Cobetia marina were studied by scanning electron microscopy (SEM) and protein measurements. XPS and FTIR analysis suggested the stable grafting of LG onto the surface of wood veneers after laccase-assisted treatment. WCA measurements showed that the hydrophobicity of the wood veneers significantly increased after the enzymatic treatment. Protein measurements and SEM pictures showed that enzymatically-hydrophobized wood veneers modified the pattern of bacterial attachment and remarkably reduced the bacterium colonization. Thus, the results observed in the present study confirmed the potential efficiency of laccase-assisted treatments to improve the marine antifouling properties of wood.The protection of wood in marine environments is a major challenge due to the high sensitivity of wood to both water and marine microorganisms. Besides, the environmental regulations are pushing the industry to develop novel effective and environmentally friendly treatments to protect wood in marine environments. The present study focused on the development of a new green methodology based on the laccase-assisted grafting of lauryl gallate (LG) onto wood to improve its marine antifouling properties. Initially, the enzymatic treatment conditions  and the effect of the wood specie  were assessed by water contact angle (WCA) measurements. The surface properties of the enzymatically modified wood veneers were assessed by X-ray photoelectron spectroscopy (XPS), Fourier transform-infrared spectroscopy (FTIR). Antifouling properties of the functionalized wood veneers against marine bacterium  Wood is a renewable, cheap, and biodegradable bioresource with interesting mechanical properties which make it a competitive material in construction applications. In marine environments, wood is used as a raw material for the construction of waterfront structures, e.g., groynes, jetties, dolphins  and clasThe biofilm formation starts with the adsorption of free organic material onto the surface of the substrata . Then, bpara-position that could provide stable hydrophobic properties if grafted onto wood. In fact, these compounds have been enzymatically grafted on different lignocellulosic substrates for such purpose ) and time of reaction, were assessed to optimize the enzymatic hydrophobization process on beech veneers. The hydrophobicity of the treated beech veneers is showed as a function of the water contact angle (WCA).It was expected that long reaction time combined with high [LG] improved the hydrophobicity of the beech veneers. Nevertheless, long reaction times (higher than 3 h) did not improve the surface hydrophobicity of the wood veneers. In fact, the time of reaction necessary to achieve the highest WCA on the surface of beech veneers was between 2–3 h for the different [LG] and laccase doses studied . It is w2 of wood) was set as the optimum value considering the small differences in the WCA in the tested range. Thus, the experimental design permitted to identify the conditions to improve the hydrophobicity of treated beech veneers while minimizing the requirements of energy and both chemical and enzymatic reagents.Regarding the laccase dose, the results observed suggest that the higher the laccase dose the higher the WCA of the enzymatically treated beech veneers. The oxidation of the phenolic moieties of lignin present in the beech veneers enables the stable grafting of LG. Hence, the higher the laccase dose the higher the lignin oxidation and, therefore, a higher grafting of LG could be achieved. A medium laccase dose (5 U/cmThe WCA of the beech veneers treated with laccase and LG was compared with those samples treated only with laccase or LG. As noted in 6–C3) which differ one each other in their methoxy substitutions on the aromatic ring, e.g., guaiacyl (G), syringyl (S) and p-hydroxyphenyl (H). The S/G ratio in eucalyptus wood is proximately to 6.25 which is much higher than the S/G ratio in beech wood, 0.71 [ortho-position. Otherwise, the radicals produced by laccase in S units should be more stable than those produced in G units, since S units have two electro-donating groups (methoxy) in ortho-position instead one methoxy group which possess G units [ortho-position in G units apparently favored the laccase-mediated grafting of LG.Regarding the different wood species tested , enzymatically treated beech veneers showed a WCA slightly higher than pine and a much higher hydrophobicity than eucalyptus veneers . Nonetheod, 0.71 ,28. In aod, 0.71 ,30. Thus G units . This ex G units . HoweverX-ray photoelectron spectroscopy (XPS) analysis was performed to obtain a better knowledge of the surface chemistry of the enzymatically hydrophobized beech veneers. Such technique has been previously used to study the surface chemistry of several wood species ,34, pulpThe elemental composition results confirmed that beech samples treated enzymatically with LG showed a clear increase of carbon atoms (4.8%) and a decrease of oxygen (5.9%) which were related to the presence of the aliphatic tail of LG onto the veneers surface . In addiFour different components were obtained after deconvolution of C1s spectra. C1 was related to C-C or C-H bonds; C2 comprised a carbon bonded to single non-carbonyl oxygen atom (C-O); C3 corresponded to a carbonyl group (C=O) or a carbon bonded to two non-carboxyl oxygen atoms (O-C-O); C4 was assigned to a carbon bonded to a carbonyl group and non-carbonyl oxygen (O-C=O). Beech veneers treated with laccase showed a remarkable increase of C3 component which was related with the oxidation of hydroxyl groups in the phenolic moieties of lignin to form carbonyl groups. The presence of hydrophobic compounds on wood surface could be related with both a low O/C ratio and a high C1/C2 ratio . Samples−1 which were related with the stretching of methyl (-CH3) and methylene (-CH2-) groups of the 12-carbon aliphatic chain of LG .The biofilm assay was performed for the assessment of the potential antifouling properties provided by the laccase-mediated grafting of LG onto the surface of pine veneers surface. Pine wood was the specie used for the antifouling assay due to its high hydrophobicity  but alsopara-substituent. In addition, it was proved that LG has antibacterial properties, specifically against Gram-positive bacteria due to inhibition of their membrane respiratory chain [C. marina) in this study is a Gram-negative bacterium which suggest that the potential antifouling properties of the enzymatically-hydrophobized pine veneers were mostly related with their hydrophobicity. It was showed that the antifouling properties of a substrate are directly linked to its surface energy [C. marina adhesion onto pine veneers.Regarding the antimicrobial properties of LG,  found thry chain . Howevere energy . It was C. marina was studied to assess its hydrophilicity degree. Hence, C. marina was suspended in a solution containing both aqueous and organic phases and the migration of the bacteria to the organic phase was measured. According to Karunakaran and Biggs [C. marina remained in the aqueous phase. Therefore, the surface chemistry of the C. marina strain (CECT4278) was mostly hydrophilic.At the same time, bacterial surface chemistry is also an important parameter since its hydrophilicity/hydrophobicity character will determine its chemical compatibility and the strength of its initial attachment with the hydrophobized pine veneers. Therefore, the surface chemistry of nd Biggs , a bacteC. marina (v/v) for 1, 3 and 5 days. The amount of bacteria onto the veneers surface was indirectly quantified by measuring the protein content on the surface of the wood samples. Such protein measurement of the colonized wood samples showed that the laccase-assisted grafting of LG reduced substantially the C. marina adhesion  were effectively hydrophobized through the enzymatic treatment. It was observed that the reaction conditions played an important role on the extent of hydrophobization, but the treated wood species were also a major factor. Based on these results, pine wood was selected to study the impact of the laccase-mediated hydrophobization on the marine antifouling properties of wood. SEM pictures and protein measurements confirmed that the hydrophobized wood veneers modified the colonization pattern of


This cross-sectional study investigates the number of US Food and Drug Administration–approved cancer drugs that displaced standard-of-care therapies between 2016 and 2021. How many cancer therapies approved by the US Food and Drug Administration between 2016 and 2021 displaced standard-of-care therapies vs being added to existing therapies in the metastatic, adjuvant, and maintenance settings?In this cross-sectional study, between May 1, 2016, and May 31, 2021, there were 207 cancer drug approvals in oncology and malignant hematology. Of these therapies, 14% displaced cancer therapies that were previously approved and considered to be the standard of care for their indication; most therapies (42%) were approved as later-line drugs in the second-, third-, or later-line settings.Although there has been a high volume of new cancer drug approvals in oncology and malignant hematology, only a minority of these drug approvals displace the current standard-of-care therapy, suggesting that newer drugs may benefit patients with few alternatives but could add to the cost of care because competition in the drug markets is crucial to lowering drug prices. Although several cancer drugs receive US Food and Drug Administration (FDA) approval each month, it is unclear how many of these cancer drugs transform the treatment landscape significantly by tumor group. Specifically, it remains unclear how many of these newly approved cancer drugs displace the existing standard-of-care therapies for their indication vs being added to existing therapies.To examine how many cancer drugs displace the standard-of-care therapies vs being added to existing therapy or filling breaks in systemic treatments in the metastatic setting, adjuvant setting, or maintenance setting.Retrospective cross-sectional study using landmark trials leading to FDA approval of cancer drugs between May 1, 2016, and May 31, 2021. The study evaluated all FDA approvals for cancer drugs between May 1, 2016, and May 31, 2021, using the FDA Oncology (Cancer)/Hematologic Malignancies Approval Notifications website. All clinical trials leading to FDA approval of cancer drugs during this period were examined.first-line displacing if a drug was approved for use in the first-line setting and displaced the prior standard-of-care drug for an indication, first-line drug alternatives/new if a drug was approved for use in the first-line setting but did not displace the standard of care at the time of approval or was a new drug that was first of its class for an approved indication, add on if a drug was approved in combination with a previously approved therapy for a disease or if a drug was approved for use in the adjuvant or maintenance settings, and later line if a drug was approved for use in the second-, third-, or later-line settings.A drug was determined to have displaced the prior standard-of-care therapy by evaluating the comparator arm (or lack thereof) in the clinical trial leading to the drug’s approval and also by reviewing National Comprehensive Cancer Network Guidelines. Cancer drug approvals were categorized as Between May 1, 2016, and May 31, 2021, there were 207 FDA cancer drug approvals in oncology and malignant hematology. Of these 207 approvals, 28 drugs (14%) were first-line displacing therapies. A total of 32 drugs (15%) were first-line drug alternatives/new drugs. A total of 61 drugs (29%) were add-on therapies. Finally, 86 drugs (42%) were approved as later-line therapies.In this study, most cancer drug approvals between 2016 and 2021 were in the later-line settings as opposed to displacing the current standard-of-care therapy for the approved indication. These later-line drugs may benefit patients with few alternatives but add to the