bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural studies reveal unique features of nsp16 from SARS‐CoV‐2, a protein essential for immune system evasion and a possible drug target
Authors: Rosas‐Lemus Monica , Minasov George , Shuvalova Ludmilla , Brunzelle Joseph , Satchell Karla
Published: May 2022

RNA viruses have several mechanisms to modify the viral RNA genome to protect from host immune surveillance. SARS‐CoV‐2, the etiological agent of COVID‐19, and other coronaviruses encode capping proteins in its genome that modify the 5’ untranslated region of the viral genome and mRNA. Capping viral RNAs promotes translation, prevents degradation, and reduces the activation of host immune responses. This process is initiated by the viral proteins nsp13 and nsp12, which cleave the 5’ phosphate group and transfer a GMP from GTP to the 5′ end of the nascent (+)ssRNA. The nsp14‐nsp10 complex then catalyzes the transfer of a methyl group from S‐adenosylmethionine (SAM) to the N7 position of the GMP cap, generating Cap‐0. Finally, the nsp16‐nsp10 complex utilizes SAM to catalyze the 2′‐O‐methylation of the first ribonucleotide, generating Cap‐1. Several structures of the nsp16‐nsp10 complex have been solved previously for SARS‐CoV, MERS and SARS‐CoV‐2 with SAM, Cap‐0 and Cap‐0‐RNA bound to the complex as well as the products of the reactions. Recently, we determined the first structure of nsp16‐nsp10 with Cap‐0‐RNA and Cap‐1 RNA, revealing that Mn2+ coordinates the first four nucleotides of the bound RNA to orient the 2’‐OH of the ribose of the first nucleotide toward the methyl group of SAM for catalysis. Herein, we also show the first apo‐crystal structure of nsp16 determined in which neither SAM nor RNA substrates bound. This new structure and comparison to structures with substraces and products bound, will reveal critical movements of the enzyme for binding substates. This study shows an important set of structures of the viral 2’‐O‐methyltransferases revealing unique features of this complex that could be used for molecular dynamics studies and designing coronavirus‐specific inhibitors.
Utilizing Isothermal Titration Calorimetry to Measure Enzyme Activity in Dairy Fluids
Authors: Jarrard Tyler , Kenealey Jason
Published: May 2022

β‐galactosidase activity can be directly measured by Isothermal Titration Calorimetry (ITC) in complex opaque solutions such as milk and whey. As many existing techniques of food analysis rely on colorimetry, ITC offers a robust method for measuring reactions directly in food matrices. The specific aim of this research is to find high‐activity β‐galactosidases for use in sweet or acid whey permeates. To measure the reaction in the ITC, 4uL β‐galactosidase solution was injected into milk, whey, and whey permeate at 60°C for β‐galactosidase fromAspergillus oryzae and 40°C for β‐galactosidase fromKluyveromyces lactis. β‐galactosidase from Kluyveromyces lactis and for Aspergillus oryzae activity were tested at enzyme concentrations of 444.4, 888.9, and 1333.3 ug/mL with 500mM‐lactose concentration added to each dairy solution. In addition,1111.1 ug/mL β‐galactosidase was added to the dairy solutions with an additional 100, 300, and 500mM lactose. The addition of the β‐galactosidase to the dairy solution induced an exothermic reaction that was dependent on the β‐galactosidase concentration. The β‐galactosidase from Kluyveromyces lactis demonstrated no enzyme activity under the acidic conditions of acid whey and acid whey permeate. β‐galactosidase from Aspergillus oryzae did have activity under acidic conditions and had activity in more neutral solutions. The β‐galactosidase from Aspergillus oryzae appeared to go to completion much faster than the β‐galactosidase from Kluyveromyces lactis. The data presented here demonstrate that ITC is an effective tool capable of directly measuring enzyme activity in dairy fluids and distinguishing the unique features catalytic action of enzymes derived from different species.
Correlation between Intermittent Fasting and Mental and Physical Fatigue in High‐fat‐induced Obese and Non‐obese Rats
Authors: Medavaka Sai , Niepoetter Paige , Butts‐Wilmsmeyer Carolyn , Gopalan Chaya
Published: May 2022

Intermittent fasting (IF) is an alternate pattern of eating and restricting food intake. This study evaluated mental and physical fatigue secondary to IF in high‐fat diet‐induced (HFD) obese male rats. Fifty‐four rats were randomly assigned to a HFD (n=28) or a standard diet (SD; n=26). After six weeks, the HFD rats were divided into one of four groups: obese HFD ad libitum (OB‐HFD‐AL), obese HFD intermittent fasting (OB‐HFD‐IF), obese SD ad libitium (OB‐SD‐AL), and obese SD IF (OB‐SD‐IF). Similarly, non‐obese controls were grouped into HFD ad libitum (C‐HFD‐AL), non‐obese HFD IF (C‐HFD‐IF), non‐obese SD ad libitum (C‐SD‐AL), and non‐obese SD IF (C‐SD‐IF). The IF groups fasted for 18 hours per day, seven days a week. After two weeks, their physical and mental fatigue was measured using open field (OF) and novel object recognition (NOR) paradigms. Regardless of the diet type, rats on IF gained weight at a slower pace (p< 0.05) and had lower glucose levels (p < 0.01) compared to the AL group. In non‐obese rats, ketone levels were higher in the IF‐HFD group compared to the IF‐SD (p<0.05) and AL‐SD (p<0.01) groups. Obese rats exhibited higher ketones in IF‐SD conditions versus AL‐SD rats (p<0.01). In both obese and non‐obese groups, AL‐HFD rats had higher ketone levels than AL‐SD rats (p<0.05). Increased ketone levels could be correlated with greater distance traveled during OF. IF protected against recognition memory deficit during NOR. Overall, this study demonstrates that increased ketone levels, whether caused by diet type or IF, correlate to decreased physical fatigue and protect against mental fatigue.
FXYD3 increases Na+ transport across human airway epithelia
Authors: Cano Portillo Camilo , Villacreses Raul , Thurman Andrew , Pezzulo Alejandro , Zabner Joseph , Thornell Ian M.
Published: May 2022

