PCTAIRE1 promotes mitotic progression and resistance against antimitotic and apoptotic signals.

PCTAIRE1 (also known as CDK16) is a serine-threonine kinase implicated in physiological processes like neuronal development, vesicle trafficking, spermatogenesis and cell proliferation. However, its exact role in cell division remains unclear. In this study, using a library screening approach, we identified PCTAIRE1 among several candidates that resisted mitotic arrest and mitotic cell death induced by polyomavirus small T (PolST) expression in mammalian cells. Our study showed that PCTAIRE1 is a mitotic kinase that localizes at centrosomes during G2 and at spindle poles as the cells enter mitosis, and then at the midbody during cytokinesis. We also report that PCTAIRE1 protein levels fluctuate through the cell cycle and reach their peak at mitosis, during which there is an increase in PCTAIRE1 phosphorylation as well. Interestingly, knockdown of PCTAIRE1 resulted in aberrant mitosis by interfering with spindle assembly and chromosome segregation. Further, we found that PCTAIRE1 promotes resistance of cancer cells to antimitotic drugs, and this underscores the significance of PCTAIRE1 as a potential drug target for overcoming chemotherapeutic resistance. Taken together, these studies establish PCTAIRE1 as a critical mediator of mitotic progression and highlight its role in chemotherapeutic resistance. This article has an associated First Person interview with the first author of the paper.

Simultaneous Measurement of Single-Cell Mechanics and Cell-to-Materials Adhesion Using Fluidic Force Microscopy.

The connection between cells and their substrate is essential for biological processes such as cell migration. Atomic force microscopy nanoindentation has often been adopted to measure single-cell mechanics. Very recently, fluidic force microscopy has been developed to enable rapid measurements of cell adhesion. However, simultaneous characterization of the cell-to-material adhesion and viscoelastic properties of the same cell is challenging. In this study, we present a new approach to simultaneously determine these properties for single cells, using fluidic force microscopy. For MCF-7 cells grown on tissue-culture-treated polystyrene surfaces, we found that the adhesive force and adhesion energy were correlated for each cell. Well-spread cells tended to have stronger adhesion, which may be due to the greater area of the contact between cellular adhesion receptors and the surface. By contrast, the viscoelastic properties of MCF-7 cells cultured on the same surface appeared to have little dependence on cell shape. This methodology provides an integrated approach to better understand the biophysics of multiple cell types.

Polo-like kinase-1 triggers histone phosphorylation by Haspin in mitosis.

Histone modifications coordinate the chromatin localization of key regulatory factors in mitosis. For example, mitotic phosphorylation of Histone H3 threonine-3 (H3T3ph) by Haspin creates a binding site for the chromosomal passenger complex (CPC). However, how these histone modifications are spatiotemporally controlled during the cell cycle is unclear. Here we show that Plk1 binds to Haspin in a Cdk1-phosphorylation-dependent manner. Reducing Plk1 activity decreases the phosphorylation of Haspin and inhibits H3T3ph, particularly in prophase, suggesting that Plk1 is required for initial activation of Haspin in early mitosis. These studies demonstrate that Plk1 can positively regulate CPC recruitment in mitosis.

A phospho/methyl switch at histone H3 regulates TFIID association with mitotic chromosomes.

Histone methylation patterns are correlated with eukaryotic gene transcription. High-affinity binding of the plant homeodomain (PHD) of TFIID subunit TAF3 to trimethylated lysine-4 of histone H3 (H3K4me3) is involved in promoter recruitment of this basal transcription factor. Here, we show that for transcription activation the PHD of TAF3 can be replaced by PHDs of other high-affinity H3K4me3 binders. Interestingly, H3K4me3 binding of TFIID and the TAF3-PHD is decreased by phosphorylation of the adjacent threonine residue (H3T3), which coincides with mitotic inhibition of transcription. Ectopic expression of the H3T3 kinase haspin repressed TAF3-mediated transcription of endogenous and of reporter genes and decreased TFIID association with chromatin. Conversely, immunofluorescence and live-cell microscopy studies showed an increased association of TFIID with mitotic chromosomes upon haspin knockdown. Based on our observations, we propose that a histone H3 phospho-methyl switch regulates TFIID-mediated transcription during mitotic progression of the cell cycle.

Grayscale median analysis of primary stenosis and restenosis after carotid endarterectomy.

