bluuebunny/crossref_metadata_2025 huggingface dataset:
PAR4 Primarily Mediates Thrombin‐induced Morphological Changes in MEG‐01 Cells through PI3K/Akt and RhoA/ROCK Signaling Pathways
Authors: Heo Yunkyung , Namkung Wan
Published: May 2022

Platelet activation is critical in hemostasis and thrombosis. Thrombin most potently activates platelets via two protease‐activated receptors (PARs): PAR1 and PAR4. While PAR1 activation results in a rapid and transient signal required for the initiation phase of platelet aggregation, PAR4 activation induces a sustained signal associated with later phases responsible for stable thrombus formation. The present study focused on the differential signaling pathways by PAR1 and PAR4 involved in thrombin‐induced intracellular events in human megakaryoblastic leukemia cell line, MEG‐01. Interestingly, we found that thrombin triggers not only Gq/PLC‐mediated calcium signaling, but also G12/13/RhoA/ROCK‐mediated morphological changes in MEG‐01 cells via PAR1 and PAR4, which showed close similarity to those observed in platelets. We developed a new image‐based assay to quantify the morphological changes in living cells and extensively investigated on the underlying mechanism for thrombin‐induced morphological changes in MEG‐01 cells. Selective blockade of PAR1 and PAR4 by vorapaxar and BMS‐986120, respectively, indicated that PAR4 activation primarily mediates thrombin‐induced morphological changes in MEG‐01 cells. Moreover, treatment of a set of kinase inhibitors showed that the PAR4‐mediated morphological changes were predominantly mediated via PI3K/Akt and RhoA/ROCK signaling pathways. As in the platelets, PAR4‐mediated signaling pathways are significant in thrombin‐induced morphological changes in MEG‐01 cells. Based on the close similarity to thrombin‐induced signaling in platelets, MEG‐01 cell line is a useful complementary in vitro model that can be used in research on platelets and thrombosis.
A biochemistry platform to incorporate replication forks, DNA lesions, and nucleosome arrays to single‐molecule experiments
Authors: Lin Jason , Simonetta Marco , Gates Evan , Dandpat Shiba , de Groot Mattijs , Groen Bas , Simpson Trey
Published: May 2022

Fundamental biological processes such as DNA replication, DNA repair, and chromatin organization are precisely regulated in space and time by sophisticated multiprotein molecular machinery. The high spatiotemporal resolution of optical tweezers combined with single molecule fluorescence microscopy enables real‐time visualization of these processes while simultaneously measuring their enzymatic activity via detection of mechanical manipulation of the DNA substrates. Our biochemistry platform provides a set of tools to incorporate replication forks, DNA lesions, and specific sequences (e.g. nucleosome arrays) in DNA molecules tethered between two beads of the optical tweezers. Importantly, our tools also allow labeling of exact positions within these DNA substrates with fluorophores. This enables precise localization of the imaged proteins in the DNA sequence during processes including DNA scanning, as well as recognition and enzymatic manipulation of specific DNA structures or lesions. Our next developments aim to combine multiple DNA features in a single DNA molecule, such as a replication fork in the presence of DNA lesions or in the context of a nucleosome array. These advances will allow more complex experimental set ups and increase the capability to mimic cellular biological processes at a single molecule level.
Measuring Cytotoxicity of the Designed Antimicrobial Peptides DesHDAP1 and DesHDAP1‐C on Human Embryonic Kidney Cells
Authors: Hu Anna E. , Darling Louise E. , Elmore Donald E.
Published: May 2022

Antimicrobial peptides (AMPs) have emerged as a promising alternative to traditional antibiotics, particularly in light of multi‐antibiotic resistant strains of bacteria. Histone derived AMPs (HDAPs) are an integral part of this toolset, with multiple mechanisms of action, strong antimicrobial effects, and protein design potential. Our lab has previously designed two robust antimicrobial peptides: DesHDAP1 and the subsequent DesHDAP1‐C, which has an additional cysteine residue to facilitate conjugation to other molecules for fluorescence imaging or attachment to active moieties. Previous work demonstrated that the cysteine mutation did not alter antibacterial activity of the DesHDAP1 peptide. However, it is also critical to determine whether this change affects potential toxicity against eukaryotic cells. Using dye‐based assays, we quantified cellular metabolism of human embryonic kidney cells as a proxy for cell viability. By optimizing this process and the data analysis, I was able to compare cytotoxicity of the two designed peptides to that of conventional antibiotics. This approach can be applied to other designed peptides, providing a blueprint for future toxicity studies on eukaryotic cells.
The Influence of Identified Key Residues on Catalytic Activity and Substrate Specificity of Taurocyamine Kinases from Arenicola brasiliensis, An Enzyme in the Phosphagen Kinases Family
Authors: Kelly Omar , Snider Mark , Fraga Dean
Published: May 2022

