Human checkpoint proteins hRad9, hHus1, and hRad1 form a DNA damage-responsive complex

Human cells have evolved protective mechanisms such as DNA repair and cell cycle checkpoints in order to promote stability of the genome. Studies on hereditary instability syndromes associated with a higher incidence of malignancies like Xeroderma pigmentosum or Nijmegen breakage syndrome demonstrated that genetic defects and subsequent dysfunction of a specific DNA repair mechanism trigger the development of cancer. Within the last years, the investigation of cell cycle checkpoints gained increasing importance in cancer research. Checkpoints are signaling cascades that halt the cell cycle in response to DNA damage, thereby providing time for repair and preventing accumulation of DNA alterations. While the p53-dependent G1-S checkpoint has been extensively investigated, little is known about other checkpoints in humans such as the G2-M or the S-phase progression checkpoint. Studies on the human cancer syndrome ataxia telangiectasia (AT) showed that AT cells fail to induce several checkpoints in response to ionizing radiation (IR), indicating that a checkpoint gene defect is responsible for the AT-associated cancers. The responsible gene (ATM) has significant sequence homology to the checkpoint kinase gene sprad3 in the fission yeast Schizosaccharomyces pombe (S. pombe). In S. pombe, spRad3 regulates G2-M checkpoint activation in response to DNA damage. Defects in the sprad3 gene, like defects in ATM, sensitize the organisms to radiation and radiomimetic drugs, suggesting conservation of checkpoint pathways from yeast to humans as well as a potential role of the G2-M checkpoint in carcinogenesis. The discovery of G2-M checkpoint-deficient yeast mutants led to the cloning of additional checkpoint genes in yeast and their human homologs. This group of novel human genes includes homologs of sprad9 (hRAD9), sphus1 (hHUS1), and sprad1 (hRAD1). In S. pombe, these genes are required for activation of spRad3, and defects in one or more of these genes render the yeast more sensitive to genotoxic agents. Mutations within the human rad genes may bring about an increased rate of mutations and genomic instability as shown for p53 or AT and may be responsible for inherited predisposition to cancer. In view of this potential importance of human rad genes in the process of carcinogenesis, we have undertaken a cellular and molecular analysis of the novel human checkpoint proteins hRad9, hHus1, and hRad1 in the leukemia cell line K562 and in human keratinocytes. Using specific antibodies to the hRad9, hHus1, and hRad1 proteins we demonstrated with co-immunoprecipitation and Western-blot experiments that the human checkpoint proteins hRad9, hHus1, and hRad1 associate in a biochemical complex similar to the spRad9-spHus1-spRad1 complex reported in fission yeast. To generate a model system of checkpoint protein function amenable to biochemical analysis, we prepared epitope-tagged expression vectors for hRad9, hHus1, and hRad1, which were transfected into K562 cells by electroporation, resulting in transient expression of epitope-tagged protein. By simultaneous expression of hRad9, hHus1, and hRad1 we showed that transiently expressed epitope-tagged checkpoint proteins hRad9, hHus1, and hRad1 recapitulate complex formation of endogenous proteins. Immunoprecipitation studies with lysates of hRad9-overexpressing cells revealed that hRad9 undergoes complex post-translational modifications. Co-expression of hRad9 with hHus1, and hRad1 resulted in a large increase of the amount of a highly modified form of hRad9, suggesting that hRad1 and hHus1 either promote formation of, or stabilize the modified form of hRad9. Previously, a direct correlation between checkpoint protein phosphorylation and activation of DNA damage checkpoints in yeast was proposed. In this study, we show that hRad9 is phosphorylated in response to DNA damage, and that phosphorylated hRad9 interacts with hHus1 and hRad1 as well. The present results suggest that the hRad9-hHus1-hRad1 complex actively participates in an evolutionarily conserved DNA damage-induced signaling cascade. hRad1 seems to possess exonuclease activity. The presence of a putative DNA-metabolizing protein in the multimolecular checkpoint complex, coupled with genetic data that place spRad9, spHus1, and spRad1 early in the response pathway of checkpoint activation suggests that the complex may function as a sensor that scans the genome for damaged DNA. Once damaged DNA is detected, this complex may initiate endonucleolytic processing of the lesions and trigger interactions with downstream signaling elements, or may link unknown damage recognition components to downstream signal-transducing pathways that include the ATM kinase, which is implicated in actively enforcing cell cycle arrest after DNA damage. Potential goals of checkpoint research include the implementation of screening tests to identify familial cancer predisposition and treatment of checkpoint gene defects by gene transfer. Another aim of checkpoint research is the development of checkpoint-based cancer therapy. More than 50% of all human malignant tumors contain mutated p53, and p53-deficient tumor cells lack induction of the G1-S checkpoint in response to DNA damage. One emerging hypothesis is that selective inhibitors of the compensating G2-M checkpoint would preferentially radiosensitize p53-deficient tumor cells. Thus, the investigation of checkpoint function in humans provides further targets for chemotherapeutic agents and will help to design future strategies in cancer therapy

