Beauveria bassiana is currently used as a biocontrol agent (BCA) of insects. Fifty-seven Beauveria bassiana isolates -53 from Spain- were characterized, integrating group I intron insertion patterns at the 3'-end of the nuclear large subunit ribosomal gene (LSU rDNA) and elongation factor 1-alpha (EF1-α) phylogenetic information, in order to assess the genetic structure and diversity of this Spanish collection of B. bassiana.The entomopathogenic anamorphic fungus B. bassiana s.s. (sensu stricto) group and separate them into 5 molecular subgroups, all of which contained a group I intron belonging to the IC1 subtype at the Ec1921 position. A number of parameters such as thermal growth or origin  were also examined but in general no association could be found.Group I intron genotype analysis was based on the four highly conserved insertion sites of the LSU . Of the 16 possible combinations/genotypes, only four were detected, two of which were predominant, containing 44 and 9 members out of 57 isolates, respectively. Interestingly, the members of the latter two genotypes showed unique differences in their growth temperatures. In follow, EF1-α phylogeny served to classify most of the strains in the B. bassiana isolates (77.2%) are grouped into a major phylogenetic subgroup with word-wide distribution. However, high phylogenetic diversity was also detected among Spanish isolates from close geographic zones with low climatic variation. In general, no correlation was observed between the molecular distribution and geographic origin or climatic characteristics where the Spanish B. bassiana isolates were sampled.Most Spanish  Beauveria bassiana  Vuill. (teleomorph: Cordyceps bassiana) is the most widely used mycopesticide for the biological control of insect pests [The anamorphic fungus ct pests ,2, formuct pests . Howeverct pests . For prect pests -7.Beauveria. Morphological and molecular studies have shown that the broad patterns of diversity in Beauveria have been accurately predicted in previous morphological studies. However, they have also shown that these approaches are insufficient to investigate species such as B. bassiana [B. bassiana is a species complex with several cryptic species and have corroborated their link to Cordyceps teleomorphs [Beauveria and the existence of at least two lineages within B. bassiana s.l. (sensu lato), and also that EF1-α sequences provide adequate information for the inference of relationships in this genus [B. bassiana are crucial for the development of molecular tools for their monitoring in the natural environment [The morphological features of conidia are common tools for identification in bassiana . Moleculeomorphs -12. In tis genus . Studiesironment .B. bassiana isolates.Minisatellite loci , random B. bassiana genetic groups and host affiliation [B. bassiana contains generalist enthomopathogens with no particular phylogenetic association with their insect host [B. bassiana [B. bassiana genetic groups and Canadian [Although some molecular studies have correlated iliation ,19, moreect host ,18, envibassiana . In thisCanadian , BraziliCanadian  climaticBeauveria brongniartii LSU genes [Beauveria. For example, Neuvéglise et al. [B. brongniartii, two of B. bassiana, and one of Metarhizium anisopliae from several geographic origins. Coates et al. [Beauveria isolates. Wang et al. [B. bassiana isolates in 13 different genotypes. In that study, their sequence analyses confirmed that the introns were invariably inserted in specific target sequences, and a strong correlation between specific insertion sites and intron subgroups was also observed. In addition, the features and behaviour of these group I intron were also detected in related genera such as Cordyceps [Metarhizium [Entomopathogenic species displayed a high degree of variability-mainly attributed to the presence of group I introns- at specific sites of the coding regions of small and large subunits of nuclear ribosomal RNA genes (SSU rDNA and LSU rDNA). Group I introns in entomopathogenic fungi were initially reported in SU genes . Work ade et al.  found 14s et al.  found 12g et al.  analyzedordyceps  and Metaarhizium .B. bassiana, obtained from different substrates or insect hosts, and 4 isolates from other European countries. The insertion patterns of group I introns at the 3'-end of the LSU rDNA genes and EF1-α phylogenetic distribution were integrated in order to explore any possible correlation between genetic groups and geographical/climate origin, and habitat or insect host.The present study was undertaken to investigate the genetic variability existing in a collection of 53 Spanish isolates of B. bassiana isolates . Insert1,84: 57.20°C; P < 0.001) and highest  growth temperatures, which were significantly lower in the genotype B1B2B3A4, with Topt and Tmax values of 24.3 and 33.9°C, respectively, than those obtained for A1B2B3A4, with Topt of 26.7 and Tmax 35.6°C (data not shown).There was a significant correlation between belonging to an intron genotype and the mean of the optimal , and was 502 bp long. This intron shared 99 and 98% identity with two sequences previously detected at the same position in the LSU of and 1121 ,25. A 38a ECBL16 .B. bassiana intron sequences exhibited the typical characteristics of group I and no ORFs were detected. These intron sequences from B. bassiana were compared with other fungal intron sequences available in databases for their placement in previously reported subgroups [B. bassiana intron sequences representing different subgroups to examine their polymorphisms (data not shown). Intron size and nucleotide identity differences were observed but P, Q, R and S motif elements, which are needed for the formation of the secondary structure of group I introns [The seven different ubgroups . The intubgroups -27, thos introns , were hiB. bassiana isolates and another 24 GenBank-deposited sequences, which represent intron sequences from M. anisopliae, B. bassiana and Cordyceps profilica, together with the subsequent phylogenetic analysis are shown in Figure Cordyceps and Metarhizium were separated from Beauveria sequences and formed an independent group, supported by a bootstrap value of 100%. In addition, the five different Beauveria sequences obtained here were separated into two of the four observed groups at this position, supported by bootstrap values of 94% and 60%. This separation was in accordance with the two sequence sizes detected: 443 and 427-bp in length. However, the four different sequence types detected for 443-bp-sized introns were not separated after phylogenetic analysis.The MP tree obtained after an alignment of the 7 different intron sequence types identified from 57 B. bassiana s.s. (sensu stricto), B. brongniartii and B. bassiana clade C [B. bassiana s.s. clade, and 5.4% (3 isolates) in clade C, which includes B. cf. (uncertain taxonomy) bassiana isolates. Within B. bassiana s.s., the 53 isolates analyzed in this study were separated in five subgroups , supported by bootstrap values higher than 50%.With the exception of isolate Bb49, where no amplification was observed, all isolates afforded PCR products of 1.1 kb for the EF1-α gene with the primers tef1fw and 1750-R. Eleven different EF1-α gene sequences were identified among the 56 isolates. The alignment and comparison of these 11 sequences and another 18 GenBank-deposited sequences, representing different lineages from  clade C , produceB. bassiana s.s. isolates showed an intron IC1 inserted at position 4. However, the IE intron inserted at position 1 was only present in the 10 isolates from subgroup Eu-7 and 33 out of 39 isolates from subgroup Wd-2. In particular, this subgroup included most of the Spanish isolates of B. bassiana forming an EF1-α phylogenetic group with isolates 681 from Romania and 792 from the USA [B. cf. bassiana isolates from clade C.In order to assess the phylogenic distribution of the different intron configuration types, they were mapped on the EF1-α tree Figure . All 53 No correlation between EF1-α phylogenetic groups and insect host was observed. Although Eu-7 subgroup did not included isolates of insect origin, the Wd-2 subgroup grouped isolates collected from Diptera, Hymenoptera, Lepidoptera and Orthoptera. Moreover, Wd-2 isolates from Orthoptera displayed different intron insertion models .B. bassiana s.s. were collected from subtropical Mediterranean climate zones and were distributed in the Eu-7, Eu-3, Wd-2 and Eu-8 subgroups. Two Spanish isolates, Bb52 and Bb53, were collected from continental climate locations and were placed within subgroups Eu-7 and Wd-2, respectively. The only B. bassiana s.s. isolate from a humid oceanic climate included in this work, Bb51 from Santander, displayed a characteristic intron insertion model and formed the EF1-α subgroup Eu-9. In addition, Bb51 produced smaller conidia than the rest of B. bassiana isolates, this morphological feature being statistically significant (data not shown). Nevertheless, other isolate from the same climatic zone, Bb50, was grouped with other European isolates in B. cf. bassiana clade C.Forty-nine Spanish and one Portuguese isolates of B. bassiana genotypes and phylogenetic subgroups in a collection of 57 isolates of this fungus, based on intron insertion patterns and EF1-α phylogenies, respectively.In the present study, we have identified different B. bassiana LSU rDNA identified 99 introns among the 57 isolates analyzed. Four specific sites of intron insertion have been described previously in Beauveria species [The variability in group I introns from rDNA genes has been used as a molecular tool for the identification of polymorphisms in entomopathogenic fungi ,30,31. O species ,25, but B. bassiana s.s. isolates had an intron inserted at position 4. This position was also preferential for intron insertion (84.4%) in a population of 125 B. bassiana isolates [B. bassiana isolates by Wang et al. [M. anisopliae isolates by Márquez et al. [Cordyceps by Nikoh and Fukatsu [B. bassiana collection while greater variability was found in other studies: 13, 7 and 9 insertion patterns within 125 B. bassiana [M. anisopliae [B. brongniartii [Particularly, our study shows that 100% of z et al. , and the Fukatsu . Howeverisopliae  and 47 Bgniartii  isolatesB. bassiana introns are separated from Metarhizium and Cordyceps introns, and two B. bassiana IC1 sequence sizes were located in two different sub-clades, supported by high bootstrap values.The MP tree based on intron sequences shows that they were distributed in four large groups, with bootstrap values of 100%, corresponding to four insertion positions  and, excepting those corresponding to B. bassiana (A and C), they are closely to species previously described on the basis of their morphology, and ii) B. bassiana s.s. (A) was determined almost entirely from nucleotide variation at EF1-α. Further phylogenetic studies carried out with nuclear and/or mitochondrial DNA regions of B. bassiana from all continents have served to resolve lineage diversity within this species [B. bassiana s.s. isolates as Eu-7, Eu-8 and Eu-9. The results obtained from MP analyses , and three isolates grouped in three different phylogenetic subgroups within B. cf. bassiana (C). As in a previous study [B. bassiana s.s. was separated in five phylogenetic subgroups. However, only isolate Bb38, sampled from insects, was grouped with one (Eu-3) of the five phylogenetic species proposed by those authors working with a Danish collection of B. bassiana s.s. [Rehner and Buckley's study  based on species ,12,18,21 species , we follB. bassiana EF1-α phylogeny . According to Meyling's study [B. bassiana s.s. could be explained by the untilled habitats where most of them were sampled .The genetic diversity of Spanish ter view , no geneter view  has prov's study  allopatrnt study  concludeB. bassiana genotypes [B. bassiana s.s. from a humid oceanic climate was the singular isolate Bb51. The fact that isolates from Mediterranean or continental climates overlapped in different phylogenetic subgroups, could be due to lower differences among the abiotic conditions existing in Spain, a country covering far