In this retrospective cohort study, we examined whether the concentration of indoor PM2.5 affected pregnancy outcomes. Additionally, we evaluated biomarkers of pregnancy-related complications caused by fine dust. We collected clinical information and data based on residential addresses from the Air Korea database to assess PM2.5 exposure levels. As a multicenter prospective cohort study, we measured the indoor PM2.5 concentration and inflammatory and oxidative stress markers. The PM2.5 concentration of the low-birth-weight (LBW) delivery group was 27.21 μg/m3, which was significantly higher than that of the normal-birth-weight (NBW) group (26.23 μg/m3) (p = 0.02). When the newborns were divided by sex, the PM2.5 concentration of the LBW group was 27.89 μg/m3 in male infants, which was significantly higher than that of the NBW group (26.26 μg/m3) (p = 0.01). In the prospective study, 8-hydroxy-2-deoxyguanosine significantly increased in the high-concentration group ; in the high-concentration group, the rates of preterm birth (PTB) and small size for gestational age significantly increased . This study showed an association between PM2.5, oxidative stress, and fetal growth, with the PTB group being more vulnerable.Particulate matter 2.5 (PM Low birth weight , small for gestational age , and preterm birth  are complications of pregnancy that directly affect the prognosis of newborns ,5,6,7. T2.5, particles with an aerodynamic diameter of ≤2.5 μm), one of the major air pollutants, has been reported to be associated with various adverse pregnancy outcomes [2.5 and LBW, PTB, and SGA infants have been reported [2.5 and pregnancy outcomes [Particulate matter (PM) 2.5  modeling system. PM2.5, from CMAQ modeling data, was estimated using meteorological research and forecasting models comprising three overlapping weather data sources at 3, 9, and 27 km for a specific time period. PM2.5 exposure levels during pregnancy were determined based on residential address . For each address, daily PM2.5 concentrations were matched to each individual according to their delivery date. Each individual’s outdoor air quality data were collected for each pregnancy trimester, extending to data from the week of delivery. PM2.5 data were collected from 2009 to 2015, depending on the pregnancy period of the study participants. The first study was a hospital-based retrospective cohort study of 1880 pregnant women who delivered live babies between 2010 and 2015 at the Ewha Womans University Mokdong Hospital (EUMC 2020-07-043). We collected information on maternal age; body mass index (BMI); gestational age at birth (GAB); neonatal sex; weight and height at birth; appearance, pulse, grimace, activity, and respiration (APGAR) score; and place of residence at the time of delivery. The Air Korea database was used for PMt-test (We stratified the groups by birth weight and height and performed Student’s t-test a. LBW waA multicenter prospective cohort study of air pollution in pregnant women (APPO) was conducted to investigate the effects of PM on mothers and fetuses by recruiting > 1200 participants between January 2021 and December 2023 in seven university hospitals across Korea. Each hospital was located in a metropolitan area, an industrial complex, or a mountainous area. The participants were singleton pregnant women with no underlying diseases before 28 weeks of gestation. In the second study, 333 women who had delivered were selected as participants b. We colThis study was approved by the Ethical Research Committee of Ewha Womans University Mokdong Hospital (EUMC 2021-04-032), Yonsei University Severance Hospital (4-2021-0414), Kangwon National University Hospital (KNUH-B-2021-04-012-008), Keimyung University Dongsan Medical Center (2021-04-073), Korea University Guro Hospital (2021GR0233), Ewha Womans University Seoul Hospital (2021-04-022), and Ulsan University Hospital (2022-04-020). All participants provided written informed consent.2.5 concentration was measured by placing a fine dust meter at breathing height in the living room of a pregnant woman’s house. An AirGuard K  instrument was used for measurements using the sensor-based method. The indoor PM2.5 was measured online at 1-min intervals. The measured indoor PM2.5 data were stored in an indoor air quality monitoring platform (IAQ Station) using Long-Term Evolution. Measurements were performed for at least 1 week, and the measured concentration values were recorded in real time using the Internet of Things and Information and Communication Technology. For indoor PM2.5, after removing outliers, the average value of measurements for each trimester of pregnancy was derived, and the average of the two or three trimesters was defined as PM2.5. The participants were divided according to the PM2.5 exposure level as follows: <10 µg/m3 (low PM2.5 group) and ≥10 µg/m3 (high PM2.5 group) concentration groups, which are the World Health Organization’s annual limit standard concentrations .The indoor PM2.5 concentrations were collected from a nearby urban atmospheric measurement network based on the residential addresses of the study participants. The Urban Air Monitoring Station data used in this study were obtained from the Air Korea database (https://www.airkorea.or.kr/web (accessed on 1 May 2021)) of the Korean Ministry of the Environment. Outdoor PMWe collected 5 mL maternal venous blood and 15 mL urine. Blood and urine samples were collected at regular follow-up visits. Whole blood samples collected in ethylenediaminetetraacetic acid tubes were transferred to cryotubes, and urine was stored in one cryotube. Samples were transferred to the institution  on the same day as collection under refrigeration to prevent deterioration (−80 °C). The oxidative stress markers 8-hydroxy-2-deoxyguanosine (8-OHdG) and malondialdehyde (MDA) were measured in urine samples collected during the second trimester of pregnancy. The collected urine samples were transported directly to the laboratory and stored at −80 °C before processing. The urine samples were filtered with a 0.2-µm filter  for the assay and diluted at 1:20 to measure the concentration of 8-OHdG using enzyme-linked immunosorbent assay (ELISA) kits . The concentration of MDA in urine was measured using an MDA ELISA kit . Among the inflammatory markers, the WBC count was measured using an XN-9000  according to the manufacturer’s protocol, from the participant’s whole blood sample, through an automated complete blood cell count. The hs-CRP level was measured using a particle-enhanced immunoturbidimetric assay according to the manufacturer’s protocol using a Cobas 8000 C702 analyzer . t-test or Mann–Whitney U test. Logistic regression model was used to estimate the associations between LBW and PM2.5 exposure (1 μg/m3) in each trimester with adjustment for covariates . Statistical significance was defined as p < 0.05. All statistical analyses were performed using the Statistical Package for the Social Sciences . Clinical characteristics were analyzed according to continuous variables (age and BMI) and categorical variables . Pregnancy outcomes were analyzed according to continuous  and categorical variables . Pregnancy complications were defined using the following criteria  and analyzed as categorical variables. Oxidative stress (8-OHdG and MDA) and inflammatory markers (hs-CRP and WBC count) were analyzed as continuous variables. Categorical variables were expressed as frequencies (percentages) and analyzed using chi-square and Fisher’s exact tests. Continuous variables were expressed as means ± standard deviations (SDs) and were compared using the p < 0.01); however, there were no significant differences in maternal age, pre-pregnancy BMI, and neonatal sex , but there was no significant difference in female infants  and 311 had LBW. The two groups showed significant differences in GAB, birth weight, height, and APGAR scores  and preterm low birth weight (PLBW) groups showed significant differences between the first and second trimesters, respectively , with more significant differences in the male group  of 1.06   a. The cop = 0.02, r2 = 0.010)   b. There = 0.010) . There w= 0.010) . The mea = 0.01) .2.5 and LBW was confirmed through a retrospective cohort study, and its effect was found to be more pronounced in the male and PTB groups. Furthermore, a multicenter prospective cohort study revealed the relationship between PM2.5 and birth height, SGA, and PTB by measuring the actual indoor PM2.5. The oxidative stress marker 8-OHdG, which was used to explore the pathogenesis of these pregnancy complications and identify biomarkers, showed a positive correlation with PM2.5. In this study, the association between PM2.5 and fetal growth and PTB [2.5 affects fetal growth, the effects of oxidative stress, placental inflammation or dysfunction, endothelial dysfunction, and blood coagulation have been reported [2.5 measurement. Our results are similar to those of previous studies that showed an association between PM and PTB ,28,29,30reported ,19. The reported ,32. Manyreported ,11,13, w2.5 remains controversial [2.5, especially in male infants. To understand the sex differences, it is necessary to understand the biological mechanisms underlying PM2.5 [2.5 actually affects fetal growth to continuously recruit participants in the future. Several studies have shown that male fetuses are vulnerable to intrauterine growth restriction ,33 and Poversial ,16,33, tng PM2.5 . Male feng PM2.5 . Anotherng PM2.5 , which ang PM2.5 ,38. The 2.5 and environmental pollutants and oxidative stress, this study showed a relationship between PM2.5 and 8-OHdG levels [2.5 exposure. In particular, urine samples have the advantage that they can be collected from patients non-invasively, and since 8-OHdG shows high stability in urine, it can be used as a biomarker [Similar to previous studies showing the relationships between PMG levels . In brieG levels . The resiomarker . By veriiomarker . PM not iomarker ,43. 