bluuebunny/crossref_metadata_2025 huggingface dataset:
Student Approach to Learning and Visuospatial Ability as Independent Predictors of Academic Performance in Human Anatomy
Authors: Wang Catherine , McWatt Sean
Published: May 2022

Several indices have been investigated for their accuracy as predictive metrics of students’ performance in anatomy courses. Both student approaches to learning (SAL) and visuospatial ability (VA) have been shown to be predictive of learning outcome achievement. However, far less anatomy‐specific evidence exists for SAL than for VA, and their respective predictive strength has yet to be examined.

The present study investigated the relationship between SAL and VA as predictive metrics for student academic performance, as measured by laboratory‐based assessments. In addition, the predictive strength of each metric was evaluated to determine which of the two was most effective at quantitatively assessing anatomy learning environments. It was hypothesized that the variation in grades could be partially explained by both SAL and VA, and that the latter would have a stronger relationship to performance.

Undergraduate students enrolled in an anatomy course at McGill University were surveyed for this study (total n = 138; 76.8% female; average years of age = 19.46, SD = ±1.04). Students were from kinesiology (58.0%), physical therapy (19.6%), occupational therapy (19.6 %), and other (2.9%) programs. In addition to collecting demographic data, the survey included the Revised 2‐factor Study Process Questionnaire to collect SAL on deep and surface scales, and the Mental Rotations Test to quantify VA.

A multiple linear regression model was used to assess deep and surface approach to learning scores and VA as covariates that influence grades, while controlling for age, sex, and program of study [F(6,131) = 7.741, P < 0.001, adjusted R2 = 0.228]. Data analyses revealed that both VA (β = 0.802, SEM = 0.284; P = 0.006) and deep approach scores (β = 0.776, SEM = 0.246; P = 0.002) had significant positive correlations with grades; however, surface approach scores (β = ‐0.399, SEM = 0.280; P = 0.157) were not significantly correlated at P ≤ 0.05. Based on the coefficients, a one‐point increase on the deep approach scale was slightly more beneficial than the same point‐increase on the Mental Rotations Test; however, the advantage was minimal (+0.026%).

These findings assert that SAL and VA can independently predict performance on laboratory‐based assessments in human anatomy – with VA representing a personal presage factor, and SAL representing a process factor. A deeper understanding of these metrics allows for better quantitative assessment of success when implementing curricular changes intended to improve anatomy learning environments. Therefore, both metrics should be used for a more comprehensive approach to evaluating educational interventions in anatomy.
Endothelial Pannexin 3 – B Cell Lymphoma‐6 Interactions Protect Against Oxidative Stress
Authors: Wolpe Abigail G. , Luse Melissa A. , Johnstone Scott R. , Xue Jianxiang , Sabapathy Vikram , Wakefield Brent , Sharma Rahul , Barr Kevin , Beier Frank , Laird Dale , Redemann Stefanie , Columbus Linda , Penuela Silvia , Isakson Brant E.
Published: May 2022

Pannexin channel isoforms (Panx1‐3) are thought to release nucleotides into the extracellular milieu and have been shown to effect vascular hemodynamics. For this reason, we examined their mRNA and protein expression in hypertensive humans and genetically‐inbred hypertensive mice. In both mouse and humans, we found a significant reduction in Panx3 expression in resistance artery endothelium. Thus, we hypothesized Panx3 may be a regulator of vascular function. In en face endothelial preparations from 3rd order mesenteric arteries, we localized Panx3 to the Golgi Apparatus as opposed to Panx1 which localized to the plasma membrane. Next, we generated an inducible, endothelial cell Panx3 knockout mouse (Panx3ECKO). Radiotelemetry revealed a renin‐independent spontaneous hypertension, with unremarkable immune infiltration in the kidney. There was no change in cytoplasmic or released ATP. To understand how Panx3 may regulate blood pressure, we examined whether Panx3 interacted with B Cell Lymphoma 6 (BCL6), a potential binding partner. En face proximity ligation assays demonstrated an interaction between Panx3 and BCL6 in the Golgi. Panx3ECKO mice exhibited significantly decreased BCL6 protein, hinting that Panx3 may stabilize BCL6 by binding at the BCL6 ubiquitin sites. In silico “threading” of the Panx3 sequence onto the cryo‐EM structure of Panx1 confirmed this site of interaction. BCL6 is a NFκB repressor, thus its degradation in Panx3ECKO mice caused an increase in NFκB activity with IκBα and p100 significantly upregulated. A novel mimetic peptide designed to block Panx3‐BCL6 interactions was administered into C57Bl/6J mice, which recapitulated these results. In addition, Panx3ECKO mice had increased endothelial NOX4 (but not NOX1 or NOX2), likely due to increased NFκB activity—this correlated with a significant increase in plasma H2O2, nitrotyrosine (NT3), and 4‐hydroxynonenal (4‐HNE). In line with this observation, 3rd order mesenteric arteries from Panx3ECKO mice constricted (not dilated) to acetylcholine, which was rescued with the H2O2‐scavenger catalase (1000U/mL), suggesting that vascular oxidative stress drives hypertension in Panx3ECKO mice. Interestingly, Panx3ECKO mice also exhibit a significant increase in IL‐4 receptors on endothelium, and increased IL‐4 cytokines in bone marrow lysates. Because IL‐4 can drive BCL6 expression in other cell types, we suggest a possible homeostatic immune‐endothelial signaling axis. These data elucidate a novel Golgi‐localized oxidative signaling pathway in endothelium with a potential immune‐derived negative feedback loop.
Analysis of Cancer‐associated Mutations in the Novel PKC Theta
Authors: Hodapp Stefanie , Newton Alexandra
Published: May 2022

