The m7G cap marks the 5′ end of all eukaryotic mRNAs, but there are also capped ends that map downstream within spliced exons. A portion of the mRNA transcriptome undergoes a cyclical process of decapping and recapping, termed cap homeostasis, which impacts the translation and stability of these mRNAs. Blocking cytoplasmic capping results in the appearance of uncapped 5′ ends at native cap sites but also near downstream cap sites. If translation initiates at these sites the products would lack the expected N-terminal sequences, raising the possibility of a link between mRNA recapping and proteome complexity. We performed a shotgun proteomics analysis on cells carrying an inducible inhibitor of cytoplasmic capping. A total of 21 875 tryptic peptides corresponding to 3565 proteins were identified in induced and uninduced cells. Of these, only 29 proteins significantly increased, and 28 proteins significantly decreased, when cytoplasmic capping was inhibited, indicating mRNA recapping has little overall impact on protein expression. In addition, overall peptide coverage per protein did not change significantly when cytoplasmic capping was inhibited. Together with previous work, our findings indicate cap homeostasis functions primarily in gating mRNAs between translating and non-translating states, and not as a source of proteome complexity. The capFunctional analysis of cytoplasmic capping required the development of tools to selectively disrupt this process without affecting nuclear 5′ end processing. One approach targets cytoplasmic m7G cap methylation by overexpressing a truncated form of RNMT in the cytoplasm (ΔN-RNMT) with a mutation in the SAM binding site. ΔN-RNMT competes with cytoplasmic RNMT for binding to both CE and RAM, and its overexpression results in loss of mRNAs with improperly methylated caps . AnotherThe current study addressed this using an isogenic system consisting of tetracycline-inducible U2OS cells stably transfected with a transgene expressing the K294A cytoplasmic guanylylation inhibitor ,15,17,192.2.1.The parental cell line for much of our work consists of U2OS osteosarcoma cells that stably express the tetracycline repressor protein (U2OS-TR cells ). That m2.2.As noted in the Introduction, one can inhibit cytoplasmic capping by interfering with GMP addition or with cap methylation. The current study employed the former approach, using a dominant-negative form of capping enzyme (termed K294A) to block the cytoplasmic guanylylation step. Twenty-four-hour induction of K294A causes prolonged inhibition of recapping that in turn results in the accumulation and/or loss of uncapped forms of cytoplasmic capping targets .Total cellular protein was recovered from triplicate cultures of cells treated ± doxycycline, reductively alkylated, trypsinized and subjected to label-free shotgun proteomics as described in Material and methods. To increase the possibility of detecting quantitative changes in peptide representation, analysis was performed with a more sensitive mass spectrometer than that used to examine the effect of doxycycline on the parental cell line, coupled to improved front-end separation (UPLC and ion mobility). Differential peptide and protein expression were determined by comparing peptide spectral matches (PSMs) and filtering for false discovery rate (FDR) less than 0.01. This identified 21 875 peptides corresponding to 3565 proteins ; this represents about one-third of the total proteins that might be expressed by this cell line . This nuTwenty-nine proteins  showed a significant increase when cytoplasmic capping was inhibited . This isa). A similarly small number of proteins  were significantly decreased when cytoplasmic capping was blocked (b), but again one cannot draw conclusions based on such a small number of differentially expressed proteins. None of these proteins correspond to transcripts in ,,9], we pin vivo single-molecule translational dynamics that showed cycling of mRNAs between translating and non-translating states [In summary, the accumulated data do not support a role for cytoplasmic capping in proteome complexity, but instead support the model of cap homeostasis put forward in , where dg states ,30. The 3.3.1.2 and were discarded after no more than 10 passages. Cells were grown in McCoy's 5A medium (Thermo Fisher 116600) supplemented with 10% tetracycline-free fetal bovine serum . Triplicate cultures of parental U2OS-TR or K294A-expressing cells at 70–80% confluence were switched to medium without or with 1 µg ml−1 of doxycycline for 24 h. Prior to harvest cultures were washed three times with phosphate-buffered saline (PBS) and lysed using ice-cold lysis buffer  and one tablet of phosphatase inhibitor . The cell lysates were sonicated using Sonic Dismembrator Model 100 (Fisher Scientific) for three cycles of alternating 30 s bursts followed by 30 s rest followed by centrifugation at 16 000× g for 15 min at 4°C. The protein concentration of the collected supernatant was determined by a bicinchoninic acid (BCA) protein assay kit (ThermoFisher Scientific).Tetracycline-inducible U2OS (U2OS-TR) cells and tetracycline-inducible U2OS cells stably transfected with pcDNA4/TO/myc-K294DNLS + NES-Flag (U2OS-K294A) were described previously . Cells w3.2.−1 trypsin (1 : 200 w/w) supplemented with 1 µl of 1% ProteaseMAX surfactant (Promega) and incubating at 37°C for 3 h. Trypsin was inactivated by addition of trifluoroacetic acid (TFA) to a final concentration of 0.5%. The digestion products were centrifuged 16 000g for 10 min, and the supernatants were collected and evaporated to dryness.Four hundred milligrams of lysate was reductively alkylated by first incubating for 1 h at 37°C with 10 mM dithiothreitol, followed by 30 min alkylation (in the dark) at 25°C with 55 mM iodoacetamide (Sigma Aldrich). Samples were diluted sixfold with 50 mM ammonium bicarbonate to reduce the concentration of guanidine hydrochloride to less than 1 M. Tryptic digestion was performed by adding 2 µl of 1 µg µl3.3.3.3.1.b) was performed by LC-MS/MS on a Waters nanoACQUITY UHPLC system  coupled to a Thermo LTQ-Orbitrap Elite hybrid mass spectrometer via an Easy Spray ion source . Peptides (0.5 µg) reconstituted in 0.1% formic acid was injected and loaded onto a Waters Symmetry C18 trap column  for desalting at a flow rate of 20 µl min−1. The analytical separation was achieved on a C18 reversed-phase column  at pH 2.4 which was equilibrated to initial conditions of 98% (v/v) A1 and 2% (v/v) B1. The subsequent separation was achieved at 35°C where the % B1 was maintained at 2% for 5 min; 2–35% over 75 min; 35–45% over 10 min and 45–85% over 10 min at a flow rate of 0.3 µl min−1. The column was held at 85% (v/v) B1 for 5 min before reaching initial conditions after 10 min. The heated capillary temperature and electrospray voltage on the Orbitrap Elite were 200°C and 1.5 kV, respectively, using top 15 data-dependent acquisition in positive ion mode. The MS scans were acquired at a resolution of 120 000 with a automatic gain control (AGC) target value of 1 × 106 for a scan range of 400–1600 m/z. Collision-induced dissociation (CID) spectra were obtained in the ion trap with AGC target of 1 × 104, maximum ion injection time (IT) of 50 ms, 1 m/z isolation width, normalized collisional energy (NCE) of 35 and ion activation time of 10 ms. The transfer tube S-lens RF was 49% and dynamic exclusion was set at 15 s with a repeat count of 1 for an exclusion list size of 500.Analysis of the impact of doxycycline on the U2OS cell proteome U2OS-TR, b was per3.3.2.−1 using a linear gradient from 2% to 25% solvent B1 over 90 min, 25% to 37% over 10 min, 37% to 80% over 10 min and maintained for 10 min for total separation method time 120 min. Data acquisition on the timsTOF Pro used the parallel accumulation serial fragmentation (PASEF) acquisition mode. Instrument settings included default imeX mode, mass range 100 to 1700 m/z, capillary voltage of 1.6 kV, dry gas 3 l min−1 and dry temp of 180°C. PASEF settings included 10 ms ms−1 scans at 1.18 s total cycle time, scheduling target intensity of 20 000, charge range 0–5, active exclusion release after 0.4 min, and CID collision energy 42 eV.Samples from U2OS-K294A cells were analysed using a nanoElute coupled to a timsTOF Pro equipped with a CaptiveSpray source . Peptides (0.2 µg) was separated on a 25 cm × 75 µm analytical column, packed with 1.6 µm C18 beads . The column temperature was maintained at 50°C using an integrated column oven . Separation was achieved using 0.1% formic acid (A1) and acetonitrile with 0.1% formic acid (B1) as mobile phases. The column was equilibrated with four column volumes of 100% solvent A1 before loading sample at a maintained pressure of 800 bar. Peptide separation was achieved at 0.4 ml min4.4.1.H. sapiens proteome (as of 15 June 2018 with 20 292 entries). Search parameters included precursor mass tolerance of 10 ppm and 50 ppm for the Orbitrap Elite and timsTOF Pro, respectively, and fragment mass tolerance of 0.6 Da and 0.05 Da, respectively. Database searches used trypsin (full) cleavage with a maximum of 2 missed cleavages. Cysteine carbamidomethylation was set as a fixed modification while oxidation of methionines and protein N-terminal acetylation were set as variable modifications. Percolator was used for estimation of the PSM (peptide spectrum match) level FDR of peptide identification. Proteins were identified at a 0.01 FDR for PSM and 0.01 protein FDR. Protein groups were filtered only to include peptides with 99% confidence.The data files from the timsTOF Pro were converted into mgf-files using MSConverGUI (ProteoWizard). Protein identifications for the Orbitrap Elite and timsTOF Pro data were obtained via the Thermo Proteome Discoverer software (v. 1.4.1.14) using the Sequest search algorithm and the Uniprot Swissprot canonical 4.2.2 transformed and differential enrichment analysis was carried out using linear models combined with empirical Bayes statistics function in Limma. False discovery correction was applied using the Benjamani-Hochberg method and data were visualized using volcano plots with significant p-values ≤0.05 and heatmaps.Label-free relative quantification was carried out using the Limma package in R  using pe5.Homo sapiens as the select organism. Pie chart illustrations of gene ontology annotations for molecular function of proteins with significantly different expression are presented in Gene ontology analysis was performed using PANTHER  Classification System ,33, v. 1


