bluuebunny/crossref_metadata_2025 huggingface dataset:
Difficulties Learning Bones and Muscles? Study at Home with 3D‐Printed Bones
Authors: Cevallos Manuel , Butler Jessica , Stabile Diana
Published: April 2020

Some schools of Medicine are giving 3D‐skull models to students. Those models can be taken home to improve the association, memorization, and integration of structures and function in the learning process. On the other hand, studies conducted on Healthcare students reported that the most common learning styles are kinesthetic and visual.

The problem

The course BIO 201 Laboratory (Anatomy and Physiology I) offers multiple topics to support lectures in a span of 15–16 weeks but with typically only one 2–3 hour session per week. In the chapters of Bones, Muscles, and Joints, the time in the lab in which the student is exposed to those materials is about three to five Labs in the semester. At that time, the student must memorize multiple structures and relate them with their function (physiological movements). Students are studying with the help of 3D models (skeleton and isolated bones) in the Lab, and 2D‐images (book or computer). In some institutions, BIO 201 labs can have digital support with 2D‐images projected to a 3D‐image, a significant advance. However, the learning style plays an essential role in healthcare students.

The Survey

A voluntary survey was conducted in the BIO 201 class in Fall 2019. Of 118 students in the class, 74 students answered the survey. The primordial Learning style was 66.2% visual followed by a 25% kinesthetic. Moreover, from all the groups, 76.4% reported having difficulties in learning Bones, muscles, and joints. Interestingly, from the students having difficulties in the learning, 70.9% answered having problems to transform 2D‐images into 3D structures (visualize and identify structures in bones and 3D models). Let us remember, in most institutions, 3D models only are accessible in Lab time, and schedules on labs are not always ideal for students for extra‐reviews. In our survey, 70.5% students are studying at home with 2D‐image (book and or computer) and 19.7% from videos. Then, how can we bring bones, joints, and muscles to students with those difficulties?

The feasible solution

Real 3D‐bones are expensive and bulky. The alternative is to use a 3D printer as a tool to prepare a set of bones for students. The quality these days is fantastic; bones can show features and students can mark them to identify origin and insertion of muscles. A 3D‐printed bones set can be reused and easily repaired in case of loss. 97.3% of students are willing to use them at home, and 98.6% of the group consider it an excellent tool for helping them to learn.

Conclusion

We believe that 3D‐printed bone support in the BIO 201 course can help students with difficulties in the transformation from 2D‐images to 3D‐structures. It is an easy access tool and accepted by students, cheap, reusable and replaceable in case of loss. Students can easily identify a structure in the 3D models.

Studying Bones, Joints and Muscles with 3D‐Printed Models at Home.

Figure 1
The Filament Forming Mechanism of SgrAI Endonuclease‐Structural and Kinetic Analysis
Authors: Horton Nancy C.
Published: April 2020

Filament formation by enzymes has recently emerged as a new paradigm of enzyme regulation
1.

Wide‐spread reversible self‐assembly of enzymes in cells has also been shown to occur in all branches of life and diverse metabolic and biological pathways. We use the model system, SgrAI, to investigate the filament forming enzyme mechanism.

Filaments formed by SgrAI have been structurally characterized using cryo‐electron microscopy
2 and the full reaction pathway including all rate constants has been determined using single turnover enzyme reactions, FRET, and global kinetic modeling
3–
4.

The structural studies uncover the mechanism of activation of enzyme activity in SgrAI by filament formation, as well as the origin of the unusual expansion of substrate specificity in SgrAI within the filament. A conformational change in the SgrAI dimer, stabilized by protein‐protein and protein‐DNA contacts in the filament, leads directly to a shift in the active site, hypothesized to lead to stronger binding of a second catalytic Mg2+ ion. DNA structure and energetics is hypothesized to control substrate specificity and filament formation by SgrAI such that the secondary class of DNA sequences are cleaved by SgrAI only upon joining a filament formed by SgrAI bound to a primary class of DNA sequences. The full kinetic model showed a slow second order step in filament formation which is shown to control enzyme activity such that only invading phage DNA is cleaved, leaving the host genome untouched. This model also shows that the filament forming mechanism is superior in fast activation of enzyme activity upon initial recognition of primary DNA sequences.

Using the SgrAI endonuclease as a model system to study filament forming enzymes, we have discovered that the enzyme is activated by stabilization of an activated conformation by contacts made within the filament. Control of the equilibrium between a low activity, non‐filamentous state and the high activity, filamentous state by DNA structural energetics gives rise to the unusual apparent expansion of DNA sequence specificity of SgrAI by filament formation. Simulations using the full kinetic pathway reveal an advantage in the filament mechanism over other non‐filamentous mechanisms in the speed of activation in response to invading phage DNA, giving strong evolutionary advantage to this filament forming mechanism.

