Phosphorylation and acetylation of sarcomeric proteins are important for fine-tuning myocardial contractility. Here, we used bottom-up proteomics and label-free quantification to identify novel post-translational modifications (PTMs) on β-myosin heavy chain (β-MHC) in normal and failing human heart tissues. We report six acetylated lysines and two phosphorylated residues: K34-Ac, K58-Ac, S210-P, K213-Ac, T215-P, K429-Ac, K951-Ac, and K1195-Ac. K951-Ac was significantly reduced in both ischemic and nonischemic failing hearts compared to nondiseased hearts. Molecular dynamics (MD) simulations show that K951-Ac may impact stability of thick filament tail interactions and ultimately myosin head positioning. K58-Ac altered the solvent-exposed SH3 domain surface – known for protein–protein interactions – but did not appreciably change motor domain conformation or dynamics under conditions studied. Together, K213-Ac/T215-P altered loop 1’s structure and dynamics – known to regulate ADP-release, ATPase activity, and sliding velocity. Our study suggests that β-MHC acetylation levels may be influenced more by the PTM location than the type of heart disease since less protected acetylation sites are reduced in both heart failure groups. Additionally, these PTMs have potential to modulate interactions between β-MHC and other regulatory sarcomeric proteins, ADP-release rate of myosin, flexibility of the S2 region, and cardiac myofilament contractility in normal and failing hearts. PTMs have been shown to alter the canonical structure, function, localization, and half-life of modified sarcomeric proteins . These mMYH7, the predominant isoform of myosin in the adult human heart. The massive size of β-MHC, ~223 kDa, imposes limitations with current technological approaches and challenges our ability to obtain complete sequence coverage. In a study by Kawai et al. examining PTMs on MYH6, the predominant murine myosin isoform,, and troponin I (TnI) by cAMP-dependent protein kinase (PKA), and myosin regulatory light chain (RLC) by myosin light-chain kinase (MLCK) . Several isoform, phosphor isoform,. In addi isoform,. Overall isoform,.While many earlier studies were conducted in animals of varying species, examining changes in PTMs on sarcomeric proteins in the context of human heart disease may provide clues toward potential interventions. Identification of PTMs on β-MHC is the first step in discovering whether these modifications provide beneficial or adverse impacts on cardiac function and disease progression. With mass spectrometry techniques increasing in sensitivity, we are becoming more successful at uncovering even low-abundance PTMs that may have a significant impact on cardiac outcomes. As new reports emerge documenting the presence of PTMs in human sarcomeric proteins, the ensuing challenge remains: testing their in vivo functional consequences. Given the emerging importance of understanding the potential role of PTMs on cardiac muscle performance, we used LC/MS and MD simulations to investigate novel PTMs sites in key functional regions of β-MHC in human hearts in healthy and diseased states. This article provides insight into the potential of these modifications to fine-tune cardiac myofilament performance.The patient groups from whom explanted heart tissues were collected included explanted hearts from healthy donors (nonfailing (NF)) and end-stage heart failure patients, who were reported to have either ischemic heart failure (I-HF) or non-ischemic heart failure (NI-HF). The demographics for the patients included information on age, gender, and race. Patients' ages ranged from 41 to 69 years with each condition represented   and wereUnique high-confidence peptides bearing PTMs were identified using bottom-up mass spectrometry data from NF, ischemic, and nonischemic failing human hearts  and 3. TA schematic of the β-MHC protein is included in m/z 766.36) was detectable as an isotope dot product (idotp) with a value below 0.5, and therefore was unquantifiable (Residue K34 is located between the SH3-like domain and the converter domain . K58 is tifiable . The acem/z 974.49). The MS/MS spectrum of the trypsin-digested common IRP is shown in To investigate the potential significance of these newly identified PTMs for cardiac function, we assessed their abundance in the human heart samples we analyzed. The relative abundance of the modifications was determined by calculating peak areas of modified peptide and normalizing to IRP peak area  of PTMs that existed in functionally significant regions of the protein seen in a–g in which positions a and d are hydrophobic residues that form a ‘knobs in holes’ interlocking structure that promotes a well-packed hydrophobic core  increased in the presence of the PTM  expression and activity plays a role in heart failure development and progression by increasing class I HDAC activity . The rod region is perhaps the more conformationally consistent portion of the myosin protein that is accessible to HATs but also HDACs. Therefore, one interpretation of the tendency for lower acetylation of K951 (p<0.05) and K1195 (not significant) in the failing heart samples is that they are not in the globular domain and may be more accessible to HDACs. It is unknown whether HDAC-inhibition would preserve acetylation at these sites and whether maintenance of this PTM is functionally important. Recent studies have explored the functional significance of sarcomeric protein acetylation to understand the impact of HDAC on cardiac function . The levactivity . Adminisactivity . HDAC inactivity  and relaactivity  as well activity . It remaWhile the remaining simulated modifications did not have significantly altered abundance in diseased states, the proximity of these PTMs to critical functional sites in myosin merited investigation. K58 acetylation is markedly reduced in the I-HF hearts, and its localization near the SH3 domain may also be slightly more exposed, with potentially greater HDAC activity in ischemic hearts. The other acetylated residues K34 and K429 are located further into the interior of the myosin head and may be less affected by increased HDAC activity in diseased hearts. Preservation of acetylated lysines in β-MHC may be beneficial to normal heart function, and pathological conditions favoring greater HDAC activation may disrupt crucial constitutively acetylated residues hastening heart failure development. The K58-Ac simulations indicated that K58-Ac caused minimal structural perturbations overall compared to the nonmodified protein. Instead, the greatest impact of K58-Ac is the decrease in electrostatic potential and structure on the outer surface of the SH3 domain. Lowey et al. and others have provided evidence that an interaction between the ELC and actin is mediated by the SH3 domain of myosin S1 . In lighThe doubly modified peptide K213-Ac/T215-P was deemed to contain high-confidence PTM sites; however, K213-Ac/T215-P were found to be detectable but not quantifiable. A set of studies on loop 1 sequences  demonstrSeveral studies have provided insight on how phosphorylation of cardiac sarcomeric proteins alters myofilament performance . Kawai eMYH7 variants are implicated in roughly one-third of diagnosed familial HCM cases , stained with Coomassie blue, and bands that corresponded to β-MHC (~223 kD) were excised as shown in In-gel digests were performed for each excised sample using the ProteoExtract All-in-One Trypsin Digestion Kit  according to the manufacturer’s instructions. Briefly, excised gel pieces were destained in wash buffer and dried at 90℃ for 15 min. Gel pieces were rehydrated in trypsin digestion buffer and treated with a reducing agent for 10 min at 37℃. Samples were cooled to room temperature and then incubated in blocking reagent for 10 min at room temperature. Trypsin was added to a final concentration of 8 ng/µl and incubated for 2 hr at 37℃ on an orbital shaker. Peptides were eluted in 50 µl 0.1% formic acid.m/z) at 60,000 resolution. MS2 were acquired in centroid mode at 15,000 resolution. Ions with single charge or charges more than 7 as well as unassigned charge were excluded. Raw data were searched with Proteome Discoverer 2.2 (Thermo Fisher Scientific) using Sequest HT and Mascot search engines and percolator as the PSM validator with species-specific FASTA database. Phosphorylation and acetylation were used as a dynamic modification in SequestHT and Mascot and PTMs were scored by ptmRS node in Proteome Discoverer 2.2. MS1-based quantification of peptides was performed using Skyline 4.0. The mass spectrometry data generated in this study has been deposited at Dryad and can be found at doi:10.5061/dryad.s4mw6m97g.Sample peptides were processed using an externally calibrated high-resolution electrospray tandem mass spectrometer  in conjunction with a Dionex UltiMate 3000 RSLCnano System (Thermo Fisher Scientific). 5 µl of sample peptide was aspirated into a 50 µl loop and then loaded onto the trap column . Separation on an analytical column  was conducted with a flow rate of 300 nl/min. A 60 min linear gradient from 3% to 45% B (0.1% formic acid in acetonitrile) was performed. The LC eluent was directly nanosprayed into Q Exactive HF mass spectrometer (Thermo Scientific). During the chromatographic separation, the Q Exactive HF was operated in a data-dependent mode and under direct control of the Thermo Excalibur 3.1.66 (Thermo Scientific). The MS data were acquired using the following parameters: 20 data-dependent collisional-induced dissociation (CID) MS/MS scans per full scan (350–1700 The area under the curve (AUC) for each peptide sequence was measured in Skyline 20.1, an open-source Windows client application software for targeted proteomics data analysis and bioinformatics . To detehttps://www.rcsb.org; PDB ID: 4DB1, 2.6 Å, residues 1–777; Modeller (leap module of AMBER. There were two modified variants of 4DB1: 4DB1-K58-Ac (corresponding to acetylation of Lys 58) and 4DB1-K213-Ac/T215-P (corresponding to simultaneous acetylation of Lys 213 and phosphorylation of Thr 215). There was one modified variant of 2FXM: 2FXM-K951-Ac (corresponding to acetylation of Lys 951). For 2FXM, K951 residues in both chains A and B were modified. Because both K951 residues were modified, our simulations sample the ‘most aggressive’ effects of acetylation at this site: diminished effects may be found for singly modified systems.Starting coordinates for the wildtype (WT) β-MHC motor domain simulations were obtained from an X-ray crystallography structure of the post-rigor, ATP state of β-MHC in the Protein Data Bank . After minimization, systems were heated to 310 K during three successive stages. In each stage, the system temperature is increased by ~100 K over 100 ps  using the NVT  ensemble. During all heating stages, 25 