bluuebunny/crossref_metadata_2025 huggingface dataset:
TMED‐A001 Attenuates Transforming Growth Factor β1‐Induced Epithelial to Mesenchymal Transition in Human Renal Proximal Tubular Cells
Authors: Thipboonchoo Natechanok , Soodvilai Sunhapas
Published: May 2022

Fibrosis is a final common outcome of almost progressive kidney disease; it strongly correlates with kidney insufficiency. Initiation of fibrosis is stimulated by sustain activation of injurious stimuli. This process leads to induction of epithelial to mesenchymal transition (EMT) of tubular cells. Transforming growth factor β1 is the master regulator of EMT and fibrosis. The aim of this study is to investigate the pharmacological effects of TMED‐A001, a natural product isolated from fungi, on EMT of human renal proximal tubular cells. The morphology of human renal proximal tubular cells was examined and photographed under light microscope. The protein expression of E‐cadherin, fibronectin, Smad7, phosphorylated Smad2, Smad3, and extracellular signal‐regulated kinase (ERK) 1/2 were evaluated by Western blot analysis. Incubation RPTEC/TERT1 cells, an immortalized human renal proximal tubular cells, with 10 ng/ml transforming growth factor β1 (TGF‐β1) led to change in cell morphology of renal epithelial to fibroblast‐like structure. Co‐incubation the cells with TGF‐β1 and TMED‐A001 (50 and 100 µM) prevented the morphology change. TMED‐A001 maintained the epithelial characteristics by preserving protein expression of E‐cadherin with reduction of fibronectin protein expression. TMED‐A001 significantly reduced downstream of TGF‐β1 signaling pathway including phosphorylated Smad2 and Smad3 protein expression. In contrast to Smad2 and Smad3, TMED‐A001 increased Smad7 protein expression which is the negative regulator of TGF‐β1‐Smad signaling pathway. In addition, TMED‐A001 also significantly reduced phosphorylated ERK 1/2 protein expression, a TGF‐β1‐non‐Smad signaling pathway. These results suggest that TMED‐A001 prevents EMT in human renal proximal tubular cells through the inhibition of TGF‐β1‐Smad and non‐Smad signaling pathways. In summary, TMED‐A001 presents anti‐EMT property of renal proximal tubular cells by counteracting TGF‐β1 signaling pathway. TMED‐A001 might be the therapeutic potential agent for renal fibrosis in the progressive kidney diseases.
Investigating Phosphorylation of the Cardiac Potassium Channel hERG using MALDI‐TOF Mass Spectrometry
Authors: Bocetti Julie , Darling Louise E. O.
Published: May 2022

Mutations in the voltage‐gated cardiac potassium channel proteins hERG and KvLQT1 are known to be associated with a cardiac condition called Long QT Syndrome (LQTS), which can result in arrhythmia, fainting, and/or sudden cardiac death. Previous research from the Darling lab suggests that hERG and KvLQT1, alpha subunits of two distinct ion channels, are capable of direct protein‐protein interactions, which can be regulated by PKA‐mediated phosphorylation.. They also found that phospho‐null mutants of hERG do not interact with KvLQT1 to the same extent as wild‐type hERG, leading to the hypothesis that the phosphorylation state of hERG dominates the regulation of interactions between hERG and KvLQT1. Thus, understanding the phosphorylation pattern of wild‐type hERG would help us characterize these protein‐protein interactions as well as increase our knowledge about the complex biogenesis and trafficking of hERG. In order to elucidate this baseline phosphorylation state, the goal of this project is to use matrix‐assisted laser desorption ionization time of flight mass spectrometry (MALDI‐TOF MS) to characterize which of the four known PKA‐phosphorylation sites of hERG are in fact modified when the protein is synthesized in heterologous model cells. In order to do this, the project aims to determine whether MALDI‐TOF MS can sensitively and reproducibly detect phosphorylation patterns in hERG that is expressed in model cell systems.
The Trunk of Henle and its Variants
Authors: Streicher Johannes , Kiehling Felix , Lechner Peter
Published: May 2022

Ojective

In colon, pancreatic and hepatobiliary surgical research, an increasing interest developed in the Gastrocolic Trunk of Henle (GTH) due to its broad variability and its importance especially for right hemicolectomy with complete mesocolic excision. The purpose of this study was to further corroborate details of the anatomical variants of the Gastrocolic Trunk, in particular with respect to pancreaticoduodenal veins and the jejunal drainage into the superior mesenteric vein.

Methods

In 15 cadaveric bodies the venous drainage into the superior mesenteric vein was dissected first in a ventral, in situ approach as well as by a subsequent dorsal approach following careful abdominal exenteration.

Results

The most frequent configuration was the tripodal gastropancreaticocolic trunk (GPCT) with an occurrence of 54.5%, followed by the gastropancreatic trunk (GPT) with 36.4% (figure 1). The right gastroepiploic vein was a constant structure draining into the trunk of Henle in 100% of the cases. During the dissection of the pancreatic drainage, a previously undescribed, middle anterior pancreatic vein, was found in 46.67% of the specimens, which drained in 85.71% into the trunk of Henle.