The diacylglycerol‐regulated novel protein kinase C theta is highly druggable, but how to target this kinase (as an oncogene or as a tumor suppressor) has not been established. Cancer‐associated mutations in Ca2+‐regulated (conventional) PKC isozymes are generally loss‐of‐function, reframing PKC as having a tumor suppressive property, but whether this is the case for PKC Theta (θ) has not been addressed. Here we examine several cancer‐associated mutations in PKCθ, focusing on those at domain interfaces that are likely to impair autoinhibition, to begin to explore whether this isozyme of the PKC family functions to promote or suppress oncogenic signaling. PKCθ is selectively expressed in hematopoietic cells, namely platelets and T lymphocytes, where it regulates inflammatory signaling. Unsurprisingly, deregulated PKCθ has been implicated in many diseases including cancer where dozens of mutations have already been identified. One such residue was R145 in the autoinhibitory pseudosubstrate segment of PKCθ, whose mutation to His or Cys has been reported for two different cancers. This Arg interacts with Asp465 and Asp508 in the substrate binding cavity to maintain effective autoinhibition in the absence of the allosteric activator, diacylglycerol. We determined the effect of R145H and R145C mutation on the cellular activity and stability of PKCθ. Specifically, we used a genetically‐encoded FRET‐based reporter, C kinase activity reporter 2 (CKAR2) to measure the basal and agonist‐evoked activity of PKCθ. Whereas wild‐type PKCθ was effectively autoinhibited and required addition of agonist (here the phorbol ester, PDBu) for activation, both the R145H and R145C mutants were almost maximally activated under basal conditions. However, addition of cycloheximide to inhibit protein synthesis revealed that the half‐time of turn‐over of the mutants was 3‐ and 8‐fold faster than that of wild‐type enzyme, respectively. Taken together, these data suggest that the R145H and R145C mutants fail to autoinhibit resulting in a PKC with constitutive activity that is unstable and subject to the quality‐control degradation we have previously reported for Ca2+‐regulated PKC isozymes. Thus, these pseudosubstrate mutations in PKCθ are paradoxically loss‐of‐function because the constitutively active kinases are sensitive to degradation.
The receptor for advanced glycation endproducts promotes allergen driven neutrophil dominant airway inflammation via NLRP3 inflammasome activation
Authors: Killian Katherine N. , Kosanovich Jessica L. , Lipp Madeline A. , Empey Kerry M. , Oury Tim D. , Perkins Timothy N.
Published: May 2022

Asthma is a major public health burden, affecting nearly 400 million people worldwide, with its prevalence steadily increasing. A growing number of populations with more severe disease are resistant to conventional therapies and are in need of novel preventative and therapeutic intervention strategies. Asthmatics with increased neutrophilia, usually have worse symptoms, more frequent exacerbations, and are insensitive to corticosteroids. Recent studies have demonstrated a critical role for the NLRP3 inflammasome in driving severe steroid‐resistant neutrophilic airway disease (SSRNAD). We have previously demonstrated a critical role for the receptor for advanced glycation endproducts (RAGE) in eosinophilic asthma, however, clinical studies indicate a potential role for RAGE in neutrophilic asthma. Therefore, in this study we have examined the role of RAGE in promoting experimental allergen‐driven SSRNAD. Here, mice are subjected to an experimental model of SSRNAD using complete freund’s adjuvant with Alternaria alternata extract (AA) during sensitization, followed by intranasal challenge with saline or AA. Exposure to AA induced strong neutrophil‐dominant inflammation as well as elevated levels of Th1/Th17 cytokines in the bronchoalveolar lavage fluid (BALF) of wild‐type (WT) but not RAGE‐KO mice. In addition, administration of NLRP3 and Caspase‐1 inhibitors also significantly reduced these features in the lungs. AA exposure elevated levels of NLRP3 and cleaved caspase‐1 in whole lung and BALF cells and increased levels of IL‐1b and IL‐6 in the BALF of WT but not RAGE‐KO mice. Interestingly, we found that circulating levels of IgE and IgG1 were increased in both WT and RAGE‐KO mice and were not affected by treatment with NLRP3 or Caspase‐1 inhibitors, suggesting humoral responses are independent of both RAGE and NLRP3. Moreover, while AA exposure increased lung levels of IL‐17A‐producing CD4 and CD8 T cells, there was a ten‐fold higher increase in a population of IL‐17A‐producing non‐conventional T cell/non‐B cells, suggesting that innate immune responses may be the driving factor of this neutrophilic inflammation. We also found elevated levels of citrullinated histone H3, which is a marker of neutrophil extracellular traps (NETs), in the BALF of WT, but not RAGE‐KO mice and inhibition of NETs reduced airway neutrophilia. Overall, these studies demonstrate that RAGE is a critical mediator of the innate immune response to allergens, promoting NLRP3 inflammasome activation and Th1/Th17‐driven inflammation, making RAGE an intriguing therapeutic target to reduce aberrant neutrophilia in airway disease.
Nitric oxide synthase inhibitor is an effective therapy for ischemia‐reperfusion injury in mice
Authors: Wisen William P. , Evans Wesley R. , Sure Venkata N. , Sperling Jared A. , Sakamuri Siva S. , Mostany Ricardo , Katakam Prasad V.
Published: May 2022

Background

Inhibition of nitric oxide synthase (NOS) has been shown to paradoxically protect against ischemic brain injury by an unknown mechanism. Recently, we found that NOS inhibitor, N (gamma)‐nitro‐L‐arginine methyl ester (L‐NAME), protects primary cultures of endothelial cells and neurons against oxygen‐glucose deprivation‐reoxygenation injury by preventing NOS uncoupling. Thus, the objective of the present study was to demonstrate the effectiveness of locally administered L‐NAME against ischemia‐reperfusion (I‐R) injury.

