bluuebunny/crossref_metadata_2025 huggingface dataset:
A Simple, Robust, Hands‐On MALDI‐TOF Laboratory Activity for Undergraduate Students
Authors: Lawrencee Jim
Published: May 2022

Mass spectrometers are widely implemented in both industrial and academic research laboratories as well as clinical laboratories. Both the hardware and software associated with current mass spectrometers can be difficult to understand, operate and utilize. The learning curve for first time users is so steep as to be nearly insurmountable. For these reasons, as well as others, undergraduate students rarely have an opportunity to have hands‐on access to high quality mass spectrometers. Therefore, these students may not be well prepared for the research environments that await them after graduation. In an attempt to correct this situation and better prepare undergraduate students for future careers, we have developed a laboratory activity to introduce students to mass spectrometry. This activity was designed to introduce students to basic mass spectrometric hardware and software operations. This activity was also designed to be accomplished in a teaching university setting with limited reagent and time availability. A full lab report, including questions about the activity and technology, was written by the students within 48 hours of the lab exercise. Finally, we believe that this individual activity can be used as a foundation for other mass spectrometry based labs, including capstone‐type, semester long laboratory classes. In this report, we describe the organization, implementation and outcomes of this activity.
Investigating a Ligand‐receptor Role for Glutathione in Angiogenesis
Authors: Mackay Ryan P. , Delgadillo Luisa F. , Rashdan Nabil A. , Pattillo Christopher B.
Published: May 2022

Introduction

In addition to its classic role in redox chemistry, glutathione (GSH) also acts as an allosteric regulator of the calcium‐sensing receptor (CaSR). CaSR expression has been found in a variety of cell types including vascular smooth muscle and endothelial cells (ECs), where its activity has been linked to EC adhesion and migration. Although a relationship between GSH and CaSR has been discovered, no link has been established between GSH‐CaSR interaction and regulation of blood vessel development. We hypothesize that a novel mechanism for GSH regulation of blood vessel development occurs through an extracellular interaction with CaSR as a part of normal physiology, with potential extensions to pathological angiogenesis.

Methods

Matrigel tube formation and under agarose migration assays were used to measure EC activity in primary human aortic endothelial cells (HAECs). Fura‐2 AM was used with cells from an hTERT immortalized aortic endothelial cell line (tHAECs) to measure changes in intracellular calcium. Cell surface expression assay (CelLISA) and flow cytometry were used to measure surface CaSR expression. Endothelial and endothelial‐mesenchymal transition (EndoMT) markers were measured using real‐time PCR.

Results

Extracellular application of GSH increased endothelial tube length and branching in tube formation assay, while treating cells with selective CaSR inhibitor (NPS‐2143) abrogated these effects, indicating a role for CaSR in vascular modeling. Extracellular GSH influenced EC migration as measured by under agarose migration assay. GSH or a CaSR agonist (R‐568) reduced surface CaSR expression, with little/no change in intracellular calcium. RT‐PCR results suggest that extracellular GSH alters endothelial phenotype along the endothelial‐mesenchymal spectrum, promoting endothelial differentiation.

Conclusion

In addition to its classic antioxidant role, GSH appears to act extracellularly to influence EC activity. GSH caused significant increases in EC branching as measured by the tube formation assay. With the use of a selective CaSR inhibitor, we were able to confirm the contribution of CaSR to this effect. Additionally, GSH influenced EC migration in the under agarose assay. These results could be explained by GSH‐CaSR‐mediated alterations in endothelial differentiation. These results suggest a role for GSH in vascular development and angiogenesis, through a ligand interaction with the CaSR G‐protein coupled receptor, which represents a novel mechanism of action for physiological and pathological blood vessel development.
In Vitro Evaluation Of The Antimicrobial Activity Of Monolaurin Against Borrelia burgdorferi,The Lyme Disease Spirochete
Authors: Bhalla Shubhang , Trinh Benjamin , Barragan Jose , Cervantes Jorge
Published: May 2022

In this study, we evaluated the antimicrobial activity of monolaurin against Borrelia burgdorferi, the Lyme disease spirochete.

B. burgdorferipossesses adaptive mechanisms that allow it to evade retrieval by host immune response. Novel therapeutic modalities to address Lyme disease complications are widely being sought. Monolaurin, a glycerol monoester of lauric acid derived from coconut oil, has demonstrated a wide‐range of antimicrobial activity through emulsification.

We hypothesize that monolaurin will reduce the viability of B. burgdorferi in a dose response manner.

B. burgdorferistrain B31 was cultured under serially diluted monolaurin conditions. Resazurin assay was utilized to assess bacterial viability and establish an approximate minimum inhibitory concentration (MIC). A live‐dead assay was conducted to evaluate monolaurin’s antimicrobial activity.

Results

We observed a MIC value of monolaurin against B. burgdorferi to reside between 75 µg/mL and 150 µg/mL. The live‐dead assay narrowed the antimicrobial range from 75 µg/mL to 125 µg/mL. There was a trend of the proportion of dead spirochetes with increased monolaurin concentrations that was statistically significant (p‐value<0.0001, Two‐way ANOVA).

