The COVID-19 pandemic has the potential to impact long-standing efforts to increase adherence to cancer screening guidelines. Healthcare workers (HCWs) experienced significant hardship, but generally have greater access to preventive services, making them a particularly relevant population in which to understand cancer screening behaviors during the pandemic. We report data from 794 HCWs enrolled in the NCI-funded Serological Sciences Network for Coronavirus Associations and Longitudinal Evaluation Study from December 2020 to April 2021. Participants reported lifestyle and screening behaviors during relevant look-back periods which included the pandemic timeframe. Among women between the ages of 40 and 74, 25.7% were overdue for mammographic breast cancer screening. Among participants 50–75 years old, 38.9% were overdue for colorectal cancer screening. The proportion over-due varied according to race/ethnicity. Lifetime low-dose computed tomography lung cancer screening among HCWs age 50–80 years who were smokers was 10.9%. Strategies to address screening disruptions are needed to minimize the impact of later stage of diagnosis. Health care services across the globe have been profoundly affected by the COVID-19 pandemic. The suspension of cancer screening programs, cancellation or rescheduling of non-elective procedures, a pivot toward virtual clinical visits, and stay-at-home orders may have impacted adherence to cancer screening . Data frHealthcare workers (HCW) experienced significant hardship during the pandemic , but genN = 794). The healthcare workers represent employees with diverse job functions and the sample was composed of 37% nurses, 32% physicians, 5% medical technicians, 4% hospital administration, 2% nursing assistant, and 20% from various other occupations.Active employees working at multiple sites comprising the Cedars-Sinai Health System, located in the diverse metropolis of Los Angeles County, California were eligible to participate. The Cedars-Sinai organization includes two hospitals  in addition to multiple clinics in the Cedars-Sinai Medical Delivery Network. The SeroNet-CORALE Study recruited HCWs from vaccine clinics at Cedars-Sinai Medical Center and through study notifications in mass emails between December 2020 and April 2021. Participants were invited to complete questionnaires, donate blood specimens and engage in long-term follow-up. Cancer screening behaviors were measured using modified versions of standardized questionnaires , 6. We rThrough the study survey, participants were asked “Have you had any of the following screenings?” Screening tests and accompanying details were listed, including “Mammogram (an x-ray of each breast to look for breast cancer),” “blood stool test ,” “sigmoidoscopy ,” “colonoscopy ,” “CT or CAT scan .” Participants were able to respond “yes,” “no,” or “not sure” to each of the screening tests listed.via a survey question that asked “How long has it been since you had your last mammogram?” The response categories included “within the past year (anytime <12 months ago),” “within the past 2 years (1 year but <2 years ago),” “within the past 3 years (2 years but <3 years),” “within the past 5 years (3 years but <5 years ago),” “5 or more years ago,” “never,” “not sure.”The U.S. Preventive Services Task Force (USPSTF) recommends biennial screening with conventional mammography for women aged 50–74 years. Screening between ages 40–49 is considered an individual decision and there is no recommendation over the age of 75 years ,” “within the past 2 years (1 year but <2 years ago),” “within the past 3 years (2 years but <3 years),” “within the past 5 years (3 years but <5 years ago),” “5 or more years ago,” “never,” “not sure.” The screening nature of the exams were ascertained via three questions which asked “Why did you have a blood stool test/sigmoidoscopy/colonoscopy?” Participants were instructed to “select all that apply” for the following response categories “Family history of colorectal cancer,” “part of regular check-up/routine screening,” “age,” “race,” follow-up on a problem,” “follow-up of colorectal cancer treatment,” “other (please specify).” Tests were only considered screening tests if the participant selected “part of regular check-up/routine screening.”For early detection of colorectal cancer, current recommendations by the USPSTF offer several screening modalities depending on preferences . We defivia low-dose computed tomography (CT) (n = 6). Instead, the proportion ever screened for lung cancer was calculated for all smokers (former and current combined regardless of pack-years) age 50–80 years. Duration since lung cancer screening was ascertained via a question that asked “How long has it been since you had your last CT or CAT scan?” The response categories included “within the past year (anytime <12 months ago),” “within the past 2 years (1 year but <2 years ago),” “within the past 3 years (2 years but <3 years),” “within the past 5 years (3 years but <5 years ago),” “5 or more years ago,” “never,” “not sure.” The screening nature of the exam was ascertained via a question that asked “Why did you have a CT or CAT scan?” Participants were instructed to “select all that apply” for the following response categories “part of prior hospitalization,” “part or regular check-up/routine screening,” “follow-up on lung problem,” “follow-up of lung cancer treatment,” “not sure,” “other (please specify).” The test was only considered a screening test if the participant selected “part of regular check-up/routine screening.”At the time of the survey, the USPSTF recommended annual lung cancer screening to those age 50–80 years who had a 20 pack-year smoking history phy (CT) . GuideliBreast and colorectal cancer screening were examined descriptively by selected patient characteristics including age group and race/ethnicity. Logistic regression models with sociodemographic predictors were implemented for breast and colorectal cancer separately. Because of insufficient sample size, some categories within variables were combined in the models. All analyses were performed using SAS software . Participants provided informed consent and the study was approved by the Cedars-Sinai IRB.Baseline characteristics for the study population are shown in N = 381), 25.7% were over-due for breast cancer screening  0.31–0.94] , 38.9% were over-due for screening. The proportion over-due among Hispanic (25.0%), non-Hispanic Asian (37.5%), and non-Hispanic White (36.8%). HCWs was lower than those among non-Hispanic Black (63.2%) HCWs. In bivariate and multivariate models, none of the predictors were statistically significantly associated with colorectal cancer screening , lifetime CT lung cancer screening was 10.9%. Lifetime screening was lowest in Hispanics compared to other ethnic/racial groups.Among men and women age 50–80 years who were either current or former smokers  . The proLimitations of our analysis include missing data and the cross-sectional nature of the study. Our sample size for lung cancer screening is also limited by the small number of HCWs who smoked.These results have important public health implications. Effective communication about how the pandemic has affected cancer screening, in particular minority populations is needed. Use of home-based stool tests for colorectal cancer could also help “catch-up” strategies . More reThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Cedars Sinai Institutional Review Board. The patients/participants provided their written informed consent to participate in this study.GD: conceptualization, methodology, supervision, writing—original draft, and writing—review and editing. ML: data curation, formal analysis, methodology, writing—original draft, and writing—review and editing. S-JS: methodology, writing—original draft, and writing—review and editing. SH: funding acquisition, methodology, writing—original draft, and writing—review and editing. SG: writing—review and editing. AM and NM: funding acquisition, resources, and writing—review and editing. KR: funding acquisition, resources, methodology, writing—original draft, and writing—review and editing. JF: conceptualization, funding acquisition, investigation, resources, supervision, writing—original draft, and writing—review and editing. All authors contributed to the article and approved the submitted version.This work was supported by the National Cancer Institute (U54CA260591). The funder had no role in the execution or reporting of this research study.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.


Rhus verniciflua, and Dipterx lacuniferaToxicodendron vernicifluum, Dahlia, Bidens tripartite , is one of the most important natural flavonoids. This compound is known to have numerous biological activities; among these, the antioxidant activity has not been thoroughly studied yet. In this study, the hydroperoxyl scavenging activity of SFR was examined by using density functional theory calculations. SFR is predicted to be an excellent HOO• scavenger in water at pH = 7.40 with koverall = 4.75 × 107 M−1 s−1, principally due to an increase in the activity of the anionic form following the single-electron transfer mechanism. Consistently, the activity of the neutral form is more prominent in the non-polar environment with koverall = 1.79 × 104 M−1 s−1 following the formal hydrogen transfer mechanism. Thus, it is predicted that SFR exhibits better HOO• antiradical activity than typical antioxidants such as resveratrol, ascorbic acid or Trolox in the lipid medium. The hydroperoxyl radical scavenging of SFR in the aqueous solution is approximately 530 times faster than that of Trolox and similar to ascorbic acid or resveratrol. This suggests that SFR is a promising radical scavenger in physiological environments.Sulfuretin (SFR), which is isolated from  This cocunifera , antimutcunifera , antiplacunifera , anti-cacunifera ,9, anti-cunifera ,10, livecunifera , anti-agcunifera , anti-obcunifera  and anticunifera –15.Figu− and HO• radicals [et al. [•+  and HO• radical scavenging activity that is higher than butylated hydroxytoluene (BHT).Jung and co-workers  reportedradicals . Chen et [et al.  also rep• antiradical activity of SFR by a combination of thermodynamic and kinetic calculations. This study also considered the effects of solvents on the antioxidant properties of SFR in comparison with some typical antioxidants.Although the antioxidant activity of SFR is broadly examined experimentally ,14, ther. 2All calculations were carried out with Gaussian 09 suite of programs . M06–2X/. 3. 3.1. 3.1.1For SFR that contains OH and moieties, the antioxidant activity may follow either of the four main mechanisms: the formal hydrogen transfer (FHT), the sequential proton loss electron transfer (SPLET), the single-electron transfer proton transfer (SETPT) and radical adduct formation (RAF) ,42. The −1. This value is lower than that of natural antioxidants such as viniferifuran  [−1) [−1) [−1) [As per l mol−1) , resvera−1) [−1) , puerari−1) [−1)  and vani−1) [−1) . The low• antiradical activity of SFR, the Gibbs free energy of the SFR + HOO• reaction was calculated according to each of the four mechanisms: FHT, single-electron transfer , sequential proton  and RAF −H bonds and RAF at the C8 position (ΔGo < 0), whereas the RAF reaction at C2 with ΔGo = 1.1 kcal mol−1 cannot be clearly excluded based on thermodynamics alone and therefore it was also included in the kinetic study. The other reactions are clearly not spontaneous with high positive ΔGo values. The ΔGo values for the reactions following the SP and SET pathways are much higher than those of the FHT mechanism. Thus, the calculated data suggest that the HOO• antiradical activity of SFR may follow either FHT or RAF mechanism (at O3′(4′)−H and C2/C8 positions, respectively), and these pathways should be investigated in the kinetic study.To confirm that FHT is indeed the preferred pathway of the HOO and RAF . It was . 3.1.2• reaction in the gas phase was investigated for the thermodynamically favourable positions and mechanisms according to the QM-ORSA protocol [Based on the above results, the kinetics of the SFR + HOOprotocol , and the• antiradical activity of SFR occurs mostly by the H-abstraction of the O4′−H bond . That is more than three times higher contribution than the hydrogen abstraction of the O3′−H bond . By contrast, the addition of the radical does not make any contribution (Γ = 0%) at either the C2 or C8 positions. This result is in good agreement with previous studies in phenolic compounds [• antiradical activity of SFR is dominated by the FHT mechanism at the O3′(4′)–H bond; therefore, this is further analysed in physiological environments.It is apparent that the HOOompounds –48. We c. 3.2. 3.2.1• antiradical activity of phenolic compounds in the aqueous solution [−, 46.0%) forms. Therefore, in the physiological environments, these states were used for the kinetic investigation.Previous studies showed that the deprotonation of the OH bonds plays a key role in the HOOsolution ,34,49. Tsolution , and the. 3.2.2• antiradical activity in non-polar environments was modelled by the hydrogen transfer mechanism at the O3′(O4′)−H bonds. In the aqueous environment, the SET mechanism was also investigated for the deprotonated state of SFR. The overall rate constants  were computed following the QM-ORSA protocol [Based on the results of the kinetic calculations in the gas phase, the HOOprotocol ,33,  in the radical scavenging activity of SFR. The H-abstraction of the anion state contributes about 13.7% to the overall rate constants. The rate constants for the H-abstraction of O3′(O4′)−H bonds against HOO• radical are kf = 4.97 × 103 and 2.86 × 103 M−1 s−1, respectively; however, these reactions do not make any contributions (approx. 0%) to the activity of SFR. Based on the results, SFR is better HOO• radical scavenger than typical antioxidants Trolox, ascorbic acid and resveratrol in lipid phase  [k = 9.97 × 107 M−1 s−1) [k = 5.62 × 107 M−1 s−1) [As shown in  M−1 s−1 , koveral M−1 s−1  and kove M−1 s−1 , respectM−1 s−1)  and fairM−1 s−1)  and resvM−1 s−1) . Thus, t. 4• antiradical activity with koverall = 4.75 × 107 M−1 s−1 in water at pH = 7.40 by the SET pathway of the anion state, and good/moderate HOO• scavenging activity in lipid environment  by the FHT mechanism via the O3′(O4′)–H bonds. The hydroperoxyl antiradical activity of SFR is better than Trolox, ascorbic acid and resveratrol in the lipid medium. This activity of SFR is approximately 530 times faster than that of Trolox and relatively similar to ascorbic acid and resveratrol in the polar environment. Thus, SFR can be a useful natural antioxidant in physiological environments.The hydroperoxyl radical scavenging activity of SFR was investigated using DFT calculations. The results showed that SFR has excellent HOOClick here for additional data file.


