Role of hilar mossy cells in the CA3-dentate gyrus network during sharp wave-ripple activity in vitro

Der Gyrus dentatus (DG) des Hippokampus wird als Eingangsstation für Informationen aus dem entorhinalen Kortex betrachtet. In das DG-Netzwerk sind zwei exzitatorische Zelltypen eingebettet: Körnerzellen, die Signale von dem entorhinalen Kortex empfangen, und Hilus-Mooszellen (MCs), die Signale von Körnerzellen als auch von feedback-Projektionen von CA3-Pyramidenzellen (PCs) empfangen. Postsynaptische Ziele von MC-Projektionen umfassen DG Körnerzellen und verschiedene Interneurone in der selben und in der kontralateralen Hemisphäre des Gehirns. Die Rolle von MCs während rhythmischer Populationsaktivität, und insbesondere während Sharp-Wave / Ripple-Komplexen (SWRs), ist bisher weitgehend unerforscht. SWRs sind prominente Ereignisse im Hippocampus während des Tiefschlafs (Slow wave sleep) und des ruhigen Wachzustandes, und sie sind an der Gedächtniskonsolidierung beteiligt. In der vorliegenden Arbeit, untersuchen wir mithilfe eines in-vitro-Modells von SWRs, inwieweit Mooszellen an SWRs in CA3 beteiligt sind. Mit CA3-Feldpotential-Ableitungen und gleichzeitigen ‚cell-attached‘ Messungen von einzelnen MCs konnten wir beobachten, dass ein wesentlicher Anteil von MCs (47%) während der SWRs in das aktive neuronale Netzwerk rekrutiert werden. Darüber hinaus fanden wir in MCs SWR-assoziierte synaptische Aktivität, bei denen sowohl die exzitatorischen als auch die inhibitorischen Komponenten phasenkohärent und verzögert zur Ripple Oszillation in CA3 auftreten. Simultane Patch-clamp Messungen von CA3-Pyramidenzellen und MCs zeigten längere exzitatorische und inhibitorische Latenzzeiten bei MCs, was die Hypothese einer von CA3 ausgehenden Feedback-Rekrutierung unterstützt. Unsere Daten zeigen zusätzlich, dass das Verhältnis exzitatorischer zu inhibitorischer Aktivität in MCs höher ist als in CA3-Pyramidenzellen, wodurch die MCs mit höherer Wahrscheinlichkeit während SWRs überschwellig aktiviert werden. Schließlich zeigen wir, dass ein signifikanter Anteil (66%) der getesteten Körnerzellen SWR-assoziierte exzitatorische Signale erhalten, im Vergleich zu MCs zeitlich verzögert, was auf eine indirekte Aktivierung von Körnerzellen durch CA3 PCs über MCs hinweist. Zusammengefasst zeigen unsere Daten die aktive Beteiligung von Mooszellen an SWRs und deuten auf eine funktionelle Bedeutung als Schaltstelle für das CA3- Gyrus dentatus Netzwerk in diesem wichtigen physiologischen Netzwerkzustand hin.The dentate gyrus (DG) is considered as the hippocampal input gate for the information arriving from the entorhinal cortex. Embedded into the DG network are two excitatory cell types –granule cells (GCs), which receive inputs from the entorhinal cortex, and hilar mossy cells (MCs), which receive input from GCs and feedback projections from CA3 pyramidal cells (PCs). The postsynaptic targets of MC projections are the GCs and hilar interneurons in both ipsilateral and contralateral hemispheres of the brain. The role of MCs during rhythmic population activity, and in particular during sharp-wave/ripple complexes (SWRs), has remained largely unexplored. SWRs are prominent field events in the hippocampus during slow wave sleep and quiet wakefulness, and are involved in memory consolidation and future planning. In this study, we sought to understand whether MCs participate during CA3 SWRs using an in vitro model of SWRs. With simultaneous CA3 field potential– and cell-attached recordings from MCs, we observed that a significant fraction of MCs (47%) are recruited into the active neuronal network during SWRs. Moreover, MCs receive pronounced, compound, ripple-associated synaptic input where both excitatory and inhibitory components are phase-coherent with and delayed to the CA3 ripple. Simultaneous patch recordings from CA3 pyramidal neurons and MCs revealed longer excitatory and inhibitory latencies in MCs, supporting a feedback recruitment from CA3. Our data also show that the excitatory to inhibitory charge transfer (E/I) ratio in MCs is higher than in the CA3 PCs, making the MCs more likely to spike during SWRs. Finally, we demonstrate that a significant fraction (66%) of tested GCs receive SWR-associated excitatory inputs that are delayed compared to MCs, indicating an indirect activation of GCs by CA3 PCs via MCs. Together, our data suggest the involvement of mossy cells during SWRs and their importance as a relay for CA3-dentate gyrus networks in this important physiological network state

