bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of fictional characters in the context of artificial intelligence and big data: taking A Dream of Red Mansions for example
Authors: Yuan Ying
Published: June 2021

This article uses artificial intelligence and big data to process the dialogs of the characters in A Dream of Red Mansions through Chinese psychoanalysis system, and obtains the prediction scores of the characters’ big-five personality. Then, the validity of this method is determined by examining the verification of the prediction scores by literature and fiction. Through the ecological identification and analysis of the personality characteristics in A Dream of Red Mansions, we find that Bao-Yu Jia in the novel has obvious openness, agreeableness, and extroversion; Dai-Yu Lin has very low extroversion, strong emotionality, and agreeableness; and Bao-Chai Xue has strong extroversion, agreeableness, and conscientiousness. The above prediction results are supported by relevant research literature and the novel itself. This shows that in the context of artificial intelligence and big data, the prediction of fictional characters’ personalities through literary intelligence analysis is helpful for us to understand the humanistic spirit or social idea behind literary works in a deeper and objective way. This research method is a beneficial supplement to the traditional literature and art research.
“Well Then, Carry On”: Piercing Recalcitrant History in LaShonda Katrice Barnett’sJam on the Vine
Authors: McKible Adam
Published: June 2021

In her 2015 novel, Jam on the Vine, LaShonda Katrice Barnett dramatizes the opposed but intertwined histories of African American modernity and US racial revanchism during the Jim Crow era. While the trajectory of her female protagonist, Ivoe Williams, can be understood as a progress narrative, Barnett explores the corrosive nature of deepening American segregation and the pervasiveness of anti-black racism through the plight of her black male characters, particularly Ivoe’s father and brother. Through these men’s experiences, we see the persistent exploitation of black male bodies, which has continued from slavery through Jim Crow and on to the twenty-first-century prison-industrial complex. This essay attends to Barnett’s development of two inverse narrative arcs: first, Ivoe’s growth, which embodies the rising consciousness and agency of the African American activists and journalists who challenged racial segregation domestically and internationally, and second, the African American male characters, who must endure the ever-increasing pressures of Jim Crow during the same period. Ultimately, Barnett connects these narrative arcs by drawing on two literary-historical forebears, Ida B. Wells and Ernest Hemingway, to create a protagonist who learns how to “carry on” and thrive during the seemingly unending era of legal inequality.
Investigation of the Relationship Between Frequency of Blast Exposure, mTBI History, and Post-traumatic Stress Symptoms
Authors: Lieb David A , Raiciulescu Sorana , DeGraba Thomas , Sours Rhodes Chandler
Published: June 2021

Introduction

Post-traumatic stress disorder (PTSD) and mild traumatic brain injury (mTBI) are common conditions among military personnel that frequently co-occur. This study investigated relationships between self-reported blast exposure, mTBI history, and current post-traumatic stress (PTS) symptoms in a population of active duty service members (n = 202) from the Intensive Outpatient Program at the National Intrepid Center of Excellence.

Materials and Methods

Participants were divided into four mTBI groups (0, 1, 2, and 3+) and four blast exposure groups (0-10, 11-100, 101-1,000, and 1,000+). Self-reported lifetime mTBI and blast history were obtained via the Ohio State University TBI Identification Method. PTSS severity was obtained via the PTSD Checklist-Civilian version (PCL-C). Several secondary measures of depression, anxiety, chronic mTBI symptoms, and sleep were also assessed.

Results

The total PCL-C scores differed significantly between mTBI groups, with significant differences detected between the 0/1 mTBI groups and the 3+ mTBI groups. Similar group differences were noted across the three PCL-C subgroup scores (avoidance, re-experiencing, and hyperarousal); however, when comparing the proportion of group participants meeting DSM-IV criteria for each symptom cluster, significant differences between mTBI groups were only noted for avoidance (P = .002). No group differences were noted in PTS symptom severity or distribution between blast groups.

Conclusions

This study demonstrates an association between lifetime mTBI history and PTS symptom severity and distribution but failed to identify the significant group in self-reported symptoms between the blast exposure groups. Results suggest that additional research is needed to understand the neurobiological mechanism behind these associations and the need for the development of precise assessment tools that are able to more accurately quantify significant lifetime sub-concussive and blast exposures experienced by service members in training and combat operations.
Extirpation of Recurrent Petrous Apex Cholesterol Granuloma Through the Zygomatic Approach: 2-Dimensional Operative Video
Authors: Eisenberg Mark , Ibn Essayed Walid , Al-Mefty Ossama
Published: June 2021

Petrous apex cholesterol granulomas are believed to result from blockage of the normal aeration of the petrous air cells, resulting in a repetitive cycle of mucosal engorgement, hemorrhage, and granuloma formation.
1
The lesion usually progressively expands causing compressive symptoms. The thick granulomatous wall envelopes various ages of breakdown products, including a cholesterol-containing fluid, which is typically hyperintense on T1 and T2 weighted magnetic resonance imaging. Drainage procedures, regardless of the route (endoscopic, endonasal, or transtemporal), with or without stenting or marsupialization, will only temporarily drain this cholesterol-containing fluid, with consequently frequent recurrences.
2-5
A total exoneration of the granuloma and obliteration of the cavity with vascularized tissue will assure a more durable outcome.
1
The extradural zygomatic/middle fossa approach provides a short distance to the petrous apex and is purely extradural. By sectioning the zygoma, temporal lobe retraction is avoided.
6
We present a case of a 29-yr-old male who presented in the year 2000 with progression of a left petrous apex cholesterol granuloma despite 2 previous drainage and stenting procedures.

