bluuebunny/crossref_metadata_2025 huggingface dataset:
Cyp1a2‐knockout increases the systemic exposure of a serotonin and norepinephrine reuptake inhibitor duloxetine in mice
Authors: Qin Xuan , Xie Cen , Hakenjos John M. , Mackenzie Kevin M. , Barzi Mercedes , Nyshadham Pranavanand , Khalil Saleh M. , Bissig Karl‐Dimiter , Gonzalez Frank J. , Matzuk Martin M. , Li Feng
Published: May 2022

Duloxetine (DLX) is a dual serotonin and norepinephrine reuptake inhibitor for the treatment of major depressive disorder, mainly metabolized by CYP1A2 and CYP2D6 in human. Despite DLX has a good tolerance, serious adverse events (e.g., liver injury) have been reported in rare cases. The role of DLX metabolism in its toxicity remains unclear. Here, we evaluated the effects of systemic Cyp1a2 loss‐of‐function on the pharmacokinetics of DLX using Cyp1a2‐knockout (Cyp1a2‐KO) and counterpart wild‐type (WT) mice. At the clinically relevant dose (12 mg/kg, p.o.), the AUC and Cmax of DLX in Cyp1a2‐KO mice increased by 56% and 106% compared to those in WT mice, which is in line with the clinical observation: Co‐administration of CYP1A2 inhibitor increases the AUC and Cmax of DLX in human subjects. Meanwhile, the AUC of one major circulating metabolite, glucuronide of hydroxylated DLX, decreased by 23% in Cyp1a2‐KO mice, in which CYP1A2 mediates the hydroxylated DLX formation. Unexpectedly, the hepatic level of DLX in Cyp1a2‐KO mice was 21% lower than that in WT mice. But the excretion of DLX in Cyp1a2‐KO mouse feces increased by 78.9%. At subtoxic dose (50 mg/kg, p.o.), the Cmax of DLX increased over 5‐fold and AUC over 10‐fold compared to those at the dose of 12 mg/kg in both WT and Cyp1a2‐KO mice. However, the Cmax and AUC of DLX shown the similar levels in WT and Cyp1a2‐KO, but the hepatic level of DLX in Cyp1a2‐KO mice slightly increased by 12%. In summary, the findings indicated that the loss of Cyp1a2 activity increased DLX systemic exposure at clinical dose and different DLX and metabolite pharmacokinetic profiles were observed in WT and Cyp1a2‐KO mice. The liver toxicity of DLX in Cyp1a2‐KO mice will be pursued in the future studies to determine the role of Cyp1a2‐mediated metabolism in DLX hepatoxicity.
Ferric Uptake Regulator (Fur) Regulates Intracellular Iron Homeostasis via reversible binding of a [2Fe‐2S] cluster in Escherichia coli
Authors: Ni Xialong , Fontenot Chelsey R. , Yang Jing , Kluchka Olivia R. , Ding Huangen
Published: May 2022

It has generally been assumed that when intracellular “free” iron content is elevated in bacteria, a global transcription factor Fur (Ferric uptake regulator) binds Fe(II) to regulate expression of its target genes and achieve intracellular iron homeostasis. Fur is highly conserved among bacteria, and purified Fur proteins from different bacteria may exist as a homodimer or tetramer with each monomer containing three metal binding sites. While two of the metal binding sites (site 1 and site 2) in purified Fur proteins are often occupied by Zn(II), the iron‐bound Fur has never been isolated from any bacteria. Here we report that Escherichia coli Fur binds a [2Fe‐2S] cluster at site 3 (via Cys‐93 and Cys‐96) in the E. coli mutant cells in which intracellular “free” iron content is elevated due to deficiency of iron‐sulfur cluster biogenesis. Depletion of intracellular “free” iron content effectively removes the [2Fe‐2S] cluster from Fur, indicating that binding of a [2Fe‐2S] cluster in Fur is determined by intracellular “free” iron content. Fur homolog from Haemophilus influenzae also binds a [2Fe‐2S] cluster at site 3 when expressed in E. coli cells, suggesting that binding of a [2Fe‐2S] cluster in Fur is highly conserved. While the Zn(II)‐bound E. coli Fur has a basal binding activity for its target DNA sequence known as Fur‐box, binding of a [2Fe‐2S] cluster significantly enhances its Fur‐box binding activity. Similarly, binding of a [2Fe‐2S] cluster is required for the strong binding activity of H. influenzaeFur for Fur‐box in vitro. Mutation of Cys‐93 to Ala results in an E. coli Fur mutant that fails to bind the [2Fe‐2S] cluster, loses the enhanced binding activity for Fur‐box, and is inactive to complement the physiological function of E. coli Fur in E. coli cells. Our results suggest that E. coli Fur and H. influenzae Fur binds a [2Fe‐2S] cluster to regulate intracellular iron homeostasis in response to elevation of intracellular “free” iron content in cells.
Binding mechanisms of Shikonin derivatives targeting SARS‐CoV‐2 main protease
Authors: Woo JooHan , Das Raju
Published: May 2022

