Lens culinaris) lectin (GSLL) exhibits hemagglutinating activity, and possesses some properties distinct from those of other lentil lectins  that deserve further investigation. This study aims to investigate the basic properties  and biological activities  of GSLL.The green speckled lentil seed  in Dulbecco’s modified Eagle’s medium with fetal bovine serum, and examined by using the MTT assay, flow cytometry, and western blotting after treatment of nasopharyngeal carcinoma CNE1 and CNE2 cell lines with the lectin.L. culinaris lectin (LcL), especially higher coverage of the β-chain of LcL. A 48-h treatment with GSLL exerted antiproliferative effects on nasopharyngeal carcinoma CNE1 and CNE2 cell lines with significant inhibition at 0.125 mg/mL (P < 0.001) and 1 mg/mL (P = 0.004), respectively, and these effects were attenuated in the presence of glucose and mannose. GSLL induced apoptosis in nasopharyngeal carcinoma CNE1 cells, with detectable phosphatidylserine externalization, mitochondrial depolarization, and cell cycle arrest. Western blot analysis suggested that GSLL triggered the extrinsic apoptotic pathway involving caspase 3, 8, and 9 in CNE1 cells.GSLL appeared as a 21-kDa band in non-reducing SDS-PAGE. It was composed of two subunits with molecular sizes of 17 and ~4 kDa. It exhibited specificity in binding to glucose and mannose, as well as glucosides and mannosides. Mass spectrometry and N-terminal amino acid sequencing revealed similarity of GSLL to GSLL possessed some different properties from LcL , and increased caspase 3, 8, and 9 activity in CNE1 cells. Lens culinaris) seeds contain high contents of proteins (~25 % dry weight) and minerals  [spleen (pi) and stomach (wei), and relieve dampness (shi) of the body [Lentil  , 2. Theyn, etc.) , have hin, etc.) , and posthe body , 7.L. culinaris lectin (LcL) or hemagglutinin (LcH) [d-mannopyranosyl and α-d-glucopyranosyl residues of glycoproteins and glycolipids [Lentil seeds contain a carbohydrate-binding protein, in (LcH) . The seqin (LcH) , and thecolipids . LcLs cacolipids , 12. Thecolipids , 14.Phaseolus vulgaris) had prominent antiproliferative activity on hepatoma HepG2 cells [P. vulgaris) inhibited breast cancer MCF7 cells and nasopharyngeal carcinoma CNE1 cells more strongly than it inhibited HepG2 cells [P. vulgaris) was devoid of anticancer activity [Lentils are differentiated into many different cultivars worldwide, such as the green speckled lentil in the United States. Although not manufactured in bulk quantities, the product of edible dry green speckled lentil seeds can be found in a number of supermarkets. Similar to other lentil seeds, green speckled lentil seeds contained a lectin that exhibited hemagglutinating activity . AlthougG2 cells . MeanwhiG2 cells , and theactivity .This study aims to investigate the basic properties  and biological activities  of the green speckled lentil seed lectin (GSLL).g and 4 °C for 25 min in an Avanti J-E High Speed centrifuge . The 800-mL supernatant obtained constituted a crude extract of the seeds. This extract was loaded onto an SP-Sepharose column  (5 cm × 15 cm) previously pre-equilibrated with the same buffer. The unbound fraction containing GSLL was collected and loaded onto an Affi-gel blue gel column  (5 cm × 15 cm). The unbound materials were removed by washing with the buffer. The adsorbed materials were eluted with 1 M NaCl in 10 mM Tris–HCl buffer (pH 7.6) and collected as the bound fraction. The bound fraction was dialyzed against double-distilled water, and lyophilized into powder form.Green speckled lentil seeds were purchased from a local supermarket in Shatin, New Territories, Hong Kong. The seeds were authenticated using a DNA analysis method, which was carried out by Prof. Shiu-Ying Hu, Honorary Professor of Chinese Medicine, The Chinese University of Hong Kong. The seeds (80 g) were soaked in 10 mM Tris–HCl buffer (pH 7.6) overnight, homogenized in a blender, and centrifuged twice at 30,000×The powder was resuspended into a 15 mg/mL protein solution in 10 mM Tris–HCl buffer (pH 7.6), and injected into a Mono Q 5/50 GL column  for fast protein liquid chromatography (FPLC) with an AKTA purifier . An increasing NaCl concentration was utilized to elute the column. Fractions of the first adsorbed peak corresponding to the lectin were collected, dialyzed, and lyophilized.The powder was resuspended into a 5 mg/mL protein solution in 10 mM Tris–HCl buffer (pH 7.6), and loaded onto a Superdex 75 10/300 GL column  for size-exclusion FPLC with the AKTA purifier. The last peak eluted at the 37th minute contained purified GSLL .Loading buffer with (for reducing SDS-PAGE) or without (for non-reducing SDS-PAGE) β-mercaptoethanol was added to the fractions obtained at different steps during the purification of GSLL. The samples were loaded onto a polyacrylamide gel , and subjected to SDS-PAGE at a constant voltage of 120 V for 70 min. The gel was stained with Coomassie Brilliant Blue for 1 h, and destained with 10 % acetic acid overnight .® Costar® 96-Well U-plate , twofold serial dilutions of a 50-µL protein sample in phosphate-buffered saline (PBS) were prepared. The same volume of 2 % rabbit red blood cell suspension was added to the wells. After approximately 1 h, the red blood cells in the control (without protein sample) sank to the bottom of the well and appeared as a red spot. The presence of proteins with hemagglutinating activity would cause the cells to clump together and form a plaque [Using a Corninga plaque .N-acetylgalactosamine, α-methyl-d-glucoside, α-methyl-pyranoside, raffinose, mannitol, xylitol) were added. The same volume of 2 % rabbit red blood cell suspension was added to the wells. A reduction in hemagglutinating activity indicated that the carbohydrate was specific in its interaction with the lectin, resulting in competitive inhibition of lectin binding with the red blood cells [Twofold serial dilutions of a 50-µL protein sample in PBS were prepared in a 96-well U-plate, and different carbohydrate solutions  at a constant voltage of 120 V for 70 min. The gel was stained, and the area containing the protein band was cut into small pieces. The gel was destained, dehydrated in acetonitrile, and air-dried. A minimal amount of trypsin was added to the gel and incubated at 37 °C for 1 h to allow digestion. The solution containing the tryptic fragments was transferred to a tube, and sent to the laboratory in the Prince of Wales Hospital, Hong Kong, for mass spectrometry analysis according to a previously described procedure .® SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad) at a constant voltage of 15 V for 40 min. The membrane was sent to the central laboratory of Beijing Agricultural University, Beijing, for N-terminal amino acid sequencing using a previously described procedure [Purified lectin was subjected to SDS-PAGE in a polyacrylamide gel  at a constant voltage of 120 V for 70 min, and then transferred onto a polyvinylidene difluoride (PVDF) membrane using a Trans-Blothttp://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome) analysis was conducted using the results from the mass spectrometry and N-terminal amino acid sequencing to search for known proteins with similarity to GSLL. The proteins showing the highest scores (most closely related to GSLL) were marked, and the differences in their amino acid sequences were identified.An NCBI BLAST  overnight. Different concentrations of GSLL were added to the cells and incubated at 37 °C for 24, 48, or 72 h. The effects of addition of 62.5 mM glucose or mannose on the GSLL treatment of CNE1 cells were examined to investigate the inhibitory effects of the carbohydrates on the action of GSLL on CNE1 cells. Briefly, after the wells were washed with PBS, 25 µL of MTT (5 mg/mL) in PBS was added and incubated for 4 h. Next, 150 µL of DMSO was added to the wells, and the optical density at 580 nm was recorded with a SpectraMax i3 Multi-mode Plate Reader . The IC50 values for GSLL treatment of the cells were determined as the GSLL concentrations causing 50 % inhibition of the cells.Human nasopharyngeal carcinoma CNE1 and CNE2 cells were purchased from the Sun Yat-sen University of Medicinal Sciences (China). The cells were cultured in Dulbecco’s modified Eagle’s medium containing 10 % fetal bovine serum and 100 U/mL penicillin and streptomycin, and maintained at 37 °C in a humidified incubator under 5 % COCNE1 cells were seeded onto 6-well plates overnight. Different concentrations of GSLL were added to the cells and incubated for 48 h. The cells were then trypsinized and washed with PBS. For assessment of phosphatidylserine externalization, 250 µL of binding buffer containing 1.25 µL of Annexin V-FITC and 0.5 µL of propidium iodide (PI) was added to the cells, followed by incubation in the dark for 15 min. For assessment of mitochondrial depolarization, 250 µL of PBS containing 2.5 µg/mL JC-1 was added to the cells, and incubated in the dark for 15 min. For cell cycle analysis, the cells were fixed in 70 % ethanol at −20 °C for 2 h and washed with PBS, followed by addition of 250 µL PBS containing 5 µL of PI and incubation in the dark for 15 min. The cells were examined with a BD LSRFortessa™ Cell Analyzer . The data were analyzed using BD FACSDiva 7.0 software (BD Biosciences).g and 4 °C for 30 min, and the supernatants were collected as the protein fractions. SDS-PAGE was performed, followed by semi-dry transfer onto a PVDF membrane using a Trans-Blot® SD Semi-Dry Transfer Cell (Bio-Rad) at a constant voltage of 15 V for 40 min. The PVDF membrane was blocked with 5 % milk in Tris-buffered saline containing 0.1 % Tween-20 (TBST) for 1 h, incubated with a primary antibody (1:1000 in 5 % milk in TBST) at 4 °C overnight, washed with TBST, and incubated with the corresponding horseradish peroxidase (HRP)-associated secondary antibody (1:1000 in 5 % milk in TBST) at room temperature for 2 h. Primary antibodies used included: Rabbit anti-caspase 3 antibody ; Rabbit anti-cleaved caspase 8 antibody ; Rabbit anti-caspase 9 antibody . Secondary antibody used included HRP-linked anti-rabbit antibody . The presence of the target proteins was visualized with an electrochemiluminescence detection system using an Amersham ECL Western Blotting Detection Kit .CNE1 cells were seeded onto 90-mm petri dishes and treated with different concentrations of GSLL for 48 h. The cells were trypsinized, washed with PBS, and lysed with RIPA buffer on ice for 2 h. The cell lysates were centrifuged at 20,000×P-values for each data point were calculated by a two-tailed Student’s t test using SPSS 16.0 . Data points with P-values below 0.05 were determined as significant results.Cell culture experiments were performed three times. The percentage inhibition obtained in each trial, and the mean percentage inhibition and standard deviation of the three trials were obtained using Microsoft Office Excel 2003 . The current protocol used for purification of GSLL from green speckled lentil seeds comprised ion exchange chromatography , affinity chromatography (Affi-gel blue gel), and size exclusion chromatography (Superdex 75). After the first two chromatographic steps (non-adsorption on SP-Sepharose and adsorption on Affi-gel blue gel), GSLL was purified by about 5.6-fold Table . A majorBoth ion exchange chromatography steps were carried out in a pH 7.6 buffer. GSLL was adsorbed on Mono Q, but not on SP-Sepharose, indicating that GSLL was anionic at this pH, and had a pI value lower than pH 7.6. However, GSLL was eluted from Superdex 75 at about 18.5 mL. Based on the Superdex 75 calibration curve for size determination, GSLL should have a molecular size of approximately 2.6 kDa. The actual size of GSLL should be much larger (≥21 kDa) than the observed size, meaning that GSLL was not eluted from Superdex 75 in keeping with its molecular size.Mass spectrometry after trypsin digestion  of GSLL was performed, and the mass values obtained were searched using NCBI BLAST to find matching peptides. In the NCBI BLAST analysis, GSLL showed the highest similarity to LcL with 275 amino acids  , 0.125 mg/mL (P < 0.001), and 0.031 mg/mL (P = 0.004), respectively, and their respective IC50 values were 0.95, 0.28, and 0.06 mg/mL . Slight inhibition of 18 % was detected after treatment with 1 mg/mL GSLL for 48 h (P = 0.004). The activity was more obvious when the duration of GSLL treatment was lengthened to 72 h, and the inhibitory effect was significant starting from 0.063 mg/mL GSLL (P = 0.044), with an IC50 value of 0.75 mg/mL  and 74.52 % (P = 0.036), respectively. After treatment with 0.5 mg/mL GSLL, the inhibitory effects were reduced from 29.25 to 71.05 % (P < 0.001) and 69.11 % (P < 0.001), respectively, in the presence of glucose and mannose. The IC50 values after GSLL treatment were also elevated from 0.28 to 1.11 mg/mL and 1.79 mg/mL with the addition of glucose and mannose, respectively.In the MTT assay, GSLL inhibited cell proliferation. GSLL strongly inhibited nasopharyngeal carcinoma CNE1 cells, and slightly inhibited CNE2 cells. Treatment with 0–1 mg/mL GSLL resulted in antiproliferative effects on CNE1 cells. After treatment with GSLL for 24, 48, and 72 h, significant inhibition was observed for the GSLL concentrations of 0.125 mg/mL  was decreased, and the population located at the lower right quadrant  was increased. The cell population at the upper right quadrant (late apoptotic or necrotic cells) was also increased, indicating that GSLL was cytotoxic toward the cells  with N-terminal amino acid sequences similar to GSLL have not been reported to elicit apoptosis in tumor cell lines or exhibit antiproliferative activities. To our knowledge, GSLL is the first lectin from the Lens genus to exhibit proapoptotic activities on tumor cells.There are no previous reports on the induction of apoptosis in tumor cells by lectins from P. sativum lectin (pea lectin) triggered apoptosis in tumor cells [1 phase , and increased caspase 3, 8, and 9 activity in CNE1 cells.


