Handwashing is the most fundamental way to prevent the spread of infectious diseases. Correct handwashing can prevent 50 to 70% of water-infections and foodborne-infections. We report the results of a fact-finding study on general handwashing attitude and practice in the Republic of Korea by analyzing habits and awareness among adults and students (grades 4 to 12) based on the 2006 to 2014 National Handwashing Surveys and observational surveys.The awareness survey was performed by telephone interviews with adults and students in 16 municipalities and provinces sampled by quota for region, sex and age. The observational survey was performed in subway, railway, and other public restrooms in seven municipalities selected through systematic sampling.Adults and students washed their hands with soap/sanitizer an average of 6.6 and 5.2 times daily, respectively, in 2014, an increase and decrease compared to 2006 (4.8) and 2013 (6.8). Their average daily handwashing frequency in 2014, 9.8 and 8.3, was higher than in 2006 (7.6), but lower than in 2013 (10.3).The percentage of participants handwashing with soap after using the restroom (29.5%) has been increasing since 2009, but remain slower than in other countries (42% to 49%). The percentages of participants handwashing with water in 2014, 2013, and 2011 were 57.5%, 72.6%, and 71.4%, respectively.Handwashing with soap is an important national public health issue, and national projects promoting it should be given high priority. Research support is necessary to provide scientific evidence of the importance of handwashing with soap and to develop and implement evidence-based policies. The United Nations Children’s Fund reports that 1.7 million children under the age of five die annually of diarrhea and pneumonia .Staphylococcus aureus .-23.18-23.1%. TheyAnalysis of home handwashing habits revealed that “after bathroom use” was most common (88.0%) among adults, followed by “before eating” (71.9%) and “before preparing food” (70.2%). Among students, “before preparing food” (89.1%) was most common, followed by “after bathroom use” (86.1%) and “before eating“ (58.7%).Among daily activities involving potential sources of infection, adult participants washed their hands more frequently “after waste disposal” (87.1%) and “after cleaning” (80.0%), and less frequently “after touching money” (18.1%) and “after coughing and sneezing” (17.6%). Student participants washed their hands more frequently “after cleaning” (79.5%) and “after waste disposal” (78.2%), and less frequently “after nosing or touching the nose” (27.3%) and “after touching money” (24.7%).The reported frequency of handwashing among adults “after using public restrooms” was 88.0%, followed by “when returning home” (78.1%), “before eating in restaurant” (57.1%), and “use of wet towel before eating” (56.2%). The handwashing frequencies among students were “after using public restrooms” (86.2%), “when returning home” (70.6%), “before eating in a restaurant” (60.8%), and “use of wet towel before eating” (27.0%)  18-23].-23.18-23The results of the awareness survey revealed that 90.1% and 92.3% of adult and student respondents, respectively, were aware that handwashing helps prevent infectious diseases. Adults did not wash their hands for the following reasons: “no habit yet” (31.4%), “no place to wash” (24.7%), and “cumbersome” (23.4%). Students reported “cumbersome” (35.7%) and “no habit yet” (22.9%) as the main reasons, followed by “no place to wash” (4.9%).Most adults (59.1%) and students (65.2%) reported washing their hands more often during domestic or international pandemic outbreaks.When asked whether public restrooms have good handwashing conditions, 29.8% of adults responded that they were “not sufficient”. The proportion of “not sufficient” responses in 2014 was lower than in 2011 (48.2%) and 2013 (31.4%); in contrast, student opinions of the conditions in public restrooms did not improve over time   18-23].-23.18-23Results of the survey on exposure to education on correct handwashing behaviors over the previous year revealed that only 12.9% of adults reported having experienced handwashing-related education, whereas 40.6% of students had participated in handwashing education programs. Compared to 2013, adults showed a negligible increase and students a slight decrease in exposure .Adults reported an average of 2.5 handwashing-related educational experiences (n=647) in the previous year. The largest proportion (47.5%) had participated in handwashing education sessions only once, and 3.6% had experienced educational programs four times. In comparison, students (n=365) participated in an average of 1.7 handwashing educational experiences in the previous year, with “once” the most frequent response (57.0%). The most common session duration among adults and students were “<10 minutes” (51.3%) and “30 to 60 minutes”(43.6%), respectively.The most common method of adult education was “lecture” (40.0%), followed by “audio-visual media such as video” (30.0%), “pamphlets such as community notice” (16.8%), and “practice” (12.7%). Students were most often exposed to “audio-visual media” (38.4%), “lecture” (29.0%), and “practice” (8.2%).While 95.2% of adults and 93.2% of students felt that handwashing education was necessary in 2013, only 80.6% and 77.7%, respectively, found the handwashing education programs necessary in 2014.More than half of the adults  reported seeing Ads or pamphlets about correct handwashing. Similarly, 56.6% of students (n=509) reported seeing promotional material. Adults most frequently encountered Ads via “terrestrial TV” (47.7%), followed by “community health center and hospital plasma display panel (PDP)” (31.9%). Students mostly encountered Ads/pamphlets via “community health center and hospital PDP” (37.7%) and “terrestrial TV” (37.5%).When asked whether the contents of the Ads/pamphlets were easy to understand, 94.1% of adults answered affirmatively, versus 3.5% who answered negatively, compared to 94.5% and 5.5% in 2013. Among students, 91.4% reported finding the contents easy to understand. Regarding their reactions to the Ads/pamphlets they had seen, 84.0% of adults reported that the Ads/pamphlets led them to consider behavioral changes including handwashing habits in 2014, a slight increase compared to 2013 (83.5%). 76.2% of students indicated that they would consider behavioral changes including handwashing after seeing the Ads/pamphlets.Most adults (90.2%) reported having learned to wash their hands correctly after seeing the Ads/pamphlets, compared to 7.8% who did not, a slight increase from 2013 (88.5%). Nearly nine in 10 students (87.6%) answered affirmatively, vs. 9.6% who did not  18-23].-23.18-23Handwashing was observed in 29.5% of individuals after using the restroom, increases of 6.0% and 10.4% from 2013 (23.5%) and 2011 (19.1%), respectively. The highest percentage of handwashing was observed in 2009, a year with an outbreak of a novel influenza strain. Handwashing after restroom use decreased from 72.6% in 2013 to 71.4% in 2014, but handwashing with soap increased from 32.3% to 41.3%. Females washed their hands more often than males .The duration of handwashing was 1 to 5 seconds in 39.0% of observed individuals, 6 to 10 seconds in 33.1%, 11 to 15 seconds for 12.9%, 16 to 20 seconds in 7.4%, and ≥21 seconds in 7.6%. The average duration increased slightly from 8 seconds in 2013 to 9 seconds in 2014.Almost all restroom users (99.4%) turned off the faucet with their bare hands, while 0.1% used a paper towel. After handwashing, hand dryers were used most frequently 23.4%), followed by paper towels (16.3%) and personal handkerchiefs (2.9%)  18-23].-23.18-23.4%, follHandwashing with soap is a fundamental and economical way to prevent infectious diseases, reducing the incidence of waterborne and foodborne diseases by 50% to 70% and pneumonia, impetigo, and diarrheal diseases by 40% to 50%. Handwashing is effective in preventing many infectious diseases associated with global warming, such as SARS, influenza, cold, cholera, dysentery, and contagious eye disease.About 80% of individuals were aware that handwashing helps prevent infectious diseases. However, their handwashing habits remain weak. Moreover, although 75% reported using soap in the awareness survey, only 29.5% of restroom users did so in the observational survey, demonstrating the discrepancy between self-assessment and actual practice. Continuous fact-finding surveys are necessary to provide information on handwashing-related awareness, attitudes, and practice of the general population as the basis for handwashing campaigns.A meta-analysis of 42 papers on handwashing and its health benefits worldwide reported that only 19% of the global population washes their hands with soap after defecation (after restroom use or diaper change), with the rate increasing from 13% and 17% in underdeveloped and developing countries to 42% to 49% in developed countries. New Zealand had the highest rate (72%), followed by the UK (52%), the Netherlands (50%), and the US (49%). The rate of handwashing with soap after restroom use in the current study (29.5%) was similar to those of Thailand (25%) and Ethiopia (22%), the least rate of handwashing with soap was Tanzania (5%).In Korea, the KCDC, National Handwashing Campaign Center, municipal authorities, and the Ministry of Food and Drug Safety conduct and washing campaigns based on their individual objectives. However, to our knowledge, no previous study has evaluated the efficacy and validity of these programs, and only the National Handwashing Campaign Center has performed fact-finding surveys, which were recently discontinued. The survey items lacked clarity in standardization and planning, and were not systemized as a whole. The survey periods and durations were not regular, even though seasonal factors can greatly influence survey results, and sample sizes also varied between surveys. The reliability of the results is limited to simple nationwide handwashing-related statistics without the ability to adjust survey items according to previous survey results or as sociate the results with campaign programs or cooperation with other industrial sectors.Handwashing campaigns and programs should be designed to increase awareness and good habits, and should be informed by systematic fact-finding surveys. Fact-finding surveys can assess the outcomes of handwashing surveillance and campaign programs; they can also identify points for improvement and gather respondent feedback. Well-designed programs based on this information should be used to promote handwashing with soap. These campaigns should both attract societal attention and approach individuals. Moreover, factors related to environmental conditions, understanding of campaign targets, and project background and orientation necessary for program operation must be considered, and policies should reflect these factors. Agenda frameworks should include factors useful for social marketing activities, and contain sub-frameworks aligned with the overall campaign strategies and goals.Handwashing-with-soap campaigns should be recognized as an important public health issue and given high priority in implementation of public healthcare programs. To this end, an integrated collaboration system should be established among public and private sectors as well as related government departments and organizations. A strategic framework for supporting systematic fact-finding surveys and studies should be established in order to consolidate the scientific grounds to justify expansion of handwashing-with-soap campaigns .This study had several limitations. First, the pre-2013 surveys had irregular survey periods and a different survey design from the post-2013 surveys in terms of participants, sample size, and survey sites, posing problems for reliable comparisons of survey results. Second, the representativeness could not be established because budget limits also sample sizes. Third, heterogeneous indicators among countries prevented observational study-based inter-county comparisons of the rates of handwashing with soap after using restrooms.Despite these limitations, the value of this study is the nationwide monitoring and evaluation of handwashing status by municipality, which provides basic data to improve handwashing programs in individual municipalities.


