Single-Cell RNA Sequencing of Mutant Whole Mouse Embryos: From the Epiblast to the End of Gastrulation.

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, a robust pipeline was designed to perform single-cell and nuclei analysis on mouse embryos from embryonic day E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to wild-type perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, a methodology is presented designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with a same-day genotyping protocol (3 h) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. This method further includes guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate that this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula.

Single-cell RNA sequencing of mutant whole mouse embryos: from the epiblast to the end of gastrulation.

Over the last decade, single-cell approaches have become the gold standard for studying gene expression dynamics, cell heterogeneity, and cell states within samples. Before single-cell advances, the feasibility of capturing the dynamic cellular landscape and rapid cell transitions during early development was limited. In this paper, we designed a robust pipeline to perform single-cell and nuclei analysis on mouse embryos from E6.5 to E8, corresponding to the onset and completion of gastrulation. Gastrulation is a fundamental process during development that establishes the three germinal layers: mesoderm, ectoderm, and endoderm, which are essential for organogenesis. Extensive literature is available on single-cell omics applied to WT perigastrulating embryos. However, single-cell analysis of mutant embryos is still scarce and often limited to FACS-sorted populations. This is partially due to the technical constraints associated with the need for genotyping, timed pregnancies, the count of embryos with desired genotypes per pregnancy, and the number of cells per embryo at these stages. Here, we present a methodology designed to overcome these limitations. This method establishes breeding and timed pregnancy guidelines to achieve a higher chance of synchronized pregnancies with desired genotypes. Optimization steps in the embryo isolation process coupled with FAST genotyping protocol (3 hours) allow for microdroplet-based single-cell to be performed on the same day, ensuring the high viability of cells and robust results. We also include guidelines for optimal nuclei isolations from embryos. Thus, these approaches increase the feasibility of single-cell approaches of mutant embryos at the gastrulation stage. We anticipate this method will facilitate the analysis of how mutations shape the cellular landscape of the gastrula. SUMMARY: We establish a pipeline for high-quality single-cell and nuclei suspensions of gastrulating mouse embryos for sequencing of single cells and nuclei.

Virtual Molecular and Precision Medicine Clinic to Improve Access to Clinical Trials for Patients With Metastatic Breast Cancer: An Academic/Community Collaboration.

PURPOSE: There is a demand for improved care delivery surrounding genomic testing and clinical trial enrollment among patients with metastatic breast cancer (MBC). We sought to improve the current process via real-time informal consultation and prescreening assessment for patients with MBC treated by community and academic medical oncologists by implementing a virtual molecular and precision medicine (vMAP) clinic. METHODS: The vMAP program used a virtual referral system directed to a multidisciplinary team with precision medicine expertise. Providers contacted vMAP regarding patients with MBC, and on receipt of referral, the vMAP team engaged in discussion to identify if further diagnostics were needed (including genomic testing) and to identify potential clinical trials or standard treatment options. Recommendations were then sent to the referring provider within 72 hours. Pre-/postsurveys were issued to network physicians to assess for barriers, clinical trial access, and vMAP referral experience. Program implementation was evaluated with the Squire 2.0 reporting guidelines for quality improvement in health care as a framework. RESULTS: Eighty-one cases from 22 providers were referred to vMAP over a 26-month period. The average response time to the referring provider with a finalized recommendation was 1.90 ± 1.82 days. A total of 86.4% of cases had clinical trial options on vMAP prescreen, with 40.7% initiating formal screening assessments and 27 patients (33.3%) ultimately enrolling on trials. On resurvey, 92% of survey responses across community oncology referring providers said that they were very likely to use vMAP again. CONCLUSION: In the initial 2-year period, vMAP demonstrated an efficient means to offer real-time interpretation of genomic testing and identification of clinical trials for patients with MBC, with effective clinical trial enrollment and high rates of referring provider satisfaction.

Navigating between punishment, avoidance, and instruction: The form and function of responses to moral violations varies across adult and child transgressors.

