Mycobacterium tuberculosis protein tig (Rv2462c) is present in membrane fraction along with the cytosol. It has been previously reported to assist in the folding of nascent polypeptides. We have investigated the role of Mycobacterium tuberculosis tig in stress adaptation; for example, it exhibits roles in thermal and oxidative shock, survival within macrophages, biofilm formation and immunological responses. Our findings establish the contribution of tig to tolerance and adaptation to host-generated stresses, implying its involvement in the infection cycle of Mycobacterium tuberculosis. These attributes of Tig highlights its significance in Mycobacterium tuberculosis virulence and make it an important target for novel therapeutic interventions against tuberculosis.Mycobacterium tuberculosis (M.tb)-encoded factors protect it against host-generated stresses and support its survival in the hostile host environment. M.tb possesses two peptidyl-prolyl cis-trans isomerases and a probable trigger factor encoded by Rv2462c which has an FKBP-like PPIase domain. PPIases are known to assist the folding of peptidyl-prolyl bonds and are involved in various cellular processes important for bacterial survival in host-generated stresses. In this study, we aim to functionally characterize Rv2462c of M.tb. Our data suggest that the trigger factor of M.tb exhibits chaperone activity both in vitro and in vivo. Heterologous expression of M.tb-Rv2462c locus into Mycobacterium smegmatis enhanced its survival within macrophages, adaptation to oxidative stress and biofilm formation. M.tb-trigger factor has strong immunomodulatory potential and modifies the cytokine profile of the host towards the proinflammatory axis. Mycobacterium tuberculosis (M.tb) is a human peril causing millions of deaths every year due to tuberculosis (TB). Evasion of host defences helps it to survive in hostile macrophages [M.tb has been marked as a global priority by WHO [M.tb infection and may suggest novel therapeutic interventions.Despite our best efforts, rophages . The Worrophages . Effortsy by WHO . Variousy by WHO . HoweverTrigger factors across diverse bacteria are essential for the co-translational folding of many nascent polypeptides . The bacM.tb encoded protein responsible for interactions between host and pathogen, stress adaptation, and immunomodulation, we identified and characterized Rv2462c locus of M.tb. It has an FKBP-like or PPIase domain which may confer chaperone-like activity and is postulated to be involved in cellular processes [M.tb [M.tb genome. Prior studies have revealed that M.tb-Rv2462c is involved in the maintenance of open conformations of newly synthesized proteins, which indicates its chaperonic nature [M.tb-Rv2462c. It has been further reported that M.tb-Rv2462c may contribute significantly to the pathogenicity of M.tb, making it significant for further studies [In an attempt to identify rocesses . This pres [M.tb . The perc nature . It has c nature . Inorgan studies ,14.M.tb-Tig (Rv2462c) and explored its immunomodulatory potential and role in promoting M.tb survival under intracellular stresses by triggering the host immune system and modifying the cytokine secretion profile of the host, highlighting the significance of the protein in M.tb pathogenesis. Here, we have demonstrated that the recombinant Tig (Rv2462c), when expressed in E. coli, displays chaperone-like activity and significantly improves survival chances under high-temperature conditions. When expressed in M. smegmatis, it results in enhanced biofilm formation and increased survivability under oxidative stress. Our observations point towards the involvement of M.tb-Tig in stress adaptation, which forms an important component of the pathogenicity of M.tb.In this study, we have characterized the E. coli BL-21 strain from Novagen was used for protein expression. M.tb-H37Rv genomic DNA was a gift from Prof. Rakesh Bhatnagar’s laboratory in JNU, New Delhi, India. THP-1 cell was obtained from National Centre for Cell Sciences, Pune, India.M. smegmatis mc2 155 was initially procured from ATCC (Cat#700084). 7H9 broth with 10% OADC and 0.05% tween-80 was used to grow mycobacterium strains.M.tb-Rv2462c gene locus was amplified by using forward (CTAGCTAGCGTGAAGAGCACCGTCGAGCAGTT) and reverse primers (CCGCTCGAGTCGAGTCACGTTGTCGCTTCGTC) and cloned in pET28a vector [M.tb-Rv2462c gene was expressed in BL21 cells. Recombinant protein was purified using the Ni-NTA column as described earlier [a vector . From ta earlier . Then, t earlier . The hiscis-trans isomerization of Ala-Pro bond. The trans form is taken as substrate by coupled enzyme α-chymotrypsin to produce chromogenic product. Thus, isomerization of Ala-Pro bond was seen as an enhancement in the absorbance at 390 nm at 15 °C. Absorbance was documented every 1.5 s for 100 s and plotted graphically against time. Control reaction was used in absence of rTig [Enzyme activity of recombinant trigger factor (rTig) was measured using a spectrophotometric assay used previously . In summM.tb-Rv2462c protein, 200 ng of the pET22b vector was digested by NdeI with and without thermally aggregated NdeI at 37 °C for 2 h. Heat-denaturation of NdeI was achieved by submerging 10 U of the enzyme at 60 °C for 10 min. The digested plasmid was then electrophoresed in agarose gel containing ethidium bromide stain and visualized by the Gel Doc system  [Restoration of enzyme activity of heat-denatured proteins is considered a key function of chaperones. To determine if enzyme activity of thermally aggregated NdeI can be recovered by the ton, UK) ,17.M.tb-rTig, ANS probe was used and monitored by fluorescence spectroscopy [To explore the hydrophobicity surface of troscopy ,18. Brietroscopy . The fluE. coli was given as described earlier [Heat-shock of  earlier . Fold su earlier .Rv2462c on the survivability of mycobacterium inside the host, we assayed recombinant M. smegmatis strains, expressing M.tb-Rv2462c, for growth within THP-1 monocyte cells, in vitro. The E. coli-mycobacterium shuttle vector pSTKT having UV15 promoter with tetO sequence (inducible with anhydrotetracycline) was used to construct pST-Rv2462c. pST-Rv2462c was electroporated into Mycobacterium smegmatis mc2 155 as described earlier [To ascertain the impact of  earlier .Mycobacterium smegmatis strains  were grown in 7H9 broth containing 10% OADC to an OD600 of 0.8–1.0. The grown cultures were diluted to a ratio of hundred 1:100. Then, the cultures were grown to an OD600 of 0.4 and treated with 7 mM H2O2 for 3 h. The cultures were diluted 1:100 and plated on 7H10 agar plates.2. M. smegmatis cultures  were then used to infect the monolayers of THP-1 for 4 h at a 50 MoI. Then, the infected THP-1 cells were treated with Gentamicin (20 μg/mL) for 30 min. Further, the THP-1 cells were washed twice with RPMI medium and fresh medium was added and incubated for 72 h in CO2 incubator. The cells were then taken out carefully at different time points and centrifuged for 3 min at 1000 rpm. The cells were washed two times in RPMI medium and lysed in sterile water. The lysates were plated on 7H10 agar plates and kept at 37 °C for 4 days.Human THP-1 monocyte cells cultured in RPMI 1640 medium were differentiated into macrophages by phorbol 12-myristate 13-acetate (PMA)  and growM. smegmatis strains  were grown in 7H9 media supplemented with 0.05% Tween 80, 10% OADC and 0.001% glycerol. The cultures were diluted to an OD600 of 0.08 and incubated at 37 °C in a test tube. Further, 20 ng/mL of anhydrotetracycline was added to all of the M. smegmatis strains. To allow pellicle development at the liquid-air interface, M. smegmatis strains were subsequently incubated as static culture for 7 days in growth medium without Tween 80.Biofilm formation was quantified using the crystal violet assay . AnhydroM.tb-tig was performed with a tool developed by the Immunomedicine group of UCM, Spain. This tool utilizes the method described to determine the antigenic peptides [In silico analysis of the antigenicity of peptides . The occ2 at 37 °C [4 cells/cm2) were treated with M.tb-tig protein (2.5–20 µg) for 48 h. LPS was used as positive control while heat-denatured M.tb-tig was used as negative control. The culture supernatants were used to quantify the levels of cytokines using ELISA as described earlier [The presence of immunogenic proteins induces the secretion of cytokines from macrophages. Cytokine secretion levels were assayed by Enzyme-Linked Immunosorbent Assay (ELISA) method as previously reported ,24. THP-at 37 °C ,25,26. T earlier .M.tb rTig was found to be 50 kDa on 10% SDS-PAGE  strain after exposure to heat stress. There was no significant difference in the CFU count between uninduced or vector control strains  has no impact on the growth pattern of M. smegmatis compared to controls   A.M.tb-Tig on the survival of the pathogen inside host cells, we compared CFU of M. smegmatis strains  after infecting THP-1 cells. A steady rise in the CFU count of Ms-Tig was observed up to 72 h (p < 0.001) while a decline in the CFU was observed for Ms_WT and Ms_VC after 24 h  was exhibited by Ms_Tig  of M.tb-Tig. The dose-dependent increased levels of TNF-α and IL-6 were observed by the treatment of M.tb-Tig. These results revealed that M.tb-Rv2462c elicited an increased secretion of proinflammatory cytokine by macrophages  into a conformation suitable for translocation [M.tb. The binding of molecular chaperones to misfolded proteins is guided by surface hydrophobicity. The distinctive blue shift in the emission maxima, and enhanced absorbance by ANS-binding to rTig suggests the presence of surface hydrophobicity similar to chaperones. Earlier studies have reported that the trigger factor protects against thermal denaturation in E. coli [M.tb tig protects the cells against high-temperatures and aids in the restoration of enzyme activity of thermally denatured proteins. Incubation with rTig restores enzyme activity of NdeI, pointing towards its expected role in protein refolding. After getting satisfactory evidence of rTig’s chaperone-like nature, in vitro, we proceeded to investigate the activity of the protein, in vivo. Additionally, indeed, E. coli transformed with rTig displays a significantly increased survival after heat shock as compared to the controls.The trigger factor, encoded by the location ,32. It h E. coli . Our curM.tb trigger factor in E. coli and M. smegmatis expression systems was carried