EGFRm+) non-small cell lung cancer (NSCLC). The EGFR-tyrosine kinase inhibitor osimertinib is a substrate of transporters ABCB1 and ABCG2 and metabolized by CYP3A4. We investigated relationships between single nucleotide polymorphisms (SNPs) ABCB1 3435C>T, ABCG2 421C>A and 34G>A, and CYP3A4∗22 and CNS treatment efficacy of osimertinib in EGFRm+ NSCLC patients.Central nervous system (CNS) metastases are present in approximately 40% of patients with metastatic epidermal growth factor receptor-mutated . For patients with no or unknown baseline CNS metastases, the primary endpoint was CNS disease-free survival . Relationships between SNPs and baseline characteristics with CNS-PFS and CNS-DFS were studied with competing-risks survival analysis. Secondary endpoints were relationships between SNPs and PFS, overall survival, severe toxicity, and osimertinib pharmacokinetics.Patients who started treatment with osimertinib for ABCG2 34GA/AA and/or ABCB1 3435CC --present in 35% of patients-- was significantly associated with decreased CNS-DFS  in the multivariate analysis. This remained significant after applying a Bonferroni correction and internal validation through bootstrapping. ABCG2 421CA/AA was related to more severe toxicity .From 572 included patients, 201 had baseline CNS metastases. No SNP was associated with CNS-PFS. Genotype ABCB1 3435C>T are predictors for developing new CNS metastases during osimertinib treatment, probably because of diminished drug levels in the CNS. ABCG2 421C>A was significantly related with the incidence of severe toxicity. Pre-emptive genotyping for these SNPs could individualize osimertinib therapy. Addition of ABCG2 inhibitors for patients without ABCG2 34G>A should be studied further, to prevent new CNS metastases during osimertinib treatment.ABCG2 34G>A and No funding was received for this trial. In vitro research showed that osimertinib is a substrate of the efflux transporters ABCB1 and ABCG2. This was further confirmed in mice. Intracerebral osimertinib accumulation was limited by both transporters, which was demonstrated in ABCB1/ABCG2 knockout mice which had six-fold higher osimertinib exposure in the brain. Osimertinib is a substrate of CYP3A4 in vitro and in vivo. When CYP3A was knocked-out in mice however, no difference in osimertinib pharmacokinetics was observed. Nevertheless, in humans with the CYP3A4∗22 variant allele, the exposure to other substrate drugs (including some tyrosine kinase inhibitors) was increased. For osimertinib, this has not been studied yet. Single nucleotide polymorphisms (SNPs) in ABCB1, ABCG2, or CYP3A4 have not been correlated with clinical endpoints before in patients who are treated with osimertinib.We performed a structured search in PubMed and Embase for preclinical and clinical studies until November 21, 2022, written in English, with the search terms “Osimertinib AND (ABCB1 OR ABCG2 OR CYP3A4)”. This resulted in 16 and 65 records respectively. ABCB1 3435C>T and/or ABCG2 34G>A were significantly correlated with the development of central nervous system (CNS) metastases in patients without CNS metastases at the start of osimertinib treatment. Furthermore, ABCG2 421C>A was significantly related with the incidence of severe toxicity.This is the first study to correlate SNPs in relevant efflux transporters to osimertinib effectiveness and severe toxicity in patients with non-small cell lung cancer (NSCLC). More specifically, SNPs ABCB1 and/or ABCG2, patients can be identified who are at higher risk of developing CNS metastases during osimertinib treatment. Osimertinib therapy could be individualized when pre-treatment genotyping is implemented after prospective validation of these results. Ultimately, ABCB1 and ABCG2 could function as promising therapeutic target in future trials to prevent the development of new CNS metastases.Patients with CNS metastases have a lowered overall survival and quality of life compared to patients without CNS metastases. Using the predictive SNPs in ,EGFRm+), this number is similar or slightly higher.,,,Central nervous system (CNS) metastases are present in almost 40% of patients with metastatic non-small cell lung cancer (NSCLC).,Systemic treatment of brain metastases is often complicated by the limited drug penetration across the blood–brain barrier. Its physiologic properties prevent large molecules to cross, and efflux transporters --predominantly ABCB1 and ABCG2-- actively transport molecules (back) into the blood, resulting in pharmacologic failure and intracerebral drug resistance.de novo occurrence of brain metastases-- was significantly lower in patients treated with osimertinib compared to patients treated with erlotinib and gefitinib (23% versus 44% after 12 months).EGFRm+ advanced NSCLC.The third generation EGFR-TKI osimertinib has the highest blood–brain barrier penetration --and hence intracerebral drug accumulation-- compared with first and second generation EGFR-TKIs.in vivo yet. Most frequently described are the 3435C>T SNP in ABCB1  and the 421C>A and 34G>A SNPs in ABCG2 .Osimertinib and its active metabolites are both substrates of ABCB1 and ABCG2.Furthermore, the effects of genetic variations in ABCB1 and ABGCG2 on systemic osimertinib concentrations are unknown. Osimertinib is mainly metabolized by cytochrome P450 isoenzyme 3A4 (CYP3A4).ABCB1, ABCG2, and CYP3A4 could influence CNS efficacy of osimertinib, c.q. intracerebral progression-free survival (CNS-PFS) and occurrence of new CNS metastases, as shown in For this study, we hypothesized that SNPs in EGFRm+ NSCLC between November 2014 and June 2021. Patients were excluded when no plasma or whole blood for SNP analysis was available. Data cut-off was January 2022 to ensure a minimal follow-up time of 6 months. ABCB1 3435C>T, ABCG2 421C>A and 34G>A, and CYP3A4∗22. To ensure single-blinding of the investigators, all clinical data were collected before any genotyping took place.This is a retrospective, cross-sectional observational cohort study, performed in six large cancer centres in the Netherlands. Patients were eligible for inclusion if they were ≥18 years and treated with osimertinib for c.q. after 2 weeks) at a dosage of 80 mg once daily were used for comparison. Furthermore, since osimertinib is unstable in whole blood and plasma at room temperature,,Baseline imaging was performed according to local practice, which was mostly within 4 weeks prior to start of osimertinib treatment. Imaging of the brain was performed according to local practice, and was mainly performed in patients with known CNS metastases or symptoms suggestive of CNS metastases. Regular imaging of the brain was not performed. Dose modifications, interruptions and discontinuation due to toxicity and the frequency of radiologic treatment evaluation were at the treating physicians’ discretion. Normally, the first radiological evaluation took place with a computed-tomography (CT)-scan after four to eight weeks, and thereafter once every 8–12 weeks until disease progression. Central nervous system metastases were followed-up with magnetic resonance imaging (MRI). Baseline characteristics and clinical data were recorded and anonymised. Since the variants of interest are germline SNPs, the timing of blood withdrawal for genotyping was not relevant. For pharmacokinetic evaluation, the time between last osimertinib administration and blood withdrawal had to be known. Only plasma concentrations at the time of steady state . The digital droplet PCR analyses were performed in the T100 Thermal Cycler and detected with the QX200 Droplet Reader. QuantaSoft (BioRad) was used for DNA quantification and subsequent genotyping the For the primary analyses, patients were divided in two cohorts, depending on the presence of CNS metastases at start of osimertinib treatment. CNS metastases were present at baseline when proven by MRI, CT-scan or pathologic diagnostics of cerebral spinal fluid --in case of leptomeningeal metastases-- prior to start of osimertinib treatment. Baseline CNS metastases were only defined as absent if these were not present on MRI, since with (contrast-enhanced) CT scanning micro-metastases and leptomeningeal metastases could be missed. All other patients had unknown CNS metastases at baseline.de novo occurrence of CNS metastases .In the cohort with known CNS metastases at baseline, primary outcome was the relationship between the four SNPs with CNS-PFS, defined as the time from treatment start until progressive CNS disease according to the Response Assessment in Neuro-Oncology Brain Metastases (RANO-BM) criteriai.e., time from treatment start until death from any cause), PFS independent of disease site, severe toxicity, and osimertinib pharmacokinetics  in the total cohort. In the PFS analysis, an event was disease progression according to the Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1,Secondary endpoints were the relationships between presence of the four SNPs and OS . For the CNS-DFS analysis, death from any cause was also considered to be a competing risk additional to other reasons for treatment stop. Internal validation of all SNPs and statistically significant parameters in the multivariable analyses of the primary endpoints was performed by bootstrapping 1000 samples.For the primary endpoints, CNS-PFS and CNS-DFS were related with every SNP and patient characteristics as single parameter using univariate proportional hazards models for the subdistribution , as described by Fine and Gray.min was calculated with the earlier published methods.,min is considered to be not-normally distributed.For the secondary endpoints OS and PFS, every SNP and patient characteristics were first tested as single parameter with the Kaplan–Meier method and log-rank test. Thereafter, Cox proportional-hazards regression analysis was used for parameters with a p-value <0.10. With the chi-square test and competing risk analyses,c.q. wild-type versus heterozygote and homozygote variants). The other variants were tested in both dominant and recessive (c.q. wild-type and heterozygote