Background

L. decemlineata is an exotic invasive insect pest, and invaded in Xinjiang Uygur autonomous region in China in the 1990s from Kazakhstan. It is a notorious defoliator of potato throughout most of the northern Xinjiang in current, and often causes extremely large yield losses of potato.

Results

The expression stability of nine L. decemlineata house-keeping genes (Actin, ACT1 and ACT2; ADP-ribosylation factor, ARF1 and ARF4; TATA box binding protein, TBP1 and TBP2; ribosomal protein RP4 and RP18; translation elongation factor 1α EF1α) was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) in seven developmental stages, three larval tissues and two insecticide treatments. The results were analyzed using three software programs: geNorm, NormFinder and BestKeeper. Although there was no consistent ranking observed among the house-keeping genes across the samples, the overall analysis revealed that RP18, RP4, ARF1, and ARF4 were the four most stable house-keeping genes. In contrast, ACT1 and ACT2, two of the most widely used reference genes, had the least stability. Our results suggest that the combined use of the four most stably expressed genes may produce optimal normalization for qRT-PCR.

Conclusions

The expression stability of the house-keeping genes varies among different developing stages, in different tissues and under different experimental conditions. Our results will enable a more accurate and reliable normalization of qRT-PCR data in L. decemlineata.

Background

Nonalcoholic fatty liver disease (NAFLD) related to obesity has been rising in the last decades, though the morphological and metabolic determinants are remain unclear in children. The aim of this study was to analyze the morphological determinants and metabolic abnormalities in obese children and adolescents, classified either as with (P-NAFLD) or without (N-NAFLD). The sample comprised 190 individuals, aged 6 to 16 years-old, assigned into one of 4 groups according to sex and presence or absence of NAFLD. Obesity was obtained according to body mass index (BMI) cut-points. Body composition variables was estimated by Dual-Energy X-ray Absorptiometry (DEXA). Total cholesterol (TC), triglycerides (TG), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), insulin, fasting glucose (FG) and blood pressure were also analyzed. The diagnosis of NAFLD, as well as the measurement of intra-abdominal fat tissue thickness (IAF) and subcutaneous abdominal fat tissue thickness (SCF), was carried-out by ultrasound.

Results

Males and females belonging to P-NAFLD group showed, respectively, higher TFM and IAF. When data were adjusted for sex, age and total fat mass, those in P-NAFLD showed statistically higher IAF, TFM and TG.

Conclusion

Our study showed that obese youngsters who were assigned to P-NAFLD group were twice as likely to present higher concentration of triglycerides, higher levels of trunk fat, as well as intra-abdominal fat compared to their N-NAFLD counterparts even after adjustments for sex, age, pubertal stage and total body fat mass.

Background

Although high-molecular-weight (HMW) adiponectin is believed to protect against atherosclerosis, the association between HMW adiponectin and the composition of coronary plaques is unknown. We evaluated whether the HMW to total adiponectin ratio was associated with the presence of coronary plaque and its composition using multi-slice computed tomography coronary angiography (MSCTCA).

Methods

Serum total and HMW adiponectin levels were measured in 53 consecutive patients (age, 71) with >50% coronary artery stenosis detected by MSCTCA. A low-attenuation coronary plaque was defined as a plaque with a mean CT density <50 Hounsfield units. Multivariate logistic regression analyses were performed to evaluate the predictors of the presence of low-attenuation coronary plaques, which is thought to be high risk, on CT.

Results

Decreased serum levels of total as well as HMW adiponectin were significantly associated with the presence of at least one calcified or non-calcified coronary artery plaque (total adiponectin level: odds ratio 0.76, 95% CI 0.58–0.99, P = 0.048; HMW adiponectin level: odds ratio 0.65, 95% CI 0.42–0.99, P = 0.047). A low ratio of HMW to total adiponectin was significantly associated with the presence of low-attenuation coronary plaques (4.55, 1.94–21.90, P = 0.049). However, neither the total adiponectin nor the HMW adiponectin level was associated with the presence of low-attenuation coronary plaques.

Conclusion

Lower total or HMW adiponectin levels are associated with the presence of calcified and non-calcified coronary plaques, whereas a lower ratio of HMW to total adiponectin associated with the presence of low-attenuation coronary plaques (thought to be high risk). Measurement of total and HMW adiponectin levels and the HMW to total adiponectin ratio may be useful for risk stratification of coronary artery plaques.

Background

Occupational burnout is associated with severe negative health effects. While stress management programs proved to have a positive influence on the well-being of patients suffering from burnout, it remains unclear whether aerobic exercise alleviates burnout severity and other parameters related to occupational burnout. Therefore, the main purpose of this study was to pilot-test the potential outcomes of a 12-week exercise training to generate hypotheses for future larger scale studies.

