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Over the past couple of weeks leading up to Spring Training, MLB.com went around the horn to examine each area of the Rays' 2019 roster. The final installment focuses on Tampa Bay's outfield and designated hitters.

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.

Rays beat reporter Juan Toribio answers fans' questions.

Over the past couple seasons, the Rays have preached patience as the organization provided time for the top prospects to make it up to the Majors. Now, the focus has been primarily in remaining flexible and keeping positions open for the young talent arriving from the Minors.

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.

Brendan McKay is still going to be a two-way player, but the Rays are planning to narrow his focus on the hitting side by keeping him to designated hitter-only duties, a source confirmed to MLB.com's Juan Toribio on Monday.

After a pair of seasons -- and a World Series title -- with the Astros, Rays pitcher Charlie Morton is confident that his new team has what in takes to make a postseason run in 2019.

A rainy morning didn't stop the Rays' pitchers and catchers from officially taking the field Wednesday. "Other than the pitchers getting out there and getting in their legs a little bit and running some of the more casual [pitchers' fielding practice drills], everything is fine," manager Kevin Cash said.

After winning the 2018 American League Cy Young Award, Blake Snell said he doesn't feel "any pressure" in '19.

When Tyler Glasnow heard the news that the Rays were signing Charlie Morton, he couldn't help but get even more excited for the 2019 season.

The 30 prospects below all are getting very long looks this spring with an eye toward breaking camp with the parent club. Even if they start the year in the Minors, they all should get the chance to contribute at some point in the very near future.

As Emilio Pagan enters his first Spring Training with the Rays, he's looking to prove that he can perform well against hitters on either side of the plate.

Colin Poche's fastball won't light up the radar gun, but it proved to be one of the most effective pitches in the Minors last season.

After a disappointing 2018 season, Jake Faria is looking to show that last season isn't reflective of the type of pitcher he is.

Tyler Glasnow is hoping to build off a positive 2018, but his delivery is going to look a little different this season.

Who do the Rays have in the pipeline? Get scouting reports, video, stats, projected ETAs and more for Tampa Bay's Top 30 Prospects on MLB Pipeline's Prospect Watch.

Kevin Kiermaier has always played with a chip on his shoulder, but he admits that there's some extra motivation heading into Spring Training after a frustrating 2018 season.

On this opening week of Spring Training, all 30 Major League teams have one thing in common: optimism. Here's an optimism cheat sheet for each of them.

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.

While most of the attention will fall on the players who are expected to make the Opening Day roster, manager Kevin Cash and the rest of the organization will be keeping a close eye on some of the players that could find their way to the big leagues at some point within the next year.

When Yandy Diaz arrived at Rays camp on Sunday, he quickly established himself as the most muscular player inside the clubhouse. During Spring Training, his focus will be to establish himself as an everyday player.

All 66 players on the Rays' Spring Training roster took the field Monday as Tampa Bay went through its first full-squad workout.

Rays principal owner Stuart Sternberg won't make any big prediction entering the 2019 season, but he still expressed a lot of enthusiasm during Tuesday's media session.

An early exposure to lipid biochemistry in the laboratory of Konrad Bloch resulted in a fascination with the biosynthesis, structures, and functions of bacterial lipids. The discovery of plasmalogens (1-alk-1'-enyl, 2-acyl phospholipids) in anaerobic Gram-positive bacteria led to studies on the physical chemistry of these lipids and the cellular regulation of membrane lipid polymorphism in bacteria. Later studies in several laboratories showed that the formation of the alk-1-enyl ether bond involves an aerobic process in animal cells and thus is fundamentally different from that in anaerobic organisms. Our work provides evidence for an anaerobic process in which plasmalogens are formed from their corresponding diacyl lipids. Studies on the roles of phospholipases in Listeria monocytogenes revealed distinctions between its phospholipases and those previously discovered in other bacteria and showed how the Listeria enzymes are uniquely fitted to the intracellular lifestyle of this significant human pathogen.

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.

