bluuebunny/crossref_metadata_2025 huggingface dataset:
Initiation of olfactory memory in patients after successful treatment of patients with congenital hyposmia – the role of neuroplasticity
Authors: Henkin Robert I , Levy Lucian
Published: June 2021

Congenital hyposmia is a genetic disorder associated with adenylyl cyclase deficiencies. These deficiencies cause smell loss (hyposmia) in these patients. These deficiencies are manifested by lower than normal levels of cAMP and cGMP in nasal mucus which cause olfactory receptor failure. Treatment with phosphodiesterase inhibitors (e.g., theophylline) increases nasal mucus cAMP and cGMP, corrects olfactory failure and initiates detection of olfactory sensory signals. While they can then detect odors they have difficulty in recognizing character of these odors since they have no past experience or memory of these odors. These signals were not previously integrated by limbic and other brain memory systems. However, over time patients learn to associate novel odors with specific objects and learn to recognize the appropriate character of various environmental olfactory stimuli. This usually begins with sweet odors since the largest receptor number in the olfactory epithelium deals with sweet detection. This is the first report of recovery of olfactory recognition with repair of congenital hyposmia. While the olfactory system is hard wired into brain in utero correction of receptor defects is only the first step in the complex process of regaining normal smell function. The second step is activation of central nervous system neuroplasticity by which olfactory recognition occurs. These results were confirmed by functional magnetic resonance imaging studies in which both detection and brain integration of olfactory stimuli were measured.
Enhancements in mossy fibers long‐term potentiation (LTP) are neurotrophin‐specific.
Authors: Jimenez‐Nunez Esther Angelica , Thompson Kenira , Porter James
Published: June 2021

In addition to a clearly defined role of BDNF (Brain‐derived neurotrophic factor) in cell survival and differentiation, this neurotrophin has recently been implicated in hippocampal plasticity, including LTP. Interestingly, the mossy fiber projection to pyramidal cells in area CA3 of the hippocampus contains the highest concentration of BDNF in the CNS (Danzer and McNamara, 2004). Also, BDNF administration results in CA3 hyperexcitability in response to mossy fiber stimulation (H.E. Scharfman et al. 1996). BDNF dramatically enhances mossy fiber field potentials and LTP in vitro (Jiménez et al 08). In vivo BDNF infusions enhance mossy fiber LTP and result in neurogenesis at the mossy fiber synapse (Gomez‐Palacio‐Schjetnan and M. Escobar et al., 2008). Previous work found that BDNF mRNA is upregulated in the hippocampus following in vivo mossy fiber LTP (Thompson et al. 2003). In this study we addressed the question of whether other neurothrophins such as NT3 and NGF are involved in LTP at the mossy fiber synapse. Using a hippocampal slice preparation, we recorded field potentials at CA3 pyramidal cells following high frequency stimulation (HFS; 2 trains 100 Hz, 1 sec duration) of the mossy fiber pathway, and observed the effects of bath application of BDNF (concentration 200 ng/ml). In addition, we recorded field potentials at CA3 pyramidal cells following HFS of the mossy fiber pathway and observed the effects of micropipette application of NT3 (200ng/ml), a tyrosine kinase C (trkC) activator, and NGF (200ng/ml), a tyrosine kinase A (trkA) activator. We found that BDNF alone resulted in mossy fiber LTP, and this effect was further enhanced if BDNF was bath applied prior to HFS whereas enhancement of LTP was not observed following application of NGF or NT3. Our results indicate that BDNF plays a specific role on synaptic plasticity and LTP at the mossy fiber synapse.

Supported by: RCMI (8G12MD007579–27), MBRS‐RISE program (NIH‐NIGMS: R25GM082406)
Expression and functions of inositol monophosphatase (IMPA) in seawater (SW)‐acclimated euryhaline teleosts
Authors: Cramb Gordon , Kalujnaia Svetlana , Gellatly Steven , Hazon Neil , Villasenor Alfredo , Yancey Paul
Published: June 2021

Organic osmolytes are essential for cell volume regulation in SW‐acclimated euryhaline teleosts. Inositol is an essential osmolyte for osmoregulation in both eels and tilapia. De novo synthesis of inositol is dependent on inositol monophosphatase (IMPA), which de‐phosphorylates inositol phosphate produced from glucose 6‐phosphate by myo‐inositol phosphate synthase. In most species, two IMPA genes (IMPAs 1 and 2) with slightly different kinetics and tissue expression profiles have been characterised. As a result of tandem and whole genome duplication events, teleost fish express up to six IMPA isoforms, although in most species some isoforms have been lost during evolution. In the eel, three IMPA1 and two IMPA2 isoforms, and in tilapia four IMPA1 isoforms have been identified which exhibit differential tissue expression. The IMPA1.1 isoform predominates in the major osmoregulatory tissues of the eel, and its expression is upregulated when fish enter SW. Although IMPA1.3 is the most abundant isoform in all tilapia tissues, again it is only IMPA1.1 expression that is increased after SW transfer. In both species, increases in tissue inositol levels are responsible for cell volume regulation in response to the increased extracellular salinity. The volume regulatory effects of inositol are particularly important in tissues directly exposed to the SW environment, such as the gills, skin and fin.

