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Baseball season is now on the clock and the Rays are looking to get back to the postseason for the first time since 2014. That quest will begin next week as Spring Training is finally upon us. Here's a primer to get you informed on all you need to know this spring.

Here's an early look at how the Rays' 25-man roster could shape up on Opening Day.

All 30 Major League teams will wear special "MLB 150" patches on their uniforms for the entire 2019 season in honor of the 150th anniversary of the 1869 Cincinnati Red Stockings, the first openly all-salaried professional baseball team.

The next five weeks will see lots of shuffling on Major League rosters. Here are the most intriguing positional battles on each of the 30 MLB clubs.

Sulfur is essential for biological processes such as amino acid biogenesis, iron–sulfur cluster formation, and redox homeostasis. To acquire sulfur-containing compounds from the environment, bacteria have evolved high-affinity uptake systems, predominant among which is the ABC transporter family. Theses membrane-embedded enzymes use the energy of ATP hydrolysis for transmembrane transport of a wide range of biomolecules against concentration gradients. Three distinct bacterial ABC import systems of sulfur-containing compounds have been identified, but the molecular details of their transport mechanism remain poorly characterized. Here we provide results from a biochemical analysis of the purified Escherichia coli YecSC-FliY cysteine/cystine import system. We found that the substrate-binding protein FliY binds l-cystine, l-cysteine, and d-cysteine with micromolar affinities. However, binding of the l- and d-enantiomers induced different conformational changes of FliY, where the l- enantiomer–substrate-binding protein complex interacted more efficiently with the YecSC transporter. YecSC had low basal ATPase activity that was moderately stimulated by apo FliY, more strongly by d-cysteine–bound FliY, and maximally by l-cysteine– or l-cystine–bound FliY. However, at high FliY concentrations, YecSC reached maximal ATPase rates independent of the presence or nature of the substrate. These results suggest that FliY exists in a conformational equilibrium between an open, unliganded form that does not bind to the YecSC transporter and closed, unliganded and closed, liganded forms that bind this transporter with variable affinities but equally stimulate its ATPase activity. These findings differ from previous observations for similar ABC transporters, highlighting the extent of mechanistic diversity in this large protein family.

Treatment of patients with chronic lymphocytic leukemia (CLL) with inhibitors of Bruton's tyrosine kinase (BTK), such as ibrutinib, is limited by primary or secondary resistance to this drug. Examinations of CLL patients with late relapses while on ibrutinib, which inhibits BTK's catalytic activity, revealed several mutations in BTK, most frequently resulting in the C481S substitution, and disclosed many mutations in PLCG2, encoding phospholipase C-γ2 (PLCγ2). The PLCγ2 variants typically do not exhibit constitutive activity in cell-free systems, leading to the suggestion that in intact cells they are hypersensitive to Rac family small GTPases or to the upstream kinases spleen-associated tyrosine kinase (SYK) and Lck/Yes-related novel tyrosine kinase (LYN). The sensitivity of the PLCγ2 variants to BTK itself has remained unknown. Here, using genetically-modified DT40 B lymphocytes, along with various biochemical assays, including analysis of PLCγ2-mediated inositol phosphate formation, inositol phospholipid assessments, fluorescence recovery after photobleaching (FRAP) static laser microscopy, and determination of intracellular calcium ([Ca2+]i), we show that various CLL-specific PLCγ2 variants such as PLCγ2S707Y are hyper-responsive to activated BTK, even in the absence of BTK's catalytic activity and independently of enhanced PLCγ2 phospholipid substrate supply. At high levels of B-cell receptor (BCR) activation, which may occur in individual CLL patients, catalytically-inactive BTK restored the ability of the BCR to mediate increases in [Ca2+]i. Because catalytically-inactive BTK is insensitive to active-site BTK inhibitors, the mechanism involving the noncatalytic BTK uncovered here may contribute to preexisting reduced sensitivity or even primary resistance of CLL to these drugs.

