Hearing loss is one of the most prevalent chronic health conditions in older adults. Growing evidence suggests that hearing loss is associated with reduced cognitive functioning and incident dementia. In this mini-review, we briefly examine literature on anatomical and functional alterations in the brains of adults with acquired age-associated hearing loss, which may underlie the cognitive consequences observed in this population, focusing on studies that have used structural and functional magnetic resonance imaging, diffusion tensor imaging, and event-related electroencephalography. We discuss structural and functional alterations observed in the temporal and frontal cortices and the limbic system. These neural alterations are discussed in the context of common cause, information-degradation, and sensory-deprivation hypotheses, and we suggest possible rehabilitation strategies. Although, we are beginning to learn more about changes in neural architecture and functionality related to age-associated hearing loss, much work remains to be done. Understanding the neural alterations will provide objective markers for early identification of neural consequences of age-associated hearing loss and for evaluating benefits of intervention approaches. Hearing loss is the third most prevalent chronic condition affecting older adults  and is oDecades of research have characterized the impact of varying degrees of hearing loss on auditory processing, speech communication, and psychosocial well-being e.g., . The detcommon cause hypothesis  and diffusion tensor imaging (DTI). VBM has been used to investigate changes in gray matter thickness, concentration, and volume, with declines in gray matter observed in a number of chronic conditions such as depression  and demeUsing VBM, The studies by A more recent longitudinal study  offers aHearing loss appears to be a prominent trigger for changes in white matter . DTI stuIn general, studies have demonstrated reduced cortical volumes in the auditory processing areas in the superior temporal cortex and declines in white matter tract integrity of central auditory pathways, with some exceptions , in indiA handful of studies have examined functional brain changes in age-associated hearing loss. Here, we focus on studies that have used functional magnetic resonance imaging (fMRI), which measures brain activity by detecting changes associated with blood flow, and event-related potentials (ERPs), which measure electrical activity in the brain that is time locked to a cognitive process. The fMRI studies in particular have varied widely in terms of the stimuli used  and the paradigms employed. Thus, our primary goal is to summarize whether these fMRI studies have observed (i) any relationship between functional brain activation patterns and hearing ability or (ii) any differences in brain activation patterns in individuals with age-associated hearing loss as compared to normal hearing controls.The fMRI studies discussed above investigated age-associated hearing loss using auditory tasks, whereas Similar to In a subsequent study, In summary, there appear to be functional alterations in the engagement of auditory cortex in age-associated hearing impairment, especially during processing of sounds. Furthermore, emerging evidence suggests that these functional alterations are noticeable in other brain regions including frontal cortices and the limbic system. These results suggest that aging may be a major factor related to changes in the functioning of the auditory system, supporting the common cause hypothesis. However, functional alterations observed in the non-auditory regions lend support to information degradation and sensory deprivation theories, indicating that these frameworks combined might provide a better explanation of age-associated hearing loss .Estimates suggest that 35.6 million people lived with dementia worldwide in 2010 . A sharpExisting evidence indicates that there are anatomical and functional alterations in the brains of individuals with age-associated hearing loss that are independent of age effects. These changes appear to occur not only in the auditory processing regions, but also in regions related to attention and emotional processing. Future neuroimaging and neurophysiological studies involving audiometrically and cognitively well-characterized participant pools are necessary to further identify changes in neural architecture and functionality related to hearing loss. As evidence starts accumulating, current theories linking aging and hearing loss should be updated to reflect new data.Figure 1).From the public health standpoint, a critical question related to the cognitive and neural consequences observed in age-associated hearing loss is whether these changes can be reversed with interventions, in turn mitigating dementia risk. A handful of studies have suggested that correction of hearing loss with amplification  and cochAuditory training, which involves training individuals to listen to auditory input with active engagement, is another promising approach to intervention  when useRM and FH have both contributed equally to the conception, drafting, and final approval of this manuscript and both agree to be accountable to all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.The reviewer BM and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review.


Traditionally, audiology research has focused primarily on hearing and related disorders. In recent years, however, growing interest and insight has developed into the interaction of hearing and cognition. This applies to a person’s listening and speech comprehension ability and the neural realization thereof. The present perspective extends this view to oral communication, when two or more people interact in social context. Specifically, the impact of hearing impairment and cognitive changes with age is discussed. In focus are executive functions, a group of top-down processes that guide attention, thought and action according to goals and intentions. The strategic allocation of the limited cognitive processing capacity among concurrent tasks is often effortful, especially under adverse communication conditions and in old age. Working memory, a sub-function extensively discussed in cognitive hearing science, is here put into the context of other executive and cognitive functions required for oral communication and speech comprehension. Finally, taking an ecological view on hearing impairment, activity limitations and participation restrictions are discussed regarding their psycho-social impact and third-party disability. Being able to communicate with others is regarded a key element of human functioning. During oral communication individuals interact with each other, and also with their social and physical surroundings by exchanging information in form of language, signals, and behavior . As suchHearing impairment constitutes a major challenge in this respect as it generally leads to difficulties in oral communication . These chearing, listening, comprehending, and eventually communicating  as well as fast interactions between sensory and cognitive processing. For example, listening to someone can be referred to as hearing with intention and attention. As such listening often demands the expenditure of mental effort, because cognitive resources including attention and executive functions (EFs) have to be invested for goal pursuit. Besides, the information must be received and decoded in a unidirectional manner in order to be able to derive and understand meaning. This step is described as comprehension and takes place throughout conversations with others. Finally, communication involves the conversational interactions between two or more people, while transferring information, meaning and intent bi-directionally. Given the described steps in the communication pathway, successful oral communication depends not only on the ability of hearing, but also requires listening and comprehending from all participants involved. One could understand a communication situation as a dynamic system that must be carefully balanced. Difficulties in either one component, such as one communication partner being hearing impaired, would require sensitivity and flexibility by means of adaptation of the system. To maintain the flow of a conversation and to avoid interruptions, when communication problems occur, strategies for compensation and repair need be activated immediately. Such strategies could include that the speaker repeats or rephrases what was said using loud and clear voicing, or that the hearing impaired person tries to concentrate more and activates additional mental resources  or relies more on other modalities —the ability to hold information in mind (maintain) and mentally work with it (manipulate) at the same time  and interpersonal processes (e.g., social influence, group dynamics, attractions, and generation gap; e.g., Modern audiology has extended its focus from hearing to considerations of cognitive processes, aging effects and social factors in order to address the communication problems of hearing impaired individuals and to meet their expectations. In recent years, great insight has been gained into this interdisciplinary field of study. For instance research has taken into account aspects of neuro-cognitive mechanisms, age-related decrements and compensatory strategies, as well as the role of SOs and the social network related to successful oral communication and rehabilitation. Nevertheless, there is still great potential for applying this knowledge as a matter of course in aural rehabilitation e.g., and tranThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Purpose. To investigate the prevalence and risk factors of hyperplastic and adenomatous colorectal polyps in a Taiwanese general population. Methods. From January 2009 to December 2011, consecutive asymptomatic subjects undergoing a routine health check-up were evaluated by colonoscopy. The colorectal polyps were assessed, and medical history and demographic data were obtained from each patient. Logistic regression analysis was conducted to search the independent risk factors for asymptomatic hyperplastic and adenomatous colorectal polyps. Results. Of the 1899 asymptomatic subjects, the prevalences of hyperplastic polyps and adenomatous polyps were 11.1% and 16.1%, respectively. Multivariate analysis revealed that high body mass index  and current smoking  were independent predictors for hyperplastic colorectal polyps. Age over 60 years old , high body mass index , heavy alcohol consumption , and current smoking  were independent predictors for adenomatous colorectal polyps. Conclusion. High BMI and smoking are common risk factors for both adenomatous and hyperplastic polyps. Old age and alcohol consumption are additional risk factors for the development of adenomatous polyps. Colorectal cancer is one of the most common cancers worldwide. In Taiwan, the incidence of colorectal cancer is increasing as lifestyles and diets have become more westernized. In the United States, by contrast, the incidence of colorectal cancer is declining probably because of advances in cancer screening. Many studies suggested most colorectal cancers originate from a precursor benign polyp , 2, whicUntil approximately 1990, colorectal polyps were classified into two groups: adenomatous polyps  and hyperplastic polyps. Conventional adenomas are precursor lesions to colorectal carcinomas developing via the traditional adenoma-carcinoma pathway characterized by chromosomal instability . Many riHyperplastic polyps are traditionally assumed to have no malignant potential. However, recent studies showed approximately 30% of colorectal carcinomas develop via the serrated neoplasia pathway characterized by frequent BRAF mutation and widespread DNA methylation . Most naCurrently, the risk factors for developing hyperplastic polyps remain unclear, and the results of risk factors for adenomatous polyps in Asian countries are quite conflicting. We therefore conducted this cross-sectional study to investigate the risk factors for the development of colorectal hyperplastic polyps and adenomatous polyps in Taiwan.From January 2009 to December 2011, consecutive asymptomatic subjects undergoing a routine colonoscopy during health