bluuebunny/crossref_metadata_2025 huggingface dataset:
Vanadium Complex Induced Cancer Cell Death via RIPK3 Activated Necroptosis
Authors: Rana Savannah R , McCaffrey Vanessa , Rabquer Bradley James
Published: November 2023

Purpose

Cancer, at the cellular level, is characterized by uncontrolled cell proliferation caused by a loss of control of the cell's normal cycle of growth and division. This loss of control usually results from irreversible cell damage, primarily mutation of the cell's DNA, which would normally result in programmed cell death, or apoptosis. Many anti‐cancer therapies, such as cytotoxic chemotherapy, trigger activation of pathways that induce this cell death. Previous studies in our lab have shown the efficacy of using dioxovanadium complexes with substituted salicylaldehyde derivatives (VCs) to inhibit cancer cell growth, yet not the activation of the caspase 3/7 apoptosis pathway. The necroptosis pathway is a mode of inflammatory programmed cell death characterized by the phosphorylation of Receptor Interacting Protein Kinase 3 (RIPK3) and Multi Lineage Kinase Like (MLKL). Here, we hypothesized that vanadium complex treated HT‐29 cells are being terminated by means of the necroptosis pathway.

Methods and Results

HT‐29 cells were used as a model for colon cancer. Previously synthesized VCs with 5‐Br and 3‐OMe functional groups were used as treatments in vitro. Immunofluorescence assays were performed on cells treated with functional group only controls (0.5 mM), 3‐OMe (0.5mM), and 5‐Br (0.5mM) VCs for incubation periods of 24 hours and 3 hours (n=2). The treated cells were probed with anti‐phosphorylated RIPK3. An increase in phosphorylated RIPK3 was evident after both 3 and 24 hour incubation periods in the experimental groups, but not the controls, indicating activation of the necroptosis pathways. For Western blotting, cells were treated with DMSO, 5‐Br (0.5mM) and 3‐OMe (0.5mM) VCs respectively for incubation periods of 3 and 24 hours (n=3). Protein lysates were collected and western blots were run to detect the presence of total and phosphorylated receptor interacting protein kinase‐3 (RIPK3), however activation of the RIPK3 necroptosis pathway was not confirmed.

Conclusion

VCs induce HT‐29 cell death via the activation of RIPK3 and the necroptosis pathway.

Support or Funding Information

This work was supported by the Albion College Biology and Chemistry Departments, the Foundation for Undergraduate Research, Scholarship, and Creative Activity (FURSCA), and the Hewlett‐Mellon Fund for Faculty Development at Albion College.
The Role of Sirtuin 2 in the Regulation of Myogenesis
Authors: Stanton David A. , Alway Stephen E , Mohamed Junaith S.
Published: November 2023

The postnatal growth and regenerative capacity of skeletal muscle rely on the successful activation and differentiation of muscle stem cells known as satellite cells. Although the NAD+‐dependent protein deacetylase sirtuin‐2 (SIRT2) has been suggested to have a role as a redox sensor during myogenesis, and in the regulation of metabolism, microtubule dynamics, and exit from the cell cycle exit, it is not known if SIRT2 has a regulatory role in muscle stem cell proliferation or differentiation.

Purpose

The objective of this study was to investigate the role of SIRT2 in the molecular and genetic control of myogenesis.

Methods

C2C12 myoblasts were cultured in growth medium containing DMEM and fetal bovine serum. Myoblasts were treated with BML‐266, a pharmacological inhibition of SIRT2 deacetylase activity. CRISPR/CAS9 technology was used to generate a stable SIRT2 knockout C2C12 cell line. GFP positive cells that were negative for SIRT2 were collected by flow cytometry. The control and SIRT2 knockout myoblasts were placed in 2% horse serum to differentiate the myoblasts to myotubes. The cells were harvested for analysis 12 or 24 hours after initiation of differentiation.

Results

SIRT2 protein expression was upregulated during early differentiation in C2C12 myoblasts after both 12 and 24 hours after the initiation of differentiation. Inhibition of SIRT2 pharmacologically or by gene knockout, impaired myotube fusion but it did not affect proliferation of C2C12 myoblasts. We have also identified downstream targets of SIRT2 activation that appear to be involved in modulation of differentiation.

Conclusions

These data suggest that SIRT2 may be a key contributor to the regulation of myoblast differentiation. Furthermore, SIRT2 may be an important target protein for treatment strategies of muscle wasting conditions including aging, muscular dystrophy, cachexia, or sepsis.
17b‐Estradiol Rescues Low Force Potentiation in Ovariectomized Mice In Vivo
Authors: Le Gengyun , Warren Gordon L. , Lowe Dawn A.
Published: November 2023

Post‐tetanic potentiation (PTP) of force is associated with phosphorylation of myosin regulatory light chain and increased Ca2+ sensitivity of the contractile apparatus. It has been previously reported that isolated muscles from ovariectomized mice have low PTP compared to that from ovary‐intact mice, and this decrement was reversed by acute estradiol treatment in vitro. However, there is no in vivo evidence supporting this estradiol effect.

Purpose

To determine the effect of acute estradiol treatment on impairment of skeletal muscle PTP in ovariectomized mice in vivo.