Conclusion

The trunk of Henle drains blood from several abdominal organs and bundles them into a short and vulnerable structure. The most common variant is a tripodal trunk, collecting veins from the pancreas, right colon, and the stomach. Dependent on the surgical approach, the right gastroepiploic vein or the superior right colic vein may serve as guiding structures towards the trunk of Henle. Preoperative imaging in conjunction with awareness of variants is essential for a personalized surgical pocedure. In addition, the finding of a middle anterior pancreatic vein prompts for further studies regarding venous drainage of the pancreatic head.
Exploring Protein Function Through In Silico Investigation: The Role of the Nsp5 Protease in SARS‐Cov‐2 Evolution
Authors: Currier Jenna , Orellana Marilyn , Duvvuru Ashish , Pikaart Michael
Published: May 2022

The Biochemistry Authentic Scientific Inquiry Laboratory (BASIL) has provided undergraduate biochemistry students with an inquiry‐based investigation into protein structure using a combination of computational and wet‐lab experimentation. Based on the skill set developed, BASIL faculty and students were invited to join a week‐long online boot camp hosted by Dr. Stephen Burley and colleagues at the Rutgers University Institute for Quantitative Biomedicine in July 2020. This session, entitled “The Summer of the Coronaverse,” saw teams of researchers analyzing genomic data to track emergence of different variants in SARS‐CoV‐2 Nsp5 (also known as main protease or Mpro) sequence. Teams used Mol*and FoldIt to model the protease and further analyze mutations of amino acids, categorizing them as conservative/nonconservative, and by location with the protein (surface/interior/boundary). Most mutations from existing SARS‐CoV‐2 strains were located distal to the enzyme’s active site and did not greatly effect the estimated protein stability relative to the parental sequence. As a result of the week spent investing these protease mutations, undergraduate students (a) learned to use online, accessible genomic and structure analysis tools, (b) experienced partnering with a large team of investigators, and (c) contributed to ongoing science, including publishing a manuscript on their results.
Soluble Epoxide Hydrolase Inhibitors: Design, Synthesis, in vitro Profiling and in vivo Evaluation in Murine Models of Pain
Authors: Vázquez Santiago , Codony Sandra , Entrena José M. , Jora Beatrice , González‐Cano Rafael , Loza M. I. , Brea Jose , Morisseau Christophe , Bartra Clara , Sanfeliu Coral , Hammock Bruce D. , Cobos Enrique J.
Published: May 2022

Epoxyeicosatrienoic acids (EETs) are endogenous chemical mediators that show anti‐inflammatory, antihypertensive, and analgesic effects. Soluble epoxide hydrolase (sEH) converts EETs to their corresponding dihydroxyeicosatrienoic acids, whereby the biological effects of EETs are modified. Therefore, inhibition of sEH has been suggested as a novel pharmacological approach for the treatment of inflammatory and pain‐related disorders.1

Recently, we have discovered a new family of sEH inhibitors (sEHI) featuring a unique benzohomoadamantane scaffold. In a murine model of cerulein‐induced acute pancreatitis, the administration of a selected candidate significantly reduced pancreatic damage and improved the health status of the animals.2

Herein we report further structure‐activity relationships within this series of benzohomoadamantane‐derived sEHI. Most of the novel derivatives were endowed with low nanomolar or even subnanomolar IC50values at the human, murine and rat sEH. Further in vitro profiling (solubility, cytotoxicity, metabolic stability, selectivity, permeability, etc.) and pharmacokinetic studies permitted us to select a candidate for in vivo efficacy studies. This candidate reduced pain in the capsaicin‐induced model of allodynia in a dose‐dependent manner and outperformed other sEHI tested.

In summary, these novel results and the previously reported studies using other families of sEHI, strongly suggest that sEH may be a target of clinical interest for managing pain.3

References:

1‐Sun, C.‐P.; Zhang, X.‐Y.; Morisseau, C.; Hwang, S. H.; Zhang, Z.‐J.; Hammock, B. D.; Ma, X.‐C. J. Med. Chem. 2021, 64, 184–215.

2‐Codony, S.; Calvó‐Tusell, C.; Valverde, E.; Osuna, S.; Morisseau, C.; Loza, M.I.; Brea, J.; Pérez, C.; Rodríguez‐Franco, M.I.; Pizarro‐Delgado, J.; Corpas, R.; Griñán‐Ferré, C.; Pallàs, M.; Sanfeliu, C.; Vázquez‐Carrera, M.; Hammock, B. D.; Feixas, F.; Vázquez, S. J. Med. Chem., 2021, 64, 5429–5446.

3‐McReynolds, C.; Morisseau, C.; Wagner, K.; Hammock, B. D. Adv. Exp. Med. Biol. 2020, 1274, 71–99.
Cortistatin Inhibits Intestinal Ion Secretion via Activating the Somatostatin‐2 Receptor in the Submucosal Neurons of the Mouse Colon
Authors: Liu Sumei
Published: May 2022

Background and Aims

Cortistatin (CST) is a somatostatin‐like neuropeptide that is found in both the central and peripheral nervous system. CST binds with high affinity to all somatostatin receptors and also has actions not shared by somatostatin. We previously found that CST is expressed by enteric neurons in the guinea pig ileum and acts as an inhibitory neurotransmitter in the submucosal plexus. The aim of the present study was to evaluate the effect of CST on neurogenic intestinal secretion.

Methods

Mucosal/submucosal flat‐sheet preparations from mouse proximal colon were mounted in Ussing flux chambers for measurement of short‐circuit current (Isc) as an indicator of mucosal secretion. Transmural electrical field stimulation (EFS) evoked characteristic biphasic increases in Isc, with an early rapidly rising phase followed by a sustained phase.

