Time to revisit? A predator's previous successes and failures in prey capture determine its return time to patches.

In a heterogeneous environment containing multiple patches that may deplete and renew, a forager should be able to detect the quality of food resources within and among patches and choose to exploit them to best maximize returns. From the predator's perspective, the behavioral responses of the prey in a patch will be perceived as depletion when they retreat to refuge and renewal when they reemerge. A predator encountering responsive prey should manage predation risk, and thus behavioral resource depression, by optimally timing its return time to the patch based on prey behavior. We evaluated the foraging decisions of a predator that encountered patches differing in size of the refuge and prey density. We used little egrets and goldfish as predators and prey in an environment that contained three patches (pools). We manipulated prey density and refuge size and availability (using covers) and observed predator foraging behavior. When the egret had previously caught a fish it did not discriminate between the pools, and the return time was similar for all cover types. The fish densities also did not affect the egret decisions to return to pools. However, when it failed to catch fish, it returned sooner to the pool containing the small cover than the larger one. Additionally, after failing to catch fish in patches containing the highest prey density, the egrets subsequently preferred to return to such patches sooner. We show experimentally that previous failures influence the foraging decisions of a predator choosing how quickly to return to a previously visited patch.

Recurrent hyperactive ESR1 fusion proteins in endocrine therapy-resistant breast cancer.

Background: Estrogen receptor-positive (ER-positive) metastatic breast cancer is often intractable due to endocrine therapy resistance. Although ESR1 promoter switching events have been associated with endocrine-therapy resistance, recurrent ESR1 fusion proteins have yet to be identified in advanced breast cancer. Patients and methods: To identify genomic structural rearrangements (REs) including gene fusions in acquired resistance, we undertook a multimodal sequencing effort in three breast cancer patient cohorts: (i) mate-pair and/or RNAseq in 6 patient-matched primary-metastatic tumors and 51 metastases, (ii) high coverage (>500×) comprehensive genomic profiling of 287-395 cancer-related genes across 9542 solid tumors (5216 from metastatic disease), and (iii) ultra-high coverage (>5000×) genomic profiling of 62 cancer-related genes in 254 ctDNA samples. In addition to traditional gene fusion detection methods (i.e. discordant reads, split reads), ESR1 REs were detected from targeted sequencing data by applying a novel algorithm (copyshift) that identifies major copy number shifts at rearrangement hotspots. Results: We identify 88 ESR1 REs across 83 unique patients with direct confirmation of 9 ESR1 fusion proteins (including 2 via immunoblot). ESR1 REs are highly enriched in ER-positive, metastatic disease and co-occur with known ESR1 missense alterations, suggestive of polyclonal resistance. Importantly, all fusions result from a breakpoint in or near ESR1 intron 6 and therefore lack an intact ligand binding domain (LBD). In vitro characterization of three fusions reveals ligand-independence and hyperactivity dependent upon the 3' partner gene. Our lower-bound estimate of ESR1 fusions is at least 1% of metastatic solid breast cancers, the prevalence in ctDNA is at least 10× enriched. We postulate this enrichment may represent secondary resistance to more aggressive endocrine therapies applied to patients with ESR1 LBD missense alterations. Conclusions: Collectively, these data indicate that N-terminal ESR1 fusions involving exons 6-7 are a recurrent driver of endocrine therapy resistance and are impervious to ER-targeted therapies.

Use of Pregabalin - A Nationwide Pharmacoepidemiological Drug Utilization Study with Focus on Abuse Potential.

INTRODUCTION: Pregabalin is currently approved for the treatment of epilepsy, generalized anxiety disorder and neuropathic pain with a licensed dosage range of 150 mg to 600 mg/day. Growing concern about the abuse potential of pregabalin is partly based on reports of pregabalin being used in dosages that exceed the approved therapeutic range. METHODS: To identify predictors of pregabalin use above recommended dosage, we conducted a pharmacoepidemological drug utilization study using the Danish nationwide registers. We deployed 4 measures of abuse: high use (≥600 mg/day) or very high use (≥1 200 mg/day) over a 6- or 12-month period, respectively. Multiple logistic regression was used to identify patient and treatment characteristics that were associated with either abuse marker. RESULTS: Out of 42 520 pregabalin users 4 090 (9.6%) were treated with more than 600 mg/day for 6 months and 2 765 (6.5%) for more than 12 months. Male gender and prescription of antipsychotics and benzodiazepines were associated with increased risk of use of above the recommended dosage. DISCUSSION: Use of pregabalin above recommended dosages was rare but abuse may occur in susceptible patients.

