Anoxybacillus are thermophiles and, therefore, their enzymes are suitable for many biotechnological applications. Anoxybacillus ayderensis AB04T (= NCIMB 13972T = NCCB 100050T) was isolated from the Ayder hot spring in Rize, Turkey, and is one of the earliest described Anoxybacillus type strains. The present work reports the cellular features of A. ayderensis AB04T, together with a high-quality draft genome sequence and its annotation. The genome is 2,832,347 bp long (74 contigs) and contains 2,895 protein-coding sequences and 103 RNA genes including 14 rRNAs, 88 tRNAs, and 1 tmRNA. Based on the genome annotation of strain AB04T, we identified genes encoding various glycoside hydrolases that are important for carbohydrate-related industries, which we compared with those of other, sequenced Anoxybacillus spp. Insights into under-explored industrially applicable enzymes and the possible applications of strain AB04T were also described.Species of  Bacillaceae [Bacillaceae are either rod-shaped (bacilli) or spherical (cocci) Gram-positive bacteria, the majority of which produce endospores [Anoxybacillus [Bacillaceae [Firmicutes [Bacilli [Bacillales [The family illaceae , 2 is onillaceae . The Bacdospores . Anoxybabacillus , 6 is onillaceae , 2, clasrmicutes , class B[Bacilli , 9, and cillales , 10.Anoxybacillus spp. are alkalo-thermophiles with optimum growth at temperatures between 50 °C and 65 °C and at pH 5.6–9.7 [Anoxybacillus spp. are found in hot springs [Anoxybacillus has also been found in animal manure [Anoxybacillus have been described [ 5.6–9.7 . Most of springs , but Anol manure , animalsl manure , insectsl manure , and plal manure . To dateescribed , 12, 13.Bacillaceae are excellent industrial enzyme producers [Anoxybacillus exhibit the additional advantage of thermostability compared to the mesophilic Bacillaceae. It has been reported that enzymes from Anoxybacillus spp. can degrade various substrates such as starches, cellulose, fats, and proteins [Anoxybacillus spp. genomes, and some of the well-studied starch-degrading enzymes are α-amylase [Anoxybacillus spp. Apart from their hydrolytic capabilities, Anoxybacillus spp. have been proposed as agents for bioremediation of Hg2+, Cr2+, Al3+, As3+ ions [Almost all members of the roducers , 14, 15.-amylase , pullula-amylase , amylopu-amylase , CDase [-amylase , and xyl-amylase . In addi-amylase  and α-L--amylase  have bees3+ ions –25, and s3+ ions , and as Bacillaceae, intensive genome sequencing efforts have been undertaken for Geobacillus [Bacillus [Anoxybacillus are rather limited, with only 16 registered projects. At present, the genome of Anoxybacillus flavithermus WK1 is the only completely sequenced genome (BioProject accession number PRJNA59135) among the Anoxybacillus spp. [Anoxybacillus ayderensis AB04T  [Anoxybacillus sp. BCO1 (PRJNA261743) [Anoxybacillus thermarum AF/04T (PRJNA260786) [Anoxybacillus gonensis G2T (PRJNA264351) [Anoxybacillus sp. ATCC BAA-2555 (PRJNA260743), Anoxybacillus sp. KU2-6(11) (PRJNA258246), Anoxybacillus tepidamans PS2 (PRJNA214279) [A. flavithermus 25 (PRJNA258119) [A. flavithermus AK1 (PRJNA190633) [Anoxybacillus kamchatkensis G10 (PRJNA170961) [A. flavithermus Kn10 (PRJDB1085) [A. flavithermus TNO-09.006 (PRJNA169174) [Anoxybacillus sp. SK3-4 (PRJNA174378) [Anoxybacillus sp. DT3-1 (PRJNA182115) [A. flavithermus subsp. yunnanensis E13T (PRJNA213809) [Anoxybacillus spp. is essential not only to fully understand their biochemical networks, but also to discover their potential applicability in industrial processes.Among the members of the family bacillus   , AnoxybaA261743) , 32, AnoA260786) –35, AnoxA264351) , AnoxybaA214279) , A. flavA258119) , A. flavA190633) , AnoxybaA170961) –42, A. fA169174) , AnoxybaA174378) , 46, AnoA182115) , 46, andA213809) , 48. TheA. ayderensis AB04T and we present a high-quality annotated draft genome of strain AB04T. Additionally, we provide a comparative analysis of the GHs of strain AB04T and other sequenced Anoxybacillus spp. In addition, we discuss the presence of other under-explored industrial enzymes and the potential applications of the bacterium.In the present report, we describe the cellular features of A. ayderensis AB04T (= TNCIMB 13972 = TNCCB 100050) was isolated from mud and water samples from the Ayder hot spring located in the province of Rize in Turkey [T were cream-colored, regular in shape with round edges, and 1–2 mm in diameter.n Turkey . MicroscT is a Gram-positive, rod-shaped, motile, and spore-forming bacterium [T were 0.7–0.8 × 3.5–5.0 μm in size  NaCl, but it was able to grow in the absence of NaCl. Growth was inhibited in the presence of ampicillin (25 μg/ml), streptomycin sulphate (25 μg/ml), tetracycline (12.5 μg/ml), gentamicin (10 μg/ml), and kanamycin (10 μg/ml). The FAME profile showed that the major fatty acid in AB04T is C15:0iso (48.17 %), followed by C17:0 iso (20.62 %), C17:0 anteiso (9.22 %), C16:0 (9.10), C16:0 iso (7.47 %), C15:0 anteiso (3.58 %), C14:0 (1.02 %), and C15:0 (0.83 %) [Phenotypic analysis revealed that strain AB04acterium . It is aize Fig. . The str(0.83 %) .Table 1CT clusters together with Anoxybacillus sp. SK3-4 [A. thermarum AF/04T [T shares 99.6 % and 99.2 % similarity with Anoxybacillus sp. SK3-4 [A. thermarum AF/04T [The 16S rRNA-based phylogenetic tree constructed using MEGA6.0  showed tp. SK3-4 , 46 and m AF/04T –35  and incubated at 50 °C for 18 h. A single colony was transferred into Nutrient Broth (pH 7.5) and incubated at 50 °C with rotary shaking at 200 rpm for 18 h. The cells were harvested by centrifugation at 10,000 × g for 5 min using a Microfuge® 16 centrifuge . Genomic DNA was extracted using a Qiagen DNeasy Blood & Tissue Kit  according to the manufacturer’s protocol. The purity, quality, and concentration of the genomic DNA were determined using a 6 % (w/v) agarose gel, NanoDrop 1000 spectrophotometer , and Qubit 2.0 fluorometer .A. ayderensis AB04T was sequenced using the Illumina MiSeq sequencing platform  with 300-bp paired-end reads. The adapter sequences were removed and low quality regions and reads were filtered out using Trimmomatic [k-mer threshold = 3, k-mer rounds = 10, and read error correction = 0.04). Next, the reads were subjected to de novo genome assembly using IDBA-UD 1.0.9 [kmin = 35).The genome of mmomatic   [Bacillaceae such as Bacillus [Lysinibacillus [Anoxybacillus [Geobacillus spp. genomes (~50.0 %) [A. ayderensis [Bacillus genomes have less than 40 % G + C content [The genome sizes of the currently sequenced  2.86 Mb –45, 47,  2.85 Mb . The inc to date . However3.50 Mb) , 45. TheBacillus  and Lysibacillus  are at l~50.0 %) , 45 is sderensis  genome  and α-glucuronidase (KIP21917)  from Anoxybacillus sp. SK3-4 has been reported previously [Anoxybacillus spp., for instance A. flavithermus WK1 [A. flavithermus subsp. yunnanensis E13T [A. kamchatkensis G10 [A. flavithermus AK1 [A. flavithermus Kn10 [Anoxybacillus  catalyzes the isomerization of xylose to xylulose and of glucose to fructose, which is important in the industrial production of high-fructose corn syrup [A. gonensis G2T was characterized and the enzyme displays 96.8 % amino acid sequence similarity to the one identified in strain AB04T (KIP21927) [Apart from the GHs, we found that rn syrup . EarlierIP21927) .A. gonensis G2T, A. gonensis A4, and Anoxybacillus sp. PDF-1 produce esterase [KIP19922 and KIP21735) in the genome of strain AB04T, which shared 96.3 % and 96.0 % amino acid sequence similarity with the esterase from Anoxybacillus sp. PDF-1 [A. gonensis G2T [A. gonensis G2T has been described [T carries two aldolases, KIP21451 and KIP21450, which showed 95.9 % and 99.9 % amino acid similarity to aldolase from A. flavithermus WK1 [A. thermarum AF/04T [T in the current study.Previous studies have indicated that esterase –72. We insis G2T , respectrmus WK1  and A. tm AF/04T –35, respAnoxybacillus spp. efficiently reduce metal ions such as Hg2+, Cr4+,Al3+, and As3+ [T contains at least six heavy metal resistance genes. Four genes are related to mercuric ion reduction; two of these are mercury resistance (mer) operons (KIP20706 and KIP20408) and the two other genes encode mercuric reductases, which catalyze the reduction of Hg2+ to Hg0 (KIP19952 and KIP20409). In addition, strain AB04T carries genes for an arsenate reductase (KIP20402) and an arsenic efflux pump protein (KIP20401). The function of these genes will be studied in the close future.Thermophilic bacteria are highly sought after for their potential use in bioremediation processes. Several and As3+ –25. The Anoxybacillus spp. are rather limited compared to that in their closest relatives, Geobacillus and Bacillus. In the present work we presented a whole-genome sequence of A. ayderensis AB04T and its annotation. Additionally, we provided insights into several GHs, under-explored enzymes, and putative applications of strain AB04T.Knowledge on the genomics, industrial enzymes, and relevant applications of


