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Shared services organizations are ascribed with adding value to business in several ways but especially by sharing resources and leading to economies of scale. However, these gains are not automatic and in some instances, particularly healthcare, they are difficult to achieve. This article describes a project to develop a shared services information technology infrastructure across two district health boards in New Zealand. The study reveals valuable insight into the crisis issues that accompany change management and identifies emergent themes that can be used to reduce negative impact. Copyright © 2006 SAGE Publications Ltd. [ABSTRACT FROM AUTHOR]

Clathrin-mediated endocytosis is an essential cellular pathway that enables signaling and recycling of transmembrane proteins and lipids. During endocytosis, dozens of cytosolic proteins come together at the plasma membrane, assembling into a highly interconnected network that drives endocytic vesicle biogenesis. Recently, multiple groups have reported that early endocytic proteins form flexible condensates, which provide a platform for efficient assembly of endocytic vesicles. Given the importance of this network in the dynamics of endocytosis, how might cells regulate its stability? Many receptors and endocytic proteins are ubiquitylated, while early endocytic proteins such as Eps15 contain ubiquitin-interacting motifs. Therefore, we examined the influence of ubiquitin on the stability of the early endocytic protein network. In vitro, we found that recruitment of small amounts of polyubiquitin dramatically increased the stability of Eps15 condensates, suggesting that ubiquitylation could nucleate endocytic assemblies. In live-cell imaging experiments, a version of Eps15 that lacked the ubiquitin-interacting motif failed to rescue defects in endocytic initiation created by Eps15 knockout. Furthermore, fusion of Eps15 to a deubiquitylase enzyme destabilized nascent endocytic sites within minutes. In both in vitro and live-cell settings, dynamic exchange of Eps15 proteins, a measure of protein network stability, was decreased by Eps15-ubiquitin interactions and increased by loss of ubiquitin. These results collectively suggest that ubiquitylation drives assembly of the flexible protein network responsible for catalyzing endocytic events. More broadly, this work illustrates a biophysical mechanism by which ubiquitylated transmembrane proteins at the plasma membrane could regulate the efficiency of endocytic internalization., (© The Author(s) 2024. Published by Oxford University Press on behalf of National Academy of Sciences.)

Endocytosis is a fine-tuned mechanism of cellular communication through which cells internalize molecules on the plasma membrane, such as receptors and their bound ligands. Through receptor clustering in endocytic pits, recruitment of active receptors to different endocytic routes and their trafficking towards different fates, endocytosis modulates cell signaling and ultimately leads to a variety of biological responses. Many studies have focused their attention on specialized endocytic mechanisms depending on the nature of the internalizing cargo and cellular context, distinct sets of coat proteins, endocytic adaptors and membrane lipids. Here, we review recent advances in our understanding of the principles underlying endocytic vesicle formation, integrating both biochemical and biophysical factors, with a particular focus on intrinsically disordered regions (IDRs) creating weakly interconnected protein networks assembled through liquid-liquid phase separation (LLPS) and driving membrane bending especially in clathrin-mediated endocytosis (CME). We finally discuss how these properties impinge on receptor fate and signaling., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The pathways of membrane traffic within the Golgi apparatus are not fully known. This question was addressed using the yeast Saccharomyces cerevisiae, in which the maturation of individual Golgi cisternae can be visualized. We recently proposed that the AP-1 clathrin adaptor mediates intra-Golgi recycling late in the process of cisternal maturation. Here, we demonstrate that AP-1 cooperates with the Ent5 clathrin adaptor to recycle a set of Golgi transmembrane proteins, including some that were previously thought to pass through endosomes. This recycling can be detected by removing AP-1 and Ent5, thereby diverting the AP-1/Ent5-dependent Golgi proteins into an alternative recycling loop that involves traffic to the plasma membrane followed by endocytosis. Unexpectedly, various AP-1/Ent5-dependent Golgi proteins show either intermediate or late kinetics of residence in maturing cisternae. We infer that the AP-1/Ent5 pair mediates two sequential intra-Golgi recycling pathways that define two classes of Golgi proteins. This insight can explain the polarized distribution of transmembrane proteins in the Golgi., (© 2021 Casler et al.)

