bluuebunny/crossref_metadata_2025 huggingface dataset:
Selective optogenetic stimulation of RTN neurons drives ventilation and active expiration in unanesthetized rats
Authors: Souza George M. , Stornetta Ruth L. , Stornetta Daniel S. , Abbott Stephen B.G. , Guyenet Patrice G.
Published: April 2020

Background

The retrotrapezoid nucleus (RTN) consists of neurons that are chemosensitive (i.e., CO2/pH sensitive), glutamatergic, Phox2b+ and Neuromedin B+ (Nmb+). Lentiviral vectors currently used to transduce RTN neurons with various opto‐ or pharmacogenetic actuators contain a Phox2b‐activated promoter, PRSx8. When injected into the RTN, these vectors also transduce substantial numbers of nearby C1 and catecholaminergic neurons. The purpose of the present study was to determine the effects on breathing elicited by selectively activating RTN neurons.

Methods

To selectively transduce RTN neurons we microinjected a mixture of Lenti‐PRSx8‐ChR2‐mCherry and AAV5‐FLEX‐taCasP3‐TEVp into the RTN of adult TH‐Cre rats. One month after the surgery, RTN neurons were stimulated with blue light (473 nm, 10 ms pulse, 20 Hz, for 20 s). All the measurements were conducted inside a plethysmography chamber. Diaphragm (DIA) and abdominal (ABD) muscle activity were recorded in conscious conditions. Results (mean ± SD), were analyzed using one‐ or two‐way ANOVA as required and differences were considered significant at P<0.05.

Results

RTN neurons (Nmb+, TH−) were selectively transduced (96.8 % ± 4) with the PRSx8 vector, the FLEX‐caspase AAV5 having caused the apoptotic death of C1 neurons (TH+) in the area where neurons were transduced with ChR2. Selective stimulation of RTN (NMB+ neurons; n=6) increased respiratory rate during wake and NREM sleep but not during REM sleep (Δ 30 ± 7 vs. 22.7 ± 6 vs. 4 ± 4 bpm from baseline), increased tidal volume also during quiet waking and non‐REM but not REM sleep (Δ 0.15 ± 0.07 vs. 0.13 ± 0.04 vs. 0.02 ± 0.007 mL/100g) and VE also during wake and non‐REM sleep but not REM (27.8 ± 11 vs. 21.4 ± 8 vs. 3.1 ±1 mL/100g/min). Selective RTN stimulation in rats breathing room air elicited active expiration (from 8.4 ± 7 to 15.9 ± 8 % of normalized abdominal EMG recorded at 9% FiCO2, n=3). RTN stimulation produced greater active expiration in rats exposed to 6% FiCO2 (from 56.3 ± 32 to 125.3 ± 104 % of normalized abdominal EMG).

Conclusions

Selective RTN neuron stimulation drives breathing amplitude and frequency during wake, NREM but not in REM sleep, and elicits active expiration, especially at elevated levels of FiCO2.

Support or Funding Information

Supported by grants from the National Institutes of Health (HL28785, 18 HL074011) and the Congenital Central Hypoventilation Syndrome Foundation to PGG.
Muscular Anatomy of the Forelimb of the Tiger, Panthera tigris
Authors: Dunn Rachel Heather , Beresheim Amy , Meachen Julie , Bitterman Kathleen , Butaric Lauren , Miller Dustin , Roje-Jones Lorraine , Bejes Katelyn
Published: April 2020

Understanding the comparative muscular anatomy of living carnivores is imperative to the reconstruction of locomotor and prey‐catching behavior in fossil carnivores. Further, because most fossils consist of only bones, detailed knowledge of the bony origin and insertion of muscles is necessary to map these muscles onto fossils for use in functional analyses. Whereas the muscular anatomy of the lion (Panthera leo) has been well documented by scholars in the late 19th and early 20th centuries, that of the tiger (Panthera tigris) is less well known; although numerous sources describe the muscular anatomy of the lion both in words and in images, there has been no published muscle map of lion or tiger limbs depicting the origins and insertions of the hind or forelimb muscles. The objective of this study was to create a muscle map of the origins and insertions of muscles acting on the forelimb of the tiger for use in comparative and paleontological research. We dissected the left forelimb of an 18‐year‐old female Amur tiger that died at the Blank Park Zoo in Des Moines, IA, documenting bony and soft‐tissue origin and insertion of all muscles present. Muscle weights were also collected. Because there is no published muscle map for extant large cats, we compared our origin and insertion data to the recently published description of the ocelot (Leopardus pardalis). The bony pattern of muscle origin and insertion is very similar between the tiger and the ocelot, suggesting a fairly conservative morphology among large terrestrial and medium‐sized scansorial felids. Where differences were present, most concerned relative area of muscle origins, rather than location. For example, unlike that of the ocelot, the anconeus of the tiger was not divisible into superficial and deep bellies and did not fuse with the extensor carpi ulnaris, instead partially fusing with the origin of the extensor to digits I and II. The right forelimb will be dissected in the future to provide insight into potential variation in tiger musculature.
Ontogenetic Trends in Frontal Sinus Shape
Authors: Butaric Lauren , Garvin Heather , Zimmerman Kristine
Published: April 2020

Adult frontal sinus morphology has been studied in biological anthropology, both in terms of its function and variation. Overall, this structure is known to be highly variable among individuals, to such an extent that it is used to make positive identifications in the medicolegal setting. However, very little is known about the growth and developmental processes leading to the wide ranges of frontal sinus size and shape. Although several studies investigate frontal sinus ontogeny in terms of size and linear dimensions, much less is known regarding the ontogeny of its shape. This study partially addresses this gap by examining frontal sinus outlines using longitudinal radiographic data to evaluate when the frontal sinus attains its adult shape.

