Extracellular vesicles (EVs) have emerged as novel biomarkers and therapeutic material. However, the small size (~200 nm) of EVs makes efficient separation challenging. Here, a physical/chemical stress-free separation of EVs based on diffusion through a nanoporous membrane chip is presented. A polycarbonate membrane with 200 nm pores, positioned between two chambers, functions as the size-selective filter. Using the chip, EVs from cell culture media and human serum were separated. The separated EVs were analyzed by nanoparticle tracking analysis (NTA), scanning electron microscopy, and immunoblotting. The experimental results proved the selective separation of EVs in cell culture media and human serum. Moreover, the diffusion-based separation showed a high yield of EVs in human serum compared to ultracentrifuge-based separation. The EV recovery rate analyzed from NTA data was 42% for cell culture media samples. We expect the developed method to be a potential tool for EV separation for diagnosis and therapy because it does not require complicated processes such as immune, chemical reaction, and external force and is scalable by increasing the nanoporous membrane size. Extracellular vesicles (EVs), which play key roles in intercellular communication between distant cells, are classified by their sizes and biogenesis mechanisms . For exag, pρ, rρ, r, and η are the gravitational acceleration, density of the particle, density of the fluid, radius of a spherical particle, and dynamic viscosity of the fluid, respectively. The sedimentation velocity is a function of the r2, resulting in separation that is efficient for large (>1 μm) particles [D, μ, Bk, and T are the diffusion coefficient, the ratio of the particle’s drift velocity to an applied force, Boltzmann’s constant, and absolute temperature, respectively. The Equation (2) is modified to describe the diffusing spherical particles in a liquid, and it is the Stokes–Einstein equation that is widely used [η and r are the dynamic viscosity and radius of the spherical particle, respectively. The diffusion coefficient is a function of the r−1, resulting in the diffusion-based separation that is efficient for small particles. In this study, an exosome separation chip was developed based on the dominant property of diffusion over sedimentation in nano-sized particles. We diffused bovine serum albumin (BSA) through the separation chip to confirm the separation efficiency and concluded that 6 h would be enough for separating the exosomes from the analyte. After that, exosomes from cell culture media and human serum were separated and analyzed.Among the above-mentioned EV isolation methods, ultracentrifugation is the most widely used for obtaining exosomes without chemical addition. However, it shows a low recovery rate and low physical intactness of exosomes due to a high g force  that corresponds to the 101 bar of hydrostatic pressure  during sarticles . On the articles (2)D=μkBely used (3)D=kBT−1 and 1.15~1.19 g·mL−1, respectively, and 1× PBS viscosity was 0.89 kg·ms−1, from the sedimentation equation, 150 nm-sized exosome gravitational sedimentation velocity was calculated as 4.62~6.09 × 10−11 m·s−1 or 4.00~5.26 × 10−6 m·day−1, and 50 nm-sized exosome gravitational sedimentation velocity was calculated as 5.14~6.77 × 10−12 m·s−1 or 4.44~5.85 × 10−7 m·day−1 [g [−14 and 9.65 × 10−14 m2·s−1, respectively, in 1× PBS buffer at room temperature. The 1-dimensional diffusion length, i.e., −5 m for 1 h and 1.86 × 10−5 m for 30 min, respectively [−5 m, 50~150 nm-sized exosomes can cross the membrane within 1 h, theoretically. For the above reason, it was concluded that the diffusive behavior of exosomes is dominant over gravitational sedimentation.The sedimentation and diffusion of exosomes can be compared as follows. Considering that the density of serum and exosome were 1.024 g·mL m·day−1 . This caday−1 [g ,46. On tectively . ConsideA nanoporous polycarbonate (PC) membrane chip was designed and fabricated to separate EVs based on their size dependency in diffusion coefficient and sedimentation velocity . Nanopor3 [To verify separation efficiency and determine optimal separation time, BSA with dimensions of 14 × 4 × 4 nm3  was injeExosomes and apoptotic bodies were obtained from doxorubicin treated SW620 . NTA anaw/w) of Triton X-100) is required, and that the surfactant does not damage the exosomes (w/w) Triton X-100) were used for the ultracentrifuge- and diffusion-based separations, respectively, and the exosome isolation time for each method was 8 h. The diffusion-based separation with the small amount of sample showed a strong signal for both syntenin and CD63 antibodies  tibodies . Especiatibodies . The higA nanoporous PC membrane chip for size-dependent separation of EVs was developed on the basis of the fast diffusion of nanoscale particles. It can be developed in a variety of ways, such as with different pore sizes, chamber scales, etc. Using diffusion-based principles, various nano-sized particles/vesicles were successfully separated, including polystyrene particles and EVs, not only from cell-cultured media but also from serum. The diffusion coefficient increased as particle size decreased, and separation times were accordingly shortened. The chip is simple to use because it does not require any other external force or antibody, unlike other separation methods . Togethe−1) [Similar studies on EV separation using porous membranes had been performed previously. In previous microfluidic studies, an electric field of up to 200 V was perpendicularly applied to the microfluidic channel for the electrophoretic separation of negatively charged exosome ,55. Othe−1) . Compare−1) ,61. DiffIn summary, we report on a nanoporous PC membrane chip for EV separation using diffusion. This chip does not require any external force or antibody, guaranteeing that separated exosomes will be chemically and physically intact. The chip is easy to fabricate and handle and can be modified for other purposes. Because it does not require pre-processing of a sample, even unskilled personnel can use it for the separation of EVs from various samples.−2) using an MU-60 exposure system  with a pattern mask and developed using AZ-1500  , with a pore size of 200 nm was placed between the 2 PDMS chambers. The two PDMS chambers were bonded with 50 W of oxygen plasma treatment  for 1 min  . PDMS faNJ, USA) . Track-eor 1 min .Before injecting samples, the inlet and outlet chambers were filled with 150 µL and 1.5 mL of PBS, respectively, for wetting the PC membrane. After 30 min, the inlet chamber PBS was replaced with 150 µL of a sample. The sample-injected chip was placed at room temperature during separation. Access holes for injecting the sample were sealed to prevent evaporation during the separation step.v/v). Second, cell culture media-originated exosomes  were obtained . Cells were cultured in RPMI 1640 medium, supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin-streptomycin solution . Cells were maintained in a 5% CO2 incubator at 37 °C. To induce EVs from cells, they were treated with 2 μM doxorubicin hydrochloride . After 48 h of doxorubicin treatment, the culture medium was harvested and centrifuged at 400× g for 10 min and then at 10,000× g for 20 min, sequentially. Pellet was resuspended with PBS and then used as the sample of apoptotic bodies. The supernatant was ultra-centrifuged at 100,000× g for 180 min. The ultra-centrifuged pellet was then resuspended with PBS buffer and concentrated using Amicon centrifugal filter MWCO 10 kDa . The mixture of EVs and apoptotic bodies was injected into the inlet chamber. Human serum  was purchased and used in separation after being centrifugated at 2000× g for 10 min. Serum was diluted 1:1 with a PBS buffer of 0.01% (w/w) Triton X-100 to prevent aggregation.Two types of samples were prepared. First, the polystyrene nanoparticle mixture solution was composed of particles of two different sizes: 132 nm (PS02N)  and 500 nm (L3280) . Both sizes of particles have a coefficient of variation of 3% (−1) was used and the concentrations of the samples were measured by Bradford assay after the indicated time . The efficiency was normalized by injected BSA amount. NTA machine Nanosight LM10 was used to measure the size distribution of the samples . For the measurement, 400 μL of the sample was loaded onto the sample stage at room temperature. For the SEM analysis of exosomes, samples were fixed as follows. The primary fixation was carried out with 2.5% (w/w) glutaraldehyde  in 0.1 M phosphate buffer (pH 7.4) for 4 h. Then, the samples were rinsed with 0.1 M phosphate buffer (pH 7.4) on a shaker for 10 min. After rinsing, the samples were fixed again with 1% (w/w) aqueous osmium tetroxide  in 0.1 M phosphate buffer (pH 7.4). For dehydration, samples were sequentially incubated with 25% (v/v) ethanol for 10 min, 50% (v/v) ethanol for 10 min, 75% (v/v) ethanol for 10 min, 95% (v/v) ethanol for 10 min, and 100% (v/v) ethanol for 10 min. After dehydration, the sample solutions were spin-coated on a wafer substrate at 2000 rpm for 60 s. The SEM imaging was carried out in two different instruments: for v/v) trichloroacetic acid  to precipitate exosomes, soluble proteins, and other vesicles. Then, the samples were precipitated with 10,000× g of centrifugation for 30 min. The pellet was then resuspended with pH 8.0, 0.1 M HEPES buffer . Samples were loaded onto SDS-PAGE and then transferred to PVDF membrane . Anti-syntenin-1 , CD63 , HSP 70 , and calreticulin  were used.Samples were analyzed with Bradford assay, NTA, SEM, and immunoblotting. Bradford assay and nanoparticle tracking analysis were repeated 5 times. To verify separation efficiency over time, BSA solution (5 μg·mL