check-up were invited to participate in this study. The study protocol was approved by institutional review boards at Kaohsiung Veterans General Hospital, and all participants provided written informed consent. The eligible subjects were excluded if they reported symptoms of lower gastrointestinal tract disease, including rectal bleeding, marked change in bowel habits, or lower abdominal pain that would normally require medical evaluation. Other exclusion criteria were current participation in other studies, history of disease of the colon , prior colonic surgery, and colorectal examination  within the previous 10 years.A complete history and physical examination were performed for every subject undergoing health check-up. All subjects were carefully queried regarding the presence of abdomen symptoms in the previous 1 month, and subjects who responded negatively were classified as asymptomatic subjects and enrolled for this study. All the participants received anthropometric and blood biochemical tests, which included fasting plasma glucose, serum triglyceride, and high-density lipoprotein (HDL) cholesterol level, and received total colonoscopy. Colonoscopies were performed by three experienced endoscopists  with the Olympus PCF-Q240AL and PCF-Q260AL  after the subjects had fasted overnight. Bowel preparation was performed with Fleet oral saline laxative using the same protocol as that used for diagnostic colonoscopy. The patients were carefully examined for colorectal mucosal lesion. Colorectal polyp was defined as a protuberance into the lumen from the normally flat colonic mucosa. The location of the colorectal polyp was divided into the proximal colon, including the cecum, the ascending colon and transverse colon, and the distal colon, including the splenic flexure, the descending colon, the sigmoid colon, and the rectum. All visible polyps were removed and examined histologically by the pathologist. The pathology types of colorectal polyps were subsequently categorized into hyperplastic polyps, adenomatous polyps, sessile serrated adenoma, and traditional serrated adenoma.To assess the relations between clinical characteristics and asymptomatic colorectal polyps, the following data were recorded for each subject: age; gender; educational status; family history of colorectal polyps; consumption of tobacco, alcohol, coffee, tea, spicy foods, or betel nut; exercise habit; whether vegetarian or not; and longtime use of nonsteroidal anti-inflammatory drugs (NSAIDs). All variables were categorized for data analyses.P value less than 0.05 was considered significant. Significant variables revealed by univariate analysis were subsequently assessed by a stepwise logistic regression method to identify independent clinical factors predicting the presence of colorectal polyps in asymptomatic subjects.The chi-square test or Fisher's exact test was employed to investigate the relationships between the rate of colorectal polyps and clinical characteristics. These variables included the following: gender; age ; educational status ; body mass index ; NSAID use (yes or no); family history of colorectal polyps (yes or no); smoking status ; consumption of alcohol, coffee, tea, or spicy foods; exercise habit ; and betel nut habit and vegetarian (yes or no). Metabolic syndrome was defined according to the modified National Cholesterol Education Program Adult Treatment Panel III definition for South Asians and Chinese. A All statistical analyses were performed using SPSS version 17.0 .From January 2009 to December 2011, 1899 asymptomatic subjects  were recruited for this study. Among them, 520 (27.4%) had colorectal polyps . The preP < 0.001, 0.048, and 0.004, resp.). The subjects with and without hyperplastic polyps had comparable gender, education level, family history of colorectal polyps, alcohol, coffee, tea, and spicy food consumption, betel nut use, exercise habit, vegetarian, metabolic syndrome status, and NSAID use. Multivariate analysis with stepwise logistic regression showed that both current smoking  and high BMI  were independent predictors for asymptomatic hyperplastic colorectal polyps . Further, the subjects with adenomatous polyps had higher BMI than those without adenomatous polyps. However, the two groups had comparable family history of colorectal polyps, coffee, tea, and spicy food consumption, betel nut use, exercise habit, vegetarian, metabolic syndrome status, and NSAID use.Multivariate analysis revealed that age over 60 years old , high BMI , heavy alcohol consumption , and current smoking  were independent predictors for asymptomatic adenomatous polyps .The current study demonstrated that the prevalences of colorectal hyperplastic polyps and adenomatous polyps in asymptomatic Taiwanese were 11.1% and 16.1%, respectively. Smoking and high BMI are risk factors for both adenomatous and hyperplastic polyps. Old age and alcohol consumption are additional risk factors for the development of adenomatous polyps. To our knowledge, this is the first work simultaneously investigating the risk factors for adenomatous and hyperplastic polyps in an asymptomatic Asian population.In this study, smoking is independent risk factor for developing both adenomatous and hyperplastic polyps with odds ratios between 1.31 and 1.87, respectively. In previous studies, cigarette smoking has consistently been a risk factor for colorectal adenoma . NonetheThis study revealed a strong positive association between BMI and both adenomatous and hyperplastic colorectal polyps. Previous studies indicate a higher BMI considered as overweight or obesity has revealed associations with the risk of colorectal adenomas and hyperplastic polyps , 22, butOur study also showed that old age and heavy alcohol consumption were independent predictors for developing adenomatous colorectal polyps in asymptomatic subjects. Advanced age is a well-known risk factor for the development of colorectal adenomas and advanced neoplasm . ColonosRegular alcohol intake has been reported as a risk factor for both colorectal adenoma and cancer in some but not all previous studies , 34. VerRegarding serrated adenomas, the prevalences of sessile serrated adenoma and traditional serrated adenoma in the current study were 1.8% and 0.7%, respectively. Compared with previous literatures , 38, theDespite its contributions, this study had certain limitations. First, self-selection bias of the population in this trial was possible because all enrolled subjects had undergone self-paid health examination and likely had better economic status than the general population in Taiwan . Second,In conclusion, the prevalence of colorectal polyps in asymptomatic subjects is 27.4% in Taiwan. High BMI and smoking are common risk factors for both adenomatous and hyperplastic polyps. Advanced age and alcohol consumption are additional risk factors for the development of adenomatous polyps of colorectum. The findings suggest that lifestyle modification including adequate control of body weight and abstaining from smoking and alcohol may potentially decrease the risk of developing colorectal adenomatous polyps. Further studies are warranted to investigate the mechanisms underlying the association of these lifestyle factors with the risk of adenomatous and hyperplastic polyps of the colorectum and to elucidate whether abstaining from smoking and alcohol really can reduce the incidence of recurrent colorectal polyps or advanced neoplasia.


N-glycosylation site were conserved across species. We also confirmed that the modori phenomenon was avoided by CA-074, a specific inhibitor for cathepsin B. Therefore, our results suggest that natural cysteine protease inhibitor(s), such as oryzacystatin derived from rice, can apply to thermal-gel processing of horse mackerel to avoid the modori phenomenon. Meanwhile, this endogenous protease may be used for food processing, such as weaning meal and food for the elderly.An endogenous protease in fish muscle, cathepsin B, was partially purified and characterized from horse mackerel meat. On SDS-PAGE of the purified enzyme under reducing conditions, main protein bands were detected at 28 and 6 kDa and their respective N-terminal sequences showed high homology to heavy and light chains of cathepsin B from other species. This suggested that horse mackerel cathepsin B formed two-chain forms, similar to mammalian cathepsin Bs. Optimum pH and temperature of the enzyme were 5.0 and 50 °C, respectively. A partial cDNA encoding the amino acid sequence of 215 residues for horse mackerel cathepsin B was obtained by RT-PCR and cloned. The deduced amino acid sequence contains a part of light and heavy chains of cathepsin B. The active sites and an  Lysosomal cysteine proteases, cathepsins B, H, L, and so on, play an important role in intracellular protein turnover. Cathepsin B EC3.4.22.1) is a major lysosomal cysteine protease and has distinctive characteristics which act not only as an endopeptidase, but also as a carboxyl dipeptidyl peptidase [ is a majIn marine food science, cathepsin B had been studied as a cause of the fish muscle softening. Muscle softening occurs much faster in fish than in livestock, leading to reduction in commercial value ,3. It ismodori phenomenon. It is one of the urgent problems in the fisheries industry. Consequently, there are some reports about the relationship between endogenous proteases and the modori phenomenon. Among the numerous proteinases present in fish muscle, cysteine proteases have the most serious effects on texture of thermal gel because of their thermo-stability and endopeptidase activity [et al. [modori phenomenon. MBSPs were also found in the skeletal muscle of lizard fish [While cathepsin B is also considered to participate in the gel disintegration during the processing of surimi-based products. Surimi-based products are a seafood product and very popular in Asian countries. It is made from fish muscle proteins, and fish meat is minced with salt to solubilize in the monomer of myofibrillar proteins, such as myosin and actin. Then, solubilized myofibrillar proteins form the network structure by heating and, as a result, the surimi gel is made. However, during heating, formed surimi gel can be destroyed by endogenous proteases in fish muscle; subsequently, quality loss of the gel may occur. It is a general concept that endogenous proteases cause thermal gel-weakening, which is called the activity . An et a [et al.  reported [et al. . In addi [et al.  as an enard fish , white card fish , yellow ard fish , silver ard fish , and cruard fish .Trachurus japonicas, is one of the most important species on seafood processing in Japan, and in particular this species is widely utilized as ingredient for surimi-based products. However, in the horse mackerel, the characteristics of the endogenous proteases and their involvement in modori phenomenon are still not clear. In our preliminary study, cathepsin B was the most active cysteine protease in horse makerel muscle at 50 °C, suggesting that the enzyme was likely to participate in thermal gel disintegration. Therefore, the present paper attempted purification, characterization, and molecular cloning of cathepsin B in horse makerel muscle. Moreover, we also investigated if the enzyme was responsible for the modori phenomenon. Ultimately the purpose in this study is to contribute to the quality improvement of surimi-based products using natural protease inhibitors and the possible application of endogenous proteases to seafood processing.Horse mackerel, Cathepsin B was partially purified 3132-fold from 1.2 kg of horse mackerel meat with a yield of 1.7%  using amPool B was applied to Mono S cation-exchange column, and the active peak was obtained A. Since modori phenomenon on horse mackerel meat. These results supported our hypothesis that cathepsin B was involved in the deterioration of surimi-based products of the horse mackerel meat. While, in common mackerel [modori phenomenon vary depending on fish species.Horse mackerel cathepsin B was examined the effects of pH and temperature using Z-Phe-Arg-MCA as a substrate . Optimummackerel  and bluemackerel , it has Cathepsin B activity was almost completely inhibited by E-64, CA-074, and chymostatin . These iThe enzyme mainly hydrolyzed Z-Phe-Arg-MCA, cathepsin B specific substrate . While, We cloned a partial cDNA corresponding to cathepsin B from the horse mackerel muscle by RT-PCR. The nucleotide and deduced amino acid sequences of cathepsin B cDNA (647 nucleotides) are shown in N-glycosylation site of horse mackerel cathepsin B were conserved in cathepsin Bs from other species.A sequence homology search of the deduced amino acid sequence of horse mackerel cathepsin B was performed on the NCBI Protein database with BLASTP and the multiple sequence alignment of the enzyme and cathepsin Bs from other species was shown in modori gel of horse mackerel meat with or without cathepsin B inhibitors . The minced muscle was used for protein purification and RNA extraction of cathepsin B.The minced meat of horse mackerel, SP-Sepharose, Superdex 75, and Mono S were obtained from GE healthcare . Fluorogenic peptide substrates, E-64, and Pepstatin A were purchased from Peptide Institute . Pefabloc SC was purchased from Merck KGaA . Protein standards for SDS-PAGE were from Bio-rad . All other chemicals used were of analytical grades.Cathepsins B activity was assayed using Z-Phe-Arg-MCA and Z-Arg-Arg-MCA as substrates according to the method of Tan . The reag for 30 min. The obtained supernatant was as the crude enzyme and was fractionated by ammonium sulfate precipitation (50%–70% saturation). The ammonium sulfate fraction was applied to SP-Sepharose cation-exchange column. Subsequently, the cathepsin B active fraction of SP-Sepharose was purified by Superdex 75 gel filtration column and Mono S cation-exchange column. All purification steps were performed at 4 °C. Cathepsin B was examined for purity using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).Horse mackerel meat (about 1.2 kg) was homogenized with four volumes of the extraction buffer  and centrifuged at 13,000× SDS-PAGE was performed according to the method of Laemmli  using 10Protein in column eluates was determined by measuring the absorbance at 280 nm. The protein concentration was determined according to the method of Lowry  using boThe purified enzyme was applied to SDS-PAGE, and transferred to a polyvinylidene difluoride (PVDF) membrane. After brief staining with Coomassie Brilliant Blue R-250, the protein bands were excised, destained, and analyzed by PPSQ-33A protein sequencer .The fresh ordinary muscle from the horse mackerel was excised and immediately soaked in five volumes of RNAlater  to stabilize the RNA in the tissue. Then, total RNA was isolated from the tissue by ISOGEN  according to the manufacturer’s protocol.16-3′) containing adapter primer sequence. The primers were removed from the reaction mixture by Microcon-100  and finally the first-stranded cDNA pool was obtained. For RT-PCR, two degenerated primers, a sense primer (5′-AGGAGATCAGNGAYCARGG-3′) and an antisense primer (5′-CAATYTCRGAYTCDATNCCAC-3′), were designed based on the N-terminal amino acid sequences of purified cathepsin B protein and the conserved sequence in other cathepsin Bs, respectively. Veriti Thermal Cycler and AmpliTaq Gold  were used for amplification and the reaction was performed with 1 cycle of 10 min at 95 °C, 35 cycles of 1 min at 94 °C, 1 min at 55 °C, 2 min at 72 °C, and 1 cycle of 7 min at 72 °C. The PCR products separated by agarose gel electrophoresis were extracted from the gel using QIAEX II Gel Extraction kit  and cloned into pGEM T-Easy Vector . Additionally, sequences of each clone were verified by DNA sequencing.The first-stranded cDNA was synthesized as a template for RT-PCR. The RNA was denatured and subsequently reverse-transcribed with SuperScript II RT  and the oligo-dT primer (CDS-BR: 5′-GGCCACGCGTCGACTAGTAC(T)DNA sequencing was carried out by the dideoxy chain termination method using BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) with 3130 Genetic Analyzer (Applied Biosystems). Sequence analysis was performed with MacVector software , the BLAST and the domain search programs on DDBJ/GenBank/EMBL databases.surimi paste was packed into a polyvinylidene chloride tube (diameter: 27 mm) and heated in a water bath at 90 °C for 20 min as a control or at 60 °C for 120 min to induce disintegration of the gel as a modori gel. After heating, the gel was immediately cooled into cold water and kept on ice before the measurement of gel strength.Minced horse mackerel meat was ground for 3 min by Stephan vacuum cutter UM-5  with 3% sodium chloride. The Evaluation of quality of the gels was carried out by measuring their gel strength. The gels were sliced into 15 mm and their breaking force (N) on a Rheoner creep meter RE3305 equipped with a spherical plunger (diameter: 5 mm)  with a rising rate of the sample table at 6 cm/min. Triplicate measurements were made and the mean value was presented.modori gel was increased by suppression of cathepsin B activity using CA-074, from these results, cathepsin B may cause modori phenomenon. Therefore, our results suggest that natural cysteine protease inhibitor(s), such as oryzacystatin derived from rice may apply to surimi-based product processing of horse mackerel to improve the quality of thermal gels. On the other hands, the further study will also be needed to utilize endogenous proteases for soft food processing, such as weaning meal and food for the elderly.This paper was the first report that cathepsin B was purified and characterized from horse mackerel muscle. The partial cDNA sequence of it was also determined. The characteristics of the enzyme from horse mackerel were similar to other species cathepsin Bs. In addition, the gel strength of


Overall, these results provide evidence that the eCB system is differently expressed in the retina of these mammals and suggest a distinctive role of eCBs in visual processing.The endocannabinoid (eCB) system is widely expressed in various parts of the central nervous system, including the retina. The localization of the key eCB receptors, particularly CB1R and CB2R, has been recently reported in rodent and primate retinas with striking interspecies differences. Little is known about the distribution of the enzymes involved in the synthesis and degradation of these eCBs. We therefore examined the expression and localization of the main components of the eCB system in the retina of mice, tree shrews, and monkeys. We found that CB1R and FAAH distributions are well-preserved among these species. However, expression of NAPE-PLD is circumscribed to the photoreceptor layer only in monkeys. In contrast, CB2R expression is variable across these species; in mice, CB2R is found in retinal neurons but not in glial cells; in tree shrews, CB2R is expressed in Müller cell processes of the outer retina and in retinal neurons of the inner retina; in monkeys, CB2R is restricted to Müller cells. Finally, the expression patterns of MAGL and DAGL N-acyl phosphatidylethanolamine-specific phospholipase D) and DAGLα , and the degradation enzymes FAAH (fatty acid amide hydrolase) and MAGL (monoacylglycerol lipase). The cannabinoid receptors are found in many mammals and in various classes of vertebrates and invertebrates and in all major subdivisions of bilaterians, urochordates, and cephalochordates but not in the nonchordate invertebrate phyla like insects [Marijuana contains over 70 cannabinoids that mimic the endogenous ligands called endocannabinoids (eCBs) that cause global psychoactive and physiological effects. The eCB system is mainly composed of the specific G-protein-coupled receptors CB1R and CB2R, the eCBs (anandamide and 2-arachidonoylglycerol), the synthesising enzymes NAPE-PLD , in the retina of different mammals have not been studied in depth. It is therefore our aim to analyze the expression of several components of the eCB system and to characterize their distribution pattern in the distinct retinal layers and cell types of three different mammalian species: mice, tree shrews, and monkeys (vervets and macaques).In the retina, the expression of CB1R is well-preserved in many species including mice, rats, chicks, larval tiger salamanders, goldfish, and rhesus monkeys . CB1R an the OPL . Both MAer cells . The expTupaia belangeri; 3-4 months old), 3 vervet monkeys , and 2 rhesus monkeys  were used in this study. The animals were part of ongoing research projects that were approved by the University of Montreal and McGill University Animal Care and Use Committees. For all species, anterior segment of the eye and vitreous were cut away. The eyecups were bathed in 4% paraformaldehyde made in 0.1 M sodium phosphate buffer at pH 7.4 and left overnight in the solution. The retina was dissected free from the eyecup in a phosphate-buffered saline (PBS) medium. It was laid flat so that the vitreous body could be removed by blotting with filter paper and gentle brushing. Samples of the retina were taken at the center and periphery. Each sample was then cryoprotected in 30% sucrose overnight and embedded in Shandon embedding media at −65°C. The blocks were cut in 20 μm sections at −18°C with a Leica CM3050S cryostat and mounted onto gelatinized subbed glass slides, air dried, and stored at −80°C for further processing.Eyes from 3 adult mice , 2 adult tree shrews  were also used conjointly with a known specific retinal cell-type marker .Single, double, and triple labeling of the retina were performed according to previously published methods , 24, 30.e marker . The nexα [α [In this study, we were confronted with the problem concerning the specificity of some of the antibodies, especially for the tree shrew. Although knockout animals are the best way to test the specificity of antibodies, this model is available only for mice and not for tree shrews and monkeys. We therefore resorted to the use of conventional alternative methods to circumvent this methodological limitation –27. We hα , FAAH [2α , 27, GS α , 37–39, α , rhodopsα , 41, andα [α , 27, 34.μm of thickness. All photomicrograph adjustments, including size, color, brightness, and contrast, were done with Adobe Photoshop  and then exported to Adobe InDesign , where the final figure layout was completed.Immunofluorescence images were taken according to . Using aA fairly consistent retinal distribution pattern of CB1R across all six retinal layers was observed in mice, tree shrews, vervet, and rhesus monkeys, as illustrated in immunolabeled retinal sections Figures . The mosFAAH, like CB1R, is well expressed in all retinal layers and in the photoreceptor layer of all species Figures . In all In mice, NAPE-PLD is widely distributed in all layers but more intensely in the NFL  was used. This antibody has proved to be efficient to label Müller cells in the rat , mouse , and monCalbindin (CB) is a marker of cones outside the foveal region, cone bipolar cells, and a subset of horizontal cells in tree shrews and monkeys , 32. On The rhodopsin antibody was used to label rods in the retina. In the mouse, NAPE-PLD is not coexpressed with rods , and 2 endocannabinoid degrading enzymes (FAAH and MAGL) in the retina of mice, tree shrews, and monkeys. This is the first study that shows the expression pattern of all the above-mentioned eCB components in the tree shrew retina as well as the localization of the NAPE-PLD, MAGL, and DAGLα in the monkey retina , 2 endocannabinoid synthesizing enzymes  are biosynthesized from phospholipids of the cell membrane assisted by NAPE-PLD hydrolysis. In this study, we report a variation in the expression of this membrane associated synthesis enzyme, NAPE-PLD, despite its well-preserved sequence from rodents to humans [Anandamide (an endogenous agonist of the CB1R) and othero humans . In the o humans . Besideso humans , anorexio humans , and proo humans . Moreoveo humans . This paα and MAGL expressions are in the vicinity of CB2R. In the mouse retina, the DAGLα and MAGL expressions are often near or in the same cell types as CB1R and CB2R. CB1R is present in cones, horizontal, bipolar, amacrine, and ganglion cells in the rat retina [Given that the lipophilic eCBs are released and degraded close to their action site, it would be reasonable to assume that the DAGLt retina , 27. CB2t retina . This dit retina . But the


