Biphasic Manner Research Articles

Biphasic unbinding of a metalloregulator from DNA for transcription (de)repression in Live Bacteria.

Microorganisms use zinc-sensing regulators to alter gene expression in response to changes in the availability of zinc, an essential micronutrient. Under zinc-replete conditions, the Fur-family metalloregulator Zur binds to DNA tightly in its metallated repressor form to Zur box operator sites, repressing the transcription of zinc uptake transporters. Derepression comes from unbinding of the regulator, which, under zinc-starvation conditions, exists in its metal-deficient non-repressor forms having no significant affinity with Zur box. While the mechanism of transcription repression by Zur is well-studied, little is known on how derepression by Zur could be facilitated. Using single-molecule/single-cell measurements, we find that in live Escherichia coli cells, Zur's unbinding rate from DNA is sensitive to Zur protein concentration in a first-of-its-kind biphasic manner, initially impeded and then facilitated with increasing Zur concentration. These results challenge conventional models of protein unbinding being unimolecular processes and independent of protein concentration. The facilitated unbinding component likely occurs via a ternary complex formation mechanism. The impeded unbinding component likely results from Zur oligomerization on chromosome involving inter-protein salt-bridges. Unexpectedly, a non-repressor form of Zur is found to bind chromosome tightly, likely at non-consensus sequence sites. These unusual behaviors could provide functional advantages in Zur's facile switching between repression and derepression.

E. coli Single Stranded Binding Protein (SSB) Self-Regulates Wrapping of SSDNA through Competitive Binding

E. coli single-stranded DNA (ssDNA) binding protein (SSB) is a stable homotetramer that wraps ssDNA in multiple conformations depending on protein concentration and solution conditions. SSB plays an important role in DNA replication and repair and has been utilized in biochemical applications such as increasing PCR specificity and yield. Here, we use an optical tweezers system to measure the binding and wrapping of a single ssDNA substrate by SSB. At saturating concentrations (>1 nM), SSB binds to ssDNA in a biphasic manner, in which a rapid contraction of the ssDNA is followed by a slower partial elongation. Subsequent removal of free SSB from solution results in further contraction of the ssDNA and addition of free SSB again elongates the ssDNA. Oscillations between the two states are controlled by free protein concentration and are repeatable over many cycles. In contrast, low SSB concentrations (<1 nM) stably contract the ssDNA with increased amplitude. We show that these results are consistent with a generalized model in which the conformation of the SSB-ssDNA complex is regulated through competitive binding, where increased protein density destabilizes higher order wrapped states and promotes the dissociation of excess SSB from the substrate. Furthermore, the ability of SSB to form stable tetramers and interactions between the disordered C-terminal tails are critical to these functions. Our results are consistent with a functional model of SSB that maximizes its ability to protect transiently exposed ssDNA without inhibiting replication and repair processes.

Single Molecule Binding Dynamics of Line-1 ORF1P to SSDNA

Long interspersed nuclear element 1 (LINE-1) is an intragenomic parasitic element that makes up ∼ 20% of the human genome. It amplifies in the host genome by copying its RNA transcript into genomic DNA through a process called retrotransposition. L1 encodes two proteins, ORF1p and ORF2p, that associate with their parent transcript to form a ribonucleoprotein complex, an essential intermediate in L1 retrotransposition. Detailed mechanistic understanding of L1 retrotransposition is sparse, particularly with respect to the function of ORF1p, a coiled coil-mediated homotrimeric nucleic acid chaperone that can form tightly packed oligomers on nucleic acids. Here we studied three ORF1 proteins: a modern human wild type (111p), an inactive mutant (m15) wherein a single residue substitution within its coiled coil domain halts retrotransposition activity, and an active mutant (m14) with a largely modern coiled coil. We characterize ORF1p-nucleic acid interactions using an ∼ 8 kbp ssDNA molecule and measure its binding kinetics while the ssDNA is held at constant force. We show that ORF1p binds to ssDNA in a biphasic manner, in which a rapid decrease in ssDNA extension is followed by a relatively slow elongation of the ORF1p-ssDNA complex. The biphasic behavior observed here qualitatively mimics E. coli SSB binding dynamics to ssDNA. Therefore, for the first time, this study provides compelling evidence that ORF1p wraps ssDNA in multiple conformations. In addition to wrapping ssDNA, we show that the ORF1p-ssDNA complex undergoes extremely stable secondary compaction due to well-established protein-protein interactions. We present data which suggest that, although the ORF1p mutants can initially bind and wrap ssDNA in an identical manner as the wild type, the stability of the compact ORF1p-ssDNA complex is considerably weaker for the inactive m15 mutant when compared to 111p and the active m14 mutant.

