bluuebunny/crossref_metadata_2025 huggingface dataset:
Factors affecting craniofacial development in a mouse model of Crouzon Syndrome
Authors: Brakora Katherine , Percival Christopher J , Green Rebecca , Gong Siew‐Ging , Hallgrimsson Benedikt , Marcucio Ralph
Published: November 2023

Crouzon Syndrome (CS) is characterized by premature fusion of calvarial bones (craniosynostosis) as well facial malformations including midfacial hypoplasia and hypertelorism. The phenotype is variable, even when caused by the same mutation within the same family. The cause(s) of this variability remain unknown. Using a mouse model of CS, caused by a missense mutation in FGF Receptor 2, we investigate the role of age, sex, and parental genotype in the development of the craniofacial phenotype. Specimens were collected at E10.5–13.5, E17.5, P0, and at 12 weeks or more, andgenotyped. Specimens younger than 3 weeks were sexed using PCR for the SRY gene. 2D and 3D geometric morphometrics was used to compare craniofacial size and shape, using surface landmarks for embryos, and bones after birth. Wildtype and heterozygous groups were compared at each age, controlling for all other variables, to create a detailed picture of the ontogenetic progression of the CS phenotype caused by this mutation. Then, specimens were grouped by genotype and tested for differences in size and shape based on sex or parental genotypes. We identify when in development homozygous mutants, heterozygous mutants, and wildtype phenotyeps diverge in size and shape, and to what degree sex and parental genotypes affect the timing, severity, or variability of heterozygous and wildtype phenotypes at each age.

Support or Funding Information

Supported by NIH‐NIDCR grants F32DE024939 to KB and 2R01DE018234‐05A1 to RM and BH.
Effects of zoledronate regimen on the induction of BRONJ in rats
Authors: Elsalanty Mohammed , Daoudi Asma , Howie Nicole , Panos Anastasia , Kurago Zoya
Published: November 2023

Background

Bisphosphonate‐Related Osteonecrosis of the Jaw (BRONJ) has been reported in patients undergoing long‐term bisphosphonate treatement. So far, no biological mechanism has been shown to explain the condition. This study compared the induction of the clinical features of BRONJ between two zoledronic acid (ZA) regimens. We hypothesized that duration of treatment would be more important than the dose of ZA for the induction of BRONJ.

Methods

We included 80 female Sprague‐Dawley rats in four groups (20 rats‐each): Group 1 received 80μg/kg of intravenous ZA weekly for 13 weeks, group 2 received 150μg/kg ZA daily for 16 consecutive days, while groups 3 and 4 received corresponding injections of saline for the same durations. At the end of the treatment period, the first and second mandibular molars were extracted. Animals were sacrificed at 1, 2, 4, and 8 weeks. The study analyzed the therapeutic effect of treatment on vertebral bone quality, the features of BRONJ, and the toxic effects on the liver and kidneys.

Results

There was an increase in the cortical thickness and area fraction of the vertebra in the ZA‐treated animals at 1 and 8 weeks post extraction compared to the controls. This “therapeutic” effect was more pronounced in the lower ZA dose. On the other hand, All ZA‐treated animals manifested the clinical, histological, and radiographic features of BRONJ. There was a significant decline in osteoclast activity in the treatment groups at 1‐week. The higher dose had a more persistent decline in osteoclast recruitment at 8 weeks.

Conclusions

Both doses induced BRONJ in all treated animals. The shorter, high‐dose course of ZA had less therapeutic effect and induced features of organ toxicity. The longer, lower dose of ZA provides a superior translational model for BRONJ.

Support or Funding Information

This study was supported by NIA 1P01AG036675, a Georgia Regents University Extramural Success Award (ESA), the Fort Gordon Periodontology Command, the Center for Undergraduate Research and Scholarship and the Honors Program at Georgia Regents University.
Determination of Palatal Bone Density to Aid in Oral Mini‐Implant Surgical Success
Authors: Flores Alicia , Reed James Christopher
Published: November 2023

Mini‐implants are titanium alloy rods implanted in the bone of the hard palate to help secure dental prostheses like dentures, fixed crowns, and bridge installations. Recent research suggests presurgical determination of bone density quality provides increased mini‐implant surgical success rates. In replication of these methods, we evaluated 19 tomographic scans of crania using Osirix 8.5 imaging software. Bone density was recorded at 90 separate coordinates using Hounsfield units, measured at three millimeter intervals (mediolaterally and anteroposteriorly starting at the incisive foramen.) The data was then analyzed for age differences and average bone density throughout different regions of the hard palate including intra‐ and interobserver analysis. Sex differences were closely investigated in this study because prior research has indicated that women may have a higher palatal bone density than men.

Support or Funding Information

Student Summer Scholars Grant, Office of Undergraduate Research, Grand Valley State University
Extracellular Matrix Protection Factor 1 (ECPF‐1), A Novel Inhibitor of Matrix Metalloprotease 13 (MMP‐13) Is Chondroprotective In A Serum‐Free Culture Model Of Primary Human Articular Chondrocytes
Authors: Buckley Andrea Lynn , George‐Weinstein Mindy , Milton LaBraya , Datillo Victoria , Goswami Soumendro , Lozano‐Torres Xiomara , Behling Kathryn , Cho Ellen , Chmielewski Sarah , Selim Abdulhafez , Miller Lawrence , D'Angelo Marina
Published: November 2023

The pathogenic process that defines osteoarthritis (OA) is the imbalance between excess degradation of the extracellular matrix in articular cartilage and a decreased ability of chondrocytes to remodel the matrix. There are many factors that contribute to this disease including unchecked actions of matrix metalloproteinase 13 (MMP‐13), an enzyme specific for the breakdown of collagen type II which is the main component of cartilage extracellular matrix. We have developed a three‐dimensional, serum‐free culture system to rear primary, human articular chondrocytes. In this study, we tested the effects of a member of the novel class of MMP inhibitors, Extracellular Matrix Protection Factors, ECPF‐1.