Results

Application of CST‐14, CST‐17, or CST‐29 to the basolateral side of the mucosal/submucosal preparation decreased baseline Isc in a concentration‐dependent manner with an IC50 of 90.5 nM, 52.1 nM, or 102.9 nM, respectively. The somatostatin‐2 receptor (SSTR‐2) antagonist CYN 154806 (1 mM) significantly suppressed CST‐14 (100 nM), CST‐17 (100 nM), or CST‐29 (100 nM)‐evoked ΔIsc by 36.0 ± 8.2% (n=5; P < 0.05), 47.2 ± 11.2% (n=5; P < 0.01), or 51.7.0 ± 8.4% (n=5; P < 0.01), respectively. The neuronal blocker tetrodotoxin (300 nM) significantly reduced CST‐14 (100 nM), CST‐17 (100 nM), or CST‐29 (100 nM)‐evoked ΔIsc by 36.5 ± 2.7% (n=5; P < 0.01), 48.9 ± 11.2% (n=5; P < 0.01), or 49.5 ± 3.6% (n=5; P < 0.01), respectively. All of the CSTs (1 nM – 1 mM) also produced a concentration‐dependent reduction of the EFS‐evoked biphasic secretory responses. The effect of CSTs on the EFS‐evoked responses was antagonized by CYN 154806 (1 mM). Immunohistochemical staining with a CST‐17 polyclonal antibody revealed expression of CST‐17 immunoreactivity (IR) in mouse colonic submucosal neurons. CST‐17‐IR was localized in cholinergic secretomotor neurons but was absent from non‐cholinergic secretomotor neurons and calbindin‐IR neurons.

Conclusions

The results suggest that CST acts at SST‐2 receptors in the submucosal plexus to suppress secretomotor neuronal excitability and neurogenic intestinal secretion in the mouse colon.
Myosin chaperone, UNC‐45A, is a novel regulator of intestinal epithelial barrier integrity and repair
Authors: Lechuga Susana , Cartagena‐Rivera Alexander X. , Khan Afshin , Crawford Bert I. , Narayanan Vani , Conway Daniel E. , Lehtimäki Jaakko , Lappalainen Pekka , Rieder Florian , Longworth Michelle S. , Ivanov Andrei I.
Published: May 2022

The intestinal epithelium creates a protective barrier separating the luminal content and the immune system in the gut. Leakiness of the intestinal epithelial barrier and inefficient healing of mucosal wounds represent common manifestations of inflammatory bowel diseases (IBD). Integrity of the gut barrier is mediated by different adhesive structures, including adherens junctions (AJ), tight junctions (TJ) and focal adhesion (FA). Functions of these adhesive structures depend on their association with the cortical actin cytoskeleton and particularly, to a major actin motor protein, non‐muscle myosin II (NM‐II). While biochemical mechanisms that regulate activity of NM‐II in epithelial cells have been extensively investigated, little is known about assembly of the contractile myosin structures at the epithelial adhesion sites. In this study, we focused on UNC‐45A, a cytoskeletal chaperone that is essential for proper folding of NM‐II heavy chains and myofilament assembly. We found abundant expression of UNC‐45A in human intestinal epithelial cell (IEC) lines and in the epithelial layer of the normal human colon. Interestingly, protein levels of UNC‐45A were decreased in colonic epithelium of patients with ulcerative colitis in parallel to diminished expression of NM‐IIA and NM‐IIC isoforms. CRISPR/Cas9‐mediated knock‐out of UNC‐45A in HT‐29 and SK‐CO15 IEC disrupted epithelial barrier integrity, impaired AJ and TJ assembly, and cell migration. Consistently, decreased UNC‐45 expression increased permeability of the Drosophila gut in vivo. The mechanisms underlying barrier disruptive and anti‐migratory effects of UNC‐45A depletion involved disorganization of the actomyosin bundles at epithelial junctions and the migrating cell edge. Loss of UNC‐45A also decreased contractile forces at epithelial junctions and matrix adhesions. Expression of deletion mutants revealed roles for the myosin binding domain of UNC‐45A in controlling IEC barrier function, junctional assembly and migration. Our findings uncover a novel mechanism that regulates integrity and restitution of the intestinal epithelial barrier, which may be impaired during mucosal inflammation.
Medium‐chain fatty acids inhibit jejunal SGLT1 activity in vitro and suppress glucose absorption in vivo in mice
Authors: Mizoguchi Akinori , Akiba Yasutada , Nishii Shin , Kaunitz Jonathan D.
Published: May 2022

Medium‐chain triglycerides (MCT) in diet improves glucose tolerance and insulin sensitivity, compared to long‐chain triglycerides (LCT). However, the direct effects of medium‐chain fatty acids (MCFA) on glucose transport have not been studied. Luminal glucose is absorbed in the small intestine through the electrogenic sodium‐dependent glucose transporter SGLT1 and electroneutral glucose transporter GLUT2. We thus examined the effects of luminal MCFA on electrogenic SGLT1 activity and glucose absorption in mice.

SGLT1 activity was assessed in the Ussing chambered, muscle‐stripped jejunal mucosa by measuring phlorizin (Phz)‐sensitive short‐circuit current (Isc) evoked by luminal addition of glucose (10 mM). Sodium propionate (C3), caproate (C6), caprylate (C8), caprate (C10), laurate (C12) and myristate (C14) were luminally added before or after glucose application. Furthermore, the effects of C10 on glucose absorption were assessed by oral glucose tolerance test (OGTT, glucose 2g/kg body weight, ig) with or without C10 treatment (1.94g/kg, ig) in conscious mice. Moreover, the portal venous (PV) blood glucose levels were measured though the PV cannulation, followed by intraduodenal bolus injection of glucose solution (100 mM) with or without C10 (10 mM) in the anesthetized mice.

Luminal pretreatment of C10 and C12 dose‐dependently inhibited the glucose‐induced Isc increase at IC50 = 1.02 mM and 1.06 mM, respectively. C8 also inhibited the glucose‐induced Isc increase, but less effective (IC50 8.4 mM), whereas C3, C6 and C14 had little effect. C10 (10 mM) also inhibited methyl α‐D‐glucopyranoside (αMG)‐induced Isc increase. Post‐treatment of C10 and C12 (10 mM) rapidly reduced the increased Isc by glucose, mimicking the inhibitory effect of Phz (1 mM). OGTT showed that glucose levels reached the peak at 30 min, while C10 pretreatment remarkably inhibited glucose increase. Furthermore, intraduodenal injection of glucose solution increased the PV glucose levels at 10 – 20 min, while the addition of C10 in the glucose solution abolished the response.

