At a mean of 6.2 years post-first islet transplant, 90% reduction in severe hypoglycaemia was maintained in all groups, with HbA1c (mean ± SD mmol/mol) 49 ± 4 in recipients with “Good” function; 56 ± 5 ; and 69 ± 25 (“Failed”). Self-reported impaired awareness of hypoglycaemia persisted in all groups but those with “Good” function were more likely to experience symptoms during hypoglycaemia. “Marginal” function was associated with greater fear of hypoglycaemia ; “Good”: 33 ) and severe anxiety : 21 ; “Failed”: 6  “Good”: 6 ; (p = 0.079)), diabetes distress and low mood. Despite clear evidence of ongoing clinical benefit, Igls criteria ‘Marginal’ function is associated with sub-optimal well-being, including greater fear of hypoglycaemia and severe anxiety. This study provides person-reported validation that “Good” and “Marginal” graft function are differentiated by general and diabetes-specific subjective well-being, suggesting those with “Marginal” function may benefit from further intervention, including re-transplantation.Associations between islet graft function and well-being in islet transplant recipients requiring exogenous insulin remain unclear. This cross-sectional analysis compared person-reported outcome measures in 15 adults with type 1 diabetes whose islet transplants were classified according to Igls criteria as “Good” (  Following seminal success in Edmonton , intrapoThe goal of the NHS programme was to prevent further severe hypoglycaemia without the expectation of insulin independence. At the outset, the National Institute for Health and Care Excellence identified core audit criteria as confirmation of graft function through C-peptide positivity; reduction in numbers of severe hypoglycaemic events; attainment of HbA1c less than 53 mmol/mol (7%); and reduction in exogenous insulin dose , 5.In the absence of insulin independence, associations between level of islet graft function and overall health status/well-being remain unclear. Validation of the Igls classification using patient-reported outcome measures (PROMs) was advocated in the original consensus statement but has not previously been undertaken . We aimea priori to exclude recipients with insulin independent “optimal” function.The study was conducted between April and June 2022 following ethical approval (REC number 07/Q0904/11) to recruit participants who had received one or more percutaneous, transhepatic, intra-portal deceased donor pancreatic islet infusions at the Newcastle upon Tyne Hospitals NHS Foundation Trust within the NHS islet transplant programme. Inclusion criteria included ≥2 episodes of severe hypoglycaemia requiring assistance in treatment  over theA questionnaire pack was compiled for participant completion to assess hypoglycaemic episodes and impaired awareness; attitudes and behaviours towards hyper- and hypoglycaemia; diabetes-associated distress and problems; and anxiety/depression . InstrumIn parallel with questionnaire completion, demographics, transplant history and biomedical data enabling Igls graft status classification according to published criteria  were obtp < 0.05 was considered statistically significant.Statistical analysis was completed using SPSS statistics software version 28.0. Data normality was determined by Shapiro-Wilks test with age, diabetes duration and other parametric data presented as mean ± standard deviation and non-parametric data as median . Categorical data are shown as number (%). Means were compared using one-way ANOVA with post-hoc testing using Tukey’s test. Medians were compared using Kruskal-Wallis test and categorical variables using Fisher’s exact test. Twenty-one islet transplant recipients fulfilling inclusion criteria were approached for potential participation and 15 returned questionnaires following written informed consent . In partAll participants had type 1 diabetes  complicated by recurrent severe hypoglycaemia requiring assistance with treatment, with 52 ± 98 events over the 12 months prior to transplantation. Twelve (80%) were female with age 60 ± 10 years and diabetes duration 45 ± 11 years. Twelve (80%) had received islet transplants alone and three (20%) islet after kidney transplants. Questionnaires were completed at a mean of 6.2 years following first islet transplant.Ten (67%) participants were using multiple dose insulin therapy with the remaining five (33%) on continuous subcutaneous insulin infusion pumps. Eight (53%) participants were using continuous glucose monitoring and the remaining seven (47%) flash glucose monitoring. Pre-transplant total daily insulin dose in the cohort as a whole was 0.47 ± 0.19 units/kg.At the time of cross-sectional assessment, five (33%) participants’ transplants were classified as having “Good” function, four (27%) had “Marginal” function and six 40%) had “Failed.” “Good” function required absence of severe hypoglycaemia and HbA1c <7.0% (53 mmol/mol) . C-pepti0% had “FAge, duration of diabetes and number of transplants were comparable in all groups . Time siInsulin dose was significantly lower in both groups with graft function, compared to the graft failure group, with comparable dose (∼50% of baseline requirements) in individuals with Igls “Marginal” and those with “Good” function. A period of insulin independence was achieved in 60% of those with current “Good” function but in ≤25% within the other two groups.Random C-peptide was significantly lower in the “Marginal” compared with the ‘Good’ function group with C-peptide/glucose ratio falling incrementally with worsening graft function.All participants with ongoing graft function were on a comparable immunosuppression regimen (tacrolimus with/without mycophenolate mofetil). Three (50%) of those classified as ‘failed’ were no longer taking immunosuppression. Two remained on immunosuppression for a functional renal transplant and one remained on low dose tacrolimus alone immunosuppression on the active waiting list for islet retransplantation.Although only 15 individuals (71% of those who fulfilled inclusion criteria) consented to participate and completed questionnaires, they appeared representative of the overall cohort. The remaining 6 exogenous insulin-requiring islet transplant recipients in this single site cross-sectional study included a comparable distribution of those with “good,” “marginal” and “failed” function. All had received islet transplants alone. Age and duration of diabetes were comparable to the study participants and the majority were female.Across all three insulin-requiring Igls groups, most recipients self-reported unresolved impairment in hypoglycaemia awareness without significant differences in Gold or HypoA-Q “Impaired Awareness” scores in those with “Good,” “Marginal” or “Failed” graft function .In addition to HypoA-Q hypoglycaemia awareness scoring, novel analysis of HypoA-Q “Symptom Frequency”  and “Symp = 0.01) or “Failed” (p = 0.039) function  asks the participant “how low does your blood glucose usually need to be before you feel” one or more symptoms, clustered into: autonomic, neuroglycopaenic and non-specific. Higher scores are allocated to lower glucose thresholds with a maximum score of 18 . All groWorry about hypoglycaemia appeared low only in those with “Good” graft function , althougDrive to take immediate action to reduce high blood glucose (measured by the HAS) and prioritisation of hyperglycaemia avoidance  were scored highly in all groups suggesting underlying behavioural preferences which are not influenced by islet graft function. Low blood glucose preference (HAS) and asymptomatic hypoglycaemia normalised/hypoglycaemia concerns minimised (A2A) were scored relatively low by participants in all three Igls groups, consistent with the approach within the UK islet transplant programme of only listing individuals who recognise concerns regarding dangerous hypoglycaemia risk as their primary motivator for proceeding with transplantation despite the need for life-long immunosuppression.Median PAID scores of those in both the “Marginal” and “Failed” graft function groups indicated they were experiencing elevated diabetes distress (PAID score >40), contrasting with those with “Good” graft function who reported lower median PAID scores . AssesseSimilarly, self-reported generalised anxiety and depressive symptom scores were highest, and in the “severe range” in those with “Marginal” graft function , and lowFeedback on individual questionnaires and overall usefulness/burden of questionnaire completion was provided by 11 particiopants and was largely positive . All werIn this study, we set out to determine whether “Good” and “Marginal” islet graft function (defined by the EPITA/IPITA consensus) were able to differentiate person-reported experience and outcomes. Fifteen adults with (pre-transplant) C-peptide negative type 1 diabetes were studied cross-sectionally at a mean of 6 years following their first islet transplant at a single centre. All required at least low dose insulin replacement but were continuing to benefit from significantly reduced severe hypoglycaemia. Despite maintained biomedical benefit, person-reported measures of health status revealed significant concerns in those with “Marginal” graft function including persistent fear regarding hypoglycaemia, diabetes distress and overall anxiety symptoms in contrast to those with “Good” function. This provides validation of the Igls criteria in meaningfully defining overall clinical outcomes through a simple biomedical scoring system. In addition, the current study provides evidence of unmet needs in those with “Marginal” function justifying further intervention, including re-transplantation.Recurrent life-threatening severe hypoglycaemia remains the primary indication for deceased donor islet transplant, both within the NHS-adopted integrated UK programme and more widely. We and others have previously reported that significant biochemical hypoglycaemia (glucose <3 mmol/L) can be successfully avoided even in those with relatively low levels of restored C-peptide secretion following islet transplant , in keepClassification of graft function as “Marginal” in the presence of any severe hypoglycaemic events has been widely accepted. Exclusion of all with HbA1c >7% from the “Good” function category has been more contentious, even though HbA1c <7% was agreed by NICE as a key performance indicator for reimbursed islet transplantation and formed part of the primary outcome measure for the US Phase 3 trials towards islet transplant licensing . The curThe evidence that optimal primary graft function predicts long term graft function is now incontrovertible . In the Previous studies have confirmed the potential to restore counter-regulatory response and improve symptomatic response to hypoglycaemia following optimal islet engraftment . In the The factors associated with incomplete recovery of awareness even following successful biochemical hypoglycaemia avoidance remain unclear . We hypoThere have been relatively few previous studies in islet transplant recipients evaluating wider person-reported impact using validated questionnaires, and PROMs have not previously been utilised to validate Igls criteria –29. ReduAttitudes and behaviours related to high glucose levels have not previously been reported in islet transplantation and were undertaken using the validated Hyperglycaemia Avoidance Scale (HAS)  and AttiIslet transplant recipients in all groups continued to report a strong preference for low versus high blood glucose levels but neither “minimisation of concerns regarding hypoglycaemia” nor “normalisation of asymptomatic low blood glucose levels” were endorsed strongly in this cohort. This likely reflects the screening process within our programme, which requires each individual to be clear that dangerous hypoglycaemia is their over-riding concern, justifying progression to beta-cell replacement therapy despite the associated risks.Worry about and avoidance of circumstances that might lead to high glucose  was scored particularly highly in islet transplant recipients, compared with those in the HypoCOMPaSS trial , potentiReduced diabetes distress following successful islet transplantation has been reported previously . In thisSignificant anxiety and low mood were, however, reported by all groups. This may be attributable to their high-risk status and requirement for prolonged “shielding” during the COVID-19 pandemic, but nevertheless confirms that successful islet transplantation  does not completely allay ongoing concerns, given the need for ongoing close monitoring and the potential risks of concomitant immunosuppression.It is possible that high anxiety and depression scores in those with “marginal function” were contributed to by continued immunosuppression despite marginal perceived benefits. We do not, however, advocate immunosuppression withdrawal in this C-peptide positive group where biomedically meaningful graft function is maintained and given the associated risk of alloantibody sensitisation . In contAs currently configured, Igls “Good” status is dependent on meeting criteria for all four included parameters . We strongly endorse the need for graft function in all islet transplant recipients to be primarily evidenced by robust demonstration of higher C-peptide compared to pre-transplantation. We believe that the current analysis provides strong supportive evidence for classifying all with HbA1c >7% as “Marginal.” While understanding that lower HbA1c may be attainable by higher dose exogenous insulin, we conclude that there is now sufficient evidence for an intrinsic impact of C-peptide on composite hypoglycaemia/HbA1c outcomes , 18. MorLimitations of this study include relatively small numbers of participants within a single centre and the cross-sectional design without inclusion of pre-transplant PROMs. Further studies will be important to confirm the current findings in larger prospective cohorts including insulin independent recipients with “optimal” function.A key strength is the comprehensive assessment of PROMS, and the robust process that we undertook to select appropriate validated measures and to confirm face-validity and utility for implementation in people undergoing islet transplantation. Despite the effort required in completion, participants fed-back positively regarding the value of collecting and reviewing PROM data in parallel with biomedical outcomes. An unmet need to assess anxiety and depression was clearly identified.For holistic assessment following islet transplantation, we recommend a “minimum dataset” PROM pack quantifying hypoglycaemia awareness, hypoglycaemia fear, diabetes distress, anxiety and depression. Suggested specific measures are Gold score, HFS-II, PAID and HADS.In addition to providing person-reported outcome validation of cut-offs selected by the healthcare professional community for Igls islet transplant function criteria, this study has confirmed the importance of collecting, reporting and responding to more holistic assessment of wider factors necessary for overall well-being and truly optimal outcomes, using a standardised PROM questionnaire pack in parallel with interval biomedical data collection.