Longitudinal radiographs for 107 individuals (57M/50F) spanning 5–29 years of age (yoa) were obtained from the AAOF Craniofacial Growth Legacy Collection (
www.aaoflegacycollection.org). Superior borders of frontal sinus outlines were digitally traced in ImageJ; the superior orbital border demarcated inferior sinus boundaries. Age of first appearance was also recorded. A total of 749 outlines were imported into SHAPE‐software, where elliptical Fourier (20 harmonics) and principal components (PC) analyses were conducted. PC1 (49.18% variation explained) reflects height‐to‐breadth relationships, while PC2 (11.74%) reflects asymmetry in right versus left lobe dominance.

To assess when the frontal sinus attains its adult shape, Euclidean distances of PCs 1–9 (91.58% cumulative variance) between each age point and the 18‐year point were calculated within individuals; this process was repeated for the 20‐year point. Scatterplots with Loess curves for each distance plotted against age suggest adult shape is attained around 20yoa. Sexual dimorphic trends are evident in the Loess curves, whereby males exhibit greater distances from 20yoa at early ages. An analysis of variance confirms these sex‐based differences (F=7.063, P<0.0001); Tukey post‐hocs further suggest that while adult females are not significantly different in shape from adult males or younger females/males (13–19yoa), adult males are significantly different from males 15yoa and younger. T‐tests failed to find significant sex‐based differences for age‐of‐first‐appearance (t=1.482, P=0.142) and adult‐shape (PC1: t=−0.670, P=0.505; PC2: t=1.827, P=0.058). Thus, it appears that while both sexes display similar start times in sinus development and reach similar adult‐shapes, females attain their adult shape sooner. This suggests a slower developmental process in males. Additional studies are needed to determine how these ontogenetic patterns in 2D sinus‐shape relate to the overall size and 3D dimensions of the sinus.
Developing a 3D Physical Model of HIV Protease to Explore Drug Resistance to Tipranavir and Darunavir
Authors: Dyrma Sherina , Van Acker Cyndi , Nooh Asha , Palacios Alessia , Martinez-Vaz Betsy
Published: April 2020

HIV infections affect over 38 million people worldwide and cause approximately one million deaths every year. Despite advances in antiretroviral therapies and disease prevention strategies, drug resistance remains a major challenge in HIV treatment. HIV protease plays a key role in viral replication by facilitating the peptide bond hydrolysis reactions necessary for the development of functional viral particles. Diverse inhibitors have been developed to target the active site and substrate envelope of HIV protease. While these drugs have been effective at inactivating the enzyme, mutations at the active site give rise to HIV resistance strains which are insensitive to protease inhibitors. The goal of this project was to create 3D printed models of HIV protease to illustrate the mechanisms of action of two inhibitors, Tipranavir and Darunavir, and the mutations involved in the development of resistance to these drugs. Database searches and sequence alignments were performed to identify conserved amino acids and structural features important in the catalytic mechanism of HIV proteases. Details of the protein structure and its interaction with Tipranavir and Darunavir were obtained by analyzing the Protein Databank Files: 6DIF, 6DGX, 6OPS, and 6DH6. To construct the physical models, the structure files were imported into Jmol and modified into a format suitable for 3D printing using scripts created by undergraduate researchers. The 3D models feature the overall structure of HIV protease, and its inhibitor molecules, Tipranavir and Darunavir. An active site box was constructed to illustrate the biochemical interactions between the enzyme and the inhibitors. The physical models also highlight key amino acids such as Asp25, Ile50, Val82, and Val84, which are critical for catalytic activity and the development of drug resistance. Magnetic pieces were created to show the effects of mutations in inhibitor binding and enzyme activity. A Jmol tutorial was designed to complement the 3D models and assess students’ learning of the structure and function of HIV protease.

Support or Funding Information

This project is funded in part by NSF‐IUSE #1725940 for the CREST Project.
Verification of aortic arch variations through modern imaging
Authors: Hirtler Lena , Tucheslau Moritz
Published: April 2020

Introduction

The aortic arch with its vessels offers one of the biggest varieties of branching patterns, which is barely beeing mentioned in modern medical textbooks though. The last big survey on the most common varieties had been published by Lippert in 1967 and, since then, not been verified, just republished. With these varieties being of great importance in certain fields of medicine, the up‐to‐dateness of Lippert’s data has been analysed.

Aim

The main objective is the analysis of CT‐Angiographies for branching varieties of the aortic arch and comparison of frequency distributions with Lippert 1967. The secondary objective is measuring the vessel lumen of varieties and comparison with matches of the standard variant to find out if varieties have influence on the vessel’s lumen.

Material/Methods

This retrospective cross‐sectional study includes 400 patients, more specificaly their CTAs from 01.01.2017 to 31.12.2017, obtained from the University Department of Biomedical Imaging and Image‐guided therapy, General Hospital Vienna. Incusion and exclusion criteria have been differently set up for main and secondary objective. Patients for the main objective have been excluded if they have had any kind of surgery altering their varieties, like bypass‐surgery. Patients for the secondary objective have have been excluded if they were under the age of 18 or have had any kind of diseases/surgery altering the supraaortic vessel’s lumen.

The main objective has been represented with tabular comparisons. For the secondary objective patients not fulfilling the criteria have been excluded at first. Afterwards the different groups of varieties have been matched with groups of the standard variant similiar in age and distribution of sex. The vessel’s lumen then has been analysed and t‐tested.