smaller geographical surface and with much less variability than that considered in other Canadian, Brazilian or world-wide studies where phylogenetic species showed a better correlation with climate characteristics [B. bassiana genetic groups from three different habitats in Canada were associated with temperature preferences. When we explored the thermal preferences within a set of Spanish B. bassiana s.s. isolates belonging to the two main intron genotypes (A1B2B3A4 and B1B2B3A4) and four phylogenetic EF1-α subgroups (data not shown), a correlation between intron genotypes and the mean optimal and maximum temperatures for growth was observed, both growth temperatures being significantly lower in the B1B2B3A4 genotype with respect to A1B2B3A4. However, no correlation was observed between thermal preferences and the climatic origin of the Spanish B. bassiana isolates.Previous studies have suggested that the saprophytic phase of entomopathogenic fungi exerts evolutionary pressure on the genotype and that adaptation to a habitat type is associated with their environmental preferences . Recent enotypes . Our stueristics , biogeogeristics  and habieristics . In a theristics  it was dB. bassiana s.s. and B. cf. bassiana (clade C) have been identified, respectively, in a collection of 57 B. bassiana isolates -53 from Spain. The highest polymorphism was observed in introns inserted at positions 2 and 4. All B. bassiana s.s. displayed an IC1 intron inserted at position 4. Integration of intron insertion patterns and EF1-α phylogenetic distribution served to demonstrate the monophyletic origin and vertical transmission of introns inserted at the same site. In subsequent events intron speciation and diversification take place as occurs at site 4, where B. bassiana introns are separated from Metarhizium and Cordyceps introns. No general correlation was observed between the molecular data and insect host, but a tenuous correlation was detected with the geographic origins. The high phylogenetic diversity of the Spanish isolates of B. bassiana s.s. could be due to the untilled habitats where most of them were sampled.Four intron genotypes, and five and three phylogenetic subgroups within B. bassiana used in this study were selected from a Spanish collection of 960 records at the CRAF  Department of the University of Cordoba , representing different geographic origins, habitats/hosts and climates. Fifty-three Spanish isolates were studied, 51 of them being collected from subtropical Mediterranean climate zones -characterized by warm to hot, dry summers and mild to cool, wet winters- and 2 from a humid oceanic climate. Forty-five out of these 53 isolates were from soil, most of them from poorly tilled or untilled fields  and 8 were isolated from insects. Information about these isolates is provided in Table The 57 isolates of Mycelia for DNA extraction were obtained as previously described . Total DBeauveria included in the study. The distribution of putative introns was investigated using the following combinations of previously described primers: I29-I38, I31-I32, I21-I22 and E23-M1 [Beauveria EF1-α genes obtained from databases. PCR was performed in a total volume of 50 μl containing 25 ng of genomic DNA and 0.20 μM concentrations of each of the above primers, using the Taq polymerase system  and following the manufacturer's instructions. The amplification program included an initial denaturing cycle of 1 min at 94°C, followed by 35 cycles of 1 min 30 s at 94°C, 2 min (for EF1-a) or 2 min 30 sec for (LSU rDNA) at 55 (for EF1-a) or 57°C (for LSU rDNA) and 3 min at 72°C, and a final extension step of 7 min at 72°C in a PCR System 9700 Genetic Thermal Cycler . The PCR products were electrophoresed on 1% agarose gels buffered with 1 × TAE [Two nuclear gene regions, LSU rDNA and EF1-α, were amplified, sequenced and analyzed. The 3'-end of the nuclear LSU rDNA cluster was also amplified with primers I29 (5'-CTGCCCAGTGCTCTGAATGTC-3')  and M1 , following the manufacturer's protocol. DNA sequences were obtained using an automated ABI 377 Prism Sequencer  with fluorescent terminators at the Department of Microbiology and Genetics of the University of Salamanca. All PCR products were sequenced in both directions, using amplification primers and internal primers when necessary.The PCR products were purified from agarose gels using the Geneclean II kitThe intron and EF1-α sequences obtained in this study were deposited in the GenBank database. Intron and EF1-α sequence accession numbers are available in Table B. bassiana genotypes based on previously reported intron insertion patterns in the LSU rDNA gene, Wang's terminology was used [Beauveria sequences to examine their polymorphisms and to identify conserved motifs. Intron subgroups were determined by comparison with representative secondary structures from previous studies [The presence or absence of introns at the 3'-end of the nuclear LSU rDNA of each isolate was determined by detecting previously described target sequences . In ordewas used . The int studies -27,30.Beauveria, Metarhizium and Cordyceps were retrieved from GenBank and included in the alignments. Alignments were generated using the MegAlign  and the CLUSTALX 1.81 program [Naegleria sp. (AM167886) and the EF1-α gene of Cordyceps cf. scarabaeicola (AY531967) were used as outgroups in the analysis of intron and EF1-α sequences, respectively. A bootstrap full heuristic analysis consisting of 1000 replicates was performed, and a 50% majority rule tree was produced.Intron and EF1-α sequences were analyzed separately. Published sequences for isolates included within the genera  program . Phyloge program , and allB. bassiana isolates. In addition, EQM and AOU participated in genomic DNA extraction. EM conceived the design of the study and helped to write the manuscript. RH participated in the design and coordination of the study, in the sequence analyses and wrote the manuscript. All authors have read and approved the final manuscript.IGJ carried out the laboratory work related to EF1-α. MM and EAI carried out the laboratory work on introns. EQM and CSA provided the a sequences obtained in this study from 57 Beauveria bassiana isolates and EF1-α subgroupsTable of GenBank accession numbers of EF1-.Click here for file


Leishmania major, this form multiplies in the body of rodents serving as reservoirs of the disease. The parasite is then transmitted to human hosts by the bite of a Phlebotomine sand fly (Diptera: Psychodidae) that was previously fed by biting an infected reservoir. We examine the seasonal and interannual dynamics of the incidence of this ZCL as a function of surface climate indicators in two regions covering a large area of the semi-arid Pre-Saharan North Africa. Results suggest that in this area, changes in climate may have initiated a trophic cascade that resulted in an increase in ZCL incidence. We find the correlation between the rainy season precipitation and the same year Normalized Difference Vegetation Index (NDVI) to be strong for both regions while the number of cases of ZCL incidence lags the precipitation and NDVI by 2 years. The zoonotic cutaneous leishmaniasis seasonal dynamic appears to be controlled by minimum temperatures and presents a 2-month lag between the reported infection date and the presumed date when the infection actually occurred. The decadal increase in the number of ZCL occurrence in the region suggests that changes in climate increased minimum temperatures sufficiently and created conditions suitable for endemicity that did not previously exist. We also find that temperatures above a critical range suppress ZCL incidence by limiting the vector’s reproductive activity.Shifts in surface climate may have changed the dynamic of zoonotic cutaneous leishmaniasis (ZCL) in the pre-Saharan zones of North Africa. Caused by  Leishmaniases are among the most important emerging and resurging vector-borne diseases, second only to malaria in terms of the number of affected people. Leishmaniases are endemic in 98 countries and three territories worldwide and threaten about 350 million people. It is estimated that 14 million people are infected worldwide with about two million new cases occurring each year. The disease contributes significantly to the spread of poverty, because of its expensive treatment, and imposes a heavy economic burden, including loss of income [Among all leishmaniases, cutaneous leishmaniasis (CL) is the most common. There are about 214,000 cases reported each year and the estimated annual CL incidence ranges from 691,000 to 1.2 million cases  , while tThe incidence of CL has increased across the globe and urbanization is indicated as a key factor in this increase . ObservaLeishmania, which multiplies in the body of rodents serving as reservoirs of the disease. The parasite is transmitted to human hosts by the bite of a vector that was previously fed by biting an infected reservoir. There are at least three Leishmania species that can cause CL [L. infantum, L. tropica and L. major. For each of these forms, the reservoir and the vector vary from place to place. In Algeria, a country with the highest number of reported CL cases per year in the Mediterranean region between 2004 and 2008 [L. major is dominant and distributed over a wide band across the southern arid zones with about 10,000 cases recorded annually [L. tropica and L. infantum have also been isolated, but are less frequent [L. major CL by the Tell Mountains which constitute a natural barrier [L.major MON-25 and investigations into the reservoirs pointed to the (fat) sand rat (Psammomys obesus) and Shaw’s jird (Meriones shawi) as proven hosts [Leishmania strains responsible for cutaneous leishmaniasis in Algeria was recently presented [L. major (50%), seven were L. infantum (44%), one was L. killicki (6%) and none of the strains were L. tropica. Like in Algeria, in Morocco, CL is caused by the same Leishmania species [L. infantum in the northern regions and L. tropica in the central to western semi-arid regions. However, the ZCL caused by L. major is exclusively found in the Saharan regions in the south and the south-east with outbreaks moving in waves from west to east and no evidence of overlap in the region of Errachidia [Meriones shawi, but it is suggested that, as in Algeria, there is a ‘sylvatic’ reservoir system that ‘feeds’ this urban system, with Psammomys obesus as reservoir and the sandfly Phlebotomus papatasi as the main vector [Cutaneous leishmaniasis (CL) is caused by a protozoan parasite of the genus cause CL ; L. infaannually ,9. Cases barrier . Recentlen hosts . The firresented . The sturachidia . The resLeishmaniamajor, and variation in its seasonal and interannual pattern as influenced by surface climate variables. This clinical form is transmitted by the sandfly (Phlebotomuspapatasi Scopoli) vector with Psammomys obesus and Meriones shawi serving as reservoirs. As for other vector-borne diseases, important determinants of this ZCL include the vector activity and reproductive periods. These factors control the vector density and biting rate and thus the number of cases of the disease. Pathogens, reservoirs and vectors each survive and reproduce within a range of climatic conditions [No or limited isoenzymatic or DNA strain characterization has been carried out to identify the CL causative agent in these regions. However, the data selected for this study was based on lesions having a form and seasonality consistent with L. major CL, although other species may have been present. In this study we are specifically interested in the ZCL, caused by nditions : temperanditions ,8. The lThere is evidence that changes in climate contribute significantly to the increase in the number of cases and expansion of the range of ZCL ,13,14,15Changes in climate, although small, may also have an impact on the geographical distribution of ZCL reservoirs and vectors and on their density, their activity and their reproductive periods . For exWhile there have been several studies of ZCL e.g., ,5,23,24),23,245,2The study area is represented by two provinces  and was completed for 2008 and 2009 by