Among auditory stimuli, the own name is one of the most powerful and it is able to automatically capture attention and elicit a robust electrophysiological response. The subject’s own name (SON) is preferentially processed in the right hemisphere, mainly because of its self-relevance and emotional content, together with other personally relevant information such as the voice of a familiar person. Whether emotional and self-relevant information are able to attract attention and can be, in future, introduced in clinical studies remains unclear. In the present study we used EEG and asked participants to count a target name (active condition) or to just listen to the SON or other unfamiliar names uttered by a familiar or unfamiliar voice (passive condition). Data reveals that the target name elicits a strong alpha event related desynchronization with respect to non-target names and triggers in addition a left lateralized theta synchronization as well as delta synchronization.In the passive condition alpha desynchronization was observed for familiar voice and SON stimuli in the right hemisphere.Altogether we speculate that participants engage additional attentional resources when counting a target name or when listening to personally relevant stimuli which is indexed by alpha desynchronization whereas left lateralized theta synchronization may be related to verbal working memory load. After validating the present protocol in healthy volunteers it is suggested to move one step further and apply the protocol to patients with disorders of consciousness in which the degree of residual cognitive processing and self-awareness is still insufficiently understood. •EEG during an active–passive task based on first names was time-frequency analyzed.•The presented names were uttered either by an unfamiliar or a familiar voice.•Counted names elicited alpha desynchronization and left theta synchronization.•Own name and familiar voices enhanced strong right alpha desynchronization.•Alpha desynchronization reflects attentional engagement and emotional processing. Also because of its countless repetitions during lifetime, the SON is intrinsically meaningful to individuals. In fact, among auditory stimuli, the own name is considered the most powerful stimulus which captures attention without any voluntary effort, as for example demonstrated in the classical “cocktail party” phenomenon , or by iEEG studies have shown that the presentation of the SON evokes larger “P300”  or “P3” Because of its self-relevance and its emotional content, the SON is preferentially processed in the right hemisphere together with other personally relevant information . More inHowever, it has been discussed that the passive own name paradigm, in which subjects only passively listen to the presented stimuli might reflect mere automatic stimulus identification and does not allow for an inference about the level of preserved awareness . AddressIn the current study we used a modified version of the classical own name paradigm including an active “counting” as well as a familiar voice condition. The active condition, in which subjects were asked to (silently) count a specific unfamiliar name should give an important insight in the amount of top-down control and attentional resources engaged by target names and could, therefore, in future studies allow for identifying “awareness” in patients suffering from DOC, in whom behavioural assessment is often challenging and leads to high rates of misdiagnosis . The intWe will focus on on-going oscillatory activity that is not necessarily exactly time-locked to the presentation of the stimulus, like event-related potentials. In fact, time–frequency analysis, quantifying evoked as well as induced brain activity, has been shown to be more sensitive than mere evoked responses which are more prone to temporal dispersion . Further22.1t1 vs. t2 vs. t3 vs. t4; t1=0–200 ms, t2 =200–400 ms, t3=400–600 and t4=600–800 ms post-stimulus),) showed that alpha desynchronization was higher for the target than for non-targets  ×ELECTRODES (Fz vs. Cz vs. Pz)×TIME (.05) cf. , Fig. 3.F2/26=5.46, p<.05) and TIME  were revealed. Post hoc tests revealed that t3 and t4 significantly differed from t1 (t(13)=−3.88, p<.05; t(13)=−3.18, p<.05) while t3 differed from t2 (t(13)=−3.55, p<.05). Furthermore, alpha ERD was higher on the electrode Pz compared to Cz (t(13)=2.86, p<.05) indicating generally larger desynchronization in the posterior part of the scalp and in particular in the last two time windows.Additionally, main effects for ELECTRODES  and CONDITION×TIME . Post-hoc tests on the first interaction revealed that target stimuli evoke stronger alpha ERD compared to non-targets mainly over Pz (t(13)=2.51, p=0.013) while post-hoc testing of the latter indicated that alpha ERD was stronger in response to targets as compared to non-targets only in the later time windows (t3: t(13)=−2.47, p<.05; t4: t(13)=−4.32, p<0.001).The difference between the two conditions is also embedded in the interactions CONDITION×ELECTRODES  and TIME  as well as an interaction between ELECTRODES and TIME . According to post-hoc analyses electrodes Fz and Cz exhibited higher theta ERS as compared to the electrode Pz (t(13)=5.29, p<.001; t(13)=10.49, p<.001, respectively) indicating that theta ERS was most pronounced over fronto-central sites. Theta ERS was strongest 200–400 ms after stimulus onset followed by a steady decrease over time (t2>t3: t(13)= 3.50, p<.05; t2>t4: t(13)=3.36, p<.05), In addition, the interaction ELECTRODES×TIME indicated that theta ERS was systematically higher on Fz (t1: t(13)=9.45, p<.001; t2: t(13)=9.44, p<0.01; t3: t(13)=8.39, p<.001; t4: t(13)=5.65, p<0.001) and Cz in all time windows as compared to Pz (t1: t(13)=4.76, p<.001; t2: t(13)=6.07, p<0.00; t3: t(13)=5.84, p<.001; t4: t(13)=3.43, p<0.05). Results are also depicted in Theta ERS analysis revealed main effects for ELECTRODES  indicating generally higher theta ERS in the left hemisphere =2.12). The interaction CONDITION×HEMISPHERE×TIME  indicated that theta ERS is greater for targets as compared to non-target on the left side of the scalp and in the time window from 200 to 400 ms (t(12)=2.186, p<.05).Since lateralization effects were evident for theta in the active counting condition we decided to also focus on potential hemispheric differences. An ANOVA including the factors CONDITION (target vs. non target), HEMISPHERE (C3 vs. C4) and TIME for the theta frequency revealed a nearly significant main effect for HEMISPHERE , as revealed by one-sample 2.3F1/13=12.16, p<.05) delta activity was significantly higher for target names as compared to non-targets (t(13)=3.48, p<.005) over all electrodes . Additionally, the main effect TIME  indicated that delta was modulated over time with higher ERS from 200 to 600 ms after stimulus onset (t2>t1: t(13)=8.98, p<.001; t3>t1: t(13)=5.65, p<.001; t2>t4: t(13)=6.01, p<.001; t3>t4: t(13)=10.17, p<.001). . cf. .2.4t1 vs. t2 vs. t3; t1=0–200 ms, t2=200–400 ms, t3=400–600 ms post-stimulus) for alpha ERD during passive listening, only a main effect for TIME (F2/26=5.71 p<.05) was significant. Post hoc tests revealed higher desynchronization in the alpha band around 400–600 ms (t3) as compared to 0–200 ms (t1) after stimulus onset (t(13)=−2.82, p<.05). To again test for hemispheric differences, an additional ANOVA including the factors NAME (SON vs. UN), VOICE (familiar voice vs. unfamiliar voice), HEMISPHERE (P3 vs. P4) and TIME  was calculated. A significant interaction VOICE x HEMISPHERE  indicated that the right parietal electrode (P4) showed higher alpha ERD for stimuli spoken in a familiar voice as compared to stimuli spoken in an unfamiliar voice (t(13)=−3.58, p<.05). In addition, the SON as compared to UN also showed enhanced alpha ERD  over the right parietal region in the last two time windows (from 200 to 400 and from 400 to 600 ms) irrespective of VOICE (t(13)=−2.25, p<.05, t(13)=−2.59, p<.05; respectively) (cf. Concerning the ANOVA NAME (SON vs. UN)×VOICE (FV vs. UV)×ELECTRODES (Fz vs. Cz vs. Pz)×TIME  and TIME .The ANOVA NAME×VOICE×ELECTRODES×TIME (for the factor levels please refer to 2.4) for theta frequency yielded main effects for ELECTRODE (t(13)=−5.87, p<.001; t(13)=−4.74, p<.001, respectively) and that theta synchronization was strongest 200–400 ms post-stimulus (t2) (t2>t1: t(13)=3.16, p<.05; t2>t3: t(13)=3.60, p<.05).Post hoc tests revealed that the electrode Pz showed less theta ERS than both Cz and Fz  to target as compared to non-target stimuli was found. The response was localized around central and posterior sites and reached its maximum about 400–600 ms post-stimulus. This is coherent with previous findings showing that alpha desynchronization reflects general task demands including attentional processes . ConsideAlso left-lateralized theta (4–7 Hz) ERS in the active condition appeared to be higher for target than non-target stimuli. Since we controlled for names of relatives and friends, in the active condition only stimuli of comparable familiarity were involved and hence familiarity cannot account for the differences between targets and non-targets. The presentation of strictly unfamiliar names in the active condition in the current study allowed for a better differentiation of top-down attention, (i.e. instruction following and counting) from automatic attention which may be grabbed automatically by the presentation of the own name . The incIn the active condition we also found a significant effect in the delta range (1–4 Hz), with delta showing higher synchronization for target than for non-target stimuli. Previous studies reported that in tasks where internal concentration is required in order to focus attention on a specific stimulus delta increases . In addiWith the passive condition, we investigated differences between the processing of the subject’s own name as compared to unfamiliar names and additionally, we were interested in the differential activation in response to familiar and unfamiliar voices. In fact, in the right hemispheric parietal alpha desynchronization was higher in response to the SON as well as in response to familiar voices. Personally relevant information is known to be more powerful in attracting involuntary attention, as demonstrated by the increase in brain activity after self-referential sound presentation . As showConcerning the found lateralization effect, the right hemispheric dominance for the SON is again possibly related to its emotional and personal relevance , which iAdditional support for a right dominance in the processing of familiar voices come from lesion studies suggesting that an impairment recognizing familiar voices (phonanosia) is only evident in cases of damage to the right hemisphere, or more specifically right temporal lobe . Thus, tAccording to the cognitive model of voice perception by Since SON and FV stimuli in our study were simply presented to participants without further instruction to