bluuebunny/crossref_metadata_2025 huggingface dataset:
Initial Characterization of the Extra Cytoplasmic Function Sigma Factor (FpvI) and Sigma Regulator (FpvR) Anti Sigma Domain Interaction in Pseudomonas aeruginosa
Authors: Zak Peyton , Sultana Tajnin , Colbert Christopher
Published: May 2022

Objective

The objective of this study is to structurally characterize the interaction between the Pseudomonas aeruginosa PA01extra‐cytoplasmic‐function (ECF) sigma factor, FpvI, and the sigma regulator, FpvR.

Hypothesis

The extra‐cytoplasmic‐function (ECF) sigma factor, FpvI, interacts with high affinity with the N‐terminal anti‐sigma domain of FpvR. It can be purified to homogeneity and used for structural studies by X‐ray crystallography.

Transcription of the pyoverdine transport system of Pseudomonas aeruginosa PA01 is mediated by Cell Surface Signaling (CSS). This process involves binding of the siderophore ferric‐pyoverdine to the outer membrane transporter FpvA. This event triggers the regulated intramembrane proteolysis of the sigma regulator, FpvR. Release of the FpvR anti‐sigma domain (ASD) in complex with the extra‐cytoplasmic‐function (ECF) sigma factor FpvI, directs RNA polymerase to the promoter of FpvA to initiate the transcription. The molecular details of the interaction between FpvR ASD and FpvI are not known. We are using a dual expression plasmid to produce the FpvR ASD and a his‐tagged construct of FpvI. Immobilized metal affinity chromatography (IMAC) followed by size exclusion chromatography is used to purify the complex. We have characterized the complex by Circular dichroism (CD) to show it is well folded. This protein is being further characterized by sparse matrix crystal screening and Small‐angle X‐ray scattering (SAXS).
Effects of a Novel Hydroalcoholic Extract of Lens Culinaria Against the Dexamethasone‐Induced bone loss and Chemotherapy Cytotoxicity in Murine and Human Cells
Authors: Tricarico Domenico , Antonacci Marina , Di Benedetto Jacopo R. , Maqoud Fatima , Scala Rosa , Centoducati Gerado , Colonna Nicola , Leonetti Francesco
Published: May 2022

Lens culinaria showed nutritional health benefit in humans. Here, we evaluated the effects of a novel hydroalcoholic extract (70% ethanol, 1E030521DIPFARMTDA) from Lens culinaria (Terre di Altamura SRL, Altamura BA) to prevent cytotoxic damage from chemotherapeutic agents such as staurosporine, a non‐selective inhibitor of tyrosine kinases, cisplatin the antineoplastic alkylating agent, doxorubicin as antimitotic, and the glucocorticoid dexamethasone targeting osteoblast.

On renal Hek293, staurosporine caused a significant reduction from ‐20% to ‐60% (p <0.05) in cell proliferation and the extract was significantly effective in bringing these values between 70% and 100% (p <0.05) with comparable activity to that of diazoxide. Also, in the clonogenic assay, the extract was effective to prevent the reduction of colonies numbers caused by staurosporine.

Cisplatin caused a slight reduction of ‐10% in Hek293 cell proliferation and a significant reduction of ‐30% in SHSY5Y cell proliferation respectively after 48 and 72 hours of incubation time. In both cases, the extract showed a significant cytoprotective effect. On the primary culture of murine osteoblasts, doxorubicin reduced survival by ‐30%, dexamethasone reduced it by ‐15%. The extract was significantly effective in preventing cell count‐reduction induced by dexamethasone but not by doxorubicin.

A UV/VIS spectroscopy analysis was carried out. We obtained three peaks at the wavelength of 350, 260 and 190 nm which correspond to families of compounds such as flavonoids, proanthocyanins and salicylates.

Therefore, this novel extract prevented the cytotoxic damage induced by cisplatin and staurosporine on the renal cell line, and by cisplatin also on the neuronal cell line by clonogenic assay and DNA staining. The extract was significantly effective in preventing the reduction of cell proliferation induced by dexamethasone in murine osteoblasts suggesting of potential use in bone loss diseases. It is not yet clear which of these families molecules composing the extract is responsible for these effects or if there is a synergistic action.
Reverting Colitis Symptoms by Oral Administration of L. reuteri Expressing CXCL12 Acting Through Novel Pathway Demonstrated in Two Experimental Animal Models
Authors: Öhnstedt Emelie , Donas Cuadra Cristian , Lofton Tomenius Hava , Frank Peter , Vågesjö Evelina , Roos Stefan , Phillipson Mia
Published: May 2022

Introduction

In USA and Europe, it is estimated that more than 3 million people are suffering from inflammatory bowel disease (IBD) and another 1.7 million cancer patients treated with checkpoint inhibitors (ICI) are diagnosed with manifest ICI‐induced colitis. The current treatments with steroids and available biologics act systemically and possess limitations with loss of response and severe adverse events and may even limit the effect of the ICI therapy. Thus, new local‐acting therapies are needed to serve the full unmet need amongst these patients.

We have recently published that oral administration of L. reuteriR2LC to mice increases IgA production in Peyer’s patches, resulting in modified gut microbiota, altered physiology and immune cell phenotype in the colon and prophylactic administration reduced inflammation in the DSS‐induced colitis.

Methods

, the novel drug candidate, ILP100, which is a genetically modified lactic acid bacteria that express the chemokine CXCL12 were generated and evaluated as a therapeutic in two different models of colitis, the DSS‐model and a model of checkpoint inhibitor induced colitis (using aPD1 and aPDL1).