Diabetic retinopathy (DR) is diagnosed clinically by directly viewing retinal vascular changes during ophthalmoscopy or through fundus photographs. However, electroretinography (ERG) studies in humans and rodents have revealed that retinal dysfunction is demonstrable prior to the development of visible vascular defects. Specifically, delays in dark-adapted ERG oscillatory potential (OP) implicit times in response to dim flash stimuli (<-1.8 log cd·s/m²) occur prior to clinically-recognized diabetic retinopathy. Animal studies suggest that retinal dopamine deficiency underlies these early functional deficits. Here, we randomized persons with diabetes, without clinically detectable retinopathy, to treatment with either low or high dose Sinemet (levodopa plus carbidopa) for 2 weeks and compared their ERG findings with those of control (no DM) subjects. We assessed dim flash stimulated OP delays using a novel hand-held ERG system (RETeval) at baseline, 2 and 4 weeks. RETeval recordings identified significant OP implicit-time delays in persons with diabetes without retinopathy compared to age-matched controls (p<0.001). After two weeks of Sinemet treatment, OP implicit times were restored to control values, and these improvements persisted even after a two-week washout. We conclude that detection of dim flash OP delays could provide early detection of DR, and that Sinemet treatment may reverse retinal dysfunction.

Amylin, a pancreatic hormone and neuropeptide, acts principally in the hindbrain to decrease food intake and has been recently shown to act as a neurotrophic factor to control the development of AP->NTS and ARC->PVN axonal fiber outgrowth. Amylin is also able to activate ERK signaling specifically in POMC neurons independently of leptin. To investigate the physiological role of amylin signaling in POMC neurons, the core component of the amylin receptor, calcitonin receptor (CTR) was depleted from POMC neurons using an inducible mouse model. The loss of CTR in POMC neurons leads to increased body weight gain, increased adiposity, and glucose intolerance in male knockout mice, characterized by decreased energy expenditure (EE) and decreased expression of uncoupling protein 1 (UCP1) in brown adipose tissue (BAT). Furthermore, a decreased spontaneous locomotor activity and absent thermogenic reaction to the application of the amylin receptor agonist were observed in male and female mice. Together, these results show a significant physiological impact of amylin/calcitonin signaling in CTR-POMC neurons on energy metabolism and demonstrate the need for sex-specific approaches in obesity research and potentially treatment.

T cells isolated from the pancreatic infiltrates of non-obese diabetic mice have been shown to recognize epitopes formed by the covalent cross-linking of proinsulin and secretory granule peptides. Formation of such hybrid insulin peptides (HIPs) was confirmed through mass spectrometry and responses to HIPs were observed among the islet-infiltrating T cells of pancreatic organ donors and in the peripheral blood of individuals with type 1 diabetes (T1D). However, questions remain about the prevalence of HIP-specific T cells in humans, the sequences they recognize, and their role in disease. We identified six novel HIPs that are recognized in the context of DRB1*04:01, discovered by utilizing a library of theoretical HIP sequences derived from insulin fragments covalently linked to one another or to fragments of secretory granule proteins or other islet-derived proteins. We demonstrate that T cells that recognize these HIPs are detectable in the peripheral blood of subjects with T1D and exhibit an effector memory phenotype. HIP-reactive T cell clones produced Th1-associated cytokines and proliferated in response to human islet preparations. These results support the relevance of HIPs in human disease, further establishing a novel post-translational modification that may contribute to the loss of peripheral tolerance in T1D.

The purpose of this study was to investigate the protective role of Peroxisome Proliferator-Activated Receptor-alpha (PPARα) against diabetic keratopathy and corneal neuropathy. Corneal samples were obtained from diabetic and non-diabetic human donors. Streptozotocin-induced diabetic rats and mice were orally treated with PPARα agonist fenofibrate. As shown by immunohistochemistry and Western blotting, PPARα was down-regulated in the corneas of diabetic humans and rats. Immunostaining of β-III tubulin demonstrated that corneal nerve fiber metrics were decreased significantly in diabetic rats and mice, which was partially prevented by fenofibrate treatment. As evaluated using a Cochet-Bonnet aesthesiometer, corneal sensitivity was significantly decreased in diabetic mice, which was prevented by fenofibrate. PPARα^-/- mice displayed progressive decreases in the corneal nerve fiber density. Consistently, corneal sensitivity was decreased in PPARα^-/- mice relative to wild-type mice by nine months of age. Diabetic mice showed increased incidence of spontaneous corneal epithelial lesion, which was prevented by fenofibrate while exacerbated by PPARα knockout. Western blot analysis revealed significantly altered neurotrophic factor levels in diabetic rat corneas, which were partially restored by fenofibrate treatment. These results indicate that PPARα protects corneal nerve from degeneration induced by diabetes, and PPARα agonists have therapeutic potential in the treatment of diabetic keratopathy.

