bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biophysical Characterization of CowN from Gluconacetobacter diazotrophicus
Authors: Bretzing Kevin , Owens Cedric , Verde Christine Lo , Espinoza Alejandro 
Published: April 2020

Gluconacetobacter diazotrophicus is a nitrogen fixing bacterium that associates with plants and plays a crucial part in providing fixed nitrogen to many crops such as sugar cane. The enzyme responsible for reducing atmospheric nitrogen to ammonia is nitrogenase. The presence of carbon monoxide gas will inhibit nitrogen fixation by nitrogenase. Many diazotrophs have a mechanism of protecting nitrogenase in vivo. In G. diazotrophicus, a protein called CowN protects nitrogenase from inhibition by carbon monoxide. Here, we present structural and biophysical characterization of CowN. Following heterologous expression of CowN in Escherichia coli and purification by affinity and size exclusion chromatography, CowN is found in two different oligomeric states; monomer and large soluble aggregate. Dynamic light scattering indicates the monomer has a radius of 1.9 nm, whereas the aggregate is larger than 5 nm. While the monomer protects nitrogenase from carbon monoxide inhibition, the aggregate does not. To determine what causes CowN aggregation and if it represents a physiologically relevant process, we investigated the mechanism of CowN aggregation. We discovered that high salt and protein concentration increase the propensity of CowN to aggregate. We further determined that under reducing conditions the monomeric state is preferred, suggesting that oxidation of CowN’s single cysteine residue plays a role in aggregation. Together, the data suggests that CowN may be most active under oxidizing conditions and, further, that aggregation might represent a mechanism to inactivate the enzyme at high concentration.Support or Funding InformationThis research was supported by the Chapman Center for Undergraduate Excellence, USDA‐NIFA (Grant no. 2015‐67012‐22895) and the National Science Foundation (Grant no. 1905399).
The Effect of Gas6‐Axl Double Knockout on Satellite Cell Proliferation and Skeletal Muscle Regeneration After Injury
Authors: Mervis Matthew J. , Matsumura Marc S. , Olsen Zachary E. , Hirschi-Budge Kelsey M. , Reynolds Paul R. , Arroyo Juan A. , Thomson David 
Published: April 2020

Growth arrest‐specific 6 (Gas6) is an extracellular signaling molecule that is known to play a role in cell proliferation, inflammation and other processes through its interaction with Tyro3, Axl, and Mer receptors, but little is known about its role in skeletal muscle. The purpose of this study was to examine the effects of Gas6‐Axl double knockout (dKO) in skeletal muscle recovery after injury. Muscle injury was produced by injecting BaCl2 into tibialis anterior (TA) muscles of wild type (WT) and dKO mice. Muscle tissue was harvested 3, 7, and 14 days after injury. Gas6 and Axl expression over the injury timecourse was characterized by western blotting and ELISA. Muscle damage and cross‐sectional fiber areas were assessed on hematoxylin and eosin (H&E) stained sections. Muscle satellite cell content and proliferation were assessed by Pax7/Ki‐67 staining. We found that both Gas6 and Axl protein expression increased substantially after injury in WT muscles, peaked at 3 days post injury, then declined toward basal levels thereafter. TA mass was lower for dKO muscles 14 days after injury, but fiber CSA was elevated in dKO muscles, suggesting a deficit in fiber number leading to compensatory hypertrophy of existing fibers. Pax7‐positive satellite cell number was unaffected by genotype in uninjured control muscles, but was ~3X higher in WT vs. dKO muscles at 3 days post injury, suggesting that the rate of satellite cell proliferation was suppressed or delayed by dKO. These results indicate that Gas6‐Axl signaling plays a role in regulating skeletal muscle regeneration after injury. Impaired Gas6 function may be linked to excess hypertrophy and loss of muscle mass after injury.Support or Funding InformationThis work was supported by a grant from the Flight Attendant’s Medical Research Institute (FAMRI) and a BYU Graduate Research Fellowship.
Automated Sectioning, Staining, and Imaging of Histologically Preserved Tissues
Authors: Monteith Corey , Parker Kristy , Springer Joey , Farahani Navid , Rhodes Christopher 
Published: April 2020

