Drought is one of the major environmental constrains that limit plant performance worldwide. Plant apoplast which acts as connecting link between environment and plant protoplast carries multiple functions in plant metabolism and signalling. To investigate the drought induced changes in apoplast, proteome analysis in conjunction with antioxidant enzyme activity changes were studied in chilli (Capsicum annuum L). Drought induced apoplast proteome revealed augmented phenyl alanine ammonia lyase, peroxidase activities and reduced catalase activity. LC-MS analysis of apoplast proteome revealed differential expression of proteins under water stress conditions. Up-regulation of 43 protein species which encompass stress related proteins such as defensins, peroxidises, polygalaturonase inhibitor proteins, superoxide dismutase proteins were observed. Unlike control, twenty unique protein species were identified to be present in proteome of drought treated plants. Qualitative and quantitative changes in apoplast proteome emphasize the dynamics of plant apoplast and its role in drought stress. This work would provide insights into drought induced proteomic changes in apoplast and also would prove to be useful for protein phenotyping. Elite chilli genotype (S-10) was subjected to two water regimes - 100% Field Capacity (Control) and 40% Field Capacity (Drought Treated). Malate Dehydrogenase (MDH) activity and was found to be 1% in treated and 0.4% in control apoplast samples. Malate Dehydrogenase activity is widely used as a specific marker to identify degree of cell membrane integrity and level of cytosolic contamination Vitis vinifera there was an increased production of phenolics under abiotic stress conditions In the present data, 1.1A total 208 protein species were identified from the LC-MS analysis of control and treated proteomes. Among 208 protein species, eight were of different origins such as, cytoplasm (5), ribosomal (2) and mitochondrial (1) origin due to cytoplasmic contamination of apoplast fluid. LC-MS proteome data of 208 protein species was provided in Arabidopsis thaliana apoplast proteome. Based on their role in Biological process, 106 proteins were categorised into six groups  seeds were procured from Horticultural Research Station, Lamfarm, Guntur, Andhra Pradesh. Seeds of S10 genotype were grown in black trays containing a mixture of peat and vermiculite (2:1 v/v) for 45 days followed by transplantation into pots (one plant/pot) and allowed for acclimatization for one week. Plants were grown in greenhouse under control conditions- 16 h light/8 h dark photoperiod at 27 °C during the day and 21 °C at night, and watered regularly.2.2Drought stress was imposed to plants using gravimetric method 2.3g for 15mins at 4 °C. The protein sample was immediately stored at −20 °C until further analysis.Apoplastic proteins were extracted using the infiltration method described by , the supernatant was used for MDH enzyme assay. Cytoplasmic contamination was calculated as the percentage of MDH activity in the apoplast protein extract compared with activity in total leaf soluble protein extract.Apoplastic fluid was tested for the presence of cytosolic contamination using Malate Dehydrogenase  assay by comparing with whole leaf protein as a control according to method described by Alves et al., 2.5For the estimation of total phenolics, to 1ml of apoplastic extract 0.5ml of Folin-Ciocalteau reagent, 7.5ml ddH2O was added and incubated for 10 min at room temperature, and then 1.5ml of 20% sodium carbonate was added and incubated for 20 min at 400C. Solution was cooled and absorbance was recorded at 755 nm. Estimation of total phenolics (mg/g) was measured as described by Tohma et al., 2.6l-phenylalanine (12mM) was added. The rate of conversion of l-phenylalanine to trans-cinnamic acid was determined at 290nm as described by Sri deepthi et al., For the estimation of phenylalanine ammonia lyase content, to 0.3ml of apoplastic extract, 1.2ml of Tris buffer  and 1.5 ml of 2.7For the estimation of peroxidise activity, for 0.5 ml of apoplastic extract, 1.5 ml of pyrogallol solution (0.05 M) and 0.5ml of H2O2 was added. The change in absorbance was recorded at 430 nm for 3 min. POD activity was quantified according to the method described by Abhayashree et al. 2.8For the estimation of catalase activity, to 40μl of apoplastic extract, 2.5ml of potassium phosphate buffer  and 0.5ml of H2O2 were added. The rate of decomposition of H2O2 was determined at 240nm for 3 min. Catalase activity was quantified according to the method described by Huseynova et al. 2.92.9.1Protein samples (50 μg) were reduced with 50 mM DTT at 60 °C for 1 h and the cysteine-groups were blocked using a 50 mM IAA solution at room temperature for 30 min. The protein samples were then subjected to trypsin digestion by adding trypsin in 1:30 ratio (Trypsin: Protein) at 37 °C in a dry bath for 16 hours. After trypsinization, samples were dried in speed vac and reconstituted in 20 μl of Milli-Q water with 0.1% formic acid and desalting was performed and then subjected to LC-MS.2.9.2LC-MS analysis is performed in 1290 Infinity UHPLC system, 1260 infinity Nano HPLC with Chip cube, 6550 iFunnel Q-TOFs  at Sophisticated Analytical Instrument Facility (SAIF), IIT Bombay. Samples were loaded in an analytical C18 column . Mobile phase consists of solvent A: 0.1% FA in Milli-Q water, solvent B: 80:20 (ACN: Milli-Q water) + 0.1% FS. The raw LC-MS data was analyzed using Thermo Proteome Discoverer 2.2 software with Sequest-HT Uniport, capsicum annuum and plants databases.2.9.3All the samples  were collected triplicate and data were analysed with One-Way ANOVA at 5% probability. Data were represented as Mean ± SD.


The results exhibited that the catalytic properties of CeO2-MnOx composite oxides were higher than pure CeO2 or MnOx. When the Ceat/Mnat ratio was 3:7, the catalytic activity reached the best. In addition, the activities of CeO2-MnOx synthesized through different routes over benzene oxidation were also comparative researched. The result indicated that the catalytic property of sample prepared by oxalate method was better than others, which maybe closely related with their meso-structures. Meanwhile, the effects of synergistic interaction and oxygen species in the samples on the catalytic ability can’t be ignored.The catalytic activities of CeO Volatile organic compounds (VOCs) produced by industrial manufacturing are an important class of air pollutants. Among the VOCs, aromatic compounds are one of the major hazardous pollutants, in which benzene is considered to be one of the representing aromatic materials extensively applied in the industry. The complete catalytic oxidation of benzene is often studied as a model reaction, characteristic of the catalytic combustion of VOCs due to its chemical stability ,2,3,4,5.2), as a typical rare earth oxide, was investigated in heterogeneous catalysis field due to its high oxygen storage capacity. It can provide active oxygen species to ensure the catalytic reaction. More recently, CeO2-based composite oxides were employed for VOCs removal and obtain satisfied results, especially Ce-Mn composites [2-MnOx can be applied as heterogeneous catalysts for the abatement of contaminants in the liquid and gas phases, such as the catalytic reduction of NO and oxidation of acrylic acid and formaldehyde, which exhibit much higher catalytic activity than those of pure MnOx and CeO2 [Ceria  [In our previous work , Mn elem–200 °C) , even if3)3·6H2O (99%), Mn(CH3COO)2·4H2O, Na2CO3, C2H2O4·2H2O, NaOH (98%), and ethanol, were purchased from Beijing Chemicals Company . A series of Ce-Mn composite oxides were synthesized by carbonate method. Briefly, Ce(NO3)3·6H2O and Mn(CH3COO)2·4H2O in appreciate amounts were dissolved in a 100 mL H2O and mixed with a 0.24 M C2H2O4·2H2O solution under strong stirring. Then, the mixture was stirred for another 1 h. The precipitates were collected by centrifugation, washed with distilled water and ethanol several times. The obtained materials, labeled as CexMn1−x (where x refers to the Ce/(Ce + Mn) atomic ratio) were dried at 80 °C overnight and calcined at 450 ℃ for 2 h with a heating rate of 2 °C·min−1. Pure CeO2 and MnOx were also prepared using the similar process as reference. In order to compare the catalytic activity, Ce-Mn composite oxides were also synthesized through carbonate method and hydrothermal method [The chemicals used in this work, including Ce. The assignment of the crystalline phases was based on the ICSD data base . The morphology images of catalysts were recorded on a scanning electron microscopy  operating at 15 kV and 10 μA. The microstructures of catalysts were examined using transmission electron microscopy  with an accelerating voltage of 200 kV.The crystal phase of the materials was characterized on X-ray diffraction  equipped with a Cu Kα radiation source (λ = 0.154187 nm) at a scanning rate of 0.03 °/s (2SBET) was measured by physical adsorption of N2 at the liquid nitrogen temperature using an Autosorb-1 analyzer . Before measurement, the samples were degassed at 300 °C for 4 h under vacuum. Surface composition was determined by X-ray photoelectron spectroscopy  using an ESCALab220i-XL electron spectrometer from VG Scientific with a monochromatic Al Kα radiation. The binding energy (BE) was referenced to the C1s line at 284.8 eV from adventitious carbon.The BET specific surface area (2-TPR) was performed with a U-type quartz reactor equipped with Automated Catalyst Characterization System . A 50 mg sample (40–60 mesh) was loaded and pretreated with a 5% O2 and 95% He mixture (30 mL/min) at 150 °C for 1 h and cooled to 50 °C under He flow. The samples were then heated to 900 °C at a rate of 10 °C/min under the flow of a 10% H2 and 90% Ar mixture (30 mL/min). Hydrogen temperature-programmed reduction . A standard reaction gas containing 1000 ppm benzene and 20% O2 in N2 was fed with a total flow rate of 100 mL/min. The weight hourly space velocity (WHSV) was typically 60,000 mL·g−1·h−1. The reactants and the products were analyzed on-line using a GC/MS 6890N gas chromatograph  interfaced to a 5973N mass selective detector  with a HP-5MS capillary column (30 m × 0.25 mm × 0.25 μm) and another GC  with a carbon molecule sieve column. The conversion of benzene  was calculated as follows:Cbenzene (in) (ppm) and Cbenzene (out) (ppm) are the concentrations of benzene in the inlet and outlet gas, respectively.Activity tests for catalytic oxidation of benzene over Ce2, MnOx and CexMn1−x catalysts was evaluated through the oxidation of benzene. The catalytic conversion of benzene as a function of the temperature, 100–400 °C, is shown in x exhibits the least active followed by CeO2. With the Mn element adding into CeO2, the activity increases monotonically up to a Mn content of 70 at.