In Vitro Platform for Studying Human Insulin Release Dynamics of Single Pancreatic Islet Microtissues at High Resolution.

Insulin is released from pancreatic islets in a biphasic and pulsatile manner in response to elevated glucose levels. This highly dynamic insulin release can be studied in vitro with islet perifusion assays. Herein, a novel platform to perform glucose-stimulated insulin secretion (GSIS) assays with single islets is presented for studying the dynamics of insulin release at high temporal resolution. A standardized human islet model is developed and a microfluidic hanging-drop-based perifusion system is engineered, which facilitates rapid glucose switching, minimal sample dilution, low analyte dispersion, and short sampling intervals. Human islet microtissues feature robust and long-term glucose responsiveness and demonstrate reproducible dynamic GSIS with a prominent first phase and a sustained, pulsatile second phase. Perifusion of single islet microtissues produces a higher peak secretion rate, higher secretion during the first and second phases of insulin release, as well as more defined pulsations during the second phase in comparison to perifusion of pooled islets. The developed platform enables to study compound effects on both phases of insulin secretion as shown with two classes of insulin secretagogs. It provides a new tool for studying physiologically relevant dynamic insulin secretion at comparably low sample-to-sample variation and high temporal resolution.

Transient water-electrolyte disturbance after hemispherotomy in young infants with epileptic encephalopathy.

This study aimed to elucidate the clinical features of water-electrolyte disturbance (WED) as a sequela of hemispherotomy. We performed a retrospective chart review to identify the clinical features of diabetes insipidus (DI) as a complication in < 12-month-old patients who underwent hemispherectomy or hemispherotomy for severe epilepsy between 2007 and 2018. Central DI was diagnosed if a patient developed polyuria (urine output > 5mL/kg/h), abnormally high serum osmolality (> 300mOsm/kg), high serum sodium level (> 150mEq/L), either abnormally low urine specific gravity (< 1.005) or low urine osmolality (< 300mOsm/kg) or both, and effective control of polyuria with arginine vasopressin (AVP). The clinical course of post-hemispherotomy WED, complications other than WED, and seizure outcomes were analyzed. The review identified that 3 of 23 infants developed WED. All patients developed polyuria within 2days after surgery, with high serum osmolality and hypotonic urine; AVP was effective in treating these symptoms. The clinical course was compatible with central DI. Two patients subsequently developed hyponatremia in a biphasic or triphasic manner. All patients had multiple seizures that were probably related to WED. Two patients developed asymptomatic cerebral sinovenous thrombosis, possibly because of the surgical procedure and dehydration; anticoagulant treatment was provided. All patients were treated for WED for up to 2months and had no residual pituitary dysfunction. Systemic complications other than intracranial ones can occur in patients who have undergone hemispherotomy. Perioperative systemic management of young infants undergoing this procedure should include careful water and electrolyte balance monitoring.

The kidney injury caused by the onset of acute graft-versus-host disease is associated with down-regulation of αKlotho

Acute graft-versus-host disease (aGVHD) and kidney injury are the major complications after allogeneic hematopoietic stem cell transplantation (HSCT). Although the underlying mechanisms for the development of these complications are not yet fully understood, it has been proposed that emergence of aGVHD contributes to the development of kidney injury after HSCT. We have shown previously that aGVHD targets the kidney in a biphasic manner: at the onset, inflammatory genes are up-regulated, while when aGVHD becomes established, donor lymphocytes infiltrate the kidney. Here, we characterize renal manifestations at the onset of aGVHD. Mice receiving allogeneic bone marrow and spleen cells displayed symptoms of aGVHD and elevated serum levels of tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ) within 4 days. There was concurrent kidney injury with the following characteristics: (1) elevated expression of the kidney injury biomarker, neutrophil gelatinase-associated lipocalin (NGAL), (2) accumulation of hetero-lysosomes in proximal tubule epithelial cells, and (3) reductions in αKlotho mRNA and protein and increased serum levels of fibroblast growth factor 23 (Fgf23), phosphate and urea. This situation resembled acute renal injury caused by bacterial lipopolysaccharide. We conclude that the onset of aGVHD is associated with kidney injury involving down-regulation of αKlotho, a sight that may inspire novel therapeutic approaches.

Biphasic effects of methanandamide on murine gastric vagal afferent mechanosensitivity.

