bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipopolysaccharide‐Induced Inflammation does not Alter Spinal Cord Excitability in Female or Male mice
Authors: Putnam Shea , Criddle Connor , Lopez Alejandro , Wilkinson Katherine A.
Published: November 2023

Peripheral inflammation can alter the excitability of dorsal horn interneurons and increase flexor reflex strength (Dubner & Ruda, 1992), but the effect of peripheral inflammation on the stretch reflex is not as well understood. Our lab has preliminary evidence that 18 hrs of systemic inflammation induced with lipopolysaccharide (LPS; a bacterial coat protein) causes a blunted response to muscle vibration in mouse muscle spindle afferents, the sensory neurons in the stretch reflex. This would suggest that muscle stretch reflex strength following LPS would be decreased if there were no changes in spinal cord excitability. To test the hypothesis that LPS induced systemic inflammation alters spinal cord excitability, we measured the H reflex, or electrical analog of the stretch reflex, in mice receiving an injection of LPS (0.5 mg/kg) or saline (200 μl) 18 hrs before experimentation. Mice were anesthetized with an intraperitoneal injection of ketamine (100mg/kg) and xylazine (10mg/kg). The sciatic nerve was exposed and stimulated with bipolar tungsten electrodes at current strengths from H wave threshold (T) to 8T (20 × 0.1 ms pulses at 0.1Hz). Bipolar recording electrodes were placed in the 4th dorsal interossei muscle and the results compared between adult male (n=9) and female (n=10) mice given LPS or saline (n=10 for both). We measured the maximum M wave amplitude (Mmax), H wave amplitude (Hmax), and latency of both waves. The percentage of motor neurons activated by electrical stimulation of the muscle spindle afferents (Group Ia) was measured by the ratio of Hmax/Mmax. Increased spinal cord excitability would be reflected in a larger Hmax/Mmax, regardless of any concurrent changes in muscle spindle afferent excitability. No difference in Hmax/Mmax was observed with LPS treatment in either sex (M control = 0.27 +/− 0.08; M LPS = 0.26 +/− 0.08; F control = 0.31 +/− 0.19; F LPS = 0.33 +/− 0.13). Similarly, no change in latency of the H wave was observed with LPS treatment in either sex (M control = 6.9 +/−1.1 ms; M LPS = 6.2 +/− 0.8 ms; F control = 6.1 +/− 1.2 ms; F LPS = 5.9 +/− 0.6 ms). Our results indicate that spinal cord excitability of the stretch reflex is unchanged following LPS treatment. Future studies will determine whether stretch reflex strength is decreased following LPS as would be expected since sensory input is decreased but spinal cord excitability is unchanged.

Support or Funding Information

This work was supported by a CSUPERB New Investigator grant (KAW).
Genome‐wide Assessment of the Pompe (Gaa−/−) Mouse Cervical Spinal Cord Confirms Widespread Neuropathology
Authors: Turner Sara , Hoyt Aaron , Falk Darin , Byrne Barry , Fuller David
Published: November 2023

Pompe disease, caused by deficiency of acid alpha‐glucosidase (GAA), leads to glycogen accumulation, disruption of cellular architecture and neuromuscular impairments. Respiratory and lingual motor systems often decline first, and respiratory motoneurons typically show profound histopathology and glycogen accumulation at an early age. Although neuropathology is now firmly established in Pompe tissues from both humans and animal models, the associated mechanisms have not been evaluated. Therefore, we studied the cervical spinal cord of adult Pompe (Gaa−/−) and wild‐type (129SVE) mice using histological methods and mRNA analyses with the Affymetrix Mouse Gene Array 2.0ST. Pathway analyses of the mRNA data were done using the Broad Institute's Molecular Signature Database. In separate animals, cervical tissues were stained for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and ionized calcium binding adaptor molecule 1 (IBA‐1) to assess DNA fragmentation and microglial morphology, respectively. From these analyses, several themes emerged (FDR q values <0.01). First, neuronal loss is an important feature of Pompe disease, and occurs in motoneurons before the onset of overt respiratory symptoms. Putative phrenic motoneurons were TUNEL‐positive, and pathway analyses showed significant upregulation of apoptotic processes in the Gaa−/− cervical spinal cord. Second, pro‐inflammatory mRNA signaling is significantly upregulated in the Gaa−/− cervical cord, and this is associated with greater neuronal co‐localization of microglia. Third, multiple signal transduction pathways are altered in the Gaa−/− cervical spinal cord. Glutamatergic signaling pathways were downregulated, as were well‐known “synaptic plasticity pathways” including genes related to long‐term potentiation. Finally, there was a robust change in cellular metabolism pathways in the Gaa−/− spinal cord that included upregulation of macromolecule, lipid, carbohydrate, and protein metabolic pathways. These data confirm that absence of functional GAA protein triggers a complex neurodegenerative process, and suggest that alterations in synaptic transmission and neuroplasticity processes may contribute to motor phenotypes in Pompe disease. Most importantly, the results underscore the importance of therapeutically targeting neuropathology in Pompe disease.

