bluuebunny/crossref_metadata_2025_split huggingface dataset:
Single‐Molecule Optical Replication Mapping (ORM) Suggests Human Replication Timing is Regulated by Stochastic Initiation
Authors: Rhind Nick , Wang Weitao , Klein Kyle , Proesmans Karel , Yang Hongbo , Hastie Alex , Yue Feng , Bechhoefer John , Chunlong Chen , Gilbert David 
Published: April 2020

DNA replication timing is regulated by the timing of initiation across the genome. However, there is no consensus as to how initiation timing is regulated. Deterministic models contend that different initiation sites are programed to initiate at different, well‐defined times. Stochastic models posit that different initiation sites have different initiation probabilities but can fire at any time during S. Sites with a high probability of initiation are more likely to fire early and thus will, on average, initiate early in S. Ensemble replication timing assays that report only average initiation time cannot distinguish between these two types of models.To test if replication initiates deterministically or stochastically, we developed Optical Replication Mapping (ORM) a high‐throughput, genome‐wide, single‐molecule replication mapping approach that combines in vivo replication labeling with the Bionano genomic mapping technology. We have mapped replication in over 20 human cell datasets, representing over 2000‐fold coverage of the human genome, allowing us to map sites of replication initiation that are used in fewer than 0.1% of S phases.Our analyses support three major conclusions. 1) Replication initiates at many infrequently‐used initiation sites spread across initiation zones of 30 to 100 kb. We find no evidence for discrete, high‐frequency initiation sites in the human genome. These result suggest that "replication origin", in the sense of a defined genetic element, is not a useful concept in metazoans. 2) Initiation zones correlate well with regions of DNase I accessible DNA, such as promoters. G4 sequences do not appear to be predictive of sites of initiation, beyond their enrichment in DNase I accessible chromatin. These results suggest that a major driver of initiation probability is DNA accessibility. 3) We observe a low frequency of early initiation across late‐replicating regions, and these initiation events are enriched in late initiation zones. Observed initiation rates across all regions of the genome are consistent with a stochastic mechanism of replication timing and inconsistent with a deterministic replication timing program. We propose that the dominant parameter that regulates replication timing is the initiation probability across initiation zones, with a high degree of heterogeneity of initiation sites within these zones, and no spatial or temporal coordination between individual initiation events.
Comparative In Vitro Nephrotoxicity of Three 3,5‐Dihaloanilines in Isolated Renal Cortical Cells from Male Fischer 344 Rats
Authors: Rankin Gary , Hicks William M. , Ju Jae , Vidrine David , Racine Christopher , Anestis Dianne 
Published: April 2020

Halogenated anilines are widely used to manufacture dyes, pesticides, pharmaceuticals and industrial products. Toxicity induced by these compounds includes hepatotoxicity, hematotoxicity, splenotoxicity, immunotoxicity and nephrotoxicity. We have shown that among the mono‐ and dichloroanilines, 3,5‐dichloroaniline (DCA) is the most potent nephrotoxicant in vivo and in vitro. DCA nephrotoxicity in vitro is attenuated by primary antioxidants (α‐tocopherol, ascorbate) and antioxidant nucleophiles (glutathione, N‐acetyl‐L‐cysteine), suggesting a role for free radicals in DCA nephrotoxicity. The purpose of this study was to compare the nephrotoxic potential of DCA with the nephrotoxic potential of 3,5‐dibromo‐ and 3,5‐diiodoaniline (DBA and DIA, respectively) in isolated renal cortical cells (IRCC) from male Fischer 344 rats. The comparative effect of antioxidants on 3,5‐dihaloaniline (DHA) nephrotoxicity was also determined. IRCC (~4 million cells/ml; 3 ml) were incubated with shaking at 37°C under a 95% oxygen/5% carbon dioxide atmosphere with a DHA (0.25 – 1.0 mM) or vehicle (dimethyl sulfoxide) for 15–90 min with or without a pretreatment. General cytotoxicity was determined by assessing trypan blue exclusion of IRCC and measuring changes in lactate dehydrogenase (LDH) release. Among the three DHAs, DBA was the most potent nephrotoxicant, inducing LDH release at 0.25 mM by 30 min. DCA was the least potent nephrotoxicant, requiring 90 min at 0.5 mM exposure to induce cytotoxicity, while DIA was intermediate in potency by inducing cytotoxicity at 60 min with 1.0 mM exposure. Pretreatment with an antioxidant (α‐tocopherol, ascorbate) or antioxidant/nucleophile (glutathione, N‐acetyl‐L‐cysteine) attenuated cytotoxicity induced by the three DHAs (1.0 mM). These results indicate that the decreasing order of nephrotoxicity in IRCC was DBA > DIA > DCA, and that free radicals play a role in the DHA‐induced nephrotoxicity induced by the three compounds tested.Support or Funding InformationSupported in part by NIH Grant P20GM103434 to the West Virginia IDeA Network of Biomedical Research Excellence.
Nef Dimerization is Required for Antagonism of the Host Cell Restriction Factor, SERINC5
Authors: Staudt Ryan P. , Smithgall Thomas E. 
Published: April 2020