Methods

The sample consisted of 12 male participants scoring high on the MBI emotional exhaustion and depersonalization subscales. The training program took place in a private fitness center with a 17.5 kcal/kg minimum requirement of weekly energy expenditure.

Results

The key findings are that increased exercise reduced overall perceived stress as well as symptoms of burnout and depression. The magnitude of the effects was large, revealing changes of substantial practical relevance. Additionally, profiles of mood states improved considerably after single exercise sessions with a marked shift towards an iceberg profile.

Conclusion

Among burnout patients, the findings provide preliminary evidence that exercise has the potential to reduce stress and prevent the development of a deeper depression. This has important health implications given that burnout is considered an antecedent of depressive disorders.

Trial registration

ClinicalTrials.gov Identifier: ISRNCT01575743

Background

Reverse transcription quantitative PCR has become a powerful technique to monitor mRNA transcription in response to different environmental conditions in many bacterial species. However, correct evaluation of data requires accurate and reliable use of reference genes whose transcription does not change during the course of the experiment. In the present study exposure to different growth conditions was used to validate the transcription stability of eight reference gene candidates in three strains from two subspecies of Francisella noatunensis, a pathogen causing disease in both warm and cold water fish species.

Results

Relative transcription levels for genes encoding DNA gyrase (gyrA), RNA polymerase beta subunit (rpoB), DNA polymerase I (polA), cell division protein (ftsZ), outer membrane protein (fopA), riboflavin biosynthesis protein (ribC), 16S ribosomal RNA (16S rRNA) and DNA helicases (uvrD) were quantified under exponential, stationary and iron-restricted growth conditions. The suitability of selected reference genes for reliable interpretation of gene expression data was tested using the virulence-associated intracellular growth locus subunit C (iglC) gene.

Conclusion

Although the transcription stability of the reference genes was slightly different in the three strains studied, fopA, ftsZ and polA proved to be the most stable and suitable for normalization of gene transcription in Francisella noatunensis ssp.

Background

This study aimed at enhancing the transcriptomic resources for two sibling species of moths, Ostrinia scapulalis (Adzuki bean borer) and Ostrinia nubilalis (European corn borer), as a foundation for future researches on their divergence history. Previous works on these species had shown that their genetic divergence was low, while they were reproductively isolated in natura and specialized on different host plants. Comparative genomic resources will help facilitate the understanding of the mechanisms involved in this isolation and adaptation to the host plants. Despite their fundamental interest, these species still lack the genomic resources to thoroughly identify candidate genes for functions of interest. We present here a high throughput sequencing and de novo transcriptome assembly for these two sibling species in line with this objective of comparative genomics.

Results

Based on 322,504 and 307,622 reads of 454 sequencing for O. scapulalis and O. nubilalis respectively, we reconstructed 11,231 and 10,773 transcripts, of which 40% were functionally annotated by BLAST analyzes. We determined the level of completeness of both assemblies as well as the recovery level of published Ostrinia genomic resources. Gene ontology (GO) of common and species-specific de novo transcripts did not reveal GO terms significantly enriched in one or the other species. By applying stringent homology searches on transcripts common to O. scapulalis and O. nubilalis, we identified a set of homologous transcripts, with a mean nucleotide identity value of 98.1%. In this set, the most divergent transcripts revealed candidate genes involved in developmental, sensorial and pathogen defense processes.

Conclusions

This data greatly increases the genomic resources of Ostrinia species and constitute a solid skeleton for future comparative analyzes of expression or diversity, despite we show that the transcriptomes for both species have not been assembled at full completion. In addition, we provide a set of homologous transcripts together with their annotation as a source of candidate genes for comparative analyzes.

Background

Cognitive behavior therapy (CBT) is regarded as an effective treatment for social anxiety disorder (SAD) in Europe and North America. Individual CBT might be acceptable and effective for patients with SAD even in non-Western cultures; therefore, we conducted a feasibility study of individual CBT for SAD in Japanese clinical settings. We also examined the baseline predictors of outcomes associated with receiving CBT.

Methods

This single-arm trial employed a 14-week individual CBT intervention. The primary outcome was the self-rated Liebowitz Social Anxiety Scale, with secondary measurements of other social anxiety and depressive severity. Assessments were conducted at baseline, after a waiting period before CBT, during CBT, and after CBT.

Results

Of the 19 subjects screened, 15 were eligible for the study and completed the outcome measures at all assessment points. Receiving CBT led to significant improvements in primary and secondary SAD severity (ps < .001). The mean total score on the Liebowitz Social Anxiety Scale improved from 91.8 to 51.7 (before CBT to after CBT), and the within-group effect size at the end-point assessment was large (Cohen’s d = 1.71). After CBT, 73% of participants were judged to be treatment responders, and 40% met the criteria for remission. We found no significant baseline predictors of those outcomes.