Support or Funding Information

CTSA U54‐TR001012; Craig H. Neilsen Foundation 313369 (SMFT); 2R01HD052682‐06A1 (DDF, BJB)
Single Leg Plantar Flexion Elicits Greater Calf Muscle Oxygen Extraction in Peripheral Arterial Disease
Authors: Luck J. Carter , Muller Matthew D. , Ross Amanda J. , Leuenberger Urs A. , Sinoway Lawrence I
Published: November 2023

Peripheral arterial disease (PAD) is characterized by atherosclerosis in the large arteries of the lower extremity resulting in impaired oxygen delivery to the gastrocnemius muscle and exercise intolerance. However, large artery hemodynamics do not fully explain symptomology or the responses to therapy in PAD. Recent studies suggest that microvascular and/or mitochondrial dysfunction also contribute to the development of leg pain and poor walking performance. Near‐infrared spectroscopy (NIRS) is increasingly being used to noninvasively measure the oxygen hemoglobin saturation (SmO2) changes that occur within the skeletal muscle during exercise. A reduction in SmO2 during exercise is indicative of greater oxygen extraction. In this study we tested the hypothesis that single leg dynamic plantar flexion elicits enhanced oxygen extraction in PAD vs. healthy subjects. Eight patients with PAD (ankle‐brachial index: 0.64 ± 0.03) and eight age and sex‐matched healthy subjects underwent single leg dynamic plantar flexion exercise in the supine posture. Subjects exercised until fatigue or a maximum of 14 minutes at 20 contractions per minute (0.5 kg to 7.0 kg) with the most symptomatic leg. The primary outcome variable was the amount of oxygen extracted from the medial gastrocnemius during exercise. Heart rate (HR) and mean arterial pressure (MAP) were also quantified on a beat‐by‐beat basis. Changes from baseline to the last 20 seconds of exercise were calculated. Because of symptomatic leg pain, PAD patients terminated exercise at lower workloads. Gastrocnemius muscle oxygen extraction at the end of exercise was significantly greater in PAD patients (78% ± 11%; M ± SEM) than in healthy subjects (23% ± 5%, P < 0.001). Similarly, the minimum SmO2 at the end of exercise was lower in PAD compared to healthy subjects (15% ± 6% versus 60% ± 6%; p < 0.001). HR increased similarly in PAD compared to healthy subjects (15 ± 2 bpm versus 11 ± 2 bpm) whereas the increase in MAP was augmented in PAD (19 ± 2 versus 12 ± 3 mmHg; P = 0.03). The recovery phase SmO2 was blunted in PAD and full recovery was not attained within three minutes post exercise. In conclusion, a threefold increase in oxygen extraction (vs. healthy subjects) is observed in the medial gastrocnemius of PAD patients in response to plantar flexion exercise.

Support or Funding Information

Experiments supported by P01 HL096570 (Dr. Sinoway), UL1 TR00127 (Dr. Sinoway), KL2 TR00126 (Dr. Muller), R25 HL115473 (J. Carter Luck). The authors appreciate the technical support of their CRC research collaborators.
The effect of fan use and folic acid supplementation on thermal and cardiovascular strain in healthy aged humans during heat exposure
Authors: Gagnon Daniel , Romero Steven A , Ngo Hai , Poh Paula YS , Kouda Ken , Cramer Matthew N , Jay Ollie , Crandall Craig
Published: November 2023

Attenuated heat loss and altered cardiovascular adjustments during heat stress are considered important contributing factors to the increased prevalence of death in aged individuals during heat waves. Interventions aimed at reducing thermal and cardiovascular strain during heat stress in the aged are lacking. We therefore examined if fan use (by improving sweat evaporation) or folic acid supplementation (by improving cutaneous vasodilation) can mitigate thermal and cardiovascular strain during heat exposure, as well as modify the limit of thermal compensability in healthy aged humans. Six healthy older individuals (69 ± 3 y, 166 ± 12 cm, 72 ± 16 kg, 2 males/4 females) rested in a thermal chamber at a temperature of 42°C and a relative humidity initially maintained at 30%. After a 30 min baseline period, relative humidity was increased in 2% steps every 5 min until a relative humidity of 70% was reached (100 min). The protocol was performed with no intervention (CON), with a fan that provided ~4 m/s of air velocity (FAN), and following 6 weeks of folic acid supplementation (FOLIC). Esophageal (Tes) and mean skin (Tsk) temperatures, local forearm cutaneous vascular conductance (laser‐Doppler, CVC), forearm vascular conductance (Doppler ultrasound, FVC), local sweat rate (ventilated capsule), heart rate and blood pressure were measured throughout. At 70% relative humidity, fan use and folic acid supplementation both increased CVC relative to control (CON: 1.34 ± 0.24 vs. FAN: 1.95 ± 0.39 vs. FOLIC: 1.81 ± 0.35 units/mmHg, P≤0.05). However, this did not translate into different FVC responses (CON: 4.86 ± 1.95 vs. FAN: 5.08 ± 1.91 vs. FOLIC: 4.41 ± 1.78 mL/min/mmHg, P>0.05). Fan use also increased local sweat rate (0.84 ± 0.27 mg/min/cm2) relative to CON (0.60 ± 0.26 mg/min/cm2, P≤0.05), whereas FOLIC had no effect (0.62 ± 0.21 mg/min/cm2, P=0.64). Relative to CON (Tes: 37.62 ± 0.29°C, Tsk: 38.41 ± 0.25°C), esophageal and mean skin temperatures were greater during FAN (Tes: 37.94 ± 0.63°C, Tsk: 38.97 ± 0.55°C, P≤0.05) but not FOLIC (Tes: 37.58 ± 0.21°C, Tsk: 38.28 ± 0.25°C, P>0.05). In contrast, heart rate and blood pressure were similar during all conditions (both P>0.05). The critical humidity at which an inflection in esophageal temperature was observed did not differ between conditions (CON: 63 ± 2 vs. FAN: 64 ± 5 vs. FOLIC: 63 ± 4%, P=0.64). Under the conditions employed, fan use results in greater esophageal and mean skin temperatures during extreme heat exposure in healthy aged individuals while folic acid supplementation has no effect. Furthermore, neither intervention reduced cardiovascular strain nor did they modify the limit of thermal compensability.