Our findings support the use of monolaurin as an antibacterial agent against B. burgdorferi. Further studies in vivo are required to establish its pharmacodynamics so it could become available as a treatment modality for Lyme disease.
CTP:phosphocholine cytidylyltransferase alpha regulates nLD biogenesis in Caco2 cells
Authors: McPhee Michael J. , Lee Jonghwa , Dellaire Graham , Pelech Steven , Ridgway Neale D.
Published: May 2022

Recent investigations reveal that nuclear lipid droplets (nLDs) stimulate lipid metabolic enzymes in response to excess fatty acid loading of cells. For example, in oleate‐treated hepatocytes the rate‐limiting enzyme in the CDP‐choline pathway, CTP:phosphocholine cytidylyltransferase alpha (CCT alpha), is recruited to the surface of nLDs and stimulates the synthesis of phosphatidylcholine (PC). Hepatocyte nLDs derive from ApoB‐free LDs in the lumen of the endoplasmic reticulum that migrate into the nucleoplasm through type I nucleoplasmic reticulum invaginations of the inner nuclear membrane (INM). Once formed inside the nucleus, nLDs recruit proteins CCT alpha, Lipin1 and promyelocytic leukemia (PML) to their surfaces. Endothelial cells of the colon also express ApoB lipoproteins for chylomicron assembly and secretion, so we hypothesized that these cells might also form nLDs in response to excess lipid loading. Indeed, using immunofluorescence confocal microscopy we found that oleate‐treated Caco2 cells formed between 5 to 10 nLDs per cell that recruited both CCT alpha and PML to their surface. CRISPR/Cas9‐mediated CCT alpha knockout Caco2 (CCT alphaKO) cells had significantly fewer but larger cLDs and nLDs compared to wild‐type cells. The percentage of PML‐positive nLDs increased in CCT alphaKO cells, suggesting that CCT alpha and PML compete for access to the nLD surface following oleate treatment. To determine if phosphorylation of CCT alpha regulates translocation to nLDs, phospho‐mimetic and phospho‐null S315/S319 and Y359/S362 mutants were expressed in CCT alphaKO cells and localization to nLDs was measured. In vitrophosphorylation assays of recombinant, dephosphorylated CCT alpha indicated that AMP‐dependent kinase phosphorylated CCT alpha S319, which could be involved in regulating CCT alpha activity on nLDs. In summary, we have demonstrated that oleate treatment induces nLD biogenesis in Caco2 cells and that CCT alpha activity regulates the biogenesis of both nLDs and cLDs.
Limonene restores A2AAR mediated‐vascular relaxation in asthmatic mice via nitric oxide
Authors: Wyer Abigayle , Nellutla Rithvik , Rajalingam Sahith , Prasad Praveena , Ponnoth Dovenia S.
Published: May 2022

Limonene, a mono‐terpene flavonoid, has anti‐inflammatory effects in asthma by activating A2A adenosine receptors (A2AAR). Our objective was to identify the mechanism by which limonene restored A2A mediated aortic relaxation in asthmatic mice. Ex‐vivo tissue bath was used to study the effects of inhaled d‐limonene on isolated aortic rings from asthmatic mice. We used mice in which the A2A receptor was genetically knocked out (A2AKO) and C57BLJ/6 (wild‐type; WT) for the study. The mice were divided into control (CON) and allergen‐induced sensitized (SEN), control mice treated with limonene through aerosol route (CON+LIM) and allergen‐induced sensitized mice treated with limonene (SEN+LIM). Mice were sensitized i.p. on days 1, 6 with 20μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. Control groups were given an i.p injection of vehicle (1,6 days) and the aerosol challenge of normal saline (11–13 days). SEN +LIM, CON +LIM groups were given 0.01% v/v aerosolized limonene via inhalation 90 min prior to challenges. Aorta were isolated on day 14 for further experiments. Tissue responses were studied in the form of contraction and relaxations in all groups. Concentration‐response curves (CRC) for selective A2Aagonist CGS‐21680 (10‐11‐10‐6M) were obtained for all groups. eNOS inhibitor L‐NAME (10‐4M) or prostaglandin inhibitor indomethacin (10‐5M) was added 30 min prior to starting CRC to study effect of nitric oxide and prostaglandins on vascular response. CGS‐21680 induced aortic relaxation was absent in WT SEN (5.62±3.72% WT SEN Vs 66.91 ± 6.8% CON, *p<0.05). Limonene restored CGS 21680‐induced relaxation in asthmatic mice (63.8 ± 6.32% WT SEN+LIM Vs 5.62± 3.72% WT SEN). L‐NAME blocked CGS‐21680 response in CON, CON+ LIM and SEN+LIM, while indomethacin had minimal effect. In conclusion, it appears that limonene restores A2AAR‐mediated aortic relaxation induced through nitric oxide in asthmatic mice.
Loop gain is Increased and the Arousal Threshold is Decreased in Caucasian Compared to African American Males with Obstructive Sleep Apnea
Authors: Puri Shipra , Kissane Dylan , Panza Gino , Lin Ho‐Sheng , Mateika Jason H.
Published: May 2022

Objective

Race and ethnicity may impact the prevalence and treatment of obstructive sleep apnea (OSA). However, only a few studies have made direct comparisons of endotypic characteristics between African Americans and Caucasians. The comparisons completed have focused primarily on differences in anatomical traits (i.e. craniofacial morphology) that may exist between races. The present study was designed to explore if other endotypic traits [(i.e. loop gain (LG) and the arousal threshold (AT)] that influence sleep apnea differ in African American compared to Caucasian males with OSA.

Methods

26 African Americans [age = 34.9 ± 11.9 years; body mass index = 28.1 ± 4.4 kg/m2; apnea hypopnea index (AHI) = 37.7 ± 21.1 events/hour] and 26 Caucasian male participants (age = 31.2 ± 8.2 years; body mass index = 29.6 ± 3.6 kg/m2; AHI = 41.7 ± 16.5 events/hour) with untreated OSA completed a baseline nocturnal polysomnogram. Thereafter, sleep staging, respiratory events, and arousals were scored according to standard criteria. A MATLAB software program was used to quantify LG and the AT from the ventilatory and sleep measures obtained during non‐rapid eye movement (NREM) sleep throughout the night. LG at the natural frequency (LGn) of obstructive events and at a frequency of 1 cycle/min (LG1, i.e., 60 events/hour) were computed. Data was compared using Student t‐test and is presented as mean ± SD.