Enterovirus A71 (EVA71) infection can result in paralysis and may be fatal. In common with other picornaviruses, empty capsids are produced alongside infectious virions during the viral lifecycle. These empty capsids are antigenically indistinguishable from infectious virus, but at moderate temperatures they are converted to an expanded conformation. In the closely related poliovirus, native and expanded antigenic forms of particle have different long-term protective efficacies when used as vaccines. The native form provides long-lived protective immunity, while expanded capsids fail to generate immunological protection. Whether this is true for EVA71 remains to be determined. Here, we selected an antigenically stable EVA71 virus population using successive rounds of heating and passage and characterized the antigenic conversion of both virions and empty capsids. The mutations identified within the heated passaged virus were dispersed across the capsid, including at key sites associated with particle expansion. The data presented here indicate that the mutant sequence may be a useful resource to address the importance of antigenic conformation in EVA71 vaccines. Enteroviruses (EVs) are globally distributed and are associated with a range of pathologies ranging from the common cold  to lethal neurological disease . Enterovirus A 71 (EVA71) is the primary causative agent of hand, foot and mouth disease (HFMD), a generally mild, self-limiting condition affecting young children. In severe cases the disease can present with neurological complications reminiscent of poliomyelitis . The finWhile the ability of inactivated virus vaccines to control the spread of EVA71 is extremely promising, there remains a significant biosafety concern associated with the large-scale production of infectious virus required for the generation of inactivated virus vaccines. There have been several instances where the production of PV vaccine material has resulted in the accidental release of infectious virus. In 1995 the incomplete inactivation of PV in ≈120 000 vaccine doses resulted in ≈40 000 cases of muscle weakness in recipients, 51 individuals were paralysed and 5 died. Additionally, spread of the virus to close contacts of the vaccine recipients resulted in an additional 113 cases and 5 deaths . BetweenEV VLPs are composed of three viral structural proteins, VP0, VP3 and VP1, assembled as icosahedral capsids that lack infectious genetic material, rendering these particles inherently safe . EV viriHere, we selected an antigenically stable EVA71 virus population using successive rounds of heating and passage and characterized the antigenic conversion of the virion and ECs. The mutant described will be a useful resource to address the importance of antigenic conformation in EVA71 vaccines and could inform the design of stabilized EVA71 VLPs for future vaccine production.EVA71 strain MS/7423/87 was a gift from SingVax (Singapore); an EVA71 reverse genetics system was generated from this strain as previously described . HeLa ce6 p.f.u. ml−1 virus was incubated at 52.5 °C for 30 min and an aliquot stored on ice as a non-heated control. After incubation at a range of elevated temperatures the samples were directly titrated by plaque assay and inoculated into T25 flasks of confluent Vero cells. These were incubated at 37 °C until full cytopathic effect (CPE) was observed. Supernatants were clarified and used for subsequent cycles of heating and passage until the titres obtained from the unheated and heated virus populations were similar for at least three consecutive passages. This resulted in a total of nine cycles of heating and passaging. Samples from passages 4, 5, 7, 8 and 9 were analysed by next-generation sequencing (NGS). The virus isolated after nine passages with heating was subjected to five further passages in the absence of heating to assess the genetic stability of the thermally resistant virus population.To select thermally resistant virus, and similar to previously described protocols , 100 µl The genetic sequence of stabilized virus populations was determined by next-generation sequencing using previously described protocols . BrieflyAatII and BamHI within the infectious clone plasmid. The ligated plasmid was transformed in Escherichia coli DH5α and plasmids isolated for Sanger sequencing.RNA was extracted from clarified supernatant of passage 9 virus using Trizol (Qiagen) and a Direct-zol purification kit (Zymogen), per the manufacturer’s instructions. The RNA was reverse transcribed and the sequence encompassing residues 784–3888 was amplified using Q5 polymerase (NEB) per the manufacturer’s instructions. Overlap PCR was used to combine the recombinant stabilized virus amplicon with the 5′ UTR derived from our EVA71 infectious clone . The resin vitro-transcribed RNA into HeLa cells. Briefly, the EVA71 infectious clone plasmid was linearized with XhoI followed by phenol–chloroform extraction. RNA was synthesized using the RiboMAX T7 express large-scale RNA production system (Promega) and purified using RNA clean and concentrator columns (Zymo Research). RNA was electroporated into HeLa cells within 0.4 mm cuvettes at 260 V for a single pulse of 25 ms using square wave. Cells were transferred to plates and incubated at 37 °C, 5 % CO2 in a humidified chamber overnight. Plates were freeze-thawed to enhance viral release from cells, and cellular debris was pelleted at 17 000 r.c.f. for 10 min. The supernatants were collected and samples titred using median tissue culture infectious dose (TCID50).Virus was recovered as previously described , followi50, 1×104 Vero cells were seeded into each well of a 96-well plate in 100 µl of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 2 % FBS. A series of 10-fold serial dilutions were made from viral supernatants in DMEM supplemented with 2 % FBS and 100 µl of each dilution (10−2 to 10−7) was added to five replicate wells. Plates were returned to a 37 °C, 5 % CO2 humidified incubator for 5 days before being fixed and inactivated by the addition of 100 µl per well of 4 % formaldehyde for 30 min. The contents of wells were discarded and residual cells stained with crystal violet solution. Titres were determined using the Reed–Muench method and are expressed as TCID50 ml−1 . Co. Co35]. The antigenic stabilities of the ECs and virions were investigated by incubating the particles at a range of temperatures and assessing their NAg and HAg reactivity by ELISA. The WT ECs maintained HAg reactivity up to 60 °C, but this decreased at higher temperatures; consistent with this, WT ECs were not reactive in the NAg-specific ELISA . As expeSurprisingly, the recombinant stabilized ECs lost reactivity with mAb 979 at a lower temperature (55 °C) than the equivalent WT sample , suggestThus, although the WT and recombinant stabilized virions have similar NAg reactivity , the WT We have selected for a combination of mutations that provides NAg stability to ECs. We propose that capsid proteins incorporating this combination of mutations will be ideal candidates for expressing stabilized VLPs in heterologous systems. We further suggest that these particles will facilitate future research to address the NAg/HAg paradigm surrounding EVA71 vaccine efficacy.Click here for additional data file.


