openFrame: A modular, sustainable, open microscopy platform with single-shot, dual-axis optical autofocus module providing high precision and long range of operation.

'openFrame' is a modular, low-cost, open-hardware microscopy platform that can be configured or adapted to most light microscopy techniques and is easily upgradeable or expandable to multiple modalities. The ability to freely mix and interchange both open-source and proprietary hardware components or software enables low-cost, yet research-grade instruments to be assembled and maintained. It also enables rapid prototyping of advanced or novel microscope systems. For long-term time-lapse image data acquisition, slide-scanning or high content analysis, we have developed a novel optical autofocus incorporating orthogonal cylindrical optics to provide robust single-shot closed-loop focus lock, which we have demonstrated to accommodate defocus up to ±37 µm with <200 nm accuracy, and a two-step autofocus mode which we have shown can operate with defocus up to ±68 µm. We have used this to implement automated single molecule localisation microscopy (SMLM) in a relatively low-cost openFrame-based instrument using multimode diode lasers for excitation and cooled CMOS cameras.

Potential reductions in ultra-processed food consumption substantially improve population cardiometabolic-related dietary nutrient profiles in eight countries.

BACKGROUND AND AIMS: The negative effect on dietary nutrient profiles is the most obvious mechanism explaining the higher risk of cardiometabolic diseases associated with increased dietary share of UPF observed in large cohort studies. We estimate the proportion of diets with excessive energy density, excessive free sugars or saturated fat contents and insufficient fiber that could be avoided, if UPF consumption was reduced to levels among lowest consumers across eight countries, as well as the proportion of diets with multiple inadequacies. METHODS AND RESULTS: Using nationally-representative cross-sectional surveys from Brazil (2008-09), Chile (2010), Colombia (2005), Mexico (2012), Australia (2011-12), the UK (2008-16), Canada (2015), and the US (2015-16), inadequate energy density (≥2.25 kcal/g) or contents of free sugars (>10% of total energy intake), saturated fats (>10% of total energy intake) and fiber (<25 g/2000 kcal) population attributable fractions were quantified. Substantial reductions in nutrient inadequacies would be observed ranging from 50.4% in Chile to 76.8% in US for dietary energy density, from 15.5% in Colombia to 68.4% in Australia for free sugars, from 9.5% in Canada to 35.0% in Mexico for saturated fats, and from 10.3% in UK to 37.9% in Mexico for fiber. Higher reductions would be observed for diets with multiple nutrient inadequacies: from 27.3% in UK to 77.7% in Australia for ≥3 and from 69.4% in Canada to 92.1% in US, for 4 inadequacies. CONCLUSIONS: Lowering dietary contribution of UPF to levels among country-specific lowest consumers is a way to improve population cardiometabolic-related dietary nutrient profiles.

Germline V repertoires: Origin, maintenance, diversification.

In our view, Melvin Cohn (Scand J Immunol. 2018;87:e12640) has set out the logical guidelines towards a resolution of the very real enigma of the selectability of vertebrate germline Ig V repertoires under the current evolutionary paradigm…" A somatically derived repertoire scrambles this (germline VL + VH) substrate so that its specificities are lost, making it un-selectable in the germline. Consequently, evolution faced an incompatibility." It is argued here in Reply that a reverse transcriptase-based soma-to-germline process (S->G) targeting germline V segment arrays goes some considerable way to resolving fundamental contradictions on the origin, maintenance and then real-time adaptive diversification of these limited sets of V segments encoded within various V repertoire arrays.

Consumption of ultra-processed foods predicts diet quality in Canada.

This study describes food consumption patterns in Canada according to the types of food processing using the Nova classification and investigates the association between consumption of ultra-processed foods and the nutrient profile of the diet. Dietary intakes of 33,694 individuals from the 2004 Canadian Community Health Survey aged 2 years and above were analyzed. Food and drinks were classified using Nova into unprocessed or minimally processed foods, processed culinary ingredients, processed foods and ultra-processed foods. Average consumption (total daily energy intake) and relative consumption (% of total energy intake) provided by each of the food groups were calculated. Consumption of ultra-processed foods according to sex, age, education, residential location and relative family revenue was assessed. Mean nutrient content of ultra-processed foods and non-ultra-processed foods were compared, and the average nutrient content of the overall diet across quintiles of dietary share of ultra-processed foods was measured. In 2004, 48% of calories consumed by Canadians came from ultra-processed foods. Consumption of such foods was high amongst all socioeconomic groups, and particularly in children and adolescents. As a group, ultra-processed foods were grossly nutritionally inferior to non-ultra-processed foods. After adjusting for covariates, a significant and positive relationship was found between the dietary share of ultra-processed foods and the content in carbohydrates, free sugars, total and saturated fats and energy density, while an inverse relationship was observed with the dietary content in protein, fiber, vitamins A, C, D, B6 and B12, niacin, thiamine, riboflavin, as well as zinc, iron, magnesium, calcium, phosphorus and potassium. Lowering the dietary share of ultra-processed foods and raising consumption of hand-made meals from unprocessed or minimally processed foods would substantially improve the diet quality of Canadian.