Ch.agilis had been performed. Most studied populations showed a medium level of chromosomal polymorphism: on average 45±3.0% of specimens are heterozygotes with 0.52±0.01 heterozygotic inversion per larvae. Besides inversions, B-chromosomes were found in two populations. The total number of banding sequences found in banding sequence pool of Ch.agilis is 16. Three banding sequences – p’agiB3, p’agiD3, p’agiF3 – are described for the first time.Quantitative and qualitative analysis of chromosomal polymorphism in 19 natural populations of  Chironomusagilis Shobanov et Djomin, 1988 belongs to the Ch.plumosus group of sibling species. This group presents a good model for studies of the evolution of the karyotype during speciation as well as chromosomal polymorphism in natural populations. Three species with the widest ranges – Ch.plumosus (Linnaeus 1758), Ch.entis Schobanov, 1989 and Ch.balatonicus Dévai, Wülker et Scholl, 1983 – have been studied most extensively. At the same time, only a few populations were studied karyologically for other species from the group. In case of Ch.agilis the data on chromosomal polymorphism were published only for two populations – one from Eastern Europe (Rybinskoe reservoir) and one from Siberia (Berdsky pond) – where only 9 banding sequences had been found in total  followed by the name of the arm and the serial number of banding sequence in this arm (according to the order of its discovery), and prefixed by a letter indicating its geographical distribution in the genus Chironomus . Thus, for example, h’agiE1 means that while Ch.agilis itself is a Palearctic species, this banding sequences is identical to banding sequences of some other species and was found in Nearctic populations of those species, thus have a Holarctic distributionEach banding sequence is given a short designation as follows: three-letter abbreviation of the species name (agi for https://evolution.genetics.washington.edu/phylip.html) and MEGA11.0.8 (https://www.megasoftware.net). Genetic distances between populations were calculated using Nei criteria  was used for this work: microscope “Axioskop” 2 Plus, CCD-camera AxioCam HRc, software package AxioVision 4 .Ch.agilis belongs to “thummi” cytocomplex with haploid number of chromosomes n=4 and arm combination AB CD EF G. The chromosomes I (AB) and II (CD) are metacentric, III (EF) is submetacentric, and IV (G) is telocentric  in the karyotype of Ch.agilis: two are situated on the arm G , and the third one – on the arm B. Unlike in many other species from the genus Chironomus, BR on the arm B is usually well developed and easily identifiable in Ch.agilis.The species ric Fig. . There aCh.agilis banding sequence should be mapped if mapping of Ch.plumosus – reference species for mapping of all Ch.plumosus group sibling species – made by Ch.plumosus made by The revision of mapping of main banding sequences in arms A, B, C, D, E and F was presented by Golygina and Kiknadze previously . Revised mapping of these banding sequences is shown on Fig. Inversion polymorphisms were found in all chromosome arms except G, but only arms B and F show a noticeably high level of polymorphism throughout different populations. Only rare or unique inversion banding sequences were found in arms A, C, D, and E. In total, 16 banding sequences were present in the studied populations.Arm A was monomorphic in all populations with the exception of the Siberian population from the pond on the Eltsovka river where banding sequence p’agiA2 was found in a heterozygous state in a single larvae   Fig. .p’agiC1 1a-2c 6c-f 7a-d 16a-17a 6hg 11d-15e 8a-11c 6b-2d 17b-22g Cp’agiC2 1a-e 5b-4h 16h-a 7d-6c 2c-1f 5c-6b 11c-8a 15e-11d 6gh 17a 4g-2d 17b-22g CArm D was also monomorphic in most populations, but in total has three banding sequences. Both p’agiD2 and p’agiD3 banding sequences differ from p’agiD1 by simple paracentric inversions in the middle of the arm . The banding sequence p’agiD3 is new for the species and described for the first time.Arm E was monomorphic in all populations with the exception of the population from the pond on the Shadrikha river where the banding sequence p’agiE2 was found in a heterozygous state in a single larvae (Table ae Table . It diffae Table .h’agiE1 1a-3e 5a-10b 4h-3f 10c-13g C (KV)h’agiE1 1a-3a 4c-10b 3e-b 4b-3f 10c-13g C (GV)p’agiE2 1a-3a 4c-10b 3e-b 4b-3f 10c-e 12g-10f 13a-g C (GV)Arm F has three banding sequences. The main banding sequence p’agiF1 occurred in 100% of larvae from European and Far Eastern populations, and was dominant in all studied populations from Siberia  with 44.4–73.1% of heterozygotic larvae and 0.467–0.937 heterozygotic inversion per larvae, while European and Far Eastern populations were almost monomorphic  were checked for deviations from Hardy-Weinberg equation. Only two populations – from ponds on the Shadrikha river (NSK-SH) and the Koynikha river (NSK-KO) – had shown a deviation from expected equation. In both populations number of heterozygotes in arm B exceeded expected values : observed and expected frequencies of p’agiB1.2 were 49.8% and 41.1%, respectively, in NSK-SH, and 65.6% and 46.0% in NSK-KO. Unfortunately, the date on water quality in studied populations are not available so it is impossible to speculate about the cause of these deviations.The cytogenetic structure of studied populations is shown on Fig. The cytogenetic distances between populations varied from 0 to 0.155 Table . They arBesides inversion polymorphism, genomic polymorphism in a form of additional B-chromosomes was also observed in two populations from Siberia – in ponds on the Ora and the Shadrikha rivers Table .Ch.agilis shows moderate level of chromosomal polymorphism in comparison with other well-studied species from plumosus group – Ch.balatonicus, Ch.plumosus and Ch.entis. For example, an average percent of heterozygotes found in Palearctic populations of Ch.plumosus is 63.2% with 0.95 inversion per larvae  and 14.3 for Ch.balatonicus (Ch.agilis from different regions will be studied.The species r larvae , while tplumosus , 15.3 foatonicus . AlthougCh.agilis shows pattern similar to Ch.balatonicus where only two arms show high level of polymorphism in most populations, while other arms are almost completely monomorphic. At the same time, chromosome arms most heavily affected by inversion polymorphism are different in these two species: A and D in Ch.balatonicus, but B and F in Ch.agilis. In other two species – Ch.plumosus and Ch.entis – inversions are found with high frequencies in most or all chromosome arms.If we consider the distribution of chromosomal polymorphism between chromosome arms, Ch.agilis, as still not enough karyological data are available, especially for Europe, Eastern Siberia and the Far East. However, it is possible to assume that Western Siberia is the center of the species range of Ch.agilis and populations here have higher level of chromosomal polymorphism, which is also more diverse than in populations from the borders of the species range.At present, it is too early to draw a final conclusion about the characteristics of chromosomal polymorphism of


Enrichment factor (EF) and geoaccumulation index (Igeo) results showed that Cr, Mn, Fe, Co, Zn, Y, Zr, Cd and U were significantly enriched, and Th was extremely enriched. The total content of REEs (ƩREEs) ranged from 101.3 to 12,911.3 µg g−1, with an average 2431.1 µg g−1 which was higher than the average crustal value of ΣREEs. A high concentration of Th and light REEs were strongly correlated, which confirmed soil enrichment with monazite minerals. High ratios of light REEs (LREEs)/heavy REEs (HREEs) with a strong negative Eu anomaly revealed a felsic origin. The comparison of the chondrite normalized REE patterns of soil with hinterland rocks such as granite, charnockite, khondalite and migmatite suggested that enhancement of trace and REEs are of natural origin.Inductively coupled plasma mass spectrometry (ICP-MS) has been used to measure the concentration of trace and rare earth elements (REEs) in soils. Geochemical certified reference materials such as JLk-1, JB-1, and JB-3 were used for the validation of the analytical method. The measured values were in good agreement with the certified values for all the elements and were within 10% analytical error. Beach placer deposits of soils mainly from Odisha, on the east coast of India, have been selected to study selected trace and rare earth elements (REEs), to estimate enrichment factor (EF) and geoaccumulation index (I During these processes heavy minerals  ,12.Recently, increasing attention has been paid towards not only environmental radioactivity studies but also to the origin of beach placer deposits in the southwest coast of Sri Lanka , SithoniThe Odisha state is an important littoral state on the eastern coast of India, and the coastal stretch between the Rushikulya river and Gopalpur town is known as the Chhatrapur–Gopalpur beach placer deposit. The total weight percentage of heavy minerals in this beach placer deposit ranges from 2.9 to 20.4%. It includes heavy minerals such as garnet, hornblende, ilmenite, magnetite, monazite, pyroxene, rutile, sillimanite, sphene, tourmaline and zircon ,30. Due geo), to identify the degree of environmental contamination.In Odisha’s coastal soils, there is a lack of TEs and REEs data of bulk sand and soil in the Chhatrapur–Gopalpur beach placer deposits. The REEs background data could be used as baselines to identify contamination level as well as to conduct a quantitative risk assessment in soils. Therefore, analyses of TEs and REEs in soils have been carried out using inductively coupled plasma mass spectrometry (ICP-MS) to evaluate two pollution indices, the enrichment factor (EF) and geoaccumulation index (I(1)To validate analysis of TEs and REEs with certified reference materials using ICP-MS;(2)Determination of TEs and REEs in Chhatrapur–Gopalpur beach placer-deposit soils;(3)geo of TEs to evaluate natural enrichment and anthropogenic contamination in soils;Estimation of EF and I(4)To understand the origin/source of TEs and REEs in beach placer-deposit soils.