Methods

Adult female C57BL/6 mice (n = 9–15 per treatment) had a stimulating nerve cuff surgically implanted on the common peroneal nerve. Six weeks later these mice were ovariectomized or underwent a sham surgery. Four weeks later, the nerve cuff was used to electrically stimulate the anterior crural muscles immediately before and 1 h after treatment with either vehicle (OVX+vehicle) or 17β‐estradiol (OVX+E2; 0.15 mg estradiol per kg body weight) via tail vein injection. To measure PTP, baseline twitch torque production about the ankle was measured using a 0.1‐ms electrical stimulus pulse. Thirty seconds later, muscles were stimulated using 0.1‐ms pulses at 100 Hz for 1 s to produce a prolonged, isometric tetanic contraction. Starting 10 s later, two post‐tetanic twitches were elicited with 2 s between the two. PTP was calculated as the percent increase in torque from baseline to the highest torque of the post‐tetanic twitches. One‐way ANOVAs were used for data analysis. If a significant main effect was found, Holm‐Sidak post hoc tests were applied.

Results

Peak torque of the unpotentiated, baseline twitches were not different among OVX+vehicle, OVX+E2 and Sham mice (0.60 ± 0.06, 0.57 ± 0.05 and 0.51 ± 0.03 N*mm, respectively; p=0.30). Potentiated twitches generated 30–100% more torque than unpotentiated twitches. However, the extent of PTP depended on the presence of estradiol (main effect, p=0.03). PTP of Sham and OVX+E2 mice were not significantly different (53.7±5.0% vs 70.4±9.4% increase, respectively; p=0.11). PTP of OVX+E2 mice was greater than that of OVX+vehicle mice (70.4±9.4% vs 44.7±3.3% increase, respectively; p=0.01).

Conclusion

Deficiency of circulating ovarian hormones negatively impacts torque potentiation of the anterior crural muscles in anesthetized mice and acute estradiol treatment can rescue the low potentiation providing evidence estradiol can benefit skeletal muscle contractility in vivo. Supported by NIH grants R01‐AG031743 and T32‐AR050938.

Support or Funding Information

Supported by NIH grants R01‐AG031743 and T32‐AR050938.
Impairment of osmotic challenge‐induced vasopressin secretion in sepsis survivor rats
Authors: Santos‐Junior Nilton Nascimento , Costa Luis Henrique Angenendt , Catalão Carlos Henrique Rocha , Kanashiro Alexandre , Sharshar Tarek , Rocha Maria José Alves
Published: November 2023

Recent clinical and experimental studies report that sepsis survivors present long‐term immunosuppression and brain alterations. Neuroendocrine dysfunctions observed during sepsis have not yet been investigated in post‐sepsis condition. In the present study, we analysed the osmotic challenge‐induced arginine vasopressin (AVP) secretion in sepsis survivor rats. Wistar rats were subjected or not (naive) to cecal ligation and puncture (CLP). Five days after CLP surgery, the survivor animals were submitted to an osmotic challenge consisting of intraperitoneal injection of hypertonic saline. Thirty minutes after the osmotic challenge, the blood and brain were collected for determination of osmolality, nitrate, IL‐1β, IL‐6 and AVP levels and c‐fos expression analysis of hypothalamic supraoptic nuclei, respectively. All experiments were approved by the animal ethical committee of the Faculty (CEUA‐FORP). High levels of nitrate and IL‐1β, but not IL‐6, were found in the sepsis survivors plasma but this long‐term systemic inflammation was not altered by the osmotic challenge. AVP secretion (but not the osmolality) and c‐fos expression in SON were significantly attenuated in CLP survivor animals as compared with naive rats. We observed that AVP secretion dysfunction following an osmotic challenge in sepsis survivor rats is associated with hypothalamic dysfunction and this may be due to a persistent inflammatory response.

Support or Funding Information

FAPESP (2015/12125‐5) and CNPq
Kinetics of Neurotransmitter Metabolism by Monoamine Oxidase in Porcine Heart Differs by Location and is Increased with Obesity/Metabolic Syndrome
Authors: Carter Kendal , Nelson Margaret , Robidoux Jacques , Lerman Lilach , Anderson Ethan
Published: November 2023

Monoamine Oxidase (MAO), an outer mitochondrial enzyme, catalyzes the oxidative deamination of neurotransmitters and biological amines, and in the process, produces a reactive aldehyde and H2O2. Our lab has recently shown that MAO activity (i.e., H2O2 production) in human right atrial myocardium is associated with postoperative atrial fibrillation. Furthermore, our lab has shown that diabetic patients have higher MAO‐A and ‐B content and rates of activity in this tissue, compared to nondiabetics. While recent studies have explored the connection between MAO and cardiac dysfunction, the chamber‐specific content and activity of MAO within the heart is still unknown. Therefore, we characterized the regional enzymatic activity of MAO‐A and ‐B based upon the hypothesis that the atria would have the highest MAO activity due to the greater density of local sympathetic nerve innervation in this region. For this study, porcine hearts were isolated from domestic pigs, as this model closely recapitulates the physiology of the human heart. One group of pigs were fed a high fat, high sucrose diet for 16 weeks to induce obesity/metabolic syndrome (n=4). Three MAO substrates (dopamine, norepinephrine, serotonin) and two isoform‐selective inhibitors (clorgyline=MAO‐A, deprenyl=MAO‐B) were used to characterize MAO activities. Enzyme content was measured using ELISA and confirmed with immunoblots. Kinetic analysis showed that, across all substrates, when normalized to tissue protein, the left atrium has the highest maximal MAO‐A and –B activity while the other three chambers are similar. Furthermore, maximal MAO activity in the left ventricle of pigs with metabolic syndrome is significantly greater (P<0.05) than left ventricle of control group. Our findings suggest that MAO activity differs by location in the heart (i.e., chamber heterogeneity). This information will be useful to inform future experimental and clinical pharmacology applications where MAO inhibitors might be used. Moreover, increased MAO in left ventricle of pigs with obesity/metabolic syndrome is important as it provides a rationale for exploring MAO inhibitors as therapeutic strategy to mitigate cardiac complications of diabetes.