These data suggest that C10 and C12 act as the SGLT1 inhibitor. Since MCT releases C10 and C12 via digestion by pancreatic lipase, slower than glucose release by maltase/sucrase, free C10/C12 solution may be more effective therapeutics for the treatment of type 2 diabetes.
Deconvolution of Bulk RNAseq Data from Human Liver Samples Reveals a Metabolic Switch in Periportal Hepatocytes in Alcoholic Hepatitis
Authors: Manchel Alexandra , Srivastava Ankita , Bataller Ramon , Hoek Jan B. , Vadigepalli Rajanikanth
Published: May 2022

The prevalence of alcoholic liver disease (ALD) is rapidly increasing, however, few if any therapies are successful in the treatment of this disease as the development of such therapies is hampered by the lack of animal models. In this work, we aim to identify points of metabolic divergence driving the progression of ALD, which may provide therapeutic potential in the treatment of the disease. To determine the differences in metabolic landscapes across these progressing stages of ALD, bulk RNA‐seq was performed on alcoholic hepatitis (AH), progressing ALD, non‐alcoholic liver disease (NALD), and healthy control human liver samples. Hierarchical clustering of metabolic genes revealed N‐glycan metabolism, nucleotide metabolism and protein modification associated with AH samples, while oxidative phosphorylation, drug metabolism and retinol metabolism associated with healthy control and NALD samples. To further investigate which hepatic cell types may be involved in this shift in metabolic activity, we performed deconvolution of the bulk RNA‐seq data using a previously published single‐cell RNA‐seq dataset from healthy human liver tissue. Two imputed populations of hepatocytes, the periportal Hep 4 population and the mid‐lobular Hep 6 population, showed a decrease in cell proportions with increasing disease severity from healthy control to AH. Oppositely, there was an increase in cholangiocyte and hepatic stellate cell (HSCs) proportions with AH progression. Hierarchical clustering of metabolic genes from each cell type showed similar clustering to the bulk data, with groups of genes partitioned into (1) those upregulated with AH and (2) those upregulated in healthy control and NALD. Clusters of upregulated metabolic genes within the AH states from the Hep 4, cholangiocyte, and HSC populations all showed overrepresentation of metabolic pathways common to those upregulated in the bulk data, which differed from the Hep 6 population, whose enriched processes were downregulated in the bulk data. However, both hepatocyte populations displayed overrepresented metabolic pathways similar to the bulk data when analyzing the cluster of upregulated metabolic genes in the NALD and healthy states. These results indicate that the Hep 4, HSC and cholangiocyte populations act synergistically to upregulate specific metabolic genes in the AH state, while only the hepatocyte populations are responsible for the upregulation of metabolic genes in the healthy control and NALD states. It is crucial to further analyze the Hep 4 cluster of cells as they seem to be driving the metabolic switch activity separating the AH states from the NALD and healthy control disease states.
Unconventional GPCR‐PKA Signaling in the Hedgehog Pathway
Authors: Myers Benjamin , Happ John , Arveseth Corvin , Bruystens Jessica , Bertinetti Daniela , Nelson Isaac , Olivieri Cristina , Hedeen Danielle , Zhu Ju‐Fen , Capener Jacob , Broeckel Jan , Vu Lily , King C. C. , Ruiz‐Perez Victor , Veglia Gianluigi , Herberg Friedrich , Taylor Susan
Published: May 2022

The Hedgehog pathway controls tissue and organ development throughout animal evolution and drives several widespread skin and brain cancers. A pivotal step in Hh signal transduction is the activation of GLI transcription factors by the atypical G protein‐coupled receptor (GPCR) Smoothened (SMO). How SMO activates GLI has remained unclear for decades. The principal issue is that SMO appears to defy standard signaling paradigms utilized by nearly all other GPCRs.

Here we show that SMO employs a decoy substrate sequence to physically block the active site of the PKA catalytic subunit (PKA‐C) and extinguish its enzymatic activity. As a result, GLI is released from phosphorylation‐induced inhibition. Using a combination of in vitro, cellular, and organismal models, we demonstrate that interfering with SMO / PKA pseudosubstrate interactions prevents Hh signal transduction. The mechanism we uncovered echoes one utilized by the Wnt cascade, revealing an unexpected similarity in how these two essential developmental and cancer pathways signal intracellularly.

Our findings help to resolve a longstanding mystery in developmental and cancer signaling, and reveal a new mechanism of receptor‐kinase communication that may apply broadly throughout the GPCR superfamily. Our work also suggests new mechanism‐inspired therapeutic strategies to manage cancers driven by ectopic Hedgehog pathway activity.
STAT3 Nuclear Accumulation Due to Loss of RanBP2 May Contribute to Pathogenesis of Acute Necrotizing Encephalopathy
Authors: Spozarski Emilee , Resendes Karen
Published: May 2022

