bluuebunny/crossref_metadata_2025 huggingface dataset:
Nuclear Localization and Biological Function of Matrix Metalloproteinase‐2
Authors: Baghirova Sabina , Kondo Marcia , Hughes Bryan , Schulz Richard
Published: June 2021

Matrix metalloproteinases (MMPs) are zinc‐dependent proteases that are involved in intra‐ and extra‐cellular matrix remodeling associated with developmental processes and disease progression. Several MMPs including MMP‐2 have been found in the nucleus. The biological functions and substrates of nuclear MMPs are mostly unknown. We hypothesize that MMP‐2 is present in the nucleus under physiological conditions but increases during oxidative stress to proteolyse structural and DNA repair proteins. Lamin A/C, a possible nuclear MMP‐2 target, is an intermediate filament protein that provides structural support to the nuclear envelope. Cytosolic, membrane and nuclear fractions were extracted from blood‐free, isolated rat hearts. Western blots for MMP‐2, lamin A/C (nuclear marker), SERCA2 (membrane marker) and GAPDH (cytosol marker) were used to demonstrate fraction purity. This showed that the nuclear fraction was free of cytosolic and membrane contamination and MMP‐2 was detected in all three fractions. MMP‐2 activity (by gelatin zymography) showed that nuclear MMP‐2 activity was lowest compared to that in the cytosolic and membrane fractions. The presence of nuclear MMP‐2 was examined by immunofluorescence confocal microscopy in HT1080 fibrosarcoma cells. Recombinant lamin A/C was proteolysed into 50 and 25 kDa fragments by incubation with MMP‐2 (0.5h, 37oC) in vitro and this was blocked by the MMP inhibitor o‐phenanthroline. MMP‐2 is present in highly purified nuclear fractions obtained from rat hearts and in the nuclei of HT1080 cells. Ongoing experiments will determine the colocalization of MMP‐2 with nuclear bodies, its nuclear substrates and therefore its function within nuclei. Support: CIHR.
Don't Teach Biochemistry, Teach Students!
Authors: Bell J Ellis
Published: June 2021

What we teach, how we teach, who we teach, why we teach, how we assess a) whether we are successful and b) what students understand, and when we assess each of these, and how we use these assessments are all issues facing educators in general but particularly in the molecular life sciences. In the last 15‐20 years there has been increased focus on teaching “foundational concepts and skills” as well as developing more student centered teaching approaches and this has triggered discussion of how we assess student outcomes, both from the student perspective and from the discipline based education research perspective ‐ evidence based decisions about teaching approaches. The focus of the talk will be on the diversity and needs of who we teach, and will present some thoughts on how we, as an academic community, can best serve all our students. In addition, whether the way we generally assess our students understanding of the “discipline”, and ability to “do science” best serves students will be discussed.
Diminished Excitatory Neurotransmission To Brainstem Cardiac Vagal Neurons In Rats With Ascending Aortic Constriction‐Induced Heart Failure
Authors: Wang Xin , Cauley Edmund , Byrne Peter , Mendelowitz David
Published: June 2021

An imbalance in autonomic activity to the heart, increased sympathetic activation and reduced parasympathetic tone, are important pathophysiological contributors to the progression of heart failure (HF), and are associated with poor outcome in patients. However the mechanisms responsible for reduced parasympathetic activity to the heart in HF is unknown. Cardiac parasympathetic activity originates from cardiac vagal neurons (CVNs) located in the brainstem. In this study we test the hypothesis that activation of CVNs is altered in an animal model of HF. HF was established by ascending aortic banding (AAB), by banding the ascending aorta with a 3‐0 silk suture ligature tied against a 0.8mm blunt steel wire. The wire was removed, whereby the aorta was constricted to 60–70% of the original diameter. Aged‐matched control animals underwent a right thoracotomy and the ascending aorta was isolated but not constricted. After five weeks the spontaneous excitatory postsynaptic currents (EPSCs) in CVNs were significantly decreased in AAB animals. The frequency of EPSCs were diminished from an average of 3.8 ± 0.8 Hz (in control) to 0.9 ± 0.1 Hz (AAB) and the EPSCs amplitude reduced from 31.8 ± 2.6 pA (control) to 26 ± 2.1 pA (AAB). This diminished frequency and amplitude of EPSCs in CVNs would result in a decrease of parasympathetic activity to the heart, and is likely a mechanism for autonomic dysfunction that occurs in heart failure.
The extent of heat tolerance depends on the heat shock protein 72 levels in cultured primary hypothalamic neurons
Authors: Lin Kao‐Chang , Chang Ching‐Ping , Niu Ko‐Chi
Published: June 2021

We attempted to investigate whether severe heat‐induced hypothalamic cell injury and apoptosis can be affected by modulating heat shock protein (HSP) 72 levels. Cell viability was determined using 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐ H‐ tetrazolium bromide staining, whereas apoptosis was evidenced using flow cytometry. The viability or the percentage of apoptotic cells in the cultured primary hypothalamic cells were significantly (p<0.01) reduced by severe heat shock (43oC for 120 min). Cell injury and apoptosis caused by severe heat could be significantly attenuated or exacerbated by increasing cellular levels of HSP72 with mild heat preconditioning (42oC for 30 min; 8 h before the onset of heat shock) or decreasing the cellular levels of HSP72 with pSUPER/siRNA hsp72 plasmid (8 h before onset of severe heat shock), respectively. In addition, the beneficial effects of mild heat preconditioning in reducing cell injury and apoptosis could be significantly (p<0.05) reversed by silencing of selected gene and knockdown of HSP72 with pSUPER/siRNA hsp72. Our data indicated that the extent of heat tolerance of cultured primary hypothalamic cells depends on the cellular levels of HSP72.
Point‐of‐care cTnI Tests Accurately Predict Heat Stroke Severity; Proof of Concept for a Heat Stroke Field Test
Authors: Audet Gerald , Quinn Carrie , Leon Lisa
Published: June 2021

