Mesenchymal stem cells and myoblast differentiation under HGF and IGF-1 stimulation for 3D skeletal muscle tissue engineering.

BACKGROUND: Volumetric muscle loss caused by trauma or after tumour surgery exceeds the natural regeneration capacity of skeletal muscle. Hence, the future goal of tissue engineering (TE) is the replacement and repair of lost muscle tissue by newly generating skeletal muscle combining different cell sources, such as myoblasts and mesenchymal stem cells (MSCs), within a three-dimensional matrix. Latest research showed that seeding skeletal muscle cells on aligned constructs enhance the formation of myotubes as well as cell alignment and may provide a further step towards the clinical application of engineered skeletal muscle. In this study the myogenic differentiation potential of MSCs upon co-cultivation with myoblasts and under stimulation with hepatocyte growth factor (HGF) and insulin-like growth factor-1 (IGF-1) was evaluated. We further analysed the behaviour of MSC-myoblast co-cultures in different 3D matrices. RESULTS: Primary rat myoblasts and rat MSCs were mono- and co-cultivated for 2, 7 or 14 days. The effect of different concentrations of HGF and IGF-1 alone, as well as in combination, on myogenic differentiation was analysed using microscopy, multicolour flow cytometry and real-time PCR. Furthermore, the influence of different three-dimensional culture models, such as fibrin, fibrin-collagen-I gels and parallel aligned electrospun poly-ε-caprolacton collagen-I nanofibers, on myogenic differentiation was analysed. MSCs could be successfully differentiated into the myogenic lineage both in mono- and in co-cultures independent of HGF and IGF-1 stimulation by expressing desmin, myocyte enhancer factor 2, myosin heavy chain 2 and alpha-sarcomeric actinin. An increased expression of different myogenic key markers could be observed under HGF and IGF-1 stimulation. Even though, stimulation with HGF/IGF-1 does not seem essential for sufficient myogenic differentiation. Three-dimensional cultivation in fibrin-collagen-I gels induced higher levels of myogenic differentiation compared with two-dimensional experiments. Cultivation on poly-ε-caprolacton-collagen-I nanofibers induced parallel alignment of cells and positive expression of desmin. CONCLUSIONS: In this study, we were able to myogenically differentiate MSC upon mono- and co-cultivation with myoblasts. The addition of HGF/IGF-1 might not be essential for achieving successful myogenic differentiation. Furthermore, with the development of a biocompatible nanofiber scaffold we established the basis for further experiments aiming at the generation of functional muscle tissue.

Environmental DNA metabarcoding for benthic monitoring: A review of sediment sampling and DNA extraction methods.

Environmental DNA (eDNA) metabarcoding (parallel sequencing of DNA/RNA for identification of whole communities within a targeted group) is revolutionizing the field of aquatic biomonitoring. To date, most metabarcoding studies aiming to assess the ecological status of aquatic ecosystems have focused on water eDNA and macroinvertebrate bulk samples. However, the eDNA metabarcoding has also been applied to soft sediment samples, mainly for assessing microbial or meiofaunal biota. Compared to classical methodologies based on manual sorting and morphological identification of benthic taxa, eDNA metabarcoding offers potentially important advantages for assessing the environmental quality of sediments. The methods and protocols utilized for sediment eDNA metabarcoding can vary considerably among studies, and standardization efforts are needed to improve their robustness, comparability and use within regulatory frameworks. Here, we review the available information on eDNA metabarcoding applied to sediment samples, with a focus on sampling, preservation, and DNA extraction steps. We discuss challenges specific to sediment eDNA analysis, including the variety of different sources and states of eDNA and its persistence in the sediment. This paper aims to identify good-practice strategies and facilitate method harmonization for routine use of sediment eDNA in future benthic monitoring.

[Establishing and evaluating a multinational Science Academy: A new tool for fostering scientific young talents in microsurgical research - a Consensus Statement of the DAM]. / Etablierung und Evaluation einer multinationalen Wissenschaftsakademie: Ein neues Modell zur Stärkung des wissenschaftlichen Nachwuchses in der mikrochirurgischen Forschung ­ Ein Konsensus Papier der DAM.