Pathologic fracture of the distal radius in a 25-year-old patient with a large unicameral bone cyst

Background: Distal radius fractures (DRF) are often referred to as osteoporosis indicator fractures as their incidence increases from age 45. In the group of young adults, distal radius fractures normally result from high-energy trauma. Wrist fractures in young patients without adequate trauma thus raise suspicion of a pathologic fracture. In this report we present the case of a fractured unicameral bone cyst (UBC) at the distal radius in a young adult. To the author's best knowledge, this is the first detailed report in an UBC at the distal radius causing a pathologic DRF in an adult patient. Case presentation: A 25-year-old otherwise healthy male presented to our Emergency Department after a simple fall on his right outstretched hand. Extended diagnostics revealed a pathologic, dorsally displaced, intra-articular distal radius fracture secondary to a unicameral bone cyst occupying almost the whole metaphysis of the distal radius. To stabilize the fracture, a combined dorsal and volar approach was used for open reduction and internal fixation. A tissue specimen for histopathological examination was gathered and the lesion was filled with an autologous bone graft harvested from the ipsilateral femur using a reamer-irrigator-aspirator (RIA) system. Following one revision surgery due to an intra-articular step-off, the patient recovered without further complications. Conclusions: Pathologic fractures in young patients caused by unicameral bone cysts require extended diagnostics and adequate treatment. A single step surgical treatment is reasonable if fracture and bone cyst are treated appropriately. Arthroscopically assisted fracture repair may be considered in intra-articular fractures or whenever co-pathologies of the carpus are suspected

Differences in the Inflammatory Response of White Adipose Tissue and Adipose-Derived Stem Cells

The application of liposuctioned white adipose tissue (L-WAT) and adipose-derived stem cells (ADSCs) as a novel immunomodulatory treatment option is the currently subject of various clinical trials. Because it is crucial to understand the underlying therapeutic mechanisms, the latest studies focused on the immunomodulatory functions of L-WAT or ADSCs. However, studies that examine the specific transcriptional adaptation of these treatment options to an extrinsic inflammatory stimulus in an unbiased manner are scarce. The aim of this study was to compare the gene expression profile of L-WAT and ADSCs, when subjected to tumor necrosis factor alpha (TNF\textgreeka), and to identify key factors that might be therapeutically relevant when using L-WAT or ADSCs as an immuno-modulator. Fat tissue was harvested by liposuction from five human donors. ADSCs were isolated from the same donors and shortly subjected to expansion culture. L-WAT and ADSCs were treated with human recombinant TNF\textgreeka, to trigger a strong inflammatory response. Subsequently, an mRNA deep nextgeneration sequencing was performed to evaluate the different inflammatory responses of L-WAT and ADSCs. We found significant gene expression changes in both experimental groups after TNF\textgreeka incubation. However, ADSCs showed a more homogenous gene expression profile by predominantly expressing genes involved in immunomodulatory processes such as CCL19, CCL5, TNFSF15 and IL1b when compared to L-WAT, which reacted rather heterogeneously. As RNA sequencing between L-WAT and ADSCS treated with TNF\textgreeka revealed that L-WAT responded very heterogeneously to TNF\textgreeka treatment, we therefore conclude that ADSCs are more reliable and predictable when used therapeutically. Our study furthermore yields insight into potential biological processes regarding immune system response, inflammatory response, and cell activation. Our results can help to better understand the different immunomodulatory effects of L-WAT and ADSCs

Tenomodulin is essential for prevention of adipocyte accumulation and fibrovascular scar formation during early tendon healing

Tenomodulin (Tnmd) is the best-known mature marker for tendon and ligament lineage cells. It is important for tendon maturation, running performance and has key implications for the resident tendon stem/progenitor cells (TSPCs). However, its exact functions during the tendon repair process still remain elusive. Here, we established an Achilles tendon injury model in a Tnmd knockout (Tnmd(-/-)) mouse line. Detailed analyses showed not only a very different scar organization with a clearly reduced cell proliferation and expression of certain tendon-related genes, but also increased cell apoptosis, adipocyte and blood vessel accumulation in the early phase of tendon healing compared with their wild-type (WT) littermates. In addition, Tnmd(-/-) tendon scar tissue contained augmented matrix deposition of biglycan, cartilage oligomeric matrix protein (Comp) and fibronectin, altered macrophage profile and reduced numbers of CD146-positive cells. In vitro analysis revealed that Tnmd(-/-) TSPCs exhibited significantly reduced migration and proliferation potential compared with that of WT TSPCs. Furthermore, Tnmd(-/-) TSPCs had accelerated adipogenic differentiation accompanied with significantly increased peroxisome proliferator-activated receptor gamma (Ppar gamma) and lipoprotein lipase (Lpl) mRNA levels. Thus, our results demonstrate that Tnmd is required for prevention of adipocyte accumulation and fibrovascular scar formation during early tendon healing