Results

Age (p = 0.19), BMI (p = 0.20), and the AHI (p = 0.44) was similar between the two groups. LG was significantly higher in Caucasians compared to African Americans (LGn: 0.44 ± 0.07 vs. 0.40 ± 0.06, p = 0.037; LG1: 0.61 ± 0.12 vs. 0.54 ± 0.12, p = 0.039). In contrast, the AT was significantly lower in Caucasians compared to African Americans (1.22 ± 0.17 vs. 1.38 ± 0.27, p = 0.009). The difference in the AT corresponded to an increased arousal index in Caucasians compared to African Americans (74.99 ± 5.36 vs. 51.13 ± 19.34 arousals/hour, p = 0.029). Furthermore, a subgroup analysis of participants with OSA and co‐existing hypertension also showed that Caucasian males (n = 11) had a higher LG (LGn: 0.45 ± 0.06 vs. 0.39 ± 0.07, p = 0.05; LG1: 0.62 ± 0.12 vs. 0.48 ± 0.09, p = 0.007) and lower AT (1.16 ± 0.12 vs. 1.37 ± 0.27, p = 0.033) than the African American participants (n =10).

Conclusion

Despite similar anthropometric measures and a similar apnea hypopnea index, loop gain and the arousal threshold were greater in Caucasian compared to African American males with OSA. Our results suggest that these endotypic characteristics may have a more prevalent role in initiating sleep disordered breathing in Caucasians compared to African Americans. Likewise, our findings suggest that Caucasians may be more responsive to therapies that target loop gain and the arousal threshold.
The Role of the Ih Current in Rhythmic Stability of the PreBötzinger Complex
Authors: Burgraff Nicholas , Baertsch Nathan , Phillips Ryan , Severs Liza , Bush Nicholas , Ramirez Jan‐Marino
Published: May 2022

The generation of rhythmicity is a fundamental property of the nervous system. Common amongst all rhythmogenic networks is the ability to reconfigure and quickly adapt to changes in metabolic, environmental, and behavioral demands. Yet, this flexibility, and plasticity must be counter‐balanced by the need to maintain stability. A group of neurons collectively known as the preBötzinger complex (preBötC) within the ventral respiratory column of the medulla assemble in a network that is both necessary and sufficient to maintain inspiratory rhythm generation. Multiple ionic conductances contribute to rhythm generation. One known for its role in the generation of rhythmic activity across multiple neural networks is the hyperpolarization activated cation current (Ih). The Ih­ current is a voltage‐dependent mixed cationic current that is activated upon phasic hyperpolarization of neurons. The role of Ih in rhythmogenesis within the preBötC is inconclusive. Some reports find that blocking Ih has minimal effect, while others conclude that this increases the rate of rhythmic bursting throughout the network. To date, Ih was specifically studied in rhythmically active neurons with unknown transmitter phenotype. Herein we sought to determine the distribution of Ih amongst the population of tonically and rhythmically active excitatory and inhibitory neurons throughout the preBötC. Additionally, we tested whether removing Ih­ in‐vitro and in‐vivo renders the network more susceptible to perturbations. Using in‐vitro patch clamping we found that Ih showed greatest expression amongst excitatory (87% of DBX1+), and inhibitory (89% of VGAT+) tonically active cell populations. Removing Ih using ZD7288 silenced nearly all tonically spiking neurons. This led to a minimal effect on the rhythmic activity of fully synchronized population bursts, but significantly increased the incidence of less synchronized burstlet activity. Moreover, removing Ih and silencing tonic activity within the network rendered the preBötC more susceptible to suppression following opioid (DAMGO) and CNQX administration. This effect was recapitulated in‐vivo with microdialysis of ZD7288 into the preBötC, which caused normally well‐tolerated doses of morphine to become lethal via terminal apnea. In summary, Ihplays a complex role within the rhythm generating network of the preBötC. Under optimal conditions, Ih is not necessary for maintaining fully synchronized bursting within the network. Yet, Ih becomes increasingly important during perturbations that challenge network function, suggesting this ionic conductance contributes to maintaining the robustness of breathing during everchanging physiological and environmental demands.
Moving biochemistry and molecular biology courses online in times of disruption
Authors: Procko Kristen
Published: May 2022

When the COVID‐19 pandemic necessitated a rapid pivot to online instruction, a team of faculty teaching biochemistry and molecular biology courses created a resource to help other educators approach online education. The group, comprised of educators with experience teaching hybrid and fully online courses, described their strategies in areas like course design, promoting engagement, and assessing student performance. Each topic has a dedicated webpage with brief recommendations, along with a curated list of links to relevant reading, videos, and resources. The American Society for Biochemistry and Molecular Biology (ASBMB) supported the project by hosting the resource on their website (https://www.asbmb.org/education/online‐teaching). At this time, I was transitioning into a position focused on curriculum redesign; one key goal was to introduce discussion sections into a biochemistry course for majors. Drawing from the team’s ideas to promote online engagement, I designed many of these activities using a collaborative whiteboard tool. These activities have now been successfully implemented both online and in‐person, and offer flexibility to instructors and students as we continue to develop engaging activities for hybrid and blended courses.
Glycoalkaloid Solasonine Targets SHH Signaling to Block MED12 Mutant Breast Cancer Oncogenesis
Authors: Akula Shivani , Burleson Marieke
Published: May 2022