% and Ce0.3Mn0.7 is the most active among all catalysts achieving complete benzene conversion at ca. 200 °C. The MnOx and CeO2 catalysts synthesized through oxalate route also achieve full conversion at ca. 300 °C. In addition, the activities of Ce0.3Mn0.7 synthesized through different routes over benzene oxidation are comparative researched in order to identify the advantage of oxalate route  used to evaluate the performances of the catalysts are summarized in The catalytic performance of CeOte route b. The re2 (JCPDS 81-0792). For pure MnOx, the XRD pattern exhibits complex diffraction peaks which can be considered as a mixture of crystalline Mn3O4 (JCPDS 80-0382) and Mn2O3 (JCPDS 89-4836). The data demonstrates that manganese oxide is a multivalent framework manganese (2+ and 3+), which indicates the existence of various manganese oxides. For CexMn1−x catalyst, the XRD patterns exhibit more complex. The XRD patterns of the CexMn1−x mixed oxide (x ≥ 0.5) do not show any diffraction of manganese oxides, and only broad reflections attributed to CeO2 are observed, which is possible to be related with the formation of solid solution between MnOx and CeO2. The diffraction patterns of CexMn1−x mixed oxides at x ≤ 0.3 show crystallization of Mn3O4 and Mn2O3 except that of CeO2. x and CeO2 can be further evidenced by the fact that the characteristic diffraction peak of CeO2 in the composite oxides is slightly shifted to higher values of the Bragg angles, compared with the pure CeO2 (2+ (0.091 nm) and Mn3+ (0.066 nm) are both smaller than that of the Ce4+ (0.1098 nm), the incorporation of Mn2+ or Mn3+ into the CeO2 lattice to form CeO2-MnOx solid solution would result in remarkable decrease in the lattice parameter of CeO2 in the CexMn1−x composite oxide. Meanwhile the O vacancy is also easier to form in order to balance charge due to Ce4+ replaced by Mn2+ or Mn3+ in the CexMn1−x catalyst. The oxygen vacancy is beneficial to catalytic activities of CexMn1−x catalyst [The formation of solid solution between MnOure CeO2 b. Since catalyst . xMn1−x oxide catalysts are presented in 2, it can be seen that the catalyst is composed of many thin flakes, which are overlapped together to form butterfly-like structure (inset picture). The thickness of every flake is about 200 nm and the length can extend to several micrometers. In the SEM image of MnOx, a lot of grains with ellipsoid-like morphology are seen clearly and the size is ca.10 μm. At the surface of every grain, deep ravines are also obviously observed (inset picture). For CexMn1−x composite catalysts, the morphology changes gradually with the Mn content increasing. When the Mn theoretical content reaches 50% (Ce0.5Mn0.5), a few of bulk-like particles with size of several micrometers can be detected except thin flakes. When the theoretical content of Mn ion reaches to 70% (Ce0.3Mn0.7), a large number of grains possessing layered structure can be observed. In addition, it can be acquired that Ce, Mn and O elements are dispersed together homogeneously through element distribution over CexMn1−x composite catalysts. The SEM images of as-prepared CexMn1−x, it has been acquired that Ce0.3Mn0.7 presents the highest activity and preparation route is also important to the property of catalyst. Therefore, the TEM images of Ce0.3Mn0.7 prepared through different technology routes are shown so as to analyze their microstructures , V1 (888.7 eV), V2 (898.1 eV), V0′ (900.7 eV), V1′ (907.1 eV) and V2′ (916.3 eV) can be identified as characteristic of Ce4+ 3d final states [2/V2′) is attributed to the final state of Ce(IV)3d94f0O2p6, doublet V1/V1′ is originated from the state of Ce(IV)3d94f1O2p5, and doublet V0/V0′ corresponds to the state of Ce(IV)3d94f2O2p4. The character peaks of Ce3+ are also observed at 903.4/884.7 eV and 897.6/879.3 eV labeled as U1/U1′ and U0/U0′, respectively [3+ is estimated to be 11.05, 10.89, 10.05 and 5.65% for CeO2, Ce0.7Mn0.3, Ce0.5Mn0.5 and Ce0.3Mn0.7, which can be calculated according to the Equation (2). Therefore, Ce species in the CexMn1−x composite oxides exist mainly in tetravalent oxidation state.3+, A is the integrate area of characteristic peak in the XPS pattern, S is sensitivity factors (S = 7.399).The oxidation state of catalyst surface species was examined by XPS analysis. l states ,25. The ectively . The amoxMn1−x composite oxides, in which Mn 2p doublet can be distinguished. The binding energies of the Mn 2p3/2 component appear at 641.7 eV and those for Mn 2p1/2 appear at 653.2 eV. The BE values of the Mn 2p3/2 (641.7 eV) and spin–orbit splitting (11.7 eV) are well matching with the reported values of the trivalent manganese [3/2 component at 640.7 eV is attributed to Mn2+ species [4+ species (642.2–643 eV) [xMn1−x are analyzed  in Mn2O3. Therefore, the element Mn in the CexMn1−x catalysts is existed in the form of Mn3O4 and Mn2O3, which is consistent with the data of XRD. In addition, the peak of Mn 2p3/2 (641.5 eV) for CexMn1−x composites is shifted to lower binding energy comparing with pure MnOx, which may be a consequence of interaction between CeO2 and MnOx [0.7Mn0.3 is similar and less weaker compared with other CexMn1−x composites. The corresponding XPS spectrum can be seen in the anganese . The sho species . The XPS–643 eV) ,30. In oanalyzed c. The spervalent ,32. The 13 × ΔEs . The datand MnOx . It is w2 and MnOx phases [β) [xMn1−x composite catalyst with higher Mn content tends to shift toward higher BE value than that of pure CeO2 (from 529.1 eV to 529.6 eV), which suggests that the environments of oxygen change with increasing Mn content. This appearance is also attributed to the interaction between CeO2 and MnOx. In addition, the content of Oα is calculated according to the Equation (3) and listed in 0.3Mn0.7 sample possesses more lattice oxygen species, confirming that the mobility and availability of lattice oxygen species are enhanced due to the synergistic effects of CeO2 and MnOx in Ce0.3Mn0.7 [α, A is the integrate area of characteristic peak in the XPS pattern, S is sensitivity factors (S = 0.711).The XPS O1s spectra d show a d as Oα) ,30. A br Oα) [β) ,34. It i0.3Mn0.7 .(3)XOα=xMn1−x systems, TPR of all the catalysts were carried out. 2-TPR profiles of CeO2, MnOx and CexMn1−x composite oxides. Similar to previous findings [2 exhibits two reduction peaks at around 405 °C and about 719 °C. The former low-temperature reduction is due to the removal of surface oxygen and the later high-temperature reduction is related to the oxygen species in bulk CeO2. The H2-TPR profile of pure MnOx shows two strong reduction peaks at 167 °C and 330 °C, respectively, with an area ratio of the lower to the higher temperature hydrogen consumption of about 1:2.42. The actual hydrogen consumption of two reduction peaks is 0.92343 and 1.88118 mmol/g and the corresponding ratio is 1:2.04. As we known, Mn2O3 proceed two-step reduction, the low-temperature reduction peak represented the reduction of Mn2O3 to Mn3O4 and the high-temperature reduction peak referred to the further reduction of Mn3O4 to MnO [3O4 phase. This is in agreement with the XRD data, which show that the crystalline phase of pure MnOx corresponds to Mn2O3 and Mn3O4.In order to check the redox properties of the new series of Cefindings ,37,38, p4 to MnO . The the2 and MnOx, the reduction profiles of CexMn1−x catalysts are more complicated. For CexMn1−x, the TPR profiles consist of three overlapping peaks at lower temperature and one peak at higher temperature. According to the reduction characteristics of pure MnOx and CeO2, it can be deduced that the lower temperature peaks  are assigned to the two-step reduction of Mn2O3. The peaks at 693 °C or 714 °C are attributed to the oxygen species in bulk CeO2. It is worthy to note that another obvious peak at lower temperature  as shown in 2+/Mn3+ and Ce4+, which is related with CeO2-MnOx solid solution. It can facilitate the mobility of the oxygen species in the CexMn1−x composite oxide. Therefore, their catalytic activities over benzene are higher than pure CeO2 and MnOx. Additionally, the reduction temperature is lower and the peak corresponding to the oxygen species in bulk CeO2 disappear in the TPR pattern of Ce0.3Mn0.7 compared with those of Ce0.7Mn0.3/Ce0.5Mn0.5. This indicates that the reduction of the manganese oxide and the cerium oxide in Ce0.3Mn0.7 is promoted more obvious, which results in the highest activity over benzene in the CexMn1−x composite oxides. In the view of hydrogen consumption, the same conclusion can be also obtained. Ce0.3Mn0.7 consumed the most hydrogen gas (4.88447 mmol/g) among CexMn1−x composite oxides indicating that Ce0.3Mn0.7 possesses more oxygen species, which are beneficial to benzene oxidation reaction. Therefore, Ce0.3Mn0.7 presents higher activity compared with other CexMn1−x.In contrast to pure CeO0.3Mn0.7 performs excellently in catalytic oxidation of benzene among the CexMn1−x catalysts. Many factors are considered to determine catalytic performances. Firstly, the existence of Ce-Mn solid solution results in the formation of oxygen vacancies due to incorporate Mn into CeO2 crystal lattice, which can enhance their activities. Secondly, the large numbers of active sites will be introduced after removal of oxalate chains, because of the formation of porous structure which can also facilitate the adsorption and diffusion of organic molecules, thus reducing limitations of interphase mass transfer and promoting their catalytic activities. Thirdly, the better reducibility at low temperature also plays a great role in the catalytic activity. Through the analysis of TPR, it has been acquired that the reductions of CexMn1−x catalysts start at the lower temperature compared with CeO2, which indicate that the catalysts possess more highly reducible surface species such as absorbed oxygen. Additionally, the existence of special peak caused by the strong interaction between Ce and Mn compared with MnOx also result in the enhancement of reducibility. Finally, the oxidation of organic molecules over transition metal oxide or mixed metal oxide catalysts involves two identical mechanisms: a Langmuir–Hinshelwood mechanism at lower temperature and a Mars–van Krevelen mechanism with increasing reaction temperature [0.3Mn0.7 exhibits a higher content of Ce4+ and Mn3+. The high-valence of cerium and manganese ions are preferred to adsorb more active oxygen species to attend in the reaction, thereby Ce0.3Mn0.7 possessed higher activity. Through the analysis, it has been acquired that Ceperature ,40. At lxMn1−x composite oxides, CeO2 and MnOx were synthesized through oxalate method and the complete catalytic oxidation of benzene were examined. The results indicated that CexMn1−x catalysts exhibited better activities comparing with pure CeO2 or MnOx, among which the catalytic activity reached the best when the Ceat/Mnat optimum ratio was 3:7. In order to identify the advantage of oxalate route, Ce-Mn composite oxides were also synthesized through carbonate method and hydrothermal method. The results indicated that the samples prepared by oxalate route exhibited higher activities, which were probably related with the microstructure of catalyst. Additionally, the influence of oxygen vacancy and synergistic effect in the benzene catalytic oxidation can’t be also ignored.A series of Ce


