bluuebunny/crossref_metadata_2025_split huggingface dataset:
Rho5 regulates lysophosphatidic acid induced macropinocytosis in Entamoeba histolytica
Authors: Datta Sunando , Apte Achala , Manich Maria , Labruyère Elisabeth 
Published: May 2022

Entamoeba histolytica is a unicellular enteric protozoan responsible for variety of gastrointestinal pathologies. The pathogenicity of the parasite is attributed to its dynamic cytoskeleton. Rho family GTPases, a major class of molecular switches, play an indispensable role in controlling cytoskeleton remodelling. Rho GTPases are known to be activated by specific Guanine nucleotide Exchange Factors (GEF) upon stimulation by various extracellular stimuli to regulate signalling.We have established that EhRho5 is important for LPA induced micropinocytosis in the amoebic trophozoites. EhRho5 translocated from cytosol to plasma membrane and endomembranous compartments when stimulated with LPA, a bioactive phospholipid known to activate Rho subfamily members (1,2). Our findings were strengthened when FRAP (Fluorescence recovery after photobleaching) study showed that LPA treated cells show 30% faster recovery of Rho5 when compared to serum starved cells. The translocation is accompanied by activation of the GTPase as revealed by effector pull down assay(2). LPA stimulation enhanced the macropinocytic efficiency of trophozoites in a Rho5 dependent manner. We then identified the GEF for Rho5 which too, shows translocation upon stimulation with LPA. Also, depletion of the GEF leads to decreased pinocytic efficiency of the trophozoites. Using inhibitor based studies, we further demonstrated that PI3K acts as an intermediate cue for EhRho5 activation during LPA induced macropinocytosis.Moreoever, Rho5 expression showed contrary relationship with random migration of the trophozoites suggesting a pivotal role of the GTPase in mutally antagonistic cellular processes(3) in the amoeba.

Reference:
Fleming IN, Elliott CM, Exton JH. Differential Translocation of Rho Family GTPases by Lysophosphatidic Acid, Endothelin‐1, and Platelet‐derived Growth Factor, J. Biol. Chem. 1996, 271: 33067–33073
Van Leeuwen FN, Olivo C, Grivell S, Giepmans BNG, Collard JG, Moolenaar WH. Rac Activation by Lysophosphatidic Acid LPA1 Receptors through the Guanine Nucleotide Exchange Factor Tiam1, J. Biol. Chem. 2003, 278:400–406
Veltman DM, Lemieux MG, Knecht DA, Insall RH. PIP3‐dependent macropinocytosis is incompatible with chemotaxis. Journal of Cell Biology. 2014 Feb 17;204(4):497‐505.
Increased Interest in Anatomical Donation As A Result of Positive Media Coverage
Authors: Walker Cam M. , Ostervoss Tamara E. 
Published: May 2022

A 99‐year old female anatomical donor at Oregon Health & Science University (OHSU) in 2018 had an unusual anomaly: Situs inversus (SI) with levocardia [left‐facing heart]. Her SI and levocardia went mostly unnoted throughout her long life, despite several surgical interventions and five successful pregnancies. After consultation with Ms. Ostervoss (director of OHSU’s body donation program), and the donor family, a poster presentation highlighting this donor was prepared and shown at last year’s American Association of Anatomy meeting at Experimental Biology in Orlando, FL, in April 2019 (Walker and Hankin 2019). Additionally, a press release to coincide with the poster presentation was prepared at OHSU for local media consumption. The donor’s story was distributed by print, radio, and television, worldwide.Afterward, the Body Donation Program (BDP) at OHSU then experienced a surge in applications for anatomical donation. The number far exceeded equivalent applications to be an anatomical donor in past years. This has had beneficial effects for the BDP, including the ability to more fully support the needs of anatomical education, accommodate requests for tissue used in surgical simulation, residency, and specialized workshop training, and to assist other regional institutions with their need for anatomical donors. In addition, the raised awareness of a rare condition has led to a series of research opportunities that may have implications for those who have SI.The benefit to the OHSU BDP of publicizing this unusual case of SI is described in the text, along with the number of consent forms registered with the BDP between 2018‐2019. Between 2014 and 2018, annual preregistration vacillated between 350 and 400 individuals. However, after the widespread publication of the case of SI with levocardia in 2019, the number of preregistered donors surged to approximately 460 individuals, with the majority of additional registrations taking place after publication of the findings in widely viewed media sources. The OHSU BDP undertakes other forms of outreach, though none were performed shortly before or after the SI press release in 2019.After the poster presentation and widespread uptake of the story on media around the world, Dr. Walker was interviewed by approximately 20 media outlets, including newspapers, online news agencies, radio, and television. As a result, roughly 40 individuals who have SI with levocardia have come forward to tell their stories, with another 15 individuals who have SI with dextrocardia [right‐facing heart]. Collectively, this group, ALL of who have stated their willingness to provide medical records, be further interviewed, and participate in a future genetic study, comprises the largest group of individuals with SI who have been surveyed in the medical literature.
Differences in Conformational Dynamics Between a Viral Polyprotein and Its Processed Products in Functionally Relevant Regions Revealed by Solution‐State NMR Spectroscopy
Authors: Winston Dennis , Boehr David 
Published: May 2022

