bluuebunny/crossref_metadata_2025 huggingface dataset:
Harnessing antibacterial properties of biomaterials for fabricating wound healing patch
Authors: Masoud Abdul , Mills David K.
Published: May 2022

Introduction

Studies aimed at elucidating the role of infections caused by bacterial colonization in wound healing are gaining increasing recognition. Proper management of wound infections purposely aimed at keeping low levels of bacteria on wound surfaces is pivotal in enhancing the wound healing process and preventing local and systematic infections. In this research, biomaterials with inherent antimicrobial properties were exploited for fabricating a wound healing composite with an improved potential of reducing or completely inhibiting bacterial growth. The use of biomaterials with inherent antibacterial properties was necessitated by the desire to reduce the incorporated antibiotic load, thus reducing the risk of development of resistance to antibiotics by these bacteria.

Methods

Carboxymethyl cellulose (CMC), one of the most common derivatives of cellulose, chitosan (CTS), a natural, biodegradable, non‐toxic polymer that demonstrates diverse biological properties, metalized and non‐metalized halloysites (HNT & mHNT) as well as variants loaded with antibiotics (LHNT, LmHNT) were tested for antimicrobial properties against E.coli at concentrations of 2mg/ml and 0.5mg/ml in 3 repeats. CTS and CMC were then physically cross‐linked and impregnated with halloysites and metal coated halloysites. A Fourier‐transform infrared spectroscopy (FTIR) characterization test was then carried out to validate the cross‐linking of the polymers. The biocompatibility of the fabricated composite was assessed by studying the proliferation rate of human dermal fibroblasts treated with the test samples.

Results

2mg/ml and 0.5mg/ml of the biomaterials tested all inhibited growth of E. coli with metal‐coated halloysites loaded with antibiotics exhibiting the most antimicrobial effect. CTS and CMC both exhibited characteristic absorption peaks on the FTIR spectra which conforms with data reported in other studies, with cross‐linking between the two polymers characterized by broadening of the bands at 3400–3301 cm−1. The proliferation assay showed that all the samples did not inhibit fibroblast cell growth with the exception of CTS‐CMC composites incorporated with 5% HNT and 5% metalized HNT.

Discussion

CTS and CMC composites incorporated with HNTs have the potential to effectively suppress microbial growth when used to fabricate wound dressings. The dressing is easy to fabricate, and its components are tunable from a personalized medicine perspective.
Effect of Ciprofloxacin on Planktonic Pseudomonas aeruginosa
Authors: Hill Clayton J. , Heuertz Rita
Published: May 2022

Pseudomonas aeruginosa is an opportunistic human pathogen that causes nosocomial, acute, and chronic infections in patients with conditions such as cystic fibrosis, severe burns, and cancer. P. aeruginosa is known for its persistence in infections due largely to its numerous means of antimicrobic resistance, one of which is its ability to form biofilms. This organism has been placed on the World Health Organization list of Priority Pathogens for Research and Development of New Antibiotics at the critical level for many reasons, that includes its high mortality, healthcare burden, prevalence of resistance, and treatability. P. aeruginosa is resistant to many antibiotics; however, >70% of P. aeruginosa are susceptible to ciprofloxacin. For these reasons, the purpose of the current study was to establish antimicrobic levels of ciprofloxacin effective against P. aeruginosa. To accomplish this purpose, antimicrobic levels of bacteria in biofilms and those not associated with a biofilm (planktonic) need separate methods of testing. For planktonic bacteria, minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) are performed, whereas other methods are used for biofilm‐associated bacteria. To initiate inhibition studies, a model system was identified of ciprofloxacin effect on standardized strains of P. aeruginosa (ATCC strains 10145, 27853, 1744, CRM‐9027, 9721). These strains were used to establish ciprofloxacin effect, in terms of MIC and MBC levels, on planktonic bacteria. Results indicate that MIC and MBC levels of these strains are in the same range as reported values of P. aeruginosa. These results establish ciprofloxacin efficacy of growth inhibition for each ATCC strain of planktonic P. aeruginosa and will serve as baseline results for future studies, such as effect on biofilm‐associated bacteria and combination antimicrobic treatments.
Effect of Caffeic acid analogs on Transient receptor potential melastatin 2 Channel
Authors: Saravanan Tanvikhaa , Thomas Gabrielle , Chowdhury Saiful M. , Deba Farah
Published: May 2022

Transient receptor potential melastatin 2 (TRPM2) is a sub family of TRP channels which plays a central role in producing neuro pathophysiological diseases such as Alzheimer’s, Parkinson’s and other neuropathic pain which are major health concern worldwide. However, their pharmacological pathway has not been fully characterized which is a significant gap in our knowledge. TRPM2 is mainly expressed in brain, bone merrow, endocrine and endothelia cells. Under oxidative stress, TRPM2 increases intracellular Ca2+ ions due to protein oxidation that causes several neurological disorders. Reactive oxygen species (ROS) production and oxidative stress are the primary cause of many pathological disease processes. Natural compounds with medicinal properties are useful and effective source for the treatment of various diseases since ancient times. Caffeic acid and its analogs, such as caffeic, chlorogenic, salvianolic A & B, and rosmarinic acid have anti‐inflammatory, antimicrobial, antioxidant, anesthetic, and cytotoxic properties. There is a critical need for new drug planning that focuses on oxidative interaction in targeted TRPM2 ion channels and intracellular ion signaling, which will regulate neuropathological diseases by interacting other TRP channels. Our ongoing experiment demonstrate that increase in ROS was decreased by caffeic acid analogs salvianolic, and rosmarinic acid more than caffeic and chlorogenic acid which was reflected by GST inhibition activity assay. Our experiments also evaluate the analgesic effects of caffeic acid analogs on TRPM2 channel by measuring intracellular calcium. Therefore, this pilot project aims to identify caffeic acid analogs as lead compounds that target TRPM2 which is one of the key pathway players for controlling various neurodegenerative diseases.
Temporal Development of Cardiometabolic Complications in A Hyperandrogenemic Model of Polycystic Ovary Syndrome
Authors: Pruett Jacob E. , Everman Steven , Romero Damian G. , Yanes Cardozo Licy L.
Published: May 2022

