Mus musculus, a species often used as a model for NAD-biosynthesis-related physiology and disorders, such as peripheral neuropathies. Suitable assay conditions were initially assessed by exploiting the metal-ion dependence of each isozyme recombinantly expressed in bacteria, and further tested after mixing them in vitro. The variable contributions of the three individual isozymes to total NAD synthesis in the complex mixture was calculated by measuring reaction rates under three selected assay conditions, generating three linear simultaneous equations that can be solved by a substitution matrix calculation. Final assay validation was achieved in a tissue extract by comparing the activity and expression levels of individual isozymes, considering their distinctive catalytic efficiencies. Furthermore, considering the key role played by NMNAT activity in preserving axon integrity and physiological function, this assay procedure was applied to both liver and brain extracts from wild-type and Wallerian degeneration slow (WldS) mouse. WldS is a spontaneous mutation causing overexpression of NMNAT1 as a fusion protein, which protects injured axons through a gain-of-function. The results validate our method as a reliable determination of the contributions of the three isozymes to cellular NAD synthesis in different organelles and tissues, and in mutant animals such as WldS.A novel assay procedure has been developed to allow simultaneous activity discrimination in crude tissue extracts of the three known mammalian nicotinamide mononucleotide adenylyltransferase  isozymes. These enzymes catalyse the same key reaction for NAD biosynthesis in different cellular compartments. The present method has been optimized for NMNAT isozymes derived from  Eukaryotic NMNATs transfer adenylate from ATP to nicotinamide mononucleotide or nicotinic acid mononucleotide, generating NAD or deamido-NAD, respectively, and PPi . The reain silicoThis highly conserved enzyme Each mammalian NMNAT isozyme has a characteristic divalent cation dependence, affinity, and specificity The occurrence in mammalian cells of multiple, compartmentalized NMNAT isozymes strongly suggests that NAD biosynthesis is differentially regulated in different subcellular compartments S) mutation S expression markedly delays the axonal degeneration after mechanical or chemical injuries, and also ameliorates several acute or chronic neuropathies morphologically converging into progressive axon degeneration S compensates for loss of the major endogenous axonal isozyme, NMNAT2, a labile protein that is essential for axon survival S represents an extra NMNAT isoform aberrantly present within the axoplasm of the mutant strain, allowing protection through a gain-of-function whose details are beginning to emerge . An important limitation for research on this and other significant topics relating to NMNAT has been the lack of methods to distinguish the effects of individual isozyme activities, especially in the absence of highly selective inhibitors. Most conclusions have been inferred from RNA and protein quantification experiments, but these are intrinsically unable to evaluate post-translational regulation and often limited by the low quality of currently available antibodies. Thus, a discrimination assay for mouse isozymes would fill an important gap.One important example is the role of NMNAT in axon survival and neuroprotection S acts, a novel assay has been developed and validated for simultaneous activity determination of all known NMNAT isozymes. Its application to both brain and liver extracts from WldS mice illustrates the potential for studying isoform-specific biochemistry in mouse tissues, thus facilitating future studies of how NMNAT isozymes respond individually to disease, injury or ageing.To allow direct estimates of the three endogenous NMNAT isozyme activities in mouse tissues, as well as of the NAD-biosynthetic machinery where WldProcedures using live animals were authorised under Project Licence 80/2254 that was approved both by the Babraham Research Campus Animal Welfare, Experimentation and Ethics Committee (AWEEC) and the UK Home Office. Animals were humanely killed using approved (Schedule 1) methods.E. coli strains TOP10F’ and BL21(D3), SuperScript™ II Reverse Transcriptase, and Platinum® SYBR® Green qPCR SuperMix UDG were from Invitrogen. Protein Assay kit and Taq-polymerase iProofTH High-Fidelity were from Bio-Rad. The pET28 expression vectors were from Novagen. Other chemicals were from Sigma.Ni-NTA Superflow and TALON® metal affinity resins were purchased from Qiagen and Clonetech, respectively. PD-10 Sephadex™ columns were from GE Healthcare. S mouse (C57BL/WldS) were obtained from a breeding colony purchased from Harlan Laboratories (UK). Collected tissues were immediately snap-frozen in liquid nitrogen and stored at −80°C until the time of processing. Frozen mouse tissues were dipped into liquid nitrogen and ground by mortar and pestle to a fine powder. Thereafter, weighed tissue aliquots were extracted with perchloric acid for HPLC determination of endogenous NAD levels Livers and brains from wild-type (C57BL/6) and mutant WldMus musculus NMNAT (mNMNAT) isoforms 1 , 2 , 3 , and WldS , were amplified by standard high-fidelity PCR from commercial plasmids. The primers used, carrying restriction overhangs for directional cloning into the polylinker region of pET28 vectors, are listed in supplemental NdeI/EcoRI of pET28c for mNMNAT1, at NdeI/HindIII of pET28c for both mNMNAT2 and mNMNAT3, and at NheI/XhoI of pET28b for WldS. Owing to this cloning strategy, the recombinant proteins were all fused to N-terminal His-Tag tails, i.e. MGSSHHHHHHSSGLVPRGSH for the three mNMNATs, and MGSSHHHHHHSSGLVPRGSHMAS for WldS. The resulting plasmid constructs were replicated into E. coli TOP10F’, verified by sequencing for their exact match with database deposited sequences, and individually transformed into E. coli BL21(D3) for protein expression. After transformation, single colonies from kanamycin-selective plates were inoculated in 10 mL Luria-Bertani medium supplemented with 50 mg/L kanamycin, and grown at 37°C overnight under rotary shacking (200 rpm). About 5 mL of each pre-culture was inoculated in 250 mL fresh medium without antibiotic, and grown as before but at the temperature of 28°C to prevent or minimize inclusion bodies formation. At middle exponential phase , 1 mM isopropylthio-β-galactoside was added to each culture and the induction was prolonged for additional 3 h at 28°C. Cells were finally collected by mild centrifugation, washed twice with PBS, and stored at −80°C.Full-length open reading frames encoding S recombinant species, were lysed by French Press at 18,000 psi after resuspension in 10–15 mL lysis buffer composed by 50 mM Na-phosphate, pH 7.0, 300 mM NaCl, 5 mM 3-(3-cholamidopropyl)dimethylammonium-2-hydroxy-1-propanesulfonate (CHAPSO) (for mNMNAT1 and mNMNAT2), or 50 mM HEPES/KOH, pH 7.5, 500 mM NaCl, 5 mM CHAPSO (for mNMNAT3 and WldS), in either case freshly supplemented with 1 mM PMSF, 1 mM tris(2-carboxyethyl)phosphine (TCEP), and 0.05 mL/g cell pellet of protease inhibitor cocktail (Sigma #P8465). After brief incubation in the presence of lysozyme (1 mg/mL) and DNAse (10 µg/mL), the protein suspensions were clarified by centrifugation at 20,000×g for 30 min. Thereafter, the His-tagged mNMNAT3 and WldS species were purified by Ni-NTA affinity chromatography, carried out onto pre-packed columns (0.5–1 mL resin) equilibrated with 50 mM HEPES/KOH, pH 7.5, 500 mM NaCl, 1 mM TCEP, 1 mM PMSF. The washing and elution steps were carried out using 20 mM and 200 mM imidazole, respectively. Instead, the His-tagged mNMNAT1 and mNMNAT2 species were purified by TALON affinity chromatography, performed in 50 mM Na-phosphate buffer, pH 7.0, 300 mM NaCl, 1 mM TCEP, 1 mM PMSF. In this case, the washing and elution steps were carried out using 20 mM and 150 mM imidazole, respectively. Collected fractions from each chromatography were analysed by SDS-PAGE and assayed for their total NMNAT activity (see below). Active and homogeneous fractions were pooled and quickly desalted on PD-10 columns against 50 mM HEPES/KOH buffer, pH 7.5, 20% glycerol, 1 mM TCEP. Enzyme stability in solution was checked after storage in aliquots at either 4°C or −20°C. Before their use in discrimination assays, thawed samples were treated with Chelex-100 resin as above described (see Tissue extract preparation).All purification steps were performed at 4°C. Harvested bacterial cells expressing either mNMNAT1, or mNMNAT2, or mNMNAT3, or Wldi.e. NAD or its analogs . Rates were calculated as tangent lines in the linear region of plots of product accumulation versus time. Kinetic parameters were measured as described Activity rates were measured by a C18-HPLC method 2, 20 mM NaF, 1 mM DDT, 1 mM both NMN and ATP, and either ∼1 mg/mL protein tissue extract or 0.15–2.5 µg/mL each pure recombinant isozyme. The above assay, referred as “A”, was used to measure total NMNAT activity, i.e. the “reference” activity value for all different mNMNAT isoforms. For isozyme discrimination, the additional mixtures “B”, “C”, and “D” were set replacing 25 mM MgCl2 with either 50 µM MgCl2 (B) or 1.5 mM ZnCl2 (C) or 4 mM CoCl2 (D). The assays under condition “D” were carried out in the absence of DTT. Suitable parameters for subsequent calculation were obtained by parallel assaying of each recombinant isozyme under