bluuebunny/crossref_metadata_2025 huggingface dataset:
A Novel Inhibitor of Epithelial‐to‐Mesenchymal Transition Reduces Pancreatic Cancer Cell Invasion and Tumor Growth in Mice
Authors: Wang Tao , Dong Ruochen , Chen Ping , Chen Qi
Published: April 2020

Objective

Epithelial to Mesenchymal Transition (EMT) has been proposed to contribute importantly to metastasis, cancer stem cell (CSC) generation and drug resistance in many cancers. Targeting EMT may be promising to benefit cancer treatment. Our previous high throughput screening study has identified a potential EMT inhibitor (namely, C150) in pancreatic cancer cells. Here, we sought to investigate the activities of C150 in inhibiting pancreatic cancer cell invasion, migration and tumor growth in mice, as well as its mechanisms of EMT inhibition.

Methods

Cell viability was detected by MTT assay. Cell migration/invasion was detected by Boyden chamber trans‐well migration‐invasion assay, wound scratch assay and a 3‐dimension (3D) cell invasion assay. MMP zymography assay was used to indicate MMP inhibition. In vivo tumor growth was evaluated in an orthotopic mouse model of pancreatic cancer. EMT marker proteins were examined by western blot.

Results

C150 exhibited a well‐separated cytotoxicity between pancreatic cancer cells and non‐cancerous cells, with IC50 values of 1~2.5 μM in multiple pancreatic cancer cell lines and >25 μM in a noncancerous pancreatic epithelial cell line (hTERT‐HPNE). C150 was found to significantly inhibit PANC‐1 cell invasion in the 3‐dimension (3D) cell invasion model, the wound scratching assay and the Boyden Chamber trans‐well migration‐invasion assay. C150 treatment reduced the expressions of MMP‐2 and MMP‐9 in PANC‐1 cells, and inhibited MMP enzyme activity in zymography assay. In orthotopic pancreatic cancer mouse model, C150 significantly reduced tumor growth at the dose of 15 mg/kg, 3x weekly by IP injection. C150 decreased the mesenchymal markers of N‐cadherin and Snail while increased epithelial markers of ZO‐1 and Claudin‐1. Further studies revealed that C150 treatment significantly increased proteasome activity in the cell without changing the protein expression levels of proteasome subunits.

Conclusion

The novel compound C150 inhibited pancreatic cancer cell migration, invasion, and in vivo tumor growth in mice. The compound increased cellular proteasome activity, which enhanced the protein degradation of Snail, resulting in EMT inhibition in the cell. The studies of more detailed mechanisms of C150 induced EMT inhibition and proteasome activation, as well as target identification are currently undergoing.

Support or Funding Information

Institutional Development Award (IDeA) of NIH and State of Kansas Grant # 8P30GM103495, KU Cancer Center Lied Basic Science Grant 2018
Glycophorin B‐A‐B Variant GP.Mur, a Chaperone for Erythroid SLC4A Transporter, Supports Gas Exchange during Exhaustive Exercise
Authors: Hsu Kate
Published: April 2020

The main function of glycophorin A is to stabilize protein complex expression of SLC4A (band 3) and to facilitate its Cl−/HCO3− antiporter activity across human red blood cell (RBC) membrane. Anion exchange through SLC4A on the RBC surface is physiologically critical for blood CO2 transport. We previously found that glycophorin B‐A‐B hybrid protein GP.Mur, a special blood type unique to people of Southeast Asian origins, further promotes erythroid expression of SLC4A. People with GP.Mur phenotype not only express more band 3 on their RBCs but also exhibit more efficient blood CO2 expiration when challenged with a mild stepping exercise test. We have noticed a disproportional number of elite athletes in Taiwan bearing the GP.Mur phenotype. The goal of this study was thus to verify whether GP.Mur expression could indeed support the respiratory function of elite athletes during exhaustive exercise. Here we recruited GP.Mur+ and GP.Mur‐young elite/professional athletes in Taiwan and challenged them with a graded, exhaustive protocol of cardiopulmonary exercise testing (CPET). As revealed by CPET, compared to the athletes lacking the special blood type, GP.Mur+ athletes showed smaller respiratory quotient (RQ) and lower arterial CO2 tension throughout the graded test before exhaustion. This suggests relative easiness on the respiratory function for athletes with GP.Mur phenotype during exhaustive exercise. We are currently exploring the possibility of utilizing one’s GP.Mur/band 3 expression levels in personalized athletic training.

Support or Funding Information

Taiwan Ministry of Science & Technology (MOST 108‐2628‐B‐195‐001)
The comparative and functional anatomy of appendicular musculature of Humboldt’s woolly monkey (Lagothrix lagotricha): What can a (mediocre) suspensory monkey tell us about and human locomotor evolution?
Authors: Deane Andrew Stewart , Organ Jason , Evie Vereecke , Muchlinski Magdalena , Butterfield Tim
Published: April 2020

Similar to other members of the platyrrhine subfamily Atelinae, Humboldt’s wooly monkey (Lagothrix lagotricha) isa ‘tail‐assisted’ brachiator that more frequently engages in non‐suspensory forms of locomotion (i.e. quadrupedal climbing). As a group the atelines share a suite of derived forelimb skeletal characters that are specialized for brachiation and that are convergent with ‘richochetal’ brachiating apes (Hylobates, Symphalangus), quadrumanus suspensory apes (Pongo) and secondarily terrestrial apes retaining suspensory capabilities (Pan, Gorilla). The skeletal anatomy of lagothrix is similar to more suspensory atelines (Ateles, Brachyteles) and living apes, but is typically less derived for suspension and many skeletal characters are intermediate between highly suspensory and non‐suspensory anthropoids. While the functional morphology of the ateline skeleton is well documented, relatively little is known about the comparative and functional morphology of the forelimb and hindlimb musculature. Specifically, can locomotor differences between Lagothrix and more suspensory atelines and hylobatids be explained, in part, as a result of differences in muscletendon architecture?

