Randomly barcoded transposon mutant libraries for gut commensals II: Applying libraries for functional genetics.

The critical role of the intestinal microbiota in human health and disease is well recognized. Nevertheless, there are still large gaps in our understanding of the functions and mechanisms encoded in the genomes of most members of the gut microbiota. Genome-scale libraries of transposon mutants are a powerful tool to help us address this gap. Recent advances in barcoded transposon mutagenesis have dramatically lowered the cost of mutant fitness determination in hundreds of in vitro and in vivo experimental conditions. In an accompanying review, we discuss recent advances and caveats for the construction of pooled and arrayed barcoded transposon mutant libraries in human gut commensals. In this review, we discuss how these libraries can be used across a wide range of applications, the technical aspects involved, and expectations for such screens.

Phenotypic and molecular characterization of ESBL producing Escherichia coli and Klebsiella pneumoniae among Lebanese patients.

Introduction: Antimicrobial resistance is a major public health issue worldwide and became one of the principal international healthcare crises of the 21st century. The production of ESBLs is one of the resistance mechanisms in Enterobacteriaceae, and they are increasingly detected in Escherichia coli and Klebsiella pneumoniae globally. Therefore, the aim of this study was to determine the phenotypic and molecular characteristics of ESBL-producing E. coli and K. pneumoniae among Lebanese patients. Methods: A total of 152 ESBL-producing E. coli and K. pneumoniae were obtained from Geitaoui Hospital in Beirut between September 2019 and October 2020 from various clinical samples. The phenotype of ESBL producers was confirmed by a double-disc synergy test and antibiotic susceptibility was determined using the disc diffusion method. Genotypically, multiplex PCR was used to detect the ESBL genes (blaTEM, blaCTX-M and blaSHV). Results: All strains were confirmed to be ESBL producers (121 isolates were E. coli and 31 isolates were K. pneumoniae). All isolates showed resistance to cefotaxime, cefuroxime, ampicillin and piperacillin. On the other hand, they showed a low susceptibility rate to trimethoprim/sulfamethoxazole and ciprofloxacin. Almost all the isolates were susceptible to ertapenem, imipenem and amikacin. In our study, ESBL genes were detected among 48 (39.67%) E. coli isolates and 8 (58.06%) K. pneumoniae isolates, and the most prevalent gene was blaTEM (25%), followed by blaCTX-M (19.08%) and blaSHV (16.45%). Conclusion: Imipenem and ertapenem are the most effective drugs to treat ESBL producers. However, antibiotic stewardship programs must be implemented immediately to combat antibiotic resistance.

Target search by an imported conjugative DNA element for a unique integration site along a bacterial chromosome during horizontal gene transfer.

Integrative and conjugative elements (ICEs) are mobile genetic elements that can transfer by conjugation to recipient cells. Some ICEs integrate into a unique site in the genome of their hosts. We studied quantitatively the process by which an ICE searches for its unique integration site in the Bacillus subtilis chromosome. We followed the motion of both ICEBs1 and the chromosomal integration site in real time within individual cells. ICEBs1 exhibited a wide spectrum of dynamical behaviors, ranging from rapid sub-diffusive displacements crisscrossing the cell, to kinetically trapped states. The chromosomal integration site moved sub-diffusively and exhibited pronounced dynamical asymmetry between longitudinal and transversal motions, highlighting the role of chromosomal structure and the heterogeneity of the bacterial interior in the search. The successful search for and subsequent recombination into the integration site is a key step in the acquisition of integrating mobile genetic elements. Our findings provide new insights into intracellular transport processes involving large DNA molecules.

Timing of integration into the chromosome is critical for the fitness of an integrative and conjugative element and its bacterial host.

Integrative and conjugative elements (ICEs) are major contributors to genome plasticity in bacteria. ICEs reside integrated in the chromosome of a host bacterium and are passively propagated during chromosome replication and cell division. When activated, ICEs excise from the chromosome and may be transferred through the ICE-encoded conjugation machinery into a recipient cell. Integration into the chromosome of the new host generates a stable transconjugant. Although integration into the chromosome of a new host is critical for the stable acquisition of ICEs, few studies have directly investigated the molecular events that occur in recipient cells during generation of a stable transconjugant. We found that integration of ICEBs1, an ICE of Bacillus subtilis, occurred several generations after initial transfer to a new host. Premature integration in new hosts led to cell death and hence decreased fitness of the ICE and transconjugants. Host lethality due to premature integration was caused by rolling circle replication that initiated in the integrated ICEBs1 and extended into the host chromosome, resulting in catastrophic genome instability. Our results demonstrate that the timing of integration of an ICE is linked to cessation of autonomous replication of the ICE, and that perturbing this linkage leads to a decrease in ICE and host fitness due to a loss of viability of transconjugants. Linking integration to cessation of autonomous replication appears to be a conserved regulatory scheme for mobile genetic elements that both replicate and integrate into the chromosome of their host.