variants versus homozygote variants) models. If single SNPs had p < 0.20 in the primary univariate competing risk analysis, combined testing of two SNPs was considered acceptable.Germline variants with minor allele frequencies ≤0.10 were only tested in a dominant model  or Stata , and p < 0.05 was considered to be significant unless stated otherwise.www.trialsearch.who.int; trial NL8914). Thereafter, the local Medical Ethics Committee at every participating site approved the study protocol independently. Since this study was observational, non-invasive, and already collected plasma samples were used, all Medical Ethics Committees waived the obligation for written informed consent of patients. Clinical data and patients samples were obtained after inclusion in local trials: START-TKI Rotterdam and Breda , N13FPB The Netherlands Cancer Institute (IRBd19-192), Maastricht 2019-1018-A-10 and Maastricht OSIBOOST . Patients from Groningen UMC were included from the Oncolifes biobank, from which the request was approved by the local ethics committee (OLS032-202000693). Patients from Amsterdam UMC were included from the Liquid Biopsy Center biobank, from which the request was approved by the local institutional review board (UVB21-0125). Flow chart for patient samples and data, including ethical approval, is presented in The study was primarily approved by the local ethics committee  and was registered in the Dutch Trial Registry and International Clinical Trial Registry Platform , presence of the T790M mutation, and not having received prior chemo- and/or immunotherapy  in 315 wild-type patients, it was statistically impossible to incorporate this SNP in the multivariable analysis as this gave estimation problems. In order to be able to use this ABCG2 34G>A SNP . Other parameters, which were statistically significantly associated with reduced number of new occurrence of CNS metastases, were EGFR exon 19 as primary EGFR mutation and WHO performance status 0–1 at baseline  remained statistically significant after validation. Since in the AURA3 studyIn the cohort without known CNS metastases at start of osimertinib treatment, 36 patients (9.7%) experienced a CNS-DFS event, with cumulative incidences of 7.2% and 10.4% after 12 and 24 months respectively. All SNPs except the 4G>A SNP A, we ana 3435C>T B. We choCG2 34GG . In the baseline . Only thEGFR exon 19 deletion and osimertinib used as first line were associated with improved PFS and OS. The presence of TP53 at baseline and known CNS metastases at baseline were associated with significantly decreased PFS and OS. Female sex and older age were significantly associated with improved PFS, but not with OS.In 572 patients, 455 (80%) experienced progression of disease with a median PFS of 10.2 months [IQR 8.9–11.5]. Two hundred eighty-eight (50%) patients died during follow-up, with a median OS of 25.3 months [IQR 23.0–27.5]. No SNPs were statistically significantly related with either PFS or OS . Asian rABCG2 421CA/AA compared with wild-type patients . In the univariate time-to-event analysis, the same ABCG2 421C>T SNP was significantly associated with higher hazard of severe toxicity , osimertinib Cmin was 11% lower compared to CT/CC patients. No other SNP was associated with osimertinib pharmacokinetics , and to offer a specific target for future research to prevent occurrence of new CNS metastases.In this multicentre trial to study SNPs in important transporter genes, the combined genotype of ,ABCG2 34GA/AA and/or ABCB1 3435CC  to have a 72% reduced risk of developing new CNS metastases compared to patients with the ABCG2 34GG and ABCB1 3435CT/TT genotype. This large effect was predominantly because of the ABCG2 34G>A SNP , it makes sense that more severe toxicity leads to more dose interruptions and reductions, which (temporarily) decrease the intracerebral osimertinib concentrations and hence increase the chance of CNS metastases.trations . These cABCB1 3435C>T SNP. This could be caused by the impaired drug transport across the basolateral intestinal cell membrane into the systemic circulation, that results in lower drug absorption.ABCB1 3435CC genotype contributes to the reduced risk in developing new CNS metastases. However for PFS, OS, or the incidence of severe toxicity, an 11% decrease in Cmin will probably not be clinically relevant, especially since no systemic exposure-efficacy relationship for osimertinib has been shown.,Osimertinib trough levels were significantly lower in patients with an The baseline characteristics which are associated with the primary and secondary endpoints could further help clinicians to identify patients at risk for early CNS disease progression , survivaA limitation of this study was the retrospective nature of the partly prospective collected data, which could have led to loss of data. This detrimental effect of the design remains limited in our opinion, because the major events --CNS progression, PFS, OS, and severe toxicity-- have clinical consequences and were hence well-documented. Nevertheless, prospective validation must take place prior to implementation of these results. Another limitation is the lack of systematic imaging in patients without CNS metastases at baseline and during osimertinib treatment, because asymptomatic metastases or progression could have been missed. The risk of bias was low, because SNPs were genotyped after all data was collected. Furthermore, these results have not been validated externally, with an independent cohort. The here used alternative --internal validation by bootstrapping-- was considered to produce the most accurate validation results, and is statistically acceptable.ABCG2 34G>A and ABCB1 3435C>T are strong predictors for the development of new CNS metastases in patients treated with osimertinib for EGFRm+ NSCLC who have no documented CNS metastases. Furthermore, ABCG2 421C>A was significantly related with the incidence of severe osimertinib toxicity. These results encourage the use of pre-emptive genotyping these SNPs in clinical practice to further individualize osimertinib therapy, and point toward ABCB1 and ABCG2 as promising targets to prevent the development of new CNS metastases during osimertinib treatment.To conclude, c.q. inclusion of patients and data collection, including pharmacokinetic and pharmacogenetic data). G.D.M.V., E.O.-d.H., A.C.D. and R.H.J.M. analysed, verified, and interpreted the (raw) data. G.D.M.V. wrote the manuscript. All authors had full access to the data, critically reviewed the manuscript, and gave final approval for publication.G.D.M.V., A.C.D., and R.H.J.M. designed the study. G.D.M.V., R.J.B., M.J., A.J.v.d.W., I.B., L.E.L.H., S.C., C.M.J.S., E.d.J., S.L.W.K., N.S., R.H.N.v.S., E.F.S., A.C.D., and A.D.R.H. performed the research (g.veerman@erasmusmc.nl) on reasonable request until five years after date of publication. Data recipients are required to enter a formal data sharing agreement that describes the conditions for release and requirements for data transfer, storage, archiving, publication, and intellectual property with every participating centre.All datasets including deidentified participant data, which are generated during and/or analysed during the current study are available from the corresponding author  in Paris, September 2022. The abstract has been awarded a merit award by the organizing committee.G.D.M.V. reports grants from Eli Lilly; outside the submitted work. A.C.D. reports grants from Boehringer-Ingelheim; outside the submitted work (paid to institution). R.H.J.M. reports an unrestricted grant from Boehringer-Ingelheim (paid to institution) and grants from Astellas, Bayer, Cristal Therapeutics, Novartis, Pamgene, Pfizer, Sanofi, and Servier; outside the submitted work (paid to institution). L.E.L.H. has no relationship to disclose in relation to this manuscript. Outside of the current manuscript: research funding Roche Genentech, AstraZeneca, Boehringer Ingelheim, Takeda ; advisory board: BMS, Eli Lilly, Roche Genentech, Pfizer, Takeda, MSD, Merck, Novartis, Boehringer Ingelheim, Amgen, Janssen ; speaker: MSD, Lilly (institution); travel/conference reimbursement: Roche Genentech (self); mentorship program with key opinion leaders: funded by AstraZeneca; fees for educational webinars: Benecke, Medtalks, VJOncology (self), high5oncology (institution); interview sessions funded by Roche Genentech, Bayer, Lilly (institution); local PI of clinical trials: AstraZeneca, Novartis, BMS, MSD, Merck, GSK, Takeda, Blueprint Medicines, Roche Genentech, Janssen Pharmaceuticals, Mirati, Abbvie, Gilead. A.J.v.d.W. reports grants and advisory board from AstraZeneca, grants and advisory board from Boehringer-Ingelheim, advisory board from Janssen, advisory board from Novartis, grants and advisory board from Pfizer, grants and advisory board from Roche, grants and advisory board from Takeda, all outside the submitted work; all payment to the UMCG. N.S. provided consultation or attended advisory boards for Boehringer Ingelheim, Cogent Biosciences, Ellipses Pharma, Luszana. N. Steeghs received research grants from Abbvie, Actuate Therapeutics, Amgen, Array, Ascendis Pharma, AstraZeneca, Bayer, Blueprint Medicines, Boehringer Ingelheim, Bristol-Myers Squibb, Cantargia, CellCentric, Cogent Biosciences, Cresecendo Biologics, Cytovation, Deciphera, Eli Lilly, Exelixis, Genentech, GlaxoSmithKline, Incyte, InteRNA, Janssen, Kinnate Biopharma, Luszana, Merck, Merck Sharp & Dohme, Merus, Molecular Partners, Navire Pharma, Novartis, Numab Therapeutics, Pfizer, Relay Pharmaceuticals, Revolution Medicin, Roche, Sanofi, Seattle Genetics, Taiho, Takeda. All outside the submitted work, all payment to the Netherlands Cancer Institute. E.F.S. has no relationship to disclose in relation to the manuscript. Outside the submitted work : research grants from Astra Zeneca, MSD, BMS, Roche Genentech, advisory boards Astra Zeneca, BMS, Boehringer Ingelheim, Bayer, DSI, MSD, Takeda, Roche, Merck, Novartis, Amgen, Janssen.All other authors declare no competing interests.