Results

, Peroral administration of ILP100 to mice with overt colitis interrupts disease progression and improves symptoms assessed as disease activity index (DAI) and colon shortening. Further analysis showed that treatment with ILP100 increased infiltrating immune cells expressing TGF‐ß and IL‐10 as well as other tolerogenic immune cell populations including Treg and Tr1 in the colonic lamina propria. There were no changes in the small intestinal lamina propria or mesenteric lymph nodes and there was no systemic exposure detected. Further, efficacy comparison studies demonstrate a reduced severity and a faster recovery of the ILP100 treated mice compared to TNF‐α inhibitor and α4β7‐inhibitor treated mice. The findings have been confirmed over a large dose range. Even though the ICI‐induced colitis did not manifest in symptoms such as body weight loss or blood in feces, fibrosis was developed measured as a reduction of the mean colon length of 12‐18 % in the ICI‐treated groups compared to control, with a tendency to a reduced colon shortening in the ICI + ILP100‐treated groups. Histological analysis of intestines samples from the checkpoint inhibitor is currently ongoing.

In conclusion

, we demonstrate that peroral administration of ILP100 efficiently ameliorate overt colitis in the DSS‐model by inducing healing through a local shift into an anti‐inflammatory and tolerogenic immune signature in the colon mucosa. Further, our data suggest that peroral administration of ILP100 ameliorate colitis in an ICI model.
Biophysical and Structural Characterization of Glucan Phosphatase SEX4 from Zea mays
Authors: Raththagala Madushi , Frenett Merissa , Vander Kooi Craig W.
Published: May 2022

Plants store energy in starch, a glucan made of two polymers of glucose‐ amylose and amylopectin. Phosphorylation, the only known in vivo modification of starch, controls the structure, function, and physicochemical properties of the starch granule and thereby modulates the starch degradation at night. Reversible starch phosphorylation has been increasingly appreciated as a key regulatory step controlling diurnal starch metabolism. Glucan phosphatase Starch Excess4 (SEX4) is an allosteric enzyme that binds to and dephosphorylates starch at the C6 and C3 positions of glucose residues. Plants lacking glucan phosphatases exhibit excess starch and starch with increased phosphorylation. However, our understanding of how SEX4 binds to the semicrystalline starch stricture and regulates the dephosphorylation mechanism is incomplete. Previously, the x‐ray crystal structure of catalytically inactive Arabidopsis thaliana SEX4 bound to maltohexaose has been described. Here we present the x‐ray crystal structure of wild‐type SEX4 from Zea mays to a resolution of 2.9 A. Utilizing hydrogen‐deuterium exchange spectroscopy and differential scanning fluorometry, we intend to understand how SEX4 interacts with its glucan substrate for an efficient starch dephosphorylation mechanism. We also investigated the ability of SEX4 to enhance in‐vitro starch degradation. Our findings reveal that SEX4 from cereal crops such as Zea mays has the increased ability to enhance starch degradation than the SEX4 orthologs from storage crops.
The diagnostic and prognostic value of COL5A2 high expression and hypomethylation in recurrent low‐grade glioma
Authors: Duan Hubin , Hao Chunyan , Wang zhuangzhuang , Xue Wen , Li Mingli , Qiao Binjun , Zhang Jinbao , Jing Zhijie , Chen Xianghui , Zheng Juan , Qiao Xiaohong
Published: May 2022

Objection

To investigate the diagnostic and prognostic value of COL5A2 high expression and hypomethylation in recurrent low‐grade glioma.

Methods

Four independent databases, Chinese Glioma Genome Atlas(CGGA), The Cancer Genome Atlas(TCGA), Gravendeel and Rembrandt, were used to analyze the differential expression and prognostic of COL5A2 in different grades and types (primary and recurrent) of LGG. And further analyzed the differential expression and prognosis of COL5A2 and its methylation between primary and recurrent LGGs (including their pathological subtypes), and correlation between COL5A2 expression and methylation in low‐grade gliomas and their clinical phenotypes. SPSS and GraphPad Prism statistical software were used to assess the prognostic and diagnostic value of COL5A2 expression and its methylation in recurrent LGG by binary Logistic regression, Cox regression and Receiver Operating Characteristic (ROC) curve. The correlation between COL5A2 expression and immune cell infiltration in TCGA for LGG was analyzed using TIMER and TISIDB database. Gene Set Cancer Analysis (GSEA) was used to investigate the signaling pathway of COL5A2.

Results

COL5A2 is highly expressed in different grades of gliomas and recurrent LGG, and overexpression of COL5A2 is detrimental to the prognosis of LGG. The expression level of COL5A2 was negatively correlated with its methylation level, and the hypomethylation of COL5A2 was not conducive to the prognosis of LGG. COL5A2 may promote the recurrence and progression of LGG through post‐ transcriptional methylation modifications and immunoinfiltration, and may provide a reference for the diagnostic threshold of recurrent LGG.

Conclusions

Our study identified COL5A2 overexpression and hypomethylation as independent risk factors and prognostic factors for different grades of glioma and recurrent LGG, and has some reference value for the diagnosis of recurrent LGG.
Contribution of stress granules and spliceosomal components to the formation of chemoresistance in ovarian cancer cells
Authors: Shnaider Polina , Malyants Irina , Ivanova Olga , Arapidi Georgij , Lagarkova Maria , Govorun Vadim , Shender Victoria
Published: May 2022

The emergence of chemoresistance in cancer cells is one of the main reasons for the high mortality from oncological diseases. We have shown that signaling molecules from dying cancer cells contribute to a more aggressive therapy‐resistant phenotype. However, the molecular mechanisms underlying this effect remain elusive.

