Mechanisms behind protein-protein interactions in a ß-lg-legumin co-precipitate.

Interactions between pea protein and whey protein isolates in co-precipitates and blends consist of a combination of disulphide bonds, hydrophobic and electrostatic interactions. The present study aims to clarify if the two proteins with free thiols, ß-lactoglobulin (ß-lg) and legumin, played a significant role for these interactions. This study used different reagents to modify the conditions of interactions: N-ethylmaleimide (NEM) was used to block reactive thiols, while NaCl and SDS were used to prevent electrostatic or hydrophobic interactions, respectively. The effects of treatments were studied on protein solubility, structure and stability. SDS had no effect, while NEM and NaCl both had great effect, especially in combination. The results showed that interactions of ß-lg and legumin in both co-precipitates and blends are a synergism of electrostatic interactions and disulphide bonds. Thus, ß-lg and legumin are the main proteins responsible for previously observed interactions in protein isolates of whey and pea.

Global parental leave in surgical careers: differences according to gender, geographical regions and surgical career stages.

BACKGROUND: There is a lack of information regarding the provision of parental leave for surgical careers. This survey study aims to evaluate the experience of maternity/paternity leave and views on work-life balance globally. METHODS: A 55-item online survey in 24 languages was distributed via social media as per CHERRIES guideline from February to March 2020. It explored parental leave entitlements, attitude towards leave taking, financial impact, time spent with children and compatibility of parenthood with surgical career. RESULTS: Of the 1393 (male : female, 514 : 829) respondents from 65 countries, there were 479 medical students, 349 surgical trainees and 513 consultants. Consultants had less than the recommended duration of maternity leave (43.8 versus 29.1 per cent), no paid maternity (8.3 versus 3.2 per cent) or paternity leave (19.3 versus 11.0 per cent) compared with trainees. Females were less likely to have children than males (36.8 versus 45.6 per cent, P = 0.010) and were more often told surgery is incompatible with parenthood (80.2 versus 59.5 per cent, P < 0.001). Males spent less than 20 per cent of their salary on childcare and fewer than 30 hours/week with their children. More than half (59.2 per cent) of medical students did not believe a surgical career allowed work-life balance. CONCLUSION: Surgeons across the globe had inadequate parental leave. Significant gender disparity was seen in multiple aspects.

Validation of the colostomy impact score in patients ostomized for a benign condition.

AIM: The colostomy impact (CI) score is a patient-reported outcome measure assessing reduction in health-related quality of life (HRQL) due to a stoma. The score was originally developed and validated in a cohort of rectal cancer survivors with a permanent colostomy. For the CI score to be applied to patients with a colostomy after surgery for a benign condition it must be validated in this patient group. The aim of this study was to assess construct validity and known groups validity of the CI score in patients with a colostomy after surgery for a benign condition. METHOD: In a cross-sectional survey among ostomates in the Capital Region of Denmark, patients completed the CI score and the SF-36 v2 questionnaires. Construct validity was assessed by Pearson's correlation coefficients and known groups validity was assessed by t-test when dividing patients into groups of minor or major CI. RESULTS: The CI score showed a moderate negative correlation with the Physical Component Summary (PCS) of -0.41 and a weak negative correlation with the Mental Component Summary (MCS) of -0.39. The strength of the correlation depended on the underlying condition leading to stoma formation. Differences were significant between the minor and major CI groups in mean PSC and MCS with t-values of 5.32 and 3.86, respectively. CONCLUSION: The CI score is a valid instrument for assessing stoma-related impact on HRQL regardless of the underlying condition leading to stoma formation, and the CI score discriminates meaningfully between groups with known differences in stoma-related reduced HRQL.

Training and validation of a novel 4-miRNA ratio model (MiCaP) for prediction of postoperative outcome in prostate cancer patients.