Plum pox virus  have been identified in peach. However, previous studies have demonstrated that grafting a “Garrigues” almond scion onto “GF305” peach rootstock seedlings heavily infected with PPV can progressively reduce disease symptoms and virus accumulation. Furthermore, grafting a “Garrigues” scion onto the “GF305” rootstock has been shown to completely prevent virus infection. This study aims to analyse the rewiring of gene expression associated with this resistance to PPV transmitted by grafting through the phloem using RNA-Seq and RT-qPCR analysis. A total of 18 candidate genes were differentially expressed after grafting “Garrigues” almond onto healthy “GF305” peach. Among the up-regulated genes, a HEN1 homolog stands out, which, together with the differential expression of RDR- and DCL2-homologs, suggests that the RNA silencing machinery is activated by PPV infection and can contribute to the resistance induced by “Garrigues” almond. Glucan endo-1,3-beta D-glucosidase could be also relevant for the “Garrigues”-induced response, since its expression is much higher in “Garrigues” than in “GF305”. We also discuss the potential relevance of the following in PPV infection and “Garrigues”-induced resistance: several pathogenesis-related proteins; no apical meristem proteins; the transcription initiation factor, TFIIB; the speckle-type POZ protein; in addition to a number of proteins involved in phytohormone signalling.No natural sources of resistance to  Prunus) fruit trees are affected by a large number of viral diseases that can cause important economic losses [Plum pox virus (PPV), is the most significant of these diseases due to the reduced fruit quality and premature fruit drop that it causes [P. persica (L.) Batsch)—the most important Prunus species, with an annual production of 25.7 million tons in 2019 —no natural sources of resistance have been identified [P. ferganensis (Kostov and Rjabov) and P. davidiana (Carrière) Franch. Nevertheless, the transmission of resistance from these related species has been questioned, and the importance of the genetic background in the effectiveness of this strategy has been highlighted [P. dulcis (Miller) Webb) species [Stone  seedlings heavily infected with PPV can progressively reduce disease symptoms and virus accumulation . This reOn the other hand, it is well known that viruses induce different defense responses related to RNA silencing in different plant species ,16. HoweOur work aims to analyze the differential gene expression linked to the resistance to PPV transmitted to peach “GF305” via grafting with “Garrigues” almond cultivars, focusing on the differential expression patterns shown and comparing the different treatments studied.The results shown in www.rosaceae.org; accessed on 21 January 2021), while 24.2% of the clean reads were not recognised by any of the eight pseudo region-chromosomes. Alignment values in the peach genome ranged from an average of 78.7% in the peach samples to 68.2% in the almond samples. On the other hand, 2.15 million unmapped reads  were mapped in the PPV genome  across samples represented a total of 3269 different genes. From this set, 3210 were assigned with a UNIPROTKB ID, and 1115 were assigned with one or more GO terms using PANTHER . The mosThe overrepresentation analysis was carried out with PANTHER to compare our complete DEG list with the reference peach genome . In the Next, the GO analysis was refined by grouping together the filtered DEGs associated with common biological effects: control healthy “GF305” peach rootstocks (A); PPV-infected “GF305” rootstocks showing severe sharka symptoms (B); “GF305” peach rootstocks grafted with healthy “Garrigues” (C); PPV-infected “GF305” rootstocks showing sharka symptoms grafted with healthy “Garrigues” (D); healthy “GF305” peach rootstock grafted with healthy “Garrigues” which were later inoculated with PPV at the end of the first cycle of evaluation (E). “Garrigues” almond samples grafted onto healthy (F) and inoculated “GF305” (G) rootstocks. Comparisons associated with the almond grafting effect , a pathogenesis-related 1 protein (Prupe.1G091400), a no apical meristem protein (Prupe.2G204700), transcription initiation factor TFIIB (Prupe.3G255800), glucan endo-1,3-beta-D-glucosidase (Prupe.7G051200) and the endoribonuclease DICER homolog 2 (DCL2) (Prupe.7G048000). The differential expression of the gene encoding the speckle-type POZ protein (Prupe.1G488200) is of special interest, since this protein has a meprin and TRAF homology (MATH) domain, and proteins with this domain have been found to be involved in resistance to PPV. The expression trends of eight of the selected genes were similar to those shown by RNA-Seq analysis. Pearson correlation coefficients between fragments per kilobase pair of transcript per million mapped reads (FPKM) values from RNA-Seq and relative gene expression (RGE) values from RT-qPCR were higher than 0.70 for 12 of the 14 assayed genes. Transcription initiation factor-TFIIB and no apical meristem protein (NAM) are exceptions, with correlation coefficients of 0.61 and 0.70, respectively. The anomalous values of speckle-type POZ protein in sample D represent another example of the low correlation between the two analyses. The expression of most analysed genes was higher in leaves showing disease symptoms (sample B) than in non-symptomatic leaves. In addition, grafting “Garrigues” induced an increase in the expression of the following genes: EREBP-like factor, chorismate mutase, no-apical meristem protein, transcription initiation factor-TFIIB, lucan endo-1.3-beta-D-glucosidase and DCL2.Most of selected candidate genes, however, code for factors related to the pathogen response, such as a pathogenesis-related thaumatin-like protein (Prupe.8G163300), glutathione S-tThe “protection effect” of the “Garrigues” scion was clear in the “GF305” rootstock that was inoculated when “Garrigues” had already been grafted (sample E). In this case, no symptoms were ever observed on “GF305”. From the agronomical point of view, this is the most important effect, since we can graft “Garrigues” as an intermediate wood on commercial rootstocks (interstock), and later, when the scions have developed, we can graft the peach varieties. The specific combination that gives resistance to PPV should be evaluated in other peach varieties. These results also open the possibility of using “Garrigues” as a rootstock or interstock to control PPV in peach, similar to the proposed solution in grapevine  and tomaP. armeniaca L.), with 437 DEGs in only two comparisons [The PPV-resistance response induced by grafting “Garrigues” onto “GF305” is associated with a strong transcriptomic imbalance with many changes at the mRNA level, as previously described for grafting in other plant species ,25,26. Tparisons . This loOne of the DEGs of the C/A comparison that attracts the most attention is Prupe.4G091400, which is identified as the peach homolog of HUA ENHANCER 1 (HEN1). This DEG encodes an RNA methyltransferase that plays an important role in RNA silencing by methylating siRNAs . This geA recent translatome (entirety actively translated mRNAs) profiling study identified one HEN1 homolog, three DCL2 homologs, six RDR1 homologs, and other predicted RNA silencing-related genes that were up-regulated in both the phloem and non-phloem tissues of plum leaves developing PPV infection after a period of cold-induced dormancy . ActivatChorismate mutase, EREBP-like factor, CYP71A1, and Dehydration-responsive RD22  and transcription initiation factor (TFIIB), respectively. NAM proteins are included within the NAC family, a family including members involved in interactions with pathogens [TFIIB is a general transcription factor that forms part of the pre-initiation complex of RNA polymerase II. The structure of TFIIB presents two domains, an imperfect repeat of approximately 70 aa and a TFII zinc-binding domain. Proteins with this last domain have been shown to be involved in the regulation of responses to biotic and abiotic stresses and in resistance to pathogens [To explore the possible involvement of other phytohormones in “GF305” infection and its recovery by grafting with “Garrigues”, we analysed a phytohormone-related gene that did not appear to be differentially expressed in the RNA-seq experiment via RT-qPCR. Prupe.5G193400, identified as giberellin-regulated protein 4, showed consistent data in the RNA-seq and RT-qPCR analyses, revealing a notable over-accumulation in the PPV-infected non-grafted RT-qPCR analyses. This result suggests that gibberellins also play a role in the PPV infection of peach trees, and, together with the results discussed above, reflects the complex pattern of phytohormone rearrangements associated with the PPV infection of “GF305” peach and the resistance induced by “Garrigues” almond, as previously discussed by Nikbakht-Dehkordi et al. . Prupe.2athogens . These pathogens ,40. TFIIathogens . Both RNArabidopsis thaliana the MATH-TRAF gene, RTM3, encodes a factor involved in the restriction of systemic spread of different potyviruses, including PPV [ParPMC2 [MATH-TRAF genes of the peach syntenic region of the apricot PPVres locus were differentially expressed. However, the expression of Prupe.1G488200, which is also located in chromosome 1, but downstream of the PPVres-related region, was induced by PPV infection (sample B), showing lower expression levels in healthy and grafting-induced recovered peaches  domain . In Arabding PPV , and at ding PPV . More imding PPV ,46. The [ParPMC2 . In our  peaches . WhetherPrunus species under greenhouse conditions [Seedlings of peach “GF305” peach are characterised by their susceptibility to fruit viruses including PPV  and are nditions . This ma® , and RT-PCR analysis was carried out using specific primers for the coat protein coding sequence VP337 (5′ CTCTGTGTCCTCTTCTTGTG 3′) and VP338 (5′ CAATAAAGCCATTGTTGGATC 3′). The enzymes used were avian myeloblastosis virus reverse transcriptase and GoTaq®Flexi2 polymerase . The RT-PCR parameters were 42 °C for 45 min (cDNA synthesis) followed by one step at 94 °C for 2 min, and 35 cycles at 94 °C for 30 s, 55 °C for 30 s and 72 °C for 30 s, with a final extension step at 72 °C for 5 min. RT-PCR amplified products were separated by electrophoresis on 1% agarose gels in 40 mM Tris-acetate and 1mM EDTA, pH 8.0, and stained with GelRed®  [All the experiments were carried out in a greenhouse under controlled conditions. The complete evaluation procedure was described in Rubio et al. . Brieflyco, USA) .