Heat stroke (HS) remains a significant health threat for both civilians and the U.S. Armed Forces. Despite the substantial public health concern posed by HS, there is still no field or clinical test of HS severity. We suggested previously that cardiac troponin (cTnI) could serve as a robust biomarker of HS severity. In the current study, we hypothesized that cTnI band intensity on a small, portable, Lifesign MI© point‐of‐care strip (POC strip) could be used to predict overall HS severity and HS induced organ damage. In brief, conscious male rats (N=24) continuously monitored for HR, BP, and core temperature (Tc) (radiotelemetry) were taken to a TcMax of 41.9 ± 0.1°C and then allowed to recover for 24h at Ta of 20°C. Blood samples (~120µL) were taken at Tc,Max and 24hrs‐post heat via submandibular bleed and 4 drops of blood were placed on POC strips. POC cTnI band intensity was ranked using a simple 4 point scale via 2 blinded scorers, and then compared to cTnI levels measured by a clinical grade blood analyzer (iSTAT®). Blood was also analyzed for biomarkers of systemic organ damage (VetScan® Liver Profile). HS severity, as previously defined using in‐heat heart rate & blood pressure as well as 24‐h recovery Tc profile, correlated strongly with cTnI (r= 0.82) & markers of systemic organ damage (albumin r= 0.90, alanine aminotransferase r= 0.87, blood urea nitrogen r= 0.71). POC cTnI band intensity ranking accurately predicted cTnI levels (r= 0.80) and overall HS severity (r = 0.91). This suggests that qualitative cTnI POC tests can accurately determine HS severity and could serve as simple, portable, cost effective HS tests in the field. Author views not official US Army or DoD policy.
Changes in Arterial Stiffness and Blood Pressure during Various Vascular Reactivity Tests
Authors: Lim Jisok , Pearman Miriam , alkatan Mohammed , Machin Daniel , Park Wonil , Tanaka Hirofumi
Published: June 2021

Increased arterial stiffness is an independent marker of cardiovascular risk. However, some investigators have questioned the utility of arterial stiffness above and beyond the traditional blood pressure (BP) measurement as the change in arterial stiffness is often accompanied by corresponding changes in BP. Although the associations between chronic levels of arterial stiffness and BP have been fairly well studied, it is unclear if and how much arterial stiffness is influenced by acute BP changes.

PURPOSE

To determine the magnitude of BP‐dependence of arterial stiffness during acute BP perturbations.

METHODS

Thirty apparently healthy subjects (46±4 years; 17 males) were studied. BP perturbations, including head‐up tilt, head‐down tilt, mental stress, isometric handgrip exercise, and cold pressor test were used to encompass blood pressure changes induced by physical, mental, and/or physiological stimuli. Arterial stiffness was measured with carotid‐femoral pulse wave velocity (PWV).

RESULTS

PWV was significantly associated with mean BP at rest (r=0.42). Mean BP was significantly changed during each perturbation, and changes ranged from △5±4 to △22±4 mmHg. Changes in mean BP and PWV were significantly correlated during mental stress (r=0.44), handgrip exercise (r=0.55), and cold pressor test (r=0.39). Significant associations were not found during head‐up tilt and head‐down tilt.

CONCLUSION

This study demonstrates that changes in arterial stiffness, as assessed by PWV, are significantly associated with acute BP changes during some BP perturbations but BP‐dependence of arterial stiffness is not observed in all perturbations.

Supported by Fukuda Denshi.
Potassium Channels Mediate Hydrogen Sulfide‐induced Cutaneous Vasodilation in Healthy Young Adults
Authors: Kutz Jessica , Greaney Jody , Alexander Lacy
Published: June 2021

Preclinical models of cardiovascular disease suggest that hydrogen sulfide (H2S) is an endothelium‐derived hyperpolarizing factor that modulates vascular function via potassium (K+) channel activation. Exogenous H2S donors elicit cutaneous vasodilation in young adults; however, the specific mechanism(s) underlying H2S‐induced cutaneous vasodilation remain unclear. The aim of this study was to examinethe specific K+ channel(s) that mediate H2S‐induced vasodilation in the cutaneous circulation of healthy adults. Five microdialysis fibers were placed in the ventral forearm skin of 6 healthy adults (25 ± 3 yrs) for the local delivery of Ringers solution (control), 5 mM glybenclamide (GLY: KATP channel inhibitor), 1 mM senicapoc (SENI: KCa intermediate channel inhibitor), 50 mM tetraethylammonium (TEA: non‐specific KCa channel inhibitor) and GLY + SENI + TEA. Laser‐Doppler flowmetry was used to continuously measure red blood cell flux. After a stable baseline, 5 mM Na2S (fast‐acting H2S donor) was perfused through all fibers to elicit H2S‐induced vasodilation. Data were normalized to maximal cutaneous vascular conductance (%CVCmax = laser‐Doppler flux/ mean arterial pressure; 28 mM sodium nitroprusside) and expressed as a percentage increase from baseline. Na2S‐induced cutaneous vasodilation (Δbase 28±8 %CVCmax; P<0.05). TEA (Δbase 4±3 %CVCmax) and GLY + SENI + TEA (Δbase 5±2 %CVCmax) blunted Na2S‐induced cutaneous vasodilation (both P<0.05), whereas GLY (Δ base18±8 %CVCmax) or SENI alone (Δbase 22±7 %CVCmax) had no effect (both P>0.05). These data suggest that exogenous H2S‐induced cutaneous vasodilation is mediated by TEA‐sensitiveKCa channels, but not by KATP or intermediate KCa channels, in healthy humans. NIH RO1 HL093238.
Bile Acid (BA), Lithocholic Acid (LCA), Reverses Chenodeoxycholate (CDCA)‐ and Cytokine‐Induced Loss in Epithelial Barrier Function in Human Colon Carcinoma T84 Cells
Authors: Khan N , Hung D , Javed F , Shukla P , Detloff S , Ao M , Domingue J , Rao M , Sarathy J
Published: June 2021