The relationship between mosquitoes and their amphibian hosts is a unique, reciprocal trophic interaction. Instead of a one-way, predator-prey relationship, there is a cyclical dance of avoidance and attraction. This has prompted spatial and temporal synchrony between organisms, reflected in emergence time of mosquitoes in the spring and choice of habitat for oviposition. Frog-feeding mosquitoes also possess different sensory apparatuses than do their mammal-feeding counterparts. The reciprocal nature of this relationship is exploited by various blood parasites that use mechanical, salivary or trophic transmission to pass from mosquitoes to frogs. It is important to investigate the involvement of mosquitoes, frogs and parasites in this interaction in order to understand the consequences of anthropogenic actions, such as implementing biocontrol efforts against mosquitoes, and to determine potential causes of the global decline of amphibian species.  Mosquitoes (family Culicidae) are well-known pests of mammals due to the anthropophilic nature of a number of species that brings them to our attention. Lesser known are species that preferentially feed on ectothermic hosts such as frogs and snakes. Female mosquitoes require blood in order to produce eggs  and in tThere are three main ways in which blood parasites can be transmitted from an insect vector to a vertebrate host. These include one form of passive transmission in which the parasite is carried by the insect host without engaging in multiplication, and two forms of biological transmission in which the parasite undergoes development or replication in the insect host. Mechanical transmission is characterized by passive transfer of a parasite, specifically on the exterior of the feeding apparatus, into the feeding site on the vertebrate . This tyPlasmodium falciparum Welch, the causative agent of the deadliest form of human malaria. This salivary transmission involves direct deposition of parasites from the mouthparts of the insect into the feeding site on the vertebrate. Filarial worms [The most familiar mechanism of biological transmission of blood parasites is by injection of the infectious agent when the insect bites, a method employed most notably by al worms  and arboal worms  are examal worms . Gorgoderina spp., Opithioglyphe spp., Ostiolum spp. and Halipegus spp., which typically infect the internal organs such as the bladder, intestines or lungs of the frog following ingestion of infected insects. Egg stages of these parasites are passed through the intestine of the frogs and deposited with faeces. Infective stages of these parasites may be ingested by insects, swept into branchial baskets or may penetrate directly into the tissues of the insect [Hepatozoon species, occurs reciprocally through ingestion of infected frog blood by mosquitoes and ingestion of an infected mosquito by frogs [Trophic transmission is another form of biological transmission in which one animal ingests an infected animal. Parasites transmitted trophically from insects other than mosquitoes to frogs include the trematodes e insect . Howeverby frogs . Certainby frogs , similarby frogs . The estCulex territans Walker [Culex peccator Dyar and Knab [Uranotaenia lowii Theobald [Uranotaenia sappharina Osten Sacken [Deinocerites spp. [Culex peccator prefers to feed on reptiles [C. territans, U. lowii and Deinocerites spp. prefer amphibian hosts [U. sappharina may feed equally on both [Aedes aegypti Linnaeus are also reported to feed on frogs [Culex territans has a wide geographic distribution, including North America, Europe and Africa [Mosquito species that feed primarily on ectothermic hosts include s Walker , Culex pand Knab , UranotaTheobald , Uranotan Sacken  and Deintes spp. . Culex preptiles , C. terran hosts ,15, and  on both . Aedes aon frogs ; howeveron frogs . Culex td Africa , whereasd Africa , tropicad Africa  and the d Africa . C. territans, which is the subject of the majority of studies on the relationships between mosquitoes and frogs, and frogs of the families Ranidae (true frogs) and Hylidae (treefrogs). We highlight the temporal and spatial synchrony of C. territans and frogs prompted by this trophic relationship, include an overview of parasites transmitted by mosquitoes to frogs, and discuss in detail the role of the reciprocal trophic relationship in transmission of blood parasites. This paper provides an overview of the trophic interactions between mosquitoes, in particular Most mosquito species require standing water on or near which to oviposit  and may C. territans. This species is capable of digesting a blood meal at temperatures of 3.9 °C, compared to a thermal minimum of 10 °C for Culex pipiens Linnaeus and Culiseta melanura Coquillett. In temperate regions of North America, emergence of C. territans corresponds with the emergence of early spring amphibians, specifically wood frogs, Rana sylvatica LeConte [C. territans; however, C. territans may not be stimulated to oviposit in these pools following the first blood meal due to a lack of chemical cues in the water following the rapid exodus of frogs [In temperate climates, one of the earliest mosquitoes that overwinter as adults and emerge in the spring is  LeConte , which a LeConte ,24. Afteof frogs . Pseudacris crucifer Wied-Neuwied, are small hylid frogs found throughout eastern North America that serve as early spring sources of blood for C. territans [Spring peepers, erritans . Spring erritans ,26. Afteerritans . Rana clamitans Latreille, and the bullfrog, Rana catesbeiana Shaw, both of which emerge later in the spring and breed from the beginning of June until mid-August in the northern part of their range [Continuous breeders in temperate North America include the green frog, ir range ,29. Bothir range . Green fCulex territans larvae are found sympatrically with, and adults of C. territans have been recorded to feed on, all four aforementioned species of frogs [C. territans larvae highly correlates only with the presence of green frogs. In the early spring, at temperatures below 9 °C, C. territans requires more than 25 d from ingestion of blood to oviposition. If wood frogs were to serve as early blood meals during their breeding season, they most likely will have left the aquatic habitat by the time mosquitoes are ready to oviposit. Thus, vernal habitats used by wood frogs for breeding are generally not suitable for C. territans in which to lay eggs since their hosts are not spatially synchronous with an oviposition site [C. territans for a significant portion of the summer. However, wood frogs or spring peepers, including those breeding in permanent bodies of water, likely serve as the source of initial blood meal of overwintering females. For example, the first collection of C. territans larvae in New Jersey, USA, in 2004 occurred on May 6, which suggests that overwintering adults took a blood meal at the end of March or beginning of April, concurrent with emergence of wood frogs or spring peepers [of frogs ,10,22. Hion site . ConsequC. territans are attracted to olfactory cues released in water containing green frog tadpoles, despite putative competition between the two for food. As previously mentioned, presence of C. territans larvae can be correlated with, and predicted by, the presence of R. clamitans. When green frogs shift to vernal habitats in the late summer, C. territans larvae appear in these pools approximately two weeks later [C. territans, possibly due to the spatial concurrence of an extended site for oviposition in green frog breeding sites and an accessible host for blood meals [Mosquito species that do not feed on frogs may choose not to oviposit in water that contains tadpoles in order to avoid interspecific competition for overlapping resources that includes bacteria, algae and detritus . Additioks later . There iod meals . The co-Culiseta longiareolata Macquart also compete with early stage tadpoles of the toad Bufo viridis Laurenti for periphyton, and the larval development rate of Cs. longiareolata is reduced when B. viridis tadpoles are present [B. viridis tadpoles prey upon early instars of Cs. longiareolata [Hyla septrentionalis Schlegel actively feeds on larvae of Culex quinquefasciatus Say in the laboratory [Cs. longiareolata larvae will prey opportunistically on immobile, early stage tadpoles of B. viridis [Use of synchronous habitats by mosquitoes and frogs can lead to competition for resources, as well as predation, and thus trophic relationships exist not only between adult mosquitoes and frogs, but also between larvae of each. Both mosquito larvae and frog tadpoles feed on bacteria, algae and detritus, which may lead to competition for resources. Rubbo and colleagues  showed t present . Additonareolata  and Hylaboratory . Interes viridis . Thus, tRana aurora Baird and Girard, experience injury as tadpoles and decreased body mass at metamorphosis in habitats containing mosquitofish [Predation of mosquito larvae by aquatic salamanders and their larvae has been observed in the laboratory  and theruitofish . Many amuitofish . Thus, auitofish .Hepatozoon are apicomplexan blood parasites related to the Plasmodium species that cause malaria, and are found in the erythrocytes, and occasionally leukocytes, of terrestrial vertebrates. These protozoa complete sexual development in the gut or haemocoel of various haematophagous arthropods, which must then be ingested by the vertebrate host for transmission to occur. Following asexual