Miracle-dz/UniFAR-FTUs huggingface dataset:
The Substrate Recognition Domains of the N-end Rule Pathway*

The N-end rule pathway is a ubiquitin-dependent system where E3 ligases called N-recognins, including UBR1 and UBR2, recognize type-1 (basic) and type-2 (bulky hydrophobic) N-terminal residues as part of N-degrons. We have recently reported an E3 family (termed UBR1 through UBR7) characterized by the 70-residue UBR box, among which UBR1, UBR2, UBR4, and UBR5 were captured during affinity-based proteomics with synthetic degrons. Here we characterized substrate binding specificity and recognition domains of UBR proteins. Pull-down assays with recombinant UBR proteins suggest that 570-kDa UBR4 and 300-kDa UBR5 bind N-degron, whereas UBR3, UBR6, and UBR7 do not. Binding assays with 24 UBR1 deletion mutants and 31 site-directed UBR1 mutations narrow down the degron-binding activity to a 72-residue UBR box-only fragment that recognizes type-1 but not type-2 residues. A surface plasmon resonance assay shows that the UBR box binds to the type-1 substrate Arg-peptide with Kd of ∼3.4 μm. Downstream from the UBR box, we identify a second substrate recognition domain, termed the N-domain, required for type-2 substrate recognition. The ∼80-residue N-domain shows structural and functional similarity to 106-residue Escherichia coli ClpS, a bacterial N-recognin. We propose a model where the 70-residue UBR box functions as a common structural element essential for binding to all known destabilizing N-terminal residues, whereas specific residues localized in the UBR box (for type 1) or the N-domain (for type 2) provide substrate selectivity through interaction with the side group of an N-terminal amino acid. Our work provides new insights into substrate recognition in the N-end rule pathway.
Outcome of Varicocelectomy with Different Degrees of Clinical Varicocele in Infertile Male

Aim. To evaluate the prevalence and effect of varicocele repair on sperm motility, sperm concentration, testicular volume, and pregnancy rate in different degrees of clinical varicocele. Material and Methods. The case records were retrospectively evaluated for 482 infertile patients with varicocele who underwent varicocelectomy from December 2008 to December 2013. Semen analysis of patient at preoperative and 3-, 6-, and 12-month postoperative period was done. Varicocele was graded as per the World Health Organization grading (WHO 1993) system and included in the study. Testicular volume was noted by ultrasonography in pre- and postoperative period in one-year follow-up. Pregnancy rate was calculated at 3, 6, and 12 months of follow-up. Results. Significant differences were seen in sperm motility and concentration of patients with Grade 3 varicocele as compared with Grade 1 and Grade 2 varicocele. Significant increase in testicular volume was present after one year in Grade 3 varicocele in the postoperative period. There was significant correlation between the testicular volume and the sperm concentration. Percentage of spontaneous pregnancy in 1 year after varicocele treatment was 32 to 41% of cases. Conclusion. Varicocelectomy significantly improved sperm motility, concentration, and testicular volume. There were 32 to 41% of infertile patients who achieved pregnancy spontaneously.
Targeting the annexin 1‐formyl peptide receptor 2/ALX pathway affords protection against bacterial LPS‐induced pathologic changes in the murine adrenal cortex

Hypothalamo‐pituitary‐adrenocortical dysfunction contributes to morbidity and mortality in a high proportion of patients with sepsis. Here, we provide new insights into the underlying adrenal pathology. Using a murine model of endotoxemia (LPS injection), we demonstrate that adrenal insufficiency is triggered early in the disease. LPS induced a local inflammatory response in the adrenal gland within 4 hours of administration, coupled with increased expression of mRNAs for annexin A1 (AnxA1) and the formyl peptide receptors [(Fprs) 1, 2, and 3], a loss of lipid droplets in cortical cells (index of availability of cholesterol, the substrate for steroidogenesis), and a failure to mount a steroidogenic response to ACTH. Deletion of AnxA1 or Fpr2/3 in mice prevented lipid droplet loss, but not leukocyte infiltration. LPS increased adrenal myeloid differentiation primary response gene 88 and TLR2 mRNA expression, but not lymphocyte antigen 96 or TLR4. By contrast, neutrophil depletion prevented leukocyte infiltration and increased AnxA1, Fpr1, and Fpr3 mRNAs but had no impact on lipid droplet loss. Our novel data demonstrate that AnxA1 and Fpr2 have a critical role in the manifestation of adrenal insufficiency in this model, through regulation of cholesterol ester storage, suggesting that pharmacologic interventions targeting the AnxA1/FPR/ALX pathway may provide a new approach for the maintenance of adrenal steroidogenesis in sepsis. —Buss, N. A. P. S. , Gavins, F. N. E. , Cover, P. O. , Terron, A. , Buckingham, J. C. Targeting the annexin 1‐formyl peptide receptor 2/ALX pathway affords protection against bacterial LPS‐induced pathologic changes in the murine adrenal cortex. FASEB J. 29, 2930‐2942 (2015). www.fasebj. org
Effects of the JAK2 Inhibitor, AZ960, on Pim/BAD/BCL-xL Survival Signaling in the Human JAK2 V617F Cell Line SET-2*

