bluuebunny/crossref_metadata_2025 huggingface dataset:
The Great Recession and international banking reforms
Authors: Keller Eileen
Published: June 2021

This chapter discusses the French and German participation in international banking reform in the aftermath of the Great Recession. It begins with a brief overview of the origins and the spread of the global financial crisis. It then spells out key elements of the G20 reform agenda on banking regulation and it discusses the implications of bank capital requirements as well as liquidity and leverage ratios for different types of bank activities. The chapter moves on to analyse how representatives from France and Germany participated in the negotiations on Basel III and its transposition in the EU by the CRR-CRDIV reform package, studying the priorities identified in both countries. In a next step, the French and the German priorities are compared to those of representatives from the United States and the United Kingdom, given their more strongly market-based financial systems.
The French and the German financial crisis
Authors: Keller Eileen
Published: June 2021

This chapter analyses how French and German banks were affected by the crisis and how the crisis unfolded in both countries. It compares the governments’ responses and reconstructs how the crisis was diagnosed. The chapter shows that surprisingly different dynamics developed in both countries, despite an initially rather similar situation. Whereas the financial crisis became a veritable crisis of bank intermediation in the way it was widely perceived in France, in Germany it was primarily considered as a crisis of Anglo-Saxon finance capitalism and the failure of individual banks’ business models that had drifted away from traditional bank lending. While the French concluded that alternatives to bank intermediation should be developed, the Germans focused on strengthening classic bank intermediation. The chapter traces the dynamics that brought about the differing reform agendas and spells out the coalitions that supported corresponding political reforms.
The nature of crisis responses
Authors: Keller Eileen
Published: June 2021

The final chapter concludes by discussing the policy implications of the findings for the future management of the financial sector and banking crises. It highlights major developments in banking since the financial crisis and discusses the nature of the political responses to it. The insights from the previous chapters relate to the selective reconstruction of the crisis and its consequences for political reform, competitive concerns, competing policy aims, and a bias of contemporary capitalist democracies towards the promise of future growth. Given the limits of financial regulation and the expectation of future bank bailouts, the chapter encourages a broadening of the fora in which financial knowledge is constructed.
Materials and Methods
Authors: Lyman R. Lee
Published: June 2021

To determine the origin of archaeological spindle graphs, and to track the frequency of use of each of several types of graph used to diagram culture change, a sample of North American archaeological literature was examined. Numerous series of monographs and volumes of journals in both the archaeological and the paleontological literature were inspected. If a graph of biological (paleontological) or cultural (archaeological) change was included in a publication, that piece of literature was recorded along with the type of graph included. To record such data, a classification of graph types was developed based on categories of statistical graphs (e.g., bar graph, line graph, pie graph, time range, spatio-temporal rectangle). More than 900 pieces of literature on North American archaeology published between ~1880 and ~1960 were inspected, and more than 450 pieces of literature on paleontology were inspected. Because different graph types are constructed under different guidelines, they require an understanding of graph grammar—the rules for constructing, deciphering, and interpreting graphs.
Combatants, Non-Combatants, and the Question of Innocence
Authors: Coady C. A. J.
Published: June 2021

Chapter 4 tackles issues surrounding the concept of combatant/non-combatant and the related notions of guilt/innocence, and the connection of these to the soldier/civilian distinction. The investigation is partly conceptual, but it also inevitably raises moral questions and their significance, since the tactical definition’s reliance upon such concepts relates immediately to the moral assessments enshrined in the just war principle of discrimination, which prohibits the direction of lethal violence at non-combatants and reflects a wider moral principle that prohibits violence against the innocent. Whether one or both of these principles should be rejected, modified, or allow of exceptions are further questions addressed in Chapters 5 and 6. The present chapter requires extended discussion of contemporary debates within the complex just war tradition, particularly between those loosely styled “traditionalist” and “revisionist.” It offers a judgment on the debate and discusses its relation to the author’s account of the nature of terrorist acts.
Religion, War, and Terrorism
Authors: Coady C. A. J.
Published: June 2021

Chapter 8 is concerned with common views affirming strong links between religion and terrorist acts. One such view is that religion has an inherent, distinctive tendency to promote war and terrorist acts; another is that many past and present wars and terrorist acts were in fact wholly or primarily caused by religious commitments; another is that religion is unusual in inevitably promoting particularly bad features of war and terrorism, such as their ferocity and duration. The chapter argues that these common views oversimplify and often exaggerate the importance of religious elements in violent conflicts. Not only are positive aspects of religious traditions condemning wrongful resort to violence neglected, but political and non-religious ideologies that can drive war and terrorism also tend to be ignored. The chapter examines arguments by a range of theorists, both those in favor of the strong links between religion and violence and those against.
Introducing the Transformative Self
Authors: Bauer Jack
Published: June 2021

This chapter introduces the main features of the transformative self—what it is and is not. For instance, the transformative self is not a person but, rather, a self-identity that a person uses to facilitate personal growth. The person creates a transformative self primarily in their evolving life story. This growth-oriented narrative identity helps the person cultivate growth toward a good life for the self and others. The chapter provides an overview of the book’s theoretical approach and topics. The book’s first section examines the components of personal growth, narrative identity, and a good life that culturally characterize the transformative self. The second section explores the personality and social ecology of the person who has a transformative self. The third section shows how the transformative self develops over time. The final section explores the hazards and heights of having a transformative self.
Growth and a Good Life Story
Authors: Bauer Jack
Published: June 2021