HIV‐1 Nef is a virulence factor that is essential for immune escape of HIV‐infected cells, viral pathogenesis and AIDS progression. Nef lacks intrinsic biochemical activity, functioning instead by interacting with host cell effector proteins to hijack multiple signaling and endocytic pathways. Many Nef functions depend on dimerization of the protein, including enhancement of viral infectivity, replication, and immune cell receptor downregulation. Work in our lab, through both X‐ray crystallography and virological assays, has shown that distinct hydrophobic residues (L112, Y115, F121) are key in Nef dimer formation, and disruption of these residues significantly impairs many Nef functions. Pharmacologic disruption of the Nef homodimer may therefore represent a novel approach to antiretroviral therapy.Here we investigated whether Nef dimerization is required for antagonism of the host cell restriction factor SERINC5 (S5). S5 is a potent suppressor of HIV‐1 infectivity that is antagonized by Nef via binding and downregulating S5 from the surface of infected cells, preventing its incorporation into nascent virions. For our studies, HIV‐1 virions were produced in 293T cells transfected with the HIV‐1 provirus and S5, Jurkat WT and S5‐knockout cells, or donor PBMCs (which naturally express high levels of S5). Viral supernatants were harvested from these “producer” cells and normalized for p24 AlphaLISA. Infectivity of producer‐cell viruses was assessed using TZM‐bl reporter cells, which couple the HIV‐1 LTR to expression of luciferase. Mutation of conserved Nef residues essential for homodimerization (L112A/D, Y115D, F121A) reduced the infectivity of HIV‐1 produced from all three producer cell lines in a S5‐dependent manner. S5 incorporation into virions was increased in the presence of Nef dimer interface mutants, suggesting that dimerization is important in its ability to exclude S5 from newly synthesized virions. Recruitment, downregulation, and endocytic trafficking of S5 by wild type and dimerization‐defective Nef mutants were also examined via confocal microscopy. Nef dimer interface mutants also failed to induce colocalization of S5 with Rab7+ late endosomes, suggesting that Nef dimerization is essential for S5 internalization and degradation via the lyso‐endosomal pathway. By contrast, Nef mutants retained their ability to interact with both S5 and subunits of the AP‐2 clathrin adaptor protein essential for S5 endocytosis, as shown via bimolecular fluorescence complementation. Taken together, these results demonstrate that downregulation of S5 and subsequent enhancement of viral infectivity requires Nef homodimerization, providing further support for pharmacological targeting of Nef quaternary structure in HIV‐1 cure strategies.
Matrix Metalloproteinase 9 (MMP9) Inhibitor Prevents Mustard Induced Microvesication and Promotes Cutaneous Wound Healing
Authors: Chang Yoke-Chen , Muhn Selina , Hahn Rita A. , Gordon Marion K. , Gerecke Donald R. 
Published: April 2020