Tauracyamine kinases (TKs) are part of the phosphagen kinase family which act as an energy buffering mechanism in some invertebrates and unicellular organisms by catalyzing the transfer of phosphate from ATP to guanidino containing compounds, such as arginine, creatine and tauracycamine. TKs utilize tauroayamine as their primary substrate but many TKs exhibit promiscuity in substrate specificity and can also phosprylate glycocyamine or lumbricine. Multiple amino acid residues have been identified as key residues for substrate binding and/or specificity in the phosphagen kinases; specifically, amino acids residue 62‐72 have been identified as key residues within the guanidino substrate (GS) loop that play an important role in substrate binding and preference. Previous work has shown that a single substitution in this loop can ‘flip’ substrate specificity from tauracyamine to glycocyamine although with a significant drop in activity. Using site‐directed mutagenesis we have created additional substitutions in the GS loop to see if they can improve overall activity for the enzyme while still maintaining a preference for glycocyamine. We will be describing our kinetic analysis (KM, kcat, and kcat/KM) of each of the variants and the wildtype TKs to help determine whether the residues mutated are critical in the determining the enzyme’s preference and overall activity with the two substrates, glycocyamine and tauracyamine. This may lead to insight into how different substrate preferences may have evolved.
How to Make a Vampire: Part II. Getting Ready to Feed
Authors: Prufrock Kristen A. , DeLeon Valerie B. , Smith Timothy D.
Published: May 2022

The mammalian feeding system is made up of multiple components that influence facial variation, including the masticatory muscles, bony jaw apparatus, and teeth. However, it is not fully understood how the development of these components are coordinated and are impacted by life history and diet.

Bats are the only mammals that continue to nurse until they are almost adult size, and often after they become volant. Common vampire bats (Desmodus rotundus) have a relatively prolonged juvenile period among bats, can nurse up to nine months, and later solely subsist off of blood. Feeding system maturity may therefore be delayed in bats, and particularly delayed in Desmodus, as a result of their lengthy juvenile period and liquid diet. In this study, we examine the developmental maturity of the cranium and feeding apparatus in the late fetus of a common vampire bat (Desmodus rotundus) relative to an adult (its mother).

The heads of each specimen were microCT scanned twice. Conventional microCT was used to image mineralized tissues. Diffusible iodine‐based contrast‐enhanced CT (diceCT) imaging was used to image soft tissues. Cranial length and palate length measurements were acquired from unstained scans to evaluate size and jaw apparatus development. Masticatory muscles (e.g., medial pterygoid, zygomaticomandibularis) and permanent tooth crypt volumes were acquired from stained tissue scans to evaluate masticatory muscle and tooth development. Developmental maturity of these structures was assessed by calculating a percentage of the growth completed (i.e., [fetal muscle volume/adult muscle volume]*100).

In the fetus, permanent tooth crypt volume is already comparable to the adult condition despite cranial length and palate length being <75% of adult size. Masticatory muscle volume maturity is highest in the lateral pterygoid muscle (~35%), whereas other masticatory muscles are far less developed (e.g., medial pterygoid volume maturity, ~20%; temporalis profundus volume maturity, ~20%; temporalis medius volume maturity, ~6%).

These results reveal highly precocious permanent tooth development in Desmodus. Although this may facilitate transition to hematophagy, Desmodus do not fly until eight to ten weeks, so the precocious incisors may primarily aid the infant to grip its mother’s skin. Differential development of the masticatory muscles may also be influenced by the need for young bats to use their mouths to help anchor themselves to their mother early in life. This may explain the advanced maturity of the lateral pterygoid muscle, which would protrude the jaw and help to facilitate a more secure grip.
Come Roll with Me: Integrating Disability Awareness and Interprofessional Learning into a Medical Musculoskeletal Course
Authors: Brooks William S. , Carver Cathy , Lein Donald , Parish Ashley , Wylie Elizabeth
Published: May 2022

One in four Americans are affected by a disability making people with disabilities (PWD) the largest minority group in the United States. PWD experience a variety of social, economic and structural barriers that limit access to medical care and create significant health care disparities. While PWD visit health care providers at a higher frequency than those without disabilities, half of U.S. medical schools lack a disability awareness program, and 58% of medical school deans reported that a curriculum for patients with disabilities was not a high priority. These factors have led to a sense of unpreparedness amongst medical students in providing care for this population. In order to address these deficiencies, a new educational activity, Come Roll with Me (CRWM), was integrated into the Musculoskeletal & Skin course within a Southeastern allopathic medical school in 2021. Authors hypothesized that CRWM would increase disability awareness amongst medical students and enhance attitudes towards interprofessional practice. Come Roll with Me paired PWD, specifically power and manual wheelchair users, with small groups of second‐year medical and Doctor of Physical Therapy (DPT) students for a two‐hour session. Groups rotated through four stations including (1) a self‐propelled wheelchair course within the students’ educational building, (2) a discussion about accessible parking and transportation at the wheelchair users’ vehicles, (3) a skill‐development session on transfers taught by DPT students, and (4) an open dialog regarding barriers to healthcare experienced by PWD. Come Roll with Me was assessed via course evaluations and student reflection essays. Additionally, students were invited to complete the Interprofessional Attitudes Scale (IPAS) before and after the course. The study was approved by the UAB Institutional Review Board. CRWM was the highest rated instructional session in the Musculoskeletal and Skin course with a mean Likert‐scale score of 4.59/5.0 (N=109). Thematic analysis of reflection essays revealed newfound awareness of difficulties navigating a building in a wheelchair and lack of accessibility in doors, bathrooms, etc. Students expressed increased comfort in engaging with PWD and a desire for advocacy on their part. Medical students’ attitudes towards interprofessional collaborative practice improved with significant increases in the mean Teamwork, Roles, Responsibilities (39.3 vs. 42.0) and Interprofessional Biases (8.5 vs. 8.9) IPAS subscales (p<0.01). Come Roll with Me represents a novel interprofessional and patient‐centered method of embedding disability awareness and health care disparities within the preclinical medical curriculum. Integration of the basic sciences with patient encounters and social aspects of medicine are essential in the training of physicians who are competent to care for a diverse patient population.
UWRF TCIC/Century College 2021 Biofabrication Internship Program: A Regional Undergraduate Campus Collaboration Focused on Bioprinting Tumor Models to Study Cancer Biology
Authors: Lyden Timothy , Jaskens Randy , LeBeau Michelle , Thrun Lori
Published: May 2022