The Na/K ATPase localizes within the basolateral membrane of most epithelia, where Na/K ATPase activity mediates Na+ absorption. The Na/K ATPase requires its alpha subunit for ion transport and its beta subunit for proper trafficking to the plasma membrane. The non‐essential gamma subunit of the Na/K ATPase modifies Na/K ATPase activity. Datasets within the NIH Gene Expression Omnibus indicate that FXYD3, a Na/K ATPase gamma subunit, is highly expressed by human airway epithelia. However, how FXYD3 affects airway epithelial function remains unstudied. In addition to binding the Na/K ATPase, FXYD3 can bind and modify H/K ATPases in heterologous expression systems. The H/K ATPase ATP12A acidifies human airway surface liquid. Therefore, we evaluated the role of FXYD3 in both acid secretion and Na+ absorption across human airway epithelia. Single cell RNA sequencing revealed high FXYD3 expression for all the airway cell types, and immunocytochemistry revealed FXYD3 expression within the basolateral membrane of airway epithelia. Consistent with basolateral FXYD3 localization, the airway surface liquid pH, established in part by apical H/K ATPase, was unaffected by siRNA‐mediated knockdown of FXYD3. FXYD3 knockdown decreased amiloride‐sensitive short‐circuit currents (~20% in NaCl and ~40% in NaGluconate solutions), a finding that is consistent with FXYD3 increasing Na/K ATPase activity. Consequently, fluid absorption across airway epithelia was reduced by ~20% with FXYD3 knockdown. FXYD3 may be required to efficiently return the airway surface liquid to homeostatic volumes after fluid secretion into the proximal airways.
Lymphangion‐Chip Experimental Platform Supports Co‐culture and Signaling Between Lymphatic Endothelial and Mural Cells
Authors: Selahi Amirali , Jain Abhishek
Published: May 2022

The pathophysiology of several lymphatic diseases, such as lymphedema, depends on the function of lymphangions that drive lymph flow. Even though the signaling between two main cellular components of a lymphangion, Lymphatic Endothelial Cells (LECs) and Lymphatic Muscle Cells (LMCs), is responsible for crucial lymphatic functions, there are no in vitro models that have included both cell types in a physiologically relevant environment. Here, a fabrication technique (Gravitational Lumen Patterning or GLP) is developed to create Lymphangion‐Chip. This organ‐on‐chip consists of co‐culture of a monolayer of endothelial lumen surrounded by multiple and uniformly thick layers of muscle cells. The platform allows construction of a wide range of luminal diameters and muscular layer thicknesses, thus providing a toolbox to create variable anatomy. In this device, lymphatic muscle cells align circumferentially while endothelial cells aligned axially under flow, as only observed in vivo in the past. This model further demonstrated a robust sensitivity of this relative alignment of the two cells with respect to the presence or absence of co‐culture and mechanical forces (shear stress), thus suggesting that LECs and LMCs are biologically and functionally active within the chip. This system successfully characterizes the dynamics of cell size, density, growth, alignment, and intercellular gap due to co‐culture and shear. The time‐dependent decrease in the subendothelial gap strongly suggests proactive LEC‐LMC signaling as LECs and LMCs grow and proliferate within the device. Further exposure to pro‐inflammatory cytokines reveals that the device could produce the regulation of endothelial barrier function through the lymphatic muscle cells. Finally, measurement of intracellular calcium of LECs and LMCs while exposed to step and oscillatory flow profiles with various shear amplitudes revealed the bidirectional mechanism of calcium signaling within lymphatic vessel. Therefore, this organ‐chip technology allows researchers to include essential lymphatic vascular components in a tunable 3D microphysiological system that is suitable for use in preclinical research of lymphatic and blood mechanobiology, inflammation, and translational outcomes.
Emerging Microbiome Genetic Engineering Technology for Stable Levodopa Delivery in Parkinson’s Disease
Authors: Padhi Piyush , Abdalla Ahmed , Backes Nick , Zenitsky Gary , Jin Huajun , Anantharam Vellareddy , Kanthasamy Arthi , Phillips Gregory , Kanthasamy Anumantha
Published: May 2022

