A robust threshold was established as 10% nt differences within the 1248 nt genome fragment, with 4 distinct AAV genotypes identified. This distinct sub-species structure is maintained by ubiquitous recombination within, but not between, rep genes of the suggested genotypes.Adeno-associated viruses (AAVs) are a convenient tool for gene therapy delivery. According to the current classification, they are divided into the species AAV A and AAV B within the genus Dependoparvovirus. Historically AAVs were also subdivided on the intraspecies level into 13 serotypes, which differ in tissue tropism and targeted gene delivery capacity. Serotype, however, is not a universal taxonomic category, and their assignment is not always robust. Cross-reactivity has been shown, indicating that classification could not rely on the results of serological tests alone. Moreover, since the isolation of AAV4, all subsequent AAVs were subdivided into serotypes based primarily on genetic differences and phylogenetic reconstructions. An increased interest in the use of AAV as a gene delivery tool justifies the need to improve the existing classification. Here, we suggest genotype-based AAV classification below the species level based on the  Adeno-associated viruses (AAVs) are non-enveloped particles with a size of 18–26 nm. Sixty protein molecules form an icosahedral capsid. The genome is represented by a linear single-stranded DNA of approximately 4.7 thousand bases.rep, which encodes four overlapping proteins necessary for the regulation of viral DNA replication: Rep78, Rep68, Rep52, and Rep40 [cap encoding viral capsid proteins: VP1, VP2 and VP3 [The AAV genome contains two open reading frames (ORFs): 1)  rep, whiFor effective replication and reproduction in host cells, AAV requires co-infection with an auxiliary virus, e.g., adenovirus (hence the name adeno-associated), although AAV replication is also possible with herpesvirus, cytomegalovirus, and papillomavirus co-infection .The best-known hosts of AAVs are primates and humans ,10, althAAVs are a convenient tool for gene therapy ,17. SincResults of preclinical and clinical studies have demonstrated that AAV vectors are safe and effective tools in gene therapy for a range of diseases including cystic fibrosis, hemophilia B, arthritis, hereditary emphysema, muscular dystrophy, Parkinson’s disease, Alzheimer’s disease, prostate cancer, malignant melanoma, epilepsy, and others ,21,22,23https://talk.ictvonline.org/taxonomy/ accessed 25 June 2021).Current AAV classification is based on the phenotype (the shape of the virion), replication peculiarities, and the host range . AccordiAdditionally, AAVs are subdivided on the intraspecies level into serotypes. The first reports of serologically distinct AAVs date back to 1966, when the first three serotypes were identified .Further, any newly identified AAV serologically distinct from known types were assigned a new serotype in chronological order. To date, 13 AAV serotypes are known. However, there are significant ambiguities and controversies in the properties and definitions of the AAV serotypes. For instance, AAV4 was identified based on the reaction with antiserum , whereasAAV6 was assigned a new serotype based on genetic differences from the complete genomes of AAV2 and AAV3 (82% identity), as well as AAV4 (75–78% identity) . Howeverrep and cap nucleotide sequences and the predicted amino acid sequence comparisons for AAV1-8 (with the exception of AAV5 as obviously incongruent) revealed differences, primarily in the region encoding capsid proteins. AAV7 was shown to have a 63% to 85% similarity to the amino acid sequences and 68% to 84% of the nucleotide sequences of other AAV serotypes; similar results were obtained for AAV8. The serological difference of AAV7, AAV8, and other serotypes was also established based on the absence of neutralization by any antiserum other than their own .In 2002, during an investigation of the asymptomatic presence of AAV in primate tissues, Gao et al. identified AAV7 and AAV8 . The autLater, Gao et al. identified 11 phylogenetic groups based on phylogenetic analysis of cap sequences—so-called ‘clades’—from A to F, consisting of phylogenetically similar representatives from three or more sources, and five groups of clones  .AAV10 and AAV11 were also identified based on the signature region differences and characterized according to the cap sequence . The serrep and cap sequence differences [Similarly, AAV12 was identified based on the ferences . Finallyferences .Thus, since the isolation of AAV4, all subsequent AAVs were subdivided into different serotypes based primarily on genetic differences and phylogenetic reconstructions. In addition to the established 13 serotypes, one of which comprises a separate virus species (AAV5), more than 100 novel genetic variants  are notHere, we analyzed all available AAV-A sequences to test the possibility of systematically distinguishing AAVs according to genomic sequence data and to suggest a classification based on genotypes with a measurable robust threshold.All sequences with a length limited to 4000–5100 nt, which approximately corresponds to the full genome, available as of June, 2021, for Dependoparvovirus isolates were retrieved from the GenBank database . Since this study aimed to characterize genetic relationships at the intraspecies level, only sequences of the species AAV A were selected. For this purpose, a phylogenetic tree for the retrieved sequences was constructed using neighbor-joining, implemented in MEGA [https://github.com/AndreiDeviatkin/repo/blob/main/AAV.rdp, accessed on 11 May 2022). A more comprehensive recombination screening was carried out using pairwise distance deviation matrices (PDDM) and pairwise distance correspondence plots (PDCP) as described earlier [https://v-julia.shinyapps.io/recdplot_app/, accessed on 25 June 2021). Similarity plots for selected sequences were generated using Simplot 3.5.1 [The selected sequences were aligned using MAFFT online . Multipl earlier  with an ot 3.5.1 .The analysis of nucleotide and inferred amino acid sequence differences was carried out in R software.A total of 272 Dependoparvovirus sequences (4000–5100 nt length) were retrieved from GenBank. Of them, 105 sequences belonged to the AAV A species based on phylogenetic analysis . The datRecombination occurs more frequently in some fragments of the virus genome than in others. The root mean square error (RMSE) of all pairwise distances for two genomic regions from the regression line reflects the extent of phylogenetic inconsistency between these fragments of the genome. A high RMSE  were used. However, conflicts and incongruences in AAV serotype assignment have been accumulating (see above). They were further complicated by serological cross-reactivity ,50. UnceRecombination is a physical interaction of viral genomes that leads to gene combinations not existing in any parent. Natural recombination has been described in all known viruses. In the case of some, it may be relatively rare, but in many viruses, it is a regular occurrence that shapes the genetics of taxa. Recombination is commonly seen as a typical feature of RNA viruses, and distinct genome regions of a virus often have different recombination profiles ,53; howeAnalysis of the genetic sequences of several AAV clones isolated from various primates showed recombination signs in cap genes . The recOur analysis confirmed that natural recombination in AAV is ubiquitous and needs to be considered for any taxonomic implications. The effect of recombination on taxonomy may both be negative, when it shuffles genomes and precludes a definitive phylogeny and sequence distance criteria, and positive, when it mixes genes within, but not between, taxa, as seen in enteroviruses  or adenorep genotypes is objective, measurable, robust, and unambiguous. To assign viruses to a particular genotype, their genome needs to be sequenced and the sequence has to be analyzed with phylogenetic methods. The same strategy has been suggested and successfully implemented previously for adenovirus classification [The difference between the suggested AAV fication .rep genotypes. However, the biological implications of such a classification remain largely unknown, as most AAV properties have been associated with the capsid. Thus, the suggested classification is not intended to replace the classical VP1-based one, but to supplement it, and further studies are required to assay its phenotypic implications. In other viruses, it is not uncommon to have different classifications for distinct genome regions. One example is rotaviruses that have a segmented genome and feature ubiquitous reassortment [rep and cap. Satisfactory transfection and expression were shown for AAV2/4, AAV2/5, AAV2/6, AAV2/8 and AAV2/9 constructs [Genetic analysis allowed for robust segregation of AAV into sortment . Howeversortment , and DNAsortment . In thesnstructs .Knowledge about AAV genetic diversity remains fragmentary. For instance, the complete genome or rep gene of AAV9 has not been sequenced yet (the only record available in GenBank refers to the VP1 complete coding sequence). Thus, our dataset lacks this serotype for analysis (conducted based on Rep coding sequence) and we could not assign AAV9 to any of the determined genotypes. It is likely that the robust rep genotype criteria suggested here would need to be refined in the future. Additionally, although the suggested classification is robust and is explained by an obvious biological phenomenon (recombination), its practical implications require further study.rep genotypes, which are proposed as an auxiliary classification within the AAV A species: AAV A genotype 1 ; AAV A genotype 2 ; AAV A genotype 3 ; and AAV A genotype 4 (represented by AAV12).Based on genome analysis, we have identified four distinct


