Methanoculleus sp002497965 and JABUEY01 sp013314815 (a member of the Syntrophobacteraceae family). The relative abundance of JABUEY01 sp013314815 was 60 times higher in bioaugmented bioreactors compared to their unaugmented counterparts after recovery from organic overloading. Genomic analysis of JABUEY01 sp013314815 revealed its metabolic potential for syntrophic propionate degradation when partnered with hydrogenotrophic methanogens  via the methylmalonyl-CoA pathway. Our results identified at least two key species that are responsible for efficient propionate removal and demonstrate their potential applications as microbial cocktails for stable AD operation.Propionic acid (HPr) is frequently accumulated in anaerobic digesters due to its thermodynamically unfavorable degradation reaction. Here, we identify key players in HPr oxidation and organic overloading recovery from metagenome-assembled genomes (MAGs) recovered from anaerobic digesters inoculated with HPr-enriched microbial consortia before initiating organic overloading. Two independent HPr-enrichment cultures commonly selected two uncultured microorganisms represented with high relative abundance:  Among VFAs, propionic acid (HPr) is especially recalcitrant to AD because the oxidation of HPr to H2 is thermodynamically unfavorable under standard conditions  is widely used to treat various organic pollutants from wastewater because of its ability to mineralize organic matter whilst simultaneously generating energy for diverse microbial communities. Several different groups of prokaryotes carry out four metabolic steps in AD. Briefly, hydrolytic and acidogenic bacteria break down complex organic molecules into volatile fatty acids , dihydrogen  and Desulfotomaculum have been reported to use the methylmalonyl-CoA pathway, whereas only the bacterium Smithella propionica, also from the Desulfobacterota phylum, is known to use the C6 dismutation pathway  among several different biological and non-biological supplementation strategies  representing >83% of the total microbial communities in our anaerobic digesters. Our analysis of these MAGs reveals novel microbes and their metabolic potential for pathways directly responsible for the efficient removal of HPr and concomitant methane production after the anaerobic digesters received an organic shock.1) and 126 days  using glucose  at S1, respectively. Total seven samples for DNA extraction were taken from each digester at either 40 days after S1 (designated as ‘_S1’) or 30d after S2 (‘_S3’), which were considered to be the time points of complete recovery from the organic overloads. These samples were divided into four groups: control (1-no_shock_S1), group A (2-no_aug_S1 and 3-JJ_aug_S1), group B (4-aug_S1 and 5-sug_S3) and HPr-enriched cultures (7-NCT and 8-LJY) for metagenomic analysis . Matches to the references were filtered for best matching, using 40% identity and 70% sequence coverage over the alignment length as threshold values. To pinpoint the phylogenetic placement of ANIsp_017_JABUEY01 sp013314815 and the genomes of known syntrophic propionate degraders in methanogenic environment, we performed phylogenetic analyses for them. The genome sequences of known syntrophic propionate oxidizing bacteria were collected based on the list in the recently published review article  and 5 g COD/L (S2), respectively, for two different time points, using glucose  shared with its best match against GTDB. Notably, all 200 MAGs together recruited an average of 92.2 ± 7.3% of the total reads in each metagenome (via Nonpareil.We obtained 47–54 million paired-ended reads after trimming per dataset (average read length ranged between 130 and 132 bp). Notably, the estimated abundance-weighted average coverage calculated by Nonpareil, an algorithm that examines the extent of overlapping reads within a dataset to determine the coverage and diversity, ranged between 89.6–96% , suggesttagenome , revealiR2 value of 0.87657 for conditions and a value of p of <0.01, PERMANOVA). It should be noted that our sample size for each condition is small and unbalanced. Thus, the differences observed between conditions need to be further corroborated with more samples in the future.Whole-metagenome comparisons based on MASH distances  suggested a clear separation of microbial communities in group A  from those of group B  in Western Siberia, Russia . This laboratory anaerobic digester had previously been inoculated with a mixture of samples collected from twelve full-scale anaerobic digesters treating food wastewater and/or sewage sludge . Strikinddy soil , it has Syntrophobacter fumaroxidans (pct). Nevertheless, at least partial sets of three CoA transferases were found in these MAGs, and a previous study detected several CoA transferases besides Pct in proteomics data obtained from a co-culture of S. fumaroxidans in association with several different methanogens  grown with 30 mM of propionate  demonstrating that the same strain of JABUEY01 sp013314815 was present in both the bioaugmented  and HPr-enrichment systems. Phylogenetic placement of single-copy genes alignmentof ANIsp_017_JABUEY01 sp013314815 and other known propionate degraders in methanogenic systems suggested that ANIsp_017_JABUEY01 sp013314815 clustered with Syntrophobacter fumaroxidans  that were recovered from both group B samples , the relative abundance of ANIsp_017_JABUEY01 sp013314815 in group B was 60× higher than in group A . Thus, maintaining a higher relative abundance of ANIsp_017_JABUEY01 sp013314815 in the AD systems enhanced the recovery rate in our organic overloaded AD processes. This effect was so pronounced that we observed double the rate of HPr removal and recovery from organic overload in the group B bioreactors that were supplemented with our putative syntrophic HPr oxidizers ANIsp_017_JABUEY01 sp013314815 and ANIsp_003_Methanoculleus , ANIsp_021_79-D21 sp000402835 (unclassified species of Synergistetes for NCBI classification), and ANIsp_040_Mesotoga prima (unclassified species of Mesotoga for NCBI classification), showed >1% of relative abundance after organic overloading in all group B samples  but present in all group B samples , and 2 were not present in any of group B samples but were present in all group A samples . This re samples .Methanosaeta harundinacea is an acetoclastic methanogen that can digest HAc to yield CH4  and its exclusive presence in group B AD samples at >1% relative abundance, this species might play an important role in the elevated HAc degradation rate observed in group B treatments compared to group A. The increased HAc degradation rate also plays a role in the enhanced recovery rate we observed after organic overloading (Synergistetes for NCBI classification) showed a similar relative abundance pattern to that of ANIsp_019_Methanothrix_A harundinacea_D . The genome of this species was recovered from a methanogenic bioreactor degrading terephthalate , and Desulfoscipio gibsoniae butyric acid oxidation genes but were able to find only a few genes in both pathways suggesting this organism performs other functions than HPr and butyric acid oxidation in the AD processes in response to organic overloading. It cannot be excluded, however, that genes from uncharacterized organisms mediated HPr and butyric acid oxidation pathways. Mesotoga prima was reported to efficiently ferment carbohydrates to acetic acid  and was only present in another HPr-enrichment  with 0.24% of relative abundance, its relative abundance in all group B samples was higher than 1% and absent in all group A samples. This result suggested that this species may be associated with the fermentation of glucose or other carbohydrates in our AD processes. This hypothesis will require further testing in future experiments.ield CH4 . Given trloading . ANIsp_0hthalate  but thertic acid . While ASyntrophobacteraceae and hydrogenotrophic/acetoclastic methanogens) that have contributed to the enhanced HPr oxidation, and therefore rapid recovery, rates observed in the bioaugmented systems. We propose that these novel organisms would make for an effective microbial cocktail that can be added during the start-up, organic-overload, and unstable operation phases of AD processes. Furthermore, we found that all of our AD systems together with two HPr-enrichments contain ANIsp_002_Cloacimonas_acidaminovorans (Candidatus Cloacimonas acidaminovorans), which are known to be syntrophic HPr oxidizing bacteria  and methanogens  would be required to find the best consortia for various AD systems. Lastly, our results and datasets in this manuscript would serve as a community resource because these contain a total of 200 MAGs representing 83 genomospecies from AD systems and 35 of them represent previously unknown species.Key microorganisms that are responsible for HPr oxidation were recovered as MAGs from organic overloaded anaerobic digesters and HPr-oxidizing enrichments. Our results suggested at least three vital players  or in only one of these replicates. Even if it is the latter case, there is a great potential for the key players to be an effective microbial cocktail because we observed not only higher abundance of the key players  after the recovery from two different