This chapter integrates theories of the good life and the life story, showing how the two phenomena scaffold each other into existence—and form a good life story. The chapter first examines concepts of time, narrative, change, development, and growth. Metaphors that suggest growthiness are listed, but growth proper emerges in narrative meaning-making. The chapter provides an overview of how the facets of value are shaped and conveyed via narrative tone, theme, and structure. Tone conveys whether an event turns out well subjectively (i.e., value fulfillment). Theme conveys why an event is subjectively important (i.e., value orientation). Structure conveys the objective degrees of value perspectivity by which events, tones, and themes are interpreted. Like the three facets of value, narrative tone, theme, and structure combine to convey the four superordinate categories of a good life story—happiness, love, wisdom, and growth—toward which a transformative self aims.
Growth in the Hard and Soft Margins of Society
Authors: Bauer Jack
Published: June 2021

Not everyone is in the same position to live out their culture’s master narratives of a good life. This chapter explores the transformative self in the margins of society, which the chapter divides into hard and soft margins. The hard margins involve demographic categories—such as socioeconomic status, race, ethnicity, sexuality, and gender—that accompany systematic forms of prejudice, discrimination, unfreedoms, and other hindrances to eudaimonic growth. Whereas people are thrust into the hard margins, the soft margins are sought or chosen by people who reject the materialistic and mechanistic values of mainstream society and wish to pursue eudaimonic growth. The bildungsroman, as a cultural master narrative for the transformative self, maps pathways of growth often in the soft margins but in the hard margins too. The chapter concludes by examining two master narratives of progress—upward mobility versus personal growth—notably in the hard margins of race.
Jasmonate regulates the FAMA/mediator complex subunit 8-THIOGLUCOSIDE GLUCOHYDROLASE 1 cascade and myrosinase activity
Authors: Feng Qingkai , Li Liping , Liu Yan , Shao Xingfeng , Li Xiaohui
Published: June 2021

Myrosinases are β-thioglucoside glucosidases that are unique to the Brassicales order. These enzymes hydrolyze glucosinolates to produce compounds that have direct antibiotic effects or that function as signaling molecules in the plant immune system, protecting plants from pathogens and insect pests. However, the effects of jasmonic acid (JA), a plant hormone that is crucial for plant disease resistance, on myrosinase activity remain unclear. Here, we systematically studied the effects of JA on myrosinase activity and explored the associated internal transcriptional regulation mechanisms. Exogenous application of JA significantly increased myrosinase activity, while the inhibition of endogenous JA biosynthesis and signaling reduced myrosinase activity. In addition, some myrosinase genes in Arabidopsis (Arabidopsis thaliana) were upregulated by JA. Further genetic and biochemical evidence showed that transcription factor FAMA interacted with a series of JASMONATE ZIM-DOMAIN proteins and affected JA-mediated myrosinase activity. However, among the JA-upregulated myrosinase genes, only THIOGLUCOSIDE GLUCOHYDROLASE 1 (TGG1) was positively regulated by FAMA. Further biochemical analysis showed that FAMA bound to the TGG1 promoter to directly mediate TGG1 expression in conjunction with Mediator complex subunit 8 (MED8). Together, our results provide evidence that JA acts as an important signal upstream of the FAMA/MED8–TGG1 pathway to positively regulate myrosinase activity in Arabidopsis.
Imbalance Model of Heart Rate Variability and Pulse Wave Velocity in Psychotic and Nonpsychotic Disorders
Authors: Zhang Tian Hong , Tang Xiao Chen , Xu Li Hua , Wei Yan Yan , Hu Ye Gang , Cui Hui Ru , Tang Ying Ying , Chen Tao , Li Chun Bo , Zhou Lin Lin , Wang Ji Jun
Published: June 2021

Objectives

Patients with psychiatric disorders have an increased risk of cardiovascular pathologies. A bidirectional feedback model between the brain and heart exists widely in both psychotic and nonpsychotic disorders. The aim of this study was to compare heart rate variability (HRV) and pulse wave velocity (PWV) functions between patients with psychotic and nonpsychotic disorders and to investigate whether subgroups defined by HRV and PWV features improve the transdiagnostic psychopathology of psychiatric classification.

Methods

In total, 3448 consecutive patients who visited psychiatric or psychological health services with psychotic (N = 1839) and nonpsychotic disorders (N = 1609) and were drug-free for at least 2 weeks were selected. HRV and PWV indicators were measured via finger photoplethysmography during a 5-minute period of rest. Canonical variates were generated through HRV and PWV indicators by canonical correlation analysis (CCA).

Results

All HRV indicators but none of the PWV indicators were significantly reduced in the psychotic group relative to those in the nonpsychotic group. After adjusting for age, gender, and body mass index, many indices of HRV were significantly reduced in the psychotic group compared with those in the nonpsychotic group. CCA analysis revealed 2 subgroups defined by distinct and relatively homogeneous patterns along HRV and PWV dimensions and comprising 19.0% (subgroup 1, n = 655) and 80.9% (subgroup 2, n = 2781) of the sample, each with distinctive features of HRV and PWV functions.