Immoral actions can elicit a wide array of responses, ranging from pugnacious confrontation to passive distancing. What leads onlookers to react so differently to various violations? Across four studies (N = 2085), we investigated how responses vary depending on whether moral transgressions are committed by adults or by children. Findings reliably demonstrated that adult participants were more likely to avoid adult transgressors, and more likely to instruct child transgressors about why their actions were wrong. These patterns arose from varying cost-benefit structures, derived in part from asymmetries in interpersonal power between adults and children, rendering adults' direct confrontation of children both less costly and more beneficial. Although adults' transgressions were judged to be relatively more wrong, participants had greater anxiety about the negative consequences of confronting adults, and they viewed adults' personalities as less malleable, thus diminishing the effectiveness of confrontation. In contrast, 4- to 9-year-old children did not differ in their willingness to avoid or instruct adult and child transgressors. Across studies, the content of transgressions (e.g., being harmful or impure) mattered little for determining the nature of responses. Overall, diverse responses to moral transgressions were uniquely tailored to the different costs and benefits associated with confronting adult and child transgressors.

YAP1 regulates the self-organized fate patterning of hESC-derived gastruloids.

The gastrulation process relies on complex interactions between developmental signaling pathways that are not completely understood. Here, we interrogated the contribution of the Hippo signaling effector YAP1 to the formation of the three germ layers by analyzing human embryonic stem cell (hESC)-derived 2D-micropatterned gastruloids. YAP1 knockout gastruloids display a reduced ectoderm layer and enlarged mesoderm and endoderm layers compared with wild type. Furthermore, our epigenome and transcriptome analysis revealed that YAP1 attenuates Nodal signaling by directly repressing the chromatin accessibility and transcription of key genes in the Nodal pathway, including the NODAL and FOXH1 genes. Hence, in the absence of YAP1, hyperactive Nodal signaling retains SMAD2/3 in the nuclei, impeding ectoderm differentiation of hESCs. Thus, our work revealed that YAP1 is a master regulator of Nodal signaling, essential for instructing germ layer fate patterning in human gastruloids.

Association Between Neonates With Laryngomalacia and Neonatal Abstinence Syndrome.

OBJECTIVE: Laryngomalacia (LM) is the most common congenital anomaly of the larynx. The cause of LM is still largely unknown, but a neurological mechanism has gained the most acceptance. There have not been any studies examining the prevalence of LM in infants with Neonatal Abstinence Syndrome (NAS). The aim of our study is to determine if infants with NAS are more likely to be diagnosed with LM. METHODS: This study was a population-based inpatient registry analysis. We examined nationwide neonatal discharges in 2016 using the Kids' Inpatient Database (KID). Only patients listed as neonates were included. The International Classification of Diseases, 10th revision, Clinical Modification (ICD-10-CM) codes for neonatal withdrawal symptoms from maternal use of drugs of addiction (P96.1) and diagnoses denoting LM were used. To quantify associations between the LM and NAS groups, prevalence rates and odds ratios (ORs) were used. RESULTS: There were 3 970 065 weighted neonatal discharges in the 2016 KID. Among patients included in our dataset, 0.809% (32 128) had NAS and 0.075% (2974) had LM. There was an increased odds ratio for neonates with NAS and LM (OR of 2.85, 95% CI = 2.24-3.63) compared to infants without NAS. Multiple logistic regression accounting for possible confounders produced an adjusted OR of 1.68 (95% CI = 1.29-2.19). CONCLUSION: Our study found an association between NAS and LM. This suggests that prenatal exposure to opioids or possibly the sequelae of withdrawal symptoms may be risk factors for the development of LM.

Cutaneous Absidia corymbifera in a Lupus Nephritis Patient.

A 28-year-old farmer with class IV lupus nephritis presented with a two-week history of a right shin lesion. The lesion was purple in color, fungating, and indurated with a focus of deep ulceration at the inferior pole and punctate, bleeding from its surface. Three months earlier, he was started on induction immunosuppression for a relapse of his lupus nephritis. Since the diagnosis of lupus nephritis, nine years previously, he had had six flares of his disease and had been treated at different time points with cyclophosphamide, rituximab, and high-dose corticosteroids, without adverse events. Laboratory investigations showed improving kidney function (chronic kidney disease [CKD] stage IV) with reducing proteinuria, on his current immunosuppressive regimen. The differential diagnosis for this lesion was calciphylaxis, pyoderma gangrenosum, vasculitic lesion, or an infection. Histology and microbiological analysis confirmed the presence of Absidia corymbifera. He was treated with a combination of isavuconazole, reduction of his immunosuppressive agents, excision of the lesion, and skin grafting.

Phase 1b clinical trial of ado-trastuzumab emtansine and ribociclib for HER2-positive metastatic breast cancer.