kcal/mol restraints were present on the backbone heavy atoms . After the system temperatures reached 310 K, the systems were equilibrated over five successive stages using the NPT  ensemble. During each stage, the systems were equilibrated for 5.4 ns in the presence of restraints on backbone atoms. The strength of the restraints was decreased from 25 kcal/mol during the first stage to 1 kcal/mol during the fourth stage. During the final equilibration stage, the systems were equilibrated in the absence of restraints.Hydrogen atoms were modeled onto the initial structure using the Production dynamics for conventional MD simulations were then performed using the canonical NVT ensemble with an 8 Å nonbonded cutoff and 2 fs time step. Coordinates were saved every picosecond. Simulations were run in triplicate for 500 ns each. Unless specified otherwise, simulations were analyzed separately, and the results of replicate simulations were averaged together. To account for potential equilibration effects, the first 100 ns were excluded from subsequent analyses.2FXM contains a linear fragment of S2 spanning ~130 residues. Due to the length of this fragment, we performed implicit solvent simulations of 2FMX using the Generalized Born model. This approach was previously used to study S2 fragments, and our methods were chosen to best match earlier simulations . Simulatα RMSD, Cα RMSF, SASA, interatomic distances, and interatomic contacts were calculated with cpptraj  feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by a Senior Editor. The following individual involved in review of your submission has agreed to reveal their identity: Stuart Campbell (Reviewer #3).Thank you for submitting your article "Post-translational modification patterns on β-myosin heavy chain are altered in ischemic and non-ischemic human hearts" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this letter to help you prepare a revised submission.Essential revisions:(A) General alterations requested.(1) Both reviewers 2 and 3 are positive about the paper but both indicate a need for shortening of the paper.(2) In particular reviewer 2,although:(a) generally positive in stating that the paper (i) represents an enormous amount of work on identifying modifications of the major cardiac motor protein β, myosin in the failing and non-failing human heart, and (ii) tackles an important understudied problem of PTMs in the human β cardiac myosin protein.However,eLife.(b) flags up the point that much of the material might rather go to a more specialized journal. On discussion, reviewer 2 specifically requests clarification why the findings should be of interest to readers of the broader remit represented by (3) Reviewer (2):Considers that this is a thought-provoking study using an appealing strategy. Human samples increase the disease relevance and mass spectrometry here yields novel discoveries. Molecular dynamics modeling is used to speculate on the relative functional impact of each PTM. The novel PTM sites identified in this work are exciting and should stimulate and inspire other work in the field.(B) Specific alterations requested:1) Reviewer 2:a) There is a lot of information in this very long (too long in my opinion) right now, its utility is unknown. The PTMs are very rare on this molecule and there are not enough samples to have much power (even though I know how hard it is to get samples).b) The authors also make several statements about how MYH6 or α myosin is induced in human heart failure. To the best of my knowledge, the opposite is what happens in human heart failure.2) Reviewer 3:a) I found it very difficult to understand the way in which PTM abundance was presented. It was not clear what the overall percentage of PTM is for a given site, e.g. in a failing heart, what percentage of myosin K951 residues are acetylated? The first paragraph of the discussion states that "phosphorylation and acetylation PTMs are present on β-MHC in non-diseased human hearts in low abundance." Is it not possible to determine what fraction of a particular peptide is modified? Otherwise, why would the authors state that PTMs are "low abundance" (first paragraph of the discussion)? Furthermore, if this information has been obtained, why would it not be clearly represented in the manuscript? It seems as though the quantification of PTMs is shown only relative to IRP.b) Similarly, the PTM to IRP ratio is also confusing in that the stated range is 1-14. Does this mean that it is not possible for PTM abundance to be less than the IRP? Why not?c). The authors mention that one way to interpret the loss of K951 acetylation in failing hearts is that there is a shift toward α-MHC. Perhaps it was outside the scope of the study, but was there no way of testing this hypothesis in some way?d). The discussion is, in my view, far too long and wide-ranging. Some of the logic presented is also challenging. For instance, the PTMs discussed are shown to be more abundant in normal than in failing hearts. Yet, the authors draw parallels between the sites for the PTMs and the locations of cardiomyopathy-causing mutations. It seems strange to associate sites of "normal" PTMs with the locations of disease-causing mutations. Overall, the discussion is far longer than would normally be expected for the corresponding significant results, which really focused just on K951. My recommendation would be to focus the discussion drastically, state the most meaningful findings very clearly, and help the reader grasp the real significance of the work.Reviewer #2 (Recommendations for the authors):This paper tackles an important understudied problem of PTMs in the human β cardiac myosin protein. The authors found acetylation sites and phosphorylation sites and used molecular dynamics simulations to predict the impacts of these PTMs on myosin function.There is a lot of information in this very long (too long in my opinion) right now, its utility is unknown. The PTMs are very rare on this molecule and there are not enough samples to have much power (even though I know how hard it is to get samples).The authors also make several statements about how MYH6 or α myosin is induced in human heart failure. To the best of my knowledge, the opposite is what happens in human heart failure.I think this information may be better suited for a more specialized audience and journal.Reviewer #3 (Recommendations for the authors):This is a thought-provoking study that makes use of an appealing strategy. Human samples increase the disease relevance of the work, and mass spectrometry has been used here to yield novel discoveries. Molecular dynamics modeling is used to speculate on the relative functional impact of each PTM. The novel PTM sites identified in this work are exciting and should stimulate and inspire other work in the field.Some suggestions for improving the work are given below.1. I found it very difficult to understand the way in which PTM abundance was presented. It was not clear what the overall percentage of PTM is for a given site, e.g. in a failing heart, what percentage of myosin K951 residues are acetylated? The first paragraph of the discussion states that "phosphorylation and acetylation PTMs are present on β-MHC in non-diseased human hearts in low abundance." Is it not possible to determine what fraction of a particular peptide is modified? Otherwise, why would the authors state that PTMs are "low abundance" (first paragraph of the discussion)? Furthermore, if this information has been obtained, why would it not be clearly represented in the manuscript? It seems as though the quantification of PTMs is shown only relative to IRP.2. Similarly, the PTM to IRP ratio is also confusing in that the stated range is 1-14. Does this mean that it is not possible for PTM abundance to be less than the IRP? Why not?3. The authors mention that one way to interpret the loss of K951 acetylation in failing hearts is that there is a shift toward α-MHC. Perhaps it was outside the scope of the study, but was there no way of testing this hypothesis in some way?4. The discussion is, in my view, far too long and wide-ranging. Some of the logic presented is also challenging. For instance, the PTMs discussed are shown to be more abundant in normal than in failing hearts. Yet, the authors draw parallels between the sites for the PTMs and the locations of cardiomyopathy-causing mutations. It seems strange to associate sites of "normal" PTMs with the locations of disease-causing mutations. Overall, the discussion is far longer than would normally be expected for the corresponding significant results, which really focused just on K951. My recommendation would be to focus the discussion drastically, state the most meaningful findings very clearly, and help the reader grasp the real significance of the work. Essential revisions:(A) General alterations requested.(1) Both reviewers 2 and 3 are positive about the paper but both indicate a need for shortening of the paper.(2) In particular reviewer 2,although:(a) generally positive in stating that the paper (i) represents an enormous amount of work on identifying modifications of the major cardiac motor protein β, myosin in the failing and non-failing human heart, and (ii) tackles an important understudied problem of PTMs in the human β cardiac myosin protein.However,(b) flags up the point that much of the material might rather go to a more specialized journal. On discussion, reviewer 2 specifically requests clarification why the findings should be of interest to readers of the broader remit represented by eLife.Please see our comments for Reviewer 2, pt. c.