High-plex immunofluorescence imaging and traditional histology of the same tissue section for discovering image-based biomarkers.

Precision medicine is critically dependent on better methods for diagnosing and staging disease and predicting drug response. Histopathology using hematoxylin and eosin (H&E)-stained tissue (not genomics) remains the primary diagnostic method in cancer. Recently developed highly multiplexed tissue imaging methods promise to enhance research studies and clinical practice with precise, spatially resolved single-cell data. Here, we describe the 'Orion' platform for collecting H&E and high-plex immunofluorescence images from the same cells in a whole-slide format suitable for diagnosis. Using a retrospective cohort of 74 colorectal cancer resections, we show that immunofluorescence and H&E images provide human experts and machine learning algorithms with complementary information that can be used to generate interpretable, multiplexed image-based models predictive of progression-free survival. Combining models of immune infiltration and tumor-intrinsic features achieves a 10- to 20-fold discrimination between rapid and slow (or no) progression, demonstrating the ability of multimodal tissue imaging to generate high-performance biomarkers.

Efficacy of the Adductor Canal Approach to Saphenous Nerve Block for Anterior Cruciate Ligament Reconstruction With Hamstring Autograft: A Randomized Controlled Trial.

BACKGROUND: For reconstruction of the anterior cruciate ligament (ACL) with hamstring autograft, perioperative analgesia can be achieved with multimodal analgesia and intra-articular local anesthesia infiltration with or without additional regional blocks. Saphenous nerve block (SNB) via the adductor canal is commonly used in our practice, but its benefit has not been well established in the literature. PURPOSE: To assess the efficacy of SNB in ACL reconstruction with hamstring autograft. STUDY DESIGN: Randomized controlled trial; Level of evidence, 1. METHODS: Consecutive patients undergoing arthroscopic ACL reconstruction with hamstring autograft were randomized into a control group (no SNB) and an intervention group (SNB). All patients received standardized anesthetic induction and maintenance agents with perioperative analgesia, per study protocol, with local anesthetic infiltration of the graft harvest site and intra-articular infiltration. RESULTS: Sixty patients were randomized into the 2 groups (n = 30 each). There was no statistically significant difference in total opiate consumption between the groups (control, 34 mg; SNB, 31 mg; P = .40). There was no statistically significant difference in visual analog scale scores for pain at 0, 8, and 24 hours postsurgery, and no difference in overall satisfaction score. The control group had a significantly higher visual analog scale score at 4 hours postsurgery (3.0 vs 1.9, P = .04). CONCLUSION: SNB has a minimal effect on postsurgical care for ACL reconstruction with hamstring autograft in the presence of multimodal analgesia and local anesthetic infiltration.

Multimodal system designed to reduce errors in recording and administration of drugs in anaesthesia: prospective randomised clinical evaluation.

OBJECTIVE: To clinically evaluate a new patented multimodal system (SAFERSleep) designed to reduce errors in the recording and administration of drugs in anaesthesia. DESIGN: Prospective randomised open label clinical trial. SETTING: Five designated operating theatres in a major tertiary referral hospital. PARTICIPANTS: Eighty nine consenting anaesthetists managing 1075 cases in which there were 10,764 drug administrations. INTERVENTION: Use of the new system (which includes customised drug trays and purpose designed drug trolley drawers to promote a well organised anaesthetic workspace and aseptic technique; pre-filled syringes for commonly used anaesthetic drugs; large legible colour coded drug labels; a barcode reader linked to a computer, speakers, and touch screen to provide automatic auditory and visual verification of selected drugs immediately before each administration; automatic compilation of an anaesthetic record; an on-screen and audible warning if an antibiotic has not been administered within 15 minutes of the start of anaesthesia; and certain procedural rules-notably, scanning the label before each drug administration) versus conventional practice in drug administration with a manually compiled anaesthetic record. MAIN OUTCOME MEASURES: Primary: composite of errors in the recording and administration of intravenous drugs detected by direct observation and by detailed reconciliation of the contents of used drug vials against recorded administrations; and lapses in responding to an intermittent visual stimulus (vigilance latency task). Secondary: outcomes in patients; analyses of anaesthetists' tasks and assessments of workload; evaluation of the legibility of anaesthetic records; evaluation of compliance with the procedural rules of the new system; and questionnaire based ratings of the respective systems by participants. RESULTS: The overall mean rate of drug errors per 100 administrations was 9.1 (95% confidence interval 6.9 to 11.4) with the new system (one in 11 administrations) and 11.6 (9.3 to 13.9) with conventional methods (one in nine administrations) (P = 0.045 for difference). Most were recording errors, and, though fewer drug administration errors occurred with the new system, the comparison with conventional methods did not reach significance. Rates of errors in drug administration were lower when anaesthetists consistently applied two key principles of the new system (scanning the drug barcode before administering each drug and keeping the voice prompt active) than when they did not: mean 6.0 (3.1 to 8.8) errors per 100 administrations v 9.7 (8.4 to 11.1) respectively (P = 0.004). Lapses in the vigilance latency task occurred in 12% (58/471) of cases with the new system and 9% (40/473) with conventional methods (P = 0.052). The records generated by the new system were more legible, and anaesthetists preferred the new system, particularly in relation to long, complex, and emergency cases. There were no differences between new and conventional systems in respect of outcomes in patients or anaesthetists' workload. CONCLUSIONS: The new system was associated with a reduction in errors in the recording and administration of drugs in anaesthesia, attributable mainly to a reduction in recording errors. Automatic compilation of the anaesthetic record increased legibility but also increased lapses in a vigilance latency task and decreased time spent watching monitors. Trial registration Australian New Zealand Clinical Trials Registry No 12608000068369.