A greater decrease in 24-h energy expenditure (24EE) during 24h fasting defines a thriftier metabolic phenotype prone to weight gain during overfeeding and resistant to weight loss during caloric restriction. As the thermogenic response to mild cold exposure (COLD) may similarly characterize this human phenotype identified by acute fasting conditions, we analyzed changes in 24EE and sleeping metabolic rate (SLEEP) in a whole-room indirect calorimeter during 24h fasting at thermoneutrality (24°C) and during energy balance both at thermoneutrality (24°C) and mild cold (19°C) in 20 healthy volunteers (80% male, age: 36.6±11.4y, percentage body fat: 34.8±10.5%). Greater decrease in 24EE during fasting (thriftier phenotype) was associated with less increase in 24EE during COLD, i.e. less cold-induced thermogenesis. Greater decreases in plasma fibroblast growth factor 21 (FGF21) after 24h fasting and after COLD were highly correlated and associated with greater decreases in SLEEP in both conditions. We conclude that the metabolic responses to short-term fasting and COLD are associated and mediated by the liver-derived hormone FGF21. Thus, the 24EE response to COLD further identifies the thrifty versus spendthrift phenotype, providing an additional setting to investigate the physiological mechanisms underlying the human metabolic phenotype and characterizing the individual susceptibility to weight change.

Diabetic Keratopathy, a sight-threatening corneal disease, comprises several symptomatic conditions including delayed epithelial wound healing, recurrent erosions, and sensory nerve (SN) neuropathy. We investigated the role of neuropeptides in mediating corneal wound healing, including epithelial wound closure and SN regeneration. Denervation by Resiniferatoxin severely impaired corneal wound healing and markedly up-regulated pro-inflammatory gene expression. Exogenous neuropeptides CGRP, SP, and VIP partially reversed Resiniferatoxin’s effects, with VIP specifically inducing IL-10 expression. Hence, we focused on VIP and observed that wounding induced VIP and VIPR1 expression in normal (NL), but not diabetic (DM) mouse corneas. Targeting VIPR1 in NL corneas attenuated corneal wound healing, dampened wound-induced expression of neurotrophic factors, and exacerbated inflammatory responses while exogenous VIP had the opposite effects in DM corneas. Remarkably, wounding and diabetes also affected the expression of Sonic Hedgehog (SHH) in a VIP-dependent manner. Downregulating SHH expression in NL corneas decreased, while exogenous SHH in DM corneas increased the rates of corneal wound healing. Furthermore, inhibition of SHH signaling dampened VIP-promoted corneal wound healing. We conclude that VIP regulates epithelial wound healing, inflammatory response, and nerve regeneration in the corneas in a SHH-dependent manner, suggesting a therapeutic potential for these molecules in treating diabetic keratopathy.

Previous studies show that 12-week of high-fat diet (HFD) consumption caused not only prediabetes, but also cognitive decline and brain pathologies. Recently, necrostatin-1 (nec-1), a necroptosis inhibitor, showed beneficial effects in brain against stroke. However, the comparative effects of nec-1 and metformin on cognition and brain pathologies in prediabetes have not been investigated. We hypothesized that nec-1 and metformin equally attenuated cognitive decline and brain pathologies in prediabetic rats. Rats (n=32) were fed with either normal diet (ND) or high-fat diet (HFD) for 20 weeks. At week 13, ND-fed rats were given a vehicle (n=8) and HFD-fed rats were randomly assigned into 3 subgroups (n=8/subgroup) with vehicle, nec-1 or metformin for 8 weeks. Metabolic parameters, cognitive function, brain insulin receptor function, synaptic plasticity, dendritic spine density, microglial morphology, brain mitochondrial function, Alzheimer’s protein, and cell death were determined. HFD-fed rats exhibited prediabetes, cognitive decline, and brain pathologies. Nec-1 and metformin equally improved cognitive function, synaptic plasticity, dendritic spine density, microglial morphology, brain mitochondrial function, reduced hyperphosphorylated-tau and necroptosis in HFD-fed rats. Interestingly metformin, but not nec-1, improved brain insulin sensitivity in those rats. In conclusion, necroptosis inhibition directly improved cognition in prediabetic rats without alteration in insulin sensitivity.