2.5 measurements were performed in the residences of pregnant women for at least 1 week, and it is thought that this can give causality to the biological mechanism of any relationship between PM2.5 and a disease. The results of this study, using correlation analysis between indoor and outdoor PM2.5, explain that outdoor conditions significantly affect indoor air quality; however, the correlation can be explained by only 18.7% influence. This indicates that the indoor air quality can be affected by various human activities, including smoking and cooking [2.5 increases in preparation for actual outdoor PM2.5. We plan to conduct further research on active intervention measures to prevent pregnancy complications. Most existing studies have estimated fine dust exposure concentrations based on differences in air quality by region and location ,11,28,44 cooking ,17, ther2.5 concentrations between Cohorts I and II, either because of the air pollution reduction effect owing to the COVID-19 lockdown [In this study, there was a difference in PMlockdown  or becau2.5 concentrations. It was also the first study to measure oxidative stress and develop fine dust exposure evaluation indicators in pregnant women with actual indoor PM2.5 measurement.To our knowledge, this was the first study to examine the relationship between fine dust and fetal growth in pregnant Korean women with continuously measured indoor PMThe strength of this study was that it was a prospective multicenter cohort study that investigated the maternal and fetal health effects of PM on pregnancy in patients from various regions of South Korea. Compared with a previous study that measured only outdoor data, it was more reasonable to confirm the causal relationship between fine dust and pregnancy complications through fine dust concentrations measured using individual indoor air quality values. 1.0, and their health effects; however, this study focused on PM2.5 and did not analyze other air pollutants.However, this study had several limitations. Although we measured the indoor fine dust concentrations of the participants for at least 1 week, there was a limitation in that the cumulative concentration during the entire pregnancy could not be calculated. Moreover, other stressful conditions that may affect pregnancy complications, including alcohol abuse and infection, were not considered. Recent, various studies have reported on air pollutants, including PM2.5 and 8-OHdG was observed, but no correlation with actual complications was found. Therefore, in the future, we will recruit more participants and make efforts to calculate the cumulative concentrations of fine dust in pregnant women and evaluate the biological mechanisms of 8-OHdG as a biomarker. Moreover, various stressful conditions that can be confounding variables in pregnancy outcomes should be included, and the impact of other air pollutants should be considered during the analysis.A correlation between PM2.5 and LBW through a retrospective cohort study, and its effect was found to be more pronounced in the male and PTB groups. Furthermore, through a multicenter prospective cohort study, we determined the relationships between PM2.5 and birth height, SGA, and PTB by measuring the actual indoor PM2.5. The oxidative stress marker showed a positive correlation with PM2.5, suggesting its potential as a biomarker for PM2.5 exposure. However, further research on the actual mechanisms of action, efficacy, and relevance to pregnancy outcomes is needed. Furthermore, we will continue to identify interventions to lower indoor PM levels.This study confirmed the association between PM


Glossina species from Cameroon, Chad and Nigeria.Tsetse flies are cyclical vectors of African trypanosomiasis. They have established symbiotic associations with different bacteria, which influence certain aspects of their physiology. The vector competence of tsetse flies for different trypanosome species is highly variable and is suggested to be affected by various factors, amongst which are bacterial endosymbionts. Symbiotic interactions may provide an avenue for the disease control. The current study provided the prevalence of 3 tsetse symbionts in Sodalis glossinidius, Spiroplasma sp and Wolbachia using specific primers. A total of 848 tsetse samples were analysed: Glossina morsitans submorsitans (47.52%), Glossina palpalis palpalis (37.26%), Glossina fuscipes fuscipes (9.08%) and Glossina tachinoides (6.13%). Only 95 (11.20%) were infected with at least one of the 3 symbionts. Among the infected, 6 (6.31%) were carrying mixed infection (Wolbachia and Spiroplasma). The overall symbiont prevalence was 0.88%, 3.66% and 11.00% respectively, for Sodalis, Spiroplasma and Wolbachia. Prevalence varied between countries and tsetse species. No Spiroplasma was detected in samples from Cameroon and no Sodalis was found in samples from Nigeria.Tsetse flies were collected from five different locations and dissected. DNA was extracted and polymerase chain reaction PCR was used to detect the presence of Spiroplasma in tsetse in Chad and Nigeria. These findings provide useful information to the repertoire of bacterial flora of tsetse flies and incite to more investigations to understand their implication in the vector competence of tsetse flies.The present study revealed for the first time, the presence of infection by  Trypanosoma. The disease exists in two forms: The human African trypanosomiasis or sleeping sickness and the animal African trypanosomiasis. In humans, the disease is caused by two subspecies of Trypanosoma brucei: Trypanosoma brucei rhodesiense responsible for the acute form of the disease in eastern and southern Africa, while Trypanosoma brucei gambiense is responsible for the chronic form of the disease in western and central Africa [2 are still at risk of T.b. gambiense infection [Trypanosoma congolense, Trypanosoma vivax, Trypanosoma simiae, Trypanosoma uniforme, Trypanosoma godfreyi, Trypanosoma brucei brucei and Trypanosoma grayi. They cause pathogenic infections in cattle, sheep, goats, pigs, dogs, camels and horses [Trypanosomiasis is one of the major endemic diseases in sub-Saharan Africa. It is caused by trypanosomes, an extracellular flagellated protozoan parasite of the genus l Africa . Approxinfection . The anid horses -5. The dPalpalis, the savannah Morsitans and the forest Fusca [Trypanosomes are cyclically transmitted between different vertebrate hosts by tsetse flies (Diptera: Glossinidae). Thirty-one species and subspecies of tsetse flies have been described. They can be grouped into three groups or subgenera based on common characteristics and morphology due to bio-ecological and genetic similarities: the riverine st Fusca , 7. Theyst Fusca .Tsetse gut harbours a diversity of bacteria acquired from the environment or maternally transmitted . PreviouWigglesworthia glossinidia, Sodalis glossinidius, Wolbachia sp, and Spiroplasma sp that was recently established as the fourth tsetse symbiont in G. fuscipes fuscipes, G. tachinoides, and G. palpalis palpalis [Tsetse flies have established long-term associations with four vertically transmitted endosymbiotic bacteria including palpalis . They shWiggleworthia glossinidia, the primary and obligate endosymbiont. It resides intracellularly within the bacteriome in the anterior midgut and also found extracellularly in milk gland of the fly [Wiggleworthia glossinidia provides dietary supplements absent from the fly’s vertebrate blood-restricted diet, supports larval development and contributes to maturation of the adult immune system [All tsetse flies house  the fly . Wigglewe system , 14.Wolbachia endosymbionts are obligatory intracellular bacteria belonging to the Order Rickettsiales. Wolbachia infects a broad range of arthropod and filarial nematode species and is probably the most prevalent endosymbiont found in insect germlines [Wolbachia, 17 supergroups (A to Q) are currently recognized based on sequences of the five conserved genes fbpA, coxA, ftsZ, gatB, coxA, and hcpA and the amino acid sequences of the four hypervariable regions of the Wsp protein [ermlines . Within  protein , 15. The protein .Wolbachia resides mainly in reproductive tissues and is maternally transmitted from generation to generation by trans-ovarian transmission. They are also transferred horizontally among arthropods. Wolbachia infection in the tsetse host results in a variety of reproductive abnormalities such as parthenogenesis, male killing, feminization and cytoplasmic incompatibility [In tsetse, tibility -18.Wolbachia infection status: when an infected male mates with an uninfected female or a female infected with a different strain of the bacterium [Glossina morsitans has been associated with the induction of cytoplasmic incompatibility [Cytoplasmic incompatibility results in embryonic mortality in the progeny derived from matings between insects with different acterium . The pretibility . This efWolbachia infections limited mosquito-transmitted pathogens including dengue virus, chikungunya virus, Plasmodium parasites, yellow fever virus, Zika virus and filarial nematodes [Some studies have shown that ematodes -22.Sodalis glossinidius. It is a gramnegative organism belonging to the Enterobacteriaceae family. It is widely spread in numerous tissues of the fly  and can be found both intracellularly and extracellularly [Sodalis genome consists of one circular chromosome of 4.17 Mbp, three extrachromosomal plasmids designated pSG1, pSG2, and pSG3, as well as a phage, ФSG1. However, its genome sequence shows reduced coding capacity with a large number of pseudogenes [Sodalis glossinidius can be transmitted maternally via haemolymph, milk gland secretions, and horizontally during mating [The tsetse’s secondary and facultative symbiont is the commensal llularly . The Sodudogenes . Sodalisg mating , 25. In g mating .Spiroplasma belongs to the Mollicutes class, and the Tenericutes phylum. Spiroplasma are found abundantly in insects either in the gut or haemolymph where they have developed a large variety of interactions with the host that can be classified as commensal, pathogenic or mutualist [The genus utualist .Spiroplasma confers protection against pathogens e.g Drosophila neotestacea from a nematode [Drosophila hydei against a parasitoid wasp Leptopilina heterotoma [Spiroplasma [It has been shown that nematode , the peanematode  and Drosterotoma . Howeverroplasma .Spiroplasma has been established as a new class of tsetse symbiont in G. fuscipes fuscipes, G. tachinoides, and G. palpalis palpalis. The interactions between Spiroplasma and Glossina seem to be beneficial because of its ability to extend lifespan and reduce the vector competence for Trypanosoma [Recently, panosoma , 30.Current control measures against trypanosomiasis are mainly based on chemotherapy. In the absence of effective vaccine and to address the limitations associated with chemotherapy, disruption of trypanosomes transmission through vector control is crucial. Transmission of pathogens