Efficient operation of electronic nanodevices at ultrafastspeedsrequires understanding and control of the currents generated by femtosecondbursts of light. Ultrafast laser-induced currents in metallic nanojunctionscan originate from photoassisted hot electron tunneling or lightwave-inducedtunneling. Both processes can drive localized photocurrents insidea scanning tunneling microscope (STM) on femto- to attosecond timescales, enabling ultrafast STM with atomic spatial resolution. Femtosecondlaser excitation of a metallic nanojunction, however, also leads tothe formation of a transient thermalized electron distribution, butthe tunneling of thermalized hot electrons on time scales faster thanelectron–lattice equilibration is not well understood. Here,we investigate ultrafast electronic heating and transient thermionictunneling inside a metallic photoexcited tunnel junction and its rolein the generation of ultrafast photocurrents in STM. Phase-resolvedsampling of broadband terahertz (THz) pulses via the THz-field-inducedmodulation of ultrafast photocurrents allows us to probe the electronictemperature evolution inside the STM tip and to observe the competitionbetween instantaneous and delayed tunneling due to nonthermal andthermal hot electron distributions in real time. Our results revealthe pronounced nonthermal character of photoinduced hot electron tunnelingand provide a detailed microscopic understanding of hot electron dynamicsinside a laser-excited tunnel junction. A particular example is laser-excitedultrafast scanning tunneling microscopy (STM), which pursues the ultimategoal to image the ultrafast dynamics of individual atoms and moleculeson surfaces with simultaneous Angstrom spatial and femtosecond temporalresolution. Recent advances in combining ultrashort optical or terahertz(THz) pulses with low-temperature STM demonstrated the potential oflight-driven STM to reach that goal.6 Attaining ultrahigh spatiotemporal resolution in STM requires thegeneration of ultrafast, atomically localized tunneling currents.In the weak excitation regime, photoassisted tunneling of hot electronsbelow the vacuum barrier gives rise to localized ultrafast currentsthat can be used for imaging in photon-driven STM.8 In contrast,lightwave-driven STM operates in the strong-field regime, where anintense localized light field of several V/nm modulates the junctionbarrier and, hence, the tunneling current on subcycle time scales.13 The two regimes are commonly distinguished by the Keldysh parameter, 14Hot carriers harvested in solidsthrough optical excitation can drive a wide range of physical andchemical processes, the understanding of which lies at the heart ofhot carrier science and technology.16 the transient tunneling of thermalizedhot electrons is rarely considered as source for ultrafast photocurrentsin STM. However, ultrafast thermionic tunneling should coexist withboth photon-driven and lightwave-driven tunneling currents and thusaccompanies any pulsed laser-driven STM. Moreover, the dynamics ofhot electrons determine the initial step of laser heating of STM tipsilluminated by ultrashort laser pulses.18 It is thusof general interest to understand the role of hot electron dynamicsfor femtosecond heating and tunneling in photoexcited STM.Bothprocesses are distinctly different in terms of the involvedelectronic excitation: Whereas photoassisted tunneling employs thetunneling of “hot” but nonthermal electrons, lightwave-drivenSTM is mediated by adiabatic tunneling of thermal but “cold”electrons directly from the Fermi level. Herein, the terms “hot”and “cold” describe whether there is excess energy  stored in the electronic system, whereas the terms“thermal” and “nonthermal” state whetherthe electronic distribution follows Fermi–Dirac statistics.Focusing ultrashort laser pulses on a metal gives rise to light absorptionand the generation of energetic, nonthermal electrons, which rapidlyredistribute their energy via electron–electron and electron–phononscattering, leading to the formation of a thermalized carrier distributionon the 10–100 fs time scale. Hot electrons from both thermaland nonthermal distributions can tunnel through the potential barrierin STM, though with different time scales and energy distributionsof the respective tunneling currents. Although the ultrafast dynamicsand thermalization of hot electrons in metals has been intensivelyinvestigated,22 It describes the emission of electrons above the vacuum barrieroriginating from the high-energy tail of a time-dependent Fermi–Diraccarrier distribution at elevated temperatures24 and can be described by the Richardson–Dushmann equation.25 In contrast to freestanding tips, the nanometer-sizedtip–sample gap in STM allows electrons to tunnel through thebarrier at all energies down to the Fermi level; therefore, ultrafastthermionic currents can flow at much lower electron temperatures.The situation is similar to thermally enhanced field emission froma high voltage (kV) biased nanotip emitter27 operating at fields of >108 V/m,which can be described by Murphy’s and Good’s equationfor thermo-field emission.28 To understandthermionic tunneling on femtosecond time scales, the ultrafast evolutionof the electronic temperature after laser excitation has to be known.Before electron–phonon equilibration has been achieved (>1ps), different temperatures are assigned to the electronic and phononicsubsystems. Their respective time evolutions can be described by thetwo-temperature model (TTM)19 or by solving Boltzmanntransport equations20 for the coupled electron–phononsystem. Whereas photoassisted tunneling and optical-field-driven tunnelingare temporally confined to the presence of the laser pulse, thermionictunneling can occur on time scales longer than the laser pulse durationand might lead to significantly delayed photocurrents in STM due toelectron dynamics inside the tip.Transientthermionic emission has been discussed in the contextof ultrafast electron guns using laser-excited nanotips.29 Energy information could, inprinciple, be obtained in STM by varying the bias voltage, but thebidirectional tunneling of photocarriers between tip and sample complicatesinterpretation of the respective hot carrier distributions. Instead,we measure the temporal response of ultrafast thermionic currentsvia phase-resolved sampling of single-cycle ultrabroadband THz pulsesinside the tip–sample junction.30 Specifically, we measure the time-dependent THz-induced change ofphotocurrents from the laser-excited STM tip,32 which is determinedby the instantaneous THz bias Here we study the interplaybetween ultrafast thermal and nonthermaltunneling from a femtosecond laser-excited STM tip. By tracking thetemporally delayed response of thermionic currents in real-time wecan discriminate between noninstantaneous thermal and instantaneousnonthermal currents, without the need to distinguish their energydistributions as for example in photoemission experiments.2, which corresponds tolaser powers between 0.11 and 0.36 mW under our conditions. The tipis placed above the Ag(111) sample at a distance d. A positive bias of 32 Precise knowledge ofthe waveform and amplitude of the THz bias will be essential to extractthe time evolution of ultrafast noninstantaneous currents photoexcitedinside the metallic junction, which will lead to temporal distortionsof the measured THz waveform as illustrated in 2 where the resonantTHz-induced tunneling into individual molecular states during thepeak of a THz half-cycle yields a time resolution faster than theTHz half-cycle. Due to the threshold character of the resonant tunnelingprocess, this regime does not require detailed knowledge of the THzwaveform to access subcycle dynamics. In contrast, accessing the dynamicsof “nonresonant” systems  on THz-subcycletime scales demands phase-resolved sampling of the THz waveform andknowledge of the THz bias transient.33 through thebarrier from a nonthermal steplike electron distribution can occurat energies that are multiples of the NIR photon energy 29 or transient thermionic emission19 will dominate the photocurrent. At high laserintensities, photocurrents might be generated in the strong-fieldregime, for which above-threshold photoemission36 and optical-field-inducedtunneling40 have to be considered as well.The time-dependent THz field acts an additional quasi-static biasthat modulates the NIR-induced photocurrent channels according totheir respective dependence on the potential barrier between tip andsample. The THz bias thus selectively probes those photocurrent channelsthat are most sensitive to the potential barrier.The different photocurrent mechanisms that canbe operative fora photoexcited STM tip are displayed in Figure S.1. Increasingthe laser intensity leads to a broadening and continuous shift ofthe measured THz waveform 29 At laser intensities 2, a significantly reducednonlinearity of 42 The intensity at which this transition occurs is independent ofthe number of electrons per pulse , implying that space charge effects are not responsiblefor the decreasing slope. Similar data from another tip are shownin Figure S.3, where pronounced waveformchanges are observed purely in the multiphoton photoemission regime.The observed waveform changes thus emerge in both the weak- and strong-fieldregimes of photoemission. In both regimes, the photoemission processis prompt, and the emitted current will be temporally confined tothe laser pulse width. Hence, photoemission cannot explain the observedlow-pass filter behavior, which requires a “slow” photocurrentcontribution . We conclude thatan additional delayed photocurrent component, which coexists withinstantaneous weak- and strong-field photocurrents, is responsiblefor the observed waveform changes. As will be discussed below, theobserved waveform changes can be assigned to delayed ultrafast thermioniccurrents from the STM tip, whose electronic subsystem is heated onthe femtosecond time scale.First, we want to understand ultrafast heating for a free-standingtip. 