 The patient consented for surgery and photo publication.

Images in video at 2:41 © JNSPG, republished from Eisenberg et al
1
with permission.
Sexuality and Bilingualism
Authors: Cashman Holly R.
Published: June 2021

Research on sexuality and bilingualism from a sociocultural linguistic approach can and should encompass not only the diverse language practices of queer bilingual and multilingual people, but also a wide range of language contexts and a broad spectrum of research participants beyond non-heteronormative subjects. This chapter explores questions relating to language, sexuality, and bilingualism. It aims to show the quantity and scope of recent research on sexuality and bilingualism, as well as highlight areas that may warrant further research. The chapter attributes the complexity of the concepts of bilingualism and sexuality in part to migration and globalization as well as evolving understandings of identity and desire. Bilingual (and multilingual) speakers are discussed in the context of urban multilingual settings and more specifically LGBTQ communities within those settings. The chapter then features a reflection on what major issues and opportunities might arise from joining research on sexuality with research on bilingualism.
Aging and Chronic Illness in Language and Sexuality
Authors: Peel Elizabeth , Ellis Sonja J.
Published: June 2021

An aging demographic in Western societies as well as globally has made public health issues, such as dementia, subject to hyperbolic metaphor such as “tsunami” and “time bomb.” This chapter reviews the state of knowledge regarding language, sexualities, aging, and chronic illness. In particular, the discussion focuses on discursive research from across the social sciences that furthers understandings of older people’s lives and experiences. The chapter highlights research that has focused on ageism and chronic conditions impacting older people (specifically, dementia and type 2 diabetes), including empirical research on these conditions, and on manifestations of heterosexism and heteronormativity in these contexts. Using illustrative examples that emphasize the intersection of discourse and issues that relate to aging, the chapter foregrounds this area as an important element of language and sexuality scholarship. Last, future directions for the development of research focusing on these topics are indicated.
Administrative Justice in Street-Level Decision-Making
Authors: Raaphorst Nadine
Published: June 2021

Street-level bureaucrats’ discretionary powers play an increasingly important role in public service provision and law enforcement. In order to deal with societal challenges, legislators and policy-makers leave room for professional judgment by formulating open laws, rules, and policies. In making responsive decisions, however, that is, when treating different cases differently, street-level bureaucrats do not necessarily attach less value to treating similar cases alike. This chapter discusses how two notions of fairness—treating similar cases alike and treating dissimilar cases differently—are studied in street-level bureaucracy literature, and sheds light on the factors that influence how bureaucrats behave in this regard. Subsequently, it is explored how street-level bureaucrats could enhance equality of treatment when rules run out. The chapter concludes with an agenda for future research.
Implementing Digitalisation in an Administrative Justice Context
Authors: Raso Jennifer
Published: June 2021

Administrative agencies have long been sites of technological innovation. Today, government officials worldwide are intensifying digitalization efforts to cut costs and to make bureaucratic operations more efficient. This article examines how digitalization initiatives are implemented in administrative settings, using examples from the United Kingdom (Universal Credit), Canada (Ontario’s Social Assistance Management System), and Australia (Online Compliance Initiative, a.k.a. ‘Robodebt’). It draws on qualitative research, government reports, and administrative justice literature to illustrate the dilemmas common to digital government projects. For example, digitalization both hardens and virtualizes the interface between officials and the public, while obscuring the vast amounts of human labour needed to maintain digital government initiatives. To function well, digital systems require deep integration between government databases and software. Yet, the process of digitalization is often piecemeal, continuous, and reproduces dilemmas that arise whenever new technologies are used to solve institutional problems. Consequently, the promised benefits of ‘digital by default’ initiatives are rarely realized. Digitalization accelerates a shift in relations between people and the state that administrative justice scholars must take seriously. First, scholars must reconsider the internal perspective from which administrative justice theories assess an outcome’s acceptability. Digitalization compels the development of new justice models centred on the values of system users within and outside of administrative institutions. Second, scholars must reassess administrative justice theory’s procedural focus. In digitalized settings, ‘administratively just’ decision-making processes may generate substantively unjust outcomes. These challenges must be addressed if administrative justice theories are to remain relevant in an age of algorithmically-driven decision-making.
Revelation in Heidegger
Authors: Fritz Peter Joseph
Published: June 2021

Martin Heidegger provides positive impetus for fresh thinking on divine revelation. Objections could immediately be raised. While it is contested whether Heidegger observes some sort of ‘methodological atheism’, at the very least he demotes theology—serious thinking based on belief in God—as ‘ontic’ (occasional, region-specific), whereas philosophy enjoys ‘ontological’ status. Heidegger refuses the revealed idea of creation as a distortive axiom for Western thinking that prepares the way for the world’s modern, technological framing. And of course there is Heidegger’s political bias, a concern that has reignited with the publication of his Schwarze Hefte. Nevertheless, this chapter’s primary thesis holds that Heidegger can help to reinvigorate Christian understanding of divine revelation in at least three respects: (1) by centring the theology of revelation on the allied themes of fundamental truth and freedom, (2) by encouraging theologians to continue pursuing renewed interest in apocalyptic, and (3) by bringing to light the revelatory character of inconspicuous, everyday phenomena.
Time and Temporality of Change Processes
Authors: Hernes Tor , Hussenot Anthony , Pulk Kätlin
Published: June 2021