Optimal foraging of little egrets and their prey in a foraging game in a patchy environment.

We explored the behavioral game between a predator, the little egret (Egretta garzetta), and a prey, the common goldfish (Carassius auratus), in a laboratory theater containing three fish pools. We tested the hypotheses that the egrets maximize their total capture success by responding to the fish's antipredatory behavior and that the behaviors of both players respond adaptively to the density distribution of fish among the pools. One experiment presented egrets with 15 fish per pool. The second experiment used a heterogeneous environment: pools 1, 2, and 3 had 10, 15, and 20 fish, respectively. Within each pool, fish could move between a safe, covered microhabitat and a risky, open microhabitat. Only the risky habitat had food, so fish were trading off food and safety by allocating the time spent in the two habitats. Egrets spent more total time in pools with more fish and returned to them sooner. Egrets maximized the number of fish they captured by following the matching rule of the ideal free distribution. The fish used the risky but productive habitat 65% of the time during experiments without egrets, but only 9% during experiments with 15 fish and egrets present somewhere in the theater. In addition, with egrets present, fish fine-tuned their behavior by reducing their use of the risky habitat as the egrets increased the frequency of their visits.

Dye efflux studies suggest that hematopoietic stem cells expressing low or undetectable levels of CD34 antigen exist in multiple species.

We previously described a method for isolating murine hematopoietic stem cells capable of reconstituting lethally irradiated recipients, which depends solely on dual-wavelength flow cytometric analysis of murine bone marrow cells stained with the fluorescent DNA-binding dye Hoechst 33342. This method, which appears to rely on the differential ability of stem cells to efflux the Hoechst dye, defines an extremely small and homogeneous population of cells (termed SP cells). We show here that dual-wavelength analysis of Hoechst dye-stained human, rhesus and miniature swine bone marrow cells reveals a small, distinct population of cells that efflux the dye in a manner identical to murine SP cells. Like the murine SP cells, both human and rhesus SP cells are primarily CD34-negative and lineage marker-negative. In vitro culture studies demonstrated that rhesus SP cells are highly enriched for long-term culture-initiating cells (LTC-ICs), an indicator of primitive hematopoietic cells, and have the capacity for differentiation into T cells. Although rhesus SP cells do not initially possess any hematopoietic colony-forming capability, they acquire the ability to form colonies after long-term culture on bone marrow stroma, coincident with their conversion to a CD34-positive phenotype. These studies suggest the existence of a hitherto unrecognized population of hematopoietic stem cells that lack the CD34 surface marker classically associated with primitive hematopoietic cells.

Early regeneration of thymic progenitors in rhesus macaques infected with simian immunodeficiency virus.

The thymus plays a critical role in the maturation and production of T lymphocytes and is a target of infection by human immunodeficiency virus (HIV) and the related simian immunodeficiency virus (SIV). Using the SIV/macaque model of AIDS, we examined the early effects of SIV on the thymus. We found that thymic infection by SIV resulted in increased apoptosis 7-14 d after infection, followed by depletion of thymocyte progenitors by day 21. A marked rebound in thymocyte progenitors occurred by day 50 and was accompanied by increased levels of cell proliferation in the thymus. Our results demonstrate a marked increase in thymic progenitor activity very early in the course of SIV infection, long before marked declines in peripheral CD4(+) T cell counts.

Paradox of enrichment: destabilization of exploitation ecosystems in ecological time.

Six reasonable models of trophic exploitation in a two-species ecosystem whose exploiters compete only by depleting each other's resource supply are presented. In each case, increasing the supply of limiting nutrients or energy tends to destroy the steady state. Thus man must be very careful in attempting to enrich an ecosystem in order to increase its food yield. There is a real chance that such activity may result in decimation of the food species that are wanted in greater abundance.

Rat brain: effects of environmental enrichment on wet and dry weights.

Wet weight of rat cerebral cortex was increased by exposure to an enriched environment, as compared with standard colony or impoverished conditions. Dry weights and wet weights were compared and both yielded identical percentage differences between brains of animals experiencing enrichment and those experiencing impoverishment.

An animal model for acute and persistent Epstein-Barr virus infection.