During clathrin-mediated endocytosis, dozens of proteins assemble into an interconnected network at the plasma membrane. As initiators of endocytosis, Eps15 and Fcho1/2 concentrate downstream components, while permitting dynamic rearrangement within the budding vesicle. How do initiator proteins meet these competing demands? Here we show that Eps15 and Fcho1/2 rely on weak, liquid-like interactions to catalyse endocytosis. In vitro, these weak interactions promote the assembly of protein droplets with liquid-like properties. To probe the physiological role of these liquid-like networks, we tuned the strength of initiator protein assembly in real time using light-inducible oligomerization of Eps15. Low light levels drove liquid-like assemblies, restoring normal rates of endocytosis in mammalian Eps15-knockout cells. By contrast, initiator proteins formed solid-like assemblies upon exposure to higher light levels, which stalled vesicle budding, probably owing to insufficient molecular rearrangement. These findings suggest that liquid-like assembly of initiator proteins provides an optimal catalytic platform for endocytosis.

Membrane bending is a ubiquitous cellular process that is required for membrane traffic, cell motility, organelle biogenesis, and cell division. Proteins that bind to membranes using specific structural features, such as wedge-like amphipathic helices and crescent-shaped scaffolds, are thought to be the primary drivers of membrane bending. However, many membrane-binding proteins have substantial regions of intrinsic disorder which lack a stable three-dimensional structure. Interestingly, many of these disordered domains have recently been found to form networks stabilized by weak, multivalent contacts, leading to assembly of protein liquid phases on membrane surfaces. Here we ask how membrane-associated protein liquids impact membrane curvature. We find that protein phase separation on the surfaces of synthetic and cell-derived membrane vesicles creates a substantial compressive stress in the plane of the membrane. This stress drives the membrane to bend inward, creating protein-lined membrane tubules. A simple mechanical model of this process accurately predicts the experimentally measured relationship between the rigidity of the membrane and the diameter of the membrane tubules. Discovery of this mechanism, which may be relevant to a broad range of cellular protrusions, illustrates that membrane remodeling is not exclusive to structured scaffolds but can also be driven by the rapidly emerging class of liquid-like protein networks that assemble at membranes., Competing Interests: The authors declare no competing interest.

Intracellular trafficking requires extensive changes in membrane morphology. Cells use several distinct molecular factors and physical cues to remodel membranes. Here, we highlight recent advances in identifying the biophysical mechanisms of membrane curvature generation. In particular, we focus on the cooperation of molecular and physical drivers of membrane bending during three stages of vesiculation: budding, cargo selection, and scission. Taken together, the studies reviewed here emphasize that, rather than a single dominant mechanism, several mechanisms typically work in parallel during each step of membrane remodeling. Important challenges for the future of this field are to understand how multiple mechanisms work together synergistically and how a series of stochastic events can be combined to achieve a deterministic result-assembly of the trafficking vesicle., Competing Interests: Conflict of interest statement Nothing declared., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Many critical biological events, including biochemical signaling, membrane traffic, and cell motility, originate at membrane surfaces. Each such event requires that members of a specific group of proteins and lipids rapidly assemble together at a specific site on the membrane surface. Understanding the biophysical mechanisms that stabilize these assemblies is critical to decoding and controlling cellular functions. In this article, we review progress toward a quantitative biophysical understanding of the mechanisms that drive membrane heterogeneity and organization. We begin from a physical perspective, reviewing the fundamental principles and key experimental evidence behind each proposed mechanism. We then shift to a biological perspective, presenting key examples of the role of heterogeneity in biology and asking which physical mechanisms may be responsible. We close with an applied perspective, noting that membrane heterogeneity provides a novel therapeutic target that is being exploited by a growing number of studies at the interface of biology, physics, and engineering.