out. Using the vector alone and uninduced controls, as well as the fact that they do have sequence and therefore structural variations, the influence of the trigger factors of the expression systems was accounted for.To assess the role in stress adaptation and to demonstrate chaperone-like activity, the heterologous expression of M.tb has the ability to resist stress and survive inside the host. We explored the contribution of rTig in protecting M. smegmatis cells under conditions of oxidative stress. In comparison to M. smegmatis controls, we observed a significant increase in the survival of M. smegmatis strain expressing M.tb tig. Our observations lie in line with in vitro results, indicative of rTig acting as a chaperone-like protein which also contributes to stress adaptation.M. smegmatis cells transformed with rTig displayed enhanced survival in THP-1 cells. This points towards a crucial role of rTig in the pathobiology of tuberculosis. There are seemingly paradoxical role of pro-inflammatory cytokines vis a vis host benefits/survival. Pro-inflammatory cytokines could be pro-host as having anti-bacterial effects and anti-host as being toxic to host. Additionally, in case of tuberculosis, pro-inflammatory cytokines released earlier in the infection result in accumulation of immune cells around the granuloma resulting in spread of the pathogen.The overproduction of pro-inflammatory cytokines by human macrophages is considered to be a hallmark of tuberculosis. Antimicrobial activity against the pathogen is affected primarily by granulomas, whose formation and maintenance are believed to be modulated by TNF-α. Previous studies have also linked TNF-α production with intracellular viability and virulence . IL-6 isIL-6 is not a pure pro-inflammatory, but rather has an anti-inflammatory tinge as it inhibits endotoxin-induced TNF-α and IL-1 production in mononuclear cells and induce the soluble receptors for TNF-α which is its natural inhibitors . FurtherE. coli trigger factor also enhances the binding efficiency of the chaperone GroEL [Streptococcus pyogenes, the homologue of the trigger factor, RopA, is critical for the secretion and maturation of SpeB [S. mutans, was found to induce the upregulation of its trigger factor, strongly suggesting its involvement in the virulence of the pathogen [Streptococcus suis serotype 2, tig encodes a virulence regulator, associated with the expression of the pathogen’s virulence genes. The tig mutants of S. suis serotype 2 influence its adaptive capacity against environmental stresses [The ne GroEL ,39,40. I of SpeB ,42. Defipathogen ,43. In tstresses .Mycobacterium abscessus and P. aeruginosa in the production of biofilms in cystic fibrosis as well as a virulence factor in UPEC isolates. The likelihood of biofilm development within the host tissues is suggested by the occurrence of extracellular M.tb in biofilm-such as structures inside the lung lesions of guinea pigs infected with M.tb [M. smegmatis expressing rTig significantly upregulates biofilm production, in comparison to the strains lacking rTig. Our findings establish the involvement of rTig in biofilm production.Previous research demonstrated the role of ith M.tb . In the Rv2462c) has been reported in the cell wall fraction along with cytosolic fraction in different proteomic profiling [M. smegmatis, then, might have altered its physiochemical properties or working as a surface-associated adhesin-like molecule, promoting biofilm formation. For example, localization of Lap A protein to cell surface enhances the biofilm formation Pseudomonas fluorescens [Shewanella oneidensis [Staphylococcus aureus have shown role of Tig biofilm formation. Tig acts as a chaperone protein and help in folding of proteins essential in biofilm formation [Cell surface proteins play crucial role in the regulation of biofilm formation. Tig .The results of this study on M.tb trigger factor protein. Our findings also establish its contribution to tolerance and adaptation to host-generated stresses, implying its involvement in the infection cycle of M.tb. Revelations of these attributes of M.tb-Tig highlights its significance in M.tb virulence and make it an important target for novel therapeutic interventions against tuberculosis, thus fulfilling the primary aim of this study. Further studies would provide a better comprehension of the mechanisms employed by the protein to enable the intracellular lifestyle and enhanced pathogenicity of M.tb.Our study demonstrates the chaperonic nature and immunomodulatory potential of the


Abiraterone acetate and enzalutamide are novel therapies used in advanced prostate cancer. However, their outcomes and toxicities may differ based on patient–specific factors. Understanding these differences may allow clinicians to make personalized treatment decisions based on individual patients. Because clinical trials do not represent the real-world population, this study used a formal protocol to review and collate smaller, real-world samples, aiming to explain the differences in outcomes following administration of these two drugs. We found that enzalutamide typically has improved cancer response and overall survival. Enzalutamide more commonly causes neurological side effects and fatigue, while abiraterone acetate has cardiovascular complications. Abiraterone acetate leads to increased costs and healthcare needs, including hospitalizations and emergency room visits. Ultimately, this study demonstrates significant differences in patient experiences and outcomes following abiraterone acetate versus enzalutamide. Clinicians may use this information to inform their treatment choice on a patient-specific basis.Abiraterone acetate (AA) and enzalutamide (ENZ) are commonly used for metastatic prostate cancer. It is unclear how their outcomes and toxicities vary with patient-specific factors because clinical trials typically exclude patients with significant comorbidities. This study aims to fill this knowledge gap and facilitate informed treatment decision making. A registered protocol utilizing PRISMA scoping review methodology was utilized to identify real-world studies. Of 433 non-duplicated publications, 23 were selected by three independent reviewers. ENZ offered a faster and more frequent biochemical response (30–50% vs. 70–75%), slowed progression , and improved overall survival versus AA. ENZ was associated with more fatigue and neurological adverse effects. Conversely, AA increased risk of cardiovascular-  and heart failure-related  hospitalizations. Ultimately, AA was associated with increased length of hospital stay, emergency department visits, and hospitalizations . Accordingly, total costs were higher for AA, although pharmacy costs alone were higher for ENZ. Existing data suggest that AA and ENZ have important differences in outcomes including toxicities, response, disease progression, and survival. Additionally, adherence, healthcare utilization, and costs differ. Further investigation is warranted to inform treatment decisions which optimize patient outcomes. Prostate cancer (PCa) is the most commonly diagnosed non-skin cancer in men, accounting for over 20% of new cancer cases ,2,3,4. AUnfortunately, NHT clinical trials frequently exclude patients with significant comorbidities, restricting the generalizability of their findings to the broader population . By undeNo adequately powered comparative studies have yet elucidated the real-world risks and benefits of AA versus ENZ. Understanding potential differences in patient survival and cancer response rates may improve PCa outcomes. Furthermore, understanding factors such as patient adherence, healthcare resource utilization (HRU), and total costs can facilitate quality improvement. Given the availability of alternative treatment options, tailored therapy has the potential to improve treatment outcomes.Importantly, adverse drug effects (ADEs) are common with NHTs and may vary with patient and drug characteristics . LimitedThis scoping review aims to describe the differential outcomes, ADEs, and systemic costs of AA and ENZ for metastatic castration-resistant PCa (mCRPC) in a real-world population. The findings of this review may guide future clinical studies and ultimately facilitate tailored treatment based on the health condition of the patient.https://doi.org/10.6084/m9.figshare.19149227.v3 (accessed on 28 June 2022) [The scoping review followed the framework outlined by the Joanna Briggs Institute Manual for Evidence Synthesis (JBIMES), incorporating protocols established by Arksey and O’Malley along with revisions from Levac et al. and Peters et al. ,22,23,24ne 2022) .The study was intended to evaluate patient outcomes following AA and ENZ utilizing data on drug-associated toxicities, mortality, hospitalizations, HRU, costs, patient adherence, and patient comorbidities. The definition of outcomes was purposefully left broad, as a variety of disease-specific, patient-reported, and systemic outcomes are important to clinical decision making. The review focused on real-world cohort studies to create an analysis applicable to the general population. Studies with rigorous patient exclusion criteria, such as clinical trials, were excluded.The search strategy for this review was informed by prior PCa therapy research, as well as recommendations by Tawfik et al. to adapt searches to the database being utilized . An expeThe search was restricted to full-length peer-reviewed English publications through 31 January 2022. Free-standing abstracts, opinion pieces, reviews, and letters to the editor were excluded. Due to their abbreviated format, abstracts do not present all relevant data, while editorials are opinion-based.This review captured studies that compared outcomes and toxicities of AA and ENZ, focusing on retrospective institutional or population-based cohort studies. Studies investigating only one of the treatments were also excluded. If further information was required, the respective authors of the publications were contacted. The reference lists of all included articles were also searched for additional studies.Endnote 20  was employed for imported reference management and duplication removal. Studies identified by the above search strategy to satisfy the initial inclusion criteria were considered for title, abstract, and keyword screening by three independent reviewers. Articles satisfying initial screening underwent full-text screening by three