Funding: NERC, UK
Role of Reactive Oxygen Species in Intermittent Hypoxia Induced Sympathoexcitation
Authors: Eubank Wendy L , White Daniel , Moralez Gilbert , Kay Victoria , Watenpaugh Donald E , Raven Peter B
Published: June 2021

The mechanism by which OSA affects the cardiovascular system remains unknown. In healthy adults, acute intermittent hypoxic apnea (IHA) is associated with a sustained increase in muscle sympathetic nerve activity (MSNA) with and without changes in mean arterial pressure (MAP). Centrally generated oxidative stress is implicated in the pathogenesis of OSA. It is probable that the increases in central sympathetic outflow (CSO) are centrally generated reactive oxygen species (CROS) mediated by CROS scavenging the central nitric oxide (CNO) which dampens CSO.

To investigate the role of CROS in IHA induced sympathoexcitation. We tested the hypothesis that pretreatment with N‐Acetyl Cysteine (NAC) will completely suppress the IHA induced increase in CROS and blunt the increase in CSO following 20 min of IHA.

In 5 healthy adults, age 40 ± 19 yrs, ht 180 ± 14 cm, wt 89 ± 14 kg muscle sympathetic nerve activity (MSNA) was recorded from the peroneal nerve prior to during and following a 20 minute IHA induction of sympathoexcitation following oral ingestion of 7mg/Kg of N‐Acetylcysteine (NAC).

Total MSNA was not significantly different pre‐ to post‐IHA with pretreatment of NAC (2539.274 ± 489.981 au Pre‐IHA and 2728.709 ± 727.913 au Post‐IHA).

We conclude that NAC pretreatment attenuated the previously demonstrated elevation in total MSNA and burst frequency after intermittent hypoxia. NIH Grant # HL106431
Recovery of disuse‐induced loss of muscle oxidative phenotype is associated with alternative NF‐κB signaling during reloading
Authors: Remels Alexander , Pansters Nicky , Gosker Harry , Schols Annemie , Langen Ramon
Published: June 2021

BACKGROUND

Loss of skeletal muscle oxidative phenotype (OXPHEN) upon mechanical unloading is evident. However, the molecular mechanisms involved in reloading‐induced recovery of muscle OXPHEN remain unclear. Interestingly, the alternative NF‐κB pathway was recently identified as a novel positive regulator of muscle OXPHEN.

METHODS

We investigated markers and key regulators of muscle OXPHEN, including the alternative NF‐κB pathway, in m. gastrocnemius of mice upon hind limb suspension/reloading (HLS/RL).

RESULTS

Expression levels of oxidative phosphorylation (OXPHOS) sub‐units and oxidative myosin heavy chain (MyHC) isoforms I and IIa decreased after HLS and increased rapidly upon RL. Expression levels of PGC‐1α and PGC‐1β as well as mitochondrial transcription factor A (Tfam) showed a similar response to HLS/RL. Although peroxisome proliferator‐activated receptor γ (PPAR‐γ), nuclear respiratory factor 1 (NRF‐1) and sirtuin 1 (SIRT‐1) levels remained unaltered upon HLS, they were potently induced during the RL phase. Moreover, upon RL, levels of IKK‐α increased and phosphorylation and subsequent processing of P100 to P52 was elevated indicative of alternative NF‐κB activation.

CONCLUSION

RL‐induced recovery of muscle OXPHEN is associated with potent activation of the alternative NF‐κB pathway.

This study was sponsored by the Netherlands Asthma Foundation (NAF 3.2.09.068)
Stretched Fibroblasts Regulate Acetylcholine Receptor (AChR) Expression and Clustering on Skeletal Muscle
Authors: Hicks Michael R , Cao Thanh , Standley Paul
Published: June 2021

Repetitive tasks reduce fine motor control and manipulative therapy improves physical performance. Stretched muscle fascia may influence the contraction of the underlying skeletal muscle by secreting cytokines that regulate AChRs. A co‐culture was setup by seeding fibroblasts on deformable membranes and C2C12 myoblasts on non‐deformable coverslips for 24hrs before transferring the C2C12s two mm above the fibroblasts, within diffusible range of secreted cytokines. Fibroblasts were then subjected to repetitive stretch and/or manipulative therapy. Agrin‐induced myotubes in 2% horse serum and non‐strained C2C12s in co‐culture served as positive and negative controls. After 96hrs, C2C12s were analyzed for AChR expression by Western Blot (N=7) and for clustering by fluorescent imaging (130 images/treatment). Data were analyzed via ANOVA, p<0.05. AChR clusters numbered 196±21 for agrin and 65±16 for non‐strain co‐culture. Manipulative therapy did not affect cluster number 56±12, but repetitive stretch attenuated clusters 38±6. Inversely we found repetitive strain increased AChR expression by >;2‐fold vs. non‐strain, while manipulative therapy did not increase expression. Stretched fibroblasts differentially regulate AChRs on skeletal muscle cells through paracrine mediators. Opposite AChR expression and clustering results suggests that unique extrinsic factors regulate each outcome.

AOA
Endurance exercise training protects skeletal muscle against doxorubicin‐induced damage via PGC‐1α‐mediated adaptations
Authors: Kavazis Andreas N , Smuder Ashley J , Powers Scott K
Published: June 2021