The fine control of food intake is important for the maintenance of a healthy metabolic state. Gastric vagal afferents (GVAs) are involved in the peripheral regulation of food intake via signalling the degree of distension of the stomach which ultimately leads to feelings of fullness and satiety. This study provides evidence that endocannabinoids such as anandamide are capable of regulating GVA sensitivity in a concentration-dependent biphasic manner. This biphasic effect is dependent upon interactions between the CB1, TRPV1 and GHSR receptors. These data have important implications for the peripheral control of food intake. Gastric vagal afferents (GVAs) signal to the hindbrain resulting in satiety. Endocannabinoids are endogenous ligands of cannabinoid 1 receptor (CB1) and transient receptor potential vanilloid-1 (TRPV1) channels. The endocannabinoid anandamide (AEA) is expressed in the stomach, and its receptor CB1 is expressed in ghrelin-positive gastric mucosal cells. Further, TRPV1, CB1 and growth hormone secretagogue receptor (ghrelin receptor, GHSR) are expressed in subpopulations of GVA neurons. This study aimed to determine the interaction between TRPV1, CB1, GHSR and endocannabinoids in the modulation of GVA signalling. An in vitro electrophysiology preparation was used to assess GVA mechanosensitivity in male C57BL/6 mice. Effects of methanandamide (mAEA; 1-100nm), on GVA responses to stretch were determined in the absence and presence of antagonists of CB1, TRPV1, GHSR, protein kinase-A (PKA), protein kinase-C (PKC) and G-protein subunits Gαi/o , or Gαq . Low doses (1-10nm) of mAEA reduced GVA responses to 3g stretch, whereas high doses (30-100nm) increased the response. The inhibitory and excitatory effects of mAEA (1-100nm) were reduced/lost in the presence of a CB1 and TRPV1 antagonist. PKA, Gαi/o or GHSR antagonists prevented the inhibitory effect of mAEA on GVA mechanosensitivity. Conversely, in the presence of a PKC or Gαq antagonist the excitatory effect of mAEA was reduced or lost, respectively. Activation of CB1, by mAEA, can activate or inhibit TRPV1 to increase or decrease GVA responses to stretch, depending on the pathway activated. These interactions could play an important role in the fine control of food intake.

SILDENAFIL-MIL-89 FORMULATION AS POTENTIAL DRUG DELIVERY SYSTEM IN PULMONARY ARTERIAL HYPERTENSION

Pulmonary Arterial Hypertension (PAH) is an aggressive disease with poor prognosis, no available cure, and low survival rates. Currently, there are several classes of vasodilator drugs that are commonly used as treatment strategies for PAH including sildenafil, a phosphodiesterase type 5-inhibitor. Despite their clinical benefits, these therapies are hindered by their systemic side effects. This limitation could be overcome using controlled drug release with the potential for targeted drug delivery using a nanomedicine approach. In the current study, we have evaluated the use of a highly porous nano-sized preparation of iron-based metal-organic framework (MOF) commonly referred to as MIL-89 for loading with sildenafil. We have previously shown that MIL-89 is relatively non-toxic in a range of human cell types and well tolerated in vivo. Here, we report the use of a nano-formulation of MIL-89 (nanoMIL-89) as drug delivery for the phosphodiesterase type 5-inhibitor sildenafil. nanoMIL-89 was first prepared and then successfully loaded with sildenafil ([email protected]). [email protected] was shown to release sildenafil in a biphasic manner with an initial rapid release over 6 hours followed by a more sustained release over 72 hours. Finally, the vasodilator effect of [email protected] was measured over 8 hours using isolated mouse aorta. Consistent with drug release kinetics, Sil-nanoMIL-89 induced vasodilation after a lag phase of 3 hours. [email protected] is a promising nano-formulation prototype of sildenafil displaying delayed drug release and vasodilator activity. Further pharmacological assessment of [email protected] in animal models for PAH is now required and constitutes the subject of an ongoing investigation.

A 100-m Sprint Time Is Associated With Deep Trunk Muscle Thickness in Collegiate Male Sprinters.