Shikonin, a well‐known bioactive chemical present in the dried roots of Lithospermum erthrorhizon, is recognized for its broad‐spectrum activities against cancer, oxidative stress, inflammation, virus, and anti‐COVID‐19 agent. In recent discovery, the crystallographic study of Shikonin bound Main protease (Mpro) of SARS CoV‐2 revealed different conformation, suggesting the possibility of designing Shikonin derivatives as potential inhibitors of Covid. Towards the goal, the present study is carried out to explore insights of Shikonin derivatives and find out the possibility of Shikonin derivatives targeting Mpro of Covid by using molecular docking and molecular dynamics simulation. A total of 25 Shikonin derivatives are introduced in this study, from where seven derivatives showed higher binding affinity than Shikonin, while four derivatives obtained the highest binding energy in MM‐GBSA binding energy calculation. According to Molecular dynamics simulation results, all compounds interact with two conserved His41 and Cys145 residues through multiple bonding in the catalytic sites, suggesting that these derivatives may effectively terminate SARS COV‐2 progression via Mpro inhibition. Taken together, the present in silico study concluded that Shikonin derivatives might play an influential role against Mpro inhibition.
Exercise Preconditioning Preserves Cardiac Function and Enhances Cardiac Recovery Following Dobutamine Stimulation in Doxorubicin‐Treated Rat Hearts
Authors: Haverbeck Simon Lea , Garritson Jacob , Pullen Nicholas A. , Hayward Reid
Published: May 2022

Doxorubicin (DOX) is a chemotherapeutic drug used to treat a wide range of cancers, and its use is limited by cardiotoxicity. Exercise preconditioning has been shown to protect against DOX‐induced cardiac dysfunction when hearts are maintained under resting conditions. However, it is unclear whether this exercise‐induced protective effect is maintained when the heart is challenged during an acute exercise bout. It was hypothesized that preconditioning protects the heart against DOX cardiotoxicity when it is challenged with the β1‐adrenergic receptor agonist dobutamine (DOB), which mimics acute exercise stress.

Fischer 344 rats were randomly assigned to sedentary (SED) or voluntary wheel running (WR) groups for 10 weeks. At week 11, rats were treated with either 15 mg/kg DOX or saline (SAL). Five days later, ex vivo cardiac function was assessed using an isolating working heart model at baseline, during the infusion of 7.5 μg/kg/min DOB, and during recovery.

DOB infusion significantly increased left ventricular developed pressure (LVDP), maximal (dP/dtmax) and minimal (dP/dtmin) rate of left ventricular pressure development, and heart rate in all groups (p<0.05). During the recovery phase (i.e., after DOB washout) LVDP was significantly lower than baseline in all groups except for SED+DOX (baseline vs. recovery: SED+SAL: 97±2 vs. 84±5 mmHg; WR+SAL: 116±7 vs. 90±7 mmHg; SED+DOX: 83±5 vs. 76±4 mmHg; WR+DOX: 109+2 vs. 100±4 mmHg, p<0.05). SED+DOX also showed a lower baseline and recovery LVDP than WR+DOX (83±5 vs. 109±2 mmHg baseline, 76±4 vs. 100±4 mmHg recovery, p<0.05). WR+DOX showed higher dP/dtmax and lower dP/dtmin when compared to SED+DOX during DOB infusion (7311±605 vs. 5167±508 mmHg/s and ‐4059±454 vs. ‐3158±416 mmHg/s, respectively). SED+DOX dP/dtmax was significantly lower during DOB infusion and during recovery when compared to all other groups (p<0.05). During DOB and recovery, +DOX groups showed lower HR values than +SAL groups (p<0.05).

These data demonstrate that exercise preconditioning preserved cardiac function after DOX exposure even when the heart is challenged, and it appeared to preserve the heart’s ability to recovery from this functional challenge. Cardioprotection seems to extend beyond rest into conditions which challenge cardiac function. These observations support the premise that exercise training can protect against cardiac dysfunction that may occur during acute exercise in cancer patients undergoing DOX treatment.
Contribution of the Microbiome to Western Diet‐Induced Gene Expression Changes in the Intestine and Vasculature
Authors: Cavalier Alyssa N. , Weir Tiffany L. , Gentile Christopher L. , LaRocca Thomas J.
Published: May 2022

Western diet (high fat/sugar) is a core contributor to obesity, which is associated with increased risk for chronic diseases, including cardiovascular disease (CVD). The mechanisms through which Western diet contributes to CVD are heavily studied, and increasing evidence suggests that Western diet alters the gut microbiota leading to dysbiosis(i.e., disruption of microbial homeostasis and accompanying intestinal inflammation), contributing to development of vascular dysfunction, a strong predictor of future CVD. Still, the biological processes linking intestinal dysbiosis with the vasculature are incompletely understood. To identify potential mediators of intestinal‐vascular crosstalk, we investigated the role of the gut microbiome in Western diet‐induced global gene expression changes using transcriptomics. We performed total RNA sequencing on colons and arteries (aorta) from wild‐type (WT) and germ‐free (GF, no microbiome) mice fed standard or Western purified diets for 8 weeks. In the colon, we found ~100 differentially expressed genes in standard diet‐fed WT vs. GF mice, and pathway analyses showed that these genes were associated with several important biological processes including peroxisome proliferator‐activated receptor (PPAR) signaling, adipocyte lipolysis regulation, and AMP‐activated protein kinase (AMPK) signaling (genes and pathways, FDR < 0.1). In colons of Western diet‐fed WT vs. GF mice, we found 163 differentially expressed genes related to cancer signaling, mitogen‐activated protein kinase (MAPK) signaling, and viral immune response (FDR < 0.1). Interestingly, in transcriptome analyses of aortas we found significantly different expression of 314 genes in standard diet‐fed WT vs. GF mice, and these were related to complement/coagulation cascades, transforming growth factor beta (TGF‐β), and advanced glycation end product (AGE) signaling, but most of these differences were absent with Western diet (only 4 significantly different genes in WT vs. GF mice, FDR < 0.1). These data suggest that the microbiome is an important mediator of gene expression in the intestine regardless of diet, but may only be important in arteries under standard diet conditions, and that the microbiome may not be a strong mediator of intestinal‐vascular crosstalk at the protein‐coding gene expression level. We are currently performing in‐depth analyses of non‐coding RNAs (e.g., micro‐RNAs) to further elucidate the mechanisms at play.
More evident roles of nNOS for the regulation of Ca2+‐sensitivity and SERCA activity in the right than the left ventricular cardiomyocytes of rats
Authors: Jeon Young Keul , Kwon Jae Won , Kim Sung Joon
Published: May 2022