In this study, a new laccase that could catalyze AFB1 degradation was purified and identified from the white-rot fungus Cerrena unicolor 6884. The laccase was purified using (NH4)2SO4 precipitation and anion exchange chromatography, and then identified as Lac 2 through zymogram and UHPLC-MS/MS based on the Illumina transcriptome analysis of C. unicolor 6884. Six putative laccase protein sequences were obtained via functional annotation. The lac 2 cDNA encoding a full-length protein of 512 amino acids was cloned and sequenced to expand the fungus laccase gene library for AFB1 detoxification. AFB1 degradation by Lac 2 was conducted in vitro at pH 7.0 and 45 °C for 24 h. The half-life of AFB1 degradation catalyzed by Lac 2 was 5.16 h. Acetosyringone (AS), Syrinagaldehyde (SA) and  (ABTS) at 1 mM concentration seemed to be similar mediators for strongly enhancing AFB1 degradation by Lac 2. The product of AFB1 degradation catalyzed by Lac 2 was traced and identified to be Aflatoxin Q1 (AFQ1) based on mass spectrometry data. These findings are promising for a possible application of Lac 2 as a new aflatoxin oxidase in degrading AFB1 present in food and feeds.Aflatoxin B Aspergillus fungi such as Aspergillus flavus and Aspergillus parasiticus, are toxoids leading to fluorescence, human carcinogenicity and mutagenicity. The toxicity of AFs is related to the C8-C9 double bond of the difuran ring and the lactone ring within the coumarin ring  +), 346.09055 ([M + NH4] +), and 351.04626 ([M + Na] +). Compared to AFB1 (C17H12O6), this formula shows addition of only one more oxygen, and it is considered to be an AFB1 metabolite. According to the results of UHPLC-MS/MS in 1 gradually decreased to zero after incubation for 60 h. In the whole process, the AFB1 metabolite increased in the first 4 h, and then declined as AFB1 appeared. Therefore, part of AFB1 would be converted to the metabolite, which would be slowly degraded, such as that of AFB1 via Lac 2 during the following incubation.In our study, a new conspicuous fraction eluted at about 3.57 min in the UHPLC chromatogram A emerged1, AFB2, AFM1, AFG1 and AFG2, can be detoxified with laccases ) were purchased from Sigma-Aldrich . AS (Acetosyringone) was purchased from Solarbio . HBT (N Hydroxy benzorizole) was purchased from Aladdin . Acetonitrile (ACN), Methanol, formic acid (FA), and trifluoroacetic acid (TFA) used in mass spectrometry of HPLC grade were purchased from Merck . Regenerable cellulose syringe filters, 0.22 μm (size 13 mm), were obtained from Jinteng . Other chemicals used were of analytical grade from Sinopharm .Chemicals for gel electrophoresis were supplied by Solarbio  and TaKaRa . Trypsin (proteomic grade), trizol, and SA  were purchased from Thermo Fisher Scientific . AFBPhylogeny of the strain was identified by using ITS sequencing. Genomic DNA was extracted with a DNA Quick Plant System , and universal primers ITS 1 and ITS 4 were used for amplification of ITS. The PCR product was sequenced. The ITS sequence has been submitted to GenBank with the accession number MT712199. The phylogenetic analysis (with 1000 bootstraps) was performed with MEGA version 7.0 by the neighbor-joining method. Other fungal ITS sequences used in this study were from GenBank.C. unicolor 6884 was routinely grown on PDA plates at 30 °C for 4 days. The extraction of total RNA and the Illumina transcriptome analysis were both performed by Sangon. An mRNA-Seq approach was employed to gain insight into the laccase gene expression profile of C. unicolor 6884 during routine growth on PDA plates. The total number of reads was assembled with Trinity (Version 2.4.0) to conduct the Transcript and Unigene, and the Unigene of C. unicolor 6884 was submitted to NCBI with the SRA accession PRJNA644218. The whole sequences in Unigene were blasted against nine databases, including NR, NT, PFAM, KOG, Swiss-Prot, KEGG, CDD, TrEMBL and GO, to find genes encoding putative laccases. These putative laccase genes have been submitted to GeneBank with accession numbers MT720691, MT720692, MT720693, MT720694, MT720695 and MT720696, respectively.C. unicolor 6884 was grown in 50 mL medium  in 250 mL Erlenmeyer flasks at 30 °C with shaking at 200 rpm. Samples were collected at regular time intervals  for enzyme activity assays and AFB1 degradation in vitro described below. The reaction mixture of AFB1 degradation contained cultured broth and AFB1 (5 µg/mL) incubated at 45 °C over a period of 60 h. The samples were removed for HPLC analysis  as detailed underneath. The values represent means ± standard errors (n = 3).The production of laccase was carried out as previously described in . Briefly−1·cm−1) according to the procedure described in [Laccase activity was measured photometrically at 420 nm 2SO4. The precipitate was resuspended and desalted by dialysis against 20 mM Tris-HCl buffer (pH 8.5) and applied to a HiTrap DEAE column  on an ÄKTATM Purifier . Adsorbed proteins were eluted with 0.2 M NaCl in 20 mM Tris-HCl buffer (pH 8.5) and ultra-filtered with a membrane module  at 4000× g for 20 min. Electrophoretic analyses were performed according to Yang [After cultivation for 14 d, the fermentation culture was harvested, collected, centrifuged and fractionated by addition of 50% to 90% . The injection volume was 5 μL. The following linear elution gradient was used for the analytical separation: solvent B remained at 2% for 3 min at the beginning of the elution, it was then varied from 2% to 6% in 3 min, from 6% to 28% in 45 min, from 28% to 34% in 4 min; then it was increased up to 95% in 2 min and this ratio was maintained constant for the following 13 min. The percentage of B suddenly decreased at 2% and kept stable for 10 min for column reconditioning. The two reserves used were: A = 2% ACN + 98% H2O + 0.04% FA and B = 80% ACN + 20% H2O + 0.08% FA; flow rate was set at 300 μL/min. The data were acquired and analyzed by Proteome Discover .The identification of laccase from at 37 °C . DigestiC. unicolor 6884 was grown on PDA plates at 30 °C for 4 d. Total RNA was extracted with Total RNA Extractor (Trizol). The cDNA of C. unicolor 6884 was generated by RT-PCR from total RNA with RevertAid First Strand cDNA Synthesis Kit. Lac 2 was amplified by PCR with the primer pair  using total cDNA of C. unicolor 6884 as the template. PCR products were inserted into the pMD18-T vector and transformed into E. coli top10 competent cells which were then cultured on an LB plate with 100 mg/mL ampicillin. Three positive clones of each fragment were sequenced. The cDNA sequence of lac 2 studied in this paper has been submitted to GenBank with the accession number MT232811.The strain C. unicolor 6884 laccases were analyzed online BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). SignalP-5.0 was used for theoretical signal peptide determination .The sequences of 1 degradation by Lac 2, a time-course analysis was carried out in the dark. The 200 μL reaction mixture contained 30 μg/mL Lac 2 (50 U/mL) and AFB1 (5 µg/mL) in MilliQ water at 45 °C over a period of 60 h. The samples were periodically  taken for HPLC analysis.For AFB1 degradation, AFB1 (5 µg/mL) was individually incubated with Lac 2 (50 U/mL) in Na2HPO4-citric acid buffer (pH 7.0) at various temperatures ranging from 30 to 65 °C  over a period of 8 h.To investigate the effect of temperature on Lac 2-mediated AFB1 degradation by Lac 2, AFB1 (5 µg/mL) was individually incubated with or without Lac 2 (50 U/mL) for 24 h at 45 °C in buffers at different pH values, including Na2HPO4–citric acid buffers (pH 3.0–8.0), and 50 mM glycine-NaOH buffers (pH 9.0–10.0), respectively.To investigate the effect of pH on AFBKm, Kcat and Vmax) for AFB1 degradation, the initial reaction rate was investigated by monitoring the removal of AFB1  at 10 min intervals up to 50 min at 45 °C and pH 7.0 with 30 μg/mL of Lac 2 (50 U/mL). The kinetic parameters were determined by nonlinear regression of Michaelis- Menten plots using the software GraphPad Prism 8.2.1 . One unit of AFB1-degratation activity was defined as the quantity of laccase that detoxified 1 μg of AFB1 per minute.To determine the kinetic parameters  in Na2HPO4–citric acid buffer (pH 7.0) at 45 °C for 3 h, while controls did not have any mediator.To investigate the effect of LMS on AFB1 (5 µg/mL) with Lac 2 (50 U/mL) for 0 h was included as a treatment control. The residual AFB1 in all aforementioned samples was determined by FLD on HPLC . The values represent means ± standard errors (n = 4).Incubation of AFB1 was extracted from the samples with an equal volume of dichloromethane three times and evaporated under nitrogen gas at 50 °C. The samples were dissolved in an equal volume of methanol and filtered .AFBv/v/v) at a flow rate of 0.1 mL/min for 5 min at 40 °C. The sample injection volume was 2 μL. AFB1 was measured by a FLD Detector which was set at the wavelengths of 365 nm (excitation) and 450 nm (emission). The retention time of AFB1 was 2.5 min.HPLC analyses of the samples were performed on an Agilent 1260 Infinity Ⅱ Series  using a Poroshell 120 EC-C18 column . The mobile phase for elution was composed of methanol, aceto-nitrile and water  coupled with a Q Exactive mass spectrometer . NSI-MS experiments were performed in the positive ionization mode. The mobile phase, which consisted of water, methanol and acetonitrile , was used for elution at a flow rate of 0.1 mL/min for 10 min at 40 °C. An Hypersil Gold C18 column  was used for separation at a flow rate of 0.1 mL/min for 10 min at 40 °C. The sample injection volume was 2 μL. AFB1 was measured by a DAD Detector at 365 nm. The retention time of AFB1 was 5.0 min. The AFB1 degradation products were identified and analyzed with the Compound Discover .The Lac 2-mediated AFB