by vector depends on its vector competence, which can be affected by several factors, including vector endosymbionts . Due to Sodalis, Spiroplasma and Wolbachia in wild population of tsetse flies from Cameroon, Nigeria and Chad.The present study aims to investigate the presence of Glossina morsitans submorsitans: 403 (47.52%); Glossina palpalis palpalis: 316 (37.26%); Glossina fuscipes fuscipes: 77 (9.08%); Glossina tachinoides: 52 (6.13%)). For this study, 848 tsetse fly samples from three countries  were used. They belonged to four Glossina species (Wolbachia-Spiroplasma)Out of 848 flies, 95 (11.20%) were infected with at least one of the 3 endosymbionts. Among the infected, 6 (6.31%) were carrying mixed infection , Chad (259) and Nigeria (270) using a Sodalis Hemolysin gene-based PCR. The Sodalis infection rates were 2.01% in Cameroon, 1.16% in Chad and 0.0% in Nigeria  with other tic tree  showed tSpiroplasma using a 16S rRNA-based PCR approach with wspecF/wspecR primers. Only 31 samples were positive (3.66%). The prevalence varied significantly (p = 0.00) between countries , Chad (423) and Nigeria (270) were used for the molecular detection of ountries . SpiroplSpiroplasma 16S rRNA partial gene sequences found in NCBI database. The Maximum Likelihood phylogenetic tree (The sequence of our amplicons showed a high identity (> 99%) with other tic tree  showed tWolbachia with a 16S rRNA-based PCR approach using the wspecF/wspecR set of primers. The samples were collected in 3 countries . The results of the screening  between the three countries. The Wolbachia prevalence in Cameroon, Chad and Nigeria was 9.68%, 7.00% and 14.07% respectively.A total of 582 tsetse samples (gut or abdomen) were screened for the presence of creening  showed tWolbachia 16S rRNA partial gene sequences found in NCBI database. The Maximum Likelihood phylogenetic tree (The sequence of our amplicons showed a high identity (> 99%) with other tic tree  showed tOQ448931 to OQ448937 and OQ458709 to OQ458712.All 16S rRNA and hemolysin gene sequences generated in this study have been deposited into GenBank under accession numbers G. tachinoides flies that were not infected by S. glossinidius  in G.p. palpalis (6.35%) and G. tachinoides (5.77%) compared to G.m. submorsitans (1.49) and G.f. fuscipes (2.60%). For Wolbachia, the prevalence was significantly lower (p = 0.005) in G.m. submorsitans (4.79%) compared to other fly species.All the 4 species were found to be infected at different rates by the three symbiotic bacteria except sinidius . There wWolbachia infection was significantly related (p = 0.017) to G. tachinoides.When the collection site is considered , there was no significant difference in symbiont prevalence among the three tsetse species in Lac Iro. However, in Yankari collection site, the Owing to their restricted source of meal, the vertebrate blood, tsetse flies are highly dependent on their microbial flora which provide them with complementary nutrients . SymbiotSodalis, Spiroplasma and Wolbachia) were detected in 11.20% of the tsetse samples examined with varying prevalence within countries, collection sites and tsetse species. This overall symbiont infection rate is lower compared to that of other previous studies [In this study, tsetse symbiotic bacteria  was lower than the 9.0% previously reported in the same area [G.p. palpalis while the previous study screened G. tachinoides and G. m. submorsitans. Up to date in Nigeria, there is no published data on S. glossinidius prevalence in tsetse flies.The overall ountries . Howeverame area . The preame area . The varame area . MoreoveS. glossinidius has been suspected to be involved in vector competence of tsetse fly. But the confirmation is still under debate. Numerous reports showed a positive association between Sodalis and trypanosome establishment in the midgut possibly through lectin-inhibitory activity involving the production of N-acetyl glucosamine [Sodalis and that of Trypanosome [The presence of cosamine , 38, 40.panosome -37.Spiroplasma general infection rate of 3.66% obtained in the present study is much lower than 17.17% and 44.5% reported respectively in Burkina Faso for G. tachinoides and in Uganda for G. f. fuscipes [G. f. fuscipes [Spiroplasma in tsetse flies in Africa. Up to date, there is no existing data on tsetse Spiroplasma prevalence in the 3 countries where our samples were collected.The fuscipes , 41. Thefuscipes . The preSpiroplasma is known to induce reproductive abnormalities and pathogen protective phenotypes in various arthropods hosts [Spiroplasma with trypanosome infection was found in G. f. fuscipes, indicating that Spiroplasma infections may have an important effect in fly’s resistance to infection with trypanosomes [G. f. fuscipes, it was discovered that Spiroplasma infection induced changes in sex–biased gene expression in the reproductive tissues, adepletion in the availability of nutrients in pregnant females resulting in delayed larval development, and compromised sperm fitness. These findings indicate that Spiroplasma could be exploited for reducing tsetse population size and therefore, the disease transmission [ds hosts . A negatanosomes , 42. In Wolbachia global prevalence of 11.00% and countries prevalence  gene. The comparison of results generated by 16S rRNA and wsp primers by [Wolbachia infection in tsetse flies in Nigeria.The evalence  obtainedectively . In Cameectively , 38. Theimers by  did not Wolbachia has received increased interest in recent years because of its high rates of distribution in a wide range of arthropods and nematodes, its unique effects on host physiology and its potential in disease control. Concerning trypanosomiasis, there is no confirmed implication of Wolbachia in tsetse vector competence. Investigations on the tripartite association between tsetse fly, Wolbachia and trypanosomes reported contrasting results. When in G. f. fuscipes, it was found a negative association between Wolbachia and trypanosome [Wolbachia; some experimental studies did not find an association between the presence of Wolbachia and the ability of tsetse flies to harbor trypanosomes [panosome  suggestianosomes , 37, 43.Spiroplasma was found to infect the 4 Glossina species, but with a low infection on G.m. submorsitans and G.f. fuscipes. On the contrary, the investigations by [Spiroplasma infection only in palpalis group and nothing in the morsitans and fusca groups attributing this result to their frequent infection with Wolbachia which may have led to the development of competitive exclusion with Spiroplasma. Wolbachia was detected in all the 4 species with varying prevalence within species. This variation agrees with findings from similar studies on tsetse symbionts. In the present study, G. tachinoides was found to be more infected with Wolbachia than the 3 other tsetse species. Sodalis was not found in G. tachinoides. However, Sodalis is found to be infecting G. tachinoides in other studies [tions by  found Sp studies , 36.Spiroplasma in tsetse in Chad and Nigeria. The infection rates of Sodalis, Spiroplasma and Wolbachia varied between countries and collection sites probably because of environmental factors. Few tsetse flies harboured symbiont co-infections. These findings provide useful information to the repertoire of bacterial flora of tsetse flies and incite to more investigations to understand the implication of these symbiotic bacteria in the vector competence of tsetse flies. More data could help the development of environmentally friendly methods for both tsetse and trypanosomiasis control.The present study revealed for the first time, the presence of infection by Tsetse flies used in this study were collected from three countries: Cameroon, Chad, Nigeria .In Cameroon flies were trapped in Dodeo (Latitude 7° 27.994’ N and Longitude 12° 04.101′ E) located in the “Faro et Déo” division of the Adamawa region. The village is close to the Cameroon-Nigeria Border. The vegetation is predominantly characterized by gallery forest along rivers and the climate type is of Sudano-Sahelian with two seasons: A rainy season from May to October and a dry season from November to April. This area has a dense hydrographic network and offers pastures for livestock , 36.In Chad, flies were collected in Maro and Lake Iro areas, all situated in the Moyen-Chari province in southern Chad. The area has a climate of Sudano-Sahelian type with two seasons of equal duration. A rainy season from May to October and a dry season from November to April. Lac Iro is situated between the Latitude 9°59’ N and Longitude 19°26′ E, along the Salamat River. The vegetation is dominated by floodplains and dense forests containing shrubs. The area is considered as a buffer zone of the Zakouma national park where domestic and wild animals can meet . Maro  are regular [In Nigeria, flies were collected in Yankari and in Ija-Gwari. Yankari Game Reserve (9°45.240’N and 10°30.448E) is situated in the south-central part of Bauchi State in the North-East of the country. It covers an area of 2244 km regular .Biconical traps were used for this study. They were set up in various tsetse fly-favourable biotopes at distances of at least 100 m intervals. Tsetse flies were collected once a day and transported in cool boxes to the base camp. The collected flies were identified using morphological identification keys , 48, numFlies were dissected in a drop of sterile saline solution as described by , 46. TheWings were stored dry for geometric morphometrics. Legs, proboscis and salivary glands were preserved in nucleic acid preservation agent  in 1.5 mL cryotubes. Gut tissue was homogenised in 200 μL of 50 mM Tris-Cl, pH 9.0, using four 2.38 mm metal beads . Fifty microliters of the homogenate were added to 500 μL of NAPA.Most of the flies from Chad were dead. For this reason, after the removal of wings, legs and proboscis, the entire abdomen was transferred in ethanol.DNA was extracted from homogenised gut tissue in NAPA with the DNeasy Blood and Tissue Kit  according to the manufacturer’s instructions with slight adjustments (100 μL of homogenate were used for purification and 100 μL elution buffer were used at elution step).For the dead flies from Chad, DNA was extracted from the abdomen using 5% Chelex-100 Resin . Briefly, the abdomen was transferred from ethanol to a new 1.5 mL centrifuge tube and allowed to dry. The abdomen was then crushed using the micro-pipette tips. Thereafter, 100 μL of chelex 5% solution were added. The