Skin exposure to the chemical warfare agent, sulfur mustard (bis‐2‐chloroethylsulfide, SM), is characterized by severe blister formation and a prolonged inflammatory response. The mechanisms of these SM toxic effects are still poorly defined, and effective medical countermeasures against SM‐induced cutaneous injury have yet to be established. Animal models demonstrate disruption of the skin architecture at the dermal‐epidermal junction (DEJ) when exposed to the vesicant, SM; the damage may be further exacerbated by overexpression of MMP9. Studies show MMP9 degrades basement membrane (BM) components and regulates inflammation in tissue injuries; inhibition of MMP9 suppresses inflammation and promotes wound repairs in tumors and various wound models. In the present studies, ([N‐hydroxy‐3‐phenyl‐2‐(4‐phenylbenzenesulfonamido) propanamide], BiPS), a specific MMP9 inhibitor, was evaluated for its ability to prevent microvesication, suppress inflammation, and promote wound healing following SM exposure using the mouse ear vesicant model. Treatment of male CD1 mouse ear skin with SM (0.08 mg) caused a characteristic SM induced injury including edema, inflammatory cell infiltration, and the formation of microvesicles at the DEJ. Expression of MMP9 mRNA and protein in the skin progressively increased with time 24–168 h following SM exposure. Immunofluorescence studies showed disruption of the BM molecule type IV collagen (COL4), increased expression of inflammation marker, COX2, and upregulation of the skin wound marker keratin 6 (K6) in SM induced skin wounds. SM also caused macrophage (F4/80) accumulation in the tissue which persisted up to 168 h post exposure. Pretreatment of ear skin with BiPS significantly reduced SM‐induced dermal edema and maintained the integrity of the DEJ‐BM as evidenced by contiguous expression of COL4. At 168 h post SM exposure, mRNA and protein expression of MMP9 was significantly downregulated in BiPS treated mice. BiPS treatment also reduced the number of macrophages accumulating in the tissue, and suppressed expression of COX2, and downregulated expression of K6. Taken together, these data indicate that BiPS treatment was able to suppress both inflammation and degradation of BM zone in skin due to SM. This compound was effective in targeting MMP9 to protect against SM induced microvesication and promote wound repairs. MMP9 inhibitors may be useful as medical countermeasure against SM‐induced cutaneous injury.Support or Funding InformationSupported by NIH grants ES005022 and AR055073.
Cisplatin Decreases ENaC Activity Contributing to Renal Sodium Wasting
Authors: Soares Antonio Garcia , Mironova Elena V. , Abd El-Aziz Tarek M. , Stockand James D. 
Published: April 2020

Cisplatin (CDDP) is a platinum‐containing compound that has antineoplastic effects due to its ability to bind DNA and inhibit replication. While an important chemotherapy drug, CDDP results in a renal salt and water‐wasting syndrome and is nephrotoxic. The origin of the renal salt and water‐wasting caused by CDDP is obscure; though, some evidence suggests that broad inhibition of the Na+/K+‐ATPase by CDDP results in generalized decreases in tubular reabsorption causing increases in sodium and water excretion. The effects of CDDP on the epithelial Na+ channel (ENaC), which is the final arbiter of renal Na+ excretion, are unknown. We test here if CDDP affects the activity of ENaC to influence renal salt and water excretion. These studies focus on understanding the cellular mechanism by which CDDP may affect ENaC. For these studies, C57Bl/6 male mice were randomly divided into 3 groups, as follow: 1) Saline, 2) CDDP and 3) Benzamil (BZM) treated. For metabolic cages experiments, C57Bl/6 male mice received a Na+ load (100 μL of a 0.9% NaCl solution) and were treated with CDDP (25 mg/kg, i.p.) or BZM (1.4 mg/kg, i.p.). Then, water consumption, Na+ intake, urine volume, urinary Na+ and creatine excretion were evaluated every 2 h over a 10 h period. To directly quantify ENaC activity in these animals, principal cells were patch‐clamped in split‐open tubules using the cell‐attached configuration. After recording baseline ENaC activity for 5 min in these tubules, they were treated with CDDP (0.5 to 1.5 μM) with ENaC activity quantified in the presence of this drug over the next 5 min. Preliminary results show that CDDP increases excretion of a Na+ load, increases fractional excretion of sodium (FeNa) and decreases ENaC activity in isolated tubules.
Importantly, our studies show that, unlike ouabain, CDDP does not affect channel conductance in cell‐attached patches. Such observations are most consistent with CDDP not affecting the electrochemical forces driving Na+ transport across principal cells, which would change in response to changes in pump activity, but rather decreasing ENaC activity through direct effects on the channel or a channel regulator. In conclusion, these observations are consistent with CDDP actions on ENaC, independent of effects on the pump, contributing to the renal salt and water wasting caused by this chemotherapy drug.Support or Funding InformationThis research was supported by NIH/NIDDK grants R01DK113816 and R01DK117909.
A potential role of Zfra in mitigating traumatic brain injury transition to Alzheimer’s disease‐like symptom in mice
Authors: Liu Tsung-Yun , Chang Nan-Shan 
Published: April 2020

