The mutational landscape of human somatic and germline cells.

Over the course of an individual's lifetime, normal human cells accumulate mutations1. Here we compare the mutational landscape in 29 cell types from the soma and germline using multiple samples from the same individuals. Two ubiquitous mutational signatures, SBS1 and SBS5/40, accounted for the majority of acquired mutations in most cell types, but their absolute and relative contributions varied substantially. SBS18, which potentially reflects oxidative damage2, and several additional signatures attributed to exogenous and endogenous exposures contributed mutations to subsets of cell types. The rate of mutation was lowest in spermatogonia, the stem cells from which sperm are generated and from which most genetic variation in the human population is thought to originate. This was due to low rates of ubiquitous mutational processes and may be partially attributable to a low rate of cell division in basal spermatogonia. These results highlight similarities and differences in the maintenance of the germline and soma.

Somatic mutation landscapes at single-molecule resolution.

Somatic mutations drive the development of cancer and may contribute to ageing and other diseases1,2. Despite their importance, the difficulty of detecting mutations that are only present in single cells or small clones has limited our knowledge of somatic mutagenesis to a minority of tissues. Here, to overcome these limitations, we developed nanorate sequencing (NanoSeq), a duplex sequencing protocol with error rates of less than five errors per billion base pairs in single DNA molecules from cell populations. This rate is two orders of magnitude lower than typical somatic mutation loads, enabling the study of somatic mutations in any tissue independently of clonality. We used this single-molecule sensitivity to study somatic mutations in non-dividing cells across several tissues, comparing stem cells to differentiated cells and studying mutagenesis in the absence of cell division. Differentiated cells in blood and colon displayed remarkably similar mutation loads and signatures to their corresponding stem cells, despite mature blood cells having undergone considerably more divisions. We then characterized the mutational landscape of post-mitotic neurons and polyclonal smooth muscle, confirming that neurons accumulate somatic mutations at a constant rate throughout life without cell division, with similar rates to mitotically active tissues. Together, our results suggest that mutational processes that are independent of cell division are important contributors to somatic mutagenesis. We anticipate that the ability to reliably detect mutations in single DNA molecules could transform our understanding of somatic mutagenesis and enable non-invasive studies on large-scale cohorts.

The mutational landscape of normal human endometrial epithelium.

All normal somatic cells are thought to acquire mutations, but understanding of the rates, patterns, causes and consequences of somatic mutations in normal cells is limited. The uterine endometrium adopts multiple physiological states over a lifetime and is lined by a gland-forming epithelium1,2. Here, using whole-genome sequencing, we show that normal human endometrial glands are clonal cell populations with total mutation burdens that increase at about 29 base substitutions per year and that are many-fold lower than those of endometrial cancers. Normal endometrial glands frequently carry 'driver' mutations in cancer genes, the burden of which increases with age and decreases with parity. Cell clones with drivers often originate during the first decades of life and subsequently progressively colonize the epithelial lining of the endometrium. Our results show that mutational landscapes differ markedly between normal tissues-perhaps shaped by differences in their structure and physiology-and indicate that the procession of neoplastic change that leads to endometrial cancer is initiated early in life.

Mapping carbon accumulation potential from global natural forest regrowth.

To constrain global warming, we must strongly curtail greenhouse gas emissions and capture excess atmospheric carbon dioxide1,2. Regrowing natural forests is a prominent strategy for capturing additional carbon3, but accurate assessments of its potential are limited by uncertainty and variability in carbon accumulation rates2,3. To assess why and where rates differ, here we compile 13,112 georeferenced measurements of carbon accumulation. Climatic factors explain variation in rates better than land-use history, so we combine the field measurements with 66 environmental covariate layers to create a global, one-kilometre-resolution map of potential aboveground carbon accumulation rates for the first 30 years of natural forest regrowth. This map shows over 100-fold variation in rates across the globe, and indicates that default rates from the Intergovernmental Panel on Climate Change (IPCC)4,5 may underestimate aboveground carbon accumulation rates by 32 per cent on average and do not capture eight-fold variation within ecozones. Conversely, we conclude that maximum climate mitigation potential from natural forest regrowth is 11 per cent lower than previously reported3 owing to the use of overly high rates for the location of potential new forest. Although our data compilation includes more studies and sites than previous efforts, our results depend on data availability, which is concentrated in ten countries, and data quality, which varies across studies. However, the plots cover most of the environmental conditions across the areas for which we predicted carbon accumulation rates (except for northern Africa and northeast Asia). We therefore provide a robust and globally consistent tool for assessing natural forest regrowth as a climate mitigation strategy.

