Circulating tumor cells (CTCs) are putative markers of tumor prognosis and may serve to evaluate patient’s response to chemotherapy. CTCs are often detected as single cells but infrequently as clusters and are indicative of worse prognosis. In this study, we developed a short-term culture of nucleated blood cells which was applied to blood samples from breast, lung, esophageal and bladder cancer patients. Clusters of different degrees of compactness, classified as very tight, tight and loose were observed across various cancer types. These clusters show variable expression of cytokeratins. Cluster formation from blood samples obtained during the course of chemotherapy was found to be associated with disease progression and shorter overall survival. The short-term cultures offer a robust and highly reliable method for early prediction of treatment response in different cancer types. Circulating tumor cells (CTCs) are cancer cells disseminated in blood and are considered to be pivotal in the metastatic cascade2. Studies on CTCs have gained momentum since last few years. However, a deeper understanding about the biological and clinical impact of CTCs in the metastatic process is still awaiting. Isolation of pure CTC population is still a major challenge as they represent only a minor fraction of white blood cells. Enrichment methods based on physical and biological properties of CTCs have not resolved the issue of heterogeneity in size, deformability and surface marker expression among other parameters. In particular, it has been difficult to estimate the number of CTCs expected to be released in each cancer type4. Inter and intra-tumor heterogeneity introduces significant challenges to characterize these rare populations of cancer cells5. Recent studies have documented that CTC clusters, although infrequently detected, could promote their longer-term survival in the blood stream and increase their metastatic potential14. For instance, it has been shown that CTC clusters have 50 times increased metastatic potential than a single CTC12. CTC clusters have been shown to engage in epithelial-mesenchymal transition (EMT) through large amounts of TGF beta released by platelets co-aggregated in these clusters15. The acquisition of an EMT status in the CTC clusters may augment chemoresistance16. In addition, platelets will contribute to the formation of a favourable metastatic microenvironment through the recruitment of granulocytes in CTC microemboli17. Another study reported the presence of giant macrophages associated with CTC possibly promoting their dissemination and survival in the circulation, together supporting the significance of the microenvironment in tumor growth, progression and metastasis18. In our previous study, we observed that in short-term cultures CTCs could form clusters comprising other cell types including macrophages and natural killer-like cells19.Metastasis contributes to 90% of cancer deaths19. CTCs that were able to survive in short-term culture were shown to form multilayered cell clusters in association with NK cells and macrophages, possibly reflecting some association in vivo as described above. In this study, we used similar microwells, molded in agar to culture erythrocyte-lysed whole blood preparations from cancer patients having locally advanced or metastatic lung, breast, esophagus, bladder and hemangioendothelioma. We have further developed a robust method to quantify the CTC cluster formed across cancer types and during various treatment regimens. These clusters were categorized into three cluster types based on the cell density. We utilized the cluster phenotype as a predictive indicator of treatment response and to determine the therapeutic efficacy of the drugs.We recently established a short-term culture method using laser-ablated microwells that permits the expansion of CTCs from whole blood of breast cancer patients+/CD45− cells after day 14 cultures  blood samples for 21 days as shown in Fig. . At day res Fig. .Figure 1We next examined whether distinct cluster phenotypes appear in culture of blood samples from patients bearing different cancer types. We classified these clusters on the basis of their density or compactness using the fluorescence intensity values of DAPI . The clusters were categorized based on their compactness into 3 different types: very tight, tight and loose Fig. . The stath and 12th week of therapy  on 9apy Fig. . This coapy Fig. . We obseth week of gefitinib treatment (targeting mutated EGFR), produced loose clusters, however, we observed tight clusters on the 12th week of the therapy. Interestingly, a blood sample from the same patient on the follow-up after 9th month of the treatment produced loose clusters in culture. Our observation suggested that the patient responded to gefitinib , Kidwai Memorial Institute of Oncology  and confirmed that all research was performed in accordance with relevant guidelines and regulations. Informed consent was obtained from all the participants and/or their local guardian for inclusion in this study. All the blood samples were collected in sterile EDTA-coated vacutainer tubes (BD Vacutainer®) and kept on ice. The clinical treatment characteristics for the four cohorts and unique cases are summarized in Supplementary Table 4Cl + 10 mM KHCO3 + 127 μM EDTA) with gentle mixing for 10 mins. The blood was suspended in RBC lysis buffer in 1:2 ratio. It was then centrifuged to remove RBC fragments and washed once with sterile phosphate-buffered saline (PBS). Nucleated cells were re-suspended in Dulbecco’s modified Eagle medium (DMEM) (Sigma) supplemented with 10% fetal bovine serum (FBS) (Life Technologies) and 1% penicillin-streptomycin (Sigma).Fresh blood was collected in EDTA coated vacutainer tubes (BD Vacutainer®) and processed within 5 hrs of the blood collection to minimize blood clotting. The whole blood was separated by centrifugation and the remaining cell fraction was subjected to RBC lysis by RBC lysis buffer  (1% O2). The culture medium was replaced every 48–72 hrs with minimal disturbances to the microwells. Cultures were maintained for 3 weeks and imaged on day 7, 14, 21 with phase contrast microscope (Olympus IX71) and analyzed the images using ImageJ .A general strategy for the culture of nucleated cells from patient samples in ellipsoidal microwells is shown in Fig. . The mic2 and 1% O2 hypoxia  under humidified conditions. The samples were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin . Each processed sample (10 ml of whole blood) was split and seeded in three 35 mm dish containing the microwells.The samples were maintained at 37 °C in 5% (V/V) COThe threshold values to classify the clusters were calculated from the median of grey values from each cluster in a microwell, which reflects the fluorescent intensity of DAPI in clusters. In majority of cases from lung and breast CTC clusters, the median grey score obtained were more than 100, which were categorized as very tight clusters, followed by grey score ranging between 10–100 as tight clusters. In the case of esophagus and bladder CTC clusters, the median grey score was below 10 and was classified as loose clusters   for 20 mins and permeabilized with 0.1% Triton X-100  in PBS. The cells were then treated by blocking buffer  in PBS) for 1 hour. Cells were incubated with CD45 , Pan-CK  primary antibodies overnight at 4 °C and then incubated with anti-mouse secondary antibody conjugated with Alexa 568  and anti-rabbit secondary antibody conjugated with Alexa 488 . Cells were then counter-stained with DAPI   and the epifluorescent images were acquired under Olympus IX71 microscope. The images were analyzed by ImageJ [National Institutes of Health (NIH)]. The clusters were classified according to the median gray values. The median values were tested for statistical significance by 2-tailed t-test.Supplementary Dataset 1


We carried out this study to determine maternal serum level of TNF-a and it's association with body weight status in a group of Nigerian women with GDM.a cross sectional analytical study conducted among 169 pregnant women, 85 with GDM and 84 with normal gestation. Diagnosis of GDM was made between 24-28 weeks gestation according to the WHO diagnostic criteria. Maternal serum level of TNF-α was measured and compared between the study groups.maternal serum TNF-α level was significantly higher in the pregnant women with GDM than in the controls . Also when comparing the serum TNF-α levels of the pregnant women with GDM and the controls for each level of body mass index, serum TNF-α levels remained significantly higher in both the normal weight and overweight pregnant women with GDM compared to their matched controls  and  respectively.it is concluded that pregnant women with GDM in this study have higher maternal serum TNF-α level compared to the pregnant women with normal glucose tolerance regardless of body weight status. Gestational diabetes mellitus (GDM) is defined as any degree of glucose intolerance with onset or first recognition during pregnancy . The preA cross sectional analytical study that involved 85 pregnant women with gestational diabetes mellitus and 84 controls  at 24-28 weeks gestation. The study was conducted at Ahmadu Bello University Teaching Hospital, Zaria, Nigeria. Diagnosis of gestational diabetes mellitus was based on the WHO diagnostic criteria . Study subjects were recruited from the antenatal clinics of Ahmadu Bello University Teaching Hospital, Zaria and Hajiya Gambo Sawaba General Hospital Zaria, Kaduna State, Nigeria. Pregnant women with hypertension, history of pre gestational diabetes mellitus, multi foetal pregnancy, or any pre-existing illness were excluded from the study. The purpose of the study was explained fully to the participants, and written informed consent was obtained from each subject, before recruitment in to the study. The study was reviewed and approved by the Health Research Ethics Committee of Ahmadu Bello University Teaching Hospital, Zaria and Kaduna State Ministry of Health, Kaduna. History including maternal age, parity and gestational age were all obtained from the subjects at the time of enrolment. Gestational age was based on the report of ultrasound scan. Body mass index (BMI) was calculated as the ratio of weight in kilogram to square of height in meters and expressed as kg/m2. The two-hour 75g oral glucose tolerance test was performed in the morning (8:00am-10:30am) following 10-12 hours overnight fast. A fasting blood sample was drawn for measurement of fasting serum glucose and TNF-α. Two-hour blood sample after an oral glucose load was taken for measurement of 2-hour serum glucose. Serum samples were prepared and stored in a freezer (-20oC) until time of analysis.Biochemical analysis: serum glucose was measured using an enzymatic glucose oxidase method (Labkit France). Serum TNF-α was assayed using human TNF-α ELISA kit (Wkea Med Supplies Corp. China). Samples were analysed at the Chemical Pathology Laboratory of Ahmadu Bello University Teaching Hospital, Zaria.Statistical analysis: statistical analysis was performed using the SPSS version 20.0 statistical package. Data are summarised using measures of central tendency and dispersion. We compared mean differences of serum TNF-α level between groups using t-test. All p-values were 2-sided and considered significant if less than 0.05.Demographic characteristics of the study subjects are presented in et al. reported that higher maternal serum level of TNF-α is a significant predictor of developing gestational diabetes mellitus [et al. [et al. did not show significant difference between serum TNF-α levels in pregnant women with gestational diabetes mellitus compared to normal controls. Their negative findings might be due to the differences in timing of maternal blood collection, assay methods used and ethnicity of the study subjects [This study has shown that the pregnant women with gestational diabetes mellitus have higher serum TNF-α levels than the pregnant women with normal gestation. This finding of increased serum TNF-α level in pregnant women with gestational diabetes mellitus has been reported in many previous studies , 11-13. [et al. , althougsubjects . The disWe conclude that among the pregnant women in this study, those with gestational diabetes mellitus have higher serum TNF-α levels than the pregnant women with normal glucose tolerance regardless of body weight status, suggesting that alterations in the circulating level of TNF-α might be related to the pathogenesis of gestational diabetes mellitus. We recommend larger prospective studies within the population that would examine the mechanism of alteration of the maternal circulating TNF-α level and its pattern from first through third trimester in pregnant women with gestational diabetes mellitus.Gestational diabetes mellitus results from an imbalance between insulin resistance and insulin secretion capacity during pregnancy;Pro-inflammatory cytokines might cause insulin resistance by inhibiting insulin signal transduction.The findings in this present study corroborates findings in previous reports suggesting that alterations in the circulating level of the inflammatory cytokine (TNF-a) might be related to the pathogenesis of gestational diabetes mellitus regardless of body weight status;The results of the study suggested that regulation of TNF-α may provide an important target for physiological and pharmacological interventions designed to reduce the risk of adverse pregnancy outcomes related to gestational diabetes. These may include prompt treatment of disease conditions that are associated with release of inflammatory cytokines including the TNF-α e.g. urinary tract infection.The authors declare no competing interests.


