Hermetia illucens) has significant economic potential. The larvae can be used in financially viable waste management systems, as they are voracious feeders able to efficiently convert low-quality waste into valuable biomass. However, most studies on H. illucens in recent decades have focused on optimizing their breeding and bioconversion conditions, while information on their biology is limited.The black soldier fly  of H. illucen, of which 177 proteins were predicted to contain signal peptides, bioinformatics analysis and manual curation determined 88 proteins may be associated with the PM, with functions in digestion, immunity, PM modulation, and others. The ultrastructure of the H. illucens larval PM observed by scanning electron microscopy shows a unique diamond-shaped chitin grid texture.A total of 565 proteins were identified in the PM samples of H. illucens larvae to date. All the proteins identified in this work has been discussed in details, except several unnamed or uncharacterized proteins, which should not be ignored and need further study. A comparison of the ultrastructure between H. illucens larval PM and those of other insects as observed by SEM indicates that the PM displays diverse textures on an ultra-micro scale and we suscept a unique diamond-shaped chitin grid texture may help H. illucens larval to hold more food. This work deepens our understanding of the molecular architecture and ultrastructure of the H. illucens larval PM.It is the first and most comprehensive proteomics research about the PM of The online version contains supplementary material available at 10.1186/s12953-021-00175-x. The peritrophic matrix (PM) is an acellular, semi-permeable structure lining the digestive tracts of certain invertebrates to encase food particles, as first reported in lepidopteran larvae , 2. The The main components of the PM are protein and chitin. Chitin is a polysaccharide polymer of N-acetylglucosamine whose chains form strong, crystal microfibers by hydrogen bonding –14. The PM-associated proteins can be divided into four classes according to their extractability . The claHermetia illucens Linnaeus; Diptera: Stratiomyidae). BSF is thought to originate in the America and is currently widely spread in tropical, subtropical, and temperate regions [This paper examines the PM of the black solder fly  B regions . 3) BSF  regions , 25. How regions –29.In this work, mass spectrometry and scanning electron microscopy were employed to analyze the proteome and ultrastructure of the PM, to produce a more comprehensive understanding of the molecular architecture and ultrastructure of this essential structure.BSF larvae were kept in an artificial climate chamber at a temperature of 27 °C, relative humidity of 70–80%, and a photoperiod of 12 h: 12 h light:dark. All the larvae were fed with commercially available wheat bran and water. About 200 fifth instar larvae were divided into three groups and sacrificed in this work. The larvae were anesthetized on ice, the larval epidermis was cut open, and the midgut was taken out. The PM was pulled out of the midgut, then washed in sterile, pre-cooled, phosphate buffered saline solution  to remove the food debris, and then flash-frozen in liquid nitrogen. Several PMs were immersed in paraformaldehyde for scanning electron microscopy observations.The total protein of the BSF larvae was extracted with protein lysis buffer (8 M urea with appropriate protease inhibitor), and treated twice with a high-throughput tissue crusher Wonbio-96c  for 40 s. The mixture was incubated on ice for 30 min, vortexed 5–10 s every 5 min, and then centrifuged at 16000×g at 4 °C for 30 min. Protein supernatants were mixed with four- fold volumes of cold acetone for 12 h to precipitate the protein and improve the final concentration. After centrifugation at 12000×g for 20 min, the pellet was rinsed twice with 90% acetone and dried under vacuum. The acetone dry powder was resuspended in protein lysis buffer (8 M urea with appropriate protease inhibitor), incubated on ice for 30 min, vortexed 5–10 s every 5 min, and then centrifuged at 16000×g at 4 °C for 30 min. Total protein concentration was determined by the bicinchoninic acid (BCA) method with a BCA Protein Assay Kit (Beyotime Biotechnology). Protein quantification was performed according to the kit protocol.From the extracted proteins, in each group 80 μg protein were collected, and tetraethylammonium bromide (TEAB) was added to a final concentration of 100 mM. The mixture was reduced with 10 mM Tris (2-carboxyethyl) phosphine (TCEP) at 37 °C for 60 min and alkylated with 40 mM iodoacetamide (IAM) at room temperature for 40 min in darkness. Six- fold volumes of cold acetone were added to precipitate the protein at − 20 °C for 4 h. After centrifugation for 20 min at 10000×g at 4 °C, the pellet was rinsed with 90% acetone. Trypsin was added at a 1:50 trypsin-to-protein mass ratio and incubated at 37 °C overnight, then desalted with HLB and dried under vacuum.18-reversed phase column  was equilibrated with solvent A (2% ACN with 0.1% formic acid) and solvent B (80% ACN with 0.1% formic acid). The peptides were eluted using the following gradient: 0–1 min, linear gradient of solvent B from 0 to 5%; 1–41 min, linear gradient of solvent B from 5 to 23%; 41–51 min, linear gradient of solvent B from 23 to 29%; 51–59 min, 29%–100% B; 59–65 min, gradient of solvent B 100%; 65–90 min, linear gradient of solvent B from 100 to 0%. The tryptic peptides were separated at a flow rate of 300 nL/min. The Q Exactive HF-X instrument was operated in data-dependent acquisition mode (DDA) to automatically switch between full scan MS and MS/MS acquisition. The survey of full scan MS spectra (m/z 350–1300) was acquired in the Orbitrap with 60,000 resolution. The automatic gain control (AGC) target was 3e6 and the maximum fill time was 20 ms. The top 20 most intense precursor ions were selected for fragmentation by higher-energy collision dissociation (HCD) in collision cells. The MS/MS resolution was set at 15000 (at m/z 100), the automatic gain control (AGC) target at 1e5, the maximum fill time at 50 ms, and dynamic exclusion at 18 s.The trypsin-digested peptides were analyzed by online nano-flow liquid chromatography tandem mass spectrometry performed on an EASY-nLC system  connected to a Q Exactive HF-X Quadrupole Orbitrap mass spectrometer  through a nanoelectrospray ion source. Briefly, the CH. illucens L transcriptome database. The main MS/MS search parameters were as follows: Mass tolerance of 10 ppm for MS and 0.05 Da for MS/MS tolerance, trypsin as the enzyme with 2 missed cleavage allowed, carbamidomethylation of cysteine as fixed modification, and methionine oxidation as dynamic modifications. High confidence peptides were used for protein identifications by setting a target false discovery rate (FDR) threshold of 1% at the peptide level. Only proteins that had at least two unique peptides were used for protein identification. The preliminarily identified proteins were further analyzed through multiple databases, including the presence of signal peptides , subcellular location (TargetP: http:// www.cbs.dtu.dk/services/TargetP/), motif analysis (https://myhits.sib.swiss/cgi-bin/motif_scan), and annotation based on the MEROPS database, UniProt database, and NCBI BLAST analysis. Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed using OmicShare tools, which is a free online platform for data analysis (www.omicshare.com/tools).The RAW data files were analyzed using ProteomeDiscoverer  against an in-house ftp://massive.ucsd.edu/MSV000086950/All raw LC-MS/MS data are available on the Mass Spectrometry Interactive Virtual Environment (MassIVE) data repository at The PM were immersed in paraformaldehyde for three hours. Then the PM were washed with PBS and dehydrated with a gradient alcohol series , 10 min each, repeated twice. After that, the PM were critical point dried with a FEI CPD-030 dryer and sputter-coated with 3–4 nm platinum. The morphological characteristics of the PM were observed under a Hitachi SU8010 scanning electron microscope .H. illucens proteome data in the existing databases, we constructed an in-house EST contigs through transcriptome sequencing, and the raw data files from the LC-MS/MS were analyzed using ProteomeDiscoverer  against the derived protein sequences from the contigs.Due to the lack of complete H. illucens ’, ‘hemolymph coagulation GO:0042381)’, ‘regulation of body fluid levels (GO:0050878)’, ‘wound healing (GO:0042060)’, ‘innate immune response (GO:0045087)’, ‘chitin metabolic process (GO:0006030)’ etc.  are moved into the median midgut for further digestion. In the ectoperitrophic space, aminopeptidase, carboxypeptidase and amylase perform the final digestion. By compartmentalizing the midgut, damage to the enzyme catalytic sites and midgut epithelial cells caused by the accumulation of digestive intermediates are avoided, promoting food digestion –35.Uca pugilator. The site for cleavage of collagen is usually located 3/4 away from the amino end of the collagen chain. Brachyurin has a wide range of specificity for peptide bonds, but exhibits low activity for the main substrates of trypsin and chymotrypsin [In the putative PM proteome, we found twenty proteins belonging to the Chymotrypsin(S1) family, including six trypsin-like proteases, six serine proteases, two Brachyurin-like proteins, and six chymotrypsins. Trypsin is a kind of endoprotease that mainly cleaves the peptide chain at the carboxyl part of lysine or arginine. Trypsin is a major protease in the midgut of most insects  with a molecular weight of 20–35 kDa, an isoelectric point of 4–5, and an optimum pH range of 8–11 –38. Bracotrypsin . Chymotrotrypsin , 41. Theotrypsin . Serine otrypsin . In the 2+) to catalyze the hydrolysis of the peptide bond at the carboxyl terminal of the polypeptide. The members of the carboxypeptidase M14 family can be divided into carboxypeptidase A and carboxypeptidase B according to the types of amino acids released by their catalytic functions. Carboxypeptidase A is more prone to hydrolyze polypeptides with aromatic or hydrophobic amino acids at the carboxyl terminal. Carboxypeptidase B is more inclined to hydrolyze peptides with lysine and arginine at the carboxyl terminal [Helicoverpa armigera larvae and pre-pupa, and is believed to be related to apolysis [Anopheles gambiae is related to the sexual development of Plasmodium, and is a potential target for malaria prevention and control [Drosophila, such as reproductive tissues, larval and adult midguts, larval tracheae, and adult salivary glands. ACE expression has also been detected in both sexes’ reproductive tissues in other insects, indicating that it plays an important role in reproduction [In the putative PM proteome, we found two metallopeptidases, one in the carboxypeptidase (M14) family, and one in the peptidyl-dipeptidase A (M2) family. Digestive carboxypeptidase is an exonuclease, which requires divalent metal cations , SMPD is expressed in various tissues, with higher expression levels in the ovary and midgut [Anopheles gambiae infected by different bacteria suggests it may also be involved in invertebrate immune response [We found a sphingomyelin phosphodiesterase (SMPD), an adenosine deaminase, and a chitinase-like protein in the PM proteome of d midgut . Differeresponse , 68. Chiresponse .H. illucens. Iron participates in a variety of physiological activities, such as oxygen metabolism, amino acid production, and DNA biosynthesis. In some arthropods, it also participates in egg development and offspring production [A transferrin and a ferritin were found in the PM proteome of oduction , 71. Howoduction . Transfeoduction , 74. Feroduction . For insoduction .H. illucens. OBPs are soluble proteins with a small molecular weight, usually between 17 and 22 kDa, first found in the antennae of the male giant moth (Antheraea polyphemus). They can bind, solubilize, and deliver odor molecules or pheromones to their receptors [Periplaneta americana [Plutella xylostella) [We found eight odorant-binding proteins (OBP) in the PM proteome of eceptors –79. Thereceptors –82. In americana , and theostella) . Four OBostella) . These costella) , 87.Bombus terrestris), SVWC participates in the host’s antiviral immunity and is related to the expression of antimicrobial peptides [BSF is an omnivorous insect with a wide range of larval diets, so its food may carry various bacteria, viruses, fungi, or parasites that can infect the insect when it eats. In the past few decades, there have been many studies on how insects fight these infections, and the PM is considered to be the first line of defense . In factpeptides . Lysozympeptides . MD-2 repeptides . Hemocytpeptides .H. illucens. Peritrophins usually