Patient knowledge in anaesthesia: Psychometric development of the RAKQ-The Rotterdam anaesthesia Knowledge questionnaire.

The transition from in-person to digital preoperative patient education requires effective methods for evaluating patients' understanding of the perioperative process, risks, and instructions to ensure informed consent. A knowledge questionnaire covering different anaesthesia techniques and instructions could fulfil this need. We constructed a set of items covering common anaesthesia techniques requiring informed consent and developed the Rotterdam Anaesthesia Knowledge Questionnaire (RAKQ) using a structured approach and Item Response Theory. A team of anaesthetists and educational experts developed the initial set of 60 multiple-choice items, ensuring content and face validity. Next, based on exploratory factor analysis, we identified seven domains: General Anaesthesia-I (regarding what to expect), General Anaesthesia-II (regarding the risks), Spinal Anaesthesia, Epidural Anaesthesia, Regional Anaesthesia, Procedural sedation and analgesia, and Generic Items. This itemset was filled out by 577 patients in the Erasmus MC, Rotterdam, and Albert Schweitzer Hospital, Dordrecht, the Netherlands. Based on factor loadings (≥0.25) and considering clinical relevance this initial item set was reduced to 50 items, distributed over the seven domains. Each domain was processed to produce a separate questionnaire. Through an iterative process of item selection to ensure that the questionnaires met the criteria for Item Response Theory modelling, 40 items remained in the definitive set of seven questionnaires. Finally, we developed an Item Response Theory model for each questionnaire and evaluated its reliability. 1-PL and 2-PL models were chosen based on best model fit. No item misfit (S-χ2, p<0.001 = misfit) was detected in the final models. The newly developed RAKQ allows practitioners to assess their patients' knowledge before consultation to better address knowledge gaps during consultation. Moreover, they can decide whether the level of knowledge is sufficient to obtain digital informed consent without face-to-face education. Researchers can use the RAKQ to compare new methods of patient education with traditional methods.

Alternatives to the in-person anaesthetist-led preoperative assessment in adults undergoing low-risk or intermediate-risk surgery: A scoping review.

BACKGROUND: The design of the optimal preoperative evaluation is a much debated topic, with the anaesthetist-led in-person evaluation being most widely used. This approach is possibly leading to overuse of a valuable resource, especially in low-risk patients. Without compromising patient safety, we hypothesised that not all patients would require this type of elaborate evaluation. OBJECTIVE: The current scoping review aims to critically appraise the range and nature of the existing literature investigating alternatives to the anaesthetist-led preoperative evaluation and their impact on outcomes, to inform future knowledge translation and ultimately improve perioperative clinical practice. DESIGN: A scoping review of the available literature. DATA SOURCES: Embase, Medline, Web-of-Science, Cochrane Library and Google Scholar. No date restriction was used. ELIGIBILITY CRITERIA: Studies in patients scheduled for elective low-risk or intermediate-risk surgery, which compared anaesthetist-led in-person preoperative evaluation with non-anaesthetist-led preoperative evaluation or no outpatient evaluation. The focus was on outcomes, including surgical cancellation, perioperative complications, patient satisfaction and costs. RESULTS: Twenty-six studies with a total of 361 719 patients were included, reporting on various interventions: telephone evaluation, telemedicine evaluation, evaluation by questionnaire, surgeon-led evaluation, nurse-led evaluation, other types of evaluation and no evaluation up to the day of surgery. Most studies were conducted in the United States and were either pre/post or one group post-test-only studies, with only two randomised controlled trials. Studies differed largely in outcome measures and were of moderate quality overall. CONCLUSIONS: A number of alternatives to the anaesthetists-led in-person preoperative evaluation have already been researched: that is telephone evaluation, telemedicine evaluation, evaluation by questionnaire and nurse-led evaluation. However, more high-quality research is needed to assess viability in terms of intraoperative or early postoperative complications, surgical cancellation, costs, and patient satisfaction in the form of Patient-Reported Outcome Measures and Patient-Reported Experience Measures.

Caenorhabditis elegans MES-3 is a highly divergent ortholog of the canonical PRC2 component SUZ12.

