bluuebunny/crossref_metadata_2025 huggingface dataset:
Cristae in Inner Ear Hair Cells Mitochondria are Polarized Toward Cuticular Plate
Authors: Arias Kevin Daniel , Price Steve D , Perkins Guy , Lysakowski Anna
Published: November 2023

The goal of this research is to study the structure and the functionality of mitochondria located in inner ear hair cells. According to size, mitochondria can be divided into three different sub‐types: large, medium, and small. In this particular scenario, our focus rests upon a single large mitochondrion with lamellar cristae (the high energy form of cristae) located in very close proximity to the cuticular plate (CP) in a vestibular Type 1 hair cell. One criterion we have been analyzing is the number of cristae junctions (CJs) facing the CP in comparison with the amount of CJs located on the other side facing away from the CP. CJs are tubular connections promoting a direct interaction between the mitochondrial cristae and the mitochondrial inner membrane (Rabl et al. 2009). We hypothesized that the number of CJs facing the CP is higher compared to the opposite side. This would suggest a polarization of CJs on one side, such that ATP and Ca2+ can be fed to points of interest, such as stereociliar rootlets and the CP (Perkins et al. 2010). In order to identify the desired structures, we have been using IMOD, a 3d reconstruction program developed by the Univ. of Colorado. First, we examined the mitochondrion tomogram section by section and using the software, we traced individual cristae. Doing this for every crista along with the mitochondria membranes, we then meshed it and get a 3D representation of the entire mitochondria in the tomogram. We have taken these data and constructed a short video to enable us to visualize both sides. In addition, IMOD is a tool to obtain accurate topographical data, such as crista surface area, from which we can compute energy production output. This large mitochondrion can produce about 4500 zeptomoles of ATP/sec, much larger than the average amount we have observed in other mitochondria with lamellar cristae, which is about 1000 zeptomoles/sec. This ATP, in turn, produces about 140 femtojoules of energy. After this large mitochondrion was fully reconstructed, we proceeded to analyze the number of CJs. We observed twice as many CJs facing the CP as away from the CP. In conclusion, the data obtained support our hypothesis that CJs are observed in greater amount in sites requiring more ATP.

Support or Funding Information

Supported by NIH R21‐DC013181 (AL) and P41‐RR004050 (GP, ME)
Cristae Align Across Mitochondrial Membranes in Vestibular Hair Cells to Possibly Increase ATP Output
Authors: Patel Meet H , Perkins Guy , Lysakowski Anna
Published: November 2023

The main objective of this research is to study mitochondria in inner ear hair cells from a functional and structural perspective. Mitochondria are divided into three different sub‐types, according to size: large, medium, and small. Our focus is mitochondria near the cuticular plate (CP) in vestibular Type 1 hair cells. Since these mitochondria are adjacent to the CP, we also see stereociliar rootlets (SRs) in close proximity. In addition, we hypothesized that the side facing the CP has a significantly larger amount of cristae junctions (CJs) compared to the opposite side in order to satisfy energy demand of rapidly regenerating SRs. This suggests a polarization of CJs towards one side, so that they can transport ATP and Ca2+ to points of interest, such as SRs and the CP (Perkins et al. 2010). Using IMOD software created by Univ. of Colorado, we examined and accurately traced individual contours that, when meshed, gave a very detailed structure of the mitochondria. By counting CJs on either side of the mitochondria, we can determine their density relative to the CP. Using the “Get info” feature of IMOD we obtained accurate surface area measurements. Interestingly, lamellar cristae in one mitochondrion were seen to curve in the direction of the stereociliar rootlets and three mitochondria were observed to form a type of “super‐mitochondrion” by alignment of their CJs and tethering of the outer mitochondrial membranes. Stereocilia are known to construct their actin from tip to base using myosin and rate‐regulating proteins (Naoz et al. 2008). We hypothesize that mitochondria provide ATP for reconstitution of actin filaments and for maintaining the structure of the cuticular plate. The alignment of cristae between mitochondria may be able to increase ATP production. In conclusion, our results support the hypothesis that CJs are asymmetrically distributed in favor of structures of interest that require more ATP.

Support or Funding Information

Supported by NIH R21‐DC013181 (AL) and P41‐RR004050 (GP, ME).
Energy output of mitochondria located near synaptic ribbons in inner ear hair cells
Authors: Babu Vidya , Vazirian Saeed , Perkins Guy , Lysakowski Anna
Published: November 2023

The mitochondrion plays an essential role in every cell. ATP, the primary energy source in the body, is produced by ATP synthase molecules located in the cristae of the mitochondria. In this study, we utilized EM tomography to create 3D models of mitochondrial cristae from hair cells in the vestibular nervous system. Analyzing these models provided insight into the role of these cristae in activity near ribbon synapses and synaptic vesicles. When action potentials reach nerve terminals, synaptic vesicles fuse with the membrane, releasing neurotransmitters. After this, the vesicles are quickly reinternalized and refilled with neurotransmitters through endocytosis, which requires ATP (Guatimosim and von Gersdorff, 2002). In this study, we wanted to determine whether ribbon synapses and their vesicles utilized large amounts of energy from adjacent mitochondria to transmit signals. Our calculations of energy output in this mitochondrion with lamellar‐shaped cristae yielded estimates of ATP production of about 1000 zeptomoles/sec and energy production at 25–30 femtojoules, consistent with other hair cell mitochondria of similar size. Using the 3D models that we constructed, we also counted the number of crista junctions (CJs), the locations where the crista meets the inner membrane of the mitochondria, and observed that the ratio of CJs facing the ribbon synapse to those facing away was approximately 1:1. We concluded that there was no relationship between the location of the crista junctions and that of the ribbon synapse.