Support or Funding Information

Funded by the National Institutes of Health (GM‐068865).
Aging of the antioxidant/inflammatory axis in human lung epithelial cells in vitro mimics aging in animal studies
Authors: Zhou Lulu , Forman Henry Jay , Zhang Hongqiao
Published: November 2023

The balance of cellular antioxidant/inflammation shifts with aging, which results from increased pro‐inflammatory cytokines and inhibition of the Nrf2 pathway. Human small airway epithelial (SAE) cells were stimulated with lipopolysaccharide at 5 μg/ml and sulforaphane at 2 μM for 12 hours in vitro. The mRNA of cell inflammatory (NF‐κB‐dependent) and Nrf2 mediated production of antioxidant (phase II) genes were analyzed at early and late passage level of SAE. It was discovered that with the treatment of sulforaphane, phase II gene expression decreases as cell passage increases. GCLC and GCLM genes were down regulated, which may result from decreased capacity of cells to respond to electrophiles. However, upon treatment with lipopolysaccharide, NF‐kB dependent genes IL‐1 and TNF‐a were up regulated in company with inhibited phase II gene expression. The up‐regulation of pro‐inflammatory gene expression together with inhibition of Nrf2 mediated genes suggests that aging human epithelial cells gradually lose the ability to induce antioxidant defense, permitting a greater NF‐kB‐regulated increase in inflammatory cytokines. This mimics what has been observed in several previously reported studies of animal organs.

Support or Funding Information

Supported by NIH grant ES023864
Ampakines Increase Spinal Respiratory Motor Output after Cervical Spinal Cord Injury in Rats
Authors: Streeter Kristi , Gonzalez‐Rothi Elisa , Sandhu Milap , Baekey David , Greer John , Fuller David
Published: November 2023

Glutamatergic synaptic transmission is a fundamental component of the neural drive to breathe, and α‐Amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid receptor (AMPAR) mediated synaptic currents contribute to respiratory rhythmogenesis and respiratory motoneuron depolarization during inspiration. Ampakines are a class of synthetic compounds initially designed to enhance AMPAR‐mediated glutamatergic neurotransmission related to cognition, learning and memory. Ampakines are also a potent respiratory stimulant, particularly during conditions of suppressed respiratory motor output. While ampakines have been safely used in clinical studies, to our knowledge they have never been evaluated with respect to neurological disorders that compromise breathing, such as cervical spinal cord injury (SCI). Therefore, we aimed to test the hypothesis that ampakines increase respiratory motor output following cervical spinal cord injury. To test this hypothesis, we gave ampakine CX717 to urethane anesthetized rats (15–30 mg/kg, i.v.) with chronic (2–8 wks) cervical (C2) hemi‐section injury (C2Hx) or mid‐cervical (C3/C4) lateral contusion injury. In preliminary experiments, ampakines caused a rapid and robust increase in phrenic motor output ipsilateral to the injury and also increased contralateral phrenic motor output to a lesser extent. Responses were similar across time points post‐injury. In addition, we observed that i.v. ampakine caused a rapid change in the discharge of spinal interneurons below the spinal injury in the vicinity of the phrenic motor pool (C4). These initial results indicate that ampakine CX717 can stimulate respiratory motor output following cervical spinal cord injury, and also alters the activity of the mid‐cervical propriospinal network.

Support or Funding Information

Funding: 1R01NS080180‐01A1 (DF) and 1F32NS095620‐01 (KS)
Impact of Free‐Living Nutrition Intervention on Microbiome in College Students at Risk for Disease: FRUVEDomic Pilot Study
Authors: Famodu Oluremi A , Cuff Christopher F , Cockburn Andrew , Downes Marianne T , Murray Pamela J , McFadden Joseph W , Colby Sarah E , Morrell Jesse S , Olfert I Mark , Olfert Melissa D.
Published: November 2023

Objective

We tested the hypothesis that an 8 week, free‐living nutrition intervention (emphasizing increased fruits and vegetables) in 18–28 year old college students “at risk” for Metabolic Syndrome (MetS) will result in significant changes in microbiome and improve health indicators.

Methods

Thirty‐seven subjects were recruited at West Virginia University and randomized into one of three groups: Half fruit & vegetables (“Fruved”); “Fruved” plus low‐refined carbohydrate (Fruved+LowCHO); and “Fruved” plus low‐fat (Fruved+LowFat). Venous blood and stool samples were collected pre and post intervention. Alterations in the fecal microbiome were assessed using high throughput sequencing.

Results

All individuals significantly decreased empty calories (p<0.0001) and increased fiber (p<0.001) and cups of fruit and vegetable (p<0.0001) after 8 weeks, with no differences between groups. Improvements in weight, waist circumference, systolic blood pressure and body fat significantly decreased in all individuals (p<0.05, p<0.0001, p<0.01 and p<0.05 respectively). Additionally, insulin sensitivity improved (p<0.05) and total cholesterol decreased (p<0.05) after intervention. Alpha diversity was found across individuals, but no differences were seen across groups. Although not significant, individuals at “lower‐risk” for MetS at the beginning of the intervention had lower Bacteroidetes and higher Firmicutes than those considered “higher‐risk”. After the nutrition intervention, trends were seen with decreasing Bacteroidetes and increasing Firmicutes in all “at‐risk” groups, however there was no significant differences seen (p=0.109).

Conclusions and Implications

Specific profiles in phylum level microbiota for those considered “at‐risk” of MetS were identified, with individuals of “higher‐risk” having a higher Firmicutes to Bacteroidetes ratio before the intervention, typically associated with obesity and inflammation. Healthy diet produced significant microbiota changes in the “higher‐risk” versus “lower‐risk” for MetS and improved cardiovascular health in all individuals. Future analysis needs to include investigation at the family level of microbiome, as well as between treatment groups in order to identifying markers among “at risk” individuals which may help to direct precision medicine approaches and generate individually tailored nutrition prescription.