It is known that neuronal NOS (nNOS, NOS1) regulates the intracellular Ca2+ ([Ca²⁺]i) and contractility of cardiomyocytes, which was investigated only in the left ventricular myocytes (LVCM) while not in the right ventricle (RVCM). In the paced cardiomyocytes, we have recently reported more relaxed diastolic sarcomere length (SLD) and smaller shortening (∆SL) while the amplitude of Ca²⁺ transient (∆[Ca²⁺]i) were not different (Jeon et al., 2021). Here we compared the expression and the functional roles of nNOS in the RVCMs and LVCMs from Sprague‐Dawley rats. The total expression and the phosphorylated Ser1417 ratio of nNOS were not different between RVCM and LVCM. However, the treatment with nNOS‐specific inhibitor, S‐methyl‐L‐thiocitrulline (SMTC, 100 nM), induced more prominent augmentation of ∆SL along with faster contraction while paradoxical faster relaxation and decreased ∆[Ca²⁺]i in RVCM. The hysteresis curve between ∆SL and ∆[Ca²⁺]i­ showed more prominent leftward shift, i.e. sensitization of the myofilaments in RVCM by SMTC. Consistently, the speed of contraction became faster, and the longer SLD of RVCM became similar to that of RVCM by the treatment with SMTC. Intriguingly, the SMTC treatment accelerated the Ca2+ removal by SERCA in RVCM, which appears to be consistent with the decreased ∆[Ca²⁺]i and faster relaxation. As for the decreased myofilament calcium sensitivity through sGC‐cGMP‐PKG pathwy, altered phosphorylation of troponin I (TnI) was suggested. In fact, the SMTC treatment decreased the TnI phosphorylation in RVCMs, while not LVCMs. Immunoblotting revealed higher expression of nNOSβ and its phosphorylated form in the myofilament fraction from RVCM than LVCM. In summary, the nNOS‐dependent longer SLD and the paradoxical negative inotropic effect despite the inhibitory effect on SERCA might be mediated by the myofilament‐associated nNOSβ decreasing Ca2+‐sensitivity through TnI phosphorylation. Interestingly, we also found that the treatment with omecamtiv mecarbil, a recently proposed cardiac myosin activator, induced more prominent inotropic effect in RVCM, which might imply a larger functional reservoir for the RV contraction.
Investigation of Effects of Hyperglycaemia on the Lung Microbiome in Diabetic Mice
Authors: Vasiljevs Stanislavs , Baines Deborah
Published: May 2022

The lungs are constantly exposed to a diversity of microbes. On average, the human inhales between 0.7 and 7000 bacterial colony forming units (CFU) every minute. The airway epithelium and the airway surface liquid (ASL) which lines the luminal surface, play a vital role in the defence against these inhaled organisms. Glucose concentration in the ASL is much lower than that of blood (approximately 12.5 times lower). It was proposed that low glucose concentration in the ASL contributes to innate protection against the growth of pathogenic organisms which can utilise glucose for growth. Previous research demonstrated that a sustained increase in blood glucose concentration (such as diabetes) led to increased glucose concentration found in the ASL in both human and animals. We therefore hypothesised that the microbial population of the lung would change in the diabetic lung.

Seven‐week‐old female db/db (BKS.Cg‐+Leprdb/+Leprdb/OlaHsd) and non‐diabetic littermates (BKS.Cg‐(Lean)/OlaHsd) db/db mice and non‐diabetic littermate controls were purchased from Envigo (UK). Mice were maintained in standard animal housing in a 12h light/dark cycle; water and standard rodent chow available ad libitumand allowed to acclimatise for three weeks before lung microbiome collection. Mice were terminated with an overdose of pentobarbital (0.2ml of 100mg/ml i.p.). Blood was collected for glucose measurement. Bronchoalveolar lavage was performed and 1 mL of solution was used to extract bacterial DNA using QIAamp DNA Microbiome Kit (Qiagen). The V3‐V4‐region of the 16S rRNA gene was amplified and sequenced using 300 bp paired‐end reads on the Illumina MiSeq platform. Bioinformatic analysis was performed using Mothur v1.39.5 as per the MiSeq SOP pipeline. After removing of contaminant sequence reads, downstream statistical analyses were performed using R statistical software.

The bacterial diversity in BAL samples was highly variable within and between diabetic and non‐diabetic mice. Hyperglycaemia did not affect the a‐diversity of the lung microbiome (Inverse Simpson rating). However, hyperglycaemia had a significant effect on the b‐diversity of lung microbiome (analysed with AMOVA, p=0.011, n=9) with the microbiome from diabetic mice clustering together. At the genus level, bacteria of genus Staphylococcus were more abundant in the normoglycaemic mice (n=9, p=0.019). The genus Pseudomonas were more abundant in diabetic mice (n=9, p=0.028) and Corynebacterium (n=9, p=0.0018), which are frequently found in the lung microbiome as commensal organisms, were decreased.

