Cigarette smoke contains more than 4500 chemicals which have toxic, mutagenic and carcinogenic effects. Strong evidences have shown that current smokers take a significantly higher risk of cardiovascular diseases, chronic obstructive pulmonary disease (COPD) and lung cancer than nonsmokers. However, less attention has been paid to the smoking induced abnormalities in the individuals defined as healthy smokers who are normal with spirometry, radiographic images, routine physical exam and categorized as healthy control group in many researches. Actually, ‘healthy smokers’ are not healthy. This narrative review focuses on the smoking related pathophysiologic changes mainly in the respiratory system of healthy smokers, including inflammation and immune changes, genetic alterations, structural changes and pulmonary dysfunction. Cigarette smoke is a multipotent mixture of thousands of components that have toxic, mutagenic and carcinogenic properties. Numerous chemicals are added to the tobacco content, paper, and filter during the manufacturing process. Among the mainstream smoke emissions from cigarettes, polycyclic aromatic hydrocarbons, N-Nitrosamines, nickel, cadmium, chromiun, arsenic, misc organic compounds are known carcinogens. Nicotine, carbon monoxide, acrolein and reactive oxidant substances are toxins that can cause immune dysfunction. Moreover, nicotine is the predominant addictive cigarette smoke constituent . EpidemiThe analysis of the induced sputum, expired breath condensate (EBC), bronchoalveolar lavage (BAL) and biopsies of lung tissue from ‘healthy smokers’ show strong evidences of inflammatory and immune changes in the airway.The presence of neutrophils in sputum is one of the most common landmarks of inflammatory changes brought by smoking. As early as 1992, Swan et al.  found smThe phenotype of macrophages in induced sputum is definitely altered by smoking. Domagala- Kulawikan et al.  detected+T lymphocytes and a higher ratio of CD4+/CD8+ T-cells in induced sputum of healthy smokers as compared with that of nonsmokers. After 6 months-cessation, the percentage of CD8+ T-cells increased in quitters and a ratio of CD4+/CD8+ decreased [+ T lymphocytes is the facilitation of the rapid resolution of acute viral infections, the lower percentage of CD8+ T-cells suggests that smokers may have a deficit in cell-mediated immunity in the lung and may explain the increased susceptibility of smokers for viral infections. The alteration of other chemicals or cytokines that represents inflammatory and immune processes in healthy smokers were also found in their induced sputum. Takanashi with his team [Biochemical analysis also showed a lower percentage of CD8ecreased . As the his team  observedhis team , 16. Acchis team . All thehis team .Much attention has been paid to the changes brought by smoking in smokers’ exhaled breath condensate (EBC). Many studies have found lower pH values in EBC, as reflected airway inflammation, in diverse inflammatory airway diseases, including bronchial asthma, bronchiectasis and COPD . In healChronic smoking also alters the level of inflammatory markers in EBC of smokers who remain symptomless and seem to be healthy on the surface. Elevated concentrations of IL-6 in EBC, a pro-inflammatory cytokine produced by epithelial cells and macrophages in the airways, was observed in healthy smokers compared to nonsmokers . Higher 2O2 levels in EBC of subjects with COPD and smokers than non-smokers have been reported [In recent years, evidence has emerged that oxidative stress plays a crucial role in the development and perpetuation of inflammation. Higher 8-isoprostane and Hreported . IsoprosThe first paper detailing BAL dealt with normal values was published in 1974 . Over th+/CD8+ caused by higher percentage of CD8+ T-cells in BAL as compared with nonsmokers [+T lymphocytes with increased ratio of CD4+/CD8+ T-cells. One explanation would be the inflammatory microenvironment in airway lumen sampled by induced sputum is different from that in the BAL and airway epithelium sampled by bronchial biopsies [+ lymphocytes, increasing their number in the large airway wall, while reducing their number in the airway lumen [+T-lymphocytes, Yu et al. [+T-lymphocytes who are key inflammatory effector and regulatory cells have been proved to play an important role in the inflammatory process of COPD [+T-lymphocytes can be differentiated into cells that synthesize interferon-gamma (IFN-γ) but not interleukin-4 (IL-4) (Tc1 cells) or cells that synthesize IL-4 but not IFN-γ (Tc2 cells) [Typically smokers have a decrease of CD4nsmokers . The sambiopsies . Anotheray lumen . Besidesu et al.  showed a of COPD . CD8+T-l2 cells) . HoweverKuschner with his co-workers  observedAlveolar macrophages (AM) are responsible for a broad set of host defense functions including recognition and phagocytosis of pathogenic material and apoptotic cells. Various changes of smokers' alveolar macrophages have been noted in several studies. The number and proportion of AM in healthy smokers’ BAL are increased as compared with nonsmokers , 43. AndHistopathological examinations help us find inflammatory alterations in bronchial biopsies of smokers without any symptoms, including vascular hyperplasia, submucosal edema, inflammatory cell infiltrates and goblet cell hyperplasia . An abno+T cell response to a flagellated pathogen [Moreover, Wang with his team  demonstrpathogen , suggestEpidemiological data has shown that long-term smokers are taking a greater risk of developing COPD and lung cancer as compared with nonsmokers. One of the possible reasons is the smoking induced genetic alterations which modify susceptibility to lung diseases. For smokers, those up- or down-regulation of gene expressions with relevant impaired biological function accelerate the progress of respiratory disorders.Human alveolar macrophages, mostly residing on the respiratory epithelial surface, are critical components of the innate immune system. The gene expression of alveolar macrophages has been altered in active smokers when compared with nonsmokers. Table Airway epithelium, lined by a variety of specialized epithelial cells, represents the first point of contact for cigarette smoke. It not only plays a central role in the barrier function of airway tract, but also responds to environment-induced damage through the release of pro-inflammatory cytokines and chemokines . Genes iSmoking induced epigenetic changes with corresponding modulation of gene expression are demonstrated both in airway epithelium and alveolar macrophages , 91. BurSince 1957, numerous studies have proved the structural changes of airway epithelium brought by smoking . An incrSmoking is regarded as the major contribution to pulmonary dysfunction, and this deterioration of lung function in smokers is far in excess of that predicted by age . The ven+T lymphocytes and increased level of CD8+T lymphocytes in peripheral blood from heavy smokers which may need further studies.Numerous studies have shown significantly increased level of white blood cell –111, TNFMany researches have confirmed the systemic oxidative damage and overall decrease in antioxidant activity in smokers as compared with nonsmokers. Antwerpen et al.  demonstrOxidative damage brought by smoking is closely related to endothelial dysfunction. Guthikonda with his colleagues  demonstrReduced number of circulating endothelial progenitor cells (EPCs) is reported . The numCompared with nonsmokers, smokers have significantly elevated risk factors for cardiovascular diseases. Although, healthy smokers’ heart rate, blood pressure and level of serum lipid and lipoprotein are in the normal range, increases in heart rate and blood pressure are detected as compared with nonsmokers , 135, thAmong the healthy smokers who don’t have any metal illnesses including alcohol or drug abuse/dependence with normal brain MRI results, abnormalities are still detected in their nervous-mental system. Hao with his team  found thAttvall et al.  demonstrThe biochemical analysis of the induced sputum, expired breath condensate, bronchoalveolar lavage, biopsies of lung tissue and peripheral blood from healthy smokers are strongly enough to prove the exist of local and systemic inflammation. Genetic alterations detected in the lung tissue of healthy smokers may contribute to smoking-related susceptibility to lung diseases, such as emphysema and lung cancer. The structural changes in respiratory system, as well as the decline in lung function as compared with nonsmokers demonstrate again smoking induced negative effects on healthy smokers which accelerate the onset of respiratory disorders. Therefore, healthy smokers who are normal with spirometry, radiographic images and routine physical exam are not really healthy. Smoking cessation as an early intervention may lead to some reversal toward the better health of lifelong nonsmokers.


Aberrant regulation of dopamine system function is thought to contribute to psychosis in schizophrenia patients; however, the brain regions associated with this dysregulation have not been conclusively demonstrated. We have recently demonstrated that medium spiny neurons in the nucleus accumbens (NAc) receive convergent input from the ventral hippocampus (vHipp) and paraventricular nucleus of the thalamus (PVT). Furthermore, inactivation of either the vHipp or PVT is sufficient to reverse aberrant dopamine system function in rodent models of schizophrenia. Using, chemogenetic experiments we now provide conclusive evidence that the thalamic input to the NAc plays a role in the regulation of dopamine neuron activity. These data demonstrate that the vHipp and thalamus  work in concert to regulate VTA dopamine neuron population activity. Such data are important as they provide evidence that thalamic abnormalities may contribute to the aberrant dopamine system function observed in schizophrenia and suggest that the PVT may be a novel site for intervention in psychosis. To examine this, we explored the orexin system, which is known to provide a dense innervation of the PVT.Pregnant Sprague Dawley (SD) rats were treated on gestational day (GD) 17 with either methylazoxymethanol acetate  or saline. For Poly I:C, pregnant dams were treated on GD12 . Male pups weaned on post-natal day 21 in groups of 2–3 until adulthood (>60 days). For chemogenetic experiments, normal SD rats were bilaterally micro-injected with AAV2 vectors (Addgene) expressing hm3D(Gq)(pAAV-h8yn-HA-hm3D(Gq)-mcherry; 0.5μL) into the PVT or mPFC. Control rats were administered the viral vector lacking the hm3D encoding gene. Prior to testing, CNO  was injected into the nucleus accumbens. In vivo extracellular recordings were performed to measure dopamine neuron activity in the VTA. Spontaneously active VTA dopamine neurons were recorded using previously established electrophysiological criteria.NMDA activation of the PVT induces a significant increase in VTA dopamine neuron population activity. MAM- and Poly I:C-treated rats (both verified rodent models of schizophrenia) consistently display aberrant VTA dopamine neuron population activity, which is restored by pharmacological inactivation of the PVT with TTX. Chemogenetic activation of PVT neurons projecting to the mPFC do not affect VTA dopamine neuron activity; however, activation of PVT neurons projecting to the nucleus accumbens induces a significant increase in dopamine neuron population activity. This effect can be replicated in rats that receive microinjections of the endogenous orexin peptide A or B into the PVT. Consequently, dopamine neuron function can be restored in MAM-treated rats that received a systemic injection of the orexin peptide antagonist TCS 1102.We now demonstrate that orexin receptors are expressed on PVT neurons projecting to the NAc and may serve as a substrate for pharmacological manipulation of this pathway. Here, we provide evidence that both systemic and intracranial (PVT) administration of the orexin receptor antagonist, TCS1102, can normalize aberrant dopamine system function in a rodent model of schizophrenia. Collectively, these data suggest that targeting orexin signaling in the thalamus, specifically, the PVT, may represent a novel site of intervention for psychosis associated with schizophrenia.