Here we aimed to find out which proteins secreted by dying tumor cells get into recipient cells and to determine the molecular cascades triggered in response to incubation with therapy‐induced secretomes. We modified the SILAC (Stable Isotope Labeling by Amino acids in Cell culture) technology. A heavy‐labeled primary culture of ovarian cancer cells was treated or untreated with cisplatin for 48 h. Then using ultrafiltration cartridges, a fraction of extracellular vesicles (EV) were isolated from culture media. The obtained EV were resuspended in a medium containing Medium‐labeled amino acids and added to the recipient chemonaive unlabeled tumor cells for 24 h. During incubation, recipient cells absorbed EV with Heavy‐labeled proteins from donor cells and synthesized new proteins with Medium label. Then, we performed proteomic analysis of recipient cells. In total, we identified 4224 proteins. Interestingly, dying tumor cells secrete and recipient cells absorb a large number of spliceosomal and ribosomal proteins along with translation initiation complex. These proteins are often presented in stress granules. In response to EV from dying tumor cells, recipient cells upregulated the translation of genes responsible for the regulation of the cell cycle and oxidative phosphorylation.

To our knowledge, this is the first report about the secretion of stress granules and spliceosomal components into the extracellular space and their internalization in other cells. To confirm these data we performed transfection of donor cells with constructs encoding the fluorescent proteins TIA1 (protein of stress granules) and SRSF4 (protein of the spliceosome). We showed that both proteins are re‐located together from the nucleus into the cytoplasm under the cisplatin treatment. Then, we demostrated the absorption of fluorescent‐labeled proteins TIA1 and SRSF4 by recipient cells after incubation with therapy‐induced EV. Our findings demonstrate that in response to cisplatin treatment tumor cells secrete spliceosomal components into the extracellular space (presumably as a part of stress granules), then these components are absorbed by recipient chemonaive cells and trigger a restructuring of metabolism and cell cycle.
Role of RNA Binding Protein with Multiple Splicing (RBPMS) Variants in Cell Proliferation, Invasion and Angiogenesis in Ovarian Cancer
Authors: Rabelo‐Fernandez Robert J. , Noriega Ricardo , Rivera Mariela , Flores Marienid , Santana Yasmary , Valiyeva Fatma , Vivas‐Mejia Pablo
Published: May 2022

Worldwide, the number of cancer‐related deaths continues to increase due to the ability of cancer cells to become chemotherapy‐resistant and metastasize. For women with ovarian cancer, a staggering 70% will become resistant to the front‐line therapy, cisplatin. RNA binding protein with multiple splicing (RBPMS) is a member of family proteins that bind to the nascent RNA transcripts regulate their processing, which includes splicing, transport, localization and stability. Evidence indicates that RBPMS interact with c‐Fos, a member of the AP‐1 family that dimerizes with c‐Jun to stimulate AP‐1 transcriptional activity. Furthermore, RBPMS interacted with transcription factor Smad3, which bind to c‐Jun increasing AP‐1 transcriptional activity. Three major RBPMS isoforms have been described, RBPMS‐A (21.8 kDa), RBPMS‐B (22.4 kDa) and RBPMS‐C (24.2 kDa). Reports have shown that each RBPMS isoform interacts with proteins and/or RNAs responsible for cell growth, proliferation, and tumor progression. Previous reports of our laboratory have found that RBPMS expression decrease in cisplatin resistance compared with cisplatin sensitive cells. However, the specific RBPMS isoform responsible of this observation has not been elucidated and less is known about its role on invasiveness ability and angiogenesis capacity. We overexpressed each RBPMS variants (A, B and C) in the cisplatin resistant ovarian cancer cells, A2780CP20, and performed cell viability, cell proliferation and invasion assays. Significant reduction in the number of colonies and their area were observed in both isoform A and isoform C clones, compared with isoform B and control empty vector clones. However, clones of isoform A were more sensitive to cisplatin treatment in comparation with isoform B and C clones. In vivo experiments showed a significant reduction in tumor growth between RBPMS A and C when compared with the controls. Tumor tissues were subjected to immunohistochemical analysis for proliferation (Ki67) and angiogenesis (CD31). Elevated signals of both antibodies were detected in tissues from control groups when compared with RBPMS A and C overexpressing tumor tissues. These studies along with the RNAseq suggest that RBPMS, regulate transcripts involved in cell proliferation and invasion capacity of ovarian cancer cells. Moreover, RBPMS A and C represses uncontrolled proliferation, invasiveness, and angiogenesis in ovarian cancer cells. However, only the isoform A contributed to the cisplatin sensitivity of ovarian cancer cells. Future efforts on this project will be focused on determining the RBPMS isoform that represses AP‐1 (c‐Fos and c‐Jun) dependent gene regulation in cisplatin sensitive ovarian cancer cells.
Lysine‐2,3‐aminomutase and a newly discovered glutamate‐2,3‐aminomutase produce β‐amino acids to overcome salt stress in methanogenic archaea
Authors: Sadler Zoie , Tunckanat Taylan , Allen Kylie , Gendron Aleksei
Published: May 2022

