Phenylethyl isothiocyanate (PEITC) was reported as a useful antioxidant, anti-inflammatory, and chemopreventive agent. Due to technological and stability issues, it is necessary to be able to extract PEITC from its natural matrix (watercress) through sustainable and scalable methodologies. In this article, we explored, for the first time, the extractive capacity of aqueous micellar systems (AMSs) of two non-ionic surfactants. For this, we compared the AMSs with conventional organic solvents. Furthermore, we developed and optimised a new integral PEITC production and extraction process by a multifactorial experimental design. Finally, we analysed the antioxidant capacity by the oxygen radical absorbance capacity (ORAC) and ABTS methods. As results, the AMSs were able to extract PEITC at the same level as the tested conventional solvents. In addition, we optimised by response surface methodology the integrated process , which was equally effective (ca. 2900 µg PEITC/g watercress), regardless of the surfactant used. The optimal extracts showed greater antioxidant capacity than pure PEITC, due to other antioxidant compounds extracted in the process. In conclusion, by the present work, we developed an innovative cost-effective and low environmental impact process for obtaining PEITC extracts from watercress by-products. Brassicales) by the enzyme myrosinase [Some cruciferous plants are classed as “superfood”, because they may have beneficial effects in the treatment and prevention of several health disorders. This property is ascribed to their complex nutritional and phytochemical profile, which includes high concentrations of carotenoids, flavonoids, phenolic acids, and isothiocyanates . Isothiorosinase ,3. In throsinase : the reaNasturtium officinale—WC) is the richest known source of the gluconasturtiin (phenylethyl glucosinolate) derived phenylethyl isothiocyanate (PEITC) [Among the aforementioned superfoods, watercress ( (PEITC) . PEITC—o (PEITC) ,4,6. Unf (PEITC) ,7,8. BesTherefore, the production and extraction of PEITC from WC discards may represent an opportunity to extract it in a stable and bioactive form taking advantage of an important by-product, generating great value. In that sense, the reported work is scarce and, even more, the existent studies are not using a sustainable approach ,10,11. TIn recent decades, a wide range of new non-toxic, non-flammable, and biodegradable solvents have been evaluated to develop environmentally friendly and sustainable extraction methods . Among tIn this framework, we carried out a study to apply AMSs to produce extracts enriched with PEITC, developing a profitable process with low environmental impact. As a road map, we pose the following questions: Are Genapol X-080 and Tergitol 15-S-7 AMSs suitable for PEITC extractants, comparable to currently used solvents? What are the variables that most affect the extractive process of PEITC with AMSs? What are the optimal conditions that maximize PEITC extraction?The surfactants Genapol X-080 (GX080) and Tergitol 15-S-7 (Tg7) were supplied from Sigma-Aldrich . Methanol, acetonitrile (ACN), chloroform (CF), and n-hexane were of HPLC grade. All the reagents were used as received without further purification. PEITC standard  were dissolved in pure methanol until obtaining 22–660 µg/mL. All the other reagents were of analytical grade and used without further purification. Ultrapure water was used to prepare all the solutions.Fresh WC by-products  was kindly supplied by the food processing company Vitacress Portugal SA . When received, WC was dehydrated in a dryer at 50 °C for 24 h  and thenAn exploratory analysis of the efficacy of different solvents to extract PEITC was performed; 1.00 g of the pulverized WC was suspended in 15 mL of 25 mM phosphate buffer pH 7.0 for 2 h at 37 °C to allow the hydrolysis of the glucosinolates . Then, 0A), temperature (XB), and pH value (XC) and the selected response variable (Y) was the PEITC content, as above. The design resulted in an arrangement of 15 treatments, which was executed in triplicate  on successive days. The levels of the factors, coded as −1 (low), 0 , and +1 (high), are shown in An integrative process to produce and extract PEITC was developed and optimised. To do this, an experimental strategy was implemented to reduce the number of experiments necessary to optimise the process, as well as to establish the most influential factors. For this purpose, a Box-Behnken design was selected, which is a type of 3-level response surface design that includes a subset of runs of a full factorial at three levels. The factors evaluated were surfactant concentration . The separation was carried out by applying the operating conditions described by Fusari, Ramirez, and Camargo , with slblank,t=0/fluorescencecontrol,t=0. From the normalized curves, the area under the fluorescence decay curve (AUC) was calculated according to the trapezoidal method. The final AUC values were calculated by subtracting the AUC of the blank from all the results. Regression equations between net AUC and antioxidant concentration were calculated. Final ORAC values were expressed as μmol TE /mg PEITC.Oxygen radical absorbance capacity (ORAC) assay was performed following the methodology used by Coscueta et al. , with so•+) was produced from the reaction of 7 mM 2,20-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt and 2.45 mM potassium persulfate  after incubation at room temperature in the dark for 16 h. For ABTS•+ working solution (freshly prepared), the ABTS•+ was filtered with a 0.45 µm syringe filter and diluted with the solvent to an absorbance of 0.70 ± 0.02 at 734 nm for the control (20 µL solvent + 180 µL ABTS•+ working solution). To 20 µL of sample or Trolox (25–175 μM) or solvent was added 180 µL of ABTS•+ working solution. The mixture was incubated for 5 min at 30 °C, and the absorbance at 734 nm was measured with a multidetection plate reader  controlled by the Gen5 Biotek software version 3.04. The scavenging activity was expressed as % reduction in absorbance for the control. Regression equations between net ABTS scavenging and Trolox concentration were calculated and the results expressed as μmol TE /mg PEITC.ABTS scavenging assay was performed in a 96-well microplate, following the method described by Gonçalves et al.  with somEach experiment was performed in triplicate and the results were expressed as the mean values with standard deviations (SD). The major statistical analysis was carried out with the aid of RStudio V 1.2.1335.Comparison of extraction efficiency: The mean PEITC values were analysed statistically by one-way analysis of variance (ANOVA) followed by the Tukey’s posthoc test ,30. SepaAX and BX are the coded levels of the independent variables mentioned above; β0, βi, βi,I, and βi,j are the regression coefficients for the independent term, the linear, quadratic, and binary interaction effects, respectively; and ε, the residual error [Optimisation: The response values (Y), i.e., the PEITC in the supernatants from each treatment, were fitted to the following polynomial quadratic model:al error ,31. The In the extraction of isothiocyanates, liquid-liquid extraction is generally applied. The highly water-soluble glucosinolates are hydrolysed in the aqueous medium by myrosinase, producing the isothiocyanates. The highly hydrophobic isothiocyanates were extracted from the aqueous medium by an immiscible solvent (phase separation) or by decreasing polarity with a less polar miscible solvent. We tested a set of different solvent systems as PEITC extractants; we compared AMSs of two aliphatic alcohol ethoxylates (Genapol X-080 and Tergitol 15-S-7) with conventional organic solvents . SystemsAfter corroborating the extractive efficacy of the studied AMSs, we decided to develop an integrated and optimal process for the production and extraction of PEITC. To do this, we applied the conditions to produce autolysis directly in the AMS. After the incubation time, it was only necessary to separate the remaining solid material by filtration.According to literature, many variables affect the extraction process of a given substance from a solid matrix, i.e., temperature, extraction time, pH, the particle size of the solid, stirring rate, and extractant concentration . In this2adj, fitting again from the multiple regression of the data. 2, R2adj, and RSD. The new models adequately explain the variation in the response, with a maximum R2adj. Then, we recalculated the models, by eliminating the non-significant effect , 25 °C. The fitted models predicted maximum PEITC values of 3199 ± 338 µg PEITC/g WC DB and 3306 ± 312 µg PEITC/g WC DB, respectively, for Genapol X-080 and Tergitol 15-S-7. The optimal condition to apply in the process with the AMSs would be 2.0% m/m at 25.0 °C and a pH value of 9.0 for both surfactants. As shown by the extractant comparison, in the optimal conditions of the process developed for the AMSs, the predicted PEITC values are not different between Genapol X-080 and Tergitol 15-S-7, similarly to what we reported for the extraction of soy isoflavones [We analysed the antioxidant activity of the optimised extracts, using the ORAC and ABTS methodologies. The use of conventional extraction with n-hexane has led to obtaining an amount of PEITC  almost h−3 and 3.9 10−3% m/m, respectively [Nevertheless, we asked the question: Why are these AMSs so effective as organic solvents in extracting a hydrophobic molecule like PEITC? Genapol X-080 and Tergitol 15-S-7 have high effectiveness in interacting with low polar molecules in plant matrices. This appears to be closely related to the amphiphilic character of these surfactants and its ability to form micelles that can interact with hydrophobic groups of PEITC molecule. As previously discussed, it should be noticed that Genapol X-080 and Tergitol 15-S-7 have critical micellar concentrations (CMCs) of 4.6 10ectively . The conConcerning the factors that affect the extractive process, Farhana, et al., with a one-factor approach, also observed similar effects of pH and temperature on PEITC production . In thatAt this point, it is important to mention the appropriateness of using solid statistical tools in the optimisation process. Biological matrices are complex systems strongly affected by different variables and their interactions, making it practically unfeasible to predict accurate behaviours based on the classic one-factor method (one variable at a time). Both multifactorial models indicate a maximisation towards one of the vertices of the design, leaving us the mystery of what could happen in conditions beyond that vertex, that is, at factor levels beyond those analysed. However, within the tested conditions, we can talk about an interesting scenario: these predictions of a unified condition, with similar results, provide the desired scenario of flexible technology, applicable more generally and not restricted to a particular surfactant.Regarding antioxidant activity, the differences between the pure PEITC and the extracts may be due to the other phytochemicals that the AMSs were able to extract, such as phenolic compounds ,25. RodrAlthough there has been a growing interest in isothiocyanates in recent years, the information available is still scarce. There is practically no solid base of work with a technological focus and still less for the PEITC. Therefore, considering the potential of this compound and the technological relevance it may have soon, an exploratory study of scalable green methodologies for its production and extraction is of high impact.In the present study, we report the development of a methodology to extract PEITC by applying cost-effective and low environmental impact systems, reaching the extractive capacity of classical organic solvents. Thus, our exploratory and optimisation work—with robust statistical tools—allows us to contribute with an innovative precedent in the state of the art.The flexibility of selection of different surfactants  for PEITC extraction with similar results, as well as the possibility of substituting organic solvents in large-scale extraction procedures, make these AMSs suitable for industrial production of PEITC from WC.As a final consideration, the extracts obtained resulted in an interesting antioxidant product, which may synergistically combine different mechanisms of direct and cellular action against harmful oxidative states. However, this must be studied in greater depth and validated by other methodologies.