43 On longer time scales of 16 Due to the differenttime scales of 44 Both nonthermal and thermal electron distributionscan give rise to a photocurrent from the tip, which we write as thesumTo understand the observed waveformchanges we calculate the photocurrent from the tip and its modulationby the THz field. The microscopic picture of our model and the electrondynamics inside the metal tip are depicted in 45z-direction. The ultrafast thermionic current through the THz-modulatedpotentialbarrier is given by the total charge section 4 of the Supporting Information). The TTM source term 46 to account for the initial fast redistribution of energy due toballistic transport. The left panel in The THz-modulated transmission probability nnQ is thecharge emitted per unit area per pulse through channel n, n accounts for the decreasing probability for excitationinto higher multiphoton channels. However, the relative contributionfrom higher channels increases with increasing laser intensity asexpected due to n-dependent excitation probability expected for multiphoton absorptionin the perturbative regime.47 The THz-inducedchange of the nonthermal charge density, The quasi-instantaneous photocurrent due to nonthermal electronscan be written as a superposition of currents through each multiphotonchannel 31 our measurement is not sensitive to electron propagationbut only to the instantaneous THz field at the tip surface. Since At high laser fields approaching Figure S.5). The simulationswith incident laser intensities close to those estimated from ourexperimental conditions reproduce very well the measured waveformbroadening and temporal shift. The exact laser intensities requiredto observe electronic heating in the simulated waveforms vary withtip size and shape, which is consistent with the experimental variationof the laser power we require to observe waveform deformations fordifferent tip conditions. The corresponding time dependence of theelectron temperature and thermionic current are shown in 48 It is important to note thatthermionic currents alone do not sufficiently describe the waveforms,especially at low laser intensities at which Figure S.4.2). To reproduce the laser power dependenceof the waveforms in 50 which is supported by our results. We note that we can exclude thatthe delayed currents originate from laser-driven rescattering51 and delayed re-emission of electrons from thetip,21 as the waveform distortions areobserved also (and for some tips solely) in the weak-field regime,in which laser-driven electron scattering is negligible (section S3 of the Supporting Information). Theresults in 30 therefore, the THz bias measured at large gap distance (gray dashedline) applies to all distances. We note that “static”rectified THz currents are negligible due to the small THz bias amplitudeand the comparably large gap distance when operating at 10 V bias.As discussed in the Supporting Information, our measurements are not affected by quasi-static or transient(pulse-to-pulse) thermal expansion of the STM tip due to pulsed laserillumination. Starting from a distorted waveform at section S6 in the Supporting Information). Whereas at large tip–sample distances the photocurrentexhibits a nonlinearity of To examine the role of thermalizedhot electrons for photoinduced tunneling in STM, we measure the THzwaveform versus tip–sample distance as plotted in The observations in 52 which is more appropriate at nanometer gap sizes.We expect that more detailed spectroscopic information about the nonthermaldistributions might be obtained in future experiments by variationof the dc and THz bias. Moreover, it may be possible to measure theelectron thermalization process and its effect on tunneling by usingshorter THz transients to increase the time resolution.Finally, we note that our simple model can reproducethe data reasonablywell without calculation of the full nonthermal steplike distribution.The experimental data, however, shows a waveform change, within adistance variation of only 1 Å close to the set point, more rapidthan that observed in the simulations. This indicates that our modelmight underestimate nonthermal hot electron tunneling close to theFermi level, which exhibits an even higher sensitivity to the tip–sampledistance. Further improvement of the agreement between the experimentaland simulated results could be obtained by extending our model by(i) the simulation of the electron thermalization process, (ii) theinclusion of the full nonthermal distribution, and (iii) replacingthe Fowler–Nordheim barrier by the Simmons barrier,In conclusion, we investigated the competitionbetween ultrafastthermal and nonthermal photocurrents inside a laser-excited STM junctionvia phase-resolved sampling of broadband THz transients. Our resultsreveal that hot electron tunneling from a laser-excited STM tip isdominated by nonthermal electron distributions, accompanied by delayedtunneling of thermalized hot electrons. At large gap sizes, at whichphotoinduced tunneling through the barrier is negligible, thermalizedhot electrons dominate the THz-induced photocurrent from a tungstentip excited at high laser intensities. However, at small gap distance,this current contribution is exceeded by nonthermal currents as soonas the probability for photoassisted tunneling at low energies belowthe vacuum level increases appreciably. We infer the markedly nonthermalcharacter of the photoexcited electron distribution from the distinctlydifferent durations of the thermal and nonthermal photocurrents. Promptnonthermal tunneling is followed by a thermionic photocurrent thatcan be delayed by several 100 fs, as determined by the time at whichthe laser-excited STM tip reaches its peak electronic temperature,which can increase up to several thousand Kelvin. The weak THz fieldserves as a highly sensitive and selective probe for photoinducedtunneling currents in ultrafast laser-excited STM, which is a uniquecapability of THz-gated STM. Detailed information about the phaseand amplitude of the THz bias allows one to extract the ultrafastdynamics of hot electrons on time scales much shorter than a singleTHz cycle even in nonresonant systems like metals or semimetals. Ourresults provide detailed microscopic insight into the nonequilibriumdynamics of hot electrons in a laser-excited STM tip, which is ofgeneral relevance to experiments that aim to study photophysical orphotochemical processes on ultrafast time scales with pulsed-laserexcited STM. We envision that extension of our approach by the imagingcapabilities of STM will allow for the spatiotemporal investigationof ultrafast carrier dynamics at photoexcited surfaces on Angstromlength and femtosecond THz-subcycle time scales.–10 mbar). The spring-loaded STM platform is equippedwith two off-axis parabolic mirrors  for illumination with a broadbandNIR laser and broadband THz pulses, respectively. Both beams are incidentto the tip axis at an angle of 68° and are polarized along thetip axis. The THz beam enters the UHV chamber through a 500 μmthick diamond window and is focused by the Au PM. The Ag PM is usedto focus the NIR pulses for photoexcitation of the STM junction. Precisefocus alignment on the tip apex is ensured by precise positioningof the PMs in UHV, which are motorized and can be moved in the x-, y-, and z-directions(Attocube GmbH). The tip position is fixed, and the sample is movedfor coarse motion and scanning. The dc bias is applied to the sampleand the current is collected from the grounded tip. The current preamplifier(Femto DLPCA) is operated at a gain of 109 V/A at 1 kHzbandwidth. A mechanical chopper operated at a frequency of 607 Hzmodulates the power of the NIR laser beam used for THz generation,and lock-in detection is used to detect the THz-induced current. Repeatedcycles of Ar+ sputtering and annealing up to 670 K wereconducted to clean the Ag(111) sample before measuring. Electrochemicallyetched tungsten tips are transferred to UHV immediately after etching.Measurements are recorded with a customizedSTM from Unisoku (USM-1400with Nanonis SPM controller) operated at room temperature and underultrahigh vacuum conditions  delivering NIR laser pulsesof 10 fs duration at 800 nm center wavelength and with 3 μJenergy (2 μJ are available for the THz-STM setup) at Supporting Information.Numerical simulations are carried out using COMSOL Multiphysics5.6., which provides the required values of the dc field, tip-enhancedoptical field, and electronic temperature evolution inside the tip.The photoinduced thermal and nonthermal currents and their THz-inducedchange (THz waveforms) are simulated using custom Python scripts.Details about the model are described in the


A 20-year-old female presented to the emergency department for evaluation of exertional, right-sided chest pain. The patient underwent a computed tomography angiogram of her chest as part of her workup, demonstrating the right lower-lobe pulmonary artery arising from the abdominal aorta.Anomalous pulmonary arterial supply is exceedingly rare. In adult patients, it is likely to be found incidentally during workup for more common medical conditions. Symptoms may include chest pain, exertional dyspnea, or hemoptysis. The high pressure of systemic blood in a low-pressure pulmonary system can result in right heart strain, pulmonary hypertension, and high-output cardiac failure. A 20-year-old female with no previous past medical history, other than a recent evaluation for Ehlers-Danlos syndrome, presented to the emergency department for chest pain. She described exertional right-sided chest pain increasing over the previous two months. Vital signs were stable, and her physical exam was unrevealing. Electrocardiogram demonstrated poor R-wave progression in the precordial leads. Laboratory testing included an unremarkable complete blood count, comprehensive metabolic panel, and troponin. Chest radiograph was normal. Her history of possible Ehlers-Danlos syndrome prompted the emergency physician to complete a computed tomography angiogram (CTA) of the chest and abdomen for consideration of aortic dissection. The CTA revealed a pulmonary artery originating from the descending aorta above the celiac plexus, supplying the right lower lobe of the lung  and 2. OAn anomalous origin of a pulmonary artery branch is a rare congenital abnormality that comprises 0.12% of all congenital heart defects.Symptoms such as chest pain, hemoptysis, and exertional dyspnea may help identify patients with anomalous pulmonary vasculature. The high pressure of systemic blood in a low-pressure pulmonary system can result in right heart strain, pulmonary hypertension, and high-output cardiac failure.3Understanding the significance of congenital anomalous pulmonary arteries is imperative for emergency physicians to aid in identification of this abnormality. If the patient is hemodynamically stable, patients may be followed closely as an outpatient with cardiothoracic surgery and cardiology. Surgical treatment is reserved for those with severe symptoms or those who have developed pulmonary hypertension to prevent cardiomyopathy and valvular disease. Endovascular embolization has been successful in certain cases.5What do we already know about this clinical entity?Anomalous pulmonary artery is a rare congenital disease that typically presents early in life with congestive heart failure.What is the major impact of the image(s)?These images are an example of a rare disease entity as a cause of chest pain and respiratory distress, which are common presenting symptoms in the emergency department.How might this improve emergency medicine practice?Although rarely discovered in adulthood, recognition of anomalous pulmonary arterial supply can lead to expedited referral and definitive management.


