Cells encounter countless external cues and the specificity of their responses is translated through a myriad of tightly regulated intracellular signals. For this, Rho GTPases play a central role and transduce signals that contribute to fundamental cell dynamic and survival events. Here, we review our knowledge on how zebrafish helped us understand the role of some of these proteins in a multitude of in vivo cellular behaviors. Zebrafish studies offer a unique opportunity to explore the role and more specifically the spatial and temporal dynamic of Rho GTPases activities within a complex environment at a level of details unachievable in any other vertebrate organism. Rho GTPases, a subgroup of the Ras superfamily, are key molecular switches that transduce signals from the cell surface to major intracellular signaling pathways. Their first members were discovered in 1985 and since then they became the center of intensive studies, showing their implication in a wide range of fundamental cellular processes. This includes cytoskeletal dynamics and re-arrangements, cell migration and polarity, membrane trafficking, NADPH (nicotinamide adenine dinucleotide phosphate) oxidase activation and transcription. Most Rho GTPases alternate between an inactive state when bound to GDP and an active conformation when bound to GTP. The GDP-GTP cycle is mediated by GEFs (guanine nucleotide exchange factors) while GTP hydrolysis is promoted by GAPs (GTPase activating proteins). Once activated, they interact with their downstream effectors to induce diverse cellular responses .Danio rerio has emerged as a very powerful model for in vivo developmental studies. Owing to their transparency and external development, zebrafish embryos are amenable to signaling pathways studies through mRNA or DNA injection, pharmacological treatment and live imaging. Several studies, that we will detail further below, have now shown essential roles for small GTPases in embryonic development and wonderful tools have been applied to zebrafish to unravel further details regarding the temporal and subcellular activity of these Rho GTPases in vivo.Most of our knowledge regarding the dynamic activity of Rho GTPases in vertebrates derive mainly from cell culture and in vitro methods, limiting insight into how Rho GTPases function in vivo. Zebrafish or The role of Rho GTPases in development is well described in mammals and it is relatively recently that zebrafish became a chief model to study the mechanisms of Rho GTPases function in vertebrates in vivo. Thirty-two Rho genes have been identified in zebrafish that represent homology to 17 human genes , howeverAs introduced above, Rac1 is one of the most studied small Rho GTPases and a plethora of data has revealed an essential role for zebrafish Rac1 in cell cytoskeletal rearrangement and motility in different cell types including mesodermal cells, endothelial cells and neurons. In this way, Rac1 controls fundamental morphogenetic processes during development that rely on substantial cell movement and cellular reorganization.During the early development in fish and amphibians, the embryonic body is shaped through gastrulation. This involves extensive cell movements including epiboly, cell internalization and convergence-extension (CE). The first elegant demonstration of a role for Rac1 in the dorsal migration of lateral cells during zebrafish gastrulation was described by Hammerschmidt laboratory . Cell trOther studies highlighted a role for Rac1 in the extension of the embryonic dorsal axis and migration of the presomitic mesoderm in zebrafish downstream of p120 Catenin . FurtherTg(sox17:GFP-UTRN) that labels actin-based structures in endodermal cells in order to monitor their behavior. Furthermore, the authors were able to measure Rac1 activity specifically in these cells thanks to a fluorescent Rac1 probe, RFP-PBD, adding to the originality of this work [Endoderm is one of the three germ layers that also needs to internalize and expand over the entire embryo. Live analysis of endodermal cells led by Nicolas David and his colleagues, revealed an active, oriented and actin-based migration that drives these cells to their inner most position. This process was also dependent on Rac1 activity . This fohis work .Studies in mouse embryos showed an important role for Rac1 in mediating intracellular signals required for early gastrulation such as PI3K-Akt and Nap1/WAVE complex , and zebrras morphants presented bilateral lumens in the spinal cord, however, the same phenotype was not observed in rras mutants due to possible redundancy with other ras family members.Neurulation is another complex morphogenetic event that requires extensive cell movement of neural plate cells as well as a direct interaction with underlying mesoderm and surrounding extracellular matrix to coordinate this process ,13,14. WTg (EGF-actin-nos1-3′UTR). This elegant study uncovered a crucial role for Rac in controlling the amount and distribution of actin brushes within germ cells that is required for their proper migration [One of the main features of embryonic development is the correct and coordinated movement of cells in both time and space. Neural crest (NC) cells are one of the most dynamic developmental cells that travel along and explore the embryo with highly organized migratory abilities. Studies in mice have shown an important role for Rac1 in post-migratory NC cells during craniofacial and cardiac development , while Higration . Rac actigration ,20.Tol2-MPO-mcherry-PA-Rac1-polyA) and were able to photoactivate Rac in neutrophils in live zebrafish embryos [Neutrophils are also highly dynamic cells that migrate towards sites of infections or inflammation where they exert their function. Here again, Rac activity was found to be sufficient to direct the migration of neutrophils in vivo, downstream of PI3K. In this study, the authors generated a photoactivated form of Rac that is spatially and temporally controlled  . Rac1 al GTPases . A recen mutants . Furtherpression .At the subcellular level, it has been shown that the Elmo1-Dock1-Rac1 complex is required for ciliogenesis in zebrafish by influencing Ezrin phosphorylation and therefore orchestrating ciliary basal body migration, docking and positioning .P29S activating mutation were abolished by PAK and MEK inhibitors in vivo [fat1 knockdown, knowing that recessive mutations of the protocadherin FAT1 cause renal disease in humans [arhgdia (Rho GDP-dissociation inhibitor 1) knockdown in zebrafish larvae, given that ARHGDIA mutation is responsible of nephrotic syndrome, linking the latter to defective Rho GTPase signaling [As detailed above, Rac1 is critical for regulating key cellular processes such as cell migration and survival in different cell types. Clearly, these essential cellular functions mediated by Rac1 could be exploited in pathological conditions such as cancer, whereby cells rely on Rac1 to invade surrounding tissues . One suc in vivo . Indeed, in vivo . Overexp in vivo . Anothern humans . This sun humans . It was ignaling .rac2 mutant, that renders mutant embryos susceptible to infection and can be rescued by ectopic expression of Rac2 or Rac1 specifically in neutrophils, suggesting overlapping function of the two isoforms [rac2 activity in zebrafish neutrophils would reduce immunological damage caused by systemic inflammation [rac2. Indeed, neutrophil-specific miR-722 overexpression protected zebrafish from systemic inflammation and showed an increase resistance to bacterial-induced inflammation highlighting a beneficial outcome for reducing Rac2 levels to dampen neutrophilic inflammation [It is important to note that the only available zebrafish loss of function mutant of the Rac family is the isoforms . Given  is 99% identical to human CDC42 and contains nuclear localization and prenylation modification signals. The other two predicted proteins, named Cdc42l2 (Cdc42b) and Cdc42l (Cdc42c), are 77 and 90% identical to human CDC42, respectively. Expression analysis by RT-qPCR showed that cdc42a and c were expressed during development and in adult zebrafish [cdc42b was not detected at any timepoint [According to phylogenetic analysis and exon organization, three zebrafish sequences were assigned as CDC42 homologs . cdc42a imepoint .cdc42 early expression correlates with a role for Cdc42 in gastrulation. Indeed, Cdc42 is already known to control gastrulation in sea urchin embryo [cdc42, to control zebrafish somite symmetry, left-right patterning and endoderm organogenesis [n embryo  and in Xn embryo , showingn embryo , howevern embryo . Anothern embryo . Moreoveogenesis . Cdc42 iCdc42 and other Rho GTPases were found to be implicated in germ cell morphology and migration in rodents, mainly in mammalian testis . Moreoveper se for neutrophils migration but was rather essential for WASp regulated phagocytosis [Paul Martin’s laboratory took advantage of zebrafish larvae transparency and the fact that they present a largely conserved innate immune system in comparison to humans to develop new zebrafish models to image wound-triggered inflammatory response. These studies have revealed important mechanisms by which leukocytes orient towards wound signals in vertebrates. In a zebrafish model of Human Wiskott–Aldrich syndrome (WAS), the migration of macrophages to wounds was significantly reduced , howeverocytosis .One particularity of Cdc42 protein is that, among Rho GTPases proteins, it promotes the formation of cellular protrusions known as filopodia, that are important for cell morphology, cell migration and tissue patterning . A very cdc42 zebrafish morphants present tail curvature, hydrocephalus , eye defects as well as glomerular expansion, that are all features of ciliopathy [sec10 and tuba zebrafish morphants. Indeed, Sec10 is part of the exocyst