Background: New molecular biomarkers for prostate cancer (PC) prognosis are urgently needed. Ratio-based models are attractive, as they require no additional normalization. Here, we train and independently validate a novel 4-miRNA prognostic ratio model for PC. Patients and methods: By genome-wide miRNA expression profiling of PC tissue samples from 123 men who underwent radical prostatectomy (RP) (PCA123, training cohort), we identified six top candidate prognostic miRNAs and systematically tested their ability to predict postoperative biochemical recurrence (BCR). The best miRNA-based prognostic ratio model (MiCaP) was validated in two independent cohorts (PCA352 and PCA476) including >800 RP patients in total. Clinical end points were BCR and prostate cancer-specific survival (CSS). The prognostic potential of MiCaP was assessed by univariate and multivariate Cox-regression analyses and Kaplan-Meier analyses. Results: We identified a 4-miRNA ratio model, MiCaP (miR-23a-3p×miR-10b-5p)/(miR-133a×miR-374b-5p), that predicted time to BCR independently of routine clinicopathologic variables in the training cohort (PCA123) and was successfully validated in two independent RP cohorts. In addition, MiCaP was a significant predictor of CSS in univariate analysis [HR 3.35 (95% CI 1.34 - 8.35), P = 0.0096] and in multivariate analysis [HR 2.43 (95% CI 1.45-4.07), P = 0.0210]. As proof-of-principle, we also analyzed MiCaP in plasma samples from 111 RP patients. A high MiCaP score in plasma was significantly associated with BCR (P = 0.0036, Kaplan-Meier analysis). Limitations include low mortality rates (CSS: 5.4%). Conclusions: We identified a novel 4-miRNA ratio model (MiCaP) with significant independent prognostic value in three RP cohorts, indicating promising potential to improve PC risk stratification.

Identification of antibacterial peptides from endophytic microbiome.

Endophytes, microorganisms living inside plant tissues, are promising producers of lead compounds for the pharmaceutical industry. However, the majority of endophytes are unculturable and therefore inaccessible for functional studies. To evaluate genetic resources of endophytes, we analyzed the biodiversity of fungal microbiome of black crowberry (Empetrum nigrum L.) by next-generation sequencing and found that it consists mainly of unknown taxa. We then separated the host and the endophyte genomes and constructed a fosmid expression library from the endophytic DNA. This library was screened for antibacterial activity against Staphylococcus aureus. A unique antibacterial clone was selected for further analysis, and a gene En-AP1 was identified with no similarity to known sequences. The expressed, folded protein En-AP1 was not active against S. aureus, while tryptic digests exhibited antimicrobial activity. Seven out of twelve synthesized peptides, predicted antibacterial in silico, exhibited in vitro activity towards both S. aureus and Escherichia coli. We propose that the En-AP1 protein is degraded in the library host E. coli and antimicrobial fragments are released from the cell, explaining the in vitro antibacterial activity of the clone. This is the first report of a novel gene expressed in vitro derived from an endophytic microbiome, demonstrating the potential of finding novel genes and compounds from unculturable endophytes.

Automated high precision secondary pH measurements.

A new setup for high precision, automated secondary pH measurements together with a reference measurement procedure has been developed and tested in interlaboratory comparisons using buffers pH 4.005, pH 7.000, and pH 10.012 at 25 °C and 37 °C. Using primary buffers as standards, a standard uncertainty in pH better than 0.005 can be reached. The central measuring device is a one piece, thermostatted cell of PFA (perfluoroalkoxy) with a built-in Hamilton(®) Single Pore™ Glass electrode. Due to its flow-through principle this device allows pH measurements with low consumption of measurement solutions. The very hydrophobic and smooth PFA as construction material facilitates complete emptying of the cell. Furthermore, the tempering unit affords very precise temperature control and hence contributes to the low target uncertainty of the produced secondary buffer solutions. Use of a symmetric measurement sequence and the two point calibration was sufficient to reach high precision and accuracy.

Prognostic significance of aberrantly silenced ANPEP expression in prostate cancer.