Xenopus oocytes. We show that functional expression of GLUT2 in yeast requires the deletion of the extended extracellular loop connecting transmembrane domains TM1 and TM2, which appears to negatively affect the trafficking of the transporter in the heterologous expression system. Furthermore, single amino acid substitutions at specific positions of the transporter sequence appear to positively affect the functionality of both GLUT2 and GLUT3 in yeast. We show that these variants are sensitive to known inhibitors phloretin and quercetin, demonstrating the potential of our expression systems to significantly accelerate the discovery of compounds that modulate the hexose transport activity of GLUT2 and GLUT3.Human GLUT2 and GLUT3, members of the GLUT/SLC2 gene family, facilitate glucose transport in specific tissues. Their malfunction or misregulation is associated with serious diseases, including diabetes, metabolic syndrome, and cancer. Despite being promising drug targets, GLUTs have only a few specific inhibitors. To identify and characterize potential GLUT2 and GLUT3 ligands, we developed a whole-cell system based on a yeast strain deficient in hexose uptake, whose growth defect on glucose can be rescued by the functional expression of human transporters. The simplicity of handling yeast cells makes this platform convenient for screening potential GLUT2 and GLUT3 inhibitors in a growth-based manner, amenable to high-throughput approaches. Moreover, our expression system is less laborious for detailed kinetic characterization of inhibitors than alternative methods such as the preparation of proteoliposomes or uptake assays in  Transport of hexoses across plasma membranes marks the first and rate-limiting step of energy metabolism in cells of all domains of life. In humans, 14 glucose transporter family members , with differing tissue distributions, mediate the facilitative diffusion of sugar along a concentration gradient. Despite a high sequence similarity, GLUTs differ in substrate specificity and affinity , matchinKM = ∼17 mM   and eveectively ). For gl∼0.8 mM) . Under n∼0.8 mM) ; the glu∼0.8 mM)  and/or s∼0.8 mM)  are the ∼0.8 mM) . Further∼0.8 mM) . Loss of∼0.8 mM) , a rare ∼0.8 mM) .KM = 1.4 mM . Other = 1.4 mM . GLUT3 s= 1.4 mM , and tog= 1.4 mM . Accorda= 1.4 mM . Consist= 1.4 mM . In gene= 1.4 mM . Recentl= 1.4 mM . Hence, Both transporters GLUT2 and GLUT3 belong to the Class I GLUT family, therefore sharing certain structure similarities as, for example, the position of the glycosylation site in the extracellular loop between the transmembrane helices (TM) 1 and 2 . For GLUSaccharomyces cerevisiae is a widely used GRAS organism and a model organism for diverse research applications. Cells are easily manipulated and maintained and exhibit a short generation time. By deleting all endogenous hexose transporter genes  and the genes of hexose transporting maltose transporters  with the loxP-Cre recombinase system in a CEN.PK2-1C strain background (0) strain, incapable of growing on glucose or related monosaccharides, was constructed and named EBY.VW4000  mutation   or fructmutation  that affmutation . The cormutation . Murine  pathway . The lat0 yeast system. Thereby, we complete the accessibility of the well-characterized Class I GLUTs  in a coE. coli strain DH10B  was used.The construction of the strains CEN.PK2-1C , EBY.VW4E. coli. Plasmid isolation from overnight E. coli cultures was carried out using a GeneJET Plasmid Miniprep Kit (Thermo Scientific) according to the manufacturer’s instructions and sequenced at GATC Biotech . For subcloning into a vector with a dominant marker, the respective transporter sequences were amplified from the p426H7 plasmids as described, with primers exhibiting overhangs to the HXT7 promotor or CYC1 terminator, respectively, and PCR products were transformed together with the linearized pRS62K plasmid, which contains the same promotor and terminator regions, into EBY.VW4000 cells to allow for homologous recombination. To check the in vivo localization, envyGFP (HXT7 promotor and the CYC1 terminator was inserted into the low-copy CEN6/ARS4 vector pUCPY1. All plasmids used in this study are listed in DNA Sequences of GLUT2 and GLUT3 are listed in  envyGFP  was fuse2O). Cells were resuspended in ddH2O and OD600nm was adjusted to 1. Dilutions of OD600nm 0.1, 0.01 and 0.001 were prepared and 4 µl of each dilution was dropped onto the agar plate. Plates were incubated at 30°C for 5 days.For growth tests on solid medium, drop tests were performed on minimal SC -URA medium, containing the respective sugar, with cells expressing transporter constructs in the p426H7 vector backbone. Pre-cultures were grown overnight in 10 ml SCM (1% (w/v)) -URA medium at 30°C and 180 rpm, centrifuged  and washed twice in double-distilled, sterile water (ddH2O. Washed cells were used to inoculate 30 ml YEP G418 (200 µg/ml) medium with the indicated sugar to an OD600nm of 0.2 in 300 ml Erlenmeyer flasks, which were mounted onto the sensor plate. Quantification of cell growth and calculation of apparent maximal growth rates were performed with the CGQuant software (Aquila Biolabs) as previously described (Cell growth in liquid YEP medium was measured with the Cell Growth Quantifier (Aquila Biolabs) . Pre-culescribed .in vivo localization of the (modified) transporters, CEN.PK2-1C and EBY.S7 cells expressing the envyGFP-tagged constructs on the low-copy CEN.ARS plasmid pUCPY1 were grown overnight in filter-sterilized, low fluorescent SC medium in which uracil was omitted (lf-SC -URA) and 1% (w/v) maltose (for EBY.S7 cells) or 2% (w/v) glucose (for CEN.PK2-1C) was added. 500 µl cell suspension of an OD600nm between 1.5 and 3 was mixed with 500 µl lf-SC -URA medium with the respective sugar containing 1.2% (w/v) low melting agarose (Roth) to reach a suspension with 0.6% (w/v) low melting agarose for immobilization. Six microliters were applied to an object plate, sealed with a cover slip and GFP fluorescence was located with the Confocal Laser Scanning Microscope .To investigate the To confirm the activity of envyGFP-tagged transporter constructs, EBY.S7 cells expressing the respective construct were streaked out on solid SC -URA medium with 0.2% (w/v) glucose and growth was recorded after 5 days of incubation at 30°C.https://www.chemcomp.com), using as a template the crystal structure of GLUT1 (PDB ID 4PYP). The homology models for the outward-facing GLUT2 were generated with MOE from the crystal structure of GLUT3 (PDB ID 5C65 or 4ZWC). The amino acid sequence identity and similarity between GLUT2 and GLUT3 are 50% and 68%, between GLUT1 and GLUT2 are 52% and 68%, and between GLUT1 and GLUT3 are 63% and 78%, respectively, as determined with the alignment function from MOE. The homology models generated were scored with GB/VI. The mutations were performed in MOE Protein Designing function and subject to energy minimization with the Forcefield Amber10.The crystal structure of GLUT2 is unknown and only the outward-facing GLUT3 structures are available. The homology models of the inward-facing GLUT2 and GLUT3 were generated with the ‘Homology Model’ function of the program package Molecular Operating Environment ) media containing 200 µg/ml G418. Cells were washed once in YEP media containing 0.2% (w/v) glucose (YEPG) and 200 µg/ml G418 and transferred in the same media so that OD600nm ∼ 0.5 and grown further for 1 to 2 days. GLUT3S66Y expressed in EBY.S7 were grown for two days in SC-URA with 1% (w/v) maltose, then washed and transferred in SC-URA with 0.2% (w/v) glucose, followed by further growth for 1-2 days. For transport activity assay, cells were centrifuged , washed once with PBS solution , and resuspended in PBS buffer at an OD600nm ∼ 10; each assay determination contained 100 µl of this cell solution. Transport activity assay was started by adding C14-glucose (10 mM for GLUT2∆loopS_Q455R or 1 mM for GLUT3S66Y). Transport activity assay was halted after 10 min by adding 3 ml ice-chilled Quench buffer , followed by filtration through a glass fiber channel  under vacuum, and another two washes with 3 ml Quench buffer and filtration. The filtration membranes were transferred into scintillation vials with 10 ml of Scintillation Solution , and vortexed briefly. The radioactivity was determined with a scintillation counter . Phloretin and quercetin were dissolved in DMSO at 20 mM stock concentration, and inhibitor concentrations used for IC50 determination were 100x stocks so that the final concentration of DMSO in the assay was 1%. Controls for determining the relative transport activity included 1% (v/v) DMSO to account for DMSO presence due to inhibitor, and the cells transformed with the empty vector. We found that the background activity with the empty vector was comparable to the transport activity of the transporters at 200 µM phloretin. Data were analyzed with the nonlinear fit analysis of GraphPad Prism .The culturing of yeast cells was done at 30°C with shaking (180 rpm). EBY.S7 yeast cells expressing GLUT2HXT7-1–329) or terminator (CYC1) region, respectively, and co-transformation with the linearized host plasmid (p426H7) in EBY.VW4000, EBY.S7, and SDY.022 cells allowed for the plasmid assembly via homologous recombination  maltose, and incubated for three days at 30°C, resulting in the growth of approximately 1000 colonies per plate. Subsequently, cells were replica plated on SC -URA medium, containing 0.2% (w/v) glucose, to screen for cells that regained the ability to use glucose as a carbon source. EBY.VW4000 transformants did not show any growth on the solid glucose medium, even after a prolonged incubation of seven days. Most of the EBY.S7 and SDY.022 transformants grew after five days of incubation on glucose. To investigate if wild-type GLUT3 is active in these strain backgrounds, we isolated and sequenced plasmids of several colonies with various sizes. In bigger colonies, one single mutation (S66Y) was found, located in TM2. However, also unmodified GLUT3 mediated the growth of EBY.S7 and SDY.022 transformants on glucose. Re-transformation of the native GLUT3 and the modified GLUT3S66Y construct in EBY.S7 cells resulted in growth on glucose