transport complex that plays an essential role in vesicular transport through its interaction with small GTPases and Tuba is a dynamin binding protein required for ciliogenesis and acts as a GEF for Cdc42 in several processes including ciliogenesis [Cdc42 is not only required for filopodia but it can also modify cilia function. Primary cilia are microtubule-based organelles that extend on apical surface of most cells. Their importance is highlighted by a group of diseases that result from defective cilia known as ciliopathies, such as cystic kidney disease, neurodevelopmental abnormalities, blindness, obesity, and perhaps even psychiatric disorders . Zebrafiliopathy . Moreoveliopathy ,72. Similiopathy . This phogenesis .redhead and bubblehead mutant, Cdc42 acts upstream of the GEF BPix and its binding partner, Pak2 (P21 (Rac1) Activated Kinase 2), for the maintenance of vascular integrity [arhgef9b deficient mice have normal vascular structures [Several studies underscore an essential role for Cdc42 in angiogenesis similar to Rac1 function. Cerebral blood vessels are particularly fragile during development and despite animal models, the biological processes leading to hemorrhage are not well understood. Zebrafish mutants revealed novel pathways required for vascular stability during development. As demonstrated in ntegrity ,76. Wakantegrity . They unructures . Similarructures , while zructures .One interesting feature of Cdc42 activity is its ability to modulate actin filaments at the synapses. Indeed, mice lacking Cdc42 present defects in postsynaptic plasticity, while its loss of function in presynaptic sensory neurons affects synapse formation ,82. Zebrrac1, cdc42 was also identified as a target gene of Wnt5b that is activated during early stages of optic nerve regeneration in zebrafish [As discussed for ebrafish . A diffeebrafish , whereasSimilar to Rac1 and Cdc42, RhoA is one of the most studied small Rho GTPases; it regulates a wide range of cellular processes including cell growth and dynamics . ConditiMaternal RhoA expression suggested a possible role for this small GTPase in zebrafish early development and morphogenesis. In many animal species, the segregation of the germ plasm (GP), a specialized cytoplasm containing particular mRNAs and proteins , is esserhoa morphants displayed shortened body axis and were reminiscent of noncanonical Wnt mutants, placing RhoA at the center of the major noncanonical Wnt signaling required for zebrafish CE movements. However, Hsu C. and her colleagues were able to partially rescue gastrulation defects observed in p120 catenin morphants by overexpressing a dominant negative form of RhoA, highlighting the necessity of a delicate balance of RhoA activity during gastrulation [Rho GTPases are main drivers of cell movements in a myriad of morphogenetic events and RhoA is no exception. The first study to establish a direct functional interaction between RhoA and Wnt for cell dynamics regulation in vivo, was assessed in 2006 by Zhu and colleagues . Using mAs for highly migratory cells, it has been shown that maintaining RhoA activity specifically at the back of NC cells was crucial to locally inhibit Rac activity via Rock. This interaction contributes to the formation of protrusions at the front of NC cells and leads their directional migration . This waslc3a2 (solute carrier family 3 member 2) morphants defects, while the attenuation of RhoA activity rescued slc3a2 morphant phenotype. The authors propose a mechanism in which Slc3a2 inhibits RhoA and contributes to YSL organization via the microtubule network [rhoa mRNA overexpression rescued heart defects observed in kctd10 (Potassium Channel Tetramerization Domain containing 10) morphants; the latter is a member of the polymerase delta-interacting protein 1 (PDIP1). KCTD10 causes RhoA degradation, a step required for normal cardiogenesis that is likely to be conserved among vertebrates [Zebrafish embryos undergo the formation of the yolk syncytial layer (YSL) during early development that is vital for the regulation of dorso-anterior axis formation, epiboly and cardiac progenitor cell movements. Overexpression of a constitutive active form of RhoA led to YSL disorganization and mimicked  network . RhoA watebrates .rhoa morphants showed a reduced body size and length and TUNEL assay highlighted a significant increase in apoptosis following gastrulation, a phenotype that was maintained in highly proliferative tissues. The authors propose a link between intensive apoptosis phenotype and reduced body size [The suppression of apoptosis by RhoA via diverse anti-apoptotic pathways has been established following several studies. RhoA upregulates anti-apoptotic Bcl2 expression in different cell types such as T cells and osteosarcoma cells ,95. Thisody size .G12V), a constitutively active form of RhoA (RhoAG14V) or a dominant-negative form of RhoA (RhoAT19N) were created for this purpose. The study highlighted for the first time a strong crosstalk between Kras and RhoA in liver outgrowth and tumorigenesis via Akt2, p21Cip and S6 kinase. Indeed, while the expression of RhoAT19N expanded liver outgrowth, hepatocellular carcinoma development and cancer mortality, RhoAG14V expression suppressed KrasG12V driven liver overgrowth in zebrafish larvae [Abnormal RAS signaling is found in up to 30% of all human cancers , howeverh larvae . This unh larvae ,101,102,h larvae . Altogetrhoa mRNA to reduce its protein levels and contribute to spinal cord regeneration [However, a different outline is proposed during optic nerve regeneration in zebrafish in which RhoA has been reported to be inactivated, contrarily to Rac1 and Cdc42. The authors therefore hypothesize that reducing the levels of RhoA might contribute to optic nerve regeneration . This isneration . This isneration .arhgef12 that could be rescued by a co-injection of a constitutive active form of RhoA (Q63L) [Finally, RhoA activity has been linked to erythroid differentiation since an overexpression of a dominant-negative form of RhoA led to anemia. This phenotype mimicked the knockdown of the RhoA specific guanine nucleotide exchange factor A (Q63L) .Studies in zebrafish have shown that the three Rho GTPases control a wide spectrum of developmental functions during which they can play complementary, overlapping or quite distinct functions .It is known from mouse knockout studies that small GTPases are essential for early embryonic morphogenesis since Rac1 and Cdc42 KO mice show early embryonic lethality due to defects in germ layers formation ,107.rac1, rac1l, rac2, cdc42 have also been tested and used and the first CRISPR/Cas9 F0 mosaïc cdc42 mutant was created to evaluate its function in the morphogenesis of Müller glia [rac2 zebrafish mutant line and showed a functional redundancy between Rac1 and Rac2 in neutrophils behavior [Most studies regarding small GTPases function in zebrafish rely on injecting established constructs that allow either an overexpression of Rac1, Cdc42 and RhoA (via WT or constitutive active (CA) forms) or act as dominant negatives (DN) see  below. Sler glia , with nobehavior .Conditional or tissue specific manipulation of small GTPases remains the best way to selectively activate or inhibit their function, either by injecting and expressing different constructs for mosaic/single cell analysis or by establishing transgenic lines for a more global analysis in larvae and in adults. Indeed, several transgenic lines that take advantage of the Gal4/UAS system are now available and allow a tissue driven expression of either DN or CA forms of UAS-Rac1, Cdc42 and RhoA when crossed to available Gal4 transgenics under the control of different promoters. Moreover, these transgenics allow you to take full advantage of zebrafish larvae live imaging and circumvent troubles with unavailable antibodies since proteins are tagged with a fluorophore (GFP or mCherry).Zebrafish is also amenable to pharmacological studies given its external development, therefore several drugs that act as activators or inhibitors of small GTPases have been tested and used in zebrafish larvae. NSC23766 and ML141 remain the most used drugs to inhibit Rac1 activity and Cdc42/Rac1 respectively. Some other compounds have been used as activators of Rac1/Cdc42/RhoA or Rac1 inhibitors but we lack some details as to their specificity and efficacy in zebrafish .Tg(UAS:RaichuEV-Cdc42), FRET-based biosensor, (ii) Tg(UAS: RaichuEV-Cdc42 NC), negative control FRET biosensor in which Cdc42 binding domain in PAK1 sequence of RahicuEV-Cdc42 was mutated and (iii) Tg(UAS:Myr-GFP-ACK42), Cdc42-specific inhibitor, a cDNA encoding minimal Cdc42 binding domain of human ACK1  [Finally, zebrafish is a perfect model to image in vivo protein interactions using fluorescent resonance energy transfer-based probes. Several probes, named Raichu-Rac and Raichu-Cdc42 were designed and used to image these small GTPases activity . Several, ACK42) .Zebrafish is a remarkable vertebrate model for in vivo live imaging that helped us unfold several aspects of Rho GTPases function over the past decade. Work performed so far has taught us a great deal about how these small GTPases regulate dynamic changes within numerous cell types and at different developmental stages. The ability to follow Rho GTPase signaling and to locally modulate its function in real time, combined with the rapidly evolving imaging and genetic techniques, will ensure that the zebrafish remains a key genetic model for study of these small proteins in cell dynamics and signaling in vivo. Generating more zebrafish tools will help us understand not only the role of Rho, Rac and Cdc42 in development and disease but also how their regulators as well as other atypical Rho family members, that have been neglected so far, contribute to these processes.