Epithelial apoptosis and tight junction (TJ) changes contribute to intestinal barrier dysfunction in Crohn's disease and colitis. We have reported that in T84 monolayers, CDCA decreased transepithelial resistance (TER) and caused dose‐ and time‐related cytotoxicity. CDCA increased cascade blue 10kDa dextran (CB10D) flux from apical to basal surfaces and this was enhanced by pro‐inflammatory cytokines (PiC: TNFα+IL‐1ß+IFNγ; FASEB J:28, 1113.3, '14). In this study we examined if LCA, a dehydroxylated product of CDCA, had similar effects. Confluent T84 cells (TER >1000 Ω.cm2) were treated with 5‐500µM LCA for 1 and 24 hr ± PiCs. Cell viability and apoptosis (Accuri flow cytometry), toxicity (lactate dehydrogenase), and paracellular permeability (TER and CB10D fluxes) were assessed. One hour or overnight (O/N) LCA (5 – 500μM) exposure neither affected cell viability nor apoptosis. LCA (1 Hr or O/N) did not increase LDH release and therefore was not cytotoxic. Time‐dependent studies (0.5 – 18 Hr) show that LCA alone did not affect TER (18 Hr: Control: 925+15; 50µM LCA: 848+41 Ω.cm2 n=6) or CB10D fluxes. In contrast, 50µM LCA attenuated PiC‐induced decreases in TER (data not shown) and increases in CBD10 fluxes ± 500 μM CDCA (18 Hr, in μg: Control: 7±1; LCA: 5±1; PiC: 51±4; PiC+LCA: 10±1; CDCA: 110±2; PiC+CDCA: 180±2; CDCA+LCA: 63±1 PiC+CDCA+LCA: 37.8±0.3; n>3). Surprisingly, unlike CDCA, LCA neither causes cell death nor disrupts tight junction function. On the contrary, LCA restores epithelial barrier integrity that is disrupted by cytokines ± CDCA. This study suggests that LCA could be a target therapeutic drug for inflammatory and diarrheal diseases.
A novel EGFR inhibitor SP101 blocks survivin and tumor growth in the human non‐small cell lung carcinoma
Authors: Chao Jui‐I , Wang Su‐Pei , Wu Ya‐Hui , Yang Jinn‐Moon , Chen Chinpiao
Published: June 2021

Gefitinib is a tyrosine kinase inhibitor that blocks the epidermal growth factor receptor (EGFR) pathway and cell growth ability in the human non‐small cell lung carcinoma (NSCLC). Here, we show a novel gefitinib derivative, dodecyl‐4‐(4‐(3‐(4‐(3‐chloro‐4‐fluorophenylamino)‐7 ‐methoxyquinazolin‐6‐yloxy)propyl)piperazin‐1‐yl)‐4‐oxobutanoate, which is called as SP101 that displayed more potent on the inhibition of survivin and cell growth ability than gefitinib in the NSCLC cells. Using a molecular docking and EGFR kinase activity assays, SP101 could bind to EGFR kinase through docking into the ATP‐binding pocket of EGFR and inhibited the EGFR kinase activity. Interestingly, SP101 displayed more potent on the cytotoxicity and apoptosis than gefitinib in various lung cancer cell lines. SP101 inhibited survivin inhibition to mediate the activation of caspase 3 and poly ADP‐ribose polymerase (PARP) protein cleavage for induction apoptosis. Expression of a survivin‐expressed vector resisted the SP101‐induced cell death. Moreover, combination of a survivin inhibitor YM155 enhanced the SP101‐induced cell death and overcoming the gefitinib‐resistant NSCLC cells. In addition, SP101 reduced the survivin protein levels and tumor size in the xenografted human lung tumors of nude mice. Together, SP101 is a potential gefitinib derivative, which may be through the blockage of survivin to inhibit tumor growth that can be developed the NSCLC therapy.
Hepatocellular Carcinoma Specific Transcriptional Interference of c‐Myc promoter by alpha‐fetoprotein and Sendai Virosome Based dsRNA System
Authors: Zakaria Mohammad Khalid , Khan Imran , Mani Prashant , Chattopadhyay Parthaprasad , Sarkar Debi P. , Sinha Subrata
Published: June 2021

Objective

c‐Myc is one of the central players of oncogenesis in hepatocellular carcinoma (HCC) and is also required in normal development. Majority of the c‐Myc transcripts are transcribed by its P2 promoter and an upstream ME1a1 site is required in its transcriptional activation. dsRNA also recruits epigenetic silencing complexes inducing long term/heritable transcriptional repression. We devised a novel HCC targeting system and studied its mechanism.

Methods

Firstly, the alpha‐fetoprotein (AFP) promoter/enhancer system was used to express shRNA against ME1a1 site. Secondly, we transformed Sendai virus into fusion (F) virosomes for packaging and delivery of AFP promoter/enhancer driven shRNA constructs into liver cells.

Results

Following F‐virosomal delivery, shRNA induced heterochromatization (H3K9Me2 and H3K27Me3) and CpG 8,9,10 methylation at the target locus. Following c‐Myc suppression, HCC cells showed decreased cell survival and increased apoptosis. Also, c‐Myc suppressed HCC cells were sensitized to sub lethal doses of 5‐Fluorouracil (5‐FU) and Cisplatin.

Conclusion

This system can specifically and prominently repress c‐Myc in HCC cells and could also be used to introduce genes specifically in embryonic liver.