Results

The analysis and comparison of data in terms of frequency have been proved statistically in‐significant. Testing of V2_TBv with V1_TB (p<0,001; α=0,05) as well as V2_ACCS with V1_ACCS (p<0,001; α=0,05) have shown statistical significance.

Conclusion

Despite minor deviation after analysis with modern imaging the data of Lippert from 1967 can still be seen as up‐to‐date. In vascular surgery it may be neccessary to be considerate about the vessel lumen in case of any known variety of the branches of the aortic arch. Certain questions may ask for more detailed analysis.
LUDIC ANATOMY PROMOTES LONG‐THERM KNOWLEDGE RETENTION
Authors: Maia Jad Gabriele Silva , Kubrusly Bruna Sobreira , Filho Cezar Nilton Rabelo Lemos , Freitas Francisco Orlando Santos Rafael , Barreto João Erivan Facanha , Cerqueira Gilberto Santos Facanha
Published: April 2020

The project Ludic Anatomy is a learning and teaching tool used in the Medical, Pharmacology, Dentistry and Biology courses of the Federal University of Ceará. Concerning to the benefits of ludic activities, they make the teaching and learning process more dynamic and interesting, by stimulating the active studying of the muscular system and creating a playful method which is greatly different from the orthodox classes, they also have a positive influence in the memorizing development of anatomical terms. Considering the method of the ludic activity, the students used pins to mark and, then, identify fifty different muscles of the doll. Therefore, the objective of this study is to investigate if the use of toys in the anatomy’s learning and teaching process promotes long therm (6 months) knowledge retention. It was realized a descriptive exploratory study with quantitative approach with 61 medical students who participated of the project. To collect data, a semi‐structured questionnaire was applied at the time of activity and lasted six months. For statistical analysis, the Mann Whitney test was considered significant when presented the p<0,05. The mean age of the participants was 19,97 ± 0,46 for males and 19,74 ± 0,37 for females, there was not an important difference between the ages. A statistically significant difference was observed in the knowledge retention when compared the correct (7,69 ± 0,18) and wrong (1,30 ± 0,18) answers. A negative correlation (R −0,057) between the time spent studying and the numbers of questions right was observed. In conclusion, it was verified that the Ludic Anatomy promotes learning and the long‐therm knowledge retention. That project’s advantage demonstrates the importance of the ludic activity as an effective learning tool in the improvement of the traditional teaching of Anatomy.

Support or Funding Information

CNPQ
Kctd15 Regulates Nephron Segment Differentiation by Repressing Tfap2a Activity
Authors: Chambers Brooke , Clark Eleanor , Gatz Allison , Wingert Rebecca
Published: April 2020

A functional vertebrate kidney relies on structural units called nephrons, which are epithelial tubules that contain a sequence of segments each expressing a distinct repertoire of solute transporters. To date, the transcriptional codes driving regional specification, solute transporter program activation, and terminal differentiation of segment populations remains poorly understood. We demonstrate for the first time that the KCTD15 paralogs, kctd15a and kctd15b, function in concert to restrict distal early (DE)/thick ascending limb (TAL) segment lineage assignment in the developing zebrafish pronephros by repressing Tfap2a activity. During renal ontogeny, expression of these factors co‐localized with tfap2a in distal tubule precursors. kctd15 loss primed nephron cells to adopt distal fates by driving expansions in slc12a1, kcnj1a.1, and stc1 marker expression. These phenotypes were resultant of Tfap2a hyperactivity, where kctd15a/b‐deficient embryos exhibited increased abundance of this transcription factor. Interestingly, tfap2a can reciprocally promote kctd15 transcription, unveiling a circuit of autoregulation operating in nephron tubule progenitors. Concomitant kctd15b knockdown with tfap2a overexpression produced genetic synergy and uncontrolled DE/TAL differentiation. Our data indicates nephron segmentation is determined by a transcription factor‐repressor feedback module that employs tight regulation of Tfap2a‐Kctd15 kinetics during kidney development.
Investigating the Binding of Albumin and C‐peptide to Red Blood Cells
Authors: Geiger Morgan , Zinn Kurt , Spence Dana
Published: April 2020

The pancreatic β‐cells of healthy individuals secrete insulin and C‐peptide, a 31‐amino acid peptide, in a 1:1 mole ratio. However, the β‐cells of people with type 1 diabetes (T1D) are damaged or destroyed. Therefore, exogenous insulin is required for people with T1D to survive. Even with regular insulin treatments, patients develop complications such as retinopathy, neuropathy, and nephropathy. Research from our group demonstrates that C‐peptide increases microvascular blood flow, therefore, it could be a useful tool to decrease these complications due to poor blood flow. However, the unknown C‐peptide receptor and mechanism has been a major roadblock in utilizing C‐peptide as a therapeutic. Utilizing ELISA, our group has demonstrated that approximately 1,800 C‐peptide molecules bind per red blood cell (RBC) in the presence of albumin. Without albumin, there was no detectable C‐peptide binding per RBC. Rather than C‐peptide solely binding to a RBC receptor, we hypothesize that C‐peptide binds to RBCs through an albumin/C‐peptide complex receptor. Bovine serum albumin (BSA) binding per RBC was detectable through an attached gamma decay radiolabel (99mTc). A binding saturation experiment was conducted to examine the specific binding of BSA to RBCs with and without C‐peptide. The specific binding curves revealed that albumin saturates at 14,021 ± 1489 BSA molecules/RBC with a Kd of 1.07 (±0.19) × 10−7 M and a Bmax of 1.94 (±0.06) × 10−8 M, or 13,900 receptor molecules/RBC. Whereas, in the presence of C‐peptide and zinc, albumin saturates at 16,696 ± 1479 BSA molecules/RBC with a Kd of 1.91 (±0.09) × 10−7 M and a Bmax of 2.50 (±0.03) × 10−8 M, or 17,900 receptor molecules/RBC. At saturation, the additional 2,700 BSA molecules binding per RBC in the presence of C‐peptide and zinc indicates that an albumin receptor exists on RBCs as well as a separate albumin/C‐peptide complex receptor.