Support or Funding Information

Supported by NIH R21‐DC013181 (AL) and P41‐RR004050 (GP, ME).
Expression of Prolactin (PRL) in Prostate Gland: Studying Histopathology on Cadaveric Tissue Samples
Authors: Nomigolzar Soroush , Khamooshi Navid , Sandy Lisa , Nagy Gyorgy Miklos
Published: November 2023

Prolactin (PRL), secreted from the anterior lobe of the pituitary gland is best known for its actions on the mammary gland. However, PRL is synthesized not only in the pituitary gland, as originally described, but also within the central nervous system (CNS), the immune system, the uterus, and even in the mammary gland itself1. Circulating PRL is also detected in males and its receptor (PRL‐R) is expressed in the prostate gland in humans as well, suggesting that the prostate is a target of PRL2,3. It is known that cadavers in the anatomy department provide ample material to introduce pathological concepts to medical students and this material is a valuable resource for teaching histopathology. Therefore, the aim of our present study was to investigate whether PRL, a possible candidate playing a role in the development of prostate cancer, is expressed in the prostate gland using cadaveric tissue samples. Randomly selected prostate glands were collected and stained by either hematoxylin and eosin (H&E) or immunohistochemistry using Avidin‐Biotin Complex (ABC) to visualize the presence of PRL in prostate epithelium. Our results indicate that PRL is present in human prostate epithelium, as it was evidenced by marked PRL immunoreactivity in prostate epithelial cells in both transitional and peripheral zones. PRL can also be detected in prostate epithelium in a tissue sample obtained from a prostate adenocarcinoma sample. Our data confirm previous observations that PRL is present in the human prostate gland. Moreover, it indicates that locally produced PRL may have an autocrine/paracrine influence on the function of human prostate gland. It may also shed a new light on the involvement of PRL in the etiology of the adenocarcinoma, which needs to be further investigated. At the same time, this study underlines that cadaveric samples are valuable resources for teaching histopathology.
Foetal Intraperitoneal AAV8 Injections as a Valid and Promising Tool to Specifically Target Neuronal Subpopulations Within the Gastrointestinal (GI) Tract
Authors: Winckelmans Kathleen , Buckinx Roeland , Waddington Simon , Timmermans Jean‐Pierre
Published: November 2023

Adeno‐associated viral vectors (AAV) are a versatile tool for gene transfer to manipulate neuronal function in preclinical research and are potential means for gene therapy. Recent studies showed successful in vivo transduction of the mouse enteric nervous system (ENS) by AAV after neonatal and postnatal injection of the viral vector (1–5). Although the full development of the mouse ENS also continues after birth, the prenatal phase starting from E9,5 till E14 spans the period of the start and complete colonization of the GI tract by neural crest cells. In humans the equivalent period corresponds to week 4–7 of gestation. Defects in ENS development result in congenital gastrointestinal phenotypes, such as Hirschsprung's disease. Therefore, in order to better understand the underlying mechanisms of ENS development and related dysfunctions, genetic studies performed at prenatal stage are essential. To test our transduction technique, AAV8 encoding eGFP driven by the immediately early human cytomegalovirus promoter was injected intraperitoneally into E14 mouse embryos. At postnatal day 35, the transduction of cell types in the gastrointestinal tract was evaluated using fluorescence microscopic imaging of the transgene‐encoded eGFP in combination with a set of neuronal and glial markers. In the distinct gastrointestinal segments, i.e. oesophagus, gastric corpus, ileum and distal colon, eGFP was exclusively found in neurons, stained with HuC/D, indicating a highly cell type specific transduction. Immunostaining revealed viral‐encoded eGFP neurons which were either nitrergic in nature, calbindin‐immunoreactive (ir), calretinin‐ir or CGRP‐ir, indicating that several functional neuronal subpopulations were transduced. A strong eGFP signal was also seen in neuronal fibers, either of intrinsic or possibly also of extrinsic origin as systemic administration of AAVs at foetal stage leads to extrinsic transduction of the nervous system as well (6). eGFP positive glial cells could not be observed. These results demonstrate the specific and successful transduction of the prenatal ENS. However, compared to earlier results from the neonatal and postnatal injections (2–4), the transduction efficiency in the myenteric plexus in prenatal injections is still significantly lower (6% versus 20–30%). Therefore, further research is still indicated to improve the technique (e.g. exploring other AAV serotypes and promoters), but the results presented in this study clearly show the enormous potential of this technique in addition to the transgenic animal breeding.