Currently there are five known mechanisms of horizontal gene transfer (HGT): transduction, conjugation, transformation, gene transfer agents and membrane vesicle transfer. The question here is: what part did HGT play in the reorganisation of genetics during the last universal common ancestor (LUCA) epoch? LUCA is a construct to explain the origin of the three domains of life; namely Archaea, Bacteria and Eukarya. This editorial offers a general introduction to the relevance and ultimate significance of HGT in relation to the LUCA. Horizontal gene transfer (HGT) is the transfer/exchange of genetic material between extant donor and recipient cells or the active take-up of strands of “naked” nucleic acids from the environment by competent cells, which are then themselves transformed. This is opposed to vertical gene transfer (VGT), the transfer of genes from parent to progeny. HGT is a ubiquitous mechanism, particularly prevalent in microbes; it occurs between the cells of the same genus as well between different genera and even different domains of life. HGT is an important process in modern biological systems—for example it has been shown that the modification of photosynthetic apparatus in cyanobacteria is widespread in marine phytoplankton as was demonstrated by the recent identification of photosystem I (PSI) and photosystem II (PS II) genes in viruses (cyanophages) of marine cyanobacteria. In this case, cyanophages seemingly help to optimise photosynthesis by providing additional photosystem genes with different properties. Having defined the parameters of HGT, the question which we aim to address in this special issue is: “what part did HGT play during the reorganisation of genomes during the era of the last universal common ancestor (LUCA) which led to formation of the three main domains of life?”It is the focus of this special issue to consider the involvement of HGT in relation to the LUCA, beginning with the formation of nucleic acids and the subsequent emergence of the LUCA, prior to the three domains becoming distinguished as separate branches in the development of life. There is good evidence for ancient HGT events, indicating that the evolutionary history of genes within genomes is best understood in terms of networks. Thus, we invite articles that consider the extent to which HGT contributed to the evolution of LUCA and the very early evolution of life on Earth. 30 viruses in the oceans alone, which far exceeds the total number of different types of living organisms on Earth (108); although this is perhaps only a small proportion of the actual total, it is unlikely to be anywhere near as high as 1030. There are reported to be a staggering 1029 viral infections per day and even if we speculate that only one millionth of 1% of these are involved in transduction, this is still quite a feat in itself.The importance of HGT cannot be underestimated as it is probable that life on Earth as we know it today would not be likely to exist without it. One example being transduction, as viruses represent the largest pool of genetic diversity, with an estimated 10To explore the phenomenon of HGT we can begin by stating that genes, being the controller of metabolisms (at least in terms of modern biology), are preserved in all life forms. These manifestations of the “controller” molecules can be observed in the unchanging biochemistry of life since, even if the controller itself mutates, overall the molecules of life and the pathways in which they are involved, do not.de novo is exceptionally difficult because there are no known simple mechanisms by which it could be made. Some scientists believe that DNA arose at or during the first branch of the phylogenetic tree of life and that it probably arose twice independently. That is, DNA would have emerged after the appearance of RNA, and in this view RNA must have been dominant during the LUCA era. Although DNA is much more stable compared to RNA, the latter has two essential requirements which “trump” DNA, in that it can act both as a repository for genetic information and as a catalyst (ribozyme). Additional circumstantial evidence in favour of RNA being the initial controller can be gleaned from the tree as well as from RNA viruses  present at the time of LUCA. What were the initial controller molecules? There are three possibilities; either DNA or RNA, or perhaps an unknown precursor molecule—such as threose nucleic acid (TNA). We don’t know much about TNA or an analogous molecule because, if it was part of initial chemistry, it was not preserved and is lost for all eternity; the best we could do is to draw-up a list of candidates with the relevant properties which could have set in motion the route to the formation of first genes. DNA is also an unlikely precursor because today it is made from RNA by replacing ribose with deoxyribose sugar and uracil nucleobase with thymine; to synthesise DNA In fact, on the basis of biochemical/genetic analysis it can be surmised that virus particles in existence at the time of LUCA contained RNA, with both DNA and DNA replication machineries emerging later. This conclusion can be reached because some viruses share homologous capsid proteins and/or ATPase proteins for packaging, suggesting that they evolved from a virus that existed at the time of or just prior to LUCA. With this in mind, it is believed that RNAs were involved in HGT and thus hold clues to the understanding of the reorganisation of genetics that led to the emergence of the three domains of life, namely Archaea, Bacteria and Eukarya.i.e., did Bacteria lead to their origin, or did proto-eukaryotes give rise to Bacterial and Archaeal domains? Finally, Prof. Micahel Syvanen (see summary report in this issue) demonstrated that, by reworking the phylogenetic tree, the LUCA may not be an appropriate device when describing the origin of its three branches.The idea of phylogenetic tree of life was first suggested by Charles Darwin under a less fanciful title of “common ancestry” meaning that every living species on Earth arose from a single common organism, which is now referred to as LUCA. But the hard empirical evidence for the phylogenetic tree began with the pioneering work of Woese and Fox , who comThe existence of a common genetic code is one of the most compelling evidence in support of the phylogenetic tree and so in light of this there appears to be a common ancestry, leading to the prediction of at least three domains of life; these being possibly derived from viruses and LUCAs which intermingled freely and exchanged MGEs frequently. i.e., whether LUCA means the ancestor of all things alive on Earth today... or of all things that have ever lived on Earth.... In the case of latter supposition it is suggested that, perhaps, its correct title should be the “first universal common ancestor”. Such reasoning raises another question: what came before LUCA? Since RNA acts both as a repository of information and as a catalyst, perhaps there were evolving entities made purely from RNAs. So, what was the nature of LUCA? Some theoretical biologists think that LUCA was only one of several designs for early life, from which a single entity capable of evolving into prokaryotes arose. Others have questioned its existence altogether; that is, some scientists maintain that there may not even have been such an entity as the LUCA at all, and that it should no longer be considered as a relevant part of evolutionary theory—this view being the central tenet of the “metabolism first hypothesis” as opposed to the “genes first hypothesis”.The LUCA is a moot point in that it is a theoretical construct designed to explain the origin of especially the Bacteria and Archaea domains, collectively called prokaryotes. It is believed that LUCA existed at the time that these two domains became separate entities in their own right. From this it can be surmised that LUCA may have been a complex and almost fully formed living entity which probably even had DNA as a repository for information, as has been shown by various comparative genomic studies. More to the point, Prof. John Allen  proposed: what does the word “last” in “last universal common ancestor” signify? There are two lines of thought on this question, i.e., via HGT). On the balance of probability and based on the evidence derived from the mechanisms used in HGT (see below), I believe it is more than likely that there was a sort of “united nations of LUCAs” in operation at the time.However a middle ground is held by those scientists who believe that the LUCA was not a single entity, but a consortium of many “LUCA-like” entities (as was proposed by Prof. Armen Mulkidjanian—see summary report). Use of the term “LUCA-like” is deliberate because although similar to the single entity, the components of such a consortium would not have been individually “complete”—sort of proto-LUCAs, as it were. In order for these entities to move up to the next level and form a complete LUCA, they would have had to exchange genes with each other and therefore exist in close proximity, which could have been encased within “semi-permeable” bubbles of clay on the sea floor and/or floating about in small pools of water, where their concentration would have been sufficiently high enough for interaction. This would have facilitated exchange of genetic material  can either be preserved through biological history of species, or by the acquisition of new divergent properties thereby allowing speciation to occur. Such studies involve examining both similarities and differences that arise, and become fixed due to selection pressure, in the appropriate elements (as above) from different species and thus can yield information about the function and evolutionary processes that act on genomes. In turn, we gain insight into new discoveries such as non-coding functional elements of the genome, and perhaps even the threshold levels of mechanism of evolution.Which process of evolution (Lamarckian or Darwinian) was predominant at the time of LUCA? Although the required threshold for Darwinian evolution to become a viable mechanism is uncertain, it is highly probable that Lamarckian evolution was in operation during the LUCA period. Moreover, when a LUCA gains a nucleotide(s), gene or an operon etc. MGEs are important in terms of gene modification, gene duplication, gene pools, acquisition of physiological properties and/or metabolic traits that are not necessarily concordant with VGT.What are the mechanisms by which HGT occurs? Currently these include: transduction, a process whereby a viral capsule is used to transfer genetic material from one cell to another; conjugation, a process exhibited by microbes during which a plasmid or a small piece of a plasmid from one donor cell is transferred to another recipient cell ; transformation, which occurs when a competent cell takes up a “naked” strands of nucleic acid from the environment—such strands of nucleic acids may not necessarily have been exuded by living entities , they could also be from recently dead cells, as well as from long extinct organisms ; gene transfer agents (GTA), which are bacteriophage-like particles containing random cellular genomic segments intended for transduction to another living recipient cell (Prof. J. Thomas Beatty—see summary report); and membrane vesicle transfer (MVT), in which small membrane sacs emanating from the surface of a cell contain genetic material for transfer to another living recipient cell. Collectively, the genetic materials being transferred are termed mobile genetic elements (MGEs) which include strands of naked nucleic acids, transposons, phages, plasmids Studies of HGT mechanisms in evolution represent new and frontier science in which old techniques are being revised and novel techniques are being devised in order to elucidate the genetics surrounding ancient genomes—for example the discovery that a pentaribonucleotide (GUGGC) is able to carry out aminoacylation, transacylation and peptide synthesis raises the profile of RNA in relation to the major part it played in the origin of life. In other studies it has been shown that one of the major evolutionary HGTs took place when certain genes from mitochondria  and chloHGT is a ubiquitous process occurring in nature, via one of the discussed mechanisms or a combination of them. It is highly probably that “RNA-organisms” and/or LUCA entities were exchanging MGEs on an extensive scale, thereby bringing about the reorganisation of genetics which underpins all living organisms today.


