall synthesis in Herbaspirillum seropedicae.

Five thousand mutants of Herbaspirillum seropedicae SmR1 carrying random insertions of transposon pTnMod-OGmKmlacZ were screened for differential expression of LacZ in the presence of naringenin. Among the 16 mutants whose expression was regulated by naringenin were genes predicted to be involved in the synthesis of exopolysaccharides, lipopolysaccharides, and auxin. These loci are probably involved in establishing interactions with host plants.

Health care resource use and costs in opioid-treated patients with and without constipation in Brazil.

OBJECTIVE: To estimate the prevalence of constipation concomitant to opioid treatment and related resource use and costs from the private payer perspective. METHODS: In this retrospective database analysis, patients receiving opioid therapy were identified from a longitudinal insurance claims database. An algorithm was used to identify patients receiving opioid therapy with coincident constipation-related claims according to ICD-10 codes, targeted procedures, and opioid use criteria. Resource use and costs were determined for these individuals and compared with patients receiving opioid therapy without constipation, without opioid therapy with constipation, and without both conditions. Results were compared using analysis of variance with a significance level of 0.05. RESULTS: A total of 23,313 patients were classified as opioid-treated patients (2.2%) and 6678 of them had events related to constipation (29.0%). Compared with opioid-treated patients without constipation, incremental mean total costs per month per patient were 261.18 BRL (P < 0.001). The average cost per month for opioid-related constipation patients was 787.84 BRL, significantly higher than other patients (P < 0.001 for all comparisons). Among cancer patients, 24.4% was receiving opioids and 27.0% of those had constipation-related claims. As expected, the opioid therapy prevalence was significantly higher when compared to all patients (2.2% vs. 24.4%, P < 0.001). Cancer patients had, in average, higher costs than did noncancer patients in all four subgroups. CONCLUSIONS: Patients with constipation coincident with opioid treatment exhibited a significantly higher economic burden than did patients without the condition. These results indicate that reducing opioid-induced constipation could lead to potential cost savings for the health care system.

Endophytic and pathogenic isolates of the cacao fungal pathogen Moniliophthora perniciosa (Tricholomataceae) are indistinguishable based on genetic and physiological analysis.

We evaluated the genetic and physiological variability of Moniliophthora perniciosa obtained from healthy and diseased branches of cacao (Theobroma cacao) plants. The diversity of the isolates was evaluated by RAPD technique and by studies of virulence and exoenzyme production. The genetic variability of endophytic and pathogenic M. perniciosa was evaluated in association with pathogenicity assays. RAPD analysis showed eight genetic groups, which were not related to plant disease status (healthy versus diseased branches). Isolates from cacao were included in three groups, excluding isolates from other host plants. Pathogenicity and enzyme analysis showed that the virulence of the isolates is not related to exoenzyme production. This is the first evidence that M. perniciosa colonizes healthy parenchymatic tissues, showing that endophytic behavior may occur in this species.

Natural parasitism in fruit fly (Diptera: Tephritidae) and interaction with wild hosts surrounding apple orchards adjacent to Atlantic Forest fragments in Paraná State, Brazil.

The South American fruit fly, Anastrepha fraterculus (Wiedemann, 1830) (Diptera: Tephritidae), is an important pest in the subtropical region of Brazil. This insect has tritrophic relation between wild fruits and parasitoids and is associated with apple (Malus domestica Borkh.) orchards adjacent to the Atlantic Forest in Paraná. We thus investigated the degree of infestation of the fruit fly and natural parasitism in wild and cultivated fruits surrounding apple orchards. For this purpose, we collected fruits of Acca sellowiana (Berg.) Burret, Campomanesia xanthocarpa (Mart), Eugenia uniflora L., Eugenia pyriformis Cambessèdes, Psidium cattleianum Sabine, Psidium guajava (L.), Annona neosericea Rainer and Eriobotrya japonica (Thumb) in apple orchards adjacent to the Atlantic Forest located in Campo do Tenente, Lapa and Porto Amazonas counties. In total, we collected 18,289 fruits during four growing years. The occurrence of A. fraterculus depends on the susceptible period of apple fruits. A. sellowiana and P. cattleianum were considered primary fruit fly multipliers and P. guajava was secondary, all occurring after the apple harvest (IS period). The group of parasitoids with A. fraterculus was Aganaspis pelleranoi (Brèthes, 1924) (Hymenoptera: Figitidae), Opius bellus (Gahan, 1930), Doryctobracon areolatus (Szépligeti, 1911) and Doryctobracon brasiliensis (Szépligeti, 1911) (Hymenoptera: Braconidae) all of which are first records in the Atlantic Forest in Paraná. First record of O. bellus occurring in the State of Paraná, as well as, first record of the tritrophic association between host plant A. neosericea, parasitoids D. areolatus and O. bellus and fruit fly A. fraterculus. The host P. cattleianum stood out among the Myrtaceae species in regard to the high diversity of parasitoid species (81% of parasitoids). The total number of Figitidae species (76.5%) was higher than that of Braconidae species. The influence of climatic events in southern Brazil on wild fruit production should be further studied to understand the association of A. fraterculus with the tritrophic relationship.

Maternal temperature in emergency caesarean section (MATES): an observational multicentre study.

BACKGROUND: Temperature regulation in women undergoing emergency caesarean section is a complex topic about which there is a paucity of evidence-based recommendations. The adverse effects of inadvertent peri-operative hypothermia are well described. Hyperthermia is also associated with adverse neonatal outcomes, an increased risk of obstetric intervention and increased treatment for suspected sepsis. We conducted a multi-centre observational cohort study to identify the prevalence of hypothermia and hyperthermia during emergency caesarean section. S: Participants undergoing emergency caesarean section were recruited across 14 sites in the UK. The primary end point was maternal temperature in the recovery room. Temperature was measured using a zero heat-flux temperature monitoring device. RESULTS: Two hundred and sixty-five participants were recruited over a 12-month period. The prevalence of hypothermia (<36.0°C) was 10.7% and the prevalence of hyperthermia (>37.5°C) was 14.7% on admission to recovery. The prevalence of hypothermia, normothermia, and hyperthermia differed among type of anaesthesia: 71.4% of the hypothermic group had received a spinal anaesthetic whereas 76.9% of the hyperthermic group had received epidural top-up anaesthesia. There was a significant decrease in maternal temperature between the time of delivery and admission to the recovery room of 0.20°C (95% CI 0.15 to 0.25, P<0.001). CONCLUSIONS: Both hypothermia and hyperthermia are prevalent findings in mothers who undergo emergency caesarean section. Therefore, accurate temperature measurement is essential to ensure that an appropriate intra-operative temperature management strategy is employed.