(3) Reviewer (2):Considers that this is a thought-provoking study using an appealing strategy. Human samples increase the disease relevance and mass spectrometry here yields novel discoveries. Molecular dynamics modeling is used to speculate on the relative functional impact of each PTM. The novel PTM sites identified in this work are exciting and should stimulate and inspire other work in the field.eLife to submit our exciting study and why the manuscript (in its present form) should engage a broad audience. New sentences (in blue) were added throughout results and discussion to improve the flow of the manuscript after it was substantially shortened.We thank the editors and the expert reviewers for giving us the opportunity to further improve our manuscript. We have made significant changes in the manuscript by carefully following the reviewers’ suggestions and comments. In addition, we clarified the reasons why we chose (B) Specific alterations requested:1) Reviewer 2:a) There is a lot of information in this very long (too long in my opinion) right now, its utility is unknown. The PTMs are very rare on this molecule and there are not enough samples to have much power (even though I know how hard it is to get samples).We agree with the reviewer that our manuscript was very long. To address the reviewer’s concern, we have revised and shortened the manuscript substantially without affecting the quality of our study. The length of the results was cut by approximately 17% (orig. 9 pages and now 7 ½) and the discussion by 40% (orig. 9 pages and now 5 pages).We appreciate your comments and acknowledgement of the difficulty in obtaining samples. We also wish we could have obtained more samples, especially for the PTMs that had a large variation in values. These variations could result from several causes and more samples may have allowed us to realize additional significant results.b) The authors also make several statements about how MYH6 or α myosin is induced in human heart failure. To the best of my knowledge, the opposite is what happens in human heart failure.We thank the reviewer for pointing it out. We will concur that this is a misstatement. We regret the mistake, and the above statement has been removed from the text in the discussion.2) Reviewer 3:a) I found it very difficult to understand the way in which PTM abundance was presented. It was not clear what the overall percentage of PTM is for a given site, e.g. in a failing heart, what percentage of myosin K951 residues are acetylated? The first paragraph of the discussion states that "phosphorylation and acetylation PTMs are present on β-MHC in non-diseased human hearts in low abundance." Is it not possible to determine what fraction of a particular peptide is modified? Otherwise, why would the authors state that PTMs are "low abundance" (first paragraph of the discussion)? Furthermore, if this information has been obtained, why would it not be clearly represented in the manuscript? It seems as though the quantification of PTMs is shown only relative to IRP.The PTM abundance was reported as normalized to IRP  and not as the percentage of occupancy of a given site. Because the ionization efficiencies of acetylated/phosphorylated peptides are different from the unmodified peptides, we ended up not using the AUC (area under curve) data to calculate the percentage of occupancy. We could have done quantifications in this study but based upon the differences in ionization efficiencies these numerical determinations would not be highly accurate. Instead, we normalized the peptides with PTM to IRP so that we can compare the relative abundance (occurrence) of PTM across all samples. We modified the manuscript to further clarify that we reported the data in this manner. We found one confusing statement (on page 26 of the submitted manuscript) that referred to % occupancy and removed it because we are not able to accurately make these determinations.Please find b) Similarly, the PTM to IRP ratio is also confusing in that the stated range is 1-14. Does this mean that it is not possible for PTM abundance to be less than the IRP? Why not?Yes, the PTM abundance could be less than the IRP. We performed calculations of PTM relative quantification based on its ratio to IRP and the reported range of 1-14 is what we have observed. The ratio depends on the abundance of PTM relative to IRP.c). The authors mention that one way to interpret the loss of K951 acetylation in failing hearts is that there is a shift toward α-MHC. Perhaps it was outside the scope of the study, but was there no way of testing this hypothesis in some way?While this statement is accurate in context with the shifts that occur in mouse hearts which has a different dominant isoform, a-MHC (mouse) and b-MHC (human) the upregulation of a-MHC in human heart failure does not occur as stated. Therefore, we removed these several statements from the text regarding an isoform switch. A more likely scenario underlying the reduction of K951-Ac in failing hearts is upregulation of HDACs in ischemic and non-ischemic heart failure, which has been documented by a number of groups.d). The discussion is, in my view, far too long and wide-ranging. Some of the logic presented is also challenging. For instance, the PTMs discussed are shown to be more abundant in normal than in failing hearts. Yet, the authors draw parallels between the sites for the PTMs and the locations of cardiomyopathy-causing mutations. It seems strange to associate sites of "normal" PTMs with the locations of disease-causing mutations. Overall, the discussion is far longer than would normally be expected for the corresponding significant results, which really focused just on K951. My recommendation would be to focus the discussion drastically, state the most meaningful findings very clearly, and help the reader grasp the real significance of the work.We agree with the reviewer that our manuscript was very long. To address the reviewer’s concern, we have revised and shortened the manuscript substantially without affecting the quality of our study. The length of the results was cut by approximately 17% (orig. 9 pages and now 7 ½) and the discussion by 40% (orig. 9 pages and now 5 pages).Our inclusion of a Table of CM mutation was intended to demonstrate importance of altering interactions/function of these regions due to inclusion of a PTM at that site. It is intended to be merely correlative; we understand your concern about including this data for “normal” PTMs, but in our view the PTMs may represent a “gain-of-function” or promote enhanced contractility –We shortened but also rearranged the discussion so that it is now organized in a way that emphasized our major findings and highlighting the real significance of the work.Reviewer #2 (Recommendations for the authors):This paper tackles an important understudied problem of PTMs in the human β cardiac myosin protein. The authors found acetylation sites and phosphorylation sites and used molecular dynamics simulations to predict the impacts of these PTMs on myosin function.There is a lot of information in this very long (too long in my opinion) right now, its utility is unknown. The PTMs are very rare on this molecule and there are not enough samples to have much power (even though I know how hard it is to get samples).The authors also make several statements about how MYH6 or α myosin is induced in human heart failure. To the best of my knowledge, the opposite is what happens in human heart failure.I think this information may be better suited for a more specialized audience and journal.eLife.We appreciate your critique and the opportunity to improve our manuscript and make it more accessible to the general reader. We have thoughtfully and carefully considered each point that you have made. We believe that the manuscript is now greatly improved and suitable for publication in


Cancer services were seriously disrupted during the COVID-19 pandemic. Using publicly available data for England, we show that breast cancer diagnoses decreased substantially in the first year of the pandemic, but have recovered to at least pre-pandemic levels. Long term follow-up is needed to assess the impact on outcomes. The impact of the coronavirus disease 2019 (COVID-19) pandemic was significant with three ‘stay at home’ orders of varying durations issued in England in March 2020, November 2020 and January 2021 [Breast cancer in England is diagnosed through two main routes largely dependent on patient age. Patients with breast related symptoms and aged ≥16 years may be referred to secondary care breast symptomatic services either urgently, where cancer is suspected, or routinely, where cancer is not suspected . Women aWe reported previously on breast cancer diagnostic services during the first 6  of the pIn the early part of the COVID-19 pandemic, from March 2020, all breast cancer screening units in England paused routine screening invitations. Services were resumed from July 2020, and all breast screening units reported sending routine invitations by September 2020. Symptomatic services were not suspended, but there was a large decrease in referrals following the first lockdown in March 2020, which largely recovered by August 2020. Referral rates remained at least at pre-pandemic levels during the further lockdowns , 7.We report key measures of breast cancer diagnostic service activity, including the numbers of referrals, first treatments, number of screen-detected diagnoses, and the stage distribution of invasive breast cancer for the 3 financial years (April–March) of 2019/20, 2020/21 and 2021/22  was similar for the two years  [The first treatment counts are a surrogate measure of the number of new cases of invasive and non-invasive breast cancer from all routes to diagnosis, including the NHSBSP. In 2019/20, the year before the pandemic, the number of first treatments for breast cancer was 49,050. This number was 23% lower in 2020/21 and 2% higher in 2021/22. The proportion of cancers diagnosed in women aged 70+ years remained roughly a third throughout the pandemic. Among patients aged 50–69 years, there was a 26% fall in the number of new diagnoses in 2020/21 and an 8% increase in 2021/22, compared to 2019/20. This is likely to be related to the pause and later resumption of NHSBSP invitations, as 60% of patients diagnosed with breast cancer in this age group are diagnosed within the NHSBSP.Assuming 2019/20 was a ‘normal’ year for the number of breast cancer diagnoses, these data suggest that there may be ~10,300 “missing” women with breast cancer since the start of the pandemic. The NHSBSP data show 6879 fewer women were diagnosed through screening in 2020/21 compared to 2019/20 and the data for 2021/22 are awaited. This reduction in numbers of screen detected cancer may account for roughly two-thirds of these missing breast cancer diagnoses. It will remain to be seen how many of these women will be diagnosed with symptomatic breast cancer in the interval between screens, or whether they will be diagnosed following their next routine screening invitation.We show the stage distribution for invasive breast cancer for these three years of interest as reported in the RCRD, which contains proxy tumour registrations for invasive breast cancer only and is produced approximately 4 months after a diagnosis and has high proportions of missing stage data . In compA recent study has estimated that the pandemic related disruption to the NHSBSP may result in 148–687 additional breast cancer deaths but scale of the impact ultimately depends on the speed of recovery . Also ofCollectively, these data suggest that breast cancer diagnostic service activity has subsequently equalled or surpassed pre-pandemic activity levels which is reassuring. The observed annual increase in the proportion of patients with symptoms referred urgently will present capacity challenges to service providers. The impact of the COVID-19 pandemic on longer-term outcomes remains unclear.


