Presentations from the 1994 MIT/industry cooperative research program annual meeting

Cover titleMay 199

Fast, accurate, and cost-effective poultry sex genotyping using real-time polymerase chain reaction

According to The Organization for Economic Co-operation and Development (OECD), demand for poultry meat and eggs consumption is growing consistently since poultry meat and eggs are readily available and cheap source for nutritional protein. As such, there is pressing demand from industry for improved protocols to determine chicken sex, especially in layer industry since only females can lay eggs. Extensive efforts are being dedicated to avoiding male chicks culling by developing in-ovo sexing detection methods. Any established in-ovo detection method will need to be validated by embryo genotyping. Therefore, there is a growing demand for fast, inexpensive, and precise method for proper discrimination between males and females in the poultry science community. Our aim with this study was to develop an accurate, high-throughput protocol for sex determination using small volumes of blood. We designed primers targeting the Hint-W gene within the W chromosome clearly distinguishing between males and females. In the interest of establishing an efficient protocol without the need for gel electrophoresis, crude DNA extraction without further purification was coupled with qPCR. We validated the accuracy of our method using established protocols and gonad phenotyping and tested our protocol with four different chicken breeds, day-nine embryos, day-old chicks and adult chicken. In summary, we developed a fast, cost-effective, and accurate method for the genotyping of sex chromosomes in chicken

AMP-activated protein kinase inhibits K_v1.5 channel currents of pulmonary arterial myocytes in response to hypoxia and inhibition of mitochondrial oxidative phosphorylation

KEY POINTS: Progression of hypoxic pulmonary hypertension is thought to be due, in part, to suppression of voltage‐gated potassium channels (K(v)) in pulmonary arterial smooth muscle by hypoxia, although the precise molecular mechanisms have been unclear. AMP‐activated protein kinase (AMPK) has been proposed to couple inhibition of mitochondrial metabolism by hypoxia to acute hypoxic pulmonary vasoconstriction and progression of pulmonary hypertension. Inhibition of complex I of the mitochondrial electron transport chain activated AMPK and inhibited K(v)1.5 channels in pulmonary arterial myocytes. AMPK activation by 5‐aminoimidazole‐4‐carboxamide riboside, A769662 or C13 attenuated K(v)1.5 currents in pulmonary arterial myocytes, and this effect was non‐additive with respect to K(v)1.5 inhibition by hypoxia and mitochondrial poisons. Recombinant AMPK phosphorylated recombinant human K(v)1.5 channels in cell‐free assays, and inhibited K(+) currents when introduced into HEK 293 cells stably expressing K(v)1.5. These results suggest that AMPK is the primary mediator of reductions in K(v)1.5 channels following inhibition of mitochondrial oxidative phosphorylation during hypoxia and by mitochondrial poisons. ABSTRACT: Progression of hypoxic pulmonary hypertension is thought to be due, in part, to suppression of voltage‐gated potassium channels (K(v)) in pulmonary arterial smooth muscle cells that is mediated by the inhibition of mitochondrial oxidative phosphorylation. We sought to determine the role in this process of the AMP‐activated protein kinase (AMPK), which is intimately coupled to mitochondrial function due to its activation by LKB1‐dependent phosphorylation in response to increases in the cellular AMP:ATP and/or ADP:ATP ratios. Inhibition of complex I of the mitochondrial electron transport chain using phenformin activated AMPK and inhibited K(v) currents in pulmonary arterial myocytes, consistent with previously reported effects of mitochondrial inhibitors. Myocyte K(v) currents were also markedly inhibited upon AMPK activation by A769662, 5‐aminoimidazole‐4‐carboxamide riboside and C13 and by intracellular dialysis from a patch‐pipette of activated (thiophosphorylated) recombinant AMPK heterotrimers (α2β2γ1 or α1β1γ1). Hypoxia and inhibitors of mitochondrial oxidative phosphorylation reduced AMPK‐sensitive K(+) currents, which were also blocked by the selective K(v)1.5 channel inhibitor diphenyl phosphine oxide‐1 but unaffected by the presence of the BK(Ca) channel blocker paxilline. Moreover, recombinant human K(v)1.5 channels were phosphorylated by AMPK in cell‐free assays, and K(+) currents carried by K(v)1.5 stably expressed in HEK 293 cells were inhibited by intracellular dialysis of AMPK heterotrimers and by A769662, the effects of which were blocked by compound C. We conclude that AMPK mediates K(v) channel inhibition by hypoxia in pulmonary arterial myocytes, at least in part, through phosphorylation of K(v)1.5 and/or an associated protein