Support or Funding Information

NIH
Role of astrocytes in cerebrovascular protection provided by an opioid agonist, biphalin during ischemic stroke
Authors: Nozohouri Saeideh , Albekairi Thamer , Zhang Yong , Vaidya Bhuvaneshwar , Abbruscato Thomas
Published: April 2020

Biphalin, a dimeric enkephalin analog (Tyr‐D‐Ala‐Gly‐Phe‐NH‐)2 with potent, nonselective opioid receptor agonist activity, has shown to be neuroprotective in both in vitro as well as in vivo ischemic stroke models. Here we aimed to understand the role of biphalin on blood‐brain barrier (BBB) integrity during ischemic stroke. In this study, we used mouse brain endothelial cells, bEnd.3 and primary astrocytes as monoculture and co‐culture in‐vitro BBB models. Restrictive BBB properties were evaluated by measuring trans‐endothelial electrical resistance (TEER) and [14C] sucrose paracellular permeability, and the redistribution of the tight junction protein occludin. The protective effect of biphalin on BBB integrity was assessed after exposing cells to oxygen‐glucose deprivation (OGD) (1% O2, 2 hours) and glutamate (2mM). Naltrexone, a non‐selective opioid antagonist was used to determine opioid receptors dependent activity of biphalin. Further, we measured the effect of biphalin on astrocytic glutamate uptake and on the expression of excitatory amino acid transporter (EAAT) to study the indirect role of astrocytes on biphalin mediated BBB protection. It was found that combined stress (2mM glutamate and 2 hours OGD) significantly increased the [14C] sucrose paracellular permeability in bEnd.3 cells as well as in a co‐culture cell model. Biphalin (100 nM) treatment attenuated the effect of the combined stress which was reversed by the opioid antagonist naltrexone (100 nM), suggesting the role of opioid receptors in biphalin activity. It was also observed that combined stress (2mM glutamate and 2 hours OGD) condition, significantly reduced the glutamate uptake by astrocytes however, biphalin treatment increased glutamate uptake in primary astrocytes, suggesting the role of astrocytes, increased buffering capacity, in biphalin meditated protection of the BBB during ischemic stroke.

Support or Funding Information

Texas Tech University Health Science Center
MAGEA9: Driver or Passenger in CARcinogenesis
Authors: Johnson Jodiene
Published: April 2020

Type I Melanoma Antigen Genes (MAGEs) are a family of genes that are normally expressed in the spermatogonial cells of the testis and abnormally reactivated in a broad variety of cancers. Due to this expression pattern and the immune response they elicit when abnormally expressed, type I MAGEs are also known as Cancer Testis Antigens (CTAs). Melanoma Antigen Genes‐A (MAGE‐A) are CTAs whose expression in cancers have been correlated with poor patient prognosis. The biological function of several members of the MAGE‐A gene family in cancer remains unclear. Ectopic expression of type I MAGEA9 in normal cells results in increased cellular proliferation. We therefore hypothesize that MAGEA9 is needed for sustenance of proliferative phenotypes. Our goals are to elucidate the molecular mechanism by which MAGEA9 increases cellular proliferation and the mechanism(s) that regulate the expression of MAGEA9. We have employed RT‐qPCR array for cancer‐associated genes to determine signaling pathways modulated by expression of MAGEA9. Results from our RT‐qPCR array indicate that MAGEA9 expression causes an upregulation in the DNA mismatch repair genes MSH6 and MSH2 and the E3 ubiquitin ligase MDM2. To identify MAGEA9 binding partners we have used IP‐mass spectrometry, and our data indicate that MAGE‐A9 binding partners are involved in invasion. We will use a combination of in silico promoter analyses and chromatin immunoprecipitation (ChIP) to determine the transcription factor (s) regulating expression of MAGEA9. Collectively, these data provide a clearer understanding for the function of MAGE‐A9 in cancer. Elucidating the role of MAGEs in cancer may provide insight for the development of novel therapeutic interventions.

Support or Funding Information

NSF Research Initiation Award to S.R. #HRD1764201

Department of Education Award # P382G170104 (TITLE‐VII)
Usefulness of high‐fluorescence body fluid cell count (HF‐BF %) in Automated hematology analyzer SYSMEX XN‐350 for screening abnormal cells in body fluid analysis
Authors: Lim Jihyang
Published: April 2020

Background

Automated hematology analyzer Sysmex XN‐350 is operated by two analytical software, whole blood‐mode and body fluid mode. The body fluid mode can be counted the cells twice as many as than whole blood mode and it is suitable for body fluid analysis which the sample has low cell count. XN‐350 is calculated the cell counts including total nucleated cell, WBC, RBC, mononuclear(MN) cell, polymorphonuclea(PMN) leukocyte and differentiated MN%, PMN%, high‐fluorescence body fluid cell(HF‐BF)%. The cell types in HF‐BF are macrophages, mesothelial cells, synovial cells and other abnormal cells. Higher HF‐BF% means higher abnormal cells. We evaluated the correlation HF‐BF % by XN‐350 to microscopic examination.