contain more than one chitin-binding domain (CBD), which are called the peritrophin-A domain (PAD), peritrophin-B domain, and peritrophin-C domain with 6, 8, or 10 cysteine residues respectively that can form 3–5 disulfide bonds, of which PAD is the most common (2). PAD is also called type-2 chitin-binding domain (ChtBD2), and its sequence feature is CX13–20CX5–6CX9–19CX10–14CX4–14C, where X represents any amino acid except cysteine [Lucilia cuprina larvae with several CBDs. These tandem CBDs may facilitate cross-linking chitin fibrils within the PM. Peritrophin-44 and another protein, Peritrophin-48, are the main proteins involved in the construction of PM, and represent approximately 70% of the protein mass of the PM [In addition to the above-mentioned, non-structural proteins, some structural proteins, namely peritrophin, were found in the PM proteome of t common . PAD is cysteine . PAD is cysteine , 95. Perf the PM –99. In tApriona germari, and in the midgut, epidermis, and Malpighian tubules of Spodoptera exigua [Antheraea pernyi) was significantly increased when they were infected with microorganisms, so it may be related to immune response [Among the other proteins in the BSF PM proteome are hexamerins. Hexamerins can store a large amount of amino acids, which play an important role in insect metamorphosis and reproduction, and when suffering adverse environments such as food shortages . Hexamera exigua , 102. Exresponse .A large number of uncharacterized or unnamed proteins were also discovered. These proteins should not be ignored, and more research is needed on them, because they may have important significance to the molecular architecture of the PM or lead to better breeding and utilization of BSF larvae.H.illucens with other reported insects, especially the Musca domestica which also belongs to Diptera. In terms of the number of proteins identified, there are 47 species in the 5th instar Bombyx mori [M.domestica [Aedes aegypti [H.illucens and M.domestica [H.illucens. Peritrophin is a kind of important protein in the PM proteome. Only 4 peritrophin are found in A.aegypti [M.domestica, and 16 in H.illucens. This may be due to the PM of A.aegypti is usually induced following a blood meal. While the larva of M.domestica and H.illucens constantly feed before pupation, but the instar of H.illucens is much longer than that of M.domestica. From the molecular weight point of view, most of the peritrophin are between 20 and 40 kDa, and a number of peritrophin of about 16 kDa have been found in H.illucens. The function of these peritrophin needs further verification.We also compared the identified PM proteome of byx mori , 71 secromestica , and 115 aegypti . From thomestica . In addi.aegypti , 8 in M.Locusta migratoria, the midgut secretes PM every 15 min. The inner PM will move posteriorly with the food, making the PM appear as a telescope-like structure (3), as we observed in the BSF PM (Fig. Trichoplusia ni is organized in a random, felt-like structure of fibers [Ostrinia nubilalis PM in an orthogonal structure of fibers [Anomala cuprea PM in a hexagonal structure of fibers. The significance of the differences in arrangement are not clear yet. We speculate that it may be related to the feeding habits of the insects. BSF larvae are described as voracious feeders in most studies, and a diamond-shaped chitin grid is more flexible when deformed, so it may enable their PM to hold more food.The SEM results showed that the outermost layer of the PM was arranged in continuous segments, and the length of each segment was approximately the same, about 25 μm Fig. a. It wasf fibers , the Ostf fibers , and AnoThe main function of the PM is to protect the midgut epithelial cells from coarse food particles, pathogens, and toxins, and it can also compartmentalize the midgut to promote digestion. In this study, we discovered a large number of digestive enzymes bound to the BSF PM, which can digest a variety of proteins, fats, and other ingested food particles. We also discovered a variety of immune-related proteins known to play roles in the identification of and defense against pathogens. The findings of some transporters suggest that the PM maybe involved in ion transport and excretion. The findings of some odorant-binding proteins also indicate that the PM is an important place for insects to interact with their external environment. Scanning electron microscopy revealed clearly the ultrastructure of the BSF PM, and found that it is a scaffold that can hold a variety of proteins. PMs in insects display diverse textures on this ultramicro scale, but the reason for this diversification needs further study. To summarize, our work used liquid chromatography-tandem mass spectrometry and scanning electron microscopy to conduct a more in-depth study of the PM of BSF larvae, and deepen our understanding of its molecular architecture and ultrastructure.Additional file 1: Table S1. all the identified protein from peritrophic matrix sample


T. trichiura infections (59.4%) were of light intensity, while 51.7% of Ascaris lumbricoides were of moderate intensity. Multivariable logistic regression analysis indicated that those who drink rainwater and walk barefoot were 2.9 and 2.5 times more likely to have helminth infections, respectively. Results from one geographical setting might not be applied to other nearby with different climatic conditions. The use of anthelmintic drugs only will not be sufficient to bring prevalence to low levels. It is necessary to design geographically more specific intervention, with communication and interaction between different disciplines  being imperative to reduce STH infection.There is a lack of epidemiological information concerning intestinal parasitic infections, and especially in soil-transmitted helminths, occurring in some departments of Nicaragua. Up to now, this is the first study involving two nearby areas  of the Región Autónoma Atlántico Norte (R.A.A.N.). One stool sample was analyzed by Kato-Katz, formaldehyde-ethyl acetate concentration method and modified Ziehl–Neelsen technique, and a simple questionnaire concerning demographic, sanitary and behavioral data was distributed among 735 children and evaluated. Overall prevalence of infection reached 97.0%, being the highest prevalences detected in all Nicaragua. The higher protozoan prevalence appears in Siuna (94.5%), a rural interior municipality, with a typical tropical monsoon climate, while the higher helminths rates were reached in Puerto Cabezas (92.8%), the urbanized coastal capital, with a typical tropical rainforest climate. No statistical differences were found with regard to sex. However, the 6–11-year age-group children presented the highest prevalences. Most  All of this is linked to the lack of access to safe water and sanitation, poor hygiene practices and unsafe human waste disposal12.Differences in the prevalence rates of intestinal parasites can be associated with environmental factors, such as vegetation, temperature, humidity and precipitation, as well as geographical site, and a variety of cultural, economic, and social characteristics13.Nicaragua is the largest country of Central America, with approximately 6 million inhabitants, occupying a landmass of 130,967 km2, and being the second poorest country in the Latin American and Caribbean (LAC) region. This country is made up of three regions: the Pacific region, with seven departments; the Center region, with eight departments; and the Atlantic/Caribbean region including the Región Autónoma Atlántico Norte (R.A.A.N.) and the Región Autónoma Atlántico Sur (R.A.A.S.). In the 1990s, Nicaragua established massive deworming programs, with a joint effort of various actors  in such a way that an 87% coverage of the child population was reported in 2011, and nowadays deworming is included as a new category on child vaccination cards21, although this is the first study published in SCI journals involving R.A.A.N. The goal of this epidemiological study was to determine the prevalence of intestinal parasite infections and the intensity of STH, assessing which variables  among schoolchildren were related to the infection, providing the baseline for appropriate strategies against STH infections.Several studies have been conducted on the prevalence of intestinal parasitic infections in Nicaragua2) of Nicaragua, covering 26.3% of the national territory  a tropical monsoon climate (Am), with 2400 to 3000 mm annual rainfall, temperature oscillating between 24 °C and 29 °C, with a winter or rainy season (from May to January) and a summer or dry season (from February to April) with only sporadic rains, typical conditions of the municipality of Siuna; and b) a tropical rainforest climate (Af), with 2000 to 6000 mm annual rainfall, temperature oscillating between 27 °C and 39 °C, 80%-90% relative humidity, with a rainy season only  was estimated in 378 children.The survey was carried out from February to April 2015 and finally involved a total of 735 children (342 boys and 393 girls) aged 2–15 years (mean ± S.D. = 10 ± 1.2) coming from both municipalities: 318 from Puerto Cabezas , and 417 from Siuna . In both municipalities, urban schools were determined with paved ground and cobbled streets while rural schools by no urbanization and surrounded by grassland. A clean, plastic, numbered container with a snap-on lid was handed out to each child. With the aid of parents/teachers, if necessary, one stool sample per child was collected and a simple questionnaire concerning various data of the children  was filled out.23.A Kato-Katz slide was made from each stool sample and examined, within 30–60 min after preparation, to count the number of eggs of STHs. The number of eggs per gram of feces (EPG) was calculated by multiplying by 24 the total egg counts in all fields. WHO criteria  were used to classify each infection as light, moderate or heavy intensity infectionsThe rest of the fecal material of each sample was fixed in 10% formalin solution (1:3) and shipped to the Institute Politécnico de la Salud (IPS) laboratory in Managua where all samples were concentrated using the formaldehyde-ethyl acetate concentration technique (FECT). Finally, each fecal sample was microscopically examined at Department of Parasitology, Universidad de Valencia (Spain), and one aliquot of sediment obtained with FECT was stained using a modified Ziehl–Neelsen technique (MZNT). The FECT and the Kato-Katz slides were used to obtain prevalence data of STHs.p value was < 0.05.Statistical analyses were carried out using the SPSS version 19 software package for Windows . Statistical comparison of categorical variables was carried out with the Chi-squared test. Univariable analysis, OR (95%b CI) and significance levels of all independent variables were assessed. Factors identified as statistically significant at the 5% level in univariable analysis were entered into a multiple logistic regression model. All results were considered significant if the Universidad de Valencia. Estudi General granted the ethical approval of the study (H1477378643204) in accordance with the Declaration of Helsinki. Verbal informed consent was obtained from the parents/guardians of children enrolled. Diagnostic results were sent to IPS which informed the Nicaraguan Ministry of Health, being in charge of appropriate treatments.Verbal consent for children publication was obtained from the parents/guardians.Balantidium coli detected in Puerto Cabezas municipality and of Cryptosporidium sp., Taenia sp. and Strongyloides stercoralis detected in Siuna municipality are noteworthy.Table p < 0.0001).Overall prevalence of infection, with at least one parasite species, reached 97.0% in the children surveyed, either being protozoa (90.7%) or helminths (61.6%). Among the R.A.A.N. children surveyed, protozoan infections were more prevalent than helminths, with statistical differences (Blastocystis spp. (75.0%) was the most common intestinal protozoan diagnosed, followed by Giardia intestinalis (45.3%). Among STH, Trichuris trichiura, Ascaris lumbricoides and Hookworm were the most prevalent ones .p = 0.002) among both municipalities studied (99.4% in Puerto Cabezas vs 95.2% in Siuna), with a higher protozoan prevalence in Siuna municipality (94.5%) (p < 0.0001), whereas a higher helminth prevalence was reached in Puerto Cabezas (92.8%) (p < 0.0001). Among STH, T. trichiura and A. lumbricoides prevalences were higher in Puerto Cabezas than in Siuna, with statistically significant differences (p < 0.0001). In Hookworm, although higher prevalence rates occur in Siuna (11.0%), no significant differences were detected (p = 0.188).The overall prevalence of infection presents statistical significant differences  and Siuna (p = 0.036) municipalities. However, protozoa infection in