Support or Funding Information

Funding provided by the WV Clinical and Translational Science Institute (NIH/MIGMS Award Number U54GM104942) and the WVU Mountains of Excellence Pilot Grant Program
Zinc and ZIP14 (Slc39a14) are Required for Adaptation to ER stress in Mouse Liver
Authors: Kim Min‐Hyun , Aydemir Tolunay Beker , Cousins Robert J.
Published: November 2023

The accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR). UPR leads cells to adaptation against ER stress by improving protein folding capacity and by attenuating the translation of mRNA. However, if stress is too severe or cells fail to maintain cellular functions, ER stress‐mediated apoptosis is triggered. Zinc, an essential mineral, has been known to be required for normal ER function. Zinc restriction can induce the UPR in vitro, suggesting a role for zinc transport in ER stress. Zinc transporter ZIP14 (Slc39a14) is abundantly expressed in liver. ZIP14 transports extracellular and organellar zinc into the cytosol of cells. We found ZIP14 expression was highly increased in mouse liver by treatment with tunicamycin, a potent ER stress inducer. However, the precise roles of zinc and/or ZIP14 in the UPR are unclear. Here we report that Zip14−/− (KO) mice injected with tunicamycin had a higher apoptosis level with impaired adaptation to ER stress in comparison to wild type (WT) mice. During UPR activation, the liver of WT mice accumulated extra zinc. This response was not observed in KO mice. Zip14 KO mice expressed higher levels of ATF4 (Activating Transcription Factor 4) and CHOP (C/EBP homologous protein), proteins which are responsible for ER stress‐mediated apoptosis. In addition, Zip14 KO mice expressed lower levels of ER chaperones, such as GRP78 and GRP94, which help cells adapt to ER stress by improving protein folding ability. Furthermore, we injected WT mice with tunicamycin 2 weeks after feeding different levels of zinc: ZnD (<1mg Zn/kg), ZnA (30mg Zn/kg) or ZnS (180mg Zn/kg) to examine the importance of dietary zinc intake on the adaptation to ER stress. Compared to the ZnA group, mice fed with the ZnD diet showed a higher level of apoptosis with elevated expression of ATF4 and CHOP proteins. In contrast, mice fed with the ZnS diet had significantly suppressed apoptosis, suggesting zinc supplementation may be beneficial for individuals with a high risk of ER stress. In conclusion, based upon these experiments using the Zip14 KO mouse model and dietary zinc manipulation, both adequate zinc intake and functional ZIP14 are essential for adapting to ER stress by regulating the expression of proteins involved in the adaptation and apoptosis pathway of the UPR.

Support or Funding Information

Supported by grant DK94244 from the National Institutes of Health.
Platelets Direct Leukocytes to their Sites of Extravasation
Authors: Zuchtriegel Gabriele , Uhl Bernd , Puhr‐Westerheide Daniel , Pörnbacher Michaela , Lauber Kirsten , Krombach Fritz , Reichel Christoph A.
Published: November 2023

Background

Leukocyte recruitment from the microvasculature to the site of injury or infection is a key event in the inflammatory response. Whereas the principles of this highly complex process have been elucidated in the past decades, it remained poorly understood how these immune cells can ‘find’ their site of extravasation.

Objective

The objective of this study was to identify the exit points of myeloid leukocytes in the inflamed microvasculature and to characterize the mechanisms underlying the recruitment of these immune cells to these sites.

Materials and methods

Interactions of platelets, neutrophils, inflammatory monocytes, and endothelial cells were analyzed by multi‐color in vivo microscopy in the cremaster muscle of male CX3CR‐1GFP/+ mice (exhibiting fluorescence‐labeled monocytes) upon stimulation with CCL2. Expression profiles of adhesion and signaling molecules in the microvasculature were assessed ex vivo by confocal microscopy in cremasteric tissue whole mounts. Different in vitro assays were used to further characterize the underlying mechanisms.

Results

Upon onset of inflammation, circulating platelets immediately adhered at distinct sites in venular microvessels enabling these cellular blood components to capture neutrophils and, in turn, inflammatory monocytes via CD40‐CD40L‐dependent interactions. In this cellular crosstalk, ligation of leukocyte PSGL‐1 by P‐selectin initiates conformational changes in surface‐expressed leukocyte integrins via ERK1/2 MAPK which subsequently promote the successive extravasation of neutrophils and inflammatory monocytes to the perivascular tissue. Conversely, blockade of this cellular partnership resulted in misguided, inefficient leukocyte responses.

Conclusion

Here, we report a previously unrecognized role of platelets as pathfinders navigating neutrophils and inflammatory monocytes to their exit points in the inflamed microvasculature. This platelet‐directed guidance enhances the efficacy of the leukocyte transmigration process and is essential for effective leukocytes responses.

Support or Funding Information

Supported by the CRC914 (DFG)
Genetic Control of Hypertrophic Chondrocyte to Osteoblast Differentiation in Development and Skeletal Disorders
Authors: Cheah Kathryn , Au Tiffany , Wynn Sarah , Tan Tiong , Yip Raymond , Chan Danny
Published: November 2023

