Detailed imaging and genetic analysis reveal a secondary BRAFL505H resistance mutation and extensive intrapatient heterogeneity in metastatic BRAF mutant melanoma patients treated with vemurafenib

Resistance to treatment is the main problem of targeted treatment for cancer. We followed ten patients during treatment with vemurafenib, by three‐dimensional imaging. In all patients, only a subset of lesions progressed. Next‐generation DNA sequencing was performed on sequential biopsies in four patients to uncover mechanisms of resistance. In two patients, we identified mutations that explained resistance to vemurafenib; one of these patients had a secondary BRAF L505H mutation. This is the first observation of a secondary BRAF mutation in a vemurafenib‐resistant patient‐derived melanoma sample, which confirms the potential importance of the BRAF L505H mutation in the development of therapy resistance. Moreover, this study hints toward an important role for tumor heterogeneity in determining the outcome of targeted treatments.     

Deficiency of Nuclear Receptor Nur77 Aggravates Mouse Experimental Colitis by Increased NFκB Activity in Macrophages

Nuclear receptor Nur77, also referred to as NR4A1 or TR3, plays an important role in innate and adaptive immunity. Nur77 is crucial in regulating the T helper 1/regulatory T-cell balance, is expressed in macrophages and drives M2 macrophage polarization. In this study we aimed to define the function of Nur77 in inflammatory bowel disease. In wild-type and Nur77^-/- mice, colitis development was studied in dextran sodium sulphate (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced models. To understand the underlying mechanism, Nur77 was overexpressed in macrophages and gut epithelial cells. Nur77 protein is expressed in colon tissues from Crohn’s disease and Ulcerative colitis patients and colons from colitic mice in inflammatory cells and epithelium. In both mouse colitis models inflammation was increased in Nur77^-/- mice. A higher neutrophil influx and enhanced IL-6, MCP-1 and KC production was observed in Nur77-deficient colons after DSS-treatment. TNBS-induced influx of T-cells and inflammatory monocytes into the colon was higher in Nur77^-/- mice, along with increased expression of MCP-1, TNFα and IL-6, and decreased Foxp3 RNA expression, compared to wild-type mice. Overexpression of Nur77 in lipopolysaccharide activated RAW macrophages resulted in up-regulated IL-10 and downregulated TNFα, MIF-1 and MCP-1 mRNA expression through NFκB repression. Nur77 also strongly decreased expression of MCP-1, CXCL1, IL-8, MIP-1α and TNFα in gut epithelial Caco-2 cells. Nur77 overexpression suppresses the inflammatory status of both macrophages and gut epithelial cells and together with the in vivo mouse data this supports that Nur77 has a protective function in experimental colitis. These findings may have implications for development of novel targeted treatment strategies regarding inflammatory bowel disease and other inflammatory diseases.     

Impaired Visual Integration in Children with Traumatic Brain Injury: An Observational Study

Background

Axonal injury after traumatic brain injury (TBI) may cause impaired sensory integration. We aim to determine the effects of childhood TBI on visual integration in relation to general neurocognitive functioning.

Methods

We compared children aged 6–13 diagnosed with TBI (n = 103; M = 1.7 years post-injury) to children with traumatic control (TC) injury (n = 44). Three TBI severity groups were distinguished: mild TBI without risk factors for complicated TBI (mild^RF- TBI, n = 22), mild TBI with ≥1 risk factor (mild^RF+ TBI, n = 46) or moderate/severe TBI (n = 35). An experimental paradigm measured speed and accuracy of goal-directed behavior depending on: (1) visual identification; (2) visual localization; or (3) both, measuring visual integration. Group-differences on reaction time (RT) or accuracy were tracked down to task strategy, visual processing efficiency and extra-decisional processes (e.g. response execution) using diffusion model analysis. General neurocognitive functioning was measured by a Wechsler Intelligence Scale short form.

Results

The TBI group had poorer accuracy of visual identification and visual integration than the TC group (Ps ≤ .03; ds ≤ -0.40). Analyses differentiating TBI severity revealed that visual identification accuracy was impaired in the moderate/severe TBI group (P = .05, d = -0.50) and that visual integration accuracy was impaired in the mild^RF+ TBI group and moderate/severe TBI group (Ps < .02, ds ≤ -0.56). Diffusion model analyses tracked impaired visual integration accuracy down to lower visual integration efficiency in the mild^RF+ TBI group and moderate/severe TBI group (Ps < .001, ds ≤ -0.73). Importantly, intelligence impairments observed in the TBI group (P = .009, d = -0.48) were statistically explained by visual integration efficiency (P = .002).

Conclusions

Children with mild^RF+ TBI or moderate/severe TBI have impaired visual integration efficiency, which may contribute to poorer general neurocognitive functioning.     

3D Topography of the Young Adult Anal Sphincter Complex Reconstructed from Undeformed Serial Anatomical Sections

Background

Pelvic-floor anatomy is usually studied by artifact-prone dissection or imaging, which requires prior anatomical knowledge. We used the serial-section approach to settle contentious issues and an interactive 3D-pdf to make the results widely accessible.