The effect of intravenous fluids (IVF) has been investigated clinically through the assessment of post-treatment reactions. However, the responses to IVF vary from patient-to-patient. It is important to understand the response of IVF treatment to be able to provide optimal IVF care. Herein, we investigated the impact of commonly used IVFs, Dextrose, NaCl and Ringer on different human cancer (HepG2 (liver hepatocellular carcinoma) and MCF7 (breast adenocarcinoma)) and immune cell lines (U937 (lymphoma) monocyte and macrophages). The effect of IVF exposure on single cells was characterized using hemocytometer, fluorescence microscopy and flow cytometry. Quantitative data on the viability and morphology of the cells were obtained. Our results emphasize that different IVFs demonstrate important differences in how they influence distinct cell lines. Particularly, we observed that the lactated ringer and dextrose solutions altered the viability and nuclear size of cancer and immune cells differently. Our findings present valuable information to the knowledge of cellular-level IVF effects for further investigations in IVF usage on diverse patient populations and support the importance and necessity of developing optimal diluents not only for drug stability but also for patient benefits. This technique involves the application of intravenous fluids (IVF). The first usage of IVF dates to the 1830s when Shaughnessy used intravenous salt solutions to treat cholera3. Since then, IVF, have been utilized in clinical practice on a large scale through intravenously injections to patients4. Over the years, discussions regarding the choice of IVF selection8 procedures of IVF that were accepted as standard for patient treatment have shifted to personalized fluid treatment depending on patients’ conditions10. Several types of IVF including normal saline (NS), dextrose and ringer have been commonly used12. In order to investigate IVF therapy, mostly to test diluents, which are optimal for drug compatibility and stability, experiments have been presented13.The choice of IVF selection has been debated over the years. IVF therapy is one of the main treatment methods that have been used to cure water imbalance problems14. To better understand the effects of IVF on patients, quantitative analysis might contribute notably to a better comprehension of cellular individuality and heterogeneity15. As vascular endothelial cells form the primary selective barrier to plasma macromolecules, support red blood cell movement, oppose transvascular fluid flux and leukocyte adhesion, majority of the quantitative studies focused on investigating response of blood cells and endothelial cells to IVF. Carden’s group reported the influence of IVF on sickle cell disease. They measured the stiffness of the red blood cells in 0.45% and 0.9% sodium chloride solutions at single-cell level. They concluded that sodium chloride solutions alter biomechanical properties of sickle red blood cells. Consequently, patients with sickle cell disease might have localized microvascular obstruction during the vasso-oclusive crisis16. Koustova and her co-workers quantified the production of reactive oxygen species from leukocytes when they were exposed to Lactated Ringer solution. They observed elevated level of reactive oxygen species and concluded that it influenced neutrophil function and leukocyte gene expression17. Jackson and Derleth examined the effect of brief contact with various infusion solutions on red blood cells from newborn infants. They determined that isotonic amino acid solution did not cause agglutination or haemolysis difference11. Likewise, Shields et al., quantified the adhesion molecules and laminin expression to reveal influence of hypertonic saline solution to the tumor cell-endothelial cell interaction18. Dr. Schmidt’s group employed mass spectrometry to measure plasma heparan sulfate, which is associated with volume of intravenous fluids, and determined that intravenous fluid resuscitation is associated with septic endothelial glycocalyx degradation19.First human trial was performed to assess the immunologic and anti-inflammatory impact of hypertonic resuscitation in trauma patients. The results showed that hypertonic saline plus dextran leaded to a more balanced inflammatory response to hemorrhagic shock20, HepG2 human liver hepatocellular carcinoma cells21, and MCF7 human breast adenocarcinoma cells22 when they were exposed to various IVF for 15 minutes. We used immune system related cell lines, U937 monocyte and U937-differentiated macrophage due to their heterogeneity and abundance in blood23. Although clinical studies demonstrated that IVF modulates macrophage response and hypertonic saline resuscitation decreases alveolar macrophage activation, cell viability and morphologic changes have not been reported24. As cancer cell lines, we chose MCF7, which might be one the most metastatic cancer line25 and HepG2, whose dominant risk factors reflect the worldwide heterogeneous incidences such hepatitis  viruses, alcoholism, aflatoxin, obesity, diabetes, cirrhosis26. In order to quantify the influence of IVF on these human cell lines, we performed single-cell resolution viability assays using hemocytometry, fluorescent microscopy, and flow cytometry. It is important to emphasize that limited studies have been conducted to quantitatively investigate cellular, metabolic, and systematic consequences of IVF on human monocytes and epithelial tissue cells. Miltra’s group employed RNA sequencing to explore global transcriptional responses induced by hypertonic saline solution27. Their results suggested that hypertonic saline attenuates the cytokine-induced pro-inflammatory signature in primary human lung epithelia27. Likewise, Dr. Banerjee and co-workers measured inflammatory cytokines and chemokines in response to TNF-α and IL-1β on alveolar pneumocyte line (A549) and concluded that hypertonic saline or hyperosmolar media disrupt cytokine signals at distinct intracellular steps28. Similarly, this group previously reported that hypertonic saline inhibited TNF-α induced NF-κB activation in the pulmonary epithelium using immunofluorescent microscopy, flow cytometry, cell viability and western blot analysis29.The study presented here aimed to provide valuable insights into the distinct cellular responses of monocytic leukemia and carcinoma cells induced by IVF. Epithelial cells function as barrier to pathogens, express a wide variety of immunomodulatory molecules and play a crucial role in the recognition and resolution of inflammatory responses. Our study designed to interrogate the acute response of U937 monocytes, U937 monocyte-derived macrophages34.However, still most of the recent discussions focus on investigating large clinical trials to better define optimal fluid therapy in acute and critical care medicineWe observed the acute response of U937 monocytes, U937-derived macrophages, MCF7 and HepG2 cancer cells when we exposed them to IVF for 15 minutes. We quantified the number of live and dead cells using hemocytometry, inverted fluorescent microscopy and flow cytometry data as described in the materials and methods section. Our results displayed the cellular and nuclear area adaptations and viability counts of the cells when the cells were incubated in IVF, PBS and each cell’s respective medium.First, we quantified the number of live and dead cells for the U937 monocytes, U937-derived macrophages, MCF7, and HepG2 cancer cell lines after 15-minute incubation with IVF. Figure Figure We quantified the behavior of the HepG2 cells after 15-minutes IVF treatment, Fig. MCF7 cell viability and morphology assay results were demonstrated in Fig. Based on the results of the One-way ANOVA Tukey’s Multiple Comparison Test, Tables Flow cytometry experiments were performed to analyze a larger number of cells to verify single-cell analysis obtained using hemocytometer and microscopy images. We quantified the viability of the cells via Propidium Iodide (PI) staining. We analyzed morphology of the cells using forward scatter and side scatter data .39. Most IVF investigations have focused on revealing their influence on medication stability, compatibility and administration18. To date, there have been few quantitative in vitro studies about the influence of IVF on cellular level, which are mostly reported for blood cells and endothelial cells41.IVF medication has been performed for millions of patients each year. Recently, new cell-based treatment strategies have been developed in which IVF plays a crucial role. Therefore, investigations of the effect of IVF have begun to emerge. The concerns of IVF on patient outcomes has been investigated in relation to critical care practicein vitro evaluation for acute reactions of white blood cells and carcinoma cells when they were exposed to IVF for 15 minutes. Cell viability changes for 60-minutes IVF exposure were also observed, which is beyond the scope of this work  and two epithelial cell lines . We characterized cell viability, cellular area and nuclear morphologic changes using multiple techniques to yield quantitative results that might highlight some potential cautions in using these IVF diluents in clinical disease management. Our results present 37. Since dextrose is a form of glucose, which might alter cellular ability to sustain ion and water balance, intracellular homeostasis. Thus, dextrose solution might rupture the plasma membrane, causing cytoplasm leakage into the extracellular environment42. Our results showed that dextrose solution decreased the viability of the U937-differentiated macrophage cells . Nonetheless, one of the most important limitations of our study is that it exhibits in vitro behavior with IVF in cell lines that do not recapitulate exactly the interactions in the human body when the medication is delivered via IVF.Distinctly, in this study, along with investigating a very short-term acute IVF influence on cell viability, we examined the surface areas of cells and their nuclei performing image analysis on microscopy images47. Currently, organotypic culture studies are also in their infancy and there is an urgent need for their improvement to achieve in vivo likeness with a better practical usage in clinical studies48.Our results lay groundwork for further studies. As a future work, these studies might be conducted using three-dimensional (3-D) organs-on-a-chip which might provide a better understanding of biokinetics, signaling between cells and clinical insightsBy reflecting a very short-term acute influence of IVF on U937 monocytes, U937-differentiated macrophages, HepG2, and MCF7 cancer cells, it was discovered that the viability of the monocytes and macrophage has significantly decreased compared to those of breast and liver cancer cells. Moreover, IVF introduced variations in the nuclear and cellular area of the cells. Our findings should be interpreted with caution since they do not present the influence of IVF on cells in the human body. However, our study supports the urgent need for more accurate interrogation of IVF at high resolution and more physiologically relevant human body mimicking models.2 cell culturing flasks were purchased from TTP (Switzerland). Sterile IVF were taken from Polifarma (Turkey); polifleks 9% isotonic NaCl solution (308 mOsm/L) in 