Study of the initial phase of biofilm formation using a biofomic approach

Aims

The purpose of this work was to study the initial steps of formation of a biofilm using the BioFilm Ring Test® and the Crystal violet staining technique.

Methods and results

Eight strains of Pseudomonas aeruginosa were studied. The two methods revealed that four strains formed a rapid biofilm. The biofilm formed by these strains was detected after only 45 min with the BioFilm Ring Test® and after 6 h with the Crystal violet method. The enumeration of bacteria of the PA01 strain confirmed that, after 30 min, a significant amount of bacteria had attached on the bottom of the culture wells. After 48 h the Crystal violet method detected a biofilm with all strains. The four strains which rapidly formed a biofilm did not differ from the slow-forming strains by their mucoid character or their swarming motility or their synthesis of rhamnose. They showed higher swimming mobility.

Conclusions

Our results show that the BioFilm Ring Test® is a method specially suited for the study of the initial phase of the formation of a biofilm.

Significance and impact of study

The BioFilm Ring Test® is an easy and rapid alternative to the Crystal violet staining and the enumeration methods.     

American ginseng (Panax quinquefolius) prevents glucose-induced oxidative stress and associated endothelial abnormalities

Purpose

Ginseng (Araliaceae), demonstrates widespread biological effects because of its purported antioxidant and other properties. The present study was undertaken to investigate the effects of American ginseng root extract on glucose-induced oxidative stress and associated oxidative damage to human umbilical vein endothelial cells (HUVECs).

Methods

Following pretreatment with various concentrations of ginseng (alcoholic extract), HUVECs were incubated with various concentrations of d-glucose ranging from 5 to 25 mmol/l for 24 h. l-Glucose was used at a concentration of 25 mmol/l as a control.

Results

Glucose-induced oxidative stress detected by intracellular reactive oxygen species accumulation, superoxide anion generation and DNA damage in HUVECs were significantly prevented by ginseng. Treatment of HUVECs with ginseng further led to significant prevention of glucose-induced NF-κB activation. Glucose-induced increase in fibronectin (FN), EDB⁺FN (a splice variant of FN), endothelin-1 (ET-1) and vascular endothelial growth factor (VEGF) mRNAs and protein levels were also prevented by ginseng treatment.

Conclusion

These data indicate that American ginseng prevented glucose-induced damage in the HUVECs through its antioxidant properties.     

Temperature influences upon vascular dynamics in cattle measured by Doppler ultrasonography

1.

Doppler-image ultrasonography was used to document vascular changes and blood flow rates of cattle (Bos taurus) under hot (32.7 °C) and cold (8.1 °C) conditions for 24 h.     

Fasciolagigantica: Parasitological and scanning electron microscopy study of the in vitro effects of ivermectin and/or artemether

Objective

To evaluate the in vitro effects of different concentrations of ivermectin and/or artemether on Fasciolagigantica worms and to study the parasitological changes and tegumental alterations using scanning electron microscopy (SEM).

Methods

Fasciola gigantica worms were incubated in vitro for 24 and 48 h with three concentrations of either ivermectin or artemether (10, 20 and 50 μg/ml) or both in half concentration of either (5, 10 and 25 μg/ml).

Results

Exposure of Fasciola worms to 25 + 25 μg/ml of combined drug regimens or to 50 μg/ml of either ivermectin or artemether for 48 h led to 100%, 41.7% and 75% worm killing which were accompanied by a significant reduction in egg laying capacity and significant increase in dead eggs maximally recorded in combined drug regimens. SEM of the flukes incubated for 48 h with combined drug regimens showed maximal tegumental disruption with swelling of the worm body, roughness, blebbing, sloughing and complete loss of spines. Disruption to the tegument of the flukes induced by artemether was more than that of ivermectin.

Conclusions

Artemether alone or combined with ivermectin in half doses had potent fasciocidal activities. Besides, half doses of combined drug regimens had higher ovicidal effects than each drug alone. In vivo studies are recommended to explore the efficacy of combined regimens against Fasciola infection.     

Common polymorphisms in the P-selectin gene in women with recurrent spontaneous abortions

Background

To investigate possible associations of P-selectin polymorphisms with idiopathic recurrent pregnancy loss (RPL).