Doxorubicin (DOX) is an antitumor agent used in cancer treatment. Unfortunately, DOX can induce skeletal muscle dysfunction. However, exercise training prior to DOX administration protects against DOX‐induced myotoxicity. Therefore, we tested the hypothesis that exercise‐induced increases in skeletal muscle PGC‐1α can mediate alterations to downstream pathways protecting the soleus from DOX‐induced damage. Rats were assigned to sedentary or exercise groups and paired with either placebo or DOX treatment. Our data indicate that exercise increased PGC‐1α in the soleus muscle and remained elevated in exercised animals treated with DOX. In addition, exercise increased expression of the PGC‐1α‐sensitive mitochondrial fusion protein Mfn2, which also remained increased when exercise was combined with DOX. Finally, PGC‐1α can protect skeletal muscle by inhibiting FoxO transcriptional activity. In this regard, our data demonstrates that DOX results in both increased FoxO nuclear translocation and gene transcription (i.e., increased Murf1, Atrogin‐1 and BNIP3). However, exercise training prior to DOX treatment protected against FoxO activation. These findings suggest that exercise‐induced protection against DOX‐induced myopathy is associated with increases in muscle levels of PGC‐1α that regulate the expression of proteins essential for mitochondrial function and FoxO transcriptional activity.
Greater peak oxygen consumption is associated with lower bone strength in soldiers
Authors: Hughes Julie Marie , Fellin Rebecca Elizabeth , Walker Leila Anne , Frykman Peter Northrop , Seay Joseph Francis
Published: June 2021

In the military population even aerobically fit soldiers are at higher risk of stress fracture than their civilian counterparts. The purpose of this study was to determine the relationship between peak oxygen consumption (VO2) and bone strength in soldiers. Tibiae of 38 male soldiers (height 1.8 ±.08m (SD), mass 83.9 ±13.6kg, age 20.3 ±2. 9yr) were scanned using peripheral quantitative computed tomography at 38% and 66% of the length of the tibia. Bone strength (section modulus (Z) and stress strain index (SSI)) were calculated by Stratec XCT software. VO2 was measured via indirect spirometry. Pearson correlation analysis (sig., p ≤ 0.05) was used to explore relationships between VO2 (relative to body mass (BM) and lean body mass (LBM)) and bone strength. Mean VO2 was 53.4 ±0.9 ml·kg−1. Bone strength was positively correlated with BM, LBM, height, and limb length. Significant negative linear correlations between VO2 (adjusted for BM) and both SSI and Z were found at the 38% site (SSI, r = −0.42; Z, r = −0.400) and at the 66% site (SSI, r = −0.39; Z, r = −0.48). With VO2 normalized to LBM, these correlations became stronger at both the 38% site (SSI, r = −0.55; Z, r = −0.51; both p ≤ 0.001) and the 66% site (SSI, r = −0.56; Z, r = −0.58; both p < 0.001). These data confirm that bone strength is correlated with anthropometric measures; however, the surprising finding was that bone strength was inversely related to peak VO2 in soldiers.
Brachial and middle cerebral artery blood flow responses during sinusoidal leg cycling exercise
Authors: Fukuba Yoshiyuki , Kondo Ayaka , Kikugawa Yuka , Endo Masako Y , Miura Kouhei , Hayashi Naoyuki , Fukuoka Yoshiyuki , Koga Shunsaku
Published: June 2021

To examine the control of peripheral circulation to non‐active muscle and brain during leg cycling exercise, we measured the dynamics of brachial artery (BA) and middle cerebral artery (MCA) blood flow (BF) to sinusoidal (sine) work rate (WR) forcing. Nine healthy subjects performed upright leg cycle exercise with a constant WR for 30 min followed by 16‐min sine WR exercises of 4‐min period fluctuated between 20 W to 60% VO2max. During protocol, we measured pulmonary gas exchange [breath‐by‐breath], heart rate (HR) and mean arterial blood pressure (MAP) [Photoplethysmograph], blood velocity (BV) and cross sectional area of BA [Ultrasound Pulsed‐Doppler], BV of MCA [Transcranial Doppler Ultrasonograph], and forearm and forehead skin BF (SBF) [Laser Doppler] and sweating rate (SR) [Capacitance Hygrometry]. The variable was fitted as y(t)=M+A*sin((2π/T)*t‐θ), where t: time, A/M: relative amplitude, T: period, θ: phase shift. Almost variables traced the sine wave adequately. The phase of variables regarding O2‐transport such as VO2, HR followed sine WR with similar delays (the mean: VO2; 51, HR; 45 °). While the BV in MCA showed the similar phase (73 °), its A/M was small (4.5 %). Contrarily, the response of BA displayed an anti‐phase (175 °) and a large A/M (30 %). The peripheral circulatory responses to nonactive limb and brain is quite different. (Support: JSPS‐KAKENHI, 22370091, 23650390, 24700701, 24247046)
Functional sympatholysis (FS) in rats is graded with exercise but is not NO‐mediated
Authors: Coney Andrew , Wallice Rachel , Ray Clare J
Published: June 2021

FS is the exercise attenuation of sympathetic vasoconstriction. It is unclear if NO is the major mediator of FS or if the size of FS is graded to the level of exercise. Experiments were performed on anaesthetised rats (under UK legislation). Sympathetic vasoconstriction was induced by stimulation of the lumbar sympathetic chain (LSC). EDL contraction was evoked and tension recorded; femoral vascular resistance (FVR) was calculated. Vasoconstriction was evoked during baseline and graded muscle contraction (10–30% max twitch) under control conditions, after NOS inhibition (L‐NAME) and following restoration of baseline (SNP infusion). LSC‐evoked vasoconstriction (an increase in FVR) was attenuated at all levels of muscle contraction. FS was graded and correlated to the peak force at each level. L‐NAME increased baseline FVR and the vasoconstriction, but had no effect on FS at any level of muscle contraction. SNP restored baseline FVR and the vasoconstriction ‐ FS was similar to control conditions. FS was graded to the peak level of muscle contraction, not the force recorded during the sympathetic stimulation per se. Manipulation of endogenous levels of NO changes the magnitude of sympathetic vasoconstriction but does not alter the extent of FS during any of the muscle contractions used. These results are similar to human studies (no role for NO) but differ from animal studies (role for NO; see Saltin et al, 2012).
Store‐operated Ca2+ entry mechanism is involved in glucose‐induced Ca2+ responses and insulin secretion, and is critical for preventing ER stress in primary pancreatic β‐cells.
Authors: Segawa Mayuko , Oslowski Christine M , Severova Severina , Yang Bo , Deeney Jude , Bolotina Victoria M
Published: June 2021