Most recently, tumor suppressor WWOX is defined as a risk factor for Alzheimer’s disease(AD). Previously, we determined that accumulation of pS14‐WWOX in the brain hippocampus and cortex correlates with the occurrence of AD and probably other neurodegeneration. Zinc finger‐like protein that regulates apoptosis (Zfra) suppresses pS14‐WWOX accumulation and restores memory loss in triple transgenic mice (3xTg) for AD. During the acute phase traumatic brain injury (TBI), overly expressed WWOX becomes phosphorylated at Tyr33 and is accumulated in the nuclei to cause neuronal death in the impacted areas. We determined that in the novel object recognition tests, mice surviving from TBI (2.8 to 3.3 Newtonian force) significantly spent less exploring time on new objects in both long‐term and short‐term memory tests post TBI for 18 weeks, indicating the occurrence of damage in long‐ and short‐term memory. Further, control and TBI mice were subjected to Morris water maze tests post TBI for 10 weeks to determine the hippocampus‐dependent, spatial memory. Both groups showed a time‐dependent decrease in escape latency, indicating that mild TBI didn’t affect the learning capabilities. BALB/c mice received Zfra4‐10 peptide via tail vein injections prior to TBI (1.7 Newtonian force). Two weeks later, these treated mice significantly spent more time on probing the new object in the short‐term memory task compared to control mice, suggesting that Zfra peptide prevents TBI‐induced memory loss. Interestingly, by immunohistochemistry, significant upregulation of tau phosphorylation at T181 and amyloid beta aggregates was shown rapidly in death‐inducing impact in BALB/c mice (3.5 Newton), and that Zfra significantly prevented the upregulation (~20 to 33%). Together, our observations suggest the transition of TBI to AD‐like symptom in mice are likely to occur, and that Zfra4‐10 peptide prevents the transition.Support or Funding InformationMinistry of Science & Technology, Taiwan, National Health Research Institute, Taiwan, Department of Defense, USA
Hemodynamic Sex‐differences in Diastolic Function
Authors: Ogilvie Leslie M. , Edgett Brittany A. , Huber Jason S. , Platt Mathew J. , Brunt Keith R. , Simpson Jeremy A. 
Published: April 2020

BackgroundIn the past decade, cardiac research has focused on the emerging crisis of diastolic dysfunction, commonly observed in patients with heart failure with preserved ejection fraction (HFpEF) — a disorder with ineffective management strategies. Diastolic dysfunction is abnormal active relaxation and/or passive stiffness of the ventricle. Because echocardiography is non‐invasive and accessible to more specialities, it remains widely used in the diagnosis and prognostic gradation of diastolic dysfunction. However, echocardiography provides indirect estimations of diastolic indices because diastolic measures were calibrated based on direct measures of end diastolic filling pressure (the gold standard for diastolic assessment). As it is increasingly evident that the manifestation of heart failure in men/males and women/females is not the same, a focus on sex‐specific mechanisms and physiology of diastole is warranted.Experimental ApproachThe primary objective of this work is to perform a comprehensive hemodynamic analysis of diastolic function in males and females to highlight sex‐differences that exist in diastolic physiology and provide implications in cardiac pathology. Male and female Wistar rats were anesthetized with isoflurane and a pressure catheter was inserted into the right carotid artery and advanced into the left ventricle. Pressure measurements were sampled at 2kHz and digitized using custom software. Diastolic parameters – end diastolic pressure (EDP), maximum relaxation rate (dP/dtmin), isovolumetric relaxation time constant (Tau Weiss, Glantz, Logistic) – were analyzed to evaluate diastolic function.Results and ConclusionsDiastolic function in healthy males and females is not equal, suggesting there are sex‐specific differences in diastolic physiology. In a pathological state, each of the diastolic parameters follows its own unique time course until severe dysfunction is present, suggesting that diastole cannot be reconciled by any one parameter. EDP, dP/dtmin and Tau Logistic should be used together to stratify dysfunction as diastole is a dynamic process and each parameter captures a specific nuance.Support or Funding InformationCanadian Institutes of Health Research
Cardiac Regeneration Occurs Following a Puncture Wound to the Heart of the Leopard Gecko (Eublepharis macularius)
Authors: Jacyniak Kathy , Vickaryous Matthew Kenneth 
Published: April 2020

