Comprehensive Anaemia Programme and Personalized Therapies (CAPPT): protocol for a cluster-randomised controlled trial testing the effect women's groups, home counselling and iron supplementation on haemoglobin in pregnancy in southern Nepal.

BACKGROUND: Anaemia in pregnancy remains prevalent in Nepal and causes severe adverse health outcomes. METHODS: This non-blinded cluster-randomised controlled trial in the plains of Nepal has two study arms: (1) Control: routine antenatal care (ANC); (2) Home visiting, iron supplementation, Participatory Learning and Action (PLA) groups, plus routine ANC. Participants, including women in 54 non-contiguous clusters (mean 2582; range 1299-4865 population) in Southern Kapilbastu district, are eligible if they consent to menstrual monitoring, are resident, married, aged 13-49 years and able to respond to questions. After 1-2 missed menses and a positive pregnancy test, consenting women < 20 weeks' gestation, who plan to reside locally for most of the pregnancy, enrol into trial follow-up. Interventions comprise two home-counselling visits (at 12-21 and 22-26 weeks' gestation) with iron folic acid (IFA) supplement dosage tailored to women's haemoglobin concentration, plus monthly PLA women's group meetings using a dialogical problem-solving approach to engage pregnant women and their families. Home visits and PLA meetings will be facilitated by auxiliary nurse midwives. The hypothesis is as follows: Haemoglobin of women at 30 ± 2 weeks' gestation is ≥ 0.4 g/dL higher in the intervention arm than in the control. A sample of 842 women (421 per arm, average 15.6 per cluster) will provide 88% power, assuming SD 1.2, ICC 0.09 and CV of cluster size 0.27. Outcomes are captured at 30 ± 2 weeks gestation. Primary outcome is haemoglobin concentration (g/dL). Secondary outcomes are as follows: anaemia prevalence (%), mid-upper arm circumference (cm), mean probability of micronutrient adequacy (MPA) and number of ANC visits at a health facility. Indicators to assess pathways to impact include number of IFA tablets consumed during pregnancy, intake of energy (kcal/day) and dietary iron (mg/day), a score of bioavailability-enhancing behaviours and recall of one nutrition knowledge indicator. Costs and cost-effectiveness of the intervention will be estimated from a provider perspective. Using constrained randomisation, we allocated clusters to study arms, ensuring similarity with respect to cluster size, ethnicity, religion and distance to a health facility. Analysis is by intention-to-treat at the individual level, using mixed-effects regression. DISCUSSION: Findings will inform Nepal government policy on approaches to increase adherence to IFA, improve diets and reduce anaemia in pregnancy. TRIAL REGISTRATION: ISRCTN 12272130 .

Participatory Women's Groups with Cash Transfers Can Increase Dietary Diversity and Micronutrient Adequacy during Pregnancy, whereas Women's Groups with Food Transfers Can Increase Equity in Intrahousehold Energy Allocation.

Background: There is scarce evidence on the impacts of food transfers, cash transfers, or women's groups on food sharing, dietary intakes, or nutrition during pregnancy, when nutritional needs are elevated. Objective: This study measured the effects of 3 pregnancy-focused nutrition interventions on intrahousehold food allocation, dietary adequacy, and maternal nutritional status in Nepal. Methods: Interventions tested in a cluster-randomized controlled trial (ISRCTN 75964374) were "Participatory Learning and Action" (PLA) monthly women's groups, PLA with transfers of 10 kg fortified flour ("Super Cereal"), and PLA plus transfers of 750 Nepalese rupees (∼US$7.5) to pregnant women. Control clusters received usual government services. Primary outcomes were Relative Dietary Energy Adequacy Ratios (RDEARs) between pregnant women and male household heads and pregnant women and their mothers-in-law. Diets were measured by repeated 24-h dietary recalls. Results: Relative to control, RDEARs between pregnant women and their mothers-in-law were 12% higher in the PLA plus food arm (log-RDEAR coefficient = 0.12; 95% CI: 0.02, 0.21; P = 0.014), but 10% lower in the PLA-only arm between pregnant women and male household heads (-0.11; 95% CI: -0.19, -0.02; P = 0.020). In all interventions, pregnant women's energy intakes did not improve, but odds of pregnant women consuming iron-folate supplements were 2.5-4.6 times higher, odds of pregnant women consuming more animal-source foods than the household head were 1.7-2.4 times higher, and midupper arm circumference was higher relative to control. Dietary diversity was 0.4 food groups higher in the PLA plus cash arm than in the control arm. Conclusions: All interventions improved maternal diets and nutritional status in pregnancy. PLA women's groups with food transfers increased equity in energy allocation, whereas PLA with cash improved dietary diversity. PLA alone improved diets, but effects were mixed. Scale-up of these interventions in marginalized populations is a policy option, but researchers should find ways to increase adherence to interventions. This trial was registered at www.controlled-trials.com as ISRCTN 75964374.

