Using an immunofluorescence assay based on CRISPR-dCas9-gRNA complexes that selectively bind to the HIV LTR (HIV Cas-FISH), we traced changes in HIV DNA localization in primary effector T cells from ...early infection until the cells become quiescent as they transition to memory cells. Unintegrated HIV DNA colocalized with CPSF6 and HIV capsid (CA, p24) was found in the cytoplasm and nuclear periphery at days 1 and 3 post infection. From days 3 to 7, most HIV DNA was distributed primarily in the nuclear intermediate euchromatic compartment and was transcribed. By day 21, the cells had entered quiescence, and HIV DNA accumulated in the perinucleolar compartment (PNC). The localization of proviruses to the PNC was blocked by integrase inhibitor Raltegravir, suggesting it was due to chromosomal rearrangements. During the reactivation of latently infected cells through the T cell receptor (TCR), nascent viral mRNA transcripts associated with HIV DNA in the PNC were detected. The viral trans-activator Tat and its regulatory partners, P-TEFb and 7SK snRNA, assembled in large interchromatin granule clusters near the provirus within 2 h of TCR activation. As T cell activation progressed, the HIV DNA shifted away from the PNC. HIV DNA in latently infected memory T cells from patients also accumulated in the PNC and showed identical patterns of nuclear rearrangements after cellular reactivation. Thus, in contrast to transformed cells where proviruses are found primarily at the nuclear periphery, in primary memory T cells, the nuclear architecture undergoes rearrangements that shape the transcriptional silencing and reactivation of proviral HIV.
The major reservoirs for HIV in the CNS are in the microglia, perivascular macrophages, and to a lesser extent, astrocytes. To study the molecular events controlling HIV expression in the microglia, ...we developed a reliable and robust method to immortalize microglial cells from primary glia from fresh CNS tissues and commercially available frozen glial cells. Primary human cells, including cells obtained from adult brain tissue, were transformed with lentiviral vectors expressing SV40 T antigen or a combination of SVR40 T antigen and hTERT. The immortalized cells have microglia-like morphology and express key microglial surface markers including CD11b, TGFβR, and P2RY12. Importantly, these cells were confirmed to be of human origin by sequencing. The RNA expression profiles identified by RNA-seq are also characteristic of microglial cells. Furthermore, the cells demonstrate the expected migratory and phagocytic activity, and the capacity to mount an inflammatory response characteristic of primary microglia. The immortalization method has also been successfully applied to a wide range of microglia from other species (macaque, rat, and mouse). To investigate different aspects of HIV molecular regulation in CNS, the cells have been superinfected with HIV reporter viruses and latently infected clones have been selected that reactive HIV in response to inflammatory signals. The cell lines we have developed and rigorously characterized will provide an invaluable resource for the study of HIV infection in microglial cells as well as studies of microglial cell function.
In an effort to clear persistent HIV infection and achieve a durable therapy-free remission of HIV disease, extensive pre-clinical studies and early pilot clinical trials are underway to develop and ...test agents that can reverse latent HIV infection and present viral antigen to the immune system for clearance. It is, therefore, critical to understand the impact of latency-reversing agents (LRAs) on the function of immune effectors needed to clear infected cells. We assessed the impact of LRAs on the function of natural killer (NK) cells, the main effector cells of the innate immune system. We studied the effects of three histone deacetylase inhibitors SAHA or vorinostat (VOR), romidepsin, and panobinostat (PNB) and two protein kinase C agonists prostratin (PROST) and ingenol on the antiviral activity, cytotoxicity, cytokine secretion, phenotype, and viability of primary NK cells. We found that
exposure to VOR had minimal impact on all parameters assessed, while PNB caused a decrease in NK cell viability, antiviral activity, and cytotoxicity. PROST caused non-specific NK cell activation and, interestingly, improved antiviral activity. Overall, we found that LRAs can alter the function and fate of NK cells, and these effects must be carefully considered as strategies are developed to clear persistent HIV infection.