Conclusions

HRV functions are significantly impaired among psychiatric patients, especially in those with psychosis. Our results highlight important subgroups of psychiatric patients that have distinct features of HRV and PWV which transcend current diagnostic boundaries.
Evaluation of non-invasive bioforensic techniques for determining the age of hot-iron brand burn scars in cattle
Authors: Tolleson Douglas R , Schafer David W
Published: June 2021

Absract

Hot-iron branding is a traditional form of permanent cattle identification in the United States. There is a need for science-based determination of cattle brand age. Near infrared reflectance spectroscopy (NIRS) has been used to obtain information about animal tissues and healing processes. Height-width allometry and NIRS were applied to hot-iron cattle brand scars to determine if either or both of these methods can be used to non-invasively establish the interval sincethe application of hot-iron cattle brands. Length and width of a brand routinely applied to calves (~30–60 d old) were established and then the same measurements were recorded on 378 calfhood branded cattle of known age ranging from 0.5 to > 6.5 yr-of-age. Brand width and height increased over the original measurements by > 100% between calfhood application and 2.5 yr-of-age (P < 0.001). Brand size did not change dramatically between 2.5 and > 6.5 yr, however, both width and height were (P < 0.05) greater at maturity than at weaning. Near infrared spectra were collected from a) branded skin b) non-clipped (hair), non-branded skin, and c) hair clipped, non-branded skin on Bos taurus cross calves. Individual trial calibrations yielded high R2 and low SE of calibration values as well as similar cross validation performance (P < 0.001). Numerically lower but still strong performance (P < 0.001) resulted from combined data set calibrations. Cross-trial prediction of brand age was unsuccessful. One single year calibration underpredicted (P < 0.001) brand age of an independent validation set by 2.83 d, and another single year calibration underpredicted (P < 0.001) the same validation set by 9.91 d. When combined, these two datasets resulted in a calibration that overpredicted brand age in the validation set by 6.9 d (P < 0.02). Discriminant analyses for identification of skin surface type yielded success rates of 90% for branded, 99% for non-clipped, non-branded, and 96% for clipped, non-branded (P < 0.01). Discriminant analyses were also performed on samples grouped into a) less than 33 d, b) 141–153 d, and c) 169 d categories. All group membership identifications were successful at greater than 90% (P < 0.01). Preliminary results indicate that brand size could be used to indicate brand age and that NIRS can predict brand age as well as discriminate between broad brand age groups in cattle. More work will need to be done before these techniques can be used in real-world forensic applications.
Identification and Fine-Mapping of a Novel QTL, qMrdd2, That Confers Resistance to Maize Rough Dwarf Disease
Authors: Xu Zhennan , Wang Feifei , Zhou Zhiqiang , Meng Qingchang , Chen Yanping , Han Xiaohua , Tie Shuanggui , Liu Changlin , Hao Zhuanfang , Li Mingshun , Zhang Degui , Han Jienan , Wang Zhenhua , Li Xinhai , Weng Jianfeng
Published: June 2021

Maize rough dwarf disease (MRDD) is caused by a virus and seriously affects maize quality and yield worldwide. MRDD can be most effectively controlled with disease-resistant hybrids of corn. Here, MRDD-resistant (Qi319) and -susceptible (Ye478) parental inbred maize lines and their 314 recombinant inbred lines (RILs) that were derived from a cross between them were evaluated across three environments. A stable resistance QTL, qMrdd2, was identified and mapped using best linear unbiased prediction (BLUP) values to a 0.55-Mb region between the markers MK807 and MK811 on chromosome 2 (B73 RefGen_v3) and was found to explain 8.6 to 11.0% of the total phenotypic variance in MRDD resistance. We validated the effect of qMrdd2 using a chromosome segment substitution line (CSSL) that was derived from a cross between maize inbred Qi319 as the MRDD resistance donor and Ye478 as the recipient. Disease severity index of the CSSL haplotype II harboring qMrdd2 was significantly lower than that of the susceptible parent Ye478. Subsequently, we fine-mapped qMrdd2 to a 315-kb region flanked by the markers RD81 and RD87, thus testing recombinant-derived progeny using selfed backcrossed families. In this study, we identified a novel QTL for MRDD resistance by combining the RIL and CSSL populations, thus providing important genetic information that can be used for breeding MRDD-resistant varieties of maize.
Screening of an Ethyl Methane Sulfonate Mutagenized Population of a Wheat Cultivar Susceptible to Fusarium Head Blight Identifies Resistant Variants
Authors: Chhabra Bhavit , Singh Lovepreet , Wallace Sydney , Schoen Adam , Dong Yanhong , Tiwari Vijay , Rawat Nidhi
Published: June 2021

Fusarium head blight (FHB) primarily caused by Fusarium graminearum is a key disease of small grains. Diseased spikes show symptoms of premature bleaching shortly after infection and have aborted or shriveled seeds, resulting in reduced yields. The fungus also deteriorates quality and safety of the grain because of production of mycotoxins, especially deoxynivalenol (DON), which can result in grain being docked or rejected at the point of sale. Genetic host resistance to FHB is quantitative, and no complete genetic resistance against this devastating disease is available. Alternative approaches to develop new sources of FHB resistance are needed. In this study, we performed extensive forward genetic screening of the M4 generation of an ethyl methane sulfonate-induced mutagenized population of cultivar Jagger to isolate variants with FHB resistance. In field testing, 74 mutant lines were found to have resistance against FHB spread, and 30 of these lines also had low DON content. Subsequent testing over 2 years in controlled greenhouse conditions revealed 10 M6 lines showing significantly lower FHB spread. Seven and 6 of those 10 lines also had reduced DON content and fewer Fusarium-damaged kernels, respectively. Future endeavors will include identification of the mutations that led to resistance in these variants.
Sensitivity of Colletotrichum fructicola and Colletotrichum siamense of Peach in China to Multiple Classes of Fungicides and Characterization of Pyraclostrobin-Resistant Isolates
Authors: Usman Hafiz Muhammad , Tan Qin , Karim Mohammad Mazharul , Adnan Muhammad , Yin Wei-Xiao , Zhu Fu-Xing , Luo Chao-Xi
Published: June 2021

