Mitochondrial DNA insertions into the nuclear chromosomes of the maize Mo17 inbred line [abstract]

Abstract only availableMitochondria contain their own DNA separate from the nucleus; however, most of their genes have been transferred to the nucleus over evolutionary time. The lateral transfer of DNA from the mitochondria to the nucleus appears to be a continuing process and is more frequent in plants than in animals. Our laboratory has documented extensive variation in the nuclear-mitochondrial sequences (NUMTs) among maize inbred lines using total mitochondrial DNA (mtDNA) as probes onto mitotic metaphase chromosomes, a technique known as fluorescence in situ hybridization (FISH). The mitochondrial genome has been cloned into 20 cosmids, which were used to examine the insertions of individual segments. The focus of the current study was to use FISH with the 20 individual mtDNA-containing cosmids to locate mtDNA within the nuclear chromosomes of the Mo17 inbred line of maize and to compare these locations with those of the B73 line. We studied Mo17 because this line and its derivatives are used in crosses with B73-derived lines to create the most commonly used corn hybrids. Fifteen NUMTs had been detected on the Mo17 chromosomes using a mixture of 19 mtDNA-containing cosmids. However, only nine of the 15 NUMTs were seen when applying individual mtDNA-containing cosmids, suggesting that the portions of nuclear DNA corresponding to the individual mtDNA-containing cosmids were too small to be detected until many cosmids were combined. A large NUMT was previously detected on the long arm of chromosome 9 by 14 of the 20 individually tested cosmids in the B73 inbred line. However, a NUMT present at the same site in Mo17 was detected by only 2 of the 20 cosmids (5 and 20). This suggests that the major insertion in B73 is recent and that it may have inserted by homologous recombination

Circular and linear mitochondrial genomes in cytoplasmic male sterile maize [abstract]

Abstract only availableCytoplasmic male sterility (CMS) is a maternally inherited condition in which a plant has an inability to produce viable pollen. It is usually due to the production of a toxic chimeric protein within the mitochondria during the maturation of pollen grains. In maize (Zea mays), there are three types of CMS: CMS-T, CMS-C and CMS-S. The S-type of cytoplasmic male sterility (CMS-S) in maize is associated with the expression of a rearranged mitochondrial DNA region. This CMS-S-specific region includes two co-transcribed chimeric open reading frames, orf355 and orf77. The nuclear restorer-of-fertility gene, Rf3, cleaves all transcripts containing both orfs, including the CMS-S-specific linear 1.6 kb mRNA; this results in male fertility. The Lancaster Surecrop-derived inbred line A619 carries a different and weaker restorer called Rf9. Fertility restoration by Rf3 and Rf9 was compared for their effects upon the CMS-associated region of mitochondrial DNA. Unlike Rf3, Rf9 affects the organization of the CMS-S-specific region. It appears to do this by affecting recombination between linear "S" plasmids and the CMS-S-specific region of the main mitochondrial genome, which produces a linear end from which transcripts for the 1.6 kb mRNA are initiated. By reducing the amount of recombination, Rf9 reduces the amount of linear template available for transcribing the S-associated 1.6 kb RNA. A reduction in this transcript is associated with an increase in pollen survival. We have studied the effects of the two restorer-of-fertility genes from several different inbred lines on the amounts of integrated and linearized orf355/orf77 genes within CMS-S mtDNA.MU Monsanto Undergraduate Research Fellowshi

Analysis of mitochondrial DNA insertions into a nuclear chromosome of the maize B73 line

Abstract only availableMitochondrial DNA (mtDNA) is known to have integrated into the nuclear DNA of plants and animals. The purpose of this project is to investigate the on-going migration of mtDNA into the nuclear DNA of maize plants. Specific objectives are to discover the amount of DNA incorporated, whether it is the whole mitochondrial genome or sections, and to see if it has replicated after migration. The maize inbred line B73 has a particularly large mt DNA insert on chromosome 9. Using the fluorescent in situ hybridization (FISH) method, the arrangement of inserted mitochondrial DNA was examined. The FISH method uses fluorescently labeled mtDNA as probes for hybridization to chromosomes. Regions of the chromosomes that contain mtDNA can then be detected using a compound microscope with fluorescent attachments. Locations that contain more mtDNA are brighter. Three combinations of probes that cover different parts of the mitochondrial genome were employed. In order to analyze the arrangement of the DNA, the chromosomes were prepared from a stage of meiosis called pachynema in which the chromosomes are elongated and have not yet begun to condense. The results have confirmed the presence of all three probes within the large insertion of mtDNA on chromosome 9 of B73. The data suggest that either different parts of the mitochondrial genome are incorporated preferentially or that there is selective replication of portions of the mitochondrial genome after incorporation.MU Monsanto Undergraduate Research Fellowshi