Many methanogenic archaea synthesize N ε ‐acetyl‐β‐lysine and/or β‐glutamate as osmolytes that allow survival in high salinity environments. Methanococcus maripaludis C7 contains two genes, MmarC7_0106 and MmarC7_1783, encoding radical S‐adenosylmethionine(SAM) enzymes with sequence similarity to lysine‐2,3‐aminomutase (KAM). MmarC7_0106 is immediately upstream of an acetyltransferase and was expected to be the KAM involved in the biosynthesis of N ε ‐acetyl‐β‐lysine. To confirm this, the gene was cloned and expressed with a hexahistidine tag in E. coli, followed by anaerobic purification and biochemical characterization. Although the bacterial KAMs involved in lysine degradation in Clostridium subterminale and a post‐translational modification of elongation factor P in E. coli have been well‐characterized, the archaeal KAM involved in compatible solute biosynthesis had never been studied in vitro before. In the presence of L‐lysine, SAM, and dithionite at 37°C, this archaeal KAM had a k cat = 0.42 s ‐1 and a K m = 17.8 mM. The product was shown to be 3(S)‐β‐lysine, which is like the well‐characterized Clostridium KAM as opposed to the E. coli KAM that produces 3(R)‐β‐lysine. The archaeal KAM was highly specific for L‐lysine, with no activity observed with D‐lysine or most other amino acids. However, low aminomutase activity was observed with L‐arginine. We further report the function of MmarC7_1783, a putative radical SAM aminomutase with a ~160 amino acid extension at its N‐terminus. Bioinformatic analysis of possible substrate binding residues suggested a function as glutamate‐2,3‐aminomutase, which was confirmed here through heterologous expression in a methanogen followed by detection of β‐glutamate in cell extracts. Finally, we investigated the differences in amino acid compatible solute usage amongst M. maripaludis C7 (contains both aminomutase genes) and M. maripaludis S2 (contains only the canonical KAM gene) in different salt concentrations. Taken together, this work defines the biosynthetic routes for β‐amino acids in methanogenic archaea and expands the importance and diversity of radical SAM enzymes in all domains of life.
Anti‐Epileptic Drug and Teratogen Valproic Acid Induces Microglial Cell Death in a Valproic Acid Murine Model of Autism
Authors: Loyola Allison , Bhushan Alok , Awale Prabha S.
Published: May 2022

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition that affects approximately 1 in 44 children in North America, and its association with neuronal connectivity is an area of intense research. Valproic acid (VPA) is a multi‐target drug widely used to treat epilepsy. It is classified as a teratogen and as a histone deacetylase inhibitor (HDACi) and fetal exposure to VPA increases the risk of ASD. The VPA model has been well characterized for behavioral and neuronal deficits, including hyperconnectivity. The cause of hyperconnectivity is poorly understood however, it is speculated that lack of pruning may be partly responsible for this anomaly. Microglia, the principal immune cells of CNS regulate dendrite and synapse formation during early brain development. The effect of VPA on microglia during early development has not been well characterized and may provide potential hints regarding the etiology of this disorder. Preliminary studies from our lab indicate that VPA reduces microglial number during peak periods of postnatal synaptogenesis in the mouse brain primary cortex. Nevertheless, the cause of this reduction is not known. Therefore, in this study, we determined the mechanism for reduced microglial numbers in the VPA model of autism.

Pregnant BALBc mice were administered 600 mg/kg of VPA by subcutaneous injection on day 13.5 of gestation. We harvested the mice brains at embryonic (E) day 14, E17, and postnatal day 1 (PD1). Brain tissue was double immunolabeled for microglia (IBA1) and cell death marker (TUNEL). We found a gradual increase in TUNEL staining beginning E14 to PD1 compared to age‐matched controls. The mechanism for reduced microglial number may be due to cell death. Thus, our findings possibly have pathophysiological/pathogenic implications in autism and may, ultimately, lead to the discovery of new therapeutic target(s) for autism.
Downstream Branches of the Fibroblast Growth Factor Signaling Pathway Act Interdependently to Shape the Face
Authors: Hanne Nicholas J. , Hu Diane , Allen Charlie , Shakir Bilal , Liu Wei , Hallgrímsson Benedikt , Marcucio Ralph
Published: May 2022

The musculoskeletal tissues of the head develop from primordia, composed of neural crest cells, that evaginate and fuse to form the structures of the face. This occurs through a complex process controlled not only by regional differences in cellular proliferation, apoptosis, and differentiation, but also directed cellular migration and oriented cellular behavior. Disruption of normal development of the facial primordia can either prevent apposition and fusion of craniofacial primordia, leading to malformations such as cleft lip and/or palate. Previously, we found that stimulating signaling by fibroblast growth factor (FGF) in neural crest cells of chick embryos slowed proximodistal expansion of the facial primordia and widened the midface. Activation of the FGF pathway decreased cell proliferation in the frontonasal process (FNP) and disrupted cellular orientation, which were correlated with shape of the FNP. Blocking FGF signaling with a chemical inhibitor caused severe truncation of the FNP and decreased proliferation. Our previous results confirm that FGF signaling affects craniofacial morphogenesis, seemingly by altering multiple cellular functions. FGF receptors are receptor tyrosine kinases which utilize many common downstream signaling cascades that direct cellular proliferation, apoptosis, differentiation, migration, and oriented cell behaviors, but it is not clear which downstream pathway explains our previous results. In this study we used chemical inhibitors to block three pathways downstream of FGF receptor activation to determine their effect on facial morphology and cellular activity in the FNP of the chick embryo. Blocking MAPK, PLCγ, and PI3K pathways individually caused similar mild truncations of proximo‐distal growth in the FNP over 24 and 72 hours, and similar reductions in cellular proliferation. Cellular orientation was not significantly affected by any treatment. Blocking all three downstream pathways together caused a more severe proximo‐distal truncation of growth of the FNP, but not as severe as blocking Fgf signaling entirely. These results suggest that the downstream branches of the FGF pathway act inter‐dependently and in a non‐linear fashion to shape the face. Soon we will measure cellular orientation in the inhibitor cocktail treated FNPs and quantify the changes in FNP shape using three‐dimensional geometric morphometrics.
Chronic Obstructive Pulmonary Diseased Human Lung Fibroblasts Secrete Increased Matrix Metalloproteases ‐12 and ‐8
Authors: Mattioli Patrisia , Koleoso Olufela , Otto Ashley , Saini Twinkle , Shind Christine , Baus Emily , Bhatt Amy , D'Angelo Marina
Published: May 2022

