Anti-inflammatory properties of Honokiol in activated primary microglia and astrocytes.

Honokiol has been used in traditional medicine for the treatment of inflammatory diseases. Activation of glial cells plays an essential role in neurodegenerative disorders. In this study, we show that Honokiol reduces the inflammatory response to LPS of primary cultures of microglia and astrocytes through the inhibition of pro-inflammatory mediators (iNOS, IL-6, IL-1ß and TNF-α) and the simultaneous stimulation of anti-inflammatory cytokines (IL-10). Expression of KLF4 was induced in microglia and astrocytes after treatment with LPS and this response was mitigated by Honokiol. These findings extend our understanding of the anti-inflammatory properties of Honokiol on central glial cells and support its use as a therapeutic compound in neuroinflammatory disorders.

Selenium Reduces Early Signs of Tumor Necrosis Factor Alpha-Induced Meniscal Tissue Degradation.

Meniscal integrity is a prerequisite for sustained knee joint health and prevention of meniscal degeneration is a main research goal. Cartilage-protective effects of selenium have been described, but little is known about the impact on the meniscus. We therefore investigated the influence of sodium selenite on meniscal explants under tumor necrosis factor-alpha (TNFα)-stimulated proinflammatory conditions. Meniscal explant disks (3 mm diameter × 1 mm thickness) were isolated from 2-year-old cattle and incubated with TNFα (10 ng/ml) and sodium selenite (low dose, LoD 6.7 ng/ml as being found in Insulin-Transferrin-Selenium medium supplements, ITS; medium-dose, MeD 40 ng/ml described as physiological synovial concentration; high dose, HiD 100 ng/ml described as optimal serum concentration). After 3 days of culture glycosaminoglycan (GAG) release (DMMB assay), nitric oxide (NO) production (Griess assay), gene expression of matrix-degrading enzymes (quantitative RT-PCR), and apoptosis rate were determined. TNFα led to a significant raise of GAG release and NO production. LoD and MeD selenite significantly reduced the TNFα-induced GAG release (by 83, 55 %, respectively), NO production (by 59, 40 %, respectively), and apoptosis (by 68, 39 %, respectively). LoD and MeD selenite showed a tendency to reduce the TNFα-mediated increase of inducible NO-synthase (iNOS) levels, LoD selenite furthermore matrix metalloproteinase (MMP)-3 transcription levels and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 levels. LoD and less pronounced MeD selenite show a substantial impact on the early meniscal inflammatory response. To our knowledge this is the first study showing the protective influence of selenium on meniscal tissue maintenance. To understand the superior potency of low-dose selenium on molecular level future studies are needed.

The new guidelines for paternity analysis in Germany-how many STR loci are necessary when investigating duo cases?

The requirements in the new German guidelines for paternity analysis have not only changed according to the so-called Gendiagnostikgesetz, the new German law regulating human genetic as well as paternity analyses, but also regarding the minimal number of short tandem repeats (STRs) which should be investigated (15 STRs) and the minimal required average exclusion chance (99.999 %). Even in paternity analyses involving only two people (e.g., father and child or mother and child), this exclusion chance is mandatory. A retrospective analysis of 330 father-child cases from our routine investigations showed in 142 cases (43 %) an individual exclusion chance below 99.999 % when using 15 STRs as required, in our routine work provided by the Powerplex® 16 kit which is reported to have an average exclusion chance of 99.988 %. Therefore, these same 330 father-child pairs were additionally analysed using the Powerplex® 21 kit and 120 of these duos were additionally analysed using the Powerplex® ESX17 kit enabling the analysis of 20 or 16 loci respectively. Now, an individual exclusion chance of more than 99.999 % could be achieved in 95.5 % (Powerplex® 21; calculation without the results of D6S1043), 98.8 % (Powerplex® 21; calculation with the results of D6S1043, using allele frequencies established in this study for a German and a West African population) and 98.3 % (Powerplex® ESX17). These data clearly demonstrate that in duo cases, more than the required 15 STR loci have to be investigated to obtain sufficient results.

Highly reliable quantification of proteins such as members of the HSP70 superfamily based on the grey scale index via immune detection stained bands on a Western blot.

The HSP70 superfamily is a reliable biomarker for hyperthermia, hypothermia and hypoxia. The Enzyme-linked Immunosorbent Assay (ELISA) respectively immunohistochemically staining methods are the typically used techniques for the quantification of those proteins. As the costs for reagents and devices as well as the work schedule of these methods are immense it was the goal of our study to develop an easy and reliable method to quantify the concentration of specific proteins. We established a procedure to measure the relative concentration of proteins fixed on ROTI(®) PVDF membranes via Western blot, calculating the relative protein concentration in dependency to the grey scale index of the normalized and digitalized pictures of the bands on the blots.

Reliable genetic identification of burnt human remains.

The identification of severely burnt human remains by genetic fingerprinting is a common task in forensic routine work. In cases of extreme fire impact, only hard tissues (bones, teeth) may be left for DNA analysis. DNA extracted from burnt bone fragments may be highly degraded, making an amplification of genetic markers difficult or even impossible. Furthermore, heavily burnt bones are very prone to contamination with external DNA. We investigated whether authentic DNA profiles can be generated from human bones showing different stages of fire induced destruction (well preserved, semi-burnt, black burnt, blue-grey burnt, blue-grey-white burnt). DNA was extracted from 71 bone fragments derived from 13 individuals. Obtained genetic patterns (STRs and mtDNA sequences) were compared to the genetic pattern of the respective bodies. Our results show that the identification via DNA analysis is reliably and reproducibly possible from well preserved and semi-burnt bones. In black burnt bones the DNA was highly degraded and in some cases no nuclear DNA was left, leaving mitochondrial DNA analysis as an option. Blue-grey burnt bones lead only sporadically to authentic profiles. The investigation of blue-grey-white burnt bones barely led to reliable results.

A new multiplex-PCR comprising autosomal and y-specific STRs and mitochondrial DNA to analyze highly degraded material.

The analysis of short tandem repeats is one of the most powerful tools in forensic genetics. Forensic practice sometimes requires the individualization of samples that may contain only highly degraded nuclear DNA, mitochondrial DNA or PCR inhibitors that hamper DNA amplification. We designed a new multiplex PCR with reduced size amplicons (<200 bp), providing a double sex determination (amelogenin plus two Y-STRs), the detection of two autosomal markers and the amplification of mitochondrial specific fragments from the hypervariable region I (HVI). Additionally, a quality sensor was developed to check for the presence of any PCR inhibitors. The new multiplex PCR shows a reproducible detection threshold down to 25 pg and gives signals even out of highly degraded materials. All signals are reproducible and reliable as it could be shown in comparison to results from commercially available STR multiplex-PCRs. In no case DNA fragments were detectable using any other assay when the quality sensor was not detectable. There was a good correlation between detection of mitochondrial specific fragments in the multiplex-PCR and success of subsequent sequencing of HVI region. The same could be shown for STR analysis: Most samples successfully analyzed in our PCR yielded at least a partial STR profile using a commercial STR kit. We present an assay that allows an easy, reliable, and cost efficient evaluation of DNA sample quality combined with a first rough sample individualization and sex determination suitable for forensic purposes. This assay may help the forensic lab personnel to decide on further sample processing.