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Sequential genotyping for phenotype-driver mutations in JAK2 (exon 14), CALR (exon 9), and MPL (exon 10) is recommended in patients with myeloproliferative neoplasms. Yet, atypical JAK2- and MPL-mutations were described in some triple-negative patients. Whether noncanonical and/or concomitant JAK2- and MPL-mutations exist in myelofibrosis (MF) regardless of phenotype-driver mutations is not yet elucidated. For this, next-generation sequencing (NGS) was performed using blood genomic DNA from 128 MF patients (primary MF, n = 93; post-ET-MF, n = 18; post-PV-MF, n = 17). While no atypical JAK2- or MPL-mutations were seen in 24 CALR-positive samples, two JAK2-mutations [c.3323A > G, p.N1108S; c.3188G > A, p.R1063H] were detected in two of the 21 (9.5%) triple-negative patients. Twelve of the 82 (14.6%) JAK2V617F-positive cases had coexisting germline JAK2-mutations [JAK2R1063H, n = 6; JAK2R893T, n = 1; JAK2T525A, n = 1] or at least one somatic MPL-mutation [MPLY591D, n = 3; MPLW515 L, n = 2; MPLE335K, n = 1]. Overall, MPL-mutations always coexisted with JAK2V617F and/or other MPL-mutations. None of the JAK2V617F plus a second JAK2-mutation carried a TET2-mutation but all patients with JAK2V617F plus an MPL-mutation harbored a somatic TET2-mutation. Four genomic clusters could be identified in the JAK2V617F-positive cohort. Cluster-I (10%) (noncanonical JAK2mutated (mut) + TET2wildtype (wt) ) were younger and had less proliferative disease compared with cluster-IV (5%) (TET2mut + MPLmut ). In conclusion, recurrent concomitant classical and/or noncanonical JAK2- and MPL-mutations could be detected by NGS in 15.7% of JAK2V617F- and MPLW515-positive MF patients with genotype-phenotype associations. Many of the germline and/or somatic mutations might act as "Significantly Mutated Genes" contributing to the pathogenesis and phenotypic heterogeneity. A cost-effective NGS-based approach might be an important step towards patient-tailored medicine.

Background: Kcnh1 is a voltage-gated potassium channel that is primarily expressed in brain. Results: Knockdown of kcnh1 in zebrafish delays neural development and causes embryonic lethality. Conclusion: Kcnh1 is involved in cell proliferation during early zebrafish development. Significance: The finding that Kcnh1 has basic functions beyond neural signaling will help to elucidate its roles in physiology and cancer formation., The Kcnh1 gene encodes a voltage-gated potassium channel highly expressed in neurons and involved in tumor cell proliferation, yet its physiological roles remain unclear. We have used the zebrafish as a model to analyze Kcnh1 function in vitro and in vivo. We found that the kcnh1 gene is duplicated in teleost fish (i.e. kcnh1a and kcnh1b) and that both genes are maternally expressed during early development. In adult zebrafish, kcnh1a and kcnh1b have distinct expression patterns but share expression in brain and testis. Heterologous expression of both genes in Xenopus oocytes revealed a strong conservation of characteristic functional properties between human and fish channels, including a unique sensitivity to intracellular Ca2+/calmodulin and modulation of voltage-dependent gating by extracellular Mg2+. Using a morpholino antisense approach, we demonstrate a strong kcnh1 loss-of-function phenotype in developing zebrafish, characterized by growth retardation, delayed hindbrain formation, and embryonic lethality. This late phenotype was preceded by transcriptional up-regulation of known cell-cycle inhibitors (p21, p27, cdh2) and down-regulation of pro-proliferative factors, including cyclin D1, at 70% epiboly. These results reveal an unanticipated basic activity of kcnh1 that is crucial for early embryonic development and patterning.

The physiological role of KCNH1 channels has not been clarified in detail yet, but the predominant neuronal expression in mammals indicates a role in electrical signaling. The only described physiological function of KCNH1 so far is the promotion of myoblast fusion. Moreover, KCNH1 channels apparently enhance proliferation of cancer cells, thus exhibiting oncogenic potential. Many genes involved in cancerogenesis play a physiological role during embryonic development. To study KCNH1 channel function in this respect, we surveyed genomic databases of Danio rerio, a widely used vertebrate-development model organism, and found two putative kcnh1 paralogs located on chromosomes 17 (kcnh1a) and 22 (kcnh1b). The corresponding amino acid sequences show >80% identity to that of human KCNH1. We observed both kcnh1 genes to be endogenously expressed in different organs of adult fish and at early developmental stages, most prominently in neuronal tissues. We electrophysiologically analyzed the encoded channels by two-electrode voltage-clamp and patch-clamp techniques on Xenopus oocytes, showing the gene products to form functional potassium channels. The Danio rerio channels exhibited typical characteristics known from other species, such as dependence of activation kinetics on prepulse potential and extracellular Mg2+ concentration, as well as current inhibition by intracellular Ca2+/calmodulin. After morpholino-mediated knockdown of the individual kcnh1 paralogs in zebrafish embryos we observed severe effects on development. The morphants were retarded in growth, showed abnormal body curvature, exhibited complete body malformation or even died within less than 24 hrs after fertilization, depending on the degree of knockdown. The most remarkable phenotypes were alterations of brain structures like head edemas, incomplete brain growth and necrotic degeneration. Given the electrophysiological similarity of Kcnh1 channels to the human homolog, we suggest that human KCNH1 potassium channels play a similar role during early embryonic development in humans.