BACKGROUND: Novel biomarkers for prostate cancer (PC) are urgently needed. This study investigates the expression, epigenetic regulation, and prognostic potential of ANPEP in PC. METHODS: Aminopeptidase N (APN; encoded by ANPEP) expression was analysed by immunohistochemistry using tissue microarrays representing 267 radical prostatectomy (RP) and 111 conservatively treated (CT) PC patients. Clinical end points were recurrence-free survival (RFS) and cancer-specific survival (CSS), respectively. The ANPEP promoter methylation levels were determined by bisulphite sequencing or MethyLight analysis in 278 nonmalignant and PC tissue samples, and in cell lines. RESULTS: The APN expression was significantly downregulated in PC compared with nonmalignant prostate tissue samples. Aberrant promoter hypermethylation was frequently observed in PC tissue samples, and 5-aza-2'-deoxycytidine induced ANPEP expression in three hypermethylated prostate cell lines, suggesting epigenetic silencing. Negative APN immunoreactivity was significantly associated with short RFS and short CSS in the RP and CT cohort, respectively, independently of routine clinicopathological predictors. Combining APN with a known angiogenesis marker (vascular endothelial growth factor or microvessel density) improved risk prediction significantly in both cohorts. CONCLUSION: Our results suggest negative APN immunoreactivity as a new independent adverse prognostic factor for patients with clinically localised PC and, furthermore, that epigenetic mechanisms are involved in silencing of ANPEP in PC.

Light intensity and social communication between hens.

1. The effect of light intensity on social communication in laying hens was investigated experimentally by comparing the transmission and detection of social signals between familiar and unfamiliar hens of similar or unequal rank in a competition for food. 2. The experimental method consisted of mutual inspection by a pair of hens at short range (approximately 24 cm), followed by a competition at a feed trough from which only one hen could eat a favoured food. The relative rank of a hen was inferred from success in this competition. 3. The relative rank of individual hens within 5 groups, each of 6 adult laying hens, was determined to identify those of high and low rank within their home group. 4 Social communication between pairs of either unfamiliar or familiar hens of either similar or unequal rank (highest and lowest ranking within their home groups) was then assessed under light intensities of 1, 5, 20 and 100 lux with all other cues present. Only the dimmest light of 1 lux perturbed some aspects of the competition for food. 5. The findings provide scientific justification, in part, for the current legal requirement in England for 'all hens to see other hens' by specifying a minimum light intensity of at least 5 lux for hens kept in close proximity to each other.

In vivo pharmacodynamic characterization of a novel plectasin antibiotic, NZ2114, in a murine infection model.

NZ2114 is a novel plectasin derivative with potent activity against gram-positive bacteria, including multiply drug-resistant strains. We used the neutropenic murine thigh infection model to characterize the time course of antimicrobial activity of NZ2114 and determine which pharmacokinetic/pharmacodynamic (PK/PD) index and magnitude best correlated with efficacy. Serum drug levels following administration of three fourfold-escalating single-dose levels of NZ2114 were measured by microbiologic assay. Single-dose time-kill studies following doses of 10, 40, and 160 mg/kg of body weight demonstrated concentration-dependent killing over the dose range (0.5 to 3.7 log(10) CFU/thigh) and prolonged postantibiotic effects (3 to 15 h) against both Staphylococcus aureus and Streptococcus pneumoniae. Mice had 10(6.3) to 10(6.8) CFU/thigh of strains of S. pneumoniae or S. aureus at the start of therapy when treated for 24 h with 0.625 to 160 mg/kg/day of NZ2114 fractionated for 4-, 6-, 12-, and 24-h dosing regimens. Nonlinear regression analysis was used to determine which PK/PD index best correlated with microbiologic efficacy. Efficacies of NZ2114 were similar among the dosing intervals (P = 0.99 to 1.0), and regression with the 24-h area under the concentration-time curve (AUC)/MIC index was strong (R(2), 0.90) for both S. aureus and S. pneumoniae. The maximum concentration of drug in serum/MIC index regression was also strong for S. pneumoniae (R(2), 0.96). Studies to identify the PD target for NZ2114 utilized eight S. pneumoniae and six S. aureus isolates and an every-6-h regimen of drug (0.156 to 160 mg/kg/day). Treatment against S. pneumoniae required approximately twofold-less drug for efficacy in relationship to the MIC than did treatment against S. aureus. The free drug 24-h AUCs/MICs necessary to produce a stasis effect were 12.3 +/- 6.7 and 28.5 +/- 11.1 for S. pneumoniae and S. aureus, respectively. The 24-h AUC/MIC associated with a 1-log killing endpoint was only 1.6-fold greater than that needed for stasis. Resistance to other antimicrobial classes did not impact the magnitude of the PD target required for efficacy. The PD target in this model should be considered in the design of clinical trials with this novel antibiotic.

