Satureja montana herbal species belongs to aromatic medicinal plants with a significant place in traditional medicine. However, products produced with conventional procedures do not meet the requirements of the modern market which include environmentally-safe processes that provide quality, safe, and standardized products. In this study, the antiproliferative activity of S. montana extracts obtained by supercritical carbon dioxide and solid–liquid extraction followed by spray drying was investigated using the in vivo model of Ehrlich ascites carcinoma (EAC) in mice. The impact of two concentrations of extracts on the growth of tumor and the redox status of malignant cells was monitored. It was determined that the extracts induced oxidative stress in the malignant cells which was confirmed by the changes in activity of biochemical indicators of oxidative stress. The posttreatment was not an efficient approach, while the extracts applied as pretreatment and treatment resulted in an increase in the xanthine oxidase (XOD) activity, a decrease in catalase (CAT) activity, and an increase in the intensity of lipid peroxidation (LPx). Furthermore, a decrease in the values of reduced glutathione (GSH) and an increase in glutathione reductase (GR) and glutathione peroxidase (GSHPx) in EAC cells were recorded. Satureja montana L.) represents an aromatic plant from the Lamiaceae family. The benefits of S. montana have long been recognized in traditional medicine which has established the plant as a widespread folk remedy. Furthermore, winter savory is used as a spice. S. montana is rich in essential oil with oxygen-containing phenolic monoterpenes carvacrol and thymol as its most dominant components [Winter savory  in mice. Considering that winter savory possesses significant bioactive components that are polar and non-polar in character, lipophilic extracts obtained by supercritical carbon-dioxide extraction (Sc-CO2) and extracts with a more polar character obtained by solid–liquid extraction with ethanol followed by the spray drying process were investigated. To determine the potential of extracts, parameters of tumor growth and the antioxidant status of malignant cells were monitored. In addition, the impact of extracts before, at the time of, and after the implantation of EAC cells was monitored.For the aforementioned reasons, this study investigated the antiproliferative activities of S. montana extracts on the parameters of tumor growth, the changes in cell parameters were monitored—ascites volume, number of tumor cells, and tumor cells variability (2 (SC extract) was investigated in two concentrations (1% (SD1 and SC1) and 5% (SD5 and SC5)) and different impacts of the extracts on the volume of ascites were recorded  in the number of malignant cells. In the post-treated groups, there was an increase recorded after the application of all extracts .The changes in the antioxidative status of EAC cells were monitored by measuring the activity of antioxidative enzymes  and the quantity of GSH and the intensity of LPx in EAC cells. The obtained results were compared with the control group, that is, with the results of measurements of the same parameters in EAC cells .In the control group, the XOD activity in EAC cells was low, and by applying the SC extracts in pretreatments, there was a significant increase in the XOD activity in malignant cells, without a difference in their impact on this parameter (SC1 and SC5). On the other hand, the application of SD extracts did not cause any significant changes in the intensity of XOD. In the groups treated with extracts, significant changes in the activity of enzymes were determined where SC extracts intensified the XOD activity in EAC cells, as well as in the treatment with SD5. Furthermore, the treatment with a lower concentration of SD significantly decreased the activity of XOD. The application of extracts in post-treatments caused significant changes in all groups (except in the posttreatment with SD1)—a decrease in XOD activity compared to the EAC group, while the post-treatment with SD1 caused an increase in the XOD activity.Compared to the activity of CAT in the EAC control group, the application of extracts resulted in a different impact on the activity of this enzyme. In the groups pretreated with extracts, the activity of CAT in EAC cells was significantly increased with the application of SD1, while it was significantly decreased in other pretreated groups. Moreover, the highest decrease was achieved when SC5 was applied in the pretreatment. The treatment with extracts in all animal groups caused significant changes in the CAT activity. In the pretreatment, the application of SD1 increased the CAT activity, while all other extracts caused a decrease in the activity of CAT of malignant cells. The application of extracts in post-treatments in all groups caused significant changes compared to the EAC control group in terms of decreasing the CAT activity with posttreatment with SD1, and an increase in the remaining three groups of post-treated mice.The activity of Px in malignant cells compared to the control EAC group increased after the application of SC extracts and SD5 in pretreatments. The treatment with SD1 resulted in a decrease in the activity of Px, and an increase was determined in the treatments with SD5, SC1, and SC5. In post-treated groups, the SD1 extract caused changes in the intensity through the increase in the Px activity, while SD5 and SC decreased the activity of Px in EAC cells.The activity of the GR enzyme in tumor cells was significantly different after the application of extracts. SC extracts and SD5 in the pretreated groups caused an increase in the activity of GR. Moreover, the highest increase was recorded in the group of mice pretreated with SC5. By starting therapy at the moment of tumor implantation, there were significant changes in the activity of enzymes in EAC cells. The treatment with SC and SD5 resulted in an increase, while treating mice with SD1 resulted in a decrease in the activity in the GR in EAC cells. After the application of SD1 in post-treated groups of animals, an increase was recorded, while the post-treatment with other extracts resulted in a decrease in the activity of GR.The GSPHx activity in EAC cells when extracts SD5 and SC were applied prior to the implantation of tumor, increased compared to the control group. The impact of extracts that were applied as a treatment was also significant. It was determined that there was an increase in this activity in all treated groups , except for the group treated with SD1 where the GSHPx activity was significantly decreased. The post-treatment of animals with SC1, SC5, and SD5 extracts caused a decrease in the GSPHx activity, and an increase with the application of SD1.Compared to the EAC control group, the amount of GSH was reduced in all groups that were pretreated. Moreover, a significant reduction in GSH was recorded in groups in which SC extracts were applied. Furthermore, animal treatment with extracts also resulted in a decrease in the amount of GSH, and SC extracts in the treatment also led to a significant reduction in the amount of GSH compared to SD extracts. In post-treatments, a reduction was recorded only in the case of SD1, while with SC extracts and SD5, an increase in the amount of GSG was determined.The intensity of the lipid peroxidation in the group with EAC was low. In the application of extracts in the pretreatment, only SC extracts led to a significant increase in the intensity of the lipid peroxidation. The treatment with extracts did not have a statistically significant impact on the changes in intensity of LPx. By post-treating mice with extracts, the changes in the intensity of LPx were significant, so the post-treatment with SD1 resulted in an increase, while others caused a decrease in the intensity of LPx.S. montana extracts obtained by different techniques of extraction. Modern extraction techniques were selected which, once the process is established, can easily be scaled-up to an industrial level and provide high-quality extracts while protecting the environment. The Sc-CO2 extraction represents the method of choice for the isolation of lipophilic components [The study investigated the antiproliferative activity of mponents . The sprmponents ,31.2 (350 bar and 50 °C) was applied to obtain extracts that are lipophilic in character and contained the highest content of carvacrol [In the previously published studies by our research group, the procedures for obtaining extracts of winter savory with optimal physical–chemical characteristics were established ,33. Sc-Carvacrol . In addi2 is a solvent that extracts only non-polar components while the mixture water/ethanol shows an affinity toward more polar components, the content of carvacrol in supercritical extract was expectedly multiple times higher compared to the powder , according to the GC-FID (gas chromatography with flame-ionization detection) analysis [S. montana powder and supercritical extract, such as p-cymene, trans-caryophyllene, caryophyllene oxide, linalool, and terpinen 4-ol  [The antioxidative activity of extracts was investigated by using the DPPH  assay. By comparing the manifested activities, expressed as the IC4 µg/mL) ,36. The 4 µg/mL)  reported4 µg/mL) .S. montana extracts, the precise mechanism responsible for the antitumor activity has not been determined. The antiproliferative activity of different extracts of S. montana was investigated on HeLa (human cervix adenocarcinoma) [S. montana extracts was confirmed along with their antiproliferative properties that were different depending on the type of extract. The authors made an assumption that S. montana as a strong antioxidant can impact the redox condition of cells which leads to decreased cell proliferation. A low level of free oxygen species is necessary to promote cell proliferation and redox changes have a significant role in the signal traductional pathway, which is important for the regulation of growth of cells [In the studies which in their focus have the investigation of antitumor potential of rcinoma) ,20, HT-2rcinoma) , and MDArcinoma) . A stronof cells . It was of cells , as wellof cells .Furthermore, carvacrol is a confirmed antitumor agent. It was demonstrated that supplementation with carvacrol exhibits the antitumor effect on liver cancer induced with diethylnitrosamine in Wistar albino rats, most likely protecting the antioxidant defense system and preventing lipid peroxidation and damage of liver cells . Recent Ascites fluid is highly important for the development of tumor considering that it represents a source of food for its cells; therefore, the increase in volume of ascites is a significant indicator of proliferation of tumor cells . It was It can be noted that SC extracts applied as pretreatment and treatment exhibited a more dominant effect on SD. The content of carvacrol in SC extracts is significantly higher compared to SD; hence, that could be the reason for SC extracts’ higher impact on the antioxidant status of EAC cells. Furthermore, when they were applied as treatment, SC extracts resulted in the reduction in the volume of ascites  and the decrease in malignant cells. On the other hand, SC extracts applied as pretreatment did not have an impact on the decrease of number of malignant cells so they cannot be considered as cytotoxic or cytostatic agents.In the groups of animals that were post-treated with SC1, SC5, and SD5, it is possible that there was an occurrence of a higher level of development of the implanted tumor before the application of extract. Based on the results of the activity of enzymes , the amount of GSH, and intensity of LPx, as well as the results of the volume of ascites and the