Computerized Tomography Detects Pulmonary Lesions in Children with Normal Radiographs Diagnosed to have Tuberculosis

This report is based on observations during the conduct of a larger study to develop diagnostic criteria for childhood tuberculosis (TB). Of 20l children confirmed to have pulmonary or lymph node TB, 84 had normal chest radiographs. Computerized tomography (CT) of the chest was performed in nine of them, seven of whom had normal chest radiographs while two had visible calcification. Eight of the nine children had definitive lesions detected by computerized tomography of the chest. While five children had primarily hilar lymph node enlargement, three had pulmonary parenchymal lesions. The use of more sensitive diagnostic tests like computed tomography helps to detect tuberculosis lesions not otherwise visualized on chest radiographs. This report highlights the difficulty in excluding active tuberculosis in children. More studies are required on the role of CT scans in the diagnosis of tuberculosis in children

Polyisoprenylated benzophenone, garcinol, a natural histone acetyltransferase inhibitor, represses chromatin transcription and alters global gene expression

Histone acetylation is a diagnostic feature of transcriptionally active genes. The proper recruitment and function of histone acetyltransferases (HATs) and deacetylases (HDACs) are key regulatory steps for gene expression and cell cycle. Functional defects of either of these enzymes may lead to several diseases, including cancer. HATs and HDACs thus are potential therapeutic targets. Here we report that garcinol, a polyisoprenylated benzophenone derivative from Garcinia indica fruit rind, is a potent inhibitor of histone acetyltransferases p300 (IC50≈7 μM) and PCAF (IC50≈5 μM) both in vitro and in vivo. The kinetic analysis shows that it is a mixed type of inhibitor with an increased affinity for PCAF compared with p300. HAT activity-dependent chromatin transcription was strongly inhibited by garcinol, whereas transcription from DNA template was not affected. Furthermore, it was found to be a potent inducer of apoptosis, and it alters (predominantly down-regulates) the global gene expression in HeLa cells

Randomized Clinical Trial of High-Dose Rifampicin With or Without Levofloxacin Versus Standard of Care for Pediatric Tuberculous Meningitis: The TBM-KIDS Trial

Background. Pediatric tuberculous meningitis (TBM) commonly causes death or disability. In adults, high-dose rifampicin may reduce mortality. The role of fluoroquinolones remains unclear. There have been no antimicrobial treatment trials for pediatric TBM. Methods. TBM-KIDS was a phase 2 open-label randomized trial among children with TBM in India and Malawi. Participants received isoniazid and pyrazinamide plus: (i) high-dose rifampicin (30 mg/kg) and ethambutol (R30HZE, arm 1); (ii) high-dose rifampicin and levofloxacin (R30HZL, arm 2); or (iii) standard-dose rifampicin and ethambutol (R15HZE, arm 3) for 8 weeks, followed by 10 months of standard treatment. Functional and neurocognitive outcomes were measured longitudinally using Modified Rankin Scale (MRS) and Mullen Scales of Early Learning (MSEL). Results. Of 2487 children prescreened, 79 were screened and 37 enrolled. Median age was 72 months; 49%, 43%, and 8% had stage I, II, and III disease, respectively. Grade 3 or higher adverse events occurred in 58%, 55%, and 36% of children in arms 1, 2, and 3, with 1 death (arm 1) and 6 early treatment discontinuations (4 in arm 1, 1 each in arms 2 and 3). By week 8, all children recovered to MRS score of 0 or 1. Average MSEL scores were significantly better in arm 1 than arm 3 in fine motor, receptive language, and expressive language domains (P < .01). Conclusions. In a pediatric TBM trial, functional outcomes were excellent overall. The trend toward higher frequency of adverse events but better neurocognitive outcomes in children receiving high-dose rifampicin requires confirmation in a larger trial. Clinical Trials Registration. NCT02958709