Introduction

Polycystic Ovary Syndrome (PCOS) is the most common endocrine disorder in young women, being characterized by androgen excess and irregular ovulation. Insulin resistance (IR), dyslipidemia, obesity, and hypertension are highly prevalent in this population. The etiology of PCOS is unknown. Most patients begin developing symptoms around puberty. However, there is a delay in the diagnosis of PCOS, so the metabolic characteristics of PCOS early on are unclear. We have demonstrated in a hyperandrogenemic PCOS rat model that obesity, increases in blood pressure, and IR are present after 3 months of androgen excess. Using this model, we tested the hypothesis that androgen‐induced IR, obesity, and dyslipidemia would develop in a time‐dependent manner.

Methods

Four‐week‐old Sprague Dawley female rats were randomly selected for sham or dihydrotestosterone pellet (DHT, 7.5mg/90 days) implantation (n=13‐17/group). Body composition was measured by EchoMRI; 24‐hour urine was collected with metabolic cages; and fasting plasma glucose, insulin, and lipid panel were collected at 1 (early), 2 (intermediate), and 3 (late) months of DHT treatment. IR was assessed by homeostatic model assessment for IR (HOMA‐IR).

Results

After 1 month of DHT, PCOS rats had decreased fasting glycemia (122.0 ± 2.8 vs 133.6 ± 3.1 mg/dL, P<0.01) and insulinemia, leading to decreased HOMA‐IR (4.3 ± 0.4 vs 7.4 ± 0.5, P<0.001). However, there were no differences between the groups in IR at 2 months of DHT, while at 3 months both fasting insulin (21.0 ± 1.6 vs 14.2 ± 1.4 µU/mL, P<0.01) and HOMA‐IR (6.3 ± 0.5 vs 4.3 ± 0.5, P<0.05) increased in PCOS compared to controls. Low‐density lipoprotein cholesterol (LDL) was increased in PCOS compared to controls at all time points, while triglycerides were only elevated at 2 months (84.9 ± 5.4 vs 65.1 ± 2.9 mg/dL, P<0.01) and 3 months (70.7 ± 5.3 vs 54.5 ± 4.5 mg/dL, P<0.05) of DHT. Meanwhile, there were no differences in high‐density lipoprotein cholesterol throughout the study. Lean mass was elevated at all time points in PCOS compared to controls, though fat mass was only elevated after 2 and 3 months (21.6 ± 1.9 vs 16.7 ± 0.8 g, P<0.05) of DHT. Proteinuria, a marker of renal injury, was elevated in PCOS at all time points.

Conclusions

In this PCOS model, early cardiometabolic complications include increases in LDL and renal injury. Intermediate cardiometabolic complications include increases in fat mass and triglyceridemia. However, DHT had a differential effect on fasting insulin and HOMA‐IR by lowering them initially at 1 month of treatment and then gradually increasing them above control levels by 3 months of DHT. These data indicate that androgen excess has a differential effect in cardiometabolic complications with time in female rats. In women with PCOS, these data may lead to a better characterization, and more effective treatments, for the evolving metabolic profile in women with PCOS.
Pigmented and Dunkin‐Hartley Guinea Pigs as Natural Models for Brain Aging and Alzheimer’s Disease
Authors: Wahl Devin , Moreno Julie , Santangelo Kelly , Cavalier Alyssa N. , Hamilton Karyn L. , LaRocca Thomas J.
Published: May 2022

Aging is the primary risk factor for most chronic diseases, including Alzheimer’s disease (AD). Current pre‐clinical approaches for studying brain aging and AD rely primarily on transgenic models intended to mirror pathologies associated with human brain aging and neurodegeneration (e.g., by increasing production of amyloid beta (Aβ) and phosphorylated tau). While these models may be useful, none completely mimic AD and its strong age‐dependence, and there has been limited success in translating pre‐clinical results and treatments to humans. We have characterized two non‐transgenic guinea pig strains (common ‘Pigmented’ guinea pigs and the Dunkin‐Hartley strain) that may be more useful models of brain aging and AD in this context. We show that these two models share many transcriptome features with both human brain aging and age‐related AD, especially with respect to biological processes like inflammation and transcriptional dysregulation. Additionally, both Pigmented and Dunkin‐Hartley animals exhibit age‐associated increases in numerous markers of human brain aging/AD, including neurofilament light chain (NfL, a marker of neuronal damage), S100 calcium‐binding protein B (S100ß, a marker of glial activation), ionized calcium‐binding adapter molecule 1 (Iba1, a marker of reactive microglia), and both Aβ and phosphorylated tau. These age‐associated effects are generally stronger in Dunkin‐Hartley animals, suggesting that this strain may be useful to model AD, while the Pigmented strain may be useful to model normal human brain aging.
Structural Analysis of a Non‐canonical Terpene Synthase
Authors: Tararina Margarita A. , Yee Danielle A. , Tang Yi , Christianson David W.
Published: May 2022