development in the liver and other visceral organs of the vertebrate host, intracellular blood parasites are ingested along with a blood meal by the haematophagous arthropod [Parasites of the genus rthropod .Hepatozoon species are prevalent in many species of true frogs (family Ranidae) and toads (family Bufonidae), and have been reported in several other families. Thus far, the life cycles of only two of 42 species of Hepatozoon infecting anurans have been fully elucidated [Hepatozoon clamatae Stebbins and Hepatozoon catesbianae Stebbins are widespread in northeastern North America, and are prevalent in green frogs and bullfrogs, respectively, although both species may be found as mixed infections in each species of frog [Hepatozoon clamatae is also found naturally in the leopard frog, Rana pipiens Schreber. Culex territans serves as the definitive host for both species of parasite, which develop as multisporocystic oocysts in the Malpighian tubules [ucidated ,40. Hepa of frog . HepatozHepatozoon species are largely unexplored; in snakes, one study suggests that infection is largely benign in most cases [Hepatozoon species may affect growth rate, reproductive output and survival of snakes [Hepatozoon americanum Vincent-Johnson et al. [Hepatozoon moccasini Laveran is pathogenic to unnatural lizard hosts [Hepatozoon species to unnatural hosts, including frog species that do not normally harbour the parasite, could result in more severe fitness consequences. Mosquito fitness during infection by Hepatozoon species is largely unexplored; however, mortality has been observed in several species of mosquitoes recently fed on blood containing high levels (5%–10% of erythrocytes infected) of these parasites, including C. territans fed on green frogs with moderate infection levels [C. pipiens infected with Hepatozoon sipedon Smith et al., a closely related parasite of snakes, was devoid of eggs during heavy infections of the parasite [Hepatozoon species affect the survivorship and reproductive fitness of their mosquito hosts by as yet undetermined mechanisms. Overall, the effect of Hepatozoon species on their hosts is not well known, nor is the effect of any interaction they have in the parasite infracommunity of their frog hosts.The fitness consequences of infection by st cases  but anotf snakes . Hepatozn et al.  and Hepard hosts . Transmin levels . As wellparasite . Both ofTrypanosoma species are cosmopolitan flagellate blood parasites of frogs, as well as other vertebrates, and transmission of amphibian trypanosomes is generally considered to occur through the saliva of leeches; however, dipterans may also be responsible for transmission [Trypanosoma rotatorium Mayer will develop in C. territans [Culex territans has been suggested as a vector for Trypanosoma ranarum Lankester [C. territans, regardless if the female was newly engorged or gravid [C. territans in the transmission of trypanosomes remains ambiguous but potentially relevant.smission ,10,17,49erritans ; howevererritans . Culex tankester  and the Foleyella are found naturally in the blood of frogs in North America, Europe, Asia and Africa [Foleyella species develop throughout the haemocoel but primarily in the fat body of mosquitoes, and they are passed to frogs via salivary transmission when the mosquito feeds [C. territans serves to vector Foleyella flexicauda Schacher and Crans among bullfrogs in North America. Microfilariae of these nematodes are ingested when mosquitoes feed on frogs, and later developmental stages are transmitted to frogs when mosquitoes take additional blood meals [Filarial worms of the genus d Africa ,51,52. Lod meals .Foleyella species are reported to cause mortality in frogs harbouring heavy infections of microfilariae or adult worms [Foleyella species reduce egg production in Aedes and Culex species [lt worms . Additio species  and can  species . The int species  and thusCulex territans tested positive for WNV by the CDC in 2005 [C. territans, C. peccator, and U. sapphirina [C. territans may also be responsible for transmission [C. territans and other frog-feeding mosquitoes as vectors for arboviruses is not fully elucidated. However, due to the integrated nature of the relationship between C. territans and other frog-feeding mosquitoes and their ectothermic hosts, their role in the transmission of such viruses must be considered.Mechanical and salivary transmission of viruses by mosquitoes may also occur during feeding, which has prompted concern over the possibility of amphibians and reptiles as reservoirs for arboviruses, such as West Nile Virus (WNV) and Eastern Equine Encephalitis Virus (EEEV) that infect humans ,56. Cule in 2005  and EEEVpphirina ,56 althosmission . Additiosmission . The oveCulex territans has fewer sense organs overall than other Culex species, including C. pipiens and C. quinquefasciatus. Specifically, C. territans has fewer blunt type I trichodea, which are considered olfactory receptors that may mediate repulsion to certain odours. They also possess fewer pointed trichodea, known to mediate attractive olfactory activity [2 detection [2 is not an important attractant for C. territans [Culex territans must thus rely on other attractant cues in order to find and feed on their amphibian hosts.The trophic relationship between mosquitoes and frogs, and any resultant parasite transmission, is dependent on host selection by the mosquito. This involves attraction  and repuactivity . Lastly,etection . This suerritans  and the C. territans and U. lowii display phonotaxis to amphibian vocalizations [C. territans and C. peccator coincide with calling peaks of male spring peepers, gray treefrogs (Hyla versicolor LeConte) and green treefrogs (Hyla cinerea Schneider) [C. territans orients towards amphibian vocalizations played through a CD player. The vocalizations of spring peepers are the most attractive, with the calls of green frogs and bullfrogs as the least attractive, although significantly more attractive than the control sound of a CD player [C. territans [C. territans fed on green frogs (63%) as compared to spring peepers (6%) in a separate study [C. territans with green frogs in permanent ponds [Hepatozoon species.Both izations ,15. Amphizations ,64. Feedhneider) . It has D player . Using berritans . In contte study ; howevernt ponds  may inflRana vaillanti Brocchi [H. cinerea, by Corethrella species, are found only in sexually mature, male frogs [Physalaemus pustulosus Cope, during breeding season [Attraction to frog calls could also increase the ratio of infected males to females if mosquitoes are more likely to hone in on a calling male during the breeding season. There are no current studies that have been able to draw a link between the sex of frogs and presence of mosquito-borne parasites , and ind Brocchi . Howeverle frogs . These mg season ,68, simiHepatozoon species are rarely found infecting wood frogs [Seasonal shifts in host selection by mosquitoes could affect parasite host specificity; for instance, od frogs . AlthougC. territans revealed that they did not feed on cricket frogs (Acris crepitens Baird and Acris gryllis LeConte), despite the observation that these species were the most abundant at the study site [Litoria caerula White, the Australian green tree frog, produces skin secretions that are repellent to both blowflies  and mosquitoes, and two other Litoria species repelled mosquitoes as measured by an olfactometer [Litoria species, have few reported cases of Hepatozoon species [Additionally, abundance of one species does not necessarily sway host preference of mosquitoes. An analysis of blood meals in udy site . Mosquitctometer . It is u species . This maHepatozoon species, are interesting to consider in the context of evolution of the trophic relationship between mosquitoes and frogs and the unique synchrony of this reciprocal interaction. The ability of parasites to influence the phenotype of their hosts can shape the interaction of organisms in an ecosystem [Study of the trophic relationships between frog-feeding mosquitoes and their amphibian hosts provides insight into the evolution of their spatial and temporal synchrony and the life history of parasites transmitted between the two. Parasites, such as cosystem , and thecosystem ,55 and sStudying the relationships between frogs, mosquitoes and their parasites also highlights the integration of trophic levels in an ecosystem. The importance of parasites in ecosystem function is becoming increasingly well recognized ,55,69 anThe trophic relationship of and spatial synchrony between mosquito larvae and frog tadpoles is also an important area of exploration in light of the global decline of amphibian species. For example, it is postulated that frog tadpoles experiencing stress resulting from high densities of insect and fish predators, as well as resource limitation, may be more susceptible to infection with the lethal Ranavirus . Additio


