Magnetic resonance imaging (MRI) is frequently used for preclinical treatment monitoring in glioblastoma (GB). Discriminating between tumors and tumor-associated changes is challenging on in vivo MRI. In this study, we compared in vivo MRI scans with ex vivo MRI and histology to estimate more precisely the abnormal mass on in vivo MRI. Epileptic seizures are a common symptom in GB. Therefore, we used a recently developed GB-associated epilepsy model from our group with the aim of further characterizing the model and making it useful for dedicated epilepsy research. Ten days after GB inoculation in rat entorhinal cortices, in vivo MRI (T2w and mean diffusivity (MD)), ex vivo MRI (T2w) and histology were performed, and tumor volumes were determined on the different modalities. The estimated abnormal mass on ex vivo T2w images was significantly smaller compared to in vivo T2w images, but was more comparable to histological tumor volumes, and might be used to estimate end-stage tumor volumes. In vivo MD images displayed tumors as an outer rim of hyperintense signal with a core of hypointense signal, probably reflecting peritumoral edema and tumor mass, respectively, and might be used in the future to distinguish the tumor mass from peritumoral edema—associated with reactive astrocytes and activated microglia, as indicated by an increased expression of immunohistochemical markers—in preclinical models. In conclusion, this study shows that combining imaging techniques using different structural scales can improve our understanding of the pathophysiology in GB. Glial tumors or gliomas represent 80% of all malignant brain tumors ,2. GradeSeizures represent a common symptom in GB and occur in 30–60% of patients with GB ,11. Up tBetter treatments are needed, and the search for new therapies requires the use of animal models that mimic disease progression in humans. Several rodent models for GB have been developed . The synMagnetic resonance imaging (MRI) plays a crucial role in diagnosis and treatment monitoring. As optimal surgical resection or radiation therapy of tumors requires an exact knowledge of the spatial extent of tumor growth, the main goal of non-invasive imaging in brain tumor diagnosis consists of the tumor localization, determination of tumor extent and detection of brain tissue infiltrated by the tumor. Therefore, it is important to distinguish between tumor tissue and tumor-related reversible changes in normal brain tissue, such as edema . MRI witDiffusion-weighted imaging (DWI) is an advanced MRI technique with a high sensitivity to changes in the microscopic cellular environment, without the need for contrast injection . The appEx vivo MRI enables longer scan times and limits motion, boosting the resolution and SNR. However, distortions and dehydration from fixation, disrupting the integrity, will be observed, and longitudinal follow-up is ruled out ,31. MoreIn this study, anatomical changes in the syngeneic Fischer/F98 rat model of GB were compared using in vivo MRI, ex vivo MRI and histology. Moreover, in vivo MD images and immunohistochemical markers were explored to identify the contribution of peritumoral edema, and an association with reactive astrocytes and activated microglia, to the estimated abnormal mass on MRI.® CRL-2397™) rat GB cells were cultured as monolayers in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 10% fetal calf serum (FCS), 1% penicillin-streptomycin, 1% L-glutamine, and 0.1% fungizone ) and placed in an incubator at 37 °C and 5% CO2.F98 (ATCC®) at 9–10 weeks old and with a mean bodyweight of 235 ± 14 g were used. The study was approved by the animal ethics committee of the Faculty of Medicine and Health Sciences of Ghent University (ECD 17/111). All animals were kept and handled according to European guidelines (Directive 2010/63/EU) and housed under environmentally controlled conditions: 12-h light/dark cycle, temperature between 21 and 24 °C, and humidity between 55 and 65%. Food and water were provided ad libitum.Thirty male F344/IcoCrl rats  and immobilized in a stereotaxic frame. Body temperature was maintained at 37 °C by a thermoregulated heating pad. The glioma cell suspension was stereotactically injected in the right entorhinal cortex (anteroposterior (AP): −8.0 mm and mediolateral (ML): +4.5 mm relative to bregma, dorsoventral (DV): −4.1 mm relative to dura) using an insulin syringe  × 12.7 mm) mounted on a pump for automatic injection . In all animals, 20,000 F98 cells in 5 µL phosphate-buffered saline (PBS) were injected over a period of 10 min. The syringe was kept in place for five min post-inoculation, and then slowly removed. The skin was sutured, and meloxicam was administered subcutaneously  to manage post-operative pain. In the remainder of this manuscript, we refer to the inoculation day as post-inoculation day 0 (PID0).2, in-plane resolution = 109 × 109 μm2, 600 µm slice thickness, 600 µm interslice distance, 30 slices, TA = 9′45″) was performed. Additionally, diffusion images were acquired with a spin-echo, echo-planar imaging (EPI) sequence between the olfactory bulb and the cerebellum. Diffusion-weighted acquisitions were recorded using an encoding scheme of 32 gradient directions with a b-value of 800 s/mm2 and with 5 b0 images. Other diffusion scanning parameters were as follows: TR = 7400 ms, TE = 24 ms, number of averages = 1, FOV = 35 × 35 mm2, matrix = 105 × 105, in-plane resolution = 333 × 333 μm2, 600 μm slice thickness, 600 μm interslice distance, 30 slices, 18.5 min acquisition time.In 9/30 animals, in vivo 7-T micro-MRI  was acquired on PID10 to visualize tumor presence and determine the volume of the estimated abnormal mass. Animals were anesthetized with a mixture of oxygen and isoflurane  and placed in the scanner, where the isoflurane-oxygen mixture was administered through a nose cone fixed onto the animal holder. A heating pad using a warm water circuit to maintain body temperature at 37 °C was placed underneath the animal. A rat head volume coil  was placed around the head. The respiratory rate was monitored during the entire protocol. After performing a localizer scan and optimizing the magnetic field homogeneity, a TurboRARE T2-weighted anatomical scan  to extract MD images.n = 30) were euthanized at PID10 (immediately after the in vivo MRI for GB01–GB09) with an overdose of sodium pentobarbital . Next, animals were transcardially perfused with phosphate-buffered saline (PBS) followed by 4% paraformaldehyde (PFA). The brains were isolated and post-fixated in 4% PFA for 24 h.All animals (n = 30) were acquired on the same 7-T micro-MRI system, using a transmit body volume coil  and an actively decoupled rat head surface coil  to receive the signal. The brains were removed from the 4% PFA solution, patted dry and transferred to a 10 mL tube containing fomblin  that was inserted in the coils. After performing a localizer scan and optimizing the magnetic field homogeneity, TurboRARE T2-weighted anatomical scans  were performed.Ex vivo MR images of all isolated brains  at 4 °C. Subsequently, the brain tissue was patted dry again, orientated in a cryomold  filled with O.C.T. compound , and frozen in isopentane  and liquid nitrogen (−196 °C). The embedded frozen brain tissue was stored at −80 °C until further processing.For cryosectioning, the embedded frozen brain tissue was removed from the cryomold and fixated on the cryostat holder with O.C.T. compound, allowing sectioning from the back. Coronal sections of 20 μm throughout the tumor were made using a cryostat . Every 30th section (every 600 µm) was mounted directly on a glass slide, and cresyl violet staining was performed. Some additional 20 µm-thick sections containing tumor tissue were also mounted on glass slides for immunohistochemical analysis.With the slice thickness of 20 µm and the interslice gap of 580 µm, we aimed to mimic the MRI situation in which slices were imaged every 0.6 mm as closely as possible. However, it is important to note that the slice thickness used for MRI was 600 µm, whereas this slice thickness was only 20 µm for histological analyses.Volumes of the estimated abnormal mass on magnetic resonance images and tumor tissue volumes on cresyl violet-stained histological slices were determined using ImageJ . On each in vivo or ex vivo T2w image or histological slice with visible lesions, the region of the estimated abnormal mass or tumor tissue around the inoculation site in the right-sided temporal lobe was marked, and the surface area was determined  and subsequent freezing (applicable for histology), correction factors were calculated for ex vivo MRI and histology to compare them to in vivo MRI (T2w or MD images). In six animals (GB04 to GB09), in vivo MRI, ex vivo MRI and histology were performed, and the images of these six animals were used to estimate mean correction factors for ex vivo MRI and histology. Correction factors were determined based on the shrinkage of the brainstem, as this region was the most straightforward to delineate on all modalities. Brainstem surface areas were determined on all ex vivo T2w images per animal with an abnormal mass, and the average was taken. This was performed for all six animals, and the mean ex vivo T2w brainstem surface area was calculated. Brainstem surface areas were then determined on corresponding in vivo images per animal, and the average  was taken. This was again performed for all six animals, and the mean in vivo brainstem surface area was calculated. To obtain a mean ex vivo correction factor, the mean in vivo brainstem surface area was divided by the mean ex vivo brainstem surface area. Similarly, a mean histological correction factor was calculated by determining a mean histological brainstem surface area, based on brainstem surface areas on all histological images with tumor tissue among the six animals, and a mean in vivo brainstem surface area, based on corresponding in vivo T2w images and by dividing the mean in vivo brainstem surface area by the mean histological brainstem surface area. The ex vivo and histological correction factors were used to correct estimated ex vivo and histological volumes by multiplying the estimated ex vivo and histological volumes by the corresponding factor.The term “tumor mass” will be used in this manuscript to indicate the region with dense GB tumor cells, without changes in the peritumoral zone. Of course, the living tumor involves both the tumor mass and a peritumoral zone including edema, inflammatory cells, and infiltrative GB cells.2O2 in PBS for 30 