Methods

Transient focal cerebral ischemia in male mice (C57Bl/6, 2‐4 months) was achieved by filament method of MCAO with a 60 min occlusion. We employed a novel approach to inject L‐NAME (1 mg/Kg dosages at a rate of 60 ug/minute) locally into the ischemic zone by threading a catheter up the middle cerebral artery. This was timed to the onset of reperfusion by rapidly followng the removal of the occlusion with the silicon suture. Following 24‐hour reperfusion, infarct size was determined by 2,3,5‐Triphenyltetrazolium chloride (TTC) staining and neurological function was assessed by employing a standard neurological score (0‐4 range) and Rotarod performance test.

Results

Admnistration of FITC Dextran 40K and fluorescence microscopy of the brain slices 1 hour post‐ischemia confirmed the effective delivery of injected L‐NAME into the ischemic tissue. Localized injection of L‐NAME was protective against I‐R injury and promoted significant recovery of neurological and motor function (Figure).

Conclusion

Local NOS inhibition could serve as an adjuvant for reperfusion therapies of ischemic stroke.
Elucidating the Role of the Electron Transport Chain in Retinal Progenitor Cells and Neuro‐ regeneration
Authors: Rueda Elda , Poche Ross A.
Published: May 2022

In the retinas of non‐mammalian vertebrates, such as zebrafish, neural regeneration is mediated by the molecular reprogramming of MGs into progenitor‐like cells. While mammalian MGs do not exhibit the regenerative potential of the zebrafish, it has been shown that MGs of mammals display limited‐ and transient‐entry into the cell cycle in response to damage. Therefore, an intrinsic proliferative and/or regenerative block likely prevents mammalian MGs from undergoing sustained cell cycle re‐entry and acquiring a multipotential retinal progenitor‐like state. Using a gain‐of‐function strategy we bypassed the Hippo signaling pathway and induced expression of a mutant version of YAP (YAP5SA) in MGs. Lineage tracing and histological analyses of these YAP5SA‐mutant retinas showed that MGs that expressed YAP5SA entered the cell cycle and formed radial clusters of proliferative cells strikingly similar to clonally expanding MG‐derived progenitors of the regenerating zebrafish retina. This finding indicated that YAP5SA is capable of spontaneously driving MGs into the cell cycle. Whether this YAP‐mediated reprogramming of MGs is sufficient to induce differentiation of MGs into fully functional neurons is undetermined. Subsequent single cell RNA sequencing analysis of the YAP5SA‐mutant retinas revealed that the proliferative event of YAP5SA‐positive cells occurred coincident with a dramatic downregulation of genes required for mitochondrial metabolic processes indicating that a metabolic shift is part of the MG reprogramming event. In a variety of cellular contexts, mitochondrial function and the electron transport chain (ETC) activity have emerged as integral regulators of the cell cycle and cell fate. The factors regulating a mitochondrial metabolic reprogramming and cell cycle are coupled but unknown. We generated a mouse line to conditionally knock out (CKO) the mitochondrial transcription factor A (Tfam) to target the ETC. In the CKO developing retinas, loss of Tfam results in loss of ETC activity. Preliminary histological analysis of CKO developing retinas show that loss of Tfam delays progenitor cell cycle exit and reduces neurogenesis resulting in a hypoplastic adult retina. Our findings so far indicate that unless retinal progenitor cells have a functional ETC‐driven oxidative activity, they remain within the cell cycle compromising the normal neural developmental program. The overall goal of this studies will be to determine how the RPC cell cycle machinery interfaces with ETC to drive cell cycle exit and neural differentiation. A better understanding of these requirements will have broad implications for the entire field of neural regeneration.
The Role of CD44v6 in Vascular Rarefaction and Left Ventricular Diastolic Dysfunction in HFpEF
Authors: Fopiano Katie Anne , Tian Yanna , Buncha Vadym , Lang Liwei , Bagi Zsolt
Published: May 2022

CD44 is differentially spliced to form specific CD44 variants, but little is known regarding their specific contribution to cardiovascular pathologies. Specifically, CD44 variant 6 (CD44v6) is involved in abnormal angiogenesis. The hypothesis tested was that CD44v6 leads to coronary microvascular rarefaction whereby it contributes to left ventricle (LV) diastolic function in heart failure with preserved ejection fraction (HFpEF, also known as diastolic heart failure). The obese ZSF1 rat model of human HFpEF was employed and using transthoracic echocardiography, it was found that 21‐week‐old male obese ZSF1 rats exhibited diastolic dysfunction, accompanied with increased myocardial expression of CD44v6. Coronary arteriolar and capillary trees were labeled using Tomato Lectin (Lycopersicon esculentum) DyLight®594 and were imaged by fluorescent confocal microscopy to generate image stacks for 3D reconstruction. Unbiased automated tracing of the microvasculature was done using VesselLucida360 software (MBF) followed by a morphometric analysis (VesselLucida Explorer). It was found that total vessel length and the number of vessel’s branching nodes were reduced in the obese ZSF1 rats, whereas the total vessel’s volumes remained unchanged, when compared to the lean ZSF1 controls. These changes in the microvasculature were accompanied by decreased angiogenesis in the coronary arteries in the obese ZSF1 rats when compared to the lean ZSF1 rats. From these results, it was concluded that upregulation of CD44v6 plays a role in the impaired angiogenesis underlying the development of LV diastolic dysfunction in HFpEF.
TNK1 is a ubiquitin‐sensing kinase that can be targeted to block tumor growth
Authors: Andersen Joshua L. , Chan Tsz‐Yin , Egbert Christina M. , Lopez‐Palacios Tania , Maxson Julia E. , Tyner Jeffrey W. , Warner Steven L.
Published: May 2022

