bluuebunny/crossref_metadata_2025 huggingface dataset:
Association between vitamin D status, cardiometabolic risk factors and metabolic syndrome in Puerto Rican adults
Authors: Palacios Cristina , Perez Cynthia , Guzman Manuel , Ortiz Ana Patricia , Suarez Erick
Published: June 2021

Objective

Vitamin D status has been related to several risk factors in Whites. This pilot study assessed the association between vitamin D status and cardiometabolic risk factors in Hispanics from Puerto Rico.

Methods

Secondary analysis of a household survey with a representative sample of 859 adults of San Juan. Serum 25(OH)D levels were measured with HPLC‐MS and categorized as <30 or ≥30 ng/ml. Age‐adjusted partial correlations were calculated between serum 25(OH)D and blood pressure, HDL‐C, non HDL‐C, LDL‐C, triglycerides, fasting blood glucose, glycosylated hemoglobin, hs‐C reactive protein, fibrinogen and plasminogen. The association between categories of serum 25(OH)D and risk factors was assessed using test‐based methods.

Results

Preliminary results in 91 adults (53 females; 38 males) showed that mean serum 25(OH)D was 36.3±7.5 ng/mL, and it was significantly lower in females (34.8 vs 38.3 ng/mL; p=0.01). Only 17.6% had levels below 30 ng/ml. Serum 25(OH)D was significantly correlated with HDL‐C (r=0.28; p=0.03) and number of metabolic syndrome components in females (r=−0.30; p<0.05) and WHR in men (r=−0.35; p=0.04). Only females with levels <30 ng/ml had higher odds of metabolic syndrome compared to those with levels ≥30 ng/ml (OR=4.93, p=0.03).

Conclusions

These findings suggest that low serum 25(OH)D is associated with increased odds of metabolic syndrome in females.
Effects of high‐dose antenatal 3rd‐trimester vitamin D supplementation (35,000 IU/week) on maternal and newborn vitamin D status: a randomized placebo‐controlled trial in Dhaka, Bangladesh
Authors: Roth Daniel E , Al Mahmud Abdullah , Raqib Rubhana , Baqui Abdullah H
Published: June 2021

Maternal‐infant vitamin D status in South Asia may be suboptimal. Yet, high dose prenatal vitamin D supplementation dose regimens have not been widely studied. In a double‐blinded randomized controlled trial in Dhaka, 160 pregnant women were assigned to vitamin D3 35,000 IU/week (VD) or placebo (P) from 26–29 weeks gestation to delivery. The primary biochemical efficacy outcome was serum 25‐hydroxyvitamin D concentration (25(OH)D) and the primary safety measure was maternal serum calcium. Mean maternal 25(OH)D was similar in the VD and P groups at baseline (45 vs. 44 nmol/L; P=0.66), but significantly differed at delivery (134 vs. 39 nmol/L, P<0.001; N=133). Women were more likely to attain >=50 nmol/L and >=80 nmol/L at delivery with VD (100% & 97%, respectively) vs. P (22% & 5%, respectively). Cord 25(OH)D was significantly higher with VD vs. P (103 vs. 39 nmol/L; P<0.001; N=132), and significantly more newborns had cord 25(OH)D>=50 nmol/L with VD vs. P (95% vs. 19%; P<0.001). VD did not cause documented maternal hypercalcemia or other supplement‐related serious adverse events. In conclusion, prenatal 3rd‐trimester supplementation with 35,000 IU vitamin D3/week prevented perinatal vitamin D insufficiency and was well tolerated. Further trials are needed to determine if increases in 25(OH)D due to high‐dose VD supplementation are safe and lead to maternal‐infant health benefits.

Support: Thrasher Research Fund
Strictly regulated blood glucose oscillations linked to rat mortality after severe burn injury
Authors: Zhang Xing , Cai Xiaoqing , Li Jia , Yan Wenli , Gao Feng
Published: June 2021

Aim

To investigate the relation of acute hyperglycemia to rat mortality after severe burn injury.

Results

Strictly regulated blood glucose oscillations were found in rats after severe burn injury (full‐thickness third‐degree burn injury comprising >40% of total body surface area). Within 12 h post burn injury, two peaks of blood glucose were detected at 30 min (increased 13%, p<0.05) and 3 h (increased 35%, p<0.01) respectively, coupled with increased serum insulin, glucagon and corticosteroid levels. More importantly, the second peak of blood glucose was in a highly linear relation to the first one (r2=0.78). Blood glucose of rats dying within 12 h was significantly higher in both peaks compared with those still alive after 12 h (p<0.05), while no peaks has been found in rats within 12 h after slight burn injury with extremely low mortality. In addition, early insulin treatment (2.5 IU/kg, immediately after burn injury) which suppressed the first peak and then removed the second one, largely decreased mortality via enhancing insulin sensitivity.

Conclusion

These strictly regulated blood glucose oscillations are highly related to rat mortality after severe burn injury. Early insulin treatment which suppresses blood glucose oscillations exerts protective effect.