Support or Funding Information

This work was supported by the National Science Foundation under Grant No. MCB‐1410355.
Lepidium meyenii L. and Isolated Macamides Reduces Lipopolysaccharide‐Induced Inflammatory Response in THP‐1 Cells
Authors: Gonzales-Urday Ana Lucia , Martínez-Málaga Jimena Alexandra , Rondón-Ortiz Alejandro Nico , Böhlke Mark , Chocano-Coralla Edward Joel , Pino-Figueroa Alejandro
Published: April 2020

Inflammation is a vital component of the immune system response to injury and infection, and it is linked to autoinflammatory and autoimmune disorders. Toll‐like receptor 4 (TLR4) has been identified as a receptor for lipopolysaccharide (LPS), a bacterial endotoxin which can induce systemic inflammation and sepsis. The LPS/TLR4 pathway triggers downstream signaling cascades, producing proinflammatory cytokines and chemokines. Therefore, the search for novel compounds that can reduce inflammation at this level is of great interest. The plant Lepidium meyenii L., known as maca, has attracted attention in the past several years due to its traditional uses in folk medicine, which include treating anemia and gastritis, preventing cancer, high blood pressure and depression, as well as enhancing fertility and sexual performance. Macamides are secondary metabolites isolated from maca which cause pharmacological inhibition of fatty acid amide hydrolase (FAAH), and this inhibition has been found to lead to anxiolytic, anti‐inflammatory and analgesic effects. However, the anti‐inflammatory mechanisms by which these natural compounds act remain to be elucidated. The purpose of this study was to evaluate the anti‐inflammatory effects of the n‐pentane extract of Lepidium meyenii L. (PELM), N‐(benzyl) linolenamide (compound 1) and N‐(3‐methoxybenzyl) linolenamide (compound 2) utilizing THP‐1 cells. To assess these effects, THP‐1 cells were treated with either 10 μg/mL PELM, 10 μM Cmpd 1, 10 μM Cmpd 2 or 0.5 % DMSO (vehicle); then, inflammation was induced for 24 h with 30 ng/mL LPS. Media supernatants were used to evaluate protein levels of cytokines in a human protein array. From 36 cytokines, maca and macamides reduced IL‐1β and IL‐6 protein levels following a 24 h stimulation with LPS. In order to confirm these results, ELISA was utilized to determine IL‐1β and IL‐6 cytokine levels in LPS‐stimulated THP‐1 cells. After a 24 h treatment with either 10 μg/mL PELM, 10 μM Cmpd 1 or 10 μM Cmpd 2, these treatments reduced protein levels of both cytokines IL‐1β and IL‐6. The transcriptional levels of these cytokines were also monitored by RT‐qPCR; treatment with 10 μM Cmpd 1 and 10 μM Cmpd 2 significantly decreased mRNA levels of IL‐1β. But, interestingly with a 10 μg/mL treatment of PELM the reduction of IL‐1β mRNA levels was not significant. Furthermore, IL‐6 mRNA levels were significantly reduced by the n‐pentane extract and isolated macamides. Taken together, these results demonstrated that Lepidium meyenii L. and isolated macamides reduced LPS‐induced inflammatory response by decreasing pro‐inflammatory cytokine production in THP‐1 cells.

Effect of PELM, Cmpd 1 and Cmpd 2 on IL‐1β production in THP‐1 cells.

Figure 1

Effect of PELM, Cmpd 1 and Cmpd 2 on IL‐1β mRNA levels in THP‐1 cells.

Figure 2
Ground Reaction Forces generated by a Lower Body Negative Pressure Exercise Device for Space Flight
Authors: Poudel Alisha , Ashari Neeki , Kong Mitchell , Friend James , Hargens Alan
Published: April 2020

Loss of bone and muscle are leading concerns among astronauts during prolonged space flight. Lower body negative pressure (LBNP) chambers generate gravitational and cardiovascular stresses to maintain musculoskeletal strength and fitness by means of suction pressure. Ground reaction forces (GRFs) generated underneath the user’s feet are directly proportional to the level of negative pressure applied. However, the exact mechanism of GRF generation remains unresolved. We hypothesize that increasing the cross‐sectional area (CSA) of the user’s waist in contact with a flexible waist seal will increase the GRFs generated at a given vacuum pressure. To test our hypothesis, we collected GRFs generated by 6 healthy subjects under two conditions: original (unchanged) CSA of their waist and a greater waist CSA using expanded cushioning around waist of the same subjects. GRFs were measured with LBNP levels ranging from 0 (ambient pressure) to 50 mmHg with subjects lying in supine position to simulate microgravity. The original CSA of 467.7 cm2 ± 69.0 cm2 generated 118 % ± 31 % body weight at −50 mmHg. However, the larger CSA of 1,451.0 cm2 ± 331.0 cm2 generated a mean GRF of 146 % ± 31% body weight at −50 mmHg. A one‐way ANOVA test resulted in p‐values < 0.05 for all pressures, denoting statistically significant increases in larger compared with original CSA GRF. The GRF generated with larger CSA followed this equation GRF = −1.8211 (LBNP) + 0.7899 while the GRF generated with original CSA followed the equation: GRF = −1.5079 (LBNP) + 0.3995. These data support our hypothesis that GRFs generated by LBNP are directly proportional to the waist CSA. Using this new concept, astronauts may generate GRFs at lower LBNP levels to prevent pre‐syncopal symptoms during exercise in space.

Support or Funding Information

Supported by NASA grants NNX13AJ12G and NSSC19K0409.

Average weight measurements with varying pressures for six volunteers; red is original CSA of subject's waist and blue is larger CSA.

Figure 1
Serum‐derived Extracellular Vesicles as Diagnostic Biomarkers for Non‐alcoholic Fatty Liver Disease
Authors: Newman Lauren
Published: April 2020

Introduction

Non‐alcoholic fatty liver (NAFL) disease is a highly prevalent chronic liver disease with variable severity, from simple steatosis to steatohepatitis (NASH). NASH involves hepatocyte death, inflammatory infiltration and fibrosis, and may progress to cirrhosis or hepatocellular carcinoma. Liver biopsy is currently the only way to definitively diagnose NASH, but this simply provides a static image and due to the highly invasive nature of the procedure, poses risks to patients and is not practical for longitudinal evaluation. Circulating populations of small extracellular vesicles (sEVs), including exosomes (30–150nm) and microvesicles (50–1000nm), are known to exhibit alterations in concentration and molecular cargo in disease states, including NAFL and NASH. Thus, they present the lucrative opportunity to not only diagnose and stage disease in a minimally invasive manner, but also track the efficacy of therapeutic interventions.