organic overloadings  compared to control and group A samples, but also their very high relative abundance in two different HPr enrichment cultures. Further, we only have a limited number of samples to conduct proper statistical analyses . Future research with more samples is needed to reveal the specific roles of the key players and their interaction with native microbial community members in recovery from organic overloaded ADs.https://dataview.ncbi.nlm.nih.gov/object/PRJNA818325?reviewer=ct8o10fce7o8atd403pdgio1hg PRJNA818325. The MAG sequences recovered in this study are available under GenBank accession numbers JALHCI000000000-JALHJZ000000000.All metagenome sequences and biosamples for MAGs used in this study are available in NCBI, under BioProject number MK: investigation, methodology, formal analysis, and writing – original draft. CR and JS: investigation, and writing – review and editing. MW: writing – review and editing. JL: resources, and writing – review and editing. SS: conceptualization, supervision, funding acquisition, and writing – review and editing. All authors contributed to the article and approved the submitted version.This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government . This work was also supported by Korea Environment Industry & Technology Institute (KEITI) through its Ecological Imitation-based Environmental Pollution Management Technology funded by Korea Ministry of Environment .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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The current study presents evidence on the relative importance of dietary patterns and vitamin D supplementation in maintaining sufficient vitamin D and iron in pregnancy. Antenatal nutrition counselling services should be provided to pregnant women who show signs of inadequate dietary intake.Vitamin D is essential for human health. However, it is not clear if vitamin D supplementation is necessary for all pregnant women. This study examines the relative importance of dietary patterns and vitamin D supplementation frequency in determining serum 25-hydroxyvitamin D (25(OH)D) and ferritin concentrations among pregnant women in Hong Kong, China. A total of 572 healthy women were recruited from antenatal clinics at 25–35 weeks pregnant. Participants completed an electronic version of the food frequency questionnaire and a web questionnaire on supplement use. Their blood samples were tested for serum 25(OH)D and ferritin. The associations of dietary patterns and vitamin D supplementation frequency with serum 25(OH)D and ferritin concentrations were analyzed using moderated hierarchical regression. Two dietary patterns were identified. The adequate dietary intake was characterized by the high probability of meeting recommended daily food group servings, whereas the inadequate dietary intake was characterized by inadequate consumption of vegetables, fruits, meat, fish, and eggs, or alternatives. The association between adequate dietary intake and serum ferritin concentrations was independent of vitamin D supplementation frequency (β = 0.05,  Vitamin D is a steroid hormone that helps to maintain calcium homeostasis and facilitates bone mineralization in the body. Vitamin D deficiency during pregnancy can cause bone loss, preeclampsia, preterm delivery, and low birth weight . Iron-dePrevious research has shown no meaningful differences in iron status improvement among women receiving different doses of vitamin D supplementation during gestation  . While sHong Kong provides a good setting for research to examine the relative effects of diet and supplementation on vitamin D concentrations because of its small changes in sunlight intensity between months and sunlight exposure duration between individuals. Thus, when the research is conducted in Hong Kong, the effects of sunlight exposure on vitamin D status can be minimized. Furthermore, given the nutritional benefits of natural foods and the amount of nutrients needed during pregnancy, the dietary intake recommended by Hong Kong’s local health agencies for pregnant women who are in the second and third trimesters (14th to 40th week), have a normal body mass index, and weigh between 45 and 60 kg before pregnancy is characterized by five food groups including grains (3.5–5 servings per day); vegetables (4–5 servings per day); fruits (2–3 servings per days); meat, fish, eggs, or alternatives (5–7 servings per day); and dairy or alternatives (2 servings per days) . Local rAll women making a visit to the antenatal clinic of local public hospitals during the period of July 2019 to December 2020 were invited to participate if they were healthy, Hong Kong Chinese citizens, aged 18 or above, at 25 to 35 weeks gestation, residing in Hong Kong, and literate in Chinese.Data collection took place in the waiting area of the antenatal clinic. Upon providing written informed consent, participants completed demographic questionnaires including questions about the weekly frequency of vitamin D supplementation during pregnancy. The electronic version of the Food Frequency Questionnaire (eFFQ) was also administered. In addition, their peripheral blood samples were collected by trained phlebotomists. As a token of appreciation for their participation in this study, an incentive of HKD 50 supermarket voucher was offered to the participants at the end of the session. The research protocol was approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster Research Ethics Committee (UW 13-055).Participants completed the eFFQ to report their food intake over the past month. Details of the eFFQ development can be found in the previous publication . BrieflySerum was extracted from the collected peripheral blood samples. In this study, the liquid chromatography-tandem mass spectrometry (LC-tandem MS) method was adopted to determine serum 25(OH)D concentration, defined as the sum of 25(OH)D3 and 25(OH)D2 minus 3-Epi-25(OH)D3, using the QTRAP 5500 LC-MS/MS system . The method has been verified against samples from the Vitamin D External Quality Assessment Scheme with satisfactory performance  . Blood sParticipants were asked, “how often do you take vitamin D supplements over the course of your current pregnancy?”. The answer options provided included “everyday”, “6–4 times per week”, “1–3 times per week”, “less than 1 time per week”, and “never”. Participants who selected “everyday”, “6–4 times per week”, and “1–3 times per week” were grouped as the “at least 1 time per week” subgroup, whereas those who selected “less than 1 time per week” and “never” were grouped as the “less than 1 time per week” subgroup.Demographics, including chronological age, gestational age, gravidity, parity, marital and employment status at enrollment, highest education level, monthly family income, and history of chronic diseases, were self-reported by the participants.t-tests (for continuous variables) and chi-square analyses  were performed to determine whether demographics and pregnancy characteristics, including vitamin D supplementation patterns and serum 25(OH)D and ferritin concentrations, differed between the dietary pattern groups. Data of ferritin were normalized using log transformation. There were no missing values on any of the key variables. Assumptions of linear regression including normality and linearity were assessed and verified graphically. There was no evidence of outliers and multicollinearity between variables. Latent class analysis (LCA) was first applied to identify latent subgroups of pregnant women with similar dietary patterns as indicated by the dichotomous status (sufficient/insufficient) of the three food group parameters using the R package “poLCA”  [p-values < 0.05 denoting statistical significance.All data were analyzed using both the SPSS statistical software package  and the R statistical software . Descriptive statistics were calculated to summarize the characteristics of participants and their families. A series of independent Austria) . Simple Austria) . Lower vAustria) . The firp = 0.01), with a higher proportion of pregnant women in the inadequate dietary intake subgroup (27.5%) taking supplements less than once per week compared to those in the adequate dietary intake subgroup (18.6%). The number of participants with vitamin D deficiency (<50 nmol/L) was low in both subgroups (inadequate: 21 (7.6%); adequate: 14 (4.7%)).The overall sample consisted of 572 women (average age at assessment: 34 years) at 26 weeks gestation on average. Models with one through four latent classes were compared in order to select a model of multiple food groups. r = 0.14, p < 0.01). p = 0.03). This association remained significant after further adjusting for vitamin supplementation frequency . No significant interaction between vitamin D supplementation frequency and dietary patterns on serum ferritin concentrations was found. On the other hand, although serum 25(OH)D concentrations had no association with dietary patterns, its association with vitamin D supplementation frequency remained significant after adjusting for dietary patterns, with lower serum 25(OH)D concentrations observed in those who took vitamin D supplements less than once a week compared to the more frequent