Penicillium lanosum, Penicillium nalgiovense, Debaryomyces hansenii, Staphylococcus equorum, and Erwinia tasmaniensis were isolated from the surfaces of the hams by the traditional culture method. By Illumina high-throughput sequencing, three fungal phyla were identified. Ascomycota was the dominant phylum followed by Basidiomycota. At the genus level, 11 fungi were identified, of which Aspergillus was the dominant fungus, followed by Penicillium and Wallemia. These findings provide fundamental knowledge regarding the microorganisms and flavor compounds in Mianning ham, which will help industrial processors develop effective strategies for standardizing quality parameters.Mianning ham, a traditional Chinese dry-cured ham, is protected by national geographical indications. To understand the surface and internal flavor composition and microbial community structure of Mianning ham, solid phase microextraction-gas chromatography (SPME-GC-MS) technology and Illumina high-throughput sequencing were utilized. The results showed that a total of 60 flavor substances were identified in the hams. Forty-nine kinds of flavorings were identified on the surface, including 14 aldehydes, 6 ketones, 10 alcohols, 5 esters, 7 hydrocarbons, 5 acids, and 2 other compounds. Thirty-six kinds of internal flavorings were identified, including 13 aldehydes, 4 ketones, 6 alcohols, 3 esters, 5 hydrocarbons, 4 acids and 1 other type. Decanal (34.91 μg/g) was the most prevalent compound on the surface, followed by n-hexanol (24.99 μg/g), n-hexanal (20.20 μg/g), and n-octyl (16.14 μg/g). n-Hexanal (20.74 μg/g) was the most common compound internally, followed by non-aldehyde (5.70 μg/g), 1-octene-3-alcohol (3.54 μg/g), and inverse-2-octenal (2.77 μg/g).  Mianning ham is a famous fermented meat product in Mianning, Sichuan province in southwest China, and is protected by national geographical indications. Mianning hams are made by traditional local techniques and are salty, delicious, rich in aroma, and bright in color. Mianning ham is characterized by plump muscles and small legs, generally weighing 4 ∼ 6 kg . The proFlavor is an important index to evaluate the quality of ham. The flavor of ham comes from the oxidative decomposition of endogenous enzymes during natural fermentation and extracellular enzymes and metabolites secreted by microorganisms . The micTen Mianning hams were selected from a batch of hams produced at a processing plant in Mianning (China) and ripened for 24 months. A 0.2-cm-thick piece of Mianning ham surface meat was cut with a sterile scalpel and taken as the surface (BM) sample. Then, a 1 ∼ 1.5-cm-thick piece of ham surface was removed with a scalpel, and sterile scissors or scalpels were used to collect a meat sample from a depth of 3 ∼ 4 cm as an internal (NB) sample . Then, tw were measured in triplicate according to the methods described by L∗ (brightness), a∗ (redness), and b∗ (yellowness) values were measured in triplicate according to the methods described by The pH and aThe surface and internal samples of Mianning ham were cut into pieces, and 3.00 g was accurately weighed into a 15 mL headspace bottle. Then, 1 μL 2,4,6-trimethylpyridine was added to the headspace bottle as an internal standard, and the bottle was sealed . The extGas chromatography conditions: an HP-5 MS UI chromatographic column  was used; the pressure was 32.0 kPa; the column flow velocity was 1.0 mL/min; the carrier gas was helium, and splitless injection was performed. The temperature of the injection port was 250°C.Column temperature program: the starting temperature was 40°C for 15 min; the temperature was increased to 160°C at 3°C/min and maintained for 0 min; then, the temperature was increased to 230°C at 4°C/min and maintained for 5 min.Mass spectrometry conditions: an electron ionization source (EI) was used; the electron energy was 70 eV; the ion source temperature was 230°C; the temperature of the four-stage rod was 150°C; the quality scanning range was 35 ∼ 500 m/z; and the detector voltage was 350 V.Qualitative analysis: the resulting sample chromatograms were integrated, and the volatile compounds corresponding to the peaks on the matched chromatogram were retrieved from the NIST14. L library at a matching degree of 80%.Quantitative analysis: the relative contents of each component were obtained by normalizing the peak areas of the total ion flow chromatograms.–1 diluent. Then, bacterial solutions of 10–2, 10–3, and 10–4 were successively obtained according to the 10-fold dilution method. Then, 0.1 ml of each bacterial dilution was inoculated with the smear method into the following media: plate count agar medium (PCA) with incubation at 36°C for 48 h; MRS agar medium at 36°C for 72 h; mannitol fermentation medium at 36°C for 24 h; and bengal red culture medium at 28°C for 5 days.Under aseptic conditions, 12.5 g surface and internal samples were weighed and placed in a sterile homogenizer, 112.5 mL sterile normal saline was added, and the homogenizer was used to beat the samples for 4 min at 5 T/sec to make a 10Plates with microbial community numbers between 30∼300 CFU were selected, and a single colony was selected to be smeared and purified on a sterilized plate for growth. The culture temperature and time were the same as before. Individual colonies were observed and preliminarily identified by microscopy after Gram staining. After the preliminary identification, the characteristic colonies were selected for further culture.2O 19 μL. PCR conditions were as follows: predenaturation at 94°C for 4 min; followed by 30 cycles of denaturation at 94°C for 30 s, annealing at 45°C for 30 s, and elongation at 72°C for 90 s; and final elongation at 72°C for 10 min. The PCR amplification products were determined by 1% agarose gel electrophoresis and then pooled on the device for sequencing. BLAST was used to analyze the homology of genes through the NCBI database, and the sequences of different species with high homology with the target gene sequences were obtained. The bacterial colony characteristics and morphology were comprehensively considered to confirm the identities of the strains.The genomic DNA of bacteria and fungi was extracted according to the instructions of the genomic DNA extraction kit. The bacterial 16S rRNA gene was amplified with universal primers: upstream primer 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and downstream primer 1492R (5′-GGTTACCTTGTTACGACTT-3′). The general primers for fungal ITS gene amplification were as follows: upstream primer ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and downstream primer ITS4 (5′-TCCTCCGCTTATTATTGA TATGC-3′). The PCR mixture (50 μL) was mixed for 30 s and contained the following: Taq PCR Master Mix 25 L, template DNA 2 μL, primers 2 μL each, and sterile ddHTotal microbial DNA of the samples was extracted with the HiPure Soil DNA kit according to the manufacturer’s protocol. Then, the DNA concentration and purity were determined by a NanoDrop microspectrophotometer. The extraction quality of DNA was determined by 2% agarose gel electrophoresis. The purified DNA was used for amplification of fungal ITS genes. ITS 3-kyo2 (F) (5′-GATGAAGAACGYAGYRAA-3′) and ITS4 (R) (5′-TCCTCCGCTTATTATTGATATGC-3′) were used as primers for PCR amplification of the fungal ITS2 region.4, 1.5 μL 10 μM forward primer, 1.5 μL 10 μM reverse primer, 1 μL KOD enzyme, 100 ng DNA template, and distilled water to final volume. The amplification procedure was as follows: predenaturation at 94°C for 2 min, 30 cycles of denaturation at 98°C for 10 s, annealing at 62∼66°C for 30 s, and extension at 68°C for 30 s, and final extension at 68°C for 5 min. PCR products were purified by AMPure XP Beads. After purification, the PCR products were quantified with Qubit 3.0. The second round PCR amplification system (50 μL) contained the following: 5 μL 10 × KOD buffer, 5 μL 2 mM dNTPs, 3 μL 25 mM MgSO4, 1 μL 10 μM connector primer, 1 μL 10 μM PCR universal primer, 1 μL KOD enzyme, and 100 ng DNA template. The reaction was brough to final volume with distilled water. The amplification procedure was as follows: predenaturation at 94°C for 2 min, 30 cycles of denaturation at 98°C for 10 s, annealing at 65°C for 30 s, and extension at 68°C for 30 s, and final extension at 68°C for 5 min.The first round PCR amplification system (50 μL) included the following: 5 μL 10 × KOD buffer, 5 μL 2 mM dNTPs, 3 μL 25 mM MgSOAMPure XP beads were used to purify the second round of amplification products. ABI StepOnePlus Real-Time PCR System  was used for quantitative analysis. According to Novaseq 6000 PE 250 model pooling, PCR amplification products were pooled on the device used for sequencing . The QIIt-tests of the variance. The results showed that P > 0.05 was not significant. 0.05 > P > 0.01 was significant and marked with ∗; P < 0.01 was extremely significant and marked with ∗∗.Microsoft Excel 2019 was used for data statistics, and IBM SPSS 25.0 was used for L∗ (brightness) of the surface was significantly lower than internal, the a∗ (redness) of the surface was extremely significantly lower than internal, and there was no significant difference in b∗ (yellowness). Because the ham of Mianning is preserved by hanging naturally, the tissue of the ham is hard outside and soft inside. The surface moisture content was 22.6 ± 0.681 g/100 g, and the internal moisture content was 36.7 ± 0.889 g/100 g. Thus, the surface moisture content was significantly lower than the internal moisture content. A low moisture content is of great significance to prevent spoilage and improve the quality of ham. W < 0.85, most of the bacteria stop growing, while mold and a small amount of salt-consuming bacteria can also grow; the surface aW of Mianning ham was 0.735 ± 0.001, and the internal aW was 0.772 ± 0.004. W of Xuanwei ham was 0.758, which was close to the aW of Mianning ham in this study. The content of fat and protein on the surface was 39.0 ± 1.0 g/100 g and 32 ± 0.58 g/100 g, respectively, and the content of fat and protein internal was 19.6 ± 1.1 g/100 g and 24 ± 3.1 g/100 g, respectively. The content of fat and protein on the surface was significantly higher than internal. The content of malondialdehyde on the surface of Mianning ham was 5.4 ± 0.31 mg/kg, and the content of malondialdehyde inside the ham was 1.7 ± 0.10 mg/kg. Malondialdehyde is the final product of fat oxidation. The surface of the ham had sufficient contact with oxygen, which made it more easily oxidized. The nitrite contents on the surface and internal areas of Mianning ham were 6.3 ± 0.58 and 0.3 ± 0.00 mg/kg, respectively, which were all within the range of nitrite residue limits (<30 mg/kg) of meat products in China. The nitrite content on the surface was significantly higher than the internal nitrite content. The reason for this finding may be that the pickling materials were all applied on the surface of the ham; only part of the nitrite enters into the ham, and the nitrite that enters into the ham is further degraded under the action of lactic acid  and internal (NB) of Mianning ham are shown in tic acid . Dang fotic acid . Fan et The flavor compound data from the Mianning hams are shown in A large number of studies have found that the complex flavor