Methods for comprehensive chromosome screening of oocytes and embryos: capabilities, limitations, and evidence of validity

Preimplantation aneuploidy screening of cleavage stage embryos using fluorescence in situ hybridization (FISH) may no longer be considered the standard of care in reproductive medicine. Over the last few years, there has been considerable development of novel technologies for comprehensive chromosome screening (CCS) of the human genome. Among the notable methodologies that have been incorporated are whole genome amplification, metaphase and array based comparative genomic hybridization, single nucleotide polymorphism microarrays, and quantitative real-time PCR. As these methods become more integral to treating patients with infertility, it is critical that clinicians and scientists obtain a better understanding of their capabilities and limitations. This article will focus on reviewing these technologies and the evidence of their validity

Characteristics and Treatment Outcomes of Patients with MDR and XDR Tuberculosis in a TB Referral Hospital in Beijing: A 13-Year Experience

Background: Information on treatment outcomes among hospitalized patients with multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) are scarce in China. Methodology/Principal Findings: We conducted this retrospective study to analyze the characteristics and treatment outcomes in MDR- and XDR-TB patients in the 309 Hospital in Beijing, China during 1996-2009. Socio-demographic and clinical data were retrieved from medical records and analyzed. Logistic regression analysis was performed to identify risk factors associated with poor treatment outcomes and Cox proportional hazards regression model was further used to determine risk factors associated with death in TB patients. Among the 3,551 non-repetitive hospitalized TB patients who had drug susceptibility testing (DST) results, 716 (20.2%) had MDR-TB and 51 (1.4%) had XDR-TB. A total of 3,270 patients who had medical records available were used for further analyses. Treatment success rates (cured and treatment completed) were 90.9%, 53.4% and 29.2% for patients with non-MDR-TB, patients with MDR-TB excluding XDR-TB and patients with XDR-TB, respectively. Independent risk factors associated with poor treatment outcomes in MDR-TB patients included being a migrant (adjusted OR = 1.77), smear-positivity at treatment onset (adjusted OR = 1.94) and not receiving 3 or more potentially effective drugs (adjusted OR = 3.87). Independent risk factors associated with poor treatment outcomes in XDR-TB patients were smear-positivity at treatment onset (adjusted OR = 10.42) and not receiving 3 or more potentially effective drugs (adjusted OR = 14.90). The independent risk factors associated with death in TB patients were having chronic obstructive pulmonary disease (adjusted HR = 5.25) and having hypertension (adjusted HR = 4.31). Conclusions/Significance: While overall satisfactory treatment success for non-MDR-TB patients was achieved, more intensive efforts should be made to better manage MDR- and XDR-TB cases in order to improve their treatment outcomes and to minimize further emergence of so-called totally drug-resistant TB cases. © 2011 Liu et al.published_or_final_versio

Use of a Molecular Diagnostic Test in AFB Smear Positive Tuberculosis Suspects Greatly Reduces Time to Detection of Multidrug Resistant Tuberculosis

Background: The WHO has recommended the implementation of rapid diagnostic tests to detect and help combat M/XDR tuberculosis (TB). There are limited data on the performance and impact of these tests in field settings. Methods: The performance of the commercially available Genotype MTBDRplus molecular assay was compared to conventional methods including AFB smear, culture and drug susceptibility testing (DST) using both an absolute concentration method on Löwenstein-Jensen media and broth-based method using the MGIT 960 system. Sputum specimens were obtained from TB suspects in the country of Georgia who received care through the National TB Program. Results: Among 500 AFB smear-positive sputum specimens, 458 (91.6%) had both a positive sputum culture for Mycobacterium tuberculosis and a valid MTBDRplus assay result. The MTBDRplus assay detected isoniazid (INH) resistanc

Prospective evaluation of chronic pain associated with posterior autologous iliac crest bone graft harvest and its effect on postoperative outcome