Anthracnose, mainly caused by Colletotrichum gloeosporioides species complex including Colletotrichum fructicola and Colletotrichum siamense, is a devastating disease of peach. Chemical control has been widely used for years, but management failures have increased with the commonly used fungicides. Therefore, screening of sensitivity of Colletotrichum spp. to fungicides with different modes of action is needed to make proper management strategies for peach anthracnose. In this study, the sensitivity of 80 isolates of C. fructicola and C. siamense was screened for pyraclostrobin, procymidone, prochloraz, and fludioxonil based on mycelial growth inhibition at discriminatory doses. Results showed that C. fructicola and C. siamense isolates were highly resistant to procymidone and fludioxonil with 100% resistance frequencies to both fungicides, but sensitive to prochloraz, i.e., no resistant isolates were found. For pyraclostrobin, 74% of C. fructicola isolates showed high resistance, 26% showed low resistance, and all of the C. siamense isolates showed low resistance. No positive cross-resistance was observed between pyraclostrobin and azoxystrobin even when they are members of the same quinone outside inhibitor (QoI) fungicide group or between pyraclostrobin and non-QoIs. Resistant isolates to QoI fungicides were evaluated for the fitness penalty. Results showed that no significant differences except for the mycelial growth rates that were detected between high- and low-resistance isolates of C. fructicola. Molecular characterization of the Cyt b gene revealed that the G143A point mutation was the determinant of the high resistance in C. fructicola. This study demonstrated the resistance status of C. fructicola and C. siamense to different fungicides and briefly discussed implications of that resistance. Demethylation inhibitor fungicides were found to be the best option among the different chemicals studied here, to control peach anthracnose in China.
Elevated hepatic fatty acid elongase‐5 (Elovl5) attenuates fatty liver in high fat diet induced obese mice
Authors: Tripathy Sasmita , Stevens Robert D , Bain James R , Newgard Christopher B , Jump Donald B
Published: June 2021

Fatty liver develops in obese individuals because of an imbalance in hepatic triglyceride (TG) synthesis, secretion, hydrolysis and fatty acid oxidation (FAO). High fat diets induce obesity and fatty liver in C57BL/6J mice and lower hepatic C20–22 PUFA and fatty acid elongase‐5 (Elovl5), a key enzyme involved in PUFA synthesis. The role Elovl5 plays in fatty liver was assessed by using a recombinant adenoviral approach to induce hepatic Elovl5 expression in fasted obese mice. Elevated Elovl5 expression increased hepatic C20–22 PUFA, acetyl‐carnitine and several long chain (C14–20) fatty acyl carnitines as well as plasma β‐hydroxybutyrate. This treatment also decreased hepatic TG content, perilipin and ER stress markers, i.e., XBP‐1, ATF6α. Analysis of enzymes involved in TG hydrolysis and FAO revealed that elevated Elovl5 activity increased expression of ATGL, CGI58 and CTP2, but not other TG lipases or enzymes involved in FAO. Elevated Elovl5 also decreased the nuclear abundance of PPARγ2, but not other transcription factors controlling fatty acid and TG synthesis, i.e., SREBP1 & ChREBP, or enzymes involved in VLDL assembly, i.e., MTTP. Thus, increased hepatic C20–22 PUFA content in response to elevated Elovl5 activity attenuated fatty liver by increasing TG hydrolysis and FAO.
Metformin attenuates cardiomyocyte hypertrophy through mitochondria: reciprocal interaction between AMPK and angiotensin II type 1 receptor
Authors: Soto‐Hernández Jessica , Javadov Sabzali
Published: June 2021

Activation of AMP‐kinase (AMPK) reduces cardiac hypertrophy although molecular mechanisms underlying this effect remain unclear. We examined the role of the AMPK/eNOS/p53 pathway in the antihypertrophic action of metformin (Met) in cell hypertrophy induced by angiotensin II (AngII). H9c2 cardioblasts were treated with AngII for 24h in the presence of the AMPK agonist, Met, the AngII type 1 receptor (AT1R) blocker, lasartan, the pan‐NOS inhibitor, Nω‐nitro‐L‐arginine methyl ester (L‐NAME) or the SIRT1 inhibitor, splitomicin. AngII increased AT1R expression and AMPK phosphorylation by 38% (P<0.05) and 20% (P>;0.05), respectively, compared to control. Upregulation of AT1R was prevented by lasartan that was associated with further phosphorylation of AMPK by 54% (P<0.05). Likewise, Met increased AMPK phosphorylation by 99% (P<0.01) and reduced AT1R levels by 30% (P<0.03) in the presence of AngII. Met also enhanced p‐eNOS levels by 3.3 folds (P<0.01) and prevented AngII‐induced upregulation of p53. Both L‐NAME and splitomicin decreased AT1R expression in AngII‐treated cells. Beneficial effects of Met converged on mitochondria that exhibited high membrane potential (Δψm). We concluded that antihypertrophic effects of Met were associated with the AT1R downregulation preventing mitochondrial dysfunction through the eNOS/SIRT1/p53 pathway. Supported by the grant SC1HL118669 from NHLBI NIH.
Using in‐line probing to characterize pathogenic mutations in human mitochondrial tRNAs
Authors: Still Brady , Her Koua , Chihade Joseph
Published: June 2021