supplementation group . The results of moderation analysis showed that only the interaction between vitamin D supplementation frequency and dietary patterns was significant . As illustrated in Serum 25(OH)D and ferritin concentrations had a small yet statistically significant correlation with each other , whereas the inadequate dietary subgroup had a high probability of not meeting the recommendations. A previous study of 285 healthy pregnant women in Ireland also identified two dietary patterns . The unhGiven that previous research has focused on either food intake or supplementation but not both, this study expands the scope of previous research by demonstrating a link between food preferences and use of vitamin supplements during pregnancy. We found that although the adequate and inadequate dietary intake subgroups had similar demographic backgrounds, the adequate dietary intake subgroup was more likely to have regular vitamin D supplementation  than the inadequate dietary intake subgroup. This finding can be explained partly by factors such as motivation to take supplements and knowledge about healthy diets, all of which can influence supplementation frequency. Dietary supplements are intended to supplement the diets of people who eat insufficiently. However, people who eat insufficiently tend to have low health consciousness. It has been posited that pregnancy is a time when women have increased motivation to make dietary improvements . ResearcIn addition, we found that the inadequate dietary intake subgroup had lower serum ferritin concentrations compared to the adequate dietary intake subgroup, after adjusting for their vitamin D supplementation frequency status. The findings are consistent with previous studies reporting that vitamin D supplementation cannot improve serum ferritin concentrations in pregnant women ,6,7. In This study has several limitations. First, while the participants were recruited from different Hong Kong local hospitals, they were not fully representative of Hong Kong’s pregnant women. However, the cluster analysis identified two latent subgroups which are largely consistent with previous research on this topic. Second, the number of participants who were vitamin D deficient in our sample was small, and other lifestyle factors  were not assessed in this study. The findings thus may not be applicable to special subgroups such as those who lack vitamin D due to genetic factors or those who smoke. Third, dietary patterns and vitamin D supplementation frequency were self-reported by participants using questionnaires. Their reports may have recall bias. Fourth, only vitamin D supplementation frequency was examined. Future research should assess the dosage and frequency of other supplements such as multivitamins and iron supplements. In addition, more studies should be conducted to identify the best dietary composition for achieving the daily recommended dietary intake of iron and vitamin D.This study expands the current understanding of vitamin D supplementation use by showing the relative importance of dietary patterns and vitamin D supplementation frequency for pregnant women. It also replicates previous research demonstrating that dietary sources are more important than vitamin D supplementation for promoting ferritin expression. The findings are particularly useful for allocation of limited health care resources. They point to the need of simple dietary screening in early pregnancy and targeted nutrition counseling and education services at antenatal clinics to improve the dietary knowledge and behavior of at-risk pregnant women such as those who have inadequate dietary intake. More studies are needed to examine the misconceptions of food safety and nutrition in these women and their behavioral barriers to meeting dietary recommendations in pregnancy.


Spodoptera exempta Walker) using antibiotics and field-collected frass, in an attempt to understand the interactions of the gut microbiome with the nucleopolyhedrovirus, SpexNPV. We found that S. exempta individuals with supplemented gut microbiome were significantly more resistant to SpexNPV, relative to those with a typical laboratory gut microbiome. Illumina MiSeq sequencing revealed the bacterial phyla in the S. exempta gut belonged to 28 different classes. Individuals with an increased abundance of Lactobacillales had a higher probability of surviving viral infection. In contrast, there was an increased abundance of Enterobacteriales and Pseudomonadales in individuals dying from viral infection, corresponding with decreased abundance of these two Orders in surviving caterpillars, suggesting a potential role for them in modulating the interaction between the host and its pathogen. These results have important implications for laboratory studies testing biopesticides.Gut microbiota stimulates the immune system and inhibits pathogens, and thus, it is critical for disease prevention. Probiotics represent an effective alternative to antibiotics used for the therapy and prevention of bacterial diseases. Probiotic bacteria are commonly used in vertebrates, although their use in invertebrates is still rare. We manipulated the gut microbiome of the African Armyworm ( Our study examines how lab strains of a widespread insect pest species (the African Armyworm) have a distinct gut microbiome and the role that gut microbes play in affecting resistance to nucleopolyhedrovirus, SpexNPV. Through a combination of antiobiotic control and field-collected frass innoculations, we demonstrate that inoculation of the field microbiome of this pest species into lab strains increases resistance to the virus, and that this is subsequently reversed when treated with a broad spectrum antibiotic. As we work to introduce more ecological principles into immunology, natural phenomena such as multiple infections, host fitness trade-offs, and interactions with microbial symbionts are shaping how we study the interactions among hosts, microbial symbionts, and pathogens within a ‘hologenome’ concept  offer a robust model system for studying the impact of the gut microbiome on pathogen susceptibility. Spodoptera exempta is a major crop pest of sub-Saharan Africa. It is highly migratory and over multiple generations during a single outbreak season can travel thousands of kilometres    and cockroaches have examined how diet impacts the gut microbiome, and that this has downstream impacts on antimicrobial peptide generation—an important factor in pathogen resistance  Can we manipulate the insect gut microbiome composition through diet? (ii) Is susceptibility to viral infection related to the diversity of gut microflora? (iii) Are specific members of the gut microbiome responsible for a defensive symbiosis, or is it due to a complex microbial community?Spodoptera exempta were maintained on a semi‐synthetic wheatgerm‐based diet that included a broad spectrum antibiotic (streptomycin 1.1 mg g−1 diet) to reduce bacterial contamination of the diet . A further 50 genetically distinct pupae were selected to begin a sub-culture maintained according to the standard laboratory diet (hereafter defined as the ‘lab line’). Within these sub-cultures, 100 adult moths were paired at the end of each generation to maintain genetic diversity within each subculture.A colony of probiotic line was reared on the same semi-synthetic diet, but with the antibiotic removed, instead replaced with frass (40 mg g−1 diet) from field-collected S. exempta caterpillars fed on grass and maize leaves in Tanzania. Caterpillars were collected from infested fields, taken to the field-station, and fed fresh vegetation; fresh frass was then collected and immediately refrigerated until suspension for use in experiments. The frass was added to 200 ml 1x phosphate buffer solution (pH 7.4) and placed in a shaking incubator for 10 min before being added to the diet. This culture was maintained for two generations, after which we confirmed restoration of the gut microbiome through Illumina MiSeq sequencing of the 16 s rRNA bacterial gene (see below). A subsample of ‘wild’ frass was used in a sequencing run also using the 16 s rRNA gene. The lab and probiotic lines were synchronized according to egg-lay date and larval emergence date for viral bioassays (see below). Each generation of larvae used in bioassays used third instar (L3) larvae selected equally from across the genetically distinct lines within each sub-culture  and probiotic line (n = 480), with controls (i.e. sterile sugar solution as above) for each group (n = 96). Each bioassay was replicated three times.To provide an LD80 dosage (predicted to kill 80% of larvae), we used a dosage of 2.5*10³ OBs  for each individual. Following the initial virus exposure, handling deaths were discounted and viral/fungal/bacterial deaths were confirmed and counted over each 24 h period until day 8 (D8) after virus exposure. On D14, all remaining survivors were killed for microbial community analysis. Each bioassay was performed using two treatment groups: lab line fed on their semi-artificial diet without antibiotics was bioassayed with the same virus dosage. To control for potential genetic selection effects in the host, a further virus bioassay was performed on