Appetite enhancement and weight gain by peripheral administration of TrkB agonists in non-human primates

Loss of function mutations in the receptor tyrosine kinase TrkB pathway resulted in hyperphagia and morbid obesity in human and rodents. Conversely, peripheral or central stimulation of TrkB by its natural ligands BDNF or NT4 reduced body weight and food intake in mice, supporting the idea that TrkB is a key anorexigenic signal downstream of the melanocortin-4 receptor (Mc4r) system. Here we show that in non-human primates TrkB agonists were anorexigenic when applied centrally, but surprisingly orexigenic, leading to gain in appetite, body weight, fat deposits and serum leptin levels, when given peripherally. The orexigenic and pro-obesity effects of peripherally administered TrkB agonists appear to be dose dependent, not associated with fluid retention nor with evidence of receptor down regulation. Our findings revealed that TrkB signaling exerts dual control on energy homeostasis in the primates that could be targeted for the treatment of either wasting disorders or obesity.     

Mechanical unloading impairs keratinocyte migration and angiogenesis during cutaneous wound healing.

Although initially thought to improve an individual's ability to heal, mechanical unloading promoted by extended periods of bed rest has emerged as a contributing factor to delayed or aberrant tissue repair. Using a rat hindlimb unloading (HLU) model of hypogravity, we mimicked some aspects of physical inactivity by removing weight-bearing loads from the hindlimbs and producing a systemic cephalic fluid shift. This model simulates bed rest in that the animal undergoes physiological adaptations, resulting in a reduction in exercise capability, increased frequency of orthostatic intolerance, and a reduction in plasma volume. To investigate whether changes associated with prior prolonged bed rest correlate with impaired cutaneous wound healing, we examined wound closure, angiogenesis, and collagen content in day 2 to day 21 wounds from rats exposed to HLU 2 wk before excisional wounding. Wound closure was delayed in day 2 wounds from HLU rats compared with ambulatory controls. Although the levels of proangiogenic growth factors, fibroblast growth factor-2 (FGF-2), and vascular endothelial growth factor (VEGF) were similar between the two groups, wound vascularity was significantly reduced in day 7 wounds from HLU animals. To further examine this disparity, total collagen content was assessed but found to be similar between the two groups. Taken together, these results suggest that keratinocyte and endothelial cell function may be impaired during the wound healing process under periods of prolonged inactivity or bed rest.     

Wearable-Sensor-Based Classification Models of Faller Status in Older Adults

Wearable sensors have potential for quantitative, gait-based, point-of-care fall risk assessment that can be easily and quickly implemented in clinical-care and older-adult living environments. This investigation generated models for wearable-sensor based fall-risk classification in older adults and identified the optimal sensor type, location, combination, and modelling method; for walking with and without a cognitive load task. A convenience sample of 100 older individuals (75.5 ± 6.7 years; 76 non-fallers, 24 fallers based on 6 month retrospective fall occurrence) walked 7.62 m under single-task and dual-task conditions while wearing pressure-sensing insoles and tri-axial accelerometers at the head, pelvis, and left and right shanks. Participants also completed the Activities-specific Balance Confidence scale, Community Health Activities Model Program for Seniors questionnaire, six minute walk test, and ranked their fear of falling. Fall risk classification models were assessed for all sensor combinations and three model types: multi-layer perceptron neural network, naïve Bayesian, and support vector machine. The best performing model was a multi-layer perceptron neural network with input parameters from pressure-sensing insoles and head, pelvis, and left shank accelerometers (accuracy = 84%, F1 score = 0.600, MCC score = 0.521). Head sensor-based models had the best performance of the single-sensor models for single-task gait assessment. Single-task gait assessment models outperformed models based on dual-task walking or clinical assessment data. Support vector machines and neural networks were the best modelling technique for fall risk classification. Fall risk classification models developed for point-of-care environments should be developed using support vector machines and neural networks, with a multi-sensor single-task gait assessment.     