Helminths are ubiquitous and have chronically infected vertebrates throughout their evolution. As such helminths have likely exerted considerable selection pressure on our immune systems. The large size of multicellular helminths and their limited replicative capacity in the host necessarily elicits different host protective mechanisms than the immune response evoked by microbial pathogens such as bacteria, viruses and intracellular parasites. The cellular damage resulting from helminth migration through tissues is a major trigger of the type 2 and regulatory immune responses, which activates wound repair mechanisms that increases tissue tolerance to injury and resistance mechanisms that enhance resistance to further colonization with larval stages. While these wound healing and anti-inflammatory responses may be beneficial to the helminth infected host, they may also compromise the host's ability to mount protective immune responses to microbial pathogens. In this review we will first describe helminth-induced tolerance mechanisms that develop in specific organs including the lung and the intestine, and how adaptive immunity may contribute to these responses through differential activation of T cells in the secondary lymphoid organs. We will then integrate studies that have examined how the immune response is modulated in these specific tissues during coinfection of helminths with viruses, protozoa, and bacteria. Helminths are ubiquitous and have chronically infected vertebrates throughout their evolution. A number of studies have shown that they can severely impact wild vertebrate populations affecting their body weight, fecundity and their ability to survive hardship in the winter –3. In huThe helminth induced type 2 immune response includes characteristic activation of immune cells. Although these activated immune cell lineages share stimulation of common signaling pathways, they also exhibit lineage specific activation states which support their characteristic effector functions. This type 2 immune cell activation motif was originally described in CD4+ Th2 cells, but it is now clear that this characteristic activation, also referred to as alternative activation, occurs in other T cells and also B cells, innate lymphocytes, mast cells, macrophages, basophils, eosinophils, and neutrophils –12. UnraThe helminth-induced type 2 immune response thus has important wound healing and anti-inflammatory properties. However, this beneficial response that helps to mediate tolerance by mitigating tissue damage during infection with these large multicellular parasites can have a dark side as well. Many properties of this immune response can potentially reduce the effectiveness of the protective response against many microbial pathogens. As coinfection with helminths and microbes affects much of the world's population, this as yet little studied area of research has considerable real world significance. In this review we will first describe helminth-induced tolerance mechanisms that develop in specific organs including the lung and the intestine, and how adaptive imunity may contribute to these responses through differential activation of T cells in the lymph nodes. With this background, we will review studies that have examined how the immune response is modulated in these specific tissues during coinfection of helminths with viruses, protozoa, and bacteria.Nippostrongylus brasiliensis, influences lung tissue. As with several other intestinal nematode parasites, including human hookworms, N. brasiliensis larvae invade the host through skin penetration, migrate through the circulation to the lung, where they are coughed up and swallowed. Once in the intestine they mature to adults, breed and produce eggs. Following N. brasiliensis primary infection, the parasites enter the lung between 12 and 48 h after inoculation, and usually exit the lung 48 h later. Thus by 3–4 days after inoculation all the parasites have left the lung. This 2 day time interval in the lung triggers a cascade of immune responses that initially triggers acute lung injury (ALI), followed by rapid mitigation of lung damage, and finally subsequent chronic lung remodeling associated with fibrosis and emphysema. Understanding the immune components of this lung remodeling response has elucidated a number of tolerance mechanisms associated with the type 2 immune response.Extensive and specific remodeling of tissues and associated organs can occur following invasion by specific pathogens. In turn, subsequent or even coincident coinfection by a different pathogen can markedly alter the course of the response in some cases compromising resistance and tolerance mechanisms directed against either pathogen. Recent studies have begun to unravel the mechanisms through which the intestinal nematode parasite, N. brasiliensis inoculation, a pronounced increase in IL-17 triggers massive recruitment of neutrophils to the lung peaking at about 1 × 106 total cells by day 2 . As IL10 is not elevated at early stages of the response, IL-4R signaling is the major trigger and the helminth-activated macrophage also does not produce IL-10 , 20. HowN. brasiliensis inoculation. Previous studies have shown that M2 macrophages persist in the lung for at least 45 days after inoculation, and this persistent macrophage phenotype is capable of mediating acquired resistance resulting in accelerated parasite destruction upon secondary challenge (N. brasiliensis excretory/secretory (ES) products, which still induce type 2 responses, can drive fibrosis and associated impaired lung function, but not emphysema  is required for upregulation of IL-33 and the corresponding downstream type 2 immune response  molecules likely also contribute as inoculation with ES supernatants alone can promote a type 2 immune response , 48, thoreceptor . Also, Ereceptor  and IL-3receptor  signalinThus helminth infection in both the lung and in the small intestine triggers type 2 immunity in part through tissue damage, and associated release of DAMPS, and is then further modulated through the release of helminth ES products. The type 2 immune response has many components shared with wound healing responses raising the possibility that the innate type 2 immune response may have originated from a conventional wound healing response, coopted by the immune system to mitigate tissue damage during helminth infection. The development and overlay of the adaptive type 2 immune response over the innate response may have in part evolved to incorporate antigen specificity to enhance resistance against helminths . TogetheA number of components of the helminth-induced response also directly inhibit both type 1 and type 17 responses. T regulatory (Treg) cells are expanded in response to many helminths and have been shown to downregulate harmful type 1 responses and also type 2 immunity associated with allergic responses . In manyH. polygyrus is able to exert potent immunomodulatory effects and inhibit intestinal inflammation induced by IL-10 deficiency  technologies to resolve shifts in differentiation trajectories of intestinal stem cell and transit amplifying cells caused by helminth infection and the inherent intraniche heterogeneity and functional specialization of the Tuft cells and Goblet Cells compartments during helminth infection. A recent scRNA Seq study has already pinpointed an interesting dichotomy within the Tuft cell niche, which was previously characterized as having both neuronal and inflammatory gene expression programs. It appears that these two functional modules may be embodies in two distinct subpopulations, both of which express IL-25, IL-25 receptor (IL17rb) and receptors for IL4 and IL-13. Nevertheless, only one subset expressed high levels of TSLP and interestingly expressed CD45, a pan-marker of hemopoietic cells .N. brasiliensis infection, resting ILC2s residing in the intestinal lamina propria rapidly acquire an activated KLRG1+ phenotype and become mobilized to seed the lung and liver. IL-25 appears to mediate this effect, as it is sufficient to cause activation and redistribution of activated ILCs without any apparent proliferative step. Activated ILC2s gain entry into the lymphatics and blood circulation and accumulated at distal tissue sites, including the lung, based on the well-known sphingosine-1-phosphate-mediated chemotactic mechanism used by T lymphocytes to egress from lymphoid organs. In the lung, relocalization of recently activated ILC2s promoted tissue repair and prevention of acute lung injury  and naïve T lymphocytes , 70. Upog injury .H. polygyrus) infection in mice. This systemic redistribution of non-activated lymphocytes persists into the chronic stage of infection and requires the participation of the lymphoxin-beta receptor signaling. IL4 secretion by Th2 and Tfh cells during helminth infection likely promotes LTβ expression by follicular B cells which then expands the stromal cell compartment to reorganize lymph node architecture. Alternatively, expression of another alternate LTβR ligand, LIGHT by T cells and DCs in the reactive lymph node may be responsible for expansion of the stromal cell compartment to further promote humoral responses to the helminth parasite. Nevertheless, the relative depletion of the naïve lymphocyte pool at other lymphoid sites result in impaired responsive to heterologous immunization or infections with other unrelated micro-organisms (In contrast to the behavior of ILC2s, the naïve T and B cell pools become depleted from non-involved lymphoid organs and accumulated in the T helper 2-reactive mesenteric lymph node during helminth (rganisms .N. braziliensis infection, dermal dendritic cells acquire parasite material and migrate to the draining lymph nodes to prime CD4 T cells capable of making IL-4 (dull MHCIIhi phenotype and expressed Th2 promoting factors including PDL2, IRF4 and OX40L as well as CD301b (In addition to systemic shifts in lymphocyte migration, helminth infections induces dramatic alterations in the cell type distribution and functional attributes of dendritic cells in the secondary lymphoid organs. During ing IL-4 . These ds CD301b . Howevers CD301b .loCD103− dendritic cell population that may be important for the expansion of Treg cells during chronic helminth infection (lo DCs exhibited poor responsiveness to TLR activation and consequently deficient T cell activating potencies. Instead, naïve T cells stimulated by these CD11clo DCs were more likely to become Foxp3-positive Tregs. Consistent with a model where distinct DC subsets mediate helminth induction of Th2 and Treg responses, depletion of CD11chi DCs abrogated Th2 effector responses, while sparing Treg expansion.In keeping with the prevailing theme that helminths induce both Th2 and regulatory immune mechanisms, chronic gastrointestinal helminth infection has also been