and 60 min, respectively, to block endogenous peroxidase activity. Secondly, sections were incubated for 45 min with a blocking solution containing PBS, 0.2% Triton X-100 and 0.4% fish-skin gelatin. Next, primary antibodies were applied for 1 h at room temperature at the following dilutions: rabbit anti-GFAP , mouse anti-vimentin , rabbit anti-Iba1  and mouse anti-AQP4 . After extensive washing, the sections were incubated for 1 h with goat anti-rabbit-Alexa fluor 594  and goat anti-mouse-Alexa fluor 488  secondary antibodies. After thorough rinsing, DAPI (1 µg/mL) was applied for 1 min for nuclear staining. A droplet of Fluoroshield histology mounting medium  was applied to the slices and slices were coverslipped.Immunohistochemical staining for glial fibrillary acidic protein (GFAP) and vimentin, for ionized calcium-binding adapter molecule 1 (Iba-1) and for aquaporin-4 (AQP4) were performed on the brain slices from eight animals  to highlight reactive astrocytes, activated microglia and edema, respectively. Briefly, 20 µm-thick mounted slices were first treated with 0.5% and 1% HTen regions of interest (ROIs) were determined in the ipsilateral hemisphere, of which five in the tumor mass and five in the peritumoral region, as well as five ROIs in the contralateral brain hemisphere. A fixed threshold was applied using ImageJ, and the integrated density  for each ROI was calculated and averages of the five ROIs per brain region were determined.All statistical analyses were performed in SPSS  with the level of statistical significance set at 0.05. Unless otherwise stated, data are expressed as means and standard error of the mean (SEM). Paired-samples T tests were used to compare the calculated volumes between the different imaging modalities . A bivariate Pearson correlation was used to explore possible correlations between in vivo MRI, ex vivo MRI and histological data. A Kruskal–Wallis test was used to compare the integrated density of immunohistochemical markers between ROIs in the peritumoral region, the tumor mass and contralateral brain hemisphere.3) was on average four times larger (t(5) = 13.6951, p < 0.001; 3). Similarly, a significant difference in volumes (t(8) = 9.940, p < 0.001) was found between the estimated abnormal mass on in vivo T2w (15.3 ± 1.3 mm3) and ex vivo T2w (4.5 ± 0.6 mm3) images. Note that volumes of the estimated abnormal mass on the in vivo T2w images were only available in nine animals  were found when comparing volumes of the estimated abnormal mass on ex vivo T2w images (8.1 ± 0.8 mm3) with histological tumor volumes (8.5 ± 0.9 mm3), with a mean deviation of volumes on ex vivo T2w images from histology of 21.0% (p < 0.001) was found (No significant differences (t(25) = 0.986; of 21.0% , blue. Aas found . Due to 3) did not significantly differ (t(8) = 0.643; p = 0.538) from the estimated abnormal mass on in vivo T2w images (15.3 ± 1.3 mm3).Volumes of the hyperintense rim on in vivo MD images (14.5 ± 1.9 mmp = 0.004) between volumes of the hypointense regions on in vivo MD images (2.9 ± 0.5 mm3) and volumes of the estimated abnormal mass on ex vivo T2w images (4.5 ± 0.6 mm3), as well as a significant difference (t(5) = 3.134, p = 0.026) between volumes of the hypointense core (2.8 ± 0.7 mm3) and histological tumor volumes (4.2 ± 0.9 mm3), with mean deviations in volume of 66.4% and 72.7%, respectively.A significant difference was found (t(8) = 4.071, 2(2) = 7.875, p = 0.019; χ2(2) = 15.005, p = 0.001, χ2(2) = 11.285, p = 0.004 and χ2(2) = 16.640, p < 0.001, respectively). Post hoc pairwise tests revealed significant increases in the staining intensity of vimentin, Iba1 and AQP4 in peritumoral regions compared to the contralateral healthy brain tissue . Numerous GFAP/vimentin double-positive cells aligned at the tumor border . Moreover, the staining intensity of GFAP was significantly higher in peritumoral regions compared to the tumor mass (p = 0.024), where fewer GFAP-positive cells were observed in the tumor mass . Therefore, we also only used male rats during this study. In future studies, it will be necessary to study the seizure occurrence and characterize the GB tumors in female rats as well, to make the results fully applicable for therapeutic screening use and translation to humans.This study shows that, by combining imaging techniques using different structural scales, a better understanding of the pathophysiology of GB can be obtained. The results of the present study show that hyperintense regions on in vivo T2w images not only represent the tumor mass but also include peritumoral changes, such as perifocal edema including reactive astrocytes and activated microglia, as indicated by a comparison of in vivo T2w images with in vivo MD images, histological and immunohistochemical analyses. However, a more careful slice selection and more exact comparison of slices will be needed to confirm this. Caution is warranted when using in vivo T2w images for treatment monitoring in preclinical GB models, as the effect of treatments on the tumor mass may be confounded by effects on tumor-related brain tissue changes. Ex vivo T2w images allow a proper volume estimation of the tumor mass. Therefore, these scans might be used for end-stage tumor volume estimations when brain tissue is needed for other analyses, not allowing further histological processing. Moreover, as in vivo MD images display GB tumors as two regions with distinct intensities—a hypointense core and a hyperintense rim, probably reflecting tumor mass and edema, respectively—these images might be useful in the future to discriminate between the tumor mass and peritumoral edema in vivo.


In conclusion, this work elucidated that SKY is a unique hot spring due to its rich lignocellulosic material, often absent in other hot springs. The data collected from this study serves as a repository of new thermostable macromolecules, in particular families of glycoside hydrolases.The SKY hot spring is a unique site filled with a thick layer of plant litter. With the advancement of next-generation sequencing, it is now possible to mine many new biocatalyst sequences. In this study, we aimed to (i) identify the metataxonomic of prokaryotes and eukaryotes in microbial mats using 16S and 18S rRNA markers, (ii) and explore carbohydrate degrading enzymes (CAZymes) that have a high potential for future applications. Green microbial mat, predominantly photosynthetic bacteria, was attached to submerged or floating leaves litter. At the spring head, the sediment mixture consisted of plant debris, predominantly brownish-reddish gelatinous microbial mat, pale tan biofilm, and grey-white filament biofilm. The population in the spring head had a higher percentage of archaea and hyperthermophiles than the green mat. Concurrently, we cataloged nearly 10,000 sequences of CAZymes in both green and brown biofilms using the shotgun metagenomic sequencing approach. These sequences include β-glucosidase, cellulase, xylanase, α-N-arabinofuranosidase, α- Besides the essential factors of temperature and pH, physicochemical parameters, such as salinity, may govern the hot spring microbiota and its genomic and macromolecule content7. Metagenome data from over 1000 hot springs has been registered in the NCBI database and extensive microbiota research has been conducted for heated springs in Argentina8, India14, Japan7, United States16, Russia19, New Zealand21, China22, and other countries24. Microbial diversity in hot springs may vary between the sediment, soil, and water even though these samples were taken from the same hot spring25. The scientific community also pays attention to biofilms at heated places27. Among the earlier publications, only a handful of hot springs contained plant residues, such as Obsidian Pool at Yellowstone Natural Park (USA)16, Deulajhari (India)28, and Malaysia SKY Hot Springs29.Hot springs are one of the main reservoirs for thermophiles and functional macromolecules1. Recombinant novel xylanase obtained from a Spanish hot spring fosmid library, is extremely thermostable where it can retain ~ 70% of initial activity after 24 h at 70 °C4. Shotgun metagenomics using hot spring samples is an emerging technique to mine massive genes encoding functional macromolecules and bypass conventional routes. In one interesting article, Kaushal et al.3 performed shotgun metagenomic of four Indian hot springs (55–98 °C) and generated contigs that contained over 4000 putative genes encoding for carbohydrate-acting enzymes (CAZymes). Recently, Reichart et al.2 data-mined CAZymes sequences from 71 hot springs’ shotgun metagenomic datasets.Thermophiles and their thermostable enzymes are essential for white biotechnology applications30. These enzymes include glycosyl hydrolases (GHs), carbohydrate esterases (CEs), and auxiliary activities enzymes (AAs). The sugars liberated from lignocellulosic polymers can then be fermented to produce biofuels. The majority of CAZymes in hot springs are yet to be discovered for biofuel production2. In addition, previous experiments in heated lab setups or in-situ hot springs enriched with insoluble cellulosic biomass, aimed to elucidate the role of thermophiles and thermozymes in lignocellulose degradation33.The breakdown of lignocellulosic polymers requires multiple enzymes generated by the microbial consortium34. A few years ago, we encountered SKY hot spring with degrading plant litters and we performed a snapshot on the prokaryotic diversity using amplicon sequencing; yet the functional genes acting on lignocellulosic polymers remain uncertain29; therefore we will address this research gap in the current report. This work will provide new enzyme targets for future use for white biotechnology, not limited to biofuel industries.Malaysia has more than 60 known hot springs with temperatures ranging from 36 to 102 °C29. We collected an emerald- green microbial mat at a location approximately three meters away from the spring head. The green (G) microbial mat and brown (B) samples were labelled as G1, G2, G3, or B1, B2, B3, respectively, to indicate which trip the samples were collected. Two separated samples and amplicon sequencing were performed for the first trip (Nov 2019), and the data were respectively designated as G1a, G1b, B1a, and B1b. The temperature during the samplings was 58–64 °C and 71–74 °C, respectively, for water adjacent to green and brown mats. The physicochemical parameters for the water are summarized in Table The SKY hot spring is in the Malaysian woodlands. Three field trips  were conducted