Single-Spin Magnetomechanics with Levitated Micromagnets.

We demonstrate a new mechanical transduction platform for individual spin qubits. In our approach, single micromagnets are trapped using a type-II superconductor in proximity of spin qubits, enabling direct magnetic coupling between the two systems. Controlling the distance between the magnet and the superconductor during cooldown, we demonstrate three-dimensional trapping with quality factors around 1×10^{6} and kHz trapping frequencies. We further exploit the large magnetic moment to mass ratio of this mechanical oscillator to couple its motion to the spin degrees of freedom of an individual nitrogen vacancy center in diamond. Our approach provides a new path towards interfacing individual spin qubits with mechanical motion for testing quantum mechanics with mesoscopic objects, realization of quantum networks, and ultrasensitive metrology.

New classes of systematic effects in gas spin comagnetometers.

Atomic comagnetometers are widely used in precision measurements searching for spin interactions beyond the standard model. We describe a new (3)He-(129)Xe comagnetometer probed by Rb atoms and use it to identify two general classes of systematic effects in gas comagnetometers, one associated with diffusion in second-order magnetic-field gradients and another due to temperature gradients. We also develop and confirm experimentally a general and practical approach for calculating spin relaxation and frequency shifts due to arbitrary magnetic-field gradients.

Nonpeptidic, monocharged, cell permeable ligands for the p56lck SH2 domain.

p56lck is a member of the src family of tyrosine kinases and plays a critical role in the signal transduction events that lead to T cell activation. Ligands for the p56lck SH2 domain have the potential to disrupt the interaction of p56lck with its substrates and derail the signaling cascade that leads to the production of cytokines such as interleukin-2. Starting from the quintuply charged (at physiological pH) phosphorylated tetrapeptide, AcpYEEI, we recently disclosed (J. Med. Chem. 1999, 42, 722 and J. Med. Chem. 1999, 42, 1757) the design of the modified dipeptide 3, which carries just two charges at physiological pH. Here we present the elaboration of 3 to the nonpeptidic, monocharged compound, 9S. This molecule displays good binding affinity for the p56lck SH2 domain (K(d) 1 microM) and good cell permeation, and this combination of properties allowed us to demonstrate clear-cut inhibitory effects on a very early event in T cell activation, namely calcium mobilization.

Permeability measurement of macromolecules and assessment of mucosal antigen sampling using in vitro converted M cells.

INTRODUCTION: M cells are located in the epithelial layer covering the gut-associated lymphoid tissue and are responsible for delivery of macromolecules and microorganisms to the underlying lymphoid cells. It has been shown that the human colonic cell line Caco-2 can be converted to M cells in vitro following coculture with isolated lymphocytes from murine Peyer's patches. Studies were undertaken to evaluate and characterize the transepithelial transport of select macromolecules across these in vitro derived M cells. METHODS: Caco-2 cells were converted to M cells as reported previously. The morphology of Caco-2 cells and M cells was compared by transmission electron microscopy (TEM). The transport properties of macromolecules such as horseradish peroxidase, FITC-conjugated polystyrene beads, and radiolabeled dextrans were examined. The activation of murine antigen-specific T cells following transport of the antigen ovalbumin across the M-cell barrier was assessed by measuring cytokine production. RESULTS: M cells were shown to be irregular in shape and have fewer and shorter microvilli compared to the Caco-2 cell progenitors. These cells were still able to form tight junctions and monolayers on polycarbonate membranes. Time-course studies demonstrated that the transport of polystyrene beads and large-molecular-weight dextrans at physiological temperature across M-cell-containing monolayers was size dependent and more rapid than across Caco-2 cell monolayers. The transport of dextrans was also shown to be temperature and concentration dependent. Befitting the role of the M cell in mucosal defense, protein antigen could be delivered by these cells in order to be processed and presented to antigen-specific CD4+ T lymphocytes. DISCUSSION: The M-cell permeability model is a functional and practical system for evaluating the transport properties of macromolecules and assessing the potential for intestinal mucosal antigen sampling to elicit immunological responses.