Accurate definition of the structural basis of TCRs and their engagement of peptide–MHCs can provide major insights into normal and aberrant immunity, and can help guide the design of vaccines and immunotherapeutics. Given the limited amount of experimentally determined TCR–peptide–MHC structures and the vast amount of TCRs within each individual as well as antigenic targets, accurate computational modeling approaches are needed. Here, we report a major update to our web server, TCRmodel, which was originally developed to model unbound TCRs from sequence, to now model TCR–peptide–MHC complexes from sequence, utilizing several adaptations of AlphaFold. This method, named TCRmodel2, allows users to submit sequences through an easy-to-use interface and shows similar or greater accuracy than AlphaFold and other methods to model TCR–peptide–MHC complexes based on benchmarking. It can generate models of complexes in 15 minutes, and output models are provided with confidence scores and an integrated molecular viewer. TCRmodel2 is available at   T cell immunity is a key component of immune protection from viruses and pathogens , such asSeveral algorithms have been developed to perform modeling of unbound TCRs , and TCRHere, we describe the development of TCRmodel2, which is a major update of our previously released TCR modeling web server, TCRmodel . While tThe TCRmodel2 modeling pipeline was generated through several modifications of the AlphaFold pipeline and database, as noted below. These changes were separately implemented in the AlphaFold v2.2 and v2.3 codes (both downloaded from the AlphaFold GitHub repository), to enable comparative performance of the two AlphaFold models in the context of TCRmodel2.Given that the AlphaFold feature selection stage includes sequence searches against large databases containing a large variety of proteins for each input chain, we reduced the databases to contain prospective TCR and MHC hits to speed up the multiple sequence alignment (MSA) building step. The AlphaFold pipeline was run with four representative human and murine TCR and MHC sequences, using the reduced database option. All hits from the TCR and MHC sequence searches against the Small BFD [Big Fantastic Database ], UniRefThe AlphaFold template search, which utilizes an MSA built from the input sequence to search against PDB sequences to identify templates for each input chain , was fouet al. , predicted TM  score, ipTM  score and model confidence, which is a linear combination of pTM and ipTM (0.2 × pTM + 0.8 × ipTM) . Additiohttps://flask.palletsprojects.com/). Users can choose to model a TCR–pMHC complex (Class I or Class II) or an unbound TCR. In both run modes, the users can either provide the target sequences or build the sequences on the fly by selecting the target TCR or MHC genes. The latter option makes possible the modeling of sequences by the information collected from databases such as VDJdb  criteria , which aP-values were calculated with ggpubr package in R (r-project.org), and receiver operating characteristic (ROC) area under the curve (AUC) calculations were performed using the pROC package . In contrast, generation of five TCR–pMHC models using the standard AlphaFold pipeline on the same computer cluster takes ∼5–7 h, of which over 90% of the time is spent in the feature generation and MSA building stage.To benchmark the TCR–pMHC modeling accuracy of TCRmodel2, we assembled a set of nonredundant TCR–pMHC structures from TCR3d that were released after 30 April 2018, with the date cutoff selected to avoid overlap with the AlphaFold (v2.2) model training set. Nonredundancy and other criteria for benchmark case selection are detailed in the ‘Materials and Methods’ section. In total, we identified 48 test cases, including 32 Class I complexes and 16 Class II complexes . Comparihttps://github.com/deepmind/alphafold/blob/main/docs/technical_note_v2.3.0.md), we implemented TCRmodel2 with the AlphaFold 2.3 model and pipeline to test whether it would lead to accuracy improvement over TCRmodel2 with the AlphaFold 2.2 model. This was benchmarked using 20 TCR–pMHC test cases with release dates after September 2021 (to ensure no overlap with the AlphaFold 2.3 training set), which is a subset of the original benchmark set , which includes an updated training set of structures (up to 30 September 2021) and additional recycling iterations during modeling , in comparison with TCRmodel  and AlphaFold . We founr = 0.75; P < 0.001). Model confidence scores also showed significant correlations with individual accuracy metrics Fnat, L-RMSD and I-RMSD  and Class II (N = 47) complexes are shown in To further assess expected model confidence for structurally uncharacterized TCR–pMHC complexes, we used TCRmodel2 to model additional Class I and Class II complexes obtained from the VDJdb database . The disAs an example of TCR–pMHC complex modeling in TCRmodel2, the server was used to predict the structure of a human TCR in complex with an immunodominant SARS-CoV-2 nucleocapsid epitope presented by the Class I HLA-B*07:02 MHC. The complex has not been structurally characterized, nor have any complexes with TCRs targeting that epitope, and its sequence is from a set of TCRs from COVID-19 recovered and unexposed donors reported in a recent study to bind that peptide (sequence: SPRWYFYYL) and MHC . Of note+ T cell recognition of the p53 R175H hotspot mutation, we input the TCR Vα and Vβ sequences into the TCRmodel2 submission page, along with a p53 peptide sequence containing the mutant residue , and selected the HLA-DRA*01:01 and HLA-DRB1*13:01 MHC genes. The Results page from TCRmodel2 included the top-ranked model of that TCR–pMHC complex  that was found to target the common Class II MHC allele HLA-DRB1*13:01 and a p53 neoantigen with the R175H mutation . While s85-TCR1 t complex , which h complex .The TCRmodel2 web server provides the community with a deep learning method to accurately model structures of TCRs and TCR–pMHC complexes. Its TCR–pMHC accuracy is higher than AlphaFold, it runs faster and without the need for dedicated computing resources, and it provides a submission and output interface designed for TCR and TCR–pMHC modeling. TCRmodel2 is distinguished from another recently reported AlphaFold-based TCR–pMHC modeling method , as it dFuture possible developments of TCRmodel2 can address improving the accuracy of confidence estimates, and increasing the overall success rate, including the generation of near-native (CAPRI High criteria) accuracy models, through additional optimizations of the modeling pipeline. Additional testing and developments may focus on application of TCRmodel2 for related complexes of interest, such as TCR-mimic antibodies , which ehttps://github.com/piercelab/tcrmodel2) and Zenodo (https://doi.org/10.5281/zenodo.7853278). Structural models from the benchmarking reported in this manuscript are available from the authors upon request.The TCRmodel2 code is available on GitHub (gkad356_Supplemental_FileClick here for additional data file.


Marine phytoplankton produce and scavenge Reactive Oxygen Species, to support cellular processes, while limiting damaging reactions. Some prokaryotic picophytoplankton have, however, lost all genes encoding scavenging of hydrogen peroxide. Such losses of metabolic function can only apply to Reactive Oxygen Species which potentially traverse the cell membrane outwards, before provoking damaging intracellular reactions. We hypothesized that cell radius influences which elements of Reactive Oxygen Species metabolism are partially or fully dispensable from a cell. We therefore investigated genomes and transcriptomes from diverse marine eukaryotic phytoplankton, ranging from 0.4 to 44 μm radius, to analyze the genomic allocations encoding enzymes metabolizing Reactive Oxygen Species. Superoxide has high reactivity, short lifetimes and limited membrane permeability. Genes encoding superoxide scavenging are ubiquitous across phytoplankton, but the fractional gene allocation decreased with increasing cell radius, consistent with a nearly fixed set of core genes for scavenging superoxide pools. Hydrogen peroxide has lower reactivity, longer intracellular and extracellular lifetimes and readily crosses cell membranes. Genomic allocations to both hydrogen peroxide production and scavenging decrease with increasing cell radius. Nitric Oxide has low reactivity, long intracellular and extracellular lifetimes and readily crosses cell membranes. Neither Nitric Oxide production nor scavenging genomic allocations changed with increasing cell radius. Many taxa, however, lack the genomic capacity for nitric oxide production or scavenging. The probability of presence of capacity to produce nitric oxide decreases with increasing cell size, and is influenced by flagella and colony formation. In contrast, the probability of presence of capacity to scavenge nitric oxide increases with increasing cell size, and is again influenced by flagella and colony formation. Phytoplankton cells span a large size range, from picoplankton (<2μm), nanoplankton (2 to 20μm), microplankton (20 to 200μm) to macroplankton (200 to <2000μm) . Cell siPhytoplankton both produce and scavenge Reactive Oxygen Species (ROS), both within and outside the cell membrane, enzymatically and non-enzymatically. Some ROS readily cross the cell membrane, connecting intra- and extra-cellular pools. Other ROS rarely cross cell membranes and therefore intra- and extra-cellular pools are at least partially segregated .2•−), a radical anion generated through the monovalent reduction of O2 to O2•−  fu fu2•− [2rococcus  may alloransport , 142 par2•− is poorly diffusible across membranes, intracellularly produced O2•− has to be scavenged to limit detrimental reactions of O2•− [2•− scavenging enzyme SOD  were present in a few diatom species  and prokaryotes (Crocosphaera spp.). Genes encoding the enzyme Superoxide Reductase  were detected in some diatoms (Pseudo-nitzschia fradulenta WWA7 and Seminavis robusta D6), and in the haptophyte Pleurochrysis carterae CCMP456. BLAST searches support these annotations of genes for SOO and SOR in the genomes of some phytoplankters. These results should be confirmed by enzyme assays, to identify if the genes indeed encode active enzymes. Finer trends in genomic allocations to O2•− scavenging may emerge among the metallo-forms of SOD [Given that the O of O2•− . As a rezyme SOD , with ths of SOD . For exas of SOD .2•− . The negative slope does not support our Hypothesis 1 that phytoplankton do not differentially allocate a changing fraction of their total gene content to O2•− scavenging with increasing cell size. Including ‘Flagella’ and ‘Colony’ as co-variates in the regression results, however, in a slope that is not statistically different from zero , driven by the influence of ‘Flagella’  but not ‘Colony’ . O2•− metabolism in phytoplankton appears to be mediated by a nearly fixed set of core genes that do not change with increasing total gene count, thus the fractional gene allocation to O2•− decreases as cell radius, and the co-varying total gene count increases. Therefore, gene dosage does not emerge as a factor in phytoplankton O2•− metabolism. With increasing cell radius, eukaryotic phytoplankton have no cell-size associated difference in genes encoding superoxide scavenging, when normalized to total ribosomal genes, suggesting that as a fraction of housekeeping genes, cells do not increase their genomic capacity to scavenge superoxide, consistent with our hypothesis 1 .With increasing cell radius, eukaryotic phytoplankton have a smaller fraction of their total genes encoding scavenging of O2•− scavenging (−3), than do non-flagellated phytoplankton. Similarly, irrespective of size, flagellated phytoplankton have a smaller ratio of genes encoding O2•− scavenging to their total ribosomal genes . This suggests that cellular motility contributes to phytoplankton homeostasis of O2•−, possibly by supporting escape from localized extracellular pockets of O2•−. This decrease in proportional allocation to O2•− scavenging is also arithmetically consistent with the difference in the number of total genes between flagellated and non-flagellated phytoplankton, whereby flagellated phytoplankton have more total genes.Consistent with the significant influence of flagella on the regressions vs. median cell radius , flagell2•− scavenging , as well as similar ratios of O2•− scavenging genes to their housekeeping ribosomal genes  . Our results support our hypothesis that differential diffusional exchange across diatoms of different shape does not influence the fraction of total gene content encoding O2•− scavenging enzymes, because O2•− diffusion outwards is limited by the cell membrane, regardless of cell shape (Hypothesis 1). Differences between genomic patterns of pennate and centric diatoms may arise when comparing metallo-forms of SOD, noting that [Pennate and centric diatoms have similar fractions of their genomes encoding Oing that  found thMicromonas polaris, have genes encoding H2O2 producing enzymes, as they all carry gene(s) encoding the ubiquitous enzyme Superoxide Dismutase. Genes encoding oxidases producing H2O2 include copropophyrinogen oxidase, found across all eukaryotic and prokaryotic phytoplankton, with the exception of one transcriptome. Genes encoding thiol oxidase and acyl CoA oxidase are also found in nearly all eukaryotic phytoplankton, with the exceptions of three transcriptomes. Genes encoding L-aspartate oxidase are found in nearly all prokaryotes, and all green algae, but are nearly absent from other eukaryotic taxa. Sarcosine oxidase is not present in small diatoms and small green algae, but is present in nearly all dinoflagellates and haptophytes. (S)-2-hydroxy-acid oxidase (whose EC number includes glycolate oxidase) is found in most eukaryotic phytoplankton, but rarely in dinoflagellates.All prokaryotic  and euka2O2 scavenging enzymes. Some strains of Prochlorococcus and Synechococcus have lost all genomic capacity to scavenge