Surgical site infections cause significant morbidity and mortality in the postoperative period. Opening of the operating room door disrupts its filtered atmosphere, increasing contamination above the ...wound. We conducted a study of traffic in the operating room as a risk for infections. This is an observational study of recorded behaviors in the operating room. Data collected included number of people entering/exiting, the role of these individuals, and the cause for the event. A total of 3071 door openings were recorded in 28 cases. Traffic varied from 19 to 50 events per hour across specialties. The preincision period represented 30% to 50% of all events. Information requests accounted for the majority of events. Door openings increase in direct proportion to case length, but have an exponential relationship with the number of persons in the operating room. There is a high rate of traffic across all specialties, compromising the sterile environment of the operating room.
Reference frames in saccadic targeting KARN, K. S; MØLLER, P; HAYHOE, M. M
Experimental brain research,
06/1997, Letnik:
115, Številka:
2
Journal Article
Recenzirano
We attempt to determine the egocentric reference frame used in directing saccades to remembered targets when landmark-based (exocentric) cues are not available. Specifically, we tested whether ...memory-guided saccades rely on a retina-centered frame, which must account for eye movements that intervene during the memory period (thereby accumulating error) or on a head-centered representation that requires knowledge of the position of the eyes in the head. We also examined the role of an exocentric reference frame in saccadic targeting since it would not need to account for intervening movements. We measured the precision of eye movements made by human observers to target locations held in memory for a few seconds. A variable number of saccades intervened between the visual presentation of a target and a later eye movement to its remembered location. A visual landmark that allowed for exocentric encoding of the memory target appeared in half the trials. Variable error increased slightly with a greater number of intervening saccades. The landmark aided targeting precision, but did not eliminate the increase in variable error with additional intervening saccades. We interpret these results as evidence for a representation that relies on knowledge of eye position with respect to the head and not one that relies solely on updating in a retina-centered frame. Our results allow us to set an upper bound on the standard deviation of an eye position signal available to the saccadic system during short memory periods at 1.4 degrees for saccades of about 10 degrees.
Historically it has not been terribly difficult for researchers to gain access to healthcare facilities to conduct user research such as contextual inquiry and workflow analysis. Today, some ...practitioners are finding it nearly impossible to get into facilities. One reason is that hospitals are getting progressively more risk-averse regarding patient confidentiality and patient safety and are less willing to have outsiders in the hospital, especially in procedure rooms. Another reason is a lack of understanding regarding how to coordinate with offices of compliance and institutional review boards (IRBs), and the fact that IRBs typically are set up to manage clinical trials rather than human factors research. The purpose of this panel was to raise awareness of healthcare access challenges, discuss solutions, and propose some best practices.
The T box system regulates expression of amino acid-related genes in Gram-positive bacteria through premature termination of transcription. Synthesis of the full-length mRNA requires stabilization of ...an antiterminator element in the 5' untranslated leader RNA by the cognate uncharged tRNA. tRNA(Gly)-dependent antitermination of the Bacillus subtilis glyQS gene (encoding glycyl-tRNA synthetase) can be reproduced in a purified in vitro transcription system, indicating that the nascent transcript is sufficient for interaction with the tRNA. Genetic analyses previously demonstrated base pairing of a single codon in the leader RNA with the tRNA anticodon, and between the antiterminator and the tRNA acceptor end. In this study, we established conditions for specific binding of tRNA(Gly) to glyQS leader RNA generated by phage T7 RNA polymerase. Structural mapping studies revealed tRNA(Gly)-induced protection in the glyQS leader RNA at the two known sites of interaction with the tRNA, as well as at other regions between these sites. The proposed tRNA-dependent structural switch between the competing terminator and antiterminator forms of the leader RNA was demonstrated directly. Changes in tRNA(Gly) upon binding to glyQS leader RNA were detected in the anticodon loop, consistent with pairing with the specifier sequence, and in the highly conserved G19 in the D-loop, similar to effects induced by codon-anticodon interaction in the ribosome. This study provides biochemical evidence for direct interaction of tRNA(Gly) with full-length in vitro transcribed glyQS leader RNA, and an initial view of structural modulations of both RNA partners within the complex.
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