NGLY1 gene encoding N-glycanase 1 that is a hydrolase for N-linked glycosylated proteins. An induced pluripotent stem cell (iPSC) line was generated from the dermal fibroblasts of a 16-year-old patient with homozygous mutation of p.R401X (c.1201 A >T) in the NGLY1 gene. Our iPSC model offers a useful resource to study the disease pathophysiology and to develop therapeutics for treatment of NGLY1 patients.NGLY1 deficiency is a rare inherited disorder caused by mutations in the  This TRNDi010-C iPSC line presents a patient-specific disease model for studies of NGLY1 deficiency phenotype and pathophysiology and can be used as a cell-based model for drug discovery and therapeutic development to treat NGLY1 patients.NGLY1 gene which encodes a specialized enzyme called N-glycanase that removes N-linked glycan from glycosylated proteins within the body. Deficiency of this protein can lead to malfunctions of cellular functions and accumulation of misfolded proteins within cells in specific tissues or organs. The symptoms and severity of this disease can dramatically vary among affected individuals, who may have developmental delays, intellectual disability, movement disorders, seizures, liver disease, and alacrima   with a homozygous nonsense mutation of p.R401X (c.1201A > T) in exon 8 of the (3p24.2) . The iPS(3p24.2) . Individ(3p24.2) . The TRN(3p24.2) . The qua(3p24.2) . G-bande(3p24.2) . The gen(3p24.2) , consist(3p24.2) . To furtmination . The STR2 at 37 °C. Patient fibroblasts were reprogrammed into iPSCs using the non-integrating Sendai virus technology (2 and 5% O2. The cells were passaged with ReLeSR™ (STEMCELL Technologies) at generally 1:10 ratio when they reached around 70% confluency.Patient skin fibroblasts were obtained from Coriell Cell Repositories (GM26612), and cultured in DMEM supplemented with 10% fetal bovine serum, 100 units/mL penicillin and 100 μg/mL streptomycin in a humidified incubator with 5% COchnology . Human iNGLY1 gene was performed using Sanger sequencing analysis. The specific primers for gene amplification and sequencing are listed in The genome analysis of variants in NGLY1 was conducted through Applied StemCell . Briefly, genomic DNA was extracted from iPSC line TRNDi010-C using QuickExtract™ DNA Extraction Solution (Lucigen) followed by PCR amplification using MyTaq™ Red Mix . Amplifications were carried out on T00 Thermal Cycler from Bio-Rad (#1861096) using the following program: 95 °C, 2 min; 35 cycles of , 72 °C 5 min; 4 °C, indefinite. Genotyping of the homozygous for the p.R401X variant (c.1201 A > T) in exon 8 of the 2+ and Mg2+ and fixed in 4% paraformaldehyde for 15 min at room temperature. Fixed cells were washed with DPBS twice, and permeabilized with 0.1% Triton X-100 in DPBS for 15 min. After 1 h of blocking, the cells were incubated with primary antibodies, diluted in the blocking buffer, for overnight at 4 °C. Cells were washed twice with DPBS and a corresponding secondary antibody conjugated with Alexa Fluor 488 or Alex Fluor 647 was added to the cells and incubated for 1 h at room temperature  without CaThe iPSCs were dissociated by TrypLE Express enzyme (Thermo Fisher Scientific). After washing once with DPBS, cells were fixed with 4% paraformaldehyde for 10 min and were permeabilized with 0.2% Tween-20 in DPBS for another 10 min at room temperature, followed by staining with fluorophore-conjugated antibodies  for 1 h The G-banded karyotyping analysis was performed by the WiCell Research Institute  using the iPS cells at passage 6. Twenty randomly selected metaphase cells were selected for the standard cytogenetic analysis.The STR analysis of patient fibroblasts and iPSCs was performed by the Johns Hopkins University Genetic Resources Core Facility using the Promega PowerPlex 18D Kit. The ABI Prism® 3730xl Genetic Analyzer was used to electrophorese the PCR products and GeneMapper® v 4.0 software (Applied Biosystems) was used to analyze the data.The Lonza MycoAlert kit was used to assess the mycoplasma according to the instructions from the company. B/A ratio > 1.2 indicates the positive sample; 0.9–1.2 indicates the ambiguous result; < 0.9 indicates the negative sample.Using the RNeasy Plus Mini Kit (Qiagen), the total RNA was extracted. The cDNA was reverse-transcribed from 1 μg RNA by SuperScript™ III First-Strand Synthesis SuperMix (Thermo Fisher Scientific). The Platinum II Hot-Start PCR Master Mix (Thermo Fisher Scientific) was used to amplify the target sequence with a PCR program: 94 °C, 2 min; 30 cycles of 94 °C, 15 s, 60 °C, 15 s, and 68 °C, 15 s on Mastercycler pro S (Eppendorf) with the specific primers . The hum7 cells in 400 μL culture medium supplied with 25 mM HEPES (pH 7.4) and stored on ice. Then, 50% volume (200 μL) of cold Matrigel  was added and mixed with the cells. The mixture was injected subcutaneously into NSG mice (JAX No. 005557) at 150 μL per injection site. Visible tumors were removed 6–8 weeks post-injection and were immediately fixed in 10% Neutral Buffered Formalin. The fixed tumors were embedded in paraffin and stained with hematoxylin and eosin.Patient iPSCs were dissociated with 0.5 mM EDTA in PBS and were resuspended approximately 1 × 101


Aspergillus flavus colonisation of maize can produce mycotoxins that are detrimental to both human and animal health. Screening of maize lines, resistant to A. flavus infection, together with a biocontrol strategy, could help minimize subsequent aflatoxin contamination. We developed a qPCR assay to measure A. flavus biomass and showed that two African maize lines, GAF4 and KDV1, had different fungal loads for the aflatoxigenic isolate (KSM014), fourteen days after infection. The qPCR assay revealed no significant variation in A. flavus biomass between diseased and non-diseased maize tissues for GAF4, while KDV1 had a significantly higher A. flavus biomass (p < 0.05) in infected shoots and roots compared to the control. The biocontrol strategy using an atoxigenic isolate (KSM012) against the toxigenic isolate (KSM014), showed aflatoxin production inhibition at the co-infection ratio, 50:50 for both maize lines (KDV1 > 99.7% and GAF ≥ 69.4%), as confirmed by bioanalytical techniques. As far as we are aware, this is the first report in Kenya where the biomass of A. flavus from maize tissue was detected and quantified using a qPCR assay. Our results suggest that maize lines, which have adequate resistance to A. flavus, together with the appropriate biocontrol strategy, could limit outbreaks of aflatoxicoses. Aspergillus species; saprophytic fungi, that occurs widely, and grows on living and non-living substrates [Aspergillus flavus is able to infect many food crops, including cereals [A. flavus and aflatoxins [Aflatoxins are categorised as an important class of mycotoxins that negatively affect human and animal health . They arbstrates . As an o cereals . In deve cereals . Aflatox cereals . Aflatox cereals . Severallatoxins ,7,8,9. Tlatoxins ,11.A flavus biomass in maize tissues using Af2, Zmt3, INCW2-97 and α-tubulin marker genes, and studied the relationship between fungal biomass and aflatoxin accumulation. Mylroie et al. [A. flavus strain’s fungal biomass on contaminated maize.The quantification of fungal biomass is critical in understanding the interactions between host plant susceptibility or resistance to a fungal pathogen, as well as identifying the competition between individual fungal species during disease progression . Real tie et al.  moreoverA. flavus, using atoxigenic strains, is already established with registered atoxigenic strains for the reduction of aflatoxin contamination on cultivated crops available to farmers [A. flavus (AF36) and Afla-Guard successfully suppressed aflatoxin producers on cottonseed in USA [TMAflasafe NG, has been provisionally registered for commercial use on maize in Nigeria [Biocontrol of toxigenic  farmers ,19,20,21 Nigeria .A. flavus strains for biocontrol, is directly connected with methods for detecting aflatoxins, as it confirms that the biocontrol agent reduces aflatoxin contamination. The levels of aflatoxin in foods and feeds are strictly regulated [Aspergillus strains [The use of atoxigenic egulated ,23. Thisegulated . Molecul strains ,26,27,28 strains ,30. With strains . Aflatox strains ,32,33. H strains .A. flavus biomass in infected maize tissues. Furthermore we tested if the atoxigenic A. flavus strain KSM012, (NCBI_accession MG385137) could serve as a biocontrol agent to minimise aflatoxin contamination of maize kernels colonised by the toxigenic A. flavus strain KSM014, (NCBI_accession MG385138). As far as we are aware, this is the first fungal qPCR biomass study on Kenyan A. flavus isolates from infected maize tissue. This approach could be used to discriminate between inbred maize lines, that are sensitive or resistant to specific A. flavus strains, and help understand the mechanism of maize defence response to A. flavus infection.This study investigated whether the respective maize lines KDV1, and GAF4, grown in different regions of Kenya, contribute to increasing or limiting the biomass of KSM014, an aflatoxigenic strain. In this regard we developed and tested a qPCR assay to quantify the amount of A. flavus KSM014 resulted in changes in the maize plant phenotype. The infected kernels for both maize lines displayed stunted growth compared to the control kernels 14 days post infection  and β-Tub (118 bp) were specific for A. flavus but the amplification of Ef1a appeared less sensitive in comparison to β-Tub  and maize membrane protein (MEP) primers were used to develop a RT-qPCR SYBR® green assay to detect and quantify A. flavus gDNA in maize tissues. The β-Tub and MEP markers were found to have qPCR sensitivity, efficiency and linearity across a wide range of DNA concentrations of 2R = 0.9997 for β-Tub and 2R = 0.9988 for MEP respectively (The ectively . p < 0.05)  see . p < 0.05) in fungal biomass for the KDV1 maize line was observed upon infection for both the root and shoot tissue  could act as a biocontrol agent against the aflatoxigenic KSM014 strain, upon infection of maize kernels from two different lines. The atoxigenic and aflatoxigenic isolates were co-infected into KDV1, and GAF4 maize kernels, respectively followed by detection of aflatoxin production, using bio-analytical analyses.We wanted to determine whether the atoxigenic A. flavus KSM012 and KSM014 . HPLC chromatograms generated for the individual isolates had retention times for aflatoxins: AFG1-11.39-11.68; AFB1-12.72-12.84; AFG2-17.71-17.80; and AFB2-18.73-18.91 as observed in our previous work Mitema et al. [At 254 nm, there was no observable blue or green fluorescence (data not shown). Thus, the presence or absence of aflatoxins and their derivatives could be visualised and identified at 365 nm. Staining with a et al. .A. flavus. The limit of detection (LOD) and limit of quantification (LOQ) ranged from 0.01–6.8 µg/mL and 0.02–35.81 µg/mL, respectively [As expected, the non-toxin producing strain (isolate KSM012) had neither an HPLC peak nor blue fluorescence on TLC plates , lane 4.ectively .p < 0.05) in GAF4 maize