Methods

Study subjects comprised 270 consecutive RPL cases attending outpatient maternity services, and 322 multi-parous control women. P-selectin genotyping was done by PCR-RFLP and PCR-ASA methods.

Results

The P-selectin variants rs1800807, rs1800805, and rs6127, were in Hardy Weinberg equilibrium, and low linkage disequilibrium was noted between the three studied SNPs. The frequency of rs6127 A allele (P < 0.001I), but not rs1800807 C allele (P = 0.957) or rs1800805 A allele (P = 0.760), was higher in RPL cases than in control women. Significant differences in the distribution of rs6127 (P < 0.001), but not rs1800807 (P = 0.444) or rs1800805 (P = 0.391) genotypes were seen between cases and controls, and only rs6127 showed a significant association with RPL, with increments of 2.65 and 4.96 in disease risk seen for heterozygous and homozygous carriers, respectively. Among the 8 three-locus Pselectin haplotypes constructed (rs1800807/rs1800805/rs6127), increased frequency of GGG (Pc = 0.0249), CGG (Pc = 0.0256), and CAG (Pc = 0.0174) haplotypes, and lower frequency of CGA haplotype (Pc = 0.0091) were seen in RPL cases, thus conferring disease susceptibility and protective nature to these haplotypes, respectively.

Conclusions

P-selectin gene polymorphisms and haplotypes contribute to RPL development.     

Association between the epidermal growth factor rs4444903 G/G genotype and advanced fibrosis at a young age in chronic hepatitis C

Background

The epidermal growth factor (EGF) rs4444903 A > G polymorphism has been associated with the development of liver cancer, which commonly complicates cirrhosis of viral origin; however, whether this polymorphism might be associated with fibrosis progression in chronic viral hepatitis is unknown. The present study was performed to assess the allelic and genotypic frequencies of the rs4444903 A > G polymorphism in patients with chronic hepatitis C virus HCV infection and to ascertain whether this polymorphism might be an independent predictor of the degree of fibrosis.

Methods

An RFLP-PCR technique was used to genotype 645 patients (211 with cirrhosis); 528 were referred for the diagnosis and treatment of chronic hepatitis C, and 117 were transplanted for HCV-related end stage liver disease. A group of 428 healthy subjects served as a control. All the subjects were of Caucasian ethnicity.

Results

The EGF rs4444903 A > G polymorphism genotype frequencies in HCV chronic infected patients were as follows: A/A = 227 (35.3%), A/G = 328 (50.9%), and G/G = 90 (14.8%). Genotype frequencies were found to differ between patients with an Ishak staging score ? 2 (A/A = 117, A/G = 157, G/G = 34) and patients with a score > 2 (A/A = 110, A/G = 171, G/G = 56, p = 0.038). A highly significant linear relationship between increasing stage scores and EGF genotype was detected in younger patients (A/A: 2.02 ± 0.18, A/G: 2.55 ± 0.17, G/G: 3.00 ± 0.32, p = 0.008). However, no significant association was detected between the stage score and EGF genotype in older patients (A/A: 3.79 ± 0.19, A/G: 3.64 ± 0.15, G/G: 3.98 ± 0.30 p = 0.579).

Conclusions

The EGF rs4444903 A > G polymorphism may facilitate liver fibrosis progression in Caucasian patients with chronic hepatitis C, especially in younger patients.     

Detection of multiple cytokines in the urine of patients with focal necrotising glomerulonephritis may predict short and long term outcome of renal function

Background

Detection of urinary cytokines in pauci-immune focal segmental necrotizing glomerulonephritis (FSNGN) may provide valuable information about disease pathogenesis and prognosis.

Methods

Epidermal growth factor (EGF), transforming growth factor (TGF-β1) and vascular endothelial growth factor (VEGF) were measured by ELISA, and Interleukins, monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein (MIP1β) by a multiplex cytokine assay, in 38 patients with FSNGN. Their levels were correlated with severity of histological findings and renal function outcome in short and long term.