Dear Editor,Almost 3 years since its first case report, coronavirus disease 2019 (COVID-19) remains one of the most discussed topics in the world. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, infects host cells, mainly in the respiratory tracts, via immediate attachment to the angiotensin-converting enzyme type 2 receptor at the surface of host cellsTo manage fever and inflammatory-related symptoms, antipyretics, and analgesics, classified as NSAIDs, are commonly usedet al.+-channels (Kv1.3 channels) highly expressed in the T lymphocytes, which are pivotal for facilitating lymphocytes activation and proliferation. As a result, the immune response of T lymphocytes is suppressed. Pharmacologically, this inhibitory mechanism should be considered a novel strategy to block the emergence of cytokine storms in COVID-19 patientsHow actually NSAIDs exert their immunosuppressive effect? Kazama Of several pathways involved in inflammatory cascades, the MAPK pathways play a significant roleSeveral studies have reported NSAIDs’ ability to disturb the production and action of cytokines and antibodiesOne of the worldwide priorities is to ensure effective antiviral agents against emerging and re-emerging viral diseases are availableIn conclusion, relevant evidence suggested that certain NSAIDs with their combinatorial anti-inflammatory and antiviral effects could provide more efficient results in alleviating the respiratory distress symptoms and hyperinflammatory responses caused by COVID-19. Such pharmacological approach shall bring tremendous efficacy for the patients suffering from COVID-19-related cytokine storm or similar symptoms caused by other viral diseases.Not applicable.None.F.N. and T.B.E designed the study. F.N. and S.S.M. wrote the first draft. F.N., S.S.M., and T.B.E. updated the manuscript. F.N., S.S.M., and T.B.E. reviewed the final draft. All authors have critically reviewed and approved the final draft and are responsible for the content and similarity index of the manuscript.The authors declare that they have no financial conflicts of interest with regard to the consent of this report.None.Firzan Nainu and Talha Bin Emran.Not commissioned, internally peer-reviewed.


Ficus heterostyla and Ficus squamosa) in Xishuangbanna, southwest China, and aim to document what is likely to be the final stages of speciation between these species using a combination of trait data and experimental manipulation. Volatile profiles at the receptive phase, crucial for attracting pollinators, were analyzed. In total, 37 and 29 volatile compounds were identified from receptive F. heterostyla and F. squamosa figs, respectively. Despite significant interspecific dissimilarity, 25 compounds were shared. Ovipositor lengths lie well within range required for access to heterospecific ovules, facilitating hybridization. Cross introduction of wasps into figs was conducted and hybrid seeds were generated for all donor/recipient combinations. F. heterostyla wasps produce adult offspring in F. squamosa figs. While F. squamosa wasps induce gall development in F. heterostyla figs and their offspring fail to mature in synchrony with their novel host. We record limited geographic barriers, minimal volatile dissimilarity, compatible morphology, complementary reproductive phenologies, and the production of hybrid seeds and wasp offspring. These findings suggest ongoing wasp specialization and reproductive isolation, potentially applicable to other related fig species.The dynamics and processes underlying the codiversification of plant–pollinator interactions are of great interest to researchers of biodiversity and evolution. Cospeciation is generally considered a key process driving the diversity of figs and their pollinating wasps. Groups of closely related figs pollinated by separate wasps occur frequently and represent excellent opportunities to study ongoing diversification in this textbook mutualism. We study two closely related sympatric dioecious figs ( Ficus, Moraceae) and their pollinating wasps .Coevolution with pollinating animals is generally considered one of major evolutionary forces driving the diversification of flowering plants . The adaFicus species, distributed globally in tropics and subtropics  do figs of these two fig species have similar volatile profiles at receptive stage? (2) Does trait matching (ovipositor length and style length) facilitate oviposition? (3) are hybrid seeds and wasps produced, and are there fitness differences between conspecific and heterospecific crosses?analyses . In XishF. heterostyla population and a transplanted F. squamosa population consisting of approximately 200 individuals relocated from Bubeng , 48 km from XTBG.Xishuangbanna, situated in Yunnan Province, southwest China, is characterized by a tropical monsoon climate with three distinct seasons: a humid hot rainy season (May–October), a foggy cool-dry season (November–February), and a hot-dry season (March–April) . XishuanFicus squamosa is a short shrub up to 2(–3) m tall with a creeping stem. It typically grows along riverbanks or near fast-flowing streams in tropical forests, spanning regions from northern India to southern Thailand. Its figs originate on branches near the water level or even below  m tall, and occurs from southwest China to Vietnam. Its figs are located in rooting stolons near or under the soil , however, the wasp of F. heterostyla is reported to visit F. squamosa . Seven male and two female F. heterostyla trees were sampled in April and September, while seven male and three female F. squamosa trees were sampled in March. The prefemale figs were enclosed in mesh bags to prevent nonpollinating wasps from laying eggs and their development was monitored based on fig size and color. The receptive B phase . Compound identification was carried out through a step-by-step process. Firstly, the NIST Mass Spectral Search Program  was used to compare compound mass spectra with reference chemicals for initial identification. Secondly, retention indices (RI) for each compound were calculated following the method described by Ficus species and the female and male trees of the same species was computed for further analysis (Wd* (number of replications = 999), based on Welch MANOVA was used to analyze the VOC data , 15 figs from five male F. squamosa trees (3 figs per tree), 30 figs from three female F. heterostyla trees (10 figs per tree), and 28 figs from four male F. heterostyla trees (2–10 figs per tree). Each fig was cut into four equal parts and 10 florets per part were sampled randomly to measure style length. To measure wasp ovipositor length, the male phase figs were sampled and individually placed in mesh bags. After the emergence of wasps from the figs, 10 females from each fig were sampled randomly. A total of 116 wasps from 12 figs of three male F. heterostyla trees (2–5 figs per tree) and 110 wasps from 11 figs of three male F. squamosa trees (1–8 figs per tree) were collected to measure ovipositor length. Both style and ovipositor lengths were measured using a dissecting microscope  with an eyepiece graticule (32 units = 1 mm).Floret styles and wasp ovipositors were measured to compare lengths. To measure style length, 79 phase B figs were sampled: six figs from five female A linear mixed-effects model was applied to assess differences between style and ovipositor lengths, as well as style lengths between female and male figs, with fruit nested within the tree as random effects . To avoiPrefemale figs were first enclosed in mesh bags to prevent wasp entry or oviposition. When experimental figs reached receptivity, mature male figs were collected and individually placed in mesh bags to collect the female wasps that emerged from the figs. These wasps were then introduced to receptive experimental figs. Each fig was inoculated with one wasp. In total, eight introduction treatments were performed, as detailed in F. heterostyla figs and 29 from F. squamosa figs, including 25 compounds shared by both species , with most categories exhibiting normal distribution  = 21.435, p < .001; Variance homogeneity of the pairwise Bray–Curtis distances between volatile samples was demonstrated via the Levene test . However, no significant differences were found between the sexes in F. heterostyla  or F. squamosa . The NMDS graph . For males, the mean style length of F. heterostyla was significant longer than that of F. squamosa . Furthermore, the style lengths of male figs were significantly shorter than those of conspecific female figs .Models with and without random effects were compared. The model selection procedures are presented in F. squamosa pollinators had an ovipositor length of 0.76 ± 0.083 mm (n = 33), significantly longer than the style of male figs , but significantly shorter than the style of female figs . This trend was also measured in F. heterostyla–pollinating wasp association. The F. heterostyla pollinators had an ovipositor length of 0.76 ± 0.094 mm (n = 36), which did not differ significantly from that of the F. squamosa pollinators . There was no observed overlap between ovipositor and style lengths in the F. squamosa–pollinating wasp association  and heterospecific (mH–mS) wasps, respectively, with successful production of adult offspring. Regarding the treatments on females, figs introduced with conspecific wasps demonstrated slightly  or significantly