cost of care because competition in the drug markets is a key factor in leading to lower drug prices. The osimertinib arm showed significant superiority over erlotinib/gefitinib, therefore displacing these drugs to become the treatment of choice for this indication.Although several oncology and malignant hematology drugs receive FDA approval each month, it is unclear how many of these cancer drugs transform the treatment landscape significantly for each tumor group. It remains unclear how many of these newly approved cancer drugs displace the existing standard-of-care therapies for their indication compared with providing simply an alternative treatment option. For example, osimertinib first received FDA approval in November 2017 for epidermal growth factor receptor  compared with best supportive care .7 Following the results of JAVELIN Bladder 100, the standard of care for patients with urothelial cancer who do not progress while taking first-line chemotherapy is maintenance avelumab.Conversely, avelumab was approved as maintenance therapy for locally advanced or metastatic urothelial carcinoma after first-line platinum-containing chemotherapy. The JAVELIN Bladder 100 study was a randomized phase 3 trial that assigned patients with unresectable locally advanced or metastatic urothelial cancer who did not progress while taking first-line chemotherapy to receive either maintenance immune checkpoint inhibitor avelumab or best supportive care.8 Therefore, it is important to evaluate among cancer drug approvals what percentage of drugs are approved for use in settings that lead to increased competition, and ultimately the possibility of reduced drug prices, as opposed to cancer drugs that prolong survival but do not necessarily compete against each other in the market. The literature is sparse regarding analysis of cancer drug approvals that are categorized according to the potential to promote competition and ultimately lead to lower drug prices.Competition is one of the most critical factors in the drug markets that leads to lower drug prices.Given the lack of data on oncology and malignant hematology drug approvals and their positions in the treatment landscape for their indications, we sought to examine how many newly approved cancer drugs displace the standard-of-care therapies compared with becoming an alternative option in the metastatic, adjuvant setting, or maintenance setting. We also wished to evaluate how many new therapies are combined with older cancer therapies.9 to evaluate all malignant hematology and oncology drugs that were approved between May 1, 2016, and May 31, 2021. Using the FDA approval notices and National Comprehensive Cancer Network Guidelines, the 4 authors evaluated all malignant hematology and oncology drug approvals to determine which newly approved drugs displaced the existing standard-of-care drugs, which were used in combination with previously approved drugs, which were approved for use in the adjuvant or maintenance settings, and which were approved for use in the second-, third-, or later-line settings.10 Per policies at the University of California, Thomas Jefferson University, and Imperial College London, the study was not submitted for institutional review board approval because all data are publicly available and because the study did not involve personal health care information. This report followed the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for cross-sectional studies.We reviewed the FDA Oncology (Cancer)/Hematologic Malignancies Approval Notifications website18 were deemed to be add-on therapies rather than displacing therapies. These categorizations were also validated with the National Comprehensive Cancer Network guidelines to ensure validity of therapy options at the date of censoring.10Data for this cross-sectional study were collected and analyzed using Excel, version 16.2 (Microsoft Corporation). Clinical trials deemed “pivotal” for drug approval were analyzed by 2 researchers independently (A.X. and M.P.L.), with any conflicting results being resolved by consensus from a third researcher (D.J.B.). Therapies were only deemed to be displacing if they were approved on the basis of improved efficacy, such as overall survival, progression-free survival, or another surrogate end point deemed suitable by the FDA for approval over established standard-of-care therapy options in this setting. Therapies that were not directly compared, such as in a single-arm study, or that were not compared with established standard-of-care options were considered to be alternative options rather than displacing therapies. Therapies that were added to existing, established treatment options, such as pembrolizumab with cytotoxic chemotherapy in NSCLC in KEYNOTE-189,first-line displacing if a drug was approved for use in the first-line setting and displaced the prior standard-of-care drug for an indication, first-line drug alternatives/new if a drug was approved for use in the first-line setting but did not displace the standard of care at the time of approval or was a new drug that was first of its class for an approved indication, add on if a drug was approved in combination with a previously approved therapy for a disease or if a drug was approved for use in the adjuvant or maintenance settings, and later line if a drug was approved for use in the second-, third-, or later-line settings. Later-line drugs are therapies that do not necessarily compete against each other in the market and therefore cannot potentially lead to lower drug prices, whereas first-line drug alternatives/new promote competition against standard-of-care therapies in the drug market. Descriptive analyses of the collected data were then conducted. No statistical analysis was conducted owing to the nature of the study.We coded approvals by the following categories: There were 207 FDA cancer drug approvals in oncology and malignant hematology between May 1, 2016, and May 31, 2021. Of these 207 approvals, 28 drugs (14%) were first-line therapies that displaced cancer therapies previously approved and considered to be the standard of care for their indication, as indicated by 11 studied alectinib vs crizotinib in patients with previously untreated, advanced anaplastic lymphoma kinase rearrangement–positive NSCLC. The randomized, open-label, phase 3 study demonstrated superior efficacy and lower toxicity with alectinib compared with crizotinib, thus leading to alectinib becoming the first-line therapy for anaplastic lymphoma kinase rearrangement–positive NSCLC per National Comprehensive Cancer Network guidelines and displacing all other anaplastic lymphoma kinase inhibitors at the time of its approval. The IMbrave150 study12 evaluated the combination of atezolizumab and bevacizumab in the setting of unresectable or metastatic hepatocellular carcinoma and demonstrated improved overall and progression-free survival in the combination therapy group compared with sorafenib. At the time of this publication, atezolizumab and bevacizumab remain the first-line preferred therapy for unresectable or metastatic unresectable or metastatic hepatocellular carcinoma with Child-Pugh A cirrhosis. Finally, the CABOSUN trial13 evaluated the tyrosine kinase inhibitor cabozantinib vs sunitinib as initial therapy for metastatic kidney cancer. The randomized phase 2 trial demonstrated improved progression-free survival with cabozantinib compared with sunitinib.13 At the time of its approval, cabozantinib was considered a first-line therapy for metastatic kidney cancer because it had displaced the prior standard-of-care drug sunitinib.14Examples of the cancer drugs that displaced previously approved therapies include alectinib for anaplastic lymphoma kinase rearrangement–positive metastatic NSCLC, osimertinib for epidermal growth factor receptor exon 19 deletion or exon 21 L858R substitution NSCLC, atezolizumab and bevacizumab for unresectable or metastatic hepatocellular carcinoma, and cabozantinib for advanced kidney cancer. A complete list of first-line displacing drugs is provided in the eAppendix in the MET exon 14–skipping NSCLC, and avapritinib for unresectable or metastatic gastrointestinal stromal tumor (GIST) with platelet-derived growth factor receptor alpha (PDGFRA) exon 18 variant including D842V variant. A complete list of approvals in the first-line setting that are first-line alternatives or first-of-their-class therapies is available in the eAppendix in the 15 The study revealed improved overall survival at 24 months  as well as improved radiographic progression-free survival at 24 months . Although apalutamide is approved for nonmetastatic castrate-resistant prostate cancer, it is considered a first-line treatment that is an alternative to darolutamide or enzalutamide depending on clinician preference and a patient’s comorbidities, among other factors.Several cancer drug approvals were in the first-line setting but did not displace the standard-of-care therapy at the time of approval; rather, these drugs were first-line alternative therapies or first-of-their-class therapies. Examples of these drug approvals include apalutamide for nonmetastatic castrate-resistant prostate cancer, tepotinib for metastatic MET exon 14–skipping NSCLC based off results from the VISION trial,16 an open-label phase 2 study. The study showed that approximately half of the enrolled patients responded to tepotinib. Tepotinib is considered to be a first-line alternative to capmatinib, which is similarly approved for MET exon 14–skipping NSCLC. Because the VISION trial did not compare these 2 tyrosine kinase inhibitors, there is no clear preferred first-line therapy for MET exon 14–skipping NSCLC. Avapritinib, a tyrosine kinase inhibitor, was studied in the NAVIGATOR trial, which was a single-arm, open-label study evaluating efficacy in patients with unresectable GIST including those with PDGFRA D842V variant–positive GIST. Among patients with PDGFRA D842V variants, the overall response rate was 91%, with the median duration of response being 27.6 months .17 The study subsequently led to the approval of avapritinib for patients with PDGFRA D842V variant–positive GIST as the first-of-class therapy for GIST harboring a PDGFRA exon 18 variant.In February 2021, the FDA granted accelerated approval to tepotinib for 18 and with carboplatin and paclitaxel for squamous NSCLC based on data from KEYNOTE-40719) for the indication of advanced NSCLC. The chemotherapy regimens used in the treatment of nonsquamous NSCLC and squamous NSCLC remained unchanged following these 2 landmark studies, KEYNOTE-18918 and KEYNOTE-407,19 except for the addition of the immune checkpoint inhibitor pembrolizumab. Another example of a new malignant hematology drug that was approved for use in combination with previously approved therapies is venetoclax, which was approved in combination with hypomethylating agents (HMAs) in adults aged 75 years or older with newly diagnosed acute myeloid leukemia and who are not candidates for intensive induction chemotherapy. Venetoclax, a B-cell lymphoma-2 inhibitor, was studied in combination with HMAs azacitadine and decitabine in treatment-naive older patients with acute myeloid leukemia. The landmark study evaluating venetoclax in combination with HMAs found 67% of patients (n = 145) achieved complete remission.20 Because HMAs had previously been used in the treatment of frail older patients, venetoclax was approved to be used as an add-on therapy with these HMAs in this patient population with untreated acute myeloid leukemia.Eighty-six  cancer drugs were approved for use “in combination” with previously approved therapies or approved for use in the adjuvant, consolidation, or maintenance settings and effectively are add-on therapies. For example, among oncology drug approvals, pembrolizumab was approved in combination with chemotherapy  compared with patients who received placebo .21 At the time of this publication, the standard of care for patients with stage III NSCLC who