mixture was vortexed and incubated at 56° C for 30 min in a Thermomixer. After this incubation, the mixture was vortexed and incubated for an additional 5 min at 95° C. The mixture was then mixed thoroughly before brief centrifugation at 7000 rpm for 1 min and stored at −20°C.Wolbachia was carried out by amplifying a 438 bp fragment of the 16S rRNA gene with the 16S W-Spec primers (The detection of  primers  designedPCR amplifications were performed in 20 μL reaction mixture containing 1 X DreamTaq buffer (10X), 150 μM dNTPs, 0.2 μM of each primer, 0.5 U of Dream Taq polymerase and 2 μl of template DNA. The cycling condition was as described by. It started by 95°C for 5 min followed by 35 cycles of 95°C for 30 s, 30 s at 54°C, 1 min at 72°C and a final extension step of 72°C for 10 min. After amplification, the PCR products were analyzed by electrophoresis on a 1.5% agarose gel containing Stain-G and visualized under UV light.S. glossinidius was determined by PCR with Hem primers . Five microliters were visualised on 1.5% agarose gel for confirmation of the amplification. The remaining 45 μL PCR products were separated on 2% agarose gel. The amplicons bands were extracted and purified using GeneJET Gel Extraction Kit (Thermo Scientific) according to manufacturer instructions. The purified amplicons were sent for sanger sequencing to a commercial company .Obtained sequences were manually checked and edited using Geneious Pro version 5.5.9 software. The Basic Local Alignment Search Tool (BLASTn) from National Center for Biotechnology Information (NCBI) was used to confirmed the identity of the amplicons and to determine the closest related sequences in the GenBank.Wolbachia and Spiroplasma, then Hasegawa-Kishino-Yano model [Sodalis as determined by the MEGA model finder tool with 1000 bootstraps replicates.Sample sequences and reference sequences obtained from NCBI were aligned using MUSCLE alignment tool with its default setting implemented in MEGAX  which wano model  for Soda2) was used to compare symbiont prevalences between countries and collection sites. The differences were considered significant when the p-values were lower than 0.05. Statistical tests were performed using SPSS for Windows, version 20.Endosymbionts hosted by tsetse flies were expressed in percentage as symbiont prevalence. The Pearson’s chi-square test (χ

Glossina species from Cameroon, Chad and Nigeria.Tsetse flies are cyclical vectors of African trypanosomiasis (AT). The flies have established symbiotic associations with different bacteria that influence certain aspects of their physiology. Vector competence of tsetse flies for different trypanosome species is highly variable and is suggested to be affected by bacterial endosymbionts amongst other factors. Symbiotic interactions may provide an avenue for AT control. The current study provided prevalence of three tsetse symbionts in Sodalis glossinidius, Spiroplasma species and Wolbachia endosymbionts, using species specific primers. A total of 848 tsetse samples were analysed: Glossina morsitans submorsitans (47.52%), Glossina palpalis palpalis (37.26%), Glossina fuscipes fuscipes (9.08%) and Glossina tachinoides (6.13%). Only 95 (11.20%) were infected with at least one of the three symbionts. Among infected flies, six (6.31%) had Wolbachia and Spiroplasma mixed infection. The overall symbiont prevalence was 0.88, 3.66 and 11.00% respectively, for Sodalis glossinidius, Spiroplasma species and Wolbachia endosymbionts. Prevalence varied between countries and tsetse fly species. Neither Spiroplasma species nor S. glossinidius were detected in samples from Cameroon and Nigeria respectively.Tsetse flies were collected and dissected from five different locations. DNA was extracted and polymerase chain reaction used to detect presence of Spiroplasma species infections in tsetse fly populations in Chad and Nigeria. These findings provide useful information on repertoire of bacterial flora of tsetse flies and incite more investigations to understand their implication in the vector competence of tsetse flies.The present study revealed, for the first time, presence of The online version contains supplementary material available at 10.1186/s12866-023-03005-6. Trypanosoma, and is one of major endemic diseases in sub-Saharan Africa. The disease exists as Human African Trypanosomiasis (HAT) (sleeping sickness) and the animal African trypanosomiasis (AAT) (nagana) forms. In humans, the disease is caused by Trypanosoma brucei rhodesiense, responsible for acute form of the disease in eastern and southern Africa, and Trypanosoma brucei gambiense, responsible for chronic form of the disease in western and central Africa [2 are still at risk of T. b. gambiense infection [Trypanosoma congolense, Trypanosoma vivax, Trypanosoma simiae, Trypanosoma uniforme, Trypanosoma godfreyi, Trypanosoma brucei brucei and Trypanosoma grayi, and is a major constraint to Agricultural development on the continent, causing pathogenic infections in cattle, sheep, goats, pigs, dogs, camels and horses [Trypanosomiasis disease is caused by trypanosomes, extracellular flagellated protozoan parasites of the genus l Africa . Approxinfection . The AATd horses –5.Palpalis, the savannah Morsitans and the forest Fusca [Trypanosomes are cyclically transmitted between different vertebrate hosts by tsetse flies (Diptera: Glossinidae). Thirty-one species and subspecies of tsetse flies have been described. The flies are grouped into three groups or subgenera based on common characteristics and morphology due to bio-ecological and genetic similarities; the riverine st Fusca , 7. The st Fusca .Tsetse fly gut harbours diverse bacteria acquired from environment or maternally . PreviouWigglesworthia glossinidia, Sodalis glossinidius, Wolbachia species and Spiroplasma species that was recently established as the fourth tsetse symbiont in Glossina fuscipes fuscipes, Glossina tachinoides, and Glossina palpalis palpalis [Tsetse flies have established long-term associations with four vertically transmitted endosymbiotic bacteria including palpalis . They shW. glossinidia as primary and obligate endosymbiont. The W. glossinidia resides intracellularly within bacteriome in anterior midgut and extracellularly in milk gland of the fly [W. glossinidia provides dietary supplements absent in the tsetse fly vertebrate blood-meal restricted diet, supports larval development and contributes to maturation of adult immune system [Wolbachia endosymbionts are obligatory intracellular bacteria belonging to the Order Rickettsiales, infecting broad range of arthropod and filarial nematode species and probably most prevalent endosymbiont in insect germlines [Wolbachia genus, 17 supergroups (A - Q) are currently recognized based on sequences of the five  conserved genes and the amino acid sequences of the four hypervariable regions of the Wsp protein [wsp gene of Wolbachia strains detected in tsetse flies collected in ten African countries revealed that the Wolbachia strains infecting G. m. morsitans, G. m. centralis, G. brevipalpis, G. pallidipes and G. austeni belong to supergroup A, while the Wolbachia strain infecting G. p. gambiensis fell into supergroup B [All tsetse flies house  the fly . The W. e system , 14. Wolermlines . Within  protein , 15. Maj protein . PhylogeWolbachia mainly resides in reproductive tissues and is maternally transmitted from generation to generation through trans-ovarian transmission. Wolbachia is also transferred horizontally among arthropods. Wolbachia infection in the tsetse fly host results in a variety of reproductive abnormalities such as parthenogenesis, male killing, feminization and cytoplasmic incompatibility [Wolbachia infection status, where infected male mates with an uninfected female or infected female with a different strain of the bacterium [Glossina morsitans morsitans tsetse fly is associated with the induction of CI [Wolbachia infections limit mosquito-transmitted pathogens including dengue virus, chikungunya virus, Plasmodium parasites, yellow fever virus, Zika virus and filarial nematodes [In tsetse fly, tibility –18. Cytoacterium . The preon of CI , 18. Somematodes –22.S. glossinidius. It is a gram-negative organism belonging to Enterobacteriaceae family. The S. glossinidius is widely spread in numerous tissues of tsetse fly , present intracellularly and extracellularly [S. glossinidius genome consists of one circular chromosome of 4.17 Mbp, three extrachromosomal plasmids designated pSG1, pSG2, and pSG3, as well as a phage, ФSG1. However, its genome sequence shows reduced coding capacity with a large number of pseudogenes [S. glossinidius can be transmitted maternally via haemolymph, milk gland secretions, and horizontally during mating [The tsetse fly’s secondary and facultative symbiont is the commensal llularly . The S. udogenes . The S. g mating , 25. In g mating .Spiroplasma genus belongs to Mollicutes class, and Tenericutes phylum. Spiroplasma species are abundant in insects gut or haemolymph where they have large variety of commensal, pathogenic or mutualist interactions with host [Spiroplasma confers protection against pathogens such as fruit fly Drosophila neotestacea from nematode [Acyrthosiphon pisum against fungi [Drosophila hydei against a parasitoid wasp Leptopilina heterotoma [Spiroplasma [Spiroplasma recently, been established as a new class of tsetse fly symbiont in G. f. fuscipes, G. tachinoides, and G. p. palpalis. The interactions between Spiroplasma and tsetse flies seem to be beneficial because of its ability to extend lifespan and reduce t vector competence for Trypanosoma [The ith host . Spiroplnematode , pea aphst fungi  and fruiterotoma . Howeverroplasma .Spiroplapanosoma , 30.Current control measures against trypanosomiasis are mainly based on chemotherapy. In absence of effective vaccine and to address limitations associated with chemotherapy, disruption of trypanosomes transmission through vector control is crucial. Transmission of pathogens by vector depends on its vector competence, which can be affected by several factors, including vector endosymbionts . Due to Sodalis glossinidius, Spiroplasma species and Wolbachia in wild populations of tsetse flies in Cameroon, Nigeria and Chad.The present study assessed presence of Glossina morsitans submorsitans: 403 (47.52%); G. p. palpalis: 316 (37.26%); G. f. fuscipes: 77 (9.08%); G. tachinoides: 52 (6.13%) species. Distributions of prevalence of different symbiotic bacteria are presented in