Grant Funding Source: Department of Biotechnology, Govt. of India
The Development of a Highly Specific Monoclonal Antibody against Ki‐67 Useful for Immunohistochemistry
Authors: Chen Caiwei , Li Zhongwu , Yuan Kehu , Zhou Lixin , Chu Boyang , Shu Youmin , Chen Jian , Stafford Joe , Fu Wei , He Wei‐wu , Chambers Ross , Ma Donghui
Published: June 2021

Ki‐67 is a nuclear protein that is a useful marker of proliferation. Ki‐67 antibodies are used to generate a Ki‐67 labeling index, which is the percentage of cells with nuclear immunostaining in immunohistochemistry. The Ki‐67 labeling index may be useful as a prognostic and predictive marker in cancer. The Ki‐67 labeling index relies on the use of antibodies with a suitable level of sensitivity and specificity, and many Ki‐67 antibodies have been developed over the last two decades. One of the most widely used Ki‐67 antibodies is the mouse monoclonal MIB1. However, there have been many reports showing that in some cases MIB1 detects an unexpected membranous and cytoplasmic staining pattern in immunohistochemistry. Since Ki‐67 is exclusively expressed in the nucleus this raises questions about the specificity of MIB1. We have created a nearly comprehensive microarray of ~17,000 human proteins to investigate the specificity of MIB1 and identify cross reacting proteins. The protein microarray was also used to screen new panels of mouse monoclonals against Ki‐67. We have identified the antibody UMAB107 that in immunohistochemistry performs with similar sensitivity as MIB1, but unlike MIB1, UMAB107 is highly specific.
Biochemical Characterization of Heterodimeric Isocitrate Dehydrogenase I Using a Novel High‐throughput Mass Spectrometry‐based Assay
Authors: Piesvaux Jennifer , Miller Richard , McCoy Mark , Hou Yan , Spacciapoli Peter , Fischer Christian , Machacek Michelle , Fradera Xavier
Published: June 2021

In several types of human tumors, a gain‐of‐function mutation within a single allele of isocitrate dehydrogenase 1 (IDH1) results in the production of a known oncometabolite, 2‐hydroxyglutarate (2‐HG). IDH1 is enzymatically active as a dimer, and both homodimeric and wt:mutant heterodimeric proteins are produced in cells. Since the heterodimer is comprised of both the mutant and wild‐type subunits, it therefore catalyzes both the canonical (a‐ketoglutarate generating) and the neomorphic (2‐HG generating) reactions. Traditionally, IDH1 assays monitor the reduction or oxidation of NADP/H, a common co‐factor substrate for both the mutant and wild‐type subunits. This, in conjunction with an extremely low predicted NADPH Km, greatly complicates the kinetic characterization of heterodimeric IDH1. Here we present a novel mass‐spectrometry‐based IDH1 assay. Not only are we able to selectively monitor 2‐HG production by the mutant subunit, but incorporation of a unique NADPH regeneration system allows us to study the reaction at nanomolar NADPH concentrations. Using this platform we determined that the IDH1 heterodimer exhibits a significantly lower Km for both a‐KG (314 μM) and NADPH (26 nM) than the mutant homodimeric protein (a‐KG Km = 2800 μM and NADPH Km = 173 nM). To further characterize the heterodimer and determine if the wild‐type subunit catalyzes production of NADP+ and isocitrate, we assessed the activity of a catalytically impaired wild‐type subunit (Y139D) and also monitored the heterodimeric reaction by NMR. Our findings indicate that under the conditions of our assay, the wild‐type subunit makes minimal contributions to total NADP+ production.
The X‐ray Crystal Structure of Full‐length Mammalian Phenylalanine Hydroxylase
Authors: Jaffe Eileen , Arturo Emily , Ramirez Ursula , Heroux Annie , Scary Thomas , Loll Patrick
Published: June 2021

Mammalian PAH catalyzes the rate‐limiting step of phenylalanine catabolism, synthesizing tyrosine from phenylalanine. Deficiency in catalytic activity or improper regulation of PAH manifests as hyperphenylalanineamia, the most critical clinical presentation of which is phenylketonuria (PKU). PKU is a common inborn error of metabolism and can be caused by a remarkably large number of different missense mutations. The catalytic activity of PAH is uniquely regulated among the family of aromatic amino acid hydroxylases: in response to elevated levels of substrate, PAH undergoes dramatic structural changes in concert with enzyme activation. We previously proposed that allostery involves major domain‐domain reorientations such that low‐activity and high‐activity tetramers are architecturally distinct. Prior to the current structure, an understanding of disease‐associated PAH variants was limited by structures of truncated proteins that could be combined to form composite homology models. We now present the X‐ray crystal structure of full‐length rat PAH in the low‐activity conformation, at 2.9 Å resolution. This structure shows unexpected differences in the orientation of PAH subunits within the tetramer and in the conformation of the C‐terminal helixes important to tetramerization through formation of an antiparallel four‐helix bundle. Comparison of this new PAH structure with structural information from other aromatic amino acid hydroxylases allows us to refine our model for the allosteric regulation of PAH and provides an improved basis for understanding PKU.
New Insights into Mechanisms Of Vitamin D Action Revealed Through The Study Of Transgenic, Knockout, and Aging Mice
Authors: Alexandre Karlbuto , Veldurthy Vaishali , Christakos Sylvia
Published: June 2021

Vitamin D is an important regulator of calcium homeostasis. However, the role of vitamin D in the distal intestine, where 70% of calcium absorption occurs, has been unexplored. Hectorol, a vitamin D analog, which is less calcemic than the active form of vitamin D, 1,25(OH)2D3, has been suggested for the treatment of osteoporosis. We hypothesize that the distal segments of the intestine are as important as the proximal intestine in vitamin D mediated intestinal calcium absorption and that transgenic mice expressing the vitamin D receptor exclusively in the distal intestine express vitamin D target genes in the distal intestine at levels equivalent to wild type. We also hypothesize that hectorol is as effective as 1,25(OH)2D3 in the maintenance of calcium homeostasis. Intestine and kidney from WT mice, Tg, and VDR KO mice were harvested. Total RNA was isolated, and protein extracts were prepared. The expression of vitamin D target genes was analyzed by RT‐PCR. Protein expression was studied using Western blot. VDR mRNA was undetectable in all segments of the intestine in VDR KO mice and was 1.7 fold elevated in ileum, cecum and colon of Tg mice compared to WT mice. The calcium binding protein calbindin‐D9k mRNA was decreased in VDR KO mice and in Tg mice levels of calbindin‐D9k mRNA in the distal intestine were equivalent to WT. mRNA for CYP24A1, an enzyme regulated by 1,25(OH)2D3, was undetectable in intestine of VDR KO mice and was expressed at equivalent levels in the distal intestine of Tg and WT mice. Hectorol was as effective as 1,25‐D3 in inducing calbindin‐D9k mRNA in all regions of the intestine of vitamin D deficient mice.
Complementing Confocal Detection of Antibody‐labeled Lysophosphatidic Acid Receptors in Human Gingivae with Label‐free Second Harmonic Generation Confocal Microscopy Detection of Collagen
Authors: Cerutis D. Roselyn , Nichols Michael , Hironaka Michael , Miyamoto Takanari , Khan Shakeel , Ogunleye Afolabi , McVaney Timothy
Published: June 2021