Is precarious employment associated with women remaining childless until age 35 years? Results from an Australian birth cohort study.

STUDY QUESTION: Does time in casual employment (while not studying full time) affect the likelihood of a woman having a child by age 35? SUMMARY ANSWER: Duration of time spent in casual employment is associated with an increased likelihood of childlessness at age 35 years, irrespective of socio-economic background as indicated by educational level. WHAT IS KNOWN ALREADY: Precarious employment conditions have become increasingly prevalent in recent decades in Western countries. The relationship between precarious employment conditions and age at first childbirth has been examined in several European countries with varying results. STUDY DESIGN, SIZE, DURATION: A retrospective cross-sectional component (n = 663) was added to an existing study based on a cohort of women born during 1973-1975. An event history calendar instrument was used to obtain data regarding a range of life domains over a 20-year period. PARTICIPANTS/MATERIALS, SETTING, METHODS: Using data from the Life Journeys of Young Women Project carried out in Adelaide, South Australia, Cox proportional hazards models were applied to investigate the research questions. MAIN RESULTS AND THE ROLE OF CHANCE: The likelihood of childbirth by around age 35 was reduced for every year spent in casual employment, irrespective of socioeconomic status, partner's education and parents' birthplace. The likelihood was reduced by 8, 23 and 35% for 1, 3 and 5 years spent in casual employment, respectively. LIMITATIONS, REASONS FOR CAUTION: Women with longer employment histories (and greater age at first birth) had more opportunities for errors in recall, but it is unlikely that such errors were systematic and led to bias in the results. While we included variables reflecting partner's education and length of time with a live-in partner, partner's employment histories were not taken into account. WIDER IMPLICATIONS OF THE FINDINGS: Duration of time spent in casual employment is associated with an increased likelihood of childlessness at age 35 years, and this association is present across the spectrum of socioeconomic status. We suggest that upstream labour market reforms could be considered in order to reduce barriers to childbearing.

Sex differences in musculoskeletal injury epidemiology and subsequent loss of tactical readiness during Marine Corps Officer Candidates School.

INTRODUCTION: The US Marine Corps (USMC) Officer Candidates School (OCS) is a 10-week training course for Marine Officer Candidates (MOCs). OCS training is rigorous and demanding, which results in a high risk of musculoskeletal injuries (MSIs). The objective of this analysis was to describe MSIs among women and men during the USMC OCS at Quantico, Virginia, from September 2020 to November 2021. METHODS: This prospective cohort study assessed MSIs that occurred among 736 MOCs (women: 17.8% of sample, men: 82.2%). Data for the study were derived from routinely collected injury data by athletic trainers and physical therapists embedded within the training units. Injury incidence, event at the time of injury occurrence, anatomic location, injury type and disposition following injury were described. Fisher's exact tests were used to compare proportions of injured women and men. RESULTS: The cumulative injury incidence was higher among women (39.7%) compared with men (23.1%, p<0.001). When specific events associated with injuries were reported, most frequent events were the obstacle course (women: 20.9% of injuries, men: 12.9%) and the conditioning hike (women: 11.6%, men: 6.9%). Most injures affected the lower body (women: 67.4%, men: 70.8%). The most frequent body part injured was the lower leg (18.6%) in women and the knee (23.3%) in men. The most frequent injury type was strain (women: 39.5%, men: 24.3%), followed by sprain (women: 16.3%, men: 14.9%). A greater percentage of female (92.3%) compared with male MOCs (69.3%; p<0.001) were assigned light duty status following MSIs. CONCLUSIONS: Mitigation of injuries during OCS events such as the obstacle course and the conditioning hike needs further investigation. The high risk of overuse lower leg injuries among women and the higher incidence of injuries among women compared with men underscore the need for further investigation of modifiable sex-specific injury risk factors.

