bluuebunny/crossref_metadata_2025 huggingface dataset:
Utilization of fresh tissue in the Procedural Skills Laboratory at Mayo Clinic, Rochester, MN.
Authors: Regnier Terry David
Published: June 2021

The Department of Anatomy at Mayo Clinic, Rochester, MN serves medical students, allied health profession students, residents, fellows, Mayo Clinic consultants and partners with Mayo School of Continuing Medical Education with an opportunity to learn anatomy and to practice surgical procedures and approaches, to develop and improve new techniques on unembalmed fresh tissue. The Procedural Skills Laboratory's (PSL) flexible design allows diverse usage of lab space for multiple small groups as well as large groups of up to 100 participants. The PSL provides an opportunity for residency directors to incorporate surgical and procedural training in a "safe" environment from which learners can advance from traditional "passive" learning to active participation in complicated surgical procedures. An example of such activities include courses in arthroscopic and laparoscopic procedures, pelvic and thoracic dissection utilizing Da Vinci surgical robot, central line placement, airway management, and many others. This hands on active training provides all lab participants with an opportunity to acquire knowledge, skills and competencies which directly translate to increased quality of patient care.
Phosphatidylinositol 4,5‐bisphosphate (PIP2) stimulation of electrogenic Na/bicarbonate cotransporter NBCe1 variants expressed in Xenopus laevis oocytes
Authors: Wu Jianping , Liu Xiaofen , Thornell Ian M. , Bevensee Mark O.
Published: June 2021

Signaling molecules such as PKA/cAMP, PKC, and ATP modulate the activity of NBCe1. We recently discovered that cytosolic PIP2 stimulates heterologously expressed rat NBCe1‐A in inside‐out macropatches excised from oocytes. To characterize the effect of PIP2 on the activity of the three rat NBCe1 variants (‐A, ‐B, and ‐C), we used the 2‐electrode, voltage‐clamp technique to monitor repetitive HCO3−‐induced outward currents (INBC) at ‐60 mV before and after injecting NBCe1‐expressing oocytes with PIP2 (10/20 μM, estimated final). Injecting PIP2 into an oocyte expressing NBCe1‐C caused a progressive rise (over ~15 min) in INBC and a peak stimulation of 146±13% (n=10) of the initial pre‐injection INBC, followed by a partial decline. Similar results were obtained with oocytes expressing NBCe1‐B (same N terminus), but not NBCe1‐A (different N terminus), which exhibited a stimulation of only 9±1% (n=4) after 10 min. For the C variant, injecting H2O or low levels of PIP2 (~1 μM) had little effect on INBC, whereas injecting IP3 (~10/20 μM) modestly stimulated INBC by 39±15% (n=4) after ~15 min. The PIP2 stimulation of INBC was reduced in oocytes expressing an NBCe1‐C missing its N‐terminal 87 residues. In summary, PIP2 differentially modulates whole‐cell currents elicited by NBCe1 variants expressed in oocytes, and the N terminus found in the B and C variants contributes to injected PIP2 stimulation of NBCe1 activity. Altered regulation of NBCs by PIP2 during energy‐deficient conditions may contribute to changes in intracellular pH, Na+, and Ca2+ with hypoxia and ischemia. NIH NS046653; AHA 0755255B
Topology Determination of the Transmembrane Domain of the Electrogenic Sodium Bicarbonate Cotransporter NBCe1‐A
Authors: Zhu Quansheng , Kao Liyo , Liu Weixin , Newman Debra , Kurtz Ira
Published: June 2021

NBCe1‐A electrogenically cotransports sodium and bicarbonate across the basolateral membrane of proximal tubule cells. The illusive transport mechanism of NBCe1‐A requires structural analysis of the transport protein. NBCe1 has been proposed to traverse the lipid bilayer at least 10 times based on in vitro glycosylation assay. To further determine the topology of NBCe1‐A in vivo, we performed cysteine scanning mutagenesis coupled with sulfhydryl specific chemistry. Fifty six cysteine residues were individually introduced throughout a modified NBCe1‐A protein with five cytoplasmic endogenous cysteines mutated to serine. Location of introduced cysteine residues were determined by whole cell labeling with two cysteine specific reagents: membrane permeant biotin maleimide (BM) and membrane impermeant MTS‐TAMRA. An introduced cysteine labeled with BM indicates its surface location, whereas lack of labeling indicates the residue resides in the lipid bilayer or in a BM inaccessible conformation. Introduced cysteines labeled with MTS‐TAMRA indicate they are exclusively located on the extracellular surface of the protein. Residues D449, A450, T451 and D452 in proposed extracellular loop 1, N503 in loop 2, E714, T718 and R722 in loop 4 were labeled both by BM and MTS‐TAMRA, demonstrating the first half of the transmembrane domain contains 8 transmembrane segments, highly homologous to AE1. The majority of introduced cysteines in the remaining transmembrane domain were not labeled by BM, suggesting that folding of this region is highly compacted unlike AE1. Based on these results, we propose a new topology model for the transmembrane domain of NBCe1‐A.