number of cells in the mentioned groups of animals, it can be concluded that post-treatment of animals is not an adequate therapeutic approach for EAC. Therefore, the time of the application of extracts is of high importance.S. montana extract. These enzymes represent markers of oxidative stress and their increased activity suggests that due to the application of certain extracts, malignant cells were exposed to oxidative stress. As a result, it was determined that extracts of S. montana induce the production of reactive oxygen species in malignant EAC cells. However, the exact mechanism of activity was not clarified and further research is necessary.During the development of tumor, the cells become resistant due to constant exposure to oxidative stress and they develop strong mechanisms of antioxidative protection ,43. The Additionally, Satureja montana) were collected at the Institute of Field and Vegetable Crops, Backi Petrovac, Republic of Serbia, in July 2012. The collected plant material was naturally dried and then stored in paper bags at room temperature.Aerial parts of winter savory (S. montana extract (SC) by Sc-CO2 extraction was described by Vladić et al. [S. montana herbal material was conducted using Sc-CO2 at a pressure of 350 bar and a temperature of 50 °C. The extraction time was 4.5 h. The separator conditions were 15 bar and 23 °C [The detailed procedure of obtaining ć et al. . Brieflynd 23 °C .S. montana extracts via spray drying is described by Vidović et al. [S. montana liquid extracts were obtained using a 50% ethanol mixture as an extraction solvent. The extraction was carried out for five days at room temperature in a dark place. Maltodextrin (DE16) in the percentage of 10%  was used as a carrier material [The detailed procedure of obtaining ć et al. . Spray-dmaterial .Gas chromatography–mass spectrometry (GC/MS) and gas chromatography with flame-ionization detection (GC/FID) analyses were performed according to the procedures described in previous studies ,33. GC a50 value which represents the concentration of the extract which inhibits 50% DPPH radicals. All the measurements were performed in triplicate.The antioxidant activity of extracts was analyzed using the DPPH assay . DiffereAnimal care and all experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animal Resources edited by the Commission of Life Sciences, National Research Council. All procedures performed in the studies involving animals were in accordance with the ethical standards of the institution . For the purposes of this research, female mice of the Hannover National Medicinal Institute (Hann: NMRI) strain were used, aged 6–8 weeks, weighing 25 g ± 10%. Experimental animals were obtained from the laboratory of the Independent Department for Biochemistry, Laboratory Medicine Center, Clinical Center of Vojvodina . The animals were kept under strictly monitored conditions  in adequate cages and without limitations to access to food  and water.Extract SC was dissolved in olive oil in a concentration of 1% (SC1) and 5% (SC5). The SD was dissolved under sonication in water (1% (SD1) and 5% (SD5)), and filtered through A 0.45 mm membrane filter. The resulting solutions were kept refrigerated at a temperature of 4 °C. The extracts were administered intraperitoneally (i.p.).The variability in the volume of administered doses was managed by adjusting the concentration to ensure a constant volume (2 mL/kg body weight).Group EAC—animals with implanted Ehrlich ascites carcinoma (EAC) cells treated with 2 mL/kg of saline, i.p. (n = 6).Group PRETREATMENT—animals pretreated with 2 mL/kg of the investigated extract, i.p. during seven days (n = 6).Group TREATMENT—animals were treated with 2 mL/kg of the investigated extract, i.p. seven days after the implantation of EAC (n = 6).Group POSTTREATMENT—animals post-treated with 2 mL/kg of the investigated extract, i.p. during seven days, seven days after implantation (n = 6).After 14 days from the day of implementation of EAC, all animals were sacrificed and the ascites was collected for further biochemical analyses.During experimental work, mice were divided into groups of 6 mice through random selection and were treated in accordance with the following protocol:3. Cell viability was determined by the Trypan blue exclusion method and expressed as percentage of damaged cells.The ascites from the abdomen was transferred to Krebs–Ringer phosphate buffer solution  and subjected to sequential centrifuging at 4500 rpm  and 12,000 rpm  to obtain a dense cell suspension (1:1). The cell number was counted in a Neubauer chamber and expressed as number of cells/mmThe samples were diluted with Krebs–Ringer phosphate buffer and the activities of antioxidant enzymes were determined in EAC cells by standard laboratory protocols. The activity of xanthine oxidase (XOD) was determined using the Bergmayer method , catalasp < 0.05 confidence level, after performing a one-way single factor ANOVA, followed by Tukey and Bonferroni multiple comparison post hoc tests.The activities are expressed as mean ± standard deviation. Mean values between the groups in the biochemical analyses were considered significantly different at the S. montana by using in vivo mice model. The impact of extracts on malignant cells was observed by applying the extracts as pretreatment, treatment, and posttreatment. It was determined that the time of application of extracts represents a significant parameter in the treatment of EAC cells. The assumption is that there was a significant development of tumors in the groups in which extracts were applied as posttreatment and that the application of extracts did not demonstrate to be an efficient approach. On the other hand, the extracts that were applied as pretreatment and treatment exhibited a far more significant effect on malign cells. The changes in the activities of biochemical parameters that represent markers of oxidative stress indicate that the malignant were exposed to oxidative stress when extracts were applied. Therefore, the results suggest that the extracts resulted in the generation of reactive oxygen species in malignant EAC cells.This study investigated the antiproliferative activity of supercritical and spray dried extracts of S. montana in the prevention and treatment of malignant diseases. Furthermore, the results point to the necessity of the application of adequate environmentally-friendly procedures for the attainment of extracts that exhibit optimal effects and are safe at the same time. It is necessary to direct further investigation towards determining a precise mechanism of activity of extracts and their pharmacokinetic properties.The achieved results indicate the potential of


Substantial and sustained reductions in community cat populations associated with trap–neuter–return (TNR) programs have been documented in a variety of locations, including in the northeastern, midwestern, and southeastern United States, as well as Australia. The present study adds to this growing body of evidence by examining the impact of a TNR program on a population of community cats living on a two-mile section of a pedestrian trail adjacent to the San Francisco Bay. An initial population of 175 cats declined by 99.4% over the 16-year program period. Of the 258 total cats enrolled between 2004 and 2020, only one remained at the end of the program period. The results of the present study corroborate previous research findings. Recently, a growing collection of evidence that associates trap–neuter–return (TNR) programs with substantial and sustained reductions in community cat populations across a variety of environments has emerged. Peer-reviewed studies emanating from the northeastern, midwestern, and southeastern United States, as well as Australia, document such reductions. The present study expands upon this body of evidence by examining the impact of a long-term TNR program on a population of community cats residing on a pedestrian trail adjacent to an oceanic bay located on the West Coast of the U.S. A population of 175 community cats, as determined by an initial census, living on a 2-mile section of the San Francisco Bay Trail declined by 99.4% over a 16-year period. After the conclusion of the initial count, the presence of cats was monitored as part of the TNR program’s daily feeding regimen. Of the 258 total cats enrolled in the program between 2004 and 2020, only one remained at the end of the program period. These results are consistent with those documented at the various sites of other long-term TNR programs.  The use of trap–neuter–return (TNR) as a humane alternative to the lethal management of stray and feral cats, henceforth referred to as community cats , has proliferated in the USA over the past three decades after originating in Europe in the 1950s ,3. The cRecently, a number of peer-reviewed articles have been published that begin to fill the aforementioned information void. From these studies, a growing body of evidence has emerged suggesting that TNR programs of sufficient intensity  are capaThe objective of the present study was to examine the impact of a TNR program on the size and dynamics of a population of community cats residing along the Pacific Coast of the United States after a 16-year observation period. Descriptive data were collected in order to assess changes in the population as well as to compare the results with those of other long-term TNR efforts. The results of the retrospective investigation of this program, which took place on a discrete section of a coastal pedestrian trail, add to the diversity of settings where TNR has been associated with long-term declines in community cat populations, including university campuses  [The San Francisco Bay Trail (Bay Trail) is a walking and cycling path—construction of which began in 1989 and is ongoing—that upon completion will stretch 500 miles and pass through nine counties and 47 municipalities in Northern California, USA, as it engirdles the bay after which it is named . The San2) . Present2) . The subIn the early 2000s, a seemingly large yet indeterminate number of unmanaged community cats resided on or in close proximity to the aforementioned stretch of the Bay Trail. By 2004, the municipality that owns the property through which the trail passes was receiving complaints about the cats and the debris left behind by a small number of people regularly feeding the cats. At the urging of a citizen advocate, a privately funded TNR program was chosen by the city to manage the cats on the trail. The volunteer organization Project Bay Cat (PBC) was formed to carry out the trapping and returning of the cats after sterilization and vaccination as well as to manage their ongoing care. PBC operates under the umbrella of the Homeless Cat Network (HCN), a well-established local, private nonprofit TNR advocacy organization. A community education campaign was initiated to inform the public about the nascent TNR program; the city erected signs along the trail explaining TNR program protocols and publicly distributed brochures for the same purpose. Brochures were disseminated via clear weatherproof dispensers affixed to the signs that were erected on the trail by the city, given (as deemed appropriate) to trail-goers by PBC volunteers, and distributed at events by HCN. The brochures served several functions, including education of the public about TNR, to provide an easily accessible list of local resources related to pet surrender in order to discourage abandonment, and to remind the public that the program was being conducted in partnership with the city. The PBC program protocol called for cats to be trapped in humane box traps, transported by volunteers to a veterinary facility (most often one of two private practices