Dopamine replacement in the form of a levodopa/carbidopa tablet has been the gold‐standard therapy for Parkinson’s disease (PD) for over six decades. Oral tablet dosing of L‐DOPA 3‐4 times/day is quite efficacious in controlling early‐stage PD symptoms). However, beyond its ~5‐yr clinical honeymoon period, this treatment becomes increasingly associated with L‐DOPA‐induced dyskinesia (LID) and ‘ON‐OFF’ motor complications resulting from the non‐continuous, pulsatile delivery of L‐DOPA to the brain. Addressing this challenge, we devised an innovative strategy to supplement the host’s microbiota with the human probiotic E.coliNissle 1917 (EcN) genetically engineered to continuously produce L‐DOPA (EcNLDOPA) with no significant side effects. By harnessing emerging synthetic biology and genetic engineering technologies, we successfully established four generations of EcNLDOPA(1‐4) and characterized their ability to endogenously synthesize L‐DOPA from tyrosine using the recombinant hpaB/C gene. The initially designed plasmid‐based EcNLDOPA1,2 systems were further improved by developing the chromosome‐integrated and regulatable systems EcN3‐4L‐DOPA using advanced Sce‐ROPE genetic engineering. The steady‐state levels of plasma and brain L‐DOPA, dopamine and its metabolites, as well as gut colonization of EcNLDOPA, were assessed over time. Our results show that all EcNLDOPAstrains effectively generate L‐DOPA in a dose‐ and time‐dependent manner in vitro, indicating that the biotherapeutic dosage can be personalized to each patient’s needs. Our mouse pharmacokinetic studies revealed that steady‐state levels of plasma L‐DOPA and brain dopamine can be achieved by oral administration of EcNLDOPA. Importantly, our pharmacodynamic efficacy studies of the L‐DOPA response in the progressive neurodegenerative MitoPark mouse model of PD showed improved performance in motor, cognitive and mood‐related tasks. Colonization studies show that our EcNLDOPA biotherapeutic requires only once or twice daily dosing. Our novel re‐engineered, self‐replicating EcNLDOPA microbial bio‐therapeutics offers an entirely new L‐DOPA treatment paradigm capable of ensuring long‐term efficacy while minimizing side‐effects
Proteasome Shuttle Factors Regulate the Relocalization of Proteasomes to Cytosolic Granules upon Specific Stress Conditions
Authors: Roelofs Jeroen , Vontz Gabrielle , Waite Kenrick A. , Lee Stella Y.
Published: May 2022

Proteasomes are essential complexes within cells responsible for the degradation of many short‐lived and aberrant proteins. These substrates are marked for degradation by post‐translational modifications with ubiquitin. This ubiquitination enables substrate recognition by three receptors that are intrinsic subunits of the proteasome. Alternatively, shuttle factors (Rad23, Dsk2, Ddi1 in yeast; a family of orthologs in humans) act as extrinsic receptors and can deliver ubiquitinated substrates to proteasomes. As the human orthologues of Dsk2, Ubiquilin 1 and 2, have been shown to contribute to autophagic processes, we hypothesized that the shuttle factors contribute to the autophagy of proteasomes in yeast. In proliferating cells, proteasomes are largely nuclear; however, upon nitrogen starvation proteasomes exit the nucleus and are degraded in the vacuole through an autophagy pathway known as proteaphagy. While our biochemical and microscopic analyses indicated that shuttle factors are not required for proteaphagy induced by either nitrogen starvation or proteasome inhibition, we discovered an unexpected role for the shuttle factors in regulating proteasome localization under other stress conditions. Yeast grown to stationary phase or exposed to acute glucose starvation respond by sequestering proteasomes into cytoplasmic granules termed proteasome storage granules (PSGs). These granules behave like liquid‐liquid phase separated structures and they appear to protect proteasomes from autophagic degradation. We recently identified a new condition that induces PSG formation, namely mitochondrial inhibition (e.g. using sodium azide). Rad23 and Dsk2 co‐localized with PSGs and were important for their formation upon mitochondrial inhibition. Under these conditions, deletion of both genes almost completely prevented granule formation, while single knockouts displayed a partial defect. Surprisingly, neither protein is required for proteasome re‐localization to PSGs upon abrupt glucose starvation. Further distinguishing these shuttle factor‐dependent granules is their dependance on active protein translation, as cycloheximide treatment prevented their formation, while having no effect on other granule inducing conditions. Our data show granules with unique properties and requirements form depending on the cellular conditions. Finally, we show that the third shuttle factor, Ddi1, has an opposing role in regulating PSGs. The deletion of Ddi1 induced proteasome localization to granules strikingly more quickly compared to wild type cells. We propose a model where the shuttle factors Dsk2 and Rad23 play a key role in facilitating the liquid‐liquid phase separation of proteasomes.
Investigating the Role of Corticostriatal Glutamate Signaling in Methamphetamine Reward and Reinforcement
Authors: Brown Chelsea , Beltran Jackie , Fultz Elissa , Tran Tori , Barger Brooke , Williams Nyssa , Yen Waylon , Park Asher , Kippin Tod , Szumlinski Karen
Published: May 2022

Highly addictive amphetamine‐type stimulants, including methamphetamine (MA), pose significant health and socioeconomic issues, with an estimated 27 million users worldwide. Understanding the neurobiological changes underlying various stages of MA abuse is key to developing safe and effective therapies. While the majority of addiction research has implicated the dopamine system, the long‐term neuroplasticity that maintains MA Use Disorder also involves drug‐induced changes in glutamate signaling in reward regions of the brain like the nucleus accumbens (NAC) and prefrontal cortex (PFC). In order to further characterize the role of glutamate in MA reward and reinforcement, glutamate receptor scaffolding protein Homer2 expression in the NAC and activity in the glutamatergic projection PFC‐NAC were manipulated prior to place‐ and operant‐conditioning procedures. Together the results from these studies argue that while research of the functional neuroanatomy of addiction tends to focus on traditional dichotomies between the NAC core and shell or PFC prelimbic and infralimbic sub‐regions, the role of these circuits in drug abuse is more complex than the narrow scope of our experiments were able to probe. Other inputs upstream from our regions of interest may be able to compensate for our manipulations via neurobiological redundancy. This would suggest that as research technologies advance, targeting functional networks will be crucial for elucidating the neurobiological underpinnings of addiction and discovering potential therapeutics.
Developing an In Vitro Platform to Mimic the Pancreas‐Liver Endocrine Axis
Authors: Whitticar Nicholas , Nunemaker Craig
Published: May 2022