Ran Binding Protein 2 (RanBP2) contributes to the efficiency of nuclear import and export from its position at the cytoplasmic filaments of the nuclear pore complex. Here it can recruit import receptors, mediate efficient loading of cargo proteins onto importins, and serve as a docking site to efficiently recycle import and export complexes. Loss of function mutations in the RanBP2 gene are observed in many familial cases of acute necrotizing encephalopathy (ANE), a rare, sometimes fatal brain disease often occurring in infancy or early childhood following a viral infection. The influence of the RanBP2 mutation on the pathogenesis of ANE is unknown, but research points to a connection to the associated cytokine storm resulting from abnormal cytokine production. Specifically, the hyperactivation of the IL‐6/JAK/STAT3 cytokine signaling pathway has been observed in patients with ANE. We aimed to investigate a possible relationship between RanBP2 and STAT3 nuclear transport to determine if a loss of RanBP2 could influence the cellular localization of the transcription factor STAT3. Due to RanBP2’s importance in maintaining the efficiency of nuclear transport, its absence could impact either the nuclear or cytoplasmic localization of STAT3. siRNA knockdown of RanBP2 caused a 27% increase of STAT3 in the nucleus, suggesting increased STAT3 induced cytokine gene expression may occur without RanBP2. Our ongoing studies involve testing RanBP2 knockdown in conjunction with IL‐6 treatment to determine if there is an additional effect on the cellular localization of STAT3 with the activation of signaling. Accumulation of STAT3 in the nucleus alone could contribute to the onset of ANE, however STAT3 typically requires activation of signaling to increase gene expression. Our current results established increased nuclear STAT3 as a potential mechanism for mutation of RanBP2 to influence escalated cytokine production, perpetuating cytokine storm in ANE.
Leveraging AI Transfer Learning for Rapid and Accurate Identification and Quantification of Cellular Biomarkers in Microscopy Images
Authors: Harkness John H. , Theis Jacob J. , O'Keefe Will M. , Wade Grant W. , Lawton Kristy J.
Published: May 2022

Many fields of biology research and biomedical diagnostics utilize microscopy imaging to evaluate a broad range of cell types, subcellular structures, and pathology. The data from this imaging is crucial for evaluating experimental outcomes and inferring relationships between relevant biomarkers. With progress in imaging technology, researchers can now view much greater detail and gather larger datasets. Unfortunately, the ability to identify and quantify these biomarkers remains a bottleneck, often requiring significant hand annotation that is both time consuming and potentially introduces intra and inter‐rater error. While some automated solutions exist, they often still require a great deal of user involvement to correct inaccuracies. In recent years, computer vision emerged as an excellent solution to accurately detect regions of interest in microscopy images. The training of deep machine learning object detection models generally requires 1000s of user annotated images. Although this hand‐annotation is still a large upfront time investment, it produces a highly accurate model that provides much faster and more consistent analysis of frequently studied cell types and biomarkers. Regrettably, this method is less useful for researchers studying a novel cell type or biomarker with few training images available. To combat these limitations, we used a machine learning technique known as transfer learning to develop object detection capabilities for novel biomarkers using previously trained models with far fewer annotated images. We tested several different transfer learning methods with two of our existing cellular biomarker detection models, including wisteria floribunda agglutinin (WFA) and Parvalbumin (PV), to create new customized versions for different research labs. Our results indicated that several transfer learning methods worked well, with one performing consistently better than the others. We then tested this best method on a small number of androgen receptor (AR) images, a biomarker for which we did not have an existing detection model, and reached accuracy scores comparable to our original models for other biomarkers. Overall, by utilizing transfer learning we were able to reduce the number of required annotated images from several 1000s to 10s, with comparable or improved detection accuracy. Our results indicate that this computational method will greatly reduce time and resources needed to train accurate object detection models, thus broadening the reach of smart automated analysis to novel biomarkers and smaller datasets. Ultimately, this technology will allow the analysis bottleneck to catch up to the current hardware advances in the field, and rapidly advance biological research progress.
Human Hyoid Descent is Adaptive rather than Maladaptive for Swallowing Performance: An Experimental and Computational Argument
Authors: Orsbon Courtney P. , Ross Callum
Published: May 2022

Purpose

Distinctive features of the human supralaryngeal vocal tract have been argued to be adaptive for speech and maladaptive for swallowing. However, this maladaptive hypothesis has not been thoroughly tested and in fact is refuted by studies of normal human feeding physiology. This study validates a computational model of hyolingual biomechanics derived from an previously validated experimental nonhuman primate model system and examines how changes in hyolingual and craniofacial morphology over the course of human evolution and ontogeny affect hyolingual biomechanics during swallowing. In particular, we examine superoanterior excursion of the hyoid, which is often reduced in human patients with dysphagia and therefore used here as a surrogate for swallowing performance.

Methods

A computational model of hyolingual range of motion during swallowing in Macaca mulatta was developed in R using in vivo data. Model outputs were compared against in vivo macaque data to determine the model's goodness of fit using a receiver operating characteristics (ROC) curve; model predictive power was estimated using the area under the ROC curve. Several aspects of macaque craniofacial and hyoid morphology were then altered towards the human condition with consequent adjustments made to muscle lengths, and the model calculated hyoid displacement during swallowing for each condition. A model was considered viable if its range of motion included a target of hyoid excursion derived from in vivo macaque and human data.

Results

The macaque model had a sensitivity of 0.79, specificity of 0.82, and accuracy of 88% compared to in vivo data. A humanoid model predicts hyoid excursion within normal limits of healthy humans. Altering macaque morphology to approach the human condition by lowering the hyoid and making the symphysis vertical had little effect on model performance. Isolated shortening of the mandible significantly decreased hyoid excursion unless the symphysis was also made more vertical. Moreover, as the model was made more humanoid, both hyoid descent and physiologically maximal muscle shortening were necessary to approximate target hyoid excursion.