Epstein-Barr virus (EBV) is a human lymphocryptovirus that causes infectious mononucleosis, persists asymptomatically for life in nearly all adults, and is associated with the development of B cell lymphomas and nasopharyngeal carcinomas. A highly similar rhesus lymphocryptovirus naturally endemic in rhesus monkeys was used to orally infect naïve animals from a pathogen-free colony. This animal model reproduced key aspects of human EBV infection, including oral transmission, atypical lymphocytosis, lymphadenopathy, activation of CD23(+) peripheral blood B cells, sustained serologic responses to lytic and latent EBV antigens, latent infection in the peripheral blood, and virus persistence in oropharyngeal secretions. This system may be useful for studying the pathogenesis, prevention, and treatment of EBV infection and associated oncogenesis.

Memory: modification of anisomycin-induced amnesia by stimulants and depressants.

Mice were trained in a passive (foot shock)avoidance task. When administered after training, the stimulants caffeine or nicotine blocked amnesia for the task that had been produced by injections of the protein synthesis inhibitor anisomycin given prior to training. With foot shock at a higher intensity, anisomycin did not produce amnesia by itself, but the administration of the depressants chloral hydrate or sodium phenobarbital after training did cause amnesia. Stimulants and depressants did not have an appreciable influence on the overall degree of protein synthesis inhibition produced by anisomycin. The results support the hypothesis that arousal after training is an important factor in the conversion of short-term to long-term memory.

Gastrointestinal tract as a major site of CD4+ T cell depletion and viral replication in SIV infection.

Human and simian immunodeficiency virus (HIV and SIV) replicate optimally in activated memory CD4(+) T cells, a cell type that is abundant in the intestine. SIV infection of rhesus monkeys resulted in profound and selective depletion of CD4+ T cells in the intestine within days of infection, before any such changes in peripheral lymphoid tissues. The loss of CD4+ T cells in the intestine occurred coincident with productive infection of large numbers of mononuclear cells at this site. The intestine appears to be a major target for SIV replication and the major site of CD4+ T cell loss in early SIV infection.

Identification of an immunoreceptor tyrosine-based activation motif of K1 transforming protein of Kaposi's sarcoma-associated herpesvirus.

Kaposi's sarcoma-associated herpesvirus (KSHV) is consistently identified in Kaposi's sarcoma and body cavity-based lymphoma. KSHV encodes a transforming protein called K1 which is structurally similar to lymphocyte receptors. We have found that a highly conserved region of the cytoplasmic domain of K1 resembles the sequence of immunoreceptor tyrosine-based activation motifs (ITAMs). To demonstrate the signal-transducing activity of K1, we constructed a chimeric protein in which the cytoplasmic tail of the human CD8alpha polypeptide was replaced with that of KSHV K1. Expression of the CD8-K1 chimera in B cells induced cellular tyrosine phosphorylation and intracellular calcium mobilization upon stimulation with an anti-CD8 antibody. Mutational analyses showed that the putative ITAM of K1 was required for its signal-transducing activity. Furthermore, tyrosine residues of the putative ITAM of K1 were phosphorylated upon stimulation, and this allowed subsequent binding of SH2-containing proteins. These results demonstrate that the KSHV transforming protein K1 contains a functional ITAM in its cytoplasmic domain and that it can transduce signals to induce cellular activation.

Efficient generation of human T cells from a tissue-engineered thymic organoid.

Biocompatible inorganic matrices have been used to enhance bone repair by integrating with endogenous bone architecture. Hypothesizing that a three-dimensional framework might support reconstruction of other tissues as well, we assessed the capacity of a tantalum-coated carbon matrix to support reconstitution of functioning thymic tissue. We engineered a thymic organoid by seeding matrices with murine thymic stroma. Co-culture of human bone marrow-derived hematopoietic progenitor cells within this xenogeneic environment generated mature functional T cells within 14 days. The proportionate T-cell yield from this system was highly reproducible, generating over 70% CD3+ T cells from either AC133+ or CD34+ progenitor cells. Cultured T cells expressed a high level of T-cell receptor excision circles (TREC), demonstrating de novo T lymphopoiesis, and function of fully mature T cells. This system not only facilitates analysis of the T-lymphopoietic potential of progenitor cell populations; it also permits ex vivo genesis of T cells for possible applications in treatment of immunodeficiency.