The mechanistic target of rapamycin complex 1 (mTORC1) is an essential regulator of cell growth and metabolism through the modulation of protein and lipid synthesis, lysosome biogenesis, and autophagy. The activity of mTORC1 is dynamically regulated by several environmental cues, including amino acid availability, growth factors, energy levels, and stresses, to coordinate cellular status with environmental conditions. Dysregulation of mTORC1 activity is closely associated with various diseases, including diabetes, cancer, and neurodegenerative disorders. The discovery of Rag GTPases has greatly expanded our understanding of the regulation of mTORC1 activity by amino acids, especially leucine and arginine. In addition to Rag GTPases, other factors that also contribute to the modulation of mTORC1 activity have been identified. In this review, we discuss the mechanisms of regulation of mTORC1 activity by particular amino acids. Saccharomyces cerevisiae , , 136], o to RagB .Finally, glutamine indirectly participates in mTORC1 activation by acting as the key amino acid for the uptake of leucine Fig. d. The so2+ mobilization. In fact, more than two decades ago, Graves et al. reported that treatment with Ca2+ ionophore A23187 or thapsigargin, increasing intracellular Ca2+ concentration, activated S6K1, while this effect was avoided by pretreatment with a Ca2+ chelator, such as EGTA or BAPTA-AM [2+ levels affected the phosphorylation status of S6K1 but not Akt in response to mitogenic stimulation [2+ concentration by pretreatment with the Ca2+ chelators EGTA or BAPTA prevented the activation of S6K1 but not Akt. On the other hand, an increase in intracellular Ca2+ concentration induced by ionomycin or thapsigargin treatment triggered the hyperphosphorylation of S6K1, but little or no activation of Akt. Thus, intracellular Ca2+ concentrations preferentially affect S6K1 activation. Interestingly, it was reported that the addition of amino acids, especially leucine, to amino acid-starved HeLa cells resulted in an increased intracellular Ca2+ concentration [2+ levels lead to the direct binding of Ca2+/calmodulin (CaM) to the human Vps34 (hVps34), which triggers the production of PI3P from phosphatidyl inositol -dependent manner -phospholipase C beta 4 (PLCβ4) axis, although it remains unknown how leucine signals are transduced to SHP-2 [2+ release and S6K1 phosphorylation. In addition, leucine-induced cytosolic Ca2+ accumulation is sensitive to the pertussis toxin, which inhibits the Giα subunit of heterotrimeric G-proteins. These results suggest that leucine is sensed by a type of GPCR. In this regard, it is interesting to note that the umami taste 1 receptor T1R1/T1R3, a heterodimeric GPCR complex, can sense amino acid availability to regulate mTORC1 in a variety of cell lines , thus activating mTORC1. Indeed, pretreatment with nifedipine, a VDCC inhibitor, reduced mTORC1 activation by amino acids [T1R3 knockdown resulted in reduced lysosomal translocation of mTOR in response to amino acids, it remains to be determined whether inhibition of Ca2+ entry through VDCCs led to a decrease in lysosomal localization of mTOR.In C2C12 myoblasts, leucine administration was reported to induce increased intracellular Cato SHP-2 . Neverthno acids . However2+ stores. Lysosomal Ca2+ can influence mTORC1 activity, especially under stressful conditions [2+ channel, TRPML1 , might represent the missing link between lysosomal Ca2+ release and mTORC1 regulation in a context-dependent manner , which forced mTORC1 translocation to the lysosome. This phenomenon indicates that TRPML1-dependent Ca2+ release can also affect mTORC1 activity independently from the localization of mTOR. Indeed, it has been reported that TRPML1 is weakly associated with mTOR. Furthermore, CaM is also associated with mTORC1 in a Ca2+-dependent manner and binding of Ca2+/CaM to mTORC1 may increase mTORC1 kinase activity in vitro [2+ release appears to affect mTORC1 activity through several different mechanisms. Moreover, it has been reported that transient nutrient starvation can induce TRPML1-mediated lysosomal Ca2+ release [2+ release activity [2+ release. Furthermore, TRPML1 is required for mTORC1 reactivation under prolonged starvation [In addition to the ER, lysosomes also act as intracellular Canditions , 153. A ner Fig. d [152–15 release . Becauseactivity , it is pIn the current model of mTORC1 activation, signals from amino acids and growth factors are independently transduced to mTORC1, as illustrated in Fig. Intriguingly, recent studies have demonstrated that both amino acids and growth factors govern the intracellular localization of TSC2, thus controlling a key mechanism for the regulation of mTORC1 activity , 60, 61.Recent biochemical and structural analyses have dramatically improved current knowledge of the molecular mechanisms of amino acid sensing, especially leucine and arginine, which ultimately converge on Rag GTPases to regulate mTORC1 activity. As shown above, it is well established that the involvement of Rag GTPases is essential for amino acid-mediated mTORC1 regulation, but much more complex regulatory mechanisms of mTORC1 have also emerged. Undoubtedly, further studies will provide new insights into how mTORC1 activity can be regulated by the integration of a variety of input signals to maintain cellular homeostasis; this knowledge will also provide novel approaches to treat human diseases, especially those associated with aberrant mTORC1 activity.


Canavalia rosea, a seashore and mangrove-accompanied halophyte with strong adaptability to adversity in tropical and subtropical regions, is a good model for studying the molecular mechanisms underlying extreme saline-alkaline and drought stress tolerance in leguminous plants. In this study, a PIP2 gene  was cloned from C. rosea, and its expression patterns and physiological roles in yeast and Arabidopsis thaliana heterologous expression systems under high salt-alkali and high osmotic stress conditions were examined. The expression of CrPIP2;3 at the transcriptional level in C. rosea was affected by high salinity and alkali, high osmotic stress, and abscisic acid treatment. In yeast, the expression of CrPIP2;3 enhanced salt/osmotic and oxidative sensitivity under high salt/osmotic and H2O2 stress. The overexpression of CrPIP2;3 in A. thaliana could enhance the survival and recovery of transgenic plants under drought stress, and the seed germination and seedling growth of the CrPIP2;3 OX (over-expression) lines showed slightly stronger tolerance to high salt/alkali than the wild-type. The transgenic plants also showed a higher response level to high-salinity and dehydration than the wild-type, mostly based on the up-regulated expression of salt/dehydration marker genes in A. thaliana plants. The reactive oxygen species (ROS) staining results indicated that the transgenic lines did not possess stronger ROS scavenging ability and stress tolerance than the wild-type under multiple stresses. The results confirmed that CrPIP2;3 is involved in the response of C. rosea to salt and drought, and primarily acts by mediating water homeostasis rather than by acting as an ROS transporter, thereby influencing physiological processes under various abiotic stresses in plants.Aquaporins are channel proteins that facilitate the transmembrane transport of water and other small neutral molecules, thereby playing vital roles in maintaining water and nutrition homeostasis in the life activities of all organisms.  Canavalia rosea (Sw.) DC., also known as bay bean, is mainly distributed in coastal and island sandy soils in tropical and subtropical areas. It is a perennial halophyte belonging to the Fabaceae family that has adapted to extreme environments with high temperature and strong light in coastal areas and exhibits significant salt-alkali tolerance and drought resistance [C. rosea shows great ecological adaptability on tropical coral reefs, which makes it a candidate greening species in restructuring the ecological functions of marine and island ecosystems. As a sea-dispersed legume with a high level of nutrient utilization efficiency and good nitrogen-fixing capacity, C. rosea constitutes a superior wild plant resource and plays important roles in wind resistance, sand fixation, landscape greening, and ecological restoration in the vegetated areas on coral islands and in coastal zones [C. rosea offers a superior genetic resource pool to identify abiotic stress tolerance genes, especially salt/drought resistance genes involved in osmotic and water deficit tolerance, for the further abiotic-stress-related genetic improvement of leguminous plants or other glycophytic crops.sistance ,2. In paal zones . In addiAquaporins (AQPs) are highly conserved integral membrane channel proteins, also known as major intrinsic proteins (MIPs), that are mainly involved in water movement across membranes in cells ,4. AQPs Xenopus oocytes, whereas PIP1s generally have much lower or no water channel activity [PIP gene expression is differentially regulated in various tissues and is also altered under different physiological and environmental stresses, including abiotic stresses and plant hormones [PIPs can be further divided into two subclasses, PIP1s and PIP2s, based on the length of the N- and C-termini of the PIPs . In planactivity . Severalactivity ,11. The activity . PIPs plactivity ,15, pathactivity ,17, metaactivity ,19,20, gactivity ,22, or oactivity  and ureaactivity . Plant Phormones ,26, espehormones . Many rehormones , plant bhormones ,28, subchormones , proteinhormones , and prohormones ,31,32.AQPs in transgenic plants increases resistance to abiotic stresses [Atriplex canescens is a temperate halophyte with excellent saline-alkaline tolerance, and its plasma membrane intrinsic protein gene, AcPIP2, improved salt and alkali tolerance, leading to increased sensitivity to drought stress, when heterologously overexpressed in Arabidopsis thaliana [A. canescens nodulin-like plasma membrane intrinsic protein gene, AcNIP5;1, showed a totally opposite phenotype when expressed in A. thaliana [Sesuvium portulacastrum is a perennial halophyte that typically grows in coastal and inland sandy soils and exhibits excellent tolerance to salt. SpAQP1 (a PIP2 member) enhanced the salt tolerance of yeast strains and tobacco plants and promoted seed germination and root growth under salt stress in transgenic plants [Selaginella moellendorffii is a desiccation-tolerant plant, and a total of 19 AQPs were detected in the S. moellendorffii-expressed sequence tag (EST) database, while only three conserved PIPs were detected [SmPIP1;1 and SmPIP2;1 exhibited different water-channel activities, and their co-expression showed a synergistic effect on the water membrane in an oocyte system, while no synergistic effect was observed in yeast [Stipa purpurea showed great adaptability to stresses such as drought and a changing environment in the meadows of the Tibetan Plateau [SpPIP1 increased significantly under drought, and the ectopic expression of SpPIP1 in A. thaliana elevated the drought tolerance of the plants [Simmondsia chinensis) is a typical desert plant with strong tolerance to drought, salinity, and nutrient-poor soils, and ScPIP1 could improve salt and drought resistance when expressed in A. thaliana [Thellungiella salsuginea displays strong resistance to high salinity, drought, and chilling stress. The overexpression of TsPIP1;1 in rice could also enhance the salt tolerance of rice by maintaining the osmotic potential and promoting photosynthesis [NnPIP1-2 and NnPIP2-1, from the hydrophyte salt-tolerant lotus Nelumbo nucifera, could enhance the abiotic stress