Congenital hearing loss is the most frequent birth defect. The American Academy of Pediatrics and the Joint Committee on Infant Hearing established quality of care process indicators for Universal Newborn Hearing Screening starting from 1999. In a previous systematic review of Universal Newborn Hearing Screening studies we highlighted substantial variability in program design and in reported performance data. In order to overcome these heterogeneous findings we think it is necessary to optimize the implementation of Universal Newborn Hearing Screening programs with an appropriate application of the planning, executing, and monitoring, verifications and reporting phases. For this reason we propose a conceptual framework that logically integrates these three phases and, consequently, a tool (a check-list) for their rationalization and standardization.Our paper intends to stimulate debate on how to ameliorate the routine application of high quality Universal Newborn Hearing Screening programs. The conceptual framework is proposed to optimize, rationalise and standardise their implementation. The checklist is intended to allow an inter-program comparison by removing heterogeneity in processes description and assessment.The online version of this article (doi:10.1186/s13052-016-0223-1) contains supplementary material, which is available to authorized users. Sensorineural hearing loss is one of the most frequently occurring permanent congenital defects at birth with a prevalence of 0.1–0.3 % for newborns   programs   to evaluWith the aim to verify whether literature reporting experiences on hospital-based UNHS programs include sufficient information to allow inter-program comparisons according to the already available indicators/benchmarks defined by the AAP and JCIH, we performed a systematic review . We founIn order to overcome these heterogeneous findings we think it is necessary to optimize the implementation of Universal Newborn Hearing Screening programs with an appropriate application of the planning, executing, and monitoring, verifications and reporting phases. For this reason we propose a conceptual framework that logically integrates these three phases and, consequently, a tool (a check-list) for their rationalization and standardization.Deliver the protocol Action is activated, apart from the first instance, if the benchmarks are not achieved, if guidelines/recommendations are updated, or if the context specificities change. The output of this phase is the protocol for UNHS execution.A planning phase based on indications from guidelines and recommendations, specificities of the local context, benchmarks, reports from verification phase. The An executing phase where the protocol is applied and where data should be generated and managed for monitoring. The outputs of this phase are the raw data for process monitoring.Monitor action is activated periodically or upon request to aggregate data and build performance indicators. The verification is made comparing indicators with benchmarks and a report is generated with an analysis of reasons for possible deviations which can push for a redefinition of the protocol and/or for a re-organization of its execution. Reports can be used also for disseminating purposes.A monitoring, reporting and verifying phase where the The framework is structured on several phases .A more detailed description of the single phases follows in the following sections  or are pulation ).The audiological risk criteria are relevant for that: specific audiological risk may require a specific screening protocol; the timing and number of hearing re-evaluation (surveillance) for infants at risk should be customized and individualized depending on the relative likelihood of a subsequent delayed onset hearing loss.The typology and the number of tests, and the healthcare setting in which performing the examinations (before or after the discharge) – The program needs to be well balanced for sensitivity, specificity, coverage of the population and costs per subject identified. E.g., Kennedy et al. [The presence of specific protocol for neonates at higher risk  [. The set of examinations for the full audiological evaluation – E.g., the one recommended by the JCIH [The tasks to be performed to increase the percentage of enrolment and to reduce the lost to follow up (neonates referred to further examinations that do not show at the planned appointments) - With reference to the former, it has to be noted that specific actions should be done for an appropriate communication with families creating the conditions for an informed consent. With reference to the latter, a survey conducted in USA [The surveillance program for early identification of infants and children with late onset  – It is recommended to perform regular surveillance of developmental milestones, auditory skills, parental concerns, and middle-ear status to for all infants, together with an objective standardized screening of global development, at 9, 18, and 24 to 30 months of age or at any time if the health care professional or family has concern [The cooperation among all the involved operators, services and institutions - The identification of the key roles is an essential step for an appropriate management of the entire process and for monitoring purposes.Activities, detailed actions, decision nodes, workflows, roles, environmental conditions have to be identified and specified. More specifically key issues are reported. y et al.  reported TEOAEs)  - Such nthe JCIH . The tasd in USA  shows thd in USA  that sta concern . The cooUniversality– completeness of universality in both recruitment and follow-up phases;Timely detection– specification of follow-up deadlines for identification and intervention, and determination of the observed prevalence;Overreferral – Efficient use of highly specialized care.In our systematic review  the AAP For each reported indicator a reference benchmark has been identified  which reMonitor, Verify and Report Activity and it is used to make process results explicit and, as previously reported, as the basis for possible re-planning and/or reorganization.This is the output of the The proposed framework has been used as a conceptual guidance for building a checklist (Additional file As reported in the 2007 JCIH Position Statement , regularThe paper is a contribution toward a standardisation in reporting UNHS experiences which may favour the emerging of best practises.


A survey related to universal newborn hearing screening was created with permission granted, based on the following study and adapted to the Italian situation: Primary Care Physicians’ Knowledge, Attitudes, and Practices Related to Newborn Hearing Screening .First distributed in October, 2008, the survey was given to 300 pediatricians of Tuscany on a regional level, and was then adapted as an online questionnaire made available to the members of the Italian Pediatric Federation (FIMP) in April, 2009. The purpose of the survey was to verify the attitudes, experience and knowledge of family pediatricians, in regard to newborn hearing screening, hearing loss and other related arguments.The principle objectives of the study were (1) to provide an overview of the current situation of pediatricians in regard to newborn hearing screening and follow-up; (2) to evaluate the knowledge and experience of pediatricians regarding newborn hearing screening programs and (3) to identify the information gaps in order to evaluate the most effective methods of providing the resources and information necessary to assist the families whose children are diagnosed with hearing loss.The results indicated that there are knowledge gaps as to how to properly manage cases of children with hearing loss .In the past five years, the FIMP Audiology Network has performed Refresher courses on Newborn Hearing Screening for over 5000 pediatricians. A principle concentration on Late Onset, Acquired and Progressive Hearing Loss is now a fundamental objective.If Pediatricians do not intervene on a national level to identify late onset hearing loss  and do not perform that which is defined as “audiological surveillance”, the screening thereby fails in its essence of being “universal”.  In fact,Italian Pediatricians are in the position to guarantee this level of surveillance.


Alternaria brassicicola causes black spot disease of Brassica species. The functional importance of pectin digestion enzymes and unidentified phytotoxins in fungal pathogenesis has been suspected but not verified in A. brassicicola. The fungal transcription factor AbPf2 is essential for pathogenicity and induces 106 genes during early pathogenesis, including the pectate lyase-coding gene, PL1332. The aim of this study was to test the importance and roles of PL1332 in pathogenesis. We generated deletion strains of the PL1332 gene, produced heterologous PL1332 proteins, and evaluated their association with virulence. Deletion strains of the PL1332 gene were approximately 30% less virulent than wild-type A. brassicicola, without showing differences in colony expansion on solid media and mycelial growth in nutrient-rich liquid media or minimal media with pectins as a major carbon source. Heterologous PL1332 expressed as fusion proteins digested polygalacturons in vitro. When the fusion proteins were injected into the apoplast between leaf veins of host plants the tissues turned dark brown and soft, resembling necrotic leaf tissue. The PL1332 gene was the first example identified as a general toxin-coding gene and virulence factor among the 106 genes regulated by the transcription factor, AbPf2. It was also the first gene to have its functions investigated among the 19 pectate lyase genes and several hundred putative cell-wall degrading enzymes in A. brassicicola. These results further support the importance of the AbPf2 gene as a key pathogenesis regulator and possible target for agrochemical development. Alternaria brassicicola is a destructive plant pathogen and causes black spot disease on almost all plant species in the Brassicaceae  / (0.1 x 5 x 2), and Units/μg protein = (Units/ml enzyme)/(μg protein/ ml enzyme). To visualize the relative amounts of free iodine at the end of the pectate lyase reactions, we added equal amounts of Na2S2O3 to each reaction to sequester the same amount of free iodine. Finally, we added starch to visualize residual iodine.The enzyme activity of pectate lyase was measured by a titrimetric stop reaction method, following a previously described protocol with appropriate modifications . A solutB. oleracea, B. juncea, or B. campestris var. chinensis using syringes with 26G x 13 mm needles. The intercellular space became waterlogged with protein solution and the soaked tissue remained visible for several hours. After infiltration, the leaves were maintained in mini humidity chambers and the development of necrotic tissue was observed for up to one week. Infiltration experiments were conducted more than three times and the progress of tissue damage was recorded daily with a digital camera.The necrosis-inducing activity of PL1332 was examined with the PL1332 fusion proteins. The proteins were resolved in protein-elution buffer  or 10 mM Tris-HCl buffer and injected into young leaves of S1 FigThree exons are marked in yellow.(DOCX)Click here for additional data file.S2 Fig-1 = [(milliliters of titrant for blank- milliliters of titrant for test) x dilution factor x 100] / (0.1 x 2). Units mg-1 protein = (Units/ml enzyme)/(mg protein/ ml enzyme). A. Reactions for 1 hour or 16 hours at 25°C. B. Reaction under two different temperatures, 25°C and 37°C. Con: commercial enzyme control, Elu: purified MBP-PL1332 fusion protein.Enzyme activity was calculated from free iodines that were not oxidized by oligogalacturonic acids, thatoriginated from polygalacturonic acids after digestion by a commercial enzyme  or purified MBP-PL1332 fusion protein. The relative enzyme activity was calculated by relative units ml(EPS)Click here for additional data file.S3 FigPL: MBP-PL1332 fusion proteins; MBP: Maltose binding proteins; Buffer: 10 mM maltose in wash/elution buffer ; DW: deionized water.(EPS)Click here for additional data file.