Infants born to mothers with obesity have a greater risk for childhood obesity and metabolic diseases; however, the underlying biological mechanisms remain poorly understood. We used a Japanese macaque model to investigate whether maternal obesity combined with a western-style diet (WSD) impairs offspring muscle insulin action. Adult females were fed a control or WSD prior to and during pregnancy through lactation, and offspring subsequently weaned to a control or WSD. Muscle glucose uptake and signaling were measured ex vivo in fetal (n=5-8/group) and juvenile offspring (n=8/group). In vivo signaling was evaluated after an insulin bolus just prior to weaning (n=4-5/group). Maternal WSD reduced insulin-stimulated glucose uptake and impaired insulin signaling at the level of Akt phosphorylation in fetal muscle. In juvenile offspring, insulin-stimulated glucose uptake was similarly reduced by both maternal and post-weaning WSD and corresponded to modest reductions in insulin-stimulated Akt phosphorylation relative to controls. We conclude that maternal WSD leads to a persistent decrease in offspring muscle insulin-stimulated glucose uptake even in the absence of increased offspring adiposity or markers of systemic insulin resistance. Switching offspring to a healthy diet did not reverse the effects of maternal WSD on muscle insulin action suggesting earlier interventions may be warranted.

Cardiac glucose uptake and oxidation are reduced in diabetes despite hyperglycemia. Mitochondrial dysfunction contributes to heart failure in diabetes. It is unclear if these changes are adaptive or maladaptive. To directly evaluate the relationship between glucose delivery and mitochondrial dysfunction in diabetic cardiomyopathy we generated transgenic mice with inducible cardiomyocyte-specific expression of the glucose transporter (GLUT4). We examined mice rendered hyperglycemic following low-dose streptozotocin prior to increasing cardiomyocyte glucose uptake by transgene induction. Enhanced myocardial glucose in non-diabetic mice decreased mitochondrial ATP generation and was associated with echocardiographic evidence of diastolic dysfunction. Increasing myocardial glucose delivery after short-term diabetes onset, exacerbated mitochondrial oxidative dysfunction. Transcriptomic analysis revealed that the largest changes, driven by glucose and diabetes, were in genes involved in mitochondrial function. This glucose-dependent transcriptional repression was in part mediated by O-GlcNAcylation of the transcription factor Sp1. Increased glucose uptake induced direct O-GlcNAcylation of many electron transport chain subunits and other mitochondrial proteins. These findings identify mitochondria as a major target of glucotoxicity. They also suggest reduced glucose utilization in diabetic cardiomyopathy might defend against glucotoxicity and caution that restoring glucose delivery to the heart in the context of diabetes could accelerate mitochondrial dysfunction by disrupting protective metabolic adaptations.

A failure in self-tolerance leads to autoimmune destruction of pancreatic β-cells and type 1 diabetes (T1D). Low molecular weight dextran sulfate (DS) is a sulfated semi-synthetic polysaccharide with demonstrated cytoprotective and immunomodulatory properties in vitro. However, whether DS can protect pancreatic β-cells, reduce autoimmunity and ameliorate T1D is unknown. Here we report that DS, but not dextran, protects human β-cells against cytokine-mediated cytotoxicity in vitro. DS also protects mitochondrial function and glucose-stimulated insulin secretion and reduces chemokine expression in human islets in a pro-inflammatory environment. Interestingly, daily treatment with DS significantly reduces diabetes incidence in pre-diabetic non-obese diabetic (NOD) mice, and most importantly, reverses diabetes in early-onset diabetic NOD mice. DS decreases β-cell death, enhances islet heparan sulfate (HS)/heparan sulfate proteoglycan (HSPG) expression and preserves β-cell mass and plasma insulin in these mice. DS administration also increases the expression of the inhibitory co-stimulatory molecule programmed death-1 (PD-1) in T-cells, reduces interferon-+ CD4+ and CD8+ T-cells and enhances the number of FoxP3+ cells. Collectively, these studies demonstrate that the action of one single molecule, DS, on β-cell protection, extracellular matrix preservation and immunomodulation can reverse diabetes in NOD mice highlighting its therapeutic potential for the treatment of T1D.