system of meat is composed of components with flavor and aroma activity . Volatil6 CFU/g. The number of molds and yeasts was the largest and played an important role in the formation of ham flavor and quality. This is consistent with the research results of Staphylococcus, Micrococcus, and molds on the surface of Xuanwei ham was above 106 CFU/g. In this experiment, the internal samples were examined twice according to the microbial culture standard in China, but no microorganisms were cultured. It may be that the high salt content (9∼10%) and low aW in the ham inhibited the growth of microorganisms.After pure culture of surface and internal samples, it was found through plate counting results  that the+ bacteria, with a white color and a grape-like pattern and was preliminarily determined to be Staphylococcus.Plate count agar, MRS, mannitol, and Bengal red plates were used to isolate and culture the characteristic bacteria from the ham surfaces, and a total of five strains were isolated and numbered MN1, MN2, MN3, MN4, and MN5. After observation of the presence of a single colony, Gram staining was used, and the colonies were then examined under the microscope. The bacterial colony characteristics and morphology are shown in Penicillium lanosum, MN2 was Penicillium nalgiovense, MN3 was Debaryomyces hansenii, MN4 was Staphylococcus equorum, and MN5 was Erwinia tasmaniensis. P. nalgiovense is a rare species found in Shennongjia of Hubei province, Deyang of Sichuan province, Dujiangyan of Sichuan province, and Guiyang of Guizhou province. It has been reported from moldy pork, soil, pot lids and paper boxes. Penicillium italium, Penicillium simum, Penicillium citrate, Aspergillus sagrada, and Aspergillus flavus.Aspergillus pseudoglaucus, Aspergillus penicillioides, Phialosimplex caninus, Yamadazyma triangularis, Candida glucosophila, Wallemia sebi, etc. Scopulariopsis, Epicoccum, Pithoascus, Phallu, Valsa, Hypochnicium, Pithoascus Aspergillus, Cercospora, Isaria, Penicillium, and so on. No P. lanosum was detected in any of these famous hams, but P. lanosum was the dominant fungus in Mianning ham. At present, there are few reports on P. lanosum, and its effect on quality characteristics and flavor formation in ham needs to be further studied. In this study, only five kinds of microorganisms were isolated. This kind of result may be related to sample freezing treatment.The isolated bacteria were subjected to ITS and 16S rRNA sequencing, and the identification results  showed tThe alpha diversity index of fungi in Mianning ham is shown in A Venn diagram  was usedPenicillium and Wallemia were found to be the dominant fungal genera, accounting for 99.19 (surface) and 99.20%  of the total fungal population, respectively. The dominant phylum of Panxian ham is Ascomycota, and the dominant genera are Aspergillus and Penicillium, similar to those of Mianning hams .The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: NCBI SRA (ZW: experiment and writing—review and editing. LJ, TB, BH, and JZ: formal analysis. RZ: validation. LC: writing—original draft preparation and funding acquisition. WW: supervision. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


To evaluate the overall diagnostic performance of 14‐3‐3 η protein in patients with rheumatoid arthritis (RA).PubMed, EMBASE, and Web of Science were searched to acquire eligible studies. Articles published in English before 20 February 2020 were included. Quality Assessment of Diagnostic Accuracy Studies 2 was used to evaluate the risk of bias and application concern of the included articles. Pooled analysis of diagnostic indicators of 14‐3‐3 η protein for RA was conducted by using a random effects model. Subgroup analysis was used to explore the sources of heterogeneity. Deeks' funnel plot asymmetry test was used to evaluate for the presence of publication bias.A total of 13 studies (1554 positive and 1934 negative participants) were included. The pooled sensitivity and specificity were 0.73 (95% CI 0.71‐0.75) and 0.88 (95% CI 0.87‐0.90), respectively. The pooled positive/negative likelihood were 5.98 (95% CI 4.39‐8.14) and 0.28 (95% CI 0.21‐0.37), respectively. In addition, the pooled diagnostic odds ratio was 23.48 (95% CI 13.76‐40.08) and the area under curve was 0.9245. The results of subgroup analysis indicated that ethnicity and control group might be the source of heterogeneity. The results of sensitivity analysis were stable. No significant publication bias was found.The current evidence indicated that 14‐3‐3 η protein has moderate accuracy for the diagnosis of RA. The following search terms were used: (“14‐3‐3 η” OR “14‐3‐3 eta” OR “14‐3‐3*”OR “YWHAH”) AND (“rheumatoid arthritis” OR "RA"). At the same time, all the references in the retrieved literatures were manually reviewed to identify other potential relevant articles. This meta‐analysis was performed based on the Preferred Reporting Items for Systematic Reviews and Meta Analyses (PRISMA) statement.2.2Diagnosis of RA conforms to 1987 ACR or 2010 ACR/ EULAR criteriaStudies that enrolled healthy donors or patients without RA who have arthritis or other autoimmune diseases including osteoarthritis (OA), ankylosing spondylitis (AS), psoriatic arthritis (PsA), systemic lupus erythematosus (SLE), Sjögren's syndrome (SS), scleroderma (Scl), and dermatomyositis (DM).Testing of 14‐3‐3 η protein by enzyme‐linked immunosorbent assay (ELISA).All articles reporting either sensitivity or specificity of 14‐3‐3 η protein in RA or reporting data allowing calculation of sensitivity or specificity.Samples in articles were restricted to human participants.Accessible full text.2.3Reviews, comments, conference abstracts, letters, editorials, and expert opinions.Studies with insufficient data, specificity and sensitivity could not be obtained.Specimen was not serum.Non‐English articles.2.4The data were extracted by 2 investigators (Huiren Lei and Ting Yuan) independently. When a disagreement arose, the third investigator (Ding Cao) was consulted to confirm the extracted data. Author, year, gender ratio, mean age, ethnicity, case numbers, control groups, study design, and disease duration were extracted from each included study. Also, detection method, cut‐off value, diagnostic gold standard, true positive (TP) results, true negative (TN) results, false positive (FP) results, and false negative (FN) results were collected from each study.2.5Quality of each study was assessed by 2 investigators (Tingguo Tang and Haiming Huang) independently using the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS‐2) tool,2.6Q test and I2 statistic were applied to evaluate the heterogeneity.P < .05 or I2 > 50%), subgroup analysis was performed to find out the reasons, and I2 greater than 75% suggested there is substantial heterogeneity between studies.P values less than .05 were considered significant difference.All data were analyzed using Stata 15, Meta‐Disc 1.4, and Rev Man 5.3. TP, FP, FN, and TN were used to calculate the sensitivity and specificity. A random effects model was produced to obtain combined sensitivity, specificity, diagnostic odds ratio (DOR), and positive/negative likelihood (LR+/LR−). Further, summary receiver operating characteristic was constructed to testify the summarized diagnostic rate. Cochran's 3A total of 381 records were identified through databases  and by scrutinizing the reference lists of the included studies,3.1The features of the 13 included articles are summed up in Tables 3.2The risks of bias and application concerns about reference standards in all included studies were low. The index test had almost 50% high risk of bias, while the application concerns of index test was low. About the domain of flow and timing in QUADAS‐2, the risks of bias in all included studies were low. Regarding the risks of bias about patient selection, all of the studies had high risk; however, the application concerns about patient selection in our meta‐analysis had high, unclear, and low, respectively Figure .3.3I2 of sensitivity and specificity was 94.4% and 81.5%, random effects model was applied to conclude the effective size. Then, the relevant pooled diagnostic values of 14‐3‐3 η protein were calculated. The pooled sensitivity and specificity were 0.73 (95% CI 0.71‐0.75) and 0.88 (95% CI 0.87‐0.90), respectively . This result indicated that this meta‐analysis did not have a threshold effect. Next, we carried out the subgroup analysis by Meta‐Disc 1.4 based on gender ratio, mean age, ethnicity, case number of RA patients, control groups, and study design. As shown in Table The heterogeneity of diagnostic tests included threshold effect and non‐threshold effect. The most important source of heterogeneity was due to different cut offs and threshold effect in all included studies.3.5P value was .45 (P > .05), which indicated there was little publication bias in this study (Figure Deeks' Funnel Plot Asymmetry Test was performed to explore publication bias, and the 4Rheumatoid arthritis is 1 of the most prevalent chronic inflammatory diseases. It primarily involves the joints, but should be considered a syndrome that includes extra‐articular manifestations, such as rheumatoid nodules, pulmonary involvement or vasculitis, and systemic comorbidities.There was high heterogeneity among this meta‐analysis. Thus, we tried to analyze the source for heterogeneity. First, the definition of a positive or negative 14‐3‐3 η protein was defined with different cut‐off values; however, threshold effect was not found by Meta‐Disc 1.4. Second, we carried out the subgroup analysis based on gender ratio, mean age, ethnicity, case of RA patients, control groups, and study design Table . We founThis meta‐analysis has several limitations. First, some articles may have been missed which were not published in the databases we searched. Second, the current studies were mostly case‐control designs and most of the participants were clearly diagnosed. Therefore, well‐designed prospective studies with larger sample sizes are needed to confirm the diagnostic value of the 14‐3‐3 η protein for RA. Third, all patients enrolled in the trials are definite RA, with no difficult‐to‐diagnose patients included, which may lead to higher diagnostic evaluation. Fourth, a large heterogeneity in this meta‐analysis existed, so the random effects model and subgroup analysis were adopted to control the size of heterogeneity. In addition, the overall sample size of some studies was relatively small.Nevertheless, this article is the first meta‐analysis describing the overall diagnostic value of 14‐3‐3 η protein in RA patients. Compared with the included individual studies, the major strength of this present study is more accurate results by synthesizing results from current published studies to evaluate the diagnostic value of 14‐3‐3 η protein in RA patients. Considering the shortcomings of this literature, relevant prospective experiments still need to be carried out.5From the results above, we confirmed that 14‐3‐3 η protein can be used as a complementary biomarker in the diagnosis of RA.None.Figure S1Click here for additional data file.Figure S2Click here for additional data file.Figure S3Click here for additional data file.Table S1Click here for additional data file.


