bluuebunny/crossref_metadata_2025 huggingface dataset:
Inner Ear Volume in Craniosynostotic Mice
Authors: Heitman Erin Ashley , Holmes Megan A , McNulty Margaret A , DeLeon Valerie Burke , Mussell Jason C
Published: November 2023

Craniosynostosis is a congenital defect causing premature fusion of cranial sutures. These skull abnormalities can cause developmental delays, cranial venous hypertension, raised intracranial pressure, hearing impairments, and facial deformities. The temporal bone, specifically, plays a crucial role in the development in many of the features affected by craniosynostosis. The jugular foramen is an interosseous foramen formed by the temporal and occipital bones. Stenosis of the jugular foramen has been reported in individuals suffering from both syndromic and non‐syndromic complex (multi‐suture) craniosynostosis. Children with complex craniosynostosis also exhibit anomalous basicranial venous drainage in the form of persistent condylar and mastoid emissary veins uniting the sigmoid dural venous sinus with the suboccipital venous plexus and posterior auricular vein, respectively. The otic capsule is crucial for proper establishment of the sigmoid dural venous sinus during development. The petrous portion of the temporal bone is a significant branch point for dural venous sinuses and is altered in children with craniosynostosis syndromes. This study aimed to compare the volume of the otic capsule in syndromic craniosynostotic mice and their wild‐type littermates for possible effects on basicranial venous drainage. Using the otic capsule as a proxy for temporal bone volume and symmetry, we hypothesize that craniosynostosis causes a change in otic capsule volume throughout ontogeny that may also alter dural venous drainage pathways.

The Fgfr2+/C342Y mouse model of Crouzon's syndrome along with wild‐type littermates were analyzed using serial micro‐computed tomography sections of whole heads at embryonic day 17.5 (E17.5), postnatal day 14 (P14), and postnatal day 42 (P42). Further, a subset of mouse crania were immersed in potassium iodine solution (Lugol's solution) to stain soft tissue structures. Immersion was performed to enable measurement of the unossified otic capsule of the earliest stages. Amira 3D analysis software was used to reconstruct three dimensional isosurface structures and compute volumes from complete two‐dimensional CT image stacks.

While we did not find evidence of otic capsule volumetric differences in the earliest stages examined, significant differences between Fgfr2+/C342Y and WT littermates did emerge in the sub adult population (P42; p = 0.0455). Further investigations will examine the surface area of the jugular foramen, as well as, emissary foramina.
Deletion of Notch signaling in the developing mouse kidney results in a disruption of planar cell polarity in tubule epithelial cells
Authors: Kearns Donovan , Livingston Safia , Baybutt Richard , Heuvel Gregory Vanden
Published: November 2023

Cux1 is the murine homologue of the Drosophila gene cut. In Drosophila, multiple genetic interactions between Cut and the Notch and Wingless signaling pathways occur during development. Cux1 is upregulated by Notch1 activation in rat kidney epithelial cells, and interacts with the groucho homologue Grg4 to regulate p27 expression in the developing kidney. To further evaluate the regulation of Cux1 by Notch signaling, we conditionally deleted RBPJ/k, a transcription factor required for canonical Notch signaling, in the developing ureteric bud and collecting duct using Cre‐recombinase (Cre) under the direction of the Hoxb7 promoter. We confirmed the Cre activity by crossing the RBPJ/kCD mice to the membrane‐targeted Tomato/Green Fluorescent Protein (mT/mG) double fluorescent reporter mouse strain (Gt(ROSA)26Sortm4(ACTB‐tdTomato,‐EGFP)Luo/J), which carries a loxP‐flanked Tomato cassette. Newborn RBPJ/kCD mice exhibited medullary and cortical cystic like dilatations. Postnatal day 14 (P14), RBPJ/kCD mice exhibited hydronephrosis and cortical dilatations. Analysis of planar cell polarity using anti‐phospho‐Histone H3 antibodies showed a disruption of spindle orientation in the collecting ducts of RBPJ/kCD mice. Taken together, these results suggest that Notch signaling is required for the normal development of the collecting duct system in the kidney, and that reduced Notch signaling may contribute to the development of renal cysts.

Support or Funding Information

National Institutes of Health
Uterine Endometrial Reprogramming during Early Pregnancy is Accompanied by Distinct Spatiotemporal Expression Patterns of Basement Membrane Components
Authors: Jones‐Paris Celestial R , Paria Sayan , Berg Taloa , Saus Juan , Bhave Gautam , Paria BC , Hudson Billy
Published: November 2023

Uterine endometrium undergoes decidualization, a tissue reprogramming process, in response to blastocyst implantation in mice. During this process, stromal cells acquire epithelial‐like behaviors including expressing basement membrane (BM), a specialized form of extracellular matrix that influences epithelial cell identity and behaviors. The assembly and function of BM during decidualization is unknown. In this study, we investigated uterine expression and compartmentalized localization of BM components: classical components collagen IV and laminin, recently identified component peroxidasin, and a putative component Good pasture antigen‐binding protein (GPBP), in the mouse uterus during early pregnancy. Electron microscopic investigation revealed: 1) a smooth continuous layer of BM in between the epithelial and stromal layers of the preimplantation endometrium; and 2) interrupted, uneven, and thickened BM within the pericellular space of the post implantation decidua. Quantification of mRNA levels by qPCR showed changed in expression levels that were complemented by immunofluorescence localization of collagen IV, laminin, peroxidasin, and GPBP. Novel observations of BM‐associated and distinct expression patterns of the four components suggest collective pericellular functions as well as individual functions of the components in the peri implantation uterus and decidua.