Impact on birth weight and child growth of Participatory Learning and Action women's groups with and without transfers of food or cash during pregnancy: Findings of the low birth weight South Asia cluster-randomised controlled trial (LBWSAT) in Nepal.

BACKGROUND: Undernutrition during pregnancy leads to low birthweight, poor growth and inter-generational undernutrition. We did a non-blinded cluster-randomised controlled trial in the plains districts of Dhanusha and Mahottari, Nepal to assess the impact on birthweight and weight-for-age z-scores among children aged 0-16 months of community-based participatory learning and action (PLA) women's groups, with and without food or cash transfers to pregnant women. METHODS: We randomly allocated 20 clusters per arm to four arms (average population/cluster = 6150). All consenting married women aged 10-49 years, who had not had tubal ligation and whose husbands had not had vasectomy, were monitored for missed menses. Between 29 Dec 2013 and 28 Feb 2015 we recruited 25,092 pregnant women to surveillance and interventions: PLA alone (n = 5626); PLA plus food (10 kg/month of fortified wheat-soya 'Super Cereal', n = 6884); PLA plus cash (NPR750≈US$7.5/month, n = 7272); control (existing government programmes, n = 5310). 539 PLA groups discussed and implemented strategies to improve low birthweight, nutrition in pregnancy and hand washing. Primary outcomes were birthweight within 72 hours of delivery and weight-for-age z-scores at endline (age 0-16 months). Only children born to permanent residents between 4 June 2014 and 20 June 2015 were eligible for intention to treat analyses (n = 10936), while in-migrating women and children born before interventions had been running for 16 weeks were excluded. Trial status: completed. RESULTS: In PLA plus food/cash arms, 94-97% of pregnant women attended groups and received a mean of four transfers over their pregnancies. In the PLA only arm, 49% of pregnant women attended groups. Due to unrest, the response rate for birthweight was low at 22% (n = 2087), but response rate for endline nutritional and dietary measures exceeded 83% (n = 9242). Compared to the control arm (n = 464), mean birthweight was significantly higher in the PLA plus food arm by 78·0 g (95% CI 13·9, 142·0; n = 626) and not significantly higher in PLA only and PLA plus cash arms by 28·9 g (95% CI -37·7, 95·4; n = 488) and 50·5 g (95% CI -15·0, 116·1; n = 509) respectively. Mean weight-for-age z-scores of children aged 0-16 months (average age 9 months) sampled cross-sectionally at endpoint, were not significantly different from those in the control arm (n = 2091). Differences in weight for-age z-score were as follows: PLA only -0·026 (95% CI -0·117, 0·065; n = 2095); PLA plus cash -0·045 (95% CI -0·133, 0·044; n = 2545); PLA plus food -0·033 (95% CI -0·121, 0·056; n = 2507). Amongst many secondary outcomes tested, compared with control, more institutional deliveries (OR: 1.46 95% CI 1.03, 2.06; n = 2651) and less colostrum discarding (OR:0.71 95% CI 0.54, 0.93; n = 2548) were found in the PLA plus food arm but not in PLA alone or in PLA plus cash arms. INTERPRETATION: Food supplements in pregnancy with PLA women's groups increased birthweight more than PLA plus cash or PLA alone but differences were not sustained. Nutrition interventions throughout the thousand-day period are recommended. TRIAL REGISTRATION: ISRCTN75964374.

Status and determinants of intra-household food allocation in rural Nepal.

