bluuebunny/crossref_metadata_2025 huggingface dataset:
The Developing Bird Pelvis Passes Through Ancestral Archosaurian and Dinosaurian Conditions
Authors: Griffin Christopher T. , Botelho João , Hanson Michael , Fabbri Matteo , Smith‐Paredes Daniel , Carney Ryan , Norell Mark , Egawa Shiro , Gatesy Stephen , Nesbitt Sterling , Bhullar Bhart‐Anjan
Published: May 2022

Living birds (Aves) have bodies dramatically modified from the ancestral reptilian condition. The avian pelvis in particular experienced dramatic changes during the transition from early archosaurs to living birds. This stepwise transformation is well documented by an excellent fossil record; however, the ontogenetic alterations that underly it are less well‐understood. We hypothesized that the avian pelvis evolved via terminal addition—a mechanism whereby ancestral states shift to derived states during late development, resulting in retention of ancestral character states. If this is the case, then developing bird embryos should possess the same character states as taxa along the avian stem lineage (i.e., non‐avian dinosaurs and their close relatives). We used embryological imaging techniques (modified CLARITY protocol) to examine the early morphogenesis of avian pelvic tissues (Japanese Quail, Common Parakeet, Chilean Tinamou), allowing direct comparison with the fossil record. We found that many ancestral dinosaurian features (e.g., forward‐facing pubis, short ilium, pubic ‘boot’) are transiently present in the early morphogenesis (‘pre‐cartilage’) of birds and arrive at their typical ‘avian’ form after transitioning through a prenatal developmental sequence that mirrors phylogeny. Using 3D geometric morphometrics, we quantitatively demonstrate that avian pelvic ontogeny closely parallels the dinosaur‐to‐bird transition and provide evidence for an evolutionary module within the pelvis that is conserved across Archosauria. The same pattern was observed in a paleognath, a galloanseriform, and a neoavian, strongly suggesting that this is the ancestral avian condition. These indicate that the avian pelvis evolved via terminal addition, and that the presence of ancestral states in avian embryos may stem from these conserved modular relationships. This modularity suggests a previously unrecognized mechanism for the retention of ancestral states in development, hinting that terminal addition may be common across evolutionary transitions.
A Modular Approach Toward Controlling Stability of Oligonucleotide‐Stabilized Lipid Micelles
Authors: Wun Jesse , Yanoff Zenya , Garcia Enrique , Wilner Samantha
Published: May 2022

Lipid micelles represent an important class of nanoparticle with the potential to enhance the solubility and delivery of hydrophobic drugs and imaging agents. Their small size (<50 nm) makes them ideal candidates for both passive tumor uptake and deep tissue penetration. The use of lipid micelles as delivery vehicles, however, has been challenged by the fact that these particles are inherently unstable in vivo, particularly due to interactions of lipids with serum proteins. We have developed a modular platform in which lipid monomers modified with short 16‐base oligonucleotide sequences function to overcome the challenges associated with micelle‐based drug delivery. Oligonucleotide‐lipid conjugates are synthesized manually using standard phosphoramidite chemistry, and oligonucleotide‐stabilized lipid micelles (OLMs) are then assembled in aqueous media. Micelle stability is assessed in the presence of serum proteins using techniques including Förster resonance energy transfer (FRET). We present data in which we have systematically varied both lipid structure and oligonucleotide sequence to establish design rules to guide the assembly and stability of OLMs. The modularity of this approach provides a pathway toward the engineering of delivery vehicles with precisely defined structure, stability, and kinetic profiles.
In vitro antioxidant, anti‐inflammatory and antihypertensive activities of methanol leaf extract of Peasea americana
Authors: Gbadegoye Joy O. , Adejumobi Olumuyiwa A. , Omobowale Temidayo , Oyagbemi Ademola , Adedapo Adeolu , Yakubu Momoh , Ashafa Anofi
Published: May 2022

Hypertension (HBP) is a cardiovascular disease of man and animal worldwide. It is a risk factor for many other cardiovascular diseases and target organ dysfunction. HBP management with orthodox medicine is expensive and unaffordable to patients from low income countries in Africa and they are associated with side effects. P. americana leaf extract (PALE) is commonly used in the management of hypertension in Nigeria trado‐medicine. In this study, we investigated the antioxidant, anti‐inflammatory and anti‐hypertensive activities of the crude methanol extract, fractions and phytochemicals of P. americana leaf. The in vitro‐antioxidant assays were carried out to evaluate the free radicals scavenging activities of PALE on 1, 1‐diphenyl‐2‐picrylhydroxyl (DPPH), 2, 2‐azenobis‐3‐ethyl benzothiozoline‐6‐sulfonic acid (ABTS), hydroxyl and nitric oxide radicals. In addition, metal chelating and ferric reducing power assay were done. Anti‐inflammatory activities were evaluated using 15‐1ypoxygenase enzyme inhibition assay. Absorption were read spectophotometrically and 1C50 was calculated using a standard calibration curve. Phytocompound isolation was by extract fractionation, column, thin layer and preparatory thin layer chromatography. Crude methanol extracts, N‐hexane, ethyl acetate, chloroform, N‐butanol and aqueous methanol fractions had significantly higher anti‐oxidant activities when compared with gallic acid. Their anti‐inflammatory activities were comparable to that of Indomethacin and antihypertensive activity for PALE (with IC50 of 135.99±10.06) was significantly lower when compared with captopril (with IC50 41.86±2.10). Four pure phytochemicals were isolated and named LEACA, LEACB, LEACC, and LEACD. The isolated compounds had significantly higher antioxidant activities on DPPH, ABTS, and hydroxyl and nitric oxide radicals when compared with gallic acid. The anti‐hypertensive activities of the isolated phytocompounds from the ethyl acetate fraction of the leaf were significantly higher (with IC50 of 24.83±0.63, 9.48±1.45, 2.79±1.35 and 1.59±0.48 respectively) than that of captopril (with 1C50 of 41.89±2.10). The findings from this study suggest that P. americana leaf has free radical scavenging and antioxidant activities, anti‐inflammatory and anti‐hypertensive activities and can ameliorate oxidative stress and inflammation associated with hypertension.
Inhibition of Ferroptosis Using UAMC‐3203 in the Post‐stroke Period Does Not Impact Cognitive Outcomes in Diabetic Rats
Authors: Edgerton Mia , Phoenix Ashley , Chandran Raghavendar , Wolf Victoria , Abdul Yasir , Jamil Sarah , Li Weiguo , Ergul Adviye
Published: May 2022