medium, proving that no further mutations, except for the fgy1 mutation, in the yeast strain were essential for this phenotype. However, while wild-type GLUT3 was incapable of mediating growth of EBY.VW4000 cells on solid glucose medium, slight growth was observed for GLUT3S66Y-expressing EBY.VW4000 cells (S66Y) on a plasmid, revealed larger colonies for those expressing the mutated version (0 cells expressing GLUT3 or GLUT3S66Y were performed in liquid YEP medium with 0.2% (w/v) glucose, for which the GLUT3 ORFs were transferred to pRS62K plasmid backbones. Consistent with the results observed in the drop tests, both constructs enabled the growth of EBY.S7 cells, but GLUT3-expressing cells showed a significantly longer lag phase and a 1.8 times lower apparent maximal growth rate than those expressing GLUT3S66Y  with oligonucleotides having 30-40 base pair overhangs to the applied promoter  human GLUT2 sequence were generated as described above for GLUT3. However, after replica plating, EBY.VW4000, EBY.S7, or SDY.022 transformants, of which more than 1000 colonies grew on solid maltose medium, showed no growth on glucose-containing agar plates, even after prolonged incubation at 30°C for one week.Like the S66Y mutation in GLUT3, previous studies found single point mutations in the second transmembrane region of GLUT1   and GLUTN62Q) within the first extracellular loop, just in case the glycosylation disturbed the activity of the transporter in yeast cells. This modification also failed to restore hxt0 yeast cell growth on glucose.Interestingly, in GLUT3 and GLUT5, a single serine mutation to a more hydrophobic amino acid enabled or improved the functional expression of these two transporters in yeast . This oA conspicuous structural difference between GLUT2 and the other Class I GLUTs, which are active in yeast, is the extension of the extracellular loop connecting the TM helices H1 and H2 .0 yeast strains, we reasoned that shortening the extension, thereby introducing a GLUT3-like-loop, could promote GLUT2 activity in the heterologous system.Since minor sequence modifications have not led to an active expression of GLUT2 in hxt∆loop), we also constructed one chimera in which those amino acids were replaced by one serine (GLUT2∆loopS) and one in which the lysine preceding the deleted region was mutated to serine (GLUT2K54S_∆loop)  or arginine (Q455R). Also, Q455R mutation was found in one out of three GLUT2∆loopS constructs isolated from SDY.022 cells, indicating an additional beneficial effect of this mutation on the glucose uptake via GLUT2. The isolated loop constructs GLUT2∆loop, GLUT2K55S_∆loop, GLUT2∆loopS, and GLUT2∆loopS_Q455R were re-transformed in all three hxt0 strains, and their growth was analyzed by a drop test on solid glucose medium  . Comparee medium . The abing cells . EBY.VW4pS_Q455R , providi∆loopS and GLUT2∆loopS_Q455R, but not wild-type GLUT2, conferred growth of EBY.S7 cells on 0.2% (w/v) glucose  (∆loopS_Q455R compared to GLUT2∆loopS-expressing cells on 0.2% (w/v) glucose medium (1.7 times) as well as on 2% (w/v) fructose medium (1.5 times)  . In agre glucose . For EBYs medium . Interes ∼76 mM) . However ∼76 mM) . Analysi ∼76 mM)  showed a5 times) .∆loopS and GLUT2∆loopS_Q455R, and of the inactive wild-type GLUT2. The activity of these envyGFP-tagged transporters was confirmed by the growth of GLUT2∆loopS_Q455R-envyGFP and GLUT2∆loopS-envyGFP-expressing EBY.S7 cells on solid SC -URA 0.2% (w/v) glucose medium  for GLUT2∆loopS_Q455R expressed in EBY.S7  indicate a strong inhibition and confirm that yeast-expressed GLUT2 can be reliably used to identify and characterize transport inhibitors despite the deletion of the extended extracellular loop. This is supported by growth tests in the presence of phloretin. We found that 50 µM phloretin inhibited the growth of EBY.S7 cells expressing GLUT2∆loopS_Q455R significantly, leading to an extensive long lag phase of 87.4 hours, a diminished apparent maximal growth rate   . Moreovend GLUT4 . Reflectnificant . In contnificant . As all ΔloopS, GLUT2ΔloopS_Q455R, GLUT3, and GLUT3S66Y were based on the crystal structures of the inward-facing conformation for GLUT1 (PDB ID 4PYP) and the outward-facing conformation of GLUT3 (PDB ID 5C65 or 4ZWC) (The prevailing transport mechanism for the Major Facilitator Superfamily (MFS) proteins, including GLUTs, postulates that the substrate cavity of the transporter is alternately open to either side of the membrane with the transporter cycling between the so-called outward- and inward-facing conformations, through a relative rocking of the transporter N- and C- halves (domains) around the active site . The stror 4ZWC) .ΔloopS and GLUT3 improved the transport activities . Cell growth of GLUT-expressing EBY.S7 cells showed that additional single mutations in GLUT2tivities , 3: Q455-domains . Its sub-domains , but dis-domains , sterica-domains . In the -domains . Convers-domains .0. These systems enable high-throughput ligand screening for a particular GLUT and determination of the potency and selectivity of the ligand candidates relative to closely related GLUTs within class I, and also class II member, GLUT5. The co-existence of several GLUT isoforms within one tissue or cancer cell line complicates efforts to understand the role of a particular GLUT in the cell pathology or physiology. Thus, besides their medical importance as potential drugs, GLUT-selective inhibitors can also serve as investigative tools to unravel how healthy and abnormal cells adjust their energetic needs through GLUT expression, regulation or function   also seem to destabilize the inward-facing conformation while leaving the outward-facing one unaffected. More generally, the substitutions that functionalize GLUTs in yeast cells favor an open substrate cavity towards the extracellular space , either by destabilizing the N- and C-domain interface of the inward-facing conformation  . The cons 35-54) , whethers 35-54) , the oneormation  or by puormation .Evidently, the lipid composition of the yeast’s plasma membrane also impacts the heterologous expression of some human GLUTs .fgy1 mutation  and fructose (GLUT2) uptake. Importantly, they are sensitive to the known inhibitors phloretin and quercetin, which validates our system as a platform for screening and characterizing potential transport inhibitors.


H1T2/H1FNT is a germ cell-specific linker histone variant expressed during spermiogenesis specifically in round and elongating spermatids. Infertile phenotype of homozygous H1T2 mutant male mice revealed the essential function of H1T2 for the DNA condensation and histone-to-protamine replacement in spermiogenesis. However, the mechanism by which H1T2 imparts the inherent polarity within spermatid nucleus including the additional protein partners and the genomic domains occupied by this linker histone are unknown.Sequence analysis revealed the presence of Walker motif, SR domains and putative coiled-coil domains in the C-terminal domain of rat H1T2 protein. Genome-wide occupancy analysis using highly specific antibody against the CTD of H1T2 demonstrated the binding of H1T2 to the LINE L1 repeat elements and to a significant percentage of the genic regions  of the rat spermatid genome. Immunoprecipitation followed by mass spectrometry analysis revealed the open chromatin architecture of H1T2 occupied chromatin encompassing the H4 acetylation and other histone PTMs characteristic of transcriptionally active chromatin. In addition, the present study has identified the interacting protein partners of H1T2-associated chromatin mainly as nucleo-skeleton components, RNA-binding proteins and chaperones.Linker histone H1T2 possesses unique domain architecture which can account for the specific functions associated with chromatin remodeling events facilitating the initiation of histone to transition proteins/protamine transition in the polar apical spermatid genome. Our results directly establish the unique function of H1T2 in nuclear shaping associated with spermiogenesis by mediating the interaction between chromatin and nucleo-skeleton, positioning the epigenetically specialized chromatin domains involved in transcription coupled histone replacement initiation towards the apical pole of round/elongating spermatids. The production of mammalian spermatozoa is a precisely controlled, complex and dynamic developmental process involving the differentiation of diploid spermatogonia to spermatocytes and finally to haploid round spermatids. The last phase of spermatogenesis namely, spermiogenesis comprises a series of morphological transformations and extreme condensation of the chromatin to form highly condensed, polarized spermatozoa. Spermiogenesis is divided into different steps based on the three developmental processes involving condensation of the nucleus followed by the formation of the flagellum and the acrosome. During the steps 1–7, the early round spermatids are characterized by a round nucleus, with the beginning of acrosome and axoneme assembly. These early round spermatids actively transcribe many of the mRNAs necessary for the whole process of spermiogenesis, while many of these mRNAs are subjected to delayed translation until the later stages of spermiogenesis and sperm function. The step 8 comprises the acrosome polarization to one side of the nucleus and the initiation of spermatid elongation followed by the assembly of the accessory structures needed for flagella function. The sperm transcripts remain as stable as ribonucleoprotein particles (RNPs) contributing to the final sperm proteome, which can have more relevant functional roles in the zygote or early embryo . The finChromatin remodeling is a unique process occurring during spermiogenesis, wherein 90% of core histones are being replaced by the testes-specific histone variants, then transition proteins (TPs), and eventually protamines (PRMs) , 5. The Many histone variants are expressed during spermiogenesis and modulate the chromatin structure to facilitate the histone-to-protamine replacement process , 18. LinDrosophila MSt77f revealed dual roles in spermatogenesis, firstly involved in regional