Conclusion

Despite several limitations, our treatment—which comprises a 14-week, individual CBT program—seems feasible and may achieve favorable treatment outcomes for SAD in Japanese clinical settings. Further controlled trials are required in order to address the limitations of this study.

Trial registration

UMIN-CTR UMIN000005897

Background

Hybridization based assays and capture systems depend on the specificity of hybridization between a probe and its intended target. A common guideline in the construction of DNA microarrays, for instance, is that avoiding complementary stretches of more than 15 nucleic acids in a 50 or 60-mer probe will eliminate sequence specific cross-hybridization reactions. Here we present a study of the behavior of partially matched oligonucleotide pairs with complementary stretches starting well below this threshold complementarity length – in silico, in solution, and at the microarray surface. The modeled behavior of pairs of oligonucleotide probes and their targets suggests that even a complementary stretch of sequence 12 nt in length would give rise to specific cross-hybridization. We designed a set of binding partners to a 50-mer oligonucleotide containing complementary stretches from 6 nt to 21 nt in length.

Results

Solution melting experiments demonstrate that stable partial duplexes can form when only 12 bp of complementary sequence are present; surface hybridization experiments confirm that a signal close in magnitude to full-strength signal can be obtained from hybridization of a 12 bp duplex within a 50mer oligonucleotide.

Conclusions

Microarray and other molecular capture strategies that rely on a 15 nt lower complementarity bound for eliminating specific cross-hybridization may not be sufficiently conservative.

Background

Fungal RNA samples are usually isolated from fungal mycelia grown in liquid culture, which relies on prolific growth of the fungus in liquid media. The fungal biomass is then collected by vacuum filtration, which can result in low recovery for samples with reduced biomass due to poor growth in liquid media.

Findings

Here we report an alternative culturing method, based on growth on solid media which is independent of the ability of a fungus to grow in liquid culture. We show that growth on solid media overlayed with a nylon membrane is superior to other culturing methods, producing large amounts of biomass and allowing for easy harvesting of fungal mycelia. Furthermore, we show that mycelium harvested with this method yielded high-quality RNA, superior to RNA isolated from liquid grown mycelium. We also show that inclusion of a second chloroform extraction step in the procedure significantly increases RNA yield.

Conclusions

This method is particularly useful for fungal species that show poor or no growth in liquid media, but are easily cultured on solid media. Culturing can be performed on small petri dishes, which significantly reduces handling and therefore allowing growth and isolation of RNA from multiple strains in a high throughput manner. The obtained RNA samples are of high quality in sufficient quantities for several northern blot experiments or quantitative RT-PCR experiments.

Background

The aim of this study was to develop liver tissue preparation suitable for investigating toxins. Hepatocyte respiration, ATP content, urea synthesis, caspase activity and morphology were measured as a function of in vitro incubation time. Mice were anesthetized by sevoflurane inhalation. Small liver fragments were then rapidly excised and incubated at 37°C in Krebs-Henseleit buffer (continuously gassed with 95% O2: 5% CO2) for up to 6 h. Phosphorescence O2 analyzer was used to determine the rate of cellular mitochondrial O2 consumption (kc, μM O2 min-1 mg-1). Cellular ATP was measured using the luciferin/luciferase system. The caspase-3 substrate N-acetyl-asp-glu-val-asp-7-amino-4-methylcoumarin (Ac-DEVD-AMC) was used to monitor intracellular caspase activity; cleaved AMC moieties (reflecting caspase activity) were separated on HPLC and detected by fluorescence.

Findings

Respiration was inhibited by cyanide, confirming the oxidation occurred in the respiratory chain. The values of kc (mean ± SD) for 0≤ t ≤6 h were 0.15 ± 0.02 μM O2 min-1 mg-1 (n = 18, coefficient of variation, CV = 13%), ATP content 131 ± 69 pmol mg-1 (1≤ t ≤6 h, n = 16, CV = 53%), synthesized urea 0.134 ± 0.017 mg/dL mg-1 in 50 min (0≤ t ≤6 h, n = 14, CV = 13%), and AMC peak area 62,540 ± 26,227 arbitrary units mg-1 (1≤ t ≤6 h, n = 3, CV = 42%). Hepatocyte morphology and organelles were reasonably persevered.

Conclusions

The described liver tissue preparation demonstrates stable hepatocyte structure, ultrastructure and biomarkers for up to 6 h, permitting in vitro studies.

Background

Frameshift is one of the three classes of recoding. Frame-shifts lead to waste of energy, resources and activity of the biosynthetic machinery. In addition, some peptides synthesized after frame-shifts are probably cytotoxic which serve as plausible cause for innumerable number of diseases and disorders such as muscular dystrophies, lysosomal storage disorders, and cancer. Hidden stop codons occur naturally in coding sequences among all organisms. These codons are associated with the early termination of translation for incorrect reading frame selection and help to reduce the metabolic cost related to the frameshift events. Researchers have identified several consequences of hidden stop codons and their association with myriad disorders. However the wealth of information available is speckled and not effortlessly acquiescent to data-mining. To reduce this gap, this work describes an algorithmic web based tool to study hidden stops in frameshifted translation for all the lineages through respective genetic code systems.