Parental work schedules and child overweight and obesity.

OBJECTIVE: Studies in school-age children have consistently shown a positive association between maternal paid work hours and child obesity. However, there is conflicting evidence about the impact of maternal work hours scheduled at nonstandard times (for example, evenings, nights or weekends), and no previous examination of paternal work schedules and child weight. We examined the associations between maternal, paternal and combined parental paid work schedules and overweight/obesity in children at age 9 years. METHODS: Data were analysed from the most recent follow-up of 9-year-old children (n=434) in an Australian birth cohort study. Children were measured and classified as overweight/obese using the International Obesity Taskforce body mass index cutoff points. Current working conditions of parents were obtained from a structured interview with the primary caregiver. Logistic regression analyses were used to investigate the effect of parental work schedules on child overweight/obesity with adjustment for a range of sociodemographic and household factors associated with parental employment and child weight. RESULTS: At 9 years of age, 99 children (22.8%) were overweight or obese. When parental work schedules were examined separately, child overweight/obesity was significantly associated with paternal nonstandard work schedules (adjusted odds ratio (OR) 1.97, 95% confidence interval (CI) 1.08-3.61). There was no association with any type of maternal work schedule. We also found an association between child overweight/obesity and circumstances in which both parents worked nonstandard schedules; however, this was of borderline statistical significance in the adjusted models (adjusted OR 2.26, 95% CI 0.99-5.16). CONCLUSION: Work hours scheduled at nonstandard times, when worked by the father or both parents, were associated with child overweight and obesity. These findings indicate the potential importance of fathers' paid work arrangements for child overweight/obesity, which until recently has largely been ignored.

Literature-based priors for gene regulatory networks.

MOTIVATION: The use of prior knowledge to improve gene regulatory network modelling has often been proposed. In this article we present the first research on the massive incorporation of prior knowledge from literature for Bayesian network learning of gene networks. As the publication rate of scientific papers grows, updating online databases, which have been proposed as potential prior knowledge in past research, becomes increasingly challenging. The novelty of our approach lies in the use of gene-pair association scores that describe the overlap in the contexts in which the genes are mentioned, generated from a large database of scientific literature, harnessing the information contained in a huge number of documents into a simple, clear format. RESULTS: We present a method to transform such literature-based gene association scores to network prior probabilities, and apply it to learn gene sub-networks for yeast, Escherichia coli and Human organisms. We also investigate the effect of weighting the influence of the prior knowledge. Our findings show that literature-based priors can improve both the number of true regulatory interactions present in the network and the accuracy of expression value prediction on genes, in comparison to a network learnt solely from expression data. Networks learnt with priors also show an improved biological interpretation, with identified subnetworks that coincide with known biological pathways.

Added sugars and ultra-processed foods in Spanish households (1990-2010).

BACKGROUND/OBJECTIVES: To study the association between ultra-processed foods acquisitions and added sugar content of total food purchases in Spanish households in 2010. Changes over time (1990-2000-2010) in ultra-processed food purchases and added sugars content of total food purchases are also compared. SUBJECTS/METHODS: We used data from three nationally representative Household Budget Surveys (HBS) conducted in 1990, 2000 and 2010. Number of studied households was 21,012, 33,730 and 22,116, respectively. Purchased foods and drinks were classified according to NOVA food groups as ultra-processed foods, processed foods, unprocessed or minimally processed foods, or processed culinary ingredients. Linear and Poisson regressions were used to estimate the association between quintiles of energy contribution of ultra-processed foods and added sugars contents of total food purchases in 2010. Changes over time were assessed using tests of linear trend and Student's t test. RESULTS: In 2010, ultra-processed foods represented 31.7% of daily energy acquisitions and 80.4% of all added sugars. Added sugars content of food purchases raised from 7.3% in the lowest to 18.2% in the highest quintiles of energy contribution of ultra-processed foods. The risk of exceeding 10% energy from added sugars quadrupled between the lowest and highest quintiles. The percentage of ultra-processed foods on all food purchases almost tripled between 1990 and 2010 (from 11.0 to 31.7%), paralleling the increase of added sugars content (from 8.4 to 13.0%). CONCLUSIONS: Cutting down exceeding added sugars availability in Spain may require a reduction in ultra-processed food purchasing.

