Dual carbon and oxygen isotopes in Siberian tree rings as indicator of millennia sunshine duration changes.

The current lack of information on past summer sunshine duration variability from annually resolved palaeoclimatological archives is hindering progress in the understanding and modelling of the earth climate system. We show that a combination of tree-ring carbon and oxygen isotopes from Siberia provides robust information on summer sunshine duration, which we use for an annual 1505-year reconstruction of July sunshine duration variability (1,5K-SIB-JSDR). We found that the Medieval maximum is 56 % higher than the average over 1505 years. Rapid and drastic decreases in sunshine duration up to 60 % correspond to major stratospheric volcanic eruptions. Grand Solar Minima and total sunspot numbers are also well preserved in the 1,5K-SIB-JSDR. Coherency with a global air temperature composite and spring Arctic Oscillation indicate that a large-scale climate signal is retained in our sunshine reconstruction.

Passenger Gene Coamplifications Create Collateral Therapeutic Vulnerabilities in Cancer.

DNA amplifications in cancer do not only harbor oncogenes. We sought to determine whether passenger coamplifications could create collateral therapeutic vulnerabilities. Through an analysis of >3,000 cancer genomes followed by the interrogation of CRISPR-Cas9 loss-of-function screens across >700 cancer cell lines, we determined that passenger coamplifications are accompanied by distinct dependency profiles. In a proof-of-principle study, we demonstrate that the coamplification of the bona fide passenger gene DEAD-Box Helicase 1 (DDX1) creates an increased dependency on the mTOR pathway. Interaction proteomics identified tricarboxylic acid (TCA) cycle components as previously unrecognized DDX1 interaction partners. Live-cell metabolomics highlighted that this interaction could impair TCA activity, which in turn resulted in enhanced mTORC1 activity. Consequently, genetic and pharmacologic disruption of mTORC1 resulted in pronounced cell death in vitro and in vivo. Thus, structurally linked coamplification of a passenger gene and an oncogene can result in collateral vulnerabilities. SIGNIFICANCE: We demonstrate that coamplification of passenger genes, which were largely neglected in cancer biology in the past, can create distinct cancer dependencies. Because passenger coamplifications are frequent in cancer, this principle has the potential to expand target discovery in oncology. This article is featured in Selected Articles from This Issue, p. 384.

Thawing permafrost can mitigate warming-induced drought stress in boreal forest trees.

Perennially frozen soil, also known as permafrost, is important for the functioning and productivity of most of the boreal forest, the world's largest terrestrial biome. A better understanding of complex vegetation-permafrost interrelationships is needed to predict changes in local- to large-scale carbon, nutrient, and water cycle dynamics under future global warming. Here, we analyze tree-ring width and tree-ring stable isotope (C and O) measurements of Gmelin larch (Larix gmelinii (Rupr.) Rupr.) from six permafrost sites in the northern taiga of central Siberia. Our multi-parameter approach shows that changes in tree growth were predominantly controlled by the air and topsoil temperature and moisture content of the active soil and upper permafrost layers. The observed patterns range from strong growth limitations by early summer temperatures at higher elevations to significant growth controls by precipitation at warmer and well-drained lower-elevation sites. Enhanced radial tree growth is mainly found at sites with fast thawing upper mineral soil layers, and the comparison of tree-ring isotopes over five-year periods with different amounts of summer precipitation indicates that trees can prevent drought stress by accessing water from melted snow and seasonally frozen soil. Identifying the active soil and upper permafrost layers as central water resources for boreal tree growth during dry summers demonstrates the complexity of ecosystem responses to climatic changes.

Updating the dual C and O isotope-Gas-exchange model: A concept to understand plant responses to the environment and its implications for tree rings.

The combined study of carbon (C) and oxygen (O) isotopes in plant organic matter has emerged as a powerful tool for understanding plant functional responses to environmental change. The approach relies on established relationships between leaf gas exchange and isotopic fractionation to derive a series of model scenarios that can be used to infer changes in photosynthetic assimilation and stomatal conductance driven by changes in environmental parameters (CO2 , water availability, air humidity, temperature, nutrients). We review the mechanistic basis for a conceptual model, in light of recently published research, and discuss where isotopic observations do not match our current understanding of plant physiological response to the environment. We demonstrate that (1) the model was applied successfully in many, but not all studies; (2) although originally conceived for leaf isotopes, the model has been applied extensively to tree-ring isotopes in the context of tree physiology and dendrochronology. Where isotopic observations deviate from physiologically plausible conclusions, this mismatch between gas exchange and isotope response provides valuable insights into underlying physiological processes. Overall, we found that isotope responses can be grouped into situations of increasing resource limitation versus higher resource availability. The dual-isotope model helps to interpret plant responses to a multitude of environmental factors.