As breast cancer is the second leading cause of cancer deaths among American women, there is a strong need to find improved treatment strategies for this disease. Radiation and chemotherapy are the frontline choice of treatment; however, personalized treatment is rapidly on the rise as a superior treatment method. Through prior studies, it has been found that 67% of breast cancer tumors carry a mutation in the Mediator subunit MED12 thus indicating that MED12 likely has a critical tumor suppressive role in the breast. Interestingly, previous results from our lab, and others, have indicated that MED12 plays a role in restricting GLI3‐dependent SHH signaling. This finding is of importance to this study as hyper‐activated SHH signaling is known to play a role in promoting breast cancer oncogenesis. We thus hypothesize that MED12 mutations cause breast cancer oncogenenesis through hyperactivated SHH signaling, and furthermore, that drugs which target the SHH signaling pathway could prove to be beneficial for MED12 mutant breast cancer. Through proliferation assays and quantitative PCR analysis, we were able to conclude that MED12 mutations, as mimicked by a MED12 knockdown setting, enhance breast cancer cell growth and promote GLI3‐dependent SHH signaling. Through a MTT screening strategy, we found that the natural compound Solasonine can differentially target MED12 knockdown breast cancer cells. Solasonine is a known glycoalkaloid that has previously been shown to potentially target anti‐inflammatory signaling pathways. To study the effect of Solasonine on MED12 knockdown breast cancer cells, quantitative PCR was used to measure the levels of GLI3 target genes in cells treated with either vehicle or Solasonine. We found that Solasonine treatment significantly decreased the GLI3 target genes GLI1 and CREB 5 thus suggesting that Solasonine could target the GLI3‐dependent SHH signaling pathway to block breast cancer oncogenesis. These findings were further complemented by colony formation assays which showed that Solasonine treatment leads to a significant reduction in the formation of colonies in vitro. Overall, our findings suggest that Solasonine targets the SHH signaling pathway to block breast cancer oncogenesis and may therefore prove to be a superior treatment method for MED12 mutant breast cancer.
Impact of Estrogen and Hormonal Birth Control on Vascular Function in Premenopausal Women with Type 1 Diabetes
Authors: Simon Abigayle B. , Derella Cassandra C. , Blackburn Marsha , Thomas Jeffrey , Layman Lawrence C. , Harris Ryan A.
Published: May 2022

BACKGROUND

Circulating concentrations of estrogen throughout the menstrual cycle contribute to the cyclic increase in vascular function in healthy women. Evidence in animals; however, has identified a vasoconstrictive role of estrogen in the presence of diabetes. The present investigation sought to determine the role of estrogen throughout the menstrual cycle on vascular function and the impact of hormonal birth control (HBC) on vascular function in premenopausal women with type 1 diabetes (T1D).

METHODS

33 women with T1D (HbA1c=8.5±1.7%, age=26±8 years, BMI=27.1±5.6 kg/m2) and 20 healthy women (HbA1c=5.2±0.3%, age=25±7 years, BMI=23.9±3.8 kg/m2) participated in this study. A subgroup of 9 women with T1D taking oral HBC (HbA1c=8.3±2.1%, age=25±7 years, BMI=29.0±6.4 kg/m2) and 9 demographically matched women with T1D not taking HBC (HbA1c=8.3±2.1%, age=23±6 years, BMI=25.7±5.0 kg/m2), was then analyzed. In all participants, a venous blood sample was collected to determine circulating concentrations of estrogen. Flow‐mediated dilation (FMD), the gold standard for measuring endothelial function non‐invasively, was performed during menses and the late follicular phase of the menstrual cycle to represent periods of low and high estrogen, respectively. Menses was confirmed by a positive hemolytic urological multistix test, and the late follicular day was determined using an ovulation predictor kit for those not on HBC. For those on HBC, baseline was conducted during their placebo week, and the “late follicular” day was conducted during the second week of the HBC pills.

RESULTS

A similar increase was observed in estrogen from menses to late follicular in both the T1D and control group. (Δ estradiol: T1D=74.5±85.4 pg/mL, controls= 88.2±79.1 pg/mL, p=0.561). A significant interaction (p=0.044) was observed between FMD across the menstrual cycle. Specifically, FMD was significantly greater (p=0.043) during late follicular phase in controls compared to T1D, albeit having a similar (p=0.666) FMD during menses. Within the subgroup analysis, there was no interaction between FMD and menstrual cycle between both T1D groups (p=0.555); however, there was a significant (p=0.04) menstrual cycle phase main effect indicating that vascular function overall was greater in women with T1D on HBC compared to women with T1D not on HBC.

CONCLUSION

These data suggest that estrogen decreases vascular function in the presence of diabetes. In addition, HBC may reduce cardiovascular disease risk in premenopausal women with T1D.
Chemotherapy, Microvascular Function, and Angiogenesis ‐ a Longitudinal Study
Authors: Norwood Toro Laura E. , Hader Shelby N. , Terwoord Janee D. , Didier Daniela , Kong Amanda , Beyer Andreas M.
Published: May 2022

Rationale

Anti‐cancer therapies (CTx), such as anthracyclines (e.g., doxorubicin, Dox), have known anti‐angiogenic effects in vitro, and inhibition of angiogenesis has known cardiotoxic effects. Cardiovascular (CV) toxicities that result from CTx significantly contribute to the morbidity and mortality of breast cancer survivors. While the negative impact of CTx, including Dox, on isolated cardiomyocytes is well‐established, significantly less is known about the effects of CTx on microvascular (MV) function and angiogenic potential in patients. Our goal was to understand how CTx damages the microcirculation in order to further the understanding of mechanisms that contribute to systemic cardiovascular toxicity. Utilizing a longitudinal study design of neoadjuvant breast cancer patients, adipose tissue was collected before, during, and after CTx treatment.