According to the American Lung Association, 16.5 million people have Chronic Obstructive Pulmonary Disease (COPD), the third leading cause of death in the United States. COPD is a condition in which the airways of the lung are inflamed and the alveoli thicken leading to decreased oxygen exchange and the ability to breathe. The thickening of the airway is in part due to abnormal remodeling of the extracellular matrix. Dysregulation of Matrix Metalloprotease (MMP) activity leads to the breakdown of collagen type I, a necessary component of the extracellular matrix in the lung tissue. In this study, we measure MMP‐8 and MMP‐12 production by Human Lung Fibroblasts (HLFB) reared in serum‐free media (normal (NHLFB) and COPD Diseased (DHLFB)). Commercially available early passage HLFB established from lung tissue, obtained during a biopsy, were cultured and passaged in our laboratory through passage 4. Both normal and diseased HLFB cells were viable when cultured at subconfluence for 24 hours in a serum‐free medium. There were detectable levels of MMP‐12 (ng/culture) and MMP‐8 (pg/culture) in both the secreted (conditioned media) and incorporated (extracellular matrix) fractions. Overall, DHLFB produced 3‐fold more secreted MMP‐12 [150ng/culture versus 50ng/culture] and 14‐fold less incorporated MMP‐12 [20ng/culture versus 300ng/culture] than the NHLFB. In the case of MMP‐8, DHLFB produced 2‐fold less secreted MMP‐8 [30pg/culture versus 80pg/culture], while the incorporated fractions of MMP‐8 were relatively similar in both normal and diseased cultures. In line with these findings, the diseased lung fibroblasts secreted approximately 10‐fold higher levels of degraded collagen type I, confirming the diseased nature of the cells. Our findings suggest that MMP‐8 has a role in the increased degradation of extracellular matrix in COPD since collagen type I is a preferred substrate for MMP‐8. But the role of MMP‐12 in the disease process may be more indirect and involve elastolysis that could disrupt the interactions with collagen type I and facilitate collagen degradation by allowing access to the fibril by MMP‐8. Future studies will measure elastin turnover in these cultures to get a clearer picture of the dysregulation of the extracellular matrix associated with COPD.
Correlation of the pulse rate in horses at the start with the results of endurance ride
Authors: Nikitin Georgy , Semenov Boris , Guseva Veronika , Kuznetsova Tatiana , Nazarova Anna , Ladanova Maria , Khomenko Roman
Published: May 2022

The study aimed to study the relationship between endurance ride and the pulse rate in sports horses. Well‐trained horses have low heart rates. The research was carried out in 2021 at the World Championships for young riders and juniors in equestrian races in the Netherlands; the distance was 120 km. The competition was attended by 76 horses, mainly of the Arab breed, of different sexes. The rules of equestrian sport strictly limited the age of the animals. Horses no younger than 7 years old took part in the competition. The pulse counting was carried out at the start according to the rules described in the regulations of the International Equestrian Federation using a pulse oximeter. Heart rate data were compared between the two groups of horses ranked in the first and last 10 places. Statistical processing was performed with BioStat, AnalystSoft Inc., version 7, and the Mann‐Whitney U test were used. At the preliminary veterinary control at the start, the pulse rate of the animals that took the first 10 places was in the reference range of 24‐42 beats per minute, and the horses that took the last 10 places at the finish line had a heart rate in the field of 40‐48 beats per minute. Based on the research, it was found that the successfully finishing horses that took the first 10 places had a pulse rate of 37.2 ± 0.5 beats per minute, and the horses that took the last 10 places had a pulse rate of 44.4 ± 0, 93. When comparing two samples using the Mann‐Whitney U Test, we got P=0.0026. Thus, the difference between the pulse rates in the horses of the compared groups is statistically significant. Horses whose pulse rate at the preliminary veterinary inspection was in the reference interval of 24‐42 beats per minute and averaged 37.2±0.5 showed the best result and took first places. On primary veterinary control, horses with a pulse rate of 40‐48 beats per minute with an average of 44.4 ± 0.93 took the last places in equestrian distance race competitions.
Anatomical Basis of Interactions Between Locomotor and Respiratory Systems
Authors: Bezdudnaya Tatiana , Vahora Moin , Giacobbo David , Marchenko Vitaliy
Published: May 2022