Geese breeding in the Arctic have to do so in a short time-window while having sufficient body reserves. Hence, arrival time and body condition upon arrival largely influence breeding success. The green wave hypothesis posits that geese track a successively delayed spring flush of plant development on the way to their breeding sites. The green wave has been interpreted as representing either the onset of spring or the peak in nutrient biomass. However, geese tend to adopt a partial capital breeding strategy and might overtake the green wave to accomplish a timely arrival on the breeding site. To test the green wave hypothesis, we link the satellite-derived onset of spring and peak in nutrient biomass with the stopover schedule of individual Barnacle Geese. We find that geese track neither the onset of spring nor the peak in nutrient biomass. Rather, they arrive at the southernmost stopover site around the peak in nutrient biomass, and gradually overtake the green wave to match their arrival at the breeding site with the local onset of spring, thereby ensuring gosling benefit from the peak in nutrient biomass. Our approach for estimating plant development stages is critical in testing the migration strategies of migratory herbivores. Spring migration is one of the most essential parts of the annual life cycle of migratory birds. The decisions taken at stopover sites determine the refuelling potential and the arrival time at the breeding site, and, accordingly, greatly influence breeding success125678Arctic breeding geese have a narrow time window to breed. Arriving too late at the breeding site leads to a severe reduction in reproductive success or even complete breeding failure, due to the competition for high-quality territories and the time constraints to raise young to fledging3101112314The advantage of early breeding is substantial, but egg laying is mainly constrained by body stores121617181The green wave hypothesis posits that migratory geese track a successively delayed spring flush of plants on their way from the wintering grounds in the temperate zone to their Arctic breeding site62223242526Despite the pronounced effect of the onset of spring in timing the spring migration of geese suggested by previous studies23An accurate estimation of the plant green-up along the flyway is critical in testing the green wave hypothesis. Studies that interpreted the green wave as the onset of spring, often quantified the green wave using a proxy of plant phenology, namely “growing degree days” (GGD) or its derivatives calculated from the accumulated temperature above a pre-defined threshold2223272526Measuring the peak in nutrient biomass in the field is time-consuming3233343536Branta leucopsis at the stopover and the breeding sites during their spring migration on the way from the Wadden Sea in Western Europe to their breeding site in Arctic Russia. We firstly calculate the timing of the green wave (defined as either the onset of spring or the peak in nutrient biomass), based on the change rate of the herbaceous-plant phenology derived from satellite imagery. Next we extract the stopover schedules of individual Barnacle Geese from a published datasetIn this study we test the role of plant phenology in the timing of arrival of individual Barnacle Geese The phenology development of herbaceous plants in the wintering, stopover, and breeding sites of Barnacle Geese was quantified by a standardized two-band Enhanced Vegetation Index (SEVI2) . A succeThe timing of goose arrival along the flyway gradually shifted from close to the peak in nutrient biomass (RMSD = 6 days) to a good match with the onset of spring (RMSD = 4 days) at the breeding site  and 3. TAs migration progressed from the southern stopover site 1 to the breeding site 5, we found an increasing difference with the peak in nutrient biomass   and a deF = 467.187, df1 = 4, df2 = 83, P < 0.001). The plant development level at goose arrival time showed a significant lower level along the flyway from the southernmost stopover site 1 to the northernmost stopover site 4  (P = 0.827)  . Similar= 0.827) . Geese fThis study utilizes specific plant phenology development patterns derived from relatively high spatiotemporal resolution satellite imagery, combined with a reconstruction of migration timing deduced from Barnacle Geese tracking data, to test whether geese track or overtake a green wave during spring migration. We demonstrate that Barnacle Geese track neither the onset of spring nor the peak in nutrient biomass on their way to the breeding site. Rather, they gradually overtake the green wave by arriving at the southernmost stopover site at a high plant development level (close to the peak in nutrient biomass), following the successively lower level of plant development as migration progresses, and matching their arrival at the breeding site with the local onset of spring, which facilitates gosling rearing later in the season. Although geese do forage along the flyway, the development stage of plants is secondary to their aim of timely arrival at the breeding site.Anser brachyrhynchus during spring migration. However, the match between their observed and simulated departure dates based on the onset of spring rule show a poorer fit at the southern stopover site (Denmark RMSD = 14 days) than the northern ones (mid Norway RMSD = 8 days and North Norway RMSD = 6 days), which is consistent with our findings, and suggests that the onset of spring was not consistently tracked by these geese. Furthermore, the difference between the migration timing of Barnacle Geese and the onset of spring at the wintering site in the Wadden Sea has increased since the 1990s (geese delayed their departure from their wintering site) due to the competition for food and predation risk in the Baltic region27Barnacle Geese do not follow the onset of spring on the way to their Arctic breeding site. We find a decreasing difference between the goose arrival time and the onset of spring as geese move northwards, running from stopover sites with a poor match  to a reasonable match at the point that geese reached the breeding site (RMSD = 4 days). A previous studyOur estimation of the peak in nutrient biomass derived from satellite imagery (day 109 at the Wadden Sea and day 137 at Gotland) is consistent with the ground reference data measured during the same spring (day 112 at the Wadden Sea and day 145 at Gotland)In agreement with van der Graaf et al. 2006)1190611920: P < 0.001), but a similar level with the breeding site (P = 0.827). This finding is in agreement with Hubner (2006)29We found a relatively good match between goose arrival time and the onset of spring at the most northern stopover site Kanin Peninsula (RMSD = 6 days). The plant development level at goose arrival time also indicated a significant lower plant development level at the site Kanin Peninsula in comparison to the other stopover sites  and found that this year fell into the 25%–75% non-outlier range see  online, A detailed characterization of the green wave is critical in assessing whether geese surf the wave (regardless of how it is defined) or whether they overtake it; whether they are driven by forage quantity, quality or profitability; and how well they can predict spatiotemporally dynamic conditions. This study proposes an approach to quantify specified levels of plant development along the migration flyway during the course of the spring growing season which can be used to test the spring migration strategy for other herbivores.http://e4ftl01.cr.usgs.gov/MODIS_Composites/MOLT/. The 8-day composite map was produced by assembling the most cloud-free pixels (the best pixels) within this 8-day time period into a single image. The spatial range of the imagery covers the whole migratory flyway and the time period covers the same year that the Barnacle Geese were tracked. A two-band Enhanced Vegetation Index (EVI2)The Moderate Resolution Imaging Spectroradiometer (MODIS) Terra 8-day surface reflectance products MOD09Q1  and yearly global land cover products (MCD12Q1)  were downloaded from min and EVI2max are the minimum and the maximum value of each EVI2 time series.There is an inherent level of noise unrelated to vegetation development in the MODIS time-series4243n+1-SEVI2n)/8, where n ranges from 1 to 45 . A sudden increase in SEVI2 may signal the onset of substantial photosynthetic activity, which is defined as the beginning of plant growthFor each stopover site  and the breeding site at Kolokolkova Bay, a 50 km buffer zone was generated to represent the local plant development conditions. For each zone (site), the median of the SEVI2 values derived from herbaceous plant area was extracted from the 8-day interval maps, resulting in a SEVI2 time-series  to describe the phenology development of each site. The median values were selected to minimize the effect of extremely low or high values related to cloud contamination or other unknown reasons. The change rate was calculated as  loggers and visual observations at the breeding site, were derived from Eichhorn et al. (2006)The root mean square deviation (RMSD) methodThe RMSD was calculated according to Linear interpolation was used to convert the 8-day interval plant phenology data into daily plant development curves. We then extracted the specific plant development level at each site at goose arrival date. We used a generalized linear-mixed model (GLMM) with sequential Bonferroni tests to compare the level of plant development at goose arrival time across sites. Goose identity (ID 1-19) was treated as a random-effect factor to account for the repeated measures and the site  was modelled as a fixed effect.Y.S., W.F.D., P.G., R.C.Y. and H.H.T.P. designed the research. Y.S. and Q.X. analysed the data. Y.S. wrote the first draft, and the manuscript was reviewed by all the authors.Do Arctic breeding geese track or overtake a green wave during spring migration?