Viruses have evolved numerous strategies to expand protein function despite their limited genetic material, including proteolytic cleavage of polyproteins to obtain proteins with different functions than their precursors. In this way, proteolysis enables temporal control over the viral life cycle by varying the amounts of proteins in competing cleavage pathways. The 72 kD poliovirus polyprotein 3CD is involved in important protein‐protein, protein‐RNA and protein‐lipid interactions in viral replication and infection. It is a precursor to 3C protease and 3D RNA‐dependent RNA polymerase, but has different protease specificity, is not an active polymerase, and participates in different protein and RNA interactions than its processed products. Despite the existence of X‐ray crystal structures for the three proteins, the molecular mechanism for the different functionality of 3CD is not well understood due to the structure of the domains in 3CD being nearly identical to the isolated 3C and 3D proteins.It has been proposed that despite nearly identical X‐ray crystal structures, the conformational dynamics of 3CD in solution may be different than those of 3C and 3D, allowing 3CD to adopt additional conformations and perform new functions compared to its processed products. To this end, we compared the conformational dynamics of 3C, 3D, and 3CD using solution‐state NMR spectroscopy. We used Carr‐Purcell‐Meiboom‐Gill (CPMG) multiple quantum relaxation dispersion, methyl 1H chemical exchange saturation transfer (CEST), and methyl spin‐spin relaxation to probe conformational dynamics from the picosecond to second timescales. We have identified differences in conformational dynamics in functionally important regions that may be useful in understanding the differences in function between 3C, 3D, and 3CD. In particular, expansion of the conformational ensemble available to 3CD may allow it to perform additional functions not seen in 3C and 3D alone despite having nearly identical lowest‐energy structures.
Mechanisms of Substrate Selectivity and Transport by a Bacterial Methionine ABC Importer
Authors: Chen Yuchun , Yang Janet G. 
Published: May 2022

ABC transporters are central in many cellular functions including nutrient uptake, signal transduction membrane assembly, and cellular detoxification. Both structural and functional studies have revealed insights into the high‐affinity uptake mechanism of the MetNI methionine ABC transporter. Using the energy from ATP binding and hydrolysis, the MetNI‐Q system can import L‐Met, D‐Met, and other methionine derivatives against concentration gradients. Many mechanistic studies of ABC transporter propose a model in which cognate binding proteins sequester substrates in the periplasm and deliver them to the transporter. In contrast, recent in vivo and crystallographic studies of MetNI‐Q suggest that some substrates may be able to access the transporter through a solvent accessible tunnel in the cognate binding protein‐transporter complex. These studies suggest that the substrate delivery to MetNI transporter may be more flexible than other ABC transport systems.To test if the MetNI‐Q system transport system uses two mechanisms of substrate delivery, we designed a series of experiments to dissect the individual steps in the transport cycle. As the starting point, we have developed a fluorescent anisotropy assay to measure the binding affinity between the MetQ substrate‐binding protein and the MetNI transporter in the presence of different methionine derivatives. Our preliminary data show that MetNI binds to apo‐MetQ ~10 fold tighter than L‐Met liganded MetQ, and additionally may differ in the presence of D‐Met. This suggests that different methionine derivatives may utilize discrete transport models.
Small Molecule Anticancer Compound Modulates Cell Cycle DNA Damage Response Pathway and Inhibit Tumorigenesis in Triple Negative Breast Cancer
Authors: Okpechi Samuel C. , Dong Shengli , Yousefi Hassan , Guidry Jessie J. , Otohinoyi David A. , Worthylake David K. , Hicks Chindo , Alahari Suresh K. 
Published: May 2022