tolerance of transgenic plants when overexpressed in A. thaliana [PIPs from plants growing in specialized habitats can be excavated as potential target genes for enhancing plant resistance to abiotic stresses though genetic engineering.In recent years, there have been numerous reports that the overexpression of stresses ,28, therthaliana . Anotherthaliana . Sesuviuc plants . Selagindetected . SmPIP1;in yeast . Stipa p Plateau . The trae plants . Jojoba thaliana . Thellunynthesis . Both Nnthaliana . These fC. rosea, an entire cDNA library was constructed from C. rosea seedlings for yeast functional screening. A series of ESTs were previously characterized, including CrPIP2;3. In this study, this AQP gene CrPIP2;3 was further functionally characterized based on an over-expression assay in yeast and A. thaliana. Our results suggested that CrPIP2;3 was significantly induced by high osmotic stress and could enhance the salt–alkali and drought tolerance of transgenic plants by facilitating water transport and increasing the drought tolerance of the plants.To further understand the mechanism of salinity tolerance in CrPIP2;3 was isolated from the cDNA library constructed using C. rosea seedlings. The length of the CrPIP2;3 cDNA is 1254 bp, with a 51-bp 5′ untranslated region (UTR) and a 333-bp 3′ UTR (GenBank Accession No.: MT787666). The open reading frame (ORF) is 870 bp, encoding a protein with a predicted molecular weight of 31.07 kDa and a theoretical isoelectric point of 7.66. CrPIP2;3 was characterized as an aquaporin with an MIP (major intrinsic protein) domain   (The full-length cDNA of PF00230) A. The in to TM6) B. Sequen to TM6) A, which  to TM6) C. We alsCrPIP2;3 (promoter region) using PlantCARE indicated that the putative cis-acting regulatory elements were classified into three types: the transcription factor-binding site involved in drought inducibility, the hormone-responsive element, and the light-responsive element , the WUN motif (wounding inducible), and several light-responsive elements   of  element . The seqT-motif) . This reCrPIP2;3, qRT-PCR was performed to explore the expression patterns under stress or ABA treatments and normal growth conditions using the total RNA extracted from the various tissues. We checked the expression of CrPIP2;3 in the whole C. rosea plant. The result indicated that CrPIP2;3 was expressed extensively in all tested tissues/organs of C. rosea and showed a relatively high transcriptional level with respect to the reference gene CrEF-α  in all three tested tissues when compared with CK samples (the beginnings of each treatment) , while in the vine and leaf, the expression level of CrPIP2;3 remained obviously decreased until 48 h. Under alkali toxicity (150 mM NaHCO3), the expression level of CrPIP2;3 exhibited correspondingly low levels, with no major changes observed  A. The exobserved B. The hinhibited C. The ABpression D. In briifferent . CrPIP2;CrPIP2;3 was first performed with a yeast expression system. The cell culture was adjusted to an OD600 value of 1 and then gradient-diluted . Two microliters of yeast solutions were spotted onto SDG agar plates with or without NaCl, sorbitol, or H2O2. As the results showed, W303 transformed with either CrPIP2;3 or pYES2 grew normally and did not show growth differences on the SDG control plate. However, with increased NaCl, W303 transformed with CrPIP2;3 showed an obvious growth lag compared with the yeast containing the pYES2 control  were selected and applied in the following tests. Briefly, about 30 sterilized seeds were spotted on the MS medium with the addition of salt, saline-alkaline, or mannitol stress, following which the germination rate was calculated. As indicated in CrPIP2;3 OX lines could be observed, and the germination of the CrPIP2;3 OX lines was higher than that of the WT when the seeds were grown in MS medium containing 200 mM NaCl or 5, 7.5, and 10 mM NaHCO3, and the corresponding statistical analysis of the seed germination rates also showed consistent results with the germination status, with better salt and alkali tolerance  cultured on MS medium for 7 d were transplanted to MS plates with different stresses to evaluate their tolerance at the seedling growth stage. Basically, the main root length of the WT and CrPIP2;3 OX lines was suppressed with the increase in the concentration of NaCl, NaHCO3, or mannitol. Although there were no clear differences between the WT and CrPIP2;3 OX lines under 150 or 200 mM NaCl, about half of the WT seedlings died under 200 mM NaCl , the length of the WT root was slightly shorter than that of the CrPIP2;3 OX lines  was relatively consistent. There was no difference in the tolerance of the adult plants between the WT and transgenic lines  under salt (200 mM NaCl) or salt–alkali  stresses (results not shown). Apparently, CrPIP2;3 strongly improved the drought tolerance during the growth of the adult plants  encodes a highly ABA-induced protein phosphatase 2C member, which is responsible for the ABA-activated signaling pathway; the expression of RD29B (At5g52300) is induced in response to water deprivation, and this response is mediated by ABA. Under salt stress treatment (200 mM NaCl), both HAI2 and RD29B in the CrPIP2;3 OX plants showed obviously elevated expression patterns  and high osmotic (400 mM mannitol) stress both induced a higher expression of RD29B in CrPIP2;3 OX plants than in WT plants  and ANAC019 (At1g52890) encode NAC transcription factors, which are involved in ABA-mediated dehydration responses. Both genes were significantly enhanced in CrPIP2;3 OX plants under salt, high osmotic, and alkali stress treatments , CSD1 (At1g08830), APX1 (At1g07890), and FSD1 (At4g25100), were also evaluated by qRT-PCR, and the results indicated that the expression of these genes was not affected by CrPIP2;3 overexpression, even under salt, high osmotic, and alkali stress challenges , wild soybean (Glycine soja), and common vetch (Vicia sativa) are all legumes with better drought or salinity stress tolerance than standard crops. The AQP family in these three species has been systematically characterized, providing valuable information for further functional analysis to infer the roles of AQPs in adaptation to diverse environmental conditions [Simmondsia chinensis), date palm (Phoenix dactylifera), and jujube (Ziziphus jujuba) can all tolerate drought, salinity, and nutrient stress, and their AQP genes have been intensively studied to better understand their potential for genetic engineering to improve plant stress tolerance [Atriplex canescens, Sesuvium portulacastrum, and Thellungiella salsuginea all exhibit strong salt tolerance, and their PIP genes have been found to function in the response to salt stress and could be used in genetic engineering to improve plant growth under abiotic stress [AQP gene from the halophyte C. rosea, CrPIP2;3, was grouped into the PIP2 subfamily and showed high levels of homology to CaPIP2;2 and GmPIP2;5 (more than 80% identity in the amino acid sequence)  A,C, whicequence) A,B, and AQPs should provide the most immediate evidence [CrPIP2;3 is expressed in different tissues or organs at different expression levels in C. rosea plants , alkali toxicity , high osmotic conditions (300 mM mannitol), and ABA treatment (100 μM). The expression pattern of CrPIP2;3 varied with stress treatment duration, generally first decreasing and then increasing , following which the expression decreased. This indicated that alkali stress could temporarily enhance root hydraulic conductivity in C. rosea, and with the increase in alkali stress treatment, the water balance could be maintained even though the expression of CrPIP2;3 decreased in the root. This could be because the water imbalance caused by alkali toxicity was only slight. High osmotic stress strongly induced CrPIP2;3 expression (about 25 times) in C. rosea roots after 48 h, which may suggest that the high expression of CrPIP2;3 could help to improve water absorption from the outside by the roots, and the higher expression of CrPIP2;3 in the vine could help coordinate water transport to the aerial parts of C. rosea. In the leaf, the expression of CrPIP2;3 was obviously decreased under all challenges or ABA treatment, which might be a protective strategy to reduce water loss via the leaves by transpiration. In general, our CrPIP2;3 expression results indicated that CrPIP2;3 may participate in osmotic and salt-alkali tolerance in C. rosea and is therefore probably involved in the adaptation of C. rosea to tropical reefs or coasts.Although numerous studies have shown that plant PIP2s play considerable roles in abiotic stress tolerance, including drought, salt, and even cold, the expression pattern of evidence . CrPIP2;a plants . We charr region , which icreasing . Alkali CrPIP2;3 in yeast , foxtail millet SiPIP3;1 and SiSIP1;1, and date palm PdPIP1;2, which improved salt and osmotic stress tolerance in yeast [AQP1 and AQP5 caused moderate growth inhibition under salt and hyperosmotic stress when expressed in yeast [2O2 in the plant response to stress [CrPIP2;3 caused sensitivity to high salt and osmotic stress, probably due to the more rapid water loss caused by the accumulation of CrPIP2;3 in the cell membrane. This offered further evidence that CrPIP2;3 is active in water transmembrane transport in vivo. Accordingly, the sensitivity to H2O2 was due to the stronger import penetration to H2O2 and more toxic effects on yeast cells caused by CrPIP2;3 than the control yeast.As a unicellular eukaryote with a rapid growth rate, yeast has become a convenient protein expression system for protein functional identification. We found that inducing the expression of in yeast  resultedin yeast , which iin yeast ,52,53. Sin yeast , and mano stress . In thisCrPIP2;3 in abiotic stress in plants, we generated transgenic A. thaliana plants overexpressing the GFP-CrPIP2;3 fusion gene under the control of the constitutive CaMV 35S promoter , the overexpression of CrPIP2;3 in A. thaliana did not affect the expression of these genes in the CrPIP2;3 OX seedlings. These results implied that the ectopic expression of CrPIP2;3 in A. thaliana did not result in significant differences in the redox state in the CrPIP2;3 OX plants under abiotic stress. Furthermore, the phenotypic difference between the WT and CrPIP2;3 OX lines to stress tolerance was mainly caused by water absorption, not by antioxidant ability. Even though CrPIP2;3 is an active H2O2 transporter . The seeds were dried outdoors in summer, and the seedlings were cultivated outdoors with nutrient vermiculite or sandy soil until flowering and seed maturation from April to December (2019) in the South China Botanical Garden . Seedlings of C. rosea were used for stress treatment assays to detect the expression patterns of CrPIP2;3. Several stress factors and hormone treatments, including salt (600 mM NaCl), alkali , water-deficit or drought (300 mM mannitol), and abscisic acid (ABA) treatment (100 μM), were applied to the C. rosea seedlings by irrigation or spraying to challenge the plants and to induce transcriptional changes in CrPIP2;3. The aerial parts (leaves and vines) and roots of C. rosea at different timepoints  were collected separately and then immediately placed into liquid nitrogen for future use. The different tissues or organs of the C. rosea seedlings or adult plants present at SCBG were also gathered for further expression assays.The A. thaliana was grown in a growth chamber at 22 °C with a photoperiod of 16 h light/8 h darkness. Transgenic A. thaliana plants were generated using the floral dip method. The T1 generation plants were selected by Basta (30 μL of 13.5% Basta solution