Staphylococcus aureus of 1 g versus 2 g of intravenous (IV) prophylactic cefazolin in total knee arthroplasty (TKA).The aim of this study was to compare the intraosseous concentrations and the inhibitory effects on the growth of S. aureus, using high-performance liquid chromatography (HPLC) and agar disc diffusion bioassays.Eighteen patients (21 knees) with primary knee osteoarthritis were divided into two groups receiving 1 g (12 patients: 14 knees) versus 2 g (six patients: seven knees) IV prophylactic cefazolin prior to the incision in TKA. Subchondral bone samples  were taken during the operation. These samples were analyzed for intraosseous concentration of cefazolin and their inhibitory effects on the growth of p = 0.007) and distal femur (p = 0.016). There were no significant differences between the two groups in terms of mean inhibitory effects in the proximal tibia or distal femur (p > 0.05). No significant correlations were found between the intraosseous concentrations and inhibitory effects in the proximal tibia  and distal femur .The mean intraosseous concentration in the 2 g dose group was significantly higher than in the 1 g dose group in the proximal tibia (IV cefazolin at a dose of 2 g produced greater intraosseous concentrations overall than a dose of 1 g. However, the higher intraosseous concentrations did not correlate with higher inhibitory effects.Level III. Staphylococcus aureus between an IV dose of 1 g and a dose of 2 g.Prophylactic antibiotics are known to reduce the risk of perioperative and/or postoperative infection –3. HowevS. aureus of IV prophylactic cefazolin at dosages of 1 versus 2 g in total knee arthroplasty. At this point, we hypothesize that the intraosseous concentrations and the inhibitory effects in the group given cefazolin at a dose of 2 g are possibly higher than those in the group given a dose of 1 g.This study aims to compare the intraosseous concentrations and the inhibitory effects on the growth of S. aureus, which were prospectively collected from further analyses. The group with the IV cefazolin dose of 1 g consisted of nine females and three males with a mean age of 70.1 ± 4.6 years (61–79 years of age). During the same period we retained a group with an IV cefazolin dose of 2 g. This group consisted of five females and one male with a mean age of 68.4 ± 3.0 years (64–73 years of age). There were no significant differences in the mean age and genders between the two groups . The mean weights of the patients were 61.2 ± 8.4 kg for the group with a 1 g dose and 62.0 ± 8.2 kg for the group with a dose of 2 g (p = 0.84).During the period between May, 2011 and February, 2013, patients with primary knee osteoarthritis were recruited to participate in this study. The inclusion criteria were patients with primary knee osteoarthritis while the exclusion criteria included patients with post-traumatic or post-infectious knee conditions, allergies to cephalosporin or penicillin, serum creatinine levels >1.5 mg%, creatinine clearance <55 ml/min, probenecid intake, post-steroid treatment, chemotherapy, those who were post-knee arthroplasty or high tibial osteotomy, immunocompromised hosts, and those with deleterious medical conditions. Eighteen patients were recruited into our study using the inclusion and exclusion criteria. The 18 patients (21 knees) were divided into two groups in accordance with their IV prophylactic cefazolin dosages  of 1 g  versus 2 g  administered before the incision was made in their total knee arthroplasty. All patients’ baseline data were prospectively collected, including the intraosseous concentrations of cefazolin and the inhibitory effects on the growth of All patients were prepared for the TKA operation with standard, sterile technique. They were administered IV cefazolin, at a dose of 1 or 2 g, prior to tourniquet inflation and before the incision had been made. After performing knee arthrotomy and bone cutting at the distal femur and at the proximal tibia, our experienced knee surgeons, B.P., P.P., and N.T. (with the same level of experience), provided bone from the segments that had been removed from each patient to a researcher (P.K.) who extracted only subchondral bone at a size of 2.5 × 2.5 mm for further analyses while under sterile technique. The period of time between IV cefazolin injection and sample collection was recorded.Each sample was processed by the antibiotic broth elution assay . The solS. aureus (ATCC 25923) [S.aureus-seeded agar  and incubated for 18 h at 37 °C. All samples were analyzed by our researcher (N.M.) who was blinded to the IV cefazolin dose in each sample. The standard MIC of serum cefazolin would inhibit the growth of S. aureus at least 18 mm from the center of the sample. All samples were analyzed by a staff person in the laboratory who was also blinded to the IV cefazolin dose used in each sample.The bioactivity of each sample was determined using an agar disc diffusion bioassay with C 25923) . The antS. aureus growth on the agar disc (mm) between the two different groups of IV cefazolin doses (1 versus 2 g). The correlations between the intraosseous concentrations of cefazolin (µg/g) and the inhibitory effects of S. aureus growth on agar disc (mm) were analyzed and interpreted via Pearson’s correlation coefficient (r). Categorical variables were analyzed using the Chi-squared test. The level of significance was set at p < 0.05.Statistical analysis was performed using the SPSS software version 13.0 . ANOVA was used to analyze the statistical significance of the differences in the values of the intraosseous concentration of cefazolin (µg/g) and the inhibitory effect on p = 0.32; tibia, p = 0.11).The mean times between cefazolin injection and sample collection were 50.1 ± 6.5 min for femur and 53.1 ± 6.5 min for tibia in the group with IV cefazolin 1 g, compared with 58.9 ± 23.1 min for femur and 67.9 ± 23.1 min for tibia in the group with IV cefazolin 2 g. There were no significant differences in these mean times for the same corresponding areas of sample collection between the two groups  and distal femur (p = 0.016)  and distal femur .From Pearson’s correlation analyses, there were no significant correlations between the levels of intraosseous concentration and inhibitory effects seen in the proximal tibia .The total mean inhibitory effects of cefazolin were 10.0 ± 5.4 mm for the proximal tibia and 11.6 ± 5.4 mm for the distal femur. The mean inhibitory effects in the two groups  were less than 34.6 ± 0.5 mm, which was shown to be the mean inhibitory effect of the standard MIC of serum cefazolin (30 µg/ml) in this present study (Table p = 0.007) and distal femur (p = 0.016). However, the mean intraosseous concentrations of the 1 g group (22.6 µg/g) and the 2 g group (44.1 µg/g) at the distal femurs were higher than the concentrations seen in a previous study (9.2 µg/g) [Although current literature recommends administration of a dosage of intravenous (IV) cefazolin of 1–2 g [p = 0.33; distal femur, p = 0.63). Although a previous report had shown that high-dose cefazolin could be used for prophylaxis in an animal study [r = 0.18, p = 0.52) or distal femur . Higher cefazolin dosages would not provide higher inhibitory effects. At this point, we hypothesize that the standard MIC of intraosseous cefazolin may not be directly related to the inhibitory effects, which are different from the relationship between the standard MIC of serum cefazolin and its inhibitory effects via the agar disc diffusion bioassay. In addition, the intraosseous area may contain a threshold inhibitory effect of cefazolin that gives no greater inhibitory effect from a higher dose of cefazolin. Further studies are needed to test the inhibitory effects of intraosseous cefazolin directly with the standard MIC of intraosseous cefazolin rather than relative tests with the standard MIC of serum cefazolin.On the other hand, there were no significant differences between the two groups in terms of the mean inhibitory effects at either the proximal tibia or distal femur via agar disc diffusion bioassay  of cefazolin may be more beneficial than that of the lower dose (1 g) due to the longer persistence of the inhibitory effect, particularly in the final period of the operation. A previous study found that biofilm formation could develop for up to 1–2 days ; therefoIV cefazolin at a dose of 2 g provides greater intraosseous concentrations in both the proximal tibia and distal femur than does a dose of 1 g. However, its higher intraosseous concentration does not correlate with higher inhibitory effects. Surgeons have to weigh the advantages and disadvantages when considering IV cefazolin at a dose of 2 g for preoperative prophylaxis in total knee arthroplasty.


Arabidopsis, trichome formation is regulated by the interplay of R3 MYBs and several others transcription factors including the WD40-repeat protein TRANSPARENT TESTA GLABRA1 (TTG1), the R2R3 MYB transcription factor GLABRA1 (GL1), the bHLH transcription factor GLABRA3 (GL3) or ENHANCER OF GLABRA3 (EGL3), and the homeodomain protein GLABRA2 (GL2). R3 MYBs including TRICHOMELESS1 (TCL1), TCL2, TRYPTICHON (TRY), CAPRICE (CPC), ENHANCER OF TRY AND CPC1 (ETC1), ETC2 and ETC3 negatively regulate trichome formation by competing with GL1 for binding GL3 or EGL3, thus blocking the formation of TTG1–GL3/EGL3–GL1, an activator complex required for the activation of the trichome positive regulator gene GL2. However, it is largely unknown if R3 MYBs in other plant species especially woody plants have similar functions. By BLASTing the Populus trichocarpa protein database using the entire amino acid sequence of TCL1, an Arabidopsis R3 MYB transcription factor, we identified a total of eight R3 MYB transcription factor genes in poplar, namely P. trichocarpa TRICHOMELESS1 through 8 (PtrTCL1–PtrTCL8). The amino acid signature required for interacting with bHLH transcription factors and the amino acids required for cell-to-cell movement of R3 MYBs are not fully conserved in all PtrTCLs. When tested in Arabidopsis protoplasts, however, all PtrTCLs interacted with GL3. Expressing each of the eight PtrTCL genes in Arabidopsis resulted in either glabrous phenotypes or plants with reduced trichome numbers, and expression levels of GL2 in all transgenic plants tested were greatly reduced. Expression of PtrTCL1 under the control of TCL1 native promoter almost completely complemented the mutant phenotype of tcl. In contrast, expression of PtrTCL1 under the control of TRY native promoter in the try mutant, or under the control of CPC native promoter in the cpc mutant resulted in glabrous phenotypes, suggesting that PtrTCL1 functions similarly to TCL1, but not TRY and CPC.In  Arabidopsis, there are a total of seven genes encoding R3 MYBs, including TRYPTICHON  are small proteins that typically contain ~100 amino acids, largely consist of a single MYB DNA-binding repeat, and are best characterized for their regulatory roles in trichome and root hair development . R3 MYBsHON TRY; , CAPRICEICE CPC; , TRICHOMS1 TCL1; , TCL2/CAB4 CPL4; , ENHANCEC1 ETC1; , ETC2 -targeted SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) 