Introduction

Post‐stroke cognitive impairment (PSCI) contributes to significant long‐term disability in stroke victims. 30% of ischemic stroke victims in the United States also have diabetes, which increases the risk of hemorrhagic transformation as well as PSCI. Ferroptosis, an iron‐induced cell death can instigate increased oxidative stress and contribute to impaired neurovascular repair leading to PSCI in diabetes. In our previous studies, we were able to identify that treating diabetic animals with an iron chelator, deferoxamine (DFX), prevents post‐stroke vasoregression and improves functional outcomes. Furthermore, DFX prevented the activation of ferroptosis in brain microvascular endothelial cells in vitro. Untreated diabetic animals experienced progressive cognitive decline while being monitored for 8 weeks. These findings led us to speculate that endothelial ferroptosis also plays a role in vasoregression and impacts cognitive outcomes post‐stroke. Therefore, this study was designed to test the hypothesis that inhibiting ferroptosis in the post‐stroke period will improve cognitive recovery in diabetic animals.

Methods

Animals were housed in reverse light cycle. 8 weeks after diabetes onset, male rats underwent 60 min middle cerebral artery occlusion (MCAO). On Day 3, after stroke injury was confirmed by MRI, animals were randomized to UAMC‐3 (2mg/kg) or vehicle treatment for 2 weeks. Sensorimotor and cognitive behavioral tests were performed during the animals’ active hours up to 8 weeks post MCAO.

Results

(Table 1): 60 min occlusion caused significant acute neurological deficits. There were no differences between the groups in indices measured by novel object recognition (NOR), Y‐maze and sucrose preference tests. Interestingly, step through latency in passive avoidance test (PAT) was lower in the UAMC‐3203 group.

Conclusion

Treatment with a ferroptosis inhibitor for 2 weeks after stroke did not impact recognition and working memory but worsened aversive learning in diabetic male rats. Unlike our previous study in which behavior tests were performed during rats’ passive hours, there was no progressive cognitive decline in untreated animals. Further evaluation of behavior testing times as well as tissue markers of neurovascular degeneration, inflammation and ferroptosis are required to determine whether molecular and cellular markers are affected by the treatment before overt changes in behavioral outcomes.
Adaptation to High‐Salt Diet Requires H,K‐ATPase Type 2 Dependent NaCl Secretion
Authors: Cheval Lydie , Lasaad Samia , Rafael Chloé , Morla Luciana , Picard Nicolas , Crambert Gilles
Published: May 2022

Introduction

The kidney controls the extracellular volume and the blood pressure because of its ability to excrete efficiently Na+ and Cl‐ ions. These last decades in Western countries the consumption of salt has dramatically increase and the question arises as to how the kidney adapts to this chronic situation. Up to now, the mechanisms described to excrete a large load of salt consist in the inhibition of Na+ reabsorption all along the nephron. We have recently identified a novel pathway that mediates the secretion of Na+ in the collecting duct (Morla, L. et al. 2016). This system involves A‐type intercalated cells (AIC), NKCC1 and the H,K‐ATPase type 2 (HKA2), which is known to be able to transport Na+. Here, we investigated the role of HKA2 in NaCl renal secretion, as well as the physiological relevance of this secretory pathway in vivo, in response to salt load.

Material and Methods

C57BL6J mice wild type or knockout for the Atp12a gene (HKA2‐KO) were used and placed under a control (NS) or high‐Na+ (HS) diets for 3 days. Outer medullary collecting ducts (OMCD) of WT mice under NS or HS were isolated for RNAseq and RT‐PCR analysis. Type A intercalated cells were isolated by FACS (following labelling with c‐kit antibodies) from WT mice under NS or HS for RT‐PCR analysis. The mice were also placed in metabolic cages for measurements of physiological parameters (food and water intakes, urine excretion, plasma parameters). Blood pressure was assessed by tail‐cuffed measurement.

Results

The RNA seq analysis of OMCD revealed that, as expected, genes involved in Na+ reabsorption (ENaCa, SGK) are downregulated. However, we also observed that HS diet also leads to an increased expression of the Atp12a gene that encodes for the catalytic subunit of the HKA2 both in OMCD and ICA. To understand the role of the HKA2, we placed the HKA2‐KO mice under high‐salt diet and observed that those mice exhibit a salt loosing phenotype with low blood pressure that is due to a stronger down‐regulation of NKCC2 than in WT in the same context. Therefore, the absence of HKA2 is over‐compensated by the inhibition of a reabsorption pathway.