<strong>CFA-2</strong> and <strong>CFA-3</strong> (Coordination Framework Augsburg University-2 and -3); novel MOFs assembled from trinuclear Cu(I)/Ag(I) secondary building units and 3,3′,5,5′-tetraphenyl-bipyrazolate ligands

The syntheses of H₂-phbpz, [Cu₂(phbpz)]·2DEF·MeOH (CFA-2) and [Ag₂(phbpz)] (CFA-3) (H₂-phbpz = 3,3′,5,5′-tetraphenyl-1H,1′H-4,4′-bipyrazole) compounds and their crystal structures are described. The Cu(I) containing metal–organic framework CFA-2 crystallizes in the tetragonal crystal system, within space group I4₁/a (no. 88) and the following unit cell parameters: a = 30.835(14), c = 29.306(7) Å, V = 27 865(19) ų. CFA-2 features a flexible 3-D three-connected two-fold interpenetrated porous structure constructed of triangular Cu(I) subunits. Upon exposure to different kinds of liquids (MeOH, EtOH, DMF, DEF) CFA-2 shows pronounced breathing effects. CFA-3 crystallizes in the monoclinic crystal system, within space group P2₁/c (no. 14) and the following unit cell parameters: a = 16.3399(3), b = 32.7506(4), c = 16.2624(3) Å, β = 107.382(2)°, V = 8305.3(2) ų. In contrast to the former compound, CFA-3 features a layered 2-D three-connected structure constructed from triangular Ag(I) subunits. Both compounds are characterized by elemental and thermogravimetric analyses, single crystal structure analysis and X-ray powder diffraction, FTIR- and fluorescence spectroscopy. Preliminary results on oxygen activation in CFA-2 are presented and potential improvements in terms of framework robustness and catalytic efficiency are discussed

Detailed analysis of surgically treated hand trauma patients in a regional German trauma centre.

Hand and forearm injuries are the most frequent reason for consultations in German emergency departments. Therefore, full recovery has a high social and economic relevance. In this study, data on surgically treated hand injuries in a regional German trauma centre between 01.01.2019 and 31.01.2021 were collected using the new German HandTraumaRegister of the German Society for Hand Surgery. These data were retrospectively analysed and correlated with mobility data of the Bavarian population, the 7-day incidence of Covid-19 infections in Germany and the number of elective hand surgeries. We found that a fall from standing height with consecutive distal radius fracture was the most common injury in women, whereas mechanism of injury and diagnosis were more diverse in men. The populations' mobility correlated well with the number of accidents, which in turn was reciprocal to the 7-day-incidence of Covid-19 infections. The number of elective hand surgeries expectedly dropped significantly during the state-imposed lockdowns. Knowing that mainly young men and elderly women suffer from hand injuries, tailored prevention measures may be elaborated. In order to reduce socioeconomic burden, care for hand injuries and elective hand surgeries must be guaranteed according to the frequency of their occurrence

Validation of a novel animal model for sciatic nerve repair with an adipose-derived stem cell loaded fibrin conduit

Despite the regenerative capabilities of peripheral nerves, severe injuries or neuronal trauma of critical size impose immense hurdles for proper restoration of neuro-muscular circuitry. Autologous nerve grafts improve re-establishment of connectivity, but also comprise substantial donor site morbidity. We developed a rat model which allows the testing of different cell applications, i.e., mesenchymal stem cells, to improve nerve regeneration in vivo. To mimic inaccurate alignment of autologous nerve grafts with the injured nerve, a 20 mm portion of the sciatic nerve was excised, and sutured back in place in reversed direction. To validate the feasibility of our novel model, a fibrin gel conduit containing autologous undifferentiated adipose-derived stem cells was applied around the coaptation sites and compared to autologous nerve grafts. After evaluating sciatic nerve function for 16 weeks postoperatively, animals were sacrificed, and gastrocnemius muscle weight was determined along with morphological parameters (g-ratio, axon density & diameter) of regenerating axons. Interestingly, the addition of undifferentiated adipose-derived stem cells resulted in a significantly improved re-myelination, axon ingrowth and functional outcome, when compared to animals without a cell seeded conduit. The presented model thus displays several intriguing features: it imitates a certain mismatch in size, distribution and orientation of axons within the nerve coaptation site. The fibrin conduit itself allows for an easy application of cells and, as a true critical-size defect model, any observed improvement relates directly to the performed intervention. Since fibrin and adipose-derived stem cells have been approved for human applications, the technique can theoretically be performed on humans. Thus, we suggest that the model is a powerful tool to investigate cell mediated assistance of peripheral nerve regeneration