Anopheles cruzii, Psorophora ferox, Runchomyia cerqueirai, Wyeomyia incaudata, Wy. theobaldi, Sabethes chloropterus, and Sa. albiprivus only collected via PHA. Collections with ovitraps resulted in low diversity and richness, with Haemagogus leucocelaenus and Hg. janthinomys/capricornii predominating. The diverse local fauna and the abundance and ubiquity of the latter species, which are the primary vectors of YFV, indicated that this area was highly vulnerable to arbovirus transmission, especially yellow fever, highlighting the need for improved surveillance and vaccination coverage in human and captive endangered non-human primates.In 2017–2019, Brazil recorded its most severe outbreak of yellow fever due to the spread of the virus (YFV) in the country’s southeast. Here, we investigated mosquito fauna and the spatial distribution of species in a primatology center in the Atlantic Forest bioregion in Rio de Janeiro state to evaluate the risk of YFV transmission in distinct environments. Fortnightly mosquito collections were performed from December 2018 to December 2019 at 12 sites along a disturbance gradient from a modified environment to 400 m inside the forest. We used ovitraps, BG-Sentinel, and protected human attraction (PHA). A total of 9349 mosquitoes of 21 species were collected. The collection method strongly influenced the captured fauna, with species such as  Aedes aegypti in urban areas and several sylvatic Aedini and Sabethini species in forested environments [Haemagogus and Sabethes, where humans can acquire the infection during epizootics [Arboviruses (arthropod-borne viruses) are the agents of some of the emerging and re-emerging diseases with the greatest impact on human health. Yellow fever is a mosquito-borne disease caused by the yellow fever virus (YFV), which occurs in Africa and the Americas in two main transmission cycles: urban and sylvatic. These are distinguished by the ecological nature of the transmission, or, more specifically, the mosquito vector species: the anthropic ronments ,2. In Soizootics ,4,5.In 2017–2019, Brazil recorded its most serious outbreak when YFV spread in the Atlantic Forest in the southeast region, where it had not circulated for almost 80 years . Haemagogus spp. play a key role in the transmission of sylvatic yellow fever in South America, mainly due to its primatophilic habit, which facilitates its contact with viremic NHPs [Haemagogus janthinomys/capricornii and Haemagogus leucocelaenus [Sabethes, Sabethes chloropterus  and Sabethes albiprivus  are commonly found in enzootic YFV foci and were found to be naturally infected with the virus. However, Sa. chloropterus has greater local epidemiological prominence and was considered a secondary vector of YFV in the Atlantic Forest bioregion [mic NHPs ,7,8. Twoioregion .Brazil has the greatest diversity of species in the world, with more than 20% of the total species on the planet . The AtlThe occurrence of species depends on factors such as the structural heterogeneity of the biome, with more diverse habitat resources allowing for more species to coexist while also minimizing the effect of competition and, consequently, increasing local biodiversity. Variations in the number of species between communities can be represented and quantified in several ways, with the most common being through diversity indices ,14.The spatial distribution of mosquito species is mainly related to the choice of oviposition site, which is strongly influenced by climatic and environmental factors. In addition, some Culicidae species’ eggs can withstand a period out of water without hatching, which is a natural ability of mosquitoes that lay their eggs out of water. This ability, which can be influenced by extrinsic factors, allows these species to withstand periods of drought .The triggering, spread, and severity of an outbreak of a zoonotic arbovirus, such as YFV, are multifactorial. In the case of the 2017–2019 YFV outbreak in southeastern Brazil, the main drivers identified were poor vaccination coverage; increased areas with conditions favorable for NHPs; mosquito population growth in some parts of the Atlantic Forest combined with the loss of natural habitats in others; human contact with the forest; and the ecological plasticity of vectors, such as the ability of sylvatic mosquitoes to bite beyond the forest limits ,16,17.In this study, we identified and described mosquito fauna and spatial distribution of mosquito species in a fragment of the Atlantic Forest in Rio de Janeiro (RJ) state impacted by the 2017–2019 outbreak to evaluate the risk of YFV infections in distinct environments.The study protocol for the collection, capture, and transport of biological material was approved by the Brazilian Ministry of Environment’s Instituto Chico Mendes de Conservação da Biodiversidade (ICMBio-SISBIO 52472-2). All team members were vaccinated against YFV.The survey was carried out at Centro de Primatologia do Rio de Janeiro (CPRJ) , which is a breeding and research center for endangered New World NHPs located in the foothills of a coastal mountain chain called Serra do Mar in the municipality of Guapimirim, RJ, next to the former Estação Ecológica Estadual do Paraíso. The center is currently part of the Três Picos State Park and is located approximately 100 km from the city of Rio de Janeiro . This siMosquito captures were carried out at 12 sampling sites (P1–P12) located along transects that encompassed environments with different forest coverage, from an open field and the modified environment where primate enclosures and other facilities are located to inside the forest. One main transect was traced with five collection sites (P1–P5) established every 200 m. The collection site located in the middle (P1) served as a reference for two more transects, along which four additional sites were placed (P6–P9), each 200 m away from P1. Site P1 was located in the modified environment at the approximate geographic center of the CPRJ; P2 and P5–P9 were located within a radius of 200 m from P1 in an ecotone between the modified environment and the forest edge, while P3 and P4 were located inside the forest, 400 m from P1 .In five sites (P1–P5), we used traps for the collection of adults (BG-Sentinel), as well as for immatures (ovitraps). Only ovitraps were used at four sites (P6–P9). In addition, the active capture of adults through protected human attraction (PHA) was conducted at three sites designated P10–P12. P10 and P11 were located inside the forest, while P12 was in the modified environment. From December 2018 to December 2019, ovitraps [2 released by dry ice as an attractant for the adult collection with the BG-Sentinel trap. Captures of adult mosquitoes were carried out during four consecutive days per month, with collections taking place over four months in the rainy season (December 2018 to March 2019) and four months in the dry season (June to September 2019). During the four days of monthly collections, the BG-Sentinel traps were operated simultaneously in five collection sites (P1–P5)  , and theAdult mosquitoes captured with BG-Sentinels and PHA or raised in the laboratory from immature forms collected in ovitraps were immediately killed using dry ice and kept at −80 °C until tested for YFV.®, Aalen, Germany) on a cold table following Arnell (1973), Consoli and Lourenço-de-Oliveira (1994), Forattini (2002), Marcondes and Alencar (2010), and Abreu et al. (2019) [The identification of mosquitoes was carried out from the direct observation of morphological characters under a stereomicroscope (Zeiss. 2019) ,20,21,2219 ,20,21g for 10 min at 4 °C. RNA was extracted from the supernatant by using the QIAamp Viral RNA Mini Kit according to the manufacturer’s instructions. The extracted RNAs were tested in duplicate via RT-qPCR using the set primers and protocols described before [Adult mosquitoes were treated and tested for the presence of the YFV genome as previously described . This and before ,23,24.Differences in mosquito community composition at each collection site were evaluated and compared using the Shannon–Wiener Diversity Index (H’) . A t-tes® SPSS® Statistics Version 23 . Dunn’s post-test was used to assess which points and traps were statistically different from each other. Mann–Whitney analysis was used to evaluate the occurrence of significant differences between the different types of traps at the sampling points where only BG-Sentinels and ovitraps were placed, also using IBM® SPSS® Statistics Version 23.To assess the differences between mosquito populations at different capture sites and different capture methods, we used Kruskal–Wallis analysis with a significance level of 5% using IBMA total of 9349 mosquitoes were collected . Just ovCulex (Culex) spp. and Wyeomyia could not be identified at the species level. Five species were collected using all the methods : the main YFV vectors Hg. leucocelaenus and Hg. janthinomys/capricornii, as well as Aedes albopictus, Aedes terrens, and Limatus durhamii  and Hg. janthinomys/capricornii (n = 509) representing 72% and 11% of the total number of individuals collected, respectively , followed by BG-Sentinel (H’ = 2.02). The paddles of the ovitraps had the lowest diversity (H’ = 0.80) and richness (S = 5) and, consequently, a high level of dominance (D = 0.56), with the species ectively . HoweverHg. leucocelaenus (61.7%), Ae. terrens (14.0%), and Hg. janthinomys/capricornii (9.1%) (Culex urichi (52.7%) and Li. pseudomethysticus (36.9%) , with 842 adults obtained from larvae and pupae found in the water of the ovitraps. The most abundant species among the immatures collected with ovitraps were i (9.1%) . These s (36.9%) .Culex (Culex) spp. were the most abundant (82.3%) across all collections of adults. However, the composition of the fauna and abundance changed when analyzing the results of adult collections according to the method used. Culex (Culex) spp. accounted for 92.5% of the total collected with BG-Sentinel with CO2 as an attractant, but only 9.0% of the mosquitoes captured with PHA, where other species were much more abundant, such as Hg. leucocelaenus (21.6%) and Ae. scapularis (12.9%) ( (12.9%) .p = 0.01), with P3 (inside the forest) having the