The role of the store‐operated channels and Ca2+ entry mechanism in β‐cell function is poorly understood. Using mouse pancreatic islets and dissociated primary β‐cells, we tested the hypothesis that inhibition of this mechanism may lead to ER stress, and impairment of glucose‐induced Ca2+ influx and insulin secretion. We found that store‐operated cation channels (cat‐SOC) are constitutively active in primary β‐cells. Inhibition of cat‐SOC with 2‐APB or DES caused significant reduction in high glucose‐induced Ca2+ responses in individual β‐cells, and nearly total oblation of insulin secretion by intact pancreatic islets. These effects were similar to that produced by inhibitors of voltage‐gated L type Ca2+ channels. We also found that sustained inhibition of cat‐SOC caused significant ER stress and CHOP expression in intact pancreatic islets. Moreover, functional inhibition of Ca2+‐ independent phospholipase A2β (PLA2g6) mimicked the effects of inhibition of cat‐SOC on glucose‐induced Ca2+ responses, insulin secretion and ER stress. Thus, PLA2g6 and cat‐SOC are critical for major β‐cell functions, and may be used as new therapeutic targets for preservation, or restoration of a normal pancreatic function.
Factors influencing blood pressure and sympathetic nerve activity during a high fat diet in rabbits
Authors: Head Geoffrey A , Burke Sandra L , Barzel Benjamin , Eikelis Nina , Lim Kyungjoon , Armitage James A
Published: June 2021

Short‐term consumption of a high fat diet (HFD) by rabbits results in increased blood pressure (BP), heart rate (HR) and renal sympathetic activity (RSNA) at a time when adiposity is increased. It is unclear how the HFD‐induced changes in circulating insulin, glucose and leptin contribute to these cardiovascular effects. Rabbits were meal‐fed a 13.5% HFD and BP, HR and RSNA were measured in the home cage daily for 3 weeks via implanted telemeters. Plasma insulin, leptin and blood glucose concentrations were measured weekly. Baseline BP, HR and RSNA over 24h were 70±1mmHg, 205±4b/min and 7±1nu. Within 2 days of commencing a HFD, values increased by 2%,18% and 22%. BP and RSNA increased further over 3 weeks due to elongation of the postprandial phase. Thus, the changes from preprandial minimum to postprandial maximum of 4±1mmHg, 51±6b/min and 1.5±1nu were reduced by 79–127% and were associated with elevated insulin, leptin and glucose. Linear regression indicated all were strong predictors, as well as bodyweight, of HFD‐induced changes to the circadian pattern of HR, BP and to a lesser extent RSNA. In conclusion, elevation in BP, HR and RSNA occur in the first few days of a HFD due mainly to maintenance of post‐prandial increases. Increased plasma insulin, glucose and leptin are predictors of the early cardiovascular effects of a HFD. Supported by grants from National Health and Medical Research Council of Australia.
A roadmap for survey design in anatomy education research
Authors: Metzger Keith A , MacKay Robert
Published: June 2021

The transition towards evidence‐based decisions regarding educational interventions, in conjunction with the increase in venues for publication of anatomical educational research, have led to an overall increase in the amount of research related to anatomy education. Many of these studies include the use of surveys, and although the quality of survey‐based educational research is largely dependent upon the reliability and validity of the measurement instruments that are used, few of these studies describe the process used to construct and validate their surveys.

This study aims to 1) briefly review the frequency of survey use in anatomical education research and 2) provide a framework for anatomy education researchers in the design and validation of survey instruments. A survey constructed to assess the impact of an integrated course in human structure on contextual anatomical knowledge is used as an example of this design process. Description of the steps of survey development are outlined, including literature review and synthesis, development of survey items, validation by subject matter experts, cognitive interviewing, and pilot‐testing. Common pitfalls in item construction (e.g., confusing item wording, inappropriate use of anchors) are also discussed.

Grant Funding Source: Lippincott Williams Wilkins/AAA Education Research Scholarship
Guided, self‐directed learning: Building an anatomy learning module for the iPad
Authors: Haugsdal Michael Larson , Pizzimenti Marc A
Published: June 2021

We investigated the potential of iBooks Author for creating a guided, self‐directed learning module (SDLM) on the iPad. Our objective was to qualitatively assess the capacity and ease to yield an interactive, multi‐media module that introduced foundational anatomical concepts. Guided, SDLMs provide learners with an opportunity to practice autonomy and personal learning styles. Modules focus on learner‐centered approaches that provide direction for students to develop topical knowledge and life‐long learning skills. Essential steps to SDLM creation include: identification of knowledge needs, formulation of learning goals, resource selection, application of learning strategies, and evaluation of outcomes. SDLMs that follow this step‐wise process are effective in establishing foundational knowledge, which facilitates further discussion of related, but advanced concepts in lectures or small groups.