lower abortion ratios  compared to those inoculated with heterospecific wasps. These findings suggest that pollination by alternative wasp species increased the abortion ratio, thereby reducing host fitness  or significantly more seeds  than those inoculated with heterospecific wasps (Female ic wasps , suggestF. heterostyla and F. squamosa. Although these species are morphologically distinguishable and are generally pollinated by distinct wasp species, reproductive isolation is not fully realized. The weak geographical barriers, minimal volatile dissimilarity, compatible reproductive morphologies, and complementary reproductive phenologies (F. heterostyla figs cannot reproduce due to complete abortion of experimental figs, wasps of F. heterostyla developed in heterospecific F. squamosa figs.Based on chemical cues, key morphological traits, and manipulative experiments, we investigated both pre- and post-zygotic isolation in a sympatric fig pair consisting of nologies  set the Wolbachia infections can intensify postzygotic isolation (F. heterostyla can produce offspring in heterospecific F. squamosa figs.Species complexes of figs associated with genetically distinct wasps can diverge under a scenario of cospeciation. The disparity in generation time between these mutualists means that wasps should speciate faster. Thus, reproductive isolation between figs may represent steps on the road to full speciation. While closely related figs can often hybridize, reproductive success is generally not realized among multiple wasp species of a single host . Hybridisolation . Considesolation . We suggF. heterostyla and F. squamosa cooccur from southern Thailand to southwest China. Despite distinct habitat preferences, both species coexist very closely within certain sympatric areas (F. squamosa wasps (F. squamosa figs by normal wasps of F. heterostyla.Examples of heterospecific visitation between allopatric figs grown together due to human introduction  underscoic areas . Consequsa wasps  may faciF. heterostyla, F. squamosa, and closely related F. hispida confirms similarities in VOCs between F. heterostyla and F. squamosa, but also highlights substantial interspecific differences (Floral volatiles released by receptive figs are a key signal responsible for attracting specific wasps, but are also variable , 2016. Vferences .F. squamosa suggests that the wasps may visit coflowering male and female trees indiscriminately. Similarly, no significant difference in volatile profiles was found between the F. heterostyla males and females in different seasons; however, analysis was conducted on two female samples only, which may introduce bias.The enduring mutualism in dioecious fig species relies on the deceptive attraction of pollen-laden wasps to rewardless female figs; therefore, females undergo selection to chemically mimic the volatiles emitted by coflowering males to attract wasps . HoweverF. natalensis and F. burkei (F. microdictya and F. umbrae (F. hirta and F. triloba (F. auriculata complex and F. semicordata varieties (Although there was notable interspecific variance, most volatile compounds emitted by one fig were also present in the other. The specific compounds that were physiologically appealing to the wasps under study have yet to be identified. However, the extensive overlap in volatiles suggests a considerable potential for cross attraction of both wasp species. In addition, it is plausible that these two sister wasp species have not diverged substantially in their chemosensory apparatus and may be attracted to the same active compounds, which requires further confirmation. Similar observations of shared compounds and attraction of “non-target” wasp species have also been recorded in other closely related fig pairs, such as . burkei , F. micr. umbrae , and F.  triloba , as wellarieties .F. squamosa primarily produces figs in the cold and dry months, resulting in interruptions in male fig availability, while F. heterostyla trees produce fruit year-round with a well-define summer peak (F. heterostyla figs throughout the period when receptive F. squamosa figs were present, but no F. squamosa wasps were being released locally. This complementary pattern of reproductive phenologies and lack of competition appear to have facilitated colonization of F. squamosa by the normal wasp of F. heterostyla.For fig populations to sustain ongoing wasp populations, it is essential for some individuals to bear figs throughout the year . Figs hamer peak . Wasps eF. heterostyla and F. squamosa display similar morphological traits, including shape, color (F. auriculata complex (F. montana and F. asperifolia (Pleistodontes imperialis, the pollinator of F. rubiginosa, cannot produce offspring in heterospecific F. macrophylla figs, because its ovipositor was too short to reach the ovule (F. heterostyla or F. squamosa wasps can access some ovaries within female F. heterostyla ﬁgs, an absence of eggs was notable. This absence could be attributed to the well-developed synstigma, a structure by the clustering of two or more stigmas (The receptive figs of both e, color , and oste, color . Upon ene, color , and pole, color . Here, te, color . In seede, color . Relativ complex , F. monterifolia . While, he ovule . Our res stigmas . The syn stigmas , while c stigmas . Thus, tF. heterostyla and F. squamosa. Wasps pollinated heterospecific figs and hybrid seeds were produced, no matter the combination. Nevertheless, such interspecific interactions compromised host fitness, as evidenced by the increased fig abortion and decreased seed set. Oviposition and subsequent gall formation in heterospecific hosts showed no significant deviation in performance compared to interactions with conspecific hosts. The F. squamosa figs inoculated by conspecific wasps exhibited slightly or significantly lower abortion rates and increased offspring relative to those inoculated with heterospecific wasps, suggesting reduced fitness. However, given the complete abortion of experimental male F. heterostyla figs, the implications for fitness costs related to these interspecific interactions remain inconclusive. Fieldwork revealed elevated abortion rates of F. heterostyla figs during the dry season. In Xishuangbanna, F. heterostyla fig crop was lowest from March to May for female trees and from December to March for male trees (F. heterostyla figs along rooting stolons close to or beneath the soil, reduction in soil moisture and temperature may adversely affect fig development. Experimental Tree 1 inoculated in the hot-dry season under mS–fH treatment showed a noticeably higher abortion rate than Trees 2 and 3 inoculated during the foggy cool-dry season (F. heterostyla figs showed a more pronounced abortion rate in male trees than in female trees (F. heterostyla figs and may also play a role in facilitating colonization in F. squamosa figs. Hybrid seeds generation without wasp reproduction in heterospecific figs has also been observed between F. lutea and F. sur (F. montana and F. asperifolia (F. hispidioides and four closely related members from the section Sycocarpus (The experimental results confirmed the potential for hybridization between le trees . This suy season . This rele trees , Table 1d F. sur , F. monterifolia , and F. cocarpus .F. lutea × F. sur and F. lutea × F. thonningii hybrids have been shown to germinate, their rarely survive beyond the cotyledon stage (Previous studies have documented that both artificial and natural hybridizations with intra-subgenus figs can produce hybrid seeds capable of germination e.g., . Nonetheon stage . Thus, hon stage  or that on stage .F. squamosa possesses larger female and male figs than F. heterostyla (see Table 1 in F. squamosa produces figs along branches above the ground, whereas F. heterostyla bears figs on rooting stolons along or partially below ground level. Consequently, F. heterostyla wasps may tend to remain in close proximity to the ground to access potential receptive figs, including those of F. squamosa figs. In contrast, F. squamosa wasps may be more likely to fly at low heights, which may impede their ability to locate the partially covered figs of F. heterostyla. Consequently, the syconia of F. heterostyla may not be readily discoverable and available for colonization by F. squamosa wasps.Research suggests that larger figs facilitate easier wasp entry compared to smaller figs . F. squaF. heterostyla and F. squamosa may not yet be established, reflecting incomplete wasp specialization. Hybrid seeds were generated through crosses, which was accompanied fitness-reduction. Wasps laid eggs and induced gall formation in heterospecific hosts. F. heterostyla wasps reproduced adult offspring in F. squamosa figs, but the reverse was not true, as figs aborted. We recorded weak geographic barriers, minimal volatile dissimilarity, compatible morphology, complementary reproductive phenologies and the production of hybrid seeds and wasp offspring in heterospecific hosts. We suggest that F. heterostyla and F. squamosa represents an example of incomplete wasp specialization and potentially incomplete reproductive isolation, this is congruent with a cospeciation scenario potentially generalizable to other fig complexes or closely related species.Prezygotic isolation between qrad045_suppl_Supplementary_Figures_S1-S11_Tables_S1-S2Click here for additional data file.