elaborate on them, we cannot be sure whether the right hemisphere enhancement for these “emotional” stimuli (i.e. FV and SON) is purely automatic or rather reflects higher levels of processing and emotional self-awareness. On an individual subject level in the active counting condition data revealed that 81% of participants did show alpha ERD (or 100% theta ERS), but only 64% more than to the non-target (62% for theta ERS) cf. . It, theFurthermore, while the predominant role of the right hemisphere in the processing of self-relevant and emotional information (FV and SON) is already validated , the linIn both conditions the differential contribution of alpha and theta was mirrored in the differential topographical distribution in these two frequency bands. In fact, while in the active condition alpha is more pronounced on the parietal area around the midline theta is higher over left central regions. In the passive condition alpha is right lateralized. These differences in scalp distribution might, therefore, underline the involvement of different cerebral structures and source localization studies should further elucidate that.In summary, our results demonstrate that time frequency analysis allows for studying the correlates of an active task demand in combination with voice familiarity. Alpha ERD seems to reflect the release of inhibition after successful memory matching. In addition, theta ERS is pronounced when selective attention is attracted by personally relevant information and when incoming information matches long term memory representations, such as a familiar voice or a subject’s own name. Ultimately, we hope that this paper will stimulate new perspectives in order to access and assess (self) awareness also in clinical populations such as DOC patients.44.1M=25.79; SD=8.17) was recorded. All volunteers were right-handed German native speakers without any recorded history of neurological disease. Participants gave written informed consent approved by the local ethics committee and received monetary compensation for their participation.A sample consisting of 14 subjects  with age ranging from 21 to 53 . Stimuli included the subject’s own name and five commonly used Austrian names (according to statistics Austria) matched for number of syllables and the gender of the participant. Stimuli were presented via headphones at a sound pressure level of 80 db.The experiment expands the SON task as introduced by ®,  was used.The task consisted of two experimental conditions: an active condition to investigate the ability to consciously follow commands and a passive listening condition with the passive condition always preceding the active condition. Each condition consisted of 3 blocks; with each block including 13 presentations of each name . In the passive condition 6 stimuli were presented with 234 repetitions in total (about 12 min), in particular, SON uttered by a familiar or unfamiliar voice and two different unfamiliar names either spoken by a familiar or an unfamiliar voice. In the active condition only 3 different stimuli were presented (117 repetition) for about 6 min, all of them unfamiliar to participants and all uttered by a familiar voice cf. . During 4.3EEG was recorded with 32 Ag/AgCl sintered electrodes and head circumference matched Easycaps  placed according to the international 10–20 system. The following scalp EEG channels were used: FP1, FP2, F7, F3, FC5, FC1, Fz, F4, F8, FC2, FC6, T7, C3, Cz, C4, T8, CP5, CP6, P7, P3, Pz, P4, P8, O1, Oz, O2, AFz and FCz. Additional electrodes were placed on the left and right mastoids (M1 and M2). For electrooculography (EOG) two horizontal (placed at the outer canthus of each eye) and two vertical (placed above and below the right eye) electrodes were used for latter correction of blinks and saccadic eye movements.Electrodes were placed on the scalp by applying abrasive electrolyte gel, preceded by a gentle peeling  and on the face secured with plasters.EEG was recorded with a 32-channel BrainAmp EEG amplifier  and Brain Vision Recorder (Brain Products). The EEG sampling rate was set to 500 Hz. Impedances were kept below 5 kΩ. AFz electrode served as a ground electrode while FCz was the recording reference electrode; the mastoid electrodes, M1 and M2 were used for later re-referencing.Acoustic stimuli were delivered binaurally over headphones and surrounding noise was reduced to a minimum.4.4c=8, linear frequency steps) with 30 frequency steps. Subsequently the wavelets were averaged across each stimulus type.In a first step data was re-referenced to mastoids and bandpass—filtered between 0.5 and 70 Hz, a notch filter was set to 50 Hz. Ocular correction was conducted using the regression-based approach  implemenFVUN2/FVUN3) in the active condition and unfamiliar names (FVUN4/FVUN5 and UFVUN4/UFVUN5) in the passive condition were averaged together and only 50% of artefact free segments were randomly selected for further analysis.After wavelet transformation all epochs were averaged together for each participant, each condition and each stimulus type separately. In order to have comparable amounts of segments to be compared, non-target stimuli , ELECTRODES . For elimination of multiple comparisons error the false discovery rate (FDR) correction according to t1, t2, t3; t1=0–200 ms, t2=200–400 ms, t3=400–600 ms post-stimulus). Additional ANOVAs were performed post-hoc in order to specify hemispheric asymmetries apparent in the passive listening and active counting condition. For post-hoc tests we only focus on effects of interest, that is interactions with factor TARGET for the active condition and factors VOICE and NAME for the passive.Five different repeated measures ANOVAs were calculated, four with theta and alpha ERS/ERD as dependent measures and one with delta ERS. Three ANOVAs tested for effects in the active condition and focused on alpha, delta and theta ERS/ERD as dependent variables, respectively: CONDITION , TIME .


Through the years, different studies showed the involvement of Protein Kinase C (PKC) in cell cycle control, in particular during G1/S transition. Little is known about their role at G2/M checkpoint. In this study, using K562 human erythroleukemia cell line, we found a novel and specific mechanism through which the conventional isoform PKC⍺ positively affects Cyclin B1 modulating G2/M progression of cell cycle. Since the kinase activity of this PKC isoform was not necessary in this process, we demonstrated that PKC⍺, physically interacting with Cyclin B1, avoided its degradation and stimulated its nuclear import at mitosis. Moreover, the process resulted to be strictly connected with the increase in nuclear diacylglycerol levels (DAG) at G2/M checkpoint, due to the activity of nuclear Phospholipase C β1 (PLCβ1), the only PLC isoform mainly localized in the nucleus of K562 cells. Taken together, our findings indicated a novel DAG dependent mechanism able to regulate the G2/M progression of the cell cycle. On the contrary, the atypical class (PKCζ and λ/ι) is not sensible to any of them, and its activation is due to protein-protein interactions  , 3433, 35/ml. Experiments of cell counting, performed for 72 hours, showed that PKC⍺ silencing did slow-down cell proliferation of K562 cells compared to the controls. The same effects were noticed using PKC inhibitor . More than one siRNA was used for the experiments in order to exclude any off-target effects and no changes in the results were found . A Scrambled siRNA was used as control (Scrambled). 24 hours after transfection, using Nocodazole, we synchronized the cells at G2/M checkpoint. Here, we observed a strong down-regulation of Cyclin B1 only in cells characterized by PKC⍺ knock-down. PKCβII did not affect it at all Figure . Next, tnhibitor .Since PKC⍺ positively modulated Cyclin B1 levels in K562 cells, we decided to investigate if its kinase activity was necessary to this process. Then, we transiently overexpressed PKC⍺ using two different vectors, a Wild Type vector (PKC⍺ OV WT) and a Dominant Negative mutant (PKC⍺ OV DN). Empty vector (Empty) was used as control. 24 hours after the transfections, we added Nocodazole for 6 hours to drive the cells to G2/M. This partial synchronization was performed to stimulate Cyclin B1 accumulation and study if PKC⍺ overexpression could promote this process. Notably, in cells characterized by PKC⍺ WT or DN overexpression, we found the same increase of Cyclin B1 compared to the controls Figure . As a fuIn order to deeply understand how PKC⍺ could affect Cyclin B1, we decided to investigate at which level this modulation took place. We silenced or overexpressed PKC⍺ and we synchronized K562 cells as previously described. We performed gene expression analyses of Cyclin B1, but we did not find any changes in its expression levels Figure . These eIn this series of experiments, we focused on the expression and localization of Cyclin B1 and PKC⍺ during cell cycle progression. Then, we treated our cells to promote their accumulation in the desired cell cycle phases. In particular, we starved K562 cells, using HBSS for 16 hours, to obtain a G0-G1 synchronization (G0-G1). G1/S phase was referred to proliferating cells in normal and complete RPMI 1640 10% FBS (G1/S). Finally, using Nocodazole, we obtained a G2/M block (G2/M) Figure . FirstlyAs reported above, PKC⍺ and Cyclin B1 co-localize and interact, in particular during G2/M progression. Both of these molecules can accumulate into the nucleus during this phase of the cell cycle, but how this mechanism works is still not well understood . In thes3H]-glycerol and lipids were extracted. The percentage of radioactivity corresponding to DAG was analyzed . Accordingly with other studies -glycerol (10 μCi/ml/1 × 106 cells) for 90 minutes. Next, nuclei and total cells were precipitated with 10% TCA and, then, 10 volumes of chloroform/methanol/concentrated HCl (300:300:1.5) were added and lipids extracted for 20 hours at 4°C. After centrifugation, supernatants were preserved and the pellets were re-extracted twice with 10 volumes of chloroform/methanol/concentrated HCl (400:200/1.5). The combined supernatants were dried under steam of nitrogen and lipids were dissolved in 100ul of chloroform and washed three times in 4 volumes of chloroform/methanol/water (3:48:47). Finally the samples were dried under a steam of nitrogen and [3H]-labelled lipids were analyzed by TLC on silica gel 60 plates developed with ether/exane/NH4 (50:50:0.25). TLC plates were sprayed with Enhancer  and fluorographed at −80°C. Spots corresponding to lipids were scraped off, extracted with 1.5 ml of 0.6N HCl-Methanol (60:40 by volume) for 48 hours with gentle stirring and counted with a liquid scintillation counter using 9ml of Packard Pico-Fluor 40 scintillation cocktail.K562 cells were cultured and synchronized as previously described. Nuclei or intact cells were labeled with [The activity of nuclear PLCβ1 was assessed using a protocol described elsewhere .


