Resolved-Sideband Laser Cooling in a Penning Trap.

We report the laser cooling of a single ^{40}Ca^{+} ion in a Penning trap to the motional ground state in one dimension. Cooling is performed in the strong binding limit on the 729-nm electric quadrupole S_{1/2}↔D_{5/2} transition, broadened by a quench laser coupling the D_{5/2} and P_{3/2} levels. We find the final ground-state occupation to be 98(1)%. We measure the heating rate of the trap to be very low with n[over ¯][over ˙]≈0.3(2) s^{-1} for trap frequencies from 150-400 kHz, consistent with the large ion-electrode distance.

Evaluation of the ToxRTool's ability to rate the reliability of toxicological data for human health hazard assessments.

Regulatory agencies often utilize results from peer reviewed publications for hazard assessments. A problem in doing so is the lack of well-accepted tools to objectively, efficiently and systematically assess the quality of published toxicological studies. Herein, we evaluated the publicly available software-based ToxRTool (Toxicological data Reliability assessment Tool) for use in human health hazard assessments. The ToxRTool was developed by the European Commission's Joint Research Center in 2009. It builds on Klimisch categories, a rating system established in 1997, by providing additional criteria and guidance for assessing the reliability of toxicological studies. It also transparently documents the study-selection process. Eight scientists used the ToxRTool to rate the same 20 journal articles on thyroid toxicants. Results were then compared using the Finn coefficient and "AC1" to determine inter-rater consistency. Ratings were most consistent for high-quality journal articles, but less consistent as study quality decreased. Primary reasons for inconsistencies were that some criteria were subjective and some were not clearly described. It was concluded, however, that the ToxRTool has potential and, with refinement, could provide a more objective approach for screening published toxicology studies for use in health risk evaluations, although the ToxRTool ratings are primarily based on study reporting quality.

Naphthoquinone-tyrptophan reduces neurotoxic Aß*56 levels and improves cognition in Alzheimer's disease animal model.

An increasing body of evidence indicates a role for oligomers of the amyloid-ß peptide (Aß) in the neurotoxicity of this peptide and the pathology of Alzheimer's disease (AD). Several neurotoxic oligomeric forms of Aß have been noted ranging from the larger Amyloid ß-Derived Diffusible Ligands (ADDLs) to smaller trimers and dimers of Aß. More recently a dodecameric form of Aß with a 56 kDa molecular weight, denoted Aß*56, was shown to cause memory impairment in AD model mice. Here, we present for the first time a potential therapeutic strategy for AD that targets the early stages in the formation of neurotoxic Aß*56 oligomers using a modified quinone-Tryptophan small molecule N-(3-chloro-1,4-dihydro-1,4-dioxo-2-naphthalenyl)-L-Tryptophan (Cl-NQTrp). Using NMR spectroscopy we show that this compound binds the aromatic recognition core of Aß and prevents the formation of oligomers. We assessed the effect of Cl-NQTrp in vivo in transgenic flies expressing Aß(1-42) in their nervous system. When these flies were fed with Cl-NQTrp a marked alleviation of their Aß-engendered reduced life span and defective locomotion was observed. Finally, intraperitoneal injection of Cl-NQTrp into an aggressive AD mouse model reduced the level of the Aß*56 species in their brain and reversed their cognitive defects. Further experiments should assess whether this is a direct effect of the drug in the brain or an indirect peripheral effect. This is the first demonstration that targeted reduction of Aß*56 results in amelioration of AD symptoms. This second generation of tryptophan-modified naphthoquinones could therefore serve as potent disease modifying therapeutic for AD.

Distal Chevron Osteotomy vs The Simple, Effective, Rapid, Inexpensive Technique (SERI) for Mild to Moderate Isolated Hallux Valgus: A Randomized Controlled Study.