This study describes preliminary quantitative data on the muscle–tendon architecture (relative muscle mass and relative physiological cross‐sectional area or PCSA) of the fore‐ and hind‐limbs of Lagothrix lagotricha and Macacasp. (n=9). Despite infrequent brachiation, Lagothrix forelimb relative muscle mass and PCSA is identical to similar results reported for hominoids. In contrast, the relative mass and PCSA of the hind‐limb musculature is most similar to quadrupedal monkeys (i.e. Macaca). Specifically, Lagothrix resembles hylobatids in the concentration of muscle mass and force in the flexor compartments of the arm and forearm but is more similar to Macaca in similar measures associated with the extensor and flexor thigh and leg. This suggests that despite a reduced reliance on suspensory postures and brachiation, Lagothrix forelimb muscle‐tendon architecture may be, at least in part, a response to the minimum functional demands of brachiation but that similar selection pressures are not as prevalent in the hind‐limb.
The Sigma1 Receptor Antagonist CM304 Enhances the Antinociceptive Effects of the Cannabinoid Receptor Agonists, but not Mu‐Opioid Receptor Full Agonists in Mice
Authors: Patel Avi , Obeng Samuel , Burns Mallory , Intagliata Sebastiano , Mottinelli Marco , Reeves Morgan E. , Behnke Mira , Restrepo Luis F. , Taylor Victoria A. , Pennington Anna E. , Gamez Jimenez Lea R. , Williamson Morgan R. , McCurdy Christopher R. , McMahon Lance R. , Hiranita Takato
Published: April 2020

There is an overdose epidemic associated with the use of illicitly manufactured as well as prescription mu‐opioid receptor agonists. Sigma1 receptor (σ1R) antagonists in combination with other drugs may provide a viable, safer pharmacological option than opioids alone for treating pain. The present study compared pharmacological effects of the σ1R antagonist CM304 alone and in combination with high (fentanyl and morphine) and low (buprenorphine) efficacy opioid agonists and the cannabinoid CB (CP55,940 and THC) receptor agonists in C57BL/6J mice. Rectal temperature, tail withdrawal latency (10‐seconds cutoff) from warm water of various temperatures (45°C, 50°C and 55°C) and counts of unhabituated locomotor activity were measured in this order. Basal latency for tail withdrawal systematically decreased from 10 seconds at 45°C to 1.3 seconds at 55°C. Morphine, fentanyl and buprenorphine dose‐dependently increased maximum possible effects (MPEs) up to 100% at 55°C (ED50 values: 7.50, 0.068, and 1.13 mg/kg, respectively), whereas CP55,940 and THC were less effective at 55°C (Emax values: 56.6% and 58.2%, respectively). CM304 (56 mg/kg, i.p.) produced an upward shift in the CP55,940 dose‐effect function such that 100% MPE was achieved at 3.2 mg/kg CP55,940, while CM304 (56 mg/kg) produced a 3‐fold leftward shift in the dose‐effect curve of THC. On the other hand, CM304 did not enhance the antinociceptive effects of morphine and fentanyl but enhanced the effects of buprenorphine. In contrast to tail withdrawal latency, there were no consistent effects of CM304 on cannabinoid‐induced decreases in rectal temperature or locomotor activity. The present results may support the development of a σ1R antagonist as an adjunct to cannabinoid and low efficacy μ‐opioid receptor agonists for the treatment of acute pain.

Support or Funding Information

This work was supported by National Institute on Drug Abuse grants DA23205 and DA48353.
Effects of An Omega‐3 High‐Fat Diet on Skeletal Muscle Protein Degradation in Glucocorticoid‐Induced Muscle Wasting
Authors: Puppa Melissa , Brown Katie , Persinger Aaron
Published: April 2020

Treatment with synthetic glucocorticoids induces muscle atrophy and are commonly used in the treatment of a variety of inflammatory and autoimmune diseases. Omega‐3 (n‐3) polyunsaturated fatty acids elicit beneficial effects in several muscle atrophy conditions. Therefore, the purpose of this study was to determine if a high‐fat diet rich in n‐3 is protective of glucocorticoid‐induced protein degradation. Male wild type C57BL/6 mice were randomized into two groups: n‐6 (45% fat 177.5 g lard) and n‐3 (45% fat 177.5 g Menhaden oil). After 4 weeks on their diets, groups were divided to receive either daily injections of 3 mg/kg/day dexamethasone (Dex) or sterile phosphate buffered saline, for 1 week while continuing diets. At sacrifice the gastrocnemius muscle was weighed and dissected into red and white portions before being snap frozen and processed for RNA extraction and protein extraction. Dex reduced gastrocnemius weight by 12% independently of diet. Protein degradation signaling in the white gastrocnemius was altered by Dex with increased atrogin‐1 expression (p=0.004) without a change in muscle RING finger 1 (MuRF‐1) expression (p=0.15). There was a large effect of Dex to decreased phosphorylation of forkhead box transcription factor O3a (FOXO3a) (Cohen’s d=1.42), increased phosphorylation of glycogen synthase kinase 3β (GSK‐3β) (Cohen’s d=1.42). However, the negative effects of dexamethasone were not attenuated by an n‐3 high‐fat diet. There was no effect of Dex or diet on phosphorylation of FOXO3a (p=0.17) or GSK3β (p=0.60) in red gastrocnemius muscle. These data support the detrimental effects of dexamethasone on muscle atrophy and report no benefit of an n‐3 high‐fat diet.