With the mortality rate of pancreatic cancer predicted to rise over the coming years, it is essential that effective treatment strategies are developed as soon as possible. Pancreatic cancer has always proven very difficult to treat due to its fast growing and aggressive nature. Chemotherapeutic treatment has struggled to increase the survival rate of pancreatic cancer patients due to effective chemo-resistant properties that derive from the supporting tumor microenvironment and autophagy, a vital survival pathway. This review will explore how the autophagy pathway and tumor microenvironment help to sustain tumor survival under stress and expand into a metastatic state. Due to the comprehensive understanding of the autophagy pathway, we will highlight the potential chinks in the pancreatic tumor’s armor and identify potential targets to overcome chemo-resistance in pancreatic cancer. We will also present novel autophagy inhibitors that could reduce tumor survival and how they could be most effectively conceived.Pancreatic cancer is known to have the lowest survival outcomes among all major cancers, and unfortunately, this has only been marginally improved over last four decades. The innate characteristics of pancreatic cancer include an aggressive and fast-growing nature from powerful driver mutations, a highly defensive tumor microenvironment and the upregulation of advantageous survival pathways such as autophagy. Autophagy involves targeted degradation of proteins and organelles to provide a secondary source of cellular supplies to maintain cell growth. Elevated autophagic activity in pancreatic cancer is recognized as a major survival pathway as it provides a plethora of support for tumors by supplying vital resources, maintaining tumour survival under the stressful microenvironment and promoting other pathways involved in tumour progression and metastasis. The combination of these features is unique to pancreatic cancer and present significant resistance to chemotherapeutic strategies, thus, indicating a need for further investigation into therapies targeting this crucial pathway. This review will outline the autophagy pathway and its regulation, in addition to the genetic landscape and tumor microenvironment that contribute to pancreatic cancer severity. Moreover, this review will also discuss the mechanisms of novel therapeutic strategies that inhibit autophagy and how they could be used to suppress tumor progression. In 2020, pancreatic cancers (PC) had the 14th highest incidence of cancer and placed 7th for the most deaths at a mortality rate of approximately 94% . In factKirsten’s rat sarcoma viral oncogene homologue (KRAS) and tumor protein p53 (TP53) which can synergize to exert a vast range of growth and tumor supporting signals ,26912,26YAP-1 knockout or inhibition was able to deactivate PSCs and severely reduce tumor proliferation . T. T343]. A more recent study identified a new compound named SBP-7455 that demonstrated a 10-fold increased potency than SBI-0206965 and was more effective at blocking autophagic initiation in triple negative breast cancer . SBP-745In conjunction with the ULK1 complex, autophagy is initiated by the PI3KC3-C1 protein conjugate consisting of a core of Beclin-1, VPS15 and VPS34 subunits ,63. As aULK1 and ATG7 . U. U356]. SAR405 was shown to synergize with HER2 inhibitors in HER2+ breast cancer ; celecoxTSC knockout mouse embryonic fibroblasts were observed to respond to autophagy deficiency through the upregulation of macropinocytosis, a survival pathway that internalizes proteins for lysosomal degradation [radation . This stradation . Notablyradation , which din vitro evaluation. A recent study has screened the Published Kinase Inhibitor Set by Glaxo-Smith-Kline resulting in the identification of two ULK1 inhibitors (GW837331X and GW406108X) and two VPS34 inhibitors (GSK2358994A and GW429374A) [Even though inhibitors of ULK1 or PI3KC3 complexes are yet to enter PDAC clinical trials, there are more compounds being developed that await thorough 429374A) . Both se429374A) . However429374A) .In addition to synthetic small molecule agents, natural products and their derivatives have also been shown to suppress PC tumour progression via interactions with the autophagy pathway. The mechanism of action of these natural products is typically not well elucidated but does provide an understanding that various compounds can beneficially interact with autophagy in different ways. One of the common avenues natural products achieve this is through the dysregulation of apoptosis pathways (namely through Bcl-2 and caspases) to achieve cell death.Ganoderma lucidum known as Fudan-Yueyang-Ganoderma lucidum (FYGL) was found to increase autophagosome levels and prevent autolysosome fusion, indicating late-stage autophagic inhibition effects [ATG5 knockdown, the researchers observed a reduced effectiveness and concluded that alantolactone toxicity was dependent on accumulated autophagosomes [A proteoglycan extracted from  effects . FYGL al effects . However effects . When usagosomes . Alantolagosomes . Additioagosomes ,366.Curcumin is another natural compound which mediates its anti-tumor effects by increasing the ratio of BAX to Bcl-2, leading to increased rates of apoptosis . The relUrsolic acid (UA) is a natural triterpene, which is known to increase cellular stress. In PDAC cells, the increased stress upregulated autophagy, while the reduced RAGE expression was shown to induce cell cycle arrest and apoptosis . RAGE isNotably, periplocin has been found to induce caspase-dependent apoptosis through AMPK activation/mTOR inhibition . HoweverOverall, these studies suggests that while the use of natural products in autophagy-related therapy remains a promising avenue for treatment, their limited efficacy and potency and promiscuous targeting must be taken into consideration when compared to targeted synthetic small molecule inhibitors of autophagy related proteins.This review of current literature demonstrates a critical role of the autophagic pathway in key mechanisms involved in PDAC progression, such as metabolic reprogramming, immune evasion, anti-apoptosis, TME-induced stress, metastasis, perineural invasion, etc. Importantly, there is a new wave of next generation autophagy inhibitors which have shown potential to be developed as novel therapeutics for the treatment of PDAC. With poor patient outcomes after PDAC chemotherapy, the development of these promising compounds can be clinically applied to significantly improve PDAC patient survival and increase quality of life. Due to modest improvement in therapeutic outcomes using current treatments , autophagy inhibitors could be implemented into the current clinical approach to PDAC treatment. Therefore, further research to comprehensively understand the role of autophagy in PDAC progression and development of autophagy inhibition based anti-PDAC therapeutic strategies are highly warranted.


Prior to incident AMI, smoking, hypertension, hyperlipidemia and diabetes mellitus were better controlled in the prior cancer group. However post-AMI, prior cancer was associated with lower guideline-directed medical therapy usage and higher all-cause mortality . Conclusions: AMI occurred despite better control of cardiovascular risk factors and lower age-standardized estimated mean 10-year ASCVD risk among patients with prior cancer than no cancer. Prior cancer was associated with lower guideline-directed medical therapy post-AMI and higher mortality.Background: Patients with cancer are at increased risk of acute myocardial infarction (AMI). It is unclear if the Atherosclerotic Cardiovascular Disease (ASCVD) risk score at incident AMI is reflective of this higher risk in patients with prior cancer than those without. Methods: We linked nationwide AMI and cancer registries from 2008 to 2019. A total of 18,200 eligible patients with ASCVD risk score calculated at incident AMI were identified . Results: At incident AMI, age-standardized mean ASCVD risk was lower in the prior cancer group (18.6%) than no cancer group (20.9%) ( Cancer survivorship has improved greatly due to advancements in cancer detection and therapy . AlthougExisting strategies for primary prevention of cardiovascular disease include the routine use of scores for risk stratification and guidance of treatment. The well-established Atherosclerotic Cardiovascular Disease (ASCVD) risk calculator endorsed by the American College of Cardiology is one of the most commonly used risk scores for primary prevention of cardiovascular disease . HoweverTo better understand the association of ASCVD scores in patients with prior cancer, we combined data across national registries to calculate the ASCVD risk scores at incident AMI, comparing risk scores in those with prior cancer to those without. Our objectives were first to determine if the age-standardized ASCVD score calculated at incident AMI was lower in patients with prior cancer than those without cancer, and second, to compare treatment of cardiovascular risk factors prior to incident AMI between the two groups of patients. We hypothesized that prior cancer was associated with lower ASCVD scores and a lower rate of treatment of cardiovascular risk factors prior to incident AMI.In this population-based cohort study, we combined data from two national registries managed by the National Registry of Diseases Office of Singapore . The NatData between the Singapore Myocardial Infarction Registry and the Cancer Registry were linked to identify patients with prior cancer presenting with AMI over the 12 years from 1 January 2008 to 31 December 2019. The Registry of Birth and Death was then linked with this combined dataset to ascertain mortality. As the ASCVD score is used for primary prevention and is not applicable for secondary prevention, we excluded patients with a prior history of AMI, stroke, percutaneous coronary intervention or coronary artery bypass grafting surgery. We also excluded patients who developed cancer after incident AMI, as well as patients with incomplete lipid or blood pressure data, thus preventing complete calculation of ASCVD scores.Each individual had their predicted risk of cardiovascular disease calculated via the ASCVD risk score via data obtained just prior to or at incident AMI. The ASCVD estimates a 10-year composite risk of coronary death, non-fatal AMI and fatal or non-fatal stroke. It incorporates nine variables: age, sex, race, diabetes, smoking, total cholesterol, high density lipoprotein cholesterol (HDL-C), systolic blood pressure and treatment for hypertension. Individuals are classified as low-risk (ASCVD risk score < 5% 10-year risk), borderline-risk (ASCVD risk score 5% to <7.5% 10-year risk), intermediate-risk (ASCVD risk score ≥ 7.5% to <20% 10-year risk) and high-risk (ASCVD risk score ≥ 20% 10-year risk) .The primary outcome of interest was the ASCVD risk score at incident AMI. Secondary outcomes of interest included rates of treatment of cardiovascular risk factors prior to incident AMI and post-AMI mortality. AMI patients were classified into two groups: patients with prior cancer (prior cancer) and patients without prior cancer (no cancer). We excluded all missing data from the analyses via case deletion without imputation. STATA SE version 13 was used to perform all statistical analyses.Demographics, comorbidities and clinical characteristics at incident AMI were compared between those with prior cancer and those without using a chi-square test for categorical variables and Wilcoxon rank-sum test for numeric variables. As the patients with prior cancer were older than those without cancer, we standardized their age structure to those without cancer . We compared the risk distribution between the two groups by the Chi-square test. Among those with prior cancer, we further categorized risk distribution by cancer characteristics. Kaplan–Meier curves were plotted to compare the cumulative incidence of all-cause death during the study period between the two groups of patients. The hazard ratio of all-cause death among those with prior cancer compared with those without cancer was assessed by Cox regression adjusted for age, sex, smoking status, hypertension, hyperlipidemia and diabetes mellitus.We identified 90,297 patients with AMI between 1 January 2008 and 31 December 2019. Following the exclusion of patients with a history of prior AMI, stroke, percutaneous coronary intervention or coronary artery bypass grafting surgery, 71,377 patients with incident AMI without prior cardiovascular disease remained. We then further excluded patients who developed cancer after incident AMI and patients with incomplete data for calculation of ASCVD risk score. A total of 18,200 patients were available for final analysis, of which 1086 patients had prior cancer (prior cancer group) and 17,114 patients did not (no cancer group) . The medAt incident AMI, the prior cancer group had fewer current smokers . The preThere were fewer ST-segment elevation myocardial infarctions in the prior cancer group, and these patients had lower rates of revascularization . AlthougThe median ASCVD score for the entire study cohort predicted a 14.8% 10-year risk of cardiovascular events. In unadjusted analyses, the prior cancer group had a significantly higher median predicted ASCVD risk and a higher proportion of high ASCVD risk than the no cancer group, reflecting the greater age of the cancer group. This study reports on the differences in estimated cardiovascular risk via the ASCVD risk score among a national cohort of patients with and without prior cancer presenting with their first AMI. Amongst more than 18,000 patients with AMI over 12 years, the age-standardized mean 10-year ASCVD risk was 18.6% in the prior cancer group, significantly lower than the 20.9% observed in the no cancer group. Prior cancer patients were more likely to be receiving treatment for their cardiovascular risk factors prior to incident AMI but reported lower use of guideline-directed medical therapy post-AMI than the no cancer group. Prior cancer was associated with lower post-AMI survival than no cancer.The main finding of this study is that AMI occurred despite a lower age-standardized estimated mean 10-year ASCVD risk in prior cancer than no cancer. This suggests that while traditional cardiovascular risk factors are better controlled in patients with prior cancer, there may be other non-traditional risk factors contributing to AMI risk unaccounted for by the ASCVD risk score. Hence, there remain clear limitations when applying the ASCVD risk prediction score to cancer patients which may underestimate the ASCVD risk. This is likely due to several potential non-traditional risk factors which may increase the risk of adverse cardiovascular events in patients with prior cancer—first, cancer therapies are commonly associated with cardiotoxicity in nearly a third of all patients . These iWe also observed that patients with prior cancer had their traditional cardiovascular risk factors better controlled prior to incident AMI, yet were discharged with lower rates of guideline-directed medical therapy post-AMI compared with patients without cancer. The improved control of cardiovascular risk factors prior to AMI could be due to patients with prior cancer having greater contact with healthcare professionals compared with patients without cancer. Conversely, the low rates of guideline-directed medical therapy in patients with prior cancer compared with patients without cancer is likely due to treatment bias. Studies have demonstrated lower prescription of ideal cardiovascular health behaviors and therapies to patients with cancer ,25. GreaOur findings highlight that clear treatment targets for primary prevention of cardiovascular disease in cancer patients are required. Although our findings demonstrated that the traditional cardiovascular risk factors were better controlled prior to incident AMI within the prior cancer group than the no cancer group, the intensity of therapy was lower than expected for the associated ASCVD score. This further highlights the current evidence gap in which current guidelines on primary prevention do not specifically provide treatment targets for patients with cancer ,11,12. OThere are several limitations in our study. First, the ASCVD was developed primarily for use within the American population and may not be fully applicable to the multi-ethnic Singaporean population. Second, the ASCVD is meant for primary prevention in patients older than 40 years old, but we included patients younger than 40 years due to the importance of risk stratification within childhood cancer survivors who are equally of high cardiovascular risk . FinallyIn conclusion, AMI occurred despite better control of traditional cardiovascular risk factors and a lower age-standardized estimated mean 10-year ASCVD risk among patients with prior cancer than patients with no cancer. Patients with prior cancer have better control of traditional cardiovascular risk factors before AMI but less guideline-directed medical therapy after AMI and higher post-AMI mortality. Additional research to support appropriate recommendations for best clinical practice are needed to improve the prevention and management of cardiovascular disease among cancer survivors.