The Janus-associated kinase 2 (JAK2) V617F mutation is believed to play a critical role in the pathogenesis of polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis. We have characterized a novel small molecule JAK2 inhibitor, AZ960, and used it as a tool to investigate the consequences of JAK2 V617F inhibition in the SET-2 cell line. AZ960 inhibits JAK2 kinase with a Ki of 0.00045 μm in vitro and treatment of TEL-JAK2 driven Ba/F3 cells with AZ960 blocked STAT5 phosphorylation and potently inhibited cell proliferation (GI50 = 0.025 μm). AZ960 demonstrated selectivity for TEL-JAK2-driven STAT5 phosphorylation and cell proliferation when compared with cell lines driven by similar fusions of the other JAK kinase family members. In the SET-2 human megakaryoblastic cell line, heterozygous for the JAK2 V617F allele, inhibition of JAK2 resulted in decreased STAT3/5 phosphorylation and inhibition of cell proliferation (GI50 = 0.033 μm) predominately through the induction of mitochondrial-mediated apoptosis. We provide evidence that JAK2 inhibition induces apoptosis by direct and indirect regulation of the anti-apoptotic protein BCL-xL. Inhibition of JAK2 blocked BCL-XL mRNA expression resulting in a reduction of BCL-xL protein levels. Additionally, inhibition of JAK2 resulted in decreased PIM1 and PIM2 mRNA expression. Decreased PIM1 mRNA corresponded with a decrease in Pim1 protein levels and inhibition of BAD phosphorylation at Ser112. Finally, small interfering RNA-mediated suppression of BCL-xL resulted in apoptotic cell death similar to the phenotype observed following JAK2 inhibition. These results suggest a model in which JAK2 promotes cell survival by signaling through the Pim/BAD/BCL-xL pathway.
High Sensitivity Proteomics Assisted Discovery of a Novel Operon Involved in the Assembly of Photosystem II, a Membrane Protein Complex*

Photosystem II (PSII) is a large membrane protein complex that performs the water oxidation reactions of photosynthesis in cyanobacteria, algae, and plants. The unusual redox reactions in PSII often lead to damage, degradation, and reassembly of this molecular machine. To identify novel assembly factors, high sensitivity proteomic analysis of PSII purified from the cyanobacterium Synechocystis sp. PCC 6803 was performed. This analysis identified six PSII-associated proteins that are encoded by an operon containing nine genes, slr0144 to slr0152. This operon encodes proteins that are not essential components of the PSII holocomplex but accumulate to high levels in pre-complexes lacking any of the lumenal proteins PsbP, PsbQ, or PsbV. The operon contains genes with putative binding domains for chlorophylls and bilins, suggesting these proteins may function as a reservoir for cofactors needed during the PSII lifecycle. Genetic deletion of this operon shows that removal of these protein products does not alter photoautotrophic growth or PSII fluorescence properties. However, the deletion does result in decreased PSII-mediated oxygen evolution and an altered distribution of the S states of the catalytic manganese cluster. These data demonstrate that the proteins encoded by the genes in this operon are necessary for optimal function of PSII and function as accessory proteins during assembly of the PSII complex. Thus, we have named the products of the slr0144-slr0152 operon Pap (Photosystem II assembly proteins).
Nerve growth factor content is increased in the rectal mucosa of children with diarrhea‐predominant irritable bowel syndrome

Background The enteric nervous system is a complex network that includes, in the digestive mucosa, neuronal bodies and fibers interacting with the immune system and mucosal mast cells (MC). These interactions involve the secretion of messengers, such as the neurotrophin nerve growth factor (NGF), which influence colonic motility and sensitivity, both affected in irritable bowel syndrome (IBS). This study was designed to test the hypothesis that, in children with IBS, colonic mucosal innervation, NGF content, and MC infiltration are altered. We aimed to measure MC infiltration, number of neuronal bodies, distance from MC to nerve fibers, inflammation, and NGF content in rectal mucosa of pediatric patients with IBS as compared with controls.
Retinal functional alterations in mice lacking intermediate filament proteins glial fibrillary acidic protein and vimentin