250 mL package, 5% Dextrose solution (277.47 mOsm/L), and Lactated Ringer’s solution (275.52 mOsm/L) in 500 mL packages49. Stains of Hoechst (10 mg/L in water) and Propidium Iodide  were received from Life Technologies and Sigma Aldrich, respectively. Authors must ensure that their Methods section includes adequate experimental and characterization data necessary for others in the field to reproduce their work.Cell culture media and supplements including Phorbol 12-myristate 13-acetate (PMA), dimethylsulfoxide (DMSO), Dulbecco’s phosphate buffered saline (PBS), Dulbecco’s modified Eagle’s medium (DMEM), Roswell Park Memorial Institute (RPMI 1640) and Trypsin (3X) were purchased from Pan Biotech (Germany). Fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Sigma Aldrich . The 75 cm2 flasks in the incubator  maintaining a humidified atmosphere at 37 °C and 5% CO2. The U937-monocyte media was renewed every 3 to 4 days depending on cell density. The U937 monocyte-derived macrophage media was replaced every day until their usage. Both HepG2 and MCF7 cells were passaged every 3 to 4 days through a standardized trypsinization method using 0.25% (v/v) trypsin-EDTA in PBS depending on their confluency (85%) according to ATCC protocols.The immortalized human histiocytic lymphoma monocyte cell line, U937 (ATCC® CRL1593.2™), human liver hepatocellular carcinoma epithelial cell line, HepG2 (ATCC® HB-8065™), and immortalized human breast adenocarcinoma epithelial cell line, MCF7 (ATCC® HTB-22™) were purchased from the ATCC . The human histiocytic lymphoma macrophages were differentiated from the U937 monocytes through incubation of 3 × 105 cells/mL in 5 mL RPMI 1640 with 10% FBS and 5 μL working solution including 10% PMA obtained from 10 ng/mL PMA/DMSO stock solution for 5 days. All cells were kept in 75 cm5 cells/mL using their growth medium. Then we collected the cells using the centrifuge conditions stated above and resuspended the cells into 1 mL IVF; Dextrose, NaCl, Ringer and PBS, respectively. Subsequently, the tubes with different IVF were incubated for 15 minutes in the incubator. Upon incubation, viable and nonviable cells were enumerated using the hemocytometer. The experiments were conducted, as three independent experiments for each cell line. The maximum decrease in the viability of the cells was at the first 15 – minute time interval during the one-hour of IVF exposure. Therefore, our results present a very short-term acute response of the cells when the cells were incubated in IVF fluids for 15 minutes.We centrifuged and collected the U937 monocytes and U937-derived macrophages at 3000 rpm for 5 minutes, the MCF7 and HepG2 cell lines at 1800 rpm for 10 minutes. Next, using a hemocytometer , we enumerated the viable and nonviable cells based on Trypan blue staining. We arranged the cell count 1.5 × 10Hoechst (0.128 mM) and PI (0.0111 mM) solutions were used for end-point staining. Cells were stained for 20 minutes in the incubator using 1 µL of dyes from the working solutions those explained in the chemicals and reagents above.To quantify the influence of IVF at single-cell resolution, the cells were prepared as explained above. 40 µL of cell suspension from each cell solution was sandwiched between two glass slides. The cells were observed on the inverted fluorescent microscope. Images of the cells were captured with a 40× objective using a Carl Zeiss, Axio Observer Z1 motorized stage equipped with the AxioCam Mrc5 camera. Both phase and fluorescent channels were used to obtain the images at 12,5 ms and 300 ms exposure times, respectively. We used both DsRed fluorescent and DAPI channels to visualize cytoplasm and nuclei of the cells. We counted the cell number and measured the area of the nuclei and cells using ImageJ on the acquired phase and fluorescent images.We adjusted the number of cells to 2.5 × 105 cells/mL. We exposed the cells to the IVF for 15 minutes. We treated one of the samples with PBS and stained with PI, while the other samples were exposed to each IVF  and stained with PI. We performed the flow cytometry experiments using the BD LSRFortessa FACS analyzer . The intact cells were accomplished based on their forward scatter (cell size) versus side scatter (cell granularity) profiles using blue (488 nm) excitation laser. We analyzed the data using the FlowJo v10 software . We performed double cell discrimination by plotting the height against the area for forward scatter. While double cells have roughly the same height with single cells, they have double the area values of single cells. Thus, we identified and excluded double cells based on disproportions between height and area. Then, we distinguished live cells from debris and dead cells by gating on the area values of cells for forwards scatter versus side scatter plots. We excluded events found at the bottom left corner of the density plots in gating to separate debris and dead cells with lower level of forward scatter.The obtained image data was analyzed using GraphPad Prism software. We performed One-way ANOVA with Tukey’s Multiple Comparison test to compare viability, cellular and nuclear area variations of the cells when the cells were exposed to IVF for 15 minutes. The viability data was obtained from the hemocytometer, viable cells were label-free and nonviable cells were stained with Trypan blue. The area measurements of the cells were performed in ImageJ as explained above. Figures showed the data as mean ± standard deviation (SD) of three independent experiments. Statistical differences were represented with (*) for p < 0.05. The q values were the relevant critical values of the studentized range statistic.Supplementary Information.


TRPA1 gene is the thermal sensor associated with infrared detection in pit vipers. However, whether genes other than TRPA1 are also involved in the infrared perception of pit vipers remains unknown. Here, we sequenced the whole exomes of ten snake species and performed genome-wide evolutionary analyses to search for novel candidate genes that might be involved in the infrared perception of pit vipers. We applied both branch-length-comparison and selection-pressure-alteration analyses to identify genes that specifically underwent accelerated evolution in the ancestral lineage of pit vipers. A total of 47 genes were identified. These genes were significantly enriched in the ion transmembrane transporter, stabilization of membrane potential, and temperature gating activity functional categories. The expression levels of these candidate genes in relevant nerve tissues  were also investigated in this study. We further chose one of our candidate genes, the potassium channel gene KCNK4, as an example to discuss its possible role in the infrared perception of pit vipers. Our study provides the first genome-wide survey of infrared perception-related genes in pit vipers via comparative evolutionary analyses and reveals valuable candidate genes for future functional studies.Pit vipers possess a unique thermal sensory system consisting of facial pits that allow them to detect minute temperature fluctuations within their environments. Biologists have long attempted to elucidate the genetic basis underlying the infrared perception of pit vipers. Early studies have shown that the  Pit vipers are the best at sensing and utilizing infrared signals among all snakes6. They have the most sensitive infrared sensory system, which can detect minute temperature fluctuations as low as 0.001 °C in their immediate surroundings7. This peculiar infrared perception ability enables pit vipers to detect and locate warm-blooded animals with high precision in the dark, which makes them one of the most successful predators in nature10.Infrared perception is a notable evolutionary adaptation of snakes that helps snakes respond to the external environment during their nocturnal activities and prey on warm-blooded animals17. We now know that the infrared perception of pit vipers is initiated by the heat stimulation of a thin sensory membrane embedded in the facial loreal pit, a highly specialized organ located between the eye and nostril12. The sensory membrane is innervated by afferent fibres of trigeminal nerve branches that transduce infrared signals to a particular nucleus in the hindbrain15. The efferents of the nucleus then project to the optic tectum, where infrared information is integrated with visual information to construct a thermal image 17. This complex infrared sensory system must involve many genes at the genetic level. However, the genetic basis of the system that is involved in the infrared perception of pit vipers is far from defined and requires more attention. Recently, a study by Gracheva et al.18 showed that a particular gene, TRPA1, is highly expressed in the trigeminal ganglion (TG) of pit vipers. This gene encodes a calcium ion channel that can respond to temperature changes caused by infrared stimulation. Thus far, TRPA1 is the only gene that has been shown to be involved in the infrared perception of pit viper snakes.Biologists have long sought to investigate the biological basis of infrared perception in pit vipers. Previous anatomical and neurophysiological studies have accumulated substantial data explaining the detection and transduction mechanism of pit viper infrared perception6), the expression level of TRPA1 in the TG is much higher than that in the dorsal root ganglion (DRG), which is not observed in infrared-insensitive snakes18. This difference in expression was the cornerstone of the transcriptome profiling analysis that led to the discovery of TRPA1. This strategy is able to reveal direct thermal sensor proteins that are involved in the infrared perception of snakes. However, it may not be able to identify genes involved in pit organ evolution, signal amplification, or information integration (if any) during infrared perception because such genes may not exhibit considerable changes in expression in different nerve tissues.In pit vipers  belong to the family Viperidae. There are three major clades of Viperidae: true vipers (Viperinae), pit vipers , and Fea’s vipers (Azemiopinae), among which Viperinae diverged from the others first, followed by Azemiopinae and Crotalinae24. For example, the “language gene” FOXP2 underwent accelerated evolution in the human lineage after its separation from the lineage leading to chimpanzees and bonobos, our closest relatives27; the echolocation gene Prestin shows convergent evolution in echolocating bats and whales30. Such comparative evolutionary analyses are an effective strategy for searching for phenotype-associated genes. Notably, our earlier work showed that the thermoreceptor gene TRPA1 underwent strong accelerated evolution in pit vipers but not in true vipers31. In contrast, TRPV1, another TRP gene that is not associated with infrared perception, shows no difference in the rate of evolution between pit vipers and true vipers. Moreover, TRPA1 contains many amino acid (AA) residues that are unique in pit vipers but evolutionarily conserved in other snakes31. We hypothesize that if there are other genes involved in the infrared perception of pit vipers, these genes may exhibit a similar evolutionary pattern to TRPA1. This particular evolutionary pattern could be used as a screening criterion to search for potential