CRISPR/Cas9 Screen in Gastric Cancer Patient-Derived Organoids Reveals KDM1A-NDRG1 Axis as a Targetable Vulnerability.

Viability CRISPR screens have proven indispensable in parsing genome function. However, their application in new, more physiologically relevant culturing systems like patient-derived organoids (PDOs) has been much slower. To probe epigenetic contribution to gastric cancer (GC), the third leading cause of cancer-related deaths worldwide, the first negative selection CRISPR screen in GC PDOs that faithfully preserve primary tumor characteristics is performed. Extensive quality control measurements showing feasibility of CRISPR screens in primary organoid culture are provided. The screen reveals the histone lysine demethylase-1A (KDM1A) to constitute a GC vulnerability. Both genetic and pharmacological inhibition of KDM1A cause organoid growth retardation. Further, it is shown that most of KDM1A cancer-supporting functions center on repression of N-myc downstream regulates gene-1 (NDRG1). De-repression of NDRG1 by KDM1A inhibitors (KDM1Ai) causes inhibition of Wnt signaling and a strong G1 cell cycle arrest. Finally, by profiling 20 GC PDOs, it is shown that NDRG1 upregulation predicts KDM1Ai response with 100% sensitivity and 82% specificity in the tested cohort. Thus, this work pioneers the use of negative selection CRISPR screens in patient-derived organoids, identifies a marker of KDM1Ai response, and accordingly a cohort of patients who may benefit from such therapy.

Climate impacts on tree-ring stable isotopes across the Northern Hemispheric boreal zone.

Boreal regions are changing rapidly with anthropogenic global warming. In order to assess risks and impacts of this process, it is crucial to put these observed changes into a long-term perspective. Summer air temperature variability can be well reconstructed from conifer tree rings. While the application of stable isotopes can potentially provide complementary climatic information over different seasons. In this study, we developed new triple stable isotope chronologies in tree-ring cellulose (δ13Ctrc, δ18Otrc, δ2Htrc) from a study site in Canada. Additionally, we performed regional aggregated analysis of available stable isotope chronologies from 6 conifers' tree species across high-latitudinal (HL) and - altitudinal (HA) as well as Siberian (SIB) transects of the Northern Hemispheric boreal zone. Our results show that summer air temperature still plays an important role in determining tree-ring isotope variability at 11 out of 24 sites for δ13Ctrc, 6 out of 18 sites for δ18Otrc and 1 out of 6 sites for δ2Htrc. Precipitation, relative humidity and vapor pressure deficit are significantly and consistently recorded in both δ13Ctrc and δ18Otrc along HL. Summer sunshine duration is captured by all isotopes, mainly for HL and HA transects, indicating an indirect link with an increase in air and leaf temperature. A mixed temperature-precipitation signal is preserved in δ13Ctrc and δ18Otrc along SIB transect. The δ2Htrc data obtained for HL-transect provide information not only about growing seasonal moisture and temperature, but also capture autumn, winter and spring sunshine duration signals. We conclude that a combination of triple stable isotopes in tree-ring studies can provide a comprehensive description of climate variability across the boreal forest zone and improve ecohydrological reconstructions.

Efficient Correction of Oncogenic KRAS and TP53 Mutations through CRISPR Base Editing.

KRAS is the most frequently mutated oncogene in human cancer, and its activating mutations represent long-sought therapeutic targets. Programmable nucleases, particularly the CRISPR-Cas9 system, provide an attractive tool for genetically targeting KRAS mutations in cancer cells. Here, we show that cleavage of a panel of KRAS driver mutations suppresses growth in various human cancer cell lines, revealing their dependence on mutant KRAS. However, analysis of the remaining cell population after long-term Cas9 expression unmasked the occurence of oncogenic KRAS escape variants that were resistant to Cas9-cleavage. In contrast, the use of an adenine base editor to correct oncogenic KRAS mutations progressively depleted the targeted cells without the appearance of escape variants and allowed efficient and simultaneous correction of a cancer-associated TP53 mutation. Oncogenic KRAS and TP53 base editing was possible in patient-derived cancer organoids, suggesting that base editor approaches to correct oncogenic mutations could be developed for functional interrogation of vulnerabilities in a personalized manner for future precision oncology applications. SIGNIFICANCE: Repairing KRAS mutations with base editors can be used for providing a better understanding of RAS biology and may lay the foundation for improved treatments for KRAS-mutant cancers.