Introduction: One reason athletes train their trunk muscles is that the body's trunk stability has been shown to prevent injury. However, the relationship between body trunk muscle thickness, particularly that of deep muscles, and athletic performance remains to be clarified.Purpose: We aimed to explore the relationship between 100-m sprint performance and the sizes of the trunk stabilizing muscles, the psoas major muscle (PM), transversus abdominis (TA), and multifidus muscle (MM), in collegiate sprinters.Methods: Fourteen male sprinters belonging to a university athletics club participated in this study. The thicknesses of the TA and MM were measured using an ultrasonic diagnostic apparatus (ProSound C3; Aloka, Tokyo, Japan). The cross-sectional area of the PM was assessed by a magnetic resonance imaging apparatus (Vantage Elan; Toshiba Medical Systems, Tokyo, Japan). The relationship between these anthropometric parameters and the 100-m sprint time was analyzed by Spearman's correlation coefficient, multi- regression analysis, and the change-point regression model.Results: The sizes (mean ± SD) of the muscles were: PM, 43.074 ± 7.35 cm2; TA, 4.36 ± 0.72 mm; and MM, 3.99 ± 0.48 cm. The mean 100-m sprint time was 11.00 ± 0.48 s. Spearman's correlation analysis revealed that the 100-m sprint time had a significant moderate negative correlation with TA (ρ = −0.691, p < 0.01) and a low negative but not significant correlation with MM (ρ = −0.327, p = 0.28), whereas PM did not show a significant or in-negligible correlation. The change-point regression model found the change-points in the 100-m sprint time and the thickness of the TA and MM at 4.70 mm (95% CI: 4.00–5.43 mm) and 3.84 cm (95% CI: 3.28–4.31 cm), respectively. The sprint time decreased with an increase in the thickness of the muscles up to the change-points, whereas it did not change even if the muscles became thicker than the change-points. The change-points were consistently observed when the thickness of the muscles was normalized by body mass.Conclusion: Sprint performance for 100-m was found to be associated with TA and MM thickness in a biphasic manner. As muscle thickness increased, the sprint time decreased, followed by a plateau phase.

IL6 Promotes a STAT3-PRL3 Feedforward Loop via SHP2 Repression in Multiple Myeloma.

Overexpression of PRL-3, an oncogenic phosphatase, was identified as a novel cluster in patients with newly diagnosed multiple myeloma. However, the regulation and oncogenic activities of PRL-3 in multiple myeloma warrant further investigation. Here, we report that IL6 activates STAT3, which acts as a direct transcriptional regulator of PRL-3. Upregulation of PRL-3 increased myeloma cell viability and rephosphorylated STAT3 in a biphasic manner through direct interaction and deactivation of SHP2, thus blocking the gp130 (Y759)-mediated repression of STAT3 activity. Abrogation of PRL-3 reduced myeloma cell survival, clonogenicity, and tumorigenesis, and detailed mechanistic studies revealed "deactivation" of effector proteins such as Akt, Erk1/2, Src, STAT1, and STAT3. Furthermore, loss of PRL-3 efficiently abolished nuclear localization of STAT3 and reduced its occupancy on the promoter of target genes c-Myc and Mcl-1, and antiapoptotic genes Bcl2 and Bcl-xL. PRL-3 also played a role in the acquired resistance of myeloma cells to bortezomib, which could be overcome by PRL-3 silencing. Of clinical relevance, STAT3 and PRL-3 expression was positively correlated in five independent cohorts, and the STAT3 activation signature was significantly enriched in patients with high PRL-3 expression. Furthermore, PRL-3 could be used as a biomarker to identify high-risk patients with multiple myeloma that exhibited poor prognosis and inferior outcome even when treated with novel combinational therapeutics (proteasome inhibitors and immunomodulatory imide drugs). Conclusively, our results support a feedforward mechanism between STAT3 and PRL-3 that prolongs prosurvival signaling in multiple myeloma, and suggest targeting PRL-3 as a valid therapeutic opportunity in multiple myeloma. SIGNIFICANCE: IL6 promotes STAT3-dependent transcriptional upregulation of PRL-3, which in turn re-phosphorylates STAT3 and aberrantly activates STAT3 target genes, leading to bortezomib resistance in multiple myeloma.

Novel interactions between ER\u03b1-36 and STAT3 mediate breast cancer cell migration