Conclusions

Hyoid descent—which is commonly assumed to be detrimental to swallowing—may actually maintain swallowing performance as the human face has shortened in recent evolutionary history. Moreover, because mandibular morphology affects suprahyoid muscle length, selective pressure to maintain swallowing performance may have resulted in the emergence of humanity’s characteristic vertical mandibular symphysis. Geometric constraints require human suprahyoid muscles to perform at their physiological limits when the hyoid is descended. Such extreme performance requirements may leave humans vulnerable to dysphagia not because of airway morphology alone but because of physiologic constraints.
Genetic polymorphisms and their influence on pancreatic adenocarcinoma progression
Authors: Jang Christine Y.
Published: May 2022

Pancreatic adenocarcinoma (PA) is one of the most fatal cancers since it shows little to no symptoms until it has metastasized. As is true with most malignancies, an early detection and diagnosis is imperative in order to help increase survival rate. Although there are several extant treatments available, their success depends on an early diagnosis. In order to identify genetic loci that may assist in early diagnosis, we analyzed 134 DNA sequences of patients with various stages of PA. NCBI’s Gene Expression Omnibus was used to obtain PA microarray data to identify overexpressed genes. The Sequence Read Archive (SRA) database was then used to provide sequenced data of a PA cohort which was then mapped, indexed and called. With these analyses, we were able to identify single nucleotide polymorphisms (SNPs) that were specific to PA primary, secondary, or metastatic tumors. These results advance our understanding of the development of PA and highlight target sites that could influence PA tumor stages.
Localizing the expression of Piezo2 in healthy and diseased lung tissue
Authors: Hennis Lauren , Hambright Parker , Eden Kristin , Binks Andrew , LeClair Renee
Published: May 2022

Intractable air hunger remains one of the most impactful symptoms of chronic cardiopulmonary disease and current pharmacological management remains limited. However, an endogenous mechanism for air hunger relief exists. Pulmonary stretch receptors (PSRs) responding to lung inflation reduce the brainstem drive to breathe and mitigate air hunger. Harnessing this mechanism could form the basis of a novel therapy. Recent animal studies suggest Piezo2, a mechanically‐gated cation channel, causes PSR depolarization when airway smooth muscle is stretched during inflation. We have assessed whether Piezo2 is associated with PSRs in healthy and diseased human airways to determine whether Piezo2 is a potential pharmaceutical target.

Methods

Human lung biopsies from NIH Lung Tissue Research Consortium were used for identification of Piezo2 receptors in emphysema, fibrosis, bronchiolitis, and normal lung tissue. Localization of Piezo2 was determined with immunofluorescent staining and imaged with confocal microscopy (Zeiss LSM880). As PSR fiber endings are associated with airway smooth muscle, co‐staining for Smooth Muscle Antibody (SMA) and Calcitonin Gene‐Related Peptide (CGRP) was performed to determine whether Piezo2 is associated with PSR.

Summary of results

Piezo2 is associated with airway smooth muscle (as evidenced by SMA, figure 1) and neuroepithelial bodies in normal human lung tissue (evidenced by cGRP, figure 2) and expression persisted in all disease states tested.

Conclusions

Piezo2 location is consistent with that of PSR in human lungs, making it a candidate for the mechanoreceptive transducer of human PSRs as has been demonstrated in animal studies. Expression persists in disease, making Piezo2 a potential therapeutic target, but it is still yet to be determined if Piezo2 is more significantly associated with airway or vascular smooth muscle in different disease states.
Surgical Anatomy of the occipital condyle and its relation to hypoglossal canal
Authors: Chentanez Vilai , Thintharua Pakpoom
Published: May 2022

Occipital condyles (OC) are two bony structures located laterally to the foramen magnum (FM) on the inferior surface of the occipital bone. It is commonly described as an ovoid structure. Craniovertebral junction (CVJ) is an area that connects the cranium to the upper cervical spine. It is bounded laterally by the jugular foramen (JF), hypoglossal canal (HC), and OC. Lesions at CVJ are difficult to manage because of their location and complex anatomic relations. Stability of CVJ after surgery in depends mainly on the resection of OC. HC has been used as a landmark for the extent of OC resection. It was claimed to locate in the middle part of OC. OC resection should not extend beyond HC to prevent CVJ instability. However, previous study showed that the HC was not always located at the middle part of OC. This study aimed to focus on the measurement of OC and its relation to HC in dry skull collected from Thailand for improving the successful surgical outcome.

One hundred dry skulls of equal number of both sex were examined for the shape and dimension of each OC. The location of intra and extra cranial orifice of HC (iHc and eHC) related to OC length and the distance to the posterior edge of OC (OCPE‐iHC, OCPE‐eHC) were determined. The results showed that the most prevalence of OC shape was oval‐like (33%) and the least was ring like (1%). Symmetrical OC shape was found in 46% of cases. The mean OC length was 22.13+2.51mm in male and 20.50+ 2.08 mm in female with a significant difference between sex (p=000). The OC width and height were 10.41+1.48 and 7.45+ 1.14 mm in male, 10.62+ 1.34 and 7.34+ 1.13 mm in female. No statistical significant difference of OC width and OC height between sex was found. The dimension of OC did not show any significant difference between sides. The location of iHC was mostly related to middle 1/3 of OC in 54% on the left side and located at the junction between anterior and middle 1/3 of OC in 36% on the right side. In contrast, eHC was located at anterior 1/3 of OC in 81% and 67% on the left and right side, respectively. None was related to posterior 1/3 of OC. Symmetrical location of iHC and eHC was found in 36% and 64% of cases, respectively. The distance OCPE‐iHC, OCPE‐eHC was 9.16+ 1.56, 14.1.0 + 1.98 mm in male and 8.83+ 1.62, 13.30+ 2.4 mm in female. Statistical significant difference between sex was found in OCPE‐eHC (p=0.011). In conclusion, the OC length is longer in male, the eHC located more anteriorly than iHC, therefore resection of the OC should not beyond posterior 1/3 of the OC length or about 7 mm from the posterior edge of OC. These morphometric data might be a benefit for surgical planning on resection of the occipital condyle to avoid injury to neurovascular structure and reserve the stability of craniovertebral junction.
Sympathetic Responses to Chemoreflex Stress in Lean Women with Polycystic Ovary Syndrome
Authors: Chen Celine , Coovadia Yasmine , Berbrier Danielle , Adler Tessa E. , Usselman Charlotte W.
Published: May 2022