Support or Funding Information

R01HL122863
Oral Glucose Tolerance Test in Insulin Resistant Adolescents Induces Regionally Different Macro‐ and Microvascular Responses in the Cerebral Circulation
Authors: Peltonen Garrett L. , Kellawan J. Mikhail , Ward Aaron T. , Carter Katrina J. , Carrel Aaron L. , Rehm Jennifer L. , Oh Jennifer M. , Bendlin Barbara B. , Schrage William G.
Published: November 2023

Impaired cerebrovascular responsiveness to insulin may contribute to decreased cerebral blood flow (CBF) and cognitive decline associated with insulin resistance in adults. In insulin resistant adolescents (IR) however, the cerebrovascular response to insulin remains unknown. We tested the hypothesis that IR will display different cerebral macrovascular and microvascular responses to an oral glucose tolerance test (OGTT) compared to insulin sensitive adolescents (Ctrl). In 5 adolescents (15 ± 1 yrs), the Homeostatic Model Assessment of Insulin Resistance (HOMA‐IR) was used to quantify insulin resistance at baseline (2 Ctrl, HOMA‐IR = 3.3 ± 0.2; 3 IR, HOMA‐IR = 5.3 ± 0.2; P = 0.016). An OGTT (1.75g/kg, up to a maximum of 75g glucose solution) was administered to induce a physiologically relevant increase in circulating insulin. Prior to and at the 45‐minute time point of OGTT, CBF was measured with 4‐dimensional phase contrast‐vastly under sampled isotropic projection reconstruction (PC‐VIPR) MRI. Cerebral vascular conductance was calculated (CVC = CBF/mean arterial blood pressure [MAP] * 100mmHg) in 11 extracranial and intracranial cerebral arteries (Internal Carotid [ICA] x2, Middle Cerebral [MCA] x2, Anterior Cerebral [ACA] x2, Vertebral [VA] x2, Basilar [BA], and Posterior Cerebral [PCA] x2). Microvascular perfusion was measured prior to and at the 50‐minute time point of OGTT in 14 neuronal regions linked to cognition and learning, using pseudo‐continuous arterial spin labeling (pcASL) MRI. Heart rate, MAP, pulse oximetry oxygen saturation (SPO2), and end‐tidal CO2 (ETCO2) were measured at all time points. Changes in macrovascular CVC and microvascular perfusion were calculated as (ΔCVC = CVCOGTT − CVCbaseline and ΔASL = ASLOGTT − ASLbaseline). MAP (P = 0.029), and ETCO2 (P = 0.042) were higher in IR compared to Ctrl, but was unchanged with OGTT (P > 0.05). In agreement with our hypothesis, ΔCVC in response to OGTT was different in left MCA of IR compared to Ctrl (−39 ± 9 vs 6 ± 0 ml/min/100mmHg; P = 0.023). Likewise, ΔASL in response to OGTT was different in the left hippocampus (−8% ± 1% vs 6% ± 1%; P = 0.003) and right superior frontal medial gyrus (+5% ± 2% vs −4% ± 0%; P = 0.028) of IR compared to Ctrl. We conclude that IR demonstrate regionally different macrovascular and microvascular responses to an OGTT compared to Ctrl. These data are the first in IR to show alterations in the cerebrovascular responses to insulin in brain regions involved in learning, cognition, and executive function. Impaired insulin mediated perfusion of these regions may have important consequences for brain development and health as adolescents mature into adulthood.

Support or Funding Information

University of Wisconsin‐Madison Graduate School Pilot Funds
Fructose‐induced neurogenic hypertension is mediated by superoxide overexpression and stimulates p38 MAPK‐induced central insulin resistance in the central nervous system
Authors: Chen Hsin‐Hung , Cheng Pei‐Wen , Tseng Ching‐Jiunn
Published: November 2023

Type 2 diabetes are at a high risk of complications related to hypertension, and reports have indicated that insulin levels may be associated with blood pressure (BP). Fructose intake has recently been reported to promote insulin resistance and superoxide formation. The aim of this study is to investigate whether fructose intake can enhance superoxide generation and impair insulin signaling in the nucleus tractus solitarii (NTS) and subsequently elevate BP in rats with fructose‐induced hypertension. Treatment with fructose for 4 weeks increased the BP, serum fasting insulin, glucose, homeostatic model assessment‐insulin resistance, and triglyceride levels and reduced the serum direct high‐density lipoprotein level in the fructose group. Treatment with tempol recovered the fructose‐induced decrease in nitric oxide production in the NTS. Immunoblotting and immunofluorescence analyses further showed that fructose increased the p38‐ and fructose‐induced phosphorylation of insulin receptor substrate 1 (IRS1S307) and suppressed AktS473 and neuronal nitric oxide synthase phosphorylation. Similarly, fructose was able to impair insulin sensitivity and increase insulin levels in the NTS. Fructose intake also increased the production of superoxide in the NTS. The results of this study suggest that fructose might induce central insulin resistance and elevate BP by enhancing superoxide production and activating p38 phosphorylation in the NTS.

