bluuebunny/crossref_metadata_2025 huggingface dataset:
Temporal transcriptomic landscape of postnatal mouse ovaries reveals dynamic gene signatures associated with ovarian aging
Authors: Zhou Zixue , Yang Xi , Pan Yuncheng , Shang Lingyue , Chen Siyuan , Yang Jialin , Jin Li , Zhang Feng , Wu Yanhua
Published: June 2021

The ovary is the most important organ for maintaining female reproductive health, but it fails before most other organs. Aging-associated alterations in gene expression patterns in mammalian ovaries remain largely unknown. In this study, the transcriptomic landscape of postnatal mouse ovaries over the reproductive lifespan was investigated using bulk RNA sequencing in C57BL/6 mice. Gene expression dynamics revealed that the lifespan of postnatal mouse ovaries comprised four sequential stages, during which 2517 genes were identified as differentially enriched. Notably, the DNA repair pathway was found to make a considerable and specific contribution to the process of ovarian aging. Temporal gene expression patterns were dissected to identify differences in gene expression trajectories over the lifespan. In addition to DNA repair, distinct biological functions (including hypoxia response, epigenetic modification, fertilization, mitochondrial function, etc.) were overrepresented in particular clusters. Association studies were further performed to explore the relationships between known genes responsible for ovarian function and differentially expressed genes identified in this work. We found that the causative genes of human premature ovarian insufficiency were specifically enriched in distinct gene clusters. Taken together, our findings reveal a comprehensive transcriptomic landscape of the mouse ovary over the lifespan, providing insights into the molecular mechanisms underlying mammalian ovarian aging and supporting future etiological studies of aging-associated ovarian disorders.
Incorporating Research into an Undergraduate Animal Behavior Course using Zoos and Webcams
Authors: Davis-Berg Elizabeth C , Rafacz Michelle L
Published: June 2021

In our nonmajors animal behavior class, we developed a semester-long research project assignment that incorporates project-based learning (PBL) and the opportunity for Course-Based Undergraduate Research Experiences (CUREs) that introduces, assesses, and applies the course concepts. This project can easily be adapted for nonmajors biology, majors biology, or other more general survey classes, including remote courses. This student-led project involves a field trip for data collection at our local zoo, additional data collection using webcams, and writing and presenting a scientific report. Students apply the scientific method to design their research project and formulate a hypothesis. Throughout the semester, students learn about different behavioral sampling methods and how to develop and use an ethogram in class using animal webcams from zoos. At the zoo, students conduct a comparative behavior project by collecting data from their main animal and two related animals using multiple trials, so students can observe differences in behavior. At the conclusion of the project, students write a report demonstrating their data analysis, graphing, explanation, and interpretation of their own scientific data. We discuss how others can design and implement PBL and CUREs in their classes and what we have learned from our experiences.
Transfer of conservative discrete differential operators between staggered grids: construction and duality relations
Authors: Goudon Thierry , Krell Stella , Llobell Julie , Minjeaud Sebastian
Published: June 2021

This paper is concerned with the construction of a discrete conservative operator on a mesh, knowing a conservative operator on a different mesh. We analyse the possibility of defining such a transfer based on the preservation of conservation properties and provide practical procedures, which can be effectively implemented for schemes that work on staggered grids. We show that such a transfer can be applied to obtain a discrete balance equation on a mesh starting from a discrete balance equation on a different mesh, and to prove local discrete duality relations for standard discrete operators defined on staggered grids.
A Vertex Model for LLT Polynomials
Authors: Corteel Sylvie , Gitlin Andrew , Keating David , Meza Jeremy
Published: June 2021

We describe a novel Yang–Baxter integrable vertex model. From this vertex model we construct a certain class of partition functions that we show are essentially equal to the LLT polynomials of Lascoux, Leclerc, and Thibon. Using the vertex model formalism, we give alternate proofs of many properties of these polynomials, including symmetry and a Cauchy identity.
Integration of Singular Foliations via Paths
Authors: Garmendia Alfonso , Villatoro Joel
Published: June 2021

We give a new construction of the holonomy and fundamental groupoids of a singular foliation. In contrast with the existing construction of Androulidakis and Skandalis, our method proceeds by taking a quotient of an infinite-dimensional space of paths. This strategy is a direct extension of the classical construction for regular foliations and mirrors the integration of Lie algebroids via paths (per Crainic and Fernandes). In this way, we obtain a characterization of the holonomy and fundamental groupoids of a singular foliation that more clearly reflects the homotopic character of these invariants. As an application of our work, we prove that the constructions of the fundamental and holonomy groupoid of a foliation have functorial properties.
High prevalence of circulating dual-class resistant Mycoplasma genitalium in asymptomatic MSM in Tokyo, Japan
Authors: Ando Naokatsu , Mizushima Daisuke , Takano Misao , Mitobe Morika , Miyake Hirofumi , Yokoyama Keiko , Sadamasu Kenji , Aoki Takahiro , Watanabe Koji , Uemura Haruka , Yanagawa Yasuaki , Gatanaga Hiroyuki , Oka Shinichi
Published: June 2021

Objectives

To assess the prevalence and antibiotic resistance profile of Mycoplasma genitalium detected from urogenital/rectal swab samples obtained from MSM in Tokyo, Japan.

Methods

We performed PCR-based screening for M. genitalium urogenital/rectal infection in 982 asymptomatic MSM between 1 January 2019 and 5 November 2020. Mutations in the antibiotic resistance-associated genes gyrA and parC and the 23S rRNA of M. genitalium were analysed.

