Mandatory Intensivist Management Decreases Length of Stay, Facilitates an Increase in Admissions and Minimizes Closure of a Neurocritical Care Unit.

BACKGROUND: The primary objectives of this study were to identify patient and community benefits of mandatory intensivist management in a neurocritical care (NCC) unit. Our hospital recently mandated intensivist management for patients admitted to the NCC unit. As one of the only comprehensive stroke centers in Orlando, an unacceptably high number of patients were being denied admission because of overcapacity. We compared length of stay (LOS), complications, outcomes, total admissions, and emergency transfer center closure rates before and after implementation of mandatory intensivist management. METHODS: A retrospective review comparing 1551 patients admitted to a 20 bed NCC unit from November 1, 2009 to October 31, 2010 (prior to mandatory intensivist management) with 1702 patients admitted from January 1, 2011 to December 31, 2011 (after the requirement) was performed. This included examining LOS, Acute Physiology and Chronic Health Evaluation III (APACHE) scores, service line closure rates, and mortality during both time periods. RESULTS: Analysis revealed that despite comparable APACHE scores, implementation of mandatory intensivist management reduced overall NCC LOS, 4.6 versus 3.7 days, (p < 0.01) and increased the number of monthly admissions, 129 versus 142, (p = 0.02). The percentage of patients declined admission because of a closed service line was reduced from 12.36 to 5.66 %, (p = 0.02). Mortality and infection rates remained unchanged. CONCLUSIONS: Implementation of mandatory intensivist management in the NCC unit decreased LOS, increased admissions, and decreased service line closure rates, while maintaining patient care.

Over the southern solar pole: low-energy interplanetary charged particles.

The heliosphere instrument for spectrum, composition, and anisotropy (HISCALE) recorded the fluxes of low-energy ions and electrons (> 50 kiloelectron volts) when Ulysses crossed the southern solar polar region and revealed that the large-scale structure of the heliosphere to at least approximately -75 degrees was significantly influenced by the near-equatorial heliospheric current sheet. Electrons in particular were accelerated by the current sheet-produced and poleward-propagating interplanetary reverse shock at helioradii far from the Ulysses location. At heliolatitudes higher than approximately -75 degrees on the Ulysses ascent to the pole and approximately -50 degrees on the descent, small, less regular enhancements of the lowest energy electron fluxes were measured whose relations to the current sheet were less clear. The anomalous component of low-energy (approximately 2 to 5 megaelectron volts per nucleon) oxygen flux at the highest heliolatitudes was found to be approximately 10(-8) [per square centimeter per second per steradian (per kiloelectronvolt per nucleon)]; the anomalous Ne/O ratio was approximately 0.25.

Evidence of low molecular weight RNAs involved in permanent character changes in amoebae.

Previous studies have established that 'informational molecules' present in cytoplasmic fractions of A. discoides may be transferred by microinjection into A. proteus. Clones derived from injected cells showed various changers, including lowered sensitivity to growth in streptomycin and neomycin, in which respects they resembled A. discoides. These changes in response to antibiotics were transferred independently and were permanent, the information being replicated over many generations. The most 'active' material in terms of the number of clones showing character changes was found following injection of 16S ribonucleoprotein obtained after sucrose density gradient centrifugation of the mcirosomal fraction. Polyacrylamide gel electrophoresis of the 16S material showed 3 small peaks of RNA. In order to obtain adequate amounts of material, these peaks of RNA were identified in electrophoresis profiles of RNA extracted from the whole microsomal fraction, and RNA eluted from these latter gels was injected into A. proteus. Although the number of surviving clones was low, all were examined for their response to growth in either streptomycin, neomycin, erythromycin or chloroquine. After injection of RNA eluted from the 3 small peaks of RNA (slices 26-33), 8 out of 10 and 9 out of 10 clones showed lowered sensitivity to growth in streptomycin and neomycin respectively, and resembled the donor A. discoides. No changes in responses to antibiotics were obtained from clones derived from cells injected with RNA eluted from another region of the gel, or after ribonuclease treatment of the RNA from slices 26-33. The relative molecular weights of these 'informational' RNA molecules were found to be between 9 and 13 X 10(4) Daltons.

