Evaluation of formalin preserved allogeneic bone graft as a grafting material

Formalin preserved allogeneic (FPA) bone graft was used in 18 patients with benign bone cavities, nine patients with malignant bone cavities, 12 patients with atrophic nonunion of fractures, seven with gap nonunion and 18 with comminuted fractures. The benign bone cavities were obliterated in 20 weeks and malignant cavities in 24 weeks. Union was obtained in atrophic ununited fractures in 29 weeks, in gap nonunion in 40 weeks and in comminuted fractures in 27 weeks. FPA is a simple, inexpensive and easily available alternative to autologous bone and to deep frozen bone in filling bone defects and enhancing fracture union.     

Noninvasive assessment of systolic and diastolic left ventricular function in patients with chronic severe anemia: a combined M-mode, two-dimensional, and Doppler echocardiographic study.

Thirty-one patients with chronic severe anemia of more than 3 months' duration (hemoglobin less than 7 gm/dl) and no underlying heart disease were studied by means of M-mode, two-dimensional, and Doppler echocardiography; an equal number of normal control subjects was also studied. There are conflicting reports regarding the influence of chronic severe anemia on systolic myocardial function, but diastolic function has not been systematically assessed. It is also uncertain whether anemia alone can cause heart failure in a structurally normal heart. We therefore performed a detailed study of echocardiographic indexes of systolic and diastolic left ventricular function in these patients. We found that patients with anemia have significantly faster heart rates and lower diastolic and mean blood pressures than normal subjects. They also have a significantly elevated cardiac output and stroke volume and larger left ventricles. Left ventricular contractility, assessed by the end-systolic stress-dimension relationship, was enhanced. There was no systematic evidence of diastolic dysfunction by Doppler assessment of mitral inflow. There was also no clinical evidence of congestive heart failure. We conclude that chronic severe anemia leads to a hyperdynamic state with systolic hyperfunction and no impairment of diastolic function. Anemia does not lead to congestive heart failure in the absence of underlying heart disease.     

Modification of septic shock in mice by the antiglucocorticoid RU 38486.

The survival rate in murine septic peritonitis was inversely proportional to the size of the needle used for cecal puncture following ligation below the ileocecal valve. The smaller, 20-gauge needle permitted 20% survival. Only 20% of the animals survived 24 hr after cecal puncture with a 20-gauge needle compared to 90% survival after 5 days if mice had been rendered tolerant to lipopolysaccharide (LPS) prior to the induction of peritonitis. A single intravenous injection of 1 mg RU 38486, concurrent with puncture with a 21-gauge needle, lowered survival to only 15% from the control level of 71%. This same dose of the antiglucocorticoid decreased the survival rate to only 35% from 90% in the tolerant group. Tolerance to the lethal effects of endotoxins, possibly responsible for resistance to septic peritonitis, was also abolished by the antiglucocorticoid. Just 1 mg RU 38486 lowered the survival rate to 5% if it was given with 600 micrograms LPS, which permitted 95% survival in LPS-tolerant mice and 60% survival in normal controls. Whereas both 1 mg RU 38486 and 100 micrograms dexamethasone, given alone, sensitized mice to septic peritonitis (15% and 30% survival, respectively), their combined effects neutralized each other, leading to 80% survival vs. 71% in the control group. Thus receptor-mediated glucocorticoid-dependent mechanisms appear important in the pathogenesis of both endotoxin and septic shock, albeit to varying degrees and in a seemingly contradictory manner.     

Detection and phasing of single base de novo mutations in biopsies from human in vitro fertilized embryos by advanced whole-genome sequencing