9 has been shown to activate TCL1, TCL2, and TRY . In the study described here, we examined if R3 MYBs from poplar can functionally substitute for Arabidopsis R3 MYBs to regulate trichome formation in Arabidopsis plants.equenced , and it bidopsis . By usinArabidopsis R3 MYB transcription factors, the entire amino acid sequence of Arabidopsis R3 MYB transcription factor TCL1 was used in BLAST searches of the P. trichocarpa proteome (www.phytozome.net). The entire amino acid sequences of identified poplar R3 MYB transcription factors were then used in BLAST searches until no more poplar R3 MYBs were identifiable. Full-length amino acid sequences of Arabidopsis and poplar R3 MYBs were subjected to phylogenetic analysis using “One Click” mode of Phylogeny (www.phylogeny.fr) with default settings.To identify poplar homologs of P. trichocarpa was collected as described previously  background  basal medium with vitamins (Plantmedia) and 1% (w/v) sucrose. Seedlings used for phenotypic analysis were obtained either by growing seeds on 1/2 MS medium or by directly sowing seeds into soil. All plants were grown in growth rooms at 22°C with 14/10 h photoperiod, and light density of approximately 120 μmol m-2 s-1.Unless specified otherwise, Arabidopsis seedlings was isolated using EasyPureTM Plant RNA Kit (Transgene) according to the manufacturer’s instructions. All RNA samples were treated with RNase-Free DNase set (Qiagen) to eliminate possible DNA contamination.Total RNA from poplar samples was isolated using PureLink Plant RNA Reagent (Invitrogen), and cleaned with RNeasy Plant Mini Kit (Qiagen) as described previously . Total RTable 1.cDNA was synthesized using 2 μg total RNA by Oligo(dT)-primed reverse transcription using Omniscript RT Kit (Qiagen). Some of the primers used for cloning or examining the expression of corresponding genes have been described previously , and poppUC19 vector under the control of the double 35S enhancer promoter of CaMV (To generate HA (human influenza hemagglutinin)- or GD -tagged constructs for poplar R3 MYB genes, the full-length, open-reading frames (ORF) of corresponding poplar R3 MYB genes were amplified by RT-PCR using RNA isolated from poplar xylem samples, and the PCR products were then cloned in-frame with an N-terminal HA or GD tag into the  of CaMV .35S:PtrTCL1-GFP construct was cloned by fusing PtrTCL1 in frame with GFP (Green fluorescent protein) and then cloned into the pUC19 vector under the control of the 35S promoter. The TCL1p:HA-PtrTCL1, TRYp:HA-PtrTCL1, and CPCp:HA-PtrTCL1 constructs were cloned by replacing the 35S promoter in 35S:HA-PtrTCL1 with TCL1, TRY, and CPC promoters, respectively .All reporter and effector plasmids were prepared using the GoldHi EndoFree Plasmid Maxi Kit (Kangwei) according to the manufacturer’s instructions. The procedures for protoplast isolation, transfection, and GUS activity assay have been described previously . BrieflyPtrTCL1–GFP under the control of the 35S promoter was examined under an Olympus FV1000 confocal microscope. Protoplast cells isolated from the PtrTCL1–GFP transgenic plants were stained with DAPI and then examined under an Olympus FV1000 microscope.Trichomes were analyzed and photographed using a Motic K microscope equipped with a Canon digital camera. Localization of PtrTCL1-GFP proteins in transgenic plants expressing PtrTCL1, Potri.002G168900; PtrTCL2, Potri.014G096300; PtrTCL3, Potri.015G022000; PtrTCL4, Potri.007G122800; PtrTCL5, Potri.017G03700; PtrTCL6, Potri.011G026300; PtrTCL7, Potri.004G021300; and PtrTCL8, Potri.004G015100.A total of eight poplar R3 MYB transcription factors were identified, and collectively named as PtrTCL1 to PtrTCL8. Corresponding gene names for the PtrTCLs identified are as follows: Arabidopsis, nearly the entire protein of poplar R3 MYBs is made up of the single MYB domain . The amino acid signature [D/E]L×2[R/K]×3L×6L×3R, that is required for the interaction with R/B-like bHLH transcription factors . In the other four poplar R3 MYBs, D/E, the first amino acid in the amino acid signature is replaced by T/N . Similarly, the amino acid motif W×M, that has been shown to be required for the cell-to-cell movement of CPC . In the other four poplar R3 MYBs, the M in the motif is replaced by S/T .Similar to their homologs in  factors , is fullbidopsis , but is t of CPC , is cons R3 MYBs , but is Figure 1B). Together, PtrTCL1–PtrTCL3 and seven Arabidopsis R3 MYBs formed one subgroup, and PtrTCL4–PtrTCL8 formed another subgroup.Phylogenetic analysis using full-length protein sequences of poplar R3 MYBs and TCL1 showed that the clade of PtrTCL1–PtrTCL3 is most closely related to the clade of TCL1, TCL2, ETC2 and TRY , were co-transfected into Arabidopsis protoplasts. GD and GD–TCL1 were used as negative and positive controls, respectively. As expected, neither GD nor GD–TCL1 activated the reporter gene in the absence of GL3. In the presence of GL3, GD–TCL1 but not GD activated the reporter gene. Similarly, none of the eight poplar R3 MYBs activated the reporter gene in the absence of GL3; however, all of them activated the reporter gene in the presence of GL3 .Arabidopsis plants expressing HA-tagged PtrTCLs under the control of 35S promoter (35S:HA-PtrTCLs). As shown in Figure 3A, expression of PtrTCL1, PtrTCL2, PtrTCL3, PtrTCL5, PtrTCL7 and PtrTCL8 in Arabidopsis resulted in glabrous phenotypes, similar to the phenotype observed in plants over-expressing TCL1 . RT-PCR analysis showed that poplar R3 MYB genes were highly expressed in their corresponding transgenic lines , indicating that the phenotypes observed in PtrTCL4 and PtrTCL6 transgenic plants were not due to relative lower expression levels of the corresponding genes.Having shown that all the eight poplar R3 MYBs interact with GL3 in plant cells, we further analyzed if PtrTCLs regulate trichome formation by generating transgenic ing TCL1 . In contGL2 is one of the target genes of the activator complex TTG1–GL3/EGL3–GL1, and it positively regulates trichome formation in Arabidopsis. Because binding of PtrTCLs to GL3 indicated that expression of PtrTCLs in Arabidopsis resulted in the inhibition of the formation of TTG1–GL3/EGL3–GL1 activator complex, we examined the expression of GL2 in Arabidopsis transgenic plants expressing poplar R3 MYB genes. As shown in Figure 4, expression of GL2 was dramatically reduced in transgenic plants expressing any of the poplar R3 MYB genes.Figure 1). We wanted to further explore the functions of poplar R3 MYB transcription factors in the regulation of trichome formation in Arabidopsis by taking PtrTCL1 as an example. We first examined the subcellular localization of the PtrTCL1 protein by generating transgenic plants expressing PtrTCL1–GFP under the control of the 35S promoter. As shown in Figure 5A, expression of PtrTCL1–GFP in Arabidopsis resulted in a glabrous phenotype, a phenotype similar to transgenic plants expressing PtrTCL1 . This indicated that the PtrTCL1–GFP fusion protein is likely functional. By examining the transgenic plants obtained, we found that GFP florescence was mainly observed in the nucleus of epidermal cells ; examination of DAPI stained protoplast cells isolated from the PtrTCL1–GFP transgenic plants confirmed that the GFP was mainly observed in the nucleus , indicating that PtrTCL1 is a nuclear localized protein.Among the eight poplar R3 MYB transcription factors, PtrTCL1 has highest amino acid similarity with TCL1, and phylogenetic analysis also showed that the clade of PtrTCL1–PtrTCL3 is most closely related to TCL1 , resulted in a glabrous phenotype when over-expressed in Arabidopsis . Therefore, we wanted to further examine if PtrTCL1 is the functional equivalent of TCL1 by testing if PtrTCL1 could rescue the tcl1 mutant phenotype when expressed under the control of TCL1 native promoter.Previously, we identified henotype . Here, wtcl1 background to express PtrTCL1 under the control of the TCL1 native promoter (TCL1p:HA-PtrTCL1/tcl1). Previously, we showed that expression of TCL1–GFP under the control of the TCL1 promoter fully rescued the tcl1 trichome phenotype  in the try background, and under the control of CPC native promoter (CPCp:HA-PtrTCL1/cpc) in the cpc background. As shown in Figure 7, expression of PtrTCL1 under the control of TRY native promoter in a try mutant background, or under the control of CPC native promoter in a cpc mutant background resulted in glabrous phenotypes.In addition to n leaves , so we aArabidopsis R3 MYB transcription factors, and analyzed their function in trichome formation in Arabidopsis. We showed that expression of any of the eight poplar R3 MYB genes under the control of the 35S promoter in Arabidopsis resulted in either a glabrous phenotype or a great reduction in trichome numbers . These results suggest that poplar R3 MYBs act as negative regulators for trichome formation and may have overlapping functions, similar to their Arabidopsis R3 MYB homologs.In this study we identified poplar homologs of Figure 1B), is localized in the nucleus , and expression of PtrTCL1 under the control of the TCL1 promoter in the tcl1 mutant background (TCL1p:HA-PtrTCL1/tcl1) almost fully restored the trichome phenotype of the tcl1 mutant . Expression of PtrTCL1 under the control of the TRY native promoter in the try background (TRYp:HA-PtrTCL1/try), or under the control of the CPC native promoter in the cpc background (CPCp:HA-PtrTCL1/cpc) resulted in glabrous phenotypes , suggesting that PtrTCL1 may be the functional equivalent of TCL1, rather than TRY and CPC. It should be noted that ETC2 and TCL2 are also in the clade that is sister to the clade of PtrTCL1–PtrTCL3; however, because no mutant available for TCL2 and etc2 mutant does not have any trichome phenotype, we could not test whether PtrTCL1 might be functionally equivalent to TCL2 or ETC2.Among the eight poplar R3 MYB transcription factors, PtrTCL1 is in the most closely related clade to TCL1 . When tested in protoplasts, however, all the eight poplar R3 MYBs interacted with GL3 , and the interaction with GL3 may be stronger for those R3 MYBs without the fully conserved amino acid signature as judged by the GUS activities . Our results indicate that either the conserved amino acid signature is not required for the interaction of poplar R3 MYBs with GL3, or a single-amino-acid substitution (D/E > T/N) in poplar R3 MYBs does not affect their interaction with GL3. It is also possible that previous designations of conserved residues may be biased due to a smaller set of proteins analyzed. Furthermore, interaction of poplar R3 MYBs with GL3 in plant cells suggests that poplar R3 MYBs can block the formation of the activator complex required for trichome formation. In accordance with this, RT-PCR results showed that expression of GL2 is dramatically reduced in the transgenic plants expressing poplar R3 MYB genes . Since our previous results showed that R3 MYBs in Arabidopsis may regulate trichome formation in a GL2 independent manner (In  complex . In acco factors , and pro factors . Sequenct manner , we cannThe 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.