Impaired access of lymphocytes to neoplastic prostate tissue is associated with neoangiogenesis in the tumour site

Recent reports demonstrated that neovasculature of certain murine tumours inhibits migration of lymphocytes to malignant tissues. We examined the possible existence of this phenomenon in human prostate adenocarcinoma by relating extent, patterns and composition of leucocyte infiltrates in adenocarinoma specimens (N=28) to microvessel density and percentages of these vessels expressing adhesion molecules CD54, CD106 and CD62E. Specimens of nodular hyperplasia (N=30) were used as a control for nonmalignant prostate. Increased microvessel density was detected in foci of adenocarcinoma, as compared with adjacent benign areas (P=0.004) or hyperplastic specimens (P=0.001). Only CD54 was detected on prostate vasculature; percentages of CD54-expressing vessels in adenocarcinoma lesions and adjacent areas were higher than in hyperplasia (P=0.041 and P=0.014, respectively). Infiltrating leucocytes were either scattered diffusely in tissue or organised into clusters mainly composed of CD4-positive lymphocytes; smaller percentage of tissue was occupied by clustered infiltrates in adenocarcinoma foci (mean=0.7; median=0; range=0–5) than in adjacent tissue (mean=2.5; median=1; range=0–15; P=.021) and hyperplasia (mean=1.9; median=2; range=0–5; P=.006). In adenocarcinoma foci, microvessel density tended to negatively correlate with percentage of tissue occupied by an overall leucocyte infiltrate (mean=8.6; median=7.5; range=30) and negatively correlated with percentage of tissue occupied by clustered infiltrate (P=0.045). Percentage of CD54-expressing vessels positively correlated with percentage of tissue occupied by an overall (mean=12; median=10; range=30; P=0.01) and clustered (P=0.023) infiltrate in hyperplasia, whereas in carcinoma-adjacent benign areas, correlation was detected only for clustered infiltrates (P=0.02). The results indicate that impaired access of lymphocytes to malignant lesions is associated with increased numbers of newly formed blood vessels, whereas vascular CD54 likely contributes to extravasation of lymphocytes only in benign prostate tissue

Fluorescence-Tracking of Activation Gating in Human ERG Channels Reveals Rapid S4 Movement and Slow Pore Opening

Background: hERG channels are physiologically important ion channels which mediate cardiac repolarization as a result of their unusual gating properties. These are very slow activation compared with other mammalian voltage-gated potassium channels, and extremely rapid inactivation. The mechanism of slow activation is not well understood and is investigated here using fluorescence as a direct measure of S4 movement and pore opening. Methods and Findings: Tetramethylrhodamine-5-maleimide (TMRM) fluorescence at E519 has been used to track S4 voltage sensor movement, and channel opening and closing in hERG channels. Endogenous cysteines (C445 and C449) in the S1–S2 linker bound TMRM, which caused a 10 mV hyperpolarization of the VK of activation to 227.562.0 mV, and showed voltage-dependent fluorescence signals. Substitution of S1–S2 linker cysteines with valines allowed unobstructed recording of S3–S4 linker E519C and L520C emission signals. Depolarization of E519C channels caused rapid initial fluorescence quenching, fit with a double Boltzmann relationship, F-VON, with VK,1 = 237.861.7 mV, and VK,2 = 43.567.9 mV. The first phase, VK,1, was,20 mV negative to the conductance-voltage relationship measured from ionic tail currents (G-VK = 218.361.2 mV), and relatively unchanged in a non-inactivating E519C:S620T mutant (V K = 234.461.5 mV), suggesting the fast initial fluorescence quenching tracked S4 voltage sensor movement. The second phase of rapid quenching was absent in the S620T mutant. The E519C fluorescence upon repolarizatio

Localization of uPAR and MMP-9 in lipid rafts is critical for migration, invasion and angiogenesis in human breast cancer cells