Reducing adverse drug events: lessons from a breakthrough series collaborative.

BACKGROUND: In January 1996, 38 hospitals and health care organizations (for a total of 40 hospitals) in the United States came together in an Institute for Healthcare Improvement (IHI; Boston) Breakthrough Series collaborative to reduce adverse drug events-injuries related to the use or nonuse of medications. METHODS: The participants were taught the Model for Improvement, a method for rapid-cycle change and evaluation, and were then coached on how to identify their own problem areas and develop changes in practice for rapid-cycle testing. These changes could be implementation of one or more known medication error prevention practices or new practices developed. RESULTS: During a 15-month period the 40 hospitals conducted a total of 739 tests of changes. Process changes accounted for 63% of the cycles; the remainder consisted of preliminary data gathering, consensus-building, or education cycles. Eight types of changes were implemented by seven or more hospitals, with a success rate of 70%. These changes included non-punitive reporting, ensuring documentation of allergy information, standardizing medication administration times, and implementing chemotherapy protocols. DISCUSSION: Success in making significant changes was associated with strong leadership, effective processes, and appropriate choice of intervention. Successful teams were able to define, clearly state, and relentlessly pursue their aims, and then chose practical interventions and moved early into changing a process. They did not spend months collecting data before beginning a change. Changes that were most successful were those that attempted to change processes, not people. Health care organizations committed to patient safety need not regard current performance limits as inevitable.

Quality improvements in end of life care: insights from two collaboratives.

BACKGROUND: Just a few generations ago, most people died suddenly, at any age. Now, most die of serious chronic disease, after a substantial period of disability. The care system does not serve this burgeoning population well. However, two quality improvement (QI) collaboratives sponsored by the Institute for Healthcare Improvement and the Center to Improve Care of the Dying set about making substantial improvements. INSIGHTS GAINED: The participating organization teams in two Breakthrough Series collaboratives found it best to identify patients by asking "Would it be surprising for this patient to die in the next year? (or the next few months?)" All the teams used standard QI approaches, with an aim, measures, and changes to try in Plan-Do-Study-Act cycles. In the first collaborative, 42 (89%) of the 47 teams made important improvements in their care systems. Because of the strength of their changes, the high performance of their team, the administrative support they received, and their ability to partner with other agencies, 13 (27%) of the teams made substantial, measurable improvement during the collaborative. In the second collaborative, 29 (85%) of the 34 teams made key changes to their care system, and 16 (47%) of the teams made substantial, measurable improvement. Coordination across programs such as between a hospital and a long term care facility or hospice remained an elusive goal, and good care cannot become routine without financing and coverage reform. CONCLUSION: Clinical providers can reliably make substantial improvements in end of life care, within a few months, and within current financing and regulation. Coordinated efforts in two Breakthrough Series produced generalizable insights.

Sequence, purification, and cloning of an intracellular serine protease, quiescent cell proline dipeptidase.

We recently observed that specific inhibitors of post-proline cleaving aminodipeptidases cause apoptosis in quiescent lymphocytes in a process independent of CD26/dipeptidyl peptidase IV. These results led to the isolation and cloning of a new protease that we have termed quiescent cell proline dipeptidase (QPP). QPP activity was purified from CD26(-) Jurkat T cells. The protein was identified by labeling with [(3)H]diisopropylfluorophosphate and subjected to tryptic digestion and partial amino acid sequencing. The peptide sequences were used to identify expressed sequence tag clones. The cDNA of QPP contains an open reading frame of 1476 base pairs, coding for a protein of 492 amino acids. The amino acid sequence of QPP reveals similarity with prolylcarboxypeptidase. The putative active site residues serine, aspartic acid, and histidine of QPP show an ordering of the catalytic triad similar to that seen in the post-proline cleaving exopeptidases prolylcarboxypeptidase and CD26/dipeptidyl peptidase IV. The post-proline cleaving activity of QPP has an unusually broad pH range in that it is able to cleave substrate molecules at acidic pH as well as at neutral pH. QPP has also been detected in nonlymphocytic cell lines, indicating that this enzyme activity may play an important role in other tissues as well.