independent reviewers. Lack of unanimity regarding determination of study eligibility at each stage was resolved through discussion with a senior member of the research team.Three members of the research team  independently conducted data extraction. From each article, the following information was extracted: author, year of publication, title, drug treatment regimen, study type/design, study population, primary objective(s), outcome(s), and primary conclusions . Given the broad scope of the research question and wide-ranging definition of patient outcomes and limited availability of published real-world cohort studies, a narrative synthesis and reporting of results was chosen. Extracted outcomes were wide-ranging in context and form, making a systematic review and meta-analysis suboptimal. Expert clinicians (WKK and SJF), who serve as the primary stakeholders in the determinations of this study, were consulted to inform data interpretation and subsequent discussion of clinical implications.The systematized literature search retrieved 571 articles, of which 118 duplicates were removed by computer software and an additional 20 duplicates were eliminated manually. The titles, abstracts, and keywords of the remaining 433 publications were screened, and 25 records were ultimately identified for full-text review. Following full-text screening, 2 articles were excluded as they did not directly compare AA and ENZ. Hence, 23 studies were included in this scoping review. The most common reasons for exclusion were a lack of direct AA versus ENZ comparison, a review article format, and a lack of measured outcomes .The studies’ characteristics are summarized in p = 0.016) and showed that positive response independently reduced risk of both biochemical progression  and death , but still found no significant difference in all-cause time to death  [p < 0.004) and greater time to progression  were recorded by Soleimani et al. [p = 0.014) and PSA response in 70.7% of ENZ versus 53.1% of AA patients, all of which were inferior to randomized clinical trials. PSA progression occurred in 30.5% and 54.9% of ENZ and AA patients [Better mCRPC response to ENZ was widely observed, although concomitant survival benefit was only occasionally found. Jarimba et al. demonstrated higher response rates with ENZ versus AA , with increased median OS (29.63 months vs. 25.87 months) [p < 0.001 and 29 m vs. 16 m, p = 0.027), alongside more frequent PSA decline greater than 50% (p = 0.020) [Three reviewed studies found a significant overall survival (OS) difference between ENZ and AA, all in favor of ENZ. Tagawa et al. reported that ENZ patients had a 16% lower risk of death  . Scailte months) . Demirci= 0.020) .p = 0.30), nor Chang et al.  [Four other studies found the two therapies more comparable, although, notably, no studies reported the superiority of AA. A mean OS of 18.9 ± 1.5 months with no significant differences between ENZ (24 months) and AA (15 months) was found by Al-Ali et al. . Similar= 0.766) ,37. ENZ = 0.766) .p = 0.04) and Demirci et al. (p = 0.016) although refuted by Chowdhury et al., who identified de novo metastases at diagnosis in 35.0% of AA and 42.3% of ENZ patients [p = 0.02).Age, co-morbidities, and disease stage played an important role when included in multivariate models. Importantly, Miyake’s study reported that AA was more commonly selected for patients with unfavorable characteristics . Baselinpatients . Behl alGenerally, most studies indicated superior response and sustained PFS with ENZ, with three also reporting increased OS. The remaining studies cited low sample size as the primary reason for a lack of significance, but still noted that ENZ seemed to improve disease-specific outcomes. Four studies did note a trend towards AA prescription for patients with unfavorable disease factors, which confounds signals of ENZ superiority.We reviewed included articles to uncover trends in ADEs and differences in common types of toxicities between AA and ENZ, along with associations with patient co-morbidities. Jarimba et al. reported ADEs in 16.1% of AA and 11.4% of ENZ patients, while Chowdhury et al. found that 7.1% of AA and 13.5% of ENZ patients discontinued treatment due to toxicity, and death during the treatment period was seen in 7.2% of AA and 11.1% of ENZ patients .p = 0.002 and p < 0.001) [We first reviewed the association of inherent patient characteristics with treatment exposure and subsequent toxicity. Importantly, older age was not reported to affect treatment exposure for either AA or ENZ, an important negative finding which confirms post hoc analyses of clinical trials . Similar< 0.001) .Cardiovascular toxicities following AA were noteworthy; such patterns were less notable in ENZ patients. Lu-Yao et al. reported that AA was associated with higher post-treatment hospitalization rates among those with pre-existing CVD. In particular, patients with ≥3 CVD conditions had a 41% lower post-treatment hospitalization risk when treated with ENZ compared to AA after adjustment for potential confounding variables  . George p = 0.04) [Central nervous system (CNS) toxicities including amnesia, cognitive disorders, confusion, and memory impairment; fatigue; and hot flashes were more common in patients treated with ENZ ,15,41. S = 0.04) , and ano = 0.04) . Similar = 0.04) . Another = 0.04) .We sought to elucidate therapy-related factors which may impact differential adherence rates between AA and ENZ, as adherence can greatly impact outcomes in the real-world setting. The World Health Organization has reported categories which impact patient adherence, including therapy-related toxicities and complexity of treatment, among other factors such as patient beliefs and mental health, disease-related factors and co-morbidities, and the clinician–patient relationship.Generally, studies found satisfactory adherence rates for both AA and ENZ, reporting medication possession ratios of 90% and 85% and non-adherence rates of 4.8% and 6.2% for AA and ENZ, with no significant differences between the two ,36,43, Ip < 0.0001). They proposed that high rates of fatigue with ENZ may explain lower adherence. Shore et al. similarly demonstrated more dose reductions with ENZ (16% vs. 6%), and attributed this to ADEs [p < 0.001). Fewer AA versus ENZ patients required dose reduction for reasons besides disease progression , resulting in lower dose exposures, although this did not harm outcomes. Interestingly, time to progression was higher in ENZ patients requiring dose reduction [Behl et al. included a Kaplan–Meier curve demonstrating that lower dose reduction risk in AA became most evident at 3 m follow-up, with the difference becoming more pronounced at greater follow-up periods . Similar to ADEs , a findiFreedland et al. conversely found that dose reductions were not more common or intense in ENZ versus AA. Moreover, dose reductions were seen in 64.4% of all study patients and were associated with 8.8% increased risk of biochemical progression. Dose reductions are relevant given that the combination of ENZ with a CYP2C8 inhibitor can increase ENZ levels by 2.2-fold, thereby potentially requiring a lower dose .p < 0.001), and AA patients experienced fewer discontinuations  across a 24 m span [p = 0.0008) [Finally, Pilon et al. found treatment duration was significantly higher with AA  ,47.For cohorts where disease-specific outcomes, co-morbidity interactions, or toxicity sensitivities are not clearly different between AA and ENZ, systemic factors such as total cost or HRU might inform treatment choice. These factors can impact quality of life and public health, but are not commonly studied as relevant outcomes, and, hence, only three studies primarily reported such data.Al-Ali et al. indicated the importance of HRU as an outcome of relevance by remarking that only 9.4% of study patients were never hospitalized, and patients spent an average of 13% of their remaining life span in the hospital. Hospital stays were generally longer with AA versus ENZ (39.4 ± 36.8 days vs. 26.3 ± 25.8 days) . Increasp < 0.0001) and PCa-related outpatient visits . This corresponded with lower all-cause  and PCa-related  outpatient costs PPPM. Total medical plus pharmacy costs were also lower with ENZ . Overall, emergency room, cancer-related visit, cancer-related pharmacy, and all-cause costs were all lower with ENZ, and a yearly cost benefit of USD 12,000 was identified [Ramaswamy et al. found fewer all-cause outpatient or pharmacy visits and costs per patient per month (PPPM) for ENZ patients in the United States. The ENZ cohort experienced fewer all-cause inpatient , this drug expense was negated by lower total healthcare costs compared to AA. Upon adjusted differences of USD 485 for total pharmacy costs (p < 0.001) and USD 834 for index drug costs (p < 0.001), this resulted in adjusted cost savings of USD 28 (p = 0.009) for emergency department visits and USD 122 (p = 0.024) for inpatient admissions with ENZ [Schultz et al. supported these findings. ENZ patients reported fewer all-cause inpatient admissions , days of hospitalization , and outpatient visits , alongside fewer PCa-related outpatient visits  versus AA. Furthermore, within 3 m of treatment initiation, ENZ patients visited the emergency department for PCa-related concerns at 28% lower rates  and were admitted as inpatients at 24% lower rates . Although monthly pharmacy costs were USD 545 higher for ENZ (with ENZ .p = 0.019), CVD-related , and heart failure-related  hospitalizations [Focusing on cardiovascular risk, two additional studies further clarified differences in HRU. AA patients had increased risk of all-cause  such as spinal cord compressions and fractures due to bony metastases. Importantly, both AA and ENZ have been shown to significantly delay SREs, although bone modulating agents are now recommended in combination with either of these antiandrogens to further reduce incidence. Nonetheless, PCa-related complications such as SREs can contribute to post-NHT HRU, and differences in their frequency between AA and ENZ should be analyzed in future comparisons ,63.Direct healthcare costs of castration-resistant PCa range from USD 2474 to USD 67,957 annually. These costs increase five-fold upon metastasis, and HRU increases 1.5–2.5-fold . Given tThis study had several strengths. The review utilized a strong, transparent methodology based upon a previously registered protocol. A broad