AbstractTullbergia from Argentina is described and illustrated; it is differentiated from Tullbergia paranensis by the number of vesicles of postantennal organ, pseudocelli shape and its formulae and the number of dorsal sensilla on Ant. IV. In addition a key for the identification of the members of the family from Argentina is included.A new species of  Tullbergiidae is a small family with important diversity in the Southern Hemisphere, it has a total of 32 genera and 216 species in the world . These species have been found in Argentinian localities such as Buenos Aires, Entre Ríos, Neuquén and Córdoba, always associated to soils with high organic matter content. The lack of studies in other parts of the country could explain their absence in other Argentinian locations.he world ; but froPageBreakTullbergiidae is an euedaphic group of small to very small size Collembola  without pigmentation, eyes and furcula. They are very sensitive to ecological changes and so of importance for detecting the impact of different factors on soil ecology. Most important contributions to the taxonomy of the family in Argentina were done by Collembola from Argentina is summarized by Tullbergiidae which resembles Tullbergia paranensis Izarra, 1969.Specimens were obtained from soils samples that were processed by Berlese-Tullgreen funnels and preserved in 75% alcohol. Later, slides were prepared in Hoyer’s solution. Specimens were studied and measured with a phase contrast microscope. = Buenos AiresBs As,  = CórdobaCba,  = Entre RíosE R,  = La PampaL P,  = NeuquénNq,  = TucumánTuc.Abbreviations: Most of the abbreviations and terminology follows Taxon classificationAnimaliaPoduromorphaTullbergiidaeLubbock, 1876Body white (without pigment); Ant. IV without an extremely large apical papilla. Tibiotarsi without clavated setae (seldom few weakly developed ones); Ant. III organ with a maximum of 5 elements including 2-3 large sensory clubs, two of them bent towards one another; Abd. VI without crescentic ridges and additional dorso-lateral spines. Th. I-III with 1, 1-2, 1-2 pseudocelli of type I after . PAO witTullbergia antarctica Lubbock, 1876Taxon classificationAnimaliaPoduromorphaTullbergiidaehttp://zoobank.org/9D213EA9-95C4-4C94-9B5A-5E0CDB0E135734°48'S; 58°00'0"W, ex soil, grassland dominated by grasses (Poaceae) and sedge (Cyperaceae), May 2010. The specimens were extracted from soil samples in Berlese’s funnels. A. Salazar Martínez collector.Type locality: Argentina: Bs As: Punta Lara, PageBreakHolotype. Female mounted on slide. Original label: 5404/1 deposited at Colección División Entomología, Museo de la Plata. Paratypes: 2 females paratypes, 2 males paratypes, all of them mounted on slides. Original label: 5404/2-4 deposited at Colección División Entomología, Museo de la Plata. 6 females, 4 males and 11 juvenil paratypes with the same collecting data are kept at senior’s author institution with catalog number 2436.PageBreakcellar formulae 11/122/22221: one minute empodial appendix, two slightly clavate tenent hairs ventrally on each tibiotarsus.Ant. III organ with 3 thick curved dorsal sensilla, one ventral sensillum; 6 dorsal sensilla on Ant. IV; about 72 vesicles on postantennal organ; pseudoBody length (n=12) 1.2 mm (range 0.83–1.4 mm), with moderately long macrosetae 61 µm (range 56–66), and short microsetae 18 µm (range 15–23), all of them smooth and thin. Relatively uniformly distributed fine intergumentary granules, interspersed with somewhat coarser granules on Abd. VI. Antennal bases well delimited.Ratio head: antenna = 1: 0.8. The relative lengths of Ant. I: II; III; IV are as 1: 1.6; 1.9; 1.8. Ant. III and IV fused dorsally (80 µm). Sense organ of Ant. III with two small sensory rods concealed behind one of the two integumentary folds; in addition there are three thick sensilla clubs, which are rounded at tip; two strongly bend towards each other and not concealed by cuticule. At bases of cuticle, the third sensillum concealed by one tegumentary fold. There is a ventral straight sensillum protected by three setae longer than other ventral setae. Ant. IV furnished with 6 sensilla, 3 of them very thin and long, three thick and short, one distinct subapical pit, one microsensillum and one slightly trilobed apical bulb . PostantLegs chaetotaxy from I to III, coxae, 2,7,7; trochanters 5,5,5; femora, 9,9,9 ; tibiotarsi , each with 2 ventral slightly clavate tenent hairs). Tibiotarsi short, 28 µm (range 22–31). Pretarsus with two setae each. Ungues untoothed. Empodial appendage rudimentary, in shape of a minute claw-like process . Dorsal Dorsal chaetotaxy of thorax and abdomen in PageBreakPageBreakpillae which touch at their bases , the presence of a minute empodial appendix (versus none). It also differs in the pseudocellar formulae (11/122/22221 vs /111/11111). The number of dorsal sensilla on Ant. IV is also different (6 vs. 5). Tullbergia is supposed to have pseudocelli of type I (Tullbergiidae from Argentina.f type I , but it Tenent hairs are very slightly capitated and often leg one has one capitated and one acuminate tenent hairs.


Acinetobacter baumannii strains isolated from pus samples of two patients with surgical site infections at Sri Sathya Sai Institute of Higher Medical Sciences, Prasanthigram, India. The average genomic size and G+C content are 4 Mbp and 38.96% (AB28) and 4 Mbp and 38.94% (AB30), respectively.We report the draft genomes of two extensively drug-resistant (XDR)  Acinetobacter baumannii is a Gram negative, nonlactose fermenting bacillus known to cause a variety of community acquired and nosocomial infections including meningitis, endocarditis, septicemia, and ventilator-associated pneumonia while exhibiting enhanced resistance to several classes of antibiotics , carbapenems , fluoroquinolones , penicillin and beta lactamase inhibitors , extended spectrum cephalosporins , trimethoprim-sulfamethoxazole, and were found to be susceptible to tigecycline, minocycline, and colistin.De novo assembly was performed using SPADES v3.5.0  400 kit and chip 318. Sequencing generated 1,195,415 reads with a mean length of 273.67 ± 110.35 bp for AB28 and 1,192,947 reads with an average length of 263.85 ± 118.86 bp for AB30. A. baumannii.No plasmid sequence was identified when analyzed by Webcutter (v2.0) and Plasmid Finder  in both LQBT00000000 (AB28) and LQBV00000000 (AB30). The versions described in this article are the ﬁrst versions.The draft genome sequences have been deposited in DDBJ/EMBL/GenBank under the accession numbers


An H9N2 virus lethal to chickens was isolated from an acutely ill chicken flock in 2012. Phylogenetic analyses indicated that this virus was phylogenetically related to the Y280 lineage. Sequence analysis showed 1 amino acid deletion in HA1 and 3 amino acid deletions in the NA stalk region. However, no chickens that had been infected with the same dose of an earlier isolate, A/chicken/Shandong/1024/2007(H9N2), died.H9N2 influenza viruses have been widely circulating in poultry flocks in Asia and the Middle East, and outbreaks of H9N2 influenza have caused great economic losses –4. The H–5–In order to explore the genome characteristics of the strain, we sequenced the complete genome of SD/818. The results showed that the genome constitutes 8 negative-sense RNA segments: the PB2, PB1, PA, HA, NP, NA, M, and NS genes, with full lengths of 2,341; 2,341; 2,233; 1,739; 1,565; 1,458; 1,027; and 890 nucleotides, respectively. Alignment by BLAST showed that the PB2, PA, HA, NP, NA, M, and NS genes shared the highest nucleotide homologies with H9N2 viruses circulating in Eastern China. However, the PB1 gene of SD/818 shared the highest nucleotide homologies with an H6N8 strain isolated from ducks in Guangxi and an H7N9 strain isolated from a human in Zhejiang. Phylogenetic analyses indicated that the PB1, HA, NA, PA, NP, and NS genes belonged to the Y280 lineage; the M gene belonged to the G1 lineage; and the PB2 gene belonged to an unknown avian lineage.N-glycosylation sites at 29 aa, 141 aa, 218 aa, 298 aa, 305 aa, 313 aa, 492 aa, and 551 aa, respectively. Alignment by BLAST showed that the HA protein of SD/818 deleted 1 amino acid at the position of 235, whereas other H9 viruses in GenBank do not show the deletion. The NA showed 3 amino acid deletions at 63 to 65 aa of the stalk region and carried 6 potential N-glycosylation sites at 69 aa, 86 aa, 164 aa, 200 aa, 234 aa, and 264 aa. There is a substitution of S31N in the M protein, indicating that this isolate owes its resistance to amantadine (SD/818 carried the amino acid sequence PSRSSR↓GLF at the HA cleavage site, a character of LPAIV . The 226antadine . The isoIn this study, we sequenced the whole genome of an H9N2 virus lethal to chickens and analyzed the characteristic of the genome, which will contribute to exploring the molecular mechanism of the virulence of the H9N2 virus.KM285394 to KM285401.The genome sequences of A/chicken/Shandong/818/2010 have been deposited in GenBank under the accession numbers


N = 10) varying the resin (FR or UR) and quantity of glass fibers . Samples (10 × 2 × 1 mm) were submitted to flexural strength test following SEM examination. Data were analyzed by two-way ANOVA, Tukey, and Student t-test (α = 0.05). Results for flexural strength (MPa) were FR-groups: 0% (442.7 ± 140.6)C, 10% (772.8 ± 446.3)ABC, 15% (854.7 ± 297.3)AB, 20% (863.4 ± 418.0)A, 30% (459.5 ± 140.5)BC; UR-groups: 0% (187.7 ± 120.3)B, 10% (795.4 ± 688.1)B, 15% (1999.9 ± 1258.6)A, 20% (1911.5 ± 596.8)A, and 30% (2090.6 ± 656.7)A, and for flexural modulus (GPa) FR-groups: 0% (2065.63 ± 882.15)B, 10% (4479.06 ± 3019.82)AB, 15% (5694.89 ± 2790.3)A, 20% (6042.11 ± 3392.13)A, and 30% (2495.67 ± 1345.86)B; UR-groups: 0% (1090.08 ± 708.81)C, 10% (7032.13 ± 7864.53)BC, 15% (19331.57 ± 16759.12)AB, 20% (15726.03 ± 8035.09)AB, and 30% (29364.37 ± 13928.96)A. Fiber addiction in BisGMA resin increases flexural properties, and the interaction between resin and fibers seems better in the absence of inorganic fillers increasing flexural properties.This study evaluated the effect of fiber addiction in flexural properties of 30 wt% silica filled BisGMA resin (FR) or unfilled Bis-GMA (UR). Ten groups were created ( For over than 30 years fibers have been used as structural reinforcement for dental resins , includiThe effectiveness of fiber reinforcement depends on many factors, including the resin material, the quantity, length, shape, orientation, and adhesion properties of fibers . Recent Studies have shown the relationship between the amount of fibers in polymer matrix and the flexural strength of tested reinforced materials –17. AccoSince the inclusion of inorganic fillers increases resin viscosity limiting the addiction of fibers into resin, the hypothesis driven in the present research is that a pure BisGMA-based resin (without inorganic filler particles) would enable the inclusion of greater quantity of glass fibers resulting in higher flexural properties. The aim of this study was to evaluate the effect of short 3 mm long glass fiber addiction in flexural strength and flexural modulus of photopolymerizable 30%(wt) silica filled BisGMA composite resin (FR) or unfilled Bis-GMA resin (UR).N = 10) according to the interaction of factors in study: the quantity of 3 mm glass fibers added (wt%), in five