ObjectiveTissue microstructure contains a wealth of information with extraordinary value for basic research and therapy discovery. But because histology is manual and tedious, tissue protocols are not set up for highly automated, structured, data‐driven methods that prevail in other areas of life sciences research.MethodsWe adapt block‐face lamination array tomography (B‐FLAT) for automated, high‐throughput histological sectioning. We can section, stain, mount, image, and microdissect paraffin‐embedded tissue sections on tape, allowing for much higher speed, accuracy, and consistency in section capture, as well as automated imaging and image registration.ResultsWe successfully captured, deparaffinized, stained, and imaged serial histological sections on tape. Separately, we validated numerous histological and immunohistochemical (IHC) stains relevant to renal pathology and established their equivalence to conventional glass slide methods. We generated a series of at least 30 hematoxylin and eosin (H&E)‐stained mouse kidney sections, digitally imaged and registered at 20X magnification, and reconstructed entire renal glomeruli with nuclear resolution adequate for algorithmic cell counting.ConclusionsOur automation approach recapitulates the performance of conventional histology practices, but allows for much higher speed, accuracy, and consistency in section capture. This enables future development to dramatically improve section quality, throughput, and sample biases.Support or Funding InformationFunding provided by NIH grant 1R43DK120281‐01
Dietary Potassium Attenuates the Effects of Dietary Sodium on Vascular Function in Salt‐Resistant Adults
Authors: Smiljanec Katarina , Mbakwe Alexis , Gonzalez Macarena Ramos , Lennon Shannon L. 
Published: April 2020

Cardiovascular disease remains a major public health problem in the U.S. and is the result of lifestyle factors, such as nutrition, that play an important role. Excess sodium consumption can increase blood pressure (BP) while consumption of potassium has been shown to have BP lowering properties. While the effect of these two nutrients on BP is fairly well understood, their impact on vascular function, particularly any interactive effects, has received less attention. Endothelial dysfunction, characterized by impaired dilation, is an important non‐traditional risk factor for atherosclerosis. Therefore, the purpose of this study was to determine if dietary potassium can offset the deleterious effect of high sodium on endothelial function as assessed by brachial artery flow‐mediated dilation (FMD). Thirty‐three adults with salt‐resistant BP (16M,17F; 27±1yr) completed 7 days each of the following diets: 65 mmol potassium/50 mmol sodium (MK/LS); 65 mmol potassium/300 mmol sodium (MK/HS); and 120 mmol potassium/300 mmol sodium (HK/HS) in random order. On the last day of each diet, 24‐hour ambulatory BP, 24‐hour urine collection and a blood draw was performed. FMD was assessed in response to reactive hyperemia. 24‐hr BP did not differ between diets confirming that subjects were salt‐resistant. Sodium excretion increased on both HS diets compared to LS/MK (p<0.05) and potassium excretion was increased on the HK diet compared to MK/LS and MK/HS (p<0.05) confirming diet compliance. Aldosterone and plasma renin activity were elevated on MK/LS compared to both HS diets (p<0.05). FMD was impaired on MK/HS (5.4±0.5%) compared to both MK/LS (7.0±0.6%; p<0.05) and HK/HS (6.5±0.5%; p<0.05) while there was no difference between the MK/LS and HK/HS diets (p>0.05). These data suggest that potassium provides vascular protection against the deleterious effects of high sodium on conduit artery function.Support or Funding InformationThis work was supported by NIH NIGMS grant # P20GM113125.
Semaphorin‐3F, a Potential Biomarker for Lymphatic Anomalies and Bone Cell Function
Authors: Solorzano Ernesto , Kelly Michael , Safadi Fayez 
Published: April 2020