Patients with HER2+ metastatic breast cancer are often treated with a multitude of therapies in the metastatic setting, and additional strategies to prolong responses to anti-HER2 therapies are needed. Preclinical evidence suggests synergy between cyclin-dependent kinase 4 and 6 (CDK 4/6) inhibitors and anti-HER2 therapies. We conducted a phase 1b study of ribociclib and ado-trastuzumab emtansine (T-DM1) in patients with advanced/metastatic HER2-positive breast cancer previously treated with trastuzumab and a taxane in any setting, with four or fewer prior lines of therapy in the metastatic setting. A standard 3 + 3 dose-escalation design was used to evaluate various doses of ribociclib in combination with T-DM1, starting at 300 mg. The primary objective was to determine the maximum tolerated dose and/or recommended phase 2 dose (RP2D) of ribociclib in combination with T-DM1. A total of 12 patients were enrolled. During dose-escalation, patients received doses of ribociclib of 300 mg (n = 3), 400 mg (n = 3), 500 mg (n = 3), and 600 mg (n = 3). No dose-limiting toxicities were observed. The majority of toxicities were Grade 1 and 2, and the most common Grade 3 toxicities were neutropenia (33%), leukopenia (33%), and anemia (25%). After a median follow-up of 12.4 months, the median PFS was 10.4 months (95% confidence interval, 2.7-19.3). Based on the pharmacokinetic analysis, adverse events, and dose reductions, 400 mg was determined to be the RP2D for ribociclib given on days 8-21 of a 21-day cycle with T-DM1.

Predictors of a Normal Sleep Study in Healthy Children with Sleep Disordered Breathing Symptoms.

OBJECTIVE: To determine the prevalence and characteristics of children with normal elective polysomnography for obstructive sleep disordered breathing (oSDB) based on the American Academy of Otolaryngology-Head and Neck Surgery (AAO-HNS) guidelines. STUDY DESIGN: In this retrospective cohort study, we identified patients ages 2 to 18 who underwent diagnostic polysomnography (PSG) ordered by our otolaryngology department for SDB between 2012 and 2018. SETTING: All patients were seen by otolaryngologists at an urban tertiary safety net hospital. SUBJECTS AND METHODS: There were a total of 456 patients studied (average age 5.66 ± 3.19; 263 (57.7%) males, 193 (42.3%) females. Demographic factors (age, gender, race, ethnicity, language, insurance status) and clinical findings (symptom severity, tonsil size) were recorded. The data were analyzed by univariate and multivariate analysis. RESULTS: Two hundred four patients (44.7%) had no obstructive sleep apnea (OSA) based on AHI<2 on PSG. Children with a larger tonsil size had 3.18 times the odds of OSA compared to those with a medium tonsil size (95% CI 1.64, 6.19) when adjusting for symptoms, age category, and race (P = .0007). Children ages 4 to 6 years had 0.25 times the odds of OSA compared to those ages 2-3 years (95% CI 0.12, 1.54) when adjusting for symptoms, tonsil size, and race (P = .0011). White children had 0.28 times the odds of OSA compared to Black children (95% CI 0.14, 0.57) when adjusting for symptoms, tonsil size, and age category (P = .0004). CONCLUSION: Among our patient population, 44.7% had normal sleep studies. Younger children (ages 2-3) were less likely to have normal polysomnography. This research demonstrates that obtaining sleep studies in otherwise healthy children with SDB can affect management decisions, and they should be discussed with families with a focus on patient centered decision making.

Characteristics and Frequency of Children With Severe Obstructive Sleep Apnea Undergoing Elective Polysomnography.

OBJECTIVE: To determine the prevalence and demographics features of pediatric patients with severe obstructive sleep apnea (OSA) who would not undergo preoperative polysomnography (PSG) under current American Academy of Otolaryngology (AAO) guidelines. STUDY DESIGN: In this retrospective cohort study, we identified patients from the electronic medical record who underwent elective polysomnography for evaluation of sleep-disordered breathing between 2012 and 2018. SETTING: Urban tertiary safety net hospital. SUBJECTS AND METHODS: A total of 456 patients with a mean (SD) age of 5.7 (3.2) years (263 male, 193 female). Demographic factors (age, sex, race, language, insurance status) and clinical findings (symptom severity, tonsil size) were recorded. The data were analyzed by univariate analysis. RESULTS: Of 456 patients identified, 66 (14.5%) were found to have severe OSA. African American patients had 3.7 times the odds of severe OSA compared to white patients (95% CI, 1.2-10.8). Patients aged 2 to 3 years had 2.2 times the odds of severe OSA compared to patients aged 4 to 6 years (95% CI, 1.2-4.0). Sex, ethnicity, language, and insurance type were not significantly associated with severity of OSA. The presence of apneic episodes and tonsil size were not found to be statistically significant. CONCLUSION: Up to 14.5% of healthy pediatric patients with sleep-disordered breathing may have severe OSA; young age and African American race are statistically significant predictors. Clinical findings, such as tonsil size and symptom severity, were not found to be statistically significant predictors.