highest abundance of specimens sampled with ovitraps, followed by P8 (situated roughly in the intermediate ecotone between the modified environment and the forest) . Dunn’s post-test indicated that the abundances in the BG-Sentinel at these three points differed from those of the ovitrap paddles and ovitrap water.The abundance of immature forms in ovitraps  was sign forest) . The abu forest) . Samplinp = 0.50) in the abundances between captures made inside the forest (P10 and P11) and the modified environment (P12). However, when analyzing the species composition by sampling site (Adult mosquito collections made with BG-Sentinels had the greatest abundance at P5 (51.5%), located in the forest ecotone , with a ing site , P5, whiing site .Hg. leucocelaenus was more abundant than Hg. janthinomys/capricornii across the study area , there was no significant difference in the abundances of both species between the sampling sites .Although When we analyzed the similarity between the collection points (Sørensen’s similarity (IS)), we found that the highest similarity (IS > 0.81) was between the sites where captures were performed only with PHA . This maHg. leucocelaenus (n = 116), Hg. janthinomys/capricornii (57), and Ae. scapularis (73).A total of 287 adult mosquitoes caught with BG-Sentinel or PHA were screened for YFV, all of which were negative . Almost Greater awareness of the diversity of mosquito species is fundamental for assessing possible changes in their behavior and adaptations according to the different environmental conditions in areas where the environment has been subjected to and/or is undergoing anthropogenic change .In this study, 21 species of mosquitoes belonging to 12 genera were recorded in sites located on a disturbance gradient from the open and modified environment to within the Atlantic Forest at CPRJ. Intriguingly, this figure was considerably lower than the diversity of mosquitoes previously described in sites on the same slope of Serra do Mar. For instance, studies undertaken at Serra dos Órgãos National Park and Guapiaçu Reserve, located around 9 and 20 km from the CPRJ, recorded 44 and 59 mosquito species, respectively ,29.Ae. scapularis illustrated this finding since it was found only at sites where adult collections were performed, whether with BG-Sentinels or PHA. This behavior was also shared by the species of genera Runchomyia and Wyeomyia. The distribution of some species appeared to be affected more by the location of the sampling site, as was the case of the two Sabethes species, which were collected only at sites where captures were carried out with PHA (P10–P12). The use of ovitraps essentially selected species of tribe Aedini, such as Hg. leucocelaenus, Ae. terrens, and Hg. janthinomys/capricornii on the paddles, and Culex urichii and Li. pseudomethysticus in the water held in the ovitraps. Adults of Culex (Culex) spp. were abundant where BG-Sentinels were used (P1–P5) but accounted for only 9.0% of mosquitoes captured using PHA where, in contrast, primary and secondary YFV vectors, such as Ae. scapularis, Hg. janthinomys/capricornii, and Hg. leucocelaenus, were much more frequent. Hg. leucocelaenus represented 21.7% of mosquitoes captured with PHA, followed by Ae. scapularis (12.9%). The latter species was considered a secondary vector of yellow fever during the recent epidemic in Rio de Janeiro [The collection method considerably influenced the results as they related to mosquito species diversity, richness, and abundance across the sampling sites. Species that do not breed in open containers, such as ovitraps, were not collected at points where only this collection method was used (P6–P9). The distribution of  Janeiro , was alr Janeiro ,31, and  Janeiro ,33.Haemagogus species considered to have been the primary YFV vector in RJ and other outbreaks in southeastern and southern Brazil [Interestingly, around 70% of immatures collected with ovitraps, regardless of their location, belonged to the two n Brazil ,38,39,40Hg. leucocelaenus is more abundant than Hg. janthinomys/capricornii. However, there was no difference in the abundance of both species across the sampling sites in the transects. That is, they occurred in similar abundance from the open field and modified environment to 400 m into the forest. This remarkable aspect of the species’ distribution had a major impact on YFV transmission.Altogether, the results of adult and immature collections indicated that Hg. leucocelaenus and Hg. janthinomys/capricornii, as observed in the CPRJ, illustrate how people can become exposed to YFV-infected sylvatic mosquito bites in rural, peri-urban, and even urban areas adjacent to the Atlantic Forest.These two vectors can move for several kilometers , coverinThe failure to detect natural infections in the tested mosquitoes from the CPRJ area was consistent with the local epidemiological data. Our collections started in December 2018 and lasted until December 2019, a period during which no human cases were diagnosed in the entire transmission season from July 2018 to June 2019 across RJ. Moreover, the last suspected infections in NHPs were reported five months earlier (July 2018) and were considered remnants of the previous season. The only and last record of YFV circulation in the state was of a dead howler monkey found approximately 90 km from the CPRJ .Flaviviruses and other arboviruses were reported in captive NHPs born at CPRJ [Our findings on mosquito diversity and spatial distribution at CPRJ, particularly concerning the primary and secondary vectors of YFV, also indicated that the captive NHPs were similarly exposed to their bites and the risk of mosquito-borne agents, including arboviruses, regardless of the location of the outdoor enclosures. The local fauna includes several species indicated as vectors of several arboviruses in Brazil . Antibod at CPRJ . Altoget at CPRJ ,44. The  at CPRJ . The negHg. leucocelaenus, Hg. janthinomys, Ae. scapularis, and Sa. chloropterus, and even the occurrence of the urban vector Ae. aegypti. These findings highlight the need to improve monitoring for the emergence of febrile diseases and vaccine coverage in humans and captive NHPs of endangered species.Considering that the abundance of adult mosquitoes was practically uniform regardless of location and the greatest richness and diversity were recorded when captures were made via PHA, whether inside the forest or the modified environment, we concluded that the study area has a high potential for arbovirus transmission. This is particularly concerning due to the ubiquitous spatial distribution of primary and secondary YFV vector species, such as


The caregivers, being mothers and having at least a secondary education, demonstrated positive attitudes and practices. Meanwhile, more urban caregivers (79.8%) followed a complete malaria treatment course of artemisinin-based combination therapies than the rural (63.3%), but in view of insecticide treated net use, more rural caregivers presented positive attitudes (85.3%) and practices (70.1%) than the urban . (4) Conclusions: For better protection of children under-5 against lethal malaria, it is essential to provide better guidance at the community level for their caregivers, especially young mothers, in order to reduce some misconceptions and inappropriate behaviors. An increase in education and employment opportunities for women, establishment of an accessible community-based malaria counselling service, and construction of an effective communication channel are also needed.(1) Background: Children under 5 years of age are the most vulnerable to malaria infection, and they suffer serious complications. Sierra Leone is one of the countries with the highest malaria burden in the world. This study aimed to assess the knowledge, attitudes, and practices (KAPs) toward malaria control and prevention among caregivers of children under 5 in the Western Area of Sierra Leone. (2) Methods: A cross-sectional survey was conducted among caregivers of children under-5 visiting the out-patient department of six selected hospitals/community health centers. Data were collected via questionnaire interviews with 350 caregivers. (3) Results: A total of 97.1% of the respondents were women and the majority of them were young mothers; 46.3% of respondents were unemployed; and 27.1% received no education. Only 1.4% accessed malaria related information from the internet/social media. This KAPs survey indicated that a misconception of the cause, transmission, and clinical symptoms of malaria; unawareness of its lethality and its severity; and inappropriate prevention and treatment behaviors, such as self-medicating, were still in existence among some caregivers. However, a positive correlation in knowledge–attitudes (r Anopheles mosquitoes. Plasmodium falciparum is responsible for over 90% of malaria cases and all the severe types of the disease in Sierra Leone [Malaria remains globally endemic in 85 countries, and it is mostly concentrated in the underdeveloped tropical and subtropical regions even though its occurrences vary with geographic–climatic environmental conditions and cultural–hygienic social factors ,2. Compara Leone ,3.In the postwar and the post-Ebola periods, malaria control efforts in Sierra Leone are hampered by inadequate capacity due to the country’s weak health care system, the unhealthy hygiene practices of people, and other poverty-related social and environmental conditions. In remote rural areas and in swampy regions, health facilities are less accessible to inhabitants, with consequent devastations to their health due to malaria . AlthougOver the past years, Sierra Leone has made some progress in controlling malaria. Such progress has included the development of a National Malaria Control Programme (NMCP) Strategic Plan 2016–2020 to ensure that the implemented program is evidence-based , the WHOHowever, there still exists such incomplete knowledge and misconceptions as well as misconduct regarding malaria, such as increased exposure to the