Natural products generated by biosynthetic enzymes provide complex structural diversity. Several fungal species have evolved to obtain this diversity by combining several core enzymes in a biosynthetic pathway, including polyketide synthases (PKS), nonribosonal peptides synthases (NRPS), and terpene cyclases (TC) along with accessorizing enzymes such as transferases and oxidoreductases. Combinations of different core enzymes in hybrid gene clusters affords structurally complex scaffolds unachievable with a single core enzyme. Recently, the Tang laboratory characterized a nine‐gene cluster from the fungal species, Aspergillus flavus, which encodes for two TCs, FlvE and FlvF, along with a single‐module NRPS, FlvI. Additionally, the flvfcluster contains multiple redox enzymes, including the short‐chain reductase FlvB, two P450s (FlvC and FlvD), and a didomain enzyme (FlvA) comprising a PLP‐dependent lyase and a non‐heme Fe α‐ketoglutarate‐dependent oxygenase. Other enzymes in the cluster include FlvG, a homolog of ornithine decarboxylase, and FlvH, a lysine N‐methyltransferase. Together, the flvf pathway produces flavunoidine, an alkaloidal terpenoid containing a tetracyclic and oxygenated sesquiterpene core connected to dimethylcadaverine and acylated with 5,5‐dimethyl‐L‐pipecolate. Notably, a unique feature of the flvf pathway is the enzyme, FlvF. Although annotated as a terpene cyclase, FlvF catalyzes the stereospecific C‐N bond formation attaching dimethylcadaverine to the sesquiterpene core, an unusual reaction given that FlvF has been proposed to take a secondary carbocation as substrate. The mechanism underlying this reaction has not been reported in any other terpene cyclase and is not well understood. Herein, we report the first crystal structure of FlvF determined at 2.6 Å resolution. The initial electron density map was phased using a search model generated by AlphaFold2 (with access provided by ColabFold) and refined with an Rwork/Rfree of 0.22/0.27, respectively. The structure adopts an alpha‐fold of class I terpene cyclases, and a search for structural homologs using the DALI server identifies the closest matches as the terpene cyclases FgGS, PaFS, and aristolochene synthase. Notably, FlvF does not contain the canonical metal‐binding motifs observed in class I cyclases. A bound molecule of Tris forms cation‐pi interactions with surrounding aromatic residues at the base of the active site, which might similarly stabilize a carbocation intermediate. Further analysis of the active site indicates multiple possibilities for general acid catalysis. Future structural and mechanistic studies are necessary to elucidate the mechanism of this enzyme.
Dysregulation of Angiopoietin‐like Proteins is a Potential Link Between Vitamin D Deficiency and Dyslipidemia
Authors: Ahmad Abdur Rahman , Ali Maryam A. , Abu‐Farha Mohamed
Published: May 2022

Vitamin D deficiency (VDD) is one of the prime nutritional problem globally. Low levels of vitamin D have been shown to correlate with obesity and dyslipidemia, but the mechanism(s) of these effects has not been well studied. Angiopoietin like proteins (ANGPTLs) are a group of proteins known to be involved in lipid metabolism by affecting the lipoprotein lipase activity. A few studies in adults have indicated that vitamin D deficiency regulates ANGPTL3,4 and 8 secretion but the effects of developmental vitamin D deficiency (DVDD) has not been well investigated. In this study, we investigated the effects of DVDD on ANGPTL3, 4 and 8 levels in rat pups. Wistar rat pups were made vitamin D deficient at different developmental stages, namely during gestation (dG group), during lactation (dL group) and during gestation and lactation (dGL group). Liver tissues and serum samples from control (C) and the three DVDD groups were used in this study. Gene expression of ANGPTL3, 4 and 8 in liver tissues were analyzed by RT‐PCR, whereas levels of ANGPTL3 and 4 in serum samples were assessed by using sandwich ELISA. One way ANOVA test revealed that gene expression of ANGPTL3 in dGL and dL groups were significantly increased (p<0.05) compared to control and dG groups. However, gene expression of ANGPTL4 in dGL and dG were significantly decreased (p<0.05) compared to control and dG groups. In addition, gene expression of ANGPTL8 was significantly increased in the dGL group, compared to control (p<0.01). The levels of ANGPTL3 in serum samples showed a significant increase in dGL groups only compared to control (p<0.05) whereas levels of ANGPTL4 were significantly decreased in dL and dGL groups compared to control (p<0.01). These results suggest that combined prenatal and postnatal vitamin D deficiency in rat pups increase the levels of ANGPTL3 and 8 and decrease expression of ANGPTL4. The modulations of these ANGPLs due to vitamin D deficiency may be involved in dyslipidemia and its subsequent health effects. Further research is needed to investigate the mechanisms of ANGPTLs‐modulating effects of vitamin D.
Peripheral Chemoreflex Sensitivity is Augmented in Human Type 2 Diabetes
Authors: Lis Eric C. , Ott Elizabeth P. , Harper Jennifer L. , Manrique‐Acevedo Camila , Limberg Jacqueline K.
Published: May 2022

Introduction

The peripheral chemoreceptors have been implicated in the pathogenesis of type 2 diabetes. Our main objective was to examine peripheral chemosensitivity in individuals with type 2 diabetes, as well as the contribution of the peripheral chemoreceptors to basal cardiovascular control. We hypothesized individuals with type 2 diabetes exhibit exaggerated peripheral chemoreflex sensitivity to hypoxia compared to healthy, non‐diabetic controls. We further hypothesized the peripheral chemoreceptors contribute to cardiovascular dysfunction in type 2 diabetes.

Methods

Eight adults with diagnosed type 2 diabetes (3M/5F, 54±3 yrs, HbA1c 8.1±0.6%) and nineteen healthy controls (8M/11F, 50±3 yrs, HbA1c 5.3±0.1%) participated (IRB #2010295 and #2018486). Ventilatory and hemodynamic responses to acute hypoxia were assessed to determine peripheral chemosensitivity [i.e., hypoxic ventilatory (HVR), heart rate (HHRR) and blood pressure (HBPR) response]. Separately, heart rate, blood pressure and forearm blood flow (Doppler ultrasound) were measured while breathing normoxic or hyperoxic air (100% O2; Modified Dejours test) to acutely attenuate peripheral chemoreceptor activity. Forearm blood flow was normalized for mean arterial blood pressure for measures of vascular resistance.