Polycomb Repressive Complex 2 (PRC2) catalyzes the mono-, di-, and trimethylation of histone protein H3 on lysine 27 (H3K27), which is strongly associated with transcriptionally silent chromatin. The functional core of PRC2 is highly conserved in animals and consists of four subunits. One of these, SUZ12, has not been identified in the genetic model Caenorhabditis elegans, whereas C. elegans PRC2 contains the clade-specific MES-3 protein. Through unbiased sensitive sequence similarity searches complemented by high-quality structure predictions of monomers and multimers, we here demonstrate that MES-3 is a highly divergent ortholog of SUZ12. MES-3 shares protein folds and conserved residues of key domains with SUZ12 and is predicted to interact with core PRC2 members similar to SUZ12 in human PRC2. Thus, in agreement with previous genetic and biochemical studies, we provide evidence that C. elegans contains a diverged yet evolutionary conserved core PRC2, like other animals.

Caenorhabditis elegans LET-413 Scribble is essential in the epidermis for growth, viability, and directional outgrowth of epithelial seam cells.

The conserved adapter protein Scribble (Scrib) plays essential roles in a variety of cellular processes, including polarity establishment, proliferation, and directed cell migration. While the mechanisms through which Scrib promotes epithelial polarity are beginning to be unraveled, its roles in other cellular processes including cell migration remain enigmatic. In C. elegans, the Scrib ortholog LET-413 is essential for apical-basal polarization and junction formation in embryonic epithelia. However, whether LET-413 is required for postembryonic development or plays a role in migratory events is not known. Here, we use inducible protein degradation to investigate the functioning of LET-413 in larval epithelia. We find that LET-413 is essential in the epidermal epithelium for growth, viability, and junction maintenance. In addition, we identify a novel role for LET-413 in the polarized outgrowth of the epidermal seam cells. These stem cell-like epithelial cells extend anterior and posterior directed apical protrusions in each larval stage to reconnect to their neighbors. We show that the role of LET-413 in seam cell outgrowth is likely mediated largely by the junctional component DLG-1 discs large, which we demonstrate is also essential for directed outgrowth of the seam cells. Our data uncover multiple essential functions for LET-413 in larval development and show that the polarized outgrowth of the epithelial seam cells is controlled by LET-413 Scribble and DLG-1 Discs large.

Dose-dependent functions of SWI/SNF BAF in permitting and inhibiting cell proliferation in vivo.

SWI/SNF (switch/sucrose nonfermenting) complexes regulate transcription through chromatin remodeling and opposing gene silencing by Polycomb group (PcG) proteins. Genes encoding SWI/SNF components are critical for normal development and frequently mutated in human cancer. We characterized the in vivo contributions of SWI/SNF and PcG complexes to proliferation-differentiation decisions, making use of the reproducible development of the nematode Caenorhabditis elegans. RNA interference, lineage-specific gene knockout, and targeted degradation of SWI/SNF BAF components induced either overproliferation or acute proliferation arrest of precursor cells, depending on residual protein levels. Our data show that a high SWI/SNF BAF dosage is needed to arrest cell division during differentiation and to oppose PcG-mediated repression. In contrast, a low SWI/SNF protein level is necessary to sustain cell proliferation and hyperplasia, even when PcG repression is blocked. These observations show that incomplete inactivation of SWI/SNF components can eliminate a tumor-suppressor activity while maintaining an essential transcription regulatory function.

Tissue polarity and PCP protein function: C. elegans as an emerging model.

Polarity is the basis for the generation of cell diversity, as well as the organization, morphogenesis, and functioning of tissues. Studies in Caenorhabditis elegans have provided much insight into PAR-protein mediated polarity; however, the molecules and mechanisms critical for cell polarization within the plane of epithelia have been identified in other systems. Tissue polarity in C. elegans is organized by Wnt-signaling with some resemblance to the Wnt/planar cell polarity (PCP) pathway, but lacking core PCP protein functions. Nonetheless, recent studies revealed that conserved PCP proteins regulate directed cell migratory events in C. elegans, such as convergent extension movements and neurite formation and guidance. Here, we discuss the latest insights and use of C. elegans as a PCP model.