do not have disease progression after chemoradiotherapy (and with no contraindications to immunotherapy) is now up to 12 months of consolidation durvalumab.With regard to examples of maintenance therapy that we categorized as add-on therapy to induction or primary treatment, durvalumab was approved as maintenance therapy for unresectable stage III NSCLC without progression after concurrent platinum-based chemotherapy and radiation therapy. The PACIFIC trial22 in the setting of relapsed or refractory indolent or aggressive lymphoma. The study included 33 patients with indolent lymphoma and 51 patients with aggressive lymphoma, with follicular lymphoma (48.5%) being the most common histological subtype. The objective response rate among patients in the indolent lymphoma cohort was 43.7% (n = 14 of 32). The study was conducted with a single arm and therefore did not compare copanlisib with any other therapies. Copanlisib was subsequently approved for use in the third-line setting and remains a third-line therapy for relapsed or refractory follicular lymphoma.22 Moxetumomab pasudotox-tdfk, a recombinant CD22-directed cytotoxin, was studied in a single-arm phase 3 trial23 in patients with relapsed or refractory hairy cell leukemia with at least 2 or more prior systemic therapies. The overall complete remission rate was 41.3% in this heavily pretreated patient cohort.23 Moxetumomab pasudotox-tdfk was approved for use in the third line for hairy cell leukemia and remains a third-line therapy option. Most second-, third-, or later-line therapies were studied in single-arm studies or vs placebo and therefore did not displace any therapies because of their approvals.Most cancer drug approvals (n = 86) were in the later-line settings. Examples of drugs that were approved for the later-line setting include copanlisib, which is approved for patients with relapsed follicular lymphoma who received 2 prior therapies, and moxetumomab pasudotox-tdfk, which is approved for patients with relapsed or refractory hairy cell leukemia who received 2 prior systemic therapies. A phase 2 study evaluated PI3K inhibitor copanlisibOf note, there were disparities among the drug approvals depending on tumor type. Lung-related tumors received the most number of approvals (n = 37), followed by genitourinary tumors (n = 28), leukemia (n = 25), lymphoma (n = 22), breast cancer (n = 19), gastrointestinal cancers (n = 14), plasma cell dyscrasias (n = 12), gynecologic cancers (n = 10), hepatobiliary tumors (n = 8), nonmelanoma skin cancer (n = 5), melanoma (n = 5), soft-tissue cancers (n = 5), tumor agnostic (n = 4), head and neck cancers (n = 3), and neuroendocrine cancer (n = 2), as shown in 24 The remaining 61 drug approvals (29%) came in the adjuvant or maintenance settings used in combination with previously approved therapies.In this cross-sectional study, between May 1, 2016, and May 31, 2021, most of the 207 FDA drug approvals for oncology and malignant hematology drugs were for therapies to be used in the second-, third-, or later-line setting, with 86 drug approvals (42%) falling in this category. Approximately 1 in 6 newly approved drugs  displaced the therapies that were considered to be the standard of care for their indication at the time of approval, while 32 drugs were approved for use in the first-line setting as alternative first-line options or new first-of-their-class drugs. Both of these categories may provide market competition and work to lower cancer drug prices; however, practical examples of competition on the basis of price are rare in oncology.Four new approvals (2%) were in the tumor agnostic setting, including neurotrophic tyrosine receptor kinase inhibitors entrectinib and larotrectinib, both approved for patients with neurotrophic tyrosine receptor kinase variants who have no satisfactory alternative treatments, and 2 approvals for the anti–programmed cell death 1 drug pembrolizumab in cancers when patients have no satisfactory alternative treatment options and have either a high tumor variant burden or have high levels of microsatellite instability or mismatch repair deficiency. These new therapies did not displace any existing therapies, thereby becoming viable new therapies in the second line and beyond.While there is justified enthusiasm for the high volume of new cancer drug approvals in oncology and malignant hematology, these approvals must be evaluated in the context of their use . It is evident that further research and cancer drug development is required to drastically change the treatment landscape for many oncologic and malignant hematologic conditions.This study has several limitations. Some drugs may displace later lines of therapy, although we did not include them in this study. For example, avelumab used as first-line maintenance therapy in urothelial cancer may displace second-line immune checkpoint inhibitor use. Therapies may also displace alternative treatment modalities; for example, radium-223 in prostate cancer may displace external beam radiotherapy but was deemed beyond the scope of this study. In addition, the potential number of patients affected by—and, by extension, the market impact—from each drug approval varies considerably. However, this analysis was deemed beyond the scope of this study.This study found that 14% of new drug approvals displaced existing standards of care, and an additional 15% provided market competition. At the same time, 29% were add-on or maintenance drugs that can only increase the cost of care. Forty-two percent were drugs approved for patients who had exhausted other treatment options. These later-line drugs may benefit patients with few alternatives but also add to cost of care and further delay palliative and comfort services. Further incentives to maximize competition in the cancer drug space warrant consideration.


TP53 missense variants often predispose to a wide spectrum of tumors characterized by Li-Fraumeni syndrome (LFS) but a subset of variants is also seen in families with exclusively hereditary breast cancer (HBC) outcomes. We have developed a logistic regression model with the aim of predicting LFS and HBC outcomes, based on the predicted effects of individual TP53 variants on aspects of protein conformation. A total of 48 missense variants either unique for LFS (n = 24) or exclusively reported in HBC (n = 24) were included. LFS-variants were over-represented in residues tending to be buried in the core of the tertiary structure of TP53 (p = 0.0014). The favored logistic regression model describes disease outcome in terms of explanatory variables related to the surface or buried status of residues as well as their propensity to contribute to protein compactness or protein-protein interactions. Reduced, internally validated models discriminated well between LFS and HBC  curve), had a low risk for over-fitting and were well calibrated in relation to the known outcome risk. In conclusion, this study presents a phenotypic prediction model of LFS and HBC risk for germline TP53 missense variants, in an attempt to provide a complementary tool for future decision making and clinical handling.Rare germline pathogenic  TP53 variant [TP53 alterations of 20–40% in LFL as compared to 70% in LFS [TP53 variants has increased, and up to 1% of families with exclusively hereditary breast cancer (HBC) have been shown to carry a germline TP53 variant [Li-Fraumeni syndrome (LFS) is a rare heritable extreme tumor risk syndrome characterized mainly by premenopausal breast cancer, soft tissue sarcoma, brain tumors, osteosarcoma and adrenocortical carcinoma, and was first described in 1969 [ variant . As moreTP53 variants makes tumor risk assessment difficult and genetic counseling challenging in these patients and families. Moreover, 7–20% of constitutional TP53 alterations are de novo [TP53 variant carriers are exposed to unnecessary examinations [The wide range of phenotypic presentation associated with germline inations ,9.The TP53 protein is a transcription factor that binds as a tetramer to DNA, and activates a large number of genes that promote DNA repair mechanisms or apoptosis including cell cycle regulatory proteins and members of the Bcl-2 family ,11. EachTP53 variants are single site missense changes, predominantly located in the DBD [TP53 variant thus possessing both wild-type and variant monomers allowing formation of hetero-tetramers that result in a dominant-negative functional effect of some variants [About two thirds of reported germline  the DBD . Carriervariants . It has variants . Moreovevariants . TP53 variants [TP53 variants on conformational aspects of TP53 and its functionality could contribute to differences in phenotypes .The TP53 DBD consists of a beta-sandwich tertiary structure with two antiparallel beta-sheets, that serve to orientate and stabilize the loop-sheet-helix DNA-binding motif . Contactvariants ,20. Intevariants . Differevariants . It is tTP53 variants can be accounted for by the differential effects of the variants on TP53 protein conformation as well as whether variant associated protein conformation changes can be used to predict disease outcome.In this study we investigate whether the phenotypic outcome observed for different TP53 variants that were defined as LFS in our cohort were identified through the IARC database, and were not found to be reported in a HBC-family there or elsewhere. According to the IARC database the families thus fulfilled the classic LFS criteria [Included variants and their clinical characteristics were selected from publicly available databases and publications as described below. All criteria  upon scrcriteria  and 2 frcriteria . HoweverTP53 missense variants used in this study is summarized in TP53 variants unique for LFS were selected by evaluation of 408 TP53 variants in the IARC database  [n = 24) to represent the LFS-variants used in the study and the amino acids involved are referred to as LFS-residues (The selection process for the ry 2020) . Out of residues . TP53 variants was selected from the IARC database, from the Fortuno et al. [TP53-related HBC without a history of LFS and from the Kharaziha et al. [TP53 cohort. Since there was no specified HBC-class in the IARC database, we selected the HBC-variants from FH-class, noFH-class and other-class and excluded those that overlapped with the LFS-class, LFL-class and TP53-Chompret-class. Further, the selected HBC TP53 missense variants were exclusively reported in breast cancer. Out of 73 identified germline variants in Fortuno et al. [TP53 variants were identified, of which 6 missense variants were specifically found in HBC including two that were not present in the LFS-class, LFL-class or TP53-Chompret-class in the IARC database. The 24 resulting TP53 variants were included in the study and the amino acids involved are referred to as HBC-residues  was used to display HBC- and LFS-residues in the context of the tertiary structure of the protein and to identify different residue classes, using customized scripts adapted from those available in the PyMOL script library [2 defined approximately 30% of residues as Buried and was used to classify residues for statistical analysis. The interfaceResidues script [2 was used. The ss script [A published X-ray crystal structure of a tetrameric TP53 derivative containing the DBD fused to the oligomerization domain bound to the natural p21 TP53-response element (PDB accession number 3TS8) was used . Details library . The fin library  was adaps script  was adaps script  was adapExplanatory variables were a priori restricted to variables reflecting the effects of missense variants on different aspects of TP53 protein conformation . Bur is 2). Bur is a categorical variable including Buried, Surface (>cutoff at 11Å2) and unknown (not included in the TP53 tertiary structure used). The Espritz predictor [The findSurfaceResidues script from Pymol software, version 2.3.1 . was adaredictor  was trairedictor  algorithredictor  predictsredictor ,34 prediThe _dif