that provided services to PBC at no cost or to an in-house clinic at the Peninsula Humane Society) to be sterilized, vaccinated against rabies and viral rhinotracheitis/calicivirus/panleukopenia (FVRCP), and tested for feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) before being returned to the trail. Cats enrolled in the program were ear-tipped (to indicate that they had been sterilized) and received medical care as warranted. Beginning in 2007, all cats brought to the clinic for sterilization, as well as those recaptured for medical treatment, received a microchip for future identification purposes.An initial census of the cats residing on the trail was conducted by the founder of PBC over a three-week period in the spring of 2004. She walked the area daily  in order to identify locations where cats were congregating and to record the presence of individual cats. Cats were identified by their physical characteristics,  as well as by behavioral traits, such as approachability and their amenability to human touch.After weeks of surveying the area, no new cats were being identified as part of the daily counts; thus, an initial population of 175 cats was documented. Over the next several months, feeding stations were installed as close as practicable to the locations where groups of cats were regularly observed. Because the San Francisco Bay Area is part of the Pacific Flyway for migratory birds , the locThe maximum number of feeding stations installed across the program area was 11. Due to the configuration of the shoreline along the two-mile section of the trail that made up the program area, placement of the feeding stations occurred in three distinct zones (A–C). In Zone A, where much of the trail is flanked by either a two-lane road or marshland and the bay, feeding stations and shelters were hidden in the marsh until the rainy season when they were moved to inconspicuous spots in the rocks along the water. In Zones B and C, where most of the trail is abutted on one side by a strip of grassy meadow covered in native flora and rocky shore on the other, feeding stations and shelters were concealed amidst the foliage in the meadow. Four feeding stations were located in Zone A, one in Zone B, and six in Zone C. The feeding stations were spaced between 50 feet (~15 meters) and 0.5 miles (0.8 km) apart; placement was determined by the terrain, locations known to be frequented by most cats, and the proximity of such spots to sensitive avian habitats.After completion of the initial census, cats were recorded and their presence tracked as they appeared at feeding stations or at adjacent locations within the program area. Descriptions of each cat’s physical characteristics and personality traits were recorded (photographs were taken as practicable). Names were assigned to most of the cats, each of whom was tracked by location; this information was recorded in notebooks and later transferred to Microsoft Word documents created for each of the feeding stations. The age category , sex, and sterilization, vaccination, and FIV/FeLV testing status of most of the cats were recorded. Such information was not recorded for cats found dead or for those immediately adopted from the program. Additional notes describing medical treatment or other details were recorded for most of the cats. However, in some cases, such details were missing from the available records.The feeding station census documents described above and annual summary reports provided to the municipality (extant only for 2009–2019), which included a synopsis of arrivals and departures during the previous 12 months and a review of historical population trends, were analyzed as part of the present study. Semistructured interviews were conducted with past and present leadership of PBC to provide additional context and fill information gaps .As has been performed as part of previous studies of TNR programs ,29, descA total of 258 cats were recorded in the program area during the 16-year program period ending in June 2020. Approximately half (130/259) of the cats resided in Zone A, 9.7% (25/259) in Zone B, and 40.2% (104/259) in Zone C; one cat spent time living in two zones, migrating from Zone B to Zone C after 1.8 years. Overall, of cats whose sex was identified, 50.5% (107/212) were male and 49.5% (105/212) were female; the sex of 46 cats (17.8%) was undocumented. Of cats for whom an age category was assigned at enrollment, 74% (154/208) were adults and 26% (54/208) were kittens; no age category was recorded for 50 cats (19.4%). The average number of cats enrolled in the program per year was 15.2 ; however, when the 175 cats included as part of the initial census are excluded, the average number of cats enrolled on an annual basis was 5.2 . Of the 83 cats who were enrolled after the initial census, 63% (41/65) of those for which an age category was recorded were adults and 37% were kittens (24/65); no age category was recorded for 18 of these cats.As of June 2020, 1 of the 258 cats enrolled in the program (<1%) remained on-site ; 107 41.Cats enrolled in the program spent an average of 4.5 years on-site . Those not adopted within six months of enrollment (211), regardless of their ultimate disposition, remained an average of 5.5 years on-site . Of the 47 cats adopted within six months of enrollment, 42 (89.4%) were kittens.n = 233) remained on-site for an average of 4.7 years , whereas intact cats (n = 25) remained on-site for an average of 2.4 years . These findings generally correspond with those from a study conducted in Rishon LeZion, Israel, demonstrating the favorable health effects of sterilization on free-roaming cats [Sterilized cats  tested positive for FeLV  and 12 cats (4.7%) tested positive for FIV. Cats who tested positive for FIV were returned to their locations of origin and monitored for health concerns (and treated as necessary); 4 were eventually adopted or relocated to a working cat program after an average of 7.5 years  on-site; 8 others were euthanized after residing in the program area for an average of 3 years . From the start of the program in 2004 to June 2020, the total community cat population across the entirety of the program area declined by 99.4% (from 175 to 1). Between 2009 and 2020, the period for which relevant data are available, the Zone A population declined by 97.5% (from 40 to 1), and the Zone B and Zone C populations were entirely eliminated . Ten of the 11 total feeding stations were eliminated due to the decline in the community cat population: 3 of 4 in Zone A, the single station in Zone B, and 6 of 6 in Zone C.Overall, 233 (90.3%) of PBC’s 258 cats were sterilized ; these cats spent an average of 4.7 years on-site . Of the 25 cats who were not sterilized, all but one were adults; after an average of 2.4 years , 44% (11) of these cats disappeared, 28% (7) were euthanized for serious illness or injury, 16% (4) died, and 12% (3) migrated out of the program area before they could be trapped.No kittens were known to have been born in the program area after 2006. All but one of the kittens who were born in the program area, as well as those who were abandoned on or who emigrated to the trail (most often with their mothers), were eventually adopted (four after being sent to foster care); one kitten was found dead on the side of the road adjacent to the trail .The purpose of the present study was to determine the impact of a 16-year TNR program on a community cat population located on a two-mile section of a pedestrian trail adjacent to the San Francisco Bay. Population size, disposition of cats from the program, and population dynamics were examined. A number of variables  make direct comparisons to other similar programs difficult. Nevertheless, notably, the results of the PBC program appear to be consistent with, or in some cases more favorable than, those recorded at sites of other long-term TNR efforts. As has occurred elsewhere ,12,13,14The census conducted at program inception in 2004 revealed an initial population of 175 cats residing in the program area. The 99.4% decline in population (down to a single cat) over the ensuing 16 years exceeds results from four other long-term TNR efforts: on a university campus in Orlando, FL, USA (85% reduction in population size over 28 years) ; on a unAlthough the available data for some of the programs shown in Similarly, the percentage of the total number of cats enrolled in the program who remained on-site at the end of the observation period (<1%) was less than what was observed at the other four locations mentioned above. A review of the existing literature revealed that only the decline in population associated with a TNR program conducted on the waterfront in Newburyport, MA, USA, where an estimated population of 300 resident cats was eliminated over a 17-year period  , slightlFeeding stations were merged or completely eliminated as the population of cats declined, as has occurred elsewhere ,14. By tAt the end of the program period, of the 258 total cats enrolled in the program, one remained on-site. The adoption of sociable cats and socializable kittens, some of whom first moved into foster care in preparation for adoption, contributed considerably to the observed reductions in population size and was the most common outcome for cats enrolled in the program. Adoption is widely accepted as a fundamental component of TNR best practices , in largThe percentage of cats who disappeared from the program area (23%) also closely matched what occurred in Orlando, FL, USA (24%) , but wasThe percentage of cats euthanized due to serious illness or injury 14%) and the percentage who were known to have died (12%) other than by means of euthanasia were consistent with what was observed in Sydney, Australia , and Key% and theThe sustained commitment to the sterilization of community cats by PBC, beginning in 2004, resulted in a sharp decline in the number of kittens in the program area. Over the course of the 16-year program, of the cats who were assigned to an age category at enrollment (208/258), 26% (54/208) were kittens; enrollment of 79.6% (43/54) of the kittens occurred during the first two years of the program, as opposed to 20.4% 11/54) afterward (2006 to 2020). The dramatic reduction in the number of kittens enrolled in the program over time is consistent with what occurred in Orlando, FL, USA . As stat/54 afterThe prevention of kitten births enhances the welfare of a community cat population  by minimThe limitations of the present study include those common to retrospective investigations. The absence of certain data  limited the types of analysis that could be performed. The lack of access to annual summary reports  for years 2004 through 2008 contributed to this absence of data. In addition, the PBC program was designed without control group colonies to allow for direct comparison of results. Such control groups, as established by Nutter ,12,13,14The results of the present study add to the mounting evidence indicating that TNR of sufficient duration and intensity is capable of greatly reducing a population of community cats and that, with adequate ongoing management, such results appear to be sustainable over long periods and in a variety of contexts ,12,13,14


