Exploring ex vivo biofilm dynamics: consequences of low ampicillin concentrations on the human oral microbiome.

Prolonged exposure to antibiotics at low concentration can promote processes associated with bacterial biofilm formation, virulence and antibiotic resistance. This can be of high relevance in microbial communities like the oral microbiome, where commensals and pathogens share a common habitat and where the total abundance of antibiotic resistance genes surpasses the abundance in the gut. Here, we used an ex vivo model of human oral biofilms to investigate the impact of ampicillin on biofilm viability. The ecological impact on the microbiome and resistome was investigated using shotgun metagenomics. The results showed that low concentrations promoted significant shifts in microbial taxonomic profile and could enhance biofilm viability by up to 1 to 2-log. For the resistome, low concentrations had no significant impact on antibiotic resistance gene (ARG) diversity, while ARG abundance decreased by up to 84%. A positive correlation was observed between reduced microbial diversity and reduced ARG abundance. The WHO priority pathogens Streptococcus pneumoniae and Staphylococcus aureus were identified in some of the samples, but their abundance was not significantly altered by ampicillin. Most of the antibiotic resistance genes that increased in abundance in the ampicillin group were associated with streptococci, including Streptococcus mitis, a well-known potential donor of ARGs to S. pneumoniae. Overall, the results highlight the potential of using the model to further our understanding of ecological and evolutionary forces driving antimicrobial resistance in oral microbiomes.

The Oral Host-Microbial Interactome: An Ecological Chronometer of Health?

An increasing number of studies reveal that host-microbial interactome networks are coordinated, impacting human health and disease. Recently, several lines of evidence have revealed associations between the acquisition of a complex microbiota and adaptive immunity, supporting that host-microbiota symbiotic relationships have evolved as a means to maintain homeostasis where the role of the microbiota is to promote and educate the immune system. Here, we hypothesize an oral host-microbial interactome that could serve as an ecological chronometer of health and disease, with specific focus on caries, periodontal diseases, and cancer. We also review the current state of the art on the human oral microbiome and its correlations with host innate immunity, and host cytokine control, with the goal of using this information for disease prediction and designing novel treatments for local and systemic dysbiosis. In addition, we discuss new insights into the role of novel host-microbial signals as potential biomarkers, and their relevance for the future of precision dentistry and medicine.

SCRT1 is a novel beta cell transcription factor with insulin regulatory properties.

Here we show that scratch family transcriptional repressor 1 (SCRT1), a zinc finger transcriptional regulator, is a novel regulator of beta cell function. SCRT1 was found to be expressed in beta cells in rodent and human islets. In human islets, expression of SCRT1 correlated with insulin secretion capacity and the expression of the insulin (INS) gene. Furthermore, SCRT1 mRNA expression was lower in beta cells from T2D patients. siRNA-mediated Scrt1 silencing in INS-1832/13 cells, mouse- and human islets resulted in impaired glucose-stimulated insulin secretion and decreased expression of the insulin gene. This is most likely due to binding of SCRT1 to E-boxes of the Ins1 gene as shown with ChIP. Scrt1 silencing also reduced the expression of several key beta cell transcription factors. Moreover, Scrt1 mRNA expression was reduced by glucose and SCRT1 protein was found to translocate between the nucleus and the cytosol in a glucose-dependent fashion in INS-1832/13 cells as well as in a rodent model of T2D. SCRT1 was also regulated by a GSK3ß-dependent SCRT1-serine phosphorylation. Taken together, SCRT1 is a novel beta cell transcription factor that regulates insulin secretion and is affected in T2D.

Caries-Associated Biosynthetic Gene Clusters in Streptococcus mutans.

Early childhood caries (ECC) is a chronic disease affecting the oral health of children globally. This disease is multifactorial, but a primary factor is cariogenic microorganisms such as Streptococcus mutans. Biosynthetic gene clusters (BGCs) encode small molecules with diverse biological activities that influence the development of many microbial diseases, including caries. The purpose of this study was to identify BGCs in S. mutans from a high-caries risk study population using whole-genome sequencing and assess their association with ECC. Forty representative S. mutans isolates were selected for genome sequencing from a large-scale epidemiological study of oral microbiology and dental caries in children from a localized Alabama population. A total of 252 BGCs were identified using the antiSMASH BGC-mining tool. Three types of BGCs identified herein-butyrolactone-like, ladderane-like, and butyrolactone-ladderane-like hybrid (BL-BGC)-have not been reported in S. mutans. These 3 BGCs were cross-referenced against public transcriptomics data, and were found to be highly expressed in caries subjects. Furthermore, based on a polymerase chain reaction screening for core BL genes, 93% of children with BL-BGC had ECC. The role of BL-BGC was further investigated by examining cariogenic traits and strain fitness in a deletion mutant using in vitro biofilm models. Deletion of the BL-BGC significantly increased biofilm pH as compared to the parent strain, while other virulence and fitness properties remained unchanged. Intriguingly, BL-BGC containing strains produced more acid, a key cariogenic feature, and less biofilm than the model cariogenic strain S. mutans UA159, suggesting the importance of this BL-BGC in S. mutans-mediated cariogenesity. The structure of any BL-BGC derived metabolites, their functions, and mechanistic connection with acid production remain to be elucidated. Nevertheless, this study is the first to report the clinical significance of a BL-BGC in S. mutans. This study also highlights pangenomic diversity, which is likely to affect phenotype and virulence.