RM) cells residing in the lung and lung airways. TRM are elicited by a diverse set of pathogens penetrating mucosal barriers and broadly identified by extravascular staining and expression of the activation and adhesion molecules CD69 and CD103. Interestingly, lung TRM fail to express these molecules, which could limit tissue retention, resulting in airway expulsion or death with concomitant loss of heterologous protection. Here, we make the case that respiratory infections uniquely evoke a form of natural immunosuppression whereby specific cytokines and cell–cell interactions negatively impact memory cell programming and differentiation. Respiratory memory is not only short-lived but most of the memory cells in the lung parenchyma may not be bona fide TRM. Given the quantity of microbes humans inhale over a lifetime, limiting cellular residence could be a mechanism employed by the respiratory tract to preserve organismal vitality. Therefore, successful efforts to improve respiratory immunity must carefully and selectively breach these inherent tissue barriers.The yearly, cyclic impact of viruses like influenza on human health and the economy is due to the high rates of mutation of traditional antibody targets, which negate any preexisting humoral immunity. However, the seasonality of influenza infections can equally be attributed to an absent or defective memory CD8 T cell response since the epitopes recognized by these cells are derived from essential virus proteins that mutate infrequently. Experiments in mouse models show that protection from heterologous influenza infection is temporally limited and conferred by a population of tissue-resident memory (T Respiratory infections continue to be one of the leading causes of morbidity and mortality worldwide . Approximem) capable of responding rapidly upon challenge  located primarily in lymph nodes and effector memory cells (TEM) which circulate through lymphoid and non-lymphoid tissues . TRM have a core transcriptional profile that distinguishes them from their TCM and TEM counterparts  which are upregulated on TRM in both humans  infection  generate skin-resident TRM that mediate protection against subsequent VacV infection  , 53RM  , 63 is uver time , hosts wvia tissue segregation. Indeed, liver TRM are exclusively segregated from tissue stroma, retained within the sinusoids  signaling within the respiratory tract. mTOR is responsible for regulating cellular metabolism, proliferation, and differentiation , includiCM pool . While rpool (CM , TRM forolecules . To datehe blood  similar he blood . In addianzyme B . While ianzyme B  and Tmemregs populate this tissue and contribute to significant IL-10 post-influenza infection (+ T cells (−/− versus wild-type mice are preferentially TEM (+ T cell pool, thereby restricting developing TRM. Thus, while enhanced PD-1 expression within the respiratory tract may be important for regulating inflammation, this may create an environment that is inhospitable to the formation of TRM.The lung environment is inherently immunosuppressive. In the steady state, a large reservoir of Tnfection . Moreovenfection  and inflnfection . In addinfection , 84. Botnfection , 86 and nfection  by tuninally TEM . ReciproRM programming via inhibition of CD103 and CD69 expression, which negatively affects the formation and/or retention of TRM cells in the respiratory tract. Constitutive expression of TGF-β in mucosal sites such as the gut (RM through induction of CD103 expression (RM retention but aid in their development or survival via tissue positioning. However, high levels of TGF-β in the respiratory tract can be detrimental, leading to the development of cystic fibrosis within the lung (RM formation. In fact, the TRM in peripheral sites can cause semi-permanent scarring in tissues that worsens after TRM re-activation and production of IFN-γ in situ (RM long term may be inherently limited to maintain host fitness. If this is the case, promoting TRM formation within the respiratory tract could have severe consequences for host respiratory health. Therefore, by reducing TGF-β, and coordinately CD103 expression, lung memory precursor cells would perhaps be ill positioned to receive homeostatic signals responsible for the development, survival, and/or retention of TRM and could be either be lost or assimilated into the TEM pool.It is also possible that respiratory infections alter T the gut  is crucipression . Epithelpression , thus hithe lung . Althougthe lung , 93, it  in situ . Since h in situ , in addi in situ , the retRM cells confer protection against secondary influenza infection, they rapidly wane, leaving only parenchyma resident TRM and circulating TEM to maintain protection against subsequent infection. However, TEM also wane over time (bona fide TRM in the lung parenchyma is limited (Figure bona fide TRM within the lung tissue. While the exact mechanisms that underlie altered TRM formation within the respiratory tract are still not fully understood, future efforts to improve the maintenance and stability of this population must bear caution due to potentially negative, long-term effects on the host. Moreover, in developing vaccines against respiratory pathogens, it will be important to identify strategies that will prevent re-infection with respiratory viruses without compromising host respiratory health.While airway-resident Tver time  and the d Figure . These iAll animal studies were conducted under guidelines approved by the Institutional Animal Care and Use Committee of the University of Georgia.Both KK and KR conceived and wrote the perspective.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.


Volcanic eruptions provide tests of human and natural system sensitivity to abrupt shocks because their repeated occurrence allows the identification of systematic relationships in the presence of random variability. Here we show a suppression of Nile summer flooding via the radiative and dynamical impacts of explosive volcanism on the African monsoon, using climate model output, ice-core-based volcanic forcing data, Nilometer measurements, and ancient Egyptian writings. We then examine the response of Ptolemaic Egypt (305–30 BCE), one of the best-documented ancient superpowers, to volcanically induced Nile suppression. Eruptions are associated with revolt onset against elite rule, and the cessation of Ptolemaic state warfare with their great rival, the Seleukid Empire. Eruptions are also followed by socioeconomic stress with increased hereditary land sales, and the issuance of priestly decrees to reinforce elite authority. Ptolemaic vulnerability to volcanic eruptions offers a caution for all monsoon-dependent agricultural regions, presently including 70% of world population. The degree to which human societies have responded to past climatic changes remains unclear. Here, using a novel combination of approaches, the authors show how volcanically-induced suppression of Nile summer flooding led to societal unrest in Ptolemaic Egypt (305–30 BCE). Egypt provides a unique historical laboratory in which to study social vulnerability and response to abrupt hydroclimatic shocks. As one of the Ancient World’s great “hydraulic civilizations”2, its prosperity was overwhelmingly tied to the annual cycle of Nile summer flooding, with diminished flooding (Nile failure) often associated with major human impacts through its many millennia of recorded history3. Of all Ancient Egyptian history, that of Ptolemaic Egypt  cropping, as well as grain storage, acted to mitigate the impacts of the mercurial Nile flood  and the Ethiopian Highlands (mainly via the Blue Nile and Atbara rivers)11. While most studies of the climatic effects of volcanism have focused on temperature changes, contemporary and historical societies were also vulnerable to hydrological changes12. Hydroclimate is harder to reconstruct and model, but studies are increasingly noting global and regional hydroclimatic impacts from explosive volcanism20. Volcanic aerosols influence hydroclimate through multiple mechanisms. Aerosol scattering of solar radiation to space reduces tropospheric temperatures; if lower-tropospheric relative humidities remain unchanged, the mass of water converged by a given wind distribution decreases, and precipitation minus surface evaporation (P-E) is thus reduced21. This thermodynamic effect may represent the principal means by which equatorially symmetric aerosol distributions from tropical eruptions alter P–E15. In addition, extratropical eruptions increase sulfate aerosols on one side of the equator, cool that hemisphere, and may thus alter tropical P–E primarily by changing winds. In particular, a high-latitude energy sink in one hemisphere forces an anomalous Hadley circulation, shifting the intertropical convergence zone (ITCZ) away from that energy sink22. Aerosol cooling of northern high latitudes can thus force a southward shift of northern hemisphere (NH) summer monsoon precipitation, promoting drought in the northern parts of monsoon regions23. These energy-budget arguments provide a more fundamental perspective on the controls on tropical rainfall than arguments based on land-ocean temperature contrast because large-scale tropical circulations are driven by horizontal gradients in the total (sensible plus latent) energy input to the atmosphere24. The hypothesis that a decrease in land-ocean temperature contrast will cause monsoon rainfall to weaken has been disproven by the observation that continental monsoon regions are cooler during years of enhanced monsoon precipitation25, and by the fact that monsoon winds weaken as land-ocean temperature contrast strengthens in projections of next-century warming26.Explosive eruptions can perturb climate by injecting sulfurous gases into the stratosphere; these gases react to form reflective sulfate aerosols that remain aloft in decreasing concentrations for approximately one to two years27 and the Islamic Nilometer (622–1902 CE)28, allow us to establish the Nile’s physical response to explosive volcanism. We replicate this response in earlier centuries using Ptolemaic era flood quality descriptions29, and further employ the period’s abundant documentary record to examine the role of repeated volcanic flood suppression in Ptolemaic political and social history.In what follows, modeling of twentieth century explosive eruptions, together with newly revised ice-core records of volcanic climate forcing19. An ensemble of climate models (Methods section) forced by these eruptions  rank on average only 132nd among the 283 largest volcanic perturbations since 500 BCE27, thereby undersampling the variability of historical volcanic aerosol climate forcing, and motivating our study of historical volcanism using ice-core-based volcanic forcing reconstructions with long temporal span . To further establish the association between eruptions and Nile summer flood heights, we compare ranked flood heights for eruption vs. non-eruption years as a function of ranked percentile , again indicating the persistence of volcanic impacts on the Nile in the Ptolemaic period.The great socioeconomic importance of the Nile summer flood ensured its frequent mention (directly and indirectly) in ancient writings before the 622 CE start of the Islamic Nilometer data. Using these writings, qualitative flood quality indications can be placed on an ordinal scale for 96 years in the Ptolemaic period, 305–30 BCE (Methods section)les Fig. , consist27, these allow us to compile indexes of socioeconomic and political activity (Methods section) to identify potential top-down (state/elite level) and bottom-up responses to volcanically induced hydrological shocks. We thus examine the timing and frequency of Ptolemaic interstate warfare and the issuance of priestly decrees (primarily top–down responses) relative to eruption years, as well as the timing and frequency of revolt onset against Ptolemaic rule and hereditary land sales (primarily bottom-up responses).For the first time in Egyptian history, well-dated records, especially papyri, survive in large numbers during the Ptolemaic period. In tandem with newly revised and extended ice-core volcanic forcing data30, as during the great 20-year Theban revolt starting 207 BCE  also suggests a multi-year or lagged response, with revolt onset in some instances plausibly delayed or potentially prevented by short-term coping strategies such as Cleopatra’s release of state-reserved grain during documented Nile failure after eruptions in 46 and 44 BCE , suggesting initiation is driven mainly by other factors such as succession disputes33. Mapping contested territories against regional rainfall patterns suggests that Ptolemaic access to Nile-independent rainfed territories was one additional strategic motivation  thus plausibly contributed to