Taken together, these data indicate that sustained hyperglycaemia modifies the lung microbiome, decreasing the abundance of commensal bacteria and promoting the growth of glucose‐utilising bacteria such as Pseudomonas which may include potential pathogenic species such as P. aeruginosa.
Towards an understanding of S100A8 and S100A9 post‐translational regulation
Authors: Polakowska Magdalena , Steczkiewicz Kamil , Wysłouch‐Cieszyńska Aleksandra
Published: May 2022

S100A8 and S100A9 are small (10.8 kDa and 13.2 kDa), single‐domain, α‐helical proteins belonging to the S100 family of Ca2+‐binding proteins. They are moonlighting proteins exerting many important intra‐ and extracellular, physiological and pathophysiological roles related to signaling, regulation and defense. It has been a long‐term challenge to distinguish the properties of different S100A8 and S100A9 proteoforms related to various biological functions in the human organism. Many limitations have been encountered in the characterization of their structures. Most structural studies were performed for either truncated or mutated variants, especially with removed cysteine residues. The single cysteine’s of S100A8 and S100A9 are reactive, and in vivo undergo post‐translational modifications (PTMs) i.e., S‐glutathionylation or S‐nitrosylation, regulating the proteins activities in inflammatory processes.

In this work, we aimed to provide the missing conformational analysis for full length, non‐mutated S100A8 and S100A9 proteins. using, in particular HDX‐MS method. Our poster presents study of consequences of S‐nitrosylation and S‐glutathionylation on cysteines reactivity and structural conformation of S100A8 and S100A9 proteins.
Neurokinin 1 and 2 Receptors Differently Modulate PEG2‐ and Citric Acid‐Induced Cough and Ventilatory Responses
Authors: Zhuang Jianguo , Gao Xiuping , Zhao Lei , Wei Wan , Xu Fadi
Published: May 2022

Inhalation of aerosolized citric acid (CA) or prostaglandin E2 (PGE2) provokes distinct cough patterns (Type I vs. II) and ventilatory responses (substantial hyperventilation vs. slight tachypnea) in humans and guinea pigs. Substance P (SP) and neurokinin A (NKA) are peripherally released mainly by vagal sensory fibers and capable of affecting CA‐induced cough and ventilatory response via preferentially activating neurokinin 1 and 2 receptors (NK1R and NK2R) respectively. The goal of this study was to determine the impacts of CA and PGE2 exposure on pulmonary SP and NKA levels and the roles of NK1R and NK2R in CA‐ and PGE2‐evoked cough and ventilatory responses. In unanesthetized guinea pigs, we determined: (1) pulmonary SP and NKA contents after CA or PGE2 exposure for 10 min; (2) effects of vehicle, CP‐99994 and SR‐48968 (a NK1R and a NK2R antagonist) given by intraperitoneal injection (IP) or aerosol inhalation (IH) on CA‐ and PGE2‐evoked cough and ventilation; and (3) immunoreactive expression of NK1R/NK2R in vagal sensory neurons labeled by TRPV1 (responsible for Type I cough) or EP3 receptors (responsible for Type II cough). CA and PGE2 exposure evoked Type I and II cough respectively associated with different degree of increases in pulmonary SP and NKA. CP‐99994 and SR‐48968 via both IP and IH similarly depressed the cough responses to CA with less impact on the cough response to PGE2 exposure and substantially inhibited or blocked the evoked ventilatory responses to both CA and PGE2. Moreover, NK1R and NK2R co‐expressed in vagal C‐neurons labeled by TRPV1 or EP3 receptors. These results suggest that the endogenously released SP and NKA play an important role in generating the cough and ventilatory responses to CA and maintaining the ventilatory response to PGE2, at least in part, via activation of vagal C‐neurons expressing NK1R and NK2R.
Neonatal opioid withdrawal syndrome disrupts the ventral swallow pattern generator in germline‐GCaMP6F mouse
Authors: Mellen Nicholas , Lane Leo , Iceman Kimberly , Cai Jun , Gourévitch Boris , Pitts Teresa
Published: May 2022

Opioid exposure during gestation can lead to neonatal opioid withdrawal syndrome (NOWS) at birth. A feature of NOWS is dysphagia. To better understand the contribution of dysregulated central control to dysphagia in NOWs infants, we compared fictive swallow in neonate (P2‐P6) control mice and mice exposed to methadone from E14.5 onwards. Propriobulbar interneuronal and cranial premotoneuronal networks that coordinate orofacial and laryngeal activity during feeding, as well as constituents of the ventral respiratory column were recorded optically in mice expressing the genetically encoded Ca2+ indicator GCaMP6F in the germline. In the sagittally‐sectioned isolated brainstem, swallow can be evoked by electrical stimulation of command neurons along the dorsomedial edge of the preparation. In vitro swallow is defined as a burst at the hypoglossal nerve (XIIn) unaccompanied by phrenic (C4) motor output. In control animals evoked swallow was accompanied by sequential activation of networks dorsal and medial to the facial nucleus (VIIn), a nucleus in the intermediate reticular formation implicated in vibrissal control (vIRT), and (pre‐)motoneurons along the nucleus ambiguus (NA). In opioid‐exposed pups fictive swallow could reliably be evoked, but only networks dorsomedial to VIIn were reliably activated, while vIRT and NA networks were silent. The distribution of ChAT+, phox2b+, and SST+ neurons was the same in control and MTD‐exposed mice, as were the networks active during inspiration. These findings suggest that dysphagia may at least in part be due to network‐level dysregulation of central networks mediating swallow, but not to gross developmental abnormality of these networks. Because neonate feeding behaviors are highly conserved in mammals, these findings suggest that the sagittally‐sectioned mouse hindbrain provides a platform to investigate the contribution of central networks to dysphagia in NOWS infants.
Using Donor and Family Statements to Promote Humanism in the Anatomy Lab
Authors: Hielscher Abby C.
Published: May 2022