C. elegans Runx/CBFß suppresses POP-1 TCF to convert asymmetric to proliferative division of stem cell-like seam cells.

A correct balance between proliferative and asymmetric cell divisions underlies normal development, stem cell maintenance and tissue homeostasis. What determines whether cells undergo symmetric or asymmetric cell division is poorly understood. To gain insight into the mechanisms involved, we studied the stem cell-like seam cells in the Caenorhabditis elegans epidermis. Seam cells go through a reproducible pattern of asymmetric divisions, instructed by divergent canonical Wnt/ß-catenin signaling, and symmetric divisions that increase the seam cell number. Using time-lapse fluorescence microscopy we observed that symmetric cell divisions maintain asymmetric localization of Wnt/ß-catenin pathway components. Our observations, based on lineage-specific knockout and GFP-tagging of endogenous pop-1, support the model that POP-1TCF induces differentiation at a high nuclear level, whereas low nuclear POP-1 promotes seam cell self-renewal. Before symmetric division, the transcriptional regulator RNT-1Runx and cofactor BRO-1CBFß temporarily bypass Wnt/ß-catenin asymmetry by downregulating pop-1 expression. Thereby, RNT-1/BRO-1 appears to render POP-1 below the level required for its repressor function, which converts differentiation into self-renewal. Thus, we found that conserved Runx/CBFß-type stem cell regulators switch asymmetric to proliferative cell division by opposing TCF-related transcriptional repression.

Cell Polarity: Getting the PARty Started.

Polarity establishment is a key developmental process, but what determines its timing is poorly understood. New research in Caenorhabditis elegans demonstrates that the PAR polarity system extensively reconfigures before becoming competent to polarize. By inhibiting membrane localization of anterior PAR proteins, AIR-1 (aurora A) and PLK-1 (polo kinase) prevent premature polarization.

Developmental Control of the Cell Cycle: Insights from Caenorhabditis elegans.

During animal development, a single fertilized egg forms a complete organism with tens to trillions of cells that encompass a large variety of cell types. Cell cycle regulation is therefore at the center of development and needs to be carried out in close coordination with cell differentiation, migration, and death, as well as tissue formation, morphogenesis, and homeostasis. The timing and frequency of cell divisions are controlled by complex combinations of external and cell-intrinsic signals that vary throughout development. Insight into how such controls determine in vivo cell division patterns has come from studies in various genetic model systems. The nematode Caenorhabditis elegans has only about 1000 somatic cells and approximately twice as many germ cells in the adult hermaphrodite. Despite the relatively small number of cells, C. elegans has diverse tissues, including intestine, nerves, striated and smooth muscle, and skin. C. elegans is unique as a model organism for studies of the cell cycle because the somatic cell lineage is invariant. Somatic cells divide at set times during development to produce daughter cells that adopt reproducible developmental fates. Studies in C. elegans have allowed the identification of conserved cell cycle regulators and provided insights into how cell cycle regulation varies between tissues. In this review, we focus on the regulation of the cell cycle in the context of C. elegans development, with reference to other systems, with the goal of better understanding how cell cycle regulation is linked to animal development in general.

Local microtubule organization promotes cargo transport in C. elegans dendrites.

The specific organization of the neuronal microtubule cytoskeleton in axons and dendrites is an evolutionarily conserved determinant of neuronal polarity that allows for selective cargo sorting. However, how dendritic microtubules are organized and whether local differences influence cargo transport remains largely unknown. Here, we use live-cell imaging to systematically probe the microtubule organization in Caenorhabditiselegans neurons, and demonstrate the contribution of distinct mechanisms in the organization of dendritic microtubules. We found that most non-ciliated neurons depend on unc-116 (kinesin-1), unc-33 (CRMP) and unc-44 (ankyrin) for correct microtubule organization and polarized cargo transport, as previously reported. Ciliated neurons and the URX neuron, however, use an additional pathway to nucleate microtubules at the tip of the dendrite, from the base of the cilium in ciliated neurons. Since inhibition of distal microtubule nucleation affects distal dendritic transport, we propose a model in which the presence of a microtubule-organizing center at the dendrite tip ensures correct dendritic cargo transport.