line  and KSM014 (aflatoxigenic strain) were grown on both aflatoxin inducing and non-inducing medium: Yeast Extract Sucrose (YES) and Yeast Extract Peptone (YEP) respectively and incubated in the dark for seven days at 30 °C. Conidia were harvested with cotton swabs and suspended in aqueous Tween 20 (0.2%). The working concentration was adjusted to spore suspension 1 × 106 conidia/mL measured using a haemocytometer. The inoculum was stored at 4 °C and used within 1 week or stored as described earlier [The aflatoxigenic eviously . Two A.  earlier .http://drylandseed.com). GAF4 is a Striga tolerant variety developed by the Kenya Agricultural Research Institute in Kibos, Kisumu county. It is grown in parts of western Kenya: Kisumu, Homa Bay and Busia [Kenya Dry land Varieties KDV1 and GAF4 were purchased from Kenya Agricultural and Livestock Research Organisation (KALRO), Nairobi, Kenya. The varieties were selected based on the agroecological region in which they were cultivated and their drought tolerance. KDV1 is an open-pollinated variety that is recommended for low to medium altitude. It matures early, is drought tolerant and flowers between 45–52 days after germination. It is commonly grown in Makueni and Homa Bay (nd Busia .Murashige and Skoog medium (MS), phytagel, glycine, nicotinic acid, thiamine hydrochloride, pyridoxine hydrochloride, myo-inositol, potassium hydroxide were purchased from Sigma-Aldrich (USA). MS vitamins; 250 mg nicotinic acid, 5 g myo-inositol, 500 mg pyridoxine-HCl, 100 mg glycine, and 500 mg thiamine-HCl was prepared in sterile water, filter sterilised and stored at −20 °C until used. The MS media was prepared by dissolving 2.15 g MS salts in sterile water, then 10 mL of MS vitamin stock was added and adjusted to the pH 5.7 with 1 M KOH, and the volume adjusted to 1 L with sterile water. Five grams of phytagel was added to MS media prepared and microwaved to dissolve the salts. Media (50 mL) was dispensed into tissue culture vessels, autoclaved, and allowed to cool in a biosafety cabinet (BSC) level 2 (Contained Air Solutions (CAS) BioMAT2, Pocklington York, UK) for approximately one hour before being inoculated. Potato dextrose agar, yeast extract, sodium chloride, ammonium acetate, tryptone, mycological peptone, malt extract agar, agar, chloroform, acetone, ethanol, methanol, dichloromethane, acetonitrile, formic acid (>98%) and trifluoracetic acid (99.8%) were from Sigma-Aldrich. Mycotoxin reference standards of aflatoxin B + G mixture dry concentrate containing 5.8 µg AFB1, AFG1 and 1.7 µg AFB2, AFG2/mL were from Sigma Aldrich . Pure and ultrapure grade water was processed by Milli Q water purification system . 6 conidia ml−1). The tubes containing seeds were sealed, para filmed and kept at 30 °C for 30 min in a shaking incubator. Control seeds were treated with 20 mL sterile water instead of a spore suspension and incubated under the same conditions. Inoculated seeds were left to dry in Petri dishes overlaid with Whatman No.1 filter paper overnight in a hood. Subsequently, the seeds were inoculated onto tissue culture media (MS) and germinated at 28 °C in a plant growth chamber, Conviron . Growth was monitored for 14 days, then plant tissue (shoots and roots) were harvested and stored at −80 °C after being flash frozen in liquid nitrogen for DNA/RNA extraction.Twenty seeds were sterilised (in triplicates) by soaking in 20 mL of 95–100% ethanol for 1 min with brief shaking for 15 s. The ethanol was discarded and replaced with 20 mL of 2.5% sodium hypochlorite. The seeds were left to stand for 15 min, then shaken for 30 s and the liquid discarded. Seeds were washed 5 × with sterile water (20 mL) with intermittent shaking between each wash. A volume of 50 mL of sterile water was added and left to stand for 1 h at room temperature (RT). The water was replaced with 20 mL of 2% Tween 20, and shaken for 30 s. The seeds were inoculated (in triplicates) with 20 mL conidia suspensions  polyvinylpyrrolidone (PVP) was prepared. Four hundred and fifty microliters of TES buffer and 5 µL RNase (10 mg/mL) was added to a 2 mL microtube containing the tissues and homogenised with a microtube pestle or vortex for 15 min. 20 µL Proteinase K (1 µg/µL) was added, vortexed for 1 min, and then incubated at 60 °C for 1 h. 160 µL of 5 M NaCl (0.3 vol.), 70 µL 10% CTAB (0.1 vol.) was then added and subsequently incubated for 10 min at 65 °C. Chloroform/isoamyl-alcohol (24:1) (750 µL) was added, vortexed for 5 min and again incubated for 30 min on ice and then centrifuged for 10 min at 14,000 rpm. The aqueous phase was transferred to a new 2 mL microtube, 300–350 µL isopropanol (0.55 vol.) added and then mixed gently for 30 s and left to stand at room temperature (RT) for 30 min. The mixture was centrifuged at 14,000× g rpm for 10 min. The supernatant was discarded, the pellets rinsed twice with chilled 70% ethanol (700 µL), gently mixed without and centrifuged again for 2 min at 14,000× g rpm. Ethanol was discarded, pellets air dried and then dissolved in 40 µL TE buffer  or nuclease free water. DNA integrity was assessed on a 1% agarose/EtBr gel and the concentration quantified on a Nano-DropTM 1000 spectrophotometer . DNA was diluted to 10 ng/µL for further analysis. The chemicals used were purchased from .DNA was extracted from 100 mg of each of the following samples: ethod of  with modβ-tubulin, Elongation factor 1 alpha (Ef1α) and Membrane protein (MEP) were used in this study. β-tubulin was designed in Primer3 ver. 4.0 programme [Ef1α and MEP were obtained from Dr. Shane Murray, MCB lab. 227 (pers. Comm). Potential secondary structure formation was assessed in DNAMAN software ver. 6.03  and further verified in OligoAnalyzer Tool (Integrated DNA Technologies). The PCR and melt curve analysis were used to identify both specific and non-specific amplification. Three sets of primers ; β-tubulrogramme , whereas2, 0.5 µL of 10 μM dNTPs (Bioline), 1 µL of 10 μM reverse and forward primers, 1 µL of 10 ng DNA template, 0.2 µL Kapa Taq and sterile water. Cycling conditions were performed according to the following protocol: 1 cycle at 94 °C for 5 min followed by 35× . A final elongation step was for 7 min at 72 °C and the reaction mixture was held at 4 °C until analysed. The PCR products were assessed on a 2% agarose/EtBr gel in 1 X TAE buffer (Tris–acetate 40 mM and EDTA 1.0 mM). Fermentas 100 bp DNA ladder was used as a molecular size marker.Conventional PCR amplification was performed in a volume of 25 µL and consisted of 10× reaction buffer, with MgCl−1 AFB1, 50 µg L−1 AFB2, 200 µg L−1 AFG1 and 50 µg L−1 AFG2 and intermediate solutions stored in amber bottles at −20 °C for three months and/or −80 °C for longer storage.The mycotoxin reference standards; aflatoxin B + G mixture dry concentrate was prepared and stored in a freezer at −20 °C according to manufactures recommendations . A working stock solution for bioanalytical analysis was prepared in a one-fold dilution containing 200 µg LA. flavus were used to generate standard curves. For each dilution, the threshold cycle (Ct) values were plotted against the logarithm of the starting quantity of the template. Efficiencies of amplification were generated from the slopes of the standard curves slopes [A ten-fold serial dilution of pooled 10 ng genomic DNA extracts from control plants and s slopes ,54. LineThe amount of target DNA in an unknown sample was extrapolated from the respective standard curves.β-tub and EF1α. DNA from control plant tissues and pure fungal cultures (A. flavus) were pooled together, diluted to 10 ng/µL and used to estimate the amount of fungal DNA template in the infected plant tissue. The final fungal DNA template concentrations were 1, 5 × 10−1, 2.5 × 10−1, 1.25 × 10−1, 6.25 × 10−2, 3.125 × 10−2 ng/µL. These dilutions were used to determine the detection limits of the β-tub and EF1α primer pair in the infected plant tissues. A serial dilution of DNA extracted from healthy maize tissue also was prepared to measure the detection limits of the MEP. To normalise the gene quantification between different samples, the amount of fungal DNA as calculated by the Ct value for β-tub and/or EF1α was divided by the amount of maize DNA as calculated by the Ct values for MEP. Rotor Gene 6000 2 plex HRM  was used to evaluate the gene expression profiles. Master mix, Kapa SYBR Fast Kit , containing DNA polymerase, dNTPs, reaction buffers and 3 mM MgCl2 were used for each PCR reaction. Final concentrations of 1× Kapa SYBR green, 10 μM gene specific primers (0.2 µL forward and 0.4 µL reverse), and 1 µL of DNA template were prepared to a total volume of 20 µL, using nuclease-free water. Primer sets of specific genes (Isolated DNA (10 ng) from healthy and infected maize shoots and roots were used to test the specificity of the primers. To exclude false negative results, the template DNA samples from fungi were tested for PCR amplification with primer pairs ic genes  were useβ-tub and Ef1α.For assessing the integrity and quality of the isolated DNA, samples from control and infected tissues of the plant, and saprophytic fungi were subjected to PCR analysis with the reference genes under the following amplification conditions: 95 °C for 10 min; 35 cycles of 95 °C for 3 s, 64 °C for 20 s, 72 °C for 1 s for MEP and at Ta 62 °C for both A. flavus strains using different solvents. Briefly, fresh mycelia (200–400 mg) was scraped off the culture plate and placed into a screw capped disposable vials containing, and/or not approximately four glass beads of 4 mm in diameter . Aflatoxins and other metabolites were extracted ultrasonically for 15 min by using, 2–10 mL of extraction solvent consisting of methanol dichloromethane, ethyl acetate (MeOH:DCM:EtOAc (1:2:3)) in 1% formic acid. Extracts were centrifuged at 14,000× g rpm for 15 min at 4 °C and 500 µL transferred to sterile 2 mL tubes. The samples were dried with a Savant SpeedVac Plus SC210A Concentrator  for 12 h. The residue was reconstituted in 400 µL methanol with 0.6% (v/v) FA, 0.02% (v/v) HCl and 2.5% (v/v) water. The reconstitution was carried out in an ultrasonic bath sonicator for 10 min at RT. Samples were centrifuged at 14,000× g rpm for 15 min at 4 °C and 250 µL transferred to glass vials for TLC and HPLC analysis. Fungal metabolites were extracted from v/v) was used as the mobile phase. Ten microliters of the aflatoxin standard mix containing 5.86 µg/mL of AFB1 and AFG1, and 1.70 µg/mL of AFB2 and AFG2 and 20 µL of test samples were spotted on TLC plates and run for 70–90 min in a TLC tank at RT. The plates were left to air dry in the fume hood at RT for approximately 30 min. Dried plates were either observed under UV light (wavelength 254 and 366 nm), or sprayed with p-anisaldehyde solution, vanillin solution or exposed to an iodine vapour. The intensity of the sample spots against the standard aflatoxins were compared and aflatoxins concentrations calculated based on the equation:E = aflatoxins (µg/kg)S = µL of aflatoxin standard equal in fluorescence to sample spotC = aflatoxin standard concentration in µg/mLV = final dilution of the