levels, and the type of resinous material, in two levels, as seen in 2-radio-opacity-filler in BisGMA . Resins were used as provided by manufacturer and then the groups were created by manual incorporation of 3 mm short glass fibers into resins.A factorial design was employed to create experimental groups . After that, specimens were finished manually on 600, 1000, and 1200-grit SiC sandpapers with running water, for 10 seconds on each side [Bar-shaped specimens (10 mm × 2 mm × 1 mm) were made in a half-split stainless steel mold between transparent Mylar sheets . For theach side . Samplesach side .P · L/2w · h2, FM = S · L3/4w · h3, where P: maximum load at fracture, L: test span (8 mm), w: specimen width , h: specimen thickness (1 mm), S: the stiffness (N/m). S: P/d, d: deflection corresponding to P applied load at the straight-line portion of the load-deflection curve.A universal testing machine  was employed for a three-point bending test, accomplished with 8 mm test span, at 0.5 mm/min, with a central load application until fracture takes place . The maxFractured specimens were silica dehydrated and sputter-coated with gold  for observation in the Scanning Electron Microscope .t-test (pairwise comparisons). For all tests, groups were considered statistically different at α = 0.05.Data were submitted to a factorial analysis with a general linear model procedure to analyze the interaction between factors in study. After that statistical analysis was completed with Kolmogorov-Smirnov test of normal distribution and 1-way analysis of variance (ANOVA) followed by Tukey HSD test or Student P = 0.0001). Further analysis showed that within FR groups the 20%wt glass fiber addiction resulted in the highest flexural strength, which was similar to 10-FR and 15-FR. The 0-FR (control group) showed the lowest results, followed by 30-FR and both were similar to 10-FR group, but just the 30-FR was similar to 15-FR. UR groups showed an increase on flexural strength according to the percentage of glass fiber addiction. The 0-UF group showed the lowest results, followed by 10-UR; the 30-UR showed the highest flexural strength, which was similar to 15-UR and 20-UR.Tables P < 0.05).Paired comparisons between FR and UR groups with similar wt% of glass fibers showed that, only at 10%wt addiction of glass fibers, UR and FR groups were similar to each other. The UR without fiber reinforcement (control group) had lower FS than FR; however all the other fiber reinforced UR groups achieved statistically higher flexural strength than FR groups .Paired comparisons between FR and UR groups with similar wt% of glass fibers showed the same behavior on flexural strength: 10%wt glass fiber UR and FR groups were similar to each other and all the other groups were considered statistically different  , 29 and Poor fiber impregnation may be a result of using a more viscous resin, leaving empty spaces that accumulate oxygen, which inhibits resin polymerization. This fact produces a flexural weak FRC . In the When comparing the results of this work with data from literature it is possible to conclude that fracture strength was significantly improved. By using 22%wt of 3 mm long glass fibers in BisGMA-TEGDMA (60% bisphenol-A-glycidyl dimethacrylate-40% triethylenglycoldimethacrylate), Garoushi et al. 2006  showed 1The optimum glass fiber/resin/filler proportion was not investigated on this research and needs further work. As seen, high values for flexural strength and flexural modulus can be obtained if inorganic fillers are not included in the composite but several other properties need to be investigated for establishing good mechanical properties and handling characteristics.The inclusion of short glass fibers (3 mm long) in a BisGMA resinous matrix increases the flexural strength and flexural modulus of the material, and the absence of inorganic filler promotes better interaction/homogeneity with the fibers, leading to greater increase in the analyzed parameters. Inclusion of 30 wt% of fibers into filled resin reduced mechanical properties but the same into unfilled resin had the opposite behavior.


Candidatus Profftella armatura” strain YCPA from Diaphorina citri in Guangdong, China, was sequenced. The strain has a chromosome of 457,565 bp, 24.3% G+C content, 364 predicted open reading frames (ORFs), and 38 RNAs, and a plasmid, pYCPA54, of 5,458 bp with 23.9% G+C content and 5 ORFs.The genome of “ Diaphorina citri) transmit the pathogen of citrus Huanglongbing  which is threatening citrus production worldwide  at South China Agricultural University, Guangzhou, Guangdong, China. DNA was extracted from a single psyllid using a DNeasy blood and tissue kit . To meet the quantity requirement for sequencing, whole-genomic DNA was amplified through an Illustra GenomiPhi V2 DNA amplification kit  that yielded in 20 µg of DNA. A sample of 10 µg DNA was subjected to sequencing using Illumina MiSeq format .For sequencing the “7 reads with a mean of 251 bp per read were generated. De novo assembling was performed with CLC Genomics Workbench 7.5. Among the 83,161 large contigs  generated, 2  were identified to be associated with “Ca. Profftella armatura” by standalone BLAST (version 2.2.30)    and CP012592 (plasmid). The versions described in this manuscript are the first versions, CP012591.1 and CP012592.1.This complete genome sequence has been deposited in DDBJ/EMBL/GenBank under the accession numbers


P < 0.05 all, ANOVA). Only aprepitant pretreatment improved heart functions; it significantly decreased end systolic diameter, improved fractional shortening, and increased peak aortic flow velocity . Pre- or posttreatment with fasudil did not significantly impact disease manifestations. These findings indicate that SP contributes to cardiac-remodeling and dysfunction following ECMV infection via its high affinity receptor, but not through the Rho-A pathway. These studies suggest that SP-receptor antagonism may be a novel therapeutic-option for patients with viral-myocarditis.Viral-myocarditis is an important cause of heart failure for which no specific treatment is available. We previously showed the neuropeptide substance P (SP) is associated with the pathogenesis of murine myocarditis caused by encephalomyocarditis virus (EMCV). The current studies determined if pharmacological inhibition of SP-signaling via its high affinity receptor, NK1R and downstream G-protein, Ras homolog gene family, member-A (RhoA), will be beneficial in viral-myocarditis. Aprepitant (1.2 mg/kg), a SP-receptor antagonist, or fasudil (10 mg/kg), a RhoA inhibitor, or saline control was administered daily to mice orally for 3 days, prior to, or 5 days following, intraperitoneal infection with and without 50 PFU of EMCV, following which disease assessment studies, including echocardiogram and cardiac Doppler were performed in day 14 after infection. Pretreatment and posttreatment with aprepitant significantly reduced mortality, heart and cardiomyocyte size, and cardiac viral RNA levels ( Viral-myocarditis is an important cause of heart failure among adolescents and young adults . MyocardSubstance P (SP) is a neuropeptide involved in pain transmission especially associated with inflammation –13. SubsRho is a member of the Ras superfamily of small GTP-binding proteins that play a central role in diverse biological processes such as actin cytoskeleton organization, microtubule dynamics, gene transcription, oncogenic transformation, cell cycle progression, adhesion, and epithelial wound repair , 22.The SP receptor, neurokinin 1 receptor (NK1R), is a G-protein coupled receptor. Binding of SP to NK1R leads to activation of upstream regulators of RhoA activity , 24. SigThis study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Baylor College of Medicine (IACUC protocol number-AN209). All efforts were taken to ameliorate animal suffering. Mice were housed under BSL2 biohazard facility conditions. The mice were observed twice daily for the duration of the experiments. Mice that became moribund were considered to have reached the end point of the experiment and were humanely euthanized.Male C57BL/6J mice that were used for the EMCV infection studies were purchased from The Jackson laboratory. The EMCV model of murine myocarditis used in these studies was as previously described . AprepitFor determination of the inflammatory score, hearts were fixed in 4% paraformaldehyde, embedded in paraffin, stained with hematoxylin and eosin, and examined microscopically at 200x magnification. The inflammatory score was determined as described by Kanda et al.  and was Evidence of cardiomyocyte hypertrophy was determined by measuring diameter of cardiomyocytes following histopathological processing of the harvested hearts. Briefly, hearts were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined microscopically at 200x magnification. Cardiac hypertrophy was determined by measuring mean cardiomyocyte diameter as determined by measuring the diameter of 50 myocytes using NIH IMAGE v.1.62 software.Viral quantification was determined by real time PCR analysis using the SYBR Green Superscript One-Step Kit  and 100 ng of RNA and specific primers: for EMCV . in vivo cardiac function and morphology were assessed. Mice were anesthetized in an induction chamber using 2.5% isoflurane and then transferred to a heated ECG platform for heart rate monitoring during the imaging procedure. Standard B-mode (2D) and M-mode images were taken in the short axis position at the level of the papillary muscles for each animal. Data analysis was performed by an experienced imager using the Visualsonics Vevo 770 cardiac analysis package. 