Supported by the State Key Program of National Natural Science Foundation of China (No. 81030005)
Loss of β‐catenin in hepatocytes leads to paradoxical increase in DEN/PB induced HCC
Authors: Awuah Prince
Published: June 2021

Activating β‐catenin mutations are implicated in 30% of hepatocellular cancers (HCC). Here, we utilized hepatocyte‐specific β‐catenin conditional knockout (KO) mice to address hepatocarcinogenesis in response to diethylnitrosamine (DEN) and phenobarbitol (PB). PB is a tumor promoter administered after DEN & has been shown to confer growth advantage to β‐catenin mutated cells during hepatic tumorigensis. Male KO (n=9) and littermate controls (WT, n=15) were followed for 8 months after DEN/PB treatment at which time they were sacrificed. Paradoxically, a significantly higher tumor burden was observed in KO mice after DEN/PB (p<0.05), which was similar to previous findings in the KO mice administered DEN only. Histology revealed an increased cell death, increased hepatic fibrosis that was accompanied by stellate cell activation and enhanced cell proliferation in KO only. The tumors in KO were β‐catenin and glutamine synthetase negative and unremarkable for HGF/Met, EGFR & IGFR signaling. However, a significant increase in total PDGFRα along with Y720‐PDGFRα (p<0.05) was identified in tumor bearing KO versus tumor bearing WT mice. Thus, HCC in response to DEN/PB in mice lacking β‐catenin in hepatocytes occurs due to enhanced cell death, fibrosis and ensuing regeneration that appears to be driven by PDGFRα activation. PDGFRα has been independently implicated in experimental and clinical HCC. Thus, β‐catenin may be responsible for maintaining hepatic homeostasis and its loss leads to aberrant cellular and molecular events, especially after genotoxic stress and this may culminate into hepatic growth dysregulation. Funded by CATER fellowship (NIH T32 EB001026‐05) to PA; 1R01DK62277 and 1R01CA124414 to SPM
Bringing “PhUn” into the Community in Burlington, North Carolina
Authors: Uno Jennifer K.
Published: June 2021

Service learning connects academic content to community‐based projects. Research on service learning pedagogy documents the many benefits that students receive from community activities and recent work supports the idea that service learning can increase the students’ learning of course material. Using the APS sponsored PhUn week as a platform, undergraduates engaged in community outreach aimed at teaching k‐12 children basic physiology of the heart and bones. The goal was to increase critical thinking, communication and global understanding of physiology using community based service learning to reinforce material covered in class. Students were asked design and develop age appropriate physiology modules with clearly stated learning objectives. Mock‐lessons were used to test modules with fellow students prior to community activities were implemented. Following PhUn week activities, students were asked to reflect upon their experiences. Both the development of the physiology modules and teaching proved to be a positive experience for the undergraduate students as well as the k‐12 children that had the opportunity to participate in hands‐on physiology activities.
Integrating emerging technologies into formal education for assessment
Authors: Clase Kari Lynn , Halverson Kristy , Rickus Jenna , Heyden Robin
Published: June 2021

Our project used an interdisciplinary approach integrating computer science and biology to explore the use of emerging technology in the classroom and address the following research questions:

How do college students represent a genome when provided interactive instruction via a virtual world environment (VWE)?

How do students react to using a VWE as an instructional intervention for improving visual comprehension of a genome?

We integrated interactive virtual world learning activities into an interdisciplinary classroom composed of students from multiple STEM majors. Each student engaged in activities related to visualizing genomes, kept records of their experiences in an electronic lab notebook, and posted about their experiences in a blog. Following this course, we interviewed students to understand how they learned to represent a genome and assess their interaction with the VWE.

We found that the VWE offered new ways to explore questions of representation that were transparent in the types of representations students later generated. The collaborative and social environment also contributed to student learning. In conclusion, our investigation provides evidence that learning outcomes valued in science education can be taught through VWE.

This research was supported by a visionary Grant from the Gordon Research Conference on Visualization in Science And Education (2009) and by the HHMI SEA.
Localization of connexin 43 in mitochondria of ovarian cancer cells
Authors: Lee Vaughan , Sun Jingfen , Linch Charles
Published: June 2021

Connexin 43 (Cx43) is a gap junctional protein typically known for its channel forming role in intercellular communication between many types of cells. It has been reported that Cx43 proteins are localized in mitochondrial membranes from cardiovascular cells, Sca‐1+ stem cells, and rat brain tissues. In those various cell types Cx43 seemingly has a cytoprotective role during physiological stress. The present studies were undertaken to determine whether Cx43 protein is present in mitochondria and could possibly play a similar role in human ovarian cancer cells (OVCAR‐3 and SK‐OV‐3 cell lines). Western blot analysis demonstrated that the relative amount of Cx43 in mitochondrial pellets was enriched over that in other fractions. This was validated by comparison with patterns of established mitochondrial proteins such as voltage‐dependent anion channel and cytochrome c oxidase subunit IV. Immunogold transmission electron microscopy confirmed localization of Cx43 in the mitochondria. Additional findings that the level of mitochondrial Cx43 channel protein was specifically reduced with induction of apoptosis by etoposide are compatible with the possibility that it has a role in mitochondrial homeostasis in these ovarian cancer cells. This project was funded by the TTUHSC Department of Cell Biology and Biochemistry and the South Plains Oncology Consortium.
3‐dimensional imaging and quantification of cell adherence on curved surfaces of medical grade devices using confocal lasers‐canning microscopy
Authors: Prater Keith Eric , Prater Roni D , Biggerstaff John P
Published: June 2021