The landscape of somatic mutation in normal colorectal epithelial cells.

The colorectal adenoma-carcinoma sequence has provided a paradigmatic framework for understanding the successive somatic genetic changes and consequent clonal expansions that lead to cancer1. However, our understanding of the earliest phases of colorectal neoplastic changes-which may occur in morphologically normal tissue-is comparatively limited, as for most cancer types. Here we use whole-genome sequencing to analyse hundreds of normal crypts from 42 individuals. Signatures of multiple mutational processes were revealed; some of these were ubiquitous and continuous, whereas others were only found in some individuals, in some crypts or during certain periods of life. Probable driver mutations were present in around 1% of normal colorectal crypts in middle-aged individuals, indicating that adenomas and carcinomas are rare outcomes of a pervasive process of neoplastic change across morphologically normal colorectal epithelium. Colorectal cancers exhibit substantially increased mutational burdens relative to normal cells. Sequencing normal colorectal cells provides quantitative insights into the genomic and clonal evolution of cancer.

A shared 'vulnerability code' underpins varying sources of DNA damage throughout paternal germline transmission in mouse.

During mammalian spermatogenesis, the paternal genome is extensively remodelled via replacement of histones with protamines forming the highly compact mature sperm nucleus. Compaction occurs in post-meiotic spermatids and is accompanied by extensive double strand break (DSB) formation. We investigate the epigenomic and genomic context of mouse spermatid DSBs, identifying primary sequence motifs, secondary DNA structures and chromatin contexts associated with this damage. Consistent with previously published results we find spermatid DSBs positively associated with short tandem repeats and LINE elements. We further show spermatid DSBs preferentially occur in association with (CA)n, (NA)n and (RY)n repeats, in predicted Z-DNA, are not associated with G-quadruplexes, are preferentially found in regions of low histone mark coverage and engage the remodelling/NHEJ factor BRD4. Locations incurring DSBs in spermatids also show distinct epigenetic profiles throughout later developmental stages: regions retaining histones in mature sperm, regions susceptible to oxidative damage in mature sperm, and fragile two-cell like embryonic stem cell regions bound by ZSCAN4 all co-localise with spermatid DSBs and with each other. Our results point to a common 'vulnerability code' unifying several types of DNA damage occurring on the paternal genome during reproduction, potentially underpinned by torsional changes during sperm chromatin remodelling.

The global potential for increased storage of carbon on land.

Constraining the climate crisis requires urgent action to reduce anthropogenic emissions while simultaneously removing carbon dioxide from the atmosphere. Improved information about the maximum magnitude and spatial distribution of opportunities for additional land-based removals of CO2 is needed to guide on-the-ground decision-making about where to implement climate change mitigation strategies. Here, we present a globally consistent spatial dataset (approximately 500-m resolution) of current, potential, and unrealized potential carbon storage in woody plant biomass and soil organic matter. We also provide a framework for prioritizing actions related to the restoration, management, and maintenance of woody carbon stocks and associated soils. By comparing current to potential carbon storage, while excluding areas critical to food production and human habitation, we find 287 petagrams (PgC) of unrealized potential storage opportunity, of which 78% (224 PgC) is in biomass and 22% (63 PgC) is in soil. Improved management of existing forests may offer nearly three-fourths (206 PgC) of the total unrealized potential, with the majority (71%) concentrated in tropical ecosystems. However, climate change is a source of considerable uncertainty. While additional research is needed to understand the impact of natural disturbances and biophysical feedbacks, we project that the potential for additional carbon storage in woody biomass will increase (+17%) by 2050 despite projected decreases (−12%) in the tropics. Our results establish an absolute reference point and conceptual framework for national and jurisdictional prioritization of locations and actions to increase land-based carbon storage.

Three-Year Outcomes Following Permissive Cardiotoxicity in Patients on Trastuzumab.