Terebratalia transversa, we have identified orthologs of genes involved in mesoderm development in other taxa and investigated their spatial and temporal expression during the embryonic and larval development of T. transversa.Brachiopods undergo radial cleavage, which is distinct from the stereotyped development of closely related spiralian taxa. The mesoderm has been inferred to derive from the archenteron walls following gastrulation, and the primary mesoderm derivative in the larva is a complex musculature. To investigate the specification and differentiation of the mesoderm in the articulate brachiopod T. transversa. Five genes, Tt.twist, Tt.GATA456, Tt.dachshund, Tt.mPrx, and Tt.NK1, were found to have expression throughout the archenteron wall at the radial gastrula stage, shortly after the initiation of gastrulation. Three additional genes, Tt.Pax1/9, Tt.MyoD, and Tt.Six1/2, showed expression at this stage in only a portion of the archenteron wall. Tt.eya, Tt.FoxC, Tt.FoxF, Tt.Mox, Tt.paraxis, Tt.Limpet, and Tt.Mef2 all showed initial mesodermal expression during later gastrula or early larval stages. At the late larval stage, Tt.dachshund, Tt.Limpet, and Tt.Mef2 showed expression in nearly all mesoderm cells, while all other genes were localized to specific regions of the mesoderm. Tt.FoxD and Tt.noggin both showed expression in the ventral mesoderm at the larval stages, with gastrula expression patterns in the archenteron roof and blastopore lip, respectively.Orthologs of 17 developmental regulatory genes with roles in the development of the mesoderm in other bilaterian animals were found to be expressed in the developing mesoderm of T. transversa. Expression of multiple mesodermal factors in the archenteron wall during gastrulation supports previous morphological observations that this region gives rise to larval mesoderm. Localized expression domains during gastrulation and larval development evidence early regionalization of the mesoderm and provide a basis for hypotheses regarding the molecular regulation underlying the complex system of musculature observed in the larva.Expression analyses support conserved roles for developmental regulators in the specification and differentiation of the mesoderm during the development of The online version of this article (doi:10.1186/s13227-015-0004-8) contains supplementary material, which is available to authorized users. Drosophila, and Caenorhabditis elegans, where the molecular basis for mesoderm development has been investigated in the greatest detail [The mesoderm is frequently considered the ‘third germ layer’ in metazoans. As its name suggests, the mesoderm is a ‘middle’ layer, located between the ectoderm and endoderm. The mesoderm gives rise to tissues including muscle, parenchyma, cartilage, hemolymph, and somatic gonads, as well as forms the lining of coelomic cavities . The mest detail -6. Orthot detail -9, MyoD t detail -12, and t detail -15 have t detail .Although mesoderm formation has been studied in detail in these and other model systems, comparatively little is known about mesoderm specification and differentiation in members of the large protostome clade Spiralia ,18. ThisWhile the internal evolutionary relationships of the Spiralia remain a subject of investigation, nearly all recent phylogenies resolve brachiopods (a.k.a. lamp shells) as having evolved from within the clade for which spiral cleavage was plesiomorphic ,26-29. TTt.tropomyosin, which is expressed in the anterior and lateral regions of the archenteron wall in the asymmetric gastrula and early larval stages . RACE amplification was performed on cDNA libraries synthesized from mixed-stage embryonic RNA with the SMARTer RACE Kit . Amplified fragments were cloned into pGEM-T vector  and verified by Sanger sequencing. Sequences for cloned genes are available in GenBank  were collected by dredging in San Juan Channel, between San Juan Island and Shaw Island, WA, USA, between October and January in 2008, 2010, and 2012, and were maintained in flow-through seawater aquaria at Friday Harbor Laboratories. In vitro fertilization was performed by manual dissection and maceration of gonads. Prior to fertilization, oocytes were maintained in clean seawater until germinal vesicle breakdown and shedding of follicle cells were observed (between 4 and 8 h after stripping of gonads). Sperm were activated with seawater buffered to pH 9.8 with Tris. Following fertilization, embryos were reared in 1-L glass beakers with daily water changes. Embryos were fixed with 4% paraformaldehyde in filtered seawater for 1 h, washed four times in phosphate-buffered saline with 0.1% Tween-20, rinsed with distilled water, and subsequently dehydrated and stored in 100% methanol until in situ hybridization.Adult In situ hybridization of transcripts for cloned genes was performed using protocols established for chromogenic detection in the cnidarian Nematostella vectensis [in situ protocol and mounting for imaging. In all cases, staining was highly consistent within stages. Embryos were cleared and mounted in 80% glycerol, and imaging was performed on a Zeiss AxioSkop microscope equipped with Plan-Apochromat 20×/08 N.A. objective and differential interference contrast optics . Images were acquired with a Zeiss AxioCam HRc digital camera and Zeiss AxioVision v4.8 software .ectensis . A detaiT. transversa for putative homologs of the transcription factor genes Forkhead C (FoxC), Forkhead D (FoxD), Forkhead F (FoxF), GATA4/5/6, MEF2, Mox, mesoPrx (mPrx), MyoD, NK1, paraxis, Pax1/9, Six1/2, and twist, the nuclear protein genes dachshund, eyes absent, and Limpet, and the BMP inhibitor gene noggin. Orthology assignments were verified by Bayesian analysis of phylogenetics using inferred amino acid sequences of the cloned transcripts and representative sequences from other metazoan taxa .Mesodermal expression was observed for all 17 genes listed above, and representative photomicrographs of the radial gastrula, asymmetric gastrula, early larval, and late larval stages are presented Figures , and 9. Tt.twist is expressed symmetrically throughout the archenteron wall, which is fated to form the mesoderm, but is absent from the archenteron roof, which will form the endoderm . During the bilateral gastrula stage, the Tt.noggin expression domain shifts to the dorsal ectodermal surface of the embryo and decreases in intensity .Mesodermal expression of two genes, Tt.eya is strongly expressed throughout the archenteron wall in the asymmetric gastrula and in the anterior mesoderm of the larval stages, there is limited evidence for mesodermal expression in other members of the Spiralia. Expression of an eya ortholog has been reported from the mesodermally derived photophore (or light organ) of the bobtail squid Euprymna scolopes [eya appears to be part of a redeployment of the Pax-Six-Eya-Dachshund network from eye specification to facilitate acquisition of light sensitivity in the photophore [Schmidtea, expression of eya is restricted to the eyes during embryonic development and regeneration [eya is more widely reported in ecdysozoans and deuterostomes, with orthologs playing key roles in myogenesis in both Drosophila [Branchiostoma [Although scolopes . Howeverotophore . In the neration ,106. A mosophila  and vertosophila  and showhiostoma .Tt.FoxF expression to the anterior mesoderm of the asymmetric gastrula and larval stages of Terebratalia may be comparable to expression in the anterior mesoderm of the mollusk Patella [Patella and the annelid Capitella, there was also expression in the posterior mesoderm [Terebratalia. More broadly, the expression of Tt.FoxF surrounds the larval endoderm, which may share an evolutionary origin with expression in the visceral mesoderm of Drosophila [Saccoglossus [The restriction of  Patella . Howevermesoderm , for whiosophila , the hemoglossus , and theoglossus .Tt.Mox and Tt.paraxis show very similar expression patterns in the mantle and pedicle lobes of the larval stages. Mox expression in the ventral mesoderm appears to be a conserved feature in Spiralia, given that orthologs in the annelid Platynereis [Haliotis [Mox is further supported by expression of orthologs in the ventral mesoderm of Drosophila [Saccoglossus [paraxis; however, most available data support a conserved role in mesoderm development. Comparable to the expression of Tt.paraxis, in the annelid Platynereis, paraxis is in the ventrolateral mesoderm [Branchiostoma, paraxis is in developing somites as they form at the posterior of the embryo [paraxis, which is required in somite formation [scleraxis, which is required for the initial specification of mesoderm as well as for subsequent chondrogenesis in tissues derived from the somites [Drosophila, where expression of the ortholog CG33557 (previously CG12648) is restricted to neural cells [tynereis  and the Haliotis  show comosophila  and the oglossus . In choroglossus -118. Lesmesoderm , and in e embryo . In mousormation , and scl somites . The oneal cells .Tt.Limpet and Tt.Mef2 is very similar with both first detected anteriorly in the early larva and then expanding throughout nearly all mesodermal tissues by the late larval stage. These patterns of expression suggest that both genes likely have roles in myogenic differentiation, as their localization closely matches that of musculature labeled by phalloidin in slightly older competent larvae. This included the notable absence of expression in the medial and posterior mesoderm where there is no apparent muscle formation. Expression has not been described of either of these genes on taxa in the Spiralia, but the myogenic function of Mef2 has been well characterized in both Drosophila [Isodiametra [Mef2 are its apparent lack of a developmental function in C. elegans [Limpet in other taxa in the Spiralia, but orthologs in Drosophila and C. elegans are expressed in subsets of the mesoderm [Limpet genes in deuterostomes, but the FHL family in vertebrates appear to be the most closely related, having lost the PET domain characteristic of