The freezing and thawing of construction concrete is becoming an increasingly important structural challenge. In this study, a shrinkage-compensating concrete based on a double expansive admixture was developed and its frost resistance was assessed through rapid freezing and thawing cycling. The frost resistance of the concrete was derived through the measurement and calculation of the relative dynamic modulus of elasticity (RDME) and the mass loss rate (MLR), and the freezing- and thawing-cycle microstructures and products of concretes with different expansive agents were analyzed using scanning electron microscopy (SEM). It was shown that changes in the properties of the concrete under freezing and thawing could be divided into three stages: slow-damage stage, fast-damage stage, and stable stage. Compared to concrete without an expansive agent, a single-expansive-agent concrete demonstrated excellent frost resistance during the slow-damage stage, but the frost resistance rapidly decreased during the fast-damage age. After 150 cycles (the stable-damage stage), the concrete with a U-type expansive agent (UEA): MgO expansive agent (MEA) mix proportion of 2:1 had the best frost resistance, with RDME and MLR values 17.35% higher and 25.1% lower respectively, than that of an expansive-agent-free concrete. These test results provide a basis for the study of frost resistance in large-scale hydraulic concrete structures. Over thng 10 cm . TherefoThe frost resistance of concrete is influenced by many factors, including the water-cement ratio, compactness of the concrete, pore structure, etc. Many cold regions, including northern Europe, North America, and the former Soviet Union, have attached great importance to frost resistance starting as early as the 1940s. Conventional methods for improving the frost resistance of concrete include the reduction of the water-cement ratio  and the The results of these previous studies reveal that adding only a single expansive agent to concrete results in limited shrinkage compensation. For example, CSA-based expansive agents such as UEA can compensate for only the early shrinkage of concrete but have little effect on later-stage compensation. Other expansive agents, such as MEA, are characterized by delayed expansion, which has a good effect on compensating the later-stage shrinkage of mass concrete. However, MEA concretes show higher initial shrinkage and are subject to continuous shrinkage over the long term, which can result in numerous cracks. Most hydraulic concrete problems arise from shrinkage cracking, which not only destroys the integrity of concrete but also worsens a number of concrete properties such as frost resistance and impermeability. Given growing requirements in terms of hydraulic concrete structure quality and service life, single expansive agents that are only effective at a specific age have limited functionality. This makes it necessary to develop compound expansive agents that exhibit large shrinkage in the early ages but slight shrinkage over the long term to improve the frost resistance of the concrete and extend its service life. The objective of this study is to develop a double expansive resource admixture based on UEA and MEA and analyze its effects on the frost resistance of concrete structures. Four different UEA: MEA mix proportions  were subjected to rapid freezing and thawing cycling tests. The effects of various expansive agent percentages on frost resistance were explored by measuring the relative dynamic moduli of elasticity (RDME) and mass loss rate (MLR) of the respective concretes. The microstructures of the shrinkage-compensating concretes were imaged using a scanning electronic microscope (SEM). Based on these results, an optimal compound ratio of the two expansive agents was proposed to provide a theoretical basis for the development of relevant test procedures for concrete subjected to freezing and thawing cycles.3, which was composed of two particle types with different diameters: 40% of the aggregate had a particle diameter of 5–20 mm; 60% had a particle diameter of 20–40 mm. Natural river sand with a fineness modulus (FM) of 2.8 and density of 2640 kg/m3 was used as the fine aggregate. Type I fly ash (FA) at a dosage of 20% was used as mineral additive; the properties of fly ash are listed in The cement used in this study was ordinary Portland cement 42.5R, which conforms to Chinese standard GB175-2007 . The coa3A·3CaSO4·32H2O) and Mg(OH)2 are produced by UEA and MEA hydration, respectively. In the study, the formation hydrates were used to fill the pores to improve the pore structure and make the concrete highly compact. The properties of the UEA and MEA are listed in UEA and MEA were used to develop the shrinkage compensating concrete. The mechanism of these expansive agents can be explained in terms of their respective hydration reactions, which are given by the following equations ,21,23:  is the fundamental transverse frequency after n cycles of freezing and thawing, and fi0 (Hz) is the fundamental transverse frequency at the beginning of the freezing and thawing process. A higher relative dynamic modulus of elasticity corresponds to better frost resistance.The freezing and thawing cycles test was generally used to simulate the damage to buildings caused by freezing and thawing, because of its many advantages, such as simulating the real damage environment, shortening the experiment time. Furthermore, the results obtained through the freezing and thawing cycles test can be used to effectively guide the engineering practice. Therefore, a rapid freezing and thawing cycling test was performed in accordance with GB/T 50082-2009 . The tesniW is the mass loss rate of the concrete after n cycles of freezing and thawing, 0iW (in g) is the mass of the concrete at the beginning of the freezing and thawing process, and niW (g) is the mass of the concrete after n cycles of freezing and thawing. A smaller mass loss rate corresponds to better frost resistance.The MLR of the shrinkage-compensating concrete is calculated as follows:2 could be distinguished. The microstructures of the concretes were imaged with a SEM every 25 cycles. The SEM analysis was used to explore the correspondence between the microstructural changes and the concrete macroscopic properties. Using the gray scale differences in the image, C–S–H, hydration products such as ettringite and Mg(OH)In this experiment, a core sample was drilled through the concrete core drilling machine from the concretes that have subjected to freezing and thawing cycles, which was used to conduct SEM test. The specific sampling procedures are as follows.(1)Drill core sample: a concrete core drilling machine was used to drill a core sample with a diameter of 20 mm and a height of 100 mm at the center of the bearing surface of the concrete specimen, as shown in (2)Cut thin slices: the thin slices with a thickness of 2 mm were cut at different longitudinal positions  from the erosion surface of the cylindrical core sample by using a cutter, as shown in (3)Spray gold: the thin slices with a thickness of 2 mm that were cut at different longitudinal positions from the erosion surface of the cylindrical core sample in step (2) were sprayed gold. First, the cut samples were placed in an energy-saving box type electric furnace for drying about 12 h. It is considered that the drying was completed when the quality of the samples no longer changes. After the sample surface was dried, the sample was placed in an SBC-12 type ion sputtering apparatus to spray gold to the sample. The sample that was sprayed gold is shown in (4)2, and other carrier gas.Observe: After the gold spray was completed, the sample was placed in the scanning electron microscope sample chamber for observation. Observations were conducted in an airtight reactor without adding O10 was higher than the other samples, and the MLR was lower, which show that the performance was better. However, with the development of freezing and thawing cycles, the RDME of UM10 decreases fast, with a rate of 16.2%. After 75 cycles, the rate of change of RDMEs of UM11, UM41, and UM21 were 8.3%, 10.9%, and 7.4%, respectively. It is shown that except PC control, the change of RDMEs of all samples was high between the 50 cycles and 75 cycles. But after 75 cycles, with the development of cycles, the change of RDMEs was slowly decreasing. The change of MLRs was same as RDMEs. Therefore, based on these RDMEs and MLRs patterns, the process of freezing and thawing cycling can be divided into three stages: (1) A slow-damage stage during cycles 0–50; (2) a fast-damage stage from cycles 50–75; and (3) a stable stage after cycles 75. The RDME and MLR patterns for the respective concretes during each stage were then analyzed separately.Each concrete sample was subjected to 150 freezing and thawing cycles, with the analysis results revealing developmental trends that differed by percentages of UEA and MEA. The RDME and MLR results over the course of the freezing and thawing cycles are shown in 2 crystals within the first 180 d of hydration [3A·3CaSO4·32H2O) generated by UEA hydration filled the internal pores to improve the compactness of the concrete. Therefore, when the water in the pores underwent freezing and thawing cycles, small volume changes occurred in the concrete owing to the expansion of the expansive agents, resulting in an improved frost resistance in the shrinkage-compensating concrete relative to that of the PC control. During this stage, the RDMEs of the shrinkage-compensating concretes decreased slightly while the MLRs changed slightly. It is seen from ydration . Therefo10 > UM41 > UM21 > UM11 > PC, while the MLRs were, in the order from highest to lowest, PC > UM11 > UM21 > UM41 > UM10. These results can be attributed to a number of factors. It has been shown that frost resistance is significantly improved at a UEA mass fraction of about 10% and that, within this range, the frost resistance is positively correlated with the UEA mass fraction [10 was 10%, in which most of pores were filled by ettringite crystals produced by hydration reaction of UEA. Therefore, the frost resistance of UM10 with mass fraction of UEA of 10% was the best, with the highest RDME of 91.51% and the lowest MLR of 0.24%. Moreover, the mass fractions of the UEA in the UM41, UM21, and UM11 samples were 8, 6.6, and 5%, respectively, and the RDMEs of these concrete were 91.51, 90.30, and 87.07% and the MLRs were 0.83, 1.14, and 1.40%, respectively. The RDMEs of these concrete samples were all higher than the RDME of PC control, which indicates that the frost resistance is better than PC control. That is to say, since the mass fractions of UEA in these concretes, the frost resistance of concrete was improved in different degree.As shown in fraction . The ett2 generated by MEA hydration was also small and had a slow growth rate. As a result, the compactness of the concrete was reduced during the fast-damage age and the pore structure was not effectively improved, resulting in rapid decreases in RDME after 75 freezing and thawing cycles. Furthermore, many microcracks had already formed in the concretes during the freezing and thawing cycles of the previous age. As the cycles continued, the microcracks connected and expanded to form larger cracks. After many cycles, the mortar and gravel at the crack edges gradually fell off, resulting in rapid increases in MLR.As the freezing and thawing cycles continued, the RDMEs of the concretes decreased rapidly at an average rate of 13.65%. It is seen from 10 changed most significantly and decreased the fastest, with an average rate of decline of 20.02%. After 75 cycles, the RDMEs of the respective concretes were, in the order from highest to lowest, UM21 > UM41 > UM11 > UM10 > PC, while the MLRs were, in the order from highest to lowest, PC > UM10 > UM11 > UM41 > UM21. These results can be attributed to the fact that, by this age, the UEA hydration reaction in concrete has been essentially completed but there was a lack of other hydration products to continually improve the pore structures of the concretes. As a result, the structures were not effectively improved, and the compactness of the concrete remained low. As the amount of Mg(OH)2 produced by MEA hydration