Clinical and molecular characteristics of patients with non-amyloid light chain deposition disorders, and outcome following treatment with high-dose melphalan and autologous stem cell transplantation.

Light chain deposition disease (LCDD) is caused by a clonal plasma cell disorder in which fragments of monoclonal immunoglobulin light chains form non-fibrillary deposits in various tissues resulting in organ dysfunction. Crystal storing histiocytosis (CSH) is another light chain deposition disorder in which monoclonal light chains form intracytoplasmic crystals. Both are uncommon diseases for which there is limited treatment experience. Between 2003 and 2005, five patients with LCDD and one with CSH were treated at Boston University Medical Center with high-dose melphalan and autologous peripheral blood stem cell transplantation (HDM/SCT). Five of the six patients had predominantly renal involvement, and one patient with LCDD had biopsy-proven deposits in the myocardium. Molecular characterization revealed that the pathologic light chains were kappa in four of the six patients, and sequence analysis revealed unusual germline donor genes and high rates of amino-acid substitutions. One light chain sequence encoded a new potential N-linked glycosylation site, and another showed evidence of antigen selection. All patients are alive and five of the six patients are in complete hematologic remission at a median follow-up of 12 months (range 4-29 months) after HDM/SCT. In our experience, HDM/SCT is a feasible and effective treatment approach for these disorders.

Neonatal HIV-1 thymic infection.

The thymus is the primary site of T cell ontogeny and selection during fetal and neonatal development. Previous studies have established that the thymus is also a site of HIV-1 infection, as early as the first trimester of pregnancy. Alteration of the thymocyte maturation process by HIV-1 could impact on the peripheral T cell population and interfere with immune responses. A neonatal thymic organ culture system was established to study HIV-1 infection within the thymus. We have shown that this primary tissue isolate can support a productive HIV-1 infection. Infection occurred without detectable thymocyte cytopathology. The ability to infect the developing thymocyte within an intact micro environment will enable us to further establish the kinetics of acute HIV-1 thymic infection and its consequences on lymphocyte maturation.

Extended culture of multipotent hematopoietic progenitors without cytokine augmentation in a novel three-dimensional device.

The ability to culture multipotent hematopoietic progenitor cells for extended periods is of practical importance to both clinical and research efforts involving these cells. Conventional techniques for the extended culture of hematopoietic progenitor cells (HPCs) have proven largely ineffective in sustaining these cells and preserving their multipotency over protracted periods. To overcome barriers to extended HPC culture, numerous alternative approaches, including cytokine augmentation and co-culture with bone marrow stroma, have been explored to enhance HPC maintenance but have generally yielded mixed results. The present study examined the ability of a novel, three-dimensional, tantalum-coated porous biomaterial (TCPB) to support HPC maintenance and multipotency in long-term cultures to which no exogenous cytokines have been added. As a follow-up to previously published short-term HPC cultures in TCPB, we examined the maintenance, phenotype and multipotency of HPCs cultured for up to 6 weeks in the TCPB matrix compared to control systems, including fibronectin-coated plastic, bone marrow stroma cocultures and other three-dimensional materials. These studies indicated that TCPB supports the maintenance of immature progenitors for up to 6 weeks in the absence of supplemented cytokines. Further, the results demonstrate that the TCPB matrix facilitates and enhances HPC maintenance and leads to a 1.5-fold expansion of HPC numbers following 1 week in culture and a 6.7-fold increase in colony-forming ability following 6 weeks in culture in the absence of exogenous cytokines. Under the same conditions, control systems were less able to support progenitor viability and multipotency. These findings point to new approaches that may improve the in vitro preservation of progenitors and may have important implications in clinical areas such as progenitor expansion, bone marrow transplantation and gene therapy.

T-lymphopoietic capacity of cord blood-derived CD34+ progenitor cells.