The effects of operating conditions such as pressure, temperature and CO2 feed composition on separation performance were analyzed. The findings showed that the formulated membrane exhibited decreasing CO2 permeation trend with the increase in pressure. Conversely, the increase in operating temperature boosted the CO2 permeation. High productivity can be attained at higher operating temperatures with a reduction in product purity. Interestingly, since PSf has higher plasticization pressure, it was not affected by the change in CO2 percentage up to 70% CO2. The experimental study showed that the membrane material formulated in this study can be potentially evaluated at the field stage. Longer testing duration is needed with the real feed gas, appropriate pre-treatment based on the material limitations, and optimum operating conditions at the site to further confirm the membrane’s long-term lifetime, resistance, and stability.Membranes with a stable performance during the natural gas sweetening process application are highly demanded. This subject has been immensely explored due to several challenges faced by conventionally used polymeric membranes, especially the high tendency of plasticization and physical aging. In this study, polysulfone (PSf) hollow-fiber membrane was formulated and tested for its application in natural gas sweetening based on several compositions of CO CO2 is miscible with oil and can induce oil swelling hence reducing its viscosity. Many studies have shown that CO2 can be used for enhanced oil and gas recovery and geological storage through lab experiments, field tests, molecular and reservoir simulation [Gas separation is an attractive technology for a wide range of chemical processes. Gas separation membranes have been predicted to have a compound annual growth rate (CAGR) of 5.87% by 2027 . Asia Pamulation ,4.2 has attracted much commercial interest. However, the particular concern is to prevent and suppress the plasticization of this polymer [2 and heavy hydrocarbon.The membrane materials used for commercial application are mainly cellulose acetate, PSf, and polyimide. However, the classical polymeric membrane often suffers from plasticization, which leads to lower performance . The use polymer . Plastic polymer . Among t2/CH4 selectivity of cellulose acetate polymer often drops to less than 15 due to multiple phenomena such as aging, competitive sorption, and plasticization. This leads to marginally acceptable project economics due to the unnecessary loss of valuable methane gas [High operating pressure beyond the material plasticization pressure ,9,10,11 hane gas ,8.Plasticization makes the polymer swell, and this slight solvation can permanently damage the polymer structure. Inter-chain spacing becomes larger hence increasing the gas and vapor diffusion coefficients . Plastic2-philic groups into the polymer, enhancing polymer stiffness by including per substituted rings, incorporating bulky side groups to lessen the rotational ability, and eliminating any flexible linkages, are included [Membranologist are constantly looking for solutions to achieve an optimum gas permselective membrane while maintaining a good balance between selectivity and permeation . Currentincluded ,15,16. Aincluded  and enhaincluded . Graftinincluded , as wellincluded .2/CH4, they found that the membrane exhibited lower CO2 permeability compared to single gas testing due to competitive sorption, while CH4 permeability remained unchanged. The selectivity was reduced from 70 to 41 (40% reduction) when the 4 atm feed pressure increased to 40 atm for 6 FDA-mPDA (m-phenylene diamine). The other type of hydroxyl-functionalized polyimides, 6 FDA-DAR (diamino resorcinol) and 6 FDA-DAP (diaminophenol) also showed similar behavior with a reduction of 30% for the selectivity when the pressure was increased. The competition effect from other gases such as the one shown in this study by Alaslai, can only be observed when multi-component testing is performed [Alaslai fabricated polyimide membranes functionalized with hydroxyl groups . From therformed .2 and CH4 mixtures for four types of asymmetric membranes. It was determined that plasticizing Matrimid and cellulose acetate membranes result in a significant loss of selectivity [Visser performed some experimental studies using various COectivity . Falbo fectivity .2/CH4 solubility selectivity increased from 16 and was maintained at 17 for equimolar CO2 and CH4, while CO2/CH4 diffusivity selectivity was reduced from 2 to 1.5. This resulted in the overall CO2/CH4 perm-selectivity being reduced from 30.5 to 24.5. CO2-induced swelling caused a faster diffusion of CH4 in the mixed gas case. CTA (Cellulose Tri-Acetate) was found to be less susceptible to the detrimental effects of CO2-induced swelling, which caused a reduction of the diffusivity-selectivity since its solubility-selectivity is more dominant. CTA perm-selectivity also displayed a weaker dependence on the CO2 content in the mixture [Ricci studied the multi-component gas separation performance of cellulose acetates membrane. They found that the competitive sorption enhanced the solubility selectivity while overall perm-selectivity was reduced due to a reduction in diffusivity selectivity that was largely impacted. From 0 to 5 MPa pressure, the CO mixture .2 and H2O-induced free volume blocking was significantly greater, leading to fugacity-dependent permeabilities for CO2 and CH4 that were below those of the pure gas components. Relative to the simplified models, the new model predicts differences up to 2% and 18% in CH4 recovery at low feed flow rates, and the difference in CO2 removal can be as significant as 50% [Miandoab performed a simulation of multi-component gas, including water, in humidified versus dry feed conditions. The membrane model shows that a simplistic model that does not account for such complex effects can deviate more than 20% from a more rigorous approach. The impact of competitive sorption and plasticization by COt as 50% .4 mixed-gas solubility, that is, when partial pressures increase, the CH4 mixed-gas solubility coefficients diverge from the pure-gas values. Because the effect of competitive sorption on the solubility coefficient of CO2 is limited, they found that at 2 atm CO2 partial pressure, the CO2/CH4 solubility selectivity increased from ~5 in the pure-gas state to ~10 in the mixture [Genduso found that the permeability selectivity of the mixture strongly diverges from the pure-gas trend. At about 18 atm partial fugacity, almost 35% of the ideal permeability selectivity was lost, and it was assumed that competitive sorption was the cause for this loss of permeability selectivity. The data of mixed-gas solubilities at 50 mol% equilibrium concentration showed that competitive sorption strongly affects CH mixture .Operating conditions such as pressure, temperature, and flow rate can influence the resistance of the membrane during its application which will, in turn, impact the working lifetime of the membrane. The product purity and productivity are determined by various factors such as membrane properties, which are also helped by selecting the optimum operating conditions . The acc2 and CH4 separation using low pressure with single gas and simple binary gas. Multi-component gas mixture permeation properties were not widely studied due to the difficulty and risk of handling the operating parameters at a higher level and the need to analyze more than two components simultaneously [2 contaminant concentration may vary from field to field in the range of 15% to 70%. For less than 15% CO2, the absorption technology is usually applied.Most previous works reported were based on COaneously . Single aneously , and the2 composition  variation were investigated. The effect of operating conditions on the rubbery membrane can be compared and is referred to the study conducted by Sadrzadeh [The objective of this research was to study the performance of a formulated glassy PSf membrane when subjected to various operating conditions. The aim was to identify the optimum setting that will be used for its field application with stable operation. Experiments using pure and mixtures of five gases typically found in natural gas with various compositions were carried out. Effects of operating temperature (25 to 45 °C), pressure (20 to 50 barg), and COadrzadeh . The opeadrzadeh .U) change of this process is:The importance of operating temperature determination for the selected membrane material for gas separation application is related to its transport properties which are diffusion and solubility. One of the main vital phenomena that happens in gas separation membranes is the Joule Thompson effect. Theoretically, a gas flows through a membrane from the high-pressure side (subscript 1) to the low-pressure side (subscript 2), and it is assumed to happen adiabatically (no heat transfer (q = 0) and the whole system is isolated) and isenthalpic . Hence, the internal energy  of gas A. A0D, DE, T, and R are the pre-exponential factor, the activation energy of diffusion (in kJ/mol), the temperature (in K), and the universal gas constant, respectively. Here, the value of the R is 8.31 J/mol. The operating temperature and the gas diffusion coefficient relationship in polymeric membranes can be illustrated by the Arrhenius equation :(4)DA=DAEqually, the Van’t Hoff equation describes solubility as a function of temperature .(5)SA=SA0S and ΔSH are the pre-exponential factors and the enthalpy of the solution of gas A, respectively. The gas permeability can be stated as a function of temperature as follows:A0P and PE are the pre-exponential factor, and the activation energy of permeation, respectively.It is worth noting that as temperature rises, so do permeation and diffusion parameters, but the solubility relationship reverses .The hollow fiber membranes were prepared from a dope solution composed of PSf from Solvay Advanced Polymers, and a mixture of solvent and alcohol bought from Sigma-Aldrich  with appropriate composition. The name and composition of the chemicals used in this study are not mentioned in accordance with the company’s unpublished confidential information guideline. The polymer pellets were dried at 60 °C in a vacuum oven overnight before use, while solvents and non-solvent were used as received. FESEM  is used to study the resultant membrane morphology for the cross-sectional image. The sample is sputter-coated with an ultrathin conductive layer, namely titanium, at high vacuum evaporation to ensure the sample is electrically conductive. This pre-treatment could avoid the accretion of a static electric field at the sample induced by electron irradiation employed during the measurement process. The membrane samples are cut into square shape sheets of size 0.5 cm × 0.5 cm and attached to a sample stub using carbon tape. For cross-sectional morphology, the membranes are fractured in liquid nitrogen and placed on a sample stub using carbon tape. Scanning is conducted at a magnification between ×500 and ×10 k.The hollow fiber membrane module made from stainless steel (SS 316) is used in this study refer to . The mem2 permeance and selectivity. Mixed gas testing is later conducted with variations of feed pressure (20 to 50 barg), temperature variation (25 to 45 °C), and CO2 percentage variation  to determine the membrane performance close to real field environment conditions. Both pure and mixed gas measurements are determined using a permeation apparatus that allows the measurement of permeability and selectivity at various operating conditions. For the mixed gas testing, the feed, non-permeate and permeate stream gaseous composition are analyzed using gas chromatography. In this study, Micro GC Agilent 490  is used for the gas composition measurement. Mass flow controller is also needed for the mass balance calculation.The