Ligands for the tyrosine kinase p56lck SH2 domain: discovery of potent dipeptide derivatives with monocharged, nonhydrolyzable phosphate replacements.

p56lck is a member of the src family of tyrosine kinases. Through modular binding units called SH2 domains, p56lck promotes phosphotyrosine-dependent protein-protein interactions and plays a critical role in signal transduction events that lead to T-cell activation. Starting from the phosphorylated dipeptide (2), a high-affinity ligand for the p56lck SH2 domain, we have designed novel dipeptides that contain monocharged, nonhydrolyzable phosphate group replacements and bind to the protein with KD's in the low micromolar range. Replacement of the phosphate group in phosphotyrosine-containing sequences by a (R/S)-hydroxyacetic (compound 8) or an oxamic acid (compound 10) moiety leads to hydrolytically stable, monocharged ligands, with 83- and 233-fold decreases in potency, respectively. This loss in binding affinity can be partially compensated for by incorporating large lipophilic groups at the inhibitor N-terminus. These groups provide up to 13-fold increases in potency depending on the nature of the phosphate replacement. The discovery of potent (2-3 microM), hydrolytically stable dipeptide derivatives, bearing only two charges at physiological pH, represents a significant step toward the discovery of compounds with cellular activity and the development of novel therapeutics for conditions associated with undesired T-cell proliferation.

Lessons in cooperation: an update on improving the quality of hospital care.

BACKGROUND: Between 1989 and 1997 the Robert Wood Johnson Foundation (Princeton, NJ) launched a demonstration project, the Improving the Quality of Hospital Care Program, to test a consortium approach to quality improvement. As part of the project, four hospital consortia in various parts of the United States shared quality resources (for example, training) and collaborated on improvement efforts. Although cooperation was not a natural approach for enhancing quality in hospitals, the consortia mounted improvements in multiple clinical areas, such as diabetes care, the intensive care unit (ICU), prevention of wound infections, and care in rural areas. WHERE ARE THEY NOW? Of the four consortia that received implementation funding, all are continuing some explicit focus on improving quality, but only two have retained the organizational form of a consortium. Based at the University of Iowa (Iowa City), the Institute for Quality Healthcare continues to operate as a free-standing consortium with more than 40 hospital members. The Vermont Program for Quality in Health Care (Montpelier) provides information and education to improve quality of care statewide. LESSONS FROM THE PROGRAM: The program taught valuable lessons about what hospitals can do together and what they can achieve when they cooperate around quality of care issues. Sharing resources for education, providing a forum for quality improvement professionals to work together on specific issues, and identifying means of improving specific aspects of care in the group are all feasible in the consortium model. Even a chaotic environment can support cooperation.

Reducing cesarean section rates safely: lessons from a "breakthrough series" collaborative.

BACKGROUND: Almost one million cesarean operations are performed each year in the United States. The objective of this project was to test the hypothesis that a structured collaborative effort can help participating health care organizations to reduce their cesarean delivery rates safely. METHODS: Experts associated with the collaborative helped participant organizations to explore several categories of change concepts and to develop action plans for safely reducing their cesarean delivery rates. Over the course of one year participants attended three two-day learning sessions. In the interval between these sessions, collaborative participants communicated by weekly conference calls and a dedicated Internet site. RESULTS: Of 28 participating organizations, 15 percent achieved cesarean delivery rate reductions of 30 percent or more during the 12-month period of active collaborative work. An additional 50 percent achieved reductions between 10 and 30 percent. CONCLUSIONS: The Healthy People 2000 goal of a cesarean delivery rate below 15 percent by the year 2000 is attainable. Clinical leadership from doctors and nurses toward the achievement of that goal is timely, ethical, and in the best interests of childbearing women in the United States.