The endomembrane system consists of the secretory and endocytic pathways, which communicate by transport to and from the trans-Golgi network (TGN). In mammalian cells, the endocytic pathway includes early, late, and recycling endosomes. In budding yeast, different types of endosomes have been described, but the organization of the endocytic pathway has remained unclear. We performed a spatial and temporal analysis of yeast endosomal markers and endocytic cargoes. Our results indicate that the yeast TGN also serves as an early and recycling endosome. In addition, as previously described, yeast contains a late or prevacuolar endosome (PVE). Endocytic cargoes localize to the TGN shortly after internalization, and manipulations that perturb export from the TGN can slow the passage of endocytic cargoes to the PVE. Yeast apparently lacks a distinct early endosome. Thus, yeast has a simple endocytic pathway that may reflect the ancestral organization of the endomembrane system., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background: New Zealand is becoming more ethnically diverse, with rising numbers of people with limited English language proficiency. Consequently, hospital interactions are increasing where patients have insufficient English to communicate adequately with doctors or nurses for appropriate, effective, and safe care. Translation technology is rapidly evolving, but evidence is limited regarding its usefulness to clinicians., Objective: To examine the acceptability to doctors and nurses of a translation application (app) used on a tablet, in brief interactions with Korean patients., Method: An app was developed to facilitate brief conversations between patients and clinicians as part of clinical care. We used the Technology Acceptance Model 2 to develop semi-structured interview questions for 15 junior and senior doctors and nurses in an urban hospital. Participants used the app to interact with the interviewer as part of a scenario. The interviews were analysed thematically., Results: The app was easy to use, learn to use, and to memorise for future use. It was considered useful for everyday brief interactions, urgent situations where there is no time to call an interpreter, and after hours, to augment the work of interpreters. Subject to perceived usefulness, there appears to be little need for social normalisation of a translation app, other than management support for the costs, maintenance, and implementation of the app for everyday use.  CONCLUSION: Guidelines are required for the use of a translation app by doctors and nurses to augment the interpreter role. A larger study and future research on the patient's perspective are required.

Selecting optimal formulation conditions for monoclonal antibodies for first time in human clinical trials is challenging due to short timelines and reliance on predictive assays to ensure product quality and adequate long-term stability. Accelerated stability studies are considered to be the gold standard for excipient screening, but they are relatively low throughput and time consuming. High throughput screening (HTS) techniques allow for large amounts of data to be collected quickly and easily, and can be used to screen solution conditions for early formulation development. The utility of using accelerated stability compared to HTS techniques (differential scanning light scattering and differential scanning fluorescence) for early formulation screening was evaluated along with the impact of excipients of various types on aggregation of monoclonal antibodies from multiple IgG subtypes. The excipient rank order using quantitative HTS measures was found to correlate with accelerated stability aggregation rate ranking for only 33% (by differential scanning fluorescence) to 42% (by differential scanning light scattering) of the antibodies tested, due to the high intrinsic stability and minimal impact of excipients on aggregation rates and HTS data. Also explored was a case study of employing a platform formulation instead of broader formulation screening for early formulation development., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Mislocalized tail-anchored (TA) proteins of the outer mitochondrial membrane are cleared by a newly identified quality control pathway involving the conserved eukaryotic protein Msp1 (ATAD1 in humans). Msp1 is a transmembrane AAA-ATPase, but its role in TA protein clearance is not known. Here, using purified components reconstituted into proteoliposomes, we show that Msp1 is both necessary and sufficient to drive the ATP-dependent extraction of TA proteins from the membrane. A crystal structure of the Msp1 cytosolic region modeled into a ring hexamer suggests that active Msp1 contains a conserved membrane-facing surface adjacent to a central pore. Structure-guided mutagenesis of the pore residues shows that they are critical for TA protein extraction in vitro and for functional complementation of an msp1 deletion in yeast. Together, these data provide a molecular framework for Msp1-dependent extraction of mislocalized TA proteins from the outer mitochondrial membrane., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Deconvolution is typically used to sharpen fluorescence images, but when the signal-to-noise ratio is low, the primary benefit is reduced noise and a smoother appearance of the fluorescent structures. 3D time-lapse (4D) confocal image sets can be improved by deconvolution. However, when the confocal signals are very weak, the popular Huygens deconvolution software erases fluorescent structures that are clearly visible in the raw data. We find that this problem can be avoided by prefiltering the optical sections with a Gaussian blur. Analysis of real and simulated data indicates that the Gaussian blur prefilter preserves meaningful signals while enabling removal of background noise. This approach is very simple, and it allows Huygens to be used with 4D imaging conditions that minimize photodamage., Competing Interests: Competing interests: No competing interests were disclosed.