sample extract (µL)Z = sample matching the standards (µL)W = sample extract weight (mg)Thin Layer Chromatography (TLC) was carried out on a TLC silica gel 60 plate 20 × 20 cm . Acetonitrile/methanol/formic acid , maintained at 40 °C operating at a flow rate of 1.0 mL min−1. Water and acetonitrile, both containing 0.005% trifluoro acetic acid (TFA) were used as mobile phases. A gradient starting from 85% water and 15% acetonitrile used to 100% acetonitrile for 20 min maintained at 100% acetonitrile for 23 min and final 15% acetonitrile for 27 min. Sample injection volume was 15 µL. All chemicals used were HPLC grade. UV wavelength signals were set at 200, 210, 230, 270, 280, 320, 350, and 410 nm. Aflatoxins in the sample solution were identified by comparison of their retention times and peak height/area with corresponding standards in the standard solution.Optimisation and chromatographic separations were achieved on an Agilent HPLC 1200 system, comprised of a binary pump equipped with micro vacuum degasser, thermostatic auto sampler, column compartment, and Diode Array Detector . Fluorescence detection was performed at excitation and emission wavelengths from 200 to 410 nm. Separations were performed on Agilent Zorbax Eclipse XDB-C6 conidia/mL) in a level 2 biosafety cabinet with respective fungal isolates (atoxigenic and aflatoxigenic) at different ratios  in sterile vials. The controls were inoculated with 1000 µL sterile water instead of fungal spore suspension. Both infected and control vial contents were shaken for 30 s in a vortex mixer  to ensure complete and uniform coating of kernels with inoculum. Vial lids were loosened briefly, to enable gas exchange and incubated at 30 °C for 14 days in the dark. At the end of the incubation period, fungal activity was discontinued by addition of 50 mL, 80% methanol or halted by oven drying at 45 °C for one day and the contents then prepared for aflatoxin extraction and further analysis. Three biological replicas of the experiments were performed twice. The efficiency of surface sterilization, and the ability of kernels to germinate, were monitored by plating five randomly selected kernels from each vial onto a selective YES and YEP media, followed by incubation at 30 °C in the dark for 14 days. Approximately, 99% of the kernels germinated and no fungal contaminants were observed at the end of the incubation period.Undamaged kernels from KDV1 and GAF4 maize lines were surface sterilised in hot water for 45 s at 80 °C, as previously described . BrieflyThe statistical analysis was performed as previously described . AflatoxA. flavus inoculum made up by the atoxigenic isolate (A). ‘A’ is the quantity of atoxigenic strain and ‘T’ is the quantity of aflatoxin-producer. All analyses and calculations were performed in GraphPad Prism software ver. 5.0.2.Standard deviations of mean differences in aflatoxin levels were calculated as a measure of variability in efficacy. The efficiency (E) of each isolate was calculated as:


The pancreatic-islet-enriched transcription factors MafA and MafB have unique expression patterns in β cells in rodents. MafA is specifically expressed in β cells and is a key regulatory factor for maintaining adult β-cell function, whereas MafB plays an essential role in β-cell development during embryogenesis, and its expression in β cells gradually decreases and is restricted to α cells after birth in rodents. +) β cells in MafA-deficient adult mice. To elucidate how MafB functions in the adult β cell under MafA-deficient conditions, we generated MafA and MafB double-knockout (A0B0) mice in which MafB was specifically deleted from β cells. As a result, the A0B0 mice became more vulnerable to diabetes under a high-fat diet (HFD) treatment, with impaired islet formation and a decreased number of insulin+ β cells because of increased β-cell apoptosis, indicating MafB can take part in the maintenance of adult β cells under certain pathological conditions.The pancreatic-islet-enriched transcription factors MafA and MafB have unique expression patterns in β cells in rodents. MafA is specifically expressed in β cells and is a key regulatory factor for maintaining adult β-cell function, whereas MafB plays an essential role in β-cell development during embryogenesis, and its expression in β cells gradually decreases and is restricted to α cells after birth in rodents. However, it was previously observed that MafB started to be reexpressed in insulin-positive (insulin Diabetes mellitus (DM) is a serious chronic disease that causes the deaths of millions of people throughout the world. DM occurs when blood glucose homeostasis is impaired because either the pancreas is no longer producing insulin, which is called type 1 diabetes (T1DM), or the cells in the body have difficulty responding to insulin properly, which is called type 2 diabetes (T2DM) (+) cell numbers; this phenomenon disappears soon after birth  mice  mice . We haveWT) mice . Based o+ cells in CKO mice was observed at E18.5. Furthermore, this change was reversed after birth, because MafB is no longer expressed in β cells during postnatal development under normal conditions  compared to the control group , the insulin content did not show any significant change at that age   and C. FP < 0.05)  . A0B0 mi < 0.05) . These rIns1 and Ins2were reduced in A0B2 and A0B0 islets in comparison to WT islets, while they were comparable within the groups  . The ratThe phenotype of MafA knockout mice in our study, which had a C57BL/6J background, was relatively mild compared with that of mice with an ICR background. In mice from the ICR strain, a deficiency of MafA contributed to fasting hyperglycemia at an early stage of adulthood . Two of them died because of this severe condition.We next analyzed the pancreatic islets histologically by hematoxylin-and-eosin (H&E) staining . The num+ cells per islet were as follows: WT, 76% ± 3.2%; A0B2, 59% ± 8.2%; A0B0, 37% ± 7.9%)  and cleaved caspase 3 (an apoptotic-cell marker) antibodies, respectively. Ki67 signaling could be observed at E18.5 in insulin+ cells , as the cells are highly proliferative during embryonic development. However, there were no detectable Ki67+ cells in the islets of any mouse group with 5-month HFD treatment . Cleaved caspase 3+ cells, on the other hand, were significantly increased by proportion in the islets of the A0B0 group compared to those of the WT group    and B. T ± 8.5%)  and D, i ± 8.5%) . This mi ± 8.5%) .In this study, we generated and analyzed mice with single and compound knockouts of MafA and MafB to determine the role of MafB in adult β cells under MafA knockout conditions. Several main observations on pancreatic function and islet morphology are reported here. First, A0B0 adult mice displayed more severely impaired glucose tolerance than A0B2 mice. This phenomenon was further aggravated with HFD treatment, which caused diabetes in the A0B0 mice. Second, deficiency of MafA has a destructive effect on normal islet structure, and moreover, the abnormality becomes more significant with the deletion of MafB. Third, a notable reduction in islet cell numbers, together with an increase of β-cell apoptosis, was detected in HFD-fed A0B0 mice. Therefore, our findings provide evidence for a functional role of MafB in maintaining mature β-cell features under some specific pathological conditions.Ins1 and Ins2, it also important for maintaining a normal expression level of key β-cell genes, such as Pdx1 and Glut2, since deletion of MafB resulted in a reduction of expression of the above-mentioned genes  mice resulted in the same phenotype as MafAΔβ mice with impaired islet structure, and their glucose tolerance was not rescued by MafB expression . Adaptive β-cell proliferation has been reported to start within the first week of HFD treatment . The C57BL/6J mouse strain from the Jackson Laboratory was used in this study.flox/flox (MafA-deficient (A0) mice were generated as previously described . Pancrealox/flox , 35 micelox/flox , which pMafA single-knockout (A0B2) and MafA and MafB double-knockout (A0B0) mice and their WT littermates were fed an HFD consisting of 62.2% fat, 19.6% carbohydrate, and 18.2% protein content on a caloric basis  from 6 to 8 weeks of age.Pancreatic tissues obtained from WT, A0B2, and A0B0 mice were fixed in 4% paraformaldehyde overnight at room temperature and embedded in paraffin. The sections were then deparaffinized in xylene  3 times for 5 min each, followed by rehydrating in graded ethanol . They were placed in tap water for 5 min and then in phosphate-buffered saline (PBS) for 5 min (3 times) and blocked with PBS containing 10% goat serum with 0.03% preservative sodium azide for 1 h at room temperature. Then, they were incubated with guinea pig anti-insulin , rabbit antiglucagon , rabbit anti-MafA , rabbit anti-Pdx1, rabbit anti-Arx , rabbit anti-MafB , rabbit anti-Ki67 , and rabbit anti-cleaved caspase 3  overnight at 4°C. After rinsing, the slides were incubated with Alexa Fluor-conjugated secondary antibodies  for 2 h at room temperature. Tissue specimens were mounted with Fluoromount (Diagnostic Biosystem). All images were acquired on a fluorescence microscope .+ cells within the islets, the number of hormone+ cells per islet was manually determined using ImageJ software and divided by the total number of Hoechst+ nuclei from the same islet.Following immunofluorescence staining, the different cell types in each islet were manually counted in the islet microscopy images. For each cell type, 10 to 30 representative islets from 3 or 4 mice per group were counted. To calculate the fraction of hormonePancreatic tissue 5-μm paraffin sections obtained from WT, A0B2, and A0B0 mice were stained by routine H&E staining. Islets in each section were manually counted in the islet microscopy images. Islet diameters were measured using ImageJ software.Whole pancreases obtained from the WT, A0B2, and A0B0 mice were weighed and chopped into small pieces in 6 ml ice-cold acid-ethanol (1.5% HCl in 75% ethanol). The tissues were then sonicated for 30 s  and stored overnight at 4°C, followed by a second round of sonication the next day. The supernatants were collected after centrifuging the samples at 2,400 rpm for 30 min at 4°C. The total protein concentration was measured with a Coomassie protein assay reagent (Thermo Scientific). Insulin and glucagon contents were measured by enzyme-linked immunosorbent assay (ELISA) . Each pancreatic content was normalized by the total protein concentration per sample.Mice were fasted for 16 h and given glucose (2 g/kg body weight) by intraperitoneal injection. Blood samples were obtained from the tail vein at the indicated times  and then measured with a glucometer .Mice were fasted for 16 h and given glucose (3 g/kg) by intraperitoneal injection. Blood was collected into heparinized tubes from the facial vein at 2 min after glucose injection. Plasma insulin levels were measured with an insulin ELISA kit (Morinaga mouse insulin ELISA kit).Pancreatic islets were isolated from 9-month-old female WT, A0B2, and A0B0 mice as previously described  from isolated pancreatic islets of female WT, A0B2, and A0B0 mice. cDNA was synthesized according to the protocol of the QuantiTect reverse transcription kit (Qiagen). Real-time PCRs were performed in duplicate on a Thermal Cycler Dyce real-time system (Takara). The expression levels of all the target genes were normalized to that of t test, and comparisons among three groups were made using one-way analysis of variance (ANOVA) with post hoc Tukey honestly significant difference (HSD) tests. A statistically significant difference was defined as a P value of <0.05.Results were expressed as means and standard deviations (SD). Statistical comparisons between two groups were made using Student's