distribution of chromatin beneath the stripe of microtubules in histone-based nuclei, and secondly, it is essential for nuclear shaping through the stabilization of microtubules  . Prediction of coiled-coil structures was predicted using the online tool  [Amino acid alignments were performed online using Clustal X, a graphical interface for the ClustalW multiple sequence alignment program of Jalview (_parser) , 115. Se_parser) .To generate antibody against the CTD of rat H1T2 protein, rabbits were immunized with synthetic peptide, EQQYVSAKEQEYVRTKEQEC and a 12 weeks protocol was used for the generation of the antibody. The custom made antibody from the Abgenex Company  was used for the present study.2, 0.1% Nonidet P-40, 1 mM PMSF, 10 mM NaHSO3, 10 mM sodium butyrate, protease inhibitor mixture, phosphatase inhibitor mixture). The homogenate was filtered through four layers of bandage cloth and centrifuged at 1500 × g for 10 min at 4 °C. Histones were extracted from the pellet of crude nuclei by suspending purified nuclei in 0.4 N H2SO4 as prescribed previously [g at 4 °C to pellet the residual chromatin. The extracted proteins were precipitated at 4 °C from the supernatant by making it to 30% with respect to TCA. After keeping on ice for 30 min, the proteins were recovered by centrifugation. The pellet of proteins so obtained was sequentially washed with cold acetone containing 0.05% HCl and ice cold acetone. The pellet recovered was dried, dissolved in water and stored in aliquot at − 20 °C.Rat testes (35–50 days old) were decapsulated and homogenized in 5 volumes of cell lysis buffer  was spotted on the nitrocellulose membrane, dried and subjected to western blot analysis using the anti-H1T2 antibody.The proteins resolved by SDS-PAGE were electrophoretically transferred onto nitrocellulose membranes presoaked in transfer buffer, using a semidry transfer technique. After the transfer, the membrane was blocked using the wash buffer, PBST (1 × PBS with 0.05% Tween 20), containing 5% BSA or milk powder for 1 h at room temperature. It was then washed with the same buffer lacking BSA or milk powder. A suitable dilution of the primary antibody was added in PBST supplemented with 1% BSA or milk powder and incubated for overnight on a rocking platform. The unbound antibody was removed by washing the membrane thrice for 5 min each using wash buffer. A suitable dilution of an appropriate secondary antibody (anti-rabbit/anti-mouse IgG) conjugated to horse radish peroxidase (HRP) in PBST with 1% BSA or milk powder was added and incubated for 1 h. After the antibody incubation, the membrane was washed thrice with the wash buffer for 5 min each. The membrane was placed in the chemiluminescence substrate solution and incubated for 2 min and developed using the ECL kit (Thermo Scientific). For the peptide competition assay, 50-fold molar excess of the peptide were added to the antibody solution and mixed for 3 h at 4 °C before the addition to the blot. The source of the antibodies is given in Table g for 10 min at 4 °C, were washed with PBS and fixed with 4% paraformaldehyde (in PBS) and permeabilized with 0.1% Triton X-100 (in PBS) for 15 min. Non-specific sites were blocked with 1% BSA in PBS. Smears were incubated with selected primary antibodies, anti-H1T2 and counterstained with corresponding secondary antibodies conjugated with Alexa fluor 488 or Alexa Fluor568. Nuclei were stained with DAPI (Sigma) and images were acquired and analyzed in a Zeiss confocal laser scanning  microscope.Rat testes were decapsulated and total testicular cells were decondensed by resuspending in decondensation buffer  and incubated at 4 °C for 1 h in an end to end rotor . Cells obtained after centrifuging at 1500 Chromatin immunoprecipitation (ChIP) was carried out using the published protocol . BrieflyRattus norvegicus genome (rn6) using Bowtie2 (version 2.2.3) with “no mixed”, “-very sensitive” and “no discordant” parameters. All aligned files were then made free from PCR duplicates using samtools. Replicates of both the control and treated were merged, respectively, into a single BAM file. Thereafter, the merged BAM file of the input samples and the merged BAM file of treated samples were used for the subsequent peak calling by MACS1.4.2 with p-value cutoff of 1e-05. Read enrichments/peaks were first called for each replicate with the uniquely mapped reads by MACS1.4.2 with default parameters except the effective genome size corrected for rn6. Chromosome-wise distribution plots were generated using custom R script. H1T2 peak distribution was identified at genomic locations enriched in CpG islands, repetitive elements, exons, introns, intergenic regions, 3′UTR and 5′UTR using HOMER v4.7 (for annotation). Further subclassification of LINE L1 repeat elements was made using the repeat masker data of UCSC table browser. Box plots were created using the R 3.3.2 statistical package. De novo motif identification was done using 300 bp regions surrounding overlapping peak summits as target set and shuffled set of same overlap sequences as background set with MEME software.The input and ChIP DNA libraries were prepared using the NEBNext Ultra II DNA library preparation kit. Sequencing was performed with Illumina HiSeq X Ten system using paired-end reads of 150-nt length. Sequenced reads containing adaptor sequences were trimmed out and filtered with Phred quality threshold as 20. Read pairs falling below 20 bp after clipping were removed. Clean reads were uniquely aligned against UCSC 0C overnight, subjected for RNase  and proteinase K  treatment and DNA were extracted by phenol–chloroform method. Input was prepared from 5% of the sonicated chromatin. 2 μL input or ChIP sample from the recovered DNA suspension of 50 μL was used for performing the qPCR analysis. Specific primers were designed against the HIT2 peaks obtained from ChIP-sequencing experiment  and directly proceeded for MNase digestion by suspending the nuclei in MNase buffer . Sperm nuclei were digested with MNase in MNase buffer for 5 min 37 °C. Reaction was stopped with the addition of 10 mM EDTA on ice. The soluble nucleosome fraction was prepared by centrifugation at 650 × g for 10 min at 4 °C, mixed with LSDB250 buffer  and proceeded to immunoprecipitation protocol.MNase immunoprecipitation was done according to the previous method . BrieflyThe oligonucleosomes fraction prepared by MNase digestion was incubated with either anti H1T2 antibody or pre-immune IgG for overnight at 4 °C in a rotating platform. Protein a/G dynabeads were added on the next day and LSDB250 buffer was used as the wash buffer for the MNase-IP studies. Proteins bound to the beads were extracted using the elution buffer from the Pierce Co-IP kit. The eluted protein complexes were resolved on a 15% SDS gel and run for about 1 cm, then the gel was subjected to Coomassie staining. The Coomassie stained wells corresponding to IP and IgG lanes were excised and subjected to downstream analysis. To determine the proteins associated to H1T2-bound chromatin, we have outsourced the samples for LC–MS/MS analysis which was carried out at the Taplin Biological Mass Spectrometry Facility, Cell Biology Department, Harvard Medical School.3 pieces. As described previously [Excised gel bands were cut into approximately 1 mmeviously , gel pieeviously . Gel pieeviously . All dat2SO4. After precipitation of histone proteins with 30% trichloroacetic acid, the histone extracts were loaded onto a 12% SDS gel and resolved completely. 5–25 kDa portion that contained all core histones were excised from each gel (H1T2 IP and IgG IP lanes) and sent for LC–MS/MS analysis of the histones (PTMs).For the mass spectrometric analysis of core histone PTMs in the immunoprecipitated chromatin complex, the final eluate was subjected to histone extraction using 0.4 n HAdditional file 1: Table S1. H1T2 ChIP-seq data: MACS 1.4.2 output file with annotations. H1T2 ChIP peaks MACS 1.4.2 with p-value cutoff of 1e-05. A total of 11,570 peaks were identified as overlapping peaks between the two replicates and the H1T2 peaks were annotated using HOMER v4.7.Additional file 2: Table S2. Chromosome-wise distribution of H1T2 peaks. Representative excel file of the number of H1T2 peaks across different chromosomes.Additional file 3: Figure S1. Chromosome-wise peak length distribution of H1T2 ChIP peaks. Box plot representing the H1T2 associated average peak length (y-axis) across different chromosomes (x-axis).Additional file 4: Table S3. Representative excel file of the peak length of H1T2 peaks across the chromosomes.Additional file 5: Figure S2. Fold enrichment of H1T2 peaks. Box plot showing the fold enrichment of H1T2 peaks (y-axis) across different rat chromosomes.Additional file 6: Table S4. Representative excel file of the fold enrichment of H1T2 peaks across chromosomes.Additional file 7: Table S5. Repeat elements associated with H1T2. Excel file representing the different classes of repeat elements associated with H1T2.Additional file 8: Table S6. LINE elements subclasses associated with H1T2. Representative excel file of the subclasses of LINE repeats associated with H1T2.Additional file 9: Table S7. Motif identification. Table represents the significant motifs identified from the overlapping peak summits using MEME analysis.Additional file 10: Table S8. GO enrichment analysis of the genes whose promoter-TSS is occupied by H1T2. Excel file related to Fig. Additional file 11: Table S9. GO enrichment analysis of the genes whose promoter-TSS is occupied by H1T2. Excel file related to Fig. Additional file 12: Table S10. H1T2 IP/MS analysis. List of H1T2 associated protein identified by mass spectrometric analysis (≥ 4 number of unique peptides).Additional file 13: Table S11. GO enrichment analysis of the H1T2 IP/MS identified proteins. Excel file related to Fig. Additional file 14: Table S12. GO enrichment analysis of the H1T2 IP/MS identified proteins. Excel file related to Fig. Additional file 15: Table S13. GO enrichment analysis of the H1T2 IP/MS identified proteins. Excel file related to Fig. Additional file 16: Table S14. List of primers used for H1T2 ChIP-PCR analysis.