Pseudomonas species that are responsible for outbreaks in pepper and tomato both in transplant-houses and in production fields in Georgia, USA. Phylogenomic analyses and whole genome sequence indices demonstrated that the pepper and tomato strains belonged to P. capsici. The whole-genome comparison revealed that 13 Pseudomonas strains from diverse isolation sources that were curated in NCBI were indeed P. capsici indicating a potential wide-host range for this bacterial species. Our greenhouse-based host-range assay also indicated that P. capsici strains were pathogenic on pepper, tomato, eggplant, cabbage, lettuce, and watermelon corroborating a wide-host-range. A phylogenetic tree inferred from the whole genome sequence data showed that the P. capsici strains from Georgia (pepper and tomato) were genetically diverse, and were closely related to tomato P. capsici strains from Florida. Genomic presence of traditional bacterial virulence factors in P. capsici strains was also determined. Pseudomonascapsici strains encode one set of type I secretion system, two sets of type II secretion systems, one set of type III secretion system, two sets of type V secretion systems, three sets of type VI secretion systems, and various secondary metabolite gene clusters including lipopeptides. In in-vitro assays, it was demonstrated that six out of seven P. capsici strains (pepper and tomato strains from Georgia) were not sensitive to 0.8 mM CuSO4. When the genomes of copper-tolerant strains were compared with the copper-sensitive strains, it was observed that the former strains encode a cluster of genes related to copper tolerance, which were absent in the genomes of copper-sensitive strains. Considering the ability of P. capsici strains to infect a range of vegetable hosts and possession of a wide range of bacterial virulence factors, secondary metabolites, and copper-tolerance genes, we envision that the management of this pathogen might potentially be a challenge.Disease outbreaks of bacterial leaf spot and blight of pepper and tomato often occur in both transplant- and field-production systems worldwide. In some cases, the outbreaks are caused by novel bacterial species. Characterization of these novel bacterial species are critical in developing diagnostic assays and identifying management options for pathogen monitoring and sustainable production, respectively. We characterized strains belonging to novel  However, they are vulnerable to several Pseudomonas species, including P. corrugata, P. syringae pv. tomato, and P. syringae pv. syringae, P. viridiflava, P. mediterranea, which have been known to cause diseases in these crops in the United States particularly in Georgia and Florida   .Pseudomonas species, P. capsici, that caused typical bacterial symptoms on pepper foliage (leaf spots and blights) under greenhouse conditions . These results will provide insights into the molecular mechanisms underlying P. capsici virulence, and may prove useful in developing new strategies for managing this destructive pathogen.We previously reported a new nditions . The patpotatoes . In this2.2.1.2O). The resulting tissue macerates were then streaked onto the nutrient agar (NA) medium. The NA plates were incubated at 28°C for 2 days. The dominant colonies on the isolation plates were selected and streaked onto new NA plates to obtain pure cultures. In addition, three P. capsici strains  isolated from pepper in Georgia (Pseudomonas strains (GEV417 and GEV1127) isolated from tomato in Florida  obtained from the National Collection of Plant Pathogenic Bacteria (NCPPB) were also included and characterized in this study. The location, host, and year of isolations for these strains are shown in In 2020, tomato foliage samples exhibiting symptoms were collected from Cook County and Grady County in Georgia, United States. For bacterial isolation, the margins of healthy and symptomatic tomato leaf tissues were excised using a sterile scalpel and then macerated in sterile distilled water  at 28°C and 200 rpm for around 16 h. After centrifugation at 16,100 x g for 1 min, the supernatants were then removed, and the resulting pellets were resuspended in sdH2O. The bacterial concentrations were then adjusted to an optical density of 0.3 at 600 nm, which corresponds to approximately 108 colony-forming units (CFU/mL), using a Biophotometer .The 2.2.2.2.1.Pseudomonas strains. First, single colonies from each strain were transferred from NA plates to 4 mL nutrient broth and cultured overnight in a shaking incubator . Then, a Monarch Genomic DNA Purification Kit  was used to extract genomic DNA from 1 mL of overnight culture. A NEBNext Ultra II DNA Library Prep Kit for Illumina was used to prepare genomic libraries and the Illumina Novaseq 6,000 platform was used to sequence the libraries by Novogene Co., Ltd. . Raw sequences were filtered using fastp v 0.20.0  divided by overall HSP length] of the genome-to-genome distance calculator 2.1 (To determine the taxonomic classification of the twelve ator 2.1  were utiator 2.1 . Additioator 2.1 .2.2.3.P. cichorii and 12 P. capsici strains with genome assemblies that were available on NCBI (accessed on Dec 07 2022) were screened using the TYGS. Based on the results, strains that were confirmed as P. capsici, along with P. cichorii ATCC 10857T (RefSeq assembly accession: GCF_900104015.1) as an outgroup, were chosen for phylogenetic analysis based on their core genomes using the M1CR0B1AL1Z3R web serverP. capsici strains in Pseudomonas cichorii ATCC 10857T was included as an outgroup.The species identity of all 49 2.2.4.gyrB and rpoD were extracted from the genomes of 12 P. capsici strains and selected strains using BLASTN and analyzed in Geneious Prime (v2019.2.3). The individual gene sequences were aligned using MAFFT (v7.294b) (gyrB (487 bp) and rpoD (474 bp) were used to construct a neighbor-joining phylogenetic tree. The robustness of the tree topology was estimated using 1,000 bootstrap replicates. The tree was visualized using the tvBOT (The sequences of housekeeping genes v7.294b)  and trimhe tvBOT .2.3.Brassica rapa ssp. chinensis) cv. Rubicon, eggplant (Solanum melongena) cv. Nadia, lettuce (Lactuca sativa) cv. Dragoon, broccoli  cv. Arcadia F1, and endive (Cichorium endivia) cv. Curlesi. Seedlings (n = 10 seedlings per host per experiment) were grown in plastic pots filled with commercial potting mix and maintained at a temperature of 28°C in a greenhouse. Two independent experiments were conducted. To inoculate the seedlings, leaves of four to six-week-old seedlings of each host were infiltrated with bacterial suspensions of Pseudomonas strains in 6 CFU/mL using a syringe. Seedlings inoculated with sdH2O were used as negative controls. The symptom was evaluated qualitatively (presence/absence) in the inoculated seedlings at 7 days post-inoculation (dpi). The bacteria were re-isolated from the symptomatic tissues and the identities of the isolated bacteria were confirmed by BOX-PCR  was prepared following published protocols  server . The subst’ flag .3.3.1.Pseudomonas sequenced genomes are summarized in Pseudomonas genomes are shown in The general features of the 12 3.2.Pseudomonas genomes, their dDDH and ANIb values were calculated by comparing them with the P. capsici type strain Pc19-1T. All strains displayed dDDH values ranging from 77.0 to 100.0% when compared with P. capsici Pc19-1T, exceeding the cut-off value of 70% for species delineation based on dDDH  of all 25 t values . These ot values .3.3.gyrB and rpoD, which had a total length of 961 nucleotides. Fifteen validly described Pseudomonas type strains were included in the analysis, with P. graminis DSM 11363T serving as an outgroup. The resulting phylogenetic tree showed that the strains claimed to be a new phylogroup of P. cichorii, as well as 12 P. capcisi strains whose genomes were sequenced in this project including P. capsici type strain Pc19-1T, clustered together and formed a separate clade from the P. cichorii clade , but are absent in the remaining five strains . Interestingly, two types of copper tolerance gene clusters were observed in the seven copper-tolerant strains listed above  / Ni (II) efflux transporter DmeF, and a metal/formaldehyde-sensitive transcriptional repressor  in Florida was initially identified as P. cichorii based on the LOPAT scheme and sequence similarity (Cucurbita pepo L. var. pepo) in Tennessee were identified as P. cichorii based on the LOPAT scheme , tomato, soybean (Glycine max), wild soybean (Glycine soja), chrysanthemum (Chrysanthemum sp.), tobacco (Nicotiana tabacum), wild celery (Apium graveolens), pepper, cabbage (Brassica oleracea), stevia, hibiscus (Hibiscus sp.), basil (Ocimum basilicum), Ficus pandurata, pumpkin, F. lyrata, and crown of thorns (Euphorbia milii). Furthermore, the results of our artificial inoculation assays revealed that 12 P. capsici strains tested were pathogenic on pepper, tomato, Chinese cabbage, eggplant, and lettuce, but non-pathogenic on broccoli and endive. These observations indicate a diverse host range for P. capsici strains across different plant families, which may have practical significance in choosing crops for rotation or for companion cropping systems. However, it is interesting to note that although P. capsici is closely related to P. cichorii it is not pathogenic on endive, which is an original source of isolation for the P. cichorii type strain (ATCC 10857T).Through phylogenomic and phylogenetic analyses , 2, it wP. capsici strains from pepper and three out of four Georgia P. capsici strains from tomato exhibited copper tolerance in-vitro. The copper-tolerant strains possess at least two different types of copper-tolerant gene clusters, which were absent in the copper-sensitive P. capsici strains. The copper-tolerant strains were predominantly from the recent outbreaks of pepper and tomato in Georgia , but not from outbreaks in or before 2011. The acquisition of these clusters probably provided a selective advantage to these strains resulting in recent outbreaks that were difficult to manage with a standard copper-based spray program.Copper-based sprays have been extensively utilized in agriculture for many years for the control of bacterial and fungal diseases. As a result copper-tolerance have been reported in bacterial pathogens affecting multiple agricultural crops including pepper, tomato, and apple. The presence of these copper-tolerant bacterial strains reduces the efficacy of copper-based bactericides . Our resP. capsici strains. This may suggest that the sources of the copper tolerance in tomato and pepper strains are distinct. The genes in both clusters encode for efflux RND transporter permease subunit, efflux RND transporter periplasmic adaptor subunit, copper homeostasis membrane protein CopD, copper homeostasis periplasmic binding protein CopC, heavy metal sensor histidine kinase, heavy metal response regulator transcription factor, heavy metal translocating P-type ATPase, cation transporter, copper resistance system multicopper oxidase, and copper resistance protein B. The specific contributions of the individual genes within these clusters remain unclear. Further research is also necessary to understand the regulation of these genes and the distribution of these genes in general P. capsici populations.We also observed two distinct copper-tolerant gene clusters in seven copper-tolerant P. capsici strains has revealed the presence of several virulence factors and secondary metabolites that may enable them to infect plant tissues and evade plant defense mechanisms. However, mere presence of these factors does not associate them with virulence unless they are validated using mutational and functional analysis. Future studies will focus on characterizing these potential virulence factors in pepper and tomato. The genus Pseudomonas is widely distributed and plays a crucial role in environmental processes  can be found at: MZ: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing – original draft, Writing – review & editing. RG: Conceptualization, Writing – review & editing. BD: Conceptualization, Writing – review & editing, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Visualization, Writing – original draft.


