Bok is a genuine multi-BH-domain protein that triggers apoptosis in the absence of Bax and Bak.

The pro-apoptotic multidomain Bcl-2 proteins Bax and Bak (also known as BAK1) are considered the gatekeepers of the intrinsic pathway of apoptosis by triggering the mitochondrial release of cytochrome c The role of the third Bax- and Bak-homologous multidomain protein Bok, however, is still unresolved. As cells doubly deficient for Bax and Bak are largely resistant to various apoptotic stimuli, it has been proposed that Bok is either dispensable for apoptosis or that its role is dependent on Bax and Bak. Here, we demonstrate, in several cell systems, that Bok efficiently induces cytochrome c release and apoptosis even in the complete absence of both Bak and Bax. Moreover, modulation of endogenous Bok levels affects the apoptosis response. By RNA interference and targeted deletion of the Bok gene, we demonstrate that Bok can significantly influence the apoptotic response to chemotherapeutic drugs in ovarian carcinoma cells. Hence, our results not only establish Bok as a Bak- and Bax-independent apoptosis inducer, but also suggest a potential impact of Bok expression in ovarian cancer therapy.

Interrogating Substrate Selectivity and Composition of Endogenous Histone Deacetylase Complexes with Chemical Probes.

Histone deacetylases (HDACs) regulate the function and activity of numerous cellular proteins by removing acetylation marks from regulatory lysine residues. We have developed peptide-based HDAC probes that contain hydroxamate amino acids of various lengths to replace modified lysine residues in the context of known acetylation sites. The interaction profiles of all human HDACs were studied with three sets of probes, which derived from different acetylation sites, and sequence context was found to have a strong impact on substrate recognition and composition of HDAC complexes. By investigating K382 acetylation of the tumor suppressor p53 as an example, we further demonstrate that the interaction profiles reflect the catalytic activities of respective HDACs. These results underline the utility of the newly established probes for deciphering not only activity, but also substrate selectivity and composition of endogenous HDAC complexes, which can hardly be achieved otherwise.

A second hotspot for pathogenic exon-skipping variants in CDC45.

Biallelic pathogenic variants in CDC45 are associated with Meier-Gorlin syndrome with craniosynostosis (MGORS type 7), which also includes short stature and absent/hypoplastic patellae. Identified variants act through a hypomorphic loss of function mechanism, to reduce CDC45 activity and impact DNA replication initiation. In addition to missense and premature termination variants, several pathogenic synonymous variants have been identified, most of which cause increased exon skipping of exon 4, which encodes an essential part of the RecJ-orthologue's DHH domain. Here we have identified a second cohort of families segregating CDC45 variants, where patients have craniosynostosis and a reduction in height, alongside common facial dysmorphisms, including thin eyebrows, consistent with MGORS7. Skipping of exon 15 is a consequence of two different variants, including a shared synonymous variant that is enriched in individuals of East Asian ancestry, while other variants in trans are predicted to alter key intramolecular interactions in α/ß domain II, or cause retention of an intron within the 3'UTR. Our cohort and functional data confirm exon skipping is a relatively common pathogenic mechanism in CDC45, and highlights the need for alternative splicing events, such as exon skipping, to be especially considered for variants initially predicted to be less likely to cause the phenotype, particularly synonymous variants.

Plural molecular and cellular mechanisms of pore domain KCNQ2 encephalopathy.

KCNQ2 variants in children with neurodevelopmental impairment are difficult to assess due to their heterogeneity and unclear pathogenic mechanisms. We describe a child with neonatal-onset epilepsy, developmental impairment of intermediate severity, and KCNQ2 G256W heterozygosity. Analyzing prior KCNQ2 channel cryoelectron microscopy models revealed G256 as a node of an arch-shaped non-covalent bond network linking S5, the pore turret, and the ion path. Co-expression with G256W dominantly suppressed conduction by wild-type subunits in heterologous cells. Ezogabine partly reversed this suppression. G256W/+ mice have epilepsy leading to premature deaths. Hippocampal CA1 pyramidal cells from G256W/+ brain slices showed hyperexcitability. G256W/+ pyramidal cell KCNQ2 and KCNQ3 immunolabeling was significantly shifted from axon initial segments to neuronal somata. Despite normal mRNA levels, G256W/+ mouse KCNQ2 protein levels were reduced by about 50%. Our findings indicate that G256W pathogenicity results from multiplicative effects, including reductions in intrinsic conduction, subcellular targeting, and protein stability. These studies provide evidence for an unexpected and novel role for the KCNQ2 pore turret and introduce a valid animal model of KCNQ2 encephalopathy. Our results, spanning structure to behavior, may be broadly applicable because the majority of KCNQ2 encephalopathy patients share variants near the selectivity filter.

There is no F in APC: Using physiological fluoride-free solutions for high throughput automated patch clamp experiments.

