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Scientific studies aimed at unlocking the genetic code of all humans have had problems with their data: it was mostly collected from people with European heritage, leaving wide gaps in the study of DNA from populations around the world.

To close the gap, the authors of one recent study turned to military veterans, a group whose health and genetics are well studied during and after military service, and whose genetics come from a wide swath of the world.

Researchers working with the Department of Veterans Affairs were granted access to the VAs database of DNA known as the Million Veteran Program. Using the DNA database from the VA, researchers found genetic markers for prostate cancer, anemia, Alzheimers dementia and cirrhosis, according to a study published in Science.

Most of the genetic data available to researchers are still derived from individuals of European descent, researchers wrote. This shortcoming limits both the biological insights that can be gleaned from these data and their clinical applications to non-European patients, who may not match up well with the traditional study participants.

The Million Veteran Program was launched in 2011 as a research effort to improve veteran health care. Its also one of the largest biobanks in the world, collecting DNA and other health information on veterans for medical research. Military veterans have long been a more racially and genetically diverse group than the U.S. as a whole.

The recent VA-funded study was done in collaboration with the Department of Energy in order to use their supercomputers to run thousands of genetic-disease analyses using MVP data.

According to Anurag Verma, a researcher at the Corporal Michael J Crescenz VA Medical Center, most genomic studies rely heavily on European ancestry DNA which limits the accuracy of research results.

If we include more diversity in these studies, then we are able to overall improve the risk prediction, said Verma, a lead author of the study and an assistant professor of medicine at the University of Pennsylvania.

In the study with Veteran DNA, researchers were able to find 101 traits including hemolytic anemias, sarcoidosis, keloid scarring and susceptibility to gout among veterans with African ancestry that were twice as prevalent than in veterans with European ancestry. The research also validated previous studies on African ancestral populations which found a higher prevalence of traits linked to prostate cancer, reduced white blood count levels and kidney-related conditions such as end-stage renal disease.

Among veterans with East Asian and Admixed American ancestry (a term that typically encompass those who self-identify as Hispanic or Latino), researchers found 18 traits with at least twice the prevalence of veterans with European ancestry including Alopecia areata in Admixed American and viral hepatitis B in East Asian ancestry.

This is an example where the donation that the million-plus veterans made to this program, its really a gift to the world, said Sumitra Muralidhar, director of the Million Veteran Program.

In most genetic association studies, research teams study one disease and determine which genetics are associated with it or a researcher identifies one genetic marker and they try to link conditions or health conditions that are associated with that specific marker, Muralidhar said.

By taking 42 million-or-so-plus genetic markers and about 2,000 health traits all at once and looking at this, weve already completed the first step so-to-speak for a number of health traits, she said. Now other researchers can really take this as a jumping point and expedite discovery and move it towards translation much faster.

The lack of diverse DNA in genomic research has been well-documented in published studies and news reports. A systematic review of existing genome-wide association studies from January 2024, found that 82% of 123 studies looking at neurodegenerative disease connections to DNA predominantly featured participants with European ancestry.

Access to veteran DNA, however, is helping to close that gap.

With the MVP study, which began in 2018, researchers were able to use veteran data from the VAs biobank, which at the time was just over 638,000 individuals and about 29% had non-European ancestry.

Not only the percentage is high, but absolute number of the individuals in this study is also massive in comparison to whatever has been published in the past, Verma said.

The VA has since reached more than one million participants and as of July 24, MVP had 1,037,886 participants. In the current breakdown, around 25% of the cohort are racially diverse (non-European ancestry) with 18% African ancestry and 7% other racial minorities; 8% ethnically diverse; namely, Hispanic.

By conducting a diverse, cross-population analysis, researchers were able to identify 834 previously unreported variant-trait associations and 15 signals from coding variants that are either rare or not observed in non-European populations.

With a substantial amount of African ancestry data, researchers also found numerous pleiotropic genes, which are genes that control more than one trait. A common example of pleiotropic genes is phenylketonuria, a disorder caused by an enzyme deficiency that can result in multiple characteristics like mental retardation, eczema, and lighter skin pigments.

This highlights the substantial contribution conferred by including diverse populations in genetic research, the study states. At the same time, cross-population heritability analyses, fine mapping, and heterogeneity analyses demonstrated substantial similarities in the genetic architecture between population groups driven by variants common across populations.

Genome-wide association studies have long been the foundation of research into complex biological traits and drug development.

People carry different genes and some genes where we call variants are more common in one population compared to another population and so how this plays out is in drugs, said Katherine Liao, a lead researcher of the study and staff physician at the VA Boston Healthcare System. There are certain drugs that if you carry a certain genetic defect, youre gonna have a really bad side effect.

Liao gave an example of 1.5% of one population carrying a specific gene and another population where 10% carry the gene.

If you were to give everybody the same drug, the population where they dont have the gene that gives you a side effect, nothing happens. So if you only test on that population, you think Oh, this drug is really safe, but in another population, 10% are having some kind of massive side effect, Liao, also a professor at Harvard Medical School said. Thats where it really matters.

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Genome-wide association studies are also helping in the field of precision medicine where doctors look at genetics, environment, and lifestyle to select the best treatment for a patient.

The promise of precision medicine is finding the best drug for the patient and also how do we manage it the best. If theres only that one drug, its not like we want to avoid it, but its like how do we deal with that?, Liao said. How do we tell the patient watch out for rash or these are the issues you need to watch out for.

A number of other studies are underway using MVP data, with researchers looking at links between DNA and prevalence of certain types of cancers, diabetes, and cardiovascular disease, as well as substance abuse and mental health disorders.

But MVP has already led to some of the largest studies ever done, said M, MVP director.

One study that looked at the genetics of PTSD, had 165,000 veterans which had never been done before and another, which was the largest study on genetics of anxiety used data from 200,000 veterans.

During the pandemic, a research team observed African Americans with COVID-19 were dying of acute kidney disease at much higher rates than the rest of the population. By diving into MVP data, researchers found a gene called APOL1 that increased African Americans risk of death. With their findings, Muralidhar said, pharmaceutical companies can develop drugs that target the gene and reduce mortality risk.

While the study does note that MVP data is ancestry-diverse, its veteran population is predominantly male and older, making the research less well-powered to study conditions more prevalent in females or younger populations. But even if only 10% of MVP is made up of female veterans, the absolute number equals 100,000 female participants which MVP officials and the studys researchers said is larger than the majority of existing biobanks.

Muralidhar said MVP has launched a couple of campaigns aimed at enrolling more women veterans. During one marketing campaign, MVP doubled the number of women participants and are developing focused campaigns for different races, ethnicities, genders, ages and even geographies for groups like rural veterans who are harder to reach. As part of the MVP sign-up process, veterans have to give a blood sample at a VA facility, but in order to expand the enrollment, MVP has started to mail a kit home for blood specimens.

Participation in MVP is voluntary and requires consent from each veteran. Enrollees have to complete online or mail surveys on their health, lifestyle habits, military experience, personal and family history, give a blood sample for genetic analysis, and agree to future contact from MVP.

The altruism of veterans has made this possible really without which we would never have been here, Muralidhar said. They really look at this as another opportunity to serve.

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The work of British chemist Rosalind Franklin (19201958) played an integral role in the discovery of the structure of DNA, but it took many years for Franklins contributions to be fully recognized. In 1962, four years after Franklins death, James Watson, Francis Crick, and Maurice Wilkins won the Nobel Prize for the discovery of the famous double helix. Wilkins, a colleague of Franklin, gave Watson and Crick several images that she produced before she published them. In his book years later, Watson actively downplayed Franklins role.

The episode is compelling evidence of sexism in the history of science, writes Michelle G. Gibbons in Philosophy of Science. But she argues that Franklins story has additional significanceit forces us to reevaluate our notions of how science works.

Gibbons describes how Franklin refined the process of x-ray crystallography, in which x-rays are directed at a molecule with a photographic plate behind it and the diffraction pattern is captured by the plate. Franklin developed new methods for extracting samples, designed a new camera, and came up with techniques for hydrating and dehydrating samples. She produced high-quality images, showing that DNA molecules appeared differently depending on the level of hydration: an A form and B form. The most famous image was Photograph 51, which clearly displays an X-shaped pattern produced by the B form of DNA.

The X-shape was evidence of a helical structure. Once they saw Photograph 51, Watson and Crick rushed to publish a paper on their model, incorporating the image. But the pattern in the A form images wasnt as clear, and Franklin refrained from claiming that DNA always possessed a helical structure. Gibbons argues that their approaches represent two distinct ways of coming to a discovery.

Rosalind Franklins research strategy was to avoid exactly the sort of speculation that Watson and Crick freely engaged in, Gibbons writes. Watson and Crick relied on creating possible models and modifying them. Franklin wanted to find the structure of DNA in the data.

Gibbons writes that Franklins method doesnt match up with common philosophical models of discovery. Philosophers such as Karl Popper and the positivists thought about scientific discovery as an unfathomable leap of insight, she writes. In this view, discovery is something that happens solely in the mind.