Optogenetic dissection of mitotic spindle positioning in vivo.

The position of the mitotic spindle determines the plane of cell cleavage, and thereby daughter cell location, size, and content. Spindle positioning is driven by dynein-mediated pulling forces exerted on astral microtubules, which requires an evolutionarily conserved complex of Gα∙GDP, GPR-1/2Pins/LGN, and LIN-5Mud/NuMA proteins. To examine individual functions of the complex components, we developed a genetic strategy for light-controlled localization of endogenous proteins in C. elegans embryos. By replacing Gα and GPR-1/2 with a light-inducible membrane anchor, we demonstrate that Gα∙GDP, Gα∙GTP, and GPR-1/2 are not required for pulling-force generation. In the absence of Gα and GPR-1/2, cortical recruitment of LIN-5, but not dynein itself, induced high pulling forces. The light-controlled localization of LIN-5 overruled normal cell-cycle and polarity regulation and provided experimental control over the spindle and cell-cleavage plane. Our results define Gα∙GDP-GPR-1/2Pins/LGN as a regulatable membrane anchor, and LIN-5Mud/NuMA as a potent activator of dynein-dependent spindle-positioning forces.

Tumor suppressor APC is an attenuator of spindle-pulling forces during C. elegans asymmetric cell division.

The adenomatous polyposis coli (APC) tumor suppressor has dual functions in Wnt/ß-catenin signaling and accurate chromosome segregation and is frequently mutated in colorectal cancers. Although APC contributes to proper cell division, the underlying mechanisms remain poorly understood. Here we show that Caenorhabditis elegans APR-1/APC is an attenuator of the pulling forces acting on the mitotic spindle. During asymmetric cell division of the C. elegans zygote, a LIN-5/NuMA protein complex localizes dynein to the cell cortex to generate pulling forces on astral microtubules that position the mitotic spindle. We found that APR-1 localizes to the anterior cell cortex in a Par-aPKC polarity-dependent manner and suppresses anterior centrosome movements. Our combined cell biological and mathematical analyses support the conclusion that cortical APR-1 reduces force generation by stabilizing microtubule plus-ends at the cell cortex. Furthermore, APR-1 functions in coordination with LIN-5 phosphorylation to attenuate spindle-pulling forces. Our results document a physical basis for the attenuation of spindle-pulling force, which may be generally used in asymmetric cell division and, when disrupted, potentially contributes to division defects in cancer.

Two populations of cytoplasmic dynein contribute to spindle positioning in C. elegans embryos.

The position of the mitotic spindle is tightly controlled in animal cells as it determines the plane and orientation of cell division. Contacts between cytoplasmic dynein and astral microtubules (MTs) at the cell cortex generate pulling forces that position the spindle. An evolutionarily conserved Gα-GPR-1/2Pins/LGN-LIN-5Mud/NuMA cortical complex interacts with dynein and is required for pulling force generation, but the dynamics of this process remain unclear. In this study, by fluorescently labeling endogenous proteins in Caenorhabditis elegans embryos, we show that dynein exists in two distinct cortical populations. One population directly depends on LIN-5, whereas the other is concentrated at MT plus ends and depends on end-binding (EB) proteins. Knockout mutants lacking all EBs are viable and fertile and display normal pulling forces and spindle positioning. However, EB protein-dependent dynein plus end tracking was found to contribute to force generation in embryos with a partially perturbed dynein function, indicating the existence of two mechanisms that together create a highly robust force-generating system.

A dual transcriptional reporter and CDK-activity sensor marks cell cycle entry and progression in C. elegans.