the third war’s ultimate cessation by 241 BCE, providing a suggestive case of how eruption-related Nile failure may have more broadly acted to constrain interstate warfare.Historical sources are helpful in examining how this may occur, here in the context of major eruptions in 247 and 244 BCE36. We thus hypothesize that decrees acted as instruments by the Ptolemaic ruling elite to re-affirm control during crises. Compositing decrees relative to eruptions reveals a statistically significant incidence in eruption years  systems, in which an interplay of influences are seen to contribute to any given societal response to a climatic or environmental shock45. We thus interpret our results as identifying a role for volcanically induced Nile failure as a trigger for revolt in Ptolemaic Egypt, and a constraint on Ptolemaic interstate conflict, against a background of multiple interacting and enabling societal stressors, or primers. These include ethnic tensions between Egyptians and Greek elites35, growing demographic and fiscal pressures46, burdensome state taxation30, the mounting costs of large semi-permanent military mobilizations47, and increasing urban and export demand for drought-vulnerable free-threshing wheat48. We similarly interpret a correspondence between priestly decrees and eruptions as a state response to related instability, and interpret increased extra-familial land sales as symptomatic of multi-year socioeconomic stress from volcanically induced Nile failure.Our results demonstrate a systematic Nile flood suppression from historical eruptions using the multi-century Islamic Nilometer and earlier written records; we find this suppression consistent with theory and modeling of volcanic monsoon impacts. We further identify statistically significant associations between eruptions and the onset of hitherto poorly understood revolts in Ptolemaic Egypt, as well as the cessation dates of Ptolemaic interstate warfare with the great Near Eastern Seleukid Empire. We stress that care must be taken to avoid engaging in and propagating a recently resurgent environmental determinism when interpreting or assigning causality  to associations observed between environmental and societal phenomena50, within the context of the political and socioeconomic pressures outlined above, alongside heavy losses of external Nile-independent rainfed territory post-195 BCE32. The volcanically perturbed 160’s BCE , which contrast markedly with the quiescent “Roman optimum” of the first two centuries CE 27, closely following a 46 BCE extratropical NH eruption27.Our results advance the understanding of Ptolemaic state and societal responses to hydrological shocks. These now warrant consideration for their potential contribution to the state’s declining political stability after the successful reigns of Ptolemy I to III . Each spatial grid point of the response to the true eruptions is considered statistically significant only if it is either less than the 2.5th percentile or greater than the 97.5th percentile of the distribution of synthetic responses at the same grid point. Similar analyses of CMIP5 data were conducted by19 and58.Our analysis uses data from 21 models  papyri and inscriptions. We draw from the chronology of revolts reconstructed by A.E. Veïsse  to distinguish revolt onset dates from dates potentially part of already ongoing revolts. The final cessation dates of revolts are generally ambiguous in comparison to onsets, and are therefore not included in our analysis. For the start and cessation dates of “Syrian Wars” between the Ptolemaic and Seleukid states, we use the compilation by Grainger 60. For land sales, we use the corpus of Egyptian (Demotic) texts assembled by Manning37.Information in all documents  are assessed for general reliability and authenticity through historical source and textual criticism. We focus on well-dated documents, distinguishing between the date of a text and the  events described therein. To convert ancient dates in (and of) documents to the Gregorian calendar, we apply Egyptian civil calendar conversions at Frank Grieshaber, Date convertor for Ancient Egypt, 227. This window was chosen to correspond to the average interval between volcanic eruptions. It thus represents the longest interval before it becomes probable that a secondary volcanic eruption will occur within the composite window. If a second eruption does occur during a given window, then the values associated with the year of the secondary eruption and the two following years are removed (set to NaN) before compositing. Statistical significance  for the binary time series  are calculated using Barnard’s exact test61. The 95% confidence threshold for each lag  is also estimated using Barnard’s exact test, accounting for the actual number of observations at each lag after accounting for the masking (noted above) of some non-focal years due to secondary eruptions. Land sale counts are not binary and we thus estimate significance using the 2-sample student’s t-test. For our revolt onset dates, we note that the 145 BCE onset date is ambiguous, with 141 BCE representing a potential alternative30. However, we find that the frequency of revolt onsets in both eruption years and in the second years following is statistically significant at (at least) the P < 0.05 level, regardless of whether we use only the 145 BCE date, the 141 BCE date, both dates  based upon observations of present-day eruptions63. For the majority of events, the deposition start year also equates to the year of maximum annual sulfate concentrations in the polar ice cores , though for the largest eruptions, particularly in the tropics , maximum annual sulfate deposition may occur one year after the start of deposition on the polar ice-sheets. This can be explained by the longer stratospheric residence times of aerosols from tropical eruptions as compared to high-latitude eruptions64. Our forcing estimates represent the time-integrated  volcanic forcing over the full time-span of sulfate deposition for each event, which is at a first order proportional to the NH peak forcing centered at our start dates. Northern Hemispheric (NH) volcanic forcing values are estimated from the global volcanic forcing values reported by27, dividing values for tropical eruptions by two, and those from NH extratropical eruptions by one. All volcanic events reported by27 for which time-integrated volcanic forcing of the NH falls below a threshold of −1 W/m2  1883, for comparison) are employed in our analyses.“Eruption years”, in all statistical tests, refer only to those years in which an eruption is estimated to have occurred. All other years are deemed “non-eruption years”. Our “eruption year” dates represent the year in which volcanic sulfate deposition associated with each eruption is first registered in Greenland and Antarctic ice cores, based upon recently refined ice-core chronologies28. The two-sample t-test comparing the distribution of flood heights between eruption and non-eruption years, and the ranked flood analysis, thus use 61 samples from the eruption-year distribution and 1030 samples from the non-eruption year distribution. One additional eruption was excluded from the composite analysis  atmospheric injection of sulfate. Our general finding of Nile summer flood height suppression is, however, robust to other threshold choices. Indeed, the magnitude of the observed suppression is larger if a lower cutoff threshold is chosen. For example, if we restrict our analysis to eruptions with a forcing of below 0, −1, −2, −3, −4, or −5 W/m2, we observe an average eruption-year decrease in Nile summer flood height of 12, 22, 29, 39, 49, and 61 cm, respectively. These results also act as a general long-period validation of the Islamic Nilometer, and the association between explosive volcanism and Nile summer flood heights, demonstrated here, has diagnostic potential in advancing future efforts to examine the historical basis of the surviving Islamic Nilometer record, a subject meriting further study51.The <−1 W/m29, covering 96 years in the Ptolemaic period, 305–30 BCE. Coverage is intermittent because of variable document survival, with particularly high survival for the area of the Fayyum oasis . The papyri variably provide precise measurements of Nile summer flood heights and qualitative assessments of flood extent , augmented by inferences from reported rupturing of dikes and similarly indicative evidence, often attested in multiple documents. We allow a minor dating uncertainty of ±1 year, e.g., arising from multiple co-existing calendars and instances wherein only regnal years are provided, hampering conversion to specific Gregorian years. Following Bonneau, Nile flood quality is ranked on an ordinal scale from 1, “poor” (“mauvaise”) to 8, “strong” (“forte”). See also the treatment of D. Bonneau’s compilation for the post-Ptolemaic era in ref. 65.We derive qualitative Nile flood quality indications from D. Bonneau’s compilation of (primarily) Greek papyriData underlying the results published in this article are available accompanying the cited literature, in the Supplementary Information


Introduction. Diagnosis of invasive aspergillosis is challenging and the gold standard for definite diagnosis remains histopathological tissue examination. However, invasive procedures such as lung biopsy are often not feasible in critically ill patients. The detection of fungal cell wall components like Aspergillus galactomannan in broncho-alveolar lavage remains a key component of the diagnostic procedure. False-positive of the Aspergillus galactomannan assay is not frequent.Case presentation. We report a case of positive galactomannan in broncho-alveolar lavage fluid after enteral nutrition aspiration without signs of invasive aspergillosis. Galactomannan was positive in the enteral nutrition solution.Conclusion. Physicians should be aware of this previously unrecognized cause of false-positive galactomannan in broncho-alveolar fluid which can result in unnecessary treatments. BAL, broncho-alveolar lavage; GM, galactomannan; IA, invasive aspergillosis; ICU, intensive care unit; VAP, ventilator-associated pneumonia.We describe the first report of a false-positive broncho-alveolar galactomannan due to aspiration of enteral nutrition. As enteral nutrition solutions are prescribed daily in critically ill patients and aspiration pneumonia is a common complication in intensive care, physicians should consider this possibility in case of broncho-alveolar lavage positive galactomannan assay under artificial enteral feeding.We believe this finding will be of great interest to microbiologists and physicians, especially to those in contact with patients in intensive care.Pseudomonas aeruginosa, a primary Enterococcus faecalis bacteremia and a severe herpetic stomatitis. A new respiratory deterioration at day 23 led to suspicion of a new episode of VAP treated by meropenem. Bronchoscopy revealed an important aspiration of enteral nutrition solution  due to incorrect position of the nasogastric tube. The procedure was followed by a pneumothorax, with pleural effusion, which required chest tube drainage. The pleural liquid had the same macroscopic aspect as the enteral nutrition solution. Aspergillus galactomannan (GM) assays performed in broncho-alveolar lavage (BAL) and pleural fluid, using the one-stage commercialized immunoenzymatic sandwich microplate assay , were highly positive in both fluids . Serum GM OD index was 0.08  and fungal cultures of both BAL and pleural fluid remained sterile. We initiated voriconazole treatment and completed the work up with chest and sinus CT scans; both showed no signs suggestive of invasive fungal infection . Considering these inconsistent findings for an invasive aspergillosis (IA), we performed Aspergillus GM detection in the enteral nutrition solution, which proved highly positive (index >6) whereas fungal culture remained negative. We therefore tested two other commercialized enteral nutrition solutions [Isosource standard fibre(Nestlé) and Fresubin original fibre (Fresenius Kabi)] also used in our institution and confirmed that all were also higher than the upper detection threshold (index >6). We therefore stopped voriconazole after 8 days of treatment. Evolution was favourable without any further fungal infection.A 65-year-old man was admitted in our intensive care unit (ICU) for pneumonia with septic shock. He was a heart transplant recipient since 1998 and had a renal graft in 2015 because of immunosuppressive treatment toxicity. Current immunosuppressive therapy consisted of methylprednisolone, mycophenolate mofetil and cyclosporine. Septic shock was associated with acute renal failure and severe acute respiratory distress syndrome requiring mechanical ventilation. No infectious pathogen