Siuna municipality was higher in the adolescent group with statistical differences (p = 0.019) and it was higher in the municipality of Puerto Cabezas in the youngest age group with statistical differences (p = 0.049).The distribution of total infection prevalence according to age groups revealed significant differences. Total prevalence increased, always being highest in the group of 6–11 years-old children, both in Puerto Cabezas (p = 0.0003). In particular, A. lumbricoides prevalence rates obtained in urban schools were higher than those obtained in rural schools (p < 0.0001), while Hookworm prevalence rates were higher in rural schools (p = 0.005). No differences between the two areas were detected in the case of T. trichiura infections (p = 0.367), with similar rates in urban (49.7%) as well as rural (46.3%) schools.The total infection rate of helminths in urban schools was significantly higher than in rural schools (p < 0.0001) to monoinfection. This monoinfection resulted more frequent in Siuna (11.6%) than in Puerto Cabezas, with statistical differences (p = 0.007).Most infected children presented polyparasitism (86.9%) with statistical differences , two (19.2%) and four (18.5%) different species, although children harboring five to ten species were also detected . Only one 8-year-old boy from Siuna municipality harbored 10 species: p < 0.0001), among the youngest children studied.The extent of polyparasitism in the entire study according to gender, age groups and urban/rural school area is shown in Fig. T. trichiura, A. lumbricoides and Hookworm median (range) infection intensity in R.A.A.N. was 960 (24–88.752), 18.096 (24–149.544) and 24 (24–264) eggs per gram (epg) of feces, respectively (Table ly Table .Table 3ST. trichiura infections were light intensity (59.4%), while 51.7% of A. lumbricoides were moderate-intensity infections. No differences in infection intensity classes appeared among the two municipalities studied in any STH species.Table T. trichiura, light-infection intensity in girls and in the urban schools was higher with statistical differences than in boys (p = 0.003) and in the rural schools (p = 0.0007). In the case of A. lumbricoides, moderate and heavy infection intensities were higher, with statistical differences  in the 6–11-year-old age group.Hookworm infection intensity classes were of light infection intensity (100%) independent of municipality, sex, age group and urban/rural schools studied. In the case of T. trichiura and A. lumbricoides standing out.Table p = 0.001). However, double and triple STH infections appear more frequently in Puerto Cabezas than in Siuna municipality, with significant differences . Significant positive associations were reached in the co-infection analysis of T. trichiura with respect to other STH detected in this study, but no associations were observed between A. lumbricoides and Hookworm co-infections.Only single Hookworm infections appear more frequently in Siuna municipality, with significant differences  and in those walking barefoot (p = 0.025). On the other hand, the odds of helminth infection were lowest among children of the urban schools (p < 0.0001).The odds of helminth infection were highest among 6–11-year-old children, in those drinking rain water  has its climatic thresholds for growth and development. In fact, altered climatic parameters due to climate change are bound to influence the biological development of STH.Each of the infective stages of STH . Likewise, significant regional differences in the prevalence and intensity of STH infections have been found in other LAC countries such as Ecuador32, no differences of infection between boys and girls were found. However, prevalence of infection was directly related to age, being highest in the 6–11-year-old group, related to starting playing outdoors and getting exposed to infections26. Moreover, polyparasitism, the most common form of infection, was also age-related. These results agree with previous work in Nicaragua28 and other countries34.Similar to other LAC countries35. The light intensity of T. trichiura is normal in endemic areas where most students harbor low or moderate burdens, while only a few individuals harbor a heavy burden36. Moreover, the moderate intensity of A. lumbricoides appears mostly in children of 6–11 years of age. In agreement with our results, surveys from other parts of the world also found light intensities of T. trichiura and hookworm infections, while values for A. lumbricoides were of light and moderate intensities35.Disease caused by STH is directly related to infection intensity. Although prevalence is used in STH epidemiological surveys, intensity of infection is more important as a determinant of morbidity11. The odds of STH infection among children of R.A.A.N. who had safe water sources at their disposal were lower compared to those who had to rely on unsafe water sources (rain water). This finding corroborates previous LAC study37 and it is in line with the result of a systematic review and meta-analysis which stated that "access to piped water was associated with lower odds of A. lumbricoides and T. trichiura infection"38, as having a water tap means easy access to water for hygienic practices.Adequate sanitation facilities may be important when explaining the spatial variation of helminth infection prevalence39.The odds of STH infection increase with age since there is an age-related change in exposure to STHs, considering that as they get older they are able to play on their own in outdoor activities. This trend was also observed in other studies conducted elsewhere41. The prevalence of Hookworm infection in our work is not the most commonly STH infection detected, but the lack of shoes is actually being a surrogate marker of lower socioeconomic status, which therefore increases the odds of total STH infection.There has been an increase in attention to wearing shoes for preventing soil-related disease conditions as well as STH infection. Previous research estimates that shoe-wearing lowers the odds of Hookworm infection42. In accordance with WHO guidelines8, the children from R.A.A.N. are exposed to a high risk of STH infection  and should thus continue to be treated with anthelmintics twice a year. Moreover, there is a need to increase people’s knowledge on proper and environmental hygiene, mainly in those communities with lack of water sanitation and hygiene (WASH) such as Puerto Cabezas and Siuna. Results obtained in our work can be used by policy makers in Nicaragua to design geographically more specific intervention programs to reduce STH infections.Effective strategies for controlling STH infection would have to center on reducing risk factors together with regular deworming, as anthelminthic drugs kill the parasite within the host but do not prevent reinfections and will not be sufficient to bring prevalence to low levelsCollaborations between research disciplines and between researchers and workers in the field responsible for individual and community health  is imperative to improve outcomes for people at risk from these STH infections.S. stercoralis larvae may not be considered definitive because the specific etiological technique for the detection of this nematode was not applied; and also, the low number of completed questionnaires, as well as the veracity of their responses.The results of our study should be interpreted in the light of some limitations, mainly: only two municipilaties have been studied out of the eight that comprise R.A.A.N.; the used of Kato-Katz technique, which can miss some egg count in an area with light-intensity infections; the prevalence obtained for


All FLs, FNs, and FPs were lowest during the third decade (20–29 years) of life. FLs were elevated consistently after that decade, and FNs were elevated sharply after the seventh decade. FPs were relatively stable after the fourth decade (30–39 years). Mixed-effect regression analyses in subjects 40 years and older showed that older age was associated with worse FLs (p < 0.0001) and FNs (p < 0.0001) but not FPs (p = 0.4126). Aging affects FLs and FNs with different modes but had minimal effects on FPs. Decreased VF sensitivity, deteriorated macular function, and technical difficulties with testing may be mechanisms of age-related changes in FLs and FNs.Relationships between age and visual field (VF) reliability indices were investigated using a large real-world dataset . All VFs tested and stored at Shimane University Hospital between 1988 and 2019 were exported. Correlations between age, mean deviation (MD), pattern standard deviation (PSD), and reliability indices including fixation losses (FLs), false negatives (FNs), and false positives (FPs) were analyzed. The mean ± standard deviation age was 65.0 ± 15.1 years; MD—−6.9 ± 8.1 decibels (dB); PSD—6.3 ± 4.6 dB; FL—8.6 ± 11.7%; FN—5.3 ± 8.3%; and FP—2.6 ± 5.0%. Univariate analyses showed strong associations between age and FNs (correlation coefficient, ρ = 0.20,  The use of semiautomated perimeters in ophthalmology is essential, especially in the management of patients with glaucoma. Early detection of visual field (VF) progression and determining the estimated progression rate are important for managing glaucoma . RetinalThe study adhered to the tenets of the Declaration of Helsinki; the institutional review board (IRB) of Shimane University Hospital reviewed and approved the research (study no. 20080911-1). IRB approval did not require that each patient provide written informed consent for publication; instead, the study protocol was posted at the study institutions to notify participants about the study. In order to perform real-world analysis, all VF data obtained during 42,421 VF tests using the Central 30-2 program Humphrey Visual Field Analyzer , tested with SITA-standard (82%) or full-threshold program (18%), and stored at the Department of Ophthalmology, Shimane University Hospital, between 1988 and 2019 were exported. These VF data, which included 199 cases in which patient identification was unknown, comprised 11,525 eyes of 5930 Japanese subjects . For analyses, subjects’ identification, age at VF testing, mean deviation (MD), pattern standard deviation (PSD), and rates of FL, FNs, and FPs were collected.The data were expressed as the means ± SD for continuous variables. Possible correlations among age and VF indices  were assessed using the Spearman’s rank correlation test. VF reliability parameters  were compared between age groups stratified by 10-year increments  by one-way analysis of variance followed by the post-hoc Tukey honesty significant difference (HSD) test for the adjustment of multi-pair comparisons. In order to further assess the effect of aging on VF reliability, mixed-effect regression analyses were performed in subjects 40 years or older with age, MD, and PSD set as the fixed effect and subject identification and tested eye (right or left) set as the random effect. All statistical analyses were calculated using JMP Pro statistical software version 14.2 .Subject demographic data and their distributions are shown in p < 0.0001); strong correlation was observed between age and FN  or MD (ρ = −0.21).p < 0.0001) and FNs (p < 0.0001) but not with FPs (p = 0.4126).The effect of age on reliability indices was assessed further in subjects 40 years and older by mixed-effect regression analyses to adjust for possible confounding effects derived from the difference in severity of VF defects and from the inclusion of repeated measurements or both eyes of a subject . Older aWe analyzed the relationship between age and reliability indices in a large dataset of perimetry tests. We found that all FLs, FNs, and FPs were lowest in subjects in the third decade of life, FLs were elevated consistently after the third decade of life, FNs were elevated sharply after the seventh decade of life, and FPs were relatively stable after the fourth decade of life. In addition, FLs and FNs were associated with aging, while FPs were not.VF testing is a probabilistic rather than deterministic examination . Due to Considering the data in The limitation of this study was the absence of the consideration of some physical factors such as fatigue and loss of concentration, which may affect the results of reliability indices in that the eye tested second has a greater amount of variability ,32,33. HThis study described the relationships between ages and each reliability indices; FLs are consistently elevated after the third decade of life, and FNs elevated sharply after the seventh decade, while FPs were relatively stable after the third decade of life. These changes are thought to have resulted from the decline in VF sensitivity, macular function deterioration, and technical difficulties that were tested, which were caused by aging.