Cutaneous Metastases: A Case Study on Clinical Care for Patients.

Cutaneous metastases (CMs) signal the spread of a primary tumor to the skin and dermis, particularly in patients with melanoma or with breast, lung, or gastrointestinal cancers. Although these lesions may present as superficial and painless, some CMs may lead to ulceration, drainage, and discomfort, causing distress to patients. Oncology nurses require knowledge about the clinical presentation of CMs, including incidence, pathophysiology, diagnostic evaluation, and complex symptomatology, as well as standard treatment and care for patients. In addition, nurses can provide psychosocial interventions to assist patients experiencing distress from CM lesions.

Research Biopsies: An Integrative Review of the Experiences of Patients With Cancer.

BACKGROUND: Research biopsies (RBs) are essential to understanding tumor biology and mechanisms of resistance and to advancing precision medicine. However, RBs have associated risks and may not benefit the patient. OBJECTIVES: The purpose of this integrative review is to summarize and synthesize the current literature on the experience, attitudes, and understanding of patients with cancer related to RBs. METHODS: Articles from January 2010 to February 2017 were retrieved via a search of MEDLINE®. Articles included reported on the willingness, perceptions, understanding, attitudes, and/or experience of patients with cancer related to RBs. FINDINGS: Nine of 216 identified studies were selected. Studies exploring patient willingness to undergo RBs (n = 6) identified RBs as a potential barrier to clinical trial participation. Studies exploring patient understanding and informed consent (n = 3) revealed variable patient knowledge of the risks and benefits of RBs.

Gain-of-function mutations in GATA6 lead to atrial fibrillation.

BACKGROUND: The genetic basis of atrial fibrillation (AF) and congenital heart disease remains incompletely understood. OBJECTIVE: We sought to determine the causative mutation in a family with AF, atrial septal defects, and ventricular septal defects. METHODS: We evaluated a pedigree with 16 family members, 1 with an atrial septal defect, 1 with a ventricular septal defect, and 3 with AF; we performed whole exome sequencing in 3 affected family members. Given that early-onset AF was prominent in the family, we then screened individuals with early-onset AF, defined as an age of onset <66 years, for mutations in GATA6. Variants were functionally characterized using reporter assays in a mammalian cell line. RESULTS: Exome sequencing in 3 affected individuals identified a conserved mutation, R585L, in the transcription factor gene GATA6. In the Massachusetts General Hospital Atrial Fibrillation (MGH AF) Study, the mean age of AF onset was 47.1 ± 10.9 years; 79% of the participants were men; and there was no evidence of structural heart disease. We identified 3 GATA6 variants (P91S, A177T, and A543G). Using wild-type and mutant GATA6 constructs driving atrial natriuretic peptide promoter reporter, we found that 3 of the 4 variants had a marked upregulation of luciferase activity (R585L: 4.1-fold, P < .0001; P91S: 2.5-fold, P = .0002; A177T; 1.7-fold, P = .03). In addition, when co-overexpressed with GATA4 and MEF2C, GATA6 variants exhibited upregulation of the alpha myosin heavy chain and atrial natriuretic peptide reporter activity. CONCLUSION: Overall, we found gain-of-function mutations in GATA6 in both a family with early-onset AF and atrioventricular septal defects as well as in a family with sporadic, early-onset AF.

Continuous Venovenous Hemodialysis Via Extracorporeal Membrane Oxygenation Pump for Treatment of Hyperammonemia Secondary to Propionic Acidemia in Monochorionic Diamniotic Twin Boys.