AbstractPageBreakXiphinema were found and analysed. The genus Xiphinema is a large group of the phylum nematoda which constitutes more than 260 species. They are polyphagous root- ectoparasites of many crop plants and some species of this genus cause damage by direct feeding on root tips and transmit nepoviruses. The delimitation and discrimination of two species in the genus is presented, described herein as Xiphinemaelongatum and Xiphinemapachtaicum. Morphological and morphometric data were done using light microscopy and results of both species were fit within the previously described nematode species of Xiphinemaelongatum and Xiphinemapachtaicum. 18S rDNA were analysed using Bayesian inference (BI) method to reconstruct phylogenetic relationships of the studied Xiphinema sp. (KP407872Xiphinemaelongatum and KP407873Xiphinemapachtaicum) with other Xiphinema species. The 18S rDNA sequence of Xiphinemapachtaicum was alike to previously described species from the GenBank but Xiphinemaelongatum exhibited very small levels of nucleotides differences (0.4%) which might be possible intra-specific divergence. Though this region of rDNA has less resolution on complex species, its combination with morphological and morphometric analyses, suggests these species as Xiphinemaelongatum and Xiphinemapachtaicum with the GenBank accession number of KP407872 and KP407873, respectively. Short notes, morphological measurements, illustrations, and molecular data are given to these species. These species are reported for the first time from Ethiopia and it provides new geographical information of these organisms.A total of six soil samples were collected around rhizosphere of citrus plants during 2010 from Melkassa Agricultural Research Center experimental station, Ethiopia. From these samples two most important ecto-plant parasitic nematodes of the genus  Xiphinema is amongst the ten most economically important plant parasitic nematode genera  were found and are herewith described using morphology, morphometric data and molecular phylogenetic analyses. These findings represent new records from Ethiopia as well it represents new geographical information.Both samples were collected from main horticultural crop production fields (mainly from citrus plantation) in the Rift valley basin of Ethiopia. Two 39°21'E, 8°24'N) in 2010. A total of six bulk samples of each 1–1.5 kg consisted of 10–15 cores taken from the top 10–40 cm of soil. The samples were kept cool in plastic bags during transportation to the laboratory for nematode extraction. Nematodes were extracted from 200g soil of sub-samples using the modified Baermann method : 50 mM KCl; 10 mM Tris-Cl pH 8.3; 2.5 mM MgCl2; 0.45% NP 40 (Tergitol Sigma); and 0.45% Tween 20) and frozen at -80 °C for at least 10 minutes. To each tube it was added 1 µl of proteinase K (60 µg ml-1) prior to incubation at 65 °C for 1 hour followed by enzyme deactivation at 95 °C for 10 minutes. To amplify the 18S region, 2.5 µl of gDNA suspension was used as template in a 25 µl PCR reaction mix  following the manufacturer’s protocol. The primers used were G18S4 (5’- GCT TGT CTC AAA GAT TAA GCC - 3’) & 4F (5’-CAA GGA CGA WAG TTW GAG G-3’) and the reverse primers were 18P (5’- TGA TCC WRC RGC AGG TTC AC - 3’), & 4R (5’- GTA TCT GAT CGC CKT CGA WC-3’)  and 0.25 M disodium EDTA, saturated with NaCl, pH 8.0 . This waA WC-3’) . The PCRA WC-3’) . SequencFor phylogenetic analysis, the sequences were aligned with related sequences from GenBank, using ClustalW  providedPageBreakTaxon classificationAnimaliaDorylaimidaLongidoridaeSchuurmans Stekhoven & Teunissen, 1938Female. Body ‘J’ shaped, cylindrical, tapering towards the anterior end but more to the posterior end. Cuticle smooth, 1.6–2.3 μm thick at neck region, 2.3–3.1 μm at mid body and 5.5–6.3 μm at tail region. Lip region, well demarcated. Amphidial aperture on lip region 50–59% of lip width. Amphidial fovea stirrup–shaped. Guiding ring, about 1/4th of the total odontostyle length from the base of odontostyle. Odontostyle, 1.6 μm diameter, 66±3 (63–73) % of total stylet length and furcated at base. Odontophore well developed with prominent basal flanges 10.9–11.7 μm wide. Lip width 3.1 ±0.5 (2.5–4.2) % of total stylet length. Female reproductive system amphidelphic, didelphic, branches equally developed. Ovaries reflexed. Pars dilatata oviductus separated from the uterus by a very robust sphincter muscle. No uterus differentiation. Vagina about half body width and perpendicular to the body axis. Vulva, 41% of body length from anterior end. Tail, conoid to dorsally convex conoid, non-digitate, terminal hyaline portion about 27% of tail length Longidoruspachtaicus Tulaganov, 1938: Xiphinemapachtaicum : Kirjanova 1951.Xiphinemamediterraneum Martelli & Lamberti, 1967: Siddiqi and Lamberti 1977.Xiphinemaneoelongatum Bajaj & Jairajpuri, 1977: PageBreakFemale. Body ‘C’ shaped after fixation, tapering to both end but more to the anterior. Cuticle smooth under light microscope. Lip region, distinctly offset by constriction. Amphid aperture post labial, fovea stirrup shaped and about two-third of lip width. Odontostyle robust, poorly forked, 1.56 μm thick, 66±3 (63–73) % of total stylet length and odontophore with weak flanges with width of 10±3 (7–12) μm. Basal Guiding ring 110±6 (104–115) μm from anterior end. Pharynx includes one anterior dorsal nucleus and two posterior subventral nuclei, pharyngeal gland length 94±5 (91–99) μm. Vulva, posterior to mid-body, a transverse slit in ventral view, one-third of the corresponding body width. Female genital branches, didelphic, reflexed, equally developed, generally short. Ovaries, with bacterial endosymbiont, uterus without Z-differentiation, sphincter not clear. Tail short, conical with narrow rounded end ; Asia ; Africa ; North America ; South America (Chile) and Australia. This species has not been recorded as a vector of plant viruses  from Ethiopia is grouped with maximal support with XiphinemaelongatumAY297824 which was submitted from Brazil (Xiphinemapachtaicum (KP407873) and the Slovakian isolate XiphinemapachtaicumAM086682 had identical sequences. The phylogeny analysis of Xiphinemapachtaicum from Spain by The PCR amplification of 18S SSU rDNA region of target nematodes with a universal primer were successfully amplified and yielded a single fragment of 1786 bp ree Fig. . This an species  with maxm Brazil . HoweverThe topology of the tree by other regions of rDNA and position of taxa agrees with previously phylogenetic analysis based on SSU rDNA by van Xiphinema.This information combined with morphological data can assure the species identity and provide new information on the geographical distribution of the genus Xiphinema from Ethiopia using both morphological and molecular analysis. The morphometric values of Xiphinemaelongatum and Xiphinemapachtaicum described from Ethiopia were similar to previously described species with slight difference in both species in ‘a’ values, but they agree with the range of the population previously recorded by Xiphinema species is difficult due to overlapping of many characteristics and their plasticity. Hence, the combination of morphology, morphometric, and molecular results can provide reliable identifications. Based on the congruence of morphological analyses and a SSU rDNA based molecular phylogeny, the Ethiopian Xiphinema species were identified as Xiphinemaelongatum and Xiphinemapachtaicum.This is the first intensive study on the genus


Leukemia relapse and nonrecurrence mortality (NRM) due to leukemia stem cells (LSCs) represent major problems following hematopoietic stem cell transplantation (HSCT). To eliminate LSCs, the sensitivity of LSCs to chemotherapeutic agents used in conditioning regimens should be enhanced. Curcumin (CUR) has received considerable attention as a result of its anticancer activity in leukemia and solid tumors. In this study, we investigated the cytotoxic effects and underlying mechanisms in leukemia stem-like KG1a cells exposed to busulfan (BUS) and CUR, either alone or in combination. KG1a cells exhibiting BUS-resistance demonstrated by MTT and annexin V/propidium iodide (PI) assays, compared with HL-60 cells. CUR induced cell growth inhibition and apoptosis in KG1a cells. Apoptosis of KG1a cells was significantly enhanced by treatment with CUR+BUS, compared with either agent alone. CUR synergistically enhanced the cytotoxic effect of BUS. Seven apoptosis-related proteins were modulated in CUR- and CUR+BUS-treated cells analyzed by proteins array analysis. Importantly, the antiapoptosis protein survivin was significantly downregulated, especially in combination group. Suppression of survivin with specific inhibitor YM155 significantly increased the susceptibility of KG1a cells to BUS. These results demonstrated that CUR could increase the sensitivity of leukemia stem-like KG1a cells to BUS by downregulating the expression of survivin. In 19+CD38− . Notably+CD38− , 12. KG1The alkylating agent BUS is commonly applied in different conditioning regimens for HSCT, to eliminate the underlying leukemia cells and exert an immunosuppressive effect. However, BUS is associated with severe toxicities, including liver, lung, and skin toxicities, hemorrhagic cystitis, diarrhea, and mucositis , 14. TheCurcumin (CUR) is a polyphenol derived from the rhizomes of turmeric, which has received considerable attention as a result of its chemopreventive, chemotherapeutic, and chemosensitizing activities in leukemia and various solid tumors, via targeting multiple signaling pathways –19. CUR In this study, we explored the cytotoxic efficiencies and molecular mechanisms of CUR and BUS alone and in combination in KG1a cells.Reagents include RPMI-1640 , fetal bovine serum , penicillin and streptomycin , CUR , DMSO , BUS , 3--2,5-diphenyltetrazolium bromide , hydroxypropyl methylcellulose , anti-CD34-PE/CD38-FITC , FITC Annexin V Apoptosis Detection Kit I , CycleTEST Plus DNA Kit , anti-PARP , anti-caspase-3 , anti-survivin , ym155 , Human Apoptosis Antibody Array Kit , electrophoresis apparatus trophoresis (Tanon EPS200), and LI-COR Odyssey Scanner (USA).2, which were kindly presented by Miaorong She .Human acute myeloid leukemia KG1a cells and human acute promyelocyte leukemia HL-60 cells were cultured in RPMI-1640 with 10% inactivated fetal bovine serum, penicillin, and streptomycin at 37°C under 5% CO5 cells/mL were incubated in 96-well plates in the presence or absence of the indicated test samples in a final volume of 0.2 mL for 24 h or 48 h at 37°C under 5% CO2. 20 μL MTT solution ) was then added to each well and incubated for 4 h at 37°C, followed by the addition of 200 μL DMSO. Finally the plates were shaken and examined at 490 nm using a microplate reader . Each assay was performed in triplicate. Cells viability was calculated as follows: survival ratio (%) =  × 100%.Cells viability was estimated by MTT assay. KG1a and HL-60 cells in logarithmic phase at 5 × 106 of KG1a and HL-60 cells were washed in PBS containing 2% fetal calf serum (FCS). The cells were resuspended in PBS and incubated for 30 min at 4°C with antibodies to surface antigens CD34 and CD38. Mouse IgG isotype was used as a control. The cells were then analyzed by flow cytometry.Single-cell suspensions of 1.0 × 102. Colonies (>50 cells) were counted and photographs were taken under a light microscopy after 14 days. All the samples were analyzed in triplicate.Approximately 500 treated or untreated cells per well were cultured in RPMI 1640 medium supplemented with 0.9% methylcellulose and 20% fetal bovine serum (FBS) in a final volume of 1 mL at 37°C under 5% CO6 cells in 6-well plates were treated with various concentrations of the indicated test samples at 37°C under 5% CO2 for 48 h. The cells were then harvested to analyze apoptosis. Cells were washed twice with cold PBS and then resuspended in 1x Binding Buffer at a concentration of 1 × 106 cells/mL and 100 μL of the solution (1 × 105 cells) was transferred to a 5 mL culture tube and then 5 μL of FITC annexin V and 5 μL PI were added and the cells were gently vortexed, followed by incubation for 15 min at room temperature (25°C) in the dark. Finally, 400 μL of 1x Binding Buffer was added to each tube and the cells were then analyzed by flow cytometry.The apoptotic rates of KG1a and HL-60 cells were determined by annexin V binding assays, according the manufacturer's instructions. Briefly, approximately 1.0 × 106 cells in 6-well plates were treated with various concentrations of the indicated test samples at 37°C under 5% CO2 for 48 h. Cell cycle analysis was performed by flow cytometry using the CycleTEST Plus DNA Kit (BD Biosciences), according to manufacture's instructions.Approximately 1.0 × 10Total cellular proteins were isolated with lysis buffer (RIPA). Equal amounts of protein were subjected to 10% or 15% polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. After blocking with 5% skim milk, the membranes were incubated with primary antibodies  over night at 4°C and then incubated with horseradish peroxidase-conjugated anti-mouse secondary antibody at room temperature for 1-2 h. The protein bands were imaged using a chemiluminescence reagent  and densities value of the bands was analyzed using Image J software, with glyceraldehyde 3-phosphate dehydrogenase  as the internal reference.The expression of 43 apoptosis-related proteins was analyzed using a Human Apoptosis Antibody Array Kit . Briefly, according to instructions, each of the capture antibodies was printed on the membranes, followed by addition of the treated or untreated cell lysate. After extensive washing, the membranes were incubated with a cocktail of biotin-conjugated anti-apoptotic protein antibodies. After incubation with the infrared fluorescent agent-streptavidin, the fluorescence signals were visualized using a LI-COR Odyssey Scanner.P < 0.05 was considered statistically significant. Compusyn software was used to evaluate the synergistic effects of drug combinations. The combination index (CI) was generated by Compusyn software, where CI < 1, CI = 1, and CI > 1 indicated synergism, additive effect, and antagonism, respectively.The data ware represented as the mean ± standard deviation (SD) and analyzed using SPSS 13.0 and Graphpad Prism 5 software. Means of different groups were compared using one-way ANOVA followed by Bonferroni multiple comparison to evaluate the differences between two groups under multiple conditions. If the date failed the normality test, the Kruskal-Wallis one-way ANOVA on ranks was used for data that failed the normality test. A value of +CD38− cells were 92.3% in KG1a cells, but no CD34+CD38− cells were detected among the HL-60 cells  for 24 and 48 h and the cytotoxic effects were detected by MTT assay. CUR exhibited dose- and time-dependent cytotoxic effects in KG1a cells  using annexin V/PI. Apoptosis was significantly increased in CUR + BUS group, compared with CUR- or BUS-alone groups . For insn groups . These rμM to 80 μM, 16 μM to 160 μM, and 32 μM to 320 μM, resp.) for 48 h. Synergistic effects were estimated using Compusyn software. Cotreatment with all doses exhibited synergistic effects in KG1a cells , caspase-3, and HTRA) were upregulated and four proteins , survivin, and X-linked inhibitor of apoptosis (XIAP)) were downregulated in CUR group and combination group    was simi90%) μM, . Suppres+CD38− phenotype [+CD38− KG1a cells maybe thus provide an ideal model of LSCs, in accord with previous studies [LSCs were a rare population of cells in patients with leukemia. They possess characteristics of self-renewal, chemotherapy resistance, and immune resistance –22. LSCshenotype , 24, 25,henotype . CD34+CD studies , 26.CUR and its analogs have been showed to suppress the growth of various leukemia cells, including U937 cells , 28, K56 in vitro or in vivo [+CD38−ALDHint leukemia cells, which were highly correlated with subsequent clinical relapse [Insensitivity of LSCs to conditioning chemotherapeutic drugs such as BUS is a major reason for leukemia relapse after HSCT. In this study, KG1a cells displayed resistance to BUS, indicated by a lack of apoptosis induction. We there explored the effects of the combination of CUR and BUS on apoptosis in KG1a cells. Encouragingly, CUR markedly enhanced BUS-induced apoptosis, as confirmed by annexin V/PI and western blot analysis. Similarly, the combination of various concentrations of CUR and BUS produced a synergistic antiproliferation effect in KG1a cells. Accumulating evidence suggests that CUR potentiates the effect, including enhancing the antiapoptotic effects of chemotherapeutic drugs such as 5-fluorouracil, bortezomib, FOLFOX, and paclitaxel in vivo –39. The  in vivo . However relapse . Combine relapse . The res relapse –44. The The results of apoptosis arrays showed that seven apoptosis-related proteins were significantly modulated in KG1a cells treated with CUR and CUR + BUS ; Table 1This study also demonstrated that survivin expression was downregulated by CUR and CUR + BUS Figures ; Table 1In summary, this study demonstrated underlying new mechanisms whereby CUR may overcome BUS insensitivity by downregulating survivin in leukemia stem-like KG1a cells. CUR, alone or in combination with BUS, could be a potential anti-LSCs agent for preventing leukemia relapse and reducing the NRM after HSCT. BUS is currently still widely used in the pretreatment of HSCT, but it shows significant side effects and carcinogenicity in patients undergoing HSCT, resulting in danger of being replaced by other conditioning regimens. CUR may solve these issues by combining BUS in the conditioning regimen.