Primary human articular chondrocytes were isolated from patients who underwent surgery for total knee arthroplasty (TKA). The medial and lateral aspects of the joint samples were kept separated and labeled as greatest or least pathology. The number of lives cells were determined and plated at 2.5 × 106 cells per 35 mm well. Cells were either treated with 250nM ECPF‐1 at time of culture, day 0, and again on day 2 of culture or left untreated as serum‐free control. Day 2 and day 5 conditioned media and day 5 cell‐associated matrix was isolated, concentrated with centrifugation and immunoassays conducted to measure collagen type I degradation telopeptides. Treatment with 250nM ECPF‐1 resulted in statistically significant reduction in collagen type I degradation products present in the conditioned media and in the cell‐associated matrix of cells isolated from the side of greatest pathology when compared with untreated controls following five days in culture. Cells isolated from the side of least pathology produced significantly lower amounts of collagen degradation products when compared to the side of greatest pathology.

This study demonstrates that human articular chondrocytes reared in serum‐free, primary culture are responsive to the novel therapeutic, ECPF‐1. Our culture model is a straightforward and cost effective system in which to test potential OA therapeutics.

Support or Funding Information

Center for Chronic Disorders of Aging Pilot Grant, PCOM Cooper Foundation Grant, Cooper Health System
Laterality of Pancreatic Metastases from Renal Cell Carcinoma: An Anatomical Perspective from the Left Kidney and Tail of the Pancreas
Authors: Raffoul Adam J , Hartley Christopher Kirk , Johnson Marjorie , Gomez Jose A , Pautler Stephen , McAlister Vivian
Published: November 2023

Introduction

Surgical findings have shown left kidney renal cell carcinoma (RCC) spreads to the pancreas in an isolated fashion. The pancreas is not a common metastatic site for cancers known to spread via the systemic circulation or lymphatics so no anatomical explanation for these findings has been described. Current literature dictates that rapidly growing neoplasms can transgress renal fascial planes. We hypothesize that there is both a lymphatic and vascular track traversing and within Gerota's fascia which provides local communication between the left kidney and tail of the pancreas. The lymphatic and vascular systems in the perirenal and pararenal spaces are undocumented.

Objectives

The objective of this study is to propose an anatomical explanation for the spread of RCC from the left kidney to the proximal tail of the pancreas and establish its laterality in regards to preferential metastatic spread.

Methods

Four fresh‐frozen cadaveric specimens and 20 fixed cadaveric specimens are in the process of retroperitoneal dissection. Left sided perirenal and pararenal lymphatic vessels and lymph nodes will be identified using GEWF (glacial acetic acid, ethanol, distilled water, and formaldehyde) and immunohistochemistry; monoclonal antibody D2–30 (a lymphatic endothelial marker). Vasculature will be identified under a surgical microscope and further characterized using immunohistochemistry; polyclonal antibody CD31(a pan‐endothelial marker). A chart review of patients with RCC metastasis (from the London Health Sciences Centre, London Ontario) is currently being developed into a retrospective case‐control study to determine frequency of RCC metastasis to the pancreas.

Results

Dissection of fresh‐frozen cadavers has revealed vascular bundles that emerge from the fibrous kidney capsule to enter the fat‐filled perirenal space. These bundles contain arteries, veins and lymphatics. Microdissection has revealed a high concentration of these vascular networks in the superior pole of the left kidney, converging on the left adrenal gland. These networks are also seen to traverse the perirenal space, contained within Gerota's fascia. Small venules are seen piercing Gerota's fascia and running within this multilaminar connective tissue. We predict that the chart review will show left kidney tumours metastasizing more often to the tail of the pancreas possibly by means of these described networks.

Conclusions

Understanding the vascular and lymphatic pathways between the left kidney and the tail of the pancreas will help determine dissection planes and the extent of radical resection surgery with a curative intent. The anatomical description of existing pathways between these structures and the perirenal and pararenal spaces may aid in the anatomical understanding of the spread of RCC. It may also have implications where local resection of tumours is considered.
Pig (Sus suis): a Half‐Sized, Dismantabled, “Ecorché” Anatomical Model of the first part of the 20th Century
Authors: LE FLOCH‐PRIGENT PATRICE , GILLOT Jean‐Bernard
Published: November 2023

A half‐sized model of a common pig (Sus suis) belonged to the Brieux‐Gillot's scientific library in Paris. It was dismantabled and écorché on the left half and represented the cutaneous aspect on the right side which was fixed on a wooden, 25 × 70 cm, basis. The two ears were removable without any detail, the anterior left leg and the left half of the head were separately removable and the left trunk and the posterior leg made the largest dismantabled part with inside the representation of the main viscera. The datation looked to be the first part of the 20th century. Each element especially the muscular ones were numbered corresponding to a list which was not known. The only label on the basis was the cardinal arrow of a compass. A large red number was painted on the superior face of the basis. This kind of pig's model is quite common for the animal studies, it looked anatomically correct and conform to classical textbooks for instance Barone, edited by Masson in Paris.
The Lumbar Facet Joint Capsule Thickens Following Spinal Segmental Hypomobility Induced Osteoarthritis
Authors: Benckendorf Adam N. , Merlo John A. , Cramer Gregory D. , Little Joshua W.
Published: November 2023