Ca1.1  influences aqueous humor outflow facility, but the contribution of auxiliary β-subunits to KCa1.1 activity in the outflow pathway is unknown.The large-conductance calcium-activated potassium channel Kβ-subunit genes in anterior segments of C57BL/6J mice (Kcnmb1-4) and in cultured human trabecular meshwork (TM) and Schlemm's canal (SC) cells (KCNMB1-4). We also measured expression of Kcnma1/KCNMA1 that encodes the pore-forming α-subunit. Using confocal immunofluorescence, we visualized the distribution of β4 in the conventional outflow pathway of mice. Using iPerfusion, we measured outflow facility in enucleated mouse eyes in response to 100 or 500 nM iberiotoxin  or 100 nM martentoxin . MarTX selectively blocks β4-containing KCa1.1 channels, whereas IbTX blocks KCa1.1 channels that lack β4.Using quantitative polymerase chain reaction, we measured expression of Kcnmb4 was the most highly expressed β-subunit in mouse conventional outflow tissues, expressed at a level comparable to Kcnma1. β4 was present within the juxtacanalicular TM, appearing to label cellular processes connecting to SC cells. Accordingly, KCNMB4 was the most highly expressed β-subunit in human TM cells, and the sole β-subunit in human SC cells. To dissect functional contribution, MarTX decreased outflow facility by 35%  relative to vehicle-treated contralateral eyes, whereas IbTX reduced outflow facility by 16% .β4-subunit regulates KCa1.1 activity in the conventional outflow pathway, significantly influencing outflow function. Targeting β4-containing KCa1.1 channels may be a promising approach to lower intraocular pressure to treat glaucoma.The  With decreasing facility, IOP becomes elevated, and elevated IOP is the major risk factor for glaucoma. The trabecular meshwork (TM) and inner wall endothelium of Schlemm's canal (SC) regulate outflow facility.Ca1.1  regulates cell volume and contractility in smooth muscle cells,,–Ca1.1 channel opener, NS1619, increases outflow facility in porcine anterior segments.Ca1.1 with iberiotoxin (IbTX) potentiates the facility decrease in response to hypotonic salineCa1.1 is thus involved in outflow facility regulation, apparently by regulating the volume or contractility of SC or TM cells.The large-conductance calcium-activated potassium channel KCa1.1 is composed of four α-subunits that form the potassium-selective transmembrane pore. Membrane depolarization or increased cytosolic calcium may activate KCa1.1, leading to channel opening, potassium efflux, and membrane depolarization.α and auxiliary β-subunits, of which there are four types (β1-4),Ca1.1 sensitivity to voltage and calcium. β-subunits are differentially expressed in a tissue-specific manner and affect the pharmacology of KCa1.1. For example, β4 renders KCa1.1 relatively resistant to IbTX but sensitive to martentoxin (MarTX), which has a smaller effect on KCa1.1 channels lacking β4.–Kβ-subunits of KCa1.1 in mouse anterior segments (Kcnmb1-4) and in human TM and SC cells (KCNMB1-4). We focus on β4, examining its localization in the TM and inner wall endothelium of SC. Using MarTX, we examine the influence of β4 on outflow facility in enucleated mouse eyes. We also examine the effect of IbTX to investigate the influence of KCa1.1 channels that lack β4 on outflow facility.In this study, we examine the expression of genes encoding the Kcnma1 and Kcnmb1-4 in mouse anterior eye segments and KCNMA1 and KCNMB1-4 in cultured human TM and SC cells. For mouse anterior segments, C57BL/6J mice  were euthanized by cervical dislocation. Eyes were enucleated, trimmed of extraocular tissue, and hemisected at the equator. The lens was removed, and the anterior segments homogenized in TRIzol using a rotor-stat tissue homogenizer . Total RNA was extracted using PureLink RNA spin columns following manufacturer protocols . RNA content was measured using a spectrophotometer , and 2 µg of RNA was used to synthesize cDNA by reverse transcription . qPCR was carried out using TaqMan master mix and primers . Expression level relative to GAPDH/Gapdh was calculated using the ΔCt method. Homogenized mouse brain tissue, which expresses Kcnma1 and Kcnmb4,Ca1.1 channels,We used quantitative polymerase chain reaction (qPCR) to measure the expression of mers see . GAPDH/G–KCNMA1 and KCNMB4 expression.Human TM and SC cells were isolated and characterized from human donor eyes following established techniques.Whole globes from three adult mice (C57BL/6J) were fixed in 4% paraformaldehyde  for 4 hours and washed in PBS. The eyes were hemisected at the equator, and 10 µm sagittal cryostat sections were cut. The sections were incubated in BLOTTO´s Blocking Buffer (ThermoFisher Scientific) at room temperature for 1 hour to reduce nonspecific staining. Following three washes in Tris-buffered PBS, specimens were incubated with the primary antibody  at 4°C o4 and dehydrated. Whole eyes were embedded in Epon resin and 1 µm semithin sagittal sections were cut using an ultramicrotome . Semithin sections were stained with toluidine blue and examined with a Keyence Biorevo BZ9000 microscope. Ultrathin sections through the TM were then cut, stained with uranyl acetate and lead citrate, and viewed with a transmission electron microscope . Four regions were examined per eye.For electron microscopy, one MarTX perfused mouse eye (100 nM) and its paired contralateral vehicle perfused eye, were postfixed in OsOWe measured the effect of IbTX and MarTX on outflow facility in enucleated eyes from C57BL/6J mice . We used a paired experimental design, in which one eye was perfused with IbTX or MarTX and the contralateral eye perfused with vehicle. We measured the relative difference in outflow facility between treated and untreated paired eyes. All procedures on living mice were carried out under the authority of a United Kingdom Home Office project license and adhered to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.IbTX was purchased from Tocris Bioscience . MarTX was custom synthesized by Peptide Protein Research Limited  with the sequence FGLIDVKCFASSECWTACKKVTGSGQGKCQNNQCRCY, as determined by Ji et al.N = 4 pairs), 500 nM IbTX (N = 5), and 100 nM MarTX (N = 12).For perfusions, mice were humanely culled by cervical dislocation. Eyes were enucleated, affixed to a support platform using tissue glue, and submerged in PBS at 35°C. Using a micromanipulator and dissection microscope, the anterior chamber was cannulated within 10 to 15 minutes of death using a 33-gauge beveled metal needle  connected to the iPerfusion system .Q and stable pressure P were calculated over the last 4 minutes of each step, and the Q − P data were fit by a power-law relationship of the formIOP was set to 9 mm Hg for 1 hour to pressurize and acclimatize the eye to the perfusion environment. Flow into the eye was then measured over 7 increasing pressure steps from 6.5 to 16.5 mm Hg. The flow rate at each step was considered to have reached stability when the ratio of the flow rate to pressure changed by less than 0.1 nL/min/mm Hg per minute over a 5-minute window. Three pressure steps from three perfusions failed to reach stability and were excluded from further analysis. The stable flow rate rC, which represents the value of outflow facility at a reference pressure rP of 8 mm Hg, and b that characterizes the nonlinearity of the Q-P relationship.rC between treated and untreated contralateral eyes, defined as the ratio of rC in the treated eye relative to that in the contralateral control eye minus unity. We tested whether the relative difference in facility was statistically different from zero using a weighted t-test on the log-transformed data, as previously described.The fitting yields Kcnma1, Kcnmb1, and Kcnmb4 in mouse anterior segments  for Kcnma1 and Kcnmb4 were similar for mouse anterior segments , whereas ∆Ct for Kcnmb1 was greater (10.1), indicating roughly four-fold lower expression .By qPCR, we detected positive expression (Ct < 35) of s N = 3; A. Kcnmb2pression A. HomogeKCNMA1, KCNMB1, and KCNMB4, but low levels of KCNMB2 and undetectable levels of KCNMB3  from primary human TM cells deposited by Porter et al.xpressed C. These β4 within the conventional outflow pathway of C57BL/6J mice using confocal immunofluorescence  for the present data (unpaired two-tailed t-test). rC for IbTX-treated eyes was 5.0  nL/min/mm Hg versus 5.9  nL/min/mm Hg for vehicle-treated eyes.We then examined the effect of two Kβ4-containing KCa1.1 channels.P < 0.0001, n = 12; rC for MarTX-treated eyes was 4.3  nL/min/mm Hg compared with 6.7  nL/min/mm Hg for vehicle-treated eyes. The effect of MarTX on rC was significantly greater than that of IbTX .MarTX is a potent and selective blocker of To examine whether MarTX induces cellular toxicity in the outflow pathway, we used transmission electron microscopy to visualize the ultrastructure of the TM in a pair of eyes perfused with MarTX or vehicle. There were no obvious morphological differences between MarTX and vehicle-perfused eyes. In both cases, the inner wall of SC appeared continuous, and the juxtacanalicular connective tissue (JCT) contained loose extracellular matrix interspersed by JCT cells that extended connections to the inner wall and TM beams A, 3C. ThCa1.1 is a known regulator of outflow facility. Opening KCa1.1 with NS1619 increases outflow facility in porcine anterior segments.Ca1.1 with IbTX prevents the increase in outflow facility following treatment with nitric oxideCa1.1, with either IbTX or MarTX, independent of nitric oxide or any other stimulus, decreases outflow facility in enucleated mouse eyes. Thus across different species, opening the KCa1.1 channel coincides with increasing outflow facility, whereas blockade of the KCa1.1 channel corresponds with decreasing outflow facility.The large-conductance calcium-activated potassium channel KCa1.1 may modulate outflow facility by influencing cell contractility. Opening KCa1.1 leads to potassium efflux and membrane hyperpolarization that inactivates voltage-gated calcium channels.Ca1.1 leads to relaxation in TM and ciliary muscle cells and a reduction in calcium-induced actin polymerization.,,,KCa1.1 channel opening may lead to water efflux across the cell membrane and a reduction in TM and SC cell volume.,,Ca1.1 with IbTX inhibits both the effect of nitric oxide on TM and SC cell volume and on outflow facility.,,Ca1.1 channel opening appears to increase outflow facility by promoting cellular volume reduction and/or cell relaxation in the outflow pathway. Conversely, blocking KCa1.1 with IbTX or MarTX should decrease outflow facility by inhibiting relaxation and/or cell volume decrease. In addition, because cell volume regulation is tightly coupled to mechanical stretch and contractility experienced by TM cells,Ca1.1 to cell volume or contractility alone.Alternatively, KCa1.1, have similar effects on outflow facility. Opening the ATP-sensitive inward rectifier potassium channel 11 (Kir6.2) with cromakalim,ir6.2.ir opening by CKLP1 may affect distal outflow.Other potassium ion channels, in addition to KCa1.1 is regulated by auxiliary β-subunits that affect channel sensitivity to intracellular calcium and membrane voltage.,β-subunits, we show that β4 is the most highly expressed in the anterior segment of the mouse eye. In cultured human SC cells, we show that β4 appears to be the sole β-subunit, and β4 is the primary β-subunit expressed by human TM cells. The β-subunit expression profile influences the pharmacology of the KCa1.1 channel. IbTX and MarTX, for example, are venomous scorpion toxins that are evolutionarily selected to be highly specific for particular β-subunit combinations of KCa1.1. IbTX completely blocks ion channels composed solely of pore-forming α-subunits that are present in all KCa1.1 channels, but IbTX only partially blocks β1-containing KCa1.1 channels (by ∼50%) and ineffectively blocks β4-containing KCa1.1 channels (by ∼20%).β4-containing KCa1.1 channels, with an IC50 value of 21 to 78 nM,,Ca1.1 channels containing only α-subunits.β1-containing KCa1.1 channels for concentrations up to 400 nM with low cytoplasmic calcium.β1-containing KCa1.1 channel activity with an EC50 of 495 nM.