Insulin is secreted from pancreatic islets in a pulsatile manner due to alternating periods of cellular activity and rest. The resulting pulses of insulin are critical for proper hormonal signaling and blood glucose regulation by the liver. To recreate the interaction between the endocrine pancreas and the liver using primary murine tissue, we have designed 3D printed microfluidic platforms that hold hepatocytes downstream of islets. Islets no longer oscillate in sync once isolated from the pancreas, so the cumulative insulin released is non‐pulsatile as demonstrated by taking the average intracellular calcium levels of multiple islets (Figure A, top). To force islets to secrete synchronized insulin pulses in vitro, we use programable syringe pumps that flow alternating high (11mM) and low (3mM) glucose solutions over isolated mouse islets. The syringe pump system aligns the oscillations which results in synchronized insulin release (Figure A, bottom). Next, we exposed primary murine hepatocytes to either 100nM pulses of insulin in three‐minute periods or constant 50nM insulin in a microfluidic system (Figure B). Both groups were exposed to the same amount of insulin, just in a different pattern. Hepatocytes treated with pulsatile insulin for 4 hours contained significantly more glycogen compared to those treated with continuous 50nM insulin (P<0.05, N=4). Future experiments will add hepatocytes downstream of the islets on the same platform to investigate the effect of pulsatile insulin and glucagon secretion on liver function. Exploring the interaction between pancreatic islets and hepatocytes will lead to a deeper understanding of the loss of pulsatile hormone secretion during the progression of type 2 diabetes.
Optogenetic stimulation of PICo neurons and the respiratory‐sympathetic coupling in in vivoCIH and control mice
Authors: Amarante Marlusa K. , Huff Alyssa , Ramirez Jan M.
Published: May 2022

The resting respiratory activity is generated by synchronized activity of a complex located within the brainstem. This contains a primary oscillator essential for the respiratory rhythmicity and is composed of pacemaker inspiratory neurons. However, recent studies suggest the postinspiratory complex (PiCo) is a novel respiratory oscillator which is critical for the postinspiratory activity in vivo and contains cholinergic and glutamatergic neurons. In addition to the generation of the respiration, studies suggest that the respiratory neurons also interact with the presympathetic neurons involved with the generation of sympathetic activity, contributing to the excitation of sympathetic activity in chronic intermittent hypoxia (CIH) conditions and promoting changes inthe respiratory‐sympathetic coupling. To investigate the contribution of PiCo in the respiratory‐sympathetic coupling, we used an optogenetic in vivo mouse preparation previously exposed to CIH for 21 days (5% O2 – 80 bouts/day). In the end of the protocol, the ventral surface of the brainstem in spontaneously breathing, anaesthetized (Urethane 1.5mg/kg) ChATCre/Ai32 adult and Vglut2cre/Ai32 mice was exposed, and an optical fiber positioned bilaterally over PiCo, while recording the diaphragm muscle (DIA), hypoglossal (HN), cervical vagus (cVN) and cervical sympathetic nerves (cSN). Our data showed that CIH protocol increased the respiratory frequency, post‐I vagus activity and sympathetic activity in the baseline conditions. Random light stimulation (10s) of PiCo ChAT neurons (~ 9mV, 447nm) in significantly increased the cSN discharge during postinspiration in control group. Although the sympathetic activity was increased during all respiratory phases in CIH mice, the stimulation of cholinergic neurons at PiCo did not change the correlation between post‐I and E2 phase. The stimulation of Vglut2 neurons at PiCo area evoked post‐inspiratory discharge of cSN in both groups. Our data demonstrates that during resting conditions, mice exposed to CIH for 3 weeks increased the baseline respiratory frequency and sympathetic activity in vivo and the optogenetic stimulation of PiCo neurons blunts the phasic respiratory –sympathetic coupling in CIH mice, increasing the post‐inspiratory discharge of the sympathetic nerve.
Novel approach to analyzing steady‐state intracellular pH and the recovery from NH4+ ‐induced acidosis in rat hippocampal neurons and astrocytes
Authors: Ruffin Vernon A. , Boron Walter
Published: May 2022

Optimal function in the brain, especially in hippocampus—an area involved in learning and memory—requires tight regulation of intracellular pH (pHi) within neurons and neuroglial. The Na‐H exchangers (NHEs) are the major family of acid/base proteins involved in regulating pHi in the absence of CO2/HCO3. In the present study, we used the pH‐sensitive dye BCECF to examine the regulation of steady‐state pHi and the recovery of pHi from NH4+ ‐induced intracellular acid loads in HC neurons and astrocytes, co‐cultured from embryonic (E18‐20) Sprague Dawley rats, and studied in CO2/HCO3 −‐free HEPES buffered (“HEPES”) solutions. After at least 14‐days in a CO2/HCO3 – incubator, cells were removed, loaded with BCECF, and placed in a recording chamber with flowing HEPES. At the beginning of each experiment, we measured pHi (checkpoint A) after allowing pHi to stabilize for 5 minutes (checkpoint C), and reported mean “initial pHi”/SEM for neurons as 7.351/0.0597; N=37 (astrocytes: 7.189/0.0118, N=25) the value at checkpoint C = (pHi)C. After using the twin paired NH4+ ‐pulse protocol to acid load cells, we find that—after the pHi recovery from the first acid load—the average neuronal steady‐state pHi (now at checkpoint E; (pHi)E) is 6.953/0.0601(astrocytes: 7.037/0.0081). After the second NH4+ pulse the neuronal steady‐state pHi (now at checkpoint F; (pHi)F) in neurons is 6.937/0.010 (astrocytes: 7.020/0.0062). The recovery from acidosis is fit with a double exponential (DExp) which we replot as dpHi/dt vs pHi. With this traditional approach, dpHi/dt, the fit as it approaches the asymptotic pHi, becomes slightly non‐linear. To exploit the mainly linearity portion of the dpHi/dt vs. pHi plot (from the DExp fit) of the double exponential, we fit these dpHi/dt vs. pHi points with a DExp with a quasi‐ single exponential (SExp) to produce a quasi–single‐exponential rate constant (kqSExp) measured as dpH/dt. This analysis—when transformed to the pHi vs. time domain—generally produces a very good fit to the original pHi vs. time data. The mean kqSExp1 in neurons is 0.0054/ 0.0008 (astrocytes: 0.0107/0.0002) whereas the mean kqSExp2 in neurons is 0.0055/0.0008 (astrocytes: 0.0010/0.0003). We summarize the twin pHi recoveries from individual experiments in which we display as thumbnails the quasi–single‐exponential dpHi/dt line segments that represent the pHi recoveries from the first and second NH3/NH4+ pulses. These new analytical approaches may ultimately provide mechanistic insight into cell‐to‐cell heterogeneity of pHi regulation in the nervous system.
Thyroid Supplementation Reverses the Abnormal Social and Oxytocin Phenotype Produced by Developmental Exposure to PBDEs in a Sex‐Dependent Manner
Authors: Kozlova Elena V. , De Angelis Meri , Denys Maximillian E. , Bishay Anthony E. , Campoy Luis , Schramm Karl‐Werner , Curras‐Collazo Margarita C.
Published: May 2022