data obtained directly from the services of epidemiology and preventive medicine (SEMEP) for the region . For ErrIn order to compare the ZCL-seasonal and annual variations in the two provinces, we normalize the time series by their respective maxima. The time stamp of these data sets may be off by one month as cases reported at the beginning and end of the month are not distinguished.For the two provinces, monthly mean minimum, maximum and mean temperatures as well as mean relative humidity, monthly total rainfall and monthly Normalized Difference Vegetation Index (NDVI) from the Advanced Very High Resolution Radiometer (AVHRR) were obtained and analyzed in terms of their relation to the number of cases of ZCL. The NDVI is an aggregate measure of the vegetation density and health.Laboratory studies of the sandfly at constant temperature are used to establish annual temporal ranges of the vector’s active period (AP) and reproductive period (RP). To remain as close as possible to laboratory results, we use monthly composite data over the period of analysis.Both Saida and Errachidia provinces reported a significant increase in ZCL incidence during the latest decade. Since the first reported case of ZCL in 1991, 1,275 cases were recorded in Saida by the end of 2009, with the vast majority of the cases (99%) reported between 2000 and 2009. Several epidemic peaks were observed, with the highest peak of 267 cases reached in 2003 followed by a significant reduction in 2004 as shown in Because of the abrupt change in ZCL infection rate occurring around year 2000 for Saida and 2004 for Errachidia, and because the objective of this study was to analyze the relationships between climate variables and disease incidence, the twenty years of climate data were separated into two sub-periods: P1 representing the decade with low incidence rates and P2 representing the period with high incidence rates. For the province of Saida, these periods correspond to 1990–1999 and 2000–2009, respectively whereas for the province of Errachidia they correspond to 1994–2003 and 2004–2009, respectively. Because of data availability for Errachidia, the composite analysis of P2 was performed over 6 years. This separation allows us to compare climate indicators during low rates of incidence with those prevailing during the high incidence period, while the decadal scale analysis provides statistical robustness to the results. Both provinces experienced a warming trend over the period of analysis as shown in For the study area, the maximum vegetation density in a typical year occurs from February through April as indicated by monthly composite NDVI values over the 20 year study period. Because the majority of the study area precipitation occurs during the winter, summer vegetative growth is water limited. During winter, however, when water is available, low temperatures limit vegetative growth ,36. Atriplexhalimus L. (Chenopodiaceae), a widespread perennial drought-resistant C4 shrub [Our analysis shows a pattern that confirms the trophic cascade suggested by  over theC4 shrub . RodentsC4 shrub . IncreasC4 shrub ,40 and uC4 shrub .et al. [et al. [Similar to the trophic cascade proposed by Yates et al.  and Erne [et al. , we postp-value of 0.08.While the correlation between precipitations and growing season NDVI can be observed within the same annual cycle, there are time-lags in the correlation between precipitations and ZCL-incidence . In bothet al. [et al. [et al. [et al. [Ernest et al.  and Yate [et al.  found th [et al.  and Yate [et al.  are obtaThe annual ZCL cycle was modulated by climate, with extreme temperatures and moisture and their interactions playing important roles, especially during the period of transmission ,42. We eP. papatasi, has been widely studied  a,45 aP. pd  and Reproductive Period (RP) based on observed monthly composite minimum and maximum temperature data over the study region. vectors . It was  vectors  while re vectors . These tOur climate record shows that during the second decade P2, the average maximum temperature for the province of Saida during the hottest months (July and August) was approximately 36 °C with a standard deviation (SD) of 1.6 °C; while in Errachidia it reached 39.08 °C with SD = 0.86 °C. Although temperature in Errachidia reached high values during July in P2, it may not have directly impacted the sandflies as these nocturnal insects seek refuge during the hottest parts of the day. Minimum temperatures, however, which drop well below the vector’s physiologic thresholds in winters, appear to be more critical in modulating the sandflies’ seasonal cycle over the study area and period.P.papatasi, other environmental factors such as relative humidity, rainfall and photoperiod are also relevant to the life cycle of this species [Using monthly composite relative humidity, minimum and maximum temperatures rounded to the nearest degree and the thresholds values defined in  species . Except  species  of 27 ± Transmission of vector-borne diseases such as ZCL is dependent on the duration of the vector’s active period (AP). Where favorable temperatures and other conditions allow for multiple life cycles, a larger sandfly population is created and there is greater probability of contact with infected and non- infected hosts . For theTwo main differences are apparent in Errachidia. First, unlike Saida where the seasonal maximum temperature reached only around 36 °C, it was around 40 °C in Errachidia in July and August. This is believed to have significantly reduced the sandfly activity and limited the number of ZCL-cases. Indeed the seasonal variation of reported ZCL-incidence for Errachidia during P2  indicateObservations over the study region indicate that both provinces experienced periods of high ZCL incidence following a period with low incidence. In Saida, during P1 (1990–1999) observations showed one to two cases per year except for 1998 during which three cases were reported. However starting in 2000 and through 2009 (P2), the incidence increased to an annual average value of 126.3 or 99.06% of all cases compared to only 0.94% during P1 . In ErraFirst, the increase in precipitation led to denser vegetation that supported a larger rodent and sandfly population. Additionally, we argue that increase in the decadal mean minimum temperature over the region extended the reproductive period of the sandfly. These environmental conditions are suitable for both rodents and sandflies to reproduce in large numbers and survive in abundance throughout the winter diapause to the following cycle. The relationship between the decadal shift in the number of ZCL cases in the region and climate indicators suggest that changes in climate created conditions suitable for endemicity that did not previously exist and ultimately, increased and maintained high prevalence of the ZCL during the second period in both provinces. Indeed in both provinces the sandfly reproductive period extended by one month into the fall season during P2. This extension represents an increase of 25% (Saida) and 20% (Errachidia) in the length of the sandfly reproductive period and is associated with the observed increase in the minimum temperature. A paired student T-test verified the statistical significance of the difference in minimum temperatures between P1 and P2 for the AP (April-November) at the 95% confidence level for both provinces. While these preliminary results may provide some guidance as to the functional relationship between surface climate indicators and ZCL-cases, further research is needed in this populated and data sparse region in order to have a comprehensive assessment of the impact of climate on vector-borne diseases e.g., ,49,50).,50.49,50While an increase in temperature and precipitation, possibly associated with climate change, resulted in an increase in ZCL-incidence, our data also show that beyond critical temperatures, the incidence of ZCL in the region of Saida abruptly declines. Even though data did not show temperatures above the theoretical livable range for the vector, in laboratory experiments the number of offspring per female begins to decline precipitously above 28 °C . TherefoPhlebotomus papatasi is an important vector of epidemiological consequence. In Semi-arid North Africa, cold winter temperatures have limited its range and prevented its establishment in much of the temperate regions along the Mediterranean coast. Consistent warming and moistening over the last decade appears to have changed the spatio-temporal distribution of the range of this insect. Understanding how climate affects the abundance and the seasonal dynamics of this vector is paramount to understanding and predicting the spread of the potential infectious disease it may transmit, cutaneous leishmaniasis. In the pre-Saharan North Africa, ZCL is a serious public health problem with significant social consequences, especially in women, due to the indelible scars that skin lesions leave on the faces of vulnerable patients. Furthermore, this region is home to growing indigenous populations that thrive to sustain livelihood. However, not much research has been conducted to describe the potential associations between climate and the incidence of the ZCL. L. major ZCL incidence and surface climate indicators. It presents observational evidence from data in two sites that changes in climate in semi-arid pre-Saharan North Africa may be the initial catalyst of a trophic cascade that results in a 1-year delayed response in rodent and sand flies population density and an additional 1-year lag in ZCL-incidence. These relationships could prove useful in predicting elevated risks of human contraction of ZCL, especially in the study region where vegetation density is highly sensitive to changes in seasonal precipitation. These relationships also support the importance of environmental surveillance of rodent’s population expansion following rainy seasons and its use as first indicator of ZCL epidemic risk level. Our study suggests the ZCL annual cycle is modulated by climate parameters with extreme temperatures and moisture playing important roles, especially during the period of transmission. In the study region, we propose a 2-month lag between the reported infection date and the presumed date when the infection actually occurred based on climate indicators and sandfly optimum physiological temperatures and humidity levels. While this result may be culturally dependent and thus regional in scope and significance, it does point to the urgent need of public awareness and education about the disease and its symptoms. ZCL appears to thrive in areas with environmental conditions that allow the vector to have multiple life cycles creating thus larger sandfly populations and a greater probability of contact with both infected and non-infected hosts. The decadal increase in the number of ZCL occurrence in the region suggests that changes in climate resulted in a sufficient increase in minimum temperatures that allowed the establishment of new endemic foci in regions that were not previously endemic. Our data also indicated that temperatures above a critical range seem to suppress ZCL-incidence, apparently by limiting the vector’s reproductive activity. Climate conditions affect the leishmaniasis complex components (parasite-reservoir-vector) and their ability to interact, persist and establish in new ecosystems. This study describes empirical relationships between In summary, our study adds to the evidence linking ZCL incidence to changes in surface climate and suggests simple associations between them that could help establish an early warning system to local populations of these remote regions. Further work must be done with more comprehensive clinical and climate data to gain a complete picture of possible dynamic relationships between ZCL incidence and changes in surface climate. However, the results presented here suggest that regional changes in surface climate and the state of vegetation may already be playing a role in establishing favorable environmental conditions for the ZCL to expand to northern territories in the pre-Saharan regions.