Results

Peripheral chemosensitivity was augmented in adults with type 2 diabetes compared to control as assessed via the HVR (‐1.10±0.16 vs ‐0.50±0.10 L/min/%, p<0.01) and HBPR (‐0.92±0.17 vs ‐0.42±0.19 mmHg/%, p=0.06), with no differences in HHRR (‐0.68±0.22 vs ‐0.56±0.12 beats/min/%, p=0.64). Higher HVR and HBPR were associated with higher fasting insulin (R=‐0.58, p<0.01 and R=‐0.38, p=0.06) and HbA1c (R=‐0.60, p<0.01 and R=‐0.39, p=0.05). Attenuation of peripheral chemoreceptor activity with acute hyperoxia decreased heart rate (p<0.01) and blood pressure (p=0.02), with no effect on forearm vascular resistance (p=0.55); any effect of hyperoxia did not differ between adults with type 2 diabetes and controls (p>0.05).

Conclusion

Individuals with type 2 diabetes exhibit exaggerated peripheral chemoreflex sensitivity which is associated with disease severity (i.e., HbA1c). In contrast, the contribution of the peripheral chemoreceptors to basal cardiovascular function in type 2 diabetes does not differ from non‐diabetic controls. Results advance our understanding of the pathogenesis of type 2 diabetes and related complications.
Quantifying flow‐induced transport of membrane‐linked proteins in model and cell membranes
Authors: Pash Samuel , Ankrom Emily , Anthony Autumn , Ratajczak Amanda , Honerkamp Smith Aurelia , Thevenin Damien
Published: May 2022

Shear flow sensing is crucial in defining fundamental cell properties, including cell fate, self‐renewal, motility, and homing behaviors. For instance, endothelial cells lining blood vessels translate blood flow into responses that in turn regulate the tension in blood vessels, and, in tumors, flow can regulate genes that promote cancer cell migration and metastasis.

Recent studies suggest that glypican‐1 (a lipid‐anchored heparan sulfate proteoglycan at the surface of endothelial cells) may play a key role in the flow signaling pathway: its flow‐mediated redistribution at the membrane appears to mediate the production of nitric oxide (NO), a key signaling molecule produced by the NO synthase eNOS. However, the molecular mechanisms that endothelial cells use to sense changes in flow and translate this mechanical stimulus into intracellular molecular responses (e.g., activation of eNOS by glypican‐1) are currently unknown. We hypothesize that protein concentration gradients in the plasma membrane are formed by physical interactions with fluid flow. This is based on the premise that extracellular lipid‐anchored proteoglycans like glypican‐1 can be transported along the membrane by external flow, with the aqueous part of the protein acting as a molecular sail.

To investigate the effect of flow on membrane‐associated proteins and cell signaling, it is crucial to visualize and quantify the motility of membrane bound proteins in response to flow. Here, we present results in both reductionist model membrane systems and cell membranes aimed at determining the relationship between drift velocity and protein size, membrane lipid composition, and lipid anchor structure: (1) Our first approach measures the flow coupling and frictional coefficients of lipid‐anchored model proteins (monomeric streptavidin fusions) of varying size reconstituted in glass‐supported bilayers. (2) We also present the development of a unique labeling approach allowing the visualization of glypican‐1 expressed at the membrane of living endothelial cells under flow.

While glypican‐1 is specific to endothelial cells, the principles of fluid mechanics are universal. We, therefore, anticipate that our methodology and results will apply to multiple cell lines and flow conditions.
Short‐term L‐Citrulline supplementation and macro‐ and micro‐vascular function in old adults
Authors: Bisconti Angela V. , Broxterman Ryan M. , Ratchford Steve , Jarrett Catherine L. , Shield Katherine L. , Park Soung Hun , Thurston Taylor S. , Lewis Matthew T. , Wray D. W. , Richardson Russell S.
Published: May 2022

Aging is an independent cardiovascular risk factor that exhibits impaired vascular function and aging itself is associated with a decline in nitric oxide (NO) bioavailability, an essential signaling molecule involved in vascular function. L‐Citrulline (L‐Cit) supplementation, and the subsequent conversion to L‐Arginine (L‐Arg), is an effective approach to augment substrate for endothelial NO production, potentially improving vascular function. However, the effect of L‐Cit supplementation in old adults is not well understood. Therefore, this study evaluated the effect of short‐term oral L‐Cit supplementation on flow mediated‐dilation (FMD) and reactive hyperemia, indicators of macro‐ and microvascular function, respectively. Eleven old adults (72±5 yrs) underwent 7‐day L‐Cit supplementation (6g per day). %FMD assessments were performed pre‐ and post‐L‐Cit supplementation in both the brachial (BA) and popliteal (PA) arteries in addition to the measurement of reactive hyperemia area under the curve (RH AUC) for 60s following FMD cuff release. Plasma L‐Cit and L‐Arg concentrations were measured to confirm L‐Cit absorption and conversion, respectively. The ratio of L‐Arg to plasma asymmetric dimethylarginine (ADMA) was calculated as a biomarker of NO‐bioavailability. L‐Cit supplementation improved plasma L‐Cit and L‐Arg concentrations (+269%; p=0.002; +74%; p=0.0008, respectively). Although plasma ADMA levels increased post‐treatment (0.51±0.02 vs 0.56±0.02uM/l, p=0.04), the L‐Arg/ADMA ratio was also significantly greater (273±30 vs 419±50; p=0.004). In contrast, there was no effect on either brachial or popliteal %FMD (BA 2.6±0.7 vs 3.4±0.7%; PA: 6.9±2.4 vs 6.6±2.0%, respectively) or RH AUC (BA: 537±124 vs 453±93ml; PA: 612±52 vs 706±81ml, respectively). In conclusion, despite the clear pharmacological efficacy of 7 days of L‐Cit supplementation, in terms of raising NO‐substrate bioavailability, neither macro‐ nor microvascular function was altered in old adults.
Modelling the Applications of CRISPR‐Cas9 for Treating Sickle Cell Disease
Authors: Cooper Grace , Akteke Elfin , Dalvi Viggo , Delage Laura , Feberova Natalie , Fujikawa Kento , Klousseh Edem , Manoharan Harish , Mcdaniel Kieran , Powter Morgan , Seyoum Michael , Greig James , MacGillivray Brendan
Published: May 2022