Support or Funding Information

MOST104‐2320‐B‐075B‐003‐MY3

General Characteristics of the 3 Groups of Rats

Control
Fructose
Fructose + Pioglitazone

Body weight (gm)
233 ± 6.6
227.5 ± 10.3
225 ± 6.7

Systolic blood pressure (mmHg)
128.4 ± 2.6
151.9 ± 2.3*
135.9 ± 5.3†

Mean blood pressure (mmHg)
113.7 ± 0.94
135.8 ± 1.7*
117.2 ± 3.2†

Fasting serum glucose (mg/dL)
110 ± 2.35
240.3 ± 12.7*
134.7 ± 18.2†

Fasting serum insulin (pmol/L)
9.38 ± 0.36
175.7 ± 24.5*
26.69 ± 1.9†

HOMA‐IR Index
0.38 ± 0.02
13.1 ± 2.9*
1.64 ± 0.23†

dHDL (mg/dL)
68.8 ± 0.67
55.0 ± 1.00*
69.7 ± 0.88†

Triglyceride (mg/dL)
56.4 ± 3.0
111.5 ± 8.96*
47.6 ± 9.2†

Cholesterol (mg/dL)
96.6 ± 1.78
100.63 ± 3.97
89.0 ± 1.2

NTS insulin (pmol/L/g protein)
7.97 ± 0.37
18.52 ± 1.73*
8.08 ± 0.32†

NTS insulin response (mmHg)
−24.62 ± 2.4
−8.86 ± 2.17*
−24.3 ± 2.2†
Beta‐hydroxybutyrate favorably alters b‐cell survival and mitochondrial bioenergetics
Authors: Sampson MaryJane , Lathen Daniel , Dallon Blake , Draney Carrie , Ray Jason , Kener Kyle , Parker Brian , Witt Jeffrey , Gibbs Jonathan , Tessem Jeffery S. , Bikman Benjamin T.
Published: November 2023

Pharmacological interventions aimed at improving outcomes in type 2 diabetes and achieving normoglycemia, including insulin therapy, are increasingly common, despite the potential for substantial side effects. Carbohydrate‐restricted diets that result in increased ketogenesis have effectively been used to improve insulin resistance, a fundamental feature of type 2 diabetes. Additionally, limited evidence suggests states of ketogenesis may also improve b cell function in type 2 diabetics. Considering how little is known regarding the effects of ketones on b‐cell function, we sought to determine the specific effects of b‐hydroxybutyrate (bHB) on pancreatic b cell physiology and mitochondrial function. bHB treatment increased b cell survival and proliferation, while also increasing mitochondrial mass, respiration, and ATP production. Despite these improvements, we were unable to detect an increase in b‐cell or islet insulin production and secretion. Collectively, these findings have two implications. First, they indicate that b‐cells thrive in the midst of bHB, the circulating form of ketones. Second, improved mitochondrial function and proliferation may be insufficient to increase insulin production.
Antiaging Gene “Klotho” Deficiency Accelerates Stem Cells Aging by Impairing Telomere
Authors: Ullah Mujib , Sun Zhongjie
Published: November 2023

Background & Objective

Klotho (anti‐aging gene) deficiency and telomere shortening occurs with aging. Mice with Klotho deficiency, show decreased lifespan with limited tissue renewal and short telomere, which suggests that telomerase is rate‐limiting step for stem cells which regenerate our bodies. It has become evident that telomere length and telomerase activity can directly affect the regenerative capabilities of stem cells. Here, we will explore the current evidence linking telomere shortening to aging and stem cell dysfunction in the light of klotho. Understanding the molecular pathways involved in this age‐dependent deterioration of stem cell function will be critical for developing new therapies.

Methods & Results

We used adipose tissue‐derived stem cells (ADSC), as a model system, to identify the role of Klotho deficiency on stem cells and to assess their telomere length and activity. We identified the regulative and modulatory role of Klotho in the maintenance of ADSC population, their differentiation potential, pluripotency, proliferative index, and cells senescence. Klotho deficiency led to generation of reactive oxygen species and apoptosis. Further, we found the major targets, belong to TGFβ, insulin, and Wnt signaling pathways. These targets under the signaling of Klotho, takes part in the regulation of stem cells in a CD90 dependent way. Klotho knockout mice‐derived ADSC, exhibited altered osteogenic and chondrogenic differentiation. Klotho deficient ADSC showed an increased expression of TGFβ, SAMD2/3/4, BMP2, FGF2, Cdx, Cdk4, Leptin, Lpl, Crat, Asc‐1 and Apo‐A1. These targets probably play a role in stem cells aging. Klotho deficient ADSC exhibited significantly reduced expression for growth factors responsible for aging like FGF, IGF, SOX2, Pparγ, Irs1, Pgc1, Ucp‐1, and Pat2. The expression of all these targets notify the importance of Klotho in stem cells aging. Klotho deficient ADSC were also treated with telomerase activator Cycloastragenol (CAG) for telomere restoration. In result, we found that CAG stimulates telomerase activity, cell proliferation and partially rescued the regenerative abilities of ADSC.

Conclusion

Klotho gene deficiency impairs ADSC proliferation and differential potential which was markedly improved by activation of telomerase. Telomere shortening may be involved in klotho deficiency‐induced stem cell aging.
Characterizing A Rat Model to Study the Role Endo‐Immunology Plays in Diet‐Induced Non‐Alcoholic Fatty Liver Disease
Authors: Hinojosa Gabriella , Hamilton Alina , Lora Jessica , Short John D. , Dearth Robert K
Published: November 2023