To better understand how cells sense and respond to their environment, it is important to understand the organization and regulation of the phosphorylation networks that underlie most cellular signal transduction pathways. These networks, which are composed of protein kinases, protein phosphatases and their respective cellular targets, are highly dynamic. Importantly, to achieve signaling specificity, phosphorylation networks must be regulated at several levels, including at the level of protein expression, substrate recognition, and spatiotemporal modulation of enzymatic activity. Here, we briefly summarize some of the traditional methods used to study the phosphorylation status of cellular proteins before focusing our attention on several recent technological advances, such as protein microarrays, quantitative mass spectrometry, and genetically-targetable fluorescent biosensors, that are offering new insights into the organization and regulation of cellular phosphorylation networks. Together, these approaches promise to lead to a systems-level view of dynamic phosphorylation networks. Protein phosphorylation, mediated by protein kinases and opposed by protein phosphatases, is one of the most widespread regulatory mechanisms in eukaryotes. Inside the cell, protein kinases, phosphatases, and their respective substrates are organized into complex phosphorylation networks that govern nearly all cellular processes. In order to achieve signaling specificity in response to a given environmental stimulus, these networks must be precisely coordinated in cellular space and time. This involves regulation at several different levels, including at the levels of protein expression , substrate selection, and spatiotemporal regulation of enzymatic activity. Not surprisingly, disruption of these regulatory mechanisms has been implicated in many pervasive diseases, including cancer  which cellular proteins are phosphorylated by a given kinase (or dephosphorylated by a given phosphatase) under physiological conditions and (2) how are specific kinases and phosphatases—and ultimately the signaling networks of which they are a part—regulated within the native cellular environment? We will begin by first discussing the properties of protein phosphorylation that make this post-translational modification an attractive chemical signal for cellular information transfer. We will then highlight some of the traditional methods that researchers have used to study the phosphorylation status of cellular proteins before turning our attention to several emerging technologies that are beginning to provide a dynamic and systems-level view of phosphorylation networks. Together, these approaches promise to shed new light on the organization and regulation of cellular phosphorylation networks.12 years in aqueous solution  may account for up to 10% of protein phosphorylation in some eukaryotes  or cAMP-dependent protein kinase (PKA) induces conformational changes that alter their open probability at the sarcoplasmic reticulum, influencing both the intensity and the frequency of Ca2+ spikes in cardiomyocytes and striated muscle cells .In addition to modulating its activity, the phosphorylation status of a protein may also impact other cellular parameters important to its function, such as its subcellular location or its stability. For instance, hyper-phosphorylation of the regulatory domain of the transcription factor, nuclear factor of activated T cells 1 (NFAT1), promotes electrostatic interactions between negatively-charged phosphate groups and positively-charged Lys/Arg residues found within a conserved nuclear localization sequence (NLS) , plays a major role in regulating cellular physiology. Therefore, it is important to understand what factors influence protein phosphorylation inside cells.Inside the cell, the phosphorylation status of a particular phosphorylation site (phosphosite) is determined by the equilibrium between kinase-mediated phosphorylation, in the one direction, and phosphatase-mediated dephosphorylation in the other directionIn addition to being co-expressed, a protein kinase and its substrate must physically interact with one another in order for phosphorylation to occur. Therefore, information about protein localization and protein–protein interactions is critical in determining whether a given kinase-substrate relationship (KSR) is likely to occur under physiological conditions. Traditionally, researchers have employed biochemical approaches to obtain this information. For instance, subcellular fractionation, generally achieved via differential centrifugation through a density gradient, has long been used to separate cellular organelles. When combined with western blot analysis, cellular fractionation is an effective means of determining the subcellular localization of a particular protein species. More recently, fluorescence imaging techniques, such as immunofluorescence and live cell imaging, have offered insights into the subcellular localization of proteins without disrupting the cellular architecture. Importantly, in the case of live cell imaging, dynamic changes in the subcellular distribution of a fluorescently tagged protein can be visualized in real-time within single cells. The advent of real-time super-resolution microscopy techniques based on photoswitchable fluorescent proteins (FPs) has dramatically increased the resolution afforded by live cell fluorescence imaging experiments (reaching ~20 nm resolution in living cells compared to >200 nm using standard fluorescence imaging) , co-IP assays can yield a wealth of information about protein–protein interactions that occur under various cellular conditions. However, because many protein interactions occur in the context of multimeric protein complexes, in the absence of cross-linking agents, co-IPs do not provide definitive information about direct protein–protein interactions. Moreover, because the cells must be lysed prior to immunoprecipitation, weak and transient interactions are often missed using this approach.Perhaps the most popular method for assaying protein–protein interactions is co-immunoprecipitation (co-IP). The immunoprecipitation step can be achieved using antibodies raised against either a specific protein-of-interest or an epitope tag, such as the hemagglutinin (HA)- or tandem affinity purification (TAP)-tags  is present in yeast. Indeed, we and others have recently reported that many KSRs are not conserved between yeast and humans, despite the presence of orthologous kinase-substrate pairs . In order to identify the site(s) of phosphorylation on the substrate, the initial screen is often followed by mutational analysis of putative phosphorylation sites. Due to safety concerns associated with radioactive assays and the high cost of radioisotope disposal, several non-radioactive assays have been developed based on alternative detection methods , allows direct visualization of phosphorylation on protein substrates using fluorescent or chemiluminescent detection methods  of phosphorylation and, in some cases, the functional consequences of phosphate addition. Each of these parameters is important for understanding how phosphorylation-dependent signaling is achieved inside cells. However, due to the time that it takes to fully characterize a single KSR, traditional biochemical approaches are not well suited to high-throughput analysis. Therefore, to increase the rate of KSR discovery, researchers have developed phosphorylation assays based on functional protein microarrays  family members  data, such as that obtained using the approaches outlined in Section Factors Influencing Protein Phosphorylation Inside Cells, is a valuable resource for determining whether a given cellular protein is likely to be phosphorylated by its cognate kinase in vivo.Due to the large number of phosphorylated species generated during a typical microarray phosphorylation assay, sophisticated statistical analyses are often required to identify those substrates that constitute positive “hits.” First, the signal intensity of each protein spot is measured and subjected to some form of background correction, be it background subtraction or ratiometric analysis  . Indeed, we have observed that known KSRs are sometimes absent from the final hit list. Visual inspection often reveals that this is not because the substrate is not actually phosphorylated on the microarray; rather it is because its normalized signal intensity does not exceed the stringent cut-off required to be considered a positive hit. Likewise, variations in protein abundance on the microarray or impairment of kinase-substrate interactions due to misfolding or truncation of the substrates during purification can bias KSR identification. Importantly, KSRs that are dependent on auxiliary factors, such as scaffolding proteins, co-factors, or post-translational modifications, may be missed all together using functional protein microarrays and other “bottom-up” approaches.Though necessary to eliminate false positives, the use of a stringent cutoff during KSR identification likely results in a relatively high false negative rate, as well   represent powerful tools for tracking changes in phosphorylation profiles across samples. Unlike the functional protein microarrays described above, RPPA fabrication entails the immobilization of cell lysates or tissue extracts on the functionalized glass surface. The arrays are then probed with an antibody specific for the epitope-of-interest  to assess changes in its expression levels or phosphorylation status under different conditions. Because each spot contains only a few nanoliters of lysate, many arrays (each containing lysates derived from tens to thousands of unique samples) can be prepared using only a small amount of starting material .