Search

In large animals, fractures of the upper long tubular bones such as forearm and humerus as well as lower and upper leg, continue to represent a major surgical challenge. An implant and a corresponding surgical procedure for intramedullary osteosynthesis in fractures of long bones were developed at fzmb GmbH. Fixation of the implanted nail to the distal drill holes has proven to be difficult and requires a high level of X-ray monitoring. Therefore, a camera-marker system (CMS) was developed without the need for X-ray monitoring during drilling. The test was performed on 10 equine femora. The results showed that 8 of 10 implants could be securely fixed and no complications occurred in the experimental procedure. For the first two implants, the distal drill holes could not be hit in the first attempt. This is due to the circumstance, that the handling of the CMS needs to be practiced to hit the distal drill holes accurately and appropriately, resulting in a hit rate of 80 %. However, the described method has great potential and does not require X-ray exposure.

The cranial cruciate ligament (CCL) rupture is a prevalent injury in dogs. A consequence of a cruciate ligament rupture is instability in the affected knee joint. A veterinary, mostly surgical treatment of the cruciate ligament rupture is usually unavoidable. The suitability of an arthroscopic surgical method with ligament replacement material was investigated. The stability of the knee joint was determined several times during 1,200 passive robotic motion cycles with movement radius between 90° flexion and 140° extension. The stability condition was measured by triggering the drawer test. After 300 motion cycles, the drawer test could be triggered (positive drawer test). In the following movement cycles up to 1,200 cycles, the drawer test could also be triggered. However, no significant differences occurred between these triggered drawer tests. The ligament replacement material showed no damage and no loosening after the tests. The first results showed that the developed arthroscopic surgical method could be a promising approach for the surgical treatment of cruciate ligament ruptures in canines.

Gamma delta (γδ) T cells represent a minor fraction of human T cell repertoire but play an important role in mediating anti-infectious and anti-tumorous effects in the context of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We performed a prospective study to analyze the effect of different transplant modalities on immune reconstitution of γδ T cells and subsets. CD3, CD4 and CD8 T cells were analyzed in parallel. Secondly, we examined the impact of γδ T cell reconstitution on clinical outcomes including acute Graft-versus-Host-Disease (aGvHD) and viral infections. Our cohort includes 49 pediatric patients who received unmanipulated bone marrow grafts from matched unrelated (MUD) or matched related (MRD) donors. The cohort includes patients with malignant as well as non-malignant diseases. Cell counts were measured using flow cytometry at 15, 30, 60, 100, 180 and 240 days after transplantation. Cells were stained for CD3, CD4, CD8, CD45, TCR αβ , TCRγδ, TCRVδ1, TCRVδ2, HLA-DR and combinations. Patients with a MRD showed significantly higher Vδ2+ T cells than those with MUD at timepoints +30, +60, +100 (p<0.001, respectively) and +180 (p<0.01) in univariate analysis. These results remained significant in multivariate analysis. Patients recovering with a high relative abundance of total γδ T cells and Vδ2+ T cells had a significantly lower cumulative incidence of grade II-IV aGvHD after transplantation (p=0.03 and p=0.04, respectively). A high relative abundance of Vδ2+ T cells was also associated with a lower incidence of EBV infection (p=0.02). Patients with EBV infection on the other hand showed higher absolute Vδ1+ T cell counts at days +100 and +180 after transplantation (p=0.046 and 0.038, respectively) than those without EBV infection. This result remained significant in a multivariate time-averaged analysis (q<0.1). Our results suggest a protective role of γδ T cells and especially Vδ2+ T cell subset against the development of aGvHD and EBV infection after pediatric HSCT. Vδ1+ T cells might be involved in the immune response after EBV infection. Our results encourage further research on γδ T cells as prognostic markers after HSCT and as possible targets of adoptive T cell transfer strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Müller, Alasfar, Preuß, Zimmermann, Streitz, Hundsdörfer, Eggert, Schulte, von Stackelberg and Oevermann.)

Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well as protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinson's disease has led to an intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its cellular activity are needed. Here, we report the identification and characterization of the first high-affinity LRRK2-binding designed ankyrin-repeat protein (DARPin), named E11. Using cryo-EM, we show that DARPin E11 binds to the LRRK2 WD40 domain. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase., Competing Interests: Conflicts of interest SRP is a consultant for Schrodinger and Stoke Therapeutics. AP is a co-founder and shareholder of Molecular Partners who are commercializing the DARPin technology. All other authors do not have any conflicts to report with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Introduction: Aging studies in humans and mice have played a key role in understanding the intestinal microbiome and an increased abundance of "inflammaging" Gram-negative (Gn) bacteria. The mechanisms underlying this inflammatory profile in the aging microbiome are unknown. We tested the hypothesis that an aging-related decrease in colonic crypt epithelial cell anti-microbial peptide (AMP) gene expression could promote colonic microbiome inflammatory Gn dysbiosis and inflammaging. Methods: As a model of aging, C57BL/6J mice fecal (colonic) microbiota (16S) and isolated colonic crypt epithelial cell gene expression (RNA-seq) were assessed at 2 months (mth) (human: 18 years old; yo), 15 mth (human: 50 yo), and 25 mth (human: 84 yo). Informatics examined aging-related microbial compositions, differential colonic crypt epithelial cell gene expressions, and correlations between colonic bacteria and colonic crypt epithelial cell gene expressions. Results: Fecal microbiota exhibited significantly increased relative abundances of pro-inflammatory Gn bacteria with aging. Colonic crypt epithelial cell gene expression analysis showed significant age-related downregulation of key AMP genes that repress the growth of Gn bacteria. The aging-related decrease in AMP gene expressions is significantly correlated with an increased abundance in Gn bacteria (dysbiosis), loss of colonic barrier gene expression, and senescence- and inflammation-related gene expression. Conclusion: This study supports the proposed model that aging-related loss of colonic crypt epithelial cell AMP gene expression promotes increased relative abundances of Gn inflammaging-associated bacteria and gene expression markers of colonic inflammaging. These data may support new targets for aging-related therapies based on intestinal genes and microbiomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Forsyth, Shaikh, Engen, Preuss, Naqib, Palmen, Green, Zhang, Bogin, Lawrence, Sharma, Swanson, Bishehsari, Voigt and Keshavarzian.)

The evolutionarily conserved circadian system allows organisms to synchronize internal processes with 24-h cycling environmental timing cues, ensuring optimal adaptation. Like other organs, the pancreas function is under circadian control. Recent evidence suggests that aging by itself is associated with altered circadian homeostasis in different tissues which could affect the organ's resiliency to aging-related pathologies. Pancreas pathologies of either endocrine or exocrine components are age-related. Whether pancreas circadian transcriptome output is affected by age is still unknown. To address this, here we profiled the impact of age on the pancreatic transcriptome over a full circadian cycle and elucidated a circadian transcriptome reorganization of pancreas by aging. Our study highlights gain of rhythms in the extrinsic cellular pathways in the aged pancreas and extends a potential role to fibroblast-associated mechanisms.

Malfunction of the actin cytoskeleton is linked to numerous human diseases including neurological disorders and cancer. LIMK1 (LIM domain kinase 1) and its paralogue LIMK2 are two closely related kinases that control actin cytoskeleton dynamics. Consequently, they are potential therapeutic targets for the treatment of such diseases. In the present review, we describe the LIMK conformational space and its dependence on ligand binding. Furthermore, we explain the unique catalytic mechanism of the kinase, shedding light on substrate recognition and how LIMK activity is regulated. The structural features are evaluated for implications on the drug discovery process. Finally, potential future directions for targeting LIMKs pharmacologically, also beyond just inhibiting the kinase domain, are discussed.