in Nov 2019, Feb 2020, and Aug 2020. The spring head is somewhere near the fork of the Y-shaped human-made feature  was another dominant phylum that constituted 41% of the total bacteria population in sample B2. However, Fervidobacterium appeared to be less than 0.2% in B1 and B3 samples. The collected brown samples were highly heterogeneous but abundant with hyperthermophiles. On average, the Shannon index for green- and brown-mat was 3.7 and 4.0, respectively.Twenty-six bacteria phyla  were identified in all brown microbial mats. Phyla Acidobacteriota, Armatimonadota, and Bacteroidota contributed 4–17% of the brown mat community Fig. a. GeneraThe overview of archaea, after removing bacteria ASVs, is summarized in Fig. Instead of ITS, 18S rRNA marker was used as it may provide a better comprehensive view on the total eukaryotes. Fungi ASVs monopolized the reads. The primary fungi phyla in the green microbial mat were Ascomycota, dominated by Dothideomycetes, Eurotiomycetes, and Sordariomycetes classes Fig. c,f. MostHeveochlorella hainangensis (phylum Chlorophyta) and unicellular green photosynthetic Trebouxia usneae (class Trebouxiophyceae). The main detected algae phyla in the brown microbial mat were Apicomplexa, Ciliophora, Diatomea, Dinoflagellata, and Protalveolata. In SKY hot spring green microbial mat, we noticed ASVs of Amoebozoa, Ciliophora, Protalveolata, and Ochrophyta.There were two dominant algae in the green mat, including the solitary green microalgae Samples G1a, G3, B1a, and B3 have individually undergone shotgun metagenomic sequencing and reads were assembled using metaSPAdes into respectively 254,331, 107,238, 81,154 and 125,514 contigs that were larger than 500 bp. After filtering the unassigned taxa, approximately 98% of green mat contigs belonged to bacteria, while the remaining were archaea or eukaryotes. The average contigs for bacteria, archaea, and eukaryote in the brown mat were 77%, 22% and 1%, respectively , Uniprot reviewed protein database (Swiss-Prot), and InterProScan. The overall number of detected protein sequences are summarized in Table d-Glu-(1 → 4) linkages in cellulose, cellooligosaccharide or cellobiose. Primary enzymes acting on the β-d-Xyl-(1 → 4) of hemicellulase  are α-glucuronidase, xylanase, β-xylosidase, and reducing-end-xylose releasing exo-oligoxylanase. Arabinan active enzymes, for instance, α-l-arabinofuranosidase, glucuronoarabinoxylan endo-β-1,4-xylanase, exo-α-1,5-l-arabinanase, endo-α-1,5-l-arabinanase, and bifunctional β-xylosidase/α-arabinosidase, putatively target the α-d-(1 → 5)-Araf, α-d-(1 → 3)-Araf, or other linkages. Other detected protein sequences, xyloglucanase, β-mannosidase, β-mannanase, β-glucuronidase, β-N-acetylhexosaminidase, presumably conduct hydrolytic reactions at the other part of hemicellulase.One primary purpose of current work is to data-mine CAZymes, especially lignocellulosic enzymes, that may aid in the decomposition of plant litter in SKY hot spring. Open reading frames (ORFs) identified in the MetaSpades-assembled contigs were translated to protein sequences prior annotated against the CAZy database using dbCANl-arabinofuranosidase, glucan 1,4-α-glucosidase, β-mannosidase, β-xylosidase, β-glucuronidase, glycoside hydrolases and others. Forty-nine unique sequences (green mat) were associated with the following phyla: Armatimonadota, Chloroflexota, Deinococcota, Ca. Thermoflexus japonica, Cyanobacteria, Acidobacteriota and two unculturable bacteria. Thirty-two unique sequences encoding lignocellulosic-acting CAZymes that were ≥ 90% subject coverage and ≥ 90% protein sequence identify were spotted in the brown microbial mat. In addition, genes assigned to GH, CE, and AA were also noticed in different extensions from the following: Acidobacteriota, Bacteroidota/Chlorobiota group, Cyanobacteria, Firmicutes, Nitrospirota, Planctomycetota, Proteobacteria, Thermodesulfobacteriota, Ca. Thermoflexus japonica, Verrucomicrobiota, unclassified bacteria/unculturable, and several candidates from the division of Bathyarchaeota and Kryptonia. Among the listed GH enzyme sequences, only a few homologues have been characterized biochemically. For instance, protein sequence G1_212042 is a homologue to a characterized β-glucosidase AmBGL17 cloned from a soil metagenomic fosmid library36.Subsequently, we mined putative protein sequences that exhibited ≥ 90% subject coverage and ≥ 90% protein sequence identity, that were affiliated with lignocellulosic related GH families 1, 3, 5, 8, 9, 10, 16, 43, 51, 74, and 116  annotated in the green and brown mat datasets were 23 and 21, respectively. These CE families comprised esterase, polysaccharide deacetylase, acetylxylan esterase, and N-acetylglucosamine-6-phosphate deacetylase, often assist in xylan breakdown. With the same threshold, AA  sequences present in the green and brown mat dataset were 22 and 16, respectively. The identified proteins were catalase-peroxidase, oxidoreductase, glycolate oxidase and sorbosone dehydrogenase.3, the authors suggested that the sequences obtained from a metagenome assembly are considered novel if the primary sequence has ≥ 90% subject coverage and 50–70% identity to the deposited protein sequences. Using this as our threshold, we cataloged 116 and 91 non-redundant GH sequences in the green and brown samples, respectively. Approximately 80% of these sequences were related to families GH1, 3, 5, 10, and 51, while the remaining were from GH6, 8, 9, 12, 16, 26, 30, 43, 44, 48, or 116. We then selected some novel β-xylanase and cellulase and were compared with sequences obtained from various databases. Figures In an earlier reportCa. Caldatribacterium conducts anaerobic sugar fermentation and exhibited diverse glycosyl hydrolases, including endoglucanase37.Phyla Bdellovibrionota, Fusobacteriota, and Myxococcota were present in the green microbial mat but in negligible quantities in the brown mat. The unique phyla detected in the brown mat, but not in the green microbial mat, included Caldatribacteriota, Thermodesulfobacteriota, Dictyoglomota, Elusimicrobiota, Thermotogota, Candidatus Calescamantes, Fervidibacteria, Hydrothermae, GAL15 and TA06. The Candidatus Caldatribacterium , earlier named OP9 was also detected in this work. Using single-cell and metagenome sequencing, data elucidated that 38, together these factors favor the growth of the microorganisms. Chloroflexota Thermoflexus hugenholtzii39 (opt. growth temp. [OGT] 72–75 °C) constituted 31% of B1 microbiota  is the only described type strain. Bacterium HLO8T, a photosynthetic strain, formed a reddish-brown microbial mat in a Japanese hot spring41. We anticipate that the Chloroflexota associated taxon that formed reddish-brown jelly-type microbial mat in the spring head of SKY hot spring (71–74 °C) is different from strain HLO8T (OGT 55 °C) as the latter could not thrive at a higher temperature41.Cyanobacteria and Chloroflexota were the main identified phyla in the green microbial mat. Because the hot spring is almost stagnant, undisturbed, and the water surface temperature (< 64 °C) is below the maximum threshold of the bacteria photosynthesis processFervidobacterium, under the Thermotogota phylum, was a major genus in sample B2. Fervidobacterium species, for instance, F. islandicum and F. changbaicum, exhibit a broad range of CAZymes. The percentage of Fervidobacterium in hot spring microbiota would be increased if the water was enriched with switchgrass inoculum32. Some Firmicutes ASVs were detected in the brown mats. Firmicutes’ members, i.e., Geobacillus and other Firmicutes bacilli thermophiles, may dominate cellulose-degrading consortium in a heated lab setup42. Caldicellulosiruptor thermophilum, another member of Firmicutes, has been targeted as one potential thermophile for consolidated bioprocessing of lignocellulose2. We detected Caldicellulosiruptor and other Firmicutes in relatively small quantities in SKY hot spring mats.Ca. Caldiarchaeum and Ca. Nitrosocaldus , Protalveolata, and Ochrophyta. The Amoebozoa Echinamoeba thermarum was a common thermophilic protist in New Zealand geothermal springs20. E. thermarum was positively identified in the green mat but absent in the brown mat. We spotted other Amoebozoa, for instance, taxa from order Euamoebida and Leptomyxida. Additionally, thermophilic protist Protalveolata  was approximately 1% of total eukaryotes ASVs in the B1b sample. Thermophilic protist Ochrophyta (particularly class Chrysophyceae) was detected in very small quantity in brown samples.The study of eukaryotes is scarce for hot springs around the globe and is often neglected compared to prokaryotes. Oliverio et al. examined the presence of protists  in 160 New Zealand geothermal springs and suggested that the main protists possibly thrived in elevated temperatures are Amoebozoa, Archaeplastida, Alveolata, Excavata, Rhizaria, and StramenopilesChaetomium (Tmax 61 °C), Paecilomyces (55 °C), Chrysosporium (60 °C), Trichothecium (57 °C), Paecilomyces (55 °C), Torula (58 °C), Talaromyces (50 °C), Paecilomyces (50 °C), Geosmithia (55 °C), and Thermomyces (60 °C)46. We were doubtful if all detected fungi ASVs could grow pleasantly in SKY hot spring. Also, the water level was low during the first field trip, and the green mat was not submerged completely, water temperature was 58 °C, as measured ~ 5 cm below the G1 green mat. The actual temperature was expected to be lower in the floating green microbial mat; therefore, certain thermophilic fungi or some heat-tolerant mesophilic fungi may survive. However, none of the currently known thermophilic fungi can develop beyond 70 °C; therefore, detected taxa are not likely to survive without sporulation at the SKY brown mat at the spring head. Moreover, it is suspected that most of the detected mesophilic fungi ASVs were originated from fallen plant litter, and we expect them to be in their dormant form. For the first time, microscopic water bear Tardigrada (Macrobiotus hufelandi) was detected in a Malaysian hot spring. This small eight-legged animal feed on microorganisms, decomposed leaf, and survive in extreme temperatures. We also detected high background of plant phyla Phragmoplastophyta that likely originated from plant debris. Other background ASVs included flies, grasshoppers, and fringed winged insects, mites, and ticks. We confirmed that eukaryotes were the minority group in the SKY hot spring microbial mats by putting together the shotgun and amplicon data. We think that 18S rRNA primers excessively amplified the background of plant residuals