BACKGROUND/OBJECTIVES: Understanding of the patterns and predictors of intra-household food allocation could enable nutrition programmes to better target nutritionally vulnerable individuals. This study aims to characterise the status and determinants of intra-household food and nutrient allocation in Nepal. SUBJECTS/METHODS: Pregnant women, their mothers-in-law and male household heads from Dhanusha and Mahottari districts in Nepal responded to 24-h dietary recalls, thrice repeated on non-consecutive days (n = 150 households; 1278 individual recalls). Intra-household inequity was measured using ratios between household members in food intakes (food shares); food-energy intake proportions ('food shares-to-energy shares', FS:ES); calorie-requirement proportions ('relative dietary energy adequacy ratios', RDEARs) and mean probability of adequacy for 11 micronutrients (MPA ratios). Hypothesised determinants were collected during the recalls, and their associations with the outcomes were tested using multivariable mixed-effects linear regression models. RESULTS: Women's diets (pregnant women and mothers-in-law) consisted of larger FS:ES of starchy foods, pulses, fruits and vegetables than male household heads, whereas men had larger FS:ES of animal-source foods. Pregnant women had the lowest MPA (37%) followed by their mothers-in-law (52%), and male household heads (57%). RDEARs between pregnant women and household heads were 31% higher (log-RDEAR coeff=0.27 (95% CI 0.12, 0.42), P < 0.001) when pregnant women earned more or the same as their spouse, and log-MPA ratios between pregnant women and mothers-in-law were positively associated with household-level calorie intakes (coeff=0.43 (0.23, 0.63), P < 0.001, per 1000 kcal). CONCLUSIONS: Pregnant women receive inequitably lower shares of food and nutrients, but this could be improved by increasing pregnant women's cash earnings and household food security.

Smartphone tool to collect repeated 24 h dietary recall data in Nepal.

OBJECTIVE: To outline the development of a smartphone-based tool to collect thrice-repeated 24 h dietary recall data in rural Nepal, and to describe energy intakes, common errors and researchers' experiences using the tool. DESIGN: We designed a novel tool to collect multi-pass 24 h dietary recalls in rural Nepal by combining the use of a CommCare questionnaire on smartphones, a paper form, a QR (quick response)-coded list of foods and a photographic atlas of portion sizes. Twenty interviewers collected dietary data on three non-consecutive days per respondent, with three respondents per household. Intakes were converted into nutrients using databases on nutritional composition of foods, recipes and portion sizes. SETTING: Dhanusha and Mahottari districts, Nepal. SUBJECTS: Pregnant women, their mothers-in-law and male household heads. Energy intakes assessed in 150 households; data corrections and our experiences reported from 805 households and 6765 individual recalls. RESULTS: Dietary intake estimates gave plausible values, with male household heads appearing to have higher energy intakes (median (25th-75th centile): 12 079 (9293-14 108) kJ/d) than female members (8979 (7234-11 042) kJ/d for pregnant women). Manual editing of data was required when interviewers mistook portions for food codes and for coding items not on the food list. Smartphones enabled quick monitoring of data and interviewer performance, but we initially faced technical challenges with CommCare forms crashing. CONCLUSIONS: With sufficient time dedicated to development and pre-testing, this novel smartphone-based tool provides a useful method to collect data. Future work is needed to further validate this tool and adapt it for other contexts.

Experiences in running a complex electronic data capture system using mobile phones in a large-scale population trial in southern Nepal.

The increasing availability and capabilities of mobile phones make them a feasible means of data collection. Electronic Data Capture (EDC) systems have been used widely for public health monitoring and surveillance activities, but documentation of their use in complicated research studies requiring multiple systems is limited. This paper shares our experiences of designing and implementing a complex multi-component EDC system for a community-based four-armed cluster-Randomised Controlled Trial in the rural plains of Nepal, to help other researchers planning to use EDC for complex studies in low-income settings. We designed and implemented three interrelated mobile phone data collection systems to enrol and follow-up pregnant women (trial participants), and to support the implementation of trial interventions (women's groups, food and cash transfers). 720 field staff used basic phones to send simple coded text messages, 539 women's group facilitators used Android smartphones with Open Data Kit Collect, and 112 Interviewers, Coordinators and Supervisors used smartphones with CommCare. Barcoded photo ID cards encoded with participant information were generated for each enrolled woman. Automated systems were developed to download, recode and merge data for nearly real-time access by researchers. The systems were successfully rolled out and used by 1371 staff. A total of 25,089 pregnant women were enrolled, and 17,839 follow-up forms completed. Women's group facilitators recorded 5717 women's groups and the distribution of 14,647 food and 13,482 cash transfers. Using EDC sped up data collection and processing, although time needed for programming and set-up delayed the study inception. EDC using three interlinked mobile data management systems (FrontlineSMS, ODK and CommCare) was a feasible and effective method of data capture in a complex large-scale trial in the plains of Nepal. Despite challenges including prolonged set-up times, the systems met multiple data collection needs for users with varying levels of literacy and experience.