Scientific imaging forces philosophers to think about discovery differently, Gibbons argues. She suggests a hypothetical camera that a scientist uses to take an unambiguous image of a DNA double helix. In such an event, the structure of DNA would become known without any burst of insight in someones mind. To Gibbons, this means that image making can itself constitute a form of discovery.

Photograph 51 required interpretation, but it was visual evidence of a helical structure, and this constrained Watson and Cricks speculations.

Watson and Crick seemed to have subscribed to a view of science that valued heroic insight above all else, Gibbons writes. In contrast, Franklins story reveals a model of scientific discovery that involves many people, each contributing some, often small part to the process.

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By: Michelle G. Gibbons

Philosophy of Science, Vol. 79, No. 1 (January 2012), pp. 6380

The University of Chicago Press on behalf of the Philosophy of Science Association

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Rosalind Franklins Methods of Discovery - JSTOR Daily

Low predictive power of CRISPR-Cas9 mutagenesis prediction programs for plants

To assess the predictability of the CRISPR-Cas9-induced mutations in plants, we examined the performance of two widely used CRISPR mutagenesis prediction programs, FORECasT and InDephi11,12. We generated CRISPR-induced mutations at 59 sites, including 26 from Arabidopsis and 33 from Setaria, by introducing the corresponding CRISPR-Cas9 constructs into each species (Supplementary Data1). In Arabidopsis, each CRISPR-Cas9 construct was introduced using the floral dip-based stable transgenic approach. Individual seedlings of each T1 transgenic plant were collected for the CRISPR mutation analysis at each target site. In Setaria, Individual CRISPR-Cas9 constructs were transformed via transient protoplasts transfection. Transformed protoplast cells were collected after 48h for the mutation assay. Subsequently, the mutation profile of each site was obtained by using the next-generation sequencing (NGS) based assay. The indel mutagenesis rates averaged at 8.9% and 28.4% at the sites from Arabidopsis and Setaria, respectively (Supplementary Fig.1a). Additionally, the indel profile from each site was further characterized into individual insertion and deletion types for each species (Fig.1a). Notably, the 1-bp insertions represent one of the most common occurring mutation types, as previously observed in human cell lines. In Arabidopsis, 1-bp insertions were the most prevalent mutation types, accounting for an average of 44.6% of all mutations across 26 CRISPR sites (Fig.1a). For Setaria viridis, the average 1-bp insertion rate appeared to be the 4th highest at 9.6% across 33 CRISPR sites (Fig.1a).

a CRISPR-Cas9 induced mutation profiles across 59 target sites in Arabidopsis (n=26) and Setaria (n=33). X-axis represents individual indel sizes. The normalized mutation rates (Y-axis) were determined by dividing the number of reads containing mutations within each indel size by the total number of reads containing all types of mutations. The horizontal bars within boxes represent medians. The top and bottom edges of the boxes represent the 75th and 25th percentiles, respectively. The upper and lower whiskers extend to data no more than 1.5x the interquartile range from the upper and lower edges of the box, respectively. b, c Two-sided Pearson correlation analysis were performed using scatter plots to compare predicted versus experimentally observed insertion (ins.) rates for each CRISPR gRNA in Arabidopsis (n=26) and Setaria (n=33). The 95% confidence interval (CI) were indicated with gray color. The source data are provided in the Source Data file.

Simultaneously, the predicted mutation profile was generated for each target site using FORECasT and InDephi. In this study, we chose to focus on the insertion rates for correlation analyses on the predicted versus observed values for the following reasons: (1) CRISPR-induced insertions appeared to exhibit less stochastic patterns than deletions; and (2) previous studies have suggested that these prediction tools demonstrate greater predictive power for insertions compared to other indel types11,12,14. As a result, we observed no positive correlations using either FORECasT or InDelphi for both Arabidopsis and Setaria datasets (Fig.1b, c). Weak negative correlations were observed in the Arabidopsis dataset (r=0.56, p<0.0031 and r=0.4, p<0.036; Fig.1b), while no correlation was found in the Setaria dataset (r=0.18, p<0.31 and r=0.07, p<0.69; Fig.1c). Thus, our data suggested that both prediction programs developed with human datasets exhibited low predictive power for the CRISPR-Cas9-induced mutation profile in plants.

The limited predictive power from the human cell-based indel prediction tools prompted us to further examine CRISPR-Cas9-induced insertion profiles in plants. Both FORECasT and InDelphi predicted CRISPR-induced insertions primarily as 1-bp insertion events occurred at the 4th position upstream of the PAM; and most of these insertions were derived from templated insertions by duplicating the 4th nucleotide. When we analyzed the observed insertions from the Arabidopsis and Setaria target sites, 1-bp insertions were consistently predominant, accounting for averagely 95.9% of insertions across all sites (Supplementary Fig.1b). However, when the 1-bp insertion patterns were plotted according to the 4th nucleotide, the observed insertions did not consistently exhibit characteristics of templated insertions in plants (Fig.2a). When the 4th nucleotide was T, the inserted nucleotide appeared to follow the templated insertion model with 78.8% and 75.7% of insertions as T in Arabidopsis and Setaria, respectively (Fig.2a). With the 4th nucleotide as A, while A remained the predominant inserted nucleotide (58.5% and 58.7% in Arabidopsis and Setaria), the fractions of other types of insertions, termed as non-templated insertions, increased substantially (Fig.2a). In cases where the 4th nucleotide was either C or G, non-templated insertions became predominant by increasing to 61.4% and 66.0% for the 4th nucleotide C, and 98.4% and 99.5% for the 4th nucleotide G in Arabidopsis and Setaria, respectively.

a Cross-species 1-bp insertion patterns to the 4th nucleotide. The 1-bp insertions were divided into 4 groups according to the inserted nucleotide for each CRISPR gRNA. The normalized 1-bp insertion (ins.) rates were calculated by dividing the number of reads containing each type of 1-bp insertions by the number of reads with all types of 1-bp insertions and were plotted to the 4th nucleotides (T, A, C, and G) for Setaria viridis (S.v.; n=33 biologically independent samples), Arabidopsis thaliana (A.t.; n=26 biologically independent samples), and the human cell line (H.s.; n=150 biologically independent samples). Data are presented as mean valuesSEM. b. The schematic workflow to compare 1-bp insertion patterns across S.v., A.t., and H.s. line through the next-generation sequencing assay. c The CRISPR targeted sequences of iPAM_T and G. The 4th nucleotide was highlighted in red with the PAM sequence underlined. d Heatmap analyses of the proportion of each inserted nucleotide type (nt) at the 4th position of the iPAM_T and G sites across S.v. (n=3), A.t. (n=3), and H.s. (n=3). The source data are provided in the Source Data file. b Created with BioRender.com released under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International license.

To compare with 1-bp insertion patterns in human cells, we analyzed the insertion profiles of 150 target sites previously reported from the human cell lines13. The results were largely consistent with the templated insertion model, showing the 1-bp insertion pattern with the 4th nucleotide duplications, while low levels of non-templated 1-bp insertions were observed at the target sites with 4th nucleotide as C or G (Fig.2a). Taken together, our observations revealed distinct 1-bp insertion patterns between plants and human cells. The 1-bp insertion profiles from plant species exhibited a higher incidence of non-templated insertions, deviating from the templated insertion model. Notably, the rates of non-templated insertions appeared to vary depending on the 4th nucleotide, increasing in the order of T, A, C, and G.

To further explore the distinctive 1-bp insertion profiles across species, we conducted direct comparisons by targeting identical CRISPR sites in Arabidopsis, Setaria, and human cell line, HEK293. This involved initially integrating the firefly luciferase gene and subsequently expressing the CRISPR-Cas9 expression cassette in the genomes of these three species (Fig.2b). We designed two CRISPR guide RNAs (gRNAs) to target overlapping sites located on opposite strands, referred to as inverted PAM (iPAM) targets, as described in previous research13 (Fig.2c). These two gRNAs, with one 4th position as T (iPAM_T) and the other as G (iPAM_G), represented the sequence contexts for the highest and lowest templated insertion rates observed in plants (Fig.2c). In Arabidopsis, CRISPR-Cas9 constructs were assembled with the firefly luciferase reporter gene in T-DNA. The resulting constructs were transformed using the floral dip-based stable transgenic approach. Three seedlings from each T1 transgenic group were collected for CRISPR mutation analysis at each target site. For Setaria viridis, a homozygous Setaria line with the firefly luciferase reporter gene integrated into the genome was obtained from previous research19. Individual CRISPR-Cas9 constructs were then transformed into protoplast cells isolated from the luciferase gene-containing plants. Transformed protoplasts were collected after 48h for the mutation assay with 3 replications for each target site. When insertion rates were examined, both CRISPR gRNAs induced substantial 1-bp insertions ranging from 33.8% to 89.0% for the iPAM_T site and from 33.4% to 74.4% for the iPAM_G site in three species (Supplementary Fig.2).

Next, we analyzed templated versus non-templated insertion patterns at each target site. In the HEK293 cells, consistent with the templated insertion model, templated insertions were predominantly presented at both target sites with rates of 97.0% and 84.8%, respectively (Fig.2d). However, in Arabidopsis and Setaria, predominant templated insertions were primarily observed at the iPAM_T site, ranging from 73.3% to 87%. At the iPAM_G site, non-templated insertions were predominant, accounting for 72.6% to 95.8% of 1-bp insertions in both plant species (Fig.2d). Taken together, these findings corroborated the observations from 59 individual target sites, revealing distinct plant-specific 1-bp insertion profiles. These profiles exhibited either templated or non-templated dominant patterns associated by the 4th nucleotide upstream of PAM.