We report a novel presentation of deficit in anterior pituitary function with variable immune deficiency (DAVID) syndrome in a healthy young girl presenting in Addisonian crisis with raised intracranial pressure. Nearly all cases of DAVID syndrome described in the literature have presented with recurrent infections and variable immunodeficiency. Pseudotumour cerebri has not been reported in DAVID syndrome to date.2O). Cerebrospinal fluid analysis was normal. The patient’s symptoms improved with hydrocortisone replacement and acetazolamide, but the raised intracranial pressure recurred after acetazolamide was discontinued. She was subsequently found to have panhypogammaglobulinaemia, and DAVID syndrome was diagnosed. Genetic testing demonstrated a truncating mutation in the NFKB2 gene c.2557C > T, p.(Arg853*).A four-year-old girl represented to hospital with vomiting, confusion and diplopia after ten days of tiredness, neck and abdominal pain, and headache. Her cranial nerve examination demonstrated a right abducens nerve palsy and papilloedema, and she was found to have ketotic hypoglycaemia and hypocortisolaemia secondary to adrenocorticotrophic hormone (ACTH) deficiency. Her neuroimaging was consistent with pseudotumour cerebri, and her lumbar puncture opening pressure confirmed raised intracranial pressure (30–40 cmHThis case demonstrates pseudotumour cerebri as a novel neurological presentation of DAVID syndrome, highlights the rare association between adrenal insufficiency and intracranial hypertension, and shows the challenges in diagnosing isolated ACTH deficiency. We emphasise that cortisol should be checked in pre-pubertal children with pseudotumour cerebri and a diagnosis of DAVID syndrome considered in those presenting with low cortisol and neurological symptoms. NFKB2 gene which result in haploinsufficiency in the non-canonical NF-kB2 signaling pathway which regulates B cell maturation . Gluc. Gluc6].A short Synacthen test was performed two days following presentation after the patient had received high-dose intravenous hydrocortisone for treatment of the Addisonian crisis. Because of the potential for high-dose glucocorticoid therapy to interfere with the Synacthen test result, the test was repeated one month after diagnosis after stopping glucocorticoid therapy for two weeks. Stress doses of glucocorticoids were to be given if the child developed any illness but were not needed. Surprisingly, the repeat Synacthen test results showed extremely low cortisol levels suggesting progressive atrophy of the adrenal zona fasciculata and a lack of any stimulation by ACTH . This maThe diagnostic work-up for children presenting with pseudotumour cerebri is extensive and includes the differential diagnoses cerebral venous sinus thrombosis, craniosynostosis, otitis media, haematological conditions, hypercoagulable states, medications, hypervitaminosis A, obstructive sleep apnoea and chromosomal abnormalities , 11. An The pathophysiology of raised intracranial pressure in corticosteroid deficiency is poorly understood. The enzyme 11ß-hydroxysteroid dehydrogenase type 1, which converts inactive cortisone to cortisol, is expressed in the choroid plexus suggesting a role for cortisol in the regulation of CSF production . FurtherNFKB2 require longitudinal surveillance not only for infective but also autoimmune complications.The patient had evidence of impaired B cell differentiation and function with frequent URTIs in early childhood years. Her clinical phenotype may evolve over time, and patients harbouring mutations in We report the case of a four-year-old girl with DAVID syndrome presenting in Addisonian crisis associated with pseudotumour cerebri. The case demonstrates the importance of measuring cortisol in children presenting with features of pseudotumour cerebri and that immunoglobulins should be tested in individuals with isolated central hypocortisolaemia. A diagnosis of DAVID syndrome should be considered in all patients presenting with low cortisol and neurological symptoms.


A gender-based disparity in physical activity, where girls are less physically active than boys, is a persistent finding in the literature. Participation in organised sport plays a significant role for adolescents' physical activity habitsand provides an important means to achieve the recommended amount of daily physical activity. Nevertheless, participation in sport decreases with age, and to a higher degree among girls. Moreover, girls living in low socio-economic status neighbourhoods are even less represented in organised sport. This upholds a social inequality as a range of physical, mental and social benefits are associated with regular participation in sport. Empowering girls as experts on their own needs and preferences toward sport is critical. The aim of this qualitative study was to examine the experiences and perceived barriers to sports participation among adolescent girls living in low socio-economic status neighbourhoods.During July-October 2021 eleven semi-structured focus groups were carried out with adolescent girls (10-16 years) who were not engaged in organised sport. The participating girls (n = 44) were recruited through purposive sampling via four Danish schools placed in areas with high deprivation. A thematic analysis was conducted from verbatim transcripts using NVivo.Six themes were identified; 1) Competing priorities, 2) Social aspects of sports participation, 3) Perceived lack of sporting abilities, 4) Feeling discomfort, 5) Gender stereotypes and 6) Physical Education as introduction to sport. Even though there were many similarities among the girls' perceived barriers to sports participation, the data showed variations in the way these barriers were experienced.The results provide insight into barriers to sports participation among adolescent girls living in low socio-economic status neighbourhoods. The findings correspond with other studies among adolescent girls in general. However, the findings among this underrepresented group will be useful to shed light on how interventions should be designed to promote sports participation among adolescent girls living in low socio-economic status neighbourhoods. As this is perceived as a hard-to-reach group, the findings will be a contribution to the field and are highly needed to illuminate the different perspectives on gender-based disparities that exist in relation to sports participation.