−1) of TEs such as Cr, Mn, Fe, Co, Ni, Cu, Zn, Rb, Sr, Zr, Cd, Cs, Ba, Pb, Th and U and REEs  are given in In this study, geochemical certified reference materials (CRMs) such as Japan lake sediment (JLk-1) and Japan basalts (JB-1 and JB-3), supplied by the Geological Survey of Japan, were used to the validate analytical method for TEs and REEs using ICP-MS. The concentrations  which refers to the precision . The acc−1) of each sample location from the study area are summarized in The mean concentration of TEs  global limit  [−1 with an average value of 1300 µg g−1 and was less than the WHO critical value (2000 µg g−1) [−1.The Fe (iron) concentration in samples varied from 19,000 to 150,000 µg g µg g−1) . The Fe  µg g−1) . However−1 with a mean value of 390 µg g−1. The high concentration of Th in the soils is attributed to the presence of monazite minerals. U concentration varied from 1.4 to 53.2 µg g−1 with a mean value of 14.6 µg g−1. Pb concentration ranged from 16.2 to 65.0 μg g−1 with a mean value of 40.0 μg g−1. The highest Pb concentration was observed at Aryapalli, however all samples were below global limit 85 μg g−1. The presence of Pb in the human body causes damage to bones and organs such as the liver, kidneys, brain, lungs, and central nervous system. Ba concentration varied from 3.4 to 385 µg g−1 with a mean value of 142 µg g−1. The highest concentration of Ba was observed at Jagnyasala.Concentration of Th ranged from 35.0 to 900 µg g−1 with a mean value of 103 µg g−1. The highest concentration of Zn was observed at Aryapalli, Kanamana, Matikhalo and Venkatraipur. Zr concentration varied from 2.2 to 370 µg g−1 with a mean value of 102 µg g−1, which was less than the average upper continental crust (UCC) value of 190 µg g−1. Cr concentration varied from 35.6 to 180 µg g−1 with a mean value of 83 µg g−1. The mean concentration was less than the global limit of 150 μg g−1. The highest concentration of Cr was observed at Aryapalli. A high concentration of Cr causes skin related diseases. Co concentration varied from 10.4 to 75.0 μg g−1 with a mean value of 27.4 μg g−1. Ni concentration varied from 1.1 to 24.5 μg g−1 with a mean value of 12.0 μg g−1. Other trace elements were in very low concentrations—below the recommended global limits.Zn concentration varied from 27.0 to 250 µg gThe EF results of trace elements in soils are given in Cr, Mn, Fe, Co, Zn, Y, Zr, and Cd were significantly enriched in Aryapalli samples. Cs has been significantly enriched in Chhatrapur and Boxipalli. Mn, Fe and Co were significantly enriched in Kanamana samples. Cr, Mn and Co were significantly enriched in Gopalpur samples. Cr, Mn, Fe, Cu, Sr, and Cs were significantly enriched in Chattrapur samples. Cr and Co were significantly enriched in Matikhalo samples. Mn and Y were significantly enriched in Boxipalli samples. The significant enrichment of Cr may be due to the mafic-source rock present in the study area. Mn and Fe enrichment may be due to the presence of ilmenite mineral present in the study area.geo values for the elements in soils are presented in The results of I−1) concentration was about 17 times higher than the UCC value (132.5 µg g−1). On the other hand, the mean ∑HREEs (71.2 µg g−1) concentration was five times higher than the UCC value (13.9 µg g−1). The total concentrations of ∑REEs ranged from 101.3–12911.3 µg g−1 with a mean value of 2431.1 µg g−1. The mean ∑REEs concentration was 16 times higher than the UCC value (146.4 µg g−1) [The mean concentrations of light and heavy REEs (LREE and HREE) from all samples are given in  µg g−1) .−1) was in the following order: Ce (1121.5) > La (540.7) > Nd (458.5) > Pr (119.1) > Sm (66.9) > Gd (39.9) > Dy (13.4) > Er (5.4) > Yb (4.3) > Tb (4.2) > Ho (2.0) > Eu (1.9) > Lu (0.9) > Tm (0.9). The REEs concentrations exhibited the same order as for the Oddo-Harkins rule with two exceptions . This type of small exception in the order of REE concentrations has been observed in Cuban soils [The enrichment of REEs (µg gan soils . The REE2 = 0.64 to 0.90) compared to HREEs (R2 = 0.46 to 0.83). This positive correlation between Th and LREEs corroborates that Th is a high-field-strength element and strongly supports the presence of monazite minerals. REEs showed similarities in behaviour including low solubility and immobility during weathering and sedimentation [2 = 0.62 to 0.99).Pearson’s correlation coefficients (significant at the 99% level) were used to understand the relationship between Th, U and REEs. The coefficients are presented in entation . U also In this study, Leedey chondrite values  were useAEu) anomaly of the samples was estimated as follows:NSm and NGd are the concentrations of samarium and gadolinium of the bulk soils normalized with respect to the chondrite value.The europium On series . The EF The Chhatrapur–Gopalpur beach placer deposits are in the Ganjam district of Odisha, India. These areas extend 20 km length from Chhatrapur City in the north to Gopalpur Town in the south  with an average width of more than 2 km. A map showing the locations of sampling stations is given in The Chhatrapur–Gopalpur beach placer deposits overlay high-grade granulite and intrusive rocks of the EMGB. The major litho-units of the EMGB are khondalite, charnockite and migmatite. The heavy minerals in the beach placers are ilmenite (39.01 mt), garnet 29.40 mt), sillimanite (17.91 mt), rutile (1.81 mt), zircon (1.33 mt) and monazite (1.13 mt) . This st.40 mt, s2, and these samples were mixed to form a composite sample. Before collection, stones, grass, litter, roots, and shoots were removed from the surface layer. The sampling site selection was based on ambient dose rate, measured using a CsI (Tl) scintillation survey meter . Three composite samples were obtained from each sampling location. Approximately 2 kg of each of the 36 composite samples were collected from corresponding 12 sampling locations of the study area. These were brought to the laboratory and air-dried at room temperature. After manually removing remaining roots, shoots, and stones, they were sieved using a 2 mm mesh sieve. The sieved samples were oven-dried at 110 °C for 24 h. Then, all samples were pulverized using a ball mill to less than 150 µm in size prior to chemical decomposition.Soil samples were collected from a surface layer (0–10 cm depth) using a Daiki soil sampler. At each sampling point, five samples were taken from an area of about 1 m3, HF, HClO4 and HCl . The microwave digestion was carried out in two steps. In step one, a mixture of concentrated HNO3 (3 mL), HF (2 mL) and HClO4 (0.5 mL) was added, and the digestion method was operated at a temperature of 80 °C and 600 W power for 2 h, including cooling time. In step two, a mixture of HNO3 (3 mL) and HF (1 mL) was added and the method was similar to step one. The microwave-digested solution was followed by open digestion using aqua regia (HCl (3 mL): HNO3 (1 mL)) at 200 °C for 2 h in a clean fume hood. After complete evaporation of aqua regia, the residue was dissolved in 10 mL of 6 M HCl and dried completely. Finally, the sample solution was prepared in 20 mL of 3% HNO3. An experimental blank solution was also processed in the same way.About 250 mg of homogenized soil samples were ashed in a muffle furnace  to decompose organic matter. In the furnace, temperature was increased sequentially as follows: 100 °C for 2 h, 200 °C for 3 h and 600 °C for 5 h. After that it was allowed to cool down for a further 7 h. The furnace-dried samples were chemically digested using a microwave  in a closed PTFE pressure vessel with a mixture of concentrated HNOn = 5). The detection limits varied from (0.03 to 0.2) × 10−6 µg g−1 for all elements.An internal standard Rh was spiked into each diluted sample to correct the signal attenuation due to the presence of various constituents in the samples (matrix effect) as well as for possible changes during ICP-MS measurement. The concentrations of TEs  and REEs  in the decomposed samples were determined using an ICP-MS system . The ICP-MS instrument was equipped with a MicroMist nebulizer and a Peltier-cooled (2 °C) Scott-type spray chamber for sample introduction. There was also an octopole-based collision/reaction cell, located between two quadrupole analyzers. The instrument was operated in a gas mode with He (flowing at 5 mL/min) to remove polyatomic ion interferences in case of multielement analysis. The analytical procedure has been described elsewhere . The ICPgeo), were used in the present study to evaluate the level of contamination in the soils.The pollution indices are an objective tool to assess the enrichment of elements in soils. The individual indices were used to obtain information on the level of soil pollution using each element’s analysed data. The complex indices were used to determine the total pollution of an area. The simultaneous use of several indicators allows us to assess the pollution of soil with elements more accurately . The polEl” is the element under consideration, “Conc” is concentration (µg g−1), and “X” stands for the reference element [sample” and “Ref. sample” indicate their respective concentrations.In the present study, the EF was used to evaluate the influences of natural enrichment and anthropogenic contamination in the soils with respect to the reference sample in the study area. The EF was calculated using Equation (2). element . The subThe normalized EF has been applied to differentiate element sources as anthropogenic or natural . The TEsgeoI was calculated using Equation (2), proposed by [geo classification was used to determine the level of contamination.iC is the element concentration in soil, iB is the geochemical background value of an element  and 1.5 is the coefficient of variation attributed to natural rock.The posed by . The Ige−1, respectively [geoI ≤ 0; Class 0), uncontaminated to moderately contaminated , moderately contaminated , moderately to strongly contaminated , strongly contaminated , strongly to extremely contaminated , and extremely contaminated . In this study, the contamination is considered as enrichment.The geochemical background values of Cr, Mn, Fe, Co, Ni, Cu, Zn, Rb, Sr, Y, Zr, Cd, Cs, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, Pb, Th, and U are 92, 774, 79344, 17.3, 47, 28, 67, 84, 320, 21, 193, 0.09, 4.9, 624, 31, 63, 7.1, 27, 4.7, 1, 4, 0.7, 3.9, 0.83, 2.3, 0.3, 2, 0.31, 17, 10.5, and 2.7 µg gectively . There ageo results. The enrichment of Mn, Fe, Co, Zn, Y, Pb, U, Th, and REEs was observed in the multielement diagram normalized with UCC values. The high concentrations of Fe and Mn were due to the presence of ilmenite heavy mineral, U was due to the presence of zircon, and the enrichment of LREEs and Th was due to the presence of monazite in the soils. Investigation of the REEs geochemistry revealed that the sources of monazite and other heavy minerals might have been derived from charnockite, migmatite, khondalite and granite rocks of the EGMB. The enrichment of elements in the soils is natural in origin. Consequently, the present data in this study will be used as a baseline for future monitoring of TEs and REEs levels in Chhatrapur–Gopalpur beach placer-deposits soils, where it is expected that substantial economic exploration into heavy minerals will occur in the coming decades.In this study, the concentration of TEs and REEs in Odisha beach placer-deposit soils were determined. EF values showed extreme enrichment of Th, U and significant enrichment of Cr, Mn, Fe, Co, Zn, Y, Zr, Cd and Cu. The extreme enrichment of Th was followed by U, Mn, Co, and Zn, Pb and Y, a slight enrichment was observed in the I