increased, the compactness of the MEA concrete also increased, resulting in an enhanced frost resistance relative to the PC control and UM10 counterpart. It has been suggested that the frost resistance of concrete is best at an MEA mass fraction of about 3% and that excessive dosage of MEA negatively affects the frost resistance [21 was highest among the concretes while its MLR was lowest. It is seen from sistance . This co21, UM41, and UM10 were 78, 75, and 71%, respectively, while the RDME of UM10 was 68%. As shown in As the number of freezing and thawing cycles progressed beyond 75, the RDMEs of the concretes decreased slowly at an average rate of 3.84%, while the MLRs increased slowly at an average rate of only 1.13%. After 150 cycles, the RDME of the PC control was lower than 60% and the MLR was more than 5%. According to relative standards, concrete will lose its applicability when its RDME is lower than 60% or the MLR is above 5%, indicating that the PC control had become useless by the stable age. From 21 > UM41 > UM11 > UM10 > PC, while the MLRs were, in the order from highest to lowest, PC > UM10 > UM11 > UM41 > UM21. These results confirm that the frost resistances of the concretes with UEA and MEA were superior to those of the concrete without an expansion agent. As the freezing and thawing cycles continued beyond 100, the UEA hydration reaction completed while the MEA hydration continued. The Mg(OH)2 generated by the MEA hydration process continued to fill into the internal pores of the concretes, effectively reducing the porosities and improving the pore structures, which in turn enhanced the frost resistance capabilities of the concretes. Mo et al. found that the frost resistance of concrete is optimized at an MEA mass fraction of about 3% and those excessive MEA dosages can negatively affect the frost resistance [21 sample was 3.3%. Most of the pores in the UM21 concrete were filled by Mg(OH)2 particles produced by the hydration of MEA, resulting in a high degree of compactness. As a result, the RDME of UM21 was the highest among the concretes and its frost resistance was the best. The mass fraction of MEA in UM41 was 2.5%, corresponding to a frost resistance worse than that of UM21 but better than those of the others, although all compound-expansive samples had, to some degree, enhanced frost resistances. The mass fraction of MEA in the UM11 sample was 5%, far higher than the 3% optimum identified in [11, and resulted in the formation of numerous cracks in the internal structure of the concrete, a result that can potentially account for the low RDME of UM11 relative to UM21 and UM41. It is also important to note that, while the UEA was most effective at improving the frost resistance during the earlier ages, the frost resistance enhancement relative to the PC control persisted during the late stages as a result of the reduced porosity caused by the filling effect of the ettringite hydrates.After 150 cycles, the RDMEs of the respective concretes were, in the order from highest to lowest, UMsistance . The masified in . This exAn analysis of the respective concretes indicates that, after 50 cycles, the RDME and MLR values of the 1:0 UEA: MEA mixture was high and low, respectively, indicating an excellent compensation effect against frost resistance during the slow-damage age. Over the longer term, however, this effect diminished as the number of freezing and thawing cycles increased, preventing further improvement in the frost resistance. By contrast, the 2:1 UEA: MEA mix was characterized by moderate frost resistance during the slow-damage age, which effectively improved after 75 cycles, with increased RDME and reduced MLR values. It was found that the addition of an appropriate UEA could compensate shrinkage and improve the frost resistance of concrete during early ages but that the effect was reduced from a longer-term perspective; by contrast, the frost resistance of the concretes with double expansive resources admixture was better from a long-term perspective. These results indicate that the addition of UEA and MEA in appropriate proportions can effectively improve the frost resistance of concrete.10, UM21, and the PC control—to represent the concretes with compound expansive agents, single expansive agents, and no expansive agents, respectively. 10 microstructure 2 particles have been deposited in the concrete pores as a result of MEA hydration, and no obvious cracks are present. These features help to explain why the frost resistance of concrete mixed with double expansive admixture is superior to those of the UM10 counterpart and PC control.ious age b. Microc10, which gradually widen and accumulate into larger clusters (2 is produced. A large amount of Mg(OH)2 and ettringite crystallization is also observed in the pores of the UM21 2 particles produced by the MEA hydration increased only gradually. After 125 cycles, a large quantity of delayed-formation Mg(OH)2 filled the pores and cracks of the concrete, improving its compactness. As a result, the UM21 had the least amount of observable microcracks, indicating an improved frost resistance from a macro perspective.In conclusion, the micro morphologies of concretes with different expansive agents varied significantly over the course of repeated freezing and thawing cycles. The size and number of cracks in the PC control increased, leading to the loss of the application properties of the concrete. In the early-stage UMThe expansive agent was used to improve the performance of concrete in many studies. A calcium sulfoaluminate-based expansive additive  was added to compensate for the shrinkage of alkali-activated material (AAM) mortar in . The resThe results of the above studies are consistent with the research in this paper. CSA-based expansive agents can compensate for only the early shrinkage of concrete but have little effect on later-stage compensation, while MEA is characterized by delayed expansion, which has a good effect on compensating the later-stage shrinkage of mass concrete. However, the performance of concrete was improved in only a stage in their studies. A concrete with two different mix proportions of UEA and MEA was developed in our study. As a result, the performance of concrete was improved in different stages. It is expected that the methodologies proposed in this paper would better improve the performance of concrete. However, there is much room for further improvement. A main limiting factor is the definition of micro image and identification of products observed by SEM, because of the imperfect instrument and technology. Besides, the more mix propositions of UEA and MEA must be considered to study to determine the optimal mix proportion more accurately.(1)Based on the evolution of the RDME and MLR values, the change in concrete characteristics under cyclic freezing and thawing could be divided into three ages: a slow-damage age, a fast-damage age, and a stable age. In the slow-damage age, RDME and MLR values changed very little, with average decline and growth rates of 9.51% and 0.97%, respectively. By contrast, there were significant changes during the fast-damage age, with average decline and growth rates of 13.65% and 2.19%, respectively. As the freezing and thawing cycles continued into the stable age, the RDME and MLR values stabilized, with average decline and growth rates of 3.84% and 1.13%, respectively.(2)The concrete with UEA had a better frost resistance than other concrete during the slow-damage ages, with RDME and MLR values 17.80% higher and 74.49% lower respectively, than that of an expansive-agent-free concrete. But it rapidly lost its frost resistance during the fast-damage age, eventually losing its application properties. From a long-term perspective, the addition of double expansive agents in concrete was found to have a significant impact on the frost resistance and was associated with high RDMEs and MLRs. The frost resistance of the concrete was most improved at a UEA: MEA ratio of 2:1, with RDME and MLR values 17.35% higher and 25.1% lower respectively, than that of an expansive-agent-free concrete. The next-best results obtained at a ratio of 4:1.(3)21 were filled by large quantities of hydration products. As a result, the UM21 was more compact than either the UM10 or C and fewer microcracks appeared over the course of the freezing and thawing cycles.Analysis of the concrete microstructures revealed that the internal pores of the UM(4)To protect water conservancy infrastructure suffering from freezing and thawing cycles, it is suggested that a 2:1 UEA: MEA mix should be used. This mixture should effectively compensate the shrinkage and creep of the concrete and, as a result, improve the frost resistance of the hydraulic concrete and extend the service life of the infrastructure.In this study, testing based on rapid cycling of freezing and thawing was conducted to assess the frost resistance of concretes with double expansive source admixtures. The RDME and MLR values of various concretes were measured and SEM images of different mix designs were recorded. The conclusions are summarized as follows:


Pithecopus rusticus  isendemic to the grasslands of the Araucarias Plateau, southern Brazil. Thisspecies is known only from a small population found at the type locality. Here,we analyzed for the first time the chromosomal organization of the repetitivesequences, including seven microsatellite repeats and telomeric sequences(TTAGGG)n in the karyotype of the species by Fluorescence insitu Hybridization. The dinucleotide motifs had a pattern ofdistribution clearly distinct from those of the tri- and tetranucleotides. Thedinucleotide motifs are abundant and widely distributed in the chromosomes,located primarily in the subterminal regions. The tri- and tetranucleotides, bycontrast, tend to be clustered, with signals being observed together in thesecondary constriction of the homologs of pair 9, which are associated with thenucleolus organizer region. As expected, the (TTAGGG)n probe was hybridized inall the telomeres, with hybridization signals being detected in the interstitialregions of some chromosome pairs. We demonstrated the variation in the abundanceand distribution of the different microsatellite motifs and revealed theirnon-random distribution in the karyotype of P. rusticus. Thesedata contribute to understand the role of repetitive sequences in the karyotypediversification and evolution of this taxon.The monkey frog,  The clusterorganization pattern of these sequences in the karyotype may also favorrecombination, either homologous or otherwise, which indicates the potential role ofthe sites as hotspots of chromosomal rearrangement, which is an important source ofvariation during karyotype diversification n repeats in the karyotype of P.rusticus, and we demonstrate the non-random distribution of theserepeats, in association with the 45S rDNA gene.Cytogenetic data on Pithecopusrusticus paratypes collected during the fieldwork, between 2009 and2012, that led to the original description of the species , GA(15), GAA(10),CAG(10), CGC(10), GACA(4), GATA(8) –marked directly with Cy5 fluorochrome (Sigma Aldrich) at the 5’ end during thesynthesis of the DNA. The telomeric (TTAGGG)n repeats were produced by PCRamplification using telomeric primers F (5’ TTAGGG 3’) and R (5’ CCCTAA 3’),with the product of this amplification being marked directly by theincorporation of 11-digoxigenin-dUTP, following the protocol described by The microsatellites were analyzed using oligonucleotide probes –CAThe microsatellite FISH experiments were based on the protocol propose by For hybridization, each slide received a final concentration of 50ng/uL of theprobe. After 24 hours of hybridization in a wet chamber at 37°C, the slides werewashed in 2X SSC at 42°C and in PBST for 5 minutes, and then dehydrated again inan increasing alcohol series  for 3 minutes. The slideswere then incubated in NFDM buffer for 15 minutes, and the signal was detectedusing the antidigoxigenin antibody in NFDM buffer for 1 hour in a wet and darkchamber, at room temperature. The slides were then washed again, three times, in0.5%/4xSSC Tween for 5 minutes, dehydrated in the alcohol series, andcounterstained with DAPI. Ten metaphases per individual were photographed underOlympus BX-51 epifluorescence microscope.