Clustered Regularly Interspaced Short Palindromic Repeats or CRISPR‐associated protein 9 (CRISPR‐Cas9) is a genome editing tool that can remove, modify, or add sections of DNA. It is made up of two main molecules: the Cas9 protein and the guide‐RNA (gRNA), which is used to match a target gene to the CRISPR‐Cas9 molecule. The desired sequence of DNA must be between 2 and 5 nucleotides and must be followed by the protospacer adjacent motif (PAM) at the 3’ end of the gRNA. This enables the target gene to be found and cut by the Cas9 endonuclease. Once cut, the DNA can be repaired using non‐homologous end joining (where a random insertion or deletion of DNA occurs) or homology directed repair (where a homologous piece of DNA is used as a template to repair the DNA). The latter is used for genome editing, as the target sequence can be changed with the CRISPR‐Cas9 system.

Sickle Cell Disease (SCD) is an autosomal recessive hereditary disorder that affects erythrocytes and is caused by a single base substitution of adenine to thymine on the hemoglobin beta (HbB) gene. Currently two different groups are examining two different techniques to treat SCD. One group has shown that by deleting the BCL11A gene, fetal hemoglobin production can occur, which greatly diminishes the amount of Sickle hemoglobin (HbS). An example of this application of CRISPR‐Cas9 for treating SCD is the story of Victoria Gray, the first person to have received genome editing treatment for SCD.
Age‐ and sex‐specific differences in activation of the cardiac adrenergic cascade
Authors: Yusifov Aykhan , Borders Megan O. , Woulfe Kathleen C. , Bruns Danielle R.
Published: May 2022

Heart failure (HF) is a costly and devastating public health problem, which differentially impacts male and female patients of all ages across the life‐course. The adrenergic system is dysregulated with HF and is the target of front‐line therapeutics. However, the majority of the work aimed at elucidating the adrenergic signaling response has been conducted in adult males. Consequently, mechanisms of adrenergic signaling that differ by sex and age remain unclear. Here, we set out to identify mechanistic differences in the adrenergic pathway in response to isoproterenol (ISO) stimulation in juvenile (4 weeks), adult (4‐6 months) and aged (18 months) C57Bl6 mice of both sexes. ISO was delivered by mini‐osmotic pump (30 mg/kg/day) for one week. We assessed changes in β‐adrenergic receptor (β‐AR) expression, protein kinase activity, and myofilament protein phosphorylation and function. β1‐AR and β2‐AR protein expression were significantly lower in response to ISO in juvenile males with no difference in expression in juvenile females. In contrast, ISO stimulation in aged males significantly upregulated β1‐AR and β2‐AR protein expression without changes in expression in aged females. Analysis of sarcomeric proteins indicated that phosphorylation of troponin varied in an age‐ and sex‐dependent manner. Phosphorylation of the inhibitory subunit of troponin (TnI) at Ser23/24 was upregulated in response to ISO in females of all ages with no differences in males. These changes in phosphorylation decreased myofilament Ca2+ sensitivity in female hearts in response to ISO. Together, this work suggests that male hearts responded robustly to ISO stimulation as evident by activation throughout the adrenergic cascade while female hearts demonstrated activation of downstream mechanisms at the level of the sarcomere. Ongoing experiments will characterize calcium handling and myofilament mechanics to mechanistically link upstream signals to downstream changes in contractility. These data demonstrate that age and sex are significant biological factors which contribute to adrenergic‐mediated changes in cardiac performance. Understanding these differences is critical for successful therapeutic intervention within the adrenergic signaling cascade for male and female patients of all ages.
Cardiovascular and core temperature responses to acute organophosphate intoxication in rats
Authors: Pan Shiyue , Bruun Donald A. , Lein Pamela J. , Chen Chao
Published: May 2022

Introduction

Organophosphates (OP) are cholinesterase inhibitors that are used as pesticides in agriculture and as nerve agents in chemical warfare. Surprisingly, we have limited information on the time course of cardiovascular responses to acute OP intoxication at doses that cause cholinergic crisis in humans. OP‐induced QT prolongation has been shown to lead to fatal ventricular arrhythmia and delayed death, however, not all cardiac failures are associated with QT prolongation.

Methods

Rats were implanted with a BP/ECG telemetry device and 14 d later, acutely intoxicated with the OP diisopropylfluorophosphate (DFP, 4 mg/kg, sc) followed 1 min later by atropine sulfate (2 mg/kg, im) and pralidoxime (25 mg/kg, im) to block peripheral cholinergic toxicity. BP/ECG data were grouped based on the rat’s survival time: <15 min (n=11, group A), >15 min and <17 hours (n=9, group B), and >17 hours (n=4, group C).

Results

In conscious, freely moving rats, DFP injection significantly increased BP (+87±10 mmHg), HR (+177±32 bpm), core temperature (+4.0±0.7 °C), and occurrences of premature ventricular complexes (PVC). These increases were similar in all three groups. Around the peak pressor response, group A had abrupt drops in BP and HR (cardiovascular collapse) that was triggered by supra‐ventricular events (sinus bradycardia and/or AV blocks). A similar pattern of cardiovascular collapse was observed in group B, hours after DFP injections. Furthermore, after the initial pressor response, half of the rats in group B had significant lower pulse pressure and BP despite a maintained HR.