Theories of organizational change tend to consider episodic and continuous change to be ontologically incompatible. We discuss how their assumed incompatibility stems from different conceptions of time. The main contribution of this chapter is to demonstrate how, by taking an event-based view, the two types of change may be integrated with one another. Continuous change takes place in the present as events in the making, whereas episodic change is marked by events in the past or as projected upon the future. This endogenous view of time as events “from within” enables the ontological gap between episodic and continuous change to be bridged. It enables us to understand how actors evoke previous episodic changes or project future episodic changes while pursuing continuous change in the on-going present. Also, in this view, continuous and episodic change become seen as two intertwined dimensions of interplay along what we call an immanent temporal trajectory.
On Critical Localism and the Privatisation of Refuge: The Resettlement of Syrian Newcomers in Canada
Authors: Ilcan Suzan , Connoy Laura
Published: June 2021

Increases in displacement and forced migration is an enduring feature of many countries. Resettlement is a policy response to displacement, that relocates refugees from a country of asylum to a safe third country. Canada’s Private Sponsorship of Refugees Program is noteworthy. It allows non-profit organizations and volunteer groups to support newcomers during their first year in Canada and has especially aided resettlement of Syrian refugees on an international scale. We take a critical look at this programme by focusing on the social implications of private sponsorship and Syrian newcomers’ experiences of resettlement. We view private sponsorship initiatives as furthering processes that privatise decision-making, identify specific sponsorship groups as objects of policy, and transfer public authority to private citizens and non-profit organizations to encourage refugee resettlement. We argue that the privatising processes defining private sponsorship are further complicated within localised settings. Based on scholarly, policy, and programme documents, and extensive semi-structured interviews with Syrian newcomers in southern Ontario, Canada, we illuminate what we call “localising the privatisation of refuge,” which calls attention to the various networks, activities and relations of power that define and shape the local, and the processes and experiences of refuge that take place within.
Treating insomnia symptoms with medicinal cannabis: a randomized, crossover trial of the efficacy of a cannabinoid medicine compared with placebo
Authors: Walsh Jennifer H , Maddison Kathleen J , Rankin Tim , Murray Kevin , McArdle Nigel , Ree Melissa J , Hillman David R , Eastwood Peter R
Published: June 2021

Study Objectives

This randomized, double-blind, placebo-controlled, crossover study was conducted to evaluate the safety and efficacy of 2 weeks of nightly sublingual cannabinoid extract (ZTL-101) in treating chronic insomnia (symptoms ≥3 months).

Methods

Co-primary study endpoints were safety of the medication based on adverse event reporting and global insomnia symptoms (Insomnia Severity Index [ISI]). Secondary endpoints included: self-reported (sleep diary), actigraphy-derived, and polysomnography measurements of sleep onset latency (SOL), wake after sleep onset (WASO), total sleep time (TST), sleep efficiency (SE); and self-reported assessments of sleep quality (sSQ) and feeling rested upon waking. Adjusted mean differences between placebo and ZTL-101 were calculated.

Results

Twenty-three of 24 randomized participants (n = 20 female, mean age 53 ± 9 years) completed the protocol. No serious adverse events were reported. Forty mild, nonserious, adverse events were reported (36 during ZTL-101) with all but one resolving overnight or soon after waking. Compared to placebo, ZTL-101 decreased ISI (−5.07 units [95% CI: −7.28 to −2.86]; p = 0.0001) and self-reported SOL (−8.45 min [95% CI: −16.33 to −0.57]; p = 0.04) and increased self-reported TST (64.6 min [95% CI: 41.70 to 87.46]; p < 0.0001), sSQ (0.74 units [95% CI: 0.51 to 0.97]; p < 0.0001), and feeling of being rested on waking (0.51 units [95% CI: 0.24 to 0.78]; p = 0.0007). ZTL-101 also decreased actigraphy-derived WASO (−10.2 min [95% CI: −16.2 to −4.2]; p = 0.002), and increased actigraphy-derived TST (33.4 min [95% CI: 23.07 to 43.76]; p < 0.001) and SE (2.9% [95% CI: 2.0 to 3.8]; p = 0.005).

Conclusions

Two weeks of nightly sublingual administration of a cannabinoid extract (ZTL-101) is well tolerated and improves insomnia symptoms and sleep quality in individuals with chronic insomnia symptoms.

Clinical Trial

ANZCTR; anzctr.org.au; ACTRN12618000078257.
Identification of Resistant Germplasm and Detection of Genes for Resistance to Powdery Mildew and Leaf Rust from 2,978 Wheat Accessions
Authors: Jin Yuli , Shi Fengyu , Liu Weihua , Fu Xiaoyi , Gu Tiantian , Han Guohao , Shi Zhipeng , Sheng Yuan , Xu Hongxing , Li Lihui , An Diaoguo
Published: June 2021