The goal of this study was to promote a more humanistic approach, characterized by a deeper sense of respect, empathy and compassion, to anatomical dissection through use of personal information provided by cadaver/donors and their family members. It was expected that upon reading these statements that students would be able to connect with the humanity of the donors and would feel more engaged in the dissection experience. A total of 124 students matriculated into the medical program at the Larner College of Medicine at the University of Vermont and were given electronic access to all surveys and questionnaires generated using Qualtrics XM. Students were asked to complete the Interpersonal Reactivity Index (IRI), an empathy measurement tool consisting of 4 sub‐scales, prior to and at the conclusion of the anatomy course. Students were additionally asked to take a pre‐anatomy survey prior to the start of the course which was designed to assess student apprehensions about dissection and interest in and reasons for viewing the donor/family statements. At the conclusion of the course, students were asked to take a post‐anatomy survey intended to determine if and how much information was read by the students and whether this information allowed the students to connect with the donor’s humanity and/or improve their dissection abilities. Statements from donors and their family members related to the donors’ lives, family, interests, and reasons for donation. This information was compiled and placed in 4 binders which were available only in the anatomy lab. Results demonstrated that students self‐reported high empathic scores on all 4 sub‐scales of the IRI with highest results for the Empathy Concern and Perspective Taking portions and lower scores for the Personal Distress and Fantasy Scale portions of the test. These scores did not change significantly between the two tested time frames (e.g. pre‐ and the post‐anatomy lab). Results from the pre‐anatomy survey showed that while 92% of students were interested in reviewing the donor/family statements, 70% of students reported that they had read the donor/family provided statements. Most students reported that they had read the statements early on in the course and indicated that reading the donor/family provided statements improved their ability to empathize with the humanity of their donor as well as other individuals in their lives, but did not improve their ability to dissect in the lab. Results from this work demonstrate that while incorporating information provided by the donors and their family members promoted self‐reported feelings of a better connection that students had with the donors, it did not change students’ self‐reported attitudes about their empathy as assessed in the IRI.
Impact of aerobic fitness status, menstrual cycle phase, and oral contraceptive use on exercise metabolic flexibility
Authors: Olenick Alyssa , Person Regis , Jenkins Nathan
Published: May 2022

Metabolic flexibility is the ability to appropriately store or utilize substrates. Exercise such as high‐intensity interval exercise (HIIE) is also a robust test of one’s ability to shift between substrates to support metabolic demand. Menstrual cycle or oral contraceptive use may impact metabolic responses to exercise. Fitness status also plays an important role in metabolism. We assessed the impact of hormonal fluctuation and fitness status on exercise metabolism in women during HIIE. 25, healthy, active women (24.4±3.6yrs; 165.5±6.9 cm; 65.1±10.5 kg; 38.8±6.6mL/kg/min) experiencing a regular menstrual cycle (n=15) or using oral contraceptives (n=11) completed the study. Participants were split by aerobic fitness status (High: 43.7±4.7mL/kg/min; Low: 33.6±3.9mL/kg/min). Participants completed a baseline and counterbalanced HIIE sessions in the follicular (FOL) and luteal phase (LUT). FOL sessions took place 2‐7d after onset of menstruation or non‐active OC days. LUT sessions took place ~6‐8d following urine ovulation or third week of active OC pills. Exercise was performed on an electrically braked cycle ergometer. High intervals (H1‐H4) were 4x4 min intervals at the power output corresponding to 50% of the difference between the ventilatory threshold and VO2peak, separated with 3min of unloaded cycling (0W; low intervals). RPE and blood lactate assessed via finger stick were taken after each interval. There was no effect of menstrual cycle phase or oral contraceptive use on fat oxidation, carbohydrate oxidation or blood lactate. There was an effect of fitness status on fat oxidation in LUT and FOL (p=0.002). High fit women oxidized more fat during the full session (FOL: p=0.050, LUT: p=0.001), high intervals (FOL: p=0.048, LUT: p=0.001), and low intervals (FOL: p=0.032, LUT: p=0.024) during FOL and LUT, and recovery during LUT (p=0.033). Carbohydrate oxidation (g/kg; p=0.049) and AUC (p=0.024) were greater in high women during FOL, but not LUT. Blood lactate was significantly greater in low fit women during exercise interval H2 through recovery during FOL, but not LUT (p≤0.050). Total change in rate of fat oxidation (Δ g/kg/min) during HIIE intervals was greater in high vs low fitness during FOL (H2: p= 0.018, H3: p=0.004), and LUT (H1: p=0.041, H2: p=0.001, H3: p=0.001, H4: p=0.006). There was a correlation between exercise fat oxidation (g/kg) and lactate AUC (H1‐L4) during FOL (r=‐0.440, p=0.030; n=25) but not LUT (‐30 minute) (r=0.215, p=0.143; n=25). Our results reflect fitness status plays an important role in exercise fat oxidation, carbohydrate oxidation and blood lactate during HIIE in young women. High fitness women benefit from greater exercise metabolic flexibility than their low fit counterparts. These these differences may be unique to the menstrual cycle phase, with low fitness women having poorer exercise metabolic flexibility in the follicular phase.
Glucose mediates upregulation of Cck expression independent of cAMP signaling
Authors: Williams Rashaun A. , Flowers Matthew , Davis Dawn B.
Published: May 2022