Functional interaction between locomotor and respiratory networks has been shown in multiple previous studies. Here, we demonstrate the anatomical basis of these interconnections using the retrograde transsynaptic tracer, pseudorabies virus (PRV), in adult Sprague Dawley rats. In particular, PRV‐152 with a green fluorophore (GFP) was injected into the left biceps brachii muscle and PRV‐614 with a red fluorophore (RFP) was applied onto the left hemidiaphragm to map biceps and diaphragm motor circuits. Cervical spinal cord and brainstem tissue were sectioned and immunostained to enhance RFP and GFP signals and stain acetylcholinergic neurons using choline acetyltransferase (ChAT) antibodies. Double‐labeled (GFP+RFP) and triple‐labeled (GFP+RFP+ChAT) neurons were identified within the spinal cord and brainstem. In the spinal cord, double and triple‐labeled interneurons were seen from C2 cervical to T1‐T2 thoracic levels ipsilateral and contralateral to PRV labeling. Interestingly, most of these neurons were found in the Rexed lamina 7 and 10. The presence of ChAT positive shared interneurons within these laminae allows us to suggest that they could be Voc interneurons, but more studies are required to support this hypothesis. In the brainstem, the double‐labeled neurons were seen in the following structures: 1) midbrain – in all parts of Periaqueductal Gray substance (PAG), Dorsal Raphe (DR) and Deep Mesncephalic (DMe) nuclei; 2) pons – in Kölliker‐Fuse and Parabrachial Nuclei (PB/KF), in areas of noradrenergic groups of cells (Locus Coeruleus and A5) and sub‐Coeruleus areas, in oral and caudal Pontine nuclei (PnO and PnC); 3) medulla – in all parts of Raphe (Obscurus, Magnus and Pallidus), Gigantocellular Reticular (Gi), Internal Reticular (IRt), and lateral Paragigantocellular (LPGi) nuclei, in para‐Fascial/Retrotrapezoid area (pF/RTN), and in Bötzinger and pre‐Bötzinger Complexes (BC and pBC). Triple‐labeled cells were found in KF, Raphe and LPGi structures. Our work confirmed the previous electrophysiological studies revealing shared interneurons in the spinal cord and pontomedullary respiratory compartments (PB/KF, pF/RTN) and uncovered the new brainstem structures that also could play a significant role in functional interactions between locomotor and respiratory systems.
Anatomy Education‐Paradigm Shift from Passive to Active Learning‐Effects on Student Engagement, Comprehension and Retention A Review of Literature from 2012 to 2022
Authors: Gamo Joel A.
Published: May 2022

Introduction

There has been a dramatic paradigm shift from passive to active teaching and learning methods. Passive teaching is faculty‐centric, while active teaching is student‐centric which many academic scholars believe will lead to better student engagement, comprehension, and knowledge retention. Instead of just pouring information into their brains, students must be responsible for their learning, and teachers only act as facilitators rather than the sage on the stage. There is a need to formulate curriculum map designs and lesson plans that leads to higher‐order learning using Bloom's taxonomy in teaching.

Objective

The objective is to review literature that compares the passive and active teaching strategies on their advantages and disadvantages regarding student engagement, comprehension, and knowledge retention.

Methodology

A review of the literature was conducted on passive and active learning methods in anatomy education from 2012 to 2022.

Results

The faculty‐centric passive teaching with lectures and videos make students disengaged. On the other hand, in active learning, students are more engaged, have better comprehension, retention and are more responsible for their learning. Active teaching utilizes discussion fora, collaborative and peer teaching, flipped classrooms, blended classes, games, concept maps, chunking, polling, and augmented reality.

The pyramid of retention from the National Training Laboratories in Bethel, Maine, showed that passive teaching led to 30 % retention while active teaching had superior retention from 50 to 90 %.

Conclusion

Active learning methods have more advantages regarding student engagement, comprehension and, retention than passive teaching. In addition, the faculty may combine both strategies to maximize learning among the students.
Impact of Patterned Feeding of a Palatable Diet on Chronic Alcohol Drinking and Anxiety‐Like Behavior in Alcohol‐Preferring Rats
Authors: Corte Tyler , Sirohi Sunil
Published: May 2022

Previous studies from our lab have shown that patterned feeding of a palatable diet (PD; moderately high in fat) significantly reduced alcohol drinking in rats. In these studies, PD and alcohol were never presented concurrently, and alcohol testing occurred on non‐palatable diet access days (Mon, Wed, and Fri). Therefore, it is unclear if the patterned feeding of a PD would produce similar effects when alcohol is presented simultaneously in a chronic access paradigm. The present study examined the impact of patterned feeding of a PD on chronic alcohol drinking and anxiety‐like behavior in male and female alcohol‐preferring P‐rats. P‐rats (n=6/group) were allowed to drink alcohol 10% v/v in their home cages for six weeks, along with intermittent (24 hrs twice a week) PD access on Tuesdays and Thursdays. Control rats received chow on Tuesdays and Thursdays along with chronic alcohol access. Anxiety‐like behavior (Light/Dark box and Open Field testing) was assessed during acute alcohol withdrawal (24hr following alcohol access) on non‐PD access days. Water and chow were available ad libitum to all groups. Male and female data were analyzed together and separately. There were no significant between‐group differences in baseline alcohol drinking in chow and PD groups of rats. Overall, in cage alcohol drinking significantly (p<0.0001) increased with time, an effect evident in both male (p<0.001) and female (p<0.01) group of rats. Following initiation of patterned feeding, overall, a strong trend (p=0.07; main effect of treatment) towards reduced alcohol drinking was observed in rats receiving intermittent PD exposure compared to the chow controls. Further analysis revealed that alcohol drinking was only significantly reduced in males (p=0.0096), whereas alcohol drinking was identical in the female group of rats receiving PD compared to their chow controls. Similar effects were observed when alcohol drinking data on PD access days (Tuesdays and Thursdays) were analyzed, documenting reduced alcohol drinking when PD was concurrently available. Anxiety‐like behavior under acute alcohol deprivation conditions was not affected by patterned feeding of PD. Collectively, these data provide the effectiveness of patterned feeding in reducing chronic alcohol drinking and highlight striking sex differences in the ability of patterned feeding of PD in regulating these effects.
Structural analysis of crosstalk between Type I and Type III CRISPR systems
Authors: Parashar Vijay , Xia Pengjun , Dutta Anirudha , Gupta Kushol , Batish Mona
Published: May 2022