BACKGROUND: Several studies have reported that echolucent carotid lesions, as determined by grayscale median (GSM) analysis, are associated with increased perioperative embolic complications during carotid artery stenting (CAS). However, there is limited research of the predictive value of GSM analysis comparing values for primary atherosclerotic lesions in the carotid artery with those for recurrent lesions after carotid endarterectomy (CEA). METHODS: Retrospective data were collected and analyzed from all patients undergoing CAS from November 2005 to August 2010. Available preoperative images amenable to GSM analysis were processed in Adobe Photoshop (version CS4; San Jose, Calif). Statistical analysis included t-test, Fischer exact test, and generation of a receiver operating characteristic curve. RESULTS: With at least 29 days of follow-up, 212 patients underwent 228 CAS procedures. There were 189 stents placed for primary lesions (CAS for primary stenosis group) and 39 stents placed for restenosis after CEA (CAS for restenosis group). GSM analysis was feasible for 47 patients, and the mean GSM was 45.6 (n = 34; 95% confidence interval, ± 8.3) for the primary stenosis group and 20.5 (n = 13; 95% confidence interval, ±9.6) for the restenosis group (P < .01). The mean time from CEA to CAS intervention for the restenosis group was 8.6 years. There was no statistical difference in procedural individual and combined complications of ipsilateral stroke, 30-day stroke, or 30-day mortality between the CAS for primary stenosis group and the CAS for restenosis group. In the primary stenosis group, the mean GSM was lower in those with procedural complications compared with those without complications (15 ± 22 vs 49 ± 8; P = .02). CONCLUSIONS: A low GSM value was associated with increased perioperative risk when CAS was performed for native carotid lesions, but a low GSM value was not associated with higher procedural risk when carotid stenting was performed for carotid stenosis after CEA (restenosis). GSM analysis for restenosis may be altered by the time interval from CEA to restenosis.

Integrin αEß7: molecular features and functional significance in the immune system.

Alpha E beta 7 (αEß7) is an α-I domain-containing integrin that is highly expressed by a variety of leukocyte populations at mucosal sites including intraepithelial T cells, dendritic cells, mast cells, and T regulatory cells (Treg). Expression depends largely or solely on transforming growth factor beta (TGF-ß) isoforms. The best characterized ligand for αEß7 is E-cadherin on epithelial cells, though there is evidence of a second ligand in the human system. An exposed acidic residue on the distal aspect of E-cadherin domain 1 interacts with the MIDAS site in the αE α-I domain. By binding to E-cadherin, αEß7 contributes to mucosal specific retention of leukocytes within epithelia. Studies on αE knockout mice have identified an additional important function for this integrin in allograft rejection and have also indicated that it may have a role in immunoregulation. Recent studies point to a multifaceted role for αEß7 in regulating both innate and acquired immune responses to foreign antigen.

Avoiding ecosystem and social impacts of hydropower, wind, and solar in Southern Africa's low-carbon electricity system.

The scale at which low-carbon electricity will need to be deployed to meet economic growth, electrification, and climate goals in Africa is unprecedented, yet the potential land use and freshwater impacts from this massive build-out of energy infrastructure is poorly understood. In this study, we characterize low-impact onshore wind, solar photovoltaics, and hydropower potential in Southern Africa and identify the cost-optimal mix of electricity generation technologies under different sets of socio-environmental land use and freshwater constraints and carbon targets. We find substantial wind and solar potential after applying land use protections, but about 40% of planned or proposed hydropower projects face socio-environmental conflicts. Applying land and freshwater protections results in more wind, solar, and battery capacity and less hydropower capacity compared to scenarios without protections. While a carbon target favors hydropower, the amount of cost-competitively selected hydropower is at most 45% of planned or proposed hydropower capacity in any scenario-and is only 25% under socio-environmental protections. Achieving both carbon targets and socio-environmental protections results in system cost increases of 3-6%. In the absence of land and freshwater protections, environmental and social impacts from new hydropower development could be significant.

Distal revascularization and interval ligation procedure for radial-basilic forearm transposition arteriovenous fistula.

The distal revascularization and interval ligation procedure is commonly performed for steal syndrome in upper arm arteriovenous accesses and is rarely performed in the forearm. We present a case of distal revascularization and interval ligation procedure performed for a 60-year-old male who presented with a 3-month history of a nonhealing ulcer of his left middle finger as a result of ischemic steal syndrome 4 years after having a left radial-basilic forearm transposition arteriovenous fistula.

Outcomes comparison of HeRO and lower extremity arteriovenous grafts in patients with long-standing renal failure.