“Top-down” approaches to substrate identification are designed to measure changes in the phosphorylation status of cellular proteins upon activation/inhibition of endogenous signaling pathways. Traditionally, this has been achieved using polyacrylamide gel electrophoresis (PAGE)-based methods Figures . FollowiS. pombe  in the first dimension followed by SDS-PAGE in the second dimension   using trivalent metal ions such as FeTo gain insights into differential phosphophorylation of cellular proteins using 2D-PAGE, two sets of lysates must be compared . However, due to inherent technical obstacles stemming from gel-to-gel variability and differential staining of proteins, cross-gel comparisons are often difficult using this approach. To address these limitations, researchers have developed 2D-differential gel electrophoresis (2D-DIGE) methodologies Minden,  Figure . In a 2DDespite the improved sensitivity and analytical power afforded by 2D-DIGE, methods based on 2D-PAGE are still only able to identify changes in a few hundred proteins at a time  and digested using an Arg/Lys-directed protease, such as trypsin, chymotrypsin or lysyl endopeptidase Figure . PhosphoThough MS analysis has been used for decades to analyze relatively simple mixtures consisting of a few hundred proteins, recent advances have now made possible the identification of thousands of phosphopeptides in a single study. As a consequence, MS-based studies have contributed to an unprecedented increase in the number of known phosphoproteins and phosphosites, which currently stands at just under 118,500 unique phosphosites in ~16,400 non-redundant proteins across human, mouse, and several other species [PhosphositePlus database (as of 4/13/2014)]. As we discuss below, this veritable explosion in phosphosite data has been made possible by several key technological advances in MS-based analysis of the phosphoproteome, including (1) the development of phosphopeptide enrichment methods, (2) improvements in the resolution and accuracy of mass analyzers, (3) the emergence of alternative fragmentation methods, and (4) the development of computational algorithms that allow high-confidence peptide identification and phosphosite localization. Moreover, the introduction of quantitation strategies based on isotopic labeling allows researchers to track dynamic changes in global phosphorylation profiles in response to a particular cellular stimulus. Due to the existence of a number of excellent reviews on these topics,  and ziroconium dioxide (ZrO2), have also been successfully combined with MS/MS for phosphosite identification  and orbitrap instruments exhibit a mass resolution of 20,000 and 100,000, respectively. Moreover, these instruments exhibit mass accuracies in the low parts-per-million, dramatically improving the coverage that can be achieved in a single experiment. Together, the increased resolution and mass accuracy afforded by modern mass spectrometers allows much more complex mixtures to be analyzed at once, markedly improving both the throughput of a given experiment and the confidence in the resulting peptide assignments.One of the primary challenges in phosphosite detection is preservation of phosphorylation during peptide fragmentation. This is because collision-induced dissociation (CID) methods, which accelerate the ionized peptides in a collision cell and then bombard them with inert gases such as helium, nitrogen, or argon, tend to cause the cleavage of the phosphate monoester linkage on hydroxyamino acid residues , . The use of sequence tags, which are less sensitive to sequencing errors than complete de novo sequencing, can dramatically reduce the database search time by reducing the search space to only a few candidate peptides that contain the sequence tag. By comparing the experimentally determined mass spectra with the theoretical spectra associated with the sequence tag, hybrid approaches have the potential to rapidly identify phosphorylation events on peptides (as well as many other modifications).In addition to database searches, However, as alluded to above, in many cases a phosphorylated peptide may contain multiple phosphorylatable residues. In these cases, the unambiguous assignment of phosphosites is a non-trivial task. Therefore, to facilitate phosphosite identification, programs such as MASCOT and MSQuant include algorithms that assign a confidence score to each Ser, Thr, and Tyr residue present in a peptide sequence. Similar algorithms, such as the Ascore algorithm, can also be applied to MS spectra to facilitate phosphosite identification  Ong,  and isob13C-Arg and/or 13C-Lys) or its normal, “light” counterpart   variants to conduct a systems-wide analysis of yeast phosphorylation networks  generally require information about consensus phosphorylation motifs Figure . Traditiin vitro using functional protein microarrays with information about phosphorylation sites identified in vivo by MS/MS analyses . When motifs generated by M3 were compared to those generated by positional scanning peptide arrays, a high degree of similarity was observed between them.One of the primary advantages of peptide arrays for consensus phosphorylation motif discovery is that the peptides in the matrix are relatively well defined . However, because the length of the derived motif is limited by the number of residues in the peptides and post-translational modifications other than pTyr or pThr are not present in the synthetic peptides, this approach may lack important information regarding the molecular determinants of kinase specificity. Moreover, since the peptides are not in the context of the full-length protein substrate, other factors involved in substrate recognition, such as docking sites or tertiary structure, are absent. Therefore, as a complementary approach, we developed an algorithm, termed Motif discovery based on protein Microarrays and MS/MS (M3), that combines data about KSRs determined disfavored at select positions. This information may be particularly useful in understanding the substrate selectivity of kinases that have very similar motifs . That being said, because it uses an iterative approach for motif discovery, M3 requires a relatively large number of substrates/phosphosites for accurate motif prediction. Likewise, the accuracy of M3 is limited by the number of known KSRs, on the one hand, and the number of annotated in vivo phosphosites, on the other. As these data sets continue to expand, it is likely that the accuracy of M3 with continue to improve, as well.One of the greatest strengths of M3 is its flexibility. For instance, though we chose to examine 15-mers in our study in order to facilitate comparison with motifs determined using scanning peptide arrays, in theory, motifs of various lengths can be generated in a relatively straightforward manner. Likewise, because M3 includes information about the entire amino acid sequence surrounding the putative phosphoacceptor site, aside from information about which residues are preferred at a particular position, it also provides insights into which residues are E. coli. Because E. coli express only two Ser/Thr kinases, ProPeL is expected to exhibit a very low background signal when examining eukaryotic Ser/Thr kinases. As a consequence, Ser/Thr phosphorylation events mediated by the kinase-of-interest should be more readily detectable. Using this approach, which uses the bacterial proteome as surrogate substrates for the kinase-of-interest, the authors accurately identified motifs for two Ser/Thr kinases, PKA and Casein Kinase II, suggesting that the use of bacterial proteins does not alter the substrate preferences of these kinases. Though it is well known that the activity of many eukaryotic kinases is poor in bacteria, the use of bacterial lysates with recombinant eukaryotic kinases could facilitate motif discovery using MS/MS-based approaches in the future.In addition to the microarray-based strategies described above, several groups have recently developed MS/MS-based approaches for motif discovery  with contextual information about kinase-substrate pairs in cells, is 0.76%    is likely to phosphorylate a given site can be phosphorylated by a given kinase (or dephosphorylated by given phosphatase), it is important to know when and where phosphorylation is likely to occur inside the cell. Such information can provide key insights into the physiological consequences of phosphorylation. Indeed, the tight spatial and temporal regulation of cellular phosphorylation networks is believed to underlie the specificity of cellular signal transduction. Therefore, in order to gain a more comprehensive understanding of dynamic phosphorylation networks, the static maps of phosphorylation networks described in the previous section will need to be supplemented with information about the spatiotemporal regulation of the protein kinases and phosphatases within these networks.In addition to information about the sites on a substrate that Traditionally, this has been achieved by examining changes in the phosphorylation state of representative cellular substrates before and after stimulation. Changes in the phosphorylation status of these proteins are typically determined using two complementary approaches. In the first approach, cellular proteins are isolated by either subcellular fractionation or immunoprecipitation so that their phosphorylation status can be probed using one of the phosphodetection methods discussed above . While careful experimental design and the use of general phosphatase and/or kinase inhibitors allows for decent temporal resolution using this approach, its spatial resolution is often poor because cellular architecture is disrupted during the lysis procedure. Moreover, since a large number of cells are required to obtain enough protein for analysis, this approach lacks single cell resolution, potentially masking subtle, yet important, differences between individual cells. The other approach, immunofluorescence using phosphospecific antibodies, offers excellent spatial resolution and can provide insights into cell-to-cell differences within a given population. However, because the cells must be fixed prior to analysis, this approach suffers from poor temporal resolution. Importantly, neither approach is able to examine changes in phosphorylation in living cells. Therefore, these methods offer only a “snapshot” of the dynamic changes in kinase and phosphatase activities that characterize most phosphorylation-dependent signal networks under physiological conditions.In order to study the activity profiles of cellular kinases and phosphatases in single, living cells with high spatiotemporal resolution, several groups have developed genetically-targetable FRET-based biosensors  a “sensor unit,” which undergoes a conformational change in response to a given cellular stimulus  and (2) a “reporter unit,” which converts the induced conformational change into a change in FRET  required for PDK1 activation. Because PDK1 is a master regulator of many AGC family members, including Akt, PKA, SGK and several PKC isoforms, these results have important implications for understanding the regulation of PDK1-dependent signaling networks  , ataxia telangiectasia mutated (ATM), Akt, Abl, Src, aurora kinase B, ERK, c-Jun N-terminal kinase (JNK), cyclin-dependent kinase 1 (CDK1), AMP-activated protein kinase (AMPK), and the epidermal growth factor receptor (EGFR)  which cellular proteins are phosphorylated in response to a given stimulus and/or in a particular cellular context, (2) where on the protein the phosphorylation event occurs, (3) which kinases and phosphatases are mediating phosphorylation/dephosphorylation of the phosphosite, and (4) how these modifying enzymes are regulated inside the cell. In the future, it will be important to expand this knowledge-base through (1) systems-wide profiling of phosphoproteomes under different cellular conditions and disease states using quantitative MS/MS and 2D-DIGE; (2) the identification of additional KSRs  using “whole-proteome” microarrays; (3) the development of novel fluorescent biosensors and orthogonal imaging modalities; and (4) the integration of these data into predictive models of phosphorylation networks. Though much work remains to be done, these goals appear to be within reach. The next challenge will be to gain a detailed understanding of the functional consequences of specific phosphorylation events and to integrate this information with other resources, such as protein–protein interaction data sets, global expression profiles, and metabolomics data (Derouiche et al., 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.