BackgroundBreast cancer is the leading cause of cancer death in women worldwide which is closely related to metastasis. But the exact molecular mechanism of ERα-36 and STAT3 on metastasis is still not fully understood.MethodsMCF-7 and MDA-MB-231 human breast cancer cell lines and MCF-10A were overexpressioned or knockdown ERα-36 and STAT3 and tested for migration, invasion and proliferation assays. Direct interaction of STAT3 and ERα-36 were analyzed by coimmunoprecipitation assays. The effect of STAT3 and ERα-36 on MMP2/9 expression was analyzed by qPCR and western blotting. STAT3 phospholyation and acetylation by ERα-36 and p300 were observed and quantified by coimmunoprecipitation assays and western blotting.ResultsCross-talk between ERα-36 and STAT3 was demonstrated to mediate through a direct physical association between the two proteins. Furthermore, the interaction between ERα-36 and STAT3 was demonstrated to give rise to functional changes in their signaling events. Both MMP2 and MMP9 expression require the binding of the newly identified protein complex, ERα-36-STAT3, to its promoter, the second phase, which is more robust, depends on ERα-mediated recruitment of p300 onto the complex and the subsequent acetylation of STAT3. In addition, STAT3 is tyrosine-phosphorylated in a biphasic manner, and the late phase requires ERα-36-mediated p300-dependent acetylation. Furthermore, interference with acetylation of STAT3 by overexpression of acetylation null STAT3 mutant led to the loss of MMP2 and MMP9 expression. ChIP analysis and reporter gene assays revealed that ERα-36-STAT3 complex binding to the MMP2 and MMP9 promoter led to an enhanceosome formation and facilitated MMP2 and MMP9 expression.ConclusionsOur studies demonstrate for the first time that the function of MMP2 and MMP9 in breast cancer cell migration, which is mediated by interactions between ERα-36 and STAT3.

Pharmacokinetics and hemorheology of phosphocreatine and creatine in rabbits: A directly comparative study between parent drug and active metabolite

This study is to investigate pharmacokinetics (PK) and hemorheology (HR) of exogenous phosphocreatine (PCr), a cardio-protective agent, and its active metabolite creatine (Cr), with particular focus on the PK and PD comparison between PCr and Cr. A specific ion-pair reversed-phase HPLC-UV assay was used to simultaneously measure PCr, Cr and ATP concentrations in plasma and red blood cells (RBC) samples of rabbits. PK and HR parameters were calculated based on concentration-time (C-T) curves and effect-time (E-T) curves, respectively, obtained after i.v. dosing. Meanwhile the apparent pharmacological activity ratio (Rapp) and real pharmacological activity ratio (Rreal) of Cr to PCr were calculated. The PCr disappeared from plasma rapidly and in a biphasic manner; plasma PCr was converted to Cr fast and largely with the elimination rate limited metabolite disposition in vivo (Km < K). The i.v. administration of PCr led to a markedly elevated and long-lasting ATP level in RBC. After i.v. administration of preformed Cr, plasma Cr displayed similar elimination kinetics behaviors to that of Cr generated metabolically after i.v. PCr. The Cr could also raise ATP level in RBC, but to less extent than PCr. Approximately 43% of PCr-derived ATP came from Cr-derived ATP in RBC. PCr could significantly reduce whole blood viscosity and RBC osmotic fragility and Cr could do so, but weakly with estimated Rapp of 0.53–0.68 and Rreal of 0.38–0.48. PCr also inhibited platelet aggregation significantly, as opposed to Cr. The PCr-caused improvement of HR is related to the rise in ATP level in RBC. Cr is likely to partially mediate HR effect of PCr.

NOVEL ROLE OF PRL-3 PHOSPHATASE IN HEMATOLOGICAL MALIGNANCIES

Overexpression of PRL-3, an oncogenic phosphatase, was identified as a novel cluster in newly diagnosed multiple myeloma (MM) patients. However, the regulation and oncogenic activities of PRL-3 in MM warrants further investigation. Here, we report that IL-6 activates STAT3, which acts as a direct transcriptional regulator of PRL-3. Upregulation of PRL-3 increased myeloma cell viability and re-phosphorylates STAT3 in a biphasic manner through direct interaction and deactivation of SHP2, thus blocking the gp130 (Y759)-mediated repression of STAT3 activity. Abrogation of PRL-3 reduced myeloma cell survival, clonogenicity and tumorigenesis, and detailed mechanistic studies revealed a “de-activation” of effector proteins such as Akt, Erk1/2, Src, STAT1 and STAT3. Furthermore, loss of PRL-3 efficiently abolished nuclear localization of STAT3 and reduced its occupancy on the promoter of target genes c-myc and mcl-1, and anti-apoptotic genes bcl-2 and bcl-xl. We also demonstrated a role of PRL-3 in the acquired resistance of myeloma cells towards bortezomib, which can be overcome by PRL-3 silencing. Of clinical relevance, STAT3 and PRL-3 expression is positively correlated in five independent cohorts, and STAT3 activation signature was significantly enriched in patients with high PRL-3 expression. Furthermore, PTP4A3 is a biomarker to identify high-risk MM patients that exhibited poor prognosis and inferior outcome even when treated with novel combinational therapeutics (PI and IMiD). Conclusively, our results supported a feedforward mechanism between STAT3 and PRL-3 that prolongs pro-survival signaling in MM, and proposed the targeting of PRL-3 as a validated therapeutic opportunity in MM.