H2O2, and appear to rely on co-occurring hosts for H2O2 scavenging [Most prokaryotic phytoplankton  and all avenging , 84, 87.Nostoc punctiforme PCC73102 retained a full gene encoding catalase. In our analyses, only Synechococcus elongatus PCC11802 maintained a catalase encoding gene 2O2 scaverococcus . Ostreochococcus , and woukaryotic .2O2 . Including ‘Flagella’ and ‘Colony’ as co-variates did not significantly alter this pattern . The pattern of a smaller fraction of total genes for H2O2 production with increasing cell radius supports our Hypothesis 2 that larger phytoplankton counter decreasing diffusional loss of H2O2 out of cells through a lower genomic capacity for H2O2 production, whereas losses of genes encoding H2O2 producing enzymes are more costly to small phytoplankton . Including ‘Flagella’ and ‘Colony’ as co-variates did not influence the negative slope of the fraction of total genes encoding H2O2 scavenging with increasing median cell radius . A parallel analysis focusing only on small phytoplankton such as pico-cyanobacteria and pico-prasinophytes might yield different results as more such genomes are sequenced, since [2O2 added to seawater at a concentration of 1.6 mg L-1 did not affect cells with a radius larger than 1 to 1.5 μm, but differentially harmed the picoprasinophyte Micromonas pusilla.With increasing cell radius, eukaryotic phytoplankton also have a smaller fraction of their total genes encoding the capacity to scavenge Hd, since  found th10 varied with the log10 (median cell radius) across the Ochrophytes alone (2O2 decreased with increasing cell radius (Slope = -1.6×10−1 ± 9.4×10−2), although the p-value for the regression was only 1×10−1). This marginal decrease in the total number of genes encoding H2O2 production with increasing median cell radius in Ochrophytes again tends to support our Hypothesis 2, with data from within a single phylum to limit confounding influences of diverse evolutionary histories and cell biologies upon patterns.Because median cell radius co-varied with Taxa, we generally excluded Taxa as a co-variate from our regressions, in order to focus on any cross-taxon patterns driven by changing median cell radius. Nevertheless, representatives of the Ochrophyte Phylum alone spanned more than an order of magnitude in median cell radius. We therefore tested whether the loges alone . We foun2O2 production  and scavenging  allocations were steady with increasing cell size, relative to the ribosomal housekeeping gene proxy. But, genes for H2O2 production and scavenging are diluted by increasing total gene counts with increasing cell size.H−1) nor the scavenging of H2O2 . Pennate and centric diatoms also do not show statistically significant differences in the ratio of genes encoding production  nor the scavenging of H2O2 , normalized to their ribosomal gene content encoding H2O2. These results do not support our Hypothesis 5 that pennates have more genes encoding H2O2 producing enzymes due to their higher surface area to volume ratio .Pennate and centric diatoms do not show statistically significant differences in the fraction of their total gene content encoding the production , although often absent, was the most frequently occurring  encoded , but was•NO scavenging enzymes  was the most frequently occurring of the  enzymes . NOD seqfunction  has rece•NO . We re-ran the Quasi-Poisson, excluding those phytoplankton that completely lack genes encoding enzymes for •NO production , which resulted in a decreasing slope with increasing cell radius. Thus, those phytoplankton with any detected capacity to produce •NO indeed have a smaller fraction of their total genes encoding •NO production with increasing radius . Including ‘Flagella’ and ‘Colony’ as co-factors for the regression that solely looks at phytoplankton with the genomic capacity to produce •NO resulted in a slope that is no longer significantly different from zero , driven by the influence of ‘Flagella’ , but not ‘Colony’ .With increasing cell radius eukaryotic phytoplankton do not vary in the fraction of total genes encoding the capacity to produce •NO, Quasi-Poisson regression slope not significantly different from zero .With increasing cell radius, eukaryotic phytoplankton do not vary in the fraction of their total genes encoding the capacity to scavenge •NO production [•NO production from some genomes across taxonomic lineages. Alternately, •NO homeostasis may be achieved in some lineages by regulating the active cellular uptake and release of intracellular •NO, as has been recently demonstrated in humans [•NO, and the lack of •NO scavenging genes across multiple phytoplankters. More research is needed on possible contributions of NOD to the active removal of •NO, as well as the NOS sequences detected in Synechococcus that also display NOD-like activity [•NO does not warrant the active removal of •NO as long as the concentration does not exceed the toxic threshold. This explanation is plausible given that Platymonas helgolandica, Platymonas subcordiformis, Skeletonema costatum, Gymnodinium sp., and Prorocentrum donghaiense showed optimum growth in the presence of •NO concentrations between 10−9 and 10−6 mol L-1 [Non-enzymatic paths contribute to intracellular and extracellular oduction , and mayn humans . Althougn humans , there iactivity . Perhaps mol L-1 , which a•NO shows no cell size effect . Including ‘Flagella’ as a co-variate does not alter these results, but does show that flagellated phytoplankton have higher likelihood of presence of •NO production than do non-flagellated phytoplankton (−5). Including ‘Colony’ as a co-variate does not show a cell size effect, nor a difference in the likelihood of •NO production between colony and non-colony forming phytoplankton (p = 1.3×10−1).A binomial model comparing the presence or absence of genes that encode the production of •NO scavenging . This trend is not influenced by flagella (p = 1.9×10−1) nor colony formation (p = 1.3×10−1). This pattern supports our hypothesis that larger, diffusionally limited cells, have a stronger requirement for •NO scavenging (Hypothesis 2).In contrast, larger phytoplankton are more likely to have a gene encoding •NO producing enzymes, whereas most pennate diatoms do not  when normalized to total genes, and when normalized to ribosomal genes . In contrast, most centric diatoms lack genes annotated as encoding •NO scavenging enzymes, whereas most pennate diatoms carry those genes  when normalized to total genes, and p-value = 2.4×10−5 when normalized to ribosomal genes) (Most centric diatoms carry genes annotated as encoding l genes) .•NO production, and smaller fraction of genes that encode •NO scavenging enzymes, in centric diatoms (•NO production (Hypothesis 5). Given the strong contrast in annotated •NO metabolism genes, it is likely that •NO has regulatory or signaling roles that vary systematically between pennate and centric diatoms, outside any diffusional influences. For example, •NO inhibits diatom adhesion to substrate [•NO production and scavenging. Alternately, [Pseudo-nitzschia arenysensis, Pseudo-nitzschia delicatissima and Pseudo-nitzschia multistriata), so it is possible the apparent lack of •NO producing sequences in pennates is due to errors in the unsupervised annotations from eggNOG.The larger fractional gene allocation to  diatoms  countersubstrate , 154. Peubstrate , which mrnately,  identifiper se. Furthermore, the gene presence or gene count in a genome is only one influence on the potential activity of the encoded enzyme, and closely related enzymes may confer different activities in different organisms.We analyzed the fractions of the total genes in the genome that are associated with the metabolisms of three major ROS. It is important to note that the content of genes encoding specific ROS metabolizing enzymes may be caused by genetic drift, or may relate to a selective advantage linked to other metabolites of the same enzymes, rather than an enzyme role in ROS metabolism, 2O2, O2•− and •NO .O2O2 has lower reactivity, longer intracellular and extracellular lifetimes and readily crosses cell membranes. Across eukaryotic phytoplankton, the fraction of the total genes encoding H2O2 producing and scavenging enzymes decreases with increasing cell radius . Presence of flagella and colony formation do not appear to influence H2O2 metabolism (contrary to hypotheses 3 & 4)H•NO has low reactivity, long intracellular and extracellular lifetimes and readily crosses cell membranes. Neither the fraction of the total genes for •NO production nor for scavenging changed significantly with increasing cell radius, consistent with relatively low cytotoxicity and roles of •NO in taxonomically diverse regulatory systems (contrary to hypothesis 5). Pennate diatoms frequently lack genes annotated as encoding •NO producing enzymes, whereas centric diatoms frequently lack genes annotated as encoding •NO scavenging enzymes (contrary to hypothesis 5). This finding is not explicable by differential diffusional losses of •NO, but may reflect distinct roles of •NO in the regulatory systems of diatom lineages.S1 FigPoint colour corresponds to the source of the data, whether Genome or Transcriptome (‘Ome’). Violin width indicates the fraction of all datapoints occurring at a cell radius (‘log_Radius_um’) within a phylum. Citations for data sources are in (TIF)Click here for additional data file.S2 FigOcean colour corresponds to depth Citations for data sources are in S3 Table. Data used to generate world map produced from the ‘ggOceanMaps’ R package .(TIF)Click here for additional data file.S3 FigData was drawn from a subset of analyzed organisms for which both genome and transcriptome were available. Colour corresponds to the taxonomic lineage (‘Phylum’) Points are jittered to avoid overlapping, resulting in blocks around frequently occurring counts. Dashed line is at 1:1 where ‘ROSGene_count.g’ and ‘ROSGene_count.t’ would be equal. Citations for data sources are in S3 Table.(TIF)Click here for additional data file.S4 FigData was drawn from a subset of genomes and transcriptomes which were both manually and automatically annotated. Colour corresponds to the ‘Gene’ Points are jittered to avoid overlapping, resulting in blocks around frequently occurring counts. Dashed line is placed at 1:1 where Manual and Automated counts would be equal. Citations for data sources in S4 Table.(TIF)Click here for additional data file.S5 FigSymbol color corresponds to taxon lineage (‘Taxa’).(TIF)Click here for additional data file.S6 FigColour corresponds to the taxonomic lineage (‘Phylum’), whereas symbol shape corresponds to the source of the data, whether Genome or Transcriptome (‘Ome’). Citations for data sources are in S3 Table.(TIF)Click here for additional data file.S7 FigPoisson (solid line) or Quasi-Poisson (dashed line) regressions fitted to data ± Standard Error (dotted line). Regressions were run without (blue line) ‘Colony’ and ‘Flagella’ as co-variates. Citations for data sources are in S3 Table.(TIF)Click here for additional data file.S8 FigSymbol colour corresponds to taxonomic lineages (‘Taxa’). Filled data points indicate that the data obtained from that organism was sourced from a genome, and unfilled data points were sourced from a transcriptome. The size of the symbol increases with the number of members of each enzyme found within each genome or transcriptome. Symbol absence means no sequences known to encode the enzyme family of interest were found in the target genome or transcriptome. The absence of transcripts encoding SOD from the Micromonas polaris transcriptome is likely due to low expression of SOD at the time that the mRNA was harvested for sequence analyses.(TIF)Click here for additional data file.S9 FigTaxa are ordered from top to bottom along the left according to increasing median cell diameter within each taxonomic lineage. Symbol colour corresponds to the genus of the prokaryote. Filled data points indicate that the data obtained from that organism was sourced from a genome. The size of the symbol increases with the number of members of each enzyme found within each genome or transcriptome. Symbol absence means no sequences known to encode the enzyme family of interest were found in the target genome or transcriptome.(TIF)Click here for additional data file.S10 FigTaxa are ordered from top to bottom along the left according to increasing median cell diameter within each taxonomic lineage. Symbol colour corresponds to taxonomic lineages (‘Taxa’). Filled data points indicate that the data obtained from that organism was sourced from a genome, and unfilled data points were sourced from a transcriptome. The size of the symbol increases with the number of members of each enzyme found within each genome or transcriptome. Symbol absence means no sequences known to encode the enzyme family of interest were found in the target genome or transcriptome.(TIF)Click here for additional data file.S11 FigTaxa are ordered from top to bottom along the left according to increasing median cell diameter within each taxonomic lineage. Symbol colour corresponds to taxonomic lineages (‘Taxa’). Filled data points indicate that the data obtained from that organism was sourced from a genome, and unfilled data points were sourced from a transcriptome. The size of the symbol increases with the number of members of each enzyme found within each genome or transcriptome. Symbol absence means no sequences known to encode the enzyme family of interest were found in the target genome or transcriptome.(TIF)Click here for additional data file.S12 FigColony (solid line) or non-colony (dashed line) regressions fitted to data. Points along the y-axis indicate whether an organism has flagella (1) or does not have flagella (0).(TIF)Click here for additional data file.S1 Table(CSV)Click here for additional data file.S2 Table(CSV)Click here for additional data file.S3 Table(CSV)Click here for additional data file.S4 Table(CSV)Click here for additional data file.S1 File(DOCX)Click here for additional data file.