BACKGROUND: The formation of professional networks and cooperations - in addition to any qualified good education - seems fundamental for a successful career. In a number of disciplines, various symposia or conferences exist. In the field of microsurgery, however, a specific, guided and designated opportunity for junior scientists to network with one another has been missing so far. METHODS: In 2017, a science academy was initiated for the first time by the German-speaking Association for Nerves and Vessels (DAM) with the goal of bringing together and networking microsurgically researching young physicians and scientists. This was intended to happen on a small scale once a year in order to develop synergies for joint research projects. For this purpose, motivated junior researchers were individually selected by their mentors and sent to the academy by the boards of research institutions that are organized in the DAM. After getting to know each other in a relaxed atmosphere, the participants were given the opportunity to present their respective research project within the framework of thematic blocks and moderated by experienced mentors. Each presentation was followed by a round table discussion and small group work, in which knowledge and methods were exchanged and points of contact for possible later cooperation were identified. RESULTS: In the past 3 years, the DAM Science Academy proved to be an optimal format to initiate and promote networks of young researchers comprising microsurgically interested physicians and scientists. There were many lively and in-depth discussions, which were mainly due to the open working atmosphere and the obligation to confidentiality. Most of the synergies were shown i. a. in the field of angiogenesis, bioreactor, carcinoma-ADSC interactions, stem cells, AV loop model, ischemia/reperfusion, and nerve regeneration. The participants consistently gave a very positive feedback in the final evaluation with the wish to continue this academy. CONCLUSION: The DAM Science Academy can be considered a highly suitable complemental platform to the existing networking opportunities among microsurgical researchers. Experience so far suggests that this will hopefully result in long-term cooperations and a permanent transfer of knowledge among the participants.

Long-term studies on the integration of acellular porcine dermis as an implant shell and the effect on capsular fibrosis around silicone implants in a rat model.

Acellular dermal matrices have recently increasingly been used in alloplastic breast reconstruction with silicone breast implants. Among these matrices, acellular porcine dermis (APD) is frequently applied, but long-term data on tissue integration and capsular fibrosis formation are still missing. Silicone prostheses with (group A) and without (group B) APD as an implant-covering shell were implanted in male Lewis rats. At 3, 12, and 52 weeks after implantation, the constructs were explanted. Molecular biological and immunohistochemical analyses were performed afterwards. On comparing the collagenous layer and the newly formed myofibroblast-rich layer around the implants of both groups, it became apparent that in group A, these layers were thinner, followed by a lower expression of TGFß1 after 12 and 52 weeks. Further, in this group, at the endpoint of 52 weeks, a lower amount of CD68-positive cells in the collagenous and myofibroblast-rich layers were observed and the expression of TNFα was reduced, while the number of Ki67-positive cells was significantly higher with time. Furthermore, MMP1 expression in group A was lower than that in group B, and the calculated ratio of MMP1:TIMP1 expression was higher. The long-term results clearly show a reduction in inflammatory and fibrotic tissue reaction when APD is used to cover silicone prostheses. These experimental data will be of considerable importance for implant-based breast surgery, as they indicate a potential benefit in the reduction of capsular fibrosis formation of an interposition of APD between the recipient and the silicone implant.

Radioiodine therapy and thyroid-associated orbitopathy: risk factors and preventive effects of glucocorticoids.

Whether or not thyroid-associated orbitopathy is exacerbated by radioiodine therapy is a matter of controversy. Several risk factors can be listed: pre-existing active ophthalmopathy, hypothyroidism following radioiodine therapy, elevated T3 levels during radioiodine therapy, recurrent or persisting hyperthyroidism after low-dose radioiodine therapy and smoking. Recent studies and own data demonstrate that worsening of pre-existing thyroid-associated orbitopathy after radioiodine therapy can be prevented by the administration of glucocorticoids. Even in patients without evident ophthalmopathy the prophylactic use of glucocorticoids is in our opinion justified to minimize a small but known risk of the development of ocular symptoms. Larger prospective randomized studies are needed to establish the optimal dose and duration of the required anti-inflammatory therapy.

[Deterioration of endocrine ophthalmology after radioiodine therapy in Graves' disease?]. / Verschlechterung einer endokrinen Orbitopathie nach Radioiodtherapie bei M. Basedow?

AIM: Our goal was to show the development of EO in RITH and compare it with the outcome of surgery or thyreostatic therapy. METHODS: In this study 103 cases of an RITH at 82 patients were performed. The EO findings were measured before RITH and several times afterwards by the same experienced researcher according to the following criteria: 1. subjective complaints, 2. NOSPECS-classification, 3. exophthalmometry, 4. by photo. The observation period was at least 12 months. The measured dosage to the thyroid gland was on average 210 +/- 80 Gy. In 57 cases no EO and in 46 cases an EO of grades I to IV before therapy occurred. RESULTS: In 11 cases there was an improvement and in 8 cases a worsening of the EO. In 84 cases the EO findings remained unchanged. Our data pointed towards the fact that with an achieved dosage to the thyroid of less than 200 Gy, a pretherapeutic thyroid gland volume greater than 55 ml, or implementation of RITH with preexisting EO without accompaning cortison therapy can worsen the EO findings. CONCLUSION: In comparison to surgical or thyreostatic therapy there was no increased risk of EO during or subsequent to RITH under cortison.

Importance of tactile and olfactory cues to the inhibition of the grasshopper mouse's attack through toxicosis.