Low back pain (LBP) is a major medical issue and the primary cause for global disability. One common cause of LBP is osteoarthritis (OA) of the lumbar facet joints. OA involves numerous tissues that can induce pain. In particular, during facet joint OA in humans, the articular capsule thickens in response to mechanical and inflammatory stresses; this thickening is associated with LBP. We have previously reported a novel rat model using external spinal linkage to cause lumbar spinal hypomobility and induce OA the in lower lumbar facet joints (L4/5 and L5/6) that may model the progression of spinal OA in humans. This hypomobility results in significant macroscopic OA changes to the articular cartilage and subchondral bone bilaterally by 8‐weeks post linkage. The onset and progression of articular capsule changes and their relationship with pain behaviors is unexplored in this model. Here we examined if lumbar spinal segmental hypomobility in rats is associated with thickening of the ventral articular capsule (ligamentum flavum, LF) as evidenced by increases in morphometric measurements in the bilateral L5/6 facet joints. We hypothesized that 8 weeks of spinal linkage, a time point of significant facet joint OA, is associated with an increase in articular capsule thickness. We used a histological approach with morphometric analyses based upon previously reported methods to test our hypothesis in linked and time matched control rats. LF thickness measurements were performed on images obtained from a brightfield microscope (10X objective, Leica DMRB, optronics microfire camera) on formalin fixed, decalcified, paraffin embedded, Ehrlich's hematoxylin and light green stained 45 μm thick transverse sections of the bilateral L5/6 facet joints. Morphometric measurements were made (OsiriX Lite software) by drawing a line through the thickest region of the LF, perpendicular to the plane of the ventral LF surface. This morphometric approach demonstrated acceptable reliability (ICC= 0.8656). Statistical analysis was performed using an unpaired t‐test. Compared with control animals (n=4), 8 weeks of hypomobility (n=4) was associated with a significant increase in LF thickness (p<0.05). The articular capsule thickness increased at a time point previously shown to have significant OA changes, which is consistent with facet joint OA and LF thickening in human subjects. Increases in articular capsule thickness are hypothesized to contribute both to the induction or reflect the ongoing pathological processes of OA (e.g., prolonged inflammation and fibrosis). Further work is required to determine the onset, joint location, and mechanisms underlying these capsule changes. These findings suggest that lumbar spine hypomobility induces LF thickening.
Accessory Quadrate of Liver & its Implications
Authors: Chandrasekaran Chithambaram
Published: November 2023

Introduction

The liver is the largest wedge‐shaped gland in the human body. Anatomically it is divided into right lobe and left lobes with quadrate & caudate lobes. The congenital abnormalities of human liver are rare and these are rarer than almost any other organs of the body. Various congenital abnormalities of the liver as agenesis of its lobes, absence of its segments, deformed lobes, decrease in size of lobes, lobar atrophy, hypoplastic lobes, and transposition of the gall bladder, extra caudate lobe and Riedel's lobe have been reported by various researchers. The quadrate lobe is an area of the liver situated on the under surface of the right lobe, bounded in front by the anterior margin of the liver; behind by the porta hepatis; on the right, by the fossa for the gall‐bladder; and on the left, by the fossa for the umbilical vein. It is oblong in shape, its antero‐posterior diameter being greater than its transverse.

Methodology & Results

Accessory quadrate lobe has been observed towards the left end of quadrate lobe, beside ligamentum teres in our routine dissections. Its shape was Triangular in shape with its dimensions of length – 3.4 cm from its apex to base, thickness 0.9 cm, base – 1.8 cm.

Conclusion

These developmental anomalies of liver may cause confusion to clinicians during procedures like biopsy, transplantation, and lobectomies. So finding of this new variant under unique configuration of this lobe assumes more importance to anatomists including morphologists, and its knowledge may be of immense use to clinicians in the diagnosis and management of hepatic diseases and to embryologists for new developmental defect. Therefore, it is worth reporting as a new variant.

Support or Funding Information

AMERICAN ASSOCIATION OF ANATOMIST(AAA)
“You Are What You Eat” OR “You Eat What You Are?” Comparative Mammalian Gastrointestinal Anatomy
Authors: Ramalanjaona Benjamin Joelinjaka , Sorrento Cristina , Pagano Anthony S , Marquez Samuel
Published: November 2023

As mammals occupy a wide array of ecological niches and exploit various sources of nutrition, so too do they exhibit great functional and morphological diversity in the gastrointestinal tract (GIT). The GIT is a metabolically expensive organ and its associated adaptations are essential to maintaining energy balance. Balancing homeothermy and lactation require a faster metabolism among mammals relative to those of reptiles given their substantial energetic cost. This study documents morphological analogies among the GIT's of three mammalian omnivores: squirrels, pigs and humans. Similarities included a monogastric (or nonruminant) template to accommodate a dietary staple of nuts, seeds, fruits, fungi, green vegetation, and animal matter. All species presented a mesentery that serves as a conduit for arterial supply and venous and lymphatic drainage of the regional gut except that, in the squirrel, the mesentery was thinner and appeared more translucent. Further differences included a relatively large caecum (probably a vestige from an ancestor that engaged in hindgut fermentation as modern squirrels cannot digest cellulose) and a curious loop in the squirrel large intestine. Pigs and human both exhibited small intestines that appeared longer and with more intricate networks of vasae rectae. They also had considerably greater amounts of adipose tissue in their messenteries with humans possessing the most. Based on our study, the pig is remarkably similar to the human condition. Both are large‐bodied mammals with similarly wider dietary preferences than that of the squirrel. In spite of the closer phylogenetic relationship among squirrels and humans (Rodentia and Primates are both within the superorder Archonta), the GIT of pigs and humans have been subjected to similar selective pressures as a likely function of similar body mass.
Prevalence of the Anterolateral Ligament
Authors: Hinkley Jacob , Lampert Peter , Canby Craig
Published: November 2023