Support or Funding Information

This project is supported by BOF‐KP grant 32765 of the University of Antwerp.
Determining the Role of Natural Killer Cells in Immune‐Mediated Fetal Growth Restriction in Rats
Authors: Baines Kelly , Renaud Stephen
Published: November 2023

Pregnancy is a unique state in which two genetically foreign individuals – mother and fetus – coexist in immunological harmony. The mechanisms facilitating this immunological tolerance are not well understood; however, the uterus contains a unique population of immune cells belonging to the natural killer (NK) cell lineage that likely have a key role in promoting tolerance. Uterine NK cells are purported to promote uterine vascular remodeling as well as facilitate placental and fetal development during pregnancy. We hypothesize that exposure to pathogenic stimuli early in pregnancy disrupts normal functioning of uterine NK cells, thereby impacting uterine vascular remodeling and fetal growth. To test this hypothesis, the viral mimetic polyinosinic:polycytidylic acid (polyI:C) was injected into pregnant rats under control conditions or following NK cell depletion using anti‐asialo GM1 antibodies, and changes in uterine cytokine production and fetal growth were assessed. Rats were injected with 1, 5, or 10 mg/kg polyI:C on gestational day 8.5, while control rats were injected with saline. Dams injected with 10 mg/kg polyI:C exhibited a 15% decrease in fetal weight and a 4% decrease in fetal crown‐rump length, without affecting fetal viability. Decreased fetal weight correlated with increased production of inflammatory cytokines (interferon‐gamma, tumour necrosis factor‐alpha, and interleukin‐6) in both spleen and implantation sites, and increased perforin expression within implantation sites. Interestingly, immunodepletion of NK cells prior to injection of 10 mg/kg polyI:C resulted in a 38% decrease in fetal weight compared to control animals, indicating that uterine NK cells have a role in preventing fetal growth restriction caused by polyI:C exposure. In conclusion, injection of polyI:C into pregnant rats resulted in elevated cytokine production and fetal growth restriction that was exacerbated by a lack of NK cells.

Support or Funding Information

Children's Health Research Institute, Preeclampsia Foundation of Canada, Schulich Medicine and Dentistry
Identification of a Novel Silencer within an Lmx1b Mediated Cis‐regulatory Module
Authors: Gray Austin L , Haro Endika , Spady Connor D , Pira Charmaine U , Oberg Kerby C
Published: November 2023

Introduction

Lmx1b is a homeodomain transcription factor critical for limb dorsalization. Expression of Lmx1b is restricted to the dorsal limb mesoderm during development. ChIP‐seq analysis identified 2 closely associated Lmx1b bound intervals (LBI) that were highly conserved non‐coding regions located 60 kb upstream of the Lmx1b gene. Active chromatin regulatory marks were associated with these intervals supporting a role as regulatory elements. Furthermore, these LBIs contained 4 potential Lmx1b binding sites (3 in LBI1 and 1 in LBI2) that were conserved across divergent vertebrates. We hypothesized that these potential regulatory sequences worked in concert to autoregulate Lmx1b expression.

Methods

We isolated both LBIs, as individual fragments and a combined fragment. The fragments were inserted into a reporter construct containing a thymidine kinase (tk) basal promoter linked to a GFP reporter. The plasmids were then electroporated into the presumptive wing region of stage 14 chick embryos. After 48 hours, the chick embryos were harvested and photographed using UV and white light. Transfection quality was ensured by co‐electroporating a plasmid containing a β‐actin promoter linked to red fluorescent protein (RFP). The negative control consisted of the tk promoter without an insert. We also performed site directed mutagenesis of the conserved predicted Lmx1b binding sites to determine the role of Lmx1b binding in LBI activity.

Results

LBI1 demonstrated activity restricted to the limb but present in both the dorsal and ventral mesoderm. LBI2 showed no enhancer activity. Interestingly, the combined construct containing both LBI1/2 displayed activity that was restricted to the dorsal mesoderm. Site directed mutagenesis of the Lmx1b binding site in LBI2 permitted enhancer activity in the ventral mesoderm, while in a companion report, site directed mutagenesis of the Lmx1b binding sites in LBI1 ablated enhancer activity indicating a requirement for Lmx1b.

Conclusion

Our data indicate that these LBIs function collectively in the limb as an Lmx1b‐associated regulatory module (LARM) with both autoregulatory enhancer and ventral‐silencer sequences.

Support or Funding Information

Funding in part by a grant from the National Organization for Rare Disorders (NORD).
Analysis of Sp6 and Sp8 Protein‐Protein Interactions and DNA Binding during Limb Development
Authors: Ros Marian , Pérez‐Gómez Rocío , Fernández‐Guerrero Marc , Zunzunegui Sandra , Bell Sheila , López‐Giménez Juan F
Published: November 2023

Sp6 and Sp8, two members of the specificity protein (Sp) family of zinc‐finger transcription factors, are expressed in the limb ectoderm. Their importance in limb development was demonstrated by the analysis of double null mutants that exhibited a range of limb malformations that correlated with the dose of Sp6/Sp8 gene products remained (Haro et al., 2014). Thus, the progressive reduction led to predictable morphologies that transit from syndactyly, to split‐hand/foot malformation phenotype, oligodactyly, truncation and finally amelia. In addition, when digits form these show a double dorsal tip. The molecular study of these mutants revealed that Sp6 and Sp8 work together downstream to Wnt/bcatenin to activate Fgf8 and downstream of Bmp signaling, possible cooperating with Smads, to activate En1, the ventral determinant in limb patterning.

Here, to further the understanding of Sp6/8 function, we have investigated Sp6/8 suspected protein‐protein interactions by co‐immunoprecipitation (CoIP) and by bimolecular fluorescence complementation (BiFC). To this end, Sp6, Sp8 and Smads were tagged with Myc or FLAG epitopes to their N‐terminal end and with YFP full length or YFP moieties to their C‐terminal end. Our results show that Sp6/8 form homo and heterodimers and that they form complexes with BMP R‐Smads. All these interactions require the C‐terminal domain of Sps. We are currently determining Sp6/8 DNA binding activity by luciferase reporter and EMSA assays.