E~ on the half thickness ℓ of a thin current sheet (TCS) is investigated for three cases of TCSs crossings. The value of ℓ was estimated from the magnetic field data by means of Cluster observations. The obtained scaling values for TCSs, Z~=ℓ/ρT, where ρT is the thermal Larmor radius, were compared with the scaling Zμ=22E~/T, where E~ and T are the average plasma energy and the temperature of plasma, which assumes a specific dynamics (conservation of magnetic flux through the trajectory segment) of the current carriers. The comparison of Z~ and Zμ shows a good agreement.The influence of average plasma energy  The new scaling is presentedThe energy‐based scaling compared with observable thickness of TCSThe population of the TCS‐forming protons results in different scaling The information about ℓ (or λ) could be used for the estimation of accumulated (or released) energy in a TCS .In our study, we use proton temperature ℓ could be obtained from the consideration of the local electric current jM as a function of the running coordinate z , where the local electric current is given as jM(z) = dBL/dz. In this case, the half thickness ℓ is usually defined as a distance between two points at which jM reaches the value of 0.5max(jM(z)). The calculation of z is based on a simple equation z=∫t0t1VN(τ)dτ, where VN is the normal plasma velocity whereas t0 and t1 are the start time and end time of integration, respectively. Obviously, the assumptions regarding the local normal velocity VN have strong influence on the calculation of z, and nowadays there exist several experimental methods to define VN. Unfortunately, due to different restricting factors none of these methods can give precise estimates for ℓ. That is the reason why we use in our study two methods of determining VN simultaneously.The information about VN is defined as a ratio of the distance between two satellites to the delay of the TCS crossing time. This technique is widely used to determine the geometry of plasma processes =3.25T[keV]/|B|[nT]. In fact, the measured values |B| and T could be different before and after the TCS crossing. In the considered case |B|= =  nT, and T =  =  keV /2. Similarly, to simplify the treatment, also an average value for the Larmor radius ρ=(ρTbefore+ρTafter)/2 is considered; and finally, the average magnetic scaling may be defined as <Z~>=<ℓ>/ρ. In the considered case we obtain <Z~>=4.65.Since we do not know which of two methods for the estimation of f(E), the average energy E~  equally contribute the creation process of the main electric current in the TCS. In view of that, the total μ scaling of the TCS could be expressed by simple averaging <Zμ>=(Zμbefore+Zμafter)/2.The reason why we distinguish between Zμ>=4.6. That is rather close to the corresponding <Z~> obtained above.In the considered case the scaling is <2.2LMN vectors L = , M = , N =  for C1 and L = , M = , N =  for C2. The behavior of BL components (for both satellites) is presented in Figure The case 2 has start time/end time at 22:50:00/23:00:00 UT on 14 September 2001. As in the previous case, all Cluster satellites have observed the TCS ) gives ℓtim=1200 km and ℓrec=900 km.The distributions of electric currents, calculated by two methods, are presented in Figure Z~=ℓ/ρT, we compute the thermal Larmor radius for the considered case. The magnetic field measured on different sides of the TCS is |B|= =  nT  for the considered case are presented in Figure E~== keV. That finally gives the total averaged μ scaling of the TCS <Zμ>=6.2, which is close to the above estimated magnetic scaling <Z~>=5.25.The 1‐D distribution functions 2.3L = , M = , N =  for C1 and L = , M = , N =  for C2. The behavior of BL components (for both satellites) is presented on Figure The event of the TCS crossing in case 3 has start time/end time at 15:28:30/15:32:00 UT on 12 August 2001. All Cluster satellites C1,C2,C3, and C4 observed the TCS ) gives ℓtim=1100 km and ℓrec=900 km.The distributions of electric currents along the running coordinate calculated by two methods are presented in Figure B|= =  nT  for the considered case are presented in Figure E~== keV. That results in the total averaged μ scaling of the TCS <Zμ>=4.3.The 1‐D distribution functions <Z~>=2.25 and <Zμ>=4.3 (by a factor 2).This case is particularly interesting because of some disagreement in the estimations of ℓ may be not sufficiently precise. Another explanation could be that both types of proton populations, i.e., Iz=const and μ = const protons, equally contribute the main current in the TCS. In this case the scaling of the TCS should be between the corresponding ZI and Zμ. To check this possibility, we calculate additionally the TCS scaling in case of Iz=const protons. We use the following asymptotic expression ZI=(VT/VD)1/3. It corresponds the assumption of VD≫VT [Sitnov et al., Sasunov et al. [VT and VD in terms of T and E~, respectively, and finally obtain ZI=(T/2E~)1/6. For the particular parameters of the considered case, i.e., E~=8 keV, T = 3.4 keV, this scaling is ZI=0.77.One of the possible explanations is that the satellites might oscillate inside the TCS (in frame of LMN system) while gradually leaving it. The specific behavior of magnetic filed magnitude /2 = (4.3 + 0.77)/2 = 2.53. The obtained value is close to the observed scaling <Z~>=2.25, which might confirm the validity of the made assumption.Note that both 3z give sometimes different internal structure of the TCS, such as bifurcation (see Figures In this paper we present the study of three TCS crossings observed by Cluster in 2001. It turns that the different methods for determination of the running coordinate −Iz=const and μ = const conditions, respectively, result in different scaling. That enables to interpret the measured geometry features of the real TCSs without making additional assumptions regarding magnetic field profile and the corresponding renormalization. In particular, regarding the considered cases, the proposed Zμ may be used to predict the thickness of the TCS and, by comparing it with the thickness obtained from the spacecraft measurements, to judge on the type of dynamics of the current carriers which create the TCS. In that respect the results of Runov et al. [Runov et al. [μ = const protons. However, to confirm this preliminary conclusion, it will be necessary to make an additional statistical comparative study of the <Z~> and Zμ scalings.We note that different dynamic regimes of the TCS‐forming protons, which correspond to v et al.  and Runov et al.  may indiμ = const, then a graduate change of the protons' dynamic regime toward the Iz=const type will result in the decreasing ℓ due to the change of the scaling from Zμ>1 to ZI<1. The experimental check of this idea is a subject for further study. It concerns also the issue of the stability of such TCSs and their related transformations.The proposed concept of two types of the TCS‐forming proton populations may be also used to construct a scenario for the adiabatic compression of a TCS without changing of magnetic field value. For instance, if the initial TCS is created by protons with


Phlebotomus papatasi, Psammomys obesus, and Meriones spp., with transmission of zoonotic cutaneous leishmaniasis  across Libya. Here, we map risk of ZCL infection based on occurrence records of L. major, P. papatasi, and four potential animal reservoirs . Ecological niche models identified limited risk areas for ZCL across the northern coast of the country; most species associated with ZCL transmission were confined to this same region, but some had ranges extending to central Libya. All ENM predictions were significant based on partial ROC tests. As a further evaluation of L. major ENM predictions, we compared predictions with 98 additional independent records provided by the Libyan National Centre for Disease Control (NCDC); all of these records fell inside the belt predicted as suitable for ZCL. We tested ecological niche similarity among vector, parasite, and reservoir species and could not reject any null hypotheses of niche similarity. Finally, we tested among possible combinations of vector and reservoir that could predict all recent human ZCL cases reported by NCDC; only three combinations could anticipate the distribution of human cases across the country.Cutaneous leishmaniasis ranks among the tropical diseases least known and most neglected in Libya. World Health Organization reports recognized associations of  Leishmania major is the potential etiological agent associated with all ZCL cases. In many countries across North Africa, L. major is transmitted by the sand fly Phlebotomus papatasi, with rodents as likely reservoir hosts. In Libya, ZCL cases are underestimated for lack of reporting, insufficient information about the distribution of ZCL, and interactions between local environmental conditions and different disease components. This situation worsened with recent political and socio-economic changes in the country, with expansion and rapid increases in numbers of cases across the country. For management and planning of leishmaniasis control, predicting the potential geographic distribution of risk of infection with the disease is important to guide such programs. We introduced ecological niche model as a tool for risk-mapping of both ZCL cases and distributions of associated species. Our models were able to anticipate areas of highest risk with statistical significance, lending confidence that they were successful in identifying areas of transmission risk.Zoonotic cutaneous leishmaniasis (ZCL) represents a major public health problem in North Africa where  Leishmania: Leishmania major Yakimoff & Schokhor, 1914 and L. tropica Wright, 1903 (Kinetoplastida: Trypanosomatidae). Leishmania major is the etiological agent of zoonotic CL (ZCL), where the parasite is thought to circulate in small-mammal reservoirs , Gerbillus gerbillus Olivier, 1801 (Rodentia: Muridae), Psammomys obesus Cretzschmar, 1828 (Rodentia: Muridae), M. shawi Duvernoy, 1842 (Rodentia: Muridae)) and is transmitted by the sand fly Phlebotomus papatasi (Scopoli), 1786 (Diptera: Psychodidae) )http://enmtools.blogspot.com/). For successful ZCL transmission, pathogen, vector, and host species should overlap spatially and ecologically . ,57. 2,57 Morocco . Elevati Morocco . Low-ele country .G. gerbillus as a main reservoir across Libya. In our previous analysis in Egypt, however, we found marked niche similarity between P. papatasi and L. major, but none between L. major and P. sergenti in terms of geographic distribution and ecological niche [Ps. obesus co-occurred with the vector. WHO had reported that Ps. obesus was likely the main reservoir of ZCL in Libya [Meriones spp.