Maintenance of bone throughout life requires continuous renewal of osteoblasts (Ob). Osteoblasts, originating from the perichondrium, accompany vascular invasion and lay down endochondral bone to replace cartilage. Using a genetic recombination lineage tracing approach in mice, we showed recently that hypertrophic chondrocytes (HCs), rather than being terminally differentiated cells fated to undergo apoptosis, can become osteogenic cells in fetal and postnatal endochondral bones and persist into adulthood. Moreover, the chondrocyte‐to‐osteoblast transition also occurs in bone repair. However, how the transition of HCs to osteoblasts is controlled molecularly, and the contribution of dysfunction in this process to skeletal dysplasia is not known. Heterozygous human SOX9 mutations cause the skeletal malformation syndrome campomelic dysplasia (CD). Complete inactivation of the Sox9 gene in mice results in failure of cartilage formation, presumably because of failure to express SOX9 target genes such as extracellular matrix genes, Col2a1, Col9a1, Col11a2 and aggrecan. Because all the SOX9 mutations are heterozygous, are distributed throughout the gene and appear to cause loss of function, the CD phenotype has been attributed to haploinsufficiency of SOX9. However SOX9 proteins containing an intact HMG box and a truncated activation domain may act as dominant negatives by competition with the wild‐type for binding to target genes and interfere with interaction with partner factors via the transactivation domain. The mutant proteins may also gain new function (neomorphic). We aimed to understand the underlying mechanism whereby such mutations in SOX9 cause CD and asked whether the HC to Ob transition is compromised as part of the disease pathogenesis. To address these questions, we generated a conditional mouse model of CD by knocking‐in a mouse equivalent of the human CD SOX9 Y440X nonsense mutation (Sox9Y440X), leading to truncation of the protein in the transactivation domain. We found that heterozygous Sox9Y440X/+ mice, display the phenotypic hallmarks of human CD, such as skeletal malformation, inner ear defects, except sex reversal. We compared the phenotypes of mice heterozygous for the Sox9Y440X and for the null allele. We found that Sox9Y440X heterozygous mutants displayed a more severe skeletal phenotype especially with respect to campomelia (bowing of limbs) than in Sox9−/+ mice. Since Sox9 is known to inhibit chondrocyte hypertrophy and osteoblast differentiation, we asked whether the chondrocyte to osteoblast transition is affected in the CD mutants. Molecular phenotyping and lineage tracing experiments implicate abnormality in HH signaling in CD mutants that impacts on the HC to osteoblast transition resulting in abnormal osteogenesis that underlies the campomelia phenotype.

Support or Funding Information

Research Grants Council Hong Kong and University Grants Council of Hong Kong [AoE/M‐04/04 & T12‐708/12‐N]
Direct Transformation of Chondrocytes into Bone Cells: Outside of the Box
Authors: Feng Jerry
Published: November 2023

It has been widely accepted that hypertrophic chondrocytes undergo apoptosis prior to endochondral bone formation during limb development and long bone growth. Furthermore, it is generally believed that chondrogenesis is a separate physiological process from osteogenesis, although both processes are closely linked. However, new studies suggest that chondrocytes directly transform into bone cells during long bone formation using a cell lineage tracing approach. To extend the current study, we initially revealed only modest numbers of apoptotic cells but high levels of anti‐apoptotic Bcl‐2 expression in hypertrophic chondrocytes of growth plates, indicating that most of hypertrophic chondrocytes do not go apoptosis. Next, we revealed dividing hypertrophic chondrocytes by BrdU staining, and high alkaline phosphatase activity (early bone marker). Furthermore, ex vivo culture of newborn cartilage on a chick chorioallantoic membrane showed that after 5 days the cells on the periphery of the explants had begun to express Col1 (bone marker) and to produce bone‐like matrices. The cartilage‐specific cell lineage tracing approach in triple mice containing Rosa 26tdTomato (tracing marker), 2.3 Col1GFP (bone cell marker), and Aggrecan CreERT2 (one‐time Tamoxifen‐induced) or Col10‐Cre (activated from E14.5 throughout adult stage) proved the direct transformation of chondrocytes into bone cells in vivo. Importantly, deletion of Bmpr1a in chondrocytes (via crossing Bmpr1a and Aggrecan CreERT2 with one‐time Tamoxifen‐induction) leads to a lack of metaphysis, delayed ossification and malformed epiphysis. As a result, these mice displayed a short status and deformed knee joint. This finding supports the novel concept that the chondrogenesis is an initial phase of endochondrogenesis (phase one to build a health cartilage template for future bone formation), in which some of these chondrocytes go apoptosis, some divide, and many of them become bone cells. In phase two, most chondrocytes directly transform into bone. Taken together, this multi‐pronged approach not only demonstrates that a majority of chondrocytes directly transform into bone cells, but also challenge the dogma that chondrogenesis is a separated process from osteogenesis during endochondral bone formation.

Support or Funding Information

(This study was partially supported by NIH grants DE025014 and R56DE022789 to JQF).
Translating the Trypanosome Surface
Authors: Ferguson Michael A.J. , Güther Maria Lucia S.
Published: November 2023

This talk will cover our current understanding of the African trypanosome cell surface, and in particular the biosynthesis and roles of protein N‐glycosylation and glycosylphosphatidylinositol (GPI) membrane anchoring in this organism.

The peculiarities of these post‐translational modifications, and associated nucleotide sugar metabolic pathways, provide drug discovery opportunities for trypanosomiasis.

The desire to exploit these, and several other, discoveries led groups at The University of Dundee to establish a Drug Discovery Unit that both translates parasite biology through to preclinical candidates and develops drug candidates phenotypically, performing drug target deconvolution at the end of the process. Examples of both approaches will be given.

The Drug Discovery Unit capabilities established for parasitic diseases are also applied to innovative targets in several other therapeutic areas, and some of these will be briefly mentioned.

Support or Funding Information

We are particularly grateful to The Wellcome Trust, which has supported our work on trypanosomatids since 1988. We also thank all of our colleagues in the Division of Biological Chemistry & Drug Discovery and the Drug Discovery Unit who's work we are drawing on in this overview. Finally, we thank Alice and C.C. Wang for the great honor of giving this award lecture.
Piecing Together New Insight Into Glycosidases: structure and imaging
Authors: Davies Gideon
Published: November 2023

Glycosidases are key players in all aspects of glycobiology. They provide mechanistic paradigms for enzyme action, they are crucial to cellular function and they provide tractable and validated drug targets. In this lecture I will present recent work on diverse enzymes under current study in my group focusing on:

New 3‐D structures, defining the topology for new CAZy families (1), and their insights into enzyme specificity and the conformational analysis of enzyme action (reviewed in 2)

Enzyme inhibition (for example 3), including applications in disease processes

Activity‐based imaging of enzyme levels in mammalian cell systems (4)

The power of chemistry and of mechanism‐based inhibitors to probe the structure and function of eukaryotic glycosidases will be central to the talk.