Endogenous beta-cell CART regulates insulin secretion and transcription of beta-cell genes.

Impaired beta-cell function is key to the development of type 2 diabetes. Cocaine- and amphetamine-regulated transcript (CART) is an islet peptide with insulinotropic and glucagonostatic properties. Here we studied the role of endogenous CART in beta-cell function. CART silencing in INS-1 (832/13) beta-cells reduced insulin secretion and production, ATP levels and beta-cell exocytosis. This was substantiated by reduced expression of several exocytosis genes, as well as reduced expression of genes important for insulin secretion and processing. In addition, CART silencing reduced the expression of a network of transcription factors essential for beta-cell function. Moreover, in RNAseq data from human islet donors, CARTPT expression levels correlated with insulin, exocytosis genes and key beta-cell transcription factors. Thus, endogenous beta-cell CART regulates insulin expression and secretion in INS-1 (832/13) cells, via actions on the exocytotic machinery and a network of beta-cell transcription factors. We conclude that CART is important for maintaining the beta-cell phenotype.

A Microscale Technique for Gas Chromatography-Mass Spectrometry Measurements of Picogram Amounts of Indole-3-Acetic Acid in Plant Tissues.

A microscale technique has been developed for routine quantifications of picogram amounts of indole-3-acetic acid (IAA) in plant tissues by combined gas chromatography-mass spectrometry. Low- and high-resolution selected-ion-monitoring and selected-reaction-monitoring mass spectrometry techniques were compared for selectivity and precision. The best selectivity was obtained with selected-reaction-monitoring analysis, and 1-mg samples containing 500 fg of IAA could be analyzed accurately with this method. This technique was used to investigate the IAA distribution pattern along the longitudinal axis of tobacco (Nicotiana tabacum [L.]) leaves. In young, developing leaves an increase of endogenous IAA from the leaf tip to the base of the leaf was observed, whereas the level of IAA was uniform along this axis in mature leaves.

Structure of the human kappa-casein gene.

The human kappa-casein-encoding gene, Kca, was cloned and sequenced. The structural gene consists of five exons ranging from 33 to 496 nucleotides (nt) separated by introns ranging from 1146 to 2942 nt, and extends over 8821 nt. All intron/exon splice junctions conform to the GT/AG rule. The gene organization is similar to that of the bovine gene. The 5'-flanking region contains an A + T-rich sequence; TTTAATT, close to where the TATA motif is found in most other genes, a CAAT box, and an AP-1 consensus sequence. In addition, one Alu repetitive element was found in the second intron.

Structure of the human beta-casein encoding gene.

The entire human beta-casein-encoding gene, Bca, was cloned and sequenced. The gene consists of eight exons ranging from 21 to 531 nucleotides (nt) in length and extending over 10,466 nt. Exon-2 contains the translational start, the entire signal sequence and the codons for the two first amino acids of the mature protein. This corresponds to the organization found in other species. The translational stop is localized to exon-7. Exon/intron boundaries are in accordance with the AG/GT rule and conform to suggested consensus sequences. Splice junctions are located between coding triplets. In all other species analyzed, Bca has been found to consist of nine exons; however, within intron-2 of the human gene, a sequence omitted from human mRNA, but corresponding to exon-3 of other known Bca genes, was revealed.

Pharmacokinetics of extracellular-superoxide dismutase in the vascular system.

Extracellular-superoxide dismutase C (EC-SOD C) is a secretory tetrameric Cu- and Zn-containing glycoprotein which has high affinity for heparin and heparan sulfate. Upon intravenous injection into rabbits, recombinant human (rh) EC-SOD C was found to be rapidly 97-98% sequestered to the vascular wall, forming an equilibrium with the plasma phase. Recombinant EC-SOD truncation variants with reduced, T216, and without, T213, heparin affinity were found to be sequestered to a reduced extent and not at all, respectively, establishing the importance of the heparin affinity for this behaviour. The halflife of rhEC-SOD C in the vasculature was of the order of 20 h. Injection of large doses resulted in saturation of the binding of rhEC-SOD C to the vascular wall. Scatchard analysis revealed a heterogeneity in affinity of the ligands on the vascular wall. The maximal binding capacity was very high. The equilibration of rhEC-SOD C to the vascular wall of an organ, clamped during enzyme injection, and the primary equilibration phase was studied by comparing binding to a clamped and reperfused kidney with binding to the contralateral control kidney. rhEC-SOD C injected in a low dose was found to equilibrate very slowly to the reperfused kidney with a halftime of about 2 h. With higher rhEC-SOD C doses, at which evidence for saturation is seen, and with the variant rhEC-SOD with reduced heparin affinity. T216, very rapid equilibrations were found.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute supplementation with the nitric oxide precursor L-arginine does not improve cardiovascular performance in patients with hypercholesterolemia.