Conclusion

Altogether, these results demonstrate that the secretion of Na+ in OMCD is stimulated in response to high salt intake.
Sperm Competition and Its Effects on the Evolution of Prostatic Acid Phosphatase in Hominids
Authors: Dimick Brandon , Jensen‐Seaman Michael
Published: May 2022

Selective pressures and their effects on protein structure and function can be found by comparing genes or proteins from different organisms. These pressures can take varying forms. Sexual selection, a form of natural selection, occurs when an organism acquires traits that give it an advantage in mating and producing offspring. An example can be seen in hominid seminal proteins. Hominids exhibit different mating systems which has resulted in seminal proteins being affected by varying degrees of sperm competition. This can result in variation both in the sequences of these genes and the function of the gene products. One such gene, ACPP, is used to generate the protein prostatic acid phosphatase (PAP). This protein is known to dephosphorylate a number of targets in seminal plasma, and it functions as a phosphatase in the acidic pH range. It has been suggested computationally by comparing dN/dS ratios that ACPP may be under strong positive selection in chimpanzees (a species with high sperm competition) and purifying selection in humans and gorillas (both species with weaker sperm competition). We hypothesize that varying degrees of sperm competition have resulted in sequence and functional variation at ACPP/PAP among hominids. ACPPcoding sequences for human, chimp, and gorilla homologs were each inserted into a mammalian expression vector with a C‐terminal His6 tag and transfected into HEK‐293T cells. The phosphatase activities of the PAP homologs were compared using a DiFMUP fluorescent substrate assay. Each protein was tested for pH optima, phosphatase activity at a fixed substrate concentration, and kcat/km. The PAP orthologs exhibited similar pH profiles, with a pH optimum between pH 5.0‐6.0. However, it was found that chimp PAP exhibited elevated phosphatase activity when compared to the other hominids, and this was consistent across a varying pH range. These results suggest that positive selection of chimp ACPPhas resulted in a more efficient PAP protein with elevated activity when compared to human and gorilla PAP, perhaps due to strong sperm competition. Gorilla PAP showed the lowest activity, which is interesting considering that many genes that code for male reproductive proteins have become pseudogenes in gorillas, a species with very low sperm competition. Overall, the results suggest that the varying mating systems of the hominid primates have resulted in functional differences in the PAP homologs of humans, chimpanzees, and gorillas. By bridging together functional and computational techniques such as in this research, we are beginning to understand the effects of specific selective pressures on the function of male reproductive proteins.
Endometriosis Induces Colonic Inflammation and Reactive Gliosis in an Animal Model
Authors: Rivera‐Arce Luis A. , Rodriguez‐Cintron Ulises , Cruz Myrella L. , Appleyard Caroline B.
Published: May 2022

Introduction

Endometriosis is a complex gynecological disorder characterized by the ectopic growth of endometrial tissue. Symptoms of endometriosis are known to impair the quality of life of patients, among these symptoms are dysmenorrhea, chronic pelvic pain, and gastrointestinal (GI) issues. These GI issues are one of the common reasons for misdiagnosis with irritable bowel syndrome (IBS), a pain syndrome characterized by abdominal pain, painful bowel movements, diarrhea, and constipation, all of which are often present in endometriosis and persist even after treatment. Enteric glial cells (EGC) are known to play a role in pain associated with IBS, and reactive gliosis has been reported in patients with IBS, but the role of EGC in endometriosis has yet to be elucidated.

Hypothesis

We hypothesize that endometriosis will induce reactive gliosis, with increased expression of the glial fibrillary acidic protein (GFAP) and S100B, in the myenteric plexus of colonic sections in an animal model of endometriosis.

Methods

We used 15 female Sprague Dawley rats and divided them into three groups (n=5 per group): Naïve, Sham, and Endo. Endometriosis was induced in the Endo group by suturing 4 pieces of autologous uterine tissue in the intestinal mesentery, while the Sham group received only sutures without tissue. Von Frey and Hot Plate tests were done the day before surgery, two weeks after surgery, and before sacrifice to assess pain behavior. 21 days after surgery, animals were sacrificed, and endometriosis development and colonic macroscopic inflammation were assessed. Colonic tissue was collected to assess GFAP and S100B immunoreactivity, microscopic damage, and mast cell whole‐tissue cell count.

Results

We found Endo animals showed a decrease in allodynia threshold, but no hyperalgesia. All Endo animals developed endometriosis with an average area per vesicle of 13.89±1.43mm2, and we found Endo animals had a significant increase in the colonic macroscopic score (3.01±0.22;p<0.001) compared to Sham (0.35±0.12) and Naïve (0.47±0.20). Paraffin‐embedded colon sections stained with H&E didn’t show statistical differences in microscopic damage between groups, yet toluidine blue staining for mast cells showed a 36% increase in the mean number of cells in Endo compared to Naïve but didn’t reach statistical difference. GFAP whole‐mount immunostaining showed significantly increased integrated density in Endo (25655.00±2395.00) when compared to Sham (13558.00±719.80;p<0.001) or Naïve (9035.00±1362.00;p<0.001), while no statistical difference was found in S100B integrated density between groups.