Beyond survival: toward continuous improvement in medical care.

Using the scientific method, continuous improvement strives to attain unprecedented levels of performance - improved patient outcomes while maintaining or reducing costs. The needs for, and benefits of, continuous improvement are discussed along with a description of its basic elements. The approaches outlined can serve to greatly increase the pace of improvement in health care.

Lessons in cooperation: four hospital consortia relate their quality improvement experiences.

BACKGROUND: In 1989, The Robert Wood Johnson Foundation launched a demonstration project to test a consortium approach to quality improvement. As part of this project, four hospital consortia in various parts of the United States are currently sharing quality resources (for example, training) and collaborating on various improvement efforts. The purpose of the project is to demonstrate that hospitals can take on more difficult problems and accomplish more in cooperation with each other than on their own. CASE STUDIES: The Institute for Quality Healthcare (Iowa City, Iowa) has built a comparative database so that 40 member hospitals can make meaningful comparisons on various aspects of performance; The Vermont Program for Quality in Health Care has lowered the postoperative infection rate in Vermont by monitoring compliance with consensus guidelines; Interwest Quality of Care, Inc, which has member organizations in Utah, Wyoming, and Idaho, has adapted and disseminated guidelines for diabetic care; and The Public Hospital Institute, in Berkeley, California, has worked with the Joint Commission on Accreditation of Healthcare Organizations to develop a written guide to help surveyors understand the unique operational traits of public hospitals. LESSONS LEARNED: Projects such as those with champions in several member organizations and comparative data analysis lend themselves more easily to cooperative work than others. They also provide some strategies for collaboration, such as continually reinforcing the principles of collaboration, obtaining a fully informed commitment, beginning with initiatives that are likely successes, and being serious and vocal about the commitment to confidentiality. CONCLUSIONS: Collaborators in quality improvement gain important resources, such as better information, more relevant reference databases, colleagues and support for quality improvement specialists, and economies of scale in education programs, training materials, and interaction with vendors. However, the difficulties in collaboration are great. Hospitals must continually consider not only "What's in this for me," but also "What can we accomplish as a group that is greater than what each of us can do alone?"

Interaction of Sec4 with GDI proteins from bovine brain, Drosophila melanogaster and Saccharomyces cerevisiae. Conservation of GDI membrane dissociation activity.

Rab GDP dissociation inhibitor (Rab GDI), will induce the dissociation of GDP-bound rab3A from synaptic membranes and will inhibit GDP dissociation from Sec4, a member of the Rab subgroup of the Ras GTPase superfamily which is required for exocytosis in Saccharomyces cerevisiae. We report that Rab GDI releases GDP-bound Sec4 from yeast membranes. dGDI, a Drosophila homologue can similarly inhibit GDP dissociation from Sec4 and release GDP-bound Sec4 from yeast membranes. An activity partially purified from yeast cytosol dissociates GDP-bound Sec4 from yeast membranes, suggesting that yeast also possess a GDI protein that functions to recycle Sec4 from its target membrane.

The cycle of SEC4 function in vesicular transport.

Sec4 is a Ras-like GTP-binding protein required for exocytosis in yeast. Unlike Ras, it is the ability of Sec4 to cycle between the GTP- and GDP-bound forms rather than the absolute levels of the GTP-bound form that is critical for function. This cycle may be coupled to an observed cycle of Sec4 localization within the cell. Sec4 binds to secretory vesicles which then fuse with the plasma membrane in exocytosis. Sec4 can recycle from the plasma membrane through a soluble pool to rebind to a new round of vesicles. We have found an activity in yeast (Saccharomyces cerevisiae) comparable to that of the GDP dissociation inhibitor protein isolated from mammalian cells that releases GDP-bound Sec4 from membranes. DSS4-1, a dominant suppressor of the sec4-8 temperature-sensitive mutation, encodes a nucleotide exchange protein. The cycle of Sec4 may function to allow the assembly and subsequent disassembly of a set of proteins necessary for exocytosis. Candidates for members of this set of proteins are encoded by sec genes which show strong genetic interactions with sec4-8. Two of these (SEC8 and SEC15) encode large proteins which form a complex that is peripherally associated with the plasma membrane.