prepared hollow fibers are put in the bundle and potted into Swagelok stainless steel tubing. The leak test is performed to ensure the integrity of the fiber and module prepared. Pure gas testing is conducted at low pressure (3 to 7 barg) using the bubble test method to determine the membrane ideal and intrinsic CO2 permeance is calculated by using the following equations.CO3/s·cmHg·cm2)3/s)2)x, y = ComponentSelectivity is measured using the below equation.The operating parameters are selected based on the real application needs. Operating pressure of 40 barg is usually applied in commercial field applications, and 20 barg has also been tested in this study as the lowest possible operating pressure, although it is not quite practical for the natural gas sweetening process; 50 bar is provided to provide a comparison with 40 barg data, and 30 barg data can be interpolated from 20 barg and 40 barg data.This study focuses on mixed gas testing because the transport behavior of one component through the membrane is affected by the presence of other penetrants, which can be termed as coupling effect or competitive sorption. Permeation properties of multi-component gas mixtures are still lacking in the literature due to the complexity of conducting the testing, difficulty in controlling the operating parameters, and simultaneous analysis of multi-component . ComputaMixed gas compositions used in this study are listed in For the long-term performance study, the membrane is subjected to 40% mixed gas composition at 40 barg operating pressure and 45 °C operating temperature using a system that can recycle the feed gas.Relative numbers are the value which is relative to the reference value. The relative change is used in this paper basically to compare the trends. This comparison is generally expressed as the ratio and this number does not have any units. The actual membrane performance in this study is not mentioned in accordance with the company’s unpublished confidential information guideline.The integrally skinned asymmetric membrane with a spongy structure and thin dense skin layer is observed in membrane cross-section images, as shown in 2 permeance and CO2/CH4 selectivity have been studied based on several important parameters, as discussed in the following sections.The membrane performances in terms of CO2 plasticization pressure [2 exhibits properties and behaviors between liquid and gas, where the density is liquid-like while the diffusivity, surface tension, and viscosity are gas-like. Supercritical CO2 is considered an organic solvent with remarkable solvation power, which should be avoided when used with polymeric membranes.Feed pressure provides the key driving force for the gas separation membrane . The detpressure . Too hig2 permeance with respect to the CO2 composition and feed pressure changes has been investigated at a fixed operating temperature of 45 °C. As the driving force is increased from 20 to 50 barg, it can be seen in 2 permeance reduced significantly for all the trends despite the variation in CO2%. This can be explained by several phenomena that took place in the membrane during the separation process, namely competitive sorption, pore blocking, site saturation, aging, and compaction, which greatly affect the membrane performance. These phenomena have overcome the higher-pressure driving force that is supposed to enhance the permeation. There are many mechanisms leading to free volume loss, and numerous theories have been invented to define the aging process in terms of free volume reduction [2 plasticization is more dominant than aging and compaction, the CO2 permeation trend should increase as the pressure increases, as is typical for cellulose-based membranes. This is because plasticization will swell the membrane, while aging and compaction will make the membrane denser. These two effects counterbalance each other, but for the cellulose case, which has lower plasticization pressure, the plasticization effect is greater than aging and compaction, as can be seen in their increasing permeability trends during the testing or operation.In this experimental analysis, the impact of COeduction . This ha2 permeation. The permeability coefficient of gases at higher feed pressure usually does not obey the dual mode sorption model due to plasticization phenomena happening in the polymer [However, in this case, since PSf has high plasticization pressure, aging and compaction are more dominant, leading to the decreasing trend of CO polymer .2 permeance with pressure increment is subject to the polymer material. Ismail and Lorna [2 plasticization pressure. An example of a type 2 polymer is polyimide which follows dual mode sorption behavior. Type 3 polymers such as cellulose acetate and polystyrene experience an increasing permeability trend from the beginning of the testing as they have low CO2 plasticization pressure and behave as rubbery permeation behavior with low selectivity [2 percentage, increasing the pressure from 20 to 50 barg, increased the CO2 partial pressure and the CO2 solubility of the membrane material. Furthermore, the CO2 plasticization effect, dilates the membrane, which also counterbalances the shrinkage effect from aging, compaction, and site saturation. This, in turn, increases the permeability when the dilation is more than the shrinkage. In this case, it will be advantageous for Type 2 and Type 3 membrane materials if the selectivity is still within an acceptable level and does not drop significantly. The increasing trend of CO2 permeation is helped by the solubility coefficient that is increased with the increment of pressure. In this condition, membrane densification happens and hinders diffusion, but solubility can still happen through the polymer matrix. As for the PSf, it has higher CO2 plasticization pressure, thus, it follows the Type 1 trend when the pressure is increased, which explains the trend in The trend for COnd Lorna  explaineectivity . At the 2% variation, as displayed in 2 permeance due to compaction and aging and reduction of selectivity for PSf membrane that has diffusivity as the transport dominant factor.The same trend for selectivity is observed when the pressure is increased from 20 to 50 barg, where the trends show a decreasing trend for all COFeed temperature is one of the most significant parameters for membrane operation as it affects the transport properties. The impact is more prominent for diffusivity-dominan polymers, where diffusion increases with rising temperature, whereas for solubility-dominating membranes, the effect is reversed, with increasing temperature lowering solubility. As mentioned by Duthie diffusion at high temperatures is mostly contributed by Henry’s law species and plasticization effects are conquered by Henry’s law dissolution . At lowe2 removal and liquid are allowed to form during operation. The liquid is formed when condensation happens in the gas separation membrane during the separation of CO2 from natural gas. There are two effects that will result in condensation. Firstly, the bulk CO2 removal that permeates faster than the heavy hydrocarbon makes the gas heavier, and the dew point increases towards the operating temperature. Secondly, the gas cools down due to Joule Thompson (JT) effect as it passes through the membrane. The change in fluid temperature happens upon expansion (pressure decrease), causing a significant change to the temperature between the feed gas, retentate gas, and the permeate gas. The drastic temperature change results in liquid formation and greatly affects the permeation properties in which the permeance is reduced by pore blocking. On the other hand, the selectivity is enhanced due to the hydrocarbon permeation is also blocked by the presence of liquid. In many cases, polymers are contacted with liquids that penetrate their structure and plasticize it [Operating temperature also considerably impacts the robustness of the membrane if the setting is below the feed gas dewpoint after the COicize it , and in Experimental studies have been performed to explore the impact of operating temperature changes on the formulated membrane performance. By doing this investigation, the optimum temperature can be set to obtain the best performance while avoiding liquid formation by providing sufficient superheating. In this experiment, condensation cannot be simulated, and it requires heavier gases in the feed gas to demonstrate the liquid formation usually found in the field.2 permeance for all cases of CO2 percentage following the Arrhenius equation [Based on equation ,34. This2 permeance with a certain limit to control the other effect, such as aging. Further investigation is needed for the long-term effect under continuous exposure of the polymer to higher pressure and temperature. At higher operating temperatures, perm selectivity is shifted to a higher permeability value [At high temperatures, the polymer matrix is also hypothesized to increase segmental mobility resulting in easier penetration of the fast gas through the thin skin layer of an asymmetric membrane. This shows that a higher temperature can be set as this membrane operating temperature to gain the benefit of higher COty value . The besty value .Condensation can be prevented for membrane materials that are sensitive to liquid hydrocarbon by heating the feed gas and inter-step membrane refer to  to the o2% variations, as shown in 2 and CH4, as the temperature is increased. The selectivity is reduced because the CH4 gas also has higher activation energy at higher temperatures, which increases permeation. The competition impact between CH4 and CO2 has resulted in a declining trend in selectivity value as the temperature increases [2 permeability and selectivity that needs to be considered when applying high-temperature application. If the selectivity value at high-temperature operation is still at an acceptable limit, then the setting can be used for the continuous membrane operation application.When the temperature is increased from 25 to 45 °C, the selectivity decreases for all the COncreases . Low-tem2 permeance trend is quite stable for the PSf membrane formulated in this study. This happens even though the CO2 partial pressure is increased with the increment of CO2 composition [2 concentration at the same operating pressure leads to higher CO2 partial pressure, which is supposed to boost CO2 permeance to a certain extent. Plasticization is expected to happen following the CO2 concentration level as CO2 is a strong plasticizer agent. Using a polyimide membrane, Duthie found that as the concentration of CO2 is boosted to a higher value, the polymer swells due to polymer relaxation, leading to an increase in the diffusion coefficient [2 gas, as well as a stable trend as the CO2% increased.Based on position  from 15%fficient . However2 composition, the effect of plasticization will become more severe. However, since the material used in this study is PSf which is known to have very high plasticization pressure [2, resulting in 28 barg of CO2 partial pressure. This partial pressure is still below the plasticization pressure of PSf, which is around 34 barg, making the trend looks stable for both permeability and selectivity as the CO2 increased from 15% to 70%. Under this condition, most membrane materials would have experienced certain degrees of plasticization.According to Fick’s Law, diffusion increases as the concentration gradient increases, and as per Henry’s law, the solubility increases as the partial pressure increases. It is noted that at higher COpressure , the hig2 partial pressure is increased with the increment of CO2 composition [Based on position  from 15%A long-term performance study is performed to investigate the stability of the membrane performance over time. It has been well-established that thin films aged faster than thick films . As plas2 partial pressure