Conclusion

Endometriosis can alter GI homeostasis by inducing macroscopic inflammation, reactive gliosis, and some mast cell infiltration. Taken together these suggest endometriosis can mimic IBS histopathology beyond the symptomatology, reinforcing this disease’s complexity and the need to treat it beyond the gynecological setting.
Insight into L‐DOPA dioxygenase mechanism with 6‐substituted L‐DOPA derivatives
Authors: Nyamkondiwa Kudzai , Squires Trevor , Jones Paige , Colabroy Keri L. , Peterson Larryn W.
Published: May 2022

Dioxygenase enzymes are essential protein catalysts for the breakdown of catecholic rings, structural components of plant woody tissue. This powerful chemistry is used in nature to make natural products as well as degrade plant material, but we have a limited understanding of substrate space and mechanism across the superfamily. To this end, we report the syntheses, redox potentials and pKas of L‐3,4‐dihydroxyphenylalanine (L‐DOPA) derivatives substituted at the 6‐position and their characterization as substrates of L‐DOPA dioxygenase from lincomycin biosynthesis in Streptomyces lincolnensis. In particular, the spectroscopic properties of 6‐nitroDOPA provide insight into the steps of the enzymatic mechanism. The applications for dioxygenase cleavage of 6‐substituted L‐DOPA derivatives to natural product biosynthesis will be discussed.
HFpEF in CKD is Associated with Elevated TNF‐α/IL‐6 Inflammatory Signaling from the Kidney
Authors: Chade Alejandro R. , Eirin Alfonso
Published: May 2022

Background

Chronic kidney disease (CKD) is independently associated with incident heart failure with preserved ejection fraction (HFpEF), but the underlying mechanisms remain unknown. We recently developed a translational swine model of CKD‐HF that replicates many features of human HFpEF. This study tested the hypothesis that CKD‐induced HFpEF associates with increased pro‐inflammatory cytokine signaling of renal origin.

Methods

CKD and normal pigs (n=4 each) were studied for 14 weeks. Renal hemodynamics (multi‐detector CT) and cardiac morphology and function (echocardiography) were quantified in vivo. Tumor necrosis factor (TNF)‐ α and interleukin (IL)‐6 levels (ELISA) were measured in circulating, renal vein, and coronary sinus blood, and their renal and cardiac gradients quantified. Systemic TNF‐α and IL‐6 levels (Luminex) were also measured in patients with CKD and age/gender‐matched healthy volunteers (n=12 each) and correlated with biomarkers of heart failure.

Results

Pigs with CKD developed hypertension, renal failure (stage 3), left ventricular (LV) hypertrophy, and abnormal LV strain and diastolic dysfunction (E/A, E/e’ ratio) with pEF, accompanied by positive renal (renal release) and negative cardiac (cardiac retention) gradients of TNF‐α and IL‐6 (Fig. 1A). Circulating TNF‐α and IL‐6 were also higher in patients with CKD compared to normal subjects (Fig. 1B), which correlated directly with ANP and NT‐proBNP levels (Fig. 1C).

Conclusions

Our experimental and clinical data supports a crosstalk between the kidney and the heart in CKD, suggesting that inflammatory signaling led by TNF‐α and IL‐6 of renal origin may impose cardiac abnormalities towards development of HFpEF.
The CRISPR in the Classroom Network: A Support System for Instructors to Bring Gene Editing Technology to the Undergraduate Classroom
Authors: Pieczynski Jay , Wolyniak Michael , Pattison Donna , Hoage Tiffany , Carter Dawn , Olson Sara , Santisteban Maria , Ruppel Nicholas , Challa Anil
Published: May 2022

CRISPR‐Cas9 based gene manipulation technology represents a once‐in‐a‐generation advance in molecular biology that has become a mainstream technique in research. However, as is often the case with a new technology, most undergraduate instructors do not have the training or support to bring CRISPR‐Cas9 technology into their curricula. To remedy this, we have established a “CRISPR in the Classroom” network of undergraduate instructors prepared to disseminate CRISPR‐Cas9 curricular and laboratory modules to undergraduates (NSF RCN‐UBE #2120417). The main objective of this network is to facilitate a series of workshops and mentoring activities designed to provide instructors, postdocs, and graduate students the skills, support, and confidence needed to introduce and implement CRISPR‐Cas9‐based modules in undergraduate classrooms. These workshops a provide flexible, easily adapted curriculum and mentoring to instructors as well as start‐up kits to overcome the hurdles associated with learning and testing new curriculum in class. By doing this, we hope to develop both a national support system for biology educators of two‐year and four‐year undergraduate degree granting institutions dedicated to spreading effective curricular modules for the classroom and laboratory activities centered on experiential‐based work with CRISPR‐Cas9 as well as an online repository of proven CRISPR‐Cas9‐based modules across a variety of model systems for instructors with “ready‐to‐use” curricular elements to adapt for their own specific pedagogical needs using the NSF‐supported QUBES platform. Assessment data from a previous online workshop as well as two NSF‐sponsored previous in‐person workshops (Awards #1823595 and 1916486) taken several weeks after their completion suggest that the majority of workshop participants develop the skills and confidence necessary to make concrete plans to implement CRISPR‐Cas9 modules into their classes within one year of the workshop. The CRISPR in the Classroom Network represents a dynamic community of practice dedicated to providing undergraduate life science instructors with the tools and support needed to introduce this technology into their curricula.
Yeast Pah1 PA phosphatase contains a novel domain within its N‐terminal intrinsically disordered region
Authors: Stukey Geordan J. , Han Gil‐Soo , Carman George M.
Published: May 2022