Methods

Angiogenic potential was evaluated using a matrigel angiogenesis assay of patient adipose. Data are presented as a percentage of wells with capillary sprouts and area of capillary sprouting ± standard error. In addition, we investigated the expression of a subset of genes involved in angiogenesis regulation (initiation/sprouting, migration, ECM remodeling, maturation, and permeability). Freshly isolated microvessels were evaluated for dilation to flow (FMD) and acetylcholine (Ach). Cardiac function was monitored via standard clinical echocardiogram. Significance was determined using paired t‐test (p ≤ 0.05*).

Results

Percentage of wells with capillary sprouts was impaired in adipose samples collected during CTx (26.3% ± 13.6%, N=4) and one month post CTx (44.3% ± 9.1%*, N=9) compared to pre‐CTx (67.1% ± 4.7%*, N=13). However, sprouting area only changed during CTx (15.0 mm2± 8.5*, N=4) compared to pre‐CTx (68.3 mm2± 10.2, N=13). One month post CTx, the sprouting area (56.8 mm2± 19.1, N=9) was not significantly different than pre‐CTx. Supporting this change in angiogenesis function, we observe changes in expression of genes involved in initiation of angiogenesis during CTx (VEGFA, VEGFB, FLT1) that show recovery of expression post‐CTx. We also observe changes in expression of genes involved in maturation of angiogenesis post CTx (KLF4, VCAM1, MMP8). Some clinical signs of reduced cardiac function were observed post CTx after microvascular and angiogenic changes were observed.

Conclusion

Our data show pathological impairment in angiogenic potential and gene expression in adipose tissue of breast cancer patients in a longitudinal study. These data support our observed defects in MV function (response to FMD and Ach) during and after CTx treatment. We observed significant reductions in MV function and capillary sprouting during treatment with CTx in the absence of a clinically relevant reduction in cardiac function. Our findings suggest that CTx‐induced MV dysfunction precedes and may contribute to future adverse CV events.
Isolation of Multidrug Resistant Bacteria from the Lane College Soil
Authors: Wright Jayla , Allen LaDaja , Van Stry Melanie
Published: May 2022

Antibiotics are substances that eliminate or slow down the growth of bacteria. Beta‐lactam antibiotics, such as penicillin, are antibiotics that contain a beta‐lactam ring that inhibits bacterial cell wall biosynthesis. Bacteria are becoming resistant to antibiotics being that antibiotics are highly prescribed when people seek health care. My goal for this project is to isolate and characterize soil bacteria that are resistant to beta‐lactams. I hypothesize that the bacteria present in the soil on Lane College campus is resistant to beta‐lactams. Furthermore, I hypothesize that these bacteria have a beta‐lactamase gene, which is an enzyme that inactivates the bacteria by cleaving the beta‐lactam ring. I collected soil samples from Lane College campus and cultured the bacteria on two nutrient agar plates with ampicillin and cultured over night at room temperature. For further characterization, I then chose 3 different colonies from the plates (S1A, S1B, and S2A) and streaked them on to new nutrient agar plates with ampicillin. The strains were tested for drug resistance using the Kirby‐Bauer method. They were incubated overnight at room temperature. I then streaked separate LB agar plates from all 3 colonies and added antibiotic disks to each plate (ampicillin, penicillin, amoxicillin, and streptomycin) with E. coli as a control group. Strains S1A, S1B, and S2A were resistant to ampicillin, penicillin, and amoxicillin, but were sensitive to streptomycin. We then further characterized by Gram staining and morphological observation. All samples were Gram negative, and the cells were a coccus shape. I am planning to use the 16S rDNA gene to identify the bacteria using Nanopore next generation sequencing, and to look for the presence of beta‐lactamase genes by PCR.
Swallowing Kinematics in Rats Treated with Low Dose Chemotherapy Concurrently with Fractionated Radiation
Authors: King Suzanne N. , Millward Jessica , Collet Jessica , Rexing Kelsey , Deshmukh Alisha , Greenwell Evan , Kaissieh Nada
Published: May 2022

Dysphagia is a common consequence of chemotherapy and radiation treatment for oropharyngeal cancer. Injury is thought to occur due to damage to the muscle and/or nerves. This can severely impair swallowing function, leading to serious negative outcomes such as aspiration related pneumonia or tube feeding placement to maintain nutrition. Currently, there are only strategies to prevent swallowing dysfunction; however, the development of chronic radiation associated dysphagia persists effecting ~25% of head and neck cancer survivors. Therefore, there is a need to study the mechanisms associated with radiation associated dysphagia to help guide new treatments to improve swallowing outcomes. The present study investigated the effects of concurrent chemoradiation on swallowing function. We tested the hypothesis that fractionated radiation with low doses of chemotherapy alters swallowing kinematics and bolus transit compared to radiation alone or normal controls. We monitored the functional extent of chemoradiation or radiation treatment using videofluoroscopy swallow study in self feeding rats (n=16) at four weeks post treatment. Twelve rats underwent 48Gy of fractionated radiation to the submental muscles using a clinical linear accelerator given in 12 fractions of 4Gy. Irradiated rats received either cisplatin 1mg/kg or saline IP once per week for four weeks during radiation treatment. Four rats received no treatment. During videofluoroscopy, thin liquids and puree consistencies mixed with 40% w/v barium sulfate were given ad libitum. The movement trajectories of the tongue, mandible, and hyoid bone were quantitatively tracked from video data. Changes in the speed of the jaw and hyoid during swallowing were observed between treated and control rats. No differences were observed in bolus flow parameters i.e. pharyngeal transit speed, inter‐swallow interval, and swallow frequency between treatment groups. The average size of the bolus in vallecula decreased by ~4% prior to initiating pharyngeal swallow with chemoradiated rats (p < 0.05). Results suggest that low doses of chemotherapy in conjunction with radiation appear to have minimal differences in swallowing kinematics compared to radiation alone. Higher doses of chemotherapy may be needed to induce similar abnormalities in swallowing as seen clinically.
Leg microvascular function remain robust throughout the female menstrual cycle
Authors: Georgescu Vincent , Jarrett Catherine , Thurston Taylor , Weavil Josh , Amann Markus
Published: May 2022