Salix matsudana Koidz , an important greening and arbor tree species, and the genome of this species was compared with those of four other tree species in Salicaceae. The total genome sequence of S. matsudana was 655.72 Mb in size, with repeated sequences accounting for 45.97% of the total length. In total, 531.43 Mb of the genome sequence could be mapped onto 38 chromosomes using the published genetic map as a reference. The genome of S. matsudana could be divided into two groups, the A and B genomes, through homology analysis with the genome of Populus trichocarpa, and the A and B genomes contained 23,985 and 25,107 genes, respectively. 4DTv combined transposon analysis predicted that allotetraploidy in S. matsudana appeared ~4 million years ago. The results from this study will help reveal the evolutionary history of S. matsudana and lay a genetic basis for its breeding.Polyploidy is a common phenomenon among willow species. In this study, genome sequencing was conducted for  Salix. L and Chosenia Nakai of Salicaceae, include deciduous arbors and shrubs mainly distributed in temperate and frigid zones of the Northern Hemisphere. Willow tree species play important roles in energy production, afforestation, and greening. To date, over 520 willow tree species have been discovered worldwide. Asia is the center of origin for willow, and 257 willow tree species are distributed in China2. Willow tree species are resistant to a number of stresses, including salt stress3, drought stress4, water stress5, and heavy metal stress6, and provide important functions in ecological restoration7.Willows, the generic term for tree species in n = 12x = 38), triploidy, tetraploidy, pentaploidy, hexaploidy, and even dodecaploidy (2n = 12x = 228), is abundant among willow species in nature9. Most shrub willow tree species are diploid, while most arbor species are allopolyploid12. To provide the necessary genetic characteristics of willow for scientific study, the genomes of several diploid shrub willow species, including Salix suchowensis13 and Salix purpurea (available at http://phytozome.jgi.doe.gov), were sequenced successively.The genetic background of willow is complex. Chromosome ploidy, including diploidy  was mapped onto the 38 chromosomes of S. matsudana, the length of which ranged from 8.80–28.42 Mb. In total, 300.41 Mb of repeated sequences, accounting for 45.97% of the whole genome, was identified, among which the numbers of SSRs and unknown repeated sequences were 6185 and 116,059, accounting for 0.51% and 6.76% of the total genome, respectively , 321 rRNAs (belonging to 4 gene families), 1557 tRNAs (belonging to 23 gene families), and 3494 pseudogenes were discovered Fig. , while oS. matsudana and four other species in Salicaceae, namely, P. trichocarpa, P. euphratica, S. suchowensis, and S. purpurea, showed that the total number of genes in S. matsudana were 1.53 times and 2.17 times that in S. purpurea and S. suchowensis, respectively , and transcription factor regulating root and shoot growth via Pin3, were identified as expanded families, and their gene numbers were expanded to 18, 27 and 29, respectively.Further investigation of the expansion and contraction of gene families in the five tree species in Salicaceae demonstrated that the numbers of gene families and genes that were expanded in R values) of Salicaceae ranged from 1.09 × 10−9 to 0.67 × 10−9/site/year13. Similarly, a second genome-wide replication event was predicted to have occurred 36.7–59.7 MYA among species in Salicaceae, since the second peak was at 0.08, at which time differentiation between poplar and willow was estimated to have appeared. In addition, the allotetraploid was estimated to have formed 4.6–7.5 MYA in S. matsudana, since a peak appeared at 0.01 for this species.The duplication time of the genome was estimated by comparing the 4DTv mutation rates among genes in the collinear fragments of the five tree species in Salicaceae Fig. . A genomS. matsudana, 213,911, with a total length of 93,513,152 bp and accounting for 14.31% of the repeated sequences, were Copia transposons, while 227,569, with a total length of 100,397,354 bp and accounting for 15.36% of the repeated sequences, were Gypsy transposons.Among the long terminal repeat sequence (LTR) transposons in S. matsudana genome . Most shrub willows are diploid, with a genome size of ~400 Mb. For instance, the genome sizes of Salix triandra L. (ref. 25) and S. suchowensis Cheng26. are 386 and 425 Mb, respectively. Arbor willows are mainly allopolyploid12, with a genome size of >650 Mb. For example, the genome sizes of Salix babylonica L. (ref. 27) and Salix alba L. (ref. 25) are 748 and 807 Mb, respectively. S. matsudana is a tetraploid species28. A previous study by our group estimated that the genome size of S. matsudana was ~670 Mb by flow cytometry29. In this study, the genome size of this species was 656 Mb  originated initially from a hybridization event between Triticum urartu (AA) and T. aestivum L. (BB) to form the tetraploid Triticum turgidum (AABB), followed by the hybridization event between Aegilops tauschii (DD) and T. turgidum32. The origin of tetraploid Gossypium hirsutum L. (AADD) and Gossypium barbadense L. (AADD) was dated back to 1.5 MYA, which involved the formation of the A genome donated by Gossypium herbaceum L. or Gossypium arboreum L., and the hybridization event between the A genome and D genome (donated by Gossypium raimondii Ulbrich)33. The genome comparison in the present study suggested that 19 of the 38 total chromosomes in S. matsudana were consistent with those in the P. trichocarpa genome , which facilitated their survival and adaptation to different environmental conditions41. The differentiation time of Salicaceae was estimated to be 12 (ref. 42), 48 (ref. 41), and 52 MYA13 based on sequencing results for the chloroplast, plastid, and nuclear genomes of S. suchowensis, respectively. The variation in differentiation time might be caused by differences in sample selection, which could lead to variation in the correction coefficient and even the final result. The differentiation of the A and B genomes of S. matsudana .Fresh leaves of S. matsudana genome was constructed initially based on the structure prediction and de novo prediction, using LTR FINDER43, MITE-Hunter44, RepeatScout45, and PILER-DF46 software. The database was classified with PASTEClassifier47 and combined with the Repbase48 database to form the final repeated sequence database. The repeated sequences were annotated based on the final repeated sequence database using RepeatMasker49 software.The repeated sequence database of the 50 software. GENSCAN51, Augustus52, GlimmerHMM53, GeneID54, and SNAP55 were used for de novo prediction. P. trichocarpa and P. euphratica were selected for homologous species-based prediction using GeMoMa56. PASA57 was used for unigene-based prediction.Three strategies, namely, de novo prediction, homologous species-based prediction, and unigene-based prediction, were adopted for gene prediction. The prediction results from different strategies were integrated by EVM58 alignment with the NR59, COG60, and KEGG61 functional databases. Functional annotations in the GO62 database were compared with the NR database alignment result using Blast2GO63. In addition, the predicted genes were also subjected to COG, KEGG, and GO enrichment analyses.The predicted gene sequences were subjected to BLASTS. matsudana were aligned with those in related species, including S. suchowensis, S. purpurea, P. trichocarpa, and P. euphratica, to analyze gene duplication within species, gene evolution between species and the classification of species-specific genes. Clustering analysis of gene families was conducted by OrthoMCL64 software. The 4DTv mutation rate was calculated for each of the homologous gene pairs between and within species. LTR sequences with scores ≥6 were detected in the genome by LTR FINDER43 and PS SCAN65, but the repeated sequences detected by LTR FINDER were filtered. The flanking sequences of LTRs were extracted and aligned with MUSCLE66. The degrees of sequence differences were calculated by DistMat software, with the Kimura model and a 7.3 × 10−9 molecular clock.The protein sequences of S. suchowensis × S. integra), “51-5” , “2521” (S. purpurea), “52-2” (S. suchowensis × S. leucopithecia), S. integra, S. babylonica, “Yanliu 1” (S. psammophila), “795” , “287” and “Yanjiang” (S. matsudana) were collected and clipped to a length of 10 cm and diameter of 1 cm. Three biological replicates with ten cuttings per replicate were analyzed for each variety. After 15 days of hydroponic culture, the roots of each cutting were collected and stored at −80 °C. Total RNA from the roots of the ten varieties was extracted using an RNAprep Pure Plant kit (Tiangen). The expression levels of the two genes (CYP71 and TCTP1) in each biological replicate were measured by qRT-PCR using three technological replicates. A PrimeScriptTM RT Reagent Kit (Takara) was used for reverse transcription of the RNAs. Reverse transcription was performed at 37 °C for 30 min and 85 °C for 5 s. A One-Step SYBR Primer Script PLUS RT-PCR Kit (Takara) was used for qRT-PCR of the CYP71 and TCTP1 genes. The reaction was performed at 95 °C for 2 min, followed by 40 cycles of 95 °C for 5 s, 56 °C for 30 s, and 72 °C for 20 s. The Actin gene in Salix was used as the reference gene. The qRT-PCR primers are listed in Supplementary Table −ΔΔCt method. The expression level of “287” (S. matsudana) was selected as the reference level (with a relative expression of 1.0).The branches of “2345” , while the regions with significant differences were removed by Gblocks (v0.91b). Finally, supergenes were obtained by connecting all the aligned gene families in each species. ModelFinder in IQ-TREE was used for model testing, and the best model was predicted to be JTT + F + I + G4. The maximum likelihood method was used to construct a rooted phylogenetic tree. P. trichocarpa was selected as the outgroup species, and the number of bootstraps was set to 1000. Split times and the gradient and Hessian parameters were calculated by MCMCTREE in PAML (v4.9i) software. Visualization of the phylogenetic tree with split times was performed by MCMCTreeR.The A and B genomes of E value < 1e − 5 and a C score > 0.5 (C scores were filtered by JCVI software), were identified by comparing gene sequences between species using Diamond (v0.9.29.130). gff3 was used to determine whether similar gene pairs were adjacent in the genome.The similar gene pairs, with the criteria of an Supplemental materials-revised


Hylocereus polyrhizus) in Malaysia, which is known as gray blight through morphological, molecular and pathogenicity analyses. Nine fungal isolates were isolated from nine blighted stems of H. polyrhizus. Based on morphological characteristics, DNA sequences and phylogeny , the fungal isolates were identified as Diaporthe arecae, D. eugeniae, D. hongkongensis, D. phaseolorum, and D. tectonendophytica. Six isolates recovered from the Cameron Highlands, Pahang belonged to D. eugeniae (DF1 and DF3), D. hongkongensis (DF9), D. phaseolorum (DF2 and DF12), and D. tectonendophytica (DF7), whereas three isolates from Bukit Kor, Terengganu were recognized as D. arecae (DFP3), D. eugeniae (DFP4), and D. tectonendophytica (DFP2). Diaporthe eugeniae and D. tectonendophytica were found in both Pahang and Terengganu, D. phaseolorum and D. hongkongensis in Pahang, whereas D. arecae only in Terengganu. The role of the Diaporthe isolates in causing stem gray blight of H. polyrhizus was confirmed. To date, only D. phaseolorum has been previously reported on Hylocereus undatus. This is the first report on D. arecae, D. eugeniae, D. hongkongensis, D. phaseolorum, and D. tectonendophytica causing stem gray blight of H. polyrhizus worldwide.This study aimed to characterize the new fungal disease on the stem of red-fleshed dragon fruit ( Hylocereus polyrhizus) is one of the most highly demand varieties, grown in Malaysia owing to its nutritional value and attractive color. It belongs to the Cactaceae family. This exotic fruit is locally known as “buah naga” or “buah mata naga”1. It is also known as pitaya, strawberry pear, and night-blooming cereus2. In 1999, dragon fruit was first introduced in Setiawan, Perak, and Kuala Pilah, Negeri Sembilan, Malaysia. The fruit was named “dragon fruit” owing to the dragon-like scales or bracts on its surface3. Aside from having an attractive color and a pleasant taste, it is considered as a healthy fruit containing excessive amounts of vitamin C and water-soluble fiber4.Red-fleshed dragon fruit  had been infected with the stem gray