added to 100 mL Murashige and Skoog (MS) medium) on MS plates, and the seedlings were identified by polymerase chain reaction (PCR) and reverse transcription (RT)-PCR to verify the overexpression of CrPIP2;3 in A. thaliana. Three T2 homozygous lines were selected for further phenotype identification or subcellular localization analysis. The house-keeping gene AtActin2 (At3g18780) was used as an internal control.The wild-type (Col-0) C. rosea was constructed using the SMART™ cDNA Library Construction Kit , with the Saccharomyces cerevisiae expression vector pYES-DEST52 as the carrier of the cDNA library. Dozens of colonies were picked randomly and sequenced, and one of the cDNAs showed intact open reading frames (ORFs) and had high homology to other plant PIP2 genes. Combining the genomic DNA sequence results of C. rosea, this cDNA sequence was named CrPIP2;3. The obtained sequences of this PIP2 gene were submitted to NCBI (Accession No.: MT787666). The protein sequences of CrPIP2;3 and other homologous proteins were aligned using ClustalW, and a phylogenetic tree was generated using the MEGA6 program with the neighbor-joining method and 1000 bootstrap replicates. The sequence information of other plant PIPs in this study is listed in http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index). The conserved domain of CrPIP2;3 was searched using the Pfam online program (http://pfam.xfam.org/). The subcellular localization of CrPIP2;3 was also predicted using the WoLF PSORT server (https://wolfpsort.hgc.jp/) and Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/).A full-length cDNA library from CrPIP2;3 promoter region, the ATG upstream sequence (2000 bp) of the CrPIP2;3 coding region was selected from the C. rosea genomic DNA sequence data (unpublished) and analyzed with the online program PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/).To predict the motifs of the Saccharomyces cerevisiae wild-type (WT) strains (BY4741 and W303) and skn7Δ via a polyethylene glycol (PEG)-lithium acetate-based transformation protocol. The yeast wild-type (BY4741) strain and H2O2-sensitive mutant yeast strain skn7Δ  were obtained from Euroscarf (http://www.euroscarf.de/index.php?name=News). The yeast wild-type W303 was kindly provided by Zhou et al. [For a further functional bioassay, the recombinant plasmid CrPIP2;3-pYES-DEST52  and pYESu et al. .2O2. The test plates were incubated at 30 °C for 3 to 7 d.The transformed single colony was inoculated in SDG-Ura medium  overnight at 30 °C, diluted with fresh pre-warmed SDG-Ura medium, and then incubated with vigorous shaking for approximately 48 h at 30 °C to reach an optical density just higher than 1.0 at OD600. Then, the cells were serially diluted in 10-fold steps, and 2 μL aliquots of each were finally spotted onto SDG-Ura medium plates with or without NaCl, sorbitol, PEG8000, or HC. rosea and A. thaliana using HiPure Plant RNA Kits , and the cDNA was synthesized using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix  according to the manufacturer’s instructions. Quantitative reverse transcription (qRT)-PCR was conducted using a LightCycler® 480 Gene Scanning system  and TransStart Top Green qPCR SuperMix . Gene expression levels were normalized using the C. rosea reference gene CrEF-α as internal control. The primer pairs  used for qRT-PCR are listed in Total RNA was isolated from CrPIP2;3 was PCR-amplified with the primer pair CrPIP2;3OXF/CrPIP2;3OXR  were confirmed by genomic PCR with the primer pair CrPIP2;3OXF/CrPIP2;3OXR and by qRT-PCR with the primer pair CrPIP2;3RTF/CrPIP2;3RTR (AtACT2 (At3g18780) in A. thaliana using the primer pair AtACT2RTF/AtACT2RTR  transgenic seeds were germinated for further research.The ORF sequence of IP2;3OXR . The PCRant gene . After sIP2;3RTR . The reftACT2RTR . The pGECrPIP2;3 in abiotic stress tolerance in plants, particularly the response to high salinity or alkali, water deficit, or drought-stress challenges, the seed germination rate, seedling root length, and the phenotype of adult plants of three CrPIP2;3 OX homozygous lines  under salt/alkali or drought stresses were recorded and analyzed separately.To evaluate the functions of OX 3#, OX 6#, OX 10#, and WT were measured after 7 d of germination. In brief, sterilized seeds were spotted on MS plates containing 150 mM, 175 mM, and 200 mM NaCl , or 5 mM NaHCO3 plus 95 mM NaCl (pH 8.2) and 10 mM NaHCO3 plus 90 mM NaCl (pH 8.2) , or 200 mM, 300 mM, and 400 mM mannitol (hyperosmotic stress). The germination rates were counted (n = 30–50), and photographs were taken.The seed germination rates of OX 3#, OX 6#, OX 10#, and WT seedlings were also measured after 7 d of different stress challenges. In short, seeds were germinated on MS plates for 3 d under suitable conditions, following which the sprouts were transferred onto MS plates supplied with 100, 150, and 200 mM NaCl , 0.5 mM NaHCO3 plus 99.5 mM NaCl (pH 8.2), 0.75 mM NaHCO3 plus 99.25 mM NaCl (pH 8.2), and 1 mM NaHCO3 plus 99 mM NaCl (pH 8.2) , or 200, 300, and 400 mM mannitol (hyperosmotic stress). The seedling lengths were calculated, and photographs were taken.To evaluate the seedling tolerance to salt, alkali, or hyperosmotic stress, the root lengths of the A. thaliana plants. The A. thaliana seeds  were sown in vermiculite directly and were well-cultivated for 30 d. Subsequently, these adult plants were subjected to the following stress tolerance assays. For the salt tolerance assays, the plants were well irrigated with 200 mM NaCl solution for 21 d. For the alkali tolerance assays, the plants were watered with 100 mM NaHCO3 (pH 8.2) solution for 35 d. For the drought tolerance assays, the plants were maintained under continuous drought for 13 d and were re-watered for another 7 d. The survival rates were counted, and photographs were taken.Salt, alkali, and drought tolerance assays were also assessed using transgenic adult CrPIP2;3 OX lines) growing in the soil were soaked in 200 mM NaCl, 400 mM mannitol, or 50 mM NaHCO3 solutions for 24 h, with water as the control. The rosette leaves were collected before and after each treatment, and the in situ detection of H2O2 and O2− in the leaves was determined with 1 mg/mL NBT or 1 mg/mL DAB solution, respectively, for 12 h, followed by clearing in 96% ethanol.The reactive oxygen species (ROS) accumulation was also assessed with a nitro-blue tetrazolium (NBT) or 3.3′-diaminobenzidine (DAB) staining assay. In brief, three-week-old seedlings  and abiotic stress-related genes  in three-week-old A. thaliana  planted in soil, the total RNA was isolated from the rosette leaves at different time points , and cDNA synthesis was performed using the above procedure. The reference gene for the qRT-PCR was AtACT2 (At3g18780) in A. thaliana. The primers used for qRT-PCR are listed in For detecting the expression of antioxidative system-related genes (t-test) of Excel 2010 software . The significance level was defined as * (P < 0.05) and ** (P < 0.01).All of the above experiments were repeated at least three times independently, and the data shown are the mean ± SD. In this research, statistical analyses were performed using the statistical tools (Student’s


TP53 mutated AML, secondary, relapsed, and refractory AML, and in patients unfit for intensive treatment, thus highlighting an unmet need for novel therapeutic approaches. Targeting the stem cell oncoprotein BMI1 in leukemic cells may represent a promising novel treatment option for poor risk AML patients, especially in combination with other targeted therapies. Here we tested the BMI1 inhibitor PTC596 in combination with a variety of targeted therapies in AML cell lines and patient samples in vitro. In addition, we defined the biomarkers of response to the combination treatments in the leukemic cells. The combination treatment with the BMI1 inhibitor PTC596 and the MCL1 inhibitor S63845 may be an effective treatment in CD34+ adverse risk AML with elevated MN1 gene expression and MCL1 protein levels, while combination treatment with BMI1 inhibitor PTC596 and the MEK inhibitor trametinib may be more effective in CD34+ adverse risk AML with elevated BMI1 gene expression and MEK protein levels. The determination of gene and protein expression levels in leukemic cells as biomarkers of response to targeted combination therapies may be helpful to optimize treatment efficacy.Prognosis for acute myeloid leukemia (AML) patients is poor, particularly in TP53 mutated AML, secondary, relapsed, and refractory AML, and in patients unfit for intensive treatment, thus highlighting an unmet need for novel therapeutic approaches. The combined use of compounds targeting the stem cell oncoprotein BMI1 and activating the tumor suppressor protein p53 may represent a promising novel treatment option for poor risk AML patients. Experimental Design: The BMI1 inhibitor PTC596, MCL1 inhibitor S63845, and MEK inhibitor trametinib, as well as the p53 activator APR-246 were assessed as single agents and in combination for their ability to induce apoptosis and cell death in leukemic cells. AML cells represented all major morphologic and molecular subtypes including FLT3-ITD and FLT3 wild type, NPM1 mutant and wild type, as well as TP53 mutant and wild type AML cell lines and a variety of patient derived AML cells. Results: AML cell lines were variably susceptible to PTC596 and to combination treatments with PTC596 and MCL1 inhibitor S63845, MEK inhibitor trametinib, or TP53 activator APR-246, independent of TP53 mutational status. Susceptibility of patient samples for PTC596 in combination with S63845 or trametinib was significant for the majority of adverse risk primary and secondary AML with minimal efficacy in favorable risk AML, and correlated significantly with CD34 positivity of the samples. BMI1 and MN1 gene expression, and MCL1 and MEK1 protein levels were identified as biomarkers for response to PTC596 combination treatments. Conclusions: The combination of PTC596 and S63845 may be an effective treatment in CD34+ adverse risk AML with elevated MN1 gene expression and MCL1 protein levels, while PTC596 and trametinib may be more effective in CD34+ adverse risk AML with elevated BMI1 gene expression and MEK protein levels.Purpose: Prognosis for acute myeloid leukemia (AML) patients is poor, particularly in  TP53 tumor suppressor axis, thereby contributing to the blockade of differentiation and accumulation of leukemic blasts in the blood and bone marrow [Acute myeloid leukemia (AML) is a clonal blood malignancy characterized by arrested maturation and abnormal proliferation of hematopoietic precursor cells. At the cellular level, specific genetic and epigenetic alterations lead to changes in cellular signaling pathways, including the common inactivation of the e marrow .MN1) gene is expressed in CD34 positive hematopoietic stem cells and down-regulated during myeloid differentiation [MN1 gene expression in myeloid stem cells leads to enhanced proliferation and loss of myeloid differentiation [MN1 overexpression has been linked to shorter overall (OS) and disease-free survival (DFS) of patients diagnosed with acute myeloid leukemia with normal cytogenetics [MN1 with myeloid gene expression levels revealed the positive association of MN1 and BMI1, CD34, FOXP1, and MDM2 expression indicating the stem cell gene BMI1 as putative MN1 target gene [The meningioma 1 , the first functional gene in the PcG family, was identified in a mouse lymphoma in 1993. BMI1 induces self-renewal of hematopoietic (HSC) and leukemic stem (LSC) cells [BMI1 gene expression levels correlate well with progression and prognosis of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) [BMI1 expression in