Support or Funding Information

Grant BFU2014‐57216‐P to MAR from the Spanish Government.
Chondrocyte Columns are Shorter in the Growth Plate of Collagen XXIV Null Mice
Authors: Gordon Marion , Zhou Peihong , Hahn Rita , Gerecke Donald R
Published: November 2023

Collagen XXIV is fibrillar type of collagen that is a component of bone. Because its function is unknown, collagen XXIV knock out (KO) mice were generated, and their long bones were compared to those of age‐matched wild type (WT) mice. MicroCT analysis indicated that the KO mouse femurs were less mineralized. Histology of bone sections showed the growth plate to be smaller in the KO mice. At 5 months of age, the wild type mouse growth plate contained a physis of uniform thickness that stained light blue (amphophilic), and was composed of cartilage with regular columns of chondrocytes perpendicular to the physis. Secondary spongiosa continued from the primary as eosinophilic lamellar bone. However, in the KO mouse growth plate, the columns of chondrocytes were shorter, the cells within the columns appeared to be less organized, and fewer chondrocytes became hypertrophic. These features may correlate with their being unable to develop into osteoblasts. No detectable differences were seen in articular cartilage, cortical bone or the boney trabeculae in the marrow cavities.

Support or Funding Information

NIEHS P30ES005022 and T32ES007148
The Back to Sleep Campaign, Positional Cranial Deformity, and the Foramen Magnum: New Lessons Learned from Old Pachacamac Crania
Authors: Zdilla Matthew J , Koons Aaron W , Russell Michelle L , Bliss Kaitlyn N , Mangus Kelsey R
Published: November 2023

In 1992, the American Academy of Pediatrics discouraged prone sleeping positions because of its association with sudden infant death syndrome. The recommendations prompted the United States Public Health Service to begin the “Back to Sleep Campaign,” to promote the habitual practice of placing newborns to sleep in a supine or side‐lying positions. After the Back to Sleep Campaign was initiated, there was a sudden increase in the incidence of deformational plagiocephaly. Therefore, research is warranted regarding effects on the influence of extrinsic forces on the development of the cranium. This study utilized ‘traditional’ and geometric morphometric techniques to compare the foramen magnum size and shape of Peruvian crania from the Samuel George Morton Collection, largely from Pachacamac and likely to have undergone intentional cranial deformation, to those of craniofacially diverse cultures that were not reputed to practice intentional cranial deformation. The results demonstrate statistically significant differences in the sagittal dimension of the foramen magnum between groups. Likewise, geometric morphometric analysis revealed differences in foraminal shapes between groups. The results of this study suggest that extrinsic compressive forces likely alter the structure of the foramen magnum. These findings should be taken into consideration with regard to modern‐day newborn sleeping positions and positional cranial deformation.

Support or Funding Information

WV Research Challenge Fund [HEPC.dsr.14.13]
Analysis of Craniofacial Variation in cleft secondary palate‐1 Heterozygous and Homozygous Newborn Mice
Authors: Bennett Holly , Moskal Russell , Holloway Jared , Jones Jennifer , Holton Nathan , Leslie Erin , Bjork Bryan
Published: November 2023

Prdm16 is involved in normal human and mouse craniofacial development. The cleft secondary palate 1 (csp1) ENU‐induced mutation displays cleft palate, shortening of the snout, and micrognathia on a congenic FVB/NJ strain background due to reduced Prdm16 expression, which mirrors the mechanism of Pierre Robin Sequence (PRS) in human. However, overt cleft palate is not evident on a congenic C57BL6/J background (csp1‐B6). Adult heterozygous csp1‐B6 mice have significantly shortened snouts, palates, and right‐side mandibles compared to wt littermates along with variable incidence of malocclusion and hydrocephalus. Homozygous mutant csp1‐B6 mice die shortly after birth for unknown reasons. Our aim is to quantitatively assess variation in bony facial morphology in newborn heterozygous (het) and homozygous mutant (mut) csp1‐B6 and contrast this with the wild type morphology.

We compared the size, shape, and volume of the facial bones in newborn wt, het, and mut csp1‐B6 mice. Anatomical landmarks, bone surface area, and bone volume data were collected from three‐dimensional microcomputed tomography (microCT) images.

Homozygous mutant newborn mice have significantly longer left‐sided premaxillae and maxillae, but shorter left‐side mandibles than the wt and het mice. The mut mice also showed significantly reduced left‐ and right‐side premaxillae heights versus wt and het mice and smaller individual facial bone volumes and surface areas. The wt and het newborn mice did not differ significantly from each other in any contrasts.

Reduced Prdm16 expression in newborn homozygous mutant csp1‐B6 mice is correlated with overall, and asymmetric, micrognathia and undergrowth of the snout compared to the newborn wt and het mice. These findings complement our concurrent research on adult heterozygous csp1‐B6 mice, and will provide insight on the downstream effects of Prdm16 expression on craniofacial development and integration among the bones of the face.