We have found that lysophosphatidic acid (LPA) controls the healing and [Ca2+]i responses of human gingival fibroblasts via the LPA1 and LPA3 receptor subtypes, and that LPA3 may modulate their inflammatory responses. We have used immunofluorescence to detect LPA receptor (LPAR) distribution in human gingivae, but that technique does not afford visualization of the state of collagen fiber organization. We hypothesized that using the second harmonic generation (SHG) technique would enable detection of changes in the appearance, collagen fibril density, and fibril organizational structure of human gingiva, and that this technique would complement visualizing LPAR expression by confocal microscopy. Pieces of intact, paraformaldehyde‐fixed, methanol:acetone (1:1) de‐lipidated attached gingivae [~1.25 mm thick (from IRB‐approved, consented donors) normal or with moderate‐severe periodontal disease] were used. Confocal analysis using anti‐LPA1 and anti–LPA3 antibodies confirmed that LPA3 is primarily found in the epithelium, and that nuclear LPA1 expression in the sub‐epithelial connective tissue is greatly diminished in periodontal disease. SHG analysis clearly showed the normal collagen structure and subsequent reduction in the abundance and organization in the collagen fibrils of periodontal gingivae. This multidisciplinary study shows that SHG effectively complements confocal microscopy as a tool to help understand periodontal changes at the level of receptors and collagen organization.
A Preliminary Investigation of Cardiac Stem/Progenitor Cells in the Lizard Heart
Authors: Jacyniak Kathy , Hawthorne Danika , Robertson Lisa , Vickaryous Matthew
Published: June 2021

Resident stem/progenitor cell populations have been identified within many vertebrate tissues, including the hearts of teleost fish, urodeles, and mammals. Although often rare, these populations are considered crucial for normal physiological repair, and may also play a role in tissue regeneration. Here we provide preliminary evidence of the presence of resident cardiac stem/progenitor cells within the lizard Eublepharis macularius (the leopard gecko). Using Proliferating Cell Nuclear Antigen (PCNA) immunostaining, we determined that gecko cardiomyocytes continue to proliferate into adulthood. Next, we performed a 5‐bromo‐2'‐deoxyuridine (BrdU) pulse‐chase experiment to identify slow cycling cells within the normal (uninjured) heart. We found BrdU label‐retaining cells within cardiac tissues chased for 4, 6 and 20 weeks within the epicardium and myocardium of the ventricular wall and trabeculae, indicating that quiescent or slow‐cycling cells are present. Finally, we observed that select BrdU label‐retaining cells at 20 weeks were also immunopositive for the cardiac progenitor marker c‐kit. Based on these findings, we predict that putative cardiac stem/progenitor cells are present in the gecko heart.
Daily Consumption of Fructose for 15 Weeks Leads to Metabolic Disorders and Impaired Renal Function without Cause Obesity
Authors: Francisqueti Fabiane , Betti Isabella , Minatel Igor , Ferron Artur , Santos Klinsmann , Nascimento Andre , Silva Vanessa , Correa Camila
Published: June 2021

To evaluate the effect of daily consumption of fructose on body weight and urine parameters indicative of kidney damage. Male Wistar rats were divided to receive standard diet + water (C group, n=24) or standard diet + frutose in drinking water (30%) for 15 weeks. At the end of this period, we assessed the animals' weight (g), calorie intake (kcal), plasm glucose (glu) and triglycerides (TG)‐ (mg/dL), glucose, albumin (alb) and creatinine (creat) urinary (mg/dL). The groups were compared by Student t ‐test and Mann‐Whitney and the data are expressed as mean ± standard deviation or median, 1st ‐ 3rd quartiles.There was no difference in weight (C= 388.22; 341.11‐ 425.39 x F= 384.44; 331.89‐ 419.50, p= 0.77) and caloric intake (C= 280.29; 255.66‐ 334.45 x F=297.86; 264.27‐ 357.21; p= 0.17) between the groups. There was increased glu (C= 131.51 ± 16.49 x F= 142.60 ± 20.54, p= 0.04) e TG (C=87.72; 68.57‐ 106.40 x F= 108.66; 86.796‐ 136.27, p= 0.006) in animals F compared to C. In urine, F animals showed an increased alb (C= 0.07; 0.05‐ 0.10 x F= 0.09; 0.07‐ 0.13, p= 0.04), glu (C= 7.08 ± 4.88 x F= 11.51 ± 6.35, p= 0.013) and creat (C= 138.32; 85.07‐ 179.34 x F= 189.04; 146.70‐ 250.69. p= 0.002). Even without causing obesity, the daily consumption of fructose for 15 weeks leads to alterations in glucose, triglycerides and markers of renal function, which is a health risk.
Vitamin D3, 25‐Hydroxyvitamin D3, and 1α‐Hydroxyvitamin D3 Modulate Fiber Type Distribution in Bicep Femoris of Growing Rats
Authors: Park Min Young , Kim Jonggun , Whang Kwang‐Youn
Published: June 2021