Method

3D reconstructions of undeformed thin serial anatomical sections of 4 females and 2 males (21–35y) of the Chinese Visible Human database.

Findings

Based on tendinous septa and muscle-fiber orientation as segmentation guides, the anal-sphincter complex (ASC) comprised the subcutaneous external anal sphincter (EAS) and the U-shaped puborectal muscle, a part of the levator ani muscle (LAM). The anococcygeal ligament fixed the EAS to the coccygeal bone. The puborectal-muscle loops, which define the levator hiatus, passed around the anorectal junction and inserted anteriorly on the perineal body and pubic bone. The LAM had a common anterior attachment to the pubic bone, but separated posteriorly into puborectal and “pubovisceral” muscles. This pubovisceral muscle was bilayered: its internal layer attached to the conjoint longitudinal muscle of the rectum and the rectococcygeal fascia, while its outer, patchy layer reinforced the inner layer. ASC contraction makes the ano-rectal bend more acute and lifts the pelvic floor. Extensions of the rectal longitudinal smooth muscle to the coccygeal bone (rectococcygeal muscle), perineal body (rectoperineal muscle), and endopelvic fascia (conjoint longitudinal and pubovisceral muscles) formed a “diaphragm” at the inferior boundary of the mesorectum that suspended the anorectal junction. Its contraction should straighten the anorectal bend.

Conclusion

The serial-section approach settled contentious topographic issues of the pelvic floor. We propose that the ASC is involved in continence and the rectal diaphragm in defecation.     

The vanilloid transient receptor potential channel TRPV4: From structure to disease

The Transient Receptor Potential Vanilloid 4 channel, TRPV4, is a Ca²⁺ and Mg²⁺ permeable non-selective cation channel involved in many different cellular functions. It is activated by a variety of physical and chemical stimuli, including heat, mechano-stimuli, endogenous substances such as arachidonic acid and its cytochrome P450-derived metabolites (epoxyeicosatrienoic acids), endocannabinoids (anandamide and 2-arachidonoylglycerol), as well as synthetic α-phorbol derivatives. Recently, TRPV4 has been characterized as an important player modulating osteoclast differentiation in bone remodelling and as a urothelial mechanosensor that controls normal voiding. Several TRPV4 gain-of-function mutations are shown to cause autosomal-dominant bone dysplasias such as brachyolmia and Koszlowski disease. In this review we comprehensively describe the structural, biophysical and (patho)physiological properties of the TRPV4 channel and we summarize the current knowledge about the role of TRPV4 in the pathogenesis of several diseases.     

Refuge from predation, the benefit of living in an extreme acidic environment?

Organisms living in extreme habitats require costly adaptations to cope with these conditions. Among the suggested potential benefits that trade off these costs is refuge from predation. To study these interactions in extreme environments, samples were taken in the cave Cueva de Villa Luz, Tabasco, Mexico, where more than 32 subterranean springs, some H(2)S rich, rise from the floor. Hydrogen sulfide gas plus oxygen is absorbed by freshwater, and oxidation forms concentrated sulfuric acid. Snottites, whitish hollow mucous tubes, hang from the ceiling of the cave. Fluid drops from these snottites were recorded as having pH values of 0-3. We report the discovery of a new species of nematode that thrives in the highly acidic environment of the snottite. Micro CT scan of snottites reveals a complex interaction between the acidic snottite, nematodes, and abundant nematode-eating mites. The nematode adaptation to low pH probably protects them against mite predation, for which nematodes are most likely the most important source of carbon in this sulfur-driven ecosystem.     

<Emphasis Type="Italic">TRAF1/C5</Emphasis>polymorphism is not associated with increased mortality in rheumatoid arthritis: two large longitudinal studies

Introduction  

Recently an association between a genetic variation in TRAF1/C5 and mortality from sepsis or cancer was found in rheumatoid arthritis (RA). The most prevalent cause of death, cardiovascular disease, may have been missed in that study, since patients were enrolled at an advanced disease stage. Therefore, we used an inception cohort of RA patients to investigate the association between TRAF1/C5 and cardiovascular mortality, and replicate the findings on all-cause mortality. As TRAF1/C5 associated mortality may not be restricted to RA, we also studied a large cohort of non-RA patients.     

Cytokine profile of autologous conditioned serum for treatment of osteoarthritis, <Emphasis Type="Italic">in vitro</Emphasis>effects on cartilage metabolism and intra-articular levels after injection

Introduction  

Intraarticular administration of autologous conditioned serum (ACS) recently demonstrated some clinical effectiveness in treatment of osteoarthritis (OA). The current study aims to evaluate the in vitro effects of ACS on cartilage proteoglycan (PG) metabolism, its composition and the effects on synovial fluid (SF) cytokine levels following intraarticular ACS administration.     