ObjectiveTo investigate the mechanism of action and therapeutic benefits of NSC001, a small molecule anticancer compound, in the treatment of triple negative breast cancer (TNBC), a clinical subtype of breast cancer that is more aggressive, has poor prognosis. In TNBC, patients harbor tumors that lack expression of the estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (Her2).HypothesisWe hypothesize that NSC001 will have a beneficial therapeutic effect on TNBC cells through the regulation of specific tumor associated signaling pathways in vitro.Material and methodsWe screened 1360 compounds from the National Cancer Institute (NCI) Diversity and Mechanistic Set library using the cell counting kit‐8 (CCK‐8) reagent from Dojindo Molecular Technologies, Inc. The screening was performed by applying 10µM of each candidate drug upon plates harboring the aggressive TNBC cell line, MDA‐MB‐231. Treatment time for all screening was fixed at 24 hours. The efficacy and potential mechanism of action of one of the potent drug candidates identified (NSC001) was further investigated on three TNBC cell lines (MDA‐MB‐231, MDA231‐LM2‐4175, and MDA‐MB‐468) using mass spectrometry and proteomic analysis. Afterwards, the results from the proteomic analysis were validated using molecular methods including cell viability assays, apoptosis assays, cell cycle assays, and western blotting.ResultsWe identified eight compounds that reduced the viability of MDA‐MB‐231 cells by up to 80% following a 24 hour treatment time point. Next, our in vitrodata show that NSC001 reduces the cell viability of TNBC cells in a dose‐and‐time dependent manner. Cell death data show that NSC001 induces potent apoptosis in a dose dependent manner in TNBC cells. Also, the results from colony formation assay show that 100nM [NSC001] is sufficient to inhibit the proliferative capacity of TNBC cells. Mass spectrometry and proteomic data reveals that NSC001 activates the cell cycle G2/M DNA damage checkpoint regulation in all the three TNBC cell lines used. Furthermore, we confirmed this finding in our cell cycle assay where we observed a clear G2/M cell cycle phase arrest. The arrest was potently induced at a dose significantly lower than the IC50 of NSC001. Lastly, we validated our proteomic results using western blotting, which confirmed that this low dose of NSC001 induces expression of p21, a cell cycle checkpoint regulator and tumor suppressor protein which is known to trigger cell cycle arrest in most cancer cell lines.ConclusionOverall, our data suggest that NSC001 may be a potent small molecule anticancer compound which should be further investigated for its potential to be used clinically in the treatment of TNBC patients.
Changes in Oral Microbial Diversity in a Piglet Model of Traumatic Brain Injury
Authors: Jeon Julie H. , Lourenco Jeferson , Fagan Madison M. , Welch Christina B. , Sneed Sydney E. , Duberstein Kylee J. , Callaway Todd R. , West Franklin D. , Park Hea Jin 
Published: May 2022