Support or Funding Information

GJD is supported by the BBSRC, Alzheimer's Research UK, and the ERC.
Supplementation with dietary EPA/DHA influences red blood cell fatty acid desaturase estimates and reflects tissue changes in fatty acids in systemic organs
Authors: Davidson Emily Ann , Pickens Charles Austin , Fenton Jenifer
Published: November 2023

Delta‐5‐desaturase (D5D) and delta‐6‐desaturase (D6D) are critical in polyunsaturated fatty acid (PUFA) metabolism. D5D and D6D catalyze the conversion of linoleic acid (LA) into arachidonic acid (AA) and that of alpha‐linolenic acid (ALA) into eicosapentaenoic acid (EPA). Altered PUFA desaturation (i.e., increased D6D and decreased D5D) can exacerbate inflammatory responses. Supplementation with the long chain PUFAs (LCPUFAs) EPA and DHA can influence PUFA metabolism and decrease pro‐inflammatory responses. Furthermore, changes in D5D and D6D activity are associated with obesity, type‐2 diabetes, and dysregulated metabolism. The objective of this study was to determine how increasing dietary EPA and DHA alters the D5D and D6D enzyme activity estimates (EAEs) in the RBC (red blood cell) and systemic tissues. In this study, mice were fed control plus three diets with increasing percent of energy (%en) derived from EPA+DHA. Dietary levels reflected recommended intakes of fish/fish oil by the American Heart Association. These doses translated to 0.1%en, 0.675%en, and 1.8%en from dietary EPA+DHA. Phospholipid (PL) fatty acids (FAs) were isolated from heart, skeletal muscle, spleen, lung, adipose tissue, and RBCs. PL FAs were converted to FA methyl esters and analyzed using gas chromatography. D5D and D6D EAEs were calculated using the ratio of arachidonic/dihomo‐gamma linolenic acid (DGLA) and DGLA/linoleic acid, respectively. We observed a positive correlation between RBC D5D EAEs and tissue D5D EAEs across all tissues. RBC D6D EAEs were positively correlated with muscle and negatively correlated with adipose D6D EAEs. D5D and D6D EAEs varied with EPA+DHA supplementation. When comparing the control vs the 1.8%en EPA+DHA diets, a significant decrease in D5D EAE was observed in heart (−42.51%), lung (−54.99%), spleen (−51.46%), and adipose (−78.06%). No significant difference was observed in muscle D5D EAE. Muscle D6D EAE significantly decreased (−39.95%). In adipose tissue, D6D was significantly increased compared to control (+97.80%). No significant dietary effect on D6D EAE was observed in heart, lung, or spleen with supplementation. RBC desaturase EAEs are reflective of tissue desaturase EAEs. Interestingly, RBC desaturase EAEs remained reflective of tissue desaturase EAEs despite changes in mean D5D and D6D EAEs after EPA+DHA supplementation. The high correlation between RBC desaturase EAEs and tissue desaturase EAEs with increasing dietary EPA and DHA, indicates RBC D5D and D6D EAEs are accurate non‐invasive methods to assess tissue PUFA metabolism. Future studies should investigate the usefulness of RBC desaturases in assessing tissues desaturases in disease models such as obesity, type‐2 diabetes, and metabolic syndrome and how EPA+DHA may alter fatty acid metabolism to favor an anti‐inflammatory profile.

Support or Funding Information

Research Support: NIH R03CA142000
Mammary Gland Structure and Functional Changes in Mouse Model of Chronic Gestational Alcohol Exposure
Authors: Amos‐Kroohs Robyn M , Cheng Adrienne A , Clugston Robin D , Huang Ting‐Ni , Yen Chi‐Liang Eric , Blaner William S , Smith Susan M
Published: November 2023

Little research has been done regarding the effects of moderate ethanol exposure (PAE) on mammary gland development and subsequent milk production during lactation. Pregnant C57/Bl6J females were exposed to 3 g/kg alcohol (EtOH) from gestational day (GD) 12.5 to 17.5 in two half doses, two hours apart using isocaloric maltodextrin (MD) as controls. Most EtOH litters did not have milkspots at lactation day two (L2) suggesting that dam lactation or pup nursing was negatively affected after PAE. Mammary glands were collected from subsequent EtOH dams on L2 and were found to weigh less than MD dams (p<0.05). Carmine staining of mammary glands revealed decreased density of ducts and tubules compared with MD mammary glands preliminarily suggesting abnormal or delayed maturation. Further investigation using weigh‐suckle‐weigh methods discovered that milk letdown during a bout of nursing was significantly decreased in EtOH compared with MD litters at L2 (p<0.05). Mammary glands, liver, and adipose tissue of dams were collected for histology, fatty acid composition and retinoic acid stores, as numerous studies document the adverse effects of alcohol on vitamin A and fatty acid metabolism. Milk was collected from these dams in order to determine composition, particularly fatty acid, protein, and lactose. H & E staining in EtOH mammary glands revealed collapsed lobules and immature ducts, as well as decreased adipose stores shown by Oil Red O staining. However, retinoic acid and fatty acid composition were unaltered between groups. Although total protein concentration did not differ in the milk, free fatty acid composition was significantly altered (p<0.05), suggesting that PAE interferes with de novo synthesis of fatty acids into milk. Further studies will characterize that pathway after PAE as well as determine the lactose content and protein profile in milk and mammary gland. Hormonal changes will also be investigated because PAE has been shown to affect prolactin and progesterone which are essential for initiation of lactation. These studies are essential for determining how lactation after PAE affects nutrition and growth in populations where it is common for the affected child to remain with the birth mother.

