A neuroskeletal atlas: Spatial mapping and contextualization of axon subtypes innervating the long bones of C3H and B6 mice

Nerves in bone play well-established roles in pain and vasoregulation and have been associated with progression of skeletal disorders, including osteoporosis, fracture, arthritis, and tumor metastasis. However, isolation of the region-specific mechanisms underlying these relationships is limited by our lack of quantitative methods for neuroskeletal analysis and precise maps of skeletal innervation. To overcome these limitations, we developed an optimized workflow for imaging and quantitative analysis of axons in and around the bone, including validation of Baf53b-Cre in concert with R26R-tdTomato (Ai9) as a robust pan-neuronal reporter system for use in musculoskeletal tissues. In addition, we created comprehensive maps of sympathetic adrenergic and sensory peptidergic axons within and around the full length of the femur and tibia in two strains of mice (B6 and C3H). In the periosteum, these maps were related to the surrounding musculature, including entheses and myotendinous attachments to bone. Three distinct patterns of periosteal innervation (termed type I, II, III) were defined at sites that are important for bone pain, bone repair, and skeletal homeostasis. For the first time, our results establish a gradient of bone marrow axon density that increases from proximal to distal along the length of the tibia and define key regions of interest for neuroskeletal studies. Lastly, this information was related to major nerve branches and local maps of specialized mechanoreceptors. This detailed mapping and contextualization of the axonal subtypes innervating the skeleton is intended to serve as a guide during the design, implementation, and interpretation of future neuroskeletal studies and was compiled as a resource for the field as part of the NIH SPARC consortium. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Neuroskeletal effects of chronic bioelectric nerve stimulation in health and diabetes

Background/Aims: Bioelectric nerve stimulation (eStim) is an emerging clinical paradigm that can promote nerve regeneration after trauma, including within the context of diabetes. However, its ability to prevent the onset of diabetic peripheral neuropathy (DPN) has not yet been evaluated. Beyond the nerve itself, DPN has emerged as a potential contributor to sarcopenia and bone disease; thus, we hypothesized that eStim could serve as a strategy to simultaneously promote neural and musculoskeletal health in diabetes. Methods: To address this question, an eStim paradigm pre-optimized to promote nerve regeneration was applied to the sciatic nerve, which directly innervates the tibia and lower limb, for 8 weeks in control and streptozotocin-induced type 1 diabetic (T1D) rats. Metabolic, gait, nerve and bone assessments were used to evaluate the progression of diabetes and the effect of sciatic nerve eStim on neuropathy and musculoskeletal disease, while also considering the effects of cuff placement and chronic eStim in otherwise healthy animals. Results: Rats with T1D exhibited increased mechanical allodynia in the hindpaw, reduced muscle mass, decreased cortical and cancellous bone volume fraction (BVF), reduced cortical bone tissue mineral density (TMD), and decreased bone marrow adiposity. Type 1 diabetes also had an independent effect on gait. Placement of the cuff electrode alone resulted in altered gait patterns and unilateral reductions in tibia length, cortical BVF, and bone marrow adiposity. Alterations in gait patterns were restored by eStim and tibial lengthening was favored unilaterally; however, eStim did not prevent T1D-induced changes in muscle, bone, marrow adiposity or mechanical sensitivity. Beyond this, chronic eStim resulted in an independent, bilateral reduction in cortical TMD. Conclusion: Overall, these results provide new insight into the pathogenesis of diabetic neuroskeletal disease and its regulation by eStim. Though eStim did not prevent neural or musculoskeletal complications in T1D, our results demonstrate that clinical applications of peripheral neuromodulation ought to consider the impact of device placement and eStim on long-term skeletal health in both healthy individuals and those with metabolic disease. This includes monitoring for compounded bone loss to prevent unintended consequences including decreased bone mineral density and increased fracture risk

Knockout of TSC2 in Nav1.8+ neurons predisposes to the onset of normal weight obesity

OBJECTIVE: Obesity and nutrient oversupply increase mammalian target of rapamycin (mTOR) signaling in multiple cell types and organs, contributing to the onset of insulin resistance and complications of metabolic disease. However, it remains unclear when and where mTOR activation mediates these effects, limiting options for therapeutic intervention. The objective of this study was to isolate the role of constitutive mTOR activation in Nav1.8-expressing peripheral neurons in the onset of diet-induced obesity, bone loss, and metabolic disease. METHODS: In humans, loss of function mutations in tuberous sclerosis complex 2 (TSC2) lead to maximal constitutive activation of mTOR. To mirror this in mice, we bred Nav1.8-Cre with TSC2 RESULTS: By lineage tracing, Nav1.8-Cre targeted peripheral sensory neurons, a subpopulation of postganglionic sympathetics, and several regions of the brain. Conditional knockout of TSC2 in Nav1.8-expressing neurons (Nav1.8-TSC2 CONCLUSIONS: Knockout of TSC2 in Nav1.8+ neurons increases itch- and anxiety-like behaviors and substantially modifies fat storage and metabolic responses to HFD. Though this prevents HFD-induced weight gain, it masks depot-specific fat expansion and persistent detrimental effects on metabolic health and peripheral organs such as bone, mimicking the \u27normal weight obesity\u27 phenotype that is of growing concern. This supports a mechanism by which increased neuronal mTOR signaling can predispose to altered adipose tissue distribution, adipose tissue expansion, impaired peripheral metabolism, and detrimental changes to skeletal health with HFD - despite resistance to weight gain