Endothelial dysfunction based on lack of nitric oxide (NO) may contribute to several settings of cardiovascular disorder. Chronic oral supplementation with the NO precursor L-arginine counteracts the development of aortic atherosclerosis in cholesterol-fed rabbits, and i.v. infusion of L-arginine may acutely improve endothelium-dependent coronary epicardial vasodilation in patients with hypercholesterolemia (HC). To clarify whether excess NO precursor may also improve general cardiovascular performance in HC, we measured working capacity indices of myocardial ischemia, and basal and post-occlusive forearm and skin blood flow in nine patients with elevated plasma cholesterol (9.1 +/- 0.2 mumol/l) following random double-blinded administration of L-arginine (16 g i.v.) or placebo. Infusion of L-arginine raised the plasma concentration of this amino acid from 85 +/- 12 to 2460 +/- 230 mumol/l but did not change the plasma level of the major NO metabolite nitrate. Maximal working capacity, indices of myocardial ischemia, and basal and post-occlusive blood flow in the skin or forearm did not differ between the treatments. The lack of positive effect of L-arginine compared to placebo indicates that excess NO precursor did not improve microvascular endothelial function in the patients, or alternatively, that the indices measured in the present study were not dependent on endothelial microvessel function. Thus, in patients with HC, deficiency of precursor for NO formation does not seem to impair either maximal exercise capacity myocardial perfusion during maximal exercise, or maximal vasodilator capacity in skeletal muscle or skin.

Delirium before and after operation for femoral neck fracture.

OBJECTIVES: The aim of this study was to investigate the differences between preoperative and postoperative delirium regarding predisposing, precipitating factors and outcome in older patients admitted to hospital with femoral neck fractures. DESIGN: A prospective clinical assessment of patients treated for femoral neck fractures. SETTING: Department of orthopedic surgery at Umeå University Hospital, Sweden. PARTICIPANTS: One hundred one patients, age 65 and older admitted to the hospital for treatment of femoral neck fractures. MEASUREMENTS: The Organic Brain Syndrome (OBS) Scale. RESULTS: Thirty patients (29.7%) were delirious before surgery and another 19 (18.8%) developed delirium postoperatively. Of those who were delirious preoperatively, all but one remained delirious postoperatively. The majority of those delirious before surgery were demented, had been treated with drugs with anticholinergic properties (mainly neuroleptics), had had previous episodes of delirium, and had fallen indoors. Patients who developed postoperative delirium had perioperative falls in blood pressure and had more postoperative complications such as infections. Male patients were more often delirious both preoperatively and postoperatively. Patients with preoperative delirium were more often discharged to institutional care and had poorer walking ability both on discharge and after 6 months than did patients with postoperative delirium only. CONCLUSIONS: Because preoperative and postoperative delirium are associated with different risk factors it is necessary to devise different strategies for their prevention.

Evidence for an anti-aggregatory effect of adenosine at physiological concentrations and for its role in the action of dipyridamole.

The effects of physiological adenosine concentrations on platelet aggregation in vitro were studied. Furthermore, we evaluated the effect of elevated adenosine levels in vivo, produced by the administration of dipyridamole, on platelet aggregation in whole blood. Platelet aggregation in plasma was significantly inhibited in vitro by adenosine at all concentrations tested in the physiological range (0.1-1.0 microM, 14-63% inhibition). Dipyridamole by itself had no effect at a therapeutic plasma concentration in vitro. Ten patients with ischaemic cerebrovascular disease were given 100 mg dipyridamole orally, and the level of adenosine increased from 0.22 to 0.29 microM (p less than 0.05). This was accompanied by a decrease in ADP-induced platelet aggregation in whole blood (17 to 15 ohms, p less than 0.05). When dipyridamole was infused in 11 healthy subjects, the adenosine level was not significantly elevated but the platelet aggregation was inhibited (from 13 to 11 ohms, p less than 0.05). It is concluded that adenosine may be of importance in the physiological regulation of platelet aggregation. Furthermore, dipyridamole treatment is associated with an anti-aggregatory effect that is probably mediated by its effect on endogenous adenosine levels.

