Whole blood BDNF levels in healthy twins discordant for affective disorder: association to life events and neuroticism

BACKGROUND: Depression has been associated with decreased blood BDNF concentrations; but it is unclear if low blood BDNF levels are a state or a trait marker of depression. METHODS: We investigated blood BDNF concentrations in a twin population including both subjects highly predisposed and protected against affective disorder. Whole blood assessed for BDNF concentrations and correlated to risk status, neuroticism, and number of stressful life events. RESULTS: Between the groups, we found no significant difference in whole blood BDNF levels. Women at high-risk for depression who had experienced three or more recent stressful events (n=26) had decreased whole blood BDNF levels compared to high-risk women with two or less recent stressful events (n=35), 21.6+/-7.0 vs. 18.5+/-4.1 ng/ml, respectively, (p<0.05). No such association was found in low-risk women or in men. In men, however, low neuroticism scores and two or less recent stressful events were associated with decreased whole blood BDNF levels (n=50, p<0.05). LIMITATIONS: The cross-sectional design limits the possibility of drawing firm conclusions on causatility of the findings. CONCLUSION: The genetic risk of developing depression does not translate directly into whole blood BDNF levels. In females who are genetically disposed for depression and subjected to recent stressful life events whole blood BDNF levels are lower.     

Fibrinmonomer binding to macrophages mediated by fibrin-binding fibronectin fragments

Binding of 125-I-fibrinmonomer to peritoneal macrophages was investigated in dependence of plasma fibronectin and of its thrombin- or plasmin-derived fragments. Plasma fibronectin failed to enhance cell binding of 125-I-fibrinmonomer. In contrast, 30kD-fragments derived from the N-termini of the fibronectin subunits improved binding considerably. The association with the cell surface was completely inhibited by EDTA, 2-5 mM putrescine and to about 40 per cent by 0.1 mM dansylcadaverine suggesting that a transamidase-catalyzed cross-linking reaction was involved. Thrombin-derived 200kD-remnants of the fibronectin subunit chains failed to mediate cell binding of 125-I-fibrinmonomer provided they had been deprived of residual thrombin activity. Otherwise they were active and their activity was inhibited by the thrombin inhibitor hirudin. Plasmin-derived 200 kD-fragments were inactive as well.     

Human whole mitochondrial genome sequencing and analysis: optimization of the experimental workflow

AimTo evaluate critical steps in Illumina® Human mtDNA Genome assay: target enrichment, limited-cycle PCR, and library normalization, in order to optimize the protocol for analysis of whole mitochondrial genomes from human reference samples.MethodsThree long-range high-fidelity DNA polymerases (Platinum^TM PCR SuperMix High Fidelity, LA Taq® Hot Start, and PrimeSTAR® GXL) were tested for their performance in the amplification of mtDNA fragments. Sequencing results of ten samples, as well as negative controls, which underwent library preparation with 12 and 15 cycles in limited-cycle PCR were compared. Additionally, two library normalization methods were compared: bead-based normalization vs quantification and individual normalization.ResultsPrimeSTAR® GXL performed best for mitochondrial DNA enrichment. Increment of amplification cycles to 15 in limited-cycle PCR step did not affect either the sequencing process or variant calling. Library quantification combined with individual library-by-library dilution outperformed bead-based normalization.ConclusionOptimizations described herein provide beneficial insights for laboratories aiming at implementation and/or advancement of similar massively parallel sequencing workflows (eg, small genomes, PCR amplicons, and plasmids).

Library preparation in massively parallel sequencing (MPS) protocols is a sensitive process, usually consisting of multiple elaborate steps. To ensure high quality of sequencing results, it is important to optimize library preparation according to characteristics of a particular target molecule. There are several critical aspects of Illumina® Human mtDNA Genome (1) assay for analysis of whole mitochondrial DNA (mtDNA) on MiSeq® instrument: initial enrichment of the target molecule (achieved, in this case, by long-range PCR); PCR step wherein index-adapter oligonucleotides are added and libraries are amplified (termed “limited-cycle PCR” step); and normalization of libraries prior to their pooling for sequencing.In this study, we aimed to test three long-range high-fidelity DNA polymerases for their performance in amplification of mtDNA fragments, in order to determine the one best suited for Illumina® assay, in which mitochondrial genomes are amplified in two large fragments (sizes 9.1 kb and 11.2 kb) (Supplementary Figure 1(Supplementary Figure 1)).Various optimizations and evaluations have been previously published (2-6), but, to our knowledge, none of them assessed the impact of increasing the number of amplification cycles in limited-cycle PCR. Therefore, we also aimed to test and observe how sequencing results were affected by this step and whether there were any adverse effects that would impact variant calling.Lastly, we aimed to compare two library normalization methods: bead-based normalization vs quantification and individual normalization. Nextera® XT Library Prep Kit (Illumina, San Diego, CA, USA) has been known to produce uneven read depth profiles (2-4,6-8). Therefore, a great risk in this protocol is potential loss of sequence information in regions that achieve very low read depth (ie, too low for analysis and subsequent variant calling), or possibly receive no reads at all. The choice of library normalization method may affect this through distribution of reads among the sequenced libraries. So, a method that provides more uniform distribution of reads would be preferable, which we hypothesized would be the method of quantification and individual normalization rather than bead-based normalization.     