Supported by NIH
AT1R dysregulation is crucial in the hypertension of human GRK4γ A142V transgenic mice
Authors: Wang Zheng , Chen Shiyou , Asico Laureano D. , Escano Crisanto , Villar Van Anthony M. , Lu Quansheng , Coffman Thomas M. , Jones John E. , Armando Ines , Felder Robin A , Jose Pedro A.
Published: June 2021

The renin‐angiotensin system (RAS), via the AT1 receptor (AT1R), plays a critical role in the control of blood pressure (BP) by regulating peripheral resistance and sodium balance. Dopamine, via several dopamine receptor subtypes (e.g., D1R), counter regulates the RAS, including the AT1R. Recent studies have revealed an important role of G protein‐coupled receptor kinases (GRKs) in BP control by regulating several G protein‐coupled receptors, including the AT1R and D1R. Polymorphisms of GRK subtype 4 (GRK4), by themselves or via their interaction with genes regulating the RAS, are associated with essential hypertension. We now report that human GKR4γ A142V transgenic mice (A142V‐Tg) relative to hGRK4γ wild‐type transgenic mice (WT‐Tg) are hypertensive and have increased expression of AT1R, associated with increased AT1R promoter activity. A142V‐Tg mice have increased BP response to angiotensin II infusion and AT1R blockade. Moreover, A142V‐Tg mice that are deficient of AT1R have normal BP. This is the first report showing a molecular mechanism for the regulation of the AT1R gene that affects BP control. The GRK4 gene by regulating several genes involved in BP control may explain the "apparent" polygenic nature of essential hypertension.
Macula densa (pro)renin receptor abundance correlates with renal renin content
Authors: Vargas Sarah Laurin , Peti‐Peterdi Janos
Published: June 2021

The (pro)renin receptor [(P)RR] has been localized to the basolateral membrane of the macula densa (MD), adjacent to (pro)renin (PR) producing granular cells. The PRR is a new exciting element of the renin‐angiotensin system (RAS). Patho(physiological) conditions, such as dietary salt, hypertension, and diabetes mellitus (DM), have well established effects on RAS and renal PR expression. We hypothesized that under these conditions (P)RR abundance in MD cells correlates with renal PR expression. First, (P)RR immunofluorescence was performed on kidney sections after rats were placed on normal salt (4% salt, NS), salt‐deficient (0.4% salt, SD), or high salt (8% salt, HS) diet for one week. Compared to NS, less cytosolic but very intense basolateral membrane labeling was observed in MD cells in response to LS, while HS greatly reduced MD (P)RR labeling. (P)RR immunoblotting using cultured mouse‐derived MD cells (MMDD1) treated with SD medium for 15‐75 minutes presented up‐regulation of (P)RR protein. Next, western blotting was performed with renal cortical homogenates from control and STZ‐DM rats against renin and (P)RR. Up‐regulation of both renin (2.6‐fold) and (P)RR (1.4‐fold) was confirmed in DM. In summary, MD (P)RR abundance correlates with renal cortical renin content, suggesting that either high/low renin states modulate MD function via the (P)RR, or the MD (P)RR regulates renal (pro)renin synthesis.
Phospholipase Cε(PLCε)‐mediated activation of classical transient receptor potential 6 (TRPC6) increases barrier function of glomerular podocytes
Authors: Dietrich Alexander , Kalwa Hermann , Storch Ursula , Smrcka Alan , Hildebrandt Friedhelm , Gudermann Thomas
Published: June 2021

TRPC6 is activated by direct exposure to diacylglycerol (DAG) produced by isoforms of the PLC family. Mutations in TRPC6 and PLCε identified in patients with nephrotic syndrome of the kidney link these proteins to structural components of the glomerular slit diaphragm in podocytes. For that reason we investigated, if there is a functional interaction between these two proteins. We were able to coimmunoprecipitate PLCε and TRPC6 in primary murine podocytes. Furthermore, siRNA mediated downregulation of endogenous PLCε in HEK 293 cells evoked a reduction of TRPC6 specific currents. The importance of this mechanism was further emphasized by the fact that murine podocytes showed an angiotensin II‐induced increase in barrier function, which was mimicked by OAG, a membrane permeable DAG‐analogue. Most interestingly, TRPC6‐/‐ podocytes isolated from TRPC6‐deficient mice displayed a markedly decreased barrier function in comparison to wild‐type cells. Similar results were obtained in PLCε‐/‐ podocytes. These data suggest an important role of PLCε in the regulation of TRPC6 activity in glomerular podocytes, and may provide inroads into the understanding of AT1 receptor blocker treatment in nephrotic syndrome.
HYPERTENSION IN RESPONSE TO CHRONIC SFLT‐1 EXCESS DURING PREGNANCY: ROLE OF REACTIVE OXYGEN SPECIES.
Authors: TamTam Kiran , LaMarca Babbette D , Murphey Sydney , Arany Marrietta , Lee Nicole M , Fournier‐Ray Lillian , Cockrell Kathy , Granger Joey P
Published: June 2021

Preeclampsia is associated with an increase in the antiangiogenic factor, soluble fms‐like tyrosine kinase‐1 (sFlt‐1). We previously reported that hypertension in response to sFlt‐1 was associated with an increase in vascular superoxide concentration; however, the importance of reactive oxygen species (ROS) in mediating the hypertension in response to sFlt‐1 during pregnancy is unknown. Therefore, we tested the hypothesis that generation of ROS plays an important role in mediating sFlt‐1 induced hypertension in the pregnant rat. To achieve this goal we compared mean arterial pressure and superoxid levels in response to chronic sFlt‐1 infusion (1ng/kg/day) in normal pregnant rats and pregnant rats pretreated with the superoxide dismutase mimetic, Tempol (30mg/kg/day) from day 12 to day 19 of gestation. MAP increased from 101+/‐1 mmHg in NP to 115 +/‐3 mmHg in sFlt‐1 infused rats. Plasma sFlt‐1 increased from 430+/‐200 to 1237 +/‐500pg/ml. Furthermore, superoxide levels in the placenta increased from 458+/+ 60 to 519+/‐65 RLU/mg/min in sFlt‐1 infused rats. There were no differences in MAP between the Tempol treated (95+/‐3mmHg) and Tempol treated sFlt‐1 infused (101+/‐3mmHg) pregnant rats. These data support the hypothesis that ROS are important in mediating the blood pressure response to chronic sFlt‐1 excess during pregnancy.
Estrogen enhances IkBα synthesis and inhibits NF‐kB p65 binding to the promoters of inflammatory genes in rat aortic smooth muscle cells
Authors: XING DONGQI , Nozell Susan , Chen Yiu‐Fai , Feng Wenguang , Oparil Suzanne
Published: June 2021

Background

The NFκB signaling pathway is critical for the expression of a variety of genes involved in the response to vascular injury. Estrogen (17β‐estradiol, E2) inhibits expression of these genes in an estrogen receptor‐dependent manner in balloon injured rat carotid arteries and in tumor necrosis factor (TNF)‐α treated rat aortic smooth muscle cells (RASMCs) in vitro. This study tested the hypothesis that E2 inhibits NFκB signaling and defined the machanisms.