Limpet genes but sharing an organization of four-and-a-half LIM domains with them [FHL family show expression in musculature and heart [Limpet/FHL may have been in the mesoderm.Mesodermal expression of osophila ,123. Myoosophila , and an diametra . Two exc elegans  and its  elegans . Data ismesoderm -127. Theith them . Membersnd heart , suggestTt.FoxD and Tt.noggin, have patterns of early expression distinct from those of the genes discussed above, which show expression in the archenteron wall. Orthologs of Tt.FoxD and Tt.noggin show comparable expression in the ventromedial mesoderm of the annelid Platynereis [Platynereis, where the ventromedial region is described as being contractile [T. transversa, based on reconstructions of phalloidin-stained larvae  have shown commonalities across bilaterian taxa, in particular between mouse and onducted ,6,144. Aosophila , despiterulation , and in mesoderm . Later iTt.twist, Tt.GATA456, Tt.dachshund, Tt.NK1, and Tt.mPrx, likely all play roles in specification of the mesoderm as a whole, given their expression throughout the archenteron wall during the radial gastrula stage. At the same time, localized expression of Tt.Pax1/9, Tt.MyoD, and Tt.Six1/2 in specific regions of the archenteron wall suggests that the mesoderm is being regionalized even during the early phases of its specification. This regionalization is further reflected as larval development progresses and the expression of most genes is restricted to a subset of the larval mesoderm. The diversity of expression patterns for mesodermal genes during the development of T. transversa likely forms the basis for the complex musculature observed in the larva. While the majority of gene expression patterns are consistent with an endomesodermal source of mesoderm, dynamic expression of Tt.noggin at the blastopore suggests a previously unrecognized contribution of the ectomesoderm. Expression patterns of many genes, including Tt.twist, Tt.MyoD, and Tt.Mef2, are consistent with conserved roles in mesoderm differentiation and specification. Widespread mesodermal expression of Tt.dachshund and Tt.Six1/2 may be an evolutionary novelty within brachiopods associated with their secondarily derived mode of radial cleavage.The expression patterns observed suggest that several transcription factors, including


ĐM<1.09). NHOs with an unsaturated backbone are essential for polymerization to occur, while substitution at the exocyclic carbon atom has an impact on the reaction pathway and ensures the suppression of side reactions.The metal-free polymerization of propylene oxide (PO) using a special class of alkene—N-heterocyclic olefins (NHOs)—as catalysts is described. Manipulation of the chemical structure of the NHO organocatalyst allows for the preparation of the poly(propylene oxide) in high yields with high turnover (TON>2000), which renders this the most active metal-free system for the polymerization of PO reported to date. The resulting polyether displays predictable end groups, molar mass, and a low dispersity ( Moreovers (NHCs).1–R3 can easily be varied by making use of well-established synthetic routes to form N-heterocycles. Although this structural diversity has so far only been explored to a limited degree, it potentially couples NHC-like adaptability with carbanionic reactivity. These characteristics suggest that NHOs will be ideally suited for application in a field where high nucleophilicity and basicity are essential.The structural versatility of NHOs makes them exciting species that need to be explored more fully, the more so since they are closely related to so-called deoxy-Breslow intermediates, which are proposed to feature a prominent role in NHC-based organocatalysis. Importan2 adducts have also been used for the oligomerization of PO, although high catalyst loadings and high temperature are required.[Despite many recent advances in organocatalyzed polymerization, challenges remain to discover methods for the efficient polymerization of several important monomers. Propylene oxide, oligomers or polymers of which are mainly used in industry as long-chain polyether-polyol components for polyurethane formation, is a prirequired. Herein, 1–3 , a small high-molecular-weight impurity was also observed .While NHC-catalyzed polymerization has been extended to the ROP of PO, ethylene 3 Table , entry 5 3 Table . Analysi4 was synthesized, which bears two methyl groups on the exocyclic carbon atom. In addition to increased steric demands, this modification was proposed to enhance the basicity of the NHO on account of the generation of a latent tertiary carbanion (NHO 3 would generate a primary carbanion), and thus favor the anionic over the zwitterionic mechanism. Gratifyingly, under the same conditions outlined above, NHO 4 yielded PPO with low dispersity and a monomodal molecular weight distribution  and likewise the convoluted aromatic region of BnOH becomes increasingly differentiated. Both observations are again in line with increasing deprotonation, which will increase the negative charge on the oxygen atom (and therefore the shielding of the adjacent methylene moiety) and also strengthen the inductive effect on the aromatic ring. This outcome strongly emphasizes the difference in the reactivity of the studied NHOs. Since NHOs 1 and 2 do not yield PPO, but NHOs 3 and 4 do, these interactions seem critical to defining the reactivity that has to be surpassed to induce polymerization.Clearly, the interaction of NHOs c proton. In contr4 was able to produce highly defined and monomodal PPO, further investigations of the key properties of the polymerization of PO catalyzed by this NHO were undertaken. Identical but independent polymerizations were stopped after different reaction times, to correlate the conversion and molecular weight. Notably, this resulted in a perfectly linear relationship . Additionally, with the proposed role of the NHO as the catalyst and BnOH as the initiator, a change in the ratios was anticipated to have distinct consequences on the molecular weight of the resultant PPO. Although a higher loading of NHO 4 at constant BnOH/PO resulted in a more rapid polymerization, the molecular weight remained within the expected range   , most li4, reactions with very low NHO loadings were conducted, using the aforementioned optimized conditions of PO/BnOH=100:1. Interestingly, with 3000 equivalents of monomer (Table Finally, to further determine the limits of the ROP of PO by NHO er Table , entry 7er Table .4, enabled the synthesis of well-defined PPO in a highly controlled manner, while at the same time, to the best of our knowledge, this NHO emerged as the most active organocatalyst for PO polymerization reported to date. The distinct NHO carbanionic reactivity can be expected to allow access to further highly challenging fields in organopolymerization, especially since the catalysts can be readily tailored, as shown in this study.In conclusion, we have reported the first example of an NHO-catalyzed organopolymerization. Through manipulation of the NHO structure we have shown that the nature of the heterocyclic ring is a key factor in determining the activity in the polymerization of PO. Imidazolium-based catalysts show a high performance, while their saturated five- and six-membered counterparts do not polymerize PO at all. Furthermore, it was demonstrated that the exocyclic carbon atom is a decisive tuning site of the catalysts, and can be used to suppress undesired side reactions. Increased steric congestion and basicity, as present in NHO


Background. Following mass vaccination campaigns in the African meningitis belt with group A meningococcal conjugate vaccine, MenAfriVac (PsA-TT), disease due to group A meningococci has nearly disappeared. Antibody persistence in healthy African toddlers was investigated.Methods. African children vaccinated at 12–23 months of age with PsA-TT were followed for evaluation of antibody persistence up to 5 years after primary vaccination. Antibody persistence was evaluated by measuring group A serum bactericidal antibody (SBA) with rabbit complement and by a group A–specific IgG enzyme-linked immunosorbent assay (ELISA).Results. Group A antibodies measured by SBA and ELISA were shown to decline in the year following vaccination and plateaued at levels significantly above baseline for up to 5 years following primary vaccination.Conclusions. A single dose of PsA-TT induces long-term sustained levels of group A meningococcal antibodies for up to 5 years after vaccination.Clinical Trials Registration. ISRTCN78147026. Neisseria meningitidis (MenA). The observed reduction in meningococcal meningitis due to MenA has been due to the development of an affordable group A meningococcal vaccine, PsA-TT . The Meningitis Vaccine Project, a partnership between the World Health Organization (WHO) and PATH, coordinated the development, testing, licensure, and introduction of this vaccine with the aim of eliminating epidemic meningitis attributable to MenA [The “meningitis belt” of sub-Saharan Africa runs across the continent from Senegal to Ethiopia, and this region is prone to major epidemics of meningococcal meningitis, with a high case fatality rate and serious sequelae. Until recently, most epidemics were due to group A  to MenA –4.Since 2010, PsA-TT has been introduced into countries across the meningitis belt through mass campaigns , 6. PsA-www.controlled-trials.com.The study was conducted in accordance with the principles of the Declaration