Genetic Normalization of Differentiating Aneuploid Human Embryos

Early embryogenesis involves a series of dynamic processes, many of which are currently not well described or understood. Aneuploidy and aneuploid mosaicism, a mixture of aneuploid and euploid cells within one embryo, in early embryonic development are principal causes of developmental failure.^1,2^ Here we show that human embryos demonstrate a significant rate of genetic correction of aneuploidy, or "genetic normalization" when cultured from the cleavage stage on day 3 (Cleavage) to the blastocyst stage on day 5 (Blastocyst) using routine in vitro fertilization (IVF) laboratory conditions. One hundred and twenty-six human Cleavage stage embryos were evaluated for clinically indicated preimplantation genetic screening (PGS). Sixty-four of these embryos were found to be aneuploid following Cleavage stage embryo biopsy and single nucleotide polymorphism (SNP) 23 chromosome molecular karyotype (microarray). Of these, 25 survived to the Blastocyst stage of development and repeat microarray evaluation was performed. The inner cell mass (ICM), containing cells destined to form the fetus, and the trophectoderm (TE), containing cells destined to form the placenta were evaluated. Sixteen of 25 embryos (64%) [95% CI: 44-80%] possessed diploid karyotypes in both the ICM and TE cell populations. An additional three Blastocyst stage embryos showed genetic correction of the TE but not the ICM and one Blastocyst stage embryo showed the reverse. Mosaicism (exceeding 5%), was not detected in any of the ICM and TE samples analyzed. Recognizing that genetic normalization may occur in developing human embryos has important implications for stem cell biology, preimplantation and developmental genetics, embryology, and reproductive medicine. 

1)Hassold, T. et al. A cytogenetic study of 1000 spontaneous abortions. Ann Hum Genet. 44, 151-78 (1980).
2)Menasha, J., Levy, B., Hirschhorn, K., & Kardon, N.B. Incidence and spectrum of chromosome abnormalities in spontaneous abortions: new insights from a 12-year study. Genet Med. 7, 251-63 (2005)

The influence of particle size and curing conditions on testing mineral trioxide aggregate cement

Objectives: To assess the effects on curing conditions (dry versus submerged curing) and particle size on the compressive strength (CS) and flexural strength (FS) of set MTA cement. Materials and methods: Two different Portland cements were created, P1 and P2, with P1 < P2 in particle size. These were then used to create two experimental MTA products, M1 and M2, with M1 < M2 in particle size. Particle size analysis was performed according to ISO 13320. The particle size at the 90th percentile (i.e. the larger particles) was P1: 15.2 μm, P2: 29.1 μm, M1: 16.5 μm, and M2: 37.1 μm. M2 was cured exposed to air, or submerged in fluids of pH 5.0, 7.2 (PBS), or 7.5 for 1 week. CS and FS of the set cement were determined using a modified ISO 9917-1 and ISO 4049 methods, respectively. P1, P2, M1 and M2 were cured in PBS at physiological pH (7.2) and likewise tested for CS and FS. Results: Curing under dry conditions gave a significantly lower CS than when cured in PBS. There was a trend for lower FS for dry versus wet curing. However, this did not reach statistical significance. Cements with smaller particle sizes showed greater CS and FS at 1 day than those with larger particle sizes. However, this advantage was lost over the following 1-3 weeks. Conclusions: Experiments that test the properties of MTA should cure the MTA under wet conditions and at physiological pH

Finding what works: Identification of implementation strategies for the integration of methadone maintenance therapy and HIV services in Vietnam

Abstract Background Integration of methadone maintenance therapy (MMT) and HIV services is an evidence-based intervention (EBI) that benefits HIV care and reduces costs. While MMT/HIV integration is recommended by the World Health Organization and the Centers for Disease Control and Prevention, it is not widely implemented, due to organizational and operational barriers. Our study applied an innovative process to identify implementation strategies to address these barriers. Methods Our process was adapted from the Expert Recommendations for Implementing Change (ERIC) protocol and consisted of two main phases. In Phase 1, we conducted 16 in-depth interviews with stakeholders and developed matrices to display barriers to integration. In Phase 2, we selected implementation strategies that addressed the barriers identified in Phase 1 and conducted a poll to vote on the most important and feasible strategies among a panel with expertise in cultural context and implementation science. Results Barriers fell into two broad categories: policy and programmatic. At the policy level, barriers included lack of a national mandate, different structures (MMT vs. HIV clinic) for cost reimbursement and staff salaries, and resistance on the part of staff to take on additional tasks without compensation. Programmatic barriers included the need for cross-training in MMT and HIV tasks, staff accountability, and commitment from local leaders. In Phase 2, we focused on programmatic challenges. Based on voting results and iterative dialogue with our expert panel, we selected several implementation strategies in the domains of technical assistance, staff accountability, and local commitment that targeted these barriers. Conclusions Key programmatic barriers to MMT/HIV integration in Vietnam may be addressed through implementation strategies that focus on technical assistance, staff accountability, and local commitment. Our process of identifying implementation strategies was simple, low cost, and potentially replicable to other settings