Methods

IκBα expression was determined using real time RT‐PCR and Western blot analysis. Interactions between NFκB p65 and promoters of inflammatory mediators were evaluated by chromatin immunoprecipitation (ChIP) assay.

Results

RASMCs treated with TNF‐α demonstrated rapid degradation of IκBα (10‐30 min), followed by a dramatic elevation in IκBα mRNA and protein synthesis (40~60 min). E2 enhanced TNF‐α induced IκBα mRNA and protein synthesis without affect IκBα degradation (Figure A~C). ChIP assay confirmed that TNF‐α increased levels of p65 significantly at all promoters analyzed (at 1hr) and these effects were reduced by E2 (Figure D).

Conclusions

E2‐inhibited expression of inflammatory mediators in the setting of acute vascular injury may be mediated, in part, by accelerating IκBα re‐synthesis and inhibiting NFκB p65 binding to promoters of target genes.
AT1‐AA induced hypertension during pregnancy is associated with renal endothelial dysfunction and endothelin (ET‐1) type A receptor activation.
Authors: LaMarca Babbette D , Glover Porter , Parrish Marc , Fournier‐Ray Lillian , Dechend Ralf , Ryan Michael J
Published: June 2021

Agonistic autoantibodies to the angiotensin II type I receptor (AT1‐AA) may be an important link between placental ischemia, endothelial dysfunction and hypertension in women with preeclampsia. Renal cortical endothelin (ET‐1) is increased in AT1‐AA infused rats compared to controls, suggesting a role for ET‐1 in AT1‐AA induced hypertension. The purpose of this study was to determine if AT1‐AA induced hypertension is associated with endothelial dysfunction and whether the hypertension is mediated by ETA receptor dependent mechanism. To achieve this goal, we chronically infused rat AT1‐AA into normal pregnant rats (NP) in the presence or absence of the ETA receptor antagonist. Mean arterial pressure (MAP) increased from 99 + 1 mmHg in NP to 120 + 2 mmHg in AT1‐AA infused pregnant rats (P<0.001). Renal endothelial function was tested using isolated renal interlobar arteries in a pressure myograph. Vasodilatory responses to the endothelial dependent agonist acetylcholine (10−8 M) were impaired in AT1‐AA rats (74±10%) compared to NP controls (95±5%, p<0.05). The response to sodium nitroprusside was not different. ETA receptor blockade attenuated hypertension in AT1‐AA treated rats (100±1 mmHg) vs ETA controls (98 ± 2 mmHg). These data suggest that hypertension associated with AT1‐AA during pregnancy is mediated by ET‐1 type A receptor dependent mechanism and is associated with renal endothelial dysfunction.
Differential effects of TNF receptor 1 and 2 deletion on blood pressure and Ang II type I receptor (AT1R) expression
Authors: Chen Chun Cheng Andy , Abdullah Huda Ismail , Pedraza Paulina Loreto , Hao Shoujin , McGiff John C. , Ferreri Nicholas R.
Published: June 2021

Effects of either TNF receptor 1 (TNFR1), or receptor 2 (TNFR2), gene deletion were determined in a model of Ang II‐dependent hypertension. C57Bl/6J wildtype (WT), TNFR1−/−, and TNFR2−/− mice were given Ang II subcutaneously by osmotic minipump (1.6 ?g/min/kg) for 7 days. Systolic blood pressure (SBP) was measured by tailcuff and radiotelemetry. SBP was similar in untreated mice but higher in TNFR1−/− mice given Ang II (181±7 mmHg) compared to TNFR2−/− (147±3) and WT (163±6) mice. Radiotelemetry confirmed higher SBP in TNFR1−/− (149±2) compared to TNFR2−/− (143±2) and WT (143±1) mice. Food intake was similar but cumulative sodium excretion was elevated in TNFR1−/− versus TNFR2−/− and WT mice (132±9 vs 107±16 vs 118±9 ?mol/d/g body weight (BW), respectively). Urinary albumin was elevated in TNFR1−/− compared to TNFR2−/− and WT (1.35±0.17 vs 0.79±0.12 vs 0.93±0.14 ?g/mg creatinine, respectively). Water intake was higher in TNFR1−/− versus TNFR2−/− and WT mice (0.52±0.02 vs 0.45±0.01 vs 0.40±0.01 ml/d/g BW, respectively); urine volume also was higher in TNFR1−/− compared to TNFR2−/− and WT mice (0.24±0.02 vs 0.12±0.01 vs 0.15±0.01 ml/d/g BW, respectively). Renal cortical AT1R protein expression was elevated in TNFR1−/− mice compared to TNFR2−/− and WT mice. These data suggest that deletion of TNFR1 augments Ang II‐mediated increases in SBP, water intake, and sodium excretion associated with upregulation of AT1R.
Anabolic‐Androgenic Steroids Attenuates the Reflex Muscle Vasodilatation
Authors: Alves Maria Janieire N. N. , Santos Marcelo R. , Dias Rodrigo G. , Akiho Cesar A. , Laterza Mateus M. , Rondon Maria Urbana P. B. , Moreau Regina L. M. , Negrao Carlos E.
Published: June 2021

We hypothesized that anabolic‐androgenic steroids (AAS) would exacerbate symphatetic nerve activity responses and attenuate muscle vasodalation responses during mental stress in young individuals.