blight disease, which may result in its reduced production. This study could provide insights into the management of plant diseases. This study aimed to identify the causal pathogen of the stem gray blight of H. polyrhizus. Of these, three isolates  were recovered from Bukit Kor, Terengganu and six isolates  from the Cameron Highlands, Pahang, Malaysia. A species or isolate was recovered from a single lesion. In general, the fungal isolates produced whitish, grayish, or brownish colonies on potato dextrose agar (PDA) plates. Two types of conidia, namely, α- and β-conidia, were produced from the formation of pycnidial conidiomata on carnation leaf agar (CLA). α-conidia were characterized as aseptate, hyaline, and fusiform with bi- or multi-guttulate, meanwhile, β-conidia were characterized as aseptate, hyaline, filiform, straight, or more often hamate, and lack guttule. The conidiogenous cells of α-conidia were phialidic, cylindrical, terminal, hyaline, and slightly tapered toward the end. However, in this study, the structure of the conidiogenous cells for β-conidia was not observed. Conidiophore was characterized as hyaline, branched, multiseptate, and filiform. Based on the described characteristics, the fungal isolates were tentatively identified as Diaporthe species. By sorting their morphological similarities and differences, the fungal isolates were classified into five groups of Diaporthe species . Isolates DF1, DF3, and DFP4 were grouped with D. eugeniae CBS 444.82; isolates DF2 and DF12 with D. phaseolorum CBS113425 and BDKHADRA-2; isolates DFP2 and DF7 with D. tectonendophytica MFLUCC 13-0471; and isolates DF9 and DFP3 with D. hongkongensis CBS 115448 and D. arecae CBS 161.64, respectively. The result of the phylogenetic analysis was in accordance with the molecular identification based on DNA sequences [Basic Local Alignment Search (BLAST)], thus resolving the morphological identification. The isolates from group 1 were confirmed to be D. eugeniae, group 2 was D. phaseolorum, group 3 was D. tectonendophytica, group 4 was D. hongkongensis, and group 5 was D. arecae. The combined sequence matrix and phylogenetic tree were deposited in TreeBASE (http://purl.org/phylo/treebase/phylows/study/TB2:S27649).The comparison of DNA sequences based on ITS, TEF1-α, and β-tubulin demonstrated that the isolates were similar to the reference sequences of lin Fig.  . The tested isolates showed typical symptoms of gray blight on the inoculated stems of H. polyrhizus. Initially, irregular yellowish lesion surrounded by reddish border appeared on the wounded point , which gradually turned into a dark-brown sunken lesion and demonstrated dampening . As the disease progressed, the lesion became apparently dry and turned gray . Then, it expanded periodically, and tiny black pycnidia appeared on the area of the lesion . No symptoms developed on the control points.The result of pathogenicity test indicated that all isolates of the eld Fig. A1–A5. Thint Fig. B1, whiching Fig. B2. As thray Fig. B3. Then,D. eugeniae) recorded the highest lesion length (10.25 ± 0.35 cm), whereas isolate DFP3 (D. arecae) had the lowest (3.25 ± 0.35 cm) , D. phaseolorum (group 2), D. tectonendophytica (group 3), D. hongkongensis (group 4), and D. arecae (group 5), were identified to be the causal agents of the disease. The Diaporthe species may act as a plant pathogen or a saprophyte or an endophytic symbiont34, however, several studies have reported that it is the genus responsible for multiple destructive diseases, such as root and fruit rots, dieback, stem cankers, leaf spots, leaf and pod blights, and seed decay39.The present study reported on stem gray blight, which is a new emerging disease infecting Diaporthe isolates were recovered from the blighted stem of H. polyrhizus. Based on their morphological characteristics, all the isolates produced both α-conidia and β-conidia, except for the D. arecae isolate, of which β-conidia was not observed. α- and β-conidia are the key characteristics for the identification of Diaporthe40. The formation of β-conidia can sometimes be rare or absent in certain species of Diaporthe41. According to Tuset and Portilla42 and Diogo et al.43, for some Diaporthe species , the formation of β-conidia can only be observed in pycnidia on the host but not in pycnidia in the culture plate.A total of nine .34 revealed that both characteristics can be varied among the Diaporthe species. The isolates from group 1 (D. eugeniae) tended to produce bi- and multi-guttules, whereas the other isolates only produced bi-guttules of α-conidia. The size of the guttules of α-conidia varied among the groups. The isolates from groups 1 and 5 (D. eugeniae and D. arecae) produced significantly smaller guttules compared with those produced by isolates from groups 2, 3, and 5   was found to be the most virulent. The fungus can act as a pathogen or a saprophyte and was reported to cause stem-end rot on mango (Mangifera indica)47. It also occurs as a saprophyte on cloves (Eugenia aromatica)34. This study discovered a new host and disease caused by D. eugeniae. The association of D. phaseolorum with dragon fruit was not new, because recently, this pathogen was reported to cause stem rot on Hylocereus undatus in Bangladesh12. However, the symptoms described were slightly different from those observed in the present study. It appeared as a yellow spot with a chlorotic halo in the previous report, but in the present study, chlorotic halo was not observed; rather, a reddish border surrounded the lesion. Similarly, gray to black pycnidia were scattered on the surface of the lesion. Aside from the dragon fruit, D. phaseolorum was reported as a causal agent of pod and stem blight, stem canker, and seed rot on soybean and trunk disease on grapevine62. It was also found to be an endophyte on Kandelia candel by Cheng et al.63.All the tested isolates of D. eugeniae, the present study highlighted H. polyrhizus as a new host associated with D. tectonendophytica as it causes stem gray blight. Contrarily, a study by Doilom et al.46 demonstrated the role of D. tectonendophytica as an endophyte occurring on teak (Tectona grandis) in Thailand. The capability of D. hongkongensis to act as a pathogen is undeniable as the fungus has been reported to cause severe diseases on a number of host plants, such as stem-end rot on kiwifruit64, dieback on grapevine45, and shoot canker on pear65. Meanwhile, D. arecae has been reported to be pathogenic on M. indica47, Areca catechu34, and Citrus66. D. hongkongensis and D. arecae were first reported on H. polyrhizus worldwide especially in Malaysia.Similar to Diaporthe, dragon fruits in Malaysia also suffer from multiple diseases caused by other fungi. Among these diseases are anthracnose caused by C. gloeosporioides23 and C. truncatum24; stem necrosis by Curvularia lunata25; stem canker by N. dimidiatum26; stem rot by Fusarium proliferatum27 and Fusarium fujikuroi28; reddish brown spot by Nigrospora lacticolonia and N. sphaerica30; and stem blight by F. oxysporum29.The occurrence of the disease in two different locations in Malaysia indicates its possibility to spread worldwide. Aside from Diaporthe causing stem gray blight on H. polyrhizus in Malaysia. It improves our knowledge on the symptomatology of the disease and identity of the pathogens through morphological and molecular analyses. The findings may be essential to strategize effective disease management for stem gray blight on H. polyrhizus and for quarantine restrictions.This study provides overview of the five different species of H. polyrhizus were collected from Bukit Kor, Terengganu, Malaysia, and the Cameron Highlands, Pahang, Malaysia. The symptomatic samples were brought back to the laboratory for isolation. One lesion per stem exhibiting the same symptom was selected for fungal isolation. The lesion consisting of diseased and healthy parts was excised (1.5 cm2) and surface-sterilized with 70% ethanol for 3 min. Then, the samples were soaked in 10% sodium hypochlorite (1% NaOCl) for 3 min and rinsed with sterile distilled water three times consecutively for 1 min each. The sterilized samples were air-dried on the sterile filter papers before being transferred to PDA plates. The inoculated plates were incubated at 25 °C ± 2 °C for 2 to 3 days. Pure cultures of fungal isolates were obtained via hyphal tip isolation and were used for morphological and molecular analyses.In November 2017 and July 2018, nine gray blighted stems from the different plants of p < 0.05) using the IBM SPSS Statistics software version 24.Each fungal isolate obtained was cultured on PDA and incubated at 25 °C ± 2 °C for 7 days. Macroscopic characteristics, such as colony appearance and pigmentation, were recorded. CLA was utilized to induce the formation of pycnidial conidiomata, and the inoculated plates were incubated at 25 °C ± 2 °C for 7 days. The morphology of α- and β-conidia was observed from the pycnidial conidiomata. The other microscopic characteristics observed were conidiophores and conidiogenous cells. The length and width of 30 randomly selected conidia and the size of the guttules of 30 randomly selected α-conidia were measured and recorded. The differences in the length and width of conidia and the size of the guttules of α-conidia were evaluated via one-way ANOVA. In addition, the means of both parameters were compared via Tukey’s test , 8 µL of MgCl2 , 1 µL of deoxynucleotide triphosphate polymerase (dNTP) , 8 µL of each primer , 0.3 µL of Taq polymerase , 1 µL of genomic DNA, and sterile distilled water. Polymerase chain reaction (PCR) was performed using MyCycler Thermal Cycler  under the following conditions: initial denaturation at 95 °C for 4 min, followed by 35 cycles of denaturation at 95 °C for 35 s, annealing at 54 °C (ITS)/57 °C (TEF1-α)/58 °C (β-tubulin) for 1 min, extension at 72 °C for 90 s, and final extension at 72 °C for 10 min. The PCR product was separated by running it in 1.0% agarose gel  stained with HealthView Nucleic Acid Stain  at 90 V and 400 mA for 90 min. The 100 bp DNA ladder  was used as a marker to estimate the size of the amplified PCR products. The PCR products were sent to a service provider  for DNA sequencing.The identity of all the fungal isolates was further confirmed by molecular characterization. The isolates were grown in potato dextrose broth (PDB) and incubated at 25 °C ± 2 °C for 7 days. Fungal mycelia from PDB were homogenized under liquid nitrogen to obtain fine powder. A total of 60 mg fine powder was transferred into a 1.5 mL microcentrifuge tube, and the genomic DNA of the fungal isolates was extracted using the Invisorb Spin Plant Mini Kit , following the manufacturer’s protocols. The primers of ITS5/ITS470. After pairwise alignment, the BLAST algorithm (https://blast.ncbi.nlm.nih.gov/Blast.cgi) was used to compare the generated consensus sequences with other sequences in the GenBank database. The sequences obtained were deposited in the GenBank database.The obtained sequences were aligned using the Molecular Evolutionary Genetic Analysis software (MEGA7)71 was used to generate the ML trees based on a single and combined genes of ITS, TEF1-α, and β-tubulin with 1000 bootstrap replications72.The isolates in the present study and reference sequences used in the phylogenetic analysis are presented in Table H. polyrhizus for all the obtained fungal isolates. Conidial suspension was prepared by flooding the 7-day-old PDA culture with sterile distilled water, and the concentration was adjusted to 1 × 106 conidia/mL using a hemocytometer . The stems were surface-sterilized with 70% ethanol, and 0.1 mL of conidial suspension was utilized for inoculation using a disposable needle and syringe. Likewise, the control points were treated with sterile distilled water. On each stem, three points were used to inoculate fungal isolate and one point for control. Each fungal isolate was tested in three replicates, and the pathogenicity tests were conducted twice. All the inoculated plants were placed in a plant house in the School of Biological Sciences, USM, and incubated at 26–32 °C for 21 days. The progression of the disease symptom was observed daily. The lesion length was measured and recorded after 3 weeks of inoculation. The differences in the lesion length were evaluated via one-way ANOVA, and the means were compared via Tukey’s test (p < 0.05) using the IBM SPSS Statistics software version 24. For the fulfillment of Koch’s postulates, the fungal isolates were reisolated from symptomatic inoculated stems and reidentified by morphological characteristics.The pathogenicity test was conducted on 18 healthy stems of Supplementary Information.