Strain prioritization and genome mining for enediyne natural products

The enediyne family of natural products has had a profound impact on modern chemistry, biology, and medicine, and yet only 11 enediynes have been structurally characterized to date. Here we report a genome survey of 3,400 actinomycetes, identifying 81 strains that harbor genes encoding the enediyne polyketide synthase cassettes that could be grouped into 28 distinct clades based on phylogenetic analysis. Genome sequencing of 31 representative strains confirmed that each clade harbors a distinct enediyne biosynthetic gene cluster. A genome neighborhood network allows prediction of new structural features and biosynthetic insights that could be exploited for enediyne discovery. We confirmed one clade as new C-1027 producers, with a significantly higher C-1027 titer than the original producer, and discovered a new family of enediyne natural products, the tiancimycins (TNMs), that exhibit potent cytotoxicity against a broad spectrum of cancer cell lines. Our results demonstrate the feasibility of rapid discovery of new enediynes from a large strain collection. IMPORTANCE Recent advances in microbial genomics clearly revealed that the biosynthetic potential of soil actinomycetes to produce enediynes is underappreciated. A great challenge is to develop innovative methods to discover new enediynes and produce them in sufficient quantities for chemical, biological, and clinical investigations. This work demonstrated the feasibility of rapid discovery of new enediynes from a large strain collection. The new C-1027 producers, with a significantly higher C-1027 titer than the original producer, will impact the practical supply of this important drug lead. The TNMs, with their extremely potent cytotoxicity against various cancer cells and their rapid and complete cancer cell killing characteristics, in comparison with the payloads used in FDA-approved antibody-drug conjugates (ADCs), are poised to be exploited as payload candidates for the next generation of anticancer ADCs. Follow-up studies on the other identified hits promise the discovery of new enediynes, radically expanding the chemical space for the enediyne family

Sarm1 knockout prevents type 1 diabetic bone disease in females independent of neuropathy

Patients with diabetes have a high risk of developing skeletal diseases accompanied by diabetic peripheral neuropathy (DPN). In this study, we isolated the role of DPN in skeletal disease with global and conditional knockout models of sterile-α and TIR-motif-containing protein-1 (Sarm1). SARM1, an NADase highly expressed in the nervous system, regulates axon degeneration upon a range of insults, including DPN. Global knockout of Sarm1 prevented DPN, but not skeletal disease, in male mice with type 1 diabetes (T1D). Female wild-type mice also developed diabetic bone disease but without DPN. Unexpectedly, global Sarm1 knockout completely protected female mice from T1D-associated bone suppression and skeletal fragility despite comparable muscle atrophy and hyperglycemia. Global Sarm1 knockout rescued bone health through sustained osteoblast function with abrogation of local oxidative stress responses. This was independent of the neural actions of SARM1, as beneficial effects on bone were lost with neural conditional Sarm1 knockout. This study demonstrates that the onset of skeletal disease occurs rapidly in both male and female mice with T1D completely independently of DPN. In addition, this reveals that clinical SARM1 inhibitors, currently being developed for treatment of neuropathy, may also have benefits for diabetic bone through actions outside of the nervous system

Crystal structure of SgcJ, an NTF2-like superfamily protein involved in biosynthesis of the nine-membered enediyne antitumor antibiotic C-1027

Comparative analysis of the enediyne biosynthetic gene clusters revealed sets of conserved genes serving as outstanding candidates for the enediyne core. Here we report the crystal structures of SgcJ and its homologue NCS-Orf16, together with gene inactivation and site-directed mutagenesis studies, to gain insight into enediyne core biosynthesis. Gene inactivation in vivo establishes that SgcJ is required for C-1027 production in Streptomyces globisporus. SgcJ and NCS-Orf16 share a common structure with the nuclear transport factor 2-like superfamily of proteins, featuring a putative substrate binding or catalytic active site. Site-directed mutagenesis of the conserved residues lining this site allowed us to propose that SgcJ and its homologues may play a catalytic role in transforming the linear polyene intermediate, along with other enediyne polyketide synthase-associated enzymes, into an enzyme-sequestered enediyne core intermediate. These findings will help formulate hypotheses and design experiments to ascertain the function of SgcJ and its homologues in nine-membered enediyne core biosynthesis