First described by Paul Segond in 1879, the anterolateral ligament (ALL), is a fibrous band of tissue attaching to the distal aspect of the lateral femoral epicondyle and the proximal tibia just lateral to Gerdy's tubercle of the tibia. A previous study by Claes et al (2013) investigated the ALL and suggested that it had a prevalence of 97% in 41 unpaired cadaveric specimens. This study was designed to screen body donors to see if the ALL was as prevalent as previously reported. Forty‐six paired cadaveric knees were dissected from student gross anatomy courses. Of the 46, we were able to confidently examine 41 as the other five were excluded from the study either due to desiccation, knee transection, or knee arthroplasty. Our study found a prevalence of the ALL to be 51%, with males having greater prevalence than females (60% occurrence in males and 29% occurrence in females). Twenty of the body donors were found to have bilaterally distinct ALLs and one had a unilateral presence. Our study also found two different attachment sites on the distal femur possibly suggesting the ALL could be an independent structure and not just a projection of the lateral collateral ligament. Our study supports the existence of the ALL as a distinct structural entity albeit at prevalence less than previously reported.

Support or Funding Information

Supported by the Anatomy Department, Des Moines University
PENO‐SCROTAL RAPHE ABSENCE & ITS HERNIATION
Authors: DOKIPARTY KRUPA DANIEL
Published: November 2023

Introduction

The scrotum is an anatomical male reproductive structure that consists of a suspended sack of skin and smooth muscle that is dual‐chambered located under the penis. One testis is typically lower than the other, which functions to avoid compression in the event of impact. The scrotum contains the external spermatic fascia, testes, epididymis, ductus deferens. It is a distention of the perineum and carries some abdominal tissues into its cavity including the testicular artery, testicular vein and pampinform plexus. A central cordlike line running over the scrotum from the anus to the root of the penis, marking the position of the scrotal septum.(Image‐1)

Methods

In our regular dissection course we found with an peculiar absence of penoscrotal raphe which has appeared as an single chambered scrotum with unified penis as absence of left corpora cavernosa & only presence of right corpora cavernosa and corpus spongiosum. The single chambered scrotum which was been herniated with the coils of intestines especially with terminal ileum & appendix with its omenta also peculiarly found with the absence of left side testis & its spermatic cord. (Imgaes ‐ 2,3,4,5)

Conclusion

Complete absence of left side testis & its spermatic cord.

Single chambered scrotum with right testis only & herniated content.

Absence of median raphe.

Inguinal herniation should occur on right side but looping over towards the left side scrotal sac with the terminal ileum, vermiform appendix and its mesentry. (Images ‐ 6,7)

Significance

Surgical correction is necessitate for this type of herniation. Surgery is the primary treatment for herniation. It's a very common operation and a highly successful procedure done by (“open” repair) or laparoscopic surgery (done through a small scope).

Support or Funding Information

AMERICAN ASSOCIATION OF ANATOMISTS (AAA)
Transcription by the telomeric shelterin protein TRF2 is telomere‐length dependent ‐telomeric partitioning and global transcription
Authors: Chowdhury Shantanu
Published: November 2023

Our data demonstrate TRF2 occupancy at the p21, as well as other promoters tens of Mb away from telomeres, to be dependent on telomere length. Of particular interest was the finding that p21 activation, following DNA damage, was transcriptionally regulated by TRF2 in a telomere‐length dependent fashion. Earlier studies have reported transcription to be telomere‐dependent in regions close to telomeres (subtelomeric) through primarily epigenetic mechanisms known as telomere‐positioning‐effect or TPE, which is unlikely to explain telomere‐dependent transcription tens of Mb from telomeres. Our results suggest a novel model of telomere‐dependent gene transcription involving telomere length in partitioning interstitial TRF2 binding.

Support or Funding Information

Wellcome Trust ‐ Department of Biotechnology India Alliance and Council of Scientific and Industrial Research
Specificity and Activity of DNA Polymerase DinB and Polκ Loop Swap Variants
Authors: Packer Morgan Renee , Antczak Nicole , Beuning Penny
Published: November 2023

DNA damage is ubiquitous, arising from both natural and artificial sources. The presence of damaged bases in DNA requires the use of specialized Y‐family DNA polymerases, which have the ability to catalyze the insertion of a nucleotide opposite a damaged base and to continue elongation past this DNA lesion. This process of DNA replication past damaged nucleotide(s) is known as translesion synthesis. A major question is how different Y‐family DNA polymerases achieve specificity for different types of DNA damage. Previous work showed that the minor groove N2‐furfuryl‐deoxyguanosine lesion is the cognate lesion for both E. coli DinB and its human ortholog Polκ and that both DinB and Polκ are inhibited by bulky major groove lesions. It was further determined that the DinB loop1 R35A mutation enables the protein to bypass the non‐cognate N6‐furfuryl‐deoxyadenosine major groove lesion (Walsh, et al. 2013 DNA Repair 12:713). Our research probes the contributions of the loop1 region to the specificity and activity of DinB and Polκ, which is being assessed via the analysis of several loop swap variants that represent step‐wise progressive changes from the native loop1 to that of DinB, Polκ, or Dpo4. Dpo4 is of interest as it is a relatively non‐specific Y‐family polymerase. We show that DinB and Polκ have high specificity for their cognate lesion, and Polκ variants containing the DinB loop1 sequence retain activity similar to wild‐type Polκ, whereas the DinB variants do not. Thus, Polκ is more resistant to changes in its amino acid sequence.

