The role of GRAS-domain proteins in arbuscular mycorrhizal symbiosis

A major limitation to plant growth is the restricted access to nutrients in the soil. To improve nutrient acquisition, the majority of land plants enter a beneficial symbiosis with arbuscular mycorrhizal (AM) fungi. The accommodation of fungal hyphae in roots requires the extensive transcriptional reprogramming of host cells. Several GRASdomain proteins, including NSP1 (NODULATION SIGNALLING PATHWAY 1), NSP2, and RAM1 (REQUIRED FOR ARBUSCULAR MYCORRHIZATION 1), have emerged as important transcriptional regulators during mycorrhization. Interaction studies suggest that these proteins form multicomponent complexes, raising the question whether they regulate similar or different mycorrhizal processes. Here, the functions of NSP1, NSP2 and RAM1 during AM development were investigated by detailed phenotypic and transcriptional analyses of the corresponding loss‐of‐function mutants. Global gene expression profiling of nsp1‐1 revealed that NSP1 is required for the expression of a large number of genes involved in strigolactone and gibberellin biosynthesis at the pre‐contact stage of AM development. Strigolactones are known to attract the fungus to the root. In line with this, the quantification of mycorrhizal structures showed a delay in mycorrhization in nsp1‐1. Transcriptional profiling confirmed that the expression of the majority of mycorrhizal‐induced genes was delayed, but not abolished in nsp1‐1, suggesting that NSP1 only has a minor role in the transcriptional regulation once the contact between the fungus and the roots has been established. Unlike NSP1, RAM1 plays a critical role in the transcriptional regulation at later stages of AM symbiosis. Mycorrhization was strongly impaired in ram1‐1, and transcriptional profiling revealed that RAM1 is essential for the expression of several genes involved in arbuscule development and the nutrient exchange between the symbionts. Meanwhile, the exact function of NSP2 remains unclear, as no effect on mycorrhization was observed in nsp2‐2 under the conditions tested here. These findings suggest that NSP1, NSP2 and RAM1 play largely different roles in the transcriptional regulation during AM development

Intervention effects maintenance: 6-month randomized controlled trial follow-up of standard and reflexive pelvic floor muscle training

BACKGROUND To date, the focus of pelvic floor muscle training for women suffering from stress urinary incontinence has been on voluntary contractions although involuntary pelvic floor muscle contractions are crucial to guarantee continence in high-impact situations typically triggering this condition. The authors developed 2 pelvic floor muscle home training programs, one including standard voluntary pelvic floor muscle training and one including involuntary reflexive pelvic floor muscle training. OBJECTIVE This study aimed to test 2 pelvic floor muscle home training programs regarding maintenance of effects of a previous 16-week intervention in terms of stress urinary incontinence symptoms (International Consultation on Incontinence Modular Questionnaire—Urinary Incontinence short form, modified 20-minute pad test), impact on quality of life (Lower Urinary Tract Symptoms Quality of Life module), and digitally assessed pelvic floor muscle strength. STUDY DESIGN This trial was a continuation of a previously published triple-blind prospective randomized controlled trial with a 6-month evaluation endpoint with 2 intervention groups (experimental group with involuntary reflexive home pelvic floor muscle training and control group with standard voluntary home pelvic floor muscle training). RESULTS From the originally included 96 randomized and allocated participants (experimental group=46, control group=46), 33 control and 27 experimental participants completed the 6-month follow-up. From post–16-week physiotherapy intervention to 6-month follow-up (home pelvic floor muscle training), there were statistically significant improvements in pelvic floor muscle strength (control and experimental group), and no difference in the International Consultation on Incontinence Modular Questionnaire—Urinary Incontinence short form and pad test, or the Lower Urinary Tract Symptoms Quality of Life module Part B (control and experimental group) and Part A (control group). However, there was a statistically significant improvement in the Lower Urinary Tract Symptoms Quality of Life module Part A (experimental group). At no point in time (pre, post, follow-up) was there any statistically significant difference between the groups. CONCLUSION Both groups could maintain their intervention training effects. This trial investigated involuntary reflexive pelvic floor muscle training alone, which proved to be an effective alternative to standard voluntary pelvic floor muscle training for maintenance of training effects among women suffering from stress urinary incontinence

Fluorescent reporters for functional analysis in rice leaves.