was identified. The shock resolved in 4 days but respiratory conditions, only slowly improved, and the presence of a severe ICU-acquired weakness required a tracheotomy after 10 days of mechanical ventilation. The ICU stay was further complicated by a ventilator-associated pneumonia (VAP) due to Aspergillus are ubiquitous in nature, and inhalation of infectious conidia is a common event that can give rise to various clinical conditions depending essentially on the host’s immunological status. IA occurs most frequently in the setting of severe immunosuppression  [Species of the genus yndrome) .Aspergillus GM remains a key component of the diagnostic procedure. Aspergillus GM are polysaccharides released during growth of the fungus and are detected by ELISA in biological fluid. In the serum, a positive GM is a strong argument in favour of invasive aspergillosis but the test lacks sensitivity [Aspergillus GM has been proposed in the BAL and seems to have a greater sensitivity. According to a meta-analysis, BAL GM sensitivity and specificity reach 92 and 96 %, respectively with a threshold of 1.5 µg l−1 in adult haematology patients [Diagnosis of IA is challenging and the gold standard for definite diagnosis remains histopathological tissue examination. However, invasive procedures such as lung biopsy are often not feasible in patients with severe respiratory insufficiency and critical illness . Despitesitivity . Therefopatients . ResultsAspergillus GM assay is not frequent. In BAL, false-positive GM assays have been observed in case of histoplasmosis [Penicillium and Paecilomyces have shown cross-reactivity with the rat EBA-2 monoclonal antibody used in our report, which has been characterized in previous studies [A false-positive result of the lasmosis  and in olasmosis . Indeed, studies . False-p studies . Several studies  but this studies . To the This is the first report of a false-positive BAL GM due to aspiration of enteral nutrition. Physicians should consider this possibility in case of BAL positive GM assay under artificial enteral feeding.


Cancer immunotherapy represents a promising, modern-age option for treatment of cancers. Among the many immunotherapies being developed, oncolytic viruses (OVs) are slowly moving to the forefront of potential clinical therapeutic agents, especially considering the fact that the first oncolytic virus was recently approved by the Food and Drug Administration for the treatment of melanoma. OVs were originally discovered for their ability to kill cancer cells, but they have emerged as unconventional cancer immunotherapeutics due to their ability to activate a long-term antitumor immune response. This immune response not only eliminates cancer cells but also offers potential for preventing cancer recurrence. A fundamental requirement for the generation of such a strong antitumor T cell response is the recognition of an immunogenic tumor antigen by the antitumor T cell. Several tumor antigens capable of activating these antitumor T cells have been identified and are now being expressed through genetically engineered OVs to potentiate antitumor immunity. With the emergence of novel technologies for identifying tumor antigens and immunogenic epitopes in a myriad of cancers, design of “oncolytic vaccines” expressing highly specific tumor antigens provides a great strategy for targeting tumors. Here, we highlight the various OVs engineered to target tumor antigens and discuss multiple studies and strategies used to develop oncolytic vaccine regimens. We also contend how, going forward, a combination of technologies for identifying novel immunogenic tumor antigens and rational design of oncolytic vaccines will pave the way for the next generation of clinically efficacious cancer immunotherapies. Immunotherapies have steadily emerged as a powerful treatment option for patients with various types of cancers. While employing the immune system to fight cancer was first proposed in the late nineteenth century, it was only recently that the improved understanding and novel discoveries in classical and tumor immunology have led to the design of more targeted and efficacious immunotherapeutics –4. One svia major histocompatibility complex (MHC) molecules of antigen-presenting cells (APCs)  . Antigenectively . Tumor aectively , 17. Ideectively , 18. In ectively , 20. ThuIn this mini-review, we highlight the studies that have incorporated tumor antigens in OVs to enhance antitumor immune responses and consequent therapeutic benefits in the context of cancer. We discuss recent studies completed using a variety of viral systems, as well as combinations of multiple strategies used to elicit the most efficacious immune response. We also throw light on some of the challenges in this area of research and emphasize the need for combining recent, cutting-edge technologies for tumor antigen discovery with oncolytic virus research for generating more efficacious cancer treatments.via OVs represents one such strategy that makes OV-based cancer therapies more potent by driving immune responses to be directed specifically toward the tumor. OVs that are genetically modified to express tumor antigens are commonly known as “oncolytic vaccines.”The first generation of OVs primarily focused on direct killing of tumor cells. OVs can replicate preferentially in tumor cells due to deregulated signaling pathways , 21, 22 In recent times, many OVs have been found to be amenable for therapeutically desired genetic modifications. Among these, VSV has been the subject of extensive genetic manipulation and consequent investigation on antitumor immunity in the context of cancer treatment. For example, studies have shown that VSV-expressing tumor antigens human papilloma virus oncogene E7 (VSV-E7) and human dopachrome tautomerase (VSV-hDCT) can induce tumor antigen-specific CD8 cytotoxic T cell responses , 31. TheFurthermore, VSV-based oncolytic vaccines have also been indicated as promising candidates to be used in combination therapies. A recent study demonstrated that in combination with stereotactic ablative radiation therapy, VSV-ova helped control local and systemic disease in a murine oligometastatic melanoma model . In addiin vitro for immunogenic tumor antigens, and demonstrated that a combination of three specific VSV-cDNA viruses infected established melanoma tumors and induced tumor rejection via TH-17 responses  and glycoprotein oncofetal tumor antigen (5T4) transgenic mice have been widely used to study the prophylactic capacity of VV vectors. One such study demonstrated that VV-expressing CEA administered in CEA transgenic mice led to the development of CEA-specific Tinations . Upon viinations . Anotherinations . No autovia CD3. The study noted that the virus killed tumor cells and induced a bystander killing of non-infected tumor cells in vitro. The modified VV also had potent antitumor activity in vivo in a lung cancer xenograft model  element along with tumor antigen EphA2 in a VV vector . The TCEft model . Anotherft model .via viral vectors represents a promising strategy for the development of a robust antitumor immune response, and thus far, many cytokines including IL-2, TNF, IFN, and GM-CSF have been expressed through OVs  to demonstrate memory T cell responses that conferred protection against tumor re-challenge with antigen-specific and non-specific colon cancer cells in mice . The autOther viruses that have been employed to target tumor antigens include Newcastle disease virus (NDV) and HSV. A study using NDV-expressing β-galactosidase-derived antigenic peptide  found enhanced antitumor immune responses in a murine colon cancer model . The impOne of the major challenges that oncolytic vaccines face is the induction of an undesired antiviral immune response against the viral vector. This antiviral immunity reduces the efficacy of the OV treatment by clearing the virus prematurely. In the context of oncolytic vaccines, immune responses against the viral vector may dominate the response against the tumor antigen, generating a much more subdued antitumor response. To overcome this obstacle, strategies that effectively redirect the immune responses against the tumor must be employed. One such strategy that we shall discuss is the heterologous virus prime-boost strategy.The heterologous virus prime-boost strategy exploits the quick and effective immunological recall responses to redirect the maximal potential of immunity toward the tumor antigen. In the first priming step, a tumor antigen is expressed using one oncolytic virus vector, whereby the immune system “sees” and responds to the tumor and viral antigen for the first time. In the second boost step, a different viral vector is chosen to express the same tumor antigen, resulting in a primary immune response against the second viral vector while a stronger memory immune response is induced against the tumor antigen. Thus, this clever manipulation allows for the skewing of immunity toward antitumor responses over antiviral responses.In preclinical studies, adenoviruses (Ad), VSV, and VV have emerged as common viral vectors that can be adapted for evaluation of priming and boosting strategies. Using the previously discussed tumor antigen hDCT, studies have shown that priming with Ad-hDCT and boosting with Maraba virus-expressing hDCT led to increased hDCT-specific T cell responses and survival of melanoma tumor-bearing mice . AnotherUnfortunately, one of the major challenges with the heterologous virus prime-boost strategy involves the rapid killing of APCs by effector T cells before the memory T cells are engaged to produce a strong antitumor T cell response. A unique study with oncolytic vaccines addressed this issue by delivering oncolytic vaccines with B cells. Here, the immunodominant gp33 peptide derived from lymphocytic choriomeningitis virus was expressed in three different viruses VSV, Ad, and VV to demonstrate that B cells loaded with the booster virus can elicit better antigen-specific secondary T cell responses, which were dependent on antigen presentation by dendritic cells .Altogether, the heterologous virus prime-boost strategy represents an innovative approach to redirecting immune responses in oncolytic vaccine therapy and warrants further research in the areas of viral vector selection and delivery of vaccines. These strategies combined with effective oncolytic vaccine design and vector development can help maximize antitumor immune responses and the development of efficacious clinical regimens.via a combination of computational and high throughput approaches provide the best chance for selecting antigens to be targeted in tumors and represent an emerging frontier in cancer immunotherapy.Oncolytic vaccine development has come a long way from its initial stages but still has ways to go. As alluded to earlier, implementation of strategies like the heterologous virus prime-boost warrants more research. An important factor when extending such strategies to clinics is a consideration of pre-existing immunity to common viral vectors, which may lead to reduced efficacy in humans. Understanding pre-existing immunity combined with choosing the most effective routes of delivery, which depends on the type of tumor being targeted, will help achieve maximum efficacy of oncolytic vaccines in clinics. Routes of delivery employed in the reviewed studies, along with other parameters and findings, have been summarized in Table in silico prediction and identification of immunogenic tumor antigens (via peptide-based vaccines (Novel tumor antigen discovery is a fundamental cornerstone in cancer immunotherapy and provides an enormous knowledge base for oncolytic vaccine research to draw from. Cutting-edge technological advances in tumor antigen discovery have identified a wide range of potential targets that can be incorporated in the next generation of oncolytic vaccines for designing highly targeted and efficacious therapies. One of these major advances is in the field of sequencing; new and improved sequencing technologies are now available to identify and categorize mutations at the level of genes, exons, and RNA from many different cancer types –67. In aantigens –74. Finaantigens –77. One antigens –81. A revaccines . Going fvia strategies like the heterologous virus prime-boost will aid the design of the most clinically efficacious cancer immunotherapeutics (summarized in Figure Combining this unique capability of oncolytic vaccines with novel tumor antigen discovery paradigms and effective, timely immunological intervention NH, YK, and SG developed the concept. NH prepared the figures and wrote the manuscript. YK participated in writing and technical editing of the manuscript. PL and SG supervised and edited the manuscript.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.