There is a rising epidemic of non‐alcoholic fatty liver disease (NAFLD) associated with the growing prevalence of obesity. NAFLD ranges from non‐progressive, steatosis, to a more aggressive disease, non‐alcoholic steatohepatitis (NASH). NASH, which is defined as an inflamed, fatty liver, contributes to liver cancer by causing chronic liver injury. A key event in the transitional progression from steatosis to NASH is the endocrine recruitment of monocytes to the liver. These infiltrating blood monocytes become polarized liver macrophages driving disease. There are currently no drugs specifically approved to treat NASH. One of the reasons is the availability of preclinical models that replicate disease pathophysiology in humans. A recent study showed that a specialized Amylin liver NASH Model (AMLN) diet (40% kcal from fat; fructose 22% by weight; cholesterol 2% by weight) caused NASH including immune cell infiltration in mice. However, the Sprague‐Dawley (SD) rat strain has been shown to be a more representative model to study human liver physiology and disease. Therefore, the objective of this study was to create a new model of diet‐induced NAFLD in the SD rat that more accurately maps the etiology of liver disease. Forty‐nine day old male Sprague Dawley rats were separated into groups and fed normal diet or AMLN diet for 4 weeks, 8 weeks, 12 weeks, or 19 weeks. AMLN diet rats showed a significant increase in weight gain and caloric intake weekly compared to controls. Histological analysis showed macrovesicular hepatic steatosis after four weeks on AMLN. Oil Red O staining confirmed an observable increase in lipid accumulation in the treated group after 4 weeks. After 19 weeks on AMLN, immunohistochemistry (IHC) revealed a significant increase in the macrophage markers CD68 and CC‐chemokine receptor 2 (CCR2) in the liver. CCR2 has been shown to be critical for the increased hepatic macrophage accumulation that accompanies NASH. We also observed notable clusters of hepatic macrophages indicative of advanced stages of NASH. Furthermore, we assessed changes in endo‐immuno markers known to be associated with liver disease progression. In conclusion, our rat model exhibited characteristics of diet‐induced liver injury that progresses from steatosis to NASH. This included the recruitment of blood borne monocytes that invaded the liver. This model may serve as a useful tool to elucidate the complex role endo‐immunology plays in the progression of diet‐induced NAFLD. Thus, allowing for a preclinical model that could be used to develop new drugs to treat NASH.

Support or Funding Information

GH, AH, and JL were supported by the National Institute of General Medical Sciences‐award Number 1R25GM100866 (awarded to RKD and JGP).
Modeling Hypophosphatasia in Ovies aries (sheep) using CRISPR/Cas9: Development of large animal models that phenocopy rare human bone diseases
Authors: Williams Diarra , Pinzon Carlos , Oldeschulte James , Huggins Shannon , Gaddy Dana , Suva Larry
Published: November 2023

Hypophosphatasia is a rare inherited disorder that affects the development of bones and teeth caused by mutations in the tissue‐nonspecific alkaline phosphatase (TNSALP) gene. Similar to other human disease models, the current models of HPP have been engineered exclusively in rodents – specifically mice harboring loss‐of‐function missense mutations. However, although useful for modeling some aspects of HPP, murine models harboring TNSALP mutations do not faithfully represent the broad spectrum of human HPP clinical bone and tooth (odontohypophosphatasia) phenotypes. Mice have smaller long bones with thin fragile cortices and do not remodel the skeleton by Haversian remodeling as in humans nor do mice lose their deciduous teeth. However, in sheep both bone and tooth remodeling are analogous to humans. Therefore[GD1], we hypothesized that HPP mutation‐specific replacements into the sheep genome using CRISPR/Cas9 would produce a model that accurately phenocopies the bone and tooth pathophysiology in the disorder. We have recently identified an index patient with two distinct mutations in the ALPL gene [Alanine (GCC) à Threonine (ACC) in exon 5 (c.346 G>A) and Isoleucine (ATC) à Methionine (ATG) in exon 10 (c.1077 C>G)] that has a mild bone phenotype, with the primary defects being rhizomelia and early loss of deciduous teeth. However, a knock‐in mouse model of the c.346 G>A mutation presents with odontohypophosphatasia and without tooth loss, and without any deleterious effect on the long bones. Additionally, HPP patients with mutations c.571G>A at exon 6 and c.1133A>T at exon 10 have infantile lethal clinical presentations. To develop the genetically‐modified HPP sheep models, 14 single guide RNAs targeting the four distinct HPP mutation loci in exons 5, 6, and 10 of the sheep TNSALP gene were cloned into a pSpCas9(BB)‐2A‐GFP CRISPR/Cas9 plasmid, validated, and then analyzed for cutting efficiency. Primers upstream and downstream of each exon region of interest were used to capture 2kb and sequenced to verify the loci‐specific sequences. The different CRISPR/Cas9 constructs, sgRNAs, and repair DNA templates containing orthologous HPP mutations are being used to modify sheep embryos by intracytoplasmatic microinjection. These four unique allelic variants of HPP will be used to model the human individual and compound heterozygous HPP phenotypes in sheep, allowing for the characterization and comparison of interspecies differences in the HPP bone and tooth phenotypes of mouse, sheep, and human.
Association of Vitamin‐D receptor polymorphisms with degenerative disc disease in Indian population
Authors: Doraiswamy Ravichandran , Ramaswami Karthikeyan , Subramanian Rashmi , Srinivasan Dinesh Kumar , Sivasankaran Balasubramanian
Published: November 2023

The degenerative disc disease (DDD) in the lumbar spine refers to a syndrome that can lead to low back pain in elderly people. In the DDD, degeneration and herniation of disc are two main processes that contribute to low back pain. VDR is the first reported gene associated with DDD in a monozygotic twins study with Finnish population, which is located on chromosome 12q12–q14. Similar associations are later confirmed in various ethnic groups including Chinese and Japanese. There is a clear association between single nucleotide polymorphisms (SNP) and vitamin‐D receptor (VDR) gene (FokI and Taq1); however the pattern of genetic association with DDD in Indian population remains unknown. Thus, the present study was performed to validate the association of VDR polymorphisms and DDD in Indian population. In the present study, we have analyzed the polymorphisms of the VDR gene with an Indian population (48 cases and 47 controls). We identified a significant difference in the frequency of VDR gene alleles (FokI and TaqI) between cases and controls. In addition, there was a significant difference in genotypic distribution of FokI and TaqI alleles between these two groups. Overall, the distributions of VDR genotype frequencies FF, Ff, TT and Tt showed strong association with the DDD. To our knowledge, the present study reveals for the first time that polymorphism in the VDR gene is associated with disc degeneration and disc herniation in Indian population.