Support or Funding Information

NIH/NIDCR (R15DE023982)Midwestern University, College of Dental Medicine Illinois Intramural Research Grant
Cleft Palate Case‐Derived ARHGAP29 Mutation Causes Lethality and Oral Adhesions in Mice
Authors: Paul Brian J , Palmer Kristina , Sharp Jocelyn C , Pratt C. Herbert , Murray Stephen A , Dunnwald Martine
Published: November 2023

Non‐syndromic cleft lip and/or palate (NSCLP) is among the most frequent major birth defects. The etiology is complex, drawing influence from environmental and genetic factors. Previous studies have identified mutations affecting ARHGAP29 on chromosome 1p22 as a factor for increased risk of NSCLP. To investigate the effects of altered ARHGAP29 in vivo, we generated a novel murine allele by replacing Arhgap29 with a mutated sequence from a human NSLCLP patient. This single‐nucleotide variation of ARHGAP29 led to a nonsense mutation (K326X) in the 11th exon of 31 total, presumably resulting in a nonfunctional protein product. Arhgap29K326X/+ mice were crossed and embryos were harvested for analysis at various time points. We hypothesize that this allele will lead to altered craniofacial development, supporting a critical role for ARHGAP29 in this process. Our results show the absence of homozygous ArhgapK326X animals in all the analyzed litters (N=36), assessed as early as embryonic day 9.5 (e9.5). At e14.5, 54.8% of Arhgap29K326X/+ mice (N=31) exhibited the presence of oral adhesions between the maxilla and mandible or maxilla and tongue, compared to 12.5% of wild type animals (N=8) (P=0.0489). Further quantification of the oral adhesions revealed that 15.94% of the length of the oral epithelium was in improper contact with another oral epithelium in Arhgap29K326X/+ compared with only 3.26% in wild type embryos (P=0.0029). Previous reports have implicated alterations in the periderm as the potential cause of oral adhesions. We performed immunofluorescent analyses at sites of adhesions and detected the presence of p63‐negative, Keratin 17‐positive cells at those sites. This suggests that periderm cells are still present, yet whether or not they are functioning properly remains unknown. Evaluation of E‐cadherin expression at adhesion sites shows proper location at cell‐cell boundaries. Oral adhesions did not appear to impair palatogenesis, as all analyzed embryos show confluent palatal mesenchyme and epithelium at e18.5 (N=22). Collectively, our data demonstrate that ARHGAP29 is required for embryonic survival, and that heterozygosity for Arhgap29 loss of function variants increases the incidence and length of oral adhesions at a critical time point during palatogenesis. In conclusion, we validate the damaging function of the human K326X mutation in vivo and reveal a previously unknown effect of altered Arhgap29 in murine embryonic and craniofacial development.

Support or Funding Information

Partial financial support was provided by a grant from the National Institute of Health R37DE08559, R01AR067739, and by the FaceBase consortium (grants DE020052 and DE020057). Brian Paul was a recipient of a fellowship from the Iowa Center for Research by Undergraduates.
Homeostatic Neurogenesis in the Lizard Brain
Authors: McDonald Rebecca Paula , Vickaryous Matthew Kenneth
Published: November 2023

Neurogenesis – the generation of new neurons from a precursor population – has been documented as a normal or homeostatic process within various regions of the adult brain. In most instances, these precursors or endogenous neural stem/progenitor cells (NSPCs) reside in neurogenic zones located adjacent to the ventricular system. Across vertebrates, the distribution of NSPCs, and the capacity of homeostatic neurogenesis, varies greatly. Among mammals, NSPCs are restricted to only two constitutively active regions of the brain. In contrast, among teleost fish NSPCs, and constitutive neurogenesis, are widespread throughout the brain. Although less is known about reptiles, available evidence indicates that NSPCs are present within multiple continuously active regions. Here we investigate neurogenesis in an emerging amniote model of central nervous system regeneration, the leopard gecko. We have identified populations of NSPCs within various regions of the brain, including the ventricular zone (VZ) of the lateral ventricles. VZ cells of the lateral ventricle characteristically express the stem cell markers SOX2 and Musashi‐1. In addition, they share features in common with radial glia, including a lengthy radial process that contacts the pial surface, and the expression of the glial markers Vimentin and glial fibrillary associated protein. As evidenced by immunostaining for both proliferating cell nuclear antigen and phosphorylated histone H3, VZ cells of the lateral ventricles are mitotically active. Utilizing a bromodeoxyuridine (BrdU) pulse‐chase experiment, we labelled VZ cells for short‐term cell tracking. Immediately following the pulse period, BrdU+ cells were confined to the VZ. Following a 30‐day chase, we observed BrdU+ cells within the cellular layer of the cortex, a region dominated by mature neurons (immunopositive for the neuronal markers HuCD and NeuN). We conclude that the VZ of the lateral ventricles is a neurogenic zone, and provide compelling evidence of homeostatic neurogenesis in the leopard gecko brain.

Support or Funding Information

Natural Sciences and Engineering Research Council (NSERC) Discovery grant 400358
Expression of Glial Fibrillary Acidic Protein (GFAP) and Histopathological Changes in Brains of Trypanosoma brucei brucei Experimentally Infected Rats
Authors: Alawa Judith Nkiruka , Maliki Joseph Simpa , Tanko Yusuf , Alawa Clement Innocent
Published: November 2023

African trypanosomiasis, also known as sleeping sickness in humans which affects the brain, is characterized by alterations in the functions of brain regions. It is unknown if the disease affects the expression of Glial Fibrillary Acidic Protein (GFAP) which is a marker for astroglia activation. Therefore, this study was aimed at investigating the astrocytic response and histopathological changes in the striatum and hippocampus of rats experimentally infected with Trypanosoma brucei brucei (Tbb). Immunohistochemical evaluation, demonstrated reactive astrocytosis characterized by dense staining of enlarged cell bodies (astrocyte hypertrophy) with distinct cell processes in the striatum and hippocampus. Histopathological changes in the striatum of infected rats showed neuronal degeneration characterized by chromatolysis and satellitosis. Hippocampus showed gliosis in the molecular layer while axonal spheroids were observed in the white matter indicative of degenerating neuronal process. Control rats did not show any of these signs. These results demonstrate that the disease alters the expression of GFAP indicative of astroglia activation thus suggests that the astroglia cells may mediate immune response during trypanosome infection.