Chromium molybdate, which adopts the monoclinic P21/a structure under ambient conditions, was found to not undergo any crystalline-crystalline transitions up to 8.9 GPa. The orthorhombic ambient pressure polymorph of yttrium molybdate was found to undergo a phase transition to the monoclinic P21/a scandium tungstate structure below 0.13 GPa. This structure is frequently observed for related materials at low temperatures, but has never been reported for Y2Mo3O12. No additional changes in this material were observed up to 4.9 GPa. The fact that the monoclinic polymorphs of these materials do not undergo phase transitions within the studied pressure range makes them unique among A2M3O12 materials, as most isostructural compositions undergo at least one phase transition to crystalline high pressure phases.The high pressure behavior of negative thermal expansion materials continues to be of interest, as their potential use in controlled thermal expansion composites can be affected by irreversible pressure-induced phase transitions. To date, it is not possible to predict the high pressure behavior of these compounds, necessitating measurements on each composition. In this work, high pressure synchrotron powder X-ray diffraction studies of Cr NTE is only observed in the orthorhombic phase, and arises from concerted tilting motions of the polyhedra. The formation of the monoclinic and orthorhombic phases depends heavily on composition, and many compounds show a reversible transition between the monoclinic polymorph at low temperatures and the orthorhombic structure at high temperatures. The temperature at which this transition occurs varies widely with composition, and in extreme cases, materials can adopt the monoclinic or orthorhombic structures over their entire stability range. For instance, scandium tungstate, yttrium tungstate and yttrium molybdate . Variations in data quality and experimental parameters have also led to conflicting results for the same materials  method as described previously ” diamond anvil cell (DAC). Data were collected with a 2-D Perkin Elmer a-Si C-window CCD detector during two separate trips. Cr2Mo3O12 was measured at a wavelength of 0.72808 Å and a detector distance of 400 mm. Y2Mo3O12 was investigated during a subsequent trip at a wavelength of 0.72959 Å with a larger detector distance of 600 mm to allow collection of data to smaller d-spacings. Both setups allowed even subtle changes in the PXRD patterns to be seen. Data were collected by averaging six individual 5 s exposures. Anhydrous isopropanol was chosen as a pressure transmitting fluid (PTF) with a hydrostatic limit of 4.2 GPa , these PTFs are not suitable for the study of many NTE materials. Water is known to penetrate the open frameworks of several NTE compounds, and may lead to formation of crystalline hydrates as observed for ZrW2O8·H2O  and 8.9 GPa (Cr2Mo3O12) while collecting data at pressure increments of approximately 0.02 to 0.2 GPa. After the highest pressure was reached, files were collected during decompression in approximately 1.0 GPa steps. Final pressures of 2.2 and 1.9 GPa were observed at the end of the runs for Cr2Mo3O12 and Y2Mo3O12, respectively. A “decompressed” pattern was collected after opening the cell to release any residual pressure. The pressure for each scan was determined from the refined NaCl lattice parameters using the equation of state published by Birch ” membrane diamond anvil cell with diamond culet faces measuring 500 microns was fitted to a 300 micron thick steel gasket pre-indented to 100 microns with a 250 micron hole. The sample was packed into the sample chamber, flooded with anhydrous isopropanol as a PTF, and the DAC was quickly sealed. To avoid hydration of Pbcn-Yh Birch, . This apf is the Eulerian strain, v is the cell volume at pressure P, and vo is the ambient pressure volume. The value for vo was determined to be 179.5864 Å3. The calculated values for f can then be used to determine pressure using equation (2):where Ko is the bulk modulus at ambient temperature and a is a constant that depends on temperature. Literature values for Ko (239.9 kbar at 25 °C) and a (1.796 at 25 °C) were used , indicating the onset of disorder or amorphization. Many NTE materials have been reported to amorphize under pressure  and cubic NaCl (01-077-2064), and these phases were used as starting models for refinements. The patterns contained intensity spikes due to sample graininess, indicating that data quality was not optimal. Throughout the data set, several unidentified peaks at 6.6, 16.9, 19.4, 25.2, and 29.5° persisted. These peaks were of low intensity .The small discontinuity observed in the lattice constants of Cr2Mo3O12 to avoid contributions from the phase transition. Compressibility constants for α-Cr2Mo3O12 were extracted from 0.05 to 3.4 GPa, and were found to be βaα,  = 0.93 ± 0.01 × 10−2 GPa−1, βbα,  = 1.19 ± 0.01 × 10−2 GPa−1, βcα,  = 1.01 ± 0.01 × 10−2 GPa−1, and βvα,  = 3.13 ± 0.02 × 10−2 GPa−1. For γ−Cr2Mo3O12, these values changed to βaγ,  = 0.64 ± 0.02 × 10−2 GPa−1, βbγ,  = 1.51 ± 0.04 × 10−2 GPa−1, βcγ,  = 0.99 ± 0.02 × 10−2 GPa−1, and βvγ,  = 3.08 ± 0.04 × 10−2 GPa−1 for the 3.6 to 4.5 GPa pressure range, indicating that the overall volume compressibility decreased slightly. Above 4.5 GPa, stiffening of all lattice constants was observed. The compressibility of α-Cr2Mo3O12 is close to isotropic, with b slightly softer than the other axes. This is similar to other scandium tungstate materials , which is close to the hydrostatic limit for isopropanol. Peaks remained broad throughout decompression (scans 111-115) to 1.9 GPa. Integrated patterns showed broader and weaker features as pressure was increased, and the scan collected at the end of the high pressure run [1.9 GPa (decompression)] remained largely featureless except for NaCl peaks , which is slightly higher than the hydrostatic limit of isopropanol. No distinct phase transitions were observed in the scan overlay Figure , and alls Figure . Upon co2Mo3O12 compounds, and suggested that a transition to the structurally related higher density P21/a polymorph may have occurred below 0.13 GPa. This agrees with a previous Raman study by Torres Dias et al.  below 550 °C is denser than the Pbcn- and P21/a-phases (Gates and Lind, 2Mo3O12. It would be interesting to study the behavior of this material in pressure-temperature space to determine the orthorhombic-monoclinic phase boundary.The fact that the monoclinic phases of Cr1/a phase below 0.13 GPa. This is the first time that this polymorph has been structurally characterized. The monoclinic P21/a structures of both compounds are remarkably stable and do not undergo abrupt structural phase transitions upon compression. This is unexpected, as most previously investigated isostructural A2M3O12 materials underwent at least one distinct pressure induced phase transition. Changes in the evolution of lattice constants and compressibility as a function of pressure suggest that Cr2Mo2O12 may undergo a subtle structural distortion similar to what is observed for Al2Mo3O12, AlGaMo3O12, Fe2Mo3O12, and FeAlMo3O12, while no such changes were observed for P21/a-Y2Mo3O12.High pressure studies of Pbcn-yttrium molybdate and chromium molybdate were conducted up to 4.9 and 8.9 GPa, respectively. Pbcn-yttrium molybdate underwent a phase transition to a monoclinic P21/a polymorph in these compounds and thus whether other compositions may exhibit this behavior as well. While the monoclinic phases are not expected to exhibit NTE, their stability under pressure has important implications for composite fabrication, as the facile back-conversion to the orthorhombic phase during decompression or heating would reverse any detrimental changes that could occur during composite formation. These materials could find applications in controlled thermal expansion applications at slightly elevated temperatures, which is expected to favor the orthorhombic NTE phase even under pressure. While Y2Mo3O12 may convert to the denser Pba2-structure at moderate temperatures and pressures, the exceptional stability of Cr2Mo3O12 upon compression with respect to both formation of denser crystalline polymorphs and amorphization makes this material an attractive target.It is currently unclear what property of the A-site cation causes the remarkable stability of the P2CL devised the experiments, trained students on synthesis and characterization, participated in high pressure experiments, data collection and analysis as well as writing of paper. LY selected samples for high pressure experiments, participated in high pressure experiments, data collection and analysis as well as writing of paper. JG participated in high pressure experiments, data collection and analysis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Protein tyrosine phosphatase PTPN13, also known as PTP-BL in mice, is a large multi-domain non-transmembrane scaffolding protein with a molecular mass of 270 kDa. It is involved in the regulation of several cellular processes such as cytokinesis and actin-cytoskeletal rearrangement. The modular structure of PTPN13 consists of an N-terminal KIND domain, a FERM domain, and five PDZ domains, followed by a C-terminal protein tyrosine phosphatase domain. PDZ domains are among the most abundant protein modules and they play a crucial role in signal transduction of protein networks.Here, we have analysed the binding characteristics of the isolated PDZ domains 2 and 3 from PTPN13 and compared them to the tandem domain PDZ2/3, which interacts with 12 C-terminal residues of the tumour suppressor protein of APC, using heteronuclear multidimensional NMR spectroscopy. Furthermore, we could show for the first time that PRK2 is a weak binding partner of PDZ2 and we demonstrate that the presence of PDZ3 alters the binding affinity of PDZ2 for APC, suggesting an allosteric effect and thereby modulating the binding characteristics of PDZ2. A HADDOCK-based molecular model of the PDZ2/3 tandem domain from PTPN13 supports these results.Our study of tandem PDZ2/3 in complex with APC suggests that the interaction of PDZ3 with PDZ2 induces an allosteric modulation within PDZ2 emanating from the back of the domain to the ligand binding site. Thus, the modified binding preference of PDZ2 for APC could be explained by an allosteric effect and provides further evidence for the pivotal