Results

The percentage of crescents in renal biopsy had positive correlation with TGF-β1 (p = 0.004) and IL-15 urinary excretion (p = 0.01), and negative correlation with EGF (p = 0.01). Increased urinary excretion of IL-6, IL-15, VEGF and MIP-1β was associated with poor renal function outcome, but increased levels of EGF, IL-2 and IL-9 predicted a favourable prognosis. In multiple regression analysis IL-6 and VEGF urinary levels were independent predictors of no-response at the acute phase (p = 0.001 and p < 0.0001, respectively), while, IL-6 was the only factor (p = 0.03) predicted worse outcome at the end of follow-up (39.4 ± 45 months).

Conclusion

Increased urinary excretion of IL-6, IL-15, VEGF, TGF-β1, MCP-1 and MIP-1β and reduced EGF, IL-2, IL-9 may be associated with histological damage and influence response to treatment in pauci-immune FSNGN.     

Bio-guided isolation of potential antimicrobial and antioxidant agents from the stem bark of Trilepisium madagascariense

Aim

This study describes the activity-guided isolation of antimicrobial and antioxidant agents from Trilepisium madagascariense stem bark.

Methods

The methanol crude extract of T. madagascariense was partitioned sequentially into n-hexane, ethyl acetate, n-butanol and the residual aqueous fractions. The ethyl acetate fraction was subjected to column chromatography and the structures of isolated compounds were elucidated using GC-MS and/or NMR data by comparing with those reported in the literature. Antimicrobial activity was assayed by agar well diffusion and broth microdilution techniques on 8 bacteria and 10 yeasts. The antioxidant activity was determined by DPPH radical scavenging method.

Results

The bioassay-guided fractionation of the crude methanol extract of T. madagascariense afforded two known compounds [vanillic acid () and isoliquiritigenin ()] and two mixtures of fatty acids (n-hexane fraction and first column fraction of ethyl acetate fraction, F1). The fractionation of the crude methanol extract enhanced the antimicrobial activity. Compound 2 was generally more active than compound 1. For all the tested samples, the most sensitive microbes were Enterococcus faecalis ATCC 10541 (MIC range of 60-780 μg/ml) for bacteria and Candida guillermondi (MIC range of 0.01-190 μg/ml) for yeasts. The DPPH radical scavenging activity (RSa) of compound 2 (RSa₅₀ = 28.73 μg/ml) was comparable to that of the crude methanol extract (RSa₅₀ = 29.92 μg/ml).

Conclusion

The antimicrobial activities and the antioxidant properties of the methanol crude extract, fractions and compounds 1 and 2 from the stem bark of T. madagascariense are being reported for the first time. These results may justify the traditional use of this plant for the treatment of gastrointestinal disorders.     

Effects of exposure to short-term heat stress on male reproductive fitness in a soil arthropod

Ambient temperature is a key environmental factor influencing a variety of aspects of the ecology and evolution of ectotherms. Reproductive traits have been suggested to be more sensitive to thermal stress than other life history traits. This study investigated the direct and indirect effects of heat shock on male reproductive success in the widespread springtail Orchesella cincta. Male springtails were exposed to four temperature treatments: heat hardening (35.2 °C for 1 h), heat shock (37.2 °C for 1 h), heat hardening + heat shock (35.2 °C for 1 h, followed 15 h later by 37.2 °C for 1 h), and control (20 °C). The heat shock gene Hsp70 showed high expression in all the heat treatments, indicating that the treatments indeed induced thermal stress. Significant mortality was only found in the treatment with heat shock, both with and without heat hardening. A direct effect of heat treatment was found on time to first reproduction, which was significantly longer after heat shock (with or without heat hardening) than in the control treatment. There was no difference among treatments in the number of spermatophores produced in the first reproductive instar. Heat treatment also had indirect effects on male reproductive success. Females chose significantly more spermatophores from control males than from males that received heat shock, heat hardening or both. A high percentage of spermatophores produced by heat shocked males caused reproductive failure in females, but no significant differences among treatments were found.Our results suggest that not all traits were equally affected by the heat stress. Heat hardening did not protect reproductive traits against the negative effects of heat shock. The indirect effects of heat shock on reproduction may be equally important as the direct effects.     