® . The quality and quantity of the total RNA samples were assessed using a NanoDrop® 2000 spectrophotometer  and diluted at the same concentration (200 ng/µL). RNA samples (5 µg) were sent to the Centre for Genomic Regulation  for library preparation and RNA sequencing, assaying 2 cDNA libraries per treatment. The 14 cDNA libraries of poly-A RNA were sequenced using an Illumina HiSeq2000 machine to perform 125 bp paired-end sequencing.We assayed the following treatments: control (sample A) and PPV-infected “GF305” peach seedlings showing strong sharka symptoms (sample B); control “GF305” grafted with “Garrigues” almond (samples C and F) and PPV-infected “GF305” grafted with “Garrigues” (samples D and G); control “GF305” grafted with “Garrigues” almond and later inoculated with PPV (sample E) . Leaf sahttp://www.rosaceae.org/peach/genome, accessed on 21 January 2021) [http://www.rosaceae.org/, accessed on 21 January 2021) in general feature format 3 (GFF3). The presence of the PPV virus was quantified by aligning the sequences that did not map to the peach genome against the PPV 3.30RB/GF-IVIA genome sequence (KJ849228.1) . The resulting lists of differentially expressed isoforms (mRNA) were then filtered by log2 (log-fold change) >1 and <−1 and a q-value <0.05, taking into account all the identified genes in each comparison, including up- and down-regulated differentially expressed genes .A quality control step was performed for the RNA-Seq (mRNA) reads using PRINSEQ software . In the ry 2021)  using STry 2021) . Genomic49228.1) . The traed genes . The tot® Green PCR Master Mix (Applied Biosystems), 10 to 20 ng of cDNA, and 2.5 µM of each primer. The qPCR conditions were as follows: 95 °C for 10 min; 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The melting temperature in these experiments was set to 60~95 °C and increased by 0.3 °C/s. Each biological sample was implemented in triplicate. RPII, actin and expansin-A8 were used as reference genes for data normalisation [To validate the RNA-Seq analysis, RT-qPCR was performed in a new experiment with new RNA samples from the same initial experiment, assaying three replicates per treatment. Relative quantitative PCR (qPCR) experiments were executed with StepOnePlus™ real-time PCR system (Applied Biosystems). Specific primers were designed based on peach/almond sequences previously obtained using Primer3 software . A totallisation , and thelisation , taking RTM genes, NAM proteins, TFIIB, TLPs (thaumatin like protein), and dehydration-responsive proteins. These candidate “resistance” genes were over-expressed after “Garrigues” grafting. In addition, several candidate genes  up-regulated after almond grafting should be considered as “susceptible” genes that are necessary for PPV infection. This down-regulation of the susceptible gene candidates could be due to a gene methylation process mediated by an sRNA molecule. Based on these results, we can deduce the following resistant hypothesis for grafting “Garrigues” to peach. First, grafting induced a series of signal transduction genes at high expression levels, with an interesting correlation among constitutive thaumatin-like protein, PR proteins, ABA compound, and disease susceptibility to PPV. Secondly, protein kinase interacts with transcription factors or other factors and up-regulates the expression relevant susceptible genes. On the other hand, if we are ultimately able to corroborate the “Garrigues effect” at the commercial level (nurseries and orchards), we will have an effective strategy against the continuous threat of PPV for peach production.Again, our results show that resistance to PPV in “Garrigues” can be transmitted through the graft union via the phloem and protect the highly susceptible “GF305” peach. We propose that grafting provides a path for horizontal gene transfer. Although we cannot determine the specific mechanisms of resistance that regulate this surprising behavior, several candidate “resistance” genes have been identified, including defense genes such as


Ophiocordyceps sinensis fungus–Thitarodes/Hepialus larva complex. In view of culturing this complex, a method for the artificial rearing of the Thitarodes/Hepialus ghost moth hosts was established. Deterioration of the host insect population and low mummification rates in infected larvae constrain effective cultivation. Hybridization of Thitarodes/Hepialus populations may overcome this problem. Thitarodes shambalaensis and Thitarodes sp. were inbred or hybridized, and the biological parameters, larval sensitivity to the fungal infection and mitochondrial genomes of the resulting populations were investigated. Hybridization of T. shambalaensis and Thitarodes sp. allowed producing a new generation. One hybrid population  showed increased population growth as compared with the parental Thitarodes sp. population. The sensitivity of the inbred larval populations to four fungal isolates of O. sinensis differed. The complete mitochondrial genomes of T. shambalaensis, Thitarodes sp. and the hybrid population were 15,612 bp, 15,389 bp and 15,496 bp in length, respectively. A + T-rich regions were variable in sizes and repetitive sequences. The hybrid population was located in the same clade with T. shambalaensis, implying the maternal inheritance of mitochondrial DNA.The Chinese cordyceps is a valuable parasitic Ophiocordyceps sinensis fungus–Thitarodes/Hepialus larva complex, is a valuable biological resource endemic to the Tibetan Plateau. Protection of the Plateau environment and huge market demand make it necessary to culture this complex in an artificial system. A method for the large-scale artificial rearing of the Thitarodes/Hepialus insect host has been established. However, the deterioration of the insect rearing population and low mummification of the infected larvae by the fungus constrain effective commercial cultivation. Hybridization of Thitarodes/Hepialus populations may be needed to overcome this problem. The species T. shambalaensis (GG♂ × GG♀) and an undescribed Thitarodes species (SD♂ × SD♀) were inbred or hybridized to evaluate the biological parameters, larval sensitivity to the fungal infection and mitochondrial genomes of the resulting populations. The two parental Thitarodes species exhibited significant differences in adult fresh weights and body lengths but not in pupal emergence rates. Hybridization of T. shambalaensis and Thitarodes sp. allowed producing a new generation. The SD♂ × GG♀ population showed a higher population trend index than the SD♂ × SD♀ population, implying increased population growth compared with the male parent. The sensitivity of the inbred larval populations to four fungal isolates of O. sinensis also differed. This provides possibilities to create Thitarodes/Hepialus populations with increased growth potential for the improved artificial production of the insect hosts. The mitochondrial genomes of GG♂ × GG♀, SD♂ × SD♀ and SD♂ × GG♀ were 15,612 bp, 15,389 bp and 15,496 bp in length, with an A + T content of 80.92%, 82.35% and 80.87%, respectively. The A + T-rich region contains 787 bp with two 114 bp repetitive sequences, 554 bp without repetitive sequences and 673 bp without repetitive sequences in GG♂ × GG♀, SD♂ × SD♀ and SD♂ × GG♀, respectively. The hybrid population (SD♂ × GG♀) was located in the same clade with GG♂ × GG♀, based on the phylogenetic tree constructed by 13 PCGs, implying the maternal inheritance of mitochondrial DNA.The Chinese cordyceps, a parasitic  Ophiocordycepssinensis fungus–Thitarodes larva complex) is a valued biological resource endemic to the Tibetan Plateau and widely used in medicinal treatments including fatigue, asthma, respiratory and kidney diseases and as health foods, especially in many Asian countries [The Chinese cordyceps . Thitarodes armoricanus Oberthür was the first host insect of O. sinensis in China to be reported and biologically characterized [O. sinensis have been reported [O. sinensis and its host insects [O. sinensis fungus have a very narrow distribution on the Tibetan Plateau, and host insect species might vary among different mountain ranges and even from different sides and habitats of the same mountain [O. sinensis fungus, fifty-seven are considered potential host species of the fungus and are distributed throughout the Tibetan Plateau [O. sinensis have never been confirmed by a hybridization technique. The insect host species of cterized . Researcreported ,14. Comp insects . It appemountain . Differemountain ,18,19,20mountain . Of nine Plateau . HoweverO. sinensis fungus and formation of a fruiting body from the infected insect cadaver. The method for the large-scale artificial rearing of the Thitarodes/Hepialus insect hosts is established [Thitarodes/Hepialus larvae are tender roots and buds of plants of the Polygonaceae family such as Polygonum viviparum, P. sphaerostachyum and P. capitatum, as well as other plants in nine different families  [Potentilla anserina and carrots (Daucus carota) are favorite larval foods for indoor cultivation [Thitarodes species have long and unusual life cycles; it takes 263 to 494 days for Thitarodes jianchuanensis and 443 to 780 days for T. armoricanus to complete the life cycle, including egg, larval instars L1-L9, pupa and adult; the larvae can develop into pupae from the L7, L8 or L9 instar [Artificial cultivation of the Chinese cordyceps in low-altitude areas is successful and contains three important milestones: mass production of the host insects, cultivation of effective ablished ,19,20. Ceraceae) . The rootivation ,23. Thit9 instar . How to O. sinensis strains from different locations in the Tibetan Plateau have been isolated [O. sinensis with mature ascospores by artificial media without living insects has also been successfully realized [O. sinensis fungus: the larvae are routinely infected by the mature ascospores collected from the wild Chinese cordyceps or from the artificial fruiting bodies, and the larvae are injected into the hemocoel with the blastospores from the liquid culture containing maltose as a carbon source [Candida albicans and Ustilago maydis [Metarhizium rileyi [M. anisopliae and Beauveria bassiana which cause the death of their host larvae within a few days [O. sinensis fungus isolate with high mummifying potential and a Thitarodes insect host species or strain with high sensitivity to the fungal infection should contribute to overcoming this obstacle in the artificial production of the Chinese cordyceps.Various isolated ,14,25 anisolated . Stable n source . In the n source  and may n source ,27, as ro maydis  and the m rileyi . The livfew days ,31. The few days ,11. SeleThitarodes/Hepialus mitochondrial genomes are reported, including Thitarodes renzhiensis , Thitarodes yunnanensis (former Ahamus yunnanensis)  [Thitarodespui  [Hepialusxiaojinensis  [Hepialus gonggaensis  [Thitarodes sejilaensis  [Thitarodes sp.  [Thitarodes damxungensis  [Mitochondrial genome sequences have been widely used as molecular markers for diverse evolutionary analyses because of their unique features, including coding content conservation, maternal inheritance and rapid evolution . Insect ,816 bp) ,36, Thit,928 bp) ,37, Hepi,397 bp) , Hepialu,280 bp)  and Thit,362 bp) . With reThitarodes insects. In this study, the hybridization between T. shambalaensis and an undescribed Thitarodes species from two different locations in the Tibetan Plateau was demonstrated. The fitness parameters , larval sensitivity to the fungal infection and mitochondrial genomes of the resulting inbred and hybrid populations were determined to evaluate the hybridization effects. Insight into the biological and molecular characters of the inbred and hybrid populations is elementary for the effective artificial cultivation and evolutionary analysis of these Thitarodes insect populations were, respectively, from the mountains in Gongga (referred to as GG♂ × GG♀)  and Shade (referred to as SD♂ × SD♀) , Kangding in Sichuan Province, China. The pupae of two Thitarodes pupae were differentiated by their genitalia. Briefly, in the last abdominal segment, females exhibit a long longitudinal suture linked to the previous abdominal segment without papillary structures, whereas males exhibit a short longitudinal suture between two papillary structures that is not linked to the previous abdominal segment [Thitarodes populations, Cytochrome b and cox1 sequences were amplified with the primers CB1 (TATGTACTACCATGAGGACAAATATC) and CB2 (ATTACACCTCCTAATTTATTAGGAAT) [The valve pattern of the male genitalia is an important characteristic for the morphological identification of Hepialidae insects ,43. The  segment . The malTAGGAAT) ,45 and LTAGGAAT) , respectFour inbred and hybrid combinations  were created with 50 female and 75 male adults for each combination, but the population GG♂ × SD♀ could not be established due to technical issues related to climatization of the culture room. Three replicates were set up for each combination. The male and female pupae were housed in cartons  