Support or Funding Information

Ahmadu Bello University
Serum enkephalin levels correlate with EAE disease severity
Authors: Ludwig Michael D. , Zagon Ian S. , McLaughlin Patricia J
Published: November 2023

Multiple sclerosis, and the animal model experimental autoimmune encephalomyelitis (EAE), are characterized by elevated immune cell replication. The endogenous peptide, opioid growth factor (OGF), chemically termed [Met5]‐enkephalin has been shown to inhibit proliferation of activated T and B cells in vitro, and to reduce the number of T and B cells in peripheral tissues spleen, nodes) and CNS (spinal cord) following EAE immunization. Systemic OGF injection of mice with chronic EAE or relapse‐remitting EAE results in diminishment of clinical disease, reductions in activated astrocytes, and fewer relapses. The hypothesis of this research is that serum levels of OGF decline during the course of EAE, and that treatment with exogenous OGF is associated with severity of disease. The chronic mouse model of MS (Ch‐EAE) was established, with one group of mice receiving daily OGF injections beginning at the time of clinical disease onset. Clinical behavior was monitored daily; locomotor activity and thermal sensitivity were measured periodically. Blood samples were collected and enkephalin (OGF) levels measured by ELISA (MyBiosource). EAE disease onset occurred on day 9 post immunization when the mean clinical score was 1.5. Peak disease scores for saline‐injected EAE mice reached a mean of 5.7 on day 18, in comparison to a peak clinical score of 2.5 for EAE‐mice receiving OGF. Serum OGF levels in non‐diseased mice were approximately 149 pg/ml, whereas EAE mice had serum OGF levels that ranged between 8 and 128 pg/ml, suggesting that EAE corresponded to a decrease in enkephalins. Following exogenous OGF therapy, serum levels increased; higher serum OGF levels were related to reduce disease severity, as well as changes in open field behavior and hot‐plate sensitivity. These data suggest that there is a dysregulation of endogenous OGF in the disease state and that treatment with exogenous OGF restores serum OGF levels to normal. Moreover, the non‐invasive tracking of serum OGF may be useful as a marker for disease progression and response to therapy.

Support or Funding Information

Supported in part by the generous gifts from The Paul K. and Anna E. Shockey Family Foundation
Amphetamine‐induced Hyperlocomotor Activity in Heterozygous Disc1 Mutant Mice
Authors: Lai Chuan‐Ching , Lee Li‐Jen
Published: November 2023

Disrupted‐in‐Schizophrenia‐1 (DISC1) is a susceptibility gene for several psychiatric illnesses. To study the pathogenesis of these disorders, various Disc1 mutant mouse models were established. In our group, Disc1 mutant mice were generated by introducing the 129S6/SvEv 25‐bp deletion Disc1 variants into the C57BL/6J strain. In this study, a battery of behavioral tests was conducted to evaluate the behavioral phenotypes and cognitive function in the heterozygous Disc1 mutant (Het) mice. In open field test, novel object recognition test, Y‐maze test, and prepulse inhibition test, no significant differences were observed between wildtype (WT) and Het mice. However, after receiving an intraperitoneal injection of amphetamine (5 mg/kg), greater locomotor activity was noticed in Het mice comparing with WT. Moreover, the results of western blot showed lower levels of dopamine transporter (DAT), D1 dopamine receptor (DRD1) and Glycogen synthase kinase 3 (GSK3α/β) and higher level of D2 dopamine receptor (DRD2) in the striatum of Het mice, relative to the WT mice. The dendritic structures of striatal medium spiny neurons were also examined. In the Het mice, reduced dendritic complexity and spine density were noticed. Our results indicated that Disc1 deficit in one allele dose does not alter the basic behavior and cognitive performance in mice. However, lacking one WT Disc1 allele causes the animals to be more vulnerable to the challenge of psychostimulant such as amphetamine, which might be attributed by the altered structure and functions of striatal cells.
Gene expression profile analysis of immunodeficient mice after WT or mSOD1 immunoreconsitution reveals differential motoneuron death mechanisms after facial nerve axotomy
Authors: Setter Deborah Olmstead , Runge Elizabeth M , Schartz Nicole D , Sanders Virginia M , Jones Kathryn J
Published: November 2023

When a facial nerve axotomy (FNA) is performed on recombinase activating gene‐2 knockout (RAG‐2−/−) mice lacking the adaptive arm of the immune system, there is significantly greater motoneuron (MN) death relative to wild type (WT) mice. Reconstituting RAG‐2−/− mice with whole splenocytes (WS) restores MN survival to WT levels, and CD4+ T cells specifically were found to drive immune‐mediated neuroprotection. The mSOD1 mouse model of amyotrophic lateral sclerosis (ALS), a MN disease, also exhibits significantly decreased MN survival after FNA, and this model mimics the immune dysregulation described in human ALS patients. Reconstituting RAG‐2−/− mice with mSOD1 WS fails to rescue MN survival, whereas isolated mSOD1 CD4+ T cell reconstitution confers neuroprotection. This finding suggests an inhibitory factor in mSOD1 mice blocks CD4+ T cell‐mediated neuroprotection. The objective of the current study is to characterize the mechanisms of neuroprotection in a wild‐type immune system and then identify disruptions of these processes in the mSOD1 mouse model of ALS.