Methods

Five hundreds twelve body fluid samples enrolled in this study. All samples analyzed HF‐BF % by XN‐350 instrument and the results compared to microscopic examination. In preliminary study, fifty‐two body fluid samples analyzed by XN‐350. Twelve samples in all samples revealed less than 1% HF‐BF and they were all normal cells by microscopy, and then we decided the cut‐off level for manual slide review was above 1.0% HF‐BF. We analyzed abnormal cell detection rate according to HF‐BF %.

Results

In five hundreds twelve samples, two hundreds eighteen samples(42.6%) were less than 1% HF‐BF and two hundreds nighty‐four samples(57.4%) were more than 1% HF‐BF. Twenty‐seven samples in more than 1% HF‐BF were detected clinically significant abnormal cells by microscopy. In according to the grade of HF‐BF %, 1.0~4.9% HF‐BF was one hundred thirty‐two samples including three samples(2.3%) that detected abnormal cells by microscopy, 5.0~9.9% HF‐BF was fifty‐one samples including six samples(11.8%) that detected abnormal cells by microscopy, 10.0~19.9% HF‐BF was fifty‐eight samples including six samples(10.3%) that detected abnormal cells by microscopy, more than 20.0% HF‐BF was fifty‐three samples including twelve samples that detected abnormal cells by microscopy. Therefore, higher HF‐BF % assumed higher detection rate of abnormal cells by microscopy.

Conclusion

HF‐BF % in SYSMEX XN‐350 instrument is useful for screening of abnormal cells in body fluid analysis. Cut‐off level of above 1% HF‐BF can be detected majority abnormal cells and decreased microscopic examination rate.
Differential effects of opioid versus alcohol dependence on pain avoidance behavior and regional endocannabinoid system deficiency
Authors: Cucinello Jessica Ann , Berner Melissa E. , Pahng Amanda R. , Edwards Kimberly N. , Edwards Scott
Published: April 2020

In contrast to their analgesic effects, chronic use of opioids or alcohol often creates the paradoxical emergence of heightened pain sensitivity, termed hyperalgesia, during withdrawal. A hallmark symptom of hyperalgesia is the development of motivational strategies to avoid painful stimuli, and we have shown that adult male Wistar rats display pain avoidance behavior using the mechanical conflict avoidance system when in withdrawal from morphine (n=10–11/group) and alcohol (n=8–9/group). The present study seeks to identify how morphine and alcohol differentially affect pain avoidance behavior and endocannabinoid signaling within pain‐ and avoidance‐ associated brain regions, including the periaqueductal grey (PAG) and basolateral amygdala (BLA).

Morphine dependence was induced via daily subcutaneous morphine injections (escalating dose of 10–20mg/kg) over 3 weeks. Alcohol dependence was induced via exposure to chronic intermittent ethanol vapor (CIEV) for 14 hours per day for 8 weeks. Pain avoidance behavior was quantified using the MCS as the latency for rats to exit a brightly lit start chamber onto nociceptive probes of varying heights (0–5mm) to enter a dark goal box. Western blotting was conducted for quantification of endocannabinoid system‐related proteins cannabinoid receptor 1 (CB1R) and eCB synthetic and catabolic enzymes diacylglycerol lipase alpha (DAGLa), monoacylglycerol lipase (MAGL).

We found that morphine and alcohol dependence produced distinct patterns of pain avoidance behavior, where morphine dependence produced a hyperalgesic behavior and ethanol dependence produced an allodynic phenotype. This behavior negatively correlated with PAG levels of CB1R and DAGLa in morphine dependent animals and with the ratio of BLA DAGLa:MAGL in alcohol dependent animals, reflective of a decreased eCB tone in these animals. This suggests that exogenous cannabinoid treatment may be an effective therapy for substance‐induced hyperalgesia. Because substance‐induced hyperalgesia and negative affective components of pain are integral to addiction reinforcement, targeting these behaviors therapeutically could treat substance abuse.

Support or Funding Information

NIAAA: T32AA007577, R00AA020839, R01AA025996, R21AA025736, NIH‐NICHD‐2018‐05
CblX is a New Cobalamin Syndrome Affecting Craniofacial Development
Authors: Achilleos Annita , Chern Tiffany , Tong Xuefei , Hill Matthew C. , Chaudhury Arindam , Reineke Lucas C. , Neilson Joel , Sun Jenny , Ray Russel S. , Watkins David , Thiagarajan Perumal , Dasgupta Swapan K. , James Martin F. , Poché Ross A.
Published: April 2020