<p>Abstract</p> <p>Background</p> <p>uPAR and MMP-9, which play critical roles in tumor cell invasion, migration and angiogenesis, have been shown to be associated with lipid rafts.</p> <p>Methods</p> <p>To investigate whether cholesterol could regulate uPAR and MMP-9 in breast carcinoma, we used MβCD (methyl beta cyclodextrin, which extracts cholesterol from lipid rafts) to disrupt lipid rafts and studied its effect on breast cancer cell migration, invasion, angiogenesis and signaling.</p> <p>Results</p> <p>Morphological evidence showed the association of uPAR with lipid rafts in breast carcinoma cells. MβCD treatment significantly reduced the colocalization of uPAR and MMP-9 with lipid raft markers and also significantly reduced uPAR and MMP-9 at both the protein and mRNA levels. Spheroid migration and invasion assays showed inhibition of breast carcinoma cell migration and invasion after MβCD treatment. <it>In vitro </it>angiogenesis studies showed a significant decrease in the angiogenic potential of cells pretreated with MβCD. MβCD treatment significantly reduced the levels of MMP-9 and uPAR in raft fractions of MDA-MB-231 and ZR 751 cells. Phosphorylated forms of Src, FAK, Cav, Akt and ERK were significantly inhibited upon MβCD treatment. Increased levels of soluble uPAR were observed upon MβCD treatment. Cholesterol supplementation restored uPAR expression to basal levels in breast carcinoma cell lines. Increased colocalization of uPAR with the lysosomal marker LAMP1 was observed in MβCD-treated cells when compared with untreated cells.</p> <p>Conclusion</p> <p>Taken together, our results suggest that cholesterol levels in lipid rafts are critical for the migration, invasion, and angiogenesis of breast carcinoma cells and could be a critical regulatory factor in these cancer cell processes mediated by uPAR and MMP-9.</p

Characterization of Multiple Ion Channels in Cultured Human Cardiac Fibroblasts

Background: Although fibroblast-to-myocyte electrical coupling is experimentally suggested, electrophysiology of cardiac fibroblasts is not as well established as contractile cardiac myocytes. The present study was therefore designed to characterize ion channels in cultured human cardiac fibroblasts. Methods and Findings: A whole-cell patch voltage clamp technique and RT-PCR were employed to determine ion channels expression and their molecular identities. We found that multiple ion channels were heterogeneously expressed in human cardiac fibroblasts. These include a big conductance Ca2+-activated K+ current (BKCa) in most (88%) human cardiac fibroblasts, a delayed rectifier K+ current (IKDR) and a transient outward K+ current (Ito) in a small population (15 and 14%, respectively) of cells, an inwardly-rectifying K+ current (IKir) in 24% of cells, and a chloride current (ICl) in 7% of cells under isotonic conditions. In addition, two types of voltage-gated Na+ currents (INa) with distinct properties were present in most (61%) human cardiac fibroblasts. One was a slowly inactivated current with a persistent component, sensitive to tetrodotoxin (TTX) inhibition (INa.TTX, IC50 = 7.8 nM), the other was a rapidly inactivated current, relatively resistant to TTX (INa.TTXR, IC50 = 1.8 μM). RT-PCR revealed the molecular identities (mRNAs) of these ion channels in human cardiac fibroblasts, including KCa.1.1 (responsible for BKCa), Kv1.5, Kv1.6 (responsible for IKDR), Kv4.2, Kv4.3 (responsible for Ito), Kir2.1, Kir2.3 (for IKir), Clnc3 (for ICl), NaV1.2, NaV1.3, NaV1.6, NaV1.7 (for INa.TTX), and NaV1.5 (for INa.TTXR). Conclusions: These results provide the first information that multiple ion channels are present in cultured human cardiac fibroblasts, and suggest the potential contribution of these ion channels to fibroblast-myocytes electrical coupling. © 2009 Li et al.published_or_final_versio

Transcriptome dynamics of a broad host-range cyanophage and its hosts

Cyanobacteria are highly abundant in the oceans and are constantly exposed to lytic viruses. The T4-like cyanomyoviruses are abundant in the marine environment and have broad host-ranges relative to other cyanophages. It is currently unknown whether broad host-range phages specifically tailor their infection program for each host, or employ the same program irrespective of the host infected. Also unknown is how different hosts respond to infection by the same phage. Here we used microarray and RNA-seq analyses to investigate the interaction between the Syn9 T4-like cyanophage and three phylogenetically, ecologically and genomically distinct marine Synechococcus strains: WH7803, WH8102 and WH8109. Strikingly, Syn9 led a nearly identical infection and transcriptional program in all three hosts. Different to previous assumptions for T4-like cyanophages, three temporally regulated gene expression classes were observed. Furthermore, a novel regulatory element controlled early-gene transcription, and host-like promoters drove middle gene transcription, different to the regulatory paradigm for T4. Similar results were found for the P-TIM40 phage during infection of Prochlorococcus NATL2A. Moreover, genomic and metagenomic analyses indicate that these regulatory elements are abundant and conserved among T4-like cyanophages. In contrast to the near-identical transcriptional program employed by Syn9, host responses to infection involved host-specific genes primarily located in hypervariable genomic islands, substantiating islands as a major axis of phage-cyanobacteria interactions. Our findings suggest that the ability of broad host-range phages to infect multiple hosts is more likely dependent on the effectiveness of host defense strategies than on differential tailoring of the infection process by the phage