(15)  and GA(15)  were distributed abundantly in all the chromosomes and presented signals ofhybridization in the subterminal regions. Interstitial CA(15)hybridizations were also observed in the long arms of the homologs of pairs 4 and 5,and in the pericentromeric regions of the short arms of pair 5 and the long arms ofpairs 11 and 12 . Interstitial hybridizations of the GA(15)were detected in the long arm of the homologs of pair 3 .The diploid number in all specimens analyzed showed 26 chromosomes. The dinucleotidemicrosatellite probes CA(10), CAG(10), CGC(10) – and tetranucleotide – GACA(4) and GATA(8) – microsatellites presented clustered hybridization signals in thesecondary constrictions of the homologs of pair 9, involving the secondaryconstriction related to the NOR site described ((10), GACA(4) and GATA(8) presentingstronger signals . Interstitial signals of GAA(10) were also detected on the short arms of pair 2 and in one of the homologs of pair4.The trinucleotide – GAAin situ hybridization detected (TTAGGG)n sequences in all thechromosomes of the P. rusticus karyotype . Hybridization signals were also detected in thepericentromeric region of pairs 5 and 8. Intense hybridization signals of (TTAGGG)nsequences were detected in the homologs of pair 13 The in situ mapping of the different microsatellite repeatscontributed to the understanding of the chromosomal organization of this repetitiveDNA in the karyotype of Pithecopus rusticus. The results of thepresent study indicated that the dinucleotide motifs has a chromosomal distributionpattern distinct from those of tri- and tetranucleotides. The CA(15) andGA(15) microsatellites are abundant and widely distributed in thechromosomes, and are located primarily in the subterminal regions of thechromosomes.The Ololygon (Repeats of (CA)n and (GA)n appear to be the most common microsatellite dinucleotidemotifs in animal genomes  of repetitive sequenceslikely reflect distinct evolutionary events ,CAG(10), CGC(10)] and tetranucleotide [GACA(4)and GATA(8)] repeats. For example, the distribution of the (GAA)nsequence was related to chromosomal rearrangements/modifications involving primarilyNOR-bearing chromosomes, as observed in a number of different lineages of wheat,Triticum spp.  repeats in some of the P. rusticuschromosomes.While the centromere is formed primarily of repetitive DNA, none of themicrosatellite repeats were detected in this region in As expected, the (TTAGGG)n probes hybridized in all terminal regions of thechromosomes, since this sequence is highly conserved in all vertebrates . In addiP. rusticus, in addition to the presence ofinterstitial signals in the karyotypes of the other species of the familyPhyllomedusidae analyzed to date  reflects different patterns of (TTAGGG)n signals in theinterstitial regions of the chromosomes of these species n sequence is an importantcomponent of the repetitive DNA of these chromosomes in the phyllomedusids (The detection of ITSs in  species. As obsePithecopus rusticusis apparently limited to a small and isolated population, the evaluation of thecomposition and distribution of repetitive DNA in the genome is fundamental tounderstand the role of these sequences in the evolution of the karyotype of thistaxon. DNA sequences that are widely repeated in the genome are capable of evolvingindependently and also serve as a substrate for recombinations and chromosomalrearrangements (P.rusticus, evolutionary novelties may arise frequently and will be fixedrapidly in the population (P. rusticus karyotype, which may be useful, inparticular, for comparative analyses, and the understanding of evolutionarymechanisms that determine the characteristics of this taxon, in addition to themolecular cytogenetics of amphibians, in general.A few studies have investigated the cytogenetic characteristics of thephyllomedusids, including descriptions of karyotypes, the identification ofheterochromatic regions and NOR sites (


In this study, the Hilbert–Huang transform (HHT) method was used to analyze the time–frequency characteristics of the PM10 concentration data series measured at three different sites at the Xinglong Port of Zhenjiang, China, over three months. The HHT method consists of two main stages, namely, empirical mode decomposition (EMD) and Hilbert spectrum analysis (HSA), where the EMD technique is used to pre-process the HSA in order to determine the intrinsic mode function (IMF) components of the raw data series. The results show that the periods of the IMF components exhibit significant differences, and the short-period IMF component provides a modest contribution to all IMF components. Using HSA technology for these IMF components, we discovered that the variations in the amplitude of the PM10 concentration over time and frequency are discrete, and the range of this variation is mainly concentrated in the low-frequency band. We inferred that long-term influencing factors determine the PM10 concentration level in the port, and short-term influencing factors determine the difference in concentration data at different sites. Therefore, when formulating PM10 emission mitigation strategies, targeted measures must be implemented according to the period of the different influencing factors. The results of this study can help guide recommendations for port authorities when formulating the optimal layout of measurement devices.To analyze the time–frequency characteristics of the particulate matter (PM Accordi10 concentrations in ports have mainly concentrated on quantifying the impact on the ambient air quality [10 concentration measured in the ports within a specific time frame, some researchers have discovered that the PM10 concentration level has distinct seasonal variation characteristics; however, the variation is not uniform in different regions [10 concentration of the surrounding cities could be accurately determined [10 concentration data in the time domain. In dry bulk ports, because of the combined impact of loading and unloading activities, terrain, wind speed, temperature, humidity, and the location of detectors, the fluctuation in the PM10 concentration data measured within a particularized period is complex. Given the combined influence of multiple factors, the fluctuation is non-linear and non-stationary. Therefore, an essential issue that needs to be urgently solved is how to conduct in-depth analyses of these particular time-series data in order to determine the periodicity and randomness of the components influencing the PM10 concentration levels on various time scales.To date, studies of PM quality ,17,18. B regions ,20. Studtermined ,22. Howe10 concentration levels in a dry bulk port. With the PM10 concentration data measured at three different locations in the Xinglong port area of Zhenjiang, China, we mainly focused on (1) analyzing the PM10 concentration data and interpreting the potential fluctuation characteristics in the time domain, and (2) identifying the factors that influence the time–frequency characteristics of the PM10 concentration data series at different measurements to encourage port authorities to formulate targeted PM pollution control policies. The remainder of this paper is structured as follows: In Our primary objectives in this study were to investigate the temporal characteristics of the PM2, and the average annual throughput is about 5,000,000 tons.The study was conducted at Xinglong Port in the east of Zhenjiang, which is a famous port city in the Yangtze River basin, located in the south of Jiangsu, Eastern China a. Xinglo10 in the ambient air. As shown in In June 2019, to monitor the air quality of Xinglong Port according to the requirements of the Department of Ecology and Environment of Jiangsu Province, three measurement sites were selected in the port in order to measure the concentration of PM10 and meteorological parameters, such as humidity, temperature, wind speed, and wind direction, in one-minute intervals, which satisfies the requirements of China Standard HJ 655-2013 (http://www.cnemc.cn/jcgf/dqhj/). The particles entered the devices at a flow rate of 16.7 L/min through an air sampler pump supplying a constant volumetric flow rate. In this study, the sensor used for PM10 measurement  uses the principle of laser scattering to measure PM10 concentrations between 0 and 999.9 µg/m3 with an accuracy of ± 10%. The humidity and temperature sensor  can measure the relative humidity between 0% and 100% with an accuracy of ± 1.5%, and measure temperature between −40 and 105 °C with an accuracy of ± 0.1 °C.The measurement devices were set to the height of 3.5 m above the ground, as shown in 10 concentration values. Considering the impact of novel coronavirus 2019 COVID-19 on port shipping activities in 2020, the period of PM10 concentration data in this study ranged from 1 October to 30 December 2019. When the environmental supervisor checked the measurement device once per month, the power supply to the equipment needed to be stopped, which caused the loss of measurement data. Therefore, during the study, a total of 390,600 PM10 concentration values were obtained from these three measurement sites, and 2520 values were lost during the device detection process. In line with previous studies [10 concentration values were measured at each site after removing the incomplete data.The port authorities completed the installation of the measuring devices in August 2019. The environmental supervisor commissioned and calibrated the devices in September 2019, and they checked the devices once a month to ensure the quality of the PM studies ,24, the The Hilbert–Huang transform (HHT) was proven to be an effective method for analyzing non-linear and non-stationary data, and is particularly effective for decomposing complex time series data and obtaining intrinsic modal functions with specific cycles . By decoThe HHT generally comprises two sequential steps, namely: empirical mode decomposition (EMD) and Hilbert spectrum analysis (HSA). The EMD methods can decompose the time-series data into eigenmode intrinsic mode functions (IMFs) with trends, and then the instantaneous frequency data can be obtained using the HSA technique .10 concentration data, EMD can decompose the PMThen, the first component, If 10 concentration data. The Hilbert transform for any IMF component The corresponding instantaneous frequencies can be obtained through applying the Hilbert transform to the IMF components of the PM10 concentration data series Now, the Hilbert spectrum of the IMF component, Thus, from the Hilbert spectrum, Using the wind speed and wind direction data obtained from the three measurement sites in Xinglong Port, the generated wind rose plots are shown in We observed that the average wind speed of Site 1 was 2.7 m/s, and the wind was blowing predominantly from the east (E) and southeast (SE) sectors. During the sampling period, the E and SE wind directions accounted for approximately 28.9% and 33.8% of the total wind, respectively. At Site 2, the average wind speed was 1.0 m/s, and the prevailing wind directions were SE and south (S), accounting for approximately 23.8% and 52.9%, respectively. At Site 3, the average wind speed was 1.3 m/s, SE was the