Bruton's agammaglobulinemia tyrosine kinase (Btk) is a cytoplasmic tyrosine kinase important in B-lymphocyte development, differentiation, and signaling. Btk is a member of the Tec family of kinases. Mutations in the Btk gene lead to X-linked agammaglobulinemia (XLA) in humans and X-linked immunodeficiency (Xid) in mice. Activation of Btk triggers a cascade of signaling events that culminates in the generation of calcium mobilization and fluxes, cytoskeletal rearrangements, and transcriptional regulation involving nuclear factor-kappaB (NF-kappaB) and nuclear factor of activated T cells (NFAT). In B cells, NF-kappaB was shown to bind to the Btk promoter and induce transcription, whereas the B-cell receptor-dependent NF-kappaB signaling pathway requires functional Btk. Moreover, Btk activation is tightly regulated by a plethora of other signaling proteins including protein kinase C (PKC), Sab/SH3BP5, and caveolin-1. For example, the prolyl isomerase Pin1 negatively regulates Btk by decreasing tyrosine phosphorylation and steady state levels of Btk. It is intriguing that PKC and Pin1, both of which are negative regulators, bind to the pleckstrin homology domain of Btk. To this end, we describe here novel mutations in the pleckstrin homology domain investigated for their transforming capacity. In particular, we show that the mutant D43R behaves similar to E41K, already known to possess such activity.

Objective Clinical outcomes of the stand-alone cage have been encouraging when used in anterior cervical discectomy and fusion (ACDF), but concerns remain regarding its complications, especially cage subsidence. This retrospective study was undertaken to investigate the long-term radiological and clinical outcomes of the stand-alone titanium cage and to evaluate the incidence of cage subsidence in relation to the clinical outcome in the surgical treatment of degenerative cervical disc disease. Methods A total of 57 consecutive patients (68 levels) who underwent ACDF using a titanium box cage for the treatment of cervical radiculopathy and/or myelopathy were reviewed for the radiological and clinical outcomes. They were followed for at least 5 years. Radiographs were obtained before and after surgery, 3 months postoperatively, and at the final follow-up to determine the presence of fusion and cage subsidence. The Cobb angle of C2-C7 and the vertebral bodies adjacent to the treated disc were measured to evaluate the cervical sagittal alignment and local lordosis. The disc height was measured as well. The clinical outcomes were evaluated using the Japanese Orthopaedic Association (JOA) score for cervical myelopathy, before and after surgery, and at the final follow-up.The recovery rate of JOA score was also calculated. The Visual Analogue Scale (VAS) score of neck and radicular pain were evaluated as well. The fusion rate was 95.6% (65/68) 3 months after surgery. ResultsSuccessful bone fusion was achieved in all patients at the final follow-up. Cage subsidence occurred in 13 cages (19.1%) at 3-month follow-up; however, there was no relation between fusion and cage subsidence. Cervical and local lordosis improved after surgery, with the improvement preserved at the final follow-up. The preoperative disc height of both subsidence and non-subsidence patients was similar; however, postoperative posterior disc height (PDH) of subsidence group was significantly greater than of non-subsidence group. Significant improvement of the JOA score was noted immediately after surgery and at the final follow-up. There was no significant difference of the recovery rate of JOA score between subsidence and non-subsidence groups. The recovery rate of JOA score was significantly related to the improvement of the C2-C7 Cobb angle. The VAS score regarding neck and radicular pain was significantly improved after surgery and at the final follow-up. There was no significant difference of the neck and radicular pain between both subsidence and non-subsidence groups. ConclusionsThe results suggest that the clinical and radiological outcomes of the stand-alone titanium box cage for the surgical treatment of one-or two-level degenerative cervical disc disease are satisfactory. Cage subsidence does not exert significant impact upon the long-term clinical outcome although it is common for the stand-alone cages. The cervical lordosis may be more important for the longterm clinical outcome than cage subsidence