The genetic materials were classified into eight clusters according to the chemical composition of the essential oils: Cluster 1—mostly linalool and 1,8-cineole; Cluster 2—mostly linalool, geraniol, and α-trans-bergamotene; Cluster 3—mostly linalool, methyl chavicol, methyl cinnamate, and β-bourbonene; Cluster 4—mostly linalool, methyl chavicol, epi-α-cadinol, and α-trans-bergamotene; Cluster 5—mainly linalool, methyl eugenol, α-trans-bergamotene, and epi-α-cadinol; Cluster 6—mainly linalool, geraniol, and epi-α-cadinol; Cluster 7—mostly linalool and methyl chavicol; Cluster 8—mainly geranial and neral.The present study aimed to chemically characterize 31 accessions and seven cultivars of basil. The percentage composition of the essential oils of the accessions and cultivars was based on the 14 most abundant constituents: 1,8-cineole, linalool, methyl chavicol, neral, nerol, geraniol, geranial, methyl cinnamate,  Ocimum L. (Lamiaceae) comprises more than 30 species that are found in tropical and subtropical regions [ O. x citriodorum Vis., O. americanum L., O. basilicum L., O. gratissimum L., O. minimo L., and O. tenuiflorum L. [The genus regions . Due to lorum L. .Ocimum basilicum L.) is a medicinal plant traditionally used for the treatment of respiratory and intestinal problems and kidney malfunction [Basil  methyl cinnamate (19–38%); and estragole (38–95%) [A total of 27 basil cultivars were characterized according to the chemical composition of their essential oils, and the cultivars were grouped into five different types: eugenol/linalool ; linalool (52–66%); estragole/linalool ;(38–95%) .The chemical characterization of 38 basil genotypes resulted in seven groups: linalool (19–73%); linalool/eugenol ; methyl chavicol (20–72% methyl chavicol); methyl chavicol/linalool ; methyl eugenol/linalool ; methyl cinnamate/linalool ; and bergamotene (one accession with bergamotene as the major constituent) . Ocimum sp. The present study observed genotypic variations in relation to the content and yield of essential oil, and promising genotypes were noted for the development of cultivars with high content and yield of essential oil rich in linalool and other active ingredients [Morphological and agronomic characterization of 55 basil accessions showed genetic variability inredients . O. basilicum L. grown in Northeastern Brazil.These comparative studies show a high variation in the chemical composition of basil essential oil and how important it is to conduct research investigating these chemical characterizations because the identification of types of basil helps in pharmacological, medicinal, and phytopathological studies. Thus, the present study aimed to chemically characterize the volatile constituents of accessions and cultivars of O. basilicum with 31 accessions provided by the North Central Regional PI Station, Iowa State University, USA, and seven commercial cultivars donated by the companies Topseed and Johnny's Selected Seeds , using helium carrier gas with a flow of 1.2 mL·min−1. The temperature was programmed to be maintained at 50°C for 1.5 min, followed by an increase of 4°C/min up to 200°C and then at 15°C/min up to 250°C, keeping this temperature constant for 5 min. The injector temperature was 250°C, and the detector temperature was 280°C; a volume of 0.5 μL ethyl acetate was injected; the partition rate of the injected volume was 1 : 87 at 64.20 kPa column pressure. The mass spectrometry conditions were quadrupole ion detector operating by electron impact at an impact energy of 70 eV; sweep speed of 1.000; sweep interval of 0.85 fragments/s; and fragments detected in the range 40–550 Da.Oil sample analysis was performed on a Shimadzu QP5050A  system comprising a AOC-20i autosampler and gas chromatograph interfaced to a mass spectrometer (GC-MS) instrument employing the following conditions: column J&W Scientific DB-5MS  fused silica capillary column (30 m × 0.25 mm i.d. × 0.25 μm film) from Phenomenex  under the same conditions as described for GC-MS. The quantification of each component was calculated by area normalization. Compounds concentrations were calculated from the GC relative peak areas (%) and they were arranged in order of GC elution.The quantitative analysis of the chemical components was performed by flame ionization detector (FID) gas chromatography using a Shimadzu GC-17A  under the following operating conditions: ZB-5MS capillary column (5% phenyl-arylene-95%-dimethylpolysiloxane) of fused silica , injected under the chromatographic conditions described above.The essential oil components were identified by comparing their mass spectra with spectra available in the database of the equipment . These libraries allowed comparisons of the spectral data, which had a similarity index of 80%. Furthermore, the measured retention indices were compared to the indices in the literature  relativeThe chemical composition data were analyzed by two multivariate analysis methods: principal component analysis (PCA) and arrangements analysis (cluster) based on the similarity between individuals and their constituent distribution, using the Statistica software version 7.0 Statsoft company.The 14 principle compounds were identified from the essential oils of 31 accessions and seven cultivars and are listed according to elution order in  Ocimum americanum var. americanum in Brazil [ O. canum [The cultivar “Cinnamon” stood out for its high concentration of methyl cinnamate, 31%, a chemical constituent with a cinnamon aroma. This constituent was present in only three accessions and always at levels below 6% . This ren Brazil , O. canuO. canum , and “CiO. canum .α-cadinol levels above 30%; accession PI 358466 presented 46% geraniol, and accession PI 172996 presented 45% methyl chavicol  and geranial (43%) constituents , confirm species , the cul regions , 32.β-bourbonene, methyl eugenol, α-trans-bergamotene, germacrene-D, epi-α-cadinol, and δ-cadinene, which defined the formation of eight clusters according to chemical composition and differentiation by cluster analysis  [The grouping of cultivars and accessions of basil through the chemical composition of the essential oil provides the market with new groups of plants with similar major active compounds, thus broadening the sources of raw materials for the industries. Various activities of-bulk 5) . Thus, iδ-cadinene (r = 0.80) and epi-α-cadinol (r = 0.85) and negatively related to germacrene-D (r = −0.10) and methyl eugenol (r = −0.20). The second principal component represented 20.87% of the total variance and was positively related to geranial (r = 0.98), (r = 0.99) and nerol (r = 0.80). The third principal component accounted for 12.65% of the total variance and was positively related to linalool (r = 0.80) and 1,8-cineole (r = 0.70) and negatively related to methyl chavicol (r = −0.90), geraniol (r = −0.20), and methyl cinnamate (r = −0.10).According to principal component analysis , the firWe observed a correlation greater than 0.50 among some of the chemical constituents of basil essential oil in the accessions and cultivars under study . The corα-cadinol showed strong positive correlation with δ-cadinene (0.80) among Cluster 5 accessions . A moderate positive correlation was observed between 1,8-cineole and linalool (0.66) among the accessions and cultivars of Cluster 1 , and these components were the major compounds in Cluster 1.Nerol and neral as well as nerol and geranial, all present in the cultivar “Sweet Dani,” showed a correlation coefficient of 0.67 and 0.57 (moderate), respectively, for each pair. Epi-Most of the constituents showed correlation coefficients that were classified as weak or very weak, which indicates a low correlation among the variables under study.α-trans-bergamotene; Cluster 3—mostly linalool, methyl chavicol, methyl cinnamate, and β-bourbonene; Cluster 4—mostly linalool, methyl chavicol, epi-α-cadinol, and α-trans-bergamotene; Cluster 5—mainly linalool, methyl eugenol, α-trans-bergamotene, and epi-α-cadinol; Cluster 6—mainly linalool, geraniol, and epi-α-cadinol; Cluster 7—mostly linalool and methyl chavicol; Cluster 8: mainly geranial and neral. We observed that compounds such as linalool, geraniol, methyl cinnamate, geranial, neral, methyl chavicol, and epi-α-cadinol showed concentrations above 30% in some of the accessions and cultivars.The accessions and cultivars were classified according to the chemical composition of their essential oils, forming eight clusters: Cluster 1—mostly linalool and 1,8-cineole; Cluster 2—mostly linalool, geraniol, and