or chromosomal fragments from the dead organisms, spores, inactivated eggs or larva in particular mesophilic fungi or insects. Collectively, we concluded that relatively high background noise was observed using 18S rRNA primer set.18S rRNA metataxonomic sample datasets elucidated that thermophilic fungi were present only as the minority (< 1% ASVs). That included 47. Judged using contigs generated from shotgun sequencing data, a greater percentage of archaea present in the brown mat was probably related to a higher temperature at the spring head  were identified from Candidatus Bathyarchaeota, Candidatus Brockarchaeota, Thaumarchaeota, Nitrososphaeria archaeon, and Thermoprotei archaeon.We performed shotgun metagenomic sequencing using two green- and two brown microbial mats. Negligible amounts (~ 0.1%) of virus reads were detected in all the mats, and it is quite common to see a trace quantities of viruses in hot springsead Fig. . We also3. Besides, the metataxonomic described in this current study differed from Deulajhari hot springs and Obsidian Pool that contained Pandanus leaf litters and heat-tolerant plant Juncus tweedyi, respectively28. The microbial and enzyme diversity in SKY hot springs are far more complex than other heated in-situ or ex-situ studies supplemented with insoluble cellulosic biomass42.On average, more than 10,000 CAZymes ORFs were found in each type of microbial mat Table . The annRoseiflexus castenholzii, candidatus taxa, and a few unclassified Chloroflexota bacteria. Other important Chloroflexota taxa for high-temperature lignocellulosic degraders included Thermomicrobium roseum, Anaerolineae, Ardenticatenia, Caldilinea aerophile, Ca. Thermofonsia Clade 1, Chloroflexus islandicus, Thermoflexales, and Thermoflexia bacterium. According to the online CAZy genome databases, members of the Chloroflexota phylum exhibited a broad range of GH enzymes. For instance, the anaerobe photoheterotrophic thermophilic R. castenholzii DSM 13941 (NCBI genome accession CP000804.1) has 22 GH families and 3 CE families that accounted for 60 different protein sequences41.Using the threshold of ≥ 90% subject coverage and ≥ 90% protein sequence identity, the three main phyla essential for biomass degradation were Chloroflexota, Armatimonadota, and Deinococcota. The primary contributors (phylum Chloroflexota) were taxa related to 48. Currently, Chthonomonas calidirosea T49T is the only thermophilic type strain48. The complete genome of strain T49T harbored 64 glycosyl hydrolases and eight carbohydrate esterases. To the best of our knowledge, the characteristics of these enzymes are still undescribed.Armatimonadota is another phylum spotted with multiple sequences from GH1, 5, 10, 43, and 51, CE7, and AA12 (threshold: ≥ 90% subject coverage and ≥ 90% protein sequence identity). Using the metataxonomy dataset, at least four Armatimonadia ASVs were detected in SKY hot spring mats, and an ASV was closely related to class Chthonomonadetes while the rest were unresolved at the lower taxonomy level. This phylum, earlier designated as candidate division OP10, was initially found in Yellowstone National Park Obsidian PoolCalidithermus, Thermus, and Meiothermus species. The draft genome of Calidithermus timidus DSM17022 indicated that the bacterium harbor seven AA, 38 GH, 16 CE sequences49. So far, only a GH57-glycogen branching enzyme50 and GH13 amylosucrase51 from this bacterium have been analysed in detail. Meiothermus spp. may help break down plant litter in SKY because a representative, M. taiwanensis WR-220 (PRJNA205607), had enzymes such as xylanase, β-xylosidase, endoglucanase, and polysaccharide deacetylase. More than a dozen Thermus spp. have completely curated genomes in the CAZyme genome database. For an example, the genome of Thermus thermophilus (http://www.cazy.org/b12268.html) encoded sequence of 15 types of enzymes particularly from families GH1, 13, 23, 36, 57, 63 and 77; however, the essential enzyme for lignocellulose hydrolysis is missing. Therefore, Thermus spp. are sugar consumers in the SKY community.Deinococcota is the third-largest phylum with 28 sequences with ≥ 90% subject coverage and identity to the CAZy database. These putative sequences have high identity to counterpart proteins from Dictyoglomus turgidum. Both protein sequences formed a cluster with β-xylanase from Thermotoga maritima (Q60037)  Fig. . All thraritima Q0037 55. 3AMC structure has a classic TIM barrel fold that resembles endoglucanase TM1752 (1VJZ) from T. maritima, endocellulase EGPh (3W6M) from Pyrococcus horikoshii and endoglucanase (6GJF) from a synthetic construct56. Cellulase EGPh has the optimum activity at 100 °C57. Putative cellulase B3_136450 , an immunoglobulin-like module (345–428), and a C-terminal CBM46 (432–533)58. The immunoglobulin-like module with two β-sheets resembles an earlier known CBM_X2 that may be involved with cellulosome59.Subsequently, we data-mined novel cellulase sequences in the SKY hot spring dataset. Fourteen unique sequences are putative cellulases, and each of the sequences contained a GH5 domain. Putative cellulase G3_96404 was 55% homologous to cellulase Cel5A of Rhodothermus marinus60. Both sequences have a single GH12 catalytic domain. Often, that domain is similar to the concanavalin-like glucanase domain superfamily that looks like a sandwich structure with 12–14 β-strands  was found in the N-terminal while a galactose-binding domain (TIM-like barrel structure) was present at the C-terminal, where the acid/base Glu 221 and nucleophilic Glu393 are located61. Enzyme CelM2 actively hydrolysed multiple substrates, including birchwood xylan, barley glucan and cellulosic CMC, respectively having β-1,3/4-glucan and β-1,4-glucan linkages61. The binding ability of multi-substrates is possibly related to the broad and deep pocket. Due to relatively close sequence identity, G1_45801 may exhibit a similar bifunctional glucanase-xylanase activity as CelM2. Lastly, putative cellulase B3_106662 (615 aa) was detected in a brown microbial dataset. The stretch 42–238 resembles a GH114-family domain having a typical aldolase-type TIM-barrel structure  hot spring is located at 3°59′50.50′′N and 101°23′35.51′′E. The temperature was measured using a portable digital thermometer, and pH was measured at 25 °C. Water from the spring head was collected into Schott bottles for chemical analysis at Allied Chemists Laboratory Sdn. Bhd. During the first sampling (Nov 2019), the water level was relatively low with a depth of 25–30 cm, but the upper layer of the plant litter bed was sufficiently wet. Two samples of green microbial mats (labelled as G1a and G1b) (~ 20 cm apart) were collected from the upper layer of the bed that was approximately 3.5 m away from the spring head. Two samples of brown microbial mats (~ 10 cm apart at the spring head) were taken and labelled as B1a and B1b. During the second and third sampling trips (Feb 2020 and Aug 2020), the water level was higher (45–50 cm depth), and the collected green and brown microbial mat samples were designated as G2, G3, B2, and B3. All microbial mats were kept at cool during transportation and frozen at − 20 °C within 12 h.g for 5 min. The supernatant was then withdrawn and underwent the protocol suggested by the manufacturer. Extracted DNA was evaluated by a NanoDrop 1000 spectrophotometer, a Qubit 3.0 Fluorometer , and a 1% (w/v) agarose gel electrophoresis.Frozen samples were thawed at room temperature; and leaves and twigs were removed manually. Bulk genomes from green and brown microbial mat samples  were extracted using the FastDNA Spin Kit for Soil . A 500 mg biofilm sample was resuspended in sodium phosphate and MT buffers as provided in the kit. The mixture was transferred into a Lysing Matrix E tube and mechanically lysed in a TissueLyser II  set at 20 Hz for 5 × 3 min and subsequently centrifuged at 10,000×The extracted metagenomic DNA from the green samples  and the brown samples  were used as templates. The following primers were used for PCR amplification: (a) bacterial 16S rRNA V3-V4 region (341F 5′-CCTAYGGGRBGCASCAG-3′ and 806R 5′-GGACTACNNGGGTATCTAAT-3′), (b) archaeal 16S rRNA V4 region (U519F 5′-CAGYMGCCRCGGKAAHACC-3′ and 806R 5′-GGACTACNSGGGTMTCTA AT-3′), and (c) eukaryotic 18S rRNA at V4 region (528F 5′-GCGGTAATTCCAGCTCCAA-3′ and 706R 5′-AATCCRAGAATTTCACCTCT-3′).63. The process includes demultiplexing, quality-filtering, denoising, dereplication, and removal of chimeras, and clustering of the paired-end sequences. Taxonomy classification was carried out using SILVA SSU 138.1 database.Amplicon sequencing was conducted using Illumina NovaSeq 6000 platform  with paired-end 250 base pairs at NovogeneAIT Genomics (Singapore). A minimum sequencing depth of 100 K raw reads was reserved for every sample. The resulting raw paired-end reads were processed using the DADA2 plugin in QIIME 2 pipeline64. Clean paired-end reads were de novo assembled by metaSPAdes assembler v3.15.265. Taxonomy classification on the assembled contigs was carried out by Kraken2 v2.1.266. MetaQUAST v5.0.2 with default MetaGeneMark as gene predictor was used to find all the open reading frames (ORFs) from the assembled contigs67. Contigs < 500 bp were excluded. All ORFs were also subjected to Carbohydrate-Active enZYmes (CAZymes) annotation via run_dbcan v2.0.11 coupled with the latest CAZy database v0731202035. Sequences that were positive in at least one in program HMMER, Hotpep, and Diamond35 were shortlisted and further annotated by NCBI non-reductant (nr) and Uniprot reviewed protein database via Diamond v2.0.11.14968. Selected CAZymes were further analysed using InterProScan v5.52-86.0 for predicting domains, motifs, and others69. The motifs of Thermotoga maritima β-xylanase was used as the reference . Protein structure prediction was performed by AlphaFold Colab v2.052 using the default parameters and displayed using UCSF ChimeraX v1.2.5.DNA samples  were fragmented by a Covaris sonicator . Then, the fragmented DNA was used for dual-indexed, paired-end library construction following the Illumina DNA Prep kit protocol . The constructed library samples were run in Qubit 3.0 Fluorometer and Agilent Bioanalyzer 2100 . Whole metagenome shotgun sequencing was carried out in an Illumina NovaSeq 6000 with the running mode of PE 150 (paired-end 150 bp) conducted in NovogeneAIT Genomics (Singapore). A minimum of 20 Gb  output was reserved for each sample. The resulting raw paired-end reads were trimmed and filtered by SOAPnuke v2.1.6 softwareSupplementary Information.