Development, tissue homeostasis and tumor suppression depend critically on the correct regulation of cell division. Central in the cell division process is the decision whether to enter the next cell cycle and commit to going through the S and M phases, or to remain temporarily or permanently arrested. Cell cycle studies in genetic model systems could greatly benefit from visualizing cell cycle commitment in individual cells without the need of fixation. Here, we report the development and characterization of a reporter to monitor cell cycle entry in the nematode C. elegans. This reporter combines the mcm-4 promoter, to reveal Rb/E2F-mediated transcriptional control, and a live-cell sensor for CDK-activity. The CDK sensor was recently developed for use in human cells and consists of a DNA Helicase fragment fused to eGFP. Upon phosphorylation by CDKs, this fusion protein changes in localization from the nucleus to the cytoplasm. The combined regulation of transcription and subcellular localization enabled us to visualize the moment of cell cycle entry in dividing seam cells during C. elegans larval development. This reporter is the first to reflect cell cycle commitment in C. elegans and will help further genetic studies of the mechanisms that underlie cell cycle entry and exit.

Standard Clinical Protocol for Bidirectional Hyperthermic Intraperitoneal Chemotherapy (HIPEC): Systemic Leucovorin, 5-Fluorouracil, and Heated Intraperitoneal Oxaliplatin in a Chloride-Containing Carrier Solution.

BACKGROUND: Intraperitoneal chemotherapy has an established role in the treatment of selected patients with colorectal peritoneal metastases. Oxaliplatin is highly suitable as a chemotherapeutic agent for hyperthermic intraperitoneal chemotherapy (HIPEC), but its use to date has been limited because of the morbidity caused by severe electrolyte and glycemic imbalances associated with 5% glucose as its carrier solution. This report provides an overview of the development, rationale, and application of intraperitoneal chemotherapy and the use of various drugs and carrier solutions. A novel, evidence-based protocol for bidirectional oxaliplatin-based HIPEC in a physiologic carrier solution (Dianeal PD4 dextrose 1.36%) is presented, and its impact on electrolyte and glucose levels is demonstrated. METHODS: After implementation of the new protocol, the serum electrolyte (sodium, potassium, and chloride) levels, glucose levels, and intravenous insulin requirements were intensively measured in eight consecutive cases immediately before HIPEC (T = 0), immediately after HIPEC (T = 30), 1 h after HIPEC (T = 60), and 3 h after HIPEC (T = 180). RESULTS: The median sodium levels were 140 mmol/L at T = 0, 138 mmol/L at T = 30, 140 mmol/L at T = 60, and 140 mmol/L at T = 180. The respective median potassium levels were 4.6, 4.2, 3.7, and 3.9 mmol/L, and the respective median chloride levels were 112, 111, 111, and 112 mmol/L. The respective median glucose levels were 9, 11.5, 10.7, and 8.6 mmol/L. The median insulin requirements were respectively 0.5, 1.5, 1.2, and 0 U/h. None of the patients were diabetic. CONCLUSION: Using a novel protocol for bidirectional oxaliplatin-based HIPEC in Dianeal instead of 5% glucose, the observed fluctuations in this study were minimal and not clinically relevant compared with historical values for electrolyte and glycemic changes using 5% glucose as a HIPEC carrier solution. This novel protocol leads to only minimal and clinically irrelevant electrolyte and glycemic disturbances, and its adoption as the standard protocol for oxaliplatin-based HIPEC should be considered.

Multisite Phosphorylation of NuMA-Related LIN-5 Controls Mitotic Spindle Positioning in C. elegans.

During cell division, the mitotic spindle segregates replicated chromosomes to opposite poles of the cell, while the position of the spindle determines the plane of cleavage. Spindle positioning and chromosome segregation depend on pulling forces on microtubules extending from the centrosomes to the cell cortex. Critical in pulling force generation is the cortical anchoring of cytoplasmic dynein by a conserved ternary complex of Gα, GPR-1/2, and LIN-5 proteins in C. elegans (Gα-LGN-NuMA in mammals). Previously, we showed that the polarity kinase PKC-3 phosphorylates LIN-5 to control spindle positioning in early C. elegans embryos. Here, we investigate whether additional LIN-5 phosphorylations regulate cortical pulling forces, making use of targeted alteration of in vivo phosphorylated residues by CRISPR/Cas9-mediated genetic engineering. Four distinct in vivo phosphorylated LIN-5 residues were found to have critical functions in spindle positioning. Two of these residues form part of a 30 amino acid binding site for GPR-1, which we identified by reverse two-hybrid screening. We provide evidence for a dual-kinase mechanism, involving GSK3 phosphorylation of S659 followed by phosphorylation of S662 by casein kinase 1. These LIN-5 phosphorylations promote LIN-5-GPR-1/2 interaction and contribute to cortical pulling forces. The other two critical residues, T168 and T181, form part of a cyclin-dependent kinase consensus site and are phosphorylated by CDK1-cyclin B in vitro. We applied a novel strategy to characterize early embryonic defects in lethal T168,T181 knockin substitution mutants, and provide evidence for sequential LIN-5 N-terminal phosphorylation and dephosphorylation in dynein recruitment. Our data support that phosphorylation of multiple LIN-5 domains by different kinases contributes to a mechanism for spatiotemporal control of spindle positioning and chromosome segregation.