CRISPR‐Cas comprise adaptive immune systems against foreign nucleic acids in prokaryotes. However, little is known about how the expression of CRISPR‐Cas genes is regulated in response to viral infection. Type‐III CRISPR interference results in the production of cyclic oligoadenylate (cOA) second messengers, which are known to bind various CRISPR Associated Rossmann Fold (CARF) ‐containing nuclease receptors to regulate their functions. Despite conservation of the CARF domain across Csa3 transcription factors, the basis for cOA binding specificity was unclear. In this study, we extend the known receptor repertoire of cOAs to include transcriptional factors by demonstrating specific binding of cA4 to Saccharolobus solfataricus Csa3 (Csa3Sso) (KD of 5.8 ± 0.03 μM). We determined a 2.0 Å resolution X‐ray crystal structure of cA4‐bound Csa3Sso, which reveals the binding of the Csa3Sso CARF domain to an elongated conformation of cA4. Binding affinity analyses of Csa3Sso mutants targeting the observed Csa3Sso•cA4 structural interface identified Csa3Sso residues that are required for ligand binding and specificity. We determined an essential role of a conserved glutamate (Glu122) for the low affinity interaction with cA4; a mutation of this residue to Ala and Gln resulted in a ~145 and ~125‐fold increase in cA4 binding affinity by Csa3Sso. Our complementary SAXS analyses detected a cA4‐induced conformational change in Csa3Sso, involving an asymmetric quaternary rearrangement of the C‐terminal winged helix‐turn‐helix (wHTH) domains supporting an allosteric mode of Csa3 regulation by cA4. Overall, our results support cA4 and Csa3‐mediated cross‐talk between type‐III and type‐I CRISPR systems that co‐exist in several prokaryotes.
Analysis of the lake trout heart, blood, liver, and brain proteome using evolutionary proteomics
Authors: Alwine Shelby E. , Dupree Emmalyn , Crimmins Bernard , Holsen Thomas , Darie Costel
Published: May 2022

Introduction

Salvelinus namaycush (lake trout) is a top‐predator fish in the Great Lakes region. The Great Lakes Fish Monitoring and Surveillance Program (GLFMSP) use lake trout as a bioindicator of chemical stress by monitoring the concentrations of persistent, bioaccumulative and toxic (PBT) chemicals in their bodies. Studies have shown that elevated concentrations of PBTs can cause changes in transcribed genes, translated mRNAs, proteins produced, and post‐translational modifications in aquatic species. Though lake trout is used to monitor chemical concentrations for over 50 years, there is very little information available on the proteome of this species. A well‐developed protein database for the lake trout would help elucidate the effects of PBT chemicals on this species providing a direct health metric for the Great Lakes ecosystem.

Methods

In this study, heart, blood, brain, and liver samples from lake trout were analyzed by SDS‐PAGE, followed by in‐gel trypsin digestion and analysis by nanoLC‐MS/MS. The raw data was searched against different NCBI and UniProtKB databases in Mascot Daemon and the output was analyzed by Scaffold 4.3 software. Databases used include Actinopterygii, Salmonidae, Salvelinus, as well as the highly studied species Oncorhynchus mykissand Danio rerio.

Preliminary Data

Previously, our group has reported a large number of proteins for lake trout liver, heart and blood (Dupree et al., Proteomics, 2019, PMID: 31578773 and Dupree et al., Proteomics, 2021, PMID: 34676671). In lake trout liver we identified 4194 proteins using the NCBI databases and 3811 potential protein hits in the UniProtKB databases. In the blood and heart we identified 838 and 580 proteins, respectively, using NCBI databases and 1180 and 561 potential protein hits for the heart and blood, respectively in BR2. A lake trout brain proteomics analysis completed using an in‐house SDS‐PAGE, followed by in gel digestion and nanoLC‐MS/MS analysis. The Mascot database search is underway. Once protein hits are cataloged, BLAST and Scaffold‐based comparisons of the liver, heart, blood and brain will be performed. Additional search of the data against the other fish relatives will also be completed. Through this process, evolutionary relationships for the lake trout species will also be explored. The current study will add to our understanding of the lake trout proteome that will be used to understand the biochemical/physiological effects of legacy PBT chemicals in the Great Lakes ecosystem.