Late-preterm twins with propionic acidemia developed severe hyperammonemic encephalopathy at 5 days of age. Continuous venovenous hemodialysis was performed successfully for both infants via extracorporeal membrane oxygenation pump, and both rapidly improved. They were taken off continuous venovenous hemodialysis and extracorporeal membrane oxygenation and discharged with dietary therapy. At 3 years of age, neurodevelopment showed globally delayed milestones.

A qualitative study of perceptions of determinants of disease burden among young patients with type 1 diabetes and their parents in South India.

BACKGROUND: Diabetes is a leading threat to public health in India. A huge prevalence of type 1 diabetes among young patients is documented in literature; India is one of the countries with the highest number of new cases per year (10,900), of which 3-4 million face poverty along with diabetes. OBJECTIVE: The aim of this study was to explore the perceptions of determinants of the disease burden among young patients with type 1 diabetes and their parents. METHODS: In June 2014, perceptions were collected from eleven young patients and five available parents using the critical incident technique. RESULTS: Disease burdens associated with the management of type 1 diabetes can vary significantly according to the different social and economic determinants facing each household. Determinants associated with good practices in the management of childhood type 1 diabetes included socioeconomic status, unawareness of health complications, and beliefs about nutrition. Coping strategies applied via lifestyle changes included monitoring glycemia and regular checkups by diabetologists. A general lack of awareness about type 1 diabetes in Indian society, stigmatization, and limited access and systematic barriers to the delivery of optimal health care were all perceived to be factors hindering the successful management of chronic type 1 diabetes by young patients and their parents. CONCLUSION: Stigmatization, a lack of therapeutic adherence, and the financial strains placed on families, particularly on poor ones, are critical. More emphasis must be put on the prevention of acute and long-term complications and education.

Kidney transplantation in children with decreased left ventricular systolic function: a Midwest Pediatric Nephrology Consortium study.

BACKGROUND: Left ventricular (LV) systolic dysfunction is a relatively uncommon but serious complication of pediatric chronic kidney disease, and may be related to uremia and uncontrolled hypertension. There is limited information on the strategy for managing these children. In some cases, combined heart-kidney transplant may be considered or kidney transplant delayed until cardiac function improves. It is unknown whether these patients are at increased risk for poor outcomes after kidney transplantation. METHODS: We conducted a retrospective, multicenter study on the outcomes of children with severe and symptomatic cardiomyopathy who underwent kidney transplantation. RESULTS: Eleven patients receiving maintenance dialysis with systolic dysfunction underwent kidney transplantation without simultaneous heart transplant. Nine patients had congestive heart failure in the pre-transplant period. There were no identified complications post-transplant related to the underlying cardiac dysfunction. LV systolic function normalized in all patients and the mean shortening fraction increased from 19.0 ± 4.6 % to 32.0 ± 4.4 % (p < 0.0001). CONCLUSIONS: Kidney transplantation should be considered for children receiving maintenance dialysis with severe LV dysfunction.

HLA-DQA1 and PLCG2 Are Candidate Risk Loci for Childhood-Onset Steroid-Sensitive Nephrotic Syndrome.

Steroid-sensitive nephrotic syndrome (SSNS) accounts for >80% of cases of nephrotic syndrome in childhood. However, the etiology and pathogenesis of SSNS remain obscure. Hypothesizing that coding variation may underlie SSNS risk, we conducted an exome array association study of SSNS. We enrolled a discovery set of 363 persons (214 South Asian children with SSNS and 149 controls) and genotyped them using the Illumina HumanExome Beadchip. Four common single nucleotide polymorphisms (SNPs) in HLA-DQA1 and HLA-DQB1 (rs1129740, rs9273349, rs1071630, and rs1140343) were significantly associated with SSNS at or near the Bonferroni-adjusted P value for the number of single variants that were tested (odds ratio, 2.11; 95% confidence interval, 1.56 to 2.86; P=1.68×10(-6) (Fisher exact test). Two of these SNPs-the missense variants C34Y (rs1129740) and F41S (rs1071630) in HLA-DQA1-were replicated in an independent cohort of children of white European ancestry with SSNS (100 cases and ≤589 controls; P=1.42×10(-17)). In the rare variant gene set-based analysis, the best signal was found in PLCG2 (P=7.825×10(-5)). In conclusion, this exome array study identified HLA-DQA1 and PLCG2 missense coding variants as candidate loci for SSNS. The finding of a MHC class II locus underlying SSNS risk suggests a major role for immune response in the pathogenesis of SSNS.