Results

The prevalence of M. genitalium infection was 6.1%: the prevalence of rectal and urogenital infection was 4.7% and 1.4%, respectively. Among the cases, 48 were successfully analysed for 23S rRNA, 41 for parC mutations and 37 for gyrA mutations. Macrolide- and quinolone-resistance associated mutations (23S rRNA and parC mutations) were observed in 43 (89.6%) and 28 (68.3%) cases, respectively. The quinolone-resistance associated mutation-harbouring variants also harboured macrolide-resistance associated mutations. The S83I mutation in the parC gene was most commonly identified (24 cases, 58.5%), and its combination with M95I or D99N mutation in the gyrA gene was observed in 9 of 36 successfully analysed cases (25.0%). No significant association was observed between the presence of antibiotic resistance and antibiotic exposure for either macrolides or fluoroquinolones (P = 0.785 and 0.402, respectively).

Conclusions

In Tokyo, there is an alarmingly high prevalence of M. genitalium harbouring macrolide and/or quinolone resistance-associated mutations in MSM, irrespective of antibiotic exposure. The high prevalence of M. genitalium strains with both parC and gyrA mutations limits the efficacy of sitafloxacin. Therefore, suitable alternatives are required to treat such M. genitalium infections.
Effectiveness of interventions aiming to reduce inappropriate drug prescribing: an overview of interventions
Authors: Kroon Daniëlle , Steutel Nina F , Vermeulen Hester , Tabbers Merit M , Benninga Marc A , Langendam Miranda W , van Dulmen Simone A
Published: June 2021

Objective

Inappropriate prescribing of drugs is associated with unnecessary harms for patients and healthcare costs. Interventions to reduce these prescriptions are widely studied, yet the effectiveness of different types of interventions remains unclear. Therefore, we provide an overview regarding the effectiveness of intervention types that aim to reduce inappropriate drug prescriptions, unrestricted by target drugs, population or setting.

Methods

For this overview, systematic reviews (SRs) were used as the source for original studies. EMBASE and MEDLINE were searched from inception to August 2018. All SRs aiming to evaluate the effectiveness of interventions to reduce inappropriate prescribing of drugs were eligible for inclusion. The SRs and their original studies were screened for eligibility. Interventions of the original studies were categorized by type of intervention. The percentage of interventions showing a significant reduction of inappropriate prescribing were reported per intervention category.

Key findings

Thirty-two SRs were included, which provided 319 unique interventions. Overall, 61.4% of these interventions showed a significant reduction in inappropriate prescribing of drugs. Strategies that were most frequently effective in reducing inappropriate prescribing were multifaceted interventions (73.2%), followed by interventions containing additional diagnostic tests (antibiotics) (70.4%), computer interventions (69.2%), audit and feedback (66.7%), patient-mediated interventions (62.5%) and multidisciplinary (team) approach (57.1%). The least frequently effective intervention was an education for healthcare professionals (50.0%).

Conclusion

The majority of the interventions were effective in reducing inappropriate prescribing of drugs. Multifaceted interventions most frequently showed a significant reduction of inappropriate prescribing. Education for healthcare professionals is the most frequently included intervention in this overview, yet this category is least frequently effective.
Clostridioides difficile Infection in Children: The Role of Infection Prevention and Antimicrobial Stewardship
Authors: Savage Timothy J , Sandora Thomas J
Published: June 2021

There are 2 primary approaches to prevent Clostridioides difficile infection (CDI) in children: prevent transmission and acquisition of the organism and prevent the progression from colonization to disease. The most important interventions to reduce the risk of transmission include contact precautions, hand hygiene, and environmental disinfection. Glove use minimizes contamination of the hands by spores and is associated with reductions in CDI incidence. Hand hygiene with soap and water and disinfection with a sporicidal agent are recommended as the best approaches in hyperendemic settings. Because antibiotic exposure is the most important modifiable risk factor for CDI, antimicrobial stewardship focused on identified high-risk antibiotic classes (including clindamycin, fluoroquinolones, and third- and fourth-generation cephalosporins) is critical to preventing progression from colonization to infection. Despite clear evidence that antimicrobial stewardship programs (ASPs) are associated with reduced CDI rates in adults, data demonstrating the ASP impact on pediatric CDI are lacking.
A Normative Framework for the Reconciliation of EU Data Protection Law and Medical Research Ethics
Authors: Hallinan Dara
Published: June 2021

EU data protection law and medical research ethics overlap in scope and content in numerous instances in which personal data are processed in medical research. It is not always the case, however, that the conditions outlined by the two rule-sets precisely coincide. In the past few years, this lack of confluence has led to confusion as to how the two rule-sets should best relate to one another. This confusion has led to different approaches to the relationship being taken, on occasion leading to counter-intuitive conclusions. Unfortunately, there has hitherto been little effort to provide clarity to this confusion. In this regard, this article attempts to provide a general normative framework aimed at facilitating optimally cogent and just reconciliations of EU data protection law and medical research ethics.
Optimizing Visualization in Endoscopic Spine Surgery
Authors: Basil Gregory W , Kumar Vignessh , Wang Michael Y
Published: June 2021