Protocol of the Low Birth Weight South Asia Trial (LBWSAT), a cluster-randomised controlled trial testing impact on birth weight and infant nutrition of Participatory Learning and Action through women's groups, with and without unconditional transfers of fortified food or cash during pregnancy in Nepal.

BACKGROUND: Low birth weight (LBW, < 2500 g) affects one third of newborn infants in rural south Asia and compromises child survival, infant growth, educational performance and economic prospects. We aimed to assess the impact on birth weight and weight-for-age Z-score in children aged 0-16 months of a nutrition Participatory Learning and Action behaviour change strategy (PLA) for pregnant women through women's groups, with or without unconditional transfers of food or cash to pregnant women in two districts of southern Nepal. METHODS: The study is a cluster randomised controlled trial (non-blinded). PLA comprises women's groups that discuss, and form strategies about, nutrition in pregnancy, low birth weight and hygiene. Women receive up to 7 monthly transfers per pregnancy: cash is NPR 750 (~US$7) and food is 10 kg of fortified sweetened wheat-soya Super Cereal per month. The unit of randomisation is a rural village development committee (VDC) cluster (population 4000-9200, mean 6150) in southern Dhanusha or Mahottari districts. 80 VDCs are randomised to four arms using a participatory 'tombola' method. Twenty clusters each receive: PLA; PLA plus food; PLA plus cash; and standard care (control). Participants are (mostly Maithili-speaking) pregnant women identified from 8 weeks' gestation onwards, and their infants (target sample size 8880 birth weights). After pregnancy verification, mothers may be followed up in early and late pregnancy, within 72 h, after 42 days and within 22 months of birth. Outcomes pertain to the individual level. Primary outcomes include birth weight within 72 h of birth and infant weight-for-age Z-score measured cross-sectionally on children born of the study. Secondary outcomes include prevalence of LBW, eating behaviour and weight during pregnancy, maternal and newborn illness, preterm delivery, miscarriage, stillbirth or neonatal mortality, infant Z-scores for length-for-age and weight-for-length, head circumference, and postnatal maternal BMI and mid-upper arm circumference. Exposure to women's groups, food or cash transfers, home visits, and group interventions are measured. DISCUSSION: Determining the relative importance to birth weight and early childhood nutrition of adding food or cash transfers to PLA women's groups will inform design of nutrition interventions in pregnancy. TRIAL REGISTRATION: ISRCTN75964374 , 12 Jul 2013.

Microbial Fe(III) oxide reduction potential in Chocolate Pots hot spring, Yellowstone National Park.

Chocolate Pots hot springs (CP) is a unique, circumneutral pH, iron-rich, geothermal feature in Yellowstone National Park. Prior research at CP has focused on photosynthetically driven Fe(II) oxidation as a model for mineralization of microbial mats and deposition of Archean banded iron formations. However, geochemical and stable Fe isotopic data have suggested that dissimilatory microbial iron reduction (DIR) may be active within CP deposits. In this study, the potential for microbial reduction of native CP Fe(III) oxides was investigated, using a combination of cultivation dependent and independent approaches, to assess the potential involvement of DIR in Fe redox cycling and associated stable Fe isotope fractionation in the CP hot springs. Endogenous microbial communities were able to reduce native CP Fe(III) oxides, as documented by most probable number enumerations and enrichment culture studies. Enrichment cultures demonstrated sustained DIR driven by oxidation of acetate, lactate, and H2 . Inhibitor studies and molecular analyses indicate that sulfate reduction did not contribute to observed rates of DIR in the enrichment cultures through abiotic reaction pathways. Enrichment cultures produced isotopically light Fe(II) during DIR relative to the bulk solid-phase Fe(III) oxides. Pyrosequencing of 16S rRNA genes from enrichment cultures showed dominant sequences closely affiliated with Geobacter metallireducens, a mesophilic Fe(III) oxide reducer. Shotgun metagenomic analysis of enrichment cultures confirmed the presence of a dominant G. metallireducens-like population and other less dominant populations from the phylum Ignavibacteriae, which appear to be capable of DIR. Gene (protein) searches revealed the presence of heat-shock proteins that may be involved in increased thermotolerance in the organisms present in the enrichments as well as porin-cytochrome complexes previously shown to be involved in extracellular electron transport. This analysis offers the first detailed insight into how DIR may impact the Fe geochemistry and isotope composition of a Fe-rich, circumneutral pH geothermal environment.