The power of fluorescence excitation-emission matrix (EEM) spectroscopy in the identification and characterization of complex mixtures of fluorescent silver clusters.

Silver and gold clusters have received a lot of recent attention for their use in biomedical imaging. However, crude solutions of clusters are often complex mixtures, leading to discrepancies in their identification and characterization; important factors in determining their utility in biological applications. In the present study, silver clusters were separated for analysis using reverse-phase high performance liquid chromatography, which has previously been implemented in the efficient separation of gold clusters. Using fluorescence excitation-emission matrix (EEM) spectroscopy, we have demonstrated that a certain family of glutathione-protected silver clusters, previously thought to be one optically distinct species, is better described as a complex mixture of at least three distinct silver cluster species, each possessing unique optical properties. Based on these findings, EEM spectroscopy can be implemented as a powerful technique for determining the purity of complex mixtures, especially when other techniques, including mass spectrometry, fail to provide adequate characterization of a given material.

Distribution of mutations around rearranged heavy-chain antibody variable-region genes.

The mechanism of somatic hypermutation in the variable region of immunoglobulin genes expressed in mammalian B cells is a major unexplained phenomenon in the generation of diversity in the immune system. To evaluate possible mechanisms, the distribution of somatic mutations was examined for a group of five cloned, rearranged, somatically mutated VH genes generated in C57BL/6j mice. These mutated VH genes were sequenced and compared with their germ line counterparts from a point approximately 550 base pairs upstream of the transcription start site to an EcoRI site some 1,200 base pairs downstream of JH-4. The location of the transcription start (cap) sites was also precisely determined. Most (greater than or equal to 94%) of the 118 mutations scored occurred between the transcription start site and the distal end of JH-4. However, seven mutations occurred upstream of the transcribed region, and at least four were found downstream of JH-4. The target region for the mutator mechanism therefore clearly extends into the 3' nontranslated and 5' nontranscribed regions. Thus, models which propose the transcribed region of the DNA as the sole substrate for the mutation process are not ruled out but are inadequate to explain the upstream distribution of somatic mutations.

Fluticasone versus HFA-beclomethasone dipropionate for chronic asthma in adults and children.

BACKGROUND: The relative efficacy of fluticasone (FP) and beclomethasone (BDP) propelled with CFCs has been well established. The potency of HFA-BDP is thought to have been improved with new propellant and some studies suggest that it may equipotent at half the dose of CFC propelled-BDP. There is a need to revisit this question in the light of a potentially more potent new non-CFC propellant. OBJECTIVES: To determine the relative efficacy of FP and HFA-propelled BDP in chronic asthma. SEARCH STRATEGY: The Cochrane Airways Group Specialised Register was searched using pre-specified terms. Searches were current as of January 2006. SELECTION CRITERIA: Randomised controlled trials were eligible for inclusion in the review. We compared either CFC or HFA-propelled FP with HFA-propelled BDP. We made a distinction between HFA-BDP and HFA-BDP extra fine, which dispenses smaller particles of drug, leading to different, usually more peripheral distribution in the airways. Any inhaler device was considered, and there was no restriction on studies with or without spacers. We included studies which assessed HFA-BDP given via either pMDI, breath-actuated MDI, or DPI. DATA COLLECTION AND ANALYSIS: Two reviewers independently assessed studies for inclusion in the review. Data were extracted and entered in to RevMan 4.2 using standard meta-analytical techniques with predefined criteria for exploring statistical heterogeneity. MAIN RESULTS: Eight studies (1260 participants) met the inclusion criteria of the review. One study was conducted in children. Study reporting quality was fair, but all studies were of short duration (three to twelve weeks). Only studies assessing HFA-BDP extra fine in comparison with FP were identified. Lung function was not significantly different between extra fine BDP and FP when compared at the same dose in parallel studies, change in FEV1: 0.04 litres (95% CI -0.03 to 0.11 litres; three studies, 659 adults); change in am PEF: -0.69 litres (95% CI -11.21 to 9.83 litres; two studies, 364 adults). Individual studies reported non-significant findings in symptom scores and quality of life questionnaires. There was no significant difference between FP and HFA-BDP in the risk of study withdrawal, dysphonia or when data were reported as any adverse event. AUTHORS' CONCLUSIONS: There was no significant difference between FP and extra fine HFA-BDP on FEV(1) or peak flow at a dose ratio of 1:1. However, the number of studies and width of the confidence intervals in the analyses do not exclude a clinically meaningful difference between these two drugs. Difficulty in the successful manipulation of the devices studied may be a barrier to the widespread use of MDIs. One paediatric study was included in the review, so extrapolation of the findings of this review to children is limited. Further longer term studies in adults and children with moderate and severe asthma are required.