genes associated with infrared perception in pit vipers, which may compensate for the limitations of transcriptome profiling analysis.Phenotype-associated genes often exhibit a particular evolutionary pattern in taxa with that phenotype compared to their sister taxa without the phenotype37, including the genomes of members of both Viperinae and Crotalinae. However, representative genomes from Azemiopinae are lacking, hindering genome-wide comparative studies for the identification of infrared perception-related genes. In this study, we performed whole-exome capture sequencing to supplement the genomic data for the three subfamilies of Viperidae and other related snake species. Based on these new genomic data, we conducted a genome-wide survey to search for novel candidate genes related to the infrared perception of pit vipers. Our comparative evolutionary analyses identified 47 candidate genes with evolutionary patterns similar to that of TRPA1, which may be associated with infrared perception in pit vipers. Most of these genes are associated with ion transmembrane transporter activity. We hope that the genome-wide identification and evolutionary characterization of novel candidate genes will contribute to the further elucidation of the genetic basis of the infrared perception of pit vipers.A number of snake genomes have been sequenced and released in public databasesDeinagkistrodon acutus32, and the on-target percentage across the ten snake samples varied from 9.8 to 31.3%. Based on these data, we were able to reconstruct over 20,000 CDSs across all the samples  with an average CDS data completeness of 92.24%  is the sister group of Crotalinae (pit vipers), and Viperinae (true vipers) is the sister group of both of the first two groups. Notably, within Viperidae, the branch lengths from the terminal tips to the root node of Viperidae are rather uniform across the three subfamilies, suggesting that most of the protein-coding genes evolved at a similar pace among the three types of viper snakes. In contrast, in the gene tree of the infrared perception-related TRPA1 gene  than the sister branch connecting to true vipers, indicating that pit vipers have evolved considerably faster than all other snakes  than in Colubridae and Elapidae (outgroup snakes). To do so, we considered the average branch length from the common ancestral node to all termini of a given snake group as the average evolution rate of the group. For example, starting from the common ancestral node of Crotalinae + Colubridae + Elapidae, the average branch length to all Crotalinae termini Fig. .TRPA1, although the patterns are somewhat less conspicuous than that of TRPA1. For example, the KCNK4 gene displays a relatively longer length along the common ancestral branch of pit vipers, and the total evolutionary branch length is significantly longer than those of all other snake lineages; the KCNH8 gene displays a longer branch length not only in pit vipers but also in pythons (another group of snakes with infrared perception ability), which may imply a close association with their common characteristic of infrared perception ability; a hypothetical gene with the ID XB-GENE-5885309 has evolved much faster in pit vipers than in adjacent vipers. These shared evolutionary patterns similar to TRPA1 suggest that other genes might also participate in the specialized trait of pit vipers, the infrared perception.We manually inspected the gene tree of these 38 candidate genes to check for pit-viper-specific signatures of accelerated evolution  in the ancestral branch of pit vipers31. Thus, we compared the one-ratio model and the two-ratio model for all 21,331 OGGs by PAML39. The one-ratio model assumes that all branches in the phylogeny have the same ω value (dN/dS), while the two-ratio model assumes two different ω values within the phylogeny: one for the ancestral branch of pit vipers (ω1) and one for the remaining branches of the tree (ω0). If the two-ratio model is significantly better than the one-ratio model for a gene, the gene experienced significantly different selection pressure in the ancestral branch of pit vipers. The selection pressure analysis identified a total of 11 genes with significantly elevated ω values in the ancestral branch of pit vipers compared to the background branches  were identified simultaneously by both filtered schemes. Detailed information on all candidate genes is given in Supplementary Table Combining the 11 candidate genes from the selection analysis and the 38 candidate genes from the branch length analysis, we identified a total of 47 candidate genes that may be related to the infrared perception of pit vipers. These genes are TRPA1, KCNK4, KCNH8, KCNK17, KCNC2, CYBB, and CRYAB genes. Ion channel proteins are crucial in the thermosensation of vertebrates40. Because our screening scheme targeted infrared perception, these ion channel genes are unlikely to have been randomly identified. It is worth mentioning that we found that the two genes (TRPA1 and KCNK4) that were identified by both the selection pressure and branch length analyses were associated with the GO term ‘temperature-gated cation channel activity (GO: 0097604)’. This GO term was significantly enriched (468.9-fold), with the lowest corrected P-value of 4.4e−6.To further explore the possible associations between the identified genes and the infrared perception function in pit vipers, we retrieved Gene Ontology (GO) terms for the 47 candidate genes under three major categories: biological process (BP), cellular component (CC), and molecular function (MF). Additionally, we performed GO enrichment analysis to test whether our candidate genes were significantly enriched in specific GO terms. The GO enrichment analysis showed that five BP GO terms, two CC GO terms, and four MF GO terms were significantly enriched Table . A GO teTRPA1, KCNK4, WAPAL, NOC4L, Q5ZLE7, ENPP3, KCNH8, TMEM9B, XB-GENE-5885309, CLCN5, DPY19, GK5, KDRL, and ADAMTS2 genes present more than five pit viper-specific AA substitutions  in its protein sequence. Among the remaining candidate genes, the KCNK4 gene possesses the highest number of pit viper-specific AA substitutions , and the proportion is comparable to that for TRPA1  and exhibits a functional interaction with CLCN5, a gene that is also present in our candidate gene set. In summary, the functional properties of many of our candidate genes suggest that they may play a role in the infrared perception of pit vipers, but their functional mechanisms need to be explored in more detail.Residue substitutions at evolutionarily conserved positions are often signs of the functional alteration of proteins. Therefore, we calculated the number of pit viper-specific amino acid (AA) substitutions for each candidate gene and used a branch-specific site model to test whether these substitution sites are under positive selection. A pit viper-specific substitution is defined as an amino acid site that is in one state for all other snake groups but is substituted to another state in pit vipers. Thirty-six of the 47 candidate genes possess at least one pit viper-specific substitution; specifically, the ons Fig. . Most ofons Fig. . The TRPOur comparative evolutionary analyses identified a total of 47 candidate genes. The investigation of the expression levels of these candidate genes in relevant nerve tissues is necessary to elucidate their functional roles in infrared perception in pit vipers. Therefore, we performed transcriptome profiling to investigate the expression levels of the candidate genes in the cerebrum, midbrain, TG, and DRG nerve tissues of both pit vipers and infrared-insensitive snakes Fig. . In pit KCNK17, SLC5A1, and ZGC_136493) were not expressed in any of the four nerve tissues that we investigated. Among the remaining 44 genes, all but four genes  were expressed in the TG of pit vipers. When pit vipers were compared with infrared-insensitive snakes, nine genes were found to exhibit upregulated expression in the pit viper TG. They are TRPA1, KCNH8, MYOT, KCNK4, ZNF703, KDRL, SETD7, CYBB, and CRYAB  is considerably higher than in the other three nerve tissues . The expression levels of KCNH8 and MYOT in the pit viper TG and the other three nerve tissues are TG: 56.5; DRG: 15.6; midbrain: 3.8; cerebrum: 1.1 and TG: 90.5; DRG: 10.1; midbrain: 0; cerebrum: 0, respectively. The expression level of KCNK4 in the TG is also higher than the levels in the other three nerve tissues, but the expression differences are small . Detailed quantitative results are provided in Supplementary Table Among the 47 candidate genes, three genes (TRPA1), the molecular functions of five genes  show no direct association with infrared perception based on the current knowledge of human orthologous proteins. Among the remaining three genes, KCNH8 is a voltage-gated potassium channel gene involved in regulating neurotransmitter release and neuronal excitability41; MYOT encodes a cytoskeletal protein that plays an essential role in stabilizing thin filaments during muscle contraction42, and KCNK4 functions as an outwardly rectifying channel and may be involved in regulating the input threshold in sensory neurons43. All of these functions are associated with infrared perception to some extent. The relatively higher expression of these genes in the trigeminal ganglion of pit vipers implied that they might function in the infrared perception of pit vipers.Among the eight upregulated genes  surrounding the central cavity, and two pore-helices (PH1 and PH2) forming the selectivity filter  located in pore helix 1 (PH1). Protein structure simulation showed that the side chain of this amino acid residue points towards the central pore. In terms of space, the asparagine (N) side chain is bulkier than the serine (S) side chain, causing increased steric hindrance , and the noncharged residue Ser is replaced by the positively charged residue Arg (S259R). Such replacements alter the charge environment of the channel egress from a negative state, which attracts potassium ions, to a positive state, which repels potassium ions, potentially also affecting the channel gating.The protein structure simulation showed that the complete ter Fig. a, b, d44ons Fig. c, three KCNK4 channel is involved in maintaining the resting membrane potential and is closely associated with neuron excitability52. This gene is usually coexpressed with heat-sensitive and cold-sensitive excitatory channels, such as TRPV1, TRPM8, and TRPA1, and functions as a silencer of these excitatory channels in sensory neurons56. It has been reported that downregulated expression or knockout of KCNK4 in mice can increase neuronal sensitivity towards a given thermal stimulus, causing heat hyperalgesia or cold hypersensitivity in mice54. This is because the deletion of neuronal K+ channels in nociceptors will slow the depolarization of sensory neurons and prolong action potentials, thus amplifying painful sensations53. Our protein structure simulation analysis suggested that the mutations in pit viper KCNK4 channel are likely to affect channel gating, which might blunt the channel responses to certain types of gating commands or alter the thermal or mechanical sensitivity of the channel. If these mutations make the thermal activation threshold of pit viper KCNK4 increase, this channel will be