for the asymmetric hollow-fiber membranes affect the long-term performance of the membrane as compared to steady and constant CO2 exposure which will yield more stable membrane performance [Based on formance . This ca2S, heavy hydrocarbon [As shown in rocarbon , aromati2 and made the membrane performance drop [Heavy hydrocarbon contamination, even at low concentrations, is hypothesized to be the reason for some membrane failures and loss in performance. According to the dual mode sorption theory, highly condensable feed components, such as toluene, will contribute to severe competition impact due to their size and based on its critical temperature, it can be easily condensed at the operating pressure applied. Toluene can block permeation opportunities of COnce drop ,44,45,46nce drop . Heavy h2 permeation as pressure rises due to gas diffusivity being blocked. Increasing the operating temperature can help to boost CO2 diffusion and permeation. High productivity can be attained at higher operating temperatures with a reduction in product purity. At higher partial pressure conditions, when the CO2 percentage is increased, the CO2 productivity and product purity remain stable due to the rigidity and integrity of the membrane produced. Interestingly, since PSf has higher plasticization pressure, it is unaffected by changes in CO2 percentage up to 70% CO2. The present study shows that the membrane material formulated has the potential for further evaluation at the field stage. Longer testing duration at the optimum operating conditions on site is needed with the real feed gas, which includes heavy hydrocarbon and aromatic components, to confirm the actual membrane lifetime, robustness, durability, reliability, and stability.Commonly, the permeation behavior of gases through membranes is related mainly to the properties of gases and membranes along with the operating conditions . Based o


We observed that the diffraction spots at resolutions between 3 Å and 20 Å fade more slowly at liquid-helium temperatures compared to liquid-nitrogen temperatures, by a factor of between 1.2 and 1.8, depending on the specimens. If the reduction in the effective rate of radiation damage for 2D crystals at liquid-helium temperature (as measured by spot fading) can be shown to extend to macromolecular assemblies embedded in amorphous ice, this would suggest that valuable improvements to electron cryomicroscopy (cryoEM) of biological specimens could be made by reducing the temperature of the specimens under irradiation below what is obtainable using standard liquid-nitrogen cryostats.We have studied the fading of electron diffraction spots from two-dimensional (2D) crystals of paraffin (C •The fading of electron diffraction spots from 2D crystals is measured at 4–100K.•Specimens studied are paraffin, purple membrane and aquaporin 4.•Spots fade 1.2–1.8•CryoEM might be improved at temperatures below those of liquid nitrogen. The consensus conclusion was that “specimens last approximately 3 to 5 times as long when the specimen is cooled to about 150K” compared with room temperature. However, they also concluded that “we have been unable to find definite evidence that there is an improvement in radiation resistance on going from liquid-nitrogen to liquid-helium cooling”. A later review There has been a long history of publications reporting studies of the consequences of radiation damage to organic and biological molecules by observing the fading of diffraction spots from crystals. Early electron diffraction work The progressive reduction in data quality with increased electron dose is usually described using a Gaussian approximation akin to the Debye–Waller equation for thermal vibration of atoms in a lattice. Using this approximation, the decay in the intensity Alongside previous work on radiation damage using electron diffraction, there has also been a considerable body of work exploring radiation damage by X-ray diffraction of three-dimensional (3D) crystals of proteins and other macromolecular assemblies The specific purpose of this work is to make a more accurate comparison of radiation damage at temperatures between 4K and 100K by measuring spot fading in electron diffraction patterns using two different microscopes that could be operated interchangeably at liquid-helium and liquid-nitrogen temperatures. Here, we again use paraffin and purple membrane as test specimens but also include 2D crystals of aquaporin (AQP4). By using these same three specimens under identical illumination conditions in the same microscopes, and with the same detectors and the same analysis, we now show that there is indeed a useful reduction in the rate of diffraction spot fading at temperatures lower than that of liquid nitrogen. If this reduction in radiation damage can be translated to imaging vitreous specimens in cryoEM, it would have wide ranging implications: the amount of information obtainable from a given specimen before the onset of radiation damage is increased, and thus smaller particles can be detected and imaged in a specimen of a given thickness 22.1Since the objective of this work was to measure rigorously the relative rates of spot fading and to demonstrate reproducibility between microscopes and institutions, specimens were prepared independently in two countries — in the UK  and at two locations in Japan .44H90 paraffin were made by applying 244H90 in hexane (Supelco Inc.) to a film of amorphous carbon that had previously been floated off mica on to 400 mesh copper EM grids, and allowed to dry in air. Crystals of C44H90 paraffin one molecule thick formed as described in In Cambridge, specimens of CIn Nagoya/Tokyo, the method of specimen preparation for purple membrane and aquaporin was slightly different. Briefly, a film of amorphous carbon was evaporated on freshly cleaved mica, using a sufficiently low current to prevent any sparking. These conditions gave smooth and stable carbon films. The mica sheets were cut into roughly 4 mm square pieces, and the film was floated off onto distilled water and picked up with a molybdenum grid. The distilled water was exchanged with crystallisation buffer  electron microscope equipped with a liquid-helium stage Each pair of liquid helium/nitrogen measurements was done with crystals on the same grid using the same microscope and the same detector, during the same data collection session with constant electron irradiation conditions for the session. Only the temperature was changed between measurements.2.344H90 paraffin, the centres of the 5 or 6 individual spots with indices ,  and  and their corresponding Friedel mates were used to integrate the background-corrected diffracted intensity for each spot on each frame as the diffraction spots faded, using the display program Ximdisp For C344H90 paraffin is shown in Examples of diffraction patterns from the first exposure of the three different specimens, before significant radiation damage had occurred, are shown in Since the cell dimensions for purple membrane  using a gas introduction system in situ form of temperature measurement incorporated directly in the specimen or perhaps the specimen support, that was accurate in range of temperatures from 1–100K, would be highly desirable.We demonstrate a significant and reliable increase in the lifetime of diffraction spots from 2D crystals of organic or biological specimens, as their temperature is lowered from near liquid-nitrogen temperature to near liquid-helium temperature. This increase was reproduced in two different laboratories using two different electron cryomicroscopes, three specimens, and different specimen preparation methods. The measurements carried out in Cambridge  show a narrower distribution (ranging from 1.1 to 1.5) of the improvement in lifetime with the Polara liquid-helium cryostat reaching a temperature of 12 to 15K. The measurements carried out in Nagoya show a wider distribution of improved lifetimes, from 1.1 up to 2.0 . Since each liquid helium/nitrogen comparison is done with crystals on the same grid using the same microscope and the same detector, one explanation for the broader spread in radiation protection factors measured on the microscope in Nagoya is variance in specimen temperatures. The temperature of the specimen, reached with liquid-helium cooling, is not necessarily equal to that of the microscope stage (4K in Nagoya and 12–15K in Cambridge), due to the low thermal conductivity of most materials in this range. It is possible that for the microscope in Nagoya, the specimen temperature varied over a wider range (from 8 to 20K) and included temperatures lower than the 12–15K in Cambridge. The temperature range of 8K to 20K was estimated from previous observations of diffraction patterns from solid Ne and N4.2We suggest that the increased lifetime as the temperature is lowered is likely to be due to the reduced mobility of the small molecular weight radiation products ) for spot-fading in 2D protein crystals at liquid-nitrogen temperature and the 5.0 2) slope of plots of We have established that the structure of molecules of paraffin, bacteriorhodopsin and aquaporin in 2D crystals is preserved for longer in every case during irradiation at liquid-helium temperatures compared to liquid-nitrogen temperatures. None of these three specimens were embedded in frozen water or amorphous ice. The paraffin crystals consisted of dry straight-chain saturated hydrocarbons, whereas purple membranes and aquaporin 2D crystals were embedded in frozen, glassy glucose or trehalose. We cannot extrapolate directly from these observations to what will happen in frozen water. Further measurements of radiation damage are needed, especially for single particles embedded in amorphous ice. Nevertheless, we are optimistic that a similar improvement in radiation damage rate will carry over to specimens in vitrified water because the underlying mechanisms of radiation damage – ionisation, bond breaking, and diffusion of radiolysis products – are common to both. There is good agreement between the magnitude of the increase in The impact of cooling from liquid-nitrogen to liquid-helium temperature on images, rather than diffraction patterns, of biological specimens is more complex. At lower temperatures some gases, such as oxygen, nitrogen and argon condense on all cold surfaces, including the specimen. Cryo-shielding, which is already required in cryomicroscopes to prevent condensation of water vapour onto the specimen at liquid-nitrogen temperature, can also protect the specimen from the condensation of any other gases at lower temperatures, but may need to be improved. Semi-conducting specimen support films, such as carbon, become more insulating as their temperature approaches 0K. Their electrostatic charging during irradiation with electrons can degrade image quality. Noble metal supports, such as gold, do not suffer from this problem. Their conductivity increases with lower temperature, meaning electrostatic charging of the specimen, prepared on all-gold supports, during imaging at liquid-helium temperature is not a significant concern. Motion of the specimen during irradiation, in the absence of stage drift, has mainly indirect effects  on the diffraction pattern which records only the intensity of the diffraction spots. In contrast, beam-induced specimen motion causes blurring of electron micrographs, and in the worst case, can cause a complete loss of all high-resolution features in an image. Previous investigations of the effects of radiation damage and beam-induced motion have proposed tradeoffs to maximise the quality of the processed images. Bammes et al. There remains some uncertainty in the properties of amorphous water during electron irradiation at liquid-helium temperature, which can affect specimen movement at these lower temperatures. Wright et al. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.


