Novel Quorum Quenching YtnP Lactonase From Bacillus paralicheniformis Reduces Pseudomonas aeruginosa Virulence and Increases Antibiotic Efficacy in vivo

Bacterial infections have become increasingly difficult to treat due to the occurrence of antibiotic-resistant strains. A promising strategy to increase the efficacy of therapy is to combine antibacterials with agents that decrease pathogen virulence via the modulation of the quorum sensing (QS). Lactonases inhibit acylated homoserine lactone (AHL)-mediated QS in Gram-negative bacteria, including the leading nosocomial pathogen Pseudomonas aeruginosa. Here we describe the characteristics of heterologously expressed YtnP lactonase from Bacillus paralicheniformis ZP1 (YtnP-ZP1) isolated from agricultural soil using the culture enrichment method. Purified YtnP-ZP1 hydrolyzed different AHLs with preference to substrates with long acyl residues as evaluated in assays with biosensors and HPLC. The enzyme showed good thermostability and activity in a wide temperature range. YtnP-ZP1 in 50 μg mL–1 concentration reduced the amount of P. aeruginosa-produced long-chain AHLs by 85%, while it hydrolyzed 50% of short-chain AHLs. Incubation of P. aeruginosa PAO1 with YtnP-ZP1 reduced its swarming motility and elastolytic activity without bactericidal effect. YtnP-ZP1 caused the inhibition of biofilm formation and disintegration of mature biofilms in P. aeruginosa PAO1 and multiresistant clinical strain BR5H that was visualized by crystal violet staining. The treatment with YtnP-ZP1 in concentrations higher than 25 μg mL–1 improved the survival of P. aeruginosa PAO1-infected zebrafish (Danio rerio), rescuing 80% of embryos, while in combination with tobramycin or gentamicin survival rate increased to 100%. The treatment of P. aeruginosa PAO1 biofilms on infected zebrafish tail wounds with 50 μg mL–1 YtnP-ZP1 and 2 × MIC tobramycin led to infection clearing in 2 days. The extensive toxicity studies proved YtnP-ZP1 was non-toxic to human cells and zebrafish. In conclusion, novel YtnP-ZP1 lactonase with its effective anti-virulence activity could be used to increase the efficacy of clinically approved antibiotics in clearing both systemic and biofilm-associated P. aeruginosa infections

Efekti različitih vrsta organskog đubriva na veličinu tela i produkciju daphnia magna

U akvakulturi se već decenijama masovno proizvodi zooplankton kao živa hrana za gajenje mlađi riba, rakova i mekušaca. Za slatkovodne ribe se osim naupliusa artemije i rotatorija može koristiti i veliki broj drugih organizama, naročito iz grupe Cladocera (Daphnia magna, D. carinata i dr.). Korišćenje organiskog đubriva (stajnjaka) za proizvodnju žive hrane, može biti ekološki (uklanjanje nutrijenata) i ekonomski isplativo. Stajnjak je bogat izvor azota, fosfora i ugljenika kao i organskih materija i kao takav se može koristiti kao alternativni izvor nutrijenata za gajenje žive hrane za potrebe akvakulture. Cilj ovog istraživanja je bio ispitivanje efekata 4 različite vrste stajnjaka (goveđi, ovčiji, koziji i riblji) na produkciju i veličinu tela D. magna. Eksperiment je realizovan u laboratorijskim uslovima, u 1L čašama, u dozi od 1g/L stajnjaka. Pored praćenja hemijskih parametara (kiseonik, elektroprovodljivost, pH, ukupan fosfor, nejonizovani amonijak), određivana je i ukupna brojnost dafnija kao i njihova veličina iz celih uzoraka. Tri veličinske klase dafnija (mala, srednja i velika) su ispitivane da bi se dobio uvid u populacionu strukturu i reproduktivni potencijal dafnija za čije su gajenje korišćeni različiti stajnjaci. Značajno veća brojnost dafnija (P<0.05) dobijena je u tretmanu sa goveđim stajnjakom, u odnosu na tretmane sa ovčijim i kozijim stajnjakom. Između ostalih nije bilo značajnih razlika. Kada se razmatraju veličinske klase, statistički značajno najveću brojnost su imale male D. magna u svim tretmanima u odnosu na velike. Na osnovu dobijenih rezultata, može se reći da je tretman sa goveđim stajnjakom pokazao najbolje rezultate i kada je u pitanju ukupna brojnost, ali i brojnost po veličinskim klasama (prosečna brojnost malih, srednjih i velikih dafnija je bila najveća). Osim toga ovaj tretman se izdvajao od ostalih i po svim hemijskim parametrima. U njemu je izmerena najveća prosečna koncentracija kiseonika, elektroprovodljivosti i pH, a najmanja koncentracija ukupnog fosfora i nejonizovanog amonijaka. U zaključku se može reći da je goveđi stajnjak obezbedio najbolje uslove za rast i reprodukciju D. magna