Polycystic ovary syndrome (PCOS) is the most common endocrinopathy among reproductive‐aged women, affecting ~10% of premenopausal women. Women with PCOS present with myriad cardiovascular risk factors including greater muscle sympathetic nerve activity (MSNA), however this has yet to be assessed during periods of reactivity. Interestingly, there is limited evidence to suggest that testosterone (i.e. free testosterone), which is elevated in ~75% of women with PCOS, may play a role in mediating greater MSNA in PCOS. Therefore, the purpose of this study was to test the hypotheses that (a) women with PCOS would exhibit greater MSNA reactivity in response to acute stress relative to control (CTRL) women, and (b) MSNA reactivity to stress would be positively correlated with elevated testosterone levels. We tested 4 lean women with PCOS and 4 CTRL women (age: 20±1 vs. 23±3 yrs, P=0.12; BMI: 25±3 vs.24±2 kg/m2, P=0.41). Heart rate (3‐lead ECG), beat‐by‐beat blood pressure (Finapres) and MSNA (microneurography at the peroneal nerve) were measured during 3‐min of quiet rest and during a maximal voluntary end‐expiratory breath hold. Analyses were performed comparing baseline to the latter half of the apnea, in which sympathetic reactivity was the greatest. Baseline mean arterial pressure (MAP) was similar between PCOS and CTRL (87±7 vs.86±5 mmHg, P=0.97), as were MSNA burst frequency (BF) and MSNA burst incidence (BI) (BF: 7±5 vs. 10±7 bursts/min, P=0.51; BI: 10±8 vs. 16±10 bursts/100hb, P=0.50). Apnea induced increases in MAP and MSNA in both groups (main effects of condition: MAP, P<0.05; BF, P<0.01; BI, P<0.01). However, MSNA during apnea was higher in women with PCOS compared to CTRL women (BF: 55±7 vs.35±12 bursts/min, P<0.05; BI: 81±18 vs.50±13 bursts/100hb, P<0.05). When all participants were pooled together, a trend towards a positive modest correlation between serum free testosterone concentrations and MSNA BI during apnea was observed (R2=0.33; P=0.18). These preliminary data provide some evidence that elevated testosterone levels observed in women with PCOS may contribute to greater sympathetic reactivity, although this requires further study. Importantly, despite greater MSNA responses to apnea, women with PCOS demonstrated similar BP responses to the CTRL group. These findings suggest that these young lean women with PCOS may benefit from an offsetting of high MSNA, which may be mediated by blunted neurovascular transduction.
Induction of Cytochrome P450 (CYP) 1A2 and CYP3A Influences Sunitinib Metabolism and Bioactivation in Primary Human Hepatocytes
Authors: Jackson Klarissa D. , Lee Jonghwa , Beers Jessica L. , Nardone‐White Dasean T.
Published: May 2022

Sunitinib is an orally administered tyrosine kinase inhibitor used to treat renal cell carcinoma, gastrointestinal stromal tumors, and pancreatic neuroendocrine tumors. The product label for sunitinib carries a boxed warning for idiosyncratic hepatotoxicity; however, the mechanisms of this toxicity remain unclear. We have previously shown that cytochrome P450 (CYP) 1A2 and CYP3A4 catalyze the metabolic activation of sunitinib via oxidative defluorination to form a chemically reactive, potentially toxic quinoneimine, trapped as a glutathione (GSH) conjugate. The purpose of this study was to determine the effects of CYP1A2 and CYP3A induction on sunitinib bioactivation in human hepatocytes in vitro. Sandwich‐cultured human hepatocytes from a single donor were pretreated with CYP1A2 and CYP3A inducers, omeprazole (50 μM) and rifampicin (25 μM), respectively, for 72 hours followed by incubation with sunitinib (10 μM) for 24 hours. Sunitinib metabolites were analyzed by liquid chromatography – tandem mass spectrometry. CYP1A2 induction by omeprazole reduced sunitinib levels by 48% and increased formation of the primary active metabolite N‐desethylsunitinib (M1) by 2.0‐fold, compared to control without inducer. CYP3A induction by rifampicin caused depletion of sunitinib by 93% and led to a corresponding increase in M1 formation by 4.9‐fold. For defluorosunitinib (M3), omeprazole increased M3 levels by 7.4‐fold, but rifampicin decreased M3 formation by 72%, compared to control. In addition, we identified a putative quinoneimine‐cysteine conjugate (M6) of sunitinib in hepatocytes formed as a downstream metabolite of the quinoneimine‐GSH conjugate. Omeprazole increased M6 formation by 7.7‐fold, but rifampicin had a minimal effect on M6. The non‐selective P450 inhibitor 1‐aminobenzotriazole (1 mM) reduced each of these metabolites (M1, M3, M6), confirming the involvement of P450‐mediated metabolism. Collectively, these findings indicate that induction of CYP3A enhances sunitinib metabolic clearance via N‐dealkylation to M1, and CYP1A2 induction increases sunitinib bioactivation to the reactive, potentially toxic quinoneimine metabolite. Modulation of P450 activity due to enzyme induction or other environmental and genetic factors may influence the risk of metabolism‐mediated adverse reactions associated with sunitinib.
Autocrine P2X4 receptor activation in RBCs drives oxygen‐dependent hyperemic responses in mouse skeletal muscle capillaries
Authors: El‐Lakany Mohammed A. , Kowalewska Paulina M. , Sandow Shaun L. , Ellis Christopher G. , Welsh Donald G.
Published: May 2022