Thirty‐eight negative kinase‐1 (TNK1) is an understudied non‐receptor tyrosine kinase with no established function or mechanism of regulation. We recently identified TNK1 as the primary kinase driver of cell survival in subsets of patient blood cancer samples1. We discovered that one unusual feature of TNK1 is a functional ubiquitin‐association (UBA) domain that binds with high affinity to multiple poly‐ubiquitin linkages. Our recent 1.5 angstrom crystal structure of the TNK1 UBA shows an atypical five‐helix UBA domain with two predicted ubiquitin‐binding interfaces. We found that TNK1 relies on its UBA domain to home to phase‐separated poly‐ubiquitylated protein condensates and becomes fully active at these sites. To our knowledge, this feature of TNK1—a bona‐fide ubiquitin‐association domain that promotes kinase activation—makes TNK1 unique across the human kinome. Our data also suggest that 14‐3‐3 interacts with a MARK‐mediated phosphorylation at S502 of TNK1 to sequester the kinase away from ubiquitin and inhibit kinase activation. Thus, TNK1 toggles between poly‐ubiquitin bound (active) and 14‐3‐3‐bound (inactive) states. Our preliminary data suggest that the interaction with poly‐ubiquitin at condensates tethers TNK1 to substrates, facilitating its phosphorylation of substrates involved in the lysosomal degradation of condensates. Furthermore, we found that patient mutations that truncate the 14‐3‐3 binding site and UBA domain convert TNK1 into a cancer driver with an altered substrate profile, skewed toward pro‐growth substrates like STAT3. Finally, we show that mutant TNK1‐driven tumors can be targeted in vivo with a novel anti‐TNK1 small molecule. Thus, together our data suggest that TNK1 acts as a ubiquitin sensor and is a candidate for targeted therapy.

1. Chan TY and Egbert CM et al., (2021) TNK1 is a ubiquitin‐binding and 14‐3‐3‐regulated kinase that can be targeted to block tumor growth. Nature Communications(in press)
Novel insights into the fundamentals of palatal shelf elevation dynamics in normal mouse embryos
Authors: Saadi Irfan , Goering Jeremy , Moedritzer Michael , Stetsiv Marta , Isai Dona Greta , Hufft‐Martinez Brittany , Umar Zaid , Rickabaugh Madison K. , Keselman Paul , Chauhan Munish , Brooks William , Fischer Kenneth , Czirok Andras
Published: May 2022

Embryonic palate development involves bilateral vertical growth of palatal shelves – extensions from the maxillary processes – next to the tongue at embryonic day (E) 13.5. This vertical growth is followed by elevation and fusion above the tongue by E14.5. Recent models indicate that this process of elevation involves a complex vertical to horizontal remodeling of the palatal shelves. While earlier studies have implied that this is a rapid process, the precise timing has not been resolved. To further understand the dynamics of palatal shelf elevation, we employed time‐restricted pregnancies with a one‐hour resolution and magnetic resonance (MR) imaging of intermediate stages. Our data showed that in C57BL/6J mice, palatal shelves are unelevated in 100% of embryos at E14.0 and completely elevated in 80% of embryos at E14.25, which is only a 6‐hour timeframe. Interestingly, all E14.25 embryos with unelevated palatal shelves (20%) were female, suggesting a shift towards delayed elevation in female embryos. Even at E14.125, 25% of embryos had completely elevated palatal shelves suggesting that once initiated, the palatal shelves elevated rapidly with elevation completing in less than 3 hours in individual embryos. In contrast to C57BL/6J, in FVB/NJ mice, the elevation window was shifted earlier and was broader to E13.875‐E14.25 without any noticeable sex differences. In a proportion of embryos in both strains, we also captured an intermediate stage with unilateral palatal shelf elevation. Both right and left shelves were identified to be unilaterally elevated indicating that it was a random process. It remains to be determined whether palatal shelves elevate in a sequential manner, i.e., one‐at‐a‐time, with an obligate unilateral elevation stage. MR imaging of various stages showed a highly dynamic antero‐posterior remodeling of the shelves during elevation, beginning with the formation of bilateral bulges in the posterior palate. These bulges tapered anteriorly, however, palatal shelf fusion still began in the middle part of the palate. The bulge regions did not show changes in cell proliferation, consistent with the notion that rapid elevation was primarily due to cytoskeletal and extracellular matrix changes. We are using finite element analysis to identify the mechanical forces underlying these morphological changes. Thus, our data reveal critical novel insights into the rapid dynamic changes as well as strain and sex differences in palatal shelf elevation that lay the foundation for future studies of normal and abnormal palatogenesis.
The Association between Diet, Exercise, and Neurobehaviors
Authors: Lee Sienna , Quazi Nawreen , Stala Olivia , Vidrin Abegail , Marinaccio Daniel , Patrissy Cara , Qureshi Maheen , Williams Sara , Begdache Lina
Published: May 2022

