Live births from urine derived cells.

Here we report urine-derived cell (UDC) culture and subsequent use for cloning which resulted in the successful development of cloned canine pups, which have remained healthy into adulthood. Bovine UDCs were used in vitro to establish comparative differences between cell sources. UDCs were chosen as a readily available and noninvasive source for obtaining cells. We analyzed the viability of cells stored in urine over time and could consistently culture cells which had remained in urine for 48hrs. Cells were shown to be viable and capable of being transfected with plasmids. Although primarily of epithelial origin, cells were found from multiple lineages, indicating that they enter the urine from more than one source. Held in urine, at 4°C, the majority of cells maintained their membrane integrity for several days. When compared to in vitro fertilization (IVF) derived embryos or those from traditional SCNT, UDC derived embryos did not differ in total cell number or in the number of DNA breaks, measured by TUNEL stain. These results indicate that viable cells can be obtained from multiple species' urine, capable of being used to produce live offspring at a comparable rate to other cell sources, evidenced by a 25% pregnancy rate and 2 live births with no losses in the canine UDC cloning trial. This represents a noninvasive means to recover the breeding capacity of genetically important or infertile animals. Obtaining cells in this way may provide source material for human and animal studies where cells are utilized.

A Unifying Framework for Understanding Biological Structures and Functions Across Levels of Biological Organization.

The relationship between structure and function is a major constituent of the rules of life. Structures and functions occur across all levels of biological organization. Current efforts to integrate conceptual frameworks and approaches to address new and old questions promise to allow a more holistic and robust understanding of how different biological functions are achieved across levels of biological organization. Here, we provide unifying and generalizable definitions of both structure and function that can be applied across all levels of biological organization. However, we find differences in the nature of structures at the organismal level and below as compared to above the level of the organism. We term these intrinsic and emergent structures, respectively. Intrinsic structures are directly under selection, contributing to the overall performance (fitness) of the individual organism. Emergent structures involve interactions among aggregations of organisms and are not directly under selection. Given this distinction, we argue that while the functions of many intrinsic structures remain unknown, functions of emergent structures are the result of the aggregate of processes of individual organisms. We then provide a detailed and unified framework of the structure-function relationship for intrinsic structures to explore how their unknown functions can be defined. We provide examples of how these scalable definitions applied to intrinsic structures provide a framework to address questions on structure-function relationships that can be approached simultaneously from all subdisciplines of biology. We propose that this will produce a more holistic and robust understanding of how different biological functions are achieved across levels of biological organization.

Psychosocial adjustment mediates impacts of playmate positive support on body mass index and overweight risk in adolescents.

OBJECTIVES: We examined the mediation effects of psychosocial adjustment on the impact of playmate positive support throughout childhood and early adolescence (from age 54 months to 11 years) on later body mass index (BMI) and overweight risk in middle adolescence (age 15 years). STUDY DESIGN: This was a prospective cohort study. METHODS: Among 844 children and their families, positive support between child-playmate dyads was repeatedly assessed from child's age 54 months to Grade 5. Long-term positive support between child-playmate dyads throughout childhood and early adolescence was prospectively linked to child's BMI and overweight/obesity status at age 15 years. The average scores of repeated assessments of internalizing and externalizing behavior problems from Grades 3 to 6 were used as mediators. RESULTS: Significant mediations of internalizing and externalizing behavior problems were observed on pathways from positive support between child-playmate dyads to later BMI and overweight/obesity status at age 15 years. The observed mediations were mainly sustained with pronounced magnitudes in girls, but not in boys. CONCLUSIONS: Our findings demonstrated a significant mediating role of psychosocial adjustment. Future research efforts are highly encouraged to replicate our findings and further explore this underlying mediation mechanism.

Blastocyst formation, embryo transfer and breed comparison in the first reported large scale cloning of camels.