tumor cells is associated with poor prognosis. BMI1 is a novel target for cancer therapy [B-cell-specific Moloney murine leukemia virus integration site 1 (C) cells . BMI1 geia (AML) . BMI1 plia (AML) ,13. Incr therapy  and may  therapy . PTC596  therapy .TP53 is a novel approach to restore the crucial p53 tumor suppressor function. APR-246 triggers an upregulation of genes involved in cell cycle control and apoptosis in both TP53 mutant and wild type cancer cells [TP53 hematologic myeloid malignant disease (NCT 03072043). Finally, a phase I/II study to investigate the safety and clinical activity of APR-246 in combination with a BRAF inhibitor in patients with mutant-BRAF unresectable metastatic melanoma resistant to anti-BRAF/anti-MEK inhibitors has started (NCT03391050).Targeting mutated er cells . APR-246er cells . The firer cells . Three cFLT3 and TP53 [In previous studies, we observed a considerable anti-leukemic efficacy of the MCL1-inhibitor S63845 and the MEK inhibitor trametinib. Hematological cells with susceptibility to the single compounds as well as to the combined treatment were defined by elevated MCL1- and MEK-protein levels, independent of the mutational status of and TP53 .Here we investigated the combined treatment of PTC-596 with the MCL1-inhibitor S63845 and the MEK-inhibitor trametinib, as well as the p53 activator APR-246 on AML cells in order to identify a potentially effective treatment specifically for aggressive AML, in particular in patients with refractory disease and in patients unfit for intensive chemotherapy. The study might provide the rationale for initiating a clinical study in aggressive AML evaluating PTC596 combination therapies.Mononuclear cells of AML patients diagnosed and treated at the University Hospital, Bern, Switzerland, between 2005 and 2018 were included in this study. Informed consent from all patients was obtained according to the Declaration of Helsinki, and the studies were approved by decisions of the local ethics committee of Bern, Switzerland, decision number #221/15. Peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) were collected at the time of diagnosis before initiation of treatment. The AML cells were analyzed at the central hematology laboratory of the University Hospital Bern according to state of the art techniques [TP53 null) cells were supplied by the Leibniz Institute DSMZ, German Collection of Micro-organisms and Cell Cultures. AML cells were grown in RPMI 1640 media  supplemented with 20% fetal bovine serum  in a standard cell culture incubator at 37 °C with 5% CO2.OCI-AML2 ; OCI-AML3 , MOLM-13 , FLT3-ITD, TP53wt), MOLM-16 , ML-2 , FLT3wt, TP53mut), PL-21 , and HL-60 , the MCL1 inhibitor S63845, the MEK inhibitor trametinib, and the p53 activator APR-246 were purchased at MedChemExpress . Conventional induction therapy consisted of equimolar solution of cytarabine and idarubicin purchased at Sigma-Aldrich  and SelleckChem . Cell viability was determined after 20 h of treatment using the MTT-based cell proliferation kit I (Roche). This time point was selected because the cellular responses were effectual for the calculation of combination indexes after 20 h of treatment with two compounds in leukemic cells. For AML cell lines, four independent assays  with four measurements  per dosage were performed. For hematological patient samples, two independent assays with three technical replicates per dosage were performed. For the calculation of combination indexes three dosages of PTC596 and two dosages of the other compounds were applied alone and in combination. Combination indexes were calculated on Compusyn software. Data are depicted as XY graphs, column plots, or scatter plots with mean values and SD. Statistical analysis was done on GraphPad Prism  in grouped analysis and significance calculated by t-test for column graphs or Mann–Whitney test for scatter plots.For assays with AML cell lines, cells were plated at a density of 5 × 10RNA was extracted from AML cells and quantified using qPCR. The RNA extraction kit was supplied by Macherey-Nagel, Düren, Germany. Reverse transcription was done with MMLV-RT . Real-time PCR was performed on the ABI7500 Real-Time PCR Instrument using ABI universal master mix  and gene specific probes Hs01104728_m1 (ABL1), Hs00180411_m1 (BMI1), Hs00923894_m1 (CDKN2A), Hs00159202_m1 (MN1), and Hs02758991_g1 (GAPDH).Measurements for BMI1 and MN1 were normalized with ABL1 values, measurements for CDKN2A were normalized with GAPDH values (ddCt relative quantitation). Assays were performed in three or more independent experiments. Statistical analysis was done on GraphPad Prism software using two-tailed t-tests. Data are depicted in column bar graphs plotting mean with SD values.Protein extraction was done according to standard protocol. In short, cell pellets were lysed in RIPA buffer. GAPDH, MCL1, and MEK1 protein levels were determined with double-antibody Sandwich ELISA . MCL1 and MEK1 values were normalized with GAPDH values. Two independent assays with three technical replicates were performed per sample. Statistical analysis was done using averaged normalized values and linear regression on GraphPad Prism software. Data are depicted as XY graphs with linear regression.Staining for apoptosis was done using AnnexinV-CF488A  in AnnexinV buffer and Hoechst 33,342 (10 μg/mL) for 15 min. at 37 °C, followed by several washes. Propidium iodide was added shortly before imaging on the Nucleocounter NC-3000 . For cell cycle analysis, cells were incubated in lysis buffer with DAPI (10 μg/mL) for 5 min. at 37 °C and analyzed on NC-3000 imager.FLT3-ITD and FLT3 wild type, NPM1 mutant and wild type, as well as TP53 wild type, mutant, hemizygous, and null cells  yielded overall antagonistic effects in the tested AML cell lines .TP53 wild type cell lines strong response to combination treatments were detected in MOLM-13 cells with induction of G2 arrest by PTC596, induction of apoptosis and cell death when treated with PTC596 in combination with S63845, HDM-201, PKC-412, and trametinib  and TP53 wild type cells (ML-2 and OCI-AML3) when treated with PTC596 and APR-246 /CBFB-MYH11 and favorable risk, and AML 10, a TP53 mutant AML , one MDS, two B-ALL, and five normal bone marrow samples were subjected to single compound and combination treatments. In the combination of PTC596 and S63845 strong cytotoxic effects were detected in two AML and one MDS-AML: AML1, classified as FLT3 mutant adverse de novo AML with 90% blast count; AML6, a relapsed t-AML; MDS-AML5, a tant AML A,C.TP53 mutant MDS-AML2, and the B-ALL2. The FLT3 and NPM1 mutant AML samples (AML2 to 5) and the TP53 mutant AML10, as well as MDS-AML4, 7, and 8 showed moderate response to PTC596 and trametinib. MDS-AML5, the DNMT3A, TET2, NPM1 mutant secondary AML, was not susceptible to trametinib. AML7 to 9, classified as AML with mutated NPM1 or inv(16) and favorable risk showed mild cytotoxic effects (In the combination treatments with PTC596 and trametinib strong cytotoxic effects were present in four MDS-AML and one B-ALL: MDS-AML 1, 3, 6 with adverse risk, the  effects B,D. The  effects E,F and g effects G,H.BMI1 and MN1 as well as the protein levels of MCL1 and MEK1 were determined in the mononuclear cells isolated from AML patients at diagnosis (BMI1 gene expression and MEK1 protein levels were positively associated to the response to PTC596 and trametinib (MN1 gene expression and MCL1 protein levels were positively associated to the response to PTC596 and S63845 (MN1 gene expression with C34 positivity (BMI1 gene expression and MEK1 protein levels with CD34 positivity (In order to define biomarkers of response the gene expression of iagnosis , and coriagnosis A–H. BMI1ametinib B,H, whild S63845 C,E. MN1 d S63845 I. There sitivity J. There sitivity K,L.TP53 activator APR-246, a dose escalation screening was performed. Susceptibility to PTC596 varied in the tested AML cell lines with elevated susceptibility of the TP53 null cell line HL-60, the TP53 wild type AML cell line OCI-AML3, as well as the MDS-AML cell line MOLM-13, and reduced susceptibility of the TP53 mutant cell lines MOLM-16, SKM-1, and PL-21 as well as the TP53 wild type cell line ML-2. The susceptibility of TP53 null cells to PTC596 indicated a p53 independent mechanism of action. Susceptibility of MOLM-13 and OCI-AML3 cells had been previously described for the BMI1 inhibitors PTC-209 [In order to determine the susceptibility of AML cell lines to the BMI1 inhibitor PTC596 and the  PTC-209  and PTC5 PTC-209 .TP53 wild type ML-2 and the TP53 mutant MOLM-16 cells indicating a p53 independent mechanism of action. APR-246 has been described as a small molecule that restores and enhances the function of mutated or wild type p53 [TP53 wild type and TP53 mutant cancer cells. Moreover, APR-246 elicits p53-independent effects including production of ROS, thereby inducing oxidative stress, as well as upregulation of heat shock proteins and UPR response genes, thereby inducing ER stress [Susceptibility to the p53 activator APR-246 was comparable in all AML cell lines with elevated susceptibility in the type p53 ,27. APR-R stress .TP53 double mutant MOLM-16, which turned out to be resistant to various other targeted therapies in the past. Here for the first time, MOLM-16 cells were susceptible to a targeted treatment with PTC596, and the effects were enhanced in combination with S63845 or trametinib.In order to define the most effective treatment combinations, we focused on inhibitors expected to elicit synergistic cytotoxic effects in combination with PTC596 based on previous studies with FLT3-, MDM2-, MCL1-, and MEK- inhibitors ,22,23. IMCL-1 expression in AML cells and may influence expression of MCL1 inducers including MEK, ERK, AKT, STAT3, and STAT5 [MCL-1 inhibitor S63845 and the MEK inhibitor trametinib correlated to the cellular MCL1 and MEK1 protein levels [Synergistic effects of PTC596 with S63845 and trametinib were to be expected, as PTC596 reduces nd STAT5 , while Sn levels . AML celIn the current study, we found antagonistic effects in the combination of PTC596 and conventional induction therapy in various AML cell lines. Similar antagonistic effects of PTC596 and doxorubicin or cytarabine had been previously described in mantle cell lymphoma . The molThe effects of PTC596 and trametinib or S63845 alone and in combination on induction of apoptosis, cell cycle arrest, and cell death were determined in cytometric assays. PTC596 induced G2 arrest and apoptosis, as published previously . EffectsMCL-1 and induced mitochondrial apoptosis in a p53-independent manner. PTC596 effectively killed CD34 positive AML stem/progenitor cells while sparing normal hematopoietic stem/progenitor cells. In a recent phase I study, the most frequently reported PTC596-related treatment-emergent adverse events were mild to moderate gastrointestinal symptoms, including diarrhea, nausea, vomiting, and fatigue. Only one patient treated with 10.4 mg/kg experienced dose-limiting toxicity of neutropenia and thrombocytopenia, both of which were reversible [MN1 gene expression and MCL1 protein levels were biomarkers for response to PTC596 combination treatments with S63845, while BMI1 gene expression and MEK1 protein levels were biomarkers of response to PTC596 combination treatments with trametinib. The determination of gene and protein expression levels in leukemic cells as biomarkers of response to targeted combination therapies may be helpful to optimize treatment efficacy. The combination of PTC596 and S63845 may be an effective treatment in CD34 positive AML with elevated MCL1 protein levels, while PTC596 and trametinib may be more effective in CD34 