less active at lower temperatures at which TRPA1 is activated. This functional effect is similar to knockdown or knockout KCNK4 in pit vipers’ infrared sensory neurons. If our functional speculation is correct, the pit vipers seem to use a mechanism similar to that underlies heat hyperalgesia in a mouse model generated by the deletion of KCNK4 to increase their thermosensitivity. Of course, this hypothesized mechanism needs further confirmation with functional studies.Early functional works showed that the TRPA1. On the basis of whole-exome capture sequencing, we conducted the first genome-wide mining of candidate genes associated with infrared perception function in pit vipers through comparative evolutionary analyses. Our study identified a total of 47 genes that underwent accelerated evolution, specifically in the pit viper lineage, which exhibited extensive accumulated pit viper-specific amino acid changes in their protein sequences. Most of the candidate genes were enriched in GO terms functionally related to ion transmembrane transport, which is closely related to infrared perception. We also examined the expression levels of these candidate genes in four different types of nerve tissues of both pit vipers and infrared-insensitive snakes and found that eight genes exhibited upregulated expression in the pit viper trigeminal ganglion. Furthermore, we simulated the protein structure of one of our candidate genes, the potassium channel gene KCNK4, and inferred its possible functional mechanisms. This work represents the first attempt to systematically identify genes associated with pit viper infrared perception based on comparative genomic analysis. We hope that the gene sets reported in this study provide a valuable stepping stone for further functional research and shed new light on the genetic basis of infrared perception in pit vipers.Compared with other specialized sensory systems such as mammalian echolocation and primate trichromatic vision, the molecular underpinning of snake infrared perception has received relatively little attention. Before this study, researchers had only performed transcriptome differential expression analyses to identify infrared perception-related genes in pit vipers and obtained only one target gene, Crotalus mitchellii, Crotalus horridus, Protobothrops mucrosquamatus, and Deinagkistrodon acutus), one infrared-insensitive viper (Vipera berus), two infrared-insensitive colubrids , one infrared-sensitive python (Python bivittatus) and one infrared-sensitive boa (Boa constrictor). In addition, we sampled another 10 snake species to conduct whole-coding-region sequence capture. These species included four Crotalinae species  and one Azemiopinae species (Azemiops feae), one Viperinae species (Daboia siamensis), one Elapidae species (Ophiophagus hannah), one Pythonidae species (Python regius), one Xenopeltidae species (Xenopeltis unicolor) and one Boidae species (Eryx tataricus). This additional sampling supplements the missing Azemiopinae and enriches the sampling density of other families, especially pit vipers .In this study, a total of 19 snake species were sampled for comparative genomic analysis, covering all 3 subfamilies of Viperidae and representatives from five other relevant snake families  were performed in strict accordance with the guidelines and regulations developed by the China Council on Animal Care and Use. Animal processing procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat-Sen University (permit number: 2018-023).Deinagkistrodon acutus32. The hybridization enrichment experiments were performed using the SureSelectXT Reagent Kit (G9611A) according to the manufacturer’s protocol. After enrichment, hybridized libraries were captured by Dynabeads MyOne Streptavidin C1 magnetic beads (Invitrogen) and amplified. The resulting captured libraries were pooled in equal quantities and sequenced on one Illumina HiSeq X-10 lane in 150-bp paired-end mode.Genomic DNA of the 10 snake species were extracted from muscle tissues using the DNeasy Blood & Tissue Extraction Kit (Qiagen). The extracted gDNA samples were randomly fragmented by NEBNext dsDNA fragmentase (New England Biolabs) to 200–400 bp. A total of 500 ng of fragmented gDNA was used for DNA library construction with the NEBNext Illumina DNA Library Prep Kit (New England Biolabs) according to the manufacturer’s protocol. We used the Custom Agilent SureSelectXT Target Enrichment System (Agilent Technologies) to capture whole coding regions from these libraries. The custom probes were designed at twofold tiling to target 21,331 snake CDSs for a total size of 32.1 Mb, based on the predicted exome of 57, and the reads were then sorted to DNA samples based on library indexes. The filtered reads of each sample were aligned to the 21,331 Deinagkistrodon acutus reference CDSs using the MEM algorithm of Burrows-Wheeler Aligner version 0.7.1058 with the default setting (-B 4) for viper species and relaxed setting (-B 2) for other snake species. The read mapping rate and the sequencing depth for each sample were calculated with SAMtools v1.459. Based on the BWA mapping results, the consensus sequences for each sample were assembled and called using SAMtools (mpileup command with -q 5 -Q 10 -B -uf) and BCFtools v1.4 . Using this bioinformatic pipeline, we obtained the orthologous CDSs of each sample corresponding to the 21,331 Deinagkistrodon acutus reference CDSs. For each reference CDS of Deinagkistrodon acutus, the obtained orthologous CDSs from different samples had exactly the same length, and the coordinates of these CDSs matched exactly those of the reference sequence.Raw sequence reads were quality filtered, adapters were trimmed using Trimmomatic v0.33Deinagkistrodon acutus reference CDSs and used to call orthologous CDSs using the aforementioned bioinformatic pipeline.Because some snake genome data used in this study were not well annotated, we used a so-called “pseudo sequencing strategy” to identify orthologous CDSs from snake species with published genome data. Briefly, the raw genome sequences of each snake species were artificially fragmented into small ‘reads’ of 150 bp at 10 × tiling density. These “pseudo” reads were mapped to the 60 according to the translated protein sequences. The program PAL2NAL v1461 was used to convert the protein sequence alignments into corresponding codon alignments for each OGG.Finally, all the orthologous sequences for each reference CDS were put into a separate FASTA file as an orthologous gene group (OGG). The obtained OGG files were aligned using the ClustalW algorithm implemented in MEGA v662 under the GAMMA + WAGF model. Branch support was estimated by a rapid bootstrap algorithm (-f a option) with 500 replicates.To construct a genome-scale phylogeny for the 19 snake species sampled in this study, the protein alignments of all 21,331 OGGs were concatenated. The supermatrix was 11,231,292 amino acids in length with a data completeness of 90.3%. The phylogeny was reconstructed with maximum likelihood (ML) inference using RAxML v8.0ML trees were separately reconstructed for the 21,331 OGGs based on their protein alignments using RAxML v8.0 with the GAMMA + WAGF model. To make branch lengths comparable across different OGG trees, we enforced the genome tree  than in the comparative snake group, totally including three steps of comparison: the first step compares Crotalinae (pit vipers) and Colubridae + Elapidae (non IR-sensitive snakes); the second step compares Crotalinae (pit vipers) and Viperinae + Azemiopinae (non IR-sensitive viper snakes); and the third step compares the ancestor of pit vipers and extant Viperinae and Azemiopinae Fig. .The aver39. To reduce the impact of missing data, all the OGG codon alignments were first filtered to exclude species with more than 20% missing data. Only OGG codon alignments with more than three pit vipers and three non-pit snake species were used in the selection pressure analysis. For each OGG codon alignment, a separate user-tree was generated by pruning the corresponding missing species from the genome tree and labeling the ancestral branch of the pit viper lineage as the foreground branch. The two-ratio branch model  was compared to the one-ratio branch model . After filtering out extremely low dN and dS values, the statistical p-values were calculated by likelihood ratio test (LRT) with one degree of freedom and false discovery rates (FDR) were computed using the Benjamini–Hochberg procedure63. This analysis searches for genes that experienced significant selection pressure alteration in the early evolution of pit vipers compared to other snakes.Selection pressure analysis was performed using the CODEML program of the PAML packageFor genes showing signs of selection pressure alteration, we used the branch-site model in PAML  to identify positively selected sites in the pit viper lineage. The null model  was compared with the alternative model . The probable positively selected sites were identified and scored under Bayes empirical Bayes (BEB) probability.https://david.abcc.ncifcrf.gov/) using lists of all candidate genes that we identified from both selection pressure analysis and branch length analysis. Enrichment was determined using the Fisher’s exact test and all P-values were adjusted for multiple comparisons by the Bonferroni method. Enriched GO categories were called using a 0.05 significance threshold and a minimum of 2 mapping entries.To provide an overview of the over-represented functional categories of identified genes, we performed GO enrichment analyses with DAVID 6.7 . mRNA isolation, library construction, sequencing, data cleaning and quality control were performed by BGI . All libraries were sequenced on an Illumina HiSeq2000 sequencer. We obtained approximately 3.5 Gb of 90-bp paired-end read data (Q ≥ 20) for each tissue sample. RNA-seq data of peripheral nerve tissues (TG and DRG) were downloaded from a previous study18 . Reads from individual tissue libraries were mapped to the corresponding transcriptome assembly by Bowtie2 v. 2.2.665 with default parameters. Read counting for each transcript was performed by RSEM v. 1.3.066. Raw read counts were then scaled and normalized per sample by transforming to FPKM values, and cross-sample normalization was performed using a trimmed mean of M values (TMM67) for comparison between libraries and species. Scaling and normalization were performed using the Trinity scripts “align_and_estimate_abundance.pl” and “abundance_estimates_to_matrix.pl”, respectively.We generated new RNA-seq data for two types of central nerve tissues (cerebrum and midbrain) for a pit viper Deinagkistrodon acutus and true viper Vipera berus) were generated based on the structure of human KCNK4 (PDB: 3UM7), with which these proteins share approximately 62% sequence identity. The SWISS-MODEL server (https://swissmodel.expasy.org/) was used to perform homology modeling of the dimer complex. The N terminus (amino acids 1–33) and C terminus (amino acids 299–464) were removed because no reliable coordinates are available for these regions in human KCNK4. The resulting 3D structures were graphically represented using PyMOL (https://www.pymol.org) and Chimera68.The structural models of two snake KCNK4 channels (pit viper Supplementary information.Supplementary tables.