The mitogen-activated protein kinase kinase 3  is a member of the dual-specificity protein kinase group (MKK) that belongs to the mitogen-activated protein kinase (MAPK) signaling pathway. Following different forms of stressful stimuli and inflammatory cytokines, MKK3 is activated through phosphorylation on serine and threonine residues at sites Ser189 and Thr193 by MKKK proteins (MEKK 1-4) [We previously identified MKK3 in a gene expression profile array performed with p53-null H1299 cells ectopically expressing mutant p53-R175H gain-of-function (GOF), one of the most frequent alteration revealed in human cancer . Under min vitro and in vivo, and with doxorubicin and sorafenib in lung and liver cancer respectively [Our results are in agreement with other studies demonstrating that MAPK14/p38 MAPK is required for cell proliferation and survival and its inhibition leads to cell cycle arrest and autophagy-mediated cell death in colorectal tumors . Other wectively  However,ectively .In summary the results obtained by our group, according to other studies, are suggesting MKK3 as oncotarget. Indeed, its inhibition, suggestively deleterious for cancer but not normal primary cells, might constitute a novel potential and promising therapeutic strategy, that in alternative to the p38 MAPK inhibition, could potentiate the efficiency of chemotherapies in non-responder patients.


AbstractLimoniidae and Pediciidae are presented from Armenia. A total of 38 species of Limoniidae and four species of Pediciidae are listed. Of these, 27 species of Limoniidae and one species of Pediciidae represent the first records from Armenia. The first checklist of these families from Armenia is appended, containing 77 species of Limoniidae and six species of Pediciidae.Records of species of the families  Limoniidae and Pediciidae are concerned. The territory occupies the southern macro-slopes of the Great Caucasus and the mountains and plateaux as far south as the Turkish and Iranian borders. PageBreakSavchenko  is among less-investigated territories, as far as the families nko e.g.  dealing nko e.g. . A totalLimoniidae and four species of Pediciidae were identified from this material, of which 27 species of Limoniidae and one species of Pediciidae represent the first records from Armenia. Seven species of Limoniidae and one species of Pediciidae are new to the whole Transcaucasia. In addition, we append the first checklist of Limoniidae and Pediciidae from Armenia, containing 77 species of Limoniidae and six species of Pediciidae.The material we present in this paper was mostly collected in the north-western part of Armenia during a recent sampling campaign from August 26 to September 4, 2015. Thirty-eight species of Samples were collected by sweep-netting from vegetation along streams and lakes by the first, third, and fourth authors  and preserved in 75% ethanol. A list of 33 sampling sites, with coordinates and altitudes, is given in Table Taxon classificationAnimaliaDipteraLimoniidaeSavchenko, 1976Tavush: below Jukhtakvank Monastery, (site 26), 29.viii.2015, 1 ♂.So far only known from North Caucasus and Georgia. First record from Armenia.PageBreakPageBreakTaxon classificationAnimaliaDipteraLimoniidaeKotayk: between Marmarik and Aghavnadzor, tributary of Marmarik R. (site 4), 27.viii.2015, 1 ♂.Europe; Iran. First record from Armenia and Transcaucasia.Taxon classificationAnimaliaDipteraLimoniidaeLori: N of Dzoraget, tributary of Pambak R. (site 17), 1.ix.2015, 1 ♂.Europe; Azerbaijan, Turkey; Kirghizia. First record from Armenia.Taxon classificationAnimaliaDipteraLimoniidaeKotayk: Hrazdan Sewage Treatment Plant, Hrazdan R. (site 7), 27.viii.2015, 1 ♂ 3 ♀.Europe; Azerbaijan, Turkey, Israel; Central Asia, Mongolia; West Siberia. First record from Armenia.PageBreakPageBreakTaxon classificationAnimaliaDipteraLimoniidaeSavchenko, 1984Tavush: W of Dilijan, Bldan R. (site 28), 28.viii.2015, 6 ♂ 3 ♀.So far only known from Tajikistan. First record from Armenia and Transcaucasia; first record since original description.Pseudolimnophila (Pseudolimnophila) melanura and Pseudolimnophila (Pseudolimnophila) sepium the former cannot be considered a subspecies of the latter, as suggested by Due to syntopic occurrence in Armenia of Taxon classificationAnimaliaDipteraLimoniidaeTavush: W of Dilijan, Bldan R. (site 28), 28.viii.2015, 1 ♂.Europe; Morocco; Georgia, Azerbaijan, Turkey; Central Asia. First record from Armenia.Taxon classificationAnimaliaDipteraLimoniidaeKotayk: Hrazdan Sewage Treatment Plant, Hrazdan R. (site 7), 27.viii.2015, 1 ♂; Shirak: NE of Musayelyan, tributary of Akhurian R. (site 23), 2.ix.2015, 2 ♀; Shirak: Zuygaghbyur, meanders of tributary of Akhurien R. (site 25), 2.ix.2015, 3 ♂ 1 ♀; Shirak: NW of Amasia, tributary of Akhurian R. (site 24), 2.ix.2015, 5 ♂; Shirak: between Aghvoik and Ardenis, tributary of Akhurian R. (site 21), 3.ix.2015, 1 ♂ 1 ♀; Shirak: E of Torosgyugh, tributary of Akhurian R. (site 22), 3.ix.2015, 4 ♂ 1 ♀; Lori: NE of Geghasar, Pambak R. (site 19), 3.ix.2015, 2 ♂ 1 ♀.Europe, except for northern countries; Morocco; Georgia, Azerbaijan, Turkey. First records from Armenia.Taxon classificationAnimaliaDipteraLimoniidaeEdwards, 1938PageBreakShirak: NW of Amasia, tributary of Akhurian R. (site 24), 2.ix.2015, 8 ♂ 2 ♀; Shirak: between Aghvoik and Ardenis, tributary of Akhurian R. (site 21), 3.ix.2015, 1 ♀; Shirak: E of Torosgyugh, tributary of Akhurian R. (site 22), 3.ix.2015, 2 ♂.Kotayk: NW of Artavaz, Marmarik R. (site 6), 26.viii.2015, 3 ♂ 1 ♀; Kotayk: Hrazdan Sewage Treatment Plant, Hrazdan R. (site 7), 27.viii.2015, 5 ♂; Kotayk: SW of Hrazdan Reservoir, tributary of Hrazdan R. (site 12), 27.viii.2015, 1 ♂; Kotayk: near Artavaz, Marmarik R. (site 10), 27.viii.2015, 1 ♀; Kotayk: Meghradzor, behind railway, tributary of Marmarik R. (site 8), 27.viii.2015, 1 ♂; Tavush: W of Dilijan, Bldan R. (site 28), 28.viii.2015, 3 ♂; Ararat: Garni, below Garni Temple, Azat R. (site 1), 31.viii.2015, 5 ♂; Lori: Meghvahovit, road H31, small steppe brook (site 16), 2.ix.2015, 1 ♀; Lori: N of Gosh, Getik R. (site 31), 4.ix.2015, 6 ♂ 5 ♀; Shirak: NE of Musayelyan, tributary of Akhurian R. (site 23), 2.ix.2015, 5 ♂ 2 ♀; Europe; Georgia, Armenia, Azerbaijan, Turkey, Israel; Turkmenia.Taxon classificationAnimaliaDipteraLimoniidaeMeigen, 1804Kotayk: NW of Artavaz, Marmarik R. (site 6), 26.viii.2015, 1 ♂; Kotayk: between Marmarik and Aghavnadzor, Marmarik R. (site 13), 26.viii.2015, 1 ♀; Kotayk: Hrazdan Sewage Treatment Plant, Hrazdan R. (site 7), 27.viii.2015, 1 ♂; Kotayk: between Marmarik and Aghavnadzor, tributary of Marmarik R. (site 4), 27.viii.2015, 1 ♀; Tavush: W of Dilijan, Bldan R. (site 28), 28.viii.2015, 1 ♂; Tavush: below Jukhtakvank Monastery, (site 26), 29.viii.2015, 1 ♀; Gegharkunik: Tsovagyugh, nr. Sevan L. (site 3), 29.viii.2015, 1 ♀; Lori: N of Dzoraget, tributary of Pambak R. (site 17), 1.ix.2015, 1 ♂; Lori: Meghvahovit, road H31, small steppe brook (site 16), 2.ix.2015, 3 ♀; Shirak: NE of Musayelyan, tributary of Akhurian R. (site 23), 2.ix.2015, 1 ♂ 1 ♀; Shirak: NW of Amasia, tributary of Akhurian R. (site 24), 2.ix.2015, 1 ♂ 1 ♀.Europe; Georgia, Armenia, Azerbaijan, Turkey, Israel, ?Iran; Central Asia; West Siberia.Taxon classificationAnimaliaDipteraLimoniidaeLori: N of Pushkin, tributary of Dzoraget R. (site 18), 3.ix.2015, 1 ♂.Europe; Azerbaijan, Turkey, Israel, Iran; Central Asia, Mongolia; West Siberia, East Siberia. First record from Armenia.Taxon classificationAnimaliaDipteraLimoniidaeLori: Lermontov, tributary of Aghstev R. (site 15), 1.ix.2015, 4 ♂ 2 ♀.Russia (North Caucasus); Georgia, Azerbaijan, Iran. First record from Armenia.Taxon classificationAnimaliaDipteraLimoniidaeGegharkunik: Tsovagyugh, nr. Sevan L. (site 3), 29.viii.2015, 2 ♂; Lori: Lermontov, tributary of Aghstev R. (site 15), 1.ix.2015, 1 ♀; Shirak: E of Torosgyugh, tributary of Akhurian R. (site 22), 3.ix.2015, 3 ♂ 1 ♀.Europe; Georgia, Armenia, Azerbaijan, Turkey, Iran.PageBreakTaxon classificationAnimaliaDipteraLimoniidaeSavchenko, 1976Tavush: E of Haghartsin, tributary of Aghstev R. (site 29), 29.viii.2015, 1 ♂.Russia (North Caucasus); Georgia, Armenia.Possibly a valid species.Taxon classificationAnimaliaDipteraLimoniidaeShirak: NE of Musayelyan, tributary of Akhurian R. (site 23), 2.ix.2015, 2 ♂.Europe; Morocco; Georgia, Armenia, Turkey, Cyprus, Lebanon, Israel.Taxon classificationAnimaliaDipteraLimoniidaeLori: N of Dzoraget, tributary of Pambak R. (site 17), 1.ix.2015, 2 ♂.Europe; Georgia, Azerbaijan, Turkey. First record from Armenia.Taxon classificationAnimaliaDipteraLimoniidaeTavush: N of Gosh, Getik R. (site 31), 4.ix.2015, 10 ♂ 3 ♀.Europe; Morocco; Georgia, Turkey; West Siberia, East Siberia, Far East of Russia. First record from Armenia.Taxon classificationAnimaliaDipteraLimoniidaede Meijere, 1920Kotayk: NW of Artavaz, Marmarik R. (site 6), 26.viii.2015, 3 ♂; Shirak: NW of Amasia, tributary of Akhurian R. (site 24), 2.ix.2015, 2 ♂.Europe; Georgia, Azerbaijan, Turkey, Cyprus, Israel, Iran; Central Asia; as far east as Far East of Russia. First records from Armenia.Taxon classificationAnimaliaDipteraLimoniidaeSavchenko, 1978PageBreak28.viii.2015, 3 ♂ 1 ♀; Tavush: below Jukhtakvank Monastery, (site 26), 29.viii.2015, 1 ♀; Lori: Lermontov, tributary of Aghstev R. (site 15), 1.ix.2015, 4 ♂ 4 ♀.Kotayk: Meghradzor, below “Gold Mine”, tributary of Marmarik R. (site 9), 27.viii.2015, 2 ♂ 2 ♀; Tavush: W of Dilijan, Bldan R. (site 28), Ukraine, Russia (North Caucasus); Georgia, Armenia, Azerbaijan. First records since original description.Possibly a valid species.Taxon classificationAnimaliaDipteraLimoniidaeSavchenko, 1973Lori: N of Dzoraget, tributary of Pambak R. (site 17), 1.ix.2015, 1 ♂.?European Russia (southeast); Georgia. First record from Armenia.Taxon classificationAnimaliaDipteraLimoniidaeGegharkunik: Tsovagyugh, nr. Sevan L. (site 3), 29.viii.2015, 1 ♀; Lori: Lermontov, tributary of Aghstev R. (site 15), 1.ix.2015, 2 ♀; Shirak: NW of Amasia, tributary of Akhurian R. (site 24), 2.ix.2015, 1 ♂; Shirak: E of Torosgyugh, tributary of Akhurian R. (site 22), 3.ix.2015, 1 ♂.Europe; Georgia, Azerbaijan, Turkey; Tajikistan. First records from Armenia.Taxon classificationAnimaliaDipteraLimoniidaeStarý, 2004Lori: Lermontov, tributary of Aghstev R. (site 15), 1.ix.2015, 1 ♂ 1 ♀.Bulgaria, Greece, Russia (North Caucasus); Georgia, Armenia, Turkey, Lebanon. First record since original description.Taxon classificationAnimaliaDipteraLimoniidaeShirak: NW of Amasia, tributary of Akhurian R. (site 24), 2.ix.2015, 1 ♀.Nearctic ; widespread in Palaearctic, including Europe; North Africa; Georgia, Armenia, Azerbaijan, Turkey, Lebanon, Israel, Iran; Central Asia, Mongolia; as far east as North Korea, Japan and China; Oriental .PageBreakTaxon classificationAnimaliaDipteraLimoniidaeTavush: W of Dilijan, Bldan R. (site 28), 28.viii.2015, 1 ♀; Tavush: NW of Teghut, tributary of Aghstev R. (site 32), 29.viii.2015, 1 ♀.Europe; Georgia, Armenia, Azerbaijan, Iran.Taxon classificationAnimaliaDipteraLimoniidaeKotayk: between Marmarik and Aghavnadzor, tributary of Marmarik R. (site 4), 27.viii.2015, 1 ♂; Tavush: W of Dilijan, Bldan R. (site 28), 28.viii.2015, 1 ♀ 1 ♂.Europe; Turkey, Israel; Afghanistan. First records from Armenia and TranscaucasiaTaxon classificationAnimaliaDipteraLimoniidaeLackschewitz, 1941Tavush: car park on road M4, tributary of Aghstev