β1-containing KCa1.1 channels, as β1 was the second most highly expressed β-subunit after β4 in TM cells and in mouse anterior segments.Activity of Kβ4-deficient), consistent with prior reports.,β4-containing KCa1.1 channels, had a nearly two-fold larger facility reduction relative to IbTX (∼35% vs. ∼16% average reduction). Although the MarTX and IbTX experiments were performed in separate cohorts such that the data are not directly comparable, the larger facility effect observed with MarTX suggests that the majority of KCa1.1 channels involved in outflow facility regulation contain the β4-subunit. However, further study would be necessary to confirm within the same cohort whether MarTX truly has additive inhibitory effects beyond IbTX. Regardless, the magnitude of the facility reduction measured in response to MarTX was comparable to that previously reported in response to sphingosine 1-phosphate,β4-containing KCa1.1 channels appear to be centrally important for the maintenance of outflow because blocking these channels significantly disrupts outflow function.In response to IbTX, we measured a significant decrease in outflow facility in enucleated mouse eyes. This suggests that at least some of the channels involved in facility regulation are IbTX-sensitive (i.e., β4 to the juxtacanalicular TM, with punctate labeling observed along the endothelium of SC. This labeling pattern was consistent with β4 expression along cell processes that connect juxtacanalicular TM cells with inner wall cells.,,–,–,β4. Specifically, we postulate the existence of a stretch-sensitive feedback loop whereby β4-containing KCa1.1 channels are opened in response to IOP-induced stretch in the juxtacanalicular TM, triggering signaling pathways that lead to increased outflow facility to oppose the elevation in IOP. Previous studies have provided evidence that KCa1.1 channels in bovine TM are directly sensitive to stretch, independent of cytosolic calcium levels.Ca1.1 channels are not directly stretch-activated, KCa1.1 may be activated by elevated intracellular calcium that often occurs in response to stretch or other mechanical stimulation. Consequently, we hypothesize that the association with particular β-subunits influence the mechanosensitivity of KCa1.1 in the juxtacanalicular TM. For example, the β4-subunit regulates shear and stretch-mediated mechanotransduction in intercalated cells of the kidney collecting duct.,β4 may influence mechanotransduction via KCa1.1 in the juxtacanalicular TM.Using confocal microscopy, we localized expression of Ca1.1 appears to be involved in the stretch response in TM cells, it is possible that blocking KCa1.1 may suppress stretch-induced release of these compounds to oppose the facility increase. KCa1.1 is also involved in homeostatic regulation of cell volume, as occurs during the regulatory volume decrease following hypoosmotic shock. As regulatory volume decrease is associated with increasing outflow facility,Ca1.1 could potentially affect cell volume regulation in the TM to affect outflow facility.Stretch induces secretion of several compounds from TM cells that increase outflow facility, such as adenosine triphosphate,β4 is the primary β-subunit of KCa1.1 expressed in outflow pathway tissues. Blockade of β4-containing KCa1.1 channels with MarTX leads to a physiologically significant decrease in outflow facility, which appeared to be larger than observed when blocking KCa1.1 channels that lack β4 with IbTX. The β4-subunit is localized to the juxtacanalicular TM and inner wall of SC, where it may influence the response of these tissues to IOP-induced stretch or shear stress. The β4-subunit is thus important for outflow regulation, potentially by influencing mechanotransduction via KCa1.1. Compounds that modulate β4-KCa1.1 activity should therefore be explored as an approach to improve outflow and lower IOP for glaucoma therapy.We have shown that


PM10 and PM2.5 levels, water soluble ions, organic carbon (OC), and elemental carbon (EC) were also analyzed to characterize their chemical profiles. bext (light extinction coefficient) was calculated, which showed the highest in winter with an average of 232.95 ± 154.88 Mm−1, followed by autumn, summer, spring. Light extinction source apportionment results investigated (NH4)2SO4 and NH4NO3 played key roles in the light extinction under high RH conditions during summer and winter. Sulfate, nitrate and Ca2 + dominated in PM10/PM2.5 ions. Ion balance results illustrated that PM samples were alkaline, and PM10 samples were more alkaline than PM2.5. High SO42−/K+ and Cl−/K+ ratio indicated the important contribution of coal combustion, which was consistent with the OC/EC regression equation intercepts results. Principal component analysis (PCA) analyses results showed that the fugitive dust was the most major source of PM, followed by coal combustion & gasoline vehicle emissions, secondary formation and diesel vehicle emissions. Potential contribution source function (PSCF) results suggested that local emissions, as well as certain regional transport from northwesterly and southerly areas contributed to PM2.5 loadings during the whole year. Local government should take some measures to reduce the PM levels.Daily PM Atmospheric aerosols have been found to be associated with adverse influences on atmospheric visibility, human health, and global climate change . AerosolYulin , located in the Mu Us Desert, is one of the Asian Pacific Regional Aerosol Characterization Experiment (ACE-Asia) super site. However, most studies carried in Yulin have been studied to determine the chemical and physical profiles of Asian dust and its transportation . As the −1. 10 and 285 PM2.5 samples were collected onto 47-mm quartz microfiber filters  using six minivolume samplers  at 5 L/min. Before sampling, the quartz filters were pre-heated to 800 °C for 3 h to remove any residual carbon. After sampling, the filters were placed in clean plastic cassettes and stored in a refrigerator at ∼4 °C until in order to minimize the evaporation of volatile components. More details can be found in Samplings were conducted at three sites : Environ4 daily. One replicate analysis was performed for each of 10 samples.PM samples were equilibrated using controlled temperature (20–23 °C) and relative humidity (35–45%) desiccators for 24 h before and after sampling, and their mass loadings were determined gravimetrically using a Sartorius MC5 electronic microbalance . Each filter was weighed at least three times before and after sampling after 24 h equilibration period. The differences among the three repeated weightings typically were less than 10 µg for blanks and 15 µg for sample filters. 1/4 of each filter sample was extracted by 10 mL distilled-deionized water (18.2 M Ω) to analyze ions. Cations were detected by a CS12A column  and 20 mM methanesulfonate as an eluent. Anions were separated by an AS11-HC column  using 20 mM KOH as the eluent with detection limits less than 0.05 mg/L . Standar4+, Ca2+, Mg2+ have been calculated using the formula below The neutralization factor (NF) can be used to describe the interaction between cations and anions . The NF 4+, Ca2+ or Mg2+, using their equivalent concentrations (microgram per cubic meter).Where X may be NHbext), is calculated as the PM2.5 scattering  absorption  (nge (VR)  (6)\docuThe revised IMPROVE formula was described as follow : (7)\docsulfatelarge) and Small sulfate  are accumulated using the IMPROVE equation (The Large sulfate (equation : (8)\doc follows : (11)\doPotential contribution source function (PSCF) is in view of backward trajectories which connects the residence time in upwind areas with relative high concentrations of a certain species through conditional probabilities . The PSCij is an arbitrary weight function to decrease small values effect of nij. nij the number of the end points; mij is the number of trajectory end points in this grid cell whose values higher than the threshold value. In addition, the airmass backward trajectories had been previously calculated based on the National Oceanic and Atmospheric Administration (NOAA) Hybrid Single-Particle Lagrangian Integrated Trajectory model .Meteorological data, including ambient temperature, relative humidity (RH), wind speed (WS), and atmospheric pressure, were collected from Weather Underground  and EHS (35.7 µg m−3 for PM10 and 26.8 µg m−3 for PM2.5) were higher than those at EPA (32.1 µg m−3 for PM10 and 24.9 µg m−3 for PM2.5). Sulfate, the most abundance component in PM, followed by Ca2+ and NO3−.Annual PM and chemical species in Yulin are summarized in 2+, NO3−, and SO42− were shown in 10 and PM2.5 mean levels for dust dominated days were 283.4 µg m−3 and 130.4 µg m−3, which were both over 1.7 times of the spring average values. Two dust events were observed in winter and spring, leading the yearly highest PM10 Ca2+ concentrations of 19.7 µg m−3 (winter) and 12.9 µg m−3 (spring). The Ca2+ levels during dust events were over double of seasonal average values for both PM10 and PM2.5 and this phenomenon was consistent with the report of previous literatures , followed by spring (3.9), winter (3.7), and autumn (3.0), which was consistent with the value as 10 and PM2.5 were plotted as shown, respectively   . Materians (32−)  in the fns (32−) . The yea−1for both spring and summer) and temperature (13 °C for spring and 23 °C for summer) inferred higher mixing and dispersion than those during winter. As shown in bext  showed the highest with an average of 232.95 ± 154.88 Mm−1, followed the decrease order of autumn >summer >spring, which were similar in Xi’an (Daily averaged VR  was 21.6in Xi’an .4)2SO4, NH4NO3, OM, EC, the major contributors to PM. Moreover, coarse matter (CM) was important contributor to bext as a result of high PM10 concentrations 2Strations . In thisbext and chemical species mass concentrations are presented with the Mm−1 and µg m−3 unit, respectively. fL(RH) is the growth curves of sulfate and nitrate, which can be found in IMPROVE net results (fL(RH) was used in this study because sulfate and nitrate mass are distributed in droplet mode. results . fL(RH) 2.5 potential advection, PSCF analysis was conducted in this study  favorable to diffusion and low relative humidity (33% in average) unfavorable to sulfate formation. High winter and low spring sulfate were observed in Xi’an, which showed a difference seasonal pattern when compared to Yulin . Lower NO3−/SO42− ratio suggests that stationary emissions are a dominant source of PM particles which has also been reported in 3−/SO42− ratios were the lowest for both PM10 and PM2.5, which because high temperature can favor SO2 converted to SO42−, while low RH was unfavorable the NO3− formation  formation 2SO4 were the most chemical species contributing to bext. (NH4)2SO4 accounted highest in summer (34.39 ± 14.79%), while NH4NO3showed the large contribution in autumn. During winter, (NH4)2SO4 also was the highest during winter followed by CM. These results indicated that the extinction effects from (NH4)2SO4 and NH4NO3 significantly increased under high RH conditions during summer and winter.Good correlations in different seasons were found between the reconstructed bsibility . A summa10 and PM2.5 over Yulin. Moreover, local emissions and some certain regional transport were the main sources. Despite the dust transportation, the economic boom in Yulin gave rise to substantial air pollution. In order to improve the air quality, strict measurements should be launched in both local and regional areas.PSCF results have shown that dust was the most abundant components during spring, which was due to the atmospheric dust transport. Unlike winter and spring, potential source area was mainly from local emissions and low potential source areas were from northerly areas like Guanzhong Plain. This is consistent with the dominant source from coal combustion related above . PCA res2+ levels during winter and autumn should attribute to fugitive dust. High spring and summer sulfate levels should be due to different sources. Ion balance illustrated that PM10 samples were more alkaline than PM2.5. Winter bext showed the highest with an average of 232.95 ± 154.88 Mm−1, followed by autumn, summer, and spring. The regression equation intercepts for different values in PM2.5 and PM10 indicated that OC primary non-combustion emissions, such as regional background carbonaceous species, long range transport, and local biological detritus, influenced heavily on fine particles in comparison with coarse fraction. Light extinction source apportionment results inferred that the extinction effects from hygroscopic species, such as, (NH4)2SO4, NH4NO3, increased significantly under high RH conditions during summer and winter. High SO42−/K+ and Cl−/K+ ratio indicated the important contribution of coal combustion. PCA analyses results showed that the fugitive dust was the most major source of PM, followed by coal combustion & gasoline vehicle emissions, secondary inorganic aerosol and diesel vehicle emissions. PSCF results suggested that PM2.5 were mainly from both local emissions and regional transport from northwesterly and southerly areas during the whole year.The chemical species for PM were analyzed and their associated sources were identified in Yulin, China. PM levels, OC, EC were highest during winter and lowest during summer. High Ca10.7717/peerj.8447/supp-1Figure S1Click here for additional data file.10.7717/peerj.8447/supp-2Figure S2Click here for additional data file.10.7717/peerj.8447/supp-3Figure S3Click here for additional data file.10.7717/peerj.8447/supp-4Figure S4Click here for additional data file.10.7717/peerj.8447/supp-5Figure S5Click here for additional data file.10.7717/peerj.8447/supp-6Table S1Click here for additional data file.10.7717/peerj.8447/supp-7Table S2Click here for additional data file.10.7717/peerj.8447/supp-8Data S1Click here for additional data file.10.7717/peerj.8447/supp-9Supplemental Information 9Click here for additional data file.