Duranta erecta in the removal of pathogens from domestic wastewater. Two experimental and control units were configured in series. Each unit contains three bioreactors and was arranged in a cascaded configuration. The two experimental units used both plant and media, but the two control units used only media to treat the wastewater. Gravel and polyester sponge were used as media. Experimental unit 1 and control unit 1 used gravel as media; however, experimental unit 2 and control unit 2 used polyester sponges as media. The experiment was operated at hydraulic retention times of 1, 3, 5, and 7 days in a continuous mode. The performance of the hydroponic system was evaluated by characterizing the influent and effluent quality using standard methods. At optimum hydraulic retention time (7 days), the average removal of experimental units 1 and 2 was 98.7% and 89.8% for heterotrophic bacteria, 96.2% and 86.8% for total coliform, and 92.9% and 84.0% for fecal coliform, respectively. Analysis of variance showed that there was a significant difference (P < 0.05) between the two experimental and control units in removing pathogens, but no significant difference (P > 0.05) was observed between the two experimental units and between the two control units. Heterotrophic bacteria and coliforms were satisfactorily removed from domestic wastewater via a gradual hydroponic system. Hence, the hydroponic treatment system planted with Duranta erecta has a promising potential in the removal of pathogens from domestic wastewater in developing countries including Ethiopia.This study aimed to evaluate the treatment potential of gradual hydroponics planted with  Fanta and Akaki rivers have been impaired for many years due to anthropological activities. Because of an increasing population, urban farming, industrial expansion, and lack of sufficient sewage treatment, the city of Addis Ababa is suffering from serious surface water pollution [Domestic wastewater disposal has become a serious problem in urban areas of developing countries . A largeollution –12.Kilinto Prison camp, which is one of the federal prisons located on the outskirts of Addis Ababa, is being directly discharged into a small river called the Fanta River. At the time of this study, 264 m3/d of untreated effluent is being discharged to the Fanta River, which is located upstream of Big Akaki River. Big Akaki River is one of the most polluted rivers in Addis Ababa [Fanta River. The Fanta River is contaminated by untreated wastewater discharges and runoff from residential settlements in its upstream catchment. Thus, the major sources of pollution in the Fanta River are residential districts and predominantly public institutional facilities that impose environmental burdens. In vegetables, grown by the river water in the studied areas, fecal coliforms and a high colony of E. coli were identified. This might cause an increase in nonspecific diarrhea for consumers, exceeding 104 per 100 ml [amoebiasis; for example, direct contact with irrigation water polluted with wastewater triggers skin irritations such as dermatitis [Wastewater and refuse disposal is often the most intractable sanitation problem in places of detention . The wasis Ababa , 14–16. r 100 ml . The farrmatitis , 19. Indrmatitis –22.Nature-based wastewater treatment and reuse concepts are growing as a naturally focused low-cost wastewater treatment and reuse technology, where wastewater is treated biologically including constructed wetland and hydroponic technology . DetermiHydroponics is one of the recent wastewater treatment technologies that uses a symbiotic relationship between plants and microorganisms to treat wastewater , 26. It Nerium oleander, Pinus patula, Euphorbia cotinifolia, Cordyline terminalis, and Duranta erecta [Duranta erecta was designed, arranged, and operated to assess the removal potential of pathogens from domestic wastewater. Duranta erecta is the most frequently used ornamental plant in Ethiopia. It is a fast-growing and multistemmed shrub plant. It grows in tropical and warm subtropical regions and suits for gardens. In areas that have infertile natural conditions and drought, they tend to grow up to 1 m, and in watered and fertilized areas, they grow up to 3 m. Duranta erecta has a moderate growth rate, usually 0.5 m/year for the first few years. They can live for at least 15 years [Ornamental plants are very important in environmental management and beautification . There aa erecta . In this15 years . Hence, Duranta erecta in hydroponic wastewater treatment technology for the removal of pathogens. Therefore, this study aimed to evaluate the treatment potential of gradual hydroponics planted with Duranta erecta in the removal of pathogens from domestic wastewater. The technology needs further researches to be utilized as an alternative decentralized wastewater treatment mechanism for developing countries including Ethiopia.There are few studies regarding the use of gradual hydroponics for wastewater treatment . It is ao 02′01.76″ N and 38o45′18.33″ E″ and has an altitude of 2300 m. It has an average temperature, rainfall, and relative humidity of 15.9°C, 1089 mm, and 60.7%, respectively.The experimental site is located in Akaki Kality sub-city, Addis Ababa, Ethiopia, as shown in Two experimental and two control units were configured in series as shown in Where A, B, and C denote bioreactors in experimental unit 1 (E-1); D, E, and F denote bioreactors in control unit 1 (C-1); G, H, and I denote bioreactors in experimental unit 2 (E-2); J, K, and L denote bioreactors in control unit 2 (C-2); RT denotes the reservoir tank; and V, W, X, Y, and Z are sampling ports.In E-1 and C-1, gravel was used as media, but in E-2 and C-2, a polyester sponge was used as media. Healthy and young plants were collected from Afincho Ber area in Addis Ababa near the experimentation site. The root of the plants was carefully washed to remove the adhered soil material. The plant's root had been trimmed to 11 cm, and five young plants were planted per reactor in E-1 and E-2. On each reactor lid, a forestry tube was used to suspend the plant along with the media. 387.28 g of sharp gravel, which has a diameter of 1 cm, is placed in each forestry tubes on both E-1 and C-1. 29.28 g of a polyester sponge is placed in each forestry tubes on both E-2 and C-2. The porosity of gravel and polyester sponge is 0.44 and 0.68, respectively. The experimental units contain both media and plants, but the control units only contain media.After planting, acclimatization was conducted for about three months to adapt to the stress of the plants due to high organic loadings . First, Kilinto Prison camp to characterize raw wastewater. Grab sampling was used to collect samples from the influents and effluents of the hydroponic treatment system to evaluate the performance of the treatment system in terms of the removal of pathogens. For each HRT , one sample was taken from influent and four samples were taken from effluents . A triplicate water sample analysis was conducted according to standard methods. Totally, 20 triplicate sample analyses were conducted according to [Four composite samples (one sample per month) were taken from the direct discharge point of rding to  standardƞ is removal efficiency, c1 is influent concentration, and c2 is effluent concentration.The generated data were analyzed by Microsoft Excel 2013. One-way analysis of variance (ANOVA) was used to compare the treatment performance of hydroponic units for the heterotrophic bacteria, total coliform, and fecal coliform removal with a 95% confidence interval. A linear correlation was also observed to determine the correlation between HRT and removal efficiency. The removal efficiency was calculated based on the following equation:Kilinto Prison camp wastewater was characterized from August 2019 to November 2019. The average (Avg) heterotrophic bacteria (HB), total coliform (TC), and fecal coliform (FC) counts (log 10 units) in the effluent ranged from 7.94 to 8.63 CFU/100 ml, 5.70 to 5.89 CFU/100 ml, and 4.37 to 5.14 CFU/100 ml, respectively, as shown in The It can be noted that the colony of bacteria is increasing from August to November as can be seen in The presence of high coliforms in the effluent indicates the presence of pathogenic organisms that can cause health effects on humans . DysenteThe mean influent value of HB, TC, and FC counts (log 10 units) during the experiment varied between 8.76 ± 0.020 and 8.95 ± 0.016 CFU/100 ml, 5.54 ± 0.005 and 5.86 ± 0.002 CFU/100 ml, and 4.55 ± 0.020 and 4.96 ± 0.008 CFU/100 ml, respectively. At 1, 3, 5, and 7 days of HRT, the mean effluent concentration of HB, TC, and FC counts (log 10 units) during the experiment varied between 7.06 ± 0.098 and 8.34 ± 0.004 CFU/100 ml, 4.43 ± 0.128 and 5.17 ± 0.021 CFU/100 ml, and 3.80 ± 0.125 and 4.25 ± 0.047 CFU/100 ml, respectively, for E-1. For E-2, the mean effluent concentration of HB, TC, and FC counts (log 10 units) for the same HRT varied between 8.43 ± 0.005 and 7.96 ± 0.015 CFU/100 ml, 5.25 ± 0.018 and 4.99 ± 0.019 CFU/100 ml, and 4.34 ± 0.032 and 4.17 ± 0.046 CFU/100 ml, respectively. Similarly, At 1, 3, 5, and 7 days of HRT, the mean effluent concentration of HB, TC, and FC counts (log 10 units) during the experiment varied between 8.63 ± 0.003 and 8.38 ± 0.007 CFU/100 ml, 5.46 ± 0.011 and 5.55 ± 0.008 CFU/100 ml, and 4.47 ± 0.031 and 4.68 ± 0.013 CFU/100 ml, respectively, for C-1. For C-2, the mean effluent concentration of HB, TC, and FC counts (log 10 units) for the same HRT varied between 8.68 ± 0.003 and 8.56 ± 0.004 CFU/100 ml, 5.49 ± 0.012 and 5.68 ± 0.007 CFU/100 ml, and 4.50 ± 0.030 and 4.79 ± 0.011 CFU/100 ml, respectively. The mean concentration of influent and effluent from each experimental unit and control unit is described in A reduction in the concentration of HB, TC, and FC in effluents was observed than that of influents in all experimental and control units. E-1 showed higher performance in removing HB, TC, and FC followed by E-2, C-1, and C-2, respectively, in all applied HRTs. The removal of all three parameters in E-1 and E-2 might be caused by sedimentation, predation by other organisms, and natural die-off . The relP > 0.05) was observed between the two experimental units (E-1 and E-2) and between the two control units (C-1 and C-2). However, a significant difference (P < 0.05) was observed between the two experimental and control units (E-1 and C-1 and E-2 and C-2). The removal performance of the hydroponics in removing HB, TC, and FC ranged between 62.4 and 98.7%, 57.14 and 96.21%, 48.7 and 92.9%, respectively, for