the probiotic line, wherein it was crossed back onto the lab line semi-artificial diet with supplementary dosages of antibiotics (n = 480). Finally, development time is impacted by the gut microbiome  of the et al. Larvae were surface cleansed using Triton-X, then total gut content  was removed. Microbial DNA was extracted from each of the 240 caterpillar gut samples across each of the treatment groups Table  using thet al. Bacterial 16S rRNA genes were partially amplified by PCR using primer pair 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1391R (5′-GACGGGCGGTGWGTRCA-3′) (Weisburg in-house using Nanodrop (Sigma Aldrich), then frozen and shipped to the Earlham Institute  for downstream processing on Illumina MiSeq. From here, the amplicons from the first PCR were quantified using a Quant-iT™ dsDNA Assay Kit (Thermo Fisher Scientific Q33120). A second PCR was performed with the Kapa HiFi HotStart PCR kit (Roche Diagnostics 7958897001) in 50ul reactions with 20 ng of the amplicon from the first PCR, and 5ul each of an i5 and i7 Nextera XT Index kit v2 (Illumina FC-131–2001) indexed primer. After 7 cycles of PCR, the PCR products were purified with a 1x Agencourt AMPure XP bead clean up (Beckman Coulter A63882) with two 80% EtOH washes and resuspended in 25 µl of elution buffer (10 mM Tris).The targeted amplicons based on primer pair 27F-1391R were quantified The libraries were quantified using the Qubit dsDNA HS Assay Kit and sized on a PerkinElmer GX using the High Sensitivity DNA chip (PerkinElmer CLS760672). Libraries were equimolar pooled and the resulting pool was quantified by qPCR using a Kapa Library Quantification Kit (Roche Diagnostics 7960204001). The pool was diluted to 2 nM and denatured using 2 N NaOH before diluting to 20pM with Illumina HT1 buffer. The denatured pool was loaded on an Illumina MiSeq Sequencer with a 600 cycle MiSeq reagent kit v3 (Illumina MS-102–3003) at 70% loading concentration with a 20% phiX control v3 spike (Illumina FC-110-3001) as per Illumina's recommendations for low diversity amplicon sequencing.et al. et al. et al. Data were analysed in in accordance with Qiime2 (Guerrini http://trace.ncbi.nlm.nih.gov/Traces/sra) under submission SUB9585236.Sequences derived from Illumina MiSeq amplicon sequencing were deposited on the NCBI Sequence Read Archive . Variation in host response to viral infection and potential effects of gut microbial supplementation on host susceptibility to viral challenge were analysed using survival analysis  in the survival package  were also analysed with treatment group using generalized linear models (glm).Non-metric multidimensional scaling (NMDS) was used to analyse correlations between host microbial communities and host responses to viral infection and based on variation in the abundances of all members of the community  of individual members of the host gut microbiome were then analysed for potential direct roles in the host viral response using glm with (quasi-) Poisson error structure. Response variables were the read counts of microbial taxa determined by sequencing. Explanatory variables included in maximal models were: culture (lab/probiotic), viral dosage (LD80/control), antibiotic (Yes/No) and viral death (dead/survived). Extended results of all glms are presented in Sequencing of amplified DNA from caterpillar gut contents generated 9332574 raw reads with an average read length of 299 bp . Post filtering, 9109934 reads were clustered to 348 distinct OTUs. For taxonomic classification and comparison, these reads were binned into their respective treatment groups , comprising on average five classes  with the top five most abundant being Bacilli, Gammaproteobacteria, Actinobacteria, Betaproteobacteria, Alphaproteobacteria , comprising on average five classes per individual  and lacking eight classes present in the probiotic line  with the top 5 most abundant orders being Lactobacillales, Bacillales, Enterobacteriales, Actinomycetales, Pseudomonadales, and Burkholderiales .OTUs from all treatment groups were clustered into 52 bacterial orders, with six representing more than 92.6% of all the orders. The les Fig. . The labActinobacteria (0.27%) and Gammaproteobacteria (0.70%). Within the dominant class Bacilli, these comprised 18 orders, primarily the Lactobacillales (98.58%). Data on bacterial family and genera distributions are available in A subsample of ‘wild’-type faeces accounted for 49651 of the post filtering reads. These reads were clustered into 8 bacterial classes, predominately the Bacilli (98.68%), lab line with the probiotic line allows us first to determine the efficacy of our attempted microbial manipulations. NMDS showed that a three-dimensional solution was sufficient to achieve low stress values to enable us to interpret gut community composition .Comparing the probiotic line and lab line forming highly distinct microbial communities, clustering in significantly different groups in the NMDS plot , and Simpson indices , were significantly different between the probiotic and lab treatment groups, but species number (or richness) was not .NMDS community composition clusters were significantly correlated with treatment group, with the P = 0.005, Fig. 1,164 = 3.648, P = 0.057), but not for Shannon  or Simpson indices .Treatment groups receiving virus that survived the bioassays were significantly different from those that did not , and Simpson indices , were significantly different between the antibiotic treatment groups, but species number was not .The Bacillales, Lactobacillales, Enterobacteriales, and Pseudomonadales. Combined, these orders accounted for 91% of sequence reads across the data set.Four bacterial orders were the focus of statistical analysis by generalized linear models: probiotic line was significantly enriched with Bacillales in comparison to the lab line .The Limited significant effects were found with other orders of the gut microbiome within this experiment; full statistical results can be found in lab line , than in the probiotic line , suggesting a potential protective effect of the probiotic supplementation in this infection system .The negative controls  were not significantly different from each other, and showed zero non-viral deaths (Spodoptera exempta (SpexNPV) as a model system for the interaction between gut microbiome and viral infections. Through manipulating a standardized artificial diet with the addition of field-collected frass to supplement and a broad-spectrum antibiotic (streptomycin) to reduce diversity, we successfully demonstrated the ability to manipulate and study the interaction effects of gut microbiome on a commonly occurring baculovirus of a key crop pest. Specifically, we identified that the ‘wild’-type frass was primarily dominated by members of the Lactobacillales , whereas the lab-type larvae were dominated by Pseudomonadales (most commonly Pseudomonas sp.). Upon receiving treatment in the probiotic line, the gut community of S. exempta shifted away from Enterobactereales dominance to Lactobacillales. Using standardized viral bioassay techniques, we further demonstrate that this shift results in an increased resistance to SpexNPV.We designed this study to examine the interactions between host gut microbial symbionts and the nucleopolyhedrovirus of Pseudomonadales and Enterobactereales) were associated with higher viral susceptibility, suggesting possible interactions between bacteria, virus and host either increasing virulence, reducing host fitness, or co-infecting the host.We showed that an increased diversity of the gut microbiome is linked with lower viral susceptibility. This effect was consistently detected across treatment groups, and notably was lost when the gut microbiome was re-treated with a broad-spectrum antibiotic. Increased abundances of certain members of the gut microbiome .The results from our present study suggest that individual S. exempta, evidence from other invertebrate studies suggest that it is far more likely that the combined community present in the gut may have a more important role. For example, in honey bees, eleven cultured bacterial phylotypes differentially inhibit the growth of the bacterial pathogen Paenibacillius larvae in vitro, but only the microbial cocktail of all 11 bacterial phylotypes completely inhibits the growth of P. larvae in vitro and in vivo  is altered following parasitism by the tapeworm Hymenolepis diminuta  that protect them against fungal pathogens and parasitoid wasps  is an important and novel result that will have widespread impacts on viral pathogen studies. These findings may go some way towards explaining widespread result differentials between lab experiments and field trials of biopesticides (Darriet et al. Pseudomonadales and the Enterobactereales on the viral bioassays may have substantial potential as a ‘cocktail’ biopesticide.Our results demonstrate the ability to alter the gut microbiome of an insect crop pest, and the significant impacts of this on the outcome of a viral bioassay. Widespread application of SpexNPV as a biopesticide could provide a viable alternative to chemical control for armyworm control in Africa (Grzywacz fiac147_Supplemental_FilesClick here for additional data file.