Powdery mildew and leaf rust, caused by Blumeria graminis f. sp. tritici and Puccinia triticina, respectively, are widespread diseases of wheat worldwide. The use of resistant cultivars is considered the most economical, environment-friendly, and effective method to control these diseases. In the present study, a collection of 2,978 wheat accessions consisting of 1,394 advanced breeding lines, 1,078 Chinese cultivars, 291 introduced cultivars, 132 lines containing alien chromosomes, and 83 landraces was tested for reactions to powdery mildew and leaf rust. The results indicated that 659 wheat accessions (22.1%) were highly resistant to a widely prevalent B. graminis f. sp. tritici isolate, E09, at the seedling stage, and 390 were consistently resistant to the mixture of B. graminis f. sp. tritici isolates at the adult plant stage. Meanwhile, 63 accessions (2.1%) were highly resistant to leaf rust at the adult plant stage, of which 54 were resistant to a predominant and highly virulent P. triticina race, THTT, at the seedling stage. Notably, 17 accessions were resistant to both powdery mildew and leaf rust. To detect known genes for resistance to powdery mildew and leaf rust, these accessions were tested with gene-specific or tightly linked markers for seven powdery mildew genes (Pm genes; Pm2, Pm4, Pm5, Pm6, Pm8, Pm21, and Pm24) and 10 Lr genes (Lr1, Lr9, Lr10, Lr19, Lr20, Lr24, Lr26, Lr34, Lr37, and Lr46). Of the 659 powdery mildew-resistant accessions, 328 might carry single Pm genes and 191 carry combined Pm genes. Pm2 was detected at the highest frequency of 59.6%, followed by Pm8, Pm6, Pm21, Pm4, and Pm5, whereas Pm24 was not detected. In addition, 139 accessions might contain unknown Pm genes different from those tested in this study. In the 63 accessions resistant to leaf rust, four leaf rust genes (Lr genes; Lr1, Lr10, Lr26, and Lr34) were detected in 41 accessions singly or in combination, whereas six genes (Lr9, Lr19, Lr20, Lr24, Lr37, and Lr46) were not detected. Twenty-two accessions might contain unknown Lr genes different from those tested in this study. This study not only provided important information for rationally distributing resistance genes in wheat breeding programs, but also identified resistant germplasm that might have novel genes to enrich the diversity of resistance sources.
Metabolomic profiling of the liver bearing human colon cancer metastasis in superimmunodeficient NOG mice
Authors: Ohmura Mitsuyo , Handa Kan , Nishime Chiyoko , Mizushima Tomoko , Kawai Kenji , Hishiki Takako , Yamamoto Takehiro , Wakui Masatoshi , Suemizu Hiroshi , Nakamura Masato , Suematsu Makoto
Published: June 2021

Hypoglycemia is one of the important events in paraneoplastic syndrome caused by solid tumors. Although metabolic feature of solid tumors characterized by Warburg effect has been thought to account for an important factor causing hypoglycemia, roles of responses of the host against tumor development remain largely unknown. In order to examine mechanisms by which tumor development causes hypoglycemia, we have performed biochemical assessment using established liver metastasis model with superimmunodeficient NOD/scid/γnull (NOG) mice transplanted with the mutant GFP expressing human‐derived colon cancer cells. The results of PCNA immunoreactivities, systemic hypoglycemia, decreased glycogen storage in the liver and impairment of alanine‐induced gluconeogenesis, as well as data from metabolome and flux analysis using hepatic parenchymal cells with mass‐labeled compounds such as 13C‐glucose, led us to suggest that, during cancer metastasis, regenerative responses of parenchymal cells requiring DNA synthesis promote large demand for utilization of carbohydrates and amino acid‐derived nitrogen, serving as an alternative mechanism causing hypoglycemia besides increased consumption of glucose in tumor cells by themselves.
Histamine and specific histamine receptors increase normal cholangiocyte growth via differential mechanisms
Authors: Francis Heather , Gaudio Eugenio , Ueno Yoshiyuki , Venter Julie , Onori Paolo , DeMorrow Sharon , Alpini Gianfranco
Published: June 2021

Normal cholangiocytes proliferate after injury. Histamine (HA) interacts with four receptors (H1–H4). We have shown that histamine receptors (HRs) induce differential effects in cholangiocytes. We evaluated: (i) HR expression, (ii) chronic effects of HA or specific HRs in normal rats (NR) and (iii) signaling mechanisms. HR expression was evaluated in cholangiocytes and cholangiocyte cultures (NRC). In vivo, NR were treated with HA or selected HR agonists: H1, HTMT dimaleate; H2, amthamine dihydrobromide; RAMH; H4, clobenpropit or NaCl. Cholangiocyte growth was evaluated by: (i) immunohistochemistry in liver sections and (ii) PCNA expression. NRIC were treated with HA or selected HR agonists. We measured growth, IP3 and cAMP levels and phosphorylation of PKA and ERK1/2. HRs are found in NR and NRC. Administration of HA or H1 and H2 (but not H3 or H4) to NR increased cholangiocyte growth compared to NR + NaCl. In vitro, HA and H1HR increased growth (via Ca2+signaling), IP3 and ERK1/2 phosphorylation. HA and H2HR stimulation increased growth (via PKA signaling), cAMP and phosphorylation of PKA and ERK 1/2. H3 and H4 had no effect. HA induces normal cholangiocyte growth. Activation of specific HRs modulates growth via independent mechanisms. Stimulatory (H1/H2) and inhibitory (H3/H4) effects by HRs may be important in the regulation of cholangiocyte growth/loss related to cholangiopathies.
Upregulation of heme oxygenase‐1 through activation of Nrf2 by the phytochemicals in Protandim
Authors: Reuland Danielle , McCord Joe , Hamilton Karyn
Published: June 2021