3‐D confocal laser‐scanning microscopy is an ideal way to visualize a sample with a curved surface. An application of this method involves the quantification of activated blood leukocytes and platelets adhering to the surface of medical grade catheters. Human blood was perfused across treated and untreated catheters using a microperfusion system and fixed with paraformaldehyde. Portions of each catheter were immunostained and 5 random fields were imaged using a Leica SP2 microscope (20× objective; 2× optical zoom). Image stacks were loaded into image analysis software and transformed into a max intensity projection. From this 2‐D projection, area occupied by fluorescence was calculated by implementing a binary threshold layer above isotypic control. Area fluorescence was divided by the mean area of a single cell, generating a cell count per unit area. Results are presented as adherent cells/mm2 ± SEM. The treated surface showed reduced adherent cell counts (monocytes: 0.67 ± 1.03, polymorphs: 1.33 ± 3.99, lymphocytes: 0.62 ± 1.86, platelets: 1.33 ± 3.99) compared to the untreated surface (monocytes: 302.43 ± 215.55, polymorphs: 1647.56 ± 615.54, lymphocytes: 38.46 ± 29.59, platelets: 4076.23 ± 2867.3). This method can be used to capture and quantify large amounts of data that may be obscured or otherwise unavailable when imaging curved surfaces, such as in biocompatibility assessment of medical devices.
Glutamatergic Neurons of the Rat Epicardial Ganglia
Authors: Wang Ting , Miller Kenneth E.
Published: June 2021

Some epicardial ganglion neurons may have a sensory function, transmitting information from the heart surface to the ganglia. In dorsal root ganglia, all somatic and visceral sensory neurons are glutamatergic, therefore, we asked if glutamatergic markers could identify sensory neurons in the rat epicardial ganglia. We examined vesicular glutamate transporter 2 (VGLUT2) and glutaminase (GLS) immunoreactivity in the rat epicardial ganglia. Tissue sections of epicardiac fat and atria were processed for VGLUT2 or GLS immunoreactivity along with peripherin. Immunoreactive (ir) VGLUT2 and GLS neurons were present in epicardiac ganglia and contacted by VGLUT2 and GLS ir nerve fibers. VGLUT2 and GLS nerve fibers occurred in adjacent atrial myocardium. Cell bodies and nerve fibers were peripherin ir. These results indicate that many rat epicardiac ganglion neurons are glutamatergic in nature. They contain GLS, enzyme for glutamate production, and VGLUT2, transporter for glutamate storage. Investigation of the glutamatergic phenotype of epicardiac ganglion cells may help in understanding the function of epicardial neurons in physiological and pathological conditions.

Grant Funding Source : NIH AR047410
Right aberrant subclavian artery in a 12‐year old with dysphagia and thoracic outlet syndrome (TOS)
Authors: Collins James D. , Saxton Ernestina H. , Gelabert Hugh , Ahn Samuel S. , Carnes Alfred
Published: June 2021

A left aortic arch with aberrant right subclavian artery is the most common congenital aortic arch anomaly.

We have demonstrated aberrant right and left subclavian arteries on MRI of the brachial plexus in adults presenting with symptoms of TOS: arm and hand pain with paresthesias; hand muscle weakness, headache; autonomic vascular and temperature changes; whooshing sounds in the ear, ear pressure pain; jaw pain, and visual “floaters”.

A 12‐year old girl presented with complaints of problems swallowing solid foods since age seven years. She recently developed pain in the right chest wall with inspiration, in addition to common TOS complaints of headache, dizziness, hand pain, tingling and numbness, muscle pains in the neck and shoulders and back, along with jaw pain and whooshing sounds in the ears.

Bilateral MRI/MRA/MRV display venous and neurovascular compression in TOS patients without the use of contrast material. Bilateral multiplanar imaging was conducted on a 1.5 Tesla GE Signa LX (Clin. Anat.1995; 8:1–16), with the field of view modified to include the dural sinuses and demonstrated a right aberrant subclavian artery with a left aortic arch and displacement of the esophagus but no significant tracheal displacement, as seen in adults. Management of these patients will be discussed.
Correlation between undergraduate anatomy & physiology and graduate anatomy outcomes: defining predictors of academic success of PA students
Authors: Hankin Mark , Gardner April , Lane Richard , Bennett-Clarke Carol , Hogue Patricia
Published: June 2021

The University of Toledo Physician Assistant Studies (UT PA) program matriculated its first class in 1996. Admission to the UT PA program requires minimum “B” grades in undergraduate prerequisite courses, including Human Anatomy and Physiology (A&P). The UT PA curriculum includes a traditional medical gross anatomy and neuroanatomy course that consists of 120 hours of instruction, including classroom lectures and laboratory sessions. This course is taught by Anatomy faculty in the College of Medicine. Despite success in prerequisite courses, some students struggle to succeed in the UT PA anatomy course. To understand this apparent disconnect, this study uses a series of quantitative and qualitative measures to examine the relationship between success in undergraduate A&P courses and performance outcomes in the UT PA program (e.g., grades in anatomy, clinical medicine, pharmacology, and pathophysiology, and performance on the Physician Assistant National Certification Examination). The goals of this study are to identify factors that determine the success of UT PA students and to establish a set of evidence‐based predictors that will allow the selection of the best candidates for admission to the program.
Helping students help themselves: evaluating the effectiveness of undergraduate study techniques in human anatomy
Authors: Eleazer Courtney , Scopa Kelso Rebecca K.
Published: June 2021

A significant challenge of teaching undergraduate anatomy is effectively communicating to students the amount of time and effort required to perform well in such a rigorous course. While the effectiveness of study methods vary individually, it would be beneficial to guide students in maximizing their study time with the best techniques for learning the course material. In some cases, poor performance might be avoided by providing students with data from previous semesters demonstrating study methods that are associated with the retention of anatomical knowledge. Questionnaires (n=183) were administered to students enrolled in undergraduate anatomy in the Fall of 2011 at the University of Tennessee, Knoxville. This survey asked students about the study methods they used (e.g., flashcards, textbooks, diagrams), as well as time spent studying for class. These methods were correlated with scores from exams taken throughout the course. Results show that course performance was not correlated with any particular study technique but was significantly and positively correlated with the total hours spent studying. In addition, students who altered their study habits after the first exam exhibited the greatest improvements in performance. This indicates the benefit of encouraging students early in the course to utilize a greater variety of study methods and invest more time studying than they have in prior courses.