Vimentin (Vim) and glial fibrillary acidic protein (GFAP) are important components of the intermediate filament (IF) (or nanofilament) system of astroglial cells. We conducted full‐field electroretinogram (ERG) recordings and found that whereas photoreceptor responses (a‐wave) were normal in uninjured GFAP–/– Vim–/– mice, b‐wave amplitudes were increased. Moreover, we found that Kir (inward rectifier K+) channel protein expression was reduced in the retinas of GFAP–/– Vim–/– mice and that Kir‐mediated current amplitudes were lower in Müller glial cells isolated from these mice. Studies have shown that the IF system, in addition, is involved in the retinal response to injury and that attenuated Müller cell reactivity and reduced photoreceptor cell loss are observed in IF‐deficient mice after experimental retinal detachment. We investigated whether the lack of IF proteins would affect cell survival in a retinal ischemia‐reperfusion model. We found that although cell loss was induced in both genotypes, the number of surviving cells in the inner retina was lower in IF‐deficient mice. Our findings thus show that the inability to produce GFAP and Vim affects normal retinal physiology and that the effect of IF deficiency on retinal cell survival differs, depending on the underlying pathologic condition. —Wunderlich, K. A. , Tanimoto, N. , Grosche, A. , Zrenner, E. , Pekny, M. , Reichenbach, A. , Seeliger, M. W. , Pannicke, T. , Perez, M. ‐T. Retinal functional alterations in mice lacking intermediate filament proteins glial fibrillary acidic protein and vimentin. FASEB J. 29, 4815–4828 (2015). www.fasebj. org
STAT‐3 contributes to pulmonary fibrosis through epithelial injury and fibroblast‐myofibroblast differentiation

Lung fibrosis is the hallmark of the interstitial lung diseases. Alveolar epithelial cell (AEC) injury is akey step that contributes to a profibrotic microenvironment. Fibroblasts and myofibroblasts subsequently accumulate and deposit excessive extracellular matrix. In addition to TGF‐β, the IL‐6 family of cytokines, which signal through STAT‐3, may also contribute to lung fibrosis. In the current manuscript, the extent to which STAT‐3 inhibition decreases lung fibrosis is investigated. Phosphorylated STAT‐3 was elevated in lung biopsies from patients with idiopathic pulmonary fibrosis and bleomycin (BLM)‐induced fibrotic murine lungs. C‐188‐9, a small molecule STAT‐3 inhibitor, decreased pulmonary fibrosis in the intraperitoneal BLM model as assessed by arterial oxygen saturation (control, 84.4 ± 1.3%; C‐188‐9, 94.4 ± 0.8%), histology (Ashcroft score: untreated, 5.4 ± 0.25; C‐188‐9, 3.3 ± 0.14), and attenuated fibrotic markers such as diminished α‐smooth muscle actin, reduced collagen deposition. In addition, C‐188‐9 decreased the expression of epithelial injury markers, including hypoxia‐inducible factor‐1α (HIF‐1α) and plasminogen activator inhibitor‐1 (PAI‐1). In vitro studies show that inhibition of STAT‐3 decreased IL‐6‐ and TGF‐β‐induced expression of multiple genes, including HIF‐1α and PAI‐1, in AECs. Furthermore, C‐188‐9 decreased fibroblast‐to‐myofibroblast differentiation. Finally, TGF‐β stimulation of lung fibroblasts resulted in SMAD2/SMAD3‐dependent phosphorylation of STAT‐3. These findings demonstrate that STAT‐3 contributes to the development of lung fibrosis and suggest that STAT‐3 may be a therapeutic target in pulmonary fibrosis. —Pedroza, M. , Le, T. T. , Lewis, K. , Karmouty‐Quintana, H. , To, S. , George, A. T. , Blackburn, M. R. , Tweardy, D. J., Agarwal, S. K. STAT‐3 contributes to pulmonary fibrosis through epithelial injury and fibroblast‐myofibroblast differentiation. FASEB J. 30, 129‐140 (2016). www.fasebj. org
Starving Neurons Show Sex Difference in Autophagy*