Support or Funding Information

This work was supported by research grant from the Karpagam Research Foundation, Karpagam Faculty of Medical Sciences and Research, Coimbatore, India.
HERG1 Expression Increases Calpain Activity in C2C12 Myotubes
Authors: Anderson Luke B. , Pratt Evan , Wang Wen‐Horng , Hockerman Gregory Howard , Pond Amber L.
Published: November 2023

Skeletal muscle atrophy is the loss of muscle size and strength caused by an imbalance in protein degradation and protein synthesis. It occurs with normal aging, neural and skeletal muscle injuries, and with diseases such as diabetes, cancer cachexia, AIDS, Muscular Dystrophy, etc. If unabated, skeletal muscle atrophy can be very debilitating and even cause death. Three proteolytic systems are known to be upregulated in atrophic muscle: 1) ubiquitin proteasome proteolysis (UPP); 2) calcium activated calpains; and 3) lysosomal cathepsins. HERG1 is a K+ ion channel shown to be up‐regulated in atrophying skeletal muscle. We have shown that it transiently increases intracellular calcium concentration in cultured C2C12 myotubes. Thus our objective was to test our hypothesis that myotubes with increased levels of the HERG1 protein would have increased calpain activity. We transfected C2C12 myotubes with either an adenovirus encoding HERG1 or an appropriate control virus and, in both experimental groups, measured: 1) myotube size using ImageJ; 2) MuRF1 protein abundance using immunoblot; and 3) calpain activity in depolarized and non‐depolarized myotubes using a Calpain‐Glo Assay Kit (ProMega; Madison, WI). Indeed, our data reveal that, relative to controls, myotubes expressing HERG1 undergo atrophy, experiencing: 1) a 55% decrease in size; 2) an increased abundance of the UPP E3 ligase MuRF1; and 3) an 18% increase in calpain activity. We conclude that HERG1 increases proteolytic calpain activity in cultured myotubes.

Support or Funding Information

This research was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health under Award Number NIH NIAMS 1R03AR053706‐01A2 to ALP. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Development of a Specialized Yeast Strain to Monitor Recruitment Patterns of Saw1 Mediated Rad1‐Rad10 Recruitment to DNA Damage Sites through Single‐Strand Annealing
Authors: Fregoso Fred E , Fischhaber Paula L.
Published: November 2023

Damage to the structural integrity of DNA by exposure to ionizing radiation is the cause of many mutations that accumulate in the human genome, which can lead to cancer and aging. The DNA double‐strand break repair (DSBR) pathways limit this accumulation by continuously repairing such damage as it forms. Single‐strand annealing (SSA) is one mode of DSB repair that occurs in regions that have tandem arrays of DNA repeats. Upon DSB induction, the processing of broken ends exposes complementary single‐stranded regions that anneal and produce 3′ overhanging flaps deriving from the sequence originally situated between the repeats. This DNA flap structure recruits Rad1‐Rad10 endonuclease to cleave the overhanging 3′ DNA. Although recent literature reported that the Saw1 protein mediates recruitment of Rad1‐Rad10 when flaps are 500 deoxynucleotides long but not ~10, the minimum flap length for Saw1 to recruit Rad1‐Rad10 in vivo is unknown.

The ultimate aim of this project is to determine whether Saw1 is required to recruit Rad1‐Rad10 to SSA sites when 3′ overhanging flaps are slightly longer (~20 deoxynucleotides). To address this important question, we have prepared a specific yeast strain containing an inducible DSB site flanked by DNA repeats that will yield 20 bp overhang flaps upon DSB induction and repair by SSA. The strain will additionally contain a fluorescently labeled Rad10 gene (Rad10‐YFP) and DSB site (DSB‐RFP) to monitor Rad10 localization to DSB sites by fluorescence microscopy in the presence or absence of Saw1.

Cloning of the 20 bp flap yeast strain was achieved by constructing two DNA plasmids that each contained the regions required to derive the desired substrate. The two plasmids were linearized and transformed into a progenitor yeast strain. Resulting transformants were screened by PCR to confirm integration of the recombinant DNA cassettes; an ISce‐I restriction site situated between two homologous HIS3 DNA repeats however spaced away from each other by 10 nonhomologous bp. DNA sequencing and ISce‐I restriction digest further supported proper insertion of the DNA cassettes and functionality of the inducible DSB site necessary for microscopy. This specialized yeast strain will be able to be used as an SSA substrate in consequent fluorescent microscopy experiments and will determine the necessity of Saw1 in recruiting Rad1‐Rad10 to SSA sites bearing ~20 deoxynucleotide flaps.