Conclusion

Our data suggest that DFP‐induced cardiovascular collapse may be centrally mediated and that compromised cardiac function may also contribute to the cardiovascular collapse in delayed death.
Endothelial cell specific knockout of caveolin‐1 attenuates AngII/BAPN‐induced vascular remodeling in mice
Authors: Rizzo Victor , Preston Kyle , Nguyen Thinh , Kawai Tatsuo , Poltronetti Anthony , Eguchi Satoru
Published: May 2022

Caveolin‐1 null mice (Cav1‐/‐) are protected from developing vascular diseases while mice expressing Cav1 solely in the endothelium are capable of disease progression. To explore potential mechanisms through which endothelial caveolin‐1 contribute to vascular disease, we subjected Cav1 fl/fl Cre ‐ and endothelial cell specific, caveolin‐1 knockout mice (Cav1 fl/fl Cre+) to Angiotensin II (AngII: infusion at 1ug/kg/min for 4 weeks) and β‐aminopropionitrile (BAPN: administered in drinking water at 0.625mg/ml for 2 weeks) to induce vascular remodeling associated with aortic aneurysm formation. We found that the inner aortic diameter significantly increased (>50%) in Cav1 fl/fl Cre‐ mice in response to AngII/BAPN. Both outer diameter and vessel wall thickness, while trending towards increased values, were not statistically different in these mice compared to aortas from saline infused mice. These remodeling events were not observed in EC‐Cav1 null mice. Immuno‐histochemical analysis of vessel cross‐sections revealed significant enhancement of macrophage accumulation (F4/80 staining) and expression of MMP2 only within the tunica media of Cav1 fl/fl Cre‐ aortas exposed to AngII/BAPN. In addition, we found that AngII/BAPN enhanced adhesion molecule expression (ICAM‐1/VCAM‐1) on the endothelial cell surface of these but not EC‐Cav1‐/‐ mice. This observation correlated with activation of the NFkB pathway in cultured rat aortic endothelial cells in response to AngII/BAPN, an event that was abated in cells depleted of Cav1 via pretreatment with a Cav‐1 miRNA. These findings indicate that endothelial cell caveolin‐1 plays a role in vascular disease through regulation of inflammatory processes that contribute to vascular remodeling.
Cardiovascular reactivity to and recovery from cold face test
Authors: Saperova Elena V. , Dimitriev Dmitry A.
Published: May 2022

The purpose of this study was to investigate reactivity and recovery to primarily parasympathetic stimulation by cold face test.

Sixty five students with a mean age of 20.61±0.13 years take part in the study (86% women, 14% men). They were studied in the morning hours in a quiet room at a comfortable temperature. Time domain (SDNN, RMSSD, pNN50), frequency domain (TP, HF, VLF, LF and LF/HF ratio) and nonlinear (SD1, SD2, ApEn, SampEn, D2, DFA1, DFA2) parameters of HRV were obtained from all participants for 5 minutes before, 5 minutes during and 5 minutes after cold face test (0–1 °C cold compresses) in a supine position. Statistical analysis was performed using Mann‐Whitney test.

Heart rate during cold face test was significantly lower than that during rest period (74.03±1.26 vs 76.74±1.21 beat/min, p=0.006). After cold face test cessation heart rate displayed significant decrease compared to cold face test period (71.96±1.17 vs 74.03±1.26 beat/min, p=0.001). Time domain parameters were found to be higher during cold face test than that during rest (SDNN: 52.6±2.85 vs 43.61±2.23 ms, p=0.006; RMSSD: 59.87±4.08 vs 44.99±2.93 ms, p=0.001 and pNN50: 30.49±2.38 vs 22.07±2.13 %, p=0.003). During recovery period time domain parameters remain to be higher, than that during rest: SDNN – 47.97±2.29 ms, RMSSD – 52.16±3.35 ms, pNN50 – 27.24±2.38%.

There was no differences during rest period, cold face test and recovery period in TP (2097.89±249.98 vs 2517.24±301.8 vs 2176.12±204 ms2, p=0.744), VLF (83.59±9.99 vs 92.44±12.21 vs 88.33±7.85 ms2, p=0.359), LF (932.86±107.70 vs 1024.98±132.06 ms2, vs 928.54±83.35, p=0.896). High frequency parameter was significantly higher during cold face test than that during rest (1395.36±184.39 vs 1078.11±143.13 ms2, p=0.013). After cold test HF parameter was 1156.11±129.74 ms2(p>0.05).

Nonlinear measures were significantly higher during cold face test than that during rest (SD1: 42.39±2.89 vs 31.83±2.07 ms, p=0.001; SD2: 60.52±3.04 vs 52.45±2.50 ms, p=0.025). During recovery period nonlinear parameters remain to be higher, than that during rest: SD1 – 36.92±2.29 ms, SD2 – 56.34±2.44 ms, pNN50 – 27.24±2.38%. ApEn and DFA1 were significantly lower during cold face test than that during rest (respectively, 1.15±0.01 vs 1.40±0.01 and 0.87±0.03 vs 1.01±0.02, p<0.001). During recovery period ApEn was 1.37±0.01 and DFA1 was 0.96±0.03.

The other HRV parameters including D2, LF/HF ratio, pLF, pHF, SampEn, DFA2 were not significantly different.