Observations on offspring of mice made diabetic with streptozocin.

My objective was to determine the effect of streptozocin (STZ)-induced diabetes in male inbred mice on nonfasting blood glucose levels and body weights of offspring. Hyperglycemia was induced in CBA/H male mice by either multiple subdiabetogenic doses (MD) of STZ (5 doses/day of 50 mg STZ/kg body wt) or by a single high sublethal (SD) dose (200 mg STZ/kg body wt). Females were made diabetic by the multiple low-dose procedure. The following matings were set up: SD males with normal (NOR) females; MD males with NOR females; NOR males with MD females; MD males with MD females. Controls were matings of NOR males with NOR females. Among the first cohort of litters was born one female from the cross of an SD male with an NOR female who became spontaneously hyperglycemic at 5 wk of age; the female progeny of this cross had significantly lower body weights. All other progeny groups were normoglycemic (up to 5 wk) and had normal body weights. Test progeny weaned in the second and subsequent cohorts of litters were also normoglycemic. The major effect in this progeny group was on body weight; diabetic fathers (particularly MD males) mated with NOR females produced offspring with significantly higher juvenile body weights than the controls (increase of approximately 0.5 g). These body-weight distributions also appeared more homogeneous than the more variable body-weight distribution of the controls. In contrast, MD mothers (mated with NOR males) produced second-cohort offspring with significantly lower average body weights (decrease of approximately 1-2 g) than the controls.(ABSTRACT TRUNCATED AT 250 WORDS)

The reversal of diabetes by pancreatic islet transplantation.

In this paper the theoretical basis of alloreactivity and its relevance to transplantation biology is discussed prior to a review of work showing that culture of adult mouse pancreatic islets for 7 days in 95% O2 and 5% CO2 facilitates successful grafting to nonimmunosuppressed allogeneic recipients. These allografts function by reversing both chemically induced and spontaneous diabetes. The fetal mouse pancreas is more immunogenic than adult islets, and even after a culture period of 10 days in 95% O2 and 5% CO2, BALB/c allografts are consistently rejected by nonimmunosuppressed recipient mice. The immunogenicity of fetal pancreas is thought to be due to the presence of contaminating lymphoreticular cells in the mesentery surrounding the fetal pancreas. Digestion of the fetal pancreas with collagenase allows the isolation of proislets that develop into functional islet tissue on transplantation. Fetal proislets are less immunogeneic than the whole fetal pancreas and may provide a source of tissue for clinical transplantation. Established islet allografts are relatively stable and are not rejected following nonspecific stimulation of the recipient's immune system or following passive transfer of either antibody or antibody and complement. After prolonged residence in the recipient a state of allograft tolerance develops and such grafts resist rejection by specific stimulation of the recipient. The administration of donor antigen in the form of uv-irradiated cells enforces this state of allograft tolerance.

Fluticasone versus HFA-beclomethasone dipropionate for chronic asthma in adults and children.