Limited research has been conducted on the potential relationship between the dietary inflammation index (DII) and mortality, particularly in individuals with Helicobacter pylori (H. pylori) infection. This study aimed to investigate the association between the DII and H. pylori infection, as well as their respective impacts on all-cause mortality in a cohort of individuals with or without H. pylori infection.Data from the 1999–2018 National Health and Nutrition Examination Survey (NHANES) were utilized for this study, with a final of 4370 participants included. Both univariable and multivariable-adjusted logistic regression analyses were employed to explore the relationship between H. pylori infection and pertinent covariates. Cox regression analysis, as well as restricted regression cubic spline analysis, were utilized to assess the association between DII and all-cause mortality among individuals with or without H. pylori infection.The findings demonstrated a positive correlation between DII scores and H. pylori infection, even after adjusting for potential confounding factors. Moreover, higher DII scores were significantly associated with an elevated risk of mortality exclusively in individuals with H. pylori infection, while no such association was observed in the uninfected population. Additional analysis using restricted cubic spline modeling revealed a positive linear relationship between DII scores as a continuous variable and the adjusted risk of all-cause mortality specifically in H. pylori-infected patients.The results of this study indicated that DII was positively correlated with an increased risk of H. pylori infection and was associated with a heightened risk of all-cause mortality solely in individuals with H. pylori infection. Consequently, DII might serve as a useful tool for risk stratification in the H. pylori-infected population among U.S. adults. Further research is warranted to elucidate the underlying mechanisms and potential clinical implications of these findings. Helicobacter pylori (H. pylori), a gram-negative bacterium, infects the human stomach and is strongly associated with various gastric diseases, including gastritis, peptic ulcer disease and gastric cancer . Its prePrior evidence suggests that diet quality and inflammation may be linked to H. pylori infection. Unhealthy dietary patterns have been associated with a higher risk of H. pylori infection and an increased likelihood of developing H. pylori-related diseases . The dieIn addition, several studies have also investigated the association between diet and mortality. In a prospective cohort study conducted on 3521 adults of normal BMI, diet of high DII scores was associated with an increased risk of cardiovascular disease (CVD) mortality . AnotherHence, this study aims to explore the relationship between the DII score and H. pylori infection, and investigate whether the associations between DII scores and all-cause mortality are mediated by H. pylori infection using the data in the NHANES database.The NHANES project employs a sophisticated and intricate methodology, to select a representative sample of the U.S. population every two years. Its primary objective is to evaluate and assess the health and nutritional status of individuals in the United States . To ensuIn the NHANES 1999–2018 cycle, a total of 101,136 individuals took part in the study. After excluding subjects without information on H. pylori infection status or dietary recall, as well as those lost follow up, the remaining sample was used for analysis  is a comprehensive scoring system that was developed to assess the potential inflammatory effects of dietary intake. In this study, the calculation of the DII involved the inclusion of 26 nutrients. These nutrients encompassed alcohol, vitamin B12/B6, β-carotene, caffeine, carbohydrate, cholesterol, total fat, fiber, folic acid, iron, magnesium, zinc, selenium, monounsaturated fatty acids, niacin, n-3 fatty acids, n-6 fatty acids, protein, polyunsaturated fatty acids, riboflavin, saturated fat, thiamin, and vitamins A/C/E. Importantly, it should be noted that even if fewer than 30 nutrients are utilized, the DII can still be computed with reliable accuracy . In the NHANES utilized an enzyme-linked immunoassay (ELISA) to evaluate H. pylori exposure by detecting IgG antibodies . The NHAhttps://www.cdc.gov/nchs/data-linkage/mortality.htm). The median follow-up time was 235  months in H. pylori positively infected participants while in H. pylori negatively infected patients it was 236  months.The participants' mortality status and cause of death were determined by linking their records to the National Death Index public access files, with a repetition rate of less than 10%, until December 31, 2019  index and DII. Hypertension was defined as a systolic blood pressure (SBP) ≥ 140 mmHg and/or diastolic blood pressure (DBP) ≥ 90 mmHg, or as having received antihypertensive treatment . Smokingt test or Wilcoxon rank-sum test, depending on the result of Kolmogorov–Smirnov normality test. Categorical variables were presented as frequency (percentage) and compared by Chi-square test.Baseline characteristics of participants were summarized and compared by H. pylori infected and uninfected patients. Continuous variables were expressed as mean (± SD) and compared by Both univariable and multivariable-adjusted logistic regression were used to calculate the odds ratio (OR) with 95% confidence interval (CI) for the relationship between DII and H. pylori infection. The possible nonlinear relationships between DII and the all-cause mortality were further evaluated on a continuous scale with restricted cubic spline (RCS) curves based on the multivariable Cox proportional hazards models and four nodes at the fixed percentiles of 5%, 35%, 65% and 95% of the distribution of DII. To examine the association of DII with all-cause mortality, Cox proportional hazards models were used to calculate hazard ratios (HRs) with 95% CIs. To maximize the available data and limit potential overadjustment for variables that could mediate associations between DII and mortality, three models were estimated: in model 1, age, gender and BMI were adjusted; in model 2, age, gender, BMI, ethnics, education, smoke and drink were adjusted; and in model 3, total cholesterol (TC), creatinine, diabetes mellitus and hypertension were additionally added into model 2. DII scores (as continuous variable) were also categorized into tertiles and then applied in Cox proportional hazards models with tertile 1 as the reference group. The event-free survival rates among the groups were estimated by the Kaplan–Meier method and compared by the log-rank test.A two-sided p < 0.05 was considered statistically significant. All analyses were performed using SPSS version 26.0  and R (version 4.2.2) –26.A total of 4, 370 American adults were included finally, of which 1, 891 participants were H. pylori positively infected  experienced mortality. The relationship between DII and all-cause mortality was further assessed by RCS curves  for dietary assessment may have introduced inaccuracies and misclassification of dietary intakes. Additionally, participants' reports of past dietary habits may have been affected by recall bias, especially among patients whose lifestyle changes when developing symptomatic H. pylori infection. Secondly, the significant association between DII and H. pylori infection could be influenced by unmeasured or residual confounding factors . FinallyIn our sample population, high DII scores are associated with higher risks of H. pylori infection. DII is significantly correlated with all-cause mortality risk in H. pylori positively infected population while not in H. pylori negatively infected participants. These outcomes highlight that the DII holds its own predictive value and offers valuable insights for evaluating the dietary management of individuals with H. pylori infection.


Using the model invasive congeneric ascidians, Ciona robusta, and Ciona savignyi, we compared their AS responses to environmental changes and explored the potential determinants. Our findings unveiled swift and dynamic AS changes in response to environmental challenges, and differentially alternative spliced genes (DASGs) were functionally enriched in transmembrane transport processes. Interestingly, both the prevalence and level of AS in C. robusta were lower than those observed in C. savignyi. Furthermore, these two indices were higher under temperature stresses compared to salinity stresses in C. savignyi. All the observed patterns underscore the species-specific and environmental context-dependent AS responses to environmental challenges. The dissimilarities in genomic structure and exon/intron size distributions between these two species likely contributed to the observed AS variation. Moreover, we identified a total of 11 and 9 serine/arginine-rich splicing factors (SRSFs) with conserved domains and gene structures in the genomes of C. robusta and C. savignyi, respectively. Intriguingly, our analysis revealed that all detected SRSFs did not exhibit prevalent AS regulations. Instead, we observed AS control over a set of genes related to splicing factors and spliceosome components. Altogether, our results elucidate species-specific and environmental challenge-dependent AS response patterns in closely related invasive ascidians. The identified splicing factors and spliceosome components under AS control offer promising candidates for further investigations into AS-mediated rapid responses to environmental challenges complementary to SRSFs.Alternative splicing (AS), a pivotal biological process contributing to phenotypic plasticity, creates a bridge linking genotypes with phenotypes. Despite its importance, the AS mechanisms underlying environmental response and adaptation have not been well studied, and more importantly, the  Understanding how organisms functionally respond to environmental changes is fundamental for elucidating adaptation mechanisms and further predicting their future performance in ecological processes, such as global climate change and biological invasions. Alternative splicing (AS), a crucial biological process bridging genotypes and phenotypes, has been widely proposed as a mechanism that facilitates stress response and adaptive evolution by expanding transcriptomic and proteomic diversity ,2. NotabCiona robusta and C. savignyi, two congeneric tunicates with similar morphology, have long served as model organisms in multi-disciplines such as evolutionary and developmental biology. Due to their significant invasion records globally, they have become model systems for invasion science [C. savignyi invested more energy in growth to enhance the efficiency of food filtering, whereas C. robusta allocated more resources to thickening its tunic to reduce predation risks [Ciona