Methodologies for measuring the setting times of mineral trioxide aggregate and Portland cement products used in dentistry

The current standard used to measure setting time for Mineral Trioxide Aggregate (MTA) involves indentation testing with arbitrary weights. This study compared indentation testing against rheological measurements and assessed the influences of particle size and the inclusion of bismuth oxide on the setting time of experimental MTA and Portland cement (PC). Two PCs (P1 and P2) of different particle sizes were produced using the same clinker. From these two PCs, two experimental MTAs (M1 and M2) were created with the addition of bismuth oxide. Particle size distributions were assessed using laser diffraction analysis. Indentation setting time tests were performed in accordance to the Gillmore needle test. Elastic modulus was assessed using a strain-controlled rheometer at 1 rad s and an applied strain of 0.01%. P1, P2, M1 and M2 cements had median particle sizes of 6.1, 12.5, 6.5 and 13.0 μm, respectively. Using indentation testing, final setting times were ranked P1 < M1 < P2 < M2. The ranking of the final setting time corresponded with the rheological assessment of time required to reach 95% of the elastic modulus plateau. The time to reach 95% elastic modulus plateau of 9.3 min corresponds to a time close to the point where the material can be overlaid with another restorative material to give a final restoration. The 95% plateau value for elastic modulus may be a more useful parameter for determining how the setting reaction of PC and MTA cements progress over time

Deconvolution of the particle size distribution of ProRoot MTA and MTA Angelus

Mineral trioxide aggregate (MTA) cements contain two types of particles, namely Portland cement (PC) (nominally 80% w/w) and bismuth oxide (BO) (20%). This study aims to determine the particle size distribution (PSD) of PC and BO found in MTA. The PSDs of ProRoot MTA (MTA-P) and MTA Angelus (MTA-A) powder were determined using laser diffraction, and compared to samples of PC (at three different particle sizes) and BO. The non-linear least squares method was used to deconvolute the PSDs into the constituents. MTA-P and MTA-A powders were also assessed with scanning electron microscopy. BO showed a near Gaussian distribution for particle size, with a mode distribution peak at 10.48 μm. PC samples milled to differing degrees of fineness had mode distribution peaks from 19.31 down to 4.88 μm. MTA-P had a complex PSD composed of both fine and large PC particles, with BO at an intermediate size, whereas MTA-A had only small BO particles and large PC particles. The PSD of MTA cement products is bimodal or more complex, which has implications for understanding how particle size influences the overall properties of the material. Smaller particles may be reactive PC or unreactive radiopaque agent. Manufacturers should disclose particle size information for PC and radiopaque agents to prevent simplistic conclusions being drawn from statements of average particle size for MTA materials

A Quality Assessment of a Collaborative Model of a Pediatric Antimicrobial Stewardship Program

BACKGROUND: Infectious Diseases Society of America guidelines recommend that key antimicrobial stewardship program (ASP) personnel include an infectious disease (ID) physician leader and dedicated ID-trained clinical pharmacist. Limited resources prompted development of an alternative model by using ID physicians and service-based clinical pharmacists at a pediatric hospital. The aim of this study was to analyze the effectiveness and impact of this alternative ASP model. METHODS: The collaborative ASP model incorporated key strategies of education, antimicrobial restriction, day 3 audits, and practice guidelines. High-use and/or high-cost antimicrobial agents were chosen with audits targeting vancomycin, caspofungin, and meropenem. The electronic medical record was used to identify patients requiring day 3 audits and to communicate ASP recommendations. Segmented regression analyses were used to analyze quarterly antimicrobial agent prescription data for the institution and selected services over time. RESULTS: Initiation of ASP and day 3 auditing was associated with blunting of a preexisting increasing trend for caspofungin drug starts and use and a significant downward trend for vancomycin drug starts (relative change -12%) and use (-25%), with the largest reduction in critical care areas. Although meropenem use was already low due to preexisting requirements for preauthorization, a decline in drug use (-31%, P = .021) and a nonsignificant decline in drug starts (-21%, P = .067) were noted. A 3-month review of acceptance of ASP recommendations found rates of 90%, 93%, and 100% for vancomycin, caspofungin, and meropenem, respectively. CONCLUSIONS: This nontraditional ASP model significantly reduced targeted drug usage demonstrating acceptance of integration of service-based clinical pharmacists and ID consultants

Statins Disrupt CCR5 and RANTES Expression Levels in CD4(+) T Lymphocytes In Vitro and Preferentially Decrease Infection of R5 Versus X4 HIV-1