The accumulation of protein aggregates in neurons is a typical pathological hallmark of the motor neuron disease amyotrophic lateral sclerosis (ALS) and of frontotemporal dementia (FTD). In many cases, these aggregates are composed of the 43 kDa TAR DNA-binding protein (TDP 43). Using a yeast model for TDP 43 proteinopathies, we observed that the vacuole  markedly contributed to the degradation of TDP 43. This clearance occurred via TDP 43-containing vesicles fusing with the vacuole through the concerted action of the endosomal-vacuolar  pathway and autophagy. In line with its dominant role in the clearance of TDP 43, endosomal-vacuolar pathway activity protected cells from the detrimental effects of TDP 43. In contrast, enhanced autophagy contributed to TDP 43 cytotoxicity, despite being involved in TDP 43 degradation. TDP 43’s interference with endosomal-vacuolar pathway activity may have two deleterious consequences. First, it interferes with its own degradation via this pathway, resulting in TDP 43 accumulation. Second, it affects vacuolar proteolytic activity, which requires endosomal-vacuolar trafficking. We speculate that the latter contributes to aberrant autophagy. In sum, we propose that ameliorating endolysosomal pathway activity enhances cell survival in TDP 43-associated diseases. Our study highlights the pivotal role of endolysosomal pathway activity in TDP 43 proteinopathies. Whereas previous studies focused on the role of autophagy in the clearance of cytotoxic TDP 43, we propose that the endolysosomal pathway is more important than autophagy for the survival of cells accumulating detrimental TDP 43. In both pathways, protein substrates (such as TDP 43) are sequestered into vesicles, either multivesicular bodies (MVBs) or autophagosomes . These vesicles fuse with the lysosomal membrane and subsequently release their content into the lysosomal lumen, where protein degradation occurs via proteases such as Pep4/Cathepsin D. Both vesicular pathways are interconnected. In mammalian cells (but not in yeast), some MVBs fuse with autophagosomes, leading to the formation of amphisomes which then fuse with lysosomes. Moreover, the endolysosomal pathway is required for directing important lysosomal proteases, thus decisively contributing to proteolytic capacity of this organelle.Our study raises a number of questions regarding TDP 43 degradation and cytotoxicity. For instance, in cells accumulating cytoplasmic TDP 43, different types of potentially cytotoxic TDP 43 species can be expected, e.g., monomers, oligomers, and different classes of protein aggregates.Which kinds of TDP 43 species are recruited into MVBs for degradation via the endosomal-vacuolar pathway, and which kinds of TDP 43 species are sequestered into Atg8-labelled cellular compartments for elimination via autophagy?For steric reasons, one would expect that MVBs contain smaller TDP 43 species than autophagosomes, which could easily enclose large protein aggregates. It is possible that the endosomal-vacuolar pathway is involved in the clearance of more cytotoxic species than autophagy. This could explain why loss of this pathway is more detrimental for TDP 43-expressing cells than loss of autophagy.What is the molecular machinery that determines whether TDP 43 is degraded via the endosomal-vacuolar pathway or via autophagy (or via the ubiquitin-proteasome system)?It is tempting to speculate that the ubiquitylation pattern of TDP 43 plays an important role. Lysine 63 (K63) polyubiquitin chains could favor the endosomal-vacuolar pathway, while K48 polyubiquitin chains could facilitate degradation via the proteasome, and both chains could be substrates for ubiquitin-dependent autophagy.What is the molecular machinery that recruits TDP 43 into MVBs for degradation via the endosomal-vacuolar pathway?Whereas there is a significant amount of information regarding the molecular machinery involved in recruiting protein aggregates into autophagosomes, the mechanisms that enable the recruitment of cytosolic proteins into MVBs remain largely unknown.Autophagy has been shown to be a cytoprotective mechanism to eliminate deleterious protein aggregates. Therefore, pharmacological induction of autophagy has been proposed to be an efficient mechanism to prevent neuronal loss in neurodegenerative disorders. However, this approach was not successful in our yeast TDP 43 model. In contrast, it increased cytotoxicity.Does the observed inhibitory effect of TDP 43 on endosomal-vacuolar pathway activity and vacuolar proteolytic activity reprogram  autophagy towards cytotoxicity?If this holds true, a therapeutic approach that addresses both endosomal-vacuolar pathway activity and autophagic turnover should be much more effective than targeting autophagy alone.Resolving these questions in yeast TDP 43 proteinopathy models and their validations in higher model systems  will improve our understanding of the molecular mechanisms of devastating TDP 43-associated human disorders.