function of PDZ2 in the PDZ123 domain triplet within PTPN13. The  classes –11. This solvent . Recentl solvent , 14., that the binding characteristics of single domains differ from PDZ tandem and that allosteric effects can indeed regulate the binding properties of PDZ tandem domains [Tyrosine-protein phosphatase non-receptor type 13 (PTPN13), which is known as protein tyrosine phosphatase PTP-Basophil/Basophil-like (PTP-BL) in mice, is the central scaffolding component of a supramolecular protein complex that exhibits numerous domain-specific interactions , 15, and domains , 22, 23.Here, we describe the binding characteristics of the second and third PDZ domain of PTPN13, both individually and as a PDZ2/3 tandem domain through heteronuclear multidimensional nuclear magnetic resonance (NMR) spectroscopy. Our results reveal that PRK2 is an additional binding partner of the PDZ2 domain. Furthermore, we have discovered an allosteric effect due to an interaction of the PDZ3 domain with the opposite surface of the canonical PDZ2 ligand binding site, which is structurally broadcasted to the binding pocket of PDZ2. The analysis of the interaction between PDZ2 and PDZ3 as well as with their respective ligands sheds light on the pivotal role of the PDZ2 domain within the PDZ123 cassette of PTPN13 on a molecular level.15N isotopically-enriched PDZ2 and PDZ2/3 samples, respectively, and by monitoring the NMR chemical shift perturbations in a series of 2D 1H-15N heteronuclear single quantum coherence (HSQC) spectra with increasing ligand concentrations. It could be shown that all peptides bind to the canonical binding groove of PDZ domains , the PRK2 peptide only weakly binds to the PDZ2 domain (KD = 661 ± 71 μM)  in the PDZ2/3 tandem domain than for the single PDZ2 domain (KD = 286 ± 21 μM), while the extracted KD value for PDZ3 (KD = 2574 ± 770 μM) is even higher in comparison to the single PDZ3 domain (KD = 721 ± 148 μM). Binding of APC to the tandem PDZ2/3 domain occurs in the fast exchange NMR regime. At a final stoichiometric ratio between APC and PDZ2/3 of 30:1, no further chemical shift changes were observed for amide resonances originating from the PDZ2 domain. However, due to the extremely low affinity of PDZ3 for APC, PDZ3 was not fully saturated at the end of the titration and thus represented a fractionally APC-bound state (data not shown). NMR chemical shift perturbations extracted from 2D 1H-15N-HSQC NMR spectra for the PDZ2/3 titration with the APC peptide are observed for both domains. As can be seen in Figs. 1H-15N-HSQC NMR spectra of the PDZ2/3 tandem domain upon titration with the APC peptide. For example, NMR chemical shift perturbations were observed for G12, L19, T31, G63, H94, L95, L97 of PDZ2 and K152 as well as S231 of PDZ3 in the tandem domain upon titration with APC  and monitored their interaction by recording a series of 2D 1H-15N-HSQC NMR spectra  to the 1H-15N-HSQC NMR spectra could not be detected   Fig.  a, b. NM31) Fig. . This su31) Fig. . Finally31) Fig. .2  while the binding affinity of the APC peptide for PDZ3 is even lowered (KD ~ 721 μM vs KD ~ 2574 μM). The latter KD value indicates that significant binding of APC to PDZ3 in the tandem domain will most likely not occur under physiological conditions.Even though the binding behaviour of PDZ domains has been intensively studied, the functional consequences are still not completely understood. Previous studies show  that theZ domain –5. This Based on the results from the NMR chemical shift perturbation analysis and HADDOCK molecular docking calculations we conclude, that an allosteric effect modulates the APC-binding characteristics of PDZ2 from PTPN13 Fig. . AlthougOur data on the PTPN13 PDZ2/3 tandem domain are therefore consistent with and complement findings for the PDZ1/2 tandem, as the binding affinity for the APC peptide ligand is increased in the PDZ 2/3 tandem domain in comparison to the single PDZ2 domain  Table 1. ObviousThis modulation of binding affinities of PTPN13 for its ligands might also lead to the formation of supramolecular protein complexes through which PTPN13 exhibits its well established scaffolding functions in several cellular processes such as cytokinesis and actin-cytoskeletal rearrangement –37. InteTaken together, our study suggests that the PDZ2 of PTPN13 plays a central role in the triplet cassette PDZ123 and that PDZ2 is regulated by the adjacent PDZ domains 1 and 3 Fig. . This isBacterial expression plasmids pGEX-2 T-PDZ2 (PTPN13 residues 1347–1450), pGEX-2 T-PDZ3 (PTPN13 residues 1474–1580), and pGEX-2 T-PDZ2/3 (PTPN13 residues 1347–1580) were constructed by sub-cloning of PCR-generated PTPN13 cDNA fragments in-frame into appropriate pGEX-2 T vectors.E. coli BL21 (DE3) cells in isotope-enriched minimal media using 13C glucose and/or 15N ammonium chloride as carbon and nitrogen sources [As previously published, isotope-labeled proteins were prepared by growing  sources , 42–47.E. coli BL21(DE3) under ampicillin and chloramphenicol selection. In order to generate 15N isotopically-enriched proteins, cells were grown in minimal media [Microfluidics Corporation. Cell debris was removed by centrifugation at 10,200 g and at 4 °C for 45 min. Afterwards, the supernatant was incubated with Glutathione Sepharose 4B beads  at 20 °C for 60 min. The GST-tag was subsequently cleaved off through incubation with Thrombin protease  for 12 h at 4 °C. Then, the protein was eluted from the Glutathione Sepharose beads with phosphate buffered saline (PBS) at 20 °C. Protein containing fractions were pooled and concentrated in a Millipore concentrator with molecular weight cut-offs of 5000–10,000 Da, frozen in liquid nitrogen, and finally stored at − 80 °C until further use. The integrity of purified proteins was checked by SDS-PAGE and mass spectrometry (data not shown).GST-fusion proteins were expressed in al media . Proteinal media , 49. In 13C and/or 15N-enriched protein in PBS buffer at pH 7.4 including 10% D2O [www.bruker.com). The backbone assignments were obtained from the BioMagResBank accession numbers 15,199 (PDZ2/3) [15N-enriched PDZ2 at a molar ratio of 1:1 at 298 K by recording 2D 1H-15N-HSQC NMR spectra. In this study, the numbering scheme of the assigned chemical shifts for PDZ2 (5131) and PDZ23 (15199) has been shifted by one residue so that, for example, T30 according to the BMRB entry 5131 now equals T31. Amino-terminally acetylated dodecapeptides were commercially obtained from JPT Peptide Technologies (Germany). The peptide sequences KRHSGSYLVTSV (M = 1332.69 g/mol) and MFRDFDYIADWC (M = 1580.64 g/mol) correspond to the last 12 C-terminal residues of APC and PRK2, respectively. APC and PRK2 peptides were titrated with PDZ2, PDZ3, and PDZ2/3 domains that were isotopically enriched with 15N. The experiments were carried out at protein-to-ligand ratios of 1:0, 1:1, 1:3, 1:5, 1:10, 1:20, and 1:30 at 298 K and pH 7.4. For all binding studies that included a PDZ3 domain, Dithiothreitol (DTT) was added to the sample before carrying out the titration in order to avoid the oxidation of cysteines. The differences in backbone 1H and 15N NMR chemical shifts were monitored via 2D 1H-15N-HSQC NMR spectra. The weighted 1HN and 15N chemical shift differences were calculated according to the following equation [NMR spectra were acquired at 298 K on Bruker DRX 600 and AVANCE III HD 700 spectrometers. Typically, NMR samples contained 0.1–0.5 mM uniformly  10% D2O –44. All  10% D2O  and anal 10% D2O  or TopSp(PDZ2/3)  and 5131(PDZ2/3) , 44, 46.equation , 47:www.originlab.com) as previously published [D values was calculated from the individual fit of several significant chemical shift perturbations found for the amide resonances located in and/or next to the canonical PDZ binding cleft. For the PDZ2/APC titration amide resonances of G23, K50, A52, L73, Q80, and A81 of were used, for the PDZ2/PRK2 titration amide resonances of T21, G23, T30, Y43, G51, A52, S55, and D56 were used, for the ‘PDZ2 in PDZ2/3’/APC titration amide resonances of L19, D57, and Q81 were used, and, finally, for the ‘PDZ3 in PDZ2/3’/APC amide resonances of G159, F162, S163, K184, L223, and G225 were used.Averaged dissociation constants for the different complexes were calculated from least-squares fitting of the NMR chemical shift perturbations observed for several amino acids as a function of ligand concentration. NMR chemical shift perturbation data were analysed with the ORIGIN software package  software package was used to generate a model of the APC-bound PDZ2/3 tandem from PTN13 based on the differential NMR chemical shift perturbations outside the canonical binding cleft of PDZ domains between PDZ2/APC and ‘PDZ2 in PDZ2/3’/APC titration . For PDZ


Hybrid compounds 7c–d showed remarkable antifungal activity against Cryptococcus neoformans with a minimum inhibitory concentration (MIC) value of 15.6 µg/mL. Against other opportunistic fungi such as Candida spp. and Aspergillus spp., these hybrids showed MIC values of 62.5 µg/mL. Regarding their antibacterial activity, all the synthetic hybrids demonstrated little inhibition of Gram-negative bacteria (MIC ≥50 µg/mL), however, hybrid 7b displayed >50% inhibition against Klebsiella pneumoniae at 20 µg/mL and full inhibition at 50 µg/mL. Moreover, this hybrid was shown to be a potent anti-staphylococcal molecule, with a MIC value of 2 µg/mL (5 µM). In addition, hybrid 7h also demonstrated inhibition of Staphylococcus aureus at 20 µg/mL (47 µM). Hybrids 7a and 7b were the most potent against Mycobacterium tuberculosis H37Rv with MIC values of 20 and 10 µg/mL (46 and 24 µM), respectively. The 7b hybrid demonstrated high selectivity in killing S. aureus and M. tuberculosis H37Rv in comparison with mammalian cells (SI >20), and thus it can be considered a hit molecule for mechanism of action studies and the exploration of related chemical space.Eight quinoline-based hydroxyimidazolium hybrids  Acinetobacter baumannii, Pseudomonas aeruginosa, or the Gram-positive Enterococcus faecium, or Staphylococcus aureus added to the multi- and extensively- drug-resistant Mycobacterium tuberculosis. Regarding fungi, they are also a source of concern in antifungal chemotherapy because many fungi can be opportunistic pathogens seriously affecting immunocompromised patients, and some of them, such as Candida albicans, Cryptococcus neoformans, and Aspergillus spp. can cause complicated systemic infections that are associated with high mortality rates Cl) 6 in the presence of AcONa and ACN as a solvent, under ultrasound irradiation at 80 °C during 1–7 h. This straightforward procedure afforded the corresponding quinoline-based hydroxyimidazolium hybrids 7a–h in 60–91% yield. Structures of hybrids 7 were confirmed by 1H NMR  of all hybrids was determined with the clinical and laboratory standards institute (CLSI) microbroth dilution methods M27-A3 and M38-A2 [Hybrids d M38-A2 ,23. HybrC. neoformans were the most susceptible sp., with all hybrids showing some degree of antifungal activity with MIC values between 15.6 and 250 µg/mL. Instead, MIC values of 7a–h against