A tissue-specific protein purification approach in Caenorhabditis elegans identifies novel interaction partners of DLG-1/Discs large.

BACKGROUND: Affinity purification followed by mass spectrometry (AP/MS) is a widely used approach to identify protein interactions and complexes. In multicellular organisms, the accurate identification of protein complexes by AP/MS is complicated by the potential heterogeneity of complexes in different tissues. Here, we present an in vivo biotinylation-based approach for the tissue-specific purification of protein complexes from Caenorhabditis elegans. Tissue-specific biotinylation is achieved by the expression in select tissues of the bacterial biotin ligase BirA, which biotinylates proteins tagged with the Avi peptide. RESULTS: We generated N- and C-terminal tags combining GFP with the Avi peptide sequence, as well as four BirA driver lines expressing BirA ubiquitously and specifically in the seam and hyp7 epidermal cells, intestine, or neurons. We validated the ability of our approach to identify bona fide protein interactions by identifying the known LGL-1 interaction partners PAR-6 and PKC-3. Purification of the Discs large protein DLG-1 identified several candidate interaction partners, including the AAA-type ATPase ATAD-3 and the uncharacterized protein MAPH-1.1. We have identified the domains that mediate the DLG-1/ATAD-3 interaction, and show that this interaction contributes to C. elegans development. MAPH-1.1 co-purified specifically with DLG-1 purified from neurons, and shared limited homology with the microtubule-associated protein MAP1A, a known neuronal interaction partner of mammalian DLG4/PSD95. A CRISPR/Cas9-engineered GFP::MAPH-1.1 fusion was broadly expressed and co-localized with microtubules. CONCLUSIONS: The method we present here is able to purify protein complexes from specific tissues. We uncovered a series of DLG-1 interactors, and conclude that ATAD-3 is a biologically relevant interaction partner of DLG-1. Finally, we conclude that MAPH-1.1 is a microtubule-associated protein of the MAP1 family and a candidate neuron-specific interaction partner of DLG-1.

Tipping the spindle into the right position.

The position of the mitotic spindle determines the cleavage plane in animal cells, but what controls spindle positioning? Kern et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201510117) demonstrate that the microtubule plus end-associated SKAP/Astrin complex participates in this process, possibly by affecting dynein-dependent pulling forces exerted on the tips of astral microtubules.

Light-controlled intracellular transport in Caenorhabditis elegans.

To establish and maintain their complex morphology and function, neurons and other polarized cells exploit cytoskeletal motor proteins to distribute cargoes to specific compartments. Recent studies in cultured cells have used inducible motor protein recruitment to explore how different motors contribute to polarized transport and to control the subcellular positioning of organelles. Such approaches also seem promising avenues for studying motor activity and organelle positioning within more complex cellular assemblies, but their applicability to multicellular in vivo systems has so far remained unexplored. Here, we report the development of an optogenetic organelle transport strategy in the in vivo model system Caenorhabditis elegans. We demonstrate that movement and pausing of various organelles can be achieved by recruiting the proper cytoskeletal motor protein with light. In neurons, we find that kinesin and dynein exclusively target the axon and dendrite, respectively, revealing the basic principles for polarized transport. In vivo control of motor attachment and organelle distributions will be widely useful in exploring the mechanisms that govern the dynamic morphogenesis of cells and tissues, within the context of a developing animal.