Thisis interpreted as a dynamical signature of the T2 statein nitrobenzene—analogous to observations in the related nitronaphthalenesystem, and additionally supported by previous quantum chemistry calculations.The discrepancy between the TAS and TRPEI measurements is discussed,with the overall findings providing an example of how different spectroscopictechniques can exhibit varying sensitivity to specific steps alongthe overall reaction coordinate connecting reactants to photoproducts.Excess energy redistribution dynamics operating in nitrobenzeneunder hexane and isopropanol solvation were investigated using ultrafasttransient absorption spectroscopy (TAS) with a 267 nm pump and a 340–750nm white light continuum probe. The use of a nonpolar hexane solventprovides a proxy to the gas-phase environment, and the findings aredirectly compared with a recent time-resolved photoelectron imaging(TRPEI) study on nitrobenzene using the same excitation wavelength[L. Saalbach et al., J. Phys. Chem. A  Nitroaromaticspecies may also be generated during incomplete combustion processesor formed in situ during atmospheric reactions.7 Suchwidespread presence means there is considerable interest in developingan improved understanding of their photophysics and photochemistry.This is particularly important given the possible health risks posedby both the parent compounds and their photochemical degradation products.9Nitroaromatic compoundsare one of the largest and most importantclasses of chemicals in current commercial use, with applicationsincluding pesticides, pharmaceuticals, and high-energy explosives.10 we reporteda time-resolved photoelectron imaging (TRPEI) study of the nonadiabaticprocesses operating in the excited electronic states of nitrobenzeneand three of its methyl-substituted derivatives following 267 nm excitation.The use of an intense 400 nm probe to induce significant 1 + 3′ionization in these measurements offered an extended view of the overallphotochemical reaction coordinate (with an effective probe energyof 9.3 eV). Despite this, however, we were unable to see any meaningfuldifferences in the dynamics operating across all four species withinthe 200 ps observation window of our experiment. Three distinct temporalsignatures were observed in our data, attributed to (i) an extremelyrapid cascade of internal conversion steps leading from the initiallyprepared S3 (π1π1*) andS4 (π2π1*) states downthrough S2 (n2π1*) to the S1 (n1π1*) state (τ1 ≤ 30 fs); (ii) decay of the S1 (n1π1*) state via intersystem crossing (ISC) to the triplet manifoldor competing IC directly to the S0 ground state (τ2 = 160–190 fs); and (iii) further ISC on a much moreextended timescale connecting the lowest-lying state within the tripletmanifold back to S0 (τ3 = 90–160ps). Here, the state labeling notation follows that adopted by Gonzálezand co-workers.11 This overall interpretationappears to be in broad agreement with mechanisms proposed in high-levelquantum chemistry calculations undertaken for nitrobenzene14 and is consistent with findings reported in several other experimentalstudies using various different techniques and associated observables.20 The same calculations also indicated that the nonadiabatic dynamicswithin the excited state singlet and triplet manifolds are mediatedby motions predominantly localized on the NO2 functionalgroup. This appears to provide a rationale for the experimentallyobserved similarities between nitrobenzene and its methyl-substitutedderivatives—despite considerable variations in starting molecularconformation prior to UV excitation. Our earlier findings also leadto the conclusion that elimination of both NO and NO2 photoproductsoccurs on timescales in the nanosecond domain following a return ofpopulation to the vibrationally hot S0 ground state. Anydifferences in NO vs NO2 branching ratio upon site-selectivemethylation in nitroaromatic species22 are therefore likely to be a consequence of factorsassociated with dynamics occurring on this extended timeframe—includingsteric effects influencing isomerization prior to dissociation. Wealso highlight a very recent computational study by Giussani and Worthindicating that ortho methyl substituents and theenlargement of the π-system can enhance NO release through modificationof the energy barrier connecting the T1 and S0 states, with the latter factor having a stronger effect.23In a recent publication,10 A more recent article by Rodríguez-Córdobaet al. also provides an instructive perspective on the excited statedynamics and photochemistry operating in nitroaromatic species moregenerally.24 Here, we focus predominantlyon a key discrepancy between our TRPEI findings and an ultrafast electrondiffraction (UED) study reported by Zewail and co-workers.25 Following excitation at 267 nm, the UED datarevealed a process operating in nitrobenzene with an exponential timeconstant of 8.8 ps. This was attributed to ISC and structural rearrangementof the T1 state leading to NO and phenyl radical products.Although the UED and TRPEI measurements were both conducted in thegas phase using the same pump wavelength, no dynamical signature operatingon a timescale close to 8.8 ps was observed in the latter work. Totry and resolve this discrepancy and gain further insight into theexcited state photophysics of nitrobenzene, we have undertaken a newinvestigation employing ultrafast transient absorption spectroscopy(TAS). Once again using a pump wavelength of 267 nm, we now interrogatethe dynamics operating in hexane and isopropanol solvents using awhite light continuum probe (340–750 nm). The use of nonpolarhexane is expected to provide a good proxy to the gas-phase environment—particularlyin relation to any femtosecond and few-picosecond processes .Results will therefore be instructive when compared directly to theUED and TRPEI findings. We note that similar transient absorptionstudies conducted on the structurally related 1-nitronaphthalene systemin various solvents do show dynamics operating on timescales of afew picoseconds.27 This has been attributed to thedecay of an intermediary member of the triplet manifold to the T1 state along with subsequent vibrational cooling. The ≥340nm pump wavelengths used in these 1-nitronaphthalene studies were,however, significantly red-shifted from the 267 nm excitation thatis employed here. Given that nitrobenzene is the prototypical nitroaromaticsystem, it is somewhat surprising to note that a study by Yip et al.from 1984 is the only TAS measurement so far reported.20 This work assigned a ≤5 ps lifetime forthe S1 (n1π1*) state following355 nm excitation in tetrahydrofuran, although the laser pulse duration(30–40 ps) and spectral observation window (415–650nm) are limited in comparison to more modern measurements—particularlyin regard to the temporal resolution. The relatively weak magnitudeof the TAS signals reported from nitrobenzene has likely been a contributingfactor in other, more recent time-resolved studies in the solvatedphase making use of alternative transient grating or transient polarizationtechniques.19A wider review of background literaturerelevant to nitrobenzenephotochemistry may be found in our earlier publication, and the readeris directed to that work for a more expanded perspective.22.1f = 25 cm and f = −7.5 cm). Frequency conversion of the pump beamto generate the third harmonic (267 nm) is initiated by passing thefundamental through a thin nonlinear β-barium borate (BBO) crystalto produce the 400 nm second harmonic. A calcite crystal then providestiming compensation between the 400 nm and residual 800 nm pulses.A dual λ/2 waveplate is subsequently employed to reorientatethe polarization of the 800 nm beam prior to propagation through asecond BBO crystal for the generation of the 267 nm pump. For thepresent experiments, this was typically limited to an energy of approximately0.2 μJ pulse–1. Residual 400 and 800 nm pulsesare removed from the 267 nm beam using a series of UV high reflectors.Experiments wereundertaken using a newly constructed transient absorption spectrometerthat was developed in-house at Heriot-Watt University. A full descriptionof the setup is therefore provided, and a schematic diagram may alsobe seen in via a pair of variable neutral densityfilters  orientated inversely with respectto each other such that the propagating beam experiences a uniformintensity reduction. A λ/2 waveplate is incorporated to orientate the probe beam at the magic angle (54.7°)with respect to the pump, eliminating any time-dependent variationin absorption arising from rotational diffusion effects. The resultantpulses are subsequently focused by a fused silica lens (f = 5 cm) into a 1 mm CaF2 window ,where self-phase modulation produces a white light continuum (WLC)spanning the visible and near-UV spectral region. The CaF2 window is mounted on a piezo motor linear stage  and kept in continuous motion to prevent optical damage . The WLC isthen recollimated using a curved aluminum mirror (f = 10 cm). Pump and probe beams are subsequently focused using apair of concave aluminum mirrors  and overlapped within a liquidflow cell . There is a minimumfocused beam waist ratio of 3:1 between pump and probe , and a beam intersection angle of ∼6°.Given that the ionization potentials of nitrobenzene, hexane, andisopropanol all exceed 9.9 eV,29 thefocused pump intensity (approx. 6 × 1011 W cm–2) is sufficiently low thatthe probability of generating any chargedspecies is assumed to be negligible in our subsequent data analysis.In the probe beamline, 800 nm pulses are initiallyreflected offtwo right-angled mirrors mounted on a motorized linear translationstage  with 300 mm of total travel. Power controlis achieved 2 windows  separated by a 100 μmthick PTFE spacer . Althoughnot required in the present study, the flow cell is mounted on anadditional piezo motor linear stage  whichmay be used for sample translation during data collection to reducethe potential impact of bleaching and photoproduct deposition on thecell windows. Nitrobenzene  was prepared inhexane  and isopropanol  solvents at concentrations of 11.3 and 9.7 mM,respectively, and the flow rate through the cell set to 50 mL min–1. These concentrations were chosen based on preliminarydata obtained using a commercial benchtop spectrophotometer (ShimadzuUV-2550) and ensured a linear absorption regime for the pump throughthe entire pathlength of the interaction region.Circulation of liquid samples between a reservoir vessel and theflow cell is achieved using a small centrifugal impeller pump  powered by an external variable DC voltage supply. At the point of laser interaction inside the flowcell, the sample of interest passes between a pair of 2 mm thick CaFf = 10 cm) and passesthrough an optical color filter  to cutout the intense 800 nm portion of the spectrum. The remaining probebeam is then spectrally analyzed by a 1 kHz compact spectrometer  via coupling into an optical fiber  usinga further fused silica lens (f = 5 cm). The detectorwithin the spectrometer consists of