Support or Funding Information

This work was supported by National Institutes of Health Grants F31 CA165787‐03 (to C.R.J.), R25 DK096999 (to B.G.H. for S.P. and T.B.), R01 HD044741 (to B.C.P.), and R01 DK018381 (to B.G.H). This study was also supported by RT2014‐2415‐1 / Prometeo II ‐2014‐048 (to J.S).
The Role of HFE in Macrophages
Authors: Nixon Anne , Neely Elizabeth , Connor James
Published: November 2023

HFE is a fundamental protein involved in the regulation of cellular iron uptake and iron homeostasis. Mutations of the HFE gene, namely the C282Y and H63D mutations, are commonly associated with iron dysregulation and iron related diseases. Our laboratory and others have reported an increased prevalence of the H63D mutation in neurodegenerative diseases such as ALS and Alzheimer's disease. Importantly, HFE gene polymorphisms of reticuloendothelial cells, primarily macrophages, have been found to be iron deficient despite overall high levels of iron within the body and parenchymal cells. The mechanism by which HFE mutations lead to the inability of macrophages to accumulate iron is not understood. We hypothesize that the HFE/H63D gene variant alters the macrophage phenotype. To test this hypothesis, we isolated macrophages from the bone marrow of mice harboring the H67D mutation, a homolog for the human H63D mutation. We demonstrate that H67D macrophages have a similar capacity to iron load compared to wildtype bone marrow macrophages. Furthermore, there was no difference in expression of transferrin receptor and ferroportin; suggesting iron uptake and export are similar to wildtype. However, we found the H67D macrophages stored significantly more iron in the form of L‐ferritin compared to the wildtype macrophages, suggesting an alternative mechanism for iron processing in the cell. There was an increase in phagocytic activity in response to LPS stimulus in the mutant macrophages as compared to the wild type control. In addition, we observed H67D macrophages have slower migration compared to the wild type control, as measure by Boyden chamber migration assays. Together, these results suggest H67D macrophages have altered phenotype relating to iron processing and motility. These changes in macrophage function are very likely relate to alterations in inflammatory responses and the role of macrophages in disease processes. These latter concepts are under investigation.
IMPACT OF BMP‐7 ON CELL MIGRATION IN THE MENISCUS
Authors: Taylor Victor , Hutchinson Ian , Danelson Kerry , Ferguson Cristin
Published: November 2023

Introduction

The meniscus is a C‐shaped fibrocartilaginous tissue that is essential for the normal biomechanical function of the knee. Damage or loss of the meniscus is associated with osteoarthritis. Inflammatory cytokines like Interleukin‐1 (IL‐1) inhibit repair. However, Bone Morphogenetic Protein‐7 (BMP‐7) showed great regenerative potential in cartilage and may help aid repair in meniscus. The effect of BMP‐7 on cell migration in the meniscus was examined to evaluate the potential of this agent to augment meniscus repair and tissue engineering applications.

Methods

The effect of BMP‐7 on fibrochondrocyte migration was examined using a model that interfaces a collagen type I sponge treated with BMP‐7, IL‐1, and BMP‐7+IL‐1 between porcine meniscus explants in a closed system held via a cloning tube. In this model, the growth factor treated sponge serves as the constant stimulus (or bait) for cell migration away from the media. Explants were incubated for 28 days. Afterward, tissues were embedded in paraffin and stained with 4′,6‐diamidino‐2‐phenylindole (DAPI) stain. Initial data represents n=7 for Control, n=8 for BMP7, n=4 for IL‐1, and n=4 for BMP‐7+IL‐1 groups.

Results

Explant meniscus tissue histology sections were divided into scaffold, middle, and peripheral zones relative to position next to the growth factor treated sponge, and cell count was performed in each zone. There were significantly more cells in the middle zone of the BMP‐7 group compare to the other groups (p < .05). In addition, there were significantly more cells in the peripheral zone of the BMP‐7+IL‐1 group compared to the peripheral zone of the BMP‐7 group (p < .005).

Conclusion

Preliminary results suggest that BMP‐7 may induce cell migration in meniscus cells. More studies will be done to better determine the effects of BMP‐7 on the meniscus.

Support or Funding Information

Institutional Research and Academic Career Development Awards (IRACDA) (NIH/NIGMS K12 PAR‐13‐290)
Evidence that Epithelial Tight Junctions are Altered during Edwardsiella Infections
Authors: Aggarwal Priyanka , Leung Ka Yin , Guttman Julian Andrew
Published: November 2023

Bacteria from the Edwardsiella genus can kill entire fish farms within days. These infections cause an array of phenotypic alterations to fish including petechial hemorrhage in the fins and skin, rectal hernia, abscesses deep within fish musculature and most strikingly, holes within (or the liquefaction of) the fish themselves. Intercellular junctions are crucial for maintaining tissue integrity, but are also common targets of bacterial pathogens. Because Edwardsiella causes alterations to various epithelia during their infectious processes, we hypothesized that tight junctions (TJs) might be targeted by these microbes. To test this hypothesis, we localized the claudin‐3 transmembrane tight junction protein in Caco‐2 cells during Edwardsiella infections and found that it was extensively re‐arranged. This work provides the first evidence of junctional alteration during Edwardsiella infections, and suggests a possible molecular modification that could lead to the observed phenotypes in diseased fish.