Adequacy of brain and spinal blood supply with antegrade cerebral perfusion in a rat model.

OBJECTIVE: The purpose of this study was to examine whether different techniques used for antegrade cerebral perfusion could account for variation in the perfusion adequacy of the brain and spinal cord. METHODS: Selected vessels were ligated in 30 rats, recreating a selection of approaches used in aortic arch surgery for patients undergoing circulatory arrest with antegrade cerebral perfusion. Filling of spinal and cerebral vessels was mapped after cannulation and perfusion with E20, gelatin/India ink, or buffered saline/India ink. Three clinical approaches were replicated: unilateral perfusion, bilateral perfusion, and bilateral perfusion with additional left subclavian artery perfusion. Filling of the spinal arteries via the common carotid arteries or the subclavian arteries alone was examined. Penetration of the marker was analyzed histologically. RESULTS: The control experiments achieved maximal arterial filling of both brain and spinal cord at gross and microscopic levels. Unilateral and bilateral antegrade cerebral perfusion provided comprehensive arterial filling of all cerebral vessels with all vascular markers. In contrast, only bilateral antegrade cerebral perfusion provided complete spinal cord perfusion with all markers. Unilateral antegrade cerebral perfusion with a viscous marker resulted in significantly reduced spinal cord arterial filling. Examination of the relative importance of either both common carotid arteries alone or both subclavian arteries alone, in terms of their adequacy of subsequent arterial filling of the spinal cord, showed severe impairment of spinal cord perfusion with either technique. Thus perfusion of both common carotid arteries resulted in only the proximal 30% of the spinal cord arteries being filled, whereas perfusion of both subclavian arteries resulted in only the proximal 40% of the spinal cord arteries being filled. CONCLUSIONS: Approaches to antegrade cerebral perfusion using the brachiocephalic and left common carotid arteries together gave good perfusion of both the brain and the spinal cord. Brachiocephalic perfusion alone gave good cerebral perfusion but showed some significant limitation in spinal cord perfusion with one vascular marker. Complete spinal cord perfusion with all markers under conditions of antegrade cerebral perfusion required some contribution from both the carotid system and the subclavian system together. Selected perfusion of either system alone was very inadequate for spinal cord perfusion.

Cooperation of the Dam1 and Ndc80 kinetochore complexes enhances microtubule coupling and is regulated by aurora B.

The coupling of kinetochores to dynamic spindle microtubules is crucial for chromosome positioning and segregation, error correction, and cell cycle progression. How these fundamental attachments are made and persist under tensile forces from the spindle remain important questions. As microtubule-binding elements, the budding yeast Ndc80 and Dam1 kinetochore complexes are essential and not redundant, but their distinct contributions are unknown. In this study, we show that the Dam1 complex is a processivity factor for the Ndc80 complex, enhancing the ability of the Ndc80 complex to form load-bearing attachments to and track with dynamic microtubule tips in vitro. Moreover, the interaction between the Ndc80 and Dam1 complexes is abolished when the Dam1 complex is phosphorylated by the yeast aurora B kinase Ipl1. This provides evidence for a mechanism by which aurora B resets aberrant kinetochore-microtubule attachments. We propose that the action of the Dam1 complex as a processivity factor in kinetochore-microtubule attachment is regulated by conserved signals for error correction.

Reconstitution and functional analysis of kinetochore subcomplexes.

Kinetochores are multifunctional supercomplexes that link chromosomes to dynamic microtubule tips. Groups of proteins from the kinetochore are arranged into distinct subcomplexes that copurify under stringent conditions and cause similar phenotypes when mutated. By coexpressing all the components of a given subcomplex from a polycistronic plasmid in bacteria, many laboratories have had great success in purifying active subcomplexes. This has enabled the study of how the microtubule-binding subcomplexes of the kinetochore interact with both the microtubule lattice and dynamic microtubule tips. Here we outline methods for rapid cloning of polycistronic vectors for expression of kinetochore subcomplexes, their purification, and techniques for functional analysis using total internal reflection fluorescence microscopy (TIRFM).

