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Boukovalas, Stefanos (1)
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Year of Publication
Ensuring good quality rna for quantitative real-time pcr isolated from renal proximal tubular cells using laser capture microdissection
Yee, Jie Yin
Rogers, Susan Mary
Tay, Vanessa SY
Lee, Edmund JD
BMC Research Notes
In order to provide gene expression profiles of different cell types, the primary step is to isolate the specific cells of interest via laser capture microdissection (LCM), followed by extraction of good quality total RNA sufficient for quantitative real-time polymerase chain reaction (qPCR) analysis. This LCM-qPCR strategy has allowed numerous gene expression studies on specific cell populations, providing valuable insights into specific cellular changes in diseases. However, such strategy imposed challenges as cells of interests are often available in limited quantities and quality of RNA may be compromised during long periods of time spent on collection of cells and extraction of total RNA; therefore, it is crucial that protocols for sample preparation should be optimised according to different cell populations.
We made several modifications to existing protocols to improve the total RNA yield and integrity for downstream qPCR analyses. A modified condensed hematoxylin and eosin (H&E) staining protocol was developed for the identification of rat renal proximal tubular cells (PTCs). It was then determined that a minimal of eight thousands renal PTCs were required to meet the minimal total RNA yield required for downstream qPCR. RNA integrity was assessed using at every progressive step of sample preparation. Therefore, we decided that the shortened H&E staining, together with microdissection should be performed consecutively within twenty minutes for good quality for gene expression analysis. These modified protocols were later applied on six individual rat samples. A panel of twenty rat renal drug transporters and five housekeeping genes showed Ct values below thirty-five, confirming the expression levels of these drug transporters can be detected.
We had successfully optimized the protocols to achieve sufficient good quality total RNA from microdissected rat renal PTCs for gene expression profiling via qPCR. This protocol may be suitable for researchers who are interested in employing similar applications for gene expression studies.
Fast oscillations during gasping and other non-eupneic respiratory behaviors: Clues to central pattern generation
Respiratory physiology & neurobiology
The mammalian nervous system exhibits fast synchronous oscillations, which are especially prominent in respiratory-related nerve discharges. In the phrenic nerve, they include high- (HFO), medium- (MFO), and low-frequency (LFO) oscillations. Because motoneurons firing at HFO-related frequencies had never been recorded, an epiphenomenological mechanism for their existence had been posited. We have recently recorded phrenic motoneurons firing at HFO-related frequencies in unanesthetized decerebrate rats and showed that they exhibit dynamic coherence with the phrenic nerve, validating synchronous motoneuronal discharge as a mechanism underlying the generation of HFO. In so doing, we have helped validate the conclusions of previous studies by us and other investigators who have used changes in fast respiratory oscillations to make inferences about central respiratory pattern generation. Here, we seek to review changes occurring in fast synchronous oscillations during non-eupneic respiratory behaviors, with special emphasis on gasping, and the inferences that can be drawn from these dynamics regarding respiratory pattern formation.
Breathing; Motor synchrony; Gasping; Apneusis
Emotional enhancement of immediate memory: Positive pictorial stimuli are better recognized than neutral or negative pictorial stimuli
Advances in Cognitive Psychology
We examined emotional memory enhancement (EEM) for negative and positive pictures while manipulating encoding and retrieval conditions. Two groups of 40 participants took part in this study. Both groups performed immediate implicit (categorization task) and explicit (recognition task) retrieval, but for one group the tasks were preceded by incidental encoding and for the other group by intentional encoding. As indicated by the sensitivity index (dʹ), after incidental encoding positive stimuli were easier to recognize than negative and neutral stimuli. Participants’ response criterion was more liberal for negative stimuli than for both positive and neutral ones, independent of encoding condition. In the implicit retrieval task, participants were slower in categorizing positive than negative and neutral stimuli. However, the priming effect was larger for emotional than for neutral stimuli. These results are discussed in the context of the idea that the effect of emotion on immediate memory enhancement may depend on the intentionality to encode and retrieve information.
emotional memory enhancement; explicit/ implicit retrieval; intentional/ incidental encoding
In Vitro Interactions of Antimicrobial Combinations with Fosfomycin against KPC-2-Producing Klebsiella pneumoniae and Protection of Resistance Development▿
Antimicrobial Agents and Chemotherapy
Using time-kill methodology, we investigated the interactions of fosfomycin with meropenem or colistin or gentamicin against 17 genetically distinct Klebsiella pneumoniae clinical isolates carrying blaKPC-2. Synergy was observed with meropenem or colistin against 64.7 and 11.8% of tested isolates, while the combination with gentamicin resulted in indifference. All studied combinations showed improved bactericidal activity, compared to fosfomycin alone and prevented the development of fosfomycin resistance in 69.2, 53.8, and 81.8% of susceptible isolates, respectively.
Early Estimation of the Reproduction Number in the Presence of Imported Cases: Pandemic Influenza H1N1-2009 in New Zealand
We analyse data from the early epidemic of H1N1-2009 in New Zealand, and estimate the reproduction number . We employ a renewal process which accounts for imported cases, illustrate some technical pitfalls, and propose a novel estimation method to address these pitfalls. Explicitly accounting for the infection-age distribution of imported cases and for the delay in transmission dynamics due to international travel, was estimated to be (95% confidence interval: ). Hence we show that a previous study, which did not account for these factors, overestimated . Our approach also permitted us to examine the infection-age at which secondary transmission occurs as a function of calendar time, demonstrating the downward bias during the beginning of the epidemic. These technical issues may compromise the usefulness of a well-known estimator of - the inverse of the moment-generating function of the generation time given the intrinsic growth rate. Explicit modelling of the infection-age distribution among imported cases and the examination of the time dependency of the generation time play key roles in avoiding a biased estimate of , especially when one only has data covering a short time interval during the early growth phase of the epidemic.
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