Isolation OP DNA Prom Yeast by Chromatography on Hydroxyapatite

A simple procedure is described for isolation of purified non degraded total DNA from yeast cells. The procedure involves conversion of the cells into sphero-plasts by enzymatic treatment, lysis of the sphero-plasts in 8 M urea - 0.24 M sodium phosphate buffer -0.01 M EDTA (ethylendiamintetraacetic acid, sodium salt) - 1% SDS (sodium dodecyl sulphate), deproteiniza-tion of the lysate with chloroform-phenol and separation of the DNA from proteins, RNA and other contaminants by hydroxyapatite chromatography. The yield is about 90% of the DNA in the starting material (sphero-plasts).     

The equine neck and its function during movement and locomotion

During both locomotion and body movements at stance, the head and neck of the horse are a major craniocaudal and lateral balancing mechanism employing input from the visual, vestibular and proprioceptive systems. The function of the equine neck has recently become the focus of several research groups; this is probably also feeding on an increase of interest in the equine neck in equestrian sports, with a controversial discussion of specific neck positions such as maximum head and neck flexion. The aim of this review is to offer an overview of new findings on the structures and functions of the equine neck, illustrating their interplay. The movement of the neck is based on intervertebral motion, but it is also an integral part of locomotion; this is illustrated by the different neck conformations in the breeds of horses used for various types of work. The considerable effect of the neck movement and posture onto the whole trunk and even the limbs is transmitted via bony, ligamentous and muscular structures. Also, the fact that the neck position can easily be influenced by the rider and/or by the employment of training aids makes it an important avenue for training of new movements of the neck as well as the whole horse. Additionally, the neck position also affects the cervical spinal cord as well as the roots of the spinal nerves; besides the commonly encountered long-term neurological effects of cervical vertebral disorders, short-term changes of neural and muscular function have also been identified in the maximum flexion of the cranial neck and head position. During locomotion, the neck stores elastic energy within the passive tissues such as ligaments, joint capsules and fasciae. For adequate stabilisation, additional muscle activity is necessary; this is learned and requires constant muscle training as it is essential to prevent excessive wear and tear on the vertebral joints and also repetitive or single trauma to the spinal nerves and the spinal cord. The capability for this stabilisation decreases with age in the majority of horses due to changes in muscle tissue, muscle coordination and consequently muscle strength.     

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest^1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal''s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.     

Metastatic tumour cells favour the generation of a tolerogenic milieu in tumour draining lymph node in patients with early cervical cancer

Objective  We compared the immune system state in metastatic tumour draining lymph nodes (mTDLN) and metastasis free TDLN (mfTDLN) in 53 early stage cervical cancer patients to assess whether the presence of metastatic tumour cells worsen the balance between an efficacious anti-tumour and a tolerogenic microenvironment. Methods  The immune system state was measured by immunophenotypic and functional assessment of suppressor and effector immune cell subsets. Results  Compared to mfTDLN, mTDLN were significantly enriched in CD4⁺Foxp3⁺ regulatory T cells (Treg), which, in addition, exhibited an activated phenotype (HLA-DR⁺ and CD69⁺). Treg in mTDLN were also significantly enriched in neuropilin-1 (Nrp1) expressing cells, a subset particularly potent in dampening T cell responses. mTDLN tended to be enriched in a population of CD8⁺Foxp3⁺T cells (operationally defined as CD8⁺Treg) that showed a suppressor potency similar to Treg under the same experimental conditions. Plasmacytoid dendritic cells (pDC) and myeloid DC (mDC) generally show distinct roles in inducing T cell tolerance and activation, respectively. In line with the excess of suppressor T cells, the ratio pDC to mDC was significantly increased in mTDLN. Immunohistochemical testing showed that metastatic tumour cells produced the vascular endothelial growth factor, a natural ligand for Nrp1 expressed on the cell surface of Nrp1⁺Treg and pDC, and therefore a potential mediator by which tumour cells foster immune privilege in mTDLN. Consistent with the overall tolerogenic profile, mTDLN showed a significant Tc2 polarisation and tended to contain lower numbers of CD45RA⁺CD27⁻ effector memory CD8⁺T cells. Conclusions  The increased recruitment of suppressor type cells concomitant with the scarcity of cytotoxic type cells suggests that in mTDLN the presence of tumour cells could tip the balance against anti-tumour immune response facilitating the survival of metastatic tumour cells and possibly contributing to systemic tolerance.     

Ridge estimation of the VAR(1) model and its time series chain graph from multivariate time‐course omics data

Omics experiments endowed with a time‐course design may enable us to uncover the dynamic interplay among genes of cellular processes. Multivariate techniques (like VAR(1) models describing the temporal and contemporaneous relations among variates) that may facilitate this goal are hampered by the high‐dimensionality of the resulting data. This is resolved by the presented ridge regularized maximum likelihood estimation procedure for the VAR(1) model. Information on the absence of temporal and contemporaneous relations may be incorporated in this procedure. Its computational efficient implemention is discussed. The estimation procedure is accompanied with an LOOCV scheme to determine the associated penalty parameters. Downstream exploitation of the estimated VAR(1) model is outlined: an empirical Bayes procedure to identify the interesting temporal and contemporaneous relationships, impulse response analysis, mutual information analysis, and covariance decomposition into the (graphical) relations among variates. In a simulation study the presented ridge estimation procedure outperformed a sparse competitor in terms of Frobenius loss of the estimates, while their selection properties are on par. The proposed machinery is illustrated in the reconstruction of the p53 signaling pathway during HPV‐induced cellular transformation. The methodology is implemented in the ragt2ridges R‐package available from CRAN.     

Quantitative analysis of intra-fractional variation in CT-based image guided brachytherapy for cervical cancer patients

We quantified intra-fractional dose variation and organ movement during CT-based 3D-image guided brachytherapy (3D-IGBT) in cervical cancer patients. Fifteen patients who underwent CT-based 3D-IGBT were studied. For all patients, pre-delivery CT for treatment planning after applicator insertion and post-delivery CT after dose delivery without changing the applicator position were acquired. Pre- and post-delivery CT were rigidly fused by matching the inserted applicator and planned dose on pre-delivery CT (pre-delivery dose) was mapped on post-delivery CT (post-delivery dose). D2, D1, and D0.1 cm³ of the rectum and bladder were compared between pre- and post-delivery doses with contours on each CT image. Organ movement and deformation was evaluated using deformation vector fields calculated by deformable image registration between pre- and post-delivery CT. We also evaluated dose variation and DVF between with and without a catheter to control filling. Differences in all DVH parameters were <±3% in physical dose and ± 5% in EQD2. However, a > 15% dose difference was found in 13.8% of the fractions in rectum D2 cm³ and in 11.1% of those in bladder D2 cm³. The mean value of DVF for bladder was larger than that of rectum, especially for the superior-inferior (S-I) direction. Insertion catheters in bladder reduced mean dose and DVF variation compared with that of without catheters. In fraction groups with large dose increasing, DVF in the S-I direction was significantly larger than that of other fraction groups. Our results indicated that preparation is needed to reduce changes in the S-I direction affect dose variation.