In‐line probing (Regulski, E.E. and Breaker, R.R., (2008) Methods Mol. Biol. 419, 53) provides a straightforward method for probing RNA structure. We have used this technique to characterize the human mitochondrial tRNAAla and four tRNA mutants associated with mitochondrial pathologies, including MERFF (myoclonic epilepsy with ragged red fibers), ophthalmoplegia, and mitochondrial myopathy. Our data suggests that two of these mutants, C5650T and A5628G, adopt abnormal secondary structures at room temperature, which may lead to reduced aminoacylation levels. This work was supported by Carleton College and the Howard Hughes Medical Institute.
Tissue culture of the Egyptian cotton cultivars: Production and morphological heterogeneity of primary callus tissues
Authors: Younis Hassan Mahmoud , Badawy Mohamed El‐Taher Ibrahim , Darrag Hossam Moustafa Salem
Published: June 2021

The success in applying new technologies to improve cotton cultivars, G. barbadense, has been limited. Here we report results that show an increase in somatic embryogenesis efficiency of four Egyptian cotton cultivars; Giza70, Alexandria4, Bahtem101 and Hairy Free. Attempts to form calluses from roots and stem parts of the above cultivars were not successful, whereas, the use of cotyledon and hypo‐cotyledon tissues produced by the embryos of the seeds have been successful. Seeds were activated by inclusion of Verticilium dahlia in the growing media. High callus frequencies and embryogenic tissues were developed. Morphology of callus tissues has been determined using stereoscopic microscope. Four types of primary callus tissues were identified in primary culture of hypocotyls and cotyledons of the four cotton cultivars. Using the above protocol, we could produce plantlets from the above mentioned cultivars that reached up to 29.2, 28.2, 20.4 and 18.5% of their starting somatic embryos, respectively. The above results pave the way for future transformation experiments of cotton plants.
The effect of conformational changes on non‐coordinated water occupancy associated with R and T allosteric states of Carp Hemoglobin
Authors: Salameh Wes , Padilla Emelia , Tipgunlakant Pooncharas N. , Goldbeck Robert A. , Kliger David S.
Published: June 2021

The chemistry of non‐coordinated water molecules in the apolar internal environment of proteins is poorly understood. Our recent discovery of a spectral marker for the entry of noncoordinated water into the distal heme pocket of myoglobin has helped clarify this molecule's functional importance in ligand binding. We will apply our novel spectrokinetic assay to test the hypothesis that conformational changes can affect ligand binding indirectly by changing the occupancy of non‐coordinated water in apolar cavities. A comparison of the spectrokinetics between the visible and Soret spectral regions distinguishes protein relaxations from those associated with changes in internal water occupancy. Hemoglobin from Carp, Cyprinus carpio, shows differences in the putative water occupancy signal between the R and T allosteric states, which correspond to changes in ligand binding dynamics. These results suggest that conformational induced changes in water occupancy may play a role in allosteric induced changes in ligand binding dynamics. This knowledge will increase the understanding of the role these water molecules play in allosteric regulation.
The role of the C‐terminus and transmembrane segments in facilitating atlastin‐mediated endoplasmic reticulum fusion
Authors: Liu Tina Yu , Bian Xin , Sun Sha , Hu Xiaoyu , Klemm Robin W , Prinz William A , Rapoport Tom A , Hu Junjie
Published: June 2021

Generation of the tubular endoplasmic reticulum (ER) network requires homotypic membrane fusion. This is mediated in metazoans by atlastin (ATL), a dynamin‐like GTPase that consists of an N‐terminal cytosolic domain followed by two transmembrane segments (TMs) and a C‐terminal tail (CT). A GTP‐hydrolysis‐induced conformational change in the N‐terminal cytosolic domain is required for fusion, but it is unclear if this alone is sufficient for the fusion reaction. Here, we show using in vitro fusion assays that the CT and TMs are required for efficient fusion. A conserved amphipathic helix in the CT promotes fusion by interacting with and perturbing the lipid bilayer. The TMs not only serve as membrane anchors but also mediate ATL oligomerization. Point mutations in the CT or the TMs also impair ATL's ability to maintain ER membrane morphology in vivo. Our results suggest that protein‐lipid and protein‐protein interactions in the membrane cooperate with the conformational change of the cytosolic domain to achieve fusion and maintain the tubular ER network. These findings are relevant to mechanisms used by other membrane fusion proteins, such as mitofusins/Fzo1p, viral fusogens, and SNAREs.
The Basis of the Substrate Specificity of the Epsilon Isoform of Human Diacylglycerol Kinase is not a Consequence of Competing Hydrolysis of ATP
Authors: Epand Richard M. , Prodeus Aaron , Berno Bob , Topham Matthew K.
Published: June 2021

The diacylglycerol kinase from E. coli transfers some of the γ‐phosphate of ATP to water as well as to diacylglycerol. We also demonstrate that glycerol can act as an acceptor for the phosphate of ATP. We have compared this behavior with that of the only mammalian isoform of diacylglycerol kinase that exhibits acyl chain specificity, i.e. DGKε. The purpose of the study was to determine if differences in the competition between ATPase activity and lipid phosphorylation could contribute to the observed acyl chain specificity with different diacylglycerols. Neither with the highly specific substrate of DGKε, 1‐stearoyl‐2‐arachidonoyl glycerol, nor with a less specific substrate, 1‐stearoyl‐2‐linoleoyl glycerol, is there any evidence for ATP hydrolysis accompanying substrate phosphorylation. Thus, at least for this isoform of diacylglycerol kinase, water does not compete with diacylglycerol as an acceptor of the γ‐phosphate of ATP. The results demonstrate that the substrate specificity of mammalian DGKε is not a consequence of different degrees of ATP hydrolysis in the presence of different species of diacylglycerol.