As indicated by prior studies, both epigenetic and genetic factors could influence CRISPR-Cas9 induced mutation profiles15,17,20. To explore the mechanism underlying the distinctive 1-bp insertion profiles in plants, we investigated the impact of the chromatin states on these insertions. We used the multi-copy CRISPR target site (MCSite) system previously developed in Arabidopsis17. Two sets of MCSites, designated as MCSite_T and MCSite_G based on their 4th nucleotide, are located in diverse epigenetic contexts as described previously17. Individual sites within each MCsite family can be categorized into two major groups as either open and unmethylated or closed and methylated chromatin (Fig.3a, b).

Normalized 1-bp insertion rates were plotted for individual MCsite_T (a) and MCsite_G (b) sites (X-axis). The 20-bp targeted sequences with 3-bp underlined PAM sequences were shown with each plot. The normalized 1-bp insertion (ins.) rates were determined by dividing the number of reads containing 1-bp insertions by the total number of reads containing all types of indel mutations. Data are presented as mean valuesSEM from three independent plants. Heatmaps in the lower panel illustrated the proportion of each inserted nucleotide type (T, A, C, G) at the 4th position of individual MCsite_T (a) and MCsite_G (b) sites. Chromatin states of individual sites were categorized into Open and Unmethylated or Closed and Methylated groups. The source data are provided in the Source Data file.

When the 1-bp insertion rates of individual MCSites were examined, variations were found across different chromatin states as previously indicated17. For MCSite_T sites, insertion rates ranged from 7.9% to 26.8%, and for MCSite_G sites, they ranged from 41.9% to 58.9% (Fig.3a, b). In contrast, heatmap analysis of the 1-bp insertion profiles revealed a consistent pattern within each MCSite family. Specifically, for MCSite_T sites, templated insertions were predominantly observed across individual sites, regardless of their chromatin states (Fig.3a). On the other hand, all individual sites within the MCSite_G family exhibited a predominant 1-bp non-templated insertion pattern across different epigenetic contexts, ranging from 94.0% to 99.8% (Fig.3b). These results suggested that chromatin states may have limited impacts on CRISPR-Cas9 induced 1-bp insertion profile.

We then investigated the genetic factors contributing to the distinct 1-bp insertion profiles in plants. Previous studies have pointed to the X-family DNA polymerase, Pol, and its homolog as pivotal players in mediating 1-bp templated insertions in human and yeast cells13,15. A single copy of the Pol homolog was identified in both Arabidopsis and Setaria genomes through sequence homology searches21. No other X-family DNA polymerases were found in plants from the homology search. Phylogenetic analyses confirmed that this plant X-family DNA polymerase exhibited a close evolutionary relationship with Pol as opposed to other members, such as DNA Pol and Terminal deoxynucleotidyl Transferase (TdT) (Supplementary Fig.3 and Supplementary Data2).

To explore the involvement of the plant Pol homolog in CRISPR-Cas9 induced 1-bp insertions, we obtained an Arabidopsis T-DNA knock-out mutant line (atpol-1), previously characterized with no notable growth or physiological defects22,23. Using the wild type and the homozygous atpol-1 mutant Arabidopsis plants, we generated stable transgenic plants with the CRISPR-Cas9 T-DNA construct to target three distinct sites: the single-copy site in the Arabidopsis Cheletase I2 gene (AtCHLI2), as well as the MCSite_T and MCSite_G sites. Three T1 CRISPR-Cas9 transgenic plants from each genotype were used to survey CRISPR-induced mutations for each target site. The single-copy CHLI2 site would allow for a rapid assessment of the involvement of Pol in 1-bp insertions, while the two MCSites provided additional insights in different epigenetic contexts.

When we examined CRISPR-Cas9 mutagenesis at the CHLI2 site, both wild-type and mutant CRISPR-Cas9 plants displayed comparable overall mutagenesis rates, averaging 38.9% and 37.9%, respectively (Fig.4a). In wild-type plants, approximately 25.3% of indel mutations were identified as 1-bp insertions at the 4th position, with non-templated insertions being predominant at a rate of 65.2%, attributable to the G nucleotide at the 4th position in the CHLI2 site (Fig.4b; Supplementary Fig.4a). In contrast, in Pol mutant plants, the 1-bp insertion rates, encompassing both non-templated and templated insertions, were reduced to undetectable levels (0.2%; Fig.4b; Supplementary Fig.4b). Additionally, we explored the potential involvement of this Pol homolog in CRISPR-Cas9 induced deletions. As a result, we observed similar levels of deletions within three different deletion groups, 1-bp, 2 to 10-bp and more than 10-bp, between the wild-type and mutant plants (Fig.4c). Thus, the plant Pol homolog appeared to be the pivotal gene for CRISPR-Cas9 induced 1-bp insertions, operating in both templated and non-templated manners, with limited involvement in deletions.

a CRISPR-Cas9 mutation (mut.) rates between the wild type and atpol-1 mutant plants. The mutation rates (Y-axis) were determined by dividing the number of reads containing indel mutations by the total number of NGS reads. b Normalized 1-bp insertion rates between the wild type and atpol-1 mutant plants at the CHLI2 site. The normalized 1-bp insertion (ins.) rates were determined by dividing the number of reads containing 1-bp insertions by the total number of reads containing all types of indel mutations. c. Normalized deletion rates between the wild type (WT)and atpol-1 mutant plants at the CHLI2 site. The normalized proportion of deletion (Prop. of Del. as Y-axis) were determined by dividing the number of reads containing deletions within each category (1-bp, 2-10bp, or >10bp) by the total number of reads containing all types of deletions. d, e Normalized 1-bp insertion (ins.) rates between the wild type and atpol-1 mutant plants at the MCsite_T (d) and MCsite_G (e) sites. Heatmaps under the bar plots illustrate the proportion of each inserted nucleotide type (T, A, C, G) at the 4th position of individual MCsite_T (d) and MCsite_G (e) sites. Data are presented as mean valuesSEM from 3 independent plants. P-values were derived from unpaired one-tailed Students t test. The source data are provided in the Source Data file.

Furthermore, we investigated the role of this Pol homolog at additional CRISPR target sites within diverse epigenetic contexts. When examining the 1-bp insertion rates at the MCSite_T and G sites, we observed significant reductions of 1-bp insertions, both templated and non-templated, across all sites, irrespective of their chromatin states. In the MCSite_T sites, the 1-bp insertion rates decreased from an average of 19.5% in wild-type plants to 1.6% in the mutant plants, while in the MCSite_G sites, the rates were reduced from an average of 49.4% to 1.8% (Fig.4d, e). These results substantiated that the plant Pol homolog is responsible for both templated and non-templated 1-bp insertions regardless of chromatin states.

Next, we hypothesized that overexpression of Atpol could restore or even enhance the 1-bp insertion rates. To test this hypothesis, we generated stable transgenic plants by overexpressing the AtPol gene in the atpol-1 mutant plants. The AtPol coding sequence was driven under the constitutive Arabidopsis Ubiquitin-10 promoter and cloned into the final construct with a CRISPR-Cas9 expression cassette to target the CHLI2 and MCSite_T sites. Three T1 CRISPR-Cas9 transgenic plants with the atpol-1 mutant genotype were used to survey CRISPR-induced mutations for each target site. When 1-bp insertions were examined at the CHLI2 site, the AtPol overexpression plants exhibited a 1.6-fold increase compared to wild-type plants, with an average rate of 39.8% (Fig.5a). The 1-bp insertion profiles appeared similar between the AtPol overexpression and the wild-type plants, with non-templated insertions still being predominant at an average rate of 74.8% (Fig.5a). When examining the 1-bp insertions at the MCSite_T sites, overexpression of the AtPol transgene in the mutant plant appeared to restore 1-bp insertion rates to the levels observed in wild-type plants at five of seven MCSite_T sites. At the other 2 sites, sites 1 and 4, the 1-bp insertion rates exhibited substantial increases by 1.4 to 1.6 folds, respectively (Fig.5b). When comparing the 1-bp insertion profiles, similar insertion patterns were observed between the overexpression and wild-type plants with predominant templated insertions across nearly all the sites except for one site, site 8 (Fig.5b). These results confirmed that overexpression of AtPol could restore or may enhance CRISPR-Cas9 induced templated and non-templates 1-bp insertions in the knockout mutant plants, further validating its pivotal role in generating 1-bp insertions.

Normalized 1-bp insertion rates at CHLI2 (a) and MCsite_T (b) among three lines: wild-type plants(WT), Pol overexpression plants in the atpol-1 mutant(atpol OE), and Pol overexpression plants in the wild-type backgrounds(WT OE). The normalized 1-bp insertion rates (Y-axis) were determined by dividing the number of reads containing 1-bp insertions by the total number of reads containing all types of indel mutations. Heatmaps under each plot illustrated the proportion of each inserted nucleotide type (T, A, C, G) at the 4th position. Data are presented as mean valuesSEM from 3 independent plants. P-values were derived from unpaired one-tailed Students t test. The source data are provided in the Source Data file.

