CRISPR-Cas9 Editing of Human Histone Deubiquitinase Gene USP16 in Human Monocytic Leukemia Cell Line THP-1

USP16 is a histone deubiquitinase which facilitates G2/M transition during the cell cycle, regulates DNA damage repair and contributes to inducible gene expression. We mutated the USP16 gene in a high differentiation clone of the acute monocytic leukemia cell line THP-1 using the CRISPR-Cas9 system and generated four homozygous knockout clones. All were able to proliferate and to differentiate in response to phorbol ester (PMA) treatment. One line was highly proliferative prior to PMA treatment and shut down proliferation upon differentiation, like wild type. Three clones showed sustained expression of the progenitor cell marker MYB, indicating that differentiation had not completely blocked proliferation in these clones. Network analysis of transcriptomic differences among wild type, heterozygotes and homozygotes showed clusters of genes that were up- or down-regulated after differentiation in all cell lines. Prior to PMA treatment, the homozygous clones had lower levels than wild type of genes relating to metabolism and mitochondria, including SRPRB, encoding an interaction partner of USP16. There was also apparent loss of interferon signaling. In contrast, a number of genes were up-regulated in the homozygous cells compared to wild type at baseline, including other deubiquitinases (USP12, BAP1, and MYSM1). However, three homozygotes failed to fully induce USP3 during differentiation. Other network clusters showed effects prior to or after differentiation in the homozygous clones. Thus the removal of USP16 affected the transcriptome of the cells, although all these lines were able to survive, which suggests that the functions attributed to USP16 may be redundant. Our analysis indicates that the leukemic line can adapt to the extreme selection pressure applied by the loss of USP16, and the harsh conditions of the gene editing and selection protocol, through different compensatory pathways. Similar selection pressures occur during the evolution of a cancer in vivo, and our results can be seen as a case study in leukemic cell adaptation. USP16 has been considered a target for cancer chemotherapy, but our results suggest that treatment would select for escape mutants that are resistant to USP16 inhibitors

Search for CP violation in the decay B0->D*+-D-+

We report a search for CP-violating asymmetry in B0 -> D*+- D-+ decays. The analysis employs two methods of B0 reconstruction: full and partial. In the full reconstruction method all daughter particles of the B0 are required to be detected; the partial reconstruction technique requires a fully reconstructed D- and only a slow pion from the D*+ -> D0 pi_slow+ decay. From a fit to the distribution of the time interval corresponding to the distance between two B meson decay points we calculate the CP-violating parameters and find the significance of nonzero CP asymmetry to be 2.7 standard deviations.Comment: 4 pages, 3 figure

Studies of the Decay B+- -> D_CP K+-

We report studies of the decay B+- -> D_CP K+-, where D_CP denotes neutral D mesons that decay to CP eigenstates. The analysis is based on a 29.1/fb data sample of collected at the \Upsilon(4S) resonance with the Belle detector at the KEKB asymmetric e+ e- storage ring. Ratios of branching fractions of Cabibbo-suppressed to Cabibbo-favored processes involving D_CP are determined to be B(B- -> D_1 K-)/B(B- -> D_1 pi-)=0.125 +- 0.036 +- 0.010 and B(B- -> D_2 K-)/B(B- -> D_2 pi-)=0.119 +- 0.028 +- 0.006, where indices 1 and 2 represent the CP=+1 and CP=-1 eigenstates of the D0 - anti D0 system, respectively. We also extract the partial rate asymmetries for B+- -> D_CP K+-, finding A_1 = 0.29 +- 0.26 +- 0.05 and A_2 = -0.22 +- 0.24 +- 0.04.Comment: 10 pages, 2 figures, submitted to Physical Review Letter

Systematic Mutational Analysis of the Intracellular Regions of Yeast Gap1 Permease

The yeast general amino acid permease Gap1 is a convenient model for studying the intracellular trafficking of membrane proteins. Present at the plasma membrane when the nitrogen source is poor, it undergoes ubiquitin-dependent endocytosis and degradation upon addition of a good nitrogen source, e.g. ammonium. It comprises 12 transmembrane domains (TM) flanked by cytosol-facing N- and C-terminal tails (NT, CT). The NT of Gap1 contains the acceptor lysines for ubiquitylation and its CT includes a sequence essential to exit from the endoplasmic reticulum (ER).Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe

African American patients with gout: efficacy and safety of febuxostat vs allopurinol