OBJECTIVE: The Hemodialysis Reliable Outflow (HeRO) graft is becoming a recognized alternative to lower extremity arteriovenous grafts (LEAVGs) as an option for patients who have exhausted traditional upper extremity access; however, which should be applied preferentially is unclear. METHODS: A retrospective review of LEAVG and HeRO implants from January 2004 to August 2010 was performed. Patient demographics, medical history, procedural data, and outcomes were evaluated. RESULTS: Within the time periods, 60 HeROs were placed in 59 patients and 22 LEAVGs were placed in 21 patients. Demographics were similar between the two groups for many factors; however, the patients who underwent HeRO placement had significantly higher body mass index compared with the LEAVG group. Mean follow-up was 13.9 months for the HeRO group and 11.9 months for the LEAVG group. The HeRO patients underwent a mean of 6.3 previous tunneled dialysis catheter insertions and 3.1 previous AVG/arteriovenous fistula placements. The LEAVG patients underwent placement of a mean of 4.1 previous tunneled dialysis catheters and 2.6 previous AVG/arteriovenous fistulas. The principal difference was the number of interventions to maintain patency, which was 2.21 per year in the HeRO group and 1.17 per year in the AVG group (P = .003) Secondary patency at 6 months was 77% for the HeRO patients and 83% for the LEAVG patients (P = .14). The HeRO and LEAVG groups had no difference in infection rate per 1000 days (0.61 vs 0.71; P = .77) or mortality rate (22% vs 19% respectively; P = .22) at 6 months. CONCLUSIONS: In access challenged patients, LEAVG and HeRO offer similar rates of secondary patency, infection, and all-cause mortality. The LEAVG required fewer interventions to maintain patency, and the HeRO maintains the benefit of utilizing the upper extremity site of venous drainage. In our practice, we prefer the HeRO to LEAVG, especially in patients with peripheral arterial disease and in the obese population, because it preserves lower extremity access options.

Acute brain slice elastic modulus decreases over time.

A common benchmark in the brain tissue mechanics literature is that the properties of acute brain slices should be measured within 8 h of the experimental animal being sacrificed. The core assumption is that-since there is no substantial protein degradation during this time-there will be no change to elastic modulus. This assumption overlooks the possibility of other effects (such as osmotic swelling) that may influence the mechanical properties of the tissue. To achieve consistent and accurate analysis of brain mechanics, it is important to account for or mitigate these effects. Using atomic force microscopy (AFM), tissue hydration and volume measurements, we find that acute brain slices in oxygenated artificial cerebrospinal fluid (aCSF) with a standard osmolarity of 300 mOsm/l experience rapid swelling, softening, and increases in hydration within the first 2 hours after slicing. Reductions in elastic modulus can be partly mitigated by addition of chondroitinase ABC enzyme (CHABC). Increasing aCSF osmolarity to 400 mOsm/l does not prevent softening but may hasten equilibration of samples to a point where measurements of relative elastic modulus are consistent across experiments.

Pharmacological approaches to understanding protein kinase signaling networks.

Protein kinases play vital roles in controlling cell behavior, and an array of kinase inhibitors are used successfully for treatment of disease. Typical drug development pipelines involve biological studies to validate a protein kinase target, followed by the identification of small molecules that effectively inhibit this target in cells, animal models, and patients. However, it is clear that protein kinases operate within complex signaling networks. These networks increase the resilience of signaling pathways, which can render cells relatively insensitive to inhibition of a single kinase, and provide the potential for pathway rewiring, which can result in resistance to therapy. It is therefore vital to understand the properties of kinase signaling networks in health and disease so that we can design effective multi-targeted drugs or combinations of drugs. Here, we outline how pharmacological and chemo-genetic approaches can contribute to such knowledge, despite the known low selectivity of many kinase inhibitors. We discuss how detailed profiling of target engagement by kinase inhibitors can underpin these studies; how chemical probes can be used to uncover kinase-substrate relationships, and how these tools can be used to gain insight into the configuration and function of kinase signaling networks.

Release of Histone H3K4-reading transcription factors from chromosomes in mitosis is independent of adjacent H3 phosphorylation.