Given the inherent limitations of spinal endoscopic surgery, proper lighting and visualization are of tremendous importance. These limitations include a small field of view, significant potential for disorientation, and small working cannulas. While modern endoscopic surgery has evolved in spite of these shortcomings, further progress in improving and enhancing visualization must be made to improve the safety and efficacy of endoscopic surgery. However, in order to understand potential avenues for improvement, a strong basis in the physical principles behind modern endoscopic surgery is first required. Having established these principles, novel techniques for enhanced visualization can be considered. Most compelling are technologies that leverage the concepts of light transformation, tissue manipulation, and image processing. These broad categories of enhanced visualization are well established in other surgical subspecialties and include techniques such as optical chromoendoscopy, fluorescence imaging, and 3-dimensional endoscopy. These techniques have clear applications to spinal endoscopy and represent important avenues for future research.
Regulation of antibody responses against self and foreign antigens by Tfr cells: implications for vaccine development
Authors: Basto Afonso P , Graca Luis
Published: June 2021

The production of antibodies can constitute a powerful protective mechanism against infection, but antibodies can also participate in autoimmunity and allergic responses. Recent advances in the understanding of the regulation of germinal centres (GC), the sites where B cells acquire the ability to produce high-affinity antibodies, offered new prospects for the modulation of antibody production in autoimmunity and vaccination. The process of B cell affinity maturation and isotype switching requires signals from T follicular helper (Tfh) cells. In addition, Foxp3+ T follicular regulatory (Tfr) cells represent the regulatory counterpart of Tfh in the GC reaction. Tfr cells were identified one decade ago and since then it has become clear their role in controlling the emergence of autoreactive B cell clones following infection and immunization. At the same time, Tfr cells are essential for fine-tuning important features of the humoral response directed to foreign antigens that are critical in vaccination. However, this regulation is complex and several aspects of Tfr cell biology are yet to be disclosed. Here, we review the current knowledge about the regulation of antibody responses against self and foreign antigens by Tfr cells and its implications for the future rational design of safer and more effective vaccines.
A roadmap of plant membrane transporters in arbuscular mycorrhizal and legume–rhizobium symbioses
Authors: Banasiak Joanna , Jamruszka Tomasz , Murray Jeremy D , Jasiński Michał
Published: June 2021

Most land plants live in close contact with beneficial soil microbes: the majority of land plant species establish symbiosis with arbuscular mycorrhizal fungi, while most legumes, the third largest plant family, can form a symbiosis with nitrogen-fixing rhizobia. These microbes contribute to plant nutrition via endosymbiotic processes that require modulating the expression and function of plant transporter systems. The efficient contribution of these symbionts involves precisely controlled integration of transport, which is enabled by the adaptability and plasticity of their transporters. Advances in our understanding of these systems, driven by functional genomics research, are rapidly filling the gap in knowledge about plant membrane transport involved in these plant–microbe interactions. In this review, we synthesize recent findings associated with different stages of these symbioses, from the pre-symbiotic stage to nutrient exchange, and describe the role of host transport systems in both mycorrhizal and legume–rhizobia symbioses.
Investor Rewards to Climate Responsibility: Stock-Price Responses to the Opposite Shocks of the 2016 and 2020 U.S. Elections
Authors: Ramelli Stefano , Wagner Alexander F , Zeckhauser Richard J , Ziegler Alexandre
Published: June 2021

Donald Trump’s 2016 election and his nomination of climate skeptic Scott Pruitt to head the Environmental Protection Agency drastically downshifted expectations about U.S. policy toward climate change. Joseph Biden’s 2020 election shifted them dramatically upward. We study firms’ stock-price movements in reaction to these changes. As expected, the 2016 election boosted carbon-intensive firms. Surprisingly, firms with climate-responsible strategies also gained, especially those firms held by long-run investors. Such investors appear to have bet on a “boomerang” in climate policy. Harbingers of a boomerang appeared during Trump’s term. The 2020 election marked its arrival. (JEL G14, G38, G41)
Lipidomic analysis reveals disturbances in glycerophospholipid and sphingolipid metabolic pathways in benzene-exposed mice
Authors: Yu Linling , Sun Rongli , Xu Kai , Pu Yunqiu , Huang Jiawei , Liu Manman , Chen Minjian , Zhang Juan , Yin Lihong , Pu Yuepu
Published: June 2021

Benzene, a known occupational and environmental contaminant, has been recognized as the hematotoxin and human carcinogen. Lipids have a variety of important physiological functions and the abnormal lipid metabolism has been reported to be closely related to the occurrence and development of many diseases. In the present study, we aim to utilize LC–MS/MS lipidomic platform to identify novel biomarkers and provide scientific clues for mechanism study of benzene hematotoxicity. Results showed that a total of 294 differential metabolites were obtained from the comparison of benzene-treated group and control group. The glycerophospholipid pathway was altered involving the down-regulation of the levels of phosphatidylcholine and phosphatidylserine. In addition, phosphatidylethanolamine (PE) and 1-Acyl-sn-glycero-3-phosphocholine levels were increased in benzene-treated group. Based on the relationship between PE and autophagy, we then found that effective biomarker of autophagy, Beclin1 and LC3B, were increased remarkably. Furthermore, following benzene treatment, significant decreases in glucosylceramide (GlcCer) and phytosphingosine (PHS) levels in sphingolipid pathway were observed. Simultaneously, the levels of proliferation marker (PCNA and Ki67) and apoptosis regulator (Bax and Caspase-3) showed clear increases in benzene-exposed group. Based on our results, we speculate that disturbances in glycerophospholipid pathway play an important role in the process of benzene-induced hematopoietic toxicity by affecting autophagy, while sphingolipid pathway may also serve as a vital role in benzene-caused toxicity by regulating proliferation and apoptosis. Our study provides basic study information for the future biomarker and mechanism research underlying the development of benzene-induced blood toxicity.
Recombinant SARS-CoV-2 genomes circulated at low levels over the first year of the pandemic
Authors: VanInsberghe David , Neish Andrew S , Lowen Anice C , Koelle Katia
Published: June 2021