Catalysis of the microtubule on-rate is the major parameter regulating the depolymerase activity of MCAK.

The kinesin-13, MCAK, is a critical regulator of microtubule dynamics in eukaryotic cells. We have functionally dissected the structural features responsible for MCAK's potent microtubule depolymerization activity. MCAK's positively charged neck enhances its delivery to microtubule ends not by tethering the molecule to microtubules during diffusion, as commonly thought, but by catalyzing the association of MCAK to microtubules. On the other hand, this same positively charged neck slightly diminishes MCAK's ability to remove tubulin subunits once at the microtubule end. Conversely, dimerization reduces MCAK delivery but improves MCAK's ability to remove tubulin subunits. The reported kinetics for these events predicts a nonspecific binding mechanism that may represent a paradigm for the diffusive interaction of many microtubule-binding proteins.

The Ndc80 kinetochore complex forms load-bearing attachments to dynamic microtubule tips via biased diffusion.

Kinetochores couple chromosomes to the assembling and disassembling tips of microtubules, a dynamic behavior that is fundamental to mitosis in all eukaryotes but poorly understood. Genetic, biochemical, and structural studies implicate the Ndc80 complex as a direct point of contact between kinetochores and microtubules, but these approaches provide only a static view. Here, using techniques for manipulating and tracking individual molecules in vitro, we demonstrate that the Ndc80 complex is capable of forming the dynamic, load-bearing attachments to assembling and disassembling tips required for coupling in vivo. We also establish that Ndc80-based coupling likely occurs through a biased diffusion mechanism and that this activity is conserved from yeast to humans. Our findings demonstrate how an ensemble of Ndc80 complexes may provide the combination of plasticity and strength that allows kinetochores to maintain load-bearing tip attachments during both microtubule assembly and disassembly.

The diffusive interaction of microtubule binding proteins.

Microtubule-based motility is often thought of as specifically referring to the directed stepping of microtubule-based motors such as kinesin or dynein. However, microtubule lattice diffusion (also known as diffusional motility) provides a second mode of transport that is shared by a much broader class of microtubule binding proteins. Microtubule lattice diffusion offers distinct advantages as a transport mechanism including speed, bidirectional microtubule end targeting, and no requirement for direct chemical energy (i.e. ATP). It remains to be seen whether a universal binding mechanism for this interaction will be identified but electrostatic interactions appear to play a significant role. In the meantime, the well-studied subject of DNA binding proteins that diffuse along the DNA backbone provides an insightful analog for understanding the nature of microtubule-based diffusional motility.

A kinesin-13 mutant catalytically depolymerizes microtubules in ADP.

The kinesin-13 motor protein family members drive the removal of tubulin from microtubules (MTs) to promote MT turnover. A point mutation of the kinesin-13 family member mitotic centromere-associated kinesin/Kif2C (E491A) isolates the tubulin-removal conformation of the motor, and appears distinct from all previously described kinesin-13 conformations derived from nucleotide analogues. The E491A mutant removes tubulin dimers from stabilized MTs stoichiometrically in adenosine triphosphate (ATP) but is unable to efficiently release from detached tubulin dimers to recycle catalytically. Only in adenosine diphosphate (ADP) can the mutant catalytically remove tubulin dimers from stabilized MTs because the affinity of the mutant for detached tubulin dimers in ADP is low relative to lattice-bound tubulin. Thus, the motor can regenerate for further cycles of disassembly. Using the mutant, we show that release of tubulin by kinesin-13 motors occurs at the transition state for ATP hydrolysis, which illustrates a significant divergence in their coupling to ATP turnover relative to motile kinesins.

Patient safety indicators for England from hospital administrative data: case-control analysis and comparison with US data.