Table For this study, 848 tsetse fly samples from Cameroon, Chad and Nigeria were used. The flies belonged Wolbachia-Spiroplasma mixed infection.Out of 848 flies, 95 (11.20%) were infected with at least one of the endosymbionts. Among infected flies, six (6.31%) had S. glossinidius was investigated in 678 field-collected tsetse flies from Cameroon (149), Chad (259) and Nigeria (270) using a Sodalis Hemolysin gene-based PCR. The S. glossinidius infection rates were 2.01%, 1.16% and 0.0% in Cameroon Chad Nigeria respectively (Table S. glossinidius infection were similar (p > 0.05) between Cameroon and Chad. The sequence of our amplicons had > 99% similarity to Sodalis’s Hemolysin partial gene sequences  in NCBI database. Maximum Likelihood phylogenetic tree  of the samples, with. prevalence varying significantly (p < 0.001) between countries  in NCBI database.A total of 847 fly samples from Cameroon 154), Chad (423) and Nigeria (270) were used for molecular detection of 54, Chad Maximum Likelihood phylogenetic tree Fig.  revealedWolbachia using 16 S rRNA-based PCR approach with wspecF/wspecR set of primers. The samples were collected in Cameroon: 155, Chad: 157 and Nigeria: 270. The results of the screening  between the three countries. The Wolbachia prevalence in Cameroon, Chad and Nigeria was 9.68%, 7.00% and 14.07% respectively. The sequence of our amplicons had > 99% similarity to Wolbachia 16 S rRNA gene sequences  in NCBI database. Maximum Likelihood phylogenetic tree  were screened for presence of ree Fig.  revealedAll 16 S rRNA and hemolysin gene sequences generated in this study are deposited into GenBank under accession numbers OQ448931 to OQ448937 and OQ458709 to OQ458712  in G. p. palpalis (6.35%) and G. tachinoides (5.77%) compared to G. m. submorsitans (1.49) and G. f. fuscipes (2.60%). For Wolbachia, the prevalence was significantly lower (p = 0.005) in G. m. submorsitans (4.79%) compared to other fly species.All the four tsetse fly species were infected at different rates by all the endosymbionts, except ns Table . There wWolbachia infection was significantly higher (p = 0.017) in G. tachinoides.When the collection site is considered , symbiont prevalence among the three tsetse species in Lac Iro were similar. However, in Yankari collection site, the S. glossinidius, Spiroplasma and Wolbachia) were detected in 11.20% of tsetse samples examined with varying prevalence within countries, collection sites and tsetse species. This overall symbiont infection rate is lower compared to that of other previous studies [In this study, tsetse symbiotic bacteria  was lower than the 9.0% previously reported in the same area [G. p. palpalis while the previous study screened G. tachinoides and G. m. submorsitans. There is no published data on S. glossinidius prevalence in tsetse flies in Nigeria. The presence of S. glossinidius has been suspected to be involved in vector competence of tsetse fly. But the confirmation is still under debate. Numerous reports showed a positive association between Sodalis and trypanosome establishment in the midgut possibly through lectin-inhibitory activity involving the production of N-acetyl glucosamine [Sodalis and that of Trypanosome [The overall ountries . Howeverame area . The preame area . The varame area . Moreovecosamine , 38, 40.panosome –37.Spiroplasma general infection rate of 3.66% obtained in present study is much lower than 17.17% and 44.5% reported respectively in Burkina Faso for G. tachinoides and in Uganda for G. f. fuscipes [G. f. fuscipes [Spiroplasma in tsetse flies in Africa. There is no data on tsetse Spiroplasma prevalence in the three countries where our samples were collected. Spiroplasma is known to induce reproductive abnormalities and pathogen protective phenotypes in various arthropods hosts [Spiroplasma with trypanosome infection found in G. f. fuscipes, indicate that Spiroplasma infections may have an important effect in tsetse fly resistance to infection with trypanosomes [G. f. fuscipes established that Spiroplasma infection induced changes in sex–biased gene expression in the reproductive tissues, a depletion in the availability of nutrients in pregnant females resulting in delayed larval development, and compromised sperm fitness. These findings indicate that Spiroplasma could be exploited for reducing tsetse population size and therefore, the disease transmission [The fuscipes , 41. Thefuscipes . The preds hosts . A negatanosomes , 42. ExpWolbachia global prevalence of 11.00% and countries prevalence obtained in the present study are in the range of prevalence reported by Doudoumis et al. [G. m. morsitans and G. pallidipes respectively [Wolbachia specific 16 S rRNA while the previous studies used primers targeting the Wolbachia surface protein (wsp) gene. The comparison of results generated by 16 S rRNA and wsp primers by [Wolbachia infection in tsetse flies in Nigeria.The s et al. . All theectively . In Cameectively , 38. Theimers by  did not Wolbachia has received increased interest in recent years because of its high rates of distribution in a wide range of arthropods and nematodes, its unique effects on host physiology and its potential in disease control. Concerning trypanosomiasis, there is no confirmed implication of Wolbachia in tsetse vector competence. Investigations on the tripartite association between tsetse fly, Wolbachia and trypanosomes reported contrasting results. When in G. f. fuscipes there was a negative association between Wolbachia and trypanosome [Wolbachia; some studies did not find association between presence of Wolbachia and ability of tsetse flies to harbor trypanosomes [panosome  suggestianosomes , 37, 43.Spiroplasma infected the four tsetse fly species, but with a low infection rate on G. m. submorsitans and G. f. fuscipes compared to G. tachinoides and G. p. palpalis. On the contrary, the investigations by Doudoumis et al. [Spiroplasma infection only in palpalis group and none in the morsitans and fusca groups. Attributing this result to their frequent infection with Wolbachia which may have led to development of competitive exclusion with Spiroplasma. Wolbachia was detected in all the four tsetse fly species with varying prevalence within species. This variation agrees with findings from similar studies on tsetse fly symbionts. In the present study, G. tachinoides was found to be more infected with Wolbachia than the three other tsetse fly species. The S. glossinidius was not detected in G. tachinoides, but has been detected in G. tachinoides in other studies [s et al.  found Sp studies , 36.Spiroplasma in tsetse flies in Chad and Nigeria. The infection rates of S. glossinidius, Spiroplasma species and Wolbachia varied between countries and collection sites probably due to environmental factors. Few tsetse flies harboured symbiont co-infections. These findings provide useful information to repertoire of bacterial flora of tsetse flies and call for more investigations to understand the implication of these symbiotic bacteria in the vector competence of tsetse flies. More data could help development of environmentally friendly methods for tsetse and trypanosomiasis control.The present study revealed for the first time, the presence of infection by Tsetse flies used in this study were collected from Cameroon, Chad, Nigeria Fig. . In CameIn Chad, flies were collected in Maro and Lake Iro areas, all situated in the Moyen-Chari province in southern Chad. The area has a climate of Sudano-Sahelian type with A rainy season from May to October and a dry season from November to April of equal duration. Lac Iro is situated between Latitude 9°59’ N and Longitude 19°26′ E, along Salamat River. The vegetation is dominated by floodplains and dense forests containing shrubs. The area is considered as a buffer zone of Zakouma National Park where domestic and wild animals meet . Maro  are regular [In Nigeria, flies were collected in Yankari and in Ija-Gwari. Yankari Game Reserve (9°45.240’N and 10°30.448E) is situated in the south-central part of Bauchi State in the North-East of the country. It covers an area of 2244 km regular .Biconical traps, very effective in catching riverine tsetse of the palpalis group  and wideFlies were dissected in a drop of sterile saline solution as described by Ngomtcho et al. and Shaida et al., , 46. TheDNA was extracted from homogenised gut tissue in NAPA with the DNeasy Blood and Tissue Kit  according to the manufacturer’s instructions with slight adjustments (100 µL of homogenate were used for purification and 100 µL elution buffer were used at elution step).For the dead flies from Chad, DNA was extracted from the abdomen using 5% Chelex-100 Resin . Briefly, the abdomen was transferred from ethanol to a new 1.5 mL centrifuge tube and allowed to dry. The abdomen was then crushed using the micro-pipette tips. Thereafter, 100 µL of chelex 5% solution were added. The mixture was vortexed and incubated at 56° C for 30 min in a Thermomixer. After this incubation, the mixture was vortexed and incubated for an additional 5 min at 95° C. The mixture was then mixed thoroughly before brief centrifugation at 7000 rpm for 1 min and stored at -20 °C.Wolbachia was carried out by amplifying a 438 bp fragment of the 16 S rRNA gene with the 16 S W-Spec primers . Five microliters were visualised on 1.5% agarose gel for confirmation of the amplification. The remaining 45 µL PCR products were separated on 2% agarose gel. The amplicons bands were extracted and purified using GeneJET Gel Extraction Kit (Thermo Scientific) according to manufacturer instructions. The purified amplicons were sent for sanger sequencing to a commercial company .Wolbachia and Spiroplasma, then Hasegawa-Kishino-Yano model [Sodalis as determined by the MEGA model finder tool with 1000 bootstraps replicates.Obtained sequences were manually checked and edited using Geneious Pro version 5.5.9 software. The Basic Local Alignment Search Tool (BLASTn)  from Natno model  for Soda2) was used to compare symbiont prevalence between countries and collection sites. The differences were considered significant when the p-values were lower than 0.05. Statistical tests were performed using SPSS for Windows, version 20.Endosymbionts hosted by tsetse flies were expressed in percentage as symbiont prevalence. The Pearson’s chi-square test (χBelow is the link to the electronic supplementary material.Additional file 1: Sequence accession numbers. Table indicating which accession number corresponds to each sequence.