of Helsinki and in compliance with Good Clinical Practice guidelines. The clinical trial was registered (identifier ISRCTN78147026) at Haemophilus influenzae type b vaccine (Hib-TT) and 10 months later were revaccinated with 1 of these 3 vaccines (the dose of PsACWY administered was one-fifth dose). Blood samples were obtained prior to primary vaccination and revaccination, 4 weeks after primary vaccination, and 1 and 4 weeks after revaccination, the results of which were previously reported [The full details of this study have been reported elsewhere by Sow et al ; in briereported . Those sBlood samples were assayed in the serum bactericidal antibody (SBA) assay using the group A target strain F8238  as previously described [Group A–specific immunoglobulin G (IgG) levels were determined using an enzyme-linked immunosorbent assay (ELISA) , except 2-transformed titers and log10-transformed concentrations as an outcome. At 5 years after primary vaccination, the comparisons in SBA GMTs and group A–specific IgG GMCs between the vaccine groups of interest were performed by analysis of covariance adjusted for baseline titers (concentrations), sex, and time. The paired t test was used to compare the GMTs and GMCs between 2 and 5 years after primary vaccination. The percentages of subjects with SBA titers ≥128 and group A–specific IgG concentrations ≥2 µg/mL along with their exact binomial 95% confidence intervals (CIs) were provided. All immunogenicity analyses were conducted in the intention-to-treat population. Missing values were treated as missing at random. All tests were 2-sided with a significance level of .05. Data analysis was performed using SAS software, version 9.1.3.The SBA geometric mean titers (GMTs) and group A–specific IgG geometric mean concentrations (GMCs) between the vaccine groups at 1 year and 2 years after primary vaccination were compared by mixed-effects modeling adjusted for baseline titers (concentrations), age, sex, study site, time, and interaction effects of interest with logAs previously reported , 601 MalP < .0001 for all 3 pairwise comparisons).The SBA GMTs and proportion of subjects with SBA titers ≥128 for each study group are shown in Table P < .0001) than the GMTs of those who received PsA-TT followed by Hib-TT (PsA-TT/Hib-TT), with GMTs of 3722.5  and 1035.0 , respectively.The SBA GMTs of those vaccinated with Hib-TT followed by PsA-TT (Hib-TT/PsA-TT) were higher (P < .0001) and similar to those who received 1 dose of PsA-TT at revaccination, with GMTs of 4485.8 , 3722.5 (2551.4–5431.3), and 1035.0  in the PsA-TT/PsA-TT, Hib-TT/PsA-TT, and PsA-TT/Hib-TT groups, respectively.The SBA GMT at 1 year after primary vaccination in those who received 2 doses of PsA-TT (PsA-TT/PsA-TT) was statistically significantly higher than that in those who only received 1 dose of PsA-TT at primary vaccination . The GMTs of the 2 groups that received a dose of PsA-TT at revaccination were similar at 2 years after primary vaccination, with GMTs of 2720.8  and 2527.6  in the PsA-TT/PsA-TT and Hib-TT/PsA-TT groups, respectively. However, the GMT of the group that received PsA-TT only at primary vaccination was lower . At 5 years after primary vaccination, the GMT of those who had not received PsA-TT in the initial study period but received PsA-TT at the end of the initial study (Hib-TT/Hib-TT) was statistically significantly higher than that in those vaccinated with only 1 dose of PsA-TT at primary vaccination (P = .0118). At the same time point, there was no significant difference in the GMTs of the Hib-TT/Hib-TT group and those who received PsA-TT at revaccination only (Hib-TT/PsA-TT) and those who received 2 doses of PsA-TT (PsA-TT/PsA-TT). From 2 to 5 years after primary vaccination, the SBA GMTs of the Hib-TT/Hib-TT group significantly increased (P = .004).At 2 years after primary vaccination, the SBA GMT in those who had not received PsA-TT (Hib-TT/Hib-TT) was significantly lower than that of the 3 groups that had received PsA-TT  of subjects in all groups that had received PsA-TT had SBA titers ≥128. In those who had not received PsA-TT , a lower percentage of subjects had SBA titers ≥128, with 72.1%  and 86.9%  of subjects achieving this level at 1 year and 2 years after primary vaccination, respectively; the percentage increased to 100%  at 5 years after primary vaccination .The group A–specific IgG GMCs (µg/mL) and proportion of subjects with group A–specific IgG ≥2 µg/mL of each study group are shown in Table P < .0001 for all 3 pairwise comparisons at 1 year and 2 years after primary vaccination).At 1 year and 2 years after primary vaccination, the group A–specific IgG GMC in all 3 groups that received PsA-TT was significantly higher than that of those who had not received PsA-TT (Hib-TT/Hib-TT) (P = .0037).The group A–specific IgG GMC of the Hib-TT/PsA-TT group was significantly higher than that of the PsA-TT/Hib-TT group at 1 year after primary vaccination, with GMCs of 1.6  and 0.9  (P < .0001). The GMC of the 2-dose PsA-TT group was also higher than that of Hib-TT/PsA-TT at 2 years following vaccination (P < .0001), with GMCs of 3.7  and 1.2 , respectively.This effect was not found at 2 years after vaccination, though, with GMCs of 1.2  and 0.9  in the Hib-TT/PsA-TT and PsA-TT/Hib-TT groups, respectively. At 1 year postvaccination, the group that received 2 doses of PsA-TT had higher group A–specific IgG GMCs  than the Hib-TT/PsA-TT group  , with significant increases in the PsA-TT/PsA-TT (P = .0149) and Hib-TT/Hib-TT (P < .0001) groups from 2 years following primary vaccination to 5 years following primary vaccination.At 5 years after primary vaccination, the GMCs of the PsA-TT/PsA-TT and Hib-TT/Hib-TT  groups were significantly higher than the GMCs in the PsA-TT/Hib-TT and Hib-TT/PsA-TT groups  . EarlierSow et al  reportedDifference in the SBA GMTs was observed at 1 and 2 years postvaccination between those who received 1 dose of PsA-TT at primary vaccination and those who received 2 doses. However, the same difference was not observed when a single dose of PsA-TT was administered at an older age of 22–33 months, at the revaccination visit. Differences between the groups that received 1 and 2 doses were also evident with the IgG GMCs, with those who received 2 doses having higher IgG GMCs at 2 and 5 years after primary vaccination. This is in contrast to the results observed by Klein et al , which sPsA-TT has been introduced into several countries of the African meningitis belt via mass vaccination campaigns in which individuals aged 1–29 years received 1 dose. PsA-TT has been shown to have had a major impact on MenA disease and carriage in Chad . This st


Eotaxin was significantly higher in plasma, whereas G-CSF showed no difference between plasma and CSF. G-CSF (P = 0.0079), IL-8 (P = 0.0223), IP-10 (P = 0.0109), and MCP-1 (P = 0.0497) in CSF showed significant difference between HIV-CI and HIV-NC group, which may indicate their relationship to HIV associated neurocognitive impairment. In addition, G-CSF (P = 0.0191) and IP-10 (P = 0.0377) in plasma were significantly higher in HIV-CI than HIV-NC. The consistent changes of G-CSF and IP-10 in paired plasma and CSF samples might enhance their potential for predicting HAND.Although HAD is now rare due to HAART, the milder forms of HAND persist in HIV-infected patients. HIV-induced systemic and localized inflammation is considered to be one of the mechanisms of HAND. The levels of cytokines in CSF were associated with neurocognitive impairment in HIV infection. However, the changes of cytokines involved in cognition impairment in plasma have not been shown, and their relationships between CSF and plasma require to be addressed. We compared cytokine levels in paired CSF and plasma samples from HIV-infected individuals with or without neurocognitive impairment. Cytokine concentrations were measured by Luminex xMAP. In comparing the expression levels of cytokines in plasma and CSF, IFN- HIV associated neurocognitive disorder (HAND) is a prevalent and significant challenge to HIV infected populations . Its cliThus, this study aimed to firstly determine whether CSF levels of the inflammatory cytokine or chemokine were elevated in HIV infected patients with neurocognitive impairment and secondly to examine relationships between the concentrations of these cytokines in the CSF and plasma.HIV infected patients were enrolled in Henan and Yunnan provinces in China, and CSF were acquired at the time of a clinically indicated lumbar puncture. CSF and plasma samples were taken simultaneously. All the samples were centrifuged, and cell-free CSF and plasma were aliquot and immediately frozen at −80°C. The Memorial Sloan-Kettering Scale (MSK) was used to categorize the neurocognitive impairment of each patient. This study was approved by ethics committees at the Capital University of Medical Science in Beijing, and all participants provided informed consent.