The purpose of this study was to investigate a potential relationship between diet quality, lifestyle factors and neurobehaviors of individuals. It was hypothesized that eating healthy and exercising improve with mental state and self‐motivation. An anonymous online survey using the Food‐Mood and Treatment Self‐Regulation Questionnaires and the five‐point Likert Scale was administered online through community outreach and social media. The survey included questions on demographics, exercise habits, frequency of food group consumption, mental distress, and motivation. Data was collected between January to March 2021 and a total of 421 participants aged 18 and above completed the questionnaires. Spearman’s Rho correlation was used to assess the associations between different variables. Data was analyzed using SPSS version 25.0. The results indicated that there is a correlation between exercise, motivation, and mood to every other metric studied, such as positive diet behaviors. Our findings also reveal strong positive associations between nutrient‐dense food, motivation to perform and mental wellbeing (P < 0.01). Interestingly, fast‐food and sugary foods produced strong negative correlation with neurobehaviors (P < 0.01). It was concluded there may be a correlation between dietary behaviors and exercise and motivation. Further evaluation of the role dietary and lifestyle choices plays on neurobehavioral should be investigated.
Interactions of TPH2 and TPH1 in Biosynthesis Pathways
Authors: Kaur Kuljeet , Redfairn‐Ogunyemi Alexis , Awe Kehinde , Gardner Lanie
Published: May 2022

Tryptophan hydroxylase‐2 (TPH2) is mainly located in neurons of the raphe nuclei. A small percentage of TPH2 can also be found through neurons in the wall of the digestive tract. Tryptophan hydroxylase‐1 (TPH1) is mainly found in the pancreas. This protein's alpha‐fold structure determines the rate of the appearance in serotonin through biosynthesis pathways. Serotonin (5‐HT), is a key neurotransmitter that modulates neuropsychological processes and neural activity receptors. Mutations in TPH2 can lead to psychiatric disorders such as bipolar disorder and depression. These can be used as a therapeutic response to selective serotonin reuptake in creating a first‐generation drug for humans. TPH2 has the potential of being synthetically manipulated to create a greater appearance of itself, which could help reduce depression in patients. Other disorders positively affected by increased activation of TPH2 include schizophrenia, autism, aggression, and suicidal behavior. Studies are being done to further pave the way for the development of animal models for behavioral and pharmacological studies. Additionally, TPH1’s impact on serotonin synthesis production throughout the body will be discussed with regards to its involvement in neuropsychiatric disorders similar to TPH2. Identified 15 years prior to TPH2, TPH1 is TPH2’s peripheral counterpart. TPH1 is predominantly expressed during the late developmental stages in the brain and is synthesizing serotonin throughout the body. Although TPH1 is particularly responsible for serotonin synthesis in enterochromaffin cells, mammary glands, lungs, and the pancreas, it is also found in the brain’s pineal gland. TPH1 shows a greater affinity for tryptophan, an amino acid used in the production of serotonin, and has a stronger enzymatic activity than TPH2 for a developing brain stem. Due to the chemical and physical specificity of the blood‐barrier which distinguishes TPH2 in the brain from the rest of the body, TPH1 and TPH2 inhibitors will function differently in the brain than in the body.
A High‐Through Put Pro‐inflammatory screening of natural products using mouse RAW 264.7 macrophages indicate a more significant number of pro‐inflammatory natural herbal products than anti‐inflammatory
Authors: Barnes Andrew , Mazzio Elizabeth , Badisa Ramesh , Soliman Karam F.
Published: May 2022

When it comes to natural product research, there are an overarching plethora of studies focusing on and reporting “antioxidant” and “anti‐inflammatory properties,” with a relative void in the literature elucidating those that have pro‐inflammatory (immune stimulating) and pro‐oxidant properties. Furthermore, when it comes to cancer study, many anti‐cancer compounds induce apoptosis in the same cell lines (e.g., RAW 264.7 and BV‐2 cells) used to model inflammation, creating a confounding variable [cell death] leading to false positives for reported anti‐inflammatory compounds. We tested over 1,400 natural products in our past work and found only 29 confirmed natural anti‐inflammatory compounds effective against LPS treated RAW 264.7 viable cells. In the present study, we are reporting (without a confounding variable of cell death and with matrix controls run for every extract) an enormous number; 444 natural products to evoke pro‐inflammatory effects in RAW 264.7 cells producing nitric oxide at levels equal to LPS and IFN‐g, using the same library. We found these herbs' capacity to induce IFN‐g, whereas IFN‐g by itself can induce iNOS, defining the general mechanism of action. These findings suggest that many natural products trigger a massive elevation in IFN‐g by immune‐competent cells such as macrophages and could either worsen auto‐immune or inflammatory conditions or stimulate the immune system to fight infections when needed. In conclusion: there needs to be more work in the pro‐inflammatory/immune stimulation aspects of herbs and the specific influence of natural products on various immune cells and processes. It was concluded that in cancer cells, the anti‐inflammatory /antioxidant compounds might likely prevent cancer, but once cancer is established, pro‐inflammatory/ pro‐oxidant compounds may be required to re‐establish immunosurveillance.
Transcriptional Landscape Established by the Euchromatic Histone‐lysine N‐methyltransferase Pathway During Pancreas Ontogenesis and Pancreatitis
Authors: Urrutia Raul , Salmonson Ann , Urrutia Guillermo , de Assuncao Thiago Milech , Zimmermann Michael T. , Mathison Angela J. , Lomberk Gwen
Published: May 2022