Grant Funding Source : NA
Sumoylation Regulates Multiple Transcription Factors to Control Lens Differentiation
Authors: LI David , Gong Lili , Ji Weike , Deng Mi , Hu Xiao-Hui , Hu Wenfeng , Chen Peichao , Hu Shujun
Published: June 2021

The mammalian small ubiquitin‐like modifier SUMO contains three major isoforms that can be conjugated to a board range of substrates. However, whether SUMO1 and SUMO2 have similar or different roles in regulating differentiation remains unknown. Using vertebrate lens as a testing system, we demonstrate here for the first time that SUMO1 and SUMO2 display distinct roles in mediating lens differentiation. First, SUMO1 is more abundant than SUMO2 with distinct cellular localizations and conjugation patterns during mouse lens differentiation; Second, SUMO1 but not SUMO2 occupied the major crystallin gene promoters. Third, in an FGF‐induced cell differentiation model, increased SUMO1‐ but decreased SUMO2‐modifications were observed in multiple protein species and overexpression of SUMO‐2 but not SUMO‐1 inhibits FGF‐induced cell differentiation. Mechanistically, we demonstrate for the first time that SUMO‐1 and SUMO‐2 differentially regulates Pax‐6, an important transcription factor involved in control of brain and eye development. Furthermore, sumoylation also modulates SP‐1, Maf‐1 and Prox‐1 to control expression of the differentiation‐related genes. Together, our results establish distinct roles of SUMO1 and SUMO2 in regulating cell differentiation. Mechanistically, sumoylation modulates several transcription factors to regulate cell differentiation. (Supported by NIH Grant 1R01EY018380)
Development of leptin mutants with enhanced bioefficacy and stability
Authors: Park Hyunju , Kim Jongsun
Published: June 2021

Leptin is a protein hormone produced by adipocytes. It is secreted into the blood stream and plays a key role in regulating body energy homeostasis by inhibiting feeding behavior followed by weight loss. Because protein stability and bioefficacy are major problems in the biotechnology and biopharmaceutical industries, if leptin can be used for the therapeutic proteins, the stability and bioefficacy are needed to be improved. In this study, we mutanted the surface residues of leptin in a systematic way by displaying 3D structure of leptin on pymol program. We compared the biological activities of leptin and mutant leptin ex vivo using MCF7 breast cancer cell line and in vivo using a male C57Bl mouse model. Mutant leptin treated MCF7 cells displayed better cell proliferation than wild type leptin. The biological activities of leptin and mutant leptin were also tested in a semi‐quantitative STAT3 and ERK phosphorylation bioassay. A representative experiment showed at least 2‐fold higher activity in mutant leptin treated group. To assess the in vivo activity, each group of mice was treated with saline, leptin, and mutant leptin for 20 days and the differences in body weight, food intake, abdominal fat contents, and TG concentration were measured. Mutant leptin appeared to decrease the body weight and food intake in male C57Bl mice more significantly than wild type leptin. As a consequence of decreased body weight, the mutant leptin treated mouse group showed decreased abdominal fat contents and low triglyceride (TG) concentration. These results indicate that the mutant leptin is more effective than wild type leptin.
Ethanol feeding compromises cardiovascular function; a potential role for mitochondrial topoisomerases
Authors: Edwards John G. , Laurent Dimitri , Mathew Jamie , Force Alireza , Taft Mathew , Labinskyy Nazar
Published: June 2021

Alcoholism remains a significant health problem and represents the third leading cause of preventable deaths in the United States. Alcoholic cardiomyopathy (ACM) presents as decreased myocardial contractility, arrhythmias, and secondary non‐ischemic dilated cardiomyopathy leading to heart failure. Mitochondrial dysfunction is thought to have a significant role in the development and complications of ACM. To examine the impact of chronic ethanol exposure on the myocardial mitochondria, rats were assigned to control or ethanol feeding for up to 4 months. Ethanol feeding lead to significant decrease (p<.05) in M‐mode Fraction Shortening and Ejection Fraction. Chronic ethanol feeding lead to a decrease in some components of Cytochrome Oxidase. Ethanol feeding significantly increased mtDNA damage, without a change in mitochondrial copy number. Using isolated myocardial mitochondria, both mitochondrial dependent DNA cleavage and DNA relaxation were altered by ethanol feeding compared to controls. Immunoprecipitation of myocardial mitochondrial extracts using a topoisomerase I antibody partially blocked mitochondrial dependent DNA cleavage. Chronic ethanol feeding compromised mitochondrial function as a result of a decline in mtDNA integrity that appears to the consequence of altered mitochondrial topoisomerase function
Glutathione peroxidase‐1‐deficiency enhances age‐dependent vascular dysfunction
Authors: Steven Sebastian , Schuhmacher Swenja , Oelze Matthias , Bachschmid Markus , Doppler Christopher , Daub Steffen , Schuff Alexandra , Scholz Alexander , Torzewski Michael , Schulz Eberhard , Lackner Karl J. , Münzel Thomas , Daiber Andreas
Published: June 2021

Goal

We investigate, whether glutathione peroxidase (GPx‐1) deficiency enhances ageing‐dependent vascular dysfunction and the formation of reactive oxygen and nitrogen species (RONS).