the four conditions above. Then, reaction rates obtained from “B”, “C”, and “D” were divided each by the reference activity value obtained from “A”. The resulting reaction rate ratios, i.e. the coefficients b1, b2, b3; c1, c2, c3; d1, d2, d3, were substituted in the following system of three linear equations:The assays discriminating for individual isozyme activity (discrimination assays) are based on isozyme-selective metal ion dependence. The reference assay mixture  contained 30 mM HEPES/KOH, pH 7.5, 0.6 mg/mL BSA, 25 mM MgClB”) = (b1·X)+(b2·Y)+(b3·Z).Tissue NMNAT activity (condition assay “C”) = (c1·X)+(c2·Y)+(c3·Z).Tissue NMNAT activity (condition assay “D”) = (d1·X)+(d2·Y)+(d3·Z).Tissue NMNAT activity  according to the manufacturer’s instructions. First strand cDNA synthesis was performed using SuperScript™ II Reverse Transcriptase in the presence of 1 µg total RNA and oligo(d)T primer. Quantitative real time PCR was performed using Platinum® SYBR® Green qPCR SuperMix UDG and the primer pairs described in supplemental i.e. ∼80% for isoforms 1 and 3, and as high as 99% for isoform 2, with only four substitutions out of 307 total residues (not shown). After bacterial expression and one-step affinity chromatography, the different purifications yielded similar results and recovery values. Typically starting from 250 mL induced bacterial cultures, 50–80 mg protein and 0.1–0.4 U/mg total NMNAT activity were obtained in the soluble crude bacterial extract, while 0.5–2 mg protein with ∼80% activity yield were recovered in the purified enzyme preparations. Under the reference assay condition “A”  for both NMN and ATP substrates were calculated from nonlinear fitting on primary plots. As summarized in kcat values comparable to the corresponding human isozymes. The catalytic efficiencies were also similar, with the order mNMNAT1> mNMNAT2> mNMNAT3. The kinetic behaviour of the WldS chimera was remarkably superimposable to that of mNMNAT1, indicating that the N-terminal sequence of WldS mutation  and 1.5 mM Zn2+ (C”), are similar to those adopted in the previous human discrimination assay 2+ (D”) was selected because it supports a very high activity for isozyme 3 and, to a lesser extent, for isozyme 1. Furthermore, when Co2+ was used as cofactor, DTT was omitted because it interfered with the activity of all three mNMNATs leading to reaction rate reduction (not shown). In these assays, metal-free enzyme preparations obtained by Chelex treatment were used  provides a fixed reference point, and the reaction rate ratio relative to this value is obtained for each mouse isozyme using pure recombinant proteins under conditions “B”, “C”, and “D”. This indicates the nine coefficients reflecting the activity ratio exerted at each condition by each isozyme . Interestingly, total activity was almost entirely accounted for by mNMNAT1 and mNMNAT2 alone  and mNMNAT2 (50%) provided most activity in brain, while in liver mNMNAT1 (67%) and mNMNAT3 (30%) were most active . In eachS mutant mice, which constitutively express the axon-protective protein chimera WldS, endowed with NMNAT1 activity S contributions, was found to be 3–6 times higher than in the corresponding wild type tissues . Furthermore, as expected, no statistical alteration on NAD tissue content was observed in WldS mutant with respect to wild-type .Parallel activity discrimination analyses were also carried out on brain and liver extracts from C57BL/Wld tissues , while nin vitro by using recombinant isozymes, revealed properties broadly similar to their human counterparts, consistent with the structural similarities, as also recently reported by others for mNMNAT2 2+ ions. WldS and mNMNAT1 were catalytically and kinetically indistinguishable.In this work, a novel method for simultaneous activity discrimination of NMNAT isozymes in complex mixtures, like mouse tissue extracts, has been developed to study the compartmentalized cellular NAD biosynthesis and its role in physiological and pathological processes. The preliminary kinetic and catalytic characterization of mNMNATs, carried out The discrimination assay method here described exploits the distinctive metal ion dependence displayed by individual mNMNATs, and introduces for the first time a matrix substitution calculation to obtain simultaneously the unknown activity values of the three isozymes. After assessing the reliability of the method using reconstituted mixtures of pure recombinant isozymes, the procedure was tailored for assaying NMNAT isozymes in tissue extracts from mouse. As this species is frequently used to model human conditions, the ability to discriminate differential contributions to NAD synthesis in physio-pathological conditions is particularly useful.e.g. by differential centrifugation, would be not suitable for quantitative measurements essentially due to 1) cross-contamination of fractionated particles, 2) not exclusive compartmentation of isozymes, which can be present in more than one subcellular district e.g., NMNAT2 Main advantage of this method is that it allows quantitative determination of the activity of the isozymes simultaneously present in a given sample, without their prior physical separation. Isozymes separation, such as one relying on their subcellular compartmentation, A fundamental requirement of this method is the preliminary selection of appropriate assay conditions for discrimination, encompassing biochemical, analytical, and mathematical features. Biochemical features refer to the selection of assay mixtures leading to consistently dissimilar isozyme reaction rate values. Analytical requirements means that actual rate values should be relatively high, in order to minimize the intrinsic experimental error. The mathematical conditions dictate that the determinant of the matrix employed is non-zero. Our selected conditions depicted in e.g. by replacing HPLC with high-throughput NAD quantitation based on microplate readers and fluorescence cycling assays. Finally of course, it could be extended to mammalian systems other than human and mouse, and to any other enzyme existing in multiple isoforms (even four or more). The only requirement is to establish a number of assay conditions that differentially favour the various isoforms, yielding an appropriate number of simultaneous equations for matrix calculation.Compared to the previously described discrimination assay Efforts to elucidate the role played by the three mammalian NMNAT isozymes in NAD biosynthesis have included a number of expression and immunological studies on their tissue distribution and cellular localization S tissues by the present discrimination assay revealed that the expected increase in total NMNAT activity in WldSS species. The activity of the two other isozymes was unchanged in mutant tissues with respect to wild-type, thus ruling out the occurrence of compensatory changes in mNMNAT2 and mNMNAT3 expression. This is consistent with independent, non-redundant functions of these extra-nuclear NMNAT isozymes. Interestingly, in the opposite case of ∼50% NMNAT1 activity reduction, as observed in whole brain from heterozygous NMNAT1 null mice, this method showed that compensatory changes were similarly absent S are not discriminated by our assay method, as we found no assay conditions leading to consistently dissimilar reaction rates. Nonetheless, their respective individual contribution could be determined by comparing isoform 1 individual activity in wild-type and WldS tissues. Alternatively, they could be likewise determined by comparing overall NMNAT activity in wild-type and WldS tissues, once unchanged mNMNAT2 and mNMNAT3 contributions have been assessed by means of the discrimination assay. In both cases, effective profiling of all three endogenous mNMNATs and WldS enzyme activities could be achieved in WldS mutant tissues. As an example, from data in S contribution to total NAD synthesis can be estimated to account for ∼0.4 mU/mg in brain, and ∼0.55 mU/mg in liver. Though partly indirect, this evidence is only made possible by the application of the present discrimination assay.Furthermore, comparison of wild-type and WldS. Additional preliminary experiments suggest this assay can also be applied to mouse nerve tissue extracts (unpublished). The ability to analyse individual isozyme fluctuations in a number of physiological and pathological conditions will be important for understanding the mechanism by which localized NMNAT catalysis specifically contributes to axon viability, as well as in other NAD-related conditions such as ageing and metabolic disorders.In conclusion, we have developed a novel method of wide applicability to discriminate the individual activity of mammalian NMNAT isozymes. Its preliminary application validated the study of NAD biosynthesis in mouse tissues as a model for human diseases. In particular, the method enables assessment in mouse brain and liver of all known NMNAT reaction-catalysing isozymes, including mNMNAT1, mNMNAT2, mNMNAT3, and WldTable S1Synthetic oligonucleotides used in this study.(DOCX)Click here for additional data file.Table S2Composition of a typical set of discrimination assay mixtures.(DOCX)Click here for additional data file.Methods S1Example of simultaneous single-sample determination of NMNAT isozyme activities in mouse tissues.(DOCX)Click here for additional data file.Methods S2MS Excel spreadsheet used for matrix calculation.(XLSX)Click here for additional data file.