In a previous study, we investigated slow cortical potential (SCP) N1-P2 amplitudes and N1 latencies in aided and unaided conditions, with the finding that despite being set to provide 20 or 40 dB of gain, none of the hearing aids resulted in a reliable increase in SCP response amplitude relative to the unaided . The current study investigates the effects of hearing-aid processing on acoustic measures for two 1000-Hz tonal stimuli: short (60 ms) and long (757 ms), presented at three intensities  in aided and unaided conditions using three hearing aids  with two gain settings . Acoustic results indicate that gain achieved by the hearing aids, measured at 30 ms after stimulus onset, for both the short and long stimuli, was less than real-ear insertion gain measured with standard hearing aid test signals. Additionally, the digital hearing aids altered the rise time of the stimuli such that maximum gain was reached well past 30 ms after stimulus onset; rise times differed between the digital aids. These results indicate that aided SCP results must be cautiously interpreted and that further research is required for clinical application. Slow cortical potentials (SCPs) are being considered for their application in hearing aid fitting, particularly for infants –7. SCP sResearch on AEPs measured with hearing aids in place has yielded varying degrees of success. One reason for this variability may be due to the stimuli used to measure them. More specifically, although there are many commercially available test signals that may be used to assess hearing aids electroacoustically and/or via AEPs , not all stimuli are appropriate for measuring both hearing-aid processing and AEPs. For instance, complex stimuli such as speech-weighted composite noise provide better estimates of gain for speech compared with tonal stimuli –16, wherThe N1 response does not reflect stimulus changes beyond the first 20–40 ms –22, and What is not clear from many of the SCP studies is what effect hearing-aid processing had on the stimuli. Billings et al.  have sinThe purpose of this study was to measure, in the ear canals of subjects, tonal stimuli used for SCP testing before and after hearing-aid processing to determine how hearing-aid processing affected the stimuli, measured under the same conditions as the SCP testing. Of particular interest was whether there would be a difference between the gain measured with standard hearing aid test system stimuli and that measured with the stimuli used for the cortical measures. Also of interest was whether there would be a differential effect of analog and digital processing on gain, particularly within the first 30 ms after stimulus onset, even with all advanced features disabled. Five subjects participated . Four of these subjects also participated in Marynewich et al. . SubjectThe same three behind-the-ear hearing aids, coupled with Comply snap tip 9-mm foam earmolds, were used for each participant: (i) Oticon E27 , (ii) Phonak Savia 211 dSZ , and (iii) Siemens Acuris S . These were the same hearing aids and settings used in our previous study [The digital hearing aids were set, using NOAH 3 and the NOAHLink, to have two programs: one with 20 dB and the second with 40 dB real-ear insertion gain (REIG) for a 50 dB SPL 1000 Hz pure tone, as verified with the Fonix 7000 real-ear system. Both programs were set with a 1 : 1 compression ratio across the frequency range and were verified for linear processing using input/output coupler measures. All additional hearing aid features such as digital noise reduction and feedback management were disabled. Other frequencies were set to minimum gain. Settings for the digital instruments were saved in the NOAH 3 software for each subject and recalled in follow-up sessions. Gain settings for the analog hearing aid were achieved by setting the volume control to 1 (minimum) and turning the dB SPL trim-pot until the REIG was 20 dB for a 1000-Hz pure tone at a 50 dB SPL input level. To achieve the 40-dB gain setting, the volume control wheel was turned up until REIG equalled 40 dB at 1000 Hz. The volume control wheel was then marked for that setting. These gain settings were remeasured in follow-up sessions. REIG measures at 1000 Hz for all subjects and hearing aids are given in Two 1000-Hz “SCP” stimuli were used for the acoustic measures: (i) a stimulus of 60-ms total duration , the same as used by Marynewich et al. , and  and presentation order for each subject was randomly assigned prior to the test date(s). During testing, participants were asked to sit as still as possible while watching a movie of their choice in closed-captioning and no audio. Subjects sat in a reclining chair set in the upright position so that each participant was seated with their head above the chair back, the same position used for the previous SCP measurements. 2 system and averaged and analyzed by the Neuroscan Scan analysis system, using a 204.75-ms analysis time for the short stimulus (including a 70-ms prestimulus baseline) and a 960-ms analysis time for the long stimulus (including a 100-ms prestimulus baseline). The stimulus was recorded in the ear canal for each test condition until at least 100 accepted trials were obtained. Single-trial epochs were saved for offline processing, which included baseline correction across the stimulus duration and averaging of the single trials.An ER7C probe-tube output (set to provide 20 dB of attenuation) was routed through a second (passive) attenuator to Channel 1 of the Neuroscan recording system. The second attenuator ensured that input was not clipped by the recording system. The recording channel was amplified, filtered (0.05–3500 Hz), and digitized  by the Neuroscan SynampsAcoustic measures of interest were (i) gain at 30 ms after stimulus onset, (ii) maximum gain, and (iii) latency of maximum gain, or “rise time” . Actual gain values were calculated for 20- and 40-dB hearing-aid gain conditions by determining the relative amplitude differences between aided and unaided stimulus waveforms from averaged recordings in the ear canal. A measurement point of 30 ms after stimulus onset was chosen because several studies indicate this is the most effective rise time and evokes the largest N1-P2 amplitudes; increases in stimulus levels beyond 20–40 ms have little to no effect on SCP amplitudes –22. MaxiFor the short-duration (60-ms) stimulus, two repeated-measures analyses of variance (ANOVA) were conducted for each of the dependent variables: gain measured at 30 ms, maximum gain, and rise time: (i) to measure the effects of the 20-dB gain setting, a two-way repeated-measures ANOVA was conducted comparing three levels of hearing aid type  and three input levels  and (ii) to measure the effects of the 40-dB gain setting, a two-way repeated-measures ANOVA was conducted comparing three levels of hearing aid type  and two input levels (30 and 50 dB SPL). The same four repeated-measures ANOVAs were performed for the long duration (757-ms) stimulus.p < .10. Huyn-Feldt correction factors were applied to the degrees of freedom and reported where appropriate . Significant interactions were examined by analyzing the simple main effects, then conducting paired t-tests for any significant simple main effects. Neuman-Keuls post hoc analyses were performed for significant main effects not involved in an interaction. Post hoc analyses were considered statistically significant if p < .10.Due to the exploratory nature of this study, main effects and interactions for all analyses were considered significant if The following section is divided into: (i) gain and rise time results for the short stimulus and (ii) gain and rise time results for the long stimulus. Mean data for gain measured at 30 ms and maximum amplitude, along with the rise times for maximum amplitude, are provided in Acoustic waveforms for the short stimulus (60-ms duration) in both unaided and aided conditions  are presented for 30, 50, and 70 dB SPL input levels in Figures Mean gain values presented in p < .1) and the 30 dB SPL compared with the 70 dB SPL input level for all three hearing aids (p < .1). Gain was not significantly different between the 50 and 70 dB SPL input levels for any of the hearing aids (p > .1). Effect of Input Level: results from the ANOVA and post hoc analysis revealed that even though all three hearing aids were set to provide linear amplification, less gain was measured for the 30 dB SPL compared with the 50 dB SPL input level for all three hearing aids (p < .01). The DigitalA hearing aid provided significantly less gain than the Analog hearing aid for the 30 dB SPL input (p < .05). There was no significant difference between gain provided by Analog and DigitalA aids for the higher input levels, 50 and 70 dB SPL (p > .1). Effect of Hearing Aid: the DigitalB hearing aid provided significantly less gain than both the DigitalA and Analog hearing aids at every input level . There was a clear trend for the two digital aids to take longer to reach maximum amplitude than the Analog aid. Rise time for the Analog aid was about 22 ms, similar to the 20-ms rise time of the input stimulus. Rise times for DigitalA ranged from 28 to 39 ms, and DigitalB showed a markedly longer rise times than DigitalA, approximately 45 ms.p > .1). For DigitalA, measured rise time differed by input level. The measured rise time was shortest for the 50 dB SPL input level (50 versus 70: p < .05), longer for the 70 dB SPL input level, and longer still for the 30 dB SPL input level (30 versus 70: p < .05).Effect of Input Level: for the Analog and DigitalB hearing aids, rise time did not differ by input level (p < .01), with the rise time being longest for the DigitalB hearing aid, shorter for the DigitalA hearing aid, and shortest for the Analog hearing aid. Effect of Hearing Aid: at every input level, the rise time differed across hearing aids . The Analog and DigitalB aids were measured to have the same amount of gain for both 30 and 50 dB SPL input levels (p > .1).Effect of Input Level: unlike the 20-dB gain condition, where less gain was measured for the 30 dB SPL input level compared to the higher input levels, for the 40-dB gain condition, only DigitalA was measured to have less gain for the 30 than 50 dB input levels (p < .1). There was a nonsignificant trend for the DigitalA to provide less gain than the Analog hearing aid for the 30 dB SPL input (p = .11). The DigitalA and Analog hearing aids provided equivalent gain for the 50 dB SPL input (p > .1).Effect of Hearing Aid: similar to the 20-dB gain condition, in the 40-dB gain condition, the DigitalB hearing aid provided significantly less gain than the DigitalA and Analog hearing aids at both input levels , and there was no significant difference between the gain provided by Analog and DigitalA (p > .1). Effect of Hearing Aid: DigitalB hearing aid provided significantly less gain than either the Analog or DigitalA hearing aids . For both the DigitalA and DigitalB hearing aids, measured rise time was longer for 30 dB SPL than the 50 dB SPL input level (p < .1).Effect of Input Level: for the Analog aid, rise time did not differ between the two input levels (p < .05) and Analog (p < .001) hearing aids. DigitalA had a longer rise time than Analog (p < .05). At the 50 dB SPL input level, DigitalB still had a longer rise time than both DigitalA (p < .001) and Analog (p < .001), but DigitalA and Analog had equivalent rise times (p > .1). Effect of Hearing Aid: at the 30 SPL input level, DigitalB had a longer rise time than both the DigitalA (Acoustic waveforms for the long stimulus (757-ms duration) in both unaided and aided conditions  are presented for 30, 50, and 70 dB SPL input levels in Figures Mean gain values in p > .1), and the gain measured for the 70 dB SPL input was greater than both the lower input levels (p < .1). For the DigitalA hearing aid, less gain was measured for the 30 compared with the 50 and 70 dB SPL input levels (p < .05) and equivalent gain for 50 and 70 dB SPL inputs (p > .1). Finally, the DigitalB hearing aid provided less gain for the 30 compared with the 50 (p < .05) and 70 dB SPL inputs (p < .01) and less gain for the 50 compared with the 70 dB SPL input (p < .1). Effect of Input Level: again, although all three hearing aids were set to provide linear amplification, in general the measured gain increased slightly as input level increased. For the Analog aid, equivalent