Endorhizosphere of indigenous succulent halophytes: a valuable resource of plant growth promoting bacteria

The adaptability of halophytes to increased soil salinity is related to complex rhizosphere interactions. In this study, an integrative approach, combining culture-independent and culture-dependent techniques was used to analyze the bacterial communities in the endorizosphere of indigenous succulent halophytes Salicornia europaea, Suaeda maritima, and Camphorosma annua from the natural salt marshes of Slano Kopovo (Serbia). The 16 S rDNA analyses gave, for the first time, an insight into the composition of the endophytic bacterial communities of S. maritima and C. annua. We have found that the composition of endophyte microbiomes in the same habitat is to some extent influenced by plant species. A cultivable portion of the halophyte microbiota was tested at different NaCl concentrations for the set of plant growth promoting (PGP) traits. Through the mining of indigenous halotolerant endophytes, we obtained a collection representing a core endophyte microbiome conferring desirable PGP traits. The majority (65%) of the selected strains belonged to the common halotolerant/halophilic genera Halomonas, Kushneria, and Halobacillus, with representatives exhibiting multiple PGP traits, and retaining beneficial traits in conditions of the increased salinity. The results suggest that the root endosphere of halophytes is a valuable source of PGP bacteria supporting plant growth and fitness in salt-affected soils. © 2023, The Author(s)

Ultrastructural characteristics of the vascular wall components of ruptured atherosclerotic abdominal aortic aneurysm

The aim of this study was to determine the ultrastructural characteristics of cell populations and extracellular matrix components in the wall of ruptured atherosclerotic abdominal aortic aneurysm (AAA). We analyzed 20 samples of ruptured AAA. For orientation to the light microscopy, we used routine histochemical techniques by standard procedures. For ultrastructural analysis, we applied transmission electron microscopy (TEM). Our results have shown that ruptured AAA is characterized by the remains of an advanced atherosclerotic lesion in the intima followed by a complete absence of endothelial cells, the disruption of basal membrane and disruption of internal elastic lamina. On plaque margins as well as in the inner media we observed smooth muscle cells (SMCs) that posses a euchromatic nucleus, a well-developed granulated endoplasmic reticulum around the nucleus and reduced myofilaments. The remains of the ruptured lipid core were acellular in all samples; however, on the lateral sides of ruptured plaque we observed a presence of two types of foam cells (FCs), spindle- and star-shaped. Fusiform FCs possess a well-differentiated basal lamina, caveolae and electron dense bodies, followed by a small number of lipid droplets in the cytoplasm. Star-shaped FCs contain a large number of lipid droplets and do not possess basal lamina. On the inner margins of the plaque, we observed a large number of cells undergoing apoptosis and necrosis, extracellular lipid droplets as well as a large number of lymphocytes. The media was thinned out with disorganized elastic lamellas, while the adventitia exhibited leukocyte infiltration. The presented results suggest that atherosclerotic plaque in ruptured AAA contains vascular SMC synthetic phenotype and two different types of FCs: some were derived from monocyte/macrophage lineage, while others were derived from SMCs of synthetic phenotype. The striking plaque hypocellularity was the result of apoptosis and necrosis of different cell populations

Rehabilitation Protocols for Children with Dysfunctional Voiding

Dysfunctional voiding is a functional voiding disorder characterized by an intermittent uroflow rate due to involuntary intermittent contractions of the striated muscle of the external urethral sphincter or pelvic floor muscles (PFMs) during voiding in neurologically normal children. Symptoms include voiding difficulties as well as urgency, voiding frequency and, in some instances, urinary incontinence and/or nocturnal enuresis. Recurrent urinary tract infections, chronic constipation and/or fecal incontinence and vesicoureteral reflux (VUR) contribute to this condition. Urotherapy is the mainstay of the treatment. It starts with education and demystification and simple behavioral modifications. Specific measures include PFM exercises with various forms of biofeedback concentrating at the recognition of PFM function and their relaxation. However, the PFMs are part of the abdominal capsule and they act in coordination with lower abdominal muscles. These muscles need to be relaxed during voiding. Diaphragmatic breathing exercises were introduced to teach children abdominal muscle relaxation. Easy to learn exercises do not require any specific equipment and can be performed at all health care levels. Children from five years of age could benefit from these exercises. In children resistant to standard treatment, botulinum toxin type A application, intermittent catheterization and surgery in children with VUR are recommended

Expression Pattern of Long Non-Coding RNA Growth Arrest-Specific 5 in the Remission Induction Therapy in Childhood Acute Lymphoblastic Leukemia