Bacillus licheniformis B307 strain, obtained from Syrian soil, were studied. Optimization experiments were carried out under submerged fermentation conditions, and colloidal chitin was the source of carbon. Luria broth medium supplied with 0.5% colloidal chitin was the optimum medium for chitinase production. The maximum chitinase yield was obtained at 30 °C, pH6, incubation time 14 days, and 150 rpm. The optimum chitinase activity was achieved at 60 °C and pH6. The chitinase activity with unmodified medium was 1.9 U/mL which then enhanced about eight folds to reach 14.2 U/mL under optimized submerged fermentation conditions. An extracellular chitinase of Bacillus licheniformis B307 was partially purified using ammonium sulfate precipitation followed by concentration with various sizes of concentrator tubes. The chitinase was partially purified 8.24 fold and specific enzyme activity increased 2.08 fold (2 U/mg). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of partial purified chitinase exhibited a molecular weight (Mr) near to 36 and 42kDa. These results make it possible to invest in this strain to produce chitinase to be used as antifungal, food additives and other applications.The optimal conditions required for chitinase production from  Bacillus licheniformis; Optimization; Submerged culture; PurificationBiochemistry; Biotechnology; Microbiology; Molecular biology; Plant biology; Chitinase;  TherefoBacillus species are one of the largest sources of bioactive natural compounds  and it showed chitinolytic and antifungal activity against Botrytis cinerea (Bacillus strain was prepared by inoculating 20 mL of sterilized nutrient broth (NB) in 100 mL flask with loop full of pure Bacillus licheniformis B307 and incubated overnight at 30 °C and 200 rpm until the optical density at 600nm reached 0.15 which equals to 2 × 108 CFU/mL . The color of the mixture was developed by incubating it for 10 min at 100 °C. Centrifugation was performed at 7500 ×g for 10 min, then the supernatant adsorption was measured at 540nm. A standard curve was plotted using N-acetyl glucosamine . One unit (U) of chitinase activity represent the amount of released enzyme of 1μmol N-acetyl glucosamine of colloidal chitin per min under reaction conditions.Chitinolytic activity was estimated by dinitrosalicylic acid (DNS) method using colloidal chitin as a substrate according to the method described by 2.4SmF experiments were performed in 100 mL flasks that contain 20 mL of the culture medium, and inoculated with 1 mL of bacterial inoculum. Various chemical and physical parameters were studied and their effect on production of the enzyme was recorded.2.5M medium, consists of (g/L): 0.5 yeast extract, 1 (NH4)2SO4, 1 KH2PO4, 0.3 MgSO4.7H2O; and Y medium, consists of (g/L): 5casein, 5 galactose, 1 KH2PO4, 0.3 MgSO4.7H2O; Z medium, consists of (g/L):0.5 yeast extract, 2 Na2HPO4, 1 K2HPO4, 1 NH4Cl, 0.5 CaCl2.2H2O, 0.5 NaCl, 0.5 MgSO4.7H2O. Colloidal chitin was added to each of the mentioned media to a final concentration of 0.5%. All the culture media were adjusted at pH 7. The cultivation experiments were carried out in shaking incubator 150 rpm, 30 °C, for 5 days.Different types of media were implemented for optimal production of chitinase including NB medium, consists of (g/L): 1 beef extract, 2 yeast extract, 5 peptone, 5 sodium chloride; LB medium, consists of (g/L): 10tryptone, 5 yeast extract, 5 sodium chloride; 2.6Production medium was prepared with different concentrations of colloidal chitin , and incubated under shaking conditions (150 rpm) at 30 °C.2.7To determine the optimum incubation temperature for chitinase production, cultivation was carried out at different incubation temperatures ranged from 20 to 40 °C with variation of 5 °C.2.8The influence of pH on chitinase production was monitored, where the pH of production mediums were adjusted before sterilization, from pH4 to pH9 at intervals of 1 unit, using NaOH 1M and HCl 1M.2.9The optimum incubation time was determined for optimal chitinase production, inoculated flasks were incubated in a rotary shaker at 150 rpm and 30 °C. Every 24 h the culture filtrate was harvested and the enzyme activity was measured for 15 days of incubation time.2.10Optimum agitation speed was determined by incubating the production medium in Erlenmeyer flasks 100 mL at different agitation speed in shaker incubator ranging from 50 to 200 rpm with interval unit of 50 rpm.33.1To determine the optimal temperature for chitinase activity, the reaction mixtures incubated at different temperatures ranged from 20 to 80 °C and then assayed the enzyme's activity under standard conditions using colloidal chitin as a substrate. Also, optimal pH for chitinase activity was determined by measuring the activity at 60 °C within a range of pH from 4 to 9 using sodium-acetate buffer (pH4.0–6.0), sodium-phosphate buffer (pH7.0–8.0) and glycine-NaOH buffer (pH 9.0).3.2g for 10 min at 4 °C and dissolved in a volume of buffer sodium-acetate pH6 equal to the size of crude enzyme. Protein determination in each fraction was performed according to g for 30 min, then the supernatant was separated using 30kDa membrane and centrifugation 2500 ×g for 30 min, after that the supernatant was separated using 50KDa membrane and 2500 ×g for 30 min finally the total protein and enzyme activity were determined for every fractions, and each were also subjected to electrophoresis on 12% polyacrylamide gel. The gel was stained using 0.5% Coomassie Brilliant Blue R250 (Sigma).The crude enzyme was partially purified from the culture supernatant using saturated ammonium sulfate (SAS). The precipitation was carried out at 4 °C in stirring conditions using different percentage of SASs 45%, 55%, 65%, 75%, and 85%. Precipitates were collected by centrifugation at 11500 ×3.3STATISTIC program  was used for statistical analyses at 5% level (P = 0.05). The data was subjected to analysis of variance  for the determination of differences in means between treatments. The percentages were analyzed by applying normal approximation test . All the experiments were repeated three times.44.1Bacillus species  was observed when 0.5% of colloidal chitin was used  at 30 °C and 35 °C respectively, while significant reduction in the production was observed when the temperature increased or decreased from the previous values  showed significant effect on the chitinase yield produced by s values . Temperais 35 °C .Figure 34.5B. licheniformis B307 was tested. The maximum chitinase production was found to be at pH6 with the activity of 8.56 ± 0.165U∖mL, while the minimum chitinase activity 3.87 U∖mL  was observed at pH4. However, yield at pH7 to pH9 has not decreased significantly  , after that, the production began to decline. In many SmF processes, when the production reaches the highest yield, further increase in incubation time can reduce the yield due to the degradation of metabolites .4.7B. licheniformis B307 was observed at agitation speed of 150 rpm (9.63 ± 0.88 U∖mL) . The chitinase production was increased as there were increases in the agitation speeds up to 150 rpm, then decreased with increased speed  . So, when the temperature increased or decreased from 60 °C, the activity of chitinase gradually reduced. The relative activity of chitinase was 80% at 50 and 70 °C (B. licheniformis B307 was compared to chitinases produced from other strains in similar works.Reaction temperature is one of the determining factors in the effectiveness of enzyme activities. In this study, results showed that the optimum temperature for chitinase activity produced by nd 70 °C . In Tabl4.9B. licheniformis B307 chitinase was determined in the range pH4.0- pH9.0 (B. licheniformis B307 was compared to chitinases produced from other strains in similar works.Enzymes activities are affected by changes in the pH value of the reaction, and the point that achieves the highest enzymatic efficiency is known as the optimum pH. Therefore, the pH- relative activity of 0- pH9.0 , and the4.10B. licheniformis B307, the fraction of 65% SAS which gave the highest specific chitinase activity was concentrated by various concentrator tubes. The fraction containing the supernatant of tube MWCO 50kDa showed the highest chitinase specific activity 2U/mg and the specific enzyme activity was increased 2.08 fold with 169% yield from 65% SAS (Mr near to 36 and 42kDa (Bacillus of different molecular weights: 89, 76, 72, 66, 62, 59, 53, 49, 42, and 36kDa (In the first step, the enzyme was partially purified by precipitation with different concentrations of SAS. Maximum specific enzyme activity 0.97U/mg was observed in the fraction containing 65% of SAS, while in the crude enzyme it was 0.24U/mg . It was  65% SAS . SDS-PAGnd 42kDa . Many rend 36kDa .Figure 75B. licheniformis B307strain were determined using SmF method. A partial characterization was also performed, where the optimum temperature and pH were determined to obtain the best chitinase activity. A partial purification of the extracellular chitinase in the crude extract was also performed, where chitinase was purified to reach 8.24 fold comparing to the crude extract with specific activity 2U/mg for the partial purified enzyme.In this study, the optimal conditions for chitinase production from B. licheniformis B307 strain, has a potential use in the industries that have obstacles in using chitinase enzymes at pH6 and/or temperature up to 50–70 °C degrees such as pharmaceuticals, biocontrol and biotechnology applications among others.Depending on its properties, the chitinase obtained by Yasser Akeed: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.Faiza Atrash: Conceived and designed the experiments; Analyzed and interpreted the data.Walid Naffaa: Conceived and designed the experiments; Wrote the paper.This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.The authors declare no conflict of interest.No additional information is available for this paper.