BACKGROUND

The female menstrual cycle (MC) is characterized by fluctuating concentrations of estrogen and progesterone. While the impact of these vasoactive sex hormones on macrovascular function has received considerable attention, little is known about their influence on the microvasculature. Since the microvasculature ultimately determines skeletal muscle perfusion, a thorough understanding of the influence sex hormones have on microvascular function is needed. It was therefore the purpose of this study to investigate microvascular function, as assessed via the passive leg movement technique (PLM), of eumenorrheic females at three unique gonadal hormone profiles.

METHODS

Six healthy females (age: 22 ± 2 years; height: 168 ± 2 cm; weight: 59 ± 2 kg) were studied during the early follicular (EF; days 1‐4), late follicular (LF; days 8‐12), and mid luteal (ML; days 8‐10 post luteinizing hormone peak) phases of their MC. Venous blood was collected and analyzed for estrogen and progesterone concentrations. For each visit, subjects arrived at the laboratory in a fasted, rested state and at the same time of day. Passive leg movement (1 Hz for one minute; 90‐degree range of motion at the knee) was performed in a seated position. At rest and during PLM, leg blood flow (femoral artery; QL) was measured with Doppler ultrasound, while heart rate (HR), cardiac output (CO), and mean arterial pressure (MAP) were continuously measured via Finapress.

RESULTS

All females presented traditional patterns of hormonal variation throughout the MC. Estrogen (EF: 144 pg/mL, LF: 239 pg/mL, ML: 359 pg/mL) was different at each phase (p < 0.05), while progesterone was similar during EF and ML (~0.2 ng/mL; p = 0.2), but significantly higher during ML (~6.8 ng/mL, p = 0.02). The area under the curve (AUC) for QL (~170 mL, p = 0.7) during the 1 min PLM, and the increase in QL from rest to the peak response to PLM (QLΔpeak; ~540 mL/min, p = 0.3) were not different at any phase of the MC. Furthermore, MAP AUC (~1.0 mL), MAPΔpeak (~6 mmHg), and therefore leg vascular conductance (VC) AUC (~1.9 mL/mmHg) and VCΔpeak (~6.5 mL/min/mmHg), did not change throughout the MC (p > 0.3). Finally, COΔpeak (~0.6 L/min) and HRΔpeak (~7 bpm) remained similar throughout the MC (p > 0.4).

SUMMARY & CONCLUSION

Despite significant differences in venous estrogen and progesterone concentrations during EF, LF, and ML, the hyperemic response to PLM was unchanged at each phase, indicating that microvascular function remain consistent throughout the MC. In addition, both the MAP and the central hemodynamic response to PLM was similar at each stage of the MC. These findings suggest that a tight control of the MC may no longer be considered critical for studies focusing on microvascular function. These new insights might, ultimately, facilitate the involvement of females in future investigations.
Maternal undernourishment stimulates fetuin‐B and impairs placental development
Authors: Camilliere Mia , Rabadi May , Khandi Sharah , Rabadi Seham , Eldana Ahmed , Rath Asha , Wolin Michael , Ratliff Brian
Published: May 2022

Background and Aims

Low birth weight (LBW) significantly increases the risk for multiple diseases, including cardiovascular and kidney diseases. Low birth weight can be caused by a variety of factors, including maternal undernourishment (MUN) during pregnancy. In our study here, we investigated the effects of MUN on the developing placenta, with particular focus on the role of oxidative stress and fetuin‐B in placental impairment.

Methods

Using a mouse model of MUN to generate LBW offspring, in which the pregnant mother consumes reduced total calories and protein during gestation, we examined placental vascularization, vascular reactivity, and blood perfusion using immunofluorescence, myograph, and laser‐doppler flowmetry, respectively. We further examined placental pathology during MUN and pharmacological intervention through histology, immunofluorescence staining and immunoblotting. Trophoblast cell cultures were also used in experiments.

Results

We observed reduced vascular density, vascular reactivity and blood perfusion in the placenta during MUN, along with a reduction in total placental trophoblasts. Further analysis revealed that MUN significantly increases the expression and secretion of the small protein fetuin‐B in the placenta. Treatment of trophoblasts with fetuin‐B increased the activity of NF‐kB in these cells, while also stimulating their generation of reactive oxygen species. Subsequently, fetuin‐B effectively reduced trophoblast quantity by impairing their proliferative expansion, stimulating their apoptosis, and reducing their differentiation. Treatment of MUN pregnant mothers with antioxidants improved placental vascular function and helped preserve placental trophoblast pools.