Methods

Nine body builder users and 7 age‐matched body builder nonusers of AAS were studied. AAS was assessed by urine test (chromatography‐mass spectrometry). Mean blood pressure (MBP, oscillometric automatic cuff), forearm blood flow (FBF, plethysmography) and muscle sympathetic nerve activity (MSNA‐microneurography) were evaluated at rest and during mental stress (Stroop Color Word Test).

Results

Resting MSNA was significantly higher (28±3 vs. 20±1 bursts/min, P=.03, respectively) and forearm vascular conductance (FVC) significantly lower (2.04±0.19 vs. 2.86±0.31 units, P=.03) in user compared with nonuser of AAS. MBP was not different between groups. FVC responses to mental stress were significantly lower in user compared with nonuser (0.66±0.35 vs. 2.96±0.78 units, P=.01, respectively). MBP and MSNA responses were not different between groups.

Conclusion

AAS increases MSNA and reduces FBF in healthy young individuals. In addition, the reduction in muscle reflex vasodilatation during mental stress in AAS user is suggestive of impaired endothelial function in these individuals.
CBF changes in relation to the phase of ovarian cycle in guinea pig Fallopian tube: effects of estrogen and progesterone
Authors: Nishimura Ayumi , Nakahari Takashi
Published: June 2021

Ciliary beat frequency (CBF) of Fallopian tube of guinea pig was measured using a light microscope connecting a high‐speed camera (500 Hz), CBFs ranged from 10 to 15 Hz in the phases of ovarian cycle. In follicular phase, CBF increased transiently; an increase at the beginning of estradiol (E2) rise (14 Hz) followed by a rapid decrease at the peak of E2 rise (10 Hz). In ovulatory and luteal phase (high progesterone), CBF was maintained 12 Hz. β‐Estradiol‐benzoate (βE2‐benz) and medroxy‐progesterone (m‐prog) dissolved in a steroid solution were administered subcutaneously for 1.5 ‐ 4 days. A high concentration of βE2‐benz administered (20 mg/day) decreased CBF (11 Hz), while a low concentration (2 mg/day) increased it (14.5 Hz), and these βE2‐benz actions were inhibited by an estrogen inhibitor (ICI 182,780, 2 mg/day). m‐Prog administered (2‐20 mg/day) maintained CBF approximately 12.5 Hz, which was abolished by a progesterone inhibitor (mifepristone, 50 mg/day). Ciliary cells of Fallopian tube were immuno‐positively stained for estrogen and progesterone receptors. The present study demonstrated that βE2‐benz accelerates CBF at a low concentration and suppresses it at a high concentration, and that progesterone maintains CBF around 12 Hz. CBF in Fallopian tube is regulated by estrogen and progesterone in relation to the ovarian cycle.
Expression of low abundance isoforms of regulatory myosin light chain validated by a novel scoring algorithm for protein isoforms correlates with vascular maturation
Authors: Graupner Gerhart , Flenner Arjuna , Zhang Kangling , Pearce William J
Published: June 2021

Regulatory myosin light chain (rMLC20) is a key determinant of vascular contractility. We have recently demonstrated that multiple high‐abundance rMLC20 isoforms are expressed in sheep arteries. The present studies explore the hypothesis that low‐abundance vascular isoforms of rMLC20 are also expressed and exhibit considerable sequence diversity that requires improved methods of statistical validation. When performing post‐database search validation of rMLC20 isoform sequences identified by shotgun protein sequencing, we recognized that no suitable software exists to correctly assess the false discovery rate for protein isoforms. As the symmetric error distribution and the three‐codon sequence coverage found in the popular BLAST algorithm are not useful for scoring of protein isoforms with short, clustered amino acid substitutions, we developed an algorithm that encompasses asymmetric error distribution and graded penalties for isoform‐specific amino acid substitutions. The new algorithm enabled validated detection of novel low‐abundance rMLC20 isoforms. Because the expression of these low‐abundance rMLC20 isoforms varied with vascular maturation, these data support the hypothesis that postnatal changes in contractility involve shifts in patterns of rMLC20 isoform expression. Supported by USPHS Grants HL54120, HD31266, & HL64867.
The mechanism of intramembrane proteolysis
Authors: Ha Ya
Published: June 2021

Intramembrane proteolytic reactions are involved in many cellular processes. These once‐considered unusual reactions are catalyzed by highly specialized integral membrane proteases. Here I describe possible mechanisms of intramembrane proteolysis, drawing lessons from the recently solved crystal structures of the membrane protease. The role of membrane compression around the protease, and how it may facilitate the unfolding of transmembrane alpha‐helices in the substrate, will also be discussed.
D‐4F attenuates calcineurin‐induced cardiac hypertrophy and heart failure
Authors: Wei Na , Ge Zhidong , Du Jianhai , An Jianzhong , Warltier David C , Pritchard Kirkwood A , Shi Yang
Published: June 2021

D‐4F, an apolipoprotein A‐I mimetic, has been shown to improve vascular function in atherosclerosis, diabetes and a variety of inflammatory diseases. However, the potential benefits of D‐4F in cardiac hypertrophy remain unknown. Calcineurin is a calcium‐sensitive type IIB phosphatase, which has been demonstrated to play a role in human heart hypertrophy. Transgenic Calcineurin A (CnA‐TG) mouse is an established murine model of cardiac hypertrophy and heart failure. Here, we investigate whether D‐4F attenuates cardiac hypertrophy and heart failure in CnA‐TG mice. CnA‐TG and wild type mice, immediately after weaning were treated with PBS or D‐4F (1 or 3 mg/kg/day) for 8 weeks. D‐4F dose‐dependently reduced ventricular mass to body weight ratios, cardiomyocyte cell size as well as interstitial fibrosis. Echocardiography revealed that D‐4F significantly prevented left ventricular wall thickness, elevated mitral E/A ratios, decreased E wave deceleration times and cardiac arrhythmias in CnA‐TG mice. Additionally, real‐time PCR shows that D‐4F decreased expression of ANP, β‐MHC; increased expression of sarcoplasmic reticulum Ca2+ ATPase and c‐fos. Cardiac hemodynamics, plasma lipids and lipoprotein levels were essentially unchanged among the groups. In conclusion, D‐4F effectively reduces cardiac hypertrophy to protect the hearts of CnA‐TG mice from programmed failure.
Telemetric recording of gastrointestinal blood flow and the effects of thermoregulatory behavior in an ectotherm
Authors: Gräns Albin , Pitsillides Koullis , Cech Joseph J , Olsson Catharina , Axelsson Michael
Published: June 2021