In vivo selection of autologous MGMT gene-modified cells following reduced-intensity conditioning with BCNU and temozolomide in the dog model.

Chemotherapy with 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide (TMZ) is commonly used for the treatment of glioblastoma multiforme (GBM) and other cancers. In preparation for a clinical gene therapy study in patients with glioblastoma, we wished to study whether these reagents could be used as a reduced-intensity conditioning regimen for autologous transplantation of gene-modified cells. We used an MGMT(P140K)-expressing lentivirus vector to modify dog CD34(+) cells and tested in four dogs whether these autologous cells engraft and provide chemoprotection after transplantation. Treatment with O(6)-benzylguanine (O6BG)/TMZ after transplantation resulted in gene marking levels up to 75%, without significant hematopoietic cytopenia, which is consistent with hematopoietic chemoprotection. Retrovirus integration analysis showed that multiple clones contribute to hematopoiesis. These studies demonstrate the ability to achieve stable engraftment of MGMT(P140K)-modified autologous hematopoietic stem cells (HSCs) after a novel reduced-intensity conditioning protocol using a combination of BCNU and TMZ. Furthermore, we show that MGMT(P140K)-HSC engraftment provides chemoprotection during TMZ dose escalation. Clinically, chemoconditioning with BCNU and TMZ should facilitate engraftment of MGMT(P140K)-modified cells while providing antitumor activity for patients with poor prognosis glioblastoma or alkylating agent-sensitive tumors, thereby supporting dose-intensified chemotherapy regimens.

Laser ablation-miniature mass spectrometer for elemental and isotopic analysis of rocks.

A laser ablation-miniature mass spectrometer (LA-MMS) for the chemical and isotopic measurement of rocks and minerals is described. In the LA-MMS method, neutral atoms ablated by a pulsed laser are led into an electron impact ionization source, where they are ionized by a 70 eV electron beam. This results in a secondary ion pulse typically 10-100 µs wide, compared to the original 5-10 ns laser pulse duration. Ions of different masses are then spatially dispersed along the focal plane of the magnetic sector of the miniature mass spectrometer (MMS) and measured in parallel by a modified CCD array detector capable of detecting ions directly. Compared to conventional scanning techniques, simultaneous measurement of the ion pulse along the focal plane effectively offers a 100% duty cycle over a wide mass range. LA-MMS offers a more quantitative assessment of elemental composition than techniques that detect ions directly generated by the ablation process because the latter can be strongly influenced by matrix effects that vary with the structure and geometry of the surface, the wavelength of the laser beam, and the not well characterized ionization efficiencies of the elements in the process. The above problems attendant to the direct ion analysis has been minimized in the LA-MMS by analyzing the ablated neutral species after their post-ionization by electron impaction. These neutral species are much more abundant than the directly ablated ions in the ablated vapor plume and are, therefore, expected to be characteristic of the chemical composition of the solid. Also, the electron impact ionization of elements is well studied and their ionization cross sections are known and easy to find in databases. Currently, the LA-MMS limit of detection is 0.4 wt.%. Here we describe LA-MMS elemental composition measurements of various minerals including microcline, lepidolite, anorthoclase, and USGS BCR-2G samples. The measurements of high precision isotopic ratios including (41)K/(39)K (0.077 ± 0.004) and (29)Si/(28)Si (0.052 ± 0.006) in these minerals by LA-MMS are also described. The LA-MMS has been developed as a prototype instrument system for space applications for geochemical and geochronological measurements on the surface of extraterrestrial bodies.