Temporally Distinct Functions of the Cytokines IL-12 and IL-23 Drive Chronic Colon Inflammation in Response to Intestinal Barrier Impairment

Epithelial barrier defects are implicated in the pathogenesis of inflammatory bowel disease (IBD); however, the role of microbiome dysbiosis and the cytokine networks orchestrating chronic intestinal inflammation in response to barrier impairment remain poorly understood. Here, we showed that altered Schaedler flora (ASF), a benign minimal microbiota, was sufficient to trigger colitis in a mouse model of intestinal barrier impairment. Colitis development required myeloid-cell-specific adaptor protein MyD88 signaling and was orchestrated by the cytokines IL-12, IL-23, and IFN-γ. Colon inflammation was driven by IL-12 during the early stages of the disease, but as the mice aged, the pathology shifted toward an IL-23-dependent inflammatory response driving disease chronicity. These findings reveal that IL-12 and IL-23 act in a temporally distinct, biphasic manner to induce microbiota-driven chronic intestinal inflammation. Similar mechanisms might contribute to the pathogenesis of IBD particularly in patients with underlying intestinal barrier defects.

Differential Effects of Purinergic Signaling in Gastric Cancer-Derived Cells Through P2Y and P2X Receptors.

Gastric cancer (GC) is the one of the most prevalent cancers and one of the leading causes of cancer-induced deaths. Previously, we found that the expression of purinergic P2Y2 receptor (P2Y2R) is increased in GC samples as compared to adjacent healthy mucosa taken from GC-diagnosed patients. In this work, we studied in detail purinergic signaling in the gastric adenocarcinoma-derived cell lines: AGS, MKN-45, and MKN-74, and compared them to a nontumoral epithelial cell line: GES-1. In GC-derived cells, we detected the expression of several purinergic receptors, and found important differences as compared to GES-1 cells. Functional studies revealed a strong contribution of P2Y2Rs in intracellular calcium increases, elicited by adenosine-triphosphate (ATP), uridine-triphosphate (UTP), and the P2Y2R agonist MRS2768. Responses were preserved in the absence of extracellular calcium and inhibited by P2Y2R antagonists. In GES-1 cells, ATP and UTP induced similar responses and the combination of P2X and P2Y receptor antagonists was able to block them. Proliferation studies showed that ATP regulates AGS and MKN-74 cells in a biphasic manner, increasing cell proliferation at 10–100 μM, but inhibiting at 300 μM ATP. On the other hand, 1–300 μM UTP, a P2Y2R agonist, increased concentration-dependent cell proliferation. The effects of UTP and ATP were prevented by both wide-range and specific purinergic antagonists. In contrast, in GES-1 cells ATP only decreased cell proliferation in a concentration-dependent manner, and UTP had no effect. Notably, the isolated application of purinergic antagonists was sufficient to change the basal proliferation of AGS cells, indicating that nucleotides released by the cells can act as paracrine/autocrine signals. Finally, in tumor-derived biopsies, we found an increase of P2Y2R and a decrease in P2X4R expression; however, we found high variability between seven different biopsies and their respective adjacent healthy gastric mucosa. Even so, we found a correlation between the expression levels of P2Y2R and P2X4R and survival rates of GC patients. Taken together, these results demonstrate the involvement of different purinergic receptors and signaling in GC, and the pattern of expression changes in tumoral cells, and this change likely directs ATP and nucleotide signaling from antiproliferative effects in healthy tissues to proliferative effects in cancer.

Probing Single-Cell Mechanical Allostasis Using Ultrasound Tweezers.