E-1 and 54.1 and 89.8%, 49.14 and 86.75%, 37.99 and 84.01%, respectively, for E-2 at HRT of 1, 3, 5, and 7 days. Similarly, the HB, TC, and FC removal performance of hydroponics ranged between 27.2 and 73.3%, 17.14 and 51.35%, and 15.73 and 48.28%, respectively, for C-1 and 18.3 and 59.6%, 11.42 and 35.13%, and 10.14 and 32.42%, respectively, for C-2 at HRT of 1, 3, 5, and 7 days as shown in Figures Duranta erecta showed high performance in removing HB, TC, and FC. Therefore, gradual hydroponics can be developed into a technology that can be used as alternative decentralized wastewater treatment mechanisms, especially for developing countries including Ethiopia.According to The TC removal performance of this study was compared with previous studies. Yeboah and Allotey  reportedPhragmites australis. Similar removal efficiency (84%) was reported by Sehar and Naeem [Bulbophyllum reptans and Trianthema portulacastrum.The FC removal efficiency of this study was also compared with previous studies. The removal efficiency of 90% was reported by De Anda and López-López  in the tnd Naeem  in removIn all three parameters, E-1 showed a better bacteria removal efficiency than E-2. This might be due to the better growth of root biomass in E-1 than that in E-2. The growth of the root biomass enhances the secretion of root exudates. E-1 and E-2 also showed a better bacteria removal efficiency than their corresponding control units. R2 = 0.989, 0.976, 0.979, and 0.981 for E-1, E-2, C-1, and C-2, respectively. In the removal of TC, the regression analysis showed that R2 = 0.982, 0.959, 0.962, and 0.953 for E-1, E-2, C-1, and C-2, respectively. In the removal of FC regression, analysis showed that R2 = 0.917, 0.969, 0.949, and 0.934, respectively. From this, we can understand that the removal of bacteria depends on HRT, which in turn depends on HLR.A strong linear correlation was observed in regression analysis between the applied HRTs and removal efficiency of the treatment system in removing HB, TC, and FC as shown in Figures Kilinto Prison camp was treated using gradual hydroponics operated at HRT of 1, 3, 5, and 7 days. The optimum HRT in the removal of pathogens was achieved at an HRT of 7 days. The findings showed that at optimum HRT (7 days), a removal efficiency of 98.7 and 89.8% was achieved for HB, that of 96.2 and 86.8% was achieved for TC, and that of 92.9 and 84.0% was achieved for FC using E-1 and E-2, respectively. The results obtained in this study show that the experimental units of gradual hydroponics planted with Duranta erecta significantly removed HB, TC, and FC from domestic wastewater. Therefore, scaling up of this pilot gradual hydroponics has a great advantage for developing countries such as Ethiopia.The effluent from the


Most studies linking long-term consequences of adolescent underweight and obesity are limited to men.To assess the sex-specific association of adolescent BMI with cardiovascular- and non-cardiovascular-related mortality in young adulthood and midlife.A nationwide cohort.927,868 women, 1,366,271 men.Medical examination data at age 17, including BMI, were linked to the national death registry.Death attributed to cardiovascular (CVD) and non-CVD causes.2), underweight women had a lower adjusted risk for CVD mortality (Cox hazard ratio (HR) = 0.68; 95% CI 0.46–0.98) in contrast to underweight men . The latter were at higher risk for non-CVD mortality , unlike underweight women . Findings, which persisted when the study sample was limited to those with unimpaired health, were accentuated for the obese with ≥ 30 years follow-up. Both sexes exhibited similarly higher risk estimates already in the high-normal BMI range (22.0 ≤ BMI < 25.0 kg/m2) with overall no interaction between sex and BMI (p = 0.62). Adjusted spline models suggested lower BMI values for minimal mortality risk among women (16.8 and 18.2 kg/m2) than men (18.8 and 20.0 kg/m2), for CVD and non-CVD death, respectively.During 17,346,230 women-years and 28,367,431 men-years of follow-up, there were 451 and 3208 CVD deaths, respectively, and 6235 and 22,223 non-CVD deaths, respectively. Compared to low-normal BMI  contains supplementary material, which is available to authorized users. Cardiovascular disease remains the leading cause of death in the Western world , 2. OverMany of the studies included only men –8 or repAll citizens obligated for military service in Israel are required to undergo a compulsory medical assessment at age of 17 years. Figure The primary outcomes of the study were either death attributed to cardiovascular causes according to the International Classification of Diseases  or deaths attributed to non-cardiovascular etiologies. Given that deaths attributed to diabetes  are closely associated with cardiovascular deaths , we grouHealth examinations and review of the medical history were performed by trained military physicians. Standardized measurements of weight and height were undertaken by trained personnel with each participant barefoot and in underwear; BMI was calculated. Data regarding education, residential socioeconomic status, country of birth and country of origin were recorded. Education was grouped into ≤ 9, 10, 11 or 12 years of formal schooling. Socioeconomic status (SES), based on locality of residence at the time of study enrolment , was gro2 (obese), as at age 17 years adolescents have completed > 98% of their growth [BMI was treated as a continuous variable and was also grouped according to a modification of the World Health Organization classification by splitting of the normal range, with the following subgroups: BMI < 18.50 (underweight), 18.50 ≤ BMI < 22.0 , 22.0 ≤ BMI < 25.0 , 25.0 ≤ BMI < 30.0 (overweight) and BMI ≥ 30 kg/mr growth . Person-2). Multiple imputation was applied to those with missing data (1.4% of examinees) as reported previously [Several sensitivity analyses were conducted. We restricted the Cox analysis to those with unimpaired health status at baseline  , 21 to aeviously . Analyse2 for women and men, respectively.Baseline characteristics are shown in Table 2) was set as the reference category . However, the strength of the association among obese women was reduced. Limiting the study sample to participants with at least 3 decades of follow-up was associated with an equal duration of follow-up between the sexes , reduced the sex differences in the age of death and accentuated the findings  group. Nevertheless, there was no statistically significant interaction between sex and BMI for CVD death (p for interaction = 0.62).During 17,346,230 and 28,367,431 person-years of follow up among women and men, respectively, there were 451 and 3208 CVD deaths (mean ages at death 41.9 ± 10.9 years and 47.0 ± 9.1 years), respectively, and 6235 and 22,233 non-CVD deaths (mean ages at death 39.3 ± 11.8 years and 37.5 ± 12.5 years), respectively. Notably, there were sex differences in the proportions of cardiovascular mortality; coronary heart disease, 21.3% in women vs. 43.7% in men; and stroke, 21.3% vs. 13.1%, respectively , with significantly increased non-CVD mortality risk at BMI values of 20.0 and 22.0 kg/m2, respectively.Underweight women did not exhibit excess risk for non-CVD death, whereas underweight men had a small, but significant, increased adjusted risk . For this outcome, an increased risk was observed for the high-normal, overweight and obese groups in both sexes with an interaction between sex and BMI in an unadjusted model (p for interaction = 0.012) reflecting the stronger associations among women, that became less significant in the multivariable model (p for interaction = 0.069). Multivariable spline models indicated a minimum risk for cardiovascular death at adolescent BMI values of 16.8 and 18.8 kg/m2 vs. 18.8 kg/m2 for CVD mortality, and 18.2 kg/m2 vs. 20.0 kg/m2 for non-CVD mortality. There was a significant interaction between sex and BMI for non-CVD death. The association between BMI and non-CVD death was stronger for women than men in all groups except for underweight.Our study presents sex-specific associations of adolescent BMI with CVD and non-CVD mortality by mid-adulthood in a national cohort of young Israeli women and men. The main findings of the study are that underweight was protective for CVD mortality among adolescent women, but was associated with excess all-cause mortality among men. The optimal BMI was lower in women than in men; 16.8 kg/mCVD mortality has declined substantially over the past several decades , 24, but2 for women and men, respectively). Data from large meta-analyses that were limited to adults (with age at enrolment that spanned at least 4 decades) pointed to an optimal range between 23.0 and 25.0 kg/m2 for all-cause mortality, and 22.5 kg/m2 for cardiovascular mortality [2 for all-cause mortality when the sample was limited to young adults (age 35–49 years) with significantly higher risk estimates for underweight men than underweight women [The definition of abnormal BMI in childhood has garnered considerable attention due to the rise of obesity epidemic . Yet, onortality , 35, 37.ortality . A pooleht women .Several lines of evidence support associations between underweight and metabolic fitness or longevity. Caloric restriction, defined as a reduced intake of calories not causing malnutrition, has been demonstrated as an intervention that can prolong life and health span  by activ2 of about 25%) [Several limitations of the current study should be addressed. First, BMI data were collected at a single time point with no available adulthood BMI measurements, preventing us from determining whether the association presented here is independent of adulthood BMI. This is a limitation as there is evidence to support that trajectory of BMI from childhood to adulthood has affects outcomes , 51. In out 25%) . This woout 25%)  as theseout 25%) . Third, out 25%) . Neverthout 25%) . In addiout 25%) , and shoThe strengths of the study include its large sample size, systematic collection of measured anthropometric data, long follow up and large person-years database. This allowed the analysis to focus specifically on women, with adequate power despite a relatively low rate of events in women in midlife.To conclude, underweight at adolescence appears to engender a protective effect among women but not men with respect to adulthood CVD-related mortality. The lowest risk for CVD related mortality is seen with lower than normal BMI values in women and in the low-normal BMI range in men.Additional file 1. Additional tables.