BACKGROUND: The relative efficacy of fluticasone (FP) and beclomethasone (BDP) propelled with CFCs has been well established. The potency of HFA-BDP is thought to have been improved with new propellant and some studies suggest that it may equipotent at half the dose of CFC propelled-BDP. There is a need to revisit this question in the light of a potentially more potent new non-CFC propellant. OBJECTIVES: To determine the relative efficacy of FP and HFA-propelled BDP in chronic asthma. SEARCH STRATEGY: The Cochrane Airways Group Specialised Register was searched using pre-specified terms. Searches were current as of March 2005. SELECTION CRITERIA: Randomised controlled trials were eligible for inclusion in the review. We compared either CFC or HFA-propelled FP with HFA-propelled BDP. We made a distinction between HFA-BDP and HFA-BDP extra fine, which dispenses smaller particles of drug, leading to different, usually more peripheral distribution in the airways. Any inhaler device was considered, and there was no restriction on studies with or without spacers. We included studies which assessed HFA-BDP given via either pMDI, breath-actuated MDI, or DPI. DATA COLLECTION AND ANALYSIS: Two reviewers independently assessed studies for inclusion in the review. Data were extracted and entered in to RevMan 4.2 using standard meta-analytical techniques with predefined criteria for exploring statistical heterogeneity. MAIN RESULTS: Seven studies (1230 participants) met the inclusion criteria of the review. One study was conducted in children. Study reporting quality was fair, but all studies were of short duration (three to twelve weeks). Only studies assessing HFA-BDP extra fine in comparison with FP were identified. Lung function was not significantly different between extra fine BDP and FP when compared at the same dose in parallel studies, change in FEV1: 0.04 litres (95% CI -0.03 to 0.11 litres; three studies, 659 adults); change in am PEF: -0.69 litres (95% CI -11.21 to 9.83 litres; two studies, 364 adults). Individual studies reported non-significant findings in symptom scores and quality of life questionnaires. There was no significant difference between FP and HFA-BDP in the risk of study withdrawal, dysphonia or when data were reported as any adverse event. AUTHORS' CONCLUSIONS: There was no significant difference between FP and extra fine HFA-BDP on FEV(1) or peak flow at a dose ratio of 1:1. However, the number of studies and width of the confidence intervals in the analyses do not exclude a clinically meaningful difference between these two drugs. Difficulty in the successful manipulation of the devices studied may be a barrier to the widespread use of MDIs. One paediatric study was included in the review, so extrapolation of the findings of this review to children is limited. Further longer term studies in adults and children with moderate and severe asthma are required.

IgM anti-erythrocyte autoantibodies specific for buried and neo-antigens using cellular-ELISA assays.

The aim was to develop a cellular-ELISA assay to detect natural autoantibodies specific for bromelain-treated mouse red blood cells (BrMRBC). High, unexpected IgM titres against normal mouse red blood cells (NMRBC) were detected in day 7-14 sera of CBA mice treated with E. coli lipopolysaccharide (LPS). These "autoantibodies" bound to normal mouse red blood cells in the presence or absence of commonly used c-ELISA adhering agents. Such high reactivity to NMRBC was never detected using complement dependent haemolytic assays in earlier work in this system. The question whether these IgM alpha-NMRBC molecules were binding nonspecifically (via Fc) or specifically (via Fab) was answered indirectly by comparing the binding titres of LPS-stimulated serum and several purified IgM antibody preparations (alpha-PC, alpha-KLH, MOPC 104E) on the same antigen coated plates. The observed binding ratios (titre on antigen X: titre on NMRBC) varied widely between different antibody sources, indicative of specific binding. In addition no significant unequivocal binding against NMRBC could be detected in vivo (LPS-stimulated mice) nor could bound IgM antibody be detected in a suspension-c-ELISA assay (high binding titres to BrMRBC could be detected in the latter test system). In conventional c-ELISA assays, modification of normal erythrocyte by adhesion to plastic microtitre plates appears to expose or create "neoantigens" on NMRBC which are not encountered in suspension-type c-ELISA, nor in lytic or agglutination assays where the erythrocyte targets are in suspension at physiological pH and isotonicity.

Hypothesis: somatic hypermutation by gene conversion via the error prone DNA----RNA----DNA information loop.

A mechanism to explain somatic hypermutation in immunoglobulin variable region genes is proposed employing polynucleotide information transfer through the error prone DNA----RNA----DNA loop. During transcription of the rearranged V-region, the primary transcript undergoes either inappropriate termination, cleavage, or reverse transcriptase priming allowing a V-region specific reverse-transcriptase-integrase complex to synthesize a DNA copy of the rearranged V-region and integrate it, by homologous recombination, back into the normal chromosomal site. Some consequences and predictions of the hypothesis are discussed.

Characterization of bovine surfactant for infants with respiratory distress syndrome.

Exogenous surfactant treatment of surfactant-deficient disease states is now under study in a number of centers, using a variety of surfactant preparations. We have chosen one preparation because of its current and potential clinical usefulness, and we have characterized it using selected tests and assays that we thought would be necessary (although not necessarily sufficient) to justify extended clinical use. We found its lipid composition to resemble that of other surfactants derived from lung mince. There is little variation among several batches with regard to lipid composition or surface tension-lowering capability. Morphologic heterogeneity occurs in individual samples of pelleted material studied by electron microscopy. Arterial oxygenation is improved when the material is administered to animals depleted of surfactant. A low molecular weight protein was identified that reacted with antibody that specifically binds nonserum surfactant proteins in a number of animal species (including human and cow). The characteristics of this surfactant preparation should be useful for comparison as newer and simpler products become available.