CRIq_LA was significantly and positively associated with better cognitive performances at all time points (p < 0.05). Our results suggest that higher is the CRIq_LA, milder is the progression of HD in terms of functional, multidimensional and cognitive outcome.We focused on Cognitive Reserve (CR) in patients with early Huntington Disease (HD) and investigated whether clinical outcomes might be influenced by lifetime intellectual enrichment over time. CR was evaluated by means of the Cognitive Reserve Index questionnaire (CRIq), an internationally validated scale which includes three sections: education, working activity, and leisure time. The clinical HD variables were quantified at three different time points  as per the Unified Huntington’s Disease Rating Scale (UHDRS), an internationally standardized and validated scale including motor, cognitive, functional and behavioral assays. Our sample consisted of 75 early manifest patients, withclinical stage scored according to the Total Functional Capacity (TFC) scale. Our correlational analysis highlighted a significant inverse association between CRIq leisure time (CRIq_LA) and longitudinal functional impairment  ( Huntington disease (HD) is a hereditary neurodegenerative disease manifesting with neurological and mental symptoms which may begin at any age, mainly in adulthood. The gene mutation is a CAG triplet expansion that is detectable by a molecular test during the presymptomatic lifetime . UsuallyAccording to the Cognitive Reserve (CR) hypothesis, clinical expression of brain diseases might be attenuated among individuals with higher education or active lifestyles . People The CR hypothesis has been well-supported by studies involving various neurological conditions, such as Alzheimer’s disease (AD) , ParkinsOn the contrary, little is known about CR in HD. While research has begun to disclose the biological factors impacting HD development , the effAltogether, these data support the hypothesis that neuroplasticity is an intrinsic property of the living brain that enables it to reorganize its structure, function and networks in response to internal/external changes  and may represent a potential protective factor in neurodegeneration . RegionaOur study aims to evaluate CR in patients with early manifest HD by investigating whether lifetime intellectual enrichment may be beneficial to cognitive and clinical performance. For this purpose, we used an international validated questionnaire, the Cognitive Reserve Index questionnaire (CRIq), which assesses the acquired lifetime CR in three different areas: education, working activities, and leisure time activities.Starting from a sample of 177 early manifesting patients with at least three yearly clinical examinations, we selected 75 patients based on the following inclusion criteria: (1) motor age at onset above 20 years; (2) Unified Huntington Disease Rating Scale (UHDRS)—Total Motor Score (TMS) > 10 and Diagnostic Confidence Level (DCL) equal to 4 at baseline; (3) UHDRS Total Function Capacity (TFC) > 6 at baseline; (4) no history of neurological conditions other than HD, substance dependence, or developmental disorder affecting cognition at baseline assessment; and (5) no severe psychiatric manifestations. A full patient description is reported in Our study conforms with the World Medical Association Declaration of Helsinki. It received approval by the Institutional Review Board of the coordinator site ; all participants signed a declaration of informed consent.All patients were assessed by health professionals with expertise in HD in accordance with ENROLL-HD guidelines . EvaluatWe performed a comprehensive cognitive evaluation including: (1) Categorical Verbal Fluency Test (VFT); (2) Stroop Color Reading Test (SCR); (3) Stroop Word Reading (SWR); (4) Symbol Digit Modalities Test (SDMT) in the written response format; and (5) Mini Mental State Examination (MMSE), according to ENROLL-HD protocol. We calculated the composite UHDRS (cUHDRS), a multidimensional measure of motor (TMS), cognitive (SDMT and SWR) and functional (TFC) outcome, recently proposed as a sensitive tool for monitoring clinical progression in early HD .https://dpg.unipd.it/en/criq (accessed on 15 January 2019). CRIq is an efficient and reliable tool for measuring CR [CR was assessed by means of the CRIq, an internationally validated instrument that is currently available in ten different languages. The questionnaire is composed of twenty items grouped into three section subscores: (1) years of formal and informal education (CRIq_Edu); (2) working activity experience according to degree of responsibility and cognitive demands (CRIq_WA), with five different levels of working activities available dealing with the degree of intellectual involvement and personal responsibility; and (3) leisure activity, calculated based on the frequency of several activities such as reading, housekeeping, driving, hobbies, travelling etc., on a weekly, monthly, yearly, or stable basis (CRIq_LA) . The toturing CR ; specifiFirst, we conducted an exploratory Pearson’s correlation analysis in order to detect potential relationships between the CRIq scores  and the clinical and genetic variables under investigation (Age at Onset (AO), expanded CAG repeats, ∆TMS (the difference between the TMS score at t2 and t0), ∆TFC, cUHDRS, cognitive variables , and Mini Mental State Examination). The correlation analysis was performed for all time points . In addition, we investigated the correlation between the mutation size  with each clinical variable.Patients were then split into two groups depending on whether the CRIq scores obtained were normal or impaired. CRIq subscores were considered pathological when below 1 Standard Deviation (SD) from the control mean; for more details, see .All dependent variables were submitted into a one-way ANOVA comparing the performance of patients with normal and impaired CRIq subscores.Alpha level was conventionally fixed to ≤0.05. All statistical analyses were performed using IBM SPSS Statistics for Macintosh, version 25.0 .Our cohort (n = 75) included 28 females and 47 males. The demographic characteristics of the sample included age at baseline of 47.2 ± 12.5 years (range 27–78 years), education level of 11.65 ± 4.6 years (range 8–18 years), age at motor onset 47.5 ± 12.3 years (range 22–70 years), and expanded CAG repeat number of 43.7 ± 2.3 (range 40–49 CAG repeats). The clinical and demographic characteristics are summarized in p = 0.037; r = 0.242), cUHDRS_t0 , cUHDRS_t1  and cUHDRS_t2   , Table 2p = 0.001; r = 0.392), VFT , SCR , SWR  and MMSE   and MMSE  as well ; r = 0.359), VFT , SCR , SWR  and MMSE  as well , VFT , SCR , SWR  and MMSE  , CRIq_WA  and CRIq_Tot  with regard to the ∆TFC, which resulted higher in the low CRIq_LA group (−2.08 ± 0.28) in comparison to the high CRIq_LA group (−1.2 ± 0.21). Moreover, the one-way ANOVA showed a significant group effect on cUHDRS at t0 , t1  and t2 , indicating a more pronounced multidimensional progression in the low CRIq_LA than in the high CRIq_LA group (t0: 54.34 ± 8.23 vs. 92.28 ± 6.69) . No significant differences emerged for ∆TMS , SDMT , VFT , SCR  and SWR , SDMT , VFT , SCR  (39.25 ± 2.71 vs. 50.17 ± 2.63) and SWR  at 1 year follow-up, indicating poorer performance in the impaired CRIq_LA group with respect to the normal CRIq_LA group , SDMT , VFT , SCR  and SWR  at 2 years follow-up, showing poorer performance in the group with low CRIq_LA with respect to high CRI_LA  may eventually highlight such a neurobiological effect of the mutation.Our findings highlight a negative correlation between mutation size and measured CR, thus supporting the interesting hypothesis that a larger expanded CAG repeat number in However, our study has limitations. First, even if we enrolled patients without severe psychiatric manifestations, we cannot fully exclude a possible influence of depression or anxiety on cognition. Second, the observational design of our study did not allow us to make causal inferences into the correlations between leisure time and the cognitive and functional domains.Despite these limitations, our findings support the interesting hypothesis that in addition to possible genetic modifiers, the environment may consistently contribute to disease course modification and may represent a therapeutic target. Further studies on larger cohorts investigating the role of leisure time in HD are required in order to confirm our hypothesis and to address conclusions.HTT gene) on the development of HD suggest that the CR may be affected in HD, and that increased leisure time lowers HD progression as measured by functional and multidimensional variables, with more preserved cognitive status over time. These aspects could be considered in clinical management to address HD by directing patients to specific cognitive training programs and clinical trial recruitments in order to minimize potential selection bias.In conclusion, our results highlighting a combined effect of environmental and neurobiological factors (i.e., the expanded CAG repeat number in


Acacia macrostachya, an indigenous African medicinal plant, were investigated. In traditional medicine, the plant is used in the treatment of microbial infections and inflammatory conditions. A crude methanol extract obtained by Soxhlet extraction was partitioned by column chromatography to obtain the petroleum ether, ethyl acetate, and methanol fractions. Antibacterial, efflux pump inhibition and antibiofilm formation activities were assessed by the high-throughput spot culture growth inhibition (HT-SPOTi), ethidium bromide accumulation, and the crystal violet retention assay, respectively. The minimum inhibitory concentrations (MICs) of the crude extract and major fractions ranged from 250 to ≥500 μg/mL. At a concentration of 3.9–250 μg/mL, all extracts demonstrated >80% inhibition of biofilm formation in S. aureus. In P. aeruginosa, the EtOAc fraction showed the highest antibiofilm activity (59–69%) while the pet-ether fraction was most active against E. coli biofilms (45–67%). Among the test samples, the crude extract, methanol, and ethyl acetate fractions showed remarkable efflux pump inhibition in S. aureus, E. coli, and P. aeruginosa. At ½ MIC, the methanol fraction demonstrated significant accumulation of EtBr in E. coli having superior efflux inhibition over the standard EPIs: chlorpromazine and verapamil. Tannins, flavonoids, triterpenoids, phytosterols, coumarins, and saponins were identified in preliminary phytochemical studies. Stigmasterol was identified in the EtOAc fraction. This study justifies the use of A. macrostachya in the treatment of infections in traditional medicine and highlights its potential as a source of bioactive compounds that could possibly interact with some resistance mechanisms in bacteria to combat antimicrobial resistance.Microbial infections remain a public health problem due to the upsurge of bacterial resistance. In this study, the antibacterial, antibiofilm, and efflux pump inhibitory activities of the stem bark of  Antimicrobial resistance (AMR) is