Combined methylmalonic acidemia and homocystinuria (cblC type), an inherited disorder of cobalamin (vitamin B12) metabolism, is a rare metabolic and multi‐systemic disease caused by mutations in MMACHC. Patients with cblC can have severe neurodevelopmental defects including microcephaly, hydrocephaly, and seizures as well as renal, cardiac and hematological defects. Recently, two new variants of cblC were discovered and termed cblX and cblX‐like. Rather than being due to mutations in MMACHC, cblX and cblX‐like result from homozygous mutations in the transcription cofactor HCFC1 and its transcription factor partner RONIN (THAP11), respectively. Patients with either HCFC1 or RONIN mutations were shown to have a dramatic reduction in MMACHC transcription. We have known that HCFC1 is an obligatory partner for RONIN and we have previously shown that the HCFC1/RONIN transcriptional complex directly regulates mouse Mmachc expression. These findings suggest that cblX and cblX‐like disorders comprise a novel family of rare and severe cblC‐like disorders that are transcriptional in nature. To better understand the cellular and molecular mechanisms underlying the pathophysiology of these devastating neurodevelopmental diseases, we have generated Hcfc1A115V and RoninF80L mice, which have the same point mutations observed in cblX and cblX‐like patients. Both Hcfc1Y/A115V hemizygous and RoninF80L/F80L homozygous mice exhibit defects in cobalamin metabolism consistent with an inherited vitamin B12 disorder, as well as defects that recapitulate those observed in the human cblX and cblX‐like patients, including severe brain developmental defects, cardiac malformations, and anemia. Additionally, these mice also exhibit craniofacial deformities, a developmental phenotype that has not been previously linked to vitamin B12. Our data identifies, for the first time, a role for RONIN/HCFC1 in craniofacial development. Together, the phenotypic and molecular data confirm that the RoninF80L and Hcfc1A115V mouse models will serve as powerful tools to further uncover the pathophysiology of this complex family of diseases, as well as to achieve a better understanding of the complexities of craniofacial development.

Support or Funding Information

This work is supported by R01 DE028298.
PGE2 is a Candidate Distal Nephron Remodeling Factor
Authors: Zapf Ava , Grimm P.R. , Al-Qusairi Lama , Delpire Eric , Welling Paul A.
Published: April 2020

We recently discovered that DCT1 cell‐specific expression of constitutively active SPAK (CA‐SPAK) drives a remodeling process of the entire distal nephron that includes atrophy of the aldosterone‐sensitive distal nephron and parallel inhibition of ROMK and ENaC (Grimm et al, JASN ’17). The mechanism by which genetic activation of NCC drives downstream nephron remodeling remains elusive but observations that PGE2 inhibits ROMK and ENaC ex vivo raises that paracrine signaling may be involved (Jin et al, AJPR 2007; Guan et al, JCI 1998). Here, we tested the hypothesis that activation of NCC causes atrophy of the CNT through increased PGE2 synthesis and release.

We found increased levels of PGE2 in kidney cortical homogenates of CA‐SPAK mice compared to control mice on a control diet. Feeding wild‐type mice (WT) a low potassium diet (LKD) to physiologically activate NCC resulted in a comparable elevation of PGE2 and ASDN remodeling response, characterized by a decrease in ROMK/ENaC. Administration of the NCC‐blocking diuretic, hydrochlorothiazide, to CA‐SPAK mice restored PGE2 to control levels but had no effect on PGE2 of control or SPAK KO mice. Thus, PGE2 is elevated in response to super activation of NCC.

PGE2 synthesis is controlled by specific prostaglandin E synthase isoforms. As revealed in western blot analysis, the microsomal prostaglandin E synthase‐1 (mPGES1) was selectively elevated in the kidney cortex of CA‐SPAK mice and WT mice on LKD compared to control mice, and this response was blocked by thiazide diuretics.

Localization of mPGES1 by quantitative microscopy surprisingly revealed that increased protein abundance of mPGES1 was confined to the CNT and CCD of CA‐SPAK or the DCT2 and CNT of LKD treated mice. No significant changes in mPGES1 were observed in the DCT1.

In conclusion, these studies identify PGE2 as a possible remodeling factor that is released from the late DCT and ASDN in response to NCC activation. We speculate that decreased sodium delivery to the CNT and CCD when NCC is activated drives the response; similar to how low NaCl drives PGE2 synthesis in the macula densa. The paracrine/autocrine pathway provides a novel means to communicate potassium sensing in the DCT to potassium secretion in the ASDN by driving tubule remodeling of specific segments of the distal tubule.

Support or Funding Information

NIH, NIDDK T32 and Fondation LeDucq
Altered Water Handing in Mice Following Chronic Aldosterone Administration
Authors: Hasan Mahpara , Mutchler Stephanie M. , Kleyman Thomas R.
Published: April 2020

Aldosterone is a steroid hormone that is necessary for sodium and water reabsorption in the distal tubules and collecting ducts of the kidney that is released in response to changes in volume and potassium levels. While aldosterone is typically associated with fluid retention due to its role in promoting sodium reabsorption, previous work has shown that aldosterone further exacerbates urine production in a rat model of diabetes insipidus. Because of this, we aimed to understand the effects of chronic aldosterone administration in mice with no underlying pathology to further elucidate the effects of chronic aldosterone administration on water reabsorption in the kidneys. Aldosterone was administered at a dose of 240 μg/kg/day via subcutaneous minipump to C57Bl/6 male mice housed in metabolic cages. By Day 15, mice given aldosterone had increased urine output compared to control animals (Ctl 4.8 ± 1.6 mL; Aldo 8.4 ± 2.4 mL), and this difference persisted through day 28. The aldosterone treated animals trended toward drinking more water starting at day 7, however significance was not obtained. To understand whether these animals were producing concentrated urine, we measured urine osmolality by freeze‐point depression. By Day 4, there was a significant decrease in daytime urine osmolality in the aldosterone administered mice compared to the control mice (Ctl 1219.8 ± 473.4 mOsm/kg; Aldo 638.8 ± 143.8 mOsm/kg), and by day 17, there was also a significant decrease in nighttime urine osmolality of the aldosterone treated versus control mice (Ctl 905.3 ± 175.1 mOsm/kg; Aldo 522.2 ± 127.3 mOsm/kg). Mice receiving aldosterone had a higher whole blood [Na+] (Ctl 146 ± 1.9mmol/L; Aldo 152 ± 1.3mmol/L) and lower whole blood [K+] (Ctl 4.8 ± 0.56mmol/L; Aldo 3.0 ± 0.34mmol/L) as measured by iSTAT cartridge after terminal cardiac blood draw. At a molecular level, a significant increase (~3.5 fold) in Aqp4 whole kidney mRNA levels were measured in the aldosterone treated mice compared to the control mice at both 2 weeks and 4 weeks of treatment. Additionally, Aqp1 and Aqp3 mRNA levels trended toward being lower in the aldosterone treated animals although significance was not reached. Aqp2 levels were variable, but unchanged at a transcript level. Protein expression and localization of these aquaporins are currently being investigated by Western blot and immunofluorescent staining. Together, these data indicate that chronic aldosterone administration causes a diuresis with decreased concentrating ability. We are currently testing whether correction of the serum [K+] corrects the concentrating defect, and whether vasopressin is involved in these responses.
Branched Photoswitchable Tethered Ligands for Optical Interrogation of Metabotropic Glutamate Receptor‐Mediated Modulation of Prefrontal Cortex Circuits
Authors: Gutzeit Vanessa A. , Ruiz Amanda Acosta , Kristt Melanie , Liston Conor , Broichhagen Johannes , Levitz Joshua
Published: April 2020

