Tepsin binds LC3B to promote ATG9A trafficking and delivery.

Tepsin is an established accessory protein found in Adaptor Protein 4 (AP-4) coated vesicles, but the biological role of tepsin remains unknown. AP-4 vesicles originate at the trans-Golgi network (TGN) and target the delivery of ATG9A, a scramblase required for autophagosome biogenesis, to the cell periphery. Using in silico methods, we identified a putative LC3-Interacting Region (LIR) motif in tepsin. Biochemical experiments using purified recombinant proteins indicate tepsin directly binds LC3B preferentially over other members of the mammalian ATG8 family. Calorimetry and structural modeling data indicate this interaction occurs with micromolar affinity using the established LC3B LIR docking site. Loss of tepsin in cultured cells dysregulates ATG9A export from the TGN as well as ATG9A distribution at the cell periphery. Tepsin depletion in a mRFP-GFP-LC3B HeLa reporter cell line using siRNA knockdown increases autophagosome volume and number, but does not appear to affect flux through the autophagic pathway. Reintroduction of wild-type tepsin partially rescues ATG9A cargo trafficking defects. In contrast, reintroducing tepsin with a mutated LIR motif or missing N-terminus drives diffuse ATG9A subcellular distribution. Together, these data suggest roles for tepsin in cargo export from the TGN; ensuring delivery of ATG9A-positive vesicles; and in overall maintenance of autophagosome structure.

Tepsin binds LC3B to promote ATG9A export and delivery at the cell periphery.

Tepsin is an established accessory protein found in Adaptor Protein 4 (AP-4) coated vesicles, but the biological role of tepsin remains unknown. AP-4 vesicles originate at the trans -Golgi network (TGN) and target the delivery of ATG9A, a scramblase required for autophagosome biogenesis, to the cell periphery. Using in silico methods, we identified a putative L C3-Interacting R egion (LIR) motif in tepsin. Biochemical experiments using purified recombinant proteins indicate tepsin directly binds LC3B, but not other members, of the mammalian ATG8 family. Calorimetry and structural modeling data indicate this interaction occurs with micromolar affinity using the established LC3B LIR docking site. Loss of tepsin in cultured cells dysregulates ATG9A export from the TGN as well as ATG9A distribution at the cell periphery. Tepsin depletion in a mRFP-GFP-LC3B HeLa reporter cell line using siRNA knockdown increases autophagosome volume and number, but does not appear to affect flux through the autophagic pathway. Re-introduction of wild-type tepsin partially rescues ATG9A cargo trafficking defects. In contrast, re-introducing tepsin with a mutated LIR motif or missing N-terminus does not fully rescue altered ATG9A subcellular distribution. Together, these data suggest roles for tepsin in cargo export from the TGN; delivery of ATG9A-positive vesicles at the cell periphery; and in overall maintenance of autophagosome structure.

AP-4 loss in CRISPR-edited zebrafish affects early embryo development.

Mutations in the heterotetrametric adaptor protein 4 (AP-4; ε/ß4/µ4/σ4 subunits) membrane trafficking coat complex lead to complex neurological disorders characterized by spastic paraplegia, microcephaly, and intellectual disabilities. Understanding molecular mechanisms underlying these disorders continues to emerge with recent identification of an essential autophagy protein, ATG9A, as an AP-4 cargo. Significant progress has been made uncovering AP-4 function in cell culture and patient-derived cell lines, and ATG9A trafficking by AP-4 is considered a potential target for gene therapy approaches. In contrast, understanding how AP-4 trafficking affects development and function at the organismal level has long been hindered by loss of conserved AP-4 genes in key model systems (S. cerevisiae, C. elegans, D. melanogaster). However, zebrafish (Danio rerio) have retained AP-4 and can serve as an important model system for studying both the nervous system and overall development. We undertook gene editing in zebrafish using a CRISPR-ExoCas9 knockout system to determine how loss of single AP-4, or its accessory protein tepsin, genes affect embryo development 24 h post-fertilization (hpf). Single gene-edited embryos display abnormal head morphology and neural necrosis. We further conducted the first exploration of how AP-4 single gene knockouts in zebrafish embryos affect expression levels and patterns of two autophagy genes, atg9a and map1lc3b. This work suggests zebrafish may be further adapted and developed as a tool to uncover AP-4 function in membrane trafficking and autophagy in the context of a model organism.

Trends and factors affecting the US adult hematology workforce: a mixed methods study.

The current demand for adult hematologists in the United States is projected to exceed the existing supply. However, no national study has systematically evaluated factors affecting the adult hematology workforce. In collaboration with the American Society of Hematology (ASH), we performed a mixed methods study consisting of surveys from the annual ASH In-Service Exam for adult hematology/oncology fellows from 2010 to 2016 (8789 participants); interviews with graduating or recently graduated adult hematology/oncology fellows in a single training program (8 participants); and 3 separate focus groups for hematology/oncology fellowship program directors (12 participants), fellows (12 participants), and clinicians (10 participants) at the 2016 ASH annual meeting. In surveys, the majority of fellows favored careers combining hematology and oncology, with more fellows identifying oncology, rather than hematology, as their primary focus. In interviews with advanced-year fellows, mentorship emerged as the single most important career determinant, with mentorship opportunities arising serendipitously, and oncology faculty perceived as having greater availability for mentorship than hematology faculty. In focus group discussions, hematology, particularly benign hematology, was viewed as having poorer income potential, research funding, job availability, and job security than oncology. Focus group participants invariably agreed that the demand for clinical care in hematology, particularly benign hematology, exceeded the current workforce supply. Single-subspecialty fellowship training in hematology and the creation of new clinical care models were offered as potential solutions to these workforce problems. As a next step, ASH is conducting a national, longitudinal study of the adult hematology workforce to improve recruitment and retention in the field.