Histone modifications influence the recruitment of reader proteins to chromosomes to regulate events including transcription and cell division. The idea of a histone code, where combinations of modifications specify unique downstream functions, is widely accepted and can be demonstrated in vitro. For example, on synthetic peptides, phosphorylation of Histone H3 at threonine-3 (H3T3ph) prevents the binding of reader proteins that recognize trimethylation of the adjacent lysine-4 (H3K4me3), including the TAF3 component of TFIID. To study these combinatorial effects in cells, we analyzed the genome-wide distribution of H3T3ph and H3K4me2/3 during mitosis. We find that H3T3ph anti-correlates with adjacent H3K4me2/3 in cells, and that the PHD domain of TAF3 can bind H3K4me2/3 in isolated mitotic chromatin despite the presence of H3T3ph. Unlike in vitro, H3K4 readers are still displaced from chromosomes in mitosis in Haspin-depleted cells lacking H3T3ph. H3T3ph is therefore unlikely to be responsible for transcriptional downregulation during cell division.

Structure-activity relationship study of beta-carboline derivatives as haspin kinase inhibitors.

Haspin is a serine/threonine kinase that phosphorylates Thr-3 of histone H3 in mitosis that has emerged as a possible cancer therapeutic target. High throughput screening of approximately 140,000 compounds identified the beta-carbolines harmine and harmol as moderately potent haspin kinase inhibitors. Based on information obtained from a structure-activity relationship study previously conducted for an acridine series of haspin inhibitors in conjunction with in silico docking using a recently disclosed crystal structure of the kinase, harmine analogs were designed that resulted in significantly increased haspin kinase inhibitory potency. The harmine derivatives also demonstrated less activity towards DYRK2 compared to the acridine series. In vitro mouse liver microsome stability and kinase profiling of a representative member of the harmine series (42, LDN-211898) are also presented.

Structure and functional characterization of the atypical human kinase haspin.

The protein kinase haspin/Gsg2 plays an important role in mitosis, where it specifically phosphorylates Thr-3 in histone H3 (H3T3). Its protein sequence is only weakly homologous to other protein kinases and lacks the highly conserved motifs normally required for kinase activity. Here we report structures of human haspin in complex with ATP and the inhibitor iodotubercidin. These structures reveal a constitutively active kinase conformation, stabilized by haspin-specific inserts. Haspin also has a highly atypical activation segment well adapted for specific recognition of the basic histone tail. Despite the lack of a DFG motif, ATP binding to haspin is similar to that in classical kinases; however, the ATP gamma-phosphate forms hydrogen bonds with the conserved catalytic loop residues Asp-649 and His-651, and a His651Ala haspin mutant is inactive, suggesting a direct role for the catalytic loop in ATP recognition. Enzyme kinetic data show that haspin phosphorylates substrate peptides through a rapid equilibrium random mechanism. A detailed analysis of histone modifications in the neighborhood of H3T3 reveals that increasing methylation at Lys-4 (H3K4) strongly decreases substrate recognition, suggesting a key role of H3K4 methylation in the regulation of haspin activity.

Robustness of NanoBiT luciferase complementation technology in the presence of widely used kinase inhibitors.

Bioluminescence assays using luciferase enzymes are widely used in research to monitor gene expression and an array of other cell properties, and split luciferase enzymes can be used to measure protein interactions in biochemical assays and in living cells. When these methods are employed in chemical library screening efforts, it is vital that the activity of the luciferase enzyme itself is not strongly influenced by library components. Here, we developed a NanoBiT split luciferase assay to measure phosphorylation of Histone H3 peptides and used it to test the robustness of split luciferase to interference from two libraries of commonly used kinase inhibitors, including the Kinase Chemogenomic Set (KCGS). We found that NanoBiT luciferase is not significantly affected by the great majority of kinase inhibitors tested. However, the weak inhibition observed for a small minority of kinase inhibitors encourages the inclusion of suitable controls in NanoBiT (or NanoLuc) assays.

Dissecting the roles of Haspin and VRK1 in histone H3 phosphorylation during mitosis.

Protein kinases that phosphorylate histones are ideally-placed to influence the behavior of chromosomes during cell division. Indeed, a number of conserved histone phosphorylation events occur prominently during mitosis and meiosis in most eukaryotes, including on histone H3 at threonine-3 (H3T3ph). At least two kinases, Haspin and VRK1 (NHK-1/ballchen in Drosophila), have been proposed to carry out this modification. Phosphorylation of H3 by Haspin has defined roles in mitosis, but the significance of VRK1 activity towards histones in dividing cells has been unclear. Here, using in vitro kinase assays, KiPIK screening, RNA interference, and CRISPR/Cas9 approaches, we were unable to substantiate a direct role for VRK1, or its paralogue VRK2, in the phosphorylation of threonine-3 or serine-10 of Histone H3 in mitosis, although loss of VRK1 did slow cell proliferation. We conclude that the role of VRKs, and their more recently identified association with neuromuscular disease and importance in cancers of the nervous system, are unlikely to involve mitotic histone kinase activity. In contrast, Haspin is required to generate H3T3ph during mitosis.