Viral recombination can generate novel genotypes with unique phenotypic characteristics, including transmissibility and virulence. Although the capacity for recombination among betacoronaviruses is well documented, recombination between strains of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has not been characterized in detail. Here, we present a lightweight approach for detecting genomes that are potentially recombinant. This approach relies on identifying the mutations that primarily determine SARS-CoV-2 clade structure and then screening genomes for ones that contain multiple mutational markers from distinct clades. Among the over 537,000 genomes queried that were deposited on GISAID.org prior to 16 February 2021, we detected 1,175 potential recombinant sequences. Using a highly conservative criteria to exclude sequences that may have originated through de novo mutation, we find that at least 30 per cent (n = 358) are likely of recombinant origin. An analysis of deep-sequencing data for these putative recombinants, where available, indicated that the majority are high quality. Additional phylogenetic analysis and the observed co-circulation of predicted parent clades in the geographic regions of exposure further support the feasibility of recombination in this subset of potential recombinants. An analysis of these genomes did not reveal evidence for recombination hotspots in the SARS-CoV-2 genome. While most of the putative recombinant sequences we detected were genetic singletons, a small number of genetically identical or highly similar recombinant sequences were identified in the same geographic region, indicative of locally circulating lineages. Recombinant genomes were also found to have originated from parental lineages with substitutions of concern, including D614G, N501Y, E484K, and L452R. Adjusting for an unequal probability of detecting recombinants derived from different parent clades and for geographic variation in clade abundance, we estimate that at most 0.2–2.5 per cent of circulating viruses in the USA and UK are recombinant. Our identification of a small number of putative recombinants within the first year of SARS-CoV-2 circulation underscores the need to sustain efforts to monitor the emergence of new genotypes generated through recombination.
Biology Of Transmission in the Pain Pathway
Authors: Yaksh Tony L.
Published: June 2021

Acute high intensity stimuli evoke activity in small A‐ and C fibers that display “free nerve endings”. Mechanical and/or thermal energy applied to the terminal is converted by transducer proteins on the terminal into an intensity‐dependent depolarization. The stimulus response sensitivity of a given afferent fiber is defined by the constituent transducer proteins that are present on the afferent terminal. These transducer proteins are typically activated (e.g. show an increased cation current) over a narrow range of intensities of the respective stimulus modality. Thus, TRP channels may be activated by temperatures > 34–38° (TRP V3), > 43° C (TRPV1) and >52°C TRPV3. Increasing intensities activate higher threshold transducers, leading to increased terminal depolarization. Following local tissue injury/inflammation, there is spontaneous activity and an enhanced response that arises from the release of products (H+, cytokines, amines, lipids, trypsin‐like enzymes, complement) from inflammatory cells and plasma. These work upon eponymous channels/receptors to activate terminal kinases, which enhance the responsiveness of the terminal transducer protein and channels. (Funded by NIH DA02110, NS16541.)
Stimulation of the Hepatic Toll‐like Receptor Signaling Pathway after Induction of Equine Laminitis Using Gastrointestinal Models
Authors: Stokes Ashley Michelle , McGeachy Matthew , Carlisle Katherine , Keowen Michael L , Garza Frank
Published: June 2021

The toll‐like receptor (TLR) signaling pathway is important in orchestrating innate immunity in response to many factors including circulating bacterial components. In cases of GI disruption, portal blood flow may lead to strong stimulation of the hepatic TLR signaling pathway with the subsequent systemic release of inflammatory mediators known to play a role in laminitis.

Our objectives were to evaluate key mediators of the TLR signaling pathway in hepatic tissues after the use of two GI models of laminitis induction (black walnut extract (BWE) or carbohydrate overload (CHO) models). We examined TLR4, cluster of differentiation 14 (CD‐14), TLR2, phosphatidylinositol‐3‐kinase regulatory subunit 1 (PIK3R1), inhibitor of NF‐êB (INFKB), cyclooxygenase 2 (COX2), and interleukin 8 (IL‐8). Liver tissues (normal, BWE‐induced, CHO‐induced, and naturally‐acquired) were used in TaqMan RT‐PCR.

CD14, TLR2, PIK3R1 and INFKB were significantly (p<0.05) upregulated in all laminitic horses. TLR4 was significantly upregulated in both models of laminitis induction, especially those following CHO which closely mimics the clinical presentation of a high percentage of laminitis cases.

These findings demonstrate that TLR mediators are upregulated in cases of laminitis following GI disruption and systemic therapeutic intervention for laminitis prevention in these cases is warranted.
Effects of polysaccharide of polygonum multiflorum thunb on mtDNA deletion mutation and the level of CO I and ATPase 6 mRNA expression in aging model rats’ liver
Authors: Wei Xiao‐Dong , Kang Yu‐Ming , Ou Qin , Wang Zhe , Qi Zong‐Chun , Wang Shu‐Qiu , Ji Qing‐Li , Li Dian‐Gang
Published: June 2021

