From Single Cell to Plants: Mesophyll Protoplasts as a Versatile System for Investigating Plant Cell Reprogramming

Plants are sessile organisms that have a remarkable developmental plasticity, which ensures their optimal adaptation to environmental stresses. Plant cell totipotency is an extreme example of such plasticity, whereby somatic cells have the potential to form plants via direct shoot organogenesis or somatic embryogenesis in response to various exogenous and/or endogenous signals. Protoplasts provide one of the most suitable systems for investigating molecular mechanisms of totipotency, because they are e ectively single cell populations. In this review, we consider the current state of knowledge of the mechanisms that induce cell proliferation from individual, di erentiated somatic plant cells. We highlight initial explant metabolic status, ploidy level and isolation procedure as determinants of successful cell reprogramming. We also discuss the importance of auxin signalling and its interaction with stress-regulated pathways in governing cell cycle induction and further stages of plant cell totipotency

High Current CD4+ T cell count predicts suboptimal adherence to antiretroviral therapy

Date of Acceptance: 30/09/2015 Funding: A.O.P. is financially supported by the Dutch AIDS Fonds (http://www.aidsfonds.nl/), grant nrs. 2011020 and 2012025. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Comparison of droplet digital PCR and seminested real-time PCR for quantification of cell-associated HIV-1 RNA

Cell-associated (CA) HIV-1 RNA is considered a potential marker for assessment of viral reservoir dynamics and antiretroviral therapy (ART) response in HIV-infected patients. Recent studies employed sensitive seminested real-time quantitative (q)PCR to quantify CA HIV-1 RNA. Digital PCR has been recently described as an alternative PCR-based technique for absolute quantification with higher accuracy compared to qPCR. Here, a comparison was made between the droplet digital PCR (ddPCR) and the seminested qPCR for quantification of unspliced (us) and multiply spliced (ms) CA HIV-1 RNA. Synthetic RNA standards and CA HIV-1 RNA from infected patients on and off ART (N = 34) were quantified with both methods. Correlations were observed between the methods both for serially diluted synthetic standards (usRNA: R2 = 0.97, msRNA: R2 = 0.92) and patient-derived samples (usRNA: R2 = 0.51, msRNA: R2 = 0.87). Seminested qPCR showed better quantitative linearity, accuracy and sensitivity in the quantification of synthetic standards than ddPCR, especially in the lower quantification ranges. Both methods demonstrated equally high detection rate of usRNA in patient samples on and off ART (91%), whereas ddPCR detected msRNA in larger proportion of samples from ART-treated patients (p = 0.13). We observed an average agreement between the methods for usRNA quantification in patient samples, albeit with a large standard deviation (bias = 0.05±0.75 log10). However, a bias of 0.94±0.36 log10 was observed for msRNA. No-template controls were consistently negative in the seminested qPCR, but yielded a positive ddPCR signal for some wells. Therefore, the false positive signals may have affected the detection power of ddPCR in this study. Digital PCR is promising for HIV nucleic acid quantification, but the false positive signals need further attention. Quantitative assays for CA HIV RNA have the potential to improve monitoring of patients on ART and to be used in clinical studies aimed at HIV eradication, but should be cross-validated by multiple laboratories prior to wider use

Cellular Levels of HIV Unspliced RNA from Patients on Combination Antiretroviral Therapy with Undetectable Plasma Viremia Predict the Therapy Outcome