Lymphatic anomalies (LA) are rare conditions characterized by abnormal vascularization of lymphatic vessels (lymphangiogenesis). Gorham‐Stout Disease (GSD) is an aggressive type of LA which invades cortical bone and makes it “disappear”. There is little evidence elucidating the etiology of GSD and its osteolytic tendencies due to the rarity of the disease. In addition, there are no biomarkers capable of predicting GSD progression or treatment outcome. Normal bone does not have lymphatic vessels. It is believed that in GSD lymphatic endothelial cells (LEC) undergo genetic changes early in development that facilitate their migration and vessel formation in bone. LECs and their secretome content (lymph) are likely both crucial to lymphatic migration into bone and the cortical destruction seen in GSD. In this study, we focused on the LEC secretome with the goal of identifying proteins capable of modifying LEC and bone cell interactions. We initially assessed the effects of normal LEC conditioned media (LEC‐CM) on bone cell differentiation and function in vitro, and found that LEC‐CM stimulates osteoclast differentiation. Next, we subjected LEC‐CM to mass spectrometry and over 500 proteins were identified; we focused specifically on Semaphorin‐3F (SEMA3F) as SEMA3F effectively inhibits lymphangiogenesis in part, by competing with vascular endothelial growth factor‐C (VEGF‐C), for binding to the neuropilin‐2 receptor. We measured mRNA expression of both SEMA3F and VEGF‐C in untreated and Rapamycin treated LECs, a clinically effective therapy for patients with lymphatic anomalies. SEMA3F mRNA expression was upregulated while VEGF‐C was downregulated in Rapamycin treated versus untreated LEC cultures. We next assessed the effects of exogenous SEMA3F on LEC viability and on osteoblast differentiation and function. We found that SEMA3F reduced LEC viability in a dose‐dependent fashion. In addition, SEMA3F inhibited osteoblast differentiation as measured by alkaline‐phosphatase activity and matrix mineralization independent of its effects on osteoblast viability or proliferation. Experiments are currently underway to determine the expression/localization of SEMA3F, VEGF‐C and neuropilin‐2 in lymphatic anomalies (LA) tissues obtained from pediatric patients at our facility. We expect that SEMA3F expression will be downregulated while VEGF‐C will be upregulated in LA compared to normal lymphatics. In summary, our data suggest that SEMA3F might be a possible biomarker for patient with LA conditions or contributes to their pathogenesis.Support or Funding InformationLymphatic Malformation Institute, USA | Rebecca D. Considine Institute of Research, Akron Children’s Hospital, Akron, OH, USA| Integrated Pharmaceutical Medicine Program, NEOMED, Rootstown, OH, USA
Muscle sympathetic nerve activity in altitude acclimatized lowlanders during leg cycling exercise
Authors: Moralez Gilbert , Hansen Alexander B. , Amin Sachin B. , Hofstatter Florian , Tymko Michael M. , Ainslie Philip N. , Hearon Christopher M. , Lawley Justin S. 
Published: April 2020

Acute normobaric hypoxia augments muscle sympathetic nerve activity (MSNA) during dynamic cycling exercise; however, the effects of chronic hypobaric hypoxia exposure on this response remain unknown. The purpose of the present study was to determine MSNA reactivity to moderate intensity cycling exercise in lowlanders at sea‐level (344m; Kelowna, BC, Canada) and after 14–21 days of high‐altitude acclimatization (~4300m; Cerro de Pasco, Peru). MSNA (radial microneurography), mean arterial pressure (brachial artery catheter), and heart rate (electrocardiogram) were measured at rest and during cycling exercise (60% of peak workload) in six healthy male participants. Chronic exposure to hypobaric hypoxia increased resting MSNA burst frequency from 14.0 ± 3.2 burst/min at sea‐level, to 38.2 ± 4.5 burst/min (P<0.001). The increase in MSNA during semi recumbent cycling exercise during chronic hypobaric hypoxia exposure was significantly lower compared to sea‐level (Δ MSNA: sea‐level: 106 ± 46% vs. high‐altitude: 43 ± 15% vs; P<0.001). To our knowledge, this is the first investigation to compare MSNA between normobaric normoxia and chronic hypobaric hypoxia during leg cycling exercise. These data indicate that chronic hypobaric hypoxic exposure blunts the relative increase in MSNA during moderate‐intensity dynamic cycling exercise, potentially due to a sympathetic “ceiling” being reached.Support or Funding InformationWilderness Medical Society research in‐training grant (GM)
Increased Interest in Anatomical Donation as a Result of Positive Media Coverage
Authors: Walker Cam , Ostervoss Tamara L. 
Published: April 2020