Support or Funding Information

Funded by NSERC Discovery Grant
Evaluation of the Relationships Between Anatomical Variations of the Pronator Teres and Pronator Quadratus
Authors: Trimble Laura , Lewis Christina
Published: November 2023

The pronator muscles of the upper limb, pronator teres (PT) and pronator quadratus (PQ), work together to rotate the radius around the ulna along an axis parallel to the central bands of the interosseous membrane while stabilizing the involved joints. Anatomical variations that affect the rotational efficiency of the PT have been described. Previously, we have described an aberrant PQ with a possible flexor function, and also defined 4 types of anatomical variations in PQ, based on the number of bellies present, and whether it occurred unilaterally or bilaterally, suggesting that PQ is more variable than appreciated. The PT typically has 2 distinct heads of origin (ulnar and humeral) and inserts onto the lateral radius. However, several anatomical variations have been reported, including the number of heads, locations of origin and insertion points, anomalous fiber orientations, and the presence of fibrous bridges and/or arches. In the current study, we sought to determine if there are patterns of anatomical variations in the PT, and whether any such variations occurred in relationship to those of the PQ. Sixteen forearms from 8 embalmed cadavers (4F,4M ages 64–93, mean 82) were dissected, and the PT and PQ muscles observed for muscle shape, fascicle orientation, and attachment points. PT and PQ heads was described, and PT tendon length, width, and distance from the radial head and styloid process measured. Our data revealed that the PT existed as only a single muscle head (humeral), bilaterally, in 25% of the cadavers examined, while exhibiting the traditional two muscle heads (humeral, ulnar) bilaterally in 75% of cadavers. Interestingly, the PT variant with a single humeral head occurred in conjunction with Type II PQ muscles bilaterally (i.e., the presence of 2 muscle bellies). Of the six cadavers with both humeral and ulnar heads of PT, 33.3% had an occurred with Type I PQ (1 head, bilateral), 33.3% with Type II PQ (2 heads, unilateral), and 33.3% with Type III PQ (2–3 heads, bilaterally). Our data suggests that there may be a relationship between the patterns of anatomical variations between PT and PQ muscles, which may provide new insight into the biomechanical relationship between these 2 muscles. The inclusion of additional cadaveric specimens, as well as quantitative comparisons between PT and PQ muscles, including cross sectional area, volume, and line of action, will help to elucidate the potential relationship between PT and PQ muscles.
a Very Large‐sized Enlarged Anatomical Model of Human Ear in Plaster (first middle of the 20th century)
Authors: LE FLOCH‐PRIGENT PATRICE P , FROMANGER MARINE , GILLOT JEAN‐BERNARD
Published: November 2023

A very large, anatomical model of human ear represented the temporal bones with the tympanic membrane and the three ear ossicles in the middle ear. It was 38 cm long, 31 cm large, 28,5 cm high. There was no basis. The roof of the petrous part of the temporal bone was removable, the representation of the cross‐sections looked like spongious as red‐spotted. The corda tympani and the internal ear content were missing. The endo‐cranial part of the cochlea‐vestibular nerve (VIII CN) was represented in the internal acoustical meatus and colored in yellow. The internal carotid artery was represented in its intra‐cranial part and also the proximal part of the Eustachian tube. Numerous numbers and letters corresponded to legends of which the text was unknown. A well drawn auricle was present. This sample in plaster was very similar but still larger to an enlarged model which also belonged to the Brieux‐Gillot Scientific Library in Paris (France) on january 2015 (see poster #4546, EB‐2016). Their craftsman was identical probably german in the first part of the 20th century.
Lumbar Spinal Segmental Hypomobility Induced Osteoarthritis is Associated with Early Regression of Facet Joint Synovial Folds
Authors: McDowell Jacob A. , Merlo John A. , Cramer Gregory D. , Little Joshua W.
Published: November 2023

Spinal osteoarthritis of the lumbar facet joints is one of the most prevalent and burdensome causes of low back pain (LBP). This condition involves numerous tissues that can directly or indirectly induce pain (e.g., cartilage, bone, synovium, and capsule). Specifically, pathophysiological and morphological changes to the synovium, a key tissue that maintains the health of the joint articular surface, are reported to contribute to the onset and progression of OA. We have previously reported a novel rat model that uses external spinal linkage to cause lumbar spinal hypomobility and induce OA in lower lumbar facet joints (L4/5 and L5/6). This model may replicate the progression of spinal OA in humans and allow mechanistic studies of facet joint OA induced pain. We have shown that this hypomobility is associated with significant bilateral OA changes including synovial fold (SF) regression from within the joint space by 4‐weeks post linkage. The onset of this SF change is unknown. Here we examined if lower lumbar spinal segmental hypomobility in rats is associated with early onset of SF regression from the joint space in the bilateral L4/5 and L5/6 facet joints. We hypothesized that significant SF regression occurs by 1 week following spinal linkage. Analysis was performed using a histological approach based upon our previously reported methods to test our hypothesis in linked (hypomobile) and time matched control rats at 1, 2, and 4 weeks post linkage. The number of facet joint quadrants with SFs was determined using brightfield microscopy to observe formalin fixed, decalcified, paraffin embedded, Ehrlich's hematoxylin and light green stained 45 μm thick sections of the facet joints. The mean number of quadrants containing SFs was calculated for each group. This approach demonstrated acceptable inter‐ and intra‐rater reliability (κ= 0.82 and κ= 0.77). Statistical analysis was performed using a one way ANOVA with Tukey's multiple comparisons. Significance was defined as p <0.05. When compared with time matched control animals (n=4), linked rats demonstrated significant reductions of the SF along the joint surface at 2 and 4 weeks of hypomobility (n=4). No significant differences were found between control groups or at week 1 following linkage. As the synovium can contribute to the development of OA, it is possible that regression of SF in the facet joint by 2 weeks post linkage may reflect pathophysiological changes that contribute to the induction of articular cartilage and subchondral bone degeneration. Further work is required to support these findings including morphometric and mechanistic approaches to determine the functional role of the synovium in hypomobility induced OA and pain. Collectively, these findings support that lumbar spine segmental hypomobility leads to an early onset of SF regression.
The presence of a variant leg muscle, flexor digitorum accessorius longus (FDAL), causing tarsal tunnel syndrome, or posterior tibial neuralgia
Authors: Swearingen Jordan V , Kennedy Patrick M , Whipp Kylen P , Miller Kyle D , Zdilla Matthew J. , Lambert H. Wayne
Published: November 2023