In response to external stress, cells alter their morphology, metabolic activity, and functions to mechanically adapt to the dynamic, local environment through cell allostasis. To explore mechanotransduction in cellular allostasis, we applied an integrated micromechanical system that combines an 'ultrasound tweezers'-based mechanical stressor and a Förster resonance energy transfer (FRET)-based molecular force biosensor, termed "actinin-sstFRET," to monitor in situ single-cell allostasis in response to transient stimulation in real time. The ultrasound tweezers utilize 1Hz, 10-s transient ultrasound pulses to acoustically excite a lipid-encapsulated microbubble, which is bound to the cell membrane, and apply a pico- to nano-Newton range of forces to cells through an RGD-integrin linkage. The actinin-sstFRET molecular sensor, which engages the actin stress fibers in live cells, is used to map real-time actomyosin force dynamics over time. Then, the mechanosensitive behaviors were examined by profiling the dynamics in Ca2+ influx, actomyosin cytoskeleton (CSK) activity, and GTPase RhoA signaling to define a single-cell mechanical allostasis. By subjecting a 1Hz, 10-s physical stress, single vascular smooth muscle cells (VSMCs) were observed to remodeled themselves in a biphasic mechanical allostatic manner within 30min that caused them to adjust their contractility and actomyosin activities. The cellular machinery that underscores the vital role of CSK equilibrium in cellular mechanical allostasis, includes Ca2+ influx, remodeling of actomyosin CSK and contraction, and GTPase RhoA signaling. Mechanical allostasis was observed to be compromised in VSMCs from patients with type II diabetes mellitus (T2DM), which could potentiate an allostatic maladaptation. By integrating tools that simultaneously permit localized mechanical perturbation and map actomyosin forces, we revealed distinct cellular mechanical allostasis profiles in our micromechanical system. Our findings of cell mechanical allostasis and maladaptation provide the potential for mechanophenotyping cells to reveal their pathogenic contexts and their biophysical mediators that underlie multi-etiological diseases such as diabetes, hypertension, or aging.

2175-P: Isoform-Specific Roles of Prolyl Hydroxylase Domain Proteins in Regulating Beta-Cell Function

Pancreatic β-cells secrete insulin in a biphasic manner via KATP-channel dependent and independent pathways. The KATP-channel independent pathway produces a rise in the NADPH/NADP+ ratio and cytosolic α-ketoglutarate (α-KG). Prolyl hydroxylase domain proteins (PHDs) belong to the α-KG-dependent dioxygenase superfamily and regulates stability of hypoxia-inducible factor α (HIF-α). Prolyl hydroxylation of HIF-α for ubiquitin-mediated proteasomal degradation requires sufficient levels of oxygen, iron and α-KG. We have previously shown pharmacological inhibition of PHDs reduces glucose-stimulated insulin secretion (GSIS), glucose utilization, and mitochondrial function in clonal β-cells. Glucose-induced changes in cytosolic α-KG may regulate PHD and thus β-cell function. In mouse pancreatic β-cells, PHD isoforms express unique subcellular localization: PHD1 is expressed exclusively in the cytosol, PHD2 is expressed in the cytosol and nucleus, and PHD3 is mainly expressed in the nucleus. To further explore the isoform-specific roles of PHD, we employed a β-cell specific knockout (β-PHD KO) mouse model for each of the PHD isoforms. β-PHD1 KO, β-PHD2 KO and β-PHD3 KO. β-PHD KO mice show no significant in vivo defects associated with glucose tolerance and insulin resistance; however, β-PHD1 KO and β-PHD2 KO mice have significantly increased plasma insulin compared to wild type (WT) controls (p&amp;lt;0.05). Consistent with our cell culture model of PHD knockdown, PHD1 and PHD3 regulate insulin secretion as both β-PHD1 KO and β-PHD3 KO mice show impaired GSIS. Only β-PHD1 KO mice have reduced beta-cell mass (p&amp;lt;0.01). In conclusion, there are significant and unique effects observed with isoform-specific suppression of PHD. Disclosure M. Hoang: None. S.M. Janssen: None. J.W. Joseph: None. Funding Natural Sciences and Engineering Research Council of Canada; Canadian Institutes of Health Research

Development of the human principal inferior olivary nucleus: A morphometric and computerized 3D-reconstruction study

IntroductionThis study describes the prenatal development of the principal inferior olivary nucleus (PO) in humans. Material/methodsTen brains were obtained from preterm infants aged 21–43 postmenstrual weeks (PW). After fixation, the brains were processed into 30-μm serial sections, which were stained using the Klüver–Barrera method. ResultsAt mid-gestation, the dorsal and ventral lamellae were distinguishable. The dorsal lamella (DL) was composed of ballooned and folded portions, with many neurons peripherally gathered in the ballooned portion, and neurons densely packed in the folded portion. Clusters of pyknotic neurons were observed in the lateral portion of the PO at 21PW. The PO acquired thin complicated folds by 28–29 PW. Then, it regained the width of a nuclear band, and further elaborated the folds. The 3D-reconstruction models showed that the basic pattern of folding like in adults was attained at 28−29PW, and that the rostro-medial region of DL was microgyric. The nuclear volume increased exponentially with age. The total surface area increased progressively, while the surface density varied in a biphasic manner, wherein it increased initially and then decreased. The neuronal profile area increased uniformly. The total neuronal number increased uniformly, while the numerical density decreased rapidly during 21–29 PW. ConclusionAfter mid-gestation, the period of 21–29 PW may be critical, because the PO undergoes extensive folding after massive neuronal death.