disease, inappropriate implementation of interventions, and inadequate recognition of the factors contributing to the occurrence and the development of malaria, which probably hinder the correct practices against it . ResearcPrimary caregivers play key roles in the health care of children under-5, being responsible for a majority of the everyday practices and decisions that determine childrens’ health status. A caregiver’s KAPs level toward protecting their children from malaria is essential for seeking appropriate medical care in case of the onset of the disease and administering recommended treatments . Thus, i2 with a total population of approximately 1.5 million people . The Western Area is divided into two districts: Western Area Rural (WAR) and Western Area Urban (WAU).This study was carried out in the Western Area, one of four principal divisions of Sierra Leone. The Western Area comprises the oldest city and the national capital, Freetown, and its surrounding towns and countryside, and it covers an area of 557 kmWe conducted pilot investigations in WAR and WAU for selection of the suitable and the representative survey sites. Consequently, government-owned hospitals or health centers have been selected as they are strategically located within easy reach of the majority of the communities, and they provide free health services for children under-5, and they almost always have a large number of caregivers for children under-5 accessing their healthcare services. Therefore, this study was carried out in six selected health care facilities in two districts, WAU and WAR , three fCaregivers with one or more child(ren) under-5 residing in WAR and WAU who visited the out-patient departments of the selected health care facilities were included.n = sample size; z = z value 1.96 for 95% confidence level; p = proportion of malaria prevalence in children under-5 in Sierra Leone [d = absolute error allowance/precision. Hence, the sample size was calculated as follow: The sample size was calculated using Cochran’s formula (1977) (pp. 72–76) . Where: ra Leone ; d = absBased on the conceptual framework of this study, we developed a questionnaire that included items mainly referred from previous studies, validated and supplemented according to the biological characteristics of malaria epidemics ,19. The The final survey questionnaire contained open and closed-ended questions, and it was divided into four sections . SectionAfter gaining ethical clearance and permission from the Sierra Leone Ethics and Scientific Review Committee, a face-to-face interview with participating caregivers and trained investigators was performed on workdays from mid-August to late-September 2019 using the questionnaire in six selected health care facilities in WAU and WAR.Socio-demographic characteristics and KAPs data were collected via the face-to face interview questionnaire from 350 caregivers. Descriptive statistics was used to analyze the socio-demographic characteristics of the respondents, and the results were expressed in frequencies and percentages. A scoring system was utilized in the evaluation of the KAPs data  10,20,2,220,21. p ≤ 0.25 were included in the multivariate logistic regression model. Reduced subset models were developed using elimination based on the AIC (Akaike information criteria) score. The level of statistical significance was set at 0.05. All of the data analyses and figure drawing were conducted in R software (Version: 3.6.1).The association between KAPs and the socio-demographic characteristics of the respondents was evaluated by univariate analyses. All variables significant in the univariate analysis with a As shown in p = 0.858) was found between the caregivers from WAR and WAU . When asked about the symptoms of malaria, the most common responses were fever (71.1%), followed by vomiting (42.9%), loss of appetite (30.9%), and body and joint pains (26.9%), with only a few responses for headaches (9.1%). Approximately 58.6% of them stated other signs and symptoms they knew such as weakness and dizziness, dark colored urine, yellow/pale/white eyes, etc. Approximately 10.0% did not know that malaria parasites can be transmitted through mosquito bites. This study revealed that misconceptions about the causes and the mode of transmission of malaria still prevailed among the caregivers, as 0.3% of them stated that eating too much and having close contacts with a person who has malaria can cause malaria. Furthermore, 4.0% of them stated other misconceptions about the cause of malaria, such as eating too many oranges, not washing hands regularly, cold and flu, dirty hands and feet, flies, drinking too many sweet/soft drinks, etc. While 86.6% of the caregivers stated that bushes/dirty places were convenient resting/breeding places for mosquitoes, followed by stagnant water (51.1%), and dark places/sheds (11.7%).As displayed in p < 0.001). Explaining their children’s lack of bed nets, 46.2% caregivers mentioned that they could not afford them, 32.1% said they were not readily available, 6.4% did not like sleeping under bed nets, 3.8% thought it was not important, and 11.5% stated other reasons such as the statements that they and their children would have allergic reactions or sweat a lot whenever sleeping under bed nets (As summarized in bed nets . Moreovep < 0.001).As shown in p < 0.01)  . However < 0.01) . Other c < 0.01) .s = 0.13, p < 0.05) and attitudes-practices . Further univariate logistic regression analysis indicated that improved knowledge might positively affect attitudes , while positive attitudes might lead to good practices .Inferred by the Spearman correlation test , a signiThrough univariate analysis, the association of socio-demographic and malaria-related KAPs were demonstrated by comparing with referred groups . The depFurthermore, independent predictors of KAPs were inferred by a multivariate logistic regression analysis comparing referred groups . Age, reIn our study, the respondents in the Western Area in Sierra Leone received malaria related information mainly from health workers/facilities followed by radio as similar to previous studies ,18, whilWith regard to malaria, fever is the highest known symptom followed by other symptoms, such as weakness, dizziness, dark colored urine, etc., which were mostly mentioned by the respondents. Fever was recorded as the most known and common symptom of malaria in children under-5 in Cabo Verde and Zambia ,13. OtheIn the present study, malaria was known as a very serious health problem by 90% of the caregivers, similar to previous surveys in south-western Saudi Arabia and in Rwanda ,31. ThatEvery respondent in this study stated that they employed at least one method of prevention against mosquito bites, with the highest being sleeping under an ITN, followed by using mosquito repellants, and regularly doing clean-up around the house, in accordance with the records in Kenya  and ZimbA good anti-malaria practice is also supported by previous studies ,42, whicAlthough some studies have found that high knowledge about prevention is poorly reflected in practice ,47, someThe majority of the caregivers of children under-5 in this study were young mothers (˂30 year-old), including 33 younger students, and 27.1% of them lacked any educational experience. Financially, most of them were unemployed and underprivileged: even among the 44.3% employed caregivers, some were self-employed, mostly engaged in petty trading and low income businesses from which they could hardly earn enough to meet their basic daily needs. For a relatively immature parent that is socioeconomically handicapped, having the right malaria related KAPs might make up for their deficiency in maturity and their socioeconomic disadvantages . HoweverMoreover, only 1.2% of urban caregivers expressed their unawareness of the lethality of the disease, whereas the number for rural caregivers was over four times higher (5.6%). It is interesting to note that significantly more urban caregivers (79.8%) followed a complete malaria treatment course of ACTs than the rural (63.3%). However, in view of using ITNs for malaria prevention, the rural caregivers (85.3%) presented significantly more positive attitudes than the urban (69.9%), and the rural (70.1%) therefore represented better practices than the urban (52.0%).The present KAPs survey toward malaria prevention and control indicates, to some extent, that misconception of the cause, transmission, and clinical symptoms, unawareness of the lethality and severity, and inappropriate behaviors in prevention and treatment, including self-medicating, are still in existence among caregivers of children under-5 in the Western Area of Sierra Leone. The KAPs toward malaria are affected by caregivers’ educational background, occupational status, parenthood, living region, and religion. Positive correlations in knowledge-attitudes and attitudes-practices are evidently demonstrated. Therefore, in order to better protect children under-5 against severe and deadly malaria, their caregivers, especially young mothers, need such prior supports as increased education and employment opportunities. Meanwhile, there is also a need to set up an accessible community-based counselling service toward malaria and to construct an effective community-based communication channel, especially for caregivers of children under-5. The present study may facilitate the implementation of integrated malaria control strategies, especially dealing with malaria infections in children under-5.Despite its efficacy in enhancing the understanding of KAPs toward malaria, the present cross-sectional survey did not investigate the cause and the effect of malaria in children under-5. Meanwhile, the survey was conducted by self-reporting, which may cause discrepancies between the reported behaviors, participants’ responses, and actual behaviors due to interviewer bias, participant bias, or response bias. Finally, another possible bias may be derived from the selection of the chosen hospitals or health centers.