Atg5-dependent autophagy contributes to the development of acute myeloid leukemia in an MLL-AF9-driven mouse model

Acute myeloid leukemia (AML) is a hierarchical hematopoietic malignancy originating from leukemic stem cells (LSCs). Autophagy is a lysosomal degradation pathway that is hypothesized to be important for the maintenance of AML as well as contribute to chemotherapy response. Here we employ a mouse model of AML expressing the fusion oncogene MLL-AF9 and explore the effects of Atg5 deletion, a key autophagy protein, on the malignant transformation and progression of AML. Consistent with a transient decrease in colony-forming potential in vitro, the in vivo deletion of Atg5 in MLL-AF9-transduced bone marrow cells during primary transplantation prolonged the survival of recipient mice, suggesting that autophagy has a role in MLL-AF9-driven leukemia initiation. In contrast, deletion of Atg5 in malignant AML cells during secondary transplantation did not influence the survival or chemotherapeutic response of leukemic mice. Interestingly, autophagy was found to be involved in the survival of differentiated myeloid cells originating from MLL-AF9-driven LSCs. Taken together, our data suggest that Atg5-dependent autophagy may contribute to the development but not chemotherapy sensitivity of murine AML induced by MLL-AF9.Acute myeloid leukemia (AML) is a clonal hematopoietic malignancy characterized by the uncontrolled proliferation of immature myeloid cells within the bone marrow (BM), eventually suppressing normal hematopoiesis.¹ Recurrent chromosomal translocations frequently occur in AML, one of which involves the fusions of the KMT2A gene on chromosome 11 to a number of potential partners that are diagnosed as prognostically intermediate to poor.¹ Among these fusions, the MLL-AF9 fusion oncogene, resulting from the t(9;11)(p22;q23) translocation, is well studied owing to its robust phenotype in various mouse models of AML.^{2, 3, 4} It has been previously reported that BM transplantation of hematopoietic progenitors expressing exogenous MLL-AF9 leads to rapid in vivo transformation and progression of AML in a syngeneic, immunocompetent mouse model and recapitulates the poor chemotherapy response of t(9;11)(p22;q23) fusion human AML.^{2, 5}Autophagy is an evolutionarily conserved catabolic pathway by which cellular components are engulfed by double-membraned vesicles, called autophagosomes, and delivered to the lysosome for degradation and recycling. Autophagy is best characterized to be induced under stressful conditions, such as organelle damage or nutrient deprivation, and is followed by the elongation of the autophagosome membrane around its cargo. In Atg5-dependent autophagy, the conversion of LC3-I to LC3-II by lipidation is crucial for autophagosome membrane expansion, which is mediated by a series of ubiquitin-like conjugation systems.⁶ Within this pathway, the Atg5-Atg12-Atg16 complex acts as an E3-ubiquitin-ligase-like enzyme that specifically mediates the conjugation of LC3-I to phosphatidylethanolamine to form LC3-II, which inserts to the autophagosomal membrane. Autophagosome maturation is followed by fusion to lysosomes, at which time the inner compartment is degraded. The genetic ablation of Atg5 leads to a complete and highly selective inhibition of LC3-dependent autophagosome formation.^{6, 7}Autophagy is known to be implicated in cancer as both a tumor promoter and a tumor suppressor.⁸ The genetic ablation of autophagy in mouse hematopoietic stem cells (HSCs) has been shown to result in severe impairments to HSC maintenance.^{9, 10, 11, 12, 13} Autophagy dysregulation has also been implicated in AML,^{12, 13, 14} suggesting that targeting autophagy could be promising for AML treatment. As an expanding arsenal of pharmacological autophagy modulators are being developed,^{15, 16} it has become increasingly important to specifically determine whether autophagy has an important role in AML using a genetic mouse model. Therefore, we sought to dissect the role of autophagy through the in vivo homozygous deletion of Atg5 in MLL-AF9-driven murine AML. We discover in this study that Atg5 deletion during primary transplantation prolongs the survival of animals, whereas Atg5 deletion after secondary transplantation has no effect on animal survival, suggesting a role for autophagy in the initiation, but not maintenance, of AML in our model. We additionally assessed the effect of autophagy in chemotherapeutic response and found that Atg5 deletion in our MLL-AF9 model had no effect on the in vivo response to cytarabine and doxorubicin combination therapy, suggesting that autophagy does not significantly contribute to chemotherapy response in this model.     