Conclusion

MUN upregulates fetuin‐B and oxidative stress in the placenta, effects which consequently impair placental development and vascular function.
Myostatin Deletion Preserves Cardio‐Metabolic Function in Type 1 diabetes
Authors: Huff Denton R. , Nunan Emily L. , Gore Jillian L. , Wright Carson L. , Valdez Cammi , Butcher Joshua T.
Published: May 2022

Type 1 Diabetes Mellitus (T1DM) is a disease characterized by the destruction of insulin‐secreting pancreatic beta cells and results in hyperglycemia, muscle wasting, and vascular dysfunction. Patients afflicted with T1DM suffer from increased morbidity and early mortality, largely driven by an inability to appropriately maintain glucose homeostasis. Skeletal muscle is the body’s largest metabolic reservoir, absorbing significant amounts of glucose from the bloodstream. The myokine myostatin is a potent negative regulator of muscle growth and is upregulated in T1DM patients but downregulated following regular exercise. Physical exercise is also known to improve cardiovascular health and increase insulin sensitivity of muscle, but many T1DM patients are unable to exercise at a level that conveys benefit due to muscle atrophy. Thus, directly targeting skeletal muscle, independent of exercise, may prove beneficial for T1DM therapy. Our hypothesis is that genetic deletion of myostatin will preserve glucose homeostasis, maintain muscle function, and protect against vascular dysfunction and cardiometabolic effects in a mouse model of T1DM.

T1DM was induced via streptozotocin (STZ) in adult male mice with (WT) and without myostatin (MyoKO). Multiple variables were assessed including glucose homeostasis (plasma glucose, HbA1c, IGTT), fluid dynamics, muscle function (in vivo plantarflexion), and vascular function (ex vivo pressure myography of gracilis arteriole).

Myostatin deletion inhibited STZ‐induced increases in plasma glucose, preserved fluid dynamics, and prevented decreases in muscle function, independent of insulin. Further, endothelial function was protected with myostatin deletion. Taken together, this data suggests that myostatin inhibition may be a target for effective treatment and management of the cardiometabolic and skeletal muscle dysfunction that occurs with T1DM.
Baroreceptor Deafferentation Impairs the Exercise Training ‐and Drug Treatment‐Induced Adaptations in a Hypertensive Model of Menopause
Authors: Ferreira Maycon J. , do Carmo Silva Gabriel , Bernardes Nathalia , de Araujo Amanda A. , da Silva Dias Danielle , Irigoyen Maria C. , De Angelis Kátia
Published: May 2022

Aim and Hypothesis

Here, we investigate the effect of SAD on physical and hemodynamic adaptations after long‐term treatment with Enalapril and concurrent exercise training (CET) in a model of menopause. We hypothesized that the beneficial effects of exercise training combined with antihypertensive drug therapy would be reduced after the chronic absence of arterial baroreceptor reflex.

Methods

Female spontaneously hypertensive rats (90 days old) were distributed (7‐8 animals/group) into ovariectomized groups: trained treated with E (OTE) and SAD trained treated with E (SAD‐OTE). SAD procedure was performed at initial of protocol. Ovariectomy consisted of bilateral removal of the ovaries. Enalapril (3mg/kg) was administrated orally in drinking water. CET (40‐60% maximal capacity) consisted of aerobic (motor treadmill) followed by resistance exercises (ladder adapted to rats), 3 days/wk for 8 weeks. Heart rate and BP variability were analyzed using signals of direct recording of BP. Vasopressor system was evaluated by sequential injection of a vasopressin receptor antagonist, losartan and hexamethonium.

Results

Treadmill performance was reduced in SAD group (p<0.001). There were no differences for systolic, diastolic, and mean blood pressure (BP) between groups. SAD increased resting heart rate (p<0.001). In addition, SAD‐OTE group showed lower variance of pulse interval (ms²) (49.2±22.5 vs. OTE: 96.5±49.6), as well as higher systolic BP variance (mmHg²) (84.1±36.9 vs. OTE: 34.2±9.0) and low‐frequency band (mmHg²) (11.2±4.9 vs. OTE: 6.5±3.4). BP reduction (mmHg) after vasopressin receptor antagonist (‐20.5±10.5) and hexamethonium (‐87.2±17.7) was higher in SAD‐OTE (vs. OTE: ‐6.9±4.6 and ‐56.0±10.4, respectively).

Conclusion

Despite similar BP control, drug therapy‐and training‐induced adaptations are blunted in absence of baroreceptor deafferentation, suggesting that this mechanism play a crucial role to maintain physical and cardiovascular autonomic benefits.
Dietary curcumin regulates hepatic cellular senescence in aged mice via p38‐MAPK activation pathway
Authors: Lee Su‐Jeong , Chandrasekran Prabha , Mazucanti Caio H. , O’Connell Jennifer F. , Egan Josephine M. , Kim Yoo
Published: May 2022