Since Fall of 2018 the UWRF Tissue and Cellular Innovation Center (TCIC) and the Century College Biofabrication Program have been engaged in a growing cross‐institutional and cross‐border collaboration effort. This effort includes both academic and research components and reached new levels this past year in the Spring and Summer of 2021 when the TCIC hosted 5 Century College Biofabrication students in a 10‐week research‐based internship. This was followed by 2 students continuing for another 10 weeks through the summer extending and expanding our collaborative research project. The main focus of this work was to develop bioprinting as a new tool at the TCIC for the study of in‐vitro 3D artificial tumor and stromal tissues. This work successfully expanded our working library of methods and techniques for generating artificial tissues while addressing and overcoming several limitations we have previously encountered. Among these have been the relative quantification and distribution of loaded cells within or on previous matrix materials. Over the past two decades, the TCIC has successfully developed and expanded several in‐vitro cancer modeling strategies using combinations of natural decellularized/decalcified ECM materials and various formats of hydrogels made of chitosan and hyaluronic acid (HA) as well as no added matrix constructs in hanging‐drop and rocker cultures. With the exception of the hanging‐drop cultures, precision in loading cell densities as well as final cell distribution have always been relative with significant margins for uncontrollable variations. With this new bioprinting approach we were able to predictably and repeatedly generate 3D artificial melanoma and breast cancer constructs containing defined numbers of cells per unit volume and a relatively precise, even distribution of cells throughout these structures. During this internship and then the research project itself, Century College students gained a wide range of experience and training in basic cell culture methods from aseptic techniques to generating hydrogel‐based 3D constructs and then beyond to bioink printed disks and cups as well as several approaches to cellular analysis. By moving to develop this new TCIC/Century College collaboration, we are increasing the reach of both institutions and the access for our respective students to new cutting‐edge tissue engineering technology as they prepare for future careers in the growing biomedical and life‐sciences sectors of both our respective states.
The Research‐Intensive Community Model has the Necessary Properties for Successful Propagation at Research‐Extensive Universities
Authors: Quick Christopher M. , McNeely Andrew C. , Gatson Sarah N.
Published: May 2022

Novel research education models often fail to propagate widely because they typically only include two of four necessary properties to successfully propagate and maintain their impacts: scalability, adaptability, sustainability, and inclusivity. Furthermore, research education models typically address the unmet needs of one stakeholder at a time. First, undergraduates need greater access to limited research opportunities at research‐extensive universities. Second, graduate students and postdoctoral scholars need mentoring and management experience to prepare for leadership roles in the next stage of their careers. Third, faculty need to maximize research productivity and recruit future graduate students with sufficient preparation. Fourth, administrators need to stretch their limited resources to support the competing institutional missions of research and education. The Research‐Intensive Community (RIC) model developed at Texas A&M University has the properties that are both necessary and sufficient to propagate broadly at research‐extensive universities because it turns the competing needs of diverse stakeholders into opportunities. Briefly, instead of a traditional 1‐on‐1 research apprenticeship, research is performed by a diverse undergraduate research team led by a graduate student or postdoctoral scholar. Research teams are productive because they leverage each undergraduate’s unique assets (talents, skills, perspectives, and experiences) to advance the research of the graduate student or postdoctoral scholar. Team leaders meet monthly to discuss best practices for leadership and mentoring that increase their own research productivity through effective team management. Because team leaders are empowered to run their team by their faculty mentors, economies of scale are leveraged to efficiently centralize program marketing, participant registration, project advertisement, and program evaluation. Since 2016, the Aggie Research Program (ARP) has implemented the RIC model to cultivate mutually beneficial relationships to meet the diverse needs of diverse stakeholders. As the ARP has grown, team leader meetings have become a nexus for specific research leadership programs that are either interdisciplinary or focused on specific life science disciplines. The ARP exhibits the necessary and sufficient properties for successful propagation: 1) Scalability: participation has grown 30% each of the first 5 years to currently serve over 800 participants/year 2) Adaptability: research teams are distributed across diverse disciplines in 12 colleges. 3) Sustainability: administrative costs are limited to $50,000/year. 4) Inclusivity: 41% of participants belong to underrepresented groups (financially disadvantaged, first‐generation, disabled and underrepresented minorities), which matches the demographics of the undergraduate student population at Texas A&M University.
Effect of Live Dissection Versus Virtual Anatomy Lab on Student Learning in a Neuroanatomy Course
Authors: George Ian , Koontz Alexandra , Roggy Connor , Moffett Elizabeth
Published: May 2022