Due to reduced cost and accessibility, the use of genetic testing has appealed to health professionals for personalising nutrition advice. However, translation of the evidence linking polymorphisms, dietary requirements, and pathology risk proves to be challenging for nutrition and dietetic practitioners. Zinc status and polymorphisms of genes coding for zinc-transporters have been associated with chronic diseases. The present study aimed to systematically review the literature to assess whether recommendations for zinc intake could be made according to genotype. Eighteen studies investigating 31 Single Nucleotide Polymorphisms (SNPs) in relation to zinc intake and/or status were identified. Five studies examined type 2 diabetes; zinc intake was found to interact independently with two polymorphisms in the zinc-transporter gene SLC30A8 to affect glucose metabolism indicators. While the outcomes were statistically significant, the small size of the effect and lack of replication raises issues regarding translation into nutrition and dietetic practice. Two studies assessed the relationship of polymorphisms and cognitive performance; seven studies assessed the association between a range of outcomes linked to chronic conditions in aging population; two papers described the analysis of the genetic contribution in determining zinc concentration in human milk; and two papers assessed zinc concentration in plasma without linking to clinical outcomes. The data extracted confirmed a connection between genetics and zinc requirements, although the direction and magnitude of the dietary modification for carriers of specific genotypes could not be defined. This study highlights the need to summarise nutrigenetics studies to enable health professionals to translate scientific evidence into dietary recommendations.

Strains of the species Komagataella phaffii are the most frequently used "Pichia pastoris" strains employed for recombinant protein production as well as studies on peroxisome biogenesis, autophagy and secretory pathway analyses. Genome sequencing of several different P. pastoris strains has provided the foundation for understanding these cellular functions in recent genomics, transcriptomics and proteomics experiments. This experimentation has identified mistakes, gaps and incorrectly annotated open reading frames in the previously published draft genome sequences. Here, a refined reference genome is presented, generated with genome and transcriptome sequencing data from multiple P. pastoris strains. Twelve major sequence gaps from 20 to 6000 base pairs were closed and 5111 out of 5256 putative open reading frames were manually curated and confirmed by RNA-seq and published LC-MS/MS data, including the addition of new open reading frames (ORFs) and a reduction in the number of spliced genes from 797 to 571. One chromosomal fragment of 76kbp between two previous gaps on chromosome 1 and another 134kbp fragment at the end of chromosome 4, as well as several shorter fragments needed re-orientation. In total more than 500 positions in the genome have been corrected. This reference genome is presented with new chromosomal numbering, positioning ribosomal repeats at the distal ends of the four chromosomes, and includes predicted chromosomal centromeres as well as the sequence of two linear cytoplasmic plasmids of 13.1 and 9.5kbp found in some strains of P. pastoris., (Copyright © 2016. Published by Elsevier B.V.)

Simple discotic cores functionalized with reactive arms have been assembled into two- and three-tiered covalent stacks through imine formation. The targets are obtained in good yields, but competing formation of misassembled byproducts highlights some of the challenges inherent to the thermodynamically controlled assembly of rigid, compact, three-dimensional architectures. The structures comprise a central stack of arenes surrounded by a triple helix of interconnected arms. The racemization rate is strongly dependent on the number of tiers, suggesting cooperative conformational coupling in these multi-tiered structures., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

FK506-binding protein (FKBP) is a monomer that binds to FK506, rapamycin, and related ligands. The F36M substitution, in which Phe36 in the ligand-binding pocket is changed to Met, leads to formation of antiparallel FKBP dimers, which can be dissociated into monomers by ligand binding. This FKBP(M) mutant has been employed in the mammalian secretory pathway to generate aggregates that can be dissolved by ligand addition to create cargo waves. However, when testing this approach in yeast, we found that dissolution of FKBP(M) aggregates was inefficient. An improved reversibly dimerizing FKBP formed aggregates that dissolved more readily. This FKBP(L,V) mutant carries the F36L mutation, which increases the affinity of ligand binding, and the I90V mutation, which accelerates ligand-induced dissociation of the dimers. The FKBP(L,V) mutant expands the utility of reversibly dimerizing FKBP.