shown to promote the development of a CD11cnfection . InteresToxoplasma coinfection (The ability of helminths and helminth products to dampen the antigen-presenting and costimulatory functions of dendritic cells and the induction of Tregs may not be the sole mechanism for how these parasites modulate proinflammatory Th1 and Th17 responses. The production of type 2 cytokines by ILC2s, eosinophils, neutrophils and basophils instruct the formation of alternatively activated macrophages, not only at affected tissue sites but also within secondary lymphoid organs. A key difference between these alternatively activated macrophages and their classically activated counterparts is in their alternative metabolism of the amino acid arginine. IL4 induces arginase 1 which results in the formation of ornithine and urea. The elaboration of arginase can result in depletion of this essential amino acid and restrain the activation and function of T cells. T lymphocytes subjected to arginine depletion become blocked in the G1 stage of the cell cycle and subsequently downregulated mTORC1 activity, while mTORC2 mediated cell cycle arrest in these starved T cells . In relanfection . Recent nfection –84. Thusnfection , 84.Trichuris muris coopts the host microbiota to increase its own fitness and alters the microbiome in such a way that inhibits subsequent rounds of infection (From a teleological perspective, helminth parasites may have coopted the type 2 and Treg response to “optimize” their species-specific host niche (see Figure nfection . It is lIn particular, these immune regulatory mechanisms, including products directly produced by the parasite, can modulate the immune response in some cases impairing effective type 1 immunity against microbial pathogens. On the other hand these same tolerance mechanisms, including factors directly enhancing wound healing, may mitigate severity of tissue damage associated with microbial infections, raising the possibility that eradication of helminths may not only enhance resistance but also deleterious effects of type 1 inflammation leading to increased severity of disease. Understanding the multiple mechanisms through which helminths modulate immune responses and promote tissue repair may lead to new and effective targeted treatments to control harmful inflammation associated with microbial pathogens as well as noncommunicable inflammatory diseases.All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.


Second phase strengthening has been applied to high entropy alloys (HEAs) for optimizing mechanical properties. In this study, by conducting mechanical testing of a eutectic dual-phase AlCoCrFeNi HEA with homogenous distribution of body-centered cubic (BCC) and face-centered cubic (FCC) lamellar phases inside a transmission electron microscope, we found that although BCC was truly the hard phase, decreasing the proportion of BCC phase in fact increased the strength due to the existence of chemically disordered semi-coherent phase boundaries, which acted as potent impediments to dislocation motion resulting in dense dislocation storage in FCC phases. Moreover, the difficulty in dislocation glide caused massive cross-slip, and the interaction between primary slip arrays and cross-slip systems during deformation increased the rate of dislocation accumulation by forming dislocation substructures, thus making the FCC phases exceptionally strong. Our findings not only revealed the underlying strengthening mechanism of eutectic dual-phase AlCoCrFeNi HEAs, but also shed light on new ways in further optimizing the mechanical properties of HEAs. Unlike precipitation strengthening, where precipitated particles of optimum size are prepared to inhibit the dislocation motion and lead to the increase of ultimate strength7, dual-phase alloys have nearly equal fraction of two phases. As a result, considerable proportion of phase boundaries are introduced into the alloys, exerting important influence on the deformation behavior and mechanical properties9. The strengthening effect usually originates from two sides: (1) the combination of the strength of two phases, which can be simply estimated by1 and f2 are the volume fraction of two phases while σ1 and σ2 are the strength of two phases; (2) the impediment to dislocation motion due to phase boundaries10. Since FCC phases are relatively soft, stronger BCC phases are commonly added to increase the strength13. Additionally, incoherent phase boundaries normally show significant impediment to dislocation glide while coherent phase boundaries have negligible influence14.Second phase strengthening has long been used to enhance the mechanical performance of commercial alloys, such as ferritic-martensitic steel16. In contrast to the conventional strategies for alloy design, HEAs are developed with at least five metallic elements, emerging as a new frontier of research in recent years17. The chemically disordered distributions of multiple elements in HEAs have been suggested to show important influence on dislocation slip19. In particularly, the high mixture of multiple elements in dual-phase HEAs may form distinctive phase boundary structures compared to conventional ones, which could result in different influence on dislocation behaviors. However, experimental observation and analysis are still lacking for a profound understanding of the deformation mechanisms of dual-phase HEAs.Recently, new types of dual-phase high entropy alloys (HEAs) were designed under the inspiration of second phase strengthening, showing good combination of high fracture strength and tensile ductility at room temperaturein situ tensile tests inside a transmission electron microscope (TEM), a unique deformation behavior and strengthening mechanism were uncovered. The plastic deformation of the alloy was dominated by dislocation activities in FCC phases, where the frequently observed interactions between the primary and cross-slip systems generated dislocation substructures and partly made the FCC phases exceptionally strong. Importantly, the semi-coherent phase boundaries in this HEA were found to be potent obstructions for dislocation motion, which resulted in ultra-high dislocation density in FCC phases and significant hardening effect. Our findings provide new insights into the second phase strengthening mechanism, which would have significant implications for further improving the mechanical performances of HEAs.In this study, a five-component eutectic AlCoCrFeNi HEA, consisting of homogenous distributed body-centered cubic (BCC) and face-centered cubic (FCC) lamellar phases, was prepared by vacuum arc melting and casting. By conducting 2.1, which demonstrated a uniform and fine lamellar feature, a typical characteristic of eutectic alloys22. The interlamellar spacing was about 1.5 μm for the larger ones and 0.7 μm for the others. An obvious contrast of the coupled two phases was observed in bright field TEM images BCC and [011]FCC//[111]BCC, which coincides with the K-S orientation relationship. It is worth noting that a narrow band of high image intensity was clearly visible in the high-angle annular dark field (HAADF) scanning transmission electron microscopy (STEM) image BCC and [110]FCC//[100]BCC. However, in another case, when the loading geometry was changed to facilitate a crack propagation through the phase boundaries, the crack penetrated the phase boundaries via the cleavage of {110} planes with limited dislocation motion in the BCC phase while the extension of crack can transform to along another direction by activating cross-slip systems in the FCC counterpart  analysis20. The alloys were repeated melting at least five times with electromagnetic stirring for homogeneity. Then the alloys were directly solidified in a water-cooled cold copper hearth and the solidified ingots were about 30 mm in diameter and 15 mm in thickness. The in situ tensile test was conducted by a Gatan 654 single-tilt straining holder at a typical strain rate of ~1.0 μm∙s−1 inside an FEI Tecnai G2 F20 S-TWIN TEM with accelerating voltage of 200 kV. An FEI Titan G2 80–200 ChemiSTEM, equipped with Cs corrector and operated at 200 kV, was also used to characterize the atomic level structure and elements distribution. In a representative process, the sample for in situ tensile test was cut into 3 × 3 × 1 mm square pieces by wire cut electrical discharge machining and then polished down to ~40 μm in thickness before producing electron transparent regions for TEM observation using ion milling. Room-temperature tensile tests were performed using the Instron 5569 testing machine at a constant strain rate of 1 × 10−3 s−1 and the tensile test specimens cut from as-cast alloys had a diameter of 8 mm and gauge length of 40 mm. Phase analyses (microstructure and element constituents) of Ni2.1 and Ni2.4 alloys were conducted by a Zeiss Axio Imager 2 polarizing microscope and a Hitachi SU-70 SEM operating at 30 kV after sequentially polished and etched the alloys with the 10 vol. % aqua regia-90 vol. % ethanol solution at room temperature.Commercially pure elements Al, Co, Ni, Cr (99.9 wt. %) and Fe (99.6 wt. %) were alloyed by vacuum arc melting to prepare the dual-phase high entropy alloy of AlCoCrFeNiSupplementary InformationMovie S1