BACKGROUND:Combination antiretroviral therapy (cART), the standard of care for HIV-1 infection, is considered to be successful when plasma viremia remains below the detection limit of commercial assays. Yet, cART fails in a substantial proportion of patients after the apparent success. No laboratory markers are known that are predictive of cART outcome in initial responders during the period of undetectable plasma viremia. METHODOLOGY/PRINCIPAL FINDINGS:Here, we report the results of a retrospective longitudinal study of twenty-six HIV-infected individuals who initially responded to cART by having plasma viremia suppressed to <50 copies/ml. Eleven of these patients remained virologically suppressed, whereas fifteen experienced subsequent cART failure. Using sensitive methods based on seminested real-time PCR, we measured the levels of HIV-1 proviral (pr) DNA, unspliced (us) RNA, and multiply spliced RNA in the peripheral blood mononuclear cells (PBMC) of these patients at multiple time points during the period of undetectable plasma viremia on cART. Median under-therapy level of usRNA was significantly higher (0.43 log(10) difference, P = 0.0015) in patients who experienced subsequent cART failure than in successfully treated patients. In multivariate analysis, adjusted for baseline CD4(+) counts, prior ART experience, and particular cART regimens, the maximal usRNA level under therapy was the best independent predictor of subsequent therapy failure (adjusted odds ratio [95% CI], 24.4 [1.5-389.5], P = 0.024). The only other factor significantly associated with cART failure was prior ART experience (adjusted odds ratio [95% CI], 12.3 [1.1-138.4], P = 0.042). Levels of usRNA under cART inversely correlated with baseline CD4(+) counts (P = 0.0003), but did not correlate with either baseline usRNA levels or levels of prDNA under therapy. CONCLUSION:Our data demonstrate that the level of HIV-1 usRNA in PBMC, measured in cART-treated patients with undetectable plasma viremia, is a strong predictive marker for the outcome of therapy

A prospective study of physician-observed concussion during a varsity university hockey season: White matter integrity in ice hockey players. Part 3 of 4

Object: The aim of this study was to investigate the effect of repetitive head impacts on white matter integrity that were sustained during 1 Canadian Interuniversity Sports (CIS) ice hockey season, using advanced diffusion tensor imaging (DTI). Methods: Twenty-five male ice hockey players between 20 and 26 years of age (mean age 22.24 ± 1.59 years) participated in this study. Participants underwent pre- and postseason 3-T MRI, including DTI. Group analyses were performed using paired-group tract-based spatial statistics to test for differences between preseason and postseason changes. Results: Tract-based spatial statistics revealed an increase in trace, radial diffusivity (RD), and axial diffusivity (AD) over the course of 1 season. Compared with preseason data, postseason images showed higher trace, AD, and RD values in the right precentral region, the right corona radiata, and the anterior and posterior limb of the internal capsule. These regions involve parts of the corticospinal tract, the corpus callosum, and the superior longitudinal fasciculus. No significant differences were observed between preseason and postseason for fractional anisotropy. Conclusions: Diffusion tensor imaging revealed changes in white matter diffusivity in male ice hockey players over the course of 1 season. The origin of these findings needs to be elucidated

REPIMPACT - a prospective longitudinal multisite study on the effects of repetitive head impacts in youth soccer

Repetitive head impacts (RHI) are common in youth athletes participating in contact sports. RHI differ from concussions; they are considered hits to the head that usually do not result in acute symptoms and are therefore also referred to as \textquotedblsubconcussive\textquotedbl head impacts. RHI occur e.g., when heading the ball or during contact with another player. Evidence suggests that exposure to RHI may have cumulative effects on brain structure and function. However, little is known about brain alterations associated with RHI, or about the risk factors that may lead to clinical or behavioral sequelae. REPIMPACT is a prospective longitudinal study of competitive youth soccer players and non-contact sport controls aged 14 to 16~years. The study aims to characterize consequences of exposure to RHI with regard to behavior (i.e., cognition, and motor function), clinical sequelae (i.e., psychiatric and neurological symptoms), brain structure, function, diffusion and biochemistry, as well as blood- and saliva-derived measures of molecular processes associated with exposure to RHI (e.g., circulating microRNAs, neuroproteins and cytokines). Here we present the structure of the REPIMPACT Consortium which consists of six teams of clinicians and scientists in six countries. We further provide detailed information on the specific aims and the design of the REPIMPACT study. The manuscript also describes the progress made in the study thus far. Finally, we discuss important challenges and approaches taken to overcome these challenges

Immune and Viral Correlates of “Secondary Viral Control” after Treatment Interruption in Chronically HIV-1 Infected Patients