For volatile compounds extraction and identification, headspace solid-phase micro-extraction (HS-SPME) and GC–MS were used, respectively. A total number of 56 volatile compounds were revealed and identified in oil extracted by SFE, while only 40 compounds were detected in extracted oil by SPP. Acids, aldehydes, esters, ketones, furans, and other components were present in the highest ratio in oil extracted by SFE. In contrast, alcohols and alkenes were found in the highest proportion in oil extracted by SPP. In this study, it was observed that SFE showed an increase in the amounts of volatile compounds and favorably impacted the aroma of gurum seed oil. The results reveal that different extraction methods significantly impact the volatile components of gurum seed oil, and this study can help evaluate the quality of the oil extracted from gurum seeds.In this study, the volatile compound profiles of gurum seed oil were determined using two methods: supercritical CO With the globally growing demand for vegetable oils, researchers’ primary aim is to explore the utility and functional characteristics of by-products from plant sources. Gurum (Citrulluslanatus var. colocynthoide) is a wild kind of watermelon belonging to the Cucurbitaceae family and is abundantly available in African countries ,2. Gurum2 is low in surface tension and viscosity, and has a higher diffusion factor that enhances mass transfer [2 is used in oil extraction due to the complete dissolving of triacylglycerols (TGA), fatty acids, and cholesterol.Numerous conventional methods exist for oil extraction. Among these, solvent (SE) or mechanical extraction (pressing) is considered the main system used for commercial and industrial plant oil production . Supercrtransfer ,10. Resetransfer ,11,12. S2 is cheap, non-toxic, and has a high purity [2, pressure and temperature are crucial factors that may impact the oil’s yield. Again, CO2 reaches a crucial point under mild  conditions in the absence of O2, and it is effective in preserving the bioactive compounds inside [Moreover, COh purity . In comps inside .Plant oils are significant to human life owing to their contribution to providing energy, nutritional components, and exhibiting an appealing range of flavors ,15. FlavProcessing techniques also significantly impact the concentrations of volatiles and are therefore responsible for causing an alteration in plant oil flavors when different techniques are employed ,18. Many2 extraction (SFE) and the screw press process (SPP) for comparison purposes.To the best of our knowledge, no published reports mention the volatile compounds of gurum seed oil. Therefore, the present study is the first to provide the profiles of volatile compounds of gurum seed oil. Thus, the aim of this study is to determine the volatile components in extracted gurum seed oil using headspace solid-phase micro-extraction (HS-SPME), later separated and identified using GC–MS (gas chromatography–mass spectrometry). The present study used the oil extracted by supercritical COThe SFE and SPP techniques were used for the oil extraction from gurum seeds. The identification and quantification of volatile compounds were performed using GC–MS, and data from these analyses are depicted in The presence of acids in food is responsible for taste perceptions, such as cheesy and sour notes, which mainly depend on acetic acid , octanoic acid , and pentanoic acid  ,19,20,21The presence of acetic acid was expected since it is the common volatile compound in most vegetable oil and is formed in the process of the treating the seeds ,23. HoweThe presence of alcohol in food products generally produces alcoholic, fruity, sweet, balmy, and green aroma and sensations. However, these aromas depend on the molecular structure of the alcohol produced ,24. In tThese components played a greater role than other alcohol components in the oil obtained by SPP, especially 1-hexanol. The 1-hexanol compound is an important flavor compound in some vegetables; it produces a green odor, is woody and herbaceous. It is obtained from the bioremediation of UFA (unsaturated fatty acids) and serves as the basis for forming long-chain esters ,25. At tBorneol elevates the numerous medicinal properties of essential oils in the Dipterocarpaceae family . In tradAldehydes in vegetable oils are fundamentally produced by two pathways: the first is the fat oxygenase pathway (which occurs through the cell fragmentation in the oilseeds), and the second is the automatic oxidation pathway (which occurs in the production and storage process) . In thisEster compounds cover a wider spectrum of flavoring and odor effects; thus, they are widely distributed as the main component in fruit and essential oils. Esters provide a floral, fruity, honey, sweet, and flowery perception in food and other products . In addiLower amounts of alkene and alkane compounds are always found in oils . In thisIn our study, alkane components accounted for 1.66% of the total volatiles in oil obtained by SPP. They were mainly cyclopropane, pentyl (0.84%), and undecane, 2, 6-dimethyl (0.68%), while α-cubebene (2.61%) was the only alkene detected in oil obtained by SPP. This study observed that one terpene  was also detected in oil obtained by SPP. Terpenes are naturally-occurring compounds in plants and form part of the essential oils .Ketones are reportedly formed by the β-oxidation of fatty acids (FAs), which produce a few important aromatic compounds ,31. In tThrough the other compounds found in oil from gurum seeds, two compounds were classified in this group . VanilliVolatiles that are studied by humans have a considerably greater effect on the flavor of gurum seed oil. A significant difference was found in the volatile constituent’s oil from gurum seeds obtained by SFE and SPP. For the total volatile compounds studied in this work, a total of 56 volatile components were noted in the oil from gurum seeds by SFE, including 12 acids, 7 alcohols, 12 aldehydes, 8 esters, 7 alkanes, 3 alkenes, 3 ketones and hydroxy ketones, 2 furans, and 2 other compounds. Meanwhile, 40 volatile components were identified in oil from gurum seeds obtained by the SPP, including 7 acids, 18 alcohols, 5 aldehydes, 1 ester, 4 alkanes, 1 alkene, 1 terpene, 1 ketone and hydroxy ketones, and 3 furans, as shown in The results reveal that the oil obtained by SFE could result in the isolation of some characteristic volatile substances. This may explain the great odor differences between the oil obtained by SFE and SPP. Acids, aldehydes, esters, alkanes, ketones, furans, and other components were found to be present in the highest ratios in the oil obtained by SFE compared to SPP b. These Thus, oils obtained by different methods result in rich bioactive metabolites, ensuring a potential application as an antiproliferative and antioxidant agent in the pharmaceutical and food industries. Furthermore, additional studies on the differences in extraction methods and conditions are required, which could present a clearer view of the metabolic pathways connected with flavor formation.2 was employed as a solvent carrier.Gurum seeds were purchased from Dongola city in Sudan and carried to the National Engineering Research Center at Jiangnan University, China. All chemicals used in the experiments were of high purity. High-purity CO2 (SFE) system was obtained through a detailed protocol, as laid out in previous reports [Citrulluslanatus var. Colocynthoideis) seed oil was extracted by the SFE system equipped with Process Suite software  and carefully washed with ethanol. The gurum seed powders were then placed in an extraction vessel and stored for 120 min at 40 °C. The system pressure of 35 MPa was applied with the flow rate of 0.5 L min−1 of solvent . The oil was then collected and put into a vacuum drying oven  under 50 ± 1 °C for 2 h to remove any traces of ethanol. The prepared oils were then stocked at −20 °C until analysis.The oil extracted through the supercritical CO reports . BrieflyAccording to the method described in detail in our previous study , 1000 g The volatile flavor components in gurum seed oil were extracted using headspace solid-phase micro-extraction (HS-SPME), then separated and identified using volatile compounds of gurum seed oil. The gurum seed oil was separated by GC–MS  using a DBWAX column (30 m × 0.25 mm × 0.25 µm) while maintaining the injector at 250 °C for 3 min. To determine these compounds, 5 g of gurum seed oil obtained by different methods was measured and analyzed following a fully described protocol in our previous report [Data collection was performed in triplicate and presented as means ± SD through the processing of Origin-Pro 9.2 . Data comparison was performed using one-way analysis of variance (ANOVA) and Fisher’s multiple comparison tests.The present study first reported the characterization of volatile compounds obtained from gurum seed oil by SFE and SPP that were separated and analyzed using GC–MS. A total of 56 volatile compounds were analyzed and identified in gurum seed oil obtained by SFE, compared to 40 compounds obtained by SPP. Acids, aldehydes, esters, alkanes, ketones, furans, and other components were found to be the highest ratio of compounds in the oil obtained by SFE compared to SPP. Alcohols and alkenes had the highest yield in oil obtained by SPP compared to SFE. These results demonstrate that different extraction methods have a significant impact on the volatile flavor composition and overall quality of gurum seed oil.


Diabetes self-management (DSM) enables maintenance of optimal individualized glycemic control for patients with diabetes through comprehensive lifestyle, medication adherence, and self-monitoring glucose level. This study aimed to evaluate DSM and to find associated factors among Vietnamese diabetes patients by using the Vietnamese version of Diabetes Self-Management Instrument (DSMI).A cross-sectional study was conducted at a single hospital in the central Vietnam. DSM was assessed using the DSMI. The participant’s socio-demographic and clinical features were obtained through face-to-face interviews and medical records. Multivariate linear regression was used to determine independent factors associated with total DSMI.The mean total DSM score based on DSMI self-administered questionnaire scores was 88.4 ± 22.1, with a range of 47 to 140. The mean self-integration, self-regulation, interaction with health professionals, self-monitoring blood glucose, and adherence to the prescribed regime were 24.8, 22.3, 21.6, 10.2, and 9.5, respectively. 48.1% of DM patients had good HbA1c control. Sex, educational status, BMI, waist circumference, medical nutrition therapy, and sufficient physical activities were factors independently predictive of DSMI total score.This study emphasizes that the DSM situation is seen to be average among DM patients with mean DSMI score 88.4 ± 22.1 and sex, educational status, BMI, waist circumference, medical nutrition therapy, and sufficient physical activities were independently predictive factors of DSMI total score. This evidence suggests that there is a need to enhance the effectiveness of DSM education programs among diabetic patients. Globally, diabetes mellitus (DM) is recognized as one of the four major non-communicable diseases besides cardiovascular disease, cancer, and chronic respiratory diseases. According to the International Diabetes Federation, in 2019 463 million people from 20–79 years of age representing 9.3% of the world’s population are living with DM and its prevalence has been increasing annually  which arDiabetes self-management (DSM) has been defined as how people with diabetes practice self-care. It involves the knowledge, attitude, and behaviors to both maintain personal health and prevent long-term diabetes complications . DSM tarBecause DSM and patient-centered care are cornerstones of successful diabetes care, over 30 validated instruments have been developed to investigate its features, prevalence, and related factors which impact DSM. These tools include the Adherence and Self-Management Monitoring Tool (ASMMT) , DiabeteThe Diabetes Self-Management Instrument (DSMI) is one of the validated tools for assessing DSM. The DSMI was developed in 2008 by Lin and his colleagues to evaluate the self-management behaviors of patients with diabetes . It is aIn Vietnam, DM is rising at a rate of about 6.23% every year, and it has become a serious health and economic burden for Vietnamese society . TherefoFrom March 2021 to May 2021, we conducted a cross-sectional study among outpatients at the Center of Endocrinology and Diabetes, Da Nang Family Hospital, Da Nang, Vietnam. Patients with DM were invited to enroll in this study if they met the following inclusion criteria: 1) A diabetes diagnosis per the ADA 2021 criteria for diagnosis made at least 3 months prior to study entry ; 2) voluPotentially eligible participants were identified during clinic visits using convenience sampling at the Center of Endocrinology and Diabetes, Da Nang Family Hospital. Nurses were trained in administration of the DSMI and participants completed the survey during face-to-face visits. A structured questionnaire and the hospital electronic medical records system were used to collect sociodemographic and clinical information.This study was conducted in accordance with the Declaration of Helsinki. The Institutional Review Board of the Danang Family Hospital (number: 71.01–30303) approved