positive AML with elevated MEK1 protein levels, including TP53 mutant AML.Susceptibility of patient samples for PTC596 in combination with S63845 or trametinib was significant for the majority of adverse risk AML and MDS-AML samples, with least efficacy in favorable risk AML. In previous studies, BMI-1 protein levels were described to be significantly higher in patients with unfavorable cytogenetics compared with those with intermediate or favorable cytogenetics , and CD3versible . In our versible , and CD3versible , hence MBMI1 inhibitor PTC-596 alone and in combination with a variety of novel targeted agents in AML cell lines and primary AML blast cells in cell viability assays. The AML cell lines included a variety of cyto-morphologic FAB subtypes as well as molecular subtypes, including FLT3-ITD and FLT3 wild type, NPM1 mutant and NPM1 wild type, TP53 mutant and TP53 wild type cell lines. The results suggest that BMI1-inhibition by PTC-596 can induce cell cycle arrest and apoptosis in AML cells independent of TP53 status. In fact, best response to PTC-596 was detected in TP53 null cells, in minimally differentiated AML and in secondary AML post MDS. Induction of apoptosis and cell cycle arrest were analyzed. Effective combination treatments were validated in cytotoxicity assays with normal bone marrow and AML patient samples. Strong synergistic effects were detected in both TP53 wild type and TP53 mutant AML cell lines treated with PTC596 in combination with the MCL1 inhibitor S63845 or the MEK-inhibitor trametinib. In addition, mild synergistic effects were detected in both TP53 wild type and TP53 mutant AML cell lines treated with PTC596 and the TP53 activator APR-246. Strong cytotoxic effects were also detected in some AML patient cells treated with PTC596 in combination with S63845 or trametinib, in particular in adverse risk AML including a TP53 mutant MDS-AML and a relapsed t-AML. The susceptibility of leukemic cells to PTC596 in combination with S63845 or trametinib correlated significantly to CD34 positivity of the hematological samples. These results indicate that the combination of PTC596 and S63845 or trametinib may be both effective and specific treatments to target adverse risk AML, especially with elevated CD34 positivity, thus providing the rationale for initiating clinical studies evaluating these treatment combinations.In this study, we tested the


Nowadays, tuberculosis, scarlet fever, measles, influenza, and mumps are five major notifiable respiratory infectious diseases (RIDs) in China. The objective of this study was to describe, visualize, and compare the spatial-temporal distributions of these five RIDs from 2006 to 2016. In addition to descriptive epidemiology analysis, seasonality and spatial autocorrelation analysis were also applied to explore the epidemiologic trends and spatial changing patterns of the five RIDs, respectively. The results indicated that the incidence of tuberculosis, measles, and mumps presented a downtrend trend, while those of scarlet fever and influenza was in a strong uptrend across the research period. The incidences of the five diseases all peaked in spring. There were significant spatial disparities in the distribution of tuberculosis, scarlet fever, and measles cases, with the hotspots mainly located in the western plateau region, northern plain region, and southern mountainous region. To conclude, notable epidemiological differences were observed across regions, indicating that some provincial units should pay more attention to prevent and control respiratory infectious diseases. In 2015, the United Nations released sustainable development goals (SDGs), which proposed the elimination of the epidemics of AIDS, tuberculosis, and other infectious diseases by 2030 , among wTo improve the surveillance of the epidemics of RIDs, the Chinese government increased its investment in infectious disease prevention and control systems nearly fivefold in the past decade . The heaNumerous researchers have explored the epidemiology of RIDs in China. Regarding tuberculosis, Wang et al.  performeTo conclude, most of the previous research paid their attention to the temporal trends and investigation-based epidemics of RIDs, but few focused on the spatial distribution patterns and seasonality. It is no doubt that the epidemiology of RIDs differs across space, time, and season, so only a spatial–temporal analysis can reveal the RIDs’ epidemics thoroughly. Besides, few previous studies compared the epidemics of RIDs. To fill the void, this study aims to explore, visualize, and compare the spatial–temporal epidemiology of five major RIDs in China.Tuberculosis, scarlet fever, measles, influenza, and mumps are all notifiable infectious diseases under the strict monitoring of China’s CDC. We collected the provincial year-end and month-end incidence data in all the available years (2006–2016) from the public health science data center of China’s CDC. We obtained the population data from the National Bureau of Statistics (NBS). The research reports on the panel data of RIDs at the 31 provincial units in China, excluding Hong Kong, Macau, and Taiwan, due to data accessibility. The time-series analysis indicated the temporal trends of RIDs incidence  in each province and also across the nation. In the first place, the box plots are used to display the statistics , and display the overall temporal trends of the incidence of RIDs. Secondly, the growth rates of the incidence in each provincial unit during the study period (2006–2016) are exhibited, and units that displayed significant linear trends were identified by the chi-square linear-by-linear association test.Affected by the seasonal factors, RIDs have obvious seasonality ,16. SeasAll these component series have a certain characteristic or type of behavior ,20,21. ZMoran’s I is an important indicator to analyze the spatial distribution characteristics of disease cases, and has been used in a lot of studies ,24,25. IThe value of Moran’s I ranges from -1 to 1, where a 0 indicates that the RIDs cases are randomly distributed in space and no clusters are detected . A valueThe values of Moran’s I were calculated by using the software GeoDa 1.8.61 . The box plots were drawn with Microsoft Excel 2016 . The chi-square linear-by-linear association tests and the seasonal decomposition were conducted in SPSS 20.0 . The seasonal scan statistic was measured with SaTScan 9.5 . All the maps were drawn and visualized in ArcGIS 10.0 .To better understand the temporal trends of RIDs in China, we also compute the growth rate of the incidence of RIDs in each provincial unit in The seasonal decomposition of the reportable RIDs rates is displayed in p < 0.01) different infection peaks for each disease. In The temporal scan statistic identified temporal clusters during the study period (2006–2016), presented in The global spatial autocorrelation of RIDs and their test results are displayed in As the local spatial autocorrelation analysis only reveals the relative state rather than the absolute incidence in each provincial unit, we divided all the 31 provincial units in China into four classes in hierarchy maps  based onRegarding scarlet fever, the HH cluster area remained in north China, and the LL area remained in south China across the study period. From 2006 to 2011, Hebei also began to display the HH cluster feature, and the HH cluster area thus extended. Anhui and Guangxi moved out of the LH cluster area, and the LL cluster area thus shrunk. Moreover, Guizhou moved into the LL cluster area in 2016. As for measles, no provincial units were displaying any cluster feature in 2006. During the first sub-period, the HH cluster area appeared in western China, and the HH cluster area thus extended in 2016. Sichuan began to display the LH cluster feature and moved out in 2016. Guangdong remained relatively stable after 2011.In terms of influenza, Xinjiang displayed the HH cluster feature, and the LH cluster was located in Qinghai, Sichuan, and Beijing in 2006. When the time came to 2011, the HH cluster area gradually moved into central China, and the LH cluster area gradually moved into northeast China. Besides, Jilin presented the LL cluster feature. From 2011 to 2016, the LH cluster area wears off, and the HH cluster area gradually moved southward. Jilin remained the LL cluster feature. As for mumps, the HH cluster was located in a part of western China, whereas Sichuan and Guangdong displayed the LH and LL cluster features, respectively, in 2006. From 2006 to 2011, the HH and LL cluster areas wear off. Hunan moved into the LH cluster area. From 2011 to 2016, the HH cluster area of mumps moved southeastward. The LH cluster area moved northward, while Jilin displayed the LL cluster feature. This study revealed the spatial–temporal epidemiology of five major RIDs  in China, which provided insight into potential solutions to diminish the diseases in China. We want to discuss the spatial–temporal epidemiology of these five major RIDs first, and then explore future prevention and control strategies.In the first place, we compared the temporal trends of the five RIDs. Based on the incidence of RIDs between 2006 and 2016, their temporal trends had similarities and differences. Regarding the number of cases, the incidence of tuberculosis was still far above that of other RIDs, despite it having been on a declining curve from 2006 to 2016, which is consistent with the results from the studies conducted by Cao et al.  and Guo Secondly, regarding the seasonal changes, the incidence of tuberculosis and influenza both peaked in late winter and early spring. Scarlet fever showed a bimodal seasonal pattern with the larger peaks occurring in the summer and a smaller peak occurring in winter. Measles displayed a high-incidence season in spring, and mumps peak incidence occurred during the summer months with a peak of about half the size occurring in spring. Previous studies have revealed an association between the RIDs and seasons ,30,36,37Thirdly, the high-risk areas for different RIDs were mainly located in the western plateau region, northern plain region, and southern mountainous region of China. The western plateau region is the hotspot for tuberculosis and measles. Most of the western plateau regions are remote areas with high altitude, frigid climate, where the cold, dry weather may cause high herd susceptibility of tuberculosis and measles ,34,38. FThere are some limitations to this study. We only did the analysis at the provincial level as we were unable to obtain more detailed statistics for the municipal units or county units. Studies that explore the temporal and spatial distributions in smaller region units are suggested for the future.In conclusion, trends, seasonal patterns, and spatial disparities in different provincial geographical units were determined. Different RIDs display different trends. The incidence of influenza was notably ascending, while that of scarlet fever indicated a resurged trend; the epidemic of tuberculosis, measles, and mumps was under control. Higher incidences of RIDs were observed from January to June, and the climate zone is associated with this high incidence. The high-risk areas for RIDs were detected. Each high-risk region faces different challenges as there exist distinct risk factors due to the different natural environments and economic development, so every provincial unit should create an evidence-based solution that identifies the priority measures and enables meaningful inputs from all key stakeholders. In particular, the measures to deal with respiratory infectious diseases in China should not be restricted to its prevention and control.