With the onset of the COVID‐19 pandemic safety measures such as social distancing was implemented to protect against transmission of the virus. Student cadaveric dissection as part of a medical gross anatomy curriculum was one of the in‐person experiences that Rocky Vista University moved to a virtual lab format in response. Live streamed prosection demonstrations over the Zoom platform allowed students to view relevant anatomy, ask for additional views, and have questions answered. Anatomy lab evaluations were also virtual through the ExamSoft platform consisting of static pictures of dissected cadavers. We looked at student performance in the Neurosensory I System, which covers the anatomy of the head, neck, and central nervous system. We saw a significant decrease in student performance on anatomy practical exams after learning via the virtual format, even after assessment changes to match were made. These results support previous research demonstrating the value of cadaveric dissection or prosection style gross anatomy labs. While a virtual lab was acceptable during a public health emergency, it is not ideal for proper education in the structure and function of the human body.
Functional Dynamics of Hsp70/CHIP/E2‐Ub Complex in Ubiquitination of Substrate Proteins
Authors: Paththamperuma Chathura , Zhang Huaqun , Page Richard
Published: May 2022

Heat shock protein (Hsp) 70 is the main mediator of protein quality control in the cytosol of eukaryotic cells. One of the major functions of Hsp70 is mediate the ubiquitination of client proteins which subsequently leads to proteasomal degradation. This process occurs via a multiprotein complex composed of Hsp70, carboxy terminus of Hsc70 interacting protein (CHIP), ubiquitin‐conjugating enzyme (E2), and ubiquitin (Ub). This complex is thought to be flexible, however, knowledge of the degree of dynamics are still lacking and detailed experimental evidence is needed. Here we present the models derived from Electron Paramagnetic Resonance (EPR) data and small‐angle X‐ray scattering (SAXS) ensembles for the complexes of Hsp70/CHIP/E2‐Ub with ATP or ADP. The model of ATP bound Hsp70/CHIP/E2‐Ub complex has an average distance of ~120 Å between the substrate‐binding domain (SBD) and the E2‐ubiquitin active site. The same distance of the multiprotein complex is found to be decreased to ~60 Å when Hsp70 is bound to ADP. Since the ADP bound conformation is considered as the active conformation that tightly binds client proteins, the alteration of the average distance between the Hsp70 SBD and the E2‐Ub active site implies that the Lys residue of the substrate might need to be positioned within appropriate distance to span the gap in order to efficiently promote ubiquitination. Here, we used molecular dynamic simulations, double electron‐electron resonance (DEER) EPR spectroscopy and in‐vitroubiquitination assays with designed model substrate proteins that place lysine residues at variable positions. We found the dynamics of the Hsp70/CHIP/E2‐Ubiquitin complex is playing a major role in the ubiquitination of various client proteins and these dynamics are necessary to overcome the distance barrier between E2‐Ub active site and the Hsp70 SBD. Our data also suggests that the Hsp70/CHIP complex is capable of ubiquitinating a client protein substrate when lysine residues are placed at several disparate positions. In sum, our data reveals the functional dynamics of the Hsp70/CHIP/E2‐Ub complex which enable ubiquitination of a client protein.
Sexually dimorphic role of smooth muscle cell estrogen receptor alpha in aging‐associated arterial stiffness
Authors: DuPont Jennifer , Vorn Jennifer , Lu Qing , Duffy Colleen , Martin Gregory , Hill Michael
Published: May 2022

Background

Aging is associated with rising arterial stiffness (AS), an independent risk factor for cardiovascular disease. The development of aging‐associated AS follows a sexually dimorphic pattern with women developing AS later in life. Estrogen and estrogen receptor alpha (ERα) are protective in the premenopausal state, potentially due to the regulation of the vasoprotective angiotensin II type 2 receptor (AT2R), however, the role of estrogen/ERα in aging‐associated AS has not been defined and the mechanisms of the sexually‐dimorphic development of aging‐associated AS are unknown.

Methods/Results

All studies were conducted in accordance with the FASEB Statement of Principles for the use of Animals in Research and Education. We generated novel smooth muscle cell‐specific ERα‐KO mice and measured aortic pulse wave velocity (PWV), an index of AS, in 3, 12, and 18 month old male and female intact and KO mice. Male mice develop increases in AS from 3 to 12 months compared to 3 to 18 months in females (p<0.05). SMC‐ERα‐KO protected females from the aging‐associated increase in AS (p<0.05). Circulating estrogens also declined in 18 month old females vs. 3 mo. (p<0.05), suggesting a role for unliganded SMC‐ERα in aging‐associated AS. SMC‐ERα‐KO females were also protected from the aging‐associated decline in aortic AT2R expression observed in intact females (p<0.05). To further investigate the potential role of unliganded SMC‐ERα in AS, we performed ovariectomy (OVX) or sham surgeries in young female SMC‐ERα‐Intact and KO mice and measured PWV. Results demonstrated an OVX‐driven increase in AS that was prevented by SMC‐ERα‐KO (p<0.05). This was also associated with an OVX‐mediated decrease in aortic expression of the AT2R, which was prevented by SMC‐ERα‐KO. To determine the in vivo role of AT2R in the vascular protection observed in 12 month old female mice, we treated 12 month old male and female mice with AT2R antagonist PD‐123319 (3mg/kg/day) or vehicle via osmotic minipumps for 4 weeks. Chronic inhibition of the AT2R increased AS in 12 month old females (p<0.05), but not males, suggesting that AT2R‐mediated protection contributes to the delay in aging‐associated AS in female mice. In vitro studies also demonstrated that estrogen upregulates AT2R mRNA and protein in female mouse aortic SMC, supporting an estrogen‐mediated mechanism of AT2R driven vascular protection in females.