Pah1 PA phosphatase catalyzes the dephosphorylation of PA to produce diacylglycerol. In yeast and higher eukaryotes, the diacylglycerol produced in the reaction is used for the synthesis of triacylglycerol or the membrane phospholipids phosphatidylcholine and phosphatidylethanolamine. Pah1 is inactive as a phosphorylated enzyme in the cytosol and becomes active after its recruitment and dephosphorylation by the ER‐associated Nem1‐Spo7 protein phosphatase complex. Conserved N‐LIP and HAD‐like domains are required for PA phosphatase activity and a conserved tryptophan within the WRDPLVDID domain is required for its in vivo function in lipid metabolism. Intrinsically disordered regions, which are located between the conserved catalytic domains and at the C terminus, contain multiple sites for phosphorylation and regulation of Pah1 location and function. Prediction of Pah1 structure by AlphaFold identifies a novel domain contained within the N‐terminal intrinsically disordered region for which its function is unknown. A truncation mutant that lacks amino acid residues 186‐266 have been constructed and expressed in a pah1D and pah1D nem1D mutants to assess the function of the novel domain for regulating Pah1 phosphorylation, recruitment and dephosphorylation by the Nem1‐Spo7 protein phosphatase complex, and function in lipid metabolism.
Histone H3K27 Demethylase KDM6A Regulates Cardiac Aging viaInduction of HoxC4 mediated ER Stress
Authors: Chen Kai , Zhang Bo , Sun Zhongjie
Published: May 2022

Background

KDM6A mediates the removal of repressive trimethylation from H3K27me3 to activate target gene expression. It is not known, however, whether KDM6A plays a role in the regulation of cardiac aging. We hypothesized that cardiac‐specific KDM6A knockout accelerates cardiac aging.

Methods and Results

Aging decreased histone demethylase KDM6A expression and activity in human cardiomyocytes. Downregulation of KDM6A expression was also found in cardiomyocytes of aged mice. The cardiomyocytes‐specific KDM6A knockout (Myh6‐KDM6A cKO) mouse model was created by using Cre‐LoxP system. KDM6A deletion didn’t cause overt cardiac remodeling and dysfunction in normal condition. However, cardiomyocytes‐specific conditional knockout of KDM6A exaggerated ISO‐induced cardiac dysfunction and cardiac remodeling. Moreover, Myh6‐KDM6A cKO mice showed early cardiac aging at 23‐month‐old, while control mice didn’t display cardiac aging until 27‐month‐old. Therefore, KDM6A deletion accelerated cardiac aging. ChiP‐Seq analysis revealed candidate transcription factors participating in KDM6A cKO‐induced cardiac aging. In particular, HoxC4 was down‐regulated in the cardiomyocytes of Myh6‐KDM6A cKO mice. Knockdown of HoxC4 gene in H9c2 cardiomyoblast cell line caused ER stress. The ER stress further resulted in oxidative stress and cardiomyocytes apoptosis in cardiomyocytes‐specific conditional knockout of KDM6A mice.

Conclusions

Cardiomyocytes‐specific conditional knockout of KDM6A causes cardiac aging through induction of HoxC4‐mediated ER stress. These observations suggest KDM6A a potential therapeutic target for cardiac aging.
Glutathione peroxidase 4 prevents 12/15 LOX induced renal oxidative cell death and improves renal post Ischemic recovery in Male Spontaneous Hypertensive Rats (SHR)
Authors: Mohamed Riyaz , Sullivan Jennifer C.
Published: May 2022

Acute kidney injury (AKI) due to ischemia‐reperfusion (IR) is a serious and frequent complication in hospital setting with high mortality rates. The mechanisms mediating renal IR injury leading to increased risk of later developing cardiovascular and renal diseases in either sex remain poorly understood. Glutathione peroxidase 4 (GPx4) is a negative regulator of 12/15 lipoxygenase (12/15 LOX) and any alteration in 12/15 LOX and GPx4 activity has been reported to contribute to the progression of kidney diseases. The goal of the current study was to test the hypothesis that an imbalance of 12/15 LOX/GPx4 activity in males leads to impaired recovery following IR compared to females. 13‐week‐old male and female SHR were subjected to sham or 30‐minute warm bilateral IR (n=6/group). Additional male SHR were randomized to receive vehicle or the GPx4 activity mimic ebselen (10 mg/kg i.p.; n=5/group) 1 hr prior to sham/IR, 1 day and 3‐day post IR. Blood and urine were collected from all rats 1 and 7 days after IR; kidneys were harvested 7 days post‐IR for biochemical, histological, and Western blot analysis. IR increased plasma creatinine (Pcr) and blood urea nitrogen (BUN) in both male and female SHR compared to respective sham controls at 24 hrs (PIR =0.0001; Psex*IR=0.2). At 7 days post‐IR, Pcr and BUN remained elevated in male SHR but returned to baseline levels in females (Pcr: PIR =0.03; Psex*IR=0.04; BUN: PIR <0.05; Psex*IR=0.05). Histological examination of kidneys 7 days post‐IR showed greater tubular damage (PIR=0.0003, Psex*IR=0.0019) and renal cell death (PIR=0.001; Psex*IR=0.001) in male vs. female SHR. Delayed recovery of renal function in male SHR was associated with activation of renal 12/15 LOX (PIR<0.05; Psex*IR=0.008), reduced renal GPx4 protein expression (PIR=0.05; Psex*IR<0.001), activity (PIR=0.01; Psex*IR=0.0016), and intracellular glutathione (GSH; PIR=0.01; Psex*IR=0.003), and increased lipid peroxidation (PIR=0.008; Psex*IR=0.018) compared to females. Pre‐treatment of male SHR with ebselen significantly reduced 12/15 LOX induced lipid peroxidation (PIR*TxT =0.02), prevented IR‐induced renal cell death (PIR*TxT=0.04) and tubular damage (PIR*TxT=0.02) and improved renal function (Pcr: PIR*TxT =0.027; BUN: PIR*TxT =0.022) compared to vehicle‐treated IR males 7 days post‐IR. In conclusion, our data demonstrate that increased GPx4 activity enhanced renal recovery post‐IR via inhibiting 12/15 LOX induced oxidative renal cell death in male SHR.
Functional Dynamics of Deubiquitinase A
Authors: Li Ying , Fether Skyler , Kabra Ashish
Published: May 2022