The flexor digitorum accessorius longus (FDAL) muscle is a variant muscle located in the posterior compartment of the distal leg. It typically originates in the distal third of the leg and can have a variable proximal attachment to the tibia, fibula, tibia and fibula, the posterior intermuscular septum, or any of the musculature in the deep posterior compartment of the leg. The FDAL then courses posterior to the medial malleolus, enters the the tarsal tunnel (deep to the flexor retinaculum), and ends by inserting onto either the flexor digitorum longus tendon and/or the quadratus plantae muscle, which are located in the second muscular layer of the sole of the foot. The prevalence of the FDAL has been reported in 6% of asymptomatic individuals based on MRI studies or 2–12% of cadaveric studies.

The presence of the FDAL has been identified as a cause of tarsal tunnel syndrome, a compression neuropathy of the tibial nerve and/or its branches that become compressed as they course deep to the flexor retinaculum. As the FDAL traverses the tarsal tunnel, it lies immediately superficial to the neurovascular bundle and can act as a space‐occupying lesion, entrapping the tibial nerve and causing numbness, pain, and paresthesias in the foot, ankle, and toes. This variant muscle is best visualized through the use of magnetic resonance imaging (MRI). While there are conservative treatments for tarsal tunnel syndrome, most often surgical intervention is needed to remove the FDAL, and the success of this operative procedure is often based on the extent of damage to the tibial nerve prior to surgery. This presentation will include a patient case and show examples of the FDAL muscle following cadaveric dissection and within MRI studies. Radiologists and surgeons should be aware of presence of the FDAL muscle when considering the etiology of tarsal tunnel syndrome.

Support or Funding Information

The research of Jordan V. Swearingen and Kyle D. Miller was supported by the West Virginia University Initiation to Research Opportunities (INTRO) Program.
The Morphological & Biomechanical Characteristics of the Ligamentum Mucosum and its Potential Role in Anterior Knee Pain
Authors: Norris Madeleine Elizabeth , Burkhart Tim , Johnson Marjorie , Sandig Martin , Smallman Thomas , Getgood Alan
Published: November 2023

Introduction

The ligamentum mucosum (LM), or infrapatellar plica, is a non‐isometric structure of the knee joint that traverses from the intercondylar notch of the femur to the infrapatellar fat pad. The LM is composed of dense regular connective tissue, of which four different morphologies have been identified: separate, vertical septum, split, and fenestrated. Microscopically, pathological LM appears fibrotic and calcified, which can lead to impingement, and ultimately may be implicated in the pathogenesis of anterior knee pain. The specific aims of this study are to investigate the anatomical relationship of the LM to neighboring structures, determine whether the histological characteristics of the LM further define its function and its potential role in proprioception, and lastly to perform an in vitro biomechanical analysis of the LM under normal physiological loading.

Methodology

Fresh‐frozen cadaveric knee specimens (n=14) were dissected to reveal the structures of the joint capsule and to identify the morphology of the ligamentum mucosum. With the knee at 90° flexion, four measurements were recorded in order to examine the important relationships within the joint capsule. The intraclass correlation coefficient was calculated to determine the consistency and reproducibility of the measurements recorded. Maintaining the proximal and distal attachments, two specimens were harvested for histological analysis using standard hematoxylin and eosin, Masson's trichrome for collagen, and mono‐clonal mouse anti‐human neurofilament protein to identify neurons and peripheral nerve endings.

Results

Overall, 64% of the knees had a LM present. With respect to morphology, 50% of the 14 specimens had a separate type, 14% had a vertical septum type, and the remaining specimens had no LM present, which is consistent with known classification systems. The histological analysis confirmed the LM to be ligamentous, thus being composed of dense regular connective tissue. Preliminary immunohistochemistry results are inconclusive at this time for evidence of peripheral nerves and nerve endings, and the biomechanical properties of LM have yet to be determined.