Support or Funding Information

AA11085, AA22999, T32DK007665
Unlike Adipokines, Insulin Concentrations in Human Milk Surpass those in Maternal Circulation, are Higher among Overweight Women, and are Associated with Maternal Insulin Resistance
Authors: Young Bridget E , Westcott Claire , Chartier‐Logan Catherine , Hernandez Teri , Barbour Linda , Friedman Jed , Krebs Nancy F
Published: November 2023

The impact of maternal weight status and insulin sensitivity on bioactive components in human milk (HM) remains poorly understood and may have clinical ramifications on the breastfeeding infant. Breastfeeding mother/infant pairs (n=26 normal weight (NW) and n=23 overweight/obese (OW/Ob) mothers) were followed over the first 4 months of lactation. Fasting HM and maternal blood samples were collected at 2‐weeks and 4‐months postpartum. Insulin, leptin, and adiponectin were measured. Maternal HOMA‐IR was calculated from fasting serum insulin and glucose.

Insulin and leptin concentrations were higher in HM of OW/Ob women at 2‐weeks (p<0.05) but only leptin remained higher at 4‐months (p<0.0001). Both HM adiponectin and leptin were significantly lower than maternal concentrations (p<0.0001). Both leptin and adiponectin in HM were positively correlated with maternal serum concentrations at 2‐weeks and 4‐months (p<0.02), but only leptin showed a consistent correlation with maternal BMI at each time point.

In contrast, HM insulin was 98% higher than maternal serum insulin at 2‐weeks (p<0.0001) and 32% higher at 4‐months (p<0.001). This difference held among NW (p<0.01) and OW/Ob (p=0.05) mothers separately and at each time‐point. HM insulin at 2‐weeks was positively related to maternal BMI at 2‐weeks (p=0.017, R2=0.12, n=48), but more strongly related to markers of maternal insulin resistance: fasting maternal insulin (p<0.0001, R2=0.42, n=32) and HOMA‐IR (p<0.0001, R2=0.49, n=32). At 4‐months, HM insulin remained positively related to concurrent maternal BMI (p=0.032, R2=0.12, n=40) but remained more strongly correlated with fasting maternal insulin (p<0.001, R2=0.29, n=38) and HOMA‐IR (p<0.0001, R2=0.37, n=38).

This study was unique in its concurrent assessment of insulin, measured in a fasting state, in both HM and maternal circulation. The potential roles of HM leptin and adiponectin on appetite and growth of the breastfed infant has been previously postulated. The demonstrated elevated concentrations of insulin in HM represent a large exposure of this powerful, appetite suppressing, and anabolic hormone to the breastfeeding infant. This enteral exposure may be especially critical early postpartum when intact hormones in HM can be absorbed directly into the bloodstream. Maternal insulin sensitivity may play a role in regulation of HM insulin concentrations. Infant ramifications of chronic exposure to high concentrations of dietary insulin need to be researched.

Support or Funding Information

University of Colorado CCTSI UL1 RR025780; NIH‐NICHD F32‐HD0978068; NIH‐NIDDK K24‐DK083772 & T32‐DK007658‐21; Center for Women's Health Research Young Investigator Award; Thrasher Research Fund Early Career Award
Maternal high‐fructose in pregnancy adversely affects morphological aspects of the placenta
Authors: Song Aleida , Astbury Stuart , Hoedl Abha , Coursen Nicole , Dixon Walter , Hemmings Denise , Bell Rhonda
Published: November 2023

Background

We have previously reported that pregnant rats fed a high fructose solution developed insulin resistance and hypertriglyceridemia, and that their offspring were smaller at birth than offspring from control (CONT) dams. However, there has been relatively little information about how fetal restriction can be induced by maternal fructose intake and how this leads to increased risks for developing diseases in offspring later in life. We focused our work on the placenta as a key organ to regulate fetal growth since placenta is an important determinants of nutrient supply for the fetus. Therefore, this study examined the effects of high fructose intake on adhesion molecule and nutrient transporter gene expression and morphological features of the placenta at term.

Methods

Female rats received either a 10% fructose solution (Fr, n=9) or distilled water (CONT, n=8) for 3 weeks prior to and during pregnancy. On gestational day 21, placentae and fetuses were dissected free from the uterus, weighed and the ratio of placental:fetal weight was calculated. Three placentae from each rat dam were used to determine total mRNA expression of glucose transporter 1 (GLUT1), fatty acid binding protein1 (FABP1), sodium‐coupled neutral amino acid transporter 2 (SNAT2), vascular endothelial growth factor (VEGF), placental growth factor (PLGF), vascular endothelial growth factor receptor 1 and 2 (VEGFR1, VEGFR2). GAPDH was used as a housekeeping gene. Another three fixed placentae were stained by in situ hybridization and measured for GLUT1 expression between labyrinth zone(LZ)/junctional zone(JZ). P<0.05 was considered statistically significant.

Results

Fetal weights at gestational day 21 were reduced in the Fr vs. CONT group (Fr=3.38±0.13, CONT=3.86±0.16 g, p<0.05), although the fetal:placental weight ratio did not differ between groups. Total (relative) mRNA expression of VEGFR1 and VEGFR2 was significantly lower in the placentae from Fr vs CONT (VEGFR1, Fr=0.93±0.06, CONT=1.13±0.07; VEGFR2, Fr=0.98±0.15, CONT=1.66±0.13, p<0.05 for both). The GLUT1 mRNA ratio between LZ/JZ was lower in Fr than in CONT (Fr=17.19±2.1, CONT=31.06±5.97, p<0.05). Total mRNA expression of VEGF, PLGF, FABP1 and SNAT2 were not different between groups.