Chronic myelogenous leukemia (CML) with thrombocytosis and complex chromosomal translocation is extremely rare in clinical setting. Here, we reported the clinical and pathological characteristics of CML patients, which were characterized by thrombocytosis and complex Philadelphia chromosome translocation. Moreover, we also introduced our therapeutic schedule for this patient as well as review relative literature.A 24-year-old female presented with night sweating, fatigue, and intermittent fever for 1 month.Fluorescence in situ hybridization results revealed that breakpoint cluster region (BCR)-Abelson (ABL) gene fusion in 62% of the cells and karyotyping showed a complex 3-way 46, XY, t  [19/20] translocation. This patient was diagnosed with CML complicated with thrombocytosis and complex Philadelphia chromosome translocation.The patients received continuously oral imatinib mesylate tablets (400 mg) once a day.IS was less than 0.1% and achieved major molecular response. Moreover, the BCR/ABLIS of this patient achieved major molecular response. The BCR/ABLIS values at 6 months and 12 months were less than 0.01% and 0.0032%, respectively. And no BCR/ABL fusion was detected in the next 2 years follow-up period.After treatment with imatinib for 3 months, the BCR/ABLImatinib might represent a preferred therapeutic option for CML patients with rare thrombocytosis and complex chromosomal translocation. In addition, BCR/ABL fusion gene examination in patients with thrombocytosis might represent an effective strategy to avoid the misdiagnosis of this specific CML population. We herein report an extremely rare case of CML presenting with isolated thrombocytosis and the chromosomal translocation  .Chronic myeloid leukemia (CML) is myeloproliferative neoplasm with an incidence rate of 1 to 2 cases per 100,000 adults. It is characterized by the presence of the Philadelphia (Ph) translocation-t  , which results in breakpoint cluster region (BCR)/ABL in 5% to 8% of CML cases.29/L, it rapidly increased to 2300 × 109/L within 1 week after drug withdrawal. This patient therefore was transferred to our center for further diagnosis and treatment in January 2019.A 24-year-old female was admitted for night sweats, fatigue, and fever. Initial physical examination results showed that no obvious splenomegaly and abdominal distension was observed. Bone marrow immunophenotyping analysis in December 2018 revealed that platelet (PLT)  count was significantly elevated and patient was initially misdiagnosed as essential thrombocytosis in other hospital. Despite hydroxyurea intervention could effectively reduce platelet count to 200 × 109/L , red blood cells 4.1 × 1012/L , and Hb 133 g/L , and aberrantly high, platelet count of 2300 × 109/L .Routine blood test indicated normal: white blood cells 4.3 × 10W515L/K were observed. Fluorescence in situ hybridization revealed the BCR-ABL fusion in, 62% of the cells , and 2 of 15 patients who underwent physical examination or imaging exhibited splenomegaly.To the best of our knowledge, this is the first report about an extremely rare case of CML presenting with isolated thrombocytosis and 3-way complicated Philadelphia chromosomal translocation. It's well-known that the typical clinical characteristics of CML are leukocytosis and splenomegaly, but rarely presented with isolated thrombocytosis.,6 The Philadelphia chromosome translocation is observed in 90% to 95% of CML patients whereas more, complex translocations, involving a third chromosome are present in only 5% to 8% of the patients.,8 Although variant translocations might involve any chromosome, the distribution of the breakpoints is nonrandom and, usually seen at some specific chromosomal bands, including 1p36, 2p22,3p21, 4q25,5q31, 6p21, 9q22, 10q22, 11q13, 12p13, 16p13,17p13,17q21, 17q25, 19q13, 21q22, 22q12, and 22q13.–16 Additionally, Al-Achkar et al also reported a novel Ph chromosome-positive CML case lacking the BCR/ABL fusion gene on der(9) and instead harboring a new complex rearrangement formed by chromosomes 11 and 20, as well as 9 and 22. Moreover, Asif et al demonstrated a novel 4-way complex variant translocation involving chromosome 46,XY,t in a chronic myeloid leukemia patient.CML is characterized by the genetic translocation, t  , that results in the fusion of Abelson gene (ABL1) from chromosome 9q34 with the breakpoint cluster region (BCR) gene on chromosome 22q11.2. Valencia et al reported that imatinib effectively improved the prognosis of patients with variant Philadelphia chromosome similar to that of patients with the classic Philadelphia translocation. Therefore, we initiated treatment with daily imatinib 400 mg, which achieved best response in 3 months, and BCR/ABL negative status after 12 months.Our patient exhibited solitary thrombocythemia without splenomegaly, and was initially misdiagnosed as essential thrombocythemia since genetic testing was not performed. Therefore, routine detection of the BCR/ABL fusion genes in peripheral blood and bone marrow smears is recommended for patients with unexplained solitary thrombocythemia to confirm the possibility of atypical CML, which is associated with poor prognosis and a median overall survival of 24 months.In conclusion, imatinib resulted in a favorable outcome in an extremely rare case of CML presenting with isolated thrombocytosis and 3-way complicated Philadelphia chromosomal translocation t  . Moreover, patients with isolated thrombocytosis should be routinely examined for BCR/ABL fusion gene, in order to effectively avoid the misdiagnosis of this specific CML population.The authors thank the patient for his approval to publicationConceptualization: Lu Gao, Zhong Yuan.Data curation: Ming-qiang Ren, Zhi-yuan Peng.Formal analysis: Zu-guo Tian.Funding acquisition: Lu Gao.Investigation: Zu-guo Tian, Zhi-yuan Peng, Genghui Shi.Methodology: Genghui Shi.Writing – original draft: Lu Gao, Ming-qiang Ren.Writing – review & editing: Lu Gao, Zhong Yuan.


Cyclotella cryptica was cultivated for 14 days under photoautotrophic and heterotrophic conditions to define the effects on growth parameters, lipid production, total fatty acids and EPA content. Photoautotrophic conditions led to a total EPA production of 1.6% of dry weight, 12.2 mg L−1 culture and productivity of 0.9 mg L−1 day−1. The heterotrophy cultures reported a total EPA production of 2.7% of dry cell weight, 18 mg L−1 culture, a productivity of 1.3 mg L−1 day−1, which are promising values in the prospective of improving culture parameters for the biotechnological exploitation of dark cultivation. C. cryptica could be a potential candidate for the heterotrophic production of EPA, also considering its robustness, capacity to resist to bacterial contaminations and plasticity of lipid metabolism.Recently, the marketable value of ω-3 fatty acid, particularly eicosapentaenoic acid (EPA), increased considering their health effects for human consumption. Microalgae are considered a valuable and “green” source of EPA alternative to fish oils, but considerable efforts are necessary for their exploitation at an industrial level. Due to the high operation costs of photoautotrophic microalgae cultivation, heterotrophic growth represents a promising economic solution. Marine diatoms are the major ecological producers of ω-3 fatty acids. Few species of diatoms are capable to grow in the dark using organic carbon sources. The marine diatom  Eicosapentaenoic acid (EPA) represents a central nutrient for human consumption to counteract cardiovascular disease, diabetes, different types of carcinoma, diabetes mellitus and brain disorders ,2,3. EPAMany marine microalgae species naturally produce EPA and DHA as components of glycerolipids ,10, thus2 [Microalgae were mainly cultivated in phototrophic conditions in different systems, namely, open ponds, flat panel or photobioreactors . Althoug2 . Any ferChlorella protothecoides, Galdieria sulphuraria, Nitzchia laevis, Crypthecodinium cohnii and Neochloris oleoabundans [A limited number of microalgae species can grow in heterotrophic conditions, especially abundans . These sabundans . The ideabundans ,18.Nitzschia laevis represents the most studied case for which impact of cultivation parameters on heterotrophic production of EPA has been assessed [Among microalgae, diatoms represent a primary trophic source in the marine food chain, sustaining zooplankton and fish nutrition and therefore are the major ecological producers of ω-3 fatty acids . Lipid aassessed ,25, suggCyclotella cryptica has been reported as a species capable of heterotrophic growth [C. cryptica at the exponential growth phase (maximum of 4 days of cultivation), as continuous cultivations are frequently used in aquaculture facilities [C. cryptica under photoautotrophic and heterotrophic conditions.The marine diatom c growth ,27,28, tc growth . Subsequcilities ,31,32, ocilities . The aimCyclotella cryptica was grown in autotrophic and heterotrophic conditions in order to compare the growth performances and EPA production among ω-3 polyunsaturated fatty acids. Autotrophic cultivation was guaranteed by an illumination at 200 µmol (photons) m−2 s−1 with a 14:10 h (light/dark) photoperiod, whereas heterotrophic growth was carried out by cultivating cells completely in the dark and by supplementing the medium with glucose as an organic carbon source. As showed in –4 days, the growth rate reflected a rapid increase in cell division. In fact, the doubling time was 2 ± 0.05 days in heterotrophy in comparison with a value of 3.3 ± 0.2 days in autotrophy, supporting the concept that a minor amount of time is necessary to double the cell number under heterotrophy. The cultures maintained the same intense brown color under photoautotrophic and heterotrophic conditions  has been described in heterotrophic conditions [Chlorella protothecoides, C. vulgaris and C. sorokiniana [Nitzschia laevis produces major biomass under heterotrophic conditions [Biomass production was evaluated on the 7th day  and 14th day (first point of senescent phase). After 7 days, the dry biomass was 246.4 ± 15.9 mg Lnditions a. Althoutotrophy a, accordnditions . Responsokiniana ,36. The nditions , althougnditions .Nannochloropsis gaditana showed similar percentages of lipids on dry weight in autotrophic, mixotrophic and heterotrophic conditions (10.7–15.7%) [Chlorella species showed a higher lipid content in heterotrophy, in comparison to autotrophic conditions [The lipid percentage was not significantly affected under two different culture conditions, being 22.2 and 18.8% of dry cell weight (DCW) in autotrophy and heterotrophy, respectively b. Cells 7–15.7%) . Chlorelnditions .Fatty acid methyl ester (FAME) analysis using GC–MS showed a simplified framework in heterotrophy , with a C. cryptica and N. laevis grown in heterotrophy, reporting a range