Efficient Assay and Marker Significance of NAD+ in Human Blood.

Oxidized nicotinamide adenine dinucleotide (NAD+) is a biological molecule of systemic importance. Essential role of NAD+ in cellular metabolism relies on the substrate action in various redox reactions and cellular signaling. This work introduces an efficient enzymatic assay of NAD+ content in human blood using recombinant formate dehydrogenase (FDH, EC 1.2.1.2), and demonstrates its diagnostic potential, comparing NAD+ content in the whole blood of control subjects and patients with cardiac or neurological pathologies. In the control group (n = 22, 25-70 years old), our quantification of the blood concentration of NAD+ (18 µM, minimum 15, max 23) corresponds well to NAD+ quantifications reported in literature. In patients with demyelinating neurological diseases (n = 10, 18-55 years old), the NAD+ levels significantly (p < 0.0001) decrease (to 14 µM, min 13, max 16), compared to the control group. In cardiac patients with the heart failure of stage II and III according to the New York Heart Association (NYHA) functional classification (n = 24, 42-83 years old), the blood levels of NAD+ (13 µM, min 9, max 18) are lower than those in the control subjects (p < 0.0001) or neurological patients (p = 0.1). A better discrimination of the cardiac and neurological patients is achieved when the ratios of NAD+ to the blood creatinine levels, mean corpuscular volume or potassium ions are compared. The proposed NAD+ assay provides an easy and robust tool for clinical analyses of an important metabolic indicator in the human blood.

Using CRISPR-Cas9 to Dissect Cancer Mutations in Cell Lines.

The CRISPR-Cas9 technology has revolutionized the scope and pace of biomedical research, enabling the targeting of specific genomic sequences for a wide spectrum of applications. Here we describe assays to functionally interrogate mutations identified in cancer cells utilizing both CRISPR-Cas9 nuclease and base editors. We provide guidelines to interrogate known cancer driver mutations or functionally screen for novel vulnerability mutations with these systems in characterized human cancer cell lines. The proposed platform should be transferable to primary cancer cells, opening up a path for precision oncology on a functional level.

Modern aridity in the Altai-Sayan mountain range derived from multiple millennial proxies.

Temperature and precipitation changes are crucial for larch trees growing at high-elevation sites covered by permafrost in the Altai-Sayan mountain range (ASMR). To contextualize the amplitude of recent climate fluctuations, we have to look into the past by analyzing millennial paleoclimatic archives recording both temperature and precipitation. We developed annually resolved 1500-year tree-ring cellulose chronologies (δ13Ccell, δ18Ocell), and used these new records to reconstruct the variability in local summer precipitation and air temperature. We combined our new local reconstructions with existing paleoclimatic archives available for the Altai. The data show a strong decreasing trend by ca. 49% in regional summer precipitation, along with a regional summer temperature increase towards the twenty-first century, relative to the preceding 1500 years. Modern dry conditions (1966-2016 CE) in the ASMR are the result of simultaneous summer warming and decreased precipitation. Our new reconstructions also demonstrate that climate change in the ASMR is much stronger compared to the global average.

RNAi-Mediated Screen of Primary AML Cells Nominates MDM4 as a Therapeutic Target in NK-AML with DNMT3A Mutations.

DNA-methyltransferase 3A (DNMT3A) mutations belong to the most frequent genetic aberrations found in adult acute myeloid leukemia (AML). Recent evidence suggests that these mutations arise early in leukemogenesis, marking leukemic progenitors and stem cells, and persist through consolidation chemotherapy, providing a pool for AML relapse. Currently, there are no therapeutic approaches directed specifically against this cell population. To unravel therapeutically actionable targets in mutant DNMT3A-driven AML cells, we have performed a focused RNAi screen in a panel of 30 primary AML samples, all carrying a DNMT3A R882 mutation. As one of the strongest hits, we identified MDM4 as a gene essential for proliferation of primary DNMT3AWT/R882X AML cells. We analyzed a publicly available RNA-Seq dataset of primary normal karyotype (NK) AML samples and found a trend towards MDM4 transcript overexpression particularly in DNMT3A-mutant samples. Moreover, we found that the MDM2/4 inhibitor ALRN-6924 impairs growth of DNMT3AWT/R882X primary cells in vitro by inducing cell cycle arrest through upregulation of p53 target genes. Our results suggest that MDM4 inhibition is a potential target in NK-AML patients bearing DNMT3A R882X mutations.