Aim

This study aimed to evaluate the performance of miRNA, derived from global circulating sEVs or hepatocyte‐specific EVs, in distinguishing patients with NAFL and NASH from healthy individuals.

Methods

Serum was obtained from healthy volunteers and patients with NAFL and NASH and ExoQuickTM precipitation solution was used to isolate global sEV samples. Magnetic DynaBeads conjugated to anti‐asialoglycoprotein receptor 1 antibody were used for immunoprecipitation of hepatocyte‐specific EVs from global samples. Total RNA was isolated from EV samples using TRIzol reagent and reverse transcribed with TaqMan miRNA RT kit. Expression of miR ‐122, ‐192, ‐128‐3p, ‐451 and ‐16 was determined by RT‐qPCR using TaqMan small RNA assays. Predictive performance of miRNA biomarkers was evaluated by receiver operating characteristic (ROC) analysis.

Results

Expression of liver miRNA (miR ‐122, ‐192, ‐128‐3p) given as ratios to non‐specific EV miRNA (‐451, ‐16) in global circulating EVs was generally greater in patients with NAFL and NASH. In distinguishing all disease from healthy, excellent performance was exhibited by miR‐128‐3p/‐451 and miR‐192/‐451 (AUROC = 0.925 and 0.882, respectively). miR‐122 ratio expression was relatively poor in global EVs but markedly improved in liver‐specific EVs, up to AUROC = 1.000.

Conclusion

We provide a proof of principle for the use of EV‐derived miRNA ratio biomarkers in minimally invasive NAFLD diagnosis and describe a method for extracting liver‐specific EVs for this application.

Support or Funding Information

NHMRC project grant (1158210) Pfizer Investigator Initiated project grant

Receiver operating characteristic (ROC) curves for miR‐122 ratios derived from liver‐specific extracellular vesicles (EVs) in predicting all NAFLD and only fatty liver.

Figure 1
Elevated Circulating Cardiac Troponins in Chronically Catheterized Pre‐and Post‐Natal Sheep
Authors: Jonker Sonnet S.
Published: April 2020

BACKGROUND

Circulating markers of cardiac damage have been reported to be elevated in cord blood of healthy infants. This elevation is reported to be independent of the stress of labor, as elevations are also reported in the cord blood of healthy infants delivered by Caesarian section prior to labor. We hypothesized that circulating markers of cardiac damage are elevated in the late‐term fetus prior to delivery.

METHODS

Three fetal sheep were surgically instrumented with indwelling catheters in the carotid artery and coronary sinus. Sheep were recovered from surgery, and daily blood samples were taken to determine circulating levels of cardiac troponin I (cTnI), c‐reactive protein (CRP), and N‐terminal pro hormone BNP (nt‐ProBNP) using the Siemens DIMENSION VISTA, and cardiac troponin T (cTnT) using the Roche cobas e601 STAT. Data were visualized to determine trends.

RESULTS

We found that circulating cTnI and cTnT levels tend to decline in the week preceding birth. Troponins also were elevated following natural vaginal delivery. CRP and nt‐ProBNP levels were not detectable.

CONCLUSIONS

These data support our hypothesis that a cardiac process is releasing cardiac troponins to circulate 1–2 weeks prior to birth. Further investigation is required to determine if this is a transient event, or if levels are high throughout fetal development. We also found that levels decline as term approaches, but that birth re‐stimulates release of these markers of cardiac damage.

Support or Funding Information

OHSU Medical Research Foundation

Circulating markers of cardiac damage in chronically catheterized perinatal sheep. The x‐intercept represents day of surgery, while the dashed line represents the day of birth. Solid symbols = blood samples from the carotid artery. Open symbols = blood samples from the coronary sinus.

Figure 1
Non‐Enzymatic Lysine Lactoylation of Glycolytic Enzymes
Authors: GALLIGAN JAMES , Gaffney Dominique O. , Jennings Erin Q. , Anderson Colin C , Marentette John O. , Shi Taoda , Oxvig Anne-Mette Schou , Streeter Matthew D. , Johannsen Mogens , Spiegel David A. , Chapman Eli , Roede James R
Published: April 2020

Post‐translational modifications (PTMs) regulate enzyme structure and function to expand the functional proteome. Many of these PTMs are derived from cellular metabolites and serve as feedback and feed‐forward mechanisms of regulation. We have identified a novel PTM that is derived from the glycolytic by‐product, methylglyoxal. This reactive metabolite is rapidly conjugated to glutathione via glyoxalase 1, generating lactoylglutathione (LGSH). LGSH is hydrolyzed by glyoxalase 2 (GLO2), cycling glutathione and generating D‐lactate. We have identified the non‐enzymatic acyl transfer of the lactate moiety from LGSH to protein Lys residues, generating a ‘LactoylLys’ modification on proteins. GLO2 knockout cells have elevated LGSH and consequently, a marked increase in LactoylLys. Using an alkyne‐tagged methylglyoxal analog, we show that these modifications are enriched on glycolytic enzymes and regulate glycolysis. Collectively, these data suggest a previously unexplored feedback mechanism that may serve to regulate glycolytic flux under hyperglycemic or Warburg‐like conditions.