–P. papatasi system, which were able to anticipate all recent human cases. Evidence for this association has also been found in the form of high infection rates with L. major in Meriones tristrami in the most recent ZCL foci documented in Israel [Although testing niche similarity among species was unable to distinguish among hypotheses of ZCL hosts, as was possible in our previous analyses in Egypt , our anaal niche , supportin Libya ; howevern Israel ; these on Israel –62; earln Israel , but latn Israel . CertainL. major among different host subpopulations, and the dynamics and potential distribution of host and parasite at finer scales across the country. Filling these gaps as regards the disease system in Libya will promote a more detailed picture both for its ecology and for control programs.The study of the association among these organisms in both spatial and temporal dimensions is of great added values to map the ZCL risk areas across Libya, guide the control program across the country, and provided the first detailed maps for the potential distributions of organisms associated with the zoonotic transmission cycle across Libya. An early study shed light on disease ecology and possible host-pathogen associations , discussLeishmaniasis control programs should consider our findings by applying integrated approaches to combating ZCL by considering the environmental risk factors that we have explored. That is, if a particular combination of host and vector species is necessary for leishmaniasis transmission, then strategies by which to interrupt that transmission can focus on removing the pathogen, the vector, or key hosts from the system. Such measures may be implemented via educational programs in risk areas, mass drug administration in infected communities, and host or vector control programs. Our future work will focus on possible hotspots in the less-well-known areas of the country via intensive disease surveillance and sampling of all relevant organisms. More deeply, we plan to consider socioeconomic variables in tandem with the physical environmental variables for a more universal model that links physical, biological, and human factors in this complex disease system.S1 Filehttps://www.climond.org/.Details on these variables are also available via (PDF)Click here for additional data file.S2 FileModels were calibrated directly across Libya. The pink areas represent modeled suitable conditions, and gray areas were modeled as unsuitable for the species.(PDF)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 FileThe diagram shows the overall environment available across Libya (gray), and the suitable conditions for species occurrences (pink).(PDF)Click here for additional data file.S5 FileD (left column) and Hellinger’s I (right column) similarity metrics.The red vertical line represent the observed niche overlap between the two ENMs in the question. The results of the background similarity tests were based on Schoener’s (PDF)Click here for additional data file.S6 FileThese cases were reported by the local health units in each province and notified to the center for control measures based on the endemic status of each focus. These cases were identified by passive surveillance, and were not diagnosed to the species level.(PDF)Click here for additional data file.S7 FileDistricts with high incidence are shown in blue, and localities within each district is presented as a dotted points. The map of Libya at the top shows the distribution of areas with water resource management initiatives as blue circles.(PDF)Click here for additional data file.


Meanwhile, quantitative real-time PCR indicated that most of the TCS genes in cucumber were specifically or preferentially expressed in certain tissues or organs, especially in the early developing fruit. Some TCS genes exhibited diverse patterns of gene expression in response to abiotic stresses as well as exogenous trans-zeatin (ZT) and abscisic acid (ABA) treatment, suggesting that TCS genes might play significant roles in responses to various abiotic stresses and hormones in Cucurbitaceae crops.Cucumber and watermelon, which belong to Cucurbitaceae family, are economically important cultivated crops worldwide. However, these crops are vulnerable to various adverse environments. Two-component system (TCS), consisting of histidine kinases (HKs), phosphotransfers (HPs), and response regulator proteins (RRs), plays important roles in various plant developmental processes and signaling transduction in responses to a wide range of biotic and abiotic stresses. No systematic investigation has been conducted on TCS genes in Cucurbitaceae species. Based on the completion of the cucumber and watermelon genome draft, we identified 46 and 49 TCS genes in cucumber and watermelon, respectively. The cucumber TCS members included 18 HK(L)s, 7 HPs, and 21 RRs, whereas the watermelon TCS system consisted of 19 HK(L)s, 6 HPs, and 24 RRs. The sequences and domains of TCS members from these two species were highly conserved. Gene duplication events occurred rarely, which might have resulted from the absence of recent whole-genome duplication event in these two Cucurbitaceae crops. Numerous stress- and hormone-responsive  Arabidopsis, AHK2, AHK3, and AHK4 function as cytokinin receptors and respond to stress negatively in cytokinin signal via TCS. These cytokinin receptors perceived cytokinin and were autophosphorylated. Then AHPs received the phosphate group from HKs and transferred to the Rec domain of type-B RRs. Finally, type-B ARRs activated type-A ARRs from putative repressors in the nucleus. Additionally, type-B ARRs could bind to multiple cis-elements in the promoter of target genes, such as MAPKs and other stress-related genes, to participate in stress responses  via phosphorylation between histidine and aspartic-acid (His-Asp) residues has been widely reported in prokaryotes  which is a member of HK family are more sensitive to heat stress via affecting pollen carbohydrate metabolism  and watermelon (Citrullus lanatus), which belong to the Cucurbitaceae family, are economically important crops consumed worldwide. However, these crops are highly susceptible to various adverse environments, such as inappropriate temperature, drought, and pathogens. TCS play essential roles in plant stress signaling network. Thus, applying a systematic identification and functional study of these stress responsive genes is necessary to elucidate the molecular mechanisms of tolerance and susceptibility in Cucurbitaceae crops . So far, few TCS genes have been characterized in Cucurbitaceae crops . Comprehensive analysis of TCS elements in Cucurbitaceae species provided insights into the structure and evolution of this system. Moreover, the results elucidated the potential roles of the TCS elements in stress and hormone response, which would provide a framework for future functional dissection of TCS in plant hormone and stress signal transduction.Cucumber  and then used as queries to perform BLASTP searches with E-value of 1e−5 as the threshold , which were applied to identify the cucumber and watermelon TCS proteins using HMMER 3.0 software (http://hmmer.janelia.org/) with a default E-value. Subsequently, redundant sequences were omitted to retain unique TCS genes. Finally, the putative TCS members were further confirmed using SMART databases (http://smart.embl-heidelberg.de/) according to whether these members possess the structural characteristics and conserved domains of TCS elements. These domains included HisK domain, HATPase domain, receiver domain (Rec), CHASE domain, ethylene-binding domain (C2H4), His-containing phosphotransfer domain (HPt), and pseudo-HPt domain. Watermelon TCS members were identified using the same method against the watermelon proteome data set. Finally, all identified sequences were checked in Arabidopsis databases of TAIR website (http://www.arabidopsis.org/) to explore their homolog genes with the highest score.Protein sequences of all known TCS genes, particularly, 56, 52, 51, 62, 98, and 85 members from http://www.icugi.org/cgibin/ICuGI/index.cgi). The positions of these genes in cucumber and watermelon genomes were then obtained. ExPASy (http://web.expasy.org/compute_pi/) was used to calculate molecular weights and isoelectric points (PIs) of putative TCS proteins of cucumber and watermelon. Subcellular localizations were predicted using TargetP website (http://www.cbs.dtu.dk/services/TargetP/).Nucleotide sequences of all TCS genes were used as queries to perform BLASTN searches against the cucumber chromosomes (http://gsds.cbi.pku.edu.cn/). MEME (http://meme.nbcr.net/meme/intro.html) was used for motif analysis to annotate the conserved motifs in these TCS proteins. The predicted peptide sequences of conserved domain in the TCS proteins were identified using the SMART database (http://smart.embl-heidelberg.de/). Then, multiple-sequence alignment for the predicted peptide sequences of conserved domains  was generated using Clustal X v1.81 with default parameters . Phylogenetic analysis was performed using MEGA 5.0 program by neighbor-joining (NJ) method with 1000 replicates of the bootstrap based on the full-length protein sequences  was used to perform synteny analysis and detect the homologous genes in different synteny regions and segment duplications, as described in cucumber MADS gene family  and non-synonymous (Ka) substitution rates  was used to align the amino-acid sequences and corresponding CDS sequences of TCS elements, and then Ks and Ka were calculated using the Codeml procedure of the PAML online program . Divergence time of the duplicated genes and orthologous gene pairs between cucumber and watermelon were estimated using synonymous mutation rate of substitutions per synonymous site per year, as follows: T = Ks/2x . PlantCARE website (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/search_CARE.html) was adopted to identify cis-elements in the promoter regions.To investigate −2 s−1 with 16-h day length. The roots, stems, leaves, male flower buds (~1.0 cm in length), and female flower buds  according to the manufacturer-recommended protocol. The first cDNA strand was generated using the PrimerScript RT reagent kit  according to the manufacturer's instructions. Specific primers used in the qRT-PCR were designed by Primer 5 Software, and each primer was checked against cucumber DNA database to ensure