This study examined the effect that toxicosis paired with the presence of a distinctive texture had on the inhibition of the grasshopper mouse's predatory attack. The first experiment measured the context in which tactile cues would be most effective by presenting prey with various combinations of added stimuli. The combination of distinctive tactile, olfactory, and gustatory cues produced the longest lasting inhibition. Because certain manipulations also had an unintended weak odor associated with them, the second experiment measured the importance of a weak or strong odor to inhibition of a mouse's attack. A combination of strong odor and distinctive texture paired with toxicosis inhibited an attack more effectively than a weak odor and a similar texture. In a third experiment, toxicosis was paired with an acraea moth caterpillar which has a highly distinctive texture. This produced the longest lasting inhibition of mouse's attack observed thus far. The ecological significance of a combination of a distinctive texture and odor to the inhibition of the grasshopper mouse's attack is discussed.

Nerve conduction studies and vibration perception thresholds in diabetic and uremic neuropathy.

Two parameters of nerve conduction studies (nerve conduction velocities and amplitudes of the evoked sensory and motor responses) were compared with quantitative vibration perception thresholds (VPT) in patients with peripheral neuropathy (diabetes mellitus and/or end-stage renal disease). VPT measurements were made using a "two-alternative, forced-choice" method in which the patient is required to identify which of the two rods is vibrating at progressively decreasing vibration intensities. VPTs correlated significantly with nerve conduction velocities in all upper and lower extremity sensory and motor nerves tested, and with the amplitudes of the evoked motor responses in three motor nerves: median and ulnar (motor components) and tibial. For the median and ulnar nerves (motor components) the amplitudes of the evoked motor responses were more sensitive than nerve conduction velocities in correlation with VPTs. Comparison of VPT values, based upon whether or not evoked sensory and motor responses were obtained, indicated that mean VPTs were consistently higher among subjects in whom these evoked responses were not elicited. VPT measurements is thus shown to be a valid and valuable method for evaluation of severity in peripheral neuropathy. It has the advantages of being simple, quick and painless. Patient cooperation and compliance with this form of testing are excellent.

Unique anti-human immunodeficiency virus activities of the nonnucleoside reverse transcriptase inhibitors calanolide A, costatolide, and dihydrocostatolide.

(+)-Calanolide A (NSC 650886) has previously been reported to be a unique and specific nonnucleoside inhibitor of the reverse transcriptase (RT) of human immunodeficiency virus (HIV) type 1 (HIV-1) (M. J. Currens et al., J. Pharmacol. Exp. Ther., 279:645-651, 1996). Two isomers of calanolide A, (-)-calanolide B (NSC 661122; costatolide) and (-)-dihydrocalanolide B (NSC 661123; dihydrocostatolide), possess antiviral properties similar to those of calanolide A. Each of these three compounds possesses the phenotypic properties ascribed to the pharmacologic class of nonnucleoside RT inhibitors (NNRTIs). The calanolide analogs, however, exhibit 10-fold enhanced antiviral activity against drug-resistant viruses that bear the most prevalent NNRTI resistance that is engendered by amino acid change Y181C in the RT. Further enhancement of activity is observed with RTs that possess the Y181C change together with mutations that yield resistance to AZT. In addition, enzymatic inhibition assays have demonstrated that the compounds inhibit RT through a mechanism that affects both the K(m) for dTTP and the V(max), i.e., mixed-type inhibition. In fresh human cells, costatolide and dihydrocostatolide are highly effective inhibitors of low-passage clinical virus strains, including those representative of the various HIV-1 clade strains, syncytium-inducing and non-syncytium-inducing isolates, and T-tropic and monocyte-tropic isolates. Similar to calanolide A, decreased activities of the two isomers were observed against viruses and RTs with amino acid changes at residues L100, K103, T139, and Y188 in the RT, although costatolide exhibited a smaller loss of activity against many of these NNRTI-resistant isolates. Comparison of cross-resistance data obtained with a panel of NNRTI-resistant virus strains suggests that each of the three stereoisomers may interact differently with the RT, despite their high degree of structural similarity. Selection of viruses resistant to each of the three compounds in a variety of cell lines yielded viruses with T139I, L100I, Y188H, or L187F amino acid changes in the RT. Similarly, a variety of resistant virus strains with different amino acid changes were selected in cell culture when the calanolide analogs were used in combination with other active anti-HIV agents, including nucleoside and nonnucleoside RT and protease inhibitors. In assays with combinations of anti-HIV agents, costatolide exhibited synergy with these anti-HIV agents. The calanolide isomers represent a novel and distinct subgroup of the NNRTI family, and these data suggest that a compound of the calanolide A series, such as costatolide, should be evaluated further for therapeutic use in combination with other anti-HIV agents.