Pharmaceutical Affairs Act was enacted in Japan, self-medication practices have increased. Because the concept of self-medication was recently introduced in Japan, few studies exist on this topic. Therefore, it is necessary to explore how self-medication is practiced. This study examined Japanese consumers’ self-medication practices and attitudes toward over-the-counter (OTC) medicines based on their sex, age, and socioeconomic status (SES).Since 2009, when the revised Mage = 41.1 years, SD = 16.22). A quota sampling method was employed based on age group, and participants completed an online questionnaire.The participants were 403 adults  medicines when sick. They also placed more value on national brand OTC medicines than private brands (likely due to advertisements). This finding suggests individuals with a low SES do not select OTC medicines based on their effects or ingredients. Regarding attitudes toward OTC medicines, Japanese participants seemed to be unaware of the potential for abuse and side effects associated with OTC medicines. Finally, in relation to caregivers’ self-medication practices for their children, the majority of participants reported taking their children to the hospital since children tend to receive free medical care. Furthermore, caregivers with a high educational background are more confident in being able to help manage their children’s health.Our results suggest that health and medical discrepancies among Japanese consumers pose new social problems. In Japan, universal health care is available, but the cost of receiving medical care is not completely free of charge. Thus, we hope that the government will attempt to meet the various needs of patients and support their well-being. Consumers also have to be more independent and aware of their health management, as self-medication practices will continue to play a more significant role in healthcare. More research is needed to find ways to teach Japanese consumers/patients of both the benefits and risks of over-the-counter (OTC) medicines. Self-medication, which is a common self-care practice, is widespread around the world . Self-meSelf-care choices vary according to sex, age, and socioeconomic status (SES). For example, in Ireland, females report buying OTC medicines more often than do males, and younger adults report buying OTC medicines more often than older adults do . With reWhen discussing self-medication issues, the topic of self-medication for children is noteworthy. Research on this topic has been accumulating within Western countries for over a decade. In the U.S., 70% of illnesses among preschoolers were treated with non-prescription medicines . SimilarPharmaceutical Affairs Act was only recently enacted in 2009; this introduced the concept of self-medication. In 2008, a group of Japanese researchers asked approximately 1,000 adults if they knew the term “self-medication” and 60.5% did not recognize this term [In Japan, the revised his term . HoweverTherefore, the goals of the present study were to examine the following: (1) how Japanese consumers/patients practice self-medication to manage their health problems, (2) how Japanese consumers/patients select particular OTC medicines, (3) how consumers/patients’ attitudes toward OTC medicines, and (4) caregiver-initiated medication behaviors for children. This study also investigated the role of sex, age, and socioeconomic status (SES) on the preceding research questions.Mage = 41.12, SD = 16.19, range 20–79, male = 47.4%, female = 52.6%). The research company, Media Interactive Inc., recruited participants in 2011. Among the 403 participants, 331 had children.Participants were 403 adults living in the Kanto area of Japan, which consists of seven prefectures including Tokyo (A quota sampling method was employed to compare the following three age groups: a young group (20–29), middle age group (30–49) and a senior group (65 years old and above). There were an approximately equal number of male and female participants within each stratum Table . These tAll of the survey questions were uploaded online. The participants who agreed to complete the survey accessed it through a provided URL. After filling out the online survey, Media Interactive Inc. gave the participants points that could be accumulated in exchange for a gift card.Participants answered 43 questions, including information about demographics. The survey inquired about self-medication behaviors  and the reasons for their choice, the choice of OTC medicines, and attitudes toward OTC medicines .Self-medication behaviors were measured by the question: “When you have a high fever (39°C/102.2°F or higher), what do you do?” Response choices were (1) I will see a doctor and treat with prescribed drugs, (2) I will treat with OTC medicines, (3) I will rest for a while, (4) I will treat with an alternative method such as acupuncture and reflexology, or (5) Other. Next, participants were asked the reason for their choice . For their children, the question was, “When your child has a high fever (39°C/102.2°F or higher), what do you do?”The choice of OTC medicines was measured by the question: “Which OTC medicines do you want to buy when you treat your illness?” Response choices were: (A) a national brand OTC medicine often seen in a TV advertisement or (B) a private brand OTC medicine that has the same effects and ingredients but is 50% cheaper than the national brand. National brand OTC medicines refer to medicines produced by a major pharmaceutical company . On the other hand, private brand OTC medicines refer to medicines that are similar to a national brand OTC medicine but are sold at specific retailers . Examples of OTC medicines in Japan are aspirin and cough and cold medicines; antibiotics are not included.In terms of reasons for selecting a national brand OTC medicine, response choices were (1) I feel safe because I often see the item on a TV advertisement, (2) I feel safe because a well-known pharmaceutical company produces it, (3) I feel safe because I have always taken the same medicine, (4) Cheap medicines make me feel anxious, (5) Unknown pharmaceutical companies make me feel anxious, (6) More expensive medicines seem to work better, (7) I like the package design, or (8) Other. Response choices for selecting a private brand OTC medicine were (1) It is cheaper, (2) Ingredients and effectiveness will be the same as the national brand OTC medicine, (3) There will not be a big difference among OTC medicines, (4) It seems better than the national brand sold by a major pharmaceutical company, (5) I like the package design, or (6) Other.Several questionnaires developed by Bradley et al.,  TrajanovSix questions from Maiman, Becker, Cummings, Drachman, and O’Connor  were useSES was measured by annual income in Japanese yen. Possible responses were (1) less than 300 K, (2) 301–400 K, (3) 401–500 K, (4) 501–600 K, (5) 601–800 K, (6) 801–1000 K, (7) 1001–1500 K, or (8) 1501 K and above.χ2) analyses were conducted to examine the presence of any differences in self-medication behaviors and the choice of OTC medicines according to sex, age, and SES. Phi coefficients (Φ) were calculated as measures of effect size. Phi values of 0.1, 0.3, and 0.5 may be interpreted as indicating a small, medium, and large effect between groups, respectively [Various chi-square  I will see a doctor and treat with prescribed drugs.” When asked to explain their behavior, one-third of this group reported, “We are not health care professionals; thus, we should not make our own judgments.” The participants who chose, “I will treat the problem with OTC medicines” comprised 18% of the total group. When asked to explain their behavior, 20.3% of this group reported, “I want to treat the problem by myself,” and 19.6% reported, “First, I want to see if OTC medicines work for me.” For a summary of responses, see Tables However, when a child has a high fever, more than 90% of the caregivers would take their child to the doctor. The most common response was “We are not health care professionals; thus, we should not make our own judgments (63%).” The second most common response was “Medical costs are free for children” (12.6%).With the regard to the question, “Which OTC medicines do you want to buy when you treat your illness,” 51.4% chose (A) a national brand OTC medicine, and 48.6% chose (B) a private brand OTC medicine. Logistic regression was performed in the selection of either a national brand or a private brand; however, none of the independent variables  showed significant group differences. Among respondents who chose a national brand, 13.5% reported “I feel safe because I often see the item on TV advertisements,” 27.0% reported “I feel safe because a well-known pharmaceutical company produces it,” 45.9% reported “I feel safe because I have always taken the same medicine,” and 13.5% reported “Unknown pharmaceutical companies make me feel anxious.” In contrast, when respondents who chose private brands were asked to explain their behaviors, about 50% reported “Ingredients and effectiveness will be the same as national brand OTC medicines,” and the other 50% reported “It is cheaper.” The comparison could not be made for children because only a few caregivers reported that they would use OTC medicines to treat their children’s high fever.χ2 (3) = 2.18, p = 0.53, n.s. As for the choice of OTC medicines, no sex difference was found. The results suggest that participants’ self-medication behaviors and their choice of OTC medicines are not affected by their sex. This is partially consistent with findings reported by Hanibuchi [A 2 (sex) × 5 (self-medication behavior choices) chi-square analysis did not reveal statistically significant differences, anibuchi , where nχ2 (6) = 11.06, p = 0.08, n.s. A 3 (age group) × 2 (the choice of OTC medicines) chi-square analysis also did not reveal statistically significant differences, χ2 (2) = 0.88, p = 0.64, n.s. The results suggest that age group does not affect participants’ self-medication behaviors and choice of OTC medicines.A 3 (age group) × 5 (self-medication behavior choices) chi-square analysis did not reveal statistically significant differences, χ2 (20) = 49.80, p < 0.01, Φ = .60. The phi coefficient indicated a moderate effect size. In examining the standardized residuals for each cell within the chi-square analysis, a significantly larger number of younger participants chose “(I am not going to see a doctor because) it costs money” as compared to the other two age groups. In addition, a significantly large number of senior participants chose “(I am not going to see a doctor because) there are no hospitals nearby” as compared to the other two age groups. In sum, younger adults are less likely to see a doctor because of financial constraints, and transportation issues can mitigate older adults’ ability/choice in seeing a doctor.Subsequently, a 3 (age group) × 5 (reasons for not seeing a doctor) chi-square analysis revealed statistically significant age differences, χ2 (21) = 21.89, p = 0.40, n.s.A 6 (SES group) × 5 (self-medication behavior choices) chi-square analysis did not reveal statistically significant differences overall, However, in examining the standardized residuals for each cell within the chi-square analysis, results indicated that a significantly larger number of people in the “less than 300 K” group fell within the “will rest and see for a while” category as compared to the rest of the SES groups.χ2 (6) = 8.70, p = 0.19, n.s), a 6 (SES groups) × 5 (reason to pick private brand OTC medicines) chi-square analysis (χ2 (12) = 6.51, p = 0.88, n.s.), and a 6 (SES groups) × 5  chi-square analysis (χ2 (18) = 21.94, p = 0.23, n.s) did not reveal statistically significant differences. However, analyses of standardized residuals revealed that participants in the group whose annual income was less than 300 K were significantly more likely to fall into the “I feel safe because I often see the item on TV” and “Unknown pharmaceutical companies make me feel anxious” categories than the rest of the SES groups. In sum, people at lower SES levels are more likely to be influenced by advertisements when selecting OTC medicines. This is partially consistent with a U.S. study, which found that caregivers with a lower annual income  are more likely to rely on packaging than caregivers who have a higher annual income  [As for the choice of OTC medicines, a 6 (SES groups) × 2 (the choice of OTC medicines) chi-square analysis  were satisfied with the medicine they give to their children. Whereas 78.7% of the caregivers answered, “When I give child an OTC medicine, I am as careful as I am when giving prescribed medicines,” 23.1% reported “I sometimes give a child OTCs in addition to those prescribed by a doctor for a problem”; thus, the results are somewhat contradictory.χ2(15) = 27.43, p < 0.05. In examining the standardized residuals for each cell within the chi-square analysis, caregivers who held a master’s degree or above were more likely to answer “very true” for “I usually trust my own opinions about my child’s health more than a doctor’s opinion.” In other words, caregivers with a higher educational background were more confident in managing their children’s health.Caregiver-initiated medication behaviors for children were also analyzed based on caregivers’ age, SES, and educational level. Only education was a significant factor in a chi-square analysis: The goals of the present study were to examine the following: (1) how Japanese consumers/patients practice self-medication to manage their health problems and the reasons behind these practices, (2) how Japanese consumers/patients select particular OTC medicines, (3) how caregivers practice self-medication for their children, and (4) caregivers’ attitudes toward self-medication for their children. This study also investigated differences in self-medication management and attitudes across sex, age, and socioeconomic status (SES).No significant sex differences were observed, but interesting age differences emerged; younger adults were less likely to see a doctor because of medical costs, and elderly adults were less likely to see a doctor because they lacked transportation. The younger-adult results partially support previous research that found that about 50% of younger adults (20s − 30s) were very anxious about their future medical expenses .With regard to SES, participants within lower income levels were less likely to see a doctor or buy OTC medicines for their health problems. This result is also consistent with previous research showing that the number of individuals who do not see a doctor when they are sick because of costs more than doubles for individuals in lower SES groups as compared to those in higher SES groups . HoweverWhen assessing reasons why participants chose either national or private brand OTC medicines, results indicated that people in the lower SES groups placed more value on national brands than private brands. This was because they often saw national brands advertised on TV. These results suggest that OTC medicine advertisements have a powerful affect on perceptions among low SES consumers/patients. However, the influence of TV advertisements on public perceptions of OTC medicines has not been well researched. Advertisements on TV convey both intentional and unintentional messages to their audience, and consumers/patients should not rely solely on information garnered from such advertisements when choosing a product. Even though private brand OTC medicines are cheaper and have the same effects and ingredients as national brands, people among low SES groups prefer national brands; this might be because unknown pharmaceutical companies evoke some anxiety. In other words, low SES consumers might not choose OTC medicines based on relevant criteria, such as evidence of their effects or their ingredients. Even though major pharmaceutical companies produce some OTC medicines, consumers/patients still need to be aware of their potential adverse effects and use them as directed. Consumers/patients should consult a pharmacist to obtain more information, especially since pharmacists are easier to access than are doctors. A study in Ireland found that a recommendation by a pharmacist most frequently influenced consumer/patients’ choice of OTC medicines . As selfPharmaceutical Affairs Act was recently revised and enacted in Japan, it is possible that Japanese consumers/patients are not highly aware of the risks associated with OTC medicines compared to Europeans. In fact, Kawase et al. [Our study also observed that Japanese participants were less aware of the potential for abuse and side effects associated with OTC medicines when compared to participants in a previous study conducted in Ireland . Since te et al.  found thFinally, caregivers with higher educational backgrounds are more confident in managing the health of their children. As Du and Knopf  argue, “Our results suggest that individuals within different age groups and SES levels have different medical needs. This is noteworthy because dramatic changes in the Japanese social structure have occurred over the past few decades. The number of senior citizens is rapidly increasing while the birth rate remains low. In 2008, the birth rate was 1.37, a number that has not changed significantly over the past 10 years . It is eOur results also suggest that more research is needed in order to find a way to teach both the benefits and risks of OTC medicines to Japanese consumers/patients. Along with public education, pharmaceutical companies can also improve the methods they use to deliver medical information. Raynor et al.  argue thFinally, one limitation of this study is that it was cross-sectional in nature, which makes it difficult to infer causal relationships. Therefore, the generalizability of our findings is limited, and future research is required. Nevertheless, the present study brings a unique perspective to the literature by addressing differences in self-medication practices and attitudes according to sex, age, and SES among Japanese consumers/patients and caregivers.The authors declare that they have no competing interests.IA carried out data collection, participated in the design of the study, and performed the statistical analysis. SK and HH participated in the study design and helped draft the manuscript. All authors read and approved the final manuscript.