Solvent polarity-dependent structural refolding: A CD and NMR study of a 15 residue peptide

A close association between the HIV surface protein gp120 and the CD4 T cell receptor initiates the viral multiplication cycle. A 15 amino acid peptide (LAV) within the CD4 binding domain of gp 120 has been shown to retain receptor binding ability. The structural behavior of the LAV peptide has been studied by CD and NMR methods in aqueous solution and upon addition of trifluoroethanol (TFE) to emulate the relatively apolar conditions at the membrane bound receptor. Previous work has shown that the LAV peptide folds into a β-pleated structure in more polar buffer/TFE mixtures, while a concerted structural change can be observed at a concentration of 60% TFE (v/v). This abrupt, cooperative refolding from a regular β-sheet to a helical secondary structure is known as “switch” behavior. Former CD experiments with LAV sequence variants have supported the assumption that four amino acids at the N-terminus (LPCR) are indispensable for the “switch.” The tetrad has a strong β-turn forming potential. The suggestion has been formulated that the tetrad can act as a nucleation site governing the refolding. The present NMR study of the LAV peptide in TFE gives evidence for a 3₁₀-helix suggesting that the tetrad adopts a type III β-turn and promotes the formation of a similar bend in the next overlapping tetrad until the sequence is restructured into a 3₁₀-helix at a critical polarity favoring intrachain hydrogen bonds. © 1995 Wiley-Liss, Inc.     

Exploring Sources of Emotional Distress among People Living with Scleroderma: A Focus Group Study

Background

Systemic sclerosis, or scleroderma, is a chronic and rare connective tissue disease with negative physical and psychological implications. Sources of emotional distress and the impact they have on the lives of people with scleroderma are not well understood.

Objectives

To gain an in-depth understanding of the emotional experiences and sources of emotional distress for women and men living with scleroderma through focus group discussions.

Methods

Three semi-structured focus group discussions were conducted (two in English, one in French) with a total of 22 people with scleroderma recruited through the Scleroderma Society of Ontario in Hamilton, Ontario and a scleroderma clinic in Montreal, Canada. Interviews were recorded, transcribed, and then coded for emerging themes using thematic inductive analysis.

Results

Core themes representing sources of emotional distress were identified, including: (a) facing a new reality; (b) the daily struggle of living with scleroderma; (c) handling work, employment and general financial burden; (d) changing family roles; (e) social interactions; and (f) navigating the health care system. Collectively, these themes refer to the stressful journey of living with scleroderma including the obstacles faced and the emotional experiences beginning prior to receiving a diagnosis and continuing throughout the participants’ lives.

Conclusion

Scleroderma was portrayed as being an unpredictable and overwhelming disease, resulting in many individuals experiencing multiple sources of emotional distress. Interventions and supportive resources need to be developed to help individuals with scleroderma and people close to them manage and cope with the emotional aspects of the disease.     

Transposon donor plasmids, based on ColIb-P9, for use in Pseudomonas putida and a variety of other gram negative bacteria

Summary The properties of pLG221, a derivative of the ColIb plasmid carrying the transposon Tn5 are described. This plasmid can be used to introduce Tn5 by conjugation from Escherichia coli into a variety of Gram negative bacteria outside the host range for maintenance of ColIb. Plasmid pLG221, and a similar plasmid pLG223 carrying Tn10 may be of general utility as vectors for transposon-mediated mutagenesis in a variety of Gram negative bacteria.     

Microarrays in infection and immunity

Over the past decade, microarrays have revolutionized the scientific world as dramatically as the internet has changed everyday life. From the initial applications of DNA microarrays to uncover gene expression patterns that are diagnostic and prognostic of cancer, understanding the interplay between immune responses and disease has been a prime application of this technology. More recent efforts have moved beyond genetic analysis to functional analysis of the molecules involved, including identification of immunodominant antigens and peptides as well as the role of post-translational glycosylation. Here, we focus on recent applications of microarray technology in understanding the detailed chemical biology of immune responses to disease in an effort to guide development of vaccines and other protective therapies.     

Epithelial organization: may the force be with you

Multicellular organization is determined by the balance of forces between cells as much as by the expression of genes. A recent study in Drosophila combines physical modeling with experimental measurements and mechanical perturbations to shed light on the processes that influence cell patterns in vivo.