Support or Funding Information

Financial support was provided by National Institutes of Health Grants (R01 ES027593 for J.R.R.), the SENS foundation (D.A.S.), the American Diabetes Association Pathway to Stop Diabetes Grant 1‐17‐VSN‐04 (D.A.S.), and the Velux Foundations (VELUX34148, A‐M.S.O). Mass spectrometry and proteomics data were acquired by the University of Arizona Analytical and Biological Mass Spectrometry Facility supported by NIH/NCI grant CA023074 to the University of Arizona Cancer Center, UA Research Development and Innovation Office, and by the BIO5 Institute of the University of Arizona.

Figure 1
Lysyl Oxidase Internalization in Deformed Cardiomyocytes Following Myocardial Ischemia in Mice
Authors: Chu Qing , Xiao Ying , Song Xin , Kang Y. James
Published: April 2020

Lysyl oxidase (LOX), a copper‐dependent amine oxidase, catalyzes the cross‐linking of collagen and elastin in extracellular matrix. In response to myocardial ischemia, LOX is over‐produced in cardiomyocytes. The present study was undertaken to understand the relationship between the changes in LOX production and alterations in cardiomyocytes following myocardial ischemia. Male C57BL/6J mice aged 8–12 weeks old were subjected to left anterior descending (LAD) coronary artery ligation to induce ischemic injury. The hearts were collected at 1st, 4th, or 7th day after the surgery and cut into serial frozen/paraffinsections. Histological examination identified typical rod‐shaped cardiomyocytes in sham‐operated mice, which was gradually transformed into rounded‐shaped from 1st to 7th day after LAD artery ligation. Corresponding to the deformation, connexin 43 (Cx43), an important gap junction protein, was decreased in the intercalated discs in ischemic infarct area. There were two isoforms of LOX, pro‐peptide (LOX‐PP) and spliced peptide (active LOX), identified in cardiomyocytes. The active LOX, not the LOX‐PP, was colocalized with Cx43 in the intercalated discs in normal cardiomyocytes. In response to ischemia, the levels of both active LOX and LOX‐PP were increased, and moreover, active LOX was dissociated from Cx43 and relocated in the cytosol of the deformed cardiomyocytes from 1st to 7th day post myocardial ischemia. Our findings suggest that in response to ischemia, the internalization of active LOX from the intercalated discs to the cytosol of cardiomyocytes is likely responsible for the breakage of the intercellular junction, leading to the loss of cell polarity and the consequent deformation cardiomyocytes.

Support or Funding Information

West China Hospital, Sichuan University

Immunofluorescence micrographs of LOX in cardiomyocytes. Two isoforms of LOX, pro‐peptide (LOXPP) and spliced peptide (active LOX), identified in cardiomyocytes. The levels of both active LOX and LOX‐PP were increased from 1st to 7th day post myocardial ischemia. Moreover, active LOX relocated in the cytosol of the deformed cardiomyocytes.

Figure 1

Co‐immunofluorescence micrographs of LOX/LOX‐PP and Cx43. The active LOX, not the LOX‐PP, was colocalized with Cx43 in the intercalated discs in normal cardiomyocytes and was dissociated from Cx43 at 7th day after ischemia.

Figure 2
Impact of S220A and E168A mutations on M. tuberculosis Indole‐3‐Glycerol Phosphate Synthase (IGPS) catalysis
Authors: Leon Hernandez Katherine M.
Published: April 2020

The enzyme indole‐3‐glycerol phosphate synthase (IGPS) catalyzes the conversion of 1‐(o‐carboxyphenylamino)‐1‐deoxyribulose‐5‐phosphate (CdRP) to indole‐3‐glycerol (IGP) in the tryptophan biosynthetic pathway in bacterial organisms (Scheme 1). The tryptophan biosynthetic pathway is necessary for bacterial life, therefore, this enzyme represents a potential target for antibacterial drug development against pathogenic organisms. Because IGPS is absent in humans, no concerns for homolog selectivity need to be considered. Our goal is to investigate substrate/ligand binding and the catalytic mechanism of M. tuberculosis IGPS to facilitate inhibitor discovery. To study how this enzyme interacts with ligands and catalyzes the reaction, mutated plasmids that code for M. tuberculosis IGPS wildtype, as well as mutants S220A and E168A, were expressed in E. coli and purified via a His‐tag. The wildtype and the active site mutants S220A and E168A were studied via kinetic assays. Based on previously reported results, we suspected that Ser220 may be involved in ligand and substrate binding in M. tuberculosis IGPS. We hypothesized that Glu168 plays an important role in either catalysis or binding. We measured wildtype activity following the rate at which the product IGP is produced monitoring fluorescence with an excitation wavelength of 280 nm and an emission wavelength of 340 nm. We have found that the mutant E168A has no detectable activity, indicating that residue E168 plays a critical role in catalysis. We have also found that the catalytic activity of S220A is greatly reduced compared to the wildtype enzyme, indicating that S220 plays an important role as well. Understanding the roles that the active site residues play in M. tuberculosis IGPS ligand binding and catalysis will contribute to future drug discovery efforts.

Support or Funding Information

NIH grant 1R15GM126467‐01 titled “Investigation of Substrate Specificity, Mechanism, and Inhibition of IGPS”

Scheme 1. Reaction Catalyzed by MtIGPS and a Proposed Mechanism

Figure 1
NRF from Diceratella elliptica (DC.) Jonsell. as Strong and Safe Insulin Resistance Modulator and NASH Preventer
Authors: Mohammed Eman D. , Zhang Zechuan , Tian Wenfang , Gangarapu Venkatanarayana , Al-Gendy A. A. , Chen Jun , Wei Jifu , Sun Beicheng
Published: April 2020

Background

Insulin resistance (IR) and lipotoxicity were evidenced as the major nonalcoholic steatohepatitis (NASH) initiators. Absence of the effective treatment against NASH progression raised our aim to discover a new fraction rich in glucosinolates (GLS) hydrolysis products from Diceratella elliptica (DC.) Jonsell, with promising insulin sensitizing activity & anti‐NASH potentiality.