Support or Funding Information

Funding provided by University of Memphis School of Health Studies
Gut bacteria and lipocalin 2 interplay mediates inflammation and arteriosclerosis in type‐II diabetic kidney
Authors: Pushpakumar Sathnur , Majumder Suravi , Sen Utpal
Published: April 2020

Genome‐wide association studies report significant correlations of unique gut microbiota and their metabolites with type II diabetes (T2D). Further, a growing body of evidence suggest the gut microbiota plays an important role in the onset and progression of Diabetic nephropathy. Lipocalin 2 (Lcn2) is a small secretory glycoprotein that can act as a mediator or modulator of inflammatory processes in various pathologies. Recently, in addition to its role as a potential biomarker of diseases, Lcn2 was shown to contribute towards the progression of nephrectomy‐induced chronic kidney disease (CKD). High levels of Lcn2 have been reported in obesity and type II diabetes (T2D) however, its role in T2D nephropathy remains unknown. Therefore, the purpose of this study was to investigate the relationship of gut microbiota and Lcn2 in T2D and whether Lcn2 contributes to the development of renal arteriosclerosis and dysfunction. Three‐month‐old mice homozygous for Leprdb mutation (db/db) were used as type – II diabetes models and mice heterozygous for Leprdb mutation (db/+, non‐diabetic) served as controls. Lcn2 was upregulated in db/db kidney compared to db/+. There was differential expression of Lcn2 in the fecal and plasma samples of db/db mice compared to db/+ and correlated with gut and renal inflammation. In db/db mice, high Lcn2 protein levels correlated with poor renal function. Microbial DNA isolation from stools and 16S rRNA sequencing showed abundance of bacteria at phyla level in db/db mice compared to db/+ and rich in alpha and beta diversity. The db/db samples showed decreased Firmicutes/Bacteriodetes ratio and increased Proteobacteria compared to db/+. Further, Actinobateria was absent in the db/db samples. The db/db kidneys showed increased vascular adhesion molecules and inflammation, reduction of vascularity and decreased blood flow. Barium angiography revealed narrowing of renal, segmental and interlobar arteries and rarefaction of terminal branches in db/db kidneys compared to db/+. The db/db kidneys showed injury, vascular thickening and reduced elastin. Our findings of altered gut microbiome and upregulation of Lcn2 suggests a significant crosstalk between the gut and kidney and is associated with renal inflammation, arteriosclerosis and dysfunction.

Support or Funding Information

NIH: DK116591 and DK104653 to Utpal SenAHA: 15SDG25840013 to Sathnur Pushpakumar
Altered Cisplatin Pharmacokinetics during Nonalcoholic Steatohepatitis Contributes to Reduced Nephrotoxicity
Authors: Jilek Joseph Lionel , Toth Erica , Frost Kayla , Jacobus Kevyn , He Wenxi , Goedken Michael , Cherrington Nathan
Published: April 2020

Cisplatin is an alkylating antineoplastic agent that is indicated for the treatment of solid malignancies. Cisplatin is preferentially eliminated from systemic circulation via tubular secretion, whereby it exhibits dose‐limiting nephrotoxicity. Interindividual variability in xenobiotic transporter expression is a known contributor to differential cisplatin toxicity and efficacy, and may be the result of genetic, environmental, and pathological contributions. In this study, we aimed to determine if nonalcoholic steatohepatitis (NASH) alters cisplatin pharmacokinetics and if this change elicits differential nephrotoxicity. Sprague Dawley rats fed a control or methionine and choline deficient (MCD) diet to model NASH were given a single bolus dose of cisplatin and sacrificed after 72 h. The MCD diet resulted in a NASH hepatic phenotype that remained unchanged following cisplatin exposure. Drug‐naïve NASH rats also displayed no evidence of differential renal pathology relative to control rats. However, renal necrosis and inflammation were reduced in NASH by 40 and 63% following cisplatin treatment, respectively, relative to healthy controls. Furthermore, kidney weights of cisplatin‐treated control rats were increased by 31%, compared to an 18% increase in NASH. Plasma cisplatin clearance was reduced from 6.78 (control) to 4.04 mL/min in NASH, and cisplatin plasma AUC was significantly increased by 44% in NASH, relative to control. Cumulative urinary elimination of cisplatin was decreased from 73 to 34% of total dose and renal clearance was reduced from 4.64 to 1.49 mL/min in NASH, compared to control. Subsequently, renal intracellular accumulation of cisplatin after 6 h was reduced by 34% in NASH, relative to control. Supporting these findings, expression of proximal tubule cisplatin uptake transporters, Ctr1 and Ctr2, were reduced by 24 and 64%, respectively compared to healthy control rats, whereas expression of Oct1, Oct2, and Oct3 were unchanged. Interestingly, expression of cisplatin efflux transporters Mate1 and Atp7a were reduced by 52 and 31%, respectively, in NASH compared to control. Taken together, these data suggest that NASH alters renal uptake and efflux transporter expression, thereby attenuating cisplatin uptake and clearance in the kidney with a corresponding reduction in renal cell exposure and nephrotoxicity during NASH. As such, this study demonstrates that NASH can influence pharmacokinetics of drugs cleared by renal elimination, which may contribute to adverse drug reactions.

Support or Funding Information

This work is supported by a NIH grant [R01 ES028668] and the Southwest Environmental Health Sciences Center [P30 ES006694]. JLJ is supported by an institutional training grant [T32 ES007091].
Kinetensin is an Endogenous β‐arrestin‐biased Ligand of Angiotensin AT1 receptor
Authors: Filipeanu Catalin , Ogunlade Blessing , Ghita Ioana , Lazartigues Eric , Fang Yayin
Published: April 2020