Findings

This paper describes SHIFT, an algorithmic web application tool that provides a user-friendly interface for identifying and analyzing hidden stops in frameshifted translation of genomic sequences for all available genetic code systems. We have calculated the correlation between codon usage frequencies and the plausible contribution of codons towards hidden stops in an off-frame context. Markovian chains of various order have been used to model hidden stops in frameshifted peptides and their evolutionary association with naturally occurring hidden stops. In order to obtain reliable and persuasive estimates for the naturally occurring and predicted hidden stops statistical measures have been implemented.

Conclusions

This paper presented SHIFT, an algorithmic tool that allows user-friendly exploration, analysis, and visualization of hidden stop codons in frameshifted translations. It is expected that this web based tool would serve as a useful complement for analyzing hidden stop codons in all available genetic code systems. SHIFT is freely available for academic and research purpose at http://www.nuccore.org/shift/.

Background

Vancomycin is the primary treatment for infections caused by methicilin-resistant Staphylococcus aureus (MRSA). The association of vancomycin treatment failures with increased vancomycin minimum inhibitory concentration (MIC) is a well-recognized problem. A number of single-centre studies have identified progressive increases in glycopeptide MICs for S. aureus strains over recent years – a phenomenon known as vancomycin MIC creep. It is unknown if this is a worldwide phenomenon or if it is localized to specific centers.

Methods

The aim of this study was to evaluate the trend of vancomycin MIC for isolates of MRSA over a 3-year period in a tertiary university hospital in Portugal. MRSA isolates from samples of patients admitted from January 2007 to December 2009 were assessed. Etest method was used to determine the respective vancomycin MIC. Only one isolate per patient was included in the final analysis.

Results

A total of 93 MRSA isolates were studied. The vancomycin MICs were 0.75, 1, 1.5 and 2 mg/L for 1 (1.1%), 19 (20.4%), 38 (40.9%), 35 (37.6%) isolates, respectively. During the 3 year period, we observed a significant fluctuation in the rate of MRSA with a vancomycin MIC > 1 mg/L (2007: 86.2%; 2008: 93.3%; 2009: 58.8%, p = 0.002). No MRSA isolate presented a MIC > 2 mg/L.

Conclusions

We were unable to find in our institution data compatible to the presence of vancomycin MIC creep during the study period. This phenomenon seems not to be generalized; as a result each institution should systematically monitor MRSA vancomycin MIC over time.

Background

Considering the current high consumption of energy drinks, the aim of the present study is to evaluate the influence of energy drinks in removing the smear layer and exposing dentinal tubules on root surface.

Methods

Dentine root surfaces were exposed using a diamond bur. Forty movements of scaling were performed in the area prepared in order to create a smear layer. One hundred and thirty specimens were obtained from 35 teeth. Specimens were randomly distributed into 12 groups (n = 10) and divided into subgroups according to the application: topical (n = 5) and friction (n = 5). Twelve energy drinks were evaluated: RedBull™, Burn™, TNT™, Flash Power™, Flying Horse™, Sports Drink™, Ionic™, Hot Power™, Army Power™, Gladiator™ and Bug™. Distilled water was used as a control group. The specimens were analysed by scanning electron microscopy.

Results

Topical application: a significant influence of energy drinks on smear layer removal was found for FlyingHorse™ and Bug™ when compared with the control group. Friction application: significant smear layer removal was found for Burn™, FlyingHorse™, Gladiator™, SportsDrinks™, when compared with the control group. Comparing the different application forms, a statistically significant difference was found for Army Power™.

Conclusion

Considering the significant smear layer removal, energy drinks can be an important etiological factor for cervical dentine hypersensitivity.

Background

There is a high prevalence of depression in individuals with type 2 diabetes mellitus. Depressive disorders are associated with increased medical morbidity and mortality in individuals with diabetes. It has been demonstrated that there is a higher prevalence of diabetic complications among individuals with diabetes and depression compared to those without depression. Several biological alterations have been reported in individuals with depressive disorders, particularly abnormal levels of endocrine-inflammatory markers.

This study aims to determine the prevalence of major depressive disorder (MDD) in type 2 diabetes patients, the prevalence of cardiovascular events in individuals with and without MDD and to compare the endocrine-inflammatory profile between groups.