The majority of all vertebrates are ectotherms and therefore have no other means of controlling their body temperature than through thermoregulatory behaviour. The effects of voluntary adjustments of body temperature on the distribution of blood flow have never been possible to study since blood flow measurements have so far required extensive confinement of the studied animals due to short leads from implanted flow probes. In the current study a new fully implantable dual channel radio‐based Doppler blood flow biotelemetry system (Endosomatic Systems Inc, Sacramento, CA USA) was used to measure the effects of thermoregulatory behaviours on gastrointestinal blood flow in white sturgeon (Acipenser transmontanus). After feeding there was a strong correlation (R2 = 0.60, p < 0.001) between gastrointestinal blood flow and the chosen temperatures. Also, freely moving fish had a significantly lower resting heart rate (36.8 min−1) compared to a more confined, traditionally instrumented control group (44.7 min−1). This is most likely a result of reduced confinement stress and more closely reflect the true routine heart rate in this species. Our results show that the blood flow biotelemetry system opens new areas of research. With this system, it is now for the first time possible to record blood flow from one or several freely moving animals and to study the effects of different behaviours on the cardiovascular system.
The 5TM VPAC1 Isoform Blocks cAMP Signaling Evoked from the Full‐Length 7TM receptor
Authors: Wilkerson Erich Raymond , Hermann Rebecca J , Branch Donald R , Dorsam Glenn
Published: June 2021

Vasoactive intestinal peptide receptor 1 (VPAC1) couples heterotrimeric G proteins, Gαs and Gαi, that activate and inhibit adenylate cyclase cAMP production. The 7 transmembrane (TM) VPAC1 isoform, increases intracellular cAMP ([cAMP]i) upon binding its ligand, vasoactive intestinal peptide (VIP). In contrast, the 5TM VPAC1 isoform fails to increase [cAMP]i over a range of 10‐6‐10‐12 M VIP. Our hypothesis is that the dual expression of both the 5 and 7TM isoforms cause a lack of [cAMP]i elevation due to an antagonist effect by the 5TM on 7TM signaling. To investigate this, human HuT 78 cells, a malignant T cell, which express both 5 and 7TM VPAC1 isoforms. Levels of [cAMP]i were measured by a competitive ELISA after treating with increasing concentrations of VIP. Interesting, we show a VIP dependent decrease in [cAMP]i, suggesting that the 5TM VPAC1 isoform coexpressed in HuT 78 cells suppress 7 TM induced [cAMP]i levels. The ratio of the 5 and 7TM receptors on the cell membrane may explain the unique level of [cAMP]i upon VIP binding. Future studies will inhibit Gαi coupling with pertussis toxin and RNAi to knockdown the 5TM VPAC1 to measure [cAMP]i response evoked by the 7TM VPAC‐1 receptor in coexpressing cells. Research supported by NIH‐KO1 1KO1DK664828 and COBRE 2P20RR015566.
Possible gentamicin trafficking channels in guinea pig cochlear stria vascularis cells
Authors: Ma Ke‐tao , Yang Yu‐Qin , Wang Qi , Steyger Peter S , Jiang Zhi‐Gen
Published: June 2021

Aminoglycosides (AG) are important antibiotics but cytotoxic to inner ear hair cells causing hearing and balance deficits. How AGs pass through cochlea blood‐labyrinth barrier remains unknown. Using current recording and fluorescent gentamicin (GTTR) imaging on cells of in vitro stria vascularis (SV), we found: 1) Whole‐cell recording revealed four types of cells by distinct membrane properties. First type, patched from endolymphatic surface, likely the marginal cells (MC), had a resting potential (RP) ~‐50 mV, input resistance (Rin) 0.5‐1.5 GΩ, single exponential capacitive current decay and input capacitance (Cin) 20‐80 pF. 18βGA changed Rin to >1 GΩ and Cin to <~30 pF, suggesting a very limited intercellular coupling. These cells had a robust TEA‐sensitive delayed rectification and La3+‐sensitive cation conductance. Outside‐out patch recording revealed a 48 pS channel with higher Po at depolarization and a Vr near EK, and a 105 pS channel with Vr ~20 mV, consistent with a maxi‐K and a NSCC. Second type, also patched apically, showed a RP ~10 mV, Rin ~ 1.2 GΩ, Cin ~ 7 pF and a slightly inward rectifying I/V. Third ones, patched from edge or basal side of SV, showed a RP ~‐70 mV, maxi‐K, Rin ~0.2 GΩ and Cin ~300 pF but changed to ~1.5 GΩ and 30 pF by 18βGA, likely being intermediate cells. Fourth type showed a RP ~‐35 mV, Rin ~1.5 G, Cin ~8 pF and a slightly outward rectifying I/V, possibly being basal cells. 2) Half hour after single dose GTTR p.i., the SV endothelium and MCs showed a much stronger fluorescence than other SV cells. We conclude that 1) MCs express NSCC and, less possibly, hemichannels that may mediate AG flux; 2) an active AG influx transport may exist in basolateral membrane of MCs. Funded by NIDCD R01 04716 (ZGJ) R01 04555 (PSS) and P30 05983.
A fibre‐optic laser system and cell‐specific viral vectors for chronic opto‐genetic experimentation on deep brain structures
Authors: Kasparov Sergey , Danielyan Georgi L , Tang Hao R , Figueiredo Melina F , Teschemacher Anja G
Published: June 2021