BACKGROUND: Hallux valgus is a common foot deformity that leads to functional disability with serious sequelae. Minimally invasive surgery is often used to treat hallux valgus in order to reduce wound complications and improve recovery time. The objective of this study was to compare a Simple, Effective, Rapid, Inexpensive (SERI) technique with a simple Chevron technique in patients with minimum of 1-year follow-up. METHODS AND MATERIALS: Between the years 2014-2015, we performed a prospective study comparing the SERI minimally invasive technique to treat symptomatic hallux valgus with a standard chevron osteotomy technique. All procedures were performed by a single fellowship trained foot and ankle surgeon. Twenty-one patients were randomized to the SERI cohort and 15 to the standard Chevron technique. RESULTS: The mean preoperative intermetatarsal angle (IMA) of the SERI group was 14.8 ± 1.9 (11.9-22.9). The mean preoperative IMA of the Chevron control group was 13.3 ± 2.3 (10.4-18.2) (p = 0.038). The mean IMA two weeks after the surgery was 6.0 ± 2.3 (2.4-12) in the SERI group, and 6.1 ± 3 (2.6-13.1) in the control group. At the two-week and 1-year follow-up, there was no significant difference found in the IMA between the two groups (p = 0.871). The mean hallux valgus angle reduction was 11.85 ± 4.88 (3-20.8) and 11.09 ± 6.51 (- 1.1 to 22.5) in the SERI and Chevron groups, respectively (p = 0.69). Neither groups reported symptomatic transfer metatarsalgia throughout the follow-up period. The SERI group had increased metatarsophalangeal joint (MTPJ) motion (p < 0.001); however, all other parameters with similar. CONCLUSION: The SERI technique provided comparable outcomes at up to 1-year follow-up when compared with a standard Chevron osteotomy for moderate hallux valgus. This study demonstrated good reproducible results using the SERI technique for moderate hallux valgus. LEVEL OF EVIDENCE: Level II Prospective Study. TRIAL REGISTRATION: Approved by local IRB at MMC.

Enhanced antigen immunogenicity induced by bispecific antibodies.

The binding of protein antigens to APC with heterocrosslinked bispecific antibodies (HBAs) enhances their processing and presentation to Th cells in vitro. Here we have asked whether HBAs could also increase immune responses in vivo. We immunized mice with hen egg lysozyme (HEL) in the presence or absence of HBA, and followed antibody production after the primary challenge and after a secondary boost. We found that HBAs that bind antigen to MHC class I or II molecules, to Fc gamma R, but not to surface IgD, enhance the immunogenicity of HEL. HBAs that bound HEL to MHC class II molecules, for examples, decreased the amount of antigen required to elicit a primary anti-HEL antibody response in mice by 300-fold, and the amount required to prime for a secondary response by 10(3)- to 10(4)-fold. In fact, HBAs were as effective as IFA in generating antibody responses. Since adjuvants cannot be used in humans, HBAs could prove useful for immunizing people, especially in cases where, due to scarcity or toxicity, minute doses of antigen must be used.

Specific targeting of human peripheral blood T cells by heteroaggregates containing anti-T3 crosslinked to anti-target cell antibodies.

Antibody heteroaggregates have been used to render human peripheral blood T cells lytic for specified targets. The heteroaggregates contain anti-T3 covalently linked to antibodies against nominal target cell antigens. Such heteroaggregates bind target cells directly to T3 molecules on effector cells and trigger target cell lysis. Freshly prepared human PBL, when coated with anti-T3-containing heteroaggregates, are lytic without further stimulation, although brief exposure to crude lymphokine-containing supernatants or recombinant IL-2, but not recombinant IFN-gamma, enhances the activity. The effector cells are T8+, and when fully stimulated, their lytic activity approaches that of some cloned CTL. When T cells are treated with heteroaggregate, washed, and incubated at 37 degrees C in medium not containing heteroaggregate, they retain activity for at least 24 h. The results of this study suggest a strategy in which heteroaggregate-coated T cells could be used in vivo to mount a lytic response against pathogenic cells such as tumor cells or virus-infected cells.

Production of target-specific effector cells using hetero-cross-linked aggregates containing anti-target cell and anti-Fc gamma receptor antibodies.