A 99‐year old female anatomical donor at Oregon Health & Science University (OHSU) in 2018 had an unusual anomaly: Situs inversus (SI) with levocardia [left‐facing heart]. Her SI and levocardia went mostly unnoted throughout her long life, despite several surgical interventions and five successful pregnancies. After consultation with Ms. Ostervoss (director of OHSU’s body donation program), and the donor family, a poster presentation highlighting this donor was prepared and shown at last year’s American Association of Anatomy meeting at Experimental Biology in Orlando, FL, in April 2019 (Walker and Hankin 2019). Additionally, a press release to coincide with the poster presentation was prepared at OHSU for local media consumption. The donor’s story was distributed by print, radio, and television, worldwide.The benefit to the OHSU BDP of publicizing this unusual case of SI can be best seen in Figure 1, which documents the number of consent forms registered with the BDP between 2014–2019. Between 2014 and 2018, annual preregistration vacillated between 350 and 400 individuals. However, after the widespread publication of the case of SI with levocardia in 2019, the number of preregistered donors surged to approximately 460 individuals, with the majority of additional registrations taking place after publication of the findings in widely viewed media sources. This is particularly apparent in Figure 2, where preregistration numbers are compared month to month between years 2017, 2018, and 2019. For example, in June, 21 persons (2017), and 29 persons (2018) preregistered to be anatomical donors. In June 2019, 48 individuals. The disparity continued in July, when 25 individuals preregistered in 2017, 34 in 2018, and 50 in 2019. The OHSU BDP undertakes other forms of ongoing outreach as well, though none were performed shortly before or after the SI press release in 2019.Health professionals have long known the benefits of anatomical donation. Transparency in body donation is important as well. In this case, nascent medical students saw unique, but not pathological, anatomical variation that will stick in their memories. The family of the donor, after being contacted by Ms. Ostervoss, received closure and a great sense of satisfaction that their loved one contributed to the education of medical students and the public as a whole. Living individuals with SI may also benefit from a future study about the causes of SI.
Exploring the Kinetics and Cleavage Sites of Cysteine Cathepsins for Vaccine Design
Authors: Matson Shayna , Rivera Kristian , Becker Tynan , Kuhn Thomas 
Published: April 2020

Cysteine proteases are key players in antigen processing, with roles in both pathogen digestion and major histocompatibility complex class II (MHCII) formation. A selection of the cleaved peptide chains are presented to CD4+ T cells to initiate an adaptive immune response. Those peptides that elicit the strongest T cell activation are termed immunodominant.In this project, the kinetics and cleavage preference of cathepsins S and L individually and S, B & H in combination were examined using hemagglutinin (HA) protein from the A/New Caledonia/20/99 H1N1 strain of influenza as a substrate. HA was digested in solution with cathepsins at specific time points both at pH 5.0 and 6.4. Post‐digestion, we separated the products by mass and characterized the peptide fragments using mass spectroscopy.We found that specific core peptides remained intact for up to 16 hours and correspond to a sub‐set of the immunodominant or subdominant HA peptides regardless of how many enzymes were applied. The kinetics of the digestion of cathepsins S, B & H in combination differed by pH with the higher pH showing slower kinetics. Both the individual cathepsins S and L displayed kinetics more similar that of pH 6.4 than 5.0 when the mixture of 3 enzymes were applied. This provides confirmation that the peptide repertoire can be impacted by regulation of the activity of individual cathepsins in addition to the pH of the endosomes where processing occurs.Information on the cleavage sites and kinetics of cathepsins increases in‐silico predictive capacity when designing vaccines against novel pathogens.Support or Funding InformationResearch funded by NIH grant R01AI118888‐01A1 and NIH grants UL1GM118991, TL4GM118992, or RL5GM118990.
Isolation stress during adolescence increases conditioned place preference to cocaine in female rats
Authors: Rivero Carlos J. , Freire Jaime A. , Santiago Ivan , Delgado Cristian , Segarra Annabell C. 
Published: April 2020