While we did succeed in building a guided SDLM with a unique degree of interactive features, development was intensive and required advanced technological skills and use of adjunct software. Furthermore, resources and software capabilities placed limits on the implementation of desired guiding strategies.
Collaborative Key Features Problems in Anatomy: Assessment drives clinical decision making and anatomical learning.
Authors: Terrell Mark , Kalmey Jon , Kulesza Randy
Published: June 2021

This study evaluated the effectiveness of using key features problems (KFP) to drive learning and assess medical students’ abilities to use anatomical knowledge for making sound clinical decisions. KFP is a validated clinical‐decision assessment technique in which examinees select their responses from prepared lists of options, containing correct responses, synonyms, incorrect responses, and common misconceptions. Students completed a unit KFP quiz using individual efforts and then immediately retook the same KFP quiz using small group discussions. Performance on the collaborative exams was higher (P < 0.01) than on the individual‐effort exams and higher on unit examination questions that related to content learned from KFPs than compared to previous years that did not use the KFP approach. Performance on questions on the final comprehensive exam related to content learned by the KFP was predicted to be higher than on other parts of the final exam. Acceptance of the KFP approach was measured through a questionnaire. These results suggest that the KFP approach improved learning and retention of anatomical concepts and supports the hypothesis that good assessment drives learning.
Mobilizing Clinical Imaging, 3D Anatomy, and Digital Microscopy Data for Virtual Reality Learning Objects: HTML5 and Ubiquitous Learning Methods
Authors: Trelease Robert Bernard
Published: June 2021

Web deployed anatomical simulations or “virtual reality learning objects” can be produced with QuickTime VR software (QTVR), but their use for online and mobile learning is constrained by declining support for personal computer web browser plug‐ins and by unavailability of the media format on mobile computing devices like Apple iPad and Android tablets. However, easily implemented image processing methods can now create comparable VR learning objects in the new HTML5 standard format, circumventing these platform‐specific software limitations. Usable data sources include clinical imaging (e.g., MR and CT in DICOM format), existing QTVR objects, digital photographs of anatomical specimens, models, and “virtual microscopy” image arrays. Multiple types or “dimensions” of anatomical information can be embedded in these learning objects, supporting different kinds of online and computer‐resident learning applications, including interactive atlases, examination questions, and complex, multi‐structure presentations. These HTML5 VR learning objects work on mobile devices that do not support QTVR, as well as on personal computers. HTML5 VR learning objects can also be embedded in “ebook” document files, for developing new types of electronic textbooks for tablets and other mobile computing devices, that are increasingly popular and self‐adopted for ubiquitous learning (“learn anywhere”) applications.
Three‐dimensional reconstructions of human embryos: Morphogenesis and hepatic vascularization.
Authors: LHUAIRE Martin , SCHARBARG Emeric , MICARD Émilien , CENDRE Romain , DELMAS Vincent , DUPARC Fabrice , MERCIER Philippe , FELBLINGER Jacques , BRAUN Marc , AVISSE Claude , LABROUSSE Marc
Published: June 2021

OBJECTIVES

The purpose of this study was to describe the morphogenesis of hepatic gland and its vascularization in 4 human embryos using three‐dimensional reconstructions from histological sections, and to compare our results with literature.

MATERIALS AND METHODS

We investigated four embryos belonging to three prestigious historical French collections of human embryos serially sectioned: 7 mm (Carnegie Stage 14), 15 mm (CS 18), 22.5 mm (CS 21) and 27 mm (CS 23) respectively.

Staining included either Masson's trichrome or standard HES. Scanning was performed using an Epson V750 PRO ®, (resolution 3200 dpi). Registration of image stacks and export in DICOM files were performed using original software developed by IADI Laboratory (INSERM U947, NANCY). The images obtained were exploited with ORS ® Visual.

RESULTS

From the 7mm embryo to 27mm, the hepatic gland was very large and filled almost the entire abdominal cavity. The hepatic vascularization was well visualized and individualized. The portal vein and hepatic veins were distinct from the 15mm embryo to subsequent stages.

CONCLUSION

This method of three‐dimensional reconstructions of hepatic morphogenesis brings a new perspective to the study of human embryology.

Grant Funding Source: AAA
Analyzing the effects of sub‐optimal doses of raltegravir on HIV‐1 integration
Authors: Varadarajan Janani , McWilliams Mary Jane , Hazuda Daria , Hughes Stephen
Published: June 2021

Integration of the DNA copy of HIV‐1 genome into the host chromosome is required for viral replication and is, for this reason, an important drug target. The HIV‐encoded enzyme integrase (IN) catalyzes two critical steps: 3′ processing of the viral DNA ends, followed by the strand transfer reaction which inserts the viral DNA into host DNA. Raltegravir is the first approved drug that blocks the integration of the viral DNA. Using the Rous Sarcoma Virus (RSV)‐derived vector system RCAS, we previously showed that mutants in which one of the viral DNA ends was not a good substrate for IN were integrated abnormally, leading to rearrangements of the host genome including duplications, inversions and, occasionally, the acquisition of sequences from other chromosomes. Based on these results, we expected that suboptimal concentrations of IN inhibitors, like Raltegravir, which could also block one end of the viral integration reaction, would cause similar aberrant integrations. In contrast to the normal proviruses recovered from untreated cells, all of which were normal, 10–15% proviruses isolated after treatment with a suboptimal dose of Raltegravir were aberrant and these integrations were accompanied by chromosomal defects similar to those seen with the RCAS system. These rearrangements of the host DNA raise concerns for HIV‐1‐infected patients who do not consistently follow their Raltegravir regimen.
Investigation of Riboflavin binding to DNA using polymerase chain reaction
Authors: Banco Michael , Benore Marilee Ann
Published: June 2021