Support or Funding Information

The authors thank NIH grant (SC3GM093858) for funding.
Acetylpyrazine Thiosemicarbazone Inhibiting Topoisomerase II
Authors: Ngo Lana C , Stults Garrett A. , Hill Jessica , Bowman Sarah B. , Lisic Edward C. , Jiang Xiaohua
Published: November 2023

We have previously showed that Cu(II) acetylpyrazine methylthiosemicarbazone chloride ([Cu(APZ‐MTSC)Cl]) inhibits the ATP hydrolysis of human topoisomerase (Topo II) as catalytic inhibitor and share some common features as covalent poisons such as increasing TopoII‐mediate DNA cleavage which can be abolished by the presence of DTT. To further test if acteylpyrazine thiosemicarbazone metal complexes all have the similar characteristics, we synthesized a series of acetylpyrazine thiosemicarbazone metal (copper and palladium) complexes with different side chain substitutions including methyl, ethyl, turt‐butyl, benzyl, phenyl and dimethyl functional groups on the terminal nitrogen. We examined their effect on TopoII‐mediated DNA relaxation and DNA cleavage assays. Our data suggest that all metal acetylpyrazine thiosemicarbazones display similar characteristics as [Cu(APZ‐MTSC)Cl], but metal ions play a very important role in inhibiting TopoII.

Support or Funding Information

This project is funded by Faculty Development Research Grant from Tennessee Board of Regents and Faculty Research Grant from Tennessee Technological University.
Filament Formation on Double‐Stranded DNA Mediates E38K RecA's Enhanced Activity in the Absence of DNA Damage
Authors: Ritger Matthew , Cox Michael M
Published: November 2023

RecA is a DNA recombinase implicated in DNA damage repair. The wild‐type version forms a filament on single‐stranded DNA (which is present after DNA damage), and this filament activates steps in the bacterial SOS response. Such activity includes stimulating the auto‐catalytic cleavage of the SOS gene repressor LexA and a subunit of the translesion DNA Polymerase V, UmuD. The E38K mutant variant of RecA shows an enhanced ability to perform these functions in the absence of DNA damage, and its expression produces a constitutive SOS response in vivo. Using ATPase, cleavage, and pH shift bioassays, I found that the constitutive SOS repsonse is mediated by E38K RecA's ability to form a filament on double‐stranded DNA (dsDNA). In the presence of dsDNA, E38K RecA hydrolyzed ATP at a superior rate compared to wild‐type RecA. Incubating LexA and UmuD in the presence of E38K RecA and dsDNA resulted in cleavage, while the same experiment with wild‐type RecA did not. Finally, by manipulating the pH of the in vitro solution to allow wild‐type RecA to form a filament on dsDNA, then reverting the pH back to physiological conditions (the filament persists), LexA cleavage was promoted. Overall, the enhanced function of E38K RecA in the absence of DNA damage is explained by its ability to form a filament on dsDNA. In addition, dsDNA is completely competent as a cofactor for RecA‐mediated LexA and UmuD cleavage.
Differential Gene Expression and Variant Analysis of Clinical Strains of Cryptococcus neoformans
Authors: Seipelt‐Thiemann Rebecca , McClelland Erin
Published: November 2023

Cryptococcus neoformans is a fungal opportunistic pathogen that kills more than 500,000 people each year. To investigate the relationships of gene expression, genetic variant, and pathogenesis characteristics, RNA sequencing was performed on seven different clinical isolates and one laboratory strain H99S. TopHat, Cufflinks, and Cuffdiff bioinformatics tools were utilized to analyze the data. As expected, all strains showed differential expression of some genes (≤2 or ≥2 fold) compared to the laboratory strain. About 1700 up regulation differences and about 2300 down regulation differences were found compared to wildtype. When the clinical strains were compared to each other, more than 6200 up regulation differences and 7500 down regulation differences were observed in total. These data are currently being analyzed to determine common patterns of gene expression among the clinical strains, discover common biological pathways using gene ontology and gene enrichment analyses, and investigate relationships between gene expression and clinical patient data. Further, variant analyses will be undertaken to investigate genetic variation in the expressed genes of the clinical strains.

Support or Funding Information

URECA Grant at MTSU
Transgene Expression Is Increased When Cells Are Cultured on Defined Biophysical Cues as Detected by a Novel Fluorescence Quantification Method
Authors: Veen Sarah C , Youssef Amanda J , Liu Claire , Gasiorowski Joshua Z
Published: November 2023

Studies have shown that the interaction between cells and their extracellular matrix (ECM) is critical in directing migration, development, differentiation, and gene expression. Gene array studies have shown that cells seeded onto topographically‐patterned substrates that mimic biophysical ECM cues demonstrate a significantly different expression profile than those seeded onto a flat control. This led us to believe that biophysical cues may also have a significant effect on the expression level of transgenes for transfections and gene therapy experiments. However, quantifying transgene fluorescence for our experiments with commonly used techniques to measure live‐cell gene expression were limiting. Employing microscopes to measure fluorescent reporter expression are useful in observing single cell transgene expression, but macro‐scale cell populations cannot be observed as a whole unless time‐consuming stitch image acquisition is performed. This method of observation and quantification can become strenuous, inaccurate, and subjected to user bias. To address this limitation, we developed a non‐invasive method to acquire sensitive, quantitative, visual data over time of whole cell populations for transgene expression analysis. Through the use of a macro‐scale fluorescence photo‐imager with appropriate light sources and filters, we investigated the influence of surface topography on transgene expression. Mesenchymal and epithelial cell lines were transfected with a plasmid encoding GFP and seeded onto either a range of nano‐, submicro‐, and micro‐scale topographic surfaces with repeating patterns of ridges and grooves, or on a flat control. An increase in transgene expression was observed in cell populations plated onto the topographic surfaces when compared to the flat control, at multiple time points over a 48 hour time course. Cells plated onto the submicron sized patterns consistently displayed the highest amount of transgene expression. With similar trends observed in several cell types, our results suggest that defined biophysical ECM cues have a direct effect on the level of transgene expression. Additionally, our macro‐scale fluorescence imaging method is a non‐invasive, efficient way to observe and quantify transgene expression in large cell populations over extended time courses.

