Mineral fabrication and golgi apparatus activity in the mouse calvarium

There is diverse opinion about the mechanism of bone mineralization with only intermittent reports of any direct organellar role played by the golgi apparatus (juxtanuclear body). Light and laser confocal microscopy was combined with electron microscopy and elemental EDX (energy dispersive X-ray microanalysis) in comparing “young” osteocytes in situ in fresh and “slam” frozen developing mouse calvarium, with similar cells (MC3T3-E1) maintained in vitro. The distribution of “nascent” electron dense mineral was examined histochemically (von Kossa, GBHA), including tetracycline (TC) staining as a fluorescent complex with bone salt, while golgi body activity was demonstrated by transfection with a specific green fluorescent construct (GFP/mannosidase II). In tissue culture golgi body activity and mineralization were both blocked by brefeldin A (an established golgi inhibitor) and restored by forskolin, enabling an association with mineral fabrication to be quantified as changing fluorescence intensity (AU) of GFP or TC markers. Results from osteocytes in situ supported previous descriptions of intracellular electron dense objects (microspheres and nanospheres) in a juxtanuclear pattern, containing Ca, P and transitory Si, in a spectrum recapitulated in the calcifying culture after 10 days, when GFP fluorophore surged from 71.7 ± 1.4SD to 133.7 ± 2.7SD AU by 14 days (p < 0.0001). At this stage TC fluorophore mean intensity was 23.8 ± 3.7SD AU (14 days) rising to 45.0 ± 5.1SD AU by 17 days, compared to its stationary 21.7 ± 3.6SD when treated 3 days previously with BFA golgi inhibitor (p < 0.0001), until forskolin reversal. It was concluded from the changing juxtanuclear morphology, Si mineralization mediation and the variably controlled activity versus stasis that the inorganic phase of bone is a complex golgi-directed fabrication with implications for bone matrix biology and evolution

Mapping trabecular disconnection "hotspots" in aged human spine and hip

Trabecular bone disconnection is an independent factor in age-related skeletal failure where real termini (ReTm; rare in youth) may cause weakness disproportionate to tissue loss, yet their structural contribution at vulnerable locations remains uncertain. ReTm (previously recorded at the iliac crest) were mapped in "normal" aged vertebral bodies (T11-L5 autopsy; 20 females, 10 males) and corresponding proximal femora (autopsy; 10 females). Results were compared with biomechanically failed femora from orthopaedic subjects aged >. 58. yr (osteoporosis OP, 10 females; osteoarthritis OA, 10 females). A novel direct 2D/3D histological method was applied to large, thick (300. μm) slices superficially silver-stained to separate ReTm (unstained) from apparent termini (planar artefacts, brown). Light microscope field co-ordinates enabled ReTm mapping and statistical testing relative to i) sex, ii) tissue sector and iii) slicing plane. In men ReTm populations were small and random while in women they were large and sector-specific. In vertebrae they clustered anterior/superior being rare posterior/inferior; in the femoral head they concentrated distal/superior and also near the fovea, being fewer distal/inferior. A distribution polarity was evident with 100% more ReTm observed transversely (i.e., on tensile-related cross struts) than longitudinally (i.e., on compression-related vertical struts). Their numbers rose in OP (BV/TV. . 14%), remaining polarised and sector-specific in OP only. Comparative experimentation by marrow elution of an OP animal model demonstrated "floating segments" as a possible outcome. Conclusions were supported statistically that trabecular disconnection "hotspots" at vulnerable locations are sex- and sector-specific, mainly transaxial, and subject to disease modulation

T cells enhance gold nanoparticle delivery to tumors in vivo

Gold nanoparticle-mediated photothermal therapy (PTT) has shown great potential for the treatment of cancer in mouse studies and is now being evaluated in clinical trials. For this therapy, gold nanoparticles (AuNPs) are injected intravenously and are allowed to accumulate within the tumor via the enhanced permeability and retention (EPR) effect. The tumor is then irradiated with a near infrared laser, whose energy is absorbed by the AuNPs and translated into heat. While reliance on the EPR effect for tumor targeting has proven adequate for vascularized tumors in small animal models, the efficiency and specificity of tumor delivery in vivo, particularly in tumors with poor blood supply, has proven challenging. In this study, we examine whether human T cells can be used as cellular delivery vehicles for AuNP transport into tumors. We first demonstrate that T cells can be efficiently loaded with 45 nm gold colloid nanoparticles without affecting viability or function (e.g. migration and cytokine production). Using a human tumor xenograft mouse model, we next demonstrate that AuNP-loaded T cells retain their capacity to migrate to tumor sites in vivo. In addition, the efficiency of AuNP delivery to tumors in vivo is increased by more than four-fold compared to injection of free PEGylated AuNPs and the use of the T cell delivery system also dramatically alters the overall nanoparticle biodistribution. Thus, the use of T cell chaperones for AuNP delivery could enhance the efficacy of nanoparticle-based therapies and imaging applications by increasing AuNP tumor accumulation