This dataset focuses on NLP tasks, as a model that serves and delivers a satisfactory response to a user's query. We obtained data from a well- known dataset known as “The Ubuntu Dialogue Corpus” for the purpose of constructing our dataset. Which consists of about one million multi-turn conversations containing around seven million utterances and one hundred million words. We derived a context for each dialogueID from these lengthy Ubuntu Dialogue Corpus conversations. We have generated a number of questions and answers based on these contexts. All of these questions and answers are contained within the context. This dataset includes 9364 contexts, 36,438 question-answer pairs. In addition to academic research, the dataset may be used for activities such as constructing this QA for another language, deep learning, language interpretation, reading comprehension, and open-domain question answering. We present the data in raw format; it has been open sourced and publicly available at  Specifications Table•This is a dataset for question answering that is created using the chat logs or conversation histories of users. It is unique because it is the only dataset that uses user dialogues.•The dataset is a valuable resource for training and testing chatbots and chat analysis algorithms in NLP research and related fields, as well as for improving the performance of conversational AI systems in businesses.•The dataset can also be used by students to learn and practice NLP approaches, and by anyone interested in NLP and AI to explore various techniques.•By modifying this dataset, it can be utilized for the development of language models in other languages, such as 'Bengali,' as well as in other domains.•This dataset also can be used in Reading Comprehension tasks. To understand the contexts and find the answers based on questions.1Our dataset is derived from a popular dataset, The Ubuntu Dialogue Corpus is composed of about one million talks extracted from the Ubuntu chat logs, which were used to acquire technical help for a variety of Ubuntu-related issues 2A chatbot is a robot that responds with relevant responses to customer questions. Chat analysis is the process of evaluating a conversation or collecting relevant information, as well as monitoring the behaviour or sentiment of users. A valuable question-response dataset is required for the development of a chatbot and a chat analysis model. In light of this, we have derived a QA dataset from a primary dataset, The Ubuntu Dialogue Corpus. This Ubuntu Dialogue Corpus dataset only covers multi-turn conversations between people Our created dataset is available in a data repository at mendeley data and there is a folder called Dataset. In this Dataset folder there are four different files. These four files are test_data.csv, test_data_json_file.json, train_data.csv, train_data_json_file.json.Our dataset is shown to be represented in two different data formats, the first of which is data formatted in CSV, and the second of which is data formatted in JSON, as seen in figure-01. The Train section of each of these files has a total of 29,150 question-answer pairs and a total of 7323 context pairs. The Test section files contain the question-answer pairs and context. There are a total of 7288 question-answer pairs and a total of 2041 contexts in the Test section The names of the columns with brief explanations of their contents of the CSV files of the dataset are found in table-01. The names of these columns are dialogueID, which is the unique identifier of the conversation, Context, which contains the overall conversation that has been contributed by multiple users, QuestionID, which is the unique identifier for both Questions and Answers, Answer start, and Answer end, which are the starting and ending indexes of the Answer for the Question.In our dataset 3The source of the raw data that we collect is the "Ubuntu Dialogue Corpus," which consists of almost one million conversations between more than one person extracted from the Ubuntu chat logs and used to receive technical support for various Ubuntu-related problems 3.1We are testing our datasets in Hugging Face transformers pre-trained model fine tuning using pytorch with two different models Bidirectional Encoder Representations from Transformers (BERT) Our dataset is trained using two different transformer-based models derived from Hugging Face. The metrics that they use for evaluation are presented in Sabbir Hosen: Software, Validation, Data curation, Investigation, Writing – original draft, Visualization. Jannatul Ferdous Eva: Validation, Data curation, Investigation, Writing – original draft, Visualization. Ayman Hasib: Validation, Data curation, Investigation, Writing – original draft, Visualization. Aloke Kumar Saha: Supervision, Writing – review & editing, Project administration. M.F. Mridha: Supervision, Writing – review & editing, Project administration. Anwar Hussen Wadud: Writing – review & editing.The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


The resulting radiolabeled mAb ([177Lu]Lu-CHX-A″-DTPA-7F5) was characterized both in vitro and in vivo. It showed a high radiochemical purity (>95%) and stability. The labelling did not affect its binding capability. Biodistribution studies showed a high specific tumor uptake compared to most non-targeted tissues in mice bearing PSCA-positive tumors. Accordingly, SPECT/CT images revealed a high tumor-to-background ratios from 16 h to 7 days after administration of [177Lu]Lu-CHX-A″-DTPA-7F5. Consequently, [177Lu]Lu-CHX-A″-DTPA-7F5 represents a promising candidate for imaging and in the future also for radioimmunotherapy.Prostate specific membrane antigen (PSMA) is an excellent target for imaging and treatment of prostate carcinoma (PCa). Unfortunately, not all PCa cells express PSMA. Therefore, alternative theranostic targets are required. The membrane protein prostate stem cell antigen (PSCA) is highly overexpressed in most primary prostate carcinoma (PCa) cells and in metastatic and hormone refractory tumor cells. Moreover, PSCA expression positively correlates with tumor progression. Therefore, it represents a potential alternative theranostic target suitable for imaging and/or radioimmunotherapy. In order to support this working hypothesis, we conjugated our previously described anti-PSCA monoclonal antibody (mAb) 7F5 with the bifunctional chelator CHX-A″-DTPA and subsequently radiolabeled it with the theranostic radionuclide  However, each of these techniques has its limitations Lu-CHX-A″-DTPA-7F5, we determined its in vitro properties and biodistribution in mice. Moreover, we performed single photon emission computed tomography (SPECT)/CT. According to the presented data, [177Lu]Lu-CHX-A″-DTPA-7F5 represents a promising candidate for imaging and radioimmunotherapy of PCa.In the present study, we conjugated the previously described full size murine anti-PSCA-specific IgG1 mAb 7F5  with theAfter purification of the anti-PSCA mAb 7F5 from hybridoma supernatant by protein A affinity chromatography, dialyzed elution fractions were analyzed by SDS-PAGE A. As expD value of 10.5 ± 4.0 nM . As positive cytotoxic control served a treatment with Triton X-100 (2.5%). Viability was estimated using the MTS assay as described under 177Lu-labeling, p-SCN-Bn-CHX-A″-DTPA was used to couple the chelating moiety CHX-A″-DTPA to primary amino groups of the anti-PSCA mAb 7F5 via its isothiocyanate functional group. MALDI-ToF-MS analyses and HPLC-SEC profiles indicated a successful conjugation of the chelator to the anti-PSCA mAb 7F5. MALDI-ToF-MS analysis of untagged and CHX-A″-DTPA-tagged mAb 7F5 showed comparable MS profiles  resulting from excess p-SCN-Bn-CHX-DTPA from the coupling reaction was readily removed by the following centrifugation steps. Finally, a radio-HPLC-SEC was used to detect radiolabeled mAb-aggregates or fragments and to determine the radiochemical purity (RCP). The HPLC profiles of [177Lu]Lu-CHX-A″-DTPA-7F5 showed a main peak at a retention time of around 11 min before and after three spin filtration steps while [177Lu]Lu-CHX-A″-DTPA eluted around 16 min . The uptake increased 32.4% and 44.8% after 24 h and 48 h, respectively  suggests an initial hepatobiliary and renal elimination route at equal parts  of blood reached 6.6 , liver, and spleen decreased over time, uptake of [177Lu]Lu-CHX-A″-DTPA-7F5 significantly increased in PC3-P  and PSCA-negative PC3 cells (PC3). In contrast to another previously reported anti-PSCA mAb (1G8) Lu-CHX-A″-DTPA-7F5. The data measured in PBS, cell media and human serum suggest a high stability of [177Lu]Lu-CHX-A″-DTPA-7F5. These data are in line with other 177Lu-labeled Abs conjugated with the CHX-A″-DTPA moiety Lu-CHX-A″-DTPA-7F5 was slightly decreased. This could be caused by the random coupling of the chelating moieties in case coupling happened in the antigen-binding region. Still the remaining functional portion of [177Lu]Lu-CHX-A″-DTPA-7F5 is in the range of other CHX-A″-DTPA-mAb-conjugates labeled with 177Lu [177Lu]Lu-CHX-A″-DTPA-7F5 does not destroy its high binding affinity with a KD of about 30 nM. Most important, there was only a specific binding to PSCA-overexpressing PC3 cells but no binding to PSCA-negative PC3 cells as measured by flow cytometry and finally supported by the SPECT imaging mouse data. Although there was a slight loss of affinity compared to the unlabeled anti-PSCA mAb 7F5, the remaining binding affinity is just in the range that is considered to be favorable for tumor-targeting IgG antibodies since very high binding affinities of radioimmunoconjugates  might be associated with low penetration efficiency and limited diffusion rate into tumors due to the so-called binding site barrier effect, which can reduce the therapeutic efficiency LuCl3  was purchased from ITM . Radiolabeling of CHX-A″-DTPA-7F5 