G protein‐coupled receptors (GPCRs) form a large family of membrane bound receptors that initiate intracellular responses which control cellular signaling cascades to contribute to a wide range of biological functions. The metabotropic glutamate receptors (mGluRs) consist of an eight‐member family of class C GPCRs that respond to the excitatory neurotransmitter glutamate and serve as promising pharmacological targets for a range of neurological and psychiatric disorders. Studies targeting mGluR2 and mGluR3, the group II Gi/o‐coupled subfamily of mGluRs, have shown that they are strong modulators of prefrontal cortex (PFC) activity and associated behaviors. However, the lack of spatiotemporal precision and specificity of classical pharmacological and genetic approaches has limited our mechanistic understanding of these receptors. Here we report an improved chemical optogenetic toolkit that permits the reversible and repeatable activation of group II mGluRs with genetic targeting, subtype‐specificity and spatiotemporal precision in vivo. Through introduction of branches and tuning of azobenzene photophysics, we have produced a family of tethered photoswitchable glutamate compounds that enable dual control and visualization of SNAP‐, CLIP‐ or Halo‐tagged mGluRs in vivo with high efficiency. We use these tools to probe the effects of mGluR2 activation within different cell‐types and projections of the medial PFC while probing behaviors associated with anxiety, depression and cognitive disorders. Using these tools in a Grm2‐Cre mouse, we characterize the projection pattern of mGluR2‐expressing cells and find that photoactivation of mGluR2 within the prelimbic cortex enables rapid and reversible modulation of working memory, including alleviation of cognitive deficits in a pharmacological model of psychosis.
A putative GSK3β binding site in the aquaporin 1 (AQP1) C‐terminal tail controls pulmonary arterial smooth muscle cell (PASMC) function.
Authors: Yun Xin , Jiang Haiyang , Philips Nicolas , Shimoda Larissa
Published: April 2020

Pulmonary hypertension (PH) is a devastating disease, characterized by functional and structural pulmonary vascular changes. In distal, small diameter pulmonary arteries, PASMC migration and proliferation are important contributors to the development of PH. In past studies, we showed that hypoxia, a stimulus for PH, elevated the expression of the water channel, aquaporin 1 (AQP1). We also showed that AQP1 induced PASMC migration and proliferation via a mechanism involving β‐catenin protein accumulation, which required the AQP1 C‐terminal tail but was independent of water transport. In this study, we wanted to elucidate the mechanism underlying AQP1‐mediated regulation of β‐catenin in PASMCs. β‐catenin can play various roles in the cell, and its protein levels are tightly regulated by the serine/threonine kinase, glycogen synthase kinase 3 β (GSK3β). Under control conditions, GSK3β is an important factor regulating the turnover of β‐catenin, binding to β‐catenin and targeting it for degradation. Recently, we performed in silico analysis of the AQP1 C‐terminal tail, and found in the proximal portion of the tail a potential binding site for GSK3β. Thus, we explored whether mutating the putative GSK3β binding site on the C‐terminal tail of AQP1 alters PASMC migration and proliferation and, if so, whether β‐catenin was involved. Primary cultured rat distal PASMCs were harvested and used for experiments. Cells were infected with adenoviral constructs expressing wild‐type AQP1 (AdAQP1), AQP1 with the putative GSK3β binding site mutated (AdAQP1M), or green fluorescent protein (AdGFP), as a control for infection. Infection with AdAQP1 and AdAQP1M increased total and surface AQP1 protein levels compared with AdGFP. The rate of change in water flux into PASMCs, tested by a water permeability assay using calcein self‐quenching, indicates that both AQP1 constructs function similarly as a water channel. While infection with AdAQP1 increased PASMC migration and proliferation, measured by BrdU incorporation and transwell assays, respectively, infection with AdAQP1M had no effect. Our results suggest that AQP1 controls PASMCs migration and proliferation via a mechanism that requires specific residues within the tail region that may form a putative GSK3β binding site. With increased AQP1 levels, as during PH, we think GSK3β is sequestered, β‐catenin escapes degradation, translocates to the nucleus and initiates transcription of the target genes involved in cell proliferation and survival.