its specificity.EF1a gene (accession number EF446145) of C. sativus was selected as an internal control , programmed to heat for 30 s at 95°C, followed by 40 cycles of 5 s at 95°C and 45 s at 55°C, and at the end, 1 cycle of 1 min at 95°C, 30 s at 50°C, and 30 s at 95°C. Two biological and three technical replicates for each sample were performed with 15 μL of reaction volume using the SYBR Premix Ex Taq kit . The Arabidopsis, rice, maize, soybean, wheat, and Chinese cabbage. A total of 236 protein hits including 80 HK(L)s, 44 HPs, and 112 RRs, were identified in the cucumber genome database. Additionally, 53 HK(L)s, 7 HPs, and 42 RRs were predicated using the HMMER 3.0 program hmmsearch with an default value. After the redundant sequences were omitted to obtain unique putative TCS genes, the remaining hits were further filtered using Pfam and SMART according to the presence of structural characteristics and conserved domains of TCS elements. Finally, 46 TCS members consisting of 18 HK(L)s, 7 HPs, and 21 RRs were identified in cucumber  proteins were identified in cucumber and classified into 10 CsHKs and 8 diverged CsHKLs according to whether they possessed conserved His-kinase transmitter (HK) domain Table . HK protArabidopsis . CsHK2 shares a high similarity of over 70% with CKI2 but a part of Rec domain is lost. The lost sequences may generate another protein CsHK3. These two genes could be spliced and form a conserved homologous CKI2-like gene with 61% identity . The event of protein domain loss also occurred in cucumber WRKY proteins  with their counterparts AHK2-AHK4 in  Figures . Domain  Figures , and fou Figures , S2. CHA Figures . Additio Figures .Arabidopsis ETR2, which regulates seed germination during salt stress by modulating ABA signaling , a GAF, and two PAS  domains  share high identities (61–72%) to Arabidopsis AHP4. The CsHPs except for CsPHP1 and CsPHP2, have a conserved Hpt domain with a signature motif of XHQXKGSSXS  with a pseudo-HPt domain, were identified in the cucumber genome Figures . The HP is Table . Similarties 61–7% to ArabArabidopsis. Protein analysis indicated that all eight type-A RRs had a receiver domain (Rec) corresponding to two conserved motif (motif 3 and 4) s, 54 HPs, and 248 RRs from Arabidopsis and Chinese cabbage except AHP4 and BrHP4/5 form a distinct subclade with the other HPs from the other five species.All the authentic and pseudo-HPs from these seven species as above mentioned were mainly divided into two clades, namely clade I and II Figure . In the Arabidopsis  of tandem duplications and one couple segment duplicate (ClHP1 and ClHP2) were found in cucumber and watermelon, respectively  and non-synonymous substitution rates (Ka) were measured to explore the gene divergence mechanism between the two genomes. The Ks of 19 orthologous gene pairs between cucumber and watermelon were calculated to speculate the main divergence time of these TCS genes pairs between cucumber and watermelon  and segment duplication (ClHP1 and ClHP2) were analyzed. Their Ks were 0.4564, 1.4117, 0.3716, and 1.5518 corresponding to divergence times of 35.11, 108.59, 28.58, and 119.37 Mya, respectively. These duplication events occurred before the divergence of cucumber and watermelon, which confirmed recent WGD was absent and only a few segmental duplication events happened in the cucumber and watermelon genomes -, salicylic acid -, and heat stress (HSE)-responsive elements were identified in 22, 20, and 22 promoters of cucumber TCS genes, respectively  were obviously induced except CsRR4 was repressed at 4 h. In cucumber, CsHP1, CsHP2, and CsHP3 were all homologous with AHP1 but showed different expression profiles. CsHP1 was obviously upregulated by 11-folds at 2 h after high-temperature treatment. However, the expression of other HPs (CsHP2-CsHP5) was markedly downregulated in response to heat stress. Consistently, Arabidopsis AHP1, AHP2, and AHP3 genes are markedly downregulated by heat stress s, 7 HPs, and 21 RRs] and 49  TCS genes in cucumber and watermelon, respectively. A comparative analysis of diverse plant species revealed that TCS members from the seven aforementioned species showed significant sequence and domain conservation. Gene duplication events rarely occurred in cucumber and watermelon TCS genes compared with those of YH performed the experiments, analyzed the data, and drafted the manuscript. XL participated in qRT-PCR experiments and data analysis. TZ and CP collected the public dataset and assisted with data analysis, LQ and LC prepared the cucumber samples, GL conceived the study and its design and assisted with revisions to the manuscript. All authors read and consented to the final version of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Various substitution patterns of coumarins have been reported, whereas the biosynthesis of coumarins remains elusive. Ortho-hydroxylation is a key step in simple coumarin biosynthesis as a branch point from the lignin biosynthetic pathway. 2-Oxoglutarate-dependent dioxygenases (2OGDs) from plants convert cinnamate derivatives into simple coumarins through the process of ortho-hydroxylation. This review describes the 2OGDs involved in coumarin biosynthesis and their substrate specificities.Coumarins are natural plant products that have been the subject of extensive phytochemical and pharmacological research studies in the past few decades. The core structure of coumarins is derived from the respective cinnamates via  Their hydroxy group is involved in conjugation to produce glycosides  or its derivatives have effectively shown that simple coumarin formation in plants proceeds via hydroxylation of the ortho-position (ortho-hydroxylation) of respective cinnamates, the adjacent position in the benzene ring to the side chain  by addition of prenyl group  in Apiaceae and Asteraceae plants is presumed to utilize another biosynthetic pathway that does not require ortho-hydroxylation  in Leguminosae plants, which also comprises a coumarin core structure, is synthesized from isoflavonoids, circumventing the need for ortho-hydroxylation of cinnamates in its biosynthetic pathway  of cinnamates that are involved in simple coumarin biosynthesis. The distribution of the ortho-hydroxylases in plants using a database search of EST homologs will be also discussed.Due to its irreversibility, AtF6′H1 (locus: At3g13610) was found to exhibit ortho-hydroxylase activity to feruloyl coenzyme A (15: feruloyl-CoA) as a substrate, with a Km value of 36 μM, yielding an ortho-hydroxylation product, 6-hydroxyferuloyl-CoA (19) , free ferulic acid (13), or feruloyl quinate. Deficient mutation of the AtF6′H1 gene in Arabidopsis causes a significant reduction in the accumulation of scopolin, a β-glucoside of scopoletin (4), indicating that AtF6′H1 catalyzes ortho-hydroxylation. Another 2OGD (AtF6′H2) encoded by a homologous gene (locus: At1g55290) exhibits an equivalent activity against CoA thioesters of cinnamates ; however, no significant change in scopolin levels was observed in the plant.In Arabidopsis, a 2-oxoglutarate-dependent dioxygenase (2OGD) encoded by the gene Ruta graveolenes, which accumulates franocoumarins, a 2OGD (RgC2′H) was cloned as the key enzyme of coumarin biosynthesis , but also to p-coumaroyl-CoA , forming scopoletin (4) and umbelliferone (2), respectively. Furanocoumarins are formed after addition of prenyl group to umbelliferone (2), which is detected in R. graveolens, whereas no scopoletin (4) was detected. This result indicates that RgC2′H exclusively catalyzes p-coumaroyl-CoA (14), besides its activity against feruloyl-CoA (15) and p-coumaroyl-CoA (14). Regulation of substrate supply to RgC2′H enzyme is likely to determine the structures of the products, namely, umbelliferone (2) or scopoletin (4).Further studies involving cloning and functional analysis of the 2OGD genes in plants have elucidated the mechanism of coumarin formation. Using 2) in sweet potato root (Ipomoea batatas) is molecular oxygen; therefore, hydroxylase using a water molecule to introduce a hydroxy group was excluded as the candidate of ortho-hydroxylation enzyme(s) , whereas those of Ib2s catalyzed both p-coumaroyl-CoA  and feruloyl-CoA  as the substrates to yield umbelliferone (2) and scopoletin (4), respectively. Root tissues of sweet potato accumulate moderate levels of scopolin. After fungal and elicitor treatments, the production of umbelliferone (2) and its β-glucoside, skimmin, was significantly higher than that before treatment, whereas the amount of scopolin remained at a moderate level after the treatments. Fungal and elicitor treatments also resulted in an upregulation of Ib2 genes, whereas no significant induction of Ib1 genes was detected. These results indicate that Ib2s mainly synthesize umbelliferone (2) using p-coumaroyl-CoA (14), besides their bi-functional activity.The biosynthetic origin of the 1-oxygen atom of umbelliferone , to produce umbelliferone (2). Interactions among the metabolic enzymes  is still unknown, whereas approaches to biosynthesis of coumarin (1) have been performed using sweet clover  formation is also remained to be elucidated. Ib1s from sweet potato showed a trace activity to caffeoyl-CoA  or caffeate (12) esters, or their free acid, respectively. Enzymatic information of ortho-hydroxylase homologs would tell mechanism of these coumarins. There is still a possibility that other enzyme families such as flavin monooxygenases or another oxidase family would also contribute to this reaction  to esculetin (3) or daphnetin  have been detected by tracer analysis, indicating a biosynthetic grid of simple coumarin formation  from M. alba. In addition, sequences from Euphorbia spp. or Manihot esculenta, which accumulate esculetin  exhibited ortho-hydroxylation of cinnamates to form simple coumarins. However, the boundary line dividing the ortho-hydroxylase sequence involved in simple coumarin biosynthesis and the other 2OGDs remains unclear. Liriodendron tulipifera, a Magnoliales plant that arose from a more basal lineage than monocots, accumulates scopoletin (4)  (Choi et al., Kawai et al.  conducteStevens, . There iortho-hydroxylases are mainly distributed in dicots, indicating that the biosynthesis of simple coumarins is a newer pathway of plant secondary metabolism, compared to flavonoids, which extensively occur in the plant kingdom (Harborne and Baxter, ortho-hydroxylases at the molecular level would provide more details on the evolution of plant coumarins.Candidates of The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.