Does changing weight change pain? Retrospective data analysis from a national multidisciplinary weight management service.

BACKGROUND: Musculoskeletal (MSK) pain is common in obese populations. Multidisciplinary Tier 3 weight management services (WMS) are effective in reducing weight; however, MSK pain as an outcome is not routinely reported post-WMS interventions. METHODS: Following ethical approval this retrospective design study using anonymized data from a national WMS established changes in anthropometric and pain prevalence and intensity scores as well as establishing variables predictive of achieving clinically significant changes (CSC) in pain scores. RESULTS: Of the 806 patients registered to the WMS (January 2011-February 2015), 59% (n = 476; CI = 56-62) attended their reassessments at 6 months. The overall mean age was 45.1 ± 12 years and 62% (n = 294) were female. At baseline 70% (n = 281; CI = 65-75) reported low back pain (LBP) and 59% (n = 234; CI = 54-64) had knee pain. At reassessment 37.3% (n = 177) of patients lost ≥5% body weight, 58.7% (n = 279) were weight stable (5% weight loss or gain) and 4.0% (n = 19) gained ≥5% body weight. Low back and knee pain prevalence reduced significantly for those who lost ≥5% body weight. Variables predictive of a CSC in LBP numerical rating scale (NRS) score included a higher baseline NRS score, weighing more, and rating losing weight as being important (p < 0.05). Higher baseline NRS and being younger resulted in higher odds of a CSC in knee pain NRS (p < 0.05). CONCLUSIONS: Overall this WMS was effective for clinical weight loss. For those who lost most weight prevalence of knee and LBP reduced. Imbedding pain management strategies within WMS's may provide a more holistic approach to obesity management. SIGNIFICANCE: Weight loss can reduce musculoskeletal pain, particularly for those who lose more weight. Imbedding pain management strategies within these services may provide a more holistic approach to obesity management.

Retrograde Degenerative Signaling Mediated by the p75 Neurotrophin Receptor Requires p150Glued Deacetylation by Axonal HDAC1.

During development, neurons undergo apoptosis if they do not receive adequate trophic support from tissues they innervate or when detrimental factors activate the p75 neurotrophin receptor (p75NTR) at their axon ends. Trophic factor deprivation (TFD) or activation of p75NTR in distal axons results in a retrograde degenerative signal. However, the nature of this signal and the regulation of its transport are poorly understood. Here, we identify p75NTR intracellular domain (ICD) and histone deacetylase 1 (HDAC1) as part of a retrograde pro-apoptotic signal generated in response to TFD or ligand binding to p75NTR in sympathetic neurons. We report an unconventional function of HDAC1 in retrograde transport of a degenerative signal and its constitutive presence in sympathetic axons. HDAC1 deacetylates dynactin subunit p150Glued, which enhances its interaction with dynein. These findings define p75NTR ICD as a retrograde degenerative signal and reveal p150Glued deacetylation as a unique mechanism regulating axonal transport.

Musculoskeletal pain profile of obese individuals attending a multidisciplinary weight management service.

Obesity is associated with numerous chronic diseases, including musculoskeletal (MSK) pain, which affects on quality of life (QoL). There is, however, limited research providing a comprehensive MSK pain profile of an obese cohort. This retrospective study used a patient database at a national weight management service. After ethical approval, anonymized patient data were statistically analyzed to develop a pain profile, investigate relationships between pain, sleep, and function, and explore variables associated with having low back pain (LBP) and knee pain. Overall, 915 individuals attended the weight management service from January 2011 to September 2015 [male, 35% (n = 318; confidence interval [CI] = 32-38); female, 65% (n = 597; CI = 62-68); mean age 44.6]. Mean body mass index was 50.7 kg/m [class III obese (body mass index ≥40 kg/m), 92% (n = 835; CI = 91-94)]. Approximately 91% reported MSK pain: LBP, 69% (n = 539; CI = 65-72) [mean Numeric Rating Scale 7.4]; knee pain, 58% (n = 447; CI = 55-61) [mean Numeric Rating Scale 6.8]. Class III obese and multisite pain patients had lower QoL and physical activity levels, reduced sleep, and poorer physical function than less obese patients and those without pain (P < 0.05). Relationships were found between demographic, pain, self-report, psychological, and functional measures (P < 0.05). Patients who slept fewer hours and had poorer functional outcomes were more likely to have LBP; patients who were divorced, had lower QoL, and more frequent nocturia were more likely to have knee pain (P < 0.05). Multisite MSK pain is prevalent and severe in obese patients and is negatively associated with most self-report and functional outcomes. This high prevalence suggests that pain management strategies must be considered when treating obesity.

A case of new-onset cardiomyopathy and ventricular tachycardia in a patient receiving ibrutinib for relapsed mantle cell lymphoma.

Ibrutinib is a first-in-class inhibitor of Bruton's tyrosine kinase, which is approved for use in chronic lymphocytic leukemia, mantle cell lymphoma, and Waldenstrom's macroglobulinemia. Although ibrutinib has been linked to an increased incidence of atrial fibrillation, this is the first report of an association with nonischemic cardiomyopathy and ventricular arrhythmia.

Draft Genome Sequence of a Diazotrophic, Plant Growth-Promoting Rhizobacterium of the Pseudomonas syringae Complex.

We report here the draft genome sequence of Pseudomonas syringae GR12-2, a nitrogen-fixing, plant growth-promoting bacterium, isolated from the rhizosphere of an Arctic grass. The 6.6-Mbp genome contains 5,676 protein-coding genes, including a nitrogen-fixation island similar to that in P. stutzeri.