Vineyards are likely to be regionally important for wildlife, but we lack biodiversity studies in this agroecosystem which is undergoing a rapid management revolution. As vine cultivation is restricted to arid and warm climatic regions, biodiversity-friendly management would promote species typical of southern biomes. Vineyards are often intensively cultivated, mostly surrounded by few natural features and offering a fairly mineral appearance with little ground vegetation cover. Ground vegetation cover and composition may further strongly vary with respect to season, influencing patterns of habitat selection by ecological communities. We investigated season-specific bird-habitat associations to highlight the importance of semi-natural habitat features and vineyard ground vegetation cover throughout the year. Given that avian habitat selection varies according to taxa, guilds and spatial scale, we modelled bird-habitat associations in all months at two spatial scales using mixed effects regression models. At the landscape scale, birds were recorded along 10 1-km long transects in Southwestern Switzerland (February 2014 –January 2015). At the field scale, we compared the characteristics of visited and unvisited vineyard fields . Bird abundance in vineyards tripled in winter compared to summer. Vineyards surrounded by a greater amount of hedges and small woods harboured higher bird abundance, species richness and diversity, especially during the winter season. Regarding ground vegetation, birds showed a season-specific habitat selection pattern, notably a marked preference for ground-vegetated parcels in winter and for intermediate vegetation cover in spring and summer. These season-specific preferences might be related to species-specific life histories: more insectivorous, ground-foraging species occur during the breeding season whereas granivores predominate in winter. These results highlight the importance of investigating habitat selection at different spatial scales and all along the annual cycle in order to draw practical, season-specific management recommendations for promoting avian biodiversity in farmland. To counteract ongoing farmland biodiversity erosion, a wealth of evidence-based knowledge has been gathered during the last decades about wildlife in agroecosystems. Restoring farmland biodiversity requires information on the ecological requirements of different species at multiple spatial and temporal scales to capture the dynamics and resource needs of entire communities ,2.Emberiza citrinella markedly declined with the progress of the season, whilst use of cereal fields augmented, probably as a consequence of impeded food accessibility due to a growing ground vegetation within the field margins.To complete their life cycle, birds have to fulfil diverse resource requirements varying between and within seasons. For instance, finding a suitable nesting place in close proximity to good foraging locations for provisioning food to chicks is a challenge commonly faced by parents during reproduction ,3,4. In Landscape-scale heterogeneity mainly affects birds through the proportions of different habitat types present in the overall matrix. Notably, the number of crop types and the relative amounts of woodland, steppe, wetlands strongly influence bird community composition and species richness [Lanius collurio seem to harbour around 15–20% hedge cover among extensive mosaics of pasture and cultivation [Within fields, sward structure constitutes a key factor for terrestrially-foraging and ground-breeding birds because it dictates both food availability (which is abundance modified by accessibility) and nesting opportunities [Alauda arvensis probably because of enhanced food accessibility for foraging parents [Since resources selection is operated in different habitat types and at different spatial scales to fulfil the crucial ecological requirements that may vary between species, the heterogeneity of the agricultural matrix must be considered at multiple spatial scales –18. Incrrichness . Semi-narichness ,22. For tivation . Within tunities –26. A botunities  and maketunities –30, withtunities . Patchestunities –35), whitunities ,36,37. C parents ,39. HencLullula arborea has been shown to select patchy ground vegetation at the foraging site scale [The vegetation structure of vineyards also shows a great geographic and temporal variation with respect to management practice and environmental context. Vineyards can host rare and specialized plant and animal species, especially when they are managed extensively –44 and/ote scale .In this study, we focussed on bird communities occurring in vineyards submitted to a continental climate regime (large variation in conditions between seasons) within a major internal valley in Southwestern Switzerland. We investigated, at two spatial scales, the species-specific habitat associations of multiple bird species that use vineyards as breeding site, as migration stop-over site and as winter habitat. The main emphasis of this study was put on the season-specific effects of habitat characteristics on foraging activities. At the landscape scale, we studied which role marginal, (semi-)natural habitats play for birds within the agricultural matrix. At the field scale, we analysed the importance of ground vegetation structure and vineyard management for birds. By accounting for the broader landscape and assessing circannual variations in habitat selection patterns, our study aims at providing season-specific recommendations for bird-friendly vineyard management both outside and within the production area.2 [The study was carried out in the Rhone river valley in the Canton of Valais . This inner alpine, west-east oriented valley is characterized by a continental climate with little rain, hot summers and cold winters. The south-exposed slopes are dominated by vineyards covering roughly 50 km2  whereas 2 . Most vi2 . Herbici2 . The rem2 .From February 2014 to January 2015, we surveyed 10 transects evenly distributed between Fully (46°08'43.0"N 7°07'30.5"E) and Leuk  from thTM 10.2.2 , species richness and Shannon diversity  and the family of Fringillidae . Species richness refers to the total number of species encountered per transect, also beyond the above two pooled taxonomic groups. Concerning landscape explanatory variables, proportions were arcsin-square root transformed and counts (densities) log transformed. We arcsin-square-root transformed proportions and log-transformed counts or other variables that were strongly right-skewed to improve model fit and to better comply with model assumptions. If the log works better than the untransformed variable, this indicates that the  effect is rather multiplicative than additive, and vice versa. The arcsin-square-root-transformation stretches small proportion values. A difference of 1% grove between two transects A and B has not the same effect for birds if A: 50% to B: 51% or if A: 1% to B: 2%. In the first case, it represents a small, in the second case a large change. Our transects exhibited a grove cover between 0 to 11%. We used arcsin-sqrt instead of logit due to the zeros in our sample. Values were then standardized  to improve convergence of the model fitting algorithms. In case of (continuous) explanatory variables showing a Spearman correlation coefficient |rs| > 0.7, the biologically less meaningful variable was excluded from modelling. Because the number of potential models was large relative to our sample size due to various possible combinations of explanatory variables, the model selection procedure was conducted in two steps.Landscape scale analyses included the four landscape structures and circular month variables   in relatn 2.3–1. ). Since In a first step, overall habitat preferences (complete data set from the whole year) were investigated using Generalized Linear Mixed Models (GLMMs) from the ‘lme4’ R-package  with ‘tTo generate the set of candidate models (differing only in the fixed effects), all possible combinations of the four landscape structure variables  were fitIn a second step, each of the landscape variables figuring in the best model  of overall habitat preferences was used separately in combination with circular month variables to further investigate seasonal variation in habitat selection patterns . For thiTurdus species considered separately) was analysed in relation to the vegetation structure, vineyard management, distances to landscape elements and circular month variables. All explanatory variables were standardized to facilitate model fitting and comparison of effect sizes while checking for collinearity . The same two habitat selection steps as described for the landscape scale were done, but preceded by an additional step due to the larger number of predictors at the field scale compared to the landscape scale; hence the following three steps were conducted:In field scale analyses, bird occurrence  in univariate models were used in the second modelling step. The vegetation structure variables were each tested for the presence of an optimum by including their linear and quadratic effects in a single model, because of a likely trade-off between food abundance (more food in denser vegetation) and accessibility  [First, in order to reduce the number of explanatory variables we pre-selected habitat variables. Vineyard management variables and distances to landscape structures showing a trend (e birds) . QuadratIn the second step , overall habitat preferences were modelled by means of hierarchical logistic regression models (logit-link function) with the random effects ‘transect’ and ‘paired ID’ (identifying pairs of presence and pseudo-absence parcels (for vegetation and management variables) and presence and random points (for distance to landscape structures)). As in the first step of the analysis at the field scale, random effects were not suggested to be important according to AICc and amount of variance explained, neither in the full models, nor in the selected best models (see below). The set of candidate models was created by combining all vegetation structure variables (linear and squared effects retained from the first step) and other variables showing a trend in univariate models in all possible combinations; these models were then ranked according to AICc .2 and brown, hence we created two models to test for seasonal effects . This approach of partial analyses was adopted because at this stage we were specifically interested in the question whether important habitat variables showed some seasonality in their effect on the outcome variable; including several habitat variables would have led to too many candidate models.Third , habitat variables occurring in the most parsimonious model (lowest AICc) were investigated for seasonal habitat selection patterns as explained for the landscape scale analyses above. Note that each habitat variable  was tested individually; e.g. for the pooled species, the most parsimonious model included the predictors green, greenFinally, at both spatial scales, we used model averaging over the set of competitive models  to estiAll statistical analyses were conducted in R version 3.1.3 . The resIn total, we recorded 8719 individuals from 4421 observations (excluding pseudo-absences) belonging to 66 bird species within the 100-m buffer of all transects. At the field scale, 886 individuals from 298 presence parcels totalling 29 species were recorded (see A significant effect of cosmonth (depicting a contrast between summer and winter bird abundance) was noted in all competitive models for the abundance of pooled species and finches, species richness as well as for Shannon diversity. Sinmonth (contrast between spring and autumn abundance) also significantly affected pooled species, thrushes and finches abundances . For speFor pooled species and thrush abundances, species richness and Shannon diversity, grove cover (hedges and woody patches) was the only significant landscape variable occurring in most competitive models . In all Overall habitat selection (pooled species) for ground vegetation cover significantly differed across seasons Tables  and 3D. As for pooled species at the field scale, thrushes favoured parcels where green ground vegetation cover exceeded 50% in winter  positively influenced bird abundance, species richness and diversity, especially in winter. At the field scale, ground vegetation cover, which undergoes much seasonal variation, appears to constitute the most crucial habitat feature for the avifauna. Finally, bird species-habitat associations also greatly varied according to taxon , highlighting the need to consider the distinct resource needs of different guilds if not species, which bears a high relevance for drawing management recommendations for bird-friendly vineyards. While these results originate from only 10 transects and a single region in Switzerland, we are confident that the importance of natural structures and ground vegetation as well as the seasonal variability in habitat selection patterns can be generalized to at least vineyards from other regions ,50–53 anParus major often look then for weed seeds on the ground and invertebrates in the bark of the vines. Alpine chough Pyrrhocorax graculus, that make daily movements from the Alpine cliffs to lower elevations in winter, are also attracted by these food sources, exploiting left-over grapes and seeds. Secondly, in winter, many bird species aggregate into larger, intra- and interspecific flocks that move in search for suitable foraging patches [A higher bird abundance, species richness and diversity in our study area in winter can be explained by two non-mutually exclusive arguments. First, south-exposed slopes devoted to viticulture constitute the first locations released from snow in winter, rendering the soil surface of vine plantations among the most readily accessible foraging sites for birds in adverse, snowy conditions. As noted by Laiolo , woodlan patches . This soEmberiza cirlus [Emberiza cia [Motacilla flava [Despite the fact that our landscape scale analysis relies on only 10 transects while groves were scarce in the matrix (0–11%), these woody structures positively influenced overall bird and thrush abundances, species richness and diversity especially during the winter season. The general preference for grove corroborates earlier findings in a variety of agroecosystems that hedges provide foraging, nesting and sheltering opportunities, as well as song posts for the avifauna –74. The a cirlus , the rocriza cia  or the rriza cia ,23,73. Hla flava  and the la flava ,77. Hedgla flava , which mla flava  but our At the field scale, an overall seasonal shift in vineyards use by birds was observed with respect to green and brown ground vegetation cover. Vineyards with a green ground vegetation cover of 40–100% were visited more often in winter whereas from spring to late summer birds tended to select parcels with an optimum green ground vegetation cover around 40%, while they avoided 100%-vegetated parcels. The preference for brown ground vegetation cover increased in autumn and winter. The strong winter selection for ground vegetated vineyards is certainly driven by the stock of seeds that are picked up by granivorous birds directly from the weed plants ,7,8, a pDifferent vegetation structures are selected by ground-foraging bird species according to their foraging strategy . For insFor finches, preferred vineyards had ca 60% green ground vegetation cover. Five out of the six species of finches identified in this study are granivores foraging either directly from herbaceous plants or on the ground all over the year; the chaffinch, the sixth species, feeds mainly upon invertebrates in spring-summer but is absent from vineyards during the breeding season . As a coBased on these results, we first recommend to keep and rehabilitate hedges and patches of woody vegetation beyond the boundaries of the grape production area or between vine parcels, this in order to bolster avian biodiversity at the landscape scale. This is in line with the suggestion by Ceresa et al.  that eveAnother important beneficial measure, to be implemented within the vineyards themselves, is to allow ground vegetation to grow on roughly half of the ground surface, for instance within every second vine row or inter-row. This would represent the best option to sustain bird communities and populations the year-round. Whenever possible, another ideal option would of course be to remove grass mechanically instead of chemically from the «bare ground» row or inter-row, as requested by the organic and biodynamic cultivation protocols. This is because of the potential deleterious effects of herbicide application on plant and invertebrate communities , 78, witBoosting biodiversity among vineyards will probably in the future represent an important asset of grape and wine production, notably in terms of ecosystem functioning and service provisioning  ,87. ThusThis study confirms that multiple species and scales must be considered when dealing with the sheer complexity inherent to the conservation and restoration of farmland biodiversity. It also shows that the land sparing and land sharing concepts, rather than being mutually exclusive options, would greatly benefit from being applied as dual, complementary management schemes .S1 FigThe three shape-coded vs. five letter-coded regional zones regroup transects for random selection of visit order during the breeding and the non-breeding seasons, respectively. Reprinted from Swisstopo under a CC BY license, with permission from Alexandra Frank see .(PDF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S1 FileEmail-response to copyright request for satellite picture in (PDF)Click here for additional data file.