Identification of chloroplast DNA insertions in nuclear chromosomes of maize B73 line using the FISH procedure

Abstract only availableIt is known that chloroplast DNA can incorporate itself into the nuclear genome of plants. However, the sites of chloroplast (ct) DNA integration into chromosomes of maize have not yet been analyzed. This project is the first attempt to find the location of the ctDNA on the maize chromosomes. Fluorescent in situ hybridization is a technique that has proved useful in karyotyping and chromosomal mapping in maize. The FISH procedure is being used in this study to discover the location of the ctDNA in the nuclear genome of the inbred line B37. In order to develop ctDNA “probes” for FISH analysis, we have used the polymerase chain reaction (PCR) to produce fragments of ctDNA. Primers were chosen to amplify fragments of 10 kb or larger. The amplified DNAs were purified and labeled with fluorescent dyes and these probes were subsequently hybridized to chromosomes. The probes recognize and bind to the corresponding DNA sequences within the chromosomes. Root tip cells were used to prepare the slides for hybridization. Because the cells are collected during the metaphase stage of division, the chromosomes are compact and more easily visible. Chromosomes that contain ctDNA can be detected using a compound microscope with fluorescent attachments. The location of the ctDNA on the chromosomes is made visible by the fluorescent labeling of the probe. Eight of eleven regions of the chloroplast genome of the B73 line have been specifically amplified and have been observed under the microscope for FISH analysis. This information will contribute to an understanding of the extent and mechanism of transfer of organellar genomes to the nucleus.MU Monsanto Undergraduate Research Fellowshi

Spatial and Temporal Variations of Microplastics within Humboldt Bay, California

This study aimed to quantify microplastic (MP) concentration and analyze the spatial and temporal variabilities of the concentrations during the tidal cycle in Humboldt Bay, California. To get an approximation of MP concentration, both water and sediment samples were taken at five different stations, twice during one tidal cycle. Sampling was conducted during two different cruises, on the 19th and 21st of September 2020. The samples were processed in the lab using a density separation procedure and filtration. MP concentrations in the different samples were determined using an average optical microscopy count. Comparison of the water column MP concentrations during ebb and flood tides shows higher concentrations during flood tide, 49.0 particles/L ± 32.37 (flood) vs 34.4 particles/L ± 16.32 (ebb), indicating that MPs are brought into Humboldt Bay from the ocean. The comparison of the MP concentrations during lower energy and higher energy conditions indicates that concentrations in the water column were elevated when there was greater tidal kinetic energy, approximated by the covariance of the measured velocity in North Bay Channel. This result was assumed to be caused by the strong tidal currents stirring up both sediments and the settled MPs into the water column. Due to lower tidal kinetic energy on the sediment sampling cruise day, we could not confirm that assumption. Water samples indicated that MPs are heterogeneously distributed in the bay, with higher concentrations found near the Entrance Channel and lower concentrations found further north in the bay. Sediment samples also indicate a heterogeneous distribution of MPs in the bay, with the lowest concentrations near the Entrance Channel, 15 particles/kg, where high tidal currents inhibit settling of particles

Identification of chloroplast DNA insertions in B73 nuclear chromosomes [abstract]

Abstract only availableIt is known that DNA from mitochondria and chloroplasts migrates to the nucleus and incorporates into the nuclear genome. It is unknown how often this transfer and integration process occurs or if specific sites in the chromosomes are preferred. We have evidence that chloroplast DNA has integrated into multiple sites within chromosomes in the B73 inbred line of maize. This has been shown with a procedure called FISH, Fluorescence in situ Hybridization, in which fluorescent probes created from DNA hybridize with chromosomes in places where there is a match. Probes for this project were generated from chloroplast DNA. The chloroplasts were separated by centrifugation and lysed to obtain the DNA. The chloroplast genome was subdivided into 15 regions. Pieces of chloroplast DNA corresponding to 14 of the 15 regions were amplified using PCR (the polymerase chain reaction). The amplified DNA was purified and labeled with fluorescent dyes to create probes. These probes were subsequently hybridized to metaphase chromosome spreads on slides. The probes recognized and bound to DNA sequences in the chromosomes. With the completion of the chloroplast DNA mapping, the B73 inbred maize line will have a complete diagram of the locations of major chloroplast DNA insertions.MU Monsanto Undergraduate Research Fellowshi