a 2048-pixel linear CMOS imagesensor, providing a wavelength resolution of ∼0.7 nm acrossa 200–1100 nm spectral range. The combination of color filterand spectrometer yields a usable WLC between 340 and 750 nm, an exampleof which may be seen in Following interactionwith the sample, the WLC is recollimatedusing a fused silica lens (2.2I(λ)off is the time-invariant transmission spectrum of the ground stateand Ion isthe time-dependent transmission spectrum following photoexcitation(which contains contributions from both the ground and excited states).Spectra collected over a range of different pump-probe delay timesΔt display a nonlinear shift in the point ofmaximum temporal pulse overlap that increases toward longer wavelengthsλ within the WLC. This is a consequence of chirping inducedby transmission through optically dense media, and a correction forthis effect must be applied prior to the extraction of any dynamicalinformation. This is performed using the coherent artifact signal(CAS)30 obtained from a TAS measurementconducted on the pure solvents as shown for hexane in 2 windows. For thecase of both hexane and isopropanol under a 267 nm pump, the initialpart of the transient response may be reasonably modeled by a Gaussianfunction gA(λ) is the amplitudeof the Gaussian function at a given wavelength, t0(λ) is the wavelength-dependent central peak positionin time, and σ(λ) is related to the peak full width at half-maximum (FWHM) via therelationship 31  values are then supplied as inputsto the following expression33 in this instance the approach appears sufficiently robust to permitmeaningful analysis of our data across all timescales. This is illustratedby the clean removal of CAS features seen in regions of our TAS datawhere there is no spectral overlap with the nitrobenzene probe absorptionbands. Application of The raw TAS datarequires several processing steps to extract and quantify any moleculardynamical signatures. The accumulated pump-on and pump-off white lightspectra must be compared to provide a measure of the change in absorbanceof the system following UV irradiation. This is expressed as a differentialchange in optical density (ΔmOD) for each timestep interrogated34 (π2π1*) state.14 This feature is red-shifted by approximately 10 nmin hexane and 20 nm in isopropanol. At the 267 nm pump wavelengthused in our present measurements, however, we always excite at thelong-wavelength side of this feature and some simultaneous directoptical preparation of the S3 (π1π1*) is also a possibility—as suggested in the nitrobenzeneband simulations presented for the gas phase and in water by Gonzálezand co-workers.11 Our preliminary absorptionspectra also indicate that we should not expect any significant groundstate probe (340–750 nm) absorption (and associated time-dependentbleaching effects) in our current TAS measurements.As already mentioned brieflyin A chirp-corrected transient absorption spectrumobtained for nitrobenzenein hexane under 267 nm pump excitation is presented in g(Δt) is the predetermined Gaussian cross-correlation function,the Ai terms are amplitudes,dt captures any offset in the zero pump-probe delayposition, and τ12 = τ1 τ2/(τ1 + τ2), τ123 = τ12 τ3/(τ12 + τ3), and so on. The FWHM of the g(Δt) function varies between 130and 170 fs at the red and blue extremes of the probe spectrum, respectively.Application of the model described by 1–3, making a particular distinction betweenthe shortest lifetime obtained in each region .In the wavelength region between these two limits (475–575nm), no significant signal persists beyond 10 ps, and To numerically quantify the transitory dynamics,a multistep sequentialexponential fitting model was applied to our data. This took the followinggeneral form1A = 140 ± 20 fs) describes an extremelyrapid process, with the second (τ2 = 6.0 ± 0.7ps) and third (τ3 = 1.4 ± 0.4ns) components indicating the presence of much longer-liveddynamics.Of immediate note here is the lifetime associated with τ2, which is comparable to the 8.8 ps transient reported inthe UED measurements of Zewail and co-workers—as highlightedin the 1B = 60 ± 20 fs) but thisis now offset by ∼100 fs fromthe Δt = 0 pump-probe position. This reflectsthe slight temporalshift in the absorption band connecting Regions A and B seen in 2 = 6.7 ± 1.7 ps). Within experimental uncertainty, thisis well matched to the 6.0 ± 0.7 ps decay seen in thebluer spectral region. Finally, decay on a nanosecond timescale (τ3 = 1.3 ± 0.2 ns) then follows a very similar trend tothat seen in the shorter-wavelength blue band. Upon switching thesolvent from hexane to isopropanol, very similar overall behavioris observed in both spectral regions of interest, although the moreextended dynamical timescales are somewhat shorter: In the picoseconddomain, τ2 is reduced by an approximate factor of2 , and this increases to morethan a factor of 4 in the nanosecond regime (340 ± 55 and 375± 75 ps over the same selected wavelength bands). Previous studiesinvestigating nonradiative relaxation of nitrobenzene in various solventsfollowing 355 nm excitation reveal no obvious trend in lifetime vs. solvent polarity.19 In therelated 2-nitrophenol molecule, however, a comparable shortening ofthe long-time (>100 ps) dynamics has been reported under isopropanolsolvation compared to hexane at a 350 nm pump wavelength.34 In the case of the present data, a higher levelof vibrational excitation in the initially prepared excited statesof isopropanol vs hexane at 267 nm may be a contributing factor tothe faster dynamical timescales—as suggested by the red-shiftingof the absorption bands in the former solvent (see vent see 2.35). The spectra resulting from this process areshown in –1 for the hexane and isopropanol solvents,respectively. The absolute peak positions are also very similar inboth cases, with isopropanol giving rise to a small red shift of approx.50 cm–1 relative to hexane. Integrating the spectraldata over smaller time intervals within the selected limits quotedabove indicates that the relative peak amplitudes, positions, andwidths within the comb structure do not change appreciably over theentire timeframe sampled in our measurements . The peak spacing is, however, too largeto be attributed to an extended progression in the NO2 X̃state (ν2 ∼ 750 cm–1).36 The NO2Ã state, on the other hand, is known to exhibit a bending frequencymuch more closely matched to the peak spacing seen in our TAS data(ν2 ∼ 880 cm). Furthermore, the Ã←X̃transition gives rise to a progression of several vibrational bandsin the 370–460 nm region that align closely with the positionof those seen in 37 To further investigate this possibleassignment, we conducted an exploratory TAS measurement on the relatedmolecule 3,5-dimethylnitrobenzene under hexane solvation at extendedpump-probe delay times (20–250 ps). The introduction of methylsubstitutions at ring positions that are not directly adjacent tothe nitro group is expected to exert only a negligible influence onthe formation of NO2 photoproducts. It will, however, significantlyalter many of the vibrational frequencies in the parent molecule relativeto unsubstituted nitrobenzene. As seen in 2 photoproducts as the carrierof the vibrational spectrum.To quantitatively investigate the vibrational combstructure seenin Region E of 44.11A and τ1B) is in general agreement with thebehavior reported previously in our TRPEI measurements.10 We therefore attribute these to the same dynamicalprocesses. Specifically, τ1A corresponds to a cascadeddecay of the S3 (π1π1*) and S4 (π2π1*) statesinitially populated under 267 nm excitation (Region A in 2 (n2π1*) to the S1 (n1π1*) state. The subsequent decay of the S1 (n1π1*) state via ISC into thetriplet manifold and/or IC back to the S0 ground state(Region B in 1B. This is summarized in 1 (n1π1*) lifetimeand ISC interpretation is broadly consistent with the extremely strongspin-orbit interaction predicted between the relevant states,14 as well as recent transient polarization spectroscopy data obtainedfrom pure samples of liquid nitrobenzene following using a multiphoton780 nm pump.15 We note here, however, thatthe wavelength vs. time curvature of the short-livedtransient signal connecting Region A to Region B in our TAS data meansthat the actual numerical values attached to τ1A andτ1B in The observation of two extremelyrapid time constants in our TAS data (as described by τ4.22 = 3.5–6.7 ps) has no equivalent analogue in the earlierTRPEI measurements. As highlighted in 25 This points to a potentiallimitation of the TRPEI approach, where the extent to which the probeprojects into the ionization continuum impacts the overall view alongthe photochemical reaction coordinate.38 The properties of the cation state(s), in addition to the probephoton wavelength, may therefore exert a significant influence overthe dynamical information that may be extracted from a given TRPEImeasurement. An instructive starting picture here comes from Koopmans’correlations, which point to a higher propensity for ionization processesconnecting two states that exhibit similar molecular orbital configurations.As a simple example: a ππ* excitation based predominantlyon a HOMO to LUMO + 1 transition would be expected to project stronglyto the D0 (π–1) state of the corresponding cation. Alternatively, an nπ* excitationbuilt predominantly on a HOMO – 1 to LUMO configuration shouldionize preferentially into the D1 (n–1) continuum. These differences in ionization propensity will be stronglyreflected in the energetic position of bands appearing in a time-resolvedphotoelectron spectrum when tracking the nonadiabatic population transferbetween the ππ* and nπ* states. A more detaileddiscussion of Koopmans’ correlations in the analysis of TRPEIexperiments may be found elsewhere.41In stark contrast to the dynamics operating in the femtoseconddomain, the picosecond transient signal seen in our TAS data ionization scheme in our earlier TRPEI measurements , all four of the lowest-lying singletstates—namely, S4 (π2π1*), S3 (π1π1*),S2 (n2π1*) and S1 (n1π1*)—can be detected efficiently.A similar argument should also apply for the triplet analogues ofthese singlet states. A key factor to note, however, is that quantumchemistry calculations also predict a low-lying state within the tripletmanifold of nitrobenzene that is built on a dominant electronic configurationwith no low-lying singlet analogue.13 This has beenassigned as either T1 or T2, depending on theexact level of theory used, but the corresponding state within thesinglet manifold sits at much higher energy . A similar situation has also been reported in the caseof 1-nitronaphthalene, where calculations conducted using time-dependentdensity functional theory indicate the T2 state is builton a HOMO-5 to LUMO transition.43 Onceagain, there is no low-lying singlet analogue of this state.In the specificcase of nitrobenzene, the first two electronic states of the cation,D2 state in nitrobenzene preferentially ionizes toa cation state that sits energetically beyond the