Cardiovascular disease (CVD) risk prediction models for individuals with type 2 diabetes are important tools to guide intensification of interventions for CVD prevention. We aimed to assess the added value of incorporating risk factors variability in CVD risk prediction for people with type 2 diabetes.1c). Such models were compared against simpler models using single last observed values or means.We used electronic health records (EHRs) data from 83 910 adults with type 2 diabetes but without pre-existing CVD from the UK Clinical Practice Research Datalink for 2004–2017. Using a landmark-modelling approach, we developed and validated sex-specific Cox models, incorporating conventional predictors and trajectories plus variability of systolic blood pressure (SBP), total and high-density lipoprotein (HDL) cholesterol, and glycated haemoglobin (HbA1c were associated with higher CVD risk (P < 0.05). Models incorporating trajectories and variability of continuous predictors demonstrated improvement in risk discrimination  as compared with using last observed values  or means . Inclusion of SDs of SBP yielded the greatest improvement in discrimination  in comparison to incorporating SDs of total cholesterol , HbA1c  or HDL cholesterol .The standard deviations (SDs) of SBP, HDL cholesterol and HbAIncorporating variability of predictors from EHRs provides a modest improvement in CVD risk discrimination for individuals with type 2 diabetes. Given that repeat measures are readily available in EHRs especially for regularly monitored patients with diabetes, this improvement could easily be achieved. Key MessagesCardiovascular disease (CVD) risk prediction models for individuals with type 2 diabetes are important tools to guide intensification of interventions for CVD prevention and diabetes management. However, most models use single measurements of risk predictors.Greater variability in risk predictors is associated with higher CVD risk, independently of the factors predictors themselves, and may be considered as additional components in risk assessment models for individuals with diabetes.We assessed the added value of incorporating within-person variability of repeatedly measured predictors into CVD risk prediction models for people with type 2 diabetes. These individuals are regularly monitored in primary care and have more repeated measurements than the general population.We derived and validated CVD risk prediction models on 83 910 individuals with type 2 diabetes from a large population-representative electronic health records database.Incorporating variability of repeat predictors of systolic blood pressure, total and high-density lipoprotein cholesterol, and glycated haemoglobin by using standard deviations provides a modest improvement in CVD risk discrimination for individuals with type 2 diabetes.,1c)] is associated with increased CVD risk, independently of the factors themselves,The prevalence of co-morbidities amongst people with type 2 diabetes is increasing in the UKThe benefits of using electronic health records (EHRs) for CVD risk assessment and subsequent personalised healthcare decisions are well recognised.The Clinical Practice Research Datalink (CPRD) is a longitudinal primary care database of anonymised EHRs from the UK general practitioners. It covers approximately 6.9% of the UK population and is representative of the UK primary care setting with respect to age, sex, and ethnicity structure.For this proposed analysis, individuals entered the study on the latest of following dates: the date of 6 months after registration at the general practice; the date the individual turned 40 years old ; the date that the data for the practice were up to standard; IJE online) at study entry or during follow-up but without prevalent CVD events at study entry. We further excluded people who experienced incident CVD events during follow-up prior to the diagnosis of diabetes. Since our primary aim was to compare models using single vs repeated predictors and their variability, we restricted our study population to those with complete information on risk predictor variables (listed below). Thus, the analysis data set consisted of 83 910 individuals (flowchart in IJE online). We randomly split our data by practice to a 2/3 derivation data set  and a 1/3 validation data set .Among the 2 589 074 individuals with linked data during study entry and exit dates, we identified 159 730 individuals with confirmed diagnosed type 2 diabetes (diagnosis codesIJE online).The outcome of interest was incident CVD, where CVD was defined as fatal or non-fatal coronary heart disease , stroke or transient ischaemic attack. The definition matched the QRISK algorithm,,,,1c (for which details of measurements have been previously describedIJE online).Based on the most commonly used predictor variables in existing CVD risk prediction tools among people with diabetes,1c by either the last observed values, the cumulative crude means or their estimated ‘current’ values  and ‘individual-level slope’ (i.e. the degree of change in the risk predictor over time) calculated from age-specific and sex-specific multivariate mixed-effects linear regression models with fixed and random age-varying effects (details in IJE online). The standard deviations (SDs) of these risk factors were further calculated to quantify the within-person variability among individuals with at least two prior measurements. Duration of diabetes was defined as the time since age at diabetes diagnosis  to each landmark age; smoking status was defined from the last observed values at each landmark age; blood pressure-lowering medication use and atrial fibrillation were defined as ever having a related prescription or diagnosis record before the landmark age.We used a landmark approach,1c, and smoking status in the Cox model. The proportional hazards assumption was tested by examining the Schoenfeld residuals and we did not observe a clear indication of a violation of the assumption in our models. Estimated hazard ratios and the baseline hazard functions from the Cox model were then applied to the 1/3 validation data set to estimate 10-year CVD risk.In Stage 2, sex-specific ‘super landmark’ Cox models,IJE online) provides details of these performance evaluation metrics.In the validation data set, we compared the predictive performance of the Cox models using last observed values, mean values or estimated current values from multivariate mixed-effects models, with and without individual-level slopes and SDs of risk factors. The predictive performance for models with SDs of risk was assessed in subgroups of individuals with at least 2, 3, 4, …, ≥10 measurements of each predictor. Discrimination was compared using Harrell’s C-index; 1c or smoking status (IJE online).In sensitivity analyses, we refitted the multivariate mixed-effects linear regression models and super landmark models amongst 143 466 individuals who had at least one measurement of any risk factors of SBP, total cholesterol, HDL cholesterol, HbAP-value of <0.05. This study follows the TRIPOD reporting guideline (IJE online).All statistical analyses were conducted using Stata version 15.1  and R version 3.6.1 . Statistical significance was defined using a two-sided IJE online). Individuals with complete data included in our analysis were slightly older, more likely to be male and had lower CVD incidence rate than those with missing risk predictor measurements  (IJE online). A large proportion (83%) of people had at least two prior measures of each continuous risk predictor, especially measures of SBP, total cholesterol, and HbA1c, and 57% had at least five measures (IJE online).Among the 83 910 people in the analysis data set, 32 804 (39%) were women and the mean age at type 2 diabetes recorded diagnosis was 59.3 (SD = 12.0) years. Characteristics at study entry in the derivation and validation data set were similar . In totaIJE online).Estimated current values of SBP from mixed-effects models were slightly higher than last observed values but lower than the means; estimated current values of total cholesterol were similar to the last observed values but lower than the means . Importantly, the SDs of SBP, HDL cholesterol, and HbA1c were strongly associated with higher CVD risk (IJE online). The individual-level slopes  of SBP and cholesterol were not associated with incident CVD and not considered further (IJE online).Hazard ratios (HRs) for each of SBP, HDL total cholesterol, and HbA1c to all risk prediction models (IJE online). Further investigations that added the SDs of each risk predictor separately revealed that the SDs of SBP yielded the strongest impact on discriminative improvement with a largest C-index increase of 0.005  compared with results from adding SDs of total cholesterol , HDL cholesterol  or HbA1c  (IJE online). Improvements in the C-index were slightly greater in men compared with women (IJE online).Using last observed values of predictors, the C-index of the prediction model was 0.652  . Replacin models . Comparen models . This C-, 0.003) , and theIJE online). In contrast to a fairly constant C-index with increasing numbers of risk factor measurements (IJE online), we