Sampling Protein Form and Function with the Atomic Force Microscope

To study the structure, function, and interactions of proteins, a plethora of techniques is available. Many techniques sample such parameters in non-physiological environments (e.g. in air, ice, or vacuum). Atomic force microscopy (AFM), however, is a powerful biophysical technique that can probe these parameters under physiological buffer conditions. With the atomic force microscope operating under such conditions, it is possible to obtain images of biological structures without requiring labeling and to follow dynamic processes in real time. Furthermore, by operating in force spectroscopy mode, it can probe intramolecular interactions and binding strengths. In structural biology, it has proven its ability to image proteins and protein conformational changes at submolecular resolution, and in proteomics, it is developing as a tool to map surface proteomes and to study protein function by force spectroscopy methods. The power of AFM to combine studies of protein form and protein function enables bridging various research fields to come to a comprehensive, molecular level picture of biological processes. We review the use of AFM imaging and force spectroscopy techniques and discuss the major advances of these experiments in further understanding form and function of proteins at the nanoscale in physiologically relevant environments.To understand biological processes at the molecular level it is essential to identify the involved proteins and proteinaceous assemblies, to characterize their structure and function, and to unravel their interplay with other proteins and molecules (1). Techniques like x-ray crystallography, electron microscopy, nuclear magnetic resonance spectroscopy, and mass spectrometry have contributed massively to elucidate such protein properties. These techniques can easily sample the properties of a large ensemble of proteins; however, they require subjecting the sample to harsh treatments such as drying, crystallizing, or vaporizing in vacuum, thereby limiting the range of measurable dynamical properties of the sample. One powerful method that permits the investigation of molecules in their native physiological buffer condition is atomic force microscopy (AFM)¹ (2). An atomic force microscope is a microscope and force spectrometer at the same time. The imaging resolution of the atomic force microscope is comparable with that of electron microscopes, and it has the special capability to image samples in a variety of environments such as in vacuum, air, or liquid, which therefore enables studying biological specimens in their native environments (i.e. in buffer solutions) (3, 4). In addition, its ability to “touch” the sample gives it the advantage to manipulate single particles/molecules and probe their mechanical properties (5–8). However, AFM force spectroscopy is currently a technique with rather fast pulling and pushing speeds, thereby often operating out of equilibrium conditions. Improvements with ultrastable atomic force microscopes are underway to tackle this problem with promising results (9, 10). Furthermore, AFM is not well suited to apply and resolve forces at the single piconewton range due to large size tips and relatively stiff cantilevers. The issue of nonspecificity of the tip interaction with the sample is also of concern, especially in pulling experiments that require the capability to accurately recognize and select the appropriate molecule or point of interest. The current introduction of carbon nanotube tips can address the former issue (11, 12), whereas techniques in chemical functionalization can provide directed tip specificity and recognition capability (13–18), thereby further improving and widening the applicability of AFM in the future. In addition, the coupling of the atomic force microscope to fluorescence microscopes further enhances its versatility by adding (single molecule) fluorescence imaging to the AFM imaging capability (19–21), and the development of high speed systems makes it possible for AFM to probe fast dynamics of various biological processes (22–26).The applicability of AFM in proteomics is diverse and includes the characterization of the cell surface proteome (for a recent review, see Ref. 27), label-free detection and counting of single proteins (28, 29), and force spectroscopy measurements of binding and unbinding events (30, 31). In structural biology, AFM has shown to be a powerful tool for high resolution imaging of proteins in near native conditions (3, 6) and structural studies of supramolecular assemblies like protein filaments and viruses by nanoindentation methods (32, 33). These experiments show the potential of AFM to study both “form” and “function” of proteins, thereby resolving questions in proteomics and structural biology quasi-simultaneously. In the following, we will explain the principles of atomic force microscopy and its different operation modes and finally discuss examples of imaging, nanoindentation, and protein (un)binding and unfolding studies using AFM.     

Engineering of Saccharomyces cerevisiae for efficient anaerobic alcoholic fermentation of L-arabinose

For cost-effective and efficient ethanol production from lignocellulosic fractions of plant biomass, the conversion of not only major constituents, such as glucose and xylose, but also less predominant sugars, such as l-arabinose, is required. Wild-type strains of Saccharomyces cerevisiae, the organism used in industrial ethanol production, cannot ferment xylose and arabinose. Although metabolic and evolutionary engineering has enabled the efficient alcoholic fermentation of xylose under anaerobic conditions, the conversion of l-arabinose into ethanol by engineered S. cerevisiae strains has previously been demonstrated only under oxygen-limited conditions. This study reports the first case of fast and efficient anaerobic alcoholic fermentation of l-arabinose by an engineered S. cerevisiae strain. This fermentation was achieved by combining the expression of the structural genes for the l-arabinose utilization pathway of Lactobacillus plantarum, the overexpression of the S. cerevisiae genes encoding the enzymes of the nonoxidative pentose phosphate pathway, and extensive evolutionary engineering. The resulting S. cerevisiae strain exhibited high rates of arabinose consumption (0.70 g h(-1) g [dry weight](-1)) and ethanol production (0.29 g h(-1) g [dry weight](-1)) and a high ethanol yield (0.43 g g(-1)) during anaerobic growth on l-arabinose as the sole carbon source. In addition, efficient ethanol production from sugar mixtures containing glucose and arabinose, which is crucial for application in industrial ethanol production, was achieved.