Cloning, through somatic cell nuclear transfer (SCNT), has the potential for a large expansion of genetically favorable traits in a population in a relatively short term. In the present study we aimed to produce multiple cloned camels from racing, show and dairy exemplars. We compared several parameters including oocyte source, donor cell and breed differences, transfer methods, embryo formation and pregnancy rates and maintenance following SCNT. We successfully achieved 47 pregnancies, 28 births and 19 cloned offspring who are at present healthy and have developed normally. Here we report cloned camels from surgical embryo transfer and correlate blastocyst formation rates with the ability to achieve pregnancies. We found no difference in the parameters affecting production of clones by camel breed, and show clear differences on oocyte source in cloning outcomes. Taken together we demonstrate that large scale cloning of camels is possible and that further improvements can be achieved.

Demetylation of the sex-determining region Y gene promoter and incidence of disorder of sex development in cloned dog males.

Canine cloning is occasionally accompanied by abnormal sexual development. Some male donor cells produce cloned pups with female external genitalia and complete male gonadal dysgenesis, which is classified as an XY disorder of sex development (XY DSD). In this study, we examine the potential of 5-aza-2'-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, to reduce the phenotypic abnormality XY DSD in somatic cell nuclear transfer (SCNT)- derived pups. We used a 9-year-old normal male German Shepherd dog as a cell donor. Donor cells were treated with 10 nM 5-aza-dC for 4 days before being used for SCNT. At the same stage of cell development, significantly lower levels of DNA methylation of the sex-determining region Y (SRY) promoter was observed in the treated donor cells compared to that in the untreated cells (95.2% versus 53.3% on day 4 for the control and treated groups, respectively). No significant differences were observed in the control or treatment groups concerning fusion rate, pregnancy rate (30 days or entire period), the number of pups, or the incidence of XY DSD. However, more XY DSD dogs were observed in the control group (31.25%) than in the treatment group (14.29%). Hypermethylation of the SRY promoter was observed in the XY DSD cloned pups in both the treatment (84.8%) and control groups (91.1 ± 1.4%) compared to the methylation level in the phenotypically normal male pups of the treatment (23.2 ± 20.9%) and control groups (39.1 ± 20.1%). These results suggest that 5-aza-dC treatment of donor cells can reduce the methylation level of the SRY promoter in donor cells, and thus, 5-aza-dC is advantageous for reducing the incidence of XY DSD in canine cloning.

Lumbar Spinal Stenosis Severity by CT or MRI Does Not Predict Response to Epidural Corticosteroid versus Lidocaine Injections.

BACKGROUND AND PURPOSE: Epidural steroid injections may offer little-to-no short-term benefit in the overall population of patients with symptomatic spinal stenosis compared with lidocaine alone. We investigated whether imaging could identify subgroups of patients who might benefit most. MATERIALS AND METHODS: A secondary analysis of the Lumbar Epidural Steroid Injections for Spinal Stenosis prospective, double-blind trial was performed, and patients were randomized to receive an epidural injection of lidocaine with or without corticosteroids. Patients (n = 350) were evaluated for qualitative and quantitative MR imaging or CT measures of lumbar spinal stenosis. The primary clinical end points were the Roland-Morris Disability Questionnaire and the leg pain numeric rating scale at 3 weeks following injection. ANCOVA was used to assess the significance of interaction terms between imaging measures of spinal stenosis and injectate type on clinical improvement. RESULTS: There was no difference in the improvement of disability or leg pain scores at 3 weeks between patients injected with epidural lidocaine alone compared with corticosteroid and lidocaine when accounting for the primary imaging measures of qualitative spinal stenosis assessment (interaction coefficients for disability score, -0.1; 95% CI, -1.3 to 1.2; P = .90; and for the leg pain score, 0.1; 95% CI, -0.6 to 0.8; P = .81) or the quantitative minimum thecal sac cross-sectional area (interaction coefficients for disability score, 0.01; 95% CI, -0.01 to 0.03; P = .40; and for the leg pain score, 0.01; 95% CI, -0.01 to 0.03; P = .33). CONCLUSIONS: Imaging measures of spinal stenosis are not associated with differential clinical responses following epidural corticosteroid injection.

Impact of Balloon-Guiding Catheter Location on Recanalization in Patients with Acute Stroke Treated by Mechanical Thrombectomy.