Early glottic cancer can be effectively treated with radiation or surgery, but recurrence is a possibility when using any of the treatment modalities.To assess the outcome of radiotherapy as initial treatment in the control of squamous cell carcinoma of vocal cord (T1) and the effectiveness of salvage surgery (endoscopic or open) after treatment failure.A retrospective study was based on the analysis of medical records from 43 patients with T1 squamous cell carcinoma of the glottis, radiotherapy as initial treatment and follow-up period of five years.The rate of recurrence after radiotherapy was 30.2% of the cases, mean diagnosis interval was 29.5 months. As an option for salvage treatment, patients underwent open partial laryngectomy or endoscopic surgery with control rates of 77.7% and 25% respectively.Our cases showed high rates of recurrence after radiotherapy and open partial laryngectomy was more effective for the salvage surgery. Malignant laryngeal neoplasm is one of the most frequent diseases of the head and neck, and in 90% of the cases it is represented by the epidermoid carcinoma and its genesis is directly associated with alcohol drinking and smoking.It more frequently affects the glottis - 64% of the cases, and any epithelial disarray or change caused by the tumor will change the vibratory movements of the vocal folds, causing dysphonia. Of all the glottis tumors, 56% are diagnosed in their initial stages (T1)The primary glottis cancer can be effectively treated with radiotherapy or surgery (endoscopic or open) and it bears some of the best cure rates among the malignant neoplasms of the aerodigestive tract. In the series published by Davis et al.Treatment must not be restricted only to curing the cancer, which is the main objective, but one must also attempt to preserve breathing, swallowing and phonation.To assess the results of radiotherapy as the initial treatment mode in controlling the epidermoid carcinoma of the vocal fold (T1) and the efficacy of surgical salvage (endoscopic or open) after initial treatment failure.The present study was approved by the Ethics in Research Committee of the institution to which this study is associated, under registration number 0353/10.In this series we retrospectively assessed the charts from 43 patients with T1 stage glottis epidermoid carcinoma (42 men and 1 woman), submitted to radiotherapy as initial treatment, who were first seen by a doctor between 1990 and 2002.All the patients were sorted according to the TNM-UICC 2002 classification, after indirect laryngoscopy obtained by tele-laryngoscopy and/or nasal fibroscopic laryngoscopy (saved in video) and diagnostic confirmation of the epidermoid carcinoma through biopsy/frozen section.We took off the sample those patients who had been previously treated in other services, those with tumors bearing other clinical stages and those with tumors of histological lineage other than the epidermoid carcinoma.•Patient clinically ineligible for surgery (n=12/28%)•Patient's wish (n=31/72%)The routine in our service is to present all treatment options (radiotherapy or surgery) to patients with T1 glottic carcinoma, explaining the respective pros and cons; nonetheless, surgery is our treatment of choice for these cases. Radiation treatment was based on external beam at the dose of 6,000 to 6,800 rad in 30–34 fractions, indicated according to two basic criteria:•Group A - T1a: Tumor not involving the anterior commissure (n=22).•Group B - T1b: Tumor involving the anterior commissure with or without invasion of the contralateral fold anterior third (n=21).In order to analyze the results obtained by radiotherapy, the patients were grouped according to primary tumor localization on the vocal folds as follows:After radiotherapy, all the patients were followed up for a period of five years or more, and in the first six months they returned monthly to the clinic. For clinical control we did indirect laryngoscopy with the goal of identifying any suspicious lesions early on. After tumor recurrence diagnosis, the situation was recorded in the patient's chart and the patient was referred to salvage treatment as soon as possible, which first option was partial open laryngectomy  in the cases with or without commissure involvement. Patients with T1a tumors upon recurrence who were clinically unfit were submitted to an endoscopic tumor resection (transmuscular cordectomy), since it is an approach with less morbidity than open surgery.The patients submitted to radiotherapy (N=43) to treat initial glottic cancer had local recurrence in 30.2% (N=13) of the cases. Everyone who had recurrences was male with an average age of 58.9 years. The mean time between radiotherapy onset and local recurrence diagnosis was 29.5 months. The most frequent histology type was the moderately differentiated epidermoid carcinoma (76.9%), followed by the well differentiated type (23.1%).As to primary tumor location, initial radiotherapy treatment had the following recurrences: In Group A, where the initial lesion did not involve the anterior commissure, there was recurrence in 31.8% (n=7) of the cases ; in GrouAs soon as the failure in radiotherapy was confirmed, the patients were once again staged, being similar to the staging before radiotherapy with no loco regional metastasis. Four patients were referred to salvage endoscopic surgery (transmuscular cordectomy), because they were clinically unfit. Nine patients were submitted to open surgery  without neck dissection .Table 3LOf the recurrence cases treated endoscopically (n=4), only one was cured, the remaining patients (n=3) were referred to total laryngectomy because of a new recurrence. In this group, one patient died of cancer.In the cases submitted to open surgery, the cure rate was higher, and 77.7% (n=7) patients benefited from surgery. 22.2% (n=2) of the patients had new treatment failure and were referred to total laryngectomy. In this group we also had one patient dying of cancer.The treatment of laryngeal cancer must always associate oncologic efficiency with function preservation. Traditionally, initial glottic carcinomas (T1) can be effectively treated through surgery or radiotherapy, with high local control and survival ratesAccording to Bron et al.There is a current trend towards performing more conservative surgical procedures in cases of tumor recurrence after radiotherapy, instead of doing a total laryngectomy. In a study carried out by Schwaab et al.Among partial laryngectomies, the endoscopic approach in cases of post-radiotherapy recurrence must be avoided, having in mind the low rate of loco regional disease control, besides missing on an opportunity to do an open surgery with partial laryngeal preservation, as shown in the present study. This is explained by the difficulty in establishing lesion margins in an accurately way in a previously irradiated larynx, associated with the limitation of achieving broad margins.In our cases, initial glottic tumors (T1) treated by radiotherapy had high rates of recurrence. This can be a treatment option, but the patient must be aware that higher cure rates can be achieved through surgery.Open partial laryngectomy without neck dissection is an efficient option to treat glottic cancer after radiotherapy failure in patients who remain in stages T1a or T1b, bearing satisfactory cancer control without the mutilation of a total laryngectomy.


Developing better treatments that work for the majority of patients with brain metastasis (BM) is highly necessary. Complementarily, avoiding those therapeutic procedures that will not benefit a specific patient is also very relevant. In general, existing therapies for patients with BM could be improved in terms of molecular stratification and therapeutic efficacy. By questioning the benefit of whole brain radiotherapy as provided nowadays and the lack of biomarkers detecting radioresistance, we identified S100A9 and receptor for advanced glycation end-products (RAGE) as a liquid biopsy biomarker and a potential target for a radiosensitizer, respectively. Both of them are being clinically tested as part of the first comprehensive molecular strategy to personalized radiotherapy in BM. •S100A9 binding to RAGE in cancer cells activates radioresistance in BM.•S100A9 is a biomarker detectable in liquid biopsy.•RAGE inhibition radiosensitizes BMs.•A prospective multicentric observational study is evaluating S100A9 to stratify patients for radiotherapy.•A multicentric phase I/II clinical trial will test azeliragon in combination with radiotherapy in brain tumors. The microenvironment initiates this phenotype by responding to the presence of colonizing metastatic cells with the production of cytokines that induce S100A9 expression in the cancer cells. S100A9 produced by metastatic cells in situ is secreted to the extracellular space. Irradiation of metastatic cells in the brain through a yet-to-be-discovered mechanism increases receptor for advanced glycation end-products (RAGE) expression in the cancer cells. Given that the binding of S100A9 to RAGE in the cancer cell is the trigger to the activation of nuclear factor kappa B (NF-κB) signaling and JunB, which underlies the resistance mechanism to radiotherapy, this therapeutic approach paradoxically desensitizes metastatic cells to it. Although NF-κB has been reported as a general mechanism of therapeutic resistance,S100A9 among several cancer cell clusters, a specific one characterized by its stem-like properties showed the highest levels of expression. Such connection between S100A9, cancer stem cells and therapeutic resistance might involve an underlying plasticity highly influenced by the microenvironment.The contribution of the microenvironment to brain metastasis (BM) has been broadly recognized. We recently demonstrated its contribution to the induction of radioresistance in BM.Our previous study has demonstrated how S100A9 plays a central role in the radioresistance of metastatic cells in the brain.As promising as these findings are, limitations still exist such as the possibility that metastatic lines used in the experiments may not be representative of the behavior of many of the tumors we may encounter in the clinical setting. Indeed, none of the melanoma experimental models tested reproduced S100A9 levels seen in a subgroup of patients with melanoma BM. However, the possibility of using PDOCs is a good alternative to circumvent this issue as they precisely mimic not only human cancer cells directly derived from the patient but also many of the conditions that the tumor encounters in the living brain. However, they also have obvious limitations such as their limited life span or their isolation from systemic circulation, which limit the sustained access of immune components. In addition, PDOCs are obtained from selected patients with good enough prognosis to be candidates for surgical treatment but at the same time present lesions deemed unsuitable for systemic treatment or isolated radiotherapy, which could also incorporate biases. Finally, the study showing the association of S100A9 levels in peripheral blood with the local control of the disease was retrospective and could not be controlled for other clinically relevant variables.Given the secretory nature of S100A9 and the possibility of its detection in peripheral blood, we postulated it as a potential biomarker of radioresistance with significant predictive value for response and therapeutic potential given the existence of drugs blocking the downstream pathway. Thus, it is necessary to run a prospective clinical study to confirm its correlation with resistance to radiotherapy while, as far as possible, controlling for other factors that may condition the response of metastatic disease in the central nervous system or systemically.Consequently, we have designed a prospective observational study in patients with BMs from lung cancer, breast cancer and melanoma treated with radiotherapy . This stThe prospective study will analyze the presence of S100A9 in a blood sample taken at the start of radiotherapy and will be correlated with the response to treatment. Patients will be stratified depending on whether they receive holocranial radiotherapy [whole brain radiotherapy (WBRT)] or other focal radiotherapy modalities. To control for variables that might influence the levels of the biomarker, additional information such as the number of metastases, their size and whether the lesion is surgically resected will be collected. Additional variables that will be registered are relevant molecular alterations, the presence of extracranial metastases, the status of the primary tumor and the age and functional status of the patient. Our first objective is to analyze the correlation of the biomarker with local disease control and will be measured by progression-free survival in the central nervous system. The secondary objective is overall survival  are expressed and secreted by different tumor cells. These ligands interact in an autocrine manner to activate cancer cells, stimulating metastasis, proliferation, invasion and resistance to treatments.Also, from a neuro-inflammatory point of view, interleukin 6 (IL-6) has been recently identified as a pro-metastatic factor in a preclinical model of non-small-cell lung cancer BM by inducing anti-inflammatory microglia signaling via the Janus kinase 2/signal transducer and activator of transcription 3 pathway.Although the role of RAGE in BMs has been studied in more depth, it may also be important in glioblastomas. In fact, HMGB1, released from necrotic glioma cells, has been implicated in glioma formation, invasion and progression through mitogen-activated protein kinase/extracellular signal-regulated kinase pathway activation.With this meaningful rationale, RAGE inhibitors are in development in different diseases as summarized in There are currently no ongoing clinical trials with these drugs in cancer, but comparative studies have already been carried out in Alzheimer’s disease and cardiovascular diseases. Although the studies in Alzheimer’s disease were negative and in cardiovascular pathology the results are not definitive, important conclusions have been drawn, such as that these drugs are safe, with few reported side-effects, and that they do have biological effects, as we will see below.Azeliragon phenyl]imidazol-4-yl]phenoxy]-N,N-diethylpropan-1-amine) is an orally administered small molecule that crosses the blood–brain barrier and binds with multiple ligands, such as β-amyloid, HMGB1, S100B and advanced glycation end-products (AGEs).Similar inhibitors tested include alagebrium (ALT7-11), which is an AGE cross-link breaker that reduced the atherosclerotic plaque formation in humans.•in situ offers novel vulnerabilities due to the influence of the microenvironment.Study of experimental BM •S100A9 was validated in a retrospective study as a biomarker of resistance to WBRT compatible with liquid biopsy.•A multicentric observational clinical study is ongoing within the Spanish National Network of Brain Metastasis (RENACER) to evaluate the predictive value of the biomarker.•Blockade of RAGE prevents S100A9 induction of radioresistance in multiple experimental models of BMs sensitizing the tumor but without detectable toxicity.•A phase II multicentric clinical trial to use RAGE inhibitor azeliragon will test safety when combined with radiotherapy in glioblastoma.•Upon completion of the clinical trial, additional trials are planned including BMs.None declared.The authors have declared no conflicts of interest.