An experiment was conducted to examine the effects of different types of vitamin D3 on skeletal muscle development using growing rats. Three‐week‐old rats were fed a vitamin D3‐deficient diet for 4 weeks and vitamin D3‐replete diets for the next 4 weeks as each of vitamin D3 (vit D3, 1,000 IU/kg diet), 25‐hydroxyvitamin D3 (25‐OH D3, 400 IU/kg diet), and 1α‐hydroxyvitamin D3 (1α‐OH D3, 4 IU/kg diet) was supplemented. Negative control group was fed a vitamin D3‐deficient diet, and positive control group was fed a vitamin D3‐supplemented diet (1,000 IU/kg diet) for the whole experimental period, 8 weeks. In results, body weight, feed intake, and body composition (lean and fat) were not affected by vitamin D3 dose and type. Quantitative gene expression of skeletal muscle differentiation markers (MyoD, Myogenin, Mrf4, Myf5, and Myostatin) also showed no difference. However, skeletal muscle fiber type distribution (myofibrillar ATPase staining) was changed in bicep femoris (BF) of the rats fed different types of vitamin D3 supplements.In positive control, type I was increased by 2.4 fold (7.22 % vs. 2.95 %) compared to negative control. In the rats fed vit D3‐replete diets followed by 4‐weeks of vitamin D3 deficiency, both type I and IIa were increased only 1.3 fold (3.72% vs. 2.95%, and 20.79 % vs. 15.74 %) compared to negative control. Percentages of type I were 5.0‐ and 6.9‐fold increased in BF of the rats fed 25‐OH D3 (14.63 % vs. 2.95%) and 1α‐OH D3 (20.45 % vs. 2.95 %) respectively, in comparison to negative control. The present study demonstrates that vitamin D3 does not affect lean growth, but it might mediate skeletal muscle fiber type distribution in growing rats depending on the vitamin D3 types.
TMED2 is Sufficient for Trophoblast Fusion
Authors: Jerome‐Majewska Loydie , Yuan Libin , Hou wenyang , Deba Taghreed
Published: June 2021

TMED2 is essential for anterograde transport from the endoplasmic reticulum to the Golgi. We identified a protein null mutation in Tmed2. At embryonic day (E) 9.5, Tmed2 homozygous (Tmed299J/99J) mutant embryos have abnormal placental labyrinth layer formation and fail to form the functional syncytiotrophoblast cells required for placental labyrinth layer formation, and die by E11.5. Tmed2 is expressed in mouse placenta and in human placentas, suggesting that TMED2 function is conserved in human and mouse placentas. We hypothesized that TMED2 was required for syncytiotrophoblast differentiation. We used qRT‐PCR to show that expression of TMED2 significantly increased when BeWo choriocarcinoma cells are induced to form syncytiotrophoblast. Furthermore, knockdown of TMED2 in this cell line does change the fusion index in the absence of Forskolin and but results in decreased metabolic activity when measured by MTT. To test for a direct role for TMED2 in syncytiotrophoblast formation, we generated JEG‐3 stable cell lines expressing TMED2myc. JEG‐3 cells do not normally fused but we will show that co‐culture of JEG‐3 stable cell lines expressing cytoplasmic GFP or mCherry allowed visualization of fusion. Quantification of syncytiotrophoblast fusion shows a significant increase in the fusion index of JEG‐3 cells, with no changes in metabolic activity by MTT. Our data suggest that TMED2 is sufficient for syncytiotrophoblast fusion.
Reduced Transcobalamin I Gene Expression Following Roux‐en‐Y Gastric Bypass Surgery Can Contribute to B12 Deficiency
Authors: Sala Priscila , Machado Natasha , Belarmino Giliane , Ishida Robson , Guarda Ismael , Giannella‐Neto Daniel , Santo Marco Aurelio , Moura Eduardo , Sakai Paulo , Silva Ismael , Ye Jianping , Heymsfield Steven , Waitzberg Dan
Published: June 2021

The objective of this study was to examine potential mechanisms leading to the often‐observed Vitamin B12 deficiency following Roux‐en‐Y gastric bypass (RYGB). Vitamin B12 absorption begins with release from food in the upper GI tract followed by transcobalamin I binding; duodenal enzyme degradation of transcobalamin I is then followed by intrinsic factor (IF) capture; IF is next degraded within the ileal enterocyte, releasing B12 that then enters the plasma. Although IF levels are reduced with RYGB due to removal of the gastric fundus, other causes of modified B12 transport may be involved. To examine these other potential mechanisms we used balloon endoscopy to acquire GI tissue at multiple sites in 20 obese women with adult‐onset diabetes before (age, 46.5±6.6 yrs; BMI, 45.7±4.1 kg/m2) RYGB and at the identical India‐Ink marked sites three months following surgery (BMI, 37.7±3.4 kg/m2). Microarray analysis was performed on all samples using a Human GeneChip 1.0 ST array (Affymetrix, Inc., Santa Clara, CA). There was significantly reduced gene expression (2.335‐fold, p<0.05) of TCN1 in the remnant stomach following surgery (TCN1 translates transcobalamin I). These findings suggest that B12 deficiency following RYGB may be caused by a reduction in transcobalamin I in addition to the anatomical absence of IF.

FAPESP 2011/09612‐3, BEPE 2014/07742‐5.
The Effect of Endocrine Disruptors on Neuron Development
Authors: Feng Joshua , Lee Yuan , Nakayama Kensaku , Acey Roger
Published: June 2021