Elite controllers (EC), a small subset of the HIV-positive population (< 1%), suppress HIV viremia below the limit of quantification of clinical viral load assays in the absence of antiretroviral therapy (ART). However, there is a paucity of longitudinal data detailing the viral and immune dynamics or HIV reservoir seeding during acute infection in individuals that go on to become Elite Controllers.In this report, we describe a case of a 42 year old woman diagnosed during acute infection who rapidly and permanently suppressed her viremia in the absence of antiretroviral therapy (ART). Rapid antibody/antigen testing was either negative or equivocal during acute infection, despite subsequent viral load testing at that time point with 71,550 plasma HIV RNA copies/mL, making initial diagnosis challenging. The patient subsequently developed detectable anti-HIV antibodies and an increase in HIV-specific CD8+ T cell responses to overlapping subtype C HIV gag peptide; very low-level plasma viremia (0.84 RNA copies/mL) was detected by an ultrasensitive assay 2 years following infection. Subsequently, she was started on ART for multifocal furunculosis despite continued suppression of virus and stable CD4+ T cell counts. Following ART initiation, HIV specific antibody levels and CD8+ T cell responses increased, but no HIV DNA or RNA was able to be isolated from large numbers of peripheral blood CD4+ T cells.This case provides important information regarding the establishment of elite HIV control during acute infection and also demonstrates an increase in HIV-specific immune responses following ART despite undetectable peripheral blood cellular measures of HIV persistence. This case also highlights the challenges in diagnosing acute HIV infection without the use of viral load testing in this rare elite controller phenotype. Followiet point . Elite cet point –6. EliteWe identified an individual who presented in the early Fiebig stage 1 of acute HIV infection  and subsequent rapid spontaneous decay of plasma viremia leading to diagnostic and therapeutic challenges. Since it is very rare to have longitudinal data and samples available from the time of acute infection through development of control , this ca, and plasma HIV RNA at this time was detectable but below the level of quantification (< 200 copies/ml).The individual continued to follow up for routine clinical care, and plasma HIV RNA became undetectable 4 months following her initial positive test as shown in Table An asymptomatic 42-year old woman presented to our sexual health clinic 1 month following unprotected sexual intercourse with a high risk male partner whom was later presumed to be the source of infection. She denied any other high risk sexual contacts at the time. A HIV Antigen/Antibody (Ag/Ab) test was reported as negative at that time, but she represented 5 months later for a repeat screen at which time a 4th generation Ag/Ab test was reactive. HIV line immunoassay testing was consistent with recent HIV-1 acquisitionSubsequent review of the sample taken at her initial STI screen revealed that the first-line 4th generation Ag/Ab (ARCHITECT® Abbott) screening assay result was at the threshold of positivity. However, this result was not confirmed on two other 4th generation tests. The clinical suspicion was low at the time, as her partner was not known to be high risk, and no further testing was performed. Retrospective HIV RNA testing of this sample, however, revealed a viral load of 71,550 copies/ml  was performed using replicate Aptima viral load Target-Capture Transcription-Mediated-Amplification (TC-TMA) assay on the Panther system (Hologic), which detected 0.84 plasma copies/ml (3 positive replicates of 13 performed). Purified peripheral blood CD4+ T cells were tested from six samples obtained longitudinally between December 2015 and January 2017 (before and after ART initiation) for total cell-associated HIV-1 DNA and unspliced RNA testing using previously described quantitative PCR methods (10 to 20 million input cells per experiment) , 10. ExoFlow cytometry was performed to determine the frequency of markers of CD4+ and CD8+ T cell subset differentiation , activation , immune checkpoint PD-1) and CCR5 expression prior to and following initiation of ART (September 2016 and November 2016). No major changes in surface marker expression were observed before and after ART initiation with the exception of modest increases in the frequency of CD8+ T cells expressing CD69, and decreases in CCR5 expression on both CD4+ and CD8+ T cells , suppress HIV viremia below 50 copies per ml in the absence of ART . HoweverThis case provides important information regarding the establishment of HIV control during acute infection which was characterized by a relatively high initial viral load at the time of initial Ab seroconversion, as would be expected in a majority of acute infections. The peak viremia of 71,550 copies/mL measured in our participant was higher than the peak viral loads observed in the prior two reported cases . Peak viremia was followed by a relatively rapid loss in detectable viral load measurements by commercial assays. The decrease in viral load occurred around the time when non-controllers start to achieve a viral set point as a result of partial immune control of viral replication .This case is also unique in the spontaneous restriction of reservoir seeding by immune control as demonstrated by a lack of nucleic acid detection in tens of millions of peripheral CD4+ T cells, despite exogenous, in vitro activation and quantitative PCR testing of samples using a highly sensitive assay with near single-copy sensitivity across HIV subtypes as previously described , 12. GivIndividuals presenting in the first 2 weeks following infection prior to the development of an antibody response are at risk of their diagnosis being missed unless viral load testing is performed . Followi


Intramuscular fat infiltration is a critical factor in surgical decision-making and is the most important factor used to prognosticate surgical repair outcomes in patients with rotator cuff tears. Quantitative 3D assessment of total rotator cuff fat infiltration in patients with rotator cuff tears has been realized. However, a reproducible method to evaluate 3D spatial distribution of rotator cuff intramuscular fat has not been established. The objective of this study was to establish the reproducibility, change detectable beyond error, and concurrent validity of a semi-automated method to evaluate the 3D spatial distribution of fat infiltration and muscle volume in patients with rotator cuff tears.Thirteen consecutive patients diagnosed with symptomatic rotator cuff pathology and 3.0 T MRI confirmation at a single center were included. Fat-water imaging was used to quantify 3D intramuscular fat (%fat) in sagittal oblique sequences and intramuscular spatial distribution with the semi-automated technique. Each rotator cuff muscle was manually segmented yielding %fat in four axial intramuscular quartile-regions  and three sagittal  regions. Reliability and concurrent validity of %fat and whole muscle volume were calculated with intraclass correlation coefficients (ICC).Intra-rater reliability for intramuscular sagittal divisions (ICC = 0.93–0.99) and axial divisions (ICC = 0.78–0.99) was good/excellent. Inter-rater reliability for %fat (ICC = 0.82–0.99) and volume (ICC = 0.92–0.99) was good/excellent. Concurrent validity with commercialized software showed good/excellent agreement (ICC = 0.66–0.99).A new semi-automated method to assess 3-dimensional intramuscular distribution of fat infiltration in patients with rotator cuff tears using advanced MR imaging demonstrates high intra and inter-rater reliability and good concurrent validity. Minimal detectable change thresholds established facilitate clinical interpretation for future clinical application of this technique to assess change and treatment efficacy in patients with rotator cuff tears. The fat infiltration and atrophy of the rotator cuff muscles are negative predictors of surgical outcomes –3 and, tMuscle atrophy and fat infiltration are clinically evaluated in a sagittal oblique magnetic resonance image in a few slices at the Y-view Fig. .. AtrophAdvanced MRI techniques that allow for greater precision and accuracy towards quantifying intramuscular fat by using chemical shift–based water-fat techniques, such as the Dixon  or IDEALTypically quantitative techniques have assessed %fat in a single slice or several slices near the scapular y-view have demonstrated construct validity with associations to the clinical Goutallier grading of muscle  and the Thus, we seek to expand on the existing 3D imaging methods used to quantify physiological degenerative changes in rotator cuff muscles by first, establishing measurement properties using sagittal oblique slices, and second to report on the feasibility and reliability of a novel semi-automated method to assess the 3D intramuscular fat distribution to allow for detection of inhomogeneous changes. The purpose of this study is to establish the reproducibility, change detectable beyond measurement error (MDC), and concurrent validity to define 3D whole muscle volume and spatial distribution of intramuscular fat% in both the axial and sagittal planes in patients with rotator cuff tears.This study was a single-center study of patients seen by one of two fellowship trained shoulder surgeons between December 2015 and May 2016. Consecutive patients diagnosed with rotator cuff tendon pathology and confirmed with MR imaging at this institution were retrospectively identified and included in the study. This subset of patients was selected to maintain consistency in the scanner used for MR imaging and sequences available for analysis. The surgeon diagnosis of rotator cuff tendon pathology included a positive finding in at least 3 of the following clinical tests: Hawkins Test, Neer Sign, Painful Arc, External Rotation Lag Sign, Hornblower Sign, Pain with External Rotation Resistance or a Jobe Sign. Exclusion criteria included a limitation in passive shoulder range of motion in 3 or more planes consistent with adhesive capsulitis. Patients with moderate to severe glenohumeral joint osteoarthritis, prior rotator cuff repair or other shoulder surgery, and patients with history of a shoulder fracture or deformity from prior fractures were also excluded. A fellowship trained, board certified musculoskeletal radiologist with over 12 years of experience, independently read the MR images blinded to the surgeon diagnosis. Patients determined to have both symptomatic clinical diagnosis and findings on MRI of rotator cuff pathology were included. Rotator cuff tendon pathology was classified by the radiologist as no tear, partial thickness tear, and full-thickness tear small < 1 cm, medium 1–3 cm, large > 3–5 cm, or massive > 5 cm. The radiologist also qualitatively graded fat infiltration using the Goutallier Classification. Using standardized methods,  the sampMagnetic resonance images were acquired according to service institutional standards using a 3-T Siemens  magnetic resonance scanner with a 16-channel phased array shoulder coil. To ensure coverage of the rotator cuff muscles, the sagittal oblique MR imaging sequence with respect to the glenoid fossa was planned on an axial scout scan with a field of view of 18 × 18 cm, which was fit to include the medial border of the scapula. T1-weighted sequences which include fast spin-echo sequences in oblique coronal and sagittal planes, a multiple echo data image continuation (MEDIC) sequence in the axial plane and a T2*-weighted fat suppressed sequences in the axial plane, coronal oblique, and sagittal oblique planes were also performed. Finally, a 3D multi-echo two-point Dixon fat/water imaging sequence was performed in the sagittal oblique plane. This sequence is a chemical-shift imaging application producing water- and fat-only images from dual echo acquisitions with precise accuracy for %fat quantification , 23. TheThe 3D intramuscular %fat and muscle volume were quantified by manual segmentation of rotator cuff muscles on the Dixon fat-water sequences using custom software in Matlab . Two examiners without experience reading MR images were trained in two 30-min sessions by a musculoskeletal radiologist and 2 orthopedic surgeons to identify the boarders of the regions of interest. To establish inter-rater reliability, both