Support or Funding Information

This work was funded by a National Science Foundation Research Experience for Undergraduates grant and the Department of Chemistry and Chemical Biology at Northeastern University.
Investigating the mechanism of trans‐lesion synthesis by Human DNA Polymerase Kappa
Authors: Younger Zane William , Allen Taylor , Benitez Benedetta Sampoli
Published: November 2023

Our DNA is constantly under attack by both exogenous and endogenous chemicals that have the ability to damage it. If not repaired, these DNA adducts can lead to mutations to the DNA or even worse, they can halt replication, eventually leading to aging and/or cancer. Human DNA polymerase kappa (pol k) is a member of the Y‐family of DNA polymerases, known for their ability to conduct translesion synthesis of damaged DNA. Translesion synthesis allows for continued replication past lesions in the DNA that would otherwise stall or halt replication, leading to cell apoptosis. So far, the mechanism for lesion bypass discrimination by pol k is unclear. Recent studies suggested that similar to other polymerases, pol k might cycle between different open and closed conformations upon binding to DNA. Starting with one of the available crystal structures of pol k, we prepared eight different DNA/protein models and we solvated them in an octahedral water box with counter‐ions to a final ionic strength of 150 MM, using CHARMMgui. Subsequently, through molecular dynamics simulations, we tested the validity of the conformational change hypothesis by comparing the structural changes undergone by pol k in the presence of different templating lesions and with correct and incorrect incoming nucleotide. All simulations were performed using the program NAMD and were run for 10 ns each. We ran multiple simulations for the same system to ensure reproducibility of the results. Simulation of the binary system (just the protein and DNA without the incoming nucleotide) was also performed as a control system. By analyzing these different structures, we ascertained the residues crucial for fidelity and proposed a mechanism for lesion discrimination.

Support or Funding Information

This work was supported in part by a grant from the Rose M. Badgeley Residuary Charitable Trust.
The Critical Role of MAF1 in Repression of RNA Polymerase III Impacts Metabolic Efficiency in Mice
Authors: Willis Ian M , Bonhoure Nicolas , Moir Robyn D , Byrnes Ashlee , Hodroj Wassim , Praz Viviane , Preitner Frederic , Marcelin Genevieve , Chua Streamson , Martinez‐Lopez Nuria , Singh Rajat , Auwerx Johan , Willemin Gilles , Shah Hardik , Hartil Kirsten , Vaitheesvaran Bhavapriya , Kurland Irwin , Hernandez Nouria
Published: November 2023

MAF1 functions to promote metabolic economy by repressing RNA polymerase (pol) III transcription of highly abundant cellular RNAs, most notably tRNAs, under conditions of nutrient limitation and cellular stress. This function of MAF1, along with its phosphoregulation in response to TOR kinase activity and its interactions with the RNA pol III transcription machinery, is conserved from yeast to humans. In addition to its effect on RNA pol III, the mammalian MAF1 protein has been shown to repress several genes transcribed by RNA pol II. However, the scope of MAF1 in repressing transcription at the genome level is not well defined in mammalian systems and studies on the impact of ablating MAF1 in metazoans have only just begun. To address these questions, we generated a whole body Maf1 knockout (KO) mouse. Maf1 KO mice are unconditionally viable and demonstrate a striking resistance to diet‐induced obesity and non‐alcoholic fatty liver disease. These phenotypes result from reduced food intake and increased metabolic inefficiency. Gene expression profiling of adipose tissue indicates that the pol II transcriptome is not significantly affected and together with other experiments, show that the increased energy expenditure of Maf1 KO mice does not result from induction of adaptive thermogenesis. Instead the data indicate that the obesity resistance of Maf1 KO mice is achieved by novel mechanisms of energy expenditure. These include the increased synthesis and turnover (futile cycling) of tRNAs in the whole animal and increased futile cycling of hepatic lipids. Metabolism in Maf1 KO mice is altered to meet the increased demand for metabolic energy: Lipolysis is increased in eWAT; polyamine synthesis is affected in liver and skeletal muscle via down‐regulation of nicotinamide N‐methyltransferase and autophagy is activated in the liver. These changes are supported by metabolite profiling which reveal elevated levels of many amino acids and spermidine in both liver and muscle and increased levels of NAD+ in muscle. The importance of MAF1 for metabolic economy reveals the potential for MAF1 modulators to protect against obesity and its harmful consequences.

Support or Funding Information

The work was supported by National Institutes of Health grants GM085177 (I.M.W.), AG043930 (J.A.), and T32 GM07491 (A.B.); the University of Lausanne, Swiss National Science Foundation grants 31003A_132958 (N.H.) and 31003A‐140780 (J.A.) and funds from the Albert Einstein College of Medicine.
Regulation of RAP1A and RAP1B by Estrogen in Human Breast and Ovarian Cancer Cell Lines
Authors: Elder Nina
Published: November 2023