search was conducted in four major databases, and studies were evaluated independently by three reviewers. Finally, a multidisciplinary team was involved throughout the study, including expert clinicians who informed this study’s discussion of clinical implications. Nonetheless, this scoping review has various limitations, including the exclusion of abstracts and non-English articles. Given the retrospective nature of included studies, patient cohorts were heterogenous. Although individual studies performed multivariate adjustments to adjust for co-morbidities and other modulating factors, the full extent of inter-cohort differences cannot be elucidated in a scoping review, and, hence, these findings should be interpreted with caution. A further limitation includes the lack of information surrounding patient diversity, including data on patient race and ethnicity, which limits broad applicability. Finally, synthesis of results and summative statistical analysis was limited given the broad definition of outcomes and the relatively small amount of data available for each individual outcome.This is the first scoping review to compare the treatment outcome of AA vs. ENZ amongst mCRPC patients. Existing retrospective and real-world studies indicate that AA and ENZ have profoundly differing effects based on patient risk factors, as AA is associated with notable cardiovascular toxicities while ENZ has neurological adverse effects and fatigue. AA demonstrates poorer systemic outcomes including HRU and cost, while it shows superior treatment adherence. Finally, ENZ offers faster and more frequent disease response, and select studies demonstrated improved survival, although these outcomes are still unclear. Real-world outcomes data with both agents were inferior to results seen in clinical trials. These differences may become increasingly relevant as many AA and ENZ users have a higher comorbidity burden than those in clinical trials and NHT indications are expanding to earlier disease stages . The res


Spodoptera litura is an important polyphagous pest that causes significant damage to the agricultural sector. We performed RNA-seq of 15 S. litura individuals from larval (fifth and sixth instar larvae), chrysalis, and adult developmental stages. We also compared the S. litura transcriptome data with Spodoptera frugiperda across the same developmental stages, which was sequenced in our previous study.S. litura. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses indicated that S. litura may undergo active xenobiotic and detoxifying metabolism during its larval and adult stages, which may explain difficulties with current population control measures. We also found that DETs of single-copy orthologous genes between S. litura and S. frugiperda were involved in basic metabolism and development. However, energy and metabolic processes genes had a higher expression in S. litura, whereas nervous and olfactory function genes had a higher expression in S. frugiperda. Metagenomics analysis in larval S. litura and S. frugiperda revealed that microbiota participate in the detoxification and metabolism processes, but the relative abundance of detoxification-related microbiota was more abundant in S. frugiperda. Transcriptome results also confirmed the detoxification-related pathway of S. frugiperda was more abundant than in S. litura.A total of 101,885 differentially expressed transcripts (DETs) were identified in S. litura. Importantly, we also identified detoxification associated genes and gut microbiota between S. litura and S. frugiperda at different developmental stages, which will be valuable in revealing possible mechanisms of detoxification and development in these two lepidopterans.Significant changes at transcriptional level were identified during the different development stages of The online version contains supplementary material available at 10.1186/s12864-022-08613-6. Spodoptera litura (Lepidoptera: Noctuidae) is a destructive omnivorous pest with a large host range of plants, and feeds on a wide range of crops [Spodoptera litura is characterized by a short life cycle consisting of eggs, larval, chrysalises and adults [of crops . It is wof crops , 3. Spodd adults . Eggs ard adults . The gred adults , 7. TherS. litura [S. litura [S. litura demonstrated that P450s may be involved in the detoxification of fluralaner in vivo [S. litura, which may be a genetic basis for its high tolerance to pesticides [S. litura, the developmental characteristics of S. litura and the underlying changes during development that allow resistance to pesticides remains poorly understood. RNA sequencing has been widely used to obtain expression data of different developmental stages of agricultural pests and to explore the key regulated genes related to development that can be targeted for controlling pests. Pairwise comparison of four developmental stages of Athetis lepigone found that several differentially expressed genes were related to cuticle and wing formation as well as the growth and development [Ostrinia furnacalis in four developmental stages revealed genes were associated with developmental pathways, cuticularization, wing formation and olfactory recognition [S. exigua developmental stages and identified four Spodoptera-specific candidate genes for RNAi-based pest management strategies [S. litura to compare the differences in gene expression profiles at different stages to design efficient strategies to control this economic pest.Previous studies primarily evaluated the influence of different host plants on the growth and development of . litura –10 and i. litura . Given t. litura , researc in vivo . Studiessticides . Despiteelopment . Transcrognition . Simon, rategies . ConsequS. frugiperda) is phylogenetically close to S. litura and belongs to the same genus Spodoptera. Despite similar biological and morphological characteristics such as high resistance to pesticides, strong reproducibility and migration [S. frugiperda and S. litura differ in their feeding habit, reproductive behavior and damage degree [Spodoptera frugiperda prefers gramineous plants, whereas S. litura prefer dicotyledons. Another difference is that S. frugiperda has more powerful locomotion than S. litura. In addition, S. frugiperda shows higher mating frequency than S. litura. The genetic divergence between the two pests was demonstrated by comparative genomics [S. frugiperda. However, the enriched biological processes in S. litura were mainly in development, reproduction, and metabolism. Furthermore, our team studied the gene expression differences between developmental stages of S. frugiperda [S. frugiperda experienced active metabolism, detoxifying and xenobiotic metabolism throughout its life, especially in the larval stage. Considering the observed differences in feeding habit and reproductive behavior between S. frugiperda and S. litura, it is possible that there are gene expression differences between them even during the same development stage. Therefore, comparative transcriptomics between the two pests at different development stages will be helpful to better understand their genetic differences.The fall amyworm , chrysalis stage (marked as LC) and adult stage , were collected and we performed RNA-seq across stages and metagenomics on the larval stage. We identified DETs between development stages and identified metabolic pathways that changed with development. Furthermore, we collected and re-analyzed the data of S. frugiperda from Wang et al., consisting of 15 S. frugiperda individuals, including larval stage , chrysalis stage (marked as FC) and adult stage , that identified detoxifying-related metabolism and basic metabolism differences with development of S. litura and S. frugiperda [S. frugiperda guts being more diverse than S. litura, and the genes related to energy metabolism process and detoxification differed  clean reads were obtained. We then mapped the clean reads to the reference genome and the results showed that the mapping rate was 81.31% ~ 94.96%  demonstrated that the larvae, chrysalis, and adult groups of  Figure S.Through cross-comparison of DETs at three developmental stages, a total of 2,703 downregulated DETs and 4,270 upregulated DETs were identified in the larval group (LL) when compared to the chrysalis group (LC). There were 3,145 downregulated DETs and 1,847 upregulated DETs in the LC group when compared to the adult group (LA). In addition, 2,979 downregulated DETs and 3,629 upregulated DETs were identified in the LL group when compared to LA  were mainly enriched in biological process (BP) and molecular function (MF) GO terms, such as lipid catabolic process , carbohydrate metabolic process , glutathione metabolic process  and trehalose transport  in BP terms, which all related to basic metabolism processes. Many enzyme-related MF terms were enriched by these DETs, such as glutathione transferase activity  and oxidoreductase activity  , upregulated DETs were mainly enriched in development BP terms, including muscle organ development , tissue development  and chitin metabolic process . In MP terms, many DETs were associated with chitin metabolism, such as chitin binding , structural constituent of pupal chitin-based cuticle  and chitinase activity   were mainly enriched in developmental BP terms, such as axoneme assembly  and cytoplasmic translation . In MF terms, many terms that were associated with enzyme activity were enriched, including glutathione transferase activity GO:0,004,364) and serine-type endopeptidase activity  . A total of 493 downregulated DETs and 589 upregulated DETs were identified in the LC group when compared to the chrysalis group of S. frugiperda . In addition, 658 downregulated DETs and 611 upregulated DETs were identified in the LA group when compared to the adult group of S. frugiperda . A heatmap was generated using DETs of single-copy orthologous genes between S. litura and S. frugiperda across the three developmental stages  were only enriched in one MF term, structural constituent of cuticle . There was no significant KEGG pathway enriched in upregulated DETs in the LL group. The GO analysis showed that downregulated DETs in the LL group were enriched in one BP term, olfactory learning  and one CC term, synapse , and they were associated with sensory and nervous system development. There were four KEGG pathways enriched by these DETs, Rap1 signaling pathway (ko04015), axon regeneration (ko04361), adherens junction (ko04520) and tight junction (ko04530)  were mainly enriched in, for example, extracellular space GO:0,005,615), extracellular region  and serine-type endopeptidase activity . There was only one enriched KEGG pathway, fanconi anemia pathway (ko03460). Downregulated DETs in the LC group were enriched in nervous system developmental related CC terms, such as neuromuscular junction . The nervous development and cell activity KEGG pathways that were associated with these downregulated DETs included the axon regeneration (ko04361) and tight junction (ko04530)  were mainly enriched in energy related BP terms such as mitochondrial translation GO:0,032,543), ATP synthesis coupled proton transport  and aerobic respiration , as well as binding-related MF terms, including chitin binding  and heme binding  , whereas the relative percentage of Proteobacteria composition was higher in S. frugiperda  stage of S. litura and S. frugiperda metagenomes, genes were predicted from the CAZy, Humann3 and CARD databases. A total of 302 annotated genes from six families, consisting of glycoside hydrolases (GHs), glycosyltransferases (GTs), polysaccharide lyases (PLs), carbohydrate esterases (CEs), auxiliary activities (AAs), and carbohydrate binding modules (CBMs), were detected in the gut microbiota based on the Carbohydrate-Active Enzymes (CAZy) database (http://www.cazy.org). In S. litura, we obtained 170 GH subfamilies, 74 GT subfamilies, 48 CBM subfamilies, 17 CE subfamilies, 24 PL subfamilies, and 16 AA subfamilies. However, S. frugiperda samples identified 134 GH subfamilies, 64 GT subfamilies, 40 CBM subfamilies, 17 CE subfamilies, 14 PL subfamilies and 11 AA subfamilies. The most abundant CAZyme families detected were represented by subfamilies GH38 and GH85 in GH families. Moreover, LEfse identified GT31, GH13_25 and GT10 as the top three significantly more abundant CAZymes in S. frugiperda than in S. litura. While GH13_14, GH67 and GH94 were the top three significantly more abundant CAZymes in S. litura than S. frugiperda  were detected in the gut microbiomes. We detected that Spodoptera litura is an important polyphagous agricultural pest, which is difficult to eliminate because it has evolved resistance to many insecticides [S. litura showed evidence of adaptive evolution of cuticular protein genes. These genes have been sequenced and characterized and results suggested that various cuticular protein groups produce cuticle layers with different physical properties at different stages [S. litura developmental patterns. In the present study, the transcriptome analyses of S. litura at three developmental stages were conducted and identified 10,184 DETs through pairwise comparisons , embryonic pattern specification  and structural constituent of chorion , which are essential to the behaviors like mating, oviposit, and incubation. Fatty acid derivatives are involved in the synthesis of sex pheromones . This waSpodoptera litura have active xenobiotic and detoxifying metabolism in the larval and adult stages. As an important member of the metabolic enzyme superfamily, P450 plays an important role in the metabolism of xenobiotics, such as pesticides and plant secondary metabolites [S. litura larvae begin to respond to the insecticide avermectin via the P450 and glutathione-s-transferase enzymes metabolism mechanism [S. litura [S. litura chrysalis stage when there is relatively low transcript expression related to the detoxification pathways may increase insecticide effectiveness. In addition, the plant xenobiotic azadirachtin [S. litura larvae, higher detoxification enzyme activities were found at the larval stage and this may help insecticide metabolism [abolites . P450s aabolites . Glutathabolites . In our echanism . Further. litura . The detirachtin , organopirachtin  and the irachtin , may cautabolism . Larvae S. litura and S. frugiperda, we first screened single-copy orthologous genes of the two species and compared their differences. Then, the functional enrichment of the DETs of single-copy orthologous genes was undertaken. The results showed that the upregulated DETs of single-copy orthologous genes in S. frugiperda were enriched in nervous and olfactory function related pathways (FL vs. LL and FC vs. LC). This was consistent with genome level studies that showed significant genetic richness in sensory systems, such as taste stimulation, as well as in the nervous system in S. frugiperda [S. frugiperda is stronger than S. litura [S. litura were mainly enriched in energy and metabolic processes, which was opposite to genome-level results. Cui et al., conducted comparative genomic analyses between S. frugiperda and S. litura that, which identified several S. frugiperda-specific positively selected genes related to energy supply [S. litura. Among the metabolic mechanisms of resistance that have been discovered, the most common include enhanced insecticide detoxification by upregulated P450, GST and the increased activities of carboxylesterase [S. litura was higher in the larval stage than in chrysalis and adult stages, while that of S. frugiperda was highly expressed in all three stages. Therefore, different results may be achieved by using this insecticide in the chrysalis and adult stages of S. litura and S. frugiperda, which would be affected by their differing resistance to pesticides.In order to compare the differences in gene expression levels between ugiperda . Sensoryugiperda . This ma. litura . In the y supply . One posesterase . By compesterase . We founS. litura and S. frugiperda. Alphabaculovirus is an entomopathogenic virus genus [S. litura and S. frugiperda. As a lepidopteran-specific nucleopolyhedroviruses, Alphabaculovirus could be a candidate genus for RNAi-based pest management strategies [S. litura and S. frugiperda revealed different microbiota compositions. Firmicutes, Proteobacteria and Actinobacteria were the most dominant phyla in all samples. Firmicutes and Proteobacteria are crucial to maintain insect growth and development during the metabolism of secondary metabolites of host plants [Clostridium, Glutamicibacter, Streptomyces, Escherichia, and Enterobacter were significantly higher in S. frugiperda than in S. litura. These bacteria participate in food digestion, nutrition, and the detoxification of plant defense compounds. Clostridium has been shown to play a key role in the breakdown of celluloses and insecticide metabolism [Streptomyces can produce antimicrobial secondary metabolites and provide chemical defenses for insects [Enterobacter participates in the encoding of carboxylesterase and glutathione S-transferase (GST) and these enzymes are involved in inducing xenobiotic detoxification, which reinforces the supposition that gut microbes can play an important role in insecticide resistance of S. frugiperda [. The relative abundance of Pediococcus was significantly higher in S. litura than S. frugiperda. Pediococcus have the ability to decompose organic materials and metabolize several plant secondary biomass to produce phenolic compounds, which are necessary for host insects [S. frugiperda than S. litura.In this study we also investigated the gut microbiome metagenomes of us genus , and in rategies . A compat plants . The vast plants . Our fintabolism . Strepto insects . Enterobugiperda . The rel insects . Our resS. litura and S. frugiperda gut metagenome indicated that their gut bacteria may be involved in various metabolic pathways, such as the carbohydrate degradation and detoxification. CAZy database analysis showed that the gut microbes of S. litura and S. frugiperda were rich in carbohydrate degradation-related genes. GH family enzymes, including β-galactosidase, β-glucosidases, β-glucuronidase, and β-xylosidase, are involved in the utilization of a variety of carbon sources. Pathway analysis revealed that some of the unstratified pathways differed between S. litura and S. frugiperda. Pentose phosphate pathway and glycogen biosynthesis pathways were more abundant in S. litura, which indicates that S. litura might have higher production of energy within the gut compared to S. frugiperda. In addition, the monothiol biosynthesis pathway was more abundant in S. frugiperda and monothiol has antioxidant and detoxification effects [S. litura has more abundant energy production pathways, while the pathway related to detoxification was more abundant in S. frugiperda, which is consistent with the findings in our transcriptomics. The difference in energy pathways between the two species may reflect differences in resistance, since insecticide resistance consumes the host’s energy reserves and reduces the energy available for other metabolic functions [The  effects . Overallunctions .Spodoptera litura and S. frugiperda also differ significantly in terms of ARGs profiles, with S. frugiperda having more ARGs than S. litura. The most abundant ARG in all samples was DfrA42, encoding dihydrofolate reductase that confers resistance to Trimethoprim. The second most abundant ARG was Efra, heterodimeric ABC transporter efflux pumps that confers resistance to macrolide antibiotic, rifamycin antibiotic and fluoroquinolone antibiotic [S. litura and S. frugiperda. The energy metabolism related pathway was more abundant in S. litura than in S. frugiperda. On the contrary, S. frugiperda had more abundant gut bacteria associated with detoxification than S. litura. The results allowed us to comprehensively analyze the potential functions of gut bacteria contributing to food digestion, nutrition, and metabolic detoxification in S. litura and S. frugiperda.tibiotic . TherefoIt should be mentioned that there are two limitations in this study: (i) Since the adults were collected from the wild, we cannot guarantee the consistency of their rearing environment. (ii) Limitations of only using single-copy orthologous genes to compare the differences between the two pests must be considered. Biological differences may also be influenced by, for example, gene duplications, deletions, and species-specific expansions, which can lead to biological differences between the two species.S. litura. A large number of DETs were identified by pairwise comparisons between developmental stages. Several of these DETs were related to detoxification and development. The comparative analysis between S. litura and S. frugiperda revealed differences in their host adaptation and energy metabolism. We identified the