We further hypothesized that overexpression of this gene should have the potential to enhance 1-bp insertions in wild-type plants. To test this idea, we introduced the same overexpression construct to wild-type plants. Three T1 CRISPR-Cas9 transgenic plants with the wild-type background were used to survey CRISPR-induced mutations for each target site. At the CHLI2 site, we observed a similar increase in the 1-bp insertion rate between the overexpression wild-type plants and the overexpression mutant plants compared to the wild-type control plants (Fig.5a). At the MCSite_T sites, when comparing the 1-bp insertion rates between the overexpression wild-type plants and the wild-type control plants, we observed substantial increases in all seven sites by 1.2 to 2.0 folds (Fig.5b). When comparing the 1-bp insertion profiles, similar insertion patterns were observed between the overexpression wild-type plants, the overexpression mutant plants, and the wild-type control across all the sites, irrespective of their epigenetic states (Fig.5b). Taken together, these observations corroborated that overexpressing the Pol homolog in wild-type plants could further increase 1-bp insertions.

To gain insights into the mechanism(s) underpinning the distinct properties of Pol across species, we conducted protein sequence analyses by aligning AtPol with X-family DNA Polymerases in humans (Supplementary Fig.5). Previous studies have indicated two conserved motifs in human X-family DNA Polymerase that contribute to template dependency24. The first motif, identified as GSYRRG in template-dependent human DNA polymerases , features two amino acids, serine and tyrosine (SY), which are replaced by glycine and phenylalanine (GF) in the template-independent human TdT (Fig.6a and Supplementary Fig.5)24,25. The second motif, known as the YF motif, contains tyrosine and phenylalanine at the catalytically active sites of the DNA polymerases . In contrast, these two residues are changed to glycine and tryptophan (GW) in TdT (Supplementary Fig.5)24,25. When analyzing these motifs in DNA polymerase homologs from Arabidopsis, Setaria, Tobacco, and rice, the first motif was identical to the sequences in human Pol, while the second motif, characterized by alanine and tryptophan (AW), showed a closer resemblance to the GW motif found in human TdT (Fig.6a and Supplementary Fig.5). Thus, the plant Pol homologs appear to combine characteristic motifs from human Pol and TdT.

a Sequence alignment of two conserved motifs, SY and YF, across Human Pol, AtPol, SvPol, and human TdT. b Comparisons of templated versus non-templated insertion rates between the wild type AtPol and two variants, PolS366G/Y367F and PolA459Y/W460F at the CHLI2 site. The templated (indicated by orange) or non-templated insertion (indicated by green) rates (Y-axis) were determined by dividing the number of reads containing each type of 1-bp insertions by the total number of reads containing 1-bp insertions in each sample. c Normalized deletion rates between the wild type and two variants at the CHLI2 site. The normalized deletion rates (Y-axis) were determined by dividing the number of reads containing deletions within each category (1-bp, 2-10bp, or >10bp) by the total number of reads containing all types of deletions. Data are presented as mean valuesSEM from three independent plants. P-values were derived from unpaired one-tailed Students t test. The source data are provided in the Source Data file. d The proposed model for the dual activities of Pol in generating templated and non-templated 1-bp insertions. Step 1: CRISPR-Cas9 generates a blunt or staggered cut at the targeted site. Blunt-ended cleavages occur at the -3rd position upstream of the PAM (indicated by the red vertical lines) on both strands, while staggered cleavages take place with one cut at the 4th position on the non-targeted strand and the other cut at the -3rd position on the targeted strand, producing 5 1-nt overhangs. Step 2: The staggered product can be filled in by Pol with template-dependent activity. Step 3: The blunt-ended product can be processed by Pol with template-independent activity to extend 1-nt at the 3 end of each strand. After ligation and correction by c-NHEJ and mismatch repair, non-templated 1-bp insertions occur at the 4th position. Additionally, cleavage products could be processed through either perfect ligation, indicated by the curved arrowheads, or through resection to generate deletion, indicated by the purple dash lines.

The presence of both human Pol and TdT motifs could potentially contribute to the observed dual templated-dependent and independent activities in AtPol. We then hypothesized that the dual activities of AtPol could be modulated by modifying each motif individually. To test this hypothesis, we generated two variants of AtPol through site-directed mutagenesis on the respective motifs. The first variant, AtPolYF, was engineered by substituting Alanine and Tryptophan (AW) with Tyrosine and Phenylalanine (YF) at the second motif to mimic human Pol (Fig.6a). Similarly, the second variant, AtPolGF, was created to mimic human TdT by replacing Serine and Tyrosine (SY) with Glycine and Phenylalanine (YF) at the first motif (Fig.6a).

The coding sequence of each AtPol variant was cloned into the T-DNA vector described above, with the constitutive Arabidopsis Ubiquitin-10 promoter and a CRISPR-Cas9 expression cassette to target the CHLI2 site. We used an agrobacterium-mediated transient expression approach to transform individual T-DNA constructs into young seedlings of the atpol knock-out mutant, and then examined the CRISPR-Cas9 mutation profile at the CHLI2 site using the NGS assay (Supplementary Fig.6a). The average mutation rates from these samples are 17.3% (AtPolWT), 12.7% (AtPolGF) and 10.3% (AtPolYF), respectively (Supplementary6b). When analyzing templated versus non-templated 1-bp insertion patterns, the samples expressing the wild type AtPol gene exhibited higher proportions of non-templated insertions compared to those of templated insertions (57.6% non-templated insertions versus 42.4% templated insertions) consistent with the observations from the stable transgenic plants (Fig.5a, b). In contrast, the samples transformed with the AtPolYF variant demonstrated altered 1-bp insertion profiles with templated insertion proportions being significantly higher than those from the overexpression of the wildtype AtPol by 100% (86.0% versus 42.4%; Fig.6b and Supplementary Fig.6c, d). Conversely, the samples transformed with the AtPolGF variant displayed significantly higher proportions of non-templated insertions compared to the wild-type AtPol overexpression lines by 18% (67.9 % versus 57.6%; Fig.6b and Supplementary Fig.6c, d). Regarding the deletion profiles, no evident differences were observed within three different deletion groups, 1-bp, 2 to 10-bp and more than 10-bp, among AtPolWT and the two variants (Fig.6c).

Notably, the overall 1-bp insertion rates from the samples with each variant reduced to 4.4% and 5.5% compared to 31.7% in the wild-type AtPol overexpression control, suggesting the involvement of additional amino acids in regulating enzymatic activity. (Supplementary Fig.6e). Collectively, these observations align with our hypothesis that these two conserved motifs play crucial roles in modulating the dual template-dependent and independent activities of AtPol. Further investigation is required to refine the enzymatic activities of these variants.

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Dual activities of an X-family DNA polymerase regulate CRISPR-induced insertional mutagenesis across species - Nature.com

Fernandes, P. Use of antibiotic core structures to generate new and useful macrolide antibiotics. In Antibiotics Current Innovations and Future Trends (eds Snchez, S. & Demain, A. L.) (Caister Academic Press, 2015).

Bush, N. G., Diez-Santos, I., Abbott, L. R. & Maxwell, A. Quinolones: mechanism, lethality and their contributions to antibiotic resistance. Molecules 25, 5662 (2020).

Article CAS PubMed PubMed Central Google Scholar

Agouridas, C. et al. Synthesis and antibacterial activity of ketolides (6-O-methyl-3-oxoerythromycin derivatives): a new class of antibacterials highly potent against macrolide-resistant and -susceptible respiratory pathogens. J. Med. Chem. 41, 40804100 (1998).

Article CAS PubMed Google Scholar

Seiple, I. B. et al. A platform for the discovery of new macrolide antibiotics. Nature 533, 338345 (2016).

Article CAS PubMed PubMed Central Google Scholar

Pavlovic, D., Fajdetic, A. & Mutak, S. Novel hybrids of 15-membered 8a- and 9a-azahomoerythromycin A ketolides and quinolones as potent antibacterials. Bioorg. Med. Chem. 18, 85668582 (2010).

Article CAS PubMed Google Scholar

Fan, B. Z. et al. Design, synthesis and structureactivity relationships of novel 15-membered macrolides: quinolone/quinoline-containing sidechains tethered to the C-6 position of azithromycin acylides. Eur. J. Med. Chem. 193, 112222 (2020).

Article CAS PubMed Google Scholar

Barry, A. L. & Jones, R. N. Comparative in vitro activity of amifloxacin and five other fluoroquinolone antimicrobial agents and preliminary criteria for the disk susceptibility test. Eur. J. Clin. Microbiol. 6, 179182 (1987).

Article CAS PubMed Google Scholar

Yourassowsky, E., Van der Linden, M. P., Crokaert, F. & Glupczynski, Y. In vitro activity of pefloxacin compared to other antibiotics. J. Antimicrob. Chemother. 17, 1928 (1986).

Article CAS PubMed Google Scholar

Dinos, G. P. The macrolide antibiotic renaissance. Br. J. Pharmacol. 174, 29672983 (2017).