Currently, the methods available for preimplantation genetic diagnosis (PGD) of in vitro fertilized (IVF) embryos do not detect de novo single-nucleotide and short indel mutations, which have been shown to cause a large fraction of genetic diseases. Detection of all these types of mutations requires whole-genome sequencing (WGS). In this study, advanced massively parallel WGS was performed on three 5- to 10-cell biopsies from two blastocyst-stage embryos. Both parents and paternal grandparents were also analyzed to allow for accurate measurements of false-positive and false-negative error rates. Overall, >95% of each genome was called. In the embryos, experimentally derived haplotypes and barcoded read data were used to detect and phase up to 82% of de novo single base mutations with a false-positive rate of about one error per Gb, resulting in fewer than 10 such errors per embryo. This represents a ∼100-fold lower error rate than previously published from 10 cells, and it is the first demonstration that advanced WGS can be used to accurately identify these de novo mutations in spite of the thousands of false-positive errors introduced by the extensive DNA amplification required for deep sequencing. Using haplotype information, we also demonstrate how small de novo deletions could be detected. These results suggest that phased WGS using barcoded DNA could be used in the future as part of the PGD process to maximize comprehensiveness in detecting disease-causing mutations and to reduce the incidence of genetic diseases.Worldwide, more than 5 million babies (Ferraretti et al. 2013) have been born through in vitro fertilization (IVF) since the birth of the first in 1978 (Steptoe and Edwards 1978). Exact numbers are difficult to determine, but it has been estimated that currently 350,000 babies are born yearly through IVF (de Mouzon et al. 2009, 2012; Centers for Disease Control and Prevention 2011; Ferraretti et al. 2013). That number is expected to rise, as advanced maternal age is associated with decreased fertility rates and women in developed countries continue to delay childbirth to later ages. In 95% of IVF procedures, no diagnostic testing of the embryos is performed (https://www.sartcorsonline.com/rptCSR_PublicMultYear.aspx?ClinicPKID=0). Couples with prior difficulties conceiving or those wishing to avoid the transmission of highly penetrant heritable diseases often choose to perform preimplantation genetic diagnosis (PGD). PGD involves the biopsy of one cell from a 3-d embryo or the recently more preferred method, due to improved implantation success rates (Scott et al. 2013b), of up to 10 cells from a 5- to 6-d blastocyst-stage embryo. Following biopsy, genetic analysis is performed on the isolated cell(s). Currently this is an assay for translocations and the correct chromosome copy number (Hodes-Wertz et al. 2012; Munne 2012; Yang et al. 2012; Scott et al. 2013a; Yin et al. 2013), a unique test designed and validated for each specific heritable disease (Gutierrez-Mateo et al. 2009), or a combination of both (Treff et al. 2013). Importantly, none of these approaches can detect de novo mutations.Advanced maternal age has long been associated with an increased risk of producing aneuploid embryos (Munne et al. 1995; Crow 2000; Hassold and Hunt 2009) and giving birth to a child afflicted with Down syndrome or other diseases resulting from chromosomal copy number alterations. Conversely, children of older fathers have been shown to have an increase in single base and short multibase insertion/deletion (indels) de novo mutations (Kong et al. 2012). Many recent large-scale sequencing studies have found that de novo variations spread across many different genes are likely to be the cause of a large fraction of autism cases (Michaelson et al. 2012; O’Roak et al. 2012; Sanders et al. 2012; De Rubeis et al. 2014; Iossifov et al. 2014), severe intellectual disability (Gilissen et al. 2014), epileptic encephalopathies (Epi4K Consortium and Epilepsy Phenome/Genome Project 2013), and many other congenital disorders (de Ligt et al. 2012; Veltman and Brunner 2012; Yang et al. 2013; Al Turki et al. 2014). Additionally rare and de novo variations have been suggested to be prevalent in patients with schizophrenia (Fromer et al. 2014; Purcell et al. 2014), and Michaelson et al. (2012) found that single base de novo mutations affect conserved regions of the genome and essential genes more often than regions of unknown function. Current targeted approaches to PGD would miss many of these important functional changes within the embryonic DNA sequence, and even a whole-genome sequencing (WGS)–based carrier screen of both parents would not enable comprehensive preimplantation or prenatal diagnoses due to de novo mutations. As more parents delay childbirth into their mid-30s and later, these studies suggest we should try to provide better diagnostic tests for improving the health of newborns. In this study, we demonstrate the use of an advanced WGS process that provides an accurate and phased genome sequence from about 10 cells, allowing highly sensitive and specific detection of single base de novo mutations from IVF blastocyst biopsies.     

Size controlled green synthesis of gold nanoparticles using Coffea arabica seed extract and their catalytic performance in 4-nitrophenol reduction

Well crystalline gold nanoparticles (AuNPs) of different sizes were fabricated using sundried Coffea arabica seed (CAS) extract at room temperature by controlling the pH of the green extract. The size, shape and crystallinity of the nanoparticles have been studied using electron microscopy and X-ray diffraction. The presence of phenolic groups (revealed through FT-IR studies) from the CAS extract are responsible both for the reduction of Au ions and stabilization of the formed AuNPs. The efficiency of the CAS extract mediated green synthesis technique for the production of AuNPs has been compared to the conventional chemical Turkevich technique, which not only uses a toxic reductant such as NaBH₄, but also operates around the boiling point of water. It has been observed that the CAS extract mediated synthesis process produces relatively bigger AuNPs at similar pH values of the reaction mixture in comparison to the AuNPs produced in the Turkevich process. Although the AuNPs synthesized using CAS extract are relatively larger and polydisperse in nature, their catalytic efficiencies for the degradation of an aromatic nitro compound (4-nitrophenol) are found to be comparable to the chemically fabricated AuNPs. Probable mechanisms associated with the formation of AuNPs and their size control in the CAS extract mediated green synthesis process have been discussed.

Size dependent catalytic activity of AuNPs synthesized at room temperature from Coffea arabica seed extract.