<p>Abstract</p> <p>Background</p> <p>Autogenous Iliac Crest Bone Graft (ICBG) has been the "gold standard" for spinal fusion. However, bone graft harvest may lead to complications, such as chronic pain, numbness, and poor cosmesis. The long-term impact of these complications on patient function and well-being has not been established but is critical in determining the value of expensive bone graft substitutes such as recombinant bone morphogenic protein. We thus aimed to investigate the long-term complications of ICBG. Our second aim was to evaluate the psychometric properties of a new measure of ICBG morbidity that would be useful for appropriately gauging spinal surgery outcomes.</p> <p>Methods</p> <p>Prospective study of patients undergoing spinal fusion surgery with autologous ICBG. The SF-36v2, Oswestry Disability Index, and a new 14-item follow-up questionnaire addressing persistent pain, functional limitation, and cosmesis were administered with an 83% response rate. Multiple regression analyses examined the independent effect of ICBG complications on physical and mental health and disability.</p> <p>Results</p> <p>The study population included 170 patients with a mean age of 51.1 years (SD = 12.2) and balanced gender (48% male). Lumbar fusion patients predominated (lumbar = 148; cervical n = 22). At 3.5 years mean follow-up, 5% of patients reported being bothered by harvest site scar appearance, 24% reported harvest site numbness, and 13% reported the numbness as bothersome. Harvest site pain resulted in difficulty with household chores (19%), recreational activity (18%), walking (16%), sexual activity (16%), work activity (10%), and irritation from clothing (9%). Multivariate regression analyses revealed that persistent ICBG complications 3.5 years post-surgery were associated with significantly worse disability and showed a trend association with worse physical health, after adjusting for age, workers' compensation status, surgical site pain, and arm or leg pain. There was no association between ICBG complications and mental health in the multivariate model.</p> <p>Conclusion</p> <p>Chronic ICBG harvest site pain and discomfort is reported by a significant percentage of patients undergoing this procedure more than three years following surgery, and these complications are associated with worse patient-reported disability. Future studies should consider employing a control group that does not include autologous bone graft harvest, e.g., a group utilizing rhBMP, to determine whether eliminating harvest-site morbidity does indeed lead to observable improvement in clinical outcome sufficient to justify the increased cost of bone graft substitutes.</p

Comparison of conventional and CT-based planning for intracavitary brachytherapy for cervical cancer: target volume coverage and organs at risk doses

<p>Abstract</p> <p>Background</p> <p>To compare intracavitary brachytherapy (ICBT) planning methods for cervical cancer, based on either orthogonal radiographs (conventional plan) or CT sections (CT plan); the comparison focused on target volume coverage and dose volume analysis of organs at risk (OARs), by representing point doses defined by the International Commission on Radiation Units and Measurement (ICRU) and dose volume histograms (DVHs) from 3D planning.</p> <p>Methods</p> <p>We analyzed the dosimetric data for 62 conventional and CT-based ICBT plans. The gross tumor volume (GTV), clinical target volume (CTV) and organs at risk (OAR)s were contoured on the CT-plan. Point A and ICRU 38 rectal and bladder points were defined on reconstructed CT images.</p> <p>Results</p> <p>Patients were categorized on the basis of whether the >95% isodose line of the point-A prescription dose encompassed the CTV (group 1, n = 24) or not (group 2, n = 38). The mean GTV and CTV (8.1 cc and 20.6 cc) were smaller in group 1 than in group 2 (24.7 cc and 48.4 cc) (<it>P <</it>0.001). The mean percentage of GTV and CTV coverage with the 7 Gy isodose was 93.1% and 88.2% for all patients, and decreased with increasing tumor size and stage. The mean D2 and D5 rectum doses were 1.66 and 1.42 times higher than the corresponding ICRU point doses and the mean D2 and D5 bladder doses were 1.51 and 1.28 times higher. The differences between the ICRU dose and the D2 and D5 doses were significantly higher in group 2 than in group 1 for the bladder, but not for the rectum.</p> <p>Conclusion</p> <p>The CT-plan is superior to the conventional plan in target volume coverage and appropriate evaluation of OARs, as the conventional plan overestimates tumor doses and underestimates OAR doses.</p

Erratum to : Analysis of the mitochondrial maxicircle of Trypanosoma lewisi, a neglected human pathogen