Impact of renal impairment on atrial fibrillation: ESC-EHRA EORP-AF Long-Term General Registry

Background: Atrial fibrillation (AF) and renal impairment share a bidirectional relationship with important pathophysiological interactions. We evaluated the impact of renal impairment in a contemporary cohort of patients with AF. Methods: We utilised the ESC-EHRA EORP-AF Long-Term General Registry. Outcomes were analysed according to renal function by CKD-EPI equation. The primary endpoint was a composite of thromboembolism, major bleeding, acute coronary syndrome and all-cause death. Secondary endpoints were each of these separately including ischaemic stroke, haemorrhagic event, intracranial haemorrhage, cardiovascular death and hospital admission. Results: A total of 9306 patients were included. The distribution of patients with no, mild, moderate and severe renal impairment at baseline were 16.9%, 49.3%, 30% and 3.8%, respectively. AF patients with impaired renal function were older, more likely to be females, had worse cardiac imaging parameters and multiple comorbidities. Among patients with an indication for anticoagulation, prescription of these agents was reduced in those with severe renal impairment, p&nbsp;&lt;.001. Over 24&nbsp;months, impaired renal function was associated with significantly greater incidence of the primary composite outcome and all secondary outcomes. Multivariable Cox regression analysis demonstrated an inverse relationship between eGFR and the primary outcome (HR 1.07 [95% CI, 1.01–1.14] per 10&nbsp;ml/min/1.73&nbsp;m2 decrease), that was most notable in patients with eGFR &lt;30&nbsp;ml/min/1.73&nbsp;m2 (HR 2.21 [95% CI, 1.23–3.99] compared to eGFR ≥90&nbsp;ml/min/1.73&nbsp;m2). Conclusion: A significant proportion of patients with AF suffer from concomitant renal impairment which impacts their overall management. Furthermore, renal impairment is an independent predictor of major adverse events including thromboembolism, major bleeding, acute coronary syndrome and all-cause death in patients with AF

Clinical complexity and impact of the ABC (Atrial fibrillation Better Care) pathway in patients with atrial fibrillation: a report from the ESC-EHRA EURObservational Research Programme in AF General Long-Term Registry

Background: Clinical complexity is increasingly prevalent among patients with atrial fibrillation (AF). The ‘Atrial fibrillation Better Care’ (ABC) pathway approach has been proposed to streamline a more holistic and integrated approach to AF care; however, there are limited data on its usefulness among clinically complex patients. We aim to determine the impact of ABC pathway in a contemporary cohort of clinically complex AF patients. Methods: From the ESC-EHRA EORP-AF General Long-Term Registry, we analysed clinically complex AF patients, defined as the presence of frailty, multimorbidity and/or polypharmacy. A K-medoids cluster analysis was performed to identify different groups of clinical complexity. The impact of an ABC-adherent approach on major outcomes was analysed through Cox-regression analyses and delay of event (DoE) analyses. Results: Among 9966 AF patients included, 8289 (83.1%) were clinically complex. Adherence to the ABC pathway in the clinically complex group reduced the risk of all-cause death (adjusted HR [aHR]: 0.72, 95%CI 0.58–0.91), major adverse cardiovascular events (MACEs; aHR: 0.68, 95%CI 0.52–0.87) and composite outcome (aHR: 0.70, 95%CI: 0.58–0.85). Adherence to the ABC pathway was associated with a significant reduction in the risk of death (aHR: 0.74, 95%CI 0.56–0.98) and composite outcome (aHR: 0.76, 95%CI 0.60–0.96) also in the high-complexity cluster; similar trends were observed for MACEs. In DoE analyses, an ABC-adherent approach resulted in significant gains in event-free survival for all the outcomes investigated in clinically complex patients. Based on absolute risk reduction at 1 year of follow-up, the number needed to treat for ABC pathway adherence was 24 for all-cause death, 31 for MACEs and 20 for the composite outcome. Conclusions: An ABC-adherent approach reduces the risk of major outcomes in clinically complex AF patients. Ensuring adherence to the ABC pathway is essential to improve clinical outcomes among clinically complex AF patients