<p>Abstract</p> <p>Background</p> <p>African Americans are twice as likely as Caucasians to develop gout, but they are less likely to be treated with urate-lowering therapy (ULT). Furthermore, African Americans typically present with more comorbidities associated with gout, such as hypertension, obesity, and renal impairment. We determined the efficacy and safety of ULT with febuxostat or allopurinol in African American subjects with gout and associated comorbidities and in comparison to Caucasian gout subjects.</p> <p>Methods</p> <p>This is a secondary analysis of the 6-month Phase 3 CONFIRMS trial. Eligible gouty subjects with baseline serum urate (sUA) ≥ 8.0 mg/dL were randomized 1:1:1 to receive febuxostat 40 mg, febuxostat 80 mg, or allopurinol (300 mg or 200 mg depending on renal function) daily. All subjects received gout flare prophylaxis. Primary efficacy endpoint was the proportion of subjects in each treatment group with sUA < 6.0 mg/dL at the final visit. Additional endpoints included the proportion of subjects with mild or with moderate renal impairment who achieved a target sUA < 6.0 mg/dL at final visit. Adverse events (AEs) were recorded throughout the study.</p> <p>Results</p> <p>Of the 2,269 subjects enrolled, 10.0% were African American and 82.1% were Caucasian. African American subjects were mostly male (89.5%), obese (BMI ≥ 30 kg/m²; 67.1%), with mean baseline sUA of 9.8 mg/dL and mean duration of gout of 10.4 years. The proportions of African American subjects with a baseline history of diabetes, renal impairment, or cardiovascular disease were significantly higher compared to Caucasians (<it>p </it>< 0.001). ULT with febuxostat 80 mg was superior to both febuxostat 40 mg (<it>p </it>< 0.001) and allopurinol (<it>p </it>= 0.004). Febuxostat 40 mg was comparable in efficacy to allopurinol. Significantly more African American subjects with mild or moderate renal impairment achieved sUA < 6.0 mg/dL in the febuxostat 80 group than in either the febuxostat 40 mg or allopurinol group (<it>p </it>< 0.05). Efficacy rates in all treatment groups regardless of renal function were comparable between African American and Caucasian subjects, as were AE rates.</p> <p>Conclusions</p> <p>In African American subjects with significant comorbidities, febuxostat 80 mg is significantly more efficacious than either febuxostat 40 mg or allopurinol 200/300 mg. Febuxostat was well tolerated in this African American population.</p> <p>Please see related article: <url>http://www.biomedcentral.com/1741-7015/10/15</url></p

Prokineticin 2 Regulates the Electrical Activity of Rat Suprachiasmatic Nuclei Neurons

Neuropeptide signaling plays roles in coordinating cellular activities and maintaining robust oscillations within the mammalian suprachiasmatic nucleus (SCN). Prokineticin2 (PK2) is a signaling molecule from the SCN and involves in the generation of circadian locomotor activity. Prokineticin receptor 2 (PKR2), a receptor for PK2, has been shown to be expressed in the SCN. However, very little is known about the cellular action of PK2 within the SCN. In the present study, we investigated the effect of PK2 on spontaneous firing and miniature inhibitory postsynaptic currents (mIPSCs) using whole cell patch-clamp recording in the SCN slices. PK2 dose-dependently increased spontaneous firing rates in most neurons from the dorsal SCN. PK2 acted postsynaptically to reduce γ-aminobutyric acid (GABA)-ergic function within the SCN, and PK2 reduced the amplitude but not frequency of mIPSCs. Furthermore, PK2 also suppressed exogenous GABA-induced currents. And the inhibitory effect of PK2 required PKC activation in the postsynaptic cells. Our data suggest that PK2 could alter cellular activities within the SCN and may influence behavioral and physiological rhythms

Mitochondrial Changes in Ageing Caenorhabditis elegans – What Do We Learn from Superoxide Dismutase Knockouts?

One of the most popular damage accumulation theories of ageing is the mitochondrial free radical theory of ageing (mFRTA). The mFRTA proposes that ageing is due to the accumulation of unrepaired oxidative damage, in particular damage to mitochondrial DNA (mtDNA). Within the mFRTA, the “vicious cycle” theory further proposes that reactive oxygen species (ROS) promote mtDNA mutations, which then lead to a further increase in ROS production. Recently, data have been published on Caenorhabditis elegans mutants deficient in one or both forms of mitochondrial superoxide dismutase (SOD). Surprisingly, even double mutants, lacking both mitochondrial forms of SOD, show no reduction in lifespan. This has been interpreted as evidence against the mFRTA because it is assumed that these mutants suffer from significantly elevated oxidative damage to their mitochondria. Here, using a novel mtDNA damage assay in conjunction with related, well established damage and metabolic markers, we first investigate the age-dependent mitochondrial decline in a cohort of ageing wild-type nematodes, in particular testing the plausibility of the “vicious cycle” theory. We then apply the methods and insights gained from this investigation to a mutant strain for C. elegans that lacks both forms of mitochondrial SOD. While we show a clear age-dependent, linear increase in oxidative damage in WT nematodes, we find no evidence for autocatalytic damage amplification as proposed by the “vicious cycle” theory. Comparing the SOD mutants with wild-type animals, we further show that oxidative damage levels in the mtDNA of SOD mutants are not significantly different from those in wild-type animals, i.e. even the total loss of mitochondrial SOD did not significantly increase oxidative damage to mtDNA. Possible reasons for this unexpected result and some implications for the mFRTA are discussed