structure of the intestinal microbiota of S. litura and S. frugiperda based on metagenomics. Our results demonstrated that the relative abundance of gut microbiota involved in detoxification was significantly higher in S. frugiperda and the pathway related to detoxification was also more abundant than S. litura. Our study provides an important basis for further understanding of the molecular mechanisms of pest development and provides insight into the development of a novel pest control strategy. In the future, more stage-specific pesticide products can be developed through further studies on genes related to detoxification metabolism, which may provide new strategies for effective S. litura and S. frugiperda control.In the present study, the RNA-seq libraries were constructed to investigate the transcript expression patterns during three developmental stages of S. litura and S. frugiperda were obtained from cornfields in Xindu District, Chengdu, Sichuan Province, China. They were randomly captured in the cornfields by hand and sweep net. The adults were bred in captivity and artificially propagated to sample their first-generation by the Institution of Plant Protection, Sichuan Academy of Agricultural Sciences. The main food of S. litura and S. frugiperda was powdery corn. The captive environment was as follows: maintained relative humidity at 70% ± 10% and temperature at 28 °C ± 1 °C in the artificial box, the ratio of light to dark time was 18:6. All samples were the laboratory bred offspring, and the rearing and feeding conditions of captive individuals were consistent.In this study, the adults of S. litura individuals were used, including three developmental stages: fifth instar larvae (N = 3), sixth instar larvae (N = 3), chrysalises (N = 3), female adults (N = 3), and male adults (N = 3). According to the developmental stage of S. litura and consistent with the grouping in S. frugiperda study [S. frugiperda downloaded from our team’s previous study, which was collected from 15 S. frugiperda individuals, with larval stage (fifth instar larvae and sixth instar larvae), chrysalis stage and adult stage  [S. frugiperda is consistent with that of S. litura.A total of 15 da study , we combnd male) . The samTrizol reagent  was used to extract the total RNA of the samples. The quantity and purity of total RNA were determined by 2% agarose gel electrophoresis. The total RNA purity with OD260/280 was detected by Nanodrop. The RNA density was determined by Qubit and the RNA integrity was analyzed by Agilent 2100. mRNA was collected by Oligo dT enriching beads. First, fragmentation buffer was added to the mRNA to break it into small fragments. Then, the first strand of cDNA was synthesized using random hexamer primers and under the premise of regarding mRNA fragments as a template. Second-strand was synthesized by adding buffer, dNTPs, DNA polymerase I and RNase H. AMPure XP beads was used to purify double-stranded cDNA. After we performed purification, terminal repair, addition of A and ligation of sequencing adaptor, libraries were produced by performing PCR amplification. Finally, the PCR product was purified by AMpure XP beads to obtain the final library. After the library was constructed, Qubit 2.0 was used for preliminary quantification, and the library was diluted to 1.5 ng/mL. Then, Agilent 2100 was used to detect the insert size of the library. After the insert met the expectation, Q-PCR method was used to accurately quantify the effective concentration of the library to ensure library quality. The Illumina Hiseq 2000 platform at Novogene  was used to generate approximately 150 bp paired end (PE) raw reads.S. litura were downloaded from the NCBI database (https://www.ncbi.nlm.nih.gov/genome/?term=Spodoptera+litura). The clean reads of each sample were mapped to the reference genome using HISAT2 v2.1.0 software [The NGSQC Toolkit version 2.3.3 software  was emplsoftware . Samtoolsoftware  was thensoftware . The rawS. litura, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotations of mRNA sequences were performed. Based on the position annotation from the assembled GTF file of the S. litura reference genome, we extracted the mRNA sequences. To obtain comprehensive annotation information of the S. litura transcript, the above-mentioned mRNA sequences were aligned to the NCBI (ftp://ftp.ncbi.nlm.nih.gov/blast/db/) nonredundant (NR) protein databases and the Swiss-Prot database using BLASTx with E-value cutoff of 1E-5 [Drosophila melanogaster, Bombyx Mori, Bombyx mandarina, apilio machaon, Pieris rapae, Danaus plexippus, Helicoverpa armigera, Trichoplusia ni, and Plutella xylostella) were considered as background sets to annotate the best alignment sequences using the KEGG database, which was accomplished by the KEGG Automatic Annotation Server (KAAS) [Since we lacked functional annotation information in the reference genome of  of 1E-5 . The GO r (KAAS) . Furtherr (KAAS) .p values were corrected for multiple testing with the Benjamini–Hochberg false discovery rate (FDR ≤ 0.05) and were considered as DETs.The transcripts that had five or more reads in any sample that could be mapped to the reference genome were used for further analysis. All the samples were divided into different groups according to their developmental stages, these being the larval group , chrysalis group , and adult group . Then, LL versus LC, LC versus LA, and LL versus LA were analyzed using DESeq2 in R packages that used the raw read count matrix as the input . Transcripts in different compared groups with |log twofold change|≥ 2 and S. frugiperda were collected from our previous study [S. frugiperda, with the larval group , chrysalis group , and adult group . We used the raw read count matrix as the input for subsequent analyses. The OrthoFinder V2.3.117 [S. litura and S. frugiperda within the same age stage. GETMM [S. litura and S. frugiperda in the expression matrix. DETs of the single-copy orthologous genes analyses between LL versus FL, LC versus FC, and LA versus FA were performed by DESeq2 after the expression matrix was normalized, and we set the FDR ≤ 0.05 and | log 2 FC|≥ 2 as the standard to select the DETs between the two species. Functional enrichment analyses of DETs between and within developmental stages and between S. litura and S. frugiperda were performed using the GO term and KEGG pathway background data sets constructed according to S. litura mRNA sequences. In our study, the p values were adjusted by the BH method, and FDR ≤ 0.05 was considered as significantly enriched GO terms and KEGG pathways by ClusterProfiler v3.16.0 [Transcriptome data of the three developmental stages of us study . There wV2.3.117  was usede. GETMM   and extracted 0.2 μg DNA per sample that was used as input material for the DNA library preparations. Genomic DNA was tested for quality, with quality genomic DNA samples being fragmented by sonication to a size of 350 bp. DNA fragments were then endpolished, A-tailed, and ligated with the full-length adapter for Illumina sequencing, followed by further PCR amplification. After PCR products were purified by AMPure XP system , DNA concentration was measured by Qubit®3.0 Flurometer , libraries were analyzed for size distribution by NGS3K/Caliper and quantified by real-time PCR (3 nM). The DNA libraries were then sequenced on Illumina platform and paired-end reads were generated.The larval samples of S. litura and S. frugiperda genome were removed by KneadData integrated Bowtie2 tool (version 2.3.4.1) [Quality control and trimming of sequences were conducted by KneadData (version 0.7.4) toolkit. Contamination sequences of the host 2.3.4.1) . The met2.3.4.1) . Taxonom2.3.4.1) . Contigs2.3.4.1) . Cd-Hit 2.3.4.1)  was appl2.3.4.1) . To obta2.3.4.1) . Resista2.3.4.1) . The met2.3.4.1) . Linear Additional file 1. Supplementary FiguresAdditional file 2. Supplementary Tables


The aim of this study was to explore the predictive role of inflammation-related parameters in prognosis of esophageal squamous cell carcinoma (ESCC).9/L), and systemic inflammation response index (SIRI) as neutrophil count × monocyte count/lymphocyte count. The optimal cut‐off values of preoperative SII, neutrophil‐to‐lymphocyte ratio (NLR), platelet‐to‐lymphocyte ratio (PLR), lymphocyte-to-monocyte ratio (LMR), PNI, and SIRI were determined via receiver operating characteristic (ROC) analysis, and their correlations with clinical parameters and survival analyzed.A total of 370 ESCC patients subjected to curative surgery were enrolled. All patients had complete medical records and did not receive preoperative adjuvant therapy. Preoperative systemic immune-inflammation index (SII) was calculated as platelet count × neutrophil count/lymphocyte count, prognostic nutrition index (PNI) as albumin concentration (g/L) + 5 × total lymphocyte count (10P = 0.022), and PLR (P = 0.037), PNI (P = 0.017) was associated with survival status, LMR was related with gender (P = 0.034) and survival status (P = 0.01), SIRI was correlated with gender (P = 0.000), smoking history (P = 0.000) and drinking history (P = 0.004). Survival analysis indicated that high PLR (P = 0.042), low LMR (P = 0.001), and low PNI (P = 0.007) were predictive of poor prognosis of ESCC. Stratified analysis revealed the prognostic predictor roles of distinct markers in different ESCC subgroups. SII and SIRI were predominantly correlated with the clinical outcome in the lymphatic metastasis subgroup. Further univariate analysis disclosed that T stage, smoking history, lymphatic metastasis, TNM staging, PLR, LMR, and PNI potentially serve as influencing factors(P < 0.05). Multivariate analysis identified T stage , TNM staging  and LMR  as independent predictors for outcomes of ESCC.NLR was associated with gender (Low LMR could serve as an independent marker of poor prognosis in patients with ESCC. Inflammation-related markers have distinct predictive roles in ESCC subgroups with different features. Esophageal squamous cell carcinoma (ESCC) with a malignant clinical course and negative outcome accounts for ~90% of esophageal cancers in China –3. Whilevia routine blood tests  and blood biochemical indicators   . Inflammprotein) . Breast protein) . Preoperprotein) . Additioprotein) . SIRI haprotein) . Systemiprotein) . HoweverIn the present study, we further focused on prognostic factors of ESCC and evaluated the prognostic performance of inflammation-related parameters in