Survivable injuries to the heart resolve via one of two outcomes: scar formation or tissue regeneration. Among mammals, injury to the heart typically results in the loss of contractile muscle cells (cardiomyocytes) and the formation of a non‐contractile fibrous scar. In contrast, some teleost fish and salamanders are capable of tissue‐specific regeneration, thus replacing lost or damaged cardiomyocytes. Here, we investigate the regenerative capacity of the reptilian heart, focusing on the lizard Eublepharis macularius, the leopard gecko. Unlike mammals, but similar to teleost fish, the gecko heart has a single ventricle. To determine if the ventricle was capable of regeneration, we performed a cardiac puncture (cardiocentesis) – a penetrating wound that passes through the thoracic cavity and into the heart. Cardiac punctures are readily tolerated by geckos, and normal (pre‐injury) behaviours are observed within hours of the procedure. To characterize the reparative events, we used serial histology and immunostaining for markers of cell proliferation (proliferating cell nuclear antigen, PCNA) or cell death (Terminal deoxynucleotidyl transferase dUTP nick end labeling [TUNEL]), each with markers for cardiomyocytes (myosin heavy chain [MHC], alpha‐smooth muscle actin [α‐SMA]), and fibroblasts/endocardial cells (Vimentin). Prior to injury, we observed proliferating populations of both cardiomyocytes and non‐cardiomyocytes. One day post‐cardiac puncture (dpc), the wound site is characterized by cell death, a localized loss of cardiomyocytes, and the formation of a blood clot capping the puncture. Between 5 and 10 dpc, there is an increase in the number of proliferating cardiomyocytes bordering the lesion, simultaneous with an increase in proliferating non‐cardiomyocytes (cardiac fibroblasts and endothelial cells) within the wound bed itself. By 14dpc, mature cardiomyocytes have repopulated the wound site, restoring the original architecture of the myocardium. Overall, our findings reveal that regeneration of the gecko heart closely resembles that of zebrafish, and is distinct from the scar‐forming response of mammals.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants 400358
The Study of Sphingolipids Metabolism and Its Effect on Brain Disorders
Authors: Hernandez Ricardo 
Published: April 2020

Sphingolipids are molecules involved in important processes like cell growth, signaling, cell structure and apoptosis. It is known that sphingolipids metabolism is involved in the proper development of the brain and deficiency in sphingolipids homeostasis lead to psychiatric disorders Sphingolipids are also known to play a role in other disorders, such: necrosis. In this research, sphingolipids metabolism is studied using Saccharomyces cerevisiae as a model. A CSG2 deletion mutant, accumulates inositolphosphoryl ceramide (IPC), the first complex sphingolipid. When it is exposed to nitrogen starvation conditions with a fermentable carbon source present, the cells do not adapt properly to this stressor because the mitochondria cannot increase the MMP (mitochondria membrane potential), which leads to an increase in reactive oxygen species (ROS) and cell death. Although, when the same mutant is exposed to nitrogen starvation conditions with a nonfermentable carbon source present, cells grow normally. Cells are rescued by the ISC1 gene activity. ISC1human’s homologue gene codes for the protein sphingomyelinase. This protein breaks down complex sphingolipids into ceramide. It is well known that ISC1 is involved in process related to nutrient availability and it is activated under nonfermentable carbon sources. Currently, Isc1p is the only protein known that can break down complex sphingolipids in yeast. This organism has two main types of complex sphingolipids, mannosylated and non‐mannosylated. In this research, we do not only study IPC (non‐mannosylated complex sphingolipid) but also the other two more complex mannosylated sphingolipids: mannose‐inositol‐phosphoceramide (MIPC) and mannose‐(inositol‐P)2‐ceramide (M(IP)2C). It has been shown that Ipt1D, mutant that accumulates MIPC since, the gene, IPT1, that codes for the protein that turns MIPC into M(IP)2C has been deleted, survive under nitrogen starvation when a fermentable carbon source is present. Our results suggest that Isc1p cannot bind to complex sphingolipids when a mannose group is attached to them and another protein, not known yet, that carries out the function of breaking down mannosylated complex sphingolipids when a fermentable carbon source is present. Therefore, this research focuses on the study of sphingolipids metabolism and homeostasis, studying genes that are involved in the anabolism as well as the catabolism pathways. This research uses data collected form hands‐on experiment, for instance: TLC, fluorescence microscope, cell viability assay; and from computational methods. The results will lead to a better understanding of sphingolipids homeostasis and can help to target the treatment of disorders caused by these molecules.Support or Funding InformationPresbyterian College, Chemistry and Biochemistry Department