Dear Editor,Togaviridae, genus Alphavirus, is transmitted by the A. aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia , an arbovirus in the family of et al.et al.et al.et al.et al.et al.et al. et al.et al.et al.et al.et al.During the southern India Ocean epidemic in 2005, the vertical transmission of CHIKV during the perinatal period was observed in Reunion island  and a Chinese woman, were vertically infected with CHIKV. They all had significant perinatal complications including rash, fever, and neonatal hyperbilirubinemia but didn’t show obvious neurological symptoms. After treatment, they were all discharged from the hospital.Ruili of Yunnan Province is a town on the border between China and Myanmar Fig. A. It hasCase 1A 36-year-old Burmese woman (M1) was admitted to the obstetrical department of Ruili People's Hospital because of fever and myalgia for 10 h on October 8th, 2019. CHIKV infection was suspected as a CHIKV outbreak was ongoing in the city, and was validated later by RT-PCR. The same day she was admitted to hospital, she had a C-section delivery and gave birth to 2 male neonates (N1 and N2).The neonates were 36 weeks and 1 day gestational age, with birth weight of 2870 g (N1) and 2530 g (N2) respectively. They had neonatal asphyxia with Apgar scores of 6/6/9 (N1) and 6/6/8 (N2), respectively. They were immediately transferred to the neonatal intensive care unit (NICU) and placed on continuous positive airway pressure and later placed on BiPhasic non-invasive ventilator. Antibiotics (Cefperazone-Sulbactam 0.4 g q12h) were used to treat the presumable perinatal bacterial infection. On the fifth day of life, they developed disseminated maculopapular rash and fever with a peak of 39 °C and 38.9 °C respectively. Bathing with tepid water and taking acetaminophen orally didn’t reduce the body temperature. One day later, yellowish skin appeared and the maximum serum indirect bilirubin reached 113.1 μmol/L (N1) and 100.6 μmol/L (N2) respectively. While in hospital, no convulsion, screaming, sleepiness and other neurological symptoms were observed. CHIKV infection was confirmed by RT-PCR. The twin progressed well and were discharged from hospital on the 12th day after delivery  developed fever, chest rash, and arthralgia on October 9th, 2019 at 38 weeks and 3 days of gestation. She was admitted to the obstetrical department of Ruili People's Hospital on Oct, 10th, and had a C-section delivery the same day. Her serum was positive for CHIKV IgM. CHIKV infection was further confirmed by RT-PCR. Six days after delivery, the skin rash reemerged.The neonate (N3) was a female with birth weight of 3080 g. Her Apgar score was 9/10/10, but the amniotic fluid was slightly stained. She was doing well until October 13th when she developed a macular erythematous rash, yellowish skin, drowsiness, arthralgia and temperature of 39 °C. The maximum of blood indirect bilirubin was 160.8 mmol/L as recorded on Oct 14th. CHIKV infection was verified with RT-PCR. On the sixth day of her life, muscle tension was recovered but arthralgia was still present. No screaming, convulsion or other nervous system symptoms were observed while in hospital. After 6 days of antipyretic  and antiviral treatment , she progressed favorably and was discharged on her 10th day of life  is circulating in this region, all febrile patients should first be tested for DENV infection, and the negative cases will then be subjected to CHIKV or ZIKV detection by RT-PCR . All reported CHIKV cases in this study were negative for DENV and ZIKV.et al.et al.et al.et al.According to a systematic review and meta-analysis on perinatal vertical transmission of CHIKV, the symptoms of infected neonates include fever, irritability, hyperalgesia, diffuse limb edema, rashes and occasionally sepsis-like illness and meningoencephalitis  raised concerns on mother-to-child transmission of vector-borne viruses. The observation of perinatal vertical transmission of CHIKV suggested attention should be paid not only to ZIKV. Unlike ZIKV whose vertical transmission occurs during the first trimester, CHIKV is usually transmitted from mother to child at the perinatal period (Contopoulos-Ioannidis


Quercus species serve as a powerful model for studying introgression in relation to species boundaries and adaptive processes. Coexistence of distant relatives, or lack of coexistence of closely relative oak species, introgression may play a role. In the current study, four closely related oak species were found in Zijinshan, China. We generated a comprehensive genome size (GS) database for 120 individuals of four species using flow cytometry‐based approaches. We examined GS variability within and among the species and hybridization events among the four species. The mean GSs of Q. acutissima, Q. variabilis, Q. fabri, and Q. serrata var. brevipetiolata were estimated to be 1.87, 1.92, 1.97, and 1.97 pg, respectively. The intraspecific and interspecific variations of GS observed among the four oak species indicated adaptation to the environment. Hybridization occurred both within and between the sections. A hybrid offspring was produced from Q. fabri and Q. variabilis, which belonged to different sections. The GS evolutionary pattern for hybrid species was expansion. Hybridization between the sections may be affected by habitat disturbance. This study increases our understanding of the evolution of GS in Quercus and will help establish guidelines for the ecological protection of oak trees.The  Q. fabri and Q. variabilis, which belong to a different section. The pattern of GS evolution for hybrids species is GS expansion. Hybridization between the sections may be affected by habitat disturbance.We examined GS variability within and among the species and present hybridization among four species. It occurred hybridization both within the section and between the sections. A hybrid offspring produced from  At present, the vegetation of Zijin Mountains is mainly artificial and natural secondary forests, which are transitional between subtropical evergreen broad‐leaved forests and warm temperate deciduous broad‐leaved forests. The zonal vegetation is a mixture of deciduous evergreen and broad‐leaved forests that is rich in various plant resources. Q. acutissima and Q. variabilis are important deciduous tree species in this area that are widely distributed and occupy dominant positions. The samples were prepared from the fresh mature leaves of natural wild plants growing in the Zijin Mountains, Nanjing, Jiangsu Province, China, during late October 2011 , saplings (1 < DBH≤10), and trees (DBH > 10) were collected randomly and evenly in this forest stand.A total of 120 samples of 2.2Petunia hybrida (2C = 2.85 pg) from the Nanjing Forestry University nursery was included as the internal standard plant  Tween 20, and 50 mM Hepes, pH 7.2) and 2.2 μl β‐mercaptoethanol should be added to each ml of buffer before use  were calculated and the variance in GS variation was analyzed using SPSS 19.0 software  and R studio 3.6.2. We set the GSs of the individual to be a variable, calculated the Euclidean genetic distances among the 120 samples and set 4 clustering categories. Finally, the hierarchical clustering was selected to realize the analysis with R studio 3.6.2.33.1P. hybrida are shown in Figure Owing to the low nuclear yields of mature leaves, we optimized Marie's Buffer for each of the four oak species. The CV values of the four oak species were all within the normal range, and the GSs of the individual species overlapped Table . The resQ. acutissima, ranging from 1.83 to 1.91 pg , with a mean of 1.87 pg. In Q. variabilis individuals, estimates varied 1.06‐fold, ranging from 1.87 to 1.99 pg , with a mean of 1.92 pg. Estimates varied 1.05‐fold in Q. fabri individuals, ranging from 1.91 to 2.02 pg , with a mean of 1.97 pg. The GS of Q. serrata var. brevipetiolata varied 1.06‐fold, ranging from 1.93 to 2.06 pg , with a mean of 1.97 pg. Quercus fabri and Q. serrata var. brevipetiolata had the same mean values (1.97 pg). The average GSs for Q. fabri and Q. serrata var. brevipetiolata were the largest, and the average GS for Q. variabilis was only slightly larger than that for Q. acutissima. As shown in Figure Q. acutissima individuals were less than the mean GS (1.82 pg), the GSs of most Q. variabilis individuals were greater than the mean GS (1.92 pg), and the GSs of most Q. serrata var. brevipetiolata and Q. fabri individuals were less than the mean GS (1.97 pg).GS estimates varied 1.04‐fold for 3.2Q. serrata var. brevipetiolata and Q. fabri. Additionally, variations between the populations were greater than variations within the populations. The intraspecific variation decreased in the following order: Q. serrata var. brevipetiolata > Q. variabilis > Q. fabri > Q. acutissima  may be hybrids. The clade II was mostly composed of Q. acutissima individuals and a few of Q. variabilis individuals. The members of clade III are relatively complex, mainly including four oak species. Firstly, a small number of Q. acutissima individuals; secondly, Q. variabilis and Q. fabri individuals make up a large proportion of this clade, lastly the Q. serrata var. brevipetiolata individuals were scattered, without specific rules in this clade. The discontinuous cross‐distribution of four species of oaks in the clade III indicated that the GSs of these individuals had interspecific transition values. Moreover, Q. acutissima individuals did not appear in the clade which composed of Q. fabri and Q. serrata var. brevipetiolata. This confirmed that there were no interspecific hybridization between Q. acutissima and either Q. fabri or Q. serrata var. brevipetiolata.In this study, only GS variation parameters were used for cluster analysis without other indicator Figure . The res3.4It has been reported that the GSs of angiosperms are significantly non‐normal, and some lineages have extremely large GSs  and 304.46 pg   was greater than that of section Quercus (1.07‐fold). This observation may be correlated with environmental parameters. According to the study of Zhang and Li on the prediction of the habitable zone of Q. acutissima and Q. variabilis in China, we found that optimal distribution regions for two species were the Yunnan‐Guizhou Plateau and Qin‐Ba Mountains having both a high altitude and latitude  is smaller than in Q. variabilis (1.92 pg). Some of the phenotypic traits, such as plant height, seed mass, cell size, and cell cycle time, may also facilitate GS variability ; formal analysis (lead); methodology (lead); resources ; writing–original draft . Xuan Li: Formal analysis (supporting); methodology (supporting); software (lead); writing–original draft ; writing–review and editing (lead). YanMing Fang: Resources ; supervision (lead); validation ; writing–review and editing (supporting).Table S1Click here for additional data file.