Kinetensin, a nonapeptide having similarities to neurotensin and angiotensin II has been isolated from human plasma more than 30 years ago, but its patho‐physiological functions and signal transduction mechanisms remain elusive. In this work, we investigated the effects of kinetensin on G protein‐coupled receptors (GPCRs), one of the largest families of the human genome and the biological target of many peptides. Using Presto‐tango system, the effects of this peptide were tested on a panel of 178 class A GPCRs. Kinetensin (10−6M) stimulated β‐arrestin activation in HTLA cells transfected with angiotensin type 1 receptor (AT1R) by 638±45% compared to basal levels (n=16). At the same concentration, kinetensin also stimulated β‐arrestin activation by histamine H1 receptor (201+22%), melatonin MT1 (187±12%) and MT2 (183±7%) receptors, MRGPRX3 (179±14%), GPR83 (186±10%) and GPR88 (190±10% n=3 in each case), but these effects were significantly smaller compared to the effects on AT1R. In contrast, kinetensin had no significant effect on β‐arrestin activation in cells transfected with other GPCRs, including receptors related to renin‐angiotensin system like AT2, MAS1 and MAS1L, or with neurotensin receptors (NTS1 and NTS2). The effects of the peptide on AT1R‐stimulated β‐arrestin activation were reproduced in HEK293T cells using nanoBRET method. These experiments demonstrated that maximal effects of kinetensin at 10−6M were 39±8% from the effects of angiotensin II (angII, 10−6M), with an EC50 of 115+21 nM (n=4). We also tested the effects of kinetensin on [Ca2+]i (a marker of G‐protein activation) in AT1R transfected HEK293T cells and we found that its effects were only a fraction of the effects of angII (14±8%, n=4). These data indicate that kinetensin is a β‐arrestin biased agonist at AT1R. Interestingly, angiotensin 1‐7 (ang1‐7), a heptapeptide with opposite actions to angII and favorable effects on the regulation of cardio‐vascular activities, demonstrated comparable effects in cells transfected with AT1R (21+7% stimulation of β‐arrestin activation and 4±8% increases in [Ca2+]i at 10−6 M, n=4). In this respect, kinetensin also resembles the effects of novel AT1R β‐arrestin biased agonist TRV027, which is currently tested in clinical trials for the treatment of acute heart failure. Overall, our results show that the nonapeptide kinetensin differentially modulates AT1R‐induced β‐arrestin and G‐protein activation, and these effects can be relevant to target this GPCR in cardio‐vascular diseases.

Support or Funding Information

This work was supported in part by grants from National Science Foundation (Nos. 1708959 & 1924092) and Office of Naval Research (Nos. N00014‐18‐1‐2145 & N00014‐19‐1‐2602) to YF, U.S. Department of Veterans Affairs (I01 BX004294 to CMF and EL) and National Institutes of Health (HL093178 and COBRE P30GM106392), and LSUHSC‐NO Research Enhancement Program to EL.
Doxorubicin‐Induced Muscle Toxicity: A Novel Mechanism Involving Inflammation‐Mediated Pyroptosis in Soleus Muscle
Authors: Dessouki Fatima Bianca , Kukreja Rakesh C. , Singla Dinender K.
Published: April 2020

Doxorubicin (Dox) is an effective chemotherapeutic agent which is widely used clinically to treat solid tumors. Unfortunately, the use of Dox has been restricted due to serious side effects including skeletal muscle toxicity. The current dogma is that Dox‐induced muscle toxicity (DIMT) is mediated through enhanced oxidative stress, inflammation, and unwanted cell death due to apoptosis and necrosis. We hypothesized that inflammation‐mediated cell death known as “pyroptosis” plays a critical role in DIMT. To test this notion, we generated a mouse model of DIMT where C57BL/6J mice (10±2 weeks, male and female), were treated with Dox [12mg/kg cumulative dose, administered through 3 intraperitoneal (i.p.) injections on alternative days]. Mice were sacrificed 14 days following the last injection of Dox and soleus muscle (SM) was harvested. The expression of TLR4, inflammasome (NLRP3 and ASC), pyroptotic markers (caspase‐1, IL‐1β, and IL‐18), pro‐inflammatory cytokines (TNF‐α and IL‐6), pro‐inflammatory M1 macrophages (iNOS), and anti‐inflammatory M2 macrophages (CD206 and Arginase‐1) were measured using immunostaining and western blot techniques. Skeletal muscle function was measured prior to sacrifice. The results showed a significant increase (p<0.05) in the expression of inflammasome formation, pyroptotic markers, pro‐inflammatory cytokines, and M1 macrophage while a significant decrease (p<0.05) in the infiltration of M2 macrophages was observed following Dox administration as compared to saline treated controls. Furthermore, muscle strength was significantly decreased (p<0.05) in Dox‐treated mice vs. control. These data indicate that Dox induces NLRP3‐ASC inflammasome‐mediated pyroptosis, increases inflammation and M1 macrophage infiltration, while decreasing M2 macrophages thereby causing muscle weakness. We propose that novel strategies specifically targeted towards limiting pyroptosis may help in attenuating DIMT in cancer patients receiving Dox chemotherapy.
Introducing a New Classification System for the Cerebral Arterial Circle of Willis
Authors: Nestor Nicholas , Ritz Brianna , Kumar Anil , Klinkhachorn Penprapa , Zdilla Matthew
Published: April 2020

The cerebral arterial circle of Willis (CoW) is an arterial ring with an extensive range of variation and implications in cerebrovascular disease. Variations of the CoW are implicated in varied pathologies including aneurysms. This study analyzed the variations of the CoW in 177 cadavers. Each vessel in the CoW was identified as normal, hyperplastic, hypoplastic, aplastic, duplicated, triplicated, or plexiform. There were 73 total variation patterns identified. These variations were used to inform the development of a novel classification system that categorizes a CoW into one of 20 morphological types. The categorization is streamlined by assessing the anterior and posterior CoW vasculature independently. The anterior (A) variations are paired with their corresponding posterior (P) variations for a more complete description of the entire CoW variation. An unremarkable CoW is referenced as “A0P0.” The most common variant type is “A0P1” (16.95%) which has a normal anterior and a hypoplastic P1/hyperplastic P2/hyperplastic PCoA. This is followed by “A0P2” (12.99%) which has a normal anterior and a hypoplastic/aplastic PCoA. The circle is incomplete in 9.03% and bilaterally asymmetric in 49.16%. The proposed classification system will help improve the way CoW variations are classified and improve the understanding of how CoW variations are related to cerebrovascular pathology.