2  M-modes were analyzed per animal with an average being taken for each.Using a Vevo 770 ultrasound machine equipped with a 30 MHz transducer ,Aortic flow velocities were measured from the apical view using a 10 MHz pulsed Doppler probe and real time Doppler spectrum analyzer (Indus Instruments) . Animals t-test or ANOVA followed by pairwise comparison using Tukey's test, Newman-Keuls or Dunnet's test as indicated. Significance was set at P < 0.05.Values are expressed as mean ± SEM or SD. Statistical differences were determined using Student's in vivo pretreatment, with the SP specific, NK1 receptor antagonist, aprepitant, or with the Rho-A inhibitor, fasudil, will serve as novel treatment options for viral-myocarditis.In these studies, we addressed ifPretreatment with aprepitant an NK1R antagonist significantly inhibited all EMCV-induced manifestations such as mortality, increase in the heart-to-body weight ratio, cardiomyocyte hypertrophy, cardiac viral levels, and heart functional changes. When aprepitant was given after virus exposure, manifestations such as mortality, heart-to-body weight ratio increments, cardiomyocyte hypertrophy, and cardiac viral levels were significantly improved, but aprepitant posttreatment did not dramatically impact heart function. We demonstrated that although only posttreatment with fasudil reduced heart-to-body weight ratio, fasudil treatment on the whole (pre- or posttreatment) did not significantly impact mortality, cardiomyocyte hypertrophy, cardiac viral levels, or heart functional changes .n = 40; n = 15 (both pre- and postaprepitant treatment groups) and P < 0.05, for both, Kaplan-Meier analysis, A mortality rate of 65 ± 7% was seen at day 14 in untreated wild type mice following infection with 50 pfu of the EMCV virus n = 40; . Importan = 25 (fasudil pretreated) and n = 30 (fasudil posttreated); P > 0.05 for both, Kaplan-Meier analysis; There was no significant difference between the mortality rate in the sham treated mice and the fasudil pre- or posttreated mice (n = 8) versus only 5.6 ± 0.76 (n = 5) in the control uninfected mice, P < 0.05, ANOVA and Tukey's post hoc test). Importantly, pretreatment with aprepitant in infected mice reduced heart-to-body weight ratio by 30% (8 ± 1 (n = 6) versus 12.6 ± 0.7 (n = 8), P < 0.05, ANOVA and Tukey's post hoc test; n = 6) versus 12.6 ± 0.7 (n = 8), P < 0.05, ANOVA and Tukey's post hoc test; The mean heart-to-body weight ratio (expressed in terms of mg/g) following infection of 50 pfu of EMCV in mice was significantly increased in response to EMCV infection (12.6 ± 0.7 (n = 8) versus 12.6 ± 0.7 (n = 8); P > 0.05, ANOVA and Tukey's post hoc test; n = 10) versus 12.6 ± 0.7 (n = 8), P < 0.05, ANOVA and Tukey's post hoc test; EMCV-induced change in heart-to-body weight ratio was not significantly impacted with fasudil pretreatment (9.8 ± 0.5 (n = 5) versus only 0 ± 0 (n = 5) in the control uninfected mice ). Although the mean inflammatory score in infected mice with aprepitant pre- or posttreatment was lower than in infected mice without treatment, the differences were not statistically significant; aprepitant pretreatment (1.3 ± 0.3 (n = 4) versus 2.4 ± 0.2 (n = 5); P > 0.05, ANOVA and Tukey's post hoc test; n = 5) versus 2.4 ± 0.2 (n = 5); P > 0.05, Tukey's post hoc test; The mean inflammatory score following infection of 50 pfu of EMCV in mice was significantly increased in response to EMCV infection (2.4 ± 0.2 (n = 7) versus 2.4 ± 0.2 (n = 5); P > 0.05, ANOVA and Tukey's post hoc test; n = 5) versus 2.4 ± 0.2 (n = 5); P > 0.05, Tukey's post hoc test; Similarly, EMCV-induced change in cardiac inflammatory score was not significantly impacted with fasudil pretreatment (2.3 ± 0.3 (μm (n = 7) versus 9.2 ± 0.4 μm (n = 5) in the control uninfected mice ). Importantly, pretreatment with aprepitant in infected mice reduced cardiomyocyte diameter by 12% (11 ± 0.2 μm (n = 6) versus 12.5 ± 0.3 μm (n = 7), P < 0.05, ANOVA and Tukey's post hoc test; μm (n = 6) versus 12.5 ± 0.3 μm (n = 7), P < 0.05, ANOVA and Tukey's post hoc test; The mean cardiomyocyte diameter following infection of EMCV in mice was significantly increased in response to EMCV infection (12.5 ± 0.3 n = 8) versus 12.5 ± 0.3 μm (n = 7); P > 0.05, ANOVA and Tukey's post hoc test; n = 9) versus 12.5 ± 0.3 μm (n = 7); P > 0.05, Tukey's post hoc test; EMCV-induced change in cardiomyocyte diameter was not significantly impacted with fasudil pretreatment  in the WT mice. Importantly, pretreatment with aprepitant in infected mice reduced viral levels by 96% (9.6 ± 5.5 (n = 4), P < 0.05, Student's unpaired t-test; n = 3) versus 220 ± 71 (n = 3), P < 0.05, Student's unpaired t-test; The relative EMCV RNA levels in mice without treatment 14 days after infection was 220 ± 71 (n = 3) versus 220 ± 71 (n = 3); P > 0.05, Student's unpaired t-test; n = 3) versus 220 ± 71 (n = 3); P > 0.05, Student's unpaired t-test; There was no difference between the relative EMCV RNA levels in mice with and without fasudil pretreatment (182 ± 138 (n = 8) versus only 2.9 ± 0.2 mm (n = 5), in the control uninfected mice . Importantly, pretreatment with aprepitant in infected mice reduced the effects of ECMV on ESD by 24% (2.9 ± 0.07 mm (n = 4) versus 3.8 ± 0.1 mm (n = 8), P < 0.05, ANOVA and Newman-Keul's post hoc test; End systolic diameter (ESD) was significantly increased in response to EMCV infection. The mean ESD following infection in mice was increased 31%, 3.8 ± 0.1 mm (n = 4) versus 3.8 ± 0.1 mm (n = 8); P > 0.05, ANOVA and Newman-Keul's post hoc test; n = 6) versus 3.8 ± 0.1 mm (n = 8); P > 0.05, ANOVA and Newman-Keul's post hoc test; n = 7) versus 3.8 ± 0.1 mm (n = 8); P > 0.05, ANOVA and Newman-Keul's post hoc test; EMCV-induced change in ESD was not significantly impacted either with aprepitant posttreatment or with fasudil pre- as well as posttreatment. There was no difference between the mean ESD in infected mice with and without aprepitant posttreatment (3.6 ± 0.2 mm (n = 8) versus only 4 ± 0.1 mm (n = 5), in the control uninfected mice, the differences were not statistically significant . Similarly, although pretreatment with aprepitant in infected mice reduced the effects of ECMV on EDD by 8%, the differences were not statistically significant (4.2 ± 0.06 mm (n = 4) versus 4.6 ± 0.1 mm (n = 8), P > 0.05, ANOVA and Newman-Keul's post hoc test). Similarly, although posttreatment with aprepitant in infected mice reduced the effects of ECMV on EDD by 7% the differences were not statistically significant (4.2 ± 0.06 mm (n = 5) versus 4.6 ± 0.1 mm (n = 8); P > 0.05, ANOVA and Newman-Keul's post hoc test). Also, EDD was not significantly impacted with fasudil treatment. There was no difference between the mean EDD in infected mice with and without fasudil pretreatment (4.3 ± 0.2 mm (n = 6) versus 4.6 ± 0.1 mm (n = 8); P > 0.05, ANOVA and Newman-Keul's post hoc test) or fasudil posttreatment (4.5 ± 0.2 mm (n = 7) versus 4.6 ± 0.1 mm (n = 8); P > 0.05, ANOVA and Newman-Keul's post hoc test).We also measured end diastolic diameter (EDD). EDD levels were not significantly increased in response to EMCV infection. Aprepitant or fasudil treatment did not significantly impact EDD. Although the mean EDD following infection in mice was increased 13%; 4.6 ± 0.1 mm (n = 8) versus 29 ± 2% (n = 5), in the control uninfected mice . Importantly, pretreatment with aprepitant in infected mice ablated any effect of the ECMV on fractional shortening (29 ± 1% (n = 4) versus 16 ± 2% (n = 8), P < 0.05, ANOVA and Newman-Keul's post hoc test; The mean fractional shortening following infection in mice was only 16 ± 2% (n = 4)) or with fasudil pre- as well as posttreatment. There was no difference between the mean fractional shortening in infected mice with and without aprepitant posttreatment (19 ± 1% (n = 4) versus 16 ± 2% (n = 8); P > 0.05, ANOVA and Newman-Keul's post hoc test; n = 6) versus 16 ± 5% (n = 8); P > 0.05, ANOVA and Newman-Keul's post hoc test; n = 7) versus 16 ± 5% (n = 8); P > 0.05, ANOVA and Newman-Keul's post hoc test; EMCV-induced decrease in fractional shortening was not significantly impacted either with aprepitant posttreatment (19 ± 1% (n = 7) versus 90 ± 8 cm/sec (n = 7) in the control uninfected mice . Importantly, pretreatment with aprepitant in infected mice increased peak aortic flow velocity by 49% (92 ± 11 cm/sec (n = 5) versus 62 ± 8 cm/sec (n = 7), P < 0.05, ANOVA and Dunnett's post hoc test; Peak aortic flow velocity was significantly reduced in response to EMCV infection. The mean peak aortic flow velocity following infection in mice was only 62 ± 8 cm/sec (n = 5) versus 62 ± 8 cm/sec (n = 7); P > 0.05, ANOVA and Dunnett's post hoc test; n = 6) versus 62 ± 8 cm/sec (n = 7); P > 0.05, ANOVA and Dunnett's post hoc test; n = 7) versus 62 ± 8 cm/sec (n = 7); P > 0.05, ANOVA and Tukey's post hoc test; EMCV-induced change in peak aortic flow velocity was not significantly impacted either with aprepitant posttreatment or with fasudil pre- as well as posttreatment. There was no difference between the mean peak aortic velocity in infected mice with and without aprepitant posttreatment .In the current studies, we examined whether blocking of NK1R-signaling (with aprepitant) or RhoA activityWe determined whether SP mediated pathogenesis of viral-myocarditis is via NK1R-signaling and downstream RhoA activation. We determined whether blocking of NK1R-signaling with aprepitant (NK1R antagonist) will improve survival by inhibiting manifestations of myocarditis. We determined that pretreatment with aprepitant reduced mortality, prevented the increase in heart-to-body weight ratio, minimized the increase in end systolic diameter (ESD), reduced the increase in cardiomyocyte diameter, and increased fractional shortening and peak aortic flow velocity compared to that of infected, saline treated mice. We also determined that although posttreatment of aprepitant was effective in inhibiting manifestations such as mortality and increases in cardiac viral load, heart-to-body weight ratio, and cardiomyocyte diameter, it did not significantly impact heart functional parameters. Although the current studies showed a significant inhibition of the pathogenesis of disease with NK1 receptor antagonist, both pretreatment and posttreatment did not completely prevent disease. This could be possible because SP binds with high affinity to NK1R. However, it can also bind to NK2R and NK3R, although with much lower affinity. Therefore, it may be possible that even in the absence of NK1R, SP may be mediating its effects through interaction with NK2R and NK3R. Therefore, we may not have observed complete ablation