The dynamic changes in the oral microbiome in response to disease has led to significant interest in utilizing microbiome analysis as a potential diagnostic and prognostic biomarker. Oral dysbiosis has been linked to the progression of neurological diseases such as Alzheimer’s and Parkinson’s diseases, however, no studies have been conducted to evaluate microbiome changes in traumatic brain injury (TBI). This study aimed to examine the changes in the oral microbiome structure during the acute stage of TBI in a clinically translational pig model.Landrace piglets (4‐5 weeks old, male) underwent either a controlled cortical impact surgery (TBI, n=6) or sham surgery (Sham, n=6). TBI was confirmed by magnetic resonance imaging 1 day post‐TBI. Oral swabs of the inside of upper and lower lips, both cheeks, and under the tongue were collected to sample the oral mucosa pre‐surgery and 1, 3, and 7 days post‐surgery (PS). Bacterial DNA was extracted and the V3‐V4 region of 16s rRNA gene was amplified and sequenced using an Illumina NovaSeq platform. Alpha‐, beta‐diversity, and oral microbial composition at the phylum (P), family (F), genus (G), and species (S) level were examined using Qiime2 plugins. Mixed effects ANOVA was used to compare differences between the groups and time points.Significant time effects were identified in several alpha‐diversity indexes during the acute stage of TBI: The number of observed features, Faith’s Phylogenetic Diversity, Shannon Index, and Pielou’s Evenness. However, no group effects were found between TBI and Sham groups, except for Faith’s Phylogenetic Diversity. Faith’s Phylogenetic Diversity showed group and time‐by‐group effects including a significant difference between groups at 7 days PS. In both TBI and Sham groups, beta‐diversity was significantly different between time points and distinct microbial patterns were observed between TBI and Sham groups at 1, 3, and 7 days PS, with the most apparent difference at 7 days PS. Changes in oral microbial composition did not differ between TBI and Sham groups at any taxonomic level (P, F, G, and S), while significant time effects were found in several microbial taxa. Interestingly, significant time‐by‐group effects were found in P_Proteobacteria, F_Leptotrichiaceae, F_Micrococcaceae, and G_Rothia.In conclusion, species diversity as measured by Faith’s Phylogenetic Diversity was lowest at 7 days PS in TBI group compared to Sham group, and TBI and Sham groups showed significant differences in beta‐diversity. With respect to individual microbial taxa, the oral microbial composition was not significantly different between the two groups during the acute stage of TBI. Future studies are needed to investigate further changes in oral microbiome during the chronic stages of TBI.
Loss of Pkm2 Alters Myocardial Metabolism and Increases Oxidative Stress After Infarction
Authors: Lee Katie C. , Williams Allison L. , Shohet Ralph V. 
Published: May 2022

IntroductionPyruvate kinase (PKM1) directs pyruvate to the Krebs cycle for oxidative metabolism in the healthy heart. Our lab described a hypoxia‐mediated switch to the alternatively spliced isoform PKM2, enhancing pyruvate to lactate conversion. Recently, we have also found that Pkm2 knockout (KO) mice had profound depletion of basal glucose in the heart compared to control mice. Pkm2 has also been shown to reduce oxidative damage and promote cardiomyocyte cell proliferation after myocardial infarction (MI). We hypothesize that upregulation of PKM2 can alter metabolic pathways by promoting glycolysis, and that after injury, this can preserve ATP production, protecting the heart from the stresses of hypoxia and injury.MethodsGlobal Pkm2 KO mice were subjected to permanent ligation of the left anterior descending coronary artery to mimic an MI. RNA‐seq analysis of left ventricles from control (n=8) and Pkm2 KO mice (n=8) before and 3 days after sham or MI surgery was performed. Semi‐quantitative real‐time PCR was used to confirm changes in selected genes of interest.ResultsLoss of Pkm2 moderately altered gene expression at baseline (q<0.05, FDR<0.05). Notably, the mitochondrial gene COX3 was downregulated in Pkm2 KO hearts. 68 genes were differentially expressed in Pkm2 KO hearts after MI, not observed in control MI hearts. MI of Pkm2 KO hearts resulted in considerable reduction of transcripts of enzymes in the insulin signaling pathway, mitochondrial oxidative phosphorylation, mitochondrial uncoupling, fatty acid metabolism, and increase in transcripts encoding enzymes in the pentose phosphate pathway, response to oxidative stress, and apoptotic signaling. Semi‐quantitative PCR of selected genes involved in glucose metabolism confirmed RNA‐seq results.ConclusionsRNA‐seq analysis of Pkm2 KO hearts demonstrated that loss of Pkm2 altered gene expression of metabolic and mitochondrial enzymes. Pkm2 KO hearts also showed increased abundance of pro‐apoptotic markers which may be a result of increased oxidative stress.
AI/ML‐driven simulation of the skin retention of opioid analgesics following topical administration in normal skin and cutaneous lesions
Authors: Khotimchenko Maksim , Antontsev Victor , Chakravarty Kaushik , Hou Hypatia , Varshney Jyotika 
Published: May 2022