of 15–20%, depending on the salinity and silicate levels, glucose input and temperature [Chlorella zofingiensis and Galdiera sp. [Chlorella vulgaris showed an opposite trend, with a decrease in MUFA in favor of PUFA in heterotrophy, suggesting that the response of fatty acid elongation and desaturation strictly depends on the microalgal species [Similar results were obtained after 7 days of cultivation, supporting the concept that the establishment of dark lipid-phenotype occurs early and is maintained along the growth curve. Data about the EPA% on TFA at the 14th day are in agreement with previous works on the diatoms perature ,39. Overiera sp. ,41. Chlo species ,43,44.1H-NMR method was used for the quantitative assessment of Total Fatty Acids (TFA) and ω-3 fatty acids in organic extracts, by integrating the diagnostic peaks at 2.35 ppm (methylene protons in α to carbonyl group) and at 0.9 ppm (methyl protons of ω-3 fatty acids), respectively [The ERETIC ectively .N. laevis also showed an increase in TFA in the heterotrophic growth mode in comparison to photoautotrophic conditions [In heterotrophy TFA were not significantly different after 7 days of cultivation but were 3-fold higher in heterotrophy than autotrophy after 14 days, arising values of 630 µmol/g DCW a. This inditions . The ω-3−1 and productivity of 1.3 mg L−1 day−1.Considering EPA contribution to ω-3 fatty acids determined by GC–MS, the quantitative assessment of EPA production is reported in Nitzschia laevis represents the most studied case for which the impact of cultivation parameters on the heterotrophic production of EPA has been assessed [Despite the recognized role of diatoms as cell factories for high-value products, few studies have explored their capacity for growth under heterotrophic conditions. assessed ,38 sugge, N. laevis, Navicula incerta, Navicula pelliculosa and C. cryptica  led to a major accumulation of TFA but did not change the EPA% on TFA. The nutritional factors to significantly burst EPA biosynthesis have not yet been determined under heterotrophic conditions. Some environmental factors , which can modulate lipid metabolism in a massive way under autotrophic conditions, did not affect the fatty acid distribution under heterotrophic growth. Alternative cultivation strategies to improve the final yield of biomass and EPA have been developed. A combination of perfusion and bleeding systems enhance the ability of N. laevis, obtaining 6.75 g L−1 day−1 and 175 mg L−1 day−1 of biomass and EPA yield, respectively, which are among the highest values ever reported in microalgal cultures [Glucose and silicate input play a critical role in the production of EPA and TFA in marine diatoms. Although a sufficient amount of silicate appears necessary for an adequate carbon metabolism, the glucose concentration appeared to be mainly related to TFA amounts and, in minor part, to the EPA percentage. Major glucose concentrations .1H NMR spectra were recorded on a Bruker DRX 600 spectrometer  equipped with an inverse TCI CryoProbe. Peak integration, ERETIC measurements and spectrum calibration were obtained by the specific subroutines of the Bruker Top-Spin 3.1 program. Spectra were acquired with 14 ppm of spectral width (8417.5 Hz), 32 K of time domain data points, 90° pulse, 32 K spectrum size and processed with 0.6 Hz of line broadening for the exponential decay function.Cyclotella cryptica (CCMP 331) was purchased from National Center for Marine Algae and Microbiota  and was maintained in an F/2 medium. C. cryptica was grown at 20 ± 2 °C in 2 L of polycarbonate carboy in 2 L of pre-filtered sterile (0.22 μm) f/2 medium [4 cells mL−1. Cultures were gently bubbled with sterile air. In autotrophic conditions, diatoms were grown under artificial light (200 μmol photons m−2 s−1), provided by daylight fluorescent tubes  with a 14:10 h light:dark photoperiod. In heterotrophic conditions, cultures were incubated in the dark and f/2 medium was supplemented with two pulses of 1g/L glucose for each one, at day 0 and day 7. Macronutrient levels were maintained at high regimes, adding nitrates  and phosphates  every 48 h, and silicate  every 24 h. Cell growth was monitored using a microscope  and a Bürker counting chamber .2 medium  at an in−1, was calculated according to Equation (1), as follows:1 and N2 are cell numbers (cells/mL) at time 1 (t1) and time 2 (t2) at the extremes of the linear phase [d was calculated according to Equation (2), as follows:Growth rate of cultures µ, expressed as divisions dayar phase . Doublind, where td is the doubling time or time required to achieve a doubling of the number of viable cells.Number of doublings (n) at a time interval t is determined by the relation t/t−1 culture. Biomass productivity was calculated in agreement with d’Ippolito et al. [Cells (500 mL) were harvested using centrifugation with a swing-out rotor at 2300 g for 10 min . Pellets were frozen at −80°C and lyophilized with a MicroModulyo 230 . Dry weight was estimated on lyophilized biomass and expressed as mg Lo et al. .−1) as the internal standard. In particular, dry cell pellet (50 mg) was suspended with 500 µL of DHBP and 400 µL of MeOH. After vortexing, 3 mL of MTBE were added to allow extraction at room temperature for 1 h, at constant shaking. Then, 750 µL of water were added and samples were left for another 10 min at room temperature, under shaking. Organic extracts were recovered (upper phase), after centrifugation at 1000× g for 10 min. The lower phase was re-extracted with 1 mL of MTBE and 750 µL of water, after centrifugation the upper phase was combined with the previous one. The extract was dried under nitrogen flow and weighed to gravimetrically estimate the lipid content (mg L−1 culture).Lipid extraction was performed using the Methyl tert-butyl ether (MTBE) method, in agreement with Cutignano et al. , using 43/CD3OD 1:1 (v/v) and transferred to a 5-millimeter NMR tube. 1H-NMR spectra were recorded on a Bruker DRX 600 spectrometer equipped with an inverse TCI CryoProbe. Chemical shift was referred to CHD2OD signal at δ 3.34. Quantitative assessment of fatty acids was established using the ERETIC method in agreement with Nuzzo et al. [Crude microalgal extracts were dissolved in 700 µL of CDClo et al. . The EREα to carbonyl group), and between 0.99 and 0.95, centered at 0.97 ppm (methyl protons of ω-3 fatty acids) were integrated to assess the µmol of Total Fatty Acids (TFA) and ω-3 fatty acids, respectively [The diagnostic peaks in the region between 2.38 and 2.28 ppm, centered at 2.35 ppm (methylene protons in ectively .2CO3 in methanol at 40 °C for 4 h. The reaction mixture was diluted with milliQ water (to dissolve completely Na2CO3), neutralized with HCl 1M, and extracted with diethylether three times. Combined organic extracts were dried under a nitrogen stream, dissolved in MeOH at a final concentration of 1 µg µL−1 and analyzed using GC–MS (Thermo Focus GC Polaris Q) equipped with an ion-trap, EI (70 eV), a 5% diphenyl column, an injector temperature of 210 °C, a transfer line temperature of 280 °C. Elution of free fatty acid methyl esters required an increasing gradient of temperature according to the following method: 160 °C for 3 min, increase by 3 °C/min up to 260 °C, increase by 30 °C/min up to 310 °C, 7 min at 310 °C. FAMEs have been identified by the comparation of retention time and mass spectra with FAMEs’ standard mixture . Fatty acid content was expressed as a percentage of total fatty acids, according to Equation (3), as follows:The total fatty acid composition of organic extracts was determined using GC–MS on the corresponding fatty acid methyl esters (FAMEs) obtained after saponification of lipid extracts with NaEPA) was established using the GC–MS integrating area of all ω-3 fatty acids , according to Equation (4). EPA (%DCW) was assessed according to Equation (5), considering the contribution of EPA fraction to ω-3 mmol assessed using 1H-NMR on organic extract obtained from 50 mg of DCW of EPA. EPA production was calculated by considering EPA expressed as % DCW, according to Equation (6), as follows:EPA content on total ω-3 (Xt-test (p ≤ 0.05).Each experiment was made in at least three replicates. Values were expressed as mean ± standard deviation (SD). The statistical significance was evaluated through Student’s


Homonucleardipolarrecoupling is routinely used for magic-anglespinning NMR-based structure determination. In fully protonated samples,only short proton–proton distances are accessible to broadbandrecoupling approaches because of high proton density. Selective methodsallow detection of longer distances by directing polarization to asubset of spins. Here we introduce the selective pulse sequence MODIST,which recouples spins that have a modest chemical shift offset difference,and demonstrate it to selectively record correlations between amideprotons. The sequence was selected for good retention of total signal,leading to up to twice the intensity for proton–proton correlationscompared with other selective methods. The sequence is effective acrossa range of spinning conditions and magnetic fields, here tested at55.555 and 100 kHz magic-angle spinning and at proton Larmor frequenciesfrom 600 to 1200 MHz. For influenza A M2 in lipid bilayers, cross-peakscharacteristic of a helical conformation are observed. It is alsoapplicable at ultrafast MAS rates of 55–100 kHz and above,where the effects of finite pulses become important14 and a heteronuclear version of the sequence becomes possible.15 Proton–proton RFDR has been widely appliedfor protein structure determination,23 which, for fully protonated proteins, is done by measuring a densenetwork of distances including side-chain protons.Proton-detected magic-anglespinning (MAS) NMR spectroscopy can be used to determine the structureand dynamics of proteins with atomic resolution. Proton–protoncorrelations obtained by recoupling homonuclear dipolar interactionsare direct indicators of the protein fold.24 This is the consequence of high proton densityin fully protonated protein samples and forms the basis for structuredetermination involving side-chain protons.17 Measurement of longer proton distances is challenging in these samples,even with second-order recoupling schemes30 that have been widely applied at both low and high MAS rates tocorrelate carbon and nitrogen spins.A characteristicof broadband proton–proton recoupling infully protonated samples is that only particularly close spins showcorrelations, while longer distances are hardly detectable.13C33 or 1H.39 For example, Linser et al.39 reportedamide proton–proton correlations up to 10 Å for a perdeuteratedmicrocrystalline sample. Using deuteration and specific methyl protonlabeling, Huber et al.40 detected 1H–1H correlations for distances up to 6Å. The former implemented broadband