Islet transplantation is an emerging therapy for type 1 diabetes (T1D) and hypoglycaemic unawareness. However, a key challenge for islet transplantation is cellular rejection and the requirement for long-term immunosuppression. In this study we established a diabetic-humanized NOD-scidIL2R^null(NSG) mouse model of T cell mediated human islet-allograft rejection and developed a therapeutic regimen of low-dose recombinant human interleukin2(IL-2) combined with low-dose rapamycin to prolong graft survival. NSG-mice that had received renal-subcapsular human islet-allografts and were transfused with 1x10⁷ of human-spleen-mononuclear-cells (hSPMCs), reconstituted human CD45⁺ cells that were predominantly CD3⁺ T cells and rejected their grafts with a median survival time of 27 days. IL-2 alone (0.3x10⁶ IU/m² or 1x10⁶ IU/m²), or rapamycin alone (0.5-1mg/kg) for 3 weeks did not prolong survival. However, the combination of rapamycin with IL-2 for 3 weeks significantly prolonged human islet-allograft survival. Graft survival was associated with expansion of CD4⁺CD25⁺FOXP3⁺ Tregs and enhanced TGF-β production by CD4⁺ T cells. CD8⁺ T cells showed reduced IFN- production and reduced expression of perforin-1. The combination of IL-2 and rapamycin has the potential to inhibit human islet-allograft rejection by expanding CD4⁺FOXP3⁺ Tregs in vivo and supressing effector cell function, and could be the basis of effective tolerance-based regimens.

Hepatic steatosis, the excess storage of intrahepatic lipids, is a rampant clinical problem associated with the obesity epidemic. Hepatic steatosis is linked to increased risk for insulin resistance, type 2 diabetes, and cardiovascular and advanced liver disease. Accumulating evidence shows that physical activity, exercise, and aerobic capacity have profound effects on regulating intrahepatic lipids and mediating susceptibility for hepatic steatosis. Moreover, exercise can effectively reduce hepatic steatosis independent of changes in body mass. In this perspective, we highlight 1) the relationship between obesity and metabolic pathways putatively driving hepatic steatosis compared with changes induced by exercise; 2) the impact of physical activity, exercise, and aerobic capacity compared with caloric restriction on regulating intrahepatic lipids and steatosis risk; 3) the effects of exercise training (modalities, volume, intensity) for treatment of hepatic steatosis, and 4) evidence for a sustained protection against steatosis induced by exercise. Overall, evidence clearly indicates that exercise powerfully regulates intrahepatic storage of fat and risk for steatosis.

Prostate cancer (PCa) cells heavily rely on an active androgen receptor (AR) pathway for their survival. Enzalutamide (MDV3100) is a second-generation antiandrogenic drug that was approved by the Food and Drug Administration in 2012 to treat patients with castration-resistant prostate cancer (CRPC). However, emergence of resistance against this drug is inevitable, and it has been a major challenge to develop interventions that help manage enzalutamide-resistant CRPC. Erythropoietin-producing human hepatocellular (Eph) receptors are targeted by ephrin protein ligands and have a broad range of functions. Increasing evidence indicates that this signaling pathway plays an important role in tumorigenesis. Overexpression of EPH receptor B4 (EPHB4) has been observed in multiple types of cancer, being closely associated with proliferation, invasion, and metastasis of tumors. Here, using RNA-Seq analyses of clinical and preclinical samples, along with several biochemical and molecular methods, we report that enzalutamide-resistant PCa requires an active EPHB4 pathway that supports drug resistance of this tumor type. Using a small kinase inhibitor and RNAi-based gene silencing to disrupt EPHB4 activity, we found that these disruptions re-sensitize enzalutamide-resistant PCa to the drug both in vitro and in vivo. Mechanistically, we found that EPHB4 stimulates the AR by inducing proto-oncogene c-Myc (c-Myc) expression. Taken together, these results provide critical insight into the mechanism of enzalutamide resistance in PCa, potentially offering a therapeutic avenue for enhancing the efficacy of enzalutamide to better manage this common malignancy.

Prevention of aberrant cutaneous wound repair and appropriate regeneration of an intact and functional integument require the coordinated timing of fibroblast and keratinocyte migration. Here, we identified a mechanism whereby opposing cell-specific motogenic functions of a multifunctional intracellular and extracellular protein, the receptor for hyaluronan-mediated motility (RHAMM), coordinates fibroblast and keratinocyte migration speed and ensures appropriate timing of excisional wound closure. We found that, unlike in WT mice, in Rhamm-null mice, keratinocyte migration initiates prematurely in the excisional wounds, resulting in wounds that have re-surfaced before the formation of normal granulation tissue, leading to a defective epidermal architecture. We also noted aberrant keratinocyte and fibroblast migration in the Rhamm-null mice, indicating that RHAMM suppresses keratinocyte motility but increases fibroblast motility. This cell context–dependent effect resulted from cell-specific regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) activation and expression of a RHAMM target gene encoding matrix metalloprotease 9 (MMP-9). In fibroblasts, RHAMM promoted ERK1/2 activation and MMP-9 expression, whereas in keratinocytes, RHAMM suppressed these activities. In keratinocytes, loss of RHAMM function or expression promoted epidermal growth factor receptor–regulated MMP-9 expression via ERK1/2, which resulted in cleavage of the ectodomain of the RHAMM partner protein CD44 and thereby increased keratinocyte motility. These results identify RHAMM as a key factor that integrates the timing of wound repair by controlling cell migration.