Understanding Context of Use and Perceptions of Usability of Cosegregation Analysis Tool AnalyzeMyVariant

Thesis (Master's)--University of Washington, 2018Calculating the genetic risk for a disease with allelic variants of unknown significance can be a complicated task. AnalyzeMyVariant is a tool designed for genetics experts that uses pedigree data from families with genetic variants of unknown significance, to calculate likelihood ratios that a variant fits pathogenic or benign patterns. In this study, we performed a two-part evaluation to understand the context within which genetics experts might use this tool and assess their initial usability perceptions. First, we surveyed existing literature to develop an instrument to assess perceptions of usability based on constructs of usability, quality, and safety. The instrument consisted of scaled as well as open-ended questions assessing users’ perceptions relating to each of the constructs of interest, with regard to their experience with AnalyzeMyVariant. We used the instrument to collect data from 57 genetic experts and trainees who were recruited via email invitations. The second part of our evaluation was comprised of semi-structured interviews with six genetics experts to identify work contexts in which users might use the tool and further delve into issues faced in using the tool. These interviews were inductively coded and major themes identified using the constant comparative method. Based on these findings, we provide recommendations for future improvement of the tool. This work has importance in the consideration of the varying needs of genetics professionals and how they use cosegregation analysis in their work, and the difference between requirements for research- and clinically-focused work. The results could also inform the future development of other tools developed for experts in a wide area of scientific fields, particularly with regard to the attention that must be paid to experts’ context of use, background knowledge, and the intended applicability of results

Novel Resveratrol and 5-Fluorouracil Coencapsulated in PEGylated Nanoliposomes Improve Chemotherapeutic Efficacy of Combination against Head and Neck Squamous Cell Carcinoma

Increasing consumption of tobacco and alcohol has led to a steady increase in the incidence of head and neck cancers in Asia. The drawbacks associated with the existing chemotherapeutic and surgical interventions have necessitated the development of a safer alternative for therapy of head and neck cancers. In this study we have explored the synergistic therapeutic potential of a phytochemical and chemotherapeutic agent using PEGylated liposomes as a delivery vehicle. Resveratrol and 5-fluorouracil were successfully coencapsulated in a single PEGylated nanoliposome. The thermal analysis and the nuclear magnetic resonance results revealed that resveratrol localized near the glycerol backbone of the liposomal membrane while 5-fluorouracil localized closer to the phosphate moiety, which influenced the release kinetics of both drugs. The nanoformulation was tested in vitro on a head and neck cancer cell line NT8e and was found to exhibit a GI50 similar to that of free 5-fluorouracil. Further, gene expression studies showed that the combination of resveratrol and 5-fluorouracil exhibited different effects on different genes that may influence the net antagonistic effect. The coencapsulation of resveratrol and 5-fluorouracil in a liposomal nanocarrier improved the cytotoxicity in comparison with the free drug combination when tested in vitro

Adenovirus Delivered Short Hairpin RNA Targeting a Conserved Site in the 5′ Non-Translated Region Inhibits All Four Serotypes of Dengue Viruses

<div><h3>Background</h3><p>Dengue is a mosquito-borne viral disease caused by four closely related serotypes of Dengue viruses (DENVs). This disease whose symptoms range from mild fever to potentially fatal haemorrhagic fever and hypovolemic shock, threatens nearly half the global population. There is neither a preventive vaccine nor an effective antiviral therapy against dengue disease. The difference between severe and mild disease appears to be dependent on the viral load. Early diagnosis may enable timely therapeutic intervention to blunt disease severity by reducing the viral load. Harnessing the therapeutic potential of RNA interference (RNAi) to attenuate DENV replication may offer one approach to dengue therapy.</p> <h3>Methodology/Principal Findings</h3><p>We screened the non-translated regions (NTRs) of the RNA genomes of representative members of the four DENV serotypes for putative siRNA targets mapping to known transcription/translation regulatory elements. We identified a target site in the 5′ NTR that maps to the 5′ upstream AUG region, a highly conserved <em>cis</em>-acting element essential for viral replication. We used a replication-defective human adenovirus type 5 (AdV5) vector to deliver a short-hairpin RNA (shRNA) targeting this site into cells. We show that this shRNA matures to the cognate siRNA and is able to inhibit effectively antigen secretion, viral RNA replication and infectious virus production by all four DENV serotypes.</p> <h3>Conclusion/Significance</h3><p>The data demonstrate the feasibility of using AdV5-mediated delivery of shRNAs targeting conserved sites in the viral genome to achieve inhibition of all four DENV serotypes. This paves the way towards exploration of RNAi as a possible therapeutic strategy to curtail DENV infection.</p> </div

Design and characterization of the rAd-sh viruses.