Increased production of reactive oxygen species has been implicated in the pathogenesis of cardiovascular disease (CVD), with enhanced endogenous antioxidants proposed as a potential mechanism for promoting redox balance. Protandim is a well‐defined combination of 5 widely studied medicinal plants derived from botanical sources [Bacopa monniera, Silybum marianum (milk thistle), Withania somnifera (Ashwagandha), Camellia sinensis (green tea), and Curcuma longa (turmeric)]. The purpose of this study was to determine if treatment of cardiomyocytes with Protandim induces phase II detoxification enzymes, including the endogenous antioxidant heme oxygenase‐1 (HO‐1), through activation of nuclear factor E2 p45‐related factor 2 (Nrf2), and if activation of this pathway offers protection from oxidative stress. In cultured HL‐1 cells, Protandim induced a significant increase in HO‐1, mediated through activation of Nrf2. Protandim supplemented cells were protected against hydrogen peroxide‐induced apoptosis as assessed by TUNEL (40% apoptotic in untreated vs. 16% apoptotic in Protandim treated). These findings support the use of Protandim as a potential method for upregulation of antioxidant defenses and protection of heart cells against an oxidative challenge. Future studies will focus on optimizing phytochemical induction of Nrf2‐mediated antioxidant defenses in relevant in vivo models of CVD.
Effect of Bosentan on Reductive Stress Induced Endothelin‐1/eNOS Dysfunction in Cardiomyocytes
Authors: Khanderao Gaytri Deepti , Davidson Christopher J , Varadharaj Saradhadevi , Benjamin Ivor J , Soorappan Rajasekaran Namakkal
Published: June 2021

Impairments of redox balance in cells and sub‐cellular structures can lead to either oxidative or reductive stress. While oxidative stress has many known, damaging effects, specific cellular and molecular responses to a reducing environment remain elusive. Reductive stress (RS) is defined as the supra‐physiological levels of intracellular reducing power (GSH/GSSG, NAD(P)H/NAD(P), Cysteine/Cystine). In mouse hearts exhibiting protein aggregation and RS (Rajasekaran et.al, Cell, 2007), transcriptional products for endothelin (ET) and its receptors (ET‐A/ET‐B) were significantly upregulated along with a profound decrease in the endothelial nitric oxide synthetase (eNOS). Using potential oxidizing/reducing agents, we have established oxidative/reductive stress models in HL‐1 cardiomyocytes in vitro. The redox state was determined by measuring the reduced/oxidized levels of glutathione (GSH/GSSG) and reactive oxygen species (ROS) using fluorescent probes. Effects of RS on cardiomyocytes hypertrophy (ANF/BNF), contractility and cell survival/death pathways were investigated. Results indicated significant impairment of Et‐1/eNOS system under RS condition, suggesting a direct role for RS on the regulation of Et‐1/eNOS function. Further, we have used bosentan, a specific antagonist/inhibitor of ET receptors, to rescue the changes associated with RS in HL‐1 cardiomyocytes.
Renal Cystic Proteins in the Olfactory Epithelium (OE)
Authors: Pluznick Jennifer L. , Rodriguez‐Gil Diego J. , Hull Michael , Mistry Kavita , Gattone Vincent , Johnson Colin , Weatherbee Scott , Greer Charles , Caplan Michael J.
Published: June 2021

In this study, we show that several proteins associated with renal cystic diseases – polycystin 1 (PC1), polycystin 2 (PC2), and Meckel‐Gruber syndrome 1 and 3 (MKS1, MKS3) – are found in the murine OE by RT‐PCR. Immunolocalization of PC1 and MKS3 in the OE revealed that PC1 is found in both dendritic knobs (from which cilia arise) and in cilia of mature olfactory neurons. Similarly, MKS3 localizes specifically to dendritic knob membranes. Rats with a disease‐causing mutation in MKS3 have reduced expression of key ciliary signaling proteins (olfactory adenylate cyclase, AC3, and the olfactory G protein). MKS1 mutant mice showed a similar reduction in AC3 expression. MKS3 mutant rats also demonstrated a decrease in acetylated α‐tubulin staining, indicating improper cilia formation and/or maintenance. This was confirmed by electron microscopy showing (a) a reduction in the number of OE cilia in mutant animals, and (b) malformation of the cilia which are present in the mutant animals (the majority of MKS3 mutant cilia had an improper number of doublets). In vitro data demonstrate that MKS3 co‐immunoprecipitates with individual olfactory receptors (ORs; 5 of the 5 ORs tested interact), implying that MKS3 may regulate OR expression/trafficking. These data show that multiple renal cystic proteins localize to the OE, where they may regulate specific aspects of the formation or physiological activities of cilia.
Large‐scale identification of phosphorylation sites in renal cortical transporters
Authors: Feric Marina , Hoffert J D , Pisitkun T , Knepper M A
Published: June 2021

Recent advances in mass spectrometry have provided means for large‐scale phosphoproteomic profiling of specific tissues. Here we report results from large‐scale LC‐MS/MS‐based phosphoproteomic profiling of biochemically isolated membranes from the renal cortex, focusing on transporters. Final data sets were filtered (target‐decoy) to limit false‐positive identifications to <2%. A total of 7,125 unique non‐phosphorylated and 744 unique phosphorylated peptides were identified. Over half of the phosphorylation sites identified on membrane transporters are novel. Phosphopeptides found included sites in Slc (n=63), Abc (n=4), and Aqp (n=3) family proteins. Most of the Slc family proteins are apical or basolateral transporters expressed in proximal tubule cells, including proteins known to mediate transport of glucose, amino acids, organic ions and inorganic ions. In addition, we also identified potentially important phosphorylation sites for transport proteins from other nephron segments, including NKCC2 and NCC. An online database from this study provides a resource for future studies of transporter regulation.
Apical Expression and Activity of Na‐Cl Cotransporters (NCC) in MDCK I TetOff Cells Does Not Require NCC Phosphorylation
Authors: Nguyen Mien T.X. , McDonough Alicia A. , Yu Alan S.L.
Published: June 2021