suffix indicates that the variable is the difference between the value for variant TP53 and wild-type TP53 at the position of the substituted residue (variant score minus wild type score).Abbreviations: DB, database; NMR, nuclear magnetic resonance; X-ray, Xray diffraction.p-values were for two-sided tests and were adjusted for multiple testing, where appropriate, using the false discovery rate method. Associations between disease outcome (LFS or HBC) and the explanatory variables for different TP53 variants as well as their predictive potential were evaluated by logistic regression using internal 1000-fold bootstrapped validation and a backwards step-down approach to variable number reduction as implemented in the rms-package . The validate function delivers values for a number of parameters relevant for assessing the discrimination performance of models and the risk for overfitting, including the concordance statistic (C-statistic). Similarly, the calibrate function  was used to test the quality of model calibration. Further validation was performed using leave-one-out cross validation by using the validate function (arguments as above except sls = 0.16) to produce reduced models for each combination of n-1 variants. Predictions were expressed as probability of LFS . ROC curves were produced using the ROCit package. The favored model was described visually using a nomogram and its potential utility was evaluated by decision curve analysis (rmda package) [p < 0.05 was considered as the threshold for statistical significance unless stated otherwise. Data were collected and processed using R software, version 3.6. Fisher’s exact test was used to evaluate whether LFS- or HBC-residues were over- or under-represented in residue sets reflecting different structural aspects of the TP53 protein structure. package) . p < 0.0TP53 missense variants were selected from the IARC database, Fortuno et al. [A total of 48 germline o et al.  and Kharo et al.  includinThe vast majority of variants were mapped to the DBD of the TP53 protein a. SpecifVariant LFS- and HBC-residues are distributed and juxtaposed throughout the DBD located in the central part of TP53 with no apparent pattern associated with either disease outcome. We considered whether there might be associations with secondary-structure elements , tertiary structure aspects (such as Surface or Buried locations) or quaternary structure aspects (DNA interacting residues or inter-monomer protein interacting residues in the context of the TP53 tetramer). For each of the TP53 monomers , there was a significant enrichment of variant LFS-residues in the approximately 30% of residues that are Buried  in the crystal structure of the wild-type TP53 tetramer bound to DNA . A similThe enrichment of LFS-residues in the set of most Buried TP53 residues suggests that the disease associated variants might tend to alter the folded conformation of TP53 in LFS patients. The more even distribution of HBC-residues on the Surface and core of the protein structure would give a greater possibility for disease associated variants to disrupt or modify interactions between TP53 and its DNA or protein ligands in HBC patients. To investigate these aspects further we made multivariate models to predict disease outcome as a function of Buried vs. Surface status for LFS- and HBC-residues, together with a range of variables predicting the effect of the variant residues on protein conformation aspects, such as intrinsic disorder, protein backbone flexibility, propensity for tertiary structure formation, propensity for secondary structure formation and protein interaction propensity . Severaln = 2 to 4) that could reasonably be included in a final model. First a full model (mod_full), likely associated with overfitting problems, was made using 12 explanatory variables describing the effects of the LFS- and HBC-variants on different aspects of protein conformation. An internal bootstrap cross-validation procedure was then used to produce a reduced model by removing less useful variables in a stepwise manner. The reduced model produced during the cross-validation procedure contained 4 variables (mod_4v). Further variable number reduction was done manually by successively removing the variable with the least significant beta coefficient to produce models with three and two explanatory variables, mod_3v and mod_2v, respectively (Since the disease outcome is defined by a binary variable (LFS or HBC) we used a logistic regression approach. The relatively small number of variants for each outcome (n = 24 in each group) imposed limitations on the number of explanatory variables  for use in developing improved tools for clinical decision making we used decision curve analysis. The net benefit of using the mod_3v to predict LFS disease outcome at different risk thresholds is shown in TP53 have classically been associated with Li-Fraumeni syndrome (LFS), a tumor predisposition syndrome with high risk of various childhood as well as adult onset tumors. Increased genetic testing has however revealed that germline TP53 variants are associated with a broader range of phenotypes, from classical LFS to hereditary breast cancer (HBC), and the outcome may be dependent on both variant characteristics and modifier gene variants elsewhere in the genome [TP53 variants creates challenges for the genetic counseling and clinical handling of these individuals.Pathogenic germline variants in e genome . Differee genome ,42, but e genome . The widTP53 missense variants on protein conformation and their association to disease phenotype. We present a quantitative model that predicts disease outcome (LFS or HBC) as a function of localization of variant residues in the tertiary structure of the TP53 DBD and oligomerization domain together with predicted variant-associated effects on conformation of the full-length protein. In an attempt to better understand the molecular basis for the differential disease outcomes associated with different variants and to develop a prediction tool, we studied the impact of germline p = 0.0014) of the tertiary structure of one or more TP53 monomers in the DNA-bound tetramer, indicating that the set of variant LFS-residues may hypothetically have a larger impact on the folding and overall conformation of the TP53 protein than the set of variant HBC-residues. While the Buried/Surface variable relates to the predisposition of affected residues to lead to LFS or HBC, the compactness (comp_dif) variable is related to how the substitution of the variant residues is predicted to affect compactness, with enhanced compactness favoring the LFS outcome. The protein interaction propensity variable (PPI6_dif) is also positively correlated with the probability of LFS outcome suggesting the importance of protein interactions for the LFS phenotype. These protein interactions could in principle be interactions between monomers within the TP53 tetramer or interactions between the TP53 tetramer and other proteins. Comparison with the positions of residues forming intra-tetramer interactions shows that the major DBD regions predicted to have protein interaction propensity (Our results demonstrate that LFS-variants were enriched in Buried regions (opensity d containTP53 variants that are particularly strongly associated with the LFS phenotype (see Introduction). It could be speculated that a variant which stabilized structural aspects of TP53 monomers and their propensity for tetrameric interactions in relation to wild-type would facilitate the formation of hetero-tetrameric TP53 tetramers in heterozygous individuals, thereby resulting in the dominant-negative phenotype that is observed for many missense variants that are associated with LFS [Extrapolation of the modelling results suggests that variants that tend to strengthen the tertiary and quaternary structure of the TP53 tetramer would tend to favor the LFS disease outcome. This may be related to the dominant-negative phenotype associated with with LFS .TP53 variants at all. Since the number of patients and families displaying some HBC variants is limited, it is also possible that some variants may subsequently be coupled to LFS. The V157I variant, for example, that is reported in 7 individuals in 2 families was classified as HBC by both IARC and Fortuno et al.  for TP53 variants with their effects on aspects of protein conformation and function. The models also appear to have a predictive capacity that may be of practical future use in genetic counselling and management of missense variant carriers. However, there is a need to evaluate the prediction model in an independent cohort prior to any implementation in clinical practice. This study explored the relationship between germline


The prime – boost strategy with the BCG–PPD system can also be applied to the immunoprophylaxis and immunotherapy of COVID-19. The autologous anti-Corona virus B and T lymphocytes can be activated in vitro by inactivated virus or mitogens like Concanavalin A to express MHC class II molecules on their surface and pulsed with PPD for carrier targeting in vivo. Such PPD – pulsed activated (MHC-II+ve) anti-viral lymphocytes if transfused back into a patient already vaccinated with BCG during childhood or primed with BCG during adulthood 2 weeks before transfusion, could lead to a high magnitude of selective in vivo amplification of specific anti-viral lymphocytes, which can mount adequate and appropriate immune response to get rid of the virus and cure the patient from COVID-19. Conjugating antigens to PPD and injecting into BCG primed humans may also be helpful for immunoprophylaxis against COVID-19. Thus, PPD may prove to be the red herring in the BCG therapy of COVID-19.Coronavirus Disease 2019 (COVID-19) morbidity and mortality was found to be less severe in countries where Bacille Calmette – Guerin (BCG) vaccination of the population is carried out. Conjugating Purified Protein Derivative (PPD) onto tumour cells and injecting into BCG primed mice was found to enhance anti-tumour immune response. We had proposed earlier that  It has recently been reported that coronavirus disease 2019 (COVID-19) morbidity and mortality has been less severe in certain countries where childhood vaccination of the population with BCG is commonly practiced.2 The notable examples are India and some African countries. In multiple studies comparing the effect of COVID-19 worldwide, findings thus far suggest lower rates of infection and mortality in countries with universal neonatal BCG tuberculosis vaccination policies compared with countries without this practice.3 In another study, it was found that every 10% increase in the BCG index (which reflects the extent of a country’s BCG vaccination) was associated with a 10.4% reduction in mortality from COVID-19, and higher mortality rates were observed in countries with later versus early initiation of universal BCG vaccination.4Bacille Calmette – Guerin (BCG), the live attenuated mycobacterial vaccine has been in use against tuberculosis for over a century and is still being used in several countries. The BCG vaccine is given to more than 130 million babies worldwide each year to protect against tuberculosis.However, BCG alone may not be the ultimate immunotherapy of choice for COVID-19 because it causes generalised non-specific immunostimulation where anti-idiotypic and suppressor lymphocytes may also proliferate dampening the specific anti-viral immune response against the SARS CoV2 virus because of immunoregulation. Furthermore, the harmful cytokines released by the suppressor cells, the phagocytes and other cells under the influence of BCG may contribute to the cytokine storm elicited because of the Corona virus infection. These undesirable and harmful effects of the non-specific immunostimulation by the BCG can be mitigated if the specific anti-viral immunity can be scaled up by focusing the lymphokine release by the helper T-lymphocytes onto the anti-viral lymphocytes (both B- and T-cells) of the