examiners independently performed segmentation of the regions of interest on fat-water sequence MR images, blinded to subject identifiers, tear size, and each other’s results. To establish feasibility and intra-rater test-retest reliability, one examiner repeated the segmentation 6-weeks later also blind to subject identifiers; tear size, and prior segmentation results. Lastly, we examined concurrent validity of %fat and muscle volumes with manual segmentation of the regions of interest using commercially available software  with the same fat-water imaging processing parameters. This system has been used to assess intramuscular fat –15 and pAll segmentation was performed manually inside the fascial borders the defined the regions of interest in supraspinatus (SS), combined infraspinatus/teres minor (IS), subscapularis (SC) muscles in each oblique sagittal image Fig. .. The MAFat Fraction = Fat/(Fat + Water) × 100.thirds  whereby average %fat and volume of each intra-muscular region were calculated.Sequential images from the most lateral image at the level of the glenoid to the most medial image of the medial scapular boarder were processed. Thus, the number of images used in the oblique sagittal view varied by subject, due to individual subject size differences needed to capture the scapula and corresponding rotator cuff muscles. The average of all consecutive images was used to establish the 3D total muscle %fat of each muscle. The 3D volume of each muscle was calculated by using the sum of the area calculated in each slice taking into account slice thickness from the glenoid to the medial boarder of the scapula. In addition to 3D whole muscle measures, three sagittal intra-muscular tertiles  were created by dividing each muscle from the glenoid to the medial boarder of the scapula into equal The relative intra and inter-rater reliability of 3D %fat and volume of the entire muscle and intramuscular divisions were determined by calculating the Intraclass Correlation Coefficient (ICC)  and 95% Intra and inter-rater reliability was assessed in MR imaging of 13 consecutive patients  who met the inclusion criteria. These patients had a mean age of 51.0 ± 16.5 (±SD) years with 7 patients with partial-thickness tears, 2 patients with small full-thickness tears, and 4 patients with medium -sized full-thickness tears. Goutallier grade 2 fat infiltration was identified in the supraspinatus in one patient, and grade 1 fat infiltration in 4 patients. The infraspinatus showed grade 1 fat infiltration in 4 patients, and the teres minor showed grade 1 fat infiltration in one patient. The mean chronicity of patient’s symptoms was 38.5 ± 78.0 months. The intra-rater absolute reliability of spatial distribution methods evaluating 3D fat infiltration and 3D muscle volume of the rotator cuff muscles was considered very good to excellent from medial to lateral tertiles  of 0.60 for the supraspinatus, 0.65 of the infraspinatus using 4 consecutive lateral slices adjacent to the sagittal oblique “scapular y-view” were reported . AnotherTo adequately evaluate temporal changes of fat and atrophy in rotator cuff muscles in patients with tendon pathology, 3D quantification could appreciate early inhomogeneous degeneration following a rotator cuff tear before irreversible change occurs. Matsumura et al., recently demonstrated excellent reliability with 3D %fat and volume using a standardized protocol  in axial slices . Our res3 volume). The intra-rater MDC was less than 2% for each rotator cuff muscle and less than 2.2% for the intramuscular spatial distribution measures of %fat within the lateral, intermediate, and medial regions of each muscle using our method . However, our results are consistent with the precision previously established, and the time intensive process of manually segmenting each muscle in its entirety limits further efforts for this purpose. Furthermore, we did not include patients with large and massive rotator cuff tears as early detection of temporal change in muscle degeneration is most imperative in patients with less severe rotator cuff disease, such as partial thickness and small-medium full-thickness tears. In these tears, operative treatment is advocated before potentially irreversible changes in the rotator cuff muscle occurs. Thus the population we studied were those who may potentially benefit the most from assessment of temporal physiological muscle changes pre-operatively. Additionally, we combined and segmented the infraspinatus and teres minor together based on previous literature [There are several limitations to the study. Overall, the generalizability may be limited as the results were obtained using a single scanner and set of imaging parameters. Additionally, we only examined the intra-rater reliability in one rater and both raters did not have MR image reading experience. While the study exceeded our sample size estimate to provide > 80% power, sample was small (terature  and the 3D MR imaging techniques including, the multi-echo Dixon fat-water sequences allow for reliable and intra- and inter-rater quantification of the intramuscular spatial distribution of fat infiltration in patients with rotator cuff pathology. It is suggested that both atrophy and fat infiltration should be evaluated separately, given these are two different physiological processes , 35. To


Candida albicans, four major classes of repeats exist in the genome: telomeres, subtelomeres, the major repeat sequence (MRS), and the ribosomal DNA (rDNA) locus. Characterization of these loci has revealed how their structure contributes to recombination and either promotes or restricts sequence evolution. The mechanisms of recombination that give rise to genome instability are known for some of these regions, whereas others are generally unexplored. More recent work has revealed additional repetitive elements, including expanded gene families and centromeric repeats that facilitate recombination and genetic innovation. Together, the repeats facilitate C. albicans evolution through construction of novel genotypes that underlie C. albicans adaptive potential and promote persistence across its human host.Genome instability often leads to cell death but can also give rise to innovative genotypic and phenotypic variation through mutation and structural rearrangements. Repetitive sequences and chromatin architecture in particular are critical modulators of recombination and mutability. In  Considerable variability in genome organization exists across the tree of life, ranging from single circular chromosomes in bacterial species to thousands of linear chromosomes in some ferns . These cRepetitive regions of the genome often serve as hotspots of genome rearrangements and evolutionary innovation. These elements can exist as single or multi-copy loci encoding tandemly-repeated identical or near-identical sequences of variable length ranging from one to thousands of nucleotides. By their very nature, repetitive loci are prone to recombination, producing insertion/deletions (indels) and translocations between both adjacent and distal repeats. Recombination within repeats alters copy number variants in addition to promoting de novo mutations due to errors in DNA exchange and repair . In contCandida albicans possesses many of these same hallmark attributes. The diploid C. albicans genome is comprised of eight chromosomes numbered 1 through 7 by size with the exception of chromosome R (ChrR), which encodes the rDNA locus . Th. ThC. alequences .C. albicans subtelomeres. These elements, the Bermuda Triangle sequence (BTS) and the TLO recombination element (TRE), lie immediately centromeric to TLO genes (TLO recombination element (TRE). The TRE stretches ~300 bp, beginning immediately following the TLO coding sequence and extending towards the centromere [tui-class family of LTR sequences which are annotated in some subtelomeres but missing in others . LOH events result from non-reciprocal crossing over or chromosome loss and reduplication of the lost region from the remaining chromosome homolog. Rates of LOH increase along all chromosomes towards the telomeres of C. albicans isolates, indicating a general relationship between the distance from the centromere and allelic homozygosis mediated by recombination [C. albicans subtelomeres where it suppresses mitotic recombination additively with Sir2 silencing. However, some stressors, including fluconazole, promote such a strong genome instability phenotype that the protective effects of Sir2 can be masked in their presence [bination  . Its conspicuous absence in select subtelomeres is thought to be due to incomplete assembly in the current genome assembly and not the absence of γH2A [Phosphorylation of serine 129 on histone H2A, commonly known as γH2A, is a common marker for heterochromatin in revisiae . Consist of γH2A . These γ of γH2A .TLO genes and all telomere-proximal sequences through non-reciprocal recombination events among different chromosome arms [TLO genes and produced novel TLO sequences, highlighting the potential for significant sequence diversity to arise via subtelomeric recombination that can contribute to phenotypic plasticity [Subtelomeric recombination directly affects their genic repertoire. Continuous passaging of SC5314-derived strains for 30 months led to the gain and loss of specific ome arms . These nasticity . TLOs can be separated into three clades  based on sequence diversity that primarily clusters in the 3’ end of the coding sequence. The TLOβ-clade is unique in containing two major indel events in this 3’ region that distinguish it from the other TLO clades. The 3’ end of TLOγ-clade genes uniformly contains a rho LTR sequence that likely disrupted an ancestral TLOα-clade homolog which then expanded across the subtelomeres through non-reciprocal exchange of this newly-formed TLOγ-clade gene [psi LTR-containing retrotransposon insertion disrupted TLOγ4 in the SC5314 background, producing a truncated TLOγ4 gene, which is actively transcribed, and a TLOψ4 pseudogene with no detectable transcript [Genes within the subtelomeres are subject to not only increased rates of recombination, but also increased formation of indels . TLOs caade gene . Furtheranscript ,72.C. albicans chromosomes, the exception being Chr3, contain large tracts of repetitive DNA known as the MRS. These regions are comprised of large, variable arrays of the 2 kb repetitive sequence (RPS) unit that are often flanked by the non-repetitive RB2 (6 kb) and HOK (8 kb) elements [C. albicans possesses nine complete MRS loci, 14 RB2 elements, and two HOK elements , but their contributions to genetic and phenotypic diversity still remain generally unresolved. Furthermore, the importance of less well-understood repetitive sequences to genome evolution and karyotypic innovation are just beginning to emerge. What is clear is that repetitive elements within the C. albicans genome produce novel genotypes through changes in DNA sequences or karyotypes that often prove to be beneficial. Some of these changes, such as isochromosome formation during antifungal drug exposure and changes to gene family copy number, are more intuitive, whereas the function of others involving changes in MRS repeat length and rDNA copy number are less easy to grasp. It could be that repeat length modulates the probability of recombination occurring or that the presence of repeats is itself sufficient to promote genome instability. Association of additional repetitive sequences at major regions within the genome  suggest that these sites are likely under relaxed selection for new insertions that increase the likelihood of additional recombination events. In contrast, repeats scattered throughout the genome beyond these defined regions may function as back-up sites for genome instability. These cryptic repetitive sequences throughout the C. albicans genome may not only promote recombination but reduce the associated fitness costs of novel genotypes by increasing the resolution of affected sequences to only the locus (loci) that is under selection. Regardless, these repetitive sequences collectively construct a dynamic environment of common and infrequent sites of genomic instability to generate genotypic and phenotypic variants that allow C. albicans adaptation to its diverse niches across the human host and ultimately promote its success as a commensal and pathogen. Thus, repetitive sequences in the