Conclusions: In a real-world setting, in patients with non-active SPMS, the maintaining or switching to the licensed interferon beta 1b did not reduce the risk of reaching confirmed EDSS 7.0.Background: No consensus exists on the possibility to stop disease modifying therapies (DMTs) in Secondary Progressive Multiple Sclerosis (SPMS). Methods: The primary outcome was the time to reach 24-weeks confirmed Expanded Disability Status Scale (EDSS) 7.0. We enrolled all patients with a confirmed diagnosis of non-active SPMS  between 1 January 2010 and 31 December 2015, at MS centers of Catania and Foggia, Italy. Patients were divided into two groups, according to the shared decision to stop DMTs (group A) or to maintain/switch to licensed interferon beta 1b for SPMS (group B). A Cox model adjusted with an inverse probability weighted propensity score (IPTW-PS) was employed. Results: A cohort of 311 patients was enrolled, 165 were in group A and 146 were in group B. Patients in the two groups were similar for baseline characteristics. The IPTW-PS adjusted Cox model for the event time to 24-weeks confirmed EDSS 7.0 did not show differences between the two groups (ExpB 0.96, CI 0.739–1.271,  In recent years, we have witnessed impressive changes in the therapeutic armamentarium of Multiple Sclerosis (MS), but almost confined to relapsing remitting MS (RRMS) .A better understanding of the pathological mechanisms that drive neurodegeneration in individuals with MS is needed to develop therapies that will effectively treat patients in the primary and secondary progressive (SPMS) stages of the disease ,3.In early MS, the inflammatory demyelinating process triggers a cascade of events that lead to neurodegeneration and are amplified by pathogenic mechanisms related to brain ageing and accumulated disease burden ,4,5.The therapeutic possibilities for SPMS are still scarce even if great expectations are placed on new drugs ,7 but evNo universal guidelines exist about the ability to maintain or withdraw disease modifying therapies (DMTs) in patients with SPMS, and in a real-world setting, the plan of therapy is shared case by case among the patients and treating neurologists .In RRMS, extensive axonal loss occurs in acute lesions, but the clinical deficits of these inflammatory events are mostly reversible as the human brain has a remarkable ability to compensate for neuronal loss.With time and ongoing inflammatory activity, axonal loss can drive the conversion of RRMS to SPMS when the brain exhausts its capacity to compensate for further neuronal loss.However, entering into SP phase represents an insidious change. Indeed, the disability accrual becomes irreversible, and the ability to recover from clinical relapses begins to be frequently ineffective .Then inherent difficulty in diagnosing transition is combined with the difficult meaning for patients and physicians further challenging the communication.Furthermore, few data are available about the course of long-term disease on patients who maintained or switched to the licensed interferon beta 1b .Some evidence seems to suggest that the maintenance of DMTs in small cohorts of SPMS with residual clinical and radiological disease activity could help in delaying severe disability milestones, but no consensus exists, and no insights have been provided about the role of therapy in patients who did not show clinical and radiological activity,  ,12.We retrospectively investigated whether the withdrawal or maintenance/switch to licensed nterferon beta 1b could influence the risk of reaching severe disability milestones in a large cohort of patients with non-active SPMS for at least 24 months before their conversion to the progressive course.In this retrospective study, we collected data from patients at the MS centres of Catania and Foggia, Italy, who received diagnosis of SPMS from 1 January 2010 to 31 December 2015. The inclusion criteria were: (1) to be patients in the SP course according to criteria from Lorscheider et al. ; (2) havPatients were also required to have a minimum of 24 months between MS diagnosis and conversion to SPMS and 12 or more months of prospective follow-up after SPMS conversion.Patients with insufficient baseline clinical and radiological data or insufficient data quality were excluded.In our study, each patient who moved to SPMS was offered the possibility, according to Italian medicine agency rules, to be treated with interferon beta 1b, which is licensed for the treatment of SPMS.The decision to stop DMTs in the cohort of non-active SPMS patients was reached after a consultation between the treating neurologists and the patient about the risks and benefits of such a decision. Psychological support was also offered.Patients were divided into two groups according to the shared decision to withdraw DMTs (group A) or to be treated with licensed interferon beta 1b  (group B) (maintain/switching to).The study protocol was approved by the local ethics committee (Comitato Etico Catania 1) of the coordinating centre , and patients provided written informed consent. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki and with the appropriate national regulations.A retrospective analysis of longitudinally collected data was performed. ®  on 30 September 2020.Demographical, clinical and radiological data were extracted from the electronical medical records on Imedhttps://www.neurostatus.net/, accessed on 1 January 2022).Disability level was assessed by Expanded Disability Status Scale (EDSS) and recorded by trained neurologists with an online Neurostatus certification  for categorical variables, the mean standard deviation (S.D.), or median with an interquartile range (IQR) for continuous variables. A parametric or nonparametric test was employed according to results obtained from the Kolmogorov test.A survival analysis with univariate Kaplan Meier and multivariate Cox proportional were built for the event time to 24 weeks confirmed EDSS 7.0. To consider the imbalance of the 2 groups, a propensity score (PS) was calculated as the following.A logistic regression was performed to score all patients according to the group  used as an independent variable and the following covariates at baseline: age at disease progression, EDSS one year before disease progression and at the time of SP diagnosis confirmation, number of therapeutic switches before progression, and follow up duration.Inverse probability of treatment weight (IPTW) and the stabilized inverse probability of treatment weight (SIPTW) were also calculated.p-value < 0.10 in the univariate analysis were considered. The results were presented as an estimate of the hazards ratio (HR) and the corresponding 95% confidence interval (CI). A generalized Cox regression model IPTW PS-adjusted was built for the event time to 24 weeks confirmed EDSS 7.0. In the multivariable analysis, all the variables with a A sensitivity analysis was conducted for patients with at least 36 months of follow up (in group B) and without disease activity during the mentioned follow up.The model with the best statistical properties was chosen according to Akaike criterion.Statistical significance was taken to be at the two-tailed 0.05 level. All statistical analysis was performed using SPSS 21.0 .At the date of data extraction, out of 2300 patients registered in our records, 311 SPMS patients fulfilled the required criteria to be enrolled. There were 165 patients in group A and 146 in group B. The baseline data are shown in p-Value = 0.234) and the Cox proportional model IPTW PS adjusted did not show differences between the two groups for the time to 24 weeks confirmed EDSS 7.0  (p = 0.874).The survival model with Kaplan Meier analysis  . A sensiDuring the follow up, six patients (four in group A and two in Group B) had clinical relapses, with a median time of 17.6 ± 7.9 in Group A and 15.8 ± 6.8 months in group B. Seven had radiological activity (three in group A and four in group B 14.5 ± 6.5 and 15.1 ± 5.8 months).In our real-world study, interferon beta 1b did not influence the risk to reach 24-weeks confirmed EDSS 7.0 in a cohort of patients with non-active SPMS. Furthermore, no differences were observed in acute disease recurrences between the two groups. The role of DMT maintenance in SPMS is constantly debated in a real-world setting; however, it is mainly investigated in heterogeneous cohorts. We included only non-active SPMS patients who maintained therapy for a long time (90% for at least 36 months) and all analyses were adjusted for baseline characteristics with IPTW PS.A recent study from the MSBase study group shows that only patients with superimposed relapses during SPMS may benefit from continued treatment with DMTs, in accordance with the 2018 American Academy of Neurology recommendation. However, no consensus exists, as another report from the same study group has not found any benefits of DMTs use when analyses were adjusted for relapse rate ,15,16. TDMT efficacy in a cohort of SPMS patients has been investigated in a previous short report that compared a group of patients without evidence of disease activity before SPMS conversion who had withdrawn DMTs (in accordance with a treating neurologist) to another group of patients who stopped DMTs voluntarily . Here, pOverall, the clinical dilemma of whether or not to maintain DMTs in such a cohort of SPMS remains unsolved, and neurologists should carefully weight the benefit–risk ratio. It is fundamental to emphasize the criticality of the conversion from a RR to a SP disease course, not only because such a conversion is evidence of disability progression, but also because, until recently, treatments that effectively reduced disability progression in relapsing MS were not proven to be effective in SPMS. Early identification of SPMS will require tools that, together with the use of appropriate treatments, may result in better long-term outcomes for the population of patients with SPMS.We undoubtedly need a better characterization of SPMS cohorts to address our therapeutic efforts. There is a need for additional research examining the psychosocial impact of disease progression in order to inform decision-making around policy and care choices.Furthermore, the formulation of an SPMS diagnosis is a key prognostic factor that may affect decisions and planning at the health care level   and the A strength of our study is that we have employed a generalized model of IPTW PS-adjusted for baseline characteristics to mitigate unbalance among the groups, and provide a long follow up.However, our study has some limits. PS cannot balance unselected bias that could be related to the unknown characteristics of each patient, according to disease history and also to demographical/social/economic elements.As this is a retrospective study, not all participants have the same follow-up, and the characteristics of sample size warrant cautious interpretation of the data.Our findings seem to suggest that discontinued interferon beta 1b could be in non-active SPMS, but more randomized, prospectively collected data with long-term follow up are needed.