Unc-51-like kinase 4 (ULK4) is a pseudokinase that has been linked to the development of several diseases. Even though sequence motifs required for ATP binding in kinases are lacking, ULK4 still tightly binds ATP and the presence of the co-factor is required for structural stability of ULK4. Here, we present a high-resolution structure of a ULK4-ATPγS complex revealing a highly unusual ATP binding mode in which the lack of the canonical VAIK motif lysine is compensated by K39, located N-terminal to αC. Evolutionary analysis suggests that degradation of active site motifs in metazoan ULK4 has co-occurred with an ULK4-specific activation loop, which stabilizes the C helix. In addition, cellular interaction studies using BioID and biochemical validation data revealed high confidence interactors of the pseudokinase and armadillo repeat domains. Many of the identified ULK4 interaction partners were centrosomal and tubulin-associated proteins and several active kinases suggesting interesting regulatory roles for ULK4., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Despite their apparent lack of catalytic activity, pseudokinases are essential signaling molecules. Here, we describe the structural and dynamic properties of pseudokinase domains from the Wnt-binding receptor tyrosine kinases (PTK7, ROR1, ROR2, and RYK), which play important roles in development. We determined structures of all pseudokinase domains in this family and found that they share a conserved inactive conformation in their activation loop that resembles the autoinhibited insulin receptor kinase (IRK). They also have inaccessible ATP-binding pockets, occluded by aromatic residues that mimic a cofactor-bound state. Structural comparisons revealed significant domain plasticity and alternative interactions that substitute for absent conserved motifs. The pseudokinases also showed dynamic properties that were strikingly similar to those of IRK. Despite the inaccessible ATP site, screening identified ATP-competitive type-II inhibitors for ROR1. Our results set the stage for an emerging therapeutic modality of "conformational disruptors" to inhibit or modulate non-catalytic functions of pseudokinases deregulated in disease., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Alcohol increases the risk of developing colon cancer (CRC), in part via tissue inflammation and impaired barrier integrity. Circadian dyssynchrony (CD) is an understudied but common lifestyle associated factor that increases the risk of multi-organ tissue injury and number of malignancies including CRC. Our prior studies showed that the shift in light-dark cycle exacerbates barrier dysfunction and colonic inflammation in the setting of alcohol treatment, and increases the risk of CRC. Here we studied the interaction of alcohol with an abnormal eating pattern on markers of CD and colonic barrier integrity., Method: Mice were subjected to day (rest-phase = wrong-time WT) or night-time (active-phase = right-time RT) access to food in combination with access to water or 15% alcohol for total duration of 10 weeks. The food and liquid intake was measured. The locomotor activity data was recorded throughout the study, using a beam-break system. Mice were euthanized at two time points (ZT2 and ZT14). Time variation in the expression of the molecular marker of circadian clock (per2 gene) was measured in the central (hypothalamus) and intestinal (colon) tissue. Colonic protein expression of barrier markers (Occludin and Claudin-1) was studied., Results: No significant differences were present in the weight gain and alcohol intake among the groups over the study period. We observed an interaction of WT eating with alcohol on behavioral markers of circadian rhythm. Compared to the RT + Water treated animals ("reference group"), combination of WT eating and alcohol consumption (WT + Alcohol) significantly changed the per2 oscillatory pattern, that was different between the colon and hypothalamus, indicative of worsening circadian dyssynchrony. This was associated with an overall impaired expression of barrier integrity markers in the colon., Conclusions: Alcohol induces circadian dyssynchrony which is worsened by abnormal food timing, associated with impaired barrier integrity in the colon. Future studies on the interaction of alcohol and food timing could provide further insights into alcohol associated CRC pathophysiology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The consumption of dietary fibers has been implicated with a lowered risk of human colorectal cancer. Proposed mechanisms involve alterations in the stool consistency, transit time, and formation of short-chain fatty acid by dietary fiber fermentation, and the reorganization of gut microbiota. Here we show that Fibersol-2, a digest-resistant maltodextrin, not only inhibits proliferation of colorectal SW480 cancer cell lines by increasing reactive oxygen species (ROS), but decreases the numbers of the adenoma count in Multiple Intestinal Neoplasia (MIN) mice carrying a mutation in the Adenomatous Polyposis Coli gene by 84 d of age. These observations provide direct evidence that Fibersol-2 intrinsically contains anti-cancer activity, independent of the intestinal metabolism and any potential interactions with the microbiota.

Despite decades of research in defining sleep-wake properties in mammals, little is known about the nature or identity of genes that regulate sleep, a fundamental behaviour that in humans occupies about one-third of the entire lifespan. While genome-wide association studies in humans and quantitative trait loci (QTL) analyses in mice have identified candidate genes for an increasing number of complex traits and genetic diseases, the resources and time-consuming process necessary for obtaining detailed quantitative data have made sleep seemingly intractable to similar large-scale genomic approaches. Here we describe analysis of 20 sleep-wake traits from 269 mice from a genetically segregating population that reveals 52 significant QTL representing a minimum of 20 genomic loci. While many (28) QTL affected a particular sleep-wake trait (e.g., amount of wake) across the full 24-hr day, other loci only affected a trait in the light or dark period while some loci had opposite effects on the trait during the light vs. dark. Analysis of a dataset for multiple sleep-wake traits led to previously undetected interactions (including the differential genetic control of number and duration of REM bouts), as well as possible shared genetic regulatory mechanisms for seemingly different unrelated sleep-wake traits (e.g., number of arousals and REM latency). Construction of a Bayesian network for sleep-wake traits and loci led to the identification of sub-networks of linkage not detectable in smaller data sets or limited single-trait analyses. For example, the network analyses revealed a novel chain of causal relationships between the chromosome 17@29cM QTL, total amount of wake, and duration of wake bouts in both light and dark periods that implies a mechanism whereby overall sleep need, mediated by this locus, in turn determines the length of each wake bout. Taken together, the present results reveal a complex genetic landscape underlying multiple sleep-wake traits and emphasize the need for a systems biology approach for elucidating the full extent of the genetic regulatory mechanisms of this complex and universal behavior.