patient.Mycobacterium tuberculosis antigen, the sensitisation initiates in the regional lymph nodes where T-lymphocytes proliferate in response to the antigenic stimulus to give rise to specifically sensitised lymphocytes, which may exist in the circulation up to many years. Antigen is presented to T-cells by being ingested by antigen presenting cells (APC), which then present it on their surface to lymphocytes in combination with various major histocompatibility molecules once they reach local lymph nodes. Purified protein derivative (PPD) is a source of mycobacterial antigens, which consist of approximately 200 protein allergens obtained from the precipitate of M. tuberculosis culture supernatant. When these antigens are injected intradermally into the skin, activated T-lymphocytes mount immune response to these antigens. Molecular analyses of PPD has revealed that four heat shock proteins  contribute to roughly 60% of the PPD proteomic content. These chaperone proteins share a high homology and are conserved amongst most mycobacterial species.6When exposed to In vitro studies show that PPD promotes the upregulation of vascular endothelial growth factor (VEGF) expression in T-lymphocytes through MHC class II interaction with CD4+ T-lymphocyte interaction.7 The reactions are evident after 5–6 h following administration.Purified protein derivative most likely interacts with toll-like receptor 2 expressed on APCs that initiates an inflammatory response. Subsequent restimulation of the sensitised lymphocytes with the same or a similar antigen, such as the intradermal injection of PPD, evokes a local reaction mediated by these cells. This reaction is referred to as a delayed-type hypersensitivity response that includes vasodilation, edema and the infiltration of lymphocytes, basophils, monocytes and neutrophils into the site of antigen injection. The sensitised antigen-specific T-lymphocytes proliferate and release lymphokines, which mediate the accumulation of other cells at the site. M. tuberculosis grown on a protein-free synthetic medium and inactivated. The sensitisation following BCG injection occurs primarily in the regional lymph nodes. T-lymphocytes proliferate in response to the antigenic stimulus to give rise to specifically sensitised lymphocytes. After 3–8 weeks, these lymphocytes enter the blood stream and circulate for years. Subsequent restimulation of these sensitised lymphocytes with the PPD evokes a strong cellular immune response.Purified protein derivative is a cell-free purified protein fraction obtained from a human strain of 8 for example, infection with mycobacteria or vaccination with BCG. It stimulates T-helper response to itself and a high magnitude immune response  to the hapten conjugated to it because of the linked recognition of the hapten and the carrier PPD. The same principle has been exploited earlier to enhance anti-tumour immune response9 by conjugating PPD onto the tumour cells and injecting it into BCG primed mice. However, in this model, the lymphokine secretion by PPD – specific helper T-cells will be directed onto the target PPD – bearing tumour cells and the proliferation of anti-tumour lymphocytes will be the result of the bystander effect.Purified protein derivative is a complex mixture of mycobacterial peptides, which cannot initiate an immune response to itself when injected alone in the body of a naïve animal or human not sensitised with mycobacterial antigens. However, if PPD is injected in an animal presensitised with mycobacterial antigens, it elicits a strong immune response to itself. Purified protein derivative acts as a strong carrier molecule when conjugated with a hapten and injected in an animal presensitised with mycobacterial antigens,10 envisaged a focused secretion of lymphokines like IL2 by helper T-cells directly onto the effector lymphocytes (anti-tumour T-cells in our case) bringing in a high magnitude of selective in vivo amplification of desired T-cells minimising the chances of non-specific amplification of harmful lymphocytes like the anti-idiotypic or the suppressor lymphocytes and phagocytes.An alternative strategy proposed by usB-cells constitutively express MHC class II molecules and can process and present antigens to helper T-cells. Activated T-lymphocytes (including the cytotoxic T-cells) from some species of animals such as rats and humans express MHC class II molecules on their surface and can present antigenic peptides to the helper T-cells. Purified protein derivative is a complex mixture of mycobacterial peptides and can be presented by MHC II bearing cells without any further processing.10 that in vitro activated autologous anti-tumour T-cells bearing MHC II on their surface, if pulsed with PPD and re-infused in a BCG – primed patient, can activate PPD – specific helper T-cells and the focused secretion of lymphokines like the IL-2 can selectively amplify the anti-tumour T-cell response by their proliferation and activation in a specific manner bypassing the suppression exerted by the anti-idiotypic and suppressor cells . It has also been observed in various studies that distant noninjected viral warts at the site away from PPD injections also respond to the therapy indicating that the release of interferon gamma is not just local but a systemic response.11Bacille Calmette – Guerin boosts the immune system to fight not only against tuberculosis but other infections also. A 2018 study suggested that the BCG vaccine reduced yellow fever vaccine viremia by 71% in volunteers in the Netherlands. Two mice studies showed that it reduces the severity of mengovirus  infection.in vitro by inactivated virus or mitogens such as Concanavalin A to express MHC class II molecules on their surface and pulsed with PPD for carrier targeting in vivo. Such PPD – pulsed activated (MHC-II+ve) anti-viral lymphocytes if transfused back into a patient already vaccinated with BCG during childhood or primed with BCG during adulthood 2 weeks before transfusion, could lead to a high magnitude of selective in vivo amplification of specific anti-viral lymphocytes, which can mount adequate and appropriate immune response to get rid of the virus and cure the patient from COVID-19.The prime – boost strategy with the BCG–PPD System described here can also be applied to immunoprophylaxis and immunotherapy of several infectious diseases including COVID-19. The autologous anti-Corona virus B- and T-lymphocytes can be activated in vivo amplification of anti-Corona virus B and T lymphocytes capable of overpowering the pathogen. Thus, PPD could prove to be the red herring in the control of COVID-19.The inactivated SARS CoV2 virus, or its subunit fractions such as the spike proteins, synthetic peptides or new recombinant vaccines against the Corona virus can be made highly immunogenic by conjugating the antigen to PPD and injecting it into BCG vaccinated or BCG primed individuals for immunoprophylaxis. The hypothesis can be tested in an appropriate animal model employing an inactivated animal Corona virus as the vaccine candidate. The lymphokines secreted by such prime – boost immunisation may be conducive to generation of long – lived memory cells granting long-term immunity against the Corona virus. The strong carrier effect induced by linking PPD to SARS COV2 antigen and injecting in a BCG primed patient can lead to a high magnitude of selective


Φ) is ∼10−4. It has been observed that CV confined in bile-salt aggregates becomes highly fluorescent in nature. The enhancement of ‘Φ’ value of CV in bile-salt aggregates is ∼1000 fold compared to that of aqueous buffer medium. It has also been observed that in the presence of different bile-salt aggregates, CV exhibits remarkable enhancement of absorption and fluorescence emission spectral behaviour. The ground state and the excited state binding constant values of CV in the presence of different bile-salt aggregates have been determined. Moreover, the release of the dye molecule from the confined bile-salt aggregates to the aqueous medium has been executed. It has been found that addition of a very minute concentration of KCl salt (100 nm) to the bile-salt aggregates leads to extreme modification of their photophysical properties of CV. The absorption, fluorescence intensity, fluorescence quantum yield, ground state and excited state binding constant values, partition coefficient and aggregation number of CV molecules entrapped in bile-salt aggregates significantly reduces by addition of KCl. This result clearly confirms that CV releases from the confined system to the aqueous medium.In this work, the entrapment of non-fluorescent dye Crystal Violet (CV) in presence of bio-mimetic confined bile-salt aggregates has been studied. The photophysical characteristic properties of CV have been carried out by changing different kinds of hydrophilic head groups and hydrophobic skeletons of bile-salt aggregates . The main aim of this work is to modulate the solubility behaviour, fluorescence properties and elucidation of different kinds of non-covalent interaction of CV confined in bile-salt aggregates. To interpret the result, steady state absorption and fluorescence emission techniques have been employed. In aqueous buffer, the CV molecule is non-fluorescent in nature. The value of fluorescence quantum yield  in presence of bio-mimetic confined bile-salt aggregates has been studied. This bile is composed of different constituents, which are deoxycholic, cholic, chenodeoxycholic, and lithocholic acids. These acids interact with taurine or glycine, forming various complex bile salt derivatives. Bile salts are also called amphiphilic surfactants. Due to the presence of small polar head groups and flexible non-polar tail groups, bile-salts exhibited a rigid structure. Bile-salts have a convex side of their steroid moiety, which is hydrophobic in nature and a concave side, which is made up of hydrophilic polar groups . This facial amphiphilicity imparts a unique property in bile-salts which leads to selective binding with different molecules. This properties of bile-salts can be exploited in the medicinal field.Bile-salts  are easi1–3 Bile salts play an important role in assisting the digestion of lipids and fats inside human system. Apart from these physiological applications, bile salt aggregates play a pivotal role in pharmaceutical and biochemical applications as vehicles for carrying many sparingly water soluble drug molecules. With the increase in demand for eco-friendly biodegradable micro-encapsulators bile salts have been used to dissolve hydrophobic probes making them an excellent host systems.8,9 While selecting an ideal drug carrier one has to consider several biopharmaceutical factors like drug stability, solubility in the gastrointestinal fluids, metabolic stability and sufficient intestinal permeability for absorption of drug molecule. Unfortunately, more than 50% of drug molecules have low aqueous solubility.4 The enterohepatic organotropism of bile-salts convert them into intriguing drug delivery vehicles for targeting these selective drugs and enhancing bioavailability of the drug molecule by improvising their intestinal absorption and metabolic stability. Therefore such compounds are well solubilised in bile salts and thus can be easily transported to different parts of body resulting in increase of their bio-availability.4 Due to their biocompatible and biodegradable nature bile-salts have gained a lot of attention for various drugs, cosmetic materials, vitamins etc. making them very cheap, non-toxic and efficacious drug carrier vehicle for medical applications.5,6 Bile salts have tendency to form micelles and aggregation behaviour beyond their critical micelle concentration (CMC). Bile salt aggregates are typically used as supramolecular host molecules. As a result, several biological active fluorophores (guest) are entrapped in their caged structure and forms encapsulated supramolecular host–guest complex. Numerous photophysical studies of encapsulated molecules confined