Antimicrobial activity of the recombinant designer host defence peptide P-novispirin G10 in infected full-thickness wounds of porcine skin.

OBJECTIVES: The growing number of patients with impaired wound healing and the development of multidrug-resistant bacteria demand the investigation of alternatives in wound care. The antimicrobial activity of naturally occurring host defence peptides and their derivatives could be one alternative to the existing therapy options for topical treatment of wound infection. Therefore, the aim of this study was to investigate the antimicrobial activity of proline-novispirin G10 (P-novispirin G10) in vitro and in the infected porcine titanium wound chamber model. METHODS: The new derived designer host defence peptide P-novispirin G10 was tested in vitro against Gram-positive and Gram-negative bacterial strains. Additionally, cytotoxicity and haemolytic activities of P-novispirin G10 and protegrin-1 were measured. For in vivo studies, six wound chambers were implanted on each flank of Göttinger minipigs (n = 2, female, 6 months old, 15-20 kg). Eleven wound chambers were inoculated 8 days post-operatively with 5 x 10(8) of Staphylococcus aureus; one wound chamber remained uninfected as a system control. After wound infection had been established (4 days after inoculation), each wound chamber was topically treated with P-novispirin G10, protegrin-1 or carrier control. Wound fluid was harvested every hour for a total follow up of 3 h. RESULTS: P-novispirin G10 demonstrated broad-spectrum antimicrobial activity with moderate haemolytic and cytotoxic activities compared with protegrin-1. In the infected wound chamber model P-novispirin G10 demonstrated a 4 log(10) reduction in bacterial counts. CONCLUSIONS: This implicates the potential of P-novispirin G10 as an alternative in future antimicrobial wound care. However, more studies are necessary to further define clinical applications and potential side effects in greater detail.

Leg health and performance of broiler chickens reared in different light environments.

1. The effects of light source and intensity on leg health and performance of female ROSS 308 broiler chickens were investigated in a 2 x 2 experimental design (8 groups of 275 chicks) of two light sources (Osram biolux and Osram warm-white) and two light intensities (5 and 100 clux, adjusted to fowl-perceived illuminance). 2. At 41 d of age, body weight, gait-score, footpad dermatitis and hock-burn were measured on 50 birds from each light environment. In addition, weekly feed intake and body weight were determined on a group basis and mortality was recorded continuously. 3. The light environment did not affect the severity of the gait-score or hock-burns. The risk of moderate to severe lameness and hock-burns increased with body weight. Birds weighing more than 2400 g had an increased probability of moderate footpad lesions in biolux light. 4. Weight and gait-score, as well as gait-score and hock-burn were positively correlated. Podo-dermatitis was weakly correlated with hock-burn, which contradicts earlier findings. The light environment did not affect feed intake, body weight or mortality. 5. The light sources and intensities employed in this study did not adversely affect production or leg health of broiler chickens reared semi-commercially.

Improving on nature's defenses: optimization & high throughput screening of antimicrobial peptides.

Antimicrobial peptides (AMPs) are ubiquitous in nature where they play important roles in host defense and microbial control. Despite their natural origin, antimicrobial spectrum and potency, the lead peptide candidates that so far have entered pharmaceutical development have all been further optimized by rational or semi-rational approaches. In recent years, several high throughput screening (HTS) systems have been developed to specifically address optimization of AMPs. These include a range of computational in silico systems and cell-based in vivo systems. The in silico-based screening systems comprise several computational methods such as Quantitative Structure/Activity Relationships (QSAR) as well as simulation methods mimicking peptide/membrane interactions. The in vivo-based systems can be divided in cis-acting and trans-acting screening systems. The cis-acting pre-screens, where the AMP exerts its antimicrobial effect on the producing cell, allow screening of millions or even billions of lead candidates for their basic antimicrobial or membrane-perturbating activity. The trans-acting screens, where the AMP is secreted or actively liberated from the producing cell and interacts with cells different from the producing cell, allow for screening under more complex and application-relevant conditions. This review describes the application of HTS systems employed for AMPs and lists advantages as well as limitations of these systems.

Force-independent expression of c-fos mRNA by endothelin-1 in rat intact small mesenteric arteries.