R. (site 27), 4.ix.2015, 1 ♂.So far only known from North Caucasus and Georgia. First record from Armenia.Taxon classificationAnimaliaDipteraLimoniidaeKotayk: E of Hankavan, Marmarik R. (site 5), 26.viii.2015, 1 ♀; Kotayk: N of Solak, Hrazdan R. (site 11), 27.viii.2015, 1 ♂; Ararat: nr. Lanjazat, Azat R. (site 2), 31.viii.2015, 1 ♀; Shirak: NE of Musayelyan, tributary of Akhurian R. (site 23), 2.ix.2015, 1 ♀; Shirak: NW of Amasia, tributary of Akhurian R. (site 24), 2.ix.2015, 1 ♀.Europe; Morocco, Algeria; Georgia, Armenia, Azerbaijan, Turkey, Iran; Afghanistan, Mongolia, ?China.Taxon classificationAnimaliaDipteraLimoniidaeKotayk: Meghradzor, behind railway, tributary of Marmarik R. (site 8), 27.viii.2015, 4 ♂, 1 ♀.PageBreakNearctic ; widespread in Palaearctic, including Europe; Georgia, Azerbaijan, ?Syria, Iran; Central Asia, Mongolia; as far east as Far East of Russia and Japan; Oriental (India). First record from Armenia.Taxon classificationAnimaliaDipteraLimoniidaeSavchenko, 1984Kotayk: between Marmarik and Aghavnadzor, tributary of Marmarik R. (site 4), 27.viii.2015, 1 ♀; Lori: Lermontov, tributary of Aghstev R. (site 15), 1.ix.2015, 2 ♂ 1 ♀; Lori: Meghvahovit, road H31, small steppe brook (site 16), 2.ix.2015, 6 ♂ 1 ♀; Shirak: NE of Musayelyan, tributary of Akhurian R. (site 23), 2.ix.2015, 1 ♂; Tavush: car park on road M4, tributary of Aghstev R. (site 27), 4.ix.2015, 1 ♂.France (Corsica), Russia (North Caucasus); Georgia, Azerbaijan, ?Lebanon. First records from Armenia.Taxon classificationAnimaliaDipteraLimoniidaeGegharkunik: Tsovagyugh, nr. Sevan L. (site 3), 29.viii.2015, 1 ♂; Lori: NE of Geghasar, Pambak R. (site 19), 3.ix.2015, 1♀; Tavush: N of Gosh, Getik R. (site 31), 4.ix.2015, 1 ♂.Nearctic ; widespread in Palaearctic, including Europe; Georgia, Armenia, Azerbaijan, Turkey, Iran; Central Asia, Mongolia; as far east as Far East of Russia and Japan.Taxon classificationAnimaliaDipteraLimoniidaeStarý, 2009Gegharkunik: Tsovagyugh, nr. Sevan L. (site 3), 29.viii.2015, 1 ♂.Bulgaria, Greece, France (Corsica); Lebanon, Syria. First record from Armenia and Transcaucasia; first record since original description.Taxon classificationAnimaliaDipteraLimoniidaeLackschewitz, 1941Tavush: W of Dilijan, Bldan R. (site 28), 28.viii.2015, 1 ♂; Lori: W of Vahagnadzor, Zamanlu R. (site 14), 1.ix.2015, 2 ♂ 1 ♀.So far only known from North Caucasus and Georgia. First records from Armenia.PageBreakTaxon classificationAnimaliaDipteraLimoniidaeKotayk: near Artavaz, Marmarik R. (site 10), 27.viii.2015, 1 ♂; Kotayk: between Marmarik and Aghavnadzor, tributary of Marmarik R. (site 4), 27.viii.2015, 2 ♂; Lori: road H23 to Pushkin Pass, small brook (site 20), 3.ix.2015, 1 ♂.Nearctic ; Europe; Georgia, Azerbaijan, Turkey, Iran; Far East of Russia, Japan. First records from Armenia.Taxon classificationAnimaliaDipteraLimoniidaeLoew, 1871Tavush: E of Matosavank Monastery (site 30), 29.viii.2015, 1 ♀; Tavush: E of Haghartsin, tributary of Aghstev R. (site 29), 29.viii.2015, 1 ♂.Europe, except for northern countries. First records from Armenia and Transcaucasia.Taxon classificationAnimaliaDipteraLimoniidaeTavush: W of Dilijan, Bldan R. (site 28), 28.viii.2015, 1 ♂; Tavush: below Jukhtakvank Monastery, (site 26), 29.viii.2015, 1 ♂ 1 ♀; Tavush: NW of Teghut, tributary of Aghstev R. (site 32), 29.viii.2015, 1 ♂; Tavush: E of Haghartsin, tributary of Aghstev R. (site 29), 29.viii.2015, 1 ♂; Lori: W of Vahagnadzor, Zamanlu R. (site 14), 1.ix.2015, 2 ♂; Lori: road H23 to Pushkin Pass, small brook (site 20), 3.ix.2015, 1 ♀.Europe, except for northern countries; Morocco; Azerbaijan, Turkey, Iran. First records from Armenia.Taxon classificationAnimaliaDipteraLimoniidaeLori: Lermontov, tributary of Aghstev R. (site 15), 1.ix.2015, 1 ♂; Shirak: NW of Amasia, tributary of Akhurian R. (site 24), 2.ix.2015, 1 ♂.Europe; Morocco; Georgia, Azerbaijan, Turkey, Cyprus; Central Asia; as far east as Far East of Russia, and ?North Korea; Oriental (Pakistan). First records from Armenia.Taxon classificationAnimaliaDipteraLimoniidaeGegharkunik: Tsovagyugh, nr. Sevan L. (site 3), 29.viii.2015, 1 ♂; Lori: Lermontov, tributary of Aghstev R. (site 15), 1.ix.2015, 1 ♂.Europe. First records from Armenia and Transcaucasia.PageBreakTaxon classificationAnimaliaDipteraLimoniidaeKotayk: E of Hankavan, Marmarik R. (site 5), 26.viii.2015, 1 ♂.Europe; Kirghizia, Mongolia; West Siberia, East Siberia, Far East of Russia. First record from Armenia and Transcaucasia.Taxon classificationAnimaliaDipteraLimoniidaeMeigen, 1818Gegharkunik: Tsovagyugh, nr. Sevan L. (site 3), 29.viii.2015, 1 ♂.Nearctic ; widespread in Palaearctic, including Europe; Georgia; Mongolia; as far east as Far East of Russia, China, and Japan; Oriental (China). First record from Armenia.Taxon classificationAnimaliaDipteraPediciidaeTjeder, 1972Shirak: Zuygaghbyur, meanders of tributary of Akhurien R. (site 25), 2.ix.2015, 1 ♂; Shirak: E of Torosgyugh, tributary of Akhurian R. (site 22), 3.ix.2015, 1 ♂.Finland, Norway, Sweden; Kazakhstan, Tajikistan. First records from Armenia and Transcaucasia.Taxon classificationAnimaliaDipteraPediciidaeCzižek, 1931Shirak: NW of Amasia, tributary of Akhurian R. (site 24), 2.ix.2015, 1 ♂.Europe, except for northern countries; Morocco; Georgia, Armenia, Azerbaijan, Lebanon; Tajikistan.Taxon classificationAnimaliaDipteraPediciidaeLoew, 1871Tavush: vicinity of Parz L. (site 33), 28.viii.2015, 1 ♂; Tavush: below Jukhtakvank Monastery, (site 26), 29.viii.2015, 1 ♂; Lori: Meghvahovit, road H31, small steppe brook (site 16), 2.ix.2015, 1 ♂.Europe; Georgia, Armenia, Azerbaijan.PageBreakTaxon classificationAnimaliaDipteraPediciidaeLori: road H23 to Pushkin Pass, small brook (site 20), 3.ix.2015, 1 ♂.Europe; Georgia, Armenia, Azerbaijan, Turkey, Cyprus, Lebanon.Limoniidae and four species of Pediciidae are recorded from Armenia. Of these, 27 species of Limoniidae and one species of Pediciidae represent the first records for Armenia. These are the following: Paradelphomyia (Oxyrhiza) brevifurca, Paradelphomyia (Oxyrhiza) fuscula, Paradelphomyia (Oxyrhiza) senilis, Phylidorea (Phylidorea) ferruginea, Pseudolimnophila (Pseudolimnophila) melanura, Pseudolimnophila (Pseudolimnophila) sepium, Erioconopasymplectoides, Erioptera (Mesocyphona) bivittata, Hoplolabis (Parilisia) iranica, Molophilus (Molophilus) ochraceus, Molophilus (Molophilus) pleuralis, Molophilus (Molophilus) propinquus, Ormosia (Ormosia) cuspidata, Ormosia (Ormosia) hederae, Antocha (Antocha) vitripennis, Dicranomyia (Dicranomyia) circassica, Dicranomyia (Dicranomyia) longipennis, Dicranomyia (Dicranomyia) melanantha, Dicranomyia (Dicranomyia) pallidinota, Dicranomyia (Dicranomyia) pontica, Dicranomyia (Numantia) fusca, Dicranoptychalivescens, Limoniahercegovinae, Limoniamacrostigma, Limoniastigma, Metalimnobia  quadrinotata, Rhipidia (Rhipidia) maculata, and Dicranota (Dicranota) crassicauda. Seven species of Limoniidae and one species of Pediciidae are new to the whole Transcaucasia, viz. Paradelphomyia (Oxyrhiza) fuscula, Pseudolimnophila (Pseudolimnophila) melanura, Antocha (Antocha) vitripennis, Dicranomyia (Dicranomyia) pallidinota, Dicranoptychalivescens, Limoniastigma, Metalimnobia  quadrinotata, and Dicranota (Dicranota) crassicauda. Four species/subspecies are here recorded for the first time since their original descriptions, viz. Pseudolimnophila (Pseudolimnophila) melanura, Molophilus (Molophilus) stroblianusdecoloratus, Rhabdomastix (Rhabdomastix) filata, and Dicranomyia (Dicranomyia) pallidinota.A total of 38 species of Limoniidae and five species of Pediciidae were previously known to occur in Armenia (Limoniidae and six species of Pediciidae.Altogether 50 species of  Armenia . Our recspecies new to Armenia are marked with an asterisk (*)Limoniidae: LimnophilinaeAfrolimnophilaminima 1. Dicranophragma (Brachylimnophila) nemorale 2. Dicranophragma (Mixolimnomyia) rufulum 3. Eloeophilamaculata 4. Hexatoma (Cladolipes) haiasana Savchenko, 19725. Hexatoma (Hexatoma) gaedii 6. Limnophila (Limnophila) pictipennis 7. PageBreakParadelphomyia (Oxyrhiza) brevifurca Savchenko, 19768. *Paradelphomyia (Oxyrhiza) fuscula 9. *Paradelphomyia (Oxyrhiza) senilis 10. *Phylidorea (Phylidorea) ferruginea 11. *Pseudolimnophila (Pseudolimnophila) lucorum 12. Pseudolimnophila (Pseudolimnophila) melanura Savchenko, 198413. *Pseudolimnophila (Pseudolimnophila) sepium 14. *LimoniidaeChioneinaeEllipteroides (Ptilostenodes) omissus 15. Erioconopasymplectoides 16. *Erioconopatrivialis 17. Erioptera (Erioptera) fusculenta Edwards, 193818. Erioptera (Erioptera) lutea Meigen, 180419. Erioptera (Mesocyphona) bivittata 20. *Gonomyia (Gonomyia) basilobata Alexander, 197521. Gonomyia (Gonomyia) conoviensis Barnes, 192422. Gonomyia (Gonomyia) lucidula de Meijere, 192023. Gonomyia (Gonomyia) papposa Savchenko, 198324. Hoplolabis (Eurasicesa) amseliana 25. Hoplolabis (Parilisia) iranica 26. *Hoplolabis (Parilisia) yezoana 27. Idiocera (Idiocera) laterospina 28. Idiocera (Idiocera) pulchripennis 29. Ilisiamaculata 30. Molophilus (Molophilus) lackschewitzianushebetatus Savchenko, 197631. Molophilus (Molophilus) obscurus 32. Molophilus (Molophilus) ochraceus 33. *Molophilus (Molophilus) pleuralis de Meijere, 192034. *Molophilus (Molophilus) politonigrus Savchenko, 198335. Molophilus (Molophilus) propinquus 36. *Molophilus (Molophilus) stroblianusdecoloratus Savchenko, 197837. Molophilus (Molophilus) urodontus Savchenko, 197838. Ormosia (Ormosia) cuspidata Savchenko, 197339. *Ormosia (Ormosia) fascipennis 40. Ormosia (Ormosia) hederae 41. *Ormosia (Ormosia) longispina Savchenko, 198342. Phyllolabisghilarovi Savchenko, 198343. Rhabdomastix (Rhabdomastix) eugeni Stary, 200444. Rhabdomastix (Rhabdomastix) filata Stary, 200445. Symplecta (Psiloconopa) stictica 46. Symplecta (Symplecta) hybrida 47. PageBreakLimoniidae: LimoniinaeAchyrolimoniadecemmaculata 48. Antocha (Antocha) libanotica Lackschewitz, 194049. Antocha (Antocha) vitripennis 50. *Dicranomyia (Dicranomyia) circassica Lackschewitz, 194151. *Dicranomyia (Dicranomyia) didyma 52. Dicranomyia (Dicranomyia) chorea 53. Dicranomyia (Dicranomyia) longipennis 54. *Dicranomyia (Dicranomyia) lucida de Meijere, 191855. Dicranomyia (Dicranomyia) melanantha Savchenko, 198456. *Dicranomyia (Dicranomyia) modesta 57. Dicranomyia (Dicranomyia) pallidinota Starý, 200958. *Dicranomyia (Dicranomyia) pontica Lackschewitz, 194159. *Dicranomyia (Glochina) transsilvanica Lackschewitz, 192860. Dicranomyia (Melanolimonia) caledonica Edwards, 192661. Dicranomyia (Melanolimonia) morio 62. Dicranomyia (Numantia) fusca 63. *Dicranoptychafuscescens 64. Dicranoptychalivescens Loew, 187165. *Dicranoptycharecurvispina Savchenko, 197466. Limoniacaucasica Lackschewitz, 194067. Limoniaeos Stary & Savchenko, 197668. Limoniaflavipes 69. Limoniahercegovinae 70. *Limoniamacrostigma 71. *Limonianubeculosa Meigen, 180472. Limoniastigma 73. *Limoniasubaequalis Savchenko, 197974. Metalimnobia  quadrimaculata 75. Metalimnobia  quadrinotata 76. *Rhipidia (Rhipidia) maculata Meigen, 181877. *PediciidaeDicranota (Dicranota) crassicauda Tjeder, 197278. *Dicranota (Ludicia) iranensis 79. Dicranota (Paradicranota) landrocki Czižek, 193180. Dicranota (Paradicranota) subtilis Loew, 187181. Pedicia  occulta 82. Tricyphona (Tricyphona) immaculata 83. PageBreak