Article CAS PubMed PubMed Central Google Scholar

Vazquez-Laslop, N. & Mankin, A. S. How macrolide antibiotics work. Trends Biochem. Sci. 43, 668684 (2018).

Article CAS PubMed PubMed Central Google Scholar

Lin, J., Zhou, D., Steitz, T. A., Polikanov, Y. S. & Gagnon, M. G. Ribosome-targeting antibiotics: modes of action, mechanisms of resistance, and implications for drug design. Annu. Rev. Biochem. 87, 451478 (2018).

Article CAS PubMed PubMed Central Google Scholar

Schlunzen, F. et al. Structural basis for the antibiotic activity of ketolides and azalides. Structure 11, 329338 (2003).

Article CAS PubMed Google Scholar

Dunkle, J. A., Xiong, L., Mankin, A. S. & Cate, J. H. Structures of the Escherichia coli ribosome with antibiotics bound near the peptidyl transferase center explain spectra of drug action. Proc. Natl Acad. Sci. USA 107, 1715217157 (2010).

Article CAS PubMed PubMed Central Google Scholar

Bulkley, D., Innis, C. A., Blaha, G. & Steitz, T. A. Revisiting the structures of several antibiotics bound to the bacterial ribosome. Proc. Natl Acad. Sci. USA 107, 1715817163 (2010).

Article CAS PubMed PubMed Central Google Scholar

Svetlov, M. S. et al. Structure of Erm-modified 70S ribosome reveals the mechanism of macrolide resistance. Nat. Chem. Biol. 17, 412420 (2021).

Article CAS PubMed PubMed Central Google Scholar

Beckert, B. et al. Structural and mechanistic basis for translation inhibition by macrolide and ketolide antibiotics. Nat. Commun. 12, 4466 (2021).

Article CAS PubMed PubMed Central Google Scholar

Tu, D., Blaha, G., Moore, P. B. & Steitz, T. A. Structures of MLSBK antibiotics bound to mutated large ribosomal subunits provide a structural explanation for resistance. Cell 121, 257270 (2005).

Article CAS PubMed Google Scholar

Hansen, J. L. et al. The structures of four macrolide antibiotics bound to the large ribosomal subunit. Mol. Cell 10, 117128 (2002).

Article CAS PubMed Google Scholar

Svetlov, M. S. et al. High-resolution crystal structures of ribosome-bound chloramphenicol and erythromycin provide the ultimate basis for their competition. RNA 25, 600606 (2019).

Article CAS PubMed PubMed Central Google Scholar

Chen, C. W. et al. Structural insights into the mechanism of overcoming Erm-mediated resistance by macrolides acting together with hygromycin-A. Nat. Commun. 14, 4196 (2023).

Article CAS PubMed PubMed Central Google Scholar

Jenni, S. & Ban, N. The chemistry of protein synthesis and voyage through the ribosomal tunnel. Curr. Opin. Struct. Biol. 13, 212219 (2003).

Article CAS PubMed Google Scholar

Kannan, K., Vzquez-Laslop, N. & Mankin, A. S. Selective protein synthesis by ribosomes with a drug-obstructed exit tunnel. Cell 151, 508520 (2012).

Article CAS PubMed Google Scholar

Davis, A. R., Gohara, D. W. & Yap, M. N. Sequence selectivity of macrolide-induced translational attenuation. Proc. Natl Acad. Sci. USA 111, 1537915384 (2014).

Article CAS PubMed PubMed Central Google Scholar

Kannan, K. et al. The general mode of translation inhibition by macrolide antibiotics. Proc. Natl Acad. Sci. USA 111, 1595815963 (2014).

Article CAS PubMed PubMed Central Google Scholar

Sothiselvam, S. et al. Binding of macrolide antibiotics leads to ribosomal selection against specific substrates based on their charge and size. Cell Rep. 16, 17891799 (2016).

Article CAS PubMed PubMed Central Google Scholar

Almutairi, M. M. et al. Co-produced natural ketolides methymycin and pikromycin inhibit bacterial growth by preventing synthesis of a limited number of proteins. Nucleic Acids Res. 45, 95739582 (2017).

Article CAS PubMed PubMed Central Google Scholar

Franceschi, F., Kanyo, Z., Sherer, E. C. & Sutcliffe, J. Macrolide resistance from the ribosome perspective. Curr. Drug Targets Infect. Disord. 4, 177191 (2004).

Article CAS PubMed Google Scholar

Versalovic, J. et al. Mutations in 23S rRNA are associated with clarithromycin resistance in Helicobacter pylori. Antimicrob. Agents Chemother. 40, 477480 (1996).

Article CAS PubMed PubMed Central Google Scholar

Wand, G. & Taylor, D. E. Site-specific mutations in the 23S rRNA gene of Helicobacter pylori confer two types of resistance to macrolide-lincosamide-streptogramin B antibiotics. Antimicrob. Agents Chemother. 42, 19521958 (1998).

Article Google Scholar

Shallom, S. J. & Zelazny, A. M. Detection of mixed populations of clarithromycin-susceptible and -resistant Mycobacterium abscessus strains. J. Clin. Microbiol. 60, e0169421 (2022).

Article PubMed Google Scholar

McGuire, J. M. et al. Ilotycin, a new antibiotic. Antibiot. Chemother. (Northfield) 2, 281283 (1952).

CAS PubMed Google Scholar

Kirst, H. A. Introduction to the macrolide antibiotics. In Macrolide Antibiotics (eds Schnfeld, W. & Kirst, H. A.) (Birkhuser Verlag, 2002).

Iacoviello, V. R. & Zinner, S. H. Macrolides: a clinical overview. In Macrolide Antibiotics (eds Parnham, M. J. & Bruinvels, J.) (Birkhuser Verlag, 2002).

Tanikawa, T. et al. Synthesis and antibacterial activity of acylides (3-O-acyl-erythromycin derivatives): a novel class of macrolide antibiotics. J. Med. Chem. 44, 40274030 (2001).

Article CAS PubMed Google Scholar

Liang, J. H. et al. Structureactivity relationships of novel alkylides: 3-O-arylalkyl clarithromycin derivatives with improved antibacterial activities. Eur. J. Med. Chem. 49, 289303 (2012).

Article CAS PubMed Google Scholar

Magee, T. V. et al. Novel 3-O-carbamoyl erythromycin A derivatives (carbamolides) with activity against resistant staphylococcal and streptococcal isolates. Bioorg. Med. Chem. Lett. 23, 17271731 (2013).

Article CAS PubMed Google Scholar

Tang, D. et al. Design, synthesis, and antibacterial activities of novel 3,6-bicyclolide oximes: length optimization and zero carbon linker oximes. Bioorg. Med. Chem. Lett. 18, 50785082 (2008).

Article CAS PubMed Google Scholar

Bryskier, A. & Denis, A. Ketolides: novel antibacterial agents designed to overcome resistance to erythromycin A within Gram-positive cocci. In Macrolide Antibiotics (eds Schnfeld, W. & Kirst, H. A.) (Birkhuser Verlag, 2002).

Fernandes, P., Martens, E., Bertrand, D. & Pereira, D. The solithromycin journeyit is all in the chemistry. Bioorg. Med. Chem. 24, 64206428 (2016).

Article CAS PubMed Google Scholar

Capobianco, J. O. et al. Studies of the novel ketolide ABT-773: transport, binding to ribosomes, and inhibition of protein synthesis in Streptococcus pneumoniae. Antimicrob. Agents Chemother. 44, 15621567 (2000).

Article CAS PubMed PubMed Central Google Scholar

Llano-Sotelo, B. et al. Binding and action of CEM-101, a new fluoroketolide antibiotic that inhibits protein synthesis. Antimicrob. Agents Chemother. 54, 49614970 (2010).

Article CAS PubMed PubMed Central Google Scholar

Douthwaite, S. Structureactivity relationships of ketolides vs. macrolides. Clin. Microbiol. Infect. 7, 1117 (2001).

Article CAS PubMed Google Scholar

Svetlov, M. S., Cohen, S., Alsuhebany, N., Vazquez-Laslop, N. & Mankin, A. S. A long-distance rRNA base pair impacts the ability of macrolide antibiotics to kill bacteria. Proc. Natl Acad. Sci. USA 117, 19711975 (2020).

Article CAS PubMed PubMed Central Google Scholar

Ma, C. X. et al. Design, synthesis and structureactivity relationships of novel macrolones: hybrids of 2-fluoro 9-oxime ketolides and carbamoyl quinolones with highly improved activity against resistant pathogens. Eur. J. Med. Chem. 169, 120 (2019).

Article PubMed Google Scholar

Liu, X. P. et al. Design and synthesis of novel macrolones bridged with linkers from 11,12-positions of macrolides. Bioorg. Med. Chem. Lett. 68, 128761 (2022).

Article CAS PubMed Google Scholar

Hutinec, A. et al. Novel 8a-aza-8a-homoerythromycin4-(3-substituted-amino)propionates with broad spectrum antibacterial activity. Bioorg. Med. Chem. Lett. 20, 32443249 (2010).

Article CAS PubMed Google Scholar

Drlica, K., Malik, M., Kerns, R. J. & Zhao, X. Quinolone-mediated bacterial death. Antimicrob. Agents Chemother. 52, 385392 (2008).