Fluorescent reporters have facilitated non-invasive imaging in multiple plant species and thus allowed the analysis of processes ranging from gene expression and protein localization to cellular patterning. However, in rice, a globally important crop and model species, there are relatively few reports of fluorescent proteins being used in leaves. Fluorescence imaging is particularly difficult in the rice leaf blade, likely due to a high degree of light scattering in this tissue. To address this, we investigated approaches to improve deep imaging in mature rice leaf blades. We found that ClearSee treatment, which has previously been used to visualize fluorescent reporters in whole tissues of plants, led to improved imaging in rice. Removing epidermal and subtending mesophyll cell layers was faster than ClearSee and also reduced light scattering such that imaging of fluorescent proteins in deeper leaf layers was possible. To expand the range of fluorescent proteins suitable for imaging in rice, we screened twelve whose spectral profiles spanned most of the visible spectrum. This identified five proteins (mTurquoise2, mNeonGreen, mClover3, mKOκ, and tdTomato) that are robustly expressed and detectable in mesophyll cells of stably transformed plants. Using microparticle bombardment, we show that mTurquoise2 and mNeonGreen can be used for simultaneous multicolor imaging of different subcellular compartments. Overall, we conclude that mTurquoise2, mNeonGreen, mClover3, mKOκ, and tdTomato are suitable for high-resolution live imaging of rice leaves, both after transient and stable transformation. Along with the rapid microparticle bombardment method, which allows transient transformation of major cell types in the leaf blade, these fluorescent reporters should greatly facilitate the analysis of gene expression and cell biology in rice

Insulin-like Growth Factor I—Releasing Alginate-Tricalciumphosphate Composites for Bone Regeneration

Purpose: Development and characterization of an in situ-forming, osteoconductive, and growth factor-releasing bone implant. Methods: Injectable in situ-forming scaffolds were prepared from a 2% (m/v) alginate solution, tricalciumphosphate (TCP) granules, and poly(lactide-co-glycolide) microspheres (MS), loaded with the osteoinductive growth factor insulin-like growth factor I (IGF-I). Scaffolds were prepared by mixing the components followed by hydrogel formation through calcium carbonate-induced physical cross-linking of the alginate at slightly acidic pH. Physical-chemical properties and cell biocompatibility using osteoblast-like cells (MG-63 and Saos-2) of these scaffolds were investigated. Results: The addition of TCP to the alginate resulted in reduced swelling and gelation time and an increase in stiffness. Osteoblast-like cells (MG-63 and Saos-2) did not show toxic reactions and adhered circumferentially to the TCP granules surface. The addition of the IGF-I MS resulted in an up to sevenfold increased proliferation rate of MG-63 cells as compared to scaffold preparations without IGF-I MS. The alkaline phosphate (ALP) activity—a parameter for osteblastic activity—increased with increasing amounts of TCP in Saos-2 loaded composite scaffolds. Conclusions: A prototype in situ-hardening composite system for conformal filling of bone defects supporting osteoblastic activity for further clinical testing in relevant fracture models was developed and characterize

Transforming traditional physiotherapy hands-on skills teaching into video-based learning.

BACKGROUND Pandemic-induced restrictions forced curriculum transformation from on-site education to virtual learning options. This report describes this transition, the challenge of creating technology-enhanced learning for hands-on psychomotor skills teaching in physiotherapy, and students' evaluations of the new technology-enhanced learning approach in Complex Decongestive Physiotherapy. METHODS On-site theoretical background lectures were replaced with e-learning sessions. Faculty hands-on skills demonstrations for the entire class were replaced with video-recorded demonstrations. Videos included verbal and written instructions and were complemented with checklists guiding the students, training in pairs, through their learning tasks. A cross-sectional observational survey for teaching quality evaluated this new technology-enhanced learning approach and assessed students' preference for traditional or video-based hands-on skills learning. RESULTS Survey return rate was > 50% (46 participating students). Teaching quality was rated between 1.5 ± 0.5 and 1.8 ± 0.4 (Likert scale from - 2 to + 2). Most students (66.7%) preferred the new approach. They appreciated for example that videos were available all the time, enabling self-paced learning, providing an equally good view on skills demonstrations, and the convenience to be able to rewind, re-view, and use speed adjustment options. CONCLUSIONS Students preferred the new video-based learning of skills for Complex Decongestive Physiotherapy. Because in-class live skills demonstrations were omitted, faculty had more time to provide individual feedback and answer questions. The shift from teacher- to student-centered learning enabled students to control their own learning pace. The innovative program was maintained after pandemic-induced restrictions were lifted. The success of this approach should be tested in other physiotherapy settings and different educational institutions

Pharmakokinetik systemischer, regionaler und lokaler Medikamente in der Therapie intraokularer Entzündungen