Yeast cells are well suited to visualizing organelles by 4D confocal microscopy. Typically, one or more cellular compartments are labeled with a fluorescent protein or dye, and a stack of confocal sections spanning the entire cell volume is captured every few seconds. Under appropriate conditions, organelle dynamics can be observed for many minutes with only limited photobleaching. Images are captured at a relatively low signal-to-noise ratio and are subsequently processed to generate movies that can be analyzed and quantified. Here, we describe methods for acquiring and processing 4D data using conventional scanning confocal microscopy.

COPI coated vesicles carry material between Golgi compartments, but the role of COPI in the secretory pathway has been ambiguous. Previous studies of thermosensitive yeast COPI mutants yielded the surprising conclusion that COPI was dispensable both for the secretion of certain proteins and for Golgi cisternal maturation. To revisit these issues, we optimized the anchor-away method, which allows peripheral membrane proteins such as COPI to be sequestered rapidly by adding rapamycin. Video fluorescence microscopy revealed that COPI inactivation causes an early Golgi protein to remain in place while late Golgi proteins undergo cycles of arrival and departure. These dynamics generate partially functional hybrid Golgi structures that contain both early and late Golgi proteins, explaining how secretion can persist when COPI has been inactivated. Our findings suggest that cisternal maturation involves a COPI-dependent pathway that recycles early Golgi proteins, followed by multiple COPI-independent pathways that recycle late Golgi proteins.

Regulation of the size and abundance of membrane compartments is a fundamental cellular activity. In Saccharomyces cerevisiae, disruption of the ADP-ribosylation factor 1 (ARF1) gene yields larger and fewer Golgi cisternae by partially depleting the Arf GTPase. We observed a similar phenotype with a thermosensitive mutation in Nmt1, which myristoylates and activates Arf. Therefore, partial depletion of Arf is a convenient tool for dissecting mechanisms that regulate Golgi structure. We found that in arf1Δ cells, late Golgi structure is particularly abnormal, with the number of late Golgi cisternae being severely reduced. This effect can be explained by selective changes in cisternal maturation kinetics. The arf1Δ mutation causes early Golgi cisternae to mature more slowly and less frequently, but does not alter the maturation of late Golgi cisternae. These changes quantitatively explain why late Golgi cisternae are fewer in number and correspondingly larger. With a stacked Golgi, similar changes in maturation kinetics could be used by the cell to modulate the number of cisternae per stack. Thus, the rates of processes that transform a maturing compartment can determine compartmental size and copy number.

The Golgi apparatus contains multiple classes of cisternae that differ in structure, composition, and function, but there is no consensus about the number and definition of these classes. A useful way to classify Golgi cisternae is according to the trafficking pathways by which the cisternae import and export components. By this criterion, we propose that Golgi cisternae can be divided into three classes that correspond to functional stages of maturation. First, cisternae at the cisternal assembly stage receive COPII vesicles from the ER and recycle components to the ER in COPI vesicles. At this stage, new cisternae are generated. Second, cisternae at the carbohydrate synthesis stage exchange material with one another via COPI vesicles. At this stage, most of the glycosylation and polysaccharide synthesis reactions occur. Third, cisternae at the carrier formation stage produce clathrin-coated vesicles and exchange material with endosomes. At this stage, biosynthetic cargo proteins are packaged into various transport carriers, and the cisternae ultimately disassemble. Discrete transitions occur as a cisterna matures from one stage to the next. Within each stage, the structure and composition of a cisterna can evolve, but the trafficking pathways remain unchanged. This model offers a unified framework for understanding the properties of the Golgi in diverse organisms.