AIM: Wall stress-independent signalling pathways were studied for endothelin-1 (ET-1)-induced c-fos expression in rat intact mesenteric small arteries. METHODS: Arteries were kept unmounted in Krebs buffer, equilibrated for 1 h and stimulated with vasoactive substances for 15-60 min. The c-fos mRNA expression was determined by real-time polymerase chain reaction. RESULTS: Stimulation with fetal bovine serum (FBS), phorbol 12-myristate 13-acetate (PMA) and ET-1 caused about a doubling of c-fos mRNA. The ET-1-induced c-fos expression was steady (15-60 min) and was inhibited by the inhibitor of the ET(A) receptor, BQ-123. Platelet-derived growth factor-B, angiotensin II and U46619 did not cause increased c-fos mRNA levels. The broad specificity inhibitor staurosporine inhibited the response to ET-1, but inhibitors of Rho-A kinase and phosphatidylinositol 3-kinase had no effect. However, inhibitors to tyrosine kinases, the MAP kinases [extracellular signal-regulated kinase 1/2 (ERK1/2), c-Jun amino-terminal kinase, p38], and to conventional protein kinase C showed no inhibition. Consistent with these findings, ET-1 did not cause activation of ERK1/2, a finding also seen in vessels held under pressure. In contrast, ET-1-induced c-fos expression was inhibited by the calcium chelator BAPTA, suggesting a role for intracellular calcium. This possibility was supported by the finding that raising the extracellular K(+) concentration caused increased expression of c-fos in a concentration-dependent manner. CONCLUSION: The results suggest that in the absence of wall stress, ET-1 is able to induce increased expression of c-fos independent of traditional growth pathways, such as MAP kinase. The mechanism appears to be calcium-dependent.

Preparation and purification of cationic solid lipid nanospheres--effects on particle size, physical stability and cell toxicity.

Cationic solid lipid nanospheres (SLN) were prepared by the microemulsion technique with polysorbate 80 (Tween 80) and butanol as surfactants. The SLN (diameter 100-500 nm, zetapotential around +15 mV) consisted mainly of stearylamine (SA) and different triglycerides. Three different purification methods, ultrafiltration, ultracentrifugation and dialysis, were investigated and compared with the cellular toxicity and physical stability of the dispersions. The cell toxicity was dependent on both the SLN composition and the purification method. Dialysis was found to easily and efficiently remove excessive surfactant determined by dynamic light scattering (DLS), leading to reduced cell toxicity and increased physical stability of the SLN on storage. The cationic SLN might constitute a promising DNA delivery system.

Non-specific markers of neurodevelopmental disorder/delay in selective mutism--a case-control study.

Selective mutism (SM) in children is frequently associated with language disorder/delay suggesting that neurobiological factors may be involved in the development of SM. Motor co-ordination problems, reduced optimality pre- and perinatally and minor physical anomalies represent other markers for neurodevelopmental disorder/delay. The present study explores these markers in referred children with SM (n=54), non-referred matched controls (n =108) and in SM subgroups with and without a communication disorder (CoD). Children with SM differed significantly from controls in parent-reported motor developmental delay, and they obtained a higher pre- and perinatal "reduced optimality score". They scored significantly lower on a motor performance test and showed a higher frequency of minor physical anomalies compared with controls. There were no differences in these respects between SM and CoD compared with SM without CoD. The results confirm that neurobiological factors may be involved in the development of SM. Accordingly, the clinical assessment of referred children with SM should include a thorough history of motor development and an evaluation of present motor skills. The interplay of shyness/social anxiety and motor function problems has to be addressed to reduce misunderstandings of the child's behaviour and to adjust demands for motor skills to the actual level of the child.

Physical stability of redispersible dry emulsions containing amorphous sucrose.