These results suggest that cold face test has been associated with increased vagal cardiac control and decrease entropy and DFA1.
H‐NOX Signaling and Biofilm Formation in Caulobacter crescentus
Authors: Lee‐Lopez Cameron C. , Yukl Erik
Published: May 2022

Bacterial Heme‐Nitric Oxide/Oxygen binding (H‐NOX) proteins utilize heme to act as diatomic gas sensors. These proteins often interact with a signaling partner to govern communal behaviors such as quorum sensing and biofilm formation in response to nitric oxide in facultative anaerobes. H‐NOX from Vibrio cholerae (Vc H‐NOX) in the Fe(III) and Fe(II)‐NO states was demonstrated to inhibit the activity of a cognate histidine kinase (HK). Heme‐free Vc H‐NOX could also act as an inhibitor of HK signaling through reversible oxidation of cysteine residues at a zinc binding site, suggesting that H‐NOX proteins may act as redox sensors in some organisms. The zinc‐binding Cys residues are conserved across numerous species, including the aquatic organism and obligate aerobe, Caulobacter crescentus (Cc H‐NOX). Using UV‐Vis spectroscopy, our lab has characterized heme complexes from Cc H‐NOX as purified, reduced, CO‐bound, and NO‐bound. ICP‐OES has provided insight to the degree of heme binding in Cc H‐NOX samples as well as zinc content. Circular dichroism has provided an estimate of secondary structure and a melt curve using single wavelength readings with variable temperatures. Lastly, we have generated a knockout mutant to investigate the importance of H‐NOX in biofilm regulation and the impact this knockout has on the Caulobacter crescentus proteome. Our results have shown complete zinc binding for Cc H‐NOX, the formation of a stable NO‐complex, and a mild hyper‐biofilm phenotype among Caulobacter crescentus mutants. The mutant also exhibits differences in protein expression levels and phosphorylation reflecting signaling pathways altered in the absence of H‐NOX. This has led to an improved understanding of the roles of H‐NOX signaling across bacterial species.
Expression and characterization of a putative cyclooxygenase from the genome of Nitrosospira multiformis
Authors: Park Dahyun , Selinsky Barry
Published: May 2022

Cyclooxygenases are dual‐function heme proteins critical for the biosynthesis of prostaglandins in mammals. Mammalian cyclooxygenases contain both dioxygenase activity (adding molecular oxygen to lipid substrates to form lipid hydroperoxides) and peroxidase activity (reducing the hydroperoxides to alcohols). Two open reading frames in the ammonia‐oxidizing betaproteobacterium Nitrosospira multiformis are annotated as cyclooxygenases by structural prediction of the protein product. We have attempted to express and characterize one of these open reading frames to test the structural prediction. DNA encoding the desired open reading frame was synthesized and inserted into several expression vectors with N‐terminal fusion partners (his6 tag, SUMO, thioredoxin). Protein expression yielded largely insoluble protein product under many different expression conditions. Recently, we have been able to express a thioredoxin‐Nitrosospira cyclooxygenase fusion partner in soluble form. Ongoing experiments to characterize the protein structurally and enzymatically will be reported.
Modulation of glutamate transporters as a potential therapeutic intervention for neurological disorders
Authors: Reeb Katelyn L. , Mortensen Ole V. , Fontana Andreia C. K.
Published: May 2022

Excitatory amino acid transporters (EAATs) are key proteins in the CNS that regulate glutamate levels and, therefore, control excitatory neurotransmission and limit excitotoxicity. EAATs are trimers, with each protomer consisting of a scaffold domain, and a transport domain that slides along the scaffold domain in an “elevator‐like motion” to move synaptic glutamate into astrocytes and neurons. Our lab has discovered novel positive allosteric modulators (PAMs) of EAAT2, the main EAAT subtype responsible for glutamate clearance in the CNS. We hypothesize that these compounds act by altering the interactions between the scaffold and the transport domain. To test this, we used computational modeling, followed by site‐directed mutagenesis and radioligand uptake assays. This allowed us to identify mutations in specific amino acid residues on astrocytic transporters EAAT1 and EAAT2 that result in increased activity without changes in expression. This suggests that interactions between the domains are responsible for enhanced efficiency of these transporters. These gain‐of‐function mutated EAAT1 and EAAT2 are currently being studied in single molecule Förster Resonance Energy Transfer (smFRET) approaches to further understand the kinetics and dynamics of EAATs. In our work, we also evaluated the neuroprotective properties of our EAAT2 PAMs in models of excitotoxicity. Previously, our lab has demonstrated that EAAT2 PAMs are protective in an in vitro stroke model. We expanded our studies to investigate potential neuroprotective properties an in vitro epilepsy model, using live calcium signaling measured in primary neuron/glia cultures. Lastly, we explored the ability of these compounds to attenuate cocaine seeking behavior in conditioned place preference behavioral experiments. Collectively, these studies expand our understanding of the mechanisms of these PAMs and broaden their potential therapeutic indications.
Correlation between heart rate variability and polysomnography‐derived scores of severities for obstructive sleep apnea
Authors: Santos Rafael R. , Silva Thais M. , Silva Luiz E. V. , Eckeli Alan L. , Salgado Helio C. , Fazan Rubens
Published: May 2022