Oryctolagus cuniculus) rabbit model is a long‐standing surgical technique for the preclinical evaluation of materials for spinal fusion. A detailed understanding of lumbar spine anatomy and perioperative care requirements of rabbits is imperative for correct execution of the model both scientifically and ethically. This study describes the preoperative procedures and surgical techniques used in single level PLF in a NZW rabbit model as it pertains to the animal husbandry, lumbar spine anatomy, anesthesia, surgical approach, and perioperative care of rabbits in a research setting.The posterolateral lumbar fusion (PLF) New Zealand White (NZW)  between January 2016 and December 2019. The number of lumbar vertebrae per rabbit, fusion rates at 12 weeks using iliac crest autograft and complications are reported. Skeletal maturity was confirmed by preoperative fluoroscopic and radiographic documented closure of hindlimb physes.The PLF rabbit surgical model and perioperative patient care is described. PLF was performed in 868 adult female entire NZW rabbits. The majority of rabbits had seven lumbar vertebrae , followed by six , and eight . Fusion rates at 12 weeks for PLF using iliac crest autograft as assessed by manual palpation and radiographic assessment was 76.9% and 70.0%, respectively. Postoperative complications included occasional partial autograft site wound dehiscence due to self‐trauma.For PLF rabbit models, a detailed understanding of the surgical technique, rabbit lumbar anatomy including number of lumbar vertebrae, and dietary and husbandry requirements of rabbits, is essential for execution of the model and animal welfare. Descriptive report of the surgical technique and perioperative patient care for single level posterolateral lumbar fusion (PLF) in a New Zealand White rabbit model, and a retrospective account of variation in number of lumbar vertebrae in rabbits. The majority of rabbits had seven lumbar vertebrae , followed by six , and eight . Mean spinal fusion rates at 12 weeks for bilateral iliac crest autograft alone were 80/104 (76.9%) and 73/104 (70.0%) for manual palpation and radiographic assessment (Lenke Grade A), respectively. For PLF rabbit models, a detailed understanding of the surgical technique, rabbit lumbar anatomy including number of lumbar vertebrae, and dietary and husbandry requirements of rabbits, is essential for animal welfare and experimental procedures. Failure to address these aspects of the study may lead to complications and associated morbidity and/or mortality which results in increased costs and time requirements of the experiment.Palumbo et al have described detailed surgical anatomy for PLF in the rabbit.2Medical records of adult female entire NZW rabbits that underwent single level PLF (L4‐L5) between January 2016 and December 2019 were reviewed. The number of lumbar vertebrae per rabbit as determined by fluoroscopic and radiographic assessment was recorded.2.1Our experimental protocol was implemented in accordance with the ASTM Standard Guide for in vivo evaluation of rabbit lumbar intertransverse process spinal fusion models2.22.2.1Prior to study enrolment, all rabbits were physically examined by a veterinarian familiar with rabbit husbandry, anatomy, physiology, and surgery. Routine parameters were assessed including mucous membrane color, capillary refill time, heart rate, respiratory rate, presence of gastrointestinal sounds, and rectal temperature. Close attention was paid to mentation, body condition, coat condition and ambulation. All rabbits experienced a minimum 1‐week acclimatization period prior to surgical intervention to allow for adjustment to their environment, including pen mates and attending staff.2.2.2Skeletal maturity was confirmed prior to enrolling an animal for PLF by fluoroscopic and radiographic documentation of distal femoral, proximal tibial , and proximal fibular physeal closure. A portable fluoroscopy unit  was used for physeal closure screening at the time of physical examination. Immediately prior to surgery, mediolateral radiographs were taken to further document physeal closure. Radiographs were taken using a POSKOM model PXP‐60HF portable machine. Digital cassettes  were processed by an AGFA CR 75.0 Digitizer and workstation.2.2.3All rabbits were weighed prior to surgery to ensure correct calculation of all medications to be administered. The rabbits were premedicated with Buprenorphine (0.03 mg/kg) and Midazolam (0.5 mg/kg) via intramuscular injection using a 23 g needle. The rabbits were preoxygenated for 10 minutes prior to masked isoflurane induction and maintained between 2% and 3%, titrated to effect. A balanced crystalloid solution (Hartmann's solution) was administered subcutaneously (20 mL/kg) prior to commencement of surgery. Continuous multiparameter cardiopulmonary monitoring  was used throughout anesthesia and recorded every 10 minutes during the procedure. The isoflurane setting was weaned nearing the completion of the procedure. The rabbits received external heat support in the form of a heat pad. Following completion of surgery, the rabbits were wrapped in a clean towel for added warmth and received masked oxygen supplementation until righting before being returned to their housing pen. All rabbits received perioperative prophylactic antibiotics (Enrofloxacin 5 mg/kg IM or Procaine Penicillin 50 000 IU/KG IM for 5 days duration), perioperative and intraoperative opioid analgesia (Buprenorphine 0.03 mg/kg IM at induction then PRN), and postoperative nonsteroidal anti‐inflammatory medication (Meloxicam 0.85 mg/kg q 12‐24 hours for 5 days).2.2.4Prior to surgery, a portable fluoroscopy unit  was used to count the number of lumbar vertebrae and screen for any structural anomalies such as hypoplastic vertebral bodies Figure .2.32.3.1The surgical fields for PLF and iliac crest corticocancellous bone graft harvest were prepared under aseptic conditions. A wide area of fur was clipped from the dorsum of the rabbit, extending caudally from the last rib to the tail base and laterally to a level approximately a quarter of the way down the flank, ventral to the wing of the ilium. The area was aseptically prepared with chlorhexidine‐alcohol soaked gauze swabs followed by povidine‐iodine spray. The surgical field was then draped in a sterile fashion.2.3.2A linear skin incision was made along the spine of the iliac crest (left or right) in a caudomedial to craniolateral direction. Subcutaneous and fascial tissue was incised using a combination of sharp and blunt dissection. The gluteal muscles were incised along their fascial origin and elevated from the cranial and caudal aspects of the iliac crest, taking care to stay as close to the ilium as possible to minimize tissue trauma and associated hemorrhage. The authors find it useful to place a gauze swab on either side of the iliac crest using a Freer periosteal elevator2.3.3Bone marrow aspirate (BMA), when required for the study, was harvested from the proximal tibia for hydration of select bone graft substitutes prior to implantation. A 25 mm, 18 g trochar needle  was manually inserted into the cancellous bone of the proximo‐medial tibia Figure . The ins2.3.4Following appropriate draping of the surgical field, the surgeon(s) determined the L4‐L5 level for fusion via counting of the dorsal spinous processes using manual palpation based on the number of vertebrae found during the preoperative preparation. The surgeon(s) identified the lumbosacral junction, then moved cranially to L4‐L5. Intraoperative fluoroscopy can be used to clarify the L4‐L5 level if required.A dorsal midline skin incision, approximately 4 to 5 cm in length, was made centered over the L4‐L5 level. Subcutaneous tissue and fat were undermined using blunt dissection. Approximately 2 cm lateral to the midline, a white line can be visualized in the longissimus muscle. Approximately 2 to 3 mm lateral to this, a 5 cm incision was made deep through the longissimus muscle with a no. 15 scalpel blade. The longissimus muscle was then separated from the iliocostalis muscle by identifying the fascial plane between the two muscle bellies using a Freer periosteal elevator  with an M‐8 Matchstick Burr. The central portion of the transverse processes were carefully decorticated for a distance of 10 mm from the vertebral body and pars to a level where bleeding bone beds were visually present to the surgeonA muscular window was then created by carefully placing Weitlaner self‐retaining retractors between the two muscle bellies, elevating the longissimus muscle dorsomedially and the iliocostalis muscle laterally to access the middle third of the transverse processes Figure . It can 2.3.5Material  for implantation was placed into 3 cc syringes with the tip removed to provide a simple means to accurately place the materials in the prepared posterolateral beds. The material was implanted bilaterally on the decorticated transverse processes and overlying the intertransverse ligament between the L4 and L5 transverse processes Figure . Care wa2.3.6The muscle planes were closed using 3‐0 monofilament absorbable suture in a simple continuous pattern. Care was taken to take deep bites of the muscle to ensure closing of dead space to prevent graft migration and seroma formation. The superficial fascia of the longissimus and multifidus muscles was included in this layer to assist in closing the dead space. The subcutaneous layer was closed in a simple continuous buried pattern with 4‐0 monofilament absorbable material. The skin was closed with 4‐0 monofilament absorbable material in either a buried simple continuous or Ford‐interlocking pattern. A transparent adherent film spray  was applied to all surgical wounds to encourage formation of a fibrin seal and discourage self‐trauma.2.3.7Dorsoventral lumbar digital radiographs were performed at the completion of surgery to confirm appropriate material placement as well as provide immediate postoperative radiographic data on the radio‐opacity of the treated level for future comparisons. Radiographs were taken using a POSKOM model PXP‐60HF portable machine. Digital cassettes  were processed by an AGFA CR 75.0 Digitizer and workstation.2.42.4.1Immediately following harvest, the stability of the lumbar spine was assessed by manual palpation by two trained and experienced observers in a blinded manner according to Professor Boden et al.2.4.2Following harvest, the spines were immediately radiographed in the dorsoventral plane using a Faxitron  and digital cassettes (described previously). Radiographs were processed by a dedicated workstation (described previously). The DICOM data was converted to bitmap images using DICOM Works . Radiographs were reviewed by blinded observers and fusion was graded according to the Lenke scale.Fusion was also assessed by micro computed tomography and biomechanical testing (range of motion) on the day of euthanasia, and subsequently processed for histology and histomorphometry. The samples are never frozen. A discussion of these experimental endpoints is beyond the scope of this manuscript.2.52 enclosed pens, floored with hay to a depth of at least 5 cm. A wooden hutch shelter was provided for each rabbit. Cardboard boxes, toilet rolls and plastic balls were provided for environmental enrichment. Close attention was paid to conflict between rabbits, with offending rabbits singularly housed with clear vision of other rabbits or rotated between pens as required.All dietary and husbandry protocols were implemented in accordance with the Animal Research Review Panel (ARRP) guidelines for the housing of rabbits in scientific institutions.All rabbits were fed a high‐quality diet, of which the majority was high‐fiber timothy or oaten hay, supplemented with leafy greens such as silver beet, spinach, bok choy, pak choy, and broccoli. High‐quality commercial pellets, herbs  and fruits  were provided in moderation. Water was provided ad libitum via self‐automated bottles. Calm, slow‐paced music was played during daylight hours.33.1PLF was performed in 868 adult female entire NZW rabbits across 30 preclinical studies. The most common number of lumbar vertebrae was seven , followed by six , and eight  Figure , Table 13.2Bilateral autograft fusion with a 12 week timepoint was performed in 104/868 (11.9%) rabbits across 13/30 preclinical studies. The remaining 764 rabbits underwent L4‐L5 PLF with autograft only at an earlier timepoint or with various combinations of autograft and synthetic material or synthetic material only, as dictated by the study design. Typical sample size was n = 8 per group per timepoint.3.3All animals were confirmed to be skeletally mature prior to surgery as verified by closure of the distal femoral, proximal tibial and fibular physes. Representative radiographs are presented in Figure 3.4Mean spinal fusion rates at 12 weeks for bilateral iliac crest autograft alone were 80/104 (76.9%) and 73/104 (70.0%) for manual palpation and radiographic assessment (Lenke Grade A), respectively. A Faxitron radiograph of a fused L4‐L5 PLF with autograft at 12 weeks is provided in Figure 3.5In our experience with this preclinical model, PLF with unilateral autograft harvest in combination with synthetic material is an approximately 30 to 40 minutes procedure, that is comprised of unilateral autograft harvest (5‐10 minutes), PLF (20 minutes) and