Arabidopsis lyrata, we assess genome-wide heterozygosity and signatures of selection at single nucleotide polymorphisms near transposable elements across an inbreeding gradient. Under intense inbreeding, we find systematically elevated heterozygosity downstream of several TE superfamilies, associated with signatures of balancing selection. In addition, we demonstrate increased heterozygosity in stress-responsive genes that consistently occur downstream of TEs. We finally reveal that TE superfamilies are associated with specific signatures of selection that are reproducible across independent evolutionary lineages of A. lyrata. Together, our study provides an important hypothesis for the success of self-fertilizing species.Elevated levels of inbreeding increase the risk of inbreeding depression and extinction, yet many inbred species are widespread, suggesting that inbreeding has little impact on evolutionary potential. Here, we explore the potential for transposable elements (TEs) to maintain genetic variation in functional genomic regions under extreme inbreeding. Capitalizing on the mixed mating system of  Arabidopsis lyrata and reducing the probability of inbreeding depression.How highly inbred populations generate novel genetic variations upon which natural selection can act is unclear. Here, the authors reveal the effect of transposable elements on the genome-wide heterozygosity landscape across a natural inbreeding gradient of  This evolutionary paradox is exemplified by the success of self-pollinating plant species, which are particularly capable of colonizing a wide range of environments6. Arabidopsis thaliana, for instance, a self-compatible species with extreme inbreeding levels across its natural range, does not show widespread signatures of increased extinction risk but instead exhibits significant adaptive potential9. Thus, while small and genetically depleted populations frequently continue to evolve and persist, we are still far from understanding the molecular mechanisms underlying this evolutionary potential.Inbreeding has been consistently shown to reduce individual fitness, population viability and evolutionary potential13. Empirical work has shown that populations with large effective sizes in particular can be vulnerable to inbreeding depression, because a long history of outcrossing has allowed the accumulation of deleterious mutations that become visible to negative selection when inbreeding levels increase13. Small and/or self-compatible populations, on the other hand, more effectively purge deleterious genetic load, allowing them to maintain fitness during episodes of high inbreeding17, but see ref. 19.Insufficient rates of outcrossing can cause populations to suffer inbreeding depression due to recessive deleterious mutations becoming homozygous, consequently increasing population extinction risk22), purging may particularly rapidly remove genetic variation required for long-term evolution and fitness. In addition, slightly deleterious alleles can become adaptive in specific environmental conditions25, or in combination with compensatory mutations26, causing purging to remove cryptic evolutionary potential associated with conditionally deleterious alleles. While inbreeding and purging may thus mutually accelerate the loss of evolutionary potential, self-compatible species frequently appear to be relatively resilient to high levels of inbreeding32.While purging can safeguard populations from inbreeding depression, it may facilitate the depletion of population genetic variation under prolonged inbreeding. When low recombination rates prevent the segregation of neutral and adaptive genetic variation from recessive deleterious alleles  can rapidly generate genetic variation through increasing mutation rates of upstream and downstream flanking genomic regions;37. Because DNA repair is an error-prone process, this mutation-by-transposition mechanism is considered to be a major source of mutations near TEs37. Second, TEs and nearby sequences tend to be heavily methylated. This methylated DNA, cytosines in particular, is susceptible to spontaneous mutation, resulting in increased cytosine-to-thymine conversion45. As a consequence, even silenced TEs may impact local mutation rates, and the mere presence of TEs may generate functional genetic diversity where host genomes have co-opted TEs48. The efficiency of this mutation-by-methylation mechanism may vary across TEs depending on their activity in the host genome. Stress-responsive TEs, including at least several Copia elements, frequently contain genetic elements conferring downstream stress-responsiveness through activation (de-methylation) or repression (methylation) under stress51. Stress-responsive TEs are thus characterized by prolonged periods of downstream methylation during or between stress episodes, which may increase mutation rates and genetic diversity particularly downstream of these TEs and in functional genes. Similarly, miniature inverted-repeat transposable elements (MITEs) have been shown to attract siRNAs that repress downstream gene expression52, and to impact methylation of a downstream flowering gene in maize53. Whether genomic regions downstream of specific TE families are systematically characterized by increased genetic diversity and involvement in evolution remains to be elucidated.Two mechanisms can generate genetic diversity near TEs. First, TE excision and insertion during transposition events require annealing and repairing of flanking DNA by DNA repair machinery55. Experimental work has also shown that, upon TE activation, transposition can exponentially increase mutation rates in genes without harmful effects on fitness55. This suggests that enhanced TE activity has the potential to reinforce evolution through generating neutral and/or beneficial genetic variation in functional genes. Recent findings of high genetic diversity in TE-rich genomic regions of invasive ant populations56 may point to a role for genetic bottlenecks and inbreeding in driving TE activity and nearby genetic diversity. In the mixed mating species A. lyrata, self-fertilizing populations are characterized by increased TE copy numbers and higher TE frequencies than outcrossing populations, aligning with models of neutral evolution and ectopic recombination57. More specifically, the higher levels of sequence homozygosity in self-fertilizing populations reduces the rate at which heterologous sequences recombine, thereby generating harmful chromosomal rearrangements. As a consequence, self-fertilization can facilitate genome-wide TE accumulation, potentially providing considerable opportunities for evolution in genomic sequences flanking TEs. However, while inbreeding may affect TE dynamics and evolution in A. lyrata59, the impact of inbreeding on genetic variation and evolution surrounding TEs has never been explicitly tested.Co-opted TEs may increase mutation rates particularly in functional genomic regions that can benefit from genetic variation. They have correspondingly been shown to be non-randomly distributed across the genome, preferentially integrating within functional genes and away from vital core functionsA. lyrata, where populations exhibit a mixed mating system61. In addition, we assess the proportion of flanking adaptive outliers per TE superfamily and validate these TE-related evolutionary signatures in populations of an independent evolutionary lineage (European A. lyrata). We finally apply gene ontology enrichment analyses to test whether genetic diversity near TEs is associated with particular functions (e.g. related to stress responses). We hypothesize that (i) TEs can impact heterozygosity across the genome, particularly downstream, (ii) this increase in heterozygosity is functional and may thus provide a basis for evolution under inbreeding, and (iii) the role of TEs in evolution is consistent across sampling areas  and most distal to TEs from the Harbinger superfamily .In order to understand the relationship between TEs and nearby heterozygosity, we first explored the tendency of random SNPs (Supplementary Data FIS > 0.6), heterozygosity was low across most of the genome . While Copia and Harbinger elements were associated with elevated levels of heterozygosity particularly under inbreeding, LINE elements had high heterozygosity irrespective of inbreeding levels , average heterozygosity levels (across inbreeding coefficients) did not differ from those near other TE superfamilies  and negative for outliers downstream of TEs (Pearson’s r −0.49(*) and −0.57* for balancing and divergent outliers, resp.). It is quite remarkable that TE-related signatures of selection were correlated between sampling areas with distinct evolutionary histories and environmental conditions, the latter being reflected by the low number of SNPs sharing signatures of selection between the two sampling areas  did not significantly differ from background heterozygosity (H0) in outbreeding European samples with FIS < −0.3  heterozygosity is elevated downstream of TEs, (ii) this increase in heterozygosity is associated with housekeeping functions, and may thus provide a basis for evolution under inbreeding, and (iii) the role of TEs in adaptive evolution is consistent across independent evolutionary lineages. In general, TEs appear to be involved in heterozygote advantage that becomes particularly apparent under inbreeding, and in frequency-dependent balancing selection through their overrepresentation in gene ontology processes related to signaling and (a) biotic stress responses. Our findings provide important indirect evidence for TEs being responsible for much of the evolutionary trajectories of natural populations with reduced outcrossing rates, and that genomic regions downstream of TEs provide functional genetic variation as potential targets of adaptive evolution.Despite more than a century of research on the molecular basis and ecological consequences of inbreeding, we still poorly understand why many natural populations characterized by intense inbreeding can frequently continue to evolve. While mutations are the predominant driver of evolution in the absence of outcrossing, they typically arise at low rates that are unlikely to maintain evolution63, most of the SNPs identified here (65%) appear to be within reach of TEs  near functional genes, where mutations flanking TEs in particular can provide fitness benefits to the host55, and (ii) away from vital genes, where mutations more distant from TEs but residing within these vital genes can be harmful or beneficial to organismal fitness depending on their impact on gene functioning. As a result, the genome-wide distribution of proximity effect of TEs on heterozygosity likely varies with gene function and essentiality. The tendency of SNPs with signatures of balancing selection downstream of TEs to appear in essential genes  demonstrating that TEs are implicated in adaptive evolution both in inbreeding and outcrossing plant populations, and (ii) revealing a potential association between specific TE superfamilies and the sensitivity of populations to inbreeding depression. Specifically, we found TE superfamily-specific signatures of selection that could be replicated across independent sampling sites Fig. . AlthougGenetic variants downstream of TEs show a notably distinct pattern as compared to upstream signatures of selection. Specifically, where TE superfamilies are featured by a high proportion of downstream outliers in North America, they typically have a low proportion of downstream outliers in Europe, and vice versa Fig. . InteresA. arenosa across independent elevation gradients71, corroborating their involvement in climate adaptation. We suggest that the combined actions of TE mutability, purging and natural selection may represent a powerful evolutionary strategy allowing long-term persistence of naturally inbred populations. More broadly, this theory could explain the success of self-fertilizing populations across the globe. Importantly, this does not refute the idea that self-fertilizing populations frequently are evolutionary dead-ends arising from continuous drift-induced accumulations of deleterious mutations73, but see ref. 76, self-pollinating taxa that do persist through time may do so through evolving strategies counteracting inbreeding depression.While calling for further research, our results collectively suggest that each TE superfamily can facilitate evolution in particular environmental and demographic situations. Specifically, where Copia and Harbinger elements are associated with increased heterozygosity in housekeeping genes, presumably to maintain fitness under elevated levels of inbreeding, other TE superfamilies are linked to genetic diversity in functional genomic regions and may thus promote adaptive evolution under changing environmental conditions. Climate warming may, for example, drive adaptive evolution particularly downstream of Helitron elements, which were found to be associated with genetic variants involved in heat and drought responses. Helitron elements have correspondingly been shown to manifest differential