Identification of a mutation in the MP19 gene, Lim2, in the cataractous mouse mutant To3.

PURPOSE: Lim2, the gene encoding the second most abundant lens specific integral membrane protein, MP19, has recently been proposed as an ideal candidate gene for the cataractous mouse mutant, To3. The aim of this study was to screen the Lim2 gene in the To3 mutant for a genetic lesion that was correlated and consistent with the mutant phenotype. METHODS: Genomic DNA was isolated from both normal mouse parental strains as well as the heterozygous and homozygous To3 cataract mutant. PCR was used to generate overlapping fragments of the entire Lim2 gene from these DNAs. The coding regions, including splice junctions and the translational termination site, of these fragments were then sequenced. RESULTS: A single G -> T transversion was identified within the first coding exon of the Lim2 gene in the To3 mutant DNA. This DNA change results in the nonconservative substitution of a valine for the normally encoded glycine at amino acid 15 of the MP19 polypeptide. CONCLUSIONS: The identified genetic lesion in the Lim2 gene of the cataractous mouse mutant, To3, confirms Lim2 as an ideal candidate gene. Future transgenic experiments should provide proof or disproof of a causative relationship between the identified mutation and the cataractous phenotype. These studies indicate that MP19 may play an important role in both normal lens development and cataractogenesis, and warrants more intense investigation of its role within the ocular lens.

Lim2(To3) transgenic mice establish a causative relationship between the mutation identified in the lim2 gene and cataractogenesis in the To3 mouse mutant.

PURPOSE: Lim2 is the gene encoding the ocular lens-specific intrinsic membrane protein MP19. We previously reported finding a single nonconservative G->T transversion in exon two of the Lim2 gene. This mutation was linked to the cataract in the To3 (Total opacity number 3) mouse mutant, confirming Lim2 as an ideal candidate gene for the To3 cataract. The aim of the present study was to substantiate a causative relationship between the mutation in the Lim2 gene and cataractogenesis in the To3 mouse mutant. To this end a Lim2To3 transgene cassette was engineered and introduced into fertilized normal mouse embryos to test its ability to induce cataractogenic lens development. METHODS: A Lim2 genomic clone was isolated and purified from a murine 129/SvJ genomic library. A restriction endonuclease map of the gene was generated using classical Southern techniques. The murine Lim2 promoter was characterized by transfecting primary chicken lens epithelial cells with Lim2 promoter-CAT reporter constructs and assaying promoter activity and specificity. This genomic clone was then used in conjunction with PCR to generate a Lim2To3 transgene cassette. After sequencing of the PCR engineered portion, the Lim2To3 transgene was then used to generate Lim2To3 transgenic mice via pronuclear injection. Founder mice and their offspring from outcrosses and intercrosses were characterized by ophthalmic examination, PCR and Southern DNA analysis, RT-PCR mRNA analysis, and histology of lens sections. RESULTS: Two mice, from independent microinjections, were identified as positive for presence of the Lim2To3 transgene cassette as well as presence of bilateral congenital cataracts and reduced eye size and mass. One of these founders was incapable of germline transmission of the transgene to offspring and was not characterized further. The other was capable of germline transmission and was characterized as described above. PCR DNA analysis revealed a perfect concordance between presence of the Lim2To3 transgene cassette and congenital cataract in offspring of this founder. Transgenic hemizygotes exhibited cataract and a reduction in eye and lens size and mass, while transgenic "homozygotes" presented with a more severe cataract and microphthalmic reduction in eye and lens size and mass. Southern analysis revealed approximately 2 copies of the transgene cassette integrated into a single chromosomal site in the founder and all hemizygous offspring. RT-PCR analysis revealed a very low ratio of Lim2To3 transgenic mRNA compared to endogenous normal Lim2. Finally, histology revealed that lens development was abnormal in mutant transgenic animals by embryonic day E15. By E19, just prior to birth, gross disorganization of secondary fibers was observed in mutants. CONCLUSIONS: These transgenic experiments firmly establish a causative relationship between the previously identified mutation in the Lim2 gene and cataractogenesis in the To3 mouse mutant. The low levels of mutant mRNA produced by the transgene cassette as compared to endogenous levels of normal Lim2 mRNA provides evidence that this dominant mutation results in a mutant MP19 protein with altered function rather than simply loss of function.