Sex-dependent differences in adaptation to famine have long been appreciated, thought to hinge on female versus male preferences for fat versus protein sources, respectively. However, whether these differences can be reduced to neurons, independent of typical nutrient depots, such as adipose tissue, skeletal muscle, and liver, was heretofore unknown. A vital adaptation to starvation is autophagy, a mechanism for recycling amino acids from organelles and proteins. Here we show that segregated neurons from males in culture are more vulnerable to starvation than neurons from females. Nutrient deprivation decreased mitochondrial respiration, increased autophagosome formation, and produced cell death more profoundly in neurons from males versus females. Starvation-induced neuronal death was attenuated by 3-methyladenine, an inhibitor of autophagy; Atg7 knockdown using small interfering RNA; or l-carnitine, essential for transport of fatty acids into mitochondria, all more effective in neurons from males versus females. Relative tolerance to nutrient deprivation in neurons from females was associated with a marked increase in triglyceride and free fatty acid content and a cytosolic phospholipase A2-dependent increase in formation of lipid droplets. Similar sex differences in sensitivity to nutrient deprivation were seen in fibroblasts. However, although inhibition of autophagy using Atg7 small interfering RNA inhibited cell death during starvation in neurons, it increased cell death in fibroblasts, implying that the role of autophagy during starvation is both sex- and tissue-dependent. Thus, during starvation, neurons from males more readily undergo autophagy and die, whereas neurons from females mobilize fatty acids, accumulate triglycerides, form lipid droplets, and survive longer.
Lipoxin and resolvin biosynthesis is dependent on 5‐lipoxygenase activating protein

Resolution of acute inflammation is an active process coordinated by proresolving lipid mediators (SPMs) such as lipoxins (LXs) and resolvins (Rvs), which are formed by the concerted action of 2 lipoxygenases (LOs). Because the exact molecular mechanisms of SPM biosynthesis are not completely understood, we aimed to investigate LX and D‐type Rv formation in human leukocytes and HEK293T cells overexpressing leukotriene (LT) pathway enzymes. Activity assays in precursor (15‐hydroxyeicosatetraenoic acids, 17‐HDoHE)‐treated granulocytes [polymorphonuclear leukocytes (PMNLs)] showed a strict dependence of LXA4/RvD1 biosynthesis on cell integrity, and incubation with recombinant human 5‐LO did not lead to LX or Rv formation. Pharmacologic inhibition of 5‐LO activating protein (FLAP) by MK‐886 inhibited LXA4/RvD1 biosynthesis in precursor‐treated PMNLs (drug concentration causing 50% inhibition ∼0.3/0.2 μM), as did knockdown of the enzyme in MM6 cells, and precursor‐treated HEK293T overexpressing 5‐LO produced high amounts of LXA4 only in the presence of FLAP. In addition, inhibition of cytosolic phospholipase A2α (cPLA2α) interfered with LXA4/RvD1 formation from exogenous precursors in PMNLs. Furthermore, inhibition of the LT synthases LTA4 hydrolase and LTC4 synthase in PMNL/platelet coincubations augmented LXA4 levels. These findings show that several enzymes known to be involved in the biosynthesis of proinflammatory LTs, such as FLAP and cPLA2α, also contribute to LX and Rv formation. —Lehmann, C. , Homann, J., Ball, A. ‐K. , Blöcher, R. , Kleinschmidt, T. K. , Basavarajappa, D. , Angioni, C. , Ferreirós, N. , Häfner, A. ‐K. , Rådmark, O. , Proschak, E. , Haeggström, J. Z. , Geisslinger, G. , Parnham, M. J., Steinhilber, D. , Kahnt, A. S. Lipoxin and resolvin biosynthesis is dependent on 5‐lipoxygenase activating protein. FASEB J. 29, 5029–5043 (2015). www.fasebj. org
Labor and skills gap analysis of the biomedical research workforce

The United States has experienced an unsustainable increase of the biomedical research workforce over the past 3 decades. This expansion has led to a myriad of consequences, including an imbalance in the number of researchers and available tenure‐track faculty positions, extended postdoctoral training periods, increasing age of investigators at first U.S. National Institutes of Health R01 grant, and exodus of talented individuals seeking careers beyond traditional academe. Without accurate data on the biomedical research labor market, challenges will remain in resolving these problems and in advising trainees of viable career options and the skills necessary to be productive in their careers. We analyzed workforce trends, integrating both traditional labor market information and real‐time job data. We generated a profile of the current biomedical research workforce, performed labor gap analyses of occupations in the workforce at regional and national levels, and assessed skill transferability between core and complementary occupations. We conclude that although supply into the workforce and the number of job postings for occupations within that workforce have grown over the past decade, supply continues to outstrip demand. Moreover, we identify practical skill sets from real‐time job postings to optimally equip trainees for an array of careers to effectively meet future workforce demand. —Mason, J. L. , Johnston, E. , Berndt, S. , Segal, K. , Lei, M. , Wiest, J. S. Labor and skills gap analysis of the biomedical research workforce. FASEB J. 30, 2673‐2683 (2016). www.fasebj. org
Inhibition of Poly(ADP-ribose) Polymerase-1 by Arsenite Interferes with Repair of Oxidative DNA Damage*