Saccharomyces cerevisiae and Schizosaccharomyces pombe, two highly divergent ascomycete yeast models. While the global architecture of the mating response is very similar between these two species, they differ significantly both in their mating physiologies and in the molecular connections between pheromone perception and downstream responses. The use of both yeast models helps enlighten both conserved solutions and species-specific adaptations to a general biological problem.Many cells are able to orient themselves in a non-uniform environment by responding to localized cues. This leads to a polarized cellular response, where the cell can either grow or move towards the cue source. Fungal haploid cells secrete pheromones to signal mating, and respond by growing a mating projection towards a potential mate. Upon contact of the two partner cells, these fuse to form a diploid zygote. In this review, we present our current knowledge on the processes of mating signalling, pheromone-dependent polarized growth and cell fusion in  Many cell types exhibit chemotaxis or chemotropism in response to external signals, which are essential for functions as diverse as neuronal pathfinding, wound healing or pathogenesis. Unicellular yeast models are potent systems to understand the molecular interactions that generate cell polarity induced by external inputs. Indeed, yeast cells exhibit chemotropism in response to pheromones produced by partner cells during the mating process. Pheromones are recognized by specific receptors expressed on the surface of cells of the opposite mating type and this binding stimulates the activation of receptor-associated heterotrimeric G-proteins, which in turn promote the activation of a conserved mitogen-activated protein kinase (MAPK) module. By ultimately activating a specific transcription factor, MAPK cascade components modulate the expression of mating-specific genes, thus promoting cell cycle arrest, polarized morphogenesis in the direction of the partner cell (a process known as shmooing), cell–cell fusion and karyogamy to produce a diploid zygote .Figure Schizosaccharomyces pombe and Saccharomyces cerevisiae. It should be highlighted here that these two yeasts are in fact highly divergent, with an evolutionary distance estimated at close to 1 Gyr [The aim of this review is to give an overview of the mating process of the two distantly related ascomycete ‘cousins’, the yeast models to 1 Gyr . We will3.At first glance the overall process of mating appears quite similar in the two yeast models. Indeed, in both cases peptide pheromones are recognized by G-protein coupled receptors expressed on the cell surface. The receptors have a conserved structure with seven transmembrane domains, a cytoplasmic C-terminal tail mediating desensitization and pheromone-induced internalization, and an intracellular loop involved in G-protein binding. Moreover, in both cases the signal is transmitted by MAPKs to a transcription factor that activates the expression of mating-specific genes. However, a more detailed analysis reveals many differences between the two species, which is perhaps not surprising given their long evolutionary distance.3.1.S. cerevisae over the last 30 years. At the physiological level, budding yeast cells mate spontaneously on rich medium when in the presence of cells of the opposite mating type, forming stable diploids, which sporulate upon starvation (b). Pheromones  are captured by the receptors Ste3 and Ste2 , which activate the same Gαβγ heterotrimeric G-protein. Pheromone binding stimulates GDP to GTP exchange on the Gα subunit (Gpa1), which allows the released Gβγ (Ste4 and Ste18) heterodimer to activate mating signalling [a). In particular, Gβ directly interacts with key effectors: in the presence of pheromones, Gβ binds to the p21-activated kinase (PAK)-like kinase Ste20 [a).The mating process has been extensively studied in arvation b. Pheromse Ste20 , the Cdcse Ste20 –8 and thse Ste20  (figure a). After pheromone stimulus, the Ste5 scaffold is rapidly translocated to the plasma membrane by Gβγ [The central hub for mating signalling is Ste5. This scaffold protein serves to link the Gβ with the PAK kinase Ste20 and the MAPK module, and has an essential role in promoting MAPK cascade activation ,11. The figure 2e by Gβγ ,14, where by Gβγ . Ste5 mee by Gβγ ,17. Ste5e by Gβγ  and actse by Gβγ . Membrane by Gβγ . Once ace by Gβγ ,23. Actie by Gβγ ,25 and te by Gβγ  to promoe by Gβγ , and thee by Gβγ .3.2.MATa cells containing the same number of pheromone-producing and non-pheromone-producing MATα partners, MATa cells are able to discriminate between the two categories, and mate almost exclusively with pheromone-producing partners [MATa cells, helps these cells avoid each other [MATa cells orient growth towards the source of an artificial pheromone gradient [Budding yeast cells are exquisitely able to project a shmoo towards the source of a pheromone gradient, allowing them to grow towards a potential mating partner. Early important experiments showed that, in mating mixtures of partners . Neverthpartners , where tpartners . In addich other ,33. Simpgradient –37. Yeasgradient ,37. Thisgradient .a). During mating, Cdc42 regulates the PAK kinase Ste20 localization to the plasma membrane and its activation; indeed active Cdc42 (Cdc42-GTP) binds Ste20 and stimulates its kinase activity [in vivo this interaction depends on the adaptor protein Far1 [The main regulator of cell polarization is the small GTPase Cdc42. Its role in symmetry breaking to define the site of bud emergence during mitotic growth has been extensively studied –41. Cdc4figure 2activity . Consistactivity ,47. Likeactivity ,8. Howevein Far1 ,48 and ifar1 does not affect the ability of cells to shmoo per se, but impairs the displacement of polarity factors from the site of bud emergence, thus leading to the formation of a mislocalized shmoo at the bud site. Consistently, mutations that prevent the formation of a Cdc24–Far1–Gβγ complex prevent the correct orientation of shmoos towards a pheromone source [Far1, a scaffold structurally similar to Ste5 ,49,50, he source ,9,48. Ine source , to recre source . Active fus3 mRNA, thus ensuring its subsequent translation and enrichment at the shmoo site [The Cdc24–Far1–Gβγ complex is not the only molecular connection between pheromone receptors and the polarization machinery. Gα also has a positive role in promoting chemotropism in budding yeast. Indeed, Gα directly interacts with active (phosphorylated) Fus3 MAPK, thus promoting its recruitment to the shmoo site . Gα alsomoo site . This remoo site . Consistmoo site  and alsomoo site . In turnmoo site , as wellmoo site . In sum,3.3.S. cerevisiae and S. pombe, which we will describe below, these organisms exhibit major differences. The first lies in their distinct physiologies for sexual differentiation: while S. cerevisiae mates spontaneously and forms stable diploids, sexual differentiation in S. pombe is triggered by starvation, and the diploid cells formed are unstable, ensuring a strict coupling between mating and sporulation . Upon nitrogen starvation, the transcription factor Ste11 (not to be confused with its S. cerevisiae homonym) is activated in three different ways [ste11 expression during vegetative growth [ste11 expression [In fission yeast, sexual differentiation is triggered by starvation when compatible mating partners are present. This leads to arrest in G1 phase of the cell cycle, mating-type-specific pheromones and pheromone receptor production, polarized growth in the direction of pheromone source, fusion of mating partners, karyogamy, meiosis and formation of resistant spores  figure a. Upon nent ways . First, e growth ,65; secopression ; and finpression . Ste11 apression . Notablypression . As Ste1pression ,69.+h and –h cells, respectively) are bound by the receptors Mam2 and Map3 , which are presumably coupled to the same components of a still incomplete heterotrimeric G-protein. Here, the Gα protein Gpa1 is responsible for the activation of the MAPK pathway [b). It is, however, unknown whether there exists a Gβγ dimer that negatively regulates Gα: a putative Gβ subunit, Gnr1, interacts with Gpa1 in a two-hybrid assay and may inhibit Gα-mediated signalling [S. cerevisiae, ‘kelch repeat’ proteins were shown to mimic Gβ subunits and to inhibit the Gα protein Gpa2, which regulates invasive growth response and filamentous differentiation in the absence of Gγ [Kluyveromyces lactis Gβ subunit alone is able to positively activate the mating pathway in the absence of Gγ [b). Spk1 was shown to directly target the transcription factor Ste11, thus promoting its activation [Pheromones  shows homology to budding yeast Ste50 [S. cerevisiae [K. lactis, an ascomycete closely related to S. cerevisiae [Cryptococcus neoformans, a basidiomycere species that lacks a Ste5 homologue [So far no data indicate a direct interaction between the Gα and the MAPKKK Byr2, and it is also possible that unknown scaffold or linker proteins mediate Byr2 activation, although there exists no Ste5 homologue. One promising candidate is the mating-specific protein Ste4, essential for sexual differentiation , which ist Ste50 , a proterevisiae . Interesrevisiae , and is omologue ,82, thusste6 is not expressed during vegetative growth, because its transcription is regulated by Ste11 [b). Both Ste6 and Ras1 are essential for sexual differentiation [b). Surprisingly, however, the Cdc42 GEF Scd1 