Methods

2, 6 and 12 months old mice were used. Vascular function was assessed via isometric tension studies. RONS were assessed by optical, immunohistochemical and immunoblotting‐based techniques. Function of endothelial NO synthase (eNOS) was assessed by phosphorylation and S‐glutathionylation pattern.

Results

GPx‐1−/−‐mice had significantly impaired vascular function, compared to wild type‐mice. RONS in mitochondria, heart‐tissue and serum was enhanced with increased age, but not significantly different compared to wild type‐mice. eNOS dysfunction due to S‐gluthationylation and phosphorylation at Thr495 and Tyr657 was increased in GPx‐1−/−‐mice.

Conclusion

GPx‐1−/−‐mice were expected to display a higher level of H2O2. However, we could only detect minor differences between RONS in GPx‐1−/−‐mice and wild type‐mice, which could not explain the differences observed for vascular function with increasing age. The dramatic endothelial dysfunction in old GPx‐1−/−‐mice may be due to phosphorylation and S‐glutathionylation dependent inactivation/uncoupling of eNOS. Further investigations will focus on the expression of other antioxidant proteins and the role of GPx‐1 for vascular inflammation.
Prolactin Receptor loss with Leydig Cell Apoptosis after EDS treatment of Adult Rats
Authors: Singhal Barkha , Dutta Dibyendu , Park In , Sang Samuel , Mills Nathaniel
Published: June 2021

Prolactin receptors (Prlr) are reported to be localized to plasma membranes of testicular interstitial cells including Leydig cells. Prlr is also present in Sertoli cell and cells of germinal epithelium (spermatogonia, spermatocytes and elongating spermatids) in the testis. Ethylene dimethane sulfonate (EDS) eliminates Leydig cell in adult rat testes leading to testosterone depletion and germ cell loss. In this study, two Prlr transcript variants (Prl1r & Prl2r) were investigated at times 6, 15, 24 hr, and 5 and 7 days (d) following EDS using RT and qPCR. Rate of Leydig cell loss was evaluated by analysis of insulin‐like peptide 3 mRNA (Insl3), a Leydig cell‐specific marker. Prl2r and Insl3 were less than 1% of normal values in 5 & 7 d EDS‐treated rats. Similar profiles for Prl2r and Insl3 were observed at earlier time points (6, 15 & 24hr). Testosterone replacement after EDS, however, did not restore the levels of Prl2r and Insl3 mRNAs suggesting they are localized to only Leydig cells. In contrast, Prl1r only declined to 40% of normal levels in EDS‐treated rats suggesting that this receptor variant is located in cells of germinal epithelium, as well as Leydig cells.

Texas Woman's University‐Research Enhancement Program & Dept. of Biology
High selectivity purification screening of histidine‐tagged proteins using small sample preparation formats containing cobalt IMAC media
Authors: Björner Maria , Lundqvist Jon , Hedlund Helena
Published: June 2021

The histidine‐tag is commonly used to facilitate purification of recombinant proteins. For immobilized metal‐ion affinity chromatography (IMAC) the most used metal ion is nickel. However, also cobalt has been shown useful obtaining high purity with good yield of his‐tagged proteins in IMAC applications.

We have designed three formats containing cobalt IMAC media, convenient for rapid and simple small‐scale purification and screening of his‐tagged proteins. The formats, a gravity flow column, a spin column and a 96‐well plate, all allow efficient parallel purification of proteins in μg to mg scale within 40 minutes using fast and easy protocols to help ensure pure target proteins. His‐tagged green fluorescent protein expressed in E. coli was used as model protein to demonstrate the functionality of the formats. The results show that recovery and purity is > 80 % using all formats. The repeatability has proven to be high for all formats with < 10 % difference from average for each run. A comparison between cobalt and nickel IMAC media was performed and our products containing cobalt IMAC media show higher purity compared to products containing nickel IMAC media while still maintaining a high yield.
Creation of a New protein by substituting all arginine residues by its toxic analogue, canavanine
Authors: ishida yojiro , Park Jung-ho , Inouye Masayori
Published: June 2021

Complete substitution of a specific amino acid in a protein with its analogue may create a novel protein with unprecedented properties but is quite challenging, particularly if the analogue is highly toxic to living cells. Canavanine (Can), an analogue of Arginine (Arg) found in certain leguminous plants is highly toxic, in which a ε‐methylene group in Arg is replaced with an oxa group, lowering the pKa value from 12.48 to 7.04. Here, we demonstrate for the first time the complete replacement of Arg residues in a protein with Can, as all the seven Arg residues in an mRNA interferase from Bacillus subtilis, MazF‐bs were replaced with Can. To achieve this, the ACA‐less gene was engineered to produce only MazF‐bs in the presence of Can in a condensed culture of the Single‐Protein Production (SPP) system using an E. coli Arg‐auxotroph strain. We show that all Arg residues were completely replaced with Can as judged by mass spectroscopy. The CD spectrum of MazF‐bs(Can) was almost identical to that of the wild‐type MazF‐bs(Arg) but MazF‐bs(Can) was less thermo‐stable than MazF‐bs(Arg). Interestingly, MazF‐bs(Can) cleaved RNA not only at the original UUACA cleavage sites, but also at the CCACU sites. This is the first report to show that the complete replacement of Arginine with Canavanine in a protein, resulting in acquiring of a new function.
Expresso® Cloning and Expression Systems streamline recombinant protein expression
Authors: Sen Saurabh , Sternard Heather , Franz Laura , Drinkwater Colleen , Sheets Lynne , Niebauer Chris , Mead David , Godiska Ron , Steinmetz Eric J
Published: June 2021