zero-quantum recoupling usingRFDR, while the latter applied a double-quantum sequence, dipolarrecoupling enhanced by amplitude modulation (DREAM).41 However, selective labeling is not always straightforward,in particular for membrane proteins, for which amide exchange maybe inhibited.42Detection of longer distancesis elegantly achieved by selectivespin-labeling of 50 In band-selective spectral spin diffusion (BASS-SD)47 selective 1H–1H transfer occursduring a spin-lock pulse,51 while in selectivephase-optimized recoupling (SPR)46 selective 1H–1H transfers occur between spins withsymmetrical frequency offsets46 . Both methods show significantenhancement of the transferred signals with respect to RFDR. Xiaoet al. recently published theoretical investigations of SPR pulsesat low MAS rates.52 In particular, theyinvestigated the behavior of p-SPR5 pulses at differentflip angles, p = π/4, π/2, and 3π/4,and concluded that small flip angles result in a narrow selectivebandwidth.Long-distance proton–protoncorrelations can also be measuredby using selective recoupling experiments.54 elements of SPR pulses46 and modified the phase, the flip angles, andthe number of pulses in the block to maximize transfer between amidespins, minimize transfer between amide and aliphatic spins, and, crucially,retain maximal total amide signal. MODIST is constructed similarlyto π/4-SPR42, but the modified phase cycling significantlymodifies its transfer characteristics. The MODIST block consists of16 π/4-pulses with the following phase cycling: yy̅x̅xx̅xy̅yy̅yxx̅xx̅yy̅  pulse sequence, where selective transfer occurs between spinswith small differences in their offsets. We based the MODIST sequenceon the jump-returnx̅xx̅yy̅ 1A. ThetSupporting Information, where we investigate the efficiency of the methodassuming different values of dipolar coupling constants, offset differences,flip angles of the selective pulses, carrier frequency settings, phasecycle schemes, chemical shift anisotropy values, and MAS rates. Insimulations , the ratiobetween transferred and untransferred signals is inferior to SPR54, but the total transfer efficiency of MODIST is better overalldue to the high retention of the total amide signals. Although thetransfer efficiency of MODIST pulses is much less dependent on theposition of the proton carrier frequency in comparison to other selectivemethods, the position of the carrier has an influence on the widthof the selective transfer, ΔfMODIST. We define ΔfMODIST as the offsetdifference with which the transferred signal reaches at least 50%of the maximal transfer with respect to the signal with zero offsetdifference. On the basis of simulations, ΔfMODIST of amide protons is ∼0.64 kHz , when the position of the carrieris in the amide region (8.2 ppm). However, it can be increased upto ∼0.9 kHz by setting the carrier to the aliphatic region without loss of efficiency.Numerical simulations of MODIST and comparison with SPR can befound in Figures S1–S10 of the Figure S10). MODIST, shows a highernumber of amide–amide correlations than (H)N(H)HRFDR even with a short mixing of 1.152 ms  for W42 (peak V) and W41(peak III), respectively. The intensity of peaks I–III is retainedat relatively long MODIST mixing time . The longest assigned distance is 7.3 Å, between D14and M25. Peaks corresponding to these long distances likely arisedue to significant contribution of relayed transfer rather than directtransfer alone. From the SH3 structure, it is evident that directtransfer is detected for distances of at least 4.5 Å N(H)(H)NHst 4.5 Å 2. We alsmethyl–Hmethyl mixing within (H)C(H)(H)CH spectra, where the carrier frequencyposition is set to −1 ppm. Figure S14 shows the 13C–13C projection of sucha spectrum, recorded on SH3 using a 600 MHz spectrometer. As expected,the peak intensity is reduced far from the diagonal , and Hα–Hαcorrelations can be observed. Because of the relatively small frequencyseparation in the aliphatic spectrum, some mixing also occurs betweenthe alpha and methyl regions.Because MODIST has minimal dependenceon the carrier frequencyposition, the approach is expected to be suitable for simultaneousHα–Hα and HFigure S15 compares MODIST and RFDRfor deuterated SH3 using an 850 MHz spectrometer. The frequency selectivityof the method is evident in the suppression of cross-peaks to protonsnear the extreme edge of the amide region, around 7 ppm. With 30.48ms MODIST mixing, an additional peak, G51 to L33 (9.63 Å), isdetected. This correlation likely arises due to relayed transfer.13C,15N-labeled influenza A M2 membrane protein N(H)HMODIST spectra , MODIST again shows excellent retention of amide signal, whileRFDR efficiently mixes signal into the side-chain.We also evaluated MODIST spectra of the uniform  protein 3 and 4. 47 and SPR.462, or MODIST, with the proton carrier frequency set to threedifferent values . Ofthe three, MODIST shows the highest retention of the total amide signalas well as less dependence on the position of the carrier frequency. 2 strongaliphatic transfer occurs for certain conditions. Figures S18–S20 show additional 2D (H)N(H)H spectracomparing MODIST, SPR54, and π/4-SPR42. For SH3 at 100 kHz, similar peak intensities were observed forboth MODIST and BASS-SD.For M2 at 55.555 kHz, MODIST compares favorably with two selectivemethods shown at 100 kHz MAS to improve amide–amide transferwith respect to RFDR: BASS-SD15N–15N projections of 3D (H)N(H)(H)NH spectra with MODIST, BASS-SD,or SPR42 mixing . Correlations inthe (H)N(H)(H)NHMODIST spectrum (blue) have ∼2 timeshigher intensities than when employing SPR42 (green) andBASS-SD (red), and at least five correlations could only be observedwhen using MODIST. Cross-peak intensities can be compared in the slicesof the 15N–15N projection shown in MODIST spectrumwith 4.608 ms mixing, two of which were assigned to correlations separatedby three residues, which confirms the known helical secondary structure. At 4.6–5 Å, thesecontacts correspond to relatively long distances. Figure S22 shows the 15N–15Nprojection of the corresponding 3D spectrum using SPR54 (1.296 ms mixing), a double quantum mixing sequence.Comparisons of transferred signals are shown in 2 mixing 4. We cho2 and SPR54). The advantageous features of MODISTallowed the detection of 1HN–1HN correlations with up to 2-fold improvement in intensityas compared with other state-of-the-art selective dipolar recouplingsequences. The bandwidth of MODIST approximately covers the amideregion even at a magnetic field of 28.18 T (a 1200 MHz spectrometer),which is the highest magnetic field currently available for high-resolutionNMR.In summary,we described MODIST, a selective dipolar recouplingsequence, and demonstrated its performance for amide protons in fullyprotonated samples. MODIST achieves efficient selective transfersfor a broad range of carrier frequency values. We presented MODISTspectra of two fully protonated proteins, microcrystalline SH3 andthe membrane protein M2, and compared them with the broadband mixingsequence RFDR and two selective methods, BASS-SD and SPR (π/4-SPR4


This study aims to prepare a DBM/CS composite bone graft material in a moldable putty form without compromising the peculiar properties of DBM and CS. For this purpose, firstly, porcine femur was defatted using chloroform/methanol and extracted by acid for demineralization, then freeze-dried and milled/sieved to obtain DBM powder. Secondly, the α-form and β-form of calcium sulfate hemihydrate  were produced by heating gypsum (CaSO4·2H2O). The morphology and particle sizes of α- and β-CSH were obtained by SEM, and their chemical properties were confirmed by EDS, FTIR and XRD. Furthermore, the DBM-based graft was mixed with α- or β-CSH at a ratio of 9:1, and glycerol/4% HPMC was added as a carrier to produce a putty. DBM/CSH putty possesses a low washout rate, good mechanical strength and biocompatibility. In conclusion, we believe that the moldable DBM/CSH composite putty developed in this study could be a promising substitute for the currently available bone grafts, and might have practical application in the orthopedics field as a potential bone void filler.Demineralized bone matrix (DBM) is a decalcified allo/xenograft retaining collagen and noncollagenous proteins, which has been extensively used because of its osteoconductive and osteoinductive properties. Calcium sulfate (CaSO Bone defects can occur from various causes such as aging, tumor, trauma, infection, surgery, or other congenital diseases . Bone grAllograft or allogeneic transplant is medical terminology referring to the transfer of bone harvested using bone tissue collected from a genetically nonidentical donor to a recipient of the same species, whereas xenograft refers to the transfer of bone tissue between different species. These are considered to be alternatives for autografts due to the similarity in chemical components and physical structure, which manifest good osteoinductive and osteoconductive properties. However, the risks of immune responses and disease transmission remains the major drawback. Demineralized bone matrix (DBM) is a kind of acid extracted demineralized allograft derivative composed of collagens, noncollagenous proteins and growth factors . After d4·2H2O), which can be a promising raw material to produce calcium sulfate hemihydrate . When gypsum is heated to over 110 °C, water can be removed in a process known as calcination, resulting in CSH production. CSH exists in α and β forms with identical chemical properties but presenting great variation in terms of structure, crystal size, surface area, and lattice imperfections [Calcium sulfate (CS) is a kind of ceramic-based synthetic bone graft material with a long history of use for bone healing since the 19th century . Gypsum fections . CSH is fections ,15,16,17fections .CSH is very suitable for filling and repairing bone defects due to its plasticity and self-hardening properties. Although the rapid biodegradation rate limits its clinical application, some studies have provided approaches by which CS bone grafting systems can be modified with other materials as composites to be more durable with enhanced bone regenerative potential ,20. For Since they are imported from other countries, most synthetic bone graft substitutes are expensive in Taiwan; therefore, in the present study, we intended to develop a local moldable composite bone graft product which is cost-effective, easy to handle, readily available and with good biocompatibility. As DBM possess excellent osteoinductive and osteoconductive capability, and CSH provides mechanical strength with self-hardening properties, a composite DBM/CSH putty was prepared and the physical/chemical properties, setting time, mechanical strength, in vitro bioactivity, and biocompatibility were evaluated. The procedures and results provided from this study could be used potentially for the production of the very first domestic DBM/CSH putty as a bone void filler for effective bone healing. 