Conclusion

High fructose intake reduced the expression of glucose transporters in the placenta and reduced the expression of adhesion molecules with important angiogenic properties. These changes were associated with lower birth weight of the fetus. Such changes to the placenta may contribute to poor fetal growth and developmental programming of the offspring associated with increased risk for metabolic disorders in later life.

Support or Funding Information

NSERC, Faculty of Medicine & Dentistry, University of Alberta
Consequences of Maternal Iron‐Deficiency: Fetal Anemia, and Hypoxia in Select Tissues of the Conceptus
Authors: Woodman Andrew , Mansour Yael , Bourque Stephane
Published: November 2023

Iron deficiency (ID) is the most prevalent nutritional deficiency worldwide, and affects populations across the socio‐economic spectrum. The incidence of ID anemia in pregnant women is of chief concern, with rates as high as 80 % in developing countries, and a prevalence of 23 % in Canada. Maternal ID during pregnancy has been shown to impact fetal iron status and growth trajectories, although the precise mechanisms underlying this intrauterine growth restriction are unknown. We hypothesized that maternal ID causes fetal hypoxia, resulting in asymmetric growth restriction in more severe ID. Female rats (initial age 6 weeks for severe (S‐ID), 9 weeks for moderate (M‐ID)) were fed iron restricted diets throughout pregnancy. Dams were treated with pimonidazole on gestational day (GD)20 to assess hypoxia (<10 mm Hg O2). Maternal and fetal hemoglobin (Hb) levels and body weights were assessed on GD21. Pup organs were excised and weighed to assess degree of asymmetric growth restriction. Tissue immunofluorescence was carried out on GD21 pup tissues with fluorescent probes to detect covalent pimonidazole adducts. Both M‐ and S‐ID groups caused fetal anemia ( fetal Hb reduced 30.9 % and 63.8 %, respectively, compared to control of 9.27 g/dL, P<0.01), despite only S‐ID dams becoming anemic (reduction in Hb 10.9 % and 50.3 % for M‐ and S‐ID, respectively, compared to 13.60 g/dL control Hb, P<0.001). Both M‐ and S‐ID caused fetal growth restriction (reduced weight by 9.1 % and 34.9 %, respectively, compared to control weight of 5.45 g, P<0.001). S‐ID, but not M‐ID, caused asymmetrical growth restriction, wherein relative heart and brain weights increased (60 % and 9.8 %, respectively, compared to control weights of 0.005 g/g and 0.041 g/g, P<0.01), and kidney and liver size decreased (20 % and 15 %, respectively, compared to control weights of 0.005 g/g and 0.067 g/g, P<0.01). M‐ID was associated with increased staining for pimonidazole in fetal livers (P=0.01) and kidneys (P=0.01), but not in brains and placentae, versus respective controls; hypoxia staining is S‐ID fetuses is ongoing. Fetal anemia and growth restriction can occur in the absence of maternal anemia, emphasizing the need for new methods of assessment in the clinical setting. Elucidation of the mechanisms through which ID impacts fetal growth and development will enable us to develop a platform with which we can test novel therapeutics.

Support or Funding Information

AW held an Alberta Innovates Health Solutions Summer Studentship. SB Holds grants from the Canadian Institute of Health Research (CIHR), and the Women and Children's Health Research Institute (WCHRI).
The Effect of Oat Beta‐Glucan on Clinical Lipid Markers for Cardiovascular Disease Risk Reduction: A Systematic Review & Meta‐Analysis of Randomized Controlled Trials
Authors: Ho Hoang Vi Thanh , Sievenpiper John L , Zurbau Andreea L , Mejia Sonia Blanco , Jovanovski Elena , Yeung Fei Au , Jenkins Alexandra L , Vuksan Vladimir
Published: November 2023

Background

Oats are a rich source of β‐glucan, a viscous soluble fibre recognized for its cholesterol lowering properties, and are associated with reduced risk of cardiovascular disease.

Objective

To conduct a systematic review and meta‐analysis of RCTs investigating the cholesterol lowering potential of oat β‐glucan on LDL‐C, non‐HDL‐C, and apoB, for the risk reduction of cardiovascular disease.

Design

MEDLINE, Embase, CINAHL, and the Cochrane Central Register of Controlled Trials were searched. We included Only RCTs of ≥ 3 weeks duration assessing the effect of diets enriched with oat β‐glucan compared with controlled diets on LDL‐C, non‐HDL‐C, or apoB. Two independent reviewers extracted relevant data and assessed study quality and risk of bias. Data were pooled using the generic inverse variance method with random effects models and expressed as mean differences (MD) with 95% confidence intervals (CIs). Heterogeneity was assessed by the Cochran Q statistic and quantified by the I2 statistic.

Results

58 trials (N = 3952) were included in the final analysis. A median dose of 3.5 g/day of oat β‐glucan significantly lowered LDL‐C (MD = −0.19 mmol/L [95% CI: −0.23, −0.14] P < 0.00001) non‐HDL‐C (MD = −0.20 mmol/L [95% CI: −0.26, −0.15] P < 0.00001) and apoB (MD = −0.03 g/L [95% CI: −0.05, −0.02] P < 0.0001) compared to control interventions. There was evidence of considerable unexplained heterogeneity in the analysis of LDL‐C (I2 = 79%) and non‐HDL‐C (I2 = 99%).

Conclusion

Pooled analyses show that oat β‐glucan have a lowering effect on LDL‐C, non‐HDL‐C, and apoB. Inclusion of oat containing foods may be a strategy for achieving targets in cardiovascular disease reduction.