Novel Aspect

Use of four types of lake trout tissue together to develop a protein database for the species.
When molecules come to life – Using Augmented reality for studying protein structure in Cell Biology class
Authors: Agrawal Swati
Published: May 2022

Most Undergraduate Biology courses cover an incredibly large range of topics integrating complex information at molecular, cellular, genetic and at the organismal level. Translating concepts from one level of thought to another and crossing disciplines equally and fluidly largely depends on how well students understand the structure‐function relationship at the micro and macro level. A survey of the literature shows that despite having encountered these structures of biomolecules in their earlier courses, students lack an ability to visualize molecules and interactions. Most classroom curriculums use textbooks and lecture slides with straightforward two‐dimensional structures that make it hard for students to visualize these macromolecules. To improve motivation and to promote student interactivity, in Cell Biology classes I have used molecular models in Augmented reality to reform content delivery by using interactive active learning techniques. This presentation will discuss ways for instructors to implement Augmented reality in their classes to help students better understand concepts of structure and function of Biomolecules.
Multiple Conserved Cysteine Residues Are Required for Delta‐12 Fatty Acid Desaturase Function in Arabidopsis thaliana
Authors: Novotny Lauren , Dave Parth , Horn Patrick
Published: May 2022

Plants produce two essential fatty acids (FAs) for human nutrition: linoleic acid (18:2Δ9,12, an omega‐3 FA) and alpha‐linoleic acid (18:3Δ9,12,15, an omega‐6 FA). Plant FA desaturase 2 (FAD2) is a microsomal enzyme that introduces the carbon‐carbon double bond at the delta‐12 position through an oxygenated intermediate. As the structure of FAD2 has yet to be experimentally determined, we are using integrated structural modeling, sequence analyses, and biochemical approaches to better understand this essential membrane‐bound enzyme. For this project we are studying the role of cysteines (Cys), using Arabidopsis thaliana FAD2 as a model, given their potential involvement in disulfide bonds, catalysis, cofactor binding, and/or metal coordination. We have identified seven conserved Cys residues in planta that we hypothesize to play distinct roles in the structure and function of FAD2, including one located in a highly conserved catalytic histidine motif HXXXH. To elucidate their roles, each individual Cys has been mutated to alanine. Transient expression assays of FAD2 in yeast, followed by lipid compositional analysis by gas chromatography has demonstrated that all Cys mutations resulted in reduced enzymatic activity with two appearing essential. Analysis of homodimer protein‐protein interactions in yeast has identified one Cys required for dimerization and has highlighted potentially interacting regions. To expand upon these results, we are currently testing for the presence of intramolecular and intermolecular disulfide bonds and assessing protein stability over time. These results have demonstrated that several Cys are required for FAD2 function providing a new model for understanding the contribution of these residues to membrane‐bound FADs in plant systems. Ultimately, a better understanding of these enzymes will inform rational engineering strategies for producing essential lipids and high‐value bioproducts.
A Diversity of Filamenting Enzymes
Authors: Park Chad K. , Horton Nancy
Published: May 2022

We present some of the diversity of functional, biologically relevant, enzymatic filament structures. The recent advances in CryoEM and associated structural computational methods, coupled with whole genome screens, have helped identify and recognize these oligomers as important in different areas such as regulation, activation, selectivity, and substrate channeling.
Engineering Spinal Interneurons for Repair of the Injured Cervical Spinal Cord
Authors: Fortino Tara A. , Randelman Margo L. , Ponna Anish , Schardien Kayla A. , Niceforo Alessia , Hall Adam A. , Zholudeva Lyandysha , Lane Michael A.
Published: May 2022

Cervical spinal cord injuries (SCI), the most common SCI, compromise respiratory networks leading to life‐threatening impairments in breathing. Following a cervical injury, extensive damage occurs to the phrenic motor network (controlling the diaphragm) consisting of phrenic motoneurons and interneurons. Despite these devastating consequences, there is now significant evidence for plasticity and limited spontaneous recovery. Our ongoing work has identified spinal interneurons (SpINs), in particular the excitatory V2a subtype, as key therapeutic targets for promoting plasticity and functional recovery of the phrenic motor network. Efforts to enhance this plasticity have shown some promise, but it is likely that treatments capable of promoting repair (e.g., cell transplantation) will provide the best therapeutic outcome.

Building on a long history of transplanting spinal neural precursor cells (NPCs) to promote spinal cord repair, the present work harnesses advances in stem cell engineering to enrich donor NPCs with specific subsets of stem cell derived SpINs. We hypothesize that NPCs enriched for V2a SpINs will have enhanced therapeutic efficacy over NPCs alone and promote phrenic motor recovery. We will test this hypothesis using a clinically relevant model of a cervical contusion in the adult rat, and test the functional contribution of donor V2a SpINs using inhibitory chemogenetics (hM4Di).

Adult female Sprague‐Dawley rats received mid‐cervical contusion injury (Infinite Horizon impactor, intended impact force 200 kilodyne). One‐week post‐injury, aggregates of NPCs with mouse embryonic stem cell derived V2a SpINs were injected into the lesion cavity. One month post‐transplantation, animals were re‐anesthetised and traced with a retrograde, transneuronal tracer, pseudorabies virus, applied to the diaphragm muscle to assess donor‐host connectivity with the phrenic network. Three days later, animals underwent terminal diaphragm electrophysiology, at which time clozapine was injected intraspinally into the transplant to silence donor SpINs. Preliminary data revealed that clozapine‐induced suppression of donor cell activity reduced diaphragm activity ipsilateral to injury, with no measurable effect on the contralateral side. Consistent with this, immunohistochemical assessment revealed donor cells survived and synaptically integrated with the injured host phrenic network. This ongoing research offers the first in‐depth assessment of how these donor SpIN populations contribute to phrenic recovery.