Polybrominated diphenyl ethers (PBDEs) are endocrine‐disrupting chemicals (EDCs) that are widely used flame retardants added to common household products. In humans, PBDEs are associated with neurodevelopmental deficits including ones associated with social competence and learning. We have previously shown that PBDE‐exposed F1 female progeny display autism‐relevant characteristics such as deficient social novelty preference and social recognition memory (SRM), exaggerated repetitive behavior and deficient social odor discrimination (Kozlova et al 2021). In this study, we show that developmental exposure (via dam GD0‐PND21) to the PBDE mixture, DE‐71 (L‐DE‐71; 0.1 mg/kg/d), reduces oxytocin immunofluorescence density in the paraventricular nucleus of the hypothalamus (PVN) that is critical for social recognition memory in male and female F1 offspring relative to VEH/CON (0 mg/kg/d). One of the well characterized actions of PBDEs is disruption of the hypothalamo‐pituitary‐thyroid axis (HPT). Thyroid hormones (TH) are critical for nervous system development and regulate the transcription of oxytocin. We show for the first time, that L‐DE‐71 alters central thyroid hormone species (T2, rT3, T3, and T4) relative to VEH/CON in F1 offspring but with a different temporal pattern in males (PND30) and females (PND15) as measured using liquid chromatography‐tandem mass spectrometry. This coincides with reduced plasma T4 in PND15 male but not female offspring. Supplementation of pregnant dams with levothyroxine, a synthetic analogue of thyroxine (T4), during gestation and lactation (GD12‐PND21), increased plasma oxytocin in exposed dams. More importantly, supplementation normalized PBDE‐induced deficiency in social recognition memory test, and PVN oxytocin levels in exposed female but not male offspring. These changes are specific to social behavior as were no changes in passive avoidance memory. Interestingly, L‐DE‐71 female but not male F1 also receiving supplementation showed a significant increase in plasma T4. Maternal behavior (retrieval of PND4 pups) was significantly reduced by supplementation with 6‐propyl‐2‐thiouracil, a drug that blocks the production of thyroid hormone by thyroid gland, but not by DE‐71 nor DE‐71 plus levothyroxine supplementation. These results provide novel insight into neuroendocrine disruption of the central oxytocin system by maternal transfer of environmental pollutants and its possible contribution to neurodevelopmental disorders such as autism.
Association of Pre‐Living Kidney Donation Body Mass Index with Post‐Donation Estimated Glomerular Filtration Rate Decline
Authors: Tantisattamo Ekamol , Tanariyakul Manasawee , Polpichai Natchaya , Mutirangura Pornthira
Published: May 2022

Introduction

Obesity is one of the risk factors for chronic kidney disease (CKD). Whether body mass index (BMI) at pre‐living kidney donation is associated with worsening kidney function of the remaining kidney after donation is unclear. We aim to examine the association between pre‐donation BMI and change in post‐donation estimated glomerular filtration rate (eGFR) in living kidney donors.

Methods

A single‐center retrospective cohort study included 14 living kidney donors. Associations between pre‐donation BMI and percentage of eGFR decline at 2 and 7 days post‐donation were examined by multiple linear regression.

Results

Of all 14 living kidney donors, mean age±SD was 52.64±13.46 years old and 50% was female. Mean BMI was 26.90±2.66 kg/m2. The majority were overweight (72%) followed by normal weight (21%) and obese (7%). eGFR at 2‐ and 7‐day post‐donation were significantly lower than pre‐donation eGFR (55.59±12.48 vs. 55.29±11.33 vs. 93.39±14.62, p <0.001; Figure 1). Every 1 kg/m2 increase in pre‐donation BMI was associated with 0.1% greater decline in eGFR at 2‐day post‐donation, while eGFR decline was up to 1.4% at 7‐day post‐donation (β2day±SE 0.149±0.716, p 0.861, 95%CI ‐1.668, 1.966; Figure 2A and β7day ±SE 1.368±0.834, p 0.080, 95%CI ‐0.191, 2.928; Figure 2B). After adjusted for age, gender, ethnicity, pre‐donation eGFR, and interaction term between age and pre‐donation eGFR, the magnitude of eGFR decline post‐donation were greater for both 2‐ and 7‐day post‐donation; however, only the 7‐day post‐donation eGFR decline was statistically significant without the interaction between age and pre‐donation eGFR (β2day±SE 0.653±0.977, p 0.525, 95%CI ‐1.657, 2.963, pinteraction 0.045 and β7day±SE 2.068±0.800, p 0.036, 95%CI 0.176, 3.959, pinteraction 0.541).