Butyrylcholinesterase (BuChE) activity peaks during a 3 hour time period 24 hours post‐initiation of neural tissue differentiation. However, the exact biological role of BuChE during neural tissue development is still unknown. Di‐n‐butyl phthalate (DBP) and bisphenol A (BPA) are well‐documented environmental contaminants that interact with BuChE. Di‐alkyl phenyl phosphates are known irreversible inhibitors of BuChE. The goal of this research is to determine the role of BuChE in stem cell differentiation. We have developed a human umbilical cord mesenchymal stem cell (HUC‐SC) system to determine the effects of these compounds on protein expression. Differentiating cells were exposed to sub‐lethal concentrations of each inhibitor for 48 hours, collected, and homogenized. Cell lysates were analyzed by 2D‐PAGE for protein expression patterns. Relative changes in protein expression were quantified and identification of differentially expressed proteins was achieved using MALDI‐TOF MS. Regardless of the inhibitor used, we identified two significantly up‐regulated proteins in cell extracts: neuron specific enolase and estrogen specific heat shock protein β‐1 (Hsp β‐1). This suggests that BuChE is critical for the neuron differentiation pathway. The presence of heat shock protein indicates that the cells are under stress from exposure to estrogen mimics. We also identified three other developmentally regulated proteins: calmodulin is up‐regulated while cofilin and galectin are down‐regulated. Supported by grants from CSUPERB and the CSULB Mini Grant Programs
A Study of Antinuclear Antibody Binding (ANA) in Lewis Rat Kidney Injected With Human Serum Positive For ANA
Authors: Javed Najma , Kelly‐Worden Marie , Griffin Marley , Hammer Leslie , Alhammad Saad
Published: June 2021

Lupus nephritis (LN) is an inflammatory condition affecting the kidneys in systemic lupus erythematosus (SLE). Pathophysiology of LN is not clearly understood. Autoantibody deposits in glomeruli can cause progressive renal damage leading to renal failure. We proposed to study the binding patterns of ANA in the kidney. Kidneys from 8 female Lewis rats (age=6 months) were harvested to study. Rats were randomly separated into 4 groups (n=2). Control group rats were injected with 300 μl of 0.9% sterile saline through the tail vein once a week for 4 weeks; the second group was a negative control injected with 250μl of ANA free human serum plus 50μl of 0.9% saline. The third group was injected with 250 μl of ANA plus 50μl of 0.9% saline. The fourth group was injected with ANA and 4 mg/kg BW of Benadryl (antihistamine). Studies have reported, that histamine can decrease glomerular permeability. Slices of frozen sections were examined by Immuno‐fluorescent staining for ANA binding. Cellular infiltration was assessed by staining the tissue slices with H&E stain. Results of this study indicate that both the ANA group and the ANA+Benadryl group show significant differences in binding as compared to the negative control group respectively (P<0.01, P<0.001). Probability of ANA binding in the ANA group and the ANA Benadryl group was significantly greater as compared to both the negative control and saline injected group. Endothelial cells were the dominant cell type in the tissues. Binding patterns of ANA in the kidney may enhance our understanding of the underlying mechanism of SLE and hence, a better therapeutic approach in treating the disease. Furthermore, it may provide a precise biomarker for future diagnosis. Support:Ball State Research Funds.
Enhanced Linkage in the Vascular Calmodulin Network by Estrogen via a Feedforward at the G Protein‐Coupled Estrogen Receptor 1 (GPER/GPR30)
Authors: Tran Quang‐Kim , Firkins Rachel , VerMeer Mark , Giles Jennifer , Francis Sarah , Matnishian Vahe , Jasurda Jake
Published: June 2021

Plasma estrogen is strongly linked to cardiovascular health. Calmodulin (CaM) is required for the activities of numerous proteins but is insufficiently expressed for all its targets, leading to competition among CaM targets. It is completely unknown if estrogen modulates cardiovascular functions via the CaM network. We observed in endothelial cells that chronic estrogen treatment (CE2T) increases total and free CaM. Chronic treatment with G‐1, an agonist of the estrogen‐sensitive receptor GPER/GPR30 mimics this effect, while agonists of ERa or ERb do not. ICI182780, an ERa/ERb antagonist and GPER/GPR30 agonist, also increases CaM level. CE2T increases CaM binding to different categories of CaM targets, including the plasma membrane Ca2+‐ATPase (PMCA), eNOS, ERa, and GPER/GPR30 itself. For GPER/GPR30, CaM antagonism or CaM binding‐negating mutations in the receptor's multiple CaM‐binding domains prevent GPER/GPR30‐mediated ERK1/2 phosphorylation. For PMCA, CE2T‐induced stimulation of activity through enhanced CaM binding is masked by Src‐dependent phosphorylation. These effects sustain cytoplasmic Ca2+ for enhanced interactions between CaM and other targets. For eNOS, CE2T doubles CaM binding. Kinetic modeling using in‐cell and in vitro data allowed comparison of CE2T's promotion of eNOS point activity and NO accumulation via effects on determinants of eNOS function, including Ca2+, CaM, and phosphorylation. Our data indicate that CE2T improves endothelial functions via a feed‐forward mechanism in which CaM is upregulated through GPER/GPR30 activation, leading to enhanced CaM binding and functional linkage in the network of CaM‐binding proteins.
Integration of G Protein Alpha (Gα) Signaling by the Regulator of G Protein Signaling 14
Authors: Brown Nicole , Goswami Devrishi , Branch Mary Rose , Ramineni Suneela , Ortlund Eric , Griffin Patrick , Hepler John
Published: June 2021

RGS14 contains distinct binding sites for both active (GTP bound) and inactive (GDP bound) forms of Gα proteins. The N‐terminal RGS domain binds active Gαi/o‐GTP, whereas the C‐terminal GPR motif binds inactive Gαi1/3‐GDP. The molecular basis for how RGS14 binds different activation states of Gα to integrate G protein signaling is unknown. Here we explored the effects of G protein binding on the GPR motif and the RGS domain, and examined whether RGS14 can functionally interact with two forms of Gα simultaneously. Using cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gαi1‐GDP at the plasma membrane (PM), and that cytosolic RGS14 is recruited to the PM by activated Gαo‐AlF4‐. Bioluminescence resonance energy transfer (BRET) studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen deuterium exchange mass spectrometry (HDX‐MS) revealed RGS14 is a dynamic protein that undergoes allosteric conformational changes when inactive Gαi1‐GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gαo‐AlF4‐ and an AlF4‐‐insensitive mutant (G42R) of Gαi1‐GDP as observed by size exclusion chromatography and differential HDX‐MS. Finally, a preformed RGS14:Gαi1‐GDP complex exhibits full capacity to stimulate the GTPase activity of Gαo‐GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals.
Regulation of mouse liver chromatin accessibility and associated early gene responses by CAR
Authors: Lodato Nicholas , Rampersaud Andy , Waxman David
Published: June 2021