Riboflavin is water‐soluble vitamin that is a precursor to the coenzymes flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD). It is well documented that Riboflavin, also known as vitamin B2, demonstrates affinity for binding to DNA. Evidence also indicates that Riboflavin plays a role in bacterial gene expression, by binding to riboswitch sites. In our lab we are investigating the binding of riboflavin to eukaryotic DNA, in order to evaluate the affinity and specificity of the binding. We are using polymerase chain reaction (PCR) inhibition to characterize the binding of riboflavin to specific sequences in the Riboflavin Binding Protein gene.
Effects of recognition sequence variations on transcription regulation of multidrug resistance regulator Pdr1p in yeast
Authors: Nishida Nao , Kuroda Kouichi , Ueda Mitsuyoshi
Published: June 2021

For precise transcription regulation, it is important to recognize specific target genes from the genome. Pdr1p is a key transcription factor of multidrug resistance, also involves in organic solvent tolerance in yeast [1]. Pdr1p is one of the transcription factors which recognize pleiotropic drug resistance elements (PDREs). PDRE is 8 bases long, and has known 5 sequence variations, which functional differences are not known. ABC transporters responsible for multidrug resistance harbor PDREs in their promoters, while cell wall proteins responsible for organic solvent tolerance do not [2]. Some one point mutations in Pdr1p cause upregulation of downstream genes, resulting in multidrug resistance. However, from our previous studies, not all genes sharing PDRE at their promoters are regulated in the same way. We changed PDREs in a native promoter to each sequence variation and evaluated downstream transcription levels. We showed that PDRE variations have some effect on transcription levels of downstream genes. The results suggest that PDRE sequence variation may contribute to precise transcription regulation by Pdr1p, by affecting binding affinity or cofactor bindings [3].
Alternative substrate kinetics of Escherichia coli ribonuclease P: Determination of relative rate constants by internal competition
Authors: Yandek Lindsay Elyse , Lin Hsuan‐Chun , Harris Michael
Published: June 2021

A single enzyme, RNase P, processes the 5′ ends of ptRNAs in cells and organelles that carry out tRNA biosynthesis. This substrate population includes over 80 different competing ptRNAs in Escherichia coli. While the reaction kinetics and molecular recognition of a few individual model substrates of bacterial RNase P have been well described, the competitive substrate kinetics of the enzyme are unexplored. To understand how different ptRNA substrates compete for processing by RNase P, we compared the steady state reaction kinetics of two ptRNAs that differ at sequences that are contacted by the enzyme. For both ptRNAs, substrate cleavage is fast relative to dissociation. As a consequence, V/K, the rate constant for the reaction at limiting substrate concentrations, reflects the substrate association step for both ptRNAs. Reactions containing two or more ptRNAs follow simple competitive alternative substrate kinetics in which the relative rates of processing are determined by ptRNA concentration and their V/K. The relative V/K values for eight different ptRNAs, that were selected to represent a range of structure variation, were determined by internal competition in reactions in which all eight substrates are present simultaneously. The results suggest that rates of ptRNA processing by RNase P are tuned for uniform specificity and consequently optimal coupling to precursor biosynthesis.
A single‐molecule fluorescence system for studying adenylate kinase under force
Authors: Wilson Christian A.M. , Leachman Samuel M. , Cervantes Bernardo , Ierokomos Athena , Marqusee Susan , Bustamante Carlos
Published: June 2021

Single‐molecule manipulation has increasingly become a useful method for studying macromolecular dynamics. We have built a novel instrument for force spectroscopy that combines the capabilities of magnetic tweezers and single molecule Förster resonance energy transfer (smFRET). A supermagnet exerts force on 2.1 μm antidigoxigenin‐coated paramagnetic beads tethered to specific proteins under study. These fluorescently labeled proteins are functionalized with DNA handles containing biotin and immobilized on the surface of a flow chamber in a total internal reflection fluorescent (TIRF) microscope via streptavidin interactions. The distance between dyes from each molecule that is attached to a bead in the microscope's field of view can be monitored as a function of force. The model system that we are currently studying is adenylate kinase (AK). This enzyme was successfully labeled with fluorescent dyes and DNA using click chemistry and cysteine chemistry. AK was first characterized in optical tweezers and was found to unfold around 25 pN during force‐extension experiments with a fast 4 nm intermediate at 15 pN. This intermediate could correspond to the ATP binding domain unfolding independently of the rest of the protein. Preliminary fluorescence data from our instrument confirms the existence of this intermediate under force. HHMI‐CB; NIH‐SM
Structural and kinetic differences in oligomerization‐fibrillation of serum amyloid A and not the intrinsic amyloidogenicity may contribute to pathogenesis in AA amyloidosis
Authors: Srinivasan Saipraveen , Patke Sanket , Aguilera Javier J , Kane Ravi S , Colon Wilfredo
Published: June 2021