Objective: The objective of this study was to compare the variability of computed tomography perfusion (CTP) results in identical data sets of middle cerebral artery (MCA) acute ischemic stroke (AIS) generated by standard singular value decomposition (sSVD) deconvolution and tracer delay-insensitive singular value decomposition (SVD+) algorithm analyses., Methods: Whole-brain 320-detector-row CTP data sets from 9 unilateral MCA AIS cases and 9 controls were retrospectively analyzed. Computed tomography perfusion values for the combined core/penumbra, contralateral hemispheres and arterial territories were measured and compared with literature values. Simple linear regression models are provided to predict corresponding SVD+ value and sSVD CTP values., Results: In the core/penumbra, sSVD generated lower cerebral blood flow (CBF) values, higher mean transit time (MTT) values, and a broader range of CBF and MTT values as compared with SVD+. Mean transit time value differences between the core/penumbra and contralateral hemispheres were statistically significant using sSVD, whereas those of SVD+ were not. Goodness of fit between algorithms for the core/penumbra was lower for CBF (0.483) and MTT (0.494), as compared with time to peak (0.891) and cerebral blood volume (0.997)., Conclusions: In this study using identical source data for patients with MCA AIS, use of either sSVD or SVD+ analyses created statistically significant differences in the CTP value results. Tracer delay-sensitive and -insensitive algorithms impact CTP results in AIS and controls, highlighting the need to pursue additional studies that assess the variability, accuracy, and clinical implications of CTP results generated when using heterogeneous deconvolution algorithms.

Objective: Whole-brain computed tomography perfusion (CTP) data sets generated by tracer delay-insensitive singular value decomposition plus (SVD+) and standard singular value decomposition (sSVD) deconvolution algorithms were evaluated to quantify relatedness and discrepancies in CTP results., Methods: Twenty females with symmetrical hemispheric CTP maps indicative of brain tissue without apparent abnormalities were studied. Tissue-specific CTP values were analyzed., Results: Standard SVD values were higher than SVD+ for cerebral blood flow. Other CTP values had minimal differences across brain regions. All simple linear regression models were statistically significant (P < 0.05) except for cerebral blood flow in white matter (P = 0.06). Cerebral blood volume had a good model fit, and mean transit time, a poor fit., Conclusions: Corresponding fitted CTP values for sSVD and SVD+ based on regression equations for brain-tissue types are presented. Additional research is required to compare SVD+ and sSVD in disease states when significant hemodynamic brain alterations are present.

Background: Electronic referral (eReferral) from community into public secondary healthcare services was introduced to 30 referring general medical practices and 28 hospital based services in late 2007., Objectives: To measure the extent of uptake of eReferral and its association with changes in referral processing., Methods: Analysis of transactional data from the eReferral message service and the patient information management system of the affected hospital; interview of clinical, operational and management stakeholders., Results: eReferral use rose steadily to 1000 transactions per month in 2008, thereafter showing moderate growth to 1200 per month in 2010. Rate of eReferral from the community in 2010 is estimated at 56% of total referrals to the hospital from general practice, and as 71% of referrals from those having done at least one referral electronically. Referral latency from letter date to hospital triage improves significantly from 2007 to 2009 (p<0.001), from a paper referral median of 8 days (inter-quartile range, IQR: 4-14) in 2007 to an eReferral median of 5 days (IQR: 2-9) and paper referral median of 6 days (IQR: 2-12) in 2009. Specialists upgrade the referrer-assigned eReferral priority in 19.2% of cases and downgrade it 18.6% of the time. Clinical users appreciate improvement of referral visibility (status and content access); however, both general practitioners and specialists point out system usability issues., Discussion: With eReferrals, a referral's status can be checked, and its content read, by any authorized user at any time. The period of eReferral uptake was associated with significant speed-up in referral processing without changes in staffing levels. The eReferral system provides a foundation for further innovation in the community-secondary interface, such as electronic decision support and shared care planning systems., Conclusions: We observed substantial rapid voluntary uptake of eReferrals associated with faster, more reliable and more transparent referral processing.

Purpose: This study aimed to characterize the challenges in using genetic information in health care and to identify opportunities for improvement., Methods: Taking a grounded theory approach, semistructured interviews were conducted with 48 participants to collect multiple stakeholder perspectives on genetic services in New Zealand., Results: Three themes emerged from the data: (1) four service delivery models were identified in operation, including both those expected models involving genetic counselors and variations that do not route through the formal genetic service program; (2) multiple barriers to sharing and using genetic information were perceived, including technological, organizational, institutional, legal, ethical, and social issues; and (3) impediments to wider use of genetic testing technology, including variable understanding of genetic test utilities among clinicians and the limited capacity of clinical genetic services. Targeting these problems, information technologies and knowledge management tools have the potential to support key tasks in genetic services delivery, improve knowledge processes, and enhance knowledge networks., Conclusion: Because of the effect of issues in genetic information and knowledge management, the potential of human genetic variation knowledge to enhance health care delivery has been put on a "leash."