of pathogenesis in the NK1R treated mice. Currently, there are no antagonists that antagonize all 3 receptors at the same time; therefore we have to rely on studies using individual antagonists that antagonize each receptor. However, as mentioned in Our previous results  wherein  gag gene expression [ in vitro [Importantly, we demonstrated that aprepitant treatment not only inhibited the manifestations of murine viral-myocarditis, but also decreased cardiac EMCV viral RNA levels compared to aprepitant untreated, EMCV-infected mice. We do not know the mechanisms which could have resulted in decreased viral load in the aprepitant treated mice. We anticipate that it could either be due to one or more of the following reasons; substance P may be directly responsible for enhancing viral replication, or it may be that aprepitant has direct antiviral effects, or it could be possible that SP and/or aprepitant indirectly via some unknown mechanism may be causing reduced ability of the virus to reach the heart from the peritoneal cavity where it was injected, and finally it may be possible that the NK1R may be important for viral entry. We anticipate the possibility that SP may directly enhance viral replication and the possibility that aprepitant may serve as an antiviral agent to be very relevant based on other studies outlined below wherein SP has shown to enhance viral replication and aprepitant has demonstrated antiviral capabilities. Substance P has shown to enhance HIV-1 replication in latently infected human immune cells such as a latently infected promonocytic cell line (U1) and a T lymphocyte line (ACH-2) . Furtherpression . In contin vitro . Also whThe current studies demonstrate that aprepitant is effective as a preventive measure; aprepitant treatment before viral infection inhibits all manifestations of EMCV infection, whilst administration of aprepitant 5 days after infection, although effective in inhibiting manifestations such as mortality and increases in cardiac viral load, heart-to-body weight ratio, and cardiomyocyte diameter, it did not significantly impact heart functional parameters. We do not know the exact reason why heart functional parameters were not improved in spite of other manifestations showing an improvement in the mice that were administered aprepitant after infection; possible reasons could include the following: it could be possible that there may be an underlying effect that occurs in the first few days of infection that despite subsequent depletion of the virus may be already in effect in the postaprepitant treated group. Dehlin and Levick  have recA question also arises as to the reason why posttreatment of aprepitant was beneficial as demonstrated by decrease in mortality in spite of decreased heart functions. Possible explanations could include that in the EMCV model of murine infection, dilated cardiomyopathy does not set in until later in infection, possibly beginning at days 10–12 after infection. Therefore if we terminate the experiment at a later time-point than 14 days, we may observe increased mortality in the aprepitant posttreated mice specifically due to declining heart functions. Alternatively, we may determine that in the aprepitant posttreated mice, the compensatory phase leading to decreased heart functions may rectify itself leading to survival as a result of improving heart functions. In order to address if the former scenario is the explanation for lack of association between survival rate and declining heart functions in the aprepitant posttreated group, as part of our future studies, we will study the survival rate and heart functions in mice at later times following infection and also compare if there is an improvement with changes in dosage and administration regime.In the current studies, we also determined if SP mediated pathogenesis is mediated via downstream RhoA activation. We anticipated that if SP/NK1R mediated pathogenesis of myocarditis is mediated via RhoA-signaling, RhoA levels will be elevated in EMCV-infected mice and RhoA inhibitors will inhibit pathogenesis of EMCV infection. We determined that RhoA levels were significantly increased in response to EMCV infection compared to uninfected mice (results not shown). Furthermore, although heart enlargement in response to EMCV infection was significantly reduced only in fasudil posttreated mice, none of the other manifestations were improved in either the fasudil pre- or posttreated mice. The predominant inability of fasudil to prevent or treat manifestations of disease may be because there may be multiple signaling mechanisms, such as other GTPases involved in SP mediated responses. Therefore blocking RhoA alone may not have resulted in inhibition of the SP induced responses. As part of our future studies, we will look for the role in pathogenesis of other GTPases like Rac and Cdc 42 that are known to be activated by SP . Other sThe role of SP and NK1R in other experimental models of cardiac-remodeling has been addressed by few investigators , 49–51. In conclusion, these findings indicate that SP contributes to cardiac-remodeling and dysfunction following ECMV infection via its high affinity receptor, NK1R. However SP/NK1R mediated pathogenesis of EMCV-induced myocarditis is not significantly mediated via RhoA-signaling. These studies implicate that pharmacological inhibition of NK1 receptor may serve as novel treatment option for viral-myocarditis.


Eurycoma longifolia (Simaroubaceae) is a popular folk medicine that has traditionally been used in Southeast Asia as an antimalarial, aphrodisiac, antidiabetic, and antimicrobial and in antipyretic remedies. This study evaluates the effects of Eurycoma longifolia extract on cytochrome P450 (CYP) enzyme-mediated drug metabolism to predict the potential for herb-drug interactions. Methanolic extract of E. longifolia  root was tested at concentrations of 1, 3, 10, 30, 100, 300, and 1000 µg/mL in human liver microsomes or individual recombinant CYP isozymes. The CYP inhibitory activity was measured using the cocktail probe assay based on liquid chromatography-tandem mass spectrometry. E. longifolia showed weak, concentration-dependent inhibition of CYP1A2, CYP2A6, and CYP2C19. The inhibitory effects on these CYP isozymes were further tested using individual recombinant CYP isozymes, showing IC50 values of 324.9, 797.1, and 562.9 μg/mL, respectively. In conclusion, E. longifolia slightly inhibited the metabolic activities of CYP1A2, CYP2A6, and CYP2C19 but this issue requires careful attention in taking herbal medicines or dietary supplements containing E. longifolia extracts. Eurycoma longifolia jack (Simaroubaceae) is used as a herbal medicine and functional food in many Southeast Asian countries, including Indonesia, Malaysia, Thailand, Vietnam, and Laos [ E. longifolia has been reported to contain various types of secondary metabolites such as quassinoids [β-carboline alkaloids [ E. longifolia have long been used to treat fatigue [ E. longifolia extracts are commonly used as dietary supplements or alternative medicine.and Laos . E. longssinoids , canthinssinoids , squalenssinoids , and β-clkaloids . Root ex fatigue , malaria fatigue , inflamm fatigue , and per fatigue . In addi fatigue  and to i fatigue . Due to The use of traditional herbal therapies is becoming more common. Recently, the World Health Organization estimated that 80% of people worldwide rely on herbal medicines for some part of their primary health care . AccordiHerb-drug interactions may interfere with various pharmacokinetic processes: herbs have been reported to alter drug absorption, induction and inhibition of metabolic enzymes, and drug excretion . In partE. longifolia extract should be evaluated for possible herb-drug interactions, as it is likely coadministered with conventional drugs. However, to our knowledge, there has been no previous study that examined the effects of E. longifolia on CYP enzyme activities. Therefore, this study examined the inhibitory effects of E. longifolia extract on CYP450-mediated drug metabolism, using human liver microsomes and individual recombinant CYP isozymes. E. longifolia roots was provided by the Institute of Marine Biochemistry, Vietnam Academy of Science and Technology . Pooled human liver microsomes and recombinant CYP1A2, CYP2A6, and CYP2C19 isozymes were purchased from BD Gentest . Glucose-6-phosphate, β-NADP+, glucose-6-phosphate dehydrogenase, phenacetin, coumarin, diclofenac, mephenytoin, dextromethorphan, midazolam, ketoconazole, and terfenadine were obtained from Sigma Chemical Co. . All other solvents used were of HPLC grade and were purchased from J. T. Baker . Distilled water was prepared using a Milli-Q purification system .The methanolic extract of E. longifolia was dissolved in distilled water (1 mg/mL). The sample was filtered through a 0.22 μm membrane filter, and a 10 μL aliquot of the sample was injected into an HPLC system  equipped with an ultraviolet (UV) detector. The analytical column  was maintained at 30°C. The mobile phase consisted of 0.1% formic acid (solvent A) and 90% acetonitrile with 0.1% formic acid (solvent B). The flow rate was set at 1 mL/min. The gradient elution mode was used; the concentration of solvent B was gradually increased from 5% to 25% for 30 min, further increased to 90% for 5 min, maintained at 90% for 1 min, decreased to the initial condition for 1 min, and maintained for reequilibrium. The injection volume was 10 μL, and the UV detection wavelength was set at 244 nm. In the HPLC chromatogram (n = 3) of E. longifolia extract.The extract ofmatogram , eurycom E. longifolia extract ; an NADPH-generating system (NGS) containing 0.1 M glucose-6-phosphate, 10 mg/mL β-NADP+, and 1.0 U/mL glucose-6-phosphate dehydrogenase, and a substrate mixture  in 200 μL of 0.05 M potassium phosphate buffer (pH 7.4). The reaction mixture was preincubated at 37°C for 5 min without NGS and then further incubated for 30 min with NGS in a water bath. Ketoconazole (5 μM), furafylline (10 μM), methoxsalen (10 μM), sulfaphenazole (50 μM), ticlopidine (20 μM), quercetin (30 μM), and quinidine (50 μM) were tested