with moist moss at 9–17 °C and 50–80% relative humidity. When the adults emerged, they were housed in small cylindric nets  to allow mating for 3–5 days. The collected eggs from the mated females were transferred to a culture room and maintained at 9–13 °C to establish the experimental populations in the Institute of Zoology, Dongguan (43 m above sea level), Guangdong Province, China.Thitarodes populations, 600 eggs from each inbred or hybrid combination were surface-sterilized for 3 min with a solution containing 2.5 mL of 4 M NaOH, 0.5 mL of 12% NaOCl and 21.5 mL of distilled water [Potentilla anserina roots as food at 9–13 °C. For each hybridization combination, 30 containers were established. When the larvae reached the third instar, they were individualized into a plastic cup  with the same peat and food as above (15 g coconut peat and 15 g food for each cup) to avoid larval cannibalism [To evaluate the development, survival, fertility and sensitivity to the fungal infection in the resulting ed water , rinsed nibalism . Fresh fO. sinensis isolated from the fruiting bodies of wild Chinese cordyceps, respectively, from Sichuan, Yunnan, Tibet and Qinghai, China, were cultured on PPDA medium  at 13 °C. The fungal isolates were identified by using the amplified sequence from the internal transcribed spacer  of the nuclear ribosomal DNA as described by [O. sinensis isolates were preserved at −80 °C in the Institute of Zoology, Guangdong Academy of Science, Guangzhou, China.KD, YN, XZ and QH fungal isolates of ribed by . The ide2PO4, 0.5 g MgSO4, 20 mg vitamin B1 and 1000 mL distilled water) [6 blastospores per mL and kept at 4 °C for less than 3 days before use for larval infection.The fungal colonies cultured on the PPDA plates at 13 °C for 60 days were transferred to 250 mL flasks containing 150 mL liquid PM medium  . The flaO. sinensis. An aliquot of 4 µL blastospore suspension containing 1.2 × 104 blastospores was injected into each 6th instar larva by a microinjection system . One hundred and eighty larvae were used for each replicate, and three replicates were set for each injection. Larvae injected with PBS buffer or without any injection were set as controls. The injected larvae were reared at 4 °C for one week and then transferred to a culture room at 13 °C. After 90 days, about 10 µL of hemolymph of each injected larva (6th instar) was sampled to confirm the presence of the growing blastospores stained by Calcofluor White  and observed by a fluorescence microscope . The injected larvae were reared at 13 °C until the larvae became stiff and were coated with growing mycelia. The mummified larvae with head upward were then planted into soil of 55–60% humidity to induce the formation of stroma at 4 °C. The survival and mummification of the injected larvae were monthly checked. Data on larval infection of the hybrid populations could not be gathered due to an insufficient number of larvae available for fungal injection.The larvae from 2 inbred populations  were injected with KD, YN, XZ or QH fungal isolates of T. damxungensis, T. gonggaensis, T. pui, T. renzhiensis, T. sejilaensis, Thitarodes sp., H. xiaojinensis, T. yunnanensis, Napialus hunanensis, Endoclita signifer). The location and secondary structures of the 22 tRNA were predicted by tRNAscan-SE (http://lowelab.ucsc.edu/tRNAscan-SE/) (accessed on 13 November 2020). After the removal of the termination codon, the codon usage frequency and the first, second and third base use frequency of the codon were calculated using MEGA 7.0. The overlapping regions and intergenic spacers between genes were manually counted. The entire A + T-rich region was subjected to a search for the tandem repeats using the Tandem Repeats Finder program [Three male adults from GG♂ and SD♂ and three larvae from SD♂ × GG♀ in dry ice were used for mitochondrial genome sequencing by Shanghai BIOZERON Co., Ltd., with the routine method . The nuc program .Drosophila melanogaster was used as an outgroup. A maximum likelihood (ML) tree was built in MEGA 7.0 using the nucleotide sequence of 13 PCGs, based on the “find best DNA/protein models (ML)”. The “GTR + G” model was chosen for phylogenetic analysis because it produced the lowest values for both the BIC (Bayesian information criterion) and the AICc (corrected Akaike information criterion). The confidence values of the ML tree were evaluated via a bootstrap test with 1000 iterations.To construct the phylogenetic relationships within Hepialidae in Lepidoptera, 10 complete mitochondrial genomes of the above hepialid species were downloaded from GenBank. II/PI, where PI = numbers of pupae in the parental generation, and PII = numbers of pupae in the next generation [p < 0.05.The data are expressed as means ± SE. The average survival rates, fresh weight, body length and sex proportions of pupae and adults at the sample time points were determined. In addition, a population trend index (I) was calculated with I = Pneration . The datThitarodes species, by the phylogenetic analysis, although the valve pattern of the male genitalia of SD♂ × SD♀ resembled that of Thitarodes kangdingensis . The SD♂ × SD♀ population from Sichuan Province, China, was considered to be an undescribed ingensis   was significantly higher than that for the other populations, whereas the number of eggs in one milliliter (3976 ± 109) was higher and the weight of one thousand eggs (0.18 ± 0.01 g) was lower for SD♂ × SD♀ compared with other populations, indicating the smaller egg size of SD♂ × SD♀ . The hatThitarodes sp. and T. shambalaensis allowed harvesting a next generation of adults at least from the SD♂ × GG♀ population.The GG♂ × GG♀ and SD♂ × SD♀ populations were hybridized in the culture room, and the larvae successfully became pupae in the resulting hybrid populations. The fresh weights of the larvae from the inbred and hybrid populations did not vary significantly with culture times in 12 months . The surAs shown in Thitarodes populations  were a circular DNA molecule of 15,389 bp, 15,612 bp and 15,496 bp in length, respectively   . Like mo uniform .Thitarodes populations was A + T-biased, ranging from 80.87% (SD♂ × GG♀) to 82.35% (SD♂ × SD♀)  . The A +♂ × SD♀) . LikewisNADH dehydrogenase subunits , 3 cytochrome c oxidase subunits (cox1-3), 2 ATPase subunits  and one cytochrome b gene (cytb). The lengths of the 13 PCGs in the mitochondrial genomes of SD♂ × SD♀, GG♂ × GG♀ and SD♂ × GG♀ were 11,073, 11,067 and 11,067, respectively  in the mitochondrial genomes of SD♂ × SD♀, GG♂ × GG♀ and SD♂ × GG♀ were 56.91%, 56.60% and 56.62%, respectively , reflectcox1) in the three mitochondrial genomes began with a canonical start codon (ATN or NTG). More specifically, eight PCGs  started with ATA, one PCG (nad3) with ATT, three PCGs  with ATG and one PCG (cox1) with CGA  were terminated with the typical stop codon TAA, while three PCGs  located upstream of tRNAs ended with T, and one PCG (nad3) ended with TAG (All PCGs (except with CGA . For thewith TAG . rrnL (16S rRNA) and rrnS (12S rRNA) genes were identified, being 1355 bp, 1336 bp and 1336 bp and 777 bp, 778 bp and 778 bp in size, respectively, falling into the reported range for the Hepialidae  [rrnL gene was located between trnL1 (TAG) and trnV (TAC), while rrnS was located between trnV (TAC) and the A + T-rich region (trnS1 (UCU), which lacks the dihydrouridine (DHU) loop   (Table 2h region . The A +HU) loop . This feNon-coding and overlapping genes. The complete mitochondrial genomes of SD♂ × SD♀, GG♂ × GG♀ and SD♂ × GG♀ were very compact with a total of 198, 190 and 188 non-coding bp dispersed among 20, 17 and 17 pairs of neighboring genes ranging from 1 to 42 bp, 1 to 45 bp and 1 to 45 bp, respectively . nad5 and trnH. A 15 bp intergenic spacer located between the trnS2 and nad1 contained the “ATACTAA” motif, which is a common feature across lepidopteran insects [trnL1 and rrnL. The atp8 and atp6 had 4 bp overlapping nucleotides  and three genomes from the present study was constructed based on the concatenated nucleotides’ alignment of 13 PCGs or each PCG by the ML method. SD♂ × SD♀ was classified into a separate clade and GG♂ × GG♀ and SD♂ × GG♀ into another separate clade  identified by morphology  and genetic cox1 phylogeny [Thitarodes species (SD♂ × SD♀) live in different locations in the Tibetan Plateau. In the present study, interspecific hybridization between these two distinct ghost moth species was demonstrated in the laboratory. The developmental performance of the studied Thitarodes populations was influenced by the hybridization, whereas the larval sensitivity to the fungal infection of the inbred populations was affected by the parental populations.hylogeny  and an uHelicoverpa armigera and Helicoverpa assulta [Nasutitermes corniger and Nasutitermes ephratae [Coptotermes formosanus and Coptotermes gestroi [Reticulitermes flaviceps and Reticulitermes chinensis [Solenopsis invicta, black imported fire ant Solenopsis richteri and their hybrid (S. invicta × S. richteri) are present in the field in Tennessee, USA [T. shambalaensis and Thitarodes sp. do not share a habitat in the Tibetan Plateau. It is speculated that reproductive individuals of the two ghost moth species may not have the chance to hybridize in nature because of the limited flying capacity of the adults. Surprisingly, these two species could mate, and the resulting hybrids produced a next generation. Whereas the inbred SD♂ × SD♀ laboratory population was very weak (I = 0.01), the SD♂ × GG♀ population exhibited a higher population trend index (I = 0.32), implying growth potential to a certain extent from GG♂ × GG♀, whose inbred population had a higher population trend index (I = 2.25). Hybridization may thus provide an effective method to create Thitarodes/Hepialus populations with increased growth potential for the improved artificial production of the insect hosts. Hybridization between two related insect species is common in the laboratory or in the field; for instance, interspecific hybridization has been reported between  assulta , Nasutitephratae , Coptote gestroi  and betwhinensis . The redsee, USA . The stuThitarodes species can be hybridized in the laboratory remains unknown. Species are defined to be groups of interbreeding natural populations that are reproductively isolated from other such groups [Thitarodes species occurred in the laboratory, not in nature. The resulting hybrids also produced a next generation, indicating that the post-zygotic reproductive isolation may not prevent hybridization between two Thitarodes species, even in nature. Thus, the successful hybridization of these two species should depend on overcoming the pre-zygotic reproductive isolation, especially geographical habitat isolation and mating behavior isolation. Although reproductive isolation can evolve in a number of different ways, species-specific mate recognition by sex pheromones is believed to be a key element [Thitarodes species can overcome the different genitalia structure and gamete isolation in the pre-zygotic phase and reproductive isolation in the post-zygotic phase in the laboratory. These results demonstrate the complexity of reproductive isolation and provide useful cues for further study in the speciation mechanism. Why these distinct h groups . The mech groups . Pre-zygh groups . Certain element . SimilarThitarodes/Hepialus mitochondrial genomes are sequenced, including T. renzhiensis, T. yunnanensis, T. pui, H. xiaojinensis, H. gonggaensis, T. sejilaensis, an undescribed Thitarodes sp. and T. damxungensis [T. shambalenensis, and SD♂ × SD♀ was considered to be an undescribed Thitarodes species  × Huso dauricus [Thitarodes hybrid and the populations sharing the same mother have different mtDNAs in genome length, A + T content and the sizes of the A + T-rich region