Support or Funding Information

The research presented here was supported by funds from Midwestern University's College of Health Sciences and Department of Biomedical Sciences.
RXRα Phosphorylation at Threonine 167 Coordinates Energy Metabolism in Mice
Authors: Sueyoshi Tatsuya , Sakuma Tsutomu , Shindo Sawako , Kanayama Tomohiko , Moore Rick , Negishi Masahiko
Published: November 2023

Threonine 162 constitutes a PKC phosphorylation motif within the DBD of human RXRα. Studies with human RXRα WT and T162A and T162D mutants demonstrated that the phosphomimetic RXRT162D mutant modulates bindings and activities of nuclear receptors differently. For example, RXR T162D repressed PPARα and activated PPARγ in PPRE‐reporter assays in Huh7 cells. Thus, this phosphorylation at T162 may have diverse activities to regulate genes. To investigate physiological significances ofthis phosphorylation in vivo a KI mouseline bearing the mouse homologueT167A mutation (RxraT167A)was established. Western blot analysis with an anti‐phospho‐Thr peptide antibody found that RXRα becomes phosphorylated at T167 in adipocytes in response to fasting; a gene array study revealed that this mutation affects expression of several key genes in lipidmetabolism in response to fasting and feeding. These genes include Mogat2, Dgat2, Acsm3, Acc1, and Fasn. In muscle, genes that maybe involved in body temperature control (Adrb1)and glycogen synthesis (Ppp1r3b) were differentially regulated in KI mice. Conversely, none of these genes observedin adipose and muscles were affected in the liver. Taken all together, RXR phosphorylated at threonine 167 may coordinate multiple organs to regulate energy metabolism in response to fasting and/or feeding in mice.
Nutrient regulation of TATA‐Box binding protein by O‐GlcNAcylation
Authors: Hardiville Stephan , Han Guanghui , Ma Junfeng , Hu Ping , Banerjee Partha S. , Hart Gerald W.
Published: November 2023

O‐GlcNAcylation is a dynamic post‐translational modification that is responsive to nutrient avabilality via the hexosamine biosynthetic pathway and its endproduct UDP‐GlcNAc. O‐GlcNAcylation serves as a nutrient sensor to regulate the activities of many proteins involved in nearly all biological processes. Interestingly, literature points out a regulation of the basal transcription machinery by O‐GlcNAcylation. TATA‐box binding protein (TBP) is a key player in regulating transcription since it is required for the activity of all three RNA polymerases. DNA‐bound TBP acts as a dock for recruitment of basal transcription factors and the RNA polymerase II to formation of a functional pre‐initiation complex. DNA‐bound TBP can also be recognized by NC2 and BTAF1. NC2 stabilizes the chromatin interaction of TBP, whereas the association of TBP with BTAF1 promotes its release upon hydrolysis of ATP. Here, we have shown that O‐GlcNAcylated TBP is bound to DNA, and that the sugar inhibits the interaction with BTAF1. Three O‐GlcNAc sites on the N‐terminus domain of TBP have been revealed by mass spectrometry. Site‐specific O‐GlcNAc mutants have been used to point out the T114 as the main regulator of TBP/BTAF1 interaction. Owing to the fact that BTAF1 dynamically regulates the chromatin interaction of TBP, we investigated the dynamic behavior of TBP and TBP O‐GlcNAc by Strip‐FRAP and ChIP assays. Collectively, our results indicate that O‐GlcNAcylation at T114 regulates TBP dynamics on gene promoters.

Support or Funding Information

The authors are supported by R01DK61671 and P01HL107153. G.W.H. receives a share of royalties received by Johns Hopkins University on sales of the CTD110.6 antibody, which is managed by JHU.
Cell penetrating peptide‐mediated nuclear delivery of Cas9 to enhance the utility of CRISPR/Cas genome editing
Authors: Axford David S. , Morris Daniel P. , McMurry Jonathan L
Published: November 2023

CRISPR/Cas9 is a transformative technology that allows genome editing in a variety of eukaryotic cells. However, many cell types and tissues are resistant to transfection or other delivery of the CRISPR/Cas9 complex, which has limited its utility. Our novel cell‐penetrating peptide (CPP) adaptor, TAT‐CaM, allows cytoplasmic delivery and release of a wide variety of biomolecular cargos. The integration of CRISPR/Cas9 with CPP‐mediated delivery could make CRISPR/Cas9 much more widely utile. Recombinant Cas9 containing a calmodulin binding site (CBS‐Cas9) was expressed in Escherichia coli, from which it was purified using affinity chromatography on a calmodulin sepharose column. TAT‐CaM/CBS‐Cas9 complexes were assayed for cell penetrating capabilities and subcellular localization using confocal microscopy. Several other CPPs and adaptor proteins, e.g. SAP and CALL3, and model cargos with subcellular localization signals were assayed in an attempt to develop CPP‐adaptor deliveries with altered kinetics and destinations. After cell penetrating capability and subcellular localization were assessed, a nuclear localization sequence was added to the gene encoding Cas9. Enhanced nuclear localization of Cas9 would make it more effective in potentially editing genes. Future work will integrate the present results into an effective CRISPR/Cas9 delivery and gene editing system.

Support or Funding Information

This work was supported by NIH grant R15GM120691