The role for osmotic agents in children with acute encephalopathies: a systematic review

Background: Raised intracranial pressure (ICP) is known to complicate both traumatic and non-traumatic encephalopathies. It impairs cerebral perfusion and may cause death due to global ischaemia and intracranial herniation. Osmotic agents are widely used to control ICP. In children, guidelines for their use are mainly guided by adult studies. We conducted this review to determine the current evidence of the effectiveness of osmotic agents and their effect on resolution of coma and outcome in children with acute encephalopathy.Methods: We searched several databases for published and unpublished studies in English and French languages, between January 1966 and March 2009. We considered studies on the use of osmotic agents in children aged between 0 and 16 years with acute encephalopathies. We examined reduction in intracranial pressure, time to resolution of coma, and occurrence of neurological sequelae and death.Results: We identified four randomized controlled trials, three prospective studies, two retrospective studies and one case report. Hypertonic saline (HS) achieved greater reduction in intracranial pressure (ICP) compared to mannitol and other fluids; normal saline or ringer's lactate. This effect was sustained for longer when it was given as continuous infusion. Boluses of glycerol and mannitol achieved transient reduction in ICP. Oral glycerol was associated with lower mortality and neurological sequelae when compared to placebo in children with acute bacterial meningitis. HS was associated with lower mortality when compared to mannitol in children with non-traumatic encephalopathies.Conclusion: HS appears to achieve a greater reduction in ICP than other osmotic agents. Oral glycerol seems to improve outcome among children with acute bacterial meningitis. A sustained reduction in ICP is desirable and could be achieved by modifying the modes and rates of administration of these osmotic agents, but these factors need further investigation

The monoclonal antibody combination REGEN-COV protects against SARS-CoV-2 mutational escape in preclinical and human studies.

Monoclonal antibodies against SARS-CoV-2 are a clinically validated therapeutic option against COVID-19. Because rapidly emerging virus mutants are becoming the next major concern in the fight against the global pandemic, it is imperative that these therapeutic treatments provide coverage against circulating variants and do not contribute to development of treatment-induced emergent resistance. To this end, we investigated the sequence diversity of the spike protein and monitored emergence of virus variants in SARS-COV-2 isolates found in COVID-19 patients treated with the two-antibody combination REGEN-COV, as well as in preclinical in vitro studies using single, dual, or triple antibody combinations, and in hamster in vivo studies using REGEN-COV or single monoclonal antibody treatments. Our study demonstrates that the combination of non-competing antibodies in REGEN-COV provides protection against all current SARS-CoV-2 variants of concern/interest and also protects against emergence of new variants and their potential seeding into the population in a clinical setting

Erratum: Global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 1990–2017: a systematic analysis for the Global Burden of Disease Study 2017

Interpretation: By quantifying levels and trends in exposures to risk factors and the resulting disease burden, this assessment offers insight into where past policy and programme efforts might have been successful and highlights current priorities for public health action. Decreases in behavioural, environmental, and occupational risks have largely offset the effects of population growth and ageing, in relation to trends in absolute burden. Conversely, the combination of increasing metabolic risks and population ageing will probably continue to drive the increasing trends in non-communicable diseases at the global level, which presents both a public health challenge and opportunity. We see considerable spatiotemporal heterogeneity in levels of risk exposure and risk-attributable burden. Although levels of development underlie some of this heterogeneity, O/E ratios show risks for which countries are overperforming or underperforming relative to their level of development. As such, these ratios provide a benchmarking tool to help to focus local decision making. Our findings reinforce the importance of both risk exposure monitoring and epidemiological research to assess causal connections between risks and health outcomes, and they highlight the usefulness of the GBD study in synthesising data to draw comprehensive and robust conclusions that help to inform good policy and strategic health planning

Global, regional, and national comparative risk assessment of 84 behavioural, environmental and occupational, and metabolic risks or clusters of risks for 195 countries and territories, 1990-2017: a systematic analysis for the Global Burden of Disease Study 2017