ascidians in the face of adverse and harsh environments [ science , particu science . These son risks . The cumronments ,11. Prevronments ,12, epigronments ,14, tranronments ,16, protronments ,18, and ronments ,18. Remaronments ,16. Thescis-regulatory elements and trans-acting factors [Cis-regulatory elements encompass canonical splice sites as well as exonic and intronic splicing enhancers/silencers , which are primarily recognized and bound by RNA-binding proteins (trans-acting factors). These trans-acting factors include serine/arginine-rich splicing factors (SRSFs) and heterogeneous nuclear ribonucleoproteins (hnRNPs) that activate or repress splicing [cis- and trans-acting factors, genomic architecture features such as exon–intron structures and exon/intron length can significantly contribute to AS variation [cis- and trans-acting factors jointly contribute to AS changes in response to environmental challenges.AS is a complex process orchestrated by the intricate spliceosome, which precisely identifies splicing signals on pre-mRNA and catalyzes the removal of introns to form mature mRNA . A cruci factors . Cis-regsplicing ,22. SRSFsplicing ,23. Durisplicing . Thus, Ssplicing . It is nsplicing . Therefoariation . As the ariation ,27. AltoC. robusta and C. savignyi as models to assess the prevalence, types, and response patterns of AS to environmental challenges  during biological invasions. Our objectives were to determine if different AS profiles exist between closely related species exposed to varying environmental cues and to investigate the mechanisms underlying the observed AS variation, primarily from the perspectives of genomic structure architecture and SRSFs.In this study, we utilized invasive p value < 0.05; We identified five fundamental types of alternative splicing events (ASEs), namely exon skipping (ES), mutually exclusive exons (MXE), alternative 3′ splice sites (A3SS), alternative 5′ splice sites (A5SS), and retained introns (IR). These ASEs accounted for 92.2%, 5.8%, 1.1%, 0.8%, and 0.01% of all ASEs, respectively A. Given C. robusta (92.2%) compared to C. savignyi, where it accounted for 70.7% under salinity stresses and 51.8% under temperature stresses  stresses were lower than those observed in C. savignyi under both salinity  and temperature  stresses. Notably, temperature stresses induced higher ASP and ASL in C. savignyi compared to the salinity stresses , the intron length of the major peak in C. savignyi (485 bp) was notably longer than that in C. robusta (372 bp)  D.C. robusta under recurrent high salinity stresses, we identified three DASGs that are functionally associated with the AS process. These genes include hnRNPLL, hnRNPK, and CLK2 . They all contained at least one RNA recognition motif (RRM) domain, in addition to a low complexity region. Notably, the density of SR/RS repeats in the low complexity region exceeded 49% within a sliding window of 30 AAs. Importantly, this characteristic did not show any significant difference between the two species  tree. This analysis revealed five distinct SRSF gene subfamilies  to seven (SRSF12). Interestingly, while they generally shared similar exon–intron organization in orthologous counterparts, the length of most introns in C. savignyi SRSF genes was longer compared to those in C. robusta  stresses, we observed a general increase in the amplitude of gene expression response under the second stress when compared to the first. Furthermore, the overall downregulated expression of SRSF genes at 6 and 24 h shifted towards an upregulated status at the recovery stage of the second stress , Cs_SRSF7a and Cs_SRSF6b were significantly downregulated at multiple timepoints, whereas Cs_SRSF12 displayed an upregulated expression profile. Under low salinity (LS) stress conditions, the expression of Cs_SRSF1a and Cs_SRSF3 significantly decreased at 24 and 48 h, respectively, and Cs_SRSF6a consistently exhibited decreased expression at 24 and 48 h. After high salinity (HS) stress, only Cs_SRSF2 showed a significant induction at 48 h.In y stress B. After Cs_SRSF12, following 24 h of HT stress, using the 3D RNA-seq APP . These stresses . Furtherstresses . A simil stimuli . An intr stimuli , while m stimuli .cis-regulatory elements [C. robusta and C. savignyi, a comparison of their genomes has unveiled extensive variations [C. savignyi genome may contribute to its elevated ASP when compared to C. robusta. Furthermore, both the length of exons and introns are pivotal parameters that influence AS profiles [C. savignyi had shorter exons and longer introns, a pattern that could elucidate its heightened ASP and ASL in comparison to C. robusta. Additionally, the mean intron size was known to exhibit a positive correlation with ASP and ASL in a genome [Studies have demonstrated that in vertebrates, most species-specific AS patterns were primarily directed by elements . Notablyelements . In the riations ,38. Thesriations . A previriations . Therefoprofiles . Shorterprofiles . Consequprofiles . Our resa genome . HoweverC. robusta and C. savignyi resembled that of teleosts [C. robusta and 59 bp for C. savignyi, compared to 76 bp for teleosts). In contrast, the additional minor peak in the exon length distribution was not as commonly observed as that in introns, although a similar distribution mode has been reported in the human genome, with the minor peak occurring at similar positions [The additional peak in intron length distribution is evolutionarily conserved among fungi, vertebrates, and higher plant species . Howeverteleosts , albeit ositions . These dC. robusta, bringing the total count to 11 SRSFs, compared to 8 in a previous study [C. savignyi, whereas an additional serine/arginine-related protein 53 (SRRP53) was identified as a crucial trans-acting factor under AS control in the response to high- and low-temperature stresses . This suggests that these genes might represent ancient duplication and divergence events. A phylogenetic analysis tracing the evolutionary history of the SRSF4/5/6 subfamily across animals and fungi revealed that the single ancestor SRSF5 gene was not duplicated until the chordate lineage (sea squirt), followed by a subsequent duplication in vertebrates [C. robusta represents an important evolutionary node for the diversification of SRSF genes and their functions related to AS.SRSFs and hnRNPs represent critical splicing regulators that play pivotal roles in recruiting the splicing machinery to splice sites. This is especially true for SRSFs, whose involvement in AS regulation during stress responses has been experimentally validated ,44. Compus study . Nine SRstresses . A protetebrates . TherefoC. robusta and C. savignyi under any environmental conditions. Only one transcript change event was identified in Cs_SRSF12 at HT24 using the 3DRNAseq v2.0.1 tool. These findings suggest that AS regulations of SRSF genes in the stress response of congeneric Ciona species may not be as common as observed in other organisms. In addition to the important roles in constitutive and alternative splicing, SRSFs are also involved in various mRNA metabolism processes, such as mRNA export and stability, nonsense-mediated mRNA decay (NMD), translation, and many others. [trans-acting factors beyond SRSFs. These findings are expected to complement our understanding of rapid responses to environmental challenges, offering insights beyond the mechanisms centered on SRSFs.The gene expression of SRSFs has been widely validated to be regulated by environmental stresses ,49. Our  others. . InsteadInvestigations into causes for AS patterns open a window to better understand how AS plays functional roles in linking genotypes with phenotypes. In addition to genome architecture and splicing factors such as SRSFs and hnRNPs investigated in this study, AS is simultaneously controlled by the core spliceosome, splice site, RNA secondary code, RNA polymerase speed, and other epigenetic regulations . The intC. robusta, which was similar to our another previously published study [C. robusta individuals were first acclimated to a laboratory environment with a salinity of 30‰ for a week and then subjected to two rounds of high salinity stress -recovery to unstressed condition (R). Each phase lasted for 24 h, and three ascidian individuals were collected at seven timepoints, including the end of acclimatization , 6 and 24 h at the first HS stress , the end of the first recovery (R1), 6 and 24 h at the second HS stress , and the end of the second recovery (R2).To study the underlying AS regulatory mechanisms under recurrent environmental changes during the ascidian invasion process, we designed an experiment simulating recurrent high salinity stresses using ed study , with loed study ). BrieflAfter sample preparation, the total RNA was isolated using a Trizol reagent  following the manufacturer’s protocol. RNA quality was assessed using Agilent 2100 Bioanalyzer . A total of 21 strand-specific cDNA libraries were constructed according to the SMART library construction method and sequenced on an Illumina X Ten sequencing platform  using the 150 bp pair-end reads. The adaptor and low-quality sequences were first filtered out from raw reads using Trimmomatic version 0.36, and the clean reads were mapped to the reference genome (Ensembl KH.109) using Hisat v2.2.1 . The mapp value < 0.05. If at least one DASE occurred on one gene, it would be defined as differentially alternatively spliced gene (DASG). After the list of DASGs was obtained, gene ontology (GO) functional enrichment analysis of DASGs was conducted using a hypergeometric test using online Omicshare CloudTools .Similarly, we used rMATS v.4.0.2 software to detect alternatively sliced events (ASEs) of five different AS types , and theGenome-wide alternative splicing prevalence (ASP) and level (ASL) were calculated separately under different environments. ASP is the proportion of alternatively spliced genes in the whole genome, whereas ASL is the average number of ASEs per alternatively spliced gene. C. savignyi individuals were separately challenged using four types of environmental challenges, including low temperature (LT), high temperature (HT), low salinity (LS), and high salinity (HS). Individuals were sampled at 1, 24, and 48 h during each treatment, which were labeled as “treatment-timepoint” such as LT1, LT24, and LT48. All bioinformatic analyses were the same as those in C. robusta.Genome-wide gene expression and alternative splicing analysis were retrieved from our previously published results ,17. BrieC. robusta and C. savignyi, the number of exons per gene and length of exons and introns across the whole genome were extracted from the genome annotation files in Ensembl using the R v4.3 package GenomicFeatures v1.52.2, and then illustrated by ggplot2 v2.1.0 package. GO enrichment analysis of the genes with a