needs further study. The three complete mitochondrial genomes of GG♂ × GG♀, SD♂ × SD♀ and SD♂ × GG♀ differ not only in the size of the genome but also in the A + T-rich region with repeat sequences. So far, eight ungensis . Based o species , given tilaensis  to 16,28odes sp. . Likewisodes sp.  or Acipedauricus  are variT. damxungensis [T. damxungensis (545 bp without repetitive sequences), T. pui (1030 bp with five 119 bp repeat units), T. sejilaensis (484 bp with four 118 bp repeat units), T. yunnanensis (1000 bp with four 107 bp repeat units), T. xiaojinensis (634 bp with four 118 bp repeat units), T. gonggaensis (1133 bp with six 112 bp repeat units), T. renzhiensis (1358 bp with eight 113 bp repeat units) and an undescribed Thitarodes. sp. (1472 bp with nine 112 bp repeat units) [The sizes of the A + T-rich region are 787 bp with two repetitive sequences of 114 bp, 554 bp without repetitive sequences and 673 bp without repetitive sequences in GG♂ × GG♀, SD♂ × SD♀ and SD♂ × GG♀, respectively. The A + T region of SD♂ × SD♀ contains non-repetitive sequences only, just like that in ungensis . It seemt units) . The relt units) .T. shambalaensis and Thitarodes sp. Secondly, we found that the offspring produced by hybridization may display increased growth potential at least from one of the parent populations, which would greatly improve the cultivation of Thitarodes insects for the artificial production of Chinese cordyceps. Finally, our study demonstrated that the mitochondrial genome from the hybrid is different from those of its parents in several features  and maternal inheritance.In conclusion, the present study demonstrated that interspecific hybridization occurred under laboratory conditions between two allopatric and morphologically distinct ghost moth species


Meditation and acupressure-like stimulations have been shown to relieve pain. The aim of this study was to determine whether a short bout of mind-body intervention combined with meditation and acupressure-like stimulation was able to alleviate shoulder pain and improve its function in a short time window. Sixty-five adults with shoulder pain were recruited and randomly classified into two groups. One group participated in an intervention which consisted of acupressure-like stimulation and meditation over a 5 min period. The other group was instructed to rest during this time. A visual analog scale (VAS) pain score and objective constant scores were measured before and after intervention to determine shoulder pain and range of motion (ROM), respectively. A two-way repeated measures analysis of variance with Bonferroni correction and a regression analysis were performed. VAS pain, objective constant score, flexion, abduction, and external rotation score showed significant interactions between time and group. The pain intensity was significantly reduced, while flexion and abduction were significantly improved, in the experimental group compared to the control group, after the intervention. In addition, the change of flexion negatively correlated with the change of pain intensity in the experimental group, but not in the control group. These results show that a short-term application of mind-body intervention significantly alleviates shoulder pain and improves shoulder movement, suggesting its potential use as a therapy for people with shoulder pain. Shoulder pain is one of the most common musculoskeletal complaints. A significant percentage of patients seeking medical attention have shoulder pain. The reported lifetime prevalence of shoulder pain ranges from 6.7% to 66.7% of the population . The shoMeditation shows promise for alleviating chronic pain , 8. In aAcupressure has been studied extensively as a method for pain management. A meta-analysis of 15 studies showed that acupressure is effective for relieving a variety of pains including dysmenorrhea, labor pain, low back pain, chronic headache, and other traumatic pain . In a raAs described above, mind-body interventions such as meditation and acupressure have the potential to relieve pain intensity and improve joint mobility. In this study, we performed a randomized controlled trial for people with shoulder pain under the hypothesis that a short-term intervention which combined acupressure-like stimulation and meditation may help to relieve shoulder pain and improve shoulder function.t-test model (g-power software 3.1.9.7). Considering the dropout rate, a total of 65 participants were recruited. Sixty-five people volunteered for the study and were randomly divided into two groups: the experimental (N = 33) and control (N = 32) groups. Two and one individuals were dropped from each group, respectively, due to low pain intensity (VAS pain score <4) during ROM measurement . The inclusion criteria were as follows: people with shoulder pain for at least 2 months, people who consented to participate in the research voluntarily, and people who could hear the explanation of the experiment (are not hearing impaired), read, understand the manual, speak their opinions, and follow the instructions. Exclusion criteria were as follows: people with shoulder pain due to rheumatic arthritis, osteoarthritis, bone defect injury, osteoporosis, and malignant tumor and people with low pain intensity (VAS pain score <4) during shoulder movement of flexion, abduction, internal rotation, and external rotation. Based on a pilot study, a sample size of 27 participants per group was sufficient to give over 90% of power with an alpha level set at 0.05 for a two-tailed unpaired surement .All participants were given an explanation of the experiments and participated in the measurement of four subscales , and strength) of the constant score and VAS pain intensity. Constant score has been adopted as an official tool for assessing the shoulder by the European Society for Surgery of the Shoulder and the Elbow . The intBrain education meditation (BEM)  is a modernized mind-body training method which is rooted in Korean Sundo tradition and consists of several techniques including exercises which improves the connection between the brain and body, such as qigong, breathing postures, body awareness meditation, and brain wave vibration meditation with rhythmic body movement –28, and BHP meditation consists of acupressure-like stimulation and breathing/body awareness meditation. The first step in BHP meditation is to find a “healing point,” where subjects report more pain than other places when a specific part of the body is pressed. This point is called the BHP point and can be found anywhere on the body. BHP meditation first applies acupressure-like stimulation and then induces relaxed attention by body awareness and imagery meditation combined with breathing . In thisA waitlist control group design was employed. Participants in the control group were seated and asked to relax for 5 min. Data were collected from the control group on the same schedule as the experimental group. Control participants were offered the BHP mediation program only after the experiments. No further data were collected from control participants at this time point.The Constant score was divided into four subscales, including pain during the last 24 h (15 points maximum), activities of daily living for the last one week (20 points maximum), range of motion , and strength (25 points maximum) . For ROMt distribution after Bonferroni adjustment. Regression analysis was performed to confirm the relationship between the change of pain intensity and the change of each objective constant score affected by the intervention.The association between BHP meditation and shoulder pain was analyzed using two-way repeated analysis of variance (ANOVA), including the between-subjects factor of intervention condition (BHP meditation or control) and the within-subjects factor of assessment time. For the constant scores, the assessments included pretreatment scores (before the BHP meditation session) and posttreatment scores (after the BHP meditation session). Multiple comparisons were corrected using critical values from the p=0.8041, χ2 test, p=0.5161, Student's t-test). The subjective subscales of constant score, including sleep, work, recreation, position, and pain scores, were not significantly different between the two groups, as given in A total of 65 subjects participated in the current study. The distribution of gender was similar in both groups  = 51.32, p=1.31 × 10−9, ηp2 = 0.461, F  = 16.69, p=1.33 × 10−4, ηp2 = 0.218, p=3.76 × 10−4, F  = 28.36, p=1.59 × 10−6, ηp2 = 0.321, F  = 16.44, p=1.47 × 10−4, ηp2 = 0.215, F  = 21.13, p=2.26 × 10−5, ηp2 = 0.26, F  = 36.07, p=1.20 × 10−7, ηp2 = 0.38, F  = 16.04, p=1.49 × 10−4, ηp2 = 0.21, F  = 24.73, p=5.84 × 10−6, ηp2 = 0.29, F  = 21.89, p=1.69 × 10−5, ηp2 = 0.27, F  = 5.54, p=2.18 × 10−2, ηp2 = 0.08, p=7.27 × 10−5; experimental group = 143 ± 2°, control group = 120 ± 3°, p=6.36 × 10−4, respectively, with Bonferroni post hoc test).The VAS pain score was analyzed via a two-way repeated measures ANOVA with group  and time (pre/post) as factors. Pre/postmeasurement results in experimental and control groups are given in Supplementary R2 = 0.15, p=0.03) in the experimental group , BHP affected flexion and abduction, but not internal and external rotation. Each motion uses the following muscles: anterior deltoid, pectoralis major, and coracobrachialis for flexion ; supraspFurthermore, we found that pain intensity was negatively correlated with flexion in the BHP intervention group . PreviouThe short nature of the intervention in this study currently only provides insights into the short-term effects of BHP intervention. In this study, the single short-term BHP intervention contributes to shoulder pain reduction and improvement in function. We surmise that the observed effects are likely to be direct responses of BHP intervention as they occurred within a short time window . However, it is not known if BHP intervention over a longer-term period would retain its effectiveness as a treatment for shoulder pain; thus, data on the effectiveness of longer-term BHP interventions remains to be examined in a future study.The method of recruiting participants is an additional limitation in this study. The participants applied for care on their own initiative and, thus, may have a positive attitude toward the treatment. This positive attitude may have created a group with high expectations. In addition, the control group did not receive a placebo. To minimize bias, the patients were asked not to reveal information to the evaluators about the treatment to which they had been assigned. We used an intention-to-treat analysis, provided standardized information to the participants, and used reliable and valid outcome measures. However, as the control group was a rest control rather than an active control or a placebo group, we cannot confirm if the effects of the intervention would be better than the placebo or other preexisting methods.This RCT revealed that a single short-term BHP meditation not only relieves pain but also improved ROM  performance. Interestingly, flexion performance was found to be significantly related to pain reduction. These results indicate that pain reduction through BHP meditation has a direct effect on improving shoulder range of motion. Further studies utilizing active controls or comparisons against other therapies would provide more insight into the efficacy of BHP as a therapy for shoulder pain. Also, studies of long-term BHP intervention could validate that this intervention is effective in prolonging the effects of pain reduction and shoulder mobility observed here. The reduction of shoulder pain and functional improvements by BHP intervention would significantly contribute to improving the quality of life for patients with chronic shoulder pain.