prevailing wind direction with a non-negligible contribution from the S, accounting for approximately 55.5% and 17.4%, respectively. The main reason for the diversity in wind speed and direction at different measurement sites within the port may be the installation of a windbreak fence and other dust suppression measures around the harbor .10 concentration values measured at the three different sites in Xinglong Port, the waveform of the data series was obtained, as shown in Similarly, using the PM10 concentration data are shown in The statistical characteristics of the PM10 concentration per hour at Sites 1, 2, and 3 reached 346.9, 279.11, and 358.77 μg/m3, respectively, and the mean values of the average PM10 concentration per hour at Sites 1, 2, and 3 were 88.77, 84.61, and 90.91 μg/m3, respectively. The average hourly data of the PM10 concentration indicates that a specific difference existed in the PM10 levels measured at these three sites. To further determine the difference in the PM10 concentration values obtained from different measurement sites, we applied EMD and HSA to investigate the time–frequency characteristics of the data.10 concentration data measured from the three sites into several prominent IMF components and one residual component, as shown in To obtain more time scale features from the raw data series, we used EMD to decompose the PMIn 10 concentration data series.10 concentration data series, as shown in 10 concentration data series clearly showed the variation in the amplitude with time (x-axis) and the normalized frequency (y-axis). Here, we found that the variation amplitude of the PM10 concentration with time and frequency was discrete, and the range of this variation was mainly concentrated in the low-frequency band .Applying the HSA technique to the IMF components at different measurement sites, we obtained the Hilbert spectrum of the PM10 concentration data, as shown in 10 concentration was mainly concentrated in two frequency bands, corresponding to a short period (about 20 h) and a long period (about 1000 h). We further observed that the fluctuations in the PM10 concentration at the three different detection sites were almost the same in the long periods, but showed a distinct difference in the short periods.According to Equation (9), the Hilbert spectrum was integrated with the time range in order to obtain the Hilbert marginal spectrum of the PM10 concentration in the port area, and the variations in different elements had a specific time range. Among them, the long-period (approximately 40 days) factors included dry bulk loading and unloading operations . Sorte et al. [10, and these emission sources can contribute about 80% of the PM10 concentration in a port. Pérez et al. [10 in the harbor. This long-term influencing factor did not significantly contribute to the differences in the PM10 concentration data measured at the three sites. In comparison, we focused on the contribution of short-period influencing factors (less than 20 h) to variations in the PM10 concentration. As changes in wind speed or direction may only take several hours, we determined that those influencing factors may include wind force, wind direction, temperature, humidity, etc. The HSA of the PM10 concentration data obtained from Xinglong Port showed that the short-period influencing factors were the main factors causing the disparity in PM10 concentrations at the different measurement sites. However, we did not have detailed log information about the cargo handling and unloading in the port, and could not accurately determine the reliability of those speculations. Furthermore, the period of valid data used in this paper was three months, and it was impossible to judge whether seasonal meteorological characteristics would affect the decomposition of PM10 components. In future research, we should combine shipping activity logs, meteorological phenomena, and more extended PM10 concentration data in the port in order to explore the correlation among IMF components and various physical phenomena.Many factors affect the PMe et al.  reportedz et al.  conclude10 concentration data measured by devices arranged within a specific range [10 concentration values at different measurement sites. In 10 concentration data at the three measurement sites was about 7% (the difference between Sites 2 and 3). The difference was insignificant, chiefly caused by short-period factors rather than long-period factors, which will not lead to essential differences in the PM10 data series from the different measurement sites. Therefore, through the previous analysis, we can infer that the layout of these three devices in Xinglong Port is unreasonable. On the one hand, the HHT analysis of the PM10 concentration data confirms that there are no fundamental differences among the data measured at these three sites, so there is redundancy in the layout of the devices. Also, from the three-month wind direction data, these three measurement sites were all upwind of the port activities. The phenomenon demonstrates that these devices are not placed in suitable measurement sites, and it is hard to accurately measure the impact of port activities on ambient air quality. We think that the corresponding analysis of other ports could be used to determine the reason for the variability in the time–frequency characteristics of the PM10 concentration data series, and then to determine whether the layout of the measurement device could be further optimized. That is, the findings here could be adopted as one of the criteria for determining the optimal arrangement of the measurement equipment. Furthermore, to control the PM concentration level around the dry bulk port, the environmental department requires the port authorities to install a windbreak fence around the harbor, increase coverage measures for the cargo yard, and spray during the cargo handling and unloading process. In previous research [10 concentration data can be used as a useful tool to judge the effects of different measures at the port. In future research, by comparing the HHT analysis of the PM10 concentration data in various ports, we could determine whether the physical and chemical characteristics of the cargo have a significant impact on these dust suppression measures. Also, with the HHT analysis and comparison of the PM10 concentration data at different scenarios, such as adopting a specific dust suppression measure in different periods, we could reveal whether there are fundamental differences among these various dust suppression measures.Previous studies have proved that obtaining air quality information by deploying a large number of detectors is a prerequisite and necessary condition for air quality assessment and improvement ,39,40,41ic range ,41. Therresearch , we foun10 concentration. These time-series data exhibited an apparent difference, of which the differences in wind speed and direction were more noticeable compared with the differences in the mean and variance of the PM10 concentration data. The main reasons for the significant differences in wind speeds and forces at the different sites within the port area are the layout of the storage yard and the series of dust suppression measures implemented by the port authority, including the installation of the porous fence and the use of fog cannons. Then, using the Hilbert–Huang transform, we conducted time–frequency characteristic analysis to clarify the reasons for the differences among these PM10 concentration data series.In this study, we used three online measurement devices deployed in Xinglong Port to obtain a three-month data series, including wind speed, wind direction, and PM10 concentration data measured at Sites 1, 2, and 3 into nine, eight, and nine IMF components, respectively. The periods of these IMF components exhibited significant differences, with the shortest period being only 3.3 h (IMF1 of Site 1) and the longest period being 1100 h (IMF9 of Site 3). In general, the IMF components with a short period (less than one day) provided a small contribution to all IMF components. Using HSA technology for these IMF components, we found that the variation amplitude in PM10 concentration with time and frequency was discrete, and the range of this variation was mainly concentrated in the low-frequency band. By analyzing the Hilbert marginal spectrum, we found that the amplitude of the fluctuation of the PM10 concentration was mainly concentrated in two frequency bands, corresponding to a short period (about 20 h) and a long period (about 1000 h). The fluctuations in the PM10 concentrations at the three different sites were almost the same in the long period, but exhibit specific differences in the short-term. Thus, we infer that long-period influencing factors (such as cargo loading and unloading operations) determine the PM10 concentration levels in the port, and short-period influencing factors (such as wind speed and direction) determine the difference in concentration data at the different sites. As the differences were insignificant (about 7%), we think that the air quality of Xinglong Port can be measured using one instead of three measurement devices, which would reduce the acquisition cost of the PM10 concentration data. The method can also be adopted as one of the criteria for determining the optimal arrangement of the measurement equipment.EMD technology is the premise of the Hilbert–Huang transformation; using this technology, we decomposed the PM10 in the warm months are higher than those during the cold periods. However, these findings can encourage researchers to pay more attention to the analysis of the time–frequency characteristics of the PM10 concentration data.The limitation of this study is that the length of the analyzed data series was only three months, which may not enable appropriate analysis of the long-term seasonal time–frequency characteristics. Gobbi et al.  discover10 concentration data series, and the long-period influencing factors determine the PM10 concentration level in the port. Therefore, when formulating PM10 emission mitigation strategies, targeted measures must be implemented according to the period of different influencing factors. The results of this study help provide recommendations for port authorities to formulate the optimal layout of measurement devices. Future analysis of the PM10 concentration data from different ports  may further reveal other time series characteristics of these data and improve the environmental management capabilities of port authorities.In summary, we found that the short-period influencing factors are the main factors affecting the difference in the time–frequency characteristics of the PM