predicting postoperative survival in our patient cohorts.via telephone and outpatient consultation based on National Comprehensive Cancer Network (NCCN) guidelines. OS time was assessed from the date of surgery to the date of the most recent follow-up or death. The follow-up period ended in November 2021.This study was a single-center, retrospective design. In total, 370 ESCC patients were enrolled at Shanxi Province Cancer Hospital between October 2016 and May 2018. None of the subjects had received neoadjuvant chemoradiotherapy or any other cancer treatments before operative treatment. Surgery was performed in all cases and ESCC diagnosis was subsequently confirmed by pathologists. And patients with complete medical information. The following exclusion criteria were applied:  patients9/L), SIRI = neutrophil count × monocyte count/lymphocyte count.All preoperative blood routine and biochemical results were recorded. Calculations for indicators were performed as follows: NLR = neutrophil count/lymphocyte count, PLR = platelet count/lymphocyte count, LMR = lymphocyte count/monocyte count, SII = platelet count × neutrophil count/lymphocyte count, PNI = albumin concentration (g/L) + 5×total lymphocyte count . The optimal cut-off values of NLR, PLR, SII, PNI, and SIRI were determined The 370 ESCC patients enrolled consisted of 245 males (66.22%) and 125 females (33.78%). The median age was 61 years (40-81 years). In total, 47.84% of ESCC patients had exposure to tobacco consumption, and 29.73% patients harbored an alcohol drinking lifestyle. The primary location was the middle esophagus in 72.43% of cases, the lower segment in 23.51% of cases, and the upper esophagus in only 4.05% cases. Moderately differentiated ESCC accounted for 65.68% of the total cases, followed by poorly differentiated (31.89%) and well-differentiated ESCC (2.43%). Overall, 65.68% of ESCC patients were at the advanced T stage at diagnosis and only a small proportion of patients (32.32%) were diagnosed at the early T stage. In total, 43.78% of ESCC patients harbored advanced TNM stages at the time of diagnosis, and ESCC was accompanied by lymphatic metastasis in 45.41% of cases. The collective clinical, epidemiological, and pathological features are presented in χ2 = 5.255, P = 0.022), PLR  and PNI  with survival status, LMR with gender  and survival status , and SIRI with gender , smoking history  and drinking history . SII was not associated with all clinical and pathological parameters. The collective results support an association of the inflammation-related index with clinical features of ESCC.We further analyzed the correlations between six inflammation-related parameters and clinicopathological features in ESCC patients. ROC analysis revealed optimum cut-off values of NLR, PLR, LMR, PNI, SII, SIRI of 1.304, 98.7144, 3.1049, 50.6, 313.6978, and 0.4854, respectively. Accordingly, patients were divided into two groups with low or high levels of each parameter based on the optimal cutoff value for further correlation analysis. As shown in P = 0.042). Moreover, ESCC cases with low LMR and low PNI  had poorer prognosis. Our results suggest that PLR, LMR, and PNI collectively serve as predictive factors of survival in ESCC , smoking history , lymphatic metastasis , TNM staging , and PLR  may enhance survival risk of ESCC while LMR  and PNI  were identified as protective factors , TNM staging  and LMR  were identified as an independent predictors of adverse events in ESCC , consistent with the finding of Gao Y et al. . In term+ T cells (The tumor environment is the dominant setting that fosters tumorigenesis and development . In addi T cells , 21–23,  T cells , 23. Add T cells , 25. Rec T cells , 27. ThrWe further conducted stratified analysis for identifying the high-risk populations for clinical management. Previous studies suggested that lymph node metastasis can commonly occur in ESCC and is predictive of a poorer prognosis , 29. BotTobacco smoking and alcohol are lifestyle risk factors for ESCC . Recent via Cox regression analysis. Univariate analysis revealed that T stage, smoking history, lymphatic metastasis, TNM staging, and PLR are related to increased risk of ESCC while LMR and PNI serve as protective factors. Furthermore, multivariate analysis showed a 1.717-fold, 5.65-fold, and 0.511-fold risk of adverse events in relation to advanced T stage, TNM staging, and high level of LMR of ESCC. Based on the data, we propose that advanced T stage and TNM staging serve as risk factors and preoperative high LMR had a protective effect on survival outcomes of ESCC following surgery. The collective results indicate that tumor-infiltrating lymphocytes and monocytes play active roles in cancer progression. In our study, we can’t find the superiority of SII and SIRI for OS was not observed, which could be potentially attributed to the limited cohort size and insufficient follow-up period.Finally, we screened the independent risk factors were screened via multi-center investigations are therefore necessary. Secondly, longer follow-up is essential for the accurate assessment of clinical applications when grouped by different post-surgical therapy. A comprehensive evaluation of the mechanisms underlying the interplay between both these cell types and cancer should be further conducted to facilitate the development of promising treatments targeting cancer-associated inflammation.Our study has a number of limitations that should be acknowledged. Firstly, this was a single-center retrospective design. Studies on larger sample sizes and avoidance of selection bias Pretreatment peripheral parameters such as high PLR, low LMR, and low PNI are significantly correlated with adverse events in ESCC. SII and SIRI showed superiority in the prediction of outcomes, especially for the lymphatic metastasis subgroup. Furthermore, LMR may serve as an independent prognostic predictor of long-term survival for patients with ESCC. Given the simplicity, reproducibility, and low cost of analysis of inflammation-related parameters, immune cells present promising indicators of survival in the clinical management of ESCC.The 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 Medical Ethics Committee of Shanxi Province Cancer Hospital. The ethics committee waived the requirement of written informed consent for participation.XX performed the analysis and wrote the manuscript. JJ supervised the data analysis and revised the manuscript. 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.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.


During range expansion in spatially distributed habitats, organisms differ from one another in terms of their patterns of localization versus propagation. To exploit locations or explore the landscape? This is the competition-colonization trade-off, a dichotomy at the core of ecological succession. In bacterial communities, this trade-off is a fundamental mechanism towards understanding spatio-temporal fluxes in microbiome composition.Escherichia coli is a fugitive species, whereas Pseudomonas aeruginosa is a slower colonizer but superior competitor. We provide evidence highlighting the role of succession and the relevance of this trade-off in the community assembly of bacteria in spatially distributed patchy landscapes. Furthermore, aggregation-dependent priority effects enhance coexistence which is not possible in well-mixed environments.Using microfluidics devices as structured bacterial habitats, we show that, in a synthetic two-species community of motile strains, Our findings underscore the interplay between micron-scale landscape structure and dispersal in shaping biodiversity patterns in microbial ecosystems. Understanding this interplay is key to unleash the technological revolution of microbiome applications.The online version contains supplementary material available at 10.1186/s12915-022-01462-5. The goal of microbial ecology is to understand the mechanisms which maintain biodiversity  and deteThe structure of bacterial habitats as well as the ephemerality of the species inhabiting them play an important role in determining community composition, yet they are rarely considered experimentally . EcologiEscherichia coli and Pseudomonas aeruginosa in spatially distributed habitats structured at the micron-scale  in what order do the species colonize the landscape? (ii) how does this impact composition? and (iii) are metacommunity dynamics reproducible?In this work, we studied ecological succession in a bacterial meta-community of see Fig. . BuildinE. coli and P. aeruginosa demonstrate a competition-colonization trade-off as E. coli colonizes first (fast colonizer) followed by succession by the superior competitor, P. aeruginosa (slow colonizer). In patchy habitats  to study ecological succession as the community develops over 48 hours , occupancy was calculated for both P. aeruginosa, E. coli, t). Figure P. aeruginosa fractional occupancy dynamics, E. coli followed by (ii) an increasing domination by P. aeruginosa reaching a relative occupancy of P. aeruginosa dominated, coexistence; P. aeruginosa fractional abundance In these devices, we performed invasion-competition experiments using time-lapse microscopy of fluorescently labeled (RFP–GFP and GFP–RFP) co-cultures of wild-type strain pairs  while on [sensu  into nonn [sensu , which sent with  and contP. aeruginosa, right E. coli) initial colonization by pioneer cells of E. coli occurs by a low density fast traveling sensu van Vliet et al. [E. coli density reaching 35% of local occupancy . At this point, the first P. aeruginosa cells enter patches 1 and 2 . At this stage, cells can be planktonic (free swimming) or sessile (aggregated). Subsequently, the aggregation phase progresses towards its climax; a MHP full with biofilm [Following the inoculation of the patchy device inlets  demonstrates a textbook example of Skellam’s “Malthusian population in a linear habitat” [sensu [E. coli occupancy (left panel) as can be appreciated in Fig. P. aeruginosa advances, E. coli retreats its range of high occupancy while keeping less localized and less dense fluctuating sub-populations. These local community dynamics  E. coli early colonization, (ii) expansion by recruitment, (iii) competition with invading P. aeruginosa, and eventually (iv) replacement by P. aeruginosa.After initial colonization by ape Fig. A. P. aerentclass1pt{minimahabitat” . This is” [sensu ) of