Purpose

Our study aimed to extract and prepare a nitriles rich fraction (NRF) from Diceratella elliptica (DC.) Jonsell, investigate its insulin‐sensitizing & anti‐NASH potentiality.

Study Design

NRF was prepared using natural autolysis and compounds were identified. Seventy male Wistar rats were feed high fat diet (HFD) or normal pellets for 35 days. In HFD rats, Streptozotocin (STZ) has been injected in day 14th and treatment has been started on day 21st with either NRF (30, 60 and 120 mg/kg; orally) or pioglitazone (PioG) (10 mg/kg; i.p) beside HFD. While, NRF‐alone rats were treated with NRF (120 mg/kg; orally) beside the normal pellets. Body weight, glucose homeostasis, hepatopathological examinations were performed.

Methods

Both GLC/MS and HPLC were used for compounds’ identification while UV spectrophotometer was used for total glucosinolates (GLS) quantification. The biochemical and general molecular investigations concerned with liver lipotoxicity, oxidative stress, inflammation and insulin signalling pathway have been done and confirmed with the computational prediction of the major compounds’ targets.

Results

Butenyl and benzyl GLS were the major along with other volatile compounds. NRF had significantly increased the insulin sensitivity and improved NASH‐hisptopathology showing hepatoprotective effect. While, the fraction’s anti‐NASH potentiality was evidenced in the normalized hepatic steatosis markers, inflammation and oxidative stress key transcriptional factors resulting in induction of insulin receptor substrates (IRSs) phosphorylation and its downstream effectors.

Conclusion

NRF has reversed IR, stimulated leptin secretion and prevented NASH initiation showing promising anti‐NASH and anti‐fibrotic effects with no side effects. Isolation of benzyl nitrile as a single compound and re‐investigate it in IR‐NASH model is recommended.

Support or Funding Information

This work was funded by Chinese Governmental Scholarship (CSC No. 2017GBJ001933), Beijing, China.

Figure 1
The Effect of Cartilage Thickness on Reconstructions of Range of Motion and Muscle Leverage in Extinct Tetrapods
Authors: Dao Brandon , Chevalier-Horgan Christine L. , Pierce Stephanie E. , Molnar Julia L.
Published: April 2020

Investigating structure‐function relationships in the locomotor system requires an understanding of the complex interactions between bones, cartilage and muscles. This is especially true when examining how these musculoskeletal structures evolved over time and across different environments in both extant and extinct species. We aimed to quantify the effect of different assumptions about cartilage thickness on reconstructions of femur length, hip range of motion (ROM), and muscle leverage in Acanthostega gunnari, one of the earliest known tetrapods and an important species in the vertebrate water‐land transition. We began by examining the hindlimbs of two extant salamanders, Dicamptodon ensatus and Ambystoma mexicanum, using contrast‐enhanced micro‐CT in order to extrapolate the degree to which cartilage thickness might affect femur length in a fossil taxon with salamander‐like morphology. Data visualization software was used to measure the distal and proximal cartilage caps of the femora in each of the extant species. Three‐dimensional modeling software was then used to estimate the center of rotation of the hip joint of Acanthostega. Then, cartilage correction factors were applied based on the measurements from the extant taxa. The limit of ROM was defined as the point at which either the femur contacted the pelvis, or less than 50% of the femoral head remained within the acetabulum. Finally, we used biomechanical modeling software to map muscles onto the bones of Acanthostega and plotted their leverage to examine how assumptions about cartilage affect reconstructions of muscle leverage. Cartilage caps on the proximal end of the femur in Dicamptodon and Ambystoma measured 36% and 14% of femur length, respectively. Taking the most conservative estimate, we modeled Acanthostega with cartilage caps of 0, 7.5 and 13% femur length. The greatest variation in muscle leverage occurred in muscles whose axes of movement were nearly perpendicular to the femur (70–120% of the mean moment arm), as opposed to those muscles whose axes were nearly parallel (less than 10%). We also found that assuming the highest percentage of cartilage increased ROM in protraction and retraction by 15–20° and in elevation and depression by 15–30° but had little to no effect on long‐axis rotation. These results will help to predict the effects of unpreserved soft tissues in future studies that reconstruct ROM and muscle leverage in extinct animals.

Effect of different cartilage correction factors on reconstructed hip muscle leverage in the early tetrapod Acanthostega gunnari.

The y‐axis shows coefficient of variation of muscle leverage (standard deviation/mean moment arms) across cartilage correction factors of 0, 7.5 and 13% femur length. Moment arms were measured in 3 axes of movement for muscles that cross the hip joint. The greatest effects occurred in muscles with lines of action nearly perpendicular to the femur.

Figure 1
ADVANCED YOGA PRACTICE AND REHABILITATION OF RECURRENT SHOULDER DISLOCATION: A CASE REPORT
Authors: singh umesh pal
Published: April 2020

The background of being a physician, yoga practitioner, and recurrent shoulder dislocation sufferer, gave me an opportunity for developing unique and detailed insights into how the design and biomechanics of the human body works, and just as important, how it doesn’t. By successfully applying the knowledge of the structure and function of latissimus dorsi (lat) muscle into my advanced yoga practice, I could be able to rehabilitate my shoulder problem up to a great extent – You can see the difference in the time‐lapse photographs of my body showing striking improvement in my condition.