Optical control of neuronal circuits by light using expression of light‐activated ion channels such as channelrhodopsin‐2 in chronic unrestrained animals is a highly attractive method. ChR2 can be targeted to specific cellular types using viral vectors. However, convenient hardware solutions for delivery of the frequency and intensity‐modulated light pulses to the selected brain areas in a chronic experiment have to be developed. We present implantable optrodes which can be coupled to a "Deepstar" pulse‐modulated blue laser (445 nm, 50 mW, 1:100000 contrast ratio, Omicron, Germany) via a fine (200 μm core) flexible optical cable. The optrodes are manufactured of multimode quartz/quartz glass with metal coating and are secured to the optical cable with a miniature connector (A.R.T. Photonics). Optrodes with a 400 mkm core, outer diameter of 0.52 mm and NA of 0.22 have different lengths available for implantation (6‐14 mm). The fibre is coupled to the laser via an adjustable optical connector SMA‐905‐DS to allow precise focusing of the beam. Using this hardware we achieved light output from the optrodes at 36 ± 4 % (n=10) of the laser input power, this should be ample for any experiments involving stimulation of cells expressing ChR2. We have also generated several viral vectors which target ChR2 expression to different cell types in the CNS using cell‐specific transcriptionaly‐enhanced promoters.
Protractor function of the human temporal muscle: anatomic‐analytic and experimental corroboration
Authors: Duque Parra Jorge Eduardo , Moscoso Ariza Oscar Hernán , Barco Rios John Jairo , Quintero Duque Luisa
Published: June 2021

An anatomical study of the of the temporal muscle anterior fibers was carried out in human corpses. An analytical study by means of vectorial decomposition was developed; secondly, the protractor function was determined, and subsequently electromyographic during the protrusive mandibular movements was experimentally verified.
A survey of anatomical variation in medical student cadavers: a longitudinal study
Authors: Hovorka Michelle S. , Uray Nandor J.
Published: June 2021

The goal of our study is to document anatomical variations found in the gross anatomy laboratory setting. This is a long term study to note trends in variations due to changes in human donor populations over the next ten years. In this first year of the study, thirty donated human cadavers are being dissected by medical students. As dissection proceeds, variations and anomalies are photographed and recorded, as are significant pathological findings. As of this time, dissection of the upper limb, back, and thorax have been completed. Anomalies noted include four variations of the brachial plexus including one in which the posterior cord is completely absent; anomalous musculature including an axillopectoral muscle and a sternalis muscle; an azygos lobe of the lung; and five instances of absent cephalic veins. Additionally, significant pathological findings and prosthetic devices are being documented. These findings will serve to form the foundation for a longitudinal study to track trends in the population over time as represented by medical cadavers. Funding is provided by the Kirksville College of Osteopathic Medicine Graduate Program.

Grant Funding Source

Kirksville College of Osteopathic Medicine Graduate Program
Gross anatomy of a horseshoe kidney: a case report
Authors: Casey Gregory Patrick , Sedlmayr Jayc C. , Oliver Peter , Cork John
Published: June 2021

At the LSU Health Sciences Center, we discovered a horseshoe kidney in one of the cadavers during laboratory dissection in the nursing gross anatomy course. The donor was an 83 year old male with causes of death related to congestive heart failure, chronic obstructive pulmonary disease, and acute renal failure. Horseshoe kidneys are relatively rare with a reported occurrence of 1 in 400 births worldwide; although this is the first observed in our gross anatomy laboratory. It is a developmental anomaly arising from fusion of the metanephroi around the fifth week of embryonic development. A detailed in situ dissection was done on the cadaver in which major arteries and veins were observed. The horseshoe kidney in this cadaver was supplied by several arterial branches from the abdominal aorta and its bifurcation, and by several venous branches from the inferior vena cava. After initial dissection in situ, the horseshoe kidney, ureters, bladder, most of the abdominal aorta, and most of the inferior vena cava with the common iliac bifurcation were removed for further study and preservation. Although the discovery of this horseshoe kidney is unusual, it is a good example of the type of anatomical variation and medical conditions that makes the cadaver lab an excellent educational environment for nursing school.

Grant Funding Source

Department of Cell Biology and Anatomy
The Challenge of Identifying Commingled Skeletal Remains via CT Imaging and Morphometric Analysis
Authors: Hong Simon , Delman Bradley N , Maciasz Rachael , Lee Rosanna , Caceres Noel , Sawyer GJ , Marquez Samuel
Published: June 2021

Human skeletal remains were discovered at a construction site in lower Manhattan. Officially determined that the post‐cranial material was not of medicolegal significance, our team undertook an anatomically driven forensic evaluation of the remains. Post‐cranial isolated bony elements were identified (n = 52), digitally photodocumented, morphometrically assessed and CT scanned for graphical examination. Post‐cranial commingled remains consisted mostly of lower limb bones from at least 3 distinct individuals based on three left cuboid bones found. 31 foot bones were measured and classified using bony landmarks. Comparison to homologous bones of known stature individuals was used to approximate the size differences found among the unknown sample. Maximum a‐p lengths of the three left cuboids measured 44.5, 35.8 and 35.6 mm while the two left calcaneal bones measured 89.2 and 77.5 mm. Size difference was used to group the tarsal bones to either the larger or two smaller individuals. CT imaging of adult femurs revealed their intensity of femoral curvature to fall within the Caucasian spectrum while overall rugosity of the larger bones indicated that the larger individual was male. A combination of CT, comparative and morphometric assessment was the key to overcoming the challenge of cataloging and identifying commingled skeletal remains, and may be the ideal approach for similar future forensic investigations.