INTRODUCTION: Cardiotoxicity, manifest by reduced left ventricular ejection fraction (LVEF), is the most common reason for the premature discontinuation of trastuzumab. While permissive cardiotoxicity (where mild cardiotoxicity is accepted to enable ongoing trastuzumab) has been shown feasible, the longer-term outcomes are unknown. We aimed to study the intermediate-term clinical outcomes of patients who underwent permissive cardiotoxicity. MATERIALS AND METHODS: We performed a retrospective cohort study of patients referred to the cardio-oncology service at McMaster University from 2016 to 2021 for LV dysfunction following trastuzumab administration. RESULTS: Fifty-one patients underwent permissive cardiotoxicity. The median (25th-75th percentile) follow-up time from cardiotoxicity onset was 3 years (1.3-4 years). Forty-seven (92%) patients completed trastuzumab; 3 (6%) developed severe LV dysfunction or clinical heart failure (HF) while on trastuzumab and prematurely discontinued therapy. One discontinued trastuzumab by patient choice. At final follow-up after therapy completion, 7 (14%) patients still had mild cardiotoxicity, including 2 who had clinical heart failure and stopped trastuzumab early. Among those with recovered LV function, 50% had normalized LVEF or GLS by 6 and 3 months, respectively, after initial cardiotoxicity. There was no difference in characteristics between those who did or did not recover their LV function. CONCLUSIONS: Among patients exposed to permissive trastuzumab cardiotoxicity for HER2-positive breast cancer, 6% were unable to complete planned trastuzumab due to severe LV dysfunction or clinical HF. Although most patients recover their LV function after trastuzumab discontinuation or completion, 14% still have persistent cardiotoxicity by 3-year follow-up.

Sex and suicide: The curious case of Toll-like receptors.

During in vitro fertilisation (IVF), pharmacological activation of the murine X chromosome-encoded receptor proteins Toll-like receptor (TLR) 7 and TLR8 reportedly results in male-biased litters by selectively disrupting the motility of X-bearing sperm cells. Thus-in the context of agonist treatment during IVF-these receptors act as 'suicidal' segregation distorters that impair their own transmission to the next generation. Such behaviour would, from an evolutionary perspective, be strongly selected against if present during natural fertilisation. Consequently, TLR7/8 biology in vivo must differ significantly from this in vitro situation to allow these genes to persist in the genome. Here, we use our current understanding of male germ cell biology and TLR function as a starting point to explore the mechanistic and evolutionary aspects of this apparent paradox.

Successful recovery of motile and viable boar sperm after vitrification with different methods (pearls and mini straws) using sucrose as a cryoprotectant.

Vitrification of sperm by direct contact with liquid nitrogen is increasing in popularity as an alternative to conventional (slow) freezing. Although slow freezing is very challenging in boar sperm cryopreservation, this is currently the standard method used. We compared vitrification in "pearls" and in "mini straws" using the in vitro fertilization media Porcine Gamete Media with 0.3 M sucrose with the standard (slow) method used to preserve boar sperm. Both vitrification methods reduced the viability of the sperm sample more than slow freezing (42.2 ± 4.3% total motility and 71.4 ± 2.3% alive), however, both protocols allowed for the successful recovery of the sperm samples. By comparing two different methods of vitrification and two different methods of post-thaw preparation we were able to determine the optimal vitrification-thaw protocol for boar sperm. When comparing pearls and mini-straws, the smaller liquid volume associated with pearls had a positive effect on the survivability of the samples, reducing sperm DNA damage (1.2 ± 0.2% vs. 5.1 ± 0.1.7%) and preserving motility (26.15 ± 2.8% vs 9.39 ± 0.9%) after thawing. In conclusion, the pearl method was the most suitable of the vitrification techniques for use with boar sperm.

Using the Colloidal Method to Prepare Au Catalysts for the Alkylation of Aniline by Benzyl Alcohol.

Using the colloidal method, attempts were made to deposit Au NPs on seven different material supports (TiO2, α and γ-Al2O3, HFeO2, CeO2, C, and SiO2). The deposition between 0.8 and 1 wt% of Au NPs can be generally achieved, apart for SiO2 (no deposition) and α-alumina (0.3 wt%). The resultant sizes of the Au NPs were dependent on the nature as well as the surface area of the support. The catalytic activity and selectivity of the supported Au catalysts were then compared in the alkylation of aniline by benzyl alcohol. Correlations were made between the nature of the support, the size of the Au NP, and the H-binding energy. A minimum H-binding energy of 1100 µV K-1 was found to be necessary for high selectivity for the secondary amine. Comparisons of the TEM images of the pre- and post-reaction catalysts also revealed the extent of Au NP agglomeration under the reaction conditions.

Complementary Medicine Use Amongst Patients with Metastatic Cancer Enrolled in Phase III Clinical Trials.