PaPCS) was isolated and its function tested through its heterologous expression in a strain of yeast sensitive to Cd. Subsequently, the Cd sensitive and high biomass accumulating species tall fescue was transformed either with PaPCS or PaGCS (a glutamyl cysteine synthetase gene of reed) on their own (single transformants), or with both genes together in the same transgene cassette (double transformant). The single and double transformants showed greater Cd tolerance and accumulated more Cd and PC than wild type plants, and their Cd leaf/root ratio content was higher. The ranking in terms of Cd and PC content for the various transgenic lines was double transformants>PaGCS single transformants>PaPCS single transformants>wild type. Thus PaGCS appears to exert a greater influence than PaPCS over PC synthesis and Cd tolerance/accumulation. The double transformant has interesting potential for phytoremediation.The production of phytochelatins (PCs) provides an important means for plants to achieve tolerance to cadmium (Cd) toxicity. A reed gene encoding PC synthase ( Phragmites australis (Cav.) Trin. ex Steud, a most effective accumulator of Cd, Pb and Zn, and has been widely exploited as a sewage treatment wetland species Festuca arundinacea Schreb), a particularly vigorous grass species with broad climatic adaptation, could be an attractive recipient species, since turf grasses tolerate regular mowing, thus allowing for the ready disposal of the heavy metals translocated into their foliage. A number of genes involved in the uptake of and tolerance to heavy metals have been identified, and some have already been successfully transferred into plants Heavy metal pollution caused by a combination of natural leaching and anthropogenic activity is becoming a significant environmental problem. Thousands of hectares of arable land have been contaminated in China, representing a significant health hazard to the population Arabidopsis thaliana and Brassica junceaP. australis (PaGCS) is a key component of the species' heavy metal tolerance, and that when this gene is expressed in the fast-growing species Agrostis palustris, tolerance to Cd toxicity is measurably enhanced P. australis as a donor of phytoremediation genes by testing the effect of expressing PaPCS and/or PaGCS in tall fescue.The presence of heavy metals tends to induce the production of phytochelatins (PCs) in plants P. australis leaf tissue using the TRIzol reagent  formed the template for the M-MLV reverse transcriptase driven synthesis of cDNA . The resulting cDNA was amplified using the primer pair 5′-CTTCCAG(A/T)CTCA(G/A)TCGGAGC and 5′-ATTGC(G/C)ACTCCT(T/A)GACAGCA to obtain an initial PaPCS sequence. After sequencing this amplicon, further primers were derived to perform 3′-RACE and 5′-RACE (TaKaRa 3′- and 5′-Full RACE Core set), according to the manufacturer's protocols.Total RNA extracted from frozen SacI/XbaI PaPCS cDNA fragments were sub-cloned into the pYES2 vector and transformed into Saccharomyces cerevisiae mutant strain YK44  using the lithium acetate method 600 reached 0.5. The cells were pelleted by centrifugation from a 500 µL aliquot of the culture and re-suspended in deionized water in 10−1, 10−2, and 10−3 concentrations, and then inoculated into 40 µL YPGAL  medium in the presence of 100 µM CdCl2. All experiments were done in quadruplicate.XbaI/SacI PaPCS cDNA fragment was cloned into the pROKII vector . Prior to agroinfection, F. arundinacea embryogenic calli were grown for one week on MS medium The binary plasmids pROK/PaGCS, pROK/PaPCS and p3301/PG were separately introduced into the F. arundinacea rooted seedlings were transplanted into soil in a greenhouse (22±3°C under a 16 h photoperiod with a photosynthetic photon flux density of 45 µmol m−2 s−1). The seedlings were at tillering stage after being transplanted into the soil for 30 days. Then, WT and transgenic tiller clones were transferred into Hoagland's solution, and exposed for five days to 150 µM CdCl2.Transgenic and wild type (WT) PaGCS coding region was amplified as previously described PaGCS transformants. To detect candidate PaPCS transformants, a primer pair was designed to target a similar fragment of the PaPCS transgene (primer sequences were 5′-CTTCCAGACTCAGTCGGAGC and 5′-ATTGCGACTCCTTGACAGCA). Each 20 mL PCR reaction contained 0.4 µM of each primer, 400 µM dNTP, 1.5 mM MgCl2, 1.25 U rTaq DNA polymerase  and either 300 ng plant DNA or 15 pg plasmid. Each PCR consisted of a denaturation step (95°C/5 min), followed by 35 cycles of 95°C/40 s, 55°C/60 s 72°C/60 s, ending with an extension step of 72°C/10 min. PCR products were separated by 1% agarose gel electrophoresis. The presence of the transgene was also tested by Southern hybridization. For this purpose, 15 µg EcoRI- or SacI digested genomic DNA was electrophoresed through a 0.8% agarose gel and electrophoretically transferred onto a Hybond N+ membrane . The membranes were hybridized with the PaGCS or PaPCS amplicons labeled with digoxigenin using the random primer method . Hybridization, washing and signal detection were performed according to the manufacturer's instructions.Genomic DNA was isolated from the leaf of candidate transformants using the CTAB method 2 for five days was recorded. Each plant was thoroughly washed with distilled water, divided into leaves, shoots, and roots. Root samples were washed for 10 min at 4°C in 2 mM CaSO4 and 10 mM EDTA, and rinsed three times in water, and then placed into oven dried to a constant weight. A sample of 30 mg dry matter was digested in HNO3 and HClO4 (4∶1 v/v). Then it was evaporated to dryness and dissolved in 2 mol/L HNO3. The concentration of Cd2+ in the solvent was determined as described by Vitória, Lea & Azevedo The biomass of WT and transgenic tiller clones exposed to 150 µM CdCl−1 cm−1). The MDA concentrations were measured after a 0 h, 6 h, 12 h, 24 h, 48 h, 72 h and 120 h exposure to 150 µM CdCl2. All treatments and experiments were using different plants done in quadruplicate. The MDA concentration unit was µmol·g−1DW.Top most expanded leaves of transgenic and wild-type tall fescue with or without Cd treatment were collected. Malondialdehyde (MDA) content was determined by measuring the concentration of thiobarbituric acid-reacting material present, using thiobarbituric acid (TBA) Top most expanded leaves of transgenic and wild-type tall fescue with or without Cd treatment were collected. Peroxidase (POD) and superoxide dismutase (SOD) activity were measured as described by Scebba et al. 2O2 solution, 20 mM guaiacol and 0.01 mL of crude extract. Adding H2O2 and guaiacol solution at the same time started the reaction. The activity was determined by monitoring the increase of absorbance at 470 nm, as a result of guaiacol oxidation. Both SOD and POD enzyme activities were measured after a 0 h, 6 h, 12 h, 24 h, 48 h, 72 h and 120 h exposure to 150 µM CdCl2. All treatments and experiments were using different plants done in quadruplicate. POD activity unit was Δ460·min−1·g−1. SOD activity unit was Unit·mg−1.Total protein of the crude extract was measured using the method of Bradford 3, centrifuged at 20,000 g for 20 minutes and GSH was fluorometrically determined in the supernatant after 15 minutes incubation at 25°C with o phthaldehyde (OPT) and the fluorescent intensity was recorded at 420 nm after excitation at 350 nm on a Hitachi Fluorescence Spectrophotometer (Model No. 650-60). The non-protein thiol (TNP-SH) content was determined as described by Tu et al. 2. All treatments and experiments were using different plants done in quadruplicate. TNP-SH, GSH and PC content unit were µmol·g−1DW.Total glutathione (GSH) content was determined as described by Gupta et al. P<0.05 was applied to indicate statistical significance. The data were analyzed using the SAS statistical package .For each statistical test, a threshold of Zinnia elegans and tomato provided the target for the design of PCR primers. A full-length P. australis cDNA was obtained by RACE-PCR. The PaPCS (GenBank accession number JX826285) sequence length was 1497 bp, and comprised a single open reading frame encoding a 498 residue polypeptide of predicted molecular mass ∼54.9 kDa. A phylogenetic analysis showed that its nucleotide sequence shared considerable similarity with its homologs from bread wheat (84.3%), bermudagrass (84.3%) and rice (70.9%). The closest related peptide sequence was the PCS from bermudagrass , and a single copy of PaPCS-PaGCS into each of the PaPCS/PaGCS lines  and PaGCS transgenic lines (1.1–1.28 fold). In the double transformant, the ratio was 1.0–1.2 fold, which was not significantly different with PaGCS transgenic lines  pROK/PaGCS, and (B) p3301/PG. RB and LB: right and left borders of the Ti plasmid; Tnos: nos terminator (260 bp).(TIF)Click here for additional data file.Figure S2Phylogeny of deduced phytochelatin synthase polypeptides.(TIF)Click here for additional data file.Figure S3Transformation of F. arundinacea with PaPCS. (A) Callus induction, (B) Agroinfection, (C) Kanamycin selection of transgenic calli, (D) Regenerated plants, (E) Selected plants maintain their greenness in the presence of kanamycin, (F) Putative transgenic plants grown in soil.(TIF)Click here for additional data file.Figure S4Transformation of F. arundinacea with PaGCS-PaPCS. (A) Callus induction, (B) Agroinfection, (C) Kanamycin selection of transgenic calli, (D) Regenerated plants, (E) Selected plants maintain their greenness in the presence of kanamycin, (F) Putative transgenic plants grown in soil.(TIF)Click here for additional data file.Figure S5PC2 and PC3 content after short term Cd exposure/After 0–120 h, the leaves were sampled for (A) PC2 and (B) PC3. TP-1, TP-2: carriers of PaPCS; TG-1, TG-2: carriers of PaGCS; T(GP)-1, T(GP)-2: carriers of PaGCS-PaPCS; WT: wild type F. arundinacea. Vertical bars indicate the standard error of the mean (n = 4).(TIF)Click here for additional data file.