Iron isotope fractionation during microbial dissimilatory iron oxide reduction in simulated Archaean seawater.

The largest Fe isotope excursion yet measured in marine sedimentary rocks occurs in shales, carbonates, and banded iron formations of Neoarchaean and Paleoproterozoic age. The results of field and laboratory studies suggest a potential role for microbial dissimilatory iron reduction (DIR) in producing this excursion. However, most experimental studies of Fe isotope fractionation during DIR have been conducted in simple geochemical systems, using pure Fe(III) oxide substrates that are not direct analogues to phases likely to have been present in Precambrian marine environments. In this study, Fe isotope fractionation was investigated during microbial reduction of an amorphous Fe(III) oxide-silica coprecipitate in anoxic, high-silica, low-sulphate artificial Archaean seawater at 30 °C to determine if such conditions alter the extent of reduction or isotopic fractionations relative to those observed in simple systems. The Fe(III)-Si coprecipitate was highly reducible (c. 80% reduction) in the presence of excess acetate. The coprecipitate did not undergo phase conversion (e.g. to green rust, magnetite or siderite) during reduction. Iron isotope fractionations suggest that rapid and near-complete isotope exchange took place among all Fe(II) and Fe(III) components, in contrast to previous work on goethite and hematite, where exchange was limited to the outer few atom layers of the substrate. Large quantities of low-δ(56)Fe Fe(II) (aqueous and solid phase) were produced during reduction of the Fe(III)-Si coprecipitate. These findings shed new light on DIR as a mechanism for producing Fe isotope variations observed in Neoarchaean and Paleoproterozoic marine sedimentary rocks.

Evidence for equilibrium iron isotope fractionation by nitrate-reducing iron(II)-oxidizing bacteria.

Iron isotope fractionations produced during chemical and biological Fe(II) oxidation are sensitive to the proportions and nature of dissolved and solid-phase Fe species present, as well as the extent of isotopic exchange between precipitates and aqueous Fe. Iron isotopes therefore potentially constrain the mechanisms and pathways of Fe redox transformations in modern and ancient environments. In the present study, we followed in batch experiments Fe isotope fractionations between Fe(II)(aq) and Fe(III) oxide/hydroxide precipitates produced by the Fe(III) mineral encrusting, nitrate-reducing, Fe(II)-oxidizing Acidovorax sp. strain BoFeN1. Isotopic fractionation in (56)Fe/(54)Fe approached that expected for equilibrium conditions, assuming an equilibrium Δ(56)Fe(Fe(OH)3 - Fe(II)aq) fractionation factor of +3.0 ‰. Previous studies have shown that Fe(II) oxidation by this Acidovorax strain occurs in the periplasm, and we propose that Fe isotope equilibrium is maintained through redox cycling via coupled electron and atom exchange between Fe(II)(aq) and Fe(III) precipitates in the contained environment of the periplasm. In addition to the apparent equilibrium isotopic fractionation, these experiments also record the kinetic effects of initial rapid oxidation, and possible phase transformations of the Fe(III) precipitates. Attainment of Fe isotope equilibrium between Fe(III) oxide/hydroxide precipitates and Fe(II)(aq) by neutrophilic, Fe(II)-oxidizing bacteria or through abiologic Fe(II)(aq) oxidation is generally not expected or observed, because the poor solubility of their metabolic product, i.e. Fe(III), usually leads to rapid precipitation of Fe(III) minerals, and hence expression of a kinetic fractionation upon precipitation; in the absence of redox cycling between Fe(II)(aq) and precipitate, kinetic isotope fractionations are likely to be retained. These results highlight the distinct Fe isotope fractionations that are produced by different pathways of biological and abiological Fe(II) oxidation.

Preclinical correction of human Fanconi anemia complementation group A bone marrow cells using a safety-modified lentiviral vector.