Acute‐phase protein serum amyloid A (SAA), specifically the isoform SAA1.1, is implicated in murine reactive AA amyloidosis. However, the mechanism of pathogenesis remains elusive to date. The co‐expressed SAA2.1 and the hybrid SAA2.2, found exclusively in CE/J type mouse, are non‐pathogenic. The proteins show remarkable differences in oligomeric propensities, fibrillation kinetics and thermal stabilities in vitro in spite of high degree of sequence homology. Using Thioflavin T fluorescence and atomic force microscopy (AFM), we show that at 37 °C, SAA1.1 aggregates as spherical oligomers and requires several days to form protofibrils and fibrils. SAA2.1 and SAA2.2 rapidly aggregates to form protofibrils leading to fibrillation (Fig. 1). Spherical oligomers showed lipid bilayer permeabilization, not exhibited by protofibrils or fibrils. Mutations relating SAA1.1 and hybrid SAA2.2 show the important role of each of the differing residues on the overall stability, assembly kinetics and aggregation pathway of the two isoforms. These factors will likely play an important role in the diverse pathogenicity among SAA isoforms across species, opening new venues towards understanding the molecular basis of AA amyloidosis and developing novel therapeutics. This work was supported by a Grant from the NIH (R01 AG028158) to W.C.
Muscle fiber typing by identification of the myosin heavy chain using LCMS
Authors: Kasper Joseph , Johnson Daniel , Hayes Erik , King Daniel
Published: June 2021

Identifying skeletal muscle fiber types is valuable to the ongoing research efforts to understand the relationships between structure and function, particularly in humans. While existing methods of fiber typing (gel electrophoresis, histochemistry, etc.) are effective in identifying fiber types, liquid chromatography‐mass spectrometry (LCMS) has the potential for also monitoring many other proteins or metabolites simultaneously. A new method is being developed using the technology of LCMS to match peptide sequences with myosin heavy chain isoforms. Samples of chicken were digested by the enzyme trypsin, followed by analysis using reversed phase LCMS. Detected peptides were mapped to their respective myosin chains based on a theoretical digest list. Using single ion monitoring (SIM), the relative amounts of each peptide were quantified by numerical integration. A blind sample was analyzed and correctly identified. The research seemed to suggest that the use of LCMS for muscle fiber peptide quantification could be a viable alternative to currently used methods. Future research plans are for the study of human muscle samples.
Benefits of fluorescent detection for quantitative Western blotting.
Authors: Grimsby Susanne
Published: June 2021

Western blotting is a well‐established and widely used technique to confirm the identity and presence of proteins from a variety of sources. The development of highly sensitive detection reagents together with advanced imaging techniques has made Western blotting a potential tool for quantitative protein analysis. A prerequisite for obtaining quantitative Western blotting data is that the signal response is proportional to the amount of protein. Fluorescent Western blotting detection is a direct detection method where the secondary antibody is conjugated to a fluorophore. The obtained fluorescent signals are stable and directly proportional to the protein amount. Fluorescent detection systems are typically characterized by their high sensitivity and broad linear dynamic range, and are as such well adapted to quantitative Western blotting. Moreover, fluorescence detection enables simultaneous detection of more than one protein on the same blot (multiplexing), even when the proteins have similar size, by utilizing secondary antibodies conjugated to fluorophores that are spectrally differentiated from each other. This makes fluorescent Western blotting a useful tool for accurate monitoring of changes in protein abundance and for detection of posttranslational modifications.

Here we demonstrate the benefits of fluorescent detection in quantitative Western blotting using Amersham™ ECL Plex™ and Image Quant™ LAS 4010 CCD camera. Proteins are identified and expression levels determined using a multiplexed Western blotting approach..
Dietary Factors Associated with Weight Loss in a Mediterranean Intervention Study
Authors: Sidahmed Elkhansa H , Cornellier Maria , Rapai Mary , Sen Ananda , Djuric Zora
Published: June 2021

The objective of the study was to evaluate dietary factors associated with weight loss in a dietary intervention study that focused on improving diet quality while maintaining baseline energy intakes. The study randomized a total of 120 individuals who were at increased risk for colon cancer to either the Health Eating diet or the Mediterranean diet for six months. The dietary goals for both diets were based on maintaining the baseline weight. To investigate the changes in dietary intakes that lead to weight loss, those subjects who completed the study were classified into tertile groups according to their weight change from baseline to six months. The three categories were: weight gain (increase in weight 1–25 lbs.), weight maintain (with weight change between 3.2–0.7 lbs.) and weight lost (weight loss between 3.3–20.0 lbs). SPSS was used to evaluate the changes in nutrients and foods over time. Changes in dietary intakes of fruit, vegetables, total fat and saturated fat were similar in the three groups. The proportion of subjects in each diet arm did not differ significantly between groups. The weight gainers paradoxically reported a significant reduction in energy intakes. The weight loser group was unique in displaying a significant increase in total protein and animal protein intakes at 6 months. These results indicate that individuals with weight loss increased their protein intakes while maintaining their total calorie. These modest increases in protein might be a viable weight loss strategy.
Protective effect of ginseng leaf extract against UVB‐Induced photoaging in hairless mouse
Authors: Kim Mi‐Ryung , Han JeungHi , Chang Un‐Jae , Suh Hyung Joo
Published: June 2021

The object of this study was to investigate the functionality of Ginseng (Panax ginseng) leaf extract (GLE) as a cosmeceutical material. We adopted Ultraviolet‐B irradiated hairless mice model to evaluate the protective effect of GLE from UVB‐induced photoaging. Skin photodamage was induced by exposure to gradient doses of UVB (36–144 mJ/cm2, for 10 weeks on alternate days). GO groups were given daily administration of ginseng leaf (0.1% and 0.2% of drinking water, respectively) and GA groups were given topical application of ginseng leaf (0.5 mg/cm2 and 1.0 mg/cm2 skin area) to evaluate dose dependent effect. We observed deep wrinkle, erythema and thick skin on the control hairless mice. UVB irradiation increased the expression of matrix metalloproteases (MMPs), which attacked collagen and elastin fiber cross linkage. After GLE treatment, a considerable recovery from photo‐damage was observed by the treatments. GLE reduced the UVB‐induced activation and expression of MMPs via upregulating tissue inhibitor of metalloproteinase (TIMP). Taken together, these results suggest that the protective effect of GLE on skin photoaging induced by chronic UVB exposure may be due to the inhibition of MMP expression and/or the increased TIMP expression.
Effects of participation in popular weight loss and fitness programs on insulin and leptin in women
Authors: Dalton R , Lockard B , Baetge C , Levers K , Galvan E , Jagim A , Simbo S , Byrd M , Jung YP , Oliver JM , Koozehchian M , Khanna D , Sanchez B , Kresta JY , Horrell K , Leopold T , Cho M , Springer S , Rivera A , Cerda C , Rasmussen C , Kreider R
Published: June 2021