Salivary MMP-9 Levels in Chronic Periodontitis Patients with Type-II Diabetes Mellitus.

Chronic periodontitis and diabetes mellitus share a two-way relationship, the common factor being the inflammatory-mediated pathway, and various cytokines are released during this inflammatory cascade, one of which being matrix metalloproteinase-9. The aim of this study was to identify whether the levels of matrix metalloproteinase-9 are increased due to type-II diabetes mellitus in chronic periodontitis patients. It was an observational, analytical, case-control study. Thirty subjects were recruited in the test group, who were suffering from type-II diabetes mellitus and chronic periodontitis, and 30 subjects in the control group, who were subjects with chronic periodontitis but systemically healthy. Periodontal parameters, including the plaque score, gingival bleeding index, probing pocket depth and clinical attachment level of the subjects, were measured, saliva samples of all of the subjects were collected and salivary matrix metalloproteinase-9 levels were analyzed by an enzyme-linked immunosorbent assay (ELISA) technique. The statistical analysis was performed using SPSS 24. Overall, the matrix metalloproteinase-9 levels of the diabetic patients with chronic periodontitis were increased almost twofold (156.95 ± 29.80 ng/mL) compared to the levels in the controls (74.96 ± 6.32 ng/mL) (p < 0.001). Similarly, the periodontal parameters were far worse in diabetics with chronic periodontitis compared to the controls. The average gingivitis score for the test subjects was 78.45 ± 8.93%), compared to 29.32 ± 12.96% in the controls (p < 0.001). The mean probing pocket depth for the test group was 5.39 ± 0.60 mm, and, for the control group, it was 4.35 ± 0.31 mm (p < 0.001). For the test subjects, the average clinical attachment level was 5.86 ± 0.58 mm, and it was 4.66 ± 0.32 mm for the controls (p < 0.001). It was ascertained that there is a two-fold increase in the levels of salivary matrix metalloproteinase-9 in the test group compared to the control group. In addition, the level of periodontal apparatus destruction was greater in the test group. This proved that type-II diabetes mellitus influences the levels of matrix metalloproteinase-9 in humans and elevates them, causing further periodontal destruction.

Consensus Guidelines for Therapeutic Drug Monitoring in Neuropsychopharmacology: Update 2017.

Therapeutic drug monitoring (TDM) is the quantification and interpretation of drug concentrations in blood to optimize pharmacotherapy. It considers the interindividual variability of pharmacokinetics and thus enables personalized pharmacotherapy. In psychiatry and neurology, patient populations that may particularly benefit from TDM are children and adolescents, pregnant women, elderly patients, individuals with intellectual disabilities, patients with substance abuse disorders, forensic psychiatric patients or patients with known or suspected pharmacokinetic abnormalities. Non-response at therapeutic doses, uncertain drug adherence, suboptimal tolerability, or pharmacokinetic drug-drug interactions are typical indications for TDM. However, the potential benefits of TDM to optimize pharmacotherapy can only be obtained if the method is adequately integrated in the clinical treatment process. To supply treating physicians and laboratories with valid information on TDM, the TDM task force of the Arbeitsgemeinschaft für Neuropsychopharmakologie und Pharmakopsychiatrie (AGNP) issued their first guidelines for TDM in psychiatry in 2004. After an update in 2011, it was time for the next update. Following the new guidelines holds the potential to improve neuropsychopharmacotherapy, accelerate the recovery of many patients, and reduce health care costs.

Total and organic mercury in liver, kidney and muscle of waterbirds from wetlands of the Caspian Sea, Iran.

We measured and compared total and organic mercury in liver, kidney, and muscle of the Great Cormorant (Phalacrocorax carbo), mallard (Anas platyrhynchos), and coot (Fulica atra) from the Caspian Sea wetlands in Iran. For the Great Cormorant organic mercury in liver, kidney and muscle comprised 82 %, 79 % and 58 % of total mercury. In the mallard same values were 46 %, 54 %, and 64 %. For coot total mercury was: 0.1 ± 0.0, 0.1 ± 0.01, 0.03 ± 0.01 in liver kidney and muscle respectively. We detected no organic mercury. In general older birds that feed on higher trophic levels can accumulate more mercury in their tissues.

Secretoneurin promotes pertussis toxin-sensitive neurite outgrowth in cerebellar granule cells.