The Expresso® Cloning and Expression Systems use Expressioneering™ Technology, an in vivo recombinational cloning strategy, to dramatically increase the speed, efficiency and success of target gene cloning and soluble protein expression in E. coli and mammalian system. Expressioneering allows directional cloning of PCR products into pre‐processed expression vectors without sample clean‐up or enzyme treatment. Expresso System vectors were constructed with various inducible promoters, N‐ or C‐terminal 6xHis tags, and an optional cleavable SUMO tag for improved recombinant protein solubility in bacterial systems.

The Expresso T7 System uses an IPTG‐inducible T7‐lac promoter and the Expresso Rhamnose Systems features the tunable, rhamnose‐inducible, rhaPBAD promoter, and uses a single host strain for both cloning and expression.

The new Expresso CMV system for mammalian protein production utilizes the same Expressioneering technology to provide rapid, precise, and directional insertion of fragments into the cloning site. The small size of the pME‐HA vector (3.4 kb) and minimal amount of bacterial DNA sequence supports robust protein expression, higher transfection efficiency and simplifies subsequent manipulations. The Expresso CMV vector allows addition of the HA tag for immono‐detection or affinity purification.
Molecular cloning of a dithiol glutaredoxin cDNA from sweet potato
Authors: Wen Lisa , Chi Xiao-Wen , Lin Chao-Ting , Jiang Yu-Chi , Lin Chi- Tsai
Published: June 2021

Glutaredoxins (Grxs) play an important role in the reduction of protein glutathione mixed disulfides. An IbGrx cDNA encoding a putative dithiol Grx has been cloned from sweet potato (Ipomoea batatas [L.] Lam). The deduced amino acid sequence is conserved among the reported dithiol Grxs having CGYC dithiol motif at the active site. To characterize the IbGrx protein, the coding region has been subcloned into an expression vector and transformed into E. coli. The recombinant His6‐tagged IbGrx has been overexpressed and purified by Ni affinity chromatography. The purified enzyme showed a monomeric band as demonstrated by 15% SDS‐PAGE. The enzyme has relatively low KM value for HED, suggesting that it can catalyze the reduction of glutathione mixed disulfide efficiently using GSH as reductant. The enzyme has been characterized in terms of thermal stability, the influence by pHs, imidazole, or proteases sensitivity. The enzyme appears to be stable under various conditions. These properties may be beneficial for its potential medical applications considering the numerous functions of Grxs in many cellular processes. This work was supported by the National Science Council of the Republic of China, Taiwan under grant NSC 100‐2313‐B‐019‐003‐MY3 to C‐T. Lin.
A novel approach to isolate unselected recombinants facilitates markerless DNA recombineering
Authors: Lezin George , Kosaka Yasuhiro , Yost H. Joseph , Kuehn Michael R. , Brunelli Luca
Published: June 2021

Current screening methods for unselected recombinants, such as colony hybridization, are essentially in situ approaches. Using the “founder effect” (Mayr E., 1963), a classical population genetics principle, we developed an en masse screening procedure in liquid cultures to isolate markerless recombinants generated by recombineering. The founder effect states that allele frequency can be randomly increased by establishing a new sub‐population from a larger population. Thus, we sampled an E. coli culture so that it contained only a few recombinants and subdivided it into 96 sub‐cultures by plating in LB plates. Recombinant‐positive wells were detected by PCR using a multi‐channel electronic pipetter. Each positive well was founded by at least 1 recombinant cell, but the number of total cells decreased by almost 100‐fold. This implies that, if there is no selection against DNA recombinants and the DNA modification is stable, approximately 100‐fold enrichment can be achieved at every sampling/dilution cycle. Using PCR screening, we isolated pure recombinant clones containing a BAC deletion and two separate DNA replacements either from plated clones after the second cycle (max 104‐fold enrichment, 7 days) or from liquid cultures after the third cycle (max 106‐fold enrichment, 10 days). These results demonstrate that our novel approach to isolate unselected recombinants facilitates markerless DNA recombineering.
Differentiating frequency of recombination from frequency of recombinants
Authors: Lezin George , Kosaka Yasuhiro , Yost H. Joseph , Kuehn Michael R. , Brunelli Luca
Published: June 2021