Bradyrhizobium japonicum Is-1, which is incompatible with Rj2 genotype soybeans. The estimated genome size of this strain is 8.9 Mb. Genome sequence information of this strain will help to identify a causal gene for this incompatibility.We report the draft genome sequence of  Bradyrhizobium japonicum is known to form nodules on the roots of soybean (Glycine max). However, nodulation of B. japonicum strains Is-1 and USDA 122 is restricted by some soybean cultivars   class of plant resistance proteins  13. This nproteins . In contB. japonicum Is-1 was extracted by using the Wizard Genomic DNA purification kit , and fragmented to 400 bp using the Covaris S2-A system . A library for sequencing was prepared by a NEBNext DNA library prep master mix set for Illumina , and paired-end sequenced (2 × 302 bp) on a MiSeq sequencer using a MiSeq version 3 reagent kit . Raw reads were trimmed and de novo assembled using CLC Genomics Workbench version 7.5.1 . Parameters for the trimming were as follows: ambiguous limit, 2; quality limit, 0.001; number of 5′ terminal nucleotides, 10; number of 3′ terminal nucleotides, 40. Parameters for the de novo assembly were as follows: mapping mode, map reads back to contigs (slow); update contigs, yes; bubble size, 600; minimum contig length, 1,000; automatic word size, 51; perform scaffolding, yes; auto-detect paired distances, yes; mismatch cost, 2; insertion cost, 3; deletion cost, 3; length fraction, 0.8; similarity fraction, 0.95.Genomic DNA of B. japonicum Is-1 was assembled into 109 contigs, with an accumulated length of 8,983,878 bp  and an average GC content of 64.0%. The genome was annotated by the Prokaryotic Genome Annotation Pipeline (PGAP version 2.10), and a total of 8,066 coding sequences (CDSs), 3 rRNAs, and 50 tRNAs were predicted.The draft genome of Bradyrhizobium strains overcame the nodulation restriction of Rj2 genotype soybeans  structural genes in the incompatible LGUJ00000000.This whole-genome shotgun project has been deposited at DDBJ/EMBL/GenBank under the accession number


Candidatus Scalindua sp.” is still lower than those of other anammox bacteria enriched from freshwater environments. The activity and growth of marine anammox bacteria are generally considered to be affected by the presence of salinity and organic compounds. Therefore, in the present study, the effects of salinity and volatile fatty acids (VFAs) on the anammox activity, inorganic carbon uptake, and biomass yield efficiency of “Ca. Scalindua sp.” enriched from the marine sediments of Hiroshima Bay, Japan, were investigated in batch experiments. Differences in VFA concentrations (0–10 mM) were observed under varying salinities (0.5%–4%). Anammox activity was high at 0.5%–3.5% salinity, but was 30% lower at 4% salinity. In addition, carbon uptake was higher at 1.5%–3.5% salinity. The results of the present study clearly demonstrated that the biomass yield efficiency of the marine anammox bacterium “Ca. Scalindua sp.” was significantly affected by salinity. On the other hand, the presence of VFAs up to 10 mM did not affect anammox activity, carbon uptake, or biomass yield efficiency.The growth rate and biomass yield efficiency of anaerobic ammonium oxidation (anammox) bacteria are markedly lower than those of most other autotrophic bacteria. Among the anammox bacterial genera, the growth rate and biomass yield of the marine anammox bacterium “ Planctomycetes  is a microbiological process in which ammonium is oxidized to dinitrogen gas under anoxic conditions with nitrite as the electron acceptor , 31, 34.omycetes , 29, 39.ronments , 37, andronments , 21, 36.vailable , 26, 32,Ca. Brocadia anammoxidans”  rate, leading to a lower biomass yield efficiency because the maximum specific ammonium consumption rate (65 μmol NH4+ [g protein]−1 min−1) of “Ca. Scalindua sp.”  concentrations. VFAs are also oxidized by both marine and freshwater anammox bacteria with nitrate as the electron acceptor , 70 mg-N L−1 of (NH4 )2 SO4 , 70 mg-N L−1 of NaNO2 , 500 mg L−1 of KHCO3 , 27 mg L−1 of KH2 PO4 , 300 mg L−1 of MgSO4 ·7H2 O, 180 mg L−1 of CaCl2 ·2H2 O, and 1 mL of trace element solutions I and II, as described by van de Graaf et al. (2 gas for 1 h to maintain an anaerobic condition (<0.5 mg L−1 of dissolved oxygen) before the addition of nutrients.Biomass samples were obtained from an upflow column reactor inoculated with an anammox biofilm, as previously described . The ino at 20°C ; therefo at 20°C , 4. The  . Cloned 16S rRNA genes were randomly selected and the clone library was constructed. The 16S rRNA genes were sequenced at the Dragon Genomics Center . Sequences with ≥97% homology were grouped into operational taxonomic units (OTUs) using the ARB neighbor-joining (NJ) method with similarity corrections performed using the ARB software  according to the manufacturer’s instructions. To construct the clone library, 16S rRNA gene fragments were amplified using the f Pla46f  and 1390f Pla46f . The PCRsoftware . A phylosoftware . A bootset al.  was dispensed into 7-mL serum vials and sealed with butyl rubber stoppers. The headspace was replaced by repeatedly vacuuming and purging with helium gas (>99.99995%). The vials were then statically incubated at 28°C for 1 d to determine the ammonium oxidation rate and inorganic carbon uptake and for 6 h to determine the VFA oxidation rate as described by Kartal et al.  to prevent pH decreases by the addition of fatty acids. The pH was adjusted to 7.5 using 1 M NaOH or 1 M HCl after the addition of VFAs. [14C] bicarbonate was added to vials at a final concentration of 20 μCi . Anammox activity and inorganic carbon uptake were expressed as a specific ammonium consumption and [14C] bicarbonate uptake rate, respectively. The biomass yield efficiency was calculated by dividing the specific [14C]bicarbonate uptake rate by the specific ammonium consumption rate.Batch experiments were performed to investigate the influence of salinity 0.5%–4%) and VFAs 0–10 mM) on the anammox activity, inorganic carbon uptake, biomass yield efficiency, and VFA oxidation rates of “% and VFA mM on thl et al. . VFAs we14C]bicarbonate uptake was confirmed by liquid scintillation counting. The biomass was collected, washed three times with phosphate buffered saline, and mixed with the scintillation cocktail . Radioactivity was subsequently determined using an LSC-5100 liquid scintillation counter .Ammonium concentrations were determined using Nessler’s method with a UV-visible spectrophotometer . Nitrite and nitrate concentrations were determined using ion chromatography  with a Shodex Asahipak NH2P-50 4D anion column  and UV-VIS detector  following the filtration of samples through 0.2-μm pore size membranes , as previously described . The con2− and consumed NH4+ (ΔNO2−/ΔNH4+) and produced NO3− and consumed NH4+ (ΔNO3−/ΔNH4+) in batch experiments for VFAs were compared using the unpaired Welch’s t-test in Microsoft Excel. A P value of less than 0.05 was considered significant.The average nitrogen stoichiometric ratios for consumed NOThe sequence data of the partial 16S rRNA gene obtained from the upflow column reactor was deposited in the GenBank/EMBL/DDBJ databases under accession number AB900163.Ca. Scalindua sp. SH” and the clone husup-a7 was 99.9%, indicating that a single species of “Ca. Scalindua sp.” was successfully enriched at 28°C, even though the inoculum contained two distinct species of anammox bacteria belonging to the “Ca. Scalindua” group (93.1% similarity to the clone husup-a2)  . The domusup-a2)  and Sca1usup-a2)  probes, usup-a2) . These rusup-a2) . The hig at 30°C .4+ and NO2− was observed under all conditions. Higher anammox activities were observed at 0.5%–3.5% salinity, whereas anammox activity was lower at 4% salinity  and carbon uptake (anabolism). Biomass yield efficiency was calculated to be 0.010–0.042 μmol-C μmol-N−1 under 0.5%–4.0% salinity conditions   at 2–3% salinity, at which carbon uptake was high, were similar to previous findings: 0.07 for “Ca. Brocadia anammoxidans” , biomass yield efficiencies  were markedly lower than the previously reported value of 0.030 at 28°C and 3.5% salinity   among the three VFAs. The average nitrogen stoichiometric ratios for ΔNO2−/ΔNH4+ and ΔNO3−/ΔNH4+ at 1–10 mM were then compared with the ratio at 0 mM for each VFA. The stoichiometric ratio was not significantly different (P>0.05) in any VFA experiment (−1, respectively (et al. (Ca. Scalindua spp.” require more energy for osmotic pressure adjustments or cell maintenance when salinity conditions are not optimal. However, future studies need to be conducted in order to confirm this phenomenon.To investigate the effects of VFAs on anammox activity and inorganic carbon uptake, formate, acetate, and propionate, all of which are known to be oxidized by anammox bacteria with nitrate as the electron acceptor , 11, 36,ectively . No signperiment . These rperiment , 1.21 anperiment , and 1.1periment . In the periment . On the periment , althougperiment . Based oectively . These v (et al. , 32. OneCa. Scalindua sp.” were similar to those of other anammox bacteria previously reported (Ca. Scalindua sp.” in the present study and “Ca. Scalindua spp.” (Ca. Brocadia fulgida” (Ca. Anammoxoglobus propionicus” (Ca. Scalindua” species indicated that they had undergone niche adaptations to marine environments with lower ammonium and nitrite concentrations.Anammox bacteria are recognized as consumers of VFAs with nitrate as the electron acceptor , 11, 36.reported . The VFAionicus” . The higCa. Scalindua sp.” were determined by batch experiments under different salinities and VFA concentrations. We clearly demonstrated that the presence of VFAs did not affect these factors, whereas salinity significantly affected the carbon uptake of “Ca. Scalindua sp.” These results provide important new insights into the activity and growth characteristics of “Ca. Scalindua sp.” and indicate that salinity is one of the crucial factors regarding the nitrogen cycle in marine environments, especially at sites in which salinity fluctuates such as estuarine environments.The anammox activity, inorganic carbon uptake, and biomass yield efficiency of “