Rabbit anti-2,4-dintrophenyl (DNP) antibodies or their F(ab')2 fragments were chemically cross-linked to the anti-mouse Fc gamma R monoclonal antibody 2.4G2 or to its Fab fragment. P388D1 cells were incubated with heteroaggregates between 2.4G2 and anti-DNP (anti-Fc gamma R X anti-DNP) and washed. The resulting cells lysed 2,4,6-trinitrophenyl chicken erythrocytes (TNP CRBC) in a hapten-specific manner. The lysis was inhibited by free hapten but was resistant to inhibition by immune complexes. Other cells coated with antibody heteroaggregates also mediated lysis of TNP-modified target cells. For example, mouse resident peritoneal exudate cells (PEC) lysed TNP CRBC and bacillus Calmette-Guérin-activated PEC lysed both TNP CRBC and TNP tumor targets. Human neutrophils, when incubated with heteroaggregates containing the anti-human neutrophil Fc gamma R antibody 3G8 and anti-DNP also lysed TNP CRBC and TNP-modified tumor cells. To test whether linkage to Fc gamma R was required for lysis, F(ab')2 fragments from the anti-KdDd monoclonal antibody 34-1-2 were cross-linked to anti-DNP F(ab')2 fragments. P388D1 cells (which express Kd and Dd) were then incubated with these heteroaggregates and washed, and their abilities to form conjugates and lyse TNP CRBC were compared with those of P388D1 cells treated with anti-Fc gamma R X anti-DNP. In both cases, P388D1 cells formed conjugates. However, only the cells treated with anti-Fc gamma R X anti-DNP mediated lysis to a significant extent. We conclude that heteroaggregates containing anti-Fc gamma R and anti-target cell antibodies can be used to create potent effector cells against red cell and tumor targets and that bridging of effectors with target cells directly to Fc gamma R on effector cells is required for lysis.

Allospecific cytolytic T lymphocytes recognize conformational determinants on hybrid mouse transplantation antigens.

Alloreactive cytolytic T cell (CTL) lines and clones have been used to identify the sites of polymorphism of antigens of the major histocompatibility complex (MHC). Specific CTL were generated against wild-type H-2b products by cells from H-2b mutant mice that had one or a few amino acid changes in either the alpha 1 or alpha 2 domains of the Kb or Db class I molecules. These CTL populations, which might be expected to react with determinants expressed on single MHC domains, were examined for lytic activity on L cells expressing newly constructed hybrid class I molecules. Transformed cell lines expressing native class I molecules or hybrid class I molecules in which the alpha 1 and alpha 2 domains of H-2Kb had been substituted by those domains of H-2Db were lysed by H-2Db-specific CTL. Similarly, all H-2Kb-specific CTL recognized hybrid molecules in which the alpha 1 and alpha 2 domains of H-2Kb were inserted into the H-2Db molecule. In contrast, exchange of the alpha 1 domains of H-2Kb and H-2Db resulted in a total loss of recognition by Kb and Db-specific CTL. These results suggest that the allodeterminants recognized by H-2 mutant CTL are influenced by interactions between the alpha 1 and alpha 2 domains, findings similar to those seen using conventional alloreactive T cells (11). These results were compared to the binding of alloreactive mAbs, including 5 new mAbs specific for the Kb molecules. Finally, it was shown that primary and secondary CTL responses could be generated by direct sensitization against hybrid class I molecules, demonstrating that these molecules express neoantigenic determinants recognized by alloreactive CTL.

Trinitrophenyl modification of H-2k and H-2b spleen cells results in enhanced serological detection of Kk-like determinants.

Several anti-H-2Kk but not anti-H-2Dd monoclonal antibodies (mAb) exhibited enhanced binding to B10.A murine spleen cells after modification of the cells with trinitrobenzene sulfonate (TNBS). The number of antibody molecules bound to TNP-modified B10.A spleen cells increased by a factor of two or more. The same anti-2Kk mAb that exhibited enhanced binding to modified B10.A cells did not bind to unmodified C57BL/10 spleen cells, as expected, but did bind to TNP-modified C57BL/10 spleen cells. This TNP-dependent binding was not a result of cross-reactions with cell surface TNP groups nor with Fc receptors. TNP modification of a variant cell line that does not express class I H-2 products did not result in enhanced binding by these mAb. These findings can account for preferential recognition of TNP-Kk by B10.A and B10.BR CTL, and also for cross-reactive lysis by C57BL/10 CTL stimulated by C57BL/10-TNP against unmodified H-2Kk targets.