Support or Funding Information

Funding: WV Research Challenge Fund [HEPC.dsr.17.06]
Primary and Secondary Bile Acids Activate Hepatic Stellate Cells
Authors: Yarde Shari Shontelle , Cheng Xingguo
Published: April 2020

The activation and proliferation of hepatic stellate cells (HSCs) is a major etiology contributing to the development and progression of liver fibrosis, cirrhosis and hepatocarcinogenesis. In response to liver injury, such as following oxidative stress, alcohol intoxication, viral infections and obesity, quiescent HSCs are transformed into an activated state. Cholestatic liver disease, which is generally characterized by the accumulation of bile acids in serum and liver, can progress to liver cirrhosis if not treated. However, the regulation of activation and proliferation of HSCs by bile acids is not fully understood. The current study was performed to determine the effects of two unconjugated primary bile acids [cholic acid (CA) and chenodeoxycholic acid (CDCA)] and one unconjugated secondary bile acid lithocholic acid (LCA) on the activation and proliferation of cultured human LX‐2 hepatic stellate cells. Our results showed that all of three bile acids induced activation and proliferation of LX‐2 cells, evidenced by increased mRNA expression of proliferating cell nuclear antigen (PCNA) and cytoglobin. In addition, all of three bile acids are predisposed to induce fibrogenesis as evidenced by increased mRNA expression of α‐smooth muscle actin (SMA) and procollagen‐1 in LX‐2 cells. In conclusion, both primary and secondary bile acids activate hepatic stellate cells and may consequently promote liver fibrogenesis.

Support or Funding Information

Department of Pharmaceutical Sciences, St. John’s University, Queens, NY 11439, USA.
Development of basicranium in cotton‐top tamarin compared to the development of basicranium in bushbabies : Role of synchondroses and brain growth
Authors: Oladipupo Lanre , Wood Brody , Mano Nanami , Hughes Gabriel , Reynolds Rebecca , Vinyard Chris , DeLeon Valerie , Smith Timothy
Published: April 2020

The purpose of this study is to describe basicranial and brain growth in sub‐adult cotton‐top tamarin (Saguinus oedipus) and compare their growth with greater bushbabies (Otolemur spp.) based on computed tomographic (CT) imaging. Bilateral synchondroses (alisphenoid‐basisphenoid synchrondrosis, ABS) have been identified within the middle cranial fossa of bushbabies which indicate active growth centers at this stage of development. In Saguinus, the ABS fuses relatively earlier and is oriented more anteriorly than in Otolemur. In this study, we used micro‐computed tomography (CT) and imaging software (Amira) to study heads of Saguinus and Otolemur that died of natural causes at two ages: near birth and at approximately one‐month postnatal age. Using Amira, the endocranial contour of crania were manually segmented throughout serial slices, in order to generate a three‐dimensional view of an endocast. This allowed a comparison of brain shape differences between ages in each species. In both species, the occipital lobe is proportional broader and more posteriorly projected at one‐month of age. In addition, the endocast of both species are more complex in contour in the older specimens (i.e., surfaces were more rounded at birth). In Otolemur, the cerebellum and the temporal lobes are more inferiorly positioned in the older animal. In contrast, in Saguinus, the cerebellum becomes less pronounced due to a relative overgrowth of the cerebrum. Also, in Saguinus the temporal lobes are more laterally projecting and anteriorly displaced at one‐month. These results are consistent with the hypothesis that the ABS fuses early in primates, and bone modeling has a primary role in accommodating postnatal shape changes in the brain. However, we note that the ABS is oriented differently (more anteriorly) in the monkey, associated with the relatively anterior displacement of the temporal lobe.

Support or Funding Information

NSF # BCS‐1830894, BCS‐1830919, BCS‐0959438
Cigarette Smoke Exposure in Mice Impairs Force Development of Injured Fast‐Twitch Skeletal Muscles
Authors: Stevens Nicole , Davila Daniel , De-Perio Maribeth , Souresrafil Ehson , Nguyen Namphuong , Vitorino Steven , Hogan Michael C. , Breen Ellen C. , Nogueira Leonardo
Published: April 2020

The use of tobacco cigarettes is known to impair muscle contractile function and decrease muscle mass. Smokers also show a slower recovery following overuse‐induced muscle injury, but the effects of cigarette smoke exposure on muscle repair are not known. We tested whether a short‐term cigarette smoke exposure would affect the regeneration of locomotor muscles following injury by lengthening contractions. Wild‐type C57Bl/6J mice were nose‐only exposed to the smoke of 10 tobacco research cigarettes (1R6F) a day (CS, n=12 mice), or air‐exposed (Air, n=10 mice), for 5 days per week, lasting 8 weeks. CS mice gained less weight than their Air counterparts over the 8‐week period (2.0 ± 0.5 g vs 3.0 ± 0.4 g, respectively). On the seventh week of the exposure, the mice underwent an in vivo lengthening contraction procedure (LCP) to injure the tibialis anterior (TA) and the extensor digitorum longus (EDL) muscles of the right hindlimb (LCP leg), while the left hindlimb was used as a contralateral control (Control leg). During the LCP, the peroneal nerve of the right hindlimb from each mouse was electrically stimulated to evoke contractions of the anterior crural muscles and the foot was actively moved to produce plantar flexion (150 contractions with 20 s intervals). One week after the LCP, mice were euthanized and the EDL and TA muscles were harvested from both legs to test ex‐vivo contractility and to perform histochemistry of cross‐sections of the muscles. Force development in EDL muscles from the LCP legs were significantly smaller than the control legs for both groups (7 ± 4 % and 14 ± 6 % decrease, for CS Air and CS groups; P<0.01 vs control leg). Air and CS groups did not show differences in force in EDL muscles from the control legs (473 ± 25 vs 469 ± 13 kPa, respectively), but in muscles from the LCP legs, force in the CS group (398 ± 24 kPa) was significantly smaller than the Air group (444 ± 24 kPa; P<0.05). These data suggest that tobacco smoking impairs muscle regeneration when muscles are injured by lengthening contractions.