reach of the (1 + 3′) ionization schemeused in our earlier TRPEI study. This then rendered the measurementeffectively blind to this specific step in the overall reaction pathway.Such a conclusion therefore leads us to now assign the (τ2 = 3.5–6.8 ps) lifetime seen in our TAS data to populationmoving through the T2 state, which is initially populated via ISC from S1 (n1π1*). This picture is consistent with more recent theoretical modelsdeveloped for nitrobenzene by several different groups,14 as well as that proposed for the related 1-nitronaphthalene system.27 In our earlier work we highlighted that although decay back to S0 is expected to proceed from the lowest-lying T1 state, we were unable to directly comment on the potential dynamicalrole played by other members of the nitrobenzene triplet manifold.One scenario discussed was the possibility that the T2 statemay decay to T1 on a timescale faster than the incomingtransfer of population from the S1 (n1π1*) state via ISC. In this limit, any small (steady-state)levels of transient T2 population will not be efficientlydetected.44 The observation of picoseconddynamics in our TAS measurements now appears to rule out this interpretationand instead leads us toward an explanation based on inefficient T2 ionization as a consequence of Koopmans’ correlations.A more detailed theoretical exploration of this possibility is beyondthe scope of this present work but will form the basis of furtherinvestigations. Any future time-resolved experiments that monitorphotoionization-based observables in nitrobenzene should thereforebe conducted with probe photon energies that project much more deeplyinto the ionization continuum to provide a more complete map of thereaction coordinate. Examples of such measurements using XUV probesfrom free electron lasers46 and high-harmonic generationsources50 have previously been reported for several other molecular systems.This includes a study on the related 2-nitrophenol system, where picoseconddynamics were observed at relatively large electron binding energiesfollowing 260 nm excitation.47 Interestingly,a similar ionization-based measurement on the same system using 350nm pumping in conjunction with a 400 nm multiphoton probe (as employedin our nitrobenzene TRPEI study) did not capture any picosecond component—althoughit was seen in accompanying transient absorption data recorded atthe same excitation wavelength.34Given the observations outlined above, we therefore suggest thatthe T4.320 In this earlier work, the two features were observed toexhibit very similar kinetic behavior and were therefore both attributedto a single excited state transient absorbing in multiple wavelengthregions. These authors also captured the first two (longest wavelength)peaks of the five-membered vibrational comb seen in 2 andτ3 associated with the long-time dynamics are verysimilar for both solvents considered across the respective blue andred absorption band regions  method in conjunction with the aug-cc-pVDZ basis set andthe implicit SMD solvation model.52 Thepresence of two Tn ← T1 transitions withsignificant oscillator strength appear within our spectral observationwindow and are energetically well matched to the 575–750 and340–475 nm absorption bands. A broadly similar prediction isalso obtained from excited state data presented by Lin et al. usingtime-dependent density functional theory at the B3LYP/6-311 levelwith a T1 optimized geometry.17 The T1 state is believed to decay via ISC back to theS0 ground state before undergoing NO2 or NOelimination, where the relative branching yield of the two differentphotoproduct channels has been observed to vary considerably acrossthe 320–193 nm excitation region.21 A recent theoretical investigation also suggests that the NO channelmay potentially involve up to three different pathways.53 These fragmentation processes are, however,expected to occur on a timescale well beyond the limit of our currentmeasurements.17 Finally here, we note thatthe T1 state lifetime  is significantly longer than that attributed tothe T1 state of nitrobenzene at the same 267 nm excitationwavelength in our earlier TRPEI study (90 ± 10 ps).10 Such a finding is not unexpected, however, giventhe potential for solvent-induced vibrational relaxation of variousmodes on relatively extended timescales that is not a factor in thegas phase.At both the red (575–750 nm) and blue (340–475 nm)extremes of the data presented in ions see 5, also s3 to decay of the T1 state manifestingas two distinct absorption bands within our spectral observation window,it is perhaps initially surprising that the same comb is thereforeabsent in the 575–750 nm region. This could point to very differentequilibrium geometries in the two different participating upper states,leading to an extended Franck-Condon progression in one absorptionband but not the other. An alternative explanation, however, is simplythat at the red end of the spectrum, the comb sits at absorption wavelengths >750nm and is obscured by the optical color filter used to remove theintense 800 nm fundamental from which the WLC probe is derived. Infuture, experiments using an alternative driving frequency for supercontinuumgeneration may be used to further explore this suggestion. To learnmore about the possible origin of the comb structure, we employeddensity functional theory (B2PLYP/Def2-TZVP) to determine the variousvibrational frequencies present in the T1 state of nitrobenzeneunder isolated conditions, as well as in hexane and isopropanol. TheB2PLYP method is well suited for problems requiring high accuracyin the evaluation of vibrational properties.54 Our analysis was once again conducted with the Gaussian16 softwarepackage at the optimized T1 minimum energy geometry, makinguse of the SMD solvation model. The key findings are presented in –1) as well as the two nearestneighboring frequencies . All three modes correspondto large amplitude motion of the H atoms attached to the benzene ringwith only minimal distortion/displacement of the NO2 group—asdepicted for the most likely frequency carrier in 2 group displacement exhibiting a frequencywithin 100 cm–1 of the experimentally observed combspacing. Given that the key motions driving the nonadiabatic dynamicsthroughout the excited state are known to be predominantly localizedon the nitro moiety, this could potentially explain the relativelylong lifetime of the T1 state, despite the apparent highlevels of vibrational excitation within the 885–887 cm–1 mode. Furthermore, the apparent lack of vibrationalquenching in this mode also indicates a highly inefficient resonanttransfer of energy into the surrounding solvent bath. This is a surprisingresult as hexane, for example, is known to exhibit several vibrationalmodes with frequencies closely matched to the comb spacing.55 In light of this, we also reiterate the possibilitythat the observed vibrational progression may arise as a consequenceof the upper state geometry being very different from that of T1. This could then give rise to an extended Franck-Condon progressionin the absorption band that would not necessarily require any initialexcitation in a specific T1 vibrational mode . The exact originof the vibrational comb therefore remains an open question at thepresent time but may be investigated in future with more targetedmeasurements on systematically substituted nitrobenzene systems aswell as more expanded theory.We now consider the vibrational comb structure seenwithin the340–475 nm region of the TAS data, as highlighted in 54 (π2π1*) and/or S3 (π1π1*) states (τ1A) down throughS2 (n2π1*) to S1 (n1π1*)—which then undergoesISC into the T2 state of the triplet manifold (τ1B) or internal conversion directly to the S0 groundstate. The T2 state, which subsequently decays to T1 on a timescale of 3.5–6.7 ps (τ2),is known to be built on an electronic configuration that has no low-lyingsinglet analogue. This provides a rationale for the absence of dynamicalsignatures operating on picosecond timescales in a previous gas-phaseTRPEI measurement at the same 267 nm excitation wavelength (usingan effective probe energy of 9.3 eV). Our present findings also confirmthe observation of picosecond dynamics in the UED study reported byZewail and co-workers. Our interpretation of this signal  differsfrom this earlier work, however, as it does not involve isomerizationof nitrobenzene to phenyl nitrate within the triplet manifold. Finally,the T1 state subsequently repopulates the S0 ground state on a nanosecond timescale under hexane solvation, andin approximately 350 ps when in isopropanol. Overall, our findingsillustrate the value of drawing on a combination of complementaryexperimental measurements (with different associated observables)when attempting to understand the complete, and often highly complexnonadiabatic dynamics operating in polyatomic molecules.A newly developed transientabsorption setup has been used to investigatethe nonadiabatic excess energy redistribution pathways exhibited bynitrobenzene in hexane and isopropanol solvents following 267 nm excitation.A white light continuum provides a probe spanning the 340–750nm spectral region and this is sufficient to reveal dynamics operatingwith four individual exponential time constants. The first two ofthese are both of order 100–200 fs and are attributed to arapid, cascaded decay of the initially populated S


IFNL4) gene, thus preventing the expression of a functional IFN-λ4 protein. While probing the expression of IFN-λ4 in human peripheral blood mononuclear cells (PBMCs), using a monoclonal antibody that binds to the C-terminus of IFN-λ4, surprisingly, we observed that PBMCs obtained from TT/TT genotype individuals could also express proteins that reacted with the IFN-λ4-specific antibody. We confirmed that these products did not emanate from the IFNL4 paralog, IF1IC2 gene. Using cell lines and overexpressing human IFNL4 gene constructs, we obtained evidence from Western blots to show that the TT allele could express a protein that reacted with the IFN-λ4 C-terminal-specific antibody. It had a molecular weight similar if not identical to IFN-λ4 expressed from the ΔG allele. Furthermore, the same start and stop codons used by the ΔG allele were used to express the novel isoform from the TT allele suggesting that a restoration of the ORF had occurred in the body of the mRNA. However, this TT allele isoform did not induce any IFN-stimulated gene expression. Our data do not support a ribosomal frameshift that leads to the expression of this new isoform, implying that an alternate splicing event may be responsible. An N-terminal-specific monoclonal antibody did not react with the novel protein isoform suggesting that the alternate splicing event likely occurs beyond exon 2. The new isoform is glycosylated similar to the functional IFN-λ4 and is also secreted. Furthermore, we show that the ΔG allele can also potentially express a similarly frameshifted isoform. The splicing event that leads to the generation of these novel isoforms and their functional