To accomplish this goal, gene expression profiles of the axotomized facial motor nucleus were gathered from 6 experimental groups: WT, RAG‐2−/−, RAG‐2−/− + WT WS, RAG‐2−/− + WT CD4+ T cells, RAG‐2−/− + mSOD1 WS, and RAG‐2−/− + mSOD1 CD4+ T cells. Genes associated with MN regeneration, glial activation, inflammatory cytokines, and cell death pathways were examined. Our findings revealed that relative to WT mice, MN regeneration and cell death pathways were unaffected in RAG‐2−/− mice, but glial activation and cytokine expression were significantly reduced. Adoptive transfer of WT or mSOD1 WS or CD4+ T cells into RAG‐2−/− mice restored glial activation and cytokine production to WT levels. MN‐specific Fas cell death receptor expression, which is increased in mSOD1 mice after FNA, is also elevated in RAG‐2−/− + mSOD1 WS mice. These data implicate a dysregulated glial response as a causative factor for MN death in immunodeficient mice. In contrast, promotion of the Fas/Fas ligand pathway may contribute to MN death in immunodeficient mice reconstituted with mSOD1 WS. In summary, these results suggest that peripheral immune cells can directly modulate central responses to injury. Future studies based upon these results are expected to elucidate how mSOD1 immune cells promote Fas expression, and whether inhibition of the process can preserve MN.

Support or Funding Information

Supported by NIH NS40433 (KJJ and VMS)
The Ligamentum Flavum Thickens During the Advanced Stages of Hypomobility Induced Lumbar Spinal Osteoarthritis
Authors: Pulisetty Tanaya , Benckendorf Adam N. , Merlo John A. , Cramer Gregory D. , Little Joshua W.
Published: November 2023

Low back pain (LBP) is a primary cause of global disability. The lumbar facet joint is recognized as a clinically relevant pain generator. Pain can occur through the onset and progression of facet joint degeneration consistent with spinal osteoarthritis (OA). OA is now defined as a whole joint disease involving many tissues in addition to the articular cartilage, including the joint capsule. Capsular thickening due to mechanical and inflammatory stress can present clinically as a source of LBP. We have previously examined capsular thickening that occurs during OA, by using a rat model of external spinal linkage to cause lumbar spinal hypomobility and induce degeneration in the lower lumbar facet joints (L4/5 and L5/6). We have shown that this model mimics the location, progression, and histopathological changes of the articular cartilage typically reported in human spinal OA. Additionally, we found that by 8 weeks post linkage this degeneration is associated with significant thickening of the ventral articular capsule (ligamentum flavum, LF) in the cephalad portion of the joint. These articular capsule changes are unexplored at later stages of this model when degeneration is more advanced. Here we used a morphometric approach to examine if spinal hypomobility of the bilateral L4/5 and L5/6 facet joints in rats is associated with LF thickening. We hypothesized that 12 weeks of spinal linkage would be associated with an increase in articular capsule thickness in joint regions undergoing cartilage degeneration. We tested our hypothesis by comparing LF thickness in linked and time matched control rats at 12 weeks post linkage. LF thickness measurements were performed on images obtained from a brightfield microscope (10× objective, Leica DMRB, optronics microfire camera) on formalin fixed, decalcified, paraffin embedded, Ehrlich's hematoxylin and light green stained 45 μm thick sections. Morphometric measurements were made by drawing a line through the thickest region of the LF, perpendicular to the plane of the ventral LF surface. This approach demonstrated acceptable inter‐ and intra‐rater reliability (ICC= 0.94 and 0.99). Twelve weeks of hypomobility was associated with a significant increase in LF thickness (p<0.05) compared with control animals in the cephalad and caudal regions. This agrees with our recent findings that significant degeneration is present in the superior articular process (predominantly cephalad region) and inferior articular process (predominantly caudal region) by week 12. Our findings suggest that advanced cartilage degeneration is associated with LF thickening. These findings require additional study to examine the causes of LF thickening (e.g., prolonged inflammation and fibrosis) to better understand the role of the LF in the pathophysiology of OA and OA induced LBP.

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NCMIC
Blood Supply to the Human Dentate Nucleus by Cerebellar Arteries
Authors: Kim Soo‐Jung , Yang Hee‐Jun , Lee Young‐Han , Kim Hyun‐Ju , Lee Hye‐Yeon
Published: November 2023