Deubiquitinase A (DUBA) is an isopeptidase that cleaves K63‐linked and K48‐linked ubiquitin chains from substrate proteins. DUBA belongs to the ovarian tumor (OTU) subfamily of deubiquitinases (DUBs) and was initially identified as a negative regulator of the production of type I interferons. DUBA is also known to regulate the production of several other cytokines in both innate and adaptive immune cells. More recently, its role in regulation of the stability of chromatin regulators essential for human development has been revealed. DUBA is a model system for understanding how the activity and substrate specificity of DUBs are regulated by post‐translation modifications (PTMs) through modulation of the conformational ensemble that pre‐exists in the apo form of the enzyme. We have previously identified changes in the conformational ensemble of DUBA upon phosphorylation, which is essential for DUBA activity, using solution nuclear magnetic resonance (NMR) spectroscopy and activity assays. We have also characterized the conformational dynamics on the microsecond‐to‐millisecond timescales using NMR relaxation dispersion experiments. We will present NMR and activity data on several mutants of DUBA, which allowed us to identify the structural determinants of the previously observed conformational dynamics and to understand the functional roles of motions in DUBA. We will also present NMR data on the enzyme‐substrate complex, which shed light on the mechanism for the specificity of DUBA for the K63‐linked ubiquitin chain. Overall, our data suggest that the conformational dynamics of DUBA can be modulated by phosphorylation and are essential for the function of DUBA. Our study highlights the functional importance of conformational dynamics in the DUB family and the need to define dynamic properties of DUBs in order to fully understand how activity and substrate specificity can both be modulated by PTMs.
Emerald Jewel Wasp Venom Adenosine Deaminase Antagonizes Purinergic Signaling in the Cockroach Brain
Authors: Connolly Maize , Theisen Rachel , Arvidson Ryan
Published: May 2022

Ampulex compressa injects venom directly into the cerebral ganglia of the cockroach (Periplaneta americana) to induce a reversable, but long‐term compliant state of hypokinesia, marked by an increase in escape threshold and a decrease in spontaneous walking. The venom is not paralytic as stung cockroaches can still stand, groom, eat and drink, and can even run if aggressively provoked. The venom is also not necrotic or cytotoxic, and despite its long‐lasting duration the venom’s effects are reversible, as the escape response returns to normal after several days. Previous work involved generation of a comprehensive venom apparatus transcriptome and venom proteome or “venome” and described several interesting signaling pathways that appear to be targeted by venom components. This includes adenosine deaminase (ADA), which may act to antagonize purinergic signaling in the brain.

The substrates of venom ADA, adenosine (Ado) and adenosine monophosphate (AMP), are also the activating ligands of the cockroach adenosine receptor (AdoR). We hypothesize that the presence of ADA at the site of injection can reduce cell damage‐induced signaling by clearing Ado and AMP released from compromised cells. We have cloned, expressed, and characterized both venom ADA and cockroach brain AdoR and show that the presence of venom ADA can attenuate Ado induced calcium signaling in vitro. Antagonizing ADA or AdoR in vivo did not alter locomotory behavior. Thus, the action of ADA in the venom may be more neuroprotective than inhibitory of specific behaviors as is the case with other venom components.
Dysregulated Mitochondrial Respiration Occurs With Aging
Authors: Leija Roberto G. , Tovar Ashley P. , Curl Casey C. , Osmond Adam D. , Arevalo Jose A. , Duong Justin J. , Brooks George A.
Published: May 2022

Numerous metabolic changes occur in skeletal muscle with aging. At the crossroads of these changes lies the cellular energy highway, the mitochondrial reticulum. Aging is associated with altered patterns of mitochondrial dynamics and morphology, which are thought to be primarily driven by mitochondrial fragmentation. This fragmentation phenomenon may also lead to altered mitochondrial respiration with aging. We used a mouse model and measured parameters of respiration in muscle mitochondrial preparations to evaluate the hypothesis of aging‐related effects on metabolism and energy substrate partitioning. Mixed skeletal muscle of C57/Bl6 mice (n=32) was excised from single hind‐limbs of young males (YM) (n=8), old males (OM) (n=8), young females (YF) (n=8) and old females (OF) (n=8). Tissues were homogenized in an isotonic solution supplemented with 0.15% protease inhibitor. Mitochondria were isolated by differential centrifugation and their concentrations were determined by BCA analysis. Isolated mitochondria (0.1mg of mitochondria/mL of respiration medium) were then respired using a Clarke‐Type Electrode at 25°C with 100 nM of ADP and either 10 mM Pyruvate+ 2.5 mM Malate (P+M) or 40 uM Palmitoyl‐L‐Carnitine+ 1 mM Malate (PC+M) as substrates. Respiratory Control Ratios (RCR) were calculated as the quotient of State 3 (ADP‐stimulated respiration) and State 4 (recovery rate post ATP synthesis) respiratory rates (State 3/State 4) and compared between substrate and age. ADP to oxygen ratios (P:O) were calculated by dividing the amount of ADP added to the respiratory chamber by the oxygen consumption rate (nmol of O2/mg of mitochondria/minute) of state 3 respiration. RCR in both P+M and PC+M significantly decreased with aging [P+M (p<0.05): YM (4.1±0.9) vs. OM (2.7±1.1); YF (5.1±1.4) vs. OF (3.2±2)] [PC+M (p<0.05): YM (4.2±0.9) vs. OM (2.4±0.7); YF (3.4.±0.5) vs. OF (2.2±0.9)]. P:O in both P+M and PC+M did not significantly change with aging [P+M: (p=0.69): YM (2.9±0.3) vs. OM (2.9±0.2); YF: (p=0.07): (3±0.1) vs. OF (2.9±0.1)] [PC+M:(p=0.65); YM (2.9±0.4) vs. OM (2.9±0.3); (p=0.22): YF (2.9±0.2) vs. OF (2.7±0.3)]. While there were no significant changes in the P:O the age related decreases in the RCR were characterized by a lower capacity to respire derivatives of both CHO and FA. Decreased State 3 respiration and RCR typically corresponds to loss of respiratory capacity and loosening of coupling efficiency. Because exercise increases mitochondrial function and biogenesis it may be prudent to study older animals after exercise training. We plan to further investigate the structural changes in the mitochondrial reticulum and proteome that may be altering mitochondrial function in aging.
Does CADM1 Immunohistochemistry Distinguish Mycosis Fungoides from its Closest Mimickers?
Authors: Haghayeghi Koorosh , LeBlanc Robert E.
Published: May 2022