NCC is regulated in the renal DCT by trafficking between apical and subapical membranes. In NCC‐transfected oocytes and non‐epithelial cells, trafficking is regulated by the WNK4‐SPAK/OSR1 cascade leading to phosphorylation of NCC at Thr58 (NCCpT58). To investigate the connection between phosphorylation, trafficking and activity, we aimed to generate a stable renal epithelial cell line expressing apical NCC. MDCK I TetOff cells were transfected with rat NCC, then cultured to full confluency (7d) +/− Doxycycline (Dox). Dox+ had no NCC expression, while Dox− had robust expression by immunoblot with anti‐rat NCC. By immunofluorescence, NCC was expressed throughout the cell including apical membrane; additionally, a fraction of NCC was biotinylatable from the apical surface. There was no detectable NCCpT58. Thiazide‐sensitive 22Na+ uptake was detected in Dox− (not Dox+) and was not increased by acute low Cl− medium or Angiotensin II, suggesting that these responses may require NCCpT58. By RT‐PCR with canine kidney as + control: SPAK, OSR1, WNK1, WNK4, but not WNK3, were detected in transfected MDCK cells. In summary, a cell line has been generated that expresses active NCC in the apical membranes of MDCK cells, suggesting that apical localization and activity do not require NCCpT58. Activation by hypotonicity or AngII, lacking in these cells, may require reconstitution of a missing component. DK34316, DK062283.
Nedd4‐2 interacts with occludin to inhibit tight junction formation and enhance paracellular conductance in collecting duct epithelia
Authors: Raikwar Nandita , Thomas Christie P
Published: June 2021

Nedd4‐2, a E3 ubiquitin ligase, regulates epithelial sodium channel (ENaC) mediated transcellular Na+ transport in the collecting duct. We investigated the effect of Nedd4‐2 on the junctional complex and paracellular conductance in mpkCCDc14, a collecting duct cell line. We demonstrate that Nedd4‐2 co‐immunoprecipitated with and reduced the expression of occludin. This interaction was mediated via a conserved PY motif in the C‐terminus of occludin and mutation of this PY motif increased the half life of transfected occludin in HEK293 cells. We demonstrate that Nedd4‐2 ubiquitinates occludin which was not seen with a catalytically inactive form of Nedd4‐2. Overexpression of Nedd4‐2 in mpkCCDc14, reduced occludin expression at the tight junctions (TJs) and increased paracellular conductance consistent with a delay in the formation of TJs. Conversely, siRNA‐mediated knockdown of Nedd4‐2 increased occludin levels and reduced paracellular conductance. In conclusion, our data suggest that Nedd4‐2 interacts with occludin leading to its ubiquitination thereby modulating the assembly of TJs and the regulation of paracellular conductance in the collecting duct.

NHLBI and VA Merit
The circadian clock protein Period 1: a candidate master regulator of renal gene expression
Authors: Gumz Michelle L. , Lynch I. Jeanette , Greenlee Megan M. , Cain Brian D. , Wingo Charles S.
Published: June 2021

The goal of the present study was to define the role of the circadian clock protein Period 1 (Per1) in the regulation of renal epithelial transport, using a model of the cortical collecting duct (CCD). Per1 regulates expression of αENaC, the α subunit of the renal epithelial Na+ channel (Gumz et al. JCI 2009). To determine if Per1 regulates other genes important in renal epithelial transport, we performed a microarray study using mpkCCDc14 cells transfected with a non‐target siRNA or a Per1 specific siRNA. Following Per1 knockdown, 299 genes were differentially expressed (p<0.05). We focused on epithelial transporters and known regulators of epithelial transport. QPCR confirmed that Per1 knockdown resulted in decreased expression of the K+ channel Kcng1 and the ammonia transporter Rhcg. Suggesting a critical role for Per1 in the regulation of Na+ transport, Per1 knockdown led to increased expression of two inhibitors of ENaC activity, endothelin‐1 and Ube2e3. Fxyd5, a positive regulator of the Na,K‐ATPase, decreased following Per1 knockdown. These data suggest that the circadian clock protein Per1 is a candidate master regulator of genes involved in renal epithelial transport. Supported by NIH, AHA
Channel inhibitory region within the STAS domain of human SLC26A9
Authors: Chen AnPing , Romero Michael F
Published: June 2021

Slc26 gene family encodes multifunctional anion transport proteins: sensor, exchangers and channels. Structurally, Slc26 members have 10–14 predicted transmembrane spans followed by a cytoplasmic domain. This carboxy‐terminus contains a STAS (sulfate transporter anti‐sigma) domain whose function we have studied in mouse Slc26a9 (Chang et al, JBC 284: 28306, 2009). Mutations in SLC26 proteins lead to a number of diseases including diastrophic dysplasia, congenital chloride diarrhea, deafness and potentially Ca‐oxalate, kidney stones. While several groups have addressed the exchanger vs. channel nature of Slc26 proteins, elements within each Slc26 protein contributing to (or controlling) the anion transport character have not been probed. Interestingly, mapping the STAS domain conserved motifs reveals a hypervariable region. This hypervariable region ranges between 30 and 130 amino acids; no similarities exist between different Slc26 members. We made a SLC26A9 construct (Δloop), lacking this STAS subregion of SLC26A9. We expressed Δloop in Xenopus oocytes and monitored transport using ion selective microelectrodes and voltage clamping. SLC26A9 Δloop showed >5‐fold increased Cl− currents compared with the wild type SLC26A9 without obvious change in Cl−‐HCO3− exchange activity. We also made several SLC26A9 mutants to remove putative phosphorylation sites located in this loop: T620A, S621A, S623A, T626A and S628A(replacing T or S with A). All of these mutants show decreased (10–50%) Cl− currents compared with the wild type SLC26A9. These results suggest this variable loop contains a channel‐inhibitory motif and also phosphorylation sites maintaining basic SLC26A9 function. We hypothesize that this variable loop is a regulatory element collaborating with conserved STAS domain regions and that this structural interaction may confer specific transport modes for SLC26A9 and possibly other SLC26 proteins.
Genistein's Mechanism(s) Of Action On Intestinal Chloride Secretion In Mice
Authors: Sears Steven , Skinner Ryan , Batia Lyn , Al‐Nakkash Layla
Published: June 2021