<p>(A) The linear genome of the rAd-sh virus constructed for this study. In constructing the rAd-sh virus, the E1 region (dashed line) is replaced by the sh DNA expression cassette (<i>sh DNA EC</i>), consisting of the U6 promoter (rightward arrow), the shDNA insert with the sense (s) and antisense (as) arms, of 21 base pairs each, followed by of the U6 terminator (empty box). The shaded box between the ‘s’ and ‘as’ arms is a 6-base pair loop sequence. The dotted lines flanking the expression cassette represent plasmid vector sequences. Other elements of the rAd-sh genome include a ∼2.7 Kb deletion in the E3 region (ΔE3), the left (L) and right (R) inverted terminal repeats, and the packaging signal (ψ). The nt sequences of the ‘s’ strands of the sh-5b and sh-scr constructs are shown below. (B) PCR analysis of wild type AdV5 (lanes 1 & 5), rAdsh-E (lanes 4 & 8), rAdsh-5b (lanes 2 & 6) and rAdsh-scr (lanes 3 & 7) using insert-specific (lanes 1–4) and AdV5 E1-specific (lanes 5–8) primers. DNA size markers (sizes in kb shown to the left) were analyzed in lanes ‘M’. The arrows to the right denote the positions of the predicted insert-specific (upper) and AdV5 E1 region-specific (lower) amplicons. (C) RNase protection assay to detect anti-sense strand of sh-5b siRNA. A radiolabeled sense probe was digested with RNases A and T1, either before (lanes 2 & 5) or after hybridization with total RNA isolated from rAdsh-5b-infected Vero cells, harvested on either day 3 (lane 3) or day 8 (lane 6) post-infection. Protected fragments (lower arrow) were analysed on 8 M urea gel and visualized using a phosphoimager. The un-hybridized probe (upper arrow) without any RNase treatment was analysed in parallel (lanes 1 & 4). It is to be noted that in lanes 3 and 6, the cells used for total RNA preparation were challenged with DENV-2 and DENV-4, respectively, at 24 hours post rAdsh-5b infection.</p

The effect of rAd mediated shRNA expression on on-going DENV infection.

<p>(A) Vero cells in 12-well plates were sequentially infected with DENV-2 (∼25 PFU/well) and 24 hours later, with rAdsh-scr or rAdsh-5b, each at a m.o.i of 5 (top row) or 10 (bottom row). Cells were overlaid with methyl cellulose and plaques visualized as explained in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001735#pntd-0001735-g003" target="_blank">Figure 3</a> legend. Two wells, of four assayed for each sequential infection experiment, are shown. (B) Vero cells in 24-well plates were sequentially infected with DENV-2 (1000 PFUs/well), followed 24 hours later with rAdsh-5b (red curves) or rAdsh-scr (green curve), each at m.o.i. of 5 (solid curve) or 10 (dashed curve). Culture supernatants were drawn at 48 hour intervals up to 7 days post DENV infection and analyzed for the presence of NS1 antigen using BioRad's Platelia Dengue NS1ELISA kit. The data represent plots of NS1 ELISA absorbance as a function of time after DENV infection. Data shown are mean values (n = 4). The vertical bars represent SD. (C) Culture supernatants in (B) were analyzed for the presence infectious DENV using a standard plaque assay. Data shown are mean values (n = 4). The vertical bars represent SD.</p

The effect of rAd mediated shRNA expression on DENV secretion.

<p>Vero cells were pre-infected either with rAdsh-5b (red curves) or rAdsh-scr (green curves) followed 24 hours later by infection with DENV-1 (A), DENV-2 (B), DENV-3 (C) and DENV-4 (D). Culture supernatants were drawn at daily intervals up to 7 days post DENV infection and analyzed for the presence infectious DENV using a standard plaque assay. Data shown are mean values (n = 6). The vertical bars represent SD.</p