Fullerenol C60(OH)24 prevents doxorubicin-induced acute cardiotoxicity in rats

Results obtained in vitro suggested that fullerenol's antiproliferative properties and protective effects against doxorubicin (DOX) cytotoxicity are mediated by antioxidative and hydroxyl radical scavenger activity. The aim of this study was to examine the influence of fullerenol on acute cardiotoxicity after the administration of a single high dose of DOX in vivo. The experiment was performed on male Wistar rats randomly divided into five groups, each containing eight individuals, that were treated as follows: I) 0.9% NaCl, II) 10 mg/kg DOX, III) 50 mg/kg fullerenol 30 min before 10 mg/kg DOX, IV) 100 mg/kg fullerenol 30 min before 10 mg/kg DOX, and V) 100 mg/kg fullerenol. A functional, biochemical, hematological, and pathomorphological examination of the heart as well as an evaluation of oxidative stress parameters was conducted on days 2 and 14 after DOX administration. The function of the heart was investigated by monitoring heart contractility after the adrenaline infusion. Fullerenol, applied alone, did not alter basal values of investigated animals. Both doses of fullerenol, used as a pretreatment, did not alter the basal parameters of the animals. The 100 mg/kg dose of fullerenol showed better protection. Considering the mechanisms of DOX toxicity, fullerenol likely exerts its protective role as a free radical sponge and/or by removing free iron through the formation of a fullerenol-iron complex. Our results suggest that fullerenol might be a potential cardioprotective agent in DOX-treated individuals.     

Health professionals’ perceptions of intimate partner violence against women in Serbia: Opportunities and barriers for response improvement

Objective

The aim of this study was to determine the perceptions and attitudes of health professionals toward violence against women in intimate relationships, and to discuss them as opportunities and barriers for improving health professionals’ response.

Methods

Six focus groups were conducted with 71 health professionals employed in the public primary health care centers in Belgrade (Serbia). The data were analyzed according to the direct approach of the qualitative content analysis.

Results

Findings suggest that the majority of health professionals perceive IPV as an unjustifiable act. They showed an understanding for women; see their role as providing support to women and collaborating with other institutions. They are willing to help, but do not know how. However, some health professionals appeared to be judgmental in terms of what preceded violence, and would insist on extracting a woman's disclosure that violence had occurred. As barriers, they emphasized the lack of training and specific education on IPV, a weak support network, and overall social insecurity.

Conclusion

There is a gap between health professionals’ willingness to help and resources needed, along with prejudice and interrogative attitudes. Barriers appeared at individual, organizational and societal levels.

Practice implications

Proper education and protocols are priorities in strengthening health professionals’ response to IPV.     

Clinical pharmacology consultation: a better answer to safety issues of drug therapy during pregnancy?

Purpose  

Drug safety classifications give a very basic estimation of risk and should only be used as general guideline when assessing risk of pregnancy-related drug exposure or planning treatment. We conducted a study to assess the strength of association between both the clinical pharmacologists’ risk assessment and the FDA risk categorization, and adverse pregnancy outcomes.     

A randomized,double-blind,placebo-controlled phase 2 study to assess safety,tolerability, and efficacy of RT001 in patients with amyotrophic lateral sclerosis

Background and purpose

RT001 is a deuterated synthetic homologue of linoleic acid, which makes membrane polyunsaturated fatty acids resistant to lipid peroxidation, a process involved in motor neuron degeneration in amyotrophic lateral sclerosis (ALS).

Methods

We conducted a randomized, multicenter, placebo-controlled clinical trial. Patients with ALS were randomly allocated to receive either RT001 or placebo for 24 weeks. After the double-blind period, all patients received RT001 during an open-label phase for 24 weeks. The primary outcome measures were safety and tolerability. Key efficacy outcomes included the ALS Functional Rating Scale (ALSFRS-R), percent predicted slow vital capacity, and plasma neurofilament light chain concentration.

Results

In total, 43 patients (RT001 = 21; placebo = 22) were randomized. RT001 was well tolerated; one patient required dose reduction due to adverse events (AEs). Numerically, there were more AEs in the RT001 group compared to the placebo group (71% versus 55%, p = 0.35), with gastrointestinal symptoms being the most common (43% in RT001, 27% in placebo, p = 0.35). Two patients in the RT001 group experienced a serious AE, though unrelated to treatment. The least-squares mean difference in ALSFRS-R total score at week 24 of treatment was 1.90 (95% confidence interval = −1.39 to 5.19) in favor of RT001 (p = 0.25). The directions of other efficacy outcomes favored RT001 compared to placebo, although no inferential statistics were performed.

Conclusions

Initial data indicate that RT001 is safe and well tolerated. Given the exploratory nature of the study, a larger clinical trial is required to evaluate its efficacy.