Mechanism of target cell recognition by natural killer cells: characterization of a novel triggering molecule restricted to CD3- large granular lymphocytes.

In an attempt to identify a molecule in target recognition by CD3- large granular lymphocytes (LGL), we have generated a rabbit antiidiotypic (anti-ID) serum against a monoclonal antibody (mAb 36) that reacted with the cell membrane of K562. Flow cytometry analysis demonstrated that the anti-ID serum bound selectively to CD3- LGL and that F(ab')2 fragments of the anti-ID serum blocked both target cell binding and lysis by NK cells. Stimulation of CD3- LGL with F(ab')2 fragments resulted in the release of serine esterases and the secretion of interferon gamma. Furthermore, anti-ID F(ab')2 antibodies crosslinked to anti-DNP F(ab')2 mediated directed cytotoxicity of a non-natural killer (NK)-susceptible mouse target (YAC-1) via this surface ligand. These functional reactivities were only removed by adsorption with the specific idiotype. Protein analysis showed that the anti-ID serum immunoprecipitated 80-, 110-, and 150-kD proteins. Using this anti-ID, a partial cDNA was cloned and an antipeptide antiserum was made against the portion of the predicted amino acid sequence that corresponded to a portion of the ID binding region. This antipeptide serum exhibited similar functional and biochemical reactivities to those observed with the anti-ID serum. These data suggest that the cell surface moiety recognized by the anti-ID and anti-p104 is novel and is selectively involved in both recognition and triggering of NK-mediated lytic function.

Regulation of apoptosis in myeloid cells by interferon consensus sequence-binding protein.

Mice with a null mutation of the gene encoding interferon consensus sequence-binding protein (ICSBP) develop a disease with marked expansion of granulocytes and macrophages that frequently progresses to a fatal blast crisis, thus resembling human chronic myelogenous leukemia (CML). One important feature of CML is decreased responsiveness of myeloid cells to apoptotic stimuli. Here we show that myeloid cells from mice deficient in ICSBP exhibit reduced spontaneous apoptosis and a significant decrease in sensitivity to apoptosis induced by DNA damage. In contrast, apoptosis in thymocytes from ICSBP-deficient mice is unaffected. We also show that overexpression of ICSBP in the human U937 monocytic cell line enhances the rate of spontaneous apoptosis and the sensitivity to apoptosis induced by etoposide, lipopolysaccharide plus ATP, or rapamycin. Programmed cell death induced by etoposide was specifically blocked by peptides inhibitory for the caspase-1 or caspase-3 subfamilies of caspases. Studies of proapoptotic genes showed that cells overexpressing ICSBP have enhanced expression of caspase-3 precursor protein. In addition, analyses of antiapoptotic genes showed that overexpression of ICSBP results in decreased expression of Bcl-X(L). These data suggest that ICSBP modulates survival of myeloid cells by regulating expression of apoptosis-related genes.

Translational outcomes in a full gene deletion of ubiquitin protein ligase E3A rat model of Angelman syndrome.