MACC1) gene, development and clinicopathological characteristics of uterine cervical cancer, and patient survival in Taiwan. Genotypic frequencies of 5 MACC1 SNPs rs975263, rs3095007, rs4721888, rs3735615 and rs1990172 were identified for 132 patients with invasive cancer, 99 with high-grade cervical intraepithelial neoplasia and 338 normal controls using real-time polymerase chain reaction. It revealed that there were no associations of these MACC1 SNPs with cervical carcinogenesis. In the meantime, cervical cancer patients with genotype GG in MACC1 SNP rs975263 tended to display more risk to have vaginal invasion than those with AA/AG . In multivariate analysis, positive pelvic lymph node metastasis could significantly predict worse 5 years survival rate  for cervical cancer patients. In conclusion, pelvic lymph node status rather than MACC1 SNPs was the only independent parameter that could significantly predict 5 years survival rate in Taiwanese women with cervical cancer.The objectives of this study were to define the associations among single nucleotide polymorphisms (SNPs) of metastasis-associated in colon cancer-1 ( Metastasis-associated in colon cancer-1 (MACC1) gene, which is related to colon cancer, was in the beginning found by Stein et al., using genome-wide expression method for an unique gene that was differently expressed in human primary colon cancer and metastatic tissues, as well as normal tissues Uterine cervical cancer is the fourth most common cancer in women worldwide MACC1 SNPs with clinical variables of colon cancer have ever been demonstrated MACC1 SNPs on the development and clinical outcome of cervical cancer has not been explored yet. Therefore, we conducted this study to investigate the involvement of the following 5 MACC1 SNPs rs975263, rs3095007, rs4721888, rs3735615 and rs1990172 in the development and clinicopathological characteristics of cervical cancer and patient prognosis in Taiwanese women.If the shared sequence of a gene exhibits a different single nucleotide between the individuals of a species, or paired chromosomes in an individual with a frequency of more than 5 %, single nucleotide polymorphism (SNP) is defined This retrospective study was designed by consecutively recruiting one hundred and thirty-two patients with invasive cancer and 99 women with high-grade CIN of uterine cervix from the Department of Obstetrics and Gynecology in Chung Shan Medical University Hospital in Taichung, Taiwan from February 1994 to October 2014. Meanwhile, 338 normal women, who received general examination at the Outpatient Patient Department in this hospital, were recruited as controls. The diagnosis for patients with invasive cervical cancer and those with high-grade CIN were included based on pathological report from colposcopy-directed cervical biopsy. Thereafter, cervical cancer patients underwent the standard treatment protocols, revised from guidelines provided by National Comprehensive Cancer Network and those with high-grade CIN, who were known to have precancerous lesions, underwent large loop excision of transformation zone, simple trachelectomy, abdominal or vaginal total hysterectomy. The normal controls were discriminated based on the cytologic report from cervical Papanicolaou smear and the report was further clarified by normal colposcopic findings. All subjects were Taiwanese women residing in central Taiwan. The study was approved by the Chung Shan Medical University Hospital institutional review board (CSMUH No: CS18208).The laboratory staff drew the blood samples from all participants using venipuncture technique. The specimens were collected into Vacutainer tubes mixed with ethylenediaminetetraacetic acid. They were stored at 4℃ shortly. DNA was extracted from leukocytes according to previous publication MACC1 genetic polymorphisms were selected based on the data of International HapMap Project and previous work MACC1 SNPs rs975263, rs3095007, rs4721888, rs3735615 and rs1990172 were checked by ABI StepOne Real-Time PCR System , and determined with SDS vers. 3.0 software, as our previous publication Five MACC1 SNP frequencies with various clinicopathological parameters.Analysis of variance (ANOVA) was applied for the comparison of the age distribution in the studied individuals using Tukey HSD test for post hoc analysis. Hardy-Weinberg equilibrium was performed to assess the genotypic frequencies of rs975263, rs3095007, rs4721888, rs3735615 and rs1990172 in normal controls [degree of freedom (d.f.)=2]. Chi-square or Fisher exact tests were applied to explore the relationships of a variety of MACC1 genetic distributions with the cervical carcinogenesis. The odds ratio (OR), adjusted odds ratio (AOR) and their 95% confidence intervals (95% CIs) were calculated using logistic regression model or multinomial logistic regression model after age adjustment. These tests were also performed to relate MACC1 SNPs and clinicopathological characteristics for 5 years survival in univariate analysis. The log-rank test was performed to identify the differences between these curves. Cox proportional hazard model was performed to evaluate the impacts of MACC1 SNPs and various clinicopathological parameters on 5 years survival in multivariate analysis relative to survival time. The SPSS, version 12.0 and WinPepi Software, version 10.0 were applied for statistical analysis. Hazard ratios (HRs) and their 95% confidence intervals (CI) were defined by the SPSS, version 12.0. P <0.05 was regarded as statistically significant difference.Kaplan-Meier curves were applied to plot the impacts of p<0.001). Using ANOVA with Tukey HSD test for post hoc analysis, the age difference was statistically significant between patients with cervical invasive cancer and those with precancerous lesion  as well as between those with cervical cancer and control women  but not statistically significant between women with precancerous lesions and control women .There was a statistical difference for age distribution between patients with cervical neoplasia and normal control women .The minor allele frequencies of MACC1 SNP rs975263 between the patients with cervical neoplasia and the normal controls  or variant homogenous genotype GG  as well as AG/GG  could not exert statistically significant distributions after age adjustment between patients with cervical neoplasia and the normal controls  as well as between patients with cervical invasive cancer and normal controls . Additionally, there was also no statistical difference in the frequencies of variant homogenous genotype GG of MACC1 SNP rs975263 using AA/AG as references after age adjustment between patients with cervical pre-cancerous lesions and normal controls  as well as between those with cervical invasive cancer and normal controls  as compared to those with AA 0.79 (95% CI=0.70-0.88) with no significantly different 5 years survival rate  as compared to those with AA/AG 0.82 (95% CI=0.75-0.90) with no significantly different 5 years survival rate , deep stromal invasion , larger tumor diameter , positive parametrium invasion  and positive pelvic lymph node metastasis  could be significantly associated with worse 5 years survival rate for cervical cancer patients  for cervical cancer patients in Taiwan , which is a nonsynonymous SNP exchanging serine to leucine, is situated at codon 515 in exon 5. In this study, it was however not found to have an impact on cervical tumorigenesis. This may be in agreement with the finding of Schmid et al. that exchange of leucine with serine in the MACC1 gene exerted no influence on the expression level of MACC1 mRNA in colorectal cancer MACC1 SNP rs4721888 (L31V) is in exon 4, leading to leucine exchange to valine MACC1 SNPs rs975263 (S515L) and rs4721888 (L31V) have been predicted to be probably benign in characteristic MACC1 SNP rs3735615 (R804T) is in exon 7, which exchanges arginine to threonine. But, our study could not demonstrate its association with cervical cancer in Taiwanese women. Meanwhile, both rs1990172 (A29858C) and rs3095009 (C77360T) are not in coding exon but in intron regions, which exhibit no impact on coding exon of MACC1 gene MACC1 SNPs between hepatocellular carcinoma (HCC) tissues and adjacent normal tissues, which showed that MACC1 SNPs probably have no influence on the risk of development of HCC MACC1 up-regulation was in the beginning identified to promote tumor proliferation, invasion, and metastasis in colon MACC1 SNPs on the clinicopathological variables of uterine cervical cancer was then examined. Cervical cancer patients with variant homozygote GG in MACC1 rs975263 tended to have vaginal invasion, as compared to those with AA/AG. However, no statistically significant difference was reached. There was no association of rs975263 with other clinicopathological parameters. Furthermore, other MACC1 SNPs displayed no significant relationships with clinicopathological characteristics. Although MACC1 overexpression was previously demonstrated to be associated with colon cancer metastasis MACC1 SNPs were not related to clinical parameters such as stage and lymph node invasion in colorectal cancer MACC1 SNP rs4721888 were higher in Chinese women with breast cancer, as compared to genotype GG The impact of MACC1 SNPs in the prognosis of cervical cancer patients. Therefore, we related the associations of MACC1 SNPs and clinicopathological characteristics with prognosis of cervical cancer patients, 5 years survival rate. In univariate and multivariate analyses, all the studied MACC1 SNPs rs975263 (S515L), rs4721888 (L31V) and rs3735615 (R804T) as well as rs1990172 (A29858C) and rs3095009 (C77360T) were not identified to be associated with 5 years survival rate in cervical cancer patients in Taiwan. Consistent with our findings, Schmid et al. found that the identification of coding MACC1 SNPs rs4721888 (L31V), rs975263 (S515L) and rs3735615 (R804T) in primary colorectal tumors does not significantly predict patient survival compared to MACC1 expression analysis alone. Only the genotype AG in MACC1 SNP rs975263 could be identified to be correlated with a decreased survival, but restricted in young colon cancer patients in early stage MACC1 SNPs rs1990172 could be predictable of decreased overall survival in patients with colorectal cancer MACC1 SNPs was the only significantly predictive parameter of 5 years survival rate in Taiwanese patients with cervical cancer MCAA1 has been demonstrated to be associated with prognosis of a variety of cancers, such as gastric cancer MACC1 SNP rs975263 tended to have more risk to develop vaginal invasion than those with AA/AG. But, this did not reach a statistical significance. Therefore, more cases may be needed to explore the role of SNPs rs975263 (S515L) that is in coding exon 5. Secondly, the mechanisms by which SNPs affect the function of MACC1 gene should be further delineated. Thirdly, in addition to coding exon and intron regions, other SNPs that are in the promoter region or the 3'-untranslated region where microRNAs may interact, should be included to investigate.The study has some limitations. Firstly, a larger cohort of patients is necessary. Cervical cancer patients presenting genotype GG in