Type 2 diabetes mellitus (T2DM) is characterized by impaired glucose-stimulated insulin secretion and increased peripheral insulin resistance. Unremitting endoplasmic reticulum (ER) stress can lead to beta-cell apoptosis and has been linked to type 2 diabetes. Although many studies have attempted to link ER stress and T2DM, the specific effects of ER stress on beta-cell function remain incompletely understood. To determine the interrelationship between ER stress and beta-cell function, here we treated insulin-secreting INS-1(832/13) cells or isolated mouse islets with the ER stress–inducer tunicamycin (TM). TM induced ER stress as expected, as evidenced by activation of the unfolded protein response. Beta cells treated with TM also exhibited concomitant alterations in their electrical activity and cytosolic free Ca2+ oscillations. As ER stress is known to reduce ER Ca2+ levels, we tested the hypothesis that the observed increase in Ca2+ oscillations occurred because of reduced ER Ca2+ levels and, in turn, increased store-operated Ca2+ entry. TM-induced cytosolic Ca2+ and membrane electrical oscillations were acutely inhibited by YM58483, which blocks store-operated Ca2+ channels. Significantly, TM-treated cells secreted increased insulin under conditions normally associated with only minimal release, e.g. 5 mm glucose, and YM58483 blocked this secretion. Taken together, these results support a critical role for ER Ca2+ depletion–activated Ca2+ current in mediating Ca2+-induced insulin secretion in response to ER stress.

Numerous zinc ectoenzymes are metalated by zinc and activated in the compartments of the early secretory pathway before reaching their destination. Zn transporter (ZNT) proteins located in these compartments are essential for ectoenzyme activation. We have previously reported that ZNT proteins, specifically ZNT5–ZNT6 heterodimers and ZNT7 homodimers, play critical roles in the activation of zinc ectoenzymes, such as alkaline phosphatases (ALPs), by mobilizing cytosolic zinc into these compartments. However, this process remains incompletely understood. Here, using genetically-engineered chicken DT40 cells, we first determined that Zrt/Irt-like protein (ZIP) transporters that are localized to the compartments of the early secretory pathway play only a minor role in the ALP activation process. These transporters included ZIP7, ZIP9, and ZIP13, performing pivotal functions in maintaining cellular homeostasis by effluxing zinc out of the compartments. Next, using purified ALP proteins, we showed that zinc metalation on ALP produced in DT40 cells lacking ZNT5–ZNT6 heterodimers and ZNT7 homodimers is impaired. Finally, by genetically disrupting both ZNT5 and ZNT7 in human HAP1 cells, we directly demonstrated that the tissue-nonspecific ALP-activating functions of both ZNT complexes are conserved in human cells. Furthermore, using mutant HAP1 cells, we uncovered a previously-unrecognized and unique spatial regulation of ZNT5–ZNT6 heterodimer formation, wherein ZNT5 recruits ZNT6 to the Golgi apparatus to form the heterodimeric complex. These findings fill in major gaps in our understanding of the molecular mechanisms underlying zinc ectoenzyme activation in the compartments of the early secretory pathway.

T-type (Cav3) Ca2+ channels are important regulators of excitability and rhythmic activity of excitable cells. Among other voltage-gated Ca2+ channels, Cav3 channels are uniquely sensitive to oxidation and zinc. Using recombinant protein expression in HEK293 cells, patch clamp electrophysiology, site-directed mutagenesis, and homology modeling, we report here that modulation of Cav3.2 by redox agents and zinc is mediated by a unique extracellular module containing a high-affinity metal-binding site formed by the extracellular IS1–IS2 and IS3–IS4 loops of domain I and a cluster of extracellular cysteines in the IS1–IS2 loop. Patch clamp recording of recombinant Cav3.2 currents revealed that two cysteine-modifying agents, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) and N-ethylmaleimide, as well as a reactive oxygen species–producing neuropeptide, substance P (SP), inhibit Cav3.2 current to similar degrees and that this inhibition is reversed by a reducing agent and a zinc chelator. Pre-application of MTSES prevented further SP-mediated current inhibition. Substitution of the zinc-binding residue His191 in Cav3.2 reduced the channel's sensitivity to MTSES, and introduction of the corresponding histidine into Cav3.1 sensitized it to MTSES. Removal of extracellular cysteines from the IS1–IS2 loop of Cav3.2 reduced its sensitivity to MTSES and SP. We hypothesize that oxidative modification of IS1–IS2 loop cysteines induces allosteric changes in the zinc-binding site of Cav3.2 so that it becomes sensitive to ambient zinc.