Upon interruption of antiretroviral therapy, HIV-infected patients usually show viral load rebound to pre-treatment levels. Four patients, hereafter referred to as secondary controllers (SC), were identified who initiated therapy during chronic infection and, after stopping treatment, could control virus replication at undetectable levels for more than six months. In the present study we set out to unravel possible viral and immune parameters or mechanisms of this phenomenon by comparing secondary controllers with elite controllers and non-controllers, including patients under HAART. As candidate correlates of protection, virus growth kinetics, levels of intracellular viral markers, several aspects of HIV-specific CD4+ and CD8+ T cell function and HIV neutralizing antibodies were investigated. As expected all intracellular viral markers were lower in aviremic as compared to viremic subjects, but in addition both elite and secondary controllers had lower levels of viral unspliced RNA in PBMC as compared to patients on HAART. Ex vivo cultivation of the virus from CD4+ T cells of SC consistently failed in one patient and showed delayed kinetics in the three others. Formal in vitro replication studies of these three viruses showed low to absent growth in two cases and a virus with normal fitness in the third case. T cell responses toward HIV peptides, evaluated in IFN-γ ELISPOT, revealed no significant differences in breadth, magnitude or avidity between SC and all other patient groups. Neither was there a difference in polyfunctionality of CD4+ or CD8+ T cells, as evaluated with intracellular cytokine staining. However, secondary and elite controllers showed higher proliferative responses to Gag and Pol peptides. SC also showed the highest level of autologous neutralizing antibodies. These data suggest that higher T cell proliferative responses and lower replication kinetics might be instrumental in secondary viral control in the absence of treatment

Persistent HIV-1 transcription during ART: time to reassess its significance?

PURPOSE OF REVIEW: Despite suppressive antiretroviral therapy (ART), HIV-1 reservoirs persist and reignite viral replication if therapy is interrupted. Persistence of the viral reservoir in people with HIV-1 (PWH) is the main obstacle to an HIV-1 cure. The reservoirs are not transcriptionally silent, and viral transcripts can be detected in most ART-treated individuals. Here, we review the recent progress in the characterization of persistent HIV-1 transcription during ART. RECENT FINDINGS: Evidence from several studies indicates that, although cell-associated unspliced (US) HIV-1 RNA is abundantly expressed in ART-treated PWH, intact full-length US transcripts are rare and most US RNA is derived from defective proviruses. The transcription- and translation-competent defective proviruses, previously considered irrelevant, are increasingly being linked to residual HIV-1 pathogenesis under suppressive ART. Recent data suggest a continuous crosstalk between the residual HIV-1 activity under ART and the immune system. Persistent HIV-1 transcription on ART, despite being mostly derived from defective proviruses, predicts viral rebound upon therapy interruption, suggesting its role as an indicator of the strength of the host antiviral immune response that is shaping the viral rebound. SUMMARY: In light of the recent findings, the significance of persistent HIV-1 transcription during ART for the long-term health of PWH and the cure research should be reassessed

Neuroimaging in repetitive brain trauma

Sports-related concussions are one of the major causes of mild traumatic brain injury. Although most patients recover completely within days to weeks, those who experience repetitive brain trauma (RBT) may be at risk for developing a condition known as chronic traumatic encephalopathy (CTE). While this condition is most commonly observed in athletes who experience repetitive concussive and/or subconcussive blows to the head, such as boxers, football players, or hockey players, CTE may also affect soldiers on active duty. Currently, the only means by which to diagnose CTE is by the presence of phosphorylated tau aggregations post-mortem. Non-invasive neuroimaging, however, may allow early diagnosis as well as improve our understanding of the underlying pathophysiology of RBT. The purpose of this article is to review advanced neuroimaging methods used to investigate RBT, including diffusion tensor imaging, magnetic resonance spectroscopy, functional magnetic resonance imaging, susceptibility weighted imaging, and positron emission tomography. While there is a considerable literature using these methods in brain injury in general, the focus of this review is on RBT and those subject populations currently known to be susceptible to RBT, namely athletes and soldiers. Further, while direct detection of CTE in vivo has not yet been achieved, all of the methods described in this review provide insight into RBT and will likely lead to a better characterization (diagnosis), in vivo, of CTE than measures of self-report