Discussion

The role of LM in anterior knee pain is not well documented; however, studies have shown that excising the LM can relieve idiopathic anterior knee pain. With a decrease in elasticity, pathological LM may result in patellar maltracking and instability, ultimately triggering anterior knee pain. Future biomechanical testing will utilize an Instron materials testing machine to load the ligaments under tension at a constant velocity in order to determine the force at which the specimen fails. Thus, the findings from this study can be implemented in models of anterior knee pain, more specifically models of patellar instability and maltracking, to determine the proprioceptive and pathogenic mechanisms of the ligamentum mucosum.
Using ultrasound to prevent iatrogenic injury to the dorsomedial cutaneous nerve of the hallux
Authors: Miller Kyle D , Swearingen Jordan V , Zdilla Matthew J. , Lambert H. Wayne
Published: November 2023

Introduction

The dorsomedial cutaneous nerve (DMCN) of the hallux is a terminal branch of the superficial fibular (peroneal) nerve which supplies the skin of the medial aspect of the great toe. This cutaneous nerve branch is particularly vulnerable to iatrogenic injury during operative procedures involving the joints of the hallux, including hallux valgus correction, hallux ridigus correction, bunionectomy, and cheilectomy. Moreover, the DMCN of the hallux can be damaged during extensor hallucis longus (EHL) tendon transfer procedures due to the fact this nerve travels superficial to EHL tendon while traveling obliquely in the dorsum of the foot from a proximal lateral location to a distal medial position.

Methods

A first‐year medical student, with introductory‐level experience in ultrasonography (USG), performed all USG in the study in an attempt to identify the intersection point of where the DMCN of the hallux crosses the EHL tendon superficially. This study used an ultrasound machine (GE Venue 40 ultrasound machine) in conjunction with an L8‐L18i wide‐band high‐frequency linear array transducer to approximate this intersection. To check the accuracy of the DMCN location, a cruciate incision was made through the skin according to the midpoint markers on the L8‐18i probe.

Results

Successful mapping of the DMCN was gauged in binary fashion; if the DMCN was located directly beneath the center of the cruciate incision described, the method was considered successful. Of 28 feet dissected, 21 DMCN nerves (75%) were successfully located.

Conclusions

This study reports that a first‐year medical student with introductory‐level experience successfully identified the intersection of the DMCN with the EHL via USG in 75% of feet examined. Therefore, the success rate would likely improve when USG is performed by surgeons and clinicians with more experience in USG in conjunction with other examination methods, such as the four toe flexion sign. Therefore, USG may provide an inexpensive, accurate, non‐invasive means of pre‐operatively identifying the location of the DMCN of the hallux relative to the EHL tendon to prevent inadvertent nerve damage during foot surgery.

Support or Funding Information

The research of Kyle D. Miller and Jordan V. Swearingen was supported by the West Virginia University Initiation to Research Opportunities (INTRO) Program.
NURF Localizes in Gene Bodies to Regulate mRNA Processing
Authors: Landry Joseph , Alhazmi Aiman , Mack Marissa , Petencin Alexander , Nelson Humberto , Hiegel Jordan , Koparde Vishal , Scarsdale Neel , Lyons Charles , Dozmorov Mikhail
Published: November 2023

Understanding how chromatin remodeling complexes are recruited to chromatin is important to our understanding of how they regulate gene expression. Towards this end we determined the genome wide localization and sites of putative chromatin remodeling activity for the Nucleosome Remodeling Factor (NURF) by targeting its largest and essential subunit bromodomain PHD finger containing transcription factor (BPTF). From our BPTFChIP‐Seq and FAIRE‐Seq studies in BPTF knockout ESC we observe that BPTF islocalized and regulates chromatin structure broadly throughout the genome with a preference for promoters, gene bodies and terminators. A bio informatics analysis of these data sets discovers that a majority of NURF function as aregulator of gene expression is correlated with gene bodies and terminators. Abiochemical screen for binding partners of the BPTF N‐terminal PHD finger discovers specific interactions with THOC4, a mRNA processing factor which binds to an elongating polymerase. Analysis of NURF function at the BPTF‐dependent gene Ccnd1 discovers BPTF‐dependent roles in splicing out intronDNA sequences from mRNA transcripts. These defects correlate with evidence of RNA polymerase stalling, a known mechanism for down regulating gene expression with intron retention (IR). These results present model where NURF regulated gene expression can occurs through defects in retaining intron sequences.

Support or Funding Information

Jeffress Foundation

Virginia Academy of Sciences

Virginia Commonwealth University

Massey Cancer Center
A Specialized Mechanism of MicroRNA‐mediated Translation in Quiescence
Authors: Vasudevan Shobha , Bukhari Syed Irfan Ahmed , Truesdell Samuel Spencer , Lee Sooncheol , Kollu Swapna , Classon Anthony , Boukhali Myriam , Jain Esha , Yanagiya Akiko , Sadyrev Ruslan , Haas Wilhelm
Published: November 2023

Quiescence (G0) represents an assortment of reversible, cell cycle‐arrested states, associated with cancer persistence as well as development. G0 involves selective mRNA expression while decreasing canonical translation. The alternative translation mechanisms in G0 remain to be uncovered.