Many xenobiotics are metabolized in mammalian liver by pathways regulated by constitutive androstane receptor (CAR). Here, we identify early gene targets of mouse liver CAR and investigate their associated, CAR‐induced changes in local chromatin accessibility and transcription factor (TF) recruitment. Genes induced or repressed by the CAR agonist TCPOBOP were identified by RNA‐Seq after 3 or 27 hr. Chromatin sites that dynamically open or close following CAR activation (changes in DNase I hypersensitive sites, ΔDHS) were identified by DNase‐Seq. Genes dysregulated in 3 hr TCPOBOP‐treated mouse liver were enriched in KEGG pathways for retinol and drug metabolism (DAVID analysis; p<E‐15). IPA identified activated CAR and its agonists as the top predicted upstream regulators of these gene responses. After 27 hr TCPOBOP treatment, many more genes were identified and the predicted upstream regulators expanded to include cyclin D and vitamin D, indicating significant secondary transcriptional regulation. DNase‐Seq identified ~3,600 ΔDHS that were opened or closed by 3 hr TCPOBOP treatment. These ΔDHS were not randomly distributed: mapping to their putative gene targets (i.e., nearest gene within 10 kb) revealed enrichment (12.4‐fold, p<E‐45) of the TCPOBOP‐induced DHS for genes inducible by CAR. Thus, chromatin opening is associated with activation of nearby CAR target genes. Genomic sequences in the 3 hr TCPOBOP‐inducible DHS regions were enriched for TF binding motifs for CAR and for CCAAT‐enhancer binding protein (CEBP), suggesting that CEBPs cooperate with CAR to open chromatin and up regulate target gene expression. Studies designed to test this hypothesis are in progress and will be reported. Grant support: NIH ES024421.
Doxycycline Prevents Right Ventricular Dysfunction Induced by Pulmonary Embolism
Authors: Neves Evandro , Kline Jeffrey
Published: June 2021

Pulmonary embolism (PE) is commonly associated to right ventricular (RV) dysfunction, which predict poor outcome. Doxycycline pretreatment has been associated to beneficial effects after PE. However, no previous study has evaluated doxycycline posttreatment.

Male Sprague‐Dawley rats (300‐350g) were anaesthetized with ketamine and xylazine. On day 1, the PE group received 85 µm polystyrene microspheres (221.000/100 g body wt, jugular vein) suspended in 0.01% Tween 20 and the Sham group received Tween only. Treatment animals received doxycycline (Doxy, 30mg/kg by gavage) and Controls received volume‐matched saline for 5 days after PE induction. Hearts were then extirpated from anesthetized animals on day 5 and perfused via the aorta using Langendorff technique with Krebs‐Henseleit buffer. We explored the Frank‐Starling relationship of RV contractile function (RV developed pressure, dP/dtmax and dP/dtmin) by increasing RV end diastolic pressures (from 0 to 40 mmHg). P<0.05 was considered significant.

In the PE group (n=6), ex vivo RV function was decreased significantly after 5 days compared with the Sham group (n=6), including RV developed pressure (PE, from 14±3.5 to 68±5.1 mmHg) vs (Sham, from 38±1.2 to 88±2.4 mmHg), dP/dtmax (PE, from 516±114 to 1022±201 mmHg/s) vs (Sham, from 1442±113 to 1826±75 mmHg/s), and dP/dtmin (PE, from ‐401±76 to ‐667±108 mmHg/s) vs (Sham, from ‐992±71 to ‐1185±77 mmHg/s). When compared with PE group, Treatment animals (PE+Doxy, n=6) had significantly improved RV function (from 35±4.0 to 89±1.7 mmHg), (from 1411±198 to 2034±149 mmHg/s) and (‐852±126 to ‐1119±87 mmHg/s) respectively.

Doxycycline improved the intrinsic RV contractile dysfunction caused by PE. Doxycycline may help preserve RV function and decrease morbidity after PE.
Salvianolic acid B ameliorates lipopolysaccharide‐induced albumin leakage from rat mesenteric venules through Src
Authors: Han Jing‐Yan , Pan Chun‐Shui
Published: June 2021

Lipopolysaccharide (LPS) causes microvascular barrier disruption, leading to albumin leakage from microvessels resulting in a range of disastrous sequels. Salvianolic acid B (SalB) is a major water‐soluble component derived from Salvia miltiorrhiza. The present study was intended to investigate the impact of SalB on endothelial cell barrier in vivo in rat mesenteric venules as well as in vitro in human umbilical vein endothelial cells (HUVECs). Male Wistar rats were challenged by infusion of LPS (2 mg/kg/h) through left femoral vein for 90 min. SalB (5 mg/kg/h) was administrated either simultaneously with LPS or 30 min after LPS infusion through the left jugular vein. Caveolae in venular walls were observed by electron microscopy. HUVECs were incubated with LPS with or without SalB. The expression of Zonula occluden‐1 (ZO‐1), VE‐cadherin, caveolin‐1 and Src in HUVECs was assessed by Western blot and confocal microscopy, binding of SalB to Src was measured using Surface Plasmon Resonance and BioLayer Interferometry. Treatment with SalB inhibited albumin leakage from rat mesenteric venules and inhibited the increase of caveolae number in venular endothelial cells induced by LPS. In addition, SalB inhibited the degradation of ZO‐1, the phosphorylation and redistribution of VE‐cadherin, the expression and phosphorylation of caveolin‐1, and phosphoirylation of Src in HUVECs exposed to LPS. Furthermore, SalB was found able to bind to Src. This study demonstrates that protection of SalB against microvascular barrier disruption is a process involving both para‐ and trans‐endothelial cell pathway, and highly suggests Src as the key enzyme for SalB to work.