wound closure (5‐10 minutes). PLF with bilateral autograft harvest is an approximately 50 to 60 minute procedure, including bilateral autograft harvest (10‐20 minutes), PLF (20 minutes) and wound closure (10‐15 minutes). Total surgical time is dependent on the required preparation of the synthetic graft material . Reporting of surgical times for each synthetic material is beyond the scope of this manuscript.3.6All rabbits maintained or gained weight during their experimental lifetime. There were no anesthetic‐related complications. We observed occasional episodes of partial dehiscence of the autograft harvest site due to self‐trauma, that were managed conservatively with Elizabethan collars. We did not observe neuropraxia or other neurological complications. No asymmetric material implantation, fracture of transverse processes, or surgical site infections were experienced.4We describe the preoperative care and procedures, variation in lumbar vertebrae, skeletal maturity assessment, surgical approach, autograft fusion rates and perioperative care of the single level PLF in the preclinical NZW rabbit model. Originally described by Professor Boden et al,The overall mean fusion rates based on manual palpation and radiographic assessment (Lenke Grade A) for PLF with bilateral iliac crest autograft at a 12 week timepoint were 76.9% and 70.0%, respectively. Our results compare favorably with previous reports using autograft alone in PLF.A common complication of PLF is incorrect implantation of the graft material which was not experienced in this cohort. Single level spinal fusion is commonly performed bilaterally at the L3‐L4, L4‐L5, or L5‐L6 levels in preclinical rabbit models.Appropriate decortication of the transverse processes is important to encourage spinal fusion.The traditional rabbit PLF spinal fusion model utilizes the Wiltse approach that is, the intermuscular plane between the multifidus and longissimus muscles, which facilitates direct access to the pars, transverse processes, and facet joints of the lumbar spine.A detailed understanding of surgical anatomy coupled with a minimally traumatic surgical technique is imperative for correct execution of the procedure to minimize intra and postoperative complications such as hemorrhage, nerve palsy, and surgical site infection. During autograft harvest, hemorrhage can occur when the caudal third of the iliac wing is harvested due to compromise to the lumbosacral trunk and cranial gluteal neurovasculature. We avoid this region and focus our harvest on the lateral aspect of the iliac crest. For the PLF approach, respect for and identification of the fascial plane between the longissimus and iliocostalis muscles minimizes hemorrhage and the associated pain of muscle splitting. Hemorrhage may also result from damage to the segmental spinal vessels as they originate cranial to the transverse processes. Great care is taken during manipulation of the implantation site and decortication of the transverse processes to avoid disrupting these vessels. If hemorrhage is encountered in either of these regions, hemostasis is achieved with gauze swab packing or vessel ligation. Attention is paid to closing dead space at both the autograft harvest and implantation sites by the apposition of tissue planes to minimize the risk of seroma formation which may foster surgical site infection.Confirming skeletal maturity prior to enrolling an animal in a study is important for study integrity.Rabbits can present an anesthetic challenge to researchers and clinicians,The vasodilative effects of inhalation anesthetics such as isoflurane in combination with their large surface area, make rabbits extremely susceptible to hypothermia.Rabbits are monogastric herbivores that utilize hindgut fermentation for digestion. Rabbits require a high‐feed intake diet, rich in insoluble fiber to promote gastrointestinal motility and maintain their aradicular hypsodont dentition (high crowned, continuously growing teeth.Our rabbit housing facilities were developed in accordance with the guidelines of the ARRP.Pasteurella multocida, a virulent and readily transmitted gram‐negative coccobacillus, may manifest as multiple clinical syndromes in rabbits including dacrocystitis, rhinitis, pneumonia, otitis media, pyometra, orchitis, abscesses, and septicaemia.P. multocida,P. multocida has variable and inconsistent antimicrobial sensitivities. Enrofloxacin is a relatively safe, concentration‐dependent fluoroquinolone with activity against P. multocida in rabbitsPasteurella‐associated abscessation in rabbit PLF studies. For these reasons, we recently updated our perioperative antibiotic choice to Procaine penicillin, which has proven efficacy against P. multocida.P. multocida‐associated disease in experimental rabbit cohorts.5We describe an updated protocol on the long‐standing PLF model of spinal fusion from the preoperative to postoperative setting, the variation in the number of lumbar vertebrae in rabbits and 12 week autograft fusion rates. Our techniques accommodate for the unique anesthetic, dietary and husbandry requirements of rabbits, supported by meticulous surgical technique to minimize surgical complications to achieve optimal experimental outcomes. We hope this information is useful for other researchers using this model with the aim of animal welfare preservation and the principles of reduction, refinement and replacement.The authors declare no potential conflict of interest.All authors have read and approved the final submitted manuscript. The following is the author contribution: James D. Crowley, Rema A. Oliver, and William R. Walsh: substantial contributions to research design, data acquisition, analysis, interpretation of current literature, manuscript preparation and critical appraisal; John W. Rawlinson, Rebekah A. Crasto, James M. O'Connor, and Gregory J. Mitchell: animal husbandry procedures, data acquisition; Daniel J. Wills, Michael J. Dan: data acquisition, drafting of manuscript and critical appraisal; Christopher J. Tan: drafting of manuscript and critical appraisal.Appendix S1: Supporting information.Click here for additional data file.Appendix S2: Supporting information.Click here for additional data file.


These scripts are executed with root privileges at installation, update, and removal of a package, which make them critical for system maintenance. While Debian policy provides guidance for package maintainers producing the scripts, few tools exist to check the compliance of a script to it. We report on the application of a formal verification approach based on symbolic execution to find violations of some non-trivial properties required by Debian policy in maintainer scripts. We present our methodology and give an overview of our toolchain. We obtained promising results: our toolchain is effective in analysing a large set of Debian maintainer scripts and it pointed out over 150 policy violations that lead to reports  on the Debian Bug Tracking system.The Debian distribution includes more than 28 thousand maintainer scripts, almost all of them are written in P


Slowness is associated with increased disability and mortality in older people. However, the relation between ideal cardiovascular health (CVH) and slowness in community-dwelling older adults is uncertain. We examined the prevalence of ideal CVH in Korean older adults and its association with slowness in community-dwelling older adults. We analyzed 2,597 participants  without cardiovascular disease from the Korean Frailty and Aging Cohort Study. The usual gait speed over a distance of 4 m was measured using an automatic timer, and slowness was defined as a speed <1.0 m/s. Ideal CVH was described as attainment of ideal health behaviors  and optimal health factors . Multiple logistic regression analysis was used to examine the association between the CVH score and slowness. Ideal CVH was present in 785(30.2%) subjects. Considering those with poor level of CVH were as the reference group, the odds ratios [OR] for slowness were 0.55  for those with intermediate level of CVH, and 0.28  for those with ideal level of CVH after adjustment for potential confounders. Among ideal CVH components, behavioral CVH score  was significantly associated with slowness vs. the biological CVH score . This study indicates that ideal CVH is significantly associated with a lower risk of slowness in community-dwelling older adults. A better CVH may help prevent slowness.


To evaluate whether the attenuation of mitochondrial Ca CIR was induced in adult male Wistar rats (300-350 g) by occlusion of the left anterior descendent coronary artery (10 min), followed by reperfusion (120 min). Rats were treated with different doses of MCU blocker ruthenium red (RuR), administered 5 min before ischemia or reperfusion. In untreated rats, the incidences of ventricular arrhythmias (VA), atrioventricular block (AVB) and the lethality (LET) induced by CIR were 85%, 79% and 70%, respectively. In rats treated with RuR before ischemia, the incidences of VA, AVB and LET were significantly reduced to 62%, 25% and 25%, respectively. In rats treated with RuR after ischemia, the incidences of VA, AVB and LET were significantly reduced to 50%, 25% and 25%, respectively.2+ overload produced by pharmacological blockade of MCU can protect the myocardium against injuries caused by CIR. The significant reduction of the incidence of CIR-induced VA, AVB and LET produced by the treatment with RuR indicates that the attenuation of mitochondrial Ca The myocardial injuries caused by AMI usually cause the patient to have severe arrhythmias that, in many cases, culminate in the patient’s death. Therefore, researchers look for strategies that can mitigate both myocardial injuries and cardiac arrhythmias1. However, the greater the duration and/or severity of cardiac ischemia, the more extensive will be the myocardial damage, as well as susceptibility to further injury during reperfusion1. The combined damage caused by the obstruction and subsequent coronary clearing is known as a cardiac ischemia-reperfusion (CIR) injury. It is well known that these lesions compromise cardiac structure and function and are responsible for many clinical sequelae typical of AMI1.The cardiac muscle can tolerate short periods of severe and total ischemia, such as those caused by coronary vasospasms, without myocyte cell death2]c) and mitochondrial ([Ca2]m) concentrations of Ca2 in cardiomyocytes is crucial to maintain the strength and frequency of cardiac contractions. Then, dysregulation of Ca2 homeostasis in cardiomyocytes caused by CIR decisively contribute to cardiac dysfunction in AMI5. This ionic imbalance produced by CIR in cardiomyocytes, especially the unbalancing of [Ca2]c and [Ca2]m has unsurprisingly been implicated as a major cause of severe and lethal cardiac arrhythmias due to ischemic heart disease2.It is well established that the precise adjustment of cytosolic (c in cardiomyocytes, which generate a significant increase in Ca2 uptake by mitochondria, thus aggravating mitochondrial Ca2 overload in a positive feedback loop7. This Ca2 overload that collapses the mitochondrial function dramatically compromises the excitation-contraction coupling, favoring the development of fatal cardiac arrhythmias8.In healthy cardiomyocytes, cytosolic Ca2]m, in the development of most severe adverse effects, pharmacological strategies with the cardioprotective potential to attenuate these changes are highly desirable. Therefore, in this study we evaluated whether the attenuation of mitochondrial Ca2 overload produced by pharmacological blockade of MCU with Ruthenium Red (RuR) protects the myocardium against injuries caused by CIR.Due to the crucial role of ionic imbalance induced by CIR in cardiomyocytes, particularly in m indirectly, reducing [Ca2]c through inhibition of Ca2 release from the SER due to interaction of with ryanodine receptors (RyR), tubulin and Ca2-ATPases29.The ionic imbalance in CIR-affected cardiomyocytes significantly affects MCU-mediated influx of Ca2]c due to inhibition of Ca2 release from SER mediated by RyR, but higher concentrations (10 to 50 μM) produced spontaneous contractions and contracture, through mechanisms that remain unclear, but may include mitochondrial dysfunction independent of RuR’s effect on MCU.Our results also showed that administration of high doses of RuR (10 mg/kg) have no cardioprotective effect compared to the CIR group and, on the contrary, tended to increase lethality, indicating that doses above 10 mg/kg may produce cardiotoxic effects. Indeed, in isolated rat hearts or cardiomyocytes, administration of 5 μM RuR reduced [Ca2]m overload by MCU blockade could play an important role in cardioprotection against CIR damage by attenuating ionic imbalance and mitochondrial bioenergetic collapse in cardiomyocytes, thus preventing cardiac contractile function failure and fatal arrhythmias30. Therefore, drugs capable of blocking MCU may constitute an effective cardioprotective strategy in the aftermath of AMI.Attenuating [Ca2 overload produced by pharmacological blockade of MCU can protect the myocardium against injuries caused by CIR. This finding could open a new avenue in AMI treatment.The significant reduction of the incidence of CIR-induced VA, AVB and LET produced by the treatment with RuR indicates that the attenuation of mitochondrial Ca