The cellular thermal shift assay (CETSA) is a biophysical technique allowing direct studies of ligand binding to proteins in cells and tissues. The proteome-wide implementation of CETSA with mass spectrometry detection (MS-CETSA) has now been successfully applied to discover targets for orphan clinical drugs and hits from phenotypic screens, to identify off-targets, and to explain poly-pharmacology and drug toxicity. Highly sensitive multidimensional MS-CETSA implementations can now also access binding of physiological ligands to proteins, such as metabolites, nucleic acids, and other proteins. MS-CETSA can thereby provide comprehensive information on modulations of protein interaction states in cellular processes, including downstream effects of drugs and transitions between different physiological cell states. Such horizontal information on ligandmodulation in cells is largely orthogonal to vertical information on the levels of different proteins and therefore opens novel opportunities to understand operational aspects of cellular proteomes.

IntroductionBruton tyrosine kinase (Btk) is a nonreceptor tyrosine kinase belonging to the Tec family of protein tyrosine kinases (PTKs). This family consists of 5 mammalian members: Btk, Itk, Tec, Bmx, and Txk. These kinases are also present in other species. 1,2 Btk is expressed in almost all the hematopoietic cells, except T lymphocytes and plasma cells, and in the B-cell lineage, from the very early up to the mature cell stages. [3][4][5] Btk is required for Blymphocyte survival, proliferation, and differentiation in response to BCR activation. Accordingly, mutations in the gene coding for Btk lead to X-linked agammaglobulinemia (XLA) in humans 6-9 and X-linked immunodeficiency (xid) in mice. 10,11 Btk and its direct substrate, phospholipase C␥2 (PLC-␥2), are essential for the activation of the transcription factor nuclear factor-B (NF-B) in response to BCR engagement. [12][13][14] Moreover, Shinners et al recently found that Btk mediates B-cell-activating factor receptor (BAFF-R)-dependent activation of NF-B, thereby promoting B-cell survival. 15 NF-B is known to be essential for both innate and adaptive immunity. Moreover, it is crucial for the initial responses of sentinel cells to pathogens, as well as for the subsequent events that lead to T cell-and B cell-mediated antigen-specific defense. The role of NF-B in the acute, innate immune response is evolutionarily conserved. 16 NF-B has also been shown to be crucial for the development of several mammalian hematopoietic cell lineages and for the formation of secondary lymphoid-organ structures.The NF-B/Rel family of proteins include NF-B1 (p50), NF-B2 (p52), RelA (p65), c-Rel, and RelB, which can form functional homodimer or heterodimer complexes. 16,17 In resting cells, NF-B is sequestered in the cytoplasm by the inhibitory proteins of the I-B family. Following stimulation of cells by inflammatory cytokines, and bacterial (eg, LPS) and viral products, the inhibitor of B (I-B) is phosphorylated by the I-B kinases (IKKs), leading to its degradation through the ubiquitin-proteasome pathway. Thus, in the absence of I-B, NF-B dimers (p50-p65 and p52-RelB) are released, translocated to the nucleus, and subsequently bound to their cognate elements on target genes. 18 Mice deficient in individual NF-B/Rel family members have demonstrated the essential role of these transcription factors in CD40, TLR4, and IgM receptor pathways leading to B-cell proliferation. In particular, B cells from mice deficient in the NF-B members c-Rel or p65 have decreased responses to antigen cross-linking. 19,20 c-Rel Ϫ/Ϫ B cells also failed to respond to CD40 ligation. An examination of B cells in mice expressing a transdominant form of I-B␣ revealed xid-like defects, including lack of proliferation in response to anti-IgM. 21 The transcriptional regulation of the Btk gene has been studied quite extensively. Accordingly, a number of transcription factors including Sp1, Sp3, Spi-B, PU.1, [22][23][24] and OCT1/OBF1 25 have been shown to bind and activate the Btk promoter. However, most...