One of the major hurdles for the development of gene therapy for Fanconi anemia (FA) is the increased sensitivity of FA stem cells to free radical-induced DNA damage during ex vivo culture and manipulation. To minimize this damage, we have developed a brief transduction procedure for lentivirus vector-mediated transduction of hematopoietic progenitor cells from patients with Fanconi anemia complementation group A (FANCA). The lentiviral vector FancA-sW contains the phosphoglycerate kinase promoter, the FANCA cDNA, and a synthetic, safety-modified woodchuck post transcriptional regulatory element (sW). Bone marrow mononuclear cells or purified CD34(+) cells from patients with FANCA were transduced in an overnight culture on recombinant fibronectin peptide CH-296, in low (5%) oxygen, with the reducing agent, N-acetyl-L-cysteine (NAC), and a combination of growth factors, granulocyte colony-stimulating factor (G-CSF), Flt3 ligand, stem cell factor, and thrombopoietin. Transduced cells plated in methylcellulose in hypoxia with NAC showed increased colony formation compared with 21% oxygen without NAC (P<0.03), showed increased resistance to mitomycin C compared with green fluorescent protein (GFP) vector-transduced controls (P<0.007), and increased survival. Thus, combining short transduction and reducing oxidative stress may enhance the viability and engraftment of gene-corrected cells in patients with FANCA.

A younger age for ALH84001 and its geochemical link to shergottite sources in Mars.

Martian meteorite ALH84001 (ALH) is the oldest known igneous rock from Mars and has been used to constrain its early history. Lutetium-hafnium (Lu-Hf) isotope data for ALH indicate an igneous age of 4.091 +/- 0.030 billion years, nearly coeval with an interval of heavy bombardment and cessation of the martian core dynamo and magnetic field. The calculated Lu/Hf and Sm/Nd (samarium/neodymium) ratios of the ALH parental magma source indicate that it must have undergone extensive igneous processing associated with the crystallization of a deep magma ocean. This same mantle source region also produced the shergottite magmas (dated 150 to 570 million years ago), possibly indicating uniform igneous processes in Mars for nearly 4 billion years.

Microbial production of isotopically light iron(II) in a modern chemically precipitated sediment and implications for isotopic variations in ancient rocks.

The inventories and Fe isotope composition of aqueous Fe(II) and solid-phase Fe compounds were quantified in neutral-pH, chemically precipitated sediments downstream of the Iron Mountain acid mine drainage site in northern California, USA. The sediments contain high concentrations of amorphous Fe(III) oxyhydroxides [Fe(III)(am)] that allow dissimilatory iron reduction (DIR) to predominate over Fe-S interactions in Fe redox transformation, as indicated by the very low abundance of Cr(II)-extractable reduced inorganic sulfur compared with dilute HCl-extractable Fe. delta(56)Fe values for bulk HCl- and HF-extractable Fe were approximately 0. These near-zero bulk delta(56)Fe values, together with the very low abundance of dissolved Fe in the overlying water column, suggest that the pyrite Fe source had near-zero delta(56)Fe values, and that complete oxidation of Fe(II) took place prior to deposition of the Fe(III) oxide-rich sediment. Sediment core analyses and incubation experiments demonstrated the production of millimolar quantities of isotopically light (delta(56)Fe approximately -1.5 to -0.5 per thousand) aqueous Fe(II) coupled to partial reduction of Fe(III)(am) by DIR. Trends in the Fe isotope composition of solid-associated Fe(II) and residual Fe(III)(am) are consistent with experiments with synthetic Fe(III) oxides, and collectively suggest an equilibrium Fe isotope fractionation between aqueous Fe(II) and Fe(III)(am) of approximately -2 per thousand. These Fe(III) oxide-rich sediments provide a model for early diagenetic processes that are likely to have taken place in Archean and Paleoproterozoic marine sediments that served as precursors for banded iron formations. Our results suggest pathways whereby DIR could have led to the formation of large quantities of low-delta(56)Fe minerals during BIF genesis.

Myeloablative irradiation in non-human primates.

PURPOSE: We used total body irradiation (TBI) as conditioning for cord blood transplantation studies in pigtailed macaques. In these studies, different doses of TBI were explored to obtain optimal myelosuppression with acceptable radiation-related side effects. METHODS: Four macaques received TBI ranging from 800 to 1320 cGy, followed by standard post-transplant care. Hematopoietic recovery was monitored by CBC and donor contribution by variable number of tandem repeats analysis. RESULTS: Animals receiving 800 or 1020 cGy TBI tolerated the irradiation well with autologous recovery of neutrophils within 24 days. At a dose of 1200 cGy, neither autologous recovery nor extramedullary toxicity was observed. A fourth animal received 1320 cGy of TBI and suffered significant toxicity necessitating termination of the study. CONCLUSIONS: We conclude that previously considered myeloablative doses of TBI allowed for autologous recovery in the pigtailed macaque, and that a dose of 1200 cGy may be most appropriate, providing both myeloablation and acceptable non-hematopoietic toxicities.