as positive controls. After the incubation, the reaction was stopped by adding 400 μL of 0.1% acetic acid containing internal standard (0.16 μM terfenadine). For the CYP inhibition assay using each CYP isozyme, 12.5 pmol isozyme  instead of human liver microsomes and the corresponding specific substrate  were added to the reaction mixture and the rest of the procedure was performed as described above.The reaction mixtures consisted of 0.5 mg/mL human liver microsomes; various concentrations ofμL of the mobile phase (0.1% formic acid in an 85 : 15 mixture of water : acetonitrile), and a 5 μL aliquot was injected into the liquid chromatography-tandem mass spectrometry (LC-MS/MS) system.The incubation mixtures were passed through activated Sep-Pak C18 cartridges . The cartridges were eluted with methanol (1 mL) and 0.1% acetic acid (2 mL). After sample loading, the cartridges were washed twice with 1 mL of 0.1% acetic acid and eluted with 1 mL of methanol. The eluate was evaporated under nitrogen gas, the residue was reconstituted in 100 μm; Fortis Technologies Ltd., Cheshire, UK). The column temperature was maintained at 40°C. The HPLC mobile phases consisted of 0.1% formic acid in distilled water (A) and 90% acetonitrile in 0.1% formic acid (B). A gradient program was used at a flow rate of 0.2 mL/min; the composition of solvent B was initially set at 15%, gradually increased to 85% over 3 min, and maintained for 1.5 min followed by a reequilibrium for 3.5 min. The mass spectrometer was operated in positive ion mode with multiple reaction monitoring (MRM). The precursor-product ion pairs (Q1/Q3) used in MRM mode are shown in The LC-MS/MS system consisted of an Agilent 1260 series binary pump HPLC system and an Agilent 6460 Triple Quadrupole mass spectrometer  equipped with an electrospray ionization (ESI) source. Chromatographic separation was achieved using a Fortis C8 column  for each CYP isozyme was estimated based on four-parameter logistic nonlinear regression analysis using Sigma Plot version 10.0 .The half maximal inhibitory concentration (IC E. longifolia extracts on CYP enzyme activities were investigated in human liver microsomes and recombinant isozymes. The CYP inhibition assay system was tested with well-known CYP selective inhibitors as follows: ketoconazole for CYP3A4, furafylline for CYP1A2, methoxsalen for CYP2A6, quercetin for CYP2C8, sulfaphenazole for CYP2C9, ticlopidine for CYP2C19, and quinidine for CYP2D6. Formation of each CYP-specific metabolite was reduced by >95% following treatment of its corresponding inhibitor, indicating that the assay system was functioning well. E. longifolia extracts were tested at concentrations of 1, 3, 10, 30, 100, 300, and 1000 μg/mL for seven CYP isozymes. The results showed that E. longifolia extracts inhibited CYP1A2, CYP2A6, and CYP2C19 activities in a concentration-dependent manner. The effects on other isozyme activities were negligible. Subsequently, the inhibitory effects against CYP1A2, CYP2A6, and CYP2C19 isozymes were further evaluated using cDNA-expressed recombinant CYP isozymes. E. longifolia extracts showed concentration-dependent inhibition against those enzymes (50 values for CYP1A2, CYP2A6, and CYP2C19 of 324.9, 797.1, and 562.9 μg/mL, respectively. Some reports suggested a classification for CYP inhibitors [50 values ≤ 10 μM, moderate for IC50 values 10–50 μM, and weak for IC50 values >50 μM. Based on these criteria, assuming that the average molecular weight of the constituents is 500 da and their average content is approximately 5% in E. longifolia extracts, IC50 values <500 μg/mL could be of some significance. Thus, possible inhibition of CYP1A2 and CYP2C19 by E. longifolia extract should be carefully considered. However, as this implication on some potential for CYP inhibition is based on the in vitro data, there is a need to verify this in in vivo studies such as interaction studies using animal models or even human subjects.The inhibitory effects of enzymes , as obse enzymes , with IChibitors : potent  E. longifolia include quassinoids, β-carboline alkaloids, squalene derivatives, and canthinone derivatives [ E. longifolia extracts. The content of eurycomanone in E. longifolia root extracts has been reported to 5.65%~9.95% of extracts [The major components ofivatives . Among textracts . As for  E. longifolia extract, the literature contains one published study of the effect of eurycomanone, a major bioactive compound of E. longifolia, on CYP enzyme activities [50 values greater than 250 μg/mL. Thus, the authors concluded there was low risk of drug-herb interaction between eurycomanone and CYP drug substrates via CYP inhibition. Therefore, ingredients other than eurycomanone might be involved in the CYP inhibitory effects of E. longifolia extracts. However, the literature on CYP-modulating effects of other constituents, such as other quassinoids, β-carboline alkaloids, or canthinones, is rather limited. Zhao et al. reported inhibition of CYP3A4 and CYP2D6 enzyme activities by β-carboline alkaloids [ E. longifolia extracts.With regard to herb-drug interaction via CYP inhibition by the constituents oftivities . Accordilkaloids . However E. longifolia extracts. Salman et al. reported that when propranolol was administered with E. longifolia, the Cmax and bioavailability of propranolol decreased by 29% and 42%, respectively, in humans [ E. longifolia extracts increased the phase I metabolism of rosiglitazone, an antidiabetic drug, in male rat hepatocytes, which suggest the likelihood of CYP enzyme induction by E. longifolia extract [Recent studies provide some preclinical or clinical evidence for herb-drug interactions ofn humans . The aut extract .E. longifolia extract is commonly consumed as a herbal medicine or dietary supplement for its various pharmacological effects. Therefore, it is likely to be coadministered with prescription or conventional drugs, particularly in patients with chronic diseases such as diabetes mellitus, hypertension, and other cardiovascular diseases. Considering the present results and those previously reported in the literature, coadministration of E. longifolia products and conventional drugs requires careful attention.


The aim was to investigate the pharmacokinetics of oral and iv melatonin in healthy volunteers.ka, t1/2 absorption, tmax, Cmax, t1/2 elimination,AUC0-∞, and bioavailability were determined for oral melatonin. Cmax, t1/2 elimination, Vd, CL and AUC0-∞ were determined for intravenous melatonin.The study was performed as a cohort crossover study. The volunteers received either 10 mg oral melatonin or 10 mg intravenous melatonin on two separate study days. Blood samples were collected at different time points following oral administration and short iv infusion, respectively. Plasma melatonin concentrations were determined by RIA technique. Pharmacokinetic analyses were performed by “the method of residuals” and compartmental analysis. The pharmacokinetic variables: P = 0.067). Mean (SD) t1/2 absorption of oral melatonin was 6.0 (3.1) min. Mean tmax was 40.8 (17.8) min with a median (IQR) Cmax of 3550.5 (2500.5–8057.5) pg ml-1. Mean t1/2 elimination was 53.7 (7.0) min. Median absolute bioavailability was 2.5 (1.7–4.7) %. Median Cmax after short iv infusion of melatonin was 389,875.0  pg ml-1. Mean t1/2 elimination was 39.4 (3.6) min, mean Vd 1.2 (0.6) l kg-1 and mean CL 0.0218 (0.0102) l min-1 kg-1.Twelve male volunteers completed the study. Baseline melatonin plasma levels did not differ significantly between the study days .Eudra-CT number: NCT01923974 .Clinicaltrials.gov Identifier:  Furtelatonin , 6. The elatonin , 6. Accoelatonin , 6. In oThe aim of the study was to investigate the pharmacokinetics of oral and iv melatonin in a cohort of healthy volunteers.Approvals were obtained from the Capital Region’s Committee on Health Research Ethics (Protocol number: H-4-2013-013), the Danish Health and Medicines Authority  and the Danish Data Protection Agency  prior to inclusion of volunteers. Informed written- and verbal consent were provided by all volunteers. The blood samples relating to iv melatonin were obtained from a primary trial investigating analgesic and anti-hyperalgesic effects of melatonin . Data presented in the present paper have not been published previously.The study was performed as a cohort crossover study. The volunteers received either 10 mg of oral melatonin or 10 mg of iv melatonin on two separate study days. Each study session was performed from 08:00 a.m. to 04:00 p.m. The study days were separated by 3 to 9 months. Inclusion criteria were age 20–40 years and male gender. Volunteers were excluded, if they did not understand written- or spoken Danish, suffered from serious physical or mental illness, were diagnosed with a sleep disorder, worked night shifts, received daily analgesics, had participated in other clinical trials within one month from study inclusion or suffered from skin abnormalities on the lower extremities .left antecubital vein .Oral melatonin consisted of one gelatine capsule containing 10 mg melatonin. Volunteers were allowed 5 cl of tap water to facilitate oral intake. Volunteers were instructed to adhere to preoperative fasting guidelines before the study session with oral melatonin  . Volunteright antecubital vein at specified time points during each study session. The time points differed between oral and iv melatonin. Oral melatonin: at baseline (before medication = endogenous melatonin production), and 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 180, 240, 300, 360 and 420 min after oral administration. Intravenous melatonin: at baseline, and 0, 60, 120, 180, 240, 300, 360 and 420 min after short iv infusion. Before each blood sample was collected, a 3 ml volume of blood  was drawn from the peripheral venous catheter and discarded. Fractionation of blood samples was performed at 5000 r.p.m. for 5 min, and plasma samples were stored at -80 °C. Quantitative determination of plasma melatonin concentrations was performed by radioimmunoassay (RIA)-technique . Precision of the RIA kit: intra-assay coefficient of variation (CV) = 9.8–13.4 %, inter-assay CV = 8.0–13.3 %. The limit of detection was 2.3 pg ml-1. Linearity of the kit ranged between 8.5–529.0 pg ml-1. If plasma concentrations exceeded detection range of the kit, plasma samples were diluted according to manufacturer’s guidelines. All plasma samples were analysed in duplicate, and the mean value was reported.Blood samples were collected from a peripheral venous catheter inserted in the P-value < 0.05 is considered statistically significant. Data were analysed using IBM SPSS Statistics for Windows version 22.0  and Graph Pad Prism version 6.0 .Normality of data was assessed by visual inspection of residual plots and histograms. Parametric or non-parametric tests were applied according to the distribution of data. Correspondingly, data are presented as mean (SD) or median (IQR), unless stated otherwise. A not subtracted from post-treatment  levels.The baseline melatonin plasma concentrations of each study day were compared using a paired sample T-test. Baseline levels were oral and iv melatonin were performed separately.Pharmacokinetic analyses of tmax) and maximal plasma concentrations (Cmax) were assessed directly at the relevant time points. The pharmacokinetic variables: absorption constant (ka), absorption half-life (t1/2 absorption), elimination rate constant (ke) and elimination half-life (t1/2 elimination) were estimated by “the method of residuals” [AUC0-∞ was estimated as AUC0-420 min + (C420 min/ke). Bioavailability was calculated as  x 100.Time to maximal concentrations  and clearance (CL) were estimated from individual linear regression lines of log-transformed  plasma concentrations. Following standard equations were applied: t1/2 elimination = ln (2) / ke, Vd = dose / C0 min, CL = ke x Vd. “Goodness of fit” of the individual linear regression lines was assessed by the coefficient of determination, R2. AUC0-∞ IV was estimated, as described above.analysis . The pha-2, respectively. Baseline melatonin plasma concentrations did not differ significantly between the study days  pg ml-1; before intravenous melatonin = 18.3 (12.3) pg ml-1) (P = 0.067).Twelve male volunteers were included and completed the study. Mean age and body mass index (BMI) were 27.1 (5.2) years and 23.2 (2.7) kg mThe pharmacokinetic variables of oral and iv melatonin are presented in Tables ka was 0.2 (0.1) min-1, and mean t1/2 absorption of oral melatonin was 6.0 (3.1) min  min with a median (IQR) Cmax of 3550.5 (2500.5–8057.5) pg ml-1. Mean t1/2 elimination was 53.7 (7.0) min, AUC0-∞ oral 281,538.3  pg ml-1 min and median absolute bioavailability was 2.5 (1.7–4.7) %.Oral melatonin demonstrated first-order absorption and elimination kinetics. Mean as 0.2 0. min-1, aCmax after iv bolus injection of 10 mg melatonin was 389,875.0  pg ml-1. Mean t1/2 elimination was 39.4 (3.6) min, mean Vd 1.2 (0.6) l kg-1 and mean CL 0.0218 (0.0102) l min-1 kg-1. Median R2 was 0.96 (0.93–0.97). Median AUC0-∞ IV vas 14,179,767.6  pg ml-1 min.The pharmacokinetic profiles of iv melatonin demonstrated first-order elimination kinetics Fig. . Median tmax of 41 min following oral administration. Cmax and AUC varied extensively between volunteers in both administration routes. Elimination half-lives were 54 min and 39 min, respectively. Bioavailability of oral melatonin was only 3 %, but demonstrated substantial inter-individual differences.This cohort crossover study demonstrated a t1/2 absorption of 6 min, corroborate studies, applying similar oral drug formulations [tmax value of 41 min is in agreement with other studies, documenting values ranging from 30 to 60 min [tmax values [Cmax and AUC0-∞ oral values, which has been described previously [t1/2 elimination values ranging from 46 to 65 min in oral doses from 0.5 to 6 mg [in-depth pharmacokinetic-pharmacodynamic modelling.Oral melatonin was absorbed by first-order kinetics, which has previously been demonstrated in doses up to 80 mg [o 60 min . Oral adx values . Oral adeviously . The inteviously . Previou to 6 mg , which c to 6 mg . It is wCmax and AUC0-∞ IV values, which is in accordance with previous studies [t1/2 elimination values ranging between 28 and 60 min in iv doses from 0.005 mg to 2 mg [CL values of 0.013 l min-1 kg-1 (weight-corrected) [-1 kg-1 [-1 kg-1. Cavallo and colleagues also documented a VD of 1.8 l kg-1 [-1, demonstrated in our study.Previous studies investigating iv administration of melatonin have also demonstrated first-order eliminations kinetics , as obse studies . Other s to 2 mg , 13, whi[-1 kg-1 , which c8 l kg-1 , which iapproximately 10 mg) [2) to document the “goodness of fit” of the individual linear regression lines in the compartmental analysis. Our data demonstrated a R2 value of 0.96, indicating a high degree of “fit” of the first-order pharmacokinetic model, and, hence, a considerable accuracy of the derived pharmacokinetic variables.Our study is the first to perform direct comparisons of pharmacokinetics of oral and iv melatonin in doses routinely administered perioperatively y 10 mg) . The stuy 10 mg) , 10. In  mg . TheFirst, this study only included healthy male volunteers in an experimental setup. Hence, a potential gender difference in pharmacokinetic variables may exist. Furthermore, previous experimental studies indicate that the pharmacokinetics of melatonin is affected by age [d by age  and exted by age  cigarettd by age  and the d by age . Also, ad by age –19 in e.d by age , 18. Intd by age .Second, oral and iv study sessions were separated by 3 to 9 months for each volunteer. These time periods may theoretically have affected the comparability of individual pharmacokinetic variables, despite the crossover design. It, however, seems unlikely, as all volunteers were healthy young males in stable physical conditions.Third, the very low bioavailability of oral melatonin documented in our study may indicate a deficient absorption of the drug in our setup. The volunteers were allowed 5 cl of tap water to facilitate intake of oral melatonin. Hence, it can be discussed, if the restricted liquid volume, despite saliva and gastric/intestinal fluid secretions was sufficient to dissolve and present the ingested melatonin to the small intestine, where absorption is mainly achieved. We, however, chose this amount of water to standardize the experimental conditions and to imitate a clinical premedication scenario [tmax values between the volunteers were demonstrated, suggesting that an impeded absorption is rather unlikely.scenario , 7. Alsomax was achieved after 41 min. Cmax and AUC varied extensively between volunteers. Elimination half-lives following oral and intravenous melatonin administration was 54 min and 39 min, respectively. The bioavailability of oral melatonin was only 3 %, but a considerable variability between the volunteers was noted.This crossover cohort study investigated the pharmacokinetics of oral and intravenous melatonin in healthy male volunteers. Oral melatonin was rapidly absorbed, and T


Maintaining energy balance by consuming the required distribution of macronutrients  is important to support performance and health in collegiate athletes; however, less than 10% of NCAA athletes possess adequate sports nutrition knowledge or maintain nutritional status . A recent study demonstrated that a sports nutrition education intervention (SNEI) improved nutritional knowledge and nutritional status in Division I volleyball players. This study investigated the effects of an SNEI on nutritional status, knowledge, body composition, and performance in NCAA Division I baseball players.Thirty resistance trained NCAA Division I baseball players  participated in the 12-week study. Fifteen players volunteered for the SNEI while 15 players matched for position served as controls (C). All players participated in a monitored, periodized strength (4 hr/wk), conditioning 3 hr/wk), and skills (20 hr/wk) training program. The nutrition intervention group (N) received a 90 min SNEI encompassing the following topics: energy intake , carbohydrate (CHO), protein (PRO), fat, food sources, and hydration. Thereafter, N met once every three weeks with the primary researcher for educational reinforcement in groups of 5. Sport nutrition knowledge questionnaires  were administered to N at baseline (t-0) and following 12 weeks (t-12). Food intake was determined by three-day dietary logs administered to N at t-0 and t-12. Energy and macronutrient intake was calculated using Diet Analysis Plus (Cengage), and compared to nutritional requirements . Body composition (BodPod), 1 RM back squat, vertical jump, and broad jump were measured at t-0 and t-12 for C and N. Pre and post nutritional status and knowledge were analyzed using paired samples t-test for N. Changes in body composition and performance were compared between C and N using an independent groups t-test with an alpha level of 0.05 for all tests.Knowledge significantly (p < 0.05) increased from t-0 to t-12 (56 ± 11% vs. 70 ± 9%). Energy consumption was significantly (p < 0.05) less than requirements at t-0  and significantly (p < 0.05) increased to meet requirements at t-12 . CHO was significantly (p < 0.05) less than requirements at t-0 (3.6 ± 1.1 g/kg) and t-12 (3.8 ± 0.8 g/kg). PRO was significantly (p < 0.05) less than requirements at t-0 (1.7 ± 0.4 g/kg) and significantly increased (p < 0.05) at t-12 (2.2 ± 0.4 g/kg). Fat was not significantly (p > 0.05) different than requirements at t-0 (1.6 ± 0.3 g/kg) and significantly (p < 0.05) increased above requirements at t-12 (2.0 ± 0.4 g/kg). Fat free mass and body mass significantly (p < 0.05) increased  with no difference between groups. Percent body fat decreased significantly (p < 0.05) in N (Δ= -1.2 ± 2.3%) but not C (Δ = 0.3 ± 1.7%). Squat, vertical, and broad jump significantly (p < 0.05) increased  with no difference between groups.Our findings indicate that an off season SNEI is effective at improving sport nutrition knowledge and some, but not all nutrient intakes in Division I baseball players. Improvements in nutritional status were associated with decreases in body fat percentage, possibly attributable to increased protein consumption.