A rare entity of leukemia called mixed-phenotype acute leukemia exhibits co-expression of markers representing two or three lineages. These two phenotypes regarding the lineage ambiguity suggest that the fate of some leukemia retain or acquire a certain degree of plasticity. Studies using animal models provide insight into how lineage specifying transcription factors can enforce or convert a fate in hematopoietic cells. Modeling lineage conversion in normal hematopoietic progenitor cells may improve our current understanding of how lineage switch occurs in leukemia. In this review, we will summarize the role of transcription factors and microenvironmental signals that confer fate plasticity to normal hematopoietic progenitor cells, and their potential to regulate lineage switching in leukemias. Future efforts to uncover the mechanisms contributing to lineage conversion in both normal hematopoiesis and leukemia may pave the way to improve current therapeutic strategies.Leukemia is characterized by the uncontrolled production of leukemic cells and impaired normal hematopoiesis. Although the combination of chemotherapies and hematopoietic stem cell transplantation has significantly improved the outcome of leukemia patients, a proportion of patients still suffer from relapse after treatment. Upon relapse, a phenomenon termed “lineage switch” is observed in a subset of leukemia patients, in which conversion of lymphoblastic leukemia to myeloid leukemia or  Hematopoietic stem cells (HSCs) establish and maintain the hematopoietic system through differentiation into the multi-lineage progenitors and committed progenitors from which all the mature lineage cell types arise. In the classical model of hematopoiesis, long-term HSCs, short-term HSCs, and multipotent progenitors (MPPs) reside at the apex of the hierarchy –5. MPPs The increasingly narrowed lineage potential results from a precise combination of gene expression signatures and epigenetic modification. Several transcription factors have been found to be involved in the fate decision of hematopoietic progenitors , 9. AmonLeukemias develop as a consequence of mutations that cooperatively confer aberrant self-renewal capacity to leukemic cells and allow them to proliferate indefinitely without differentiation. Recent advances in high-throughput sequencing of leukemia genomes have revealed numerous mutations in cytokine signaling, epigenetic regulators, and transcription factors , 28. GenNRAS mutated bi-potent leukemia clone with T-ALL and AML potential existed  to AML switch upon chemotherapy were transplanted into SCID mice, engrafted AML cells exhibited myeloid cell markers (such as CD33) as expected, but the cells exhibited T-cell markers  similar to the T-ALL at diagnosis if the recipient mice were treated with cytokines GM-CSF or interleukin 3 (IL-3) . Interes existed .On the other hand, other studies have shown the presence of TCR rearrangements in myeloid leukemia cells upon lineage switch from lymphoid leukemias , 44. In AF4 is the most common partner  but select for a latent clone that survived the therapy, or the dominant clone may acquire additional mutations to evolve. While this has been elegantly demonstrated in relapsed AML , therapy partner . MLL–AF4 partner . Recent  partner –54. Tran lineage . Moreove lineage , 56. TheSimilar to how lineage-specific transcription factors reprogram the fate of normal hematopoietic progenitors, these transcription factors have a large impact upon the fate of leukemia cells. Leukemogenic mutations appear to tip the balance created by the network of transcription factors to block and/or bias the differentiation program toward a particular lineage. Dysregulated expression of Pu.1, Gata1, and C/ebp-α all contribute to leukemogenesis , and somIn zebrafish models, AML1–ETO upregulates Pu.1 and downregulates Gata1 to convert the fate of erythroid cells into granulocytic cells, causing a phenotypic change similar to human AML . OverexpThe local microenvironment of HSPCs regulates the maintenance of HSPCs and is often altered in hematological malignancies –64. The MLL-translocated leukemias appear to retain lineage plasticity that can be tapped to direct the differentiation toward either B-cell or myeloid lineages using different cytokines. Expression of MLL-fusion oncogenes in cord blood HSPCs induces B-ALL upon xenotransplantation . The typA recent study demonstrated that BCR–ABL1 rearranged B-ALL can be reprogrammed to a myeloid fate by myeloid-instructing and proinflammatory cytokines –73. PuriSimilar to normal hematopoietic cells, leukemia cells also exhibit lineage plasticity and reversibility aided by master transcriptional regulators that control lineage determination of normal hematopoietic progenitor cells, by the instructive cytokine milieu and also by the strong selective pressure imposed by therapies. Although some improvements in treatment outcome have been reported by the use of intensified ALL therapy followed by AML therapy upon lineage switch, treatment of these leukemia remains challenging , 75. TheTH, RM, and DN reviewed the literature and wrote the manuscript. TH designed the figures.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.


Chronic hepatitis B virus (HBV) infection puts more than 250 million people at a greatly increased risk to develop end-stage liver disease. Like all hepadnaviruses, HBV replicates via protein-primed reverse transcription of a pregenomic (pg) RNA, yielding an unusually structured, viral polymerase-linked relaxed-circular (RC) DNA as genome in infectious particles. Upon infection, RC-DNA is converted into nuclear covalently closed circular (ccc) DNA. Associating with cellular proteins into an episomal minichromosome, cccDNA acts as template for new viral RNAs, ensuring formation of progeny virions. Hence, cccDNA represents the viral persistence reservoir that is not directly targeted by current anti-HBV therapeutics. Eliminating cccDNA will thus be at the heart of a cure for chronic hepatitis B. The low production of HBV cccDNA in most experimental models and the associated problems in reliable cccDNA quantitation have long hampered a deeper understanding of cccDNA molecular biology. Recent advancements including cccDNA-dependent cell culture systems have begun to identify select host DNA repair enzymes that HBV usurps for RC-DNA to cccDNA conversion. While this list is bound to grow, it may represent just one facet of a broader interaction with the cellular DNA damage response (DDR), a network of pathways that sense and repair aberrant DNA structures and in the process profoundly affect the cell cycle, up to inducing cell death if repair fails. Given the divergent interactions between other viruses and the DDR it will be intriguing to see how HBV copes with this multipronged host system. Hepatitis B virus (HBV) is the prototypic member of the hepadnaviruses, a family of small enveloped hepatotropic viruses that replicate their tiny ~3 kb) DNA genomes through reverse transcription. HBV causes acute and chronic hepatitis B; chronic HBV infection puts more than 250 million virus carriers at a greatly increased risk to develop terminal liver disease, i.e., liver fibrosis, cirrhosis and hepatocellular carcinoma (HCC) . While a kb DNA gHBV, by contrast, is a pararetrovirus with an obligatory nuclear phase ,7,8. TheCurrent treatments for chronic hepatitis B include type I interferons for a fraction of the patients , and theThe nucleocapsids (core particles) in enveloped HB virions carry relaxed circular DNA (RC-DNA) in which the 5′ end of the minus-strand is covalently linked to the Terminal Protein (TP) domain of the viral P protein see B. Upon iHence, cccDNA is a crucial intermediate in the hepadnaviral replication cycle. Importantly, cccDNA is not directly targeted by current anti-HBV drugs. NAs can potently block reverse transcription of pgRNA and suppress production of new RC-DNA containing virions see B but notinter alia, a restriction by the structural maintenance of chromosomes (Smc) complex Smc5/6 [Nuclear cccDNA is loaded with cellular histone and non-histone proteins forming a nucleosomally organized minichromosome ,23,24 whx Smc5/6 ,32,33,34However, the pathway from nucleocapsid-borne RC-DNA to chromatinized cccDNA is obscure. While some viruses such as SV40 package their DNA genomes already as histone-associated minichromosomes , for hepHowever, the unknown dynamics of core protein replacement by histones on nuclear hepadnaviral DNA raise several issues. A practical caveat concerns chromatin immunoprecipitation (ChIP) assays which may not report exclusively on the cccDNA status, but could include chromatinized non-cccDNA forms. If full displacement of the originally bound core protein by histones is slow, the association of core protein with the minichromosome ,25 may sA more fundamental issue implied by the model in Even without the extra complexities of chromatinization, the basic mechanisms of cccDNA formation are not yet well understood, except that each cccDNA molecule arises from a series of biochemical steps that start with an RC-DNA molecule as precursor. The structural differences between the two DNA forms then define the principal modifications RC-DNA must undergo to become cccDNA ,49. Protein-primed reverse transcription of hepadnaviral pgRNA causes RC-DNA to contain several unusual molecular features see . Most obHBV’s tiny 3 kb genome encodes P protein as the only—albeit multifunctional—enzyme. At least some of the RC-DNA to cccDNA conversion steps could be performed by P protein, foremostly filling-in the gap in plus-strand DNA. However, confirming earlier studies in animal models ,51,52,53Another possibility relates to P protein release from RC-DNA. Topoisomerases relax torsional stress by incising DNA  via a reAlthough this review is largely conceptual a short detour to the bench will serve to highlight some relevant technical issues in cccDNA research. The basic dilemma is that the amounts of human HBV cccDNA in all tractable test systems are low, and that Southern blotting which allows for unambiguous distinction of cccDNA from all other viral DNA forms  is an inInfected woodchuck and duck livers may carry ≥50 copies of cccDNA per hepatocyte ,58. DHBVHuman HBV cccDNA copy numbers in infected livers appear much lower , and mayMore sensitive PCR methods for truly specific cccDNA detection are thus urgently needed but still not available. The main issue is the distinction of cccDNA from the sequence-identical non-cccDNA forms  which maAs substrate we used a 3 kb HBV plasmid either in its ccc form, or in the RC form obtained by treatment with a nickase enzyme see A. MixturEscherichia