the study protocol prior to initiation of study activities. Each participant was informed of the purposes of this study in detail via an information sheet and provided informed consent form if they agreed to join the study. Participants were free to withdraw at any time, without giving any reason for doing so and without affecting their present or future medical treatment. All participant information was kept confidential and used only for study purposes.Socio-demographic variables consisted of age , sex , occupation , family status , living area , and academic background , secondary/high school (grade 6–12), and vocational/college).2, grouped following Asia-Pacific body mass index classifications [Clinical information consisted of the types of DM , duration of living with DM , blood pressure , body mass index  completed the V-DSMI questionnaire. Among non-completers, 20 withdrew because of the COVID-19 pandemic and 10 lacked results of study blood tests.The socio-demographics of the participants are shown in The average HbA1c was 7.6%  and fasting blood glucose levels were 8.0 mmol/l . According to ADA criteria 48.1% had good HbA1c control (HbA1c < 7%) and 49.1% good fasting plasma glucose control (FPG: 4.4–7.2 mmol/l). The prevalence of overweight and obesity, android obesity and dyslipidemia were 67.7%, 33.3%. and 87.0%, respectively.The DSM and its internal consistency are shown in The independently predictive factors of the DSMI total score resulted from multivariate linear regression are shown in Diabetes self-management can help achieve good individualized glycemic control to reduce the risk of diabetes microvascular and macrovascular complications , 37. TheOur study provides insights into the self-management behavior characteristics of patients with DM, the majority of whom have type 2 diabetes, who are receiving primary care in central Vietnam. Our purpose was to examine diabetes self-management and its related factors by using the V-DSMI, a validated diabetes self-management instrument. This study showed that the mean total DSM score based on the V-DSMI self-administered questionnaire was 88.4 ±22.1 and the score of 5 domains got an average of their range. In the cohort of Vietnamese patients with diabetes in this study 48.1% had good HbA1c control (HbA1c <7%) as defined by ADA 2021 criteria for classification of glycemic control. In addition, sex, educational status, BMI, waist circumference, following a medical nutrition therapy plan, and sufficient physical activity were independently predictive factors of DSMI total score.In a comparison of DSMI scores with other regions, our outcomes are in line with the results of a study conducted in China  which shRegarding other factors positively associated with DSM, the current study’ s results reveal an association of occupation and educational level with total DSMI score. This finding may be attributed to a higher educational level translating into better knowledge, attitudes and practices related to prevention and control of DM. Higher educational levels were also associated with better adherence to diabetes medications, medical nutrition therapy, and better interactions with doctors , 44. SimNotably, this study showed that the rate of good glycemic control was 48.1%. While this result is in line with other studies in Vietnam and other countries –47. It dThe multivariate linear regression model results lead to the conclusion that DSMI total score can be predicted through sex, educational status, BMI, waist circumference, medical nutrition therapy, and sufficient physical activity. Sex plays an important role in adherence to self-management. As was the case in this study, female patients have been shown to more frequently engage in DSM, be more focused on self-care, and to search diabetes information more than males in a previous study . AbdominOur study’s strengths include the application of a validated Vietnamese version of the DSMI among a sample of patients with DM in a primary care setting in Central Vietnam  and contribution of more insights into the current status of DSM and its related factors in Vietnamese DM patients. However, there are some limitations in this study. First, a cross-sectional study at a single hospital with convenience sampling may not be generalizable to the whole picture of DSM among Vietnamese patients with DM. The sample size was 78.3% of that anticipated, largely due to the COVID-19 pandemic. We were unable to use the shortened version of the DSMI which includes 20 items because it is currently unavailable in Vietnamese. The short version may be preferable for wider scale future administration, however, use of the full DSMI did allow us to obtain interesting insights into DSM in the present study. Fourth, other related variables which might affect DSM status in people with diabetes, including prior participation in DSM education known to be essential to successfully acquiring DSM skills and knowledge, psychological illness , medical insurance status, isolation status due to the COVID-19 pandemic, and medical treatment costs were not collected in this study. These limitations highlight the need for future research on DSM and the need for diabetes self-management education and support in adults living with DM in Vietnam.The results of this cross-sectional study of the state of DSM among patients with diabetes at a single hospital in central Vietnam demonstrates that the status of diabetes self-management may be classified as average at this time, as reflected in the mean DSMI score of 88.4 ± 22.1. Female sex, higher educational status, higher BMI and waist circumference, following a medical nutrition therapy plan and regular sufficient physical activity were independently predictive factors of DSMI total score. These findings demonstrate a need for improvement in diabetes self-management in the central region of Vietnam. There is clearly a need for further research into strategies to provide diabetes self-management education and support, particularly among those who are male, have a lower educational status and are not following medical nutrition therapy and regular physical activity regimens.S1 Data(XLSX)Click here for additional data file.


Material and Method. Firstly, The Cancer Genome Atlas (TCGA) database was applied to analyze the expressions of HSPA5 in different cancer types, especially in BC. Then, the LinkedOmics database was used to screen genes coexpressed with HSPA5 in BC, presented by protein-protein interaction (PPI) and analyzed by functional enrichment analyses. Next, the Kaplan-Meier plotter was adopted to study the prognostic significance of HSPA5 and the relation between HSPA5 expression and different clinical factors in BC. Finally, the Tumor Immune Estimation Resource (TIMER) method was adopted to explore the relation between immune infiltration and HSPA5 in BC.  Breast cancer (BC) is a frequent disease in females. The heat shock 70 kDa protein 5 (HSPA5) has recently been discovered to have an important function in tumor growth. However, the biological significance of HSPA5 in BC is unknown.  HSPA5 was highly expressed in most cancers, including BC. Genes coexpressed with HSPA5 were mainly related to endoplasmic reticulum unfolded protein response, melanosome, thyroid hormone synthesis, N-glycan biosynthesis, and so on. In the survival analysis, high HSPA5 expression indicated a poor prognosis in BC, and the expression of HSPA5 in BC was elevated after the incidence of BC, changing with different clinical factors. In the immune infiltration, HSPA5 was positively correlated with most immune cells.  HSPA5 is an oncogene in BC progression, and it is connected with the prognosis and the immune infiltration in BC. Our findings suggest that HSPA5 could be an immunotherapy target and a prognostic biomarker in BC. Breast cancer (BC) is a frequent disease in females , with a HSPA5, a member of the heat shock protein 70 (HSP70) family , is the In recent years, bioinformatics  has becoTCGA database contains clinical sample information of more than 20,000 patients and 33 cancers, as well as sequencing data of various omics, such as genome, transcriptome, and epigenetics, which can effectively help 1 better understand the molecular mechanism of cancer. This time, we explored the expressions of HSPA5 in different cancer types based on TCGA database.LinkedOmics is an online analysis tool of TCGA database, including 3 analysis modules: LinkFinder, LinkInterpreter, and LinkCompare. Among them, LinkFinder is convenient for users to search and query information. LinkInterpreter is used to perform rich analysis of Gene Ontology (GO), biological path, network module, and other functional categories. LinkCompare uses visualization functions to compare the correlation results, and supports multigroup analysis of cancer types. Based on the above three modules, we first analyzed the genes related to HSPA5 in BC, the top 50 upregulated and downregulated genes, and the top 200 genes with significance that were displayed by heat maps and protein-protein network (PPI), respectively. Then, GO term and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted on these genes.To verify the relation between HSPA5 and the prognosis of BC patients, we analyzed the effects of different levels of HSPA5 on the probability of relapsed-free survival (RFS) and postprogression survival (PPS) by the Kaplan-Meier plotter. After that, the relation between HSPA5 and pathological parameters  in BC samples was compared in the UALCAN database to explore the therapeutic value of the gene for patients.P < 0.05, the results obtained were meaningful.TIMER is an online tumor immune cell assessment tool based on TCGA, which provides researchers with the infiltration status of 6 immune cells, CD4+ T cells, B cells, CD8+ T cells, macrophages, neutrophils, and dendritic cells. Herein, based on this tool, we conducted a correlation study between 6 immune infiltrating cells and the HSPA5 gene in BC. When Based on TCGA database, we verified the expressions of the HSPA5 in different cancers. According to the results of Figures Through the LinkedOmics database, we identified the positive and negative coexpressed genes associated with HSPA5 , displayP = 5.8e − 05, HR = 1.34, log-rank P = 0.013, To evaluate the clinical value of HSPA5 in BC patients, we used the Kaplan-Meier mapping tool to plot the relationship between differently expressed HSPA5, RPS, and PPS in BC patients. The results demonstrated high expression of HSPA5 that indicated poor RFS , CD8+ T cell , macrophage , neutrophil , and dendritic cell . Besides, the level of HSPA5 was negatively correlated with CD4+ T cell . The finding indicates that HSPA5 might be an immunotherapeutic target in BC.Based on TIMER, we analyzed the relation between the expression of HSPA5 and 6 immune infiltrating cells in BC tissues. The results in http://string-db.org) can critically assess and integrate protein–protein interactions (PPI), including both direct  and indirect  associations. To detect potential relationships among our initial candidate genes, we mapped all the genes to the STRING network and visualized the network using Cytoscape [The Search Tool for the Retrieval of Interacting Genes (STRING) database  dependent BC cells [Herein, we firstly found that the expression of HSPA5 was upregulated in most cancers, including BC. Then, we screened the HSPA5 coexpressed genes, mainly enriched in response to unfolded protein, melanosome, thyroid hormone synthesis, and N-Glycan biosynthesis. Unfolded or improperly folded proteins that accumulate in the endoplasmic reticulum's lumen trigger the unfolded protein response (UPR), a stress reaction (ER). Numerous human illnesses, including cancer, neurological and inflammatory disorders, and metabolism, are mechanistically caused by this unchecked activation. In breast tumors, some evidence suggests that chronic activation of UPR is related to treatment resistance and disease recurrence. It was reported that the role of abnormal UPR activation and overexpression of UPR components were resistant to apoptosis and drug therapy in BC . RuffoloBC cells . At presBC cells , which iIn the survival analysis of HSPA5 in BC, we found the high level of HSPA5 in BC patients led to poor RFS and PPS by the Kaplan-Meier plotter. To investigate whether HSPA5 could be a prognostic biomarker in BC progression, we investigated the relationship between HSPA5 and clinicopathological parameters in TCGA database. It was found the expressions of HSPA5 were differentially expressed in patients with different pathologic stage, age, HER2 status, T stage, N stage, and M stage. From these results, we can conclude that HSPA5 is highly expressed in BC tumor tissues, and the highly expressed HSPA5 has a shorter survival time than the lowly expressed HSPA5. Therefore, HSPA5 has the potential to be a new diagnostic and prognostic biomarker in BC patients.Tumor does not simply contain tumor cells, but it consists of various cells, stromal cells, and immune cells, which constitutes the tumor microenvironment . DiffereIn conclusion, the level of HSPA5 in BC tumor tissues was significantly higher, and its high expression was related to poor PFS and PPS in BC patients. In addition, HSPA5 is positively correlated with most immune infiltration cells. Based on these results, HSPA5 can be used as a potential biomarker for the diagnosis and prognosis and a new target in the immunotherapy of BC.