Understanding medical care of transplant recipients through integrated registry and pharmacy claims data.

BACKGROUND: Limited data exist on medication use aside from immunosuppression among large samples of kidney transplant recipients. METHODS: We examined a novel database wherein Organ Procurement and Transplantation Network (OPTN) registry data were linked to records from a US pharmaceutical claims clearinghouse (2005-2010 claims) to examine pharmaceutical care at the first transplant anniversary (n = 16,157). We quantified the use of the following medication types within ±60 days of the first-year OPTN report according to estimated glomerular filtration rate (eGFR): antihypertensives, lipid-lowering, bone and mineral, and anemia treatments. Adjusted associations of medication use with eGFR and other clinical factors were quantified by multivariate logistic regression. RESULTS: Requirements for multiple antihypertensive agents rose with lower eGFR, with ß-blockers comprising the most commonly used antihypertensive agent. The adjusted likelihood of vitamin D (adjusted odds ratio (aOR) 2.07, 95% CI 1.19-3.59) and especially erythrocyte-stimulating agents (aOR 19.94, 95% CI 7.01-56.00) rose in a graded manner to peak with eGFR <15 versus >90, whereas statin use was most common with eGFR 30-59 ml/min/1.73 m(2). Black race was independently associated with increased use of all classes of antihypertensives and vitamin D, but lower adjusted statin use. Rapamycin-based immunosuppression was associated with increased use of statins and erythrocyte-stimulating agents. CONCLUSIONS: Integrated registry and pharmacy fill data provide a novel tool for pharmacoepidemiologic investigations of delivered post-transplant care. Transplant recipients with reduced renal function have increased requirements for pharmaceutical care of comorbidities. Causes of racial variation in medication fills warrant further investigation.

Focal segmental glomerulosclerosis in children: multivariate analysis indicates that donor type does not alter recurrence risk.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS), the second leading cause of end stage renal disease in children, appears to be increasing. Moreover, posttransplantation FSGS recurrence is a major problem, and there is concern that children receiving kidneys from living donors (LD) have increased recurrence risk. METHODS: Data from the United Network for Organ Sharing from 1988 to 2008 were analyzed for number of de novo transplant recipients with a primary diagnosis of FSGS in children 1 to 20 years of age. Poisson regression was used for trend analysis. Univariate and multivariable logistic regression analyses were performed to examine the association of gender, race, human leukocyte antigen matching, age, and donor type with recurrence. RESULTS: Trend analysis of kidney transplantations for FSGS in children (n=2157) showed an increase in cases of 5.8% per year or 209% over 20 years (P<0.0001). Recurrence was reported in 327 (15%) cases overall, with a preponderance for white recipients (P<0.001) in younger age subgroups (P<0.01). Donor type was significant (P=0.02), with recurrence reported in 17% versus 14% of recipients of kidneys from LDs versus deceased donors. Using multivariate analysis, recipients' young age (P=0.02) and white race (P<0.001) were identified as significant risk factors for recurrence, whereas receiving a LD kidney became insignificant. CONCLUSIONS: FSGS as a cause of pediatric end-stage renal disease leading to transplantation is on the rise. FSGS recurrence is highest in young, white children, whereas receiving a LD kidney is not independently associated with increased risk of recurrence.

Scale-up of mammalian cell culture using a new multilayered flask.

A growing number of cell-based applications require large numbers of cells. Usage of single layer T-flasks, that are adequate during small-scale expansion, may become cumbersome, laborious and time-consuming when large numbers of cells are required. To address this need, the performance of a new multi-layered cell culture vessel to facilitate easy scale up of cells from single layered T-flasks will be discussed. The flasks tested are available in 3- and 5-layer format and enable culture and complete recovery of three and five times the number of cells respectively, compared to T-175 flasks. A key feature of the BD Multi-Flask is a mix/equilibration port that allows rapid in-vessel mixing as well as uniform distribution of cells and reagents within and between layers of each vessel and consistently produce cells that can be cultured in an environment that is congruent to T-175 flasks. The design of these Multi-Flasks also allows for convenient pipette access for adding reagents and cells directly into the flasks as well as efficient recovery of valuable cells and reagents and reduces risk of contamination due to pouring. For applications where pouring is preferred over pipetting, the design allows for minimal residual liquid retention so as to reduce wastage of valuable cells and reagents.