Allogeneic HCT is curative in myelofibrosis (MF) with a survival (OS) advantage compared to non-JAK-inhibitor treatments in patients (pts) with Int-2/high-risk DIPSS (Kroeger et al, Blood 2015). On the other hand, data demonstrate a OS benefit of the JAK1/JAK2 inhibitor ruxolitinib (RUX) in intermediate (Int)-2/high-risk IPSS (Cervantes et al, Blood 2013; Verstovsek et al, Haematologica 2015). Yet, comparative data to the impact of RUX and HCT on OS are lacking. This was pursued retrospectively at the University of Leipzig in light of clinical-, cytogenetic-, phenotype-driver-, and somatic mutations-based prognostic variables. Patient and Methods: RUX became available in November, 2009. MF pts (n=137; median age 58y) seen afterwards were included with the exception of MPL + pts (small number). Characteristics are shown in table 1. RUX was given for spleenomegaly and/or constitutional symptoms [n=76 (55%)]. Irrespective of RUX, a donor search was initiated in int-2 and high-risk IPSS pts < 70 y and HCT performed if a suitable donor identified. Based on this biologic randomization, 50 (37%) received HCT, 58 (42%) continued with RUX (median exposure 19 months), and 29 (21%) pts had other options. The RUX- and HCT-groups were balanced in terms of IPSS, platelets, cytogenetic-, and epigenetic molecular-risks. Treatment was initiated within the first year (median 10 months), thus probability of OS 5 years after diagnosis was calculated (SPSS20 software). Cytogenetic-risk was conducted according to Caramazza et al. Leukemia 2011. Somatic mutations detected by NGS were used to calculate molecular-risk (Vannucchi et al. Leukemia 2013). Results: Median OS time was 6.8 y. The RUX-group was older (median age 64 y) with profound spleenomegaly and a higher JAK2 V617F allele burden compared to the HCT group (median age 55 y) (p50 y (p=0.001). In the RUX group, age 60 y (p=0.02)]. No prognostic implication of anemia, WBC, peripheral blasts, and constitutional symptoms could be detected with both treatments. OS in JAK2 + pts was similar to CALR + pts in both therapy cohorts (p=0.3). Triple negativity retained its detrimental influence (p Conclusions: Our data imply that, besides allogeneic HCT, a prolonged JAK1/JAK2 inhibition could, at least partly, attenuate most of the prognostic detrimental parameters. Pre-treatment with ruxolitinib prior to HCT does not seem to have a negative impact on survival. It is the first time where outcome with a non-transplant procedure is shown to be comparable to that after HCT in MF, even in high-risk disease. This needs to be verified in prospective randomized trials to define the role of allogeneic HCT in the era of JAK1/JAK2 inhibition. Table 1. Patients characteristics ( n = 137 ) Variable Impact on OS Impact on LT Median age (years) 58 0.002 0.6 JAK2+/CALR+/Double negative (%) 63 / 20 / 17 Disclosures Roskos: Novartis: Honoraria. Al-Ali:Celgene: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding.

Twin arginine translocation (Tat) systems catalyze the transport of folded proteins across the bacterial cytosolic membrane or the chloroplast thylakoid membrane. In the Tat systems of Escherichia coli and many other species TatA-, TatB-, and TatC-like proteins have been identified as essential translocase components. In contrast, the Bacillus subtilis phosphodiesterase PhoD-specific system consists only of a pair of TatA(d)/TatC(d) proteins and involves a TatA(d) protein engaged in a cytosolic and a membrane-embedded localization. Because soluble TatA(d) was able to bind the twin arginine signal peptide of prePhoD prior to membrane integration it could serve to recruit its substrate to the membrane via the interaction with TatC(d). By analyzing the distribution of TatA(d) and studying the mutual affinity with TatC(d) we have shown here that TatC(d) assists the membrane localization of TatA(d). Besides detergent-solubilized TatC(d), membrane-integrated TatC(d) showed affinity for soluble TatA(d). By using a peptide library-specific binding of TatA(d) to cytosolic loops of membrane protein TatC(d) was demonstrated. Depletion of TatC(d) in B. subtilis resulted in a drastic reduction of TatA(d), indicating a stabilizing effect of TatC(d) for TatA(d). In addition, the presence of the substrate prePhoD was the prerequisite for appropriate localization in the cytosolic membrane of B. subtilis as demonstrated by freeze-fracture experiments.

The reactivity of heteroarylic ketones toward vinylmagnesium bromide (2) and the regiochemistry of the addition were investigated. The reactivity drastically increases when the carbonyl is conjugated with at least one aza group and the regiochemistry of the addition of the vinyl Grignard reagent depends on the carbonyl compound: in the series of di(heteroazolyl) ketones the O-alkylation product was observed as unique with di(1,3-benzothiazol-2-yl) ketone, and in different relative ratios with respect to the classic C-alkylation product with di(1,3-thiazol-2-yl) ketone, (1,3-benzothiazol-2- yl) (1,3-thiazol-2-yl) ketone, and di(1,3-benzoxazol-2-yl) ketone, whereas di(N-methylbenzimidazol-2- yl) ketone gave the exclusive formation of the carbinol. This behavior can be explained by the intervention of the delocalization power of the heterocyclic ring and this was confirmed by the results obtained from the reaction between vinylmagnesium bromide and a series of mixed (1,3- benzothiazol-2-yl) (para-substituted phenyl) ketones, that showed a relative O-/C-alkylation ratio dependent on the nature and on the electronic effect of the substituent on the phenyl ring. The results are in agreement with the existence of intermediate species bearing a negative charge on the benzylic carbonyl carbon atom, and make the O-alkylation reaction between vinyl Grignard reagents and carbonyl compounds no longer a rare case, since it was observed with a number of heterocyclic carbonyl compounds, such as (1,3-benzothiazol-2-yl) aryl ketones and di(heteroaryl) ketones of the pentatomic 1,3-heteroazolic series.