The Euchromatic Histone‐lysine N‐Methyltransferase 1/2 (EHMT1/2) complex, responsible for catalyzing mono‐ and di‐methylation of histone H3K9, has emerged as key regulator of embryogenesis and carcinogenesis. We have recently shown that EHMT2 inactivation antagonizes oncogenic KRAS‐mediated pancreatic cancer initiation and promotion by altering growth and immune gene expression networks. The current study unravels the transcriptional landscape regulated by EHMT2 during pancreatic development and acute pancreatitis. We performed conditional knockout of EHMT2 in the pancreas using the Pdx1‐Cre model, comparing perinatal (10‐day‐old) vs. young adult mice (4‐week‐old). Notably, despite carrying EHMT2 inactivation, both the whole animal and pancreas develop normally through adulthood. The transcriptional landscape resulting from EHMT2 inactivation, however, is different in perinatal mice from young adults, suggesting that this epigenomic regulator plays a role in modulating distinct gene networks during the development of this organ. Congruent with a role for EHMT2 in gene silencing, we show that its inactivation in both life stages primarily resulted in gene expression signatures dominated by upregulated transcripts. Notably, among these genes, we find pancreas‐specific clusters, including those encoding zymogen granules, during the transition to young adult, suggesting that EHMT2 facilitates acinar cell‐fate decisions. DEGs from young adults with EHMT2 loss displayed prominent activation of TP53 signaling‐related and DNA damage response gene networks that were not present in the perinatal cohort, demonstrating that cell cycle arrest accompanies this differentiation event. We also find de‐repression of genes that participate in early endodermal development, metabolism, and inflammation. Studies on acute pancreatitis, induced by repeated injections of the CCK‐agonist peptide, caerulein, show that EHMT2 inactivation results in more severe disease compared to that in controls, as measured by histological scoring of edema, inflammatory cell infiltrate, and necrosis. More importantly, and congruent with our histological assessment, EHMT2 inactivation in acinar epithelial cells regulates the expression of key inflammation‐associated genes that reprograms the non‐epithelial responses to pancreatitis. Digital cytology analysis demonstrated that the transcriptional landscape resulting from EHMT2 inactivation triggers a more aggressive inflammatory response for the pancreas. This data demonstrates that, although not critical to development of the organ, EHMT2 is involved in pancreas‐specific gene expression. Lastly, during pancreatitis, EHMT2 behaves as an important regulator of inflammation, extending our knowledge of epigenomic mechanisms underlying development, homeostasis, and diseases of the pancreas.
AT1R blockade together with AT2R stimulation prevents aortic aneurysm in mice with progressively severe Marfan syndrome
Authors: Asano Keiichi , Cantalupo Anna , Hansen Jens , Spronck Bart , Humphrey Jay D. , Iyengar Ravi , Ramirez Francesco
Published: May 2022

Angiotensin II receptor signaling regulate vascular tone by stimulating either vasoconstriction (AT1R) or vasodilation (AT2R). AT1R antagonism (via losartan) has been shown to mitigate thoracic aortic aneurysm (TAA) progression in mouse models of Marfan syndrome (MFS). Here we monitored multiple pathological readouts of TAA in MFS mice chronically treated for 90 days with either losartan, the AT2R agonist C21, or both. While C21 had no apparent effects on arterial disease by itself, it significantly enhanced losartan’s efficacy by preventing death of MFS mice from ruptured TAA during the treatment period. Specifically, the combination therapy normalized the diameters of the aortic root and ascending aorta, the architecture of the vessel wall, endothelial‐dependent relaxation, and circumferential wall strength (a strong biomechanical indicator of aneurysmal vulnerability). Computational analyses of bulk RNASeq data predicted that the combination therapy largely decreased expression of abnormally upregulated genes, principally those associated with inflammatory pathways. By contrast, these in silico analyses predicted no modifications of signaling pathways associated with dysregulated mitochondrial energy metabolism. Ongoing investigations are using single cell RNASeq to identify the different cell types targeted by the two drugs used in combination therapy, as well as potential cell‐cell interactions that underlie the efficacy of the combination therapy.
Human Cytochrome P450 8B1 Structure and Function: Supporting Drug Design for NAFLD and Diabetes Mellitus
Authors: Liu Jinghan , Scott Emily
Published: May 2022

Human cytochrome P450 8B1 (CYP8B1) performs a key step in human production of bile acids. It 12α‐hydroxylates a steroidal substrate to produce the relatively polar primary bile acid cholic acid. The absence of CYP8B1 catalytic function results in the more hydrophobic bile acid chenodeoxycholic acid. Knockouts of CYP8B1 indicated this P450 may be a good drug target for nonalcoholic fatty liver disease due to its alteration of bile acid‐mediated fat absorption. Similar studies indicate that the absence of CYP8B1 also has a favorable effect on type 2 diabetes. However, the absence of selective CYP8B1 inhibitors both restricts further study of its physiological roles and potential disease treatment. We recently generated the first CYP8B1 structure, with the nonspecific type II inhibitor (S)‐tioconazole, and are now employing a structure/function strategy to understand CYP8B1 interactions with a) its native substrate and b) a wider array of inhibitors. Within the CYP8B1 active site tioconazole is located near the B’ helix due to spatial hinderance from W281 and N286 in the I helix on the opposite side of the active site. W281 has previously been suggested as a key residue for CYP8B1 catalysis with its native steroidal substrate. To test this concept, a human CYP8B1 W281F mutant was generated. The W281F enzyme demonstrated both impaired substrate binding and significantly reduced catalytic efficiency. Thus W281 appears to be important in binding both the native substrate and the tioconazole inhibitor. Subsequently a set of additional azole ligands were evaluated to broaden the definition of ligand structures compatible with the CYP8B1 active site and potentially identify even better inhibitors. Changes in visible absorbance spectra were used to determine the binding mode and determine Kd values, while inhibition of steroid 12a‐hydroxylation was used to determine IC50 values. Most azole compounds bound CYP8B1, typically via the expected type II imidazole‐to‐heme‐iron interaction like tioconazole, and with Kd values in the nM to µM range. Miconazole and econazole bound with the highest affinity and had the lowest IC50 values, demonstrating improvements over tioconazole. The structural differences between these ligands are a relatively moderate substitution of a thienyl vs. phenyl ring and modified chlorination patterns on that ring, demonstrating CYP8B1 sensitivity to these inhibitor components. Both the CYP8B1 structure and the functional characterization should facilitate the identification increasingly effective CYP8B1 inhibitors. If selectivity can also be incorporated into these compounds, the resulting inhibitors should enable a better understanding of the role of CYP8B1 in normal physiology and facilitate a potential treatment for nonalcoholic fatty liver disease and type 2 diabetes.
Resistance profile of bacteria to antibiotics in city laboratories
Authors: CHEMSI Hicham , Haitam Abdelaziz , Hilali Farida , Sekhsokh Yassine
Published: May 2022