observed curve-linear relationships between the Brier score and the number of risk factor measurements (IJE online). Calibration slopes by age ranged from 0.17 to 1.67 and were approximately 1 between ages 60 and 70 years, and those values deviating from 1 were mostly observed in women at younger or older ages with relatively fewer CVD events (IJE online). No substantial improvements in NRI were observed across landmark ages when using the estimated current values compared with the last observed values (IJE online).Brier scores estimated using different approaches were similar , with lo1c or smoking status produced similar HRs (IJE online) to those reported above for 83 910 individuals with complete data on all risk factors. The overall C-index among this population was 0.670  (IJE online).Sensitivity analyses on 143 466 (90%) individuals with at least one measurement of any risk factors of SBP, total cholesterol, HDL cholesterol, HbA1c from longitudinal EHRs into CVD risk prediction models. We found no improvement in CVD risk prediction when merely replacing single last observed measurements of SBP, cholesterol, and HbA1c with means or current values estimated from prior longitudinal measurements. However, we did find a moderate discriminative improvement when incorporating the within-person variability (quantified by SDs) of past SBP, cholesterol, and HbA1c. Such improvement from longitudinal information was comparable with or even greater than the gains from well-established predictors (e.g. SBP and cholesterol) in our study population.The current analysis of 83 910 people with type 2 diabetes reliably assessed the added benefit of incorporating variability of repeatedly measured SBP, cholesterol, and HbA1c with CVD risk in patients with type 2 diabetes,Our results concord with previous studies identifying independent associations of long-term within-person variability of SBP, HDL cholesterol, and HbA,,Evidence from systematic reviews1c and their SDs into CVD risk prediction for people with type 2 diabetes. The added benefit in model discriminative improvement may help to better guide the implementation of intensive therapeutic interventions for type 2 diabetes patients with highest CVD risk. Such predictive ability gain may also be informative for setting serial treatment targets of blood pressure, lipid, and glucose control based on CVD risk levels to help diabetes management. The use of national representative EHRs data, the large sample size, the high number of CVD events and long period of follow-up time further enhanced the reliability of the study results for the 10-year CVD risk prediction. Moreover, the landmark framework optimised the use of repeated measurements of risk predictors by age and enabled the estimation of age-varying risk predictor levels.To our knowledge, this is the first study to investigate and compare methodologies incorporating longitudinal measurements of multiple risk predictors together, especially including HbAThis study also has several potential limitations. First, Cox models were derived amongst those with complete data on risk predictors to make direct comparisons on the use of repeated measurements with single last observed values or mean values of risk factors and assess the added value of within-person variability. This may restrict the generalisability of the results to people with missing values, who may also have different characteristics . Results of our sensitivity analyses from fitting the multivariate-mixed-effects models to people with at least one observed value of any risk factor  showed reasonable discriminatory performance. However, further work is needed to fully assess the value of within-person variability in these individuals. Second, the multivariate-mixed-effects model required additional assumptions  than simply using the last observed values or means. However, it has the advantage of handling missing values that are common in EHRs and enables the extension of our methods to a larger population with incomplete information. Third, the overall C-index in our analyses ranged from 0.65 to 0.67, which does not present an outstanding discriminative ability. Previous meta-analysis of CVD risk prediction models developed for people with diabetes also demonstrated similar results with a pooled C-index of 0.67 .1c to improve CVD risk prediction and guiding the intensitification of therapeutic interventions for people with type 2 diabetes. Incorporating the within-person variability of risk predictors provides a moderate improvement in CVD risk discrimination for individuals with type 2 diabetes. With the increasing availability and improving quality for routinely collected EHRs data, our approach may be easy and efficient to apply based on data already existing in EHRs. The added information from longitudinal risk predictor measurements can be integrated into future risk prediction models and help to improve CVD prevention as well as diabetes management for the type 2 diabetes population.Our study highlighted the benefit of utilizing information from longitudinal past measurements of SBP, cholesterol, and HbAThis study is based on data from the Clinical Practice Research Datalink (CPRD) obtained under licence from the UK Medicines and Healthcare products Regulatory Agency (protocol 162RMn2).dyac140_Supplementary_DataClick here for additional data file.


BRCA1/2. Timely diagnosis and identification of mutation carriers is of utmost importance to improve survival benefit and quality of life. Cancers that have been included into screening of BRCA1/2 associated HBOC included prostate and pancreatic cancers etc. In this case, we reported a patient who firstly presented symptoms of CRC and was finally diagnosed as BRCA1 associated HBOC with advanced peritoneal carcinoma. With strategies of cetuximab based treatment and olaparib, and debulking surgeries, she has achieved an overall survival (OS) > 35 months. The aim was to indicate that HBOC might also first present as CRC, and comprehensive next-generation sequencing analysis might be a key complement for screening and diagnose of HBOC.Hereditary breast and ovarian cancer (HBOC) syndrome has increased predisposition to breast and/or ovarian cancer, and 24% of families with HBOC were associated with the germline pathogenic variants in  BRCA1/2 associated HBOC was not identified.Hereditary breast and ovarian cancer (HBOC) syndrome is defined as an increased predisposition to breast and/or ovarian cancer , and 24%BRCA1 associated HBOC with advanced peritoneal carcinoma. With strategies of cetuximab based treatment and olaparib, and debulking surgeries, she has achieved an overall survival (OS) > 35 months. The aim was to indicate that HBOC might also first present as metastatic CRC, and comprehensive next-generation sequencing (NGS) analysis might be a key complement for screening and diagnose of HBOC or possible hereditary cancers.In this case, we reported a patient who first presented symptom of CRC and was finally diagnosed as A 62-year-old female patient was referred to our hospital on May 5, 2019 with complaints of less and narrow stool with hematochezia for 3 months. Colonoscopy showed multiple protuberant ulcer lesions in both sigmoid colon and rectum . Investigation of family history and NGS analysis  scan revealed the enlargement of rectal lesion Fig. A. The paThe patient started olaparib treatment  on July 30, 2020. She then achieved partial response and a progression-free survival (PFS) of 14 months  and CDX-2(+)/SATB2(+) were both reported to identify primary CRC with considerable accuracy , 13. WhiMSH2, MLH1, MSH6 and PMS2). A comprehensive analysis of germline and somatic gene alteration could help with the early diagnose and matching the individualized management approach. In our case, the same germline BRCA1 was detected in our patient and observed in the mother with breast cancer. No concurrent germline alterations were detected. This provided important evidence of HBOC  polymerase) inhibitor of olaparib. Including susceptive CRC into screening criteria of BRCA1 associated HBOC might be essential to give a precise management strategy.In summary, our case presented a rare case of metastatic colorectal cancer as the primary phenotype of