The present study was undertaken to investigate the effects of the polysaccharide of polygonum multiflorum thunb (PPMT) on mtDNA deletion mutation and the level of mRNA expression of CO I and ATPase 6 in aging model rats liver and explore its mechanism on anti‐aging. Forty Wistar rats were divided randomly into four groups: young, aging model, PPMT and polygonum multiflorum thunb (PMT) decoction groups. The results showed that, compared with the young group, activities of Mn‐SOD, COX and Na+‐K+ATPase in liver were lower in PPMT group and PMT decoction group, but higher than the aging group. The contents of malondialdehyde(MDA) and mtDNA deletion mutation and the level of CO I mRNA and ATPase 6 mRNA in liver were more in PPMT group and PMT decoction group, but less than that in aging group. The efficacy of PPMT and PMT decoction has no difference. PPMT and PMT decoction could increase the activities of Mn‐SOD, COX and Na+‐K+ATPase, and decrease contents of MDA, mtDNA deletion mutation and expressions of CO I mRNA and ATPase 6 mRNA in liver of aging rats induced by D‐gal.
KGF Induction of Human Peroxiredoxin 6 (Prdx6) Gene Expression is Mediated by the Transcription Factor Nrf2
Authors: Feinstein Sheldon I. , Chowdhury Ibrul , Christofidou‐Solomidou Melpo , Fisher Aron B.
Published: June 2021

Peroxiredoxin 6 (Prdx6), an enzyme that has both phospholipase A2 and peroxidase activities can reduce lipid hydroperoxides by a unique mechanism employing a single conserved cysteine. It is highly expressed in lung, both protecting against oxidative stress and regulating pulmonary surfactant phospholipid metabolism. We reported previously that rodent Prdx6 is up‐regulated in development and by oxidative stress. The goal of our experiments was to identify the mechanism by which keratinocyte growth factor (KGF) induces transcription of the human Prdx6 gene. We had previously found that a consensus Antioxidant Response Element (ARE), located between 357 and 349 nucleotides before the transcriptional start of the human Prdx6 gene was necessary for transcriptional activation of the Prdx6 promoter by KGF in the human lung cell line, A549, and in primary cultures of rat type II cells. We hypothesized that Nrf2, a transcription factor that binds to the ARE might be the activating factor. siRNA directed against Nrf2 blocked the up‐regulation of Prdx6 by KGF; a control siRNA had no effect. KGF increased Prdx6 promoter activity in mouse type II cells, but not in type II cells from Nrf2 null mice. KGF appears to trigger a migration of Nrf2 from cytoplasm to nucleus and an increased binding to the Prdx6 promoter in ChIP assays. We conclude that KGF activation of Prdx6 transcription requires Nrf2. Supported by HL P01‐75587.
Regulation of P‐gp Under Inflammatory Conditions in the BME‐UV In Vitro Model
Authors: Al‐bataineh Mohammad M. , Schultz Bruce D. , van der Merwe Deon , Malreddy Pradeep , Gehring Ronette
Published: June 2021

P‐glycoprotein (efflux pump) belongs to the ATP‐binding cassette (ABC) super‐family that may influence the bioavailability and disposition of many drugs. Mammary secretory epithelium expresses different families of drug transport proteins, including P‐glycoprotein [1,2]. Results from functional studies suggest that P‐glycoprotein is expressed on the apical membrane of the mammary epithelium. During inflammatory reactions, which can be associated with changes in mammary epithelial barrier functions, pro‐inflammatory cytokines tumor necrosis factor alpha (TNF‐α) and interleukin‐6 (IL‐6) are elevated in milk and serum [3]. In this study, the role of TNF‐α in regulating P‐glycoprotein was investigated in cultured BME‐UV cells. The mRNA expression of ABCB1, the gene encoding for P‐glycoprotein, was upregulated after 48 and 72 h of exposure to TNF‐α. After 120 h, mRNA expression decreased but still higher than the control level. P‐glycoprotein expression followed the same trend as ABCB1 mRNA expression, increasing after 24, 48, 72 h of exposure to TNF‐α and then decreasing after 120 h. Transepithelial electrical resistance (Rte) was within 15% of the control value at all the time points, except 120 h, when it increased by 50%. These results suggest that expression levels of P‐glycoprotein may be altered by inflammation thereby affecting the movement of its substrates across mammary epithelial barrier.
Intestinal epithelial barrier integrity requires Desmoglein2‐mediated adhesion
Authors: Schlegel Nicolas , Meir Michael , Heupel Wolfgang Moritz , Germer Christoph Thomas , Waschke Jens
Published: June 2021

The integrity of intercellular junctions in intestinal epithelium is crucial for sealing the intestinal barrier. While specific roles of tight and adherens junctions are well known, the contribution of desmosomal adhesion for maintaining the intestinal epithelial barrier has not been addressed. We generated a desmoglein 2 antibody directed against the extracellular domain (Dsg2 ED) to test whether impaired Dsg2‐mediated adhesion affects intestinal barrier functions in vitro. This antibody was capable to block Dsg2 interaction in cell‐free atomic force microscopy experiments. For in vitro studies of the intestinal barrier we used Caco2 cells following differentiation into enterocyte‐like epithelial monolayers. Application of Dsg2 ED to Caco2 monolayers increased cell dissociation compared to controls. Under similar conditions Dsg2 antibody significantly decreased transepithelial electrical resistance. As revealed by immunostaining, this was due to Dsg2 ED antibody‐induced loss of tight junction proteins from the cell borders. Similar results were obtained using a commercial monoclonal antibody against the extracellular domain of Dsg2 to Caco2 monolayers. Antibody‐induced effects were blocked by absorption experiments. Our data indicate that Dsg2‐mediated adhesion affects tight junction integrity and is required to maintain intestinal epithelial barrier properties.
Dynamic imaging of single microvilli in opossum kidney cells
Authors: Lanzano Luca , Fwu Peter , Giral Hector , Levi Moshe , Gratton Enrico
Published: June 2021

Reabsorption of inorganic phosphate (Pi) in the renal proximal tubule is fundamental to overall Pi homeostasis. Its regulation occurs via the apical brush border membrane (BBM) sodium gradient‐dependent Pi (NaPi) cotransport proteins. Distinct families of NaPi cotransporters show differential regulation under dietary and hormonal stimuli, but how this is accomplished at a molecular level is not yet fully understood.