Conclusions

Higher pre‐donation BMI is associated with greater eGFR decline at 7‐day, but not 2‐day, post‐donation. Pathological glomerular hyperfiltration from overweight or obesity may counteract with physiological glomerular hyperfiltration from acquired solitary kidney post‐donation leading to worsening post‐donation eGFR decline.
Long noncoding RNAs in regulation of inflammation, immune response, and glucose metabolism
Authors: Mandal Subhrangsu S. , Obaid Monira , Chini Avisankar , Guha Prarthana
Published: May 2022

Noncoding RNAs (ncRNAs) are emerging players in cell signaling and metabolism. Studies from our laboratory demonstrate that long noncoding RNA (lncRNA) HOTAIR plays critical roles in inflammation via regulation of NF‐kB activation and cytokine expression. HOTAIR also regulates glucose metabolism in macrophages during inflammation. Importantly, metabolic reprogramming is critically linked to inflammation and macrophage activation. Therefore, our findings showing the HOTAIR mediated regulation of glucose metabolism in macrophages indicate potential roles of lncRNAs in macrophage activation and metabolic reprogramming. In a recent study, we explored potential involvement of other ncRNAs in inflammation and immune signaling. Based on independent RNAseq analyses in mouse and human macrophages, we have discovered a series novel mouse and human lncRNAs which are potential regulators of cytokine expression, inflammation, and immune response. Notably, there appear to be poor conservation among the lncRNAs between mouse and human. Mechanistic studies demonstrated that some of the novel inflammatory lncRNAs are regulated via NF‐kB pathways while others are not, suggesting distinct modes of action of different lncRNAs during inflammatory response in macrophages. Here, we will present the discovery of novel mouse and human lncRNAs that are potential regulators of inflammation and immune signaling and discuss functional roles of selected lncRNAs in cytokine regulation, inflammation, and glucose metabolism in macrophages.
The trepidatious return to in‐person instruction during the COVID‐19 pandemic: valuable lessons applied from online teaching using Lt in the face‐to‐face classroom
Authors: Ford Bridget M.
Published: May 2022

The speed at which educators have embraced new technologies during the COVID‐19 pandemic has been remarkable. This pivot to virtual instruction has been particularly difficult in courses where hands‐on experiences are the norm, such as in anatomy and physiology laboratory courses. Now with the transition back to mostly in‐person instruction, the anatomy and physiology lab environment has faced a new set of challenges where the want to return to a strictly hands‐on experience is being met with the need to still maintain flexibility and accessibility for students placed in quarantine. The anatomy and physiology lab curriculum at the University of the Incarnate Word (UIW) has adapted to this varied set of needs by adopting the use of the web‐based laboratory software platform, Lt. Students surveyed during strictly virtual semesters reported a high level of satisfaction with the Lt lab software. Ratings regarding ease of use, support of learning, and overall ratings increased each semester with the highest ratings occurring during the most recent Fall 2021 semester where instruction was mostly in‐person and hands‐on. This helps to support the widely held belief that students perform best, and that instruction is more effective with face‐to‐face laboratory courses. With our continued use of Lt, we have found an effective combination of virtual and in‐person instruction that best fits our course outcomes while still supporting the flexibility and accessibility our students require.
Regulation of Endothelial Cell function by Integration of Gαi and β‐arrestin signaling at Atypical Chemokine Receptor 3
Authors: Lee Claudia , Kohlmann Taylor , Xiong Xinyu , Rajagopal Sudarshan
Published: May 2022

Atypical Chemokine receptor 3 (ACKR3), also known as C‐X‐C chemokine receptor type 7 (CXCR7), is a G protein‐coupled receptor (GPCR) implicated in several physiological processes including leukocyte trafficking, cancer, and angiogenesis. Commonly co‐expressed with C‐X‐C chemokine receptor type 4 (CXCR4), ACKR3 is classified as an β‐arrestin‐biased “scavenger” receptor due to its lack of G‐protein signaling and its ability to efficiently internalize and degrade its ligands, CXCL11 and CXCL12. However, much is still unknown regarding ACKR3 signaling mechanisms and its implications on cellular physiology. Here we describe ongoing studies to characterize the mechanisms that underlie ACKR3 signaling and endothelial cell function. We demonstrated two ACKR3 specific agonists, WW36 and WW38, promote proliferation and migration of human umbilical vein endothelial cells (HUVECs). Surprisingly, we found that ACKR3‐promoted proliferation and migration was sensitive to pertussis toxin (PTX), suggesting the involvement of Gαi/o proteins at a receptor which does not canonically signal via G proteins. We also observed similar sensitivity to PTX on ERK activation in HUVECs. We hypothesized that these results may in part be explained by the recently described coordination of Gαi and β‐arrestin (βarr) to promote Gαi:βarr complex‐specific downstream signaling. In support of this hypothesis, we demonstrated the formation of a Gαi:βarr complex upon ACKR3 activation in a dose‐dependent manner. To characterize the necessary components in Gαi and βarr, we first developed active proximal polar core mutant and proximal finger loop βarr mutant and tested these at the prototypical class B Vasopressin 2 Receptor. Both mutants lead to considerable decrease in complex formation with the finger loop mutation nearly abolishing our split signal. Within the Gαi subfamily, we observed considerable differences in the ability to form complex, with Gαi2 and Gαi3 promoting less complex formation than Gαi1 and Gαo. We developed several Gαi mutants, hypothesizing that residues that differ between Gαi isoforms may impact Gαi:βarr formation. Using NanoBRET we demonstrated that these mutants impair complex formation, suggesting that they may be involved either via direct contacts with βarr or impacting Gαi conformation such that complex formation is no longer as favorable. These studies reveal a novel role for Gαi:βarr complexes in mediating ACKR3 function that contribute to endothelial cell biology. Additionally, these findings support an emerging paradigm in which G proteins and βarr coordinate their signaling downstream of GPCRs.
Role of Digital Media in Enhancing Anatomical Education: A Survey Study Focused on the Cranial Cavity and Its Contents
Authors: Mashayekhi Mohammadsadegh , Laghaei Farimani Pedram , Kassam Farris , Doroudi Majid
Published: May 2022