Drought stress is among the major threats that affect negatively crop productivity in arid and semi-arid regions. Probably, application of some additives such as biochar and/or brassinosteroids could mitigate this stress; however, the mechanism beyond the interaction of these two applications is not well inspected. Accordingly, a greenhouse experiment was conducted on wheat (a strategic crop) grown under deficit irrigation levels (factor A) i.e., 35% of the water holding capacity (WHC) versus 75% of WHC for 35 days while considering the following additives, i.e., (1) biochar  and (2) the foliar application of 24-epibrassinolide . All treatments were replicated trice and the obtained results were statistically analyzed via the analyses of variance. Also, heat-map conceits between measured variables were calculated using the Python software. Key results indicate that drought stress led to significant reductions in all studied vegetative growth parameters (root and shoot biomasses) and photosynthetic pigments  while raised the levels of oxidative stress indicators. However, with the application of BC and/or BR, significance increases occurred in the growth attributes of wheat plants, its photosynthetic pigments, especially the combined additions. They also upraised the levels of enzymatic and non-enzymatic antioxidants while decreased stress indicators. Furthermore, they increased calcium (Ca), phosphorus (P) and potassium (K) content within plants. It can therefore be deduced that the integral application of BR and BC is essential to mitigate drought stress in plants. This stress has adverse effects on photosynthetic machinery, particularly disrupting thylakoid electron transport, stomatal conductance, CO2 assimilation and Calvin cycle5. Moreover, drought stress interrupts the balance between antioxidative defense mechanisms and production of reactive oxygen species (ROS) causing ROS accumulation and this, consequently, disorganize cell membrane lipids, protein and DNA strands6. Accordingly, plants have developed a wide range of adaptive processes during their evolution, including physiological, morphological and biochemical mechanisms allowing a proper response to water scarcity, e.g., increasing the phytohormonal signaling pathways in response of external abiotic stress7.Drought stress possesses a potential threat that hampers plant growth and productivity in arid and semiarid regions8. Moreover, BR are involved in controlling abiotic stress responses9 via: (1) increasing activities of antioxidative enzymes10; hence, lessening the production rate of superoxide anion11, (2) reducing abscisic acid (ABA) accumulation12 in spite of that these hormones increase stomatal closure under drought conditions13 and (3) increasing the osmotic permeability of root cells to take up more water from soil.Different safe approaches have been introduced for increasing plant tolerance to drought stress. For example, brassinosteroids (BR) are polyhydroxylated steroidal phytohormones, which regulates numerous processes of plant physiology and morphogenesis starting from seed germination up to the regulation of flowering and senescence14, maize15 and wheat16. This might take place via (I) increasing soil water retention, hence reducing soil irrigation demands17, (II) increasing nutrient use efficiency18, (III) stimulating auxin, gibberellin and brassinosteroids regulation19 and (IV) increasing plant chlorophyll content20, stomatal conductance, cytotoxicity and leaf K+ content21. This cost-effective carbon rich product is formed through the pyrolysis of organic residues under limited oxygen supply23 to give a product of high porous aromatic carbon content24. It is worth mentioning that this product retains longer in soil versus other organic residues such as compost; hence mitigate climate changes through carbon sequestration25.Biochar is another promising additive that can mitigate the adverse effects of drought on plant growth and productivity, e.g., sorghum26. Wheat is among the most important crops for global food security27 which contributes up to 40% to the world food demand. Its global production is estimated by 757.4 million tons in the year of 201928. Although, the modern high yielding wheat cultivars are introduced to increase wheat productivity29 and these cultivars consume massive amounts of mineral fertilizers30; yet, appropriate techniques are also needed to maximize nutrient utilization by plants in order to sustain the available limited resources. For example, nitrogen use efficiency by wheat does not exceed 33% worldwide28 and others nutrients mostly do not exceed 50%. Under drought conditions, nutrient use efficiency decreases considerably31 and this may cause further reductions in plant productivity. In this investigation, we tested the feasibility of using the combined application of exogenous BR and BC as potential safe approaches to mitigate drought stress via increasing plant capability to utilize soil nutrients as well as the enzymatic and non-enzymatic antioxidants; hence this combined application might increase considerably crop yield productivity under drought conditions. Although, many studies highlighted the positive effects of the sole application of each of exogenous BR and BC on plant growth; yet, the implications of this combined application on increasing plant tolerance to stress conditions is not so far investigated.In the next years, significant increases in global crop products will be required to fulfill the increasing human and animal food demandsThe current study is a trial to investigate the potential co-application ameliorative impacts of timber waste biochar as a soil amendment and exogenous brassinosteroids as foliar application to alleviate the stress conditions on wheat plants subjected to deficit irrigations. Specifically, we hypothesized that this combined application could effectively mitigate drought stress effects on wheat than applying each solely; consequently, the productivity of treated plants increased considerably under such adverse conditions. Wheat was selected as an experimental model plant in this study, following guidelines for testing chemicals by the Organization for Economic Cooperation and Development (OECD) guideline 208.32 and the results are presented in Table Surface soil samples (0–30 cm) were collected from the experimental farm of Government College Women University Faisalabad, then air dried, crashed and sieved to pass through a 2-mm sieve. Soil characters were investigated according to Sparks et al.Triticum aestivum L., cv. Lasani-2008) were obtained from certified seed dealer of the Government of Punjab, Pakistan. All seeds were disinfected with 95% ethanol followed by 70% sodium hypochlorite solution washing, then washed three times with distilled water. The use of plants in the present study complies with the IUCN Policy Statement on Research Involving Species at Risk of Extinction and the Convention on the Trade in Endangered Species of Wild Fauna and Flora.Wheat seeds  was crushed in a grinder and sieved through a 2 mm sieve and the fine residue was kept for the experimental procedure. Some major timber waste biochar properties are presented in Table 28H48O6 having Mw = 480.7 sourced from Sigma-Aldrich) at either 0 (distilled water), 1 (BR1) or 3 (BR2) μmol concentrations per pot starting from the third week after sowing with one day interval between foliar applications using a hand sprayer at the rate of 50 mL per plant.A pot experiment was conducted at the Botanical Garden, Government College Women University Faisalabad, Pakistan to test the hypothesis of the study. In this experiment, soil portions equivalent to 8 kg soil were mixed thoroughly with one of the following treatments: 0%  and 2% biochar  were packed uniformly in plastic pots (28 cm diameter × 20 cm height). These pots were arranged in a complete randomized block design, and each treatment was replicated trice. Seven seeds were sown in each pot, and five healthy seedlings were left by thinning at 15 days after planting till the end of the incubation period (35 days after planting). All plants were watered optimally at 75% WHC (75% of the water holding capacity) until three weeks of sowing; thereafter one group of pots (drought-stressed) was watered at only 35% WHC (35% of the water holding capacity), while the other group was watered at 75% of WHC. Plants were sprayed trice with 24-epibrassinolide .The chlorophyll a, b and total contents as well as carotenoids pigments were estimated in leaves following the Arnon protocol2O2 contents was achieved following Mukherjee and Choudhari technique34. In this method, 0.1 g leaf sample was extracted in 10 mL cold acetone, centrifuged at 10,000 rpm, then 4 mL titanium reagent and 5 mL of concentrated ammonium solution were added to the reaction mixture. The mixture was then centrifuged at 10,000 rpm for 5 min and the precipitate was dissolved in 10 mL of 2 N H2SO4. The residue was again centrifuged to remove suspended particles. Optical density was recorded at 415 nm against blank by spectrophotometer .Estimation of H35. The electrolyte leakage (EL) was determined following Anjum et al.36 protocol.Chloroplast’s lipid peroxidation was determined by estimating malondialdehyde (MDA) contents following thiobarbituric acid (TBA) reaction by Heath and Packer method37, flavonoids and anthocyanin38, phenolics39, ascorbic acid40, proteins41 and glycine betaine42 contents.To estimate osmolytes, i.e., proline and other non-enzymatic antioxidants, 50 mg dried plant samples were extracted in 10 mL ethanol (80%), then filtered followed by re-extraction in ethanol (10 mL). A final volume of 20 mL was maintained by mixing the two samples. The obtained extracted solution was used to estimate proline43 then measured by spectrophotometer . K ion concentrations in plant extracts were measured by flame photometer while Ca and Na concentrations in these extracts were estimated using Atomic Absorption Spectrum .In the same extract, Ca, Na, P and K ion concentrations were estimated as follows: molybdate/ascorbic acid blue method was used for P determinationP < 