Methods

The study was approved by the “Comité de Etica de Protocolos de Investigación del Departamento de Docencia e Investigación del Hospital Italiano de Buenos Aires” with the number “1262” and included only patients who provided written informed consent. The study was conducted in accordance with the Declaration of Helsinki and the Habeas Data law on protection of personal data (Law Nª 25326, Argentina).

Type 2 diabetes patients (n = 61) were included and they were classified as having MDD or not according to DSM-IV. Macrovascular disease was obtained from the medical history. Additionally, the intima-media thickness of the common carotid, carotid bifurcations and internal carotid arteries was measured non-invasively by two-dimensional ultrasound imaging. Fasting glucose, fasting lipid profile, inflammatory (CRP, TNF-α) and endocrine (urine free cortisol and saliva cortisol) markers. Student t tests were used to compare means for normally distributed variables and Mann-Whitney test for variables without normal distribution. Relative frequencies were calculated and a chi-square analysis was conducted. Data were expressed as mean ± standard deviation (SD) or median and interquartile range. Multivariable logistic regression was used to determine the relative odds of clinical cardiovascular disease in individuals with compared to those without depression. Differences were considered significant using a two-sided p < 0.05.

Results

21 patients (34%) had MDD and 40 patients (66%) didn’t have MDD. Diabetic patients with MDD had significantly higher CRP levels (4.1(1.9-7.6) vs 1.5(0.5-4.4) mg/l; p = 0.02) and 24-hour urine free cortisol (71.4 ± 21.3 vs 59.8 ± 29.3 ug/24 h; p = 0.03). The other metabolic and inflammatory parameters were not statistically different between groups. There was a significantly higher prevalence of cardiovascular events in individuals with MDD: 38% for the depressive group vs 15% for non-depressive group, p = 0.04). Patients with MDD had a 3.5-fold greater odd of having cardiovascular disease.

Conclusions

Diabetic patients with depression are more likely to have cardiovascular events, and different factors can determine this high association.

Background

A valid, accurate method for determining gestational age (GA) is crucial in classifying early and late prematurity, and it is a relevant issue in perinatology. This study aimed at assessing the validity of different measures for approximating GA, and it provides an insight into the development of algorithms that can be adopted in places with similar characteristics to Brazil. A follow-up study was carried out in two cities in southeast Brazil. Participants were interviewed in the first trimester of pregnancy and in the postpartum period, with a final sample of 1483 participants after exclusions. The distribution of GA estimates at birth using ultrasound (US) at 21–28 weeks, US at 29+ weeks, last menstrual period (LMP), and the Capurro method were compared with GA estimates at birth using the reference US (at 7–20 weeks of gestation). Kappa, sensitivity, and specificity tests were calculated for preterm (<37 weeks of gestation) and post-term (>=42 weeks) birth rates. The difference in days in the GA estimates between the reference US and the LMP and between the reference US and the Capurro method were evaluated in terms of maternal and infant characteristics, respectively.

Results

For prematurity, US at 21–28 weeks had the highest sensitivity (0.84) and the Capurro method the highest specificity (0.97). For postmaturity, US at 21–28 weeks and the Capurro method had a very high sensitivity (0.98). All methods of GA estimation had a very low specificity (≤0.50) for postmaturity. GA estimates at birth with the algorithm and the reference US produced very similar results, with a preterm birth rate of 12.5%.

Conclusions

In countries such as Brazil, where there is less accurate information about the LMP and lower coverage of early obstetric US examinations, we recommend the development of algorithms that enable the use of available information using methodological strategies to reduce the chance of errors with GA. Thus, this study calls into attention the care needed when comparing preterm birth rates of different localities if they are calculated using different methods.

Background

Accurate measurements of spinal movement require reliable determination of anatomical landmarks. Current methods of identifying these are not sufficiently reliable or valid for this purpose. A reliable and convenient method of placing markers on selected vertebra is needed to compare measurements between different testers, subjects and sessions.

Findings

Two testers palpated T4, T7, T10, L1 and L4 spinal processes according to established criteria. They measured the position of spinal processes between C7 and the Posterior Superior Iliac Spine (PSIS) at the Pelvis independently using a flexible ruler placed on the spine. Subjects with a wide range of body heights but without visible spinal deformities were recruited for measurements. Reliability was calculated using absolute and relative values. Mean percentage position and 95% Confidence Intervals were calculated using the mean of both testers’ measurement for all subjects.

Twenty-two subjects participated. The mean distance between C7 and the PSIS level was 50.9 cm (SD: 3.5 cm). Relative reliability for all spinal processes was almost perfect (ICC: > 0.9). Absolute reliability values showed high agreement between testers. Percentage position of T4 was found to be situated 21% along the distance between C7 and the PSIS level, T7 at 39%, T10 at 54.1%, L1 at 70.9% and L4 at 86.1% accordingly. 95% Confidence intervals around mean percentage positions had a maximum at L1 with 2.8% range from upper to lower limit.