in bile-salt aggregates are reported in literature.8–18The biological importance of bile-salts is their capability to solubilize fats, monoglycerides, and emulsification of cholesterol, as well as fat-soluble vitamins in a human digestive system.19–22 Crystal violet exhibits potent clinically useful properties and exhibits various pharmacological activities.23–26 CV is one of the most important dye molecule which is used for the histological staining, classifying bacteria (Gram's method), topical treatment of skin infections and wounds like burns, ringworm and eye infections like pink eye, due to its antiseptic, anti-fungal, anti-bacterial properties. From almost 100 years, it has been used for medicinal treatment for anti-angiogenic, anti-trypanosomal, anti-helminthic and anti-tumour activities. Recently it has also been used as a blood additive during the transfusion to inactivate Trypanosoma cruzi (causative agent), for the prevention of the transmission of Chagas' disease.15 CV can also be used for the treatment of Ichthyophthirius multifiliis, which leads to ‘white spot disease’ in fish. Crystal violet  is chemically assigned as tris(4-(dimethylamino)phenyl)methylium chloride. It is an inexpensive cationic dye  shows striking optical behaviour as it exhibits intense visible absorption bands of molar extinction coefficient (εmax) is 105 cm2 M−1 upon excitation. In aqueous medium, CV molecule exhibits weak fluorescence due to very fast rotation of its aromatic rings. The absorption spectrum of CV displays one shoulder band along with the absorption maxima. This characteristic feature of the absorption spectra of CV can be explained on the basis of the existence of two ground state isomers. The two type of ground state isomers  substrates of encapsulated crystal violet inside the aqueous liposome medium. They also confirmed that in presence of liposome, high SERS enhancement of the studied biological probe molecule are encapsulated.28 Revuelta et al. studied the encapsulation of crystal Violet molecule in a biopolymeric matrix composed of pectin and Arabic gum.29 Very limited studies on interaction of CV in confined environments have reported in literature.22,28–30 Therefore, the main aim and motivation of this work is to endeavour the interaction of CV in confinement of different kinds of bile-salt aggregates. Since, CV is non-fluorescent in aqueous medium; therefore another aim of this study is to improve the fluorescence property of CV due to supramolecular interactions in confinement of bile salt aggregates. Therefore, to get more insight and comprehend the interactions of encapsulated complex, the photophysics of CV molecule have been carried out by modulating several kinds of hydrophilic head groups and hydrophobic skeletons of bile-salt aggregates  and to rationalize the location of CV molecule in confined environment. Another major aim of this work is to release the CV molecule from encapsulated bile-salt aggregates to the aqueous medium by addition of foreign substance (non-toxic and green method). This will be possible if the studied CV molecule will exhibits strong fluorescence to non-fluorescence property or in other words, fluorescence turn-on-off property. The detection analysis of the bio-mimetic confined bile-salt aggregates on the studied biologically active CV molecule and its release phenomenon is very much important in biological model systems. Addition of KCl salt perturbs the micellization process of bile-salt aggregates. As a result, CV molecule releases from the confined environments to aqueous medium.Lum 2.−5 (M). Bile-salts such as sodium cholate (NaC), sodium deoxycholate (NaDC), sodium taurocholate (NaTC) and sodium tauroglycocholate (NATGC) were obtained from Loba Chemie. Potassium chloride (KCl) was obtained from Sigma-Aldrich, India. All the bile-salts used of high purity grade (≥97%).Crystal Violet (CV) was purchased from Loba Chemie, India and used as received. The purity of the compounds was more than 97%. Fresh solution of CV was prepared every time to avoid any problem of degradation and aggregation. The concentration of CV solution was maintained at 1 × 10λexi = 550 nm and 590 nm) two different excitation wavelengths were selected since the studied dye molecule displayed shoulder band (550 nm) followed by absorption maxima (590 nm). The emission slit widths were fixed at 15 nm and 15 nm respectively. The scan time was fixed at 250 nm per minute. Fourier transform infrared (FT-IR) spectral data were recorded by PerkinElmer Spectrum 400 instrument, USA in attenuated total reflection (ATR) mode with diamond crystal having resolution of 2 cm−1. FE-SEM image was recorded using Hitachi S4800 instrument, Japan with an acceleration voltage of 10.0 kV. All the experiments were performed at physiological pH value of 7.4 by using 0.01 M phosphate buffer solution.Absorbance measurements were performed by Specord 205 Analytik Jena spectrophotometer, India using 1 cm path length quartz cuvette. The spectra were recorded for 400–800 nm wavelength range. The fluorescence emission spectra of the experimental solution were measured by PerkinElmer LS 55 fluorescence spectrometer, USA using quartz cuvette of a 1 cm path length. Fluorescence spectra were recorded at two different excitation wavelengths  and reference (Rhodamine B), ‘Abs’ denotes absorbance, ‘A’ represents the area under the fluorescence emission, ‘n’ is the refractive index of the solvent used. The subscripts ‘S’ and ‘R’ denotes the corresponding parameters for the CV (sample) and Rhodamine B (reference) respectively. The fluorescence quantum yields of CV in different bile-salt systems were determined by using ‘Rhodamine B' as reference solution in aqueous medium (ΦR = 0.31).32Fluorescence quantum yield values are determined from the fluorescence emission intensity (integrated area) and the absorbance value at the particular wavelength of excitation. The fluorescence quantum yield can be mathematically expressed as:3.et al.27 proposed that the appearance of the shoulder band was due to formation of dimeric structure in which the dimethylamino groups overlap in a head-to-tail fashion resulting in increased hydrophobic interactions which are the driving forces for dimer formation. Garcia-Rio et al.33 explained the presence of CV shoulder band is due to the existence of two ground state isomers in the aqueous medium, one is the pyramidal form (C3 symmetry) or distorted form, which is caused by rotation of phenyl rings and another is propeller structure (D3 symmetry).In phosphate buffer solution, CV showed shoulder band ∼550 nm along with the absorption maxima at 590 nm. The origin of the shoulder band of CV has remained a topic of argument from past many years. Lueck 34,35 The specific interactions or changes in the micro-environment around the dye molecule might occur, which can cause redistribution of electron densities in the chromophore leading to the spectral changes. This result clearly suggested that electrostatic interactions takes places between the cationic dye molecule with the hydrophilic anionic parts of the respective bile-salts. The enhancement of the absorbance value signified that the extent of solubility of CV molecule confined in bile-salt aggregates significantly enhances due to hydrophobic–hydrophobic interaction. Due to the presence of strong hydrophobic aromatic group of CV, it has tendency to dissolve in hydrophobic core of the bile-salts. Therefore, both the electrostatic and hydrophobic interactions occur between CV and bile-salt aggregates. This analysis may also give a preliminary idea that the dye molecule encapsulate to the confined bile-salt aggregates from the aqueous solution. From literature reports, we have also found that various hydrophobic guest molecules are encapsulated to the hydrophobic interiors of the bile-salt aggregates, which caused difference of the absorbance values.11,16–18,34.35On gradual addition of the respective bile-salts, CV molecule undergoes significant bathochromic shift  as well The absorbance value of the CV–bile aggregates astonishingly decreases on progressive addition of lower concentration (100 nM) KCl salt . The decΦ) was very low (∼10−4) at both the excitation wavelengths (λexi = 550 nm and 590 nm). Therefore, the dye molecule present in buffer solution becomes non-fluorescent in nature. Since, the studied molecule showed shoulder band (550 nm) along with the absorption maxima (590 nm) in phosphate buffer as well as in aqueous medium. Therefore, CV molecule was excited at both the selected wavelengths to comprehend the excited state dynamics and the nature of interaction of the fluorophore entrapped in bile-salt aggregates.In phosphate buffer, the studied drug molecule (CV) displayed unstructured fluorescence emission maxima and the fluorescence quantum yield  at both the excitation wavelengths significantly enhanced. This characteristic modification of the emission spectra clearly demonstrated that the microenvironment of the studied molecule inside the bile-salt medium gets modulated compared to that buffer medium. Φ) of CV in different bile-salt aggregates significantly enhanced (∼1000 folds)  . This re31 it has been found that incorporation of salts to the bile-aggregates results more aggregation of the bile-salts, leading to enhancement of the fluorescence intensity and fluorescence quantum yield values. They also explained that addition of salts also responsible for the conformational and structural change of the bile-aggregates.36 But in our case, opposite result was found. Increasing the concentration of KCl salt beyond 100 nM, there is not found any change of the fluorescence intensity and fluorescence quantum yield values. This exciting result may be due to the reason that the studied drug molecule may disrupts CV–bile complex and release from the confined hydrophobic core of the bile-salt aggregates to the hydrophilic regions and/or to the aqueous medium. Similar kind of phenomenon was also obtained from the absorption study. Here, it is important to note that if the drug molecule (CV) releases from the confined bile-aggregates after the addition of small concentration of KCl salt, then the binding constant of the drug–bile aggregates should be significantly lowered.37The addition of lower concentration of KCl salt (100 nM) to the encapsulated bile-salts causes remarkable decrease of fluorescence intensity  and fluo38where, ‘Abuffer’ and ‘Amicelle’ are the absorption intensities of CV in buffer and respective highest micellar concentration of bile-salts. ‘K1’ is ground state 1 : 1 binding constant value of CV–bile aggregates.In order to get more insight the stability of the studied drug molecule (CV) in bile-salt aggregates, the binding constant values of CV molecule was evaluated by non-linear 1 : 1 regression analysis method:The ground state binding constant values were calculated from the absorbance data of CV with different concentration of the respective bile-salts and are tabulated in Similarly, in presence of KCl (100 nM), the binding constant values of CV with varying concentration of CV were also evaluated and tabulated in 38where, ‘Fbuffer’ and ‘Fmicelle’ are the fluorescence intensities of CV in buffer and respective highest micellar concentration of respective bile-salts.  