0.05). Logarithmic transformations for data normalization were carried out before analysis, where necessary. Pearson’s correlation analysis was performed to compute associations among various analyzed variables. The heat-map conceits between measured variables were also calculated by using the Origin software.Statistical analysis was conducted by using the analysis of variance to find significance of applied treatments in drought stress. All the treatment means were compared by Dunken’s test at 5% level of significance (Root and shoot (fresh and dry) weights of wheat plants subjected to deficient irrigations (35%) WHC were significantly lower than the corresponding ones that received 75% WHC Fig. . LikewisThe abovementioned growth parameters were improved significantly owing to BR and/or BC applications versus the control. In this concern, the foliar application of BR raised significantly plant growth parameters exceeding those attained for the application of biochar (BC), especially with increasing the level of BR application. On the other hand, the application of biochar did not noticeably improved plant growth parameters under deficit irrigation (35% WHC) versus the control (except for root fresh weight).The combination of BR and BC caused further significant increases in plant growth parameters, especially in presence of the higher application dose of BR (3 µmol). It is worth mentioning that the usage of BR + BC with 35% WHC considerably enhanced root and shoot fresh weights, dry weights and lengths of wheat plants exceeding those recorded for the control plants that were irrigated with 75% WHC. These results probably signify the value of these amendments in increasing the efficiency of water use by the grown plants.Deficit irrigations negatively affected chlorophyll a, b and the total contents as well as plant carotenes Fig.  i.e., al2O2, electrolyte leakage and Na content increased under such conditions. Table Nutrient  contents increased significantly in plants tissues owing to irrigation with water to achieve 75% of WHC comparable with the ones that were irrigated to reach only 35% of WHC (Table 2O2 and Na (sodium) contents beside of the electrolyte leakage, while increased proteins, Ca , K (potassium) and P (phosphorous) in plant tissues. It seems that the sole applications of either BR or BC were superior in decreasing these oxidative stress indicators than their integral application. Moreover, all applied treatments decreased significantly all enzymatic and non-enzymatic antioxidants under investigation  and this probably signify the success of these treatments in ameliorating drought stress effects. In this concern, BR2 + BC recorded the least enzymatic and non-enzymatic enzymes antioxidants activities.All additives (BR and BC) showed positively significant improvements in ameliorating drought stress on plants via decreasing MDA and oxidative stress indicators. Such improvements were more detectable with increasing the dose of BR foliar application. Also, these additives decreased considerably HPearson correlation analyses were conducted to highlight the relationships between plant growth parameters and the increases that took place in plant oxidative stress indicators grown under drought conditions. As mentioned above, significant improvement in plant growth parameters, i.e., root and shot weights and lengths was observed due to the applications of BR and BC. Root and shoot (fresh and dry) weights of wheat plants subjected to deficient irrigations at 35% WHC decreased significantly versus the ones that received 75% WHC Fig. .2O2, electrolyte leakage and Na content. Proline content in leaves also affected significantly and negatively all the studied growth parameters. It is worth noting that the oxidative stress indicators were correlated positively and significantly with proline in leaves and these stress parameters affected negatively pigment contents in plants  and their lengths were significantly and positively correlated with each other Fig. . Improvents Fig. .Figure 3The current study is a trial to alleviate drought stress conditions on wheat plants via the foliar application of brassinosteroids (BR) and/or timber waste biochar (BC) as a soil amendment.44. Potassium is a special case because its content did not vary significantly in tissues of stressed plants versus non-stressed ones. This is because its influx occurs via non-energetic transporters and gated channels which are highly specific for K ions45. Moreover, this nutrient is highly mobile in plant and acts directly in regulating the osmotic potential in compartments to increase their turgor46. Generally, the nutrient imbalance that occurred in drought-stressed plants may led to undesirable drop in plant pigments  and photo-assimilates47.Wheat plants absorb and accumulate high concentrations of Na in their tissues; while contents of other nutrients such as Ca and P decreased considerably. Probably, this stress down-regulates genes encoding plasma membrane intrinsic proteins in plant roots in addition to plant hydraulic conductivity48. Overall, this stress affected negatively plant growth (fresh and dry weights of plants as well as the plant heights) and such a result was verified in many previous studies, e.g., Sankar et al.49 and Hussain et al.50.Significant increases in some oxidative stress indicators, namely anthocyanin and flavonoids occurred under drought conditions while total soluble protein contents diminished. Other mechanisms may be included to cope from drought stress such as regulating stomatal aperture to maintaining tissue hydration2O2 works as signaling molecules under low concentrations; yet their accumulation within plant tissues in higher concentrations are toxic51. Further changes may takes place at both cellular and whole-organism levels, making drought stress tolerance a complex physiological phenomenon53.Application of either biochar or BR1 did not successfully increase nutrient contents within plant tissues and therefore the changes in plant pigments were not detectable versus the control. Likewise, the changes in root and shoot biomasses and heights owing to the sole application of these two treatments were not noticeable versus the control. Though, these additives lessened considerably all oxidative stress indicators such as malondialdehyde, hydrogen peroxide, electrolyte leakage via increasing the activities of antioxidant enzymes and non-enzymes. Such a result might indicate that plants synthesize these bioproducts to lessen the impacts of drought on plants prior to the synthesis of other metabolites needed for enhancing plant growth. Probably, radicals such as H46 and also acts as a secondary messenger in cell osmoregulation46.The combination between biochar and brassinosteroids ( BR1 + BC and BR2 + BC) beside of the application of the higher dose of the latter additive i.e. BR2 recorded further significant further increases in both Ca and K uptake by plants (with superiority for the combined applications versus BR2); consequently their contents raised in plant tissues; hence plant pigments upgraded. In spite of that, these treatments were of no significant effect on Na content within plant tissues; yet, these additives improved the cation balance in plant tissues, especially the Ca: Na ratio and therefore plant roots and shoots boosted notably. In this context, Ca is an integral component of cell wall7 and decrease the oxidative stress indicators in plant54. In case of biochar, it stimulates gibberellic acid synthesis in plants hence, improve their growth55. It may also be indirect through increasing soil moisture retention56 that increases the efficiency of the grown plants to utilize soil nutrients and promote plant biomass57.The possible adaptations of wheat, in case of the combined applications of BC and BR, were either direct, as BR increase photosynthetic activitiesIt is worthy to mention that the activities of enzymatic and non-enzymatic antioxidants were higher in sole applications than the combined ones. This finding did not signify that BR and BC additives antagonize each other because of the superior effect of this combination on plant growth parameters comparable with the sole applications of any of these additives. It can therefore be deduced that the optimum strategy of improving plant growth under drought conditions is through enhancing more antioxidant activities as well as improving the nutritional status of the grown plants. The above results; therefore, support the main hypothesis of the study which signify the supremacy of the combined application of BR + BC for mitigating drought stress on wheat versus their sole applications.Application of either of brassinosteroids (BR) or timber waste biochar (BC) can successfully alleviate drought stress and improve wheat growth under deficit irrigation system. Moreover, the combined application of these two additives had further positive impacts on increasing plant growth parameters via their integral role in decreasing oxidative stress indicators in plants. There is an actual need for more investigations under field level conditions to validate the results obtained herein using more deficit irrigation variations.