There are very few muscles in the body whose functionality is as important as the lats. They affect major functions throughout the body i.e., reaching, walking, and respiratory movements. Problem is that most people have uneven lat strength. Keeping them uneven, however, can cause shoulder problems of instability (subluxation and even dislocation), low back, neck, and spine issues.

Optimally strengthening and stretching the latissimus dorsi is critical for overall balance and stability ‐ that is required for everyday tasks. The problem is that lats are a very hard muscle to access and make good use of. But, via yoga practice (gravity assisted and resisted bodyweight exercises), we can keep both the lats (weak and strong) online ‐ consciously engaged – so that tension integrity can be attained and maintained (Sherington’s principles). This understanding can lead to the judicious application of interventions to improve a person’s abilities!

Gravity is the primary mode of intervention ‐ the postural reflexes provide the basis for automatic control of posture, balance, and coordination in a gravity‐based environment. Moreover, Gravity workout can increase awareness of our feelings, help release myofascial and arthrogenic fixations, and mitigate stress response through vagal stimulation by utilizing specific movements or positions, breathing practices, and meditation – you can do them throughout the day to loosen the body and calm the mind.

The exercise program presented in this article can be helpful in optimizing the function of your shoulder.

Results ‐ time‐lapse photographs of the body showing improvement of my condition

Support or Funding Information

NA

Scapular dyskinesis – we can change

Figure 1
Restoration of Dystrophin in Duchenne Muscular Dystrophy by Human iPS Cells Derived Skeletal Muscle Progenitor Cells
Authors: Xuan Wanling , Tang Yaoliang , Ashraf Muhammad
Published: April 2020

Background and Objective

Duchenne muscular dystrophy (DMD) is caused by mutations of the gene that encodes the protein dystrophin. Loss of dystrophin leads to severe and progressive muscle‐wasting in both skeletal and heart muscles. Human induced pluripotent stem cells (hiPSCs) offer an important opportunity to treat a number of diseases. Here, we investigated whether givinostat, a histone deacetylase inhibitor (HDACi), could reprogram hiPSCs into muscle progenitor cells (MPCs) for DMD treatment.

Methods and Results

hiPSCs were treated with CHIR99021(10 μM) for two days and followed by givinostat (100 nM) for 5 days in E6 medium (Fig.
1). After culture in E6 medium for additional seven days, cells were analyzed for myogenic makers (Pax7, desmin) expression. Reprogrammed cells (MPCs) from 4 human iPS cell lines expressed Pax7 and desmin. MPCs were differentiated into myotubes in N2 or DMEM medium with 2% horse serum expressing MF20. Additionally, we also generated MPCs from hiPSCs using CHIR99021 and FGF according to a previous study (Fig.
2) and referred these cells as control‐MPCs. We observed that givinostat induced MPCs exhibited superior proliferation and migration capacity using CCK‐8 assay, colony assay and migration assay compared to control‐MPCs. Moreover, we transplanted human myoblasts (1×105) and MPCs (1×105) labeled with GFP into tibialis anterior (TA) muscles of Mdx/scid mice, after injury with cardiotoxin (CTX) for one day prior to cell transplantation. One month after transplantation, dystrophin and GFP double positive fibers were analyzed. Givinostat induced MPCs showed higher engraftment capacity and restoration of dystrophin than treatment with control‐MPCs and human myoblasts. In addition, givinostat induced MPCs treated muscle contained significantly less inflammatory cells and showed limited muscle necrosis and fibrosis.

Conclusion

We successfully generated highly expandable and integration free MPCs from multiple hiPS cell lines using CHIR99021 and givinostat. Givinostat induced MPCs showed marked and impressive regeneration and restored dystrophin in injured tibialis muscle compared to control MPCs. It is concluded that hiPSCs pharmacologically reprogrammed into MPCs with a small molecule, givinostat might be effective cellular source for treatment of muscle injury and restoration of dystrophin in DMD.

Support or Funding Information

National Institutes of Health grants R01 HL134354 & R01 AR070029 (M Ashraf, Y Tang, NL Weintraub)

Schematic outline of generation of MPCs from hiPSCs using CHIR99021 and givinostat (Givi) small molecule combo.

Figure 1

Schematic outline generation of control MPCs from hiPSC.

Figure 2
Peptide barcoding for high‐throughput functional evaluation of antibodies without immobilization
Authors: Aoki Wataru , Matsuzaki Yusei , Ohtani Yuta , Kajiwara Kaho , Miyamoto Kana , Miura Natsuko , Aburaya Shunsuke , Ueda Mitsuyoshi
Published: April 2020

Background

Display technologies such as phage display and yeast display can evaluate a large number of antibodies in a high‐throughput manner, and have been used to isolate various antibody medicines. In display technologies, the immobilization of antibodies on the cell surface is essential to establish genotype–phenotype linkages, but this immobilization can affect functions of antibodies. Here, we developed a novel methodology, “peptide barcoding”, for high‐throughput quantitative measurement of binding properties of antibodies without immobilization. In the scheme of “peptide barcoding”, unique peptide barcodes are fused with each antibody, and they represent genotype–phenotype linkages. A mixture of different peptide‐barcoded antibodies is reacted with antigens, and peptide barcodes of functional antibodies are identified by mass spectrometry after cleavage by specific proteases. This methodology will enable high‐throughput development of antibodies for experimental reagents, diagnostics, and pharmaceuticals.

