Robust and fast whole brain mapping of the RF transmit field B1 at 7T

In-vivo whole brain mapping of the radio frequency transmit field B(1) (+) is a key aspect of recent method developments in ultra high field MRI. We present an optimized method for fast and robust in-vivo whole-brain B(1) (+) mapping at 7T. The method is based on the acquisition of stimulated and spin echo 3D EPI images and was originally developed at 3T. We further optimized the method for use at 7T. Our optimization significantly improved the robustness of the method against large B(1) (+) deviations and off-resonance effects present at 7T. The mean accuracy and precision of the optimized method across the brain was high with a bias less than 2.6 percent unit (p.u.) and random error less than 0.7 p.u. respectively.     

DLK1(PREF1) is a negative regulator of adipogenesis in CD105⁺/CD90⁺/CD34⁺/CD31⁻/FABP4⁻ adipose-derived stromal cells from subcutaneous abdominal fat pats of adult women

The main physiological function of adipose-derived stromal/progenitor cells (ASC) is to differentiate into adipocytes. ASC are most likely localized at perivascular sites in adipose tissues and retain the capacity to differentiate into multiple cell types. Although cell surface markers for ASC have been described, there is no complete consensus on the antigen expression pattern that will precisely define these cells. DLK1(PREF1) is an established marker for mouse adipocyte progenitors which inhibits adipogenesis. This suggests that DLK1(PREF1) could be a useful marker to characterize human ASC. The DLK1(PREF1) status of human ASC is however unknown. In the present study we isolated ASC from the heterogeneous stromal vascular fraction of subcutaneous abdominal fat pats of adult women. These cells were selected by their plastic adherence and expanded to passage 5. The ASC were characterized as relatively homogenous cell population with the capacity to differentiate in vitro into adipocytes, chondrocytes, and osteoblasts and the immunophenotype CD105?/CD90?/CD34?/CD31?/FABP4?. The ASC were positive for DLK1(PREF1) which was well expressed in proliferating and density arrested cells but downregulated in the course of adipogenic differentiation. To investigate whether DLK1(PREF1) plays a role in the regulation of adipogenesis in these cells RNAi-mediated knockdown experiments were conducted. Knockdown of DLK1(PREF1) in differentiating ASC resulted in a significant increase of the expression of the adipogenic key regulator PPARγ2 and of the terminal adipogenic differentiation marker FABP4. We conclude that DLK1(PREF1) is well expressed in human ASC and acts as a negative regulator of adipogenesis. Moreover, DLK1(PREF1) could be a functional marker contributing to the characterization of human ASC.     

Beam-induced motion of vitrified specimen on holey carbon film

The contrast observed in images of frozen-hydrated biological specimens prepared for electron cryo-microscopy falls significantly short of theoretical predictions. In addition to limits imposed by the current instrumentation, it is widely acknowledged that motion of the specimen during its exposure to the electron beam leads to significant blurring in the recorded images. We have studied the amount and direction of motion of virus particles suspended in thin vitrified ice layers across holes in perforated carbon films using exposure series. Our data show that the particle motion is correlated within patches of 0.3-0.5 μm, indicating that the whole ice layer is moving in a drum-like motion, with accompanying particle rotations of up to a few degrees. Support films with smaller holes, as well as lower electron dose rates tend to reduce beam-induced specimen motion, consistent with a mechanical effect. Finally, analysis of movies showing changes in the specimen during beam exposure show that the specimen moves significantly more at the start of an exposure than towards its end. We show how alignment and averaging of movie frames can be used to restore high-resolution detail in images affected by beam-induced motion.     

Intermediate Monocytes but Not TIE2-Expressing Monocytes Are a Sensitive Diagnostic Indicator for Colorectal Cancer

We have conducted the first study to determine the diagnostic potential of the CD14++CD16+ intermediate monocytes as compared to the pro-angiogenic subset of CD14++CD16+TIE2+ TIE2-expressing monocytes (TEMs) in cancer. These monocyte populations were investigated by flow cytometry in healthy volunteers (N?=?32) and in colorectal carcinoma patients with localized (N?=?24) or metastatic (N?=?37) disease. We further determined blood levels of cytokines associated with monocyte regulation. The results revealed the intermediate monocyte subset to be significantly elevated in colorectal cancer patients and to show the highest frequencies in localized disease. Multivariate regression analysis identified intermediate monocytes as a significant independent variable in cancer prediction. With a cut-off value at 0.37% (intermediate monocytes of total leukocytes) the diagnostic sensitivity and specificity ranged at 69% and 81%, respectively. In contrast, TEM levels were elevated in localized cancer but did not differ significantly between groups and none of the cytokines correlated with monocyte subpopulations. Of interest, in vitro analyses supported the observation that intermediate monocytes were more potently induced by primary as opposed to metastatic cancer cells which may relate to the immunosuppressive milieu established in the advanced stage of metastatic disease. In conclusion, intermediate monocytes as compared to TIE2-expressing monocytes are a more sensitive diagnostic indicator of colorectal cancer.     

Maturation in Action: CryoEM Study of a Viral Capsid Caught during Expansion

Bacteriophage HK97 maturation involves discrete intermediate particle forms, comparable to transitional states in protein folding, before reaching its mature form. The process starts by formation of a metastable prohead, poised for exothermic expansion triggered by DNA packaging. During maturation, the capsid subunit transitions from a strained to a canonical tertiary conformation and this has been postulated to be the driving mechanism for initiating expansion via switching hexameric capsomer architecture from skewed to 6-fold symmetric. We report the subnanometer electron-cryomicroscopy reconstruction of the HK97 first expansion intermediate before any crosslink formation. This form displays 6-fold symmetric hexamers, but capsid subunit tertiary structures exhibit distortions comparable to the prohead forms. We propose that coat subunit strain release acts in synergy with the first crosslinks to drive forward maturation. Finally, we speculate that the energetic features of this transition may result from increased stability of intermediates during maturation via enhanced inter-subunit interactions.     

CCL3 and viral chemokine-binding protein gg modulate pulmonary inflammation and virus replication during equine herpesvirus 1 infection

CCL3 is a proinflammatory chemokine that mediates many of the cellular changes occurring in pulmonary disease. Here, CCL3^−/− mice were used to investigate the role of this chemokine during respiratory herpesvirus infection. Compared to wild-type mice, CCL3^−/− mice infected with the alphaherpesvirus equine herpesvirus 1 (EHV-1) displayed reduced body weight loss but had higher pulmonary viral loads. Lungs from infected CCL3^−/− mice suffered a milder interstitial pneumonia, and fewer immune cells were recovered from the pulmonary airways after infection. We could also demonstrate that herpesvirus-encoded chemokine-binding glycoprotein G (gG) was capable of inhibiting the chemotactic functions of CCL3. This CCL3-mediated chemotaxis, however, was restored in the presence of gG-specific antibodies, which puts into question the advertised use of gG deletion mutants as marker vaccines. In summary, we concluded that CCL3 is a major player in controlling herpesvirus replication in the target organ, the lung, and does so by evoking a strong inflammatory response. The immunomodulatory activity of CCL3 is balanced by the expression of viral gG, whose chemokine-binding activity is mitigated in secondary infections by the production of anti-gG antibodies.