3 reagent  for 5 days , planetary ball mill , blade or diamond file. Chloroform, methanol, diethyl ester, HCl, AlCl3, Na2SO4, trypsin-EDTA solution were purchased from Sigma , and formic acid was obtained from J. T. Baker .The procedure for preparation of a DBM implant is shown in Lyophilized DBM powder (10 mg) was treated with 1 mg/mL of pepsin  in 0.01 N HCl for 96 h at room temperature until no visible matrix remained, then centrifuged at 14000 rpm for 5 min to collect supernatant for dsDNA Quantitation. ds DNA was measured by a Quant-iT™ PicoGreen™ dsDNA Assay Kit  following the manufacturer’s instruction. The samples were read by Thermo Scientific Varioskan Flash  to obtain OD values .v/v 1-propanol, 0.7 M NaOH, 0.2 M citric acid, 0.425 M sodium acetate trihydrate, and 1% glacial acetic acid) for 25 min. Five-hundred μL of Ehrlich’s solution (1 M 4-(Dimethylamino)benzaldehyde in 33% perchronic acid and 67% 1-propanol) was then added to the sample and incubated at 65 °C for 20 min. Standards (hydroxyproline) and samples were plated in triplicate in a 96-well clear, flat-bottomed plate (200 μL/well) and read in a spectrophotometer Thermo Scientific Varioskan Flash  at an absorbance wavelength of 550 nm. Chloramine-T, 1-propanol, glacial acetic acid, 4-(Dimethylamino)benzaldehyde, perchronic acid were purchased from Sigma , NaOH was from Showa  and citric acid, sodium acetate trihydrate were obtained from J.T Baker .Lyophilized DBM powder (10 mg) was treated with 1 mg/mL of pepsin in 0.01 N HCl for 96 h at room temperature until no visible matrix remained, then centrifuged at 14,000 rpm for 5 min to collect supernatant for collagen quantification. The sample was acid hydrolyzed with equal volume of 37% HCl at 110 °C overnight and treated with NaOH. Fifty μL of the resultant sample was mixed with 450 μL chloramine T reagent (0.07 M Chloramine-T in 10% 4·0.5H2O) was prepared using a wet, hot method. CSD  was heated at 132 °C for 30 min, then mixed with 30% CaCl2  solution and heated at 132 °C for 30 min again. Afterwards, boiling water was added to remove CaCl2, and the mixture was incubated at 132 °C for 30 min. The resultant product was dried in oven at 50 °C then milled by a high-efficiency ball mill for 4 min [The α-form of CSH  with 0–4% HPMC . The self-hardening, working time and setting time properties of the putty were evaluated . Representative images and particle sizes of DBM powder and CSH powder were obtained by scanning electronic microscopy (SEM) using an S-3000H microscope  under low vacuum conditions. Each specimen was covered with gold by a sputter coater . An Energy Dispersive Spectrometer (EDS) X-act, Oxford Instruments, High Wycombe, UK) was used to detect the elements from preparation of DBM powder and synthesis of CSH.The IR spectra of the samples prepared were recorded using a Nicolet AVATAR 330 Fourier Transform Infrared spectrophotometer  with KBr mixed with sample at a 100:1 ratio. X-ray diffraction  was performed to investigate the crystalline phases of synthetic CSH using Cu Kα X-rays generated at 40 kV and 30 mA at a diffraction angle (2θ) from 10° to 60° with a step size of 0.05°/step and an interval of 0.2 s/step.To prepare the samples for the compressive strength test, the powder part  and liquid part (glycerol and water) were mixed at a ratio of 0.25 (mL/g) under atmospheric conditions. Upon reaching the dough phase, the putty was spatulated into stainless steel molds and the resulting cylindrical samples had a diameter of 6 mm and a height of 12 mm for the mechanical test. The compressive strength of DBM/CSH composite putty was examined in accordance with ASTM F1633 standards. The compressive properties of cylindrical samples were measured at a loading rate of 1 mm/min with a universal mechanical testing machine . The measurements were performed three times for each group.DBM/CSH putty was soaked in phosphate-buffer saline  for 15 min and statically placed in an incubator at 37 °C for 3 days. The weight loss ratio (washout) was calculated from the formula of the weight loss of the putty divided by the weight of initial sample. The reference putty used for the wash out resistant test was Genex® putty .2 and 95% relative humidity. L929 cells  were purchased from the Food Industry Research and Development Institute, Hsinchu, Taiwan. Cells were routinely maintained in Modified Eagle Medium  supplemented with 10% fetal bovine serum  at 37 °C under 5% CO3 cells/well onto a 96-well culture plate in culture medium at 37°C overnight. After removing culture medium the next day, cells were washed with PBS and cultured in MEM supplemented with 1% FBS containing DBM/putty extract. 3--2,5-diphenyltetrazolium bromide  solution was then added to the medium, and cells were incubated at 37 °C for 2 h. The samples were read by an Enzyme-linked immunosorbent assay (ELISA) reader  with a wavelength of 570 nm to obtain OD values. Cell viability higher than 70% was considered good biocompatibility.In vitro cell viability testing of bone cement samples was conducted according to ISO10993-5. Briefly, DMB/DSH putty was extracted in 5 volumes of MEM at 37 °C for 24 h. L929 cells were plated at a density of 5 × 102O), and two demineralization reagents, namely acidic AlCl3 reagent or 0.6 N HCl, were examined in this study  and uses CS as a carrier in the form of an injectable paste. It possesses great osteoconductivity and degradability and has been widely used in clinical applications such as spinal fusion, trauma surgery, or benign bone tumors [TM, a composite paste or putty containing 40% of DBM and CS/calcium phosphate, is usually used as bone void filler. It can accelerate healing and be osteoinductive to support bone remodeling. OSTEOSET® 2 DBM is a bone graft substitute incorporating DBM (approximately 53% by volume) into OSTEOSET® CS pellets which provide a combination of osteoinduction and osteoconduction for bone repair. The DBM/CSH composite putty produced in this study consisted of around 86.4% (w/w) DBM mixed with CSH in 70% glycerol/4% HPMC as a carrier in a moldable putty form with good biocompatibility. Further study is required to verify the osteoconductive and osteoinductive properties of the composite putty for bone healing in animal models.Currently there are some commercially available DBM bone substitutes, but only very few of them are combined with CS as composite materials . Allomate tumors ,36,37,38In this study, DBM powder was obtained from porcine femur and α-/β-form CSH was successfully synthesized from CSD. We provided the optimal DBM/CSH ratio for this composite moldable putty with good biocompatibility, mechanical strength and the potential of osteoinduction/osteoconduction . We stro


Ivoria species  is described from Ivory Coast based on five females. The new species differs from the previously described congener  based on the shape of female genital shield, dorsal setae, centro-caudal part of the marginal shield and peritremes.A new  Uropodina are a very diverse group of soil-inhabiting mites, especially in tropical rainforests . Measurements are given in micrometers (μm).Specimens were cleared in lactic acid for a week and investigated with a Leica 1000 scientific microscope with a drawing tube. The photos were taken with a Keyence 5000 digital microscope. Specimens examined were stored in 70% ethanol and deposited in the Natural History Museum, Geneva  in the family Urodinychidae are small, narrow, and usually have a shorter or longer apical prolongation on the fixed digit.The robust and large chelicerae occur only in some genera within the Taxon classificationAnimaliaMesostigmataUrodinychidae46FAB210-3C0C-58A6-B2D5-3E0B6ECB3B25http://zoobank.org/B83D5B76-2A95-47D8-A1C1-A52AB3DA7964Holotype. Female. ”Afrique Occidentale, Côte d’Ivore, Dropleu, tamisage sans tronc mort” , 7°24'31"N, 8°19'14"W, 10 Oct. 1980, V. Mahnert and J.L. Peret leg. Paratypes. Four females, with same collection data as those for the holotype.h1 robust, with a short lateral branch and with numerous lateral teeth, h2, h3, and h4 narrow and marginally serrate. Palptrochanter setae v1 robust and serrate, v2 situated on small protuberance, basally serrate and apically pilose. Internal malae long and smooth with a short lateral branch.Idiosoma subpentagonal, dorsally domed, marginal and dorsal shields fused anteriorly. All dorsal setae short, with pilose margins. Five pairs of sternal setae pilose. Genital shield of female triangular, anterior margin rounded and situated between coxae IV. Peritreme hook-shaped. Tritosternum with vase-like base, apically serrate, its laciniae subdivided into two pairs of short lateral branches and one pair of long central branches. Hypostomal setae Female. Length of idiosoma 560–580, width at level of coxae IV 360–375 (N = 5), color reddish-brown. Shape of idiosoma pentagonal with vertex, dorsally domed.Dorsal idiosoma , st2, st3 and st5 longer (ca 10–12). Setae st1 inserted at level of anterior margin of coxae II, st2 at level of central region of coxae II, st3 at level of posterior margin of coxae III, st4 at level of central area of coxae III, st5 close to basal margin of genital shield. Sternal shield without sculptured pattern, one pair of poroid situated close to st3. 7–8 pairs of short (ca 21–25), marginally pilose ventral setae situated posterior to genital shield, surface around these setae covered by oval pits. 20–21 pairs of long (ca 34–36), marginally pilose setae placed on small protuberance situated on L-shaped longitudinal row from peritremes to anal opening. Ventral surface bears reticulate sculptural pattern posterior to pedofossae IV. Anal opening oval ca 10–12 long and ca 4–6 wide, anal valves narrow and with smooth surface , robust and with a short lateral branch and with numerous lateral teeth. Setae h2 (ca 35–37), h3 and h4 (ca 20–24) marginally serrate. Deutosternal region without teeth or denticulate rows. Chelicerae large and robust with internal sclerotized nodes , v2 long (ca 90–92), basally serrate and apically pilose and situated on a small protuberance. Other setae on palp segments smooth  people who are the major cultural group of the Ivory Coast.Until now, only one species had been described from this poorly-known genus. The differences between of the two species are summarized in Table Ivoria seems to be a rare, endemic genus in the West African region. The two known species occur only in Ivory Coast. The Uropodina fauna and the distribution of the known species are very poorly investigated in this region, therefore discovery of numerous additional species might be expected. A similar situation exists for the East African Uropodina genus Bloszykiella (The genus zykiella .