The completion of several genome-sequencing projects has increased our need to assign functions to newly identified genes. The presence of a specific protein domain has been used as the determinant for suggesting a function for these new genes. In the case of proteins that are predicted to interact with mRNA, most RNAs bound by these proteins are still unknown. In yeast, several protocols for the identification of protein-protein interactions in high-throughput analyses have been developed during the last years leading to an increased understanding of cellular proteomics. If any of these protocols or similar approaches shall be used for the identification of mRNA-protein complexes, the integrity of mRNA is a critical factor.We compared the effect of different lysis protocols on RNA integrity. We report dramatic differences in RNA stability depending on the method used for yeast cell lysis. Glass bead milling and French Press lead to degraded mRNAs even in the presence of RNase inhibitors. Thus, they are not suitable to purify intact mRNP complexes or to identify specific mRNAs bound to proteins.We suggest a novel protocol, grinding deep-frozen cells, for the preparation of protein extracts that contain intact RNAs, as lysis method for the purification of mRNA-protein complexes from yeast cells. Genome analyses of a range of organisms have lead to the identification of an increasing number of putative RNA-binding proteins (RBPs) whose function is still unknown. RBPs have been found to act as integral part of ribonucleoparticles (RNPs) controlling gene expression at different levels . RNPs arTrypanosoma brucei [Drosophila [Recently, research has mainly been focused on identifying protein-protein interactions using two-hybrid interactions ,5, immuna brucei , Drosophosophila  and mammosophila . It has osophila .Besides the purification method, the way to lyse cells is also crucial. In yeast, different lysis methods are in use. Glass bead milling has been applied to identify RNAs from immunoprecipitated RNPs . Both FrBreaking yeast with a glass bead mill is a common method to produce cell lysates. The principle is based on the physical rupture of the yeast's cell wall and cell due to the friction produced by glass beads rapidly moving through the cell suspension. One of the advantages of this method is the high lysis efficiency.We lysed two different yeast strains using a "bead-beater" bead mill in the presence of RNase inhibitors  as described in Methods. We used a strain where Nrp1p, a putative RNA-binding protein that contains one RRM (RNA Recognition Motif) , has beePDA1) or by direct staining of ribosomal RNAs after blotting with methylene blue  and extracted the RNA. As shown in Figure PDA1 and ASH1 mRNAs is already detected in the crude lysate. Whereas no further degradation of PDA1 mRNA is detected during subsequent centrifugation, ASH1 mRNA is heavily degraded, indicating that there might not be a protective effect of RNA-binding proteins like She2p. In addition, when the extract was incubated with the IgG-coupled beads for 2 hours degradation becomes evident also for PDA1 mRNA , since the following purification steps might become an additional source of RNA degradation. This idea is also supported by our results Figure  where, eOur data suggest that mechanical breakage of frozen yeast cells at ultra-low temperatures is the lysis method of choice for purification of intact mRNP complexes. Since in recent approaches ,16 diffeStrains used in this work are isogenic with W303. The relevant genotypes are listed in Table TAP-tagged strains were generated by transformation with PCR products using the lithium acetate method . The pri600 nm/ml of 2–3. Cultures were harvested at 4°C and washed with ice-cold sterile water.Yeast cultures were grown at 30°C in YPD media  up to an600 nm (5 l cultures with an OD600 nm/ml of 2) were harvested, washed with cold sterile water and immediately lysed or the wet pellet snap-frozen in liquid nitrogen. For lysis, harvested cells were resuspended in 30 ml lysis buffer , 0.7 μg/ml pepstatinA, 100 U/ml SuperaseIn , 20 mM Ribonucleoside Vanadyl Complex ) and immediately broken. Frozen cells were stored at -80°C until used. Then, they were thawed in 30 ml lysis buffer and immediately lysed. Cells were lysed in a glass bead mill  in 3 cycles (3 min breaking and 5 min cooling). During lysis, cells were kept cold by a water-ice bath.Cell pellets from 10000 OD600 nm (2.5 l cultures with an OD600 nm/ml of 2) were harvested, washed with cold sterile water and immediately lysed or the wet pellet snap-frozen in liquid nitrogen. For lysis, harvested cells were resuspended in 15 ml lysis buffer  and immediately broken. Frozen cells were stored at -80°C until used. Then, they were thawed in 25 ml lysis buffer and immediately broken. Lysis was performed as described [Cell pellets from 5000 ODescribed  with a c600 nm/ml of 2–3, were frozen by pressing the cell pellet through a 50 ml syringe directly into liquid nitrogen [2, 0.30% Igepal CA-630, 120 mM NaCl, 80 mM KCl, 2 mM EDTA, 1 mM DTT, 1 μg/ml aprotinin, 0.8 μg/ml bestatin, 1 μg/ml leupeptin, 0.05 mg/ml pefabloc, 0.7 μg/ml pepstatinA, 200 U/ml SuperaseIn, 40 mM RVC) per gram.Cell pellets corresponding to 3–15 l cultures, with an ODnitrogen  and stor2, 100 mM K-acetate, 10 mM Tris-HCl pH 8, 100 mM NaCl, 10 mM Mg-acetate, 1 mM EGTA, 0.1% Igepal CA-630, 0.5 mM DTT, 100 U/ml SuperaseIn, 10 mM RVC, 1 μg/ml aprotinin, 0.8 μg/ml bestatin, 1 μg/ml leupeptin, 0.05 mg/ml pefabloc, 0.7 μg/ml pepstatinA) with the addition of adjusting buffer . The resulting sample was again spun for 20 min at 20000 × g and the supernatant (S20) finally spun at 200000 × g for 20 min. The supernatant was collected (S200) and the resulting pellet (P200) was resuspended in 10 ml IgG binding buffer .After cell lysis, cell extracts were subjected to serial centrifugations at 4°C. First, the crude lysate (CL) was spun 3 min at 1200 × g to pellet cell debris. The total extract (S1) was further spun for 20 min at 7500 × g and the supernatant collected (S7). Salt and buffer concentration were adjusted to IgG binding conditions  was spun 3 min at 1200 × g to pellet cell debris. Total extract (S1) was further spun for 20 min at 7500 × g and the supernatant (S7) was collected. The resulting pellet (P7) was resuspended in 10 ml 0.5 × lysis buffer. Supernatant (S7) was again spun for 20 min at 20000 × g and the supernatant (S20) finally spun at 200000 × g for 20 min. The supernatant was collected (S200) and the resulting pellet (P200) was resuspended in 10 ml 0.5 × lysis buffer.For TAP purifications of Sub2p, total extract (S1) was directly centrifuged at 100000 × g for 1 h (S100).The lipid phase was discarded from all fractions.100–300 μl aliquots from the different fractions were extracted immediately 3–4 times with phenol:chloroform:isoamylalcohol (pH 4), precipitated with sodium acetate and ethanol and stored at -80°C until analyzed.2, 100 mM K-acetate, 10 mM Tris-HCl pH 8, 100 mM NaCl, 10 mM Mg-acetate, 1 mM EGTA, 0.1% Igepal CA-630, 0.5 mM DTT, 100 U/ml SuperaseIn, 10 mM RVC, 1 μg/ml aprotinin, 0.8 μg/ml bestatin, 1 μg/ml leupeptin, 0.05 mg/ml pefabloc, 0.7 μg/ml pepstatinA. Samples were taken before (IgG input) and after IgG immunopurification  and immediately extracted, precipitated and stored at -80°C as described above.Glycerol  was then added to those samples used for further TAP purification steps. These samples were snap-frozen in liquid nitrogen and stored at -80°C for a maximum of 2–3 days. Aliquots were taken before freezing and immediately after thawing to control for degradation during freeze-thawing. These aliquots were extracted, precipitated and stored as described above. TAP purification was performed up to the IgG immunopurification step (first step of the TAP protocol) as described , using a260 nm/A280 nm measured and equivalent amounts of RNA were loaded onto 1.2% agarose-formaldehyde gels. Samples loaded on gels for direct visualization were supplemented with 0.2 μg/μl of ethidium bromide in loading buffer. For Northern blot analysis gels were blotted onto nylon membranes, the membrane stained with methylene blue for determining transfer efficiency and probed against ASH1 and/or PDA1 mRNA .Total RNA was isolated by direct phenol:chloroform extraction from the same strains used in the study as described , precipiThe abbreviations used are: RNP, ribonucleoprotein; RBP, RNA-binding protein; TAP, tandem affinity purification; RVC, ribonucleoside vanadyl complex; RRM, RNA recognition motif; UTR, untranslated region; ORF, open reading frame; DEPC, diethylpyrocarbonate; TCA, trichloroacetic acid; TEV, tobacco etch virus protease.MLdH carried out the studies and drafted the manuscript. RPJ conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Vaccinia virus (VACV) is a promising oncolytic agent because it exhibits many characteristic features of an oncolytic virus. However, its effectiveness is limited by the strong antiviral immune response induced by this virus. One possible approach to overcome this limitation is to develop deimmunized recombinant VACV. It is known that VACV p35 is a major protein for B- and T-cell immune response. Despite the relevance of p35, its epitope structure remains insufficiently studied. To determine neutralizing epitopes, a panel of recombinant p35 variants was designed, expressed, and used for mice immunization. Plaque-reduction neutralization tests demonstrated that VACV was only neutralized by sera from mice that were immunized with variants containing both N- and C- terminal regions of p35. This result was confirmed by the depletion of anti-p35 mice sera with recombinant p35 variants. At least nine amino acid residues affecting the immunogenic profile of p35 were identified. Substitutions of seven residues led to disruption of B-cell epitopes, whereas substitutions of two residues resulted in the recognition of the mutant p35 solely by non-neutralizing antibodies. Orthopoxvirus is the most well-known genus of the Poxviridae family, which contains complex DNA viruses. All viruses of this genus replicate in the cytoplasm of infected cells, because they have their own replication and transcription regulatory mechanisms [The chanisms ,2. Some chanisms ,4,5,6,7,chanisms . Althougchanisms ,9,10. Inchanisms ,19,20,21chanisms . Howeverchanisms ,24. In tchanisms .In addition, VACV is considered a promising oncolytic agent, because VACV has many characteristics that make it an ideal platform for developing oncolytic agents. VACV has a short life cycle that takes place in the cytoplasm, which eliminates the risk of genome integration, and demonstrates notable benefits, such as efficient delivery, large transgene-encoding capacity, intravenous stability, and the ability to induce efficient immune responses ,27. MoreIn order to avoid a strong antiviral immune response, a VACV deimmunization strategy is required. One possible approach to deimmunization is to mask or eliminate the most effective neutralizing epitopes of the virus. This approach has been successful in developing antibody-escaping variants of the measles virus ,29, inflAmong several dozens of VACV proteins found in the sera of vaccinated individuals, p35 encoded by the H3L open reading frame (ORF) is one of the major immunogenic proteins ,40,41,422 at 37 °C.CV-1 cells were obtained from the cell culture repository of the Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences. Cells were grown in Iscove’s Modified Dulbecco’s Medium  containing 10% heat inactivated fetal bovine serum  and antimicotics-antibiotics solution  in an atmosphere of 5% COAttenuated vaccinia virus (VV) LIVP/GFP (TK-) strain containing an insertion of the green fluorescent protein (GFP) gene in the viral thymidine kinase gene was grown and titrated on CV-1 cells, as described previously .The sera from vaccinated donors were obtained as part of the research performed previously .3. To develop the p35Δfuse, two overlapping primers p35fuseN and p35fuseC and extension PCR were used. The resulting PCR product containing the p35Δfuse was obtained using primers H3L Bam-1+ and H3L Back12-EcoRI. All obtained PCR fragments were digested with BamHI and EcoRI and ligated into BamHI/PstI-digested expression plasmid pET32a . Escherichia coli BL21(DE3) cells were transformed with the resulting plasmids pET32-p35Δ, pET32-p35Δ3, pET32-p35Δ6, pET32-p35Δ12, and pET32-p35Δfuse and plated independently onto agar with 50 mkg/mL ampicillin and incubated at 37 °C overnight. E. coli clones were screened for the presence of the appropriate gene fragment by PCR using primers pET32a-dir (5′-TGCTAGTTATTGCTCAGCGG-3′) and pET32a-rev (5′-GGTTCTGGTTCTGGCCATA-3′), followed by sequencing. All recombinant p35 fragments were derived from H3L ORF, VACV strain LIVP (GenBank KP233807.1) and produced as fusion proteins with thioredoxin and a C-terminal His tag. Gene fragments p35Δ (1–282 aa), p35Δ3 (1–135 aa), p35Δ6 (128–282 aa), and p35Δ12 (1–239 aa) were obtained using a set of primers . In addi600 reached 0.6–0.8, protein synthesis was induced by adding 0.1 mM of isopropyl β-D-1-thiogalactropyranoside (IPTG). Then, cells were cultured for 4 h under the same conditions. After that, the cells were centrifuged for 10 min at 6000× g and resuspended in a phosphate buffered saline (PBS), containing 1 mM PMSF, pH 7.5. The cell pellet was homogenized using an ultrasonic homogenizer Sonopuls HD 2070  for 10 min at 35% amplitude, with 10 s of sonication followed by a 10 s rest period. Subsequently, the samples were centrifuged at 15,000× g for 20 min. The obtained soluble cytoplasm fractions were used for proteins purifications.In each