BACKGROUND The haemoflagellate Trypanosoma lewisi is a kinetoplastid parasite which, as it has been recently reported to cause human disease, deserves increased attention. Characteristic features of all kinetoplastid flagellates are a uniquely structured mitochondrial DNA or kinetoplast, comprised of a network of catenated DNA circles, and RNA editing of mitochondrial transcripts. The aim of this study was to describe the kinetoplast DNA of T. lewisi. METHODS/RESULTS In this study, purified kinetoplast DNA from T. lewisi was sequenced using high-throughput sequencing in combination with sequencing of PCR amplicons. This allowed the assembly of the T. lewisi kinetoplast maxicircle DNA, which is a homologue of the mitochondrial genome in other eukaryotes. The assembly of 23,745 bp comprises the non-coding and coding regions. Comparative analysis of the maxicircle sequence of T. lewisi with Trypanosoma cruzi, Trypanosoma rangeli, Trypanosoma brucei and Leishmania tarentolae revealed that it shares 78 %, 77 %, 74 % and 66 % sequence identity with these parasites, respectively. The high GC content in at least 9 maxicircle genes of T. lewisi (ATPase6; NADH dehydrogenase subunits ND3, ND7, ND8 and ND9; G-rich regions GR3 and GR4; cytochrome oxidase subunit COIII and ribosomal protein RPS12) implies that their products may be extensively edited. A detailed analysis of the non-coding region revealed that it contains numerous repeat motifs and palindromes. CONCLUSIONS We have sequenced and comprehensively annotated the kinetoplast maxicircle of T. lewisi. Our analysis reveals that T. lewisi is closely related to T. cruzi and T. brucei, and may share similar RNA editing patterns with them rather than with L. tarentolae. These findings provide novel insight into the biological features of this emerging human pathogen

Biosynthesis of Gold Nanoparticles by Foliar Broths: Roles of Biocompounds and Other Attributes of the Extracts

Biosynthesis of nanoparticles has arisen as a promising alternative to conventional synthetic methodologies owing to its eco-friendly advantages, and the involved bioprotocol still needs further clarification. This research, for the first time from the standpoint of statistics, confirmed an electrostatic force or ionic bond-based interaction between the chloroauric ions and the involved bioconstituents and manifested that reducing sugars and flavonoids were both important reductants responsible for conversion of Au(III) to Au(0). The result also demonstrated that the proteins were not the reducing agents, yet they might be protection agents in biosynthesis of gold nanoparticles (GNPs). Besides, a significant linear relationship was found between the anti-oxidant ability of the foliar broths and their capability to reduce Au(III) into Au(0). Furthermore, the preliminary investigation based on the boxplot on the size/shape distribution of the biosynthesized GNPs revealed that gold nanospheres with higher degree of homogeneity in size tended to be promoted by foliar broths containing higher content of reducing sugars/flavonoids and proteins. Otherwise, i.e., for those broths with lower content of the above biocompounds, sphere GNPs of wider size distribution or even gold nanotriangles tended to be fabricated

Inhibition of Lipoprotein-Associated Phospholipase A2 Ameliorates Inflammation and Decreases Atherosclerotic Plaque Formation in ApoE-Deficient Mice

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is thought to play modulatory roles in the development of atherosclerosis. Here we evaluated the effects of a specific lp-PLA2 inhibitor on atherosclerosis in ApoE-deficient mice and its associated mechanisms.ApoE-deficient mice fed an atherogenic high-fat diet for 17 weeks were divided into two groups. One group was administered the specific lp-PLA2 inhibitor, darapladib (50 mg/kg/day; p.o.) daily for 6 weeks, while the control group was administered saline. We observed no differences in body weight and serum lipids levels between the two groups at the end of the dietary period. Notably, serum lp-PLA2 activity as well as hs-CRP (C-reactive protein) and IL-6 (Interleukin-6) levels were significantly reduced in the darapladib group, compared with the vehicle group, while the serum PAF (platelet-activating factor) levels were similar between the two groups. Furthermore, the plaque area through the arch to the abdominal aorta was reduced in the darapladib group. Another finding of interest was that the macrophage content was decreased while collagen content was increased in atherosclerotic lesions at the aortic sinus in the darapladib group, compared with the vehicle group. Finally, quantitative RT-PCR performed to determine the expression patterns of specific inflammatory genes at atherosclerotic aortas revealed lower expression of MCP-1, VCAM-1 and TNF-α in the darapladib group. inflammation and decreased plaque formation in ApoE-deficient mice, supporting an anti-atherogenic role during the progression of atherosclerosis

Molecular detection of rifampin and isoniazid resistance to guide chronic TB patient management in Burkina Faso