Our data show that microRNAs, regulatory, non‐coding RNAs that target distinct mRNAs to alter gene expression, are important in G0, where they can associate with alternative translation factors to regulate specific mRNA translation. One subset of transcripts expressed in G0 includes specific mRNAs recruited by an FXR1a‐associated microRNP (microRNA‐protein complex) for translation activation in G0 mammalian cells and G0‐like immature Xenopus laevis oocytes. MicroRNPs predominantly mediate repression and downregulation; however, FXR1a‐microRNP lacks the conventional microRNP repressor, GW182, and instead, contains a specific RNA binding protein isoform, FXR1a. FXR1a promotes translation and is overexpressed and associated with poor prognosis in certain cancers. Activation is dependent on target recruitment by FXR1a‐microRNP in the nucleus, indicating compartmentalized mRNA recruitment for selective target activation. Our data reveal that microRNA‐mediated activation requires target mRNAs with unadenylated/shortened poly(A) tails. Polyadenylated mRNAs are repressed, potentially due to the role of PABP in enhancing microRNA‐mediated downregulation and in canonical translation that is impaired in these conditions. Consistently, PARN deadenylase is required for microRNA‐mediated activation. Low mTOR activity in G0 activates the cap complex inhibitor, eIF4E‐BP, and thereby, impairs canonical translation; instead, as previously shown, interaction of PARN with 5′ mRNA caps is increased in G0. The target mRNA 3′‐UTR recruits FXR1a‐microRNP, which associates with PARN and with p97, a homolog of the translation factor eIF4G, which recruits eIF3, and thereby, 40S ribosome subunits but lacks cap complex‐ and PABP‐interacting domains. These data reveal a specialized translation mechanism that is important for G0 maintenance and chemoresistance, where FXR1a‐microRNP connects specific, poly(A) shortened mRNAs to the ribosome through interactions with alternative factors, PARN and p97, in these conditions of reduced canonical translation.

Support or Funding Information

Cancer Research Institute, The Leukemia & Lymphoma Society, MGH funds & NIGMS (SV). Fund for Medical Discovery postdoctoral fellowship (SL).
Alternative ribosomes synthesize alternative proteins
Authors: Dow Allexa , Prisic Sladjana
Published: November 2023

In addition to the complete set of ribosomal proteins needed to form ribosomes, many bacteria have at least one duplicated (i.e., alternative) ribosomal protein. It has been suggested that alternative ribosomal proteins (AltRP) in Bacillus subtilis and Escherichia coli serve as a replacement for zinc‐binding “primary” homologs (PrimRPs) when zinc is deficient. Therefore, it has been proposed that Prim/AltRP pairs have a role in zinc storage and release depending on zinc availability. Mycobacterium tuberculosis has four alternative ribosomal proteins organized in a single operon, whose function is not known. We hypothesize that mycobacterial AltRPs have functions other than zinc storage.

In order to test if mycobacterial PrimRPs serve as a source of zinc in low zinc environment and whether or not they have some other roles, we deleted the altRP operon in M. tuberculosis. There was no growth defect in the mutant compared to the wild type when grown in zinc‐rich or zinc‐deficient medium. We have evidence that AltRPs are replacing PrimRPs in ribosomes when zinc is limited (i.e., they form “alternative” ribosomes), but this switch from PrimRPs to AltRPs did not have any measurable effect on ability of M. tuberculosis to survive under low zinc conditions. However, we observed “bulging” cell morphology in the altRP deletion mutant, which is consistent with a cell wall defect. Considering that the cell envelope defect may be caused by changes in building blocks or enzymes required for their synthesis, we used proteomics to analyze protein content in the altRP mutant vs. wild type. Indeed, proteomes of the deletion mutant (contains only “primary” ribosomes) was significantly different compared to the wild type parental strain (contains a mixture of “primary” and “alternative” ribosomes). The perturbation of the proteome caused by the lack of “alternative” ribosomes allowed us to identify a set of proteins preferably synthesized by such ribosomes. Specifically, differences in the abundance of proteins involved in cell wall synthesis or components of the cell wall may explain the difference in the cell morphology.

Our data indicate that AltRPs in M. tuberculosis are not required for growth under low zinc conditions and therefore they may have only limited role in adaptation to changes in zinc availability. Importantly, our study shows that AltRPs can replace PrimRPs in ribosomes, which in turn causes a significant change in the proteome, either directly by preferentially translating a distinct set of mRNAs or indirectly, e.g., by changing the amount of repressors or proteases. Therefore, in addition to transcriptional regulation, the shift from “primary” to “alternative” ribosomes provides another layer of regulation, which may be important for adaptability and pathogenicity of M. tuberculosis.

Support or Funding Information

This research is supported by College of Natural Sciences Startup Funds
Development of Prolyl‐tRNA Synthetase from Pseudomonas aeruginosa as a Platform to Screen for Inhibitors of Protein Synthesis
Authors: Pena Noah , Hu Yanmei , Bullard James M
Published: November 2023

Introduction

Pseudomonas aeruginosa is an opportunistic pathogen and a common cause of nosocomial infections. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis. We describe here the enzymatic characterization and development of a screening platform based on prolyl‐tRNA synthetase (ProRS) from P. aeruginosa.

Results

ProRS from P. aeruginosa was cloned and expressed in E. coli and purified to greater than 98% homogeneity. Sequence analysis shows that this protein contains the characteristic motifs of class I aminoacyl‐tRNA synthetases and when compared with E. coli exhibited approximately 70% amino acid sequence conservation. The kinetic parameters for the interaction of P. aeruginosa ProRS with its three substrates (tRNA, ATP, and methionine) were determined. Initial velocities were determined for charging of tRNA using tRNAPro concentrations between 0.8 and 3 μM. The KM and Vmax was determined to be 7 μM and 0.67 μM min−1, respectively, resulting in an observed kcat of 0.22 sec−1. This resulted in a kcat/KM value of 0.033 s−1μM−1. The ATP:PPi exchange reaction was used to monitor the interaction with ATP and proline (Pro). The KM, Vmax, observed kcat, and kcat/KM for interaction with ATP was 700, 168, 14, and 0.02 μM, respectively. Scintillation proximity assay (SPA) technology was adapted to the aminoacylation assay and then used to screen for inhibitors of activity of P. aeruginosa ProRS in a high throughput format. Using this assay, a natural product (800 compounds) and a synthetic compound (890 compounds) library was screened.