is the excited state 1 : 1 binding constant value of CV–bile aggregates.The excited state binding constant values of CV–bile aggregates in absence of KCl and in presence of KCl were also obtained from the fluorescence intensity data with varying the concentration of bile-salts using the following equation:λexi = 550 nm and 590 nm), the presence of KCl salt suppress the binding interaction between CV–bile aggregates in the excited state. From the analysis of both the ground and the excited state binding studies, it can be clearly demonstrated that addition of salt drives out the drug molecule from the confined hydrophobic region of bile-aggregates to outside. As a result, binding constant values significantly dropped both in ground state and the excited state. The high binding constant or association constant of NaTC is also supported by previously reported work by Bohne et al.39 where association rate constant of different bile salt were observed in order of NaTC > NaDC > NaC.From λexi = 550 nm) is greater compared to excitation of absorption maxima band (λexi = 590 nm). It was also noticed that the extent of binding interaction at the excitation of shoulder band (KP) of the molecule between two different phases (aqueous and confined) is mathematically expressed as following:16,40where, ‘Ct’, ‘Cm’ and ‘Cw’ represents total concentration of dye molecule, concentration of dye bile-salt aggregates and buffer medium respectively. Experimentally, the partition coefficient41 can be determined from absorbance (ground state partition coefficient) as well as fluorescence intensity (excited state partition coefficient) data of CV in buffer with varying concentration of bile-salts using the following equation:16where, ‘I0’, ‘It’ and ‘I∞’ represents the absorption and/or emission intensities of the dye molecule in aqueous buffer medium, at different concentrations  of respective bile-salts and at highest micellar concentrations. ‘KP’ is the partition coefficient value. The partition coefficient values were tabulated in To elucidate the location of the studied drug molecule (CV) at highest micellar concentration of the respective bile-salt aggregates (100 mM), the ground state and excited state partition-coefficient values were evaluated. The partition coefficient (3). This significantly higher values of partition coefficient clearly suggest that the drug molecule resides at the confined environment rather than the aqueous medium.It was observed that magnitude of partition coefficient is very high  from confined environment to the surface. Therefore, the release of drug molecule from the confined environment of bile-salts has been carried out using the fluorescence intensity data. The percentage of the release of CV molecule in different bile salt aggregates are tabulated in From the above −5 M and 100 mM respectively. 0.01% CV molecule was loading in capsules. The encapsulation efficiency was 98%. From FESEM image, the size of the capsule is 50 nm. Fig. S2It is noteworthy to mention that we have kept the concentration of CV molecule and different bile salts as 10Moreover, we have studied the release kinetics of CV molecule encapsulated in different bile salt aggregates with the addition of KCl salts . It has 38where, ‘B’ represents the highest micellar concentration of respective bile-salt at saturation, CMC is the critical micellar concentration.Aggregation numbers of different bile salt systems were calculated using the following equation:44–47 Zana et al.36 have reported that with the increase in concentration of NaCl salt, there is a prominent increases in the aggregation number due to salting out effect. As a result, the hydrophobic interaction enhances, which results decrease in CMC values and increase the aggregation number. But in our case, a completely opposite trend , high ground state and excited state binding constant as well as partition coefficient clearly depicts the dye molecule resides in bile-salt aggregates. The release of the dye molecule from the confined bile-salt aggregates to the aqueous medium was modulated by addition of non-toxic, biodegradable substance (KCl). It has been found that addition of nano-molar concentration of KCl salt to the bile-salt aggregates leads to extreme modification of the photophysical property of CV molecule. The release of the dye molecule was confirmed upon addition of KCl from the decrease in absorption, fluorescence intensity, fluorescence quantum yield, ground state and excited state binding constant values, partition coefficient, percentage of release and aggregation number of CV molecule entrapped in bile-salt aggregates. Therefore, it is perhaps not an overstatement that the work involved in this work might be valuable for potential targeted drug-delivery implications, detection analysis, sensors, and also in physiological systems.In conclusion, the present work deciphers the photophysics and supramolecular interaction of non-fluorescent drug molecule (CV) in presence of varying the hydrophilic head groups and hydrophobic skeletons of four bile-salts . From the UV-vis absorption and fluorescence emission techniques, it is clear that the studied drug molecule (CV) molecule displayed remarkable enhancement in absorption and fluorescence emission spectral behaviour. A significant increase in ‘The authors have no conflict of interest in the publication of this manuscript.RA-011-D0RA06599D-s001


Older people are subject to an increased risk of falling compared to the young, especially during obstacle negotiation. This study aimed to quantify the effects of long-term Tai-Chi Chuan (TCC) practice on the balance control during obstacle-crossing in older people in terms of the inclination angles (IA) of the body’s centre of mass (COM) relative to the centre of pressure (COP), and the rate of change of IA (RCIA). Fifteen healthy older adults who had practised TCC for at least 13 years and 15 healthy controls without any experience in TCC performed obstacle-crossing in a gait laboratory. The TCC group showed significantly greater leading and trailing toe-obstacle clearances but smaller trailing stride lengths when compared to controls. In the sagittal plane, the TCC group showed significantly smaller average anterior IA when the COM was anterior to the COP but greater average posterior IA when the COM was posterior to the COP, with significantly smaller average and peak RCIA over the crossing cycle. Long-term TCC practitioners showed an obstacle-crossing technique for less risk of tripping and better balance control, as indicated respectively by significantly increased toe-obstacle clearances and more posterior COM position relative to the COP with smaller anterior IA and RCIA during leading crossing and greater posterior IA and frontal RCIA at trailing-toe crossing. These benefits appeared to be related to the main features of TCC movements that emphasized maintaining balance during single-leg support and keeping the body weight on the trailing limb during the slow weight-shifting of double-limb support. The older adults were found to increase the foot-clearance6 and increased lead foot-clearance linearly with increasing obstacle height9, apart from slower crossing speed, and shorter step length and heel-obstacle distance compared with young adults8. Increasing foot-clearance to avoid tripping10 may lead to perturbations to balance and whole-body posture changes, which is, in turn, another major contributing factor to falls in older people11. Alterations in whole-body posture may include smaller frontal pelvic motion12, greater variabilities in the lower limb inter-joint coordination13, and altered COM-COP control14, leading to a greater risk of imbalance during obstacle-crossing. Once a trip or loss of balance occurs, failure to recover balance would lead to falls. Therefore, understanding the changes in the balance control of older people is essential in developing and evaluating methods for reducing fall risks.Tripping over obstacles is considered one of the most common causes of falls in older people17. Both the COM-COP inclination angle (IA), namely the angle formed by the COM-COP vector and the vertical vector, and the rate of change of IA (RCIA), i.e., the change of IA over a given short time interval, are sensitive indices for quantifying the actual COM-COP control19. Generally, the greater the IA, the greater the COM-COP vector deviates from the vertical, and the greater the effort  needed to reduce or maintain the IA unless accompanied by an appropriate RCIA for dynamic balance, corresponding to the position and velocity control of the COM described by Pai and Patton20. Increased toe-obstacle clearance during obstacle crossing demonstrated by older people reduces the risk of tripping13. However, the COM-COP control becomes more challenging, requiring extra joint torques to maintain the body’s dynamic stability unless accompanied by an increased RCIA. To compensate for the more difficult COM-COP control when obstacle height was increased, healthy older people tended to reduce the separation between COM and COP in order to reduce the mechanical load on the supporting limb14. Despite this control strategy however, older people still displayed greater IAs with smaller RCIAs in the sagittal plane at leading-toe crossing and demonstrated greater RCIA for the same IA as young controls during the rest of the crossing cycle5. These results suggest that ageing may compromise the ability of whole-body balance control described by the IA and RCIA in older people during obstacle-crossing. Therefore, balance training for safer obstacle-crossing may be needed for older people.The body’s balance control during locomotion has been described by the motion of the body’s COM relative to the COP in various populations during dynamic activities22, multi-factor exercises appear to be more effective in improving balance capability24. These multi-factor exercises can be structured to emphasize muscular strength/endurance training and other functions such as balance/somatosensory reaction and confidence. Tai-Chi Chuan (TCC), an ancient Chinese martial art, has been shown to improve multiple factors in older people31, and is a popular exercise among older people for enhancing their general physical condition. The practice of TCC consists of a series of slow, continuous, gentle motions transitioning from double-limb to single-limb support, thus focusing on dynamic weight shift to a narrower base of support. Participating in long-term TCC exercise can increase muscle strength30, flexibility32, balance29, sensory organization in postural control27, and reduced COP sway area during challenging tasks such as single-leg stance and tandem stance25. These changes are all critical components for preventing falls in older people34. TCC may cause practitioners to modify their gait and movement behaviours, leading them to move more cautiously. These modifications may provide some explanation for the observed reductions in falls35.Although many training programmes addressing single factors, such as resistance, endurance, and feedback training, are available to older people to improve their balance capacity and reduce the risk of falling38. Older people with TCC experience showed significantly larger forward ground reaction forces to propel the body with greater hip flexion of leading limb36, faster crossing velocity with altered pressure distribution in the trailing foot37, and longer periods of single-limb support38 while crossing obstacles. No study has reported the effects of TCC on foot-clearances and the whole-body balance control in terms of IA and RCIA in older people when crossing obstacles of different heights. To form a complete assessment of the TCC effects on balance control relevant to fall risks, data on the COM-COP motions in terms of IA and RCIA from both TCC and non-TCC practitioners are needed.Knowledge of the effects of TCC on the COM-COP motions during obstacle-crossing may help better understand the balance control strategy and plan programmes to prevent fall-related injuries in older people. However, previous studies on older people with TCC experience during obstacle-crossing have focused mainly on the gait modifications in terms of temporospatial parameters, ground reaction forces, foot pressure distribution and joint kinematicsThe purpose of the current study was to quantify the effects of long-term TCC practice and obstacle height on foot-clearances and the whole-body balance control in terms of IA and RCIA during obstacle-crossing in healthy older people. It was hypothesized that healthy older people with long-term experience in practising TCC would show more conservative COM-COP control with greater toe-obstacle clearance than