In recombineering, the frequency of recombinant cells is often used as a measure of the frequency of recombination events. We used the “founder effect” (Lezin G. et al., in preparation) and PCR screening to study the origin and propagation of mini‐lambda‐mediated recombinants in liquid cultures. Specifically, we deleted a Neo marker from an mStrawberry cassette‐containing BAC, and then replaced mStrawberry with eYFP, ZsGreen or turboGFP. Upon deletion of Neo, some recombinants were PCR positive but Neo resistant, suggesting the formation of multiple targets during BAC recombinereing. Additionally, after replacement of mStrawberry with eYFP, there was evidence of partial or complete eYFP duplication at the first two enrichment cycles. However, at the third cycle, only one eYFP copy was detected. Both aberrations are similar to previously reported data (Poteete AR at al., BMC Mol Biol 2004). Upon replacement of Neo‐less mStrawberry or eYFP with ZsGreen or turboGFP, we observed similar frequency of recombination to the first eYFP enrichment cycle (estimated via the fraction of negative wells, Luria & Delbruck, Genetics 1943). However, we were unable to isolate ZsGreen and turboGFP recombinants because PCR products became progressively smeared at subsequent enrichment cycles, suggesting delayed DNA structure rearrangement. These data show that lower frequency of recombinant cells does not necessarily imply decreased frequency of recombination, and these two variables need to be independently assessed.
Selecting target populations for community nutrition intervention using spatial analyses
Authors: Bryant Jordan E.
Published: June 2021

Target populations for community‐based interventions are often selected using subjective methods, despite interest in increasing objective, quantifiable methods in public health intervention design. The purpose of this study was to determine if spatial patterns exist among the environmental influences on childhood obesity and to establish the effectiveness of spatial analyses in determining areas in need of nutritional intervention. Three environmental risk factors for childhood obesity were selected; socioeconomic status, the proximity of grocery stores with healthy options, and the walkability of the study area. Prevalence data from each influence was mapped for each census tract in Berkeley County, West Virginia using the ArcGIS program (ESRI). The resulting maps showed clustering of risk factors and by ranking each census tract by degree of prevalence it was possible to highlight a single census tract with the greatest cumulative risk for obesity in children, with respect to the selected risk factors. In conclusion, this study has found an effective, quantifiable method with which to determine the target population in highest need for nutrition intervention.

Funding source: USDA Agriculture and Food Research Initiative
Shaping college‐age eating behavior in an institutionally derived food setting with a simple, interactive computer spreadsheet
Authors: Crowder Todd A. , East Whitfield B.
Published: June 2021

Many higher educational, institutional food settings offer set menus created by RD's yet students are allowed choices on amounts and food selections. Our institution has a goal of providing < 30% fat for each meal yet allows freedom of choice.

Purpose

Create a simple, user friendly computer spreadsheet that will assist students with higher quality food selections and enhance overall nutrition.

Methods

An interactive computer program was created, allowing students to build a daily nutrition plan based on total energy expenditure (TEE); macronutrient planning based on meat, dairy, bread, vegetables, starches, fruits, dessert; and diet tracking based on consumption during breakfast, lunch, dinner, snacks. The overall program (originally joint produced with RD) was designed to shape eating behaviors, with the goal of decreasing fat consumption, increasing fiber consumption, and consuming a more nutrient‐dense diet. Students consumed one “normal” (n) day then one “planned” (p) day using the interactive software to create the “planned” day dietary intakes by food category. A diet analysis was conducted on each day and results analyzed.

Results

Forty one college‐age students (male = 35, female =6) completed all facets of the investigation.

Additionally, the consumption of carbohydrates expressed in g/kg body mass increased for both men (5.32 to 5.8, p = .06) and women (2.87 to 3.73, p=.16).

Status
Kcal n
Kcal p
Fiber (g) n
Fiber (g) p
Fat (g) n
Fat (g) p

Men
3026
2983
25.6
40.3*
99.9
81.6#

Women
1920
2014
15.1
19.5**
66.3
65.6

p < .0001,

p < .02;

p =.17

Conclusion

An interactive, computer software program appears to assist in shaping positive nutritional behavior practices in college‐age students.
Osteocalcin is significantly higher in Caucasian pregnant women with gestational diabetes mellitus
Authors: Tabatabaei Negar , Giguère Yves , Charland Marc , Forest Jean-Claude , Rodd Celia , Kremer Richard , Weiler Hope
Published: June 2021

Lower 25(OH)D and higher osteocalcin (OC) concentrations have been observed at mid‐gestation in pregnant women with gestational diabetes (GDM) compared to those without. We aimed to investigate simultaneously 25(OH)D and OC throughout pregnancy in a nested case‐control (48–48) study in Caucasians living in Quebec City (matched for age, season, BMI and length of pregnancy). Maternal serum 25(OH)D and OC were measured by chemiluminescence and ELISA, respectively at 13–16 and 24–28 weeks gestation and at delivery. Differences in concentrations were tested using a MIXED model repeated measures ANOVA. We conclude that 25(OH)D was not significantly different (p=0.260) while OC was significantly higher (p=0.024) in GDM vs healthy pregnant women. Both 25(OH)D and OC increased significantly with time (p<0.001), but there were no interactions between health state with time (p=0.092, p=0.434, respectively). Funded by CIHR.