Flavonoids from a variety of fruits, including açaí, have beneficial antioxidant activity in several diseases, including cancer. Breast cancer is the second most prevalent cancer among Brazilian women. Studies have shown the action of flavonoids on neoplastic cells, as well as on diabetes and neurodegenerative and cardiovascular diseases.  To analyze the relationship between the consumption of açaí and the presence of chronic diseases in women residing in the rural area of São Luís, Maranhão.  A convenience sample of 150 women residing in the Maracanã neighborhood in São Luís, Maranhão, was used; the collected data included sociodemographic characteristics, habits, sexual and reproductive history, consumption of açaí, and history of cancer and other chronic diseases. The sample was divided into women who consumed açaí at least once a week (cases) and women who did not consume açaí (controls). Statistical analysis was performed to assess the relationships between those variables and the consumption of açaí.  A total of 141 women (94%) consumed açaí. Among these, 79.3% were aged between 20 and 50 years, 78.67% were farmers or housewives, 64.67% were Pardo (mixed race), 76.67% were nonsmokers, 70% were not receiving hormonal therapy, 40.67% had already undergone mammography, 28% had already undergone breast ultrasound, and 27.33% had a family history of cancer, with breast cancer being the second most prevalent cancer. There was a higher prevalence of hypertension among women who did not consume açaí than that among those who did; however, previous cancer, family history of cancer, heart disease, and diabetes were more prevalent among the consumers of açaí. There were no statistically significant relationships.  Flavonoids are known to have a beneficial effect on some types of neoplastic cells and other diseases; therefore, larger studies are necessary to better evaluate the beneficial effects of consuming foods containing flavonoids on these diseases. Euterpe oleracea Mart.) is a typical and popular fruit of the Amazon region that has gained importance in recent years because of its health benefits, which are associated with its phytochemical composition and antioxidant capacity [Açaí , whose content in fruits is related to the prevention and reduction in the risk of cancer and cardiovascular and circulatory diseases through the scavenging of free radicals [Experimental studies with transgenic mice and models of cancer mediated by carcinogens such as radiation and agents derived from exogenous or endogenous sources have shown that some phytochemicals consumed in the diet have a protective effect against cancer. This protection provided by dietary phytochemicals is the result of the induction of cellular defense mechanisms, including antioxidants, detoxification, activation of anti-inflammatory enzymes, and signaling pathways that lead to cell cycle arrest and/or cell death .Cancer is a growing health problem worldwide, resulting from the increase in life expectancy and urbanization and subsequent changes to the environment . Breast Euterpe oleracea Mart.) [Cancer-preventing phytochemicals, especially flavonoids, have been shown to suppress or prevent the progression of cancer by a variety of mechanisms. The intake of some types of flavonoids may contribute to the prevention of breast cancer , and a pa Mart.) .Plants contain natural compounds with important bioactive properties, which might be alternatives to the currently used medications and lead to the discovery of new drugs , 9. The Thus, the objective of the present study was to analyze the relationship between the consumption of the açaí fruit and the presence of chronic diseases in women residing in the rural area of São Luís, Maranhão.This cross-sectional, retrospective, and analytical study assessed the prevalence of açaí intake among women in São Luís, Maranhão, and its association with chronic diseases.The study was conducted in the municipality of São Luís among women residing in the Maracanã Ecological Park, an Environmentally Protected Area (EPA), in which the Juçara Park is located .The population selected for this study was a convenience sample with a calculated sample size of 150 individuals aged between 12 and 80 years. The study period was between November 2014 and February 2015.The inclusion criteria were women who resided in the Maracanã Ecological Park who provided their informed consent for their inclusion in the study.The exclusion criteria included women residing in the Maracanã Ecological Park with mental impairments that could hinder their understanding of the questions on the specific questionnaire and women who refused to provide their informed consent.The sample was composed of 141 women residing in the Maracanã Ecological Park who consumed açaí at least once a week (cases) and nine women who did not consume açaí (controls).Data collection was performed between November 2014 and February 2015.Interviews were conducted using a structured epidemiological questionnaire that included information on sociodemographic characteristics, habits, sexual and reproductive history, consumption of açaí, and history of cancer.The data were entered into an electronic database and analyzed using Stata/SE 9.0 for Windows . The categorical variables were expressed as absolute and percentage values and the numerical variables as means and standard deviations.t-tests were used for comparison of numerical variables (age).Chi-square test or Fisher's exact test was used to assess the relationship between categorical variables, and two-sided α) was set at 5%; i.e., the results were considered significant for p < 0.05.In all tests, the significance level . The women included in the study provided their informed consent .The study included 150 women, of which 0.67% (1/150) were less than 20, 25.33% (38/150) were between 20 and 30, 33.33% (50/150) were between 31 and 40, 15020.67% (31/150) were between 41 and 50, 10.67% (16/150) were between 51 and 60, 6.00% (9/150) were between 61 and 70, and 3.33% (5/150) were between 71 and 80 years of age. The patients' ages ranged between 12 and 80 years . There was no statistically significant difference in age between the group that consumed açaí (141 women) and the control group (9 women). With regard to years of schooling, 6.67% (10/150) were illiterate women and 9.34% (14/150), 27.33% (41/150), 43.33% (65/150), and 12.67% (19/150) of women had studied for 1 to 3, 4 to 7, 8 to 11, and more than 11 years, respectively. With regard to occupation, 40.67% (70/150) were housewives, 32% (48/150) were farmers, 14.67% (22/150) were merchants, 4.66% (7/150) were retired, and 2.00% (3/150) were students. The distributions according to marital status were as follows: 36.67% (55/150) were single; 32.67% (49/150) were married; 22.00% (33/150) lived in cohabitation, 6.66% (10/150) were widows, and 2.00% (3/150) were separated. With regard to ethnicity, 64.67% (97/150) were Pardo Brazilian, 20.67% (31/150) were Afro-Brazilian, 13.33% (20/150) were White Brazilian, 0.67% (1/150) were indigenous, and 0.66% (1/150) were Asian Brazilian. The analysis of family income showed that 15.34% (23/150) received half of the minimum salary per month, 48.00% (72/150) received one minimum salary per month, 21.33% (32/150) received two minimum salaries per month, 0–67% (1/150) received three minimum salaries per month, 11.33% (17/150) received more than three minimum salaries per month, and 3.33% (5/150) did not answer .Most women were nonsmokers  and 6.00% (9/150) were former smokers; however, most of the former and current smokers (35 women) had been or were smokers for 10 years or more . In the sample, 54.67% (82/150) of women were not alcoholics, 42.00% (63/150) consumed alcoholic beverages, and 3.33% (5/150) were former alcoholics .With regard to açaí intake, 94.00% (141/150) consumed the fruit at least once a week and 6.00% (9/150) did not consume it. The majority of those who consumed açaí  had been consuming it for more than 10 years, 51.77% (73/141) consumed açaí at least three times a week, and 86.52% (122/141) consumed it in the form of juice .With regard to reproductive characteristics, the majority of women  had attained menarche between 11 and 14 years of age and had not yet entered menopause . Moreover, 89.33% (134/150) had been pregnant, with the majority  having had two to four full-term pregnancies, 91.33% (137/150) had not had preterm pregnancies, 83.33% (125/150) never had an abortion, and 82.67% (124/150) had breastfed . HormonaIn the sample, 40.67% (61/150) of the women had undergone mammography, with 50.82% of these (31/61) having undergone an exam approximately one year before, 39.34% (24/61) having undergone an exam two or more years before, and 9.84% (6/61) not knowing when they had undergone an exam. The diagnostic result was Category I for most of these women . Most women  had not undergone a breast ultrasound. Among those who had, most  had undergone an exam approximately one year before, 35.71% (15/42) had undergone an exam two or more years before, and 9.52% (4/42) did not answer. Most women  had a Category I result. The results of the diagnostic exams were subjected to statistical analysis for correlation with açaí intake, with no statistically significant correlations .The assessment of comorbidities showed that 13.33% (20/150) of the women were diabetic, 6.67% (10/150) had heart disease, and 22.67% (34/150) were hypertensive. Statistical analysis revealed no statistically significant relationships between açaí consumption and the analyzed parameters .History of cancer revealed that 1.33% (2/150) had had cancer previously and the remaining women  had not and that 7.33% (11/150) had breast nodules. With regard to a family history of cancer, 27.33% (41/150) had cancer in the family. Of these, 31.71% (13/41) had a history of cancer in first-degree relatives , whereas 68.29% (28/41) had a history of cancer in second-degree relatives . The types of cancer in the family history included uterine , breast , ovarian , stomach, liver, bowel, prostate and penis cancers , and bone and lung cancers . One participant  did not answer this item. There were no statistically significant relationships between a previous history of cancer, presence of breast nodules, and family history of breast cancer and other types of cancer and açaí consumption .Diets rich in fruits and vegetables have been reported to increase the antioxidant capacity of plasma. In addition to containing vitamins C and E and beta-carotene, these foods contain phenolic compounds with antioxidant properties . DietaryEuterpe oleracea Mart. is a monocotyledonous species indigenous of the floodplain forests of the Amazon region, particularly in Venezuela, Colombia, Ecuador, The Guianas, Suriname, and Brazil . It is known by the population as açaí, “açaí do pará,” “açaí do baixo amazonas,” “açaí de touceira,” “açaí de planta,” “juçara,” and “juçara de touceira” [ouceira” , 15. TheThe fruit has a rounded shape, a purplish-black color, is approximately 25 mm in diameter, and contains only one large seed. The analysis of the phenolic compounds in açaí showed the presence of anthocyanin 3-glucoside, ferulic acid, epicatechin, p-hydroxybenzoic acid, gallic acid, protocatechuic acid, catechin, ellagic acid, vanillic acid, p-coumaric acid, and gallotannin .The intake of fruits and vegetables has been associated with a reduction in the risk of cancer in humans, particularly breast cancer. Dietary flavonoids, a group comprising more than 5,000 different polyphenolic compounds, have been identified as components of fruits and vegetables with the potential to prevent cancer .in vitro studies. Moreover, açaí was shown to have a higher antioxidant capacity than that of other fruits with antioxidant potential, including cranberries, blueberries, and strawberries. The antioxidant activity of açaí supplementation has been examined in in vivo studies conducted with mice. After six weeks of açaí consumption, there was an increase in the antioxidant activity of paraoxonase 1 (PON-1) in rats that received normal or cholesterol-enriched diets [The high antioxidant capacity of açaí has been confirmed in several ed diets .Epidemiological studies and systematic analyses suggest that diets rich in fruits and vegetables are associated with a reduction in the risk of developing cancer, especially cancers of epithelial origin such as mouth, colon, rectum, lung, and breast cancers . HoweverTherefore, the objective of the present study was to analyze the relationship between açaí consumption and chronic diseases. For this purpose, a survey was conducted to collect data to assess this relationship. The results showed a high prevalence of açaí consumption among the women in the sample , most of which (77.30% or 109/141) had consumed açaí for more than 10 years. Despite this habit, there were no statistically significant differences relative to the control group  with regard to comorbidities, previous history of cancer, family history of cancer, changes in imaging exams (mammography and breast ultrasound), and presence of breast nodules. The prevalence of comorbidities among the açaí consumers was higher than that among the nonconsumers . Breast cancer was the second most prevalent type of cancer included in the family history of cancer. In fact, several experimental studies have evaluated the effects of flavonoids present in extracts of açaí and other fruits on breast cancer cells , 19.Our study evidenced a tendency of açaí to reduce diabetes and hypertension in women.Açaí is rich in polyphenols that have some antidiabetic effects . They cade Bem et al. evidenced that association of exercise and the açaí seed extract in rats improved diabetic complications. Açaí reduced blood glucose, insulin resistance, leptin and IL-6 levels, lipid profile, and vascular dysfunction .Regarding antihypertensive effects of açaí, da Costa et al. evidenced that administration of the açaí seed extract (200 mg/kg) increased systolic blood pressure, decreased urea and creatinine levels, and prevented oxidative damage in animals .Polyphenols from the seed of acai increase endothelial nitric oxide production leading to endothelium-dependent relaxation, reduction of reactive oxygen species, and regulation of key targets in hypertension .This study is the first study to correlate the açaí intake and chronic disease prevention in Sa˜o Luís, MA, Brazil. The sample size was not too great, and a small control group could be difficult for statistical analysis; the results were not significant.Thus, studies on the relationship between the intake of fruits and vegetables rich in flavonoids and the risk of developing chronic diseases, including cancer, are of great value for the international literature and for the advancement of cancer treatment and prevention, especially in the case of cancers that are more prevalent and associated with higher morbidity, such as breast cancer in Brazil and worldwide. Further studies need to be performed to evaluate the relationship between açaí intake and chronic diseases.