Pharmacological mechanism responsible for the Atractylodes japonica-induced distal colonic contraction in rats

Background and aim

Atractylodes japonica Koidz (Compositae) has been commonly used to treat the gastrointestinal (GI) disorders in Korean traditional medicine, but its pharmacological roles in the regulation of GI motility have not been clarified yet.

Methods

Atractylodes japonica was sequentially partitioned with MeOH, n-hexane, CHCl₃, EtOAc and n-BuOH saturated with H₂O, and the effects of Atractylodes japonica extracts on the spontaneous contractility of GI muscle strips prepared from rats were measured.

Results

Among five different fractionations, EtOAc extracts of Atractylodes japonica (AJEA) dose-dependently increased the low frequency contraction of distal colon longitudinal muscles (DCLM), and the ED₅₀ values were revealed to be 1.71 × 10⁻⁹ g/ml. Among GI tracts, a prominent contractile response to AJEA was observed only in the DCLM. The contractile patterns produced by AJEA remarkably differed from those caused by acetylcholine and 5-HT. 4-DAMP and methoctramine at 0.5 μM significantly blocked the AJEA (1.0 μg/ml)-induced contraction of DCLM, but ondansetron, GR113808 and methysergide at 1.0 μM in combination did not change the AJEA-induced DCLM contractions. Acetylethylcholine mustard (5.0 μM) significantly diminished the AJEA-induced DCLM contractions, whereas p-chlorophenyl alanine (1.0 μM) did not affect the stimulatory effects of AJEA on the DCLM contractions.

Conclusion

The present results suggest that AJEA may specifically act on the DCLM among GI smooth muscles, and AJEA-induced DCLM contraction is likely mediated, at least, by activation of ChAT and acetylcholinergic muscarinic receptors.     

Serum steroid profiling by isotopic dilution-liquid chromatography-mass spectrometry: comparison with current immunoassays and reference intervals in healthy adults

Background

The simultaneous, rapid and reliable measurement of a wide steroid panel is a powerful tool to unravel physiological and pathological hormone status. Clinical laboratories are currently dominated by high-throughput immunoassays, but these methods lack specificity due to cross-reactivity and matrix interferences. We developed and validated an isotopic dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method for the simultaneous measurement of cortisol, corticosterone, 11deoxycortisol, androstenedione, deoxycorticosterone (DOC), testosterone, 17OHprogesterone, dehydroepiandrosterone (DHEA) and progesterone in serum, and compared it to routine immunoassays employed in our laboratory. We also established adult reference intervals in 416 healthy subjects.

Methods

0.9 ml of serum were spiked with labelled internal standards (IS) and extracted on C18 cartridges. Eluate was injected into a two-dimensional LC-system, purified in a perfusion column and separated on a C8 column during a 21 min gradient run. Analytes were revealed by atmospheric pressure chemical ionization (APCI) followed by multiple reaction monitoring (MRM) analysis.

Results

Of the four immunoassays compared with the ID-LC-MS/MS method, only the results of ElecsysE170 for cortisol, testosterone in males and progesterone > 1 ng/ml were in agreement with ID-LC-MS/MS. ElecsysE170 for testosterone in females and progesterone < 1 ng/ml, Immulite2000 for androstenedione, DSL-9000 for DHEA and 17OHP Bridge for 17OHprogesterone, respectively, showed poor agreement. Reference intervals and steroid age and fertility related fluctuations were established.

Conclusion

Our ID-LC-MS/MS method proved to be reliable and sensitive in revealing steroid circulating concentrations in adults and in highlighting the limits of routine immunoassays at low concentrations.     

Serotonin synthesis inhibition by pre-treatment of p-CPA alters sleep-electrophysiology in an animal model of acute and chronic heat stress

The effects of pre-treatment of para-chlorophenylalanine (p-CPA) on sleep–wake electroencephalograms (EEG) have been demonstrated in three age groups of rats subjected to heat stress. Each age group for both p-CPA pre-treated and untreated subjects was sub-divided into three groups: (i) acute heat stress—subjected to a single heat exposure for 4 h at 38 °C; (ii) chronic heat stress—exposed for 21 days daily for 1 h in the incubator at 38 °C; and (iii) handling control groups. Digital polygraphic sleep recordings were performed just after the heat exposure from acute stressed rats and on the 22nd day from chronic stressed rats. The analyses of results demonstrated that many changes associated with sleep-EEG (either in sleep–wake parameter or in EEG frequencies) due to acute and chronic heat stress were reversed (changes were analyzed; P<0.05 or better) in p-CPA pre-treated groups of rats. However, differential observations between acute and chronic heat stress groups of subjects were recorded, which are thought to have happened due to acclimatization of subjects to the hot environment. The results of present study supported the previous hypothesis about the significant involvement of serotonin in sleep–wake parameters and also demonstrated its participation in brain electrophysiological alterations in stressed conditions.     