Article CAS PubMed Google Scholar

Aldred, K. J., Kerns, R. J. & Osheroff, N. Mechanism of quinolone action and resistance. Biochemistry 53, 15651574 (2014).

Article CAS PubMed Google Scholar

Miotto, P., Cirillo, D. M. & Migliori, G. B. Drug resistance in Mycobacterium tuberculosis: molecular mechanisms challenging fluoroquinolones and pyrazinamide effectiveness. Chest 147, 11351143 (2015).

Article PubMed Google Scholar

Machalek, D. A. et al. Prevalence of mutations associated with resistance to macrolides and fluoroquinolones in Mycoplasma genitalium: a systematic review and meta-analysis. Lancet Infect. Dis. 20, 13021314 (2020).

Article CAS PubMed Google Scholar

Lungu, I. A., Moldovan, O. L., Biris, V. & Rusu, A. Fluoroquinolones hybrid molecules as promising antibacterial agents in the fight against antibacterial resistance. Pharmaceutics 14, 1749 (2022).

Article CAS PubMed PubMed Central Google Scholar

Pavlovic, D. & Mutak, S. Discovery of 4-ether linked azithromycinquinolone hybrid series: influence of the central linker on the antibacterial activity. ACS Med. Chem. Lett. 2, 331336 (2011).

Article CAS PubMed PubMed Central Google Scholar

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Macrolones target bacterial ribosomes and DNA gyrase and can evade resistance mechanisms - Nature.com

AGAINST LINDSAY CLANCY STANDS NOW. LINDSAY CLANCYS APPEARANCE WAS WAIVED, MEANING SHE DID NOT HAVE TO ATTEND TODAYS HEARING AT SUPERIOR COURT HERE IN BROCKTON. AND SHE DID NOT. CLANCY IS RECEIVING TREATMENT AT A STATE HOSPITAL. KEVIN REDDINGTON ON BEHALF OF THE DEFENDANT, HER ATTORNEY, KEVIN REDDINGTON, IS SEEN HERE APPEARING ON HER BEHALF. HE AND THE PROSECUTOR WERE UPDATING THE JUDGE ABOUT EVIDENCE IN THIS CASE, SPECIFICALLY DNA EVIDENCE THAT IS STILL IN THE TESTING QUEUE. ACCORDING TO THE PROSECUTOR CLANCY IS CHARGED WITH THREE COUNTS EACH OF MURDER AND STRANGULATION FOR KILLING HER. THREE CHILDREN IN THEIR THEIR HOME IN DUXBURY IN JANUARY OF 2023. HER HUSBAND MADE THE DISCOVERY. HER ATTORNEY, KEVIN REDDINGTON, HAS PREVIOUSLY STATED THAT HIS CLIENT HAD POSTPARTUM DEPRESSION AND WAS OVERMEDICATED AT THE TIME, BUT THE PROSECUTION HAS DESCRIBED LINDSAY CLANCYS ACTIONS ON THE DAY OF THE KILLINGS AS DELIBERATE AND PREMEDITATED. ITLL BE AT 2 P.M. HERE IN THIS COURTROOM. THE CASE RETURNS TO COURT ON SEPTEMBER 27TH. ON THAT DATE, LIKE TODAY, LINDSAY CLANCY IS NOT REQUIRED TO ATTEND. REPORTING IN BROCKTON TODD

Attorneys waiting on DNA evidence in case of Duxbury, Massachusetts mother Lindsay Clancy accused of killing 3 kids

Updated: 1:03 PM EDT Jul 26, 2024

A hearing was held Friday in the case of the Massachusetts mother accused of strangling her three young children with exercise bands and jumping out of the window of the family's Duxbury home last year.Lindsay Clancy is facing three counts each of murder and strangulation in the January 2023 deaths of her 5-year-old daughter Cora, 3-year-old son Dawson and 7-month-old son Callan.Her attorney, Kevin Reddington, said his client had postpartum depression and was overmedicated at the time. However, prosecutors described Lindsay Clancy's actions on the day of the killings as deliberate and premeditated.During a status hearing Friday in Brockton Superior Court, the defense and prosecution updated the judge about evidence in the case, specifically DNA evidence that is still in the testing queue, according to the prosecutor.Lindsay Clancy has pleaded not guilty and is receiving treatment at Tewksbury State Hospital while her attorneys wade through nearly 300 pages of documents that were released last year.They include 11 search warrants that sought access to collect exercise bands, medications, phones, computers, notebooks, cameras, a bloody knife and other physical evidence from inside the Clancy home. According to the documents, Lindsay Clancy's notebooks contained documentation of her medications and thoughts of suicide. There's also mention that she used her phone to document her mental state and research ways to kill. Prosecutors allege on the evening of the alleged attack, Clancy's husband, Patrick, left the couple's home to pick up medications and dinner, and when he returned, he found Clancy outside."He called 911. During this time, he asked the defendant, 'What did you do?' She responded to him, 'I tried to kill myself and jumped out the window.' During the 911 call, Patrick can be heard asking the defendant, 'Where are the kids?' He later told police that she replied 'in the basement,'" Assistant District Attorney Jennifer Sprague said during Clancy's arraignment.Patrick Clancy allegedly found his children in the basement. All three had exercise bands around their necks and died of "ligature strangulation," Sprague said.The case is scheduled to return to court on Sept. 27. Lindsay Clancy is not required to attend. If you or someone you know is struggling with suicidal thoughts or mental health matters, please call the Suicide & Crisis Lifeline at 988 or 800-273-8255 to connect with a trained counselor or visit 988lifeline.org.

A hearing was held Friday in the case of the Massachusetts mother accused of strangling her three young children with exercise bands and jumping out of the window of the family's Duxbury home last year.

Lindsay Clancy is facing three counts each of murder and strangulation in the January 2023 deaths of her 5-year-old daughter Cora, 3-year-old son Dawson and 7-month-old son Callan.

Her attorney, Kevin Reddington, said his client had postpartum depression and was overmedicated at the time. However, prosecutors described Lindsay Clancy's actions on the day of the killings as deliberate and premeditated.

During a status hearing Friday in Brockton Superior Court, the defense and prosecution updated the judge about evidence in the case, specifically DNA evidence that is still in the testing queue, according to the prosecutor.

Lindsay Clancy has pleaded not guilty and is receiving treatment at Tewksbury State Hospital while her attorneys wade through nearly 300 pages of documents that were released last year.

They include 11 search warrants that sought access to collect exercise bands, medications, phones, computers, notebooks, cameras, a bloody knife and other physical evidence from inside the Clancy home.

According to the documents, Lindsay Clancy's notebooks contained documentation of her medications and thoughts of suicide. There's also mention that she used her phone to document her mental state and research ways to kill.

Prosecutors allege on the evening of the alleged attack, Clancy's husband, Patrick, left the couple's home to pick up medications and dinner, and when he returned, he found Clancy outside.

"He called 911. During this time, he asked the defendant, 'What did you do?' She responded to him, 'I tried to kill myself and jumped out the window.' During the 911 call, Patrick can be heard asking the defendant, 'Where are the kids?' He later told police that she replied 'in the basement,'" Assistant District Attorney Jennifer Sprague said during Clancy's arraignment.

Patrick Clancy allegedly found his children in the basement. All three had exercise bands around their necks and died of "ligature strangulation," Sprague said.

The case is scheduled to return to court on Sept. 27. Lindsay Clancy is not required to attend.

If you or someone you know is struggling with suicidal thoughts or mental health matters, please call the Suicide & Crisis Lifeline at 988 or 800-273-8255 to connect with a trained counselor or visit 988lifeline.org.

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Attorneys waiting on DNA evidence in case of Duxbury mom accused of killing 3 kids - WCVB Boston

Years after the city of Tulsa began exhumations at a cemetery long believed to hold the unmarked graves of numerous Tulsa Race Massacre victims, DNA has confirmed the speculation and identified a victim.

Its the first such identification in 23 years revealing a 20-something Black man whose last known residence was in Utah.

The man C.L. Daniel had been buried in an unmarked grave at the Oaklawn Cemetery, east of downtown and about 1.5 miles away from Tulsas Greenwood District, in what was known more than a century ago as Black Wall Street.

DNA collected from Daniels living relatives and submitted to GEDmatch.com and FamilyTreeDNA.com connected his name to the remains found in burial 3, according to Intermountain Forensics, the nonprofit laboratory contracted to do the DNA analysis.

From there, researchers learned more about him. For instance, Daniel, who was Black, had been living in Ogden prior to visiting Tulsa, and he was a World War I veteran with a disability that hindered his ability to find work.

In one letter, Daniel wrote that if the government had a job he could perform, he would work it until I die, Intermountain Forensics said in a news release.

But much else about Daniel, including why he was in Tulsa, has been lost to time, like many facts about the massacre itself.

All 35 blocks of the affluent Black, Tulsa neighborhood were razed between May 31 and June 1, 1921, when, according to research posted by the nonprofit digital library JSTOR, white rioters looted and burned the district, while deputized white people arrested and interned Black people in detention camps. Planes flew low over the neighborhood, with some witnesses claiming they dropped accelerant on the already burning neighborhood.