The major route for cAMP‐mediated chloride (Cl) secretion across the intestinal epithelial apical membrane is presumed to be via CFTR, a chloride channel widely known to be activated in vitro by genistein. We have shown that daily injections of genistein (600mg/kg body weight, 600G) mediate significant increases in intestinal Cl secretion (Isc) in male mice after 2 weeks and female mice after one week, when compared to control (0G). This study aimed to determine whether genistein‐induced increases in intestinal Cl secretion were attributed to; (a) changes in CFTR protein expression using standard western blot techniques and, (b) changes in intracellular signaling mechanisms using basolateral application of: MDL‐12330A (an adenylate cyclase inhibitor), KT‐5720 (a PKA inhibitor), LY‐294002 (a PI3K inhibitor) and PD‐98059 (a MAPK inhibitor). We found no effect of 1μM KT‐5720, 20μM LY‐294002 or 10μM PD‐98059 on the basal or steady‐state forskolin‐stimulated Isc in 600G male or female mice. MDL‐12330A (10μM)) significantly reduced basal Isc in 600G females from 215.8±12.6 μA/cm2 (n=15) to 157.0±21.5 μA/cm2 (n=8, P<0.05) and decreased steady‐state forskolin‐stimulated Isc from 101.8±17.5 μA/cm2 (n=15), to 35.6±8.3 μA/cm2 (n=8, P<0.05), but was without effect in 600G males. Total CFTR protein expression normalized to total actin protein expression was unchanged in jejunum removed from any groups. These data suggest that the genistein‐mediated increases in intestinal Isc; (a) are not mediated via changes in intestinal CFTR protein expression in male or female mice and, (b) are mediated by adenylate cyclase in female but not male mice. RS and SS were supported by the Midwestern University Summer Fellowship Program. LA was supported by NIH (1R15DK071625‐01A2).
Tissue damage causes hyperthermia‐induced loss of intestinal barrier function via an oxidative stress pathway
Authors: Clanton Thomas L , Oliver S Ryan , Bakos Matt P
Published: June 2021

This study distinguished between two contrasting mechanisms for loss of the intestinal barrier during hyperthermia: tight junction opening and epithelial damage. The permeabilities of mouse small intestinal segments to fluorescent markers were monitored in vitro in everted gut sacs. Drugs known to open tight junctions (enterotoxin, palmitoyl carnitine, NOC‐12) had no effect at 37°C, though cytocholasin D (20μM) increased permeability. Electron microscopy revealed that cytocholasin D caused gross distortion of the epithelium, unrelated to tight junction opening. In contrast, LDH release (a marker of cytotoxicity) was significantly increased at 41.5°C and light microscopy revealed extensive structural damage to the epithelium. To determine whether oxidant formation could be responsible, protein oxidation was evaluated by carbonyl formation. Hyperthermia caused significant elevations in carbonyls, while co‐treatment of the segments with the antioxidants, N‐acetylcysteine (10 mM) or baicalein (200 μM) reduced carbonyl formation and greatly lowered hyperthermia induced permeability. The data are not consistent with tight junction opening alone being the primary mechanism for loss of barrier function. However, damage to the intestinal epithelial wall and perhaps other elements within the barrier may be responsible, possibly via oxidative stress mechanisms NHLB 53333.
Regional susceptibility of the small intestine to hyperthermia
Authors: Phillips Neil Andre , Novosad Veronica L , Clanton Thomas
Published: June 2021

Increases in core temperature have been associated with a decline in intestinal barrier function. The loss of this barrier is believed to be an important contributor to severe heat illness. We tested the hypothesis that the small intestine displays varied changes in hyperthermia‐induced permeability along its length. We isolated and everted segments from the small intestine of adult mice, creating ~8 × 2 cm sacs from each mouse, which were then separated into heated and control groups (42 ± 0.5°C and 37 ± 0.5°C, respectively). Permeability was measured during a 90 min exposure by the accumulation of a fluorescent‐labeled dextran (4KD) that moved from the incubation media into the sacs. The data showed a significant and relatively proportional increase in permeability in the hyperthermia treated segments as a function of the distance from the anus along the length of the small intestine (P < 0.007) but no significant relationship was found in the matched group exposed to 37°C. We conclude that in the mouse, the anterior portions of the small intestine (near the jejunum) have an increased susceptibility to hyperthermia compared to posterior regions (near the ileum). This suggests that treatments targeted to protect the upper intestine may be helpful in protecting humans from severe heat illness. NHLBI 53333
Role of gut barrier dysfunction and endotoxemia in development of colon cancer cachexia
Authors: Puppa Melissa J , White James P , Sato Shuichi , Baynes John W , Carson James A
Published: June 2021