Background

Immunohistochemistry (IHC) sometimes fails to distinguish clinically ambiguous cases of evolving patch‐stage mycosis fungoides (MF) from relatively common, non‐neoplastic inflammatory skin diseases (ISD), particularly spongiotic dermatitis (SD), psoriasis (PSO), and pityriasis lichenoides (PL). CADM1 (cell adhesion molecule 1) is a tumor suppressor gene implicated in the pathogeneses of solid skin tumors, visceral malignancies, and peripheral T‐cell lymphomas. Recent data suggest that CADM1 IHC expression could help distinguish early‐stage MF from ISD.

Design

This pilot study sought to determine whether CADM1 IHC distinguishes MF from ISD in clinically‐annotated cases from our archives. MF (n=14), PL (n=10), PSO (n=10), and SD (n=10) were stained with CADM1. Using appropriate controls and antibody optimization, two pathologists established consensus scores in each case for percentage of lymphocytes staining with CADM1 and CADM1 staining intensity. The localization of CADM1‐positive cells (epidermis including junction and dermis) was also noted.

Results

There were no differences in CADM1 expression pattern or intensity between groups. All cases showed moderate to strong nuclear CADM1 expression in ≥75% of lymphocytes in both the dermis and epidermis. Some intraepidermal lymphocyte nuclei were challenging to detect on CADM1‐stained slides given uniform moderate‐intensity staining of background keratinocyte nuclei.

Conclusion

The challenge of distinguishing evolving patch‐stage MF from ISD facilitates demand for novel and readily available diagnostic tests. Although recent data have suggested that differential expression of CADM1 distinguishes MF from ISD, there was no difference in lymphocyte CADM1 expression and distribution between any of the groups included in our pilot study. Furthermore, evaluation of CADM1 expression in intraepidermal lymphocytes was challenging given background keratinocyte expression.
Arg84/65 Within the αC‐Helix of Erk1/2 is Pivotal for Blocking Autoactivation and Oncogenicity and Distinguishes MAP Kinases from Other EPKs
Authors: Soudah Nadine , Baskin Alexey , Engelberg David
Published: May 2022

Extracellular Regulated Kinases 1/2 (ERK1/2) belong to the MAPK family and are critical for the cell's responses to a variety of stimuli. The Erk pathway is particularly essential for mediating growth factor signaling and is found to be abnormally overactive in almost all human cancers. Although all oncogenic mutations in the pathway result in strong activation of Erks, activating mutations in Erks themselves were not reported in cancers. Recently we identified a point mutation in Erks, a conversion of arginine residue located within the αC‐helix in Erks, to Ser, which was sufficient to grant Erks independence of it’s upstream activator and also rendered Erk1 (but not Erk2) oncogenic. Here we describe a thorough analysis of the mutated Arg. We show that it is invariant within all members of the MAPK family and distinguishes them from other protein kinases. It may be a key component in determining their catalytic and physiological properties, by functioning as a blocker of Erks’ autophosphorylation ability. Investigating the mechanism behind the oncogenicity of Erk1R84S in living cells, it was found that following initial steps of activity, its activity is down‐regulated via a dramatic feedback mechanism. It could be re‐activated when cells are treated with erk inhibitors, suggesting re‐evaluation of the strategy of using anti Erk inhibitors for therapy.
Localization of Cdc14 Isoforms to Ciliary Structures in Tetrahymena thermophila
Authors: Goode Cody , Milholland Kedric , Hall Mark , Forney James
Published: May 2022