Informational molecules in amoebae: an attempt to follow injected fractions using autoradiography.

Cytoplasmic fractions from Amoeba discoides contain informational molecules which, when injected into A. proteus, may influence the characters of resulting clones. An attempt was made to follow and locate these molecules by autoradiography. Using either 3H-uridine or 3H-leucine, radioactive fractions of A. discoides were microinjected into A. proteus and cells examined at varying times after injection. A. discoides were injected with the same preparations to serve as controls. Grain counts were compared for equivalent areas of nucleus and cytoplasm, and results expressed in terms of the ratio between nucleus and cytoplasm. When the light microsomal or the supernatant fractions were injected, the level of labelled molecules was higher in the nucleus than the cytoplasm in over 50% of the cells examined after 6 h, except for those cells injected with 3H-leucine-labelled supernatant material when only 15% cells showed a nuclear/cytoplasmic grain count greater than 1.1. After some purification by sucrose density gradient centrifugation, 'information-containing' peaks '13' from the light microsomal fraction, and '16' from the supernatant fraction were injected. A different pattern of results was obtained. The problems of interpreting these results in the light of known migratory molecules and strain incompatibility are discussed.

Absence of low molecular weight DNA polymerase activity from the nuclei of Amoeba discoides.

Amoeba discoides nuclear protein partially purified by passage through Sephadex G-200 showed 3 high-mol.-wt. DNA polymerase activities which eluted in and just following the void volume. No low-mol.-wt (45,000 daltons) DNA polymerase beta activity was detected. Nuclear protein layered on 5--20% sucrose gradients also showed an absence of low-mol.-wt DNA polymerase beta. The void volume enzyme showed deoxyribonuclease activity, but no low-mol.-wt nuclease activity was detected.

Characterization of a strain-specific inhibitor of cell division from Amoeba cytoplasm.

The microinjection of cytoplasm taken from one strain of large free-living amoeba into another strain is followed by an incompatibility phenomenon, the inhibition of division amongst the recipient cells. The post-microsomal supernatant fraction from Amoeba discoides (T1D13) injected into A. proteus (T1P) inhibited division in 90% of the injected cells. Further centrifugation of this fraction yielded a pellet which when resuspended and injected, inhibited division in over 95% (and sometimes 100%) of the cells. No inhibitory activity remained in the supernatant after the removal of this pellet. Treatment with 10 micrograms/ml trypsin destroyed the activity of this pellet, while 25 micrograms/ml ribonuclease reduced the inhibitory activity by approximately 40%. Passage of the resuspended post-microsomal pellet through Sephadex G-200 gave one main peak of material which eluted in the void volume. Concentration of this material by either dialysis or lyophilization followed by microinjection into A. proteus showed that this void volume peak contained the inhibitory material, although the most active preparations did not give more than 66% inhibition of division. After elution from Sephadex, the void volume material was analysed by electrophoresis under non-denaturing and denaturing conditions, and by isoelectric focusing. One problem was the loss of inhibitory activity after keeping the pellet at 4 degrees C for 4-5 days, which made further analysis by microinjection difficult. Preliminary experiments using a post-microsomal pellet prepared from Dawson's A. proteus (DP) which inhibited division in A. proteus (T1P) gave a similar profile after Sephadex chromatography and gel electrophoresis.

Inversion in Volvox tertius: the effects of con A.

During the development of Volvox tertius spheroids, a single-celled gonidium enlarges and undergoes multiple incomplete cleavages to give an embryo which is 'inside-out' with respect to the adult organism. A morphogenetic movement, termed 'inversion', turns this hollow ball of cells 'inside-out' through a hole, the phialopore. In V. tertius this phialopore possesses 4 inwardly directed lips. Normal inversion was studied in vitro in slide chambers and involved cell-shape changes accompanied by the production of pseudopodia and the bending backwards of the phialopore lips. 100 micrograms/ml Con A specifically and reversibly blocked inversion. Despite the inhibitory effect on cell division, the blocking of inversion was not due to the blocking of the last cell division some 50-100 min prior to inversion. Neither did the first cell-shape change from pear- to spindle-shape appear blocked. A feature of inhibition by Con A was the enhanced production of pseudopodia by embryos blocked at inversion, and the abnormal production of pseudopodia by embryos blocked at earlier stages. Non-inverting embryos showed internal flagella. We suggest that the Con A block to inversion, which may be reversed by alpha-methyl mannoside, arises from the prevention of backwards-bending of the phialopore lips. Fluorescein-isothiocyanate-Con A bound to embryo and cell coat, ane more strongly to the embryo at pre-inversion. SDS-polyacrylamide gel analysis of proteins isolated from embryos showed 4 glycoprotein bands, but Con A binding to these bands could not be demonstrated.