This article describes a study of a major change management project involving the establishment of a shared services organization to align the information services functions for two district health boards in New Zealand. The research uncovered a capability crisis that many people experienced when they realized the magnitude of the task they were involved in. Not everyone experienced the crisis and it seems that learning, especially in a complex healthcare environment, plays an important role in reducing the negative impact of change.

The sensitivity of different cell lines (Chinese hamster ovary, HeLa, L1210, and P388) to 43 degrees was compared Chinese hamster ovary and HeLa cells were much less temperature sensitive than were L1210 or P388 cells. This difference persisted even when HeLa and L1210 cells were grown in the same medium and suggested that this was an inherent difference between the cell lines. Of all the cell lines, 7-day L1210 ascites, maintained by transfer in mice, were the most sensitive. The greater sensitivity of these cells, as compared to 4-day L1210 ascites or cells in culture, may be explained by the difference in their growth stages. The 7-day ascites cells would be in stationary growth as compared to the exponentially growing 4-day ascites or the cells in culture. The temperature sensitivity of Chinese hamster ovary cells in different parts of the cell cycle was determined. Mid and late-S-phase cells were more sensitive than cells in mitosis or early S; G1 and G2 cells were the least sensitive. In a partially synchronized culture, the heat sensitivity of L1210 cells increased with increased percentage of cells in S phase.

When L1210 cells growing logarithmically were exposed for 8 hr to a nonlethal dose of 5-fluorouracil (FU) (0.25 microgram/ml), the percentage of cells in S phase increased from 74.9% in the asynchronous culture to 93% in the FU-treated culture. This resulted in increased cell-kill by S-phase-specific inhibitors [1-beta-D-arabinofuranosylcytosine (ara-C), 5-hydroxy-2-formylpyridinethiosemicarbazone] when they were added to a culture partially synchronized by pretreatment with FU. For example, 2 hr exposure to ara-C alone or ara-C plus FU (added simultaneously to asynchronous culture) gave 28.8 and 25.8% survival, respectively, compared to 6.8% survival when ara-C was added for 2 hr to the partially synchronized culture. Eight to 12 hr after FU removal, the culture became asynchronous, such that ara-C addition at this time did not result in increased cell-kill. Cultures pretreated with FU were also highly sensitive to vincristine and Adriamycin. Adriamycin acted synergistically with FU (after 8 hr pretreatment) in killing L1210 cells.

Cytosine arabinoside (ara-C) plus actinomycin D acted synergistically in killing L1210 and DON cells. The time course of cell-kill by the combination was different from that seen for either drug alone. The results obtained (both with L1210 and synchronous DON cells) suggest that cells blocked in G1/S with continuing unbalanced growth are particularly sensitive to the combined action of ara-C and actinomycin D.

Drug sensitivity and cell cycle parameters of L1210 cells in culture in exponential (10(5) to 4 x 10(5) cells/ml) and plateau phase (greater than 8 x 10(5) cells/ml) of growth were compared. The percentage of cells in G1, S, and G2 + M for exponential and plateau-phase cultures was 24.1, 70.0, and 5.9, and 42.5, 45.6, and 11.9, respectively. These values correspond to those reported previously for early and late L1210 mouse leukemia cells. Cell survival (measured by cloning) after drug exposure showed that actinomycin D, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, chlorozotocin, and streptozocin were all equally lethal to cells in both phases, and 1-beta-D-arabinofuranosylcytosine, high-specific-activity (20Ci/mmole) [3H]thymidine, vincristine, daunomycin, adriamycin, and 5-fluorouracil were more lethal to exponential cells than to plateau-phase cells. The percentage kill by S-phase-specific agents such as 1-beta-D-arabinofuranosylcytosine and[3H]thymidine corresponded well with the percentage of cells in S. Finally, 1,3-bis(2-chloroethyl)-1-nitrosourea was more toxic to plateau cells than to exponential cells.