This work was supported by a grant from the Natural Sciences and Engineering Council of Canada, 9848.
Phospholipase Cβ1 is Linked to RNA interference of Specific Genes through Translin‐Associated Factor X
Authors: Scarlata Suzanne , Philip Finly , Guo Yuanjian
Published: June 2021

Phospholipase Cβ1 (PLCβ1) is a G protein regulated enzyme whose activity results in proliferative and mitogenic changes in the cell. We have found that PLCβ1 binds to the RNA processing protein Translin‐Associated factor X (TRAX) and that this binding competes with G proteins. Here, we show that endogenous PLCβ1 and TRAX interact in SK‐N‐SH cells and in HEK293 cells. In HEK293 cells, TRAX over‐expression ablates Ca2+ signals generated by G protein‐PLCâ1 activation. TRAX plays a key role in protein down‐regulation by small, interfering RNA. PLCβ1 overexpression completely reverses down‐regulation of GAPDH by siRNA in HEK293 and HeLa cells as seen by recovery in both the transcript and protein levels. Also, down‐regulation of endogenous PLCβ1 in HEK293 and HeLa cells increases siRNA(GAPDH) silencing. While PLCβ1 overexpression reverses cell death caused by siRNA(LDH), it does not affect cell survival or silencing of other genes (e.g. cyclophilin, Hsp90, translin). PLCβ1 overexpression in HEK293 and HeLa cells reduces the total amount of small RNAs. LDH and GAPDH are part of a complex that promotes H2B synthesis allowing progression through the S phase. We find PLCβ1 reverses the cell death and completely rescues H2B levels caused by siRNA knock‐down of LDH or GAPDH. Taken together, our study shows a novel role of PLCβ1 in gene regulation through TRAX association. Support‐NIH GM053132.
Vac14 multimerization is required for Fab1 complex assembly and function
Authors: Ho Shannon Cheuk Ying , Alghamdi Tamadher , Botelho Roberto J
Published: June 2021

Phosphatidylinositol‐3,5‐bisphosphate (PtdIns3,5P2) is a poorly understood regulator of endolysosome function. In yeast, PtdIns3,5P2 is synthesized by the Fab1 PtdIns3P 5‐kinase and degraded by the antagonistic Fig4 phosphatase. Interestingly, Fab1 and Fig4 exist in a common protein complex containing Vac14. Vac14 is a self‐interacting protein necessary for Fab1 complex assembly. However, the exact function of Vac14 self‐interaction is not known, which we explore here. Size‐exclusion chromatography and velocity ultracentrifugation suggest that the Vac14 multimer is non‐globular and exists as a homodimer and/or homotrimer. Moreover, using sequential Vac14 truncations, site‐directed mutagenesis and co‐immunoprecipitation studies with both yeast and recombinant proteins, we identified several motifs in the C‐terminal half of Vac14 that are necessary for self‐interaction. Monomeric Vac14 mutants do not support interaction with Fab1 and Fig4, and consequently, did not rescue phenotypic defects in vac14Δ. Together, these results suggest that Vac14 multimerization is necessary and is the first step in the formation of the Fab1 complex and the synthesis of PtdIns3,5P2. Funded by NSERC.
Membrane ultrastructure modulates immune cell signaling
Authors: Pralle Arnd , Huang Heng , Simsek Muhammed
Published: June 2021

Membrane lipids modulate the cell membrane ultra‐structure by promoting nanocluster formation, and by influencing the coupling of the membrane to the cytoskeleton. These in turn modulate the cell membrane signaling. Direct observation of the interplay of signaling and lipid nanodomains is challenging due to their small size and dynamic nature (bimFCS), Monte‐Carlo‐Simulations and optical trap based thermal noise imaging (TNI), to characterize the biophysics of the lipid nanodomains Combining total internal fluorescence microscopy with fluorescence correlation spectroscopy into bimFCS, we are able to measure the diffusion of membrane receptors on multiple length scales simultaneously. We characterize the receptor‐nanodomain interaction continuously on single cells while perturbing the cell through receptor dimerization. I will show how bimFCS measurements, combined with defined TNI to confine a single membrane protein to diffuse for seconds in a small area provides sufficient data for high resolutions maps of local diffusion, local attraction potentials and membrane stiffness. Using a GPI‐anchored protein to probe the cell membrane we detect domains of increased membrane stiffness, which also show increased viscosity and are the preferred location for the GPI‐anchored protein.
Fatty Acids and their Thioester Derivatives as Potential Endogenous Ligands of LXRα
Authors: Hines Genesis Victoria , Kaliappan Alagammai , Rider S. Dean , Hostetler Heather A.
Published: June 2021