OBJECTIVE: To assess the feasibility of deriving patient safety indicators for England from routine hospital data and whether they can indicate adverse outcomes for patients. DESIGN: Nine patient safety indicators developed by the United States Agency for Healthcare Research and Quality (AHRQ) were derived using hospital episode statistics for England for 2003-4, 2004-5, and 2005-6. A case-control analysis was undertaken to compare length of stay and mortality between cases (patients experiencing the particular safety event measured by an indicator) and controls matched for age, sex, health resource group (standard groupings of clinically similar treatments that use similar levels of healthcare resource), main specialty, and trust. Comparisons were undertaken with US data. SETTING: All NHS trusts in England. PARTICIPANTS: Inpatients in NHS trusts. RESULTS: There was fair consistency in national rates for the nine indicators across three years. For all but one indicator, hospital stays were longer in cases than in matched controls (range 0.2-17.1 days, P<0.001). Mortality in cases was also higher than in controls (5.7-27.1%, P<0.001), except for the obstetric trauma indicators. Excess length of stay and mortality in cases was greatest for postoperative hip fracture and sepsis. England's rates were lower than US rates for these indicators. Increased length of stay in cases was generally greater in England than in the US. Excess mortality was also higher in England than in the US, except for the obstetric trauma indicators where there were few deaths in both countries. Differences between England and the US in excess length of stay and mortality were most marked for postoperative hip fracture. CONCLUSIONS: Hospital administrative data provide a potentially useful low burden, low cost source of information on safety events. Indicators can be derived with English data and show that cases have poorer outcomes than matched controls. These data therefore have potential for monitoring safety events. Further validation, for example, of individual cases, is needed and levels of event recording need to improve. Differences between England and the US might reflect differences in the depth of event coding and in health systems and patterns of healthcare provision.

Phosphoregulation and depolymerization-driven movement of the Dam1 complex do not require ring formation.

During mitosis, kinetochores form persistent attachments to microtubule tips and undergo corrective detachment in response to phosphorylation by Ipl1 (Aurora B) kinase. The Dam1 complex is required to establish and maintain bi-oriented attachment to microtubule tips in vivo, and it contains multiple sites phosphorylated by Ipl1 (Refs 2, 3, 4, 5, 6, 7, 8, 9, 10). Moreover, a number of kinetochore-like functions can be reconstituted in vitro with pure Dam1 complex. These functions are believed to derive from the ability of the complex to self-assemble into rings. Here we show that rings are not necessary for dynamic microtubule attachment, Ipl1-dependent modulation of microtubule affinity or the ability of Dam1 to move processively with disassembling microtubule tips. Using two fluorescence-based assays, we found that the complex exhibited a high affinity for microtubules (Kd of approximately 6 nM) that was reduced by phosphorylation at Ser 20, a single Ipl1 target residue in Dam1. Moreover, individual complexes underwent one-dimensional diffusion along microtubules and detached 2.5-fold more frequently after phosphorylation by Ipl1. Particles consisting of one to four Dam1 complexes - too few to surround a microtubule - were captured and carried by disassembling tips. Thus, even a small number of binding elements could provide a dynamic, phosphoregulated microtubule attachment and thereby facilitate accurate chromosome segregation.

In vitro and in vivo analysis of microtubule-destabilizing kinesins.

Cellular microtubules are rigid in comparison to other cytoskeletal elements (1,2). To facilitate cytoplasmic remodeling and timely responses to cell signaling events, microtubules depolymerize and repolymerize rapidly at their ends (3). These dynamic properties are critically important for many cellular functions, such as spindle assembly, the capture and segregation of chromosomes during cell division and cell motility. Microtubule dynamics are spatially and temporally controlled in the cell by accessory proteins. Molecular motor proteins of the kinesin superfamily that act to destabilize microtubules play important roles in this regulation (4).

The role of the kinesin-13 neck in microtubule depolymerization.

To ensure genetic integrity, replicated chromosomes must be accurately distributed to daughter cells-a process that is accomplished on the microtubule spindle. Kinesin-13 motors play an essential role in this process by performing regulated microtubule depolymerization. We set out to dissect the depolymerization mechanism of these kinesins, and in particular, the role of their conserved neck sequence. We used a monomeric kinesin-13 MCAK, consisting of the neck and motor core, which has strong depolymerizing activity. In the presence of a non-hydrolysable ATP analogue, this construct induced formation of rings around microtubules. The rings are built from tubulin protofilaments that are bent by the kinesin-13 motor engaged at the ATP-binding step of its ATPase cycle. Our data suggest that the ring-microtubule interaction is mediated by the neck and support the idea of a role for the kinesin-13 neck in depolymerization efficiency, acting by optimizing release of tubulin from microtubule ends.

State-dependent block of CNG channels by dequalinium.

Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels with a high permeability for Ca(2+). Not surprisingly, they are blocked by a number of Ca(2+) channel blockers including tetracaine, pimozide, and diltiazem. We studied the effects of dequalinium, an extracellular blocker of the small conductance Ca(2+)-activated K(+) channel. We previously noted that dequalinium is a high-affinity blocker of CNGA1 channels from the intracellular side, with little or no state dependence at 0 mV. Here we examined block by dequalinium at a broad range of voltages in both CNGA1 and CNGA2 channels. We found that dequalinium block was mildly state dependent for both channels, with the affinity for closed channels 3-5 times higher than that for open channels. Mutations in the S4-S5 linker did not alter the affinity of open channels for dequalinium, but increased the affinity of closed channels by 10-20-fold. The state-specific effect of these mutations raises the question of whether/how the S4-S5 linker alters the binding of a blocker within the ion permeation pathway.