and the scaffold protein Scd2 (Bem1 homologue), which promote Cdc42 activation during mating and are essential for the mating process, are not required for MAPK activation [The small GTPase Ras1, the only homologue of human Ras in fission yeast, is another regulator of the MAPK cascade ,74,83,84by Ste11  . In particular, Fus1, an O-glycosylated 1-pass plasma membrane protein [fus1 deletion both partners are still able to sense, attract and grow mating projections towards each other, and is required for vesicle positioning and clustering at the fusion site [cdc42 allele, which displays defects only in cell fusion [a).One important function of the polarization and actin apparatus is to promote the localization of fusion-specific factors critical for cell wall remodelling a. In par protein –117, dep protein ,104,111.ion site . In turnion site . Howeverion site ,116. Fusion site ,118. It ion site . Similarl fusion . Fus2, wl fusion . Fus2 all fusion ,118. Rema). Prm1 mutants degrade the cell wall between both partners as observed by electron microscopy, but cannot efficiently fuse their plasma membranes. Prm1 is a 4-pass plasma membrane protein, with two extracellular loops that, in the endoplasmic reticulum, assemble dimers stabilized by disulfide bonds [prm1Δ cells can cause cell lysis, which cannot be prevented by osmotic stabilization [prm1Δ mating pairs are unable to fuse [2+ in the medium, to promote repair mechanisms. Three other proteins are proposed to act for plasma membrane fusion: Fig1, a 4-pass transmembrane Ca2+ influx regulator, and Kex2, a Golgi-resident protease necessary for the proteolytic processing of alpha-factor, both act additively to Prm1 for membrane fusion [a). Fus1, described above for its role in cell wall remodelling, has been implicated in membrane pore opening and expansion during cell fusion, although its specific function is unknown [Cell wall remodelling allows plasma membrane contact for fusion. Plasma membrane composition and dedicated transmembrane proteins are both critical for this latter process. Bioinformatic screens for transmembrane proteins, whose expression is induced by pheromone, revealed Prm1, which localizes at the fusion site ,120 figa. Prm1 mde bonds ,122. Thrlization ; third,  to fuse . It has  to fuse , a functe fusion ,125  and ergosterols are enriched at the shmoo tip, and are required for Ste5 scaffold recruitment and MAPK activation [Plasma membrane fusion also depends on its composition. In pheromone-treated cells, the lipid bilayer at the shmoo tip is more condensed than the rest of the plasma membrane, an organization that depends on sphingolipids . In parttivation ,129. Ergtivation ,131, sug4.2.S. pombe, as these cells express a specific pheromone-dependent actin nucleator, the formin Fus1 (entirely distinct from its S. cerevisiae homonym Fus1), essential for cell fusion. In addition, fus1Δ cells are fully fusion-deficient, suggesting the fusion machinery may be less redundant in S. pombe than S. cerevisiae [The process of cell fusion has not received much attention in fission yeast. However, as for mating signalling and polarization, several observations suggest that considerable knowledge would be gained from studying cell fusion in this organism. In particular, the role of the actin cytoskeleton in cell fusion is more evident in revisiae .fus1 disrupts actin localization at the shmoo tip and blocks mating pairs at the pre-zygote stage with an intact cell wall [b). As these are also involved in cell fusion in S. cerevisiae, it suggests the actin cytoskeleton is used similarly by both organisms, but in absence of a dedicated formin in S. cerevisiae.Fus1 is targeted to the shmoo tip by its N-terminus and requires its actin nucleation activity to promote cell fusion ,134. Delell wall . Tropomyell wall . Myo51, ell wall  . The question of cell–cell fusion would merit more attention in fission yeast.g fusion . Prm1 is5.pombe Ras1, human Ras activates a conserved Raf–MAPK cascade to promote gene expression and induces cytoskeleton reorganization, which requires Rho family GTPases Cdc42 and Rac [The main proteins involved in the mating pathways of these two simple yeast models are conserved and participate in important processes in response to external signal in other organisms. In higher eukaryotes, for instance, Cdc42 is involved both in axon specification and in dendrite development in response to growth factors in neurons , and pro and Rac . Finally and Rac . Thus, aDrosophila. Here, a fusion-competent myoblast (FCM) migrates towards a founder cell. Upon contact and adhesion, a prominent actin structure, in this case dependent on Arp2/3 nucleation, forms in the FCM and recruits other factors for cell fusion [Drosophila myoblasts [S. cerevisiae [S. pombe M cells ‘take the initiative’ for mating [The process of cell fusion also underlies several important developmental events, including fertilization, muscle fibre formation, placenta development and osteoclast formation. In very few cases have the bona fide fusogens been identified . It is tl fusion . The preyoblasts ,145. Howrevisiae , yeast crevisiae , with bor mating . Future As we hope will be clear from this review, we now understand in great detail some of the molecular connections underlying the response to pheromones. However, many molecular questions remain wide open: what are the molecular connections between pheromone sensing and signalling in fission yeast? How did such divergent connections evolve in the ascomycete lineage and beyond? What are the initial steps that allow the orientation of the polarization apparatus in response to pheromones? What is the molecular nature of the apparatus mediating cell–cell fusion? Beyond the single cell response, how groups of cells interact at a system level also raises many questions: how is a pheromone landscape shaped in a cell population? How do cells make a ‘choice’ for one partner when presented with many options? How is this choice sustained during polarized growth? How are other potential partners for a mating pair ‘discouraged’? Continued investigation using these two highly divergent yeast species will undoubtedly reveal novel insights into these and other fascinating questions.6.We are grateful to Felipe Bendezú and Serge Pelet for their suggestions. Research in S.G.M.'s laboratory is supported by a Swiss National Science Foundation Research grant (no. 31003A_138177) and a European Research Council Starting grant (no. 260493).


Hevea brasiliensis) clone GT1. When light intensity increased from 20 to 1000 µmol m−2 s−1, there was no effect on the maximal quantum yield of photosystem II (PSII) photochemistry (Fv/Fm), indicating that high light intensity did not damage the structure and function of PSII reaction center. However, the effective photochemical quantum yield of PSII (Y(II)), photochemical quenching coefficient (qP), electron transfer rate (ETR), and coefficient of photochemical fluorescence quenching assuming interconnected PSII antennae (qL) were increased significantly as the light intensity increased, reached a maximum at 200 µmol m−2 s−1, but decreased from 400 µmol m−2 s−1. These results suggested that the PSII photochemistry showed an optimum performance at 200 µmol m−2 s−1 light intensity. The chlorophyll content was increased along with the increase of light intensity when it was no more than 400 µmol m−2 s−1. Since increasing light intensity caused significant increase in H2O2 content and decreases in the per unit activity of antioxidant enzymes SOD and POD, but the malondialdehyde (MDA) content was preserved at a low level even under high light intensity of 1000 µmol m−2 s−1, suggesting that high light irradiation did not induce membrane lipid peroxidation in rubber tree. Moreover, expressions of antioxidant-related genes were significantly up-regulated with the increase of light intensity. They reached the maximum expression at 400 µmol m−2 s−1, but decreased at 1000 µmol m−2 s−1. In conclusion, rubber tree could endure strong light irradiation via a specific mechanism. Adaptation to high light intensity is a complex process by regulating antioxidant enzymes activities, chloroplast formation, and related genes expressions in rubber tree.Light is one of most important factors to plants because it is necessary for photosynthesis. In this study, physiological and gene expression analyses under different light intensities were performed in the seedlings of rubber tree ( If not efficiently scavenged, these accumulated ROS will inevitably damage DNA, RNA, proteins, and lipid in organisms. To cope with elevated levels of ROS, aerobic cells have evolved a range of non-enzymatic and enzymatic antioxidant systems. For example, ascorbate (vitamin C)-glutathione cycle, superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) enzyme system, tocopherol (vitamin E), and carotenoids (xanthophyll cycle pigments) are commonly known as antioxidants Light is an important source of photosynthetic apparatus for photosynthesis and carbohydrate assimilation in plants, alga, etc. All photosynthetic organisms have to balance the efficient acquisition of light energy (light-harvesting) with protection from the adverse effects of light (photo-protection) Hevea brasiliensis Muell. Arg.) and consists of high molecular weight cis-polyisoprene produced from the isoprenoid pathway Hevea clones. Significant differences were observed in photosynthetic rate in different clones of rubber tree HbCOA, a