The neuropeptide secretoneurin (SN) is an endoproteolytic product of the chromogranin secretogranin II. We investigated the effects of SN on the differentiation of immature cerebellar granule cells derived from the external granular layer (EGL). Secretoneurin caused concentration-dependent increases in neurite outgrowth, reflecting differentiation. The maximum effect was reached at a concentration of 100 nm SN. Secretoneurin immunoneutralization using specific antiserum significantly decreased neurite outgrowth; however, neurite morphology was altered. An affinity chromatography-purified antibody significantly inhibited the outgrowth response to SN (p < 0.001) without altering the morphology. Binding studies suggest the existence of specific G-protein-coupled receptors on the surface of monocytes that recognize SN. Assuming that SN promotes neurite outgrowth in EGL cells by acting through a similar G-protein-coupled mechanism, we treated SN-stimulated EGL cultures with pertussis toxin. Exposure to pertussis toxin (0.1 micro g/mL) showed a significant inhibition of the SN-induced outgrowth. To establish a second messenger pathway we used the protein kinase C inhibitor staurosporine. We found that EGL cell viability was not enhanced following chronic SN treatment for 24 h. These data indicate that SN is a novel trophic substance that can affect cerebellar maturation, primarily by accelerating granule cell differentiation through a signalling mechanism that is coupled to pertussis toxin-sensitive G-proteins.

Intravenous drug injection habits: drug users' self-reports versus researchers' perception.

The present study was designed to obtain human data on the speed of intravenous (i.v.) injection of cocaine, heroin, and morphine as well as on the rate of onset of their subjective effects and their duration in order to improve the accuracy of animal and human experimental models of i.v. drug abuse. To that end, a questionnaire was submitted both to clients of a substitution therapy outpatient clinic and to members of the drug abuse research community. It was found that i.v. drug abusers injected cocaine, heroin, or morphine much faster and also experienced the drug effects much faster than assumed by the drug abuse researchers. The time course of the reemergence of craving was also greatly misjudged by the researchers. On the other hand, the i.v. drug users' self-reports were internally consistent and corresponded well to data obtained in several different human behavioral laboratories. Interestingly, more than half of the i.v. drug users reported that injection speed was not important when injecting cocaine (57%), heroin (72%) or morphine (73%) under conditions that guarantee a maximum effect, suggesting that the rate of the rise in the brain concentration of a drug of abuse is less important for its reinforcing effect and, thus, for its abuse liability, than previously assumed, at least within the time frame of an i.v. drug injection.

Intravenous administration of ecstasy (3,4-methylendioxymethamphetamine) enhances cortical and striatal acetylcholine release in vivo.

The effect of intravenous administration of 3,4-methylendioxymethamphetamine (MDMA), in a range of doses (0.32-3.2 mg/kg) that have been shown to maintain self-administration behaviour in rats, on in vivo acetylcholine release from rat prefrontal cortex and dorsal striatum was studied by means of microdialysis with vertical concentric probes. Intravenous administration of MDMA dose-dependently increased basal acetylcholine release from the prefrontal cortex to 57+/-21%, 98+/-20%, 102+/-7% and 141+/-14% above baseline, at doses of 0.32, 0.64, 1.0 and 3.2 mg/kg, respectively. MDMA also stimulated striatal acetylcholine release at the dose of 3.2 mg/kg i.v. (the maximal increase being 32+/-3% above baseline) while at the dose of 1 mg/kg i.v., MDMA failed to affect basal acetylcholine output. Administration of MDMA also dose-dependently stimulated behaviour. The results of the present study show that MDMA affects measures of central cholinergic neurotransmission in vivo and suggest that at least some of the psychomotor stimulant actions of MDMA might be positively coupled with an increase in prefrontal cortical and striatal acetylcholine release.

Reinforcing effects of MDMA ("ecstasy") in drug-naive and cocaine-trained rats.

3,4-Methylenedioxymethamphetamine (MDMA; "ecstasy") is one of the most prevalent illegal drugs of abuse among European adolescents, a population not generally experienced with respect to "hard" drugs such as cocaine. We, therefore, determined the reinforcing effect of intravenously self-administered MDMA in a fixed ratio 1 time-out 150 s schedule of reinforcement in rats that were truly drug naive and compared it to cocaine-trained rats. The reinforcing effect of MDMA [0.032-10 mg/(kg.injection)] did not differ between drug-naive rats and cocaine-trained ones. MDMA sensitized the animals to its own rate-increasing effect but not to that of cocaine. When MDMA was tested after cocaine, there was no carryover of cocaine's reinforcing effect to that of MDMA, suggesting that MDMA and cocaine produce distinct interoceptive stimuli in rats.