Angelman syndrome (AS) is a rare neurodevelopmental disorder characterized by developmental delay, impaired communication, motor deficits and ataxia, intellectual disabilities, microcephaly, and seizures. The genetic cause of AS is the loss of expression of UBE3A (ubiquitin protein ligase E6-AP) in the brain, typically due to a deletion of the maternal 15q11-q13 region. Previous studies have been performed using a mouse model with a deletion of a single exon of Ube3a. Since three splice variants of Ube3a exist, this has led to a lack of consistent reports and the theory that perhaps not all mouse studies were assessing the effects of an absence of all functional UBE3A. Herein, we report the generation and functional characterization of a novel model of Angelman syndrome by deleting the entire Ube3a gene in the rat. We validated that this resulted in the first comprehensive gene deletion rodent model. Ultrasonic vocalizations from newborn Ube3am-/p+ were reduced in the maternal inherited deletion group with no observable change in the Ube3am+/p- paternal transmission cohort. We also discovered Ube3am-/p+ exhibited delayed reflex development, motor deficits in rearing and fine motor skills, aberrant social communication, and impaired touchscreen learning and memory in young adults. These behavioral deficits were large in effect size and easily apparent in the larger rodent species. Low social communication was detected using a playback task that is unique to rats. Structural imaging illustrated decreased brain volume in Ube3am-/p+ and a variety of intriguing neuroanatomical phenotypes while Ube3am+/p- did not exhibit altered neuroanatomy. Our report identifies, for the first time, unique AS relevant functional phenotypes and anatomical markers as preclinical outcomes to test various strategies for gene and molecular therapies in AS.

Extrachromosomal circular DNA in eukaryotes: possible involvement in the plasticity of tandem repeats.

Extrachromosomal circular DNA (eccDNA) is ubiquitous in eukaryotic organisms, and has been noted for more than 3 decades. eccDNA occurs in normal tissues and in cultured cells, is heterogeneous in size, consists of chromosomal sequences and reflects plasticity of the genome. Two-dimensional (2D) gel electrophoresis has been adapted for the detection and characterization of eccDNA. It shows that most eccDNA consists of chromosomal tandem repeats, both coding genes and satellite DNA and is organized as circular multimers of the repeating sequence. 2D gels were unable to detect dispersed repeats within the population of eccDNA. eccDNA, organized as circular multimers, can be formed de novo in Xenopus egg extracts, in the absence of DNA replication. These findings support a mechanism for the formation of eccDNA that involves intra-chromosomal homologous recombination between tandem repeats and looping-out. Furthermore, eccDNA appears to undergo extrachromosomal replication via a rolling circle mechanism. Hence, the formation of eccDNA from arrays of tandem repeats may cause deletions, and the possible re-integration of rolling-circle replication products could expand these arrays. This review summarizes recent experimental data which characterizes eccDNA in several organisms using 2D gel electrophoresis, and discusses its possible implications on the dynamics of chromosomal tandem repeats.

Identification of secreted proteins associated with obesity and type 2 diabetes in Psammomys obesus.

OBJECTIVE: Skeletal muscle produces a variety of secreted proteins that have important roles in intercellular communication and affects processes such as glucose homoeostasis. The objective of this study was to develop a novel Signal Sequence Trap (SST) in conjunction with cDNA microarray technology to identify proteins secreted from skeletal muscle of Psammomys obesus that were associated with obesity and type 2 diabetes (T2D). DESIGN: Secreted proteins that were differentially expressed between lean, normal glucose tolerant (NGT), overweight and impaired glucose tolerant (IGT) and obese, T2D P. obesus were isolated using SST in conjunction with cDNA microarray technology. Subsequent gene expression was measured in tissues from P. obesus by real-time PCR (RT-PCR). RESULTS: The SST yielded 1600 positive clones, which were screened for differential expression. A total of 91 (approximately 6%) clones were identified by microarray to be differentially expressed between NGT, IGT and T2D P. obesus. These clones were sequenced to identify 51 genes, of which only 27 were previously known to encode secreted proteins. Three candidate genes not previously associated with obesity or type 2 diabetes, sushi domain containing 2, collagen and calcium-binding EGF domains 1 and periostin (Postn), as well as one gene known to be associated, complement component 1, were shown by RT-PCR to be differentially expressed in skeletal muscle of P. obesus. Further characterization of the secreted protein Postn revealed it to be predominantly expressed in adipose tissue, with higher expression in visceral compared with subcutaneous adipose depots. CONCLUSION: SST in conjunction with cDNA microarray technology is a powerful tool to identify differentially expressed secreted proteins involved in complex diseases such as obesity and type 2 diabetes. Furthermore, a number of candidate genes were identified, in particular, Postn, which may have a role in the development of obesity and type 2 diabetes.

Behavioral characterization of d- and l-amphetamine: neurochemical implications.