Introduction

Bacteria are the causative agents most frequently described in infections. Therefore, antibiotic resistance is a real public health problem. Thus, bacterial infections are becoming one of the major threats to public health in the world. The objective of this study is to evaluate the frequency of isolation of bacteria and to study the sensitivity of bacteria isolated in different biological samples at the level of city laboratories in the entire population (women, men and children).

Patients and methods

This is a prospective descriptive study carried out in city medical analysis laboratories. The inclusion criteria were all positive samples from the examinations performed: urinary examination, vaginal swab, urethral swab, pus, sputum, semen. The exclusion criteria concerned patients with negative examinations and other microorganisms (viruses, mycoses and parasites). Data collection was carried out using an information sheet. An antibiogram is carried out to study the resistance of bacteria. Data processing was carried out and statistically analyzed at a risk of 5%.

Results

Out of a total of 8,594 samples identified, 2,465 strains isolated were responsible for bacterial infections with a prevalence of 28.7%. A clear predominance was noted in the female sex with 1710 (69%) versus 755 (31%) in the male. The F / M sex ratio was 2.2. The most affected of the patients were those over 60 years of age with an enrollment of 832 (33.7%). 1954 strains were Gram negative bacilli or a frequency of 79.3%, 462 strains were Gram positive coccis or a frequency of 18.7% and 49 strains were Gram negative coccis or a frequency of 2%. The distribution of isolates according to the type of sample revealed a predominance of urine samples with a frequency of 1703 (69%), followed by vaginal samples with a frequency of 276 (11.2%), urethral samples with a frequency of 166 (6.8%), sputum with a frequency of 154 (6.2%) and others. The bacteriological profile was largely dominated by Escherichia coli 1410 (57.2%), Klebsiella pneumoniae 355 (14.4%), Staphylococcus sp 294 (12%), Enterococcus sp 109 (4.4%), Streptococcus sp85 (3,4%). For Enterobacteriaceae, the lowest sensitivities in out‐of‐hospital settings were recorded with Amoxicillin followed respectively by Ticarcillin, Cefalotin, and Amoxicillin‐clavulanic acid. High sensitivities have been observed for Imipenem, Colistin, Furan, and Aminoglycosides.

Conclusion

At the end of the study, the results showed an increase in the resistance of bacteria. This rise could be linked to selection pressure due to the overuse of antibiotics. Regular monitoring is essential to define effective and appropriate therapeutic strategies, limiting the emergence and dissemination of very resistant strains.
PDE4 Inhibitor Reverses Alcohol Impairment of CFTR Function and Protects Against Bacterial Pneumonia
Authors: Stanford Denise , Rasmussen Lawrence , Antony Linto , Allen Antonio , Raju S. V.
Published: May 2022

INTRODUCTION

Alcohol is a significant risk factor for respiratory infections. Excessive alcohol users suffer from more frequent lung infections, severe symptoms, and poor antibiotic response. Mucociliary clearance (MCC) is a primary lung defense mechanism against inhaled/aspirated pathogens and is impaired by alcohol use. Previously, we found diminished MCC associates with reduced CFTR‐mediated ion transport in alcohol‐fed rats. In addition, CFTR dysfunction correlated with increased cAMP degradation by phosphodiesterase‐4 (PDE4) enzymes and reduced channel gating. Here, we test whether roflumilast, a clinically used PDE4 inhibitor, can preserve cAMP levels in alcohol‐fed rats and promote CFTR function and clearance of Klebsiella pneumoniae (KP, ATCC 43816), to which alcohol users are disproportionately susceptible.

METHODS

Age and gender‐matched rats (CFTR+/+) were pair‐fed with Lieber‐DeCarli alcohol diet or isocaloric control diet for eight weeks. CFTR activity was assayed by Ussing chamber electrophysiology of tracheal explants. Rats were randomly treated with vehicle/roflumilast (5mg/kg/day) by oral gavage starting 7 days before the end of 8‐week alcohol diet. Airway surface liquid (ASL) depth, periciliary layer (PCL) height, mucus viscosity, and transport (MCT) on excised trachea were quantified with micro‐optical coherence tomography (µOCT) imaging. In addition, lung function was assessed by flexivent oscillometry.