Objective: The purpose of this study was to assess the effectiveness of a 4-week intervention in which an online personal activity monitor (Gruve-Technologies™) was used to reduce sedentary behavior among sedentary adults. Method: Eighteen, sedentary adult volunteers  were recruited to participate in the study. Time spent in sedentary activities and light-, moderate-, and vigorous-intensity physical activity and energy expenditure were assessed during waking hours using the monitor and the 7-day SLIPA Log at both baseline and post-intervention. Results: A significant decrease of 33%  was found between the time spent in sedentary activities measured at baseline (9.4 ± 1.1 h/day) and at the end of the 4-week intervention (6.3 ± 0.8 h/day). Consequent to the changes in sedentary time, significant increases were found in the amount of time spent in light- (45% (2.6 h/day), p < 0.001), moderate- (33% (1 h/day) p < 0.001), vigorous-intensity physical activity (39% (0.16 h/day), p < 0.001), and energy expenditure , p < 0.001). Conclusion: This monitor contributes to a meaningful reduction in time spent in sedentary activities and has a large effect on energy expenditure and physical activity patterns. Due to concerns about declining levels of physical activity and the increasing amount of time people spend in sedentary behavior ,2, the bAs a consequence of such research, increased attention is being paid to developing potential intervention methods focused on replacing time spent in sedentary activities with standing and other light-intensity activities of daily living ,18,19,20The aims of this study were to (1) examine the use of the Gruve online personal activity monitor over a 4-week intervention to reduce sedentary behavior and increase physical activity levels and energy expenditure among sedentary adults during free-living activities, and (2) use a self-report method to examine changes in time spent in sedentary activities and light-intensity physical activity across different life domains .All participants completed a demographic questionnaire at baseline. Twenty-four adult volunteers  were recruited to participate in the study through advertisements in local newsletters, flyers and emails at a metropolitan university in Brisbane, Australia in October 2012. Pre-screening using the International Physical Activity Questionnaire (IPAQ) self-report questionnaire was used to determine participant eligibility in relation to their volume of sedentary behavior. Only those who reported a high total sitting time, defined as >7 h/day, were invited to participate in the study ,24. WritThe data for this research emanated from a larger study which was a randomized, lifestyle intervention for adults. That intervention was designed to reduce sedentary behavior and increase physical activity levels in daily living for sedentary adults and to determine if these changes would also be associated with improvement in total wellness . This reet al. [2 > 0.95) with the Modular Signal Recorder accelerometer  for the measurement of both sitting and standing. They then suggested that the Gruve would be appropriate for use during free-living conditions.MUVE, Inc.  developed the Gruve online personal activity monitor in cooperation with the Mayo Clinic  . The monet al.  found thParticipants completed a 4-week intervention using the Gruve monitor to reduce sedentary behavior during free-living activities. The intervention simply consisted of the participants regularly logging onto the Gruve online personal activity monitor software and tracking their physical activity level during those 4 weeks. Each participant completed two visits to the university. During the first visit, participants’ height (cm) and weight (kg) were measured. The International Society for the Advancement of Kinanthropometry (ISAK) protocols were followed . Body maTime spent in sedentary activities and light-, moderate-, and vigorous-intensity physical activity and energy expenditure were measured during waking hours. The Gruve software employs commonly utilized cutoffs as follows: sedentary activity is defined as 0 to 1.5 metabolic equivalent of task (MET), light activity as 1.6 to 3 MET, moderate as 3.1 to 6 MET , and intense as 6+ MET  . Every 3‑1∙min-1 [r = 0.86, p < 0.001) and for light-intensity physical activity  was acceptable across the four daily life domains [Daily sedentary activities and light-intensity physical activity within specific behavioral life domains were measured using the 7-day Sedentary and Light Intensity Physical Activity Log (7-day SLIPA Log) . The 7-d‑1∙min-1 . Questio domains .t-tests were calculated following Cohen’s d  [p < 0.05. The sample size was calculated using the G*Power V.1.1.3 software . A target of 20 participants was set, as this was estimated to provide 85% power  to detect a 10% decrease in the amount of time spent in sedentary activities. An allowance for 10% attrition was included in determining the required sample size.All statistical analyses were carried out using SPSS statistical software version 21.0 for Windows . Descriptive data were expressed as means and SDs. To determine whether pre-post changes across the intervention differed from baseline to the end of the 4-week intervention, a paired t-test was used. Differences between the average of the amount of time spent in sedentary activities, light-, moderate-, and vigorous-intensity physical activity, and energy expenditure were assessed at baseline and at the end of the 4-week intervention and standardized for wear time using the residuals method . Paired  ≥ 0.80) . Differet (17) = 9.5, p < 0.001) (p < 0.01) was found between the amount of time spent in daily sedentary activities measured at baseline (9.4 ± 1.1 h/day) and at the end of the 4-week intervention (6.3 ± 0.8 h/day) and the effect size was large (d = 3.0). This amounts to a decrease of 33% (3.1 h/day) in sedentary activities. t (17) = −7.7, p < 0.001) was found between the amount of time spent in light-intensity physical activity at baseline (5.8 ± 1.3 h/day) and at the end of the 4-week intervention (8.4 ± 1.0 h/day) and the effect size was also large (d = 2.2). This amounts to a daily average increase of approximately 45% (2.6 h/day) in light intensity physical activity.Analysis of the amount of time spent in sedentary activities revealed a significant decrease over the intervention period compared to baseline (< 0.001) . A signit (17) = −4.8, p < 0.001) with a large effect size (d = 0.8) was found for the amount of time spent on daily moderate-intensity physical activity from baseline (0.75 ± 0.2 h/day) to the end of the 4-week intervention (1.0 ± 0.3 h/day). This difference amounts to an increase of 33% (0.25 h/day). Additionally, there was a statistically significant difference (t (17) = −3.0, p < 0.001) in the amount of time spent on vigorous-intensity physical activity from baseline (0.44 ± 0.2 h/day) to the end of the 4-week intervention (0.61 ± 0.3 h/day) and the effect size was medium (d = 0.5). This difference amounts to an average daily increase of approximately 38% (0.16 h/day) in vigorous-intensity physical activity (t (17) = −7.0, p < 0.001), representing an increase from baseline to the end of the 4-week intervention, was detected. Finally, a significant difference with a large effect size (d = 0.9) was found in RMR between baseline  and the end of the 4-week intervention . This amounts to a daily increase above the RMR of 47% .A significant difference (activity . For enework (t (17) = 3.5, p < 0.001, d = 0.4) (5.2 ± 1 h/day decreasing to 4.6 ± 1 h/day); transportation (t (17) = 3.9, p < 0.001, d = 1.0) (0.79 ± 0.4 h/day decreasing to 0.42 ± 0.2 h/day); and leisure time (t (17) = 5.9, p < 0.001, d = 1.5) (5.0 ± 0.9 h/day decreasing to 3.6 ± 0.9 h/day).Statistically significant differences in the mean amount of time spent in sedentary activities between the mean baseline to the end of the 4-week intervention were observed across the domains of work (t (17) = −4.7, p < 0.001, d = 0.9) (1.2 ± 0.7 h/day increasing to 2.1 ± 1 h/day); transportation (t (17) = −11.8, p < 0.001 d = 2.2) (0.20 ± 0.1 h/day increasing to 0.42 ± 0.1 h/day); and leisure time (t (17) = −5.1, p < 0.001, d = 3.2) (0.9 ± 0.7 h/day increasing to 2.1 ± 1 h/day). There were no significant differences between baseline and the end of the 4-week intervention for sedentary activities (t (17) = −1.6, p = 0.116) (2.1 ± 2 h/day increasing to 2.5 ± 2 h/day) or light-intensity physical activity (t (17) = 1.2, p = 0.215) (0.46 ± 0.2 h/day decreasing to 0.41 ± 1 h/day) in the household activities domain  [Currently, there are few intervention studies dedicated to reducing sedentary behavior that use objective methods for quantifying the changes induced. To some extent, the results from this study are consistent with recent research, although differences exist regarding sample demographics, intervention objectives and measurement instruments. For example, one study examined the efficacy of 4-week intervention by breaking up prolonged periods of sedentary behavior time with brief physical activity breaks . Thirty overweight and obese adults were regularly prompted via an Android smartphone (GH-T759) . The resGH-T759) . Time spGH-T759) . Adams aGH-T759)  recentlyp < 0.001). The medium effect size of this pre/post difference in a sedentary adult sample is surprising, given that the personal activity monitor was developed to promote daily non-exercise activity thermogenesis (NEAT) activities, which are composed mainly of energy expenditures related to daily physical activity of light-to-moderate intensity [Participants in the current study replaced the time spent in sedentary activities by significantly increasing light and moderate physical activity. Unexpectedly, our investigation found a significant increase in time spent in vigorous physical activity between baseline and the end of the 4-week intervention  is the most frequent evening activity among adults [While the amount of time spent in sedentary activities decreased and light-intensity physical activity increased, it is important to consider pre-post changes that occurred across the specific SLIPA-determined daily life domains , as these are likely to differ in terms of control over behavioral change ,40. The g adults ,42.The main limitation of the study is the limited generalizability due to a small sample size that was primarily comprised of younger adult participants. Although the Gruve monitor has been validated in laboratory conditions for sitting, standing and walking, it has not been fully validated in “free living” conditions. A further limitation will be the threat to the internal validity which pre-test post-test designs invoke. Such a design is helpful, however, with future hypothesis generation to assist in determining whether a more rigorous study design is warranted. Participants were asked to charge the monitor battery during sleep. The monitor battery needed to be recharged every 2–3 days, and a full charge normally took between 2 and 4 h. Therefore, if the participants were to charge the battery during waking time, this event could lead to an unintentional, yet additional, 2–4 h in the amount of time spent in sedentary activities being recorded. This study has several strengths. It is the first intervention study which has reported such a large and significant decrease in sedentary activities among sedentary adults during free-living activities using the Gruve monitor as an intervention tool. As part of the inclusion criteria, the participants’ sedentary behavior was determined via the self-report (using the IPAQ), thus they were engaged in high volumes of sedentary behavior before participating in this study. Consequently, we were confident that our participants met the most recent definitions of sedentary behaviors . In addiThis lifestyle study involving sedentary adults suggests that, when engaging with the online personal activity monitor, individuals decreased (by 33%) the amount of time they spent in sedentary activities. No previously published research has found such a large reduction in the amount of time spent in sedentary activities. Furthermore, participants increased their daily light- and moderate-intensity physical activity and energy expenditure. In addition, due to the time stamp on the Gruve output, it was evident the increased physical activity and reduction in sedentary time were most likely to occur within the daily leisure time domain. Future investigations should focus on determining whether this intervention has a sustainable impact in sedentary behavior and related health outcomes.