Plasmodium falciparum remains sensitive to artemether-lumefantrine in Mali.In Mali, since 2007, artemether-lumefantrine has been the first choice against uncomplicated malaria. Despite its effectiveness, a rapid selection of markers of resistance to partner drugs has been documented. This work evaluated the treatment according to the World Health Organization’s standard 28-day treatment method. The primary endpoint was the clinical and parasitological response corrected by a polymerase chain reaction. It was more than 99.9 percent, the proportion of patients with anemia significantly decrease compared to baseline ( Plasmodium falciparum malaria, which has positively contributed to reducing the global burden of malaria [In the past two decades, we have witnessed a revolution in the diagnosis and treatment of malaria worldwide. In sub-Saharan Africa, classical antimalarial drugs have been gradually abandoned in favor of faster and more effective molecules, mainly because of the increasing resistance of parasites ,2. Indee malaria .In Mali, malaria remains a major health problem, with more than 2 million cases and more than 1050 deaths recorded in 2017, a case-fatality rate of 0.50‰ . In addiIn order to promote evidence-based policy decisions, the WHO stressed the need to monitor regularly the efficacy of antimalarial drugs using standardized protocols .Plasmodium falciparum are essential to validate the treatment and guarantee a rapid response to the infection emergence of parasite resistance. In Mali, some studies showed a rapid selection of molecular markers of P. falciparum resistance to artemisinin partner drugs [In vivo efficacy of antimalarial as well as the evaluation of the prevalence of molecular markers associated with resistance of er drugs ,9,10,11.In Southeast Asia, reports have shown the emergence and potential spread of resistance to artemisinin ,13,14, rPlasmodium falciparum to artemether-lumefantrine in two sentinel sites of Mali’s National Malaria Control Program (NMCP) during two malaria transmission seasons 2017–2018.In an open and global world with the movement of populations, it seems appropriate to strengthen the monitoring of the effectiveness of ACTs. This study is part of the monitoring of the sensitivity of Study sites and malaria in Mali: The study was conducted according to WHO standard in vivo efficacy protocol  in two NThe work took place in three community health centers in Bougouni district  and a district hospital in the Bandiagara district between July 2017 and December 2018.Plasmodium falciparum mono-infection with asexual blood density ≥1000/μL and <200,000/μL, and the absence of severe signs of complicated malaria as defined by WHO [Patients: The study population included patients aged six months or older with uncomplicated acute malaria confirmed by microscopy. Other inclusion criteria included body weight ≥5 kg, presence of fever (axillary ≥ 37.5 °C) or a history of fever in the previous 24 h, Treatment: Eligible patients were treated with Artemether Lumefantrine (each tablet containing 20 mg artemether and 120 mg lumefantrine). The study drug used was provided to the local health facilities by the Malian Ministry of Health. The dosage and method of administration of the drugs followed the manufacturer’s instructions and the Mali NMCP malaria treatment guideline. One dose at baseline (time 0), a second dose 8 h later, the third dose 24 h later and the last three doses 12 h apart  for 3 days. The number of tablets to be taken was determined according to body weight: one tablet for children from 5 to <15 kg, two tablets for children from 15 to <25 kg and three tablets for subjects of 25 to <35 kg and four tablets for subjects over 35 kg. Drug administrations were directly observed by the investigators. When vomiting occurred within 30 min of dosing, a new dose was re-administered. For febrile subjects (fever > 37.5 °C), paracetamol was used. In the event of signs of danger or severe malaria, the patient was hospitalized and given injectable artesunate, in accordance with the National Malaria Treatment Policy [Follow-up: Follow-up visits took place on days 1, 2, 3, 7, 14, 21 and 28 after enrollment or at any time when the child was ill. Patients who withdrew their consent or participation were stopped prematurely for various reasons were followed through local guides until the end of the study. Adverse events that occurred were recorded, treated, and assessed by severity and drug-study relationship. Parasite clearance was monitored by microscopy. Microscopy slides were obtained and then stained with Giemsa later prior to each dose of AL and at each follow-up visit on days 2, 3, 7, 14, 21, and 28. The slides were examined by certified microscopists and were considered negative in the absence of parasites after examination of 200 fields in a thick smear of blood according to MRTC standard operating procedures. The parasite density was estimated by counting the number of asexual parasites in 200 white blood cells, assuming a standard count of 8000/μL. The determination of the species (and thus the confirmation of the mono-infection) was carried out on the basis of an evaluation of the thin films. As part of quality control measures, a second independent microscopist from the project team read 10% of all smears from all visits.Plasmodium merozoite surface protein 2 genes (msp2) and microsatellite (CA1 and TA87) to discriminate reinfection from recrudescence as described previously [Dried blood spots (DBS) for Polymerase Chain Reaction (PCR) analysis were collected from each patient using 3MM Whatman™ filter papers at enrolment (day 0) and at 7, 14, 21 and 28 days, on the day of treatment failure or any other unplanned visits with suspected malaria. After drying, they were stored in plastic bags containing silica gel (desiccant) and were used to distinguish the recrudescence of a new infection using the procedures recommended by WHO . For pareviously ,23. RecrStudy outcomes: The main measured outcome of the efficacy of AL was the adequate clinical and parasitological response (ACPR) at day 28 corrected by PCR. Then, early treatment failure (ETF), late clinical failure (LCF) and late parasitological failure (LPF) as defined by the WHO  were alsData management and statistical analysis: The data were recorded using electronic case report forms with the open data kits software (ODK). All survey questionnaires were captured using a digital tablet and sent to a database hosted in a server at the Malaria Research and Training Center at the University of Science, Techniques and Technologies of Bamako (USTTB), Mali. Tablets were secured by an individual password and kept under seal outside working hours. Efficacy was calculated in the protocol-treated population, which includes all patients meeting the eligibility criteria of the protocol, having completed the three-day treatment of the study drug, having adhered to all study procedures until a possible failure or at the final evaluation of 28th day. Efficacy rates were calculated by dividing the number of patients with clinical and parasitological cure at day 28 by the total number of patients that could be assessed. In addition, statistical analyses  were performed with the R version 3.4.3 software and the statistical significance level was set at 5%.Sample size calculations: Previous studies in Mali found artemether-lumefantrine efficacy of at least 95% in the different research sites ,25. The Ethical considerations: The protocol was approved by the Ethics Committee of the Faculty of Medicine and Odonto-Stomatology and Pharmacy (FMOS-FAPH) of Mali before the start of all activities (Ref N° 2017/108/CE/FMPOS) and at the amendment for the extension of the study to 2018 (Ref N° 2018/108Bis/CE/FMOS). The trial was conducted in accordance with the guidelines of good clinical practice. Written informed consent was obtained from all participants. Informed assent was also obtained from children aged 12 to 17 years.Trial profile and baseline characteristics: A total of 432 febrile patients or patients with a history of fever within 24 h were screened between July 2017 and December 2018, of whom 375 (86.8%) were recruited and 330 (95.7%) completed the study, with or without recurrent parasitemia .Plasmodium falciparum mono-infection or the presence of danger signs such as vomiting or the notion of taking other antimalarials within two weeks or during follow-up and withdrawal of consent were the main causes of exclusion. The absence of During the 28-day follow-up during the two malaria transmission seasons 2017 and 2018, 13 patients  did not successfully complete the study due to loss of follow-up, withdrawal of consent or other protocol violations such as the accidental or deliberate taking of antimalarial drugs, incoercible vomiting after inclusion or noncompliance with the visit schedule. Reported fever was the main recorded sign at the enrollment with 79.2% of the patients. This frequency decreased rapidly during the first 48 h of follow-up, to 2% or less throughout the rest of the follow-up .The treatment results by locality according to season are summarized in For both locations, 50.9% of treated patients eliminated their parasitemia at the end of the first 48 h of follow-up, while 93.1% (22/439) did it at the end of the first 60 h of follow-up, and only 0.3% did not eliminate parasites at the end of the first 72 h of follow-up .We also used a parasite clearance estimator from The Worldwide Antimalarial Resistance Network (WWARN) . The estp < 0.0001) between the study sites  who had reinfection is provided in Fourteen patients reported vomiting  during the three days of treatment in both locations . Twenty-The proportion of patients with hemoglobin below 11 g/dL at inclusion  was significantly improved on day 28 . This improvement was observed in all localities and in both seasons for the Bougouni site . HoweverThis study describes the 28-day in vivo efficacy of AL under field conditions at two NMCP sentinel sites in Mali. These two sites, one Guinean Sudanese area, the other Sahelian area, provide a good geographical representation of the variability of malaria endemicity and transmission patterns in the country. The standard protocol for evaluating the in vivo efficacy of WHO ACTs was used at both sites , includiFrom the point of view of efficacy, the cure rate corrected by PCR (>99.9%) remains high and adequate according to WHO recommendations. These results confirm those of previous studies in Mozambique and Myanmar, all of which have documented the efficacy and safety of the combination of artemether-lumefantrine ,29.The study was not designed to compare sites, so caution should be exercised in interpreting results such as late parasitological failure (LPF) levels, Es identified and the effect of treatment on hemoglobin recovery. Parasitic clearance was obtained at the sites within the first 72 h after treatment, apyrexia without analgesia was obtained 48 h after treatment as documented in 2016 in Mali , a reassDifficulties encountered were on the respect of the AL dose taking schedules, which often occurred late at night. In order to minimize the inconveniences linked to this situation, the investigators took the participants’ telephone number with their agreement and called them an hour or thirty minutes in advance or went directly to the participants’ homes with the help of the local guide if they encountered a problem in coming to the health center to take the doses. The same strategy was used for parasitological follow-up (every 12 h) until two consecutive negative thick blood smears were obtained. On the other hand, the 28-day follow-up time was also difficult because it was considered long and contributed to a relatively high number of people lost to follow-up.The molecular genotyping with msp1, msp2 and The Glutamate-Rich Protein (GLURP) is commonly used to distinguish recrudescence from reinfection in the monitoring of antimalarial drug efficacy ,34,35,36Plasmodium falciparum to antimalarial using the 53 episodes of recurrent parasitemia samples and the results will be published.In addition, efforts are underway to perform the parasite molecular resistance of Continuous monitoring of the in vivo efficacy of artemether-lumefantrine across the country is necessary to detect early signs of decreased efficacy of artemether-lumefantrine.Plasmodium falciparum malaria after more than a decade of use of this drug and was safe and well-tolerated. The findings of this study advocate for the continuous use of AL as first-line therapy for uncomplicated malaria in Mali. However, a genotyping of the parasite molecular resistance to antimalarial is needed to support these findings, and the monitoring of AL efficacy should continue as recommended by WHO.This study showed high efficacy of AL against