α2, IFN-γ-induced protein- (IP-) 10, and monocyte chemotactic protein- (MCP-) 1. The lower levels of detection were ranged between 0.2 and 10.1 pg/mL for each of the cytokines measured. All samples were assayed concurrently in duplicate. Data were collected using a Bio-Plex Suspension Array Reader . Cytokine concentrations that were lower than the lower limits of detection were reported as undetectable (0).A total of 6 cytokines were measured in paired CSF and plasma samples using human cytokine/chemokine MILLIplex kits : IL-8, eotaxin, granulocyte colony-stimulating factor (G-CSF), interferon- (IFN-) U test and Wilcoxon signed rank test were used for unmatched and matched comparisons, respectively. Spearman rank tests were used to test for correlations. P values less than 0.05 were considered significant.Univariate analyses were performed using GraphPad Prism 5 . Mann-Whitney n = 44), intravenous drug use (n = 15), blood transfusion (n = 7), paid blood or plasma donation (n = 2), mother-to-child transmission (n = 2), and unknown (n = 15). In total, 29 of 85 patients had complications, including cryptococcal meningitis (n = 21), tuberculous meningitis (n = 2), Toxoplasma encephalitis (n = 2), Pneumocystis pneumonia (n = 2), and cytomegalovirus radiculitis (n = 2). Nearly half of the HIV infected patients (43%) were receiving HAART at the time of evaluation. All were on multidrug combination ART regimens, which consisted of at least 2 NRTIs  plus an NNRTI (NVP or EFV) or a PI (LPV). None were on mono- or dual-therapy. Based on MSK classification, patients were classified into HIV infected with normal cognition  and impaired cognition groups . The HIV-CI group consisted of MSK = 0.5 (n = 24), MSK = 1 (n = 12), MSK = 2 (n = 10), and MSK = 3 (n = 6). The viral burden in CSF or plasma of HIV-CI group was higher than that of HIV-NC group, accompanied with lower CD4 cell count. However, there was no statistically significant difference whether in viral load or in CD4 cell count for two groups  and 59 (69%) were men. The risk factors for HIV infection were sexual transmission (o groups .P = 0.0079), IL-8 (P = 0.0223), IP-10 (P = 0.0109), and MCP-1 (P = 0.0497) between HIV-CI and HIV-NC group (P = 0.0191) and IP-10 (P = 0.0377) showed significant difference (α2 did not show any significant difference between these two groups either in CSF or in plasma (data not shown).HIV infected patients with neurocognitive impairment had elevated levels of cytokines in CSF and plasma in comparison to those with normal neurocognition . There wNC group . Howeverfference . The conα2, IL-8, IP-10, and MCP-1 in CSF were significantly higher than in plasma. In contrast, eotaxin was significantly higher in plasma. But there was no difference for G-CSF. The expression levels of several cytokines showed significant correlations between CSF and plasma. Among them IP-10 presented the strongest correlation in the patients with HIV-CI   .α2, IP-10, and MCP-1 were higher in CSF than plasma samples, whereas eotaxin and G-CSF were significantly higher in plasma. Taking into account the lack of correlation between CSF and plasma cytokine concentrations, these might suggest that some cytokines may be localized within the CSF which is the potential origin of immune response rather than resulting from the dissemination of cytokines via blood stream.We demonstrated that the concentrations of G-CSF, IL-8, IP-10, and MCP-1 in CSF samples from neurocognitive impaired HIV-1 infected patient were significantly higher than patients with normal cognition. This confirmed our finding in previously published study . Elevateγ-inducible protein 10, IP-10 , is expressed by astrocytes, microglia, and endothelial cells in an inflammatory lesion and cells culture supernatants, acts specifically on activated T cells and macrophages, and attracts T lymphocytes into the CSF. Expression of IP-10 in the brain is correlated closely with increased infiltrating cells in herpes simplex virus (HSV), MHV, and West Nile virus (WNV) infection . There was no significant difference in CD4 cell count between HIV-CI and HIV-NC patients; nevertheless, relative poorer immune state could exert a negative influence on inflammatory response.In conclusion, our study suggested that elevated levels of G-CSF, IL-8, IP-10, and MCP-1 in CSF may indicate HIV associated neurocognitive impairment. Consistent changes of G-CSF and IP-10 in paired plasma and CSF samples enhance their potential for predicting HAND.


Altered cerebrospinal fluid (CSF) levels of lactate have been described in neurodegenerative diseases and related to mitochondrial dysfunction and neuronal degeneration. We investigated the relationship between CSF lactate levels, disease severity, and biomarkers associated with neuroaxonal damage in patients with multiple sclerosis (MS).One-hundred eighteen subjects with relapsing-remitting multiple sclerosis (RRMS) were included, along with one-hundred fifty seven matched controls. CSF levels of lactate, tau protein, and neurofilament light were detected at the time of diagnosis. Patients were followed-up for a mean of 5 years. Progression index (PI), multiple sclerosis severity scale (MSSS), and Bayesian risk estimate for multiple sclerosis (BREMS) were assessed as clinical measures of disease severity and progression. Differences between groups and correlation between CSF lactate, disease severity and CSF biomarkers of neuronal damage were explored.CSF lactate was higher in RRMS patients compared to controls. A negative correlation was found between lactate levels and disease duration. Patients with higher CSF lactate concentration had significantly higher PI, MSSS, and BREMS scores at long-term follow-up. Furthermore, CSF lactate correlated positively and significantly with CSF levels of both tau protein and neurofilament light protein.Measurement of CSF lactate may be helpful, in conjunction with other biomarkers of tissue damage, as an early predictor of disease severity in RRMS patients. A better understanding of the alterations of mitochondrial metabolic pathways associated to RRMS severity may pave the way to new therapeutic targets to contrast axonal damage and disease severity. Multiple sclerosis (MS) is a chronic autoimmune disorder of the central nervous system (CNS), presenting with unpredictable clinical relapses and remissions and by disability progression over time . NeuropaIn the last years, mitochondrial dysfunction and subsequent energy penalty have been hypothesized to drive axonal degeneration and disease progression –7. In thRecent data indicate that other biomarkers associated with neuroaxonal damage, such as tau protein (t-tau), play an important role in modulating mitochondrial function and dynamics . To dateThe aim of our study was to investigate the following: (1) the level of CSF lactate in patients with relapsing-remitting multiple sclerosis (RRMS) compared to healthy subjects; (2) the potential value of CSF lactate to predict disease severity and progression; (3) the relationship between CSF lactate levels and both t-tau and neurofilament light (NFL) protein.The study was approved by the ethics committee of the Tor Vergata Hospital in Rome, and informed consent was obtained from all patients.We collected CSF from 118 consecutive patients admitted to the Neurology Clinic of Tor Vergata Hospital of Rome and diagnosed as RRMS according to validated criteria . SubjectOne hundred fifty-seven age- and gender-matched individuals, who underwent neurological investigation, brain magnetic resonance imaging (MRI), and LP for diagnostic purposes, were enrolled as control subjects. They resulted negative for inflammatory or degenerative disorders of the central and peripheral nervous system, systemic diseases, and of abnormal cell count and/or other abnormalities in the CSF. Subjects under treatment with drugs interfering with central nervous system (CNS) were also excluded.At the time of confirmed diagnosis, all MS patients started disease-modifying therapy. Second-line treatment was also considered for patients who experienced at least two relapses during 1 year of therapy with other approved immunomodulatory agents.After their admittance, all patients underwent CSF and blood tests, complete neurological evaluation, and brain  MRI scan. Clinical status was assessed during outpatient scheduled visits every 3 months. Demographic and clinical data were collected from medical records. MS disease onset was defined as the first episode of focal neurological dysfunction suggestive of MS. Disease duration was estimated as the number of years from onset to the most recent assessment of disability. Clinical activity was defined as the occurrence of any clinical relapse, during the follow-up period.http://www.neurostatus.net/index.php?file=start), a ten-point scale measuring neurological disability by rating nine different neurological domains [Relapses were defined as the occurrence of new or recurrent neurological symptoms not associated with fever or infection lasting for at least 24 h. The annualized relapse rate (ARR) was defined as the number of relapses per year. In addition, the number of relapses in the first 2 years of the disease course and the time until the first relapse were used as clinical indexes of inflammatory activity. Disability was determined by a trained neurologist using the expanded disability status scale (EDSS) ( domains . Sustain domains . MSSS ha domains .www.fdi.com).All CSF samples were obtained through LP performed in lateral decubitus. CSF samples were collected in polypropylene tubes using standard sterile techniques. Each CSF sample was divided in two aliquots: 2 ml of CSF sample were used for biochemistry analysis including total cell count and lactate levels; 3 ml of CSF sample were centrifuged at 1300 rpm for 10 min after withdrawal to remove cellular elements and immediately stored at −80 °C until used. Biochemistry assays were carried out using commercially available kits following the manufacturer’s instruction. NFL was detected in CSF samples using