Dromiciops gliroides) is the last living representative of the order Microbiotheria and is known to express both short torpor episodes and also multiday torpor depending on environmental conditions. However, only limited laboratory experiments have documented these patterns in D. gliroides. Here, we combined laboratory and field experiments to characterize the heterothermic responses in this marsupial at extreme temperatures. We used intraperitoneal data loggers and simultaneous measurement of ambient and body temperatures  for analyzing variations in the thermal differential, in active and torpid animals. We also explored how this differential was affected by environmental variables , using mixed-effects generalized linear models. Our results suggest that: (1) individuals express short bouts of torpor, independently of TA and even during the reproductive period; (2) seasonal torpor also occurs in D. gliroides, with a maximum bout duration of 5 days and a mean defended TB of 3.6 ± 0.9°C ; (3) the best model explaining torpor occurrence (Akaike information criteria weight = 0.59) discarded all predictor variables except for photoperiod and a photoperiod by food interaction. Altogether, these results confirm that this marsupial expresses a dynamic form of torpor that progresses from short torpor to hibernation as daylength shortens. These data add to a growing body of evidence characterizing tropical and sub-tropical heterothermy as a form of opportunistic torpor, expressed as daily or seasonal torpor depending on environmental conditions.Hibernation  is considered an adaptive strategy of mammals to face seasonal environmental challenges such as food, cold, and/or water shortage. It has been considered functionally different from daily torpor, a physiological strategy to cope with unpredictable environments. However, recent studies have shown large variability in patterns of hibernation and daily torpor (“heterothermic responses”), especially in species from tropical and subtropical regions. The arboreal marsupial “monito del monte” ( TB) approaching ambient temperature (TA) and torpor bout duration of several days or weeks. This has been classically referred as “hibernation” or “seasonal torpor”  express transient periods of metabolic depression (known as “torpor” or “heterothermy”) as adaptations to seasonal or unpredictable changes in environmental productivity . When to torpor” . NeverthTB reductions of ~20°C) to 5.1% in hibernators , displaying seasonal torpor (Elephantulus spp.) expressing characteristics of daily torpor (bout duration) and hibernation  showing flexible heterothermic phenotypes  known to express torpor bouts of up to 120 h , which have remained unclear since the first description by Here, we analyzed the natural heterothermic patterns of D. gliroides , including Andean forests where freezing temperatures are common in winter  was first described by  to the euthermic metabolic curve , the torpid animal stops passive cooling  D. gliroides is an opportunistic hibernator that could express short and multiday torpor depending on environmental conditions; (2) short torpor shifts into multiday torpor as the cold season progresses, primarily induced by shortening daylength and reduced food availability, and (3) D. gliroides has a critical torpor temperature slightly higher than zero degrees, thereby thermoregulating to control its TB above freezing temperature.In this study we analyzed a suite of torpor thermoregulatory patterns in D. gliroides individuals  over 2 years. Animals were captured in San Martin Biological Station (39°38'50.71' S 73°11'46.43' O) in February 2019. No animal was harmed in these procedures, the data loggers were safely removed, and all animals were released at the place of capture after finishing the study. We followed the guidelines of the American Society of Mammalogists for the use of wild mammals in research  and by the Bioethics Committee of the Austral University of Chile (resolution 313/2018 annex 2019). We performed the experiments in eight research . Since wn = 25) were captured in San Martin in February and maintained in enclosures, for another study. In one of the enclosures this year , one of the females gave birth to two pups of approximately 4 mm, which were discovered on December 10 , we found the whole cluster of five animals torpid, including the female and the two pups  dataloggers  into the abdomen  of four individuals in March 2019 (autumn) and removed them in November (spring). According to the manufacturer, the devices are calibrated at factory over a temperature range of −10–50°C. We calibrated the devices in a beaker with water at 40°C that was allowed to cool to room temperature (10°C), with temperature records every 2 min, using a laboratory thermometer . The linear regression between water and logger temperature (20 points) was highly significant . We set the dataloggers for hourly recordings during the first winter. The next winter, we reused them in other two animals for one-month ramp experiments designed to determine torpor patterns at a broad thermal range (see details in the “ramp experiment” section below). We removed dataloggers from these animals after 1 month of continuous measurements. For both implantation and removal, we used subcutaneous tramadol 5 mg kg−1, plus inhalation anesthesia for induction (isoflurane in oxygen 5%) and maintenance (isoflurane in oxygen 2.5%). We then administered subcutaneous meloxicam 0.5 mg kg−1. We have previously sterilized the dataloggers with formaldehyde gas and UV exposure for 15 min and then set them to record temperature hourly. The surgical approach consisted in a small incision (3 mm) on the abdominal region in their median plane. The device was delicately placed perpendicularly to the body axis, between the layers of the peritoneum. The wound was closed with a stitch using sutures that are self-absorbing, both in the muscular plane and in the skin. Every time, we maintained the animals in the clinic for 2 weeks for recovery after the surgery. No animal was harmed in any of these surgeries. Surgical procedures had a duration of 5 min per animal and were led by the third author (AE), an expert veterinarian in charge of a center for wildlife recovery (CEREFAS-UACh).We surgically implanted body temperature (D. gliroides habitat (3 of volume) and included twigs and plant material mimicking this species’ microenvironment. It had a mesh that allows light and ventilation to enter a similar way under the forest canopy, checked with a luxmeter (TASI 21®). We also included one temperature datalogger  set for recording air temperature (TA) every 15 min and located at 1 m above ground in the shadow. During the outdoor experiment , we recorded a mean monsthly TA ranged from 17.1°Cmax and 3°Cmin (the TA range for one of the enclosures is shown in Results), and the seasonal change in photoperiod was 4.5 h . Each individual was released in one different enclosure, where food was provided daily: two of the animals received “restricted” food , and the other two received ad libitum food . Food consisted of a mix of cereals, jam, and tuna that provides balanced nutrition for this omnivorous species  and body mass change from the last week. We replaced food plates every day to ensure animals received the constant amounts.Water was provided  species . Energy “ramp experiment” to determine torpor at near-zero TA. We performed the ramp experiment  and photoperiod accordingly . In a few cases, the cooling system froze, raising the set point’s temperature, which did not produce a perfect ramp. Animals did not arouse during these short TA increments. During the ramp experiment, we offered water ad libitum but removed food. Also, we maintained animals in separated cages within the incubator. After the “cold” ramp, animals remained in the incubator at 15°C for 1 week. Then, we assayed the “warm ramp,” which consisted of a similar ramping rate as before but increasing temperature from 15°C until 28°C. No animals were harmed during these ramps and were released in the capture site in spring  after checking their physiological condition by a trained veterinarian.Given that some of the dataloggers still had operational batteries, we reused them in the 2020 winter, with two newly captured animals in a controlled TBmin), we performed linear regressions between the thermal differential  and TA with the data points of the plot where TDIFF is inversely proportional to TA during torpor. That is, we amplified each graph and took every point that fell in the bottom-left edge of the figure. To estimate torpor thermoregulation regressions, we predicted the RMR as RMR = Cmin. Then, to determine the combination of variables that trigger torpor, we used TDIFF as a continuous response variable associated with fixed and random factors. We used TDIFF because we got non-convergent models when using it as a dichotomic variable . Each record was associated with the fixed factors: food (low/high), TA (°C), photoperiod (daylength in hours), delta photoperiod (difference in daylength from the day before in hours), MB (g), and ∆MB (difference MB with the previous week). Then, we fitted mixed-effects generalized linear models (GLMM) with a Gaussian error distribution, including individual ID, sampling week, and date as random effects to account for inter-individual and inter-enclosure variability along with the repeated measures in time . Here, we present the best nine models:The four individuals used for the outdoor experiment produced hourly records of  in time . We usedTDIFF ~ food + (1 | ID) + (1 | Date)→ Model 1: TDIFF ~ food + TA + (1 | week) + (1 | ID) + (1 | Date)→ Model 2: TDIFF ~ food + TA + photoperiod + (1 | week) + (1 | ID) + (1 | Date)→ Model 3: TDIFF ~ food + TA + photoperiod + ∆MB + (1 | week) + (1 | ID) + (1 | Date)→ Model 4: TDIFF ~ food × TA + (1 | week) + (1 | ID) + (1 | Date)→ Model 5: TDIFF ~ food × photoperiod + (1 | week) + (1 | ID) + (1 | Date)→ Model 6: TDIFF ~ TA × photoperiod + (1 | week) + (1 | ID) + (1 | Date)→ Model 7: TDIFF ~ TA × ∆MB + (1 | week) + (1 | ID) + (1 | Date)→ Model 8: TDIFF ~ photoperiod × ∆MB + (1 | week) + (1 | ID) + (1 | Date)→ Model 9: The model including all the interactions was discarded since it gave a variance inflation factor (VIF) = 2048.We estimated GLMM parameters and their significance using a restricted maximum likelihood approach with a Kenward–Roger approximation to estimate degrees of freedom . We perfWe detected a mean of 150 torpor bouts of 13.4 h of duration in four individuals at the outdoor experiment, with interbout euthermic periods of 13.6 h .n = 25 individuals) that increased in winter, but was never zero even in the warmest days of the summer  below, in which torpid animals start thermoregulating  = 0.62 ± 0.2 (kJh−1) – 0.15 ± 0.03 × TA . This implies that torpid individuals, once reaching the critical TB of 3.6°C, start generating 0.15 kJ per hour per every 1°C reduction. These RMR patterns were quite less variable in the chamber experiment  was RMR is a combination of food and photoperiod (ωAIC = 0.59).The multi-model analysis indicated that the most important variable explaining torpor occurrence (measured as reductions in MB) and photoperiodic changes were dropped from the models, thus not significantly contributing to the observed variation in TDIFF. According to the obtained coefficients, the most important factor explaining torpor occurrence was photoperiod’s main effect, with a coefficient of 9.9 ± 1.5  and endured sub-freezing temperatures during 24 h. These individuals generated 0.15 kJh−1, per every Celsius degree reduced . In daily heterotherms for instance, these parameters are TBmin = 8.3°C, and the slope of thermoregulation regression = 0.004 kJh−1°C−1 for the Rufous-tailed hummingbird  in winter, TBmin = 11°C and the thermoregulatory slope is 0.10 kJh−1°C−1 , which is considered an extreme hibernator, the slope of the thermoregulatory regression was 0.17 kJh−1°C−1 , Futaleufú in Chile, and Parque Nacional Los Alerces in Argentina  on a weekly basis , and by the Bioethics Committee of the Austral University of Chile (resolution 313/2018 annex 2019).RN conceived the study, designed the methodology, and wrote the first draft of the manuscript. CM and AE contributed to the field and laboratory work, and manuscript editions. ER, FB, and FF contributed with the statistical analyses and manuscript editions. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pseudomonas species. Further, several PhaC genotypes were discovered within Thaumarchaeota, an archaeal phylum with poly-extremophiles and the ability to efficiently use CO2 as a carbon source, a significant ecological group which have thus far been little studied for PHA production. Bacterial and archaeal PhaC genotypes were also observed in high salinity and alkalinity conditions, as well as high-temperature geothermal ecosystems. These genome mining efforts uncovered previously unknown candidate taxa for biopolymer production, as well as microbes from environmental niches with properties that could potentially improve PHA production. This in silico study provides valuable insights into unique PHA producing candidates, supporting future bioprospecting efforts toward better targeted and relevant taxa to further enhance the diversity of exploitable PHA production systems.The accumulation of petroleum-based plastic waste has become a major issue for the environment. A sustainable and biodegradable solution can be found in Polyhydroxyalkanoates (PHAs), a microbially produced biopolymer. An analysis of the global phylogenetic and ecological distribution of potential PHA producing bacteria and archaea was carried out by mining a global genome repository for PHA synthase (PhaC), a key enzyme involved in PHA biosynthesis. Bacteria from the phylum Actinobacteria were found to contain the PhaC Class II genotype which produces medium-chain length PHAs, a physiology until now only found within a few  Plastic waste pollution has become a significant issue, with plastic waste as of 2015 amounting to 6,300 million metric tons predicted to almost double to 12,000 million metric tons by 2050, with only 9% being recycled, 12% disposed of through incineration and 79% of which is sent to landfill or ending up in the natural environment . The disAlcaligenes latus, Ralstonia eutropha, Azotobacter beijerinckii, Bacillus megaterium and Pseudomonas oleovorans  are a group of microbially-made polyesters: 100% biodegradable, thermoplastic, insoluble in water, non-toxic and biocompatible. The major advantage for waste management is that PHA products are 100% biodegradable (compostable bioplastics) in the land and ocean, leaving no lasting waste management footprint. As such these biopolymers are well suited as a “green” alternative to petroleum-based plastics by being both biodegradable and non-toxic . Prokaryeovorans , as welleriaceae . PHAs areriaceae . The careriaceae .Pseudomonadaceae  PHAs consist of 3–5 carbon atoms, medium-chain length (MCL) PHAs with 6–14 carbon atoms and PHAs with > 14 carbon atoms are considered long-chain length (LCL) . The mecnadaceae . LCL-PHA strains  and have strains .The enzyme responsible for the polymerization of monomeric substrates during PHA biosynthesis is PHA synthase (PhaC) which is organized into four classes depending on their primary protein structure, substrate specificity and subunit composition . Class IMore than 150 unique monomers have been discovered within PHA polymer samples since the discovery of PHA . PHA polin silico bioprospecting where collections of genome sequences are searched for putative enzymes involved in the biosynthesis of secondary metabolites  and unreviewed (TrEMBL) results  and 1932 archaea (accessed 20th Feb 2021) genome sequences along with their associated taxonomic and ecosystem metadata were obtained from the IMG/M dataset , 2. Due  results . The bacThe sequences were first manually curated through visual inspection of the metadata. The following categories of sequences were removed through inspection of the protein name: (1) Names determined to not be PhaCs, (2) Names with ambiguous function, (3) Putative PhaCs and (4) Fragment PhaCs. PhaC sequences with amino acid lengths < 328 in bacteria and < 100 in archaea were also filtered out as “fragments.” Duplicate protein sequences were removed. Subsets of the PhaC sequences created according to the class listed in the protein name field from the respective metadata entry. Where the protein name entry did not identify the class, the TIGRFAM metadata entry was used to assign the PhaC class.via BlastP with e-value = 1 × 10–10 against the unclassified PhaC sequences of the respective taxonomic domain. The unclassified sequences were then classified according to the class of the query sequence with the lowest e-value. In cases where multiple hits achieved the lowest e-value, the query sequence with the highest bitscore was used for classification. The final curated protein sequence dataset used for the prediction of PhaCs in the genome datasets were 2,799 bacterial PhaCs  and 140 archaeal PhaCs . The final curated bacteria and archaea query PhaC protein sequences and associated metadata are available in BLAST + v2.10.1 was used to classify the remaining unclassified PhaCs . The com–10. A single best hit was chosen from each genome as the representative PhaC protein sequence for use in downstream phylogenetic comparisons by selecting the lowest e-value result. In cases where multiple hits achieved the lowest e-value, the hit with the highest bitscore was used as the representative predicted PhaC sequence. The UniprotKB ID of the matching query, as well as PhaC Class, were appended onto the taxonomic and ecosystem metadata of IMG genomes for identified PhaC genotypes and can be found in via Kalign 3, with default settings (6 online platform (7 (iTOL) online tool  of the genomes. From this subset of genomes with PhaC genotypes, 2339 Class I, 529 Class II and 1,229 Class III PhaCs were identified according to the class of the matching query sequence used in the BLAST search  of the Class I containing genomes with the remaining 1.8% comprising of Actinobacteria (n = 26), Bacteroidetes (n = 12), Candidatus Dadabacteria (n = 1), Chloroflexi (n = 1) and Spirochetes (n = 1). Within Class II PhaC identified genomes, Proteobacteria were also dominant appearing in 81.5% (n = 431) of the genomes with the remaining 18.5% from Actinobacteria (n = 97) and Bacteroidetes (n = 1). Class III PhaCs in comparison were identified in a much larger variety of phyla although Proteobacteria still remaining the largest contributor at 51.8% (n = 637), followed by Actinobacteria 27.7% (n = 341), Firmicutes 12% (n = 148), with the remainder consisting of established phyla 5.4%; Acidobacteria (n = 5), Bacteroidetes (n = 13), Chloroflexi (n = 9), Cyanobacteria (n = 17), Elusimicrobia (n = 3), Gemmatimonadetes (n = 2), Nitrospirae (n = 3), Planctomycetes (n = 3), Spirochetes (n = 9), Verrucomicrobia (n = 2) and from candidatus or unknown phyla 3.1%; candidate division NC10 (n = 1), Candidatus Blackallbacteria (n = 3), Candidatus Dadabacteria (n = 1), Candidatus Falkowbacteria (n = 3), Candidatus Kaiserbacteria (n = 2), Candidatus Melainabacteria (n = 3), Candidatus Moranbacteria (n = 1), Candidatus Riflebacteria (n = 1), Candidatus Rokubacteria (n = 15), Candidatus Wallbacteria (n = 1), and unclassified (n = 6).From the initial bacterial genome dataset PhaCs were identified in 24.8%  present in “Freshwater,” 30.4% (n = 232) in “Soil” and 21.8% (n = 167) from “Marine” ecosystem types. Class I were also present in other aquatic ecosystem types consisting of; 5.2% (n = 40) “Non-marine saline and alkaline,” 5.1% (n = 39) “Sediment” and 2% (n = 15) “Thermal springs.” The remaining 2.7% appeared in small amounts from terrestrial environments; (n = 5) “Agricultural field,” (n = 1) “Deep subsurface” and (n = 1) “Oil reservoir.” Within Class I bacterial genotypes, the presence of Proteobacteria were prevalent across all observed ecosystem types, with trace representations of Actinobacteria in “Soil” and “Thermal springs,” Bacteroides in “Freshwater” and “Soil,” Chloroflexi in “Marine,” Spirochetes in “Freshwater” and Candidatus Dadabacteria in “Soil” (Class I PhaC genotypes were most abundant in diverse environments with 32.8% (n “Soil” .n = 67) representation with the second and third largest percentage consisting of diverse aquatic environments, 18.4% (n = 21) “Freshwater” and 9.6% (n = 11) “Marine” ecosystem types. The rest of the Class II genotypes were found in the remaining 7% terrestrial environments; (n = 4) “Geologic,” (n = 3) “Agricultural field,” (n = 3) “Peat” and (n = 1) “Mud volcano” as well as 3.6% aquatic environments; (n = 2) “Non-marine saline and alkaline,” (n = 1) “Sediment” and (n = 1) “Thermal springs.” From the Class II genotypes, Proteobacteria was present across almost all observed ecosystem types, with Actinobacteria being the only other phylum present, found mostly within “Soil” and in trace amount across “Freshwater,” “Marine” and “Geologic” and the sole representative within “Mud volcano”  in “Soil,” 25.9% (n = 96) “Freshwater” and 16.6% (n = 61) from “Marine” ecosystem types. Class III genotypes were present in other aquatic environments with relative abundances of; 5.7% (n = 21) “Non-marine saline and alkaline,” 3% (n = 11) “Thermal springs,” 1.4% (n = 5) “Sediment” and 0.3% (n = 1) “Aquaculture” with the remaining 1.8% from terrestrial ecosystem types; (n = 2) “Agricultural field,” (n = 2) “Geologic” and (n = 1) “Cave.” Compared to Class I and II, Class III genotypes were highly diverse across the observed ecosystem types  Class I, 5.4% (n = 6) Class II and 10.8% (n = 40) Class III PhaC genotypes were found in ecosystems with extreme physicochemical conditions. The most common niche environments present across all bacterial PhaC genotypes were high salinity and alkalinity conditions found in “Non-marine saline and alkaline” ecosystem types and the unique high-temperature geothermal environments found in “Thermal springs” and ‘Mud