Precise dielectric property measurements and E-field probe calibration for specific absorption rate measurements using a rectangular waveguide.

Specific absorption rate (SAR) measurements require accurate calculations of the dielectric properties of tissue-equivalent liquids and associated calibration of E-field probes. We developed a precise tissue-equivalent dielectric measurement and E-field probe calibration system. The system consists of a rectangular waveguide, electric field probe, and data control and acquisition system. Dielectric properties are calculated using the field attenuation factor inside the tissue-equivalent liquid and power reflectance inside the waveguide at the air/dielectric-slab interface. Calibration factors were calculated using isotropicity measurements of the E-field probe. The frequencies used are 900 MHz and 1800 MHz. The uncertainties of the measured values are within ±3%, at the 95% confidence level. Using the same waveguide for dielectric measurements as well as calibrating E-field probes used in SAR assessments eliminates a source of uncertainty. Moreover, we clearly identified the system parameters that affect the overall uncertainty of the measurement system.

NOD/SCID repopulating cells contribute only to short-term repopulation in the baboon.

We have previously compared the repopulation ability of gene-modified baboon CD34+ cells in an autologous transplantation versus a xenotransplant model in irradiated nonobese diabetic/severe combined immune deficiency (NOD/SCID) mice. Baboon CD34-selected marrow cells were transduced with a gammaretrovirus vector and infused into irradiated baboons and NOD/SCID mice. A limited integration-site analysis could only detect two common retrovirus integration sites in the NOD/SCID and monkey. Here, we performed locus-specific PCR on 30 clones recovered from NOD/SCID beta2-microglobulin mice reconstituted with transduced baboon CD34+ cells. We identified five common integrants in the baboon early after transplant (2-6 weeks) but none during the long-term follow-up (6 and 12 months). These results confirm that repopulating cells in the NOD/SCID mouse contribute only to short-term repopulation in a clinically relevant large animal model.

Thermal inactivation of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in breaded pork patties.

Decimal reduction times (D-values) and thermal resistance constants (z-values) for 3 foodborne pathogenic bacteria in formulated ready-to-eat breaded pork patties were determined with thermal inactivation studies. Meat samples, inoculated with Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes cultures or uninoculated controls, were packaged in sterile bags, immersed in circulated water bath, and held at 55, 57.5, 60, 62.5, 65, 67.5, and 70 degrees C for different durations of time. The D- and z-values were determined by using a linear regression model. Average calculated D-values for E. coli O157:H7, Salmonella, and L. monocytogenes at a temperature range of 55 to 70 degrees C were 32.11 to 0.08 min, 69.48 to 0.29 min, and 150.46 to 0.43 min, respectively. Calculated z-values for E. coli O157:H7, Salmonella, and L. monocytogenes were 5.4, 6.2, and 5.9 degrees C, respectively. The results of this study will be useful to food processors to validate thermal lethality of the studied foodborne pathogens in ready-to-eat breaded pork patties.

Thermal inactivation studies of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in ready-to-eat chicken-fried beef patties.

Thermal inactivation studies were used to determine the D- and z-values of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in ready-to-eat chicken-fried beef patties. Inoculated meat was packaged in sterile bags, which were immersed in a circulated water bath and held at 55, 57.5, 60, 62.5, 65, 67.5, and 70 degrees C for different lengths of time. D- and z-values were determined with a linear regression model. Average D-values at temperatures 55 to 70 degrees C were 27.62 to 0.04 min for E. coli 0157:H7, 67.68 to 0.22 min for Salmonella, and 81.37 to 0.31 min for L. monocytogenes. The z-values were 5.2 degrees C for E. coli O157:H7, 6.0 degrees C for Salmonella, and 6.1 degrees C for L. monocytogenes. The results of this study can be used by food processors to validate their processes and help eliminate pathogenic bacteria associated with chicken-fried beef products.