The application of modern fluorescence correlation spectroscopies to the BBM of living cultured Opossum Kidney (OK) cells expressing NaPi cotransporters with different GFP constructs could provide useful information on the dynamics of the proteins, including diffusion, aggregation and interaction with other proteins. However, BBM microvilli show a relatively fast motion that makes the use of fluctuation spectroscopy difficult.

We developed an optical imaging technique called Modulation Tracking (MT) in which we track the center of mass of the microvillus at an arbitrary point along its length via orbital scanning while the laser spot rapidly oscillates perpendicularly to the surface. Using this technique we obtained high resolution images of the microvilli and applied fluctuation correlation spectroscopies in order to study protein dynamics.

Work supported in part by NIH RO1 DK066029‐01A2 (ML, EG), NIH‐P41 P41‐RRO3155 (EG, PF and LL) and P50‐GM076516 (EG, PF and LL).
Prolactin stimulates K+ secretion in isolated rat distal colon
Authors: Deachapunya Chatsri , Poonyachoti Sutthasinee , Krishnamra Nateetip
Published: June 2021

The effect of prolactin (PRL) on ion transport across the rat colonic epithelium was investigated using the Ussing chamber technique. PRL induced a sustained decrease in basal short circuit current (Isc) by 5–35% depending on the colonic segment: a maximum response in the distal colon and less in the proximal colon. In the distal colon, PRL decreased Isc in a concentration dependent manner with an IC50value of 140 ng/ml. The PRL‐induced decrease in Isc was not affected in the presence of Na+ channel blocker amiloride, but showed higher response in the presence of Cl− channel blocker NPPB or glibenclamide added to the apical solution. In contrast, the PRL response was inhibited in the presence of Na+‐K+‐2Cl− transporter inhibitor bumetanide or K+ channel blocker BaCl2, but not other K+ channel blocker apamin, clotrimazole or 293B added serosally. Among K+ channel blockers, only 293B added apically partially diminished the PRL response. In addition, the carbachol or forskolin‐activated Cl− secretion remained unchanged in the presence of PRL. Thus in the rat distal colon, PRL stimulates K+ secretion through barium‐sensitive basolateral K+ channel and bumetanide‐sensitive pathway, with no effect on Na+ or Cl− transport. This work was supported by The Srinakharinwirot University Research Grant (325/2552).
TNF is a key mediator in sepsis‐induced intestinal barrier dysfunction but is independent of enterocyte NFκB
Authors: Samocha Alexandr J. , Coopersmith Craig M. , Dominguez Jessica A.
Published: June 2021

Inhibition of enterocyte NFκB augments sepsis‐induced intestinal hyperpermeability, gut apoptosis, and TNF expression. We hypothesized that TNF is a key mediator of gut barrier dysfunction and apoptosis in sepsis.

Mice lacking enterocyte IKKβ (resultant NFκB inhibition) were created via Cre/lox recombination under control of the villin promoter. Wildtype (WT) and Vil‐Cre/IkkβF/Ä (IKKβ−/−) mice were injected 3 hr pre‐operatively with monoclonal anti‐TNF antibody or isotype control followed by cecal ligation and puncture (n=5–12/group). At 24 hr, intestinal permeability was assessed in vivo using FD4. Apoptosis was evaluated in crypts by active caspase‐3 staining. Blood was harvested for bacterial cultures.

Anti‐TNF decreased permeability in septic WT (1832±321 vs. 4678±421 ng/ml; p<0.001) and septic IKKβ −/− mice (2528±446 vs. 6650±951 ng/ml; p<0.01). Sepsis‐induced apoptosis was decreased in both anti‐TNF treated septic WT (13±1 vs. 4±2; p<0.05) and septic IKKβ −/− mice (20±2 vs. 4±1; p<0.001). Anti‐TNF decreased bacteremia in septic WT (4×103±2×103 vs. 4×105±3×105 CFU/ml; p<0.05) and septic IKKβ −/− mice (500±500 vs. 3×106±2×106 CFU/ml; p<0.01). There were no differences between anti‐TNF treated groups for any parameter.

TNF appears to be a key mediator in sepsis‐induced intestinal barrier dysfunction. However, this mechanism is independent of enterocyte NFκB. NIH GM66202 & GM082008.
Hibernation increases gut permeability and occludin expression in ground squirrel intestine
Authors: Pike Amanda C. , Carey Hannah V.
Published: June 2021

During hibernation, ground squirrels fast for several months and rely solely on adipose stores to fuel metabolism. We showed previously that ionic permeability of hibernating squirrel intestine is 1.5‐fold greater than in summer squirrels, which is consistent with studies in non‐hibernating species that fasting and total parenteral nutrition can increase gut permeability. However, whether macromolecular permeability is also elevated during hibernation is not known. To investigate the severity of the permeability change, we gavaged summer squirrels and hibernators that were fully aroused (Tb~37 °C) with FITC‐labeled dextran (MW 4kD). Appearance of label in plasma 3 h later was ~10‐fold greater in hibernating (n=6) vs. summer (n=6) squirrels (P<0.05). Western analysis showed that cecal expression of the tight junction protein occludin is 3.5‐fold greater in hibernators (n=12 each group) (P<0.01). We hypothesize that in the setting of elevated gut permeability during hibernation, upregulation of occludin expression may help restrict microbes and their products to the gut lumen, thereby minimizing the risk of mucosal inflammation and sepsis.
RhoA‐dependent tight junction regulation is associated with dysregulated Akt and ERK signaling and increased claudin‐2 expression
Authors: Gluth Markus , Pape Ulrich‐Frank , Theuring Franz , Fischer Andreas
Published: June 2021