Arsenic enhances skin tumor formation when combined with other carcinogens, including UV radiation (UVR). In this study we report that low micromolar concentrations of arsenite synergistically increases UVR-induced oxidative DNA damage in human keratinocytes as detected by 8-hydroxyl-2′-deoxyguanine (8-OHdG) formation. Poly(ADP-ribose) polymerase-1 (PARP-1) is involved in base excision repair, a process that repairs 8-OHdG lesions. Arsenite suppresses UVR-induced PARP-1 activation in a concentration-dependent manner. Inhibition of PARP-1 activity by 3-aminobenzamide or small interfering RNA silencing of PARP-1 expression significantly increases UVR-induced 8-OHdG formation, suggesting that inhibition of PARP-1 activity by arsenite contributes to oxidative DNA damage. PARP-1 is a zinc finger protein, and mass spectrometry analysis reveals that arsenite can occupy a synthetic apopeptide representing the first zinc finger of PARP-1 (PARPzf). When the PARPzf peptide is preincubated with Zn(II) followed by incubation with increasing concentrations of arsenite, the ZnPARPzf signal is decreased while the AsPARPzf signal intensity is increased as a function of arsenite dose, suggesting a competition between zinc and arsenite for the same binding site. Addition of Zn(II) abolished arsenite enhancement of UVR-stimulated 8-OHdG generation and restored PARP-1 activity. Our findings demonstrate that arsenite inhibits oxidative DNA damage repair and suggest that interaction of arsenite with the PARP-1 zinc finger domain contributes to the inhibition of PARP-1 activity by arsenite. Arsenite inhibition of poly(ADP-ribosyl)ation is one likely mechanism for the reported co-carcinogenic activities of arsenic in UVR-induced skin carcinogenesis.
FBP17 Mediates a Common Molecular Step in the Formation of Podosomes and Phagocytic Cups in Macrophages*

Macrophages act to protect the body against inflammation and infection by engaging in chemotaxis and phagocytosis. In chemotaxis, macrophages use an actin-based membrane structure, the podosome, to migrate to inflamed tissues. In phagocytosis, macrophages form another type of actin-based membrane structure, the phagocytic cup, to ingest foreign materials such as bacteria. The formation of these membrane structures is severely affected in macrophages from patients with Wiskott-Aldrich syndrome (WAS), an X chromosome-linked immunodeficiency disorder. WAS patients lack WAS protein (WASP), suggesting that WASP is required for the formation of podosomes and phagocytic cups. Here we have demonstrated that formin-binding protein 17 (FBP17) recruits WASP, WASP-interacting protein (WIP), and dynamin-2 to the plasma membrane and that this recruitment is necessary for the formation of podosomes and phagocytic cups. The N-terminal EFC (extended FER-CIP4 homology)/F-BAR (FER-CIP4 homology and Bin-amphiphysin-Rvs) domain of FBP17 was previously shown to have membrane binding and deformation activities. Our results suggest that FBP17 facilitates membrane deformation and actin polymerization to occur simultaneously at the same membrane sites, which mediates a common molecular step in the formation of podosomes and phagocytic cups. These results provide a potential mechanism underlying the recurrent infections in WAS patients.
Bovine mastitis and its associated risk factors in lactating cows in Wolayta Soddo, Southern Ethiopia.

A cross-sectional study of bovine mastitis was conducted on 275 lactating cows from November 2007 to April 2008 to estimate the prevalence of mastitis and to determine the pathogens causing mastitis with the associated risk factors. Diagnosis was based on clinical examination of the udder and milk and the use of White Side Test (WST) as a screening test for subclinical mastitis. The overall prevalence of bovine mastitis was 27.3% with clinical and subclinical mastitis contributing 9.5 % and 23.4 %, respectively. Significant (P Keywords: Bovine mastitis, White Side Test, risk factors, Wolayta Soddo, Ethiopia.