Alteration of normal ploidy (aneuploidy) can have a number of opposing effects, such as unbalancing protein abundances and inhibiting cell growth but also accelerating genetic diversification and rapid adaptation. The interplay of these detrimental and beneficial effects remains puzzling. Here, to understand how cells develop tolerance to aneuploidy, we subject disomic (i.e. with an extra chromosome copy) strains of yeast to long-term experimental evolution under strong selection, by forcing disomy maintenance and daily population dilution. We characterize mutations, karyotype alterations and gene expression changes, and dissect the associated molecular strategies. Cells with different extra chromosomes accumulated mutations at distinct rates and displayed diverse adaptive events. They tended to evolve towards normal ploidy through chromosomal DNA loss and gene expression changes. We identify genes with recurrent mutations and altered expression in multiple lines, revealing a variant that improves growth under genotoxic stresses. These findings support rapid evolvability of disomic strains that can be used to characterize fitness effects of mutations under different stress conditions. Aneuploidy  can enable rapid adaptation to stress conditions, but it also entails fitness costs from gene imbalance. Here, the authors experimentally evolve yeast while forcing maintenance of aneuploidy to identify the mechanisms that promote tolerance of aneuploidy. The extra chromosomes delay cellular growth, interfere with embryonic development, increase genomic instability, and promote aging5. Impaired proliferation is a common feature of any type of karyotype alteration, including aneuploidy6. Having an extra chromosome has been shown to extend the G1 phase and decrease the growth rate in yeast6, partly due to gene expression imbalance6. However, studies in aneuploid yeast strains have shown that the slow growth rate of the initial clonal population can be restored within as few as 20 generations of adaptive evolution. The growth restoration was ascribed to a handful of mutations providing tolerance to aneuploidy7.Whole chromosome gains and losses lead to unbalanced karyotype, a phenomenon known as aneuploidy. Aneuploidy is frequently observed in eukaryotes and has been shown to affect cellular physiology in organisms from yeast to human8, suggesting that aneuploidy can be advantageous for adaptation, if the initial challenge of gene imbalance can be overcome. Indeed, several studies indicated that aneuploidy allows cell populations to rapidly explore adaptive mechanisms by increasing the frequency of beneficial mutations and changing the mean fitness of the population11. Rapid aneuploidy-driven adaptation was observed for diverse genetic and environmental perturbations  yeast strains were shown to feature increased genomic instability, assessed by fluctuation assays using genetic markers, such as uracil (URA3) and canavanine (CAN1)Saccharomyces23, we found that the lethal effect of chromosome III, IV, VI, and VII duplications is specific to our strain background (W303), since natural occurrences of whole or partial copy number alterations could be found for each of them among 386 aneuploid WT isolates  control cells   and kanamycin (KANMX) selection to maintain disomy (each copy of the duplicated chromosome contained one selection marker). We then measured doubling time of ancestral and evolved disomes and WT cells to estimate growth rate. Consistent with previous reports20, in our hands all disomic lines improved growth rate after adaptive evolution , so that all the ancestor strains formed red-colored colonies when plated on solid selective medium. The red pigment derived from the polymerization of ribosylaminoimidazole (AIR)27 in aerobically growing cells, which causes DNA cleavage27 and leads to growth defects26. It was reported that, in these conditions, spontaneous suppressor mutations may occur in genes upstream in the adenine pathway (ADE4 through ADE8) to block AIR formation, and/or in genes involved in oxidative metabolism to prevent oxidation of AIR, resulting in white colored mutants that grow faster than their red relatives27. Indeed, at the end of our long-term adaptive growth, all three evolved WT lines formed cream-colored smooth colonies  acquired partial or whole chromosome losses and/or large segmental amplifications  and their respective ancestors. We then characterized the karyotype changes in every line, by analyzing genome-wide read coverage . This may have facilitated unequal homologous recombination of selection markers, resulting in their co-occurrence on the same chromosome V molecule, and thus allowing subsequent restoration of normal ploidy through the loss of the other copy . However, we observed many genomic reads mapped to ChrV:3169-33207. TAnother karyotype change was observed in disomes D12 and D14. Here, only a portion of the original duplicated chromosome remained, along with newly emerged segmental duplications of chromosome III and chromosome XII, respectively Fig. . Notablyp = 0.41). We computed the “apparent mutation rate” of each evolved line by dividing the number of observed de novo mutations by genome size and by the number of generations. This rate differs from the true spontaneous mutation rate, estimated in mutation-accumulation experiments with frequent bottlenecks30, in that here selective forces can alter the dynamics of variant fixation. WT cells accumulated 1.67 substitutions and indels per evolved strain, implying a rate of fixed mutations, 1.1 × 10−10 per nucleotide per generation, slightly lower than the previously reported spontaneous mutation rate of ~3 × 10−1030. The apparent mutation rate in disomic strains was higher than in WT, averaging 8.4 × 10−10 and ranging from 4.1 × 10−10 (disome D9) to 16.5 × 10−10 (disome D10)  and non-synonymous (average 3.5 × 10−10 vs 1.3 × 10−10) substitutions. Although hitchhiking in selective sweeps may also be inflating the apparent rate, our observations are consistent with the disomic strains having increased spontaneous mutation rates, as shown in other studies31. We observed bias in the localization of mutations towards noncoding regions (Table 32). The effect was statistically significant for both substitutions and indels, and was stronger for indels . These observations are consistent with purifying selection acting on coding sequences. Notably, we found that mutations in disomic lines were preferentially located on the duplicated chromosomes . The significance of this pattern is discussed later.In addition to karyotype changes, we analyzed de novo mutations accumulated over the course of laboratory evolution in both disomic and WT lines. To do this, we compared the genomes of evolved lines to their corresponding ancestral strains and focused on the variants absent in the ancestral genome. This analysis revealed 253 de novo nucleotide substitutions and 120 small insertions/deletions (indels) across the 32 evolved disomic lines (Table 10) Fig. . The hig34. However, C:G → A:T transversion was the most prominent transversion mutation 35, rather than spontaneous deamination of methylated cytosines, is the major driver of mutations in disomic strains, consistent with the observation that many disomic strains are characterized by increased levels of reactive oxygen species20. Moreover, we examined variants across the entire genome to identify potential positional effects and to determine if any of these disomic cells were affected by DNA replication stress. At the global level , there was no association between the position of mutations and the known replication timing of genomic regions. This observation suggests that none of these chromosome duplications altered DNA replication timing at the onset of S phase, which is also consistent with disomic strains having extended G1 phase but not S phase 36. Interestingly, the disomic yeast mutations resembled COSMIC signature 5  to block AIR formation, consistent with the color changes in the evolved lines . For this, we analyzed mitochondrial DNA (mtDNA) copy number and performed growth assays in the medium containing glycerol as a carbon source. We found that many evolved lines increased mtDNA quantity compared to their ancestral strains , known as nonsense suppressors40. These genes normally encode for tyrosine tRNAs, and specific mutations in the wobble position of its anticodon allow them to read stop codons and suppress nonsense mutations41. Indeed, we found that all evolved white colonies without compensatory mutations in the adenine pathway carried the same C to A mutation in the wobble position of tyrosine tRNA  that replaced Lys352 with Asn in Apn1 (K352N variant). Since it is highly improbable that the same mutation occurred de novo in all evolved lines, we hypothesized that the variant was already present in the ancestral strain population and was later driven to fixation by positive selection during adaptive growth. Indeed, we confirmed through Sanger sequencing the existence of this variant at low frequency in the ancestral disome population , and under conditions of oxidative DNA damage. First, we introduced constructs harboring mutated (K352N) and non-mutated APN1 in APN1-knockout (ΔAPN1) euploid cells