Prominins (proms) are transmembrane glycoproteins conserved throughout the animal kingdom. They are associated with plasma membrane protrusions, such as primary cilia, as well as extracellular vesicles derived thereof. Primary cilia host numerous signaling pathways affected in diseases known as ciliopathies. Human PROM1 (CD133) is detected in both somatic and cancer stem cells and is also expressed in terminally differentiated epithelial and photoreceptor cells. Genetic mutations in the PROM1 gene result in retinal degeneration by impairing the proper formation of the outer segment of photoreceptors, a modified cilium. Here, we investigated the impact of proms on two distinct examples of ciliogenesis. First, we demonstrate that the overexpression of a dominant-negative mutant variant of human PROM1 (i.e. mutation Y819F/Y828F) significantly decreases ciliary length in Madin–Darby canine kidney cells. These results contrast strongly to the previously observed enhancing effect of WT PROM1 on ciliary length. Mechanistically, the mutation impeded the interaction of PROM1 with ADP-ribosylation factor–like protein 13B, a key regulator of ciliary length. Second, we observed that in vivo knockdown of prom3 in zebrafish alters the number and length of monocilia in the Kupffer's vesicle, resulting in molecular and anatomical defects in the left-right asymmetry. These distinct loss-of-function approaches in two biological systems reveal that prom proteins are critical for the integrity and function of cilia. Our data provide new insights into ciliogenesis and might be of particular interest for investigations of the etiologies of ciliopathies.

Kindlins are focal adhesion proteins that regulate integrin activation and outside-in signaling. The kindlin family consists of three members, kindlin-1, -2, and -3. Kindlin-2 is widely expressed in multiple cell types, except those from the hematopoietic lineage. A previous study has reported that the Drosophila Fit1 protein (an ortholog of kindlin-2) prevents abnormal spindle assembly; however, the mechanism remains unknown. Here, we show that kindlin-2 maintains spindle integrity in mitotic human cells. The human neuroblastoma SH-SY5Y cell line expresses only kindlin-2, and we found that when SH-SY5Y cells are depleted of kindlin-2, they exhibit pronounced spindle abnormalities and delayed mitosis. Of note, acetylation of α-tubulin, which maintains microtubule flexibility and stability, was diminished in the kindlin-2–depleted cells. Mechanistically, we found that kindlin-2 maintains α-tubulin acetylation by inhibiting the microtubule-associated deacetylase histone deacetylase 6 (HDAC6) via a signaling pathway involving AKT Ser/Thr kinase (AKT)/glycogen synthase kinase 3β (GSK3β) or paxillin. We also provide evidence that prolonged hypoxia down-regulates kindlin-2 expression, leading to spindle abnormalities not only in the SH-SY5Y cell line, but also cell lines derived from colon and breast tissues. The findings of our study highlight that kindlin-2 regulates mitotic spindle assembly and that this process is perturbed in cancer cells in a hypoxic environment.

Multidrug resistance (MDR) in cancer arises from cross-resistance to structurally- and functionally-divergent chemotherapeutic drugs. In particular, MDR is characterized by increased expression and activity of ATP-binding cassette (ABC) superfamily transporters. Sphingolipids are substrates of ABC proteins in cell signaling, membrane biosynthesis, and inflammation, for example, and their products can favor cancer progression. Glucosylceramide (GlcCer) is a ubiquitous glycosphingolipid (GSL) generated by glucosylceramide synthase, a key regulatory enzyme encoded by the UDP-glucose ceramide glucosyltransferase (UGCG) gene. Stressed cells increase de novo biosynthesis of ceramides, which return to sub-toxic levels after UGCG mediates incorporation into GlcCer. Given that cancer cells seem to mobilize UGCG and have increased GSL content for ceramide clearance, which ultimately contributes to chemotherapy failure, here we investigated how inhibition of GSL biosynthesis affects the MDR phenotype of chronic myeloid leukemias. We found that MDR is associated with higher UGCG expression and with a complex GSL profile. UGCG inhibition with the ceramide analog d-threo-1-(3,4,-ethylenedioxy)phenyl-2-palmitoylamino-3-pyrrolidino-1-propanol (EtDO-P4) greatly reduced GSL and monosialotetrahexosylganglioside levels, and co-treatment with standard chemotherapeutics sensitized cells to mitochondrial membrane potential loss and apoptosis. ABC subfamily B member 1 (ABCB1) expression was reduced, and ABCC-mediated efflux activity was modulated by competition with nonglycosylated ceramides. Consistently, inhibition of ABCC-mediated transport reduced the efflux of exogenous C6-ceramide. Overall, UGCG inhibition impaired the malignant glycophenotype of MDR leukemias, which typically overcomes drug resistance through distinct mechanisms. This work sheds light on the involvement of GSL in chemotherapy failure, and its findings suggest that targeted GSL modulation could help manage MDR leukemias.