Conclusions

The distance of three thoracic and two lumbar spinal processes can be reliably and accurately measured by independent testers, using a flexible ruler. Percentage positions between C7 and PSIS level correspond to spinal processes for subjects without visible deformities in the sagittal and frontal plane.

Background

Yoga exercises are known to decrease stress and restore autonomic balance. Yet knowledge about the physiological effects of inversion postures is limited. This study aimed to investigate the effects of inversion postures (head below the heart) on blood pressure (BP) and heart rate variability (HRV).

Methods

Twelve healthy women and men took part in an 8-week yoga program (60 min once a week). BP was measured with an automatic Omron mx3 oscillometric monitoring device and HRV with a Holter 24-hour ECG at baseline and 8 weeks after the intervention.

Results

There was no significant effect of inversion postures on BP. Nine out of 12 participants showed a significant increase in HRV (p < 0.05) at night (2 hours) on pNN50% (12.7 ± 12.5 to 18.2 ± 13.3). There were no significant changes in other HRV measures such as NN50, LF, HF, LF/HF ratio, LF normalized units (n.u.), HF n.u. and RMSSD.

Conclusion

Eight weeks of hatha yoga improved HRV significantly which suggests an increased vagal tone and reduced sympathetic activity.

Background

As an in vitro model porcine peripheral blood mononuclear cells (PBMCs) is frequently used as for immunogenetic research with the stimulation of bacterial antigens. To investigate the immunocompetence of PBMCs for recognition of Gram-positive and Gram-negative bacteria and in order to dissect the pathogenesis of diseases, gene expression assay is most commonly used. The gene expressions are required to normalize for reference genes which have tremendous effect on the results of expression study. The reference genes should be stably expressed between different cells under a variety of experimental conditions, but recent influx of data showed that expression stability of reference genes are varied under different experimental conditions. But data regarding the expression stability of reference genes in porcine PBMCs are limited. Therefore, this study was aimed to know whether the expression stability of commonly used reference genes in PBMCs is affected by various bacterial antigens under different experimental conditions in pigs.

Results

The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined by RT-qPCR in PBMCs that were stimulated by LPS and LTA in vitro as well as cells un-stimulated control and non-cultured were also consider for this experiment. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P < 0.05). geNorm software revealed that in case of irrespective of stimulation (without considering the type of stimulation), RPL4, PPIA and B2M were the most stable reference genes in PBMCs; in case of the control group, PPIA, BLM and GAPDH were the most stable reference genes. PPIA, B2M and RPL4 were the most stable reference genes in LPS stimulated PBMCs; and YWHAZ, RPL4 and PPIA were the most stably expressed reference genes in the case of LTA stimulated PBMCs. When LPS was used combined with LTA for the stimulation, YWHAZ, B2M and SDHA remained the most stable genes. PPIA, BLM and GAPDH were found to be most stably expressed reference genes when PBMCs were not cultured. NormFinder revealed different sets of stably expressed reference genes in PBMCs under different experimental conditions. Moreover, geNorm software suggested that the geometric mean of the three most stable genes would be the suitable combination for accurate normalization of gene expression study.

Conclusion

There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms (geNorm and NormFinder). In conclusion, the geometric mean of the RPL4, B2M and PPIA seemed to be the most appropriate combination of reference genes for accurate normalization of gene expression data in porcine PBMCs without knowing the type of bacterial pathogenic status of the animals and in the case of mixed infection with Gram-negative and Gram-positive bacteria. In case of PBMCs without any stimulation, PPIA, BLM and GAPDH could be suggested as suitable reference genes.

Background

A prophylactic antibiotic is recommended to reduce infection-related complication following cesarean delivery. There is a current debate on the time of prophylactic antibiotic in cesarean delivery.

Methods

An opened randomized, controlled clinical trial was conducted at Soba hospital, Sudan to investigate the timing (pre-incision or after clamping of the umbilical cord) of ceftizoxime for elective cesarean delivery. The outcome measures were; the incidence of post-cesarean febrile and infection-related morbidity and neonatal outcomes between the two groups.

Results

Hundred –eighty women (90 women in each arm of the study) received intravenous injection of 1 g of ceftizoxime as single dose either at pre-incision or after clamping of the umbilical cord. None of the women in either group had endometritis. One woman in the pre-incision group had chest infection. There was no significant difference in the incidence of wound infection between the two groups, 8 (6.7%) vs. 3 (3.3%); P = 0.2. Two babies in the pre-incision group (P = 0.497) had a low Apgar score (< 8) at 1 min. Similar number of neonate (15 in each arm) was admitted to nursery. There were no significant difference in the neonatal jaundice between the two groups, 5 (5.5%) vs. 4 (4.4%), P = 0.2. There was no perinatal death.