Introduction

Recently, there has been an increasing effort to modify traditional learning tools to adopt them with the new demands of students by producing visual learning tools. These efforts have aimed for an equal access to high quality learning opportunities to fulfill the increasing interest in self‐paced flexible studying using digital medial technology. The emergence of COVID pandemics has highlighted these demands for these educational resources. Human gross anatomy education is one of the fields that has been impacted by recent critical situation more dramatically. Thus, it is crucial to create more educational videos in gross human anatomy and study the effects of such videos on anatomy education.

Objectives

This project aims for understanding the role of self‐paced educational videos in education of human body anatomy with a focus on cranial cavity and its contents.

Method

A video exploring the cranial cavity and its contents for learners in professional programs was filmed and edited by Camtasia 2020 software. Different features such as labels, transitions, schematic pictures, and quiz questions were added in the editing process to enhance the students’ experience. The edited video was uploaded on YouTube and shared with students taking human gross anatomy courses. The viewers were asked to complete an online survey that was designed to assess their learning experience using the video.

Link to video: https://www.youtube.com/watch?v=ROGgR7mxsr8&t=107s

Results

The video received 2429 views, 92 likes and 29 responses to the survey from Jan 2021 to Dec 2021. Participants watched the video from different countries such as Canada, US, India, and Brazil. Analysis of the data shows that the majority of the participants who completed the online survey found the video and its features enhancing their learning of the anatomy of the human body. More specifically, 82.8% of students selected “to prepare for my anatomy lab” as their main purpose for viewing the video and 93.1% choose “Strongly Agree” when asked if the videos helped them fulfill their purpose.

Moreover, on the 1‐5 scale, the average score of 4.59, 4.66, 4.41, and 4.86 were given by students for the quality of labelling, transitions, quiz questions, and overall quality of the video, respectively.

Conclusion

In conclusion, the feedback received from this survey strongly suggests that using digital media is beneficial for enhancing the anatomical learning experience of students.
The use of wet lab and computational docking experiments to elucidate idiosyncrasies associated with amino acid activation
Authors: Faustino Kalii , Iverson Douglas , Broome Payson , Dela Rosa Mylie , Mobisa Japheth , Jordan Timothy , Doppalapudi Dhathry , Mohamed Amran , Samuel Kyra , Okeke Idiuso , Ghebreigziabher Daniel , Serrano Crystal , Bell Anthony J.
Published: May 2022

Aminoacyl‐tRNA synthetases (aaRSs) are a well‐known class of enzymes that act as sentinels of genome. During protein translation, aaRSs ensure that L‐amino acids are charged or acylated onto their cognate tRNA isoacceptor(s). Hence, aaRSs help ensure that ribosomally translated proteins contain the proper sequence of amino acids. aaRS catalyze tRNA charging in a two‐step process. In step one, aaRSs selectively form a high energy amino acid adenylate. This step, refereed to amino acid activation, is the first level of selectivity in protein translation. Here, aaRSs effectively discriminate non‐cognate L‐amino acids. In step two, aaRSs transfer adenylated amino acids onto the cognate tRNA isoacceptor. This step is referred to as aminoacylation or tRNA charging. A great deal is known about aaRS reaction mechanisms but a number of confounding idiosyncrasies remain. The focus of this work aims to elucidate idiosyncrasies associated with amino acid activation. Here, a 32P‐based assay is used to measure the level to which E. coliaaRSs activate non‐cognate L‐amino acids. The purpose of these assays is to measure the plasticity of aaRS active sites to define additional underlying binding interactions that modulate amino acid recognition. This information could also be used to develop small molecule inhibitors of aaRS. Moreover, these data could reveal underlying clues to the potential origins of aaRSs. Here, three aaRSs are investigated: ArgRS, IleRS and LysRS. The activation data shows that each aaRS perhaps somewhat surprisingly activates more than five non‐cognate AAs to varying levels. Next, computer docking experiments are used to visualize the binding interaction of synthetase active site residues to each non‐cognate amino acid. These data are currently being used to construct protein‐ligand interaction maps of each aaRS active site. The resulting interactions maps can be used to more clearly elucidate idiosyncrasies associated with amino activation.
Effect of Protease Activated Receptor‐2 antagonist in the transition from endometrium to endometriosis and ovarian cancer
Authors: Ocasio‐Rivera Marinel , Rosado Solimar , Velez Jaleisha , del Mar Mendez Maria , Flores Idaliz , Suarez Edu
Published: May 2022