Distribution of roots of random real generalized polynomials

The average density of zeros for monic generalized polynomials, $P_n(z)=\phi(z)+\sum_{k=1}^nc_kf_k(z)$, with real holomorphic $\phi ,f_k$ and real Gaussian coefficients is expressed in terms of correlation functions of the values of the polynomial and its derivative. We obtain compact expressions for both the regular component (generated by the complex roots) and the singular one (real roots) of the average density of roots. The density of the regular component goes to zero in the vicinity of the real axis like $|\hbox{\rm Im}\,z|$. We present the low and high disorder asymptotic behaviors. Then we particularize to the large $n$ limit of the average density of complex roots of monic algebraic polynomials of the form $P_n(z) = z^n +\sum_{k=1}^{n}c_kz^{n-k}$ with real independent, identically distributed Gaussian coefficients having zero mean and dispersion $\delta = \frac 1{\sqrt{n\lambda}}$. The average density tends to a simple, {\em universal} function of $\xi={2n}{\log |z|}$ and $\lambda$ in the domain $\xi\coth \frac{\xi}{2}\ll n|\sin \arg (z)|$ where nearly all the roots are located for large $n$.Comment: 17 pages, Revtex. To appear in J. Stat. Phys. Uuencoded gz-compresed tarfile (.66MB) containing 8 Postscript figures is available by e-mail from [email protected]

First Detection of Mycobacterium ulcerans DNA in Environmental Samples from South America

The occurrences of many environmentally-persistent and zoonotic infections are driven by ecosystem changes, which in turn are underpinned by land-use modifications that alter the governance of pathogen, biodiversity and human interactions. Our current understanding of these ecological changes on disease emergence however remains limited. Buruli ulcer is an emerging human skin disease caused by the mycobacterium, Mycobacterium ulcerans, for which the exact route of infection remains unclear. It can have a devastating impact on its human host, causing extensive necrosis of the skin and underlying tissue, often leading to permanent disability. The mycobacterium is associated with tropical aquatic environments and incidences of the disease are significantly higher on floodplains and where there is an increase of human aquatic activities. Although the disease has been previously diagnosed in South America, until now the presence of M. ulcerans DNA in the wild has only been identified in Australia where there have been significant outbreaks and in western and central regions of Africa where the disease is persistent. Here for the first time, we have identified the presence of the aetiological agent's DNA in environmental samples from South America. The DNA was positively identified using Real-time Polymerase Chain Reaction (PCR) on 163 environmental samples, taken from 23 freshwater bodies in French Guiana (Southern America), using primers for both IS2404 and for the ketoreductase-B domain of the M. ulcerans mycolactone polyketide synthase genes (KR). Five samples out of 163 were positive for both primers from three different water bodies. A further nine sites had low levels of IS2404 close to a standard CT of 35 and could potentially harbour M. ulcerans. The majority of our positive samples (8/14) came from filtered water. These results also reveal the Sinnamary River as a potential source of infection to humans. © 2014 Morris et al

Identification of pre-leukaemic haematopoietic stem cells in acute leukaemia.

In acute myeloid leukaemia (AML), the cell of origin, nature and biological consequences of initiating lesions, and order of subsequent mutations remain poorly understood, as AML is typically diagnosed without observation of a pre-leukaemic phase. Here, highly purified haematopoietic stem cells (HSCs), progenitor and mature cell fractions from the blood of AML patients were found to contain recurrent DNMT3A mutations (DNMT3A(mut)) at high allele frequency, but without coincident NPM1 mutations (NPM1c) present in AML blasts. DNMT3A(mut)-bearing HSCs showed a multilineage repopulation advantage over non-mutated HSCs in xenografts, establishing their identity as pre-leukaemic HSCs. Pre-leukaemic HSCs were found in remission samples, indicating that they survive chemotherapy. Therefore DNMT3A(mut) arises early in AML evolution, probably in HSCs, leading to a clonally expanded pool of pre-leukaemic HSCs from which AML evolves. Our findings provide a paradigm for the detection and treatment of pre-leukaemic clones before the acquisition of additional genetic lesions engenders greater therapeutic resistance