Conclusions

There was no difference in the two regimens (pre-incision or post-clamping of the umbilical cord) of ceftizoxime as prophylactic for elective cesarean delivery.

Trial registration

NCT01347593

Background

Paratuberculosis remains today a major global problem in animal health, especially for dairy cattle. However, the diagnosis of its etiologic agent, Mycobacterium avium subsp. paratuberculosis (Map), still lacks sensitivity because of the lack of available antigens. Little is known about the virulence factors for this pathogen. In this study we have developed a method to produce and purify the heparin-binding hemagglutinin (HBHA), a major adhesin of Mycobacteria, from a culture of Map.

Findings

For this extremely slow-growing Mycobacterium, a culture was established in a 3-liter bioreactor. Using the bioreactor the amount of the Map biomass was increased 5-fold compared to a classical culture in flasks. The map-HBHA was purified from a Map lysate by heparin-Sepharose chromatography on HiTrap columns. Binding of map-HBHA onto heparin-Sepharose can be reduced in the presence of salt. Consequently, all steps of sample preparation and column equilibration were carried out in 20 mM Tris–HCl (pH 7.2). The map-HBHA was eluted by a linear NaCl gradient. High resolution mass spectrometry analyses revealed that the native form of map-HBHA has posttranslational modifications, including the removal of the initiation methionine, acetylation of the alanine residue at the N-terminal extremity and the presence of methylated lysines in the C-terminal domain of the protein.

Conclusions

An optimized culture of Map in a bioreactor was established to purify the native map-HBHA from a Map lysate by heparin-Sepharose chromatography. The availability of this antigen offers the possibility to study the structure of the protein and to examine its role in pathogenicity, in particular to better understand the specific interactions of Map with the intestinal tissue. The map-HBHA obtained in its native immunogenic form may also be useful to improve the diagnostic test, especially for the development of a new T-cell-based interferon gamma release assays.

Background

Obesity among fertile women is a global problem. 25% of pregnant Swedish women are overweight at admission to the antenatal clinic and 12% of them are considered as obese. Previous studies have shown an increased risk of delivery complications with an elevated maternal BMI. The aim of this study was to evaluate delivery outcomes in relation to maternal BMI on admission to the antenatal clinic.

A healthy group of 787 women with full-term pregnancies and spontaneous onset of labor were included in the study. Delivery outcome was assessed in relation to maternal BMI when attending the antenatal clinic.

Results

The results indicated that in deliveries where the maternal BMI was >30 a high frequency of abnormal CTG trace during the last 30 minutes of labor was shown. A blood sample for evaluation of risk of fetal hypoxia was performed in only eight percent of these deliveries. A spontaneous vaginal delivery without intervention was noted in 85.7%, and 12% of neonates were delivered with an adverse fetal outcome compared to 2.8% in the group with a maternal BMI<30 (p<0.001).

Conclusion

These results indicate an increased risk at delivery for healthy, but obese women in labor. Furthermore, the delivery management may not always be optimal in these deliveries.

Background

The current ‘gold standard’ for serotyping pneumococci is the Quellung test. This technique is laborious and requires a certain level of training to correctly perform. Commercial pneumococcal latex agglutination serotyping reagents are available, but these are expensive. In-house production of latex agglutination reagents can be a cost-effective alternative to using commercially available reagents. This paper describes a method for the production and quality control (QC) of latex reagents, including problem solving recommendations, for pneumococcal serotyping.

Results

Here we describe a method for the production of latex agglutination reagents based on the passive adsorption of antibodies to latex particles. Sixty-five latex agglutination reagents were made using the PneuCarriage Project (PCP) method, of which 35 passed QC. The other 30 reagents failed QC due to auto-agglutination (n=2), no reactivity with target serotypes (n=8) or cross-reactivity with non-target serotypes (n=20). Dilution of antisera resulted in a further 27 reagents passing QC. The remaining three reagents passed QC when prepared without centrifugation and wash steps. Protein estimates indicated that latex reagents that failed QC when prepared using the PCP method passed when made with antiserum containing ≤ 500 μg/ml of protein. Sixty-one nasopharyngeal isolates were serotyped with our in-house latex agglutination reagents, with the results showing complete concordance with the Quellung reaction.

Conclusions

The method described here to produce latex agglutination reagents allows simple and efficient serotyping of pneumococci and may be applicable to latex agglutination reagents for typing or identification of other microorganisms. We recommend diluting antisera or removing centrifugation and wash steps for any latex reagents that fail QC. Our latex reagents are cost-effective, technically undemanding to prepare and remain stable for long periods of time, making them ideal for use in low-income countries.

Background

The use of quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) has become widely applied as a method to measure transcript abundance. In order to be reflective of biological processes during health and disease this method is dependent on normalisation of data against stable endogenous controls. However, these genes can vary in their stability in different cell types. The importance of reference gene validation for a particular cell type is now well recognised and is an important step in any gene expression study.