Conclusion

These data indicate sex differences in the time course of the development of aging‐associated arterial stiffness and suggest the presence of unliganded SMC‐ERα contributes specifically to aging‐associated arterial stiffness in females via reductions in vascular AT2R. Further, these results implicate that AT2R agonist therapy may improve cardiovascular outcomes in aging women to retain the beneficial downstream effects of estrogen signaling, without the potential detrimental effects of hormone replacement therapy.
The Effects of Mild Traumatic Brain Injury (mTBI) on Serotonergic Gene Expression
Authors: Cominski Tara P. , Stiritz Victoria , Beck Kevin D.
Published: May 2022

Traumatic brain injury (TBI) is a risk factor for suicide. Approximately 1.7 million TBIs occur each year and new TBI cases in the U.S. military have more than doubled in the last 5 years. The majority of military TBIs are mild in severity and can lead to chronic symptoms including, impulsivity, emotional instability, and impaired inhibition. Impulsivity is one of the most frequent behavioral changes seen following mTBI and involves poor response inhibition and/or poor decision making. Also, impulsivity is a key risk factor for suicide. The serotonergic system is an important modulator of impulsivity and, thus, a molecular target for understanding the behavioral changes in impulsivity that occur following mTBI. One major source of serotonin in the brain is the raphe nuclei in the pons. The serotonergic neurons in the raphe nuclei project to the prefrontal cortex, frontal cortex and hippocampus and are involved in many cognitive processes related to impulsivity and suicidality. In addition, both animal and human research indicates that the brainstem is affected in mTBI, particularly, in the lateral fluid percussion model utilized in this study. This provides additional support for the notion that mTBI may lead to dysfunction of the serotonin producing raphe nuclei located in the brain stem. In this study, changes in serotonergic gene expression in the raphe nuclei and target regions including the prefrontal cortex, frontal cortex and hippocampus were assessed using qRT‐PCR. Rats sustained a single mTBI induced using the lateral fluid percussion model and were sacrificed three months following injury. Brains were extracted, flash frozen, sectioned at 300um and tissue for the raphe nuclei and target regions was collected using a 1.5 mm tissue punch. RNA was extracted from the tissue punches, reverse transcribed into cDNA and qRT‐PCR was performed. The hypothesis states that mTBI leads to altered expression of serotonin related genes in the raphe nuclei and/or raphe nuclei target regions including the prefrontal cortex, frontal cortex, and hippocampus. Results show altered expression levels of tryptophan hydroxylase and serotonin transporter genes in both the medial raphe and dorsal raphe nuclei in mTBI rats compared to SHAM control rats. Changes in expression levels of the 5HT1A and 5HT2A receptor genes were also seen in the frontal cortex, prefrontal cortex, and ventral hippocampus. The current study serves to provide evidence for a link between TBI and altered serotonin levels to better understand the molecular mechanisms underlying impulsivity and suicidality in humans following TBI.
Design of Experiment (DOE) for Optimization of PLGA Nanoparticles
Authors: Gupta Ritu , Xie Huan , Sarkar Mahua , Chen Yuan
Published: May 2022

Design of Experiments (DOE) is useful tool for formulation optimization. In present study, Design Expert® software (version 13) was used for optimization of Poly(dl‐lactide‐co‐glycolide) (50:50) (PLGA) nanoparticles (NPs). Capecitabine, an approved anticancer drug with short half‐life, was used as model drug. Capecitabine NPs can minimize dosing frequency and improve patient compliance. Effect of two numeric (drug and polymer amount) and one categoric factor (pH of W2 phase) was studied on NP Size and Entrapment Efficiency (EE). Drug amount and pH significantly influenced EE. Polymer amount significantly influenced NP size.

Methods

PLGA NPs were prepared by double emulsion technique. Aqueous drug solution (W1) was emulsified with polymer solution in ethyl acetate using probe sonicator (Misonix) for 2 minutes. Formed primary emulsion was sonicated with second aqueous phase (W2) containing stabilizer Kolliphor®P188 for 1 minute to produce double emulsion. Organic solvent and excess water were evaporated at 600 rpm, 60°C for 1.5 hr and NPs were washed thrice (14,000 rpm, 4°C for 45 min) Lyophilized NPs were sonicated with acetonitrile for 5 min and EE was analyzed using UV/Vis spectrometer at ʎmax304nm. Particle size was determined using Malvern Nano‐ZS.

Results

Experimental data were fitted into models of increasing polynomial complexity. EE data is best described by two‐factor interaction model with R² of 0.9145. ANOVA results show model is significant. Predicted R²of 0.8076 is in reasonable agreement with adjusted R² of 0.8824. Drug amount, pH of W2 phase and their interaction are significant (p<0.05). Higher drug amount decreases EE which maybe because polymer amount was not sufficient to encapsulate increased drug amount and more drug migrated to W2 phase. Lower EE at pH 4.5 might be described by drug leaching into W2 phase due to better drug solubility. Diagnostic normal plot, perturbation plots, interaction plots and 3D‐surface plot confirm model robustness. Size data was best fitted to linear model showing polymer amount being significant. Higher polymer amount might have decreased dispersion efficiency of first emulsion into W2 phase and led to fusion of semi‐formed particles to larger size.