BACKGROUND AND PURPOSE: Mechanical thrombectomy with proximal flow control and forced aspiration may improve the outcome of endovascular revascularization therapy for patients with acute stroke. The purpose of this study was to compare the impact of balloon-guiding catheter locations in patients treated for anterior circulation acute ischemic stroke using mechanical thrombectomy. MATERIALS AND METHODS: The influence of the balloon-guiding catheter location (proximal, balloon-guiding catheter tip proximal to C1 vertebral body; distal, between the skull base and the C1 vertebral body) was analyzed in patients with acute anterior circulation stroke treated with stent-retriever thrombectomy. The baseline angiographic/clinical characteristics, time intervals, recanalization rates, and clinical outcomes were compared. RESULTS: The clinical analysis included 102 patients (mean age, 69.5 ± 12.8 years; male/female ratio = 52:50). The balloon-guiding catheter was located distally in 49 patients and proximally in 53 patients for flow control and forced aspiration during stent retrieval. The puncture-to-recanalization time was shorter in the distal group than in the proximal group (40 versus 56 minutes, P = .02). Successful and complete recanalizations were more frequently achieved in the distal group compared with the proximal group (98.0% versus 75.5%. P = .003; 67.3% versus 45.3%, P = .04, respectively). Multivariate analysis showed that the distal catheterization location was independently associated with successful recanalization (adjusted OR, 13.4; 95% CI, 2.4-254.8; P = .02). CONCLUSIONS: Location of the balloon-guiding catheter has a significant impact on recanalization in patients with acute stroke. The balloon-guiding catheter should be positioned as distally as safely possible in the cervical ICA for maximally effective thrombectomy.

Sustained Posttransplantation Diabetes Is Associated With Long-Term Major Cardiovascular Events Following Liver Transplantation.

Cardiovascular disease is a leading cause of death among liver transplant (LT) recipients. With a rising burden of posttransplantation metabolic disease, increases in cardiovascular-related morbidity and mortality may reduce life expectancy after LT. It is unknown if the risk of long-term major cardiovascular events (MCEs) differs among LT recipients with varying diabetic states. We performed a retrospective cohort study of LT recipients from 2003 through 2013 to compare the incidence of MCEs among patients (1) without diabetes, (2) with pretransplantation diabetes, (3) with de novo transient posttransplantation diabetes, and (4) with de novo sustained posttransplantation diabetes. We analyzed 994 eligible patients (39% without diabetes, 24% with pretransplantation diabetes, 16% with transient posttransplantation diabetes, and 20% with sustained posttransplantation diabetes). Median follow-up was 54.7 months. Overall, 12% of patients experienced a MCE. After adjustment for demographic and clinical variables, sustained posttransplantation diabetes was the only state associated with a significantly increased risk of MCEs (subdistribution hazard ratio 1.95, 95% confidence interval 1.20-3.18). Patients with sustained posttransplantation diabetes mellitus had a 13% and 27% cumulative incidence of MCEs at 5 and 10 years, respectively. While pretransplantation diabetes has traditionally been associated with cardiovascular disease, the long-term risk of MCEs is greatest in LT recipients with sustained posttransplantation diabetes mellitus.

Dynamic Contrast-Enhanced MRI-Derived Intracellular Water Lifetime (τ i ): A Prognostic Marker for Patients with Head and Neck Squamous Cell Carcinomas.