<p>Abstract</p> <p>Background</p> <p>Drug-resistant tuberculosis (DR-TB) is considered a real threat to the achievement of TB control. Testing of mycobacterial culture and testing of drug susceptibility (DST) capacity are limited in resource-poor countries, therefore inadequate treatment may occur, favouring resistance development. We evaluated the molecular assay GenoType^®MTBDR<it>plus </it>(Hain Lifescience, Germany) in order to detect DR-TB directly in clinical specimens as a means of providing a more accurate management of chronic TB patients in Burkina Faso, a country with a high TB-HIV co-infection prevalence.</p> <p>Methods</p> <p>Samples were collected in Burkina Faso where culture and DST are not currently available, and where chronic cases are therefore classified and treated based on clinical evaluation and sputum-smear microscopy results. One hundred and eight chronic TB patients (sputum smear-positive, after completing a re-treatment regimen for pulmonary TB under directly observed therapy) were enrolled in the study from December 2006 to October 2008. Two early morning sputum samples were collected from each patient, immediately frozen, and shipped to Italy in dry ice. Samples were decontaminated, processed for smear microscopy and DNA extraction. Culture was attempted on MGIT960 (Becton Dickinson, Cockeysville, USA) and decontaminated specimens were analyzed for the presence of mutations conferring resistance to rifampin and isoniazid by the molecular assay GenoType^®MTBDR<it>plus</it>.</p> <p>Results</p> <p>We obtained a valid molecular test result in 60/61 smear-positive and 47/47 smear-negative patients.</p> <p>Among 108 chronic TB cases we identified patients who (i) harboured rifampin- and isoniazid-susceptible strains (n 24), (ii) were negative for MTB complex DNA (n 24), and (iii) had non-tuberculous mycobacteria infections (n 13). The most represented mutation conferring rifampin-resistance was the D516V substitution in the hotspot region of the <it>rpoB </it>gene (43.8% of cases). Other mutations recognized were the H526D (15.6%), the H526Y (15.6%), and the S531L (9.4%).</p> <p>All isoniazid-resistant cases (n 36) identified by the molecular assay were carrying a S315T substitution in the <it>katG </it>gene. In 41.7% of cases, a mutation affecting the promoter region of the <it>inhA </it>gene was also detected.</p> <p>Conclusion</p> <p>The GenoType^®MTBDR<it>plus </it>assay performed directly on sputum specimens improves the management of chronic TB cases allowing more appropriate anti-TB regimens.</p

Cofactor-mediated conformational control in the bifunctional kinase/RNase Ire1

<p>Abstract</p> <p>Background</p> <p>Ire1 is a signal transduction protein in the endoplasmic reticulum (ER) membrane that serves to adjust the protein-folding capacity of the ER according to the needs of the cell. Ire1 signals, in a transcriptional program, the unfolded protein response (UPR) via the coordinated action of its protein kinase and RNase domains. In this study, we investigated how the binding of cofactors to the kinase domain of Ire1 modulates its RNase activity.</p> <p>Results</p> <p>Our results suggest that the kinase domain of Ire1 initially binds cofactors without activation of the RNase domain. RNase is activated upon a subsequent conformational rearrangement of Ire1 governed by the chemical properties of bound cofactors. The conformational step can be selectively inhibited by chemical perturbations of cofactors. Substitution of a single oxygen atom in the terminal β-phosphate group of a potent cofactor ADP by sulfur results in ADPβS, a cofactor that binds to Ire1 as well as to ADP but does not activate RNase. RNase activity can be rescued by thiophilic metal ions such as Mn²⁺and Cd²⁺, revealing a functional metal ion-phosphate interaction which controls the conformation and RNase activity of the Ire1 ADP complex. Mutagenesis of the kinase domain suggests that this rearrangement involves movement of the αC-helix, which is generally conserved among protein kinases. Using X-ray crystallography, we show that oligomerization of Ire1 is sufficient for placing the αC-helix in the active, cofactor-bound-like conformation, even in the absence of cofactors.</p> <p>Conclusions</p> <p>Our structural and biochemical evidence converges on a model that the cofactor-induced conformational change in Ire1 is coupled to oligomerization of the receptor, which, in turn, activates RNase. The data reveal that cofactor-Ire1 interactions occur in two independent steps: binding of a cofactor to Ire1 and subsequent rearrangement of Ire1 resulting in its self-association. The pronounced allosteric effect of cofactors on protein-protein interactions involving Ire1's kinase domain suggests that protein kinases and pseudokinases encoded in metazoan genomes may use ATP pocket-binding ligands similarly to exert signaling roles other than phosphoryl transfer.</p