Previous studies have demonstrated that expression of either constitutively‐active (CA) or dominant negative (DN) RhoA reduced tight junction barrier function in cultured MDCK monolayers (Jou et al. 1998). However, the mechanisms by which RhoA disruption causes barrier loss have not been defined. The aims of this study were to determine whether RhoA regulates barrier function in mammary epithelia and to identify the molecular events responsible. V14 and N19 (CA and DN, respectively) RhoA were expressed in EpH4 mammary epithelial cells. RhoA (CA or DN) expression in confluent monolayers caused a dose‐dependent loss of transepithelial electrical resistance. Neither trafficking of ZO‐1 and occludin to the tight junction nor cell viability were affected by CA or DN RhoA expression, but western blots showed that ERK and Akt were phosphorylated (consistent with activation) after either CA or DN RhoA expression. Transcript content of several claudins was altered after CA or DN RhoA expression. Most notably, claudin‐2 mRNA and protein expression were increased following either CA or DN RhoA expression. These data demonstrate that RhoA regulates the barrier in mammary epithelia and suggest that increased claudin‐2 expression contributes to RhoA‐mediated tight junction regulation.

Supported by Charite Universitaetsmedizin Berlin.
Down‐regulation of CFTR is required for epithelial‐mesenchymal transition in cancer development.
Authors: Zhang Jieting , Chung Hilda , Chan Hsiao Chang
Published: June 2021

Epithelial‐mesenchymal transition (EMT) is an essential component of embryonic development, tissue remodeling, and wound repair. Many epithelial tumors also appear to undergo EMT to acquire a more invasive and metastatic phenotype. Subsequently, the metastasized tumor cells should undergo mesenchymal‐epithelial transition (MET) in order to form a new site. Using an EMT and MET colon carcinoma cell model LIM1863, we report that the cystic fibrosis transmembrane regulator (CFTR) was down‐regulated in cells undergoing EMT induced by TGF‐β alone in 5 days or by TGF‐β with TNF‐α in 24 hours. The down‐regulation of CFTR during EMT was time dependent. On the contrary, the expression of CFTR was upregulated during MET. A specific CFTR inhibitor CFTRinh‐172 induced EMT in the absence of TGF‐β or TNF‐α in the cell model, and the effect was dependent on inhibitor concentrations. MET was blocked by CFTRinh‐172 at similar concentrations. These results suggest that down‐regulation of CFTR is required for EMT, which is inhibitory for MET. Further studies along this line my reveal novel mechanism regulating the processes of EMT and MET.

This project was supported by Focused Investments of the Chinese University of Hong Kong.
Ontogenetic changes of histone modifications and polymerase II binding in the promoter region of rat intestinal sugar transporter genes in vivo
Authors: Suzuki Takuji , Douard Veronique , Mochizuki Kazuki , Goda Toshinao , Ferraris Ronald P
Published: June 2021

Expression of the intestinal glucose (G) transporter SGLT1 increases with age, that of the fructose (F) transporter GLUT5 does not unless its substrate is present at ≥ 14 d of age, indicating substrate‐inducible, age‐sensitive transcription. We have shown histone H3 acetylation with transcriptional activation of GLUT5 in Caco2. Here, we perfused intestines of 10 and 20 d old rats with either F or G then performed ChIP assays with antibodies against polymerase II (pol II) and acetylated histones H3 and H4 using primers targeting promoter regions 0 to −5100 bp of both genes. SGLT1 mRNA increased with age, GLUT5 increased only with F perfusion and age. Patterns of pol II binding on SGLT1 and GLUT5 promoters 0 to −200 bp correlated tightly with mRNA expression patterns. H3 acetylation in the GLUT5 and SGLT1 promoters increased with age, but acetylation in F was higher than in G between −1000 to −2000 bp only in GLUT5 suggesting total promoter sensitivity to age but specific regions dedicated to substrate induction. H3 acetylation and pol II binding did not change in the F‐responsive gene glucose‐6‐phosphatase regulated by mRNA stability. H4 acetylation did not change with age or perfusion in GLUT5 and SGLT1. Programmed age‐dependent increases in SGLT1 expression may be designed to meet hard‐wired increases in G consumption, but GLUT5 transcription increases only after weaning when the opportunity to consume F arises. (NSF722365)
Vascular Endothelial Growth Factor Protein is Decreased in a Mouse Model of Necrotizing Enterocolitis
Authors: Carrasco Rosa , Liu Shirley XL , Tian Runlan , Kushnir Anna , De Plaen Isabelle G
Published: June 2021