BACKGROUND AND PURPOSE: Shutter-speed model analysis of dynamic contrast-enhanced MR imaging allows estimation of mean intracellular water molecule lifetime (a measure of cellular energy metabolism) and volume transfer constant (a measure of hemodynamics). The purpose of this study was to investigate the prognostic utility of pretreatment mean intracellular water molecule lifetime and volume transfer constant in predicting overall survival in patients with squamous cell carcinomas of the head and neck and to stratify p16-positive patients based upon survival outcome. MATERIALS AND METHODS: A cohort of 60 patients underwent dynamic contrast-enhanced MR imaging before treatment. Median, mean intracellular water molecule lifetime and volume transfer constant values from metastatic nodes were computed from each patient. Kaplan-Meier analyses were performed to associate mean intracellular water molecule lifetime and volume transfer constant and their combination with overall survival for the first 2 years, 5 years, and beyond (median duration, >7 years). RESULTS: By the last date of observation, 18 patients had died, and median follow-up for surviving patients (n = 42) was 8.32 years. Patients with high mean intracellular water molecule lifetime (4 deaths) had significantly (P = .01) prolonged overall survival by 5 years compared with those with low mean intracellular water molecule lifetime (13 deaths). Similarly, patients with high mean intracellular water molecule lifetime (4 deaths) had significantly (P = .006) longer overall survival at long-term duration than those with low mean intracellular water molecule lifetime (14 deaths). However, volume transfer constant was a significant predictor for only the 5-year follow-up period. There was some evidence (P < .10) to suggest that mean intracellular water molecule lifetime and volume transfer constant were associated with overall survival for the first 2 years. Patients with high mean intracellular water molecule lifetime and high volume transfer constant were associated with significantly (P < .01) longer overall survival compared with other groups for all follow-up periods. In addition, p16-positive patients with high mean intracellular water molecule lifetime and high volume transfer constant demonstrated a trend toward the longest overall survival. CONCLUSIONS: A combined analysis of mean intracellular water molecule lifetime and volume transfer constant provided the best model to predict overall survival in patients with squamous cell carcinomas of the head and neck.

Effect of Adding Cerium on Microstructure and Morphology of Ce-Based Inclusions Formed in Low-Carbon Steel.

Intra-granular Acicular Ferrite (IAF), as one of the most well-known desirable microstructure of ferrite with a chaotic crystallographic orientation, can not only refine the microstructure and retard the propagation of cleavage crack but also provide excellent combination of strength and toughness in steel. The effect of adding cerium on microstructure and controlling proper cerium-based inclusions in order to improve properties in low-carbon commercial steel (SS400) were investigated. The type of inclusions can be controlled by changing S/O ratio and Ce content. Without Ce modification, MnS is a dominate inclusion. After adding Ce, the stable inclusion phases change from AlCeO3 to Ce2O2S. The optimum amount of cerium, 0.0235 wt.%, lead in proper grain refinement and formation of cerium oxide, oxy-sulfide and sulfide inclusions. Having a high amount of cerium results in increasing the number of inclusions significantly as a result it cannot be effective enough and the inclusions will act like barriers for others. It is found that the inclusions with a size of about 4∼7 µm can serve as heterogeneous nucleation sites for AF formation. Thermodynamic calculations have been applied to predict the inclusion formation in this molten steel as well, which show a good agreement with experimental one.

Application of peripherally inserted central catheter in acute myeloid leukaemia patients undergoing induction chemotherapy.

Increasingly, peripherally inserted central catheters (PICC) are applied in patients with haematological malignancies. The feasibility and safety of PICC for induction chemotherapy in acute myeloid leukaemia (AML) remain unclear. Medical records of 89 newly diagnosed adult de novo AML patients, who achieved complete remission, were retrospectively reviewed (PICC group, n = 43; intravenous [IV] line group, n = 46). Patients' clinical characteristics and the number of blind punctures for blood sampling were compared between these two groups, and risk factors associated with bacteraemia were identified by univariate analysis. Patients in the PICC group experienced significantly fewer blind punctures than those in the IV line group (3.3 ± 3.6 vs. 14.4 ± 6.0; p = .000); 20.9% of PICC patients had bacteraemia, compared with 23.9% in the IV line group (p = .803). Most patients (76.7%) removed their PICC because treatment was completed. PICC increased the quality of life in AML patients undergoing chemotherapy induction by reducing the number of blind blood punctures required. Bacteraemia in PICC patients was comparable to that in IV line patients. PICC is, therefore, a feasible and safe central venous device for use in AML patients.

An exploration of the applicability of the refined disease risk index and its integration with other independent risk factors for individualized prognostication.