Cdc14 protein phosphatase is highly conserved across the eukaryotic kingdom, from single‐celled yeast and protozoa to multicellular organisms including mammals. In budding yeast, where it was first studied, Cdc14 is required for mitotic exit; however, this function is not widely conserved. In humans and mice, Cdc14A mutations cause deficiencies in hearing and male fertility due to defects in cilia formation and maintenance. There is evidence for Cdc14 localization to the basal bodies at the proximal end of cilia. The large number of cilia in the free‐living protozoan Tetrahymena thermophila make it an attractive model for molecular studies of cilia structure and function. Our goal is to determine the localization of Cdc14 isoforms to gain some insight into their cellular function and to test if Cdc14 cilia localization is broadly conserved. Interestingly, T. thermophilahas a larger number of Cdc14 isoforms than most organisms. Thus far, genes encoding three of the seven T. thermophilaCdc14 isoforms have been used to create C‐terminal gene fusions with YFP that are expressed in vivo. The constructs were inserted into the T. thermophilamacronuclear genome adjacent to the RPL29 gene and cell lines were selected due to the resulting conversion to cycloheximide resistance. Expression of the transgenes was regulated by a metallothionine promoter. Cells were examined via fluorescence microscopy in the presence and absence of cadmium. All three isoforms localize along ciliary rows and in the oral apparatus after induction during vegetative growth, consistent with basal body localization. No differences in localization have been noted between the three isoforms thus far. The presence of multiple isoforms with the same localization raises questions regarding the redundancy and/or functional specialization of Cdc14 in this highly ciliated organism.
4D diSPIM Myography Reveals Changes in Endothelial Cell Shape and Ca2+ Signaling in Response to Intravascular Pressure in ex vivo Resistance‐sized Arteries
Authors: Collier Daniel M. , Al‐Aqtash Rua'a
Published: May 2022

The vascular endothelium is critical in promoting vasodilation of resistance‐sized arteries through Ca2+dependent pathways. The endothelium is exposed to mechanical stimulation through changing intravascular pressure and flow. Understanding how the endothelium integrates this information into a change in vascular function requires the ability to simultaneously measure changes vessel diameter and EC Ca2+ signaling with high spatial and temporal resolution. We hypothesized that endothelial cell shape and Ca2+ signaling is pressure dependent. To test this, we combined 4D Dual Inverted Selective Plane of Illumination Microscopy (diSPIM; 3i, CO) with pressure myography. This system allows for imaging of three‐dimensional tissue preparations over time with very low photobleaching and phototoxicity, millisecond temporal resolution, and isometric 320 nm spatial resolution over a large imaging volume (330 x 330 x 150 mm). We designed and fabricated pressure myography chambers that position ex vivo pressurized arteries in the correct orientation for acquisition of high‐resolution fluorescent data via diSPIM while using an inverted microscope base to continuously capture vessel diameter measurements via transmitted IR light. Resistance‐sized mesenteric arteries from mice expressing an endothelium specific GECI are positioned between the diSPIM objectives and maintained at physiologic temperature and pH. We captured fluorescent images of whole pressurized arteries at 80, 60, 40, and 20 mmHg before and after the generation of myogenic tone and in the absence of extracellular Ca2+. As static intravascular pressure is increased, ECs increase in width and decrease in thickness. Myogenic tone minimizes changes in EC shape with changing pressure, however increasing static intravascular pressure decreases local EC Ca2+ signaling. The data demonstrate a relationship between intravascular pressure and EC shape and Ca2+ signaling in intact resistance arteries. This technique provides a method of studying the effects of physiologically relevant mechanical stimuli and the roles of various mechanosensitive mechanisms on vascular function.
Structural and computational investigation of MMP‐3 affinity improvements with an engineered TIMP‐1 inhibitor
Authors: Coban Matt , Maina Eran , Raeeszadeh‐Sarmazdeh Maryam , Caulfield Thomas R. , Radisky Evette S.
Published: May 2022

Matrix metalloproteinases (MMPs) are a family of zinc‐dependent endopeptidases that function to mediate tissue remodeling; however, they are also implicated in pathological conditions such as cancer, inflammatory and degenerative diseases. There are 23 human MMPs and significant sequence and structural homology exists among them and with other metalloproteinases. This represents a substantial challenge in developing therapeutically viable selective inhibitors due to off‐target effects. Tissue inhibitors of metalloproteinases (TIMPs) are a family of four endogenous protein protease inhibitors that regulate MMP activity. Our lab and others have utilized TIMPs as scaffolds, engineering them to selectively target specific MMPs for potential therapeutic applications. We previously employed a yeast surface display platform to identify TIMP‐1 variants possessing enhanced MMP‐3 affinity. In the current study, we used structures of MMP‐3 co‐crystallized with either WT TIMP‐1 or an engineered TIMP‐1 variant as seeds for molecular dynamics simulations. Simulations were also conducted on unbound WT and engineered TIMP‐1 to examine MMP‐independent dynamics that distinguish the engineered variant. The simulation data were analyzed with root‐mean‐square deviation/fluctuation, time‐dependent secondary structure, temporally resolved hydrogen bonding, conformational clustering, and dynamical network algorithms to discern structural and dynamical determinants of the increased affinity. Key observations that demonstrate the increased effectiveness of engineered TIMP‐1 as an MMP‐3 inhibitor were secondary structural changes in the MMP‐binding loops in proximity to mutations, attenuated global and mutation‐adjacent dynamics, decreased conformational dynamics, and increased persistence of MMP‐3‐compatible conformations. There were also indications of greater interdomain cooperativity within the engineered TIMP‐1 variant: increased interdomain hydrogen bonding, coordinated motions of the N‐terminal region with the C‐terminal domain, and increased intercommunity information flow/motion transfer. From these observations, we hypothesize the increased interdomain interactions represent a mechanism of dynamical stabilization such that mainly cooperative motion between domains presides, rather than random independent motions. Our findings suggest propagated motions between domains may influence the MMP affinity of TIMPs. Furthermore, these data suggest future strategies to design next‐generation mutant TIMP libraries by targeting regions dynamically coupled to the MMP binding loops for variation, thus exploiting natural protein dynamics to achieve desired selectivity.