Pathogenic bacteria of the genera Mycobacterium and Corynebacterium cause severe human diseases such as tuberculosis (Mycobacterium tuberculosis) and diphtheria (Corynebacterium diphtheriae). The cells of these species are surrounded by protective cell walls rich in long-chain mycolic acids. These fatty acids are conjugated to the disaccharide trehalose on the cytoplasmic side of the bacterial cell membrane. They are then transported across the membrane to the periplasm where they act as donors for other reactions. We have previously shown that transient acetylation of the glycolipid trehalose monohydroxycorynomycolate (hTMCM) enables its efficient transport to the periplasm in Corynebacterium glutamicum and that acetylation is mediated by the membrane protein TmaT. Here, we show that a putative methyltransferase, encoded at the same genetic locus as TmaT, is also required for optimal hTMCM transport. Deletion of the C. glutamicum gene NCgl2764 (Rv0224c in M. tuberculosis) abolished acetyltrehalose monocorynomycolate (AcTMCM) synthesis, leading to accumulation of hTMCM in the inner membrane and delaying its conversion to trehalose dihydroxycorynomycolate (h2TDCM). Complementation with NCgl2764 normalized turnover of hTMCM to h2TDCM. In contrast, complementation with NCgl2764 derivatives mutated at residues essential for methyltransferase activity failed to rectify the defect, suggesting that NCgl2764/Rv0224c encodes a methyltransferase, designated here as MtrP. Comprehensive analyses of the individual mtrP and tmaT mutants and of a double mutant revealed strikingly similar changes across several lipid classes compared with WT bacteria. These findings indicate that both MtrP and TmaT have nonredundant roles in regulating AcTMCM synthesis, revealing additional complexity in the regulation of trehalose mycolate transport in the Corynebacterineae.

Contact between inflammatory cells and endothelial cells (ECs) is a crucial step in vascular inflammation. Recently, we demonstrated that the cell-surface level of endomucin (EMCN), a heavily O-glycosylated single-transmembrane sialomucin, interferes with the interactions between inflammatory cells and ECs. We have also shown that, in response to an inflammatory stimulus, EMCN is cleared from the cell surface by an unknown mechanism. In this study, using adenovirus-mediated overexpression of a tagged EMCN in human umbilical vein ECs, we found that treatment with tumor necrosis factor α (TNF-α) or the strong oxidant pervanadate leads to loss of cell-surface EMCN and increases the levels of the C-terminal fragment of EMCN 3- to 4-fold. Furthermore, treatment with the broad-spectrum matrix metalloproteinase inhibitor batimastat (BB94) or inhibition of ADAM metallopeptidase domain 10 (ADAM10) and ADAM17 with two small-molecule inhibitors, GW280264X and GI254023X, or with siRNA significantly reduced basal and TNFα-induced cell-surface EMCN cleavage. Release of the C-terminal fragment of EMCN by TNF-α treatment was blocked by chemical inhibition of ADAM10 alone or in combination with ADAM17. These results indicate that cell-surface EMCN undergoes constitutive cleavage and that TNF-α treatment dramatically increases this cleavage, which is mediated predominantly by ADAM10 and ADAM17. As endothelial cell-surface EMCN attenuates leukocyte–EC interactions during inflammation, we propose that EMCN is a potential therapeutic target to manage vascular inflammation.

Objective:

GPR40 is a G protein-coupled receptor regulating free fatty acid-induced insulin secretion. We have generated transgenic mice overexpressing the human GPR40 gene (hGPR40-Tg) under control of the mouse insulin II promoter and have used them to examine the role of GPR40 in the regulation of insulin secretion and glucose homeostasis.

Nonalcoholic fatty liver disease (NAFLD) is a risk factor for type 2 diabetes (T2D). We aimed to identify the peripheral blood DNA methylation signature of hepatic fat. We conducted epigenome-wide association studies of hepatic fat in 3,400 European ancestry (EA) participants and in 401 Hispanic ancestry and 724 African ancestry participants from four population-based cohort studies. Hepatic fat was measured using computed tomography or ultrasound imaging and DNA methylation was assessed at >400,000 cytosine-guanine dinucleotides (CpGs) in whole blood or CD14+ monocytes using a commercial array. We identified 22 CpGs associated with hepatic fat in EA participants at a false discovery rate <0.05 (corresponding P = 6.9 x 10^–6) with replication at Bonferroni-corrected P < 8.6 x 10^–4. Mendelian randomization analyses supported the association of hypomethylation of cg08309687 (LINC00649) with NAFLD (P = 2.5 x 10^–4). Hypomethylation of the same CpG was also associated with risk for new-onset T2D (P = 0.005). Our study demonstrates that a peripheral blood–derived DNA methylation signature is robustly associated with hepatic fat accumulation. The hepatic fat–associated CpGs may represent attractive biomarkers for T2D. Future studies are warranted to explore mechanisms and to examine DNA methylation signatures of NAFLD across racial/ethnic groups.