The Liver X Receptor Alpha (LXRα) is a ligand‐activated nuclear transcription factor that plays a role in lipid homeostasis. Upon binding of oxysterols, LXRα is activated and regulates the transcription of target genes involved in these processes. Additionally, LXRα can form heterodimers with the Peroxisome Proliferator Activated Receptor Alpha (PPARα), another ligand‐activated nuclear receptor involved in fatty acid (FA) metabolism. Previous studies have shown that PPARα binds FAs and their thioester derivatives (FA‐CoAs); however, little is known about the effects of these ligands on the activity of LXRα. Therefore, the objective of this study was to characterize endogenous ligand binding of LXRα to FAs and FA‐CoAs. Using circular dichroism, we were able to characterize changes in the spectra of LXRα in the presence or absence of FAs and FA‐CoAs, which suggests a novel role for these nutrients in the activation of LXRα. Additionally, docking experiments were done to examine the binding affinities of these potential ligands. Our results help further elucidate the pathways by which LXRα is activated and regulates its target genes. This work was supported by USPHS NIH grant DK77573.
Lipid binding properties of Ebola virus matrix protein VP40
Authors: Yong Sylvia , Scott Jordan L. , Stahelin Robert V.
Published: June 2021

The Ebola virus (EBOV) is a pathogen that causes hemorrhagic fevers in humans. It is most prevalent in Africa, where outbreaks with fatality rates of up to 90% have occurred. It also poses a global threat as infections can be spread rapidly. While it is known that EBOV infections function by inhibiting the immune system, the specific cellular secretion pathway of the virus has not been uncovered. As no treatment for this deadly virus exists, it is important to learn how EBOV co‐opts infected cells in order to pharmacologically target the formation and egress of new virions. VP40, one of the seven structural proteins that EBOV encodes, is essential for initiation of new virion formation at the plasma membrane. In this study, vesicular sedimentation assays were used to better understand the lipid binding properties of VP40. In addition, surface plamon resonance (SPR) was used to determine the binding specificity of the wild type VP40 and mutant VP40 A299W. VP40 WT and VP40 A299W bind phosphatidylserine (PS), a plasma membrane phospholipid, with equal affinity. VP40 is essential for EBOV cellular egress and the mechanisms by which it is targeted to the plasma membrane and subsequently recruits the virus capsid are under further investigation.

This research is supported by the NIH (AI081077).
Targeting of a mating machinery ATPase to the cell membrane
Authors: Broulidakis Matthew Peter , Laurer Stephanie J. , Berkmen Melanie B.
Published: June 2021

Conjugation or mating is the transfer of DNA between cells through the mating machinery. The DNA translocation channel is composed of several proteins located in the cell membrane. ConE is one of the proteins that make up the mating machinery of the conjugative element ICEBs1 of Bacillus subtilis. ConE fused to green fluorescent protein (ConE‐GFP) localizes to the membrane preferentially at the cell poles. One or more ICEBs1 proteins are required to recruit ConE to the membrane, as ConE‐GFP mislocalizes to the cytoplasm in the absence of ICEBs1. Fluorescence microscopy was used to analyze ConE‐GFP's localization in strains deleted for single ICEBs1 genes that encode putative membrane proteins. Prior studies indicate that the ICEBs1 membrane protein ConB is required for proper targeting of ConE‐GFP. Here, we investigated ConE‐GFP localization in a strain deleted for the mating gene conD. While a portion of ConE‐GFP was observed at the membrane in the conD strain, a significant amount was mislocalized within the cytoplasm. We hypothesize that ConB and ConD interact with and recruit ConE to the cell membrane. This research was supported by an NSF‐RUI grant to MBB.
Cyclin dependent kinase 5′s role in the development of both insulin‐positive and neuronal phenotypes
Authors: Khost Daniel Emerson , Aguanno Ann
Published: June 2021

Cyclin dependent kinase 5 (CDK5) is a member of the cyclin dependent kinase family that is unusual in that it is predominately active in terminally differentiated neurons. CDK5 has been implicated in Alzheimer's Disease (AD), where its dysregulation contributes to the formation of neurofibrillary tangles. Recently CDK5 has been associated with insulin exocytosis in the pancreas. An emerging correlation between Type II diabetes and AD suggests a connection between insulin dysregulation and neurodegeneration. As CDK5 is active in both tissues, it represents a possible common feature in these two pathologies. To study this, our lab models the nervous and pancreatic systems using PC12 and AR42J cell lines, respectively. We transdifferentiate our cells using growth factors, then assess the extent of differentiation via qualitative and quantitative methods in the presence or absence of CDK5 chemical inhibitors. For our neuronal model, we quantify morphological changes using digital photomicroscopy and CDK5 activity using kinase assays. In our pancreatic model, we evaluate transdifferentiation through RT‐PCR analysis of insulin gene expression and immunochemistry assays for insulin secretion. Our results indicate that aberrant CDK5 activity contributes both to alterations in the insulin positive phenotype and neuronal architecture, thus making CDK5 a potential common link between AD and Type II diabetes.

Funding from the US Dept. of Education, the Rose M Badgely Charitable Trust, and ASBMB Summer Undergraduate Research Award
Control of entry into meiosis and quiescence in budding yeast
Authors: Sarkar Sourav , Millar Jonathan , Arumugam Prakash
Published: June 2021

How cells sense changes in environment and mount a response is a fundamental question in biology. Nutrition starvation in budding yeast has been an useful model to study this biological trait. When yeast cells are deprived of essential nutrients they can either enter a quiescent state (referred to as G0) or enter meiosis to form spores that can stay dormant for long periods of time. Precisely how environmental signals are sensed by yeast cells to trigger quiescence/meiosis is not known. Signals from multiple nutrient‐sensing signal transduction pathways converge on a serine‐threonine kinase called Rim15 which integrates the signals to orchestrate entry into quiescence or gametogenesis. In this we have identified a signalling pathway (REP) consisting of a serine‐threonine kinase Rim15, c‐AMP protein Igo1 and protein phosphatase PP2ACdc55 which regulates entry into both quiescence and meiosis. PP2ACdc55 negatively regulates entry into meiosis and G0. On nutrient starvation, Rim15 becomes active and phosphorylates endosulfines at Serine‐64 to convert it into an inhibitor of PP2ACdc55 thereby promoting entry into meiosis and G0. Remarkably a similar pathway controls entry into mitosis in mammalian cells.
AKAP7 Regulates CaM Kinase Activation in MCF‐7 Cells
Authors: McFarland Hannah M. , Dodge‐Kafka Kimberly , Schmitt John M.
Published: June 2021