coli , but regardless of the specific enzyme it will be mandatory that efforts towards standardized protocols include all enzymatically relevant parameters such as units of enzyme per total amount of substrate DNA, DNA concentration, exact buffer composition, and incubation temperature and duration. In sum, PSD as used here did not generate a pure cccDNA template. More enzyme per DNA, longer incubation times and/or exploiting the higher sensitivity of RC- vs. cccDNA towards heat denaturation may give more favorable results; this also holds for T7 exonuclease. T5 exonuclease came closest to the desired degradation of all non-cccDNA forms but also induced a loss of cccDNA, likely via the endonuclease activity that caused complete degradation of the RC-DNA B,C. Therper se increase cccDNA specificity. Notably, even completely noncontiguous HBV DNA fragments can efficiently yield longer, contiguous PCR products via “PCR recombination” [A recent methodological advance is digital PCR which can give absolute template numbers in a sample without requiring a standard for calibration ; howeverination” , underscIn view of the problems with cccDNA quantitation several cell culture systems could provide useful workarounds, as summarized in The high production of cccDNA by DHBV even in human hepatoma cells  greatly The second kind of systems see B relies However, discrimination of HBeAg from the cccDNA-independently produced core protein is problematic owing to their largely identical amino acid sequences; furthermore, not only HBeAg but also non-enveloped capsids are found in the culture supernatant ; this hoHence, these and similar stable cell lines should become very useful tools for the screening of host factors involved in the intracellular steps of hepadnaviral replication, including cccDNA formation. E. coli. When transfected into hepatoma cells, these molecules resemble true cccDNA much more than conventional plasmid vectors [A complementary new tool is provided by the minicircle technology which allows to produce cccDNA-like molecules  in  vectors , and whe vectors . ObviousProductive HBV infection clearly depends on cccDNA formation but cell culture infection systems were restricted, until recently, to primary human  or tupaiExperimental flexibility was thus greatly expanded by the discovery of the bile acid transporter sodium taurocholate cotransporting polypeptide (NTCP) as a receptor for HBV and its satellite hepatitis D virus (HDV), which exploits HBV’s envelope to enter new host cells ,16. StabNotably though, reasonable infection rates require multiplicities of infection (MOIs) in the range of 1000 viral genome equivalents or more per cell plus additives such as dimethyl sulfoxide (DMSO) and polyethylene glycol . AlthougOne approach is finding more efficiently infectable cells. For HCV some sublines of the principally infectable Huh7 cell line exerted much higher susceptibility , and thiAlternatively, the sensitivity of infection detection may be enhanced by engineered HBV reporter viruses encoding easily traceable  molecules which are expressed as a result of productive infection see E. Such rThere is no evidence that HBV could perform the multiple steps of RC- to cccDNA conversion without exploiting host nucleic acid manipulating enzymes, the richest source for which is the cell’s DNA damage response (DDR) system. Several additional lines of evidence support such an interaction. One is the accumulating evidence that all viruses with a nuclear phase, and likely even RNA viruses with a purely cytoplasmic replication cycle , have toDNA damage describes any of a huge variety of deviations from a perfectly double-stranded DNA structure, including chemically and/or radiation-induced base-modifications, intra- and interstrand crosslinks, mismatches, abasic sites and/or covalent adducts of small or proteinaceous moieties. Each cell in our body may experience 10,000 or more such damage events per day . As all The diversity of DNA damage events calls for a matching diversity of damage recognition mechanisms (for a comprehensive overview see ) and repSmall local DNA lesions, including base modifications or mismatches from DNA replication, are repaired by base excision repair (BER), nucleotide excision repair (NER) or mismatch repair (MMR). Thereby the damaged site is excised from the DNA, often together with a few neighboring residues, followed by gap repair synthesis and strand ligation, using the undamaged strand as template  usually induce an immediate DDR. Single-strand DNA breaks (SSBs) and exposed ssDNA regions are obligatory intermediates in nearly all nucleolytic repair pathways. The major break repair mechanisms are the error-prone classical nonhomologous end-joining (c-NHEJ) and alternative EJ  and PARP1 , which recruit DNA-activated protein kinase  or the HR-mediated repair is embedded into a complex signaling network that couples DNA repair to the cell cycle . HaltingDSBs destined for HR-repair are recognized by the MRN complex which also has nuclease and signaling roles through autophosphorylation and phosphorylation of downstream targets including ATM ,112. PhoCell-death as a result of non-repairable DNA damage, via apoptosis or necroptosis , is largely induced through p53 , both viFrom the viral viewpoint the DDR resembles a self-service store for repair factors; tapping this reservoir is smart yet also risky, owing to the emerging coupling of DNA damage sensing to innate immunity ,117. FroGiven the exquisite sensitivity with which cells can detect subtle alterations in their DNA (in the range of 1 part per billion) it is not that surprising that viral genomes in the nucleus do not go undetected. DDR triggers can be unusual genome structures as such ,101,102,Several recent reviews comprehensively summarize how individual viruses cope with the cellular DDR ,125,126.Most HBV-related HCCs contain integrated viral DNA , usuallyRecent deep sequencing data confirmed the random nature of genomic integration sites , but theAn alternative is dsL-DNA circularization, which was directly demonstrated by the formation of cccDNA-like molecules from DHBV genomes with engineered defects in RC-DNA formation ,134. ThiThat also hepadnaviral dsL-DNA is circularized by NHEJ ,134 is sA related strategy for minimizing the number of free DNA ends is concatemerization. A prime example for this type of damage response are the linear (about 36 kb) dsDNA genomes of adenoviruses which, unless counteracted by the virus, are “repaired” into concatemers too large to be packaged into the viral capsids . To prevIn sum, these considerations strongly support an interaction of hepadnaviruses with cellular DNA repair, with the free ends of dsL-DNA as a likely major trigger that is shut off by integration and circularization, and perhaps by concatemerization and degradation. While RC-DNA bears less resemblance to broken DNA, its 5’ terminal protein and RNA modifications are still obvious tags for distinction from normal DNA (see below). Hence, both forms of hepadnavirus DNA could conceivably contribute to DDR activation.A connection to DNA repair was surmised early-on based on the association of HCC with chronic hepatitis B and the known correlation of cancer with improper DNA damage repair and/or failure in preventing cells with damaged DNA to proliferate. Various aspects of HBV expression were reported to impact on DNA repair ,144,145,Among the dozens of reported HBx interacting proteins  there weHowever, binding of HBx to DDB1 does not by itself establish a connection to DNA damage. Though also involved in DNA repair, DDB1 does not directly bind to damaged DNA; this function is rather due to DDB2 ,157, oneBased on structural data , HBx actThe most immediate benefit of the host DNA repair system for HBV would be the provision of enzymatic activities that help converting RC-DNA into cccDNA . As summA second recently identified host enzyme important for RC- to cccDNA conversion is DNA polymerase kappa (POLK), one of the Y-family translesion DNA polymerases ,167 thatAltogether, it is likely that the new cccDNA-dependent test systems see  will proUnderstanding the molecular details of RC-DNA conversion into the cccDNA minichromosome will be a critical asset in developing strategies for a cure of chronic hepatitis B. There are still technical obstacles hampering high-throughput approaches for the global identification of host factors involved in the process, yet several lines of evidence support a crucial role for the cellular DDR; however, at present many options are open as to how such an interaction may manifest itself for HBV. Hepadnaviral genomes are amongst the smallest animal virus genomes known, implying a particularly strong dependence on host factors, including on the DNA damage repair machinery. Conversely, they also lack the sophisticated genetic equipment that larger viruses invest in handling the challenges of a close encounter with the DDR. As outlined above, RC-DNA appears to present a sufficient number of unique molecular features to make itself conspicuous to cellular DNA surveillance and induce the respective repair activities. However, which are the cellular sensors and which of the different programs are activated, if any? With HBV infecting largely quiescent hepatocytes, does this depend on the cell cycle? Could the seemingly useless fraction of dsL-DNA have a DDR-triggering role that facilitates cccDNA formation? Typical for cellular DNA damage repair is the megabase-wide spreading of γH2AX around the break site; as recently shown, even the 36 kb adenovirus genomes are too short for this . How theWould DDR induction also induce an innate response that jeopardizes the virus ? Two recNot the least, there should be differences in the cell’s perception of RC-DNA that has freshly been released from the nucleocapsid and is just ready to be converted into cccDNA versus long-term chromatinized cccDNA. Is cccDNA as a perfectly double-stranded circle without free ends protected from cellular damage surveillance? Is the HBV minichromosome a safe store for cccDNA as it is inconspicuouly similar to cellular chromatin? Or does its small size and possible association with viral proteins cause it to be recognized? If so how does HBV prevent a permanent DDR activation and/or apoptosis?Questions like these had often tried to be addressed using the coarse experimental systems, such as transient overexpression of individual gene products, that until recently dominated the field. With further improvements, the new cell culture systems promise more physiological answers, and these will also impact on the chances for developing new curative treatments of chronic hepatitis B.