single exon and more than ten exons was conducted using online Omicshare CloudTool ., and after obtaining the list of significant GO terms of biological process category, redundant GO terms were removed by REVIGO website .To test potential genomic characteristics underlying different alternative splicing profiles in C. robusta and C. savignyi were downloaded from the Ensembl website and then submitted to EggNOG v6.0  for comprehensive functional annotation. First, we searched for SRSF genes from the annotation results and used each SRSF member in C. robusta (or C. savignyi) as a query to do BLAST against the proteomes of C. savignyi (or C. robusta) for all potential orthologs. Secondly, the Hidden Markov Model (HMM) profiles of RRM (PF00076 and PF13893) and Arginine/serine-rich protein (PF17069 and PF15996) were downloaded from Pfam database, and we searched these four domains against the proteome database of C. robusta and C. savignyi using HMMER 3.0 with an E-value < 1 × 10−6, and only the proteins with both domains were retained for subsequent analyses. The identified SRSF genes were renamed using three parts of information, species  + SRSF member + one additional lowercase letter if this SRSF member has more than one copy. All obtained candidate SRSF members were further verified for the presence of both RRM and RS domains by the SMART database. The density of RS repeats in the RS domain was tested by the LCD-Composer web server [The reference proteomes of b server .C. robusta and C. savignyi were aligned by MUSCLE, and then the maximum likelihood (ML) phylogenetic tree was constructed using MEGA X to illustrate the homologous relationship among orthologs or paralogs. The exon–intron structures of all SRSF genes were illustrated using Gene Structure Display Server 2.0 by aligning the cDNA sequences with the corresponding genomic DNA sequences [The longest peptide isoform of all identified SRSF members of equences .Gene expression and alternative splicing changes of all SRSF genes in response to environmental stresses were extracted from the above RNA-seq analysis. In addition to AS analyses by rMATS, we also used a 3D RNA-seq v2.0.1 tool to study AS at the transcript isoform level .C. robusta and C. savignyi as a model, we assessed and further compared the alternative splicing prevalence, type, and response patterns to environmental challenges  during biological invasions. Our results showed species-specific and environmental context-dependent patterns in closely related ascidians. Divergent genome architecture might underlie the observed AS variation between these two species. Surprisingly, the traditionally believed important splicing factors in SRSFs did not show prevalent AS regulations during environmental stress response, but we identified a list of other splicing factors and spliceosome components under AS control as potential functional trans-acting factors. Such a gene list provides potential candidates to further study AS-mediated rapid responses to environmental challenges complementary to SRSFs.Using congeneric invasive


A 56-year-old female with a history of diabetes, hypertension, and hyperlipidemia presents with a 6 month history of rash under bilateral breasts for which treatment with nystatin powder and ketoconazole cream demonstrated no improvement. On examination, loose atrophic skin and white-to-yellow nonfollicular papules coalescing into plaques in the bilateral inframammary region with incomplete involvement of the folds were observed . A punchQuestion 1: What is the diagnosis?A.Pseudoxanthoma elasticum (PXE)B.Pseudoxanthoma-like papillary dermal elastolysis (PXE-PDE)C.Papillary dermal elastosis (PDE)D.White fibrous papulosis (WFP)E.Mid-dermal elastolysis (MDE)Answers:A.Pseudoxanthoma elasticum – Incorrect. Papillary dermal elastosis (PXE) resembles PXE-PDE, but onsets at a younger age with ocular and cardiovascular manifestations.B.Pseudoxanthoma-like papillary dermal elastolysis – Correct. This is a rare, benign elastic tissue disorder, mainly in women with a clinical similarity to PXE without systemic manifestations.C.Papillary dermal elastosis – Incorrect. This appears like PXE-PDE with asymptomatic papules on the neck and upper back, but histology of PDE demonstrates foci of clumped elastic fibers in the papillary dermis with normal reticular dermis.D.White fibrous papulosis – Incorrect. WFP is similar to PXE-PDE but distinguished from it due to equal presence in both sexes with nonconfluent paler papules, usually on the nape of the neck, lacking the cobblestone appearance.E.Mid-dermal elastolysis – Incorrect. This is another rare elastolytic disorder presenting with 3 distinct variations: well-demarcated patches of wrinkles on the trunk and proximal extremities (type I); follicular papules on the lateral neck (type II); and reticular erythema with wrinkling.Question 2: Which of the following is NOT a proposed etiologic theory for the development of PXE-PDE?A.Ultraviolet (UV) radiationB.GeneticsC.Aging processD.Abnormal elastin synthesisE.Environmental exposureAnswers:A.UV damage – Incorrect. UV exposure is one of the proposed etiologic theories of PXE-PDE. However, UV exposure as the main cause of PXE-PDE is debated as involvement of the axillae, and inframammary folds as in our patient, can occur. In patients with increased melanophages in the dermis, a marker of UV damage in chronic heliodermatitis, the UV radiation etiologic theory may play a role in PXE-PDE development.B.Genetics – Incorrect. Although less likely, a genetic cause of PXE-PDE is supported by one report of a familial case of PXE-PDE.C.Aging process – Incorrect. The thinning of oxytalan and elastin within the papillary dermis associated with intrinsic aging has also been postulated as a cause of PXE-PDE and why it occurs in middle-aged individuals.D.Abnormal elastin synthesis – Incorrect. The dysfunctional elastin synthesis theory has the most supportive evidence with studies using immunohistochemical staining revealing immature elastic fibers in the mid-dermis with a defect in elastin rather than fibrillin-1.E.Environmental exposure – Correct. Although UV damage is a proposed theory for the development of PXE-PDE, other environmental exposures have not been elucidated as contributing to this disease process.Question 3: Which of the following is NOT a treatment option?A.Topical retinoidsB.Fraxel laserC.Clear and brilliant laserD.Oral retinoidsE.No treatmentAnswers:A.Topical retinoids – Incorrect. Most treatments for PXE-PDE are ineffective, however there have been case reports in which topical retinoids have had minimal to marked change with desirable results.B.Fraxel laser – Incorrect. Fraxel is a nonablative fractional resurfacing laser that has shown up to a 50% improved appearance of the skin after 3 sessions in 1 case report.C.Clear and brilliant laser – Incorrect. This is also a nonablative fractional resurfacing laser that may be used to treat the cosmetic concerns of PXE-PDE.D.Oral retinoids – Correct. To date, oral retinoids have not been utilized in the literature to treat PXE-PDE. Additionally, because PXE-PDE is a benign condition, the risks of adverse effects from oral retinoids outweigh the benefits of any potential improvement.E.No treatment – Incorrect. Once PXE is ruled out, the disease remains benign, and treatment is not necessary unless cosmetic concern exists.None disclosed.


BRCA genes and other homologous recombination-mediated DNA damage response (HR-DDR) genes.,BRCA-mutated patients, leading to FDA approval of this combination in the mCRPC first-line setting for HR-DDR molecular subsets.Cancer of the prostate is the most common cancer occurring in men in the US. Despite advances in diagnosis and management, more than 34,000 men continue to die of prostate cancer each year in the United States.eBioMedicine provide some additional insights into the landscape of DDR defects in mCRPC analyzing liquid biopsy and germline samples from a total of 375 men from the US and Australia using the CLIA-certified PredicineCARE™ cell-free DNA (cfDNA) panel assay which targets sequencing of 152 cancer driver genes including 27 individual DDR genes.BRCA2 (17%). Also consistent with some previous reports in BRCA2 mutated mCRPC, clinical outcomes including PSA response, PFS and OS were found to be inferior with ARPI treatments.,BRCA2 zygosity status  in cfDNA was indicative of poor clinical outcomes. This is interesting because previous data have suggested that bi-allelic BRCA altered mCRPC patients, particularly those with homozygous or bi-allelic BRCA2 deletion, exhibited the most clinical benefit from PARPi. In contradistinction, previous studies have demonstrated that PARPi responses are rarely observed in patients without bi-allelic inactivating alterations in HR-DRR genes.,,Fettke and colleagues in this issue of BRCA2 altered mCRPC have a specific genomic background of co-occurring oncogenic drivers gene mutations, many of which are potentially actionable. They demonstrated using cfDNA sequencing that BRCA2 altered mCRPC harbored mutations at approximately twice the frequency in potentially targetable pathways such as the AR, PI3K/PTEN, Fibroblast Growth Factor Receptor 1 (FGFR1) and CDK4/6 cell cycle pathways.As previously described, exploring different PARPi combination strategies will likely escalate as an important therapeutic option given recent positive results with PARPi and ARPI clinical studies.BRCA2 alterations of at least one allele within plasma cfDNA is sufficient to identify patients with inferior clinical outcomes, and that a distinct genomic landscape is present in patients with BRCA2 altered mCRPC, with a high prevalence of potentially actionable oncogenic driver co-targets. Thus, this nice work by Fettke and colleagues with cfDNA has shed additional light on these potentially actionable co-targets with PARPi in BRCA altered mCRPC patients. However, much work remains to prospectively evaluate and validate co-targeting strategies with PARPi and the optimal sequencing of these strategies to increase treatment opportunities in mCRPC.Overall, these findings from Fettke et al. support continued efforts suggesting that cfDNA approaches can reliably detect HR-DDR gene alterations with clinical implications in mCRPC patients.Literature search: S.B. and P.T.T.; Data collection: S.B. and P.T.T.; Data interpretation: S.B. and P.T.T.; Writing: S.B., P.S., M.P.D, C.H.M. and P.T.T. All authors read and approve the manuscript.CHM declares consulting fees for Bayer, Dendreon, Obseva and Astellas. PTT declares institutional grants from RefleXion Medical, Bayer and Astellas, Patent #9114158–Compounds and Methods of Use in Ablative Radiotherapy licensed to Natsar Pharm, consulting fees from RefleXion Medical, Janssen, AstraZeneca, Noxopharm, Myovant and Lantheus. The other authors declare no competing interests.