Support or Funding Information

This work was funded by HL12651, 18POST34030262
Participation of the pvd Gene Cluster in the Mechanism of Quorum Sensing and Virulence of Pseudomonas aeruginosa PAO1
Authors: Perez Sharel Pamela Diaz , Peña Aide Campos Espino , Perez Alma Laura Diaz , Alcántar Lorena Martínez , García Jesús Campos
Published: April 2020

Objective

Determination of the molecular mechanism involved in the virulence modulation of Pseudomonas aeruginosa PAO1 involving the pvdI and pvdL genes.

Methods

Supernatants of the P. aeruginosa PAO1 strain and mutants pvdI, pvdJ, and pvdL were obtained from LB liquid medium after 24 h of growth. Evaluation of virulence factors: biofilm was determined by growth of P. aeruginosa strains in 96‐well plates using violet crystal staining. Rhamnolipids production was determined by quantification of rhamnose by the orcinol method. Survival assays were carried out in Caenorhabditis elegans using supernatants of PAO1 strains.

Pseudomonas aeruginosa is an opportunistic pathogenic bacterium for humans, animals and plants. This bacterium possesses a system of perception of bacterial quorum (quorum sensing, QS), which modulates several virulence factors. The LasI/LasR QS system modifies the production of the cyclodipeptides (CDPs) cyclo(L‐Pro‐L‐Val), cyclo(L‐Pro‐L‐Phe) and cyclo(L‐Pro‐L‐Tyr). Although CDPs are molecules with bioactive properties, low elements concern in the mechanism of biosynthesis in bacteria and their role in communication with host cells. In addition, it has been described that the CDPs production in P. aeruginosa mainly depends of the multi‐modular non‐ribosomal peptide synthetases (MM‐NRPS), whose products such as the aeruginaldehyde has been involved in the bacterial QS mechanisms. In this study, was found that levels of pyocyanin (a virulence factor in P. aeruginosa), it was increased in mutant strains in the pvdJ gene, which encodes for a NPRS. The pvd gene cluster is directly related to the pioverdin synthesis, which is a fluorescent peptide‐siderophore involved in the iron acquisition, being essential in the bacterial pathogenicity on the host. Therefore, is important to elucidate the mechanism by which the CDPs of P. aeruginosa PAO1 contribute to its pathogenesis and/or virulence. Production of several virulence factors in P. aeruginosa PAO1 with different single and double mutants in pvdI, pvdJ and pvdL genes were compared. Mutants in the pvdI, pvdL and pvdL/pvdI genes decrease 50% the production of biofilm respect to the WT strain. Likewise, when evaluating rhamnolipids amount, the production of rhamnolipids was significantly increased in the single mutants pvdJ and pvdL compared with the WT strain, but not modified in the double mutant pvdJ/pvdL. Survival in the C. elegans worms indicated that the pvdJ mutant increase the death of the nematode 40% than the WT. The results suggest that the pvd gene cluster was involved in the production of CDPs in P. aeruginosa PAO1 and may to regulate the production of virulence factors (biofilm and rhamnolipids), impacting its pathogenicity on the nematode in vivo model.
The Stability and Localization of Tetrahymena thermophila UBC9 Mutants
Authors: Meeks Kaylen , Forney James
Published: April 2020

UBC9 is the sole E‐2 conjugating enzyme in the SUMOylation pathway, which is analogous to the ubiquitin pathway. SUMOylation can affect the localization of proteins or their ability to interact with other substrates. UBC9 is essential in most eukaryotes including the ciliate Tetrahymena thermophila. Tetrahymena thermophila are protozoa with two nuclei, a somatic macronucleus (MAC) and germline micronucleus (MIC). Transcription occurs in the macronucleus, but not in the micronucleus. Tetrahymena have a large increase in SUMOylation within the developing macronucleus during conjugation (sexual reproduction). In an effort to create UBC9 mutants with altered activity or cellular location, we designed site‐directed changes to the UBC9 gene. Some of these changes were analogous to mutations identified in yeast UBC9, others were directed at the N‐terminus that is conserved between Tetrahymena thermophila and other ciliates, but is substantially different from other eukaryotes. Plasmids containing YFP fusions of UBC9 with small deletions in the N‐ or C‐ terminus or single amino acid changes were transformed into Tetrahymena and viewed via fluorescence microscopy to determine localization. Constructs with the same mutations but N‐terminal FLAG tags were analyzed via Western blots to determine stability of the mutant proteins. Versions of UBC9‐YFP fusions with 13 amino acids removed at the N‐terminus were not detectable, or barely detectable by microscopy as compared with wild type UBC9‐YFP fusions. A YFP fusion with only 4 amino acids removed from the N‐terminus was visible with a similar intensity as wild type (full length) controls. In contrast, removal of 4 amino from the C‐terminus eliminated all signal from expression of a YFP‐fusion. The tagged versions of UBC9 with 13 and 4 amino acids of the protein removed at the N‐terminus produced a signal on a Western blot; while a UBC9 construct with 4 amino acids removed from the C‐terminus was not able to produce a signal on a Western blot. To our surprise, removing amino acids from the C‐terminus had a greater effect on protein stability than removing those from the N‐terminus. Analysis of two other mutations (R20A, R24D) predicted to create altered cellular location or stability of the protein showed small, if any differences from wild type UBC9 in the distribution between cytoplasm and nucleus. Additional mutations are being constructed in the C‐terminal region in an attempt to generated UBC9 with marginal stability that could be used for in vivo analysis of function.

Support or Funding Information

Department of Biochemistry, Purdue University, West Lafayette, INCollege of Agriculture (Molecular Agriculture Summer Institute), Purdue University, West Lafayette, IN