The major roles of human cerebellum are balancing and planning, initiation, and modulation of voluntary movements. In addition, the cerebellum is known to be related with the cognitive function. The dentate nucleus is the largest deep nucleus in human cerebellum. The dentate nucleus is known to have somatotopic arrangement in itself in relation with the upper limb and lower limb. Series of cognitive function tests revealed that the areas for different cognitive domains are located in specific arrangement in the dentate nucleus as well. However, the mapping of arterial supply to each regions of the dentate nucleus by cerebellar arteries has not been clarified anatomically. The aims of this study are to clarify the arterial territories in the dentate nucleus and to match them with the known somatotopy and the arrangement of cognitive area in the dentate nucleus which should be useful for research and clinical situation of various cerebellar diseases. Fourteen cerebellar hemispheres from seven adult cadavers were used in this study. Red‐colored radio‐opaque silicon dye was injected into the vertebral arteries or their branches supplying the cerebellum. Computed tomographs of the cerebellar hemispheres were taken. The cerebellar hemispheres were sliced into sections of 5 mm thickness. The sliced cerebellar hemispheres were stained by Mulligan method. The stained sections were microdissectioned under surgical microscopes. Most of the dentate nuclei were mainly supplied by three or four direct branches of superior cerebellar arteries and two or three direct branches of posterior inferior cerebellar arteries. Between the known suppliers to the dentate nucleus, the superior cerebellar artery is more dominant for dorsal side of the dentate nucleus, whereas the dominant artery for the ventral side of the dentate nucleus is the posterior inferior cerebellar artery.
Diversity of microvascular endothelial cell markers in different regions of the human brain
Authors: Mbagwu Smart , Filgueira Luis
Published: November 2023

Background

Microvascular endothelial cells in the brain have various important functions, including contribution to the blood‐brain barrier and supply of nutrients and oxygen to the brain, as well coagulation control, inflammatory responses and angiogenesis. Endothelial cells have observable morphological and molecular differences in different tissues and organs. However little is known about differences of microvascular endothelial cells in the human brain. The aim of this study was to characterize qualitatively the expression of endothelial cell markers within different parts of the brain.

Methods

Frozen sections from different anatomical regions (precentral and postcentral gyrus, hippocampus, rhinal and visual cortex) of human formalin fixed brains (n=3) were stained immunohistochemically for endothelial cell markers, including CD31, claudin 5, occluding, von Willebrand Factor, ZO‐1, as well as astrocyte (glial fibrillary acid protein) microglia (Iba1) markers.

Results

The expression patterns of the markers was heterogenous in the different regions of the brain studied. CD‐31 had the most positive expression among the different biomarkers especially in the precentral cortex. ZO‐1 and and vWF had fairly positive expressions in the pre central and post central cortices compared to other regions.

Conclusion

The expression pattern of the different endothelial markers is heterogenous in the microvasculature within the different anatomical regions of the cerebral cortex. This results indicate that the diversity in microvascular endothelial cells contribute to functional differences in the different brain brain regions.
Assessing Accreditation at the ASBMB: Surveying our Constituency
Authors: Tansey John T. , Carastro L. Michael , Dean Diane M. , Kennelly Peter , Martin Debra , Wolfson Adele J. , Provost Joseph J.
Published: November 2023

The ASBMB recently began accrediting biochemistry and molecular biology degree programs and certifying the degrees of qualified graduates from those programs. Accreditation occurs in two stages. First, programs apply for accreditation through an evaluation process, for which a program must demonstrate satisfactory infrastructure, including instructional resources, professional development and commitment to diversity; breadth and depth of faculty expertise; and curriculum, including the four core concepts ‐ energy, structure and function, information storage, quantitative skills, and two underlying concepts: evolution and homeostasis ‐ at introductory and advanced levels, and a robust experiential learning component. The second component of accreditation is the ASBMB certification exam. The exam is predominately open‐ended problem solving questions designed to test deep understanding of the core concepts. Accredited programs may administer this examination to their students, and the degrees of those passing are designated ASBMB certified. To assess the efficacy of this combination of programmatic and individual assessment, we surveyed seven different groups: students in accredited programs, students not in accredited programs, schools that have not yet applied for accreditation, ASBMB‐accredited schools, schools that have applied and not received accreditation, graduate and professional schools, and potential employers in industry. Each group was asked a unique set of questions regarding their roles in the program and their perceptions of ASBMB accreditation. Topics queried included what respondents knew about the ASBMB accreditation program, their impressions of the process, how they used it in their program, company, or career. The results of the study varied widely depending on the group queried. Most respondents valued accreditation and the accreditation process and provided suggestions for improvement. However, there were differences in the reasons respondents valued accreditation and the aspects they found most beneficial. Both those schools that had received accreditation and those that had not found the application process helpful in that they had to acquire important information and discuss curriculum with colleagues. Initial findings also indicated that the ASBMB needs to improve program promotion to students, graduate programs, and employers. Collectively, these data indicated that the accreditation and credentialing process started strongly and provided several areas for which improvement can be made.
Support and Expectations for Biochemistry and Molecular Biology Faculty Promotion and Tenure at PUI Institutions
Authors: Cortes Kimberly Linenberger , Angotti Robin L , Provost Joseph , Benore Marilee Ann
Published: November 2023

Biochemistry and Molecular Biology (BMB) faculty at Primarily Undergraduate Institutions (PUI) work in very diverse environments. PUI institutions are those defined by the NSF as U.S. four‐year, masters‐level, and small doctoral colleges and universities, that award on average no more than 2 Ph.D. or D.Sc. degrees per year in a department. BMB faculty work at private and public universities, in chemistry and biology departments, and with a varied ratio of teaching, research and service expectations. Within these parameters characterizing, comparing and developing expectations for promotion and/or tenure is challenging, with limited data to assist in important decision making. In order to assist faculty and administrators with strategic planning, goal setting, measuring productivity, and to provide guidance for internal and external program/departmental reviewers, we have initiated a survey to collect a broad set of data from PUIs. After writing and testing sample survey questions and responses, a final survey was developed to query participants about parameters such as teaching load, research support, and output. Initial survey results and plans to disseminate the results including using interactive graphical tools will be presented.