long-chain-fatty-acyl-CoA reductase gene (EC 1.2.1.50), was found to play an important role in natural rubber biosynthesis HbAPX, an ascorbate peroxidases gene, was involved in antioxidant system HbATP, encoding the β-subunit of mitochondrial ATP synthase in Hevea brasiliensis was cloned HbCuZnSOD and HbMnSOD encoded antioxidant superoxide dismutase enzymes, and their roles in drought response were confirmed in rubber tree Natural rubber is found in the latex of rubber tree , quantum yield of light-induced (ΔpH and zeaxanthin-dependent) non-photochemical fluorescence quenching (Y(NPQ)), and non-photochemical quenching of chlorophyll fluorescence (qN). We found that as the light intensity increase, Y(NPQ), NPQ, and qN were decreased sharply to reach a minimum at 200 µmol m−2 s−1, but increased from 400 to 1000 µmol m−2 s−1 ), photochemical quenching coefficient (qP), electron transfer rate (ETR), and coefficient of photochemical fluorescence quenching assuming interconnected PSII antennae (qL) were increased significantly as the light intensity increased, reached a maximum at 200 µmol m m−2 s−1 . These r m−2 s−1 . However m−2 s−1 . The hig−2 s−1. However, when light intensity increased to 1000 µmol m−2 s−1, the total chlorophyll content decreased slightly. Moreover, the increase rate of Chl a content was lower than that of Chl b, which resulted in the decrease of Chl a/b ratio from 100 to 1000 µmol m−2 s−1. These indicated rubber tree leaves in the dark green stage could endure strong light irradiation. A steadily increase was observed in the β-carotene content as the light intensity increase. These results showed different responses of chlorophyll and β-carotene to strong light irradiation. The β-carotene (xanthophyll cycle pigments) content was increased to scavenge the accumulated ROS and quenching light under high light intensity. The formation of chlorophyll was used to develop new chloroplasts. Since more light were absorbed by light harvesting chlorophyll protein complex, the formation rate of Chl b was higher than that of Chl a.Chloroplast development needs formation of chlorophyll (Chl) and β-carotene. In order to find out whether exciting energy would be used as a light signal to regulating the chlorophyll and β-carotene synthesis, we measured the pigment composition under different light intensities. As showed in 2O2 content. Usually, the oxidative burst causes damage on lipid membranes. MDA is one of the most frequently used indicators of lipid peroxidation. Under stress conditions, the accumulation of MDA usually leads to the damage of cell membrane in plant and animal. As the light intensity increased, the MDA content underwent a complicated variation. It was increased at 100 µmol m−2 s−1, but decreased at 200 µmol m−2 s−1, then increased slightly at 400 µmol m−2 s−1, followed by a sharp decrease at the high light intensity of 1000 µmol m−2 s−1. Along with the increase of light intensity, the per unit activity of SOD enzyme was decreased dramatically. Meanwhile, the per unit activity of POD enzyme was increased at 100 µmol m−2 s−1, but decreased sharply from 200 µmol m−2 s−1. Since low light intensity was not strong enough to induce the formations of chlorophyll and antioxidant enzymes in rubber tree, MDA content was increased at 20 to 100 µmol m−2 s−1. These results indicated that high light intensity increased ROS contents, more antioxidant enzymes were formed and the amounts of enzymes were increased at the meantime, resulting in per unit activity of enzymes was decreased. So it did not induce membrane lipid peroxidation under high light intensity in rubber tree.Changes of physiological indices including the ROS and malondialdehyde (MDA) contents, and antioxidant enzymes activities were systematically analyzed under different light intensities. As showed in HbCOA, HbCOAL and HbCOXI with the same pattern. Their expressions reached the highest level at 400 µmol m−2 s−1, and increased 1.92-, 765-, and 3-fold comparing their expressions at 20 µmol m−2 s−1, respectively. For the three genes, this increase was followed by a strong decrease at 1000 µmol m−2 s−1. Transcript level of HbCAX was significantly up-regulated by the increase of light intensity, and reached the maximum level at 200 µmol m−2 s−1   . With th m−2 s−1 . Howeverecreased . The HbA m−2 s−1 . For the m−2 s−1 . These sHbAPX, HbCAT, HbCuZnSOD, and HbMnSOD. They reached a maximum at 400 µmol m−2 s−1, and increased 34.9-, 15.2-, 6.31-, and 2.29-fold over that at 20 µmol m−2 s−1, respectively. All of the four genes expressions were decreased at the light intensity of 1000 µmol m−2 s−1   . High va2O2 content was significantly increased with the increase of light intensity  seedlings were grown in the plastic pots in the chamber with vermiculite and turfy soil (1∶3) at the experimental farm of the Chinese Academy of Tropical Agricultural Sciences in Danzhou city, Hainan province, China . In growing season, the average temperature was about 30°C, precipitation was about 180 mm, and humidity was around 97.5%. Leaves samples in dark green stage were collected from seedlings under the indicated light intensity treatment provided by a 1000 W tungsten bulb. A water tank with recycled water was used between radiation source and samples to absorb heat.Chlorophyll was extracted with 80% ice cold acetone from 0.1 g leaves sample. The extract was measured at 475, 645, and 663 nm with spectrophotometer , respectively. Specific chlorophyll and β-carotene contents were determined according to the reported method 0) in dark-adapted state was measured by the measuring modulated light, which was sufficiently low (<0.1 µmol m−2 s−1) not to induce any significant variable fluorescence. To determine the minimal fluorescence level during illumination (F0′), a black cloth was rapidly placed around the leaf and the leaf-clip holder in the presence of far-red light (7 µmol m−2 s−1) in order to oxidize the PSII center fully. Upon darkening the leaf, fluorescence dropped to the F0′ level and immediately rose again within several seconds. The maximal fluorescence level in the dark-adapted state (Fm) and the maximal fluorescence level during natural illumination (Fm′) were measured by a 0.8-s saturating pulse at 8000 µmol m−2 s−1. Fm was measured after 30 min of dark adaptation. Fm′ and Fs were measured when photosynthetic photon flux densities (PPFDs) were approximately 200 and 1400 µmol m−2 s−1, respectively. Other parameters were calculated based on measured parameters above.Modulated chlorophyll a fluorescence measurement was made in attached leaves at mid-day with a PAM-2500 portable fluorometer  connected to a computer with data acquisition software Pam-Win3 . The experimental protocol described by −1 cm−1.MDA content was determined by the thiobarbituric acid (TCA) reaction as described by −2 s−1 (placing the test tubes under two 15 W fluorescent lamps). A complete reaction mixture without enzyme, which gave the maximal colour, was served as the control. The reaction was stopped by switching off the light and putting the tubes into dark. A non-irradiated complete reaction mixture was served as a blank.SOD (EC 1.15.1.1) was prepared by first freezing 0.5 g of leaves sample in liquid nitrogen to prevent proteolytic activity, followed by grinding with 5 ml extraction buffer . Brie was centrifuged for 20 min at 15000 g and the supernatant was used as an enzyme. The soluble proteins concentration in the supernatant were determined using the method of Bradford with bovine serum albumin (BSA) as standard POD (EC 1.11.1.7) activity was determined with spectrophotometer. 0.5 g leaves sample was extracted with 5 ml 100 mM phosphate buffer (pH 6.0). Homogenate was centrifuged at 4000 g for 10 min. Reaction mixture was 50 ml 100 mM phosphate buffer (pH 6.0) with 23 mM guaiacol and 1.8 mM hydrogen peroxide. 1 ml supernatant was added into 3 ml reaction mixture. The change of OD was recorded at 470 nm. The per unit activity of enzyme was defined as the increase of 0.1 ΔOD per minute.2O2 content was conducted according to the reported method 2O, shaken vigorously again and placed in an ice box for 5 min. After stratification of liquid solution, the tube centrifuged at 6500 g for 5 min. Took 5 ml of the upper aqueous phase, added 0.5 mL 5% TiSO4 and 1 ml of concentrated ammonia water in a centrifuge tube. Shaking mixture to make precipitation with ample color, centrifuged at 10000 g for 10 min, discarded supernatant. The deposition was dissolved by 5 ml of 2 M H2SO4, and filtered with filter paper. Filtrate was measured OD at 415 nm with spectrophotometer.Measurement of HTotal RNA was extracted from leaves according to the methods of Tang et al. (2007) and Xiao et al. (2009) 18S rRNA (Hb18SRNA) was used as a house-keeping gene  according to the manufacturer's instruction. The cDNA was diluted 1∶20 with nuclease-free water. Aliquots of the same cDNA sample were used for real-time PCR with primers designed for the selected genes, and ing gene . The PCRP<0.05 (probability level) was considered significant difference. Figures were drawn by Origin data analysis and graphing software, Origin Pro 9.0 . For real-time PCR analysis, each value was the average of two biological replicates tested in triplicate, and for other analysis, 6 replicate samples tested in replicate were used.All data were analyzed with IBM-SPSS analytical software package version 20.0 . One-way ANOVA and Tukey text were used to assess the different level.