The objective of the present study was to estimate the stability of redispersible dry emulsions containing amorphous sucrose. Dry emulsions were prepared by spray drying liquid o/w-emulsions in a laboratory spray dryer. The effect of hydroxypropyl methylcellulose (HPMC) on the glass transition temperature T(g) of spray dried sucrose-HPMC mixtures, relative to the T(g) of amorphous sucrose, was investigated. For the sucrose-HPMC mixtures the values of T(g) followed the ideal Gordon-Taylor equation up to 30% HPMC. For dry emulsions containing 40% HPMC, 30% lipid and 30% sucrose, the T(g) was increased by 12 degrees C relative to the T(g) of amorphous sucrose. The stability of the dry emulsions was investigated by a conventional stability study and by an enthalpy relaxation study. The measured enthalpy recovery of amorphous sucrose below T(g) was used to calculate molecular relaxation time parameters based on the Williams-Watts equation. The molecular mobility of amorphous sucrose at temperatures 50 degrees C below T(g) was low and negligible with respect to the shelf life stability. It was concluded that the dry emulsions are physically stable with respect to the lifetime of a pharmaceutical product when stored in dry condition and at temperatures up to 28 degrees C.

MCMI-II personality traits and symptom traits in parents of children with selective mutism: a case-control study.

The etiology of selective mutism (SM) in children is unknown but is regarded as multifactorial. SM is frequently associated with social anxiety and language disorder or delay. The present study addresses SM and social anxiety as a familial phenomenon by comparing self-reported personality traits and symptom traits (Millon Clinical Multiaxial Inventory) in parents of 50 SM children with control parents. Personality and symptom traits reflecting social anxiety are also explored separately in the parents of SM children with and without a comorbid communication disorder. The results confirm SM and social anxiety as a familial phenomenon. Assessment and treatment planning should take this into account. The family data also suggest different family transmissions of SM and social anxiety in the SM group with and without a comorbid communication disorder.

Comparison of the lymphatic transport of a lipophilic drug from vehicles containing alpha-tocopherol and/or triglycerides in rats.

The applicability of alpha-tocopherol as a lymphotropic carrier for a highly lipophilic drug has been evaluated. Transport to the intestinal lymph of the highly lipophilic model drug, Lu28-179, in rats after administration to the stomach in an alpha-tocopherol emulsion was compared with lymphatic transport after administration of a sesame oil emulsion and an alpha-tocopherol/sesame oil emulsion. Lymphatic transport of the triglycerides and of alpha-tocopherol was determined. A conscious rat model was used, and the mesenteric lymph was collected. There was no significant difference between the cumulative masses of triglyceride from the two emulsions containing triglyceride 24 h after administration. Administration of an alpha-tocopherol emulsion seemed to induce mobilization of endogenous triglyceride. The lymphatic transport of alpha-tocopherol was less than 1 mg 24 h after administration of both emulsions containing alpha-tocopherol. The absorption of Lu28-179 from the alpha-tocopherol emulsion was very low, with a lymphatic recovery of 0.05%. When administered in an alpha-tocopherol/sesame oil emulsion, the recovery of Lu28-179 increased sevenfold to 0.35%. However, after administration of Lu28-179 in a sesame oil emulsion, the lymphatic recovery increased a further 13-fold to 4.5%. In conclusion, the study showed that alpha-tocopherol did not promote lymphatic absorption of Lu28-179 and thus was not a good lymphotropic carrier, as compared with sesame oil. Alpha-tocopherol in combination with sesame oil was not a good lymphotropic carrier either. The non-absorbed alpha-tocopherol fraction in the intestine might be able to prevent the absorption of Lu28-179.

Comparison of total oral bioavailability and the lymphatic transport of halofantrine from three different unsaturated triglycerides in lymph-cannulated conscious rats.

The lymphatic transport and the portal absorption of the lipophilic drug halofantrine were investigated in a conscious rat model. The rats were dosed with 0.1 g with triolein, trilinolein or trilinolenin containing 2 mg halofantrine. Following oral administration of the triglycerides, the mesenteric lymph and plasma samples were collected. The lymphatic transport for halofantrine was 11.1+/-1.2 after administration of trilinolein, 9.0+/-3.5 for trilinolenin and 8.6+/-2.2 for triolein and the total amount of halofantrine transported in the lymph was linear proportional with the amount of triglyceride in the lymph. The absorption of halofantrine directly into the blood showed a trend towards a higher AUC for trilinolien and trilinolenin compared to triolein, but no statistical difference could be found. The statistically analysis of the mean total bioavailability therefore shows that the absorption of halofantrine was largely independent on triglyceride unsaturation.