P < 0.05), which indicated that absorption of ciprofloxacin and sulfamethoxazole in RLQ pretreated rats was significantly affected. It indicated the coadministration of RLQ would have an influence on the efficacy of ciprofloxacin and sulfamethoxazole, and the doses of ciprofloxacin tablet and compound sulfamethoxazole tablet need adjustment.Relinqing granule (RLQ) is the best-selling Chinese patent drug for treatment of urinary system diseases. In this study, the effects of RLQ on the pharmacokinetics of ciprofloxacin, sulfamethoxazole, and trimethoprim in SD rats were investigated. Rats were randomly divided into control group 1, control group 2, RLQ group 1, and RLQ group 2. RLQ group 1 and RLQ group 2 were treated orally with RLQ for 7 days, and rats were treated with the same volume of water in control group 1 and control group 2. Then, RLQ group 1 and control group 1 were given intragastrically ciprofloxacin on day 8, while RLQ group 2 and control group 2 were given intragastrically sulfamethoxazole and trimethoprim on day 8. Blood samples were collected and determined. There was no significant influence of pharmacokinetic parameters of trimethoprim on two groups. But some pharmacokinetic parameters of ciprofloxacin and sulfamethoxazole in RLQ pretreated rats were evidently altered ( Polygonum capitatum plant and some other excipients, which has been approved by the Chinese State Food and Drug Administration , a well-known Miao's medicinal plant, belongs to the Polygonaceae family and has been used for many years in traditional medicine, where it plays an important role in the treatment of various urological disorders, including urinary calculus and urinary tract infections [ P. capitatum mainly included fatty acid esters, triterpenoids, steroids, flavonoids, gallic acid, and its analogs, as well as other phenolic compounds [fections . Aqueousompounds –9, whichompounds , 11. Altompounds , 13, it ompounds –18. Moreompounds , 20.Ciprofloxacin is useful for the treatment of a number of bacterial infections, which belongs to a second-generation fluoroquinolone antibiotic . Its spe P. capitatum water extract [Our previous study indicated that CYP450 activity was induced by treatment with extract . However extract . TheoretCiprofloxacin (product number 20130326), sulfamethoxazole (product number 20130406), and trimethoprim (product number 20130609) were purchased from Dalian Meilun Biology Technology Co., Ltd. ; the internal standard (IS), phenacetin (product number 81105), was obtained from Dr. Ehrenstorfer GmbH (Germany). Ciprofloxacin hydrochloride tablets (250 mg per tablet) were produced by Guangzhou Bai Yun Shan Pharmaceutical General Factory . Compound sulfamethoxazole tablets (sulfamethoxazole 400 mg and trimethoprim 80 mg per tablet) were produced by PKU International Healthcare Group Southwest Pharmaceutical Co., Ltd. . Relinqing granules were produced by Guizhou Warmen Pharmaceutical Co., Ltd. . High-performance liquid chromatography- (HPLC-) grade acetonitrile and formic acid were supplied by Merck . Distilled water was obtained from Watsons Group Co., Ltd. (Hong Kong). All other reagents were of analytical grade and obtained from Kermel Technology Co., Ltd. .24 male Sprague-Dawley rats (weighing 200–240 g) were provided by the Animal Supply Center of Guiyang Medical University . All animals maintenance and experimental study were based on the guidelines of the National Institutes of Health for the Care and Use of Animals, as well as under the approval of the Experiment Animal Center of Guiyang Medical University. All rats were kept in house under controlled temperature (22–24°C) and relative humidity (55–60%). Animals are maintained on a 12 h light/dark cycle and free access to food and water. They were fasted for 12 h, but they can drink freely before experiment.n = 6 for each group). RLQ group 1: there was oral administration of RLQ . Control group 1: rats were treated with the same volume of water twice daily for 7 continuous days. Fasting was carried out with free access to water on day 7, and oral administration of 0.108 g/kg ciprofloxacin (suspended in 0.5% CMC-Na) was conducted on day 8. Then, 0.15 mL of blood obtained from the caudal vein was collected at 5 min, 15 min, 30 min, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 4 h, 6 h, 8 h, and 12 h. The samples were injected into centrifuge tubes with heparin and centrifuged at 5000 rpm for 5 min. 50 μL of rat plasma samples was collected and frozen at −80°C prior to analysis.Twelve male rats were randomly divided into 2 groups (n = 6 for each group). RLQ group 2: there was oral administration of the RLQ . Control group 2: rats were treated with the same volume of water twice daily for 7 continuous days. Fasting was performed on the evening of day 7 with free access to water. Oral administration of 0.13 g/kg sulfamethoxazole and 0.026 g/kg trimethoprim (suspended in 0.5% CMC-Na) was carried out on day 8. Then, 0.15 mL of blood was collected at 5 min, 15 min, 30 min, 45 min, 1 h, 1.5 h, 2 h, 3 h, 4 h, 6 h, 12 h, 24 h, and 36 h and centrifuged to obtain plasma. All samples were frozen at −80°C before analysis.Twelve male rats were randomly divided into 2 groups . A Waters TQD Quantum triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source was used for mass analysis and detection. Chromatographic separation was achieved on a Waters Acquity BEH C18 column .The chromatographic analysis was performed using an ACQUITY UPLCμg/mL to 25 μg/mL, and the regression equation was Y = 6.5217X − 0.0503 with the mean correlation coefficient of 0.9939. The concentration ranges for sulfamethoxazole and trimethoprim were 0.1~500 μg/mL and 0.1~30 μg/mL, respectively, and the regression equations Y = 0.027X + 0.086 and Y = 1.9878X + 0.0716 were with the mean correlation coefficient of 0.9994 and 0.9991.The retention times of trimethoprim, sulfamethoxazole, and phenacetin (IS) were 1.10, 1.42, and 1.54 min, respectively. The mean recoveries of ciprofloxacin, sulfamethoxazole, and trimethoprim were between 86 and 110%, and the intraday and interday precision were less than 10%. Besides, ciprofloxacin, sulfamethoxazole, and trimethoprim in analyzed samples were stable within 6 h at room temperature, 30 days at −20°C, and three freeze-thaw cycles. All validation experiments of these methods met the requirements of the Guidance for Industry Bioanalytical Method Validation Document of the American Food and Drug Administration (FDA). For ciprofloxacin, the calibration curve was linear over the concentration range from 0.125 μL plasma was transferred to 1.5 mL centrifuge tube, and 10 μL of internal standard working solution  and 50 μL of 3 mol/L formic acid were added. The mixture was vortexed for 1.0 min and then 300 μL of methanol was added. This was then vortexed for another 1.0 min and centrifuged at 20,000 ×g for 10 min. The upper organic phase was transferred into tubes and evaporated to dryness under a gentle stream of nitrogen at 48°C. The residue was dissolved in 400 μL of the mobile phase and centrifuged at 15000 rpm for 5 min, and 1 μL of the solution was injected into UPLC-MS.For ciprofloxacin analysis, 50 μL.Analysis was carried out with an elution gradient of (A) acetonitrile and (B) water (both containing 0.1% formic acid) at a flow rate of 0.35 mL/min as follows: 0–2.5 min (5–95% A) and 2.5–3.5 min (95–5% A). The column and autosampler were maintained at 45°C. The injection volume was 1 m/z 332.4 for ciprofloxacin and m/z 180.2 for phenacetin. Cone voltage was 35 V for ciprofloxacin and 30 V for phenacetin. The mass spectrometer was operated in positive mode. Desolvation temperature was set to 350°C; nebulizer gas (N2) and source heater were adjust to 650 L/h and 120°C, respectively. The scan time for each analyte was set at 0.05 s. Data acquisition and processing were conducted on Masslynx™ v4.1 and Quanlynx™ v4.1 software .ESI(+) was selected as an ionization source, and the detection was conducted using selected ion recording mode (SIR), m/z 254.3, trimethoprim was m/z 291.3, and phenacetin was 180.2. The cone voltage was 25 V for sulfamethoxazole, 35 V for trimethoprim, and 30 V for phenacetin.Plasma sample pretreatment method and gradient elution condition were the same as those used for ciprofloxacin analysis. And the settings of MS detector were the same as those used for ciprofloxacin analysis. The selected ion to sulfamethoxazole was Data were presented as mean values ± standard deviation (SD). Pharmacokinetic parameter calculations were carried out using the DAS 2.0 pharmacokinetic program  and generated by a noncompartmental model . Statistically significant differences in the pharmacokinetic parameters of RLQ groups and control groups were assessed by one-way analysis of variance (ANOVA) followed by Tukey test with the level of statistical significance setting at 0.05.tz1/2, CLz/F, and Vz/F that were ×1.86, ×1.89, and ×3.17 higher; AUC∞)(0– and Cmax decreased by 50.0% and 29.3% (P < 0.05), respectively. These results indicated that preadministration of 4.0 g/kg RLQ (twice a day) substantially changed the pharmacokinetics of ciprofloxacin in rats, leading to the reduction of the ciprofloxacin AUCt) and after repeated administration of RLQ is shown in z/F and Vz/F for sulfamethoxazole were ×2.50 and ×2.94 higher and AUC∞)(0– and Cmax decreased by 36.79% and 35.79% (P < 0.05), respectively. However, there were no substantial differences in relevant parameters of trimethoprim between control group 2 and RLQ group 2. Results from the pharmacokinetic experiment showed that preadministration of RLQ significantly influenced the pharmacokinetics of sulfamethoxazole in rats, leading to the elevation of the sulfamethoxazole CLz/F and Vz/F and the reduction of AUC∞) and after repeated administration of RLQ are shown in Cmax of ciprofloxacin in RLQ group 1 significantly decreased; simultaneously, tz1/2, CLz/F, and Vz/F in RLQ group 1 were significantly increased. AUC∞). If the plasma concentration of antibiotic goes down lower than the MIC, it will possibly lead to failure of therapy even drug resistance. RLQ reduced plasma concentrations of sulfamethoxazole and ciprofloxacin, which were combined clinically with RLQ in common usage. It would perhaps lead to reducing antibacterial action of ciprofloxacin and cotrimoxazole (coadministration of trimethoprim and sulfamethoxazole). Therefore, clinicians should adjust the dosage of ciprofloxacin and cotrimoxazole in patients who take RLQ.