Mutations lowering the kinase activity of Drosophila Doubletime (DBT) and vertebrate casein kinase Iepsilon/delta (CKIepsilon/delta) produce long-period, short-period, and arrhythmic circadian rhythms. Since most ckI short-period mutants have been isolated in mammals, while the long-period mutants have been found mostly in Drosophila, lowered kinase activity may have opposite consequences in flies and vertebrates, because of differences between the kinases or their circadian mechanisms. However, the results of this article establish that the Drosophila dbt mutations have similar effects on period (PER) protein phosphorylation by the fly and vertebrate enzymes in vitro and that Drosophila DBT has an inhibitory C-terminal domain and exhibits autophosphorylation, as does vertebrate CKIepsilon/delta. Moreover, expression of either Drosophila DBT or the vertebrate CKIdelta kinase carrying the Drosophila dbt(S) or vertebrate tau mutations in all circadian cells leads to short-period circadian rhythms. By contrast, vertebrate CKIdelta carrying the dbt(L) mutation does not lengthen circadian rhythms, while Drosophila DBT(L) does. Different effects of the dbt(S) and tau mutations on the oscillations of PER phosphorylation suggest that the mutations shorten the circadian period differently. The results demonstrate a high degree of evolutionary conservation of fly and vertebrate CKIdelta and of the functions affected by their period-shortening mutations.

Continuous disruption of circadian rhythms, as seen in human shift workers, has been associated with the development of a number of adverse mental and physiological conditions. However, scientific evidence linking circadian disruption to overall health, particularly in animal models, is not well documented. In this study, we have demonstrated that exposing C57BL/6J mice to 12-h phase shifts every 5 days for 3 mo had no effect on body weight or intestinal physiology. However, when animals were further challenged with dextran sodium sulfate to induce colitis, chronic shifting of the light-dark cycle led to a dramatic increase in the progression of the colitis as indicated by reduced body weight, abnormal intestinal histopathology, and an exacerbated inflammatory response. These data indicate that circadian disruption is an important predisposing factor that may provoke the onset or worsening of various disease states such as inflammatory disorders. This study provides further evidence for continued investigations using animal models of circadian disruption to examine the consequences of circadian disruption on health when organisms are faced with a "challenging" environment.

A mutation (K38R) which specifically eliminates kinase activity was created in the Drosophila melanogaster ckI gene (doubletime [dbt]). In vitro, DBT protein carrying the K38R mutation (DBT(K/R)) interacted with Period protein (PER) but lacked kinase activity. In cell culture and in flies, DBT(K/R) antagonized the phosphorylation and degradation of PER, and it damped the oscillation of PER in vivo. Overexpression of short-period, long-period, or wild-type DBT in flies produced the same circadian periods produced by the corresponding alleles of the endogenous gene. These mutations therefore dictate an altered "set point" for period length that is not altered by overexpression. Overexpression of the DBT(K/R) produced effects proportional to the titration of endogenous DBT, with long circadian periods at lower expression levels and arrhythmicity at higher levels. This first analysis of adult flies with a virtual lack of DBT activity demonstrates that DBT's kinase activity is necessary for normal circadian rhythms and that a general reduction of DBT kinase activity does not produce short periods.

In both mammals and fruit flies, casein kinase I has been shown to regulate the circadian phosphorylation of the period protein (PER). This phosphorylation regulates the timing of PER's nuclear accumulation and decline, and it is necessary for the generation of circadian rhythms. In Drosophila melanogaster, mutations affecting a casein kinase I (CKI) ortholog called doubletime (dbt) can produce short or long periods. The effects of both a short-period (dbt(S)) and long-period (dbt(L)) mutation on DBT expression and biochemistry were analyzed. Immunoblot analysis of DBT in fly heads showed that both the dbt(S) and dbt(L) mutants express DBT at constant levels throughout the day. Glutathione S-transferase pull-down assays and coimmunoprecipitation of DBT and PER showed that wild-type DBT, DBT(S), and DBT(L) proteins can bind to PER equivalently and that these interactions are mediated by the evolutionarily conserved N-terminal part of DBT. However, both the dbt(S) and dbt(L) mutations reduced the CKI-7-sensitive kinase activity of an orthologous Xenopus laevis CKIdelta expressed in Escherichia coli. Moreover, expression of DBT in Drosophila S2 cells produced a CKI-7-sensitive kinase activity which was reduced by both the dbt(S) and dbt(L) mutations. Thus, lowered enzyme activity is associated with both short-period and long-period phenotypes.

The genome of bacteriophage P1 harbors a gene coding for a 162-amino-acid protein which shows 66% amino acid sequence identity to the Escherichia coli single-stranded DNA-binding protein (SSB). The expression of the P1 gene is tightly regulated by P1 immunity proteins. It is completely repressed during lysogenic growth and only weakly expressed during lytic growth, as assayed by an ssb-P1/lacZ fusion construct. When cloned on an intermediate-copy-number plasmid, the P1 gene is able to suppress the temperature-sensitive defect of an E. coli ssb mutant, indicating that the two proteins are functionally interchangeable. Many bacteriophages and conjugative plasmids do not rely on the SSB protein provided by their host organism but code for their own SSB proteins. However, the close relationship between SSB-P1 and the SSB protein of the P1 host, E. coli, raises questions about the functional significance of the phage protein.