Craniospinal irradiation (CSI) is part of the treatment of central nervous system (CNS) tumors and is associated with cardiovascular disease in adults. Global myocardial strain analysis including longitudinal peak systolic strain (GLS), circumferential peak systolic strain (GCS), and radial peak systolic strain (GRS) can reveal subclinical cardiac dysfunction.Retrospective, single-center study in patients managed with CSI vs. age-matched controls. Clinical data and echocardiography, including myocardial strain analysis, were collected at early (< 12 months) and late (≥ 12 months) time points after completion of CSI.p = 0.0002), GCS was − 22.5 ± 5.2% vs. -21.3 ± 3.4% (p = 0.28), and GRS was 21.8 ± 11.0% vs. 26.9 ± 7.7% (p = 0.07). For late, CSI vs. control: GLS was − 16.2 ± 5.4% vs. -21.6 ± 3.7% (p < 0.0001), GCS was − 20.9 ± 6.8% vs. -21.9 ± 3.5% (p = 0.42), and GRS was 22.5 ± 10.0% vs. 27.3 ± 8.3% (p = 0.03). Radiation type (proton vs. photon), and radiation dose (< 30 Gy vs. ≥ 30 Gy) did not impact any parameter, although numbers were small.Echocardiograms were available at 20 early and 34 late time points. Patients at the late time point were older (21.7 ± 10.4 vs. 13.3 ± 9.6 years) and further out from CSI (13.1 ± 8.8 vs. 0.2 ± 0.3 years). Standard echocardiographic parameters were normal for both groups. For early, CSI vs. control: GLS was − 16.8 ± 3.6% vs. -21.3 ± 4.0% (Subclinical cardiac systolic dysfunction by GLS is present both early and late after CSI. These results argue for future studies to determine baseline cardiovascular status and the need for early initiation of longitudinal follow-up post CSI. Each year there are over 1.7 million new cases of cancer in the United States, including almost 16,000 pediatric patients (< 20-years-old). Advances in treatment have led to 5-year survival of > 80% for all pediatric cancer types, which amounts to more than 450,000 total survivors of pediatric cancers . Few stuThis was a retrospective study performed at a single-center, Cincinnati Children’s Hospital Medical Center. Patients diagnosed with CNS malignancy and managed with CSI between the years 1986–2018 were identified by the Neuro-Oncology Program and included in the study if they had at least one echocardiogram post therapy with digitized images available for review by speckle tracking. Patients who received anthracyclines were excluded from the study. Transthoracic echocardiograms were performed on 1 of 3 ultrasound systems used at our institution during the study period: Vivid 7 , iE33 , and Sequoia 512 . Demographic, treatment , relevant medical history, relevant lab values, and standard echocardiographic data were collected from the electronic medical record for two time periods: “early” representing an echocardiogram performed at < 12 months from the end of therapy, and “late” representing an echocardiogram performed at ≥ 12 months from the end of therapy. Of note, very few patients had echocardiography performed prior to CSI so this time point was not included. For any missing echocardiographic data , measurements were made retroactively. These data were compared to well-established population normal values.post-hoc analysis was performed at early and late time points. In brief, Digital Imaging and Communications in Medicine (DICOM) data were analyzed using vendor-independent clinical echocardiographic software . Since there are no available normative data for two-dimensional strain in pediatric patients specific to this software, an age-matched control group was generated from individuals referred for echocardiogram for a variety of reasons  and ultimately found to have no cardiac pathology. Because these individuals had normal echocardiograms, in almost all cases no further laboratory data or work up was pursued and thus there was no evaluation for factors that may affect strain such as hemoglobin. However, it was assumed these patients, as a group, were normal regarding medical history and laboratory data.Myocardial strain is a relatively angle-independent measure of ventricular function, noninvasively measured by echocardiogram or cardiac MRI . Strain Data were examined for completeness, distributions were examined for shape, and outliers were checked for feasibility. Cross-checks were done, including by scatterplots, and contingency tables. Any questionable data points were referred back to the source, and any typographical, or other errors in reporting were corrected. Patient characteristics and demographics were displayed in contingency tables, and as means ± standard deviations for continuous variables. A control group was established for each time point by selecting control echocardiographic studies to match patients as closely as possible by age. Comparisons between controls and patients were done using Fisher’s exact tests for categorical data, and continuous variables were tested using Wilcoxon rank-sums analyses. Boxplots were constructed to display differences between control and patient results. Comparisons between early and late results were done using paired t-tests.A total of 51 patients treated with CSI for CNS malignancies were identified from our database for the years 1986–2018, including 67% (34/51) male and 88% (45/51) white race. The most common diagnosis was medulloblastoma (45/51), with the remaining cases including atypical teratoid, ependymoma, and glioma. Most patients received cisplatin (44/51) and cyclophosphamide (39/51). While almost all patients received craniospinal radiation, 1 received only spinal radiation and 1 received no spinal radiation. The latter patient was excluded from further analysis. Photon therapy was used more often than proton, and more than half of the patients received ≥ 30 Gy of spinal radiation , and time between end of therapy and echocardiogram was greater  (Table p = 0.0002), GCS was − 22.5 ± 5.2% vs. -21.3 ± 3.4% (p = 0.28), and GRS was 21.8 ± 11.0% vs. 26.9 ± 7.7% (p = 0.07). At late, CSI vs. control: GLS was − 16.2 ± 5.4% vs. -21.6 ± 3.7% (p < 0.0001), GCS was − 20.9 ± 6.8% vs. -21.9 ± 3.5% (p = 0.42), and GRS was 22.5 ± 10.0% vs. 27.3 ± 8.3% (p = 0.03)  and late  time points demonstrated depressed GLS in the presence of normal left ventricular EF and SF, which is evidence of subclinical myocardial dysfunction. This is in line with previous studies showing GLS as a marker of dysfunction in patients receiving chemotherapy with otherwise normal EF. For other strain parameters, GCS was no different at any time point and GRS only showed a decrease at the late time point. Analyses were also performed to determine whether dose or type of radiation were important in these changes in strain. There were no significant differences based on these parameters, however the numbers available for analysis in the sub-groups were small making definitive conclusions suspect.One potential confounding factor in the present analysis is the concomitant use of chemotherapy with CSI, particularly cyclophosphamide which is known to be cardiotoxic. Although there are certain young patients with specific tumors that may be treated with high dose chemotherapy and stem cell transplant rescue, radiation is the standard of care in this population and in our sample the vast majority also received chemotherapy. Analysis of patients who did not receive radiation as part of their treatment may help resolve how much of a role chemotherapy played in this cohort, however in this retrospective study numbers were expected to be too small for meaningful analysis.The diagnosis and management of cardiovascular injury in pediatric patients undergoing CSI may be delayed due to the misconception that this particular cancer-directed therapy results in only long-term cardiovascular side effects due to the absence of studies documenting the clinical and advanced imaging manifestations from CSI. Indeed, current guidelines in pediatrics for the surveillance of cardiovascular disease in patients undergoing CSI call for screening many years after completion of therapy. Strain analysis during the past decade has facilitated significant advances in noninvasive myocardial mechanics and cardiac function assessment . MyocardThe current study has several limitations. First, the patients included were treated over a 2-decade period. Therapeutic approach in that time has evolved, and an overall decreased scatter in radiation doses has limited the incidence of cardiotoxicity, and this could decrease the signal for cardiotoxicity in patients treated in the recent era . No signSubclinical left ventricular systolic dysfunction demonstrated by GLS is present both early and late after CSI. There was not a clear association between dose and/or type of radiation therapy, however numbers in these sub-groups were small and may not have been enough to find a difference if present. These results argue for future prospective studies to determine baseline cardiovascular disease burden and need for early initiation of longitudinal follow-up in CNS tumor patients post CSI. Coupled with cardiac biomarkers, this may allow a more complete phenotype of injury related to treatment of CNS tumors in pediatric patients.