The C-type lectin receptors (CLRs) form a family of pattern recognition receptors that recognize numerous pathogens, such as bacteria and fungi, and trigger innate immune responses. The extracellular carbohydrate-recognition domain (CRD) of CLRs forms a globular structure that can coordinate a Ca2+ ion, allowing receptor interactions with sugar-containing ligands. Although well-conserved, the CRD fold can also display differences that directly affect the specificity of the receptors for their ligands. Here, we report crystal structures at 1.8–2.3 Å resolutions of the CRD of murine dendritic cell-immunoactivating receptor (DCAR, or Clec4b1), the CLR that binds phosphoglycolipids such as acylated phosphatidyl-myo-inositol mannosides (AcPIMs) of mycobacteria. Using mutagenesis analysis, we identified critical residues, Ala136 and Gln198, on the surface surrounding the ligand-binding site of DCAR, as well as an atypical Ca2+-binding motif (Glu-Pro-Ser/EPS168–170). By chemically synthesizing a water-soluble ligand analog, inositol-monophosphate dimannose (IPM2), we confirmed the direct interaction of DCAR with the polar moiety of AcPIMs by biolayer interferometry and co-crystallization approaches. We also observed a hydrophobic groove extending from the ligand-binding site that is in a suitable position to interact with the lipid portion of whole AcPIMs. These results suggest that the hydroxyl group-binding ability and hydrophobic groove of DCAR mediate its specific binding to pathogen-derived phosphoglycolipids such as mycobacterial AcPIMs.

In cyanobacteria, metabolic pathways that use the nitrogen-rich amino acid arginine play a pivotal role in nitrogen storage and mobilization. The N-terminal domains of two recently identified bacterial enzymes: ArgZ from Synechocystis and AgrE from Anabaena, have been found to contain an arginine dihydrolase. This enzyme provides catabolic activity that converts arginine to ornithine, resulting in concomitant release of CO2 and ammonia. In Synechocystis, the ArgZ-mediated ornithine–ammonia cycle plays a central role in nitrogen storage and remobilization. The C-terminal domain of AgrE contains an ornithine cyclodeaminase responsible for the formation of proline from ornithine and ammonia production, indicating that AgrE is a bifunctional enzyme catalyzing two sequential reactions in arginine catabolism. Here, the crystal structures of AgrE in three different ligation states revealed that it has a tetrameric conformation, possesses a binding site for the arginine dihydrolase substrate l-arginine and product l-ornithine, and contains a binding site for the coenzyme NAD(H) required for ornithine cyclodeaminase activity. Structure–function analyses indicated that the structure and catalytic mechanism of arginine dihydrolase in AgrE are highly homologous with those of a known bacterial arginine hydrolase. We found that in addition to other active-site residues, Asn-71 is essential for AgrE's dihydrolase activity. Further analysis suggested the presence of a passage for substrate channeling between the two distinct AgrE active sites, which are situated ∼45 Å apart. These results provide structural and functional insights into the bifunctional arginine dihydrolase–ornithine cyclodeaminase enzyme AgrE required for arginine catabolism in Anabaena.