BACKGROUND: Complementary medicines (CM) are frequently used by patients with cancer. Controversy exists over the effectiveness and risk that CM may add to conventional cancer therapy. The incidence of CM use among patients enrolled in phase III clinical trials is unknown. METHODS: Medication lists from 6 international phase III clinical trials were retrospectively reviewed to identify patients using CM. Patients had metastatic breast, colorectal, or lung cancers. Quality of life, adverse events, overall survival, and progression-free survival were compared between CM users and non-users. Baseline differences between groups were adjusted with propensity score matching groups. RESULTS: Seven hundred and six of 3446 patients (20.5%) used at least one CM. CM use was highest among patients with breast cancer (35.6%). CM users had more favorable baseline prognostic factors (ECOG 0-1, non-smoking status, younger age, and fewer metastases). CM use was associated with lower rates of adverse events (50% vs. 62%, P = .002) and quality of life was similar between both groups. After adjustment with propensity score matching, CM use was also associated with longer overall survival in patients with lung cancer (adjusted hazard ratio 0.80, 95%CI, 0.68-0.94, P =.0054). However, several key control variables like EGFR status were not available. CONCLUSION: One in 5 patients in phase III clinical trials report using CM. CM was not associated with worse cancer-specific outcomes. However, CM users had more favorable baseline prognostic factors, and likely other confounders that may have contributed to improved outcomes observed in the lung cohort. Physicians should monitor for CM use and potential interactions with clinical trial drugs.

Enzyme kinetic approach for mechanistic insight and predictions of in vivo starch digestibility and the glycaemic index of foods.

BACKGROUND: Starch is a principal dietary source of digestible carbohydrate and energy. Glycaemic and insulinaemic responses to foods containing starch vary considerably and glucose responses to starchy foods are often described by the glycaemic index (GI) and/or glycaemic load (GL). Low GI/GL foods are beneficial in the management of cardiometabolic disorders (e.g., type 2 diabetes, cardiovascular disease). Differences in rates and extents of digestion of starch-containing foods will affect postprandial glycaemia. SCOPE AND APPROACH: Amylolysis kinetics are influenced by structural properties of the food matrix and of starch itself. Native (raw) semi-crystalline starch is digested slowly but hydrothermal processing (cooking) gelatinises the starch and greatly increases its digestibility. In plants, starch granules are contained within cells and intact cell walls can limit accessibility of water and digestive enzymes hindering gelatinisation and digestibility. In vitro studies of starch digestion by α-amylase model early stages in digestion and can suggest likely rates of digestion in vivo and expected glycaemic responses. Reports that metabolic responses to dietary starch are influenced by α-amylase gene copy number, heightens interest in amylolysis. KEY FINDINGS AND CONCLUSIONS: This review shows how enzyme kinetic strategies can provide explanations for differences in digestion rate of different starchy foods. Michaelis-Menten and Log of Slope analyses provide kinetic parameters (e.g., K m and k cat /K m ) for evaluating catalytic efficiency and ease of digestibility of starch by α-amylase. Suitable kinetic methods maximise the information that can be obtained from in vitro work for predictions of starch digestion and glycaemic responses in vivo.

Correction: A Genetic Basis for a Postmeiotic X Versus Y Chromosome Intragenomic Conflict in the Mouse.

[This corrects the article DOI: 10.1371/journal.pgen.1002900.].

Inhibition of the facilitative sugar transporters (GLUTs) by tea extracts and catechins.

Tea polyphenolics have been suggested to possess blood glucose lowering properties by inhibiting sugar transporters in the small intestine and improving insulin sensitivity. In this report, we studied the effects of teas and tea catechins on the small intestinal sugar transporters, SGLT1 and GLUTs (GLUT1, 2 and 5). Green tea extract (GT), oolong tea extract (OT), and black tea extract (BT) inhibited glucose uptake into the intestinal Caco-2 cells with GT being the most potent inhibitor (IC50 : 0.077 mg/mL), followed by OT (IC50 : 0.136 mg/mL) and BT (IC50 : 0.56 mg/mL). GT and OT inhibition of glucose uptake was partial non-competitive, with an inhibitor constant (Ki ) = 0.0317 and 0.0571 mg/mL, respectively, whereas BT was pure non-competitive, Ki  = 0.36 mg/mL. Oocytes injected to express small intestinal GLUTs were inhibited by teas, but SGLT1 was not. Furthermore, catechins present in teas were the predominant inhibitor of glucose uptake into Caco-2 cells, and gallated catechins the most potent: CG > ECG > EGCG ≥ GCG when compared to the non-gallated catechins (C, EC, GC, and EGC). In Caco-2 cells, individual tea catechins reduced the SGLT1 gene, but not protein expression levels. In contrast, GLUT2 gene and protein expression levels were reduced after 2 hours exposure to catechins but increased after 24 hours. These in vitro studies suggest teas containing catechins may be useful dietary supplements capable of blunting postprandial glycaemia in humans, including those with or at risk to Type 2 diabetes mellitus.

The impact of replacing wheat flour with cellular legume powder on starch bioaccessibility, glycaemic response and bread roll quality: A double-blind randomised controlled trial in healthy participants.