The Tulsa Historical Society and Museum said that around 300 people were killed in the massacre many buried in unmarked graves.

The massacre began with an encounter between a Black man, Dick Rowland, and a white woman, Sarah Page, in an elevator in downtown Tulsa on May 30. Rowland was arrested.

The details of what followed vary from person to person, according to the Tulsa Historical Society. Accounts of an incident circulated among the citys white community during the day and became more exaggerated with each telling.

The next day, an inflammatory Tulsa Tribune article stoked tensions, and racially segregated armed mobs faced off in front of the courthouse, where Rowland was jailed.

Shots were fired and the outnumbered African Americans began retreating to the Greenwood District, according to the historical society. And the white rioters followed. Greenwood was left in charred shambles.

Dick Rowland, Sarah Page and an unknown gunman were the sparks that ignited a long smoldering fire, the historical society said. Jim Crow, jealousy, white supremacy, and land lust, all played roles in leading up to the destruction and loss of life on May 31 and June 1, 1921.

In 2001, Oklahoma officials brought together a commission to investigate the massacre, culminating in a 200-page document that outlined how the massacre began and its repercussions. Daniel was the first victim identified since that report was released, according to a news release.

The city of Tulsa began exhuming graves in the Oaklawn Cemetery in 2018, in hopes to learn more about those killed in the massacre. Daniels remains were exhumed in 2021.

C.L. Daniel was a veteran who served our country in World War I, who was killed in the 1921 Tulsa Race Massacre, and whose family did not know where he had been buried for the last 103 years - until [now], Tulsa Mayor G.T. Bynum said in a news release. The Salt Lake Tribune was unable to contact Daniels family members.

In addition to DNA, analysts also verified Daniels identity using physical evidence, including notes from relatives and one from a Georgia congressman, a representative from the state where Daniels mother was living at the time.

That letter stated, C. L. Daniel was killed in Oklahoma some time shortly after his discharge Another letter, written by an attorney representing Daniels mother and sent to the U.S. Veterans Administration, stated Daniel died in a race riot in Tulsa Oklahoma in 1921.

Records show Daniel was drafted in the Army in 1918 and was discharged in 1919. He wasnt married and didnt have any children.

Letters also indicate that prior to his death, Daniel had been in Utah working to find a job and a way back home to his mother in Georgia, according to a news release.

Daniels remains will stay where theyve been buried at the Oaklawn Cemetery until his family members decide on a proper burial. Meanwhile, excavators have begun the next round of exhumations at the cemetery in hopes to identify more victims, according to a news release.

Intermountain Forensics continues to analyze DNA taken from the burial sites. They are also working to see if any of Daniels other relatives were connected to Tulsa or the massacre.

More here:
Utahn is first ever Tulsa Race Massacre victim identified through DNA - Salt Lake Tribune

A web of roads encircles the Earth and stretches 40 million miles. In Crossings, a new book by environmental journalist Ben Goldfarb, tarmac is exposed for the planet-shaping force it is one that has polluted rivers and the air, emptied soils and woodlands, and struck fear into wild animals.

Roads are one of the most ubiquitous man-made features, existing on every continent and in most habitats. Their effects dont end at the paved periphery either. While roads cover 1% of land in the US, their ecological effects disruptive noise, foul air and habitat fragmentation, to name a few extend over 20% of the country, according to Goldfarb.

Furthermore, transport is among the fastest-growing contributors to climate change, wildlife collisions with vehicles cause more than 59,000 human injuries in the US each year, and road design disproportionately burdens the health and welfare of low-income and minority communities.

As an ecologist who studies their effect on wildlife, I applaud Goldfarb for weaving such a rich commentary on roads. Crossings could not be more timely: the biggest expansion of infrastructure in history is underway and road networks are growing fastest of all.

An estimated 25 million miles of new road lanes will be built worldwide by 2050. Roadbuilding will have to change drastically for any hope of preserving biodiversity and halting climate change.

But first, we must face some uncomfortable truths.

Roads bring us into contact with animals we rarely see. Unfortunately, theyre usually dead.

Despite roadkill being a common sight, its consequences are easily ignored. Vehicles directly kill more land-based vertebrate animals than anything else humans do, be it poaching, hunting, trapping or causing fires.

Roads and their effects are so pervasive that they even leave a mark in DNA. For example, after years of flying in between and over cars, cliff swallows have evolved shorter wings to nimbly avoid getting hit by them.

Swifts are, in one sense, a success story. Other species havent been so lucky. Throughout Crossings, roads are described as knives, scalpels and guillotines that carve up the landscape. Nor are waterways spared: culverts (tunnels that carry streams and rivers under roads) are so ubiquitous and faulty that they have thwarted fish migrating upstream to breed, and caused populations to collapse.

Even the sounds that roads create can dramatically change animal lives. Robins, wrens and great tits raise the frequency of their calls to be heard above cars. One study found that some birds spent so much additional energy around noisy roads, trying to listen for predators, that they were too tired to forage and starved.

Highways and dirt roads enable deforestation, hunting, urban sprawl and tourism. More than 50 studies have shown what this means for wildlife. Elk, bears and wolves have learned to associate cars with the hunters they carry. These animals avoid roads not because of the vehicles, but because of the people inside them.

Roads have created a new landscape of fear, according to ecologists; one that governs how animals behave in an environment. Species may avoid being killed by remaining on one side of a busy road but by clinging to safety like this, they increase the rate at which natural habitats are being broken up.

Goldfarb also explores the racist legacies of interstate highways that were bulldozed through predominantly Black and Latino neighbourhoods in his native US, dividing families and causing extensive economic damage. One such neighbourhood in the Bronx is tightly bound by three expressways. Here, asthma kills three times more people than the national average.

And a paper published by the Paris regional health agency calculated that even the noise from roads shortens the lifespan of some Parisians by up to three years.

Solutions in road ecology have, to date, been more reactive than proactive. This is because many roads were built long before their harmful effects were understood. For example, the US Forest Service has only recently started to remove a proportion of the 370,000 miles of road it manages.

The difficulty with mitigating the effects of roads is in changing driver behaviour. Road signs rarely slow drivers down. In fact, some people intentionally swerve to hit animals. The best solutions remove choice altogether.

In one example, Goldfarb praises the SP-139 highway in Carlos Botelho state park in southern Brazil. When I worked there in 2015 and 2016, I saw how this highway was closed at night, and that the road was designed to wind and undulate, forcing drivers to slow down. The Brazilian government dared to inconvenience drivers, and wildlife was the better for it.

The dream of road ecology is a dream of connectivity. Wildlife crossings, bridges and other man-made structures that go over or under roads offer an opportunity for animals to cross safely. They are, as Goldfarb says, the work of literal and metaphoric bridge-building. In Banff National Park, Canada, 44 wildlife crossings have helped cut the number of collisions between cars and large mammals by more than 80%.

Goldfarb proposes building more of these. But, while there are several case studies demonstrating their success, I doubt that crossings are a panacea for the coming infrastructure tsunami.

Research shows that what works for a handful of species may not work for others. Foxes and feral cats in Australia used road crossings on average three times more frequently than scientists expected. In stark contrast, 40% of surrounding animal species were not detected at a crossing at all. Whether effective or not, wildlife crossings could become a new form of greenwashing that excuses more roads and more destruction.

To wildlife, roads spell death and division. People are the cause but they can also be the solution. I share Goldfarbs optimism and hope road ecology will grow into a global campaign for a kinder, more connected world.

In the meantime, I urge you to read Crossings.

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How roads are reshaping and scarring our planet, and even changing animals DNA - The Conversation

When considering human settlements on the Moon, Mars and further afield, much attention is given to the travel times, food and radiation risk. Well undoubtedly face a harsh environment in deep space and some thinkers have been pointing to genome editing as a way to ensure that humans can tolerate the severe conditions as they venture further into the solar system.

In January, I was fortunate to attend a much-anticipated debate between astronomer royal Lord Martin Rees and Mars exploration advocate Dr Robert Zubrin. The event at the British Interplanetary Society took on the topic of whether the exploration of Mars should be human or robotic.

In a recent book called The End of Astronauts, Lord Rees and co-author Donald Goldsmith outline the benefits of exploration of the solar system using robotic spacecraft and vehicles, without the expense and risk of sending humans along for the ride. Dr Zubrin supports human exploration. Where there was some agreement was over Reess advocacy of using gene editing technology to enable humans to overcome the immense challenges of becoming an interplanetary species.

Our genome is all the DNA present in our cells. Since 2011, we have been able to easily and accurately edit genomes. First came a molecular tool called Crispr-Cas9, which today can be used in a high school lab for very little cost and has even been used on the International Space Station. Then came techniques called base and prime editing, through which miniscule changes can be made in the genome of any living organism.

The potential applications of gene editing for allowing us to travel further are almost limitless. One of the most problematic hazards astronauts will encounter in deep space is a higher dosage of radiation, which can cause havoc with many processes in the body and increase the longer-term risk of cancer.

Perhaps, using genome editing, we could insert genes into humans from plants and bacteria that are able to clean up radiation in the event of radioactive waste spills and nuclear fallout. It sounds like science fiction, but eminent thinkers such as Lord Rees believe this is key to our advancement across the solar system.