Endogenous proteases are associated with the ovulation process and inflammation, among other human physiological processes. Still their role in the transition from normal tissue to cancer development remains uncertain. For example, the Protease activated receptor 2 (PAR‐2) is a G Protein Couple receptor expressed in mostly in epithelial cells, including the female reproductive system. Previous work from our lab showed that the PAR‐2 antagonist‐FSLLRY‐NH2 inhibited the cell‐mediated inflammation in the airways. The aim of this study is to evaluate the effect of the PAR‐2 antagonist when a normal cell progresses to a cancerous one, using trypsin as a gold standard enzyme for Par‐2 activation. In this case, the transition from normal endometrium, to endometriosis, ovarian. We hypothesized that PAR‐2 antagonist will decrease the activation of its receptor reducing the transition to endometriosis and ovarian cancer. To test this, we performed western blot to determine PAR‐2 expression in endometriosis (12Z) cells, human endometrial stromal cells (HESC) cells, ovarian cancer (A2780) cells, and breast cancer (MCF‐7) cells. In addition, we quantified the activation of PAR‐2 receptor using a Fluorogenic intracellular calcium assay in human normal endometrial (uterine) epithelial cells (HEUEC), ovarian cancer (Caov‐3), and MCF‐7 cells as internal positive control. We also performed the BrdU proliferation assay on these cells. Our results showed a higher expression (p<0.05) of PAR‐2 in A2780 cells (ovarian aggressive cancer) when compared with the other cells lines. The intracellular calcium assay resulted in a significant reduction (p<0.05) once exposed to the PAR‐2 antagonist in the HEUEC cells and Caov‐3 when compared to cells treated with trypsin (p<0.05). In contrast, there was not significant difference in the MCF‐7 cell line upon exposure to treatments. We observed a significant decrease in MCF‐7 proliferation when treated with the PAR‐2 antagonist in comparison to trypsin treatment. Our results suggested that PAR‐2 antagonist can be used as treatment or adjunct therapy for specific female reproductive cancers. Next steps of this study will include the active full‐length PAR‐2 recombinant and test other concentrations of the antagonist to determine the LD50 in the selected cells. Proliferating markers will be measured for all the previous cell lines as well for endometrial cancer.
Tibial shaft fractures in children: flexible intramedullary nailing in growing children especially weighing 50 kg (110 lbs) or more
Authors: Hanf-Osetek Dorota , Bilski Paweł , Łabądź Dawid , Snela Sławomir
Published: May 2022

Currently the gold standard in surgical treatment of displaced tibial shaft fractures in children with open growth cartilage is elastic stable intramedullary nailing (ESIN). The purpose of this study is the analysis of indications, complications, and duration of treatment using intramedullary flexible nails in children who are still growing but especially weighing 50 kg or more. Hospital records from 2017 to 2020 were retrospectively reviewed to identify the children from 4 to 17 years of age with displaced tibial shaft fractures admitted to the hospital. Only children with open growth cartilage, with a minimum of 6 months of follow-up and complete clinical data, were included. Studies of 91 children xwere analyzed. The average patient age at the time of the injury was 10.88 ± 2.82 years. In the entire group, 31.9% children weighed 50 kg or more, and 68.1% of the children were below this weight. All children were treated using ESIN. The mean time to nail removal was 8.4 ± 4.09 months in the whole group of children stabilized with ESIN. There were no differences in the two groups depending on the weight (P = 0.637). Only two adverse events were observed. This study demonstrates that the use of ESIN in displaced tibial shaft fractures in growing children weighing 50 kg or more is acceptable and safe. The discussion to be made is whether it is still an acceptable method of treatment for this type of fracture due to the progressive obesity epidemic in children and adolescents.
Actionable Mutation Profile of Sun-Protected Melanomas in South America
Authors: Hsieh Ricardo , Nico Marcello M. S. , Camillo Cláudia M. C. , Oliveira Kátia K. , Carraro Dirce M. , Sangueza Martin , Lourenço Silvia V.
Published: May 2022

Melanomas that arise in sun-protected sites, including acral and oral mucosal melanomas, are likely under the control of unique, specific mechanisms that lead to mutagenesis through various pathways. In this study, we examined somatic mutations in tumors by targeted sequencing using a custom Ion Ampliseq Panel, comprising hotspots of 14 genes that are frequently mutated in solid tumors. Tumor DNA was extracted from 9 formalin fixation, paraffin-embedded sun-protected melanomas (4 primary oral mucosal melanomas and 5 acral lentiginous melanomas), and we identified mutations in the NRAS, PIK3CA, EGFR, HRAS, ERBB2, and ROS1 genes. This study reveals new actionable mutations that are potential targets in the treatment of photo-protected melanomas. Additional studies on more of these melanoma subtypes could confirm our findings and identify new mutations.
The Role of Reflectance Confocal Microscopy in the Evaluation of Pigmented Oral Lesions and Their Relationship With Histopathological Aspects
Authors: Luppi Juliana Bronzato , Pereira de Souza Renata , Florezi Giovanna Piacenza , Nico Marcello Menta Simonsen , Lourenço Silvia Vanessa
Published: May 2022

Oral pigmentations are a heterogeneous group and can be the result of physiological activity of oral mucosal melanocytes, secondary to exogenous causes, associated with systemic or local diseases, or due to proliferative activity of melanocytes. Their diagnosis is critical because these lesions can be markers of internal diseases or, in the case of melanocytic proliferative processes, they may represent a malignant neoplasm. In the past decade, the use of reflectance confocal microscopy, a noninvasive imaging tool, has aided the analysis of such lesions, but the establishment of firm criteria in their evaluation is still lacking. This study evaluated a series of 19 cases of pigmented oral lesions and correlated the reflectance confocal microscopy findings with histopathological classical criteria. We found 13 cases of melanotic macule, 1 of them associated with Peutz–Jeghers syndrome and 2 with Laugier–Hunzinker syndrome; 1 melanocytic nevus; 2 lentigo maligna; 2 pigmented actinic cheilitis; and 1 case of postinflammatory pigmentation secondary to a lupus erythematosus oral discoid lesion. The main difference between benign and malignant lesions was the presence of atypical proliferation in lentigo maligna. Langerhans cells with thick dendritic processes, which may be present in other benign and inflammatory pigmentations is one of the main reasons for diagnostic pitfalls.