Background The Global Burden of Diseases, Injuries, and Risk Factors Study (GBD) 2017 comparative risk assessment (CRA) is a comprehensive approach to risk factor quantification that offers a useful tool for synthesising evidence on risks and risk–outcome associations. With each annual GBD study, we update the GBD CRA to incorporate improved methods, new risks and risk–outcome pairs, and new data on risk exposure levels and risk–outcome associations. Methods We used the CRA framework developed for previous iterations of GBD to estimate levels and trends in exposure, attributable deaths, and attributable disability-adjusted life-years (DALYs), by age group, sex, year, and location for 84 behavioural, environmental and occupational, and metabolic risks or groups of risks from 1990 to 2017. This study included 476 risk–outcome pairs that met the GBD study criteria for convincing or probable evidence of causation. We extracted relative risk and exposure estimates from 46 749 randomised controlled trials, cohort studies, household surveys, census data, satellite data, and other sources. We used statistical models to pool data, adjust for bias, and incorporate covariates. Using the counterfactual scenario of theoretical minimum risk exposure level (TMREL), we estimated the portion of deaths and DALYs that could be attributed to a given risk. We explored the relationship between development and risk exposure by modelling the relationship between the Socio-demographic Index (SDI) and risk-weighted exposure prevalence and estimated expected levels of exposure and risk-attributable burden by SDI. Finally, we explored temporal changes in risk-attributable DALYs by decomposing those changes into six main component drivers of change as follows: (1) population growth; (2) changes in population age structures; (3) changes in exposure to environmental and occupational risks; (4) changes in exposure to behavioural risks; (5) changes in exposure to metabolic risks; and (6) changes due to all other factors, approximated as the risk-deleted death and DALY rates, where the risk-deleted rate is the rate that would be observed had we reduced the exposure levels to the TMREL for all risk factors included in GBD 2017. Findings In 2017, 34·1 million (95% uncertainty interval [UI] 33·3–35·0) deaths and 1·21 billion (1·14–1·28) DALYs were attributable to GBD risk factors. Globally, 61·0% (59·6–62·4) of deaths and 48·3% (46·3–50·2) of DALYs were attributed to the GBD 2017 risk factors. When ranked by risk-attributable DALYs, high systolic blood pressure (SBP) was the leading risk factor, accounting for 10·4 million (9·39–11·5) deaths and 218 million (198–237) DALYs, followed by smoking (7·10 million [6·83–7·37] deaths and 182 million [173–193] DALYs), high fasting plasma glucose (6·53 million [5·23–8·23] deaths and 171 million [144–201] DALYs), high body-mass index (BMI; 4·72 million [2·99–6·70] deaths and 148 million [98·6–202] DALYs), and short gestation for birthweight (1·43 million [1·36–1·51] deaths and 139 million [131–147] DALYs). In total, risk-attributable DALYs declined by 4·9% (3·3–6·5) between 2007 and 2017. In the absence of demographic changes (ie, population growth and ageing), changes in risk exposure and risk-deleted DALYs would have led to a 23·5% decline in DALYs during that period. Conversely, in the absence of changes in risk exposure and risk-deleted DALYs, demographic changes would have led to an 18·6% increase in DALYs during that period. The ratios of observed risk exposure levels to exposure levels expected based on SDI (O/E ratios) increased globally for unsafe drinking water and household air pollution between 1990 and 2017. This result suggests that development is occurring more rapidly than are changes in the underlying risk structure in a population. Conversely, nearly universal declines in O/E ratios for smoking and alcohol use indicate that, for a given SDI, exposure to these risks is declining. In 2017, the leading Level 4 risk factor for age-standardised DALY rates was high SBP in four super-regions: central Europe, eastern Europe, and central Asia; north Africa and Middle East; south Asia; and southeast Asia, east Asia, and Oceania. The leading risk factor in the high-income super-region was smoking, in Latin America and Caribbean was high BMI, and in sub-Saharan Africa was unsafe sex. O/E ratios for unsafe sex in sub-Saharan Africa were notably high, and those for alcohol use in north Africa and the Middle East were notably low. Interpretation By quantifying levels and trends in exposures to risk factors and the resulting disease burden, this assessment offers insight into where past policy and programme efforts might have been successful and highlights current priorities for public health action. Decreases in behavioural, environmental, and occupational risks have largely offset the effects of population growth and ageing, in relation to trends in absolute burden. Conversely, the combination of increasing metabolic risks and population ageing will probably continue to drive the increasing trends in non-communicable diseases at the global level, which presents both a public health challenge and opportunity. We see considerable spatiotemporal heterogeneity in levels of risk exposure and risk-attributable burden. Although levels of development underlie some of this heterogeneity, O/E ratios show risks for which countries are overperforming or underperforming relative to their level of development. As such, these ratios provide a benchmarking tool to help to focus local decision making. Our findings reinforce the importance of both risk exposure monitoring and epidemiological research to assess causal connections between risks and health outcomes, and they highlight the usefulness of the GBD study in synthesising data to draw comprehensive and robust conclusions that help to inform good policy and strategic health planning