Antibodies are widely used as cancer therapeutics, but their current use is limited by the low number of antigens restricted to cancer cells. A receptor tyrosine kinase, receptor tyrosine kinase-like orphan receptor 2 (ROR2), is normally expressed only during embryogenesis and is tightly down-regulated in postnatal healthy tissues. However, it is up-regulated in a diverse set of hematologic and solid malignancies, thus ROR2 represents a candidate antigen for antibody-based cancer therapy. Here we describe the affinity maturation and humanization of a rabbit mAb that binds human and mouse ROR2 but not human ROR1 or other human cell-surface antigens. Co-crystallization of the parental rabbit mAb in complex with the human ROR2 kringle domain (hROR2-Kr) guided affinity maturation by heavy-chain complementarity-determining region 3 (HCDR3)-focused mutagenesis and selection. The affinity-matured rabbit mAb was then humanized by complementarity-determining region (CDR) grafting and framework fine tuning and again co-crystallized with hROR2-Kr. We show that the affinity-matured and humanized mAb retains strong affinity and specificity to ROR2 and, following conversion to a T cell–engaging bispecific antibody, has potent cytotoxicity toward ROR2-expressing cells. We anticipate that this humanized affinity-matured mAb will find application for antibody-based cancer therapy of ROR2-expressing neoplasms.

Fucosylation of the innermost GlcNAc of N-glycans by fucosyltransferase 8 (FUT8) is an important step in the maturation of complex and hybrid N-glycans. This simple modification can dramatically affect the activities and half-lives of glycoproteins, effects that are relevant to understanding the invasiveness of some cancers, development of mAb therapeutics, and the etiology of a congenital glycosylation disorder. The acceptor substrate preferences of FUT8 are well-characterized and provide a framework for understanding N-glycan maturation in the Golgi; however, the structural basis of these substrate preferences and the mechanism through which catalysis is achieved remain unknown. Here we describe several structures of mouse and human FUT8 in the apo state and in complex with GDP, a mimic of the donor substrate, and with a glycopeptide acceptor substrate at 1.80–2.50 Å resolution. These structures provide insights into a unique conformational change associated with donor substrate binding, common strategies employed by fucosyltransferases to coordinate GDP, features that define acceptor substrate preferences, and a likely mechanism for enzyme catalysis. Together with molecular dynamics simulations, the structures also revealed how FUT8 dimerization plays an important role in defining the acceptor substrate-binding site. Collectively, this information significantly builds on our understanding of the core fucosylation process.

Type IV filaments (T4F), which are helical assemblies of type IV pilins, constitute a superfamily of filamentous nanomachines virtually ubiquitous in prokaryotes that mediate a wide variety of functions. The competence (Com) pilus is a widespread T4F, mediating DNA uptake (the first step in natural transformation) in bacteria with one membrane (monoderms), an important mechanism of horizontal gene transfer. Here, we report the results of genomic, phylogenetic, and structural analyses of ComGC, the major pilin subunit of Com pili. By performing a global comparative analysis, we show that Com pili genes are virtually ubiquitous in Bacilli, a major monoderm class of Firmicutes. This also revealed that ComGC displays extensive sequence conservation, defining a monophyletic group among type IV pilins. We further report ComGC solution structures from two naturally competent human pathogens, Streptococcus sanguinis (ComGCSS) and Streptococcus pneumoniae (ComGCSP), revealing that this pilin displays extensive structural conservation. Strikingly, ComGCSS and ComGCSP exhibit a novel type IV pilin fold that is purely helical. Results from homology modeling analyses suggest that the unusual structure of ComGC is compatible with helical filament assembly. Because ComGC displays such a widespread distribution, these results have implications for hundreds of monoderm species.

Ole Schmitz
Dec 1, 2004; 53:S233-S238
Section V: The Incretin Pathway

Maria Sörhede Winzell
Dec 1, 2004; 53:S215-S219
Section V: The Incretin Pathway

Mandeep Bajaj
Dec 1, 2012; 61:3078-3080
Commentary

Oct 1, 1992; 41:1368-1369
System[egrave] International (SI) Units Table

Jens Juul Holst
Dec 1, 2004; 53:S197-S204
Section V: The Incretin Pathway

Jennifer Vogel
May 1, 2020; 69:1032-1041
Pharmacology and Therapeutics

Marc Y. Donath
Dec 1, 2005; 54:S108-S113
Section III: Inflammation and beta-Cell Death

Juli Bai
Jun 1, 2019; 68:1099-1108
Perspectives in Diabetes

Errol B. Marliss
Feb 1, 2002; 51:S271-S283
Section 6: Pusatile and Phasic Insulin Release in Normal and Diabetic Men

Miriam Cnop
Dec 1, 2005; 54:S97-S107
Section III: Inflammation and beta-Cell Death