RESULTS

Alcohol administration reduced CFTR‐mediated ion transport in rat airways. µOCT image analysis of excised trachea from these rats indicated decreased PCL depth and increased mucus viscosity. Despite the normal ciliary beating, there was a noticeable delay in MCT in alcohol‐fed rats. Roflumilast treatment significantly increased CFTR activity in both control and alcohol‐administered rats. Increased CFTR activation by roflumilast resulted in augmented PCL depth, reduced mucus viscosity, and significantly accelerated MCT rate. While roflumilast partially improved clearance of KP, there was pronounced protection against increased airway resistance and lung injury. Histopathological examination of lung sections indicates roflumilast reduced the severity of multifocal pneumonia with edema, fibrin deposition, and numerous bacteria in alveolar spaces. These improvements were more evident in control rats over those administered with alcohol. Ongoing studies are exploring how roflumilast altered immune cell recruitment and inflammatory signaling to reduce infectious injury.

CONCLUSIONS

Acquired (non‐genetic) CFTR dysfunction may be a suitable target to improve mucus clearance impaired by alcohol use. Administration of PDE4 inhibitors such as roflumilast may boost lung host defense against bacterial pathogens and improve pneumonia outcomes in individuals affected by excessive alcohol use.
3D Hyolingual Kinematics Reveal New Insights Into the Biomechanical and Neural Control of Chewing and Swallowing in Macaque Primates
Authors: Ross Callum F. , Feilich Kara , Gidmark Nicholas , Laurence‐Chasen JD , Orsbon Courtney
Published: May 2022

Hyolingual kinematics are difficult to measure, but are central to hypotheses regarding musculoskeletal mechanisms and motor control. Recent application of biplanar videoradiography to the study of hyolingual kinematics in nonhuman primates provides new insight into tongue movements during chewing and swallowing, and into the role of hyoid movement in tongue base retraction and the oral phase of swallowing. During grape chewing, complex shape changes in the tongue were dominated by a combination of flexion in the tongue’s sagittal planes and roll about its long axis. As hypothesized for humans, in macaques during tongue retraction the molar region of the tongue rolls to the chewing side simultaneous with sagittal flexion, while the tongue tip flexes to the balancing side. Twisting and flexion reach their maxima early in the fast close phase of chewing cycles, positioning the food bolus between the approaching teeth prior to the power stroke. The muscular drivers of these movements are likely to include a combination of intrinsic and extrinsic tongue muscles. Nerve block of tactile sensation from trigeminal innervated oral structures decreased feeding performance, and the fast open phase of the gape cycle became significantly longer, relative to the other phases. The tongue made similar shapes in both the control and nerve block conditions, but the pattern of tongue‐jaw coordination became significantly more variable after the block. Disruption of oral somatosensation impacts feeding performance by introducing variability into the typically tight pattern of tongue‐jaw coordination. During primate swallowing, tongue base retraction (TBR) drives the food bolus across the oropharynx towards the esophagus but the mechanics of TBR are poorly understood. XROMM of four macaque monkeys falsifies both the hypothesis that extrinsic tongue muscle shortening pulls the tongue base posteriorly, and the muscular hydrostat hypothesis that intrinsic muscle shortening displaces the tongue base posteriorly by increasing tongue length. Our data suggest a novel hydraulic mechanism of TBR: shortening and rotation of suprahyoid muscles compresses the tongue between the hard palate, hyoid and mouth floor, squeezing the midline tongue base and food bolus back into the oropharynx. Hyoid elevation and protraction are powered by concentric activation and rotation of mylohyoid and digastric, followed by concentric activation of geniohyoid. This research suggests that structure, function, and coordination of mylohyoid and geniohyoid muscles are especially important determinants of swallowing performance in macaques, and probably humans.
Dietary Protein Quality Influences Liver Circadian Rhythms in a GCN2‐dependent Manner
Authors: Levy Jordan L. , Mirek Emily T. , Anthony Tracy G.
Published: May 2022

Circadian disruption correlates with a wide range of metabolic and neurological disorders. General control non‐derepressible 2 (GCN2) activation, the primary sensory mechanism of amino acid insufficiency, also plays an integral role in maintaining the circadian clock in peripheral tissues such as the liver. The objective of this work is to examine the regulation of the circadian clock via GCN2‐mediated sensing of dietary protein quality. We hypothesized that low protein quality feeding will alter the circadian clock in a GCN2 dependent manner. To test this, wild type (WT) and whole body GCN2 knockout (GCN2KO) mice were housed in total darkness within an environmentally‐controlled indirect calorimetry cabinet and provided a diet either devoid of leucine (LeuD) or amino acid replete (Control) for 8‐days and then killed at 2 circadian time (CT) points 12‐hours apart (CT3, CT15). Following the treatment period, LeuD‐fed GCN2KO mice displayed a 40% reduction in the circadian oscillation of their energy expenditure (p<.001) relative to Controls whereas no such difference was present in their WT counterparts. Measurements of the 12‐hour expression pattern of various circadian oscillatory genes within the liver revealed that GCN2 is necessary for circadian oscillation of Bmal2 mRNA expression independent of diet (p<0.01). Furthermore, LeuD feeding alters the expression pattern of the clock‐controlled genes (CCGs) Ciart, Klf10and Noctby over 5‐, 10‐, and 2‐fold respectively (p<0.05) in a GCN2 dependent manner. Together, these findings demonstrate that dietary protein quality plays a role in the maintenance of circadian rhythms within the liver. Furthermore, GCN2 is required to maintain whole body metabolic rhythms when challenged with a diet of poor protein quality. In observing the alterations in the circadian expression pattern of these CCGs with feeding a low protein quality diet, our findings expand our understanding of the role of dietary amino acid intake and the maintenance of circadian metabolic rhythms within the liver.