Numerical Optimization

The desired goals were set to minimize polymer amount (to increase drug amount per unit weight of NPs), to minimize drug amount and the pH was set at 7.4 to maximize EE. Predicted optimum responses were checked experimentally. Batch prepared with 120mg polymer and 25mg capecitabine has average size of 111.3 nm (predicted value 116.8nm) and EE of 38% (predicted value 40%), both with 5% coefficient of variance.

Conclusion

DOE gives an opportunity to look at individual and combined impact of various factors. It can provide valid conclusions with minimal experimental runs, time, and cost. Similar DOE approaches can be used for other PLGA‐drug formulations optimization.

Conflict of Interest

Authors declare no conflicts of interest.
A Community of Medical Educators Coming together to lend an “Anatomical” Hand: Showing the Value of an In‐Person Anatomy Lab based Curriculum
Authors: Marquez Samuel , Katz Joanne , Pagano Anthony S. , Dannenhoffer Raymond , Smith Michael , Laitman Jeffrey T. , Mtui Estomih P.
Published: May 2022

Anatomy education has suffered greatly during the Covid‐19 pandemic, forcing medical institutions to switch from in‐person to remote learning. This entails sacrifice of the 3D learning component of the laboratory and creates a major experience gap in the curriculum. At the School of Health Professions of SUNY Downstate Health Sciences University, anatomy courses were delivered remotely over the past two summers for the physical therapy, physician assistant, and occupational therapy programs. Student evaluations reveal the perception of their respective cohorts having “lost out” on the value of in‐person experience in the anatomy lab compared to upper classmen who took this course before the pandemic lockdown. To bridge this gap, medical educators came together as a community to offer an in‐person, hands‐on anatomy experience to 94 health professional students. Weill Cornell Medicine hosted the event and provided 20 donor dissection bodies with a total of 16 participating faculty that included anatomy directors from SUNY Albany, University at Buffalo, Icahn and NYU Schools of Medicine, in addition to Emergency department and Rehabilitative Medicine Attendings and former students who are now in private practice. A review of the musculoskeletal system was offered in three distinct categories that involved demonstration of movements, the neurovascular elements involved, and clinicopathologies. The follow up survey reveals positive student responses in all three categories. They also credited the event with providing an appreciation of the three‐dimensionality of the region, citing that remote learning did not offer a full understanding of contiguous anatomical structures in 3D space. What was not measured in the survey were unexpected losses of corollary benefits to other courses that learning human dissection conferred. Program directors reported that some of these students underperformed in their respective programs which was unexpected based on their admissions profile compared to students who had the in‐person anatomy lab experience. What is missing when students do not have a 3‐D appreciation for the musculoskeletal system is their understanding of elements such as direction of muscle fibers (parallel vs pennate), line of pull, and eccentric versus concentric contractions. Human gross anatomy in the laboratory, aside from providing 3D learning, lays the foundation for the problem‐solving skills and professionalism that will be expected of them throughout their health professional careers. Thus, gross anatomy education is not solely a course to “memorize” the names of soft‐tissue structures in 3D space but instead also serves to develop and fortify professional behaviors that will prepare students beyond the laboratory and into the workplace.
Unparalleled Approaches to Directly Visualize DNA‐Binding Proteins and Biomolecular Condensates
Authors: Dandpat Shiba , Gates Evan , Lin Jason , Balassy Zsombor , Simpson Trey , Groen Bas , Driessen Rosalie , Llauró Aida
Published: May 2022

The structure, regulation, and maintenance of DNA are critical to cellular function. Detailed molecular insights into these processes are essential for understanding their associated pathological conditions, establishing and validating biological models, and developing novel therapeutics. As a result, direct, real‐time observations of individual proteins and biomolecular condensates interacting with DNA are needed to obtain a thorough understanding of their mechanisms and dynamics, and to validate and complete the current biological models. Technologies, such as the C‐Trap, offer an unique opportunity to meet these challenges by enabling researchers to observe DNA processes in real‐time at the single‐molecule level. Here, we present our efforts for further enabling discoveries in the field of DNA‐protein interactions using the combination of optical tweezers with correlative confocal fluorescence microscopy. We present several examples in which our technologies enhance the understanding of DNA repair mechanisms, chromatin structure and stability, DNA replication, DNA editing tools (e.g. CRISPR/Cas), and biomolecular condensates. Importantly, we show that advances in single‐molecule technologies can be turned into easy‐to‐use instruments that enable discoveries to all biologists previously reserved for the world’s most specialized biophysics labs. This approach complements current structural or cell biology methods and provides key insights to complete the molecular mechanisms and models describing essential DNA‐binding processes.
Estimation of Shear Stress Heterogeneity along Capillary Segments in Angiogenic Rat Mesenteric Microvascular Networks
Authors: Hu Nien‐Wen , Lomel Banks , Balogh Peter , Murfee Walter L.
Published: May 2022