The refined disease risk index (DRI) is a powerful prognostic model based solely on the disease type and stage for predicting survival outcomes of various hematological malignancies after allogeneic transplant. Here, we analyzed our series of 690 patients transplanted over the past 15 years, and showed that besides overall survival (OS), the refined DRI is also able to segregate event-free survival and relapse mortality in our cohort of largely Southeast Asian patients with a long and complete follow-up. Stratification by refined DRI remains statistically significant even when broken down by specific diseases each with a smaller number of patients, as well as for a small subset of patients younger than 18 years old, providing a robust model for prognostication. Multivariable analysis shows that refined DRI, age, year of transplant and donor type are independent risk factors for OS. We further demonstrated here that prognostication for a given patient with a specific disease can be made more discriminating by integrating independent risk factors such as age and donor type with the refined DRI. The future development of prognostic system incorporating the refined DRI with patient- and transplant-related risk factors will provide a more precise estimate of transplant outcome.

Isomorphic coalescence of aster cores formed in vitro from microtubules and kinesin motors.

We report fluorescence microscopy studies of the formation of aster-like structures emerging from a cellular element-based active system and a novel analysis of the aster condensation. The system consists of rhodamine labeled microtubules which are dynamically coupled by functionalized kinesin motor proteins cross-linked via streptavidin-coated quantum dots (QDs). The aster-shaped objects contain core structures. The cores are aggregates of the QD-motor protein complexes, and result from the dynamic condensation of sub-clusters that are connected to each other randomly. The structural specificity of the aster core reflects a configuration of the initial connectivity between sub-clusters. Detailed image analysis allows us to extract a novel correlation between the condensation speed and the sub-cluster separation. The size of the core is scaled down during the condensation process, following a power law dependence on the distance between sub-clusters. The exponent of the power law is close to two, as expected from a geometric model. This single exponent common to all the contractile lines implies that there exists a time regime during which an isomorphic contraction of the aster core continues during the condensation process. We analyze the observed contraction by using a model system with potential applicability in a wide range of emergent phenomena in randomly coupled active networks, which are prevalent in the cellular environment.

Recurrent trichosporonosis with central nervous system involvement in an allogeneic hematopoietic stem cell transplant recipient.

Trichosporon is an ubiquitous yeast that has emerged as an opportunistic pathogen in the immunocompromised host. We describe a case of invasive trichosporonosis in an allogeneic hematopoietic stem cell transplant (allo-HSCT) recipient while on caspofungin antifungal prophylaxis. She developed disseminated trichosporonosis in the pre-engraftment period and was successfully treated with voriconazole. She later developed 2 further episodes of invasive trichosporonosis involving the central nervous system. This case highlights the challenges of managing trichosporonosis in allo-HSCT recipients and suggests the need for lifelong therapy in some patients.

Evaluation of Focal Cervical Spinal Cord Lesions in Multiple Sclerosis: Comparison of White Matter-Suppressed T1 Inversion Recovery Sequence versus Conventional STIR and Proton Density-Weighted Turbo Spin-Echo Sequences.