Cachexia, accounts for ~30% of all cancer‐related deaths, and up to 50% of deaths in patients with gastrointestinal cancers. ApcMin/+ mice have been used as a model of colorectal cancer that become cachetic at 4–5 months of age. Gut barrier dysfunction (GBD) is an increase in the permeability of intestinal epithelium which can allow bacteria to breach the gut barrier and induce an inflammatory response. The purpose of this study was to determine if GBD is associated with cachexia in the ApcMin/+ mouse. Mice were administered FITC‐dextran, FD4, (MW 4000 Da) by gavage and blood sampled 1h later via retraorbital stick. Plasma endotoxin was measured with the limulus assay at 12wk and 19wk of age. There was a 27% decrease in body weight of the ApcMin/+ mice at 20 wks compared to the C57/BL6 wild type. Plasma FD4 was increased in the ApcMin/+ mice at 20wk compared to 14wks (6.9±2.4 μg/ml, n=10 vs 0.5±.5μg/ml, n=7). Endotoxin was greater at 19wks than 12wk ApcMin/+ mice (10.5±3EU/ml, n=9 vs 2.02±.8EU/ml, n=4). Mesentaric lymphs were taken at the 20wks were significantly larger in the ApcMin/+ mice compared with wild type mice (38±2.4mg, n=6 vs 24.4±1.7mg, n=9). Tumor number between 12 and 20 weeks was not different but, 20wk ApcMin/+ mice had an 8 fold greater number of large tumors. From these results, we hypothesize that GBD and resultant endotoxemia induce the pro‐inflammatory state that leads to development of cachexia in ApcMin/+ mice.
Effect of ezrin knockdown on the architecture of gastric epithelia
Authors: Asano Shinji , Tetsui Takahito , Kobayakawa Yuka , Tamura Atsushi , Tsukita Sachiko , Mukaisho Kenichi , Hattori Takanori
Published: June 2021

Ezrin, an adaptor protein that cross‐links plasma membrane‐associated proteins with actin cytoskeleton, is highly concentrated on the apical surface of many epithelial cell types especially in microvilli in small intestines and stomachs. In stomachs, ezrin is predominantly expressed on apical canalicular membrane of parietal cells. The ezrin knockdown (Vil2kd/kd) mice showed achlorhydria due to the impairment of membrane fusion of tubulovesicles with apical membrane. Here, we newly showed the involvement of ezrin in the architecture of gastric glands using the Vil2kd/kd mice. The Vil2kd/kd mice showed hypergastrinemia and hyperplasia of gastric surface epithelial cells. Atrophy was observed in the gastric glands with the number of parietal cells and chief cells being decreased, and the number of mucus neck cells being increased. The expression level of gastric proton pump was significantly decreased whereas the expression level of chloride channel CFTR was increased. These results demonstrate that ezrin is involved in the development of gastric epithelial cells and expression of transport proteins involved in gastric acid secretion.
Loss of Sonic Hedgehog in the adult stomach results in foveolar hyperplasia and delayed differentiation of the zymogenic cell lineage
Authors: Xiao Chang , Roland Joseph , Nam Ki Taek , Goldenring James R , Zavros Yana
Published: June 2021

Objective

Parietal cell‐expressed Sonic Hedgehog (Shh) regulates function and differentiation of the gastric epithelium. Correlative evidence shows that loss of Shh expression associated with Helicobacter pylori infection results in the disruption of the normal gastric epithelium. We sought to identify whether Shh regulates gastric epithelial cell differentiation in the absence of inflammation.

Methods

A parietal cell‐specific, tamoxifen‐inducible deletion of Shh mouse model was developed (HKCreInd/ShhKO). Epithelial cell proliferation and migration was studied using BrdU and Ki67 co‐immunostaining. Cell lineage changes were studied using markers specific for surface mucous (Ulex europaeus, UEAI), mucous neck (Griffonia simplicifolia, GSII) and zymogen (intrinsic factor, IF) cells.

Results

Compared to Controls, HKCreInd/ShhKO mice developed foveolar hyperplasia reflected by an expansion of BrdU/UEAI positive cells. HKCreInd/ShhKO mice also showed an expansion of GSII/IF co‐expressing cells at the base of the gastric gland. Co‐localization of BrdU and Ki67 revealed loss of normal migration of the epithelial cells within HKCreInd/ShhKO mouse stomachs.

Conclusion

Loss of Shh in the adult stomach results in the development of foveolar hyperplasia and delayed differentiation of the zymogenic cell lineage independent of inflammation.

(DHC Pilot and Feasibility Project Award, Zavros)
Linking a Newly Discovered Hormone with Orphan Receptor Candidates: The Search for a Cognate Ligand
Authors: Miller Geoff , Salvatori Alison S , Yosten Gina LC , Samson Willis K
Published: June 2021

Neuronostatin is a newly identified peptide hormone cleaved from the somatostatin pre‐prohormone. Since neuronostatin is encoded in the somatostatin gene, early candidate receptors included the five known somatostatin receptors (SSTRs). Using luciferase readout in CHO cells expressing the five SSTRs, we have confirmed that neuronostatin does not signal through the classical somatostatin receptors. During the search for neuronostatin‐responsive tissues, a transcriptional response to neuronostatin was observed in KATO‐III cells, an immortalized, gastric cancer cell line. When KATO‐III cells are transfected with an SRE‐luciferase construct, luciferase read‐out is a convenient assay to determine receptor activation. Based on the small size of neuronostatin and the requirement for c‐terminal amidation for activity, we hypothesize that neuronostatin signals through a G‐protein coupled receptor. Currently, there are 22, known, non‐olfactory orphan G‐protein coupled receptors. We examined the expression of these receptors in KATO‐III cells by RT‐PCR. Only 6 of the 22 putative receptors are expressed in KATO‐III cells. We are confirming expression of these receptor candidates in other neuronostatin‐responsive tissues (myocytes, neurons) and will employ systematic knock‐down of each candidate to identify a single, putative neuronostatin receptor.