Fluid shear stress has been identified as an important player in regulating endothelial cell behavior and angiogenesis, defined as the sprouting of new blood vessels from existing vessels. However, the correlative or functional linking of shear stress to vessel‐specific endothelial cell dynamics requires a better understanding of the shear stress values at the capillary level in microvascular networks. Critical questions remain unanswered. What is the shear stress experienced by endothelial cells along capillaries? Do all capillaries experience similar shear stress magnitudes? Can variations in shear stress along different capillary segments explain vessel‐specific responses? The objective of this project was to estimate shear stress values in regions of angiogenic microvascular networks. Rat mesenteric tissues were harvested from adult male Wistar rats post the stimulation of angiogenesis via 48‐80 mast cell degranulation. Microvascular networks were identified by perfusion of a 40kDa fixable dextran prior to harvesting and immunolabelling for PECAM. Dextran presence identified perfused segments along the hierarchy of microvascular networks, which were characterized by increased vessel density indicative of angiogenesis. Two representative network regions with arteriole input and venule output vessels were selected for analysis. Vessel segments were defined by nodes, branch order position, and the measurements of lengths and diameter. Assuming an inlet pressure of 75 mmHg and outlet pressure of 10 mmHg, a network segmental Newtonian flow model was used to computationally estimate vessel‐specific shear stresses and velocities. Viscosity was set to 4 cP and assumed to be constant. For region 1, shear stresses along capillaries (diameters less than 10 microns) ranged from 0.7 to 479 dyne/cm2 (average = 57± 69 dyne/cm2). For region 2, shear stresses along capillaries ranged 0.1 to 1032 dyne/cm2 (average = 75± 94 dyne/cm2). For both regions, the maximum shear stress along capillary segments was greater than the maximum shear stress in the initial arterial branches. The high variability at the capillary level in both network regions suggests the potential for vessel‐specific stimuli. The maximum shear stresses in proximal capillary segments connecting the high‐pressure arterial sides to the lower pressure venous sides in a network were increased compared to stresses for segments in more distal network regions. The results of this study highlight the heterogeneity of shear stress experienced by endothelial cells at the capillary level in angiogenic microvascular networks and motivate questions related to how vessel‐specific hemodynamic environments regulate endothelial cell function.
Novel Therapeutics Modulate Cardiac Leukocyte Populations Following Myocardial Infarction
Authors: Staffenson Melanie , Kumar Sushil , Sepe Joseph , Coussens Lisa , Habecker Beth
Published: May 2022

Myocardial infarction (MI) triggers an inflammatory response that transitions from pro‐inflammatory to reparative over time, a progression that is essential for cardiac healing. Sympathetic nerve loss in the heart after MI predicts the probability of serious ventricular arrhythmias, and restoring nerves prevents arrhythmias. Although noradrenergic transmission is pro‐inflammatory in several contexts, acetylcholine (ACh) can be anti‐inflammatory; and cardiac sympathetic nerves release ACh along with norepinephrine after MI. Recent studies have discovered sympathetic regulation of global inflammatory responses, and our lab has shown previously that treatment with either Intracellular Sigma Peptide (ISP) or the small molecule HJ‐02 following ischemia‐reperfusion (IR) injury restores sympathetic innervation to the cardiac scar and prevents arrhythmias. However, whether sympathetic reinnervation modulates leukocyte homing and function in the heart post‐IR was unknown.

C57BL/6J mice were treated on days 3‐10 after IR with ISP or HJ‐02, and we used quantitative multiplex immunohistochemistry (mIHC) to identify the leukocyte phenotypes present in the heart two weeks following IR. Reinnervated hearts had decreased inflammatory (M1‐like) macrophage levels [Percent of total macrophages ± SEM: VEH, 47.29%±1.446; ISP, 37.97%±1.590; HJ‐02, 29.72%±1.226] and elevated reparative (M2‐like) macrophages [VEH, 52.71%±1.446; ISP, 62.03%±1.590; HJ‐02, 70.28%±1.226]. HJ‐02 stimulated a significantly greater shift from inflammatory to reparative cell types compared to ISP.

However, it is unknown whether it is a direct effect of the treatments on immune cells or the restoration of sympathetic nerves in the infarct causing the changes in leukocyte populations. To answer whether the treatments are the instigator, we isolated and cultured primary peritoneal macrophages from unoperated C57BL/6J mice, treated with ISP or HJ‐02, and examined macrophage marker expression using flow cytometry. Preliminary results suggest that both ISP and HJ‐02 expand isolated peritoneal macrophages in situ, with a predominantly M2‐like phenotype. M2‐like macrophages promote wound‐healing, suggesting these novel therapeutics may be beneficial for the heart after MI.
Myristic Acid‐Trans‐Activator of Transcription Dual Conjugation Improves Intracellular Delivery of Protein Kinase C Beta II Peptide Inhibitor Cargo in Isolated Rat Polymorphonuclear Leukocytes
Authors: Singh Sunit G. , Verwoert Alexis , Nair Arjun , Johnson Devani , Humayun Annam , Clair Logan , DiLisi Taylor , Harrell Kayla , Dean Tameka , Chen Qian , Barsotti Robert , Young Lindon
Published: May 2022

Protein kinase C beta II (PKCβII) activation promotes polymorphonuclear (PMN) superoxide (SO) production by phosphorylating serine and threonine amino acid residues on NADPH oxidase (NOX‐2). In previous studies, cell‐permeable myristic acid conjugated PKCβII inhibitor (myr‐PKCβII‐) significantly attenuated PMN SO release induced by phorbol 12‐myristate 13‐acetate (PMA), a diacylglycerol mimetic. Myr‐PKCβII‐ was determined to be superior to unconjugated peptides and nontreated controls, suggesting enhanced intracellular delivery of cargo. We hypothesize that the simple diffusion of myr‐conjugation combined with the endocytotic mechanism of trans‐activator of transcription (Tat) would optimize the intracellular delivery of PKCβII‐ cargo compared to myr‐conjugation alone.