Bacillus subtilis transcriptomic study) deposited at NCBI’s SRA database, one read has 497,027 identical copies. Instead of storing them as separate entries, one can and should store them as a single entry with the SeqID_NumCopy format (which I dub as FASTA+ format). The second is the proper allocation of reads that map equally well to paralogous genes. I illustrate in detail a new method for such allocation. I have developed ARSDA software that implement these new approaches. A number of HTS files for model species are in the process of being processed and deposited at http://coevol.rdc.uottawa.ca to demonstrate that this approach not only saves a huge amount of storage space and transmission bandwidth, but also dramatically reduces time in downstream data analysis. Instead of matching the 497,027 identical reads separately against the B. subtilis genome, one only needs to match it once. ARSDA includes functions to take advantage of HTS data in the new sequence format for downstream data analysis such as gene expression characterization. I contrasted gene expression results between ARSDA and Cufflinks so readers can better appreciate the strength of ARSDA. ARSDA is freely available for Windows, Linux. and Macintosh computers at http://dambe.bio.uottawa.ca/ARSDA/ARSDA.aspx.Two major stumbling blocks exist in high-throughput sequencing (HTS) data analysis. The first is the sheer file size, typically in gigabytes when uncompressed, causing problems in storage, transmission, and analysis. However, these files do not need to be so large, and can be reduced without loss of information. Each HTS file, either in compressed .SRA or plain text .fastq format, contains numerous identical reads stored as separate entries. For example, among 44,603,541 forward reads in the SRR4011234.sra file (from a  The researchers predicted that, in some species where the splice site signals are strong, E7 skipping would disappear. This prediction is readily tested and confirmed with existing HTS data, i.e., E6-E8 mRNA was found in species with weak splice signals flanking E7, and E6-E7-E8 mRNA in species with strong splice signals flanking E7 (High-throughput sequencing (HTS) is now used not only in characterizing differential gene expression, but also in many other fields, where it replaces the traditional microarray approach. Ribosomal profiling, traditionally done through microarray , althougIn spite of the potential of HTS data in solving practical biological problems, severe underusage of HTS data has been reported . One majThe explosive growth of HTS data in recent years has caused serious problems in data storage, transmission, and analysis , but conEscherichia coli K12 in the file SRR1536586.sra (where SRR1536586 is the SRA sequence file ID in NCBI/DDBJ/EBI). The file contains 6,503,557 sequence reads of 50 nt each, but 195,310 sequences are all identical (TGTTATCACGGGAGACACACGGCGGGTGCTAACGTCCGTCGTGAAGAGGG), all mapping exactly to sites 929–978 in E. coli 23S rRNA genes . There are much more extreme cases. For example, one of the 12 HTS files from a transcriptomic study of E. coli (SRR922264.sra), contains a read with 1,606,515 identical copies among its 9,690,570 forward reads. There is no sequence information lost if all these 1,606,515 identical reads are stored by a single sequence with a sequence ID such as UniqueSeqX_1606515 . Such storage scheme not only results in dramatic saving in data storage and transmission, but also leads to dramatic reduction in computation time in downstream data analysis. At present, all software packages for HTS data analysis will take the 1,606,515 identical reads separately, and search them individually against the E. coli genome (or target gene sequences such as coding sequences). The SeqID_CopyNumber storage scheme reduces the 1,606,515 searches to a single one.HTS data files do not need to be so huge. Take, for example, the characterized transcriptomic data for Saccharomyces cerevisiae) genes is significantly associated with ribosome density and occupancy , nematode (Caenorhabditis elegans), fruit fly (Drosophila melanogaster), zebrafish (Danio rerio), and mouse (Mus musculus), and deposited the resulting reduced data at coevol.rdc.uottawa.ca in the form of BLAST databases. BLAST reduces sequences further by representing tetranucleotides AAAA, AAAC, ..., TTTT by 0, 1, ..., 255 so that each tetranucleotide takes only 1 byte in storage. The sequence ID in these BLAST databases are in the form of SeqID_CopyNumber. These files reduce the computation time for quantifying gene expression from many hours to only a few minutes (>2 min for my Windows 10 PC with an i7-4770 CPU at 3.4 GHz and 16 GB of RAM). This eliminates one of the key bottlenecks in HTS data analysis /2, where i = 1, 2, …, L, and Q1 and Q2 are quality scores from the two reads of the same sequence.One may ask how quality scores are treated when reads with identical sequences are grouped into the form of SeqID_CopyNumber format. Let me first highlight two observations. First, different reads with the same sequence have similar quality scores. For example, sequence “TGTTATCACGGGAGACACACGGCGGGTGCTAACGTCCGTCGTGAAGAGGG” occur many times in file SRR1526586.sra. I took the first six reads with this 50-nt sequence, and computed Pearson correlation among the associated quality scores . The correlation coefficients are all high and positive . The same.g., SRR922267  that can be directly compared among different genes and among different experiments with different total number of matched reads . Now for those reads that match the two genes equally well, the proportion of the reads contributed by paralogous gene 1 may be simply estimated asP1 to paralogous gene 1, and (1−P1) to paralogous gene 2. In the extreme case when paralogous genes are all identical, then UN.1 = UN.2 = 0, and we will assign 1/2 of these equally matched read to genes 1 and 2. We should modify Equation (1) to make it more generally applicable as followsUN.1 = UN.2 = 0, then a read matching equally well to these paralogous genes will be equally divided among the two paralogues.We need a few definitions to explain the allocation. Let L1 = L2. If paralogous gene 1 is much longer than the other, then UN.1 is expected to be larger than UN.2, everything else being equal. One may standardize UN.1 and UN.2 to number of unique matches per 1000 nt, designated by U.iSN = 1000U.iN/Li (where i = 1 or 2) and replace U.iN in Equation (2) by U.iSN as follows . I outline the approach below for dealing with gene families with more than two members.One might, mistakenly, think that it is quite simple to extend Equation (3) for a gene family of two members to a gene family with With three or more paralogous genes, one may benefit from a phylogenetic tree for proper allocation of sequence reads. I illustrate the simplest case with a gene family with three paralogous genes A, B, and C, idealized into three segments in AP, BP, and CP, respectively. For the 20 reads that matched B and C equally well, we allocate 20*6/(6 + 12) to B and 20*12/(6 + 12) to C. This gives the estimated number of matches to each gene asOne could apply maximum likelihood or least-squares method for the estimation, but ARSDA uses a simple counting approach by applying the followinge.g., the 20 reads shared by genes B and C in BP = 6/(6 + 12) = 1/3 and CP = 12/(6 + 12) = 2/3. Now genes B and C will have 12.66667 (= 6 + 20*BP) and 25.33333 (= 12 + 20*CP) assigned reads, i.e., U.BN = 12.66667 and U.CN = 25.33333. Once we have done with reads shared by two paralogous genes, we go through reads shared by three paralogous genes, e.g., the 23 reads shared by genes A, B, and C in U.AN = 7, U.BN = 12.66667, U.CN = 25.333333, and N = U.AN + U.BN + U.CN = 45, so we haveFile S1 was obtained in this way.In practice, one can obtain the same results without actually undertaking the extremely slow process of building trees for paralogous genes. One first goes through reads shared by two paralogous genes . Instead, all N paralogous genes will get a share of the read according to sequence similarity reflected in e-value and BitScore. The simplest scheme based on BitScore is to allocate such a read to paralogous gene i according toe-value isE is e-value and K is a scaling factor to ensure that ∑ip = 1. Equation (10) allocates shared reads more to the paralogous gene with a small e-value than those with large e-value. In practice, Equation (9) is often close to equal allocation, whereas Equation (10) results in more biased allocation favoring the best-match paralogous genes.There are alternative approaches for read allocation among paralogous genes. ARSDA also has an alternative allocation scheme based on BitScores and E. coli wild type . Although there are rpmA, rpmB, …, rpmJ genes in E. coli, they are not paralogous. One particular read “AGTGCCGAGCCGAAGCATAAACAGCGCCAAGGCTGATTTTTTCGCATATT” occurs 2684 times in SRR1536586.sra. It matches perfectly to the 36 nt at the 3′ end of rpmJ and 14 nt immediately downstream. However, Cufflinks output reported a count of only 2114 reads for rmpJ instead of 6426  reads to cspF and (58 + 28) reads to cspH, with relative proportions of 0.7719 and 0.2281 for cspF and cspH, respectively. Twelve reads match cspF and cspH equally well , so we assign them proportionally to the two genes, i.e., 12*0.7719 to cspF and 12*0.2281 to cspH. The final counts for cspF and cspH are 300.2626 and 88.7374, respectively. However, Cufflinks output shows counts of 2 and 63 for cspF and cspH, respectively. The discrepancy is particularly striking given that gene expression from ARSDA and Cufflinks are mostly concordant . The BLAST databases derived from HTS reads for several model species, in which sequence IDs are in the format of SeqID_CopyNumber, are deposited at coevol.rdc.uottawa.ca. One can use these BLAST databases with ARSDA to characterize gene expression or other analysis. The QuickStart.PDF file available at the same site details the use of ARSDA, either alone or in conjunction with the free DAMBE software (https://github.com/xuhuaxia/ARSDA.ARSDA is freely available at software , 2017. Twww.g3journal.org/lookup/suppl/doi:10.1534/g3.117.300271/-/DC1.Supplemental material is available online at Click here for additional data file.


The current study partially characterizes the enzymes’ molecular structures as well as their catalytic performance on different substrates which can be used to improve their potential for lignocellulosic biomass conversion.The high crystallinity of cellulosic biomass myofibrils as well as the complexity of their intermolecular structure is a significant impediment for biofuel production. Cloning of 6 antiparallel helices and right-handed β-helices were the folds identified for cel12B, cel8C, and peh28, respectively, in their corresponding protein model structures. Purifications of 17.4-, 6.2-, and 6.0-fold, compared to crude extract, were achieved for cel12B and cel8C, and peh28, respectively, using specific membrane ultrafiltrations and size-exclusion chromatography. Avicel and carboxymethyl cellulose (CMC) were substrates for cel12B, whereas for cel8C catalytic activity was only shown on CMC. The enzymes displayed significant synergy on CMC but not on Avicel when tested for 3 h at 45 °C. No observed β-glucosidase activities were identified for cel8C and cel12B when tested on p-nitrophenyl-β-d-glucopyranoside. Activity stimulation of 130% was observed when a recombinant β-glucosidase from Pcc was added to cel8C and cel12B as tested for 3 h at 45 °C. Optimum temperature and pH of 45 °C and 5.4, respectively, were identified for all three enzymes using various substrates. Catalytic efficiencies (kcat/Km) were calculated for cel12B and cel8C on CMC as 0.141 and 2.45 ml/mg/s respectively, at 45 °C and pH 5.0 and for peh28 on polygalacturonic acid as 4.87 ml/mg/s, at 40 °C and pH 5.0. Glucose and cellobiose were the end-products identified for cel8C, cel12B, and β-glucosidase acting together on Avicel or CMC, while galacturonic acid and other minor co-products were identified for peh28 action on pectin.