Renal effects of i.v. adenosine infusion in humans.

The effects of systemic intravenous (i.v.) infusion of adenosine on renal blood flow and glomerular filtration in eight healthy, awake females have been examined. Renal blood flow and glomerular filtration rate were measured as the clearance of para-aminohippuric acid (PAH) and inulin, respectively. Following basal sampling adenosine was infused i.v. at successive rates of 60 and 80 micrograms kg-1 min-1 for 30 min at each rate. Plasma clearance of PAH showed a minor, but non-significant, increase from 697 +/- 41 to 775 +/- 97 ml min-1. However, the arterial plasma concentration of PAH decreased by 17 +/- 4% (P < 0.001), indicating that there was a small increase in renal blood flow. Inulin clearance was reduced from 123 +/- 14 to 88 +/- 11 ml min-1 1.73 m-2 (P < 0.01). The resulting filtration fraction was reduced from 18 +/- 1 to 11 +/- 1 (P < 0.001). The K+/Na+ excretion ratio increased from a basal value of 10 +/- 1 to 42 +/- 11 (P < 0.01) at the highest dose of adenosine, and renal oxygen consumption decreased from 17 +/- 2 to 9 +/- 1 ml min-1 (P < 0.001). In conclusion, i.v. infusion of adenosine in healthy, awake subjects causes a minor increase in total renal blood flow and a marked reduction in glomerular filtration. This shows that adenosine also exerts a vasodilatory effect in the renal circulation, primarily on postglomerular arterioles. In addition, adenosine may produce an aldosterone-like effect on salt excretion, and a reduction in renal oxygen consumption.

Theophylline increases coronary vascular tone in humans: evidence for a role of endogenous adenosine in flow regulation.

To elucidate the role of adenosine in coronary vasoregulation, we studied the effects of adenosine antagonism (by theophylline) on coronary blood flow at different levels of adenosine formation (stimulated by hypoxia and exercise). Six healthy subjects were studied. Coronary sinus (CS) blood flow (thermodilution) and cardiac oxygen extraction [(A-CS)O2D] were determined while breathing room air at rest, and 12% oxygen, both at rest and during light exercise, on two occasions. One of the experiments was performed during infusion of theophylline. The basal CS flow was 118 (67-168) mL min-1 (mean and 95% confidence interval), and the (A-CS)O2D was 125 (111-142) mL L-1. Inhalation of 12% O2 decreased the arterial haemoglobin oxygen saturation to 83 (80-86)% at rest and to 77 (73-81)% during exercise. CS flow increased to 167 (93-214) and 261 (179-343) mL min-1, respectively, and (A-CS)O2D decreased to 102 (85-119) and 94 (77-111) mL L-1, respectively. Theophylline, at a dose lacking effects on myocardial work, markedly attenuated the coronary flow response to exogenous adenosine, and decreased CS flow to 89 (58-119), 120 (79-161) and 190 (162-218) mL min-1 at normoxic rest, hypoxic rest and hypoxic exercise, respectively. The overall decrease amounted to 23% (P < 0.05). The calculated coronary vascular conductance also decreased by 23% (P < 0.05) and (A-CS)O2D increased by 15% (P < 0.001). In conclusion, the data support the hypothesis that endogenous adenosine is involved in regulation of human coronary tone.

Hypoxia elicits liberation of anti-aggregatory substances from isolated rabbit hearts.

The hypothesis was investigated that myocardial hypoxia stimulates the production of platelet anti-aggregatory substances in the heart. Rabbit hearts were perfused under normoxic or hypoxic conditions and the coronary and interstitial effluents from the hearts were separated. The occurrence of anti-aggregatory activity (AAA) in the interstitial effluent was detected in vitro from its capacity to inhibit ADP-induced platelet aggregation. The AAA in the effluent was deemed to be prostacyclin (PGI2) if its release was abolished by administration of indomethacin (5 X 10(-5) M) to the heart, and to be adenosine if it was abolished by incubation of the effluent with adenosine deaminase. During normoxic perfusion, only a minor efflux of AAA appeared from the heart; neither was the efflux appreciable during mild hypoxia (30 or 60% O2). Severe hypoxia (venous pO2 below 5 kPa), on the other hand, was associated with a marked release of AAA. Incubation of hypoxic effluent with adenosine deaminase resulted in a small loss of activity, indicating that the major part of the AAA was not ascribable to adenosine. After indomethacin treatment, significant amounts of AAA still appeared in the effluent during hypoxia. However, unlike the case before indomethacin, this AAA was completely destroyed by adenosine deaminase. From these data, we conclude that myocardial hypoxia can mobilize either of two independent mechanisms for protection against platelet aggregation: an activation of the synthesis and release of prostacyclin, and a more complete breakdown of ATP, leading to an increased formation and efflux of adenosine.