Hydrolysis of GTP by Sec4 protein plays an important role in vesicular transport and is stimulated by a GTPase-activating protein in Saccharomyces cerevisiae.

Sec4, a GTP-binding protein of the ras superfamily, is required for exocytosis in the budding yeast Saccharomyces cerevisiae. To test the role of GTP hydrolysis in Sec4 function, we constructed a mutation, Q-79----L, analogous to the oncogenic mutation of Q-61----L in Ras, in a region of Sec4 predicted to interact with the phosphoryl group of GTP. The sec4-leu79 mutation lowers the intrinsic hydrolysis rate to unmeasurable levels. A component of a yeast lysate specifically stimulates the hydrolysis of GTP by Sec4, while the rate of hydrolysis of GTP by Sec4-Leu79 can be stimulated by this GAP activity to only 30% of the stimulated hydrolysis rate of the wild-type protein. The decreased rate of hydrolysis results in the accumulation of the Sec4-Leu79 protein in its GTP-bound form in an overproducing yeast strain. The sec4-leu79 allele can function as the sole copy of sec4 in yeast cells. However, it causes recessive, cold-sensitive growth, a slowing of invertase secretion, and accumulation of secretory vesicles and displays synthetic lethality with a subset of other secretory mutants, indicative of a partial loss of Sec4 function. While the level of Ras function reflects the absolute level of GTP-bound protein, our results suggest that the ability of Sec4 to cycle between its GTP and GDP bound forms is important for its function in vesicular transport, supporting a mechanism for Sec4 function which is distinct from that of the Ras protein.

A mammalian inhibitory GDP/GTP exchange protein (GDP dissociation inhibitor) for smg p25A is active on the yeast SEC4 protein.

Evidence is accumulating that smg p25A, a small GTP-binding protein, may be involved in the regulated secretory processes of mammalian cells. The SEC4 protein is known to be required for constitutive secretion in yeast cells. We show here that the mammalian GDP dissociation inhibitor (GDI), which was identified by its action on smg p25A, is active on the yeast SEC4 protein in inhibiting the GDP/GTP exchange reaction and is capable of forming a complex with the GDP-bound form of the SEC4 protein but not with the GTP-bound form. These results together with our previous findings that smg p25A GDI is found in mammalian cells with both regulated and constitutive secretion types suggest that smg p25A GDI plays a role in both regulated and constitutive secretory processes, although smg p25A itself may be involved only in regulated secretory processes. These results also suggest that a GDI for the SEC4 protein is present in yeast cells.

Binding and hydrolysis of guanine nucleotides by Sec4p, a yeast protein involved in the regulation of vesicular traffic.

The 23.5-kDa Sec4 protein is required for vesicular transport between the Golgi apparatus and the plasma membrane in Saccharomyces cerevisiae. In order to analyze its biochemical properties, we have purified the soluble pool of the wild-type protein from an overproducing yeast strain. At 30 degrees C, Sec4p bound [35S] guanosine 5'-O-(thiotriphosphate) (GTP gamma S) with a rate of 0.18 min-1 in a reaction requiring micromolar concentration of free magnesium ions. The protein had high affinity for guanine nucleotides with Kd values for GTP gamma S and GTP of 3.7 nM and 3.5 nM, respectively, and that for GDP of 77 nM. The dissociation of [3H] GDP from Sec4p occurred with a rate of 0.21 min-1 suggesting that the association of GTP gamma S was the result of exchange for prebound GDP. The release of GTP from Sec4p was slow and correlated with a low inherent GTPase activity of 0.0012 min-1. By analogy with other classes of GTP binding proteins, both the nucleotide exchange and hydrolysis activities of Sec4p may be modulated in vivo to facilitate its role in the regulation of intercompartmental membrane traffic.