C. albicans were in the range of 62.5 to (>250 µg/mL) with two hybrids (7e and 7h) showing to be inactive (MIC >250 µg/mL). Regarding Aspergillus spp., they were the less sensitive spp, since 5 hybrids  (of the 8 hybrids tested) were inactive.Results in 7 against C. neoformans, the percentages of inhibition of C. neoformans for each hybrid 7a–h at concentrations from 250 to 3.90 µg/mL (obtained by two-fold dilutions) were determined. Results are recorded in 7 against C. neoformans can be clearly observed.In order to have a deeper look at the antifungal behavior of hybrids 7a–d) were more potent than those from 2-oxo-quinolines (7e–h), and this can clearly be seen in 7a–d possess higher anti-cryptococcal activity than 7e–h since the inhibition percentages of 7a–d at concentrations ≤31.25 µg/mL are clearly higher than those of 7e–h.The dotted lines show the percentages of inhibition of the hybrids at 31.25 µg/mL and is useful to clearly observe the behavior of the hybrids at lower concentrations that are considered highly active. In 7a), induced a relative lower activity than the methyl group in the same position (hybrid 7b), as shown in 7c seemed to confer a significant increase in antifungal potency compared with the methyl group in position C-6 of hybrid 7b. (iii) Hybrids 7c and 7d showed the same MIC (15.62 µg/mL) values, although 7c seemed to be more potent than 7d, as evidenced by the much stronger inhibition displayed by 7c at 3.9 µg/mL, (see 7d decreased the activity or the methyl group in position C-8 (hybrid 7c) increased the antifungal effect, or that both played a significant role in the activity. (iv) The same detrimental trend by the chlorine atom in position C-7 on the antifungal activity was also observed when comparing the N-butylated hybrids 7f (6-Me) (MIC = 125 mg/L) with 7g (7-Cl) (MIC = 250 mg/L. (v) Nevertheless, the methyl group in position C-6 (hybrid 7b) induced a lower activity even than the chlorine atom at position C-7 in 7d, affording MIC values of 15.62 and 31.25, respectively, O-butylated hybrid 7b (MIC = 31.25) with the N-butylated hybrid 7f (MIC = 62.5), both possessing a 6-Me substituent, indicated that not only this substituent and its position contributed to the activity of the synthesized hybrids 7.From the above results, some preliminary structure–activity relationships can be drawn: (i) The methoxy group in position C-6 , as shown in E. coli, K. pneumoniae, and S. aureus, and isoniazid for mycobacteria. All the synthetic hybrids showed little activity against the Gram-negative organisms with MIC values ≥50 µg/mL. Against K. pneumoniae, hybrids 7a and 7b showed to be the most potent, as evidenced by their moderate growth inhibition at high concentration . The hybrid 7h also demonstrated a significant inhibition against S. aureus with a MIC value of 20 µg/mL (47 µM) and should also be considered as a hit molecule for exploring related chemical space. Hybrids 7a and 7b showed significant inhibition against M. tuberculosis H37Rv with MIC values of 20 and 10 µg/mL (46 and 24 µM), respectively. The remaining hybrids 7d, 7e, and 7h displayed moderate growth inhibition against the virulent H37Rv strain with MIC values of 50 µg/mL (–115 µM). A similar trend of activity was found against M. bovis BCG, confirming their antimycobacterial effect.The hybrids ntration . The hybM. bovis BCG and the virulent M. tuberculosis H37Rv was compared  to our best results. Against Mycobacterium tuberculosis, the hybrids of quinolone and pyrazole displayed inhibitory values (MIC 12.5–50 mg/L) similar to our results [The anti-mycobacterial response of the hybrids towards the non-virulent compared . A correterature ,25, and terature , showed  results . However results  and were7b represents a promising molecule showing significant antibacterial effect against S. aureus (5 µM) and M. tuberculosis H37R (24 µM), for which the study of its mechanism of action should be undertaken to assess its potential development as an anti-infective agent. In this direction, some previous reports indicated that quinoline hybrids, along with other nitrogen-containing heterocycles have shown to bind to heme and hemozoin unities, but also DNA, acetylcholinesterase, or to affect prostaglandin production [In summary, the hybrid oduction ,31,32,337a–h was evaluated by an MTT -2,5-diphenyl-2H-tetrazolium bromide) assay on VERO Cells (ATCC® CCL-81™) with peroxide as a positive control and dimethyl sulfoxide (DMSO) as a negative control. VERO cell line growth was expressed as half-lethal concentration (LC50) values and are summarized in The cytotoxic activity of the hybrids 7a–h on the green monkey VERO cell line showed, in general, a low effect with LC50 values higher than 125 µM (7a–d) showed to be slightly less cytotoxic (LC50 ~ 189–277 µM) than the 2-oxo-quinolines (7e–h) (LC50 ~ 125–235 µM). The selectivity index (SI) was calculated as the ratio between LC50 on VERO cells and MIC against bacteria. As the cytotoxic concentrations were relatively similar, the hybrids with lower MIC values such as 7a, 7b, and 7h, showed higher selectivity. Specifically, the hybrid 7b showed high SI values of 117 and 23.4 for S. aureus and M. tuberculosis H37Rv. The SI is an indirect measure of the therapeutic window, and it can serve as a predictor of safety during in vivo trials for a given bacterial infection.Examination of cytotoxic activity of the hybrids n 125 µM . The 2-aC. albicans ATCC 10231, C. neoformans ATCC 32264, A. flavus ATCC 9170, A. fumigatus ATTC 26934, A. niger ATCC 9029. Strains were grown on Sabouraud-chloramphenicol agar slants for 48h at 30 °C, maintained on slopes of Sabouraud-dextrose agar  and sub-cultured every 15 days to prevent pleomorphic transformations. Inocula were obtained according to reported procedures [3 cells with colony-forming units (CFU)/mL.For the antifungal evaluation, standardized strains from the American type culture collection , and CEREMIC  were used: N-morpholino)propanesulfonic acid). Microtiter trays were incubated at 35 °C for yeasts and Aspergillus spp. MICs were visually recorded at 48h for yeasts, and at a time according to the control fungus growth, for Aspergillus spp. For the assay, stock solutions of the hybrids were 2-fold diluted with RPMI-1640 from 250 to 3.90 µg/mL  and a final DMSO concentration ≤1%. A volume of 100 µL of inoculum suspension was added to each well, with the exception of the sterility control where sterile water was added to the well instead. Amphotericin B  was used as positive control. Endpoints were defined as the lowest concentration of drug resulting in total inhibition (MIC) of visual growth compared to the growth in the control wells containing no antifungal drug.Minimum inhibitory concentration (MIC) of each hybrid was determined by using broth microdilution techniques according to the guidelines of the Clinical and Laboratory Standards Institute for yeasts (M27-A3)  and for C. neoformans ATCC 32264. For the assay, compound test wells (CTWs) were prepared with stock solutions of each hybrid in DMSO (maximum concentration ≤1%), diluted with RPMI-1640, to final concentrations of 250–3.90 μg/mL. An inoculum suspension (100 μL) was added to each well . A growth control well (GCW)  and a sterility control well (SCW)  were included for each fungus tested. Microtiter trays were incubated in a moist, dark chamber at 30 °C for 48h for both yeasts. Microplates were read in a Versa Max microplate reader . Amphotericin B was used as positive control. Tests were performed in triplicate. Reduction of growth for each compound concentration was calculated as follows: % of inhibition = 100−(OD 405 CTW−OD405 SCW)/(OD405 GCW−OD405 SCW).This determination was performed with the yeast M. tuberculosis H37Rv in a biosafety level 3 laboratory of the National Health Institute in Bogota. A stock solution of the hybrids was prepared in DMSO at a concentration of 200 mg/mL. Dilutions of the compounds were prepared in DMSO in 24 well plates, 2 mL of each dilution was dispensed in each well at 200, 100, 50, 20, and 10 mg/mL. The final volume was 2 mL of molten Middlebrook 7H10 medium  supplemented with 0.5% glycerol and 10% oleic acid, albumin, dextrose, and catalase  were added to the wells. An inoculum having a cell density of 106 CFU/mL was prepared from a 2-week culture of M. tuberculosis H37Rv (ATCC 27294) grown in Löwenstein–Jensen medium slants at 37 °C. Two microliters of the diluted inoculum were dispensed in the middle of the agar from each well, and the plates were incubated for 2–3 weeks at 37 °C. Isoniazid was included as a positive control at 10, 1, 0.1, 0.05, and 0.01 μg/mL concentrations. After the incubation period, the plates were observed, and the MIC was determined as the minimum concentration on which growth was not observed. The experiment was repeated on a different day observing exactly the same results. The SPOTi agar dilution method was also employed for MIC determination against M. bovis BCG. The cells were passaged first in Middlebrook 7H9 and then spotted into Middlebrook 7H10 medium with supplement albumin, 0.5% glycerol, dextrose, and sodium chloride (NaCl). The plates were incubated for 1 week at 37 °C. The concentrations tested were 200, 100, 50, 20 and 10 μg/mL and isoniazid was used as a positive control.The agar dilution spot culture growth inhibition (SPOTi) assay ,35 was pE. coli  was cultured in Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% bovine fetal serum (FBS) and 1% streptomycin-penicillin and passaged twice before the assay in 21 cm2 cell culture Petri dishes at 37 °C in 5% CO2 incubator and 100% humidity. The cells were then cultured in 96 well plates for 24h before the assay to a cell density of 90.000 cells per well. A 10 mg/mL hybrid stock solution was prepared in DMSO, following 2-fold serial dilution until 0.01 mg/mL. Finally, 5 μL were transferred to each plate containing the VERO cells and incubated for 48h. Hydrogen peroxide was used as a positive control and DMSO as a negative control under the same dilution conditions. After 24h of incubation, culture media was changed for 100 μL of DMEM media without FBS freshly prepared with MTT solution at 5 mg/mL, and the plates were further incubated for 2h. The media was then removed, and 100 μL of DMSO was added to each well. After 30 min incubation, the absorbance was read at 540 nm on a microplate reader (FLUOstar Omega) [50 values were determined by interpolation from the mean absorbance data of 100% viability (negative control) and 0% viability (positive control).The VERO cell line (ATCCr Omega) . The exp7a–h against panels of fungal and bacterial pathogens, including mycobacteria. Hybrid compounds 7c–d showed the highest antifungal activity against C. with MIC values of 15.6 µg/mL each one. Furthermore, all hybrids showed promising antibacterial activity, although, compound 7b presented the strongest activity against S. aureus and M. tuberculosis H37Rv with MIC values of 2 µg/mL (5 µM) and 10 µg/mL (24 µM), respectively.In summary, we report the antimicrobial activity of eight quinoline-based hydroxyimidazolium hybrids