Detection of paternally inherited fetal point mutations for β-thalassemia in maternal plasma using simple fetal DNA enrichment protocol with or without whole genome amplification: an accuracy assessment

Objective: To design and evaluate a noninvasive protocol for prenatal diagnosis (PND) of β-thalassemia, using cell free fetal DNA (cff-DNA) in maternal circulation. Traditional current PND which is mainly based on chorionic villous sampling (CVS), amplification refractory mutation system and sequencing holds as gold standard.

Methods: Ten thalassemia trait couples with distinct mutations for the husband and wife were included in this study. The mutations in carrier fathers were IVSI-1, IVSI-5, FR8/9 and CD44. After maternal plasma isolation and free DNA extraction, all samples subjected to designed protocol including DNA size separation on agarose gel, elution of DNA from the gel slices using a simple and efficient manual purification method, with or without whole genome amplification and the detection method was allele-specific real-time PCR.

Results: Presence or absence of the paternal mutant allele was correctly determined in all of cases and the accuracy of designed protocol was determined 100%.

Conclusions: The protocol described here is very simple, inexpensive and easy to perform, but with satisfactory accuracy in detection of paternal mutations in cff-DNA. Due to the risk of fetal loss with current invasive sampling for PND, a noninvasive alternative is highly demanded in clinical setting.     

Widespread ripples synchronize human cortical activity during sleep,waking, and memory recall

Declarative memory encoding, consolidation, and retrieval require the integration of elements encoded in widespread cortical locations. The mechanism whereby such “binding” of different components of mental events into unified representations occurs is unknown. The “binding-by-synchrony” theory proposes that distributed encoding areas are bound by synchronous oscillations enabling enhanced communication. However, evidence for such oscillations is sparse. Brief high-frequency oscillations (“ripples”) occur in the hippocampus and cortex and help organize memory recall and consolidation. Here, using intracranial recordings in humans, we report that these ∼70-ms-duration, 90-Hz ripples often couple (within ±500 ms), co-occur (≥ 25-ms overlap), and, crucially, phase-lock (have consistent phase lags) between widely distributed focal cortical locations during both sleep and waking, even between hemispheres. Cortical ripple co-occurrence is facilitated through activation across multiple sites, and phase locking increases with more cortical sites corippling. Ripples in all cortical areas co-occur with hippocampal ripples but do not phase-lock with them, further suggesting that cortico-cortical synchrony is mediated by cortico-cortical connections. Ripple phase lags vary across sleep nights, consistent with participation in different networks. During waking, we show that hippocampo-cortical and cortico-cortical coripples increase preceding successful delayed memory recall, when binding between the cue and response is essential. Ripples increase and phase-modulate unit firing, and coripples increase high-frequency correlations between areas, suggesting synchronized unit spiking facilitating information exchange. co-occurrence, phase synchrony, and high-frequency correlation are maintained with little decrement over very long distances (25 cm). Hippocampo-cortico-cortical coripples appear to possess the essential properties necessary to support binding by synchrony during memory retrieval and perhaps generally in cognition.