Methods

Unique peptide barcodes were designed to have high detectability for mass spectrometry. Peptide‐barcoded nanobodies (anti‐CD4 or anti‐GFP) were produced by Pichia pastoris. A mixture of peptide‐barcoded nanobodies were reacted with antigen‐coated magnetic beads. Peptide barcodes of functional nanobodies were cleaved by a specific protease, enterokinase, and released peptides were identified by mass spectrometry. Precise dissociation constants of nanobodies were measured by BIACORE T200.

Results & Discussion

We designed peptide‐barcoded anti‐CD4 nanobody or anti‐GFP nanobody, and successfully produced them using P. pastoris. We measured the values of dissociation constants of the peptide‐barcoded nanobodies using surface plasmon resonance, and confirmed that addition of peptide barcodes didn’t affect affinities of these nanobodies. To demonstrate high‐throughput evaluation of free nanobodies, we produced peptide‐barcoded nanobody mutants, and mixed them with antigen‐coated magnetic beads. Released peptide barcodes from functional nanoboides were quantified by selected reaction monitoring, and we successfully ranked nanobody mutants in order of affinity. This methodology has potential scalability and could evaluate affinities of hundreds of antibodies in one shot [
1,
2].

Support or Funding Information

This research was supported by JST, CREST (grant number JPMJCR16G2), Japan.
Genome‐wide Association Study of Sperm Resistance to Freezing in Horses
Authors: Nikitkina Elena , Kudinov Andrey , Atroshchenko Mikhail , Dementeva Natalia , Pozovnikova Marina , Larkina Tatiana , Shcherbakov Yuri , Krutikova Anna , Musidray Artem
Published: April 2020

The use of cryopreserved sperm has become a routine in horse breeding. Genetic factors affecting cryoresistance remain poorly understood. The aim of the study was to search for genome‐wide associations with a decreasing in sperm motility after freezing. Semen of 96 horses was frozen. Sperm motility was evaluated by computer‐assisted semen analysis (CASA)( ArgusSoft, Russia) before and after freezing. The evaluation was carried out using an Axio Imager microscope (Carl Zeiss Microscopy GmbH, Germany). DNA was isolated from sperm samples for genotyping on Affymetrix Equine HD array 96 SNP chips. Calculations were performed using the PLINK and EMMAX programs. The Genome‐Wide Association Studies (GWAS) was used for the analysis of genomic associations. As a result 45 significant associations were found. 14 are on the first chromosome in region 1: 166861063‐167517944 in the vicinity of the LOC11769038 gene. It is assumed that the polymorphisms found are associated with the formation of functional resistance of spermatozoa to cryopreservation. Studies have opened up the possibility of studying the changes in the cells after freezing. Found SNPs markers will be used to select males whose semen is suitable for cryopreservation.

Support or Funding Information

The study was supported by the Russian Science Foundation (project No. 18‐16‐00071)

Manhattan plot of GWAS

Figure 1
Structure, Function and Mechanism of AztD, a Zinc Metallochaperone
Authors: Yukl Erik T. , Neupane Durga Prasad , Fullam Stephanie , Chacón Kelly
Published: April 2020

Zinc is an essential element that is toxic in excess. Thus, zinc homeostasis is a critical function for all organisms. In bacteria, it is generally maintained by transcriptional regulation of zinc import and export proteins by zinc‐responsive transcriptional regulators. Disruption of zinc homeostasis in bacterial pathogens can result in dramatic attenuation of virulence, making these systems potential targets for the development of novel antibiotics. In particular, zinc importers of the ATP binding cassette (ABC) type are critical for the survival of many bacteria under zinc starvation conditions. Bacterial ABC transporters rely on a periplasmic or lipoprotein solute binding protein (SBP) to bind zinc with high affinity and specificity and deliver it to the membrane permease for import into the cytoplasm. Recently, the participation of other extracellular zinc‐binding proteins (e.g. metallochaperones) in metal import through ABC transporters has been demonstrated in some species. We are studying zinc homeostasis in Paracoccus denitrificans, an organism with two zinc‐specific ABC transporter operons znuABC and aztABCD. These are under transcriptional control of the zinc uptake regulator (Zur), are highly upregulated during zinc deprivation, and are required for normal growth under these conditions. Further, both operons are highly conserved in several human pathogens, including Klebsiella pneumoniae, a member of the carbapenem‐resistant Enterobacteriaceae (CRE) considered an “Urgent Threat” by the Centers for Disease Control. The aztABCD operon encodes a periplasmic metallochaperone (AztD) capable of transferring zinc to the associated SBP (AztC) through a specific, associative mechanism that can be followed in vitro using intrinsic tryptophan fluorescence. Crystal structures of AztD and AztC combined with kinetic data and mutational studies are beginning to reveal the function of AztD in zinc homeostasis and the molecular mechanism of zinc transfer. The observation that AztD is conserved in more than 500 genomes from diverse bacterial taxa suggests that this protein is an important component of zinc homeostasis and a newly identified family of zinc metallochaperones.