There is evidence that heart rate variability (HRV) is reduced in major depressive disorder (MDD), although there is debate about whether this effect is caused by medication or the disorder per se. MDD is associated with a two to fourfold increase in the risk of cardiac mortality, and HRV is a robust predictor of cardiac mortality; determining a direct link between HRV and not only MDD, but common comorbid anxiety disorders, will point to psychiatric indicators for cardiovascular risk reduction.anticipation), or comorbid panic and posttraumatic stress disorders .To determine in physically healthy, unmedicated patients whether (1) HRV is reduced in MDD relative to controls, and (2) HRV reductions are driven by MDD alone, comorbid generalized anxiety disorder (GAD, characterized by anxious A case-control study in 2006 and 2007 on 73 MDD patients, including 24 without anxiety comorbidity, 24 with GAD, and 14 with PD/PTSD. Seventy-three MDD and 94 healthy age- and sex-matched control participants were recruited from the general community. Participants had no history of drug addiction, alcoholism, brain injury, loss of consciousness, stroke, neurological disorder, or serious medical conditions. There were no significant differences between the four groups in age, gender, BMI, or alcohol use.HRV was calculated from electrocardiography under a standardized short-term resting state condition.HRV was reduced in MDD relative to controls, an effect associated with a medium effect size. MDD participants with comorbid generalized anxiety disorder displayed the greatest reductions in HRV relative to controls, an effect associated with a large effect size.Unmedicated, physically healthy MDD patients with and without comorbid anxiety had reduced HRV. Those with comorbid GAD showed the greatest reductions. Implications for cardiovascular risk reduction strategies in otherwise healthy patients with psychiatric illness are discussed. Major depressive disorder (MDD) and cardiovascular disease (CVD) are leading burdens of disease worldwide, and there is increasing recognition that the two are related. Up to 50% of CVD patients suffer from depression without comorbid physical illness. Using meta-analysis, we recently reported HRV reductions in depressed patients without CVD Research has shown that depressed patients with CVD display lower HRV than non-depressed patients Another body of evidence suggests that anxiety – a condition frequently comorbid with MDD in more than 60% of cases This study examined the impact of MDD and comorbid anxiety disorders on HRV to identify psychiatric indicators for cardiovascular risk reduction. Our hypothesis was that HRV would be reduced in MDD patients relative to age- and sex-matched controls in an independent, physically healthy, and unmedicated sample. Further, we sought to determine whether MDD without comorbidity, MDD with GAD or MDD with PD and/or PTSD display the greatest reductions in HRV. This is an important issue given the frequent comorbidity of MDD with anxiety disorders and competing accounts on the impact that anxiety with specific features  may have on HRV www.brainresource.com). The study was approved by University of Sydney, Sydney West Area Health Service, University of Adelaide and Flinders University human research ethics committees, and all participants provided written informed consent in accordance with the Australian National Health and Medical Research Council guidelines.Seventy-three patients with a primary diagnosis of MDD and 94 age- and sex-matched controls were included in this study. Participants were recruited from the general community via self-referral from advertisements and collaborating clinicians. We obtained their data from the Brain Resource International Database . All participants were free from anti-depressant medication for at least five half-lives (70% of whom were drug-naive). Twenty-four participants with MDD had comorbid GAD without other diagnosis (“anxious apprehension”). Fourteen participants with MDD had either comorbid PD (n = 7), PTSD (n = 4) or both (n = 3) . No patient with comorbid PD and/or PTSD had GAD.Participants were seated in a sound- and light-controlled room at 24°C and two 2-minute electrocardiogram (ECG) recordings were collected during resting state. The data was sampled at 500 Hz, with 22-bit resolution digitization using a Compumedics Neuroscan NuAmps amplifier and SCAN software, version 4.3. Experimental studies have shown that 2-minute recordings are predictive of coronary heart disease and mortality http://kubios.uku.fi) for calculation of time, frequency and nonlinear measures. The two time-domain measures calculated on the R–R intervals include the standard deviation of R–R intervals (SDNN), and the square root of the mean squared differences between successive R–R intervals (RMSSD), a measure based on comparisons of lengths of adjacent cycles. Two frequency domain measures were quantified: high frequency , a measure of parasympathetic activity; and the LF/HF ratio, a measure of sympathovagal balance. We also examined two nonlinear domain measures: the standard deviation of the Poincaré plot perpendicular to the line of identity (PCSD1), and detrended fluctuation analysis . The Poincaré graph plots each R–R interval as a function of the next R–R interval, and the PCSD1 measure represents the short-term variability of the nonlinear dynamic system and reflects parasympathetic nervous system activity. DFA measures the correlation within the signal, and the DFAα1 measures the short-range correlation between successive R–R intervals. White Gaussian noise  is reflected in a value of 0.5; a Brownian noise signal  is reflected in a value of 1.5 All data was manually inspected prior to analysis: data quality was high, consistent with resting-state recording conditions. ECG was analyzed using custom-developed software to perform semi-automated pre-processing to remove noise from the ECG, allowing for the identification of the R-peaks based on established methods d) relating to the difference between patient groups and controls were determined.Statistical analysis was conducted using predictive analytics software (PASW) statistics, version 18. Independent-samples t-tests on controls versus MDD, and one-way ANOVAs on controls versus the three MDD groupings were conducted to determine differences on age, gender, body mass index (BMI), and alcohol use , comorbid PD and/or PTSD , MDD without anxiety comorbidity, and healthy control groups . Our hypothesis that HRV is reduced in MDD relative to controls (CTL) was confirmed by an overall MANOVA, including all HRV measures  . Strikind = −0.48) and SDNN (at trend levels) (F (165) = 3.57, p = 0.06, Cohen's d = −0.29). In frequency-domain measures, the groups differed on HF (F (165) = 8.712, p = 0.004, Cohen's d = −0.46) and LF/HF ratio (F (165) = 7.71, p = 0.006, Cohen's d = 0.42). Although the LF/HF ratio was increased in MDD relative to CTL, this finding is interpreted as an increase in sympathetic relative to parasympathetic activity in MDD, consistent with a decrease in HRV. In nonlinear-domain measures, the groups differed on PCSD1 (F (165) = 9.35, p = 0.003, Cohen's d = −0.48) and DFAα1 (F (165) = 11.30, p = 0.001, Cohen's d = 0.52). Although the DFAα1 measure is increased in MDD relative to CTL, this finding indicates that the MDD group displays a less random signal than CTL, consistent with a reduction in HRV.These results are shown in p = .003, η2 = .09] and SDNN (at trend levels) . Post-hoc comparisons revealed that RMSSD is reduced in MDD patients with GAD . No other patient groups differed from each other or from controls.These results are shown in p = .004, η2 = .08] and LF/HF ratio . Post-hoc comparisons revealed that HF  was reduced and LF/HF ratio , increased in MDD patients with GAD, relative to controls. MDD patients with no anxiety also displayed increased LF/HF ratio relative to controls at trend levels . No other significant differences were observed.In frequency-domain measures, ANOVA revealed a statistically significant difference for HF  and DFAα1 . Post-hoc comparisons revealed that PCSD1  decreased and DFAα1 ratio  increased in MDD patients with GAD, relative to controls. MDD patients with PD and/or PTSD also displayed a statistical trend towards increased DFAα1 ratio  No other significant differences were observed.In nonlinear-domain measures, ANOVA revealed a statistically significant difference for PCSD1 [F = 4.7, per seRecent debate GAD is characterized by anxious apprehension and worry, involving pre-attentive biases to threat information, and rigid and inflexible response patterns There are a number of explanations for why HRV may be reduced in depression and in those with comorbid GAD in particular. One potential explanation proposed for the link between anxiety, HRV and CVD is the inability to disengage threat detection, which serves to perpetuate worry and hypervigilance, even when no real threat exists It is possible that the observed HRV reductions were due to potential lifestyle factors not assessed in the current study, such as smoking or low physical activity levels in the depressed patients. While it is possible that these factors could explain the differences observed between MDD patients and controls, it is unlikely they could explain the differential effects observed for patient sub-groupings relative to controls. Recent work also highlights a relationship between HRV and inflammatory markers In conclusion, this study has provided strong evidence that clinicians should consider comprehensive cardiovascular risk-reduction strategies for MDD patients with comorbid GAD in particular. Further research examining intermediate (HRV)  is required to confirm whether such strategies are needed for patients with MDD alone, and MDD with comorbid PD and/or PTSD.


The present method enabled visualization of cell composition during apoptosis and captured complicated molecular events. For example, apoptotic cells that lost Δψm did not always externalize PS, while some late apoptotic cells had polarized Δψm. The findings of unchanged PS-externalization and aberrant cell death suggest that there is no relationship of PS externalization and apoptosis with an unknown apoptotic mechanism. Based on PS-externalization, sensitivity to staurosporine, and the combination of cell lines and triggers, the apoptotic process was classified into 2 types. Importantly, most of our findings could not be observed by PS–PI and Δψm assays when independently performed. Our method may be useful for examining mitochondrial-related apoptosis and death signalling pathways, as well as screening novel apoptosis-inducing cancer drugs.Recently, it has been proposed that novel methodologies are needed to re-evaluate apoptotic cell death, as studies of apoptosis have shown it to be a complex process. Since mitochondria are key regulators in cell death pathways, we developed a simultaneous 3-parameter flow cytometric analysis that incorporates the change in mitochondrial membrane potential (Δψ Collapse of Δψm during apoptosis has been reported in a number of studies, leading to the general notion that depolarization of mitochondria is one of the first events to occur during apoptosis and a prerequisite for cytochrome-c release , TMRE (tetramethylrhodamine), JC-1, DiOC6(3), DilC1(5), and rhodamine 123  of analysis  on dying cells using Annexin-V in combination with propidium iodide (PI) (PS–PI assay) , 2 leukemic cell lines (K562 and THP1), and 2 B-cell lines (Ramos and SKW6.4). The ATL cell lines KK1, ST1, and LM-Y1 were established in our laboratory  and betulinic acid (BEA) were purchased from Calbiochem . They were dissolved in DMSO and STS to make stock solutions of 100 μM and 5 mg/mL, respectively. Anti-Fas was purchased from MBL  and dissolved in RPMI1640 medium to make a stock solution of 1 μg/mL. TRAIL was purchased from BIOMOL Research Laboratories  and dissolved in RPMI1640 medium to make a stock solution of 20 μg/mL. Z-VAD-fmk was purchased from MBL.Jurkat cells were treated with STS , anti-Fas (2.5 ng/mL) ] , and Annexin-V and PI  assays. When the mitochondrial membrane potential (Δψm) is intact, the dye becomes concentrated in the mitochondrial matrix and fluorescence intensity is determined using flow cytometry. Disruption of Δψm significantly compromises DilCl(5) accumulation, which is detected as a decrease in fluorescence intensity  are considered to be apoptotic , PS+/PI− (early apoptotic cells), PS+/PI+ (late apoptotic or necrotic cells), and PS−/PI+ (dead or unknown cells) areas were coloured red, blue, green, and violet, respectively  staining  or late (green) apoptotic cells  and late apoptotic (green) cells co-existed for a long period of time. Interestingly, dead (or unknown) cells (violet) had 2 populations that were distributed into polarized and depolarized areas  area. Thus, it is reasonable that these populations were distributed into polarized and depolarized areas.Jurkat cells treated with various death triggers under conditions that caused cell death after 24 h became uniform Fig. d, then tm as the x-axis and PI as the y-axis, incorporating the 4 colours from the PS–PI assay. We focused on the cell compositions in polarized and depolarized areas, and observed the time course of changes in colour for each area. For example, in STS-treated Jurkat cells, viable cells (red) in polarized areas disappeared after 6 h. Similarly, early apoptotic cells (blue) in depolarized areas changed to late apoptotic cells (green). We found this time-course analysis of 3-parameter cytogram findings useful to understand the cell death pattern in relation to loss of Δψm.Figure The diverse apoptotic patterns could be classified into 2 major types when we focused on STS-treated cells  and then turned late apoptotic (green)  Fig. a, left. en) Fig. a, right.en) Fig. b, left. en) Fig. b, right.