Microvascular blood flow is tightly coupled to tissue function and when disturbed, organ insult ensues. Changes in red blood cell (RBC) oxygenation are thought to be key to the initiation of robust microvascular responses, a process mechanistically linked to ATP release from RBCs and conducted signalling. The RBC signaling cascade governing ATP release was the focus of this study, and of note is the role of ATP‐activated P2X4 receptors. Using an in‐vivo mouse muscle preparation (extensor digitorum longus, EDL) and a focally delivered oxygen stimulus via a rapid gas exchange chamber, we probed capillary hemodynamics, prior to and following purinergic receptor antagonism. Dropping oxygen tension from 7% to 2% reduced RBC oxygen saturation in surface capillaries while increasing RBC supply rate. Incubating the EDL with PPADS (non‐selective purinergic receptor antagonist, 30 µM) or 5‐BDBD (selective P2X4 antagonist, 20 µM) reduced baseline RBC supply rate by ≍ 40% and 47%, respectively, and attenuated the capillary responses to the oxygen stimulus by ≍ 5 and 3 RBC/s, respectively. This blunting predictably enhanced the drop in capillary RBC oxygen saturation, as more oxygen was drawn per RBC to meet tissue demand. Immunofluorescence labeling revealed robust expression of P2X4 receptors in RBCs but not endothelial cells. Together, this data begins to elucidate a role for RBC P2X4 receptors, potentially in a positive feedback loop with pannexin channels, to dynamically regulate demand‐supply coupling in skeletal muscle.
Autophagic State Confers Facultative Stem Cell Capacity in The Intestinal Epithelium
Authors: Parham Louis R. , Johnson Nicolette , Na Jeeyoon , Klochkova Alena , Williams Patrick , Li Ning , Whelan Kelly A. , Lengner Christopher J. , Hamilton Kathryn E.
Published: May 2022

Background

The intestinal epithelium is a highly proliferative tissue that undergoes complete turnover in 3 to 5 days. The presence of intestinal stem cells expressing the canonical Wnt target gene Lgr5, known as active intestinal stem cells (a‐ISCs), are responsible for generating daughter cells that differentiate into specialized epithelial cells to carryout various functions in the intestine. The proliferative nature of the intestinal epithelium renders it susceptible to DNA damage‐inducing injury, such as high‐dose irradiation and chemotherapy. Although a‐ISCs and early daughter cells are killed by these insults, a radio‐resistant population of facultative intestinal stem cells (f‐ISCs) can regenerate lost a‐ISCs to restore homeostasis following injury. The functional characteristics of f‐ISCs are becoming increasingly understood, however, whether any epithelial cell can act as an f‐ISC is less clear. One reason for this ambiguity is a heavy reliance on Cre‐driven reporter mouse models with low recombination efficiency. Our objective was to find a functional marker to identify f‐ISCs based on cellular state rather than gene expression. The autophagy pathway has been shown to protect against DNA damage and irradiation‐induced apoptosis in the intestinal epithelium. Furthermore, recent studies demonstrate a requirement for the autophagy pathway during cellular de‐differentiation in both gastric chief and pancreatic acinar cells following metaplasia‐inducing injury. Given that autophagy plays roles in both radio‐resistance and cellular plasticity, two integral features of f‐ISCs, we hypothesized that autophagic activity could serve to identify cells with f‐ISC capacity.

Methods and Results

Using the autophagic vesicle tracer dye CytoID combined with fluorescence‐activated cell sorting, we demonstrate that intestinal epithelial cells with high levels of CytoID exhibit increased organoid‐formation efficiency (OFE) compared to CytoID ‘low’ cells. Single cell sequencing reveals that the CytoID high population is largely composed of secretory cells including Paneth, Goblet, Tuft, and Enteroendocrine cells, whereas the CytoID low population consisted mainly of a‐ISCs and absorptive Enterocytes. Using reporter mice and antibodies against cell surface receptors, we observe that CytoID can identify cells with high OFE within Enteroendocrine, Paneth, and Goblet cell lineages. Finally, we demonstrate that autophagy is required for the enhanced organoid‐formation observed in CytoID high cells by plating these cells in the presence of the lysosomal inhibitor Bafilomycin A1.

Conclusions

Our new data support the notion that epithelial cells in a high autophagic state are biased to secretory lineages and that autophagy status functions as a lineage agnostic marker of f‐ISCs. Furthermore, our data suggests that autophagy is required for organoid formation in these lineages.
Decreasing tracheal system volume during intermolt development in grasshoppers
Authors: Sheahan Andrew J. , Kirkton Scott D.
Published: May 2022

One major reason for the evolutionary success of insects is their unique tracheal respiratory system. However, during development when oxygen demands are high, the tracheal system may not be able to deliver adequate oxygen to the tissues. For example, in developing American locusts (Schistocerca americana), body mass can double during the intermolt period. Also during the intermolt period, jumping performance decreases while jumping muscle lactate production increases, suggesting increased oxygen delivery problems at the end of the instar. We hypothesized that decreased oxygen delivery during the instar is due to increasing body mass within the rigid exoskeleton compressing the air‐filled tracheal system. To test this hypothesis, we identified early and late female sixth instar S. americana. Similar to previous work, body mass increased during the instar from 0.73g to 1.3g (78% increase) while femur length remained constant at 19.3mm. Using a Bruker SkyScan1272 micro‐computed tomography (uCT) system, we scanned early and late sixth instars (n=5). uCT image (TIF) files were reconstructed using NRecon and the tracheal system was segmented using Amira 3D and Biomedisa. Total body and tracheal volumes were analyzed using Amira 3D. Although total body volume increased by 53% during the instar, tracheal volume decreased by 145%. Early instar tracheal volume was 0.31 mL, while late instar tracheal volume was 0.05 mL. Therefore, the relative proportion of tracheal volume to body volume ratio decreases from 44% to 5.2% during the instar. Our finding that tracheal volume decreases during the intermolt period supports previous work showing that late stage grasshoppers rely more on anaerobic metabolism during repeated jumping.