commercial ELISA kit . The levels of NFL in CSF were measured by fitting data to a four-parameter standard curve using GraphPad Prism Software Package .CSF levels of t-tau were quantified with standard procedures, using commercially available ELISA test . The biomarker concentrations were calculated using a standard sigmoid curve equation   and control  groups (not shown).CSF lactate was higher in RRMS patients compared to controls (08) Fig. . Lactaters = −0.21; p = 0.008). In contrast, lactate levels did not correlate with clinical disability assessed by the EDSS at the time of CSF withdrawal. At long-term follow-up, there were significant correlations between lactate levels at diagnosis and BREMS , PI , and MSSS  scores among all MS cases  and MSSS . Conversely, after correction for age and gender, CSF lactate correlation with BREMS lost its significance.After entering age and gender as control variables in a partial correlation analysis, CSF lactate levels at the time of LP still showed a significant correlation with PI (n = 60) or the presence  of contrast-enhancing lesions at baseline MRI. Our analysis showed that CSF levels of lactate were similar in the Gd+ group compared to the Gd− group (p = n.s.), suggesting no involvement of this biomarker in the acute stage of inflammation. In addition, non-significant correlations were found between CSF lactate levels, CSF leukocyte count, or relapse rate. Furthermore, lactate levels were similar among patients categorized according to the presence of oligoclonal bands  (not shown).To better investigate the correlation between lactate and the inflammatory process typical of MS, RRMS subjects were categorized according to the absence  and NFL , well-recognized markers of neuronal damage  studies showed that MS patients have higher levels of CSF lactate , 13. MRINo significant correlation was observed between CSF lactate levels and the number of contrast-enhancing lesions, IgG index, CSF leukocyte count, or relapse, suggesting that increased anaerobic pathways and acute inflammation are independent events and supporting the hypothesis that focal inflammation and neurodegeneration may disconnect early in the disease course .Interestingly, CSF lactate correlated significantly with PI, MSSS, and BREMS scores, suggesting an association between altered energy metabolism and MS severity, that may start during the relapsing-remitting phase of the disease. Moreover, we observed that CSF lactate concentrations significantly correlated with age both in patients and controls, indicating an age-related increase in CSF lactate independent of MS pathology . Age at We found no correlation between CSF lactate levels and neurologic disability as assessed by the EDSS in accordance to previous studies .It is well established that CSF tau and NFL levels represent biomarkers of acute neuroaxonal damage . ExperimRemarkably, inflammation and myelin loss in the CNS in MS increase the energy demand of a neuron due to ineffective nerve conductance and thus challenge the mitochondrial machinery . PresumaAlthough not yet proven, it is likely that accumulated neuro-axonal damage may influence per se mitochondrial injury and energy deficiency over time, throughout a potential tau-dependent amplification mechanisms, as reported in other neurodegenerative disorders and tauopathies . The invInterestingly, NFL reflects acute axonal loss due to inflammatory mechanisms . In factEarly mitochondrial dysfunction may be reversible and a valuable new target for treatment . The res


Radial forearm free flap is the most reliable flap for intraoral soft tissue reconstruction after cancer ablation surgery. However, unesthetic scar of the donor site and the need for a second donor site for skin graft are major disadvantages of the forearm flap. The purpose of this study was to report the clinical results of double-layered collagen graft to the donor site of the forearm free flap without skin graft.Twenty-two consecutive patients who underwent oral cancer ablation and forearm reconstruction between April 2010 and November 2013 were included in this study. Male to female ratio was 12:10, and average age was 61.0 years old (27–84). Double-layered collagen was grafted to the donor site of the forearm free flap and healed for secondary intention. Upper silicone had been trimmed at the periphery during secondary intention, and dry dressing was used. Postoperative scar healing and esthetic results and function were evaluated.An average follow-up period was 34.9 months. The scar area was decreased to 63.9 % in average. The complete healing was obtained between 1.5 and 3 months according to the defect size. There was no functional defect or impairment 3 months after operation. All patients were satisfied with the esthetic results. Three patients died of recurred cancer.Double-layered collagen graft was successfully performed in this study. Without the thigh skin graft, patients had experienced less painful postoperative healing periods and discomfort. Since the early 1980s, the radial forearm free flap (RFFF) has been used for head and neck reconstruction due to its thin, hairless skin, pliable soft tissue, consistent anatomy, and long vascular pedicle . HoweverThe purpose of this study was to evaluate clinical results of double-layered collagen graft technique for radial forearm donor site reconstruction without skin graft.Twenty-two consecutive patients between April 2010 and September 2014 were included in this study. Institutional review board from Asan Medical Center issued an exemption to this study because of the use of collected existing data in such a manner that subjects cannot be identified, directly or through identifiers linked to subjects. Male to female was 12:10 and average age was 61 years old. The age of the patients ranged from 27 to 84 years. The mean follow-up period was 34.9 months. Only one patient was revealed melanoma and the others were squamous cell carcinoma histopathologically.RFFF was elevated by subfascial plane under general anesthesia. After the elevation of RFFF, adjacent muscles covered exposed the brachioradialis tendon. A MatriDerm®  sheet on the bottom layer and a Terudermis®  sheet on the surface layer fitted to the defect site. Double-layered collagens were fixed with 2-0 silk temporary sutures, and a tie-over dressing was applied. A wrist-around splint was performed for 1 week to stabilize the graft and to minimize wound contractures Figs. , and 4. Before the elevation of RFFF, the length and width of the flap were measured, and the length multiplied by width was recorded as initial scar area. Tie-over dressing was removed a week after surgery. The scar area was measured when the patients came to the outpatient clinic for follow-up checks. The area change had been recorded, and the ratio before and after operation was calculated.2, and the mean defect size was 31 cm2 (Table 2 except one case of tendon exposure patient. The mean of final scar size was 11.6 cm2. The original donor defect of the tendon exposure case was 42 cm2, but 2 months later, the scar size was measured to 40.5 cm2 approximately. On average, the final scar area was decreased to 36.1 % size in average compared to the original donor defects. The complete healing was obtained between 1.5 and 3 months according to the defect size in 21 patients (Figs. Donor site complications including graft rejection, graft failure, infection, impaired hand manipulation, and swelling of hand were not noted in any patient. There was no functional defect or impairment 3 months after operation. One patient had suffered from tendon exposure at follow-up visit 2 months from surgery. The other 21 patients were satisfied with the esthetic results. Three patients died of recurred buccal mucosa cancer 17, 22, and 30 months after operation, respectively (patient nos. 2, 13, and 15 in Table ts Figs. . The scats Figs. , and 19.Many investigators have reported that closing donor sites with FTSG had resulted satisfactory outcomes functionally and esthetically . But, FTIn this study, we tried to replace epidermis with silicon-contained bilayer artificial dermis. We used MatriDerm of inner layer and Terudermis of outer layer for the coverage of the forearm donor site. Terudermis has a bilayer structure consisting of an outer silicone layer and an inner layer of mixed atelocollagens . The inn2. Therefore in our cases, the costs for the substitution ranged between 90 and 216US$ in the USA [MatriDerm, which was made commercially available in 2004, minimizes contracture of scar and increases hand and wrist movement . MatriDeIn our study, without the second donor site morbidity caused by elevation of split-thickness skin, we evaluated the effectiveness of this technique in consideration of decreased scar sizes and patient’s comfort during healing period. It is a major advantage of this procedure to skip of secondary surgery for donor site coverage. The other disadvantage of using artificial dermis without STSG is a delayed healing period. The healing period of an STSG is typically 4 to 6 weeks . In the Double-layered collagen graft to the donor site of the forearm flap was successfully performed in this study. Patients were satisfied with the esthetic and functional results. Without the thigh skin graft, patients had experienced less painful postoperative healing periods and discomfort. Therefore, double-layered collagen graft without STSG represents a viable option for radial forearm donor site reconstruction.