and tested their ability to grow on the MMS-containing medium. We observed that the mutated APN1 conferred improved viability against MMS genotoxicity  and non-mutated APN1 in disomic and WT cells and tested for their ability to grow on the MMS-containing medium. We observed that the mutated APN1 improved viability for all disomic lines except D11, D15 and D16 ; a protein kinase that is part of the growth control pathway (SCH9); essential components of the RSC chromatin remodeling complex and condensin complex proteins (RSC8 and SMC4); a putative protein of unknown function with similarity to sialidase (YMR317W); and a mitochondrial protein with unknown function (FMP27). Although mapping to the same genes, these mutations were at different loci in different disomic strains, indicating that they emerged independently in the various lines.We further observed that some genes accumulated distinct coding sequence mutations in multiple evolved disomic strains (Supplementary Data ERT1); an essential nuclear protein required for maturation of 25S and 5.8S rRNAs (MAK16); a protein involved in ribosome biogenesis (SDA1); a histone-fold protein (MHF2); and, once again, the SCH9 kinase. Among them, the SCH9 gene presented both promoter mutations in evolved D2 and D15 lines, and coding region mutations in evolved D10 and D13 lines. SCH9 is the ortholog of mammalian S6 kinase (required for TOR1-mediated regulation of ribosome biogenesis and translation) and it was previously found to stabilize the tetraploid phenotype in yeast cells46. We hypothesized that SCH9 acted in the translational regulation of disomic strains to decrease the proteotoxic effects of duplicated chromosomes, and decided to test the effect of mutations observed in evolved disomes on proliferation. We focused on coding sequence mutations in SCH9, W377C and F760L, which were found in D10 line 3 and D13 line 1, respectively. We employed fragments harboring either non-mutated or mutated SCH9 to replace the endogenous copy in the genome of disomic strains and monitored their growth. We could assay 9 disomic strains, in which we successfully replaced the endogenous copy with the mutated SCH9. While expression of both mutant versions of SCH9 caused a significant growth delay in WT cells, the response of disomic lines was diverse ; or it may effectively be a neutral variant, located on SCH9 simply by chance (which we deem highly unlikely).We also considered mutations in intergenic regions, which we assigned to gene promoters based on their distances to transcription start sites (see “Methods”)  and histone-fold (MHF2) proteins may affect chromatin condensation and, ultimately, gene expression regulation in disomic strains. Our results support the idea that mutations recurrently fixed in the same genes represent adaptive responses in transcriptional and translational regulation to gene imbalance caused by aneuploidy and point to specific pathways as potential drivers of genetic adaptation to aneuploidy stress. Our study also shows that aneuploid yeast can be used for future studies to capture adaptive mutations under different stress conditions, since rapid evolvability drives adaptation with strong positive selection in these strains.In addition to 20. We further compared the transcriptional profiles of evolved lines with their corresponding ancestral strains, reflecting the changes that occurred during evolution of aneuploid strains. Even after adaptive evolution, many evolved lines maintained a pattern of chromosome-specific elevated gene expression . These genes segregated into functional categories , and 189 genes were downregulated and enriched for the amino acid synthetic pathway (adjusted p = 1.57 × 10−18), endonuclease activity (adjusted p = 4.85 × 10−30) and endopeptidase activity (adjusted p = 3.83 × 10−19) . We then compared ancestor disomic lines with the WT line to outline the effect of aneuploidy on transcription. As expected, each ancestor disomic strain showed a dramatic increase in the expression of genes on the duplicated chromosome, with a clear karyotype-specific pattern of gene expression Fig. . The norion Fig. 7,20. We p = 1.49e-5); the remaining 257 negatively correlated with the shift towards WT, and were enriched for amino acid biosynthesis (adjusted p = 1.79e-20), endonuclease activity (adjusted p = 9.48e-24) and endopeptidase activity (adjusted p = 1.29e-25)  . We used this metric to compare evolved disomic strains with WT, normalizing by the initial distance of ancestor disomes with WT. We observed that the great majority of evolved strains  decreased their distance to WT compared with their ancestors  Fig. . We notiLEU3 (adjusted p = 7.9e-03), involved in branched chain amino acid biosynthesis; MOT3 (adjusted p = 9.9e-04), regulates wide range of genes including glucose transporters; GCR1 (adjusted p = 4.3e-09), regulating the expression of glycolytic and ribosomal genes; XBP1 (adjusted p = 4.0e-09), regulating the expression of stress or starvation induced genes; and HSF1 (adjusted p = 5.3e-04), activating multiple genes in response to stresses that include hyperthermia. Many of the shift-associated genes possessed motifs for more than one of these TFs  and XBP1  . Strikingly, our analysis revealed that deletion of LEU3 significantly increased growth rate in six out of seven disomic strains, while XBP1 or MOT3 deletion had no significant effect  may be relevant to mitigate the burden of aneuploidy on gene expression. Still, further investigation is required to address their direct role, and their mechanism of altered regulation. Altogether, our gene expression analysis revealed common regulatory mechanisms, including changes in protein translation, amino acid biosynthesis, and stress response, as potential key factors in the adaptation to aneuploidy.Finally, we searched for transcription factor (TF) binding motifs enriched in the promoter sequences of the genes associated with expression shift, aiming to identify TFs involved in the response to aneuploidy. Our analysis revealed significant enrichment of 5 TFs: In this study, we subjected lines of disomic yeast to long-term adaptive evolution to reveal mechanisms of tolerance to various chromosomal aneuploidies. In our evolution experiment, we chose a strategy of progressive dilution, rather than single colony bottleneck, to maximize the cumulative supply of beneficial mutations. Using selective markers to force the maintenance of aneuploidy, we were able to investigate the patterns of fixed mutations, karyotype alterations, and gene expression changes. This experimental strategy allowed us to extensively characterize responses to aneuploidy, uncovering several associated molecular mechanisms. Our analyses recapitulated well-known characteristics of aneuploidy and provided many new insights. Disomic cells featured increased genomic instability, i.e., evolved disomic lines sustained diverse karyotype alterations, and concomitantly presented an apparent mutation rate on average 10-fold higher than that of WT cells. The gene expression profiles of aneuploid cells showed marked differences with normal cells: genes on the duplicated chromosomes increased their expression levels 1.8-fold on average. The doubling time of ancestor disomic lines was clearly deteriorated compared to euploid cells.However, adaptive evolution over ~1,200 generations partially restored the growth rate, sometimes approaching that of control cells Fig. . We thenOur analyses identified diverse mechanisms related to aneuploidy response. The most evident events involved large chromosomal rearrangements. Some strains (lines 2 and 3 of D5) managed to practically bypass our selection scheme to force continuous disomy, and reverted to the euploid status. Facilitated by the proximity of highly repetitive sequences, unequal homologous recombination brought the selection markers to the same chromosome molecule, subsequently allowing the loss of the extra copy. Similar events allowed other disomic lines to lose large portions of duplicated chromosomes, while retaining selection makers , in some cases concomitantly with the formation of novel small chromosome bodies .p-values two-sided t-tests > 0.6). Based on this, we favor relaxed purifying selection as most likely explanation for the biased localization of mutations on disomic chromosomes.Disomic lines accumulated a number of mutations in coding and non-coding sequences during evolution. Notably, we found that these variants were preferentially located on the chromosome with an extra copy. This observation may be explained by either relaxed purifying selection on genes with a redundant copy, or by increased positive selection for variants compensating the imbalance in gene dosage. In the latter scenario, we would expect mutations to be fixed preferentially in the genes with the most extreme expression imbalance in disomic strains. Yet, mutated genes are indistinguishable from non-mutated in their distribution of expression fold change, both when we compare ancestral disomes with WT, or evolved lines with WT . This kinase is involved in growth control through regulation of ribosome biogenesis and translation and has been previously implicated in the stabilization of tetraploidy46. We experimentally investigated two coding mutations in SCH9 53. A previous study reported that, even though disomic strains suffer from proteotoxic stress, they did not activate the UPR49. In contrast, our results suggest that the UPR is indeed activated in disomic lines during adaptive growth. HSF1 is also linked to the UPR: it was shown to regulate HSP90-mediated protein folding, and increased levels of HFS1 counteract the adverse effects of aneuploidy54. Furthermore, the activity of HSF1 is known to be compromised in aneuploidy cells. Our discovery of XBP1 and HSF1 motifs in commonly regulated genes points to a key role of the UPR during adaptation to aneuploidy, presumably to handle proteotoxic stress. On the other hand, GCR1 and MOT3 are known to regulate glycolytic gene expression and glucose transporters. Several adaptive mutations in MOT3 were shown to increase fitness of aneuploid cells55. The presence of binding motifs for GCR1, MOT3, as well as LEU3 (which activates branched chain amino acid synthesis) points to potential strategies for metabolic adaptation.58. Notably, the same also applies to the evolution of cancers in humans, which are under constant stress from the immune system, scarce oxygen and ATP availability. Aneuploidy is believed to play a key role in enhancing stress-mediated adaptation and survival ability of cancer cells59. The disomic yeast strains we used in this work are haploid and sensitive to chromosome copy number alterations, so that it is unclear whether the mechanisms of aneuploidy tolerance highlighted here are relevant for other aneuploid genomes including