Mechanochemical Coupling and Junctional Forces during Collective Cell Migration

Cell migration, a fundamental physiological process in which cells sense and move through their surrounding physical environment, plays a critical role in development and tissue formation, as well as pathological processes, such as cancer metastasis and wound healing. During cell migration, dynamics are governed by the bidirectional interplay between cell-generated mechanical forces and the activity of Rho GTPases, a family of small GTP-binding proteins that regulate actin cytoskeleton assembly and cellular contractility. These interactions are inherently more complex during the collective migration of mechanically coupled cells because of the additional regulation of cell-cell junctional forces. In this study, we adapted a recent minimal modeling framework to simulate the interactions between mechanochemical signaling in individual cells and interactions with cell-cell junctional forces during collective cell migration. We find that migration of individual cells depends on the feedback between mechanical tension and Rho GTPase activity in a biphasic manner. During collective cell migration, waves of Rho GTPase activity mediate mechanical contraction/extension and thus synchronization throughout the tissue. Further, cell-cell junctional forces exhibit distinct spatial patterns during collective cell migration, with larger forces near the leading edge. Larger junctional force magnitudes are associated with faster collective cell migration and larger tissue size. Simulations of heterogeneous tissue migration exhibit a complex dependence on the properties of both leading and trailing cells. Computational predictions demonstrate that collective cell migration depends on both the emergent dynamics and interactions between cellular-level Rho GTPase activity and contractility and multicellular-level junctional forces.

Biphasic Functional Interaction between the Adenovirus E4orf4 Protein and DNA-PK.

The adenovirus (Ad) E4orf4 protein contributes to virus-induced inhibition of the DNA damage response (DDR) by reducing ATM and ATR signaling. Consequently, E4orf4 inhibits DNA repair and sensitizes transformed cells to killing by DNA-damaging drugs. Inhibition of ATM and ATR signaling contributes to the efficiency of virus replication and may provide one explanation for the cancer selectivity of cell death induced by the expression of E4orf4 alone. In this report, we investigate a direct interaction of E4orf4 with the DDR. We show that E4orf4 physically associates with the DNA-dependent protein kinase (DNA-PK), and we demonstrate a biphasic functional interaction between these proteins, wherein DNA-PK is required for ATM and ATR inhibition by E4orf4 earlier during infection but is inhibited by E4orf4 as infection progresses. This biphasic process is accompanied by initial augmentation and a later inhibition of DNA-PK autophosphorylation as well as by colocalization of DNA-PK with early Ad replication centers and distancing of DNA-PK from late replication centers. Moreover, inhibition of DNA-PK improves Ad replication more effectively when a DNA-PK inhibitor is added later rather than earlier during infection. When expressed alone, E4orf4 is recruited to DNA damage sites in a DNA-PK-dependent manner. DNA-PK inhibition reduces the ability of E4orf4 to induce cancer cell death, likely because E4orf4 is prevented from arriving at the damage sites and from inhibiting the DDR. Our results support an important role for the E4orf4-DNA-PK interaction in Ad replication and in facilitation of E4orf4-induced cancer-selective cell death.IMPORTANCE Several DNA viruses evolved mechanisms to inhibit the cellular DNA damage response (DDR), which acts as an antiviral defense system. We present a novel mechanism by which the adenovirus (Ad) E4orf4 protein inhibits the DDR. E4orf4 interacts with the DNA damage sensor DNA-PK in a biphasic manner. Early during infection, E4orf4 requires DNA-PK activity to inhibit various branches of the DDR, whereas it later inhibits DNA-PK itself. Furthermore, although both E4orf4 and DNA-PK are recruited to virus replication centers (RCs), DNA-PK is later distanced from late-phase RCs. Delayed DNA-PK inhibition greatly contributes to Ad replication efficiency. When E4orf4 is expressed alone, it is recruited to DNA damage sites. Inhibition of DNA-PK prevents both recruitment and the previously reported ability of E4orf4 to kill cancer cells. Our results support an important role for the E4orf4-DNA-PK interaction in Ad replication and in facilitation of E4orf4-induced cancer-selective cell death.