Adolescence is a developmental period characterized by neuronal growth. Environmental factors such as maternal neglect, social stress and drugs of abuse have a direct impact on future behavioral patterns. Because rats are highly social animals, we decided to investigate if isolation stress during adolescence increases conditioned place preference (CPP) to drugs of abuse. Since lower D2 receptor expression in the mesocorticolimbic system is known to increase cocaine conditioning in adult animals, and because adolescents and adults differ in dopaminergic connectivity and D2 receptor populations, we investigated if D2 receptors are altered during these conditions. Female rats were weaned at postnatal day 23 and housed singly or in pairs. At day 34 rats were tested in an open field, at day 35 in an elevated plus maze and at day 36 they were tested for CPP to cocaine (15mg/kg). At post‐natal day 48, rats were euthanized, brains collected and stored at −80°C. Single‐housed rats showed greater conditioning to cocaine when compared to group‐housed rats. This suggest that housing rats singly increases the rewarding properties of cocaine. Single‐housed rats also show lower D2 receptor immunoreactivity in the Nucleus accumbens (NAc) and the Prefrontal cortex (PFC) when compared to group‐housed rats. This correlates with the greater conditioning observed in single housed rats. Isolation stress had the effect of lowering D2 receptor populations in the NAc and PFC thus augmenting the rewarding properties of cocaine in adolescent female rats. Chronic stress during adolescence thus can lower D2 receptors and could be a determining factor in substance abuse and relapse.Support or Funding InformationFinancial assistance was provided by the Office of International Science and Engineering (OISE) of NSF through the Partnerships for Research and Education (PIRE) Program (OISE‐#1545803).
A rare finding of suspected synovial chondromatosis
Authors: Beger Aaron W. , Dudzik Beatrix , Millard Jonathan A. , Ford Stephen , Thompson Brent J. 
Published: April 2020

IntroductionHuman gross anatomy laboratory dissections provide a rich learning environment, sometimes including rare anatomical variation or pathological findings. Here we describe a rare knee joint abnormality, which provided an opportunity to review pathological processes unique to this region and expand the learning experience beyond the standard curriculum.FindingsDuring a routine lower limb dissection of an 88‐year‐old formalin‐fixed Caucasian male donor, first‐year medical students discovered an enlarged semimembranosus bursa in the superomedial corner of the right popliteal fossa. Opening of the bursa revealed nine irregular‐shaped osteochondral bodies bathed in synovial fluid, with an average length of 1.76 cm and an average width of 1.25 cm. The donor exhibited no obvious trauma, however bilateral knee joint dissections revealed advanced osteoarthritis. Glenohumeral and acetabulofemoral joint cavities and their surrounding bursae were explored bilaterally, with the only significant finding being bilateral glenohumeral osteoarthritis. Histological studies supplemented by a review of the literature suggests a diagnosis of secondary synovial chondromatosis.DiscussionSynovial chondromatosis outlines a rare, benign process characterized by the metaplasia of synovial tissue into chondrocytes within joint cavities, tendinous sheaths, or bursae. It can be further classified into one of two categories: primary, where the proliferation of hyaline cartilage in synovial membranes is seemingly spontaneous, or secondary, in which osteocartilaginous bodies are generated as a result of trauma or inflammatory joint pathologies. The condition most often affects males, has a monoarticular presentation, with a proclivity for the knee joint, followed by the shoulder and hip. Cases in the ankle, elbow, wrist, and temporomandibular joints have also been described. Although it is classified as benign and non‐metastatic, pain, swelling, and functional deficits have been reported. Treatment most often consists of surgical excavation of loose bodies with open arthrotomy and synovectomy.ConclusionAnomalies discovered through anatomical dissection provide unique opportunities to research pathological processes that may not otherwise be covered in standard anatomical curricula.