Obstructive sleep apnea (OSA) is one of the most common sleep disorders and affects nearly a billion people worldwide. Furthermore, it is estimated that many patients with OSA are underdiagnosed, which contributes to the development of comorbidities, such as cardiac autonomic imbalance, leading to high cardiac risk. Heart rate variability (HRV) is a non‐invasive, widely used approach to evaluate neural control of the heart. This study aims to seek the relationship between HRV indices and the presence and severity of OSA. Polysomnography (PSG) exams of 157 patients were classified into four groups: OSA‐free (N=26), mild (N=39), moderate (N=37), and severe OSA (N=55). The electrocardiogram was extracted from the PSG, and a 15‐min beat‐by‐beat series of RR intervals was generated every hour during the first 6 h of sleep. Linear and nonlinear HRV approaches were employed to calculate a variety of indices of HRV. Specifically, time‐ and frequency‐domain, symbolic analysis, entropy measures, heart rate fragmentation, acceleration and deceleration capacities, asymmetry measures, and fractal analysis (detrended fluctuation analysis). Results with indices of sympathovagal balance reinforced previous findings that patients with OSA have sympathetic overactivity. Nonlinear indices showed that these patients suffer from a loss of physiologic complexity, contributing to their higher risk of development of cardiovascular disease. Moreover, as shown in the table below, many HRV indices presented statistically significant correlation with PSG's clinical scores: apnea/hypopnea index (AHI), microarousal index (MI), percentage of the total sleep time that the patient was with oxygen saturation below 90% (T90), and oxygen saturation nadir during sleep (SatMin). Therefore, a complete set of HRV indices, especially the nonlinear ones, can bring valuable information about the presence and severity of OSA, suggesting that HRV can be helpful in a quick diagnosis of OSA, potentially reducing the development of comorbidities.
Hypoxemia Augments the Local Metabolic Error Signal and Improves Coronary Pressure‐Flow Autoregulation
Authors: Warne Cooper M. , Essajee Sal I. , Dick Gregory M. , Tune Johnathan D.
Published: May 2022

The local metabolic hypothesis proposes that myocardial oxygen tension, indexed by coronary venous PO2 (CvPO2), determines the degree of coronary pressure‐flow autoregulation by increasing the production of vasodilator metabolites as coronary perfusion pressure (CPP) is reduced. We tested this hypothesis by examining the extent to which exaggeration of the metabolic error signal influences coronary autoregulatory capability. Experiments were performed in anesthetized, open chest swine (n = 8) in which the left anterior descending coronary artery was cannulated and connected to a servo‐controlled roller pump system. This allowed CPP to be reduced from 140 to 40 mmHg in increments of 10 mmHg before and during hypoxemia (PaO2 from 138 ± 5 to 34 ± 1 mmHg). Under control‐normoxic conditions, CvPO2 decreased from 33 ± 1 to 20 ± 1 mmHg and coronary blood flow fell from 0.81 ± 0.09 to 0.35 ± 0.04 ml/min/g as CPP was reduced from 140 to 40 mmHg. Hypoxemia augmented myocardial oxygen consumption (P < 0.01), increased coronary blood flow (P < 0.0001), and reduced CvPO2 (22 ± 1 to 14 ± 1 mmHg; P < 0.0001) over the same range of CPPs. Increases in coronary blood flow during hypoxemia were sufficient to maintain myocardial oxygen delivery at values equivalent to normoxic conditions (P = 0.20). Calculation of closed‐loop autoregulatory gain (Gc) over a CPP range of 120 to 60 mmHg (value of 1 represents perfect autoregulation) demonstrated that Gc was improved from 0.18 ± 0.05 to 0.45 ± 0.14 under normoxic vs. hypoxemic conditions respectively (P = 0.02). Gc was also inversely related to CvPO2 and the slope increased ~4‐fold by hypoxemia. These findings support that coronary pressure‐flow autoregulatory capability is augmented by hypoxemia‐induced increases in the local metabolic error signal.
Mechanistic Insights of Sulfonamide‐Based NLRP3 Inhibitors for the Treatment of Neurodegenerative Diseases
Authors: Blevins Hallie , Xu Yiming , Biby Savannah , Zhang Shijun
Published: May 2022

Neuroinflammation has proven to play an important role in the pathogenesis of neurodegenerative diseases by encouraging the expression and maturation of pro‐inflammatory regulators. Studies have shown the upregulation of pro‐inflammatory cytokines interleukin (IL)‐1β and IL‐18 in neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), multiple sclerosis (MS), traumatic brain injury (TBI), and many more. The NLRP3 (NOD‐, LRR‐ and pyrin domain‐containing protein 3) inflammasome is the key regulator in the maturation and release of these inflammatory cytokines and is therefore an attractive target for suppressing inflammation. Upon assembly and activation of the multimeric NLRP3 inflammasome complex comprised of NLRP3, ASC protein, and pro‐caspase‐1, autoproteolytic cleavage of pro‐caspase‐1 occurs. Activated caspase‐1 is then able to cleave pro‐IL‐1β and pro‐IL‐18 and release their biologically active forms, IL‐1β and IL‐18, consequently leading to an inflammatory cell death known as pyroptosis (Figure 1). Our recent medicinal chemistry campaign has led to a series of novel small molecule inhibitors that are suggested to selectively target NLRP3 and show both in vitro and in vivo activity to inhibit the release of IL‐1β and IL‐18 with sub‐micromolar potencies. Through various assays and photoaffinity labeling experiments using a chemical probe, we have revealed and validated the mechanisms and binding of our lead compound, YQ128, to NLRP3. YQ128 is able to inhibit caspase‐1 and GSDMD cleavage as well as ASC speck formation in mouse macrophages; however, it has no effect on the NLRP3 expression, suggesting its target is upstream of ASC speck and caspase‐1 but downstream of priming. Through target identification studies using a photoaffinity labeling (PAL) technique, we found that YQ128 specifically interacts with NLRP3, but not other components of the NLRP3 inflammasome complex. Competition studies with the parent compound and other known NLRP3 inhibitors suggested the specific and unique binding of YQ128 to NLRP3. The results from other biological assays proposed its binding to the NACHT domain of NLRP3. Using our chemical probe in a pull‐down assay, we also discovered other potential targets within the inflammasome pathway, including a protein that was recently termed “undruggable”. These results suggest a novel mechanism of inhibition by our compounds and further studies are currently underway to improve these inhibitors as potential therapeutics for neuroinflammation in neurodegenerative diseases.