Ripples are brief high-frequency oscillations that have been well-studied in the rodent hippocampus during non-rapid eye movement sleep (NREM), when they mark the replay of events from the previous waking period, and are critical for memory consolidation in the cortex (1–4). Recently, ripples were found in rat association cortex but not primary sensory or motor cortices during sleep, with increased coupling to hippocampal ripples in sleep following learning (5). An earlier study reported ripples in waking and sleeping cat cortex, especially NREM (6). In humans, cortical ripples have recently been identified during waking and were more frequently found in lateral temporal than in rolandic cortex. Hippocampal sharpwave-ripple occurrence and ripple coupling between parahippocampal gyrus and temporal association cortex increase preceding memory recall in humans (7, 8), possibly facilitating replay of cortical neuron firing sequences established during encoding (9). In rats, ripples co-occur between hippocampus and ∼1 mm² of parietal cortex in sleep following learning (5), in mice, ripples propagate from the hippocampus to retrosplenial cortex (10), and in cats, ripple co-occurrence is reportedly limited to short distances (6).We recently reported, using human intracranial recordings, that ∼70-ms-long, ∼90-Hz ripples are ubiquitous in all regions of the cortex during NREM as well as waking (11). During waking, cortical ripples occur on local high-frequency activity peaks. During sleep, cortical ripples typically occur on the cortical down-to-upstate transition, often with 10- to 16-Hz cortical sleep spindles, and local unit firing patterns consistent with generation by pyramidal-interneuron feedback. We found that cortical ripples group cofiring within the window of spike-timing-dependent plasticity. These findings are consistent with cortical ripples contributing to memory consolidation during NREM in humans.While there is thus an emerging appreciation that hippocampal and cortical ripples have an important role in human and rodent memory, nothing is known of the network properties of cortical ripples. Specifically, it is not known if ripples co-occur or phase-synchronize between cortical sites and, if so, whether this is affected by distance or correlated with the reconstruction of declarative memories. These would be critical properties for cortical ripples to participate in the binding of different elements of memories that are represented in disparate cortical areas, the essence of hippocampus-dependent memory (12).The binding of disparate elements of a memory is a specific case of a more general problem of how the various contents of a mental event are united into a single experience. Most often addressed is how different visual qualities of an object (e.g., color, shape, location, and texture) are associated with each other (13), but the “binding problem” generalizes to how the contents of awareness are unified in a single stream of consciousness (14). Modern accounts often rely on hierarchical and multimodal convergence. However, cortical processing is distributed, and it would be difficult to represent the combinatorial possibilities contained in all potential experiences with convergence, leading to the suggestion that temporal synchrony binds cortical areas (15). This hypothesis was first supported by phase-locked unit firing and local field potentials (LFPs) at 40 to 60 Hz evoked by simple visual stimuli in the anesthetized cat primary visual cortex at distances <7 mm (16). Although some further studies found similar results in other cortical areas, behaviors, and species, as would be expected under the binding-by-synchrony hypothesis (17, 18), others have been less successful (19). Synchronous high-gamma oscillations have also been criticized as providing no mechanism for neuronal interaction beyond generic activation (19, 20).Here, using human intracranial stereoelectroencephalography (SEEG) recordings, we find that ripples co-occur and, remarkably, phase-synchronize across all lobes and between both hemispheres, with little decrement, even at long distances. Furthermore, ripple co-occurrence is enhanced between cortical sites as well as between the cortex and hippocampus preceding successful delayed recall. Corippling was progressively above that expected as it involved a larger proportion of sites, and this led to progressively stronger phase locking. Single-unit firing increased during, and phase-locked to, cortical ripples, providing a basic requirement for ripples to enhance communication via gain modulation and coincidence detection. Enhanced communication was supported by our finding of increased high-frequency correlation between even distant corippling regions. These characteristics suggest that distributed, phase-locked cortical ripples possess the properties that may allow them to help integrate different components of a memory. More generally, ripples may help to “bind” different aspects of a mental event encoded in widespread cortical areas into a coherent representation.     

Billing and Reimbursement for a Bedside Toxicology Service at a Tertiary Care Academic Center During Its First Fiscal Year

A bedside toxicology consult service may improve clinical care, facilitate patient clearance and disposition, and result in potential cost savings for poisoning exposures. Despite this, there is scant data regarding economic feasibility for such a service. Previously published information suggests low hourly reimbursement at approximately $26.00/h at the bedside for toxicology consultations. A bedside toxicology consultant service was initiated in 2011. Coverage was available 24 h a day for 50 out of 52 weeks. Bedside rounding on toxicology consult patients was available 6/7 days per week. The practice is associated with >800 bed teaching institution in a large upstate NY region with elements of urban and suburban practice. Demographic and billing data was collected for all patients consulted upon from July 1, 2011 to June 31, 2012. In charges of $514,941 were generated during the period of data collection. Monthly average was $42,912. Net reimbursement of charges was 29 % of overall charges at $147,792. In terms of total encounters, net collection rate in which something was reimbursed or “paid” against charges for that encounter was 82.6 % of all encounters at 999/1,210. Average encounter time for inpatients, including critical care, was 1.05 h, and the average time spent for outpatients was 1.18 h. Reimbursement rates appear higher than previously reported. Revenue generated from reimbursement from toxicology consultation can result in recouping a substantial portion of a toxicologist’s salary or potentially fund fellowship positions and salaries or toxicology division infrastructure.     

Diabetes mellitus aggravates ranolazine-induced ECG changes in rats

Journal of Interventional Cardiac Electrophysiology - Diabetes mellitus (DM) is known to affect the pharmacokinetics of drugs. In this study, we evaluated the effect of DM on the liver content of...