Grant Funding Source

None
The melanocortin‐1 receptor (MC1R) gene and color polymorphism in Plethodon cinereus
Authors: Hyatt Carly J. , Kimble Danielle , Pytel Barbara
Published: June 2021

The salamander Plethodon cinereus can be found in two different color phases; the red‐back phase and the lead‐back phase. The molecular basis behind these different color morphs is not fully understood. In this study, the gene for the melanocortin receptor (MC1R) was sequenced to see if mutations in this gene are responsible for these color differences. MC1R is a G protein‐coupled receptor that has seven transmembrane domains. It plays an important role in pigmentation and melanin production in skin, hair and feathers and mutations in this protein appear to correspond to light or dark phenotypes in many different animals. It was hypothesized that mutations in MC1R are responsible for the polymorphism in Plethodon cinereus. Frozen tissue from salamanders caught from Helderberg Escarpment was used to isolate, amplify and sequence 692 base pair fragment of MC1R. Sequencing this gene for MC1R will hopefully help to better understand the molecular basis for the color morphs in Plethodon cinereus. Supported by Union College, Biology Department.
Paraspinal muscular defects in Lfng segmentation mutant mice
Authors: Smith Heather F , Kusumi Kenro , Rawls Alan , Wilson‐Rawls Jeanne , Fisher Rebecca
Published: June 2021

Mutations in several regulatory genes along the Notch signaling pathway, including Lfng, are associated with vertebral defects and segmentation disorders. However, the role of these genes in regulating myogenic morphology is unknown. The objective of this study was to assess the role of Lfng in determining the morphology of paraspinal musculature. Nine mice that were homozygous null for Lfng and nine wild‐type specimens were dissected and their paraspinal musculature analyzed. Four Lfng null specimens exhibited variation in the iliocostalis muscle, ranging from unilateral reduction to complete bilateral absence. For multifidus, eight Lfng null specimens exhibited variation, including unilateral and bilateral cranial shifts, caudal and cranial reductions, elongations, and one bilateral absence. A χ2 test revealed a statistically significant difference between the Lfng mutant specimens and wild‐types in the frequency of defects in the iliocostalis and multifidus. These results will be compared to similar findings from Dll3 mutants. The results support the hypothesis that genes in the Notch signaling pathway, Lfng in particular, may influence paraspinal muscular development, although with an incomplete penetrance. These findings have implications for studies of muscular involvement in idiopathic scoliosis and segmentation disorders.

This research was partly supported by NIH RO1 AR050687 (KK).

Grant Funding Source

University of Arizona
Exploring the anti‐diabetic effect of duodenojejunal bypass: a novel mouse model
Authors: Woods Michael Gregory , Lan Zhu , Wang Jingming , Wang Rennian
Published: June 2021

Obese patients with Type 2 diabetes who underwent bariatric surgery reverted to normal blood glucose and insulin levels and developed a dramatic increase in insulin sensitivity without continued use of diabetic medication. The mechanisms involved in this reversal are not known although the data suggest that alteration of factors such as glucagon‐like peptide‐1 (GLP‐1) produced by the upper intestinal tract is involved. Currently, no studies have focused on a non‐diabetic DJB animal model to determine its importance of the duodenum and proximal jejunum in maintaining normal glucose homeostasis. Normal C57BL/6J mice underwent duodenojejunal bypass (DJB), a surgery in which the distal end of the jejunum was connected to a small portion of the duodenum just distal to pylorus and bilopancreatic secretions where directed to the distal jejunum. Normal‐fed mice undergoing DJB display diabetic‐like pancreatic morphology. These mice showed a decrease in the percent area of beta‐cells (33%) as well as a decrease in beta‐cell proliferation (50%) compared to sham‐operated controls. However, surviving beta‐cells showed similar levels of pancreatic duodenal homeobox‐1 (Pdx‐1), GLUT‐2 expression, and similar beta‐cell size between DJB and sham‐operated groups. These results suggest the importance of the duodenum in maintaining an anti‐diabetic state in the normal mouse. Funded by Lawson Health Research Institute.

Grant Funding Source

Lawson Health Research Institute
Spectrin is sequestered to Enteropathogenic Escherichia coli pedestals
Authors: Ruetz Tyson , Guttman Julian
Published: June 2021

Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that colonizes the small intestine of humans, causing serious diarrhea that can result in death. Upon attachment to host cells, EPEC injects effector proteins that hijack numerous sub‐cellular components, which leads to the generation of actin‐rich pedestals located within the host cells beneath the bacteria. A key effector responsible for pedestal formation is the translocated intimin receptor (Tir). Because EPEC alters the functions of all major cytoskeletal components examined thus far (actin filaments, intermediate filaments and microtubules) we investigated another cytoskeletal protein, spectrin, to test the hypothesis that the spectrin cytoskeleton is also modified by these pathogens. Spectrin was immunolocalized during in vitro infections with wild‐type EPEC and compared to mutants defective in effector secretion as well as Tir‐specific bacterial mutants. Surprisingly, spectrin was sequestered to wild‐type EPEC pedestals, but not to sites of bacterial attachment using EPEC effector mutants. Western blots identified that spectrin levels were equivalent in both infected and control cells. Our findings provide the first evidence of spectrin involvement in pathogenic E. coli‐mediated disease, whereby spectrin is re‐localized to the pedestals of EPEC.