volcano” ecosystem types as well as “Hydrothermal vents” found in “Marine” ecosystem types. Bacteria PhaC genotypes were also found in acidic, desert, saline or permafrost environments, as well as ecosystems with petroleum contamination: “Oil reservoir” and “Creosote-contaminated soil.” The distribution of phyla across these niche ecosystem settings for Class I, II and III bacterial genotypes can be found in Environmental bacterial PhaC genotypes also investigated to determine whether they were recovered from unusual or niche settings . Within n = 338) of the samples (n = 1) belonged to the phylum Euryarchaeota. Class III was the dominant PhaC genotype appearing within the archaea genomes with 65.4% belonging to Euryarchaeota (n = 217), 30.1% from Thaumarchaeota (n = 100), 3% from Crenarchaeota (n = 10) and the remaining 1.5% unclassified (n = 5).In the maximum-likelihood tree, most of the archaeal PhaC protein sequences belonged to Class III PhaCs, with the few Class I and unclassified Class PhaCs sequences appeared to group closely together apart from the Class III PhaCs . Due to  samples . The soln = 60) “Marine,” 27.4% (n = 56) “Rock-dwelling” and 21.6% (n = 44) “Non-marine saline and alkaline” ecosystem types. Contrary to the bacteria PhaCs, archaea Class III PhaCs genotypes were less represented in the diverse ecosystem with abundances of 8.3% (n = 17) in “Freshwater” and 4.9% (n = 10) from “Soil” with the remaining found in 3.4% (n = 7) “Thermal springs,” 3.4% (n = 7) “Geologic” and 1.6% (n = 3) from “Sediment” ecosystem types. Euryarchaeota were present across all observed ecosystem types, with Thaumarchaeota present in “Freshwater,” “Marine,” “Thermal springs” and “Soil,” Crenarchaeota present solely in “Marine,” and unclassified archaeal phyla within “Marine” and “None-marine saline and alkaline” (Within the archaeal PhaC identified genomes, only Class III PhaC genotypes had representative samples from environmental settings according to the GOLD metadata listed in lkaline” .Over half of the archaea Class III genotypes were found in extreme settings with 57.8% of all archaea PhaC environmental genomes from ecosystems with extreme physicochemical conditions . The twoin silico microbial bioprospecting method presents a broad categorization method that inexpensively searches publically available data for PHA producing candidates across multiple bacterial and archaeal phyla. Through this approach, we were able to visualize the presence of different PhaC classes amongst various phyla and ecosystems. The resulting data provided insights on the general distribution of PhaC genotypes including ecological “hot spots,” as well as presenting microbial candidates that could improve current PHA production systems by utilizing cheaper feedstock or providing more efficient operating conditions.The genome mining effort has provided an encompassing view of the phylogenetic and ecological distribution of bacterial and archaeal PhaC genotypes. This 8 through the standardization of bacterial taxonomy (in silico observations.PhaC genotypes appeared in a broad number of bacterial and archaeal genomes, consisting of 24.8 and 17.5%, respectively of the genome datasets, suggesting that PHAs are a relatively conserved form of carbon energy storage within bacteria  and archtaxonomy . Class Itaxonomy . Combinetaxonomy . The finphaC has been identified as the hub gene and plays a major role in cluster formation in prokaryotes (The dominance of Proteobacteria across Class I and II and roughly half of Class III PhaC genotypes suggest that PHA production is highly conserved within the phylum. The prevalence of Proteobacteria as PHA producers was similarly observed in a study investigating PHA accumulating organisms in a mixed microbial culture, with Proteobacteria accounting for nearly 88% of the microbial community when the PHA content was at its peak level . Proteobkaryotes . FurtherPseudomonas which consists of almost all of the Proteobacteria PhaC II genotypes that were found in this study (Pseudomonas PHA production, meaning cheaper compatible feedstocks were needed to be found (A potential MCL-PHA producing alternative the study discovered is bacteria from the phylum Actinobacteria, which was amongst the Class II PhaC genotypes that were identified in our studies. Class II PhaCs are of particular interest as they are the only PhaC class capable of producing MCL-PHAs and bacteria that generate MCL-PHAs are mainly is study . MCL-PHAbe found , or recobe found . Actinobbe found . Since rbe found .2 fixation pathway is highly efficient even in nutrient-limited conditions within Thaumarchaeota, meaning it could potentially utilize CO2 as a cheap and sustainable feedstock for PHA production (Roughly two-thirds of the archaea PhaC genotypes found in this study were from the phylum Euryarchaeota, of which most of the constituents were class Halobacteria. The abundance of Halobacteria PhaC genotypes and the favourability of extremophiles to work in broad conditions is reflected in the almost exclusive focus on haloarchaea in the study of archaea PHA producers . Our seaoduction . With a The environment with extreme conditions that PhaC genotypes from both bacteria and archaea were observed to appear most in was the “non-marine saline and alkaline” ecosystem type. Soda lakes are the prime example of this ecosystem type being saline, high pH environments with high amounts of available carbon in the form of carbonates and bicarbonates, often with limited levels of nitrogen compounds—conditions that favor the production of PHAs . DespiteAnother extreme physicochemical environment of note from our findings into the ecological distribution of PhaC genotypes were geothermal ecosystems such as hydrothermal vents or hot springs. Hydrothermal vents are areas of the ocean floor where seawater percolates through cracks in the crust and are superheated by rocks in contact with magma . The areHaloferax mediterranei, a species of halophilic archaea, changing stressors such as increasing salinity (Over half of the archaea PhaC genotypes were found in extreme environments, which may indicate that taxa that commonly contain extremophiles may have developed physiological strategies utilizing PHA production. Within extreme environments, PHA production in microbial extremophiles acts not only as a carbon energy reserve but may also provide a protective role by maintaining cellular and membrane integrity against extreme physiochemical conditions . PHA hassalinity  or makinsalinity  can promPhaC genotypes discovered from these extreme environments could prove beneficial for cost reduction in industrial PHA production. Factors that contribute to the high cost of PHA production include maintaining a closed, contamination-free environment for fermentation/growth, expensive feedstock and difficult PHA extraction processes . UtiliziJanthinobacterium sp. isolates recovered from Antarctica were found to contain hitherto uncharacterized putative PHA synthase genes that were phylogenetically divergent from the known PhaC classes and tentatively labeled as “Class V” PhaCs (phaC genes (in silico based approaches are effective for diversifying PHA production methods (in silico screening methods for directing future strategies toward selecting promising candidates and novel synthases for PHA production strategies.Studies screening for PHA synthase sequences recovered from niche and under-represented environments has yielded success in novel insights toward diversifying PHA production methods. Genomes from V” PhaCs . The firaC genes . A study methods . The afoin silico screening methods to screen for putative PHA and β-galactosidase genes in a select number of microorganisms from literature to find PHA producers that could hydrolyze lactose for utilizing excess whey produced from the dairy industry as a cheap feedstock alternative. In comparison, our study has presented a broader scope by using genomes from large-scale data repositories in the public domain, allowing researchers access to a wider array of candidates which can be sorted by PhaC class, taxonomy and ecological information. Through this resource, users can filter for candidates of interest for and can locate the resulting genome for further analysis of metabolic activity relevant to the production method being investigated.The creation of a categorized genomic list of potential PHA producing microbes presents a valuable resource that directs researchers to explicit candidates, as well as access to potential metabolic pathways of interest. A similar bioprospecting strategy was utilized successfully by 9 and the GOLD metadata system by utilized by IMG/M being examples of creating standards for robust metadata reporting. The continual growth of online sequence repositories within the public domain and improvement of standards for metadata reporting presents a massive pool of data freely available to exploit. The mining of large-scale data provides a broader view into patterns in environmental or taxonomic distribution of biosynthetic products, which only improves over time as more data is constantly accumulated from studies conducted across the globe.This study is a proof of concept that contextual information generated from large-scale genome mining is highly valuable for use in developing bioprospecting strategies with regards to both ecological and taxonomic data. This approach can be applied to any target functional protein or gene of interest to create a shortlist of microbial candidates and their potential ecosystems for further in-depth analysis or experimentation. The effectiveness of this approach is dependent on not only the volume of the available sequence data, but also on prudent reporting and ready access to metadata. Accompanying information such as physiochemical conditions, geolocation and other ecologically contextual data are often missing from sequence data recovered from environments, which is critical for the eventual recovery of isolates . To addrin silico identification of PhaC genotypes. Through the categorization of genotypes via phylogenetic and ecological information, we can find insight through the discovery of less-studied taxa as well as microbes from environmental niches with potential properties that can improve on current PHA production techniques. Although this approach is no substitute for protein expression assays, it presents an overview of the trends inherent in PHA producing microbes. The information gained from this study could be utilized for directing bioprospecting ventures for more effective discovery of potential PHA producing microbes.Genome mining allows for the large-scale discovery of potential microbial PHA producers through The original contributions presented in the study are included in the article/PV and DL performed the research under the guidance of PK, DM, MW, and AW. PV wrote the manuscript with contributions from DL, PK, DM, MW, and AW. All authors read the manuscript and approved the content.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.