Results

Cultured primary human choroidal and retinal endothelial cells were treated with the immunostimulant polyinosinic: polycytidylic acid or untreated. qRT-PCR was used to quantify the expression levels of 10 commonly used endogenous control genes, TBP, HPRT1, GAPDH, GUSB, PPIA, RPLP0, B2M, 18S rRNA, PGK1 and ACTB. Three different mathematical algorithms, GeNorm, NormFinder, and BestKeeper were used to analyse gene stability to give the most representative validation. In choroidal endothelial cells the most stable genes were ranked as HPRT1 and GUSB by GeNorm and NormFinder and HPRT1 and PPIA by BestKeeper. In retinal endothelial cells the most stable genes ranked were TBP and PGK1 by GeNorm and NormFinder and HPRT1 by BestKeeper. The least stable gene for both cell types was 18S with all 3 algorithms.

Conclusions

We have identified the most stable endogenous control genes in intraocular endothelial cells. It is suggested future qRT-PCR studies using these cells would benefit from adopting the genes identified in this study as the most appropriate endogenous control genes.

Background

Colonoscopes are designed with balance between flexibility, required to negotiate angulations, and stiffness, required to counteract the propensity for looping in unfixed sections of the colon, which retards advancement of the instrument. Colonoscopy can be challenging with old instruments that have lost native stiffness and become less responsive to torquing.

A new intraluminal stiffening device has become available in two grades of stiffness. However, there is no published evidence of its effectiveness. This randomized, controlled trial was designed to determine the effectiveness of the stiffening wires in improving cecal intubation rate and time following routine application. A secondary analysis determines effectiveness of application only after intractable failure with the unaided colonoscope.

Methods

The colonoscope tested was an Olympus CF-100TL, approximately fifteen years old. Patients were randomly assigned to the unaided colonoscope or the standard or firm wire introduced routinely on entry into transverse colon. Each phase of colonoscopy was timed. Failure to advance the colonoscope for 5 minutes (despite usual manipulations to minimize looping) required switching to another intervention according to a prescribed methodology and the originally assigned intervention was recorded as failed.

Results

The study was terminated after accrual of 112 participants (target sample size 480) because the colonoscope required repairs (no damage attributable to stiffening wires) which would have been uneconomical. There were no statistically significant differences between per-protocol cecal intubation rates (81.1, 71.1 and 70.3 percent respectively), a finding which persisted after multiple imputation for a virtual sample size of 480. Similarly, there were no statistically significant differences between per-protocol cecal intubation times (15, 16.2 and 13.9 minutes). However, a statistically significant improvement in cecal intubation rate (from 81.1% to 97.3%, P = 0.0313) was achieved when the wires were applied after intractable failure of the unaided colonoscope in the first intervention group.

Conclusions

Routine application of either stiffening wire does not improve caecal intubation rate nor time compared to the unaided colonoscope. However, application of the stiffening wires after intractable failure of the unaided colonoscope enabled a statistically significant improvement in cecal intubation rate.

Trial Registration

clinicaltrials.gov Identifier: NCT01115010

Background

Accurate measurements of mRNA expression levels in tissues or cells are crucially dependent on the use of relevant reference genes for normalization of data. In this study we used quantitative real-time PCR and two Excel-based applets (geNorm and BestKeeper) to determine the best reference genes for quantification of target gene mRNA in a complex tissue organ such as the guinea pig cervix.

Results

Gene expression studies were conducted in cervical epithelium and stroma during pregnancy and parturition and in cultures of primary cells from this tissue. Among 15 reference gene candidates examined, both geNorm and BestKeeper found CLF1 and CLTC to be the most stable in cervical stroma and cervical epithelium, ACTB and PPIB in primary stroma cells, and CLTC and PPIB in primary epithelial cells. The order of stability among the remaining candidate genes was not in such an agreement. Commonly used reference such as GAPDH and B2M demonstrated lower stability. Determination of pairwise variation values for reference gene combinations using geNorm revealed that the geometric mean of the two most stable genes provides sufficient normalization in most cases. However, for cervical stroma tissue in which many reference gene candidates displayed low stability, inclusion of three reference genes in the geometric mean may improve accuracy of target gene expression level analyses. Using the top ranked reference genes we examined the expression levels of target gene PTGS2 in cervical tissue and cultured cervical cells. We compared the results with PTGS2 expression normalized to the least stable gene and found significant differences in gene expression, up to 10-fold in some samples, emphasizing the importance of appropriately selecting reference genes.

Conclusions

We recommend using the geometric mean of CFL1 and CLTC for normalization of qPCR studies in guinea pig cervical tissue studies, ACTB and PPIB in primary stroma cells and CLTC and PPIB in primary epithelial cells from guinea pig.