Various doses of d- and l-amphetamine affect the temporal pattern of rat behavior in the following ways: First, the patterns of activity produced by d- and l-amphetamine are similar but out of phase; that is, the response to d-amphetamine has a relatively shorter latency whereas the effects of l-amphetamine persist for longer periods of time. Second, d-amphetamine is approximately five times as potent as l-amphetamine in its effects on both the total amount of locomotor activity and the duration of stereotypy. Both amphetamine-induced locomotion and stereotypy may be mediated by the same neurochemical mechanisms.

Thyroid state: effects on pre- and postsynaptic central noradrenergic mechanisms.

For hypothyroid rats, spontaneous motor activity was less than that in matched normal controls, and the specific activity of tyrosine hydroxylase in the midbrain was significantly greater than that in controls. Rats made hyperthyroid with thyroxine became hyperactive and showed increased sensitivity to the behaviorally activating effects of norepinephrine administered intraventricularly. In hyperthyroid rats, the specific activity of tyrosine hydroxylase in the midbrain remained within the normal range. These results are consonant with studies that suggested both receptor "tuning" and feedback regulation of activity of enzymes involved in biosynthesis of presynaptic neurotransmitter as methods of regulation of the central catecholamine synapse. These results may also help explain the reported potentiation by thyroid hormone of the antidepressant effects of imipramine.

Endorphins: profound behavioral effects in rats suggest new etiological factors in mental illness.

The endogenous morphinomimetic brain peptides Met5-enkephalin and alpha-, beta-, and gamma-endorphins have been evaluated in rats after intracerebrospinal fluid injection. beta-Endorphin produces marked, prolonged muscular rigidity and immobility similar to a catatonic state, counteracted by the opiate antagonist naloxone; this effect occurs at molar doses 1/100 to 1/400 that at which the other peptides or morphine block the response to painful stimuli. All peptides evoked dose-related, naloxone-reversible, wet-dog shakes in rats that had not been exposed to drugs. beta-Endorphin produced hypothermia, whereas gamma-endorphin produced hyperthermia. Such potent and divergent responses to naturally occurring subtances suggest that alterations in their homeostatic regulation could have etiological significance in mental illness.

Cycloheximide: its effects on activity are dissociable from its effects on memory.

Cycloheximide, when injected subcutaneously or intracerebrally, produces changes in the activity level of mice. Isocycloheximide, injected intracerebrally, produces identical effects on activity, but it does not produce inhibition of cerebral protein synthesis or amnesia. Amphetamine, in doses that can antagonize the amnesic action of cycloheximide, does not antagonize the effect of cycloheximide on activity. Effects of cycloheximide on activity do not appear to be responsible for its amnesic action.

Strain differences during intraventricular infusion of norepinephrine: possible role of receptor sensitivity.

Two rat strains previously shown to differ with respect to behavioral activity, regional brain tyrosine hydroxylase activity, and norepinephrine-elicited accumulation of adenosine 3', 5'-monophosphate exhibited differential behavioral responsiveness during the intraventricular infusion of norepinephrine. The results are interpreted in terms of differential catecholamine receptor sensitivity.

Multiple daily amphetamine administration: behavioral and neurochemical alterations.

In rats, multiple daily amphetamine injections (2.5 milligrams per kilogram of body weight, injected subcutaneously every 4 hours for 5 days) resulted in a progressive augmentation in response, characterized by a more rapid onset and an increased magnitude of stereotypy. By contrast, offset times of both the stereotypy and the poststereotypy hyperactivity periods were markedly shortened. When the animals were retested with the same dose of amphetamine 8 days after the long-term treatment was discontinued, the time of offset of the stereotypy and hyperactivity phases had recovered to values found with short-term amphetamine treatment, whereas the more rapid onset of stereotypy persisted. Brain monoamine and amphetamine concentrations and tyrosine hydroxylase activity were determined in comparably treated rats at times corresponding to the behavioral observations. The behavioral data indicate that enhanced responsiveness to amphetamine following its repeated administration may contribute to the development of amphetamine psychosis.