Conclusion

ProRS identified in P. aeruginosa was cloned, expressed characterized and developed into a screening platform to identify compounds that have the potential for development as an antibacterial agent against pathogenic organisms.

Support or Funding Information

Research was funded by NIH grant 1SC3GM098173‐01A1. Partial student support was from a Departmental Grant from the Robert A. Welch Foundation (Grant No. BG‐0017), the NIH UTPA RISE program grant # 1R25GM100866‐01 and the Howard Hughes Medical Institute (HHMI) Precollege and Undergraduate Science Education Program grant #52007568.
Integrating reductive and synthetic approaches in biology using man‐made cell‐like compartments
Authors: Aoki Wataru , Komura Shun , Tamiya Eiichi , Ueda Mitsuyoshi
Published: November 2023

We propose ‘synthetic genetics’ as a novel methodology that integrates reductive and synthetic approaches used in life science research [1]. Life science research seeks to elucidate the relationships between genotypes and phenotypes. This typically involves reductive (genetic or omics researches) and synthetic (synthetic biology) approaches. Genetic and omics research seeks to identify genes involved in a biological subsystem of interest such as transcription, translation, signal transduction, genome repair, and metabolism. This approach enables identifying individual genes in a target biological subsystem, although the entirety of the subsystem is not readily characterized. To overcome this, synthetic approaches are used, in which a known set of genes is isolated and combined to reconstruct a target biological subsystem with the aim of a complete proof for the entirety of the subsystem. Life science research combines these two methodologies. However, it is often difficult to fill in information gaps in a target subsystem on the basis of these approaches, and tremendous amounts of time and effort are required to completely understand a subsystem's functions.

‘Synthetic genetics’ enables determinations of sets of genes required for the functioning of any biological subsystem [2]. This method utilizes artificial cell‐like compartments, including a randomly introduced whole gene library, strictly defined components for in vitro transcription and translation, and a reporter that fluoresces ‘only when a particular function of a target biological subsystem is active.’ The set of genes necessary for the target biological subsystem can be identified by isolating fluorescent artificial cells and multiplex next‐generation sequencing of genes included in these cells. The importance of this methodology is that screening for the set of genes involved in a subsystem and reconstructing the entire subsystem can be done simultaneously. This methodology can be applied to any biological subsystem of any species and may remarkably accelerate life science research.
Chemical Genetic Analysis Of Mast‐Cell Activation
Authors: August Avery
Published: November 2023

Mast cells play critical roles in allergic responses, and calcium signaling controls the function of these cells, and a role for actin in regulating calcium influx into cells has been suggested. Using chemical genetic approaches, we identify the actin reorganizing protein Drebrin as a target of the immunosuppressant BTP, which inhibits calcium influx into cells and IgE‐mediated allergic responses. We also show that Drebrin−/− mice exhibit reduced IgE‐mediated allergic responses. Furthermore, Drebrin−/− mast cells exhibit defects in actin cytoskeleton organization and calcium responses downstream of the FcɛRI, and agents that relieve actin reorganization rescue mast cell FcɛRI induced degranulation. Drebrin thus represents a novel regulator of mast cell function by regulating the actin cytoskeleton.

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AI108598
Are Enzymes Required for the Extraction and Isolation of DNA in Conjunction with Mechanical Homogenization?
Authors: Easparro Brandon A. , Garrett Shari , Atwood James
Published: November 2023

Enzymes are biological catalysts that play a role in the isolation of nucleic acids from tissue and microbial sample types. In 1973, Maria Gross‐Bellard was one of the first researchers to use Proteinase K in the isolation of DNA from mammalian cells1. Today, enzymes are commonly provided in DNA isolation kits to enable cellular and organelle disruption and for the removal of contaminating proteins. DNA kit enzymes vary based on the target sample. While Proteinase K is commonly used in the isolation of DNA from mammalian cells and tissues, lyticase and lysozyme are enzymes used to degrade the cell walls of yeast and bacteria and are frequently included in microbial DNA isolation kits. While effective, enzymes represent a significant percentage of DNA kit cost and require special storage and handling conditions. While standard lysis buffers coupled with enzymes can be used to disrupt soft tissues and cells, mechanical homogenizers are routinely employed for disruption of tougher tissues and cells with robust cell walls. In these cases, are enzymes necessary to obtain high concentrations of DNA?