Lack of reinforcing effect of the benzodiazepine and tricyclic antidepressant combination of diazepam and dothiepin.

Fixed combinations of a tricyclic antidepressant (TCA) with a benzodiazepine (BZD) for the treatment of depressive syndromes enjoy remarkable acceptance among patients and prescribing physicians. In order to investigate if the widespread use of one such fixed TCA-BZD combination might be due to its high positive reinforcing effect, we tested each drug alone and in combination in an operant conditioning paradigm (fixed ratio 1 time-out 150 s) of intravenous self-administration in rats and compared their reinforcing effects to that of cocaine. Diazepam proved to be of only moderate reinforcing strength. Dothiepin alone was ineffective as a reinforcer but essentially abolished the reinforcing effect of diazepam when given in combination with it. These data indicate that the widespread acceptance of the fixed diazepam-dothiepin combination by the therapeutic community is not due to an increase in the positive reinforcing effect of diazepam by dothiepin but that, in contrast, addition of dothiepin might even decrease diazepam's moderately positive reinforcing effect.

Alterations within the endogenous opioid system in mice with targeted deletion of the neutral endopeptidase ('enkephalinase') gene.

The biological inactivation of enkephalins by neutral endopeptidase (enkephalinase, NEP, EC3.4.24.11) represents a major mechanism for the termination of enkephalinergic signalling in brain. A pharmacological blockade of NEP-activity enhances extracellular enkephalin concentrations and induces opioid-dependent analgesia. Recently, knockout mice lacking the enzyme NEP have been developed [Lu et al., J. Exp. Med. 1995;181:2271-2275]. The present study investigates the functional consequences and biochemical compensatory strategies of a systemic elimination of NEP activity in these knockout mice. Using biochemical and behavioural tests we found that the lack of NEP activity in brain is not compensated by enhanced activities of alternative enkephalin-degrading enzymes. Also no change in enkephalin biosynthesis was detectable by in situ methods quantifying striatal proenkephalin-mRNA levels in NEP-deficient mice compared with wildtype. Only a 21% reduction of mu receptor density in crude brain homogenates of NEP knockout mice was observed, while delta- and kappa-opioid receptor densities were unchanged. This receptor downregulation was also confirmed functionally in the hot-plate paradigm. NEP knockouts developed normally, but showed enhanced aggressive behaviour in the resident-intruder paradigm, and altered locomotor activity as assessed in the photobeam system. Thus, although NEP plays a substantial role in enkephalinergic neurotransmission, the biochemical adaptations within the opioid system of NEP-deficient mice are of only modest nature.

Retinoic acid treatment enhances the acetylcholine contents in the human teratocarcinoma cell line NTera-2.

Human NTera-2/clone D1 teratocarcinoma cells are induced by retinoic acid (RA) to differentiate into postmitotic cells with morphological and biochemical characteristics of embryonic human neurones. Currently only limited information concerning peptide-contents and neurotransmitter pools of these cells is available. Zeller and Strauss [Int. J. Dev. Neurosci. 1995;13(5):437] described an increase in choline acetyltransferase (ChAT) activity in RA-treated, but not in untreated NTera-2 cells, suggesting the induction of a cholinergic phenotype during treatment with RA. In the present study we investigated the effect of RA-differentiation on the amount of the neurotransmitters acetylcholine (ACh), and dopamine in NTera-2 in order to specify the transmitter phenotype induced by RA-differentiation. We found that a 4-week treatment of NTera-2 cells with 10 microM RA markedly increased the ACh-content of these cells, while dopamine levels were unchanged. Depolarisation with potassium (60 mM) enhanced ACh-outflow in the differentiated cells in a Ca(++) dependent way. Also neuropeptides like substance P and NPY were detectable in the undifferentiated NTera-2 cells, while vasointestinal peptide (VIP) could not be found in either precursor or RA-differentiated cells. Differentiation was accompanied by a marked reduction of neutral endopeptidase enzyme activity and aminopeptidase activity. From these observations it was concluded that RA induces a cholinergic neurochemical differentiation of this human teratocarcinoma cell line, and that these cells might provide a model system to investigate cholinergic properties of human origin.

Margatoxin and iberiotoxin, two selective potassium channel inhibitors, induce c-fos like protein and mRNA in rat organotypic dorsal striatal slices.