Informational molecules in amoebae studies of template utilization by the DNA polymerase activities of Amoeba discoides.

Studies have shown that certain strain-specific characters of large, free-living amoebae may be influenced by the microinjection of non-homologous low molecular weight RNAs. To investigate the mechanisms involved in 'information' transfer, the template preferences of partially purified DNA polymerase activities isolated from Amoeba discoides have been studied. After passage through Sephadex G-200, DNA polymerase activities from whole homogenates could utilize both 'activated' calf thymus DNA and the synthetic ribohomopolymer poly rA oligo d(pT)10 as templates. Fractionation using different saturations of (NH4)2SO4 showed that there was no detectable poly A d(pT)10-directed DNA polymerase activity in the extra-mitochondrial cytoplasm, and the ability to utilize this template appeared to be located in the nuclei. Nuclear protein passed through short (30 cm) columns of Sephadex G-200 showed DNA polymerase activities which could use both synthetic and natural RNA as templates, but little activity was detected when using longer (70 cm) columns, although DNA-directed DNA polymerase activities were more clearly defined. Use of DEAE-cellulose only revealed a low (1--3%) activity with poly A d(pT)10 compared to the activity observed using 'activated' calf thymus DNS. DNA polymerase activities of amoebae showed a response to added RNA templates which depended on the purity of the enzyme preparation, and the possibility that these enzymes are involved in 'information' transfer cannot be ruled out.

A method for studying the effect of inhibitors on the development of the isolated gonidia of Volvox tertius.

During asexual reproduction in Volvox tertius, a single cell, the gonidium, contained within the parental spheroid, undergoes enlargement followed by multiple cleavages. A method has been developed for the isolation and maintenance of gonidia in vitro in paraffin chambers. In these chambers development of the gonidium to a multicellular spheroid and subsequent inversion was apparently normal. The paraffin chambers allowed the monitoring of the effects of inhibitors on development without the possible interference of the parental spheroid. Enlargement and division of isolated gonidia was prevented by actinomycin D, cycloheximide and streptomycin. Division but not enlargement was inhibited by 5-fluorouracil, mitomycin C and ethidium bromide. Colchicine at high concentrations prevented inversion, as did streptomycin, cycloheximide and actinomycin D.

The effect of antrycide of patterns of RNA synthesis in Amoeba discoides.

Antrycide is an aminoquinaldine whose inhibitory action on the growth of Trypanosoma and Crithidia is not fully understood at the cellular level. The growth of Amoeba discoides in concentrations of antrycide between 0.5 and 2 microgram/ml was reduced considerably, while cells failed to divide in 4 microgram/ml. The effects on growth rate were reversible at least up until 7 days in antrycide. In order to assess the action of this synthetic drug on RNA synthesis in amoebae, the pattern of synthesis in normal cells was investigated using polyacrylamide gel electrophoresis. The profile of high molecular weight RNAs observed depended on the length of time in [3H]uridine, and was only fully developed after 66 h, when 5 peaks could be seen. The relative molecular weights of these peaks (I--V) were 2.45, 1.55, 1.13, 0.8 and 0.52 X 10(6) Daltons respectively. Those of 1.55 and 0.8 X 10(6) corresponded to ribosomal RNAs, the identity of the other peaks is unknown. After growth in 2 microgram/ml antrycide for 4 days, no high molecular weight RNA was found. Use of [14C]adenine/[3H]uridine showed that after 17 h in antrycide there was a loss of ribosomal RNA and increased levels of low molecular weight RNAs, due either to lack of synthesis or to degradation of newly synthesized material. Incorporation of [3H]leucine into hot acid-precipitable protein was inhibited in antrycide-treated cells by at least 50%. A possible explanation of the effect of antrycide on A. discoides was the inhibition of mRNA synthesis for ribosomal proteins, leading to degradation of newly synthesized rRNA. Reduced growth would continue on pre-existing ribosomes and previously synthesized long-lived mRNAs.