Topical drug administration in the treatment of skin disorders is known to have high efficacy with a favorable safety profile in clinical settings. Disposition of the active pharmaceutical ingredient (API) in the skin layers plays a critical role for the dose regimen calculator required for desired therapy efficiency. Experimental measurements of the API skin concentration are complicated and inaccurate and can be replaced with physiologically based pharmacokinetic modeling predicting local and systemic drug exposure. Opioid analgesics are commonly used in treating severe pain and it is frequently associated with putative adverse effects. Topical applications of opioids were shown to have high efficacy with a favorable safety profile in clinical settings. Clinical investigations of transdermal drug formulations, especially in patients with skin lesions, are often hindered due to the patient intolerance, high cost of the studies, and ethical aspects.BIOiSIM, a hybrid artificial intelligence computational platform integrating machine learning optimization with mechanistic modeling was implemented to accurately simulate in vivo plasma concentration profile and predict partition coefficients and API skin retention for two opioid analgesics in healthy skin and simulated skin lesions. Publicly available drug parameters and physiological data were used for the model building.The platform successfully predicted API systemic exposure and skin concentration following the epicutaneous application of central opioid agonist buprenorphine and peripheral opioid agonist oxycodone in healthy human skin and simulated subjects impaired skin. Experimentally observed plasma concentration for both drugs demonstrated validity of the in‐silico methods. The results indicated that buprenorphine permeates rapidly through the stratum corneum and partitions into the dermis, where its concentration slowly declines as the reservoir in the compound vehicle is depleted. Thus, the impact of lesions is not as significant. In contrast, the hydrophilic oxycodone, where the rate‐limiting stratum corneum layer prevents a significant portion of the compound from permeating deeper in healthy patients. Hence, concentration of oxycodone sharply declines over the first hours of exposure in deeper indicating poorer accumulation capacity in the skin in the presence of lesions.An AI/ML‐driven PBPK model was applied for accurate simulation skin concentrations of two opioid analgesics – buprenorphine and oxycodone. The results have confirmed that both these drugs have good skin retention and can stimulate opioid receptors located in skin. The results suggest that buprenorphine showing substantial skin retention capacity can be applied topically for the treatment of skin disorders in in patients.
Comprehensive analysis of NFAT5 associated gene expression in the renal collecting duct
Authors: Edemir Bayram , Petrillo Federica , Chernyakov Dmitry , Fenton Robert 
Published: May 2022

BackgroundThe renal cortico‐medullary osmotic gradient is a crucial factor for the urine concentration mechanism and is mainly generated by sodium chloride and urea. Due to this, the cells in the renal inner medulla are challenged with a hypertonic environment. The adaptation of these cells is mediated by the action of the tonicity‐responsive enhancer‐binding factor (TonEBP, also known as nuclear factor of activated T cells 5, NFAT5). NFAT5 induces the expression of genes that lead to accumulation of organic osmolytes within the cells. The cortico‐medullary osmotic gradient also induces a specific spatial gene expression pattern within the kidneys. So far, a systematic analysis of the contribution of NFAT5 on gene expression in the kidneys was missing, which was accomplished here.MethodsWe performed next generation RNA sequencing (NGS) using NFAT5 deficient and wild type control primary cultivated inner medullary collecting duct (IMCD) cells. As a second approach, we used renal cortex and renal inner medulla from principal cell specific NFAT5 deficient mice and control mice for NGS analysis. Differentially expressed genes were analyzed by pathway, functional and gene enrichment analysis.ResultsIn primary IMCD cells hyperosmolality induced massive changes in gene expression. Aquaproin‐2 (Aqp2) was one of the transcripts with the highest level of induction. Loss of NFAT5 abolished hyperosmolality‐induced expression of Aqp2. Most of the hyperosmolality induced transcripts had the highest expression in the inner medulla. Loss of NFAT5 affected the expression of more than 3,000 genes in renal cortex and more than 5,000 genes in the inner medulla, compared to control kidneys. Again, loss of NFAT5 was associated with decreased expression of Aqp2. We observed increased expression of kidney injury marker genes, like lipocalin‐2 or kidney injury molecule 1. Gene enrichment analysis also indicated that loss of NFAT5 is associated with a renal inflammation‐like phenotype.ConclusionThis is the first study addressing the role of NFAT5 on hypertonicty‐induced changes in gene expression in the kidney. NFAT5 is the master regulator of osmolality induced Aqp2 gene expression and in vivoloss of function induces a kidney injury‐like phenotype through currently unknown mechanisms.