those who did not practise TCC and that these results were not affected by obstacle heights.39 for two-way mixed-design analysis of variance (ANOVA) with one between-subject factor (group) and one within-subject factor (obstacle height) for the comparison of IA and RCIA determined that a projected sample size of 14 subjects for each group would be needed with a power of 0.8 and a medium to large effect size (Cohen's d > 0.5) at a significance level of 0.05. Thus, 15 subjects for each group were considered adequate for the main objectives of the current study.All experiments of the current study were conducted with the approval of China Medical University Hospital Institutional Review Board (IRB No. DMR98-IRB-072). All the experiments and procedures conformed to the Ethical Principles for Medical Research Involving Human Subjects . Fifteen healthy older adults who practised TCC at least 60 min a day and five days a week for thirteen or more years , and fifteen healthy controls who did leisure exercises  for the same period, and matched with the TCC group for sex, age and BMI  participated in the current study with informed written consent. They were all free of neuromusculoskeletal dysfunction and with normal or corrected vision. An a priori power analysis based on pilot results from four subjects for each group using G*POWER18. Three-dimensional marker trajectories were measured using a seven-camera motion capture system  at 120 Hz, and the ground reaction forces (GRF) and the centre of pressure (COP) were measured at 1080 Hz using two forceplates  placed on either side of the obstacle. The test conditions included crossing obstacles of 10%, 20%, and 30% of the subject’s leg length—defined as the distance between the ipsilateral ASIS and medial malleolus—in a random order, with each lower limb leading40. The greatest obstacle height of 30% leg length was selected to cover the range of obstacles one might normally encounter in the daily environment because obstacles higher than 30% leg length are rare in daily activities41. Three obstacle heights enabled the trend of height effects to be determined44. A trial was defined as unsuccessful if the subject hit the obstacle during crossing. In the current study, all the subjects were able to cross the obstacles successfully without hitting the obstacle. Data for three complete crossing trials, each lasting about 30 s, for each lower limb leading were obtained for each obstacle height for each subject. Therefore, a total of 25–30 min was needed for the obstacle-crossing experiment for each subject. A 5-min break was allowed when changing obstacle-height conditions.In a hospital-based gait laboratory, each subject walked on a 10-m walkway at his/her preferred walking speed and crossed an obstacle made of an aluminium tube placed horizontally across a height-adjustable metal frame. The position and height of the obstacle placed in the middle of the walkway were defined by two infrared-retroreflective markers placed on each end of the tube. The poses of the body segments were tracked using 39 infrared-retroreflective markers placed on specific bony landmarks45. The motions of the COM relative to the COP were described using the inclination angles (IA) in the sagittal and frontal planes as follows:PCOM-COP was the vector pointing from the COP to the COM in the laboratory coordinate system  with X pointing in the direction of progression. The RCIA was calculated by differentiating the time trajectories of IA using the GCVSPL method46. The leading limb was taken as the reference limb; thus, positive sagittal and frontal IA indicate a COM position anterior to the COP and away from the COP towards the trailing limb, respectively.The body’s COM position was calculated as the mass-weighted sum of the positions of the COMs of all the body segments using the marker data and segmental inertial properties, which were obtained using a model-based optimization method that minimizes errors between model-predicted and measured COP positions during a number of calibration posturesThe GRF and marker data were used to determine the gait events for the definition of a crossing cycle beginning at the toe-off of the leading limb before crossing T1) followed by the heel-strike of the leading limb after crossing (T3) and toe-off of the trailing limb before crossing (T4), and ending at the heel-strike of the trailing limb after crossing (T6), as well as two critical events of crossing when the leading toe was above the obstacle (T2), and when the trailing toe was above the obstacle (T5)  with crossing speed as a covariate would be used instead for the analysis of the IA- and RCIA-related variables. All significance levels were set at α = 0.05. SPSS version 20  was used for all statistical analyses.A two-way mixed-design analysis of variance (ANOVA) with one between-subject factor (group) and one within-subject factor (obstacle height) was performed to analyze the spatio-temporal parameters, namely crossing speed, stride length, toe-obstacle distance, heel-obstacle distance and toe-obstacle clearance. Previous studies have shown that crossing speed may affect the magnitudes of the IAs during locomotionThere were no statistical interactions between group and obstacle height factors for any of the tested variables. Therefore, only the main effects are reported here. The TCC group showed significantly greater leading and trailing toe-obstacle clearances but smaller trailing stride lengths when compared to the control group Table . No signIn the sagittal plane, both Control and the TCC group showed similar patterns in the IA and RCIA curves but with significant differences in the magnitudes Fig. . The TCCIn the frontal plane, similar patterns in the IA and RCIA curves were found in both Control and the TCC group Fig. . QuantitThe current study aimed to determine the effects of long-term TCC practice and obstacle height on foot-clearances and whole-body balance control during obstacle-crossing in older people, in terms of IA and RCIA of the COM relative to COP. The TCC group was found to cross obstacles with significantly greater leading and trailing toe-obstacle clearances and significantly more posterior COM position relative to the COP throughout the crossing cycle, showing specific COM-COP control. The current results suggest that long-term TCC practitioners crossed obstacles with a more conservative COM-COP control strategy for less risk of tripping and better balance control compared to non-practitioners of TCC.37. Accompanied with the greater toe-obstacle clearance, the TCC group adopted a specific balance control strategy that involved a significantly more posterior COM position relative to the COP throughout the crossing cycle, showing greater posterior IA during the first half of leading-limb crossing (T1–T2), smaller anterior IA during the second half of leading-limb crossing (T2–T3) and DLS (T3–T4), and greater posterior IA during trailing-limb crossing (T4–T5)  with greater toe-obstacle clearance, the TCC group kept the position of the body’s COM more posteriorly relative to the COP, which effectively increased the posterior IA. However, the anterior RCIA did not increase correspondingly, which would require greater joint torques in the stance limb to provide support to the body for the smooth COM-COP movement, as well as for precise control of the swing foot42. During this period, the older TCC practitioners also kept the position of the body’s COM more posteriorly relative to the COP with smaller average anterior IA and smaller posterior RCIA, suggesting a well-controlled, more conservative COM-COP motion for a smoother bodyweight transfer while maintaining dynamic balance48. Note that these observed COM-COP control features in the older TCC practitioners compared to non-TCC controls were not affected by obstacle height as both groups maintained the same crossing speed when crossing obstacles of increasing height. The smooth and stable transfer of the body weight over DLS during obstacle-crossing, the more posterior COM-COP in T2 and T5, and increased foot clearance suggest long-term TCC practitioners have better dynamic balance control during obstacle crossing than non-practitioners, in addition to a reduced risk of tripping.During the DLS phase between the leading heel-strike and trailing toe-off (T3–T4), the COM was controlled within a relatively small range while the COP travelled from the trailing to the leading limb for the bodyweight transfer49. It has been reported that the sum of the percentage duration of fixing, forward, and backward movements was about 60%, and that of single-limb support was almost 70% in TCC training50. These movements emphasize maintaining balance during single-leg support and keeping the body weight more on the trailing limb during the slow weight-shifting of double-limb support, helping increase the muscle strength in the lower extremities and improving whole-body balance30. The current findings in the long-term TCC practitioners suggest that they had an improved ability for the dynamic balance and endpoint (foot) control during obstacle-crossing, most likely related to the training effects of TCC movements.The observed COM-COP control strategies in the TCC group who kept the position of the body’s COM more posteriorly relative to the COP during obstacle-crossing appeared to correspond to the main features of TCC basic movements, which emphasize maintaining the trunk in the upright position with the COM at a more posterior position when moving forward36 and improved standing and walking balance33, no studies have provided quantitative descriptions of the dynamic control (COM relative to the COP) during each phase of the crossing cycle to reveal the general control strategy employed by experienced older TCC practitioners, nor has the current literature reported on such COM-COP controls associated with increased leading and trailing toe-obstacle clearances. Nonetheless, the current study was limited to the dynamic balance control when negotiating fixed obstacles in healthy older adults with long-term TCC experience. It is also worth investigating the benefits of TCC in older people when negotiating unexpected obstacles that are commonplace in real-life conditions and are more challenging to cross. Further studies will be needed to determine the efficacy in older adults with compromised neuromusculoskeletal function, such as frailty, neurological diseases, and/or joint degeneration. Future longitudinal studies will be needed for determining direct correlations between muscle strength and the observed COM-COP control during obstacle-crossing in TCC practitioners. Studies on the joint kinematics and kinetics of the locomotor system associated with the observed COM-COP control strategies adopted by the TCC subjects will be needed to reveal the underlying mechanisms.The current study was the first to document the effects of long-term TCC practice on foot-clearances and the whole-body balance control in terms of IA and RCIA in older people when crossing obstacles of different heights during walking. Although previous studies have reported the positive benefits of TCC training, including increased foot-obstacle clearance during obstacle crossingLong-term TCC practitioners displayed an obstacle-crossing technique with a lesser risk of tripping and better balance control, as indicated respectively by significantly increased toe-obstacle clearances and a more posterior COM position relative to the COP with smaller IA and RCIA during leading-limb crossing, and greater posterior IA and frontal RCIA during trailing-limb crossing compared to non-practitioners of TCC. The observed between-group differences in the COM-COP control appeared to be related to the main features of TCC movements that emphasize maintaining the trunk in the upright position with the COM at a more posterior position when moving forward and keeping the body weight more on the trailing limb during the slow weight-shifting of double-limb support.