Fluoride has been used in the internal recording solution for manual and automated patch clamp experiments for decades because it helps to improve the seal resistance and promotes longer lasting recordings. In manual patch clamp, fluoride has been used to record voltage-gated Na (NaV) channels where seal resistance and access resistance are critical for good voltage control. In automated patch clamp, suction is applied from underneath the patch clamp chip to attract a cell to the hole and obtain a good seal. Since the patch clamp aperture cannot be moved to improve the seal like the patch clamp pipette in manual patch clamp, automated patch clamp manufacturers use internal fluoride to improve the success rate for obtaining GΩ seals. However, internal fluoride can affect voltage-dependence of activation and inactivation, as well as affecting internal second messenger systems and therefore, it is desirable to have the option to perform experiments using physiological, fluoride-free internal solution. We have developed an approach for high throughput fluoride-free recordings on a 384-well based automated patch clamp system with success rates >40% for GΩ seals. We demonstrate this method using hERG expressed in HEK cells, as well as NaV1.5, NaV1.7, and KCa3.1 expressed in CHO cells. We describe the advantages and disadvantages of using fluoride and provide examples of where fluoride can be used, where caution should be exerted and where fluoride-free solutions provide an advantage over fluoride-containing solutions.

Contribution of BH3-domain and Transmembrane-domain to the Activity and Interaction of the Pore-forming Bcl-2 Proteins Bok, Bak, and Bax.

Central to intrinsic apoptosis signaling is the release of cytochrome c from mitochondria, which depends on the pro-apoptotic effector proteins Bax, Bak or Bok. These pore-forming effector proteins share four Bcl-2 homology (BH) domains, a functionally essential and conserved sequence of hydrophobic amino acids in their BH3-domain and a C-terminal transmembrane-domain whose specific function remains rather unknown. To elucidate the molecular basis of Bok-mediated apoptosis we analyzed apoptosis induction by transmembrane-domain deficient BokΔTM compared to the respective Bax and Bak proteins and proteins in which the first leucine in the BH3-stretch was mutated to glutamic acid. We show that deletion of the C-terminal transmembrane-domain reduces the pro-apoptotic function of each protein. Mutation of the first leucine in the BH3-domain (L78E) blocks activity of Bak, while mutation of the homologue residues in Bax or Bok (L63E and L70E respectively) does not affect apoptosis induction. Unexpectedly, combined mutation of the BH3-domain and deletion of the transmembrane-domain enhances the pro-apoptotic activity of Bok(L70E)ΔTM by abolishing the interaction with anti-apoptotic proteins, especially the primary Bok-inhibitory protein Mcl-1. These results therefore suggest a specific contribution of the transmembrane-domain to the pro-apoptotic function and interaction of Bok.

Comparison of the non-invasive Nexfin® monitor with conventional methods for the measurement of arterial blood pressure in moderate risk orthopaedic surgery patients.

OBJECTIVE: Continuous invasive arterial blood pressure (IBP) monitoring remains the gold standard for BP measurement, but traditional oscillometric non-invasive intermittent pressure (NIBP) measurement is used in most low-to-moderate risk procedures. This study compared non-invasive continuous arterial BP measurement using a Nexfin® monitor with NIBP and IBP monitors. METHODS: This was a single-centre, prospective, pilot study in patients scheduled for elective orthopaedic surgery. Systolic BP, diastolic BP and mean arterial blood pressure (MAP) were measured by Nexfin®, IBP and NIBP at five intraoperative time-points. Pearson correlation coefficients, Bland-Altman plots and trending ability of Nexfin® measurements were used as criteria for success in the investigation of measurement reliability. RESULTS: A total of 20 patients were enrolled in the study. For MAP, there was a sufficient correlation between IBP/Nexfin® (Pearson = 0.75), which was better than the correlation between IBP/NIBP (Pearson = 0.70). Bland-Altman analysis of the data showed that compared with IBP, there was a higher percentage error for MAPNIBP (30%) compared with MAPNexfin® (27%). Nexfin® and NIBP underestimated systolic BP; NIBP also underestimated diastolic BP and MAP. Trending ability for MAPNexfin® and MAPNIBP were comparable to IBP. CONCLUSION: Non-invasive BP measurement with Nexfin® was comparable with IBP and tended to be more precise than NIBP.

Colloid-polymer mixtures in solution with refractive index matched acrylate colloids.

Colloid-polymer (CP) mixtures extend between two limiting cases, the colloid limit with the polymer coil size small compared to the colloid radius Rcol and the protein limit with the colloidal particles much smaller in size than the radius of gyration of the polymer chains Rg. In the present work, model systems are developed for the protein limit. The colloid-solvent pairs are optimized in terms of their isorefractivity in order to facilitate the characterization of large polystyrene chains in suspensions of small colloids. The degree of isorefractivity of colloidal particles was successfully evaluated in terms of a reduced scattering intensity. Two polystyrene samples with radii of gyration of Rg = 96 nm and Rg = 78 nm, respectively, are used. The radii of the colloidal particles are close to Rcol = 12 nm, leading to size ratios of Rg/Rcol = 8 and Rg/Rcol = 6.5. Four colloid solvent systems were found to be suitable for polymer characterization by light scattering, one based on silica particles and three systems with acrylate particles. The present investigation is focused on the three acrylate systems: poly(methyl methacrylate) in ethyl benzoate (ETB) at 7 degrees C, poly(ethyl methacrylate) in toluene at 7 degrees C and poly(ethyl methacrylate) in ETB at 40 degrees C. Characterization of PS chains is for the first time performed in colloid concentrations up to 2.5% by weight. In all cases, the size and shape of the polymer chains remain unchanged. A slight mismatch of the colloid scattering or a limited colloid solubility prevented investigation of PS chains at higher colloid concentration.