In all mammals, two RAS‐like GTPase genes produce the proteins RAP1A and RAP1B, which act in cell adhesion, motility, and junction formation, and are upregulated in a variety of cancers. Recent research has shown that in the presence of estrogen, Myc protein binding to the RAP1B gene promoter is lost. The effect of estrogen on the expression of other RAS‐related genes has been studied, but has yet to be fully elucidated in RAP1. Previous studies have also shown that estrogen‐stimulated increases in gene expression disappeared after the addition of mithramycin. Mithramycin inhibits Sp1 proteins, and the RAP1 gene promoters contain several Sp1 binding sites. Based on these findings, we expected the addition of mithramycin alone to effect RAP1 expression, and potentially to change estrogen‐regulated RAP1 expression. In human breast and ovarian cancer cell lines RAP1A and RAP1B gene expression were measured, and mithramycin and estrogen‐mediated effects quantified using qRT‐PCR. Any potential interaction between estrogen and mithramycin was also explored. Results showed that the ovarian cancer cell lines tested expressed more RAP1A and RAP1B than did the breast cancer cell lines. We found estrogen deprivation caused a decrease in RAP1 across cell lines, and the time‐course of RAP1 regulation by estrogen appears to vary among cell lines, as does the effect of reintroduction. This exploration of RAP1 expression in breast and ovarian cancer cell lines may help to elucidate its role as a conditional oncogene.
Expression Analysis and Characterization of Tetratricopeptide Repeat Doman 39c (Ttc39c) in Skeletal Muscle
Authors: Waddell David , Menke Jacob
Published: November 2023

Ttc39c has been identified as a novel gene in skeletal muscle that is differentially regulated under conditions of neurogenic atrophy in wild‐type mice compared to Muscle RING Finger 1 (MuRF1) knockout mice. MuRF1 is a ubiquitin E3 ligase that is known to be an important regulator of muscle wasting; however, evidence presented here suggests that MuRF1 may also function as a modulator of atrophy‐induced gene expression. Western blot analysis confirms that Ttc39c is expressed at the protein level in undifferentiated C2C12 myoblasts but is down‐regulated as cells differentiate to myotubes. Bioinformatic analysis of the Ttc39c gene locus reveals two alternative variants with distinct transcription initiation sites, which is an architecture that is conserved from rodents to humans. The transcriptional regulation of the shorter splice variant was examined by cloning the putative regulatory region and fusing it to a reporter gene. This reporter construct was then transfected into muscle cells along with an expression plasmid for MuRF1. MuRF1 overexpression repressed Ttc39c reporter activity, while overexpression of a MuRF1 RING domain mutant failed to repress the Ttc39c reporter. Furthermore, overexpression of myogenin, which is a myogenic regulatory factor (MRF) that regulates the expression of muscle‐specific genes by binding to E‐box elements, also repressed Ttc39c reporter gene activity. A conserved E‐box element in the Ttc39c proximal promoter was identified, mutated and analyzed for its role in the regulation of Ttc39c expression. The mutation of the conserved E‐box rendered the Ttc39c reporter gene inactive, demonstrating that the element is likely necessary for proper Ttc39c transcriptional regulation. The identification and characterization of novel genes that are activated during neurogenic atrophy helps improve our understanding of the molecular genetic events that lead to muscle wasting and could eventually lead to new therapeutic targets in the future.

Support or Funding Information

University of North Florida Transformational Learning Opportunity Grant
Transcriptional and translational regulation of CCAAT/Enhancer Binding Protein β expression by P53
Authors: Hu Biao , Wu Zhe , Bai David , Sheng Susanne , Phan Sem H
Published: November 2023

The transcription factor CCAAT/enhancer‐binding protein β (C/EBPβ) is highly expressed in various tissues during development, including liver, adipose tissue, blood cells, lung and the endocrine pancreas. Two isoforms of C/EBPβ namely liver‐enriched activator protein (LAP) and liver‐enriched inhibitor protein (LIP) translated from the same mRNA have been described. The LAP and LIP isoforms share the same C‐terminal DNA binding domain except LAP has an extra N‐terminal activation domain. Probably due to its higher affinity for its DNA cognate sequences, the LIP can counteract the effects of LAP isoforms even at substoichiometric levels. In vitro and in vivo experiments with cultured cells and knockout mice demonstrated that C/EBPβ play a critical role in the regulation of myofibroblast differentiation and bleomycin induced pulmonary fibrosis. However, the regulatory mechanism of C/EBPβ gene expression is unclear. Analysis of the C/EBPβ promoter sequence identified a conserved P53 response element (P53RE) in the C/EBPβ gene promoter. Gel shift mobility and chromatin immunoprecipitation assays confirmed that P53 binds to the P53RE. Promoter reporter assays and real time PCR analysis of samples from fibroblasts transiently transfected with P53 or dominant negative P53 expression plasmids indicated that P53 was a repressor of C/EBPβ gene transcription. Western blot analysis with protein extracts from similarly transfected fibroblasts or P53 deficient fibroblasts showed that P53 differentially regulated translation of the C/EBPβ LAP and LIP isoforms through the regulation of eIF4E and eIF4E‐BP1. Further studies with constructs containing the LAP 5′ untranslated region or the LIP 5′ untranslated region showed that the 5′ untranslated region is important in differential control of C/EBPβ LAP and LIP translation. Thus P53 could regulate C/EBPβ gene expression at both transcriptional and translational levels.