Refining the distinction between heat tolerant and intolerant individuals during a Heat tolerance test

Background

The heat tolerance test (HTT) is a standardized physiological test that constitutes one of the considerations in the Israel Defence Forces (IDF) for return to duty after a heat injury. The HTT consists of a 2 h controlled exercise-heat stress with a threshold of maximal rectal temperature (Tc) and heart rate (HR) values above which subjects are referred to as heat intolerant; the dynamics of the HR and Tc during the test, which tend to plateau during the 2nd hour of the test, is also considered. Since “tendency to plateau” is a subjective measure, this study aimed to quantify the tendency to plateau during a HTT.

Material and methods

The physiological results of 102 HTT subjects (83 normal and 19 heat-intolerant) served as the database for analysis. The first 28 subjects, who were considered heat tolerant (HT) by an experienced examiner, served to evaluate a “normal” dynamic of Tc and HR during a HTT. Then, we applied the results on seven heat tolerant and seven heat intolerant (HI) subjects in order to determine which of the two variables (dTc or dHR) in different time intervals (t_120–0, t_60–0, t_120–60, and t_120–100) may best distinguish heat tolerant from heat intolerant subjects. During the 3rd stage of the study 60 random test results (post factum: 48 heat tolerant and 12 heat intolerant subjects) were evaluated. Post-hoc results of heat tolerance determined by dTc were compared to the expert diagnosis of the test.

Results

A rise of less than 0.45 °C in Tc during the 2nd hour of the HTT was found acceptable to define a tendency to plateau of the Tc with a 100% sensitivity and specificity. We did not find a numerical value of HR increase over time during the HTT acceptable to define a tendency of the HR to plateau.

Conclusion

During the last hour of the heat tolerance test, there is a tendency to plateau in Tc in heat tolerant individuals. It is concluded that a rise in Tc of less than 0.45 °C during this period (Tc_120–60) can be used as a supporting measure to distinguish between heat tolerant and heat intolerant individuals.     

Effect of the acute heat stress on the rat pituitary gland. Morphological and stereological study

1.

Morphological and stereological changes were examined in the pituitary gland of the rats exposed to heat stress (38 °C) for 60 min.     

Chemical composition and therapeutic effects of Lippia alba (Mill.) N. E. Brown leaves hydro-alcoholic extract in patients with migraine

Background

There is no universally accepted and effective prophylaxis of migraine headache episodes. Thus we aimed to investigate the effects of Lippia alba (Mill.) N. E. Brown, an herb with many effects on central nervous system, on pain frequency and intensity of migraine patients.

Methods

Patients were enrolled in a prospective, phase 2, non-controlled cohort study to orally receive hydro-alcoholic extract of L. alba leaves. Headache intensity and frequency of episodes were recorded before and after 30-60 days of treatment. We also studied the chemical composition of its essential oil by gas chromatography-mass spectrometry.

Results

We described for the first time a particular L. alba chemotype with geranial and carvenone as major compounds. With treatment, both frequency and intensity of pain episodes significantly decreased from baseline to first reassessment date. More than 80% of patients experienced a minimum 50% reduction on pain intensity and frequency. No side effects were reported.

Conclusions

Treatment with a geranial plus carvenone chemotype of L. alba hydro-alcoholic extract is a cheap, widely available, highly effective therapy to reduce both the intensity and the frequency of headache episodes of migraine patients with no side effects.     

Clinical significance of plasma CD146 and P-selectin in patients with type 2 diabetic nephropathy

Objective

To investigate the levels of plasma CD146 and P-selectin in patients with type 2 diabetic nephropathy at different stages.