Aging is a physiological decrease of several biological activities in an organ that all organisms go through. The presence of a gradual deterioration of cell functioning, due to damage accumulation in metabolic organs, accelerates biological aging. Recently, dietary interventions with food‐active compounds have been linked to suppressing the accumulation of senescent cells and senescence‐associated secretory phenotype (SASP). Curcumin has potent biochemical and biological activities, including antioxidant and anti‐inflammatory actions. However, it largely remains unclear how curcumin has anti‐aging properties such as protection of DNA damage and cell survival/cell fate decisions. The objective of this study is to examine the regulatory effect of dietary curcumin on hepatic cellular senescence in the aged mouse model. Aged (18‐20 months old) male C57BL/6 mice were fed a normal chow diet (NCD) or NCD containing 0.4% (w/w) curcumin ‐ equivalent to 2g/day for a 60 kg adult ‐ (NCD+CUR), high fat high sugar diet (HFHSD) or a HFHSD+CUR (N=7‐9 per group) for 15 weeks. Mice given an HFHSD supplemented with curcumin displayed a different metabolic phenotype compared to mice given an HFHSD alone. To examine the phenotypic plasticity led by transcriptomic alteration, we used RNA‐Seq and analyzed differential gene expression in Gene Ontology (GO) terms and KEGG pathway analysis. There were 1687 and 3794 number of genes that showed a significant change with curcumin in NCD and HFHSD groups compared to their respective control groups. There were at least 8‐fold higher uniquely upregulated and 6‐fold uniquely downregulated genes in the HFHSD+CUR group when compared with their NCD counterparts. To this extent, curcumin supplementation altered hepatic gene expression profiling, especially in senescence pathways and their associated genes. Thus, to validate downregulation in senescence pathway involved genes from the RNA‐Seq data results, we compared the distribution of hepatic senescent cells by β‐gal staining in HFHSD groups. It revealed that HFHSD+CUR mice showed less density of the hepatic senescent cell (Figure A). We then mechanistically sought how curcumin regulates the hepatic senescence pathway. We found that curcumin supplementation decreased senescence effectors, specifically p38 and JNK protein expression levels in the liver (Figure B, C). Our findings suggest that the multifaceted therapeutic potential of curcumin can be used as a protective agent for age‐induced metabolic diseases.
Investigations of BRAF Regulation through Auto‐Inhibition and HRAS Interaction
Authors: Trebino Tarah , Markusic Borna , Wang Zhihong
Published: May 2022

BRAF is a key member in the MAPK signaling pathway essential for normal cell growth, reproduction, and differentiation. For this reason, BRAF is also the cause of numerous cancers when misregulated or mutated. Upstream of BRAF is the small, compact GTPase, RAS—a protein implicated in nearly a third of all cancers, yet extremely difficult to target due to its shape and shallow binding pocket. Upon interaction with RAS through the RAS binding domain (RBD) and membrane recruitment, BRAF transitions from an inactive, auto‐inhibited monomer to an active dimer and subsequently phosphorylates MEK to propagate the signal. The auto‐inhibited conformation is thought to be stabilized through interactions between the Cysteine Rich Domain (CRD) and the Kinase Domain (KD) of BRAF. Despite its central role in cellular signal transduction, the order of BRAF’s activation steps have yet to be experimentally validated. We propose that by studying the inter‐ and intra‐molecular interactions of BRAF, we will begin to unveil the fine details of RAF regulation, important for developing new strategies towards therapeutic intervention. We performed biophysical, biochemical, and cell‐based assays to further investigate the role of the regulatory regions in BRAF activation and auto‐inhibition. We quantified in vitro binding between the N‐terminal domains of BRAF and the KD as well as binding between the N‐terminal domains of BRAF and HRAS through Surface Plasmon Resonance (SPR). We show that the interaction between RAS and regulatory regions of BRAF disrupt the interaction between the regulatory regions and catalytic region of BRAF, providing direct evidence that RAS binding to BRAF relieves the auto‐inhibitory interactions among BRAF domains. Furthermore, we examined the effect of Raf‐mimetic peptides in disrupting the RAS‐RAF interaction and relief of auto‐inhibition. The studies outlined here are leading to promising new insights into the activation mechanism of BRAF kinase.
Mutations in Caveolin Binding Motif Alter Human Follicle Stimulating Hormone Receptor Signaling
Authors: Zahedi Katarina , Cohen Brian
Published: May 2022

Globally, there are about 48 million couples and 186 million individuals of reproductive age that are affected by infertility. Some cases of infertility in both men and women have been attributed to impaired follicle stimulating hormone (FSH) signaling. Lack of proper function of the cognate receptor for FSH (FSHR) could contribute to infertility since the biochemical signal generated by FSH binding to FSHR stimulates the production of a sperm‐stabilizing protein in males and follicle maturation in females. It has been demonstrated that human FSHR (hFSHR) localizes to lipid rafts, which are rigid and detergent‐resistant microdomains in the cell membrane. Their structure is in part due to having higher concentrations of cholesterol and sphingolipids compared to the surrounding plasma membrane. Additionally, some lipid rafts are characterized by the presence of the protein caveolin. Signaling pathways for some cell surface receptors have been demonstrated to be regulated by residency in lipid raft domains either by separating the related molecules inhibiting premature signaling or by co‐localizing the related molecules to facilitate signaling. Based on evidence that FSHR forms direct protein‐protein interactions with caveolin, it was hypothesized that disruption of the caveolin binding motif (CBM), a conserved peptide sequence present in proteins associated with caveolin, would affect hFSHR signaling. In hFSHR this sequence is found in transmembrane helix 4 between amino acids 479‐489. The current study investigated the effect of mutations in the CBM on hFSHR signaling. The mutants were created via site‐directed mutagenesis and expression vectors were transfected in HEK293 cells. Cells stably expressing the mutant FSHRs underwent western blot analysis for qualitative comparison of signaling. Mutations in all four conserved phenylalanine residues of the CBM resulted in much higher signaling compared to the wild‐type and at a faster rate of activation. Elevated signaling at shorter FSH treatment times suggest that the mutated receptor might be more sensitive to FSH and may hint at the role of the caveolin interaction in regulating hFSHR signaling. Because current infertility treatments are costly, tend to have low success rates, and are not accessible to everyone, a greater understanding of FSH signaling pathways could lead to the design of more successful infertility treatment methods.