Support or Funding Information

This work was supported by grants HL052285 and HL112880 from the National Institute of Health.
2‐Aminopurine is a Reporter for the Formation of the C·A+ Wobble Base Pair in U6 snRNA
Authors: Grossenbacher Max
Published: November 2023

The spliceosome is a multimegadalton complex that is found in all eukaryotes and is involved in the excision of introns and ligation of exons to create mature RNA from pre‐mRNA. The U6 subunit of the spliceosome contains a highly conserved internal‐stem loop (ISL), which is essential to the activation and catalytic function of the spliceosome. The ISL has a 1X2 bulge that consists of a C67 ·A79+ wobble base pair (pKa 6.5) and an unpaired U80 nucleotide. The formation of C67· A79+ results in the base flipping of U80 out of the RNA helix, thus, stabilizing the formation of a continuous A‐form helix. At a pH above 6.5, U80 is stacked within the RNA helix, resulting in a bend in the helix that favors ion interactions. We examined the structural dynamics of U6 RNA using 2‐aminopurine (2‐AP), a fluorescent analog of adenine and guanine, that was substituted into U6 RNA constructs for A78. Thermal denaturation and fluorescent studies were used to measure the effects of pH and Mg2+ on the stability of U6 wild type RNA. Additionally, several modified constructs, including a control helix, were utilized to examine the role of nearby sequences on the formation of the C67 ·A79+ wobble base pair. Experiments were performed in 1M KCl and 10mM MgCl2 buffers in a pH range of 5.5 – 8.0. Thermal denaturation of U6 RNA constructs shows that the formation of C67 ·A79+ contributes ~1.0 kcal/mol of stability to U6 wild type RNA. Additionally, U6 wild type RNA shows a 2.2 kcal/mol increase in stability in 10mM MgCl2 buffer at pH 7.5, as compared to U6 wild type RNA in 1M KCl buffer.

Support or Funding Information

Neena Grover & Colorado College Department of Biochemistry
The Interplay between S‐Palmitoylation and Gamma‐COP Binding of MAN1B1 Manages Alpha‐1‐Antitrypsin Quality Control
Authors: Young Evelin , Sifers Richard N.
Published: November 2023

Genetic information is transformed into biological activity through the correct folding and proper deployment of encoded proteins. Numerous diseases result from complications caused by the failure of newly synthesized proteins to achieve these milestones. In Alpha‐1‐Antitrypsin Deficiency (AAT‐D), the toxic accumulation of aberrant AAT polymers in the hepatocyte secretory pathway is responsible for the development of childhood liver disease. Misfolded AAT monomers, which function as the direct precursor of toxic polymers, are subjected to proteasomal degradation in response to a poorly understood non‐enzymatic role of alpha‐1,2‐mannosidase, Class 1B, Member 1 (MAN1B1). In support of a role for MAN1B1 as a disease modifier, we have reported that a single nucleotide polymorphism in the corresponding gene that leads to the reduced expression of MAN1B1 is responsible for promoting the development of infantile end‐stage liver cirrhosis, the most severe form of AAT‐D. Importantly, MAN1B1, which can traffic between the Golgi complex and ER in response to the binding of gamma‐COP, promotes proteasomal degradation of the misfolded AAT monomer, designated NHK. However, it is presently unclear how the gamma‐COP‐mediated trafficking of MAN1B1 is regulated in order to govern the rate of ER‐associated degradation (ERAD) substrate recruitment. In the present study, we have uncovered that the amino‐terminal cytoplasmic tail of MAN1B1 is subjected to S‐palmitoylation. Mutation of the cysteine attachment sites enhanced the capacity of transfected MAN1B1 to degrade NHK, which coincided with an increased interaction with gamma‐COP. A novel posttranslational regulatory model is proposed in which the S‐palmitoylation of nearby cysteine residues functions to antagonize gamma‐COP binding, thereby hindering the retrograde trafficking and availability of MAN1B1, and reducing the proteasomal degradation of NHK by ERAD. Determining how to tip the balance of MAN1B1 to favor enhanced degradation of the misfolded AAT monomer is vital to the development of new therapeutic strategies for AAT‐D patients.

Support or Funding Information

IRACDA K12‐GM084897 and Alpha‐1 Foundation
Development of Arginyl‐tRNA Synthetase from Pseudomonas aeruginosa as a Platform to Screen for Inhibitors of Protein Synthesis
Authors: Cantu Daniel , Salazar Humberto , Bullard James M
Published: November 2023

Introduction

Pseudomonas aeruginosa is a common cause of nosocomial infections and the leading cause of mortality in patents with cystic fibrosis. Aminoacyl‐tRNA synthetases (aaRSs) are a class of enzymes that catalyze the covalent attachment of amino acids to their cognate tRNAs during protein biosynthesis.

Results

P. aeruginosa arginyl‐tRNA synthetase (ArgRS) was overexpressed in Escherichia coli cells, and purified to homogeneity. ArgRS from P. aeruginosa is a discriminating synthetase with respect to the requirement for the presence of tRNAArg to produce a stable arginyl‐AMP intermediate. The kinetic parameters for interaction with tRNA were determined and the KM and Vmax were 4.0 μM and 11.7 μM min−1, respectively. The observed kcat was calculated to be 0.32 s−1 resulting in a kcat/KM value of 0.08 s−1μM−1. Scintillation proximity assay (SPA) technology was adapted to the aminoacylation assay and then used to screen for inhibitors of activity of P. aeruginosa ArgRS in a high throughput format. Using this assay, natural product (800 compounds) and synthetic compound (890 compounds) libraries were screened to detect compounds with the ability to inhibit function of the enzyme. Four compounds were identified which inhibit greater than 50% of enzymatic activity. Compounds BM06E07, BT04F10, BT10B04 and BT11F03 inhibit the activity of P. aeruginosa ArgRS with IC50 values of 29.2, 35.4, 31.6 and 8.1 μM, respectively. All four of these compounds exhibited promising MICs against Gram+ bacteria and moderate activity against Gram‐ bacteria causing respiratory infections.