Umbilical cord blood contains an abundance of CD34+ hematopoietic progenitor cells and has been used in transplantation as an alternative to adult bone marrow or mobilized peripheral blood. Although efficient myelomonocytic, erythroid, and B lymphoid differentiation has been shown in highly purified cord blood CD34+ mononuclear cells lacking expression of lineage-specific antigens, generation of functional T cells has not been previously documented. Exploiting two recently developed, complementary thymic stromal monolayer systems, we show here that immature hematopoietic progenitor cells (CD34+lineage-) from human and rhesus monkey cord blood mononuclear cells undergo T lymphopoiesis in a manner that recapitulates T cell ontogeny in vivo. After 2 weeks of proliferation, cultures contained myeloid [corrected] cells and discrete populations of CD4+CD8+ (double-positive) immature T lymphocytes, followed after an additional 2 weeks by the appearance of single-positive CD4+CD8- and CD4-CD8+ T cells that coexpressed CD3. The T lymphoid phenotype was confirmed at the transcriptional level by the presence of the lymphoid-restricted genes RAG-2 and T cell receptor. T cells generated from cord blood progenitors in these systems exhibited immunofunction as assessed by alloreactive responses in mixed lymphocyte reactions. These findings were comparable between human and rhesus progenitor cells and closely resemble previous data using adult bone marrow CD34+ cells in these models. Together, these observations demonstrate that cord blood contains abundant lymphoid progenitors that undergo T lymphopoiesis in vitro, suggesting the full multipotentiality of this stem cell source and its validity in investigating T lymphoid differentiation.

Selective thymocyte depletion in neonatal HIV-1 thymic infection.

OBJECTIVE: To determine the impact of HIV-1 infection on thymocyte development, and the role of thymic infection on the pathogenesis of neonatal HIV-1 infection. DESIGN AND METHODS: The consequences of thymic infection by HIV-1 were examined by comparative histologic and molecular analyses of an asymptomatic, HIV-1-seropositive 3-day-old subject, versus age- and treatment-matched controls. The presence of replicating virus was established by in situ hybridization with specific molecular probes to HIV-1. The distribution of thymocyte subsets was determined by quantitative flow cytometry following staining with antibodies to CD4 and CD8 cell surface proteins. RESULTS: The results show clear evidence of severe thymic involution, HIV-1 infection of thymocytes, and selective depletion of thymocyte subpopulations. The consequences of HIV-1 infection were a marked depletion of CD3+CD4+ CD8hi and CD3+CD4+CD8- cells. The phenotype of the residual thymic lymphoid population was predominantly that of immature CD3-CD4-CD8- double negative and CD3+CD4+CD8lo cells. CONCLUSION: Changes in the distribution of thymocyte subsets suggests a role for thymic involvement in the pathogenesis of HIV-1 infection in neonates.

Effects of dietary choline on memory and brain chemistry in aged mice.

The purpose of this study was to investigate in more detail the characteristics of the age-related extension of the retrograde amnesia gradient previously demonstrated in a passive avoidance task [6]. In Experiment 1, it was found that while 2-3 month old mice were susceptible to the amnesic effects of anisomycin (ANI) only when given prior to 15 min post-training, memory of 14-16 month old mice was susceptible to disruption when ANI was given as late as 20 min post-training, and retention of 17-20 month old mice was impaired when ANI was injected even as late as 30 min after training. Experiment 2 examined whether the age-related change in susceptibility to the effects of ANI could be ameliorated by chronic pretreatment with a choline-enriched diet. Results showed that ANI injected 20 min after training did not induce amnesia in choline treated mice (14.5 month old), but did induce amnesia when injected 15 min post training. Subsequent assay of choline acetyltransferase (ChAT) and tyrosine hydroxylase (TH) activity showed that choline treatment significantly reduced ChAT activity but did not affect TH activity. It appears that dietary choline treatment can render new long-term memories less susceptible to disruption following training.

Recombinant simian immunodeficiency virus expressing green fluorescent protein identifies infected cells in rhesus monkeys.

We engineered recombinant derivatives of simian immunodeficiency virus (SIV) to express enhanced green fluorescent protein (EGFP). Replacement of vpr sequences with EGFP resulted in a genome that did not produce detectable levels of replication-competent virus. Replication-competent virus and bright fluorescence of infected cells were obtained with two other constructs, one in which SIV nef sequences were replaced by EGFP and another in which EGFP was inserted into the SIV nef locus and HIV-1 nef sequences were expressed by downstream placement of an internal ribosomal entry site. These strains were infectious in rhesus monkeys and green fluorescing cells were detected in the tissues of infected monkeys by FACS analysis and by direct microscopic visualization. EGFP sequences were absent from recovered virus by 8 weeks following infection. We conclude that recombinant SIV that is engineered to express EGFP can be used to directly detect productively infected cells and aid in the immunophenotypic characterization of these cells within the first 2 weeks of infection of rhesus monkeys.