Support or Funding Information

Tobacco‐Related Disease Research Program
Chemical Biology Approaches for Genome‐Wide Screening of GPCR Trafficking
Authors: Lobingier Braden , Tsvetanova Nikoleta , von Zastrow Mark
Published: April 2020

G protein‐coupled receptor (GPCRs) are the largest family of membrane‐bound signal transduction molecules. Many of these receptors have a complex lifecycle which includes residence at various intracellular organelles: endoplasmic reticulum, Golgi, plasma membrane, endosomes, and lysosomes. Movement of the receptor between these organelles is essential for GPCR function and mis‐trafficking can alter, blunt, or entirely disrupt GPCR signaling. Given the complexity of GPCR trafficking, we sought to develop chemical biology methods which would allow forward genetic screens to be performed in mammalian cells. Here we describe a novel approach, based on a genetically encoded peroxidase called APEX, allowing for quantitative fluorescent detection of GPCR expression. The fluorogenic substrate used in this method is membrane permeable while the fluorescent oxidation product is stably deposited into cells, allowing for subsequent single‐cell analysis and sorting. We demonstrate that APEX is protease‐labile and its activity is rapidly quenched upon trafficking of the GPCR‐APEX transgene to the lysosome. Specific chemical or genetic perturbation of GPCR trafficking to lysosomes preserves the enzymatic activity of APEX, which opens the door to forward genetic analysis. We combine this chemical biology method with genome‐wide CRISPRi screening to identify genes involved in the trafficking of the delta opioid receptor from ER (synthesis) to lysosome (degradation). Together with our previous work using APEX for high‐resolution proximity labeling, we present a comprehensive pipeline for proteomic and genomic characterization of GPCR cell biology.

Support or Funding Information

NIH DA043607
Optimization of Cell‐Free Protein Synthesis Through Identification of Rate‐Limiting Factors
Authors: Smith Philip E. J. , Burrington Logan R. , Levine Max Z. , Watts Katharine R. , Oza Javin P.
Published: April 2020

We have implemented the Genetic Code kit into classrooms at Cal Poly, San Luis Obispo for a hands‐on inquiry‐based module of transcription and translation. Our Genetic Code kit is based on cell‐free protein synthesis (CFPS) which captures the transcription and translation machinery required for protein synthesis in vitro, allowing for direct manipulation of protein production. By obviating the use of a living cell, CFPS allows early‐career STEM students to observe protein synthesis. To observe the process, superfolder green fluorescent protein (sfGFP), is used as a reporter protein. The detectable fluorescence of sfGFP gives students real‐time feedback of protein synthesis occurring. However, this process takes upwards of three hours for complete synthesis and maturation. To be more applicable in an educational setting, CFPS needs to occur in a more time‐efficient manner. Thus, this work focuses on identifying the rate‐limiting factors of CFPS. These rate‐limiting factors can be organized into two classes. The first class consists of energy sources and small molecules, and the second class consists of protein‐based translation factors. Direct titrations of these small molecules and proteins into CFPS reactions were performed. Here, we report our findings of key additives that increase the translation rate in cell‐free. Additionally, miscellaneous small molecules, such as osmolytes and detergents, have also been found to increase the reaction rate. The translation factors currently being studied are: prokaryotic initiation factors, elongation factors, release factors, ribosome recycling factor and the methionyl transferase. By optimizing the process of CFPS, the platform can become time‐efficient for a single lab setting. To date we have decreased reaction time by 30%, significantly improving the broad applicability of the Genetic Code Kit in classrooms.

Support or Funding Information

Our research is funded by the Center for Applications in Biotechnology, the Bill and Linda Frost Fund, and NSF‐1708919.
Pro‐Resolving Lipid Mediators and Anti‐Angiogenic Therapy Exhibit Synergistic Anti‐Tumor Activity via Resolvin Receptor Activation
Authors: Hallisey Victoria M. , Kipper Franciele C. , Moore Justin , Gartung Allison , Bielenberg Diane R. , Petrik Jim , Lawler Jack , Panigrahy Dipak , Serhan Charles N.
Published: April 2020

Inflammation and angiogenesis are interdependent hallmarks of cancer. Tumor growth is angiogenesis‐dependent and inflammation is a risk factor for many cancers. Inflammation is regulated by endogenous specialized pro‐resolving lipid‐autacoid mediators (SPMs), including resolvins, protectins, and maresins, which are present in multiple tissues. Unlike the majority of anti‐inflammatory agents, SPMs are non‐immunosuppressive, and non‐toxic. These lipid autacoids inhibit angiogenesis and clear cellular debris by macrophages resulting in reduced localized inflammatory cytokines. SPMs mediate their pro‐resolution and anti‐inflammatory activity through at least five known human G‐coupled protein receptors (GPR32, GPR18, ChemR23, GPR37, and LGR6) for resolvin (Rv) D1, RvD2, RvE1, protectin D1, and maresin 1, respectively, as well as a murine RvD1 receptor ALX/FPR2. We show that stimulating resolution of inflammation (RvD4 or RvD5) and anti‐angiogenic therapy (e.g. the thrombospondin (TSP)‐1 peptide 3TSR or anti‐VEGF via DC101) induced synergistic antitumor activity in human xenograft models including ovarian cancer (e.g. 36M2) compared with either treatment alone. The triple therapy 3TSR, anti‐VEGF, and resolvins displayed additive anti‐tumor activity. As the role of SPM receptors in cancer is unknown, we screened various stromal cells in the tumor microenvironment (e.g. macrophages, pericytes, fibroblasts, and neutrophils) for expression of SPM receptors. SPM receptors were expressed in various clinical specimens including breast cancer, head and neck cancer (e.g. oral squamous cell carcinoma), and brain cancer. Flow cytometry and double immunohistochemistry staining confirmed that SPM receptors were expressed in non‐tumor cells in the tumor microenvironment (e.g. macrophages, fibroblasts, pericytes and blood vessels) in vivo. Accordingly, SPMs inhibited tumor cell proliferation in vivo. Thus, SPMs exhibit anti‐tumor activity via the tumor stroma. Loss of SPM receptor expression may be associated with cancer progression in clinical cancer specimens. Notably, resolvins (RvD4 or RvD5) inhibited tumor growth at doses 10,000 times lower than anti‐inflammatory agents such as aspirin and NSAIDs. Thus, stimulating the expression or activity of SPM receptors may enhance current anti‐angiogenic therapy for various cancers.
Role of the Renal Nerves in Regulating SGLT2 inhibitor‐induced Diuresis and Natriuresis in rats with Heart Failure
Authors: Katsurada Kenichi , Nandi Shyam S. , Sharma Neeru M. , Patel Kaushik P.
Published: April 2020