E. centclass1pt{minimaentclass1pt{minimaentclass1pt{minimas), the long-term pattern of local community structure can be appreciated. In Fig. i) fully occupied patches 100% dominated by P. aeruginosa, i.e., succession climax, point ; (ii) fully occupied patches 100% dominated by E. coli, point ; and (iii) extremely low density patches 100% dominated by E. coli, point P. aeruginosa, E. coli, Considering all patches  a case where E. coli dominates, P. aeruginosa; (ii) a P. aeruginosa dominated case where E. coli is mostly excluded, only coexisting in low numbers, iii) a case where E. coli persists at significant levels, In Fig. P. aeruginosa affects the dynamics of E. coli. Between P. aeruginosa “chases” a wave of E. coli. At time E. coli gets trapped at patch P. aeruginosa in patch In Fig. E. coli colonizing first followed by P. aeruginosa out-competing E. coli locally and sometimes globally. Coexistence of alternative local community states can be observed even in adjacent patches as a consequence of stochasticity. In most experiments however, there is remarkable determinism, which emerges as the statistical pattern we can appreciate in Fig. As in all experiments, what is reproduced here is the general pattern of E. coli aggregating in corridors which can disrupt later arrivals. Fragmentation events act as priority effects changing the course of succession  and reflects back as well as diffracting , givingibiotics  and highE. coli micro-colonies are unable to fragment the landscape. Here, coexistence is due to the emergence of a neutral ecology where species reach a similarly high level of long-term  expansion front invading a patchy landscape and displacing E. coli (magenta) contrasting the result for a flat landscape , a statistical comparison was made regarding the spatial correlations between ed sensu  renderinWhen comparing virtual and real patches in the flat and patchy landscapes, respectively, we observe a near neutral, but slightly positive correlation describing average interactions Fig. C. While Comparing the distributions of virtual and real corridors, differences become qualitative Fig. D. For viThe competition-colonization (CC) trade-off  has beenWe consider sites on a lattice representing a local community: vacant (state 0), occupied by competitor state 1), colonizer state 2), or both  a coexistence phase, where both persist; and (III) an interference phase, where the competitor wins. In Fig. coexistence  if the difference is of intermediate magnitude, coexistence phase; and (iii) if small differences are considered, interference phase. When no differences exist, neutral coexistence as the lottery has no bias (For no priority effects (interference and coexistence and (ii) coexistence and scramble. Thus, at intermediate values of priority effects, we can expect coexistence for a pair of strategies with a small difference in colonization, E. coli coexist with P. aeruginosa by a localization and fragmentation mechanism, thus highlighting the importance of priority effects occurring at the corridors.Scanning along a transect E. coli is a fugitive species while P. aeruginosa is a superior competitor enacting a competition-colonization trade-off in patchy environments. Aggregation-dependent priority effects deterring competitor dispersal provide a mechanistic explanation for coexistence and a textbook example of niche pre-emption [Combining theory and experiments, we demonstrate that, in a synthetic two-species community of motile strains, -emption .In patchy environments, bacterial life history resonates with periodic habitat structures , 32 and P. aeruginosa which in some cases ultimately can become fragmented. From the perspective of succession, coexistence is transient as waves of competitors will eventually break through  and P. aeruginosa (PUPa3-G and PUPa3-R) labeled with green and red fluorescent proteins (GFP and RFP). Strain details are listed in Additional file Experiments were performed using wild-type strains of E.coli and P.aeruginosa were grown from P. aeruginosa, E. coli (P. aeruginosa) were back-diluted 1:1000 (1:500) in Single colonies for both Cultures of JEK1037/36 and PUPa3-G/R were grown overnight until stationary phase and back-diluted 1:1000. Once back-diluted and cultures had reached an E. coli) or antibiotics (50 P. aeruginosa). Overnight cultures were back-diluted 1:1000, incubated to grow until reaching E. coli and P. aeruginosa colonies can easily be distinguished based on colony morphology. Results are shown in Additional file JEK1037 and PUPa3-G strains were grown overnight until stationary phase  were also tested on the same day. Each experimental set-up was performed with 3 replicates. Flasks were placed in a shaking incubator set at 30E. coli . A water tight wall around the perimeter of the sealed device using four Prior to inoculation, the devices were wettened with LB + i) flat landscape and patchy landscape—single inlet, (ii) patchy landscape—both inlets, (iii) flat landscape, and patchy landscape—distributed across the entire landscape.Alternative initial conditions were also considered , Andor Neo sCMOS camera (Andor Technology plc.), and LUMEN 200 Pro metal arc lamp (Prior Scientific Ltd.). A Prior Proscan II motorized stage (Prior Scientific Ltd.) was used for scanning. The NIS Elements Ar software (Nikon Inc.) was used for image acquisition and data processing. Image analysis was carried out using ImageJ and Python. Fluorescence intensity is a poor estimation for biomass due to differences in expression among cells. To avoid this problem we use a custom script in ImageJ to convert all images to occupancy data for each of the two strains used in an experiment.E. coli (P.aeruginosa) occupancy for a MHP indexed by k is denoted as P. aeruginosaE. colik at time t as P. aeruginosa or E. coli respectively.For every timestep, background correction is performed on each image and a threshold pixel value is calculated based off the corresponding auto-fluorescence for each of the color channels used (red and green). Above this threshold the pixel  trade-off. Table S1. Symbols. Table S2. Glossary. Table S3. Bacterial strains.Additional file 2: Supplementary Note 1. Alternative initial conditions inoculating with a 1:1 mixture of species. Supplementary Note 2. Stochastic spatial model. Supplementary Note 3. Patch occupancy and the competition-colonization trade-off.


Our study thus rejects the Orthogastropoda concept showing that the selection of the representative species and use of sufficient informative sites greatly influence the analysis of deep gastropod phylogeny.The Gastropoda contains 80% of existing mollusks and is the most diverse animal class second only to the Insecta. However, the deep phylogeny of gastropods has been controversial for a long time. Especially the position of Patellogastropoda is a major uncertainty. Morphology and some mitochondria studies concluded that Patellogastropoda is likely to be sister to all other gastropods (Orthogastropoda hypothesis), while transcriptomic and other mitogenomic studies indicated that Patellogastropoda and Vetigastropoda are sister taxa (Psilogastropoda). With the release of high-quality genomes, orthologous genes can be better identified and serve as powerful candidates for phylogenetic analysis. The question is, given the current limitations on the taxon sampling side, how many markers are needed to provide robust results. Here, we identified single-copy orthologous genes (SOGs) from 14 gastropods species with whole genomes available which cover five main gastropod subclasses. We generated different datasets from 395 to 1610 SOGs by allowing species missing in different levels. We constructed gene trees of each SOG, and inferred species trees from different collections of gene trees. We found as the number of SOGs increased, the inferred topology changed from Patellogastropoda being sister to all other gastropods to Patellogastropoda being sister to Vetigastropoda + Neomphalina (Psilogastropoda  Gastropl vents) . A coupll vents) . Severalon, 1855 , golden 22, etc. . All the22, etc. , make ga22, etc. .e.g., in Vetigastropoda , and a close relationship of Neomphalina, Cocculiniformia, and Vetigastropoda has been supported  between-proteome and N within-proteome pairs using MMseqs2  , Fig. 1.C. consors is missing in most of the SOGs   to 33%   among the rest of the 15 species, increasing SOG numbers 2.15 times. Then we used the 847 SOGs for phylogenetic reconstruction using a concatenation method and compared it with Furthermore, when examining the gene coverage of each species we found that the species the SOGs . For exan = 911) ; allowin= 1,505) . We thuse.g., e.g., transcriptomic studies  orthologs gene sets, especially on a large species scale. As expected, the numbers of detected SOGs shared by all gastropod species decreased as the number of quarried species increased, which highly influenced the results of the phylogenetic inference . WithoutC. consors led to a significant decrease in the number of shared SOGs and influenced the following phylogenetic inference. This might be caused by the fragmentation and incomplete assembly of C. consors   , which ius, 1758  and Lautin, 1791 .L. gigantea, which is the only available Patellogastropoda, although it showed long-branch attraction and challenged deep gastropod mitogenomics  is highlighted in red. Branch length is marked with blue numbers.Click here for additional data file.10.7717/peerj.13285/supp-2Supplemental Information 2posterior probability (PP) is highlighted in red. Branch length is marked with blue numbers.Click here for additional data file.10.7717/peerj.13285/supp-3Supplemental Information 3posterior probability (PP) is highlighted in red. Branch length is marked with blue numbers.Click here for additional data file.10.7717/peerj.13285/supp-4Supplemental Information 4posterior probability (PP) is highlighted in red. Branch length is marked with blue numbers.Click here for additional data file.10.7717/peerj.13285/supp-5Supplemental Information 5The best-fit model according to BIC: JTT+F+R5. AIC, w-AIC: Akaike information criterion scores and weights. AICc, w-AICc: Corrected AIC scores and weights. BIC, w-BIC: Bayesian information criterion scores and weights. Plus signs denote the 95% confidence sets. Minus signs denote significant exclusion.Click here for additional data file.10.7717/peerj.13285/supp-6Supplemental Information 6Click here for additional data file.