expression in A. lyrata. While lethal TEs are rapidly purged from a population41, slightly or conditionally deleterious alleles arising near TEs are more difficult to purge, and may reach higher frequencies resembling signatures of balancing selection77. The lack of low-frequency signatures of balancing selection  and associative overdominance , both arising from purifying selection at linked recessive deleterious loci, may also contribute to genome-wide signatures of balancing selection. Both types of selection are covered by our analysis of purifying selection  were sampled from field sites and grown in standard greenhouse conditions to produce leaf tissue for DNA extraction82. Only confirmed diploid populations are used83. Where seeds were not available, dried leaf tissue sampled from the field was used82. RAD-seq genotyping using Illumina sequencing (via Edinburgh Genomics) was then performed to generated single nucleotide markers (SNPs) for 91 individuals from 13 North American populations  and 2–3 individuals from each of 18 Northern European populations . For analyses, we randomly selected only one SNP per RAD locus and only SNPs present in all individuals (so unlinked SNPs without missing data). We limited the dataset to highly qualitative SNPs through maintaining (i) minimum allele frequencies of 0.05, (ii) no missing data, and (iii) an average read depth of 73.18 (European samples) and 60.09 (North American samples) per SNP , using the TEannot pipeline of REPET. We calculated the genomic distance of each SNP to its nearest TE and the orientation of the SNP relative to its nearest TE (within TE vs. downstream vs. upstream). Through focusing on nearest neighbors, the probability of non-reference TE insertions  was calculated per sample using the R package “hierfstat”   > 0.5 representing ‘substantial’ evidence for selection88. Bayescan decomposes FST coefficients into population-specific (beta) and locus-specific  effects, with negative alphas pointing to balancing or purifying selection. The false discovery rate (FDR) was set at 0.05. For each TE superfamily, we calculated the proportion of SNPs with signatures of balancing (low FST) vs. divergent selection (high FST).The genome-wide inbreeding coefficient  as candidates of purifying selection if their minimum allele frequency (MAF) was (i) smaller than the MAF of neutral SNPs, and (ii) smaller for non-synonymous as compared to synonymous codon usage. In any other cases, we considered SNPs with negative alpha values as candidates of balancing selection. Codon usage (4-fold and 0-fold positions in the A. lyrata reference genome) of individual SNPs was obtained using the NewAnnotateRef.py script91.To assess the role of purifying selection in producing negative alpha values, we explored the tendency of SNPs with negative alpha values to be associated with low-frequency non-synonymous variants. If alleles at these loci were rarer than expected under neutrality, we considered these SNPs candidates of purifying selectionN = 13 levels) by a simple TE grouping variable . TE effect thus refers to the potential of specific TE superfamilies (here Copia and Harbinger elements) to maintain nearby heterozygosity under inbreeding. This new grouping variable distinguishes between TE superfamilies that significantly impact heterozygosity under inbreeding (HTE) and all other TEs (H0). To test whether this effect was exclusively associated with high inbreeding levels, the explorative model was run on a subset of the samples characterized by predominant self-fertilization and with inbreeding coefficients exceeding 0.6 (Model 0a), and compared to a model with subset of the samples characterized by predominant outcrossing and with inbreeding coefficients below 0.6 (Model 0b). The probability of a locus to be heterozygous was consistently modeled with the random effect “Relatedness”, a categorical variable correcting for genetic relatedness among samples as revealed by the multivariate PCadapt analysis. Throughout our study, the probability of a locus to be heterozygous was modeled using a binomial distribution with cloglog link within the glmmTMB R package92. Analysis of variance tables were produced using the lmer R package.We followed a stepwise modeling approach that allowed testing our hypotheses consecutively without running complex models with many parameters. We first ran an explorative mixed model  caused deviations from the expected negative relationship between heterozygosity and inbreeding, and whether this deviation was associated with signatures of selection. The variable “Selection” indicated whether a SNP was an outlier of balancing selection or of divergent selection rather than a neutral SNP (three levels). To test whether the patterns observed for Model 2a are unique to the TE superfamilies identified in Model 1, we compared the Model 2a outcomes to a competing background model (Model 2b) ran on all other TE superfamilies. We expected the inbreeding coefficient to show a stronger correlation with H0 (Model 2b) than with THE . We expected that (i) increased heterozygosity near TEs would be associated with signatures of balancing selection facilitating heterozygosity under inbreeding, particularly downstream of TEs and for HTE, and (ii) some TE superfamilies may facilitate divergent selection driving SNPs towards fixation (i.e. reduced heterozygosity) but this effect should be more pronounced for HO than for HTE.Model 2a Table  relates A. lyrata lineage that is characterized by an outcrossing mating system. If the role of particular TEs in evolution is particularly important under inbreeding, the reinforcing effects of these TEs on heterozygosity across inbred populations should be absent across outbred populations. In addition, if TEs are involved in evolution irrespective of inbreeding levels, signatures of selection associated with TEs identified across the North-American samples should be predictive for evolutionary signatures in other populations . We thus modeled the “TE effect” on heterozygosity across European A. lyrata populations, while accounting for potential effects of SNP orientation relative to nearest TE. We expect no TE effect on HEurope (i.e. HO = HTE). To disentangle mating strategy effects from demographic effects on the inbreeding coefficient, we explored this expectation on two subsets that share the same outcrossing mating system but differ in inbreeding coefficients (FIS > −0.3 vs. FIS < −0.3), presumably due to strong demographic bottlenecks experienced by the most inbred European individuals93. If mating system strategy is the driver of TE effects, than H0 = HTE for both subsets. If demography drives TE effects, than H0 = HTE only for the subset with FIS < −0.3. Second, the proportion of SNPs under selection across TE superfamilies identified in the North-American dataset should be comparative for the European dataset. While very different outliers of selection are expected in Europe , the general tendency of TE superfamilies to be involved in specific evolutionary processes may be similar between the North-American and the European samples. To test this, we compared the proportion of outliers of each TE superfamily between the North-American and the European dataset (Pearson correlation). We applied the same outlier detection approaches used for the North-American dataset to the European accessions.First, All analyses were performed at the superfamily level because (i) the number of replicate TEs within families was too low to statistically analyze heterozygosity at the TE family level, and (ii) it allows to elucidate genome- and superfamily-wide relationships between TEs and nearby genetic diversity.HTE), and (iii) SNPs upstream vs. downstream of TEs. We hypothesized that outliers of divergent selection, and SNPs downstream TEs are enriched for stress-related processes . Outliers of balancing selection and SNPs underpinning HTE are hypothesized to be enriched for fitness-related and housekeeping processes .We used Blast2go to map and annotate the RAD loci containing the SNPs against all plant genomes to obtain gene ontology terms. With Fisher’s exact tests (R package “TopGO”), we then assessed which biological processes and molecular functions were particularly overrepresented in (i) outliers of selection, (ii) SNPs near TE superfamilies that significantly increase heterozygosity under intense inbreeding , which is expected to be lower for more essential genes54. An analysis of variance (ANOVA) was used to compare these measure of gene essentiality between signatures of selection  and depending on orientation relative to TE .For all SNPs within genes, we calculated two proxies of gene essentiality and tested whether SNPs near TE superfamilies are particularly essential to organismal functioning, depending on their signature of selection. First, we assessed the size of the gene family, with single copy genes tending to be more essential due to the lack functional redundancyFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6Supplementary Data 7Supplementary Data 8Supplementary Data 9Reporting Summary


Mycobacterium tuberculosis (Mtb), an etiological agent of tuberculosis (TB), render TB the world’s deadliest infectious disease before the COVID-19 outbreak. Our outdated TB treatment method is designed to eradicate actively replicating populations of Mtb. Unfortunately, accumulating evidence suggests that a small population of Mtb can survive antimycobacterial pressure of antibiotics by entering a “persister” state . The formation of drug-tolerant Mtb persisters is associated with TB treatment failure and is thought to be an adaptive strategy for eventual development of permanent genetic mutation-mediated drug resistance. Thus, the molecular mechanisms behind persister formation and drug tolerance acquisition are a source of new antibiotic targets to eradicate both Mtb persisters and drug-resistant Mtb. As Mtb persisters are genetically identical to antibiotic susceptible populations, metabolomics has emerged as a vital biochemical tool to differentiate these populations by determining phenotypic shifts and metabolic reprogramming. Metabolomics, which provides detailed insights into the molecular basis of drug tolerance and resistance in Mtb, has unique advantages over other techniques by its ability to identify specific metabolic differences between the two genetically identical populations. This review summarizes the recent advances in our understanding of the metabolic adaptations used by Mtb persisters to achieve intrinsic drug tolerance and facilitate the emergence of drug resistance. These findings present metabolomics as a powerful tool to identify previously unexplored antibiotic targets and improved combinations of drug regimens against drug-resistant TB infection.Suboptimal efficacy of the current antibiotic regimens and frequent emergence of antibiotic-resistant  Unlike other omics techniques, metabolomics reveals the biochemical state of Mtb as metabolites are the final products of enzymatic, posttranslational, allosteric, and environmental integration.High-persistence forms of Mtb have complicated chemotherapeutic options because they are likely to evolve drug-resistant Mtb strains.Mtb in a persistent state shifts its metabolic networks to mitigate the production of antibiotic-induced reactive oxygen species and accompanied cellular damage, whereby Mtb persisters evade mycobactericidal activity of antibiotics.Multiple metabolic networks within Mtb persisters’ central carbon metabolism and their carbon fluxes are simultaneously shifted to mitigate antibiotic effects.Metabolic shifts required to form Mtb persisters are also observed in drug-resistant TB clinical isolates, all of which are a source of new antibiotic targets to efficiently control drug-resistant TB infection.The introduction of antibiotics into clinical medicine has drastically changed the societal impact of infectious diseases from fatal bacterial infections into predictably curable maladies . The cliThe advent of unbiased systems-level disciplines has helped predict DR emergence in clinically isolated pathogens . MetabolAlthough tuberculosis (TB) has afflicted humans for more than 4,000 years, it remains a major public health issue in the top 10 leading causes of human death worldwide . DespiteMycobacterium tuberculosis (Mtb) persister cells (persisters) possess intrinsic drug tolerance that is phenotypically reversible and free of genetic mutations  remodeling during Mtb persister formation as the major mechanism of their adaptive strategy and altered biochemical states . IntrinsThe genome, transcriptome, and proteome are constructed by polymerization of certain sets of building blocks. Unlike these macromolecules, the metabolome—a collection of metabolites—is not constrained by simple polymerization, which gives rise to vast structural and functional variation and diverse physiological properties . Due to The metabolic phenotype provides a clear readout of Mtb’s biochemical state, as metabolites are the final products of enzymatic, posttranslational, allosteric, and environmental integration under a particular