Ultrafast measurements of mode-specific deformation potentials of Bi2_2Te3_3 and Bi2_2Se3_3

Quantifying electron-phonon interactions for the surface states of topological materials can provide key insights into surface-state transport, topological superconductivity, and potentially how to manipulate the surface state using a structural degree of freedom. We perform time-resolved x-ray diffraction (XRD) and angle-resolved photoemission (ARPES) measurements on Bi$_2$Te$_3$ and Bi$_2$Se$_3$, following the excitation of coherent A$_{1g}$ optical phonons. We extract and compare the deformation potentials coupling the surface electronic states to local A$_{1g}$-like displacements in these two materials using the experimentally determined atomic displacements from XRD and electron band shifts from ARPES.We find the coupling in Bi$_2$Te$_3$ and Bi$_2$Se$_3$ to be similar and in general in agreement with expectations from density functional theory. We establish a methodology that quantifies the mode-specific electron-phonon coupling experimentally, allowing detailed comparison to theory. Our results shed light on fundamental processes in topological insulators involving electron-phonon coupling

Hundreds of variants clustered in genomic loci and biological pathways affect human height

Most common human traits and diseases have a polygenic pattern of inheritance: DNA sequence variants at many genetic loci influence the phenotype. Genome-wide association (GWA) studies have identified more than 600 variants associated with human traits, but these typically explain small fractions of phenotypic variation, raising questions about the use of further studies. Here, using 183,727 individuals, we show that hundreds of genetic variants, in at least 180 loci, influence adult height, a highly heritable and classic polygenic trait. The large number of loci reveals patterns with important implications for genetic studies of common human diseases and traits. First, the 180 loci are not random, but instead are enriched for genes that are connected in biological pathways (P = 0.016) and that underlie skeletal growth defects (P < 0.001). Second, the likely causal gene is often located near the most strongly associated variant: in 13 of 21 loci containing a known skeletal growth gene, that gene was closest to the associated variant. Third, at least 19 loci have multiple independently associated variants, suggesting that allelic heterogeneity is a frequent feature of polygenic traits, that comprehensive explorations of already-discovered loci should discover additional variants and that an appreciable fraction of associated loci may have been identified. Fourth, associated variants are enriched for likely functional effects on genes, being over-represented among variants that alter amino-acid structure of proteins and expression levels of nearby genes. Our data explain approximately 10% of the phenotypic variation in height, and we estimate that unidentified common variants of similar effect sizes would increase this figure to approximately 16% of phenotypic variation (approximately 20% of heritable variation). Although additional approaches are needed to dissect the genetic architecture of polygenic human traits fully, our findings indicate that GWA studies can identify large numbers of loci that implicate biologically relevant genes and pathways.

Exposure to secondhand tobacco smoke and lung cancer by histological type: A pooled analysis of the International Lung Cancer Consortium (ILCCO)

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/108359/1/ijc28835.pd

Nucleolin Inhibits G4 Oligonucleotide Unwinding by Werner Helicase

The Werner protein (WRNp), a member of the RecQ helicase family, is strongly associated with the nucleolus, as is nucleolin (NCL), an important nucleolar constituent protein. Both WRNp and NCL respond to the effects of DNA damaging agents. Therefore, we have investigated if these nuclear proteins interact and if this interaction has a possible functional significance in DNA damage repair.Here we report that WRNp interacts with the RNA-binding protein, NCL, based on immunoprecipitation, immunofluorescent co-localization in live and fixed cells, and direct binding of purified WRNp to nucleolin. We also map the binding region to the C-terminal domains of both proteins. Furthermore, treatment of U2OS cells with 15 µM of the Topoisomerase I inhibitor, camptothecin, causes the dissociation of the nucleolin-Werner complex in the nucleolus, followed by partial re-association in the nucleoplasm. Other DNA damaging agents, such as hydroxyurea, Mitomycin C, and aphidicolin do not have these effects. Nucleolin or its C-terminal fragment affected the helicase, but not the exonuclease activity of WRNp, by inhibiting WRN unwinding of G4 tetraplex DNA structures, as seen in activity assays and electrophoretic mobility shift assays (EMSA).These data suggest that nucleolin may regulate G4 DNA unwinding by WRNp, possibly in response to certain DNA damaging agents. We postulate that the NCL-WRNp complex may contain an inactive form of WRNp, which is released from the nucleolus upon DNA damage. Then, when required, WRNp is released from inhibition and can participate in the DNA repair processes