Spring arctic oscillation as a trigger of summer drought in Siberian subarctic over the past 1494 years.

Rapid changes in the hydrological and temperature regimes over the past decades at the northern latitudes enhance significantly permafrost degradation accelerating carbon release, increase the frequency of drought events and extensive wildfires. However, the mechanisms and dynamics driving drought events and their influence on Siberian forests are currently the subject of numerous research activities. Newly developed and annually resolved stable carbon and oxygen isotope chronologies of larch tree-ring cellulose (δ13Ccell and δ18Ocell) for the period 516-2009 CE allowed the reconstruction of July precipitation and Arctic Oscillation (AO) in May, respectively. Unprecedented drought events occurred towards twentieth-twenty-first centuries as indicated by the July precipitation reconstruction. Positive AO phases in May were most pronounced during the second part of the first millennium, but also increased in frequency in the modern period of the twentieth-twenty-first centuries. Negative AO phases are associated with cold anomalies and show a remarkable decrease in the nineteenth century caused by a series of major volcanic eruptions. Our findings help explaining the increased frequency of Siberian forest fires over the past decades in Central Siberia consistent with a reduction of summer precipitation, triggered by a positive phase of the Arctic Oscillation in May.

The influence of decision-making in tree ring-based climate reconstructions.

Tree-ring chronologies underpin the majority of annually-resolved reconstructions of Common Era climate. However, they are derived using different datasets and techniques, the ramifications of which have hitherto been little explored. Here, we report the results of a double-blind experiment that yielded 15 Northern Hemisphere summer temperature reconstructions from a common network of regional tree-ring width datasets. Taken together as an ensemble, the Common Era reconstruction mean correlates with instrumental temperatures from 1794-2016 CE at 0.79 (p < 0.001), reveals summer cooling in the years following large volcanic eruptions, and exhibits strong warming since the 1980s. Differing in their mean, variance, amplitude, sensitivity, and persistence, the ensemble members demonstrate the influence of subjectivity in the reconstruction process. We therefore recommend the routine use of ensemble reconstruction approaches to provide a more consensual picture of past climate variability.

Can we really teach cognitive behavioral therapy with a massive open online course?

A better training in psychotherapy is needed for psychiatry trainees. Online Cognitive Behavioural Therapies (CBT) could be a good solution. Free and wide audience course like Massive Open Online course (MOOCs) increase dissemination and accessibility of the training. However, the engagement needs to be improved. A hybrid approach seems relevant with the MOOC as an incentive. Beyond the promotion of the topic, a MOOC can be a promotion tool for the provider. The economic model of the MOOC needed to be taken into account to allow sustainability. To explore these elements, we take into account a survey taken during the 1st European Psychiatric Association MOOC about CBT.

Compound-specific carbon isotope patterns in needles of conifer tree species from the Swiss National Park under recent climate change.

Elevated CO2 along with rising temperature and water deficits can lead to changes in tree physiology and leaf biochemistry. These changes can increase heat- and drought-induced tree mortality. We aim to reveal the impacts of climatic drivers on individual compounds at the leaf level among European larch (Larix decidua) and mountain pine (Pinus mugo) trees, which are widely distributed at high elevations. We investigated seasonal carbon isotope composition (δ13C) and concentration patterns of carbohydrates and organic acids in needles of these two different species from a case study in the Swiss National Park (SNP). We found that average and minimum air temperatures were the main climatic drivers of seasonal variation of δ13C in sucrose and glucose as well as in concentrations of carbohydrates and citric acid/citrate in needles of both tree species. The impact of seasonal climatic drivers on larch and mountain pine trees at the needle level is in line with our earlier study in this region for long-term changes at the tree-ring level. We conclude that the species-specific changes in δ13C and concentrations of carbohydrates and organic acids are sensitive indicators of changes in the metabolic pathways occurring as a result of climatic changes.

Human primary liver cancer-derived organoid cultures for disease modeling and drug screening.