Sodium‐glucose cotransporter 2 (SGLT2) inhibitors promoting glucose excretion are widely used to treat type 2 diabetes. Recently, the clinical trial in both diabetic and non‐diabetic heart failure (HF) patients with reduced ejection fraction demonstrated that SGLT2 inhibitor prevents worsening HF. However, the underlying mechanisms have not been fully elucidated. The present study was conducted to assess whether the effects of SGLT2 inhibitor on urine flow, sodium and glucose excretions are changed in HF. To address the underlying mechanisms the mRNA and protein levels of renal SGLT2 are investigated. HF was induced in rats by left coronary artery ligation. Further, to assess the contribution of renal nerves in the regulation of renal SGLT2, four weeks after ligation surgery, surgical bilateral renal denervation (RDN) was performed. Acute experiments were performed one week after RDN (five weeks after coronary ligation). Urine flow, sodium and glucose excretion responses to SGLT2 inhibitor dapagliflozin (0.5 mg/kg, i.v.) were greater in HF than Sham (Urine flow 15.8 ± 1.8 vs. 8.3 ± 1.1 μl/min/gkw, Sodium excretion 2.23 ± 0.35 vs. 1.13 ± 0.18 μEq/min/gkw, Glucose excretion 0.84 ± 0.06 vs. 0.52 ± 0.08 mg/min/gkw at 120 min, P<0.05, n=5–7). RDN significantly attenuated these responses to dapagliflozin in HF (Urine flow 9.8 ± 1.2 vs. 15.8 ± 1.8 μl/min/gkw, Sodium excretion 1.31 ± 0.17 vs. 2.23 ± 0.35 μEq/min/gkw, Glucose excretion 0.66 ± 0.04 vs. 0.84 ± 0.06 mg/min/gkw at 120 min, P<0.05, n=5–7). Western blot analysis indicated that SGLT2 protein expressions in the renal cortex were higher in HF than Sham (SGLT2/GAPDH 0.83 ± 0.06 vs. 0.58 ± 0.05, P<0.05, n=6). RDN significantly reduced enhanced protein levels of SGLT2 in the renal cortex of rats with HF (SGLT2/GAPDH 0.60 ± 0.07 vs. 0.83 ± 0.06, P<0.05, n=6). In contrast, RT‐PCR analysis indicated that RDN increased mRNA levels of SGLT2 in the renal cortex of rats with HF (SGLT2/18sRNA 1.08 ± 0.28 vs. 0.23 ± 0.06, P<0.05, n=6), possibly a compensatory change to SGLT2 protein expressions. Immunohistochemistry of renal sections confirmed the enhanced staining for SGLT2 in HF. These findings suggest that increases in urine flow, sodium and glucose excretion responses to SGLT2 inhibitor are enhanced in HF due to the upregulation of renal SGLT2. Renal sympathoexcitation in HF might enhance the expression and functional activity of renal SGLT2, thereby contribute to subsequent pathophysiology of renal sodium and water retention associated with HF.

Support or Funding Information

Supported by Japan Heart Foundation and NIH grants DK 114663 and HL62222.
GAL3 Regulates Smooth Muscle Cell Survival in PAH via its NWGR Motif
Authors: Haigh Stephen , Li Xueyi , Bordan Zsuzsanna , Chen Feng , Meadows Mary Louise , Stepp David , Barman Scott , Fulton David
Published: April 2020

Pulmonary arterial hypertension (PAH) is a complex and fatal disorder characterized by an unrelenting increase in pulmonary arterial pressure. Excessive proliferation of pulmonary artery smooth muscle cells (PASMC) leads to increased vascular resistance and eventually right ventricular failure and death. Our laboratory has identified a key role for Galectin‐3 (GAL‐3 or Lgals3) in driving PASMC proliferation and elevated pulmonary pressures. However, the mechanisms by which GAL‐3 alters PASMC behavior remains poorly understood. GAL‐3 contains a BH1 like domain that has an NWGR sequence with high homology to the anti‐apoptotic protein, BCL2. Our central hypothesis is GAL‐3 upregulation plays an important role in driving PASMC proliferation through repression of apoptosis via the NWGR domain. In order to test the role of this domain we have made an adenoviral vector encoding the GAL‐3 G182A mutation and have shown that it expresses equally to a WT GAL‐3. We have also made a GAL‐3 KO rat and have isolated GAL‐3 PASMCs from these animals (RPASMC). Our in vitro studies show that overexpression of GAL‐3 G182A cause a decrease in viability using flow cytometry upon treatment overnight with 0.5% serum containing media(p<.001). However there was no significant difference in survival(p=.840) when cells were conditioned in normal media. Proliferation experiments similarly showed no significant difference between GAL‐3 WT and G182A in KO human aortic endothelial cells(HAEC) and human PASMC (p>.05). When exposed to apoptotic stimuli (10ng/mL TNFα and 1μg/ml Cycloheximide) GAL‐3 G182A overexpressing GAL‐3 KO RPASMCs had significantly (p<.001) decreased vitality as compared to GAL‐3 WT overexpressing cells. Overall our data suggests that the NWGR domain of GAL‐3 is important in regulating PASMC survivability, and our future work will investigate the role of GAL‐3 mediated apoptosis in animal models of PAH.