Necrotizing enterocolitis (NEC) is a severe intestinal disease affecting premature infants. Its pathogenesis remains poorly understood. Vascular endothelial growth factor (VEGF) is an angiogenic protein that plays a role in several pathologic processes. While VEGF polymorphisms have been associated with NEC, the role of VEGF in the pathogenesis of NEC remains unknown. Here, we examined the developmental expression of VEGF in the mouse intestine and in a neonatal mouse NEC model. To determine VEGF developmental expression, pups were sacrificed at embryonic day (E) 16, postnatal day (D) 0 or 3. To examine intestinal VEGF in our NEC model, animals were submitted to the NEC protocol for 48 hours or allowed to be dam fed. Intestinal VEGF protein was assessed by western blot followed by band densitometry (values were normalized to β‐actin). Compared to D0, only a low amount of VEGF was detected in the mouse intestine at E16 (2.6±0.42 % of D0 value; p<0.05). At D3, there was a marginal increase in VEGF compared to D0 (194 ±20 %), although this was not statistically significant. In pups exposed to the NEC protocol, intestinal VEGF was not detectable while it was strongly present in dam fed controls. In conclusion, we found that intestinal VEGF is increased after birth in dam fed pups, but undetectable at 48 hours in our neonatal mouse NEC model. Therefore, we speculate that VEGF plays a role in the pathogenesis of this disease.
Neck position affects the dimensions of the rat cervical esophagus
Authors: Lorber Mortimer
Published: June 2021

Might esophageal structure allow dimensional alterations? The rat esophagus has elastic fibers, skeletal muscle and keratinized epithelium. Will they shorten as the neck flexes and lengthen as it hyperextends? In eight adult female rats under pentobarbital six dots of dye were placed rostrocaudally on the cervical esophagus. When prone on a flat surface the neck was flexed to about 6° short of perpendicular (84° ± 4° s.d.) and maintained by a head clamp. Greater flexion caused the chin to mask the most rostral dot. Interdot measurements were made using a dividers and digital micrometer. A 1 cm diameter vial was then placed under the shoulders. The measurements were repeated with the neck hyperextended to about 6° below 180°. Paired “t”‐tests compared distances between adjacent dots. The paired measurements (mm) were: 1.71 ± 0.26 vs 2.58 ± 0.40; 1.69 ± 0.21 vs 2.48 ± 0.29; 2.69 ± 0.40 vs 3.71 ± 0.61; 1.99 ± 0.26 vs 2.43 ± 0.23; 1.95 ± 0.25 vs 2.34 ± 0.31. As it lengthened the esophagus narrowed. At the level of the rostralmost and caudalmost dots its widths were 2.91 ± 0.28 vs 2.67 ± 0.23 and 2.76 ± 0.19 vs 2.21 ± 0.16. All differences but the rostralmost width were significant (P < 0.001). The cervical esophagus changes length and width as the neck moves. This is also likely in man. On looking down at one's plate while eating, esophageal dimensions and motility might differ from when one swallows while looking up at the waiter.
Plasticity of the noradrenergic response in gastric vagal circuits
Authors: Browning Kirsteen N , Travagli R. Alberto
Published: June 2021

Catecholaminergic inputs from the nucleus tractus solitarius pars centralis (cNTS) to dorsal motor nucleus of the vagus (DMV) neurons exert profound, vagally mediated effects on gastrointestinal motility.

We have shown that opioid and pancreatic polypeptides modulate glutamatergic, but not GABAergic, transmission between NTS and DMV. Elevation of cAMP levels within the brainstem, however, allows transient modulation by these peptides of the otherwise unresponsive GABAergic synapse.

The aim of this study was to ascertain whether this short‐term plasticity is a generalized phenomenon also relevant to modulation by catecholamines.

Whole cell recordings were made from identified gastric‐projecting rat DMV neurons. Electrical stimulation of the NTS evoked either paired IPSC or EPSC. Norepinephrine and/or the α2 agonist UK 14,304 decreased the amplitude of EPSC; these effects were blocked by yohimbine. The amplitude of the IPSC was, however, unaffected by either agonists unless the levels of cAMP were elevated by pretreatment with agents, such as forskolin, known to increase cAMP levels.

These results suggest that, as with opioids and pancreatic polypeptides, the effects of catecholamines on GABAergic transmission depend upon the levels of cAMP within gastric vagal circuits. This short‐term plasticity appears to be a generalized phenomenon within the NTS to DMV GABAergic synapse.

Supported by DK 55530
Induction of cellular responses and signaling pathways by bioassayable growth hormone
Authors: Tracy Andrea C , Ramos Yamil , Herrera Gloria V , Serrano Michelle N , Cooper Rasalin D , Gosselink Kristin L
Published: June 2021

Bioassayable growth hormone (BGH) is an exercise‐responsive pituitary growth factor that stimulates bone growth through cartilage cell proliferation. In vivo testing for BGH activity requires large sample volumes and numerous experimental animals, limiting our ability to rapidly screen samples from experiments seeking to purify and characterize the BGH molecule. Therefore, our goal was to determine the viability of an in vitro bioassay for quantifying BGH activity and examining the cellular signaling pathways it utilizes. RCJ3.1C5.18 chondrogenic cells were grown to 10d post‐confluence, and stimulated under serum‐free conditions with whole or fractionated BGH‐containing samples. Proliferation was assessed by BrdU incorporation, and levels and activation of ERK and MEK signaling molecules evaluated by Western blot. Cellular proliferation correlated with the amount of BGH shown previously to be present in the samples, and BGH activity was highest in low molecular weight fractions (<10kD). These same fractions increased ERK expression and phosphorylation, and tyrosine phosphorylation, with few consistent effects on active or inactive MEK. The data demonstrate that BGH can stimulate cartilage cell division in vitro, through the induction of well‐known signaling cascades associated with growth factor receptor activation. Support: APS Frontiers, NIH 5G12 RR008124, NIH 5R25 GM0490, HHMI 52005908