BACKGROUND: Statins have previously been shown to reduce the in vitro infection of human immunodeficiency virus type 1 (HIV-1) through modulation of Rho GTPase activity and lipid raft formation at the cell surface, as well as by disrupting LFA-1 incorporation into viral particles. PRINCIPLE FINDINGS: Here we demonstrate that treatment of an enriched CD4(+) lymphocyte population with lovastatin (Lov), mevastatin (Mev) and simvastatin (activated and non-activated, Sim(A) and Sim(N), respectively) can reduce the cell surface expression of the CC-chemokine receptor CCR5 (P<0.01 for Sim(A) and Lov). The lowered CCR5 expression was associated with down-regulation of CCR5 mRNA expression. The CC-chemokine RANTES protein and mRNA expression levels were slightly increased in CD4(+) enriched lymphocytes treated with statins. Both R5 and X4 HIV-1 were reduced for their infection of statin-treated cells; however, in cultures where statins were removed and where a decrease in CCR5 expression was observed, there was a preferential inhibition of infection with an R5 versus X4 virus. CONCLUSIONS: The results indicate that the modulation of CC-chemokine receptor (CCR5) and CC-chemokine (RANTES) expression levels should be considered as contributing to the anti-viral effects of statins, preferentially inhibiting R5 viruses. This observation, in combination with the immunomodulatory activity exerted by statins, suggests they may possess more potent anti-HIV-1 activity when applied during the early stages of infection or in lowering viral transmission. Alternatively, statin treatment could be considered as a way to modulate immune induction such as during vaccination protocols

Functional genomic screening identifies dual leucine zipper kinase as a key mediator of retinal ganglion cell death

Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations

Sequence Homology at the Breakpoint and Clinical Phenotype of Mitochondrial DNA Deletion Syndromes

Mitochondrial DNA (mtDNA) deletions are a common cause of mitochondrial disorders. Large mtDNA deletions can lead to a broad spectrum of clinical features with different age of onset, ranging from mild mitochondrial myopathies (MM), progressive external ophthalmoplegia (PEO), and Kearns-Sayre syndrome (KSS), to severe Pearson syndrome. The aim of this study is to investigate the molecular signatures surrounding the deletion breakpoints and their association with the clinical phenotype and age at onset. MtDNA deletions in 67 patients were characterized using array comparative genomic hybridization (aCGH) followed by PCR-sequencing of the deletion junctions. Sequence homology including both perfect and imperfect short repeats flanking the deletion regions were analyzed and correlated with clinical features and patients' age group. In all age groups, there was a significant increase in sequence homology flanking the deletion compared to mtDNA background. The youngest patient group (<6 years old) showed a diffused pattern of deletion distribution in size and locations, with a significantly lower sequence homology flanking the deletion, and the highest percentage of deletion mutant heteroplasmy. The older age groups showed rather discrete pattern of deletions with 44% of all patients over 6 years old carrying the most common 5 kb mtDNA deletion, which was found mostly in muscle specimens (22/41). Only 15% (3/20) of the young patients (<6 years old) carry the 5 kb common deletion, which is usually present in blood rather than muscle. This group of patients predominantly (16 out of 17) exhibit multisystem disorder and/or Pearson syndrome, while older patients had predominantly neuromuscular manifestations including KSS, PEO, and MM. In conclusion, sequence homology at the deletion flanking regions is a consistent feature of mtDNA deletions. Decreased levels of sequence homology and increased levels of deletion mutant heteroplasmy appear to correlate with earlier onset and more severe disease with multisystem involvement

SNCA Triplication Parkinson's Patient's iPSC-derived DA Neurons Accumulate α-Synuclein and Are Susceptible to Oxidative Stress

Parkinson's disease (PD) is an incurable age-related neurodegenerative disorder affecting both the central and peripheral nervous systems. Although common, the etiology of PD remains poorly understood. Genetic studies infer that the disease results from a complex interaction between genetics and environment and there is growing evidence that PD may represent a constellation of diseases with overlapping yet distinct underlying mechanisms. Novel clinical approaches will require a better understanding of the mechanisms at work within an individual as well as methods to identify the specific array of mechanisms that have contributed to the disease. Induced pluripotent stem cell (iPSC) strategies provide an opportunity to directly study the affected neuronal subtypes in a given patient. Here we report the generation of iPSC-derived midbrain dopaminergic neurons from a patient with a triplication in the α-synuclein gene (SNCA). We observed that the iPSCs readily differentiated into functional neurons. Importantly, the PD-affected line exhibited disease-related phenotypes in culture: accumulation of α-synuclein, inherent overexpression of markers of oxidative stress, and sensitivity to peroxide induced oxidative stress. These findings show that the dominantly-acting PD mutation is intrinsically capable of perturbing normal cell function in culture and confirm that these features reflect, at least in part, a cell autonomous disease process that is independent of exposure to the entire complexity of the diseased brain