case, a confirmed positive colony was grown in 100 mL of LB medium containing 50 μg/mL of ampicillin at an agitation rate of 180 rpm at 37 °C. When the optical density ODRecombinant proteins rp35Δ, rp35Δ3, rp35Δ6, rp35Δ12, and rp35Δfuse were purified from cytoplasm using Ni-NTA agarose . For chromatography, PBS was used with the addition of NaCl up to 300 mM (wash buffer). Nonspecific proteins bound with Ni-NTA were eluted with a wash buffer, containing 50 mM imidazole, after which the truncated p35 protein was eluted with a buffer containing 250 mM imidazole. After purification, protein dialysis was performed using a storage buffer, containing 50 mM Tris-HCl, 150 mM NaCl, pH 7.5. Proteins were concentrated to 1–2 mg/mL in storage buffer, using centrifuge concentrator «Vivaspin turbo 15 PES» with a 10 kDa cutoff .Three-month-old male BALB/c mice were obtained from the animal care facility in ICBFM, Novosibirsk. Animals were housed under a normal light–dark cycle; water and food were provided ad libitum. All animal procedures were carried out in accordance with the recommendations for the protection of animals used for scientific purposes (EU Directive 2010/63/EU). All experiments with animals were approved by the Inter-institutional Bioethics Committee of Institute of Cytology and Genetics Siberian Branch of Russian Academy of Sciences, Novosibirsk, Russia.Mice were immunized intraperitoneally (i.p.) with 50 μg of truncated proteins per mouse, three times with an interval of 14 days between immunizations. Incomplete Freund’s adjuvant was used for the first immunization, whereas incomplete Freund’s adjuvant was used for the second and third immunizations. Each experimental group of mice included 10 animals; mice from the control group were immunized with 0.9% NaCl. Blood samples were taken from the facial vein 2 week after the final immunization.VACV or recombinant proteins were resolved by 12.5% SDS-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane . After blocking with 5% dry skim milk, the membrane was incubated with a 1\10 dilution of human or mice pooled sera in PBS, containing 0.1% Tween 20% for 1 h at 37 °C. Then, the membrane was incubated with alkaline phosphatase-conjugated anti-human IgG rabbit antibodies  or alkaline phosphatase-conjugated anti-mouse IgG rabbies antibodies . Protein–antibody interactions were visualized by incubating the membrane in a mixture of nitro blue tetrazolium  and 5-bromo-4chloro-3-indolyl-phosphate  for 20 min at room temperature. The serum of a volunteer who had never been vaccinated nor infected by orthopoxviruses and normal mouse serum were used as corresponding negative controls.For indirect ELISA, the wells of 96-well microtiter plate were independently coated with 200 ng of truncated VACV p35 proteins per well or a control antigen in PBS, pH 7.4. In the study, the trx-NS1 protein of the tick-borne encephalitis virus , produceDepletion of specific antibodies from anti-Trx-p35Δ mice sera was performed using Dynabeads m270 Epoxy  according to the manufacturer manual. For this purpose, 100 μg rp35Δ, rp35Δ3, rp35Δ6, rp35Δ12, or rp35Δfuse was covalent bound to 5 μg paramagnetic beads for 20 h at 37 °C in phosphate buffer pH 7.5 with slow tilt rotation, and after a washing step with PBS, 500 μL of 5-fold diluted serum was added and incubated for 3 h at 37 °C. Thereafter, magnetic beads were pelleted by magnetic force and depleted sera were obtained. Bound antibodies were eluted from beads using 0.1 M citrate buffer pH 3.1. Three rounds of antibody depletion were performed similarly. The absence of specific antibodies was confirmed by ELISA with the antigen being used for depletion.0 − Vn)/V0 × 100%, where V0 is the number of plaques in control wells without serum, and Vn is the number of plaques in tested wells.VACV suspension diluted in IMDM and supplemented with 10% FBS was mixed with an equal volume of serial two-fold dilutions of sera or depleted sera (starting serum dilutions were 1:10). The mixtures were incubated at 24 °C for 2 h and then added to confluent CV-1 cell monolayers in 24-well culture plates  and incubated for 1 h at 37 °C. Then, the mixtures were removed and the cells were washed with IMDM and overlaid with IMDM containing 10% FBS. Two days after infection, the viable cells were stained with 2% crystal violet with 10% formalin, and clear plaques were visualized. The normal mouse serum was used as a control of nonspecific neutralization. Neutralization titer was calculated according to N =  according to the manufacturer’s recommendations. Mutated PCR fragments were inserted into the expression plasmid pET32a and sequenced. Mutant rp35Δfuse proteins were obtained as described in 50. Differences were considered significant when the p value was less than 0.05. Data were analyzed with GraphPad Prism software, version 7. Two-tailed t tests were used to compare antibody titers in sera and PRNThttp://zhanglab.ccmb.med.umich.edu/I-TASSER/, access date 14 April 2022) and molecular coordinates for VACV p35 H3 protein (PDB 5EJ0) obtained from the Protein Data Bank [E. coli.Several recombinant VACV-derived p35 antigens for immunoassay, neutralization, and depletion analysis were produced and purified as described in ata Bank . More spata Bank . In addiata Bank . The TrxTo confirm that all recombinant p35 proteins contain epitopes that are recognized by human polyclonal antibodies, the proteins were subjected to Western blot analysis with human sera from VACV-LIVP-vaccinated volunteers. Before the experiment, anti-VACV polyclonal antibodies from vaccinated volunteers were individually tested by Western blot analysis for their binding with VACV-LIVP proteins. Six serum samples that had exhibited the most effective binding b were seFive groups of ten BALB/c mice were immunized with purified rp35Δ, rp35Δ3, rp35Δ6, rp35Δ12, and rp35Δfuse. In each group, serum samples were taken 2 weeks after the final immunization and pooled, resulting in five pooled sera. The antibody response induced by rp35Δ rp35Δ3, rp35Δ6, rp35Δ12, and rp35Δfuse was tested in indirect ELISA a. Pooled serum samples obtained from mice immunized with rp35Δ, rp35Δ6, rp35Δ12, and rp35Δfuse demonstrated positive signals in binding with the corresponding immunogen, diluted to 1:10,000 or more. The antibody response induced by rp35Δ3 was lower and anti-rp35Δ3 polyclonal antibodies revealed this antigen to be diluted to 1:1000 a. In addTo study the contribution of various epitopes in eliciting neutralizing antibodies, pooled serum samples obtained from mice immunized with rp35Δ, rp35Δ3, rp35Δ6, rp35Δ12, and rp35Δfuse were tested for their ability to neutralize VACV in PRNT. It was shown that anti-rp35Δ and anti-rp35Δ12 mice polyclonal antibodies reduced the number of plaques by more than 50% (PRNT50) after being diluted to 1:320, whereas anti-rp35Δfuse antibodies neutralized VACV at a dilution of 1:160 . Anti-rpThe pooled anti-rp35Δ mice serum sample was depleted with rp35Δ, rp35Δ3, rp35Δ6, rp35Δ12, and rp35Δfuse. Depletion was controlled by ELISA with corresponding depletion antigens; depletion was considered complete if the ELISA threshold was less than 5% residual reactivity a.The effect of the removal of distinct antibody subsets was then determined in PRNT. Pooled anti-rp35Δ mice sera after depletion with rp35Δ or rp35Δ12 almost completely lost their neutralizing activity and did not reduce the number of VACV plaques by more than 50% even after being diluted to 1:20 b. DepletE. coli BL21(DE3) cells and purified as described previously.Since rp35Δfuse is the shortest of the obtained truncated p35 variants (46 aa) that elicit anti-VACV neutralizing antibodies, a panel of rp35Δfuse mutants was developed using error-prone PCR. The obtained PCR-fragments were inserted into a pET32a plasmid and their nucleotide sequences were determined. Four unique mutant genes were identified . Mutant Purified rp35Δfuse2, rp35Δfuse4, rp35Δfuse11, and rp35Δfuse18 proteins were subjected to Western blot analysis with anti-VACV polyclonal human antibodies from the pooled sera of VACV-vaccinated volunteers. Only rp35Δfuse4 was bound by the specific anti-VACV polyclonal human antibodies similar to rp35Δfuse, whereas rp35Δfuse2, rp35Δfuse11, and rp35Δfuse18 were not recognized by them . The obtThe mutant protein rp35Δfuse4 recognized by the sera of VACV-vaccinated volunteers was used for mice immunization, as was described in VACV is a promising candidate for the development of new cancer therapies. However, its effectiveness is limited by the strong antiviral immune response induced by VACV. One possible approach to overcome this limitation is to develop low-immunogenic recombinant VACV by masking epitopes that elicit neutralizing antibodies. Here, this approach was used for the deimmunization of VACV p35, which is a major target for neutralizing antibodies in humans.3–228–239 aa) were examined. This artificial antigen elicited neutralizing antibodies in mice and bound neutralizing antibodies in anti-rp35Δ sera in depletion experiments. These characteristics in rp35Δfuse were slightly reduced as compared with these characteristics in rp35Δ and rp35Δ12, which can be explained by the structure of rp35Δfuse, which did not fully correspond to the conformation of the appropriate regions in rp35Δ. Previously, a discontinuous epitope recognized by a neutralizing human monoclonal antibody was localized on the p35 of CPXV within two p35 regions, i.e., 15–19 aa and 232–237 aa [First, the p35 region of VACV that exposes most neutralizing epitopes was determined. It was confirmed that most, or even all, neutralizing epitopes are localized between 1 and 239 aa, and they are exposed on rp35Δ12 (1–239 aa) for neutralizing polyclonal antibodies from sera similar to, or even better than, on rp35Δ (1–282 aa). Unexpectedly, the N- and C-terminal parts of rp35Δ, namely, rp35Δ3 (1–135 aa) and rp35Δ6 (129–282 aa), did not elicit strong neutralizing antibodies when being used for mice immunization. In addition, they did not deplete anti-rp35Δ neutralizing polyclonal antibodies. Considering that 3D modeling showed that rp35Δ3 and rp35Δ6 maintained their structures similar to the corresponding parts of rp35Δ A, we con2–237 aa . Our res13VIDRLPSETFPNVHEHINDQKF34 and 231DNAAKYVEH239, which are available for antibodies, as shown in the 3D structure of the VACV p35 fragment [Second, we determined which aa are involved in forming the epitope recognized by anti-VACV neutralizing antibodies. The obtained data demonstrated that aa R16, P17, P18, E20, T21, V25, and A233 are crucial and their substitutions led to the formation of epitopes unrecognizable by human anti-VACV polyclonal antibodies. The aa necessary for binding with polyclonal antibodies are localized on loops fragment . We assuTwo other substitutions, V13I and D15N, affected B-cell immunity in a more complicated way. They did not affect the recognition of the epitope containing them by anti-VACV human polyclonal antibodies; however, the mutant protein with these substitutions only elicited non-neutralizing antibodies in mice. Thus, some p35 substitutions can eliminate B-cell epitopes on the protein, whereas other substitutions can only mask epitopes from neutralizing antibodies.Our data, which were obtained by random mutagenesis, are partly consistent with those from a previous study of linear p35 epitopes, which was performed using alanine scanning investigation of epitopes . In thisIn conclusion, an epitope that elicits strong neutralizing antibodies in humans was localized on the VACV p35 surface. At least nine aa in p35 were shown to be crucial. Substitutions of seven of them prevent binding of anti-VACV human polyclonal antibodies with mutant p35, whereas substitutions of two aa result in the recognition of the mutant p35 solely by non-neutralizing antibodies. The identified aa could be useful for the development of low-immunogenic recombinant VACV.