Cholecystokinin (CCK) is a gut peptide hormone that is also produced by pancreatic β‐cells under conditions of metabolic stress and obesity. Treatment with exogenous CCK can alleviate obesity‐related diabetes in animal models without significant side effects. We have established that CCK is expressed primarily in the pancreatic β‐cell and that CCK is highly upregulated in ob/ob mice. We have also shown that elevated cellular cAMP stimulates β‐cell CCK expression and secretion. However, the specific islet cell types that express CCK and the regulation of Cck transcription in the islet has not been thoroughly explored. A more recent study from our lab showed that high glucose concentrations can upregulate Cck expression in mouse islets and human islets. We hypothesize that Cck expression is upregulated in response to various diabetogenic conditions and that CCK upregulation by glucose can occur independent of cAMP activation.

Islets from 16‐week‐old C57BL/6J mice were harvested for use in a dose‐dependent 24‐hour study using different glucose concentrations: No glucose, 2.5mM, 7mM, 11mM & 25mM. After 24‐hour treatment, islets were collected for RNA isolation and cDNA synthesis to identify changes in Cck mRNA expression after incubation with different glucose concentrations. Cck mRNA levels were elevated 4‐8 fold in islets treated with 25 mM glucose compared to islets treated with 0 to 11mM glucose. Notably, the hypoglycemic treatment of no glucose and 2.5mM showed a 4‐fold increase when compared to the 7mM and 11mM treatments. Following this experiment, we wanted to determine if high glucose would still increase Cck mRNA expression after inhibiting cAMP. Islets were pre‐incubated for 45 minutes with 10nM sulprostone, which activated prostaglandin E3 (EP3) receptor and inhibits adenylate cyclase. After inhibition of cAMP, we incubated the islets at 2.5mM and 25mM glucose concentrations like the prior experiment and observed changes in Cck mRNA expression. After treatment with sulprostone, we are still able to see a significant increase in Cck mRNA expression in islets treated with 25mM glucose compared to those treated with 2.5mM glucose, even with cAMP inhibition.

This data suggests that there are alternative pathways that could lead to CCK upregulation in the pancreatic islet independent of cAMP. It also shows that hypoglycemia may also increase Cckexpression, which suggests that CCK is upregulated in response to several forms of metabolic stress. We can further explore the effectiveness of glucose to increase CCK expression by treating islets with cycloheximide and deoxyglucose to determine if glucose metabolism is required for upregulation of Cck.This information will help with our general understanding of how CCK functions in pancreatic islet and how Cck transcription is regulated through signaling pathways and transcriptional networks that regulate β‐cell function and identity under diabetogenic conditions.
Searching for Novel sRNA‐Binding Proteins in Chlamydia trachomatis
Authors: Jo Sungeun , Williams Anne L. , Hussain Salina , Larsen Aleah M. , Berry Katherine E.
Published: May 2022

Non‐coding small RNAs (sRNAs) are regulators of mRNA expression, and sRNA‐mediated gene expression plays an important role in bacterial processes such as stress responses, metabolism, and virulence, providing a promising field of study to understand bacterial behavior. In the well‐studied case of E. coli, global RNA‐binding proteins such as Hfq and ProQ are important for supporting the stability and function of sRNAs. However, many bacterial species that produce sRNAs do not encode either of these RNA‐binding proteins. This raises the intriguing possibility that there may be as‐of‐yet‐undiscovered RNA‐binding proteins in the subset of bacteria that do not possess an Hfq or ProQ protein. Among such bacteria, Chlamydia trachomatis is a human pathogen that causes a sexually transmitted infection and is the most common cause of blindness worldwide. C. trachomatis has been shown to express numerous sRNAs (Elwell et al., 2016; Klepsch, 2020), but its genome does not encode Hfq or ProQ. In this study, we are employing a bacterial three‐hybrid (B3H) assay to search for new RNA‐binding proteins encoded in the genome of this pathogenic bacterium. This B3H assay, developed by Berry and Hochschild (2018) to assess RNA‐protein interactions, was derived from a bacterial two‐hybrid (B2H) assay, which has been used with a genomic‐fragment library from C. trachomatis to identify a new protein‐protein interaction in Rao et al. (2009). To screen for new sRNA‐protein interactions in C. trachomatis, four sRNAs (ctrR1, ctrR2, ctrR0332, and IhtA), previously identified by Albrecht et al. (2010), were cloned into the B3H system’s pBait (RNA moiety) and transformed into reporter cells with a pPrey (protein moiety) genomic‐fragment library from C. trachomatis. Some candidate sRNA‐binding peptides and proteins have been found through this screening process. And, we are conducting β‐galactosidase assays to quantify the interactions of these candidates with sRNAs, along with computational analysis to map the candidate fragments back to the context of the Chlamydia genome and proteome. In the future, we hope to confirm the underlying mechanisms of interactions through site‐directed mutagenesis and to expand our screens to the genomes of additional bacterial pathogens such as Mycobacterium tuberculosis. By finding novel sRNA‐binding proteins in C. trachomatis and M. tuberculosis, we would expand our understanding of RNA‐based regulation in these bacteria and potentially provide new pharmaceutical targets in the future.
Sex‐differences and cell type‐specific alterations in brain Bcl2 expression after transient focal cerebral ischemia in aged mice
Authors: Griffiths Brian , Xu Lijun , Stary Creed
Published: May 2022

Introduction

Stroke remains the second leading cause of death worldwide and remains the primary cause of long‐term disability in the US. To date, the only pharmacological treatment for stroke remains tissue plasminogen activator. A major factor for translational failure is likely overwhelming use of young male rodents despite the fact that stroke occurs predominantly in aged populations and is a sexually dimorphic disease with sex differences in incidence, prevalence, and outcomes. The Bcl2 family is known to play an important role in the evolution of injury following cerebral ischemia. Overexpression of pro‐survival Bcl2 protects against cerebral ischemia in vivo and in vitro. In the present study we explored whether Bcl2 could play a role in known age‐ and sex‐related differences in stroke.