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F31HL147428

R01HL125926‐03
Angiotensin II‐Induced Increase in Blood Pressure in Mice Consuming High Salt and High Fructose Diet: Females Not Protected
Authors: Fan Liming , Vallejo Jessica , Sung Austin , Rouch Alexander
Published: April 2020

Hypertension presents major risk factors for cardiovascular and renal disease. High salt and high fructose (HSHF) consumption contribute to hypertension via salt retention and obesity. Sex differences in blood pressure regulation are well known. Some studies have shown protection for females from angiotensin II (AngII)‐induced and dietary‐induced increase in blood pressure. Our results indicate that HSHF consumption increases blood pressure in male and female mice. Also, AngII administration increased blood pressure in male and female mice while consuming normal diet with water, but no sex differences were noted (Rouch, A. FASEB J. 32(1) A904.6, 2018). The objective of this study was to determine the effects of AngII on blood pressure, sodium excretion, and urine flow rate in male and female CD‐1 mice while consuming a HSHF diet. Four groups of mice included male‐vehicle, male‐AngII, female‐vehicle, and female‐AngII (n=6/group). Mice at four weeks of age were placed in metabolic cages for a 5‐day baseline period followed by a ten‐day AngII period where AngII was administered via an Alzet® osmotic minipump containing either saline (vehicle) or AngII to yield 1mg/kg/min. All mice consumed a diet of 4% salt with fluid containing 1% NaCl and 20% fructose ad libitum. Systolic blood pressure was measured daily via the tail‐cuff method (CODA, Kent Scientific). Excessive fluid retention evidenced by a significant increase in body weight of 4–5 g occurred in the AngII‐treated mice of both sexes in the first 5 days of the AngII period. One mouse of each AngII‐treated group died before the end of the AngII period while the other mice recovered to their normal body weight by the end of the ten‐day period. AngII increased systolic blood pressure (mmHg) in females from 108.0 ± 3.4 to 130.0 ± 6.1 (p<0.0001) and in males from 101.1 ± 1.3 to 127.5 ± 3.2 (p<0.0001). In both AngII‐treated groups, daily urine flow rate and sodium excretion demonstrated biphasic patterns of significant decreases in the first 5 days of the AngII period followed by increased levels to achieve fluid and sodium balance. We conclude that AngII‐infusion during HSHF consumption in mice presents with potentially fatal salt and water retention. No sex differences in the AngII‐induced increase in SBP or renal handling of sodium were measured.

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OK‐INBRE SURP
Inhibiting Progesterone induced blocking factor induces markers of endothelial dysfunction and inflammation in pregnant rats
Authors: Amaral Lorena M. , Witcher Alexis C. , Comley Kyleigh , Cottrell Jesse N. , Cunningham Mark W. , Ibrahim Tarek
Published: April 2020

Preeclampsia (PE) is a leading cause of maternal and perinatal morbidity in the U.S. While the pathogenesis remains unclear, PE is characterized by new onset hypertension associated with progesterone deficiency, elevated cytolytic natural killer cells (cNK), inflammation, and endothelial dysfunction. Progesterone is essential in the initiation and maintenance of pregnancy. We have shown that progesterone supplementation regulates endothelial function and suppresses endothelin‐1 (ET‐1) and stimulates nitric oxide (NO) in response to placental ischemia during pregnancy. In addition, progesterone signals the synthesis and release of progesterone induced blocking factor (PIBF) from lymphocytes in order to regulate the proinflammatory balance of early pregnancy. However the role of PIBF in PE pathology is not well examined. This study was designed to test the hypothesis that inhibition of PIBF causes inflammation and increases markers of endothelial dysfunction and hypertension during pregnancy. Rabbit anti‐PIBF IgG (0.25, low dose‐LD or 0.50 mg/mL, high dose‐HD) was administered intraperitoneal on gestation day (GD) 15 to normal pregnant Sprague Dawley (NP) rats, on GD 18 carotid catheters were inserted and on GD 19 blood pressure (MAP) and samples were collected. MAP in NP rats (n=7) was 99± 3 mmHg, which increased to 116± 2 in NP+ anti‐PIBF LD (n =10) and 113±4 mmHg in NP+ anti‐PIBF HD (n=6), p<0.05. Neither placental weight nor pup weight was affected by PIBF blockade. Plasma TNF‐alpha levels were 35±8 pg/mL in NP rats and increased to 84±21 pg/mL in NP+ Anti‐PIBF HD (n=4), p<0.05. Circulating total NK cells were 67± 11 in NP rats (n=4), which decreased to 36±4 in NP+ Anti‐PIBF HD however, cytolytic NK cells were 0.6 ± 0.2 in NP which were increased to 3.0±1 in NP+ Anti‐PIBF HD, p<0.05. Importantly, circulating NO levels were 44± 11 μM in NP rats (n=5), which significantly decreased to 21±1 μM in NP+ Anti‐PIBF (n=6) HD, p<0.05. Moreover, renal cortex PPET‐1 levels increased 15 fold in NP+ Anti‐PIBF (n=6) HD compared to NP rats (n=5). Collectively, our study demonstrates that inhibition of PIBF causes hypertension, increased TNF alpha while causing signs of endothelial dysfunction, all of which are associated with PE, thus indicating the importance of progesterone signaling pathways for the maintenance of endothelial function and inflammation during healthy pregnancy.

Support or Funding Information

This work was supported by National Institutes of Health grants RO1HD067541‐06, P20GM121334 and AHA 19CDA34670055