Anidulafungin and micafungin were quantified in cerebrospinal fluid (CSF) of critically ill adults and in cerebral cortex of deceased patients. In CSF, anidulafungin levels (<0.01 to 0.66 μg/ml) and micafungin levels (<0.01 to 0.16 μg/ml) were lower than those in plasma concentrations  drawn simultaneously. In cerebral cortex, anidulafungin and micafungin levels were 0.21 to 2.34 and 0.18 to 2.88 μg/g, respectively. Candida strains exceed concentrations in CSF and in brain.Anidulafungin and micafungin were quantified in cerebrospinal fluid (CSF) of critically ill adults and in cerebral cortex of deceased patients. In CSF, anidulafungin levels (<0.01 to 0.66 μg/ml) and micafungin levels (<0.01 to 0.16 μg/ml) were lower than those in plasma concentrations  drawn simultaneously. In cerebral cortex, anidulafungin and micafungin levels were 0.21 to 2.34 and 0.18 to 2.88 μg/g, respectively. Thus, MIC values of several pathogenic  The echinocandins anidulafungin and micafungin are recommended for treatment of invasive candidiasis , 2. CandPost hoc consent was obtained from patients who were incompetent at the time of enrollment. Autopsy samples were taken from deceased patients who, on admission, had permitted scientific use of residual specimens taken for clinical laboratory tests.The study was approved by the local ethics committee and performed in accordance with the Declaration of Helsinki and Austrian law. Written informed consent for scientific use of CSF and blood samples was granted by competent patients. CSF was taken during diagnostic lumbar puncture (LP) or via external ventricular drain (EVD) from critically ill adults treated with anidulafungin or micafungin. Simultaneously, we took 2-ml arterial blood samples. CSF and plasma were stored at –80°C.0–24) was computed with the log-linear method when the concentration in a trapezoid decreased or with the trapezoidal method when the concentration increased.Anidulafungin and micafungin concentrations were quantified by high-performance liquid chromatography and UV detection (HPLC-UV) as described previously . Noncompg. For calibration, we used porcine brain samples spiked with anidulafungin or micafungin and homogenized and proceeded in the same manner. Both echinocandins were quantified by HPLC-UV at 306 nm , homogenization with a Precellys homogenizer  at 4,500 rpm , and centrifugation for 5 min at 8°C and 4,665 × U test using IBM SPSS statistics 24.0. PR was the ratio between the AUC0–24 in CSF and that in plasma over 24 h (AUC0–24 CSF/AUC0–24 plasma), when multiple samples were drawn via EVD. For single samples drawn by LP, PR was the ratio between the concentration in CSF and that in plasma (CCSF/Cplasma).The significance of the difference between CSF and plasma concentrations was assessed by Wilcoxon matched-pairs test, and the difference between the penetration ratio (PR) of anidulafungin and PR of micafungin was calculated by Mann-Whitney P < 0.05 and P < 0.01, respectively) (P = 0.40). In the cerebral cortex of four deceased patients, anidulafungin had reached concentrations of 0.21 to 2.34 μg/g. Brain concentrations of micafungin were <0.10 to 2.88 μg/g (n = 6). Anidulafungin and micafungin were measurable even 13 and 10 days, respectively, after treatment (CSF samples were obtained from three patients on anidulafungin and three patients on micafungin. One patient on anidulafungin (patient 2) and one patient on micafungin (patient 5) had undergone EVD ; Fig. 1.ctively) ; Fig. 1.reatment .Candida species   . In the  strains , 7. Howects MICs . HoweverDuring cerebral aspergillosis, the 2-fold maximum dose of micafungin  achieved CSF concentrations of <0.02 μg/ml . After i3H]caspofungin brain concentrations were ≤0.16 μg eq/g


In the literature, there are many reports about patients who developed thrombosis with coexistent iron deficiency (ID) or ID anemia (IDA) ,5,6,7,8.Moreover, the frequency of severe anemia  and IDA The predilection to hypercoagulation in ID/IDA was predicted to be due to reactive thrombocytosis, microcytosis, dehydration, infections, alterations in laminar flow, formation of turbulence, corruption of the oxidant/antioxidant balance, increases in platelet aggregation, increased procoagulants, and hypoxia ,5,6,7,8.Herein we aim to provide laboratory evidence of the propensity to thrombosis in IDA using thromboelastography, which can qualitatively determine the status of coagulation as hyper- or hypocoagulation, and to state whether the abnormality stems from any pathology in primary hemostasis, secondary hemostasis, or the fibrinolytic system or any effects of anticoagulants or inhibitors within 30 min. With thromboelastography, the formation, strength, elasticity, and firmness of a clot can be shown using parameters such as reaction (R) time, clot formation (K) time, alpha (α) angle, maximum amplitude (MA), maximum lysis (LY30), and coagulation index (CI). Their functions and implications are presented in ® 5000 Thromboelastograph® Hemostasis Analyzer).Blood samples from 34 IDA patients between the ages of 3.5 and 191 months and from 39 healthy children of 12 to 191 months of age were studied using the flat cup test in thromboelastography  or infectious/inflammatory diseases, high c-reactive protein (CRP) levels, obesity, hypertension, smoking habit, hyperuricemia, liver or renal function abnormalities, vitamin B12 or folic acid deficiencies, and self or family history of thrombosis or bleeding were excluded from the study, as were those on any drug therapy.The thromboelastographic measurements observed in the IDA and control groups were as follows: K, 1.4±0.6 vs. 1.8±1.1 min (p=0.03); MA, 70.6±4.9 mm vs. 66.9±8.3 mm (p=0.05); LY30, 3.8±4.4 vs. 2.0±3.2 (p=0.12); R, 3.9±1.4 vs. 4.0±1.4 min (p=0.78); α, 53.0±8.9° vs. 53.0±9.6° (p=0.91); and CI, 1.0±1.4 vs. 0.3±2.1 (p=0.19)  and 1B.Significant decrease in K and increase in MA with borderline significance compared to the controls implied hypercoagulability, which was possibly due to increased fibrinogen levels and/or to a lesser extent increased thrombocyte functions . InflammOur findings revealed a positive linear relationship between serum iron levels and α  and between red blood cell distribution width (RDW) and α , and an inverse linear relationship between RDW and K  in the control group.In the IDA group, there was a positive and weak linear relationship between ferritin and α , a positive linear relationship between mean corpuscular volume (MCV) and MA , and an inverse linear relationship between thrombocyte count and K .Although the positive linear relationship of ferritin with α and of MCV with MA pointed at hypocoagulability, the inverse linear relationship between thrombocyte count and K pointed at hypercoagulation, being correlated with the severity of thrombocytosis, the latter of which is a usual finding in IDA. These conflicting results of the correlation studies may be due to the limited number of patients.In another study similar to ours that investigated the effect of IDA by rotational thromboelastometry (ROTEM), normal coagulation test results were also revealed, whereby maximum clot firmness in ROTEM, equivalent to MA in thromboelastography, was increased in the IDA group and  clot formation time, equivalent to K in thromboelastography, was decreased in the IDA group, both implying hypercoagulability. This study also revealed similar thrombocyte counts in the IDA and control groups despite a negative correlation between thrombocyte count and CFT [equivalent to K in thromboelastography] , as in oThe fact that none of our patients in this report had developed thrombosis suggests that additional determinants may be required for the development of thrombosis. Moreover, the real incidence of thrombosis in IDA may be too low to be established in a small cohort of patients such as ours. The duration and the severity of anemia may be other factors for the initiation of thrombosis. In a review of 54 patients who developed thrombosis on the basis of ID/IDA, the majority had “severe” IDA , while SIndeed, of the 54 patients with coexistent ID/IDA and thrombosis in the literature , 18% hadWe furthermore did not have an opportunity to compare thromboelastography values before and after iron therapy. However, our findings showed a propensity to hypercoagulation in patients with IDA and confirm the results of previous studies ,2,8,9.Although further laboratory evaluations are required with larger numbers of patients and the exclusion of accompanying thrombophilic factors, IDA seems to be a new candidate among thrombotic factors.


The use of natural dietary supplements in aquaculture has received a great deal of attention in recent years. This article provides data describing body weight and length of rainbow trout juveniles fed with natural dietary supplements dihydroquercetin, arabinogalactan or a mixture of both in an aquaria experiment. Before feeding trial, rainbow trout were tagged to identify individuals. Fish grown in tanks were fed one of four diets in duplicate: a basal diet without any supplements (control diet) or a basal diet supplemented with dihydroquercetin , arabinogalactan  or a mixture of both . Our dataset could be used to evaluate the effect of dihydroquercetin, arabinogalactan or a mixture of both on the growth performance of cultivated rainbow trout. Specifications TableValue of the Data•The presented data give details on the effect of dietary dihydroquercetin (25 and 1000 mg kg⁻¹ of diet), arabinogalactan (50 and 2000 mg kg⁻¹ of diet) or their combination (25+50 and 1000+2000 mg kg⁻¹ of diet) on the weight and length of rainbow trout in an aquaria experiment.•These data could be taken into account by fish farmers in the estimation of the effect of natural dietary supplements such as dihydroquercetin and arabinogalactan on rainbow trout growth. These data will be helpful for researchers involved in aquaculture nutrition assessments and related research.•These data support the development of further studies aimed to reveal concentrations of dihydroquercetin and arabinogalactan that may affect the growth performance of farmed rainbow trout.•These data confirm the current knowledge on the growth pattern of rainbow trout juveniles.Fig. 1The dataset presented in this article as a supplementary file (.xls) provides raw data on the growth of rainbow trout individuals fed with dihydroquercetin, arabinogalactan or a mixture of both in an aquaria experiment. It has 7 columns: tag number, measurement date, weight (g), length (cm), group , the concentration of supplements (mg kg⁻¹ of feed), and tank number. We applied multilevel linear modeling for repeated measures data, which revealed no difference in growth parameters (body weight and length) between the control and experimental groups. Data extracted from this experiment are summarized in 22.1All animal handling procedures were approved by the Ethics and Animal Care Committee of the Institute of Biology, Karelian Research Center of the Russian Academy of Sciences, following EU-established norms and procedures.O. mykiss, juveniles (age: 6 months) obtained from a commercial trout farm  were transported to the Laboratory for Environmental Biochemistry at the Institute of Biology, Karelian Research Center of the Russian Academy of Sciences . Fish were randomly stocked into 8 glass tanks (250 - 270 L capacity) with 14–17 fish per tank. Tanks were continuously supplied with aerated water with the flow rate set at 0.16 L min⁻¹, water temperature 12 ± 1 °C, dissolved oxygen 7.5–8.5 ppm, total ammonia nitrogen < 0.1 mg L − 1, nitrite nitrogen < 0.1 mg L − 1 and nitrate nitrogen < 10.0 mg L − 1, under natural photoperiod. On November 19, 2018 fish were PIT tagged intraperitoneally to identify individuals. Fish were anesthetized using a clove oil bath (25–30 mg L⁻¹) before tagging as well as before all subsequent body weight and length measurements. On November 19, 2018 stocking density was 3.24, 3.25, 3.39, 3.24, 3.12, 3.60, 3.29, 3.37 kg m − 3 for tanks from 1 to 8, respectively. Before the start of the feeding trial, all fish were given a basal diet, EFICO Alpha 717R , containing 22–25% lipid, 40–43% protein, 20–23% carbohydrate, 2.8–5.8% fiber, 0.9% total P, 4–7% ash and 22–25 MJ kg⁻¹ total energy. Tagged fish were acclimatized to the experimental conditions for 19 days prior to the start of the feeding trial.On November 1, 2018 rainbow trout, 2.2Dihydroquercetin, a bioflavonoid,  and arabinogalactan, a polysaccharide,  were purchased from Ametis (Russia). Distilled water was used to dissolve supplements; then the solution was heated up to 45 °C and stirred continuously for 1 h. Visual control of the dissolution process made it possible to make sure that there was no sediment. The solution with supplements was sprayed onto feed pellets from a spray gun directly on the day of feeding. Then the feed pellets were dried at room temperature for an hour. In the control group, distilled water alone was added to the feed.The feeding trial was started on December 8, 2018. Fish were fed one of four diets in duplicate tanks: a basal diet without any supplements (control diet) or a basal diet supplemented with dihydroquercetin , arabinogalactan  or a mixture . The supplement concentrations listed above were recommended by the manufacturer  for fish farming. Fish were fed these diets until March 20, 2019. From March 21, 2019 until May 15, 2019, fish were fed diets with a 40-fold increase of the initial supplement concentration: a basal diet without any supplements (control diet), or a basal diet supplemented with dihydroquercetin , arabinogalactan  or a mixture of both . High concentrations of dihydroquercetin  have been used previously in the experiment with gilthead seabream with no toxicity for fish 2.3m − 3 for tanks from 1 to 8, respectively. During the study, some healthy fish were lost as a result of accidents . These accidents involved fish with tags nos. 330 (01.01.2019), 486 (11.01.2019) and 677 (18.01.2019) from tank no. 2, fish with tag no. 913 (08.01.2019) from tank no. 3, and fish with tag no. 924 (06.12.2019) from tank no. 5. The remaining fish having two measures  were used for biochemical analysis.Rainbow trout body weight and length measurements were taken on November 19, 2018; December 7, 2018; January 31, 2019; March 4, 2019; April 26, 2019; and May 16, 2019 with a digital balance  and a 300 mm liquid crystal display (LCD) digital Vernier caliper , respectively. Up to 10 fish from each tank were sacrificed on December 7, 2018 for further biochemical analysis , so for these fish, growth parameter data is only available for two dates . On December 8, 2018 stocking density was 2.16, 1.52, 2.34, 2.00, 1.82, 2.12, 2.21, 2.15 kg 2.4The analysis of repeated measures data was performed utilizing a multilevel linear model (MLM). The data were analyzed with the lmer function from the lme4 package The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article.