gain was measured for the 30 and 50 dB SPL inputs (p < .01). The DigitalA and Analog hearing aids provided equivalent gain for all input levels (p > .1). Effect of Hearing Aid: the DigitalB hearing aid provided significantly less gain than both the DigitalA and Analog hearing aids at every input level . Effect of Input Level: for all three hearing aids, increasing amounts of gain were measured with increases in input level (p > .1).Effect of Hearing Aid: at each input level, there was no difference among hearing aids in the amount of gain measured . For DigitalA, the rise time measured for 30 was longer than that measured for 50 and 70 dB SPL input levels (p < .1). For Analog, rise time was equivalent across input levels.Effect of Input Level: for DigitalB, the rise time measured for 30 dB was slightly shorter than the rise time measured for the 50 dB SPL input level (p < .01), with the rise time being longest for the DigitalB hearing aid, shorter for the DigitalA hearing aid, and shortest for the Analog hearing aid. Effect of Hearing Aid: at every input level, the rise time differed across hearing aids . Both the DigitalA and Analog hearing aids provided equivalent gain across the two input levels (p > .1). Effect of Input Level: the DigitalB hearing aid provided less gain for the 30 than 50 dB input levels (p < .01), and there was no significant difference in gain provided by Analog and DigitalA at either input level (p > .1). Effect of Hearing Aid: once again the DigitalB hearing aid provided significantly less gain than both other hearing aids at both input levels . Analog had equivalent rise times at the two input levels (p > .1).Effect of Input Level: both DigitalA and DigitalB had longer measured rise times for the 30 than 50 dB SPL input level (p < .05). Effect of Hearing Aid: for both input levels, DigitalB had a longer rise time than both DigitalA and Analog, and DigitalA had a longer rise time than Analog . When measuring the hearing aids with the stimuli used for the SCP measures, however, all of the hearing aids were measured to have less gain, particularly the two digital aids. The amount of gain reduction differed between the two digital hearing aids, with DigitalB showing much less gain than DigitalA in almost every condition. The two digital hearing aids, DigitalB in particular, reached their maximum gain well past 30 ms after stimulus onset. When the hearing aids were measured with a long stimulus at their maximum gain, there were no longer any differences among hearing aids in the amount of gain measured, but all three hearing aids demonstrated about 3–5 dB less gain than the gain measured with conventional REIG procedures.The first main finding was that the maximum gain of all of the hearing aids was about 3–5 dB less than nominal gain even when a long stimulus was used. Although the conventional and SCP acoustic measures were made with different sets of equipment, different signals, and in different rooms, these effects cannot account for reductions in measured gain. Both acoustic measures  were insertion gain; thus, gain was always calculated as the aided response minus the unaided response. Additionally, the stimulus for both measures was a 1000-Hz pure tone, at the same input levels. Insertion gain is robust to differences in probe-tube insertion depth, measurement bandwidth, and small changes in room acoustics, particularly if the hearing aid has linear processing –28, whicIt was a consistent finding that less gain was provided by digital hearing aids for the 30 dB SPL input level; this is likely due to the low-level expansion in both hearing aids, which could not be changed or disabled in the programming. Gain for a 30 dB SPL input was not measured with the standard hearing aid test system, as signals that low are not provided. However, reduced gain for low-level inputs cannot explain why nominal gain was not achieved even at higher input levels.The second main finding was that both digital hearing aids altered the rise times of the stimuli such that there was a significant delay for both hearing aids to reach their maximum gain, and the amount of delay differed significantly between the two digital aids. This might be expected because of the commonly reported delays associated with digital processing , 29–35. These changes in altered rise time are also unlikely to be due to hearing aid processing parameters. All of the hearing aids were set (and subsequently verified) to linear processing. Any compression processing, had it remained on, would be expected to have the opposite effect as found here; that is, compression would be associated with faster rise times than measured here due to the overshoot that results from compression attack time , 37. AllIt is worth noting that the issues identified in this study are unlikely to be problematic only when using tonal stimuli, even though tonal stimuli have proven to be troublesome for measuring digital hearing aid processing , 39, 40.In our previous study , we demoTo determine whether any of the measured acoustic parameters could predict the SCP response, we conducted an analysis of the group mean data from both studies. Although a thorough answer to this question would require a larger-scale parametric investigation of the relationship between acoustic variables and the SCP responses, some initial exploration of the findings can be instructive. Specifically, we used the group-mean SCP amplitude from Marynewich et al.  and deveμV) and slope of onset (average error: 1.08 μV) predicted the SCP amplitude equally well. The greatest amount of error was seen for maximum stimulus amplitude (average error: 1.35 μV). The analysis is limited because it was performed on group mean data for two different groups of subjects. However, it is likely that the group analysis is representative of individual analysis for several reasons: four participants were in both studies; the hearing aids were set for individual ears; all participants had normal hearing; the acoustic measures generally had low variability. We can cautiously interpret this analysis to mean that the effect of hearing-aid processing on the onset characteristics of the stimulus had a greater influence on SCP amplitude than did the effect of hearing-aid processing on maximum stimulus amplitude. Recall that gain at 30 ms was generally much lower than the nominal gain, and particularly for DigitalB often measured close to 0 dB gain for the 20-dB gain condition. Thus, if the SCP was responding to the first 30 ms after stimulus onset, it is not surprising that primarily there was often little to no difference between aided and unaided acoustic measures, especially for the digital aids. These results are consistent with the view that approximately the first 30 ms of stimulus onset largely determines SCP N1 presence and amplitude [The results of this analysis showed that both stimulus amplitude at 30 ms  of Panel (b) of Finally, in Panel (c), there is an example where neither stimulus amplitude at 30 ms nor SNR seems to be good predictors of the SCP response. In this example, comparing 70 dB SPL unaided to DigitalB 50 dB SPL input +40 dB gain, these measures have similar stimulus levels and similar (good) SNRs, yet the SCP response for DigitalB is much lower than unaided. In this case, neither stimulus level nor SNR can explain the different SCP responses observed.Altogether, this set of examples shows that SCP amplitudes in our data set may be accounted for by changes to rise time in some conditions, SNR in some conditions, and some other, as yet unidentified, acoustic parameter in other conditions. In the present study, we attempted to determine why several recent studies, including our own study , have beIn light of findings from the present study, it is likely that prior studies using speech or tonal stimuli , 4–9, 41Prior studies on hearing-aid processed click and brief-tone stimuli  reported considerable variability among hearing aids in terms of gain provided to onset and steady-state portions of transient stimuli , 13, thuThe less-than-expected measureable gain resulting from hearing-aid processing for SCP stimuli suggests that SCP stimuli do not provide appropriate measures of hearing aid gain. Our acoustic analysis shows that changes to rise time, particularly in ways that affect stimulus amplitude at 30 ms, may explain our previous findings . HoweverAlthough our studies included only participants with normal hearing, the acoustic alterations of the stimuli that we measured are independent of hearing status. The concerns raised by these studies indicate that much is unknown about the application of SCP measures in hearing aid fitting. Future research might involve (i) additional hearing aid measures to determine the source of alteration to rise time and (b) parametric study of the relationship between the stimulus acoustic measures and the SCP responses. Future research might also explore different/more-appropriate SCP stimuli or presentation paradigms for hearing aid measures to determine under what conditions aided SCP measures would be valid.


An error occurred in the legend of Figure 1 during the preparation of this article for publication. A reference is missing from the sentence beginning "The area framed by thick gray lines with downward arrows represents a simple rule of thumb[?]...". The sentence should instead read:"The area framed by thick gray lines with downward arrows represents a simple rule of thumb [14] according to which a glycerol test should be considered as a diagnostic option when a patient with suspected Menière’s disease has a mean low-frequency hearing loss between 30 and 70 dB and a mean high-frequency hearing loss that does not exceed the mean low-frequency loss.


Background and Objectives. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by loss of motor neurons in the brainstem, motor cortex, and spinal cord. Oxidative stress and neuroinflammation have been implicated in the pathophysiology of ALS. Members of the family of damage-associated molecular patterns, including reactive oxygen species, high-mobility group box 1, and eosinophil-derived neurotoxin (EDN), may participate in pathological conditions. In this study, we aim to discover new biomarker for detecting ALS. Materials and Methods. We examined 44 patients with ALS, 41 patients with Alzheimer's disease, 41 patients with Parkinson's disease, and 44 healthy controls. The concentration of serum EDN was measured using an enzyme-linked immunosorbent assay. Results. EDN levels were significantly increased 2.17-fold in the serum of patients with ALS as compared with healthy controls (P < 0.05). No correlation between the levels of serum EDN and various clinical parameters of ALS was found. Moreover, the levels of serum EDN in patients with Parkinson's disease and Alzheimer's disease and healthy controls were similar. Conclusion. A higher level of serum EDN was found specifically in patients with ALS, indicating that EDN may participate in the pathophysiology of ALS. Amyotrophic lateral sclerosis (ALS) is the most common and devastating adult-onset neurodegenerative disease . The undα, which stimulates local inflammatory responses and neuronal degeneration [Currently, the etiology of ALS is unclear. However, several lines of evidence suggest that neuroinflammation , glutamaneration , 20. Furneration , 22. Micneration –25. Therneration , 27. BecEDN, also known as RNase2, is a member of the ribonuclease A superfamily . EDN is ECP is a paralog of EDN in humans, and they share 70% similarity at the protein level. They are both secreted by activated eosinophils during pathogenic stimulation and inflammatory processes . In pati Because EDN and ECP participate in the induction of inflammatory diseases and because both serve as disease biomarkers, we examined the serum levels of EDN and ECP in patients with ALS. The demographic information for the normal control individuals and the patients with ALS is given in  Venous blood (10 mL) was collected from patients with ALS, AD, and PD as well as controls; all participants were free of acute infection and acute stress at the time of collection. Serum collected using yellow-stopper clot-accelerating tubes was harvested by centrifugation at 3000 ×g for 20 min, divided into aliquots, and frozen at −30°C until use. The serum levels of EDN and ECP were measured using commercially available ELISA kits for human EDN