Estrogen (E2) activates calcium/calmodulin‐dependent protein kinases (CaM Kinases) in MCF‐7 breast cancer cells. In particular E2 activates a CaM KK, CaM KI, and ERK pathway to promote proliferation. CaM Kinase activation of ERK may be blocked by PKA in certain cell types through direct phosphorylation and inhibition of CaM KK. The ability of PKA to phosphorylate its cellular targets may be dictated by protein kinase A anchoring proteins (AKAPs). Hormones that elevate cAMP and activate PKA may utilize AKAPs to regulate signal transduction. Our goal was to evaluate the role of AKAPs in regulating E2 activation of the CaM KK and CaM KI pathway in MCF‐7 cells. We also examined the ability of vitamin D (VitD) working through cAMP and PKA to block CaM KK signaling in breast cancer cells. Our results suggest that E2 activates CaM KK and CaM KI within 5 minutes. VitD promoted PKA‐dependent phosphorylation of CaM KK. VitD and epinephrine treatment of cells triggered a potent increase in cAMP levels. Interestingly, purified GST‐RII pulled down both CaM KK and CaM KI. Similarly, purified AKAP7 but not AKAP5 bound CaM KK and CaM KI an effect that is enhanced with E2. Endogenous AKAP7 and CaM KK associated in E2‐stimulated but not in VitD‐treated cells and VitD also blocked CaM KK activation in MCF‐7 cells. Our results suggest that VitD blocks E2 activation of CaM Kinases and their association with AKAP7 in MCF‐7 cells.
The Bcl‐2 family members Mcl‐1 and Bim modulate apoptosis in glutamine‐deprived Sp2/0‐Ag14 mouse cells
Authors: Gauthier Eric R. , Zhou Catherine , Woodley Jenna , Harnett Curtis
Published: June 2021

In highly proliferating cells, metabolic reprogramming results in a cellular dependence toward L‐glutamine (Gln), a phenomenon of marked therapeutic interest. However, the molecular events linking Gln to cell survival are still poorly defined. Our group has shown that Sp2/0‐Ag14 (Sp2/0) cells are acutely sensitive to Gln starvation, undergoing apoptosis via the intrinsic pathway after only 2 hours of Gln deprivation. The current study examined the relative contribution of proteins of the Bcl‐2 family in this phenomenon. Immunoblotting revealed that Mcl‐1, Bax, Bim and Puma are the major Bcl‐2 family proteins expressed in Sp2/0 cells. While Sp2/0 cells with an RNAi‐induced decrease in Puma levels behaved like the wild‐type cell line, reducing Bim expression led to a significant increase in cell survival following Gln deprivation. Western blot analysis revealed that Gln starvation did not affect Bim levels, but did lead to a reduction in Mcl‐1. We found that Sp2/0 cells are highly sensitive to obatoclax, a Mcl‐1‐specific chemical inhibitor. Furthermore, obatoclax increased the sensitivity of Sp2/0 cells to glutamine starvation, an effect prevented by the constitutive expression of Bcl‐xL (which is not targeted by the drug). All together, our data place Mcl‐1 and Bim at the core of the apoptotic machinery triggered by Gln starvation in Sp2/0 cells. Funded by NSERC‐CANADA (to ERG).
Pasteurella multocida toxin (PMT) activates mTOR and inhibits PP2A via Gαq/11/PLCβ/PKC
Authors: Oubrahim Hammou , Shi Yilan , Chock P, Boon
Published: June 2021

PMT, a potent mitogen, activates several signaling pathways via deamidation of a conserved Gln residue in the α subunit of G‐proteins. However, its detailed mechanism is unknown. We show that PMT induces cell proliferation in serum‐starved Swiss 3T3 cells. Concomitantly PMT induces phosphorylation of ribosomal S6 kinase (S6K1) and its substrate, S6 protein (rpS6). The PMT mediated phosphorylation is inhibited by rapamycin and Torin1, two mTORC1 specific inhibitors. The mTOR activation occurs in MEF WT but not in Gαq/11 knockout cells, consistent with the notion that PMT‐induced mTORC1 activation proceeds via the deamidation of Gαq/11 leading to the activation of PLCβ to generate diacylglycerol (DAG) and inositol trisphosphate (IP3), two activators of PKC pathway. PMT‐induced rpS6 phosphorylation is inhibited by PKC inhibitor, Gö 6976. The prolonged mTOR activation may involve inhibition of phosphatases. We show that PMT treatment leads to the phosphorylation of the protein phosphatase type 2 (PP2A) at Y307 and inactivates it. PMT‐mediated PP2A phosphorylation is inhibited by rapamycin and Gö 6976, and occurs in MEF WT but not in Gαq/11 knockout cells. Together, our findings reveal that PMT activates mTORC1 through Gαq/11/PLCβ/PKC pathway leading, by an unknown mechanism, to phosphorylation/inhibition of PP2A. This research was supported by the Intramural Research Program of NHLBI, NIH.