Methods

All studies were conducted in accordance with National Institutes of Health guidelines for the use of experimental animals, with protocols approved by the Stanford Animal Care and Use Committee. Transient focal cerebral ischemia was induced by suture occlusion of the middle cerebral artery (MCAO) for 1h in 20 month‐old male and female C57BL/6 mice. Sham surgery consisted of anesthesia and internal carotid exposure without suture occlusion (n=6 per group). Histological assessment: Animals were sacrificed at post‐MCAO days 1, 3, and 30 (n=6 per group, per time point), and brains fixed with 4% phosphate‐buffered paraformaldehyde for stereological analysis. Fixed sections were stained for Bcl2, the astrocyte marker glial fibrillary acidic protein (GFAP), and the mature neuronal marker NeuN. An observer blinded to conditions quantified the cell‐type specific relative intensity of Bcl2 from maximum projection Z‐stack images.

Results

In sham animals Bcl2 expression was significantly (p<0.05) higher in both female astrocytes and neurons relative to male. After MCAO male neuronal Bcl2 expression was significantly depressed from days 1 until 30, while female neuronal Bcl2 remained unchanged. Conversely in astrocytes, female Bcl2 expression decreased significantly at post‐injury days 1 and 3, but recovered by day 30, while male astrocyte Bcl2 decreased by day 3 and remained significantly repressed at day 30.

Conclusions

Baseline differences in Bcl2 expression between aged males and females, and cell‐type specific differences in post‐injury Bcl2 expression may account for age‐ and sex‐related differences in injury outcome from stroke. Bcl2 may represent a therapeutic target for stroke for both sexes in clinically‐relevant aged populations.
Fingerprinting Small Molecule Modulators of Nucleolar Biophysics
Authors: Donlic Anita , Brangwynne Clifford P.
Published: May 2022

Ribosomal RNA (rRNA) is the most abundant RNA in cells, reflecting its fundamental role in enabling protein synthesis in all living organisms. Nucleolus is a membraneless and multicompartmentalized nuclear body that governs rRNA biogenesis, a process that is misregulated in various diseases. Recent research has identified several small molecule therapeutics that alter rRNA biogenesis and subsequently nucleolar morphology, challenging the accepted mechanisms of action for these compounds and simultaneously establishing the nucleolus as an attractive therapeutic target. Nevertheless, there are currently no comprehensive and quantitative frameworks that capture and relate various drug‐induced changes in nucleolar morphology and its material properties to its function. We hypothesized that we can establish such a structure‐function framework by screening a panel of nucleolus‐perturbing small molecule drugs and measuring changes in biophysical properties of the nucleolus in cells from microscopy images. Here, we combine morphometric, thermodynamic, and kinetic measurements of drug‐treated nucleoli to reveal “fingerprints” for drugs that modulate distinct stages of rRNA biogenesis. We anticipate that this framework provides a starting point for robustly determining and predicting structure‐function relationships for nucleolar phenotype perturbations as well as for developing high‐content screening platforms to expedite the discovery of novel nucleolus‐targeting therapeutics.
The Evolution of Language‐Related Long Association Pathways: A Comparative Study Between Humans and Rhesus Macaques
Authors: Alatorre Warren Jose Luis , Drak Lilit , Shalwani Saba , Jung Homoon , Ortug Alpen , Takahashi Emi
Published: May 2022

The human brain stands out from other primates for its large size. After the split with our last common ancestor (LCA) with chimpanzees—our closest living relative—, the hominin brain expanded threefold, and the frontal and parietal operculum underwent substantial morphological reorganization, likely reflecting language acquisition in our species. The increase of brain size was not homogeneous: the inferior frontal and the parietal areas had a disproportionally large expansion while the occipital lobe retained its absolute size. These evolutionary changes are even more pronounced when human brains are compared to those of Old World monkeys. However, the amount of change in long association pathways within these highly modified regions is not well understood.

This study aims to identify differences and similarities in homologous pathways of the human language network in the macaque brain. We hypothesize that language‐related pathways projecting to/from the inferior frontal and parietal areas will exhibit substantial reorganization in humans.

Using state‐of‐the‐art, publicly available diffusion MRI data of a healthy (in vivo) adult human (MGH‐USC) [1] and 6 ex vivo rhesus macaque (Oxford University, PRIME‐DE) [2] brains, we reconstructed and virtually dissected in DSI Studio three association pathways: the arcuate fasciculus (AF), the frontal aslant tract (FAT), and the parietal‐projecting portion of the extreme capsule (pEmC). We show that the course and terminal projections of the FAT (inferior frontal and supplementary motor area) and the pEmC (superior parietal lobule and frontal cortical areas) are highly evolutionarily preserved in both species despite their crucial role in language in humans. In stark contrast, the AF underwent a major modification in its posterior segment, as previously reported.