Background: Long non-coding RNA growth arrest-specific 5 (GAS5) is deregulated in many cancers because of its role in cell growth arrest and apoptosis. Additionally, GAS5 interacts with glucocorticoid receptor, making it a potential pharmacotranscription marker of glucocorticoid (GC) therapy. In this study, we aimed at analysing GAS5 expression in the remission induction therapy phase of childhood acute lymphoblastic leukemia (ALL), in which GCs are mandatorily used, and to correlate it with therapy response. Methods: GAS5 expression was measured in peripheral blood mononuclear cells taken from 29 childhood ALL patients at diagnosis, on day 15 and day 33 ofremission induction therapy using RT-qPCR methodology. Results: Our results have shown interindividual differences in GAS5 expression at all time points. For each ALL patient, GAS5 expression was higher on day 15 in comparison to its level at diagnosis (p<0.0005). On day 33, the level of GAS5 expression decreased in comparison with day 15 (p<0.0005), but it was still significantly higher than at diagnosis for the majority of patients (p=0.001). Patients whose number of blasts on day 8 was below 100 per mL of peripheral blood had a higherGAS5 expression at diagnosis (p=0.016), and lower ratio day 15/diagnosis (p=0.009). Conclusions: Our results suggest that the expression level of GAS5 could be a potential marker of therapy response in remission induction therapy of childhood ALL

Effect of remote ischaemic conditioning on clinical outcomes in patients with acute myocardial infarction (CONDI-2/ERIC-PPCI): a single-blind randomised controlled trial.

BACKGROUND: Remote ischaemic conditioning with transient ischaemia and reperfusion applied to the arm has been shown to reduce myocardial infarct size in patients with ST-elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI). We investigated whether remote ischaemic conditioning could reduce the incidence of cardiac death and hospitalisation for heart failure at 12 months. METHODS: We did an international investigator-initiated, prospective, single-blind, randomised controlled trial (CONDI-2/ERIC-PPCI) at 33 centres across the UK, Denmark, Spain, and Serbia. Patients (age >18 years) with suspected STEMI and who were eligible for PPCI were randomly allocated (1:1, stratified by centre with a permuted block method) to receive standard treatment (including a sham simulated remote ischaemic conditioning intervention at UK sites only) or remote ischaemic conditioning treatment (intermittent ischaemia and reperfusion applied to the arm through four cycles of 5-min inflation and 5-min deflation of an automated cuff device) before PPCI. Investigators responsible for data collection and outcome assessment were masked to treatment allocation. The primary combined endpoint was cardiac death or hospitalisation for heart failure at 12 months in the intention-to-treat population. This trial is registered with ClinicalTrials.gov (NCT02342522) and is completed. FINDINGS: Between Nov 6, 2013, and March 31, 2018, 5401 patients were randomly allocated to either the control group (n=2701) or the remote ischaemic conditioning group (n=2700). After exclusion of patients upon hospital arrival or loss to follow-up, 2569 patients in the control group and 2546 in the intervention group were included in the intention-to-treat analysis. At 12 months post-PPCI, the Kaplan-Meier-estimated frequencies of cardiac death or hospitalisation for heart failure (the primary endpoint) were 220 (8·6%) patients in the control group and 239 (9·4%) in the remote ischaemic conditioning group (hazard ratio 1·10 [95% CI 0·91-1·32], p=0·32 for intervention versus control). No important unexpected adverse events or side effects of remote ischaemic conditioning were observed. INTERPRETATION: Remote ischaemic conditioning does not improve clinical outcomes (cardiac death or hospitalisation for heart failure) at 12 months in patients with STEMI undergoing PPCI. FUNDING: British Heart Foundation, University College London Hospitals/University College London Biomedical Research Centre, Danish Innovation Foundation, Novo Nordisk Foundation, TrygFonden

Glycosylation and pH stability of penicillin G acylase from providencia rettgeri produced in Pichia pastoris

Penicilin G acilaza (PAC) je jedan od najšire korišćenih enzima u industrijskoj sintezi polusintetskih antibiotika. U ovom radu dobijeni nivo ekspresije PAC gena iz Providencia rettgeri u ekspresionom sistemu Pichia pastoris iznosio je 2.7 U/ml. Rekombinantni enzim je prečišćen i određen je njegov glikozilacioni status. Nađeno je da osim što su obe subjedinice enzima (α i β) N-glikozilovane, β subjedinica sadrži još i O-glikane. Takođe je ustanovljeno da je rekombinantna PACP. rett. stabilna u širokom pH opsegu što ju je, zajedno sa predhodno ustanovljenom visokom termostabilnošću, učinilo izuzetno privlačnim biokatalizatorom sa industrijske tačke gledišta.Penicillin G acylase (PAC) is one of the most widely used enzymes in industrial synthesis of semi-synthetic antibiotics. The Providencia rettgeri pac gene was expressed to a level of 2.7 U/ml using the Pichia pastoris expression system. The recombinant enzyme was purified and its glycosylation status was determined. It was found that both subunits (α and β) of the enzyme were N-glycosylated, while the β-subunit also contained O-glycans. It was also observed that rPACP.rett. was stable in a wide range of pH, which, in addition to the previously proved high thermostability, makes it an attractive biocatalyst from an industrial point of view