Grant Funding Source

CIHR, NSERC
Co‐localization of nodal in hypoxic regions of tumours as seen using confocal microscopy and stereoscopic 3D reconstruction methods
Authors: Stock Tamara , Wilson Timothy D , Postovit Lynne‐Marie
Published: June 2021

Metastatic breast cancer is associated with a poor clinical prognosis. Understanding the factors that promote metastasis is required so that new treatments can be derived. Studies have shown that low oxygen levels (hypoxia) characterize the microenvironment of many tumours and that tumour hypoxia is correlated with increased metastatic potential. Nodal, an embryonic growth factor, is associated with tumour progression in breast cancer. Previous work indicates that Nodal is up‐regulated in response to hypoxia. The aim of this study was to investigate the co‐localization of Nodal with areas of hypoxia within three‐dimensional (3D) tumour spheroids. A poorly metastatic, well differentiated, human breast cancer cell line (MCF‐7) was grown as mammospheres and fixed using paraformaldahyde. Nuclei, hypoxia, and Nodal were simultaneously detected using immunofluorescence. Stained mammospheres were whole mounted for optical sectioning using a confocal microscope. Images were obtained at a slice thickness of 1‐2µm. Images were compiled and rendered into a 3D image using digital segmentation software. The co‐localization of Nodal and areas of hypoxia within the mammospheres were observed. Early results indicate that there may be pockets of hypoxic regions within the mammospheres and it is anticipated that Nodal will be up‐regulated in these areas. Research support: Canadian Institute of Health Research.

Grant Funding Source

Internal
Differential Distribution of Podocan Isoforms
Authors: Trubin Paul Albert , Klotman Paul , Hyink Deborah
Published: June 2021

Podocan is a relatively novel member of the small leucine‐rich repeat family of proteins. First isolated from podocytes of HIV‐1‐infected kidney tissue, this protein seems to be involved in a number of biological pathways. Initial experiments in podocan‐knockout mice revealed increases in bone density and in vascular smooth muscle cell proliferation in response to injury when compared to the phenotypes of genetically unaltered mice. In addition, protein labeling of human kidney biopsies and vascular lesions has shown both intra and extracellular distribution of podocan. To determine if multiple isoforms of the protein exist, we began a bioinformatics study to align all reported isoforms of human podocan. We found that the previously reported 613 amino acid isoform had a strong N‐terminal signal peptide, as expected for a secreted protein. However, three other isoforms had additional amino acids in front of the signal peptide. We hypothesized that additional sequences upstream of the signal peptide would lead to retention of podocan in the cytoplasm. To test this hypothesis, we fused EGFP to the N‐ or C‐ terminus of secreted podocan. These constructs were subsequently transfected into renal 293T cells. We examined the cells by fluorescent microscopy and western blotting 48 hours later. As predicted, N‐terminal GFP‐podocan prevented efficient secretion of podocan. Our studies have led us to posit that podocan is involved in differential signaling, and this area will be the focus of future investigation. We have generated 3 C‐terminal His‐tagged podocan isoforms that have differing lengths of N‐terminal sequences upstream of the signal peptide and will use these to confirm our GFP‐fusion findings. We have also developed a series of isoform‐specific primers to help us determine whether these isoforms are differentially expressed in normal or diseased human tissue.

Grant Funding Source

American Association of Anatomists
The Expression and Functional Role of Syndecan 4 in Breast Cancer
Authors: Lendorf Maria Elisabeth , Couchman John R , Wewer Ulla M , Multhaupt Hinke A.B.
Published: June 2021

The syndecans are a four‐member family of transmembrane cell surface heparan sulfate proteoglycans (HSPG) expressed in both developing and adult tissue. They are believed to have regulatory roles in cell adhesion, proliferation and differentiation. In cancer, syndecan expression has been shown to regulate tumour cell behaviour and serve as a prognostic marker. Syndecan‐4 plays a role in cell adhesion and in the regulation of cell migration. It is the second most abundant HSPG produced by most breast carcinoma cell lines. However, little is known about the role of syndecan‐4 in malignancy which is under investigation by a combination of in vivo and in vitro studies. Epithelial cell surface syndecan‐4 in clinical breast carcinoma samples was detected by immunoperoxidase staining at varying intensity levels. Using immunofluorescence microscopy, breast carcinoma cells localized syndecan‐4 to discrete, circular structures which may correspond to the morphological organelles known as podosomes or invadopodia. These structures are thought to be involved in the migratory and invasive behaviour of cancer cells. Double‐staining for podosome/invadopodia markers showed at least partial co‐localization with syndecan‐4. Markers included integrins and cytoskeletal proteins such as cortactin, and src‐family kinases. These results suggest that syndecan‐4 plays a role in breast cancer cell migration and invasion.
Keratin Biomaterials Activate Schwann Cells via Integrin‐Mediated Signaling and Promote Regeneration of Large Peripheral Nerve Defects in a Rabbit Model
Authors: Sierpinski Paulina , Apel Peter J , Tawfik Bernard , Smith Tom , Koman L. Andrew , Atala Anthony , Van Dyke Mark
Published: June 2021

Previous work in our lab has shown that alpha and gamma keratins can be extracted from human hair and processed into a hydrogel that promotes rapid regeneration of peripheral nerves in a mouse model. We have shown that this regeneration is partly due to activation of Schwann cells but the mechanism by which this happens is not known. Our aim in this study was to (1) evaluate the primary AA structure of known HHKs, (2) devise fractionation schemes that allow separation based on structural criteria, (3) investigate the effect that structural characteristics had on SC activity at a mechanistic level, and (4) assess the ability of a keratin gel to regenerate large peripheral nerve defects. Here we show that keratins can be effectively separated based on structural criteria, and that these structural characteristics have an effect on the receptor mediated‐signaling of Schwann cells. Cells grown on alpha, gamma and basic gamma keratin substrates had a 16‐18 fold upregulation in integrin αV over uncoated dishes. The acidic alpha fraction showed a 2.4‐fold increase in the pMAPK to MAPK ratio compared to the basic alpha fraction. Furthermore, we show that a keratin gel composed of alpha and gamma keratins promotes regeneration across a critical sized defect in a rabbit model. At 3 months, keratin treated nerves had significantly better conduction delay (4.5% vs. 21%) and improved CMAP (51% vs. 36%) over empty conduit controls.