The global rise in obesity and type 2 diabetes has generated significant interest in regulating the glycaemic impact of staple foods. Wheat breads (white or wholemeal) are popular staples, but have a high-glycaemic index, due to the highly digestible wheat starch. Reducing the glycaemic potency of white bread is challenging because the bread-making conditions are mostly conducive to starch gelatinisation. Cellular legume powders are a new source of type 1 resistant starch, where the starch is encapsulated by dietary fibre in the form of intact plant cell walls. The starch in these cell powders is less susceptible to gelatinisation and digestion than starch in conventional legume flours. However, legume cell resilience to baking conditions and the effects of this ingredient on glycaemic responses and product quality are unknown. Here we show that the integrity of cell wall fibre in chickpea powder was preserved on baking and this led to a ~40% reduction in in vivo glycaemic responses (iAUC120) to white bread rolls (~50 g available carbohydrate and 12 g wheat protein per serving) when 30% or 60% (w/w) of the wheat flour was replaced with intact cell powder. Significant reductions in glycaemic responses were achieved without adverse effects on bread texture, appearance or palatability. Starch digestibility analysis and microscopy confirmed the importance of cell integrity in attenuating glycaemic responses. Alternative processing methods that preserve cell integrity are a new, promising way to provide healthier low glycaemic staple foods; we anticipate that this will improve dietary options for diabetes care.

Value assessment of oncology drugs using a weighted criterion-based approach.

BACKGROUND: Globally, the rising cost of anticancer therapy has motivated efforts to quantify the overall value of new cancer treatments. Multicriteria decision analysis offers a novel approach to incorporate multiple criteria and perspectives into value assessment. METHODS: The authors recruited a diverse, multistakeholder group who identified and weighted key criteria to establish the drug assessment framework (DAF). Construct validity assessed the degree to which DAF scores were associated with past pan-Canadian Oncology Drug Review (pCODR) funding recommendations and European Society for Medical Oncology Magnitude of Clinical Benefit Scale (ESMO-MCBS; version 1.1) scores. RESULTS: The final DAF included 10 criteria: overall survival, progression-free survival, response rate, quality of life, toxicity, unmet need, equity, feasibility, disease severity, and caregiver well-being. The first 5 clinical benefit criteria represent approximately 64% of the total weight. DAF scores ranged from 0 to 300, reflecting both the expected impact of the drug and the quality of supporting evidence. When the DAF was applied to the last 60 drugs (with reviewers blinded) reviewed by pCODR (2015-2018), those drugs with positive pCODR funding recommendations were found to have higher DAF scores compared with drugs not recommended (103 vs 63; Student t test P = .0007). DAF clinical benefit criteria mildly correlated with ESMO-MCBS scores (correlation coefficient, 0.33; 95% CI, 0.009-0.59). Sensitivity analyses that varied the criteria scores did not change the results. CONCLUSIONS: Using a structured and explicit approach, a criterion-based valuation framework was designed to provide a transparent and consistent method with which to value and prioritize cancer drugs to facilitate the delivery of affordable cancer care.

Large climate mitigation potential from adding trees to agricultural lands.

While improved management of agricultural landscapes is promoted as a promising natural climate solution, available estimates of the mitigation potential are based on coarse assessments of both agricultural extent and aboveground carbon density. Here we combine 30 meter resolution global maps of aboveground woody carbon, tree cover, and cropland extent, as well as a 1 km resolution map of global pasture land, to estimate the current and potential carbon storage of trees in nonforested portions of agricultural lands. We find that global croplands currently store 3.07 Pg of carbon (C) in aboveground woody biomass (i.e., trees) and pasture lands account for an additional 3.86 Pg C across a combined 3.76 billion ha. We then estimate the climate mitigation potential of multiple scenarios of integration and avoided loss of trees in crop and pasture lands based on region-specific biomass distributions. We evaluate our findings in the context of nationally determined contributions and find that the majority of potential carbon storage from integration and avoided loss of trees in crop and pasture lands is in countries that do not identify agroforestry as a climate mitigation technique.

Spermatid Development in XO Male Mice With Varying Y Chromosome Short-Arm Gene Content: Evidence for a Y Gene Controlling the Initiation of Sperm Morphogenesis

The journal and the authors apologise for an error in the above titled article published in this journal (vol 144, pp 433­445). The authors inadvertently presented duplicate sperm images for XY and XESxrbO mouse testes of Fig. 6 (bottom panels). This error does not change the findings of the paper, as this figure does not give a quantitative breakdown of the proportions of different shapes.