100 sedentary women (46±11 yr, 45.8±5% body fat, 35.2±5 kg/m2) were randomized to participate in a no diet or exercise control group (C) or the Curves Complete® 90‐day Challenge (CC), Weight Watchers® Points Plus (WW), Jenny Craig® (JC), or Nutrisystem® Advance Select™ (NS) weight loss programs for 12‐wks. Participants in the diet groups were encouraged to exercise (WW, JC, NS) while those in the CC group participated in a structured circuit‐style resistance training (3 d/wk) and walking (3 d/wk) program. Fasting blood samples were obtained at 0, 4, 8, & 12 wks. Changes from baseline to 12‐wks intervention for fasting insulin, the glucose to insulin ratio, homeostatic model assessment (HOMA), and leptin were analyzed by one‐way ANOVA. Participants in the CC group tended to experience greater changes in fasting insulin (C 0.8±6.9; CC −7.5±14; WW − 2.9±8.1; JC −3.8±6.3; NS −1.2±8.3 uIU/ml, p=0.10), the glucose to insulin ratio (C −1.3±4.1; CC 7.0±14; WW 3.3±5.4; JC 4.8±7.4; NS −6.0±21, p=0.01), HOMA (C 0.1±1.6; CC −2.3±4.4; WW − 0.8±1.7; JC −1.0±1.9; NS −0.4±2.2, p=0.07) and leptin (C 4.3±16; CC −17.9±21; WW −13.0±16; JC −12.2±25; NS −3.5±26 ng/ml, p=0.03) compared to some of the other diet and exercise interventions. Results indicate that participation in different diet and exercise programs may have variable effects on markers of insulin resistance and leptin.
Relationship of total water intake and specific components of water intake to hydration measures: National Health and Nutrition Examination Survey (NHANES) 2001–2010
Authors: Papanikolaou Yanni , Fulgoni Victor L , Lieberman Harris R , Guelinckx Isabelle
Published: June 2021

NHANES data were used to assess the relationship of usual intake of total water, water from food, fluid intake, and specifically water in the diets of those 19+ years of age to hydration measures: plasma osmolality (Posm), urine osmolality (Uosm) and specific gravity (USG). Intake was determined using the National Cancer Institute method and quartiles of intake were determined. Covariate adjusted regression analyses were used to assess p‐for‐trend of intake variables to hydration measures. Posm (N=22,662) was inversely (p<0.001) related to total water, fluid and water intake; Posm decreased 0.36 ± 0.05 mmol/kg, 0.32 ± 0.05 and 0.24 ± 0.05 mmol/kg for each quartile increase in total water, fluid and water intake respectively. Food moisture was not related to Posm. Uosm (N=5,126) and USG (N=4,615) measured on a spot urine collection were inversely (p<0.001) related to total water, fluid and water intake. For each quartile increase in total water intake, fluid intake and water intake, Uosm decreased 41.8 ± 6.1, 40.7 ± 5.9 and 31.2 ± 5.7 mOsm/kg, respectively and USG decreased 0.0010 ± 0.0002, 0.0009 ± 0.0002 and 0.0007 ± 0.0002, respectively. Food moisture was modestly related to Uosm (−12.9 ± 3.9; p=0.0015) and USG (−0.0003 ± 0.0001; p=0.0118). Given the physiological range of variation of Posm, Uosm and USG, Uosm appears to be the most sensitive marker of total water intake and best measure of hydration in the general population. Support: Danone Research.
A whole foods diet with omega‐3 and vitamin D supplementation is associated with improved health outcomes in children with ASD
Authors: Franck Jennifer , Kansy Matt , Noel Sabrina , Tucker Katherine L
Published: June 2021

Children on the autism spectrum have numerous metabolic abnormalities that are often not well controlled and can lead to early chronic disease. Children with ASD often consume very limited diets that tend to be high in processed, simple carbohydrate type foods, which has been associated with an increase in chronic disease risk factors including obesity, dyslipidemia, and elevated insulin secretion/resistance. We pilot tested an intervention with children admitted to our residential program for severe autism. The intervention included a diet of predominantly whole foods, low carbohydrates proportionally (30–45%), plant based, with higher levels of healthy fats (i.e. monounsaturated, pastured animal products: approx 35%), with supplementation of omega 3 fatty acids (350mg EPA, 230mg DHA QD), and vitamin D3 (500–2,000IU QD) for a mean of 12 months. Here we present the results for 5 of these children (boys only) ages 16 to 19 y. After a mean of 9 months on the intervention diet, we saw decreases in total cholesterol and triglycerides, which approached significance (p=0.06), with no significant change in body weight. These results suggest that provision of a mainly whole foods diet with supplementation of omega 3 fatty acids and vitamin D3 could have an important effect on children on the autism spectrum and may reduce their risk of chronic disease. Further research in this population is warranted.