Methods

A total of 80 patients with type 2 diabetes mellitus were enrolled in the present study. According to 24 h urinary albumin excretion ratio and renal function, they were further divided into group of diabetes without microalbuminuria (DN0, n = 20), microalbuminuria group (DN1, n = 20), macroalbuminuria group (DN2, n = 20) and renal insufficiency group (DN3, n = 20). Another 20 healthy subjects were enrolled as control group (non-DM). Plasma CD146 and P-selectin were measured by ELISA.

Results

Plasma CD146 and P-selectin were significantly increased in patients with type 2 diabetes with microalbuminuria (DN1) compared with health control (CD146: 415.3 ± 29.0 vs. 243.5 ± 14.7 ng/ml, P < 0.05; P-selectin: 66.8 ± 3.4 vs. 45.3 ± 2.7 ng/ml, P < 0.001). With the development of diabetic nephropathy, both plasma CD146 and P-selectin level progressively rise, with the highest levels in patients with significant renal insufficiency (DN3: 515.9 ± 36.9 and 81.5 ± 5.1 ng/ml respectively, P < 0.001). Moreover, the increase in CD146 is positively co-related to the rise of P-selectin in patients with type 2 diabetes.

Conclusion

Expression of CD146 and P-selectin in patients with type 2 diabetes is elevated, and they are positively correlated with severity of diabetic nephropathy.     

A re-investigation of saliva collection procedures that highlights the risk of potential positive interference in cortisol immunoassay

Background

The use of saliva for measurement of cortisol permits non-invasive study of adrenal function, but collection can be technically difficult, particularly in small infants. Saliva collection can be assisted by citric acid to increase saliva flow, or by the use of cotton or polyester swabs in the mouth.

Aim

To determine whether different methods of saliva collection affect cortisol radioimmunoassay (RIA) performance.

Experimental

Cortisol was measured in saliva collected from 16 adults using intra-oral cotton swabs or polyester swabs, compared with saliva dribbled directly into a pot either alone (plain saliva) or after citric acid had been placed on the tongue. An in-house RIA, without prior extraction, was used to measure cortisol with an encapsulated sheep antibody.

Results

Mean (median) salivary cortisol was 10.9 (10.5) nmol L⁻¹ in plain saliva, 10.4 (8.4) nmol L⁻¹ in citric acid stimulated saliva; 25.3 (25.1) nmol L⁻¹ in saliva collected on cotton swabs, and 27.9 (27.3) nmol L⁻¹ collected on polyester swabs. Cortisol in saliva collected using citric acid was not significantly different from plain saliva (p = 0.997), but cortisol in saliva collected using cotton and polyester swabs was significantly higher than that of plain saliva (p < 0.01).

Conclusion

The use of cotton or polyester swabs for collection of saliva can result in spuriously high levels of cortisol when measured by RIA.     

Testicular gene expression in male mice divergent for fertility after heat stress

Fertility losses in male mice occur approximately 18-28 d after heat stress. The objective of this study was to identify gene expression differences in males highly versus lowly fertile after heat stress. Mature male mice were exposed to heat stress (35 ± 1 °C; n = 50) or thermoneutral (21 ± 1 °C; n = 10) conditions for 24 h (Day 0) and hemicastrated (Day 1) to collect tissue for gene expression analyses. Males were subjected to a mating test from Days 18 to 26 when variation in fertility was anticipated. A fertility index was used to rank heat-stressed males and identify those males resistant and susceptible to heat stress, respectively. Microarray analyses were conducted on testis tissues from control (n = 5), heat stress resistant (n = 5), and heat stress susceptible (n = 5) males, and 225 genes were observed to be differentially expressed (P < 0.05), including genes involved in chaperone (Canx, Hspcb1, and Tcp1) and catalytic (Fkpb6, Psma7, and Idh1) activity. Expression patterns of these genes were confirmed using real-time RT-PCR. Male progeny from selected sires were similarly divergent in fertility after heat stress. Testicular expression levels of Canx, Hspcb, and Tcp1 genes were determined in these progeny. Hspcb expression was moderately heritable (0.31 ± 0.25); however, expression patterns of Canx and Tcp1 were not heritable.