Our results suggest that evolutionary changes in the regional volumetric morphology of the brain do not necessarily imply similarly large changes in the relative volumes and trajectories of underlying white matter pathways. In addition, the current results suggest that the structural basis of human language functions already existed, at least to some extent, in the LCA with macaques. Functional and behavioral studies are needed to understand the role of this white matter network in both species.

References

[1] Wang, F., Dong, Z., Tian, Q., Liao, C., Fan, Q., Hoge, W.S., Keil, B., Polimeni, J.R., Wald, L.L., Huang, S.Y. and Setsompop, K., 2021. In vivo human whole‐brain Connectom diffusion MRI dataset at 760 µm isotropic resolution. Scientific Data, 8(1), pp.1‐12.

[2] Milham, M.P., Ai, L., Koo, B., Xu, T., Amiez, C., Balezeau, F., Baxter, M.G., Blezer, E.L., Brochier, T., Chen, A. and Croxson, P.L., 2018. An open resource for non‐human primate imaging. Neuron, 100(1), pp.61‐74.
Role of Rho‐associated kinase (ROCK) in the different speed of relaxation between pulmonary arteries and mesentery arteries of rats
Authors: Oh Seoung beom , Cho Suhan , Kim Sung Joon
Published: May 2022

Pulmonary arteries (PA) are exposed to hemodynamic environment and contractile stimuli different from those of systemic arteries. Previous studies reported that in situapplication of sodium nitroprusside (SNP, NO donor) lowered the PA pressure, which was less prominent than the response of systemic arteries. Here, we investigated the relaxation speed and the in vitro effects of NO‐dependent signaling modulators to compare between isolated PA and mesenteric arteries (MA) of rats. In the dual‐wire myography study, PA showed significantly slower relaxation after the response to 80 mM KCl (80K) or to pharmacological agonists (U46619 and PGF2a). Surprisingly, addition of 30 μM SNP could not prevent the 80K contraction in PA while significantly attenuated the contraction of MA. The pretreatment with soluble guanylate cyclase (sGC) inhibitor (ODQ, 10 μM) markedly further slowed the relaxation of PA after 80K or agonist‐induced contraction. Perplexingly, immunoblot assay revealed higher expression of sGC‐a with similar level of sGC‐b1 in PA than MA. The expression of RhoA‐kinase (ROCK) was higher while myosin light chain phosphatase (MYPT) appeared lower in PA than MA. The treatment with ROCK inhibitor (Y27632, 10 μM) could abolish the different of relaxation response between PA and MA. Taken together, we speculate that despite the higher expression and the functional importance of NO/sGC pathway in PA, the higher level of ROCK and relatively low MYPT in PA are responsible for the characteristic slow relaxation after removal of stimuli.
Small molecule inhibitors of Nek2 kinase using a whole animal Nek2 overexpression model
Authors: Musayev Rafael , Hauter Lamia , Bhuiyan Ashif I. , Santha Asha R. , Dickson Anna A. , Finkelstein Alan J. , Talele Tanaji T. , Pathak Sanjai K.
Published: May 2022

Nek2 kinase, a prominent member of the Never in Mitosis (NIMA) Related Kinases (Neks), is overexpressed in a wide variety of highly aggressive cancers. These include triple‐negative breast, cervical, testicular, ovarian, and prostate cancers. Aberrant Nek2 function as a result of cellular overexpression has been associated with drug resistance and cancer metastasis. Despite this, no clinical agent targeting Nek2 kinase has been developed thus far. Using a Nek2 overexpression model in Drosophila melanogaster, we have shown that the overabundance of Nek2 kinase can dysregulate intracellular signaling to promote highly invasive cellular migratory and proliferation pathways (e.g. activation of Akt/PI3K pathway). Using the same model system, we also identified a novel, non‐toxic drug‐like quinoline‐based pharmacophore. In this work, we report on the development of a library of potential Nek2 inhibitory compounds derived from this pharmacophore. We anticipate that the lead compounds derived from this study will result in the development of highly effective Nek2‐targeting anti‐cancer agents with reduced toxicity.
Tau Mutants Alter the Rheological Properties of Phase‐Separated Tau Condensates
Authors: McDonald Charles J. , Wajid Mahnoor , Elbaum‐Garfinkle Shana
Published: May 2022

Tau has been implicated in many neurogenerative diseases called “tauopathies” where tau self‐assembles into neurofibrillary tangles. Alzheimer’s disease, the most common tauopathy, affects over 50 million people causing a dire need for therapeutics to be generated. However, the cellular mechanism of Alzheimer’s disease and related tauopathies remains poorly understood. Tau has recently been discovered to phase separate into liquid‐like droplets, or condensates, a phenomenon with growing significance in biology with the potential to inspire new therapeutic avenues. To gain insight into the putative role of phase separation in tau pathology, here we quantify the impact of pathological tau mutations on the phase behavior and material properties of tau condensates. We combine DIC and confocal microscopy with particle tracking microrheology measurements in order to characterize droplet morphology and quantify droplet viscoelasticity, respectively. We find that WT full‐length tau readily phase separates with polyU RNA to form condensed viscous fluids. Interestingly, while some pathological mutations have a pronounced effect on droplet viscosity and droplet aging dynamics, others display no significant effect on droplet properties. These results demonstrate an important, albeit complex relationship between tau phase separation, aggregation and pathology. Continued parsing of the complex landscape of tau assembly states, coupled with genetic and cellular analysis has the potential to reveal the roots of tau pathology and its ultimate therapeutic targeting.