and ECP .P value of the ANOVA reaches the significance level, Bonferroni multiple comparison test was used to determine the significant difference among the groups of control, AD, PD, and ALS. The Spearman rank correlation was used to correlate the levels of EDN or ECP versus the subgroups of age, ALSFRS-R, disease duration, or onset types. The serum concentrations of EDN and ECP were compared among patients with ALS, AD, and PD and controls using an ANOVA. The one-way ANOVA test was used to determine the difference among all of the test groups. When the P < 0.005; The concentration of serum EDN in patients with ALS, AD, and PD and in healthy controls was measured using a commercial ELISA kit. The average EDN levels in patients with ALS and controls were 45.7 ng/mL  and 21.0 ng/mL , respectively . The levNext, the ALSFRS-R, disease duration, age, and disease onset among patients with ALS were analyzed and statistically correlated with EDN levels. The clinical indicators were not correlated with EDN levels .The levels of ECP were not significantly different in the sera from patients with ALS , AD , and PD  and healthy controls  curve was used to determine the performance of EDN and ECP correlated among ALS, AD, and PD. EDN showed the best performance with 88.53% accuracy, 77.27% sensitivity, and 84.09% specificity when the cut-off concentration was set at 23.43 ng/mL for ALS. ROC curve analysis also showed that EDN had the highest area under the curve (AUC) value of 0.8264. As expected, values for AD and PD were 0.5294 and 0.5538, respectively, indicating a nearly random distribution . For ECPDamage-associated molecular patterns (DAMPs) play an important role in stimulating macrophages and T lymphocytes . Recent We also observed no interaction between the level of EDN and the age of patients with ALS. In the elder population, chronic inflammation increases, and inflammatory stimulation is upregulated during aging , 53. TheNeuroinflammation is critical in the pathogenesis of ALS . High nuγ, tumor necrosis factor-α, and nitric oxide are elevated in the serum of patients with ALS [Although macrophages, mast cells, and T cells are reported to induce neuroinflammation in the cortex and spinal cord in ALS  and althwith ALS , 62, no


COL2A1, COL11A1 and COL11A2 , and COL9A1 and COL9A2 . The auditory phenotype in Stickler syndrome is inconsistently reported. Therefore we performed a systematic review of the literature to give an up-to-date overview of hearing loss in Stickler syndrome, and correlated it with the genotype.Stickler syndrome is a connective tissue disorder characterized by ocular, skeletal, orofacial and auditory defects. It is caused by mutations in different collagen genes, namely English-language literature was reviewed through searches of PubMed and Web of Science, in order to find relevant articles describing auditory features in Stickler patients, along with genotype. Prevalences of hearing loss are calculated and correlated with the different affected genes and type of mutation.COL11A1 (82.5%) and COL11A2 (94.1%) seem to be more frequently associated with hearing impairment than mutations in COL2A1 (52.2%).313 patients (102 families) individually described in 46 articles were included. Hearing loss was found in 62.9%, mostly mild to moderate when reported. Hearing impairment was predominantly sensorineural (67.8%). Conductive (14.1%) and mixed (18.1%) hearing loss was primarily found in young patients or patients with a palatal defect. Overall, mutations in Hearing impairment in patients with Stickler syndrome is common. Sensorineural hearing loss predominates, but also conductive hearing loss, especially in children and patients with a palatal defect, may occur. The distinct disease-causing collagen genes are associated with a different prevalence of hearing impairment, but still large phenotypic variation exists. Regular auditory follow-up is strongly advised, particularly because many Stickler patients are visually impaired. Stickler syndrome or hereditary progressive arthro-ophthalmopathy . In order to avoid selection bias , some calculations were repeated in an analysis where all families were equally weighted. In the subtypes of Stickler syndrome where sufficient audiograms of different patients could be collected, an average audiogram or even an age-related typical audiogram (ARTA) of the respective subtype was calculated.Calculations were performed using SPSS version 19 . Where appropriate, statistical tests were used to assess statistical significance. Exons of The study was conducted taking into account the instructions of the PRISMA statement for reporting systematic reviews and of tA flow diagram of the search process is depicted in Figure Mean age of the individual Stickler patients was 26.4 years , and gender distribution was 55.2% female versus 44.8% male persons (gender was provided in 210 patients).Hearing loss was reported in 197 of the 313 patients  was present in 42.6% 121/284) of the studied population. Of the patients with a palatal defect, 73.6% (89/121) were hearing-impaired, compared to 54.0% (88/163) without palatal defect . As seen in Table 21/284 ofSTL1 occurred in 224 cases (71.6%), while this was 40 (12.8%) for STL2, 34 (10.9%) for STL3, 7 (2.2%) for STL4 and 8 (2.6%) for STL5. For each Stickler gene, the distinct percentage and type of hearing loss is shown in Figure The mutations of 36 STL1 patients were expected to result in a dominant-negative effect, while these of 174 STL1 patients were supposed to induce nonsense-mediated decay (mutation effect was available in 210 STL1 patients). Hearing loss was present in 47.2% of the first group versus 52.9% of the second , and a palatal defect occurred in 16.7% versus 46.9% . No statistical significance was observed between location of the mutation in the collagen gene (exon/intron number) and the presence of hearing loss , but differences were seen between mutation location and palatal defects .When the 111 different families are equally weighted, regardless of the number of affected family members, the prevalence of hearing loss was 60.1%. Within these hearing-impaired subjects, the percentages of sensorineural, mixed and conductive hearing loss were 60.4%, 18.3% and 21.3%, respectively. The prevalence of palatal defects was 51.5%. STL1 occurred in 85 families (76.6%), while this was 17 (15.3%) for STL2, 5 (4.5%) for STL3, 3 (2.7%) for STL4 and 1 (0.9%) for STL5.Hearing loss is a common feature in Stickler syndrome, expressed in 63% of all patients. About two-thirds of these hearing-impaired patients express a purely sensorineural loss. This prevalence increases with advancing age, most probably not only due to age-related hearing loss. Conductive and mixed hearing loss is mostly found in children with Stickler syndrome and in patients with a history of a palatal defect, but can also be present in adults. These results are consistent with those of other authors studying hearing features in a large group of Stickler patients,63. As tCOL2A1. This type has the best prognosis concerning hearing: 52.2% of the affected persons exhibit hearing loss, which is mostly sensorineural. As in the other dominant Stickler types, conductive and mixed hearing loss may also be present, especially in young children and with a palatal defect. The sensorineural hearing loss is often reported to affect mainly the higher frequencies. Progression of hearing impairment is described, but it is unclear whether the high-frequency hearing loss in STL1 progresses beyond presbyacusis. It is generally mild to moderate and does not evolve to severe hearing impairment.About 75% of all Stickler patients have type I Stickler syndrome, caused by mutations in COL2A1 is present in numerous inner ear structures, which may explain why mutations in this gene can cause hearing loss[ing loss. StickleCOL11A1) is the second most common type of Stickler syndrome. It is estimated to be present in about 15% of the probands with Stickler syndrome. Hearing loss is found in 82.5% of these patients; most of them have sensorineural loss, but again, also conductive and mixed hearing impairment have been described. Hearing loss in STL2 seems to be more pronounced than in STL1, and is already apparent at young age. It is not clear whether the higher frequencies are predominantly affected and whether or not the hearing loss progresses with age.Type II Stickler syndrome , hearing loss was found in 94.1% of the patients. It can even be suggested that all STL3 patients have some degree of hearing loss, because the two subjects not exhibiting hearing problems, were not audiometrically tested[In type III Stickler syndrome  and mild to moderate in STL5 (COL9A2). None of these patients showed palatal defects.Four different families with autosomal recessive Stickler syndrome have been described, all exhibiting a mutation in genes encoding type IX collagen,9,41. ThThese results are consistent with animal studies, which demonstrated type IX collagen, in addition to type XI collagen, as an important factor in maintaining the integrity of collagen fibers in the tectorial membrane. Type IXBesides Stickler syndrome, mutated type IX collagen can also result, inter alia, in multiple epiphyseal dysplasia (MED), a skeletal dysplasia not associated with hearing loss. However, MED has been attributed to heterozygous splice site mutations in type IX collagen genes, resulting in in-frame exon skipping situated in the third collagenous domain (COL3) and effectuating a dominant-negative effect,73. In cD tympanograms were obtained[Another, rather specific, auditory finding in Stickler syndrome is hypermobility of the tympanic membrane, measured by tympanometry. In up to 46% of Stickler patients, type Aobtained,11,14. Mobtained. Hypermoobtained and is tobtained.In Stickler patients, language and speech may be affected by hearing impairment and by the typical facial morphology, including cleft palate, especially if these problems are not recognized early and optimally treated. However, speech audiometry showed perception and discrimination scores consistent with the obtained pure tone thresholds.Little attention is paid to the vestibular system of Stickler patients. Ocular and skeletal manifestations can contribute to instability complaints and may interfere with some of the balance tests. Five teTemporal bone imaging in Stickler syndrome mostly does not reveal middle or inner ear structural anomalies ; STL2: Stickler syndrome type II (COL11A1); STL3: Stickler syndrome type III (COL11A2); STL4: Stickler syndrome type IV (COL9A1); STL5: Stickler syndrome type V (COL9A2); OSMED: Otospondylomegaepiphyseal dysplasia; ARTA: Age-related typical audiogram; DFNA: Deafness, autosomal dominant; DFNB: Deafness, autosomal recessive; MED: Multiple epiphyseal dysplasia.STL1: Stickler syndrome type I (The authors declare that they have no competing interests.FA collected the data, performed the statistical analysis and drafted the manuscript. ID and EDL participated in the design of the study, reviewed the data collection independently and made contributions to the draft of the manuscript. FM was involved in genetic data interpretation and in drafting the manuscript. All authors read and approved the final manuscript.Table S1. List of articles included in the meta-analysis. This table shows the articles from which data were extracted to use in the results section. For each article, the number of patients and families included, and the type of Stickler syndrome is mentioned, as well as some methodological features. References are numbered according to and can be found in the original article.Click here for file