Zusammenfassung: In der Therapie intraokularer Entzündungen gibt es ein breites Spektrum entzündungshemmender Medikamente mit unterschiedlichen Wirkmechanismen. Die mit Abstand häufigste Anwendung finden Kortikosteroide. Die Mechanismen sind bei den meisten Medikamenten vor allem aus tierexperimentellen Studien recht gut verstanden. Schaut man sich demgegenüber die pharmakokinetische Evidenz dieser Medikamente am Auge an, so ist man überrascht, wie wenig fundierte Daten und Informationen es hierzu für das menschliche Auge im Allgemeinen und das entzündete Auge im Besonderen gibt. Denn es ist zu erwarten, dass die Bioverfügbarkeit eines Medikaments in einem entzündeten Auge durch die Schrankenstörung anders ist und schneller niedrigere intraokulare Wirkspiegel als in einem nicht entzündeten Auge durch den besseren Abtransport entstehen können. Dieser Beitrag soll einen aktuellen Überblick zu dieser Problematik und den aktuellen Kenntnisstand zunächst für Lokaltherapeutika, dann intravitreal/parabulbär verabreichte Medikamente und schließlich der immunsuppressiven Systemtherapie vermitteln

Pharmacological Validation of Trypanosoma brucei Phosphodiesterases as Novel Drug Targets

The development of drugs for neglected infectious diseases often uses parasite-specific enzymes as targets. We here demonstrate that parasite enzymes with highly conserved human homologs may represent a promising reservoir of new potential drug targets. The cyclic nucleotide-specific phosphodiesterases (PDEs) of Trypanosoma brucei, causative agent of the fatal human sleeping sickness, are essential for the parasite. The highly conserved human homologs are well-established drug targets. We here describe what is to our knowledge the first pharmacological validation of trypanosomal PDEs as drug targets. High-throughput screening of a proprietary compound library identified a number of potent hits. One compound, the tetrahydrophthalazinone compound A (Cpd A), was further characterized. It causes a dramatic increase of intracellular cyclic adenosine monophosphate (cAMP). Short-term cell viability is not affected, but cell proliferation is inhibited immediately, and cell death occurs within 3 days. Cpd A prevents cytokinesis, resulting in multinucleated, multiflagellated cells that eventually lyse. These observations pharmacologically validate the highly conserved trypanosomal PDEs as potential drug target

In vitro and in vivo investigation of bisphosphonate-loaded hydroxyapatite particles for peri-implant bone augmentation

Locally applied bisphosphonates, such as Zoledronate, have been shown in several studies to inhibit peri-implant bone resorption and recently to enhance peri-implant bone formation. Studies have also demonstrated positive effects of hydroxyapatite particles on peri-implant bone regeneration and an enhancement of the anti-resorptive effect of bisphosphonates in the presence of calcium. In the present study, both hydroxyapatite nanoparticles (nHA) and Zoledronate were combined to achieve a strong reinforcing effect on peri-implant bone. The nHA-Zoledronate-combination was first investigated in vitro with a pre-osteoclastic cell assay (RAW 264.7) and then in vivo in a rat model of postmenopausal osteoporosis. The in vitro study confirmed that the inhibitory effect of Zoledronate on murine osteoclast precursor cells was enhanced by loading the drug on nHA. For the in vivo investigation, either Zoledronate-loaded nHA or pure nHA were integrated in hyaluronic acid hydrogel. The gels were injected in screw holes that were predrilled in rat femoral condyles before insertion of miniature screws. MicroCT-based dynamic histomorphometry and histology revealed an unexpected rapid mineralization of the hydrogel in vivo through formation of granules, which served as scaffold for new bone formation. The delivery of Zoledronate-loaded nHA further inhibited a degradation of the mineralized hydrogel as well as a resorption of the peri-implant bone as effectively as unbound Zoledronate. Hyaluronic acid with Zoledronate-loaded nHA, thanks to its dual effect on inducing a rapid mineralization and preventing resorption, is a promising versatile material for bone repair and augmentation

Intra-session test-retest reliability of pelvic floor muscle electromyography during running

Introduction and hypothesis: The prevalence of female stress urinary incontinence is high, and young adults are also affected, including athletes, especially those involved in "high-impact” sports. To date there have been almost no studies testing pelvic floor muscle (PFM) activity during dynamic functional whole body movements. The aim of this study was the description and reliability test of PFM activity and time variables during running. Methods: A prospective cross-sectional study including ten healthy female subjects was designed with the focus on the intra-session test-retest reliability of PFM activity and time variables during running derived from electromyography (EMG) and accelerometry. Results: Thirteen variables were identified based on ten steps of each subject: Six EMG variables showed good reliability (ICC 0.906-0.942) and seven time variables did not show good reliability (ICC 0.113-0.731). Time variables (e.g. time difference between heel strike and maximal acceleration of vaginal accelerator) showed low reliability. However, relevant PFM EMG variables during running (e.g., pre-activation, minimal and maximal activity) could be identified and showed good reliability. Conclusion: Further adaptations regarding measurement methods should be tested to gain better control of the kinetics and kinematics of the EMG probe and accelerometers. To our knowledge this is the first study to test the reliability of PFM activity and time variables during dynamic functional whole body movements. More knowledge of PFM activity and time variables may help to provide a deeper insight into physical strain with high force impacts and important functional reflexive contraction patterns of PFM to maintain or to restore continenc