Grant Funding Source : Canadian Institutes of Health Research
High protein intake improves insulin sensitivity but exacerbates bone resorption in immobility
Authors: Heer Martina , Smith Scott M. , Frings-Meuthen Petra , Zwart Sara R. , Baecker Natalie
Published: June 2021

Inactivity, like bed rest (BR), causes insulin resistance (IR) and bone loss even in healthy subjects. High protein intake seems to mitigate this IR but might exacerbate bone loss. We hypothesized that high protein intake (animal:vegetable protein ratio: 60:40), isocaloric, compared to the control group plus high potassium intake would prevent IR without affecting bone turnover. After a 20‐day ambulatory adaptation to controlled confinement and diet, 16 women participated in a 60‐day, 6° head‐down‐tilt BR and were assigned randomly to one of the two groups. Control subjects (CON, n=8) received 1g/kg body mass/d dietary protein. Nutrition subjects (NUT, n=8) received 1.45g/kg body mass/d dietary protein plus 7.2g branched chain amino acids per day during BR. All subjects received 1670 kcal/d. Bed rest decreased glucose disposal by 35% (p<0.05) in CON. Isocaloric high protein intake prevented insulin resistance, but exacerbated bed rest induced increase in bone resorption markers C‐telopeptide (> 30%) and N‐telopeptide (>20%) (both: p<0.001). Bone formation markers were unaffected by high protein intake. We conclude from these results that high protein intake might positively affect glucose tolerance, but might also foster bone loss. Further long‐duration studies are mandatory before high protein intake for diabetic patients, who have an increased fracture risk, might be recommended.
Comparison of vegetarian and non‐vegetarian diets to 2010 Dietary Guidelines
Authors: Farmer Bonnie , Larson Brian , Keast Debra R , Fulgoni Victor L
Published: June 2021

The 2010 Dietary Guidelines recommend that Americans increase vegetable and whole grain intake, minimize intake of added sugars and solid fats, and control calorie intake. Typical American diets fall short of goals for healthy eating patterns, but vegetarian diets are thought to be more consistent with dietary guidelines. Dietary intake from the National Health and Nutrition Examination Survey (2001 – 2008) was used to determine usual intake of foods, as MyPyramid equivalents, for vegetarians (V) and non‐vegetarians (NV), using the National Cancer Institute method. Vegetarians were defined as those who did not report consuming meat, poultry, or fish on day one of the survey, representing a lacto‐ovo vegetarian dietary pattern. In subjects 19 years and greater (N=18,954), calorie intake was 1697±41 kcal for V (n=1,138) and 2213±10 kcal for NV (n=17,816). Total vegetable intake was higher for NV; however, intake of white potatoes was twice as high for NV than for V (p<0.001). When compared on a calorie adjusted basis (per 1000 kcal), total vegetable intake for V was 0.77 cup equivalents vs 0.75 for NV. Whole grain and legume consumption was higher for V than for NV (p<0.001), and was twice as high per 1000 kcal. Added sugar, discretionary solid fat, and discretionary oil intakes were higher for NV than for V, although per 1000 kcal, added sugar for V was 9.5 tsp equivalents vs 8.8 for NV, and discretionary oil for V was 9.3g vs 8.7g for NV. While deficits exist for both V and NV, the V diet pattern is more advantageous in terms of 2010 Dietary Guidelines recommendations.
Metabolites in human breastmilk identified as markers for sample storage quality
Authors: Hartmann Peter , Lui James K , Piper Kristin , Rawlinson Catherine , Maker Garth L , Trengove Naomi J , Trengove Robert D
Published: June 2021

Detailed understanding of how storage temperature affects specific breastmilk metabolites is scarce. Current storage recommendations of expressed breastmilk for either scientific or domestic use is based exclusively on studies of bacterial growth and immuno capacity. Metabolomic profiling could reveal significant changes in metabolite levels in breast milk due to different exposure conditions that may affect the overall transfer of metabolites from the mother to the infant, ultimately affecting the health of the newborn.

A pilot study was conducted using gas chromatography coupled with mass spectrometry (GC‐MS) to determine the milk metabolite profile under different storage temperatures over a 24‐hour period. Milk expressed from 5 lactating mothers, between 3 to 7 months post‐partum was collected, aliquoted, then stored immediately at room temperature, 4°C, −20°C or −80°C, before sample processing and analysis by GC‐MS.

Levels of specific metabolites in stored milk samples were found to have changed at room temperature and/or 4°C but not at −20°C or −80°C in the first 24 hours. These results established the need for breastmilk storage at < −20°C for both scientific and/or domestic use. Moreover, changes in metabolite levels could potentially serve as biomarkers/indicators for breast milk sample integrity.

Funded by Medela AG. Switzerland.
Concept of slowly released dietary glucose: a focus on starch digestion at the mucosal α‐glucosidase level
Authors: LIn Amy Hui-Mei , Nichols Buford L. , Hamaker Bruce R.
Published: June 2021

The aim of our research is to control dietary glucose delivery rate to the body for potential health benefit. There are six enzymes, two α‐amylase and four small intestinal mucosal glucosidases, involved the digestion of starch. The convention view is that α‐amylase is the key enzyme that determines digestion rate, and mucosal α‐ glucosidases have a more simple function of rapidly converting the small molecules generated from α‐amylase digestion to glucose. We investigated the digestion patterns of four recombinant α‐ glucosidases on various starches in an in vitro system. The notable finding was that starches with different molecular structures have different digestibilities by the mucosal α‐glucosidases following through digestion by α‐amylase, and that various starch molecules have different susceptibilities to the individual α‐glucosidases. Furthermore, a small fraction of starch molecules was digested at a very low rate, indicating structures that are additionally difficult to digest at the brush border level. Our group has further showed that slowly released glucose causes reduced gastric emptying in a dose response manner. To take benefit of slowly released glucose, the mucosal α‐glucosidases is a potential control point for controlling glucose delivery to the body. This work was supported by internal funding from the Whistler Center for Carbohydrate Research of Purdue Univ.