Reducing adverse impacts of Amazon hydropower expansion.

Proposed hydropower dams at more than 350 sites throughout the Amazon require strategic evaluation of trade-offs between the numerous ecosystem services provided by Earth's largest and most biodiverse river basin. These services are spatially variable, hence collective impacts of newly built dams depend strongly on their configuration. We use multiobjective optimization to identify portfolios of sites that simultaneously minimize impacts on river flow, river connectivity, sediment transport, fish diversity, and greenhouse gas emissions while achieving energy production goals. We find that uncoordinated, dam-by-dam hydropower expansion has resulted in forgone ecosystem service benefits. Minimizing further damage from hydropower development requires considering diverse environmental impacts across the entire basin, as well as cooperation among Amazonian nations. Our findings offer a transferable model for the evaluation of hydropower expansion in transboundary basins.

Regulation of mitotic chromosome cohesion by Haspin and Aurora B.

In vertebrate mitosis, cohesion between sister chromatids is lost in two stages. In prophase and prometaphase, cohesin release from chromosome arms occurs under the control of Polo-like kinase 1 and Aurora B, while Shugoshin is thought to prevent removal of centromeric cohesin until anaphase. The regulatory enzymes that act to sustain centromeric cohesion are incompletely described, however. Haspin/Gsg2 is a histone H3 threonine-3 kinase required for normal mitosis. We report here that both H3 threonine-3 phosphorylation and cohesin are located at inner centromeres. Haspin depletion disrupts cohesin binding and sister chromatid association in mitosis, preventing normal chromosome alignment and activating the spindle assembly checkpoint, leading to arrest in a prometaphase-like state. Overexpression of Haspin hinders cohesin release and stabilizes arm cohesion. We conclude that Haspin is required to maintain centromeric cohesion during mitosis. We also suggest that Aurora B regulates cohesin removal through its effect on the localization of Shugoshin.

Haspin: a newly discovered regulator of mitotic chromosome behavior.

The haspins are divergent members of the eukaryotic protein kinase family that are conserved in many eukaryotic lineages including animals, fungi, and plants. Recently-solved crystal structures confirm that the kinase domain of human haspin has unusual structural features that stabilize a catalytically active conformation and create a distinctive substrate binding site. Haspin localizes predominantly to chromosomes and phosphorylates histone H3 at threonine-3 during mitosis, particularly at inner centromeres. This suggests that haspin directly regulates chromosome behavior by modifying histones, although it is likely that additional substrates will be identified in the future. Depletion of haspin by RNA interference in human cell lines causes premature loss of centromeric cohesin from chromosomes in mitosis and failure of metaphase chromosome alignment, leading to activation of the spindle assembly checkpoint and mitotic arrest. Haspin overexpression stabilizes chromosome arm cohesion. Haspin, therefore, appears to be required for protection of cohesion at mitotic centromeres. Saccharomyces cerevisiae homologues of haspin, Alk1 and Alk2, are also implicated in regulation of mitosis. In mammals, haspin is expressed at high levels in the testis, particularly in round spermatids, so it seems likely that haspin has an additional role in post-meiotic spermatogenesis. Haspin is currently the subject of a number of drug discovery efforts, and the future use of haspin inhibitors should provide new insight into the cellular functions of these kinases and help determine the utility of, for example, targeting haspin for cancer therapy.

Studies of haspin-depleted cells reveal that spindle-pole integrity in mitosis requires chromosome cohesion.

Cohesins and their regulators are vital for normal chromosome cohesion and segregation. A number of cohesion proteins have also been localized to centrosomes and proposed to function there. We show that RNAi-mediated depletion of factors required for cohesion, including haspin, Sgo1 and Scc1, leads to the generation of multiple acentriolar centrosome-like foci and disruption of spindle structure in mitosis. Live-cell imaging reveals that, in haspin-depleted cells, these effects occur only as defects in chromosome cohesion become manifest, and they require ongoing microtubule dynamics and kinesin-5 (also known as Eg5) activity. Inhibition of topoisomerase II in mitosis, which prevents decatenation and separation of chromatids, circumvents the loss of cohesion and restores integrity of the spindle poles. Although these results do not rule out roles for cohesin proteins at centrosomes, they suggest that when cohesion is compromised, spindle-pole integrity can be disrupted as an indirect consequence of the failure to properly integrate chromosome- and centrosome-initiated pathways for spindle formation.