a global menace that has jeopardised the effective prevention and treatment of infectious diseases . In bactAcacia macrostachya (Rchb. ex DC) of the family Leguminoseae is one of such useful plants in African traditional medicine. A. macrostachya is a prickly shrub growing to about 5 m high with pubescent twigs, armed with rose-like recurved thorns. The plant is widely distributed in savanna areas in West Africa [A. macrostachya. Other works report the presence of oleanane triterpene saponins in the roots as well as phenolic acids and flavonoids with antioxidant activity in the leafy stems [A. macrostachya (Leguminoseae) were studied.t Africa . Traditit Africa , 8. The t Africa  and for t Africa –9. The rt Africa , 8. The t Africa –9 and ast Africa , 8. Prevt Africa , antidiat Africa , antioxit Africa , anti-int Africa , antiplat Africa  and antit Africa  activitify stems , 16. In fy stems , 18, theChemicals used were purchased from Sigma and Aldrich chemicals, UK. Ciprofloxacin and fluconazole were from Denk Pharma GmbH & Co., Germany. Organic solvents were of analytical grade from BDH laboratory supplies, England.A. macrostachya was collected from Kwahu Asakraka in the Eastern region of Ghana in November 2019 . The plant sample was identified and authenticated by Dr. G. H. Sam, a botanist at the Department of Herbal Medicine, Faculty of Pharmacy and Pharmaceutical Sciences (FPPS), Kwame Nkrumah University of Science and Technology (KNUST). A voucher specimen with reference number KNUST/HM1/2019/S026 was deposited at the Herbarium of the Faculty of Pharmacy and Pharmaceutical Sciences.The stem bark of ww) subsequently referred to as the “crude extract” or “ACF” in this report.The plant material was washed under running water, cut into smaller chunks, sun-dried for 5 days, and coarsely milled. About 2 kg of the powdered stem was Soxhlet extracted using 6 L of methanol (MeOH) at 60°C until the material was completely exhausted (lasted for approximately 8 h). The extract obtained was concentrated under reduced pressure by means of a rotary evaporator. The concentrate was oven-dried at 40°C to yield a dark brown extract weighing 217 g (yield = 10.37% ww), EtOAc , and MeOH  fractions. The crude extract and organic fractions were kept in a desiccator until ready for use.The crude extract (100 g) was adsorbed onto silica gel 60 (70–230) and partitioned into three main fractions by successive elution with petroleum ether , ethyl acetate , and methanol  by column chromatography. This afforded the pet-ether  was used as the reference substance. TPC was expressed as gallic acid equivalent (GAE) in mg/g of dried extract.The total phenolic content of the leaf and stem bark extracts were determined by the Folin–Ciocalteu method . Gallic μg/mL) was used as positive control. A standard curve was prepared using quercetin and the TFC of the extracts extrapolated from this curve. TFC was expressed as quercetin equivalent (QCE) in mg/g of dried extract.The total flavonoids in the leaf and stem bark extracts were quantified by the aluminum chloride colorimetry method . Quercet1H, 13C NMR, and mass spectral data with published data. Details of the isolation procedure are presented in Supplementary Material  was further fractionated by column chromatography (CC) using silica gel 60 (70−230 mesh). Thin-layer chromatography (TLC) was performed using precoated silica gel 60 TLC plates . Mixtures of pet-ether, EtOAc, and MeOH were used for elution in a gradient manner. Pure isolate was identified by comparing Material .Staphylococcus aureus (ATCC 25923) and Streptococcus pyogenes  and two Gram-negative bacteria Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853). These were provided by the Cell Culture Laboratory of the Department of Pharmacology, KNUST. The microorganisms were cultured on nutrient agar as agar slants in falcon tubes. Standardized cultures were prepared by subculturing in sterile nutrient broth and incubating at 37°C for 24 h. Working cultures were prepared by serial dilution with normal saline to achieve an initial cell count of approximately 1 × 105 CFU/mL.The microorganisms used for this study included two Gram-positive bacteria μg/mL in 2% dimethyl sulphoxide, DMSO) was done in a PCR half-skirted plate to give a concentration range of 7.8–500 μg/mL. Aliquots of each dilution (100 μL) were dispensed into corresponding wells of a 96-well plate and mixed with 200 μL of molten agar (stabilized at 45°C). The plates were swirled to mix, allowed to set, and then spotted with 2 μL of standardized microbial suspension of the test organism. The plates were allowed to stand for 20 minutes, sealed, and incubated at 37°C for 24 h. Ciprofloxacin was included as the positive control and 2% DMSO, negative control. The presence or absence of growth was determined by visual examination comparing with the control wells.The antibacterial activity of the crude extract and its organic fractions were determined using the High Throughput Spot Culture Growth Inhibition Assay (HT-SPOTi) as described by Danquah et al. and the method description partly reproduced their wording . BrieflyS. aureus, E. coli, and P. aeruginosa was investigated using the microplate crystal violet stain retention method previously described by Abidi et al. and the method description partly reproduces their wording [S. aureus, E. coli, and P. aeruginosa prepared in tryptone soy broth  were used for the assay. The test samples were solubilized in DMSO (2%) and reconstituted in TSB to achieve a concentration range of 3.9–250 μg/mL. Aliquots of the microbial culture (180 μL) and the test samples (20 μL) were pipetted into a flat bottom 96-well microtiter plate and incubated at 37°C for 24 hours. Negative controls were included in the plate. The experiment was performed in triplicate. After incubation, the supernatant was aspirated and each well was washed with phosphate buffer saline  to remove planktonic cells. The adherent biofilms were fixed by drying at 50°C for 30 minutes and stained with 200 μL of 0.1% w/v aqueous solution of crystal violet (CV) for 10 minutes at room temperature. The wells were carefully washed with sterile water and the stain bound to cells solubilized with 200 μL of 95% ethanol. Absorbance was measured at 600 nm using an automated microplate reader (BioTek Synergy H1 Hybrid Multi-Mode Reader: 271230). The mean absorbance was determined, and the percentage inhibition of biofilm was calculated asThe effect of the crude extract and fractions on biofilm formation by  wording . OvernigS. aureus, E. coli, and P. aeruginosa were prepared in Mueller Hinton broth (MHB) and incubated at 37°C with shaking at 150 rpm until an optical density (OD600) of 0.8–1 was achieved. The OD of the cultures was adjusted to 0.4 in 10 mL MHB and centrifuged at 3000 rpm for 15 minutes. Pellets were washed and vortexed with phosphate buffer saline (PBS). Aliquots (500 μL) of the bacteria suspension and PBS (500 μL) were pipetted into separate Eppendorf tubes to serve as the test and blank respectively. Filter sterilized glucose solution (0.4%) and different volumes of the extracts and fractions prepared at ½ MIC were added to the test and blank Eppendorf tubes. Aliquots of 100 μL from each mixture were pipetted into a 96-well microtiter plate to which 5 μL of EtBr (0.5 mg/L) was added. Florescence was measured at an emission wavelength of 585 nm and excitation wavelength of 530 nm every 5 minutes over a period of 60 minutes at 37°C using a microplate reader . Verapamil and chlorpromazine served as reference drugs and DMSO as the negative control.Efflux pump inhibitory activity was investigated by the ethidium bromide (EtBr) accumulation assay described by Prasch et al. and the method description partly reproduces their wording . BrieflyAll experimental results were analysed using GraphPad prism .A. macrostachya revealed the presence of various classes of phytochemicals including tannins, saponins, triterpenoids, phytosterols, flavonoids, and coumarins. Alkaloids were not detected  and was expressed as gallic acid equivalent (GAE). TFC was determined from a standard curve of quercetin using equation y = 0.007687x + 0.1365; (R2 = 0.9106) and was expressed as quercetin equivalent (QCE) . Both phenolic and flavonoid contents varied with respect to the solvent used for extraction with methanol extracting the highest amounts of phenolic and flavonoid constituents. The results are summarized in Quantitative determination of the total phenolic content (TPC) and total flavonoid content (TFC) for the stem bark was also determined. TPC was obtained from a standard curve of gallic acid using the equation 1H and 13C NMR data .Fractionation of the bioactive EtOAc fraction by chromatography resulted in the isolation of a known phytosterol, stigmasterol , identifμg/mL . S. aureus was resistant to ciprofloxacin at the highest concentration tested (10 μg/mL).The crude extract and its fractions showed varying degrees of antimicrobial activity against Gram-positive and Gram-negative bacteria in the HT-SPOTi test. The MICs for susceptible bacteria ranged from 250 to ≥500 μg/mL . The cruS. aureus, E. coli, and P. aeruginosa at a concentration range of 3.9–250 μg/mL. The percentage biofilm inhibition for all samples is demonstrated in Figures S. aureus, all samples demonstrated remarkable inhibition with the highest effect given by the ethyl acetate fraction (AEF). The percentages of biofilm inhibitions recorded against S. aureus were AEF (84–90%) > AMF (71–90%) > ACF (63–83%) > APF (43–90%). In P. aeruginosa, the ethyl acetate fraction (AEF) demonstrated the highest activity with 59–69% inhibition of biofilm formation  (The crude extract (ACF) and major fractions  were tested for biofilm formation inhibitory effect against ormation . Much lo(45–67%) .S. aureus, E. coli, and P. aeruginosa in the presence of the crude extract (ACF), MeOH (AMF), EtOAc (AEF), and pet-ether (APF) fractions measured over 60 mins. This was compared to the action of two standard efflux pump inhibitors (EPIs): verapamil (VP) and chlorpromazine (CP).The potential efflux pump inhibitory effect of the crude extract and fractions was assessed in the ethidium bromide (EtBr) accumulation assay. S. aureus, AMF and ACF (at ½ MIC) displayed remarkable efflux pump inhibition resulting in high EtBr fluorescence  resulted in significant accumulation of EtBr with AMF having superior efflux inhibition over the standard EPIs: CP and VP  retention assay against three biofilm-forming bacteria: S. aureus, E. coli, and P. aeruginosa. This assay is based on the ability of CV, a basic dye, to nonspecifically bind to an extracellular polymeric substance secreted by bacteria for biofilm formation. The extent of binding indicates active biofilm formation which can be verified spectrophotometrically [The production of biofilms is a passive resistance strategy characterized by the formation of a polysaccharide matrix around the bacterium, which consequently leads to an obstruction of the passage of antibiotics, thus making the bacterium highly resistant to these molecules . The