Support or Funding Information

National Institutes of Health 1R01GM122819‐01A1

Docking model of the zinc transfer complex between the metallochaperone AztD (green) and the solute binding protein AztC (magenta)

Figure 1
Deep Learning‐Based Point‐Scanning Super‐Resolution Imaging
Authors: Manor Uri , Fang Linjing , Howard Jeremy , Monroe Fred , Weiser Sammy , Kastner Kyle , Kirk Lyndsey , Harris Kristen , Pekkurnaz Gulcin , Yoon Blenda , Schiavon Cara , Zhang Tong
Published: April 2020

Point scanning imaging systems (e.g. scanning electron or laser scanning confocal microscopes) are perhaps the most widely used tools for high resolution cellular and tissue imaging. Like all other imaging modalities, the resolution, speed, sample preservation, and signal‐to‐noise ratio (SNR) of point scanning systems are difficult to optimize simultaneously. In particular, point scanning systems are uniquely constrained by an inverse relationship between imaging speed and pixel resolution. Here we show these limitations can be mitigated via the use of deep learning‐based super‐sampling of undersampled images acquired on a point‐scanning system, which we termed point‐scanning super‐resolution (PSSR) imaging. Oversampled, high SNR ground truth images acquired on scanning electron or Airyscan laser scanning confocal microscopes were ‘crappified’ to generate semi‐synthetic training data for PSSR models that were then used to restore real‐world undersampled images. Remarkably, our EM PSSR model could restore undersampled images acquired with different optics, detectors, samples, or sample preparation methods in other labs. PSSR enabled previously unattainable 2 nm resolution images with our serial block face scanning electron microscope system. For fluorescence, we show that undersampled confocal images combined with a multiframe PSSR model trained on Airyscan timelapses facilitates Airyscan‐equivalent spatial resolution and SNR with ~100x lower laser dose and 16x higher frame rates than corresponding high‐resolution acquisitions. In conclusion, PSSR facilitates point‐scanning image acquisition with otherwise unattainable resolution, speed, and sensitivity.

Linjing Fang1

Fred Monroe2

Sammy Weiser Novak1

Lyndsey Kirk3

Cara R. Schiavon1

Seungyoon B. Yu4

Tong Zhang1

Melissa Wu1

Kyle Kastner5

Yoshiyuki Kubota6

Zhao Zhang7

Gulcin Pekkurnaz4

John Mendenhall3

Kristen Harris3

Jeremy Howard8

Uri Manor1

1. Waitt Advanced Biophotonics Center, Salk Institute for Biological Studies, La Jolla, CA, USA

2. Wicklow AI Medical Research Initiative, San Francisco, CA, USA

3. Department of Neuroscience, Center for Learning and Memory, Institute for Neuroscience, University of Texas at Austin, Austin, TX, USA

4. Neurobiology Section, Division of Biological Sciences, University of California San Diego, La Jolla, CA, USA

5. Montreal Institute for Learning Algorithms, Université de Montréal, Canada

6. Division of Cerebral Circuitry, National Institute for Physiological Sciences, Okazaki, 444‐8787 Japan

7. Texas Advanced Computing Center, University of Texas at Austin, Austin, TX, USA

8. Fast.AI, University of San Francisco Data Institute, San Francisco, CA, USA

Figure 1
WWOX possesses N‐terminal cell surface‐exposed epitopes WWOX7‐21 and WWOX7‐11 for signaling cancer growth suppression and prevention in vivo
Authors: Chang Nan-Shan , Wang Wan-Jen , Ho Pei-Chuan , Nagarajan Ganesan , Chen Yu-An , Kuo Hsiang-Ling
Published: April 2020

Membrane hyaluronidase Hyal‐2 supports cancer cell growth. Inhibition of Hyal‐2 by specific antibody against Hyal‐2 or pY216‐Hyal‐2 leads to cancer growth suppression and prevention in vivo. By immunoelectron microscopy, tumor suppressor WWOX is shown to be anchored, in part, in the cell membrane by Hyal‐2. Alternatively, WWOX undergoes self‐polymerization and localizes in the cell membrane. Proapoptotic pY33‐WWOX binds Hyal‐2, and TGF‐β induces internalization of the pY33‐WWOX/Hyal‐2 complex to the nucleus for causing cell death. In contrast, when pY33 is downregulated and pS14 upregulated in WWOX, pS14‐WWOX supports cancer growth in vivo. Here, we investigated whether membrane WWOX receives extracellular signals via surface‐exposed epitopes, especially at the S14 area, that signals for cancer growth suppression and prevention. By using a simulated 3‐dimentional structure and generated specific antibodies, WWOX epitopes were determined at amino acid #7 to 21 and #286 to 299. Synthetic WWOX7‐21 peptide, or truncation to 5‐amino‐acid WWOX7‐11, significantly suppressed and prevented the growth and metastasis of melanoma and skin cancer cells in mice. Time‐lapse microscopy revealed that WWOX7‐21 peptide potently enhanced the explosion and death of 4T1 breast cancer stem cell spheres by ceritinib. This is due to rapid upregulation of proapoptotic pY33‐WWOX, downregulation of prosurvival pERK, prompt increases in Ca+2 influx, and disruption of the IκBα/WWOX/ERK prosurvival signaling. In contrast, pS14‐WWOX7‐21 peptide dramatically increased cancer growth in vivo and protected cancer cells from ceritinib‐mediated apoptosis in vitro, due to a prolonged ERK phosphorylation. Further, specific antibody against pS14‐WWOX significantly enhanced the ceritinib‐induced apoptosis. Together, the N‐terminal epitopes WWOX7‐21 and WWOX7‐11 are potent in blocking cancer growth in vivo. WWOX7‐21 and WWOX7‐11 peptides and pS14‐WWOX antibody are of therapeutic values in suppressing and preventing cancer growth in vivo.

Support or Funding Information

Ministry of Science and Technology, Taiwan, National Health Research Institute, Taiwan, and Department of Defense, USA

WWOX peptides regulate breast cancer cell sensitivity to ceritinib.

Figure 1