cancer cells, which mostly start as diploid. In this regard, future research in near-haploid cancer cell lines61 may be informative.Our study provides insights into how cells adapt to tolerate chromosomal imbalance, allowing to employ the characteristic genomic instability of aneuploidy for rapid adaptive stress responses, while attenuating its detrimental effects due to impaired gene dosage. Dissecting these mechanisms is essential for understanding the rapid adaptation of unicellular organisms to stress environments, which is often characterized by aneuploid states18. All experiments were performed in Yeast Nitrogen Based synthetic media supplemented with all amino acid except histidine (YNB-His) and supplied with 200 µg/ml Kanamycin (G418). This setup forces the maintenance of disomy, since each copy of the disomic chromosome harbors a selection cassette (HIS3 and KANMX). The parental WT W303 strain was also grown under these conditions, as selection marker cassettes were integrated into its genome18. The laboratory evolution experiment was carried out by serial dilution. Single colonies of each disomic and WT cells were grown overnight at 30 °C with moderate shaking and then diluted by a factor of 1:500 with fresh YNB-His medium into three individual cultures to generate evolved lines for each strain. These lines were diluted daily by a factor of 1:500 (∼9 generations per dilution for WT cells) for 134 days. This procedure was repeated for a course of an estimated 1200 generations for WT, and aneuploid lines were grown for the same time duration. Due to their slower growth rate, this corresponded to fewer than 1200 generations, so that mutation rates are slightly underestimated for aneuploid lines. At the end of experimental evolution, whole populations were used to isolate DNA and RNA for genome and transcriptome sequencing.The W303 derivatives of yeast disomic strains were reported previously600 measurements were taken at 15 min intervals using a Biotek-Epoch2 instrument until cells reached early stationary phase. Doubling time was calculated with R script by analyzing fitting spline function from growth curve slopes62. The maximum slope of the spline fit was used as an estimate for the growth rate and doubling time for each evolved line.Cells were grown at 30 °C, and ODAPN1 gene was amplified with PCR from S288c genome and cloned into pBluescript SK (+) plasmid at the SpeI-XhoI restriction sites. This construct was used to introduce K352N mutation by PCR, and mutation was verified by Sanger sequencing. Both mutated and non-mutated APN1 was cut from pBluescript SK (+) and ligated into low copy p413ADH vector containing the HIS3 and hygromycin (HYG) selection markers, with APN1 expression controlled by constitutive ADH promoter. Both ΔAPN1 cells from yeast knockout library and Δ7 cells were transformed with plasmids to express mutated APN1, non-mutated APN1 or an empty vector as control. Clones were selected on YNB -HIS, +HYG plates and grown overnight in liquid YNB -HIS, +HYG medium. On the next day, the OD was adjusted to 0.5, and 10-fold dilutions were spotted on YNB plates to test for resistance of ΔAPN1 cells to various concentration of MMS. Viability of Δ7 cells was also tested with both spot assay and growth measurement. Plates were incubated for 4 days at 30 °C and pictured. A region encompassing 1 kb upstream and 250 bp downstream of the SCH9 gene was amplified with PCR from S288c genome and cloned into p42Nat 1-F GW (https://www.addgene.org/58549/) plasmid, which harbors natMX6 cassette for Natamycin resistance in yeast. This construct was used to introduce W377C and F760L mutations individually. Both mutated and non-mutated SCH9 were amplified along with natMX6 cassette, and in total 6 kb PCR fragments were used for replacement of the endogenous SCH9 locus in WT and disomic strains. Colonies were selected on YNB -HIS, +NAT plates after 4 days and integration of PCR fragment was verified by PCR and Sanger sequencing. Individual colonies were grown overnight from each strain to test the effect of introduced mutation on proliferation next day.The Genomic DNA libraries were prepared using NextEra genomic DNA kit according to the manufacturer’s manual. Sequencing of libraries were performed on the Illumina Hi-Seq platform with 75 bp paired-end read option. PFGE was conducted using a BioRad Contour-clamped homogeneous electric field (CHEF) Mapper XA system and genomic samples were prepared using the CHEF genomic DNA plug kit. Agarose-embedded chromosomal DNA preparation and running conditions were performed according to the kit manual.63 and the recently published high-quality genome for the W303 laboratory strain (GenBank: LYZE00000000.1) was used as a reference64. We used the Genome Analysis Toolkit (GATK)65 to calculate the depth of coverage for each chromosome. The “DepthOfCoverage” tool was applied to sorted *.bam files to generate a genome-wide table of sequencing depth for karyotype analysis. The average depth of coverage was 50x per strain; however, single evolved lines of disomes D10 and D13 were covered below 10x and were eliminated from further analyses. In addition, single lines of D14 and D16 were also eliminated due to sample contamination during growth. We normalized coverage by the total number of reads per sample. The genomic read densities were plotted with the ggplot2 package within the R statistical environment (https://www.r-project.org/). In addition, the ShortRead package66 was used to identify chromosome breakpoints, which were then verified by manually visualizing bam files on Tablet software. Mutations were analyzed with both GATK65 and Free Bayes67. Mutations identified by both software packages were considered for further analysis, and some of the mutations were verified by analyzing bam files with the Tablet software as well68. Mutations on duplicated chromosomes were analyzed with GATK -L option with at least 5 paired reads covering the corresponding mutation loci. Mutations were called if ≥40 and 80% reads mapped to the mutation on the duplicated and non-duplicated chromosomes, respectively, and mutations commonly identified in every ancestral strain and the corresponding evolved lines were eliminated, since these mutations accumulated in the ancestral strain prior to the evolution experiment.Genomic reads were mapped using Bowtie 2 v2.1.069. RNA-seq (FASTQ) files were mapped to the yeast reference genome; Ensembl build R64-1-1 was downloaded from the Illumina iGenome resource at support.illumina.com/sequencing/sequencing_software/igenome.html. Mapping was performed using Bowtie 2 v2.1.053 and read counts per gene were calculated using Feature Counts70. To filter out genes with the low number of reads, only the genes with at least 1 count per million (cpm) in at least half of the strains were retained, resulting in an expression set of 6059 genes across 32 evolved lines . Filtered data was then subjected to RLE normalization71. Differential gene expression analysis was performed using the R package EdgeR72, with FDR of 10% to increase the statistical power of subsequent functional enrichment analyses. When searching for genes with common expression changes across all evolved strains, a single statistical model with factorial variable representing shift from the ancestor to evolved strains was used. When searching for genes associated with a transcriptional shift towards WT, a single statistical model with numeric variable equal to the expression distance from the ancestor strain towards WT (see below) for each evolved strain was used. FDR threshold was set to 0.173.RNA-seq libraries were prepared with using Cell-Seq2 methodu/(n*g), where u is the estimated rate, n is the number of bases in the genome, and g is the number of generations74. The distribution and relationship with replication timing of mutations were analyzed according to a previously described method75.We compared the variants predicted in each evolved disomic line and WT cells to the corresponding ancestral strains. The common mutations identified in both ancestral and evolved lines were eliminated to identify unique de novo mutations in the evolved strains. Apparent mutation rate was calculated by dividing the number of detected de novo mutations by the total number of bases in the reference genome sequence and by the number of generations that each strain was propagated. Mutation rates for the different mutation types were calculated by further normalizing mutations from AT base pairs by the AT fraction of the genome and mutations from GC base pairs by the GC fraction of the genome. Standard errors for apparent mutation rates were calculated as the square root of http://snpeff.sourceforge.net/). Intergenic (non-coding) mutations were attributed to the promoters of their closest gene if their transcription start sites were located within 750 bp with a custom Python script  analysis was performed using gprofiler tool76. TF binding motif search and related TF network were generated using yeastract tools77.Mutations on protein coding genes were annotated as either missense, nonsense or silent using SnpEff  as the Euclidean distance of their expression levels in log space . To focus on changes relevant for aneuploidy, at each comparison we considered only those genes that were altered in the ancestral disomes, using a threshold of absolute fold change ≥ 1.5 . We then normalized the distance between WT and each evolved disomic line by the distance between WT and their corresponding ancestor disome. This resulted in the “normalized distance to WT” values shown in Supplementary Fig. 10a. For practical purposes, we then reformulated this as “normalized shift towards WT” . We used a two-sided binomial test to assess the significance of the differential distribution of mutations (substitutions and indels) in coding and non-coding regions. The number of mutations in target regions was considered as the number of successes, whereas the total number of mutations was considered as the number of trials. The probability of success was set to the proportion of the target sequences in the genome (74% for coding regions). We used the same approach to test if the fraction of mutations were preferentially located on the duplicated chromosome. Probabilities under null hypothesis were calculated based on the ratio of the corresponding chromosome length to the sum of lengths of all chromosomes. Obtained p-values were adjusted for multiple comparisons by the Benjamini-Hochberg procedure.All statistical analyses were performed in the R statistical environment (Further information on research design is available in the Supplementary InformationReporting SummaryDescription of Additional Supplementary FilesSupplementary Data 1Supplementary Data 2Supplementary Data 3Supplementary Data 4Supplementary Data 5Supplementary Data 6