anttrically .S. aureus, P. aeruginosa, and E. coli. varied according to the type of solvent fraction and was not concentration-dependent. The complexity of mixtures of active phytoconstituents distributed in the various solvents used for extraction could result in this remarkable, yet scattered antibiofilm activity observed for the test samples. This approach of investigating bioactivity based on various solvent fractions is appropriate as it can lead to the identification of specific bioactive compounds [A. macrostachya, a previous study by Tchatchedre et al. revealed that the EtOAc and EtOH extracts of A. macrostachya stems significantly inhibited quorum sensing (QS) in C. violaceum and P. aeruginosa [A. macrostachya in this study could be attributed to possible quenching of quorum sensing mechanisms in the bacteria. Extracts from medicinal plants have been shown to interrupt bacteria biofilm formation through mechanisms such as damaging microbial membrane structures, inhibiting peptidoglycan synthesis [Acacia species including A. nilotica and A. dudgeon have demonstrated remarkable antibiofilm-forming effects against S. aureus, E. coli, and P. aeruginosa [Acacia [β-sitosterol have demonstrated remarkable antibiofilm activities in pathogenic bacteria [From the results, the antibiofilm-forming activity of the test samples against ompounds . Though ruginosa . Studiesruginosa , 33. Theynthesis , and/or ynthesis . Acacia ruginosa , 37. The [Acacia  makes th [Acacia . Flavono [Acacia . Pentacybacteria , 40.A. macrostachya demonstrated remarkable efflux pumps inhibition in both Gram-positive and Gram-negative bacteria comparable to standard EPIs. Some Acacia species have shown resistance modifying activities towards multidrug-resistant strains which produce efflux pumps. A. Senegal restored the antibiotic effects of chloramphenicol and florphenicol against multidrug-resistant strains of E. coli  and K. aerogenes  that overproduce AcrAB or FloR pumps [A. polyacantha extracts and compounds also inhibited the growth of multidrug-resistant Staphylococcus bacterial strains overexpressing efflux pump [The intracellular accumulation of ethidium bromide (EtBr) has been used severally to confirm efflux pump inhibition using the whole-cell method , 41, 42.oR pumps . A. polylux pump , 44.A. macrostachya extracts to inhibit quorum sensing [A. macrostachya in this study highlights the plant's potential as a source of bioactive compounds that could act as both EPIs and biofilm inhibitors to curb antibacterial resistance.Studies demonstrated a correlation between the QS signalling system and the control of the expression of the genes involved in active efflux systems . The abi sensing  could be sensing . The antA. macrostachya stem bark. Further studies to ascertain its safety as well as identifying specific constituents responsible for the observed activities through chromatographic purification and spectrophotometric methods are considered in future works.The current study demonstrates remarkable antibacterial activity by Acacia macrostachya. This gives scientific credence to the traditional uses of the plant for managing infections and highlights A. macrostachya as a potential source of bioactive compounds to both antibiofilm and efflux pump inhibitory activity.This study has provided the first evidence of the antibiofilm formation and efflux pump inhibitory potentials of the stem bark of


Background: Vancomycin is widely used for empirical antimicrobial therapy in critically ill patients with sepsis. Continuous infusion (CI) may provide more stable exposure than intermittent infusion, but optimal dosing remains challenging. The aims of this study were to perform population pharmacokinetic (PK) analysis of vancomycin administered by CI in intensive care unit (ICU) patients to identify optimal dosages. Methods: Patients who received vancomycin by CI with at least one measured concentration in our center over 16 months were included, including those under continuous renal replacement therapy (CRRT). Population PK was conducted and external validation of the final model was performed in a dataset from another center. Simulations were conducted with the final model to identify the optimal loading and maintenance doses for various stages of estimated creatinine clearance (CRCL) and in patients on CRRT. Target exposure was defined as daily AUC of 400–600 mg·h/L on the second day of therapy (AUC24–48 h). Results: A two-compartment model best described the data. Central volume of distribution was allometrically scaled to ideal body weight (IBW), whereas vancomycin clearance was influenced by CRRT and CRCL. Simulations performed with the final model suggested a loading dose of 27.5 mg/kg of IBW. The maintenance dose ranged from 17.5 to 30 mg/kg of IBW, depending on renal function. Overall, simulation showed that 55.8%  of patients would achieve the target AUC with suggested dosages. Discussion: A PK model has been validated for vancomycin administered by CI in ICU patients, including patients under CRRT. Our model-informed precision dosing approach may help for early optimization of vancomycin exposure in such patients. Staphylococcus aureus (MRSA) and ampicillin-resistant enterococci [In critically ill patients, infection due to Gram-positive bacteria remains a major cause of mortality . Vancomyerococci . Vancomyerococci ,4. Moreoerococci . The traerococci ,7. Indeeerococci ,11,12,13erococci  (TDM), werococci .In addition to the advantages cited above, CI of vancomycin can facilitate AUC-based therapeutic drug monitoring that is now recommended . Indeed,However, optimal dosing of vancomycin administered by CI in critically ill patients remains challenging. Clinicians must adjust the initial loading and maintenance doses to the patient’s needs before TDM can be performed and used for subsequent individualization.While there are some recommendations to use a 20–35 mg/kg loading dose, there is no formal recommendation regarding the maintenance dosing regimen. Some studies have suggested to use a higher dosage of vancomycin in case of continuous infusion ,19.Therefore, this study aimed to perform a population pharmacokinetics analysis of vancomycin administered by continuous infusion in critically ill patients, and to determine a dosing regimen to better achieve target exposure over the first 48 h of therapy.Patient characteristics recorded on hospital admission  are described in In the learning set, 335 vancomycin concentrations were available from 78 patients hospitalized in ICU. The mean TDM follow up time was 4.1 ± 2 days. Mean vancomycin loading and maintenance dosages were 22.7 ± 7.5 mg/kg and 28.6 ± 9.4 mg/kg/day respectively. A total of 22 out of 78 patients (28.2%) of patient were under CRRTIn the validation group, 417 samples were obtained from 84 patients. The mean TDM follow-up time was 5.0 ± 1.9 days. Mean vancomycin loading and maintenance dosages were 22 ± 5.8 mg/kg and 25.2 ± 7.6 mg/kg/day, respectively, and 4 out of 84 patients (4.8%) of patients were under CRRT.The model that best described the data was a two-compartment model with a proportional residual error. Mean vancomycin clearance (CL), central volume (V1), intercompartment clearance (Q), and peripheral volume (V2) were 0.79 L/h, 27.3 L, 6.1 L/h, and 63.1 L, respectively. All parameters were estimated with an acceptable standard error, as shown in EFR were retained as influential covariates in the final model. Vancomycin CL was modeled as a function of CRCL in patients without CRRT, and as a function of CRRTEFR in patients on renal replacement therapy. V1 was allometrically scaled to ideal body weight. Population and individual predictions from the model correlated well with observations, and bias and imprecision were low, as shown in Creatinine clearance, IBW, and CRRTObserved concentrations (Y axis) versus predicted concentrations (X axis) are shown for population predictions (left panel) and individual predictions (right panel). The black line is the identity line (Y = X) and the red line is the linear regression line. Bias is the mean error of prediction. MAE = mean absolute error and RMSE = root mean squared error.Concentrations of vancomycin (Y axis) are plotted against time after the first dose (X axis). Black dots represent observed concentration data. Blue lines represent the 10th, 50th (median) and 90th percentiles of observations. Blue areas represent the 95% prediction interval of the 10th and 90th simulated percentiles, while the magenta area represents the 95% prediction interval of the simulated median.Observed concentrations (Y axis) versus predicted concentrations (X axis) are shown for population predictions (left panel) and individual predictions (right panel). The black line is the identity line (Y = X), the red line is the linear regression line. Bias is the mean error of prediction. MAE = Mean absolute error, RMSE = Root mean square errorResults of dosing simulations targeting AUC24–48 h between 400 and 600 mg·h/L are summarized in 24–48 > 400 are shown in Results of the second set of simulations targeting AUCCL, and CRRT were found to influence vancomycin PK. Simulations performed with the final model provided initial dosage regimens optimizing achievement of the AUC target in this clinical setting.This study investigated the population PK of vancomycin administered as continuous infusion in critically ill patients. The final model adequately described the data and was externally validated. Ideal body weight, CRVancomycin remains widely prescribed as a probabilistic antibiotic therapy to treat sepsis in critically ill patients . However−1 (of TBW) and median volume of distribution of 0.864 L/kg (TBW). Those values are higher than the estimates from our study, with a typical CL of 0.0185 L h−1 kg−1 (TBW) and a typical V1 of 0.43 L kg−1 (TBW). However, we reported a larger coefficient of variation of V1 (63.8%) while the coefficient of variation of CL in our cohort was similar to previous study results [p = 0.067). The effect of inotropic agents on vancomycin PK has been described elsewhere [In a recent review on vancomycin population pharmacokinetics model , the aut results . It has  results  reportedlsewhere .We found that IBW was a better descriptor of vancomycin V1 compared with TBW. Alternative body size descriptors have been thoroughly studied in pharmacokinetic studies in pediatric and obese adults . HoweverCL based on IBW) and CRRT, as expected considering the renal transport of vancomycin… Darren et al. reported a great variability of vancomycin pharmacokinetics in patients receiving CRRT [EFR as a quantitative descriptor of vancomycin CL clearance instead of setting CRRT as a binary variable. Overall, the use of CRRTEFR and CGIBW resulted in the best estimation of vancomycin clearance in our cohort.Vancomycin clearance was influenced by renal function (CRing CRRT . It has ing CRRT . This isPrevious studies recommended maintenance dosing regimens of vancomycin ranging from 20 to 35 mg/kg (TBW) to achieve target plateau concentration of 20–25 mg/L ,23. DosaThis study has several limitations. First, it was performed in routine clinical conditions and some errors may have occurred in blood sampling and data recording. Second, blood sampling was sparse, which may alter the precision of estimation for some PK parameters, such as V1 and Q. Finally, information on other drugs co-administered with vancomycin was not recorded.Overall, our model performed well with low bias and acceptable precision. External validation yielded similar results, suggesting robust application in critically ill patients. Continuous vancomycin infusion appear safer than intermittent infusion ,34. Our This study was carried out in patients hospitalized in two ICU centers during two different periods.