In this study, DNA extractions were performed using commercially available DNA extraction kits from three sample types: bacteria, yeast and tissues. DNA was extracted per the kit manufacturer's instructions in conjunction with mechanical homogenization via bead milling. The analysis was then repeated without the use of enzymes. DNA concentration and quality was evaluated by spectrophotometry and electrophoresis respectively for each sample type and processing method. High quality DNA was recovered with and without the use of enzymes. Although samples with enzyme digestion brought higher yields of DNA, the integrity of DNA was equivalent in samples without the enzyme digestion. Enzyme requirement in DNA isolation is based on down stream analysis.
Dissection of DNA Synthesis in crystallo Reveals a Third Mg2+ in the Transition State
Authors: Gao Yang , Yang Wei
Published: November 2023

A conserved two‐metal‐ion mechanism has been proposed for all DNA and RNA polymerases. However, a third divalent cation coordinated by the reaction products was recently observed during in crystallo DNA synthesis by Pol η and Pol β (ref. 1, 2). Using time‐lapse X‐ray crystallography, we show that the well established binding of two Mg2+ or Mn2+ ions by Pol η leads to alignment of the DNA substrate and the incoming nucleotide for catalysis, but acquisition of the third metal ion is necessary for the phosphoryl‐transfer reaction. The third metal ion binding site is absent in the enzyme‐substrate complexes and relies on thermal energy for its formation. Pol η appears to acquire the third metal ion as a cofactor in the transition state to overcome the energy barrier for product formation. Identification of the non‐enzyme bound but essential third metal ion in the Pol η catalysis leads us to anticipate that acquisition of additional catalytic cofactors in transition states may be a general feature of enzyme catalysis.

Support or Funding Information

This work is funded by NIH intramural program (DK036146‐08, W.Y.).
IN VITRO EVALUATION OF CYTOTOXIC AND ANTIOXIDANT ACTIVITY OF ESSENTIAL OIL AND TERPENES OF Satureja macrostema
Authors: Salgado‐Garciglia Rafael , Torres‐Martínez Rafael , Hernández‐García Alejandra , Saavedra‐Molina Alfredo , López‐Meza Joel Edmundo , Ochoa‐Zarzosa Alejandra
Published: November 2023

Satureja macrostema is a Mexican medicinal plant known as nurhitini té or nurite, whose antioxidant properties are mainly attributed to the volatile compounds from aerial part. Therefore, the goal of the present study was to investigate the in vitro cytotoxic and antioxidant potential of essential oil and some related terpenes of S. macrostema. Essential oil from fresh aerial parts of S. macrostema plants were prepared by hydrodistillation and characterized by GC‐MS, finding that the major terpenes were caryophyllene, limonene, linalool, menthone, pulegone and thymol. The effects of essential oil and terpenes from S. macrostema on bovine umbilical vein endothelial cells were determined. Different concentrations of essential oil and terpenes (1, 10, 100, 1000 μM) were added to cultured cells and incubated for 24 h. Cell survival was evaluated using the MTT‐based cytotoxicity assay. The essential oil and linalool, menthone and pulegone did not affect the BUVEC viability (> 86%), while limonene, caryophyllene and thymol showed toxicity at 1000 μM, causing less than 60% viability. Nitric oxide (NO) was determined using Griess reaction method in BUVEC induced with Staphylococcus aureus and treated with essential oil and terpenes from S. macrostema. The essential oil and terpenes at 1, 10 and 100 μM showed a diminution of NO production, indicative of the antioxidant activity. In addition, antioxidant effectiveness of essential oil and terpenes was examined by two different radical scavenging methods, DPPH (2,2‐Diphenyl‐1‐picrylhydrazyl) and ABTS (2,2′‐azino‐bis‐3‐ethylbenzthiazoline‐6‐sulfonic acid), testing 1, 10, 100 and 1000 μM. The essential oil showed the highest antioxidant capacity, reaching 53.10% capture of radicals in the DPPH and 92.13% in the ABTS method. Thymol was the terpene with higher antioxidant activity (94%) at 1 μM determined with both methods. The results obtained show that essential oil and the major terpenes of S. macrostema have antioxidant activity, at concentrations that do not compromise BUVEC viability.

Support or Funding Information

Financial support grant from CONACYT (RTM) and CIC/UMSNH (Project 2.10 to RSG and 14.1 to AOZ).
Small Molecule Binding Sites Explored in Variable Heavy Chain Antibodies
Authors: White Brandy R , Gee Shirley J , Hammock Bruce J , Brooks Cory R
Published: November 2023

Polybrominated diphenyl ethers (PBDEs) are chemical flame retardants that have been used extensively over the last 40 years in furniture, plastics, wiring, and electronics. Some PBDEs have been classified as possible human carcinogens. PBDEs have been widely found in human blood, breast milk, and body fat. While PBDEs have recently been placed on the California Biomonitoring Program and have been banned by the EPA, these PBDEs persist in the environment. 2,2′,4,4′‐tetrabrominated diphenyl ether (BDE‐47) and Tetrabromobisphenol A (TBBPA) are two of these chemical flame retardants that are persistent in the environment and detecting them with an VHH‐based method has been developed. However, it is not well understood how these detection methods work and it is difficult to improve on the methods without this understanding. The variable heavy chain domain antibodies (VHH) from camelids have many advantages compared to conventional monoclonal antibodies including increased stability and ease of recombinant production. Although it is well established how VHH interact with proteins, it is less well understood how these antibodies interact with small molecules and haptens. There are currently four known binding modes available for VHH to interact with small molecules including: combining VHH's three CDR loops, domain swapping utilizing a 2:1 binding stoichiometry, and tunneling under the CDR1 loop. Using VHH specific for BDE‐47 and TBBPA as a model for PBDEs we aim to examine VHH small molecule interactions. We have successfully completed expression tests of seven VHH and purified 2 VHH. Crystallization trails have yielded preliminary crystals. Future work will focus on crystal optimization and structure determination.

Support or Funding Information

‐BW was supported with funds from the Undergraduate rGrant from The Associated Students, Inc. at California State University, Fresno.