significance remains to be elucidated.The TT allele of the dinucleotide variant rs368234815 (TT/ΔG) abolishes the open reading frame (ORF) created by the ancestral ΔG allele of the human interferon lambda 4 ( IFNL4 by Prokunina-Olsson and others showed that the TT allele abolishes the ORF, but can express inactive isoforms such as p124 and p143 that are unrelated to IFN-λ4  is a newly discovered type III IFN that has been associated with hepatitis C virus and other infections  homozygous individual was cloned in pcDNA3.1+ (Invitrogen) by using primers listed in IF1IC2, a 1.5 kb gene [from start to stop codon (including the premature stop codon) with all exons and introns] was similarly cloned from human genomic DNA by using the primers listed in A full-length (exons and introns only) 1.4 kb All clones were confirmed by Sanger sequencing of the plasmid DNA. The wild-type (wt) IFN-λ4 cDNA clone, pIFNL4-Halo, was a gift from Ludmila Prokunina-Olsson (NIH), while the codon optimized IFN-λ4 cDNA was commercially synthesized . The TT mutation-addition was introduced into the wt cDNA clones using SDM. Green fluorescent protein  was cloned in either pCDNA+ vector with or without the ATG start codon. GFP without the ATG start codon was also cloned immediately before the TGA stop codon of TT (in pCDNA+) along with a GGGS linker sequence to express a fusion product with the protein translated from the TT allele.Human genomic DNA was extracted from whole blood using a QIAamp Blood DNA Mini Kit (Qiagen). PCR was used to amplify a 1.4 kb amplicon from the genomic DNA, using the primers listed in 2. Cells were seeded into 6-well [for Western blot (WB)] or 12-well (for qPCR) plates and allowed to reach a 70%–80% confluency (∼24 h). Cells were transfected with pcDNA3.1 or pIF1IC2, TT or ΔG, or the cDNA constructs with Lipofectamine LTX and Plus (Invitrogen). Unless mentioned otherwise, 2 μg/well of the constructs was transfected for WB experiments. Twelve hours (for qPCR) or 24 h (for WB) post-transfection, cells were collected and processed for WB/qPCR.HEK293, C33A  , and Huh7.5  cells were maintained in Dulbecco's modified Eagle's medium (Himedia) in addition to 10% FBS and penicillin/streptomycin  at 37°C and 5% COFor fluorescence experiments, HEK293 cells seeded in clear bottom white plates  were transfected with 100 ng of the different constructs (in pCDNA+) at near confluency and the fluorescence intensity was read after 24 h in the EnSight multimode plate reader (PerkinElmer) using Kaleido data acquisition and analysis program.2. After 5 h, cells were either treated or not for 24 h with phorbol 12-myristate 13-acetate , polyI:C , and lipopolysaccharides . After 24 h, cells were collected and processed for WB.The NIBMG Bioethics Committee approved this study with the certificate No. NIBMG/2022/1/0018. Five milliliters of blood was drawn from volunteers after obtaining informed consent. Peripheral blood mononuclear cells (PBMCs) were isolated using the Ficoll density gradient method (Sigma). Isolated PBMCs were plated or not (fresh PBMCs) in 6-well plates with RPMI Medium-1640 (Gibco) along with 10% FBS and incubated at 37°C and 5% COIFNL3 gene : MX1, OAS1, ISG15, and IFITM3 were measured using SYBR Green after amplifying the cDNA using the primers described previously . cDNA was synthesized from 1 μg of RNA using the Verso cDNA Synthesis Kit (Thermo Scientific). qPCR was performed in the QuantStudio 5 (Applied Biosystems) using custom-made isoform-specific IFNL4 TaqMan probes and primers as described previously for the p179, p131, and p107 isoforms, and for the eviously . GAPDH wThe proteins were run on 15% SDS-PAGE gels, and WB analysis of human recombinant IFN-λ4 was carried out using the rabbit anti-IFN-λ4  or mouse anti-IFN-λ4  or anti-HA . Following overnight treatment with the primary antibody, the blots were developed with a secondary HRP-linked goat anti-rabbit antibody  or goat anti-mouse HRP-conjugated secondary antibody . For beta-actin (Santa Cruz Biotechnology), primary antibodies at 1:10,000 dilution were used. The blots were developed and imaged in ChemiDoc MP imaging system (Bio-Rad).Deglycosylation was done by using PNGase F . For deglycosylation, 9 μL of cell lysate along with 1 μL of 10 × denaturing buffer was heated at 100°C for 10 min. After denaturation, 2 μL of glyco buffer, 2 μL of 10% NP-40, 1 μL of PNGase F, and 5 μL of deionized water were added. This mixture was incubated at 37°C overnight and then proceeded with WB.Trichloroacetic acid was added to the clarified media in 1:4 ratio and incubated at 4°C for 30 min. The sample was centrifuged at 14,000 rpm for 10 min at 4°C. The supernatant was decanted, and the precipitated protein pellet was washed thrice with 200 μL of cold acetone by centrifugation at 14,000 rpm for 5 min at 4°C. Pellet was dried at 95°C for 5 min. Lysis buffer was added to the pellet and proceeded to WB.In the beginning of this study, we were interested to look for the expression of IFN-λ4 in human PBMCs. For the initial experiment, we identified two individuals who were homozygous for the major (TT) and minor (ΔG) alleles  at rs368After incubation, the cells were lysed and probed in WB with a rabbit monoclonal antibody (RAB) that is raised against the C-terminus of IFN-λ4, and that reacts only against the human IFN-λ4 protein by recognizing amino acids 128–152  and of IFNL3 in both freshly isolated PBMCs (without plating) and PBMCs that were cultured and treated, as in IFNL4 isoforms were used  above, we subjected the fresh PBMC lysates from all three genotype individuals to WBs using a mouse monoclonal antibody (MAB) that binds to the N-terminus of IFN-λ4  above, when we queried, we saw that  Premzl,  has spec Premzl, , leading Premzl,  that couIF1IC2 gene in a pcDNA3.1+ (Invitrogen) expression vector; similarly, we constructed another clone, pIFNL4 (TT) (referred to as TT hereafter), that contained the full-length IFNL4 gene  obtained from a TT/TT homozygous individual. We used two different well-characterized  construct to the ΔG allele (referred to as ΔG hereafter) and repeated the experiment along with the TT allele construct . Only thWe simultaneously ran products from a PBMC sample obtained from a TT/TT individual along with the products from overexpressed TT or ΔG constructs in HEK293 cells and saw that at least some of the bands expressed from the ΔG construct moved at similar positions to that from the TT/TT PBMC sample . We alsoInterestingly, these HMW products reacted with the MAB, similar to the results that we saw from PBMC experiments  and S3B.IFN-λ4 is known to be heavily glycosylated (Chen et al., IFNL4 so that the GFP could be expressed only as a fusion protein along with the product of the TT allele (The results show that the novel TT allele isoform cannot express an RAB reacting protein if the start codon is mutated, and it also produces an HA tag suggesting the translation of an in-frame protein beyond exons 4 and 5 (T allele  bottom. T allele .Furthermore, the novel isoform is secreted more efficiently than the products of the ΔG allele in C33A cells, but is secreted to a similar extent in Huh7.5 cells .IFNL4 TT allele that has a restored reading frame beyond the dinucleotide DNA polymorphism rs368234815. Restoration of the reading frame could be due to a ribosomal frameshift, or it could be due to a hitherto unreported splicing event in the body of the mRNA that leads to the reading frame restoration. To address both possibilities, we introduced the TT mutation-addition to the ΔG allele in a p179 expressing cDNA construct. We used two different cDNA constructs, one the wt and another a codon optimized version. However, no RAB reactive products were produced in either of the TT allele carrying versions of the overexpressing cDNA constructs (The above results clearly indicate that a novel protein is produced from the nstructs . This woTo understand the origin of the novel TT allele isoform, we introduced frame changing mutations next to the start codon and subjected the overexpressed products to WBs using the RAB . These i+” protein from the TT allele also raises the possibility of a similar event happening with the ΔG allele (isoform referred to as p123+; However, our results from a + 1 frame-shifted ΔG allele construct (IFNL4 gene regardless of the alleles present at rs368234815, leading to the generation of novel transcripts. PCR and qPCR products generated with cDNA from TT and ΔG allele overexpressed cells were indistinguishable between the two alleles (This hypothesis could be tested to see if an RAB reacting product is generated from onstruct ; indeed,onstruct . These r alleles ; sequenc alleles , table; IFNL4 gene, were identified by RNA-seq and the subsequent molecular analysis (Prokunina-Olsson et al., At least 10 mRNA isoforms, 5 each from the ΔG and TT alleles of human We began this study with an aim to examine the expression of IFN-λ4 in human PBMCs . Our obsIFNL4 mRNA is the most expressed IFN mRNA in bone marrow stem cells of healthy individuals (Gadalla et al., A recent report has shown that


Error in Figure/TableIn the published article, there was an error in The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.Text CorrectionMaterials and methods section and one spelling error in the Results section.In the published article, there were three errors. There were two unit errors in the 2 Materials and methods, 2.2 Liquid chromatography–mass spectrometry, paragraph three. This sentence previously stated:A correction has been made to “injection volume, 2 ml”The corrected sentence appears below:“injection volume, 2 μL”2 Materials and methods, 2.8 16S rRNA sequencing, paragraph four. This sentence previously stated:A correction has been made to “DNA was diluted to 1 ng/L in sterile water according to the concentration.”The corrected sentence appears below:“DNA was diluted to 1 ng/μL in sterile water according to the concentration.”3 Results, 3.8 Ferulic acid ameliorates glucose and lipid metabolism disorders in PCOS mice, paragraph two. This sentence previously stated:A correction has been made to “the high FA dose significantly increased the HDL-C levels while decreased the TC levels.”The corrected sentence appears below:“the high FA dose significantly increased the HDL-C levels while decreased the TG levels.”The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.Error in Figure/Table LegendIn the published article, there was an error in the legend for “ Analysis of gut microbial diversity was performed on the basis of 16S rRNA sequencing and was presented by rarefaction curves and rank abundance curves.”The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.Incorrect Supplementary MaterialIn the published article, there were two errors in “ DHEA or sesame oil were given to female mice for 20 days.”Figure 2 legend, “FA instead of FC days.” The correct material statement appears below.“ Estrus cycles of the control, PCOS and FA groups (n = 5/group).”The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.