® device [Data of the first population of patients (learning dataset) were collected between December 2013 and April 2015 in center 1 . Inclusion criteria were as follows: age ≥ 18 years, hospitalization in ICU, administration of continuous IV vancomycin, vancomycin therapy initiated in the ICU unit, vancomycin TDM with at least one measured vancomycin concentration, and hemodynamic monitoring with the Picco® device . The latData of the second population of patients  were retrospectively collected from February 2019 to September 2020 in center 2  as part of vancomycin routine TDM. As we performed a retrospective analysis of anonymized data collected in routine care, patients’ consent and ethics approval were not required for this part, as stated in French regulations for clinical research .In both populations, TDM was performed as part of routine patient care, in accordance with guidelines . In mostCL) estimated by the Cockcroft–Gault equation with total (CGTBW), ideal (CGIBW), and adjusted body weight (CGAjBW). If the patient had anuria or was undergoing CRRT, the value of CRCL was set to 0 mL/min. The use of CRRT was also collected, as well as the CRRT effluent flow rate (CRRTEFR) calculated according to the CRRT technique. It is important to note that all patients were administered continuous RRT. As patients were under invasive hemodynamic monitoring PiCCO® , pulmonary vascular permeability index (PVPI), extravascular lung water index (EVLWI), and cardiac index were also collected. Finally, information regarding vancomycin therapeutic drug monitoring (TDM) including dosing history and administration times were collected.Vancomycin assays were performed by an immunoturbidimetry method on an Abbott Architect C8000 analyzer. In plasma, the lower limit of quantification was 1.1 μg/mL, and the linear range of the assay was 1.1 to 100 μg/mL. In the population used for model building, several variables were collected from the computerized patient files, including anthropometric measurements such as age, sex, total body weight (TBW) measured every day, height, body temperature, SOFA, BMI, and the presence of septic shock at admission time as defined by the Sepsis 3 guidelines . AlternaData used in routine vancomycin TDM were collected in another group of critically ill patients in another center of the University Hospitals of Lyon from February 2019 to September 2020.X is the parameter value , popX is the population value of X, COV is the covariate value normalized by the median value in the population (medianCOV), and α is the coefficient of the power relationship.Population pharmacokinetic (PPK) modeling was performed using Monolix software . We used a nonlinear mixed effect modeling approach and the Stochastic Approximation of Expectation maximization algorithm (SAEM) implemented in Monolix. We assumed a log normal distribution of PK parameters to describe inter individual variability (IIV). Continuous covariates were introduced into the model as follows (1):X is the pharmacokinetic parameter, COV is the binary covariate value (0 or 1), popX is the population value of X when COV = 0, and βCOV is the coefficient of the covariate effect.The model for categorical covariate was as follows (2):All covariates collected were tested to study their influence on PK parameters using a stepwise procedure, based on changes observed in the objective function value (OFV) computed by Monolix. OFV was assumed to follow a chi-squared distribution. P-values of 0.05 and 0.01 were considered statistically significant for forward selection and backward deletion of covariates, respectively. The best structural error and covariate model was identified based on several classical criteria and diagnostics including the objective function value, parameter value along with their relative standard error, plots of predicted vs observed concentration and residuals, and simulation-based diagnostics with prediction-corrected visual predictive checks (VPC).External validation was performed with the validation dataset. Predictive performance was assessed by comparing observed vs predicted concentration plots and computation of bias (mean error of prediction), mean absolute error (MAE) and root mean squared error (RMSE) in both datasets. Details about validation criteria in population pharmacokinetics have been presented elsewhere ,42.The final model was then implemented in Simulx  to perform simulations of various loading and maintenance dosing regimens.CL influenced vancomycin clearance in the final model, simulations were performed for various stages of CRCL ranging from 10 to 150 mL/min, with a 10 mL/min increment. In addition, two values of CRRTEFR of 30 and 20 mL/min were considered for dosing in patients under CRRT. As IBW was found to influence vancomycin volume of distribution, weight-based dosing was used in all simulations. For each stage of renal function, dosages ranging from 5 to 40 mg/kg of IBW were simulated, with a 2.5 mg/kg increment.Because CRFor each set of simulations, 500 virtual patients were simulated and their vancomycin PK profiles were estimated. Each simulation was run 20 times, in order to account for variability in random sampling. The endpoint considered was the AUC24–48 h on the second day of therapy, because the loading dose resulted in higher vancomycin exposure on the first day. The AUC24–48 h target on day 2 was based on 2020 IDSA guidelines . We assuConsidering that an alternative goal of initial therapy may be to maximize the effect in some patients with severe infection, a second set of simulations was performed to identify the dosage that would minimize underexposure, so maximizing the proportion of AUC24–48 h > 400 mg·h/L. A proportion ≥ 90% was considered as acceptable.This is the largest population pharmacokinetic study of vancomycin administered by CI in critically ill patients. Our final model provided good predictive performance in external validation. Our results support IBW as a covariate, influencing vancomycin PK and dosing. Based on dosing simulations, we suggest a loading dose of 27.5 mg per kg of IBW in all patients. Maintenance dose should be adjusted to renal function or CRRT intensity. Those regimens should optimize the achievement of AUC target of 400–600 mg·h/L over the first two days of therapy. TDM remains necessary for individualization throughout therapy in those unstable patients.


However, pine stands located in western and central Poland, and in the northwest near the Baltic Sea, exhibited higher Hg concentrations in foliage than in eastern regions. Hg content in foliage depends on the mean temperature of the driest quarter, as well as on Hg content in soils. This indicates that the periods of drought observed in recent years in Poland may affect Hg concentrations in pine foliage.This work evaluates current mercury (Hg) contamination in Poland, represented by the Hg concentrations in Scots pine foliage. Samples were collected over 295 investigation plots in monitoring grids throughout Poland, from pines aged between 12 and 147 years. Analyses were conducted with consideration of bioclimatic factors and soil properties. Concentrations in the pine foliage did not exceed the values characteristic of an ecosystem unaffected by industrial pollution, ranging from 0.0032 to 0.0252 mg kg Mercury (Hg) pollution is one of the most serious environmental problems, mainly due to its toxicity to environmental and human health . Long-teHg is emitted from natural sources—such as volcanic eruptions, ocean evaporation, geothermal processes, and weathering of minerals containing Hg ,4—or as Poland’s Hg emissions are among the highest in Europe , due to Due to the threats posed to ecosystems and human health, both local and global Hg monitoring are critical to meeting the international Hg treaty, as well as to most national policies . One mea0 volatilized from the soil. Soils have different potentials for Hg storage, depending on texture, soil organic matter content, and pH [The Hg in plant foliage comes mainly from the air, because Hg uptake from the soil tends to be accumulated in the belowground biomass , and the translocation of this element above ground is poor ,19. For , and pH ,22, so dPinus sylvestris L.) is one of the most commonly used tree species in biomonitoring in temperate climate zones, due to its wide geographical range, broad ecological amplitude, and high tolerance to pollution. A good indicator of environmental pollution is the pollutant content in foliage [Scots pine  with 25 m resolution and vertical accuracy (random mean square error) of +/− 7 m. Local microclimate was characterized by aspect and slope position. The local topographic wetness index (TWI) and slope position were derived from the DEM data . ElevatiThe soils of the research plots were characterized by a diversity of parent rock types  and groups  .2O in mineral layers  ranged from 3.5 to 8.7. Cation-exchange capacity (CEC) also showed a large variation, ranging from 0.76 to 60.77 cmol(+) kg−1. The carbon content in surface soil layers (0–10 cm) ranged from 0.3 to 47.7% [−1; in 0–10 cm layers was 0.02 mg kg−1; and in both the 10–40 cm and 40–100 cm layers was 0.01 mg kg−1  was selected. From the first branch located in the southwest exposure of the crown, 25 pairs of one-year-old needles were collected from the seventh whorl, counting from the apex. The samples were taken during two vegetation seasons in 2015 and 2016 .−1, and uncertainty of 0.0022 mg kg−1. The ERM analysis was performed at the beginning, and again at the end, of each experimental run (number of samples = 50). The ERM and each sample were measured twice, with acceptable differences between the measurements < 10% (recoveries above 90%).In the laboratory, the needle samples were dried at 65 °C, ground, and then the Hg content was determined using a DMA-80 Hg analyzer (Milestone), via drying and thermal decomposition . Qualityp = 0.05 and p = 0.01. The significance of individual independent variables in multiple regression equations was tested using the t-test at a significance level of p < 0.05. The statistical analyses were carried out using STATISTICA 13.1 software   .p = 0.05). The spatial interpolation analysis for visualization of Hg concentration distribution in pine foliage in Poland was performed using the IDW module of QGIS 3.10 software (QGIS Development Team)  .