β-Jelly roll topology, (α/α)This study provides some insight into which parameters should be optimized when application of cel8C, cel12B, and peh28 to biomass conversion is the goal. The injector was operated in a split mode with a split ratio of 1:10, injection port temperature of 250 °C, and injection volume of 1 µl. Data acquisition was done using MSD ChemStation software . The total ion current (TIC) chromatogram of GC elution and selective fragment ion (SFI) spectra of MS were used for sugar-identification where the SFI intensity and TIC retention time were used throughout for each eluted fragments as separated by GC–MS. Standard sugar oximes and/or TMS derivatives at 0-2.0 mg/ml concentrations of anhydrous d-(+)-glucose, d-(+)-galactose, d-(+)-mannose, d-(+)-xylose, l-rhamnose, d-(−)-fructose, sucrose, d-(+)-cellobiose, and d-(+)-galacturonic acid  were formed by the same procedure and used for peak identification. The mass spectrum of each standard was verified with the corresponding mass-spectrometry data of the National Institute of Standards and Technology (NIST) (http://webbook.nist.gov/cgi/cbook.cgi).Derivatization of hydroxyl groups of reducing sugar products via their reported . Oxime dreported . Parametreported . For oxiData in triplicate sets were analyzed using GraphPad Prism 6.0  and were compared using one-way analysis of variance (ANOVA) and Tukey post-test analysis as offered by the program.celB, celC, and peh into E. coli, encoding for cel12B, cel8C, and peh28, respectively, has been previously reported [Pcc polygalacturonase and cellulases as reported in accession numbers (Acc. No.) AAC02965.2, ZP_03832232.1 and AAA03624.1 of NCBI’S database [Pcc, Acc. No. gb|L32172 was 99% based on NCBI-BLASTP analysis [Successful cloning of full length DNA of analysis , 40  [Enzyme molecular weights of 29.5, 40, and 41.5 kDa were previously determined using SDS-PAGE for cel12B, cel8C, and peh28, respectively, [fischeri , pI 9.18mium sp.  and pI 7rzianum) . Understrzianum) .Table 1IN-glycosylation sites were similarly predicted for the cel12B and peh28 sequences, unlike cel8C where only one site was detected  [Other molecular target motifs were also noted for peh28 such as Cystovorum) . The conAspergillus sojae, who suggested that stability of the enzyme might be negatively affected by their purification due to the possible synergistic effect from other proteins found in solution with the desired enzyme components as originally proposed by Naidu and Panda [The crude protein extracts, partially purified with B-PER accessory reagents, were desalted and concentrated by multiple phases of ultrafiltration using PES membranes with different MWCs. The concentrated fractions were subjected to further purification by gel filtration using Sephadex G-100. A summary of each purification step is depicted in Table nd Panda . Thus, tp-NPG, respectively. The soluble cellulose derivative, CMC, is commonly used as substrate for endolytic-cellulase activities [Trichoderma reesei [p-NPG and, thus, the enzymes’ β-glucosidase function can be dismissed. Similar findings have been reported for endoglucanases from different sources tested on p-NPG and cellobiose as substrates [Exoglucanase, endoglucanase, and β-glucosidase activities were determined for cel12B and cel8C using 20 mg/ml of Avicel, 20 mg/ml of CMC, and 10 mM of tivities , while Ativities . Both amtivities . The dattivities . The acta reesei . The prea reesei . The laca reesei  also repa reesei , 71. Tabbstrates , 73.TablVmax, Km, and kcat and kcat/Km, given in Table Vmax values. However, the Km value of cel8C with CMC, 35 mg/ml, was about twofold higher than that of cel12B on CMC but the Km values of both enzymes on CMC are lower than those reported for other cellulases by Kim et al. [kcat/Km, 0.14 ml/mg/s, was 17-fold lower than that of cel8C on CMC  but consideration of it will be left to a future study in order to incorporate mass-transfer effects to and from the solid surface into the model, as discussed by Cruys-Bagger et al. [Enzyme kinetic parameters, m et al.  and Lin m et al.  for modia et al.  for a rer et al. , and alsr et al. , 78.TablKm of peh28 with polygalacturonic acid, 0.87 mg/ml, is similar to those of commercial polygalacturonases [Vmax of peh28 on polygalacturonic acid, 2.01 µmol/ml/min at 40 °C and pH 5.0, is higher than those of Ortega et al. [Bacillus subtilis at 40 °C and pH 8.0. Such variations in Vmax might be due to the dissimilar reaction conditions, including enzyme molar concentration; the better comparator would be kcat, if those concentrations were known. Activities of polygalacturonases are generally affected by the substrate-esterification, substrate surface charges, and the enzyme pI values [Verticillium alboatrum [Table uronases . On the a et al. , for coma et al. , for a mI values . Low sublboatrum . Exo-pollboatrum . Thus, tlboatrum  at pH 8.lboatrum  at pH 9.E. coli K12 strain, and Maisuria et al. [Pcc-BR1 and Aspergillus niveus, respectively. Figure T. reesei, a GH-12 xyloglucanase from Fusarium graminearum and a GH-8 cellulase mutant from E. coli K12 strain, as reported by Karlsson et al. [The optimum pH values for enzyme activities were investigated over a broad pH range of 3.0–10.0. Reactions were conducted for 1 h at 45 °C using 25 mg/ml of CMC for cel12B and cel8C, and at 40 °C using 4 mg/ml of polygalacturonic acid for peh28. The pH profile for the enzymes is shown in Fig. a et al.  and Malla et al.  for GH-2n et al. , Habrylon et al. , and Linn et al. , respectTemperature optima for enzyme activities were determined over a range of 20–80 °C at pH 5.4, using 25 mg/ml of CMC for cel12B and cel8C, and 4.0 mg/ml of polygalacturonic acid for peh28. The temperature profiles for the enzymes shown in Fig. Fusarium lini and Erwinia chrysanthemi, respectively.An experiment was carried out to examine the potential synergy of cel12B and cel8C to achieve maximum hydrolysis on CMC and Avicel substrates. Total quantities of reducing sugars formed in separate and combined reactions of cel12B and cel8C with the substrates were measured and compared. The synergetic response was also investigated in the combined activities of cel12B and cel8C on each substrate in the presence of β-glucosidase. All synergies were investigated at intervals throughout a 3 h period of incubation at 45 °C and pH 5.4 using CMC and Avicel at 25 mg/ml as shown in Fig. E. chrysanthemi, as due to the inability of CelY to utilize the soluble degradation products cellotetraose and cellopentaose which could be readily utilized by CelZ. Products averaging 10.7 glucosyl units were reported by the authors for the action of CelY, while average fractions of 3.6 glucosyl units arose by the combined action of CelZ and CelY. The lower activity found for cel12B by itself on CMC is similar to that of CelZ from E. chrysanthemi. Moreover, the CelY from E. chrysanthemi was assigned to the same GH-8 family ascribed to the present cel8C. These observations suggest relative substrate preferences as a possible mechanism for the enzyme synergy observed with cel12B and cel8C. Zhou and Ingram also reported that sequential hydrolysis of CMC by their two enzymes, CelZ and CelY, improved the synergy between them, when CelY was used first. They suggested that CelY increased the substrate digestibility for the ensuing action of CelZ on the partially hydrolyzed CMC. Thus, the low synergy observed herein might be improved if similar sequential hydrolysis had been used, cel8C first, then cel12B. Further investigation of the complementary actions of cel8C and cel12B is a promising focus for future research.The synergism displayed by cel12B and cel8C on CMC may be correlated with their retention and inversion modes of action, respectively, on the substrate as investigated earlier , explainOn the other hand, no detectable activity was shown on Avicel in the case of cel8C over all periods of incubation unlike cel12B Fig. b. The maE. chrysanthemi, for their lack of synergy on Avicel [The insignificant activity observed for cel8C on Avicel and/or its lack of synergy with cel12B might be attributed to the formation of long insoluble products of six or more glucosyl units by cel8C that tend to not be further hydrolyzed and, in turn, must be removed by centrifugation prior to analysis. A similar explanation has been given for CelY and CelZ from n Avicel . On the n Avicel , similarActivity stimulation of 28–30% was observed when β-glucosidase was added to the mixture of cellulases using CMC or Avicel as substrates Fig. a, b. SimInvestigation of cel8C and cel12B and β-glucosidase combined actions and the hydrolytic products formed over 3 h on Avicel or CMC has been carried out using gas chromatography coupled with mass spectrometry (GC–MS) as shown in Fig. E. chrysanthemi, and their combined activities on Avicel and CMC [Due to the GC limitations in quantification of the tri- and higher-oligomers, glucose and cellobiose were seen as dominant hydrolytic products from Avicel or CMC using the defined enzyme cocktail, as shown in Fig.  and CMC . This isGlucose and cellobiose were the hydrolysis products found in various combination reactions of other enzymes, such as CelY and CelZ , or indiGlucose concentration increased steadily in the CMC reaction over 2 h then leveled off Fig. a. CellobE. chrysanthemi acting on Avicel [On the other hand, cellobiose and glucose concentrations were essentially constant over the time course for the enzyme cocktail acting on Avicel Fig. b. This cn Avicel .Trichoderma cellulase (Novozyme® 50013) and β-glucosidase (Novozyme® 50010) in their initial activities on Avicel which was attributed to the utilization of the easily accessible amorphous cellulose on the substrate surface [The extent of substrate conversion was calculated for CMC and Avicel as 11.4 and 4.0%, respectively, based on the corresponding glucose and cellobiose products accumulated during the course of the cocktail activity on both substrates Fig. a, b. It  surface . However surface ) observePeh28 activity on pectin over 4 h was also investigated by GC–MS, and the hydrolysis product concentrations are given in Fig. T. reesei  Δlgd1and A. niger ΔgaaB) and Rhizopus oryzae RPG1, respectively. Formation of monogalacturonate products during the enzymatic reaction on pectin might be correlated with its processive action while continuously bound to the substrate, as explained by Mertens and Bowman [96 in the corresponding peh28 model structure, similar to what was previously described by van Pouderoyen et al. [Formation of monogalacturonate as a major hydrolytic product has been previously reported by Kuivanen et al.  and Mertd Bowman . This sun et al.   on cellulose and pectin substrates. The enzymes were assigned for their molecular similarity to glycoside hydrolase families 12, 8, and 28, respectively, and their catalytic domain residues were identified based on the analysis of their model structures. These outcomes suggest that some residues of cel12B and peh28 related to conformational and thermal stability are targets for further analysis. The presence of a CBD-II site in the cel12B sequence could partially explain the enzyme’s apparent function on the crystalline cellulose domains of Avicel. The relative thermal instability of cel8C at higher temperatures could also be predicted from its structural similarity to other GH-8 cellulases. The high catalytic activity of cel8C on CMC and the absence of similar activity on Avicel are correlated with typical endoglucanase characteristics. On the other hand, the lower CMCase activity of cel12B and its apparent activity on Avicel indicate atypical endoglucanase behavior. Enzyme processivity was concluded for peh28 from its close similarity to endo-polygalacturonase I from A. niger and from the existence of monogalacturonate as its dominant hydrolytic end-product. Moreover, the close sequence similarity to that of endo-polygalacturonase-I and pectate lyase-6 domains suggests the multi-domain activities of peh28. The relative substrate conversion values in terms of glucose and cellobiose formation from CMC and Avicel, and monogalacturonate from pectin for the current-modified system, suggest the enzymes’ candidacy for biofuel production. Site-directed mutagenesis, to promote sequential cel12B and cel8C hydrolysis and integration with other cellulolytic systems, is suggested for improving the cellulolytic synergy of the applied mixture. Overall, this study provides justification for further optimization of the enzymes’ catalytic performance in saccharification of lignocellulosic materials in future work.The study provided some molecular characterization as well as biochemical analysis for the behavior of recombinant cel12B, cel8C, and peh28 enzymes, alone and in certain combinations, from