The aim of this study was to determine the clinical significance of the results of screening of newborn hearing and the incidence of deafness-susceptibility genes. One thousand newborn babies in the Handan Center Hospital  underwent screening of hearing and deafness-susceptibility genes. The first screening test was carried out using otoacoustic emissions (OAEs). Babies with hearing loss who failed to pass the initial screening were scheduled for rescreening at 42 days after birth. Cord blood was used for the screening of deafness-susceptibility genes, namely the GJB2, SLC26A4 and mitochondrial 12S rRNA (MTRNR1) genes. Among the 1,000 neonates that underwent the first hearing screening, 25 exhibited left-sided hearing loss, 21 exhibited right-sided hearing loss and 15 cases had binaural hearing loss. After rescreening 42 days later, only one of the initial 61 cases exhibited hearing loss under OAE testing. The neonatal deafness gene tests showed two cases with 1555A>G mutation and two cases with 1494C>T mutation of the MTRNR1 gene. In the SLC26A4 gene screening, four cases exhibited the heterozygous IVS7-2A>G mutation and one case exhibited heterozygous 1226G>A mutation. In the GJB2 gene screening, two cases exhibited the homozygous 427C>T mutation and 10 exhibited the heterozygous 235delC mutation. The genetic screening revealed 21 newborns with mutations in the three deafness-susceptibility genes. The overall carrier rate was 2.1% . The association of hearing and gene screening may be the promising screening strategy for the diagnosis of hearing loss. Hearing loss is the most common human birth defect, with an incidence of approximately one case among 1,000 newborns. The American newborn hearing screening project began in 1964 and has gradually spread globally. Newborn hearing screening has been gradually implemented in large and middle-sized cities across China and an increasing number of children with hearing loss are diagnosed shortly after birth. The program has a profound effect on the language development, communication, cognition, mental health and career planning of the children if early intervention is achieved  mutations (such as 12S rRNA gene 1555A>G and 1494C>T) also lead to undetected hearing loss at birth, whereas late-onset hearing loss occurs later  between November 2010 and October 2011 were studied. The GJB2, SLC26A4 and MTRNR1 genes were tested simultaneously in 532 males and 468 females. This study was conducted in accordance with the Declaration of Helsinki and with approval from the Research Ethics Committee of the Central Hospital of Handan City . Written informed consent was obtained from all guardians/parents of the participants.Hearing screening was performed in the maternity ward and the newborns were tested under quiet natural sleeping conditions using ambient noise <30 dB. An AccuScreen Screening Instrument  was used for the hearing test. At the third day after birth, every newborn was tested at different frequencies and volumes using otoacoustic emissions (OAEs). If the newborn failed the test, the OAE test was repeated at 42 days after birth in a hearing diagnostic laboratory.Approximately 2 ml of cord blood was collected from each newborn and then stored in an EDTA-anticoagulated vacutainer for gene screening . The parDNA was extracted from the cord blood to screen for the GJB2, SLC26A4 and MTRNR1 genes. A MassARRAY system  was used to screen for the deafness mutation sites of GJB2 and SLC26A4 genes. The MTRNR1 gene was screened for the 1449C>T and 1555A>G mutation sites through direct sequencing.Genomic DNA was extracted using a kit  according to the manufacturer’s instructions. A NanoDrop 1000 spectrophotometer  was used to detect the concentration and purity of the extracted DNA. Following completion of the extraction process, a final concentration of 10 ng/μl genomic DNA was achieved from all samples in the 384-well plates and preserved at −20°C.The Assay Designer package  was used to design the polymerase chain reaction (PCR) primers and single-base extension primers for the deafness loci on the GJB2 and SLC26A4 genes. The primer probe design was strictly in accordance with the requirements of the MassARRAY system and the probes were synthesized by Shanghai Invitrogen Biotechnology Co., Ltd. . A 14-plex PCR amplification reaction was performed in one well, and the detected point mutations are shown in Genomic DNA  and 4 μl of PCR mixture (Sequenom Inc.) were added into 384-well plates. The reaction conditions were as follows: 95°C for 2 min; 95°C for 30 sec, 56°C for 30 sec and 72°C for 1 min, for 45 cycles; then 72°C for 5 min.Shrimp alkaline phosphatase  reaction mixture (2 μl) was added into 384-well plates for the SAP reaction. The reaction conditions were as follows: 37°C for 40 min and 85°C for 5 min. After completing the reaction, the mixture was cooled to room temperature and stored at 4°C.The extension reaction conditions were as follows: 94°C for 30 sec; 94°C for 5 sec, 52°C for 5 sec, 80°C for 5 sec, for 40 cycles; 72°C for 3 min. The products were then preserved at 4°C.Approximately 16 μl of deionized water and 6 mg of resin were added to the 384-well plates for desalination and then were analyzed with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). Final results were read by the MassARRAY RT real-time sotware sustems. Genotype analyses were completed by the MassARRAY Typer software.http://bioinfo.ut.ee/primer3-0.4.0/primer3/. Accessed: October 23, 2013). The following parameters were set to improve the effectiveness and specificity of PCR amplification: the GC content in the primers was set between 40 and 60%, the annealing temperature was set between 55 and 60°C, the 3′-end of the primers were generally set to end with G or C to improve the integration of the primers into the template strand, and the amplified fragment was generally set between 200 and 500 bp to facilitate sequencing. The sequences of the mitochondrial primers were as follows: forward primer, 5′-CAACCTCACCACCTCTTGCT-3′ and reverse, 5′-GTAAGGTGGAGTGGGTTTGG-3′. The fragment length was 497 bp. The primers were synthesized by Shanghai Invitrogen Biotechnology Co., Ltd.The two mutation points of the MTRNR1 gene, 1494C>T and 1555A>G, were combined to design the primers. The online software Primer3  was used for designing the mitochondrial amplification primers  was used to verify the results of the PCR amplification. The bands obtained were clearly visible and did not overlap .SAP mixture (1.5 μl) and the PCR product (1 μl) were added to 384-well plates. The reaction conditions were as follows: 37°C for 60 min and 80°C for 20 min. After the completion of the reaction, the reaction mixture was cooled to room temperature and stored at 4°C.BigDye Terminator  and a DNA sequencing of the PCR-amplified product was performed bidirectionally on an ABI3730XL automated sequencer  using the same primers. Sequence data were analyzed by evaluating samples for alignment with the National Center for Biotechnology Information reference (NCBI) sequence of MTRNR1 (NC_012920.1) using Sequencher Demo 3.0 and Mutation Surveyor Demo V4.0.Among the 1,000 newborns tested, 996 cases were rescreened. Only one of the 61 cases who failed the initial hearing screening test also failed the single-ear rescreening test. All 61 cases were screened for the three genes, and only the infant who failed the rescreening exhibited homozygous 427C>T mutation of the GJB2 gene. All other subjects passed the genetic screening, which indicated that no disease-causing mutation was present.The two cases with the 1555A>G mutation and the two cases with the 1494C>T mutation of MTRNR1 and the other 16 cases that carried pathogenic mutations in the GJB2 and SLC26A4 genes passed the newborn hearing screening test.In the initial screening of the hearing of 1,000 newborns, 939 (93.9%) of the newborns passed the initial OAE screening, whereas 25 cases failed the left-ear hearing test and 21 cases failed the right-ear hearing test, for total of 46 (4.6%) cases. Fifteen (1.5%) cases failed both the right- and the left-ear hearing tests. A total of 61 (6.1%) newborns failed the initial screening. At 42 days after birth, only one of the 61 cases who failed the initial hearing screening also failed the single-ear rescreening. Thus, one (0.1%) case did not pass OAE rescreening.A total of 1,000 newborns were screened for mutations in the GJB2, SLC26A4 and MTRNR1 genes. Ten cases exhibited a heterozygous 235delC mutation of the GJB2 gene, and two cases exhibited a homozygous 427C>T mutation. Four cases exhibited heterozygous IVS7-2A>G mutation of the SLC26A4 gene and one case exhibited heterozygous 1226G>A mutation. Two cases exhibited homogeneous 1494C>T mutation of the MTRNR1 gene and two cases exhibited homogeneous 1555A>G mutation. The overall carrier rate was 2.1% . The specific carrier rates of the three genes among the 1,000 cases were then analyzed.GJB2 gene screening indicated that 10 cases had miscellaneous 235delC mutations and two cases had homozygous 427C>T mutations. The pathogenic carrier rate was 1.2% , among whom 11 cases passed the initial hearing screening test. One case did not pass the primary screening and the rescreening, and was identified to carry a homozygous 427C>T mutation  and 3.For the SLC26A4 gene screening, four cases carried heterozygous IVS7-2A>G mutations of the SLC26A4 gene and one case carried a heterozygous 1226G>A mutation. The pathogenic carrier rate was 0.5% . The five newborns with these mutations passed the initial hearing screening test. The distributions of the mutations are shown in MTRNR1 genetic screening revealed that four of the 1,000 cases carried gene mutations [pathogenic carrier rate of 0.4% ]. Two of the four cases exhibited homozygous 1494C>T mutations, whereas the other two cases exhibited homozygous 1555A>G mutations. The mutations detected by the forward sequencing were identified via reverse sequencing. The four cases passed the initial hearing screening test. The forward sequencing results are shown in In the present study, a program for the simultaneous screening of newborn hearing and genes was implemented, that is, the hearing of the newborns was tested and GJB2, SLC26A4 and MTRNR1 genes were screened simultaneously. Of the 999 newborns that passed the newborn hearing screening, 20 exhibited mutations in deafness-associated genes. The carrier rate of disease-causing mutations reached 2.1%, which is 20 times higher than the incidence of congenital deafness (0.1%) from hearing screening. Notably, these cases were not identified through hearing screening. Ten cases exhibited a heterozygous mutation of GJB2 235delC  and all passed the hearing screening test. The possibility of late-onset type hearing loss among these 10 cases is significantly increased during development compared with that of newborns with the normal gene. Four cases of IVS7-2A>G heterozygous mutation were identified during SLC26A4 gene screening. Individuals with this mutation should avoid strenuous exercise, trauma and collision, and follow-up examinations should be conducted at regular intervals. The aforementioned 14 newborns should also avoid marriage with carriers of the same genotype since their offspring would have a 25% chance of deafness. Two cases exhibited homozygous 427C>T mutation of GJB2 and one of these cases failed the hearing screening test in 1998. The 427C>T mutation was first reported in the GJB2 gene, with the mutation causing the amino acid at position 143 to change from arginine to tryptophan, thereby causing autosomal recessive deafness  genotyping, gene expression, copy number variation, gene methylation analysis, pathogen typing and prenatal diagnosis in one platform. The system integrates the high sensitivity of PCR and high accuracy of mass spectrometry . The advThe present study initially explored the distribution of deafness-susceptibility genes in newborn hearing screening and analyzed the conditions of newborn deafness gene carriers. The results demonstrate the necessity and feasibility of genetic screening for deafness in newborns. Newborn hearing screening combined with deafness-susceptibility gene screening may be a promising strategy for the early diagnosis of prelingual hearing loss, for individuals with a high risk of delayed-type deafness or deafness gene carriers . In the