BACKGROUND AND PURPOSE: Conventional MR imaging of the cervical spinal cord in MS is challenged by numerous artifacts and interreader variability in lesion counts. This study compares the relatively novel WM-suppressed T1 inversion recovery sequence with STIR and proton density-weighted TSE sequences in the evaluation of cervical cord lesions in patients with MS. MATERIALS AND METHODS: Retrospective blinded analysis of cervical cord MR imaging examinations of 50 patients with MS was performed by 2 neuroradiologists. In each patient, the number of focal lesions and overall lesion conspicuity were measured in the STIR/proton density-weighted TSE and WM-suppressed T1 inversion recovery sequence groups. Independent side-by-side comparison was performed to categorize the discrepant lesions as either "definite" or "spurious." Lesion contrast ratio and edge sharpness were independently calculated in each sequence. RESULTS: Substantial interreader agreement was noted on the WM-suppressed T1 inversion recovery sequence (κ = 0.82) compared with STIR/proton density-weighted TSE (κ = 0.52). Average lesion conspicuity was better on the WM-suppressed T1 inversion recovery sequence (conspicuity of 3.1/5.0 versus 3.7/5.0, P < .01, in the WM-suppressed T1 inversion recovery sequence versus STIR/proton density-weighted TSE, respectively). Spurious lesions were more common on STIR/proton density-weighted TSE than on the WM-suppressed T1 inversion recovery sequence (23 and 30 versus 3 and 4 by readers 1 and 2, respectively; P < .01). More "definite" lesions were missed on STIR/proton density-weighted TSE compared with the WM-suppressed T1 inversion recovery sequence (37 and 38 versus 3 and 6 by readers 1 and 2, respectively). Lesion contrast ratio and edge sharpness were highest on the WM-suppressed T1 inversion recovery sequence. CONCLUSIONS: There is better interreader consistency in the lesion count on the WM-suppressed T1 inversion recovery sequence compared with STIR/proton density-weighted TSE sequences. The focal cord lesions are visualized with better conspicuity due to better contrast ratio and edge sharpness. There are fewer spurious lesions on the WM-suppressed T1 inversion recovery sequence compared with STIR/proton density-weighted TSE. The WM-suppressed T1 inversion recovery sequence could potentially be substituted for either STIR or proton density-weighted TSE sequences in routine clinical protocols.

Early relapse post autologous transplant is a stronger predictor of survival compared with pretreatment patient factors in the novel agent era: analysis of the Singapore Multiple Myeloma Working Group.

The clinical outcome of multiple myeloma is heterogeneous. Both the depth of response to induction and transplant as well as early relapse within a year are correlated with survival, but it is unclear which factor is most relevant in Southeast Asian patients with multiple myeloma. We retrospectively analyzed outcomes of 215 patients who were treated with upfront autologous transplant in Singapore between 2000 and 2014. In patients who received novel agent (NA)-based induction, achieving only partial response (PR) post-induction was associated with poorer OS (HR 1.95, P=0.047) and PFS (HR 2.9, P<0.001), while achieving only PR post-transplant was strongly correlated with both OS (HR 3.3, P=0.001) and PFS (HR 7.6, P<0.001), compared with patients who achieved very good partial response (VGPR) or better. Early relapse was detected in 18% of all patients, although nearly half had initially achieved VGPR or better post-transplant. Early relapse after NA-based induction led to significantly shorter OS (median 22 months vs not reached, P<0.001), and was strongly associated with OS (HR 13.7, P<0.001). The impact of suboptimal post-transplant response and early relapse on survival may be more important than pretransplant factors, such as International Staging System or cytogenetics, and should be considered in risk stratification systems to rationalize therapy.

Higher tacrolimus concentrations early after transplant reduce the risk of acute GvHD in reduced-intensity allogeneic stem cell transplantation.

There is significant variability in the serum concentrations of tacrolimus attained early post transplant due to drug interactions and genomic variation. We evaluated whether tacrolimus concentrations early post transplant correlated with incidence of acute GvHD in 120 consecutive patients allografted with a uniform reduced-intensity conditioning regimen. All patients received standard prophylaxis with oral tacrolimus and IV methotrexate. The primary variable of interest was mean weekly tacrolimus concentrations in the initial 4 weeks post transplant. In multivariate analysis, week 1 tacrolimus concentration was an independent predictor of acute grade 2-4 GvHD (hazard ratio (HR), 0.90; 95% confidence interval (CI), 0.84-0.97; P<0.01). This association was driven by a lower risk of acute grade 2-4 GvHD in patients with week 1 tacrolimus concentrations >12 ng/mL (HR, 0.47; 95% CI, 0.25-0.88; P=0.02). Week 1 tacrolimus concentrations were not associated with chronic GvHD, relapse or overall survival. Lower tacrolimus concentrations at weeks 2, 3 and 4 were not associated with a higher incidence of GvHD. In summary, we found that higher tacrolimus concentrations during the first week after allografting with a reduced-intensity conditioning regimen were associated with significantly reduced risk of acute grade 2-4 GvHD without increasing risk of relapse.