condition . Analysivia permissive carbon/nitrogen source determination, but also to interact with the host immune system as well as with neighboring populations  glucose to fully label all CCM intermediates with 13C. Then, the filters were transferred to media containing unlabeled glucose to reversely wash only CCM intermediates with unlabeled carbon fraction, ultimately producing Mtb bacilli with unlabeled CCM and a fully 13C-labeled carbohydrate core of cell wall glycolipids. This 13C pulse chase labeling allowed tracing of glycolipid carbon fluxes during adaptation to various environmental stresses such as hypoxia. The filter culture-based method also allows for simultaneous collection of intracellular metabolites and extracellular secretome. To this end, Eoh and Rhee replaced the underlying agar media with a plastic inset containing chemically equivalent liquid media in direct contact with the underside of the Mtb-laden filters. Growth atop this liquid medium within the inset was indistinguishable from that achieved on adjacent agar medium and enabled timed start–stop measurements of the secretome or antibiotic uptake by sampling cell-free spent liquid media , a state of permanent DR against the antibiotic. Delayed lag time after treatment with antibiotics was also detected in Mtb , an active DNA mutagen . Thus, tturnover . Nonetheturnover . De novonditions . Most banditions .As previously mentioned, antibiotics kill Mtb by accumulation of ROS, which causes irreversible damage on cellular processes essential for Mtb growth and survival. As a countermeasure, Mtb acquires drug tolerance by entering a persistent state which is triggered by metabolic remodeling to mitigate the ROS-mediated cellular damage and subsequently evade mycobactericidal activity Figure 2The hallmark of latent TB infection (in a state of persistence) is its ability to survive various antimicrobial environments inside hosts. Mtb replicates even under a fully functioning immune system: soon after recruitment to the site of infection, immune cells release ROS, nitric oxide (NO), and reactive nitrogen intermediates (RNI), and in response, Mtb slows or ceases its growth rates, which enhances drug tolerance levels within the population . During The high level of drug tolerance from non-heritable DR in Mtb persisters is the main factor that destabilizes effective chemotherapies . This isMtb CCM plays an essential role in enzymatic transformation of carbon substrates through the activities of glycolysis, gluconeogenesis, the pentose phosphate pathway (PPP), trehalose metabolism, the tricarboxylic acid (TCA) cycle, and TCA cycle-branched pathways such as the glyoxylate shunt and the methylcitrate cycle (MCC) Figure 2+ levels act as an intrinsic factor to trigger a metabolic shift during Mtb persister formation, as NAD+ is a crucial cofactor involved in a broad range of cellular processes from redox cellular homeostasis to catabolism and energy production  regulatory circuits in Mtb CCM allows the bacilli to co-catabolize glycolytic and gluconeogenic carbon sources simultaneously during replication, a metabolic capacity that confers a survival advantage in an environment where nutrients are scarce . This reoduction . The absoduction . The reloduction . Antibiooduction . For exanhibitor . Intrigunhibitor . Catalytnhibitor . These fMtb persisters collected after exposure to biochemical growth-limiting stresses that resemble the host environment or from the sputum of active pulmonary TB patients have both long been popular models used to understand the adaptive strategies of Mtb. However, these findings were focused on transcriptional analysis due to suboptimal culture methods and analytical tools. Only recently has research begun to focus on the metabolism of intrinsic drug tolerance. The following are reported examples of metabolic networks shifted during Mtb persister formation.tgs1 gene using transposon (Tn) mutagenesis while screening for genes whose Tn insertion resulted in Mtb growth or survival advantage under hypoxic stress (tgs1 in Mtb (Δtgs1) allows bacilli to continue replication even under hypoxia, indicating the essential role of Tgs1 for the slowly replicating or non-replicating phenotype of Mtb persisters. Metabolomics analysis of Δtgs1 showed that acetyl-CoA-mediated carbon flux towards the biosynthesis of TAG was prevented, with its flux redirected towards the biosynthesis of intermediates in the oxidative arm of the TCA cycle including citrate and α-ketoglutarate (α-KG). This pathway is a major biosynthetic source of NADH, an initial substrate of Mtb ETC. As a result, the carbon flux shift in Δtgs1 significantly increases antibiotic susceptibility against a broad range of clinically relevant antibiotics  even under persister-inducing environments and within mouse models. The mechanistic basis underlying the antibiotic susceptibility of Δtgs1 was supported by comparing the drug tolerance and growth pattern of Mtb overexpressing citrate synthase (CitA), an enzyme that condenses oxaloacetate (OAA) and acetyl-CoA in the oxidative arm of the TCA cycle. This study indicates that environmental stress-induced rewiring of acetyl-CoA-mediated carbon flux is an adaptive response of Mtb to environmental stress used to regulate its growth rate and drug tolerance  led to reduced growth rates accompanied by drug tolerance. TAG, located at the outer leaflet of the outer membrane, functions as a structural barrier while also providing a source of ATP via the β-oxidation pathway of TAG acyl-chains. Thus, intrabacterial TAG levels are metabolically associated with bacterial ATP biosynthesis, another biosignature for bacterial persisters. Like acyl-chains of phthiocerol dimycocerosates (PDIM)  , TAG servia its new role in antioxidant defense rather than its canonical role in fatty acid metabolism. Work by Nandakumar et al. showed that Mtb changes its carbon fluxes through the ICL-mediated glyoxylate shunt as a method to evade antibiotic effects (icl-deficient Mtb (Δicl) became hypersensitive to treatment with INH, RIF, and STR, and the normal level of antibiotic sensitivity was restored by supplementation with a chemical antioxidant such as thiourea. These findings collectively suggest that the catalytic shift from the canonical TCA cycle towards biosynthesis of glyoxylate shunt intermediates serves as an adaptive strategy used to evade antibiotic effects. This is accomplished by mitigating the production of respiratory radicals arising from canonical TCA cycle-mediated NADH production and ETC activity (Recent observations have identified isocitrate lyase (ICL) as a specific mediator to produce the metabolic shifts of Mtb persisters . ICL has effects . In this effects . icl-deficl, both the mRNA expression of ald and the enzymatic activity of its translated product were increased while entering the hypoxia-induced persister state. Intriguingly, Δald showed minor survival disadvantages under in vitro hypoxia, but a significant lag phase delay upon resuming growth after reoxygenation. Ald is proposed to maintain optimal NAD+ recycling during initial regrowth after reoxygenation. Two independent studies separately identified an essential role of succinate metabolism in the ability of Mtb persisters to survive hypoxic stress  was not functionally associated with reduced succinate secretion and impaired membrane potential. Eoh and Rhee used stable 13C isotope tracing metabolomics of Mtb persisters under hypoxia using [U-13C] acetate. This work showed that, upon exposure to hypoxia, Mtb persisters slowed TCA cycle activity and increased succinate production via ICL in the glyoxylate shunt. By fermenting fumarate to succinate, Mtb persisters are capable of sustaining both membrane potential and ATP synthesis under low oxygen levels -deficient Mtb  . First, c stress . Watanab13C isotope tracing metabolomics of Δsdh1 confirmed that succinate oxidation was downregulated during aerobic growth but uncontrolled during adaptation to hypoxic conditions. As a result, Δsdh1 failed to survive during the stationary phase or during chronic infection in the C3HeB/FeJ mouse model due to several impaired bioenergetic activities arising from a mismatch between glyoxylate shunt-mediated succinate biosynthesis and ETC respiration.In Mtb persisters, succinate biosynthesized by either the reverse reductive branch of the TCA cycle or the glyoxylate shunt is further catalyzed by succinate dehydrogenase (Sdh1), the enzyme that couples growth processes controlled by the TCA cycle with energy production resulting from the ETC. Hartman et al. characterized the impact of genetically deleting the membrane anchor subunit of Mtb Sdh1 (Δsdh1). Sdh1 oxidizes succinate to biosynthesize fumarate and deliver two electrons to ETC membrane electron carriers such as menaquinone . Stable These studies identified enzymes involved in the glyoxylate shunt and succinate metabolism—malate dehydrogenase, succinate dehydrogenase, and ICL—as potential sources of new drug targets to eradicate Mtb persisters.via trehalose synthase (TreS) activity. A treS-deficient Mtb (ΔtreS) was unable to adapt to, or recover from, incubation under hypoxia stress. These results were similarly reproduced by treatment with validamycin A, a validated chemical inhibitor of TreS. These changes were dissociated from TCA cycle remodeling, as confirmed by inclusion of NO3, a physiologically relevant terminal electron acceptor of ETC capable of supporting near-aerobic levels of canonical TCA cycle activity under hypoxic conditions  and fructose 1, 6-bisphosphate (FBP) . This renditions   and to trigger persister formation. Mtb persisters were generated in the biofilm culture in pelleted form at both the wall–media border and air–media interface , approaching nearly complete protection against second-line TB antibiotics such as BDQ. These findings indicate that the trehalose-mediated catalytic shift is an adaptive strategy to primarily sustain ATP levels and additionally serve as a source of antioxidant biosynthesis to protect Mtb persisters from antibiotic-induced antimicrobial damage.As alluded, Lee et al. supported the crucial role of the trehalose-mediated catalytic shift as an adaptive strategy of Mtb persisters used to survive many clinically relevant biochemical stresses and antibiotic effects . This stnterface . Floatinnterface . This meRecently, Ruhl et al. reported that another set of glycolipids in Mtb, termed sulfolipid-1 (SL-1), plays a role as a nociceptive neuron activating molecule . The hosin vitro growth arrest or in vivo mouse lung infection. Lim et al. recently reported that hypoxia-induced TAG biosynthesis is mediated by a metabolic shift involving downregulation of glycolytic intermediates such as PEP , which catalyzes the first committed step in Mtb gluconeogenesis  could not be recapitulated when culturing ΔpckA on fatty acid media or in resting or immune-activated mouse bone marrow-derived macrophages (BMDMs)  and PEP (a product of PEPCK) as compared to those of wild-type Mtb under the same condition (A gene termed ogenesis . Transcr (BMDMs) . Similarnditions . Computaondition . This me13C isotope tracing metabolomics using Δicl in the presence of [U-13C] acetate or [U-13C] propionate as a single carbon source and confirmed that ICL also functions as a methylisocitrate lyase (MCL), the last enzyme in the MCC, which catabolizes propionate after β-oxidation of odd-chain fatty acids  supported the growth of both drug-sensitive and DR TB clinical isolates at a similar level. In vitro growth patterns of all TB clinical isolates in media containing butyric acid were enhanced by supplementation with trehalose. Intriguingly, the improved growth of DR TB clinical isolates was suppressed by treatment with validamycin A or a trehalose analog, 6-azido-6-α,α’-trehalose (6-treAz), while the enhanced growth of drug-sensitive TB clinical isolates was relatively unaffected even after treatment with validamycin A. The specific sensitivity of DR TB clinical isolates to treatment with a TreS-specific inhibitor indicated that DR TB clinical isolates, unlike drug-sensitive clinical isolates, rely on TreS activity in managing their CCM activities. These findings were further confirmed by metabolomics and lipidomics profiling. Chemical inhibition of the trehalose-mediated catalytic shift afforded more rapid and vigorous depletion in CFUs of DR TB clinical isolates against BDQ by ameliorating its weak early bactericidal activity  mutants of genes in fatty acid metabolism and CCM have been isolated after treatment with bactericidal concentrations of RIF and STR , American Lung Association Innovation Award, and Donald E. & Delia B. Baxter Foundation to HE.We would like to thank all Eoh lab members for reading and commenting on the manuscript. We are grateful to C. Stallings for kindly sharing her unpublished manuscript. We apologize that many papers could not be discussed owing to the lack of space.