with 177Lu was achieved by mixing about 50 MBq of the radionuclide solution with the same volume of 0.05 M HEPES  to obtain a pH between 5 and 6. Then, the solution was added to 1 nmol of CHX-A″-DTPA-7F5 conjugate and allowed to react for 60 min at 37 °C. An amount of 1 nmol CHX-A″-DTPA was added in order to complex free 177Lu for 10 min at 23 °C. The reaction mixture was then separated via spin filtration with a 30 kDa MWCO Amicon Ultra-0.5 mL centrifugal filter (Merck).Non-carrier added [177Lu in the [177Lu]Lu-CHX-A″-DTPA-7F5 preparation was determined by using instant thin layer chromatography (iTLC) with a silica gel-impregnated glass fiber support and a mobile phase of 10 g/L DTPA in H2O  and autoradiographic analysis . Radiochemical purity was determined using SEC-HPLC with a TSKgel SuperSW mAb HR column  and 0.2 M sodium phosphate buffer  as mobile phase at 0.8 mL/min flow. HPLC was performed using a Knauer HPLC-System  with a Ramona detector  for radioactivity flow monitoring.Residual free 177Lu]Lu-CHX-A″-DTPA-7F5 or [177Lu]Lu-p-SCN-CHX-A″-DTPA was mixed 1:10 with PBS, cell medium, or human serum and maintained at 37 °C. After 1, 24, and 48 h, samples were mixed 1:10 with protein A binding buffer and thereafter absorbed on a pre-equilibrated protein A affinity column. Released radioactive constituents were compared with the bound activity that was then eluted from the column. Ab integrity was further determined using SDS-PAGE  with subsequent autoradiography  and silver staining  of the gel.Approximately 100 kBq of [177Lu]Lu-CHX-A″-DTPA-7F5 was assessed using the method of Lindmo et al. [177Lu]Lu-CHX-A″-DTPA-7F5 for 2 h at 37 °C, filtered through glass fiber filters , and finally treated with 0.3% polyethylenimine for 90 min using a cell harvester . After washing with ice-cold PBS, the radioactivity accompanied with the cells harvested on individual filter punches was measured using an automatic gamma counter  together with standards containing the total activity. Data were plotted as reciprocal of the cell concentration (X axis) against the quotient of totally applied and bound radiolabeled mAb (Y axis) and fitted with linear regression using the nonlinear regression analysis software GraphPad Prism 8 . The Y intercept value gave the reciprocal of the IRF (%).The immunoreactive fraction (IRF) of Lu-CHX-A″-DTPA-7F5  for 0 h (5 min), 1, 4, 24, and 48 h at 37 °C, respectively. Adjacent samples received 0.5 to 1 µM of cold anti-PSCA mAb 7F5 1 h before radioligand application to obtain the non-specific binding. After each time interval, the incubation medium was removed, and the cells were washed three times with ice-cold PBS (containing Mg2+ and Ca2+). Surface-bound activity was stripped with 4 °C-cold acid-wash buffer  for 5 min. Cytosolic activity was determined after treatment with cell lysis buffer (1% SDS in 0.1 M NaOH) for 30 min at 23 °C. Cell surface and cytosolic activities were measured separately in a gamma counter.Cells were incubated with [177Lu]Lu-CHX-A″-DTPA-7F5 (0.5 to 70 nM) for 2 h at 37 °C. Free Ab was removed by washing three times with ice-cold PBS (containing Mg2+ and Ca2+). The cells were detached from the culture dish by incubation with cell lysis buffer for 30 min at 23 °C. Activity of the cell suspensions was measured in a gamma counter. Specific binding was calculated and normalized to the protein content of the individual samples measured using bicinchoninic acid assay at 562 nm  with a spectral multimode well-plate reader . Data were fitted with the saturation binding model and the KD value was calculated using the nonlinear regression analysis software GraphPad Prism 8.After treating a part of the cell samples with an excess of cold anti-PSCA mAb 7F5 (0.5 µM) for 1 h to obtain the non-specific binding, all cell samples of a plate were incubated with eight concentrations of Lu-CHX-A″-DTPA-7F5 (molar activity 47 GBq/µmol) was evaluated in male NMRI nude mice bearing a PC3-P tumor on their flank. An amount of 0.28 ± 0.02 MBq  of [177Lu]Lu-CHX-A″-DTPA-7F5 was administered into tumor-bearing mice via tail vein injection. Organ distribution of [177Lu]Lu-CHX-A″-DTPA-7F5 was measured 1 h and 48 h after injection . The mice were sacrificed under deep anesthesia by heart puncture and cervical dislocation; organs and tissues of interest were excised and weighted. The [177Lu]Lu-CHX-A″-DTPA-7F5 uptake was measured in comparison to three reference samples in a gamma counter and high activity values in a cross calibrated dose counter. The decay corrected activity concentration in organs and tissues was expressed as percent injected dose per g tissue (%ID/g) and standardized uptake value (SUV = (activity in the organ/weight of the organ)/). Accumulated activity counts (%ID) were measured in all fully extractable organs. Intestine and stomach were measured with content (w.c.), and urine plus feces activity was calculated as the difference between the activity recovery and the injected activity.Biodistribution of [177Lu]Lu-CHX-A″-DTPA-7F5  was injected into a lateral tail vain of female NMRI nude mice bearing both PC3 and PC3-P tumors on their flanks under general desflurane anesthesia (10% desfluran in a 30% oxygen/air mixture). SPECT/CT studies were performed between 1 and 144 h after [177Lu]Lu-CHX-A″-DTPA-7F5 injection . Imaging data were presented as maximum intensity projection (MIP). Anatomical reference images were captured before SPECT acquisition using CT-subsystem of the nanoSPECT/CT scanner. Imaging data were reconstructed by Tera-TomoTM 3D SPECT iterative reconstruction (Mediso) using CT images for attenuation correction. The images were prepared using ROVER  and illustrated as MIP.Lu-CHX-A″-DTPA-7F5 via the excretory organs. Due to low, if any, uptake in non-targeted tissues, the 177Lu labeled Ab might be effective in visualizing and treatment, especially of metastases. The stable surface binding of the radiolabeled anti-PSCA antibody 7F5 and its specific accumulation at PSCA-positive tumor sites is favorable for application of the anti-PSCA antibody 7F5 and its radiolabeled derivates for imaging, immunotherapies, endoradionuclide therapies, and combinations thereof.The anti-PSCA mAb 7F5 can be successfully radiolabeled with


Festuca arundinacea, to year-round warming (+3 °C) and cool-season drought  in a factorial field experiment to test the hypotheses that: (i) drought and warming increase carbon allocation belowground and shift root traits towards greater resource acquisition and (ii) increased belowground carbon reserves support post-drought aboveground recovery. Drought and warming reduced plant production and biomass allocation belowground. Drought increased specific root length and reduced root diameter in warmed plots but increased root starch concentrations under ambient temperature. Higher diameter and soluble sugar concentrations of roots and starch storage in crowns explained aboveground production under climate extremes. However, the lack of association between post-drought aboveground biomass and belowground carbon and nitrogen reserves contrasted with our predictions. These findings demonstrate that root trait plasticity and belowground carbon reserves play a key role in aboveground production during climate stress, helping predict pasture responses and inform management decisions under future climates.Sustaining grassland production in a changing climate requires an understanding of plant adaptation strategies, including trait plasticity under warmer and drier conditions. However, our knowledge to date disproportionately relies on aboveground responses, despite the importance of belowground traits in maintaining aboveground growth, especially in grazed systems. We subjected a perennial pasture grass,  Drought and warming can reduce forage production by altering belowground carbon allocation. However, a trade-off between belowground production and root trait plasticity can offset negative effects on aboveground plant productivity. Global climate models predict an increase in mean surface temperature ranging from 2.6 °C to 4.8 °C by the end of this century, along with an increase in the intensity and frequency of drought conditions . EmpiricfBNPP; belowground net primary production to total plant production ratio)  and nitrogen (N) to root and crown storage. In grasses, the crown is the tissue that joins the roots with the shoots, bears shoot meristems and stores carbon (C) and N reserves . These bAn increase in the intensity and frequency of drought in a future climate is predicted to reduce natural and managed grassland production in many ecoregions by negatively affecting plant physiological functioning . HoweverWarming can limit plant performance either directly by determining the rate of photosynthesis and respiration  and year-round warming (ambient +3 °C) using a factorial field manipulation experiment, PAstures and Climate Extremes (PACE), at Western Sydney University, Australia. We hypothesized the following. (H1) Both drought and warming reduce plant production (above- and belowground) and standing root biomass while increasing the relative allocation of biomass belowground (fBNPP and RMF). (H2) Fine root traits exhibit plasticity in response to drought and warming, with increasing SRL and decreasing RTD and mean root diameter, reflecting a shift towards more acquisitive trait values that may reduce the effects of climate extremes by promoting soil resource acquisition. (H3) Drought increases and warming decreases belowground storage (NSCs and N), with consequences for post-drought recovery of aboveground productivity. During drought, the senescence of shoots and reallocation of C and N reserves to crowns and roots is expected; however, higher maintenance costs with warming are expected to reduce belowground C storage.A large proportion of temperate