The relationships between short-term ozone exposure and the acute exacerbations of asthma in adults have not been fully studied. Existing studies commonly ignored the effects of ozone on mild or early asthma exacerbations.To investigate the associations between short-term ozone exposure and asthma exacerbations in Chinese adults.We administered health management for adult asthma patients through the Respiratory Home Platform and required them to monitor their lung function every morning and evening by themselves. Finally, a total of 4,467 patients in 18 Chinese cities were included in the current analyses, with 79,217 pairs of lung function records. The maximum daily 8-h average ozone concentrations were collected from fixed-site air quality monitoring stations. We calculated diurnal peak expiratory flow (PEF) variation using morning and evening measurements of PEF and then defined different severity of asthma exacerbations with diurnal PEF variations >10, 15, and 20%, respectively. A binomial distributed generalized additive mixture model combined with distribution non-linear models was applied to examine the association of ozone with asthma exacerbations. We further conducted stratified analyses by sex, age, season of lung function tests, and region.3 increment in ozone concentration was associated with odds ratios of 1.010 , 1.014 , and 1.017  for asthma exacerbations that were defined by diurnal PEF variation over 10, 15, and 20%, respectively. The associations remained significant after adjusting for other pollutants, and became unstable when using 24-h average ozone concentration. We also found that the associations were relatively stronger in males, those aged 45 years and older, and in the warm season.We found that short-term ozone exposure was independently associated with an elevated risk of asthma exacerbations defined by lung function and the effects could last for about 2 days. At lag 0–2 days, each 10 μg/mOur results suggest that short-term ozone exposure can increase the risk of asthma exacerbations, even in the early stage of exacerbation. Male and older asthma patients may be more vulnerable to ozone air pollution, especially in the warm season. By 2019, ~262 million people worldwide suffered from asthma, one of the most common respiratory diseases, and nearly half a million died from asthma each year , 4. Rese3 in ozone concentration were 1.014 for asthma hospitalization and 1.006 for asthma emergency room visits  believes that lung function measures, such as peak expiratory flow (PEF), are more responsive and reliable than symptoms  in reflecting asthma exacerbation during the acute phase . In addiTherefore, we conducted this multi-city longitudinal study with repeated measurements to investigate the possible associations of short-term ozone exposure with asthma exacerbations defined by individual lung function in Chinese adults. We further assessed whether these associations were modified by sex, age, the season when lung function was tested, and the region where the participants lived.This is a dynamic longitudinal study with repeated measurements in adults with asthma from the Respiratory Home Platform. The entire study began on January 1, 2017 and ended on December 31, 2020. A total of 11,599 physician-diagnosed mildly asthmatic patients registered on this platform and received daily follow-ups for asthma management. The median duration of follow-ups was 31 days. All patients were instructed to use a portable spirometer to consecutively monitor their lung function twice daily. Baseline information on individual characteristics  and residential address was also obtained through the platform. As described previously , 17, we Each registered patient was assigned a portable and smart spirometer  for daily monitoring of their lung function. These devices meet the requirements of American Thoracic Society and European Respiratory Society (ATS/ERS). After receiving technical training from their physicians, all participants were able to use the spirometer correctly to measure their lung function by themselves in strict accordance with the standard procedures . ParticiIn the present study, we downloaded data on PEF from the Respiratory Home Platform and calculated the diurnal PEF variation according to the following equation :m and PEFe refer to the highest PEF value measured in the morning and the evening on the same day, respectively.where PEFWe then defined asthma exacerbation based on the calculated diurnal PEF variation. In many previous studies, diurnal PEF variation has been used to indicate the severity of asthma , 19. TheAmbient ozone concentrations were measured by the state-controlled air quality monitoring stations in real-time and were uploaded on the China's National Urban Air Quality Real-time Publishing Platform. All monitoring stations are located far away from buildings, factories, and major traffic roads, and thus are not affected by local pollution sources and can truly reflect ambient ozone levels in the city. We obtained hourly ozone data from this platform and assigned data from the monitoring station nearest to the participants' residential address (median distance of 3.3 km) to the corresponding participants. Given that the diurnal PEF variation reflected the variability of lung function throughout the day, we used the maximum daily 8-h average concentration as a daily exposure metric for ozone, which was widely used in epidemiological studies , 22. We 2.5), nitrogen dioxide (NO2), sulfur dioxide (SO2), and carbon monoxide (CO). We calculated 24-h average concentrations for these pollutants. Meteorological data were obtained from the China Meteorological Data Sharing Service, including 24-h average temperature and relative humidity. In statistical analyses, we excluded outliers of the meteorological data and pollutant data when they are greater than (mean + 3 × standard deviation) or less than (mean – 3 × standard deviation).We also obtained other air pollutants data from the platform, including particulate matter with an aerodynamic diameter <2.5 μm  models with binomial distribution were employed to estimate the associations of ozone exposure with asthma exacerbation. In the main analysis, we introduced three dichotomous variables of asthma exacerbation defined by diurnal PEF variation as a dependent variable, separately. Participants' ID number was included as a random effect intercept. We fitted a distributed non-linear model (DLNM) to generate a cross-basis matrix of ozone, and then incorporated it in the GAM models as an independent variable. The DLNM has an advantage of exploring the associations between ozone and asthma exacerbation along two dimensions of exposure variation and lag, simultaneously. Specifically, we used a linear function to characterize the exposure-response dimension, since most previous studies have demonstrated the linearity of the health effects of ozone –25, and 2.5, NO2, SO2, and CO. Second, we controlled for temperature and relative humidity using alternative lag periods, including the average values on the present day of lung function tests (lag 0 day) and moving averages from the present day to the previous 3 days (lag 0–3 days). Third, to examine whether our findings were affected by the choice of exposure metrics, we replaced the maximum daily 8-h average ozone concentration with the 24-h average concentration in the main models.We performed three sensitivity analyses to test the robustness of our results. First, as in previous environmental epidemiological studies, we fitted two-pollutant models to additionally control for possible confounding by PMThen, we performed subgroup analyses by sex , age (18–44 and 45–88 years), season of lung function tests (warm and cool), and region (southern China or northern China) to examine their potential effect modifications. We then assessed the statistical significance of between-group differences in the associations of ozone with asthma exacerbation by calculating their 95% confidence intervals (CIs), as in previous studies , 26. In Last, we plotted the exposure-response curves between ozone and asthma exacerbation. To fully capture the possible non-linear associations, we used natural cubic splines with 2 degrees of freedom for both lag-response dimension and exposure-response dimension in the DLNM. Other parameters in the main model remained unchanged.p < 0.05 were regarded statistically significant. The results were shown as odds ratios (ORs) of asthma exacerbation and their 95% CIs in relation to a 10 μg/m3 increase in ambient ozone concentration.All statistical analyses were conducted in R software (Version 4.3.1). All tests were two-sided and 2 . Of the 4,467 patients, 59.7% were female. A total of 24.4, 13.6, and 8.2% of lung function records met the broad, moderate, and strict definitions of asthma exacerbation in this study, i.e., diurnal PEF variation of more than 10, 15 and 20%, respectively. The proportions of records with diurnal PEF variation >10, 15 and 20% were similar between males and females. The proportions of the three indicators were larger in the people aged between 45 and 88 years than in the people aged between 18 and 44 years . The concentration of ozone was higher in the warm season than in the cool season, and higher in the southern China than in the northern China , 1.014 , and 1.017  for asthma exacerbations that were defined by diurnal PEF variation over 10, 15, and 20%, respectively .However, our study still has some limitations. First, our study failed to distinguish between patients with a single asthmatic condition and those with asthma combined with other diseases , who may have a different response to short-term ozone exposure . Second,Our results suggest that short-term ozone exposure can increase the risk of asthma exacerbations, even when the exacerbation was in the early stage, and also suggest that male and older asthma patients may be more sensitive to ozone air pollution. The effect of ozone on asthma exacerbation was more evident in the warm season. These findings will have important implications for the health management of asthma patients, especially for vulnerable populations in the warm season.niuy@fudan.edu.cn.The datasets presented in this article are not readily available because individual data after deidentification only will be made available to researchers whose proposed use of the data has been approved by an independent review committee. Requests to access the datasets should be directed to YN, The studies involving human participants were reviewed and approved by the Institutional Review Board in School of Public Health, Fudan University, reviewed and approved the study protocol (IRB#2021-04-0889). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin.XF: data curation, formal analysis, writing—original draft preparation, and visualization. SH: project administration, investigation, and data curation. YZ: methodology, investigation, and data curation. JL: methodology and data curation. YX: conceptualization and methodology. YN: conceptualization, writing—reviewing and editing, and funding acquisition. RC: conceptualization, supervision, and funding acquisition. All authors read and approved the final manuscript.