−1 to 0.0252 mg kg−1 (mean: 0.01 mg kg−1). Relatively higher Hg concentrations (> 0.01 mg kg−1) were found in central (area I) and western Poland (area II). In addition, Hg concentrations in pine foliage exceeding 0.02 mg kg−1 were found in the vicinity of Zielona Góra (HS I), in the copper-exploitation region (HS II), and in the vicinity of Janów Lubelski (HS III) (Hg concentrations in Scots pine foliage ranged from 0.0032 mg kg(HS III) .−1) and II (0.0129 mg kg−1) were significantly higher than in area III (0.0075 mg kg−1) (We determined that the average Hg concentrations in areas I (0.0128 mg kgmg kg−1) .Hg content in pine foliage correlated positively with the mean temperature of the driest quarter (BIO9), annual precipitation (BIO12), precipitation in the driest month (BIO14), precipitation in the driest quarter (BIO17), and precipitation in the coldest quarter (BIO19). Foliar Hg content correlated negatively with mean diurnal range (BIO2), temperature seasonality (BIO4), maximum temperature of the warmest month (BIO5), annual temperature range (BIO7), mean temperature of the wettest quarter (BIO8), and precipitation seasonality BIO15; .No significant correlation was found between foliar Hg content and stand ages or soil properties . The lack of correlation with soil properties indicates that Hg content in pine needles does not depend on the soil parent material or geological strata, because different parent materials and geological strata have different properties. No significant correlation was found between foliar Hg content and elevation, aspect, slope position, or TWI index.However, multiple regression analysis showed that foliar Hg content was related to the BIO9 and Hg content in the 0–100 cm soil layer. The determination coefficient explains 53% of the variation in foliar Hg content  [−1 in the plant’s low-impact zone, up to 0.0615 mg kg−1 in the high-impact zone) [−1 [−1 [Although Hg content in Scots pine foliage in Poland did not exceed the values found in ecosystems outside industrial emission areas, this study examined the spatial variation in mercury content across the country. According to Gworek et al. , plants mg kg−1) , and thect zone) . In urbaone) [−1 . However [−1 [−1 , which f−1 overlaps with the emission levels estimated, for 2015, above 10 g km−2 year−1, and Hg deposition above 15 g km−2 year−1 [−2 year−1, and Hg deposition above 30 g km−2 year−1 [Spatial variation in Hg concentrations in Scots pine foliage is consistent with the estimates of spatial emission and deposition of Hg in Poland by Bartnicki et al. ,38. It c2 year−1 . However2 year−1 ,39. Ther2 year−1 . The abs2 year−1 . −1) in pine foliage occurred in three plots: The high Hg content in the first point (around Polkowice) resulted from the nearby Żelazny Most copper mining flotation waste landfill, 500 m to the south. The Żelazny Most flotation waste landfill is the largest of its kind in Europe, and has a negative impact on adjacent ecosystems through wind erosion and transport of pollutants—including trace elements—to both ground and surface waters [−1 according to Gworek et al. [The highest Hg concentrations ; howevek et al. ,43. The 2, i.e., ~60% of the sea area) were 4300 ± 1600 kg, and accounted for less than 5% of Hg emissions across Europe. According to Kuss and Schneider [−2 yr−1, and was higher in summer than in winter [Interestingly, higher Hg concentrations in pine foliage were found in the vicinity of the Baltic Sea. Seas are an important sink for mercury deposition. Evaporation from the seas is one of the main factors of natural Hg emission to the environment, and also results from the re-emission of earlier depositions of anthropogenic origin. The sea is a Hg sink in winter, and an emission source during the growing season . It was chneider , the hign winter . The decn winter . The sean winter . For the0 volatilized from the soil. The Hg content in the air often increased with increasing temperature, which was likely caused by temperature-dependent surface emissions [Atmospheric Hg reaches plants via both dry and wet deposition ,49. Precmissions . The stumissions  estimatemissions . Howevermissions . At verymissions . This caNo significant correlations were noted between Hg concentration in pine foliage and soil properties, including Hg content in soils, confirming the limited translocation of this element from the roots to the aboveground biomass ,19. Such−1 overlap with the emission levels estimated for 2015 above 10 g km−2 year−1 and Hg deposition above 15 g km−2 year−1. However, based on Hg concentrations in Scots pine foliage, no “hot spots” were found in Upper Silesia, with its significantly higher emissions and the negative environmental effect of industrial pressure in recent years. Hg concentrations in pine foliage were positively correlated with the mean temperature of the driest quarter and annual precipitation. This correlation may indicate that Poland’s recent droughts may affect Hg re-emissions from stock accumulated in ecosystems from previous anthropogenic emissions.Hg concentrations in Scots pine foliage did not exceed the values typical for ecosystems without industrial pollution, which are considered non-threatening. Spatial variation in Hg concentrations in Scots pine foliage did not overlap with spatial variation in Hg contamination in Polish soils, but was consistent with the estimates of Hg emission and deposition. It can be assumed that Hg concentrations in pine foliage above 0.01 mg kg


Pyrus pyrifolia) to facilitate its genetics and genomics as well as breeding programs, in which a variety 'Nijisseiki' with superior flesh texture has been used as a parent for most Japanese pear cultivars. De novo assembly of long sequence reads covered 136× of the Japanese pear genome and generated 503.9 Mb contigs consisting of 114 sequences with an N50 value of 7.6 Mb. Contigs were assigned to Japanese pear genetic maps to establish 17 chromosome-scale sequences. In total, 44,876 high-confidence protein-encoding genes were predicted, 84.3% of which were supported by predicted genes and transcriptome data from Japanese pear relatives. As expected, evidence of genome-wide duplication was observed, consistent with related species. This is the first chromosome-scale genome sequence analysis reported for Japanese pear, and this resource will support breeding programs and provide new insights into the physiology and evolutionary history of Japanese pear.We analyzed the genome sequence of a Japanese pear ( Pyrus spp.) is a genus of the Malinae subtribe of the Rosaceae that includes European pear (P. communis), Chinese white pear (P. × bretschneideri), Japanese pear (P. pyrifolia), and apple . The Japanese pear variety ‘Nijisseiki’ (a Japanese term referring to the 20th century) was discovered in Matsudo  in 1888.Pear (P. betulifolia and (P. ussuriensis × communis) × spp.) that are used for root stocks.,,Genome information can enhance breeding programsLong-read sequencing, also known as single-molecule real-time sequencing  and nanopore sequencing , has several advantages over short-read technologies based on a sequencing by synthesis method .2.1.P. pyrifolia), variety ‘Nijisseiki’, which is planted at the orchard of Kyoto Prefectural University , was used for genome sequencing analysis. Genome DNA was extracted from the young leaves by a modified sodium dodecyl sulphate method.A single tree of Japanese pear (2.2.AP012207 and KY563267),A short-read sequence library was prepared using a PCR-free Swift 2S Turbo Flexible DNA Library Kit  and converted into a DNA nanoball sequencing library with an MGI Easy Universal Library Conversion Kit (MGI Tech). The library was sequenced on a DNBSEQ G400RS (MGI Tech) instrument in paired-end, 101-bp mode. The obtained reads were used to estimate the genome size with Jellyfish after removing low-quality bases  with PRINSEQ, adaptor sequences (AGATCGGAAGAGC) with fastx_clipper in FASTX-Toolkit, and reads from organelle genomes ,Protein-encoding genes were predicted by a MAKER pipeline. Two presets of Augustus and SNAP for Arabidopsis were used as training data for Repetitive sequences in the PPY_r1.0.pmol assembly were identified with RepeatMasker, using repeat sequences registered in Repbase and a de novo repeat library built with RepeatModeler. The repeat elements were classified into nine types: short interspersed nuclear elements (SINEs), long interspersed nuclear elements (LINEs), long terminal repeat (LTR) elements, DNA elements, Small RNA, satellites, simple repeats, low complexity repeats, and unclassified, in accordance with RepeatMasker.3.1.K-mer distribution analysis indicated that the ‘Nijisseiki’ genome was highly heterozygous, and that the estimated haploid genome size was 537.1 Mb . Of these, reads ≥15 kb in length  were selected and assembled into 750.0 Mb contigs (495 sequences with an N50 of 5.9 Mb). The contig sequences were resolved into primary contigs and haplotigs, and polished to correct potential errors. The sizes of the resultant sequences were 745.8 Mb for the primary contigs and 113.3 Mb for the haplotigs, which represented genome sequences of one haploid genome sequence and another haploid of highly heterozygous regions in the diploid genome, respectively. As this primary contig size was longer than the estimated haploid size, we doubted the existence of duplicate sequences. Hence, 257 haplotype duplications in the primary contigs (241.6 Mb) identified by a read depth distribution pattern of Purge_Dups  were exc3.2.,Two previously established genetic maps for Japanese pearThe structure of the pseudomolecule sequences was compared with those of apple, European pear, and Chinese white pear to reveal one-to-one synteny relationships at chromosome level between the species . Further3.3.Initial predictions suggested 85,013 likely genes in the genome assembly. This gene number was resolved to 44,876  after reThe genome positions of 37,835 (84.3%) of the 44,876 genes were covered with those of transcripts of Japanese pear and/or peptide sequences predicted from the genomes of apple, European pear, and Chinese white pear . FunctioRepetitive sequences occupied a total of 280.3 Mb (55.6%) of the PPY_r1.0.pmol (503.8 Mb) assembly. The repeats consisted of nine major types in varying proportions . The domPyrus genus.Pyrus spp.,Pyrus genomics. Genome structures and genome-wide duplication events were conserved among pears and apples (,Here, we present the first reported genome sequence analysis of Japanese pear, complementing the existing available genome sequences of other members of the d apples  despite The ‘Nijisseiki’ variety of Japanese pear was used for the genome analysis. Although the parents of the ‘Nijisseiki’ variety are unknown, its favourable flesh textureThe Japanese pear genome will provide new insights into the physiology and evolutionary history of Malinae and will facilitate Japanese pear breeding programs. Further analysis will perhaps contribute to identify the relevant genetic mechanisms for favourable flesh texture of ‘Nijisseiki’ and its progeny cultivars.dsab001_Supplementary_DataClick here for additional data file.