Identifying and then inserting genes into humans that slow down aging and counter cellular breakdown could also help. We could also engineer crops that resist the effects of exposure to radioactivity as crews will need to grow their own food. We could also personalise medicine to an astronauts needs based on their particular genetic makeup.

Imagine a future where the human genome is so well understood it has become pliable under this new, personalised medicine.

Tardigrades are microscopic animals sometimes referred to as water bears. Experiments have shown that these tiny creatures can tolerate extreme temperatures, pressures, high radiation and starvation. They can even tolerate the vacuum of space.

Geneticists are eager to understand their genomes and a paper published in Nature sought to uncover the key genes and proteins that give the miniature creatures this extraordinary stress tolerance. If we could insert some of the genes involved into crops, could we make them tolerant to the highest levels of radiation and environmental stress? Its worth exploring.

Even more intriguing is whether inserting tardigrade genes into our own genome could make us more resilient to the harsh conditions in space. Scientists have already shown that human cells in the lab developed increased tolerance to X-ray radiation when tardigrade genes were inserted into them.

Transferring genes from tardigrades is just one speculative example of how we might be able engineer humans and crops to be more suited to space travel.

Well need much more research if scientists are ever to get to this stage. However, in the past, several governments have been keen to enforce tight restrictions on how genome editing is used, as well as on other technologies for inserting genes from one species into another.

Germany and Canada are among the most cautious, but elsewhere restrictions seem to be relaxing.

In November 2018, the Chinese scientist He Jiankui announced that he had created the first gene edited babies. He had introduced a gene into the unborn twins that confers resistance to HIV infection.

The scientist was subsequently jailed. But he has since been released and allowed to carry out research again.

In the new space race, certain countries may go to lengths with genome editing that other nations, especially in the west where restrictions are already tight, may not. Whoever wins would reap enormous scientific and economic benefits.

If Rees and the other futurists are right, this field has the potential to advance our expansion into the cosmos. But society will need to agree to it.

Its likely there will be opposition, because of the deep-seated fears of altering the human species forever. And with base and prime editing now having advanced the precision of targeted gene editing, its clear that the technology is moving faster than the conversation.

One country or another is likely to take the leap where others pull back from the brink. Only then will we find out just how viable these ideas really are. Until then, we can only speculate with curiosity, and perhaps excitement too.

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If we want to settle on other planets, well have to use genome editing to alter human DNA - The Conversation

Feral DNA may help domestic rabbits thrive in the wild, a new analysis suggests, shedding new light on the evolution of an animal that can cause major environmental destruction.

Publishing in Nature Ecology & Evolution, researchers looked at the relationship between rabbit genetics and feralization, an evolutionary process in which the descendants of domestic rabbits that live in the wild shed characteristics that helped them survive in human settings, taking on those of feral animals instead.

The researchers sequenced DNA from 297 rabbits in six populations in South America, Europe and Australia, all places where rabbits were introduced within the past 200 years. They compared the genetic information with the DNA of other wild and domestic rabbits.

To their surprise, the researchers discovered that all of the rabbits studied had a mixture of wild and domestic DNA.

This was not what we had expected to find, Leif Andersson, a professor of veterinary integrative biosciences at the Texas A&M University School of Veterinary Medicine and Biomedical Sciences and a co-author of the study, says in a news release. We expected that feral rabbits were domestic rabbits that have somehow relearned how to live in the wild. But our findings show us that these rabbits already had a portion of wild DNA helping them survive in nature.

The researchers found that the descendants of domestic rabbits quickly shed the docility and coat colors that humans prefer in pet bunnies, trading them for characteristics that help them thrive in the wild.

That might explain why rabbits in Australia, a continent now overrun with wild bunnies, didnt immediately take over when domestic rabbits were first introduced. The rabbit population surged only after 1859, when the introduction of just 24 wild and domestic rabbits began a population boom that continues to this day.

Today, there are at least 150 million feral rabbits in Australia. The animals are considered invasive pests, competing with livestock and native animals, destroying native plants and crops, and even affecting groundwater absorption.

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Domesticated rabbits can rewild thanks to feral DNA, study finds - The Washington Post

The road to Olympic gold might seem paved with sweat and genetics, but for three-time gymnastics champion Gabby Douglas, the reality is more complex.

During her most recent comeback attempt, she encountered a fascinating paradox: While dedication and diet have undoubtedly shaped her athletic potential, the precise influence of her own genetics remained a mystery, leaving Douglas to wonder how she can optimize her performance moving forward. Douglas has joined forces with Ancestry and 17 world-class athletes to unlock the secrets her genes hold, aiming to optimize her performance and conquer the podium once again.

Douglas first set foot in the world of gymnastics courtesy of her sister, Arielle. A triple threat with a background in cheerleading, gymnastics, and ballroom dancing, Arielles skills captivated Douglas, sparking a fire that would soon become a lifelong passion.

At just three years old, Douglas was already tumbling into the world of gymnastics, her tiny hands and feet pattering against the mat as she chased after her sisters lead. Arielle, delighted to share her own love for the sport, took Douglas under her wing and began teaching her the basics.

As Douglas grew, so did her skills. By the time she was six years old, she was already taking matters into her own hands or rather, her own feet teaching herself new tricks and stunts like cartwheels and backflips. By eight, she won her first competition.

Douglas would go on to have an illustrious career, securing three Olympic gold medals and making history as the first Black American gymnast to win gold in the Olympic all-around in London in 2012. She also became the first U.S. gymnast to win gold in both the individual all-around and team competitions at the same Olympics cementing herself as one of the greatest gymnasts ever at 20 years old.

But Douglas road was not easy. During her career, Douglas endured vitriolic media criticism, including racist, sexist, and bigoted comments about her hair. In 2016 she stepped away from competition to prioritize her mental health, a pivotal decision that would ultimately allow her to reclaim her identity, rewrite her narrative (on her own terms), and rekindle her passion for gymnastics.

This year, Douglas began her comeback to competition after an eight-year hiatus, only for her attempt to be derailed by a serious ankle injury before the U.S. National Championships. But this injury is not stopping the 28-year-old gymnastics legend. Despite the setback, Douglas is determined to continue competing, telling theGrio that while bittersweet, this obstacle marks the end of a chapter, but not the end of her gymnastics career. Shes set her sights on the 2028 Olympics, and if successful, at age 32, Douglas would make history as the oldest gymnast to compete in the Olympic Games. To defy the odds, though, shes on a mission to unlock the secrets her genes hold, seeking answers on how to optimize her athletic potential and reach the peak of her game once again.

Douglas is not unique in her quest to repeat or return to athletic dominance, but a lingering question persists: What drives the competitive fire within her, and how do genetic factors influence an athletes ability to acquire new skills and re-master old ones as they navigate the physical changes that come with age?

Douglas athleticism and competitive nature appear to run deep, with a great-great-grandfather who served as a porter for the 1912 Mens National Gymnastics team. Modern science backs this up: a recent DNA test by Ancestry reveals genes linked to athleticism in Douglas. These include a natural inclination towards team sports, alongside qualities essential for elite gymnasts like flexibility, strength, and a high overall athletic potential.

Still, the test did highlight areas for improvement, including balance and technique. I was surprised to see that I wasnt naturally good at something like balance, said Douglas who reflected on the results during our interview, but when I think about my career, I wasnt very good at the beam. I had to work hard to be consistent and solid, she continued.

The test results also revealed that Douglas has a remarkably high pain tolerance a cringeworthy finding, given the unfounded and long-standing myth that Black women experience pain differently than women of other races. However, Douglas own experiences confirm this notion.

I have a high pain tolerance, she said. In 2015, I hyperextended my leg while vaulting and heard a loud snap. Despite being compromised, I continued to compete. It wasnt until after returning from Scotland that I realized I needed to get [my injury] checked out and it turned out I had a significant break in my leg. Douglas has since learned the importance of addressing injuries promptly, acknowledging that the intensity of competition can lead to ignoring or downplaying injuries. However, she is now prioritizing the healing of her current foot injury, ensuring it is fully recovered before returning to competition.

As Douglas moves forward, she plans to use her newfound insights to focus on improving her weaknesses and refining her skills, but her primary focus is on supporting her teammates as they compete in this years Paris Olympics. Theres no doubt, however, that the flying squirrel (a nickname given to her by national team coordinator Martha Karolyi), armed with new knowledge, her trademark work ethic, and determination, is well-poised to make a triumphant comeback to the sport she loves. Well be watching.

Dr. Shamard Charles is the executive director of graduate studies in public health at St. Francis College and sits on the Medical Advisory Board of Verywell Health (Dot Dash-Meredith). He is also host of the health podcast, The Revolutions Within Us. He received his medical degree from the Warren Alpert Medical School of Brown University and his Masters of Public Health from Harvards T.H. Chan School of Public Health. Previously, he spent three years as a senior health journalist for NBC News and served as a Global Press Fellow for the United Nations Foundation. You can follow him on Instagram @askdrcharles or Twitter @DrCharles_NBC.

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How Gabby Douglas is using DNA tech in her comeback bid - TheGrio