In this study, we tested the concentration‐dependent effects of a dual myr‐Tat conjugated PKCβII‐ (myr‐Tat‐PKCβII‐; N‐myr‐Tat‐CC‐SLNPEWNET) on intracellular delivery compared to myr‐PKCβII‐, scrambled myr‐Tat‐PKCβII‐ (myr‐Tat‐PKCβII‐ scram), unconjugated PKCβII‐, and 0.5% dimethyl sulfoxide (DMSO) vehicle control group. Rat PMNs were incubated for 15 min at 37°C with either unconjugated PKCβII‐ (20μM), myr‐Tat‐PKCβII‐ (2μM, 5μM, 7.5μM, 10μM, and 20μM), or myr‐Tat‐PKCβII‐scram (2μM, 5μM, 7.5μM, 10μM, and 20μM). PMN SO release was calculated by the change in absorbance at 550 nm over 390 sec via ferricytochrome c reduction after PMA stimulation (100nM). The efficacy of intracellular drug delivery was evaluated by the magnitude of PMA‐induced PMN SO release attenuation with the PKCβII‐ cargo. Data were analyzed with ANOVA Fisher’s PLSD post‐hoc analysis.

Myr‐Tat‐PKCβII‐ 5μM (n=12, 0.392±0.04), 7.5μM (n=11, 0.397±0.05), 10μM (n=5, 0.211±0.05) and 20μM (n=5, 0.121±0.02) demonstrated a concentration‐dependent increase in intracellular delivery compared to DMSO vehicle control (n=84, 0.496±0.02, all p<0.05). Myr‐PKCβII‐ only significantly increased intracellular delivery at the 20μM concentration (n=27, 0.303±0.02, p<0.05) compared to DMSO vehicle control. Intracellular delivery of myr‐Tat‐PKCβII‐ 2μM (n=10, 0.436±0.06) and all concentrations of myr‐Tat‐PKCβII‐scram were not significantly different from DMSO vehicle controls.

Results suggest that myr‐Tat dual conjugation is superior to myr‐conjugation alone at intracellular delivery of cell impermeant cargo. Future studies will investigate the concentration‐dependent effects of PKCβII‐ peptide conjugates on PMA‐induced PKCβII activity and translocation to membrane targets, such as NOX‐2, using immunocytochemistry and western blot analysis.
Development of DNA Aptamers Against Structural Proteins of SARS‐CoV‐2
Authors: Velázquez Roig Rosalba , Rodríguez‐Martínez José A.
Published: May 2022

The severe acute respiratory syndrome‐coronavirus‐2 (SARS‐CoV‐2) is a positive‐sense single strand (ss) RNA virus responsible for causing the COVID‐19 disease. With the continuous incidence of infections by SARS‐CoV‐2 and the appearance of strains that may evade existing vaccines and treatments, it is imperative to implement strategies that allow an effective and rapid diagnosis of this disease. Due to their high‐abundance in viral particles, structural proteins (i.e., nucleocapsid (N) and spike glycoprotein (S)) are logical targets for diagnostic tests. Aptamers are single‐stranded DNA or RNA oligonucleotides, ~100 bases long, that interact with high‐affinity binding and specificity with a wide variety of molecules including proteins. Aptamers can be generated through an in vitro selection process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX). This research aims to use SELEX process to select DNA‐aptamers targeting structural proteins of SARS‐CoV‐2. The developed aptamers resulting from this work will be incorporated on a sensitive and cost‐effective rapid antigen test to detect the presence of SARS‐CoV‐2.
3D mitochondrial remodeling during postnatal heart development
Authors: Kim Yuho , Bleck Christopher K. , Glancy Brian
Published: May 2022

The heart is featured by high mitochondrial volume, by which continuous contractile activity can be maintained. However, it is unclear how mitochondrial structures are matured during postnatal development. Using FIB‐SEM, we thus sought to identify 3D mitochondrial structural characteristics in the heart of mice at postnatal (P) day 1, 7, 14, and 42, respectively. Here, matured mitochondria (P14‐42) were shown to have larger and more spherical structures and were highly connected with adjacent mitochondria as compared to early postnatal days (P1‐7). Nevertheless, the immature mitochondria appeared to have more intense interaction with other subcellular organelles including lipid droplets and sarcotubular structures (i.e., SR/T‐tubules). Thus, our study suggests that initial mitochondrial development can be augmented by crosstalk with adjacent mitochondria as well as other subcellular components and that it would be important for the further mitochondrial maturation process in the heart muscle.
Design and characterization of Eco‐friendly composite for bone tissue engineeing
Authors: Zo Sunmi
Published: May 2022

It has got a lot of attention that a substitute based on calcium phosphate (CaP), an essential component of bone, Among bio‐engineering materials for the development of scaffolds for bone tissue engineering. The methods to obtain CaP‐based ceramics are two ways; chemical synthesis such as hydroxyapatite (HAP) and biological apatite obtained from tooth or bone. The latter are biocompatible, easy to be prepare, owing to be similar with human bones. WPU (waterborn polyurethane), used as a scaffold for bone tissue engineering, is synthesized as polyols and isocyanates, petrochemical. it related with environmental problems. In this study, livestock waste(premolars and molars) were used as bioapatite, and followed by incorporated a CMCS(carboxymethyl chitosan)/WPU support using WPU replaced with a biomass castor oil‐based polyol. And compared with the HAP‐based CMCS/WPU scaffold, the mechanical properties were measured. after seeding osteoblasts, biocompatibility testing was performed.