Transformation in Naegleria gruberi: patterns of RNA synthesis examined by polyacrylamide gel electrophoresis.

Naegleria gruberi were grown on bacteria and methods were devised to free the cellular RNA from bacterial RNA contamination. Use of actinomycin D and cycloheximide showed that the transformation of Naegleria from amoeba to flagellate required RNA synthesis for 30 min and protein synthesis for 40 min after the initial stimulus of distilled water. Comparison of the patterns of RNA synthesized during transformation with those during growth indicated a considerable amount of new RNA produced during the phenotypic change. Most marked was the increase in RNA co-migrating on polyacrylamide gels with the small ribosomal sub-unit RNA, together with RNAs between the latter and transfer RNA. These results were compared with other published results using axenically-grown cells cells and sucrose density gradient centrifugation. Cells placed in 80 mM NaCl instead of distilled water fail to transform but the pattern of newly-synthesized RNAs was not significantly different from that seen in transforming cells. This suggested that high salt concentrations inhibit transformation by inhibiting synthesis and/or assembly of certain proteins rather than RNA synthesis. Eluted material from various regions of polyacrylamide gels containing RNA extracted from transforming cells was used in a cell-free system. Incorporation of 3H-glutamic acid but not 3H-tryptophan was stimulated by material extracted from the 18S regions of the gels.

Inhibition of cell division in amoebae: the incorporation of tritiated precursors into Amoeba proteus after the injection of non-homologous cytoplasm.

The injection of non-homologous cytoplasm into any strain of large free-living amoebae leads to a 60% inhibition of division amongst recipient cells. When the post-microsomal supernatant fraction of Amoeba discoides was injected into A. proteus, this inhibition of division was as high as 95%. The incorporation of tritiated precursors, either [3H]uridine or 3H-amino acids, into these inhibited amoebae was studied at various times after the injection of the inhibitory material using autoradiography. When cells were grown in [3H]uridine, autoradiographs indicated that RNA synthesis had ceased 2 days after the injection of non-homologous material. However, if [3H]uridine was injected into the inhibited cells, some synthesis of RNA could be detected up to 4 days after the injection of inhibitor. These results suggested that uptake of [3H]uridine was impaired and that one site of action of the inhibitory molecules was RNA synthesis for membrane components. Experiments with a variety of 3H-amino acids suggested that protein synthesis continued for at least 9 days after the injection of non-homologous cytoplasm, and that in these cells some informational RNA molecules were long-lived. There seemed to be accumulation of material containing [3H]lysine in the nuclei of control cells taken at random from cultures, and this was seen in the nuclei of inhibited cells 1 day after injection. However, 2 days after the injection of inhibitor, no accumulation of [3H]lysine-containing material was found in the nuclei.

Studies on transformation in Naegleria gruberi: effects of ions and bacterial suspensions.

The presence of electolytes inhibited the transformation of Naegleria gruberi from amoeba to flagellate, the molarity required varying with the salt used, namely 80 mM NaCl, 90 mM KCl, 50 mM CaCl2 or 60 mM MgCl2. Non-electrolytes also prevented this transformation at 250 mM for either sucrose or glucose, and this is known to be an osmotic effect. That the effect of ionic solutions was different was demonstrated by varying the time at which the environemnt was changed from distilled water to salt solution. Experiments with suspensions of either living or heat-killed bacteria in distilled water, together with the supernatants obtained when bacteria were removed by centrifugation, showed that the inhibition of transformation which occurred in bacterial suspensions was not due to any factors produced by the bacteria and present in solution. It appeared that this inhibition was brought about by the physical presence of the bacteria, either living or heat-killed, and some possible interpretations of this 'contact' phenomenon are discussed.