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Intimate partner aggression (IPA) is a prevalent public health concern that is associated with multiple negative consequences. Rates of IPA in the U.S. have increased since the onset of the Coronavirus Disease 2019 (COVID-19) pandemic, likely due to stress associated with the pandemic. Socioeconomic deprivation is associated with COVID-19 outcomes as well as IPA. However, whether socioeconomic deprivation interacts with COVID-19 stress in predicting IPA remains unclear.Using a sample of 510 individuals recruited via Qualtrics Research Services in April 2020, the present study tested whether socioeconomic deprivation moderates the association between COVID-19 stress and IPA perpetration and victimization. Participants completed a questionnaire battery that included measures of COVID-19 stressors and physical and psychological IPA perpetration and victimization. In addition, participants reported their residential zip codes, which were subsequently matched with scores on the Social Deprivation Index, a composite measure of seven demographic variables from the 5-year American Community Survey.Sequential generalized linear models in Mplus Version 8.7 showed that the effects of COVID-19 stress on physical IPA perpetration and psychological IPA victimization can be best understood through its interactive effects with socioeconomic deprivation. Higher COVID-19 stress was associated with higher levels of physical IPA perpetration and psychological IPA victimization when socioeconomic deprivation was low but not when socioeconomic deprivation was high. Importantly, however, overall rates of IPA were higher among individuals with higher socioeconomic deprivation than among individuals with lower socioeconomic deprivation, regardless of the amount of COVID-19 stress they experienced.The present analyses implicate COVID-19 stress as a critical correlate of IPA and show that the association between this stress and physical IPA perpetration and psychological IPA victimization may be particularly salient among individuals who live in areas of lower socioeconomic deprivation. Furthermore, our results clearly pinpoint the detrimental effects of socioeconomic deprivation more broadly, showing that individuals who live in more deprived areas tend to have high levels of IPA regardless of their level of COVID-19 stress. These findings call for public health policies at the community and societal level that target not only COVID-related stress but also the impacts of socioeconomic inequality. Intimate partner aggression (IPA), including both physical and psychological aggression, is highly prevalent, with more than 10 million adults in the United States (U.S.) experiencing IPA every year , 3. In UCOVID-19 stress) directly resulting from the pandemic’s profound impacts ) . Future The present results implicate COVID-19 stress as a critical correlate of IPA and show that the association between this stress and physical IPA perpetration as well as psychological IPA victimization may be particularly salient among individuals who live in areas of lower socioeconomic deprivation. Furthermore, our findings clearly pinpoint the detrimental effects of socioeconomic deprivation more broadly, as individuals who lived in more deprived areas had high levels of IPA regardless of their level of COVID-19 stress. Because the economic, social, and psychological stress associated with COVID-19 are likely to endure even after the pandemic phase has ended, studies are needed that examine the proximal and causal association between COVID-19 stress, socioeconomic deprivation, and IPA. Replication of the present findings, potentially using experimental or longitudinal designs, would provide strong justification that interventions which target stress could be useful in reducing IPA.Although not intended to directly inform IPA policy or intervention efforts during a pandemic, the present findings suggest that focusing on couples’ stress will be critical. Our results show that individuals living in areas of lower socioeconomic deprivation are particularly vulnerable to the detrimental effects of COVID-19 stress on relationship functioning in that their levels of IPA resemble those of individuals living in areas of greater deprivation when stress is high. These findings call for public health policies at the community and societal level that target not only COVID-related stress but also the impacts of socioeconomic inequality more broadly. Given IPA’s vast economic impact , socioec


Precise genome editing with CRISPR/Cas paves the way for many biochemical, biotechnological, and medical applications, and consequently, it may enable treatment of already known and still-to-be-found genetic diseases. Meanwhile, another rapidly emerging field—structural DNA nanotechnology—provides a customizable and modular platform for accurate positioning of nanoscopic materials, for e.g., biomedical uses. This addressability has just recently been applied in conjunction with the newly developed gene engineering tools to enable impactful, programmable nanotechnological applications. As of yet, self-assembled DNA nanostructures have been mainly employed to enhance and direct the delivery of CRISPR/Cas, but lately the groundwork has also been laid out for other intriguing and complex functions. These recent advances will be described in this perspective.  Genetics; Nanotechnology; Nanostructure The CRISPR-Cas system is a revolutionary molecular tool, enabling rapid, cheap, and targeted double-strand break (DSB) induction for varied applications. CRISPR  describes the arrangement of genomic DNA motifs within bacteria and archaea that possess this form of adaptive immunity. CRISPR RNAs guide the CRISPR-associated-protein (Cas nuclease) to the target sequence (invading virus or plasmid) to cleave and destroy it through targeted DSB induction . The natCutting of a target DNA sequence has many applications from analysis of gene function to generation of new disease models and gene therapies . HoweverDifferent natural and engineered variants of the CRISPR-Cas system have been discovered or developed in the years since its advent to optimize and customize this tool for different applications . NaturalIn addition, Cas engineering efforts have focused on increasing nuclease specificity to reduce off-target cutting, essential for clean genome engineering . ConventDespite the overwhelming success of this technology in the decade since its description, challenges remain. Delivery of the large CRISPR-Cas9 ribonucleoprotein (RNP) complex to target cells is primary amongst them, particularly for human gene therapy . Potenti7-nt-size discrete/finite structures  RNP system that aimed to reduce cholesterol serum levels in mice.Somewhat earlier, in a combination of similar ideas as those later employed by 20-grafted polycaprolactone (T20-g-PCL)).Another comparable delivery strategy was conceived by HhaI enzyme cleavage sites. The DNA strands were compressed into nanoparticles using DNAzyme cofactor Mn2+, and the particles were further equipped with acid-degradable polymer-coated HhaI enzymes. The acidic environment in lysosomes could then trigger polymer decomposition allowing Hhal to cut off the cleavage sites and release both sgRNA/Cas9 and DNAzymes for gene expression regulation in breast cancer cells.Finally, In addition to the more straightforward delivery systems, the combination of DNA nanostructures and CRISPR/Cas has also yielded other kinds of intriguing applications, such as studying the fundamental interactions between CRISPR/Cas and DNA. To this end, In a comparable study, CRISPR/Cas has also enabled the post-processing of DNA origami into otherwise impossible configurations and for inducing dynamic conformational changes as was demonstrated by In a twist to the previously introduced delivery applications, In a very recent accomplishment, In this article, we have introduced a number of recently developed techniques for merging the realms of CRISPR/Cas systems and rationally designed DNA nanostructures  analysis, the authors uncovered that with 3′ ssDNA overhang, RNase H works as a processive exoribonuclease that continuously degrades RNA from 5′ to 3′. Comprehensively, these results suggest the possibility of triggering the RNase H-mediated disassembly of nanostructure components containing DNA:RNA hybrid duplexes, by varying/modulating the RNase H binding site within a hybrid DNA:RNA nanostructure.In a very recent breakthrough article, HhaI), introduction of structural defects in the triangle (lacking only four staples) activates otherwise unreactive sites, with a site-to-defect distance of nearly 50 nm. The results – fully consistent with the behavior of ten restriction enzymes on the same DNA nanostructure – show that nucleolytic action on a DNA origami can be regulated in a digital fashion through local structural control of DNA-enzyme recognition. In particular, the presence or absence of a DNA nick can allosterically control the reactivity of an adjacent restriction site.This aforementioned approach would simplify nanostructure design, chemical composition, and therefore, synthesis. In contrast, however, it would require overcoming other limitations such as the steric inhibition of enzymatic reactions within DNA nanostructures . Stopar HinP1I endonuclease (an isoschizomer of HhaI), obtaining good agreement with the experimental data. According to this study, the endonuclease action was regulated by both global and local mechanical properties of a DNA origami triangle linked to the existence of metastable conformations that significantly change in nanostructure variants containing even small defects because of the increase of global fluctuation. These results introduce the possibility of varying the allosteric properties of DNA nanostructures to regulate biomolecular recognition and reactions, which is a new concept in DNA nanotechnology. Pursuing this direction will therefore require further investigations to identify structural determinants and antideterminants of Cas enzymes under nanoscale confinement.Despite the work proposing an empirical model accounting for the mechanical rigidity around restriction sites, accurate interpretation of the results would require computational modeling to describe the details of protein-DNA nanostructure interactions. For example, Integrating all these features challenges nucleic acid nanostructure design, whereas multistage robotic cargo release approaches could offer more feasible solutions . For insAnother direction to integrate CRISPR/Cas systems and programmable DNA nanostructures has been recently signposted by the disruptive approach . Their pFinally, in the shorter term, building on genomic integration of a scaffold-like transgene by involving transgene-dependent staples, the next step could be developing a universal strategy for transgene incorporation by freeing (or minimizing) DNA staple design, based on a programmable, transgene nanocarrier that remains inert during the DNA integration process.


Despite a prospective consecutive evaluation of a large homogenous cohort  in the index study, certain points require authors’ comments and clarification to enhance the lucidity of their findings.Dear Editor, I read with great interest the Carrascal et al.-4. Moreover, the authors also fail to delineate their institutional perioperative glucose management protocol.While preoperative diabetes mellitus did not emerge as a baseline risk-predictor of CS-AKI , the lack of elucidation of perioperative glucose homeostasis in the present study captivates attention, particularly when poor glucose control has been linked to an enhanced propensity for CS-AKI owing to an augmented oxidative stress, endothelial dysfunction and systemic inflammatory response aimed at developing the ‘Acute Kidney Injury in Cardiac Surgery’ (AKICS) score, which adequately outline the superiority of postoperative CVP and low cardiac output as independent risk factors in predicting CS-AKI over other hemodynamic variables. Across the cardiac surgical population, the risk of AKI doubles once the postoperative CVP reaches a threshold value of 14 mmHg. Ahead of the fact that estimated CVP at 6 h postoperatively has been found to be significantly associated with CS-AKI in the non-congested cardiac surgical subset, possible links should definitely not be overlooked in valvular heart disease patients included in the present study who can be particularly prone to postoperative systemic venous congestion.Talking of the hemodynamic predisposition to CS-AKI, there are additional caveats that merit elaboration. It is noteworthy that systemic venous hypertension has been demonstrated to be associated with poor renal outcomes in cardiac surgical patients manifesting as a congestive renal failure, as an elevated central venous pressure (CVP) is potentially transmitted backwards, reflecting as an accentuated renal venous pressure (RVP) which reduces effective renal perfusion pressure , accelerating AKI progression. The aforementioned is heralded by the results of the study by Palomba et al.Lastly, the lack of comparative account for inotropes-vasopressors in the AKI and non-AKI groups or a standardized inotrope-vasopressor study protocol requires consideration while interpreting the findings.