The isolated single organotypic slice model allows to investigate the effects of drugs and toxins on the expression of transcription factors in the striatum without dopaminergic and glutamatergic interactions. In this study the effects of margatoxin and iberiotoxin on the expression of c-fos mRNA by in situ hybridization as well as on c-fos like protein by immunohistochemistry in isolated dorsal striatum after 10 days in culture were investigated. C-fos mRNA dose-dependently increased 30 min after incubation with margatoxin and iberiotoxin. Expression of c-fos like protein was transiently detected 3h afterwards. This effect is independent from extrinsic neuronal circuitry as dopamine neurons were found to be absent in the cultured slices. It is concluded that inhibition of voltage-gated as well as calcium-activated (Slo) potassium channels leads to activation of gene transcription in striatal neurons which may trigger long-term changes in transmitter plasticity.

Neutral endopeptidase and alcohol consumption, experiments in neutral endopeptidase-deficient mice.

Alcohol consumption was investigated in mice which were rendered deficient in the peptide-degrading enzyme neutral endopeptidase (EC 3.4.24.11) (NEP-/-) by gene targeting and compared to alcohol consumption in corresponding wild type mice (NEP+/+). Mice were offered a free choice to drink tap water or 10% alcohol. The NEP-/- mice consumed significantly more alcohol ( approximately 42%) than the NEP+/+ mice, whereas no significant differences were observed in the total fluid consumption. The daily food consumption of alcohol naive NEP-/- animals was elevated ( approximately 29%). Furthermore, the activities of peptidases closely related to neutral endopeptidase were analysed ex vivo in several brain regions from NEP-/- and NEP+/+ mice not treated with alcohol. There was no obvious compensation for the total loss of neutral endopeptidase by the functionally related peptidases angiotensin-converting enzyme and aminopeptidase N. In vitro, the degradation of exogenously applied [Leu(5)]enkephalin was not reduced in membrane preparations of those brain regions assayed in NEP-/- mice. A small reduction in [Leu(5)]enkephalin degradation was detected in striatal membrane preparations of NEP-/- mice, if aminopeptidase N was additionally blocked by bestatin or amastatin.

Videotape preparation of patients before hip replacement surgery reduces stress.

OBJECTIVE: Elective surgery represents a considerable source of stress for the patient. Many attempts have been made to prepare patients before surgery with the aim of reducing stress and improving outcome. This study used a novel approach to fulfill this aim by showing a videotape of a patient undergoing total hip replacement surgery, covering the time period from hospital admission to discharge, that strictly keeps to the patient's perspective. METHODS: Before elective total hip replacement surgery, 100 patients were randomly assigned to a control group or a preparation group; the latter group was shown the videotape on the evening before surgery. Anxiety and pain were evaluated daily for 5 days, beginning with the preoperative day, by means of the State-Trait Anxiety Inventory and a visual analog scale. Intraoperative heart rate and blood pressure, as well as postoperative intake of analgesics and sedatives, were recorded. Urinary levels of cortisol, epinephrine, and norepinephrine were determined in 12-hour samples collected at night for 5 nights, beginning with the preoperative night. RESULTS: Compared with the control group, the preparation group showed significantly less anxiety on the morning before surgery and the mornings of the first 2 postoperative days, and significantly fewer of them had an intraoperative systolic blood pressure increase of more than 15%. The pain ratings did not differ significantly between the two groups, but the prepared patients needed less analgesic medication after surgery. Prepared patients had significantly lower cortisol excretion during the preoperative night and the first 2 postoperative nights. Excretion of catecholamines did not differ significantly between groups. CONCLUSIONS: We conclude that use of the videotape decreased anxiety and stress, measured in terms of urinary cortisol excretion and intraoperative systolic blood pressure increase, in patients undergoing hip replacement surgery and prepared them to cope better with postoperative pain.

Signal transduction efficacy of the highly potent mu opioid agonist 14-methoxymetopon.

In search of a truly high-efficacy (i.e., tau > 100) mu opioid analgesic, we determined the efficacy (tau) and apparent in vivo affinity (KA) of the high-potency alkoxymorphinan 14-methoxymetopon. However, in the present study, 14-methoxymetopon's efficacy proved to be only 1.5-fold higher than that of morphine (tau, 19 vs. 12). KA values were 2,900 nmol/kg for 14-methoxymetopon and 46,000 nmol/kg for morphine (Ki for [3H]DAMGO binding, 0.33 vs 3.4 nmol/l). Thus, the 24-fold higher potency of methoxymetopon could be fully accounted for by its 16-fold higher apparent in vivo affinity and its only 1.5-fold higher efficacy. Furthermore, the 10-fold higher affinity of 14-methoxymetopon for the mu opioid receptor - as previously determined in radioligand binding assays - was confirmed in the present behavioral tests of thermal antinociception.