This study aimed to observe the application value of dezocine and ketorolac tromethamine in patient-controlled intravenous analgesia (PCIA) of patients undergoing laparoscopic cholecystectomy (LC).n = 77) and group B (n = 77). Group A was given dezocine and group B was given ketorolac tromethamine. The analgesia, sedation, comfort, and adverse reactions of the two groups were closely observed at 4, 8, 12, and 24 h after surgery.A total of 154 patients who underwent LC surgery in our hospital and received PCIA after surgery from September 2020 to September 2021 were selected, they were divided into group A (P < 0.05). At 4, 8, 12, and 24 h after surgery, the Ramsay scores in group B were higher than those in group A (P < 0.05). At 4, 8, 12, and 24 h after surgery, there was no significant difference in Bruggrmann comfort scale scores between the two groups (P > 0.05). There was no significant difference in the incidence of adverse reactions between the two groups (P > 0.05).At 4, 8, 12, and 24 h after surgery, the visual analog scale scores in group B were lower than those in group A (Both dezocine and ketorolac tromethamine have high clinical application value in patients who underwent LC surgery and received PCIA, with higher patient comfort and fewer adverse reactions. But compared with dezocine, ketorolac tromethamine can achieve better sedative and analgesic effects, which is worthy of clinical promotion. Laparoscopic cholecystectomy (LC) has the advantages of short surgery time, good surgical vision, less trauma, less blood loss, easy recovery, and low infection rate, among others. It has been favored and accepted by clinicians and patients and has become the routine surgery of cholecystectomy . HoweverRelieving postoperative pain of patients undergoing LC is one of the important tasks for clinicians. At present, patient-controlled intravenous analgesia (PCIA) after LC has been widely used in clinics. PCIA refers to when patients feel pain, they can adjust the timing and dosage of intravenous injection of painkillers according to their needs, so as to meet the patient's analgesic needs . This meIn recent years, a variety of analgesic drugs have been continuously and widely developed in the PCIA field. Dezocine and tromethamine ketorolac are both favored for PCIA in clinics. Both of them have certain analgesic effects, but there is limited clinical research on the difference of analgesic effects between these two analgesic methods. In this study, 154 patients who underwent LC surgery and were given PCIA after surgery were selected as the research object. We investigated the analgesic and sedative effects, comfort, and safety of dezocine and tromethamine ketorolac for PCIA.n = 77) and group B (n = 77). (1) Inclusion criteria were: compliance with surgical indications; normal mental and cognitive function; and complete case data. (2) Exclusion criteria were: already received second surgery; used drugs within 24 h of surgery that could affect the efficacy of the study drug; important organ dysfunction; drug dependent and/or drug addict; with severe immune system diseases; with severe hypertension and diabetes; and allergic to research drugs.In this study, 154 patients who underwent LC surgery in our hospital and received PCIA after surgery from September 2020 to September 2021 were selected, they were divided into group A  Analgesia: the visual analog scale (VAS) score was used. In brief, we took a 10 cm scale ruler and asked the patient to mark a scale that can represent their pain degree on the top using a scale of 10 points, with 0 point being painless and 10 points being severe pain. VAS score was assessed by medical staff who were unaware of the patient's condition. (2) Sedation: the Ramsay score was used with the following indicators: 1 point: Sober but anxious; 2 points: Quiet cooperation; 3 points: Sleepy but awake; 4 points: Shallow sleep could wake up; 5 points: Deep sleep but slow response; and 6 points: Deep sleep, no response to call. (3) Comfort: the Bruggrmann comfort scale (BCS) was used with the following scores: 0 point: Persistent pain; 1 point: Painless when quiet, heavy pain when breathing deeply or coughing; 2 points: Painless when quiet, slightly painful when breathing deeply or coughing; 3 points: Painless breathing; 4 points: Cough or other actions are painless; and 5-6 points: Excessive sedation. (4) Adverse reactions were recorded whether there were any adverse reactions such as nausea, vomiting, dizziness, dry mouth, skin pruritus, and urinary retention within 3 days after surgery in both groups.SD. Paired sample t-test was used for intra-group comparison, while an independent-sample t-test was used for inter-group comparison. The counting data were expressed by %, and the χ2 test was used for comparison. P < 0.05, the difference was statistically significant.For statistical analysis, SPSS 22  was used for data processing. The measurement data conforming to the normal distribution were expressed as mean ± P > 0.05; There was no significant difference in gender, age, intraoperative blood loss, and surgery time between the two groups (P < 0.05; At 4, 8, 12, and 24 h after surgery, the VAS scores in group B were lower than those in group A (P < 0.05; At 4, 8, 12, and 24 h after surgery, the Ramsay scores in group B were higher than those in group A (P > 0.05; At 4, 8, 12, and 24 h after surgery, there was no significant difference in BCS scores between the two groups (P > 0.05; There was no significant difference in the incidence of adverse reactions between the two groups (2 pneumoperitoneum, patients after LC are prone to pain symptoms, which affects their physical and mental health and prognosis recovery (Laparoscopic cholecystectomy surgery has a significant positive effect as an effective method for the clinical treatment of cholecystitis, cholecystolithiasis, and other gallbladder diseases . Howeverrecovery . Postoperecovery , 11. TheDezocine is a potent opioid analgesic with both exciting and antagonistic effects . On the Ketorolac tromethamine is a non-steroidal anti-inflammatory drug with the best analgesic effect at present, and its analgesic effect is 7.5 times that of morphine, which is suitable for short-term treatment of acute moderate, and severe pain . It has The commonly used multimodal analgesia in clinical practice is sufentanil combined with dezocine. Sufentanil has a longer sedative effect time and less inhibitory effect on respiratory function. Sufentanil has strong lipophilicity and a higher rate of passing through the blood-brain barrier, thereby increasing the binding rate to plasma proteins, subsequently achieving an analgesic effect. The results showed that within 24 h after surgery, the VAS score of group B was lower, the Ramsay score of this group was higher, and there was no significant difference in BCS score and incidence of adverse reactions between group A and group B. We believe that both dezocine and ketorolac tromethamine have high clinical application value in patients who underwent LC surgery and received PCIA, with higher patient comfort and less adverse reactions. But compared with dezocine, ketorolac tromethamine can achieve better sedative and analgesic effects. At present, the common clinical adverse reactions are nausea and vomiting, and serotonin receptor blockers and glucocorticoids are the first-line drugs to deal with nausea and vomiting. If both are ineffective, droperidol can be used. In addition, other possible adverse reactions include dizziness, dry mouth, skin pruritus, and urinary retention, which generally do not require special treatment. It is worth mentioning that there are still some shortcomings in this study, such as the following: the scheme is a single-center study, and hence multi-center research needs to be carried out in the follow-up study. The infusion rates of both groups were 2 ml/h, and only one PCIA infusion rate was used. The PCIA infusion rate should be adjusted individually according to the needs of patients. The doses of dezocine and ketorolac tromethamine have not been classified hierarchically, and we still need to improve the research .Both dezocine and ketorolac tromethamine have high clinical application value in patients who underwent LC surgery and received PCIA, with higher patient comfort and fewer adverse reactions. However, compared with dezocine, ketorolac tromethamine can achieve better sedative and analgesic effects, which is worthy of clinical promotion.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author.The studies involving human participants were reviewed and approved by the Ethics Committee of the Second Affiliated Hospital of South China. The patients/participants provided their written informed consent to participate in this study.YY is the mainly responsible for the writing of the article. SF is mainly responsible for research design. ZZ and XQ are mainly responsible for data analysis. ZC is responsible for the guidance of the entire research. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.