−/PI− areas . These results suggest that neither the depolarization of Δψm not inhibited by Z-VAD nor activation of caspases in STS-treated Jurkat cells was due to a lack of Z-VAD.Next, we focused on inhibition of caspases by Z-VAD in STS-treated Jurkat cells. The results of the PS–PI assay indicated that Z-VAD attenuated apoptotic changes, remaining as a viable (red) cell population in PSeas Fig. a, b. In ies Fig. c, d. Furies Fig. e, it wasies Fig. f. These m assays are predominantly used in analyses of apoptosis, likely because they provide concise results. In the present study, we developed a simple methodology by combining conventional methods for apoptotic estimation based on mitochondrial functions. We conducted 3-parameter flow cytometric analyses with 4 colours and evaluated the apoptotic process from various aspects.Although numerous studies have investigated apoptosis, the expansion of apoptotic research has made it difficult to understand common concepts, including cell death patterns. Therefore, it is necessary to re-evaluate cell death patterns and present novel methodology from a new conceptual viewpoint  cells in depolarized areas (wide or sharp peak). These results indicate that substantially different events occurred in response to each trigger in the same cell line. Thus, 3-parameter histogram findings may be suitable for examining the mechanism of apoptosis.m using 3-parameter cytogram findings. Despite diversity or differences in response patterns between cell type and type of death trigger, diverse death patterns shown by the 3-parameter cytogram were classified into Types 1 and 2. Interestingly, the features of Types 1 and 2 were not dependent on cell type, but rather were flexible. ST1, KK1, and LYM1 cells were Type 1 when triggered by DRs, and then changed into Type 2 when using STS. Another characteristic of the 3-parameter cytogram findings was detection of aberrant cell populations based on localization site and/or colour. We found late apoptotic (green) cells in polarized areas among BEA-treated Jurkat cells and STS-treated LM-Y1 cells. These cells likely employ an unknown death signalling pathway different than mitochondrial membrane depolarization and such an aberrant cell death is not verified yet  is not revealed by a common Δψm assay. In this regard, the individual weaknesses of the PS–PI and Δψm assays seem to be covered by the strength of the other one in our 3-parameter analysis. Interestingly, novel cell death modes such as parthanatos and netosis, which showed irreversible Δψm dissipation but not PS exposure, were reported and were considered as a kind of regulated necrosis and/or autophagic cell death  cells among STS-treated cells in depolarized areas. It is known that exposure to PS does not occur in early apoptotic stages and is scarcely detectable in some types of cells by Annexin-V  cells were distributed in depolarized areas and those cells harboured active caspase-3. These results are not consistent with previous reports that suggested that early loss of Δψm occurs independently of caspase activation (Ly et al. m assay have been rarely reported (Fox and Aubert We conducted another 3-parameter analysis using a caspase activity assay instead of Δψm assays seem to cover for the disadvantages of the other one in 3-parameter assays, which suggests that this is a novel methodology comprised of conventional methods. In future studies, 3-parameter assays may be useful for screening mitochondrial-related apoptosis and various regulated cell death patterns, as well as novel apoptosis-inducing anti-cancer drugs.Apoptosis is a representative cell death pattern and aberrant apoptosis is causatively associated with diseases. Most chemotherapeutic drugs have been shown to activate common apoptotic pathways in target cells. Since our methodology enables visualization of cell composition during the process of apoptosis, it captures and screens complicated molecular events. In this study, we observed some previously unnoticed cell populations, though some findings are left unexplained or raise further questions. It is interesting that the PS–PI and Δψ


Eragrostis tef) provides a major source of human nutrition in the Horn of Africa, but biotechnology has had little impact on its improvement to date. Here, we report the elaboration of an in vitro regeneration protocol, based on the use of immature zygotic embryos as explant. Explant size was an important determinant of in vitro regeneration efficiency, as was the formulation of the culture medium. Optimal results were obtained by culturing 0.2–0.35 mm embryo explants on a medium containing KBP minerals, 9.2–13.8 μM 2,4-dichlorophenoxyacetic acid, 6 mM glutamine, and 0.5% Phytagel. Although this protocol was effective for both the improved cultivar “DZ-01-196” and the landrace “Fesho”, the former produced consistently more embryogenic tissue and a higher number of regenerants. An average of more than 2,800 shoots could be obtained from each “DZ-01-196” explant after 12 weeks of in vitro culture. These shoots readily formed roots, and plantlets transferred to soil were able to develop into morphologically normal, fertile plants. This regeneration and multiplication system should allow for the application of a range of biotechnological methods to tef.Tef ( Eragrostis tef (Zucc.) Trotter] is indigenous to Ethiopia, which is its centre of diversity and site of domestication [n = 4x = 40), facultatively self-pollinating C4-plant [−1 [injera. Lodging is a major barrier to increasing productivity and has yet to be overcome by conventional breeding approaches or interspecific hybridization, so probably other strategies, such as mutagenesis or genetic transformation, are needed. To date, little investment in biotechnology has been applied to tef. In vitro plant regeneration from explants such as roots, young leaf bases, or seeds has been demonstrated [The small-grained cereal tef [tication . The speC4-plant , 3, and nstrated –7, but tin vitro gynogenesis has been described by Gugsa et al. [de novo shoots can be regenerated. The very small size of the tef caryopsis (1.0–1.6 mm) means that the excision of the immature embryos is technically challenging [A viable method of doubled-haploid formation using a et al. , but whallenging —this expllenging . in vitro culture system for immature tef embryos which has the capacity to differentiate somatic embryos able to form multiple shoots and viable regenerant whole plants. Here, we detail the elaboration of an Grain of the tef land race “Fesho” and the widely grown improved variety “DZ-01-196” (cv. “Magna”) were germinated in peat-soil mix in a glasshouse at ~24°C. Plantosan fertilizer  was applied at 12 g per 16 L pot three weeks after planting. Around 7–10 d after the onset of anthesis, panicles were cut at the peduncle and each placed in a 50 ml screw-capped plastic centrifuge tube to allow surface sterilization of the immature caryopses by shaking for 15 min in 1%  w/v sodium hypochlorite, followed by rinsing 3-4 times in sterile doubled distilled water. The immature embryos were recovered from the detached caryopses  by squeeμM NaFeEDTA, 250 mM maltose (as sole carbohydrate source), 9.2 μM 2,4-dichlorophenoxyacetic acid , pH 5.8, and solidified with 0.4% Phytagel , if not stated otherwise. Except for the Phytagel, which was autoclaved as 1.5% stock, the above components were all filter-sterilized before preparation of the media. The small and intermediate-sized “DZ-01-196” embryos required two weeks of culture to form somatic embryos, while the large ones required three weeks. The “Fesho” embryos, irrespective of their size, needed to be transferred to fresh callus induction medium after two weeks and held for a further two weeks, because somatic embryos emerged later than from “DZ-01-196” explants. Comparative experiments were also conducted to explore the benefit of varying the concentration of 2,4-D , glutamine , and Phytagel , as well as determining the effect of embryo size. Small (0.1–0.2 mm), intermediate-sized (0.2–0.35 mm), and large (0.35–0.75 mm) embryos were sorted on the basis of size and plated scutellum side up at 7–10 embryos per 3.5-cm Petri dish Figures  containiresponding embryos) were transferred to 9 cm Petri plates containing 15 mL antibiotic-free K4NB regeneration medium [Explants which developed callus and/or somatic embryos  and transferred to K4NB regeneration medium, with subcultivation and further fragmentation being conducted fortnightly over a period of eight weeks. Where necessary, nonrooted shoots were cultivated on the same medium to induce root formation. Rooted plantlets were potted in soil and grown in a greenhouse, as described above for the donor plants. Three independent replicates were set up in experiments designed to optimize the callus induction medium, with each replicate consisting of two or three Petri dishes with 7 to 10 zygotic embryos each. For the validation of the optimized protocol, each replicate comprised four embryos per embryo size class. The software package SigmaStat v3.0  was used for all statistical calculations. The data were interpreted either by conventional ANOVA, or—where the data failed either a normality or an equal variance test—by the nonparametric Kruskal-Wallis test. Pairwise multiple comparisons of the different treatments were conducted using the Student-Newman-Keuls method. de novo shoot regeneration from both genetic stocks, with the latter medium giving significantly the best response (The large (0.35–0.75 mm) “Fesho” and “DZ-01-196” explants cultivated on media containing N6 and L3 minerals displayed a low level of somatic embryogenesis and very little, if any, differentiation of shoots. In contrast, both MS and KBP minerals were permissive of callus formation, somatic embryogenesis, and response . KBP is μM) was more effective than either the lowest or the highest concentrations tested  were eventually validated by an experiment based on “DZ-01-196” explants. All the intermediate-sized explants regenerated multiple shoots. On the K4NB medium, not only did shoots and roots develop, but also proliferation continued and further somatic embryogenesis occurred (The optimized conditions (callus induction medium containing KBP minerals, 6 mM glutamine, 9.2 occurred , resultioccurred . Throughoccurred . All roooccurred . in vitro culture and can be induced to form either callus and/or somatic embryos. The optimized protocol allows many regenerants to be rapidly produced from a single explant, with no sign of frequent morphological abnormalities. We believe that this protocol will find applications such as the rapid multiplication of valuable germplasm, embryo rescue in wide crosses, mutation breeding, haploid technology, and genetic transformation; a set of technologies which can now be brought to bear to overcome the major breeding bottlenecks affecting the productivity and end-use quality of tef.We have demonstrated that immature tef embryos, even though they are rather small, are still amenable to