Glucagon secretion is regulated by circulating glucose, but it has turned out that amino acids also play an important role, and that hepatic amino acid metabolism and glucagon are linked in a mutual feed-back cycle, the liver-alpha cell axis. On this background, we hypothesized that hepatic steatosis might impair glucagon’s action on hepatic amino acid metabolism and lead to hyperaminoacidemia and hyperglucagonemia.

We subjected 15 healthy lean and 15 obese steatotic male participants to a pancreatic clamp with somatostatin and evaluated hepatic glucose and amino acid metabolism during basal and high physiological levels of glucagon. The degree of steatosis was evaluated from liver biopsies.

Total RNA sequencing of liver biopsies revealed perturbations in the expression of genes predominantly involved in amino acid metabolism in the obese steatotic individuals. This group was also characterized by fasting hyperglucagonemia, hyperaminoacidemia and an absent lowering of amino acid levels in response to high levels of glucagon. Endogenous glucose production was similar between lean and obese individuals.

Our results suggest that hepatic steatosis causes resistance to the effect of glucagon on amino acid metabolism resulting in increased amino acid concentrations as well as increased glucagon secretion providing a likely explanation of fatty liver-associated hyperglucagonemia.

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.

To ensure fetal lipid supply, maternal blood triglyceride (TG) concentrations are robustly elevated during pregnancy. Interestingly, a lower increase in maternal blood TG concentrations has been observed in some obese mothers. We have shown that high-fat (HF) feeding during pregnancy significantly reduces maternal blood TG levels. Therefore, we performed this study to investigate if and how obesity alters maternal blood TG levels. Maternal obesity was established by prepregnant HF feeding (ppHF), which avoided the dietary effect during pregnancy. We found that maternal blood TG concentrations in ppHF dams were not only remarkably lower than control dams, but the TG peak occurred earlier during gestation. Hepatic TG production and intestinal TG absorption were unchanged in ppHF dams, but systemic lipoprotein lipase (LPL) activity was increased, suggesting that increased blood TG clearance contributes to the decreased blood TG concentrations in ppHF dams. Although significantly higher levels of UCP1 protein were observed in iBAT of ppHF dams, Ucp1 gene deletion did not restore blood TG concentrations in ppHF dams. Expression of the angiopoietin-like protein 4 (ANGPTL4), a potent endogenous LPL inhibitor, was significantly increased during pregnancy. However, the pregnancy-induced elevation of blood TG was almost abolished in Angptl4^-/- dams. Compared with control dams, Angptl4 mRNA levels were significantly lower in iBAT, gWAT and livers of ppHF dams. Importantly, ectopic overexpression of ANGPTL4 restored maternal blood TG concentrations in ppHF dams. Together, these results indicate that ANGPTL4 plays a vital role in increasing maternal blood TG concentrations during pregnancy. Obesity impairs the rise of maternal blood TG concentrations by reducing ANGPTL4 expression in mice.

Despite considerable progress, development of glucose-responsive insulins (GRI) still largely depends on empirical knowledge and tedious experimentation – especially on rodents. To assist the rational design and clinical translation of the therapeutic, we present a Pharmacokinetic Algorithm Mapping GRI Efficacies in Rodents and Humans (PAMERAH), built upon our previous human model. PAMERAH constitutes a framework for predicting the therapeutic efficacy of a GRI candidate from its user-specified mechanism of action, kinetics, and dosage, which we show is accurate when checked against data from experiments and literature. Results from simulated glucose clamps also agree quantitatively with recent GRI publications. We demonstrate that the model can be used to explore the vast number of permutations constituting the GRI parameter space, and thereby identify the optimal design ranges that yield desired performance. A design guide aside, PAMERAH more importantly can facilitate GRI’s clinical translation by connecting each candidate’s efficacies in rats, mice, and humans. The resultant mapping helps find GRIs which appear promising in rodents but underperform in humans (i.e. false-positives). Conversely, it also allows for the discovery of optimal human GRI dynamics not captured by experiments on a rodent population (false-negatives). We condense such information onto a translatability grid as a straightforward, visual guide for GRI development.

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.