Human liver cancer research currently lacks in vitro models that can faithfully recapitulate the pathophysiology of the original tumor. We recently described a novel, near-physiological organoid culture system, wherein primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumors. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumor, allowing for discrimination between different tumor tissues and subtypes, even after long-term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumorogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo. PLC-derived organoids are amenable for biomarker identification and drug-screening testing and led to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized-medicine approaches for the disease.

Site-specific water-use strategies of mountain pine and larch to cope with recent climate change.

We aim to achieve a mechanistic understanding of the eco-physiological processes in Larix decidua and Pinus mugo var. uncinata growing on north- and south-facing aspects in the Swiss National Park in order to distinguish the short- and long-term effects of a changing climate. To strengthen the interpretation of the δ(18)O signal in tree rings and its coherence with the main factors and processes driving evaporative δ(18)O needle water enrichment, we analyzed the δ(18)O in needle, xylem and soil water over the growing season in 2013 and applied the mechanistic Craig-Gordon model (1965) for the short-term responses. We found that δ(18)O needle water strongly reflected the variability of relative humidity mainly for larch, while only δ(18)O in pine xylem water showed a strong link to δ(18)O in precipitation. Larger differences in offsets between modeled and measured δ(18)O needle water for both species from the south-facing aspects were detected, which could be explained by the high transpiration rates. Different soil water and needle water responses for the two species indicate different water-use strategies, further modulated by the site conditions. To reveal the long-term physiological response of the studied trees to recent and past climate changes, we analyzed δ(13)C and δ(18)O in wood chronologies from 1900 to 2013. Summer temperatures as well as summer and annual amount of precipitations are important factors for growth of both studied species from both aspects. However, mountain pine trees reduced sensitivity to temperature changes, while precipitation changes come to play an important role for the period from 1980 to 2013. Intrinsic water-use efficiency (WUEi) calculated for larch trees since the 1990s reached a saturation point at elevated CO2 Divergent trends between pine WUEi and δ(18)O are most likely indicative of a decline of mountain pine trees and are also reflected in decoupling mechanisms in the isotope signals between needles and tree-rings.

Grinding and polishing instead of sectioning for the tissue samples with a graft: Implications for light and electron microscopy.

A broad use of the graft replacement requires a detailed investigation of the host-graft interaction, including both histological examination and electron microscopy. A high quality sectioning of the host tissue with a graft seems to be complicated; in addition, it is difficult to examine the same tissue area by both of the mentioned microscopy techniques. To solve these problems, we developed a new technique of epoxy resin embedding with the further grinding, polishing, and staining. Graft-containing tissues prepared by grinding and polishing preserved their structure; however, sectioning frequently required the explantation of the graft and led to tissue disintegration. Moreover, stained samples prepared by grinding and polishing may then be assessed by both light microscopy and backscattered scanning electron microscopy. Therefore, grinding and polishing outperform sectioning when applied to the tissues with a graft.

Do centennial tree-ring and stable isotope trends of Larix gmelinii (Rupr.) Rupr. indicate increasing water shortage in the Siberian north?

Tree-ring width of Larix gmelinii (Rupr.) Rupr., ratios of stable isotopes of C (delta(13)C) and O (delta(18)O) of whole wood and cellulose chronologies were obtained for the northern part of central Siberia (Tura, Russia) for the period 1864-2006. A strong decrease in the isotope ratios of O and C (after atmospheric delta(13)C corrections) and tree-ring width was observed for the period 1967-2005, while weather station data show a decrease in July precipitation, along with increasing July air temperature and vapor pressure deficit (VPD). Temperature at the end of May and the whole month of June mainly determines tree radial growth and marks the beginning of the vegetation period in this region. A positive correlation between tree-ring width and July precipitation was found for the calibration period 1929-2005. Positive significant correlations between C isotope chronologies and temperatures of June and July were found for whole wood and cellulose and negative relationships with July precipitation. These relationships are strengthened when the likely physiological response of trees to increased CO(2) is taken into account (by applying a recently developed delta(13)C correction). For the O isotope ratios, positive relationships with annual temperature, VPD of July and a negative correlation with annual precipitation were observed. The delta(18)O in tree rings may reflect annual rather than summer temperatures, due to the late melting of the winter snow and its contribution to the tree water supply in summer. We observed a clear change in the isotope and climate trends after the 1960s, resulting in a drastic change in the relationship between C and O isotope ratios from a negative to a positive correlation. According to isotope fractionation models, this indicates reduced stomatal conductance at a relatively constant photosynthetic rate, as a response of trees to water deficit for the last half century in this permafrost region.