We point out that mild post deposition treatments lead to the conversion of CsPb2Br5 and Cs4PbBr6 into CsPbBr3 due to its higher thermodynamic stability. Conversion results into smooth and pinhole-free CsPbBr3 films with good light absorption and emission properties. We demonstrate the suitability of obtained films for planar devices by preparing perovskite-based pure-green light emitting diodes, thus promoting Single Source Thermal Ablation as a promising alternative deposition technique for all-inorganic perovskite-based devices.Hybrid organo-lead halide perovskites are becoming the benchmark material for next generation photovoltaics and a very important player for other applications such as photodetectors and light emitting diodes. Nevertheless, the most important issue hindering the large-scale application of these materials remains their intrinsic instability due to the organic cation. Although the substitution with inorganic cesium (Cs) enhances stability, in most cases solution deposition methods of fully inorganic perovskites result in high surface roughness and poor surface coverage. This work reports on the evaporation of the CsPbBr In the last few years, the introduction of metal halide perovskites in solution processable solar cells led to impressive advances that today make perovskite solar cells (PCSs) the fastest developing solar technology  include in the A-site organic cations such as methylammonium (MA) and formamidinium (FA), which make these materials suffer from environmental degradation under moisture, oxygen, heat, and illumination exposure, so that long-term stability remains the main issue hindering perovskite real-life application  perovskites  was lost after the thermal treatment, thus making these films not suitable for light emitting devices.An alternative to dual source evaporation is the single source vapor deposition (SSVD) technique where powders of either raw precursors or preformed perovskites are placed into an alumina thermal source that is heated by a rapid increase of the work current. This method was used to evaporate CsPbX3. Due to the high vapor pressure of MAI, high quality films require not only high currents to make MAI and PbI2 evaporation as simultaneous as possible, but also a MAI excess in the precursor (up to MAI to PbI2 molar ratio of 2.0)  in DMSO under continuous stirring. The test tube was then sealed and removed from the glove box. In order to improve reproducibility, the subsequent operations were performed in rooms where temperature was maintained in the 19–22°C range and relative humidity (RH) was kept below 20%. Hereafter, these environmental conditions will be referred as “DH atmosphere.”A 0.45 M precursor solution was prepared in a nitrogen-filled glove box by dissolving at room temperature equimolar amounts of CsBr (Aldrich) and PbBrGlass substrates were cleaned by sequential rinsing in 1% Hellmanex, de-ionized water, hot acetone, and isopropanol for 15 min. After drying in blowing nitrogen, up to four substrates were placed on a sample holder that was mounted into the evaporation chamber at a fixed vertical distance of 8 cm from the evaporation source. The evaporation system was tailor made  that was then placed on a hotplate at 80°C covered by a Petri dish. Temperature was then raised to 100°C to promote DMSO evaporation. This procedure was repeated by adding another 40 μl of the precursor solution. As the final step, the boat was heated at 120°C for 5 min to remove the excess solvent, finally producing a pale orange layer inset in  that wastructure  . The chamber was then opened, substrates were replaced by virgin others and the chamber was pumped again to vacuum. Then, a second evaporation was performed by applying 150 W power until the boat became incandescent (step 2).Thermal annealing treatments were performed into a laboratory oven in DH atmosphere.α radiation. Diffraction patterns were collected by 0.05° 2θ steps and counting times of 5 s per step. In some cases, counting times as low as 2 s per step were used in order to minimize measurement duration despite an increase of the background noise.All the film characterizations were carried out in DH atmosphere. In particular, the structural quality of the films was studied by X-ray diffraction (XRD) measurements in a Siemens (D500) powder diffractometer, with CuKFilm absorbance spectra were measured by a Jasco UV–vis V-530 spectrometer. Morphological characterization was performed by atomic force microscopy (AFM) using a Veeco Dimensions 3100 SPM and by scanning electron microscopy (SEM) using a Zeiss Auriga field emission microscope (FESEM) operated at 5 kV. Selected Area Electron Diffraction (SAED) and High-Resolution transmission Electron Microscopy (HREM) were performed in a Jeol 2200FS Transmission Electron Microscope (TEM).−2, spot area 0.5 cm2). Emission decay time were determined with the single photon counting technique by means of the same Edinburgh FLS980 spectrometer using a laser diode as excitation source  and a Hamamatsu MCP R3809U-50 (time resolution 20 ps) as detector  . An Edinburgh Xe900 450 W Xenon arc lamp was used as exciting light source. Corrected spectra were obtained via a calibration curve supplied with the instrument  doped with cesium atoms. A 10 nm thick neat film of pure BPhen after the emissive layer was also included as hole-blocking layer. The structure was closed with a cathode of 120 nm thick silver (Ag).Concerning PeLEDs fabrication, before the deposition of the organic compounds, Indium Tin Oxide (ITO)-covered glass substrates were cleaned in acetone, isopropanol, and deionized water for 10 min at 60°C in an ultrasonic bath. On top of the oxide anode, the hole injection film consists of 40 nm of spin-coated poly polystyrene sulfonate (Pedot:PSS). Then, the n-doped layer, following CsPbBr−8 mbar.The small molecules materials were deposited by thermal evaporation in a Kurt J. Lesker organic thin film and metallization deposition system, at a base pressure around 10The fabricated PeLEDs have been characterized by electrical-optical measurements performed under vacuum with an Optronics OL770 spectrometer, coupled, through an optical fiber, to the OL610 telescope unit for the luminance measurements. The whole system, NIST calibrated using a standard lamp, was directly connected by RS232 cable to a Keithley 2420 current-voltage source meter.2 in DMSO, as explained in the Experimental methods. Soon after the deposition, films obtained by this kind of precursor layers exhibit a full coverage of the substrate  and rhombohedral Cs4PbBr6 (ICSD 254272). Noteworthy, obtained films show pronounced, although partial (010), orientation of the crystallites, likely induced kinetically by the growth conditions.Powder XRD patterns collected immediately after removing the sample from the evaporation chamber  reported in 3, MAI starts evaporating before PbI2 after the initial perovskite decomposition  is about 3 V, a value very near to the thermodynamic limit given by the energy gap per unit charge, suggesting that the voltage drop due to interfaces does not have an important role in the light emission.The above results show that smooth, compact, and pinhole-free CsPbBrntensity . Two set3, centered at 522 nm with a full width at half-maximum (FWHM) of 16 nm that equals the smallest value reported in the literature for green PeLEDs (Zhang F. et al., Interestingly, although the efficiency is rather low , Figure 3 precursors, showing by XRD measurements that as-prepared films consist of a mixture of CsPbBr3, CsPb2Br5, and Cs4PbBr6 phases, due to vertical composition gradient following the partial decomposition of the precursor during evaporation. We showed that deposited films turn into pure CsPbBr3 phase in low humidity atmosphere, either spontaneously at room temperature or by mild thermal treatments. This conversion affects also film absorbance, whose spectra show an absorption onset at about 530 nm that is ascribed to CsPbBr3 and whose height increases with time until saturation is achieved. Morphology changes associated to the conversion into CsPbBr3 lead to polycrystalline films, whose features depend on the conversion procedure. Compact, smooth and pinhole-free films were achieved by aging in DH atmosphere at room temperature for 24 h or by annealing at 100°C after overnight aging in DH atmosphere. The suitability of obtained CsPbBr3 films for planar devices was assessed by fabricating PeLEDs with a very narrow green electroluminescence, thus confirming the potential of the obtained CsPbBr3 films. Reported results show how SSTA can be used profitably to achieve planar heterostructure devices based on all-inorganic halide perovskites.We investigated the preparation of films by single source thermal ablation of CsPbBrThe datasets generated for this study are available on request from the corresponding authors.RM, MM, and AL designed the project. LN performed the AFM and TEM investigation. DC performed the SEM characterization. FMe performed the XRD characterization. PF contributed technically to the film deposition and XRD characterization. RM supervised the film deposition, contributed to the XRD characterization, performed the absorbance characterization, and wrote the first draft of the manuscript. AL performed the steady state and time resolved photoluminescence measurements. FMa and MM contributed in the fabrication and characterization of the electroluminescent devices. All authors contributed to the discussion and revision of the manuscript and approved the final version.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.