Frequencies, clinical characteristics, and outcome of somatic CALR mutations in JAK2-unmutated essential thrombocythemia.

Calreticulin (CALR) mutations were recently identified in patients with essential thrombocythemia (ET) and primary myelofibrosis (PMF) devoid of JAK2 and MPL mutations. We evaluated the clinical, laboratory, and molecular features of a Taiwanese population of patients with ET. Among 147 ET patients, CALR mutations were detected in 33 (22.5 %), JAK2V617F in 94 (63.9 %), and MPL mutations in 4 (2.7 %). Sixteen (10.9 %) patients were negative for all three mutations (CALR, JAK2V617F, and MPL; triple negative). Interestingly, one patient with the type 2 CALR mutation also harbored a low allele burden (0.025 %) of JAK2V617F mutation. Furthermore, we found a novel CALR mutation, with the resultant protein sharing an identical amino acid sequence to the type 6 CALR mutant. Compared to those with JAK2 mutation, CALR-mutated ET patients were characterized by younger age, lower leukocyte count, higher platelet count, and decreased risk of thrombosis. CALR mutations had a favorable impact on thrombosis-free survival (TFS) for ET patients, whereas the respective TFS outcomes were similarly poorer in JAK2-mutated ET and PV patients. Multivariate analysis confirmed that younger age (<60 years), presence of CALR mutations, and a lower platelet count (<1,000 × 10(9)/L) were independently associated with a longer TFS in ET patients. The current study demonstrates that CALR mutations characterize a special group of ET patients with unique phenotypes that are not discrepant from those seen in Western countries.

Clinical characteristics and treatment outcome in a Taiwanese population of patients with Epstein-Barr virus-positive diffuse large B-cell lymphoma.

OBJECTIVE: Epstein-Barr virus-positive diffuse large B-cell lymphoma is a provisional entity in the 2008 World Health Organization classification of tumors of hematopoietic and lymphoid tissues. Reports on the characteristics and clinical outcome of this disease in different geographic regions showed great disparities. METHODS: To define the clinical characteristics as well as the prognostic impact of Epstein-Barr virus infection on diffuse large B-cell lymphoma in Taiwan, we retrospectively investigated the Epstein-Barr virus status of 89 patients with newly diagnosed diffuse large B-cell lymphoma in our institute. RESULTS: Using a cutoff point of positive nuclear staining of Epstein-Barr virus-encoded RNA-1-in situ hybridization in ≥20% of the examined cells, we identified 15 cases (16.9%) of the entire study cohort as Epstein-Barr virus-positive diffuse large B-cell lymphoma. The clinical and laboratory features were not different between Epstein-Barr virus-positive and -negative diffuse large B-cell lymphoma patients. Univariate analysis showed patients with diffuse large B-cell lymphoma that were either Epstein-Barr virus-positive or had activated B-cell-like features had an inferior overall survival. Older age, advanced stage and lymphoma with activated B-cell-like features or Epstein-Barr virus-encoded RNA positivity were independent prognostic factors affecting overall survival on multivariate analysis. Patients with two or three of these adverse-risk factors were considered high risk and fared far worse than patients with no or only one adverse factor. CONCLUSIONS: Taken together, we demonstrated that a higher frequency of Epstein-Barr virus association was detected in a Taiwanese cohort of diffuse large B-cell lymphoma patients, and Epstein-Barr virus-encoded RNA positivity was shown to add important prognostic value in these patients.

Glycosylation and S-palmitoylation regulate SARS-CoV-2 spike protein intracellular trafficking.

Post-translational modifications (PTMs), such as glycosylation and palmitoylation, are critical to protein folding, stability, intracellular trafficking, and function. Understanding regulation of PTMs of SARS-CoV-2 spike (S) protein could help the therapeutic drug design. Herein, the VSV vector was used to produce SARS-CoV-2 S pseudoviruses to examine the roles of the 611LYQD614 and cysteine-rich motifs in S protein maturation and virus infectivity. Our results show that 611LY612 mutation alters S protein intracellular trafficking and reduces cell surface expression level. It also changes S protein glycosylation pattern and decreases pseudovirus infectivity. The S protein contains four cysteine-rich clusters with clusters I and II as the main palmitoylation sites. Mutations of clusters I and II disrupt S protein trafficking from ER-to-Golgi, suppress pseudovirus production, and reduce spike-mediated membrane fusion activity. Taken together, glycosylation and palmitoylation orchestrate the S protein maturation processing and are critical for S protein-mediated membrane fusion and infection.

BMP4 is a peripherally-derived factor for motor neurons and attenuates glutamate-induced excitotoxicity in vitro.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor-beta (TGF-ß) superfamily, have been shown to play important roles in the nervous system, including neuronal survival and synaptogenesis. However, the physiological functions of BMP signaling in the mammalian neuromuscular system are not well understood. In this study, we found that proteins of the type II bone morphogenetic receptors (BMPRII) were detected at the neuromuscular junction (NMJ), and one of its ligands, BMP4, was expressed by Schwann cells and skeletal muscle fibers. In double-ligated nerves, BMP4 proteins accumulated at the proximal and distal portions of the axons, suggesting that Schwann cell- and muscle fiber-derived BMP4 proteins were anterogradely and retrogradely transported by motor neurons. Furthermore, BMP4 mRNA was down-regulated in nerves but up-regulated in skeletal muscles following nerve ligation. The motor neuron-muscle interactions were also demonstrated using differentiated C2C12 muscle cells and NG108-15 neurons in vitro. BMP4 mRNA and immunoreactivity were significantly up-regulated in differentiated C2C12 muscle cells when the motor neuron-derived factor, agrin, was present in the culture. Peripherally-derived BMP4, on the other hand, promotes embryonic motor neuron survival and protects NG108-15 neurons from glutamate-induced excitotoxicity. Together, these data suggest that BMP4 is a peripherally-derived factor that may regulate the survival of motor neurons.

Agrin induces association of Chrna1 mRNA and nicotinic acetylcholine receptor in C2C12 myotubes.

In the mammalian central nervous system transcripts of certain synaptic components are localized near the synapse, allowing for rapid regulation of protein levels. Here we test whether an mRNA localization mechanism also exists in the postsynaptic specialization induced by agrin in C2C12 myotubes. RT-PCR showed that Chrna1 was co-purified with nicotinic acetylcholine receptor (AChR) isolated by affinity column or by ultracentrifugation. In addition, Stau1 was found to interact with Chrna1 mRNA, and knocking down of Stau1 by RNAi resulted in defective AChR clustering. These results suggest that mRNA localization also participates in the formation of mammalian neuromuscular junction (NMJ).