grasslands occur in areas that experience temperatures well above their thermal optima for productivity, such that rising temperatures may significantly reduce their growth . In addiThis study was conducted from the austral winter in June 2019 to the autumn of March 2020 at the PACE (PAstures and Climate Extremes) field facility on the Hawkesbury campus of Western Sydney University in Richmond, New South Wales, Australia . This site receives a mean annual rainfall of 806 ± 37 (SE) mm with a large inter-annual variability ranging from 500 mm to 1400 mm over the past 30 years . Summer (December–February) is typically the wettest season, and winter (June–August) is generally the driest. Climate models predict a decrease in cool-season (winter and spring) rainfall under most scenarios, along with an increase in the frequency and severity of drought throughout south-eastern Australia . The meaThe PACE experimental facility consists of six polytunnel shelters constructed from galvanized steel frames and covered with a single layer of polyethylene 180 μm plastic  to intercept all ambient rainfall. Each shelter is 48 m long × 8 m wide × 4.6 m in height, oriented along a SW–NE axis with the open ends facing the direction of the prevailing wind. In addition, the long sides of the shelter are open to a height of 1.5 m to allow free airflow that minimizes shelter effects on microclimate. Each shelter has eight plots (4 m × 4 m) that are further subdivided into four subplots (2 m × 2 m each) with different pasture species growing at the subplot level. In each shelter, plots were assigned to drought and warming treatments in a factorial design, which allowed us to evaluate interactions between co-occurring climate treatments and root trait plasticity responses to these treatments by comparing them with control. Plots were established during 2017–2018 from seed. This study reports data from the 2019 cool-season drought and the following recovery period. Before sward establishment, the surface soil in each shelter was rotary tilled to a depth of 12 cm to homogenize the upper soil profile. Root barriers  were installed to a depth of 90 cm around all 4 × 4 m plots to ensure hydrological isolation between treatments. The 2 × 2 m subplots were separated by a 30 cm deep root barrier  to minimize root intrusion among pasture types. Furthermore, there was a buffer area of 1 m between each plot and 0.5 m between subplots, which included pavers aboveground to enable plot access.3 pasture grass F. arundinacea (Quantum II MaxP cultivar). Festuca arundinacea is native to large regions of Europe, temperate Asia, and North Africa . The following 6 month summer and autumn period represents the post-drought recovery period when all plots received the control irrigation amount. The 60% reduction in the amount of cool-season rainfall represents the upper end of climate model drought predictions for south-eastern Australia at the end of the century under RCP8.5 . We collThe warming treatment  comprised a year-round +3 °C increase in canopy temperature relative to ambient temperature (aT). Warming was achieved using a heating array that included eight 1000 W ceramic heaters  installed 1.4 m above the ground surface in each heated 4 × 4 m plot . ExperimEach shelter had data loggers which recorded the real-time environmental conditions under each climate treatment. Time Domain Reflectometers  and soil temperature probes  recorded soil volumetric water content (0–15 cm depth) and soil temperature (at 0–5 cm), respectively, at 15 min intervals. Air temperature (Series RHP-2O3B) and relative humidity  sensors mounted in force-ventilated radiation shields were installed at 0.6 m height inside and outside shelters to record air temperature and relative humidity, respectively, every 5 min. Quantum sensors  installed inside (3 m height) and outside (6 m height) the shelter recorded photosynthetically active radiation (PAR) at 5 min intervals.–2 per 6 months). The harvest method mimics the cell-grazing technique, a form of rotational grazing. During cell-grazing, small paddocks are intensively grazed, followed by a long recovery period , and weighed. The timing of harvests was based on grazing recommendations from the pasture industry, harvesting at the peak of vegetative growth at ~30 cm height (y period . The smaAboveground biomass recovery following the 6 month drought (which ended on 30 November 2019) was measured in mid-March 2020. Post-drought aboveground vegetation was harvested 5 cm above the soil surface, in the same way as harvests during the cool-season drought period. This harvest captured 3.5 months of growth during the summer, where all plots received control precipitation amounts under both warming and ambient temperature. We excluded data from one of the six replicate shelters from aboveground biomass recovery analyses due to the destruction of this shelter during a severe storm at the end of November 2019.–2 per 6 months) for each soil depth. However, our open ingrowth cores did not capture root crowns due to their small size. Here we show belowground production data for 0–20 cm after summing the two depths. We chose to sample roots at a depth of 0–20 cm since the majority (>80%) of root biomass occurred at this depth in our study.BNPP was measured during the 6 month cool-season drought using open ingrowth cores ; root and crown data are presented separately.Standing root biomass was sampled at the end of the 6 month cool-season drought period, in November 2019, at two depths (0–10 cm and 10–20 cm) using a 5cm diameter soil auger; here we present summed data for 0–20 cm depth. Four randomly located soil cores were collected from each replicate plot and pooled to make a composite sample. Root samples were processed as described above. At the same time, root crowns were sampled in a 30 cm × 30 cm area in a similar position in all fBNPP was calculated as the ratio of belowground net primary production to total plant net primary production.The The RMF was calculated as the fraction of plant biomass allocated to roots.f BNPP and RMF do not include crown biomass.–1) was calculated as the ratio of root length to root dry mass, and RTD (g cm–3) as the root dry mass to the root volume ratio.Washed fine root samples from ingrowth cores were scanned using an Epson Perfection 4990 scanner at 800 DPI  within 24 h of sampling. Scanned images were analysed using Winrhizo™ software  to determine root length, diameter and volume. Scanned root samples were oven-dried (at 70 °C for 3 d) and weighed. SRL  in plant roots produced during the cool-season drought and in crowns sampled at the end of drought were measured using methanol:chloroform:water extraction and a colorimetric phenol–sulfuric acid assay . In brie–2). All statistical analyses were performed using R 4.2.2  function from the ‘car’ package  in relation to belowground traits.All biomass and productivity data were scaled to dry mass per unit ground area , warming (–9%), and their combination (–51%), relative to control . SimilarfBNPP although there were no significant interactions between treatments  ; Table 1Here, we addressed a knowledge gap relating to belowground trait plasticity responses to future warmer, drier climates and their relationships to aboveground production using a factorial field experiment. The effects of drought and warming on above- and belowground biomass production and the fraction of biomass allocated to belowground were negative, independent, and thus additive. However, the combined effects of drought and warming resulted in a shift in root trait values towards more acquisitive combinations, notably higher SRL, lower mean diameter and higher N concentrations. Greater mean root diameter and higher root soluble sugar and crown starch concentrations were predictors of plant aboveground production during drought and warming. However, in contrast to our expectations, there were no relationships between post-drought aboveground biomass and pre-existing belowground NSC and N reserves.F. arundinacea. However, the greater drought sensitivity of BNPP relative to ANPP observed in our study supports findings from grassland studies elsewhere . The possible explanations for this response may be an overall reduction in C fixation under warming, and thus a decrease in available C to allocate belowground or increased C loss via belowground maintenance respiration . The observed root trait plasticity, such as an increase in SRL or reduction in mean root diameter, might be due to increased root branching or an increase in the production of first-order roots rich in N driven by lower SWC in the combined drought and warming treatment compared with the warmed (eT–C) plots  plots  (OuimettC) plots . The lacC) plots .F. arundinacea  and higher root N to support metabolic activity might reduce the requirement for C investment in belowground production. Again, the lower root diameter in plots with lower belowground production supports our alternative hypothesis that reduced plant development in the combined drought and warming treatment was the key reason for observed differences in root traits.F. arundinacea , there were no relationships between post-drought aboveground biomass and plot-level crown biomass or crown NSC and N reserves. In addition, these findings were also in contrast to other studies, which have shown the importance of belowground C and N storage in post-drought recovery  mixed plant communities.In conclusion, our findings highlight the sensitivity of The following supplementary data are available at Fig. S1. Climate treatments and environmental conditions under each climate treatment during the experimental period.Table S1. Summary statistics for post-drought recovery biomass from Wilcoxon signed rank test.erad021_suppl_supplementary_fig_S1_table_S1Click here for additional data file.