Trichosanthes kirilowii Maximowicz (common name Tian Hua Fen). In this study, we extended the potential medicinal applications of TCS from HIV, ferticide, hydatidiform moles, invasive moles, to breast cancer. We found that TCS manifested anti-proliferative and apoptosis-inducing activities in both estrogen-dependent human MCF-7 cells and estrogen-independent MDA-MB-231 cells. Flow cytometric analysis disclosed that TCS induced cell cycle arrest. Further studies revealed that TCS-induced tumor cell apoptosis was attributed to activation of both caspase-8 and caspase-9 regulated pathways. The subsequent events including caspase-3 activation, and increased PARP cleavage. With regard to cell morphology, stereotypical apoptotic features were observed. Moreover, in comparison with control, TCS- treated nude mice bearing MDA-MB-231 xenograft tumors exhibited significantly reduced tumor volume and tumor weight, due to the potent effect of TCS on tumor cell apoptosis as determined by the increase of caspase-3 activation, PARP cleavage, and DNA fragmentation using immunohistochemistry. Considering the clinical efficacy and relative safety of TCS on other human diseases, this work opens up new therapeutic avenues for patients with estrogen-dependent and/or estrogen-independent breast cancers.Breast cancer ranks as a common and severe neoplasia in women with increasing incidence as well as high risk of metastasis and relapse. Translational and laboratory-based clinical investigations of new/novel drugs are in progress. Medicinal plants are rich sources of biologically active natural products for drug development. The 27-kDa trichosanthin (TCS) is a ribosome inactivating protein purified from tubers of the Chinese herbal plant  Trichosanthes (family Cucurbitaceae) comprises vines which own good potential as the target for anticancer drug discovery Trichosanthes kirilowii Maximowicz is the most well-known medicinal species for there is a long history for the clinical application as well as experimental studies of its tubers T. kirilowii tuber, well-known by its vernacular name ‘Tian Hua Fen’, is considered as a ‘wonder drug’ against some gynecological conditions/illnesses. It was used as an abortifacient and for the treatment of hydatidiform moles, invasive moles, and ectopic pregnancy T. kirilowii tubers. The mature form of TCS contains 247 amino acids, but its preprotein consists of 289 amino acids with a 23-residue N-terminal signal peptide and a 19-residue C-terminal propeptide Trichosanthes and Momordica, including trichomislin (27.2 kDa) and karasurin-C (27.4 kDa) from T. kirilowii, as well as MAP30 and the N-glycosidase part of Momordica charantia lectin/MCL purified from M. charantiaAmong 145 families of plants, the plant genus in vitro and in vivo with different molecular mechanisms. First, TCS is useful for treating choriocarcinoma as it can produce reactive oxygen species (ROS) and initiate apoptosis in human choriocarcinoma (Jar) cells Besides the ability to ablate the replication of human immunodeficiency virus (HIV) in vitro and in vivo studies. The potential antitumor mechanisms were also unveiled. This work broadens the medicinal applications of TCS which may serve as a novel treatment against human breast cancer.Breast cancer is one of the most prevalent and severe health problems for women T. kirilowii (origin: Guangdong Province in China) were purchased from a local vendor and authenticated by Prof. Shui-Ying Hu, Honorary Professor of Chinese Medicine at the Chinese University of Hong Kong (CUHK). TCS was purified according to the methods reported by Maraganore et al. Dried pieces of the root tubers of The two estrogen-dependent breast cancer cell lines used were MCF-7 cells, obtained from the American Type Culture Collection (ATCC) The effect of TCS on cell viability was performed by MTT assay as described before MCF-7 cells  and MDA-MB-231 cells (TCS: 10 µM and 20 µM) were treated with TCS at the abovementioned concentrations for 24 h. Subsequently, cells were harvested and fixed with ice-cold 70% ethanol for 2 h. Cells were then washed with PBS twice and stained with propidium iodide medium  2) for 30 min. Afterward, PI  was added followed by incubation for another 15 min After culture with TCS for 24 h, MCF-7 cells  and MDA-MB-231 cells (TCS: 10 µM and 20 µM) were harvested and stained with 0.5 mg/ml Annexin V in binding buffer  were inoculated subcutaneously into the right flanks of mice. Once the tumors were palpable, mice were randomly assigned into 2 groups . The TCS group received an intraperitoneal injection of sterile TCS in PBS at a dose of 5.0 mg/kg every two days. The control group was treated with the same volume of PBS instead. Tumor diameters were serially recorded with an electronic caliper every 2 days, and tumor volumes were calculated with the formula: tumor volume (mm3) = 0.5×length (mm) ×width2 (square mm). Body weights were recorded every 2 days as an indicator of TCS toxicity. Mice were sacrificed on the 12th day of treatment.Female BALB/c nude mice  were obtained from the Chinese University of Hong Kong Laboratory Animal Services Center, and procedures of all animal experiments had been approved by the Chinese University of Hong Kong Animal Research Ethics Committee. The estrogen-independent cell line MDA-MB-231 was employed for establishing the xenograft model following the procedures previously established for other tumor cells Immunostaining of tumor tissues was performed as reported elsewhere 50 values were calculated by Sigmaplot software. SPSS 11.0  was applied for data analysis. All the data are shown in mean ± standard deviation (S.D.) from at least two independent experiments. For both in vitro experiments and in vivo mouse experiments, a two-sided Student t test was applied for comparison of continuous variables between two groups. Differences were considered significant when the p values were<0.05.The IC50s for the MCF-7, MDA-MB-231and BT-474 tumor cells were 31.6 µM, 20.5 µM, and 130 µM, respectively. After treatment with TCS for 48 h, The IC50s for the MCF-7, MDA-MB-231 and BT-474 tumor cells were 25.7 µM, 12.4 µM, and 42.5 µM, respectively. Based on the IC50 values, MCF-7 and MDA-MB-231 cells were chosen for further study. Hep G2 cells served as a positive control for the MTT assay as it has been reported to be inhibited by TCS 50 of TCS toward Hep G2 cells was 28.6 µM, a value that corroborated previous reports TCS was purified following the method described previously p<0.05, To investigate the potential molecular mechanisms, the effects of TCS on breast cancer cell proliferation were analyzed. Cell cycle analysis showed that 24 h co-culture with TCS induced arrest of proliferation . TCS treAs the accumulation of cells in sub-G1 phase is indicative of cell death p<0.05). Accordingly, there were increased levels of both activated caspase-8 and caspase-9 in TCS-treated group compared with control. It has been proved that apoptosis-inducing agents trigger translocation of caspase 9 from the mitochondria to the nucleus which is a pre-requisite for caspase 9 activation, and apoptosis may be caused by the loss of mitochondrial membrane potential in other mechanisms p<0.05 in both MCF-7 and MDA-MB-231 cells, Apoptosis is always executed in a caspase-8-regulated plasma membrane extrinsic pathway and/or caspase-9-regulated cell damage intrinsic pathway in vitro, we went on to determine whether the effect was also observed in vivo. After subcutaneous inoculation with MDA-MB-231 cells into the right flanks of nude mice for 4 days, tumor xenografts were detectable and the mice were assigned to two groups with 6 in each group: control treated with PBS, and TCS-treated group . The TCS dose applied had no detectable toxicity in both groups since there were no statistically significant effects on body weight , behavior, and appearance (th day of TCS treatment (day 16 post-tumor injection) (p<0.05)  . On day (p<0.05) . A reprep<0.05).Immunohistochemistry analysis of tumor tissues further corroborated that necrosis and apoptosis had occurred in TCS-treated tumors . In compBreast cancer is a leading gynecological disease, and though its mortality has been significantly lessened due to adjuvant therapies, the increased incidence, high relapse as well as metastasis after treatment make breast cancer still a major clinical hurdle Momordica charantia seeds (RNase MC-2) manifested the same effects on MCF-7 cells Three breast cancer cell lines, including two estrogen-dependent breast cancer cell lines are MCF-7 cells and highly metastatic BT-474 cells, and estrogen-independent MDA-MB-231 cells, were employed in this study. TCS inhibited cell viability in all three tested cell lines in the following ranking of potency:MDA-MB-231>MCF-7>BT-474 cells. MDA-MB-231 and MCF-7 cells were chosen for further studies. It was revealed that TCS exhibited both cytostatic  and cellM. charantia lectin (MCL). This type-II RIP has been reported to induce apoptosis by activation of both caspase-8 and caspase-9 regulated pathways in nasopharyngeal carcinoma cells Caspases are the principal effectors of apoptosis involved in pathways such as caspase-8-regulated extrinsic and caspase-9-regulated intrinsic pathways. The caspase-9 pathway links mitochondrial damage to caspase activation, and serves as an index of damage in mitochondrial membrane function in vivo systems was substantiated by results from MDA-MB-231 bearing nude mice. Every other day, TCS was administered intraperitoneally at a dose of 5.0 mg/kg body weight. Compared with control, TCS treatment abated both tumor volume as well as tumor weight from the 6th day of treatment. This in vivo antitumor effect correlated with increased levels of apoptosis in the tumors of TCS-treated group, as supported by the increase of activated caspase-3, cleaved PARP, and DNA fragmentation (TUNEL-positive cells). Interestingly, necrosis was also detected in TCS-treated group compared to the control group. These data suggest that reduction of tumor proliferation by induction of apoptosis is a mechanism of the in vivo antitumor activity of TCS toward MDA-MB-231 xenograft. More importantly, previous clinical studies on the application of TCS against other human diseases showed that it was also effective, and the effective dose level was safe and relatively well tolerated, though with some acceptable side effects The antitumor efficacy of TCS in M. charantia which manifestsvsequence homology with TCS to some extent in vivo in a CNE-2 xenograft tumor model. It inhibited tumor growth by inducing apoptosis of tumor cells In particular, several natural RIPs have been the subject of studies that have documented potential medicinal applications of RIPs. The 30-kDa MAP30 is a type I RIP isolated from An interesting question is whether the apoptosis-inducing activity of TCS is attributed to its ribosome inactivating activity. Based on the information provided by previous studies on other RIPs, it seems that the apoptosis-inducing activity of a RIP could be contributed by ribosomal damage, but not restricted by its ribosome inactivating activity. When investigating the apoptosis-inducing activity of the well-known ricin on U937 cells, Komatsu and colleagues found that ribosome inactivation may be responsible for the apoptotic events induced by ricin, since the application of different protein synthesis inhibitors did not significantly alleviate the effects It is probable that an RIP may trigger multiple death signaling pathways, and our current results clearly indicate that both extrinsic and intrinsic caspase cascades were involved in TCS-induced apoptosis in breast cancer cells. Whether inactivation of ribosomes was another cause of cell death needs further investigation. Furthermore, the specificity of TCS on tumor cells and its cellular entry mechanism have also been studied previously. The way for TCS to enter into the tumor cells is associated with the binding on two receptor proteins , and/or depending on a2-macroglobulin receptor/low density lipoprotein receptor related protein (LRP)-mediated endocytosis Trichosanthes cucumerina Linn) displayed anticancer effects in breast cancer cells in vitro and/or in vivo experiments. Proteolytic processing of initiator caspases as well as executor caspase, and subsequent spawned apoptosis contributed to TCS-induced apoptosis. Besides the function as an abortifacient, and application in the treatment of hydatidiform moles and invasive moles, TCS may provide a plethora of treatments or even for the development of a novel therapy combined with other chemotherapeutic measures for both estrogen-dependent and estrogen-independent breast cancers. Further clinical trials are warranted.In this study, TCS was administered intraperitoneally at a dose of 5.0 mg/kg body weight. The dose applied is acceptable since the mice did not lose weight during the course of treatment. Recently, a large clinical study indicated that TCS was effective against ectopic pregnancy in 140 women  at the dose of 1.8 mg/person using intramuscular injection. There were no significant side effects for most of the patients and it seems to have no effects on their subsequent pregnancy as well as giving birth to a healthy baby