Conclusion

ArgRS 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).
Characterization of the HECT Domain Ubiquitin Ligase Nedd4‐2
Authors: Todaro Dustin , Augustus‐Wallace Allison , Klein Jenny , Haas Arthur
Published: November 2023

Nedd4‐2 is a WW domain‐containing HECT (Homologous to E6AP C‐Terminus) ligase best known for its role in targeting cell surface receptors, including ENaC in the distal kidney, for Lys63‐linked polyubiquitin chain‐mediated endocytic uptake and lysosomal degradation. The Nedd4‐like ligases, including Nedd4‐2, also function in the pathway for viral budding of Ebola and Marburg viruses, among others. By employing rates of 125I‐polyubiquitin chain assembly as a functional readout of ligase activity, we quantitatively explore the mechanism of Nedd4‐2 in biochemically‐defined assays. We demonstrate that Nedd4‐2 exhibits cooperative allosteric kinetics (n=1.8±0.7) with varying Ubc5B~125I‐ubiquitin thioester substrate concentrations ([S]1/2=103±29 nM; kcat=0.07±0.01 s−1), requiring a priori that the enzyme exist as an oligomer. Gel filtration analysis reveals that the active form of Nedd4‐2 migrates as a trimer of 250 kDa, larger than the predicted 110 kDa monomer, confirmed by dynamic light scattering. Successive addition of a dominant negative Nedd4‐2C922A active site mutant inhibits wild type Nedd4‐2 to limiting 30% activity, consistent with polyubiquitin chain assembly requiring intersubunit interactions within an intact trimer. Based on sequence and structural homology of the predicted Nedd4‐2 trimer to E6AP, we identify Glu646 and Arg604 as conserved putative catalytic residues by point mutagenesis. Our studies demonstrate Ca2+‐independent polyubiquitin chain assembly, contrary to previous reports of Ca2+‐dependent C2 domain inhibition of Nedd4‐2 activity. These findings led us to further investigate the role of the N‐terminal C2 domain on Nedd4‐2‐catalyzed chain assembly. Truncation of the C2 domain of Nedd4‐2 to yield Nedd4‐2ΔC2 leads to a 2‐4‐fold increase in kcat and loss of cooperativity. Exogenous C2 domain non‐competitively inhibits Nedd4‐2ΔC2 (Ki=68±0.93 μM) without restoring cooperativity, consistent with a modest Ca2+‐independent allosteric autoinhibition by the C2 domain via intersubunit interactions. Using the proteasomal subunit S5a as a model substrate in vitro, we demonstrate that Nedd4‐2 catalyzes S5a conjugation with apparent hyperbolic kinetics (KM=254±0.078 nM; kcat= 0.020±0.002 s−1), requiring the polyubiquitin chain assembly and substrate conjugation half reactions to be conformationally independent. The good agreement between kcat for S5a conjugation (0.020±0.002 s−1) and that for free polyubiquitin chain assembly (0.07±0.01 s−1) suggests that Nedd4‐2 does not discriminate between lysine donors and that both conjugation half reactions share similar transition states. Parallels to our recent observations with E6AP suggest mechanistic conservation with subtle differences based on distinct sequence and structural variations for Nedd4‐2. These findings support mechanistic conservation within the HECT ligase superfamily.

Support or Funding Information

[Supported by GM034009 to A.L.H]
Uptake and Lysosomal Delivery of Recombinant Human Alpha‐N‐Acetylglucosamine‐6‐Sulfatase to Mucopolysaccharidosis IIID Fibroblasts
Authors: Moen Derek R , Wang Daniel M , Zhang Xiaoyi , Kan Shih‐hsin , Wood Jill , Ekins Sean , Chou Tsui‐Fen , Dickson Patricia I
Published: November 2023

Sanfilippo syndrome (mucopolysaccharidosis type III; MPS III) is a devastating neurodegenerative lysosomal storage disorder of childhood. There is no cure or effective treatment available. The fundamental cause of MPS III is an inherited mutation in one of the 4 enzymes required to catabolize heparan sulfate (HS), a glycosaminoglycan which plays important structural and functional roles in the brain and elsewhere. We now propose to develop an enzyme replacement treatment (ERT) for MPS IIID that will ameliorate or reverse the catastrophic and fatal neurologic decline caused by this disease. The symptoms of MPS IIID are largely localized to the brain, therefore, our strategy proposes to deliver recombinant human alpha‐N‐acetylglucosamine‐6‐sulfatase (rhGNS) intrathecally to effectively treat the underlying causes of the neurologic symptoms that dominate MPS III pathology. We have purified ~100 ug rhGNS from 1500 mL media of CHO cells (specific activity >10,000 nmol/hr/mg), demonstrated maximal enzymatic activity at pH 5.6 (low activity at neutral pH), which is closer to lysosomal pH, demonstrated good enzymatic activity at 37°C and showed it was stable for over one month at 4°C in artificial cerebrospinal fluid storage buffer. Additionally we have demonstrated intracellular enzymatic activity of rhGNS in MPS IIID fibroblasts when rhGNS is added to the media, shown it colocalizes with lysosomal markers using confocal microscopy, and confirmed radiolabelled HS is diminished (33–65%) to WT levels in MPS IIID fibroblasts treated with rhGNS. These results demonstrate scale‐up is feasible in preparation for proof of concept studies in the MPS IIID knock‐out mouse. Our ultimate goal is bring this ERT to the clinic which will likely have a transformational effect on patients and families.

Support or Funding Information

This work was funded by Grant R41 NS089061‐01 from NIH NINDS